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LABORATORY 
TECHNIQUE 

IN 

BIOLOGY  AND  MEDICINE 


LABORATORY 
TECHNIQUE 

IN 

BIOLOGY  AND  MEDICINE 


BY 

E.  V.  COWDRY 

Research  Professor  of  Anatomy  and  Director  Wemae 

Cancer  Research  Laboratoru,  Washington 

University,  St.  Louis 


THIRD  EDITION 


BALTIMORE 

THE  WILLIAMS  &  WILKINS  COMPANY 

1952 


Copyright,  1952 
The  Williams  &  Wilkins  Company 


Made  in  the  United  States  of  America 


Published  1943 

Second  Edition  1948 

Third  Edition  1952 


First  Edition  known  as 
Microscopic  Technique  in  Biology  and  Medicine 


Composed  and  Printed  at  the 
WAVERLY  PRESS.  INC. 

FOR 

The  Williams  &  Wilkins  Compant 
Baltimore,  Md.,  U.  S.  A. 


PREFACE  TO  THE  THIRD  EDITION 

The  request  by  The  Wilhams  &  Wilkins  Company  to  prepare  a  Thh-d  Edition 
so  soon  after  the  Second  Edition  was  pubhshed  (1948)  was  unexpected.  It  would 
have  been  a  simple  matter  for  them  simply  to  reprint  the  Second  Edition  in 
whatever  number  they  thought  that  they  could  sell  within  a  reasonable  period. 
This,  however,  would  have  been  a  short-sighted  policy  for  there  have  been  great 
advances  in  laboratory  technique  since  the  material  for  the  Second  Edition  was 
assembled  in  1947  prior  to  publication  in  1948,  which  advances  should  be  in- 
cluded. In  fact  the  advances  made,  or  reported,  in  the  four  years  1948,  1949, 
1950  and  1951  are  probably  greater  than  those  accomplished  in  any  previous 
10  year  period.  Though  some  of  the  modifications  of  standard  techniques  that 
proved  useful  during  the  war  as  well  as  altogether  new  techniques  were  published 
before  1948,  many,  while  known  to  specialists  in  various  fields,  had  not  been 
gathered  together  in  convenient  form  from  which  laboratory  workers  as  a 
whole  could  select  those  most  hkely  to  be  helpful  in  their  particular  problems. 
It  is  hardly  necessary  to  note  that  the  vast  amount  of  new  work  in  the  four  years 
mentioned,  reflecting  the  appreciation  of  the  value  of  research  in  medicine 
and  biology  gained  in  the  war,  and  implemented  by  an  unprecedented  outpour- 
ing of  funds  for  research,  has  resulted  in  the  discovery  of  new  and  better  means 
of  revealing  the  structure  of  organisms  from  the  highest  to  the  lowest  in  health 
and  disease.  The  electron  microscope  has  found  its  way  into  about  300  labora- 
tories. Satisfactory  methods  for  cutting  the  extremely  thin  sections  required 
have  been  devised.  There  is  a  rapprochement  between  what  one  can  see  at 
very  high  magnification  and  increasing  knowledge  of  molecular  structure  and 
orientation  determined  by  several  methods.  The  phase  microscope  likewise 
has  been  produced  in  quantity  in  this  country.  The  quality  of  moving  pictures 
of  living  cells  has  thereby  been  greatly  unproved.  Thanks  to  the  Atomic 
Energy  Commission  radioactive  isotopes  have  been  made  available.  Plastics 
have  been  introduced  in  great  variety.  Microscopic  localization  of  enzyme 
activities  has  been  advanced.  Microchemistry  has  leaped  ahead.  Quantitative 
analyses  of  extremely  small  amounts  of  material  reduce  the  gap  between  chem- 
istry and  microscopy.  The  separation  and  collection  en  masse  in  a  condition 
suitable  for  analysis  of  certain  cellular  components  has  been  most  helpful.  And 
so  on  ahnost  without  end. 

Obviously  no  single  individual  can  authoritatively  present  these  new  tech- 
niques, as  well  as  mjTiads  of  others  of  great  value,  because  he  cannot  have 
personal  experience  with  all  of  them.  Partly  to  compensate  for  such  limitations 
I  have  included  descriptions  of  some  of  the  key  techniques  written  by  the  in- 
vestigators who  introduced  them,  or  by  others  who  have  had  extensive  experience 
in  their  use.  Most  of  these  descriptions  are  new  while  others  are  revisions  of 
accounts  given  in  previous  editions  of  this  book.     The  name  of  each  contributor 


Vl  PREFACE   TO   THE  THIRD    EDITION 

is  given  followed  by  the  address  (so  that  questions  can  be  asked  if  desired)  and  the 
date  (so  that  it  can  be  seen  at  a  glance  when  it  was  last  checked  for  improve- 
ments). To  all  of  these  friends  I  am  very  grateful.  Proofs  of  their  contributions 
have  not  been  submitted  to  them  for  corrections,  because  there  are  so  many  such 
accounts — ^most  of  them  very  brief — and  the  material  presented  by  them  was 
clear  and  concise.  For  typographical  errors  the  responsibility  is  to  be  shared 
by  me  and  the  publisher.  Most  of  the  text  I  have  written  or  revised  and  in 
doing  so  I  have  relied  heavily  on  the  reference  books  listed  on  page  xxxi. 

It  is  hardly  necessary  to  explain  that  the  policy  is  to  provide  brief  accounts 
of  techniques,  and  leads  to  others,  and  to  make  this  information  quickly  available 
by  alphabetical  arrangement.  It  is  better  to  give  some  data  not  required  by 
experts  than  it  is  to  write  mainly  for  well-informed  but  limited  groups.  Ob- 
viously the  said  experts  must  approach  fields  other  than  their  own  as  beginners 
handicapped  by  unfamiliarity  with  speciahzed  technique  and  equipment.  Be- 
cause the  several  biological  and  medical  sciences  have  so  much  to  offer  each  other 
in  the  way  of  laboratory  technique  this  exposure  of  opportunities  may  facilitate 
inter-specialty  cooperation.  My  thanks  are  due  to  Mrs.  Theresa  Bresnahan  for 
help  in  preparing  the  manuscript. 

E.   V.   COWDRY 

St.  Louis 


COOPERATION 

My  friends  have  generously  contributed  techniques  written  by  them  as 
follows : 

G.  Adolph  Ackerman,  Hinsman  Hall,  Ohio  State  University,  Columbia  10, 
Ohio,  May  24,  1951. 
Auer  Bodies. 
Paul  M.  Aggeler,  University  of  California  Medical  Center,  San  Francisco  22, 
California,  November  15,  1951. 
Blood  Platelets. 
Glenn  H.  Algire,  U.  S.  Public  Health  Service,  Bethesda  Maryland,  June  15, 
1950. 
Transparent  Chamber  Techniques. 
James  W.  Bartholomew,  Department  of  Bacteriology,  University  of  Southern 
California,  Los  Angeles  7,  California,  July  9,  1951. 
Gram  Stains  Mechanism. 
H.  W.  Beams,  Department  of  Zoology,  University  of  Iowa,  Iowa  City,  Iowa, 
September  27   1951. 
Ultracentrifuges. 
R.  Bogoroch,  Department  of  Anatomy,  McGill  University,  Montreal,  Canada, 
September  12,  1951. 
Radioautographic  Technique  (With  C.  P.  Leblond). 
L.  R.  Boling,  Department  of  Anatomy,  Washington  University  School  of 
Dentistry,  St.  Louis  10,  Missouri,  December  16,  1950. 
Teeth  Blood  Vessels. 
Teeth  Decalcification. 
Geoffrey  Bourne,  Department  of  Histology,  London  Hospital  Medical  School, 
London,  England,  November  5,  1951. 
Golgi  Apparatus. 
Mitochondria. 
Marian  Pfingsten  Bryan  (Mrs.  WilhamT.  K.  Bryan),  Department  of  Otolaryn- 
gology, Washington  University  School  of  Medicine,  St.  Louis  10,  Missouri, 
September  20,  1951. 
Ear  Cell  Smears. 
Nasal  Cell  Smears. 
E.  J.  Carey,  Department  of  Anatomy,  Marquette  University  School  of  Medi- 
cine, Milwaukee,  Wisconsin,  1942  (now  deceased). 
Carey's  Method  for  Motor  End  Plates. 
Christopher  Carruthers,  Division  of  Cancer  Research,  Washington  Uni- 
versity, St.  Louis  10,  Missouri,  May  12,  1950. 
Oxidation-Reduction  Potential. 
Vitamins. 
Polarographic  Technique. 

vii 


67B79 


VUl  COOPERATION 

Jane  E.  Cason,  Department  of  Pathology,  Medical  College  of  Alabama,  Bir- 
mingham, Alabama,  January  27,  1951. 
Mallory-Heidenhain  Rapid  One-step  Stain  for  Connective  Tissue. 
Robert  Chambers,  Department  of  Biology,  Washington  Square  College  of  New- 
York  University,  New  York,  New  York,  May  15,  1950. 
Micromanipulation. 
E.  R.  Clark,  Department  of  Anatomy,  University  of  Pennsylvania,  and  the 
Wistar  Institute  of  Anatomy  and  Biology,  Philadelphia,  Pennsylvania,  No- 
vember 28,  1951. 
Transparent  Chamber  Technique. 
Barry  Commoner,  The  Henry  Shaw  School  of  Botany,  Washington  University, 
St.  Louis  5,  Missouri,  November  27,  1951. 
Microspectrophotometry. 
A.  H.  Coons,  Department  of  Bacteriology  and  Immunology,  Harvard  Medical 
School,  Boston,  Massachusetts,  August  31,  1951. 
Antigen  Localization. 
W.P.  CovELL,  Departments  of  Anatomy  and  Otolaryngology,  Washington  Uni- 
versity School  of  Medicine,  St.  Louis  10,  Missouri,  October  26,  1951. 
Ear. 

E.  W.  Dempsey,  Department  of  Anatomy,  Washington  University  School  of 
Medicine,  St.  Louis  10,  Missouri,  February  26,  1951. 

Cholinesterase. 

Dehydrogenase. 

Enzymes. 

Esterases. 

Nucleases. 

Phosphatases. 
W.  T.  Dempster,  Department  of  Anatomy,  University  of  Michigan,  Ann  Arbor, 
Michigan  and  R.  C.  W^illiams,  Department  of  Biochemistry,  University  of 
California,  Berkeley,  California,  June  9,  1950. 

Shadow  Casting. 
0.  H.  Duggins,  Department  of  Anatomy,  Washington  University  School  of 
Medicine,  St.  Louis  10,  Missouri,  May  18,  1950. 

Hairs  (With  Mildred  Trotter). 

F.  Duran-Reynals,  Department  of  Microbiology,  Yale  University  School  of 
Medicine,  New  Haven,  Connecticut,  October  8,  1951. 

Spreading  Factors. 
W.  R.  Earle,  National  Cancer  Institute,  Bethesda,  Maryland,  July  10,  1951. 

Tissue  Culture. 
LXrus  Einarson,  Normal-Anatomisk  Institut,   Aarhus  Universitet,  Aarhus, 
Denmark,  February  27,  1951. 

Gallocyanin-Chromalum  Staining  of  Basophilic  Cell  Structures. 


COOPERATION  IX 

Joseph  A.  Falzone,  Department  of  Anatomy,  Washington  University  School 
of  Medicine,  St.  Louis  10,  Missouri,  October  26,  1951. 
Desoxyribose  Nucleic  Acid,  Method  for  Determination  In  Isolated  Hepatic 

Nuclei. 
Differential  Centrifugation  of  Cell  Particulates. 
Honor  B.  Fell,  Strangeways  Research  Laboratory,  Cambridge,  England,  June 
8,  1951. 
Organ  Culture  in  Vitro. 

F.  H.  J.  FiGGE,  Department  of  Anatomy,  University  of  Maryland  Medical 
School,  Baltimore,  Maryland,  October  10,  1951. 

Porphyrins. 
Hematoporphyrin. 
H.   L  FiRMiNGER,  Pathology  Section,  National  Cancer  Institute,  Bethesda, 
Maryland,  February  9,  1951. 
Carbowax  Embedding. 
E.  D.  Gardner,  Department  of  Anatomy,  Wayne  University  School  of  Medi- 
cine, Detroit,  Michigan,  June  15,  1950. 
Articular  Nerve  Terminals. 
David  Glick,  Department  of  Physiological  Chemistry,  University  of  Minne- 
sota Medical  School,  Minneapolis  14,  Minnesota,  October  17,  1951. 
Linderstr0m-Lang,  Kaj.  U.,  and  Holter  Heinz,  Histochemical  Advances. 
Morris  Goldman,  Department  of  Parasitology,  School  of  Hygiene  and  Public 
Health,  Johns  Hopkins  University,  Baltimore,  Maryland,  January  29,  1951. 
Iron  Hematoxylin  Single  Stain. 

G.  Gomori,  Department  of  Medicine,  University  of  Chicago,  Chicago,  lUinois, 
May  7,  1950. 

Gomori's  Method  for  Reticulum  and  Acid  Phosphatase. 
A.  R.  Gopal-Ayengar,  Barnard  Free  Skin  and  Cancer  Hospital,  St.  Louis, 
Missouri,  September  10,  1946  (now  Tata  Memorial  Hospital,  Bombay). 
Chromosomes. 
Hyaluronic  Acid. 
H.  S.  N.  Greene,  Laboratory  of  Pathology,  Yale  University  School  of  Medi- 
cine, New  Haven,  Connecticut,  September  11,  1951. 
Anterior  Chamber  Transplantation. 
Joan  Haberman,  Parkland,  Washington,  March  10,  1951. 

Anethole  Clearing  Agent. 
J.  D.  Hamilton,  Department  of  Medical  Research,  University  of  Western  On- 
tario, London,  Canada,  February  13,  1951. 
Cell  Measurement,  The  EUiptometer. 
N.  L.  Hoerr,  Department  of  Anatomy,  School  of  Medicine,  Western  Reserve 
University,  Cleveland  6,  Ohio,  November  28,  1951. 
Frozen -Dehydration  Method. 
Isolation  of  Mitochondria. 


X  COOPERATION 

R.  D.  HoTCHKiss,  The  Rockefeller  Institute  for  Medical  Research,  New  York 
21,  New  York,  November  14,  1951. 
Polysaccharides. 
M.  H.  Knisely,  Department  of  Anatomy,  University  of  South  Carolina,  Charles- 
ton, South  Carolina,  June  27,  1950. 
Quartz  Rod  Technique  for  Illuminating  Living  Organs. 
N.  B.  KuRNiCK,  Department  of  Medicine,  Tulane  University,  New  Orleans  12, 
Louisiana,  January  31,  1951. 
Aceto-Orcein-fast  Green. 
Edward  L.  Kuff,  Department  of  Anatomy,  Washington  University  School  of 
Medicine,  St.  Louis,  Missouri,  December  18,  1951. 
Nucleic  Acid-Dye  Interactions. 
A.  I.  Lansing,  Department  of  Anatomy,  Washington  University  School  of 
Medicine,  St.  Louis  10,  Missouri,  October  5,  1951. 
Collagenic  Fibers. 
Elastic  Fibers. 
A.  Lazarow,  Department  of  Anatomy,  Western  Reserve  University  School  of 
Medicine,  Cleveland,  Ohio,  November  28,  1951. 
Separation  of  Cell  Components  by  DiflFerential  Centrifugation. 
C.  P.  Leblond  and  R.  Bogoroch,  Department  of  Anatomy,  McGill  University, 
Montreal,  Canada,  September  12,  1951. 
Radioautographic  Technique. 
R.  D.  LiLLiE,  Division  of  Pathology,  National  Institute  of  Health,  Bethesda, 
Maryland,  May  8,  1950. 
Azure  or  Toluidin  Blue  Eosin. 

A.  M.  Lucas,  U.  S.  Regional  Poultry  Research  Laboratory,  East  Lansing,  Michi- 
gan, August  10,  1951. 

Cilia  (with  M.  S.  Lucas). 
M.  S.  Lucas,  Department  of  Biological  Science,  Michigan  State  College,  East 
Lansing,  Michigan,  August  10,  1951. 
Cilia  (with  A.  M.  Lucas). 
Plastics. 

B.  J.  LuYET,  Department  of  Biology,  St.  Louis  University,  St.  Louis,  Missouri, 
January  15,  1951. 

Revival  after  Ultra  Rapid  Cooling. 

C.  C.  Macklin,  Department  of  Anatomy,  University  of  Western  Ontario,  Lon- 
don, Ontario,  Canada,  November  28,  1951. 

Alveolar  Epithelium.  Gash   Irrigation  Recovery  Method 

Alveolar  Fluid.  for  Lung  CeUs  (GO- 

Alveolar  Foam  Cells.  Heart  Failure  Cells. 

Alveolar  Pores.  Lungs,  Uncollapsed,  Fixation. 

Ammoniacal  Silver.  Pneumonocytes. 

Aquax.  Silver  Lineation  on  Pulmonary  Al- 

Bronchiolar  Epithelium.  veolar  Walls. 


COOPERATION  XI 

Carmine  Dusting.  Tissue  Phagocytes. 

Dust  Cells.  Vacuoloids. 

Wash-out  Recovery  Method. 

Paul  Masson,  Department  of  Pathology,  University  of  Montreal,  Montreal, 
Canada,  October  24,  1951. 
Masson's  Trichrome  Stain. 
Morris  Moore,  Barnard  Free  Skin  and  Cancer  Hospital,  St.  Louis,  Missouri, 
December  10,  1951. 
Fungi. 
Norman  Moskowitz,  Department  of  Zoology,  University  of  Pennsylvania, 
Philadelphia,  Pennsylvania,  January  24,  1951. 
Protein  Silver  for  Staining  Protozoa. 
J.  L.  O'Leary,  Department  of  Neuropsychiatry,  Washington  University  School 
of  Medicine,  St.  Louis  10,  Missouri,  May  8,  1950. 
Golgi-Cox  Method. 
Golgi  Method,  Quick. 
O'Leary's  Brazilin  Method. 
Jean  Oliver,  Department  of  Pathology,  State  University  of  New  York,  Brook- 
lyn 2,  New  York,  September  4,  1951. 
Kidney. 
Donald  L.  Opdyke,  Department  of  Anatomy,  Washington  University  School 
Medicine,  St.  Louis  10,  Missouri,  November  8,  1951. 
Keratohyalin  Granules,  Separation  and  Analysis. 
Robertson  Pratt,  University  of  California,  College  of  Pharmacy,  San  Fran- 
cisco, January  29,  1951. 
Triphenyltetrazolium  Chloride. 
Eugene  Roberts,  Division  of  Cancer  Research,  Washington  University,  St. 
Louis  10,  Missouri,  July  15,  1951. 
Paper  Chromatography. 
T.  B.  Rosenthal,  Department  of  Anatomy,  Washington  University  School  of 
Medicine,  St.  Louis  10,  Missouri,  June  6,  1951. 
Fluorescence  Microscopy. 
Radioactive  Isotopes. 
Electron  Microscopy. 
C.  H.  Sawyer,  Duke  Hospital,  Durham,  North  Carolina,  December  16,  1950. 

Safranin-Light  Green. 
Francis  0.  Schmitt,  Department  of  Biology,  Massachusetts  Institute  of  Tech- 
nology, Cambridge,  Massachusetts,  May  19,  1950. 
Polarization  Optical  Method. 
Gordon  H.  Scott,  Department  of  Anatomy,  Wayne  University  School^of  M«di-^ 
cine,  Detroit,  Michigan,  January  16,  1951.  yWo^'^^'  '"^  '--    /^ 

Altmann-Gersh  Frozen  Dehydration  Method.  /^,      ^s  Hq     "f^py 

Microincineration.  !>•  ^  ^  \  '^ 

Cryostat.  /  ^^'  /   i  r* 

-if   \  ■        ;■  ■'•         /    ^~: 


Xll  COOPERATION 

W.  M.  Shanklin,  American  University  of  Beirut,  Beirut,  Lebanon,  March  30, 
1951. 
Pineal. 

Silver  Diaminohydroxide  after  Sensitizing  with  Sodium  Sulfite  for  Neu- 
roglia. 
R.  E.  Stowell,  Department  of  Oncology,  University  of  Kansas  Medical  Center, 
Kansas  City  2,  Kansas,  January  19,  1951. 
Photoelectric  Microphotometer. 
Charlotte  M.  Street,  Department  of  Anatomy,  Cornell  University  Medical 
College,  New  York,  New  York,  May  21,  1951. 
Papanicolaou  Techniques  in  Exfoliative  Cytology. 
Lloyd  E.  Thomas,  Department  of  Biochemistry,  University  of  Missouri  School 
of  Medicine,  Columbia,  Missouri,  July  8,  1951. 
Arginine  Reaction. 
Mildred  Trotter  and  O.  H.  Duggins,  Department  of  Anatomy,  Washington 
University  School  of  Medicine,  St.  Louis  10,  Missouri,  May  8,  1951. 
Hairs. 
L.  F.  Wicks,  Veterans  Administration  Hospital,  Jefferson  Barracks,  Missouri, 
February  1,  1951. 
Hydrogen  Ion  Indicators. 
Piccolyte  Resins. 
G.  B.  WiSLOCKi,  Department  o  Anatomy  Harvard  Medical  School,  Boston  15, 
Massachusetts,  March  8,  1951. 
Placenta. 
R.  C.  Williams,  Department  of  Biochemistry,  University  of  Cahfornia,  Berkeley, 
California,  June  9,  1950. 

Shadow- Casting  (with  W.  T.  Dempster) 


PREFACE  TO  THE  FIRST  EDITION 

What  appeared  altogether  impossible  twenty-five  years  ago  has  in  several 
cases  been  attained  by  improvements  in  technique.  Who  would  have  believed 
at  that  time  that  ultramicroscopes  would  now  be  manufactured  in  quantity, 
built  without  any  optical  lenses,  and  capable  of  revealing  a  world  of  structures 
quite  beyond  their  ken?  Who  would  have  thought  that  a  whole  series  of  dif- 
ferent atoms  could  be  tagged  and  their  distribution  to  the  several  tissues,  when 
introduced  into  the  body,  accurately  measured?  Who  would  have  anticipated 
the  significant  and  unexpected  new  developments  which  have  been  made  in 
polarization  optical  methods?  Had  we  been  told  twenty-five  years  ago  that  the 
cell  itself  can  be  broken  up  into  parts  several  of  which  can  be  collected  in  quantity 
and  chemically  analyzed,  we  would  have  been  incredulous.  All  this  and  more 
has  been  achieved  as  a  result  of  team  work  between  the  biological  and  physical 
sciences.     And  we  may  believe  that  more  surprises  are  in  store. 

Yet  some  of  us  individually  are  still  extraordinarily  conservative  in  the 
methods  we  use.  The  possibilities  of  improving  old  techniques,  of  replacing 
some  of  them  by  new  ones  and  of  reljdng  more  upon  microchemical  and  physical 
procedures  are  not  explored  as  they  should  be.  The  purpose  of  this  book  is  to 
extend  the  horizon  by  exposing  in  an  introductory  way  a  few  of  the  many  oppor- 
tunities awaiting  workers  in  biology  and  medicine  interested  in  the  minute  struc- 
ture of  living  things.  Success  will  depend  upon  ability  to  anticipate  and  meet 
the  needs  of  those  likely  to  consult  it.  Definite  information  about  specific 
matters  is  likely  to  be  more  in  demand  than  general  statements.  The  latter  are 
limited  to  a  few  pages  and  deal  with  "choice  of  methods"  and  "organization  of 
laboratory." 

Some  may  turn  to  the  names  of  the  structures  in  which  they  happen  to  be 
most  interested  at  the  moment — Nissl  Bodies,  Nerve  Fibers,  Capillaries  and  so 
forth — on  the  off  chance  of  finding  some  useful  hints  as  to  methods  better  adapted 
for  their  microscopic  study,  the  most  likely  experimental  errors  and  so  on.  Be- 
cause the  range  of  cells,  parts  of  cells,  tissues,  organs  and  systems  is  obviously 
so  immense,  mention  is  only  possible  of  a  small  proportion  of  them  so  that  much 
depends  on  the  selection  made. 

Others  may  seek  information  under  the  headings  of  elements  such  as  Iron, 
Potassium  and  Calcium,  of  enzymes  Hke  Pepsin  and  Phosphatase  and  of  many 
other  components  of  living  material.  It  is  difficult  to  draw  the  line  but  most  of 
those  that  can  be  localized  microscopically  are  mentioned,  likewise  techniques 
for  the  determination  of  permeability,  viscosity,  pH  and  other  properties  of 
tissues. 

It  often  happens,  however,  that  data  are  required  about  a  particular  technique, 
which  the  workers  are  using  or  expect  to  use,  and  which  is  known  to  them  by 
the  names  of  those  who  discovered  it,  as  for  example  the  methods  of  Giemsa  and 

xiii 


XIV  PREFACE   TO   THE    FIRST   EDITION 

Mallory.  Consequently  information  also  must  be  supplied  under  various  names 
though  this  is  usually  less  satisfactory  than  under  subjects.  A  very  annoying 
handicap  is  the  host  of  synonyms  for  dyes.  Being  ignorant  of  chemistry,  I 
have  with  confidence  listed  those  given  by  Dr.  H.  J.  Conn.  Many  more  will  be 
found  in  The  Colour  Index  of  the  Society  of  Dyers  and  Colourists. 

Since  all  are  busy  people,  time  is  a  factor  and  they  will  wish  to  dig  out  what 
they  want  as  directly  and  quickly  as  possible.  It  is  for  this  reason  that  every- 
thing is  listed  alphabetically.  Obviously  this  book  can  be  nothing  more  than 
a  brief  entr6  to  microscopic  technique.  Therefore,  numerous  references  to  the 
literature  are  supplied  for  follow  up.  Again  to  save  time,  these  are  given  each 
in  its  appropriate  place,  thus  avoiding  the  necessity  of  turning  the  pages  and 
locating  them  in  a  large  bibliography.  But  no  attempt  is  made  to  trace  the 
techniques  to  their  original  exponents  and  to  apportion  credit  for  numerous 
modifications.  Often  the  most  recent  and  accessible  reference  is  provided  re- 
lying on  the  author  to  state  history  fairly.  Evidently,  in  order  to  keep  up  to 
date  as  to  methods,  the  reader  must  repeatedly  consult  the  latest  issues  of  many 
journals.  Stain  Techn.;  J.  Lab.  &  Clin.  Med.;  J.  Tech.  Meth.;  Bull.  d'Hist. 
Appl.;  and  Zeit.  f.  mikr.  Tech.  are  particularly  valuable. 

Finally  I  wish  to  thank  my  colleagues  for  their  help,  particularly  Drs.  L.  R. 
Boling,  C.  Carruthers,  William  Cramer,  Morris  Moore,  J.  L.  O'Leary,  W.  L. 
Simpson,  R.  E.  Stowell,  Lester  Wicks  and  Dr.  H.  J.  Conn,  Chairman  of  the 
Biological  Stain  Commission,  who  very  kindly  read  the  manuscript  and  made 
several  useful  suggestions. 


CHOICE  OF  METHODS 

The  selections  will  depend  on  several  considerations.  The  first  is  what  one 
wants  to  discover.  Many  investigators  in  biology  and  medicine  wish  to  obtain 
more  information  about  structural  components  of  the  body  whether  gross,  micro- 
scopic or  submicroscopic  in  size.  They  desire  to  obtain  further  data  on  the 
physical  and  chemical  properties  of  these  components  whether  solid  or  fluid  in 
different  physiological  stages  of  activity  and  in  disease  in  both  man  and  in 
animals.  They  are  interested  in  subjects,  not  personalities,  so  that  in  this 
alphabetically  arranged  presentation  the  names  of  individuals  are  seldom  listed. 
Emphasis  is  given  to  subjects.  After  all  the  problems  continue  while  the  names 
of  generation  after  generation  of  individuals  fade  out. 

Those  who  perchance  may  consult  this  book  will  need  help  at  two  levels.  It 
may  be  a  simple  question  of  the  nature  of  some  dye,  or  the  composition  of  some 
solution,  or  the  making  of  a  well  known  test,  or  it  may  be  the  selection  of  a 
technique  to  be  employed  in  a  comprehensive  series  of  experiments.  In  the 
latter  case  it  involves  a  major  decision.  It  is  necessary  not  only  to  choose  the 
technique  most  likely  to  lead  to  the  answer  but  also  give  some  thought  to  the 
equipment  required  and  the  training  demanded  for  its  proper  use. 

A  few  leading  references  to  the  most  recent  utilization  of  the  technique  in 
question  may  be  helpful.  But  to  discuss  the  history  of  its  development  and  to 
assess  priority  is  not  attempted.  A  complete  discussion  of  the  literature  may  in- 
deed constitute  a  handicap  by  providing  an  excuse  for  doing  nothing.  To  be 
stuffed  with  information  may  give  a  feeling  of  frustration. 

Some  chemical  and  physical  procedures  are  well  within  the  reach  of  people 
who  are  neither  chemists  nor  physicists  while  others  will  simply  lead  them 
beyond  their  depth  into  futility.  It  is  equally  true  that  well  trained  chemists 
and  physicists  are  Ukely  to  fail  to  reahze  the  complexity  of  vital  processes  and  to 
make  little  progress  through  ignorance  of  physiology  and  pathology.  Conse- 
quently one  should  never  hesitate  to  seek  advice  from  friends  in  other  depart- 
ments. 

1.  To  Examine  Directly  in  Vivo 

The  ideal  arrangement  is  to  look  into  the  body  and  to  study  its  parts  as  they 
function  without  causing  any  disturbance.  With  protozoa  and  certain  small 
transparent  invertebrates  this  is  relatively  simple.  The  web  of  a  frog's  foot  is 
thin  and  can  easily  be  looked  through  without  seriously  interfering  with  the 
frog.  Some  other  parts  of  the  bodies  of  various  aquatic  lower  forms  lend  them- 
selves to  direct  examination  in  vivo ;  but  there  are  definite  limitations  in  such  a 
study  of  what  is  going  on  in  the  human  body.  It  is  possible  to  peer  into  the 
various  apertures  but  to  get  close  enough  to  the  living  tissues  to  use  high  mag- 
nifications is  not  feasible.     The  cornea  and  lens  of  the  eye  are  transparent  and 

XV 


XVI  CHOICE    OF   METHODS 

much  valuable  information  can  be  secured  by  direct  examination  of  the  retinal 
blood  vessels.  Even  here  their  distance  from  the  surface  is  considerable  and 
magnification  is  therefore  limited.  As  far  as  we  know  at  present  the  best  that 
can  be  done  is  to  take  advantage  of  a  discovery,  made  by  Lombard  (W.  P.,  Am. 
J.  Physiol.,  1911-12,  29, 335-362)  that  the  epidermis  can  be  rendered  transparent 
by  the  addition  of  a  little  highly  refractile  oil  without  noticeably  injuring  it  or 
disturbing  the  underlying  tissues.  By  this  means  the  blood  vessels  of  the  dermal 
papillae  in  the  fold  of  skin  over  the  nail  bed,  which  are  very  near  to  the  surface, 
can  be  studied  directly  at  fairly  high  magnification  and  over  long  periods  of  time 
thus  permitting  the  making  of  excellent  pictures.  See  review  of  literature  by 
Wright,  I.  S.  and  Duryee,  A.  W.,  Arch.  Int.  Med.,  1933,  52,  545-575. 

That  the  lymphatics  in  the  human  skin  can  be  made  visible  in  vivo  by  the 
injection  of  small  amounts  of  Patent  Blue  V  has  been  demonstrated  by  Hudack, 
S.  S.  and  McMaster,  P.  D.,  J.  Exp.  Med.,  1933,  57,  751-774.  The  vessels  in 
the  ears  of  living  mice  can  readily  be  seen  without  any  surgical  procedure.  It 
is  even  possible  to  directly  watch  the  dye,  Chicago  blue,  after  intravenous 
injection  elsewhere  in  the  body,  leak  out  into  the  tissues  especially  through  the 
walls  of  the  venules  (Smith,  F.  and  Rous,  P.,  J.  Exp.  Med.,  1931,  54,  499-514). 
Ideas  as  to  the  relative  hydrogen  ion  concentrations  of  some  of  the  tissues  visible 
from  without  can  be  secured  by  the  injection  of  Hydrogen  Ion  Indicators  (Rous, 
P.,  J.  Exp.  Med.,  1925,  41,  739-759).  The  opportunities  are  many  especially 
in  animal  experimentation. 

Another  way  to  examine  structure  in  vivo  is  to  record  the  structure  by  x-ray 
photographs  and  to  magnify  the  photographs,  see  Microradiographic  examina- 
tion. 

2.  To  Examine  through  Windows  in  Vivo 

The  construction  of  windows  in  the  skin  or  body  wall  through  which  the 
tissues  can  be  examined  in  vivo  is  a  less  ideal  technique  because  it  involves 
surgical  interference  with  the  body.  In  the  most  used  of  these  techniques  a  hole 
is  made  through  a  rabbit's  ear  from  one  surface  to  the  other.  A  glass  chamber 
is  then  sewed  into  the  hole  in  such  a  way  that  a  blood  vessel  is  included  between 
a  thin  layer  of  glass  (serving  as  a  cover  glass)  and  a  thicker  one  serving  as  a  slide. 
After  a  time  the  epidermis  adheres  to  the  edges  of  the  chamber  and  blood  vessels, 
nerves  and  other  tissues  grow  into  it  where  they  can  be  studied  under  oil  immer- 
sion objectives.  This  technique  was  first  reported  by  Sandison  (J.  C,  Anat. 
Rec,  1924,  28,  281)  working  under  Dr.  E.  R.  Clark  at  the  University  of  Penn- 
sylvania. It  has  since  been  very  greatly  improved  (Clark,  E.  R.,  et  al.,  Anat. 
Rec,  1930,  47,  187-211  and  Abell,  R.  G.,  and  Clark,  E.  R.,  Anat.  Rec,  1932, 
53,  121-140)  by  the  introduction  of  "round  table"  and  "moat"  chambers. 

To  place  a  window  in  the  wall  of  the  skull  and  to  observe  what  is  going  on 
within  has  been  done  with  more  or  less  success  on  several  occasions.  The  tech- 
nique devised  by  Forbes  (H.  S.,  Arch.  Neurol,  and  Psych.,  1928,  19,  75)  permits 
direct  observation  at  low  magnification  of  the  blood  vessels  over  the  cerebral 


CHOICE    OF   METHODS  XVU 

convolutions  with  so  little  injury  that  their  behaviour  in  various  experimental 
conditions  can  be  investigated  (see  also  Clark,  E.  R.,  and  Wentsler,  N.  E.,  Proc. 
Assoc.  Res.  Nerv.  and  Ment.  Dis.,  1937,  18,  218-228).  Through  a  window  in 
the  thoracic  wall  Wearn  and  his  associates  (Weam,  J.  T.  et  al..  Am.  J.  Physiol., 
1934,  109,  236-256)  have  similarly  studied  the  pulmonary  arterioles  and  capil- 
laries.    They  employed  a  fused  quartz  cone  to  conduct  light  to  the  tissue. 

Other  investigators  have  availed  themselves  of  the  natural  window,  the 
cornea,  through  which  what  goes  on  immediately  within  it  in  the  anterior  cham- 
ber of  the  eye  can  be  observed.  Several  tissues  have  been  successfully  trans- 
planted into  this  chamber.  Perhaps  the  most  dramatic  is  the  behavior  of  trans- 
planted uterine  mucosa  in  the  rhesus  monkey.  In  it  the  menstrual  changes 
can  be  seen  in  detail  and  the  influence  of  hormones  noted  (Markee,  J.  E.,  Con- 
trib.  to  Embryol.,  Carnegie  Inst,  of  Washington,  1940,  28,  219-308).  For  some 
kinds  of  work  the  fact  that  the  tissue  fluid  (aqueous  humor)  in  this  chamber 
differs  from  others  in  the  same  animal  by  the  absence  of  certain  species  specific 
growth  inhibiting  factors  is  a  priceless  asset.  Thus  Greene  (H.  S.  N.,  Science, 
1938,  88,  357-358)  was  able  to  grow  pieces  of  human  cancers,  which  ordinarily 
quickly  die  in  other  species,  in  the  anterior  chambers  of  the  eyes  of  some  mam- 
mals. The  existence  of  a  barrier  protecting  this  fluid  against  the  entry  of  anti- 
bodies from  blood  plasma  and  thus  making  possible  the  growth  of  tumor  trans- 
plants, while  all  other  tissues  are  resistant  to  their  growth,  has  recently  been 
emphasized  (Saphir,  O.,  Appel,  M.  and  Strauss,  H.  A.,  Cancer  Res.,  1941,  1, 
545-547). 

In  order  to  view  the  less  accessible  living  tissues,  techniques  have  been  devised 
that  include  opening  the  body  and  partly  withdrawing  the  organ  so  that  it  can 
be  placed  on  the  stage  of  a  microscope  but  with  circulation  and  nerve  supply 
intact  and  adequate  regulation  of  temperature  and  humidity.  Particularly 
fruitful  has  been  the  direct  observation  through  oil  immersion  objectives  of 
secretion  by  acinous  cells  of  the  Pancreas  by  Co  veil  (W.  P.,  Anat.  Rec,  1928, 
40,  213-223)  and  of  islet  cells  by  O'Leary,  (J.  L.,  Anat.  Rec,  1930,  45,  27-58). 
Thus  the  influence  of  drugs  on  the  secretory  process  can  now  be  followed  in 
minute  detail. 

Knisely  (M.  H.,  Anat.  Rec,  1936,  64,  499-523;  65,  23-50)  has  perfected  a 
technique  for  the  study  of  the  living  Spleen  at  somewhat  lower  magnification. 
The  essential  features  are  slight  displacement  of  the  spleen  so  that  it  can  be 
transilluminated  by  light  delivered  through  a  quartz  rod.  This  allows  for  the 
first  time  direct  examination  of  the  behavior  of  the  venous  sinuses.  Undoubt- 
edly the  Quartz  Rod  technique  will  be  of  great  service  in  providing  light  for 
similar  examination  of  other  organs. 

3.  To  Study  the  Arrangement  of  Parts  in  the  Body 

Since  the  body  is  structurally  so  very  complex  it  is  often  illuminating  to  view 
parts  of  it  in  their  normal  shape  and  size  but  unobscured  by  all  the  neighboring 
components.     There  are  several  ways  by  which  this  can  be  accomplished. 


XVlll  CHOICE    OF   METHODS 

The  first  method  of  Reconstruction  from  serial  sections  is  well  known.  Briefly 
stated  the  particular  tissue,  organ  or  system  is  outlined,  as  it  appears  in  section 
after  section,  at  the  desired  magnification  on  sheets  of  material  of  uniform  and 
carefully  selected  thickness.  The  outlined  areas  are  then  cut  out  and  when 
superimposed  they  constitute  a  reconstruction  of  the  original  structure.  This 
technique  is  tedious  but  it  may  reveal  topographical  relations  that  can  be  dis- 
covered by  no  other  means. 

The  second  kind  of  technique  is  to  make  casts  of  vascular,  respiratory  and 
other  lumina.  Woods'  metal,  formerly  used  for  this  purpose,  has  now  been 
almost  displaced  by  Celloidin  and  other  substances.  The  surrounding  tissue  is 
freed  from  the  cast  by  digestion  in  concentrated  hydrochloric  acid  and  gentle 
brushing  away  in  a  stream  of  water.  Very  beautiful  Corrosion  preparations  of 
the  lungs  and  kidneys  have  been  obtained  by  this  method  which  should  be  more 
widely  employed. 

The  third  is  by  Maceration  to  soak  the  organs,  without  previous  preparation, 
in  fluids  that  either  digest  away  the  tissues  which  it  is  desired  to  eliminate  or 
loosen  their  connections  with  those  under  investigation,  which,  latter,  can  then 
be  individually  examined.  Techniques  of  this  sort  are  the  only  available  means 
for  the  isolation  of  individual  seminiferous  and  renal  tubules.  Oliver's  researches 
on  the  kidney  illustrate  the  value  of  reconstruction  and  maceration  in  pathology. 
Only  three  other  examples  will  be  submitted.  Thyroid  follicles  can  be  isolated 
by  maceration  (Jackson,  J.  L.,  Anat.  Rec,  1931,  48,  219-239).  Their  study  as 
individuals  provides  data  as  to  size  and  shape  only  obtainable  otherwise  by  the 
tedious  examination  of  serial  sections.  The  Epidermis  is  so  tightly  bound  to 
the  underlying  dermis  that  separation  is  extremely  difficult;  but,  after  treatment 
of  skin  with  dilute  acetic  acid,  the  attachment  is  loosened  and  the  epidermis  can 
readily  be  removed  as  a  complete  sheet  of  tissue  which  can  be  stained,  made 
transparent  and  examined  as  a  whole  mount.  Opportunities  are  thus  afforded 
for  the  detection  of  regional  differences  which  might  not  be  located  even  by  pains- 
taking study  of  sections  and  the  making  of  mitotic  counts  is  greatly  facilitated. 
By  macerating  in  the  same  fashion  the  nasal  mucous  membrane  covering  the 
septum  can  also  be  removed  for  study.  Perhaps  still  other  epithehal  sheets  can 
be  similarly  isolated.  However  such  sheets  are  of  Httle  value  for  chemical 
analysis  because  of  the  action  of  the  acetic  acid.  Fortunately  it  has  been  found 
that  the  epidermis  may  also  be  quickly  loosened  by  simply  heating  the  skin 
to  50°C.  when  it  can  be  peeled  off  like  the  covering  of  a  scalded  tomato  (Baum- 
berger,  J.  P.,  Suntzeff,  V.  and  Cowdry,  E.  V.,  J.  Nat.  Cancer  Inst.,  1942,  2, 
413-423). 

There  is  still  another  alternative.  Instead  of  simply  omitting  the  unwanted 
material  by  reconstructing  only  the  structures  chosen  for  demonstration,  or  of 
removing  the  material  by  corrosion  or  maceration,  it  can  be  left  in  and  rendered 
transparent  so  that  it  does  not  obstruct  the  view.  After  marking  the  particular 
structures  by  vital  dyes  or  other  means  the  whole  tissue  is  cleared  by  the  method 
of  Spalteholz  or  Schultze.     These  techniques  give  admirable  results  in  the  study 


CHOICE    OF   METHODS  XIX 

of  Cartilaginous  Skeletons,  Ossification  centers,  Blood  Vessels  and  so  on  almost 
without  end. 

4.  To  Employ  the  More  Routine  Method  of  Fixation  and  Staining 

Here  there  is  wide  latitude  of  choice.  For  some  purposes  thin  Smears  are 
just  fixed  and  stained  without  resort  to  sectioning.  In  the  case  of  the  denser 
tissues  which  must  be  cut  in  sections  one  first  has  to  decide  which  of  many 
Fixatives  is  likely  to  give  the  best  results.  Then,  whether  fixation  is  to  be  by 
immersion  or  injection  has  to  be  determined. 

The  purpose  of  fixation  by  vascular  injection  is  to  bring  the  fixative  into  close 
contact  with  the  tissues  as  they  exist  in  the  freshly  killed  animal  without  sub- 
jecting them  to  mechanical  trauma  or  disturbing  their  topographic  relations  one 
to  another.  In  choosing  this  procedure  it  is  well  to  remember:  (1)  That  it  is 
usually  necessary  first  to  wash  out  most  of  the  blood  by  perfusion  with  physio- 
logical salt  solution  for  otherwise  the  fixative  often  clogs  the  vessels.  This  wash- 
ing unfortunately  also  facilitates  chemical  change.  (2)  That,  even  when  it  is 
not  done,  the  concentration  of  the  fixative  about  the  cells  is  gradually  increased 
and  at  different  rates,  rapidly  in  highly  vascularized  tissues  (kidney,  liver,  etc.) 
and  very  slowly  in  avascular  ones  (epidermis,  cornea  and  cartilage).  The  time 
for  chemical  change  before  fixation  is  therefore  variable  depending  upon  the 
tissue.  (3)  That  the  pressure  may  bring  about  an  unnatural  swelling  of  the 
tissues  so  located  that  they  can  enlarge,  especially  the  abdominal  organs  as 
compared  with  brain  and  bone  marrow  which  are  confined  within  rigid  walls. 

Fixation  by  immersion  is  the  usual  and  easiest  method.  If  small  pieces  or 
thin  slices  are  used  the  preservation  is  quicker  and  more  uniform  than  by  vascu- 
lar injection.  The  cells  are  suddenly  killed  while  active.  The  factor  of  slow 
death  at  uneven  rates,  present  in  supravital  examinations,  does  not  have  to  be 
reckoned  with;  but  many  precautions  are  required.  Under  Fixation  is  given  a 
general  account  of  the  procedure.  Under  the  several  organs,  Lungs,  Small 
Intestine,  Skin,  etc.,  some  special  suggestions  are  provided.  There  are  many 
fixatives  to  choose  from.  For  routine  purposes  Zenker's  Fluid  as  originally 
described  or  in  one  of  its  numerous  modifications  is  suggested.  Bouin's  is  also 
a  very  popular  fixative  especially  among  dermatologists.  Formalin  is  an  ex- 
cellent one.  It  is  good  practice  to  set  aside  some  tissue  in  formalin  for  examina- 
tion as  may  be  needed  later.  Both  formalin  and  alcohol  are  the  most  useful 
fixatives  preliminary  to  microchemical  determinations.  When  preparations 
must  be  made  very  quickly,  Alcohol  Formalin  and  Caraoy's  Fluid  are  suggested 
(see  also  Frozen  Sections).  For  microincineration,  formalin-alcohol  is  ordinarily 
employed;  but  the  Altmann-Gersh  method  of  freezing  and  drying,  by  which 
contact  with  fixatives  is  altogether  dispensed  with,  is  much  less  open  to  criticism. 
Osmic  acid  containing  fixatives  penetrate  poorly  and  are  therefore  only  useful 
for  very  small  pieces  of  tissue.  Regaud's  fluid  with  subsequent  mordanting  in 
bichromate  is  the  best  for  mitochondria.  Heat  fixation  is  useful  for  blood  cells. 
Fixation  in  various  vapors  is  called  for  in  special  cases.     See  Fixatives. 


XX  CHOICE   OF   METHODS 

After  fixation  some  Washing  of  the  tissue  in  water  is  necessary  unless  it  has 
been  fixed  in  alcohol,  Carnoy  or  similar  mixtures.  The  next  step  is  Dehydration 
and  a  choice  must  be  made  between  slow  and  rapid  methods.  Sometimes  a 
substitute  for  alcohol  is  indicated.  If  Imbedding  is  to  be  in  celloidin  Clearing 
in  a  xylol-like  fluid  is  omitted  and  heating  is  unnecessary.  There  are  many  ways 
of  clearing  preliminary  to  paraffin  imbedding.  In  Sectioning  the  thickness 
depends  upon  the  purpose  in  view.  Thick  sections  may  be  as  necessary  as  thin 
ones  and  serial  sections  are  often  required.  In  the  Mounting  of  sections  on 
slides  the  use  of  water  must  occasionally  be  avoided.  Numerous  techniques  are 
applicable  to  the  sections  and  are  given  individually  later  either  under  the  head- 
ing of  the  substance  or  structure  to  be  demonstrated  or  under  the  name  of  the 
technique  or  its  introducer.     For  choice  see  Staining. 

Many  beautifully  stained  sections  of  well  fixed  tissue  are  of  but  little  value, 
because  the  investigator  failed  to  note  the  exact  location  in  the  organ  or  tissue 
whence  they  were  excised  and  omitted  to  have  the  sections  cut  in  the  most 
favorable  plane. 

5.  To  Mark  Selected  Individual  Cells  or  Tissues  in  Vivo 

FOR  Later  Examination 

In  this  connection  we  at  once  think  of  the  vital  stains,  trypan  blue,  carmine, 
India  ink  (carbon)  and  hundreds  of  others,  which,  when  injected  into  the  body, 
are  phagocytosed  by  the  reticulo-endothelial  cells  (or  macrophages).  Pieces  of 
tissue  can  then  be  excised  and  the  accumulations  of  stains  can  be  studied  within 
the  still  living  cells,  that  is  supravitally,  for  unless  cultured  the  cells  are  slowly 
dying.  But,  if  desired,  the  tissues  can  be  fixed  and  the  results  observed  at 
leisure  in  sections. 

It  has  long  been  known  that  bone  laid  down  in  the  presence  of  Madder  fed  to 
the  animals  is  marked  by  the  madder  and  can  thus  be  distinguished  from  bone 
deposited  beforehand  and  afterwards.  In  the  same  way  dentine  can  be  marked 
in  vivo  with  Alizarin  Red  S. 

Another  example  of  in  vivo  marking  is  the  deposition  of  Prussian  Blue.  Thus 
a  slightly  hypertonic  solution  (potassium  ferrocyanide  0.5  gm.,  iron  ammonium 
citrate,  0.5  gm.  and  aq.  dest.  50  cc.)  injected  into  the  subarachnoid  space  of  the 
spinal  cord  is  useful  in  the  localization  of  the  pathways  of  drainage  of  cerebro- 
spinal fluid,  because  of  the  marking  secured  when  the  tissues  are  fixed  in  40% 
formalin  plus  1%  concentrated  hydrochloric  acid  by  the  deposition  of  Prussian 
blue  (Weed,  L.  H.,  J.  Med.  Res.,  1914,  26,  21-117). 

The  tissues  of  animals  recently  killed  or  under  anesthesia  can  be  selectively 
marked  with  various  dyes  by  Perfusion  of  the  blood  vessels  with  dilute  solutions 
of  dyes.  The  outstanding  methods  in  this  group  have  been  devised  by  Bensley 
(R.  R.,  Am.  J.  Anat.,  1911,  12,  297-388)  for  histological  analysis  of  the  epithelial 
components  of  the  pancreas  and  stomach.  Dilute  solutions  of  the  dyes  in  physio- 
logical saline  are  injected  into  the  thoracic  aorta  of  an  animal  killed  by  bleeding. 
Pieces  of  pancreas  and  gastric  mucous  membrane  are  then  removed  and  examined 


CHOICE   OF   METHODS  XXI 

fresh.  Neutral  red  picks  out  the  Islets  of  Langerhans  of  the  pancreas,  pyronin 
the  duct  system  of  the  pancreas,  naphthol  blue  the  parietal  cells  of  the  Stomach 
and  so  on.  In  the  same  way  Nerve  Fibers  can  be  marked  for  subsequent  study 
by  vascular  perfusion  with  methylene  blue  and  degenerating  nerve  fibers  in 
poUomyelitis  (and  presumably  in  other  conditions)  can  be  sharply  differentiated 
from  uninjured  ones  by  the  fact  that  they  take  up  neutral  red  (Covell,  W.  P. 
and  O'Leary,  J.  L.,  Arch.  Neurol.  &  Psych.,  1932,  27,  518-524).  It  has  long 
been  known  that  the  best  way  to  mark  renal  glomeruli  is  to  perfuse  in  the  same 
fashion  with  a  dilute  solution  of  janus  blue.  The  glomeruli  stand  out  clearly  in 
the  fresh  kidney  by  their  deep  blue  color  in  a  red  background  (Cowdry,  E.  V., 
Contrib.  to  EmbryoL,  Carnegie  Institution  of  Washington,  1918,  No.  25,  39- 
160).  A  similar  selective  staining  in  less  brilhant  colors  is  obtainable  with  janus 
green.  Relatively  permanent  preparations  can  be  made  of  some  of  these 
specimens. 

The  same  dyes,  and  many  others,  can  also  be  applied  in  dilute  solutions  to 
cells  freshly  removed  from  the  body  and  which  are  still  living.  Such  methods 
have  become  very  popular  in  hematology.  However,  the  cells  thus  colored  live 
only  for  a  limited  time  and  it  is  important  to  cut  short  the  observations  before 
they  are  vitiated  by  approaching  death. 

It  is  feasible  to  employ  a  wide  variety  of  Tracer  Techniques,  that  is  substances 
can  be  traced  through  the  body  by  the  markings  given  to  them.  The  largest 
group  is  made  up  of  Radioactive  Isotopes.  Because  of  their  radioactivity  they, 
and  substances  in  which  they  are  chemically  combined,  can  be  quantitatively 
measured  by  a  Geiger  Counter.  Wherever  they  go  in  the  body,  they  are  ap- 
parently accepted  by  the  tissues  and  play  their  roles  in  metabolism  in  the  same 
way  as  if  they  were  not  radioactive.  Thus  Radiocalcium  is  found  to  be  stored 
almost  entirely  in  bone  and  the  amount  taken  in  in  a  given  time  is  an  indication 
of  the  amount  of  nonradioactive  calcium  given  out  in  conditions  in  which  the 
total  amount  of  calcium  is  not  changed.  The  turnover  of  calcium  can  therefore 
be  estimated.  Radioiodine  tends  to  be  stored  in  the  thyroid,  and,  again,  when 
the  total  amount  of  iodine  does  not  change,  the  amount  stored  in  a  given  time 
balances  the  amount  lost  and  is  a  measure  of  the  iodine  replacement. 

By  the  technique  of  Autoradiography  the  exact  location  of  the  radioelements 
can  be  determined  by  holding  a  section  of  the  tissue  in  contact  with  a  photo- 
graphic film.  The  images  on  fine  grained  films  can  then  be  magnified.  Con- 
sequently, by  selection  of  radioelements  based  on  information  as  to  where  they 
are  stored  in  largest  amounts  and  by  their  use,  heavy  radiation  can  be  brought 
to  bear  upon  several  kinds  of  tissues  leaving  others  influenced  but  little  or  not  at 
all.  An  excellent  account  of  Isotopes  in  Nutrition  Research  is  given  in  Borden's 
Review  of  Nutrition  Research,  1945,  6,  Nos.  8  and  9. 

6.  To  Employ  Culture  Methods 

The  common  feature  in  these  techniques  is  to  plant  cells,  tissues  or  organisms 
in  new  and  different  fluid  environments  and  to  observe  their  behavior  therein. 


XXll  CHOICE   OF   METHODS 

Thus  cells  can  be  grown  in  Tissue  Cultures  of  chemical  composition  suited  to 
their  requirements.  Mixed  cultures  are  those  containing  several  types  of  cells 
and  pure  cultures  those  containing  but  one  sort.  This  technique  affords  un- 
rivalled opportunities  for  experimentally  changing  the  fluid  environments  of 
cells,  for  the  study  of  nutritional  factors,  growth  stimulating  and  growth  in- 
hibiting factors,  and  the  influence  of  cells  on  one  another.  Individual  cells  can 
be  observed  at  high  magnification  and  the  phenomena  of  motility,  phagocytosis, 
mitosis,  cell  death,  etc.  can  be  recorded  by  moving  pictures  so  that  the  analysis 
of  form  and  function  is  possible  with  a  high  degree  of  accuracy. 

The  limitation  of  the  method  is  the  obvious  one  that  the  fluid  environments 
are  artificial  and  must  be  changed  at  intervals  to  keep  the  strains  of  cells  alive. 
Consequently  tissue  cultures  are  unsatisfactory  for  the  investigation  of  inter- 
cellular materials,  like  fibers,  hyaline  deposits  and  so  on.  Moreover  the  cells 
cannot  properly  organize  to  form  tissues  and  organs  as  they  do  in  vivo  since  they 
are  isolated  from  normal  influences  by  other  tissues  of  the  body.  But  they 
make  the  effort.  Methods  have  recently  been  advocated  for  the  culture  of 
organized  tissues,  bones,  teeth,  etc.  (Fell,  H.  B.,  J.  Roy.  Micr.  Soc,  1940,  60, 
95-112). 

In  selecting  the  technique  of  tissue  culture  for  the  solution  of  any  problem 
it  is  well  to  remember  that  considerable  equipment  and  several  years  training 
are  required  to  realize  its  full  usefulness.  For  this  reason  valuable  time  will  be 
saved  by  learning  the  technique  from  an  expert. 

The  new  and  highly  productive  technique  of  analysing  cellular  responses  by 
their  observation  in  Motion  Pictures  offers  more  attractive  leads  when  applied 
to  living  cells  in  tissue  cultures  than  to  cells  viewed  in  other  situations.  In 
tissue  cultures  they  can  be  photographed  at  high  magnification,  both  by  direct 
illumination  and  in  ths  dark  field,  because  they  occur  as  individuals  or  as  thin 
clumps  in  the  fluid.  Moreover,  their  behavior  can  be  followed  in  successive 
photographs  over  long  periods  of  time  and  it  is  possible  directly  to  observe  how 
this  is  modified  by  a  host  of  different  influences  experimentally  brought  to  bear 
on  them.  For  teaching  Motion  Pictures  are  helpful,  but  can  be  used  too  much. 
Easy  come,  easy  go  is  true  of  instruction.  Unless  learning  is  combined  with 
some  sort  of  effort  it  will  be  of  very  transitory  value. 

Transplantation  of  tissue  from  its  original  location  to  a  new  and  different 
position,  such  as  the  Anterior  Chamber  of  the  Eye,  is  also  a  culture  method  of 
value  in  the  solution  of  certain  problems.  The  factors  that  condition  the  growth 
and  the  behavior  of  the  transplant  are  of  importance. 

Some  organisms  can  best  be  grown,  and  viruses  increased  in  amount,  by 
implanting  them  into  the  Chorioallantoic  Membrane  of  chick  embryos.  This 
technique  has  abundantly  proved  its  worth.  The  feasibility  of  culture  in  this 
membrane  depends  essentially  on  the  lesser  development  of  growth  inhibiting 
factors  in  young  tissues  than  in  older  ones. 

Viruses  will  "take"  and  increase  in  amount  in  some  locations  better  than  in 


CHOICE   OF   METHODS  Xxiii 

others.     Intracerebral  and  intratesticular  inoculations  are  often  made  and, 
again,  young  animals  are  in  general  most  susceptible. 

The  culture  of  Bacteria  and  Protozoa  has  for  generations  been  a  fine  art  based 
on  meticulous  study  of  their  needs.  These  relatively  simple  organisms  provide 
wonderful  material  for  the  investigation  of  the  most  basic  of  vital  phenomena. 

7.  To  Investigate  Composition  by  Chemical  Means 

This  cannot  be  done  blindly — by  just  taking  a  chunk  of  tissue  and  analysing 
it.  The  investigations  must  be  guided  by  knowledge  of  the  structure  and  func- 
tion of  the  materials  analysed.  Blood  can,  for  example,  be  collected  in  suf- 
ficient volume  for  routine  chemical  analysis;  but  the  results  will  differ  depending 
upon  whether  it  is  arterial  blood,  portal  venous  blood  from  the  intestines,  or 
venous  blood  from  the  extremities.  Analyses  of  whole  skin  are  practically 
worthless  because  the  skin  is  a  structure  made  up  of  two  parts:  avascular  epi- 
dermis of  ectodermal  origin  and  underlying  dermis  made  up  of  connective  tissue 
differing  in  vascularity,  fiber,  fat,  tissue  fluid  and  gland  contents  in  various 
regions  of  the  body.  Only  since  a  technique  has  been  devised  whereby  whole 
Epidermis  freed  from  dermis  can  be  obtained  in  a  condition  suitable  for  analysis, 
not  having  been  exposed  to  any  fluids,  has  progress  been  possible. 

Results  of  direct  chemical  analysis  of  any  tissue  may  be  misleading  unless 
interpreted  in  terms  of  its  structural  make  up  and  of  what  has  happened  to  it 
since  it  existed  in  vivo.  Among  the  experimental  errors  to  be  guarded  against 
are  variability  in  sacrificing  the  animal,  or  the  manner  of  death  of  the  patient, 
in  excision  of  tissue  allowing  more  or  less  blood  and  other  fluids  to  drain  out  or 
evaporate,  in  time  and  in  temperature,  in  age,  sex,  and  in  conditions  before 
death. 

The  extracellular  and  intracellular  fluids  or  phases,  are  large  in  volume,  w^hen 
all  are  taken  together,  but  difficult  to  get  at  directly.  To  obtain  data  "the 
deducive  histochemical  method"  is  suggested.  This  is  described  by  Lowry, 
0.  H.  and  Hastings,  A.  B.  in  Cowdry's  Problems  of  Ageing,  1942,  728-755. 

Those  wishing  to  analyse  extremely  small  volumes  of  fluid  which  by  contrast 
can  be  collected  for  direct  determinations  cannot  do  better  than  to  familarize 
themselves  with  the  techniques  elaborated  by  A.  N.  Richards  and  his  associates 
at  the  University  of  Pennsylvania  for  the  study  of  glomerular  urine. 

By  the  useful  technique  of  Microincineration  minerals  which  are  not  volatilized 
at  high  temperature  can  be  directly  studied  in  the  tissues  in  the  positions  which 
they  previously  occupied  in  living  organisms.  They  appear  as  shining  particles 
when  viewed  by  the  Dark  Field  Microscope.  Microincineration  is  truly  a 
microchemical  method  for  the  localization  of  structure  which  is  microscopic 
in  its  fineness. 

Quite  a  number  of  Microchemical  Reactions  capable  of  demonstrating  the 
precise  location  in  the  cells  of  minerals,  fats,  carbohydrates  and  proteins  are 
available. 


XXIV  CHOICE   OF   METHODS 

By  a  Photoelectric  Microphotometer  it  is  possible  to  estimate  quantitatively 
reactions  like  that  of  Feulgen  for  Thymonucleic  Acid  which  give  distinctive 
colors  and  numerous  stains  which  are  specific  for  tissue  components  and  can 
be  standardized  in  their  action.  But  the  data  obtained  are  relative,  that  is 
it  can  be  said  that  the  reaction  is  say  60  per  cent  greater  in  one  specimen  than 
in  another.  The  absolute  amount  of  the  component  demonstrated  per  gram 
of  tissue  cannot  yet  be  arrived  at. 

Several  Enzymes  (phosphatase,  dopa-oxidase,  arginase)  can  now  be  micro- 
scopically identified  and  their  position  within  cells  determined.  By  close  com- 
parison of  enz5Tnatic  properties  with  the  cellular  composition  of  tissues,  the 
localization  of  many  others  can  be  inferred. 

In  the  case  of  these  and  other  microchemical  methods  the  treatment  of  the 
tissue  after  excision  and  before  the  special  procedures  are  commenced  is  of  con- 
sequence. Even  in  the  preparation  of  routine  frozen  sections,  and  much  more 
so  when  the  specimens  are  fixed,  dehydrated,  cleared,  imbedded  and  sectioned, 
there  are  many  opportunities  for  the  loss  of  chemical  substances  and  of  change 
in  their  position  in  the  tissue  and  within  cells.  The  best  way  to  hold  the  com- 
ponents in  the  positions  they  occupy  in  the  living  state  is  to  instantaneously 
freeze  the  tissue  and  dehydrate  in  vacuum  while  still  frozen,  thus  avoiding  all 
fixatives,  by  the  Altmann-Gersh  technique.  Moreover,  the  reagents  used  in 
testing  must  contact  all  the  tissue  equally  for  unequal  contact  may  well  be 
followed  by  stronger  reactions  in  some  areas  than  in  others. 

Quite  recently  chemical  analysis  has  been  accurately  focussed,  not  merely 
on  cells,  but  on  parts  of  cells.  Nuclei,  Mitochondria  and  many  other  cellular 
components  including  even  Chromatin  Threads  can  now  be  collected  en  masse 
by  Centrifugation  of  broken  up  cells  and  analysed.  This  is  a  departure  of  con- 
sequence. 

Finally  standard  qualitative  chemical  methods  are  often  appUcable  on  a 
microscopic  basis.  The  reader  wishing  to  do  so  may  well  consult  Chamot,  E. 
E.,  and  Mason,  C.  W.,  Handbook  of  Chemical  Microscopy.  New  York:  John 
Wiley  &  Sons,  1940,  vol.  2,  439  pp.  Another  book  that  will  be  found  of  service, 
especially  for  analysis  on  microscopic  slides,  is  Benedetti-Pichler,  A.  A.,  In- 
troduction to  the  Microtechnique  of  Inorganic  Analysis.  New  York:  John 
Wiley  &  Sons,  1942,  302  pp.  Sometimes  one  is  held  up  by  having  to  deal  with 
some  unfamiliar  chemical  substance  in  which  case  aid  may  be  secured  from  the 
large  and  comprehensive  "Dictionary  of  Organic  Compounds"  edited  by  Heil- 
bron  and  published  in  3  volumes,  1934,  1936  and  1938,  by  Oxford  University 
Press,  New  York.  No  attempt  is  made  in  this  dictionary  to  include  dyes  but 
thousands  of  other  organic  compounds  are  conveniently  arranged  in  alphabetical 
order.  If  the  wanted  material  is  some  sort  of  medical  preparation  seek  informa- 
tion in  the  following  reference  books.  (1)  New  and  Nonofficial  Remedies, 
1946.     Chicago:  Am.   Med.   Assoc,   770   pp.;   (2)    The   National  Formulary. 


CHOICE   OF   METHODS  XXV 

VII.     Washington:  Am.  Pharmaceutical  Assoc,  1942,  690  pp.;  (3)  The  Phar- 
macopoeia of  the  U.  S.  XII.     Easton:  Mack  Printing  Co.,  1942,  880  pp. 

8.  To  Employ  Physical  Techniques  in  the  Investigation 

OF  Composition 

Chemistry  is,  at  rock  bottom  physics  so  that  the  distinction  here  made  is 
convenient  but  without  vaUdity.  Hydrogen  Ion  Indicators  and  Oxidation- 
Reduction  Potential  could  come  under  either  heading. 

Histospectrography  is  a  quick  and  reliable  method  to  gain  information  on 
the  presence  or  absence  of  many  minerals.  It  is  a  kind  of  survey  technique, 
for  the  absorption  lines  of  many  elements  can  be  obtained  in  a  single  spectrogram. 
The  density  of  the  lines  can  be  determined  photometrically  but  data  obtained  on 
concentration  of  a  particular  element  are  relative  (more  in  one  tissue  than  in 
another)  but  not  absolute  (in  mgm.  per  gm.  of  tissue).  Ultraviolet  Absorption 
Spectra  have  been  employed  to  advantage  by  Caspersson  and  others  in  the 
intracellular  determination  of  certain  components  but  the  technique  requires 
elaborate  and  costly  instrumentation.  It  gives  promise,  however,  of  being 
of  great  value  in  the  solution  of  fundamental  problems. 

Utilization  of  physical  techniques  in  biology  and  medicine  is  now  the  order 
of  the  day  and  the  limitations  thereof  cannot  be  envisaged.  In  this  elementary 
survey  only  a  few  others  can  be  mentioned  briefly  in  passing  as  examples.  By 
Electrophoresis  measurements  the  electric  charge  on  particles  can  be  determined. 
The  Polarization  Optical  Method  is  of  surpassing  value  and  Fluorescence  Micros- 
copy, supplemented  by  fluorescence  spectrography,  is  coming  into  its  own. 
Surface  Tension  measurements  can  be  made  in  numerous  ways.  Particle 
size  can  be  measured  by  a  flock  of  different  techniques  from  which  the  one  must 
be  chosen  that  best  suits  the  material.  The  simplest  way  is  to  compare  the 
objects  with  rulings  of  a  micrometer  slide.  DiflTraction  methods  are  labor  saving 
and  often  preferable.  Filters  of  d^erent  porosity  are  available  so  that  the 
sizes  of  particles  passing  through  can  be  roughly  gaged.  To  employ  Ultra- 
centrifugation  techniques  are  among  several  other  possibilities.  There  are 
now  Microscopes  of  many  varieties  to  choose  from. 

The  Electron  Microscope  is  a  physical  tool  which  can  be  used  only  by  a  spe- 
cially trained  individual,  and  it  has  the  limitation  that  the  cells  and  other  ma- 
terials must  be  very  thin,  sections  not  more  than  about  j  of  a  micron.  See 
Burton,  E.  F.  and  Kohl,  W.  H.,  The  Electron  Microscope.  New  York:  Rein- 
hold  Publishing  Corporation,  1946,  325  pp. 

In  biology  and  medicine  it  is  clearly  evident  that  the  techniques  of  physics 
and  chemistry  are  so  revealing  that  some  knowledge  of  these  basic  sciences  is 
necessary.  A  little  knowledge  can  however  be  a  dangerous  thing  often  leading 
to  half  baked  conclusions.  Cooperation  with  real  physicists  and  chemists  is 
essential  and  team  work  must  take  the  place  of  isolated  individual  endeavor, 
moreover  a  laboratory  of  whatever  kind  must  be  well  organized  to  be  effective. 


XXVI  CHOICE   OF   METHODS 

An  untidy  laboratory  is  not  a  sign  of  industry  but  an  indicator  of  carelessness, 
and  sometimes  a  source  of  actual  danger  to  the  occupants. 

9.  To  Detect  Deviations  from  Normal 

The  Normality  of  a  tissue  or  organ  is  often  in  doubt.  There  is  no  single 
technique  capable  of  yielding  an  unqualified  answer.  Since  some  properties 
may  be  normal  while  others  are  abnormal  (pathological)  we  need  first  to  be  told 
the  property  under  consideration.  If  it  is,  for  instance,  the  amount  of  contained 
pigment,  this  can  be  said  to  be  normal  when  it  is  the  amount  usually  present  in 
a  particular  tissue  under  the  same  conditions.  By  the  word  "usually"  is  in- 
tended in  the  majority  of  cases,  that  is  in  51  per  cent  or  in  any  higher  percentage. 
The  phrase  "same  conditions"  means  that  the  conditions  likely  to  influence  the 
amount  of  pigment  are  so  nearly  alike  as  to  be  not  responsible  for  any  difference 
observed  between  the  property  of  the  tissue  where  normality  is  in  question  and 
that  of  others  of  the  same  kind.  Thus,  we  could  say  with  reasonable  assurance 
that  the  amount  of  pigment  is  normal  if  it  is  that  usually  demonstrated  by  the 
same  technique  in  tissues  of  the  same  kind  of  animals  of  the  same  species,  sex 
and  age  living  under  the  same  conditions.  Judgment  is  necessary  in  specifica- 
tion of  possibly  modifying  conditions  which  will  depend  to  some  extent  on  the 
property  under  consideration  and  on  the  number  of  observations  necessary  to 
establish  the  percentage  within  the  limits  of  probability.  It  would  not  do  to 
compare  the  amount  of  pigment  in  the  specimen,  the  normality  of  which  is  in 
question,  with  that  in  too  few  others.  This  is  the  statistical  definition  of  nor- 
mality which  is  not  universally  accepted  but  which  is  useful  and  easily  under- 
stood. 

Only  a  few  samples  of  the  various  kinds  of  technique  have  been  mentioned  in 
this  survey  as  a  kind  of  menu  from  which  to  make  a  selection  or  to  obtain  clues 
to  other  methods  that  may  fit  the  case.  Many  of  them  are  very  ingenious  and 
were  only  discovered  after  wisely  conceived  attempts  to  overcome  practical 
difficulties.  This  overcoming  of  obstacles  is  a  pleasant  experience.  It  calls  for 
actual  work  and  experiment  and  appeals  to  many  of  our  best  minds.  The 
techniques  may  be  regarded  as  keys  by  which  scientific  treasure  can  be  unlocked. 
Unused  they  are  worthless. 


STANDARDIZATION  OF  STAINS* 

In  the  use  of  stains  one  encounters  a  multitude  of  names,  many  of  which  are 
synonyms,  and  it  is  difficult  to  be  sure  of  their  meaning.  Two  comprehensive 
dye  indexes  have  been  pubhshed.  One,  "Schultz'  Farbstofftabellen",  is  now  in 
its  7th  edition  (1928  to  1939)  but  confusion  is  created  by  the  fact  that  the  index 
numbers  of  the  dyes  given  in  it  do  not  correspond  to  those  in  the  earlier  editions. 
The  other,  the  "Colour  Index  of  the  Society  of  Dyers  and  Colourists",  was  edited 
by  F.  M.  Rowe  and  published  in  1924.  It  was  followed  in  1928  by  a  supplement, 
but  there  has  been  no  second  edition.  This  Colour  Index  gives  (1)  the  com- 
mercial name,  or  much  more  frequently  names  for  there  are  so  many  synonyms; 
(2)  the  formula,  (3)  the  preparation,  (4)  the  discoverer  and  (5)  the  properties  of 
a  vast  assemblage  of  dyes.  It  is  the  standard  of  reference  in  the  United  States 
and  other  English-speaking  countries.  When  one  wishes  to  be  specific  it  is 
customary  to  list  after  the  dye  used  its  colour  index  number,  for  example  vital 
red,  C.  I.  No.  456. 

The  most  recent  Year  Book  of  the  American  Association  of  Textile  Chemists 
and  Colorists,  New  York:  Hawes  Publishing  Co.  1945,  743  pp.  is  often  of  as- 
sistance. It  provides  an  alphabetical  list  of  over  6,000  American  made  dyes  with 
classification,  manufacturer  and  Colour  Index  Number  if  any.  A  listing  of 
American  made  Dyes  arranged  by  Colour  Index  Numbers  is  also  useful.  For 
example,  if  one  is  interested  in  Orange  II,  CI.  151,  it  will  be  seen  that  this  is  avail- 
able under  26  names  from  12  different  makers.  In  another  place  the  foreign 
prototype  names  of  dyes  without  Colour  Index  numbers  are  listed  alphabetically 
with  the  corresponding  American  dyes  and  their  manufacturers  so  that  the 
available  American  substitutes  for  foreign  dyes  can  be  found.  This  Year  Book 
is  unfortunately  often  lacking  in  medical  school  libraries  but  it  is  usually  on 
hand  in  the  better  Public  Libraries  like  that  of  St.  Louis. 

Much  aid  is  given  to  investigators  by  the  Biological  Stain  Commission  and 
particularly  by  its  distinguished  Founder,  Dr.  H.  J.  Conn.  This  commission 
is  concerned  with  the  inspection  and  standardization  of  stains,  not  with  their 
manufacture  as  is  sometimes  supposed.  It  was  found  in  1920,  while  the  post- 
war embargo  on  dyes  was  still  in  effect,  that  American  scientists  were  being  sup- 
plied with  dyes  from  three  or  four  different  stain  companies  and  that  their 
products  were  not  sufficiently  uniform  to  be  reliable.  Accordingly,  through  the 
cooperation  of  the  National  Research  Council  and  of  several  national  scientific 
societies,  the  Commission  on  Standardization  of  Biological  Stains  (now  the 
Biological  Stain  Commission)  was  established.  The  Commission  is  now  an 
independent  organization  but  includes  in  its  membership  representatives  of 
eight  societies  with  which  it  cooperates.  The  work  of  the  Commission  is  two- 
fold.    First,  by  cooperation  of  biologists  and  chemists  it  gathers  information 

*  Kindly  revised  by  Dr.  H.  J.  Conn. 

xxvii 


XXVlll  STANDARDIZATION   OF   STAINS 

concerning  the  nature  of  dyes  as  related  to  their  use  in  microscopic  technique ; 
secondly,  by  working  with  the  manufacturers  and  dealers  it  endeavors  to  see 
that  the  supply  of  available  stains  in  America  is  of  the  highest  possible  quality 
as  judged  by  their  performance  in  actual  laboratory  use.  The  first  of  these 
purposes  has  inspired  a  useful  book  on  "Biological  Stains"  by  Conn,  now  in  its 
fifth  (1946)  edition,  and  at  the  same  time  has  led  to  the  pubhcation  by  the 
Commission  of  a  quarterly,  "Stain  Technology."  The  second  object  is  being 
brought  about  by  the  certifying  of  stains. 

The  certification  plan  has  been  adopted  because  of  the  great  difficulty  of 
drawing  up  any  chemical  or  physical  standards  to  determine  which  stains  are 
satisfactory'-  and  which  are  not.  If  such  standards  were  formulated,  it  would 
be  possible  to  prepare  specifications  with  which  manufacturers  of  stains  ^^■ould 
be  expected  to  comply.  In  the  early  work  of  the  Stain  Commission  an  attempt 
was  made  to  draw  up  such  specifications  and  they  were  published,  in  provisional 
form,  for  a  few  stains  in  the  first  edition  of  "Biological  Stains."  Full  specifica- 
tions are  given  in  the  current  edition  and  in  the  National  Formulary. 

Instead  of  drawing  up  specifications,  therefore,  the  Stain  Commission  instructs 
the  manufacturers  of  stains  to  submit  samples  to  it  of  every  batch  manufactured 
of  any  of  the  stains  that  are  on  the  certification  basis.  The  Commission  submits 
these  samples  to  certain  definite  tests  which  have  now  been  formulated  and 
published  (see  Conn,  pp.  246-276).  The  methods  in  question  include  chemical, 
spectrophotometric,  and  biological  tests,  and  only  those  dyes  are  certified  which 
are  satisfactory  in  all  these  tests.  Such  dyes  the  manufacturers  are  allowed  to 
sell  with  a  special  label  on  the  package  indicating  approval  by  the  Stain  Com- 
mission. 

The  certification  label  on  any  bottle  of  stain  means,  therefore,  that:  (1)  a 
sample  of  the  batch  bearing  the  label  has  been  submitted  to  the  Commission  for 
testing  and  a  portion  of  the  sample  is  permanently  on  file  in  the  chairman's 
oflBce;  (2)  the  sample  proves  true  to  type,  as  judged  by  spectrophotometric  tests; 

(3)  its  dye  content  is  up  to  specification  and  is  correctly  indicated  on  the  label; 

(4)  it  has  been  tested  by  experts  in  the  procedures  named  on  the  label  and  has 
been  found  satisfactory  by  them ;  and  lastly,  (5)  no  other  batch  can  be  sold  under 
the  same  certification  number  except  by  such  a  flagrant  breach  of  confidence  on 
the  part  of  the  company  as  to  risk  losing  the  good  will  of  the  Commission.  At 
present  (1950)  the  following  stains  have  been  placed  on  the  certified  list.  In 
descriptions  of  their  use  the  names  should  be  followed  by  C.C.,  indicating  that 
the  products  were  Commission  Certified,  for  instance,  alizarin  red  S  (C.C.). 

Eight  companies  in  the  United  States  are  now  submitting  their  stains  to  the 
Commission  for  certification  before  putting  them  on  the  market.  It  must  be 
realized,  however,  that  no  one  of  these  concerns  necessarily  manufactures  all 
the  stains  which  it  thus  submits ;  but  in  the  case  of  any  stain  which  is  manufac- 
tured elsewhere,  the  company  takes  responsibility  for  its  performance  as  a  bio- 
logical stain,  on  the  basis  of  tests  made  to  show  its  adequacy,  and  in  many  in- 
stances carries  out  a  certain  degree  of  purification  or  other  processing  before 


STANDARDIZATION    OF   STAINS 


XXIX 


Alizarin  red  S 

Anilin  blue,  water  soluble 

Auramine  O 

Azocarmine  G 

Azure  A 

Azure  B 

Bismarck  brown  Y 

Brilliant  cresyl  blue 

Brilliant  green 

Carmine 

Chlorazol  black  E 

Congo  red 

Cresyl  violet 

Crystal  violet 

Eosin,  bluish 

Eosin,  yellowish 

Erythrosin  B 

Ethyl  eosin 

Fast  green  FCF 

Fuchsin,  acid 

Fuchsin,  basic 

Giemsa  stain 

Hematoxylin 

Indigo  carmine 

Janus  green  B 

Jenner's  stain 


Light  green,  S.F.,  yellowish 
Malachite  green 
Martius  yellow 
Methyl  green 
Methyl  orange 
Methyl  violet  2B 
Methylene  blue  chloride 
Methylene  blue  thiocyanate 
Methylene  violet 
Neutral  red 
Nigrosin 
Nile  blue  A 
Orange  G 
Orange  II 
Orcein 
Phloxine 
Pyronin   P 
Resazurin 
Rose  bengal 
Safranin  O 
Sudan  III 
Sudan  IV 
Sudan  black  B 

Tetrachrome  stain  (MacNeal) 
Thionin 

Toluidine  blue  O 
Wright's  stain 


putting  the  stain  on  the  market.  One  of  these  companies  puts  on  the  market 
every  stain  now  on  the  certification  list.  Two  other  companies  submit  samples 
of  over  half  the  stains  thus  listed,  while  the  other  companies  merely  request 
certification  of  one  or  two  dyes  in  which  they  speciaUze.  No  dyes  have  yet 
been  certified  by  the  Stain  Commission  submitted  by  any  foreign  concern 
except  for  one  located  in  Montreal.  Cooperation  among  the  Americas  is 
increasing  (Conn,  H.  J.,  Stain  Techn.,  1942,  17,  5-6). 

In  several  recent  editions  of  the  National  Formulary,  published  by  the  Ameri- 
can Pharmaceutical  Association,  a  section  has  been  included  in  which  formulae 
of  staining  solutions  are  given.  Originally  there  was  no  agreement  between 
these  formulae  and  the  ones  recommended  by  the  Stain  Commission.  Begin- 
ning in  1937,  however,  it  was  decided  that  the  National  Formulary  Committee 
and  the  Biological  Stain  Commission  should  cooperate  in  this  matter.  Accord- 
ingly, the  chairman  of  the  latter  was  made  a  member  of  the  former  and  a  member 
of  the  National  Formulary  was  put  on  the  Board  of  Trustees  of  the  Commission. 
This  interlocking  membership  is  assurance  that  the  work  of  preparing  staining 
formulae  for  each  edition  of  the  National  Formulary  is  carried  on  in  close  coopera- 
tion with  the  Stain  Commission.  This  cooperation  has  resulted  in  two  important 
steps : 

1.  Specifications  of  the  most  important  stains  now  on  the  certification  basis 
have  been  published  in  the  National  Formulary  (1942,  1946).     These  specifica- 


XXX  STANDARDIZATION   OF   STAINS 

tions  are  partly  chemical  and  spectrophotometric,  but  also  contain  detailed  state- 
ments as  to  how  the  stains  should  be  tested  as  to  their  behavior  for  biological 
purposes  and  state  the  results  to  be  expected  from  these  tests.  In  every  case 
these  specifications  have  been  made  to  harmonize  with  the  tests  as  actually  per- 
formed by  the  Stain  Commission. 

2.  The  formulae  given  in  the  National  Formulary,  in  "Biological  Stains"  and 
in  the  "Manual  of  Methods  for  the  Pure  Culture  of  Bacteria,"  published  by  the 
Society  of  American  Bacteriologists,  have  been  compared  and  critically  studied 
with  the  object  of  making  them  identical  in  all  three. 

The  years  since  the  second  World  War  have  seen  more  progress  in  stain 
standardization  than  during  any  preceding  similar  period.  Dr.  E.  H.  Stotz, 
Biochemist,  of  University  of  Rochester  Medical  School,  has  been  made  an  officer 
of  the  Stain  Commission  and  is  now  in  charge  of  its  research  and  assay  labora- 
tory at  Rochester,  N.  Y.  This  laboratory  is  making  a  comprehensive  survey  of 
nearly  every  stain  sample  that  has  ever  been  submitted  for  certification,  making 
a  systematic  comparison  between  their  physical  and  chemical  characteristics 
(notably  spectrophotometric)  and  behavior  in  staining.  If  it  is  ever  going  to  be 
possible  to  draw  up  specifications  that  correlate  with  staining  properties,  such 
a  survey  should  furnish  the  necessary  data. 


ABBREVIATIONS  XXXI 

ABBREVIATIONS 

1  fi  (Greek  letter  for  micron)  =  l/lOOOth  part  of  a  millimeter  (mm.)  =  0.001  mm.  =  10~' 

mm.  =  10,000  A  =  approximately  l/25,000th  of  an  inch. 
1  m^  (millimicron)  =  1/lOOOth  part  of  a  micron  =  l/l,000,000th  part  of  a  mm.  =  10~*  mm. 

=  0.001  M  =   10  A. 
1  A  (Angstrom  unit)  =  0.1  m/ii  =  0.0001  n  =  10"^  mm. 
1  MM  (micromicron)  =  1/1, 000,000th  part  of  a  micron  =  1/1, 000, 000, 000th  part  of  a  mm.  = 

10-»  mm.  =  0.000,001  a«  =  lO""  A. 
1  Kg.  =  approximately  2.2  lbs. 

1  gm.  =  10-'  Kg.,  0.001  K.,  1000  mgm.,  1,000,000  ng. 
1  mgm.  =  10-«  Kg.,  10-»  gm.,  1000  Mg- 
1  /ig.  =  I7  =  10^  Kg.,  10-8  gm.,  10-'  mgm. 
m^g  =  1/1,000,000  mgm. 

A^  NaCl  is  normal  solution  of  sodium  chloride,  see  Normal  Solution. 
M  HCl  is  molecular  solution  of  hydrochloric  acid,  see  Molecular  Solution. 
M  =  mole. 
mM   =>   millimole. 
ME  =  milligram  equivalent. 
1  ml  (milliliter  =  l/l,000th  part  of  a  liter  =  1  cc.  (approx.)  that  is  1  cc.  +  0.000027  cc.  at 

40°C. 
M»  =  1/1,000  ml 

1  ml  (milliliter)  =  l/l,000th  part  of  a  liter  =  1  cc.  (approx.). 
CI  76  means  that  the  number  of  a  dye  is  76  in  the  Colour  Index  of  the  Society  of  Dyers  and 

Colourists. 
CC.  given  after  a  dye  signifies  that  it  has  been  certified  by  the  Biological  Stain  Commission 

The  following  publications  are  simply  referred  to  by  author,  or  senior  author, 
or  editor's  name  and  page  number  (cf.  Conn,  p.  26). 

Bensley,  R.  R.  and  S.  H.,  Handbook  of  Histological  and  Cytological  Technique,  Univ. 
Chicago  Press,  1938,  167  pp. 

BouBNE,  G.,  Cytology  and  Cellular  Physiology,  Oxford:  Clarendon  Press,  1942,  296  pp. 
(Second  Edition,  1951,  524  pp.) 

Conn,  H.  J.,  Biological  Stains,  Geneva,  N.  Y.:  Biotech  Publications,  1940,  308  pp. 

CowDRY,  E.  v.,  Textbook  of  Histology,  Philadelphia:  Lea  &  Febiger,  1938,  600  pp.,  3rd 
Edition,  1950,  640  pp. 

Craig,  C.  F.,  Laboratory  Diagnosis  of  Protozoan  Diseases,  Philadelphia:  Lea  &  Febiger 
1942,  349  pp. 

Downey,  H.,  Handbook  of  Hematology,  New  York:  Hoeber,  1938,  3136  pp. 

Emig,  W.  H.,  Stain  Technique,  Lancaster:  Science  Press  Printing  Co.,  1941,  75  pp. 

Glasser,  O.  (Editor),  Medical  Physics,  Chicago:  Year  Book  Publishers,  1944,  1744  pp. 

Glick,  David,  Techniques  of  Histo-  and  Cytochemistry.  New  York:  Interscience  Pub- 
lishers, Inc.,  1949,  531  pp. 

Lee,  Bolles,  The  Microtomists'  vade-mecum.  Philadelphia:  P.  Blakiston's  Son  &  Co. 
(Tenth  Edition,  Edited  by  J.  B.  Gatenby  and  T.  S.  Painter,  1937,  784  pp.  Eleventh 
Edition  edited  by  J.  B.  Gatenby  and  H.  W.  Beams,  1950,  753  pp.) 

LiLLiE,  R.  D.,  Histopathologic  Technic.     Philadelphia:  Blakiston,  1948,  300  pp. 

LisoN,  L.,  Histochemie  Animale,  Paris:  Gauthier-Villars,  1936,  320  pp. 

Mallory,  F.  B.,  Pathological  Technique,  Philadelphia:  Saunders,  1938,  434  pp. 

McClung,  C.  a.,  Microscopical  Technique,  New  York:  Hoeber,  1938,  698  pp.,  2nd  Edition 
by  Ruth  McClung  Jones,  1950,  790  pp. 


TECHNIQUES 


A-V  Bundle,  see  Todd,  T.  W.,  Cowdry's 
Special  Cytology,  1932,  2,  1173-1210. 

Abopon.  For  mounting  amyloid  stains 
(Leib,  Am.  J.  Clin.  Path.,  1947,  17,  413). 

Absorption.  Every  solid  surface  attracts 
other  substances  more  or  less.  This 
holding  is  referred  to  as  absorption. 
The  finer  the  structure  of  the  solid  the 
greater  the  combined  surface  area  of 
the  constituent  particles  and  conse- 
quently the  greater  the  degree  of  ab- 
sorption. An  interferometer  is  an  in- 
strument employed  to  measure  change 
in  concentration  by  absorption.  There 
are  many  other  ways  of  obtaining  this 
information .  See  Water  Absorption  and 
fat  absorption  after  previous  coloration 
of  fat  with  Sudan  III  or  Sudan  black 
(see  Vital  Staining).  See  X-ray  Ab- 
sorption. 

Absorption  Spectra.  Methods  are  avail- 
able for  the  determination  of  absorption 
spectra  of  cell  structures.  Caspersson 
(T.,  J.  Roy.  Micr.  Soc,  1940,  60,  8-25) 
has  described  apparatus  for  absorption 
from  intracellular  objects  larger  than 
1  micron  such  as  Nissl  bodies.  This 
line  of  investigation  is  just  developing 
and  is  likely  to  be  productive  of  im- 
portant results.    See  Histospectroscopy. 

Acacia,  properties  as  a  macromolecule 
(Hueper,  W.  C,  Arch.  Path.,  1942,  33, 
267-290).     See  V.  Apathy's  Syrup. 

Acanthocephala,  see  Parasites. 

Acarina,  see  Parasites,  Ticks. 

Acetic  Acid  (L.  acetum,  vinegar).  Widely 
used  as  a  component  of  fixatives.  The 
undiluted  solution  is  often  termed 
"glacial  acetic  acid."  This  contains 
99.5%CH3COOH.  Causes  a  distinctive 
swelling  of  fresh  collagenic  fibers. 
Employed  in  dilute  solution  to  destroy 
red  blood  cells  so  that  whites  can  be 
examined.  In  1%  solution  separates 
epidermis  from  dermis.     See  Epidermis. 

Acetic-Osmic-Bichromate  fixative  of  Bens- 
ley.  2%  osmic  acid,  2  cc;  2.5%  aq. 
potassium  bichromate,  8  cc;  glacial 
acetic  acid,  1  drop.  Excellent  for 
mitochondria  but  very  small  pieces  of 
tissue  must  be  used  because  the  fluid 
penetrates  poorly.  The  best  stain  is 
Anilin-Fuchsin  Methyl  Green,  see  also 
Copper  Chrome  Hematoxylin. 

Acetin  Blue  R  (CI,  560)— Induline  Alcohol 
Soluble — a  basic  dye  of  light  fastness  4. 
Paraffin  sections  of  plant  tissues  color 
dull  light  blue  (Emig,  p.  58). 

Acetic-Carbol— Sudan  III,  see  Sudan  III. 


Aceto-Carmine  (Schneider's).  Add  10  gms. 
carmine  to  100  cc.  45%  aq.  glacial  acetic 
acid.  Dissolve  with  heat  and  bring  up 
to  boiling.  Cool,  filter,  and  store  as 
stock  solution.  Used  for  smears  this 
combines  fixation  with  staining;  but 
it  causes  a  swelling  of  some  cellular 
elements  and  is  not  recommended. 

Aceto-Orcein-Fast-Green. — Written  by  Dr. 
N.  B.  Kurnick,  Dept.  of  Medicine, 
Tulane  University,  New  Orleans  12. 
January  31,  1951 — This  modification  of 
La  Cour's  aceto-orcein  stain-fixative 
for  chromosomes  permits  a  one-step 
difi"erential  staining  of  tissues.  The 
introduction  of  fast  green  and  NaCl 
(to  prevent  overstaining  by  the  former) 
provides  a  green  counterstain  for  the 
reddish-brown  chromatin.  The  intensity 
of  this  counterstain  may  be  modified  by 
varying  the  salt  concentration  (increas- 
ing the  salt  concentration  reduces  the 
intensity  of  green  staining),  but  the 
method  described  here  has  proved  satis- 
factory for  most  materials.  The  stain 
mixture  may  be  used  as  a  stain-fixative, 
as  for  dipteran  salivaries,  some  plant 
materials,  and  for  the  study  of  isolated 
chromosomes  and  nuclei,  or  as  a  stain 
following  other  fixatives.  Flood  ma- 
terial for  few  minutes  in  following  solu- 
tion: 27  ml.  1%  orcein  in  45%  acetic 
acid,  3  ml.  1%  fast  green  in  95%  alcohol, 
2  ml.  2M  NaCl;  cover  with  cover  slip, 
press  out  on  filter  paper,  if  desired. 
Paraffin  sections  must  be  brought  to 
water  before  staining  Permanent 
mounts  may  be  prepared  by  rinsing 
the  stained  material  successively  in 
70%,  95%,  100%  alcohol,  xylene,  and 
mounting  in  Clarite.  Cytoplasm,  col- 
lagen, and  nucleoli  are  green,  chromatin 
is  reddish-brown  (Kurnick,  N.  B.,  C^old 
Spring  Harbor  Symp.  Quant.  Biol., 
1947,  12,  191;  Kurnick,  N.  B.  and  Ris, 
Hans,    Stain    Tech.,    1948,    23,    17-18). 

Acetone,  see  Dehydration  of  Tissues,  as 
fixative  for  Phosphatases  and  Lipases. 

Acid  Alcohol  is  used  for  the  differentiation, 
or  decolorization,  of  certain  stains. 
It  is  usually  made  by  adding  1  cc. 
hydrochloric  acid  to  99  cc.  70%  ethyl 
alcohol.  It  is  also  employed  for  clean- 
ing cover  glasses. 

Acid  Alizarin  Blue  (1)  G.R.  (CI,  1048).  An 
acid  anthraquinone  dye  called  for  in 
Buzaglo's  Method  which  the  author  pro- 
poses as  substitute  for  Van  Gieson. 

(2)  B.B.  (CI,  1063)  likewise  an  acid 
anthraquinone  dye  little  used,  if  at  all. 


ACID  ALIZARIN  GREEN  G 


ACID  FAST  BACILLI 


Acid  Alizarin  Green  G  (CI,  1049),  a  direct 
mordant  dye  of  color  fastness  1.  Use 
for  staining  blue  green  and  green  algae 
and  paraffin  sections  of  animal  tissues 
after  mordanting  in  1%  aq.  ferric  alum 
is  described  (Emig,  p.  63). 

Acid  Blue  B  (CI,  736),  an  acid  dye  of  light 
fastness  5  gives  light,  fugitive  and  in- 
distinct coloration  of  tissue  (Emig, 
p.  52). 

Acid  Blue  G  (CI,  712)— Brilliant  Acid  Blue 
V — an  acid  dye  of  light  fastness  5  (Emig, 
p.  52). 

Acid   Bordeaux,  see   Bordeaux  Red. 

Acid  Congo  R,  see  Vital  Red. 

Acid  Dyes,  see  Staining. 

Acid  Fast  Bacilli.  Of  these  the  organisms 
of  tuberculosis  and  leprosy  are  the  most 
important. 

1.  In  smears  apply  Carbol  Fuchsin 
gently  heat  3-5  min.  or  stain  room 
temperature  15  min.;  decolorize  95% 
ethyl  alcohol  containing  3%  of  cone, 
hydrochloric  acid  until  only  slight  pink 
color  remains;  wash  in  water;  counter- 
stain  sat.  aq.  methylene  blue  or  Loef- 
fler's  Alkaline  Methylene  Blue;  wash 
and  dry. 

2.  In  sections  the  organisms  can  be 
stained  red  in  paraffin  sections  after 
almost  any  fixation  (formalin-Zenker 
preferred).  First  color  with  Harris 
hematoxylin.  Wash  in  water  and  per- 
haps decolorize  a  little  in  Acid  Alcohol. 
Wash  again.  Stain  with  warmed  carbol 
fuchsin  1  hr.  or  more.  Decolorize  in 
acid  alcohol.  Wash  carefully  in  water 
plus  few  drops  ammonia.  95%  ale, 
abs.  ale,  xylol,  balsam.  A  critique  of 
the  methods  has  been  published  (Fite, 
G.  L.,  Am.  J.  Path.,  1938,  14,  491-508). 
To  color  the  organisms  blue,  fix  3-5  days 
or  more  in  equal  parts  10%  formalde- 
hyde and  95%  alcohol.  Stain  sections 
in  new  fuchsin  0.5  gm.;  phenol  crystals, 
5.0  gm.;  alcohol  methyl  or  ethyl,  10  cc. 
+  aq.  dest.  to  make  100  cc.  at  60°  C. 
over  night,  12-24  hrs.  or  at  room  tem- 
perature 24-48  hrs.  Longer  for  M. 
leprae.  Freshly  distilled  aq.  formalde- 
hyde 5-30%,  5  min.  (Note  that  this 
formalin  must  not  be  alkaline  and  that 
it  is  safer  to  have  it  faintly  acidified.) 
2%  hydrochloric  acid  in  95%  alcohol, 
5  min.  1%  aq.  potassium  permanganate 
2-5  min.  (until  brown).  2%  aq.  oxalic 
acid,  1  min.  Harris'  hematoxylin  2 
min.  Stain  in  acid  fuchsin,  0.1  gm.; 
picric  acid,  0.5  gm.;  aq.  dest.  to  make 
100  cc.  Without  washing,  dehydrate  in 
alcohol,  clear  in  xylol  and  mount  in 
balsam.  Nuclei,  brown;  connective 
tissue  fibers,  red;  muscle,  yellow;  acid 
fast  bacilli,  dark  ultramarine  blue. 
Good  for  photography  (Fite,  G.  L.,  J. 
Lab.  &  Clin.  Med.  1939,  25,  743-744;  re- 


vised by  G.  L.  Fite,  U.  S.  Marine  Hos- 
pital, Carville,  La.   May  13,  1946.). 

3.  Mr.  J.  M.  Albrecht  employs  the 
following  method  in  our  laboratory. 
Deparaffinize  5-6  n  sections  of  10% 
formalin  or  Regaud  fixed  tissues.  Wipe 
off  excess  water  around  sections  and 
cover  with  strip  of  filter  paper.  Flood 
filter  paper  with  carbol  fuchsin  (Phenol 
crystals,  8  gm.;  basic  fuchsin,  4  gm.; 
95%  ethyl  alcohol,  20  cc;  aq.  dest.,  100 
cc).  Steam  for  3  min.  and  then  allow 
to  stand  for  30  min.  adding  more  stain 
if  necessary.  The  filter  paper  prevents 
deposition  of  ppt.  of  dye  on  sections. 
Flush  off  stain  with  aq.  dest.  Partly 
differentiate  in  1  cc.  cone  hydrochloric 
acid  in  100  cc.  70%  alcohol,  sections  be- 
coming deep  pink.  Wash  in  aq.  dest. 
Stain  Harris'  Hematoxylin  10  min., 
wash  in  aq.  dest.  Complete  differentia- 
tion of  both  fuchsin  and  hematoxylin  in 
50  cc.  70%  ale  -f  4-5  drops  hydrochloric 
acid,  sections  becoming  light  pink. 
Wash  in  aq.  dest.  Neutralize  in  6  drops 
cone  ammonia  +  50  cc.  aq.  dest. 
Wash,  dehydrate,  clear  and  mount  as 
usual. 

4.  In  frozen  sections  (Krajian,  A.  A., 
Am.  J.  Clin.  Path.,  Techn.  Suppl.,  1943, 
7,  45-47).  Transfer  frozen  sections  of 
leprous  tissue  to  slides.  Dehydrate, 
blot  with  filter  paper,  dip  in  celloidin. 
Blow  over  surface  till  dry.  Wash  in  tap 
water.  Apply  Carbol  Fuchsin  steaming 
gently  for  3  min.  Pour  off  and  wash  in 
tap  water.  Differentiate  with  1  gm. 
arsenic  acid  in  100  cc.  60%  alcohol  ap- 
plied by  medicine  dropper.  Again  wash 
in  tap  water  and  counterstain  with 
Loeffler's  methylene  blue  2  min.  Wash 
in  tap  water,  dehydrate  with  3  applica- 
tions of  anhydrous  isopropanol  or 
absolute  ethyl  alcohol.  Apply  imme- 
diately equal  parts  anhydrous  iso- 
propanol or  abs.  alcohol  and  beechwood 
creosote.  Agitate  slide  removing  ex- 
cess blue  color.  Blot  with  filter  paper, 
clear  with  xylol  and  mount  in  damar. 

See  Tubercle  and  Leprosy  Bacilli, 
Fluorescence  Microscopy,  also  paper  by 
Richards,  O.  W.,  Kline,  C.  K.  and 
Leach,  R.  E.,  Am.  Rev.  Tubere,  1941, 
44,  255-266.  Efficiency  of  Ziehl-Neel- 
sen  and  fluorescence  techniques  com- 
pared. The  latter  superior  (Van  Dyke, 
A.  E.,  Am.  J.  Clin.  Path.,  Techn. 
Suppl.,  1943,  7,  6-8.)  For  acid  fast 
bacilli  in  urine  see  Kelso,  R.  E.  and 
Galbraith,  T.  W.,  Am.  J.  Clin.  Path., 
Techn.  Suppl.,  1943,  7,  8-11. 

Less  is  known  about  the  conditions 
that  determine  acid  fastness  than  those 
which  determine  Gram  positiveness 
(see  Gram  Stain).  The  facts  are  well 
stated  for  mycobacteria  in  general  and 


ACID  FUCHSIN 


ADENOSINASE 


especially  for  the  Tubercle  Bacillus  by 

Dubos,  R.  J.,  The  Bacterial  Cell. 
Harvard  Univ.  Press,  1945,  460  pp. 
There  is  present  in  the  tubercle  bacillus 
mycolic  acid  which  is  acid  fast  even 
after  isolation  in  the  pure  state;  but 
the  property  of  acid  fastness  is  lost  by 
the  bacilli  under  conditions  that  do  not 
destroy  this  acid.  These  conditions 
involve  destruction  or  impairment  of 
structure  of  the  organisms  by  mechani- 
cal, chemical  or  enzymatic  means. 
Apparently  the  cell  surface  must  be 
intact.  Dubos  quotes  Yegian  et  al.  as 
showing  that  tubercle  bacilli  stained  in 
absence  of  electrolytes  are  uniformly 
colored  rods,  that  addition  of  electro- 
lytes causes  a  beaded  appearance  and 
that  treatment  with  ethyl  alcohol  re- 
stores uniform  solid  staining  to  beaded 
organisms  which  means  that  the  change 
from  beaded  to  uniform  state  is  a  re- 
versible process.  This  dependence  of 
microscopic  appearance  on  experi- 
mental conditions  of  technique  is  ob- 
viously a  matter  of  great  consequence 
in  leprosy  as  well  as  in  tuberculosis. 
The  investigator  has  to  check  carefully 
by  study  of  living  unstained  bacilli. 

Acid  Fuchsin  (CI,  692) — acid  magenta,  acid 
rubin,  fuchsin  S,  SN,  SS,  ST  or  S  Ill- 
Commission  Certified.  Since  this  is  a 
sulfonated  derivative  of  basic  fuchsin, 
and,  because  there  are  4  possible  pri- 
mary basic  fuchsins,  Conn  (p.  118)  points 
out  that  at  least  a  dozen  primary  acid 
fuchsins  are  possible  and  samples  are 
usually  mixtures  of  several.  Acid 
fuchsin  is  employed  is  so  many  ways 
that  to  enumerate  them  would  be  both 
futile  and  unnecessary.  See  New 
Fuchsin. 

Acid  Green,  see  Light  Green  SF  yellowish. 

Acid  Green  O,  see  Naphthol  Green  B. 

Acid  Hemalum,  see  Hemalum. 

Acid  Magenta,  see  Acid  Fuchsin. 

Acid  Orange  II,  Y  or  A,  see  Orange  II. 

Acid  Phosphatase,  see  Phosphatase. 

Acid  Phloxine  GR,  see  Chromotrope  2R. 

Acid  Rubin,  see  Acid  Fuchsin. 

Acid  Violet.  Several  triphenyl  methane 
dyes  come  under  this  heading.  Conn 
(p.  132)  says  that  the  term  "acid 
violet"  is  too  indefinite  for  identifica- 
tion. This  is  unfortunate  because  dyes 
bearing  this  label  have  been  used  in 
several  combinations  as  in  Bensley's 
Neutral  Safranin  acid  violet.  Bailey, 
P.,  J.  Med.  Res.,  1921,  42,  349-381  and 
Maurer,  S.  and  Lewis,  D.  D.,  J.  Exp. 
Med.,  1922,  36,  141-156,  working  in 
Bensley's  laboratory,  used  it  for  the 
pituitary.  Acid  violet  is  one  of  the 
stains  employed  by  Weiss,  E.,  J.  Inf. 
Dis.,  1928,  43,  228-231  to  stain  flagella 


and  spirochetes  (J.  Lab.  &  Clin.  Med., 
1928-29,  14,  1191-1193). 

Acid  Yellow,  see  Fast  Yellow. 

Acid  Yellow  R,  see  Metanil  Yellow. 

Acidity,  see  Hydrogen  ion  indicators. 

Acidophilic,  see  Staining. 

Acids,  see  under  first  name,  Acetic  Acid, 
Hydrochloric  Acid,  etc  . 

Ackerman,  see  Auer  Bodies. 

Acridine  Dyes.  As  the  name  suggests  they 
are  formed  from  acridine  which  is  re- 
lated to  xanthene.  Examples:  acri- 
flavine,  neutral  acriflavine  and  phos- 
phine.  Phosphine  3R  is  employed  as  a 
fluorochrome  for  lipids. 

Acridine  Orange  (CI,  788),  a  basic  dye  of 
light  fastness  1  to  2.  Gives  clear  brown 
or  dark  orange  coloration  of  plant  tis- 
sues of  exceptional  fastness.  Tech- 
nique described  (Emig,  p.  55). 

Acridine  Red  3B  is,  according  to  McClung, 
Microscopical  Technique,  1950,  p.  73, 
not  an  acridine  dye  but  a  pyromin 
closely  related  to  Pyronin  Y.  It  has 
been  employed  by  Gomori,  G.,  Am.  J. 
Path.,  1936,  12,  655-663  mixed  with 
methyl  green  to  reveal  calcium  salts 
and  phosphatase  activity. 

Acriflavine  (CI .  790) .  A  yellow  fluorchrome . 
It  is  useful  as  a  vital  stain  for  nuclei. 
Farr,  R.  S.,  Anat.  Rec,  1946,  94,  16, 
has  employed  acriflavine  hydrochloride 
to  label  transfused  leucocytes  and  to 
determine  how  long  they  remain  in  the 
circulation. 

Actinomyces.  Mallory's  stain  for  actino- 
myces  in  sections  (Mallory,  p.  279). 
For  the  organisms,  fixation  in  alcohol 
or  in  10%  formalin  is  preferable;  but 
for  the  lesions,  Zenker's  fluid  is  better. 
Stain  deparaffinized  sections  in  Alum 
Hematoxylin  3-5  min.  After  washing 
in  water  stain  in  2.5%  aq.  phloxine  or 
in  5%  aq.  eosin  in  paraffin  oven,  15  min. 
After  again  washing,  stain  in  Stirling's 
or  Ehrlich's  aniline  crystal  violet  (see 
Anilin  Crystal  Violet), 5-15  min.  Wash 
in  water  and  treat  with  Gram's  Iodine, 
1  min.  Wash  in  water,  blot  and  destain 
in  aniline  oil  until  no  further  color 
comes  out.  Rinse  in  xylol  and  mount 
in  balsam.  Branched  forms,  blue; 
clubs,  pink  to  red. 

Actinosphaerium,  see  McClung,  Microscopi- 
cal Technique,  1950,  p.  469. 

Addis  Count  to  provide  quantitative  data 
on  number  of  red  blood  cells  and  casts 
in  the  urine  is  critically  described  by 
C.  J.  Gentzkow  and  H.  A.  Van  Auken 
in  Simmons  and  Gentzkow,  p.  32. 

Adenosinase.  A  method  for  analysis  of 
adenosinase  in  lymphocytes  and  poly- 
morphonuclear leucocytes  (neutro- 
philes)  is  given  by  Barnes,  J.  M.,  Brit. 
J.  Exp.  Path.,  1940,  21,  264-275. 


ADENYLPYROPHOSPHATASE 


AGONAL^CHANGES 


Adenylpyrophosphatase.  The  technique  of 
localization  of  this  inaportant  enzyme 
in  cytoplasmic  granules  has  been  de- 
scribed and  used  in  extracts  of  chick 
embryos  by  Steinbach,  H.  B.  and  Moog, 
F.,  J.  Cell  and  Comp.  Physiol.,  1945, 
26,  175-183.  These  authors  are,  how- 
ever, not  sanguine  about  the  feasibility 
of  its  localization  by  histochemical 
methods  (Science,  1946,  103,  144)  as 
reported  by  Glick  and  Fischer,  Science, 
1945, 102,  429^30.  However,  Malngwyn- 
Davies,  E.  D.  and  J.  S.  Friedenwald, 
J.  Nat.  Cancer  Inst.,  1950,  10,  1379, 
recently  reported  at  the  Histochemical 
Society  that  specific  localizations  were 
achieved  when  unfixed  frozen  sections 
were  incubated  in  muscle  adenosine 
triphosphate  substrates. 

Adermin,  see  Vitamin  B6. 

Adhesiveness,  or  stickiness  of  cellular  sur- 
faces is  a  phenomenon  of  great  im- 
portance in  connection  with  movement, 
phagocytosis  embryological  develop- 
ment and  other  processes.  There  is  no 
standard  technique  to  measure  it,  ex- 
cept in  special  circumstances  as  when 
it  ia  manifested  by  agglutination  of 
bacteria  and  sedimentation  of  red  blood 
cells.  The  way  leucocytes  stick  to  the 
endothelial  wall  of  a  small  blood  vessel, 
shown  by  Motion  Pictures,  is  impres- 
sive. Adhesion  tests  have  been  intro- 
duced as  means  of  diagnosis  of  various 
trypanosomes.  A  fine  general  discus- 
sion of  this  phenomenon  is  provided  by 
Beams  and  King  in  Calkins,  G.  N.  and 
Summers,  F.  M.,  Protozoa  in  Biologi- 
cal Research.  New  York:  Colombia 
University  Press,  1941,  1148  pp. 

Adrenal.  For  routine  purposes  fix  in 
Zenker's  Fluid  and  stain  paraffin  sec- 
tions with  Hematoxylin  and  Eosin. 
There  are  many  techniques  for  Lipids. 
The  Chromaffin  Reaction  is  often  used 
for  adrenalin  but  Cramer,  W.,  J.  Path. 
&  Bact.,  1937,  44,  633,  considers  black- 
ening with  osmic  acid  vapor  as  more 
specific.  Silver  methods  for  vitamin 
C  are  difficult  to  apply  but  are  appar- 
ently reliable.  They  are  given  under 
Vitamins.  The  Schultz  cholesterol  test 
gives  excellent  results.  A  selection 
may  be  made  from  several  methods  for 
Reticular  Fibers.  Corner,  G.  W.,  Con- 
trib.  to  Embryol.,  Carnegie  Inst.,  1920, 
9,  87-93,  employed  for  reticulum  the 
Bielschowsky-Maresch  silver  method 
exactly  as  specified  by  Ferguson,  J.  S., 
Am.  J.  Anat.,  1912,  12,  277-296.  The 
Bodian  protargol  method  for  nerve 
fibers  has  been  adjusted  to  the  adrenal 
by  MacFarland,  W.  E.,  and  Davenport, 
H.  A.,  Stain  Techn.,  1941,  16,  53-58, 
also  Cajal's  chloral  hydrate  method. 
If  one  contemplates  ultracentrifugation 


and  the  demonstration  of  the  Golgi 
apparatus  consult  Guyer,  M.  F.,  and 
Claus,  P.  E.,  Anat.  Rec,  1939,  73, 
17-27. 

Method  proposed  by  Bennett,  S.  H., 
Am.  J.  Anat.,  1940,  67,  151-227  for  keto- 
steroid  cortical  hormone  said  by  Go- 
mori,  G.,  Proc.  Soc.  Exp.  Biol.  &  Med., 
1942,  51,  133-134  not  to  be  specific  but 
to  indicate  merely  location  of  lipids 
having  keto  or  aldehyde  groups.  A 
technique  for  microscopic  study  of 
living  grafts  of  adrenal  cortex  (Wil- 
liams, O.,  Anat.  Rec,  1945,  91,  307). 

Adrenalin,  see  Chromaffin  Reaction. 

Aerosol,  a  detergent  used  in  preparing  bac- 
teria for  staining  (Sineszko,  S.  F., 
Science,  1942,  96,  589). 

Affixatives  are  materials  used  to  fix  sections 
to  slides.     See  Albumen-Glycerin. 

Agar,  as  matrix  for  cutting  plant  material 
with  freezing  microtome  (Evenden,  W. 
and  Schuster,  C.  E.,  Stain  Techn., 
1938, 13, 145^146).  Lillie  (p.  42)  says  that 
infiltration  of  tissues  from  water  in  2% 
aq.  agar  at  55-60°C.  for  2-4  hrs.  is  useful 
for  holding  friable  tissues  and  exudates 
in  place  before  cutting  frozen  sections. 
The  Agar  does  not  color  appreciably 
with  the  usual  stains. 

Age  Changes  are  as  manifold  as  life  itself. 
Some  are  detectable  by  structural 
modifications  while  others  can  only  be 
measured  by  decrease  in  performance. 
Many  old  tissues  can  easily  be  dis- 
tinguished from  new  ones  as  for  example 
Bone.  Some  accumulate  definite  prod- 
ucts with  age  like  Lipofucsin.  The  age 
of  tissue  and  of  cellular  components, 
that  is  the  time  they  endure,  can  be 
determined  by  attaching  Tracer  Sub- 
stances to  them  so  that  their  rates  of 
Replacement  can  be  measured.  With 
the  passage  of  time  colloids  age,  become 
less  elastic  and  more  granular.  Old 
Elastic  Fibers  can  be  distinguished  from 
young  ones.  Now  that  the  ultra  struc- 
ture of  Collagenic  Fibers  has  been  re- 
vealed by  the  electron  microscope  we 
may  hope  for  more  accurate  means  of 
estimating  their  condition  in  relation 
to  age.  Numerous  physical  techniques, 
including  the  Polarization  Optical 
Method,  may  well  bring  to  light  sig- 
nificant age  changes.  Obviously  many 
methods  of  chemical  analysis  and  of 
enzyme  activity  provide  data  on  the 
modes  of  run  down  of  vital  activities. 
Aggeler,  see  Blood  Platelets. 
Agonal  Changes  are  particularly  difficult 
to  avoid  in  villi  of  small  intestine. 
They  are  evidenced  by  a  ballooning  of 
the  epithelial  cap  most  marked  when 
absorption  of  ordinary  food  stuffs  is 
active.  The  ballooning  phenomenon 
can  be  produced  in  the  living  animal  by 


ALBERT'S  STAIN 


ALDOLASE 


ligating  arteries  of  supply  or  by  em- 
ploying fixatives  which  induce  forcible 
contraction  of  smooth  muscle  (Macklin, 
C.  C.  and  M.  T.,  Chapter  on  Intestinal 
Epithelium  in  Cowdry's  Special  Cy- 
tology, N.  Y.,  Hoeber,  1932,  1,  235). 

Albert's  Stain  for  Diphtheria  Bacilli,  which 
see. 

Albumen-Glycerin  for  mounting  paraffin 
sections.  Egg  white  50  cc,  glycerin 
50  cc,  sodium  salicylate  1  gm.  This  is 
"Mayer's  Albumen".  Shake  together 
and  filter  during  several  days.  See 
also  Starch  Paste  and  Masson's  Gelatin 
Glue. 

Alcohol.  Unless  indicated  to  the  contrary 
the  word  "alcohol"  as  employed  in  this 
book  refers  to  the  ethyl  variety.  Alone 
it  is  a  good  fixative  preliminary  to  tests 
for  Amyloid,  Copper,  Fibrin,  Glycogen, 
Gold,  Hemofuscin,  Hyaline,  Iron,  Lead, 
Palladium,  Phosphatase,  Potassium 
and  Thallium,  which  see.  It  is  also 
employed  in  the  demonstration  of  Nissl 
bodies  by  Gallocyanin,  of  mucus  by 
Mucicarmine,  of  proteins  by  the  Ro- 
mieu  Reaction,  etc.  In  combination 
with  other  chemicals  alcohol  is  also  much 
used  as  a  fixative,  see  Alcohol  Formalin, 
Carney's  Fluid  and  many  others. 

Alcohol  of  70%  is  a  good  preservative 
and  celloidin  blocks  can  be  stored  in  it. 
Absolute  alcohol  is  supposed  to  contain 
not  more  than  1%  by  weight  of  water. 
It  is  considered  to  be  100  per  cent.  A 
very  rough  test  for  absolute  alcohol  is  to 
mix  with  it  a  few  drops  of  turpentine. 
If  it  becomes  milky  it  contains  too  much 
water.  To  make  a  lower  per  cent  from  a 
higher  one  by  dilution  take  the  number 
of  cc.  corresponding  to  the  percentage 
required  and  add  aq.  dest.  to  make  in  cc. 
the  percentage  of  the  alcohol  diluted. 
Thus  to  make  30%  from  70%  take  30  cc. 
of  70%  and  add  aq.  dest.  to  make  70  cc. 
Alcohol  is  the  best  dehydrating  agent 
for  tissues.  It  is  sometimes  not  easy  to 
purchase  absolute  alcohol  so  that  it  must 
be  prepared.  Take  say  10  liters  of  95% 
alcohol,  add  400  gms.  freshly  ignited 
calcium  oxide.  Leave,  with  occasional 
shaking,  24  hrs.  until  most  of  the  water 
is  absorbed  by  the  oxide.  Pour  off 
fluid  (leaving  oxide  at  bottom  of  con- 
tainer) and  distill  using  appropriate 
precautions.  Keep  the  "absolute"  as 
nearly  so  as  possible  by  using  a  tight 
glass  stopper  for  the  bottle,  or  in  place 
of  the  stopper  an  absorption  tube  con- 
taining calcium  chloride  so  that  any 
water  in  entering  air  will  be  absorbed 
and  will  not  reach  the  alcohol.  See 
Dehydration,  also  Amyl,  n-Butyl,  Ter- 
tiary  Butyl,  Isopropyl,  n-Propyl  and 
Polyvinyl  Alcohols. 


Alcohol -Formal  in  is  a  fixative  containing  9 
parts  of  absolute  alcohol  and  1  part  of 
formalin.  Since  it  penetrates  quickly 
and  dehydration  can  be  commenced  in 
absolute  alcohol  immediately  after  fixa- 
tion, skipping  the  lower  grades  of  alco- 
hol, permanent  preparations  can  be 
made  within  a  few  hours'  time.  For 
routine  purposes  3-6  hrs.  fi.xation  will 
suffice  but  as  a  preliminary  to  Micro- 
incineration 24  hrs.  is  recommended. 
Alcohol-formalin  is  recommended  for 
Fibrin,  Glycogen,  Indigo-Carmine 
stains  and  Peroxidase.  It  is  employed 
with  acetic  acid  in  Bodian's  Method 
for  nerve  fibers. 

Aldehyde  Green  (CI,  676a) — Aniline  Green, 
Benzaldehyde  Green— a  basic  dye  of 
light  fastness  4,  employed  as  counter- 
stain  for  Biebrich  Scarlet,  Acid  Fuch- 
sin.  On  xylene  and  sclerenchyma  gives 
rather  brighter  shade  than  Malachite 
Green  (Emig,  p.  48). 

Aldehydes.  The  bound  form  of  aldehyde 
has  been  called  "plasmalogen".  From 
this  the  loosely  bound  "plasmal"  form 
is  developed  on  treatment  with  mercuric 
chloride  or  by  acid  hydrolysis  (the  Schiff 
Reaction  for  aldehydes).  This  is  the 
basis  of  the  Feulgen  reaction  for  Thymo- 
nucleic  Acid.  According  to  Oster,  K. 
A.  and  Oster,  J.  G.,  J.  Pharmacol,  and 
Exper.  Therap.,  1946,  87,  306-312,  the 
fuchsin  sulphurous  acid  reagent  em- 
ployed in  this  reaction  when  "true"  is 
specific  for  aldehydes  while  other  car- 
bonyl  compounds  sometimes  give  a 
"pseudo"  reaction.  Oster,  K.  A.  and 
Mulinos,  M.  G.,  J.  Pharmacol,  and 
Exper.  Therap.,  1944,  80,  132-138  report 
that  the  purple  of  the  "true"  reaction 
can  be  decolorized  with  dilute  sodium 
hydroxide  and  restored  with  hydro- 
chloric acid,  whereas  the  reddish  tint 
of  the  "pseudo"  reaction  cannot  be 
restored  in  this  way  after  decoloriza- 
tion  (see  Glick  p.  65).  See  Thymo- 
nucleic  Acid  (desoxyribonucleic  acid) 
and  Carbonyl  Compounds. 

Aldolase.  Triose  phosphate  was  used  as 
a  substrate  for  the  Gomori  phosphatase 
procedure  by  Allen,  R.  S.  L.  and  G.  J. 
Bourne,  J.  Exp.  Biol.,  1943,  20,  61-64. 
The  phosphate  ion  liberated  by  enzymic 
activity  was  precipitated  in  an  alkaline 
medium.  The  enzyme  was  not  in- 
hibited by  fluoride  in  concentrations 
which  blocked  alkaline  phosphatase 
activity.  The  pH  of  the  mixture  was 
not  stated,  but  was  presumably  about 
9.5.  The  method  was  not  completely 
satisfactory  because  of  spontaneous 
precipitates;  nevertheless,  concentra- 
tions of  enzyme  were  noted  in  striated 
and  cardiac  muscle,  kidney  and  liver. 
Little  or  none  was  seen  in  the  lung. 


ALGIRE 


ALPHAZURINE  2G 


Aldolase  +  Isomerase  are  known  as 
Zymohexase. 

Algire,  see  Transparent  Chamber  Tech- 
niques. 

Alizarin  (CI,  1027)  a  little  used  acid  an- 
thraquinone  dye. 

Alizarin  No.  6,  see  Purpurin. 

Alizarin  Blue  RBN,  see  Gallocyanin. 

Alizarin  Carmine,  see  Alizarin  Red  S. 

Alizarin  Cyanine  R  (CI,  1050),  an  acid  mor- 
dant dye  which  is  not  stable  in  solution, 
and  on  heating  yields  reddish  ppt. 
(Emig,  p.  64). 

Alizarin  SX,  or  anthrapurpurin,  use  same 
as  Alizarin  Red  S. 

Alizarin  Green  G  (CI,  917),  an  acid  mordant 
dye  of  light  fastness  1.  After  mor- 
danting in  1%  aq.  ferric  alum  stain  for 
30  min.  at  50°C.  in  0.1  gm.  of  dye  in 
100  cc.  1%  aq.  ammonium  acetate. 
The  green  color  obtained  is  the  clearest 
given  by  a  mordant  dye.  Additional 
directions  are  supplied  (Emig,  p.  59). 

Alizarin  Line  Test  for  new  bone  and  vitamin 
D  (Martin,  G.  J.,  J.  Lab.  &  Clin.  Med., 
1940,26,714-719).    See  Line  Test. 

Alizarin  Purpurin,  see  Purpurin. 

Alizarin  Red  S  (CI,  1034) — alizarin  red 
water  soluble,  alizarine  carmine — Com- 
mission Certified.  By  far  the  most  used 
of  all  the  alizarin  stains.  An  important 
ingredient  in  Benda  Method.  Much 
superior  to  Madder  for  the  staining  of 
bone  and  dentine  laid  down  while  it  is  in 
the  circulation.  Schour  has  employed 
it  extensively.  The  technique  is  de- 
scribed in  detail  by  him  and  his  asso- 
ciates (J.  Dent.  Res.,  1941,  20,  411-418). 
He  employed  an  Alizarin  red  S  (CI, 
1034)  obtained  from  Coleman  and  Bell 
Co.  The  effective  dose  for  rat,  rabbit, 
guinea  pig,  cat,  monkey  and  human  in- 
fant is  between  50^100  mg.  per  Kilo, 
conditioned  by  species,  age  and  weight. 
For  newborn  white  rats  he  recommends 
0.2  cc.  2%  Alizarin  and  for  rats  weighing 
100-200  gms.  5-I  cc.  given  intraperi- 
toneally.  Colors  are  retained  in  speci- 
mens fixed  in  10%  neutral  formalin  or  in 
95%  ale.  As  in  the  case  of  Madder 
staining  of  bone,  tissues  can  be  cleared 
and  examined  as  whole  preparations,  or 
ground  sections  can  be  prepared  for 
microscopic  study.  Decalcification 
spoils  the  color.  Age  factor  in  alizarin 
staining  (Ercoli,  N.  and  Lewis,  M.  N., 
Anat.  Rec,  1943,  87,  67).  See  Ossifica- 
tion and  Line  Test. 

Alizarin  Red  Water  Soluble,  see  Alizarin 
RedS. 

Alizarin  Sapphire  BN  (CI,  1054)  of  NAC,  a 
direct  mordant  dye  of  light  fastness  2 
(Emig,  p.  64). 

Alkali  Blue  6  B  (CI,  703),  an  acid  dye  of 
light  fastness  4  to  5  and  of  little  value 


for     permanent     preparations     (Emig, 
p.  51). 

Alkali  Green  (CI,  665),  an  acid  dye  of  light 
fastness  5  gives  very  fugitive  pale  dull 
green  color  (Emig,  p.  47). 

Alkaline  Methylene  blue,  see  Loeffler's. 

Alkaline  Phosphatase,  see  Phosphatase 
and  Kidney. 

Alkalinity,  see  Hydrogen  Ion  Concentration. 

Allantoin,  colorimetric  method,  Borsook, 
J.  Biol.  Chem.,  1935,  110,  481-493. 

Allen's  Fluids  are  modifications  of  Bouin's 
often  containing  urea.  They  are  excel- 
lent for  chromosomes.     See  McClung. 

Allergy,  see  Pollens. 

Allochrome  Procedure,  a  differential 
method  segregating  tissues,  collagen, 
reticulum  and  basement  membranes 
into  two  groups  (Lillie,  R.  D.,  Am.  J. 
Clin.  _Path._,  1951,  21,  484-488).  The 
term  is  derived  from  G.  allochroos,  a 
different  or  changing  color,  since  colla- 
gen and  some  related  substances  during 
the  procedure  changes  from  red  to  blue. 
See  Lillie's  paper  for  details  of  this 
Periodic  Acid  Schiff,  picro-methyl  blue 
technique. 

Alloxan  Reaction.  1%  alcoholic  solution  of 
alloxan  gives  red  color  with  a  aminoacids. 
Romieu  (M.,  Bull.  d'Hist.  appl.,  1925, 
2,  185-191)  employs  a  cold  neutral  solu- 
tion. Giroud  (A.,  Protoplasma,  1929, 
7,  72-98)  uses  heat  but  states  that  great 
care  is  necessary  in  interpretation.  See 
Lison,  p.  129. 

This  reaction  is  described  as  follows 
by  Serra,  J.  A.,  Stain  Techn.,  1946,  21, 
5-18.  Fix  tissue  as  given  under  Nin- 
hydrin  Reaction.  "An  alcoholic  1% 
solution  of  alloxan  gives  with  amino 
acids  and  proteic  compounds  a  pink 
coloration,  after  a  long  time  at  room 
temperature,  or  rapidly  if  the  reaction 
is  activated  by  heating  in  a  boiling 
water  bath.  In  our  experience,  this 
test  is  relatively  insensitive;  besides 
this,  the  coloring  formed  diffuses 
easily,  so  that  the  reaction  can  be  in- 
distinctly localized.  With  fixed  mate- 
rials the  reaction  is  weak. 

"The  test  must  be  carried  out  in 
neutral  solutions;  this  is  attained  by 
addition  of  a  phosphate  buffer,  as  de- 
scribed for  the  ninhydrin.  This  reac- 
tion is  not  specific  for  amino  acids  and 
proteins,  as  it  is  also  given  by  com- 
pounds with  free  NH2  and  perhaps  SH 
groups  (see  Winterstein,  1933)." 

Almkvist-Christeller,  see  test  for  Mercury. 

Alpha  Naphthol,  see  Oxidase. 

Alpha  Particle,  see  Gross,  J.,  Bogoroch,  R., 
Nadler,  N.  J.,  and  Leblond,  C.  P.,  Am. 
J.  Roentgenol.  Rad.  Therap.,  1941, 
65,  420-458. 

Alphazurine  2G  see  Patent  Blue. 


ALTMANN'S  FLUID 


ALUMINUM  CHLORIDE  CARMINE 


Altmann's  Fluid.  Equal  parts  of  5%  aq. 
potassium  bichromate  and  2%  aq.  osmic 
acid.  Employed  in  his  method  as  well 
as  for  staining  with  Copper  Chrome 
Hematoxylin.  It  gives  good  surface 
fixation  but  penetrates  very  badly. 

Altmann's  Method  of  anilin  fuchsin  and  pic- 
ric acid  for  mitochondria.  Fix  small 
pieces  not  more  than  2  mm.  in  diameter 
24  hrs.  in  Altmann's  Fluid.     Wash  for 

1  hr.  dehydrate,  clear  imbed  in  paraffin 
and  cut  sections  i/i.  Pass  down  to 
water.  Stain  in  anilin  fuchsin  (20% 
acid  fuchsin  in  anilin  water)  6  min. 
Blot  with  filter  paper.  Differentiate 
and  counter  stain  by  flooding  the  sec- 
tions with  1  part  sat.  ale.  picric  acid  and 

2  parts  aq.  dest.  Rinse  rapidly  in  95% 
ale,  dehydrate  in  abs.  ale,  clear  in 
xylol  and  mount  in  balsam.  The  mito- 
chondria are  stained  crimson  against  a 
bright  j^ellow  background.  Altmann's 
magnificent  original  plates  should  be 
examined  (Altmann,  R.,  Die  Elementar- 
organismen  und  ihre  Beziehungen  zu  den 
Zellen.  Leipzig:  Veit  Co.,  1894,  160 
pp.).  If  these  are  not  available  see 
Meves,  F.,  Arch.  f.  mikr.  Anat.,  1913, 
82,    (2),  215-260. 

Altmann-Gersh  frozen-dehydration  method 
(Gersh,  I.,  Anat.  Rec,  1932,  53,  309- 
337). — Account  written  by  Dr.  Gordon 
H.  Scott,  Dept.  of  Anatomy,  Wayne 
University  School  of  Medicine,  De- 
troit, Mich.  This  method  has  proved 
to  be  of  much  value  in  the  preparation 
of  tissues  for  microchemical  proce- 
dures. It  has  also  been  used  as  a  pre- 
liminary treatment  for  tissues  destined 
for  examination  by  the  electron  micro- 
scope (Wyckoff,  R.  W.  G.,  Science, 
1946,  104,  21-26).  Tissues  are  frozen 
in  liquid  nitrogen  or  in  liquid  oxygen 
and  dehydrated  in  vacuo  at  low  tem- 
peratures. The  tissue  sample  remains 
frozen  at  such  a  temperature  that  little 
or  no  chemical  change  can  take  place. 
It  is  believed  that  the  only  significant 
revision  in  cellular  organization  takes 
place  during  the  freezing  process.  This 
is  occasioned  by  possible  shifts  in  pro- 
teins, etc.,  during  ice  crystal  formation. 
Some  users  of  the  method  believe  that  it 
is  possible  to  freeze  small  tissue  samples 
at  speeds  which  will  actually  prevent 
ice  crystal  formations.  Efforts  in  this 
direction  have  been  made  by  freezing 
in  cooled  iso-pentane  (technical) 
(Hoerr,  N.  L.,  Anat.  Rec,  1936,  65,  293- 
317;  Simpson,  W.  L.,  Ibid.,  1941,  80, 
173-189). 

For  many  reasons  it  has  been  found 
desirable  to  dehydrate  at  lower  tem- 
peratures than  were  first  thought  neces- 
sary.    Now  the  standard  procedure  is 


to  dehydrate  in  vacuo  from  40-65°C. 
Apparatus  of  special  design  has  been 
constructed  a  number  of  times  to  meet 
various  needs.  In  general  the  prin- 
ciples are  the  same.  What  is  needed 
is  a  vacuum  system  with  high  pumping 
speed  and  with  provision  for  keeping 
the  frozen  tissue  at  constant  tempera- 
ture. Several  of  these  have  been  de- 
scribed, each  with  its  adaptation  to  the 
needs  of  the  case. 

For  general  use  in  histochemistry  the 
device  described  by  Packer  and  Scott 
(J.  Tech.  Methods,  1942,  22,  85-96)  and 
by  Hoerr  and  Scott  (Medical  Physics, 
Otto  Glasser,  1944,  Year  Book  Pub- 
lishers) is  both  easy  to  operate  and  re- 
liable. It  has  the  distinct  advantage 
that  tissues  can  be  infiltrated  with 
paraffinjwithout^exposure  to  air.  This 
apparatus  can  also  be  used  for  the 
preparation  of  tissues  for  electron 
microscopy.  For  this  use  only  the  de- 
hydration device  described  by  Wyckoff 
is  probably  more  suitable. 

Recently  developed  equipment  will 
permit  drying  frozen  tissues  in  5  hours. 
(Stowell,  R.  E.,  Stain  Techn.  1951,  in 
press) 

Alum.  The  alums  are  double  salts  of  sul- 
phuric acid.  Aluminum  potassium  sul- 
phate, or  potassium  alum,  unless  other- 
wise stated  is  the  one  used  in  making  up 
hematoxylin  solutions.  Aluminum  am- 
monium sulphate,  or  ammonia  alum, 
should  not  be  used  as  a  substitute  unless 
called  for.  Ammono-ferric  sulphate,  or 
iron  alum  is  used  as  a  mordant  and  differ- 
entiator in  the  iron  hematoxylin  tech- 
nique and  for  other  purposes.  The 
crystals  are  of  a  pale  violet  color.  Their 
surfaces  oxidize  readily  and  become  use- 
less. The  surface  should  be  scraped  off. 
Only  the  violet  crystals  are  of  any  use. 

Alum-Carmine  (Grenacher).  Boil  1-5%  aq. 
ammonia  alum  with  0.5-1%  powdered 
carmine.  Cool  and  filter.  Does  not 
penetrate  very  well  and  hence  is  not 
suitable  for  staining  large  objects  in 
bulk.  But  it  is  useful  and  does  not 
overstain    (Lee,  p.   140). 

Alum-Cochineal,  see  Cochineal. 

Alum  Hematoxylin.  Many  hematoxylin  so- 
lutions contain  alum,  see  Delafield's, 
Ehrlich's,  Harris',  Mayer's. 

Aluminium  Chloride  Carmine  (Mayer). 
Dissolve  1  gm.  carminic  acid  and  3  gm. 
aluminium  chloride  in  200  cc.  aq.  dest. 
Add  an  antiseptic  as  formalin  or  0.1% 
salicylic  acid.  Employ  in  same  way  as 
carmalum.  Gives  blue  violet  color. 
Very  penetrating  but  not  so  specific  for 
chromatin  as  carmalum   (Lee,  p.   142). 


ALVEOLAR  EPITHELIUM  OF  LUNGS   8 


ALVEOLAR  PORES 


Alyeolar  Epithelium  of  Lungs 

1.  Gold  sodium  thiosulphate  (Bensley, 
R.  D.  and  S.  H.,  Anat.  Rec,  1935,  64, 
41-49).  Inject  a  mouse  intravenously 
through  the  tail  vein  with  100  mg.  of 
gold  sodium  thiosulphate  in  1  cc.  aq. 
dest.  The  mouse  dies  in  about  20  min. 
from  asphyxia.  Fix  pieces  of  lung  in 
10%  neutral  formalin,  dehydrate  with- 
out washing  in  water,  clear  and  imbed 
in  paraffin.  Deparaffinise  sections  and 
stain  in  1%  aq.  toluidin  blue  (tested 
for  polychromatism)  and  examine  in 
water.  The  epithelium  is  raised  by  in- 
crease in  volume  of  ground  substance 
which  is  stained  metachromatically 
pink  while  the  cells  and  their  nuclei  are 
blue.  The  color  of  the  ground  sub- 
stance can  be  changed  to  blue  by  alco- 
hol and  back  again  to  pink  by  water. 
To  mount  protect  against  reversing 
action  of  alcohol  by  treating  with  equal 
parts  freshly  prepared  5%  aq.  am- 
monium molybdate  (Kahlbaum  or 
Merck)  and  1%  aq.  potassium  ferro- 
cyanide.  Dehydrate,  clear  in  xylol 
and  mount  in  balsam.  (Revised  by 
R.  D.  and  S.  H.  Bensley,  Dept.  of 
Anatomy,  University  of  Chicago,  Chi- 
cago, 111.,  April  18,"  1946.) 

2.  Silver  nitrate  (Bensley,  R.  L).  and  S. 
H.,  Anat.  Rec,  1935,  64,  41-49).  Use 
guinea  pigs.  Silver  Citrate  sol.  (which 
see)  is  injected  into  lung  substance  by 
hypodermic  syringe,  the  roots  of  the 
lung  being  first  ligated,  until  the  lung 
is  moderately  distended.  Cut  out 
pieces,  fix  in  10%  formalin,  imbed  in 
paraffin  or  celloidin,  section,  develop 
with  dilute  photographic  developer  and 
counterstain  or  examine  unstained. 
The  margins  of  the  cells  are  blackened. 
For  the  most  delicate  results  a  slow 
acting,  fine  grain  developer  such  as  the 
following  should  be  used:  phenyl  hy- 
drazine hydrochloride,  1  gm.,  sodium 
sulphite  (anhydrous),  10  gm.;  aq.  dest., 
100  cc.  Caution:  Phenyl  hydrazine 
hydrochloride  is  extremely  toxic  to 
some  people  producing  skin  reactions. 
(Revised  by  R.  D.  and  S.  H.  Bensley, 
April  18,  1946.) 

Alveolar  Fluid.  Method  for  collecting, 
Terry,  R.  J.,  Anat.  Rec,  1926,  32,  223- 
304;  1936,  64,  75. 

Alveolar  Foam  Cells. — Written  by  C.  C. 
Macklin,  Dept.  of  Histological  Re- 
search, The  University  of  Western 
Ontario,  London,  Canada.  November 
28,  1951. — These  represent  nonphago- 
cytic  pneumonocytes  which  have  be- 
come free  in  the  alveoli  and  air  tract 
of  the  lungs.  They  may  be  obtained 
by  the  "gash-irrigation"  and  "wash- 
out" techniques  (which  see).  Macklin 
found    in    them    refractile    vacuolelike 


bodies  or  vacuoloids  (which  see)  readily 
demonstrable  in  fresh  mounts  by  bright- 
or  dark-field  illumination  (Proc  6th 
International  Congress  of  Experimental 
Cytology,  Stockholm,  1947,  published 
1949,  pp.  383-387).  In  ordinary  sec- 
tions these  bodies  appear  empty  or  with 
a  very  small  granule  within  them. 
When  foreign  particles  appear  in  them 
they  are  known  as  "Dust  Cells"  (which 
see).  Mitochondria  in  foam  cells  are 
mainly  in  the  frothy  perivacuoloidal 
cytoplasm  (Macklin,  C.  C,  Biol.  Bull., 
1949,96,  173-178). 

Alveolar  Phagocytes  of  Lungs,  see  Dust 
Cells. 

Alveolar  Pores  of  the  lung.^Revised  by 
C.  C.  Macklin,  Dept.  of  Histological 
Research,  The  University  of  Western 
Ontario,  London,  Canada.  November 
28,  1951— Formalin  (10%)  and  Zenker- 
formalin  are  among  the  fixatives  sug- 
gested. The  fixative  is  injected  into 
the  trachea  or  bronchus  at  a  gravity 
pressure  of  4-6  inches  until  the  lungs 
are  moderately  distended.  During  this 
operation  they  are  covered  with  physio- 
logical salt  solution.  The  lungs  are 
then  immersed  in  fixative  for  days  or 
even  weeks.  Slices  about  1  cm.  thick 
are  cut,  imbedded  in  soft  paraffin  and 
sections  are  made  at  100/i  or  more. 
Resorcin-fuchsin  and  other  stains  may 
be  used.  The  blood  in  the  capillaries 
is  a  useful  guide.  The  pores  can  be 
identified  by  their  rounded  edges 
(Macklin,  C.  C,  Arch.  Path.,  1936,  21, 
202-216). 

In  lungs  fixed  by  immersion  of  the 
flayed  intact  thorax  (IIT)  or  perfusion 
of  the  pulmonary  capillaries  of  the  in- 
tact thorax  (PIT)  the  pores,  in  thick 
sections,  appear  as  short  narrow  tunnels 
with  funnel-shaped  entrances  joining 
neighboring  alveolar  spaces.  Their  ex- 
tremely thin  walls  are  composed  of  parts 
of  the  contiguous  capillaries.  (See 
"Fixation  of  the  Uncollapsed  Lung" 
and  "Dust  Cells".)  Thus  seen  a  pore 
is  the  empty  sheath  or  vagina  from 
which  a  process  of  a  pneumonocyte 
(septal  cell,  alveolar  wall  cell,  etc.)  has 
been  withdrawn.  When  such  a  process 
cannot  be  withdrawn  because  it  is  bul- 
bous the  cell  is  seen  as  a  dumbbell - 
shaped  structure  with  the  thin  con- 
necting part  in  the  pore.  In  its  func- 
tional state  this  vagina  is  occupied  by 
a  part  of  a  pneumonocyte,  and  the  latter 
is  thus  placed  where  nutrition  from  the 
capillaries  is  constantly  available. 
Any  good  fixative  suffices  to  show  pores 
by  these  methods.  When  pieces  of 
lung  tissue  are  fixed  by  immersion  the 
ensuing  contraction  usually  closes  the 
pores    to    that    they    cannot    be    seen 


ALVEOLAR  SIZE 


9 


AMLXE  OXIDASE 


(Macklin,  C.  C,  Jour,  of  Thor.  Surg., 
1938,  7,  53&-551,  Macklin,  C.  C,  The 
Lancet,  Feb.  24,  1951,  432-435).  See 
also  Loosli,  C.  G.,  Arch.  Path.,  1937,  24, 
743,  and  Loosli,  C.  G.,  Amer.  J.  of  Anat., 
1938,  62,  375.  When  the  entire  fresh 
collapsed  lungs  are  fi.xed  b}^  filling  them 
with  preservative  via  the  trachea,  or 
when  parts  of  the  lung  are  so  filled  via 
a  bronchus,  the  alveolar  walls  are  com- 
pressed and  flattened,  and  here  the 
pores  appear  as  holes  punched  in  thick 
paper;  and  if  the  distention  has  been 
great  the  morphology  of  the  pore  and 
its  relation  to  the  environing  capillary 
is  not  obvious  (Macklin,  C.  C.,  Trans. 
Roy.  Soc.  Can.,  Sect.  V,  1934,  28:  p.  28; 
J.  Anat.  [pt.  2]  1935,  69,  188;  J.  Assoc. 
Am.  Med.  Coll.,  1935,  10,  265;  Arch. 
Path.,  1936,  21,  202).  Pores  are  well 
seen  "en  face"  in  frozen  sections  which 
have  been  dried  on  the  slide. 

The  foregoing  description  refers  to 
true  pores.  A  second  type  of  communi- 
cation between  adjoining  alveoli  occurs 
in  "medical  emphysema"  or  alveolar 
ectasia  and  is  due  to  the  wasting  of 
the  alveolar  sidewalls  or  bases.  These 
openings  may  be  very  large  and  numer- 
ous, and  the  walls  are  then  said  to  be 
riddled  with  them.  Pathologists  are 
familiar  with  this  type  of  "false  pore". 
The  true  type  of  pore  is  usually  un- 
discernible  in  the  collapsed  lung  tissue 
examined  by  pathologists. 

Alveolar  Size,  in  the  lungs.  For  techniques 
used  in  determining  alveolar  size  in  ten 
mammalian  types  see  the  papers  of 
W.  S.  Hartroft  and  C.  C.  Macklin  in 
the  bibliography  of  the  26th  Lewis  Linn 
McArthur  Lecture  of  the  Frank  Billings 
Foundation;  by  C.  C.  Macklin  in  Proc. 
of  the  Institute  of  Medicine  of  Chicago, 
1950,  18,  78-95. 

Alveolus  of  the  Lungs.  The  morphologj', 
in  sections,  can  best  be  demonstrated 
in  small  animals  by  immersion  of  the 
fresh  skinned  intact  thorax  in  a  fixative, 
such  as  Bouin's  fluid.  See  Fixation  of 
the  uncollapsed  lung. 

Alzheimer's  Modification  of  Mann's  eosin- 
methyl  blue  for  neuroglia  and  degenerate 
nerve  fibers  as  given  by  Mallory  (p.  245) 
is  abbreviated.  Fix  thin  slices,  14  days, 
in  Weigert's  Neuroglia  Mordant  -f  10% 
of  formalin.  Wash  8-12  hrs.  in  running 
water.  Mordant  lO^i  frozen  sections 
2-12  hrs.  in  sat.  aq.  phosphomolybdic 
acid.  Wash  2  changes  aq.  dest.  Stain 
in  Mann's  Eosin  Methyl  Blue  1-5  hrs. 
Wash  quickly  in  aq.  dest.  until  color 
"clouds"  are  no  longer  given  off. 
Treat  with  95%  alcohol  until  gray  matter 
becomes  light  blue  and  white  matter 
pink  or  bright  red.  Dehydrate  quickly 
in  absolute  alcohol,  clear  in  xylol  and 


mount  in  balsam.  Normal  axis  cylin- 
ders, purple  or  deep  blue ;  degenerating 
ones,  red;  neuroglia  fibers,  dark  blue; 
and  neuroglia  cytoplasm,  pale  blue. 
Mallory  states  that  change  from  blue  to 
red  staining  of  axis  cylinders  occurs  as 
soon  as  48  hrs.  after  experimental  lesion. 

Amanil  Garnet  H.,  see  Erie  Garnet  B. 

Amaranth  (CI,  184) — azo  rubin,  Bordeaux, 
Bordeaux  SF,  fast  red,  naphthol  red  S, 
C  or  O,  Victoria  rubin  O,  wool  red — An 
acid  mono-azo  dye  used  long  ago  by 
Griesbach,  H.,  Zeit.  wis.  mikr.,  1886, 
3,  358-385  to  color  axis  cylinders. 

Amebae.  The  techniques  that  have  been 
and  can  be  employed  for  the  organisms 
are  almost  endless.  In  brief,  these  are 
their  examination  in  the  living  condi- 
tion unstained  using  ordinary,  phase 
and  dark  field  microscopes,  imple- 
mented or  not  by  supravital  stains.  As 
a  beginning,  the  method  of  Meyers  is 
suggested:  washed  amebae  in  a  clean 
vessel  are  allowed  to  settle  to  the 
bottom.  They  are  then  fixed  in  warm 
Bouin's  Fluid,  concentrated  in  a  centri- 
fuge tube  and  stained  with  Grenacher's 
borax  carmine  and  Indulin  (Meyers, 
E.  H.,  Trans.  Am.  Micr.  Soc,  1933,  52, 
58).  For  showing  cytoplasmic  com- 
ponents of  Amoeba  Proteus,  see  Mast, 
S.  O.  and  Doyle,  W.  L.,  Arch.  f.  Pro- 
tistenk.,  1935,  86,  155.  To  determine 
the  density  of  amebae  a  starch  Density 
Gradient  is  recommended  by  Lovtrup, 
S.  C.  rend.  Lab.  Carlsberg,  Ser.  Chim., 
1950,  27,  137-144.  For  determination 
of  permiability  of  nucleus  see  Monn6, 
L.,  Proc.  Soc.  Exp.  Biol.  &  Med.,  1935, 
32,  1197.  The  technique  for  enucelation 
of  fresh  water  amebae  advised  by 
Holter,  H.  and  Kopac,  M.  J.,  J.  Cell, 
and  Comp.  Physiol.,  1937,  10,  423  is 
recommended.     See  Entameba. 

Amethyst  Violet  (CI,  847)— heliotrope  B, 
iris  violet — It  is  a  basic  azin  dye  of 
little  importance  to  histologists. 

Amine  Oxidase.  Oster,  K.  A.  and  Schloss- 
man,  N.  C,  J.  Cell.  Comp.  Physiol., 
1942,  20,  373-378.  As  explained  by 
Glick,  p.  93,  the  method  is  based  on 
detection  of  aldehyde  formed  by  amine 
oxidation.  The  Fuelgen  sulphurous 
acid  agent  (see  Thymonucleic  Acid)  is 
employed  for  microscopic  visualization 
of  aldehyde.  Binding  of  naturally 
occurring  aldehydes  and  "plasmal"  is 
accomplished  with  bisulphite  so  that 
they  do  not  invalidate  the  method. 
Because  the  color  produced  diffuses, 
localization    is   subject    to    criticism. 

Place  frozen  sections  of  fresh  tissue 
in  2%  aq.  sodium  bisulphite  at  37°C.,  24 
hrs.  wash  thoroughly.  Control  sec- 
tions immersed  in  Feulgen  agent  should 
remain     colorless     showing     that    free 


AMIDONOPHTHOL  GR 


10 


AMYL  ACETATE 


aldehyde  has  been  bound.  Incubate 
these  sections  in  0.5%  tyramine  hydro- 
chloride in  M/15  phosphate  buffer  pH 
7.2  at  37°C.  24  hrs.  Incubate  other 
control  sections  in  same  solution  minus 
tyramine.  Immerse  both  test  and  con- 
trol sections  in  Feulgen  agent.  Ex- 
amine when  quickly  produced  blue 
color  seems  to  be  at  maximum.  Foci 
of  enzymatic  activity,  blue  in  con- 
venient contrast  with  reddish  purple  of 
"plasmal"  (see  Aldehydes). 

Amidonaphthol   GR,   see   Azophloxine   GA. 

Amino  Acids,  see  Alloxan  Reaction,  also 
Schmidt,  C.  L.  A.,  The  Chemistry  of 
the  Amino  Acids  and  Proteins.  Spring- 
field, Charles  C  Thomas,  1938,  1031  pp. 
See  paper  chromatography  added  by 
Roberts. 

Aminoacridines,  some  are  strong  antiseptics, 
do  not  stain  skin  (Albert,  A.  and 
Ritchie,  B.,  J.  Soc.  Chem.  Ind.,  1941, 
60,  120). 

Amitosis  is  direct  nuclear  division  by  con- 
striction without  formation  of  a  chro- 
matin thread.  No  special  technique 
required.  Study  of  embryonic  mem- 
branes and  of  bladder  of  mouse  (Dogiel, 
A.  S.,  Arch.  f.  Mikr.  Anat.,  1890,  35, 
389-406)  is  suggested. 

Ammonia  Carmine  (Ranvier).  A  suspen- 
sion of  carmine  in  water,  with  slight 
excess  ammonia,  is  allowed  to  evaporate 
in  air.  If  it  putrefies  so  much  the 
better.  Dissolve  the  dry  deposit  in 
aq.  dest.  and  filter  (Lee,  p.  145). 

Ammoniacal  Silver  for  branchioles — Written 
by  C.  C.  Macklin,  Dept.  of  Histological 
Research,  The  University  of  Western 
Ontario,  London,  Canada.  November 
28,  1951 — This  is  useful  to  demonstrate 
the  two  types  of  epithelium  of  the  finest 
bronchioles,  as  in  the  albino  mouse. 
There  are  two  stock  solutions.  Solu- 
tion 1:  Five  grams  of  silver  nitrate  are 
dissolved  in  300  ccms.  of  distilled  water 
and  dilute  aqua  ammoniae  is  added 
until  the  precipitate  is  nearly  redis- 
solved;  filter;  add  water  to  make  500 
ccms.  Solution  2:  One  gram  of  silver 
nitrate  is  dissolved  in  a  small  quantity 
of  water  and  poured  into  half  a  liter  of 
boiling  water.  Rochelle  salt  (0.83  gm.) 
is  dissolved  in  a  small  quantity  of  water 
and  added  to  the  boiling  solution,  which 
is  then  boiled  for  half  an  hour  till  a  gray 
precipitate  gathers  at  the  bottom  of  the 
flask.  Filter  hot;  add  water  to  make 
500  ccms.  In  using,  equal  parts  of  the 
two  solutions  are  mixed.  The  separate 
solutions,  kept  in  the  dark,  remain  good 
for  a  month  or  two. 

The  etherized  animals  are  exsangui- 
nated, the  lungs  collapsed  by  carefully 
nicking  the  diaphragm  and  filled  via 
the  trachea  with  the  ammoniacal  silver 


solution.  At  the  end  of  two  minutes 
the  lungs  are  emptied,  refilled  with  10% 
formalin,  and  the  trachea  is  tied.  The 
material  is  placed  in  10%  formalin  for 
a  day  or  more.  Frozen,  paraffin,  or 
celloidin  sections  are  cut  and  exposed 
to  direct  sunlight  or  weak  photographic 
developer.  Flattened  frozen  sections 
are  useful  in  affording  surface  views  of 
lengths  of  bronchiolar  epithelium.  The 
following  description  is  from  C.  C. 
Macklin  (Canad.  Jour,  of  Research,  D, 
1949,  27,  50-58— Bibliography) :  "Two 
definite  types  of  cells,  dark  and  light, 
are  revealed  in  the  terminal  bronchiolar 
epithelium  by  this  supravital  silveriza- 
tion.  The  dark  cells  are  ciliated  and 
scattered  among  the  light  unciliated 
cells  as  singles,  pairs,  triads  or  larger 
groups  to  form  a  striking  and  charac- 
teristic surface  pattern.  The  cuticles 
of  the  dark  cells,  far  outreached  by  the 
neighboring  light  cells,  appear  en  face 
as  dense  crowds  of  small  uniform  golden 
brown  granules.  Viewed  laterally  these 
show  as  pairs  in  vertical  alignment,  and 
make  two  layers  with  a  thin  lighter  band 
between.  The  sides  and  bases  are  in- 
dicated by  deposits  of  black  grains. 
Near  the  sharply  marked  margin  of  the 
definitive  epithelium  the  dark  cells  are 
shorter,  more  dispersed,  and  formed 
like  truncated  pyramids  with  narrow 
densely  impregnated  apices  on  some  of 
which  are  single  delicate  points  instead 
of  discrete  cilia.  The  light  cells  have 
bulging  villuslike  free  ends  and  make 
flutings  on  the  contiguous  dark  cells. 
Small  silverized  particles  sparsely 
stipple  their  air  surfaces,  and  rounded 
plaques  crown  their  summits.  A  simi- 
lar dicellular  picture  is  found  in  the 
terminal  bronchiolar  epithelium  of  the 
golden  hamster." 

Ammonium  Molybdate,  as  mordant  for 
Mann's  stain  and  Weigert-Pal  (Perdrau, 
J.  R.,  J.  Path.  &  Bact.,  1939,  48,  609- 
610.  Recommended  by  Bethe  as  a 
fixative  for  supravital  methylene  blue, 
see  Lillie,  p.  245. 

Amoeba,  see  Entameba. 

Amphinucleolus  (G.  amphi  on  both  sides). 
A  nucleolus  which  is  double  consisting  of 
both  acidophilic  and  basophilic  parts, 
the  former  is  usually  a  central  core  ana 
the  latter  plastered  on  its  surface. 

Amphioxus,  as  an  ancestor  of  vertebrates 
of  interest  to  all,  see  the  technique  of 
Conklin,  E.  G.,  J.  Morph.,  1932,  54,  69. 

Amphophilic,  see  Staining. 

Amy!  Acetate,  as  solvent  for  imbedding 
tissues  (Barron,  D.  H.,  Anat.  Rec, 
1934,  59,  No.  1  and  Suppl.,  1-3);  as  a 
clearing  agent  for  embryological  material 
(Drury,  H.  F.,  Stain  Techn.,  1941, 
16,  21-22). 


AMYL  ALCOHOL 


11 


AMYLOID 


Amyl  Alcohol.  Merck  lists  3,  commercial, 
normal  and  tertiary.  It  mixes  with 
95%  alcohol  and  with  xylol.  HoUande 
(A.  C,  C.  rend  Soc.  de  Biol.,  1918,  81, 
223-225)  was  the  first  to  recommend 
amyl  alcohol  as  a  substitute  for  absolute 
alcohol  in  the  dehydration  of  specimens 
sta,ined  by  the  Romanovsky  and  Giemsa 
techniques. 

Amyl  Nitrite.  McClung  (p.  620)  says  that 
this  may  serve  as  a  dilator  of  peripheral 
capillaries  when  a  complete  injection 
of  small  blood  vessels  is  required.  Add 
it  to  the  ether  at  time  of  anesthetization. 

Amylase,  micromethod  for  (Pickford,  G.  E. 
and  Dorris,  F.,  Science,  1934,  80,  317- 
319).  This  was  later  used  with  marked 
success  by  Dorris  (F.,  J.  Exp.  Zool., 
1935,  70,  491-527)  in  a  study  of  relation 
between  enzyme  production  and  histo- 
logical development  of  gut  of  ambly- 
stoma.  An  extract  is  made,  adjusted 
to  proper  pH,  applied  to  slides  coated 
with  a  starch-agar  solution  and  incu- 
bated. The  slides  are  then  washed,  the 
coating  fixed  in  formalin  and  colored 
with  dilute  iodine  solution.  Sites  of 
amylase  activity  are  clear  or  pink  stain- 
ing spots.  For  necessary  details,  see 
author's  description,  van  Genderen 
andEngel  (H.andC.,  Enzymologia,  1938, 
5,  71-80)  localized  this  enzyme  by 
analysis  of  horizontal  sections  through 
the  intestinal  wall.  It  was  found  that  it 
is  present  in  rabbits  in  maximum  amounts 
in  Brunner's  glands.  Holtfir  and  Dogle 
(C.  R.  Lab.  Carlsberg,  S6r.  Chim.,  1938, 
22,  219-225)  observed  that  in  amebae  it 
is  concentrated  in  association  with  the 
mitochondria  which  they  assume  to  be 
carriers  of  amylase.  See  Barnes,  J.  M., 
Brit.  J.  Exp.  Path.,  1940,  21,  264-275 
for  identification  of  amylase  in  lympho- 
cytes and  polymorphonuclear  leuco- 
cytes . 

Salivary  amylase  digests  glycogen  in 
sections,  but  does  not  alter  mucus, 
cartilage  matrix  and  other  mucins  which 
give  similar  histochemical  reactions. 
Lillie,  R.  D.,  Stain  Tech.,  1947,  22,  67- 
70,  recommends  a  commercial  prepara- 
tion of  diastase  for  this  purpose,  but 
Dempsey  and  Wislocki  prefer  saliva, 
since  the  commercial  preparations  ap- 
parently contain  traces  of  mucinase 
which  attacks  mucus,  cartilage  and 
mast  cell  granules. 

Amyloid  (G.  arnylon,  starch  and  eidos,  re- 
semblance), a  substance  which  accumu- 
lates in  pathological  conditions  in  the 
tissue  fluids  between  cells  particularly 
in  chronic  infections.  Methods  for  its 
detection  are  fully  described  by  Mallory 
and  Parker  (McClung,  pp.  417-419). 
From  nimaerous  tests  the  following  are 
selected : 


1.  Iodine  and  sulphuric  acid:  Stain 
section  lightly  with  Lugol's  iodine. 
Place  in  1-5%  aq.  or  cone,  sulphuric  or 
hydrochloric  acid.  Color  of  amyloid 
changes  quickly  from  red  through 
violet  to  blue  or  it  may  become  deep 
brown . 

2.  Methyl-violet:  Treat  frozen  sections 
of  fresh,  formalin  or  alcohol  fixed  tissue 
with  1%  aq.  methyl  violet,  3-5  min. 
Wash  in  1%  aq.  acetic  acid,  and  remove 
acid  by  washing  carefully  in  water. 
Examine  in  glycerin  or  water.  Amyloid 
is  violet  and  tissue  blue.  Colors  will  be 
retained  longer  if  sections  are  mounted 
in  Levulose  Syrup. 

3.  Iodine  green:  Fresh  or  hardened 
sections  are  stained  24  hrs.  in  0.3% 
aq.  iodine  green.  Wash  in  water  and 
examine  in  water  or  glycerin.  Amyloid 
is  stained  violet  red  and  tissue,  green. 

4.  Mayer's  stain:  Transfer  paraffin 
sections  immediately  after  cutting  to 
0.5%  aq.  methyl  violet  or  gentian  violet 
at  40°C.  for  5-10  min.  Rinse  in  water 
and  differentiate  in  1%  aq.  acetic  acid 
for  10-15  min.  Wash  thoroughly  in  wa- 
ter. Change  to  ^  sat.  aq.  alum  and  wash 
it  off  in  water.  Place  section  on  slide  and 
let  water  evaporate.  Remove  paraffin, 
clear  in  xylol  and  mount  in  balsam. 
Crystal  violet  and  iodine  green  can  be 
employed  in  the  same  way. 

A  Congo  red  test  has  been  described 
(Taran,  A.,  J.  Lab.  &  Clin.  Med.,  1936- 
37,  22,  975-977)  and  a  polysaccharide 
has  been  isolated  from  amyloid  bearing 
tissues  which  closely  resembles  chon- 
droitin-sulphuric  acid  obtained  from 
infantile  cartilage  (Hass,  G.,  Arch. 
Path.,  1942,  34,  92-105). 

As  pointed  out  by  Highman,  B.,  Arch. 
Path.,  1946,  41,  559-562  the  staining 
methods  for  amyloid  are  in  general 
satisfactory  when  employed  by  skilled 
workers.  However,  when  stained  sec- 
tions are  mounted  in  glycerin  Apathy's 
syrup,  or  some  such  medium,  they  tend 
to  fade  quickly,  or  the  stain  diffuses  out 
into  the  surrounding  tissue,  or  mount- 
ing medium,  and  the  nuclei  are  seldom 
sharply  colored.  Highman  therefore 
recommends  staining  of  deparaffinized 
sections  of  formalin  fixed  tissues  in  iron 
hematoxylin  5  min.,  washing  in  water, 
staining  in  0.5%  crystal  violet  or  methyl 
violet  in  2.5%  aq.  acetic  acid,  washing 
again  in  water  and  mounting  in  Lillie 's 
Apdthy's  syrup  modified  by  addition 
of  50  gm.  potassium  acetate  or  10  gm. 
sodium  chloride  to  100  cc.  of  syrup. 
He  also  gives  a  technique  for  mounting 
in  clarite. 

See  Mallory-Heidenhain  rapid  one 
step  stain. 


AMNIOTIC  FLUID 


12 


AXILIN  BLUE 


Amniotic  Fluid,  technique  for  study  of  ab- 
sorption from,  Wislocki,  G.  B.,  BulL 
Johns  Hopkins  Hospital,  1921    32,  93. 

Anaplasma  is  a  small  spherical  body  found 
within  red  blood  cells  in  anaplasmosis 
diseases.  There  are  two  types  A  margi- 
nale  and  A  centrale  depending  upon 
whether  the  bodies  are  situated  near 
the  margin  or  in  the  centers  of  the  cells. 
The  bodies  are  supposed  to  be  parasites 
consisting  of  nuclear  material  with  little 
if  any  cytoplasm.  Anaplasmosis  is  im- 
portant economically  as  a  group  of  tick 
borne  diseases  of  domestic  animals. 
For  demonstration  stain  blood  smears 
by  the  methods  of  Giemsa  or  Wright. 

Anethol  is  anise  camphor  suggested  as  a 
medium  in  which  to  soak  tissues  before 
making  frozen  sections  (Stephanow, 
Zeit.  wiss.  Mikr.,  1900,  17,  181). 

Anethole  Clearing  Agent.— Written  by  Joan 
Haberman,  Parkland,  Washington. 
March  10,  1951 — Anethole  is  a  useful 
agent  in  clearing  segments  of  brain 
tissue  because  of  its  refractive  index. 
The  basic  principle  is  that  homogeneous 
tissue,  which  has  been  bleached  and 
dehydrated,  may  be  cleared  by  placing 
in  an  oil  of  similar  refractive  index  was 
discussed  by  Groat,  R.  A.,  Stain  Techn., 
1941,  16,  111-117.  Brain  tissue  consists 
primarily  of  protein  and  lipids  and  is 
therefore  not  homogeneous  as  are  other 
types  of  tissue  such  as  muscle.  Since 
protein  forms  the  framework  of  the 
brain,  it  necessitates  that  the  lipids  be 
removed  to  obtain  a  homogeneous 
tissue. 

The  lipid  composition  of  the  brain 
tissue  must  be  known  in  order  to  per- 
form an  adequate  extraction.  Accord- 
ing to  Koch,  as  quoted  by  A.  P. 
Mathews,  Physiological  Chemistry. 
Baltimore:  Williams  and  Wilkins  Co., 
1939,  the  lipid  composition  is  as  follows: 
phospholipids— 27.3%  of  dry  matter; 
cerebrosides— 13.6%  of  dry  matter; 
cholesterol— 10.9%  of  dry  matter.  The 
phospholipids,  according  to  G.  A.  Hill 
and  K.  Kelly,  Organic  Chemistry. 
Philadelphia:  Blakiston  Co.,  1943,  are 
composed  mainly  of :  lecithin— 48  p. p.m., 
soluble  in  ether  and  in  alcohol;  cepha- 
lin — 200  p. p.m.,  soluble  in  ether,  in- 
soluble in  alcohol;  sphingomyelin— 56 
p. p.m.,  insolublejin  ether,  soluble  in  hot 
alcohol.  These  are  all  insoluble  in 
acetone.  The  cerebrosides  are  soluble 
in  hot  alcohol,  insoluble  in  ether. 
Cholesterol  can  be  extracted  with  ether 
and  alcohol.  (The  data  on  solubilities 
are  taken  from  Harrow,  B.,  Textbook 
of  Biochemistry.  Philadelphia:  W.  B. 
Saunders  Co.,  1943). 

Brain  tissue  must  first  be  dehydrated 
by  using  the  alcohol  series  starting  at 


35%  or  higher  if  one  wishes  to  risk  more 
shrinkage  and  changing  every  few  hours 
depending  upon  the  size  of  the  tissue. 
The  lipids  are  then  removed  by  placing 
the  tissue  in  100%  alcohol  at  60  degrees 
centigrade  for  1-2  days  with  frequent 
changes  in  solution.  The  remaming 
lipids  are  extracted  with  ether  for  2 
days  or  longer  depending  upon  the  size 
of  the  tissue.  The  procedure  results 
in  a  tissue  that  consists  largely  of  pro- 
tein and  gives  a  refractive  index  of 
1.560  as  determined  on  the  Abb6  re- 
fractometer. 

By  placing  the  tissue  in  an  oil  of  very 
similar  refractive  index  it  will  soon  be- 
come transparent  when  brightly  illum- 
inated. Anethole  (C10H12O)  having  a  re- 
fractive index  of  1.5614  was  found  to 
be  the  oil  of  choice.  It  is  colorless, 
very  slightly  soluble  in  water,  soluble  in 
alcohol  and  ether,  and  is  liquid  at  ordi- 
nary temperatures.  It  can  be  obtained 
from  the  Eastman  Kodak  Company, 
Rochester,  New  York. 

After  the  tissue  has  been  placed  in 
anethole  the  container  should  be  left 
uncovered  so  that  the  displaced  ether 
may  escape. 

The  transparency  of  the  tissue  de- 
pends upon  the  completeness  of  the 
lipid  extraction.  Larger  pieces  of 
tissue  such  as  4  x  6  x  3  cm  must  be  kept 
in  100%  alcohol  for  a  week  and  in  ether 
for  1-2  weeks.  Frequent  changes  of 
solution  are  advisable.  This  block  of 
tissue  will  still  appear  only  translucent 
when  cleared  in  anethole.  However, 
sections  of  §  to  1  cm.  in  thickness  cut 
from  this  block  will  be  sufficiently  clear 
to  insure  visualization  of  injected 
vessels  etc.  under  the  binocular  with 
good  illumination.  It  is  probable  that 
a  large  piece  of  tissue  could  be  cleared 
satisfactorily  if  the  alcohol  and  ether 
extraction  were  carried  out  by  means  of 
an  intermittent  siphoning  distillation 
extractor. 

Angina,  see  Vincent's  Angina. 

Angstrom  Unit.  lA  =  0.1  m/*  =  O.OOOIm  = 
10-' mm. 

Anhydrase,  see  Carbonic  Anhydrase. 

Anilin  Blue  Alcohol  Soluble,  see  Spirit  Blue. 

Anilin  Blue,  WS  (CI,  707)— China  blue, 
cotton  blue,  marine  blue  V,  soluble  blue 
3M  or  2R,  water  blue  (Wasserblau)— A 
mixture  of  trisulphonates  of  di-phenyl 
rosanilin  and  tri-phenyl  pararosanilin. 
Conn  (p.  135)  explains  that  this  desig- 
nation (like  acid  fuchsin)  applies  not 
to  a  single  compound  but  to  a  group 
of  dyes.  Anilin  blue  is,  nevertheless, 
the  best  stain  for  Collagenic  Fibers  and 
is  employed  for  many  other  purposes. 


ANILIN  CRYSTAL  VIOLET  1. 

Anilin  Crystal  Violet  1.  Ehrlich's.  Shake 
up  5  cc.  anilin  oil  with  95  cc.  aq.  dest. 
Filter  and  to  84  cc.  of  filtrate  addl6  cc. 
Bat.  ale.  crystal  violet.  Leave  24  hrs. 
before  using.  After  about  10  days  stain- 
ing potency  decreases  (Mallory,  p.  89). 
2.  Stirling's.  Crystal  violet,  5  gm.  ; 
abs.  ale,  10  cc;  anilin  oil,  2  cc,  aq. 
dest.,  88  cc.  Keeps  well  (Mallory,  p. 
90). 

See  Anilin  Crystal  Violet  and  Gentian 
Violet. 

Anilin-Fuchsin  Methyl  Green  method  for 
mitochondria.  This  technique  is  based 
on  Altmann's  method.  It  was  used  by 
Bensley  to  stain  tissues  fixed  in  his 
Acetic-Osmic-Bichromate  fluid.  Cow- 
dry  recommends  instead  fixation  in  the 
better  penetrating  Regaud's  fluid. 

Fix  small  pieces  in  freshly  prepared 
Regaud's  fluid  (3%  aq.  potassium  bi- 
chromate 4  parts,  commercial  formalin 
1  part).  Ordinarily  it  is  not  necessary 
to  neutralize  the  formalin  before  hand 
by  saturating  it  with  magnesium  car- 
bonate. Keep  in  ice  box  and  change 
the  fluid  every  day  for  4  days.  Pour 
off  fixative  and  mordant  in  3%  aq.  po- 
tassium bichromate  8  days  changing 
every  second  day.  Wash  in  running 
water  over  night  or  in  several  changes  of 
water.  Dehydrate  iu  alcohol,  clear  in 
xylol,  imbed  in  paraffin  and  cut  sections 
about  4  fi  thick.  Pass  mounted  sec- 
tions through  xylol  and  alcohol  to 
water.  Dry  the  slide  with  a  cloth  ex- 
cept area  covered  by  sections.  Pour 
on  anilin  acid  fuchsin  and  heat  to 
steaming  over  a  spirit  lamp.  (To  make 
this  saturate  125  cc.  aq.  dest.  with 
anilin  oil  by  shaking  the  two  together. 
Filter  and  add  15  gms.  acid  fuchsin 
to  100  cc.  of  filtrate.  Allow  to  stand 
24  hrs.  before  using.  It  lasts  about  a 
month.)  Allow  to  cool  and  stain  about 
6  min.  Pour  stain  back  into  bottle. 
Remove  most  of  remainder,  except  from 
sections,  with  a  cloth  or  filter  paper. 
Rinse  in  aq.  dest.  about  1  min.  Allow 
1%  aq.  methyl  green,  added  with 
a  dropper,  to  flow  over  sections  and 
counter  stain  them.  This  usually  takes 
about  5  sec.  but  the  time  must  be 
determined  by  trial.  Wash  off  excess 
methyl  green  in  95%  alcohol,  dehydrate 
quickly  in  absolute,  clear  in  toluol 
(or  xylol)  and  mount  in  balsam.  The 
mitochondria  are  stained  crimson  and 
the  nuclei  green.  For  colored  illustra- 
tions see  Cowdry,  E.  V.,  Contrib.  to 
Embryol.,  Carnegie  Inst,  of  Washing- 
ton 1917,  No.  11,  27-43.  If  the  methyl 
green  does  not  stain  intensely  enough 
treat  the  sections,  before  coloration  with 
fuchsin,  with  l%aq.  potassium  perman- 
ganate 30  sec.  followed  by  5%  oxalic  acid 


13  ANTERIOR  CHAMBER 

TRANSPLANTATION 

30  sec.  and  wash  in  water.  More  methyl 
green  can  be  retained  by  blotting  the 
sections  after  staining  in  it  with  filter 
paper  and  by  then  passing  directly  to 
absolute  alcohol.  If  the  time  of  fixation 
and  mordanting  is  reduced  much  below 
that  specified  the  fuchsin  itself  may  not 
color  with  sufficient  intensity.  Such 
preparations  hold  their  colors  for  a  year 
or  more  unless  they  have  been  unduly 
exposed  to  sunlight,  or  the  balsam  is  acid. 

Anilin  Fuchsin  Picric  Acid,  see  Altmann's 
method  for  mitochondria. 

Anilin  Fuchsin  Toluidine  Blue  and  Aurantia, 
see  Champy-Kull  method  for  mito- 
chondria. 

Anilin  Gentian  Violet  usually  credited  to 
Ehrlich.  Rarely  is  its  composition 
given  exactly  the  same  by  any  two 
people.  The  "emended  formula"  (Soc. 
Am.  Bact.)  is  A:  2.5  gm.  crystal  violet 
(85  per  dye  content)  -f  95%  ethyl  alco- 
hol, 12  cc.  B:  anilin  oil  2  cc.  -f  aq. 
dest.  98  cc.  (shake,  leave  few  minutes, 
filter).  Mix  A  and  B.  (McClung, 
p.  137.) 

Anilin  Oil.  A  good  product  is  easily  obtain- 
able. It  is  much  used  in  the  making  of 
stains  (cf.  anilin  fuchsin)  and  to  clear 
tissues  from  95%  alcohol  and  even  sec- 
tions from  70%.  Lee  (p.  71)  says  that 
it  should  not  be  employed  after  fixation 
in  osmic  acid  and  that  unless  removed 
by  chloroform  or  xylol  it  will  give  the 
tissues  and  mounting  medium  a  brown 
coloration . 

Anilin  Red,  see  Basic  Fuchsin. 

Anilin-Safranin  (Babes).  Aq.  dest.,  98 
cc. ;  anilin  oil,  2  cc. ;  excess  of  safranin  O. 
heat  in  flask  in  hot  water  bath  at  70- 
80°C.  Cool,  filter  and  use  filtrate. 
This  is  an  excellent  nuclear  stain,  but 
it  must  be  freshly  prepared  every  month 
or  so.  It  colors  very  promptly.  Dif- 
ferentiation is  in  95%  ale  or  even  in 
Acid  Alcohol. 

Anilin  Yellow,  see  McClung,  1950,  p.  439. 

Anterior  Chamber  Transplantation — 
Written  by  Harry  S.  N.  Greene,  Labora- 
tory of  Pathology,  Yale  University 
School  of  Medicine,  New  Haven.  Sept. 
11,  1951 — The  anterior  chamber  of  the 
eye  possesses  many  advantages  over 
other  bodily  regions  as  a  transplanta- 
tion site.  The  technique  of  transfer  is 
simple,  a  high  percentage  of  takes  is 
obtained,  and  the  growing  tissue  can 
be  followed  by  direct  visual  observa- 
tion or  even  subjected  to  microscopic 
examination.  A  further  advantage  de- 
rives from  the  fact  that  the  chamber 
supports  the  growth  of  heterologous 
embryonic  or  cancer  tissue  whereas  in 
other  bodily  sites  such  tissues  generally 
fail  to  survive. 
The  technique  varies  somewhat  with 


ANTERIOR  CHAMBER 
TRANSPLANTATION 

the  size  of  the  animal  used.  Animals 
larger  than  mice  are  immobilized  on  an 
operating  board  with  tie  strings  in  such 
a  manner  that  the  desired  eye  presents 
to  the  operator.  The  cornea  is  anaes- 
thetized by  contact  with  a  5%  aqueous 
solution  of  cocaine  administered  with 
a  medicine  dropper.  An  incision  is 
made  at  approximately  the  midpoint  of 
the  upper  border  of  the  limbus  utilizing 
a  sharp  double-edged  corneal  knife. 
The  knife  is  directed  slightly  forward 
so  that  the  blade  enters  the  anterior 
chamber  without  damage  to  the  iris. 
The  pressure  necessary  to  pierce  the 
cornea  is  sufficient,  even  with  a  sharp 
knife,  to  rotate  the  eyeball  beneath  the 
lower  lid  and  conceal  the  operative 
field.  A  short,  quick  thrust  of  the 
knife,  however,  results  in  an  adequate 
opening  and  the  temporary  obscurity 
is  inconsequential.  Withdrawal  of  the 
blade  is  accompanied  by  the  escape  of 
a  small  amount  of  aqueous  humor,  but 
unless  the  iris  has  been  cut  by  a  mis- 
directed  knife,   there   is  no   bleeding. 

Transfer  of  the  tissue  is  effected  by 
means  of  a  trocar  commensurate  in 
diameter  with  the  corneal  incision. 
The  trocar  should  be  equipped  with  a 
tight-fitting  plunger  and  a  short  bev- 
elled mouth  with  all  edges  filed  to 
smoothness.  A  small  fragment  of  tissue 
is  placed  in  the  mouth  of  the  trocar 
and  forced  into  the  barrel,  a  step  greatly 
facilitated  by  withdrawing  the  plunger 
to  produce  suction.  The  tip  of  the 
trocar  is  inserted  through  the  incision 
a  short  distance  so  that  the  bore  is 
entirely  within  the  chamber  and  the 
fragment  is  expelled.  It  is  important 
that  all  manual  pressure  about  the  eye 
and  head  of  the  animal  be  released 
before  withdrawing  the  trocar  to  pre- 
vent escape  of  the  fragment.  Finally, 
the  fragment  is  forced  into  the  inferior 
angle  of  the  iris  by  applying  light  pres- 
sure along  the  corneal  surface  with  a 
blunt  instrument.  The  incision  is  not 
closed. 

The  knife  should  be  sharp  and  of  such 
width  that  a  stab  wound  will  admit  the 
trocar.  The  use  of  a  narrow  knife 
necessitates  side  cutting  to  obtain  a 
sufl&cient  opening  and  this  may  be 
attended  by  iris  damage  with  conse- 
quent bleeding.  Pointed  surgical 
blades  or  single-edged  corneal  knives 
are  undesirable,  for  the  triangular  cut 
produced  heals  slowly  and  may  result 
in  herniation  of  the  iris. 

The  fragments  of  tissue  to  be  trans- 
planted should  be  cut  with  sharp  instru- 
ments and  should  not  exceed  1  mm.  in 
diameter.  The  careful  selection  of 
fragments  is  of  the  utmost  importance 


14  ANTERIOR  CHAMBER 

TRANSPLANTATION 

in  the  heterologous  transplantation  of 
human  cancer.  In  addition  to  essential 
parenchyma,  all  human  cancers  contain 
stroma  and  many  are  infiltrated  with 
desmoplastic  connective  tissue.  Such 
tissues  are  adult  in  nature  and  will  not 
grow  on  heterologous  transfer.  In  fact, 
their  presence  in  quantity  will  give  rise 
to  a  foreigh  body  reaction  in  the  alien 
host  and  result  in  death  of  the  trans- 
plant. It  is  essential,  therefore,  that 
selection  be  based  on  the  content  of 
tumor  parenchyma  and  some  knowledge 
of  gross  pathology  is  necessary  for  such 
a  differentiation.  Frozen  sections  ob- 
tained from  different  areas  of  the  tumor 
mass  are  of  considerable  aid  in  some 
cases.  It  is  obvious  that  necrotic 
tumor  or  normal  tissue  adjacent  to  the 
tumor  are  not  suitable  materials  for 
transfer. 

The  placing  of  the  fragment  in  a 
wedged  position  in  the  inferior  angle  of 
the  iris  is  important  for  early  vasculari- 
zation. Occasionally,  fragments  so 
placed  work  loose  shortly  after  opera- 
tion and  an  examination  with  the  re- 
quired readjustment  is  desirable  before 
returning  the  animals  to  the  colony. 
Fixation  occurs  within  a  few  hours  and 
further  check  is  unnecessary. 

A  slight  modification  of  the  technique 
is  desirable  when  applied  to  mice  or  to 
newborn  animals  of  larger  species. 
General  anesthesia  is  preferable  and 
may  be  effected  with  ether  or  nembutal. 
Both  hands  are  employed  in  the  opera- 
tion and  preliminary  loading  of  the 
trocar  is  necessary.  The  trocar  is  made 
by  shortening  the  bevel  at  the  tip  of 
a  20-gauge  hypodermic  needle.  A  suit- 
able, tight-fitting  plunger  can  be  manu- 
factured or  obtained  simply  be  select- 
ing a  wire  stylet  of  proper  size  from  the 
stock  supplied  with  the  needles  and  ap- 
plying a  knob  of  plastic  material  to  one 
end. 

The  mouse  is  held  loosely  in  the  left 
hand  and  the  lids  of  the  right  eye  forced 
apart  with  the  thumb  and  index  finger. 
Slight  pressure  with  the  fingers  causes 
the  eye  to  protrude  sufficiently  to  allow 
adequate  exposure  for  the  operation. 
An  incision  is  made  at  the  upper  border 
of  the  limbus  using  a  double-edged  knife 
of  the  same  caliber  employed  in  larger 
animals. 

The  trocar,  held  between  the  thumb 
and  middle  finger  of  the  right  hand,  is 
inserted  into  the  chamber  through  the 
incision  and  the  fragment  expressed 
by  pushing  the  plunger  with  the  index 
finger.  In  order  to  prevent  extrusion 
and  escape  of  the  fragment  through  the 
incision,  all  pressure  exerted  on  the 
animal  by  the  left  hand  should  be  re- 


ANTHROCOSIS 


15 


ANTIGENS,  HISTOCHEMICAL 


leased  before  withdrawing  the  trocar. 
Trouble  may  be  encountered  in  pre- 
venting the  escape  of  soft,  slippery 
tissues  such  as  embryonic  brain.  This 
difficulty  may  be  circumvented  by  in- 
cising the  iris  as  well  as  the  cornea  at 
the  limbus  and  directing  the  trocar 
behind  the  superior  half  of  the  iris, 
through  the  pupil  and  into  the  inferior 
portion  of  the  anterior  chamber.  With 
withdrawal  of  the  trocar,  the  fragment 
is  almost  invariably  caught  at  the 
pupillary  border  and  retained  in  the 
chamber. 

The  tissue  concerned  may  be  trans- 
planted immediately  or  stored  at  ice- 
box temperature  for  several  days  before 
use.  It  is  essential  that  the  material 
be  free  of  infection  and  that  surgical 
sterility  be  maintained  throughout  all 
manipulations. 

Aside  from  careful  technique,  the 
success  or  failure  of  anterior  chamber 
transfer  depends  on  the  nature  of  the 
tissue  used  and  the  species  of  the  re- 
cipient host.  Adult,  embryonic  and 
cancer  tissues  grow  on  homologous 
transfer,  while  benign  tumors  and  pre- 
cancerous tissues  fail  to  survive,  and 
heterologous  transfer  is  successful  only 
in  the  case  of  embryonic  tissue  and 
cancer.  In  the  selection  of  recipient 
species  for  the  heterologous  transplan- 
tation of  cancer,  it  should  be  noted  that 
transfer  between  species  with  the  same 
type  of  Vitamin  C  metabolism  (man 
and  guinea  pig)  is  comparatively  easy, 
while  transfer  between  species  with 
different  types  (man  and  mouse)  is 
difficult. 

Takes  are  first  recognized  by  increase 
in  size  and  vascularization  of  the  trans- 
planted fragment.  The  time  required 
varies  within  wide  limits;  1  day  in  the 
case  of  homologous  embyronic  tissues 
and  3  months  in  the  case  of  a  hetero- 
logous glioblastoma  multiforme.  In 
the  former  instance,  the  growing  trans- 
plant may  fill  the  chamber  in  a  week 
while  in  the  latter,  6  to  8  months  may 
be  required.  Serial  transfer  is  readily 
effected  with  fragments  of  the  first 
generation  growth.  For  details  see 
Greene,  H.  S.  N.,  Cancer  Res.,  1943,  3, 
809-822;  1947,  7,  491-501,  and  Yale  J. 
Biol.   &  Med.,  1950,  6,  611-620. 

Anthracosis.  The  deposition  of  carbon, 
usually  in  lungs  and  mediastinal  lymph 
nodes,  distinguished  by  its  resistance 
to  solvents  and  bleaching  agents  and 
by  its  blackness.     See  Carbon. 

Anthrapurpurin,  see  Alizarin  SX. 

Anthraquinone  Dyes.  Derivatives  of  an- 
thracene through  anthraquinone.  Acid 
alizarin  blue  GR  and  BB,  alizarin, 
alizarin  red  S,  purpurin. 


Antibiotics,  influence  on  dehydrogenase. 
Systems  of  bacteria,  see  Triphenyl- 
tetrazolium  Chloride. 

Anticoagulant  Solutions  have  been  very  care- 
fully studied  by  Leichsenring,  J.  M., 
et  al.,  J.  Lab.  &  Clin.  Med.,  1939-40,  25, 
35-44.  They  found  that  1.6%  potassium 
oxalate  prepared  from  dried  salt  is  most 
nearly  isotonic  for  human  blood.  Win- 
trobe,  M.  M.,  Clinical  Hematology, 
Philadelphia,  Lea  &  Febiger,  1942,  792 
pp.  advises  0.06  gms.  of  ammonium 
oxalate  and  0.04  gms.  of  potassium  oxa- 
late for  5  cc.  of  blood.  He  dissolves  1.2 
gm.  ammonium  oxalate  and  0.8  gm. 
potassium  oxalate  in  100  cc.  aq.  dest. 
and  adds  1  cc.  formalin  to  prevent  de- 
terioration. Then  he  measures  out  with 
a  burette  0.5  cc.  into  each  of  the  con- 
tainers and  lets  it  dry  before  taking  into 
each  5  cc.  of  fresh  blood.  Heparin  is 
also  advised  but  it  is  much  more  expen- 
sive. 0.075  gm.  will  prevent  coagula- 
tion of  5  cc.  of  blood.    See  citrate. 

Antigens,  Histochemical  Identification  Of — 
Written  by  A.  H.  Coons,  Dept.  of  Bac- 
teriology and  Immunology,  Harvard 
Medical  School,  Boston,  August  31, 
1951 — The  localization  of  antigenic 
substances  in  tissue  cells  can  be  carried 
out  by  the  use  of  specific  antibody  con- 
jugated with  fluorescein.  The  method 
furnishes  a  means  for  localization  and 
identification  with  all  the  specificity  of 
immune  reactions.  Suitably  prepared 
tissue  sections  containing  an  antigenic 
substance  which  it  is  desired  to  study 
are  flooded  with  a  solution  containing 
antibodies  against  the  substance  pre- 
viously conjugated  with  fluorescein 
isocyanate.  The  antibody  molecules 
precipitate  over  those  sites  in  the  tissue 
section  containing  the  specific  antigen, 
the  excess  of  fluorescent  proteins  is 
washed  away,  and  the  tissue  section 
mounted  in  glycerol.  When  examined 
under  the  fluorescence  microscope,  the 
brilliant  yellow-green  fluorescence  of 
fluorescein  is  visible  over  those  areas 
where  the  immune  reaction  has  taken 
place. 

The  preparation  and  assay  of  immune 
sera  is  described  in  standard  works  on 
immunology.  Two  such  recent  ones 
are  Kabat,  E.  A.,  and  Mayer,  M. 
M.,  Experimental  Immunochemistry, 
Thomas,  Springfield,  Illinois,  1948; 
Boyd,  W.  C.,  Fundamentals  of  Im- 
munology, 2nd  Edition,  Interscience, 
New  York,  1947.  Whenever  possible, 
it  is  best  to  start  with  a  purified  antigen, 
since  antibodies  against  any  impurities 
present  in  the  material  injected  may  be 
represented  by  antibody  in  the  result- 
ing serum.  These  antibodies  against 
biological  impurities  may  react  in  tissue 


ANTIGENS,  HISTOCHEMICAL 


16 


ANTIGENS,  HISTOCHEMICAL 


sections  with  antigenic  components 
other  than  that  under  investigation. 
However,  it  is  possible  to  remove  such 
interfering  antibodies  by  absorbing  the 
anti-serum  with  material  containing  the 
*  antigenic  impurities,  but  not  contain- 
ing the  antigen  it  is  desired  to  study. 
Pains  should  be  taken  to  secure  as  high 
a  titer  of  antibody  as  is  feasible.  Pro- 
longed courses  of  immunization  and  the 
use  of  adjuvants  may  be  necessary. 

Concentration  of  antibodies.  It  is 
often  advantageous  to  concentrate  the 
globulin  fraction  of  such  immune  sera, 
either  by  precipitation  of  the  globulins 
with  half-saturated  ammonium  sul- 
phate, or  the  use  of  low  temperature, 
buffers,  and  alcohol  after  the  method 
of  Nichol  and  Deutsch  (J.  Am.  Chem. 
Soc,  1948,  70,  80).  If  ammonium 
sulphate  is  used,  the  ammonium  ion 
must  be  carefully  dialyzed  from  the 
final  globulin  solution  as  otherwise  it 
will  interfere  seriously  with  subsequent 
procedures. 

Conjugation  with  fluorescein  isocyanate. 
The  derivatives  of  fluorescein  leading 
to  the  isocyanate  are  not  as  yet  com- 
mercially available.  Fluorescein  amine 
may  be  synthesized  and  converted  to 
the  isocyanate  for  conjugation  to  pro- 
tein by  the  procedures  desci-ibed  by 
Coons  and  Kaplan  (J.  E.xp.  Med.,  1950, 
91,  1). 

Conjugation  of  antibody  solution  with 
fluorescein  isocyanate.  The  protein  con- 
tent of  the  serum  or  isolated  globulin 
fraction  to  be  conjugated  must  be  de- 
termined. It  should  be  at  least  1.7%. 
A  convenient  amount  of  protein  for  one 
run  is  from  300  to  600  mgm.  The 
amount  must  be  known. 

Fix  a  small  beaker  in  an  ice-bath  and 
equip  it  with  good  mechanical  stirring. 
Put  reagents  by  volume  into  it  in  the 
following  order: 
Saline  (0.9%  NaCl)     to  make  100%. 
Carbonate    -   bicar- 
bonate buffer  (0.5 

M,  pH9.0) 15% 

Dioxane      (distilled 

from    and    stored 

over  sodium) 15% 

Acetone 7.5%  (minus  2  ml.) 

When  the  solution  is  4°C.  or  below, 
and  the  isocyanate  solution  is  ready, 
add 

Protein  solution,  such  that  the  pro- 
tein concentration  in  the  final  mixture 
is  1%. 

For  example,  there  are  30  ml.  of  a 
concentrated  antibody  solution  con- 
taining 2.0%  total  protein.  It  is  de- 
sired to  conjugate  500  mgm.  (25  ml.), 
holding  the  remainder  for  control  pur- 


poses.    The  reaction  mixture: 

Saline 6.75  ml. 

Buffer 7.5  ml. 

Dioxane 7.5  ml. 

Acetone 1.25  ml. 

Protein  sol 25  ml. 

Total 48  ml. 

An  amount  of  one  of  the  two  fluores- 
cein amine  isomers  such  that  there  is 
0.05  mgm.  of  amine  per  mgm.  of  protein 
(in  the  example,  25  mgm.)  is  dissolved 
in  dry  acetone  and  treated  with  phos- 
gene. This  procedure  should  be  carried 
out  in  a  good  chemical  hood  with  forced 
exhaust.  Phosgene  is  led  out  of  the 
tank  through  concentrated  sulphuric 
acid,  thence  to  a  vessel  containing  15 
ml.  of  dry  acetone  and  fitted  with  a 
dropping  funnel,  thence  to  an  empty 
vessel  which  serves  as  a  trap,  thence 
through  20%  sodium  hydroxide  to 
destroy  the  excess  phosgene,  and  finally 
through  a  trap  with  a  controlled  leak 
to  a  water  suction  pump.  The  whole 
reaction  train  should  be  maintained 
at  a  pressure  slightly  below  atmospheric 
by  means  of  the  controlled  leak.  Phos- 
gene is  turned  on  and  allowed  to  bubble 
through  to  remove  air  and  to  saturate 
the  acetone  in  the  reaction  flask.  At 
the  end  of  a  few  minutes,  the  amine  dis- 
solved in  5  cc.  of  dry  acetone  is  added 
slowly  from  a  dropping  funnel  to  the 
vessel  containing  the  acetone  saturated 
with  phosgene.  When  all  the  amine- 
containing  solution  has  been  added  to 
the  reaction  vessel,  phosgene  is  allowed 
to  continue  bubbling  through.  During 
this  time  the  color  of  the  solution  in  the 
reaction  flask  slowly  changes  from  a 
fluorescent  green  to  a  pale  yellow.  A 
small  amount  of  heat  is  generated  dur- 
ing the  reaction  of  the  amine  with 
phosgene.  At  the  end  of  about  15  min- 
utes the  reaction  flask  is  transferred  to 
a  vacuum  still,  immersed  in  a  water 
bath  at  about  45°C.,  and  the  acetone 
boiled  off  under  reduced  pressure.  Small 
pieces  of  dry  anthracite  can  be  used  as 
antibumping  chips.  This  step  removes 
the  phosgene  still  dissolved  in  the  ace- 
tone. When  the  reaction  flask  is  warm 
and  dry,  a  greenish  brown  gum  is  vis- 
ible on  the  wall;  this  should  be  dis- 
solved in  2  ml.  of  acetone.  This  solu- 
tion is  added  in  toto,  drop  by  drop,  to 
the  stirred,  chilled,  buffered  protein  so- 
lution. (This  additional  acetone  brings 
the  total  acetone  concentration  up  to 
7.5%.)  Stirring  is  allowed  to  continue 
for  16  hours  in  the  cold.  The  solution 
is  poured  into  a  cellophane  sac  and 
dialyzed  against  repeated  changes  of 
saline   buffered   with    0.01  molar  phoa- 


AXTIGENS,  HISTOCHEMICAL 


17 


ANTIGENS,  HISTOCHEMICAL 


phate  at  pH  7.0  in  the  cold  until  the 
dialysate  outside  the  sac  shows  fluores- 
cence of  less  than  1  part  in  20  million. 
This  can  be  roughly  determined  by  eye 
using  a  known  solution  of  fluorescein  as 
a  standard. 

The  antibody  solution  may  be  further 
purified  by  one  or  more  precipitations 
with  half  saturated  ammonium  sulphate 
followed  by  re-solution  and  dialj^sis, 
and  by  precipitation  with  cold  acetone 
(Coons,  et  al.,  J.  Immunol.,  1942,  45, 
159)  or  by  40%  alcohol  (Marshall,  J. 
Exp.  Med.,  1951,  94,  21).  Merthiolate 
(Eli  Lilly  Co.)  should  be  added  as  a 
preservative  (1:100  of  a  1%  solution). 

Despite  these  chemical  purification 
procedures,  substances  remain  in  such 
fluorescein-protein  conjugates  which 
stain  some  tissue  elements.  It  has  been 
found  necessary  to  shake  such  con- 
jugates with  dry  acetone-powder  de- 
rived from  animal  tissues,  usually  liver 
powder  of  the  species  whose  tissue  it 
is  proposed  to  study.  Such  powder 
may  be  prepared  by  homogenizing  the 
tissue  in  a  Waring  blendor,  washing 
several  times  with  distilled  water  in 
the  centrifuge,  suspending  the  product 
in  saline  and  precipitating  it  with  four 
volumes  of  acetone.  The  acetone  pre- 
cipitate can  be  harvested  on  a  Buchner 
funnel  and  washed  with  dry  acetone  to 
remove  water.  Such  a  powder  is  added 
to  a  small  aliquot  of  conjugate  in  the 
proportion  of  100  mgm.  of  powder  to 
each  ml.  of  conjugate,  the  paste  allowed 
to  stand  for  an  hour  at  room  temper- 
ature, and  the  powder  separated  in  the 
centrifuge.  The  yield  is  higher  if  the 
centrifugation  is  carried  out  in  the  cold 
at  18,000  rpm.  Two  such  absorptions 
are  often  necessary  to  remove  "non- 
specific staining."  Merthiolate  should 
be  added  again  as  above. 

The  preparation  of  tissue  sections  for 
use  ivith  fluorescent  antibody.  The  prob- 
lem of  preparing  tissue  sections  retain- 
ing the  antigenic  activity  of  the  ma- 
terial sought  varies  with  the  antigen  in 
question.  The  bacterial  polysaccha- 
rides which  have  so  far  been  studied 
survived  fixation  in  Rossman's  picric 
acid-alcohol-formalin  followed  by 
paraffin  embedding.  Such  sections  are 
deparafRnized  and  hydrated  and  then 
stained  with  the  appropriate  antibody 
solution.  Care  must  be  taken  not  to 
wash  out  the  antigen  during  the  pro- 
cedures preceding  the  flooding  of  the 
section  with  labeled  antibody.  In  the 
case  of  bacterial  polysaccharides,  it  is 
necessary  to  remove  the  picric  acid  in 
70%  alcohol,  in  which  these  polysac- 
charides are  insoluble. 

In  the  case  of  less  stable  materials, 


for  example  proteins,  fixation  of  the 
tissue-block  is  unsatisfactory;  sections 
must  be  prepared  from  unfixed  material 
and  fixation  carried  out  on  the  indi- 
vidual section.  There  are  two  methods 
available  for  the  preparation  of  such 
sections  from  unfixed  tissue,  one  that 
of  Linderstr0m-Lang  and  Mogensen, 
the  other  that  of  Altmann-Gersh,  and 
others. 

With  either  of  these  two  methods, 
it  is  necessary  to  fix  the  tissue  section 
in  some  appropriate  reagent  before 
applying  labeled  antibody  solutions 
lest  the  material  looked  for  be  dissolved 
out  of  the  section  during  exposure  to 
conjugated  antibody.  A  certain 
amount  of  experimentation  is  necessary 
with  each  new  antigen  in  order  to  find 
the  appropriate  fixative.  For  mumps 
and  influenza  A  virus  and  the  virus  of 
infectious  canine  hepatitis,  acetone  is 
satisfactory.  This  is  used  at  room 
temperature  for  15  to  30  minutes  fol- 
lowed by  drying  of  the  section  in  an 
incubator.  For  proteins,  95%  ethanol 
by  volume  (start  with  absolute  ethanol) 
at  37°C.  for  30  minutes  is  satisfactory. 
For  ACTH  in  the  anterior  lobe  of  the 
hog  pituitary,  absolute  methanol  has 
been  shown  to  be  a  satisfactory  fixative 
(Marshall,  J.  Exp.  Med.,  1951,  94,  21). 
In  general,  these  organic  solvents  are 
best  removed  by  evaporation. 

These  fixatives  and  exposure-times 
are  cited  as  examples  only.  Any  anti- 
gen-antibody system  chosen  for  study 
must  be  investigated  from  this  as  well 
as  from  other  points  of  view. 

The  use  of  labeled  antibody  solutions 
on  tissue  sections.  A  small  drop  of 
fluorescein-antibody  solution  is  placed 
over  the  tissue  section,  and  allowed  to 
react  for  from  10  minutes  to  48  hours, 
depending  on  the  system  under  study. 
Thirty  minutes  is  usually  satisfactory. 
During  the  reaction,  evaporation  must 
be  minimized.  Usually  the  reaction 
can  be  carried  out  at  room  temperature, 
although  incubator  temperatures  or 
refrigerator  temperatures  can  be  em- 
ployed. Following  exposure  to  the 
fluorescent  antibody  solution,  the  sec- 
tion should  be  placed  in  a  Coplin  jar 
containing  0.9%  saline  buffered  at  pH 
7.0  with  0.01  molar  phosphate,  and 
washed  with  very  gentle  motion  for 
10  minutes.  At  the  end  of  this  time, 
the  slide  should  be  wiped  dry  except 
for  the  area  of  the  section  and  the  sec- 
tion itself  mounted  under  a  cover  slip 
in  glycerol  containing  a  trace  of  buffer 
at  pH  7  (commercial  glycerol  is  slightly 
acid),  and  examined  under  the  fluores- 
cence microscope. 

Control     of    specificity    of    staining. 


ANTIGENS,  HISTOCHEMICAL 


18 


ANTIGENS,  HISTOCHEMICAL 


Because  of  the  ever-present  hazard  of 
"nonspecific"  staining,  careful  controls 
of  specificity  must  be  carried  out  at 
every  step.  When  the  antigenic  material 
sought  is  foreign  to  the  tissue  under 
investigation,  control  is  relatively 
simple  since  the  conjugate  should  fail 
to  stain  normal  tissue  sections.  Im- 
munologically specific  staining  can  be 
greatly  diminished  or  altogether  pre- 
vented by  pretreatment  of  parallel  sec- 
tions with  unlabeled  homologous  anti- 
body, thereby  blocking  the  antigen. 
However,  replacement  of  unlabeled  by 
labeled  antibody  occurs  and  the  time 
of  staining  must  usually  be  decreased 
in  order  to  carry  out  this  control  suc- 
cessfully. Failure  to  stain  the  tissue 
under  investigation  with  heterologous 
labeled  antibody  is  also  a  suitable 
control,  although  the  degree  of  purifica- 
tion of  conjugates  varies  and  it  is  diffi- 
cult to  establish  that  any  two  conju- 
gates are  strictly  comparable.  There 
are  so  many  possible  variations  that 
controls  appropriate  to  each  situation 
must  be  carefully  planned. 

Fluorescence  microscopy.  Fluores- 
cence microscopy  is  described  in  an- 
other section.  The  amount  of  de- 
posited antibody  is  quite  small,  and  the 
fluorescence  therefore  faint.  Light 
sources  adequate  for  the  study  of 
brightly  fluorescent  materials  present 
in  high  concentration  are  not  appropri- 
ate for  use  with  labeled  antibody. 
Either  a  10  amp.  carbon  arc  or  a  high 
pressure  water  cooled  mercury  vapor 
arc  (General  Electric  Co.,  AH -6)  are 
satisfactory.  At  this  level  of  bombard- 
ment, fluorescence  of  optical  elements 
becomes  important.  It  is  necessary  to 
use  a  quartz  condenser  since  glass  ones 
often  fluoresce  in  the  ultraviolet  beam. 
Definition  is  improved  at  the  cost  of 
intensity  by  the  use  of  a  cardioid 
darkfield  condenser. 

Photography.  Findings  revealed  by 
this  method  can  be  recorded  photo- 
graphically using  35  mm.  film  (either 
high  speed  panchromatic  film  or  fast 
green-sensitive  film,  e.g.,  "Photoflure" 
Eastman).  Exposure  times  vary  with 
the  amount  of  fluorescent  antibody 
deposited  and  the  autofluorescene  of 
the  background,  varying  at  "high  dry" 
(400  X)  from  3  to  10  minutes.  Focus- 
ing on  ground  glass  is  very  difficult 
because  of  the  low  degree  of  intensity 
of  the  projected  image,  but  can  be 
carried  out  by  the  use  of  a  magnifying 
lens  focused  on  a  clear  area  in  the 
ground  glass. 

Anyone  attempting  to  carry  out  these 
procedures  is  advised  to  consult  the 
original  papers  to  which  reference  has 


been  made  (cf.  Federation  Proc,  1951, 
10,  558). 

Frozen  Sections  by  the  Method  of 
hinderstr^m-Lang  and  Mogensen 
(Compt.-Rend.  Trav.  Lab.  Carlsberg, 
s6rie  chim.,  1938,  23,  27;  Coons  ,et  al., 
J.  Exp.  Med.,  1951,  93,  173).  The  prin- 
ciple of  this  method  is  quite  simple. 
It  involves  the  quick-freezing  of  tissues 
at  low  temperatures,  and  cutting  them 
while  frozen  in  a  cryostat  maintained 
at  —20.  This  cold  chamber  should  be 
equipped  with  adequate  lighting,  an 
insulated  window  through  which  the 
operator  can  see,  and  gloved  armholes 
through  which  he  can  work.  Sections 
are  cut  on  a  good  rotary  microtome, 
the  knife  of  which  is  equipped  with  a 
glass  guide  to  keep  the  sections  from 
curling  as  they  are  cut.  Convenient 
improvements  in  the  method  are  de- 
scribed by  Coons,  et  al.  Tissues  may 
be  quick-frozen  by  placing  thin  slices 
on  the  side-walls  of  test  tubes  which 
are  then  stoppered  and  plunged  but 
not  submerged  into  alcohol  cooled  to 
the  temperature  of  dry  ice.  They  can 
then  be  stored  in  a  deep  freeze  below 
—  20,  or  put  in  the  cold  cryostat  for 
immediate  sectioning.  Long  storage 
results  in  the  growth  of  ice  crystals. 
The  tissue  is  mounted  by  putting  a 
small  drop  of  water  on  a  previously 
chilled  tissue  holder  and  touching  the 
tissue  to  it.  Within  a  few  seconds  the 
tissue  block  will  be  tightly  frozen  to 
the  holder.  Care  must  be  taken  to 
avoid  ice  formation  near  the  cutting 
area.  The  knife  blade  should  be  set 
at  an  angle  of  approximately  20°  from 
the  plane  of  movement  of  the  tissue 
block.  The  glass  guide  should  be  ad- 
justed so  that  its  upper  edge  is  parallel 
to  and  at  the  height  of  the  cutting  edge 
of  the  knife.  Each  section  as  cut  is 
removed  with  forceps  from  the  knife 
blade,  placed  gently  on  a  cold  slide, 
and  thawed  by  placing  a  finger  under 
the  section.  It  can  then  be  dried  in 
the  air  stream  from  a  fan  at  room  tem- 
perature. Appreciable  movement  of 
tissue  components  during  this  momen- 
tary thawing  has  not  been  troublesome, 
although  it  could  be  serious  in  some 
situations. 

An  adhesive  is  necessary  on  the  slides; 
the  one  in  use  by  the  authors  quoted 
has  been  formalinized  gelatin.  Acid 
cleaned,  dry  glass  slides  are  dipped  in 
0.5%  gelatin,  and  placed  on  end  to 
drain  and  dry.  When  dry,  they  are 
dipped  in  10%  formalin,  drained,  and 
allowed  to  dry.  They  can  then  be 
stored  for  long  periods  until  required. 

Good  sections  10  ju  thick  are  easy  to 
cut  by  this  method,  and  with  practice 


ANTIRACHITIC  VITAMIN 


19 


ARGENTAFFINE  REACTION 


sections  about  4  ju  thick  can  be  cut  as  a 
routine. 

Antirachitic  Vitamin,  see  Vitamin  D3. 

Antiscorbutic  Vitamin,  see  Vitamin  C. 

Antimony  Trichloride,  see  Carr-Price  Re- 
action. 

Aorta,  see  Arteries  and,  for  an  account  of 
technique  for  measuring  elastic  proper- 
ties, Saxton,  J.  A.,  Arch.  Path.,  1942, 
34,  262-274. 

Aortic  Paraganglion  (Glomus  aorticum). 
Technique  for  blood  supply  and  innerva- 
tion is  provided  by  Nonidez,  J.  F.,  J. 
Anat.,  1936,  70,  215-224.  Negative  re- 
sults in  application  of  the  chromaffin 
reaction  to  the  rabbit  and  guinea  pig 
are  described  by  the  same  author.  Am. 
J.  Anat.,  1935,  57,  259-293.  Carotid 
glomus  is  very  similar. 

V.  Apathy  Syrup  has  been  modified  by 
Lillie,  R.  D.  and  Ashburn,  L.  L., 
Arch.  Path.,  1943,  36,  432-435.  Dis- 
solve acacia  (gum  arable)  50  gm.  and 
cane  sugar  50  gm.  in  aq.  dest.  100  cc. 
shaking  frequently  at  55-60°C.  Add 
aq.  dest.  to  make  up  original  volume 
and  merthiolate  (sodium  ethylmercuri- 
theosalicylate)  15  gm.,  or  thymol  100 
gm.  to  act  as  a  preservative.  This 
syrup  is  recommended  as  a  mounting 
medium  for  frozen  sections  stained 
with  Sudan  III,  or  other  alcohol  soluble 
dyes. 

Aposiderin,  see  Lillie,  p.  127. 

Aquax — Written  by  C.  C.  Macklin,  Dept. 
of  Histological  Research,  The  Univer- 
sity of  Western  Ontario,  London, 
Canada.  November  28,  1951 — A  water- 
soluble  wax  for  embedding  purposes 
produced  by  George  T.  Gurr,  136,  New 
King's  Road,  London,  S.W.  6.  It  has 
been  found  useful  for  the  demonstra- 
tion of  osmiophil  granules  in  pneumo- 
pneumonocyies  (which  see)  after  levu- 
lose  mounting  (See  Levulose  Syrup). 
Its  merit  is  the  retention,  in  sections, 
of  materials  which  tend  to  diffuse  away 
in  alcohol,  clearing  fluids  and  par- 
affin. 

Aqueous  Humor,  see  Anterior  Chamber  of 
Eye. 

Arachnids,  sectioning  is  facilitated  by 
methods  intended  to  soften  Chitin. 
See  also  Fleas,  Ticks. 

Archelline  2B,  see  Bordeaux  Red. 

Argentaffine  gastrointestinal  cells  (entero- 
chromaffin  cells).  Rare  even  in  duo- 
denum. Occur  singly,  usually  in  deep- 
est parts  of  crypts  and  may  be  free  from 
epithelium.  Cytoplasmic  argentaffine 
granules  are  of  small  size,  often  closely 
packed  together  and  acidophilic.  It  is 
said  that  they  cannot  be  found  in  bodies 
autopsied  as  late  as  4-5  hrs.  after  death 
(Hamperl,  H.,  Ztschr.  f.  Mikr.-anat. 
Forsch.,  1925,  2,  506-535). 


Fluorescence  Microscopy  brings  out 
the  argentaffine  cells  sharply  because 
the  cytoplasmic  granules  fluoresce  a 
yellow  color.  This  color  is  not  abol- 
ished by  fat  solvents  and  is  attributed 
to  pterins  (McClung,  Microscopical 
Technique,  1950,  p.  682).  See  Eros, 
G.,  Zentralbl.  f.  Allg.  Path.  Anat., 
1932,  54,  385;  Jacobson,  W.,  J.  Path. 
&Bact.,  1939,49,  1). 

Two  specific  methods  are  advised  by 
Jacobson,  W.,  J.  Path.  &  Bact.,  1939, 
49,  1-19.  For  both  fix  in  10%  formol- 
saline,  or  10%  neutral  formol,  dehydrate 
in  alcohol,  clear  in  cedarwood  oil  or  in 
methyl  benzoate  -\-  2%  celloidin  and 
imbed  in  paraffin.  In  the  first  wash 
deparaffinized  sections  10  mm.  in  2 
changes  glass-dist.  water.  Transfer  for 
12-24  hrs.  to  Fon tana's  sol.  prepared  by 
adding  NH4OH  to  5%  AgNO,  until  ppt. 
is  dissolved,  then  AgNOs  drop  by  drop 
until  fluid  exhibits  slight  presistent 
opalescence.  Wash  in  glass-dist.  water, 
1  min.,  5%  NajSjOs,  1  min.  and  tap 
water  10  min.  Counterstain  with  car- 
malum.  Dehydrate,  clear  and  mount 
in  balsam.  Granules  of  argentaffine 
cells  appear  black.  In  the  second  more 
rapid  method  dissolve  small  amount 
p-nitro-methyloxybenzene  diazotate  in 
aq.  dest.  producing  light  yellow  solution 
alkalinize  with  a  little  Li2C03.  After 
about  I5  min.,  when  pH  10-11  is  reached, 
color  has  changed  to  dark  orange-yellow. 
Immerse  sections  brought  down  to  aq. 
dest.,  in  this  30-40  sec.  Then  wash  in 
aq.  dest.,  1  min.  Granules  of  argen- 
taffine cells  appear  dark  red  in  yellow 
background.  Counterstain  with  hema- 
lum  if  desired. 

Since   Dawson,   A.   B.,   Anat.    Rec, 

1944,  89,  287-294  has  found  that  a  larger 
number  of  argentaffine  cells  are  demons- 
trable in  the  rat's  stomach  by  Bodian's 
technique  than  are  reported  after  silver 
impregnations  like  those  of  Masson- 
Hamperl,  it  is  important  to  try  the 
Bodian  Method  in  the  manner  suggested 
by  Dawson.     Sharpies,  W.,  Anat.  Rec, 

1945,  91,  237-243  used  the  Bodian 
Method  successfully  in  study  of  human 
stomach. 

Argentaffine  Reaction.  This,  according  to 
Lison  (p.  147)  is  given  by  polyphenols, 
aminophenols  and  polyamines  in  ortho 
and  para  position.  It  is  a  reduction  of 
ammoniated  silver  hydroxide  into  me- 
tallic silver.  He  recommends  Masson's 
method  for  sections  :  Fix  in  Bouin's  fluid 
or  other  fixative.  Deparaffinize  sec- 
tions and  wash  2  hrs.  in  aq.  dest.  Treat 
for  3&-40  hrs.  in  Fontana's  fluid  in  dark- 
ness and  in  a  sheltered  place.  Wash  in 
much  aq.  dest.  Tone  with  0.1%  aq. 
gold   chloride    (few   minutes).    Fix   in 


ARGINASE 


20 


ARGININE  REACTION 


5%  aq.  sodium  hyposulphite.  Counter- 
stain  with  alum  carmine,  mount  in 
usual  way.  To  make  Fontana's  fluid 
add  ammonia  drop  by  drop  to  5%  aq. 
silver  nitrate  until  ppt.  formed  is  ex- 
actly redissolved;  then  carefully  drop 
by  drop  5%  aq.  silver  nitrate  until 
appearance  of  persistent  cloudiness  and 
the  liquid  does  not  smell  of  ammonia. 
Decant  before  employing.  See  also 
Clara,  M.,  and  Canal,  F.,  Zeit.  f.  Zellf. 
u.  Mikr.  Anat.,  1932,  15,  801-808;  Clara, 
M.,  Ergeb.  d.  Anat.  u.  Entw.,  1933,  30, 
240-340. 
Arginase.  It  is  possible  to  localize  arginase 
in  the  cytoplasm  and  nuclei  of  liver  cells 
by  Behren's  technique  (Zeit.  Physiol. 
Chem.,  1939,  258, 27-32).  Finely  ground 
tissue  is  dried  to  powder  in  frozen  condi- 
tion. It  is  then  suspended  and  cen- 
trifuged  in  different  mixtures  of  benzene 
and  carbon  tetrachloride.  The  nuclei 
only  are  found  in  the  lowest  layer,  next 
comes  nuclear  debris  and  above  this 
cytoplasmic  debris.  His  analysis 
showed  arginase  present  in  the  same  con- 
centration in  the  nuclei  as  in  the  cyto- 
plasm. Blaschko  and  Jacobson  (Bourne, 
p.  217)  remark  that  this  is  the  first  in- 
stance of  the  demonstration  of  an  enzyme 
in  the  cell  nucleus. 
Arginine  Reaction.  The  method  of  Serra, 
J.  A.,  Stain  Techn.,  1946,  21,  5-18  is 
detailed  by  him  as  follows:  Prepare 
tissue  as  described  under  Ninhydrin 
Reaction. 

"1.  Before  the  reaction  the  pieces  or 
sections  are  hardened  with  10%  for- 
maldehyde during  12-24  hours,  the 
formalin  being  afterwards  well  washed 
out.  (If  the  fixative  contains  formalin 
this  step  can  be  omitted.) 

"2.  Immerse  the  pieces  for  15  minutes 
in  a  mixture  consisting  of  0.5  ml.  of 
diluted  a-naphthol ;  0.5  ml.  of  N  NaOH ; 
and  0.2  ml.  of  40%  aqueous  urea  solu- 
tion. The  diluted  a-naphthol  is  pre- 
pared at  the  moment  of  use  by  diluting 
a  stock  solution  (1%  crystallized  n- 
naphthol  in  96%  alcohol)  1 :  10  with  40% 
alcohol.  The  watch  glass  containing 
the  liquids  is  placed  in  an  ice-bath  and 
the  temperature  of  the  reaction  fluid 
inside  it  must  be  0.5°C. 

"3.  After  12-15  minutes  add  0.2  ml. 
of  a  2%  solution  of  NaOBr.  This  re- 
agent is  allowed  to  act  for  3  minutes  and 
the  solution  must  be  well  stirred  during 
this  time.  The  2%  NaOBr  must  be 
freshlj'  prepared  by  pouring  2  g.  (or 
approximately  0.7  ml.)  of  liquid  bro- 
mine into  100  ml.  of  5%  NaOH,  with 
agitation  and  cooling. 

"4.  Add  another  0.2  ml.  of  40%  urea 
solution,  stir,  and  immediately  after- 
ward, 


"5.  Add  another  0.2  ml.  of  2%  NaOBr 
and  stir  well.  The  coloration  attains 
its  maximum  after  3-5  minutes  and 
would  last  only  for  a  short  time  if  it 
were  not  stabilized.  To  stabilize  the 
coloration: 

"6.  Take  the  pieces  out  of  the  reac- 
tion mixture  and  immerse  in  pure 
glycerin  for  2-3  minutes  and  then  trans- 
fer to  fresh  glycerin.  Repeat  the  opera- 
tion another  two  or  three  times.  The 
passage  through  4  glycerin  baths  is 
sufficient  to  stabilize  the  coloration 
for  some  months,  even  if  the  pieces  are 
left  at  room  temperature.  (We  have 
not  mentioned  this  improvement  in 
any  previous  publication.) 

"Besides  this  procedure,  which  we 
may  call  the  normal  method,  there  is 
also  another  method  which  results  in 
stronger  colorations  and  very  satisfac- 
tory preparations.  To  accomplish  this, 
after  step  5  the  pieces  are  taken  off  the 
reaction  liquid  and  immersed  in  NaOBr 
solution  for  not  more  than  3  minutes. 
Afterwards  the  coloration  is  stabilized 
in  glycerin,  as  in  the  normal  procedure. 
The  pieces  are  mounted  and  observed 
in  pure  glycerin. 

"This  reaction  is  specific  for  guani- 
dine  derivatives  in  which  only  one  H- 
atom  of  one  amino  group  is  substituted 
by  a  radical  of  the  alkyl  or  fatty  acid 
type.  In  proteic  compounds  it  is 
specific  for  arginine.  As  all  proteins 
hitherto  analyzed  possess  arginine  in 
their  molecules,  the  reaction  may  be 
used  to  demonstrate  the  presence  of 
proteins  in  general,  other  compounds 
with  a  reactive  guanidine  group  being 
rare.  The  test  may  also  be  used  to 
characterize  the  basic  proteins." 
Arginine  Reaction.  The  method  of  Thomas, 
Lloyd  E.,  Stain  Techn.,  1950,  25, 143-148, 
has  been  modified  as  follows  (unpub- 
lished) written  byL.  E.  Thomas,  Dept. 
of  Biochemistry,  University  of  Missouri, 
Columbia.  July  8,  1951 — Bouin's  fixa- 
tive has  given  the  best  results.  Formal- 
dehyde (4%)  is  almost  as  good.  Carnoy's 
(6:3:1)  may  be  used.  After  fixation  pre- 
pare paraffin  sections. 

1.  Remove  the  paraffin  from  the  slide 
with  xylene  and  pass  it  through  the 
alcohols  to  70%. 

2.  Place  the  slide  in  a  0.3%  aq.  of 
8-hydroxyquinoline  (oxine)  in  a  Coplin 
jar  for  15  min.  at  room  temperature. 
This  solution  is  prepared  by  diluting 
with  water  a  1.0%  stock  solution  of 
oxine  in  absolute  alcohol. 

3.  Move  the  slide  with  a  quick  motion 
{not  allowing  it  to  drain)  into  a  0.15  N 
sodium  hypochlorite  solution  (0.015  N 
with  respect  to  potassium  hydro.xide). 
This  solution  is  prepared  by  standardiz- 


ARGON 


21 


ARTERIES 


ing  Clorox  or  other  suitable  hypochlorite 
solution  and  using  it  as  a  stock  solution. 
Leave  the  slide  in  the  hypochlorite 
solution  exactly  60  sec.  at  room  temper- 
ature, holding  it  stationary. 

4.  Move  quickly  [without  draining) 
into  a  solution  containing  per  100  cc: 
15  gm.  urea,  70  cc.  tertiary  butyl  alcohol 
and  potassium  hydroxide  to  make  the 
concentration  of  the  latter  0.015  N. 
Move  the  slide  gently  in  the  solution 
for  10  sec,  then  transfer  to  another  jar 
of  the  same  solution  for  2  min. 

5.  Transfer  to  100%  tertiary  butyl 
alcohol,  moving  the  slide  gently  for 
10  sec,  then  place  in  a  second  jar  of  the 
same  reagent  for  3^  min. 

6.  Pass  through  three  changes  of 
xylene  for  10  seconds,  1  minute  and 
2  min.,  respectively. 

7.  M.ount  with  Permount  containing 
aniline.  This  reagent  is  prepared  by 
dissolving  0.1  cc.  of  aniline  in  100  cc. 
of  xylene.  One  volume  of  this  solution 
is  mixed  with  four  volumes  of  Permount. 

Reagents  2,  3  and  4  must  be  made 
fresh  daily.  It  is  best  not  to  let  any 
water  accumulate  in  the  tertiary  butyl 
alcohol. 

The  three  arginine  methods  are  all 
based  on  the  Sakaguchi  reaction,  which, 
in  biological  materials,  is  specific  for 
arginine,  galegine  and  agmatine.  The 
latter  two  are  extremely  rarely  en- 
countered and  so  for  most  purposes,  it 
is  specific  for  arginine.  The  histo- 
chemical  methods  are  specific  for  pro- 
tein-bound arginine,  since  free  arginine 
is  inevitably  removed  by  the  pro- 
cedures. 

Argon,  see  Atomic  Weights. 

Argyrophilic  Fibers.  Because  of  their  affin- 
ity for  silver,  Reticular  Fibers  are  often 
called  argyrophilic. 

Arneth  Count  of  lobes  of  granular  leucocytes 
as  a  basis  for  estimation  of  their  rela- 
tive age.     See  Leucocyte  Counts. 

Arsenic  1.  Use  10%  neutral  formalin  in  aq. 
dest.  after  test  with  hydrogen  sulphide 
shows  absence  of  trace  of  metals.  To 
100  cc.  add  2.5  gm.  copper  sulphate. 
Fix  small  pieces  of  tissue  5  days.  Wash 
24  hrs.  in  running  water.  Imbed  in 
paraffin.  Direct  examination  of  section 
after  removal  of  paraffin  shows  arsenic 
as  well  defined  green  granules  of  hydro- 
arsenite  of  copper  (Scheele's  green). 
If  neutral  acetate  of  copper  is  employed 
in  place  of  the  sulphate  the  green 
granules  are  of  acetoarsenite  of  copper 
(Schweinf urth's  green ) . 

2.  Fix  pieces  of  tissue  12-24  hrs.  in 
abs.  ale  50  cc. ;  chloroform,  50  cc. ;  pure 
hydrochloric  acid,  3  cc.  saturatea  by 
passage  of  pure  hydrogen  sulphide.  In 
sections  the  arsenic  ppt.  appears  as  yel- 


low granules.  Double  coloration  with 
hematein-eosin  is  possible.  Both  tech- 
niques have  been  devised  by  Castel 
(P.,  Bull.  d'Hist.  Appl.,  1936,  13,  106- 
112).  He  lias  described  the  histologic 
distribution  of  the  arsenic.  See,  how- 
ever, paper  by  Tannenholz ,  H .  and  Muir, 
K.  B.,  Arch.  Path.,  1933, 15,  789-795  who 
employed  a  somewhat  similar  method 
ana  were  unable  to  conclude  that  the 
yellow  crystals  were  in  fact  those  of 
arsenic  trisulphide.  They  considered 
them  more  probably  a  sulphur-protein 
combination. 

Consult  the  detailed  account  of  Os- 
borne's method  for  arsenic  given  by 
Heuper,  W.  C,  Occupational  Tumors 
and  Allied  Diseases.  Springfield: 
Thomas,  1942,  896  pp.  (p.  50).  This 
releates  particularly  to  localization  of 
arsenic  in  the  skin. 

The  distribution  to  the  several  tissues 
of  radioactive  arsenic  injected  intra- 
venously into  rabbits  as  sodium  arsenate 
has  been  investigated  by  duPont,  O., 
Irving,  A.  and  Warren,  S.  L.,  Am.  J. 
Syph.  etc.,  1942, 26, 96-118.  It  is  impor- 
tant to  determine  whether  the  results 
conform  with  those  given  by  the  micro- 
chemical  techniques. 

Arsphenamines.  The  specificity  of  the 
silver  reaction  of  Jancs6,  N.,  Ztschr. 
f.  d.  Ges.  exper.  Med.,  1929,  65,  98  is 
questioned  by  Gomori,  G.,  J.  Mt. 
Sinai  Hosp.,  1944-45,  11,  317-326  since 
it  may  demonstrate  other  reducing  sub- 
stances beside  the  arsphenamines. 

Artefacts,  see  Artifacts. 

Arteries.  If  one  wishes  an  elastic  artery 
take  a  large  trunk  near  the  heart  such  as 
the  aorta,  innominate  or  subclavian;  if, 
on  the  other  hand,  a  typical  muscular 
artery  is  required  select  one  further 
afield  like  the  radial  or  external  carotid. 
Arterial  walls  are  seldom  examined 
microscopically  in  vivo  because  they  are 
relatively  large  and  difficult  to  get  at 
without  injury.  An  exception  in  man  is 
the  retinal  artery  which  can  be  seen 
by  ophthalmoscopic  examination.  To 
closely  observe  excised  pieces  of  arteries 
is  all  too  frequently  neglected.  The 
tissue  elements  are  so  tightly  bound  to- 
gether that  to  tease  them  apart  for  study 
at  high  magnification  is  rather  unsatis- 
factory. However,  when  the  adventitial 
adipose  and  connective  tissue  is  stripped 
off  from  a  fresh  specimen,  the  remainder 
of  the  wall  can  very  advantageously  be 
made    translucent    by    treatment  with 

Eure  glycerin  for  1-2  hrs.  as  described 
y  Winternitz,  M.  C,  Thomas,  R.  M. 
and  LeCompte,  P.  M.  in  their  book  "The 
Biology  of  Arteriosclerosis",  Spring- 
field: Thomas,  1938,  142  pp.  Since  the 
color  of  the  blood  is  preserved  within 


ARTERIOLES 


22 


ARTIFACTS 


the  intramural  vessels  their  arrangement 
can  be  studied  (see  Vasa  Vasorum). 
Fatty  substances  can  also  be  located 
because  they  are  not  removed  by  the 
glycerin. 

Chief  reliance  is  ordinarily  placed  in 
the  appearance  of  arterial  walls  when 
seen  in  sections  of  fixed  tissue.  It  is 
important  to  remember  that,  when  carry- 
ing blood  during  life,  the  lumina  are 
larger  and  the  walls  less  folded  than  in 
the  fixed  condition.  The  difference  has 
been  graphically  demonstrated  by  Gallo- 
way, R.  J.  M.,  Am.  J.  Path.,  1936,  12, 
333-336.  His  figures  should  be  exam- 
ined. For  routine  purposes  fixation  in 
Formalin-Zenker  followed  by  Mallory's 
Connective  Tissue  Stain  supplemented 
by  Resorcin  Fuchsin  or  Orcein  for 
elastic  tissue  is  satisfactory.  Special 
methods  may  be  needed  for  Lipids ;  and 
for  minerals,  see  Calcium,  Iron  and 
Microincineration.  Innervation,  like- 
wise, is  to  be  studied  by  methods  em- 
ployed to  demonstrate  Nerve  Endings 
in  other  tissues.  See  Vasa  Vasorum. 
Much  literature  on  techniques  is  given 
by  various  authors  in  Cowdry,  E.  V., 
Arteriosclerosis,  New  York:  Macmillan, 
1933,  617  pp.  The  investigation  of 
arterial  walls  is  apt  to  be  one  sided 
limited  only  to  structure  and  composi- 
tion demonstrated  microscopically.  It 
is  high  time  that  these  lines  of  study 
are  supplemented  by  accurate  meas- 
urement of  the  physical  properties  of 
pulse  wave  velocity,  sound  production, 
elasticity  and  so  on  of  the  same  vessels 
by  methods  described  by  Bramwell  in 
the  above  mentioned  volume. 
Arterioles,  capillaries  and  venules,  in  con- 
trast to  the  much  larger  arteries  and 
veins,  can  readily  be  examined  in  experi- 
mental animals  microscopically  in  the 
living  state.  Since  they  are  linked 
together  a  single  preparation  by  Sandi- 
son's  rabbit  ear  method  shows  all  three, 
or  they  may  be  viewed  in  the  living 
tadpole's  tail  or  other  transparent  tissue 
of  lower  forms.  For  convenience,  how- 
ever, it  seems  best  to  briefly  mention 
the  microscopic  techniques  for  each 
separately.  There  is  much  to  choose 
from.  Information  is  frequently  de- 
manded on  the  condition  of  the  arterio- 
lar walls.  This  can  best  be  supplied 
by  staining  paraffin  sections  of  Forma- 
lin-Zenker fixed  material  with  Mal- 
lory's Connective  Tissue  stain  or  with 
Masson's  Trichrome  stain  which  is 
closely  related  to  it.  Weigert's  Re- 
sorcin Fuchsin  is  satisfactory  for  elastic 
tissue.  The  Silver  Citrate  technique  is 
capable  of  yielding  valuable  data  on  arte- 
rioles and  capillaries.  Because  arte- 
rioles  contain  a   higher  percentage  of 


muscle  than  any  other  blood  vessel  their 
appearance  will  vary  greatly  with  the 
degree  of  contraction  or  relaxation  of 
muscle.  According  to  Kernohan,  J.  W., 
Anderson,  E.  W.  and  Keith,  N.  M., 
Arch.  Int.  Med.,  1929,  44,  395-423  in 
fixed  preparations  from  normal  persons 
the  average  ratio  of  thi  ckness  of  arteriolar 
wall  to  width  of  lumen  is  1:2. 

Arteriovenous  Anastomoses  are  direct  con- 
nections between  arteries  and  veins 
vrithout  intervening  capillaries.  No 
special  histological  technique  is  required 
for  their  demonstration  in  sections  but 
one  should  look  for  them  where  they  are 
particularly  numerous,  as  in  rabbits  at 
the  tip  of  the  nose  (diameter,  80-100/i) 
and  in  humans  in  the  palms  of  the  hands, 
the  soles  of  the  feet  and  near  the  ends 
of  the  fingers  where  their  diameter  is 
about  35m  (Grant,  R.  T.  and  Bland, 
E.  F.,  Heart,  1930,  15,  385-411).  The 
best  way  is  to  study  them  in  vivo  (Clark, 
E.  R.  and  E.  L.,  Am.  J.  Anat.,  1934,  55, 
407-467). 

Arteriosclerosis.  The  arteries  in  this  condi- 
tion show  changes  well  demonstrated 
by  Mallory's  Connective  Stain  and  its 
modifications  as  well  as  by  Weigert's 
Resorcin  Fuchsin.  In  addition,  tech- 
niques for  Lipids,  Calcium  and  Iron  are 
indicated.  Methods  for  the  measure- 
ment of  physical  properties  of  arteries 
might  well  be  applied  to  arteries  most 
and  least  prone  to  develop  arterio- 
sclerosis. These  are  summarized  by 
Bramwell,  C,  in  Cowdry's  Arterio- 
sclerosis. New  York:  Macmillan  Co., 
1933,  617  pp. 

Artifacts.  Webster  defines  an  artifact  as 
being  "in  histology,  a  structure  or 
appearance  in  a  tissue  or  cell  due  to 
death  or  to  the  use  of  reagents  and  not 
present  during  life."  The  degree  of 
artifact  is  proportional  to  the  difference 
between  the  structure  existing  normally 
in  the  living  body  and  the  structure  in 
the  condition  directly  studied. 

1.  In  the  case  of  living  tissues,  ob- 
served with  blood  and  nerve  supply 
intact,  there  is  a  possibility  of  artifact. 
It  is  at  a  minimum  in  the  Rabbit  Ear 
Chambers  and  rather  more  to  be  reck- 
oned with  when  tissues  must  be  dis- 
placed in  order  to  supply  the  necessary 
illumination.  With  increase  in  time 
modifications  due  to  changes  in  light, 
temperature,  hydrogen  ion  concentra- 
tion, etc.  are  likely  to  also  increase. 

2.  In  living  cells  removed  from  the 
body  and  examined  in  Tissue  Cultures 
the  possibility  of  artifact  is  again  at  a 
minimum ;  but,  though  the  cells  in  suc- 
cessive generations  in  suitable  media  go 
on  living  indefinitely,  their  environ- 
ments are  different  from  those  existing 


ARTICULAR  NERVE  TERMINALS 


23 


ATABRINE 


within  the  body.  When  after  Vital 
Staining  or  Supravital  Staining  still 
living  cells  are  examined  in  approxi- 
mately isotonic  media,  there  is  a  grave 
danger  of  artifact  if  the  study  is  pro- 
longed because  the  cells  are  slowly  dying. 
3.  In  fixed  tissues  the  degree  of  di- 
vergence from  the  normal  living  condi- 
tion is  obviously  much  greater  than  in 
the  case  of  still  living  ones.  However 
death  has  been  sudden  so  that  artifacts 
due  to  gradual  death  are  eliminated.  If 
the  technique  has  been  carefully  stand- 
ardized the  same  fixative  applied  to  the 
same  type  of  cell  in  the  same  physiologi- 
cal state  is  likely  to  yield  similar  results. 
Among  common  artifacts  are:  1.  The 
shrinkage  and  increased  affinity  of  cells 
near  the  surface  for  stains  due  to  allow- 
ing the  surface  of  the  tissue  to  dry  be- 
fore fixation.  2.  The  glassy  appearance 
of  nuclei  and  cytoplasm  sometimes  oc- 
casioned by  overheating  in  imbedding 
or  in  spreading  out  sections.  3.  Mate- 
rial within  blood  vessels  faintly  resem- 
bling organisms  caused  by  coagulation 
of  blood  proteins.  4.  Extraneous  sub- 
stances either  present  in  the  albumen 
fixative  used  to  mount  the  sections  or 
deposited  as  dust  from  the  air.  Careful 
focussing  is  required.  See  Agonal  and 
Postmortem  changes,  Ice  Crystal  Arti- 
facts. A  paper  by  Bensley,  R.  R.,  Exp. 
Cell  Res.,  1951,  2,  1-9  on  "Facts  versus 
Artifacts  in  Cytology:  the  Golgi  ap- 
paratus" is  illuminating.  See  Agonal 
and  Postmortem  changes.  Ice  Crystal 
Artifacts,  Normality,  Nucroscopic. 
Articular  Nerve  Terminals. — Written  by 
Dr.  E.  D.  Gardner,  Wayne  University 
School  of  Medicine,  Detroit.  June  15, 
1950— Gardner,  E.  D.,  Anat.  Rec, 
1942,  83,  401-419,  adapted  silver 
methods  to  the  demonstration  of  nerve 
terminals  in  the  knee  joints  of  mice 
1-60  days  old.  Subsequently,  J.  Comp. 
Neur.,  1944,  80,  11-32,  similar  methods 
were  applied  to  the  knee  joints  of  33- 
and  46-day-old  cat  fetuses.  The  Bodian 
method  was  used,  but  the  necessary 
protargol  seems  no  longer  to  be  avail- 
able. It  appears,  however,  that  similar 
results  can  be  obtained  with  Romanes' 
staining  method  (J.  Anat.,  1950,  84, 
104-115) .  A  variety  of  fixatives  are  satis- 
factory, particularly  Bouin  and  acetic- 
formol-alcohol.  Decalcify  in  20% 
sodium  citrate  and  50%  formic  acid, 
equal  volumes,  prior  to  embedding. 
Sections  are  placed  overnight  at  56 °C. 
in  the  following  solution:  Distilled 
water  50  ml.,  0.1%  silver  nitrate  2.9 
ml.,  and  0.1%  sodium  chloride  1.0  ml. 
To  this  are  added  a  few  drops  of  very 
weak  ammonia  sufficient  just  to  turn 
phenol  red  paper  pale  pink.  Subsequent 


treatment  is  similar  to  that  of  the 
Bodian  method.  Silver  methods  are 
excellent  for  following  nerves  in  thin, 
serial  sections,  for  tracing  nonmyeli- 
nated fibers  and  for  small  nerve  end- 
ings. But  large,  proprioceptive  endings 
such  as  occur  in  the  posterior  region  of 
the  knee  joint  capsule  are  best  demon- 
strated by  methylene  blue.  Excellent 
results  may  be  obtained  by  using  0.05% 
methylene  blue  in  normal  saline.  It 
may  be  perfused,  injected  into  the 
joint  cavity  or  pieces  of  capsule  can  be 
removed  and  placed  in  methylene  blue 
at  37°C.  for  10-15  minutes.  They  are  then 
exposed  to  air  for  10-15  minutes,  placed 
in  8%  ammonium  molybdate  in  normal 
saline  for  at  least  1-2  hours  and  then 
dehydrated  rapidly  in  cold  alcohols. 
Whole  mounts  are  made  after  clearing 
in  xylol.  This  method  shows  better  than 
any  other  the  grouping  and  distribution 
of  large  endings,  and  the  preparations 
are  also  satisfactory  for  small  fibers, 
particularly  those  in  vascular  plexuses. 

Artificial  Fever,  influence  on  adrenal  (Bern- 
stein, J.  G.,  Am.  J.  Anat.,  1940,  66, 
177-196).  See  Cramer,  W.,  Fever, 
Heat  Regulation  and  the  Thyroid- 
Adrenal  Apparatus.  London:  Long- 
mans, Green  &  Co.,  1928,  153  pp. 

Asbestos,  see  Lillie  p.  135. 

Ascorbic  Acid.  Colorimetric  method  of 
Lowry,  O.  H.,  Lopez,  J.  A.  and  Bessey, 
O.  A.,  J.  Biol.  Chem.,  1945,  160,  609- 
615  for  blood  serum  in  volumes  of  10 
ix\  up.  Pijoan,  M.  and  Gerjovich,  H.  J., 
Science,  1946,  103,  202-203  advise  cau- 
tion in  use  with  tissues.  See  titrimetric 
technique  of  Click,  D.,  J.  Biol.  Chem., 
1935,  109,  433-436.  See  Vitamin  C. 

Ascorbic  Acid,  see  Vitamin  C. 

Aspirated  Sternal  Marrow,  method  for 
preparing  smears  and  sections  (Gordon, 
H.,  J.  Lab.  &  Clin.  Med.,  1940-41,  26, 
1784-1788). 

Astra  Violet,  see  Leishmania. 

Astrocytes.  These  cells  make  up  one  of  the 
two  divisions  into  which  neuroglia  is 
usually  divided.  They  also  pass  under 
the  names  of  "Classical  neuroglia" 
and  "macroglia."  They  are  star  shaped 
cells  with  processes  radiating  in  all 
directions  some  of  which  form  peri- 
vascular expansions  on  the  surface  of 
small  blood  vessels.  The  most  selective 
methods  for  their  demonstration  usually 
involve  some  form  of  metallic  impregna- 
tion. For  details  see  Chapter  by  Pen- 
field  and  Cone  in  McClung's  Micro- 
scopical Technique,  1950,  p.  407  and 
Lillie's  Histopathologic  Technic,  1948, 
p.  237.  In  this  book  see  Neuroglia. 

Atabrine,  anti-malarial  agent;  fluorescence 
microscopical  localization  of  atabrine 


ATHEROMA 


24 


AUER  BODIES 


in   the   body    (Jailer,   J.   W.,  Science, 
1945,  102,  258-259. 
Atheroma,  see  methods  for  Calcium,  CoUa- 
genic    Fibers,    Elastic    Fibers,    Lipids, 

Atomic'  Weights.  See  G.  E.  F.  Lundell, 
Chairman,  Report  of  Committee  on 
Atomic  Weights.  J.  Am.  Chem.  Soc, 
1949,  71,  1141-1142. 

International  Atomic  Weights 
1949  Revision 

Actinium Ac  227 

Aluminum Al  26.97 

Americium Am  [241] 

Antimony Sb  121.76 

Argon A  39.944 

Arsenic As  74.91 

Astatine At  [210] 

Barium Ba  137.36 

Beryllium Be  9.013 

Bismuth Bi  209.00 

Boron B  10.82 

Bromine Br  79.916 

Cadmium Cd  112.41 

Calcium Ca  40.08 

Carbon C  12.010 

Cerium Ce  140.13 

Cesium Cs  132.91 

Chlorine CI  35.457 

Chromium Cr  52.01 

Cobalt Co  58.94 

Columbium  (see  Niobium) 

Copper Cu  63.54 

Curium Cm  [242] 

Dysprosium Dy  162.46 

Erbium Er  167.2 

Europium Eu  152.0 

Fluorine F  19.00 

Francium Fr  [223] 

Gadolinium Gd  156.9 

GalUum Ga  69.72 

Germanium Ge  72.60 

Gold Au  197.2 

Hafnium Hf  178.6 

Helium He  4.003 

Holmium Ho  164.94 

Hydrogen H  1.0080 

Indium In  114.76 

Iodine I  126.92 

Iridium Ir  193.1 

Iron Fe  55.85 

Krypton Kr  83.7 

Lanthanum La  138.92 

Lead Pb  207.21 

Lithium Li  6.940 

Lutetium Lu  174.99 

Magnesium Mg  24.32 

Manganese Mn  54.93 

Mercury Hg  200.61 

Molybdenum Mo  95.95 

Neodymium Nd  144.27 

Neptunium Np  [237] 

Neon Ne  20.183 

Nickel Ni  58.69 


Niobium Nb  92.91 

Nitrogen N  14.008 

Osmium Os  190.2 

Oxygen O  16.0000 

Palladium Pd  106.7 

Phosphorus P  30.98 

Platinum Pt  195.23 

Plutonium Pu  [239] 

Polonium Po  210 

Potassium K  39.096 

Praseodymium Pr  140.92 

Promethium Pm  [147] 

Protactinium Pa  231 

Radium Ra  226.05 

Radon Rn  222 

Rhenium Re  186.31 

Rhodium Rh  102.91 

Rubidium Rb  85.48 

Ruthenium Ru  101.7 

Samarium 8m  150.43 

Scandium Sc  45. 10 

Selenium Se  78.96 

Silicon Si  28.06 

Silver Ag  107.880 

Sodium Na  22.997 

Strontium Sr  87.63 

Sulfur S  32.066 

Tantalum Ta  180.88 

Technetium Tc  [99] 

Tellurium Te  127.61 

Terbium Tb  159.2 

Thallium Tl  204.39 

Thorium Th  232. 12 

Thulium Tm  169.4 

Tin Sn  118.70 

Titanium Ti  47.90 

Tungsten  (see  Wolfram) 

Uranium U  238.07 

Vanadium V  50.95 

Wolfram W  183.92 

Xenon Xe  131.3 

Ytterbium Yb  173.04 

Yttrium Y  88.92 

Zinc Zn  65.38 

Zirconium Zr  91.22 

A  value  given  in  brackets  denotes  the  mass  number 
of  the  most  stable  known  isotope. 

Auditory  System,  see  Ear. 

Auer  Bodies. — Written  by  G.  Adolph  Acker- 
man,  Hinsman  Hall,  Ohio  State  Uni- 
versity, Columbus  10,  Ohio.  May  24, 
1951 — Auer  bodies  are  rod-like  struc- 
tures occasionally  present  in  the  cyto- 
plasm of  leukemic  myelocytes  and 
monocytes;  they  are  less  than  1  micron 
in  width  and  vary  in  length  from  1-10 
micra.  Although  the  number  of  Auer 
bodies  observed  within  a  single  leukemic 
cell  is  frequently  limited  to  one  or  two, 
more  than  fifteen  have  been  observed. 
Auer  bodies  are  formed  bj'  the  coas- 
cervation  of  the  cytoplasmic  granules 
in  young  leukemic  cells.  As  the  cell 
matures,  the  Auer  bodies  undergo  dis- 
solution    into     cytoplasmic     granules. 


AUER  BODIES 


AUER  BODIES 


1.  Unstained  moisl  preparations:  A 
drop  of  blood  or  bone  marrow  is  placed 
on  a  clean  cover  slip,  inverted  on  a  slide 
and  rimmed  with  vaseline  or  suitable 
substance  to  prevent  drying.  The  prepa- 
ration is  examined  using  the  phase 
microscope.  Auer  bodies  appear  as  dis- 
crete cytoplasmic  structures. 

2.  Supravital  technic:  The  supravital 
technic  is  carried  out  in  the  same  man- 
ner as  indicated  above  (No.  1.)  for  un- 
stained moist  preparations  with  the 
exception  of  the  fact  that  the  slide  has 
been  previously  coated  with  neutral 
red  and  janus  green.  Preparations  may 
be  examined  with  the  bright  field,  dark 
field  or  phase  microscope.  Under  the 
bright  field  microscope,  the  Auer  bodies 
appear  as  discrete  structures  which 
stain  with  neutral  red.  The  color  of  the 
Auer  bodies  varies  from  a  deep  red 
(acidic  reaction)  in  young  cells  to  tan 
(less  acidic  reaction)  in  the  more  mature 
leukemic  cells.  The  staining  reaction 
of  the  Auer  bodies  with  neutral  red 
closely  parallels  that  of  cytoplasmic 
granules.  Auer  bodies  stain  blue-purple 
in  supravital  preparations  prepared 
with  brilliant  cresyl  blue  (Dameshek, 
W.  Arch.  Int.  Med.,  1932,  50,  579). 

3.  Romanowsky  stains:  The  Roman - 
owsky  dyes  may  be  used  to  demonstrate 
Auer  bodies.  Due  to  the  solubility  of  the 
azurophilic  component  of  the  Auer 
body  in  water  and  alcohol,  this  technic 
is  not  as  satisfactory  as  the  supravital 
and  the  unstained  moist  technics. 

4.  Swlan  black  B  technic:  Dry  blood 
and  bone  marrow  films  are  fixed  with 
either  formalin  vapor  or  with  a  1%  solu- 
tion of  formalin  in  95%  ethyl  alcohol 
for  20-60  seconds.  The  dried-fi.xed  films 
are  placed  in  a  saturated  solution  of 
Sudan  black  B  (National  Aniline  Co., 
New  York)  in  70%  ethyl  alcohol  for 
20-30  minutes.  The  solution  of  Sudan 
black  must  be  prepared  at  least  2  weeks 
prior  to  using.  The  blood  films  are  then 
dipped  in  70%  ethyl  alcohol  and  rinsed 
in  50%  ethyl  alcohol  and  washed  in  dis- 
tilled water,  dried  and  mounted  with 
"permount."  Auer  bodies  are  sudano- 
philic  and  appear  black  or  black-brown. 
This  technic  is  an  excellent  method  for 
demonstrating  Auer  bodies. 

5.  Periodic  Acid-Schiff  {PAS)  re- 
action: Dried  films  are  fi.xed  with  forma- 
lin vapor  for  20-60  seconds.  The  fixed 
preparations  are  immersed  in  an 
aqueous  0.5%  solution  of  periodic  acid 
for  5  minutes,  washed  thoroughly  with 
distilled  water  and  immersed  in  Schiff's 
reagent  10  minutes,  followed  by  3  suc- 
cessive changes  in  sulfurous  acid,  3-5 
minutes  each,  washed  in  distilled  water, 
dried  and  mounted  with  "permount." 


Auer  bodies  are  PAS-positive  appear- 
ing a  moderate  pink. 

6.  Toluidine  blue  ami  Thionin:  Dried 
films  may  be  fixed  with  formalin  vapor, 
4%  aqueous  basic  lead  acetate  or  other 
suitable  fixatives.  Preparations  are 
washed,  dried  and  immersed  in  a  dilute 
solution  (0.2-0.5%)  toluidine  blue  or 
thionin,  washed  in  distilled  water,  dried 
and  mounted  with  "permount."  Auer 
bodies  stain  lavender  following  the  use 
of  this  technic. 

7.  Plasinal  reaction:  Dried  films  are 
fixed  with  formalin  vapor  for  20-60 
seconds,  washed  in  distilled  water  and 
placed  in  a  1%  aqueous  solution  of 
mercuric  chloride  for  5  minutes,  washed 
in  distilled  water  and  placed  in  Schiff's 
reagent;  the  films  are  placed  in  three 
successive  changes  of  sulfurous  acid, 
washed  in  distilled  water,  dried  and 
mounted  with  "permount."  In  staining 
control  preparations,  the  same  pro- 
cedure is  followed  with  the  exception 
of  the  fact  that  distilled  water  is  sub- 
stituted for  mercuric  chloride.  If  the 
control  exhibits  a  positive  reaction,  the 
length  of  exposure  to  Schiff's  reagent 
must  be  reduced.  Following  the  plasmal 
reaction,  the  Auer  bodies  appear  a  very 
pale  pink,  and  may  best  be  identified  by 
means  of  the  phase  microscope  followed 
by  bright  field  observation  of  the 
stained  bodies. 

8.  M.  nmli  reaction:  Dried  blood  and 
bone  marrow  films  may  be  fi.xed  either 
with  formalin  vapor  or  1%  formalin  in 
95%  ethj'l  alcohol,  washed  with  dis- 
tilled water  and  dried.  The  fi.xed  films 
are  immersed  in  a  mixture  containing 
equal  parts  of  1%  alkaline  solution  of 
alpha-napthol  and  1%  aqueous  solution 
of  p-aminodimeth_ylanaline  (Eastman 
Kodak,  P2147)  for  1-5  minutes,  washed 
in  distilled  water  and  mounted  in  water. 
Auer  bodies  stain  deep  blue  following 
the  M.  nadi  reaction.  Preparations  fade 
rapidly. 

9.  Sato  and,  Sekeya  Peroxidase  Technic: 
Dried  blood  and  bone  marrow  films  are 
placed  in  a  0.5%  aqueous  solution  of 
copper  sulfate  for  30  seconds,  washed 
briefly  in  50%  ethyl  alcohol  and  placed 
in  a  benzidine-hydrogen  peroxide  mix- 
ture (0.1  gm.  benzidine  in  100  ml.  dis- 
tilled water  and  2  drops  of  3%  hydrogen 
peroxide)  for  2  minutes,  washed  with 
distilled  water,  dried  and  mounted  with 
"permount".  Counterstaining  with  1% 
aqueous  safranin  is  optional.  Auer 
bodies  are  peroxidase-positive  and  ap- 
pear blue  or  j'ellow. 

Auer  bodies  are  soluble  in  water, 
saline  and  many  organic  solvents  and 
are  readily  digested  bj'  ribonuclease. 
Digestion  by  ribonuclease  may  be  pre- 


AUERBACH'S  PLEXUS 


26 


AZURE  DYES 


vented  by  immersing  the  Auer  bodies  in 
an  aqueous  solution  of  lanthanum  ace- 
tate for  2  hours  prior  to  exposure  to  the 
enzyme.  Wright's  stain  may  be  used  to 
demonstrate  the  basophilic  properties 
of  the  Auer  rods.  Auer  bodies  are  not 
demonstrable  following  the  Bauer- 
Feulgen,  Lugol's  aqueous  iodine,  Go- 
mori  acid  and  alkaline  phosphatase  and 
lipase  reactions. 

Auerbach's  Plexus.  Supravital  staining 
by  injecting  methylene  blue  through 
the  aorta  is  apparently  improved  by 
addition  of  hydrogen  acceptors.  Scha- 
badasch,  A.,  Bull.  d'Hist.  AppL,  1936, 
13,  1-28,  72-89,  137-151  advises  0.03- 
0.05  gm.  per  liter  of  p-amidophenol, 
0.02-0.07  of  p-phenylenediamine,  0.02- 
0.05  of  pyrocatechine  or  0.05-0.9  of 
resorcin.  The  methylene  blue  must  be 
of  high  quality  and  free  from  metallic 
salts.  He  obtained  in  5  min.  intense 
staining  of  the  plexus  in  a  cat  which  re- 
ceived 1200  cc.  of  fluid  of  the  following 
concentration  :  aq.  dest.,  1000  cc. ;  NaCl, 
7  gm. ;  resorcin,  0.15  gm.  and  methylene 
blue  (chlorzink  free,  Hoecht)  0.2  gm. 

Aural  Smears,  see  Ear  Smears. 

Auramin  (CI,  655) — canary  yellow,  pyok- 
tanin  yellow,  pyoktaninum  aureum — 
This  basic  diphenyl  methane  dye  may 
be  of  use  in  fluorescence  microscopy. 
Auramine  O  is  Commission  Certified. 
It  is  one  of  the  substances  which  arrests 
mitosis  in  the  metaphase,  an  action 
which  has  been  carefully  studied  by 
Ludford,  R.  J.,  Arch.  f.  Exper.  Zellf., 
1935-6,  18,  411-441.  Tubercle  bacilli 
treated  with  auramin  give  golden  yellow 
fluorescence  (Hageman,  P.  K.  H., 
Munch.  Med.  Woch.,  1938,  85,  1066). 

Aurantia  (CI,  12) — imperial  yellow — An 
acid  nitro  dye  employed  in  Champy- 
Kull  method.  Aurantia  is  explosive 
and  it  can  cause  severe  dermatitis. 
All  those  using  it  should  be  warned  of 
the  danger. 

Aurin  or  rosolic  acid  (CI,  724). 

Autoradiography,    see   Radioactive   Isotopes. 

Axenfeld  Reaction.  Giroud  (A.,  Proto- 
plasma,  1929,  7,  72-98) :  Add  to  prepara- 
tion few  drops  of  formic  acid,  then  3-4 
drops  0.1%  aq.  gold  chloride  and  heat 
slowly.  A  rose  color  appears,  then  vio- 
let. Lison  (p.  129)  says  that  the  reaction 
is  very  little  characteristic  of  proteins 
since  analogous  reactions  are  given  by 
creatine,  urea,  uric  acid,  glycogen.  Its 
employment  is  contraindicated. 

Axis  Cylinders.  These  are  the  cytoplasmic 
cores  of  the  nerve  fibers.  Mitochondria 
can  often  be  seen  in  them  unstained 
and  after  supravital  coloration  with 
Janus  Green.  The  best  method  to 
demonstrate  mitochondria  in  fixed  tis- 
sues is  Anilin  Fuchsin  Methyl  Green 


after  Regaud  fixation.  Silver  methods 
show  Neurofibrils.  Alzheimer's  modi- 
fication of  Mann's  eosin-methyl  blue 
method  is  recommended  to  show  early 
degenerative  changes .  De  Renyi ,  G .  S . , 
Cowdry's  Special  Cytology,  1932,  3, 
1370-1402  has  fully  described  use  of 
methods  of  microdissection.  See  Ama- 
ranth. 

Azan  Stain,  see  Heidenhain's. 

Azidine  Blue  3B,  see  Trypan  Blue. 

Azidine  Scarlet  R,  see  Vital  Red. 

Azins.  Azin  dyes  are  those  formed  from 
phenazin.  Two  benzene  rings  are  joined 
by  2  nitrogen  atoms  forming  a  third  ring. 
Examples  :  amethyst  violet,  azocarmine 
G,  indulin  alcohol  and  water  soluble, 
Magdala  red,  neutral  red,  neutral  violet, 
nigrosin  water  soluble,  phenosafranin, 
safranin  O. 

Azo  Blue  (CI,  463) — benzoin  blue  R  and 
direct  violet  B — This  acid  dis-azo  dye 
is  one  of  those  microinjected  vitally 
into  cytoplasm  against  the  nucleus  of 
amebae  to  ascertain  whether  the  nucleus 
can  be  vitally  colored  (Monn6,  L., 
Proc.  Soc.  Exp.  Biol.  &  Med.,  1934-35, 
32,  1197-1199).  Butt,  E.  M.,  Bonynge, 
C.  W.  and  Joyce,  R.  L.,  J.  Inf.  Dis., 
1936,  58,  5-9  report  that  azo  blue  can 
be  substituted  for  India  ink  in  the  nega- 
tive demonstration  of  capsular  zones 
about  hemolytic  streptococci. 

Azo-Bordeaux,  see  Bordeaux  Red. 

Azocarmine  G  (CI,  828) — azocarmine  GX, 
rosazine,  rosindulin  GXF — This  basic 
azin  dye  is  used  in  place  of  acid  fuchsin 
in  Heidenhain's  Azan  stain.  Azocar- 
mine B  is  CI,  829. 

Azocarmine  GX,  see  Azocarmine  G. 

Azo  Dyes.  Chromophore — N=N — uniting 
naphthalene  or  benzene  rings.  See 
Mono-azo,  Dis-azo  and  Poly-azo  Dyes. 
Lipophilic  substitutions  in,  and  slight 
curative  effect  claimed  in  tuberculosis 
and  leprosy  (Bergmann,  E.,  Haskelberg, 
L.  and  Bergmann,  F.,  J.  Am.  Chem.  Soc, 
1941,63,2243. 

Azo-fuchsin.  Seven  are  recognized  in  the 
Colour  Index.  Acid  mono-azo  dyes  re- 
lated to  Bordeaux  red  and  orange  G. 

Azolitmin,  see  Hydrogen  Ion  Indicators. 

Azophloxine  G.  A. — Fast  crimson  GR., 
Amidonaphthol   G— an   acid   Azo   dye. 

Azo  Reaction  for  phenols.  Formation  of 
azo  color  by  action  of  diazonium  salt  on 
tissue  phenol  (Lison,  p.  140).  See 
Lison,  L.,  C.  Rend.  Soc.  de  Biol.,  1933, 
112,  1237-1239). 

Azo  Rubin,  see  Amaranth. 

Azure  Dyes.  These  are  basic  thiazin  stains 
of  great  usefulness.  The  description 
given  by  Conn  (pp.  76-80)  should  be 
consulted.  It  is  here  summarized. 
Azure  I  (Giemsa)  is  a  trade  name  for  a 
secret  preparation  apparently  a  variable 


AZURE  OR  TOLUIDIN  BLUE-EOSIN       27 


BACTERIA 


mixture  of  Azure  A  and  B.  Azure  II 
is  an  intentional  mixture,  in  equal  parts, 
of  Azure  I  and  methylene  blue.  It  is 
the  main  constituent  of  Giemsa's  stain. 

1.  Azure  A  is  asymmetrical  dimethyl 
thionin  and  has  been  Commission  Certi- 
fied for  some  time.  It  is  considered  as 
the  most  important  nuclear  staining 
component  of  polychrome  methylene 
blue  by  MacNeal,  W.  J.,  J.  Inf.  Dis., 
1925,  36,  538-546.  This  dye  has  been 
used  as  a  nuclear  stain  following  eosin 
and  after  phloxine,  see  Phloxine-Azure 
(Ilaynes,  R.,  Stain  Techn.,  1926,  1, 
68-69,  107-111). 

2.  Azure  B  is  the  tri -methyl  deriva- 
tive of  thionin.  It  is  specified  by 
Jordan,  J.  H.  and  Heather,  A.  H.,  Stain 
Techn.,  1929,  4,  121-126  as  a  stain  for 
Negri  bodies.  Roe,  M.  A.,  Lillie,  R.  D. 
and  Wilcox,  A.,  Pub.  Health  Reports, 
1940,  55,  1272-1278  recommend  its  in- 
clusion in  Giemsa's  stain. 

3.  Azure  C  is  mono-methyl  thionin. 
French,  R.  W.,  Stain  Techn.,  1926,  1, 
79  has  described  a  method  for  its  use 
followed  by  Eosin  Y  and  orange  II  in 
staining  sections  of  formalin  fixed  mate- 
rial; but  Haynes,  R.,  Stain  Techn., 
1927,  2,  8-16  doubts  whether  it  is  sig- 
nificantly better  than  Azure  A  and 
thionin. 

Azure  or  Toluidin  Blue-Eosin. — Written 
by  Dr.  R.  D.  Lillie,  Division  of  Pathol- 
ogy, National  Institute  of  Health, 
Bethesda,  Md.  May  8,  1950.— Prepare 
a  1/1000  solution  of  Azure  A,  Azure  C 
or  Thionin  (85-90%  dye  content)  or  a 
0.15%  solution  of  Toluidin  Blue  (60% 
dye  content)  and  a  1/1000  solution  of 
Eosin  Y  or  Eosin  B  (a  redder  shade). 

Bring  paraffin  sections  to  water  as 
usual,  including  an  iodine,  thiosulfate 
sequence  for  material  fixed  with  mer- 
curic chloride  mixtures.  Stain  1  hour  in 
stock  azure,  Toluidin  Blue  or  Thionin 
4  cc,  stock  eosin  4  cc,  Mcllvaine  buffer 
of  desired  pH  level  2  cc,  acetone  5  cc. 
and  distilled  water  25  cc.  Rinse,  de- 
hydrate with  acetone,  clear  with  50:50 
acetone  xylene  and  2  changes  of  xylene, 
mount  in  synthetic  resin  (polystyrene, 
permount,  clarite,  HSR  or  the  like). 
The  procedure  has  been  used  for  techni- 
con  staining.  The  stain  mixture  is  made 
fresh  weekly  in  this  case. 

For  neutral  formalin  or  Orth  fixa- 
tions, use  pH  4.0-4.5,  for  acid  formalin 
pH  4.5  is  better,  for  Zenker  or  Helly 
pH  5.0,  for  Bouin  pH  5.5-6.0  (less  satis- 
factory than  others  as  the  picric  acid 
seems  to  interfere),  for  Carnoy,  alcohol 
and  similar  fluids  4.8-5.5. 

Color  values  are  deep  blue  for  nuclei, 
bacteria,  and  rickettsiae,  violet  to 
purple  for  mast  cell  granules  and  carti- 


lage matrix,  lighter  blues  for  cyto- 
plasms, varying  pinks  for  muscle,  ery- 
throcytes, fibrin,  necrotic  cytoplasm 
and  oxyphil  inclusion  bodies. 

This  has  been  modified  somewhat 
from  Histopathologic  Technic,  Lillie 
(Blakiston,  Phila.,  1948),  which  gives 
further  details. 

Azure  II  Eosin  and  Hematoxylin  (Maximow, 
A.,  J.  Inf.  Dis.,  1924,  34,  549),  gives, 
in  addition  to  coloration  of  chromatin 
by  hematoxylin,  a  granule  stain  some- 
thing like  that  provided  by  Giemsa's 
method.  Make  up:  (1)  azure  II  eosin: 
A.  eosin  water  soluble  yellowish,  0.5 
gm.;  aq.  dest.,  500  cc.  B.  azure  II, 
0.5  gm.;  aq.  dest.,  500  cc.  Mix  10  cc. 
A,  100  cc.  aq.  dest.,  and  10  cc.  B.  (2) 
hematoxylin  (Delafield's)  1-2  drops, 
aq.  dest.,  100  cc.  to  make  a  pale  violet 
solution. 

Formalin-Zenker  fixed  tissues  (sec- 
tions, smears,  spreads)  are  stained  up- 
right in  hematoxylin  washed  in  aq.  dest. 
and  counter-stained  with  azure  II  eosin 
24  hrs.  each.  Transfer  to  95%  ale, 
differentiate  and  dehydrate  in  abs.  (2 
changes);  clear  in  xylol  and  mount  in 
balsam.  Care  must  be  taken  to  use 
pure  aq.  dest.  The  proportions  of  A 
and  B  can  be  varied  slightly  to  suit  the 
tissue.  In  order  to  hold  the  azure  II 
eosin  colors  the  balsam  should  be  neu- 
tral or  nearly  neutral  as  when  Giemsa's 
stain  is  employed. 

To  appreciate  the  beauty  of  this 
method  see  numerous  colored  illustra- 
tions marked  "ZF,  Ham,  EAz"of  agreat 
many  organs  and  tissues  by  Maximow,  A. 
Section  on  Bindegewebe  und  Blutbil- 
dende  Gemebe  in  Mollendorff's  Handb. 
d.  mikr.  Anat.  d.  Menschen,  1927,  2, 
(1)  232-583. 

Babes'  see  Anilin-Safranin. 

Bacillus  Typhosus,  technique  for  dark  field 
study  of  flagella  (Pijper,  A.,  J.  Path. 
&  Bact.,  1938,  47,  1-17).  See  9  plates 
by  author. 

Bacteria.  Methods  employed  for  the  micro- 
scopic identification  of  bacteria  and  to 
demonstrate  their  structure  are  legion. 
The  Committee  on  Bacteriological  Tech- 
nique of  the  Society  of  American  Bac- 
teriologists has  prepared  a  useful  leaflet 
entitled  "Staining  Procedures"  pub- 
lished in  Geneva,  N.  Y.  (Fifth  Edition 
1934)  to  supplement  their  "Manual  of 
Methods  for  the  Pure  Culture  of  Bac- 
teria" (1923).  A  detailed  account  of 
Bacteriological  methods  by  H.  J.  Conn, 
F.  B.  Mallory  and  Frederic  Parker,  Jr., 
is  contained  in  McClung's  Microscopical 
technique  to  which  reference  should 
also  be  made.  Bergey's  "Manual  of 
Determinative  Bacteriology"  (Balti- 
more: Williams  &  Wilkins,  1948),  which 


BACTERIA.     BIOCHEMICAL  TESTS       28 


BACTERIA.    MEDIA 


is  a  key  to  identification  of  bacteria,  is 
often  useful. 

Motility,  agglutination,  lysis  under 
influence  of  bacteriophage,  ingestion  by 
leucocytes  and  many  other  phenomena 
can  best  be  observed  by  examination  of 
living  bacteria  by  direct  illumination  or 
in  the  darkfield.  Smears,  usually  fixed 
by  heat,  are,  however,  most  often  used. 
A  choice  must  be  made  from  many  well 
known  stains  including :  Anilin  Gentian 
Violet,  Loeffler's  Methylene  Blue, 
Giemsa,  Gram  and  Carbol  Fuchsin. 
Others  are  best  listed  under  the  particu- 
lar structures  to  be  demonstrated 
Spores,  Flagella,  Capsules.  In  some 
cases  search  for  bacteria  in  Milk,  Soil, 
Cheese,  Sputum,  etc.  is  indicated. 
When  bacteria  are  so  few  in  number  that 
they  may  be  missed,  or  large  numbers 
are  required  separated  from  the  tissues 
for  chemical  analysis,  Concentration 
methods  may  be  useful.  Accurate 
localization  of  bacteria  requires  their 
study  in  sections.  See  Giemsa's  stain, 
Gram-Weigert  stain,  Goodpasture's 
stain  (!MacCallum's  modification),  Mal- 
lory's  Phloxine-Methylene  blue  and 
Acid  Fast  Bacilli.  The  darkfield 
examination  of  stained  preparations  is 
said  to  be  an  advantage  (Goosemann, 
C,  J.  Lab.  and  Clin.  Med.,  1935-36, 
21,  421-424).  Appearance  when  viewed 
at  high  magnification  with  electron 
microscope  (Mudd,  S.,  Polevitsky,  K., 
and  Anderson,  T.  F.,  Arch.  Path.,  1942, 
34,  199-207).  See  Fluorescence  micros- 
copy, Negative  Strains,  Dead  bacteria, 
Tubercle  bacilli,  Leprosy  bacilli,  Mito- 
chondria and  Bacteria  in  same  cells, 
Rickettsia,  Gonococcus,  Diphtheria  Ba- 
cilli, Bacterium  Tularense,  Bacterium 
Monocytogenes. 
Bacteria.  Biochemical  Tests.  Given  in 
greater  detail  by  H.  R.  Livesay  in 
Simmons  and  Gentzkow,  387-389. 

1.  Indicators  of  pH.  Incorporate  in 
basic  culture  of  medium  measured 
amounts  of  0.02%  aq.  phenol  red,  0.04% 
aq.  bromcresol  purple,  or  0.1%  aq. 
bromthymol  blue.  Their  pH  ranges 
and  colors  are  given  under  Hydrogen 
Ion  Indicators. 

2.  Indoltest.  Use  Bohme's  reagents. 
To  5  day  culture  in  1%  aq.  peptone  add 
1  cc.  ether,  shake  and  settle.  Let  1  cc. 
of  following  run  down  inside  tube :  p- 
dimethylaminobenzaldehyde,  4  gm.; 
95%  ethyl  alcohol,  380  cc;  cone,  hydro- 
chloric acid,  80  cc.  If  after  1  min.  no 
color  develops  add  1  cc.  sat.  aq.  po- 
tassium persulf ate .  Positive ,  pale  pink 
to  deep  magenta. 

3.  Ilosvay's  Nitrate  reduction.  To 
5  day  culture  at  37°C.  in  broth  +  0.1% 
HNO3  add  1  cc.  of  following  solution. 


Dissolve  1  gm.  a-naphthylamine  in  22 
cc.  aq.  dest.  Filter  and  add  180  cc.  of 
dilute  acetic  acid  (sp.  gr.  1.04).  Then 
1  cc.  of  sulfanilic  acid  (0.5  gm.  in  150  cc. 
dilute  acetic  acid).  Positive,  pink, 
red  or  maroon;  negative,  no  color. 

4.  Ammonia.  To  5  day  peptone 
water  culture  add  0.5  cc.  Nessler's  Re- 
agent. Positive,  brown;  negative,  faint 
yellow. 

5.  Hydrogen  sulfide.  Inoculate  or- 
ganisms on  lead  acetate  agar  made  by 
sterilizing  extract  broth  containing  4% 
peptone  +  2.5%  agar  and  equal  volume 
0.1%  aq.  basic  lead  acetate.  Positive, 
brown  or  black;  negative,  no  color. 

6.  Reductase.  To  a  24  hr.  broth  cul- 
ture add  1  drop  1%  aq.  methylene  blue. 
Incubate  at  37°C.  Positive,  complete 
decolorization;  weakly  positive,  green 
color;  negative,  no  decolorization. 

7.  Catalase.  Pour  1  cc.  HjOj  over 
24  hr.  agar  slant  culture  incubated  at 
37''C.  holding  tube  on  incline.  Posi- 
tive, gas  bubbles;  negative,  none. 

8.  Methyl  red.  To  4  day  culture  in 
glucose  phosphate  medium  at  37°C. 
add  5  drops  0.04%  methyl  red  in  60% 
alcohol.  Positive,  red;  negative,  yel- 
low. 

9.  Voges-Proskauer.  To  4  day  cul- 
ture in  glucose  phosphate  medium  at 
37°C.  add  5  cc.  10%  aq.  KOH.  After 
18-24  hrs.  positive,  pink  fluorescence; 
negative,  no  color. 

10.  Oxidase.  To  surface  of  colony 
add  loop  full  or  1-2  cc.  fresh  1%  aq. 
dimethylparaphenylenediamine  hydro- 
chloride. Positive,  color  change  from 
pink  to  maroon  to  black. 

Bacteria  Flagella.  Electron  microscopic 
technique  reveals  the  ultrastructure  of 
bacterial  flagella  as  composite  struc- 
tures made  up  of  a  central  coiled  trypsin 
resistant  filament  and  a  peripheral 
sheath  probably  nonresistant  (De 
Robertis,  E.  and  Franchi,  C.  M.,  Exp. 
Cell  Res.,  1951,  2,  295-298). 

Bacteria.  Media.  The  following  are  brief 
summaries  of  culture  media  as  described 
by  H.  R.  Livesay  in  Simmons  and 
Gentzkow,  388-403. 

(Glucose  phosphate.  Witte  or  Difco 
proteose  peptone,  0.5  gm.;  K2HPO4,  0.5 
gm.;  glucose,  0.5  gm.;  aq.  dest.,  100 
cc;  pH  7.5.) 

Meat  extract  broth  (routine).  Add 
to  1000  cc  aq.  dest.,  beef  extract,  3  gm. ; 
peptone,  10  gm.;  sodium  chloride,  5  gm. 
Dissolve  by  stirring  with  heat  (water 
bath  65°C.).  Make  up  weight  loss  with 
aq.  dest.  and  make  pH  7.2-7.4.  Boil 
over  flame,  cool  to  25°C.,  again  make 
up  weight  loss,  clarify  and  check  pH. 
Place  in  flasks  or  tubes,  autoclave  15 
lbs.,  15  min. 


BACTERIA.    MEDIA 


29 


BACTERIA.     MEDIA 


Meat  extract  broth  (for  water  anal- 
ysis). As  above,  using  beef  extract, 
3  gm.;  peptone,  5  gm.;  aq.  dest.  1000 
cc.  pH  6.4-7. 

Meat  extract  agar  (routine).  Dis- 
solve 20-30  gms.  powdered  agar  in 
1000  cc.  meat  extract  broth  stirring 
over  flame  and  titrate  to  pH  7.4.  Cool 
to  50°C.,  add  stirred  eggs,  heat  gently 
till  egg  material  is  firmly  coagulated. 
Remove  coagulum  with  fine  wire  mesh 
strainer,  filter  through  cotton,  make 
up  filtrate  to  original  weight  with  aq. 
dest.  and  make  pH  7.2-7.4.  Tubes  or 
flasks.     Autoclave  15  lbs.,  15  min. 

Meat  extract  agar  (for  water  anal- 
ysis). Add  15  gm.  best  quality  agar 
to  1000  cc.  of  above  meat  extract  agar 
and  make  pH  6.4-7. 

Meat  infusion  broth.  Mix  500  gms. 
ground  fat-free  beef,  or  veal  round,  in 
1000  cc.  aq.  dest  in  ice  box  18-24  hrs. 
Heat  over  small  flame  in  Arnold  steri- 
liier,  1  hr.,  add  5  gm.  sodium  chloride 
and  10  gm.  peptone.  Dissolve  our 
flame,  filter,  add  aq.  dest.  to  1000  cc, 
titrate  to  pH  7.4,  tube  or  flask,  and 
autoclave  15  lbs.,  15  min. 

Meat  infusion  agar.  Add  20  gm.  agar 
to  1000  cc.  Meat  infusion  broth  and 
continue  as  in  making  meat  extract 
agar,  pH  to  7.4. 

Gelatin,  Nutrient.  Add  120  gm. 
gelatin  to  1000  cc.  meat  extract  broth 
in  double  boiler,  weigh,  dissolve  by 
heat,  titrate  to  pH  7.4  and  add  aq.  dest. 
to  make  original  weight.  Add  1  egg 
clarified  by  mixture  with  small  volume 
of  aq.  dest.,  heat  slowly  till  egg  is 
coagulated,  filter  through  cotton  and 
sterilize  filtrate  in  10  cc.  portions  in 
tubes  in  Arnold  20  min.  3  successive 
days. 

Huntoon's  hormone.  Add  500  gm. 
fresh  finely  ground  beef  heart,  10  gm. 
peptone,  5  gm.  sodium  chloride,  1 
whole  egg,  20  gm.  agar  (Bacto)  to  1000 
cc.  aq.  dest.  in  enamel-ware  dish,  heat 
and  stir  constantly.  Make  pH  8. 
Cover,  place  in  Arnold  1  hr.  Remove, 
separate  clot  from  sides  and  return  to 
Arnold  IJ-hr.  Remove,  let  stand  in- 
clined, room  temperature,  10  min. 
Remove  clear  part  and  filter  it  through 
fine  wire  sieve  into  tall  cylinders.  Al- 
low to  stand  15-20  min.  and  skim  off 
fat.  Clear  further  by  passing  through 
glass,  or  asbestos  wool,  or  by  centrifug- 
ing.  Tube  in  10  cc.  lots,  sterilize  in 
Arnold  30  min.  on  3  successive  days. 

Glucose  agar.  Add  10  gm.  glucose 
to  1000  cc.  meat  extract  or  meat  in- 
fusion agar  and  dissolve  by  slowly 
heating.  Adjust  pH  to  that  of  original 
agar.  Pour  in  tubes,  or  flasks,  and 
sterilize  in  Arnold  3  successive  days. 


Blood  agar.  Add  5-10%  of  sterile 
defibrinated  blood  (preferably  horse) 
to  meat  infusion  or  meat  extract  agar 
which  first  has  been  melted  and  cooled 
to  45°C.  Pour  into  plates  or  into  tubes 
and  slant,  then  incubate  to  prove 
sterilit}'. 

Chocolate  blood  agar.  Add  5%  of 
sterile  defibrinated  blood  to  meat  in- 
fusion agar  at  50-55°C.  mix  avoiding 
bubbles,  slowly  increase  to  75°C.  Pour 
into  plates,  or  into  tubes  and  slant, 
then  incubate  to  prove  sterility. 

Serum  agar.  Add  100  cc.  sterile 
normal  horse  serum  to  1000  cc.  melted 
meat  infusion  agar,  pour  into  plates, 
or  tubes,  and  slant,  then  incubate  to 
prove  sterility. 

Liver  infusion  agar  (for  Br.  abortus). 
Mix  500  gm.  ground  beef  liver  with  500 
gm.  aq.  dest.  in  cool  place  24  hrs.,  strain 
through  cheesecloth  and  collect  500  gm. 
resulting  infusion  (1) .  Add  20  gm.  agar 
and  500  gm.  aq.  dest.  and  autoclave 
15  lbs.  pressure,  30  min.  (2).  Dissolve 
10  gm.  peptone  and  5  gm.  sodium  chlo- 
ride in  No.  1,  beef  infusion  (3).  Add 
aq.  dest  to  2  and  3  combined  to  make 
up  weight  lost  by  evaporation,  adjust 
pH  to  7  and  cool  to  50°C.  Add  10  gm. 
egg  albumin  (first  dissolved  in  10  cc. 
aq.  dest.),  heat  to  100°C.  IJ-hrs.,  strain 
through  fine  wire  sieve,  filter  through 
clean  glass  wool,  adjust  pH  to  7,  tube 
in  15  cc.  lots  and  autoclave  at  15  lbs., 
30  min.  When  required  melt  and  pour 
plates,  or  make  slants. 

Trypagar.  Put  500  gm.  fat  free, 
finely  ground  beef  or  veal  ground  in 
1000  cc.  aq.  dest.  in  container  adding 
20%  aq.  NaOH  until  slightly  alkaline  to 
litmus.  Cook  at  75°C.,  5  min.,  cool  to 
37°C.  and  add  0.5  gm.  trypsin  (Bacto). 
Incubate  37.5°C.,  5  hrs.  If  trypaniza- 
tion  is  complete  5  cc.  liquid  -|-  5  cc.  in 
NaOH  +  1  cc.  dil.  aq.  CuSO^  will  give 
pink  color.  If  not  incubate  again  1  hr. 
and  re-test.  When  complete,  slightly 
acidify  with  glacial  acetic  acid,  slowly 
bring  to  boiling  point  and  hold  15  min. 
Filter  through  wet  paper,  add  20  gm. 
agar  and  5  gm.  sodium  chloride.  Dis- 
solve agar  with  heat,  clear  with  an  egg, 
adjust  to  pH  7.6  and  autoclave  15  lbs., 
15  min. 

Veal  infusion  brain  broth  (for  Strep- 
tococci and  anaerobes).  With  large 
bore  pipette  insert  about  50  cc.  ground 
fresh  calf  brain  in  bottom  200  x  25  mm. 
tube  and  add  35  cc.  veal  infusion  broth 
pH  7.6.  Autoclave  15  lbs.,  20  min. 
Remove  10  cc.  test  reaction,  pH  7.4- 
7.6  being  satisfactory,  if  a  change  has 
taken  place  adjust  to  pH  7.6  and  esti- 
mate from  titration  of  this  10  cc. 
amount  needed  to  bring  to  this  figure 


BACTERIA.    MEDIA 


30 


BACTERIA.    MEDIA 


bulk  and  correct  the  whole.  Fill  tubes 
with  similar  amounts,  then  incubate  at 
37°C.  to  prove  sterility. 

Robertson's  (for  Anaerobes).  To 
500  gm.  ground  fat,  fascia  and  blood 
vessel-free  fresh  beef  heart,  add  10  gm. 
peptone  and  1000  cc.  aq.  dest.,  bring  to 
boil  and  adjust  to  pH  8.  Continue  sim- 
mering 1^-hrs.  and  again  adjust  reac- 
tion. Separate  broth  from  meat,  place 
former  in  flasks,  autoclave  15  lbs.,  15 
min.  Dry  meat  on  filter  paper  in  oven 
56°C.  48  hrs.  Place  desired  amounts 
of  meat  plus  10  cc.  broth  in  tubes. 
Autoclave  cool,  remove  broth  and  re- 
titrate.  Adjust  to  desired  pH,  finally 
fill  tubes  same  quantity  meat  and  broth 
and  autoclave  15  lbs.  30  min.  Final 
pH  should  be  7.4-7.6. 

Calcium  carbonate  broth  (for  Pneu- 
mococci).  Dissolve  10  gm.  glucose  in 
1000  cc.  meat  infusion  broth  by  heating, 
make  pH  7.6.  Place  clean  marble  chips 
(CaCOs)  in  bottom  of  tubes  pour  in 
broth,  sterilize  in  Arnold  15  min.  3  suc- 
cessive days. 

Blood  culture  (Kracke).  Add  500 
gm.  finely  ground  fat-free  beef  heart 
muscle  to  1000  cc.  aq.  dest.  in  ice  box 
over  night.  Press  through  4  layers 
gauze  cloth,  heat  extract  to  boiling, 
filter  through  small  mesh  wire  gauze. 
Add  250  gm.  ground  beef  brain  to  500 
cc,  treat  in  same  way  but  do  not  filter 
this  suspension.  Mix  800  cc.  extract, 
110  cc.  suspension,  1  gm.  sodium  citrate, 
10  gm.  dextrose  (Bacto),  10  gm.  pro- 
teose peptone  (Difco),  2  gm.  disodium 
phosphate  and  4  gm.  sodium  chloride 
and  place  50  cc.  lots  in  tubes  or  flasks. 
Autoclave  15  lbs.,  20  min. 

Bile  (For  typhoid  group).  Combine 
900  cc.  ox  bile,  100  cc.  glycerol  and  20 
gm.  peptone  by  heating  over  water 
bath.  Pour  in  bottles  or  small  flasks 
and  autoclave. 

Brilliant  green  lactose  bile.  Dissolve 
10  gm.  peptone  and  10  gm.  lactose  in 
500  cc.  aq.  dest.  add  200  cc.  fresh  ox  bile, 
or  20  gm.  dehydrated  ox  bile  dissolved 
in  200  cc.  aq.  dest.,  the  latter  having 
pH  7.4.  Add  13.3  cc.  0.1%  aq.  brilliant 
green  and  aq.  dest.  to  make  1000  cc. 
Filter  through  cotton,  place  in  fer- 
mentation tubes,  sterilize  after  which 
pH  by  potentiometer  (not  colorimeter) 
should  be  7.1-7.4. 

Le vine's  eosin  methylene  blue  agar 
(Standard  for  water  analysis).  Dis- 
solve 10  gm.  peptone,  2  gm.  K2HPO4, 
and  15  gm.  agar  in  1000  cc.  aq.  dest.  by 
boiling.  Add  aq.  dest.  to  compensate 
for  evaporation  and  distribute  meas- 
ured amounts  in  flasks.  Immediately 
before  use  to  each  100  cc.  add  5  cc.  20% 
aq.  lactose  (sterile),  2  cc.  2%  aq.  eosin 


and  2  cc.  0.5%  aq.  methylene  blue. 
Mix,  pour  into  Petri  plates,  harden  and 
incubate  to  prove  sterility. 

Endo's  (Standard  for  water  analysis). 
Add  5  gm.  beef  extract,  10  gm.  peptone 
and  30  gm.  agar  to  1000  cc.  aq.  dest.  in 
container  and  weigh.  Boil  till  dis- 
solved, restore  lost  weight  with  aq. 
dest.,  place  in  vessel  with  straight  walls 
and  autoclave  15  lbs.,  15  min.  Let  agar 
harden,  remove  en  masse  to  clean  paper, 
cut  away  and  discard  debris  from  bot- 
tom. Melt  clean  agar,  make  pH  7.8- 
8.2,  pour  in  100  cc.  or  larger  amounts 
and  autoclave  15  lbs.,  15  min.  To  each 
100  cc.  of  this  stock  agar  add  5  cc.  20% 
aq.  C.P.  lactose  (sterilized  by  fractional 
method),  0.5  cc.  10%  basic  fuchsin  in 
95%  alcohol  (from  filtrate  of  super- 
natant fluid  having  let  stand  24  hrs.). 
Mix  carefully,  pour  into  sterile  Petri 
dishes,  let  agar  set  at  room  tempera- 
ture and  harden  over  night  in  incubator. 
Check  sterility. 

Agar,  sodium  desoxycholate.  Dis- 
solve 10  gm.  peptone  in  1  Kg.  water, 
bring  to  pH  7.3-7.5  with  sodium  hy- 
droxide, boil  few  minutes  and  pass 
through  filter  paper.  Add  12-17  gm. 
agar.  After  soaking  15  min,,  melt  by 
boiling.  To  each  1000  cc.  add  6  cc. 
1  N  sodium  hydroxide  plus  ferric  am- 
monium citrate,  2  gm.  dipotassium 
phosphate  and  1  gm.  sodium  desoxy- 
cholate. Titrate  with  phenol  red  in- 
dicator to  pH  7.3-7.5  and  add  3  cc. 
1%  aq.  neutral  red.  Sterilize  in  flowing 
steam  only  sufficient  to  kill  vegetable 
cells  (15  min.  enough  for  tubes  with 
10-15  cc.  medium). 

Selenite-F  enrichment.  Use  mono- 
sodium  and  disodium  phosphates  in 
exact  proportions  which  experiment 
shows  that  with  particular  lot  of  pep- 
tone and  brand  of  sodium  selenite  will 
give  pH  7.0-7.1.  Dissolve  with  heat 
10  gm.  these  phosphates  (anhydrous), 
4  gm.  this  sodium  hydrogen  selenite 
(anhydrous),  5  gm.  peptone,  4  gm.  lac- 
tose in  aq.  dest.  to  make  1  Kg.     Boil. 

Russell's  double  sugar  agar.  Mix 
1000  cc.  melted  meat  extract  agar,  40  cc. 
25%  aq.  lactose  (sterile)  and  4  cc.  25% 
aq.  glucose  (sterile)  and  adjust  to  pH 
7.2.  Add  50  cc.  0.02%  aq.  phenol  red, 
filter  if  necessary,  tube  and  autoclave 
8  lbs.,  25  min.  Slant  with  deep  butt. 
Check  reaction  of  medium  with  known 
E.  coli  and  E.  iyphosa. 

Simmons'  citrate  agar.  Dissolve  5 
gm.  sodium  chloride,  0.2  gm.  MgS04, 
1.0  gm.  (NH4)H2P04,  2.28  gm.  sodium 
citrate  (2H2O)  in  1000  cc.  aq.  dest.  and 
add  20  gm.  agar.  Heat  to  dissolve 
agar,  make  pH  7.2,  and  add  10  cc.  1.5% 
alcoholic     bromthymol     blue.       Filter 


BACTERIA.    MEDIA 


31 


BACTERIA.    MEDIA 


through  cotton,  tube,  autoclave  15 
lbs.,  15  min.  Slant  with  deep  butt. 
Check  reaction  of  medium  with  known, 
E.  coll.,  A.  aerogenes,  S.  schottmuelleri 
and  E.  typhosa. 

Jordan's  tartarate  agar.  Dissolve  by 
heating  20  gm.  agar,  10  gm.  peptone, 
10  gm.  sodium  potassium  tartarate, 
5  gm.  sodium  chloride  in  1000  cc.  aq. 
dest.  Adjust  pH  to  7.4  and  add  12  cc. 
0.2%  alcoholic  phenol  red.  Tube  in 
10  cc.  lots,  autoclave  15  lbs.,  15  imn. 
Check  reaction  of  medium  with  known 
S.  aertrycke,  S.  enteritidis,  S.  paratyphi 
and  S.  schottmuelleri. 

Lead  acetate  agar  (for  H2S  test).  To 
100  cc.  sterile  meat  extract  agar  add 
following  sterile  Seitz-filtered  solutions: 
4  cc.  25%  aq.  glucose,  4  cc.  25%  aq. 
lactose  and  1  cc.  0.5%  aq.  lead  acetate. 
Tube  aseptically  and  incubate  to  prove 
sterility.  Check  reaction  of  medium 
with  known  S.  paratyphi  and  S.  schott- 
muelleri. 

Dieudonne's  alkaline  blood  agar  (for 
Vibrio  comma).  Make  700  cc.  nutrient 
agar  and  neutralize  to  litmus  about  pH 
6.8.  Ivlix  150  cc.  defibrinated  beef  blood 
and  150  cc.  in  1  AT  KOH  and  steam  in 
Arnold  30  min.  Add  this  to  blood  agar 
in  proportion  of  3  to  7.  Pour  Petri 
plates,  let  harden  uncovered  (but  pro- 
tected by  paper)  placing  strips  sterile 
filter  paper  between  dish  and  protection 
to  take  up  ammonia  and  moisture. 
Incubate  15  hrs.  at  37°C.  before  use. 

Carbohydrate  broth  (for  fermenta- 
tion tests).  Inoculate  1000  cc.  infusion 
broth  with  active  E.  coli  and  incubate 
18  hrs.  at  37.5°C.  Boil  few  minutes  to 
kill  organisms.  Put  in  large  mortar 
20-30  gms.  purified  talc.  While  grind- 
ing add  broth  and  thoroughly  mix. 
Pass  through  wet  filter  paper  till  clear. 
Titrate  and  adjust  pH  to  7.3.  Weigh 
broth  and  add  1%  of  desired  ferment- 
able substances  dissolved  in  a  little  hot 
water.  Then  add  45  cc.  0.04%  aq. 
bromcresol  purple  per  liter.  Sterilize 
in  Arnold  20  min.  on  3  successive  days, 
or  autoclave  7  lbs.,  10  min. 

Lactose  broth  (Standard  for  water 
analysis).  Add  0.5%  lactose  to  nu- 
trient extract  broth  and  adjust  reac- 
tion to  pH  6.4-7.  Autoclave  15  lbs. 
15  min.  restricting  total  heat  exposure 
to  30  min. 

Clark  and  Lubs.  Dissolve  5  lbs. 
each  of  peptone,  dextrose  and  dipo- 
tassium  phosphate  in  1000  cc.  aq.  dest. 
using  heat.  Filter  through  paper,  add 
water  lost,  tube  in  10  cc.  lots,  sterilize 
in  Arnold  20  min.,  3  successive  days. 

Bendick's  saccharose  peptone-water 
(for  Vibrio  comma).  Add  1  gm.  an- 
hydrous sodium  carbonate  to  1000  cc. 


peptone  solution  neutralized  to  phenol- 
phthalein.  Boil,  filter  and  to  filtrate 
add  5  gm.  saccharose  +  5  cc.  sat.  phe- 
nolphthalein  in  50%  alcohol.  Tube 
10  cc.  lots,  sterilize  in  Arnold  15  min., 
3  successive  days. 

Dunham's  peptone  solution  (for  indol 
test).  Dissolve  10  gms.  bacto-tryp- 
tone  (Difco)  +  5  gm.  sodium  chloride 
in  1000  cc.  aq.  dest.  with  heat.  Make 
pH  7.6  and  filter  if  necessary,  tube  10  cc. 
lots,  autoclave  15  lbs.  15  min. 

Nitrate  broth  (for  nitrate  reduction 
test).  Dissolve  10  gms.  peptone  +  1 
gm.  potassium  nitrate  (nitrite-free)  in 
1000  cc.  aq.  dest.  (ammonia-free)  with 
heat.  Filter  through  paper,  tube  10  cc. 
portions  and  sterilize  in  Arnold  20  min., 
3  successive  days. 

Bromcresol  purple  milk.  Remove 
cream  and  heat  remainder  in  cylinder 
in  Arnold  20  min.  Again  skim  off  fat 
and  to  each  liter  remaining  add  40 
cc.  0.04%  aq.  bromcresol  purple.  Tube 
10  cc.  lots,  sterilize  in  Arnold,  20  min., 
3  successive  days.  Prove  sterility  by 
incubation. 

Loeffler's  (for  C.  diphtheriae) .  Col- 
lect beef  blood  in  large  glass  vessels 
and  let  clot  without  moving.  Loosen 
clot  from  wall  with  sterile  glass  rod  and 
place  in  refrigerator.  To  3  parts  clear 
serum  removed  by  pipette  add  1  part 
meat  infusion  broth  containing  1% 
glucose  pH  6.8-7.  Mix  by  stirring,  in- 
spissate on  slant  raising  temperature 
gently  to  approximately  85°C.  Main- 
tain temperature  till  coagulated  firmly. 
Sterilize  in  Arnold  20  min.,  3  successive 
days,  paraffinize  cotton  plugs  and  test 
sterility. 

Hiss'  serum-water  (for  fermentation 
tests).  Add  3  parts  aq.  dest.  to  1  part 
clear  serum,  mix,  heat  in  Arnold  15  min. 
Add  1%  desired  carbohydrate  dissolved 
in  small  quantity  hot  aq.  dest.  Add 
50  cc.  0.02%  aq.  bromthymol  blue  to 
each  1000  cc,  tube,  sterilize  in  Arnold 
20  min.,  3  successive  days  and  prove 
sterility  by  incubation. 

Glycerol  agar.  Add  30  cc.  pure 
glycerol  to  1000  cc.  melted  infusion 
agar,  adjust  to  pH  7.2,  tube,  autoclave 
15  lbs.,  15  min.  and  slant. 

Petroff's  (for  M.  tuberculosis).  In- 
fuse 500  gm.  beef  or  veal  in  500  cc. 
15%  aq.  glycerol.  After  24  hrs.  put  in 
sterile  press  and  collect  extract  in 
sterile  vessel.  Place  washed  eggs  in 
70%  alcohol,  10  min.  Take  out  with 
sterile  tongs,  flame  and  remove  con- 
tents to  sterile  vessel.  To  2  parts  egg 
add  1  part  meat  extract.  Add  1%  alco- 
holic gentian  violet  to  make  final  con- 
centration 1:10,000.  Mix  and  continue 
as  with  Loeffler's  medium. 


BACTERIA.     MEDIA 


32 


BALSAM 


Cystine  blood  agar  (for  P.  tularensis) . 
To  1000  cc.  beef  or  veal  infusion  broth 
add  15  gm.  agar,  10  gm.  peptone  and 
5  gm.  sodium  chloride.  Autoclave 
15  lbs.,  15  min.  Before  use  add  1  gm. 
cystine  (or  cystine  hydrochloride)  and 
10  gm.  glucose.  Dissolve  by  heating  in 
Arnold  and  sterilize  30  min.  Cool  to 
50°C.  add  50  cc'  sterile  horse  blood, 
tube  aseptically  in  10  cc.  lots,  slant  and 
incubate  to  prove  sterility. 

Noguchi's  leptospira  medium.  Com- 
bine sterile  80°C.  0.9%  aq.  sodium 
chloride,  100  cc.  fresh  rabbit  serum, 
100  cc.  2%  aq.  agar  (melted  pH  7.4)  and 
10-20  cc.  rabbit  hemoglobin  (1  part 
blood,  3  parts  aq.  dest.).  Tube  asepti- 
cally in  10  cc.  lots.  Incubate  to  prove 
sterility. 

Tryptone  glucose  extract  milk  agar. 
Combine  15  gm.  agar,  3  gm.  beef  ex- 
tract, 5  gm.  tryptone,  1  gm.  glucose  and 
1000  cc.  aq.  dest.  by  boiling  over  free 
flame.  Make  up  volume  lost  with  aq. 
dest.,  adjust  to  pH  7,  add  10  cc.  skim 
milk,  place  measured  volumes  in  flasks 
or  tubes  and  autoclave  15  lbs.,  15  min. 

Tellurite  (for  C.  diphtheriae).  Melt 
infusion  agar,  or  0.2%  dextrose  agar 
and  cool  to  50°C.  To  each  10  cc.  add 
1  cc.  citrated,  or  defibrinated,  blood 
-f  1  cc.  sterile  2%  aq.  potassium  tellu- 
rite, mix  and  pour  into  Petri  dishes. 

Bismuth  sulfite  agar  (Wilson  and 
Blair  for  E.  iyphosa).  Mix  20  gm.  agar, 
5  gm.  beef  extract  and  10  gm.  peptone 
in  sufficient  hot  aq.  dest.  to  make 
1000  cc.  Dissolve  by  autoclaving  15 
min.  Store  in  refrigerator.  (A).  Dis- 
solve 6  gms.  bismuth  ammonium  citrate 
scales  in  50  cc.  boiling  aq.  dest.  (1),  20 
gm.  anhydrous  sodium  sulfite  in  100  cc. 
boiling  aq.  dest.  (2),  and  10  gms.  dex- 
trose in  50  cc.  boiling  aq.  dest.  (3). 
Mix  1  and  2,  boil  and  add  10  gms.  an- 
hydrous disodium  phosphate  while 
boiling.  Cool  and  add  3.  Add  water 
to  restore  lost  weight.  Store  in  closely 
stoppered  pyrex  container  in  dark  at 
room  temperature  (B).  Dissolve  1  gm. 
ferric  citrate  in  100  cc.  aq.  dest.  using 
heat  and  add  12.5  cc.  1%  aq.  brilliant 
green.  Store  likewise  in  pyrex  vessel 
in  dark.  With  1000  cc.  hot  (A)  thor- 
oughlj'  mix  200  cc.  (B)  and  45  cc.  (C). 
Immediatel}'  pour  into  porous-top  petri 
dishes  each  15-20  cc.  After  2  hrs.  at 
room  temperature  store  in  refrigerator 
and  use  within  4  days. 

Chocolate  agar  (for  Neisseria). 
Grind  strips  lean  meat  of  5-6  beef 
hearts.  To  each  500  gm.  add  1000  cc. 
tap  water,  infuse  in  refrigerator  over 
night,  strain  and  press  through  course 
gauze.  Add  10  gm.  proteose  peptone 
No.  3  (Difco)  per  liter,  heat  to  50°C. 


1  hr.  and  boil  10  min.  Strain  through 
gauze,  dissolve  5  gm.  sodium  chloride 
per  liter  and  titrate  to  pH  7.6.  Boil 
lightly  10  min.  pour  off  measured  quan- 
tities in  flasks,  autoclave  15  lbs.,  15 
min.  Cool  to  60°C.,  add  5%  human  or 
horse  blood,  heat  slowly  on  water  bath 
to  80-85''C.  rotating  to  get  even  mix- 
ture.    Cool   to  55°C.  and  plate. 

Bacterial  Pigments.  These  cannot  be  meas- 
ured microscopically  but  a  method  has 
been  devised  for  doing  so  with  spectro- 
photometer and  photoelectric  colorim- 
eter (Stahly,  G.  L.,  Sesler,  C.  L.  and 
Erode,  W.  R.,  J.  Bact.,  1942,  43, 
149-154). 

Bacterial  Polysaccharides.  Solutions  of 
reduced  bases  and  leuco  bases  of  penta- 
and  hexa-methyl  triamino-triphenyl- 
methane  and  tetramethyl  diamino- 
triphenylmethane  and  certain  other 
triphenylmethanes  react  with  staphylo- 
coccal polysaccharides  and  may  be 
useful  in  their  detection  (Chapman, 
G.  H.  and  Lieb,  C.  W.,  Stain  Techn., 
1937,  12,  15-20). 

Bacteriophage  Localization  by  Electron 
Microscopy,  see  Hennessen,  W.,  Zeit. 
f.  wis.  Mikr.,   1951,  60,   172-180. 

Bacteriostatic  Titration  of  Dyes.  (Reed, 
M.  V.  and  Genung,  E.  F.,  Stain  Techn., 
1934,  9,  117-128). 

Bacterium  Monocytogenes.  Intravenous 
injections  of  this  organism  in  rabbits 
produce  a  marked  increase  in  the  num- 
ber of  circulating  monocytes  and  there- 
fore provide  an  important  experimental 
method  (Murray,  E.  G.  D.,  Webb,  R.  H. 
and  Swan,  M.  B.  R.,  J.  Path,  and  Bact., 
1926,  29,  407-439). 

Bacterium  Tularense  in  sections.  Add  10 
cc.  sat.  aq.  nile  blue  sulphate  and  6  cc. 
l%aq.  safranin  to60  cc.aq.  dest.  Stain 
sections  over  night.  Wash  quickly, 
dehydrate  in  alcohols,  clear  in  xylol 
and  mount  (Foshay,  L.,  J.  Lab.  &  Clin. 
Med.,  1931,  17,  193-195). 

Balances.  Ordinary  balances  need  no  de- 
scription but  for  weighing  very  small 
amounts  special  balances  are  essential. 
See  review  of  literature  by  Gorbach, 
G.,  Mikrochemie,  1936,  20,  254-336.  The 
torsion  balances  of  Roller-Smith  Co., 
Bethlehem,  Pa.,  are  sensitive  to  ap- 
proximately 2  Atgm.  The  quartz  fiber 
balances  are  still  more  sensitive.  See 
Click,  pp.  189-191. 

Balantidium.  Celloidin  embedding  and  sec- 
tioning (Scott,  M.  J.,  J.  Morph.  & 
Physiol.,  1927,  49,  417). 

Balsam  for  mounting  sections  is  usually 
satisfactory  as  purchased.  To  make, 
mix  equal  parts  dry  balsam  and  sodium 
bicarbonate  and  grind  in  mortar.  Add 
sufficient  xylol  to  make  clear  solution. 


BARBER  AND  KOMP 


33 


BASOPHILE  LEUCOCYTE 


After  few  days  filter  and  heat  gently 
(avoiding  flame)  to  bring  to  suitable 
consistency.  The  best  mounting  me- 
dium when  neutrality  is  essential  is 
Clarite  or  the  cedar  oil  used  for  oil 
immersion  objectives.  The  latter  sets 
more  slowly  than  balsam  and  it  is  ordi- 
narily not  necessary  to  employ  it.  See 
Mounting  Media. 

Barber  and  Komp  thick  film  for  malaria 
Plasmodia  (Barber,  M.  A.  and  Komp, 
W.  H.  W.,  Pub.  Health  Rep.,  1929,  44, 
2330)  is  described  by  Craig,  p.  290-291 
as  the  most  used  and  satisfactory  of  the 
thick  film  techniques.  His  account  of 
the  method  abbreviated:  Place  large 
drop  blood  on  clean  slide  and  smear  with 
needle  over  area  about  half  size  of  that 
usually  covered  by  a  thin  blood  smear. 
Dry  in  incubator  at  37°C.,  1-1|  hrs. 
Stain  in  1  part  good  Giemsa  and  6  parts 
neutral  or  slightly  alkaline  aq.  dest., 
about  30  min.  Partly  decolorize  in 
aq.  dest.  5  min.  (If  films  have  back- 
ground deep  blue  and  leucocytes  almost 
black  they  may  be  worthless;  but  leav- 
ing in  aq.  dest.  longer  may  help). 
Drain    thoroughly,    dry    and    examine. 

Barium,  spectrographic  analysis  of,  in  retina 
(Scott,  G.  H.  and  Canaga,  B.,  Jr.,  Proc. 
Soc.  E.xp.  Biol.  &  Med.,  1940,  44,  555- 
556).  Barium  chloride  and  formalin  are 
advised  as  fi.xative  for  Bile  Components. 
Barium  sulphate  emulsion  injections  are 
recommended  by  Woollard,  H.  H.  and 
Weddell,  G.,  J.  Anat.,  1934-35,  69,  25-37 
to  demonstrate  arterial  vascular 
patterns.  The  emulsion  should  be  of 
such  consistency  that  it  cannot  easily  be 
forced  beyond  the  small  arterioles  by  a 
pressure  of  1 .5  atmospheres.  Fi.x  tissues 
by  hypodermic  injection  of  formalin 
and  subsequent  immersion  in  it.  Take 
x-ray  photographs  of  the  radiopaque 
barium.  See  Cretin's  Test  for  Calcium 
and    barium   under   Calcium   5. 

Bartholomew,  see  Gram  Stains  Mechanism. 

Bartonella,  Try  Giemsa's  Stain  for  sections. 

Basal  Bodies  of  cilia  (Wallace,  H.  M., 
Science,  1931,  74,  369-370).  Fix  in 
Zenker  (containing  acetic)  or  in  Zenker- 
formalin  (90  cc.  Zenkers  +  10  cc.  10% 
formalin).  Mount  paraffin  sections  5/x 
thick.  After  very  light  staining  with 
hematoxylin  and  thorough  washing  in 
tap  water  dip  in  0.5%  aq.  eosin 
(Grubler's  wasserlich.  If  not  available, 
use  Eosin  Y.)  ^  min.  and  wash  quickly 
in  large  volumes  of  water.  Make  up 
stain  by  adding  9  parts  sat.  aq.  methyl 
violet  (Grubler's  6B  only.  If  not 
available,  use  CC.  which  is  2B.)  to  1 
part  abs.  alcohol  33  cc. ;  aniline  oil  9  cc. 
-f  methyl  violet  in  excess.  Stain  is 
best  3-8  days  after  mixing  but  the  two 
solutions  can  be  kept  separately.     After 


staining  sections  for  2  hrs.  wash  well  in 
tap  water,  treat  with  Lugol's  iodine 
10-15  min.  and  repeat  the  washing. 
Blot  with  filter  paper.  Differentiate  in  1 
part  aniline  oil  -f  2  parts  xylol.  Wash 
in  several  changes  of  xylol  and  mount  in 
balsam.  Basal  bodies  deep  purple, 
nuclei  dark  blue.  Good  also  for  intra- 
cellular bacteria  and  fibrin. 

Basement  Membranes  of  epithelia.  Suggest 
Mallory-Heidenhain's  connective  tissue 
stain  as  modified  by  Schleicher,  E.  M., 
Am.  J.  Clin.  Path.",  1943,  7,  3&^39  and 
Schiff  Reagent  method  as  detailed  by 
McManus,  J.  F.,  Am.  J.  Path.,  1948, 
24,  643. 

Basic  Brown,  G,  GX,  or  GXP,  see  Bismark 
Brown  Y. 

Basic  Dyes,  see  Staining. 

Basic  Fuchsin — anilin  red,  basic  rubin,  and 
magenta  (CI  676  or  677) — Commission 
Certified.  The  tri-amino  tri-phenyl 
methane  dyes  bearing  this  name  are 
mixtures  of  pararosanilin,  rosanilin  and 
magenta  II  in  varying  proportions. 
They  are  employed  for  a  great  many 
purposes.  Basic  fuchsin  in  a  cytologi- 
cal  technique  for  anterior  pituitary  is 
described  bj'  Faire,  W.  R.,  and  Wolfe, 
J.  M.,  Anat.  Rec,  1944,  90,  311-314. 
New  fuchsin  (CI  678)  is  a  different  com- 
pound. It  is  the  deepest  in  color  of  4 
dyes  and  pararosanilin  is  the  lightest. 

Basic  Lead  Acetate  used  as  fixative  for 
Tissue  Basophiles. 

Basic  Rubin,  see  Basic  Fuchsin. 

Basket  Cell  Arborizations,  technique  for, 
Cajal,  S.  R.,  Trav.  Lab.  Rech.  Biol. 
Univ.  de  Madrid,  1926,  24,  217. 

Basophila  Erythroblasts,  see  Erythrocytes, 
developmental  series. 

Basophile  Leucocyte  (mast-leucocyte,  blood 
mast  cell).  Least  numerous  granular 
leucocyte ;  percentage  about  0-1 ; 
slightly  smaller  (8-10m)  than  other 
types;  nucleus  spherical  or  slightly 
lobated,  faintly  staining  and  centrally 
placed;  specific  granules  only  slightly 
refractile,  basophilic,  large,  variable  and 
less  numerous  than  in  other  types; 
function  unknown.  This  cell  is  difficult 
to  study  in  fresh  preparations  of  pe- 
ripheral blood  because  it  is  so  scarce. 
Smears  colored  by  the  usual  methods 
(Giemsa,  Wright,  etc.)  are  satisfactory. 
The  basophilic  granules  appear  to  be 
particularly  soluble  in  water.  Doan 
and  Reinhart  (C.  A.  and  11.  L.,  Am.  J. 
Clin.  Path.,  1941,  11,  Tech.  Suppl.  5, 
1-39,  with  beautiful  colored  plates) 
recommend  supravital  staining  with 
neutral  red  and  janus  green.  There  is 
difference  of  opinion  as  to  whether  the 
oxidase  and  peroxidase  reactions  are 
positive   (Michels,  N.  A.  in  Downey's 


BASOPHILIC 


34 


BERBERIAN'S  METHOD 


Hematology,  1938,  1,  235-372).  See 
Tissue  Basophiles. 

Basophilic,  see  Staining. 

Bauer  Negative  and  positive  substances, 
for  method  see  Glycogen. 

Beams,  see  Ultracentrifuges. 

Bell's  Method  for  fixing  and  staining  of  fats 
as  described  by  the  Bensleys  (p.  114). 
Intracellular  fats  are  mobilized  by  heat 
to  form  droplets  which  are  chromated 
and  later  stained.  Consequently  the 
preparations  show  these  fats,  in  addition 
to  other  microscopically  visible  fat,  but 
not  their  true  distribution  in  the  cells. 
Fix  for  10  days  at  45-50 °C.  in  10%  aq. 
potassium  bichromate  100  cc.  +  5  cc. 
acetic  acid.  Imbed  and  make  paraffin 
sections  as  usual.  Pass  them  down  to 
absolute  alcohol.  Stain  with  freshly 
prepared  Sudan  III  10  min.  Rinse  off 
in  50%  alcohol  and  pass  to  water  to  arrest 
action  of  alcohol.  Counter-stain  with 
Delafield's  Hematoxylin.  Wash  in 
water,  differentiate  in  acid  alcohol,  wash 
in  water  again  and  mount  in  Glycerine 
Jelly. 

Benda's  Method  of  crystal  violet  and 
alizarin  for  mitochondria.  Fix  in  Flem- 
ming's  fluid  8  days  (see  Flemming's 
Fluid).  Wash  in  water  1  hr.  Then 
half  pyroligneous  acid  and  1%  chromic 
acid,  24  hrs.  2%  potassium  bichromate, 
24  hrs.  Wash  in  running  water  24  hrs. 
Dehydrate,  clear,  imbed  in  paraffin  and 
cut  sections  at  4ju.  Pass  down  to  water 
and  mordant  in  4%  iron  alum  24  hrs. 
Stain  amber-colored  sol.  sodium  sulpha- 
lizarinate  made  by  adding  sat.  ale.  sol. 
to  water,  24  hrs.  Blot  with  filter  paper 
and  color  in  equal  parts  crystal  violet 
sol.  and  aq.  dest.  (The  sol.  consists  of 
sat.  crystal  violet  in  70%  ale.  1  part, 
ale.  1  part  and  anilin  water  2  parts.) 
Warm  until  vapor  arises  and  allow  to 
cool  5  min.  Blot  and  immerse  in  30% 
acetic  acid  1  min.  Blot,  plunge  in  abs. 
ale.  until  but  little  more  stain  is  ex- 
tracted, clear  in  xylol  and  mount  in 
balsam.  The  mitochondria  are  stained 
deep  violet  in  a  rose  background.  The 
colors  are  more  lasting  than  in  Altmann 
preparations.  This  is  one  of  the  classi- 
cal techniques  of  histology  but  it  is 
difficult.  For  colored  illustrations  see 
Duesberg,  J.,  Arch.  f.  Zellforsch.,  1910, 
4,  602-671. 

Benda's  stain  for  fat  necrosis.  See  Fisch- 
ler's  modification. 

Bensley's  Neutral  Safranin.  For  mitochon- 
dria and  secretion  antecedents  especially 
in  the  pancreas.  Fix  in  2.5%  aq.  potas- 
sium bichromate,  100  cc;  mercuric 
chloride,  5  gms.  24  hrs.  Wash,  dehy- 
drate, clear,  imbed  and  section.  To 
prepare  stain  slowly  add  sat.  aq.  acid 
violet  to  sat.  aq.  safranin  0  in  a  flask 


until  ppt.  ceases  when  a  drop  of  mixture 
on  filter  paper  gives  not  an  outside  red 
rim  of  safranin  but  a  solid  neutral  color. 
Filter.  The  filtrate  should  be  as  nearly 
as  possible  colorless.  Dry  ppt.  on  filter 
paper  and  make  of  it  a  sat.  sol.  in  abso- 
lute alcohol.  Pass  sections  through  2 
changes  each  of  toluol  and  absolute 
alcohol,  then  down  through  lower  alco- 
hols to  aq.  dest.  (Bleach  chrome  and 
osmium  fixed  tissues  in  permanganate 
and  oxalic  acid,  as  described  under 
Anilin  Fuchsin  Methyl  Green  and 
mercury  fixed  ones  in  Lugol's  solution, 
10  sec.  finally  washing  in  aq.  dest.) 
Dilute  alcoholic  neutral  safranin  with 
equal  volume  aq.  dest.  and  stain  5min.- 
2  hrs.  Quickly  blot  with  filter  paper. 
Plunge  into  acetone  and  immediately 
pass  to  toluol  without  draining.  Exa- 
mine and  if  further  differentiation  is 
needed  treat  with  oil  of  cloves.  If  this 
is  not  enough  rinse  in  abs.  ale,  flood 
momentarily  with  95%  ale.  and  pass 
back  through  absolute  to  toluol.  Wash 
in  2  changes  toluol  and  mount  in  balsam. 
This  is  a  difficult  method  but  the  results 
are  worth  it.  (see  Bensley,  R.  R., 
Am.  J.  Anat.,  1911,  12,  297-388). 

Benzamine  Blue  3B,  see  Trypan  Blue. 

Benzene-Azo-a-Naphthylamine.  A  mono- 
azo  dye  used  by  Carter,  J.  S.,  J.  Exp. 
Zool.,  1933,  65,  159-179  as  a  vital  stain 
for  Stenostomum. 

Benzo  Blue  SB,  see  Trypan  Blue. 

Benzo  New  Blue  2B,  see  Dianil  Blue  2R. 

Benzo  Sky  Blue,  see  Niagara  Blue  4B. 

Benzoazurine  G  (CI,  502),  a  direct  dis-azo 
dye  of  light  fastness  4  sometimes 
polychromatic  (nuclei  red,  cytoplasm 
blue  to  blue-violet).  Applied  after 
treating  blue-green  algae  with  copper 
sulphate,  spores  orange  red,  vegetative 
cells  dark  blue  or  violet  (Emig,  p.  41). 

Benzene  ring  compounds  distinguished  from 
pyrrols  by  Nitro  Reaction. 

Benzoin  Blue  R,  see  Azo  Blue. 

Benzopurpurin  4B  (CI,  448) — cotton  red, 
diamin  red,  dianil  red.  Sultan  and 
direct  red,  all  4B — An  acid  dis-azo  dye 
no  longer  used. 

Benzoyl  Perioxide  treatment  is  recom- 
mended by  McClung,  Microscopical 
Technique,  1950,  p.  71  for  revival  of 
staining  capacity  of  old  smears  and 
spreads. 

Benzyl  Benzoate  is  employed  in  the  Spalte- 
holz  Method  of  clearing. 

Benzyl  Violet.  Conn  (p.  132)  states  that 
this  term  relates  to  a  group  of  violets 
which  are  pararosanilins,  some  acid  and 
some  basic,  with  benzyl  substitution  in 
one  or  more  amino  groups. 

Berberian's  Method.  Berberian,  D.  A., 
Arch.  Dermat.  &  Syphil.,  1937,  36,  1171- 
1175,  has  developed  a  method  for  stain- 


BERBERINE  SULPHATE 


35 


BILE 


ing  fungi  in  epidermal  scales  and  hair, 
which  differentiates  epithelial  cells, 
blood  cells,  bacteria  and  'mosaic 
fungi'.  The  following  account  was 
written  by  D.  A.  Berberian,  American 
University  of  Beirut,  Beirut,  Lebanon, 
June  22,  1946: 

Fix  small  pieces  of  scales  or  hair  on  a 
slide  with  50%  aq.  glacial  acetic  acid 
by  drying  in  an  incubator.  Defat, 
clear,  hydrate,  and  wash  off  the  acid  as 
follows:  Cover  the  preparation  with 
ether  2-3  times,  20-30  sec.  each;  flood 
twice  with  absolute  acetone,  30-60  sec. 
each;  and  then  flood  consecutively  with 
absolute,  95,  70  and  50  per  cent  alcohol. 
Stain  for  3-5  min.  with  Martinotti's 
solution  (aq.  dest.,  75  cc;  lithium  car- 
bonate, 0.5  gm.;  and  toluidine  blue, 
1  gm.  After  the  stain  dissolves,  add 
20  cc.  glycerin  and  5  cc.  95%  alcohol). 
Wash  gently  in  water  and  differentiate 
with  0.5%  acetic  acid.  Dehydration  is 
best  carried  out  by  3-4  changes  of 
absolute  acetone  kept  2-3  minutes  each 
time.  Pass  through  xylol  and  mount 
in  Euparol  or  any  other  neutral  mount- 
ing agent.  Success  of  preparation 
depends  largely  on  proper  differentia- 
tion, dehydration  and  de-acidification. 
See  Fungi. 

Berberine  Sulphate.  An  alkaloid  used  as  a 
fiuorochrome  for  malarial  parasites 
(Metcalf,  R.  L.  and  Patton,  R.  L., 
Stain  Techn.,  1944,  19,  11-22). 

Bergamot  Oil  is  sometimes  used  for  clearing 
because  it  will  mix  with  95%  alcohol. 

Berlin  Blue  is  another  name  for  Prussian 
Blue  (a  metallic  pigment).  It  is  em- 
ployed for  microchemical  detection  of 
Iron.  Kremer,  Zeit.  f.  wiss.  mikr., 
1938,  54,  429-432  suggests  proceeding  as 
follows:  Fix  in  absolute  alcohol.  De- 
paraffinize  10/x  sections.  Bleach  in  3-5% 
H2O2  3-5  days.  Wash  carefully  in  aq. 
dest.  Quickly  darken  in  (NH4)2S. 
Transfer  to  K  ferrocyanide  and  HCl. 
Iron  gives  blue  color. 

Curiously  enough  when  injection  of 
blood  vessels  is  demanded  this  mineral 
pigment  is  usually  called  for  as  Berlin 
blue.  Thus  the  Bensleys  (p.  153)  give 
directions  for  making  up  Tandler's 
Berlin  blue  gelatin.  Soak  and  melt  5 
gms.  pure  gelatin  in  100  cc.  aq.  dest. 
Add  sufficient  Berlin  blue  to  give  desired 
color  and  then  5-6  gms.  potassium  iodide 
and  a  crystal  of  thymol  as  a  preservative. 
Inject  this  mass,  which  is  fluid  above 
17°C.  Fix  tissues  in  5%  formalin  which 
preserves  it  even  through  decalcifica- 
tion. 

Beryllium.  In  various  forms  and  dosages 
in  production  of  osteogenic  sarcomata 
in   rabbits    (Hoagland,   M.   B.,   Grier, 


R.  S.,  and  Hood,  M.  B.,  Cancer  Rea., 
1950,  10,  629-635. 

Best's  Carmine.  Griibler's  carminum  ru- 
brum  optimum,  or  some  other  good 
carmine,  2  gm. ;  potassium  carbonate, 
1  gm.;  potassium  chloride,  5  gm.;  aq. 
dest.,  60  cc.  Boil  gently  until  color 
darkens,  cool  and  add  20  cc.  cone,  am- 
monia. Allow  to  ripen  24  hrs.  This 
is  stock  solution.  Used  to  stain  Glyco- 
gen. See  Bensley,  C.  M.,  Stain  Tech., 
1939,  14,  47-52. 

Beta  Particles,  influence  in  making  radio- 
autographs,  see  McClung's  Microscopi- 
cal Technique,  1950,  p.  707. 

Beyer  Brown,  a  diazo  dye,  stains  in  aq.  or 
alcoholic  solution  like  a  good  Ehrlich's 
hematoxylin  (H.  G.  Cannan,  J.  Roy. 
Micr.  Soc,  1941,  61,  88-94). 

Biaxial  Bodies,  see  McClung's  Microscopical 
Technique,  1950,  p.  605. 

Bichromate-Chromic-Osmic  mixture,  see 
Champy's  Fixative. 

Blebrich  Scarlet,  water  soluble  (CI,  280) — 
croceine  scarlet,  double  scarlet  BSF. 
Ponceau  B,  scarlet  B  or  EC — An  acia 
dis-azo  dye  much  used  in  histology. 
See  Bowie. 

Blebrich  Scarlet  and  Picro-Anilin  Blue, 
as  a  differential  stain  for  connective 
tissue  and  muscle  (Lillie,  R.  D.,  Arch. 
Path.,  1940,  29,  705).  Deparaffinize 
sections  of  material  fixed  in  formalin, 
Zenker's  or  Orth's  fluid.  Stain  for  5 
min.  in  following:  Dissolve  1  gm. 
hematoxylin  in  95%  ale.  and  4  cc.  29% 
aq.  FeCla  in  95  cc.  aq.  dest.  -f-  1  cc. 
cone,  hydrochloric  acid.  Mix  and  use 
while  fairly  fresh.  Wash  in  tap  water. 
Stain  for  4  min.  in  0.2  gm.  Biebrich 
scarlet  +  100  cc.  1%  aq.  acetic  acid. 
Rinse  again  inaq.  dest.  Stain  for  4  min. 
in  0.1  gm.  anilin  blue  W.S.  (CC.)  + 
100  cc.  sat.  aq.  picric  acid.  Wash  for 
3  min.  in  1%  aq.  acetic  acid.  Dehydrate 
in  acetone  or  alcohol.  Clear  and  mount 
in  salicylic  acid  balsam.  Connective 
tissue,  glomerular  basement  membrane 
and  reticulum,  deep  blue;  muscle  and 
plasma,  pink;  erythrocytes,  scarlet. 
(Checked  by  R.  D.  Lillie,  National  In- 
stitute of  Health,  Bethesda,  Md.,  April 
22,  1946.) 

Bielchowsky  Silver  Methods.  These  are 
designed  for  the  nervous  system  and 
consist  essentially  of  formalin  fixation, 
silver  impregnation,  washing,  treating 
with  ammoniacal  silver  solution,  wash- 
ing and  reduction  in  formalin.  Several 
useful  modifications  are  detailed  by 
Addison  (McClung,  pp.  463-466).  See 
Nervous  System,  Silver  Methods. 

Bile.  This  frequently  comes  in  for  micro- 
scopic examination  of  centrifuged  sedi- 
ment. Stitt  (p.  761)  says  that  one  must 
be  on  the  lookout  especially  for:  (1) 


BILE  CAPILLARIES 


36 


BIOLOGICAL  STANDARDS 


Pus  cells  (neutrophiles),  scattered 
through  the  specimen  and  bile  stained, 
which,  when  occurring  in  fair  numbers, 
indicate  cholecystitis.  Unstained  pus 
cells  associated  with  mucus  are  generally 
from  the  mouth.  (2)  Bile  colored  epi- 
thelial cells  and  cellular  debris  suggest 
chronic  cholecystitis.  (3)  Cholesterin 
crystals  are  identifiable  as  opaque  or 
translucent,  flat,  rhombic  plates  or 
irregular  masses.  (4)  Large  amounts  of 
light  brown  granules  or  dark  black- 
brown  ppt.  of  calcium  bilirubinate  are 
suggestive  of  gall  stones.  (5)  Tiny  gall 
stones  (bile  sand)  are  identifiable  by 
their  concentric  lamination.  Negative 
findings  are  not,  he  is  careful  to  point 
out,  conclusive  of  absence  of  lesions. 
Bile  Capillaries.  1.  Hematoxylin  staining. 
Clara,  M.,  Zeit.  f.  mikr.  Anat.  Forsch., 
1934,  35,  1-56  advises  treatment  of  cel- 
loidin  sections  of  pieces  of  liver  fixed  in 
Alcohol  Formalin,  formalin — absolute 
alcohol — acetic  acid  (20:80:1)  and  other 
mixtures  by  the  Stolzner  Holmer  tech- 
nique and  his  own  method.  According 
to  the  former,  mordant  the  sections  in 
liquor  ferri  sesquichlorati  (try  10% 
aq.  ferric  chloride)  30-45  min.  Wash 
quickly  in  aq.  dest.  Stain  in  ripe  0.5% 
aq.  hematoxylin,  20-30  min.  Wash 
quickly  in  water.  Differentiate  in  much 
diluted  liquor  ferri  sesquichlorati. 
Wash  again  quickly  in  water.  Blue 
with  dilute  aq.  lithium  carbonate. 
Wash  in  spring  water  (tap  water  will  do ) . 
Dehydrate,  clear  and  mount.  According 
to  Clara,  mordant  the  sections  in  equal 
parts  A  and  B  at  40-50  °C.  for  24  hrs. 
(A  =  potassium  bichromate,  2.0  gm. ; 
chrome  alum,  1  gm.,  aq.  dest.,  30  cc. 
B  =  ammonium  molybdate,  2.5  gm.; 
chromic  acid,  0.25 gm.;aq.  dest.,  100  cc.) 
Wash  briefly  in  aq.  dest.  Stain  in 
Kultschitzky's  Hematoxylin.  Wash  in 
spring  water.  Dehydrate,  clear  and 
mount  in  balsam.  See  Clara's  illustra- 
tions. 

2.  Rio  Hortega  silver  carbonate  method 
adapted  by  Mclndoe,  A.  H.,  Arch. 
Path.,  1928,  6,  598-614.  Fix  small  pieces 
normal  human  liver  at  least  20  days  in 
10%  formalin.  Heat  gently  but  do  not 
boil  and  cool  several  times  thin  frozen 
sections  for  20  min.  in  silver  bath  until 
they  are  uniformly  of  a  golden  brown 
color.  (To  make  the  bath  combine  30 
cc.  10%  aq.  silver  nitrate  and  10  cc.  sat. 
aq.  lithium  carbonate.  Wash  ppt.  re- 
peatedly with  doubly  distilled  water, 
decanting  washings.  Add  100  cc.  doubly 
distilled  water  to  ppt.  Dissolve  ^i 
of  it  by  adding  ammonia  water  drop  by 
drop.  Filter  supernatant  fluid  into 
opaque  bottle  and  store  in  dark  where  it 
can  be  kept  2-4  weeks.    For  use  take 


5  cc.  of  this  stock  solution  and  add  5  cc. 
aq.  dest.  and  2-3  drops  pyridine.) 
Wash  quickly  in  aq.  dest.  Place  in  20% 
neutral  formalin,  1  min.  Fix  in  2%  aq. 
sodium  thiosulphate,  ^-1  min.  Wash 
thoroughly  in  tap  water,  2-3  days  adding 
a  little  neutral  formalin.  Dehydrate 
in  95%  and  abs.  ale,  clear  in  carbol- 
xylol  and  mount  in  balsam.  Canaliculi, 
black. 

Bile  Components  in  hepatic  cells.  Place 
small  pieces  of  liver  in  3%  aq.  barium 
chloride  for  6  hours ;  fix  18  hours  in  10% 
formalin;  dehydrate  rapidly  in  alcohol, 
clear  in  benzol  and  embed  in  paraffin. 
The  bile  components,  precipitated  by 
barium  chloride,  can  be  stained  with 
acid  dyes  especially  the  acid  fuchsin  in 
Mallory's  connective  tissue  stain  (Fors- 
gren,  E.,  J.  Morph.,  1929,  47,  519-529). 

Bile  Pigments.  Histochemical  reaction. 
Fix  in  10%  formalin  or  in  alcohol.  Pro- 
longed fixation  is  contraindicated.  Fix 
paraffin  sections  to  slides  with  egg 
albumen.  Deparaffinize  and  immerse 
in  2  or  3  parts  Lugol's  solution  and  1 
part  tincture  of  iodine,  6-12  hrs.  Wash 
in  aq.  dest.  and  cover  with  sodium  hypo- 
sulphite (5%  aq.)  15-30  sec.  until  de- 
colorized. Wash  in  aq.  dest.  and  stain 
with  alum  carmine  1-3  hrs.  Wash  in 
aq.  dest.,  dehydrate  in  acetone,  clear 
in  xylol  and  mount  in  balsam.  Bile 
pigment  granules  emerald  green  (Stein, 
J.,  C.  R.  Soc.  de  Biol.,  1935,  120,  1136- 
1138).    See  Gmelin's  Test. 

Bilharzial  Cercariae.  For  intra  -  vitam 
staining  examine  in  serum  plus  a  little 
neutral  red.  For  permanent  prepara- 
tions fix  in  hot  lactophenol  (equal  parts 
lactic  acid,  carbolic  acid,  glycerin  and 
aq.  dest.).  Stain  with  alcoholic  borax- 
carmine.  Mount  in  following:  dissolve 
by  boiling  gum  tragacanth  3  parts  and 
gum  acacia  1  part  in  aq.  dest.  100  parts. 
Add  equal  parts  lactophenol  and  use 
filtrate.  (Marshall,  A.,  Lab.  J.,  1937, 
7,  565-569). 

Bilirubin,  a  reddish  bile  pigment  which  is 
isomeric  or  identical  with  Hematoidin 
and  which  by  oxidation  can  be  converted 
into  the  green  Biliverdin,  see  Bile 
Pigments,  Urobilin  and  Van  den  Bergh 
Test. 

Biliverdin,  a  green  bile  pigment  produced 
by  oxidation  of  Bilirubin.  See  Bile 
Pigments. 

Bindschedler's  Green  (CI,  819).  A  basic 
indamin  dye  easily  reduced  to  a  sub- 
stituted diphenylamine.  See  use  as  a 
Redox  dye  in  study  of  metabolism  of 
tumor  tissue  (Elliott,  K.  A.  C.  and 
Baker,  Z.,  Biochem.  J.,  1935,  29  (2), 
2396-2404). 

Binnennetz,  see  Golgi  Apparatus. 

Biological  Standards.  Vitamins,  antitoxins, 


BIOTIN 


37 


BISMUTH  PIGMENTATION 


hormones  and  other  substances  adopted 
internationally  are  critically  considered 
by  Irwin,  J.  O.,  J.  Hyg.,  1950,  48,  215- 
238. 

Biotin,  see  Vitamins. 

Bird's  Eye  Inclusions.  Some  of  these 
bodies,  and  the  so-called  Plimmer's 
Bodies,  seen  in  cancer  cells  are  ap- 
parently greatly  enlarged  Centrosomes. 
Methods  and  results  are  given  by  Le- 
Count,  E.  R.,  J.  Med.  Res.,  1902,  7 
(N.S.  2),  383-393. 

Birefringence,  see  Polarization  Optical 
Method. 

Bismark  Brown  Y  (CI,  331) — basic  brown, 
G,  GX,  or  GXP,  Excelsior  brown, 
leather  brown,  Manchester  brown, 
phenylene  brown,  Vesuvin — A  mixture 
of  basic  dis-azo  dj'es  of  different  shades. 
Quite  widely  employed,  see  Blaydes, 
G.  W.,  Stain  Techn.,  1939,  14,  105-110 
for  use  with  plant  tissue.  See  Weissen- 
berg's  method,  as  described  by  McClung, 
1950,  p.  184,  for  the  preservation  in 
sections  of  Bismark  brown  employed 
for  supravital  staining  of  eggs. 

Bismiocymol  (see  Pappenheimer,  A.  M. 
and  Maechling,  E.  H.,  Am.  J.  Path., 
1934,  10,  577-588. 

Bismuth.     Microchemical  detection  of : 

1.  Method  of  Christeller-Komaya. 
Make  frozen  sections  of  formalin  fixed 
tissues.  A  =  quinine  sulphate,  1  gm.; 
aq.  dest.,  50  cc;  nitric  acid,  10  drops. 
B  =  potassium  iodide,  2  gm.,  aq.  dest., 
50  cc.  Immediately  before  use  mix 
equal  parts  A  and  B  and  add  2  drops 
nitric  acid,  C.P.  After  treating  sec- 
tions with  this  for  1  min.  wash  very 
quickly  in  10  cc.  aq.  dest.  +  2  drops 
nitric  acid.  Mount  section  on  slicle. 
Dry,  counterstain  with  gentian  violet. 
Bismuth  appears  as  dark  brown  grains 
(Lison,  p.  98).  See  Komaya,  G.,  Arch, 
f.  Dermat.  u.  Syph.,  1925,  149,  277-291 
(good  colored  figures)  and  Califano,  L., 
Zeit.   f.   Krebsf.,   1927-28,  26,   183-190. 

2.  Another  modification  of  the 
Komaya  method  is  given  by  Castel,  P., 
Arch.  Soc.  d.  Sci.  Med.  et.  biol.  de 
Montpellier,  1934-35,  16,  453-456  as 
follows  :  Dissolve  1  gm.  quinine  sulphate 
in  50  cc.  aq.  dest.  with  aid  of  a  few  drops 
of  sulphuric  acid.  Dissolve  2  gm. 
potassium  iodide  in  50  cc.  aq.  dest. 
Mix,  apply  to  section,  gives  red  ppt. 
of  salts  of  bismuth  in  form  of  iodo- 
bismuthate  of  quinine  or  double  iodide 
of  bismuth  and  quinine.  See  Pappen- 
heimer and  Maecnling's  (Am.  J.  Path., 
1934,  10,  577-588)  study  of  nuclear 
inclusions  in  the  kidney. 

3.  Wachstein,  M.  and  Zak,  F.  G., 
Am.  J.  Path.,  1946,  22,  603-611  have  in 
turn  improved  the  Castel  method.  See 


Glick,  Techniques  of  Histo-  and  Cyto- 
chemistry, 1949,  p.  31. 

Reagents:  A.  Modified  Castel:  After 
dissolving  0.25  gm.  brucine  sulphate  in 
100  cc.  aq.  dest.  add  2  gm.  aq.  dest. 
Store  in  brown  bottle  and  filter  before 
using.  B.  Diluted  Castel:  One  part  A 
to  3  parts  aq.  dest.  C.  Levulose:  Dis- 
solve 30  gm.  in  20  cc.  aq.  dest.  at  37°C. 
for  24  hrs.  and  add  1  drop  of  B.  D. 
Counterstain:  Add  1  cc.  1%  aq.  light 
green  S.  F.  (Hartman-Leddon  Co., 
Philadelphia)  to  100  cc.  of  B  and  filter 
before  use. 

Technique:  Treat  frozen  sections  or 
deparaffinized  formalin  fixed  sections 
on  slides  for  few  sec.  with  several  drops 
30%  hydrogen  peroxide  (Superoxol, 
Merck)  thus  removing  black  sulfide. 
Wash  in  tap  water  and  treat  with  A  1 
hr.  Transfer  to  B  and  shake  slightly 
to  detach  precipitates.  Counterstain 
in  Z)  4  min.  Blot  and  mount  in  C.  Bis- 
mith  appears  as  an  orange  yellow  de- 
posit. 
Bismuth  Pigmentation.  Histochemical 
identification  as  advised  by  Wachstein, 
M.  and  Zak,  F.  G.,  Am.  J.  Path.,  1946, 
22,  603-611  depends  on  ability  of  hydro- 
gen peroxide  to  decolorize  bismuth 
sulfide  instantaneously  and  of  a  slightly 
modified  Castel  reagent  to  change  bis- 
muth sulfate  into  an  orange  red  deposit. 

Treat  deparaffinized,  or  frozen,  sec- 
tions with  few  drops  superoxol  (30% 
hydrogen  peroxide,  Merck)  from  dark 
bottle  kept  in  refrigerator.  In  a  few 
seconds  black  of  bismuth  sulfide  dis- 
appears. Wash  thoroughlj'  in  tap  wa- 
ter and  place  in  Coplin  jar  containing 
modified  Castel  reagent  made  as  fol- 
lows: Dissolve  0.25  gm.  brucine  sulfate 
(Merck  or  Eastman  Kodak)  in  100  cc. 
aq.  dest.  plus  2  or  3  drops  concentrated 
sulfuric  acid.  Then  acid  2  gm.  potas- 
sium iodide,  keep  in  a  brown  bottle  and 
filrer  before  use.  After  1  hr.  transfer 
sections  to  another  jar  containing  some 
of  reagent  diluted  with  3  parts  aq.  dest., 
and  shake  gently  to  remove  precipi- 
tates. Remove  most  of  fluid  from  sec- 
tions by  blotting  and  cover  with  levu- 
lose solution  made  by  dissolving  30  gm. 
levulose  in  20  cc.  aq.  dest.  at  37°C.  for 
24  hrs.  to  which  drop  of  diluted  Castel 
reagent  has  been  added.  To  counter- 
stain color  4  min.  with  freshly  filtered 
100  cc.  nondiluted  reagent  plus  1  cc. 
1%  aq.  light  green  S  F  (Hartman-Led- 
don Co.). 

Method  also  can  be  used  for  study  of 
fresh  tissues  and  gross  specimens.  Add 
cone,  hydrogen  peroxide  drop  by  drop 
to  pigmented  area.  Decolorization  is 
rapid  if  bismuth  sulfide  is  present. 
Wash  thoroughly  in  running  water  to 


BISMUTOSE 


38 


BLOOD  CELL  VOLUME 


remove  excess  hydrogen  peroxide.  Ap- 
ply modified  Castel  reagent  to  surface 
and  examine  for  orange  ppt.  See  de- 
scription by  authors  of  distribution  of 
bismuth  pigmentation  in  the  tissues 
and  comparison  with  other  pigments. 

Bismutose,  a  compound  of  bismuth  and 
albumen  which  on  application  becomes 
concentrated  in  the  area  of  the  Golgi 
apparatus  (Kredowsky,  Zeit.  f.  Zellf., 
1931,  13,  1). 

Biuret  Reaction.  Described  as  follows  by 
Serra,  J.  A.,  Stain  Techn.,  1946, 21,  5-18: 
Prepare  tissue  as  described  under  Nin- 
hydrin  Reaction.  "The  pieces  are  im- 
mersed in  strong  NaOH  or  KOH  solu- 
tion in  a  watch  glass  and  some  drops  of 
a  1%  aqueous  solution  of  CuSO^  are 
then  added  with  stirring.  A  blue- 
violet  coloration  indicates  the  presence 
of  peptides  or  proteins. 

"The  reaction  is  given  by  the  peptide 
linkage  when  the  peptides  are  composed 
of  at  least  three  amino  acids.  The 
color  is  more  reddish  with  the  simpler 
peptides.  For  cytological  or  histologi- 
cal work,  the  reaction  has  the  disad- 
vantage of  requiring  a  strong  alkaline 
reaction,  which  tends  to  dissolve  the 
protoplasm.  To  avoid  a  serious  dis- 
solution the  tissues  must  be  hardened, 
for  instance  with  formalin  (10%  for- 
maldehyde during  24  hours,  followed 
by  a  thorough  washing).  This  reaction 
has  also  the  disadvantage  of  being  in- 
sensitive." 

Blastomeres  of  eggs,  see  separation  of  in 
McClung's  Microscopical  Technique, 
1950,  p.  557. 

Blastomycosis.  The  differentiation  of  Zy- 
monema  (Blastomyces)  dermatitidis ,  the 
cause  of  blastomycosis,  from  Crypto- 
coccus  hominis,  the  cause  of  crypto- 
coccosis or  torulosis,  is  best  accom- 
plished by  w^et  India  ink  technique  of 
Weidman,  F.  D.  and  Freeman,  W., 
J.A.M.A.,  1924,83, 1163.  Stir  suspected 
material  in  a  drop  of  india  ink,  place  on 
a  clean  slide  and  cover.  Use  a  small 
drop  so  as  to  form  a  thin  film.  Work 
rapidly  before  the  ink  dries  out.  In 
blastomycosis  the  wall  of  the  organism  is 
thick  and  presents  a  double-contoured 
appearance.  Cryptococciis  hominis  is 
surrounded  by  a  thick  mucoid  capsule 
which,  against  a  dark  background,  shows 
up  as  a  clear  halo  surrounding  the  fun- 
gus. Spinal  fluid  usually  dilutes  the 
ink  making  a  lighter  background.  See 
Fungi. 

Bleaching  of  tissue  fixed  in  osmic  acid  mix- 
tures can  be  brought  about  by  immers- 
ing in  dioxan  for  as  long  as  3  daj^s.  Sec- 
tions require  only  a  few  minutes  (Asana, 
J.  J.,  Stain  Techn.,  1940,  15,  176). 

Blendors,   Micro-Waring   for   low   temper- 


ature use  (Sorof,  S.  and  Cohen,  P.  P., 
Exp.   Cell  Res.,  1951,  2,  299-300). 

Blood.  Microscopically  considered  blood 
is  the  field  of  the  hematologist  (see 
Downey's  Hematology,  N.  Y.,  Hoeber, 
1938  in  4  volumes).  Any  conception  of 
the  formed  elements  of  the  blood  is 
artificial  and  inadequate  unless  it  is 
based  upon  knowledge  of  their  appear- 
ance and  behavior  in  vivo.  To  examine 
circulating  blood  in  the  web  of  a  frog's 
foot  is  helpful  but  it  is  better  to  use 
mammals.  In  the  latter,  the  methods 
devised  by  Covell  and  O'Leary  for  study 
of  the  living  Pancreas  are  recommended 
for  blood  cells  also.  Probably  the  best 
technique  is  that  of  Sandison  for  direct 
e.xamination  of  contents  of  small  blood 
vessels  and  capillaries  in  transparent 
chambers  inserted  into  rabbits'  ears. 
Living  blood  cells  can  be  observed 
in  vitro  at  high  magnification  in  Tissue 
Cultures;  but,  of  course,  circulation  is 
lacking. 

When  blood  cells  are  removed  from  the 
body  and  mounted  on  slides  in  approxi- 
mately isotonic  media,  they  can  be 
studied  for  a  short  time  before  they  be- 
come seriously  injured  and  die. 
Examination  in  the  dark  field  and  after 
Supravital  Staining  may  be  helpful. 
It  is  important  in  interpreting  the 
results  to  remember  that  the  conditions 
are  very  abnormal,  that  the  cells  are 
often  more  flattened  than  in  vivo,  and 
that  the  actual  speed  of  movement  is 
not  that  seen,  but  is  that  observed  di- 
vided by  the  magnification  because  the 
distance  travelled  per  unit  of  time 
naturally  appears  greater  than  it 
actuallj^  is.  The  motion  picture  tech- 
nique has  great  potentialities. 

Examination  is  usually  limited  to  fixed 
and  stained  Blood  Smears  but  valuable 
data  can  also  be  secured  from  sections. 
Normal  values  for  blood  cells  during  first 
year  of  life  (Merritt,  K.  K.,  1933,  46, 
990-1010).  For  details,  see  Blood  Pro- 
tein (coagulated).  Bone  Marrow,  Chylo- 
microns, Erythrocytes,  Erythrocyte 
Counts,  Fibrin,  Hematoidin,  Hemato- 
porphyrin,  Hemofuscin,  Hemoglobin, 
Hemosiderin,  Leucocytes,  Leucocyte 
Counts,  Lymphocytes,  Monocytes, 
Platelets,  Parhemoglobin,  Reticulo- 
cytes, Sulfmethemoglobin. 

Blood  Agar,  see  Bacteria,  Media. 

Blood  Cell  Volume.  Dry  Evans  Blue 
(Merck)  at  100 °C.  Dissolve  400-800 
mg.  in  1  liter  aq.  dest.  Put  0.5-1  cc.  in 
tube  3-4  cc.  capacity  and  evaporate  to 
dryness  at  70  °C.  Collect  blood  to 
contain  2.0-2.5  units  heparin  or  0.2% 
ammonium  oxalate.  Centrifuge  and 
transfer  1  cc.  plasma  to  tube  containing 
dye.    Remove  0.1  cc.  dyed  plasma  to 


BLOOD  FLOW 


39 


BLOOD  PLATELETS 


9.9  cc.  saline  in  photoelectric  colorimeter 
tube.  Make  blank  without  plasma. 
Compare  in  Evelyn  or  Klet-Summerson 
colorimeter  using  filter  to  pass  only  light 
of  about  620  m/x.  Calculate  as  directed 
for  the  colorimeter  (Shohl,  A.  T.  and 
Hunter,  T.  H.,  J.  Lab.  &  Clin.  Med., 
1941,  26,  1829-1837).  See  also  earlier 
cell  opacity  method  (Shohl,  A.  T.,  J. 
Lab.  &  Clin.  Med.,  1939-40,  25,  1325- 
1332). 

Blood  Flow,  technique  for  local  measure- 
ment of,  using  radioactive  sodium 
(Semple,  R.,  McDonald,  L.  and  Ekins, 
R.  P.,  Am.  Heart  J.,  1951,  41,  803-809). 

Blood  Grouping  technique  does  not  properly 
come  in  the  scope  of  this  book;  but  since 
it  is  involved  in  fundamental  medical 
and  biological  problems  the  following 
leading  reference  is  given:  Schiff,  F., 
and  Boyd,  W.  C,  Blood  Grouping 
Technic.  New  York:  Interscience  Pub- 
lishers, Inc.,  1942,  248  pp. 

Blood  Platelets— Written  by  Paul  M.  Ag- 
geler,  University  of  California  Medical 
Center,  San  Francisco  22,  California. 
November  15,  1951 — It  is  believed  by 
most  authorities  today  that  the  plate- 
lets originate  from  megakaryocytes, 
chiefly  in  the  bone  marrow  but  perhaps 
also  in  the  lungs.  The  platelets  are 
thought  to  be  detached  fragments  of  the 
cytoplasm  of  mature  megakaryocytes. 
In  man  the  platelets  usually  vary  be- 
tween 2  and  3  microns  in  length,  al- 
though microplatelets  of  less  than  one 
micron  and  macroplatelets  as  long  as  25 
to  50  microns  have  been  observed. 
They  vary  in  thickness  from  0.5  to  1.0 
micron.  In  rapidly  fixed  blood  they 
usually  assume  the  shape  of  an  oval  disc 
or  lentil  although  a  variety  of  forms 
may  be  encountered.  In  unfixed  blood, 
even  in  the  presence  of  an  isotonic 
anticoagulant  many  degenerative  forms 
may  occur.  These  may  appear  shrunken 
or  "exploded"  and  there  may  be  numer- 
ous spinelike  projections  from  the  sur- 
face of  the  platelet.  In  dry  smears  of 
imperfectly  fixed  blood  stained  with 
Wright's  stain  the  platelets  appear  to 
be  divided  into  two  zones:  the  clear 
blue  hyalomere  and  the  chromomere 
made  up  of  purple  staining  granules. 
This  separation  into  two  zones  is  prob- 
ably an  artefact  produced  by  changes 
in  distribution  of  the  granular  material 
of  the  platelet  after  leaving  the  circula- 
tion, for  when  blood  is  rapidly  fixed 
the  granules  are  evenly  distributed 
through  the  body  of  the  platelet. 
Macroplatelets  found  in  the  blood  in 
periods  of  abnormal  blood  regeneration 
often  take  a  deeper  stain  and  the 
granules  are  coarse  and  do  not  show, 
even  in  slowly  dried  preparations,  the 


clear    separation    between    hyalomere 
and  chromomere. 

Platelets  are  found  in  the  circulating 
blood,  particularly  in  the  capillaries  of 
the  liver  and  lung,  in  the  bone  marrow 
and  in  the  spleen,  both  in  the  sinuses 
and  between  the  cells  of  the  pulp. 
They  are  not  found  in  the  lymph  or  in 
the  thoracic  duct.  More  platelets  are 
found  in  arterial  than  in  venous  or 
capillary  blood. 

Physiological  decreases  in  the  plate- 
let count  are  said  to  occur  during  the 
first  day  of  menstruation  and  increases 
have  been  found  after  violent  exercise 
and  following  a  change  to  a  high  alti- 
tude. An  increased  platelet  count 
(thrombocytosis)  may  be  found  in  in- 
fectious diseases,  trauma,  fractures, 
asphyxiation,  surgical  operations,  acute 
blood  loss,  chronic  myelocytic  leu- 
kemia, Hodgkin's  disease  and  erythre- 
mia. A  decreased  platelet  count 
(thrombocytopenia)  is  the  basic  defect 
in  idiopathic  thrombocytopenic  purpura 
and  also  occurs  secondarily  in  certain 
acute  infectious  diseases  of  the  blood 
and  blood  forming  organs,  diseases  of 
the  spleen,  allergies,  sensitization  reac- 
tions to  certain  drugs  and  chemicals, 
and  following  the  use  of  certain  toxic 
agents  such  as  benzol  or  ionizing  radia- 
tions. 

The  platelets  are  thought  to  survive 
in  the  circulation  of  the  normal  subject 
for  from  three  to  five  days.  There  is 
recent  evidence  to  suggest  that  in 
idiopathic  thrombocytopenic  purpura 
they  are  destroyed  at  a  much  more 
rapid  rate.  The  reduction  of  the  plate- 
let count  in  this  disease  had  previously 
been  thought  to  be  due  either  to  failure 
of  production  of  the  platelets  or  to 
abnormally  rapid  removal  from  the 
circulation  by  phagocytosis  in  the 
spleen.  The  mechanism  of  reduction 
of  the  platelet  count  in  secondary 
thrombocytopenic  purpuras  may  be: 
(1)  destruction  of  megakaryocytes  as 
in  ionizing  radiation;  (2)  splenic  in- 
hibition of  maturation  of  megakaryo- 
cytes as  in  congestive  splenomegaly  or; 
(3)  a  direct  inhibition  of  maturation 
of  megakaryocj'tes  as  in  sensitivity 
reactions  to  drugs. 

The  platelets  are  concerned  in  the 
coagulation  of  the  blood,  in  retraction 
of  the  blood  clot,  in  the  formation  of 
thrombi  and  in  the  maintenance  of 
capillary  continuity.  The  most  signifi- 
cant property  of  platelets  is  the  readi- 
ness with  which  they  agglutinate  in 
shed  blood  or  when  exposed  to  a  for- 
eign surface.  Agglutination  is  gener- 
ally followed  by  fusion  and  lysis. 

Direct  Methods:  The  principle  of  all 


BLOOD  PLATELETS 


40 


BLOOD  PLATELETS 


direct  methods  of  platelet  counting 
consists  in  the  accurate  dilution  of  a 
measured  quantity  of  blood  with  a 
suitable  fluid.  The  diluted  blood  is 
placed  in  a  counting  chamber  and  the 
number  of  platelets  in  a  circumscribed 
volume  is  counted.  These  methods  of 
counting  platelets  involve  the  use  of 
the  same  apparatus  as  is  employed  in 
the  erythrocyte  count.  The  Thoma 
diluting  pipette  is  generally  used.  It 
consists  of  a  capillary  tube  graduated 
in  tenths,  which  opens  into  a  bulb  con- 
taining a  glass  bead.  The  bulb,  when 
filled  to  the  mark  101  above  it,  will 
hold  100  times  the  quantity  of  fluid 
contained  in  the  10  divisions  of  the 
capillary  tube.  The  counting  chamber 
is  a  heavy  glass  slide,  with  two  central 
ruled  platforms.  They  are  separated 
from  each  other  by  one  moat,  and  from 
elevated  shoulders  on  each  side  by 
transverse  moats.  These  lateral  shoul- 
ders are  so  ground  that  a  cover-slip 
resting  on  them  lies  exactly  0.1  mm. 
above  each  platform.  A  ruled  area  of 
3  mm.  by  3  mm.  (9  sq.  mm.)  is  engraved 
on  each  side.  This  area  is  divided  into 
9  large  1  mm.  squares.  The  central 
square  is  divided  into  intermediate- 
sized  squares  of  1/25  sq.rnm.,  each  of 
which  is  further  divided  into  smaller 
squares  of  1/400  sq.  mm. 

In  making  a  count,  blood  is  drawn  by 
suction  into  the  Thoma  pipette  to  the 
0.5  mark.  The  blood  adhering  to  the 
outside  of  the  pipette  is  wiped  off  and 
the  diluent  is  drawn  in  until  it  fills  the 
bulb  and  reaches  the  mark  101.  While 
drawing  in  the  diluent,  the  pipette  is 
revolved  between  the  finger  and  the 
thumb,  in  order  to  mix  the  blood  thor- 
oughly with  the  diluent.  After  the 
desired  quantity  of  solution  has  been 
drawn  into  the  pipette,  it  is  held  hori- 
zontally and  shaken  for  one-half  niin- 
ute,  or  it  may  be  placed  in  a  mechanical 
shaking  device,  in  order  to  secure 
thorough  mixing.  The  coverglass  is 
next  placed  on  the  chamber.  Several 
large  drops  of  fluid  are  expelled  from 
the  pipette  and  discarded,  and  a  small 
quantity  of  diluted  blood  is  allowed  to 
run  under  the  coverglass.  The  plat- 
form should  be  completely  covered  with 
fluid,  and  none  should  run  over  into 
the  moats. 

The  preparation  is  allowed  to  stand 
10  minutes  to  allow  settling  of  the  cells, 
then  the  number  of  platelets  seen  in 
the  entire  central  1  mm.  ruled  area  is 
counted.  This  number  is  multiplied 
by  2000  [1/10  (depth)  X  1/200  (dilu- 
tion)]. 

There  may  be  errors  due  to  uncleanli- 
uess     of     the     glassware,     inaccurate 


calibration  of  pipettes  or  chambers, 
imperfect  dilution,  failure  to  mix  the 
blood  and  diluting  fluid  thoroughly, 
imperfect  filling  of  the  chamber  and 
improper  sampling,  such  as  might  occur 
if  a  free  flow  of  blood  is  not  obtained 
from  the  finger  or  ear  when  capillary 
blood  is  employed,  or  from  improper 
sampling  if  venous  blood  is  used.  The 
above  sources  of  error  can  be  avoided 
by  an  experienced  technician,  so  that 
their  contribution  to  the  inaccuracy  of 
the  platelet  count  is  minimal.  How- 
ever, there  are  additional  sources  of 
error  when  this  method,  which  was 
originally  devised  for  erythrocyte 
counting,  is  used  for  the  enumeration 
of  platelets.  Olef,  I.  (J.  Lab.  &  Clin. 
Med.,  1935,  20,  416)  has  listed  the  fol- 
lowing: 1)  clumping  of  the  platelets 
during  drawing  of  the  blood  into  the 
pipette,  2)  the  impossibility  of  visualiz- 
ing the  smaller  platelets  with  the  high 
dry  lens,  3)  the  failure  to  visualize 
some  of  the  platelets  in  the  100  micron 
space  that  exists  between  the  bottom 
of  the  counting  chamber  and  the  over- 
lying coverglass,  4)  the  sticking  of  the 
platelets  to  the  side  of  the  pipette  and 
to  the  parts  of  the  hemacytometer, 
and  5)  the  confusion  of  foreign  particles 
or  precipitates  with  platelets. 

Platelet  clumping,  the  adhesion  of 
platelets  to  glassware  and  the  occur- 
rence of  morphological  changes  in  the 
platelets  can  be  prevented  only  by  the 
immediate  mixing  of  the  blood  with  a 
diluting  fluid  which  contains  both  an 
anticoagulant  and  a  fixative.  It  is 
not  generally  appreciated  that  de- 
generative changes  occur  quite  rapidly 
in  platelets,  even  in  the  presence  of 
an  anticoagulant,  unless  a  fixative  is 
also  incorporated  in  the  diluting  fluid. 
The  anticoagulants  which  have  been 
employed  are  sodium,  potassium  or 
ammonium  oxalate,  sodium  citrate, 
sodium  metaphosphate,  sodium  sulfate, 
magnesium  sulfate,  heparin,  peptone 
and  gelatin.  Of  these,  3.8  per  cent 
sodium  citrate  appears  to  be  the  most 
satisfactory,  since  it  is  isotonic  with 
the  blood  plasma,  does  not  form  crystals 
or  precipitates  with  the  plasma,  and  is 
not  highly  conducive  to  bacterial 
growth.  The  fixatives  most  commonly 
employed  are  mercury  bichloride,  osmic 
acid,  solution  of  formaldehyde,  and 
methyl  or  ethyl  alcohol.  Of  these, 
the  solution  of  formaldehj^de  is  prefer- 
able because  it  does  not  produce  clump- 
ing of  the  erythrocytes,  does  not  form 
protein  precipitates  with  the  plasma, 
and  does  not  produce  secondary  changes 
in  the  platelets.  In  order  to  avoid 
hemolysis  from  the  formation  of  formic 


BLOOD  PLATELETS 


41 


BLOOD  PLATELETS 


acid,  the  diluting  fluid  should  be  made 
fresh  every  week  and  only  38  per  cent 
neutral  formaldehyde,  U.S. P.  should 
be  employed.  A  concentration  of  0.1 
per  cent  of  formaldehyde  in  the  dilut- 
ing fluid  is  sufficient.  Higher  con- 
centrations may  hemolyze  the  erythro- 
cytes and  form  precipitates  with  the 
plasma. 

It  is  generally'  conceded  that  the  in- 
corporation into  the  fluid  of  a  dye,  such 
as  brilliant  cresyl  blue,  methyl  violet, 
methylene  blue,  toluene  red,  crystal 
violet  and  nile  blue,  is  superfluous, 
since  it  does  not  assist  in  the  differenti- 
ation of  platelets  from  artefacts  and 
may  even  itself  be  the  source  of  arte- 
facts. Diluting  fluids  containing  no 
anticoagulant,  such  as  Tyrode's  or 
Ringer's  solution,  produce  many  arte- 
facts and,  in  addition,  cause  rapid 
disintegration  of  platelets  unless  a 
fixative,  such  as  solution  of  formalde- 
hyde, is  incorporated  in  the  solution. 
A  satisfactory  diluting  fluid,  therefore, 
is  the  one  proposed  by  Rees,  H.  M., 
and  Ecker,  E.  E.  (J.  A.  M.  A.,  1923, 
80,  621)  without  the  brilliant  cresyl 
blue  dye.  This  solution  is  prepared 
by  adding  0.22  ml.  of  neutral  formalde- 
hyde (38  per  cent  U.S. P.)  to  100  ml. 
of  3.8  per  cent  sodium  citrate.  The 
solution  should  be  kept  in  a  well-stop- 
pered bottle  in  a  refrigerator  and  should 
be  filtered  each  time  just  before  use. 
Tocantins,  L.  M.  (Arch.  Path.,  1937, 
23,  850)  also  recommends  sterilizing 
the  solution  in  order  to  reduce  the  num- 
ber of  artefacts  caused  by  bacterial 
contamination. 

Despite  the  use  of  a  satisfactory  dilut- 
ing fluid,  the  adhesion  of  platelets  to 
each  other  and  to  the  walls  of  the 
pipette  is  almost  impossible  to  avoid 
when  capillary  blood  is  used.  Drawing 
the  diluting  fluid  to  the  0.5  mark  on  the 
pipette  and  subsequently  drawing  the 
head  of  the  column  of  fluid  to  the  1.0 
mark  while  aspirating  the  blood  into 
the  pipette  is  unsatisfactory,  since 
there  is  little  opportunity  for  the  blood 
to  mi.\  with  the  diluting  fluid  within 
the  narrow  confines  of  the  capillary 
stem  of  the  pipette.  Furthermore, 
even  the  very  short  time  required  to 
draw  the  exact  quantity  of  blood  into 
the  pipette  and  to  mix  it  with  the  dilut- 
ing fluid  is  sufficient  in  many  instances 
to  allow  marked  clumping  of  the  plate- 
lets to  occur.  The  clumping  of  plate- 
lets can  best  be  avoided  by  mixing  the 
blood  with  the  diluting  fluid  before  it 
is  drawn  into  the  capillary  pipette. 
This  can  be  accomplished  by  the  use  of 
venous  blood.  The  blood  may  be  drawn 
into  a  silicone-lined  syringe  and  then 


immediately  transferred  to  a  tube  con- 
taining the  diluting  fluid  or,  preferably, 
a  given  amount  of  blood  is  drawn  into 
a  syringe  containing  a  measured  amount 
of  diluting  fluid.  Needles  varying  in 
size  from  No.  18  to  No.  26  have  been 
employed  without  any  apparent  effect 
on  the  quality  of  the  prep.-iration.  A 
satisfactory  method  for  obtaining  ve- 
nous blood  is  as  follows.  Place  exactly 
1  cc.  of  diluting  fluid  in  a  5  cc.  syringe. 
Attach  a  sterile  dry  No.  24  needle  and 
aspirate  1  cc.  of  blood  from  the  vein, 
without  stasis,  by  withdrawing  the 
plunger  to  the  2  cc.  mark.  Withdraw 
the  needle  from  the  vein  and  aspirate 
the  blood  contained  in  it  into  the  syr- 
inge. Remove  the  needle  and  thor- 
oughly mix  the  diluted  blood  in  the 
syringe  and  expell  it  into  a  collecting 
bottle.  It  is  important  to  use  a  syr- 
inge whose  plunger  and  needle  fit 
snugly,  in  order  to  avoid  the  leakage  of 
any  air  into  the  syringe  while  aspirat- 
ing the  blood.  A  1  to  200  dilution  of 
the  blood  can  be  made  by  drawing  this 
mixture  to  the  1.0  mark  on  the  Thoma 
pipette  and  subsequently  filling  it  to 
the  101  mark  with  the  diluent.  If  this 
procedure  is  carried  out  carefully, 
it  is  unnecessary  to  resort  to  methods, 
such  as  those  suggested  bv  Aynaud, 
M.  (Compt.  Rend.  Soc.  Biol.,  1910 
68,  1062)  and  Preiss,  W.  (Zeitsch.  Ges. 
Exp.  Med.,  1932,  84,  1932)  in  which  no 
attempt  is  made  to  obtain  an  accurate 
initial  dilution  in  the  syringe,  and  con- 
sequently the  final  dilution  of  blood  in 
the  counting  chamber  must  be  deter- 
mined b}'  establishing  the  ratio  of  an 
erythrocyte  count  done  on  the  platelet 
preparation  with  an  independent  eryth- 
rocyte count  done  in  the  usual  manner. 
There  is  still  another  limitation  in 
the  accuracy  of  the  direct  platelet 
count,  imposed  by  the  relatively  small 
concentration  of  platelets  in  the  count- 
ing chamber.  The  dilution  of  1  part 
blood  in  200  parts  of  fluid  is  required 
because  with  any  greater  concentration 
the  platelets  would  be  obscured  by  the 
erythrocytes.  However,  since  there 
are  only  appro.ximately  5  per  cent  as 
many  platelets  as  erj^throcytes  present, 
the  statistical  error  is  much  greater 
than  that  of  the  erythrocyte  count, 
even  if  all  the  platelets  in  the  entire  1 
square  mm.  central  ruled  area  are 
counted.  Attempts  have  been  made 
to  overcome  this  difficulty  by  increas- 
ing the  concentration  of  platelets  in 
preparations  from  which  the  erythro- 
cytes have  been  eliminated.  In  some 
methods,  such  as  that  of  Brecher,  G. 
and  Cronkite,  E.  P.  (J.  Applied  Phj's- 
iol.,  1950,  3,  365)  the  erythrocytes  are 


BLOOD  PLATELETS 


42 


BLOOD  PLATELETS 


hemolyzed.  However,  the  use  of  hemo- 
lytic diluting  fluids,  such  as  potassium 
cyanide,  urea,  or  1  per  cent  ammonium 
oxalate  is  to  be  discouraged  since 
hemolyzed  erythrocytes  may  be  the 
source  of  artefacts.  Furthermore,  if 
such  diluting  fluids  contain  no  fixative, 
dissolution  of  some  of  the  platelets 
may  also  occur.  Another  means  of 
attaining  a  higher  concentration  of 
platelets  in  the  counting  chamber  is 
to  do  the  platelet  count  on  plasma. 
In  Reimann's  modification  of  Thorn- 
sen's  method  (J.  Exper.  Med.,  1924, 
40,  553)  0.9  CO.  of  blood  is  drawn  into 
a  tuberculin  syringe  containing  0.1 
cc.  of  10  per  cent  sodium  citrate.  The 
needle  is  removed  and  the  open  end 
of  the  syringe  is  closed  with  a  piece  of 
rubber  tubing  fastened  to  the  barrel 
with  rubber  bands.  The  plunger  is 
removed  and  the  syringe  is  placed  in 
a  vertical  position.  After  sedimenta- 
tion has  occurred,  a  1  to  20  dilution  of 
the  supernatant  plasma  is  made  with 
physiologic  sodium  chloride  solution. 
The  platelets  in  the  mixture  are  then 
counted  in  the  counting  chamber  in 
the  usual  manner.  If  the  platelets 
seen  in  five  intermediate  sized  squares 
(80  small  squares)  are  counted,  the 
number  is  multiplied  by  1000  [1/5  (area) 
X  1/20  (dilution)  X  1/10  (depth)],  in 
order  to  find  the  number  of  platelets 
per  cubic  millimeter  of  plasma.  A 
further  procedure  has  been  designed  to 
determine  the  number  of  platelets  per 
cubic  millimeter  of  blood.  The  syringe 
containing  the  blood  is  centrifugalized 
for  20  minutes  at  2000  r.p.m.  The 
relative  amount  of  plasma  and  packed 
cells  is  recorded  and  the  number  of 
platelets  per  cubic  millmeter  of  blood 
is  determined  thus: 


Number  of 
platelets  per 
cubic  milli- 
meter of 
blood 


Number  of 
platelets  per 
cubic  milli- 
meter of 
plasma 


Amount  of  plasma 


Total     amount 
mixture 


of 


To  the  result,  10  per  cent  of  the  total 
number  is  added  to  offset  the  original 
dilution  in  the  syringe.  The  determi- 
nation of  the  platelet  count  in  plasrna 
by  this  method  has  the  advantage  of 
increasing  the  concentration  of  plate- 
lets per  unit  volume  in  the  counting 
chamber  approximately  ten  times. 
However,  there  is  some  question  as  to 
the  stability  and  uniformity  of  the  sus- 
pension of  platelets  in  plasma  during 
the  time  required  for  sedimentation 
of  the  erythrocytes.  Furthermore,  the 
determination  of  the  relative  plasma 
and  packed  cell  volumes  by  the  method 
given  is  inadequate,  since  even  at  a 
centrifugalization  speed  of  3000  r.p.m. 


at  least  15  per  cent  of  the  plasma  is 
retained  in  the  packed  cell  mass. 

In  order  to  avoid  the  errors  inherent 
in  the  Thomsen  method  and  still  in- 
crease the  concentration  of  platelets 
in  the  counting  chamber,  methods  have 
been  devised  for  eluting  the  platelets 
from  the  whole  blood.  In  the  method 
of  Villarino  and  Pimentel  (Am.  J. 
Clin.  Path.,  1942,  12,  362)  a  1  to  2  dilu- 
tion of  venous  blood  with  Aynaud's 
fluid  is  made  in  the  syringe.  Four  cc. 
of  pooled  eluate  from  0.2  cc.  of  this 
mixture  are  obtained  by  four  separate 
centrifugations  for  1  minute  at  1500 
r.p.m.  Only  an  insignificant  number  of 
platelets  can  be  recovered  by  further 
washing.  A  similar  method  has  been 
devised  by  Scheff,  G.  I.  and  Ralph, 
P.  H.  (Am.  J.  Clin.  Path.,  1949,  19, 
1113)  using  an  angle  centrifuge  at  a 
speed  of  700  r.p.m.,  and  dark  field 
illumination.  Although  it  would  ap- 
pear that  practically  all  of  the  plate- 
lets are  recovered  by  these  methods 
since  only  an  insignificant  number  can 
be  recovered  by  further  washing  and 
only  a  very  few  can  be  found  in  stained 
smears  of  the  washed  sediment,  there 
is  some  question  as  to  whether  some  of 
them  may  not  have  been  destroyed 
during  the  process  of  repeated  centri- 
fugalization. 

Regardless  of  the  source  of  the  speci- 
men, type  of  diluting  fluid  or  concentra- 
tion of  platelets  per  unit  volume  of 
fluid  in  the  chamber,  there  are  certain 
difficulties  in  differentiating  platelets 
from  artefacts  when  high  dry  magnifica- 
tion and  bright  field  illumination  are 
used.  Under  these  circumstances,  a 
platelet  is  defined  simply  as  "a  small 
refractile  body".  According  to  Tocan- 
tins,  L.  M.  (Arch.  Path.,  1927,  23,  850) 
"Only  forms  from  1  to  3  microns  or 
larger  in  size,  rod  or  comma-like  if  seen 
sidewaj^s,  and  thin,  translucent  and 
disclike  if  flat  on  the  floor  of  the  count- 
ing chamber,  should  be  counted.  Gran- 
ules 0.8  micron  in  diameter  or  smaller, 
jerkily  moving  about  more  or  less  ac- 
tively, globules  of  oil,  irregularly 
shaped  debris  floating  on  the  upper 
layers  of  the  fluid,  strings  of  cocci, 
and  other  minute  objects  may  be  dis- 
tinguished from  platelets  after  a  little 
practice.  The  error  of  counting  too 
few  platelets  may  be  equaled  only  by 
the  error  of  counting  every  particle  in 
the  field  as  a  platelet."  That  experi- 
enced observers  differ  significantly, 
even  when  using  the  same  method,  is 
shown  by  the  following  results  of  cu- 
taneous platelet  counts  on  normal 
adult  subjects,  using  the  Rees  and  Ecker 
method. 


BLOOD  PLATELETS 


43 


BLOOD  PLATELETS 


Author 

Number 

of 
Subjects 

Mean 

Stand- 
ard 
Error 
of  Mean 

Stand- 
ard 

Devia- 
tion 

Tocantins,    L.    M., 
Arch.  Path.,  1937, 
23,850 

40 

64 
80 

250,000 

409,000 
241,000 

7458 
8500 

68,500 

68,000 
50,000 

Aggeler,  P.  M.,  How- 
ard, J.  and  Lucia, 
S.  P.,  Blood,  1946, 
1,472 

Sloan,  A.  W.,  J.  Clin. 
Path.,  1951,4,  37 

The  author  (Aggeler)  is  aware  of  the 
fact  that  the  platelet  counts  reported 
by  him  and  his  associates  could  be 
higher  than  those  observed  by  Tocan- 
tins and  Sloan  because  certain  arte- 
facts were  consistently  counted  as 
platelets.  On  the  other  hand  there  is 
no  proof  that  the  latter  authors  did 
not  mistake  some  of  the  platelets  for 
artefacts.  It  is  begging  the  point  to 
demonstrate  that  any  single  observer 
may  obtain  consistent  results.  This 
means  only  that  he  is  constant  either 
in  identifying  platelets  as  platelets, 
platelets  as  artefacts,  or  artefacts  as 
platelets.  It  does  not  prove  that  the 
particular  observer  is  always  identify- 
ing platelets  as  platelets  and  artefacts 
as  artefacts.  Furthermore,  the  fact 
that  several  observers  using  the  same 
naethod  may  obtain  reasonably  con- 
sistent results  does  not  prove  the  valid- 
ity of  the  method,  since  both  observer 
may  be  committing  the  same  error. 
The  ultimate  cause  for  the  great  varia- 
tion in  the  reported  results  of  different 
observers  using  the  same  method  is 
that  platelets  cannot  be  distinguished 
from  artefacts  with  certainty  in  any 
method  in  which  the  magnification  and 
resolution  is  not  considerably  better 
than  that  which  can  be  achieved  with 
the  high  dry  lens  and  bright  field  il- 
lumination. Some  improvement  can 
perhaps  be  gained  by  the  use  of  dark 
field  illumination,  as  recommended  by 
Scheff  and  Ralph  (Am.  J.  Clin.  Path., 
1949,  19,  1113)  or  by  the  use  of  phase 
contrast  illumination,  as  advocated 
by  Brecher,  G.  and  Cronkite,  E.  P. 
(J.  Applied  Physiol.,  1950,  3,  365). 

Nothing  is  gained  by  the  use  of  ocu- 
lars giving  a  magnification  greater 
than  lOX  since,  in  the  absence  of  any 
mprovement  in  resolution,  the  in- 
creased magnification  only  serves  to 
increase  the  confusion.  Attempts  have 
been  made  by  Helber,  E.  (Arch.  f. 
Klin.  Med.,  1904,  81,  316)  and  by 
Maixner  and  Von  Decostello  (Med. 
Klin.,  1915, 11, 14)  to  increase  magnifica- 


tion and  resolution  by  the  use  of  the 
Zeiss  D  water  immersion  lens.     With 
this  equipment,  magnification  of  lOOOX 
was  attained.     Approximately  the  same 
magnification    with    better    resolution 
can  be  achieved  with  an  oil  immersion 
lens.     However,  with  these  lenses,  it  is 
necessary  to  use  a  specially  constructed 
counting  chamber  of  0.02  mm.  depth 
and  a  cover  slip  not  more  than  0.25  mm. 
in  thickness.    Because  of  the  reduced 
depth,  a  1  to  40  dilution  of  blood  is 
used  in  order  to  obtain  the  same  num- 
ber of  cells  per  unit  area  of  the  counting 
chamber  as  are  present  when  using  a 
1  to  200  dilution  in  the  0.1  mm.  depth 
chamber.     The  author  and  his  associ- 
ates  have  found  no  difficulty  in  dis- 
tinguishing   artefacts    from    platelets 
with  a  chamber  and  coverslip  of  this 
type*  used  in  conjunction  with  a  Spen- 
cer binocular  microscope  equipped  with 
1.8    mm.,    97X    (oil    immersion)    N.A. 
1.25,  medium  dark  contrast  phase  ob- 
jective, standard  condenser  lower  ele- 
ment, standard  N.   A.   1.25  condenser 
top  element,  97X    annular  diaphragm 
and    lOX    Hugenian    eyepieces.     Even 
the  morphologic  characteristics  of  small 
platelets  were  clearly  visible.     Most  of 
the  platelets  appeared  as  flat,  round  or 
oval  discs,  about  2  microns  in  diameter 
and  0.5  microns  thick.     The  cytoplasm 
showed    a    uniform,    fine    granulation. 
Some   of  the  platelets   had   a  slightly 
irregular  shape,  most  had  a  single  short 
slightly    curled    filament,    others    had 
more  than  one  filament,  and  a  few  had 
none.     Occasionally  two  platelets   at- 
tached by  a  long  streamer  of  cytoplasm 
were  observed.     Rarely  was  an  "explo- 
sive"   degeneration    form    of    platelet 
seen,  and  none  of  the  platelets  had  the 
multiple    sharp    spinelike    projections 
which  always  appear  when  no  fixative 
is  employed  in  the  diluting  fluid.     The 
principal  artefacts  observed  were  bits 
of   amorphous   debris   which   could   be 
distinguished  by  their  irregular  shape 
and    heavy    granulation;    erythrocyte 
fragments,  which  were  colored  yellow, 
had  no  granulation  and  were  intensely 
refractile;     and    clumps    of    bacteria, 
which  were  identified  by  their  regularly 
spaced    heavy   granules    separated    by 
fine     dark     filaments.     Unfortunately, 
while  the  problem  of  differentiation  of 
platelets  from  artefacts  is  solved  by  this 
systern  of  microscopy,  extreme  differ- 
ences in  the  platelet  counts  on  aliquots 
of  the  same  venous  blood  specimen  were 
encountered,  so  that  the  advantage  of 

•  Manufactured  by  the  American  Optical  Co.,  Buf- 
falo, N.  Y.,  through  the  courtesy  of  Mr.  C.  E.  Guellich, 
Manager  of  Product  Salea,  Scientific  Division. 


BLOOD  PLATELETS 


44 


BLOOD  PLATELETS 


better  visualization  of  the  platelets  was 
nullified.  The  variations  in  the  platelet 
count  appear  to  be  due  to  differences 
in  the  volume  of  fluid  contained  in  the 
chamber  caused  by  upward  lifting  or 
downward  bending  of  the  coverslip. 
Before  this  technique  can  be  perfected, 
it  will  be  necessary  to  develop  methods 
for  insuring  scrupulous  cleanliness  of 
the  glassware.  In  addition,  a  radical 
change  in  the  design  of  the  chamber  to 
prevent  downward  bending  of  the  cover- 
slip  due  to  intense  capillary  attraction 
will  be  required. 

With  regard  to  the  established  direct 
methods  of  platelet  counting,  there- 
fore, it  appears  that  most  of  the  sources 
of  error  can  be  minimized,  except  that 
of  accurate  identification  of  the  plate- 
lets. This  error  can  be  made  a  constant 
for  a  given  method  by  an  experienced 
observer,  but  may  lead  to  large  in- 
consistent variations  in  platelet  counts 
done  by  inexperienced  individuals. 

Indirect  Methods:  One  of  the  principal 
advantages  of  indirect  methods  of 
platelet  counting  is  that  microscopic 
objectives  giving  higher  magnification 
and  greater  resolution  can  be  employed. 
Furthermore,  the  blood  can  be  mixed 
immediately  with  the  diluting  fluid 
and  the  platelets  fixed  before  it  is  neces- 
sary for  them  to  come  in  contact  with 
any  foreign  surface,  except  perhaps 
momentarily  with  the  skin  or  a  veni- 
puncture needle.  In  all  indirect 
methods  of  platelet  counting  unknown 
quantities  of  blood  and  diluent  are 
mixed.  The  number  of  platelets  seen 
per  1000  erythrocytes  is  multiplied  by 
the  number  of  thousands  of  erythro- 
cytes found  in  an  independent  erythro- 
cyte count  done  in  the  usual  manner. 
There  are  three  methods  of  determining 
the  ratio  of  platelets  to  erythrocytes: 
in  a  counting  chamber,  a  wet  slide 
preparation  or  a  dry  smear. 

Methods,  such  as  that  of  Kemp,  G.  T. 
and  Calhoun,  H.  (Brit.  Med.  J.,  1901, 
2,  1539)  in  which  capillary  blood  is  em- 
ployed and  the  ratio  of  erythrocytes 
to  platelets  is  determined  in  the  count- 
ing chamber,  are  subject  to  the  same 
error  involved  in  identifying  the  plate- 
lets as  is  inherent  in  all  direct  methods 
of  platelet  counting. 

Methods  such  as  those  of  Aynaud,  M. 
(Compt.  Rond.  Soc.  Biol.,  1910,  68, 
1062)  and  Preiss,  W.  (Zeitsch,  Ges. 
Exp.  Med.,  1932,  84,  810)  referred  to 
above,  in  which  venous  blood  is  used 
are  sometimes  referred  to  as  indirect 
methods.  They  are  not,  since  in  these 
methods,  the  ratio  of  the  erythrocyte 
count  in  the  platelet  preparation  to  the 
erythrocyte   count   done   in   the   usual 


manner  only  serves  to  establish  the  dilu- 
tion of  blood  employed  in  the  plate- 
let count.  The  number  of  platelets 
counted  per  unit  volume  is  an  absolute 
not  a  relative  value.  The  additional 
step  of  establishing  the  ratio  of  the  two 
erythrocyte  counts  could  be  avoided 
by  accurate  measurement  of  the  degree 
of  dilution  of  the  blood  in  the  syringe. 
These  methods  are  also  subject  to  the 
errors  involved  in  identifying  platelets 
with  the  high  dry  lens  in  the  standard 
counting  chamber. 

Some  of  the  objections  to  the  indirect 
method  of  platelet  counting  in  which 
the  ratio  of  platelets  to  erythrocytes 
is  determined  in  a  wet  preparation  or 
a  dry  smear  have  been  summarized  by 
Tocantins  (Arch.  Path.,  1937,  23,  850). 
1)  The  mixture  of  blood  and  diluting 
solution  is  seldom,  if  ever,  uniform  and 
not  the  same  each  time.  2)  Platelets 
and  erythrocytes  are  not  distributed 
evently  through  the  preparation,  since 
no  provision  is  made  for  shaking  before 
counting.  3)  The  method  has  defects 
intrinsic  in  any  determination  done 
indirectly,  that  is,  in  relation  to  another 
equally  changeable  element.  4)  The 
greatest  source  of  error,  however,  is 
in  the  assumption  that  platelets  and 
erythrocytes  keep  an  even  proportion 
in  numbers  toward  each  other  between 
the  two  main  steps  of  the  method. 
The  markedly  different  physical  proper- 
ties (adhesiveness,  specific  gravity, 
size  and  others)  of  platelets  and  eryth- 
rocytes lead  to  continuous  changes  in 
this  ratio.  5)  The  proportion  of  plate- 
lets to  erythrocytes  varies  at  the  same 
time  in  different  portions  of  the  circula- 
tion and  this  variation  is  even  more 
marked  within  short  spaces  of  time  in 
capillaries  than  venules. 

The  term  "indirect  platelet  count" 
has  come  to  imply  that  the  count  is 
done  on  capillary  blood.  Curiously 
enough  it  seems  never  to  have  occurred 
to  anyone  to  do  an  indirect  platelet 
count  on  accurately  diluted  venous 
blood.  This  would  overcome  some  of 
Tocantins'  objections  since  it  would 
allow  for  a  constant  uniform  dilution 
of  the  blood,  shaking  of  the  diluted 
blood  before  mixing  and  the  determina- 
tion of  the  erythrocyte  count  on  the 
same  specimen  as  is  used  for  the  plate- 
let count. 

Olef  (J.  Lab.  &  Clin.  Med.,  1935,  20, 
416)  has  also  raised  objections  to  the 
technique  employed  in  certain  indirect 
methods  of  platelet  counting.  In  those 
in  which  a  drop  of  blood  is  allowed  to 
fall  into  the  diluting  fluid  on  a  glass 
slide  or  contained  in  a  special  vessel 
(Pratt,  J.H.J.  A.  M.  A.,  1905,  45,  1999). 


BLOOD  PLATELETS 


45 


BLOOD  PLATELETS 


the  undiluted  blood  is  allowed  to  come 
into  contact  with  the  surface  of  the 
skin  and  with  the  external  air,  thus  al- 
lowing the  platelets  to  clump.  In 
methods  in  which  the  finger  is  punctured 
through  a  drop  of  fluid  (Fonio,  A., 
Deutsche  Ztschr.  f.  Chir.,  1912,  117, 
176)  the  first  drop  of  blood  must  be 
used.  This  procedure  is  said  not  to 
yield  accurate  counts  because  the  blood 
contains  disintegrated  products  of 
crushed  and  injured  cells  and  is  also 
diluted  by  an  admixture  of  Ij'^mph. 

With  regard  to  methods  in  which 
the  blood  and  diluent  are  mixed  on  the 
finger,  Olef  stated,  "This  technic  in- 
volves a  number  of  inaccuracies.  Dur- 
ing the  process  of  stirring  the  mixture 
there  is  unavoidable  contact  of  blood 
and  skin  with  resulting  destruction  and 
clumping  of  platelets.  If  the  blood  is 
flowing  freely,  as  it  should  if  a  correct 
count  is  to  be  obtained,  the  blood  and 
diluting  fluid  form  a  very  large  drop  in 
which  it  is  rather  difficult  to  obtain  a 
uniform  distribution  of  the  blood  and 
which  frequently  rolls  off  the  finger, 
especially  in  women  in  whom  the  surface 
at  the  tip  of  the  finger  is  small.  Fur- 
thermore, freely  flowing  blood  mixed 
with  only  one  drop  of  preserving  fluid 
yields  preparations  too  thick  for  ac- 
curate platelet  counting.  Dameshek 
does  not  stir  the  blood-diluent  mixture 
at  all ;  he  places  a  drop  of  the  preserving 
fluid  over  the  puncture  wound  after  the 
first  drop  or  two  of  blood  has  been  wiped 
away,  than  allows  some  blood  to  escape 
into  the  overljdng  drop  of  diluent  and 
by  applying  a  cover  slip  to  the  mixture 
carries  some  of  it  away.  This  pro- 
cedure is  inaccurate  because  the  plate- 
lets, being  very  light,  quickly  rise  to 
the  surface  of  the  drop  of  diluting  fluid 
before  the  considerably  heavier  eryth- 
rocytes have  become  uniformlj^  dis- 
tributed. The  fluid  on  the  cover  slip, 
therefore,  contains  a  relatively  larger 
number  of  platelets  than  red  cells." 
Olef  has  also  pointed  out  that  all  glass- 
ware must  be  scrupulously  clean  since 
hemolysis  may  occur  with  soiled  glass- 
ware. For  use  in  wet  preparations,  he 
advocates  a  1  to  5  dilution  of  blood, 
since  in  very  thin  preparations  both 
the  platelets  and  erythrocytes  are 
likely  to  be  destroyed,  whereas  in  thick 
preparations  the  erythrocytes  may  ob- 
scure some  of  the  platelets.  Olef's  ob- 
jection to  dilutions  of  blood  of  greater 
than  1  to  5  on  the  grounds  that  this 
maj'  cause  dissolution  of  the  platelets 
is  not  valid  if  a  fixative  is  contained 
in  the  diluting  fluid. 

In  Olef's  method,  the  first  drop  or 
two  of  blood  is  wiped  away.     A  drop 


of  diluting  fluid  is  then  placed  over 
the  puncture  wound  before  the  blood 
reaches  the  surface  of  the  skin,  and  the 
hand  is  quickly  turned  over  so  that  the 
palmar  surface  is  directed  downward. 
After  a  sufficiently  large  drop  has  es- 
caped, the  entire  mixture  is  applied  to 
the  surface  of  a  small  quantity  (three 
to  four  drops)  of  diluting  fluid  contained 
in  a  paraffin  cup.  The  entire  drop  on 
the  finger,  consisting  of  approximately 
equal  parts  of  blood  and  diluent,  drops 
off  into  the  cup.  The  contents  of  the 
cup  are  then  stirred  gently  with  a 
wooden  applicator,  the  end  of  which 
is  coated  with  paraffin.  The  mixture 
is  allowed  to  stand  for  a  minute  or  two, 
stirred  again,  and  then  is  transferred 
by  means  of  a  clean  paraffin-coated  ap- 
plicator to  a  glass  slide.  Usually  three 
preparations  are  made.  A  coverslip 
is  placed  over  each  drop  and  after  the 
preparations  have  been  allowed  to  stand 
for  ten  to  fifteen  minutes,  a  relative 
platelet-erythrocyte  count  is  made, 
using  the  oil  immersion  lens.  While 
this  method  has  certain  advantages  over 
other  indirect  methods,  it  does  not 
overcome  the  principal  objection  raised 
by  Tocantins,  i.e.,  that  the  platelets 
and  erythrocytes  may  not  keep  an  even 
proportion  in  numbers  toward  each 
other  between  the  two  main  steps  of  the 
method.  That  they  do  not  can  be  easily 
demonstrated  by  counting  both  the 
erythrocytes  and  platelets  in  successive 
microscopic  fields  in  different  parts  of 
the  same  preparation.  It  will  be  found 
that  the  platelets  maintain  a  fairly  even 
distribution  despite  large  differences 
in  the  erythrocyte  concentration.  This 
leads  to  variation  in  the  ratio  of  plate- 
lets to  erythrocytes  in  different  parts 
of  the  same  preparation.  The  observer 
must  arbitrarily  choose  areas  to  be 
counted,  but  because  of  unconscious 
bias  in  selection  he  will  tend  to  count 
only  those  which  he  thinks  have  an 
average  distribution  of  erythrocytes. 
However,  in  this  type  of  preparation  it 
is  impossible  to  determine  what  the 
average  distribution  is.  This  bias  in 
sampling  greatly  increases  the  sub- 
sampling  error.  The  uneven  distribu- 
tion of  erythrocytes  occurs  regardless 
of  whether  the  blood  is  capillary  or 
venous,  or  whether  it  has  been  thor- 
oughly shaken  in  a  pipette  or  simply 
mixed  with  a  stirring  rod  before  the 
preparation  is  made.  This  maldistribu- 
tion of  erythrocytes  cannot  be  avoided 
since  it  is  caused  by  physical  phe- 
nomena which  occur  during  the  very 
act  of  placing  the  coverslip  on  the  drop 
of  blood. 
Dry  slide  preparations  made  from  a 


BLOOD  PLATELETS 


46 


BLOOD  PLATELETS 


TABLE  1 

Characteristics  of  various  direct  methods  of  platelet 
enumeration 


.    •-»  1 

^ 

■0 

P4 

(3 

(L, 

Cb 

W 

w 

« 

H 

X 

w 

if 

5 

J3 

(U 

V 

4.J 

3 

td 

_  O 

a. 

M 
^ 

.£) 

a 

c3 

11 

T3 

a 

to 

% 

T3 

2j 

a 

d 

a 

o  5? 

d 

K 

'•t-J 

a 

'a 

J3 

CO 

i 

r& 

lo" 

C3 

(J 
o 

o 

0) 

)-4 

(J 

J^ 

2 

Pi 

H 

H 

P3 

H 

> 

W 

a 

1.  Diluting  fluids 

With  dye 

X 

Without  dye. 

X 

X 

X 

X 

X 

X 

X 

With  fixative. 

X 

X 

X 

X 

Without  fixa- 

tive  

X 

X 

X 

X 

With     hemo- 

lytic prop- 

erties   

X 

Without 

hemolytic 

properties... 

X 

X 

X 

X 

X 

X 

X 

2.  Source  of  speci- 

men 

Capillary 

blood 

X 

X 

Venous  blood. 

X 

X 

X 

X 

X 

X 

3.  Type  of  prepa- 

ration 

counted 

Diluted  whole 

blood 

X 

X 

X 

X 

X 

Plasma 

X 

Eluate    from 

whole  blood 

X 

X 

4.  Counting  cham- 

ber em- 

ployed 

Standard  0.1 

mm.  depth. 

X 

X 

X 

X 

X 

X 

Special     0.02 

mm.  depth 

X 

X 

5.  Microscope  lens 

employed 

High  dry 

X 

X 

X 

X 

X 

X 

X 

Water 

immersion. 

X 

6.  Microscope  illu 

mination 

employed 

Bright  field.. 

X 

X 

X 

X 

X 

X 

Dark  field... 

X 

Phase  contras 

t 

X 

1  to  2  dilution  of  blood,  stained  in  the 
usual  manner,  and  examined  with  the 
oil  immersion  lens  have  the  advantage 
of  a  somewhat  more  uniform  distribu- 
tion of  erythrocytes  and  platelets,  but 
there  is  question  as  to  whether  some  of 
the  platelets  and/or  erythrocytes  may 
not  be  destroyed  during  the  process  of 
making  the  smears. 

The  above  multiplicity  of  methods, 
and  numerous  other  variations  not  men- 
tioned, is  not  the  result  of  idle  inven- 
tion but  rather  of  a  persistent  effort  on 
the  part  of  each  investigator  to  over- 
come the  errors  inherent  in  the  methods 
of  his  predecessors.  In  many  instances 
however,  the  successful  solution  of  one 
difficulty  has  only  served  to  give  rise 
to  other  sources  of  error.  The  im- 
possibility of  achieving  a  uniform  dis- 
tribution of  erythrocytes  and  platelets 
makes  it  unlikely  that  a  satisfactory 
indirect  method  can  be  devised.  On 
the  other  hand  the  inadequate  visualiza- 
tion of  platelets  with  the  high  dry  lens 
makes  all  direct  methods  in  which  the 
standard  0.1  mm.  depth  counting  cham- 
ber is  employed  unreliable.  The  only 
solution  to  the  problem  appears  to  be 
the  perfection  of  a  technique  for  using 
an  oil  immersion  lens  in  conjunction 
with  a  chamber  of  0.02  mm.  depth  and 
phase  contrast  lighting. 

There  is,  at  the  present  time,  no 
standard  procedure  for  the  enumeration 
of  blood  platelets.  Numerous  methods 
have  been  devised,  which  vary  in  the 
following  manner:  1)  Manner  of  count- 
ing— direct  or  indirect;  2)  type  of  dilut- 
ing fluid — with  or  without  a  variety  of 
dyes,  fixatives  and  anticoagulants — 
with  or  without  hemolytic  properties; 
3)  source  of  specimen — capillary  or 
venous  blood;  4)  material  on  which  the 
count  is  made — whole  blood,  plasma  or 
eluate  from  whole  blood;  5)  type  of 
counting  chamber — standard  0.1  mm. 
depth  or  special  0.02  mm.  depth  with 
thin  coverslip;  6)  microscopic  mag- 
nification and  resolution — 430X  (high 
dry)  to  lOOOX  (water  or  oil  immersion) ; 
7)  microscopic  illumination — bright 
field,  dark  field  or  phase  contrast  (see 
tables  1  and  2). 

Reports  of  average  normal  platelet 
counts  in  man  have  varied  from  200,000 
per  cu.  mm.  (1)  to  800,000  per  cu.  mm., 


"J.  A.  M.  A.,  1923,80,621. 

*  .\rch.  Path.,  1937,  23,  850. 

«  S.  Gynec.  et  Obst.,  1927,  15,  436. 
^  J.  Applied  Physiol.,  1950,  3,  365. 
'  Exper.  Med.,  1924,40,  553. 
/Am.  J.  Clin.  Path.,  1942,  12.  362. 
9  Am.  J.  Clin.  Path.,  1949,  19,  1113. 

*  Arch.  f.  Clin.  Med.,  1904,  81,  316. 


BLOOD  PLATELETS 


47 


BLOOD  SMEARS 


TABLE  2 

Characteristics  of  various  indirect  methods  of  platelet 
enumeration 


u 

-a 

a 
a 

Wv. 
Hi 

-a 

6 

a 
•a 
a 
a 

o 

Q 

< 

o 
'c 

(2 

m 

rtpq 

1.  Diluting  fluids 

With  dye 

X 

X 

X 

X 

X 

Without  dye. 

X 

X 

X 

With  fixative. 

X 

X 

X 

Without  fixa- 

tive  

X 

X 

X 

X 

X 

With  anti- 

coagulant... 

X 

X 

X 

X 

X 

X 

Without  anti- 

coagulant. . . 

X 

X 

2.  Source  of  speci- 

men 

Capillary 

blood 

X 

X 

X 

X 

X 

X 

X 

Venous  blood . 

X 

3.  Type  of  prepa- 

ration 

counted 

Dry  smear 

(stained)... . 

X 

Wet  prepara- 

tion  

X 

X 

X 

X 

X 

Counting 

chamber 

X 

X 

4.  Microscope  lens 

employed 

High  dry 

X 

X 

X 

Oil  immersion 

X 

X 

X 

X 

X 

5.  Microscope 

illumina- 

tion em- 

ployed 

Bright  field... 

X 

X 

X 

X 

X 

X 

X 

X 

Dark  field.... 

Phase  contrast 

«  Arch.  Path.,  1937,  23,  850. 

*J.  A.  M.  A.,  1923,80,621. 

'  J.  A.  M.  A.,  1905,  45,  1999. 

''Arch.  Int.  Med.,  1932,  50,  579. 

•  Deutsche  Ztschr.  f.  Chir.,  1912,  117,  176. 

/J.  A.  M.  A.,  1921,76,427. 

ePrit.  Med.  J.,  1901,2,  1539. 

''Lancet,  1929,  1,992. 

depending  upon  the  methods  employed. 
The  range  of  reported  normal  platelet 
counts  in  man  has  varied  from  a  mini- 
mum of  130,000  per  cu.  mm.   (2)  to  a 


maximum  of  900,000  per  cu.  mm.  (3) 
Since  all  methods  so  far  devised  are 
subject  to  inherent  errors,  there  is  no 
sound  basis  for  a  choice  between  the 
mutually  exclusive  standards  of  normal- 
ity which  have  been  reported.* 

Blood  Protein.  Coagulated  blood  protein 
within  the  vascular  lumina  of  stained 
sections  of  fixed  tissues  is  an  artifact 
in  the  sense  that  its  appearance  has  been 
greatly  modified  by  the  technique.  It 
is  sometimes  made  up  of  particles  of 
quite  uniform  size  and  has  been 
mistaken  for  masses  of  microorganisms ; 
but  it  docs  not  exhibit  both  acidophilic 
and  basophilic  staining  reactions  sug- 
gestive of  cytoplasmic  and  nuclear 
components. 

Blood  Smears.  These  should  be  made  on 
slides  rather  than  on  cover  glasses  for 
several  reasons.  A  larger  film  of  blood 
is  thereby  provided  for  examination. 
Smears  on  slides  are  easier  to  make  and 
to  handle.  They  can  be  studied  without 
covering  them  whereas  a  smear  on  a 
cover  glass  cannot  be  moved  about  on 
the  stage  of  the  microscope  unless  it  is 
mounted  smear  side  down  on  a  slide. 
The  colors  are  often  more  permanent  in 
smears  on  slides  which  are  not  covered 
with  cover  glasses.  A  good  way  is  to 
spread  a  thin  film  of  immersion  oil  over 
them.  This  dries  much  more  quickly 
than  balsam  or  any  other  medium  under 
a  cover  glass. 

Slides  of  good  quality  with  ground 
edges  and  scrupulously  clean  are  neces- 
sary (Cleaning  Glassware).  A  finger 
tip  or  ear  lobule  is  first  cleaned  with  95% 
alcohol.  As  soon  as  the  surface^  has 
dried  a  small  puncture  is  made  with  a 
previously  sterilized  needle.  Special 
needles  with  lance  shaped  cutting  ends 
are  better  than  ordinary  pointed  ones. 
A  small  droplet  of  blood  should  appear 
on  slight  pressure.  The  first  is  wiped 
away  with  sterile  gauze  and  the  second 
and  following  ones  are  used.  Unless 
the  blood  is  very  strongly  pressed  out, 
the  differential  count  of  white  cells  will 
not  be  affected.  Some  advise  holding 
the  fingers  in  hot  water  beforehand  to 
produce  a  temporary  hyperemia  in  them 
but  this  is  seldom  advisable.  A  droplet 
of  size  sufficient  to  produce  a  smear  of 
the  desired  thickness  (determined  by 
trials )  should  be  touched  to  the  surface  of 
a  slide  about  3  cm.  from  one  end  conven- 
iently placed  on  a  table.  Immediately 
the  end  of  a  second  slide,  with  its  edge 

•  Original  observations  and  confirmation  of  published 
data  contained  in  tliis  paragraph  were  done  under  a  con- 
tract (No.  AT-ll-l-GEN-10,  Project  2),  between  the 
United  States  Atomic  Energy  Commission  and  the  Uni- 
versity of  California. 


BLOOD  SPECIES  CHARACTERISTICS   48 


BODIAN  METHOD 


squarely  across  the  first  slide  is  brought 
in  contact  with  the  blood  on  the  remote 
side  of  the  drop  from  the  nearest  end  of 
the  first  slide.  The  blood  spreads 
quickly  along  this  edge  toward  the  sides 
of  the  slide  on  the  table  which  is  steadied 
with  the  left  hand.  The  end  edge  of  the 
second  slide  is  slowly  but  steadily 
pushed  the  length  of  the  first  slide  and 
the  blood  is  drawn  out  in  a  thin  layer 
after  it.  The  angle  of  inclination  of  the 
second  to  the  first  slide  determines  the 
thickness  of  the  smear.  It  is  well  to 
make  the  first  smear  at  an  angle  of  about 
45  degrees;  increase  it  for  a  thicker 
smear  and  decrease  it  for  a  thinner  one. 
In  the  making  of  smears  it  is  important 
to  have  plenty  of  elbow  room.  To  rnake 
good  smears  is  a  fine  art  and  a  credit  to 
the  individual. 

Blood  smears,  whether  simply  dried 
by  waving  in  air  or  thereafter  fixed  by 
gently  heating,  retain  their  staining 
properties  for  a  few  days  but  they 
should  be  colored  without  undue  delay. 
However  they  can  be  kept  unstained  or 
stained  if  protected  by  dipping  in 
melted  paraffin  (Queen,  F.  B.,  Am.  J. 
Clin.  Path.,  Techn.  Suppl.,  1943,  7,  50). 
It  is  both  wasteful  and  undesirable  to 
cover  the  whole  slide  with  stain.  Part 
of  the  slide  will  have  to  be  used  for 
record  written  with  a  diamond  pencil. 
Therefore  draw  two  lines  across  the 
slide  near  each  end  with  a  wax  pencil  or 
a  piece  of  paraffin.  The  stain  added 
with  a  dropper  will  cover  only  theinter- 
vening  part.  For  stains  see  Giemsa, 
Wright,  Ehrlich,  Oxidase,  Peroxidase 
and  Gordon's  Silver  Method. 

For  electron  microscopy  of  blood 
cells,  see  Bessis,  M.,  Blood,  1950,  5, 
1083-1098. 
Blood  Species  Characteristics.  References 
to  the  literature  on  the  blood  of  many 
different  kinds  of  animals  and  data  on 
their  differential  counts,  total  counts, 
hemoglobin  concentrations  and  so  on 
are  often  found  of  great  service  (Win- 
trobe,  M.M.,  Clinical  Hematology. 
Philadelphia:    Lea    &    Febiger,    1942, 

703  pp.). 
Blood  Vessels.  These  comprise  structures 
of  different  sorts,  existing  in  a  wide 
variety  of  environments,  which  can  be 
investigated  from  many  angles.  Con- 
sequently to  present  examples  of  avail- 
able techniques  under  the  expected 
headings  involves  a  lot  of  mind-reading. 
The  blood  vessels  of  the  Skin  are  of 
course  the  most  accessible.  Detailed 
methods  for  their  direct  and  indirect 
study  are  presented  by  Sir  Thomas 
Lewis  (The  Blood  Vessels  of  the  Skin 
and  their  Responses.  London:  Shaw 
&  Sons,  1927,  322  pp.). 


But  to  microscopically  examine  all 
the  blood  vessels  of  any  particular  organ 
is  not  possible  in  the  living  state  because 
of  lack  of  accessibility,  thickness  and 
other  mechanical  obstacles.  Resort  is 
therefore  made  to  various  devices  for 
viewing  the  vessels  by  themselves 
unobscured  by  surrounding  tissue.  The 
unwanted  tissue  is  removed  by  corrosion 
when  the  vessels  are  demonstrated  by 
Neoprene  injection.  It  is  simply 
passed  over  when  x-ray  photographs 
are  examined  after  the  vascular  injec- 
tion of  radiopaque  substances  like 
Bismuth  Sulphate  and  Diotrast.  It  is 
rendered  transparent  when  the  vessels 
are  filled  with  easily  visualized  materials 
such  as  Carmine  or  Berlin  Blue,  or  is 
relatively  colorless  after  their  walls  are 
selectively  stained  by  Janus  Green, 
Silver  Citrate  or  Silver  Chloride  Di- 
chlorfluoresceinate.  See  red  lead  and 
glue  method  for  blood  vessels  of  nerves 
(Epstein,  J.,  Anat.  Rec,  1944,  89,  65- 
69). 

Though  the  larger  blood  vessels  are 
too  thick  and  cumbersome  for  micro- 
scopic study  in  vivo,  this  is  not  so  with 
the  smaller  ones.  Indeed  excellent 
moving  pictures  can  be  made  of  them. 
A  film  entitled  "Control  of  Small  Blood 
Vessels"  by  G.  P.  Fulton  and  P.  R. 
Lutz  of  Boston  University  is  very  help- 
ful. The  supravital  method  of  studying 
Nerve  Endings  with  methylene  blue 
must  be  combined  with  careful  dissec- 
tions (Woollard,  H.  H.,  Heart,  1926,  13, 
319-336)  in  order  to  gain  an  impression 
of  the  innervation  of  blood  vessels.  See 
Arteries,  Arterioles,  Capillaries,  Sinus- 
oids, Venous  Sinuses,  Venules,  Arterio- 
venous Anastomoses,  Veins,  Vasa 
Vasorum,  Valves,  Perfusion.  See 
Quartz  Rod  Technique. 
Bodian  Method.  For  staining  nerve  fibers 
in  paraffin  sections  (Bodian,  D.,  Anat. 
Rec,  1937,  69,  153-162;  MacFarland, 
W.  E.  and  Davenport,  H.  A.,  Stain 
Tech.,  1941,  16,  53-58).  The  following 
details  of  this  very  useful  technique 
have  been  supplied  by  Dr.  J.  L.  O'Leary. 
Fix  by  vascular  perfusion,  with  80% 
alcohol  containing  5%  formol  and  5% 
acetic  acid,  or  by  immersion  in  10% 
formalin  or  Bouin's  fluid.  For  boutons 
terminaux,  perfuse  tissue  with  10% 
chloral  hydrate  and  extract  tissue  with 
alcohol  for  several  weeks.  Run  paraffin 
sections  (15/i  or  less)  to  aq.  dest.  Place 
in  1%  Protargol  (Winthrop  Chemical 
Co.)  with  4-6  gms.  of  metallic  copper 
per  100  cc.  (This  can  be  used  only 
once.)  Wash  in  redistilled  water  1 
change.  Transfer  for  10  min.  to  :  hydro- 
chinone,  1  gm.;  sodium  sulfite,  5  gm.; 
aq.  dest.,  100  cc.    Wash  in  redistilled 


BOEDEKER'S  METHOD 


49 


BONE 


water  1  change.  Tone  in  1%  gold  chlo- 
ride with  3  drops  of  glacial  acetic  acid 
per  100  cc,  5-10  min.  Wash  in  re- 
distilled water  1  change.  If  sections 
do  not  have  a  light  purple  color  place 
in  2%  oxalic  acid  until  the  entire  section 
has  the  slightest  blue  or  purplish  tinge. 
Pour  off  as  soon  as  tissue  gets  slightly 
blue.  Remove  residual  silver  salts  in 
5%  sodium  thiosulfate  5-10  min.  Wash, 
dehydrate,  clear  and  mount.  Note : 
the  Coplin  jars  used  must  be  cleaned  in 
Cleaning  Fluid.  The  Bodian  method 
has  been  adjusted  for  the  demonstra- 
tion of  melanin  by  Dublin,  W.  B.,  Am. 
J.  Clin.  Path.,  1943,  7  (Technical  Sec- 
tion), 127. 

Boedeker's  Method,   see   Enamel   matrix. 

Bogoroch,  see  Radioautographic  Technique. 

Boling,  see  Teeth,  Decalcification. 

Bollinger  Bodies,  see  Borrel  Bodies. 

Bone.  A  good  account  of  methods  is 
provided  by  Shipley  (McClung,  pp. 
344-352).  Examination  without  decal- 
cification involves  the  cutting  and 
grinding  of  thin  sections.  The  instru- 
ments used  by  dentists  for  the  making 
of  sections  of  undecalcified  teeth  are  of 
the  greatest  service  and  should  be  pur- 
chased or  borrowed.  If  they  are  not 
available  Grieves'  method  for  dental 
tissues  is  suggested.  In  order  to  de- 
termine the  structure  of  bone  with 
organic  material  removed,  Shipley  ad- 
vises cutting  away  all  soft  parts  after 
which  the  bone  may  or  may  not  be  split. 
Place  in  tap  water,  or  in  a  2%  aq.  gelatin, 
to  which  a  loop  full  of  culture  of  B.  coli 
has  been  added.  After  5-6  days  wash 
in  running  water  24-48  hrs.  in  a  stink 
cupboard.  This  will  dissolve  and  wash 
away  all  organic  material.  Sterilize  the 
bone  by  boiling  or  immersion  in  alcohol. 
Saw  into  sections,  grind  these  to  the 
necessary  thinness  and  polish.  De- 
hydrate in  ether.  Dry  thoroughly  and 
mount  in  balsam.  Routine  examination 
includes  some  method  of  fixation,  de- 
calcification and  staining.  Hematoxylin 
and  eosin  are  recommended,  likewise 
phosphomolybdic  acid  hematoxylin  and 
Mallory's  connective  tissue  stain. 

For  different  structural  components 
special  techniques  are  reauired.  Bone 
corpuscles  may  be  isolated  by  putting  a 
thin  section  of  bone  in  concentrated 
nitric  acid  for  a  few  hours  to  a  day. 
Then  place  the  section  on  a  slide,  cover. 
Pressure  on  the  cover  glass  will  squeeze 
out  ellipsoidal  bone  cells  with  their 
processes  (Shipley).  Bone  lamellae  ca,n 
be  peeled  off  easily  when  decalcified 
bone  has  been  allowed  to  simmer  in 
water  for  several  hours  (Shipley). 
Lacunae  and  canaliculi.  The  easiest 
method    is    to    impregnate    sections   of 


ground  bone  with  0.75%  aq.  silver 
nitrate  for  24  hours.  Wash,  polish  the 
sections  on  a  fine  hone  to  remove  preci- 
pitated silver,  dehydrate  in  alcohol, 
clear  in  xylol  and  mount  in  balsam. 
The  lacunae  and  canaliculi  appear  black 
in  a  yellowish  brown  background.  To 
impregnate  thin  sections  with  acid 
fuchsin,  dry  them  after  extraction  with 
alcohol.  Place  them  in  watch  glasses 
in  a  20%  aq.  sol.  of  acid  fuchsin  in  a 
desiccator  connected  with  a  suction 
apparatus.  Extract  air  for  about  an 
hour  and  close  the  dessicator.  After 
24  hrs.  the  solution  will  have  dried. 
Rub  off  ppt.  on  a  hone,  pass  through 
xylol  and  mount  in  damar  or  balsam 
(Shipley). 

Linings  of  lacunae  and  canaliculi. 
(Schmorl's  method  modified  by  Ship- 
ley.) Employ  a  fixative  not  containing 
mercury.  Decalcify  in  Miiller's  fluid, 
wash  in  running  water,  embed  in  cel- 
loidin  and  section  not  over  10  microns. 
Stain  in  thionin  solution  alkalinised 
by  2  drops  ammonia.  Transfer  with 
glass  needle  to  sat.  aq.  phosphotungstic 
or  phosphomolybdic  acid.  Leave  until 
blue,  gray  or  green.  Place  in  water 
until  sky-blue.  Ammonium  hydroxide 
1  cc.  and  aq.  dest.  10  cc,  3-5  min. 
Several  changes  90%  alcohol.  95%  ale. 
Clear  in  carbolxylol  and  mount  in 
damar  (or  balsam).  This  method  is 
suggested  for  bones  of  children. 

Processes  of  young  osteoblasts  in  grow- 
ing bone.  Shipley  suggests  following 
treatment  of  slices  of  bone  of  a  rickety 
animal.  4%  aq.  citric  acid  20^30  min. 
in  the  dark.  Rinse  in  aq.  dest.  1%  aq. 
gold  chloride  in  the  dark,  20-30  min. 
3%  formic  acid  in  the  dark,  48  hrs. 
Rinse  in  aq.  dest.  and  preserve  in  pure 
glycerin.  Make  frozen  sections,  mount 
in  glycerin  and  ring  with  damar,  balsam, 
paraffin  or  cement.  Keep  specimens  in 
dark  when  not  is  use. 

To  determine  relative  age  of  deposi- 
tion the  following  method  has  proved 
useful  in  senile  osteoporosis.  Saw  sec- 
tions of  bone  not  more  than  0.5  cm. 
thick  and  fix  in  4%  formalin  2-4  days. 
Decalcify  in  6%  isotonic  formalin,  40 
cc,  85%  formic  acid,  60  cc,  and  sodium 
citrate,  5  gm.  changing  every  second 
day  for,  say,  a  week,  that  is  until  they 
become  flexible  and  can  be  penetrated 
by  a  fine  needle.  Embed  in  celloidin 
(slow  method).  Prepare  stain  by  dis- 
solving 30  gm.  potassium  alum  in  1  liter 
hot  water  and  by  adding  1.5  gm.  hema- 
toxylin crystals.  Cool  and  add  1  gm. 
chloral  hydrate.  Ripening  in  sunlight 
to  rich  dark  color  is  hastened  by  addition 
of  crystal  of  potassium  hydroxide. 
Stain   celloidin   sections  about   2   days 


BONE  MARROW 


50 


BOTANICAL  TECHNIQUE 


checking  by  microscopic  examination 
until  some  areas  are  definite  violet  azur, 
others  lighter  or  colorless.  Wash  in  tap 
water  24  hrs.  Stain  in  100  cc.  aq.  dest. 
+  2-3  drops  1%  aq.  eosin  1-2  days 
(uncolored  areas  become  dark  rose 
color).  Dehydrate,  clear  in  xylol  and 
mount  in  balsam.  Old  bone  azur;  new- 
bone  bright  rose  (Belloni,  L.,  Arch. 
Ital.  Anat.  e  Istol.  Path.,  1939,  10, 
622) .  See  Madder  staining  of  new  bone, 
Alizarin  Red  S  staining  of  dentine, 
various  tests  for  Calcium,  and  Ossifica- 
tion, Line  Test  for  vitamin  D  potency. 
Polarized  light  is  excellent  for  the 
demonstration  of  bone  camellae. 

The  micro-diffraction  technique  per- 
mits obtaining  diffraction  patterns  of 
small  areas  such  as  a  single  Haversian 
system.  Using  it  Enstrom,  A.  and 
Zetterstrora,  R.,  Exp.  Cell  Res.,  1951, 
2,  268-274,  found  that  the  orientation 
of  mineral  salts  is  relatively  unchanged 
in  different  physiological  conditions. 
Bone  Marrow.  Microscopic  examination  of 
bone  marrow  in  vivo  has  not  been 
achieved  because  of  the  obvious  techni- 
cal difficulties  involved.  The  best  that 
can  be  done  is  to  study  still  living  cells 
removed  from  bone  marrow  unstained 
or  supravi tally  stained.  The  methods 
are  essentially  the  same  as  for  blood. 
From  humans  samples  can  be  obtained 
by  sternal  puncture  (Young,  R.  H.  and 
Osgood,  E.  E.,  Arch.  Int.  Med.,  1935, 
55,  186-203,  and  many  others).  Pri- 
mitive cells  of  the  erythrocytic  and 
leucocytic  series  can  only  be  identified 
when  hemoglobin  and  specific  granules 
respectively  appear  within  them.  Mi- 
crochemical  tests  for  Hemoglobin  should 
be  more  used.  For  the  granules  the 
methods  of  Giemsa,  Wright,  Ehrlich 
and  others  are  the  best  available. 
Special  techniques  have  been  described 
for  Megakaryocytes  particularly  in  rela- 
tion to  platelet  formation.  The  normal 
megakaryocyte  concentration  is  as- 
pirated human  bone  marrow  is  described 
by  Ebaugh,  F.  G.  Jr.  and  Bird,  R.  M., 
Blood,  1951,  6,  75-80.  To  demonstrate 
the  vascular  pattern  special  methods 
are  required  (Doan,  C.  A.,  Johns  Hop- 
kins Hosp.  Bull.,  1922,  33,  222-226). 
To  reveal  the  nerve  supply  is  par- 
ticularly difficult.  Glaser  (W.,  Ztsch. 
f.  Anat.  u.  Entw.,  1928,  87,  741-745)  has 
described  a  fine  network  accompanying 
the  vessels  but  Doan  and  Langworthy 
(Downey,  p.  1852)  were  less  successful. 
Sternal  bone  marrow  during  first  week 
of  life  (Shapiro,  L.  M.,  and  Bessen,  F. 
A.,  Am.  J.  Med.  Sci.,  1941,  202,  341- 
354).  Bone  marrow  of  normal  adults 
(Plum,  CM.,  Acta  Med.  Scand.,  1941, 
107,  11-52).  See  chapters  by  Sabin  and 


Miller  and  by  Doan  in  Downey's  Hand- 
book of  Hematology,  New  York, 
Hoeber,  1938,  3,  1791-1961  for  details. 
A  method  for  studying  numerical  and 
topographic  problems  in  the  whole 
femoral  marrow  of  rats  and  guinea  pigs, 
with  the  use  of  undecalcified  sections 
(Mayer,  E.  and  Ruzicka,  A.  Q.,  Anat. 
Rec,  1945,  93,  213-231).  A  technique 
for  the  quantitative  estimation  of  mast 
cells  in  bone  marrow  is  advanced  by 
Mota,  Ivan,  Blood,  1951,  6,  81-83. 

Borax  Carmine  (Grenacher).  Make  con. 
sol.  of  carmine  in  borax  (2-3%  carmine 
in  4%  aq.  borax)  by  boiling  for  30  min. 
Allow  to  stand  2-3  days  with  occasional 
stirring.  Dilute  with  equal  volume  70% 
ale,  again  allow  to  stand  and  filter. 
Much  used  for  staining  tissues  in  bulk. 
They  are  colored  for  days  if  necessary, 
transferred  directly  to  acid  ale.  (70% 
ale.  100  cc,  hydrochloric  acid  4  drops) 
in  which  they  assume  a  bright  red  trans- 
parent appearance.  Then  wash  in  alco- 
hol, mount  as  whole  specimens  or  embed 
in  paraffin  and  cut  sections.  Borax 
carmine  can  also  be  employed  to  stain 
sections   (Lee,  p.  146). 

Borax  Ferricyanide,  see  Weigert's. 

Bordeaux,  see  Amaranth. 

Bordeaux  Red  (CI,  88) — acid  Bordeaux, 
archelline  2B,  azo-Bordeaux,  cerasin  R, 
fast  red  B,  BN  or  P — An  acid  mono-azo 
dye  very  widely  employed  in  histology. 

Bordeaux  SF,  see  Amaranth. 

Boron,  see  Atomic  Weights. 

Borrel  Bodies  (Bollinger  bodies)  in  fowl  pox. 
References  to  earlier  staining  methods 
and  directions  for  applying  the  microin- 
cineration technique  with  figures  show- 
ing the  comparative  results  are  given  by 
Danks,  W.  B.  C,  Am.  J.  Path.,  1932,  8, 
711-716.  See  microincineration  of  Mol- 
luscum  bodies  (Van  Rooyen,  C.  E.,  J. 
Path.  &  Bact.,  1939,  49,  345-349). 

Borrelia  Vincenti,  see  Vincent's  Angina. 

Borrel's  Stain.  Fix  in  osmic  acid,  2  gm.; 
platinum  chloride,  2  gms. ;  chromic  acid, 
3  gm. ;  glacial  acetic  acid,  20  cc.  and  aq. 
dest.,  350  cc.  for  24  hrs.  Wash  in  run- 
ning water  several  hours.  Dehydrate, 
clear,  embed  and  section.  Stain  sections 
in  l%aq.  magenta  Ihr.  Then  in  sat.  aq. 
indigo-carmine,  2  parts  and  sat.  aq. 
picric  acid,  1  part.  Wash  in  ale,  dehy- 
drate, clear  and  mount.  The  above  has 
been  partly  taken  from  Lee's  Vade 
Mecum,  p.  433.  Other  more  convenient 
fixatives  will  do  equally  well.  The  stain 
has  been  used  for  the  Borrel  bodies  in 
fowl  pox. 

Botanical  Technique.  Many  of  the  methods 
used  in  animal  histology  are  applicable 
also  in  plant  histology  and  vice  versa. 
Details  are  given  in  a  chapter  by  W.  R. 


BOUIN'S  FLUID 


51 


BRAZILIN-WASSERBLAU 


Taylor  in  McCIung,  p.   155-245.     See 
Plants. 

Bouin's  Fluid.  Sat.  aq.  picric  acid,  75  cc; 
formalin,  25  cc;  acetic  acid,  5  cc.  For 
mammalian  tissues  fix  24  hrs.,  wash  in 
water,  dehydrate  and  embed  in  the  usual 
way.  This  is  the  most  generally  useful 
of  all  fixatives  containing  picric  acid. 
Almost  any  stain  can  be  used  after  it. 
The  picric  acid  need  not  be  altogether 
washed  out  because  it  serves  as  a  desir- 
able mordant.  Giemsa'a  stain  gives 
good  coloration  of  protozoan  parasites 
after  fixation  in  Bouin's  fluid  (Cowdry, 
E.  V.  and  Danks,  W.  B.  C,  Parasitology, 
1933,  25,  1-63) .  The  use  of  this  fixative 
is  specified  under  Argentaffine  Reaction, 
Bodian's  Method,  Elementary  Bodies, 
Foot's  Method,  Gold,  Johnson's  Neu- 
tral Red  Method,  Laidlaw's  Method, 
Liebermann-Burchardt  Reaction,  Mas- 
son's  Trichrome,  Purkinje  Cells,  Tape- 
worm Proglottids,  etc.  It  is  a  fixative 
rapidly  gaining  in  popularity  and  there 
are  naturally  many  modifications.  The 
application  of  Davenport's  silver  tech- 
nique to  Bouin  fixed  tissues  is  described 
by  Foley,  J.  O.,  Stain  Techn.,  1938,  13, 
5-8. 

The  cytological  action  of  Bouin's  fluid 
has  been  investigated  at  the  University 
of  Pennsylvania.  Three  formulae  are 
particularly  recommended  by  McClung 
and  Allen  (McClung,  p.  561).  (1) 
Allen's  fluid:  sat.  aq.  picric  acid,  75  cc; 
formalin  C.P.,  15  cc. ;  glacial  acetic  acid, 
10  cc;  urea,  1  gm.  (2)  The  same  plus 
1  gm.  chromic  acid.  (3)  The  original 
formula  plus  2  gms.  urea  and  1.5  gms. 
chromic  acid.  For  details  regarding  use 
in  study  of  cell  division,  shrinkage,  etc. 
see  Allen,  Ezra,  Anat.  Rec,  1916,  10, 
565-589. 

Bourne,  see  Golgi  Apparatus,  Mitochondria. 

Boutons  Terminaux.  For  this  special  type 
of  nerve  ending  the  methods  given 
under  Nerve  Endings  are  useful,  partic- 
ularly that  of  Bodian.  These  terminal 
buttons  or  swellings  can  be  visualized 
and  their  behavior  determined  in  living 
tadpoles  by  techniques  introduced  by 
Speidel,  C.  C,  J.  Comp.  Neurol.,  1942, 
76,  57-73.  Several  special  methods  for 
their  demonstration  in  fixed  tissues  are 
recommended  by  Gibson  (McClung, 
1950,  pp.  389-398).  Distinction  be- 
tween normal  and  degenerating  in  the 
inferior  olive  of  the  cat  made  by  silver 
methods  (Blackstad,  T.,  Brodal,  A. 
and  Walberg,  F.  Acta  Anat.,  1951,  11, 
461-477). 

Bowie's  Ethyl  Violet-Biebrich  Scarlet  stain 
for  pepsinogen  granules  (Bowie,  D.  J., 
Anat.  Rec,  1935-36,  64,  357-367).  Dis- 
solve 1  gm.  Biebrich  scarlet  in  250  cc. 
aq.  dest.  and  2  gms.  ethyl  violet  in  500 


cc.  Filter  the  former  through  a  rapid 
filter  paper  into  a  beaker  and  then  the 
latter  into  the  same  beaker.  The  end 
point  of  neutralization  is  when  a  little 
of  the  mixture  placed  on  filter  paper  does 
not  show  any  scarlet  color.  Collect  the 
ppt.  of  neutral  dye  by  filtering  and  dry 
it.  To  make  stock  solution  dissolve  0.2 
gm.  in  20  cc.  95%  alcohol.  To  make 
staining  solution  add  1-5  drops  to  50  cc. 
of  20%  alcohol.  Stain  paraffin  sections 
of  Regaud  fixed  gastric  mucosa  in  this 
for  24  hrs.  Wipe  dry  around  edges  and 
blot  with  smooth  filter  paper.  Differ- 
entiate by  covering  section  with  equal 
parts  clove  oil  ancl  xylol.  This  takes 
about  30  min.  and  should  be  observed 
under  microscope.  Pass  through  several 
changes  of  xylol,  rinse  in  benzol  and 
mount  in  benzol  balsam.  Pepsinogen  of 
pepsin-forming  cells,  violet ;  and  parietal 
cells,  red.  Bowie  also  makes  a  crystal 
violet-orange  G  stain  which  does  not 
differ  materially  from  Bensley's  Neutral 
Gentian. 

Brandt's  glycerin  jelly.  Melted  gelatin,  1 
part;  glycerin  I5  parts  plus  few  drops 
carbolic  acid  to  serve  as  a  preservative. 
See  Kaiser's  glycerin  jelly  under  gly- 
cerin. 

Bryan,  see  Ear  Cell  Smears,  Nasal  Cell 
Smears. 

Brazilin  (CI,  1243)  is  a  substance  produced 
from  red  wood  of  Brazil.  Its  formula 
is  like  that  of  hematoxylin  minus  1 
hydroxyl  group  and  in  its  use,  as  well 
as  its  origin,  it  resembles  hematoxylin. 
Ripening  may  be  required  for  both. 
Thus  we  have  an  iron  brazilin  method 
(Hickson,  S.  J.,  Quart.  J.  Micr.  Sci., 
1901,  44,  469-471)  and  O'Leary's  Bra- 
zilin for  myelin  sheaths.  See  also 
Brazilin-Wasserblau  technique  of 
Bensley. 

Brazilin-Wasserblau  for  secretion  ante- 
cedents of  thyroid  gland  (Bensley,  R. 
R.,  Am.  J.  Anat.,  1916,  19,  37-54)  as 
described  later  by  the  Bensleys  (p.  SO) 
is :  To  make  up  the  Brazilin  stain  dis- 
solve 0.05  gm.  in  a  little  aq.  dest.  with 
aid  of  heat  and  add  this  to  100  cc.  1% 
aq.  phosphotungstic  acid.  Ripen  by 
addition  of  2  drops  hydrogen  peroxide. 
Solution  should  not  be  employed  after 
3  days.  Run  paraffin  sections  of  forma- 
lin-Zenker  fixed  thyroids  down  to  aq. 
dest.,  mordant  briefly  in  a  fresh  aq. 
ammonium  stannic  chloride,  and  stain 
in  above  solution  1  or  more  hrs.  Wash 
in  water  and  treat  for  1-5  min.  with  aq. 
dest.,  100  cc.  +  1.0  gm.  phosphomolyb- 
dic  acid  and  0.2  gm.  Wasserblau  (anilin 
blue).  Wash  quickly  in  water,  dehy- 
drate in  absolute  alcohol,  clear  and 
mount.  See  colors  in  R.  R.  Bensley's 
plate.    Nuclear  chromatin,  red;  secre- 


BRAZILWOOD 


52 


BUFFERS 


tion  antecedents  in  pale  blue  droplets; 
mitochondria,  reddish-purple;  connec- 
tive tissue,  blue;  erythrocytes,  orange- 
red;  etc. 

Brazilwood.  The  true  brazilwood  is  of  the 
tree,  Caesalpina  echinata  and  its  varie- 
ties. It  yields  a  dye  stuff  formerly 
much  used  after  an  aluminum  mordant 
for  fabrics,  except  silk,  to  which  it  gave 
a  bright  red  color.  After  potassium 
bichromate  as  a  mordant  the  color  ob- 
tained was  purple  red.  The  term 
"brazil"  is  from  the  arable  word 
"braza"  meaning  fiery  red.  Leggett 
writes  that  increased  use  of  brazilwood 
in  Europe  resulted  from  the  delivery 
of  Asiatic  brazilwood  directly  to  Lisbon 
made  possible  by  Vasco  da  Gama's  dis- 
covery of  an  all  water  route  from  India 
around  the  Cape  of  Good  Hope  and, 
further,  that  three  years  later  a  Portu- 
guese expedition  bound  for  India  missed 
the  mark  and  landed  on  the  north  east- 
ern bulge  of  South  America  where  the 
voyagers  found  many  brazilwood  trees 
so  they  called  the  land  "Terra  de 
Brazil"  (Leggett,  W.  F.,  Ancient  and 
Medieval  Dyes,  Brooklyn:  Chemical 
Publishing  Co.,  Inc.,  1944,  95  pp.). 

Breast,  see  Mammary  Glands. 

Brilliant  Alizarin  Biue(CI,  931),  a  basic  dye 
of  light  fastness  3  to  4.  Gives  darker 
color  than  New  Methylene  Blue  (Emig, 
p.  61). 

Brilliant  Blue  C,  see  Brilliant  Cresyl  Blue. 

Brilliant  Congo  R,  see  Vital  Red. 

Brilliant  Congo  Red  R,  see  Vital  Red. 

Brilliant  Cresyl  Blue  (CI,  877)— brilliant 
blue  C,  cresyl  blue  2RN  or  BBS- 
Commission  Certified.  This  basic  oxa- 
zin  dye  is  used  for  making  Platelet 
Counts  and  for  many  other  purposes. 

Brilliant  Dianil  Red  R,  see  Vital  Red. 

Brilliant  Fat  Scarlet  B,  see  Sudan  R. 

Brilliant  Green  (CI,  662) — diamond  green, 
ethyl  green,  malachite  green  G,  solid 
green  JJO — Commission  Certified.  This 
di -amino  tri-phenyl  methane  dye  is  used 
to  color  culture  media. 

Brilliant  Pink  B,  see  Rhodamine  B. 

Brilliant  Ponceau  G,  see  Ponceau  2R. 

Brilliant  Purpurin  R  (CI,  454).  An  acid 
dis-azo  dye.  Conn  (p.  62)  says  that 
this  is  the  dye  which  Gutstein,  M.,  Zeit. 
f.  Ges.  Exp.  Med.,  1932,  82,  479-524 
called  "brilliant  purpur  R"  and  used  as 
a  vital  stain  for  yeasts. 

Brilliant  Vital  Red.  Use  in  determination  of 
plasma  volume  is  justified,  since  the  dye 
is  not  taken  into  the  erythrocytes  (Gre- 
gersen,  M.  I.,  and  Schiro,  H.,  Am.  J. 
Physiol.,  1938,  121,  284-292).  See  Vital 
Red. 

Brilliant  Yellow  (CI,  364),  an  acid  dis-azo 
dye  of  light  fastness  3  apparently  of 


little  use  as  a  stain  for  paraflRn  sections. 
In  acid  solutions  colors  resinous  tissues 
bright  yellow,  and  in  alkaline  solutions, 
blue-green  algae  a  clear  yellow  (Emig, 
p.  39). 

Bromcresol  Green.     See  Hydrogen  Ion  Indi- 
cators. 

Bromcresol  Purple.  See  Hydrogen  Ion  In- 
dicators. 

Bromine.  According  to  Lison  (p.  110) 
bromine  has  not  been  investigated  histo- 
chemically  in  animal  tissues.  For  its 
detection  in  plants  consult  Mangenot, 
H.  G.,  Bull.  d'Hist.  Appl.,  1927,  4, 
52-71. 

Bromphenol  Blue.  See  Hydrogen  Ion  Indi- 
cators. 

Bromphenol  Red.  See  Hydrogen  Ion  Indi- 
cators. 

Bromthymol  Blue.  See  Hydrogen  ion  Indi- 
cators. 

Bronchial  Aspirates,  see  Papanicolaou  Tech- 
niques. 

Bronchiolar  Epithelium — Written  by  C.  C. 
Macklin,  Dept.  of  Histological  Re- 
search, The  University  of  Western 
Ontario,  London,  Canada.  November 
28,  1951— For  the  dark  and  light  cells, 
as  revealed  by  supravital  silverization, 
see  Ammoniacal  Silver.  For  a  means  to 
demonstrate  the  continuation  of  the 
network  of  surface  silver  lines  from  the 
bronchiolar  epithelium  to  the  alveolar 
walls,  in  en  face  views,  see  Silver  Linea- 
tion.  Mitochondria  in  the  "villus" 
cells  are  abundant  and  often  apparently 
merged  (Macklin,  C.  C,  Anat.  Rec, 
1949,  103,  550;  Rev.  can.  de  Biol.,  1949, 
8,  328;  and  Proc.  Instit.  of  Med.  of 
Chicago,  1950,  18,  78-95— the  26th 
Lewis  Linn  McArthur  Lecture).  They 
are  well  demonstrated  by  Altviann^s 
method  of  anilin  fuchsin  and  picric  acid 
(which  see).  Tumor  formation  in  the 
bronchiolar  epithelium  of  rats  that  have 
been  subjected  to  urethane  (which  see) 
has  been  described  by  Rosin  (Cancer 
Res.,  1949,  9,  583). 

Bronz  Bromo,  see  Eosin  Y. 

Brown  Salt  R,  see  Chrysoidin  Y. 

Brownian  Movement.  Calculation  of  cyto- 
plasmic viscosity  through  measurement 
of  displacement  of  particles  in  Brownian 
movement  gives  results  not  very  differ- 
ent from  determinations  by  the  centrif- 
ugation  method  (Danielli  in  Bourne, 
p.  31). 

Brucella  Ring  Test,  see  Triphenyltetra- 
zolium  Chloride. 

Buffalo  Garnet  R,  see  Erie  Garnet  B. 
Buffers.  For  many  purposes  it  is  essential 
to  use  solutions  buffered  at  a  certain  pH. 
Details  concerning  numerous  buffers  are 
given  by  Clark,  W.  M.,  The  Determina- 
tion of  Hydrogen  Ions.  Baltimore: 
Williams    &    Wilkins,    1928,    717    pp. 


BUNDLE  OF  HIS 


63 


CADMIUM 


French,  R.  W.,  Stain  Techn.,  1930,  5, 
87-90  (see  correction,  1932,  7,  107-108) 
recommends  Sorensen's  phosphate  mix- 
tures and  Palitzsch's  borax-boric  acid 
mixtures  each  over  certain  ranges  of  pH. 
He  emphasizes  the  fact  that  the  addition 
of  buffer  salts  is  known  to  have  a  decided 
influence  in  some  cases  on  the  behavior 
of  the  dyes  irrespective  of  pH.  See 
also  Clark  and  Lubs  BuflFers,  also 
Veronal  Acetate  BuflFers. 

Petrunkevitch,  A.,  Anat.  Rec,  1937, 
68,  267-280  explains  that  aqueous  solu- 
tions of  stains  at  certain  pH's  are  more 
selective  than  alcoholic  ones  and  that 
the  greatest  differentiation  is  obtained 
with  the  former  ones  with  pH  suit- 
ably adjusted  by  addition  of  HCl  or 
NaOH.  Next  in  desirability  come 
stains  dissolved  in  acetate,  phosphate 
and  borate  buffers.  Citrate  buffers  are 
in  his  experience  less  suitable  because  a 
more  diffuse  staining  results  while 
phthalate  buffers  should  not  be  used. 
He  gives  specific  directions  for  the 
preparation  of  solutions  at  pH  of  maxi- 
mum staining  of  acid  fuchsln,  aniline 
blue,  aurantia,  benzoazurine,  eosin  Y, 
light  green,  metanil  yellow,  methylene 
blue,  orange  G,  toluidin  blue,  Wrights 
stain  and  eosin  methylene  blue. 

For  safranin  O,  see  Sawyer,  C.  H., 
Stain  Techn.,  1940,  15,  3-7  and  for  hema- 
to.xylin,  malachite  green  and  eosin  Y, 
Craig,  R.  and  Wilson,  C,  ibid,  1941,  16, 
99-109.  Levine,  N.  C,  ibid,  1940, 
15,  91-112  contributes  useful  data  on 
buffered  stains  in  relation  to  isoelectric 
point  of  cell  components.  Obviously 
the  maximum  intensity  of  staining 
depends  not  only  on  pH  but  also  on 
properties  of  substances  stained  and 
their  treatment  from  beginning  to  end 
of  the  technique.  Lillie,  R.  D.,  Stain 
Techn.,  1941,  16,  1-6  employed  McU- 
vaine  citric  buffers  in  order  to  improve 
Romanowsky  staining  (see  Toluidine 
Blue  Phloxinate)  after  various  fixatives. 
See  McJunkin-Haden  Buffer.  Use  of 
buffered  thionin  as  Nissl  stain  (Windle, 
W.  F.,  Rhines,  R.  and  Rankin,  J.,  Stain 
Techn.,  1943,  18,  77-86).  For  buffering 
in  connection  with  silver  impregnation 
see  Davenport,  H.  A.,  McArthur,  J. 
and  Bruesch,  S.  R.,  Stain  Techn.,  1939, 
14,  21-26;  Silver,  M.  L.,  Anat.  Rec, 
1942,  32,  507-529.  When  accuracy  is 
essential  check  the  actual  pH  of  the 
solution  to  which  buffers  have  been 
added  by  the  glass  electrode  method 
which  anyone  can  learn  to  use  in  a  few 
hours  and  which  gives  the  answer  very 
quicklv.  See  Hydrogen  Ion  Indicators. 
Bundle  of  His,  see  Todd,  T.  W.,  Cowdry's 
Special  Cytology,  1932,  2,  1173-1210. 


Burettes.  For  accurate  volume  determina- 
tions use  Microliter  Burettes. 

Burns.  Methods  of  experimental  produc- 
tion, vital  staining  with  trypan  blue, 
and  histological  changes  (Ham,  A  W., 
Ann.  Surg.,  1944,  120,  689-697. 

Butter  Fat,  reactions  in  tissue  to  fat  stains 
after  various  fixations  (Black,  C.  E., 
J.  Lab.  &  Clin.  Med.,  1937-38,  23, 
1027-1036). 

Butyl  Alcohol,  see  n-Butyl  and  Tertiary 
Butyl. 

Buzaglo's  Connective  Tissue  Stain.  (Bu- 
zaglo,  J.  H.,  Bull.  d'Hist.  Appl.,  1934, 
11,  40-43).  This  method  is  intended  to 
replace  that  of  Van  Gieson.  Solutions 
required:  (1)  Gallocyanin  (Hollborn, 
2264).  Boil  0.1  gm.  in  100  cc.  5%  aq. 
chrome  alum  for  10  min.  After  cooling 
make  up  to  100  cc.  with  aq.  dest.,  filter 
and  adci  a  little  formalin  to  filtrate.  (2) 
Orcein  (Hollborn,  2466).  Dissolve  1 
gm.  in  100  cc.  acid  alcohol  (70%  alcohol, 
100  cc.  +  1  cc.  hydrochloric  acid  stand- 
ard). (3)  Acid  alizarin  blue  (Hollborn, 
2559).  Boil  for  10  min.  5  gm.  in  100  cc. 
10%  aq.  aluminum  sulphate.  After 
cooling  make  up  to  100  cc,  filter  and  add 
formalin.  (4)  Alizarine-viridine  (Holl- 
born, 2035).  Dissolve  0.2  gm.  in  100 
cc.  aq.  dest.  acidulated  to  pH  5.8  with 
hydrochloric  acid.  He  advises  fixation 
in  formalin,  Maximow's  fluid,  Susa  or 
Hoffker  (of  which  he  does  not  give 
composition).  Pass  sections  (presum- 
ably paraffin)  down  to  aq.  dest.  Stain 
nuclei  in  gallocyanin  as  deeply  as  pos- 
sible 5  times,  24  hrs.  Rinse  twice  in 
aq.  dest.  Stain  elastic  fibers  in  orcein, 
then  aq.  dest.,  3  times,  5  min.  Stain 
muscle  in  acid  alizarin  blue,  7  min.,  aq. 
dest.  twice.  Differentiate  in  5%  aq. 
phosphomolybdic  acid  25-30  min.,  aq. 
dest.  twice.  Stain  collagen  in  alizarine 
viridine  7  min.  Blot  with  4  layers  filter 
paper.  95%  ale  96%  ale  Carbol- 
xylol,  2  changes  xylol.  Balsam.  Nu- 
clei, dark  blue ;  elastic  fibers,  red  brown ; 
muscle  and  epithelium,  pale  blue  violet ; 
collagen,    mucus,    cartilage,    shades    of 

§reen;  myelin  sheaths,  rose;  axis  cylin- 
ers,  dark  blue;  erythrocytes,  red 
brown . 

Cabot  Rings.  Ring-shaped  formations  in 
erythrocytes  which  color  red  with 
Wright's  Blood  Stain.  The  color  in 
the  concavity  of  the  ring  is  the  same 
as  that  in  the  rest  of  the  cell.  Oc- 
casionally observed  especially  in  per- 
nicious anemia  and  lymphatic  leukemia. 

Cadmium.  The  chloride  is  employed  in 
fi.xation  of  Golgi  apparatus  prior  to  silver 
impregnation  (Aoyama,  F.,  Zeit.  wiss. 
mikr.,  1929,  46,  489-191).  See  comment 
by  Baker  (Bourne,  p.  19)  on  this  and 
use  by  Ciaccio  of  cadmium  nitrate  to 


CAJAL'S 


54 


CALCIUM 


render  phospho-  and  galactolipines  less 
soluble.  Bourne  (p.  106)  refers  to 
Joyet-Lavergne's  claim  that  cadmium 
lactate  reacts  with  glutathione  in  the 
cell  producing  a  cadmium  glutathione 
compound  which  is  microscopically 
visible. 

Cajal's.  Properly  the  name  should  be  listed 
as  Ramon  y  Cajal.  1.  Brom-formol- 
silver  method  for  neuroglia.  Details 
supplied  by  Dr.  J.  L.  O'Leary.  Fix 
small  fresh  pieces,  3-15  days,  in:  aq. 
dest.,  85  cc;  formalin,  15  cc;  ammo- 
nium bromide,  2  gm.  Cut  25/i  frozen 
sections  and  return  to:  aq.  dest.,  50  cc; 
formalin,  6  cc;  ammonium  bromide, 
3  gm.  for  4-6  hrs.  at  30-38°C.  or  for  8-10 
hrs.  at  room  temperature.  Wash  for  a 
few  seconds  in  aq.  dest.  Place  in  the 
following  fluid  in  a  porcelain  dish  and 
heat  over  the  flame:  aq.  dest.,  10-15  cc; 
ammoniacal  silver  oxide,  5  cc;  pyridine 
C.P.,  4-5  drops.  (To  prepare  silver 
oxide  solution:  Take  10  cc.  10%  silver 
nitrate,  add  12  drops  40%  NaOH.  Col- 
lect the  ppt.,  wash  5-6  times  with  aq. 
dest.,  then  add  ppt.  to  a  beaker  con- 
taining 60-70  cc.  aq.  dest.  Redissolve 
with  least  quantity  of  ammonia  neces- 
sary. If  too  much  ammonia  is  added, 
results  are  bad.)  Remove  when  sec- 
tions have  reached  a  tobacco  brown 
color.  Wash  through  2  changes  aq. 
dest.  not  more  than  5  sec.  in  all.  Re- 
duce in  5%  formalin  for  2-3  min.  Tone 
with  0.2%  aq.  gold  chloride  and  fix  in 
5%  aq.  sodium  hyposulfite.  After 
washing  carry  to  95%  alcohol,  carbol- 
xylol,  xylol  balsam.  See  Microglia  and 
Oligodendroglia. 

2.  Chloral  hydrate  method  as  de- 
scribed by  Willard,  D.  M.,  Quart.  J. 
Micr.  Sci.,  1935-36,  78,  475-485  for 
innervation  of  adrenal.  Fix  for  24  hrs. 
in :  chloral  hydrate,  2.5 gm. ;  95%  alcohol, 
40  cc. ;  aq.  dest.,  40  cc. ;  pyridine,  20  cc 
Wash  in  aq.  dest.  until  smell  of  pyridine 
disappears.  97%  alcohol,  24  hrs.  Wash 
again  in  aq.  dest.  and  transfer  to  2.5% 
aq.  silver  nitrate  at  37°C.  for  9-12  days 
(longer  times  better  for  nerve  cells). 
Wash  for  1  min.  in  aq.  dest.  Reduce  for 
12-24  hrs.  in:  hydroquinone,  1  gm.; 
neutral  formol,  10  cc;  aq.  dest.,  90  cc. 
Dehydrate  rapidly,  embed  in  paraffin 
and  cut  15-30m  sections.  Nerve  fibers, 
black;  background,  yellow. 

Cajal  Silver  Methods.  These  depend 
mainly  on  silver  impregnations  reduced 
by  photographic  developers  such  as 
hydroquinone.  They  have  all  been 
very  greatly  improved  by  a  preliminary 
fixation  and  in  other  ways  and  have 
played  a  leading  r61e  in  neurology.  See 
Ranson  pyridine  method  and  other 
modifications  given   by   Addison    (Mc- 


Clung,  pp.  452-463).  Many  techniques 
spring  from  a  combination  of  Cajal  and 
Bielchowsky  methods. 

Calcareous  deposits.  Vital  staining  with 
Alizarin  Red  S  (Ham,  A.  W.,  Arch. 
Path.,  1932,  14,613-626). 

Calciferol,  see  Vitamin  D2. 

Calcium.  There  is  no  absolutely  specific 
microchemical  test  for  calcium  in  sec- 
tions. A  critical  account  by  Cameron 
(G.  R.,  J.  Path,  and  Bact.,  1930,  33, 
929-955)    affords    instructive    reading. 

1.  von  Kossa  test.  Sections  are  trans- 
ferred from  aq.  dest.  to  10%  silver 
nitrate  and  exposed  to  bright  light  for 
30  min.  or  more.  Wash  carefully  in  aq. 
dest.  Mount  in  glycerin,  or  dehydrate 
clear  and  mount  in  balsam.  Inorganic 
material  in  most  cases  calcium  phos- 
phate or  carbonate  is  deep  black.  See 
comments  of  Gomori,  G.,  J.  Mt.  Sinai 
Hosp.,  1945,  11,  317-326.  Test  has  been 
adapted  to  investigation  of  bone  by 
McLean,  F.  C.  and  Bloom,  W.,  Anat. 
Rec,  1940,  78,  333-359. 

2.  Alizarin.  Sections  from  aq.  dest. 
are  stained  in  1%  aqueous  alizarin  S 
(sodium  alizarin  sulphonate)  or  in  1% 
alcohol  tetra-hydroscy-anthraquinon  (or 
anthrapurpurin)  for  an  hr.  or  more. 
They  are  then  differentiated  in  1  part 
concentrated  ammonia  and  9  parts 
absolute  alcohol.  This  is  followed  by 
rapid  washing  in  acid  alcohol  (hydro- 
chloric acid  5  cc,  95%  alcohol  95  cc). 
It  may  be  desirable  to  alternate  alkali 
and  acid  alcohols  2  or  3  times.  Wash 
thoroughly  in  aq.  dest. ;  dehydrate  clear 
and  mount.  The  alizarin  forms  a  fast 
compound  with  earthy  salts  especially 
calcium  more  easily  in  young  than  in 
old  bones.  Substances  may  exist  in  the 
tissues  that  inhibit  the  combination 
(see  Bone,  Madder  staining). 

3.  Hematoxylin.  This  is  not,  as  is 
generally  supposed,  a  stain  for  calcium 
though  it  may  color  calcium  as  well  as 
other  materials  when  mordanted  with 
chromium  salts  or  alum.  According  to 
Cameron,  in  bone,  "staining  with 
hematoxylin  is  dependent  on  the  essen- 
tial ground  substance  and  the  presence 
of  certain  heavy  metals  especially  iron 
chromium  and  aluminum ;  it  has  no 
direct  relation  to  calcium  salts."  He 
thinks  that  areas  of  pathological  calci- 
fication which  stain  deeply  with  alum 
hematoxylin  do  so  because  of  the  pres- 
ence of  free  iron. 

4.  Fluorescence  x-radiation.  Used  for 
thin  sections  of  undecalcified  bone.  It 
is  not  feasible  to  magnify  much  but  the 
method  is  said  to  be  almost  specific  for 
calcium  (Dershem,  E.,  Proc  Nat.  Acad. 
Sci.,  1939, 25,  6-10). 

5.  Cretin,    A.,    Bull.    d'Hist.    Appl., 


CALCOZINE  RED  CG 


55 


CANNULAS 


1924,  1,  125-132  has  proposed  a  blue 
color  reaction  with  trioxymethylene  and 
gallic  acid.  In  comparison  with  calcium 
strontium  and  barium  show  green,  mag- 
nesium rose  and  iron  brownish  violet. 
See  Lillie  p.  251  for  details. 

6.  With  magnesium,  but  free  from  all 
other  minerals  in  muscle,  by  electron 
microscope  (Scott,  G.  H.  and  Packer, 
D.  M.,  Anat.  Rec,  1939,  74,  17-45). 

7.  Sulfuric  and  oxalic  acid.  Lillie  (p. 
251)  refers  to  the  granular,  opaque  and 
white  appearance  of  unstained  calcium 
deposits.  If  the  material  is  mounted 
under  a  cover  glass  in  water  and  sulfuric 
acid  is  drawn  through  by  removal  of 
water  from  one  side  with  filter  paper  the 
deposits  dissolve  but  colorless  mono- 
clinic  calcium  sulphate  (gypsum)  crys- 
tals are  formed  while  if  5-10%  oxalic 
acid  is  used  typical  cubic  calcium 
oxalate  crystals  appear.  Carbonates, 
on  the  contrary,  are  dissolved  by  run- 
ning through  acetic  acid  with  the  forma- 
tion of  gas  bubbles.  Phosphates  are 
dissolved  by  the  acetic  acid  without 
gas  bubbles.  The  development  of  these 
crystalline  calcium  salts  is  of  course 
specific. 

8.  Microincineration.  To  distinguish 
between  the  dense  white  ashes  of  cal- 
cium and  magnesium  it  is  suggested  that 
a  "microdrop"  of  0.1  N  hydrochloric 
acid  be  added,  plus  a  similar  drop  of 
0.1  N  sulphuric  acid  in  order  to  produce 
needle  like  calcium  sulphate  crystals 
(Moreau,  P.,  Bull,  histol.  appl.  physiol. 
et  path,  et  tech.  microscop.,  1931,  8, 
245-248).  As  follow  up  on  the  above 
microchemical  methods  a  curvette 
colorimetric  by  Sendary,  J.  Jr.,  J.  Biol. 
Chem.,  1942,  144,  243-258;  1944,  152, 
539-556  and  a  titrimetric  technique  by 
Sobel,  A.  E.  and  Kaye,  I.  A.,  Ind.  Eng. 
Chem.,  and  Ed.,  1940,  12,  118-120  are 
possibilities.  By  the  latter  as  little  as 
4  Mgm.  of  calcium  can  be  measured  in 
a  5  ml.  burette.     See  Glick,  p.  273. 

Calcozine  Red  CG,  see  Rhodamine  CC. 

Camphor,  see  Sandarac. 

Camsal  is  a  mixture  of  camphor  and  salol 
used  by  McClung  in  making  San- 
darac. 

Canada  Balsam,  see  Balsam. 

Canary  Yellow,  see  Auramin. 

Cancer.  Because  cancer  and  other  malig- 
nant tumors  can  develop  in  so  many 
organs  and  tissues  that  contain  inter- 
mitotic  or  reverting  postmitotic  cells 
(Cell  Classification),  techniques  de- 
signed to  compare  the  malignant  cells 
with  their  non-malignant  prototypes 
are  altogether  too  numerous  to  mention. 
They  will  be  found  under  the  several 
tissues:  Pancreas,  Connective  Tissue 
and  so  forth. 


There  is  no  known  technique  which 
will  reveal  a  structure  or  a  substance  in 
cancer  cells  wholly  absent  in  normal 
cells  of  the  sort  from  which  the  particu- 
lar cancer  cells  have  originated. 
Neither  can  the  reverse  be  demon- 
strated, that  is  something  absent  in 
cancer  cells  and  present  in  normal  ones. 
Available  methods  are  only  capable  of 
demonstrating  quantitative  differences 
in  properties  exhibited  by  normal  and 
malignant  cells.  Properties  of  cancer 
cells  have  been  systematically  reviewed 
by  Cowdry,  E.  V.,  Arch.  Path.,  1940, 
30,  1245-1274.  Yet  the  Dopa  Reaction 
is  of  service  in  diagnosis  of  Melano- 
carcinoma. 

Frequently  it  is  helpful  to  excise  can- 
cers and  transplant  them  into  other 
situations  such  as  the  Anterior  Chamber 
of  the  Eye  where  they  can  conveniently 
be  studied.  The  Tissue  Culture 
method  is  of  great  service,  likewise 
Motion  Pictures  made  of  cancer  cells. 
The  most  recently  developed  line  of 
investigation  is  by  Radioactive  Isotopes. 
See    also    Papanicolaou   Techniques. 

Candida  Albicans.  Method  for  demonstrat- 
ing this  parasite  with  fat  soluble  dyes 
in  frozen  sections  by  Fuentes,  C,  J. 
Bact.,  1946,  51,  245-246. 

Cannulas.  Glass  cannulas  are  required  for 
insertion  into  blood  vessels  in  the  Per- 
fusion technique.  To  make  one  of  about 
the  size  for  guinea  pig's  thoracic  aorta 
file  and  break  6  mm.  outside,  4  mm. 
inside  diameter  soft  glass  tubes  into 
pieces  about  15  cm.  long.  (Pyrex  of 
this  size  will  do.  It  requires  a  little 
more  heating.)  Take  one  of  these, 
place  middle  in  gas  flame  rotating  it  so 
as  to  heat  it  evenly.  When  fairly  soft 
remove  from  the  flame,  draw  the  ends 
apart  to  a  distance  of  about  50  cm.  and 
hold  until  solid.  File  and  break  in  the 
middle.  With  a  little  practice  this  will 
give  two  tubes,  each  tapering  evenly 
from  the  6  mm.  outside  diameter  to 
about  2-3  mm.  within  a  distance  of 
approximately  3  cm.  Next  bring  the 
tube  where  it  has  a  diameter  of  2-3  mm. 
near  to  a  fine  flame,  like  that  of  a  small 
alcohol  lamp.  Let  it  get  soft  and  pull 
just  enough  to  produce  a  slight  narrow- 
ing to  be  used  later  to  prevent  the 
thread  employed  to  tie  the  cannula  in 
the  vessel  from  slipping.  Then  fracture 
with  file  and  break  off  the  thin  end 
about  4  mm.  beyond  the  constriction 
and  distant  from  the  wide  part  of  the 
tube.  If  this  break  can  be  made  at  an 
acute  angle  to  the  length  of  the  tube,  so 
much  the  better;  because  then  one  rim 
of  the  small  end  of  the  tube  will  project 
out  beyond  the  rest  which  will  facilitate 
its  insertion  into  the  vessel  to  be  cannu- 


CAPILLARIES 


56 


CAPRI  BLUE 


lated.  When  the  break  is  made  across 
the  tube,  at  right  angles,  the  rim  on  one 
side  can  be  ground  down  on  a  water  stone 
so  as  to  produce  a  similarly  projecting 
lip.  In  either  case  it  is  necessary  to 
remove  sharp  cutting  edges  from  both 
ends  of  the  cannula  by  smoothing  in  a 
flame.  The  6  mm.  wide  body  of  the 
cannula  should  be  3-4  cm.  long  for  con- 
venient attachment  of  rubber  tube. 
Obviously  larger  cannulas  are  required 
for  larger  vessels.  Those  for  Micro- 
injection are  very  much  smaller,  made 
of  hard  glass  and  do  not  require  to  be 
tied  in. 
Capillaries.  In  living  humans  these  can 
best  be  seen  in  the  skin  by  the  method 
of  Capillaroscopy.  Render  the  epider- 
mis at  the  root  of  the  finger  nail  trans- 
lucent by  addition  of  a  drop  of  highly 
refractive  oil  and  examine  directly  at 
fairly  high  magnification  the  capillary 
loops  in  the  dermal  papillae.  It  is 
possible  to  record  their  changes  by 
making  moving  pictures  through  a  long 
period  of  time.  See  review  by  Wright, 
I.  S.  and  Druryee,  A.  W.,  Arch.  Int. 
Med.,  1933,  52,  545-575.  See  also 
Gingiva. 

In  living  mammals  the  most  favorable 
site  in  which  to  watch  capillaries  at  high 
magnification  is  in  the  transparent 
chambers  of  the  Sandison's  Technique. 
For  shorter  periods  they  can  be  studied 
in  the  displaced  but  living  pancreas  by 
the  methods  of  Covell,  W.  P.,  Anat. 
Rec,  1928,  40,  213-223  and  O'Leary,  J. 
L.,  ibid,  1930,  45,  27-58.  Some  changes 
in  Permeability  of  living  capillaries  are 
evidenced  by  the  trypan  blue  capillary 
permeability  test.  If  microdissection 
is  intended  and  a  shift  to  the  tongues 
and  nictitating  membranes  of  frogs  is 
made  consult  Zweifach,  B.  W.,  Anat. 
Rec,  1934,  59,  83-108,  and  Am.  J.  Anat., 
1937,  60,  473-514.  The  results  have 
been  recorded  in  moving  pictures. 
Supravital  staining  of  the  tissues  just 
mentioned  with  janus  green  (Bensley, 
R.  R.,  and  Vimtrup,  B.,  Anat.  Rec, 
1928,  39,  37-55)  affords  beautifully  clear 
views  of  the  muscular  elements  of 
arterioles  grading  into  capillaries.  See 
Perivascular  Cells,  Rouget  Cells. 

For  investigations  on  the  topographic 
arrangement  of  capillaries  arterial  injec- 
tions with  Carmine,  Berlin  Blue  or 
some  other  easily  recognizable  material 
followed  by  clearing  by  the  Spalteholz 
method  may  be  helpful.  When  however 
any  fluid  is  injected,  under  great  pres- 
sure, into  a  fresh,  relaxed  tissue  that  can 
easily  swell  there  is  a  chance  that  an 
exaggerated  idea  of  the  capillaries  will 
be    conveyed.     In    resting    muscle  for 


instance    a    large    proportion    of    the 
capillaries  are  collapsed  (Krogh). 

The  structure  of  the  endothelial 
capillary  wall  is  relatively  uncompli- 
cated. The  outlines  of  the  endothelial 
cells  are  nicely  revealed  in  pink  by  the 
Silver  Chloride  Dichlorfluorescineate 
technique  or  in  black  by  simply  treating 
with  silver  nitrate.  Nuclear  and  cyto- 
plasmic structure  can  be  brought  out  by 
methods  used  for  other  tissues.  Nerve 
fibers  closely  accompany  most  capil- 
laries. The  existence  of  actual  nerve 
endings  on  the  wall  is  debated.  The 
most  convincing  looking  preparations 
of  human  tissues  have  been  secured  by 
Stohr,  Ph.,  Zeit.  f .  Zellf .  u.  Mikr.  Anat., 
1926,  3,  431-448  who  employed  a  modifi- 
cation by  Gros  of  the  Bielchowsky 
silver  technique  (see  particularly  his 
Fig.  2).  See  Sinusoids. 
Capillaries    of    brain.     Lepehne-Pickworth 

Eeroxidase  method  simplified  by  Camp- 
ell  and  Alexander  (Mallory,  p.  257). 
Fix  for  1-3  weeks  in  10%  formalin.  To 
make  required  solution  dissolve  0.1  gm. 
benzidine  in  0.5  cc  glacial  acetic  acid 
and  add  20  cc.  aq.  dest.  Dissolve  0.1 
gm.  sodium  nitroprusside  in  10  cc.  aq. 
dest.  and  add  benzidine  solution.  Add 
aq.  dest.  to  100  cc.  In  case  a  ppt.  forms 
filter  it  out.  Solution  must  be  fresh. 
Cut  frozen  sections  200-300/:(  and  wash 
in  aq.  dest.  H  hr.  Change  to  above 
described  solution  for  5  hr.  at  37 °C. 
agitating  often.  Wash  in  aq.  dest.  10 
sec.  and  transfer  to  100  cc.  aq.  dest.  + 
2-3  drops  30%  hydrogen  peroxide  for 
i  hr.  at  37°C.  shaking  frequently. 
Wash  in  aq.  dest.  and  dehydrate  in  70%, 
95%  and  absolute  alcohol.  Clear  in 
xylol  and  mount  in  balsam.  Blood  ves- 
sels black  in  almost  colorless  back- 
ground. This  method  has  the  advantage 
of  not  involving  vascular  perfusion. 
See  comparison  of  injection  and  red  cell 
staining  methods  for  quantitative  study 
of  capillaries  of  central  nervous  system 
(Drummond,  S.P.,  Anat.  Rec,  1944,  89, 
93-106).  Microinjection  of  capillaries, 
Chambers,  R.  W.  and  Kopac,  M.  J., 
McClung's  Microscopical  Technique, 
1950,  p.  530. 

Capillary  Fragility  Tests.  Discussion  (Gold- 
man, L.  and  Corrill,  E.  M.,  J.  Invest. 
Dermat.,  1945,6,  129-147). 

Capillary  Respirometry.  The  development 
of  the  techniques  is  described  bv  Tobias, 
J.  M.,  Physiol.  Rev.,  1943,  23,  51-75. 
A  differential  respirometer  is  described 
by  Cunningham,  B.  and  Kirk,  P.  L., 
J.  Gen.  Physiol.,  1940,  24,  135-149. 
Whole  problem  is  discussed  by  Glick, 
pp.  314-326. 

Capri  Blue  (CI,  876),  a  basic  dye  of  light 


CAPSULE  STAIN 


57 


CARBOL-THIONIN 


fastness  3.  0.1  gm.  in  95  cc.  aq.  dost. 
+  5  cc.  5%  aq.  ammonium  alum  +  0.5 
cc.  acetic  acid  stains  plant  tissues  blue 
or  black.  Can  be  employed  in  prefer- 
ence to  Cyanine.  Should  stain  well 
after  Erythrosin  (Eniig,  p.  58). 
Capsule  stain.  1.  Hiss'  method  for  smears 
(McClung,  p.  145).  Dry  organisms  in 
ascitic  or  serum  medium  on  slides. 
Stain,  slightly  heated  in  5-10  cc.  satu- 
rated ale.  gentian  violet  or  basic  fuchsin 
made  up  to  100  cc.  aq.  dest.,  few  sec. 
Wash  off  dye  with  20%  aq.  copper  sul- 
phate crystals.  Dry  by  blotting.  See 
also:  Huntoon,  F.  M.,  J.  Bact.,  1917,  2, 
241.     See  Pasteurella. 

2.  W.  H.  Smith's  method  for  sections 
(Mallory,  p.  275).  Cover  deparaffin- 
ized  sections  of  Zenker  fixed  material 
with  Anilin  Crystal  Violet  (either 
Ehrlich's  or  Stirling's).  During  few 
seconds  warm  by  passing  slide  through 
flame  2  or  3  times.  Wash  in  Gram's 
Iodine  solution  followed  by  formalin 
(commercial).  Decolorize  in  95%  ale. 
Quickly  wash  again  in  Gram's  iodine. 
Cover  with  aniline  green  eosin  and  heat 
as  before.  To  make  this  shake  1  part 
aniline  green  with  200  parts  3-6%  aq. 
eosin  yellowish  W.S.  and  after  1-2  hrs. 
remove  ppt.  bj'  filtering.  Wash  in  aq. 
dest.  Dehydrate  in  95%  and  abs.  ale, 
clear  in  xylol  and  mount  in  balsam. 
Bacterial  capsules,  red;  Gram  positive 
bacteria,  blue.  Mallory  says  that  a 
stronger  iodine  may  be  desirable  (iodine, 
1  gm.,  potassium  iodide,  2gm.  ;aq.  dest., 
100  cc.)  and  that  the  times  must  be 
suited  to  each  preparation. 

3.  Churchman's  (S.  Bayne-Jones  in 
Simmons  and  Gentzkow,  p.  385). 
Flood  air-dried  films  with  Wright's 
stain  and  leave  until  almost  evaporated 
to  dryness.  Original  blue  of  stain  is 
replaced  by  pinkish  color.  Wash 
quickly  in  water,  or  in  Clark  and  Lubs 
buffer  pH  6.4-6.5.  Do  not  blot  but  dry 
with  fan.  Body  of  organisms,  blue; 
capsular  material,  purplish-pink;  often 
surrounded  by  capsular  membrane  or 
peripheral  zone,  deep  purplish-pink. 

Capsule  Substance.  This  obviously  is  un- 
der investigation  in  many  sorts  of  cells 
and  the  methods  introduced  for  one 
kind  may  well  be  of  service  for  others. 
See  Cell  Membrane  for  physical  proper- 
ties, thickness,  etc.  See  Adhesiveness 
and  Acid  Fast  Bacilli.  Under  Gram 
Stains  is  a  description  of  the  mechanism 
of  their  action  which  includes  data  ob- 
tained by  use  of  the  enzyme,  ribonu- 
clease,  on  the  nature  of  walls  of  Gram 
positive  bacteria.  Under  Enzymes,  see 
enzymatic  destruction  of  capsules  of 
pneumococci. 

Carbanthrene  Blue  GO  (CI,  1113),  Carban- 


threne  Brilliant  Orange  RK,  Carban- 
threne Jade  Green  (CI,  1101),  Carban- 
threne Red  BN  (CI,  1162)  Carbanthrene 
Red  BN  (CI,  1162)  and  Carbanthrene 
Violet  2R  (CI,  1104)  all  of  NAC  are 
referred  to  by  Emig,  p.  64. 

Carbohydrates,  see  Starch. 

Carbol-Anilin  Fuchsin  methylene  blue 
method  for  Negri  bodies  (Goodpasture, 
E.  W.,  Am.  J.  Path.,  1925,  1,  547-582). 
Fix  in  Zenker's  fluid  24  hrs.  (not  Helly's 
fluid).  Color  for  10-30  min.  in  mixture 
made  by  adding  1  cc.  of  pure  phenol  and 
1  cc.  of  anilin  oil  to  100  cc.  of  stock  0.5% 
basic  fuchsin  in  20%  alcohol.  Wash  in 
running  water,  blot  with  filter  paper  and 
decolorize  with  95%  alcohol  until  sec- 
tions become  pink.  Then  wash  in  water 
and  stain  with  Loeffler's  methylene 
blue,  15-60  sec.  Wash  again  in  water. 
Dehydrate  and  destain  for  few  sec.  in 
absolute  alcohol,  clear  in  xylol  and 
mount  in  balsam.  Negri  bodies,  crim- 
son; background,  blue.  Also  excellent 
for  Borrel  Bodies. 

Carbol-Crystal  Violet.  Because  the  solu- 
tions as  prescribed  in  Nicolle's  original 
formula  for  carbol  gentian  violet  tend 
to  gelatinize,  the  following  formula  is 
recommended  by  Conn,  H.  J.,  Stain 
Techn.,  1946,  21,  31-32:  Mix  solution  of 
0.4  gm.  crystal  violet  C.  C.  in  10  cc. 
95%  ethyl  alcohol  with  solution  of  1  gm. 
phenol  in  100  cc.  aq.  dest. 

Carbol-Fuchsin,  The  original  formula  of 
Ziehl  has  been  much  modified.  Ziehl- 
Neelsen  is  sat.  ale.  basic  fuchsin,  10  cc. ; 
5%  aq.  carbolic  acid,  90  cc.  Verhoeff 
(F.  H.,  J.A.M.A.,  1912,  58,  1355)  advises 
basic  fuchsin,  2  gm.;  abs.  ale,  50  cc; 
melted  carbolic  acid  crystals,  25  cc. 
McClung  (p.  136)  suggests  mixing  10  cc. 
3%  basic  fuchsin  (90%  dye  content) 
with  95  cc.  5%  aq.  phenol.  The  im- 
portant thing  is  the  character  of  the 
fuchsin  not  its  concentration  relative  to 
carbolic  acid.  Carbol-fuchsin  is  em- 
ployed in  stains  for  Acid  Fast  Bacilli. 
DeipoUi,  G.  and  Pomerri,  G.,  Mon. 
Zool.  Ital.,  1938,  49,  123-124  have  ad- 
vised its  use  as  follows  for  Nissl  Bodies. 
Fix  small  pieces  in  95-98%  alcohol  or  in 
10%  formalin  water  or  in  physiological 
saline  24  hrs.  or  longer.  Stain  deparaf- 
finized  sections  3-4  min.  in  carbol- 
fuchsin  (basic  fuchsin,  0.2  gm.;  cone, 
phenol,  1  cc;  95%  ale,  2  cc. ;  aq.  dest. 
20  cc.)  2.5  cc. ;  aq.  dest.,  100  cc. ;  glacial 
acetic  acid,  0.5  cc.  Wash  rapidly  in  aq. 
dest.  and  destain  in:  aq.  dest.,  100  cc; 
formalin,  1  cc. ;  glacial  acetic  acid,  1  cc. 
Wash  in  aq.  dest.,  dehydrate  in  alcohols, 
clear  in  xylol  and  mount  in  neutral 
balsam.  Nissl  bodies  and  nucleoli  dark 
red,  rest  unstained. 

Carbol-Thionin,  see  King's. 


CARBOL-XYLOL 


58 


CARBOWAX  EMBEDDING 


Carbol-Xylol.  Xylol  saturated  with  car- 
bolic acid  crystals.  After  using  it  for 
clearing  celloidin  sections,  wash  quickly 
in  xylol  before  naounting  them  in 
balsam. 

Carbon  from  inspired  air  occurs  abundantly 
in  lungs  and  bronchial  lymph  nodes. 
It  may  be  transported  to  the  great  blood 
filters  (spleen  and  liver)  where  it  is 
distinguishable  by  its  black  color  and 
by  its  insolubility  in  cone,  sulphuric 
acid  which  dissolves  all  other  body 
pigments.  Fine  suspensions  of  carbon 
are  of  great  service  as  vital  stains  to 
demonstrate  phagocytosis.  See  Hig- 
gins'  Ink  and  Lampblack. 

Carbon  Monoxide,  Measurement  of,  see 
Scholander,  P.  F.,  and  Roughton,  F. 
J.  W.,  J.  Biol.  Chem.,  1943,  148,  551- 
563,  or  Glick,  p.  334. 

Carbonic  Anhydrase.  This  can  be  localized 
in  the  oxyntic  (or  parietal)  cells  of  the 
fundus  of  the  stomach.  Davenport, 
H.  W.,  Am.  J.  Physiol.,  1940,  128, 
725-728;  129,  505-514  employed  an 
adaptation  of  Linderstr0m-Lang's  tech- 
nique and  observed  that  in  rats  and  cats 
the  parietal  cells  contain  5  to  6  times  as 
much  of  the  enzyme  as  red  blood  cells 
while  the  peptic  cells  are  free  from  it. 
A  microspectroscopic  method  for  demon- 
stration of  carbonic  anhydrase  within 
erythrocytes  depends  on  the  action  of 
methemoglobin  as  an  indicator  which 
changes  both  its  color  and  pattern  of 
absorption  spectrum  with  change  of  pH 
from  6.5-9.5  (Keilin,  D.  and  Mann,  T., 
Nature,  1941,  148,  493-496).  For  data 
on  the  distribution  of  this  enzyme  in 
lower  forms,  see  Blaschko  and  Jacobson 
(Bourne,  p.  200). 

Carbonyl  Compounds,  water  insoluble  alde- 
hydes and  ketones,  see  critical  state- 
ment by  Glick,  pp.  69-72.  Bennett, 
H.  S.  (Am.  J.  Anat.,  1940,  67,  151-228) 
regarded  his  phenylhydrazine  reaction 
for  carbonjd  compounds  as  indicative 
of  ketosteroids  in  the  adrenal  cortex. 
Gomori,  G.  (Proc.  Soc.  Exper.  Biol.  & 
Med.,  1942,  51,  133-134)  however  does 
not  agree  unless  there  is  additional  evi- 
dence. Glick  is  of  the  same  opinion 
that  in  the  absence  of  such  evidence  the 
carbonyl  reactions  only  indicate  lipid 
aldehyde  or  ketone.  According  to 
Albert,  S.  and  Leblond,  C.  P.  (Endo- 
crinology, 1946, 39, 386-400)  it  is  plasmal- 
ogen  instead  of  ketosteroid  which  is 
demonstrated  by  the  phenylhydrazine 
reaction. 

1.  Bennett's  phenylhydrzine  reac- 
tion. Place  frozen  sections  of  fresh 
tissue  from  microtome  into  M/10  ace- 
tate buffer,  pH  6.0-6.5.  If  sections  of 
fixed  tissue  are  employed  place  in  water. 
Add  1%  iodine  in  ale.  drop  by  drop  till 


pale  straw  yellow  color  persists.  Let 
stand  15  min.  Add  1%  aq.  sodium 
thiosulphate  drop  by  drop  till  color  is 
lost  and  a  small  amount  more  is  added. 
Let  stand  5  min.  Wash  sections  re- 
peatedly in  aq.  dest.  Transfer  sections 
to  buffered  phenylhydrazine  solution 
just  prepared  by  mixing  equal  volumes 
of  2%  aq.  phenylhydrazine  hydro- 
chloride and  the  acetate  buffer  and  by 
removing  oxygen  by  gently  bubbling 
through  carbon  dioxide  for  15  min. 
This  solution  is  to  be  poured  into  glass- 
stoppered  bottles  so  that  with  the  sec- 
tions added  no  air  bubbles  remain  under 
the  stopper.  Control  slides  are  taken 
through  the  same  procedures  but  minus 
the  phenylhydrazine  treatment. 

2.  Albert  and  Leblond's  2,4-Dinitro- 
phenylhydrazine  reaction.  Saturate 
2,4-dinitrophenylhydrazine  (No.  1866 
Eastman  Kodak  Co.)  in  30%  ale.  and 
bring  pH  to  neutrality  by  addition  of 
0.2  N  sodium  acetate.  This  is  the  re- 
agent. Fix  tissue  in  formalin  neutra- 
lized with  magnesium  carbonate  for 
48  hrs.  and  wash  in  running  water  for 
24  hrs.  Cut  frozen  sections  10-15  n 
and  transfer  to  17%  ale.  4  hrs.  Place 
them  in  the  reagent  over  night  and 
wash  in  17%  ale.  20  min.  Change  to 
aq.  dest.  and  mount  in  glycerol  gelatin 
(see  Glychrogel).  Yellow  color  indi- 
cates positive  reaction. 

3.  Seligman,  A.  M.  and  Ashbel,  R. 
(Cancer,  1951,  4,  579-596):  Frozen  sec- 
tions, 10  to  20  M,  are  cut  from  formalin- 
fixed  tissues  and  attached  to  slides  by 
air-drying,  after  which  the  formalin  is 
washed  out  wath  several  changes  of 
water.  Sections  are  incubated  for  one 
or  more  hours  at  room  temperature  in 
a  0-1%  solution  of  3-hydroxy-2-naph- 
thoic  acid  hydrazide  in  50%  aldehyde- 
free  alcohol  and  5%  acetic  acid.  Re- 
move e.xcess  reagent  by  washing  for 
2  hrs.  in  50%  alcohol  followed  by  2  hrs. 
in  several  changes  of  water. 

A  blue  pigment  is  produced  at  the 
sites  of  carbonyl  reactivity  by  immer- 
sion in  a  solution  prepared  from  equal 
volumes  of  absolute  ale.  and  an  aqueous 
solution  containing  two  parts  water 
and  one  part  1/15  M  phosphate  buffer 
(pH  7-2),  followed  by  the  addition  of 
tetrazotized  o-dianisidine  in  powered 
form  (50  mg.  for  50  cc.  of  solution). 

The  development  of  a  blue  color 
reaches  a  maximum  in  one  or  two  min. 
The  sections  are  washed  in  several 
changes  of  water  (acidified  with  a  few 
drops  of  acetic  acid)  and  are  mounted 
in  glycerogel. 
Carbowax  Embedding — Written  by  Dr. 
H.  I.  Firminger,  Pathology  Section, 
National   Cancer  Institute,  Bethesda, 


CARBOWAX  EMBEDDING 


59 


CAREY'S 


Maryland.  February  9,  1951 — Carbo- 
wax  (Carbide  and  Carbon  Chemical 
Division,  30  East  42nd  Street,  New- 
York  17,  N.  Y.),  a  water  soluble  wax, 
is  an  excellent  embedding  medium  for 
tissues.  It  circumvents  the  long  de- 
hydration process  necessary  for  em- 
bedding in  paraffin  or  celloidin  and  yet 
sections  prepared  by  this  method  show 
excellent  preservation  of  cytological  de- 
tail. Sections  of  Carbowax  embedded 
tissue  are  easy  to  cut  at  from  1  to  10  ^i 
in  thickness  which  makes  this  embed- 
ding method  well  suited  both  for  the 
study  of  minute  cytological  details  and 
for  routine  histological  examinations. 
In  contrast  to  paraffin  and  celloidin 
embedded  tissues,  lipids  can  be  studied 
in  sections  of  the  same  tissue  block 
(Firminger,  H.  I.,  Stain  Tech.,  1950, 
25,  121-123).  Any  type  of  fixation  may 
be  used  and  almost  all  staining  pro- 
cedures can  be  performed  on  Carbowax 
sections  with  the  exception  of  osmic 
acid.  Staining  with  osmic  acid  is 
better  after  the  conventional  methods. 

Carbowax  is  not  miscible  with  fats 
and  penetrates  adipose  tissue,  brain  or 
spinal  cord  only  very  slowly.  Such 
tissues  can  sometimes  be  embedded 
after  long  periods  of  infiltration  in  the 
oven  with  resultant  distortion  or  by 
previous  removal  of  lipid  with  fat  sol- 
vents. Other  disadvantages  are  the 
difficulty  in  mounting  ribbons  on  the 
slide  because  of  the  solubuility  of  the 
Carbowax  in  the  aqueous  bath  used  for 
floating  the  sections,  and  the  difficulty 
of  making  sections  stick  on  the  slide 
during  the  staining  procedure.  Blank 
et  al.  (Blank,  H.  and  McCarthy,  P.  L., 
J.  Lab.  and  Clin.  Med.,  1950,  36,  776- 
781)  recommend  placing  sections  on  an 
aqueous  bath  containing  gelatin  and 
potassium  dichromate,  floating  sec- 
tions onto  a  clean  slide  and  drying 
thoroughly.  An  alternate  procedure 
which  avoids  exposure  to  chromates 
(undesirable  for  certain  staining  pro- 
cedures) is  to  cover  the  sections  with 
thin  colloidin  prior  to  staining.  Blank 
has  also  suggested  mounting  ribbons 
directly  on  a  slide  wet  with  the  gelatin- 
dichromate  solution. 

The  solution  recommended  by  Blank 
et  al.  for  floating  and  affixing  sections 
to  slides  is  made  by  dissolving  0.2  gm. 
of  potassium  dichromate  and  0.2  gm.  of 
gelatin  in  1000  cc.  aq.  dest.  The  mix- 
ture is  boiled  in  daylight  for  five 
minutes,  cooled  and  filtered. 

Embedding  in  Carbowax  ordinarily 
employs  a  mixture  of  Carbowax  "4000" 
and  Carbowax  "1500".  The  exact  pro- 
portions of  the  components  must  be 
varied    to    suit    climatic    conditions. 


Pure  Carbowax  "4000"  may  be  neces- 
sary in  hot  climates.  In  cooler  climates 
one  can  use  a  mixture  composed  of  85 
gms.  of  Carbowax  "4000"  and  15  gms. 
Carbowax  "1500".  For  use,  the  Carbo- 
waxes  are  combined  in  the  proper  pro- 
portions, heated  to  175°C.  for  30  seconds 
and  placed  in  an  oven  at  56°C.  This 
solution  should  be  kept  in  the  oven.  If 
the  temperature  of  the  oven  should  drop 
and  the  Carbowax  mixture  solidifies, 
it  requires  reheating  above  56°C.  to 
remelt  it.    To  embed: 

1.  Place  fixed  tissue  in  small  Stender 
dish  containing  the  above  Carbowax 
mixture  in  oven  at  56°C.  1-3  hrs. 

2.  Agitate  dish  and  contents  every  10 
min.  to  aid  penetration.  All  tissues, 
including  lung  tissue,  should  sink  before 
further  embedding. 

3.  After  sufficient  infiltration  remove 
tissue  and  place  in  fresh  mixture  in 
another  Stender  dish. 

4.  Cover  dish  and  place  in  icebox  to 
harden  at  5°C.  to  avoid  crystal  forma- 
tion. 

5.  Trim  block  with  hot  knife. 

6.  Block  on  hot  object  holder;  then 
cool  object  holder  with  ice,  taking  care 
that  water  or  ice  do  not  come  in  con- 
tact with  block. 

7.  Cut  sections  (1  to  10  n).  If  satis- 
factory ribbons  are  not  obtained  the 
procedure  is   not  working  properly. 

8.  (a)  Float  sections  on  a  water  bath 
at  room  temperature  to  which  2  drops 
of  a  detergent  or  the  first  ribbons  of 
Carbowax  have  been  added  to  prevent 
"explosion"  of  the  sections  by  the  sur- 
face tension.  Stain  sections  before 
mounting  on  slide;  or,  float  sections 
onto  the  slide  from  the  water  bath, 
cover  carefully  with  a  few  drops  of  a 
mixture  of  equal  parts  of  absolute  al- 
cohol and  ether.  Pour  off  excess  and 
cover  with  thin  collodion;  harden  in 
70%  ale.  or  water.     Stain. 

(b)  Or,  place  sections  on  a  bath  con- 
taining 0.02  gm.  %  gelatin  and  0.02  gm. 
%  potassium  dichromate.  Float  onto 
clean  slide,  permit  to  drj^  thoroughly 
and  stain. 
Carey's  method  for  motor  end  plates  is  an 
adaptation  for  his  study  of  their  ameboid 
motion  (Carey,  E.  J.,  Anat.  Rec,  1941, 
81,  393-413)  of  Wilkinson's  (H.  J.,  Med. 
J.  Austral.,  1929,  2,  768-793).  Modifica- 
tion of  Ranvier's  gold  chloride  technique. 
— Written  by  the  late  Dr.  E.  J.  Carey, 
Dept.  of  Anatomy,  Marquette  Uni- 
versity School  of  Medicine,  Milwaukee, 
Wis. 

1.  Remove  any  muscle  from  rat  or 
chameleon  from  its  origin  to  insertion 
while  the  animal  is  under  ether  ornemo- 
butal  anesthesia.     Using  a  very  sharp 


CAREY'S 


60 


CARMINE  DUSTING 


knife  cut  the  muscle  quickly  into  pri- 
mary pieces,  0.5  cm.  long,  and  0.5  cm. 
thick,  following  the  long  axis  of  the 
muscle  fibers.  Then  cut  the  primary 
pieces  longitudinally  into  thin  strips 
1  to  2  mm.  wide. 

2.  Soak  strips  in  freshly  prepared 
filtered  lemon  juice  for  5  to  10  min. 
until  they  become  clear  or  translucent. 
Rinse  in  cold  tap  water  4  to  5  times. 

3.  Place  strips  in  1%  aq.  gold  chloride 
at  30°C.  using  at  least  10  times  the 
volume  of  gold  chloride  solution  to  each 
volume  of  muscle.  While  muscle  is  in 
gold  chloride  solution,  stir  at  least  once 
a  min.  The  time  for  the  optimum  im- 
pregnation of  gold  varies  in  the  different 
muscles  of  the  same  animal  at  a  rela- 
tively constant  rate,  for  example,  the 
sternocleido-mastoid  muscle  of  the 
normal  rat  requires  16  min.;  the  pec- 
toralis  major,  adductors  of  the  thigh, 
and  biceps  femoris,  13  min.;  and  the 
gastrocnemius,  tibialis  anterior,  and 
the  intercostal  muscles  10  min.  After 
these  muscles  have  been  in  the  gold 
chloride  solution  for  the  proper  length 
of  time,  they  assume  a  yellowish-tan 
color  and  have  a  firm  consistency.  It 
is  highly  important  that  this  variability 
in  the  reaction  of  different  muscles  in 
the  same  animal  to  gold  impregnation 
be  realized.  This  may  have  been  one 
of  the  factors  that  led  to  the  discarding 
of  the  gold  technique  because  it  could 
not  be  rigidly  standardized. 

4.  Pour  off  gold  chloride  solution  and 
rinse  the  tissue  with  tap  water  until  the 
water  remains  clear.  Then  place  muscle 
in  25%  aq.  formic  acid  in  the  dark  16  to 
24  hrs.  Too  little  time  gives  incomplete 
reduction  of  the  gold  and  too  long  time 
excessive  softening  and  maceration. 

5.  Quickly  rinse  in  tap  water  5  or  6 
times  to  remove  as  much  of  the  formic 
acid  on  the  surface  of  the  muscle  as 
possible.  Even  small  amounts  of  for- 
mic acid  in  the  preserving  fluid  may 
cause  ultimate  maceration  of  the  tissue. 

6.  Store  the  muscles  until  they  are 
teased  in  a  mixture  of  §  glycerine  and 
I5  70%  alcohol.  (The  muscles  have 
been  preserved  in  a  good  condition  for 
teasing  in  this  mixture  for  7  years.) 

7.  To  tease  the  muscle  cut  from  one 
edge  with  a  flat  bladed  teasing  needle  a 
piece  1  mm.  thick  and  the  full  length  of 
the  muscle  fiber  of  short  muscles.  The 
edge  of  the  teasing  needle  may  be  flat- 
tened by  hammering  the  needle  after  it 
has  been  placed  in  a  Bunsen  flame  until 
the  needle  is  red  hot.  Orient  this  strip 
of  muscle  in  a  drop  of  glycerine  on  a 
clean,  1x3  slide.  Gently  add  a  clean 
cover  slip.  Lightly  press  down  with 
the    teasing    needles,    using    a    gentle 


lateral  movement  at  right  angles  to  the 
long  axis  of  the  muscle  fibers.  The 
muscle  fibers,  by  this  means,  are  gently 
rolled  out  so  that  the  preparation  is  one 
muscle  fiber  thick.  Check  with  micro- 
scope. Such  a  preparation  will  keep 
without  any  sealing  of  the  cover  slip 
for  at  least  7  years.  Any  of  the  usual 
cements,  however,  used  for  glycerine 
mounts,  may  be  used  to  make  the  prepa- 
ration permanent.  We  have  success- 
fully used  clarite. 

8.  When  cross  or  longitudinal  sections 
are  desired  reduce  the  gold  by  placing 
muscle  in  a  mixture  of  formalin  10%  for 
its  hardening  effect,  and  in  formic  acid 
3%  for  the  reduction  of  the  gold.  The 
gold  may,  likewise,  be  reduced  by  strong 
electric  light  for  16  to  24  hrs.  The  rou- 
tine method  for  celloidin  embedding  is 
then  used.  After  the  tissues  have  been 
cut  in  sections,  the  nuclei  can  be  coun- 
terstained  by  various  techniques. 

Carmalum  (Mayer).  Dissolve,  if  necessary 
with  heat,  1  gm.  Carminic  acid  and  10 
gms.  ammonia  alum  in  200  cc.  aq.  dest. 
Filter  and  to  filter  add  1  cc.  formalin  as 
a  preservative.  The  tissues  stained 
should  not  be  alkaline  (Lee,  p.  141). 

Carmine  has  been  very  widely  used  as  a 
stain.  Most  of  the  formulae  for  stain- 
ing of  fixed  tissues  were  proposed  40  or 
more  years  ago  chiefly  by  Ranvier  and 
Mayer.  Now  aniline  dyes  are  more 
popular  but  carmine  is  still  of  great  use 
for  staining  small  animals  m  toto,  for 
staining  tissues  in  bulk  which  are  later 
sectioned,  as  the  best  counterstain  for 
blue  vital  dyes  like  trypan  blue,  as  the 
most  specific  stain  for  Glycogen  and  for 
Mucus  in  the  form  of  mucicarmine,  for 
coloring  gelatin  used  to  inject  blood 
vessels  and  as  a  vital  stain.  Karsner, 
H.  T.  and  Swanbeck,  C.  E.,  J.  Med. 
Res.,  1920,  42,  91-98  employed  15-25 
cc.  of  fairly  thick  suspension  for  intra- 
pleural injections  in  cats.  At  present 
carminic  acid  is  available  and  can  be 
employed  instead  of  powdered  carmine. 
The  only  advantage  is  that  the  acid  is  of 
more  uniform  composition.  See  Aceto- 
carmine  (Schneider),  Alum  Carmine 
(Grenacher),  Aluminum  Chloride-Car- 
mine (Mayer),  Ammonia  Carmine 
(Ranvier),  Best's  Carmine  for  glycogen, 
Borax  Carmine  (Grenacher),  Carma- 
lum (Mayer),  Lithium  Carmine  (Orth), 
Mucicarmine  for  mucus,  Para-Carmine 
(Mayer),  Picro-Carmine  (Ranvier). 
Many  more  carmine  combinations  are 
given  by  Lee   (pp.  139-149). 

Carmine  Dusting  of  the  Lungs— Written 
by  C.  C.  Macklin,  Dept.  of  Histological 
Research,  The  University  of  Western 
Ontario,  London,  Canada.  November 
28,  1951 — Mice  or  other  mammals  are 


CARMINE-GELATIN  INJECTIONS 


61 


CARR-PRICE  REACTION 


exposed  in  a  closed  compartment  to  air 
laden  with  the  dust  of  dry  powdered 
carmine.     Agitation  is  by  fan  or  blast 
from    air-main.     Atropinization    facili- 
tates entry  into  the  lung  alveoli.     One 
hour  suffices  to  mark  the  alveolar  phago- 
cytes     (phagocytic     pneumonocytes — 
which    see)    with    red    particles.     The 
cytological    picture    varies    with    the 
time   elapsing   after    cessation    of    the 
dusting  (Macklin,  C.  C,  The  Lancet, 
Feb.  24, 1951,  432-435).     See  Dust  Cells. 
Carmine-Gelatin  Injections  of  blood  vessels. 
Methods  have  been  reviewed  by  Moore, 
R.  A.,  J.  Tech.  Methods,  1929,  12,  55- 
58.    He     proposes     a     more     accurate 
technique  for  preparation  of  the  gelatin 
mass.    Allow  80  gms.  gelatin  to  take  up 
200  cc.  cold  water  and  heat  to  complete 
the  gel .     Suspend  20  gms .  carmine  in  100 
cc.  water  and  add  ammonia  until  dis- 
solved.    Mix  the  gelatin  and  carmine 
solutions  and  add    15  gms.   potassium 
iodide  to  reduce  gelation  point  to  less 
than    25°C.     Place    in    water    bath    at 
25  °C.  and  immerse  a  prepared  platinum 
electrode  in  it.     Pass  electrolytic  hydro- 
gen from  a  tank  over  the  electrode  and 
agitate  the  gelatin  with  a  motor  stirrer. 
Read  electrical  potential  by  balancing 
against  a  standard  cell.     Add  acetic  acid 
cautiously  until  reading  of  voltage  corre- 
sponds to  pH  7.2. 

Two  other  techniques  are  listed  by 
Moore:  1.  Dissolve  40  gms.  carmine  in 
40  cc.  strong  ammonia  and  add  water. 
Allow  to  stand  12-24  hrs.  and  filter 
through  paper.  Boil  filtrate  until  it  is 
ammonia  free.  Precipitate  the  carmine 
as  a  colloidal  gel  by  adding  95%  alcohol. 
Filter,  wash  well  with  alcohol  and  dry 
material  collected.  Dissolve  2  gm.  in 
5  cc.  water  and  add  5  cc.  100  percent 
gelatin  in  water  thus  making  the  product 
20%  carmine  and  50%  gelatin  (Bensley, 
R.  R.,  personal  communication  to  Dr. 
R.  A.  Knouff).  2.  Triturate  40  gms. 
carmine  Merck  NFIV  with  40  cc.  strong 
ammonia  and  add  water  to  200  cc .  After 
standing  24  hrs.  filter  through  paper. 
Boil  filtrate  down  to  100  cc,  add  water 
to  200  cc.  and  repeat.  Add  70  gms. 
gelatin  dissolved  in  water  and  make  up 
with  water  to  1  liter  (MacCallum,  D.  B., 
Am.  J.  Anat.,  1926,38,  153-175). 
Carmoisine,  see  Chromotrope  2  R. 
Carnoy-Lebrun  fixative  for  insects  and  ticks. 
Equal  parts  chloroform,  absolute  alcohol 
and  acetic  acid  saturated  with  mercuric 
chloride.  See  Slifer-King  Method. 
Carnoy's  Fluid  in  abs.  ale,  6  parts;  chloro- 
form, 3  parts;  and  glacial  acetic  acid,  1 
part.  Also  known  as  Van  Gehuchten's 
mixture.  A  very  quick  fixative.  Do 
not  wash  in  water  but  in  95%  ale.  It  is 
employed     for     many     purposes.    See 


Fibrin,  Foot's  Method,  Glycogen  Neu- 
rofibrils. 
Carotene  (Carotin),  put  green  leaves  in  sat. 
aq.  KOH,  1  part;  40%  ethyl  alcohol,  2 
parts  and  tap  water  3  parts  in  wide 
mouthed  bottle  with  tight  glass  stopper 
to  prevent  absorption  of  CO2  from  air 
or    seal    with    vaseline.     Keep    several 
days  in  dark  until  tissue  is  yellow  and 
fluid  is   green.     Change   pieces  to  aq. 
dest.     several     hours.     Remove    small 
pieces,  dry  on  slide  with  filter  paper. 
Add    1    drop    cone.    H2SO4.     It    turns 
green,    then    blue.     Under    microscope 
carotene    crystals    appear    dark    blue 
(Steiger,    A.,    Microkosmos,    1941,    8, 
121-122).     Carotene   is   a  precursor  of 
Vitamin  A. 
Carotinalbumins.     Combinations    of    caro- 
tinoid  pigments  with   protein.     Rather 
uncommon.     As  an  example  Lison    (p. 
245)   cites  the  blue  carotinalbumin  in 
the    carapace  of  the   lobster   which  on 
boiling  is  split  into  a  protein  and  a  red 
carotinoid. 
Carotinoids.     Pigments     which     are     non- 
saturated    and    nonnitrogenous    hydro- 
carbons.    Entirely  different  chemically 
from   fats,   they  are  nevertheless  only 
present    in    vivo    as    solutions    within 
lipoids.     They  generally  appear  yellow, 
orange  or   brown   in   unstained   frozen 
sections  mounted  in  syrup  of  levulose. 
Lison  (p.  244)  indicates  that  tissues  con- 
taining these  pigments  can  sometimes 
be  embedded  in  paraffin,  because  they 
are  only  slowly  soluble  in  cold  alcohol. 
They  are  however  more  quickly  soluble 
in  chloroform,  acetone  petroleum  ether 
and  toluol.     According  to  Lison  (p.  245) 
they  are  always  easily  identifiable  by 
the  fact  that  when  treated  with  concen- 
trated sulphuric  acid  they  turn  intense 
blue  before  being  destroyed.     Treated 
with     solution    of    iodine-iodide     (say 
Gram's,  Lugol's)  they  give  a  black  green 
or    brown    color.     When    treated    with 
solution  of  chromic  acid  they  lose  their 
color  more  or  less  quickly.     See  Lipids, 
tabular  analysis,  also  Carotin. 
Carr-Price  Reaction  for  vitamin  A.    When 
frozen    sections    of    liver   are    plunged 
directly    into   a    solution   of   antimony 
trichloride  in  chloroform  and  immedi- 
ately   examined    therein    mitochondria 
take  bright  blue  color  which  fades  within 
30  min.     (Bourne,  G.,  Austral.  J.  Exp. 
Biol.  &  Med.  Sci.,   1935,  13,  238-249). 
Antimony  trichloride  is  said  not  to  be 
specific  for  vitamin  A  since  it  also  gives 
blue    color    with    carotinoid    pigments 
(Bourne,  p.  106).     Sterols  yield  by  this 
reaction   a   red   color    (Raoul,   Y.    and 
Meunier,  R.,  J.  Pharm.  Chim.,  1939,  29, 
112-118). 


CARRUTHERS 


62 


CASPERSSON 


Carruthers,  see  Oxidation  Reduction  Poten- 
tial, Vitamins. 

Cartesian  Diver  Manometry,  see  detailed 
description  of  apparatus  and  technique 
by  Glick,  pp.  342-393. 

Cartilage.  This  is  one  of  the  most  awkward 
tissues  of  the  body  to  examine  in  the 
living  state  because  of  the  mechanical 
difficulties  involved  in  separating  its 
component  parts  sufficiently  thinly  for 
examination  at  high  magnification  in 
approximately  isotonic  media.  But  the 
differentiation  of  cartilage  in  tissue 
cultures  has  been  studied  to  advantage 
(Fell,  H.  B.,  Arch.  f.  exper.  Zellf., 
1929,  7,  390-412)  and  an  account  of  the 
direct  investigation  of  living  cartilage 
in  Sandison  transparent  chambers  in- 
serted in  the  ears  of  rabbits  (Clark,  E. 
R.,  and  E.  L.,  Am.  J.  Amt.,  1942,  70, 
167-200)  sounds  very  promising.  The 
varieties  of  cartilage  (hyaline,  articular, 
elastic  and  fibrous)  depend  upon  the 
quantitative  and  qualitative  differences 
in  the  three  chief  components — cells, 
fibers  and  ground  substance. 

When  the  cartilage  is  fixed  to  bone, 
which  is  also  to  appear  in  the  sections, 
it  is  obviously  necessary  to  employ 
decalcification,  see  Bone.  Otherwise 
cut  thin  slices,  2-4  mm.  thick,  and  fix  by 
immersion.  Fixation  by  perfusion  is 
not  a  great  help  because  cartilage  is 
practically  avascular.  The  choice  of 
fixatives  and  stains  will  depend  upon 
what  it  is  desired  to  demonstrate.  For 
routine  purposes  Zenker's  Fluid  is 
satisfactory  followed  by  coloration  of 
paraffin  sections  with  Hematoxylin  and 
Eosin  or  Mallory's  Connective  Tissue 
stain.  But  many  prefer  Celloidin  sec- 
tions. Resorcin  Fuchsin  is  recom- 
mended for  the  elastic  fibers  of  the 
matrix.  Since  the  fibers  are  somewhat 
obscured  by  the  ground  substance  in 
hyaline  cartilage  dark  field  and  polarized 
light  may  be  useful  as  employed  by 
Lubosch,  W.,  Zeit.  f.  mikr.  Anat., 
Forsch.,  1927,  11,  67-171.  A  paper  by 
Dawson,  A.  B.,  and  Spark,  C,  Am.  J. 
Anat.,  1928,  42,  109-137  also  contains 
useful  information.  If  it  is  desired  to 
show  the  Golgi  apparatus  in  the  cells 
follow  the  technique  used  by  Fell,  H. 
B.,  J.  Morph.,  1925,  40,  417-459.  See 
Chondriotin  Sulphuric  Acid  and  Phos- 
phatase as  components  of  cartilage. 
The  specific  staining  of  cartilage  cells 
with  crystal  violet  has  been  reported  by 
Hass,  G.  M.,  Arch.  Path.,  1942,  33, 
174-181.  The  characteristic  basophilia 
of  the  ground  substance  is  the  basis  for 
the  following  excellent  method  for  the 
demonstration  of  cartilage  in  whole 
mounts. 

Van  Wijhe^s  methylene  blue  (Noback, 


G.  J.,  Anat.  Rec,  1916-17,  11,  292-294). 
This,  by  demonstrating  cartilage  in  blue 
in  transparent  whole  mounts,  supple- 
ments very  nicely  the  vital  coloration  of 
growing  bone  by  Madder  feeding  or 
Alizarin  injections.  Use  embryos,  or 
bones  of  young  animals  like  rats  or  mice, 
long  bones,  ribs,  chrondocranium,  etc. 
Fix  in  10%  formalin  a  day  or  more.  1% 
hydrochloric  acid  in  67%  alcohol  several 
days  or  a  week.  Same  solution  +  0.25% 
methylene  blue  or  toluidin  blue  1  or  2 
weeks  until  thoroughly  stained.  De- 
colorize in  Acid  Alcohol.  Change  alco- 
hol when  it  becomes  much  colored  or 
every  1  or  2  days.  Continue  until  only 
the  cartilage  retains  deep  blue  color. 
Wash  several  days  in  82%  ale.  Dehy- 
drate in  95%  and  abs.  Equal  parts  abs. 
and  benzene.  Benzene  change  twice. 
Leave  in  this  or  mount  in  xylene  damar 
which  is  better  than  balsam  because  of 
its  light  color. 

R(^ioactive  gold  distribution  in  carti- 
lage (Ekholm,  R.,  Acta  Anat.,  1951, 
Suppl.  15  to  11,  75  pp.,  a  first  rate  study 
especially  on  the  knee  joint). 

Cartilaginous  Skeleton  of  mammalian  fe- 
tuses. A  modification  of  the  Wijhe, 
Lundvall  and  Schultze  techniques  used 
in  the  Department  of  Embryology, 
Carnegie  Institution  of  Washington  is 
given  by  Miller,  C.  H.,  Anat.  Rec, 
1921,  20,  415-419.  Wash  formalin  fixed 
material  over  night  in  water  plus  few 
drops  ammonia.  Transfer  to  70%  alco- 
hol and  leave  7-14  days  changing  alcohol 
daily  for  first  five.  Stain  for  3-10  days 
in:  toluidin  blue  (Grubler),  1  gm.; 
70%  alcohol,  400  cc;  and  hydrochloric 
acid,  4  cc.  Decolorize  for  7-10  days 
until  decolorizer  is  but  slightly  tinged 
with  the  dye  in:  70%  alcohol,  100  cc. 
plus  hydrochloric  acid,  1  cc.  Then  80% 
and  95%  alcohol,  3  days  each.  Transfer 
to  2%  potassium  hydroxide,  in  aq. 
dest.  and  leave  2-3  days  until  cleared. 
Change  to  20,  40,  60,  and  80%  glycerin 
in  aq.  dest.  2  days  or  more  in  each. 
Store  or  mount  in  pure  glycerin  plus  few 
crystals  of  thymol.  Obviously  length 
of  times  depends  chiefly  upon  size  of 
specimen.  This  staining  of  cartilage 
with  toluidin  blue  can  be  combined  with 
the  coloration  of  bone  with  Alizarin 
Red  S  to  make  very  contrasty  prepara- 
tions (Williams,  T.  W.,  Stain  Techn., 
1941,  16,  23-25). 

Carycinel  Red  is  l-amylaminoanthraqui- 
none,  an  oil  soluble  dye,  recommended 
by  Lillie,  R.  D.  Stain  Techn.,  1945,  20, 
73-75  as  a  stain  for  fat  which  it  colors 
deep  red.  Employ  as  described  for 
Coccinel  Red. 

Caryospora,  see  Coccidia. 

Caspersson,  see  Absorption  Spectra. 


CASEATION 


63 


CELL  MEASUREMENT 


Caseation  (L.  caseus,  cheese).  This  change 
follows  local  Necrosis.  It  is  charac- 
terized by  grayish  or  light  yellow  cheesy 
masses  of  tissue  which  look  amorphous 
and  have  lost  their  original  structure. 
Identification  is  morphological.  Almost 
any  good  staining  method  is  satisfactory. 
In  some  cases  fibrin  is  present. 

Cason,  see  Mallory-Heidenhain  Rapid  One- 
Step  Stain  for  Connective  Tissue. 

Catalase.  Method  for  demonstration  in 
elementary  bodies  of  vaccine  virus 
(Macfarlane,  M.  G.,  and  Salaman,  M. 
H.,  Brit.  J.  Exp.  Path.,  1938,  19,  184; 
Hoagland,  C.  L.  et  al.,  J.  Exp.  Med., 
1942,  76,  163-173).  See  Holter,  H.  and 
Doyle,  W.  L.,  J.  Cell  Comp.  Physiol., 
1938,  12,  295-308. 

Cataphoresis.  Most  solid  particles  sus- 
pended in  water  move  under  electric 
stress.  A  positively  charged  one  moves 
toward  the  cathode  and  a  negatively 
charged  one  toward  the  anode.  Micro- 
cataphoretic  cells  are  employed  to  de- 
termine and  measure  the  movement 
which  obviously  has  an  important 
bearing  on  bacterial  agglutination. 
Electrophoresis  is  a  better  term  than 
cataphoresis.  (Holmes,  H.  N.  in  Glas- 
ser's  Medical  Physics,  257-263)  see 
Coagulation. 

Cataract,  see  Optic  Lens. 

Cathepsin.  A  method  for  analysis  of 
cathepsin  in  lymphocytes  and  poly- 
morphonuclear leucocytes  (neutro- 
philes)  is  given  by  Barnes,  J.  M.,  Brit. 
J.  Exp.  Path.,  1940,  21,  264-275. 

Cebione,  see  Vitamin  C. 

Cedar  Oil,  see  Clearing,  Immersion  Oils 
and  Mounting. 

Celestin  Blue  B  (CI,  900)— coreine  2R— A 
basic  quinone-imine  dye  employed  by 
Proescher,  F.  and  Arkush,  A.  S.,  Stain 
Techn.,  1928,  3,  28-38  and  by  Lendrum, 
H.  C,  J.  Path.  &  Bact.,  1935,  40,  41&- 
416  as  a  nuclear  stain. 

Cell  Classification  according  to  manner  of 
life.  lutermitotic  cells  live  from  the 
mitosis  which  gives  them  birth  to  the 
mitosis  by  which  they  divide  to  produce 
two  other  cells.  They  thus  cease  life  as 
individuals  by  division  not  by  ageing, 
degeneration  and  death.  There  are  2 
kinds  of  intermitotic  cells:  First,  the 
vegetative  intermitotics  some  of  which 
continue  a  sort  of  vegetative  life  con- 
stituting a  reservoir  of  undifferentiated 
cells  on  which  the  body  can  draw  in 
some  cases  as  long  as  it  lives.  They  are 
found  in  the  epidermis  bone  marrow 
and  other  places.  Second,  the  differ- 
entiating intermitotics,  which  exist  in 
series,  one  building  up  a  certain  degree 
of  differentiation,  which,  when  it  di- 
vides, it  passes  on  to  its  daughter  cells. 
The   progeny   of  these   daughter   cells 


differentiate  still  further  and  pass  on 
this  higher  level  of  specialization  to 
their  successors.  Good  examples  are 
myeloblasts  and  myelocytes  in  leuco- 
cytogenesis.  But  the  first  differentiat- 
ing intermitotic  in  any  line  of  differen- 
tiation is  produced  by  division  of  a 
vegetative  intermitotic.  One  of  the 
daughter  cells  of  this  division,  or  in 
some  instances  both  daughter  cells  from 
mitosis  of  a  dividing  vegetative  inter- 
mitotic, achieve  no  further  differentia- 
tion than  their  parent  cells,  for 
otherwise  the  reservoir  of  vegetative 
intermitotics  would  not  be  maintained 
but  would  differentiate  itself  out  of 
existence. 

Postmitotic  cells,  on  the  other  hand, 
are  cells  whose  lives  are  postmitotic  in 
the  sense  that  they  perform  their  duty, 
age  and  die.  They  are  the  culminations 
of  the  various  lines  of  differentiation. 
Again,  two  sorts  are  recognizable: 
First  the  reverting  postmitotics,  which 
are  capable  of  full  functional  activity 
and  usually  go  on  to  death,  yet,  on 
occasion,  some  of  which  can  revert  and 
divide.  Hepatic  and  renal  cells  are 
examples.  Second,  the  fixed  postmi- 
totics, which  are  different  insofar  that 
they  are  incapable  of  mitosis  so  that 
aging  and  death  is  for  them  inevitable 
as  for  instance  nerve  cells  of  adults, 
sperms  and  polymorphonuclear  neutro- 
phile  leucocytes.  In  contrast  with  the 
other  3  kinds  these  fixed  postmitotics 
have  lost  the  potentiality  of  malignant 
transformation  (Cowdry,  E.  V.,  Prob- 
lems of  Aging.  Baltimore,  Williams 
&  Wilkins,  1942,  626-629). 

Cell  Components  can  be  examined  by  tech- 
niques too  numerous  to  list  including 
Staining,  Supravital  and  Vital  Staining, 
Impregnation,  Microdissection,  Micro- 
manipulation, Microinjection,  Centrif- 
ugation,  many  Microchemical  Reac- 
tions, and  Indicators  by  at  least  6  differ- 
ent kinds  of  Microscopes.  Methods  for 
many  of  these  components  are  given 
under  Capsule  Stains,  Mitochondria, 
Zymogen,  Nissl  Bodies,  etc. 

Cell  Division,  see  Mitosis,  Amitosis  and 
series  of  papers  on  chemistry  of  cell 
division  (Mauer,  M.  E.  and  Voegtlin, 
C,  Am.  J.  Cancer,  1937,  29,  483-502). 

Cell  Enlargement,  see  Giant  Cells. 

Cell  Injury  detected  by  fluorescence 
(Herick,  F.,  Protoplasma,  1939,  32, 
527-535).     See  Dead  Cells. 

Cell  Measurement,  The  Elliptometer — 
Written  by  Dr.  J.  D.  Hamilton,  Dept. 
of  Medical  Research,  University  of 
Western  Ontario,  London,  Canada. 
February  13, 1951 — The  geometric  shape 
of  the  section  of  many  histological 
structures  is  circular  or  elliptical.     The 


CELL  MEASUREMENT 


64 


CELL  MEASUREMENT 


cross  sections  of  cell  nuclei,  the  bound- 
aries of  many  cells  such  as  sea  urchin 
eggs,  nerve  cells,  the  central  zone  of 
cells  undergoing  division,  ovarian  fol- 
liculi,  glomeruli,  blood  vessels  and 
ducts,  may  all  be  described  in  terms 
of  their  geometrical  parimeters. 

A  quantitative  measurement  of  the 
area,  eccentricity,  and  geometric  center 
of  these  structures  may  often  be  made 
by  means  of  an  elliptometer  (Hamilton, 
J.  D.  and  Barr,  AL  L.,  Stain  Tech., 
1948,  23,  123). 

Essentially  the  elliptometer  method 
provides  a  means  of  fitting  an  ellipse  of 
light  to  a  camera  lucida  drawing  of  the 
cell  or  structure.  Alternatively  in 
principle,  the  camera  lucida  drawing 
board  may  be  replaced  by  a  ground 
glass  screen  and  an  ellipse  of  light  of 
suitably  controlled  intensity,  angle, 
and  size  matched  to  the  structure  as 
viewed  directly  in  the  ocular  of  the 
microscope.  In  both  methods,  by 
direct  graphic  means,  the  axes  of  their 
equivalent  ellipse  or  ellipse  of  best  fit 
is  then  determined. 

In  the  instrument  a  variable  circular 
iris  such  as  a  camera  diaphragm,  is 
used  to  define  a  cone  of  light  arising 
from  a  point  source.  Point  sources  of 
light  provided  by  a  zirconium  or  con- 
centrated arc  lamp  are  quite  suitable 
and  make  possible  the  construction  of 
the  apparatus  without  the  use  of  a  con- 
densing lens  system  to  define  a  point 
source.  The  cone  of  light  falls  upon 
a  screen  upon  which  is  mounted  a  grid 
of  graph  paper.  An  alternative  to  the 
above  design  uses  a  lens  between  the 
illuminated  diaphragm  and  the  screen. 
The  lens  provides  an  image  of  the 
diaphragm  in  the  perpendicular  plane 
of  the  screen. 

The  screen  maj^  be  rotated  by  means 
of  an  attached  arm  which  rotates  in  a 
sleeve  bearing  lying  parallel  to  the  hori- 
zontal lines  of  the  grid.  By  tilting 
the  screen  out  of  the  vertical  plane  it 
is  thus  possible  to  create  a  family  of 
ellipses.  The  minor  axes  will  be  con- 
trolled and  fixed  by  the  diameter  of 
variable  diaphragm.  The  points  at 
which  the  ellipse  touches  the  horizontal 
and  vertical  grid  lines  may  be  deter- 
mined and  the  axes  calculated  by  addi- 
tion. 

In  practice  the  camera  lucida  draw- 
ing is  placed  flat  on  the  screen  and  the 
elliptometer  aperture  and  screen  angle 
adjusted  to  give  the  best  fit.  The  two 
ends  of  the  major  axes  are  marked  on 
the  drawing  for  future  reference,  and 
the  grid  values  noted. 

Derived  Measurements:  a)  Area:  If  A, 
and  B,  are  the  major  and  minor  diam- 


eters of  the  ellipse  then  the  area  is 
K  TT  A,B,/4  n^  where  K  is  a  propor- 
tionately constant  depending  on  the 
grid  system  used  and  the  magnifica- 
tions entering  into  the  camera  lucida 
tracing.  In  our  experience  with  motor 
nerve  cells  (Barr,  M.  L.  and  Hamilton, 
J.  D.,  J.  Comp.  Neur.,  1948,  89,  93) 
the  areas  determined  by  elliptometer 
and  planimeter  measurements  agree 
within  five  per  cent  on  an  absolute  basis 
in  comparing  individual  cells,  and  the 
statistical  parameters  of  populations 
examined  by  the  two  methods  of  meas- 
urement are  of  equal  value  when  con- 
trol and  experimental  populations  are 
compared.  Area  measurement  using 
the  elliptometer  are  more  rapid  and  less 
fatiguing  than  planimeter  measure- 
ments. 

b)  Center  and  Foci:  The  center  and 
foci  of  elliptic  sections  may  be  readily 
determined  by  using  a  rule  or  by  graphic 
methods.  If  the  plane  of  section  is 
transverse,  sagittal,  or  coronal,  a  refer- 
ence line  running  dorsal-ventral  or 
antero-posterior  or  medio-lateral  may 
be  drawn  through  the  center  of  the 
elliptic  figure.  The  orientation  of  the 
ellipse  or  the  location  of  inclusions  or 
other  features  of  the  figure,  may  then 
be  measured  with  respect  to  the  axes 
of  the  body  as  a  whole.  By  this  type 
of  orientation  and  definition  for  ex- 
ample, it  has  been  possible  to  show  that 
the  axone  hillock  of  motor  cells  of  the 
dorso-lateral  group  of  the  ventral  horn 
arise  predominantly  in  the  ventral 
medial  quadrant  of  the  cell. 

c)  Eccentricity:  The  eccentricity  is 
the  ratio  of  the  minor  to  the  major 
axis  B/A.  The  eccentricitj^  of  indi- 
vidual sections  may  possess  some  value 
as  a  descriptive  measurement.  The 
average  eccentricity  of  populations  may 
be  of  value  in  following  the  time  course 
of  changes  of  shape,  as  for  example  in 
hypertrophy,  cell  division,  or  blastula 
development. 

d)  Cell  Shape  and  Value:  Consider 
an  ellipsoid  having  major,  intermediate, 
and  minor  diameters,  A,  >B,  -^  C, 
lying  at  random  in  space  related  to  the 
plane  of  section.  The  eccentricities  of 
sections  drawn  from  an  ellipsoidal 
population,  homogenous  in  respect  to 
shape  (but  not  necessarily  size)  will 
range  from  a  maximum  C/B  (which 
may  be  unity  if  the  body  is  an  ellipse 
of  "revolution)  to  a  minimum  C/A. 
This  distribution  of  eccentricities  is 
true  even  if  the  plane  of  section  does 
not  pass  through  the  center  of  the  el- 
lipsoid. 

The  eccentricity  data  may  be  used 
to  secure  an  estimate  of  the  shape  and 


CELL  MEMBRANES 


65 


CELLOIDIN  IMBEDDING 


volume  of  the  average  body.     Accord- 
ing   to    experience,    eccentricities    are 
placed  in  several  classes,  from  1.0  to 
0.4,   say,   with   class   intervals   of  0.2. 
From  the  first  class,  1.0  to  0.8,  an  esti- 
mate of  the  eccentricity  C/B  can  be 
made,  since  ellipses  of  this  class  are 
primarily  of  the  type  C,  B,  particularly 
if  B  and  C  are  appro.ximately  equal  and 
somewhat  less  than  A.     Similarly  eccen- 
tricities of  the  last  class  interval  0.6 
to  0.4  will  provide  an  estimate  of  C/A. 
Combining    these    two    estimates    the 
average  ratio  B/A  may  be  determined. 
The   minor   axes   of   sections   of   the 
first  and  last  eccentricity  groups  are 
representative  of  the  C  or  minor  axes 
of  the  space  figure.     Hence  an  estimate 
of  the  average  value  of  C  may  be  made, 
and   using   the   eccentricitj^   ratios   al- 
ready determined  the  average  values  of 
A  and  B  can  be  calculated.     The  aver- 
age volume  V  equals  iir  ABC  can  then 
be  calculated. 
Cell  Membranes  do  not  require  any  special 
technique  for  their  demonstration.     Al- 
most any  good  fixative  will  do  and  they 
can  be  stained  a  host  of  different  colors. 
There  is  however  some  difference  in  the 
interpretation  of  what  we  see  with  the 
microscope.     The   essential    component 
of  the  walls  of  all  cells  is  called  the 
plasma  membrane.    This  conditions  per- 
meability and  its  integrity  is  essential 
to  the  life  of  the  cell.     It  is  said  to  con- 
sist   of    a    continuous    layer   of   lipoid 
molecules    (phosphatides,  sterols,  fats) 
not  more  than  2-4  molecules  thick  on 
which  proteins  are  adsorbed,  the  lipoids 
give    permeability    and    the    proteins 
elasticity  and  great  mechanical  strength. 
The  evidence  is  critically  presented  by 
Danielli  (Bourne,  pp.  68-98).     He  says 
that  it  is  improbable   that  the  lipoid 
layer  is  ever  thicker  than  10  m/x  and 
that  the  whole  membrane  is  between  Ifx 
and  1  m/i  thick.     Consequently  in  many 
cases  we  cannot  expect  to  visualize  the 
plasma  membrane  itself  directly  with 
visible    light    because    the    theoretical 
limit  of  visibility  is  a  particle  size  of 
0.25m.     However    the    position    of    the 
plasma  membrane  is  made  clear  by  the 
difference  in  properties  of  the  cytoplasm 
which  it  limits  and  the  fluid  without 
and  also  in  the  dark  field  by  the  light 
reflected  from  its  surface.     In  addition 
it  is  often  backed  internally  by  a  thin 
layer  of  cytoplasmic  cortex  (ectoplasm) 
which  is  typically  free  from  cytoplasmic 
granules.     The  plasma  membrane  may 
be  supplemented  externally  by  special 
membranes  such  as  the  myelin  sheaths 
about    nerve    fibers.     There  are   many 
special  techniques  for  its  investigation. 
Some    are    briefly    referred    to    under 


Lysis,   Permeability,   Surface  Tension 
and  Wetting  Properties,  Nuclear  Mem- 
brane, PinocytosiB. 
Cell  Shape.    The  shape  of  epithelial  cells, 
and  of  all  cells  for  that  matter,  is  deter- 
mined   by    perfectly    definite    causes. 
Obviously  those  suspended  in  fluid  tend 
to   be   spherical    (lymphocytes)    unless 
their  internal  organization  conditions 
some  other  shape  (erythrocytes) .     Con- 
tact with  a  surface  generally  promotes 
flattening  on  that  surface.     Epithelial 
cells  are  sessile.     The  study  of  their 
rnorphology  is  not  complicated  by  mo- 
tility.    When  disposed  in  a  single  layer 
and  subjected  to  lateral  pressure  from 
their  neighbors  they  take  a  distinctive 
shape   which  has   been   analyzed  in   a 
convincing  way  by  F.  T.  Lewis  (Am. 
Scientist,  1946,  34,  357-369,  and  many 
earlier    papers).     In    sections    of    the 
laj^er  parallel  to  the  surface  it  may  be 
seen  that  most  of  the  cells  are  six-sided, 
or   hexagonal.     They    form    a   mosaic, 
the  character  of  which  can  easily  be  re- 
membered by  students  forced  to  dream 
of  the  benzene  "ring"  with  its  6  carbon 
atoms.     By  drawing  many  such  chemi- 
cal symbols  side  by  side  a  similar  mosaic 
is  formed.     As  Lewis  points  out,  the 
intersections  are  three-raj'ed  not  four- 
rayed  as  might  be  the  case  if  the  cross- 
sections    were    squares.     Mechanically 
this  is  a  great  advantage.     When  the  de- 
ithelium  is  stratified  provision  must  be 
made  for  contact  with  cells  on  all  sides. 
Nature  adheres  to  the  same  three-rayed 
intersection  and  molds  the  cells  in  that 
shape  which  provides  the  smallest  sur- 
face area  for  closely  crowded  bodies. 
Lewis  found  that  this  could  be  deter- 
mined   mathematically    as    a    14-sided 
figure  and  by  careful  reconstruction  of 
actual  cells  proved  that  they  were  all 
primarily  tetrakaidecahedral  in  shape. 
Examination  of  his  clear  illustrations 
will  be  more  helpful  than  pages  of  de- 
scription.    The  same  architectural  prin- 
ciples    apply     to     many     other     cell 
aggregates,    like    fatty    tissue    for    ex- 
ample.    No    longer    is    the    histologist 
justified  in  vaguely  referring  to  such 
cells  as  polyhedral.     Evidently  in  the 
construction  of  epithelial  surfaces  the 
cells  are  fitted  together  in  a  much  more 
effective  way  than  bricks  in  the  building 
of  a  wall.     Except   for  the   reference, 
the  above  paragraph  is  quoted  from  the 
Second  Edition  of  Cowdry's  Histology, 
Philadelphia :  Lea  &  Febiger ,  1938.     See 
technique  for  three-dimensional  study 
of  cell  shape  in  plants,  Holtzman,  D.  H., 
Am.  J.  Bot.,  1951,  38,  221-234. 
Celloidin  Imbedding.     Celloidiu  is  a  kind 
of  generic  term  covering  various  cellu- 
lose compounds,  nitrocellulose,  soluble 


CELLOIDIN  IMBEDDING 


66 


CELLOIDIN  INJECTIONS 


gun  cotton,  etc.,  employed  for  imbed- 
ding. The  collodions  are  solutions  of 
pyroxylin  made  as  specified  in  the 
U.S.P.  Pyroxylin  U.S.?.  XI  consists 
chiefly  of  cellulose  tetranitrite  (Merck 
Index,  p.  465).  Obviouslj'^  a  purified, 
nonexplosive  form  of  pyroxylin  is 
necessary.  There  are  several  in  the 
market  of  which  Parlodion  (Mallinck- 
rodt)  is  the  one  used  in  our  laboratory. 
The  Bensleys  (p.  37)  use  as  celloidin 
"RS  5  sec.  low  viscosity  nitrocellulose 
30  per  cent  solvent  in  absolute  alcohol" 
obtained  from  the  Hercules  Powder  Co., 
Gillespie,  N.  J._  To  make  20%  stock 
solution  they  dissolve  140  gms.  nitro- 
cellulose in  250  cc.  ether  and  210  cc. 
absolute  alcohol.  This  requires  4-5 
days  shaking  occasionally.  It  is  diluted 
with  ether  alcohol  to  make  10  and  5% 
solutions  respectively.  Nitrocellulose 
is  much  used  especially  in  neurological 
technique.  It  is  abbreviated  L.V.N. 
Some  advantages  over  "celloidin"  are 
claimed  for  it  by  Davenport,  H.  A., 
and  Swank,  R.  L.,  Stain  Techn.,  19^4, 
9,  137-139. 

Celloidin  imbedding  is  less  popular 
than  it  used  to  be  owing  to  certain 
advantages  of  Paraffin  Imbedding  rein- 
forced by  the  mania  for  speed.  But 
celloidin  imbedding  is  in  some  respects 
superior.  It  yields  sections  in  which 
the  affinity  of  the  tissue  components  for 
dyes  is  often  greater.  Clearing  of  the 
tissue  in  xylol  and  similar  fluids  is  not 
required  and  it  need  not  be  subjected  to 
heat.  The  tissue  usually  shrinks  less 
and  seldom  becomes  so  brittle.  Brain 
specimens  can  easily  be  cut  in  celloidin 
even  after  long  mordanting.  When 
sections  are  required  of  large  pieces  of 
tissue  in  which  cavities,  such  as  the 
lumina  of  the  paranasal  sinuses,  alter- 
nate with  stout  bony  walls  this  method 
is  indicated  because  the  celloidin  in  the 
spaces  gives  more  support  than  paraffin 
(see  also  Double  Imbedding). 

The  slow  method,  which  is  the  best, 
requires  for  tissue  slices  not  more  than 
5  mm.  thick,  at  least  1  day  each  in  95% 
alcohol,  absolute  alcohol,  and  in  half 
absolute  and  ether.  This  is  followed  by 
1  day  in  thin  celloidin  (about  4%  dried 
strips  of  celloidin — Parlodion,  Mal- 
linckrodt— dissolved  in  equal  parts 
absolute  alcohol  and  ether)  and  1  or 
more  weeks  in  thick  8%  celloidin. 
The  tissue,  with  some  celloidin  about  it, 
is  then  mounted  on  a  fiber  block,  hard- 
ened in  chloroform  1-2  hrs.  and  stored 
in  80%  alcohol. 

Mallory  (p.  60)  gives  the  following  as 
a  rapid  method.  Fix  thin  tissue  pieces 
12-18  hrs.  in  Formalin-Alcohol.  Then 
95%  alcohol,  2  changes,  2  hrs.;  absolute 


alcohol,  2  changes,  3  hrs.;  alcohol -ether, 
3  hrs. ;  thick  celloidin  12-15  hrs. ;  mount 
and  harden  in  chloroform,  1  hr.;  80% 
alcohol. 

A  still  quicker  technique  has  been 
proposed  (Richardson,  G.  D.,  J.  Tech. 
Meth.,  1934,  13,  81) :  To  make  celloidin 
solution,  add  1100  cc.  absolute  ethjd 
alcohol  to  8  oz.  celloidin  (dried  in  air) 
and  leave  over  night.  Add  1100  cc. 
ether.  Let  stand  several  days.  It  is 
ready  when  celloidin  is  dissolved. 
Fix  tissue  in  10%  formalin,  2  hrs.; 
acetone,  2  hrs.;  oil  of  cloves  ^-2  hrs.  or 
until  clear;  celloidin  6  hrs.  at  room 
temperature  or  ^-3  hrs.  in  water  bath 
at  55 °C.  (being  careful  to  keep  away 
from  flame).  Block  and  harden  in 
chloroform  j-2  hrs. 

Another  so  called  hoi  celloidin  method 
is  proposed  with  all  steps  in  the  tech- 
nique at  an  elevated  temperature 
(Koneff,  A.  A.,  and  Lyons,  W.  R.,  Stain 
Techn.,  1937,  12,  57-59).  Fix  pieces 
not  thicker  than  2-3  mm.  in  10%  neutral 
formol,  Bouin  or  Siisa.  Wash  in  aq. 
dest.  several  changes  (1  hr.  each)  at 
room  temperature.  Dehydrate  at  50°C. 
70,  80,  95  and  abs.  ale.  2  changes  ^  hr. 
each.  Equal  parts  abs.  ale.  and  ether 
1  hr.  Infiltrate  at  56 °C.  in  (1)  10% 
nitrocellulose  (R.S.  ^  second,  viscosity 
■ia~z^>  Hercules  Powder  Co.)  in  equal 
parts  abs.  ale.  and  ether,  1  hr.  (2)  25% 
in  45  cc.  ale.  +  55  cc.  ether,  over  night. 
(3)  50%  in  40  cc.  ale.  +  60  cc.  ether 
2-3  hrs.  Then  transfer  tissue  to  micro- 
tome block  moistened  with  ether-alcohol. 
Add  50%  nitro-cellulose  and  the  tissue. 
Harden  in  2  changes  chloroform  during 
1  hr.  Then  pass  through  3  changes  80% 
ethyl  alcohol  and  cut.  The  authors 
mention  fixation  in  "Carnoy  II"  and 
removal  of  mercury  with  iodized  alcohol 
in  case  a  fixative  containing  mercuric 
chloride  was  employed.  Obviously 
every  precaution  must  be  taken  to  avoid 
explosion. 

Store  celloidin  blocks  in  80%  ale.  See 
special  methods  for  imbedding  Teeth 
and  Bone. 
Celloidin  Injections  of  lungs.  For  smaller 
vessels  and  bronchi  use :  acetone,  100  cc. ; 
celloidin,  4  gm.;  and  camphor,  3  gm. 
For  larger  vessels  and  bronchi  employ : 
acetone,  100  cc,  sheet  celloidin,  20  gms., 
and  camphor,  15  gm.  In  place  of  sheet 
celloidin  old  x-ray  films  can  be  used  if 
first  the  emulsion  is  removed  by  washing 
in  warm  water  and  they  are  then  dried 
and  cut  into  strips.  If  colors  are  desired 
employ  oil  paints.  If  Roentgenograms 
are  to  be  made  of  the  corrosion  specimens 
add  10-12%  sodium  iodide  or  barium 
sulphate  to  a  30%  suspension.  In  case 
of  the  vessels  wash  out  blood  first  by 


CELLOIDIN  SECTIONS 


67 


CENTRIFUGATION 


forcing  physiological  saline  solution  into 
vena  cava  thence  through  right  heart 
and  via  pulmonary  arteries  to  lungs 
evacuating  by  pulmonary  veins.  Allow 
injected  lung  to  stand  in  running  water 
over  night  thus  hardening  celloidin. 
Immerse  in  concentrated  hydrochloric 
acid  to  digest  away  tissues  leaving 
celloidin  cast.  This  usually  takes  24 
hrs.  Wash  thoroughly  in  gentle  stream 
of  water.  Mount  dry  or  mount  wet  in 
solution  made  up  as  follows :  Boil  for  10 
min.  100  cc.  aq.  dest.  +  20  cc.  glycerin. 
When  cool  add  formalin  to  2%  and  filter 
until  clear  (Marquis,  W.  J.,  J.  Tech. 
Methods,  1929,  12,  59-64).  See  illus- 
trations of  Marquis  and  arrangement  of 
pressure  bottles.  A  celluloid  corrosion 
technique  for  the  kidney  is  described  by 
N.  W.  Baker,  J.  Tech.  Methods,  1929, 
12,  65-68. 

Celloidin  Sections.  Cut  side  of  celloidin 
block  to  smooth  plane  surface.  Moisten 
this  and  surface  of  microtome  block 
holder  with  alcohol-ether.  Add  drop 
thick  celloidin.  Press  together,  harden 
in  chloroform  and  cut  in  80%  alcohol  on 
a  sliding  microtome  with  knife  at  an 
angle.  Keep  surface  of  knife  and  block 
wet  with  80%  alcohol  from  overhead 
dropping  bottle.  (A  method  has  been 
described  for  treating  block  with  cedar 
oil  and  cutting  dry  with  rotatory  micro- 
tome, Walls,  G.  L.,  Stain  Techn.,  1936, 
11,  89-92).  Sections  are  usually  cut  at 
a  thickness  of  10-16  p.  (It  is  possible  to 
arrange  the  sections  serially  but  it  is  a 
tedious  business.  If  serial  sections  are 
needed,  paraffin  should  be  selected  in 
place  of  celloidin.)  The  sections  un- 
mounted can  be  stained  without  remov- 
ing the  celloidin  after  which  they  are  to 
be  dehydrated  and  cleared  before  mount- 
ing. The  object  is  not  to  remove  the 
celloidin  but  to  soften  it.  The  following 
mixture  is  recommended  by  Lee  (p.  108) 
in  place  of  xylol,  toluol  or  benzol :  creo- 
sote, 40  cc. ;  Bergamot  oil,  30  cc. ;  xylol, 
20  cc.  and  origanum  oil,  10  cc. 

Cellosolve  is  ethylene  glycol  monoethyl 
ether.  It  mixes  with  water,  acetone, 
alcohol,  ether  and  dissolves  many  oils, 
waxes,  etc.  Employed  by  Lendrum 
(A.C.,  J.  Path.  &  Bact.,  1939,  49,  590- 
591). 

Cellulose,  microchemical  reaction  for.  Solu- 
tion A :  Dilute  20  cc.  of  2%  iodine  in  5% 
aq.  potassium  iodide  with  180  cc.  aq. 
dest.,  add  0.5  cc.  glycerin  and  mix  by 
shaking.  Solution  B :  Saturate  15  cc. 
aq.  dest.  with  lithium  chloride  at  80°C., 
cool  and  use  supernatant  solution. 
Tease  out  section  or  fibers.  Apply  2-3 
drops  "A"  by  glass  rod  and  leave  10  sec. 
Blot  with  filter  paper  and  dry.  Add 
drop  "B",  cover  and  examine.     Cellu- 


lose blue,  green,  yellow  depending  upon 
its  source  (Post,  E.  E.  and  Laudermilk, 
J.  D.,  Stain  Techn.,  1942,  17,  21-26. 
See  Polysaccharides. 
Cements.  W.  C.  Tobie  (in  Simmons,  and 
Gentzkow,  p.  356)  gives  two  useful 
types : 

Vacuum  wax  for  ordinary  vacuum 
seals  not  subjected  to  high  temperature 
is  made  by  melting  together  equl  parts 
of  beeswax  and  rosin.  It  is  pliable  and 
easily  removed  with  hot  water. 

Acid  resisting  cement  is  made  by 
mixing  asbestos  powder  and  sodium 
silicate  solution  (water  glass)  into  a 
paste  of  desired  consistency.  Will  dry 
in  24  hrs. 

For   ringing   specimens   mounted    in 
glycerin,  etc.  see  Kronig's  Cement  and 
Mounting  Media. 
Centigrade  temperature  to  Fahrenheit 

1.  Above  0°C.  multiply  by  9,  divide  by  5, 
add  32.  Example:  37°C.  =  37  X  9  = 
333  -^  5  =  66.6  -f  32  =  98.6°F. 

2.  Between  -17.77  and  0°C.  multiply  by 
9,  divide  by  5  subtract  from  32.  Ex- 
ample: -12°C.  =  12  X  9  =  108  -T-  5  = 
21.6;  32  -  21.6  =  10.4°F. 

3.  Below  -17.77°C.  Multiply  by  9,  di- 
vide by  5,  subtract  32.  Example: 
-18°C.  =  18  X  9  =  162  -^  5  =  32.4  - 
32   =   0.4°C. 

Central  Body,  see  Centrosome. 

Centrifugation.  To  even  sketch  in  outline 
the  techniques  that  come  under  this 
heading  is  difficult  because  the  centrifu- 
gation of  so  many  materials  and  tissues 
is  helpful  and  the  instruments  vary  from 
simple  hand  driven  machines  to  power- 
ful ultracentrifuges  which  may  weigh 
several  tons  and  which  certainly  require 
experts  to  care  for  them.  See  Svedberg, 
T.  and  Pedersen,  K.  O.,  The  Ultra- 
centrifuge,  Oxford,  Clarendon  Press, 
1940,  478  pp. 

The  centrifuge  has  long  been  of  help 
in  the  displacement  of  certain  com- 
ponents of  cells  (especially  marine  eggs) 
in  order  to  determine  their  functional 
r61es.  It  has  also  proved  invaluable  in 
the  investigation  of  cytoplasmic  and 
nuclear  Viscosity,  which  see. 

In  recent  years  centrifugation  has 
opened  a  new  chapter  in  microchemistrj' 
by  the  part  which  it  has  played  in  the 
collection  of  cellular  components  in 
sufficient  volume  for  analysis.  Pioneer 
work  was  done  with  the  liver.  The 
Bensleys  (p.  6)  give  instructions  which 
are  in  part  as  follows.  First  perfuse  the 
abdominal  organs  of  a  guinea  pig  with 
about  1000  cc.  0.85%  aq.  sodium  chloride 
(see  Perfusion).  This  removes  a  good 
deal  of  the  blood.  Excise  liver  and 
grind  up  thoroughly  in  a  mortar.  Place 
the  resulting  thick  fluid  in  large  centri- 


CENTRIFUGE  MICROSCOPE 


68 


CENTROSOMES 


fuge  tubes,  add  about  twice  the  volume  of 
0.85%  aq.  sodium  chloride  and  balance 
the  tubes  with  more  as  may  be  necessary. 
If  complete  separation  of  mitochondria  is 
desired  centrifuge  for  1  min.  at  3000 
r.p.m.  which  results  in  stratification. 
In  first  and  lowest  stratum,  at  the  bot- 
tom of  the  tubes,  will  be  found  liver  cells, 
cell  debris  and  connective  tissue  ele- 
ments ;  in  the  second,  nuclei  and  red 
blood  cells;  in  the  third  mitochondria 
and  small  cell  fragments;  and  in  the 
fourth  and  uppermost,  free  fatty  drop- 
lets. The  materials  in  any  of  these 
layers  can  then  be  collected  by  drawing 
up  in  a  pipette,  suspended  again  in  salt 
solution  and  purified  by  further  cen- 
trifugation. 

For  the  isolation  of  ellipsin  (structural 
protein)  and  mitochondria  see  Bensley, 
R.  R.  and  Hoerr,  N.  L.,  Anat.  Rec, 
1934,  60,  251-266  and  449-455.  Since  it 
is  in  the  mitochondrial  fraction  resulting 
from  centrifugation  that  vitamin  A  is 
found  the  Goerners  have  greatly  ex- 
tended the  usefulness  of  the  method  in  a 
series  of  studies  on  tumors  (Goerner) 
A.,  J.  Biol.  Chem.,  1937-38,  122,  529- 
538  and  A.,  and  M.  M.,  ibid,  1939,  128, 
559-565).  The  technique  has  been 
further  improved  by  Claude  (A.,  Sci- 
ence, 1938, 87,  467-468 ;  Cold  Spring  Har- 
bor Symposia  on  Quantitative  Biology 
1941,  9,  263-270)  who  used  18000  r.p.m. 
See,  particularly,  standardized  tech- 
niques in  his  1941  paper.  Beams, 
H.  W.  and  King,  R.  L.,  Anat.  Rec, 
1940,  76,  95-101,  and  in  a  series  of 
other  papers,  have  greatly  contributed 
to  the  use  of  ultracentrifugation  in  the 
solution  of  biological  problems.  See 
Lucas,  A.  M.,  Am.  J.  Path.,  1940,  16, 
739-760  on  intranuclear  inclusions. 
Centrifuge  Microscope.  By  this  ingenious 
combination  of  microscope  and  cen- 
trifuge it  is  possible  to  observe  living 
cells  with  the  highest  dry  objectives 
while  they  are  actually  being  cen- 
trifuged.  Cells  or  organisms  to  be 
examined  are  placed  in  isotonic  media 
of  appropriate  density  in  special  slides 
constructed  so  that  the  centrifugal 
force  derives  them  into  approximately 
the  focus  of  the  objective.  The  clear- 
ness of  the  2  dimensional  image  is  not 
conditioned  by  the  speed  of  rotation. 
The  slide  is  fixed  into  the  centrifuge 
head  remote  from  the  axis  of  rotation. 
Strong  light  is  focussed  by  condensing 
lens  from  above  onto  the  slide.  A  prism 
below  the  slide  in  the  centrifuge  head 
directs  the  light  toward  the  axis  of 
rotation  directly  through  an  objective. 
When  received  at  the  axis  of  rotation  it 
is  directed  upward  by  reflecting  prisms 
into  an  ocular  in  position  above  the 


center  of  the  centrifuge  head.  Not  only 
can  stages  in  displacement  of  intra- 
cellular components  be  watched  but 
permanent  records  are  easily  made  in 
the  form  of  motion  pictures.  Since  its 
introduction  by  Harvey  and  Loomis  in 
1930  several  structural  improvements 
have  been  achieved.  A  commercial 
design  is  made  by  Bausch  and  Lomb 
Optical  Company  (Harvey,  E.  N.,  in 
Glasser's  Medical  Physics  1944,  147). 

Centriole,  see  Centrosome. 

Centrosomes  (G.  Kentron,  center;  soma, 
body),  sometimes  called  a  "central 
body",  is  a  minute  spherule  which  is  a 
dynamic  center  of  some  sort  involved  in 
cell  division.  It  is  sometimes  called  a 
centriole  though  Conklin  (Cowdry's 
General  Cytology,  pp.  542  and  544)  says 
that  a  central  body,  the  centriole, 
appears  within  the  centrosome  during 
mitosis.  When  the  centrosome  is  double, 
that  is  consists  of  two  minute  bodies 
side  by  side,  it  is  designated  a  diplosome. 
About  the  centrosome,  or  diplosome, 
there  is  usually  a  clear  area  which  is 
known  as  a  centrosphere.  The  centro- 
some, or  centriole  plus  the  clear  area  is 
called  the  cytocentrum.  For  terminology 
see  Wilson,  E.B.,  The  Cell.  New  York: 
Macnaillan  Co.,  1925,  1232  pp.  For 
functional  significance  see  Fry,  H.J., 
Biol.  Bull.,  1929,  57,  I3I7I5O.  Giant 
centrospheres  in  degenerating  cells  are 
described  by  Lewis  and  Lewis  (Cow- 
dry's  General  Cytology,  p.  427)  and 
multiplication  of  centrioles  in  striated 
muscle  tumors  by  Wolbach,  E.  B.,  Anat. 
Rec,  1928,  37,  255-273. 

Centrosomes  are  not  easily  demon- 
strated in  tissue  sections.  The  tech- 
nique originally  used  by  Heidenhain 
(Arch.  f.  mikr.  Anat.,  1894,  42,  665)  ap- 
pears to  be  the  best.  It  consists  of 
fixation  in  a  Sublimate  Acetic,  or  Sub- 
limate Alcohol  Acetic,  and  of  staining 
the  sections  24  hours  in  a  dilute  aq.  sol. 
of  Bordeaux  red  or  of  anilin  blue  fol- 
lowed by  iron  hematoxylin  in  the  usual 
way.  The  centrosomes  are  stained 
black  or  gray  with  a  tinge  of  red  or  blue. 
In  glandular  epithelial  cells  look  for 
them  in  the  cytoplasm  between  the 
nucleus  and  the  lumen. 

To  reveal  centrosomes  in  non-dividing 
nerve  cells  is  difficult,  probably  because  1 
they  are  seldom  present.  Hatai  (S., 
J.  Corap.  Neurol.,  1901,  11,  25)  was  able 
to  stain  them  in  certain  nerve  cells  of 
adult  rats.  He  fixed  in  sat.  mercuric 
chloride  in  formalin,  30  cc;  glacial 
acetic  acid,  50  cc.  and  physiological  salt 
solution,  15  cc.  for  6-12  hrs.,  then 
washed,  4-5  hrs.  in  running  water,  im- 
bedded in  paraffin,  stained  in  sat.  aq. 
toluidin  blue  or  thionin,  dehydrated, 


CEPHALIN 


69 


CHAMPY-KULL'S  METHOD 


cleared  and  mounted  the  sections.  Rio 
Hortega  (P.,  Trab.  Lab.  Invest.  Biol. 
Univ.  Madrid,  1916,14,  117)has  obtained 
beautiful  silver  preparations  of  centro- 
somes.  Addison  (McClung,  1950  p.  378) 
advises  fixation  in  Flemming's  Fluid 
or  in  Allen's  chromic-urea  modification 
of  Bouin's  Fluid  followed  by  staining 
with  Heidenhain's  Iron  Hematoxylin. 

A  detailed  investigation  of  the  effects 
of  a  great  many  fixatives  on  the  mitotic 
figure  in  chaetopterus  eggs  has  been 
made  by  Fry  (Fry,  H.J.  Biol.  Bull.,  1933, 
65,  207-237).  He  concluded  (1)  that 
acetic  acid,  picric  acid,  formaldehj'de 
and  alcohol  and  certain  combinations  of 
them  are  most  useful  as  fixatives  (2) 
that  anesthetics  like  chloroform  and 
ether  and  inorganic  fixatives  are  to  be 
avoided;  (3)  that  the  fixatives  must  be 
diluted  to  about  10%  of  the  original  con- 
centration with  aq.  dest.  or  better  with 
sea  water.  Comparable  information  for 
human  tissues  is  lacking. 

Cephalin,  a  phosphatide,  is  a  compound  of 
phosphoric  acid,  glycerol,  2  fatty  acid 
molecules  and  amino  ethyl  alcohol.  It 
differs  also  from  lecithin  in  being  only 
very  slightly  soluble  in  alcohol,  see 
Lipoids. 

Cerasin  R,  see  Bordeaux  Red. 

Cerasln  Red,  see  Sudan  HL 

Cerebrosides  are  galactosides,  that  is  com- 
pounds of  fatty  acid,  galactose  and 
sphingosine,  without  phosphorus,  sol- 
uble in  benzene,  pyridine  and  hot 
alcohol  and  almost  insoluble  in  ether, 
see  Lipoids. 

Cerebrospinal  Fluid.  Total  cell  count  is 
best  made  in  a  Fuchs-Rosenthal  count- 
ing chamber.  In  making  smears  for 
the  differential  count  it  may  be  neces- 
sary first  to  add  a  little  albumin  fixa- 
tive to  the  slides  to  get  the  cells  to 
stick  (C.  J.  Lind  in  Simmons  and  Gentz- 
kow,  p.  91). 

Ceresin  Imbedding.  Ceresin  is  purified 
ozokerite,  a  mixture  of  hydrocarbons, 
with  melting  point  61-78°C.  used  as  a 
substitute  for  beeswax  and  for  other 
purposes.  Waddington,  C.  H.  and 
Kriebel,  J.,  Nature,  1935,  136,  685  ad- 
vise for  hard  objects  like  feathers  addi- 
tion of  ceresin  to  a  paraffin  of  slightly 
lower  melting  point  than  that  usually 
employed.  The  whole,  when  cooled,  has 
a  very  fine  texture.  See  the  methyl 
benzoate  celloidin  ceresin  method  of 
'Espinasse  for  imbedding  hard  objects 
in  a  suitable  condition  for  sectioning  as 
described  by  Lee  (p.  96)  and  Waterman, 
H.  C,  Stain  Techn.,  1939,  14,  55-62. 
Ceresin  can  be  obtained  from  Shell  Oil 
Co.,  melting  point,  82-85°C. 

Cerium,  see  Atomic  Weights. 

Ceroid.     This  is  a  wax  like  endogenous  pig- 


ment (G.  keros,  wax  -f  eidos,  resem- 
blance). It  is  greenish  yellow  to  golden 
brown  in  color,  fluorescent,  acid  fast, 
stains  with  fat  dyes  as  well  as  with 
methyl  green,  and  is  relatively  insoluble 
in  fat  solvents.  Ceroid  has  oeen  sepa- 
rated from  the  tissues  of  rats,  collected 
en  masse  and  analyzed  by  Moore,  T. 
and  Wang,  Y.  H.  (Brit.  J.  Nutr.,  1947, 

I,  53-64).  Mason,  K.  E.  and  Emmel, 
A.  F.  (Anat.  Rec,  1945,  92,  33-59)  con- 
sider its  demonstration  in  muscle  to 
be  reliable  indication  of  vitamin  E 
deficiency.  Grenados,  H.,  Mason,  K. 
E.  and  Dam,  H.  (Acta  Path.,  1947,  24, 
86-95)  present  evidence  in  rats  of  a 
metabolic  relationship  between  vitamin 
E  and  unsaturated  fatty  acids.  A  help- 
ful summary  of  the  distribution  of 
ceroid  pigment  in  human  tissues  is 
supplied  by  Pappenheimer,  A.  M.  and 
Victor,  J.  (Am.  J.  Path.,  1946,  22, 
395-412).  The  properties  of  ceroid  pig- 
ment in  relation  to  experimental  necro- 
sis in  rats  and  mice,  as  revealed  by  many 
histochemical  techniques,  are  presented 
by  Lee,  C.  S.    (J.  Nat.  Cancer  Inst., 

II,  339-347).  The  important  point  is 
made  that  there  are  easily  noticeable 
differences  between  the  ceroids  of  these 
two  species.  An  interesting  technique 
for  the  study  of  ceroidlike  substances 
is  to  produce  them  in  vitro  and  in  vivo 
from  certain  lipids  and  erythrocytes 
as  has  been  done  by  Hartroft,  W.  S. 
(Science,  1951,  113,  673-674). 

Cerotine  Ponceau  3B,  see  Sudan  IV. 

Cerulein  MS  (CI,  783) — Anthracene  Green, 
Coerulein  MS — a  mordant  dye  of  light 
fastness  3  to  4  gives  unsatisfactory 
coloration  of  animal  tissues.  Direc- 
tions for  plants  (Emig,  p.  55). 

Cervical  Swab-Smears,  see  Paparnicolaou 
Techniques. 

Cesares-Gil  flagella  stain  evaluated, 
Thatcher,  L.  M.,  Stain  Techn.,  1926, 
1,  143-144. 

Cesium,  spectrograph  c  analysis  of,  in 
retina  (Scott,  G.  H.  and  Canaga,  B., 
Jr.,  Proc.  Soc.  Exp.  Biol.  &  Med.,  1939, 
40,  275). 

Cestoda,  see  Parasites,  Taenia. 

Cevitamic  Acid,  see  Vitamin  C. 

Chalkley  ratio  method  for  determining  rela- 
tive volumetric  composition  of  tissue 
sections  (Chalkley,  H.  W.,  J.  Nat. 
Cancer  Inst.,  1943,  4,  47). 

Chambers,  see  Micromanipulation. 

Champy-KuH's  Method  of  anilin  fuchsin, 
toluidine  blue  and  aurantia  for  mito- 
chondria. Fix  in  Champy's  fluid  (3% 
potassium  bichromate,  7  cc. ;  1%  chromic 
acid,  7  cc. ;  2%  osmic  acid,  4  cc.)  24  hrs. 
Wash  in  aq.  dest.  Place  in  pyrolig- 
neous  acid,  1  part  and  1%  chromic  acid, 
2  parts  20  hrs.     Wash  aq.  dest.  30  min. ; 


CHAMPY'S  FLUID 


70 


CHLORAZOL  PAPER  BROWN  B 


mordant  3%  aq.  potassium  bichromate, 
3  days.  Wash  running  water  24  hrs., 
dehydrate,  clear,  imbed  and  section 
at  4ju.  Remove  paraffin  from  sections. 
Stain  with  anilin  acid  fuchsin  (acid 
fuchsin  10  gms.,  anilin  water  100  cc.) 
heated  over  spirit  lamp  and  allow  to  cool 
6  min.  Rinse  in  aq.  dest.  Counter- 
stain  in  0.5%  aq.  toluidine  blue  1-2  min. 
Rinse  inaq.  dest.,  then  0.5%aurantiain 
70%  alcohol  20-40  sec.  Differentiate  in 
95%  alcohol,  dehydrate,  clear  and 
mount.  Mitochondria  red,  nuclei  blue 
and  ground  substance  yellow. 

Champy's  Fluid  is  3%  potassium  bichro- 
mate, 7  parts ;  1%  chromic  acid,  7  parts ; 
and  2%  osmic  acid,  4  parts.  It  is  an 
excellent  fixative  for  cytologic  details. 

Charcot-Leyden's  Crystals.  Octahedral 
phosphate  crystals  found  in  stools  of 
persons  infected  with  Endameba  his- 
tolytica and  in  a  variety  of  other  condi- 
tions. See  description  and  illustration 
by  Craig,  p.  58. 

Cheese.  Bacteria  in,  see  Hucker,  G.  J., 
N.  Y.  Agric.  Exp.  Sta.  Tech.  Bull. 
1921,87(McClung,  p.  147). 

Chelidonium,  see  Fluorescence  Microscopy, 
shows  "secondary"  fluorescence. 

Chicago  Blue,  see  escape  from  venules  after 
intravenous  injection  (Smith,  F.  and 
Rous,  P.,  J.  Exp.  Med.,  1931,  54,  499- 
514). 

Chick,  see  Chorioallantoic  Membrane. 

China  Blue,  see  Anilin  Blue. 

Chitin.  This  is  a  keratoid,  or  keratin-like, 
substance  which  is  an  important  part 
of  the  exoskeleton  and  hard  parts  of 
invertebrates  (G.  chiton,  a  coat).  It  is 
soluble  in  hot  concentrated  sulphuric 
and  hydrochloric  acids;  relatively  in- 
soluble in  alcohol,  ether,  dilute  acids 
and  alkalies.  There  are  several  color 
reactions  for  chitin.  Lillie  (p.  147) 
applies  to  chitin,  thoroughly  washed 
after  treatment  with  potassium  hydrox- 
ide, 33%  aq.  zinc  chloride  to  which  3-5 
drops  cone.  aq.  iodine  potassium  iodide 
have  been  added  to  each  10  cc.  Chitin 
becomes  brown  on  the  surface,  violet 
within.  Chitin  is  colored  red  violet 
by  a  solution  containing:  iodine,  50 
mg.,  potassium  iodide  50  mg.,  calcium 
chloride  16  gm.  and  aq.  dest.  4  cc. 

1.  A  method  for  softening  of  chitin  in 
formalin  fixed  insects  (Murray,  J.  A., 
J.  Roy.  Micr.  Soc,  1937_,  57,  15).  Fix 
primarily  in  10%  formalin  in  0.8%  aq. 
sodium  chloride,  or  indefinitely.  Fix  sec- 
ondarily and  dehydrate  in  equal  parts 
absolute  alcohol,  chloroform  and  glacial 
acetic  acid  +  corrosive  sublimate  to  satu- 
ration (about  4%).  Warm  together  equal 
parts  chloral  hydrate  and  phenol  until 
they  fuse  and  form  an  oily  liquid  which  is 
fluid  at  room  temperature .     Leave  speci- 


mens in  this  12-24  hrs.  or  longer.  Clear 
in  chloroform,  xylol  or  carbon  disul- 
phide.     Imbed  in  paraffin. 

2.  According  to  Hennings  (see  Lee, 
p.  597)  fixation  of  insects  in  the  following 
mixture  softens  the  chitin  sufficiently 
to  permit  the  making  of  paraffin  sections: 
nitric  acid,  16  cc. ;  5%  aq.  chromic  acid, 
16  cc;  sat.  corrosive  sublimate  in 60% 
alcohol,  24  cc;  sat.  aq.  picric  acid,  12 
cc;  and  abs.  ale,  42  cc.  Fixation  is 
12-24  hrs.  followed  by  washing  in  iodine 
alcohol.  An  older  method  is  to  soften 
chitin  by  treatment  with  a  solution  of 
hypochlorite  of  soda  (Lee,  p.  249). 
See  Diaphanol,  N.  Butyl  Alcohol,  In- 
sects, and  Ticks. 

Chloral  Hydrate,  as  a  fixative  for  peripheral 
nerves  (Bank,  E.  W.  and  Davenport, 
H.  A.  Stain  Techn.,  1940,  15,  9-14). 
Chloral  hydrate  is  also  recommended  as 
a  macerating  medium  for  the  separation 
and  isolation  of  epithelial  and  lining 
cells  by  the  Bensleys  (p.  5).  Accord- 
ing to  their  instructions  remove  small 
pieces  alimentary  tract  of  pithed  or 
freshly  killed  frog  and  leave  them  in 
5%  aq.  chloral  hydrate  12-48  hrs.  Then 
tease  with  fine  needles  and  examine. 
See  Cajal's  chloral  hydrate  method. 

Chlorazol  Black  E  (CI,  581)  of  British  Dye- 
stuffs  Corporation — Erie  black  G  X  00 
(National  Aniline  and  Chemical  Com- 
pany), Pontamine  black  E  (I.  E.Du  Pont 
deNemours  &  Co.) — an  acid  poly-azo 
dye.  First  described  as  a  new  biological 
stain  by  Cannan  (H.  J.,  Nature,  1937, 
139,  549).  Review  of  its  uses  (Cannan, 
H.  J.,  J.  Roy.  Micr.  Soc,  1941,  61, 
88-94).  As  a  vital  dye  (Baker,  J.  R., 
Nature,  1941,  147,  744).  Stains  chro- 
matin black,  cytoplasm  greenish  gray 
after  Zenker  fixation  (Darrow,  M.  A. 
Stain  Techn.,  1940,  15,  67-^8).  As  an 
acetocarmine  auxiliary  stain  for  chro- 
mosomes (Nebel,  B.  R.,  Stain  Techn., 
1940, 15,  69-72) .  As  a  simple  connective 
tissue  stain  (Levine,  N.  D.,  and  Morril, 
C.  C,  Stain  Techn.,  1951  16,  121-122) 
it  is  excellent  for  arterial  elastic  tissue. 

Chlorazol  Blue  3B,  see  Trypan  Blue. 

Chlorazol  Fast  Pink  used  as  anticoagulant 
in  experiments  designed  to  influence 
growth  of  transplants  of  lymphosar- 
comas (Williams,  W.  L.,  Cancer  Re- 
search, 1946,  6,  344-353).  Toxicity  as 
an  intra  vitam  stain  is  presented  by  Wil- 
liams, W.  L.  and  Hodge,  H.  C,  Anat. 
Rec,  1943,  87,  181. 

Chlorazol  Paper  Brown  B.  A  sat.  aq.  sol. 
of  this  dye  has  been  employed  to  stain 
plant  tissues.  It  is  differentiated  in 
1%  aq.  nitric  acid.  Acetone  is  used 
for  dehydration  and  Euparal  for  mount- 
ing (Verdcourt,  B.,  Stain  Techn.,  1947, 
22,  155-156). 


CHLORAZOL  PINK  Y 


71 


CHLORIDE 


Chlorazol  Pink  Y,  see  Thiazine  Red  R. 

Chloride.  In  1908  Macallurn  reviewed  the 
older  literature  and  described  his  silver 
test  for  chloride  (Macallum,  A.  B., 
Ergeb.  d.  Physiol.,  1908,  7,  552-652). 
The  possibility,  which  has  not  yet  been 
finally  answered,  is  that  at  some  stage 
in  the  technique  there  is  a  shift  in  the 
position  of  chloride.  The  mere  applica- 
tion of  the  silver  reagent  may  conceiv- 
ably withdraw  chloride  from  the  cell. 
For  these  reasons  prior  treatment  of  the 
tissue  by  the  Altmann-Gersh  freezing 
and  drying  method  which  reduces  the 
chance  of  movement  of  chloride  to  a 
minimum  is  recommended. 

1.  Gersh  (Gersh,  I.,  Anat.  Rec,  1938, 
70,  311-329)  gives  details  of  the  proced- 
ure on  which  the  following  instructions 
are  based.  Tissues  frozen  in  liquid  air, 
dried  in  vacuum,  embedded  in  paraffin 
and  sectioned  at  15m  are  mounted  near 
one  edge  on  chemically  clean  large  cover 
slips  by  simply  pressing  down  with  a 
finger,  just  melting  over  a  flame  and 
pressing  down  again.  Immerse  cover- 
slips  with  attached  sections  in  anhydrous 
petroleum  ether  (b.p.  20-40°C.)  freshly 
distilled  over  sodium  in  a  watch  glass 
covered  by  another  at  all  times  except 
during  actual  manipulations.  This  re- 
moves the  paraffin.  Remove  and  burn 
off  the  ether  quickly  by  a  flame  and  allow 
to  cool  to  room  temperature.  Then  treat 
two  coverslips  with  attached  sections 
differently. 

A.  Cover  for  few  seconds  with  drop 
of  60%  aq.  silver  nitrate  diluted  with 
sufficient  quantity  of  cone,  phosphoric 
acid  to  prevent  precipitation  of  rather 
large  concentrations  of  phosphates  and 
then  saturate  with  silver  chloride.  After 
filtering  2-3  drops  aq.  dest.  are  added  to 
every  10  cc.  before  using. 

B.  Cover  similarly  with:  60%  aq. 
silver  nitrate  saturated  with  silver  phos- 
phate and  silver  chloride  and  dilute 
after  filtering  in  the  same  way. 

Decant  fluids  from  both  coverslips. 
Add  to  each  1  drop  chemically  pure 
glycerin  and  mount  with  section  plus 
glycerin  down  on  chemically  clean  slides. 
Expose  both  to  carbon  arc  radiation  for 
same  length  of  time  but  at  a  distance 
not  to  warm  the  specimens.  Examine 
immediately  the  reduced  silver  by  direct 
illumination  or  in  the  dark  field.  A. 
shows  specifically  only  the  chloride  and 
B.  the  same  amount  of  chloride  plus 
maximal  concentrations  of  phosphate 
and  some  carbonate. 

2.  Dichlor fluorescein  method  (Bens- 
ley,  R.  D.  and  S.  H.,  Anat.  Rec,  1935, 
64,  41-49).  For  the  lung  of  a  rabbit. 
Inject  1%  aq.  dichlorfluorescein  intra- 
venously   until    the    animal    becomes 


quite  yellow.  Then  kill  it  and  inject 
10%  aq.  silver  nitrate  or  Silver  Citrate 
solution  either  intratracheally  or  di- 
rectly into  the  lung  substance  by  a  hy- 
podermic syringe  until  the  lung  is 
moderately  distended.  In  about  20 
min.  the  color  reaction  reaches  its 
maximum.  The  silver  chloride  becomes 
pink  owing  to  adsorption  of  the  dichlor- 
fluorescein on  the  positively  cliarged 
silver  chloride  molecule.  Then  fix 
pieces  of  lung  in  10%  neutral  formalin 
and  make  frozen  sections.  Examine 
immediately  for  best  color  reaction. 
Dehydrate  the  sections,  clear  in  absolute 
alcohol  and  iso-safrol  and  mount  in  bal- 
sam. The  color  reaction  is  not  perma- 
nent but  is  masked  and  finally  lost  by 
the  browning  and  blackening  of  the  sil- 
ver. It  is  not  a  true  microchemical 
test;  but  it  does  detect  the  presence  of 
chlorides  though  they  are  mobilized  by 
the  silver  and  tend  to  move  to  the  per- 
iphery of  the  cell.  The  alveolar  epi- 
thelial cells  are  outlined  by  pink  stip- 
pling and  their  cytoplasm  is  also  stippled 
and  the  nuclei  are  richly  stippled. 
Mesothelial  and  endothelial  cells  are 
brilliantly  and  completely  outlined  in 
pink.  The  technique  was  first  sug- 
gested by  David  M.  Ritter. 

The  location  of  chloride  is  a  matter  of 
great  importance.  Lowry,  O.  H.  and 
Hastings,  A.  B.  in  Cowdry's  Problems 
of  Ageing,  Baltimore:  Williams  &  Wil- 
kins,  1942,  936  pp.  cite  the  following  as 
evidence  for  the  extracellular  position 
of  chloride  in  skeletal  muscle : 

(1)  Direct  microscopic  studies  show- 
ing that  chloride  is  exclusively  extra- 
cellular (Gersh,  I.,  Anat.  Rec,  1938, 
70,311-329). 

(2)  Perfusion  experiments  showing 
that  chloride  can  be  removed  without 
apparently  affecting  the  intracellular 
phase  (Amberson,  W.  R.  et  al..  Am.  J. 
Physiol.,  1938,  122,  224-235). 

(3)  Variations  in  amount  of  chloride 
and  in  acid  base  balances  of  tissues  can 
only  be  accounted  for  by  assuming 
an  extracellular  position  for  chloride 
(Hastings,  A.  B.  and  Eichelberger,  L., 
J.  Biol.  Chem.,  1937,  117,  73-93). 

(4)  Isolated  tissues  equilibrated  in 
vivo  against  solutions  of  varying  chloride 
concentrations  retain  chloride  in  pro- 
portion to  the  concentration  in  the 
medium  but  at  a  very  much  lower  level 
(Fenn,  W.  O.,  Cobb,  D.  M.  and  Marsh, 
B.  S.,  Am.  J.  Physiol.,  1934,  110,  261- 
272;  Eggleton,  M.  G.  and  P.  and  Hamil- 
ton, A.  M.,  J.  Phvsiol.,  1937,  00,  167- 
182). 

(5)  Conclusion  that  in  many  tissues 
for  all  practical  purposes  all  radioactive 
sodium  and  radioactive  chloride  remain 


CHLOROPHENOL  RED 


72 


CHLOINESTERASE 


outside  the  cells  (Manery,  J.  F.  and 
Bale,  W.  F.,  Am.  J.  Physiol.,  1941,132, 
215-231;  Manery,  F.  W.  and  Haege, 
L.  F.,  ibid,  134,  83-93). 

See,  however,  Heilbrunn,  L.  V.  and 
Hamilton,  P.  G.,  Physiol.  Zool.,  1942, 
15,  363-374  for  demonstration  of  chloride 
in  muscle  fibers. 

If  chloride  is  always  extracellular  in  all 
tissues  it  is  possible  accurately  to  meas- 
ure the  amount  of  extracellular  fluid 
and  a  new  chapter  in  histochemistry  is 
opened.  Lowry  and  Hastings  give  an 
example.  If  rat  muscle  is  found  to 
contain  10.5  milliequivalents  of  chloride 
per  kilogram  of  tissue  and  the  serum  of 
the  same  animal  105.2  milliequivalents 
of  chloride  per  kilogram  of  serum  water, 
in  view  of  the  Donnan  effect  on  chloride 
distribution  it  can  be  calculated  that  a 
kilogram  of  extracellular  fluid  contains 
109.7  milliequivalents  of  chloride.  Con- 
sequently the  sample  of  muscle  contains 

10  5 
rrr^  X  1000  =  96  gms.  of  extracellular 

fluid  per  kilogram.  When  the  extra- 
cellular fluid  contains  coUagenic  and 
elastic  fibers,  collagen  and  elastin  must 
be  determined  and  the  necessary  correc- 
tions made  as  well  as  for  blood  and  fat 
when  these  are  present.  When  the  in- 
tracellular phase  is  chiefly  composed  of 
a  single  type  of  cell  as  in  skeletal  or 
cardiac  muscle  the  further  evaluation  of 
intracellular  components  is  not  diffi- 
cult. Taking  every  known  precaution, 
evidence  can  apparently  be  collected  of 
the  relative  composition  of  extracellular 
and  intracellular  phases. 

If  it  is  desired  to  determine  chloride 
in  very  small  amounts  of  fluid,  as  in 
the  analysis  of  glomerular  urine,  a 
method  described  by  Westfall,  B.  B., 
Findlev,  T.  and  Richards,  A.  N.  (J. 
Biol.  Chem.,  1934,  107,  661-672)  is  sug- 
gested. Glick  (p.  200)  says  this  per- 
mits chloride  determination  in  a  frac- 
tion of  a  m1  of  fluid  containing  1  Mgni. 
or  less  of  the  choride.  The  technique 
of  Sendroy,  J.  (Jr.,  J.  Biol.  Chem. 
Chem.,  1942,  142,  171-173)  is  adequate 
for  10  (mI  of  serum.  There  are  in  addi- 
tion several  titrimetric  methods  for 
chloride  which  should  likewise  be  con- 
sidered as  possibilities.  See  Glick,  pp. 
281-283. 

Chlorophenol  Red.  See  Hydrogen  Ion  Indi- 
cators. 

Chlorophyll.  The  green  pigment  of  plants 
is  a  mixture  of  2  substances  chlorophyll 
a  and  b,  of  which  many  derivatives  are 
known.  In  man  several  fluorescent 
chlorophyll  porphyrins  are  identifiable 
in  feces  and  urine.  A  detailed  account 
of  chlorophyll  is  provided  by  Rothe- 


mund.  P.,  in  Glasser's  Medical  Physics, 
1944,  154-180. 

Chloroplasts.  Isolation  and  collection  en 
masse  from  spinach  leaves  by  centri- 
fugation  (Menke,  W.,  Zeit.  f.  Physiol. 
Chem. ,  1938-39,  257,  43.     See  Glick. 

Chloroprene,  see  Neoprene. 

Chlorothymols,  as  preservatives  of  gelatin, 
glues,  starches,  etc.  (Law,  R.  S.,  J.  Soc. 
Chem.  Ind.,  1941,  60,  66). 

Chocolate  Blood  Agar,  see  Bacteria,  Media. 

Cholesterol  (esters)  =  cholesterides.  In 
unstained  frozen  sections  mounted  in 
syrup  of  levulose  they  show  no  color  of 
their  own;  but  the  Liebermann-Bur- 
chardt  Reaction  in  frozen  sections  of 
formalin  fixed  tissue  is  positive.  Digi- 
tonine  Reaction  in  similar  sections 
yields  a  complex  in  which  the  esters,  if 
present,  will  color  with  Sudan  III  and 
lose  birefringence  in  polarized  light. 
See  Lipids  tabular  analysis,  see  Schultz 
test  for  cholesterol  and  its  esters. 
Technique  for  determination  in  buffer 
is  given  by  Nieman,  C.  and  Groot,  E.  H., 
Acta  Physiol,  et  Pharmacol.  Neerland., 
1950,  1,  488-501. 

Cholesterols  (free).  In  unstained  frozen 
section  mounted  in  syrup  of  levulose, 
they  show  no  color  of  their  own.  Lie- 
bermann-Burchardt  Reaction  in  frozen 
sections  of  formalin  fixed  tissue  is  posi- 
tive :  blue,  purple  or  violet  then  becom- 
ing green.  Digitonine  Reaction  in  simi- 
lar sections  yields  strongly  birefringent 
cr3-stals  and  rosettes  which  do  not  stain 
with  Sudan  III.  See  Lipids,  tabular 
analysis. 

Choline.  See  Florence's  Reaction  for  Semi- 
nal Stains. 

Choline  Deficiency.  Use  of  fluorescence 
microscopv  in  (Popper,  11.  and  Chinn, 
H.,  Proc.  Soc.  Exp.  Biol.  &  Med.,  1942, 
49,  202-204).     See  Vitamin  B  complex. 

Cholinesterase — Written  bj'  E.  W.  Demp- 
sey,  Dept.  of  Anatomy,  Washington 
University,  St.  Louis,  February  26, 1951 
— Important  since  it  is  implicated  in 
nervous  transmission.  It  is  present  in 
high  concentration  in  the  neuromuscu- 
lar junction  and  in  the  electric  organ  of 
eels,  and  has  also  been  detected  in  the 
axis  cj4inder  of  nerves.  Anfinsen,  C. 
B.,  O.  H.  Lowry  and  A.  B.  Hastings 
(J.  Cell,  and  Comp.  Physiol.,  1942, 
20,  231-237)  have  developed  a  method 
whereby  the  same  section  of  rat  brain 
cortex  can  be  stained  for  microscopic 
examination  and  thereafter  used  for 
enzyme  measurement.  Recently,  a 
number  of  techniques  have  been  pro- 
posed for  its  detection  on  slides.  Go- 
mori,  G.  (Proc.  Soc.  Exp.  Biol,  and 
Med.,  1948,  68,  354-358)  reported  that 
long-chain  fatty  acid  esters  of  choline 
were  hydrolyzed  by  tissue  sections,  and 


CHONDRIOSOMES 


73 


CHOR'S  MODIFICATION 


that  the  fatty  acids  could  then  be 
visualized.  However,  hydrolysis  of 
such  substrates  is  slow  and  probably 
caused  by  a  non-specific  esterase. 
Koelle,  G.  B.  and  J.  S.  Friedenwald 
(Proc.  Soc.  Exp.  Biol,  and  Med.,  1949, 
70,  617-622)  proposed  the  use  of  acetyl- 
thiocholine  iodide  as  a  substrate,  and 
obtained  beautiful  localizations  within 
the  central  nervous  system.  These  au- 
thors have  also  differentiated  between 
the  true  and  pseudocholinesterases  by 
means  of  specific  inhibitors  (J.  Nat. 
Cancer  Inst.,  1950, 10, 1364).  Seligman, 
A.  M.,  M.  M.  Nachlas,  L.  H.  Man- 
heimer,  O.  M.  Friedman  and  G.  Wolf 
(Ann.  Surg.,  1949,  130,  333-341)  have 
proposed  a  naphthyl  derivative  of  ace- 
tylcholine as  a  substrate.  After  hydro- 
lysis, the  naphthyl  moiety  is  visualized 
by  diazotization. 

Chondriosomes,  see  Mitochondria. 

Chondriotin  Sulphuric  Acid.  Present  in 
cartilage  and  bone,  stains  metachro- 
matically  with  basic  dyes,  described  in 
detail  by  Lison,  L.,  Arch,  de  biol., 
1935,  46,  599-668.     See  Muccproteins. 

Chorioallantoic  Membrane.  1.  Vital  stain- 
ing of  virus  lesions  in  membrane  (Cooke, 
J.  V.  and  Blattner,  R.  J.,  Proc.  Soc. 
Exp.  Biol.  &  Med.,  1940,  43,255-256). 
Place  1  cc.  0.5%  aq.  trypan  blue  directly 
on  membrane  through  window  in  shell. 
Rotate  egg  gently  and  return  to  incu- 
bator, 10-30  min.  Small  lesions  require 
longer  time  to  stain  than  large  ones. 
Remove  membrane,  wash  it  gently  in 
physiological  saline  and  fix  flat  in  10% 
formalin,  a  few  minutes.  Make  up 
glycerin  jelly  by  soaking  5  gms.  gelatin 
in  44  cc.  aq.  dest.  Then  add  50  cc. 
glycerin  and  1  cc.  phenol.  Heat  gently 
and  stir.  Flatten  membrane  on  a 
2  X  2.5  in.  slide,  warm  glycerin  jelly  to 
about  70°C.  Add  drop  by  drop  to  mem- 
brane until  well  covered.  Flame  a  cover 
glass  and  apply  with  slight  pressure  until 
it  has  begun  to  set.  Remove  hardened 
jelly  around  edges  and  seal  with  balsam. 
Foci  of  virus  increase  are  sharply  marked 
by  clumps  of  deep  blue  stained  cells. 

2.  Cultivation  of  microorganisms. 
The  membrane  has  been  shown  to  be  an 
excellent  medium  for  the  cultivation  of 
viruses  by  Goodpasture,  E.  W.,  Wood- 
ruff, A.  M.  and  Buddingh,  G.  J.,  Am.  J. 
Path.,  1932,  8,  271-282  and  many  others. 
Its  usefulness  has  been  extended  to 
Rickettsiae  and  spirochetes  by  Good- 
pasture, E.  W.,  Am.  J.  Hyg.,  1938,  28, 
111-119,  to  fungi  by  Moore,  M.,  Am. 
J.  Path.,  1941,  17,  103-125  and  to  acid- 
fast  bacteria  by  Moore ,  M . ,  Am .  J .  Path . , 
1942, 18,  827-847.  This  method  of  inocu- 
lation has  the  advantage  over  laboratory 
animal  inoculation  in   that  lesions  will 


develop  in  the  former  within  5-8  days  as 
compared  to  weeks  or  months  in  the 
latter;  most  organisms  will  produce  defi- 
nite and  usually  characteristic  lesions  in 
the  chick  membrane,  whereas  they  may 
have  no  effect  on  experimental  animals, 
often  requiring  human  subjects;  and 
because  the  lesions  are  so  readily  visible 
and  traceable  the  chlorioallantois  serves 
well  as  a  means  of  virulence  deter- 
mination. 

The  technique  is  essentially  that  of 
Goodpasture  and  Buddingh  (E.  W.and 
G.  J.,  Am.  J.  Hyg.,  1935,  21,  319-360) 
with  some  slight  changes.  Fertile  eggs 
are  incubated  12  days  in  an  electrical 
thermostat-controlled  incubator  regu- 
lated to  maintain  a  temperature  of  98°F. 
The  eggs  are  turned  twice  daily.  A  cm. 
square  window  is  cut  in  the  shell  above 
the  embryo,  exposing  the  chorioallantoic 
membrane.  The  position  of  the  em- 
bryo is  determined  by  candling.  The 
membrane  is  then  inoculated  directly 
with  the  fungus  and  the  window  is  cov- 
ered with  a  sterile  coverslip  and  sealed 
with  a  paraffin-vaseline  mixture  (9  parts 
vaseline,  1  part  paraffin).  After  inocu- 
lation, the  eggs  are  set  in  a  bacteriologic 
incubator  and  maintained  at  a  tempera- 
ture of  approximately  33°C.,  without 
turning.  The  membrane  is  watched 
daily  through  the  window.  When  the 
inoculated  area  has  shown  marked 
change,  the  shell  is  cut  below  the  window 
and  the  membrane  exposed.  The 
chorioallantois  is  cut  with  a  pair  of  fine 
curved-end  scissors,  removed,  fixed  in 
Zenker's  solution  (with  5%  glacial 
acetic).  After  washing,  dehydrating, 
clearing  in  xylol,  and  imbedding  in 
paraffin,  it  is  sectioned  and  stained. 
Various  staining  techniques  can  be  used 
depending  on  the  organism  inoculated. 
In  general,  for  fungi,  Loeffler's  meth- 
ylene blue  and  eosin  have  given  satis- 
factory results.  For  experimental  tech- 
nique of  growing  mouse  sarcoma  in 
chorio-allantoic  membrane,  see  Jacoby, 
F. , McDonald,  S.  and  Woodhouse.D.  L., 
J.  Path,  and  Bact.,  1943,  55,  409-417. 
Chor's  Modification  of  Ranson's  pyridine 
silver  method  was  worked  out  in  our 
laboratory  to  show  alterations  in  motor 
end  plates  in  biceps  and  triceps  of  mon- 
keys in  experimental  poliomyelitis 
(Chor,  H.,  Arch.  Neurol.  &  Psychiat., 
1933,  29,  344-357).  Fix  in  1%  ammonia 
water  (28%  Merck)  in  95%  alcohol  for 
24  hrs.  Wash  in  aq.  dest.,  ^  hr.  Pyri- 
dine, 48  hrs.  Wash  in  8  changes  aq. 
dest.  during  24  hrs.  2%  aq.  silver  nitrate 
in  dark  at  room  temperature,  72  hrs. 
Reduce  6-8  hrs.  or  over  night  in :  pyro- 
gallic  acid,  4  gm.;  aq.  dest.,  95  cc; 
formalin,  5  cc.     Dip  in  water  and  trans- 


CHROM  BLUE  GCB 


74 


CHROMOPHIL 


fer  immediately  to  95%  alcohol  for  a  few 
seconds.  Place  tissue  on  slide  with 
longitudinal  markings  of  fibers  visible. 
Add  a  second  slide  and  squeeze  gently. 
Trim  edges  with  sharp  knife  so  that  neat, 
flat  blocks  result.  95%  alcohol,  30  min. 
Absolute  alcohol,  2  changes,  over  night. 
Xylol,  10-12  hrs.  until  blocks  are  clear. 
Imbed  in  paraffin  8  hrs.  changing  re- 
peatedly each  hr.  for  first  five.  Cut 
serial  sections  10/it.  Mount  in  neutral 
balsam.  Nerves,  dark  brown  or  black; 
muscle  and  connective  tissue,  yellow. 

Chrom  Blue  GCB,  see  Gallocyanin. 

ChromaflBn  Reaction  (chromic  salts  +  L. 
ajjinis,  akin).  Brown  coloration  when 
treated  with  fixatives  containing  bi- 
chromate. In  adrenal  medulla  adrenalin 
is  revealed  by  this  brown  color  but  the 
reaction  can  also  be  elicitated  by  po- 
tassium iodate  and  is  not  altogether 
specific  for  adrenalin.  Lison  (p.  147) 
advises  fixation  in  Formol-Miiller  or  in 
6%  potassium  iodate  containing  10%  of 
formol.  After  the  usual  fixations  chro- 
maffin substances  can  be  demonstrated 
simply  by  treating  the  sections  for  a  few 
hours  with  3%  aq.  bichromate  or  iodate 
of  potassium  (Lison).  See  Vulpian Re- 
action and  Osmic  Acid. 

Chromatin  Filaments.  The  studies  of 
Claude,  A.  and  Potter,  J.  S.,  J.  Exper. 
Med.,1943,77,  345andof  Mirsky,  A.  E., 
and  Pollister,  A.  W.,  Biological  Sym- 
posia., 1943,  10,  247-260  indicate  that 
chromatin  is  almost  wholly  made  up  of 
fibrous  nucleoprotein.  By  extraction, 
precipitation  and  centrifugation  the 
chromatin  filaments  can  be  collected. 
They  are  of  very  uniform  diameter, 
like  chromosomes,  are  very  resistant  to 
deforming  mechanical  injury,  can  easily 
be  stained  with  acetocarmine  are  given 
a  positive  Feulgen  reaction.  Mirsky 
and  Pollister  favor  the  view  "that 
chromatin  is  largely,  if  not  entirely,  a 
complex  of  highly  polymerized  desox- 
yribose  nucleic  acid  with  a  basic  protein 
of  either  the  protamine  or  histone 
type." 

Chromatin  Stains.  The  most  specific  stain 
for  basic  chromatin  is  methyl  green. 
Bismark  brown  is  less  so.  Safranin  is 
useful  for  chromatin  if  a  red  coloration 
is  desired  as  in  the  safranin-light  green 
combination.  Tests  for  Iron  and  Thymo- 
nucleic  Acid  are  listed  separately. 
See  Idiochromatin,  Linin,  Chromosomes 
and  Nucleolus. 

Chromatolysis  of  nerve  cells  investigated  by 
absorption  spectra  of  Nissl  bodies 
(Gersh,  I.,  and  Bodian,  D.,  Biological 
Symposia,  1943,  10,  163-184). 

Chromatophores.  These,  when  present  in 
the  dermis,  are  also  called  melanoblasts, 


see   Dopa   Reaction   for   their   demon- 
stration. 

Chrome  Violet  CO  (CI,  727).  A  carboxyl 
derivative  of   pararosolic  acid. 

Chromic  Acid  is  purchased  as  the  red  crys- 
tals of  chromic  anhydride  which  dissolve 
easily  in  water  forming  chromic  acid. 
The  crystals  should  be  kept  in  a  bottle 
with  closely  fitting  glass  stopper  because 
they  are  highly  deliquescent.  Alone  in 
very  dilute  solution  chromic  acid  is 
helpful  in  Maceration.  When  applied  in 
aqueous  solutions  of  about  1%  to  a  slice 
of  fresh  adrenal  it  produces  a  brown  color 
in  the  medulla  known  as  the  chromaffin 
reaction.  In  mixtures  with  other  chem- 
icals it  was  more  used  as  a  fixative  50 
years  ago  than  today  but  in  Perenyi 
Fluid  it  is  recommended  strongly  by 
Lee  (p.  32)  for  embryos,  segmenting  eggs, 
etc.  It  is  also  a  component  of  Flem- 
ming's  fluid. 

Chromidial  Substance,  a  designation  often 
applied  to  basophilic  cytoplasmic  ma- 
terial supposed  to  be  of  nuclear  origin 
and  therefore  to  resemble  theextranu- 
clear  chromatin  (chromidia)  of  protozoa. 
It  is  nongerminal  chromatin  or  tropho- 
chromidia  in  contrast  to  germinal  or 
idiochromidia  (G.  idios,  individual, 
one's  own).     See  Nissl  bodies. 

Chromolipoids.  In  contrast  to  the  caro- 
tinoids,  which  are  hydrocarbons,  the 
chromolipoids  are  fats  or  derivatives  of 
fats  themselves  colored.  They  occur 
frequently  especially  in  nerve  cells,  in- 
terstitial cells  of  the  testicle  and  in  the 
adrenal,  and  are  easily  distinguishable 
from  carotinoids  because  they  do  not  give 
the  color  reactions  with  sulphuric  acid 
and  iodine-iodide.  From  melaninsthey 
are  to  be  distinguished  by  not  dissolving 
in  alkalies,  by  staining  with  sudanand 
scharlach  and  by  not  reducing  am- 
moniacal  silver  nitrate.  The  following 
method  of  Hueck  is  given  by  Lison : 
Stain  with  nile  blue.  Treat  the  sections 
for  24  hrs.  with  aq.  dest.  oxygenated 
3%  (=  commercial  hydrogen  peroxide 
diluted  with  12  volumes  water).  This 
leaves  the  chromolipoids  blue,  themela- 
nins  decolorized.  Lison  concludes  that 
distinction  from  pigments  of  hema- 
togenous origin  is  not  so  easy  because 
some  chromolipoids  contain  iron.  See 
Lipids,  tabular  analysis. 

Chromophil  (G.  chroma,  color  and  phileo, 
I  love),  a  loose  term  applied  to  almost 
any  granule,  cell,  or  tissue  which  has  a 
pronounced  affinity  for  stains.  Baso- 
philic cytoplasmic  materials  in  gland 
cells  and  in  nerve  cells  (Nissl  bodies)  are 
sometimes  called  chromophil,  moreover 
chromophil  reaction  is  unwisely  used  to 
designate  the  chromaffin  reaction  of 
epinephrin  producing  tissues. 


CHROM-OSMIC-ACETIC 


75 


CHROMOSOMES 


Chrom-Osmic-Acetic  fixative,  see  Lillie's. 

Chromosomes. — Written  by  A.  R.  Gopal- 
Ayengar,  Barnard  Free  Skin  and  Cancer 
Hospital,  St.  Louis,  Sept.  10,  1946— 
(Now  Pata  Memorial  Hospital,  Bom- 
bay) .  These  are  discrete  bodies  usually 
constant  in  number  in  the  cells  of  a 
given  species  and  frequently  having 
distinctive  structure  into  which  the 
chromatin  material  of  a  nucleus  re- 
solves itself  during  the  mitotic  process 
(see  Mitosis).  From  a  chemical  stand- 
point the  chromosome  is  a  protein  fiber 
like  silk  or  hair,  presumably  depending 
on  a  peptide  chain  linkage — C — C — N — . 
On  this  structural  framework  the  per- 
manent hereditary  units,  the  genes 
(which  may  be  considered  as  the  atoms 
of  heredity),  are  located  at  definite 
loci.  In  a  sense,  therefore,  the  chromo- 
some may  be  considered  a  giant  mole- 
cule (Darlington,  C.  D.,  Nature,  1942, 
149,  66-69,  Astbury,  W.  T.,  Proc.  7th 
Int.  Genet.  Congress.,  1939  (Camb.), 
1940,  49-51).  It  is  generally  stated 
that  the  chromosomes  of  sperm  cells 
consist  of  basic  proteins,  such  as  pro- 
tamines or  histones,  in  combination 
with  highly  polymerized  desoxyribose 
nucleic  acid  (Mirsky,  A.  E.,  Advances 
in  Enzymology,  1943,  3,  1-34). 

Smear-Squash  technique.  The  rapid 
and  spectacular  advances  in  our  knowl- 
edge of  cytology  and  cytochemistry 
during  the  last  fifteen  years  have 
greatly  altered  our  ideas  of  chromo- 
some structure  and  behavior.  Progress 
in  this  direction  has  been  possible 
through  the  introduction  of  newer  tech- 
niques, such  as  smears  and  squashes, 
which  have  largely  supplanted  the 
classical  methods  of  paraffin  sectioning. 
In  order  to  obtain  the  most  satisfactory 
results,  it  is  imperative  to  employ  cyto- 
logical  techniques  calculated  to  bring 
out  fine  structures  and  details  as 
quickly  and  accurately  as  possible. 
Most  of  the  smear-squash  methods,  de- 
spite their  extreme  simplicity,  give 
superior  results  because  of  instantane- 
ous fixation  and  rapidity  of  staining  and 
finishing  schedules.  Moreover,  it  is 
possible  to  examine  thin  layers  of  cells 
or  even  isolated  cells  and  their  parts. 
They  are  especially  useful  in  the  anal- 
ysis of  chromosome  complexes  and 
associations  and  in  the  exact  establish- 
ment of  numerical  relations.  Squash 
preparations  have  special  advantages. 
Individual  chromosomes  can  be  recog- 
nized more  readily  and  easily,  inter- 
relations between  chromosomes  become 
clearer,  the  preparations  are  almost 
two-dimensional,  which  is  of  undoubted 
value  in  chromosome  measurement 
since  it  obviates  errors  of  measurements 


due  to  differential  focussing  of  three 
dimensional  structures.  However,  there 
are  a  few  disadvantages  resulting 
from  the  disturbances  of  the  natural 
relationships  of  the  chromosomes. 
These  are  more  than  offset  by  the  merits 
of  the  method. 

Of  course  a  uniform  application  of  the 
same  technique  is  not  likely  to  prove 
suitable  for  every  kind  of  material,  but 
the  general  principles  are  valid,  requir- 
ing a  few  modifications  depending  upon 
the  particular  type  of  material.  No 
single  method  can  be  recommended 
which  would  prove  adequate  for  all 
stages  of  development.  The  methods 
that  have  proved  particularly  satisfac- 
tory and  have  given  the  best  results  are 
the  Feulgen-smear-squash  technique  of 
Coleman,  L.  C,  Am.  J.  Bot.,  1940,  27, 
887-895;  Coleman,  L.  C,  Genetics, 
1943,  28,  2-8;  Hillary,  B.B.,  Bot.  Gaz., 

1939,  101,  276-300;  Hillary,  B.B.,  Ibid, 

1940,  102,  225-235;  Heitz,  E.,  Ber.  d. 
biol.  Ges.,  1936, 53,  870-878;  Darlington, 
C.  D.,  and  LaCour,  L.  F.,  The  Handling 
of  Chromosomes,  1942.  New  York: 
The  Macmillan  Company;  and  McClin- 
tock,  B.,  Stain  Tech.,  1929,  4,  53-56. 

In  my  experience  of  making  prepara- 
tions for  the  study  of  somatic  and  meio- 
tic  chromosomes,  the  method  given  in 
the  schedule  here  has  proven  most  satis- 
factory. It  is  essentially  a  modifica- 
tion of  the  technique  developed  by  Dr. 
Coleman  and  his  associates  of  the  Uni- 
versity of  Toronto  and  may  be  applied 
to  a  wide  variety  of  materials,  both 
plant  and  animal.  The  tissues  are 
fixed  in  Carnoy,  one  of  the  Navashin 
modifications  or  Flemming-Heitz.  One 
of  the  best  fixatives  for  small  mam- 
malian tissues  is  that  given  by  LaCour, 
L.  F.,  Proc.  Roy.  Soc.  Edin.  B.,  1944, 
62,  73-85.  It  is  a  mixture  of  methyl 
alcohol  (15  parts),  5%  acetic  acid  (5 
parts),  formalin  (1  part),  and  water 
(5  parts).  The  proportions  may  need 
to  be  varied  according  to  the  material. 
For  a  study  of  the  morphology  of  mouse 
chromosomes  the  liver  tissue  of  a  new- 
born mouse  is  particularly  good. 

Chromosome  structure.  The  nuclear 
cycle,  whether  in  plants  or  animals,  is  an 
alternation  between  two  stable  states — 
spiralization  and  despiralization.  The 
metaphase  chromosomes  usually  repre- 
sent a  state  of  maximum  spiralization 
and  the  chromosomes  in  the  resting 
nucleus,  the  maximum  of  despiraliza- 
tion or  minimum  of  spiralization.  For 
demonstration  of  the  spiral  structure 
of  the  chromosomes  and  unravelling  of 
the  coils,  fixation  should  be  preceded 
by  some  form  of  pretreatment.  Differ- 
ent methods  such  as  hydration,  dessi- 


CHROMOSOMES 


76 


CHYLOMICRONS 


cation,  exposure  to  acid  vapors  or  sub- 
jection to  dilute  solutions  of  alkalis, 
treatment  with  dilute  solutions  of  salts 
of  strong  alkalis  and  weak  acids  such 
as  KCN,  NaCN,  in  fact  anything  that 
tends  to  change  the  pH,  have  been  em- 
ployed to  bring  out  the  real  structure 
(Nebel,  B.  R.,  Zeitschr.  Zellf.  u.  Mikr. 
Anat.,  1932,  16,  251-284;  Kuwada,  Y. 
and  Nakamura,  T.,  Cytologia,  1934,  5, 
(2),  244-247;  Sax,  K.,  and  Humphrey, 
L.  M.,  Bot.  Gaz.,  1934,  96,  353-362; 
Huskins,  C.  L.,  and  Smith,  S.  G.,  Ann. 
Bot.,  1935,  49,  119-150;  LaCour,  L.  F., 
Stain  Tech.,  1935,  10,  57-60;  Oura,  G., 
Zeit.  f.  Wiss.  Mikr.,  1936,  53,  36-37; 
Kuwada,  Y.,  Shinke,  N.,  and  Oura,  G., 
Zeit.  f.  Wiss.  Mikr.,  1938,  55,  8-16;  Cole- 
man, L.  C,  and  Hillary,  B.  B.,  Am.  J. 
Bot.,  1941,  28,  464-469;  Gopal-Ayen- 
gar,  A.  R.,  Genetics,  1942;  Coleman, 
L.  C,  Genetics,  1943,  28,  2-8;  Ris,  H., 
Biol.  Bull.,  1945,  3,  242-257).  Some  of 
the  best  results  have  been  obtained  by 
treatment  with  K  or  NaCN  21-^  to  21-6 
mol.  solutions  for  periods  varying  with 
the  material. 

Stain  with  aceto-carmine,  acetic 
orcein,  acetic  lacmoid  or  Feulgen.  If 
Feulgen  is  used  a  counter  stain  with 
fast  green  in  acetic  acid  may  be  used 
if  desired.  The  cells  are  squashed  on 
the  slide  after  staining.  The  amount 
of  pressure  needed  is  determined  by 
experience.  The  following  schedules 
of  treatment  for  mouse  chromosomes 
may  be  applied  mutatis  mutandis  in  the 
study  of  chromosomes  from  other 
tissues. 

Fix  pieces  of  liver  from  a  newborn 
mouse  in  a  mixture  of  methyl  alcohol- 
formalin-acetic  acid  of  LaCour  for  15 
min.  Wash  in  70%  ale.  Transfer 
small  piece  of  material  on  to  a  slide  and 
add  first  drop  of  acetocarmine  or  acetic 
orcein  and  then  coverslip.  Gently  tap 
with  the  rubber  tipped  end  of  a  pencil 
until  the  cells  are  loosened  up  and  are 
more  or  less  one  layer  in  thickness. 
Squeeze  out  gently  the  excess  of  stain. 
Apply  pressure  on  the  coverslip  with 
thumb  or  by  carefully  rolling  a  round 
edged  pencil  over  it,  taking  care  to  see 
that  the  coverslip  does  not  slide  during 
the  process.  The  amount  of  pressure 
needed  is  judged  by  experience.  If  air 
bubbles  get  in  add  a  drop  or  two  of  the 
stain  at  the  edge  of  coverslip  and  repeat 
the  process  if  necessary.  Seal  edge  of 
coverslip  with  beeswax  and  vaseline. 
If  it  is  desired  to  make  slides  permanent 
follow  McClintock's  method  (McClin- 
tock,  B.,  Stain  Tech.,  1929,  4,  53-56). 

For  Feulgen-squash  preparations  fix 
material  as  in  the  preceding  outline. 
Wash  in  water  thoroughly.     Hydrolyse 


in  N.HCl  at  60°C.  for  6-8  min.  Trans- 
fer to  Leuco-basic  fuchsin  for  20  min. 
to  ^  hr.  Pour  off  stain  and  add  SO2 — 
water  and  allow  it  to  remain  for  3  min. 
Change  2-3  times.  Place  a  small  piece 
of  material  in  a  drop  of  45%  acetic  acid 
on  a  slide  and  add  a  coverslip.  Gently 
tap  and  squeeze  out  excess  of  stain  as 
described  above.  Flatten  out  the  cells 
by  applying  pressure  with  thumb  or  by 
rolling  a  round  edged  pencil  over  the 
coverslip.  Transfer  slide  into  large 
petri  dish  containing  dioxan  until  cover- 
slip  floats  off.  The  cells  will  adhere 
either  to  the  coverslip  or  slide.  Use 
dioxan  balsam  as  mounting  medium. 
Chlorazol  black  E  +  acetocarmine 
(Nebel,  B.  R.,  Stain  Techn.,  1940,  15, 
69-72).  Fixation  in  cold  Flemming's 
fluid  plus  urea  (Hance,  R.  T.,  Anat. 
Rec,  1917,  12,  371-382).  Microincin- 
eration of  (Barigozzi,  CI.,  Bull.  d'Hist. 
Appl.,  1938,  15,  213-219).  Method  of 
localization  of  genes  by  experimental 
deletions,  distribution  of  protein  and 
nucleic  acid,  classification,  etc.  (Pain- 
ter, T.  S.,  J.  Roy.  Micr.  Soc,  1940,  60, 
161-176).  Feulgen  stain  for  chromo- 
somes (Mensinkai,  S.  W.,  J.  Roy  Micr. 
Soc,  1939,  59,  82-112).  Aceticorcein 
is  advocated  as  a  new  stain-fixative  for 
chromosomes  (LaCour,  L.,  Stain 
Techn.,  1941,  16,  169-174).  Demonstra- 
tion of  alkaline  phosphatase  in  chromo- 
somes (Krugelis,  E.  J.,  J.  Cell.  &  Comp. 
Physiol.,  1942,  19,  376-379). 

Chromotrope  2R  (CI,  29) — acid  phloxine 
GR,  chromotrope  blue  2R,  fast  fuchsin 
G,XLcarmoi3ine6R — An  acid  mono-azo 
dye  employed  by  Lendrum,  A.  C,  J. 
Path.  &  Bact.,  1935,  40,  415-416  in  a 
study  of  breast  carcinoma  and  skin 
lesions  as  counterstain  for  celestin  blue. 

Chromotrope  Blue  2R,  see  Chromotrope  2R. 

Chrysamine  G  (CI,  410)  an  acid  dis-azo  dye 
of  light  fastness  5  of  no  value  as  a  tissue 
stain  (Emig,  p.  40). 

Chrysoidin  Y  (CI,  20)— brown  salt  R,  dark 
brown  salt  R — A  basic  mono-azo  dye 
suggested  by  Conn  (p.  46)  as  a  substi- 
tute in  some  techniques  for  Bismark 
brown.  Used  as  stain  for  mitochondria 
and  Golgi  apparatus  viewed  in  polarized 
light  (Monne,  L.,  Protoplasma,  1939, 
32,  184-192). 

Chryosomonadina.  Fixation  and  staining 
for,  Doflein,  F.  (Arch.  f.  Protistenk., 
1922,  44,  149),  also  Wenrich,  D.  H.  and 
Diller,  W.  F.,  in  McClung's  Microscopi- 
cal Technique,  1950,  p.  470. 

Chrysophenine  (CI,  365),  a  direct  dis-azo  dye 
of  light  fastness  4  to  5,  for  paraffin  sec- 
tions too  light  and  fugitive  a  color 
(Emig,  p.  39). 

Chylomicrons  (lipomicrons).  These  tiny 
fatty  droplets  are  easily  demonstrated  by 


CIACCIO 


77 


CILIA 


dark  field  examination  of  blood  of  a  person 
or  animal  fed  butter  or  cream.  The 
increase  begins  about  1  hr.  there  after 
and  reaches  a  maximum  at  4  hrs.  after 
which  the  number  of  chylomicrons  de- 
clines. By  contrast  a  carbohydrate  meal 
of  rice  and  sugar  or  a  protein  meal  of 
whites  of  boiled  eggs  and  salt  does  not 
result  in  an  increase.  For  details  see 
Gage,  S.  H.  and  Fish,  P.  H.,  Am.  J. 
Anat.,  1924-25,  34,  1-86;  also,  Had- 
jiolofT,  A.,  Bull.  d'Hist.  Appl.  1938, 
15,  81-98. 

Ciaccio,  methods  for  lipoids.  One  of  the 
simplest  is :  Fix  small  pieces  2  days  in : 
5%  potassium  bichromate,  80  cc;  for- 
malin, 30  cc,  acetic  acid,  5  cc.  3% 
potassium  bichromate  for  5-8  days. 
Running  water  24  hrs.  Ascending  alco- 
hols, 24  hrs.  Abs.  alcohol  2  hrs.,  xylol, 
1  hr.,  xylol -paraffin  at  60°C.,  1  hr. 
Paraffin  1-1 J  hrs.  Pass  sections  down 
to  70%  alcohol,  stain  ^1  hr.  at  30 °C. 
in:  80%  alcohol,  95  cc,  acetone  5  cc. 
saturated  at  50°C.  with  sudan  III  then 
cooled  and  filtered.  Rinse  in  50%  alco- 
hol, wash  in  water,  counterstain  with 
hemalum.  Mount  in  syrup  of  Apathy 
(or  glycerin).  Lipoids  yellow  orange. 
Lison  (p.  206)  questions  specificity  for 
lipoids  and  gives  in  addition,  with  useful 
comments,  several  other  methods  of 
Ciaccio. 

Cilia — written  by  Alfred  M.  Lucas  and 
Miriam  S.  Lucas,  U.  S.  Regional  Poultry 
Research  Laboratory  and  Biological 
Science  Department,  Michigan  State 
College,  East  Lansing,  Michigan.  Au- 
gust 10,  1951 — Ciliary  activity  can  be 
studied  in  isolated  cells,  in  rows  of  cells, 
and  in  broad  epithelial  surfaces.  Cilia 
and  flagella  are  found  throughout  most 
of  the  animal  kingdom  except  in  nema- 
todes and  arthropods.  They  perform 
many  functions,  such  as  locomotion  of 
whole  organisms,  driving  fluids  through 
tubes,  and  propelling  sheets  of  mucus 
across  epithelial  surfaces. 

The  simplest  method  of  study  is  to 
collect  isolated  cells  by  gently  scraping 
a  ciliated  surface  and  observing  the 
vibrations  of  the  cilia  in  a  suitable 
fluid  on  a  slide  under  the  microscope. 
However,  numerous  special  techniques 
are  required  to  answer  problems  relat- 
ing to  the  shape  of  cilia  during  active 
and  passive  phases  of  their  cycle,  ciliary 
coordination  and  how  much  work  they 
can  do.  The  beats  of  cilia  may  oc- 
casionally be  counted  by  eye;  for  ex- 
ample, Lucas,  A.  M.,  and  Doublas,  L. 
C.  (Arch.  Otolaryng.,  1935,  21,  285- 
296)  reported  2.2  to  5.5  vibrations  per 
second  for  cilia  on  epithelium  of  the 
turtle  trachea,  at  about  30°C.  More 
rapid  vibrations  require  special  devices. 


Martius  (Arch.  f.  Physiol.,  1884,  456- 
460)  who  was  the  first  to  use  the  strobo- 
scope, found  10  to  17  vibrations  per 
second  for  the  cilia  of  the  frog's  pharynx 
and  values  as  high  as  32  to  42  vibrations 
per  second  are  given  by  Plammond, 
J.  C.  (Science,  1935,  82,  68-70)  for  the 
membranelle  of  the  protozoan,  Episty- 
lis.  The  use  of  the  stroboscope  and 
moving  pictures  have  been  the  methods 
most  used,  not  only  to  determine  the 
frequenc}'^  of  vibrations  but  also  the 
succession  of  shapes  taken  by  the  cilia 
or  flagella  during  the  cycle  of  vibration. 
Martius  emphasized  a  fundamental  cau- 
tion in  using  the  stroboscope  that  one 
should  reduce  the  speed  until  all  mul- 
tiples of  the  ciliary  frequency  have  been 
eliminated.  Hammond,  on  the  other 
hand,  recommends  using  a  known  har- 
monic of  the  frequency.  The  shapes 
taken  by  vibrating  cilia  and  flagella  have 
often  been  subject  of  study.  One  of  the 
simplest  methods  is  to  reduce  the  rate  of 
movement  by  cooling  the  fluid  in  which 
they  are  beating.  Gray,  J.  (Proc  Roy. 
Soc,  B,  1930,  107,  313-332)  and  Jenni- 
son,  M.  W.,  and  J.  W.  M.  Bunker 
(J.  Cell,  and  Comp.  Physiol.,  1934,  5, 
189-197)  used  both  photographic  and 
stroboscopic  methods  to  study  the  form 
of  the  isolated,  giant  compound  ab- 
frontal  cilia  of  the  mollusc  gill.  It  is  a 
peculiarity  of  this  cilium  to  swing  usu- 
ally through  an  arc  of  only  90°  instead 
of  180°  as  characteristic  of  most  cilia 
and  thus  it  may  be  atypical  material 
for  study.  Gray  noted  that  in  these 
cilia  the  active  stroke  was  longer  than 
the  recovery  stroke.  Brown,  H.  P. 
(Ohio  J.  Sci.,  1945, 45,  247-301)  has  given 
an  extensive  analysis  of  types  of  ciliary 
and  flagellar  movement.  He  tested  his 
theories  in  various  practical  ways  and 
one  was  the  construction  of  a  mecha- 
nical model  which  would  swim  through 
the  water.  It  was  composed  of  a  test 
tube  which  contained  a  strong  bent  wire 
holding  a  rubber  band.  The  rubber 
band  as  it  unwound  rotated  a  wire  pass- 
ing through  a  cork  stopper.  The  free 
end  of  the  wire  revolving  in  the  water 
propelled  the  tube. 

Couch,  J.  N.  (Am.  J.  Bot.,  1941,  218, 
704-713)  recommends  the  use  of  dark 
field  to  study  movement  of  individual 
cilia  and  flagella.  The  three  common 
types  of  dark  field,  the  use  of  a  disc 
stop  below  the  ordinary  condenser,  the 
Zeiss  cardioid  condenser  and  even  mov- 
ing the  diaphragm  laterally  to  give 
oblique  light  worked  satisfactorily. 
With  dark  field  are  often  produced  sha- 
dow effects  of  one  cilia  lying  above  and 
across    others,    which    sometimes    re- 


CILIA 


78 


CILIA 


semble  cross-striations  and  may  be  con- 
fusing. 

Metachromism  is  shown  best  by  cili- 
ated cells  arranged  in  rows.  The  most 
studied  examples  are  the  lateral  cilia 
of  the  mollusc  gill,  especially  Mytilus 
and  Modiolus,  because  in  these  species 
the  gills  are  primitive  and  simple.  The 
location  and  shape  of  the  cells  are  shown 
in  Modiolus  (Lucas,  A.  M.,  J.  Morph., 
1931,  51,  147-193;  and  J.  Morph.,  1932, 
53,  265-276).  Atkins,  D.  (Quart.  J. 
Micr.  Sci.,  1938,  80,  331-344)  has  made 
a  comparative  study  of  these  cells  for 
many  families  and  genera  of  lamelli- 
branchs.  Gray,  J.  (Proc.  Roy.  Soc, 
B.  1930,  107,  313-332)  observed  that  the 
waves  were  a  succession  of  spikes  and 
semicircles  and  with  the  stroboscope, 
which  caused  them  to  appear  to  stand 
still,  he  measured  the  relative  time  con- 
sumed by  the  active  and  recovery 
phases  of  the  beat.  The  ratio  was 
about  1  to  5.  A  photographic  set-up 
for  obtaining  a  permanent  record  of 
coordination  of  lateral  cilia  has  been 
described  in  some  detail  by  Lucas,  A. 
M.  (J.  Morph.,  1932,  53,  243-263). 
However  by  this  method  stroboscopic 
effects  of  the  movie  camera  shutter  may 
give  false  impressions  of  the  rate  of 
wave  propagation. 

The  most  commonly  used  material 
for  studies  on  ciliary  activity  is  the 
frog's  pharyngeal  membrane,  yet  it  is 
poorly  adapted  for  practically  all  the 
problems  for  which  it  has  been  used 
as  will  appear  later.  Many  earlier 
workers  set  up  recording  devices  by 
which  records  of  ciliary  activity  were 
recorded  on  smoked  kymograph  drums; 
the  cilioscribe,  devised  by  Dixon,  W. 
E.,  and  Inchley,  O.  (J.  Physiol.,  1905, 
32,  395-400)  is  a  good  example  and  still 
has  merit  for  classroom  demonstration. 
The  apparatus  consists  of  a  vertical 
spindle  against  which  is  gently  pressed 
the  ciliated  surface  of  a  strip  of  frog 
epithelium.  The  cilia  cause  the  spindle 
to  rotate  and  this  in  turn  moves  a  light 
weight  drum  on  which  an  interval  timer 
marks  rate  of  movement.  The  chief 
difficulty  is  the  variable  amount  of 
mucus  which  collects  on  the  spindle 
interferes  with  its  smooth  rotation. 

Many  investigators  have  used  the 
ciliated  epithelium  of  the  frog's  pharynx 
as  a  delicate  indicator  to  show  the  re- 
sponse of  living  tissue  to  a  wide  variety 
of  chemical  substances,  therapeutic 
compounds,  and  biological  extracts,  yet 
it  was  not  recognized  until  the  work  of 
McDonald,  J.  R.,  Leisure,  C.  E.,  and 
Lenneman,  E.  E.  (Am.  Acad.  Ophthal. 
and  Oto-Laryng.,  1928,  318-354)  that 
the  cilia  of  the  frog's  pharynx  are  under 


definite  nervous  regulation.  A  few 
years  later  Seo,  A.  (Jap.  J.  Med.  Sc. 
Ill  Biophysics,  1931,  2,  47-75)  estab- 
lished clearly  that  cilia  respond  to 
stimuli  through  the  glossopharyngeal 
nerves  and  that  the  motor  fibers  are 
carried  along  the  palatine  nerves. 
Methods  which  had  been  used  thus 
far  involved  placing  fluids  or  particles 
on  the  surface  but  two  years  later  Lu- 
cas, A.  M.  (Arch.  Otblaryng.,  1933, 
18,  516-524)  devised  a  method  whereby 
ciliary  activity  could  be  followed  di- 
rectly under  very  low  powers  of  the 
microscope  focusing  on  the  high-lights 
reflected  from  the  surface  of  the  moving 
ciliary  waves.  Application  of  this 
method  the  same  year  to  the  frog 
(Lucas,  A.  M.,  Proc.  Soc.  Exp.  Biol, 
and  Med.,  1933,  30,  501-506)  demon- 
strated conclusively  that  the  cilia  in 
the  frog's  mouth  are  normally  not  ac- 
tive and  are  completely  at  rest.  They 
become  active  when  particles,  no  matter 
how  fine  and  light  they  may  be,  are 
added  to  the  surface;  even  the  addition 
of  clear  saline  solutions  has  the  same 
effect.  Thus,  the  normal  baseline  for 
ciliary  activity  in  the  frog's  pharynx 
is  zero  instead  of  some  positive  value 
derived  by  the  use  of  foreign  stimulating 
agents.  Recently  Steward,  W.  C.  (Am. 
J.  Physiol.,  1948,  152,  1-5)  although 
recognizing  these  facts,  sought  to  estab- 
lish some  arbitrary  baseline  values  for 
cilia  which  had  been  stimulated  by  discs 
and  weights  placed  on  the  mucous  sur- 
face. His  experiments  gave  a  mean 
velocity  of  0.75  mm/sec.  but  he  found 
that  even  very  light  stimuli  on  the 
membrane,  well  removed  from  the  route 
covered  by  the  test  object,  would  in- 
crease the  velocity  142  to  415  per  cent. 
Lucas,  A.  M.  (Am.  J.  Physiol.,  1935, 
112,  468-476)  designed  a  moist  chamber 
in  which  threshold  levels  of  the  nerve- 
cilia  and  nerve-muscle  preparations 
could  be  determined  simultaneously. 
He  found  that  the  threshold  current 
which  would  stimulate  the  cilia  by  way 
of  the  palatine  nerve  was  15.4  times 
stronger  than  that  required  to  activate 
the  muscles  of  the  toe.  The  behavior  of 
of  excised  mucous  membrane  is  quite 
similar  to  that  found  in  vivo,  the  cilia 
are  normally  quiescent  and  become  ac- 
tive only  when  stimulated. 

What  is  needed  is  a  different  biologi- 
cal material  for  studies  where  chemicals 
are  to  be  tested  in  which  the  cilia  beat 
incessantly,  as  in  the  oviduct  of  birds. 
This  was  one  of  the  materials  chosen 
when  ciliary  studies  were  just  beginning 
(Purkinje  and  Valentin,  Muller's  Arch. 
Anat.  and  Physiol.,  1834,  1,  391-400). 
They  used  macerated  black  pigments 


CILIA 


79 


CILIA 


of  the  retina  suspended  in  fluid  to  fol- 
low ciliary  movement.  The  oviduct 
or  trachea  of  turtles  or  other  reptiles 
would  be  useful  where  it  is  not  conven- 
ient to  control  the  temperature  of  tis- 
sues from  a  warm  blooded  animal.  The 
ciliature  of  the  avian  and  reptilian 
oviducts  has  been  described  by  Parker, 
G.  H.  (Phil.  Trans.  Roy.  Soc.  B,  1931, 
219,  381-419).  Umeda,  T.  (Acta  Der- 
matologica,  1929,  6,  629-646)  used  ox 
trachea  which  provides  large  ciliated 
areas.  He  employed  it  to  study  the 
effects  of  temperature,  sugar  solutions, 
alkaloids,  alcohol,  x-ray,  and  ultra- 
violet light  radiation.  The  underlying 
tracheal  cartilage  was  removed  with  the 
ciliated  epithelium  in  pieces  about  2  x 
3  cm.  and  pinned  out  beneath  a  layer  of 
Ringer's  solution  at  38°C.  It  was  fixed 
at  a  slant  of  10  to  15  degrees  to  facili- 
tate the  movements  of  millet  seeds 
placed  on  the  surface.  There  is  ob- 
viously need  for  someone  to  recheck  the 
reactivity  of  the  frog's  pharyngeal  cilia 
against  several  other  kinds  of  ciliated 
epithelium  and  sort  out  the  effects  due 
to  the  type  of  biological  material  used 
from  the  real  effects  of  chemicals  or 
other  agents  being  tested. 

In  the  frog's  pharynx  and  in  the  ten- 
tacle of  the  snail  (Merton,  H.,  Pfluger's 
Arch.  Ges.  Phys.,  1923,  198,  1-28), 
nerves  are  responsible  for  activation 
of  ciliary  movement;  whereas,  in  the 
Ctenophore  swimming  plates  (Gothlin, 
G.  Fr.,  J.  Exp.  Zool.,  1920,  31,  403-441) 
and  the  velar  cilia  of  the  nudibranch 
veliger  (Carter,  G.  S.,  Brit.  J.  E.xp. 
Biol.,  1926,  4,  1-26)  the  nerves  are  re- 
sponsible for  inhibition  of  ciliary  ac- 
tivity. The  procedures  used  by  these 
authors  are  well  adapted  to  differentiate 
between  inherent  ciliary  activity  and 
nerve  regulation.  In  this  same  cate- 
gory should  be  included  the  work  of 
Copeland,  M.  (Biol.  Bull.,  1922,  42, 
132-142)  who  studied  the  r61e  played 
by  nerves  in  regulating  the  ciliary  ac- 
tivity on  the  foot  of  the  snail. 

Polarity  and  ciliary  reversal  have 
been  problems  of  considerable  academic 
interest.  Coonfield,  B.  R.  (Biol.  Bull., 
1936,  70,  460-471)  described  his  opera- 
tive techniques  for  reversing  rows  of 
swimming  plates  in  Ctenophores  and 
Twitty,  V.  C.  (J.  Exp.  Zool.,  1928,  50, 
319-344)  and  later  Luther,  W.,  (Roux 
Arch.  Entw.  Organ.  1934,  131,  532-539) 
reported  the  relatively  simple  methods 
used  in  reversing  small  segments  of  the 
ciliated  epidermis  of  the  amphibian 
larva.  Induction  effects  on  ciliary  po- 
larity have  been  worked  out  recently  by 
Twitty,  V.  C.  and  Bodenstein,  D.  (J. 
Exp .  Zool . ,  1941 ,  86, 343-379) .    Reversal 


of  tracheal  epithelium  in  dogs  was  ac- 
complished by  Isayam,  S.  (Zeit.  f.  Biol., 
1924,  82,  155-156)  who  found  no  reversal 
of  direction  of  ciliary  beat.  Ciliary 
reversal  is  a  well  known  reaction  in 
some  ciliated  protozoa,  such  as  Para- 
mecium and  minimal  techniques  are 
required  for  study  of  its  physiology 
(Oliphant,  J.  F.,  Physiol.  Zool.,  1942, 
15,  443-452).  Ciliary  reversal  in  Meta- 
zoa  is  rare  indeed.  Parker,  G.  H.  (Am. 
J.  Physiol.,  1905,  14,  1-6)  employed 
simple  techniques  to  demonstrate  its 
existence  in  the  labial  cilia  of  sea- 
anemones.  Equally  simple  procedures 
were  followed  by  Matthews,  S.  (J.  Exp. 
Zool.,  1928,  51,  209-262)  to  demonstrate 
that  the  seeming  reversal  of  the  cilia- 
ture of  the  pelecypod  palp  did  not  actu- 
ally exist.  Atkins,  D.,  (J.  Marine  Biol. 
A.  United  Kingdom,  1930,  16,  919-970) 
observed  permanent,  natural  reversal 
in  frontal  cilia  of  the  gill  filaments  of 
Mytilus  following  injury. 

Numerous  investigators  have  pre- 
sented diagrams  of  the  direction  of  cili- 
ary movement  in  a  field  such  as  Irving, 
L.  (J.  Exp.  Zool.,  1924,  41,  115-124)  who 
plotted  the  circulation  of  fluids  and 
particles  within  the  coelom  of  the  star- 
fish. Another  good  example  is  the  work 
of  Atkins,  D.  (Quart.  J.  Micr.  Sci., 
1936-1937,  79,  181-308,  339-373,  375-421) 
who  worked  out  carefully  the  direction 
of  ciliary  movements  in  a  great  many 
molluscs  and  evolved  a  phylogenetic 
tree  based,  in  part,  on  the  lateral- 
frontal  cilia  (Quart.  J.  Micr.  Sci.,  1938, 
80,  345-436).  Also  the  study  made  by 
Meyer,  A.  (Biol.  Zentralbl.,  1936,  56, 
532-548)  on  Nephthys  hombergli  illus- 
trates a  careful  approach  to  this  type 
of  problem.  Barclay,  A.  B.,  Franklin, 
K.  J.,  and  MacBeth,  R.  G.  (J.  Physiol., 
1937,  90,  347-348)  observed  that  mucus 
is  moved  in  a  clockwise  direction  up 
through  the  mammalian  trachea. 
Hilding,  A.  (Arch.  Otolaryng.,  1932, 
15,  92-100)  plotted  the  direction  of  cili- 
ary movement  in  the  human  nose  by 
watching  the  direction  of  drainage  of 
the  mucous  sheet  with  a  speculum  after 
dusting  with  face  powder  and  observed 
in  the  posterior  two-thirds  of  the  nose 
a  new  mucous  layer  about  every  10 
minutes  and  about  once  an  hour  in  the 
anterior  third.  Lucas,  A.  M.  (Am.  J. 
Anat.,  1932,  50,  141-177)  and  Lucas, 
A.  M.  and  Douglas,  L.  C.  (Arch.  Oto- 
laryng., 1934,  20,  518-541)  used  carbon 
particles  to  plot  the  drainage  pattern 
for  monkey,  rat,  mouse,  rabbit,  cat, 
cow,  and  sheep.  The  potential  error 
resulting  from  following  mucous  flow 
rather  than  direct  observation  of  cilia 
is  shown  on  some  wound  experiments 


CILIA 


80 


CILIA 


on  the  frog's  palate  by  Lucas,  A.  M. 
(Arch.  Otolaryng.,  1933,  18,  516-524). 
Hill,  L.  (Lancet,  1928,  2,  802-805)  had 
previously  shown  that  cuts  across  the 
trachea  arranged  in  alternating  fashion 
completely  stopped  the  flow  of  mucus 
and  also  showed  a  high  sensitivity  to 
ultra  violet  light.  A  combination  of 
both  direct  and  indirect  techniques 
gives  the  most  accurate  results.  The 
double  nature  of  the  mucous  layer  and 
the  three  ways  in  which  ciliary  move- 
ment and  mucous  flow  could  act  to- 
gether or  independently  were  suggested 
by  Lucas,  A.  M.,  and  Douglas,  L.  C. 
(Arch.  Otolaryng.,  1934,  20,  518-541). 

Most  ciliary  activity  can  be  defined 
in  terms  of  work.  Bowditch,  H.  P. 
(Boston  Med.  and  Surg.  J.,  1876,  15, 
159-164),  one  of  the  first  to  study  this 
problem,  permitted  a  sheet  of  ciliated 
epithelium  from  the  frog  to  climb  up  an 
inclined  plane  set  at  various  angles  and 
carrying  various  loads.  He  cooncluded 
that  cilia  are  capable  of  doing  only 
about  one-thirty-fifth  the  work  that  the 
heart  does  in  pumping  blood.  Parker, 
G.  H.  (J.  Exp.  Zool.,  1914,  16,  443-446) 
placed  a  tube  in  the  osculum  of  the 
sponge  and  ascertained  that  the  pres- 
sure developed  in  the  system  by  the 
collar  cells  was  only  a  few  millimeters 
but  that  the  volume  turn-over  was 
large.  A  similar  technique  was  applied 
by  Hecht,  S.  (J.  Exp.  Zool.,  1916,  20, 
429-434)  to  the  large  Bermuda  ascidian. 
A  100  gm.  animal  could  circulate  about 
173  liters  of  sea  water  in  a  day.  Hecht 
gives  his  technique  for  getting  the  glass 
tube  into  the  excurrent  siphon.  Galt- 
soff,  P.  S.  (Bull.  U.  S.  Bur.  Fish.  1928 
(1930)  44,  1-39)  has  gone  farther  than 
anyone  thus  far  in  designing  apparatus 
to  measure  rate  of  flow  of  water  due  to 
ciliary  action.  He  used  the  oyster  and 
collected  data  on  feeding  and  effects 
of  temperature  and  seasonal  and  diurnal 
cycles.  Hilding,  A.  C.  (Trans.  Am. 
Acad.  Opth.  and  Otolaryng.,  1944,  367- 
378)  found  that  the  trachea  of  the  hen 
was  particularly  well  suited  to  measure 
the  positive  and  negative  pressures  de- 
veloped at  the  two  ends  of  the  tube. 
He  collected  mucus  from  other  hens 
and  used  it  to  form  a  plug  at  the  lower 
end  of  the  trachea  being  tested.  A 
negative  pressure  of  34  mm.  and  a  posi- 
tive pressure  of  32  mm.  of  water  was 
developed  at  the  two  ends  of  the  tube. 
The  techniques  used  are  simple  but 
effective.  The  procedures  devised  by 
White,  H.  L.  (Am.  J.  Physiol.,  1929, 
90,  689-704)  to  measure  the  pressure 
developed  by  the  nephostomal  cilia  in 
the  Necturus  kidney  is  a  good  example 
of  how  the  microscopic  methods  can  be 


applied.  In  general,  in  vivo  studies  of 
ciliary  movement  have  been  rather  few 
but  a  nice  technique  was  used  by  Ernst, 
A.  M.  (Arch.  Internat.  Pharmacodyn. 
et  de  Th^rap.,  1938,  58,  207-212)  in 
which  periodic  roentograms  were  made 
of  the  cat's  thorax  to  show  the  rate  of 
elimination  of  "Neobar"  powder 
(Merck)  which  had  previously  been 
blown  into  the  trachea  and  bronchial 
spaces.  Normally  the  trachea  was 
cleared  in  an  hour  and  from  this  base- 
line the  effect  of  various  anesthetics 
could  be  determined.  X-ray  radiation 
in  itself  has  very  little  effect  on  ciliary 
activity  as  shown  by  Goldhaber,  A., 
and  Black,  A.  (Proc.  Soc.  Exp.  Biol, 
and  Med.,  1941,  48,  150-151)  who  ob- 
served that  the  oral  epithelium  of  the 
toad  transplanted  as  a  tissue  culture 
required  1,000,000  to  1,400,000  r  to  stop 
ciliary  activity.  The  essential  details 
of  their  x-ray  set-up  are  given. 

Numerous  papers  mention  the  pres- 
ence of  cilia  lining  various  parts  of  the 
central  canal  of  the  nervous  system  and 
suggestions  have  been  offered  concern- 
ing the  function  they  perform  but  only 
one  author  (Chu,  H.  Y.,  Am.  J.  Physiol., 
1942,  136,  223-228)  to  our  knowledge 
has  selected  a  suitable  material  (trans- 
lucent stages  of  amphibian  larva)  and 
thus  been  able  to  study  this  activity 
in  situ.  He  followed  the  movements 
of  naturally  occurring  pigment  granules 
within  the  ventricles  and  also  injected 
red  cells. 

Satisfactory  methods  for  examination 
of  ciliary  movement  in  tubular  organs 
such  as  the  lungs  are  not  always  easy. 
SoUmann,  T.,  and  Gilbert,  A.  J.  (J. 
Pharmacol,  and  Exp.  Therap.,  1937,  61, 
272-285)  filled  the  excised  organ  with 
10  per  cent  gelatin  in  Ringers  and  then 
chilled  it  in  iced  Ringers.  Free-hand 
sections  were  pinned  on  rings  of  cork 
and  studied  in  shallow  dishes  contain- 
ing Ringer's  warmed  to  37 °C.  Cilia 
were  vigorously  active  up  to  about  8 
hrs. 

A  method  for  testing  the  phagocytic 
properties  of  ciliated  epithelium  was 
employed  by  Ropes,  M.  W.  (Contrib. 
to  Embryol.  #128,  Carnegie  Inst., 
Wash.,  1930,  22,  77-90).  He  used  the 
tracheal  mucosa  of  the  rabbit.  In- 
haled carbon  particles  were  taken  up  by 
the  cytoplasm  and  could  be  recognized 
in  the  living  ciliated  cells. 

The  older  editions  of  Lee's  Vade 
Mecum  suggest  boric  acid  methods  for 
maceration  of  ciliated  epithelia.  This 
technique  has  been  omitted  from  some 
of  the  more  recent  editions  but  is  men- 
tioned again  in  the  last  edition  (11th). 
The  method  was  revived  by  Goodrich, 


CILIA 


81 


CILIA 


E.  L.  (Quart.  J.  Micr.  Sci.,  1942,  83, 
245-258)  as  a  new  technique  with  slight 
modifications.  Lucas,  A.  M.  (J. 
Morph.,  1931,  51,  147-193)  used  sodium 
borate  to  which  was  added  a  trace  of 
iodine  or  eosin.  The  cells  swelled  as 
they  separated.  Poska-Teiss,  L.  (Zeit. 
f.  wiss.  Mikr.,  1934,  51,  238-243)  used 
Ranvier's  1/3  alcohol  method  to  isolate 
the  cells.  Loginoff,  W.  J.  (Anat.  Anz., 
1911,  38,  353-361)  who  worked  with 
horses,  cows  and  sheep,  discovered  that 
preliminary  fixation  of  ciliated  cells  of 
the  trachea  for  30  min.  in  1%  formalin 
followed  by  1/3  alcohol  for  24  hrs.  gave 
well  preserved  cells  which  could  be 
stained  under  the  coverglass  with  picro- 
carmiue. 

In  vitro  explants  of  ciliated  epithelium 
show,  eventually,  dedifferentiation  and 
loss  of  cilia.  More  often  short  term 
studies  on  surviving  cells  have  been 
resorted  to.  Isolated  cells  from  the 
frog's  palate  under  tissue  culture  condi- 
tions beat  continuously  since  they  are 
no  longer  regulated  by  the  nervous  sys- 
tem and  are  suitable  for  testing  pur- 
poses (Ishikawa,  S.,  ActaDermat.,  1927, 
9,  339-364).  Verne,  J.  (Compt.  Rendu 
Assoc.  Anat.,  1932, 27, 564-578)  using  well 
known  tissue  culture  techniques  cul- 
tivated lung  tissue  of  15  to  17  day  old 
chick  embryos.  In  this  case  the  cil- 
iated epithelium  did  not  undergo  meta- 
plasia but  remained  active.  Proetz,  A. 
W.,  and  Pfingsten,  M.  (Arch.  Otolar- 
yng.,  1939,  29,  252-262)  combined  cul- 
ture of  guinea-pig  nasal  epithelium  with 
motion  picture  photography. 

Umeda,  T.  (Acta  Dermat.,  1930,  1, 
13-38)  tested  some  700  dyes  for  their 
vital  staining  characteristics  and  found 
that  thionin  blue  and  Nile  blue  gave 
satisfactory  results  with  frog  ciliated 
epithelium.  Vital  methylene  blue  has 
frequently  been  used  to  stain  lining 
ciliated  cells.  Carter,  G.  S.  (Brit.  J. 
Exp.  Biol.,  1926,  4,  1-26)  observed  that 
it  was  the  region  of  the  ciliary  rootlets 
which  showed  granules  staining  strongly 
with  this  dj'e  and  Coonfield,  B.  R. 
(Biol.  Bull.,  1936,  70,  400-471)  found 
the  greatest  staining  in  Nephthys  where 
the  rootlets  converged  near  the  nucleus. 

Most  of  the  usual  histologic  or  cyto- 
logic techniques  will  demonstrate  cilia. 
Cilia  are  stained  chiefly  with  acid  dyes 
but  may  hold  Heidenhain's  iron  hema- 
toxylin if  the  tissues  have  been  only 
lightly  destained  with  iron  alum. 
When  staining  the  very  short  sensory 
ciliary  hairs  on  olfactory  epithelium, 
the  iron  hematoxylin  without  counter 
stain  or  only  a  light  counter  stain  makes 
them  stand  out  quite  well. 

A  cuticular  border  is  present  on  some 


types  of  ciliary  cells  and  absent  on 
others.  It  is  refractile  to  most  stains 
and  oftentimes  appears  as  a  very  nar- 
row clear  space  between  the  line  mark- 
ing the  surface  of  the  cuticle  and  the 
underlying  cell  membrane.  If  the  over- 
lying mucus  is  stained  with  mucicar- 
mine,  the  outer  surface  of  the  cuticle 
is  clearly  delimited.  Frequently,  the 
lower  margin  of  a  nonciliated  striated 
cuticular  border  looks  like  a  row  of  basal 
bodies  and  Heidenhain's  iron  hema- 
toxj'lin  gives  the  best  differentiation. 
Likewise  it  is  the  stain  usually  chosen 
to  show  flagellate  diplosomes  and  stages 
in  ciliogenesis. 

Ciliary  rootlets  are  as  difficult  to 
demonstrate  in  the  stained  as  in  the 
living  condition.  In  order  to  see  them 
clearly  it  is  important  to  select  the  right 
kind  of  cell — one  which  has  a  clear 
protoplasm  containing  a  minimum  of 
pigmented  opaque  or  refractile  gran- 
ules. Even  living  unstained  ciliated 
cells  of  the  mammalian  nose  when  iso- 
lated on  a  slide  show  clearly  the  root- 
lets in  the  narrow  clear  zone  of  proto- 
plasm just  beneath  the  cell  membrane 
and  in  relatively  transparent  cells  they 
can  be  observed  converging  toward  the 
nucleus  as  an  inverted  truncated  cone. 
In  the  latero-frontal  ciliated  cells  of 
the  mollusc  gill  the  cilia  form  two  rows 
transversely  across  the  surface  of  the 
cells  so  that  the  rootlets  lie  in  rows  one 
behind  the  other  and  under  these  condi- 
tions they  show  up  very  easily.  Grave, 
C,  and  Schmitt,  F.  O.  (J.  Morph.  and 
Physiol.,  1925,  40,  479-515)  recom- 
mended the  Ehrlich-Biondi  triple  mix- 
ture as  a  counterstain  to  Heidenhain's 
iron  hematoxylin  and  this  works  very 
well  although  sometimes  more  than  one 
attempt  is  necessary  to  obtain  a  satis- 
factory mixture  of  Ehrlich-Biondi 
stain. 

Considerable  latitude  is  permitted  in 
the  use  of  fixatives.  Allen's  Bj  and  Bu 
are  good  fixatives  although  the  alveolar 
appearance  that  picric-containing  fixa- 
tives give  to  the  appearance  of  the  cyto- 
plasm may  be  a  disturbing  factor.  Del- 
linger,  O.  P.  (J.  Morph.,  1909,  20,  171- 
209)  made  an  informative  comparative 
study  of  the  fixatives  and  the  basic  con- 
stituents which  go  into  them.  He  found 
that  HgCli  emphasized  fibrillar  struc- 
tures and  enhanced  the  staining  and  so 
fixatives  such  as  Zenker's,  with  formalin 
or  acetic  acid,  are  often  used  for  preser- 
vation of  ciliated  tissues.  These  are 
vigorous  fixatives  and  may  result  in 
some  cell  distortion.  Bellinger  recom- 
mended 0.4  to  2%  osmic  acid  as  best  for 
cilia.  Thanhoffer,  L.  von.  (Zeit.  Anat. 
Entwickl.,  1929,  90,  713-724)  employed 


CILIA 


82 


CILIA 


Heidenhain's  "Susa  mixture"  in  his 
study  of  ciliogenesis.  Mihalik,  P.  von. 
(Anat.  Anz.,  1935, 79,  259-268)  preferred 
the  "Susa  mixture"  for  the  study  of 
intracellular  ciliated  cysts  in  the  rabbit 
oviduct.  Meves,  Fr.  (Arch.  mikr. 
Anat.,  1897, 50, 110-114)  used  Hermann's 
osmic  mixture  for  the  study  of  sperm 
flagella  of  the  salamander.  Kindred, 
J.  E.  (J.  Morph.  and  Physiol.,  1927, 
43,  267-297)  recommended  Meves'  fluid 
as  giving  the  most  constant  results  in 
his  investigation  of  ciliogenesis  in  the 
pharynx  of  the  frog.  Flemming's  fluid 
also  gave  good  results  but  he  did  not 
find  2%  osmic  acid  satisfactory.  Ap- 
plication of  Da  Fano's  modification  of 
Cajal's  silver  method  by  Cowdry,  E.  V. 
(Anat.  Rec,  1921,  22,  289-299)  revealed 
flagella  present  on  the  thyroid  follicles 
of  the  dogfish.  The  blackened  flagel- 
lum  showed  up  nicely  against  a  yellow- 
ish background.  The  collars  and  fla- 
gella of  sponge  choanocytes  are  difficult 
to  preserve  for  microscopic  study.  Rob- 
bertsou,  M.  and  Minchin,  E.  A.  (Quart. 
J.  Micr.  Sci.,  1910,  N.S._55,  611-640) 
found  Hermann's  fluid  satisfactory  and 
stained  with  Heidenhain's  iron  hema- 
toxylin and  light  green. 

Cilia  and  flagella  of  protozoa  can  be 
demonstrated  in  fresh  preparations  by 
first  introducing  strands  of  some  ma- 
terial such  as  cotton  fibers  or  frayed 
lens  paper  beneath  the  cover  glass  which 
will  limit  activity  of  the  organisms. 
Maier,  H.  N.  (Arch.  f.  Protist.,  1903, 
2,  73-179)  drew  1%  osmic  acid  and  sub- 
sequently 5%  soda  solution  beneath  the 
cover  glass  to  study  cilia  and  this 
method  is  still  used.  Noland,  L.  E. 
(Science,  1928,  67,  535)  used  a  gentian 
violet  stain  (20  mg.  gentian  violet  to 
1  cc.  aq.  dest ;  plus  80  cc.  sat.  aq.  phenol ; 
20  cc.  of  40%  formaldehyde;  4  cc.  gly- 
cerin) which  he  mixed  with  a  drop  of 
ciliates  or  flagellates  in  culture.  Don- 
aldson, R.  (Lancet,  1917,  2,  571-573) 
demonstrated  protozoa  in  feces  by  using 
0.1  to  0.5%  aq.  of  eosin,  or  with  iodine 
solutions  of  various  strengths,  or  with 
a  mixture  of  the  eosin  and  dilute  iodine. 
These  solutions  are  also  good  for  dem- 
onstrating cilia  and  flagella. 

Fixation  of  flagella  by  cupro-picro- 
formol  acetic  mixture  and  staining  by 
eosin  and  phosphomolybdic  acid  ac- 
cording to  A.  C.  Hollande's  (Arch,  de 
Zool.  exper.  et  gen.,  1920,  59,  Notes  et 
Revue,  75-77)  method  is  satisfactory. 
This  technique  is  described  in  detail  by 
Wenrich,  D.  H.  (McClung's  Micr.  Tech- 
nique, 1937,  Hoeber,  p.  547). 

Relief  staining  demonstrates  ciliary 
rows  and  other  surface  markings  on 
protozoa:  B.  M.  Klien's  silver  method 


(Zool.  Anz.,  1926,  67, 160-162)  opal  blue, 
china  blue,  and  other  stains  dried  on 
the  protozoa  (Bresslau,  E.,  Arch.  f. 
Protist.,  1921,  43,  467-480)  and  uigrosin 
(Coles,  A.  C,  Watson's  Micr.  Rec, 
1927,  23-25). 

Owen,  H.  M.  (Trans.  Amer.  Micr. 
Soc,  1947,  66,  50-58)  maintains  that  the 
"brush"  or  "flimmer"  effect  produced 
on  flagella  of  bacteria  by  Loeffler's 
stain  is  an  artifact  due  to  interaction  of 
heavy  metals  and  mordant.  He  recom- 
mends a  fixative  which  does  not  produce 
the  "brush"  on  flagella  (10  parts,  2% 
osmic  acid  to  1  part  20%  formalin). 

Ciliogenesis  can  be  observed  occur- 
ring in  a  number  of  protozoa.  Lucas, 
M.  S.  (Arch.  f.  Protist.,  1932,  77,  407- 
423)  used  vom  Roth's  platinic  chloride 
fixative  followed  by  Heidenhain's  iron 
hematoxylin  stain  on  smears  of  Cya- 
thodinium  from  the  guinea  pig  intestine, 
demonstrated  formation  of  new  basal 
bodies  and  cilia  within  cysts  which  later 
evert  upon  the  protozoan's  surface. 

When  cilia  or  flagella  are  to  be  studied 
under  the  electron  microscope,  it  is 
necessary,  of  course,  that  they  be  dried 
under  high  vacuum.  This  may  intro- 
duce distortion.  A  critique  on  the  ad- 
vantages, limitations,  and  possible  arti- 
facts of  electron  microscope  techniques 
has  been  given  by  Williams,  R.  C. 
(Growth  Symposium,  1947,  11,  205-222). 
Baylor,  M.  R.  B.,  Nalbandov,  A.,  and 
Clark,  G.  G.  (Proc.  Soc.  E.xp.  Biol,  and 
Med.,  1943,  54,  229-232)  studied  the 
sperm  head  and  axial  filament.  They 
observed  that  fresh  sperm,  dried  and 
dehydrated  showed  a  fraying  out  of  the 
axial  filament  into  its  component  fibrils 
whereas  stained  sperm  did  not  and  they 
conclude  that  the  stain  is  sufficiently 
adhesive  to  prevent  the  separation. 
Brown,  H.  P.  (Ohio  J.  Sci.,  1945,  45, 
247-301),  in  his  study  of  flagella,  gives 
his  technique  for  preparation  of  his 
material  in  great  detail,  including  the 
results  from  his  tests  on  numerous  fixa- 
tives and  stains  suitable  for  use  with  the 
electron  microscope.  Jakus,  M.  A.  and 
Hall,  C.  E.  (Biol.  Bull.,  1946,  91,  141- 
144)  studied  the  trichocyst  and  cilia  of 
Paramecium  and  used  the  shadow-cast- 
ing technique. 

Microdissection  has  been  used  oc- 
casionally to  study  the  mechanisms  of 
ciliary  movement  and  coordination. 
The  techniques  of  microdissection  are 
well  known  from  various  books  and 
articles.  It  was  used  by  v.  R6nyi,  G. 
(Zeit.  f.  Anat.  u.  Entwick,  1926,  81, 
692-709)  to  determine  if  cilia,  separated 
from  their  basal  bodies,  were  contrac- 
tile. Carter,  G.  S.  (Proc.  Roy.  Soc. 
B,  96,  115-122)  made  use  of  this  tech- 


CINEPHOTOMICROGRAPHY 


83 


CLEARING 


nique  to  demonstrate  that  the  latero- 
frontal  cilia  of  Mytilus  are  compound 
and  that  the  separate  components  can 
beat  independently  of  the  others. 
Peterfi,  T.,  and  Woerderman,  M.  W. 
(Biol.  Zentralbl.,  1924,  44,  264-268), 
Grave,  C.  and  Schmitt,  F.  O.  (J.  Morph. 
and  Physiol.,  1925,  40,  479-515),  Lucas, 
A.  M.  (J.  Morph.  1931,  51,  147-193)  and 
Worley,  L.  G.  (J.  Cell,  and  Comp. 
Physiol.,  1941,  18,  187-197)  employed 
this  method  to  gain  information  con- 
cerning ciliary  coordination. 

Cinephotomicrography.  A  convenient  illus- 
trated account  of  apparatus  and  meth- 
ods is  given  by  Tuttle,  H.  B.,  in  Glas- 
ser's  Medical  Physics,  183-194.  See 
Motion  Pictures. 

Cinnamon  Oil  (Cassia  oil)  resembles  clove 
oil  and  is  particularly  recommended  by 
Lee  (p.  70)  for  clearing.  Two  kinds  are 
given  in  Merck  Index.  The  U.S.P.XI 
variety  contains  80-90%  cinnamalde- 
hyde. 

Citrate  of  sodium  can  be  used  as  an  antico- 
agulant in  the  proportion  of  18  cc.  of  2% 
aq.  sodium  citrate  to  100  cc.  of  blood. 

Clarite  X  (Neville  Co.,  Pittsburg)  60%  in 
toluol  is  suggested  as  substitute  for 
balsam  owing  to  its  neutral  reaction, 
lack  of  yellow  color  and  quickness  oi 
hardening.  Clarite,  also  called  Nevil- 
lite  V,  is  useful  if  added  to  paraffin  when 
one  wishes  to  obtain  thin  sections  when 
it  is  not  convenient  to  imbed  in  a  very 
high  melting  point  paraffin.  Wehrle, 
W.,  Stain  Techn.,  1942,  17,  131-132  ad- 
vises imbedding  in  a  mixture  of  90% 
paraffin  (m.p.  53°C.),  5%  bleached  bees- 
wax and  5%  clarite  and  the  elimination 
of  electrical  charge  when  ribbons  are 
cut  by  a  spark-coil  device  described  by 
Blandau,  R.  J.,  Stain  Techn.,  1938,  13, 
139-141. 

Clark  and  Lubs  Buffers  (Clark,  W.  M.  The 
Determination  of  Hydrogen  Ions,  Balti- 
more: Williams  &  Wilkins,  1928,  717 
pp.).  Prepare:  (1)  A  solution  contain- 
ing M/5  boric  acid  and  M/5  potassium 
chloride  made  by  dissolving  12.368  gms. 
of  H3BO3  and  14.912  gms.  of  KCl  in  aq. 
dest.,  and  diluting  to  1  liter.  (2)  A 
M/5  sodium  hydroxide  (carbonate  free) 
solution  by  dissolving  50  gms.  of  NaOH 
in  50  ml.  (cc).  aq.  dest.  in  a  Pyrex 
flask.  Let  stand  overnight  to  allow  the 
sodium  carbonate  to  settle,  or  filter 
through  a  Gooch  or  sintered  glass  cruci- 
ble. (Exclude  air  to  prevent  formation 
of  more  carbonate  by  atmospheric  CO2.) 
Keep  the  strong  alkaline  solution  in  a 
paraffin-lined  glass  bottle.  Dilute  with 
aq.  dest.  which  has  been  boiled  to  re- 
move the  excess  CO2  so  that  the  solution 
is  about  1  N.  Then  make  an  approxi- 
mately M/5  solution  of  the  alkali  which 


can  be  accurately  standardized  against 
potassium  acid  phthalate. 

To  make  buffer  of  the  desired  pH  add 
to  50  cc.  of  (1 )  M/5  HjBC-KCl  the  desig- 
nated amount  of  (2)  M/5  NaOH  and 
dilute  to  200  cc.  with  aq.  dest.  Or 
combine  the  two  in  similar  proportions 
but  in  larger  amounts  to  minimize  error 
in  measurement. 


pH 

CO.  of  M/6  NaOH 

7.8 

2.61 

8.0 

3.97 

8.2 

6.90 

8.4 

8.60 

8.e 

12.00 

8.8 

16.30 

9.0 

21.30 

9.2 

26.70 

9.4 

32.00 

.6 

36.85 

.8 

40.80 

10.0 

43.90 

Cleaning  Glassware.  Pulverize  20  gms. 
potassium  bichromate.  Dissolve  this  in 
200  cc.  aq.  dest.  with  aid  of  a  little  heat. 
Add  slowly  20  cc.  sulphuric  acid  C.P. 
Before  treating  beakers,  graduates, 
bottles,  etc.  with  this  acid  cleaning  solu- 
tion first  wash  them  in  soap  and  hot 
water.  Rinse  in  water  to  remove  the 
soap.  Leave  in  cleaning  solution  2  hrs. 
or  more.  Rinse  in  running  tap  water  and 
dry  with  opening  downward  on  drying 
racks  as  in  biochemical  laboratories  if 
possible  in  a  dust  free  cupboard.  For 
new  slides  and  cover  glasses  wash  in  the 
same  way  and  after  final  rinsing  in  tap 
water  store  in  fresh  95%  alcohol  in 
covered  dishes  until  they  are  required 
for  use  when  they  should  be  wiped  with 
gauze.  For  old  slides  and  cover  glasses 
soak  in  xylol  to  permit  separation  and 
removal  of  most  of  balsam.  Then  leave 
in  waste  alcohol  several  days.  Soak  for 
a  day  or  more  in  strong  soap  solution. 
Wash  in  running  water.  Clean  in  clean- 
ing solution.  Wash  in  water  and  store 
in  95%  alcohol.  Unless  strict  economy 
is  necessary  it  is  hardly  worthwhile  to 
use  slides  and  covers  twice  especially 
when  the  former  have  been  marked  with 
diamond  pencils. 

Clearing  is  a  process  in  microscopic  tech- 
nique which  is  required  in  three  different 
situations. 

1.  As  the  step  following  dehydration 
in  paraffin  imbedding.  The  tissue  be- 
comes translucent  but  this  is  not  the 
essential  feature  of  the  process.  What 
is  necessary  is  for  the  alcohol,  which  is 
not  a  paraffin  solvent,  to  be  removed  by 
the  clearing  agent  before  the  tissue  is 
infiltrated  with  paraffin.  Consequently 
the  agents  must  mix  freely  with  alcohol 
on  the  one  hand  and  with  paraffin  on  the 


CLOTH  RED  B 


84 


COAGULATION 


other.  Of  them  xylol  is  by  far  the  most 
widely  used  and  rightly  so.  Two 
changes  of  half  absolute  alcohol  and  xylol 
within  1  hr.  and  2  changes  of  xylol  within 
the  next  3-4  hrs.  are  usually  sufficient 
for  slices  of  tissue  4-6 mm.  thick,  but  the 
time  should  not  be  extended  beyond 
that  needed  to  attain  translucency  be- 
cause so  doing  causes  a  hardening  and  a 
shrinkage  of  the  tissue. 

Several  other  substances  can  be  used 
in  place  of  xylol.  Cedar  wood  oil  is  ac- 
cording to  Lee  (p.  80)  the  very  best 
clearing  agent  for  paraffin  imbedding. 
It  penetrates  rapidly,  does  not  make  the 
tissues  brittle,  and,  when  not  entirely 
displaced  by  paraffin,  does  not  seriously 
interfere  with  sectioning.  First  treat 
the  tissue  with  ^  absolute  and  xylol  for 
about  2  hrs.  The  time  required  in  the 
oil  of  cedarwood  is  however  a  little  longer 
than  in  the  case  of  xylol  used  alone,  say 
12  hrs.  Some  recommend  2  changes  of 
xylol  (about  30  min.)  after  the  oil  of 
cedarwood  before  entering  ^paraffin and 
cedarwood  oil. 

Methyl  benzoate  is  now  quite  popular. 
Pass  the  tissue  from  absolute  alcohol 
through  2  changes  of  pure  methyl  ben- 
zoate within  12-24  hrs.  When  it  has 
been  definitely  cleared  remove  benzoate 
by  2  changes  of  benzol  (^-1  hr.)  before 
passing  into  paraffin,  or  half  benzol  and 
paraffin. 

Chloroform  penetrates  pxaorly  and 
should  not  be  employed  unless  called  for. 
It  has  the  further  disadvantage  that 
unless  completely  removed  in  the  paraf- 
fin bath,  it  will  make  the  final  paraffin 
block  soft  and  unfit  for  cutting.  The 
usual  practice  is  to  clear  very  small 
pieces  for  about  12  hrs.  in  2  changes,  or 
as  long  as  may  be  necessary  to  make 
them  transparent,  and  in  the  imbedding 
to  use  4  changes  of  paraffin. 

A  more  rapid  method  is  to  pass  di- 
rectly from  the  fixative,  Bouin  or  forma- 
lin, without  washing,  to  3  changes  of 
pure  dioxan  within  4  hrs.  and  thence 
into  3  changes  of  paraffin  as  advised  by 
Graupner,  H.  and  Weissberger,  A., 
Zool.  Anz.,  1931,  96,  204-206.  Stowell, 
R.  E.,  Stain  Techn.,  1941,  16,  67-83 
confirms  and  extends  earlier  work  of 
Seki  which  shows  that,  although  xylol 
shrinks  tissues  more  than  dio.xan,  in 
placing  in  hot  paraffin,  the  final  shrink- 
age is  greater  in  tissues  after  dioxan. 
When  great  haste  is  necessary  Mallory 
(p.  54)  suggests  acetone  \-2  hrs. ;  benzol, 
15-30  min. ;  and  paraffin  3  changes,  30-90 
min.  The  shrinkage,  however,  is  very 
marked  and  it  would  probably  be  better 
to  use  Frozen  Sections. 

By  the  Altmann-Gersh  technique, 
which  is  at  once  very  time  consuming 


and  very  valuable  for  special  purposes, 
fixation,  alcoholic  dehydration  and  clear- 
ing can  be  side  stepped  and  the  dried 
tissue  directly  impregnated  with 
paraffin. 

2.  As  the  step  following  dehydration 
of  sections  before  mounting.  The  clear- 
ing is  of  course  easier  and  much  quicker 
owing  to  the  thinness  of  the  tissue. 
Again  xylol  comes  first  and  will  probably 
not  be  displaced  though  some  prefer 
toluol.  It  is  not  necessary  to  protect 
against  shrinkage  and  brittleness. 
When  desired,  abs.  ale.  can  be  omitted 
and  the  clearing  be  done  from  90  or 
even  80%  ale.  with  terpineol,  clove  oil, 
anilin  oil,  beech  wood  creosote,  Bergamot 
or  some  other  substance. 

3.  As  a  means  of  rendering  clearly 
visible  certain  structures  in  embryos  or 
whole  tissues.  Clearing  is  generally 
done  by  the  Spalteholz  method.  See 
Cartilaginous  Skeleton  and  Ossification 
centers.  When  glycerin  mixtures  are 
employed  as  Mounting  Media  they  also 
clear  the  tissues.  See  Groat,  R.  A., 
Stain  Techn.,  1941,  16,  111-117  for  clear- 
ing tissues  with  mixtures  of  tributyl 
and  tri-o-cresyl  phosphates. 

Cloth  Red  B  (CI  262).  A  sulfonated  azo 
dye.  For  formula  and  influence  on 
mouse  tumors,  see  Stern,  K.,  Cancer 
Res.,  1950,  10,565-570. 

Cloudy  Swelling.  This  is  a  marked  swelling 
and  granulation  of  the  cytoplasm  of 
cells.  It  is  sometimes  observed  post- 
mortem in  acute  febrile  conditions 
especially  in  the  kidneys,  liver  and  m5'o- 
cardium.  An  almost  meaningless  syno- 
nym, often  used,  is  Parenchymatous 
Degeneration.  The  extent  of  cloudy 
swelling  that  may  occur  in  vivo  and 
from  which  the  cells  may  recover  is 
not  known.  The  fatty  droplets  present 
can  be  demonstrated  in  Sudan  stained 
frozen  sections  of  formalin  fixed  mate- 
rial. Special  stains  for  Fibrin,  Myo- 
fibrils and  Mitochondria  may  be  de- 
sirable. 

Coacervates  (L.  acervus,  a  cloud  or  swarm) 
are  masses  of  particles  clumped  together 
(but  encased  in  a  little  water)  by  a 
change  in  their  electrical  charge  while 
in  colloidal  suspension  in  water  or 
by  dehydration  with  resultant  loss 
of  loosely  bound  water.  Ilirsch  (G. 
C,  Form  und  Stoffwechsel  der  Gol- 
gi-Korper.  Protoplasma  Monographs, 
Berlin,  1939)  has  likened  the  Golgi 
apparatus  to  a  coacervate.  See  Bensley, 
R.  R.,  Anat.  Rec,  1937,  69,  341-353  for 
critical  consideration  of  suggestion  that 
mitochondria  are  coacervates. 

Coagulation.  A  phenomenon  frequently 
encountered  in  the  case  of  blood  and 
lymph  is  of  wide  occurrence  and  is  in- 


COBALT  NITRATE  SILVER 


85 


COLLAGEN IC  FIBERS 


fluenced  by  many  factors.  Small  quan- 
tities of  many  electrolytes  cause  coagu- 
lation of  colloids.  Some  ions  are  much 
more  powerful  in  this  respect  than 
others  and  certain  "protective  col- 
loids" such  as  gelatin  protect  colloidal 
suspensions  against  coagulating  action 
of  electrolj'tes.  Peptization  is  dis- 
integration of  the  coagulum  into  col- 
loidal particles.  Water  is  employed  as 
a  peptizing  agent  in  dissolving  glue, 
agar,  and  similar  materials  (Holmes, 
H.  N.,  Glasser's  Medical  Physics,  257- 
263). 

Cobalt  Nitrate  Silver  for  Golgi  Apparatus. 

Cocarboxylase,  see  Method  of  Westen- 
brink,  H.  G.  K.,  Enzymologia,  1940,  8, 
97-107,  capable  of  determining  as  little 
as  0.01  fjL  gm.  of  carboxylase. 

Coccidia.  These  sporozoa  include  many 
parasites  of  great  importance  not  only 
to  physicians  and  veterinarians  but  also 
to  cj'tologists  who  are  interested  in 
their  intracellular  behavior.  Conse- 
quently the  volume  by  Becker,  E.  R., 
Coccidia  and  Coccidiosis  of  domesti- 
cated, game  and  laboratory  animals 
and  of  man.  Ames:  Collegiate  Press, 
Inc.,  1934, 147  pp.  will  contain  numerous 
helpful  leads  on  the  coccidia  of  the 
digestive  tracts  of  vertebrates  and  in- 
vertebrates. See  also  McClung,  1950, 
p.  455. 

Coccinel  Red  is  1, 5-diamylaminoanthra- 
quinone,  an  oil  soluble  dye,  recom- 
mended by  Lillie,  R.  D.,  Stain  Techn., 
1945,  20,  73-75  as  a  stain  for  fat  which 
it  colors  deep  orange  red.  Make  up 
stock  solution  of  4.2%  in  absolute 
(99%)  isopropanol.  Dilute  this  down 
to  30  or  40%  isopropanol  with  water  and 
treat  frozen  sections  of  normal  cat  kid- 
ney and  human  adrenal  with  resulting 
solution  for  10-20  min.  This  solution 
is  only  usable  for  several  hours.  Coc- 
cinel red  is  a  good  counterstain  after 
hematoxylin. 

Coccinine  (CI,  120),  an  acid  monoazo  dye, 
light  fastness  3  to  4,  which  colors  sec- 
tions pale  pink  not  equal  to  Biebrich 
Scarlet  (Emig,  p.  31). 

Cochineal  (CI,  1239).  This  crimson  dye 
was  in  use  by  the  Aztecs  before  the 
Spanish  conquest.  It  is  derived  from 
an  insect  which  feeds  on  a  cactus.  So 
highly  prized  was  it  that  Montezuma 
took  as  yearly  tribute  from  the  State  of 
Huaxyacas  (Now  Oaxaca)  20  sacks  of 
cochineal.  The  invading  Spaniards 
were  not  slow  to  note  the  superiority  of 
cochineal  over  Kermes,  the  crimson 
stain  in  use  at  home  (1523  A.D.). 
Charles  V  of  Spain  commanded  Cortez 
to  inform  him  immediately  "whether 
what  has  been  reported  is  true  that 
Kermes  were  to  be  found  in  abundance 


in  New  Spain  and,  if  so,  could  be  sent 
with  advantage  to  Spain".  So  coch- 
ineal figured  largely  in  the  Aztec 
tributes  to  Cortez  and  the  industry 
became  a  Spanish  monopoly.  In  1858 
A.D.  aniline  red  became  a  competitor, 
depressed  the  sales  of  cochineal,  which, 
latter  as  a  commercial  dye,  was  defi- 
nitely replaced  when  azo  dyes  came  into 
the  market  about  A.D.  1880.  (Leggett, 
W.  F.,  Ancient  and  Medieval  Dyes. 
Brooklyn:  Chemical  Publishing  Co. 
Inc.,  1944,  95  pp.).  See  Kermes,  Lac. 
In  microscopy  cochineal  is  used 
mostly  for  staining  in  tola  of  small  in- 
vertebrates. Mayer's  alcoholic  cochi- 
neal is  a  popular  preparation  made,  ac- 
cording to  Lee  (p.  149),  by  powdering 
5  gm.  cochineal  with  5  gm.  calcium 
chloride  and  5  gm.  aluminum  chloride  to 
which  100  cc.  50%  alcohol  and  8  drops  of 
nitric  acid  (sp.  gr.  1.20)  are  added. 
Heat  to  boiling  point,  cool,  shake  oc- 
casionally during  several  days  and  filter. 
Before  staining  bring  objects  to  70% 
alcohol,  destain  if  necessary  in  70% 
alcohol  containing  0.1%  hydrochloric 
acid.  Dehydrate,  clear  and  mount  in 
balsam.  Nuclei  are  colored  red  and 
other  structures  a  variety  of  colors  from 
red  to  deep  purple.  In  some  respects 
it  is  better  than  carmine.     Neither  fade. 

Cochlea,  see  Ear. 

Coelestin  Blue,  see  Skyblue. 

Coeline,  see  Skyblue. 

Coeruleum,  see  Skyblue. 

Colchiceine,  different  from  colchicine,  see 
Ludford,  R.  J.,  Arch.  f.  exper.  Zellf., 
1935-36,  18,  411-441. 

Colchicine,  see  Mitosis  Counts. 

Collagenase.  An  enzyme  which  specifically 
destroys  collagenous  fibers.  Bidwell, 
E.  and  W.  E.  van  Heyningen,  Biochem. 
J.,  1948,  42,  140-151,  report  on  its  isola- 
tion and  purification. 

Collagenic  Fibers — Written  by  A.  I.  Lans- 
ing, Dept.  of  Anatomy,  Washington 
University,  St.  Louis  10,  Mo.  October 
5, 1951 — These  are  widely  distributed  in 
the  body,  occur  in  large  bundles  in 
tendons  and  are  best  viewed  in  spreads 
of  loose  connective  tissue.  Unlike  elas- 
tic fibers  which  are  yellow  and  highly 
refractile,  coUagenic  fibers  are  white 
and  poorly  refractile,  form  broad  wavy 
bands  which  do  not  branch,  have  great 
tensile  strength,  and  are  attacked  by 
dilute  acids  and  alkalis.  On  boiling 
they  yield  gelatin.  The  fact  that  they 
pass  from  the  gel  to  the  sol  state  on 
alkalinization  or  warming  is  the  basis 
for  methods  of  separating  epidermis 
from  dermis.  Collagenic  fibers  are 
readily  digested  by  pepsin  in  acid  solu- 
tion but  resist  trypsin.  A  specific  col- 
lagenase has  as  yet  not  been  found. 


COLLAGENIC  FIBERS 


86 


COLOR  PRESERVATION 


Amino  acid  analyses  (see  Elastic  Fi- 
bers) indicate  an  absence  of  aromatic 
amino  acids,  an  abundance  of  proline, 
hydroxylysine  and  particularly  hy- 
droxyproline,  the  latter  being  almost  a 
diagnostic  feature.  The  isolectric 
point  of  collagen  is  about  pH  7.0. 

Although  tinctorial  reactions  would 
imply  that  reticulum  and  coUagenic 
fibers  differ,  recent  studies  indicate  that 
reticulum  is  but  a  finer  unit  of  collagen. 
Both  have  a  marked  affinity  for  aniline 
blue.  They  differ  in  that  reticulum  is 
strongly  argyrophilic  while  collagenic 
fibers  are  not;  the  former  is  weakly 
acidophilic  (in  H  &  E)  while  the  latter 
takes  up  much  eosin. 

Electron  microscopy  and  x-ray  dif- 
fraction studies  have  done  much  to  re- 
veal the  submicroscopic  structure  of  col- 
lagenic fibrils  (Gross,  V.,  J.  Gerontol., 
1950,  5,  343).  These  are  apparently 
composed  of  parallel  polypeptide  chains 
approximately  10  A°  in  diameter  which 
are  bonded  together  laterally.  Elec- 
tron microscopy  reveals  considerable 
fine  structure  in  fibrils.  There  is  a 
regular  periodicity  of  640  A°  with  much 
intra-period  detail. 

A  protein  termed  procollagen  may  be 
prepared  by  extraction  with  citrate  at 
pH  4.  Such  procollagen  can  form  fibrils 
with  regular  periodicity  by  the  addi- 
tion of  monovalent  salts  or  mucoprotein 
(Highberger,  J.  H.,  J.  Gross  and  F.  O. 
Schmitt,  Proc.  Nat.  Acad.  Sci.,  1951, 
37,  286). 

The  best  stain  for  collagenic  fibers  in 
sections  after  Zenker  fixation  is  aniline 
blue  in  Mallory's  Connective  Tissue 
Stain  and  in  Masson's  Trichrome  Stain. 
Phosphomolybdic  Acid  Hematoxylin 
also  gives  a  fine  coloration  of  collagenic 
fibers.  See  Van  Gieson,  Buzaglo,  Bieb- 
rich  Scarlet  and  Picro-Aniiin  Blue  of 
Lillie  and  Curtis'  Substitute  for  Van 
Gieson. 

Lillie,  R.  D.  (J.  Tech.  Methods,  1945, 
No.  25,  45  pp.)  has  performed  a  very 
useful  service  in  testing  the  effective- 
ness of  a  large  series  of  dyes  as  colla- 
genic fiber  stains  in  the  Van  Gieson, 
Mixed  Masson-Van  Gieson  and  Masson- 
Mallory  procedures.  He  found  the  best 
to  be  Naphthol  blue-black  (CI,  246), 
Fast  Green  FCF,  Acid  Fuchsin  (CI, 
692),  Methyl  Blue  (CI,  706),  Anilin  Blue 
(CI,  707),  Wool  Green  S  (CI,  737)  and 
Volamine  R  (CI,  758) .  For  photometric 
histochemical  determination  see  Sto- 
well,  R.  E.,  J.  Invest.  Derm.,  1945,  6, 
183-189. 

The  technique  of  microincineration 
as  adapted  to  collagenic  fibers  is  de- 
scribed by  AUara,  E.,  Bull.  d'Hist. 
AppL,  1938,  15,  220-242.    See  Tendons. 


Collargol,  as  negative  stain  for  spirochetes 
(Harrison,  Brit.  Med.  J.,  1912,  2,  1547). 

Collodions.  There  are  several.  See  U.S.P. 
XI.    All  are  solutions  of  Pyroxylin. 

Colloxylin,  see  Pyroxylin. 

Colophonium,  usually  dissolved  in  turpen- 
tine is  employed  to  mount  sections. 
Not  advised. 

Color  Estimation.  Accuracy  in  reporting 
differential  stains  and  micro-chemical 
reactions  yielding  colors  is  highly  de- 
sirable. The  same  holds  for  colors 
determined  by  naked  eye  inspection. 
A  monograph,  Ridgway,  R.,  Color 
Standards  and  Color  Nomenclature, 
Washington,  D.  C,  1912  with  53  colored 
plates,  is  the  accepted  standard  for 
comparison.  In  general,  however,  it  is 
desirable  to  achieve  some  measure  of 
uniformity  by  limiting  oneself  when- 
ever possible  to  use  of  the  terms  recom- 
mended in  the  National  Formulary  VII. 
Washington:  American  Pharmaceutical 
Association,  1942,  690  pp.,  a  publication 
which  is  available  in  most  medical 
libraries: 

pink  yellow  greenish  blue 

red  olive-brown  blue 

reddish  orange        greenish  yellow    purplish  blue 
reddish  brown        olive  bluish  purple 

orange-pink  yellow-green  purple 

orange  olive-green  reddish  purple 

brown  yellowish  green     purplish  pink 

yellowish  orange    green  red-purple 

yellowish  brown     bluish  green         purplish  red 
blue-green 
For  accurate  measurement  of  color 
employ    Photoelectric    Colorimeter    or 
Photoelectric     Microphotometer.       See 
Hemoglobin  Estimation. 

Color  Index,  p.  xxvii. 

Color  Preservation  in  museum  specimens. 
Fix  24  hrs.  or  less  in  10%  formalin. 
Wash  in  running  water  3-6  hrs.  Stand 
in  2%  aq.  ammonia  5-10  min.  which 
hastens  return  of  original  colors.  Run- 
ning water  another  hour.  Mount  for 
permanent  exhibition  in  mixture  made 
as  follows:  Filter  a  sat.  sol.  antimony 
trioxide  in  aq.  dest.  (about  5  gm.  per 
liter).  To  each  1000  cc.  filtrate  add  100 
gm.  potassium  acetate,  100  gm.  chloral 
hydrate  and  50  cc.  glycerin.  Stir  until 
completely  dissolved  (Meiller,  F.  H., 
J.  Tech.  Methods,  1938,  18,  57-58). 

Mallory  (p.  380)  recommends  for  this 
purpose  the  methods  of  Kaiserling  and 
Jores. 

There  are  3  Kaiserling  solutions : 
1.  For  fixation:  Formalin,  40  cc;  tap 
water,  2000  cc;  potassium  nitrate,  30 
gm.  and  potassium  acetate,  60  gm. 
Small  specimens  require  1-14  days. 
Large  ones  can  be  more  uniformly  fixed 
by  vascular  Perfusion.  Sometimes  it 
is  advisable  to  inject  fixative  into  central 


COLORS 


87 


CONCENTRATION 


parts  of  the  tissue  with  a  hypodermic 
syringe  and  long  needle.  Do  not  use 
too  much  pressure  and  be  careful  not  to 
let  any  of  the  fixative  spurt  back  into 
one's  face.  Before  the  next  step  wash 
in  running  water  for  about  12  hrs. 

2.  For  color  restoration:  Place  the 
tissue  in  80%  ethyl  alcohol  for  10-60 
min.  and  watch  for  optimum  coloration. 
If  left  too  long  in  the  alcohol  the  colors 
fade.  Rinse  in  water  and  transfer  to 
No.  3. 

3.  For  final  preservation:  Change  to 
glycerin,  500  cc. ;  1%  aq.  arsenious  acid, 
200  cc;  tap  water,  2300  cc;  potassium 
acetate,  250  gms.;  thymol,  2.5  gm.  To 
obviate  difficulty  of  dissolving  the 
arsenious  acid  and  to  sterilize  add  25 
gms.  arsenic  trioxide  to  2500  cc.  water 
+  the  thymol  crystals  first  ground  up 
in  a  mortar  and  place  in  steam  sterilizer 
for  6  hrs.     Then  add  other  substances. 

There  are  2  J  ores  solutions. 

1.  For  fixation:  Chloral  hydrate,  50 
gms.;  artificial  Carlsbad  salts  (sodium 
sulfate,  22  gm.;  sodium  bicarbonate,  20 
gm. ;  sodium  chloride,  18  gm. ;  potassium 
nitrate,  38  gm.;  potassium  sulphate,  2 
gm.),  50  gm.;  formalin,  100  cc ;  tap 
water,  1000  cc  Allow  to  act  2-14  days 
depending  on  size,  wash  12  hrs.  in  run- 
ning water. 

2.  For  final  preservation:  Potassium 
acetate,  300  gm.,  glycerin,  600  cc;  aq. 
dest.,  1000  cc. 

Mai  lory  suggests  fixation  in  Jores' 
first  solution  and  preservation  in  Kaiser- 
ling's  third  solution. 

Colors,  Interference,  see  Interference 
Colors. 

Columbium,  see  Atomic  Weights. 

Concentration.  1.  Tubercle  bacilli  in  spu- 
tum. Nagy  (A.H.,  J.  Lab.  &  Clin. 
Med.,  1939,  25,  67-71)  having  critically 
evaluated  several  techniques  recom- 
mends Pottenger's  Dilution-Flotation 
method.  Shake  equal  parts  sputum  and 
0.5%  aq.  sodium  hydroxide  for  10  min. 
Digest  in  water  bath  at  56°C.  for  30 
min.  Add  1  ml.  (=  1  cc.)  hydrocarbon 
(gasoline  or  xylene),  then  200  ml.  aq. 
dest.  and  shake  10  min.  Allow  hydro- 
carbon to  collect  at  top  15-20  min. 
Take  up  hydrocarbon  layer  in  rubber 
bulbed  pipette.  Keep  in  vertical  posi- 
tion until  supernatant  fluid  separates 
from  hydrocarbon,  5-10  min.  Make 
smears  from  hydrocarbon  and  dry. 
Remove  hydrocarbon  by  washing  with 
ether.  Stain  with  carbol  fuchsin  3  hrs. 
or  longer.  Decolorize  with  acid  alco- 
hol 30  sec.  or  less.  If  further  decolor- 
ization  is  required  employ  10%  aq. 
sodium  sulphite.  Counterstain  with  1% 
aq.  picric  acid  or  with  methylene  blue. 
The  concentration  of  bacilli  is  about  33 


times.  Perhaps  a  modification  of  the 
method  could  be  used  for  leprosy  or- 
ganisms in  emulsions  of  tissues.  See 
also  Pottenger,  J.  E.,  Am.  Rec  Tuberc, 
1939,  40,  581.  Concentration  of  tuber- 
cle bacilli  in  spinal  fluids  (Hanks,  J.  H. 
and  Feldman,  H.  A.,  J.  Lab.  &  Clin. 
Med.,  1939,  25,  886-892).  It  is  often 
necessary  to  concentrate  for  micro- 
scopic study  objects  which  are  not 
present  in  abundance  and  which  might 
otherwise  be  overlooked.  See  exami- 
nation of  Feces  for  ova  of  parasites,  of 
Urine  for  sediment. 

2.  Leprosy  bacilli  for  chemical  analy- 
sis. Ra void's  method  for  leprosy  bacilli 
can  perhaps  be  used  for  others.  Rela- 
tively large  masses  of  bacilli-laden  cells 
are  dissected  away  from  neighboring 
uninvolved  tissue  and  from  necrotic 
tissue  when  present  in  the  centers  of 
the  nodules.  They  are  placed  in  a 
Wueller  press  without  addition  of  any 
fluid.  On  exertion  of  pressure  many 
of  the  cells  are  ruptured  and  the  tissue 
fluid,  together  with  cytoplasm,  nucleo- 
plasm and  some  entire  cells,  passes 
through  minute  holes  in  the  press  and  is 
collected,  leaving  most  of  the  fibrous 
elements  behind.  Then  a  little  saline 
solution  is  added  and  the  material  is 
ground  up  in  sand  and  made  up  to  a 
volume  of  about  50  cc.  The  sand  is 
allowed  to  sediment  out  at  the  bottom 
of  a  centrifuge  tube.  The  supernatant 
fluid  is  then  centrifuged  at  low  speed 
(300  r.p.m.).  This  throws  all  the  rest 
of  the  debris  to  the  bottom  while  the 
bacilli  remain  in  suspension.  The 
supernatant  fluid,  containing  the  bacilli, 
is  again  decanted  and  centrifuged  at  high 
speed  (3500  r.p.m.)  in  an  angle 
centrifuge  for  1  hr.  The  supernatant 
fluid  is  discarded  and  the  pasty  material 
at  the  bottom  of  the  tube,  made  up  of 
bacilli,  is  diluted  and  washed  by  re- 
peated centrifugation  in  some  experi- 
ments with  saline  solution  and  in  others 
with  distilled  water. 

Beginning  with  a  large  nodule  or  with 
several  small  ones  it  is  a  simple  matter 
to  collect  in  4  or  5  hrs.  billions  of  bacilli. 
The  pasty  bacterial  mass  can  be  desic- 
cated and  weighed  in  grams.  For  our 
experiments  we  used  only  the  wet 
bacilli.  When  viewed  en  masse  they 
appear  dense  white  with  a  faint  shade 
of  gray.  They  are  not  yellow  or  even 
yellowish.  Examination  of  a  thick 
smear,  made  after  washing  in  saline, 
shows  myriads  of  bacilli  without  any 
trace  of  cellular  material.  The  bacilli 
retain  to  a  remarkable  degree  their 
characteristic  morphology,  as  seen  in 
sections  and  in  smears  of  fresh  tissue, 
and  their  acid-fast  properties  are  not 


CONDENSER  MANOMETERS 


88 


COPPER 


interfered  with.  After  washing  in 
distilled  water  until  the  supernatant 
fluid  gave  no  precipitate  when  added 
to  an  aqueous  solution  of  silver  nitrate, 
the  bacilli  do  not  fuse  together  but  still 
remain  discrete  bodies  though  their 
shape  is  different.  (Cowdry,  E.  V., 
Ravold,  A.  and  Packer,  D.  M.  Proc. 
Soc.  Exp.  Biol.  &  Med.,  1939,  41,  341- 
345).  See  Floatation  Techniques  for  in- 
testinal parasites. 

Condenser  Manometers  in  differential  pres- 
sure measurements  (Hansen,  A.  T.  and 
Warburg,  E.,  Acta  Physiol.  Scand., 
1951,22,  211-215). 

Congo  Blue  3B,  see  Trypan  Blue. 

Congo  Corinth  G  or  GW,  see  Erie  Garnet  B. 

Congo  Red  (CI,  370).  Synonyms:  Congo, 
cotton  red.  A,  B  or  C,  direct  red  C,  R  or 
Y.  An  acid  dis-azo  dye  which  is  an 
excellent  indicator  and  a  useful  stain. 
Matsuura,  S.,  Fol.  Anat.  Jap.,  1925,  3, 
107-110  has  obtained  very  fine  coloration 
of  the  skin  which  he  has  illustrated  in 
colors.  Congo  red  is  the  basis  of  Kra- 
jian's  stain  for  elastic  fibers.  See  also 
Blackman,  V.  H.,  New  Phytol.,  1905, 
4,  173-174  (uredineae);  Merton,  H., 
Arch.  Protistenk.,  1932,  76,  171-187; 
Cumley,  R.  W.,  Stain  Techn.,  1935, 
10,  53-56  (negative  stain  for  bac- 
teria), etc. 

Connective  System.  Provides  both  for  the 
binding  together  of  parts  and  for  their 
separation  one  from  another  by  capsules, 
membranes  and  other  structures  (see 
Cowdry,  p.  429-466).  It  ranges  all  the 
way  from  Loose  Connective  Tissue 
and  Fatty  Tissue  through  Fibrous 
Connective  Tissue  and  Tendons  to 
Cartilage  and  Bone.  Neuroglia  is  a 
special  form  of  it.  In  general  there  are 
three  components.  Fibroblasts,  Fibers 
and  Tissue  Fluid  (ground  substance). 
Cells  of  hematogenous  and  lymphatic 
origin  may  be  present  since  blood  vessels 
and  lymphatics  run  in  connective  tissue 
pathways.  See  techniques  under  these 
headings,  also  Masson's  Trichrome 
Stain,  Mallory's  Connective  Tissue 
Stain,  Phosphomolybdic  Acid  Hema- 
toxylin, Van  Gieson,  Buzaglo,  Mallory- 
Heidenhain  rapid  one  step  stain,  etc. 

Connective  Tissue  Cells,  preservation  of 
trypan  blue  and  neutral  red  in  those  of 
loose  connective  tissue.  Inject  sub- 
cutaneously  5  cc.  fresh  sterile  1%  aq. 
vital  trypan  blue  (Coleman  and  Bell 
Co.)  into  a  mature  white  rat  weighing 
about  90  gras.  and  wait  48  hrs.  Make  up 
0.02%  certified  neutral  red  (National 
Aniline  in  0.9%  NaCl).  After  slight 
etherization  exsanguinate  the  animal. 
Inject  neutral  red  into  subcutaneous 
tissue  of  groin  in  several  places  near 
original  puncture.     After  3-5  min.  re- 


move small  blobs  of  edematous  tissue. 
Tease  these  out  on  clean  slides  with  aid 
of  needles  and  filter  paper.  When 
corners  are  dry  spread  is  ready  for 
direct  observation  under  cover  glass  or 
for  fixation.  Make  up  10%  formalin. 
Test  it  by  addition  of  a  drop  or  two  of 
neutral  red.  If  this  turns  orange  add 
a  little  N/10  HCl  until  it  becomes  red. 
Fix  in  this  formalin  over  night  or  for 
several  days.  Rinse  in  aq.  dest.  Coun- 
terstain  in  1%  fast  green  F.C.F.  (War- 
ner-Jenkinson  Co.)  in  2%  aq.  acetic 
acid  for  ^1  min.,  pass  through  suc- 
cessive changes  dioxan,  3-5  min.  each. 
Agitate  slightly.  Mount  in  dioxan 
employing  medium  hardened  diaphane 
(Will  Corp.),  redissolve  in  dioxan  or 
pass  through  xylol  and  mount  in  balsam. 
Avoid  alcohols.  Note  blue  granules  in 
macrophages  and  fine  red  granules  in 
mast  cells  (Snook,  T.,  Stain  Techn., 
1939,  14,  139-142).  See  Connective 
System. 

Contraction  Bands,  or  waves,  demonstration 
of  in  smooth  muscle.  Remove  intestine 
of  freshly  killed  cat,  expose  to  air  of  room 
or  rub  with  blunt  end  of  scalpel.  When 
preparations  are  made  numerous  con- 
traction bands  will  be  seen.  Contrast 
with  this  intestine  of  cat  killed  with 
chloroform  and  not  excised  until  rigor 
mortis  begins  in  which  muscle  fibers 
are  fully  extended  (Dahlgren,  McClung, 
p.  430). 

Coons,  see  Antigen  Localization. 

Copper.  1.  Microchemical  tests.  Fix  in 
formalin  or  alcohol,  use  same  hema- 
toxylin or  methylene  blue  stain  as  for 
Lead.  With  former  copper  hemofuscin 
is  blue  and  hemosiderin  (iron  pigment) 
is  black,  while  with  latter  copper  pig- 
ment is  pale  blue  and  the  iron  pigment 
uncolored  (Mallory,  F.  B.  and  Parker, 
F.,  Jr.,  Am.  J.  Path.,  1939,  15,  517-522). 
See  also  Okamoto,  K.,  Utamura,  M.  and 
Mikami,  G.,  Acta  Sch.  Med.,  Univ. 
Imp.  in  Kioto.,  1939,  22,  335-360  (il- 
lustrated in  colors);  Mendel,  L.  B.  and 
Bradley,  H.  C,  Am.  J.  Physiol.,  1905, 
14,  313-327  (bromine  test  for) ;  Claude, 
A.,  Cold  Spring  Harbor  Symposia  on 
Quantitative  Biology,  1941,  9,  263-270 
(copper  of  respiratory  pigment) ;  Hoag- 
land,  C.  L.  et  al.,  J.  Exper.  Med.,  1942, 
76, 163-173  (copper  and  other  substances 
in  virus  elementary  bodies).  Water- 
house,  D.  F.,  Bull.  Council  for  Scientific 
and  Industrial  Research,  Melbourne, 
Australia,  1945,  No.  191,  20  pp.:  add  1 
drop  0.1%  aq.  sodium  diethyl  dithio- 
carbamate  to  fresh  tissue  followed  by 
1  drop  cone,  hydrochloric  acid  which 
yield  yellow  product  with  copper.  Iron 
interferes  with  this  result  but  the  sen- 
sitivity for  copper  is  much  greater  than 


COPPER  CHROME  HEMATOXYLIN 


89 


CORONARY  ARTERIES 


for  iron  (quoted  from  Click  p.  23). 
When  search  is  necessary  for  traces 
of  copper  without  need  for  microscopic 
localization  an  emission  spectrograph 
may  give  the  information  qucikly,  see 
Histospectrography.  If  quantitative 
determinations  of  copper  in  small 
amounts  of  tissue  are  required  use  the 
polarographic  technique  elaborated  by 
Carruthers,  C,  Indust.  and  Engin. 
Chem.,  1945,  17,  398-399.  Details  for 
determination  of  copper  in  epidermis 
are  given  bj^  Carruthers,  C.  and  Sunt- 
zeff,  v.,  J.  Biol.  Chem.,  1945,  159,  647- 
661. 

2.  As  vital  stain.  Intravenous  in- 
jections of  colloidal  solutions  of  copper 
in  rabbits  are  described  by  Duhamel, 
B.  G.,  C.  rend.  Soc.  de  Biol.,  1919,  82, 
724-726. 

Copper  Chrome  Hematoxylin  (Bensley's) 
for  mitochondria.  Fix  very  small  pieces 
in  Altmann's  Fluid  or  in  Acetic-Osmic- 
Bichromate  fixative  of  Bensley  12-24 
hrs.  Wash,  dehydrate,  clear,  imbed  in 
paraffin  ana  cut  sections  at  4  or  5  mi- 
crons. Deparaffinize.  Sat.  aq.  copper 
acetate,  5  min.  Several  changes  aq. 
dest.,  1  min.  0.5%  aq.  hematoxylin,  1 
min.  After  rinsing  in  aq.  dest.  pass  to 
5%  aq.  neutral  potassium  chromate,  1 
min.  which  should  turn  sections  dark 
blue-black.  If  they  are  only  light  blue, 
rinse  in  aq.  dest.  again  place  in  copper 
acetate  and  repeat  if  necessary  several 
times  until  no  increase  in  color  is  ob- 
tained. Wash  in  aq.  dest.  and  treat  for 
few  sec.  with  copper  acetate.  Wash  in 
aq.  dest.  and  differentiate  under  the 
microscope  in  Weigert's  borax-ferri- 
cyanide  mixture  (borax,  1  gm. ;  potas- 
sium ferricyanide,  1.25  gm.;  aq.  dest. 
100  CO.)  diluted  with  twice  the  volume 
aq.  dest.  Wash  in  tap  water,  6-8  hrs. 
Dehydrate,  clear  and  mount  in  balsam. 
The  mitochondria  appear  a  beautiful 
deep  blue  against  a  yellowish  back- 
ground. It  is  important  to  have  good, 
ripe  hematoxylin.  It  is  usually  made 
by  dilution  from  a  10%  sol.  in  abs.  al- 
cohol. This  method  of  staining  should 
be  tried  after  fixation  in  Regaud's  fluid. 

Coproporphyrin  of  megaloblasts  in  embryos, 
see  Porphyrins.  Coproporphrin  III  can 
be  isolated  from  human  tubercle  bacilli 
by  chromatographic  and  fluorescence 
analysis  (Crowe,  M.O'L.,  and  Walker, 
A.,  Brit.  J.  Exp.  Path.,  1951,  32,  1-6). 

Coreine  2R,  see  Celestin  Blue  B. 

Corinth  Brown  G,  see  Erie  Garnet  B. 

Coriphosphine  O  (CI.  787).  An  acridine  dye 
used  as  a  fluorchrome  (Metcalf,  R.  L. 
and  Patton,  R.  L.,  Stain  Techn.,  1944, 
19.  11-27). 

Corn  Blue  B,  see  Victoria  Blue  R. 

Corn  Blue  BN,  see  Victoria  Blue  B. 


Cornea.  This  is  a  difficult  tissue  to  prepare 
in  stained  sections  because  of  its  curva- 
ture and  avascularity.  A  valuable  sil- 
ver method  is  minutely  described  by 
Pullinger,  B.  D.,  J.  Path,  and  Bact., 
1943,  55,  97-99. 

Fix  anterior  and  posterior  surfaces 
in  10%  aq.  neutral  formalin  before  ex- 
cision of  eye,  if  possible,  by  flooding 
anterior  surface  with  fixative  and  by 
injecting  fixative  into  anterior  chamber 
through  a  hypodermic  needle  at  the 
same  time  withdrawing  fluid  from  the 
chamber  likewise  bj'  hypodermic.  Re- 
move eye,  inject  fixative  into  vitreous 
at  same  time  removing  fluid  from  it. 
Leave  in  fixative  24  hrs.  Excise  cornea 
along  corneo-scleral  margin,  detach 
iris,  ciliary  body  and  lens.  Fix  latter 
separately  and  cornea  for  further  3 
days,  4  altogether.  Indicate  location 
future  sections  by  nick  in  opposite  edge. 
Transfer  cornea  to  aq.  dest.  avoiding 
metal  instruments  then  and  thereafter. 
Wash  and  leave  over  night  in  aq.  dest. 
+  3  drops  ammonia  (S.  G.  0.88)  per,  say, 
50  cc.  After  washing  in  2  or  3  changes 
aq.  dest.,  and  pouring  off  last  aq.  dest., 
filter  onto  cornea  through  cotton  wool 
moistened  with  aq.  dest.  20  cc.  del  Rio- 
Horiega's  solution.  To  make  this  add 
300  cc.  5%  aq.  sodium  bicarbonate  to 
100  cc.  10%  aq.  silver  nitrate  in  brown 
glass  stoppered  bottle.  Add  few  drops 
ammonia  waiting  each  time  for  smell 
of  ammonia  to  disappear  until  almost 
but  not  all  ppt.  is  dissolved.  They 
add  250  cc.  aq.  dest. 

Place  container  with  cornea  plus  fil- 
tered solution  in  incubator  at  37°C. 
4  hrs.  Pour  off  solution  and  wash 
cornea  in  several  changes  aq.  dest. 
Then  reduce  by  pouring  onto  cornea 
10%  aq.  neutral  formalin,  15  min.  Cut 
away  "dome"  of  cornea  with  knife  and 
support  its  concave  surface  with  the 
lens,  freeze  and  section  at  about  15  /x. 
parallel  to  surface.  Take  sections  into 
aq.  dest.;  mount  at  once  in  glycerin 
jelly  or  pass  through  alcohols  to  abso- 
lute, clear  in  creosote  and  mount  in 
balsam.  Collagen  pale  yellow,  nuclei 
and  cytoplasm  well  shown  and  espe- 
cially Descemets  membrane. 

Cornyebacterium  Diphtheriae.  Evaluation 
of  methods  for  staining  metachromatic 
granules  (Morton,  H.  E.,  Stain  Techn., 
1942,  17,  27-29).  See  Gobar,  M.  A.,  J. 
Bact.,  1944,  47,  575,  also  Diphtheria 
Bacilli. 

Coronary  Arteries.  Their  distribution  may 
be  demonstrated  by  injection  of  the 
easily  recognizable  fluids  listed  under 
Blood  Vessels.  Owing  however  to  their 
great  importance  it  is  well  to  mention 
two  special  adaptations  of  the  said  fluids. 


CORPORA  AMYLACEA 


90 


GROSSMAN'S 


Gross  (L.,  The  Blood  Supply  of  the 
Heart  in  its  Anatomical  and  Glinical 
Aspects.  New  York:  Iloeber,  1921) 
employed  injections  of  barium  sulphate 
suspensions  in  gelatin  followed  by  x-ray 
photographs;  while  Spalteholz  (W., 
Die  Arterien  der  Herzwand,  etc., 
Leipsig:  Herzel,  1924)  used  injections 
of  cinnabar  and  other  pigments  likewise 
in  gelatin  which  were  later  cleared  by 
his  method.  Ehrich,  Ghapelle  and  Gohn 
(W.,  C.,  and  A.  E.,  Am.  J.  Anat.,  1931, 
49,  241-282)  found  the  latter  technique 
preferable.  Celloidin  injections  also 
give  good  results.  Histological  demon- 
stration of  the  blood  supply  of  the 
coronaries  is  described  under  Vasa 
Vasorum. 

Corpora  Amylacea,  see  Prostate. 

Corpuscles,  see  those  of  Pacini,  Meissner, 
Krause,  etc. 

Corrosion  Preparations.  In  them  the  struc- 
tures to  be  demonstrated  are  left  while 
all  the  surrounding  tissue  is  corroded 
and  washed  away,  for  instance  Celloidin 
and  Neoprene  injections. 

Corrosive  Sublimate,  see  Mercuric  Chlo- 
ride. 

Corti,  organ  of,  see  Ear. 

Cortin  (interrenalin),  hormone  of  adrenal 
cortex. 

Cotton  Blue,  see  Anilin  Blue,  Methyl 
Blue.     See  Fungi. 

Cotton  Corinth  G,  see  Erie  Garnet  B. 

Cotton  Red,  see  Safranin  O. 

Cotton  Red,  A,  B,  or  C,  see  Congo  Red. 

Cotton  Red  4B,  see  Benzopurpurin  4B. 

Covell,  see  Ear. 

Cover  Glasses,  see  Cleaning. 

Creatine  and  Creatinone,  see  colorimetric 
methods  of  Borsook,  H.,  J.  Biol.  Chem., 
1935,  110,  481-493  and  of  Sure,  B.  and 
Wilder,  V.  M.,  J.  Lab.  Clin.  Med., 
1941,  26,  874-878. 

Cresol  Red.     See  Hydrogen  Ion  Indicators. 

Creosote  (Beechwood)  is  an  important 
clearing  agent  for  celloidin  sections. 
It  is  a  mixture  of  phenols,  mainly 
guaiacol  and  creosol. 

Cresyl  Blue  2RN,  or  BBS,  see  Brilliant 
Cresyl  Blue. 

Cresyl  Violet — cresylecht  violet  (cresyl  fast 
violet) — Commission  Certified.  A  basic 
oxazin  dye.  A  technique  for  its  use 
(or  that  of  toluidin  blue)  in  studies  on 
the  cytoarchitectonics  of  the  nervous 
system  is  proposed  by  Landau,  E., 
Bull.  d'Hist.  Appl.,  1934,  11,  44^6. 
As  a  stain  for  nerve  cells  in  celloidin 
sections  (Tress,  G.,  and  M.,  Stain  Tech., 
1935,  10,  105-106).  Wash  low  viscosity 
nitrocellulose  (celloidin)  sections  of  10% 
formalin  fixed  tissues  in  aq.  dest.  Stain 
for  30  min.  at  50°C.  in  cresyl  violet, 
0.5  gm.;  aq.  dest.,  100  cc. ;  glacial  acetic 
acid,   4  drops    (filtered   before   using). 


Wash  in  aq.  dest.  Differentiate  in  70% 
alcohol  until  most  of  stain  leaves  cel- 
loidin. Completely  immerse  for  2-5 
min.  in:  chloroform,  60  cc;  abs.  ale, 
10  cc;  and  ether,  10  cc.  Almost  no 
destaining  of  cells  occurs  but  stain  is 
removed  from  background.  Differen- 
tiate in  100  cc.  95%  ale  +  4  drops  1% 
aq.  hydrochloric  acid  but  stop  while 
cells  are  a  little  darker  than  desired. 
Neutralize  sections  in  90%  alcohol  + 
a  little  sodium  bicarbonate.  Wash  in 
95%  alcohol  to  remove  the  bicarbonate. 
Complete  dehydration  in  2  changes  n 
butyl  alcohol.  Clear  in  4  changes  xylol 
and  mount.  See  Kallichrom.  Note: 
There  are  two  different  dyes  sold  as 
cresyl  violet:  (1)  The  CC.  product 
(Nat.  Anilin,  Mfgrs.;  see  Conn,  1940, 
p.  93)  which  is  good  in  biopsy  work; 
(2)  The  Grtibler  product  (also  sold  by 
Coleman  and  Bell,  but  not  on  the  market 
during  the  war)  which  is  needed  in 
neurological    work,    cf.    Tress,    above. 

Cresylecht  Violet,  intensification  of  meta- 
chromatic properties  (Williams,  B.  G. 
R.,  J.  Lab.  &  Clin.  Med.,  1934-35,  20, 
1185-1187).  A  new  domestic  cresylecht 
violet  has  been  made  available  by  Cole- 
man and  Bell  and  is  stronglj'-  recom- 
mended by  Banny,  T.  M.  and  Clar,  G., 
Stain  Techn.,  1950,  25,  195-196. 

Cretin's  Test,  see  Calcium. 

Crime  Detection  Techniques.  These  are  of 
course  legion.  Many  of  them  are  mi- 
croscopic and  involve  identification  of 
materials.  See  for  example,  Hair, 
Semen  Stains  and  Hemoglobin.  In  re- 
spect to  the  latter  the  object  is  to  deter- 
mine whether  blood  is  human  by  pre- 
cipitin tests  and  to  which  group  it  be- 
longs by  detection  of  agglutinins  as  is 
well  described  by  Schiff,  F.  and  Boyd, 
W.  C,  Blood  Grouping  Technic  New 
York:  Interscience  Publishers,  Inc., 
1942,  248  pp.  The  identification  of 
metals,  such  as  chips  from  a  razor  blade, 
by  spectroscopic  examination  is  often 
conclusive,  see  Histospectrography. 
Cracks  in  metal  surfaces  can  be  de- 
tected with  astonishing  delicacy  by  the 
Magnaflux.  An  interesting  elementary 
account  of  Crime  Detection  Techniques 
is  provided  by  Hoover,  J.  E.,  Scientific 
Monthly,  1945,  60,  18-24. 

Croceine  Scarlet,  see  Biebrich  Scarlet, 
water  soluble. 

Grossman's  modification  of  Mallory's  con- 
nective tissue  stain  (Grossman,  G., 
Anat.  Rec,  1937,  69,  33-38).  Deparaf- 
finize  sections  of  Zenker  fixed  material. 
Lugol's  iodine,  5  min.  Rinse  in  70% 
alcohol,  several  changes.  Wash  10  min. 
in  running  water.  Overstain  nuclei  in 
Mayer's  acid  Hemalum  or  Weigert's 
Iron   Hematoxylin.    Wash   in   running 


CRYOSTAT 


91 


CURTIS'  SUBSTITUTE 


water  10  min.  Stain  for  1  min.  or  more 
in:  acid  fuchsin  (C.C.),  1  gm.;  orange 
G  (CO.),  0.4  gm.;  aq.  dest.,  300  cc; 
thymol,  0.2  gm.;  glacial  acetic  acid,  3 
cc.  Rinse  in  aq.  dest.  Decolorize  in 
fresh  1%  aq.  phosphotimgstic  or  phos- 
phomolybdic  acid  until  arterial  media 
is  red  and  adventitia  is  colorless.  Rinse 
very  quickly  in  aq.  dest.  Counterstain 
in  2%  aq.  anilin  blue,  W.S.  (CC.) 
100  cc.  +  glacial  acetic  acid,  2  cc.  or  in 
1%  aq.  light  green  SF  yellowish  (CC.) 
100  cc.  +  glacial  acetic  acid,  1  cc.  Rinse 
in  aq.  dest.  Decolorize  in  1%  acetic 
acid  under  microscope.  Rinse  in  aq. 
dest.  Dehydrate  in  3  changes  abs.  ale. 
Clear  in  3  changes  .xylol  and  mount. 
Like  original  method  but  nuclei  brown  or 
black  and  collagen  blue  or  green  de- 
pending on  counterstain. 

Cryostat.  Written  by  Dr.  Gordon  H.  Scott, 
Dept.  of  Anatomy,  Wayne  University, 
School  of  Medicine,  Detroit,  Mich. — 
This  apparatus  is  one  which  is  designed 
to  dehydrate  tissues  at  low  tempera- 
tures. A  detailed  description  has  been 
given  by  Packer  and  Scott  (J.  Tech. 
Methods,  1942,  22,  85-96)  and  by  Hoerr 
and  Scott  (Medical  Physics,  Otto  Glas- 
ser,  1944,  Year  Book  Publishers).  Tis- 
sues frozen  in  liquid  air  or  nitrogen  are 
placed  in  a  chamber  which  is  connected 
with  a  fast  pumping  vacuum  system. 
Water  vapor  which  is  released  from  the 
tissues  is  trapped  by  P2O5  as  well  as  by 
a  cold  trap.  In  the  Packer-Scott  ap- 
paratus the  relative  amount  of  water 
vapor  in  the  system  is  determined  by 
its  flow  past  a  spaced  pair  of  ionization 
gauges  between  which  is  placed  the 
P2O5  trap.  When  these  gauges  are  in 
balance  it  is  assumed  water  vapor  is  no 
longer  being  released  in  quantity,  and 
therefore  the  tissues  are  dry.  As  soon 
as  the  tissues,  placed  on  a  container 
of  solid  out-gassed  paraffin,  are  dry, 
the  paraffin  is  melted  and  the  tissues 
are  infiltrated.  This  latter  procedure 
is  accomplished  without  the  necessity 
of  breaking  the  vacuum.  This  small 
but  important  step  provides  tissues 
which  have  been  frozen-dried  and  pre- 
pared for  cutting  without  their  having 
been  partially  rehydrated  by  exposure 
to  air  at  ambient  pressure  and  temper- 
ature. See  Altmann-Gersh  frozen  de- 
hydration method. 

Cryptococcus  Hominis,  see  Blastomycosis. 

Cryptosporidium,  see  Coccidia. 

Crystal  Violet  as  vital  stain  for  fibroblast 
nuclei  (Bank,  O.  and  Kleinzeller,  A., 
Arch.  exp.  Zellforsch.,  1938,  21,  394- 
399).  See  Anilin  Crystal  Violet  and 
Gentian  Violet. 

Crystal  Violet-Acid  Fuchsin.  This  is  one 
of  R.  R.  Bensley's  neutral  stains  espe- 


cially advocated  for  the  demonstration 
of  secretion  antecedents  in  gland  cells. 
The  technique  is  described  by  the 
Bensleys  (p.  97).  To  make  stain  add 
filtered  sat.  aq.  acid  fuchsin  to  similar 
solution  crystal  violet  until  precipita- 
tion is  complete.  Collect  ppt.  on  fdter 
paper,  wash  through  once  with  aq.  dest. 
Dry  and  dissolve  in  absolute  alcohol  to 
saturation.  For  staining  add  5  cc.  of 
above  stock  solution  to  45  cc.  20% 
alcohol  made  from  absolute.  In  this 
color  paraffin  sections  of  Formalin- 
Zenker  fixed  material  for  5  min.  Blot 
with  filter  paper  in  one  hand  and  add 
with  other  hand  absolute  alcohol  from 
a  pipette,  flood  with  absolute.  Blot 
immediately.  Add  few  drops  clove  oil. 
When  differentiation,  observed  under 
microscope,  is  optimum  transfer  to 
pure  benzol  and  mount  in  balsam. 

Crystal  Violet  and  Alizarin,  see  Benda's 
Method  for  Mitochondria. 

Crystals.  These  are  encountered  in  many 
forms,  see  Charcot-Leyden,  Ice,  Sul- 
fonamides, Hemin,  Florence,  Virchow's, 
Spermin,  Lubarsch,  Neumann's,  Teich- 
mann's,  Mineral  residue  of  Microincin- 
eration, Polarization  Optical  methods. 
Numerous  Microchemical  Reactions 
especially  for  minerals,  yield  crystal- 
line materials.  Fat  crystals  are  often 
distinctive  as  for  beef,  duck,  lard,  etc. 
(Schneider,  A.  The  Microbiology  and 
Microanalysis  of  Foods.  Philadelphia: 
P.  Blakiston's  Son  &  Co.,  1920,  262  pp.) . 
Study  of  crystals  is  really  a  problem  for 
experts.  For  the  best  techniques  con- 
sult Section  I  on  "Identification"  in 
Bunn,  C.  W.,  Chemical  Crj^stallog- 
raphy,  Oxford  University  Press,  1946, 
234  pp.  Comparison  of  the  crystals  to 
be  diagnosed  with  some  of  the  234 
figures  in  the  book  may  result  in  prompt 
recognition. 

Culture  Media,  see  Bacteria,  Leishmania, 
Protozoa,  Tissue  Culture,  Trypano- 
somes,  NNN  Medium. 

Curcumine,  see  Brilliant  Yellow. 

Curettings,  gelatin  method  for  rapid  frozen 
sections  of  (Meeker,  L.  II.,  J.  Techn. 
Meth.  &  Bull.  Int.  Assoc.  Med.  Mu- 
seums,  1936,   16,  41-42). 

Curtis'  Substitute  for  Van  Gieson  stain  as 
modified  by  Leach,  E.  H.,  Stain  Techn., 
1946,  21,  107-110.  Use  any  desired 
fixative.  Bring  sections  to  water  and 
treat  with  iodine  and  hypo  (sodium 
thiosulfate)  if  necessary.  Stain  for  5- 
10  min.  in  Weigort's  hematoxylin.  To 
make  this  mix  (just  before  use)  1  part  1% 
hematoxylin  in  absolute  alcohol  with 

1  part  of  mixture  containing  30%  aq. 
ferric  chloride  4  cc,  cone,  hydrochloric 
acid,  1  cc.  2%  acetic  acid  100  cc.  and  add 

2  parts  aq.  dest.    Wash  for  5  min.  in 


CYANOCHIN 


92 


DARK-FIELD  MICROSCOPE 


running  water.  Stain  2-4  min.  in  Cur- 
tis' substitute:  2%  Ponceau  S,  CI,  282, 
(National  Aniline)  5  cc;  sat.  aq.  picric 
acid,  95  cc. ;  2%  acetic  acid,  2  cc.  Rinse 
in  96%  alcohol,  dehydrate,  clear  and 
mount.  Chromatin,  black;  cytoplasm, 
yellow;  collagen  and  reticular  fibers, 
red.  Red  and  yellow  colors  are  said 
to  be  purer  than  those  given  by  the 
Van  Gieson  technique  and  too  heavy 
staining  with  red  is  less  likely.  In 
original  account  volumes  are  given  in 
ml.  which  are  of  practically  the  same 
value  as  cc. 

Cyanochln  of  Bresslau,  E.,  Arch.  f.  Protis- 
terik.,  1921,  43,  467,  see  Cyanosine. 

Cyanosine,  see  Phloxine  B. 

Cyclohexanone  has  been  recommended  for 
dehydration  and  clearing  instead  of 
absolute  alcohol  and  xylol  by  Bourdon, 
P.,  Bull.  d'Hist.  Appl.,  1942,  19,  55. 
After  dehydrating  tissue  in  95%  alco- 
hol, 12  hrs.;  pass  to  cyclohexanone, 
4  hrs.;  then  to  another  lot  of  cyclo- 
hexanone, 2  hrs.;  and  impregnate  with 
paraffin  2  baths  2  hrs.  or  le.ss  each.  For 
pieces  more  than  3  nmi.  thick  longer 
times  are  necessary.  This  saturated 
cyclic  ketone  has  density  similar  to 
water,  mixes  with  organic  solvents  and 
paraffin  and  does  not  harden  tissue. 
From  Review  by  Jean  E.  Conn  in  Stain 
Techn. 

Cyclospora,  see  Coccidia. 

Cytocentrum,  centrosome  plus  centrosphere. 

Cytochrome.  This  is  the  name  given  by 
Keilin  (D.,  Proc.  Roy.  Soc,  1925,  B, 
98,  312-339)  to  hemin  compounds  of  a 
reddish  color  which  occur  in  oxidized 
or  reduced  condition  in  almost  all  living 
cells.  Blaschko  and  Jacobson  (Bourne, 
p.  192)  have  summarized  our  knowledge 
about  them.  They  say  that  the  red 
color  of  cytochrome  can  be  observed 
when  a  slice  of  brain  tissue,  from  which 
the  blood  has  been  carefully  washed  out, 
is  suitably  illuminated  by  transmitted 
light.  A  thick  suspension  of  yeast  and 
the  thoracic  muscles  of  insects  are  also 
recommended  as  material.  There  are 
4  cytochromes :  a,  b,  c  and  as  recog- 
nizable spectroscopically. 

Cytochrome  Oxidase.  Cytochrome  is  ox- 
idized by  cytochrome — oxidase  which  is 
identical  with  indophenol  o.xidase  and 
Warburg's  respiratory  enzyme.  See 
study  of  cytochrome  oxidase-cyto- 
chrome system  in  kidne}^  (Flexner,  L. 
B.,  J.  Biol.  Chem.,  1939,  131,  703-711). 
See  Oxidase. 

Cytolipochrome  Pigment,  see  Lillie,  p.  127. 

Cytophaga  Group  of  organisms,  enrichment 
cultures,  pure  culture  techniques, 
methods  of  examination  and  identifica- 
tion (Stanier,  R.  Y.,  Bact.  Rev.,  1942, 
6,  143-197). 


Cytoplasmic  Inclusions  caused  by  viruses. 
They  are  more  diversified  in  size,  shape 
and  chemical  composition  than  the 
Nuclear  Inclusions.  Frequently,  as  in 
the  case  of  large  Negri  Bodies,  they 
contain  both  acidophilic  and  basophilic 
components  (Trachoma  Bodies).  Gly- 
cogen tests  for  Trachoma  inclusion 
bodies  are  described  by  Thygeson,  P., 
Am.  J.  Path.,  1938,  14,  455-462.  The 
techniques  mentioned  for  Nuclear  In- 
clusions may  be  employed.  See  de- 
scription by  Goodpasture,  E.  W.  and 
Woodruff,  A.  M.,  Am.  J.  Path.,  1930, 
6,  699-711 ;  713-720  of  the  reactions  of 
fowl-pox  inclusions  to  potassium  hy- 
droxide and  other  chemicals  and  the 
nature  of  the  particles.  See  also  Borrel, 
Guarnieri  and  KurlofiT  bodies.  Rickett- 
sia are  not  to  be  listed  as  cytoplasmic 
inclusions  but  Giemsa's  stain  is  the 
best  for  them. 

In  plant  cells,  as  in  animal  ones,  cer- 
tain cytoplasmic  inclusions  are  indica- 
tive of  virus  action.  They  are  of  two 
sorts:  (1)  X  bodies,  which  are  rather 
amorphorus  structures,  and  (2)  crystal- 
line inclusions.  The  latter  are  best 
seen  in  the  dark  field  and  in  polarized 
light  and  are  made  up  chiefly  of  virus. 
For  technique  employed  to  demonstrate 
the  relationship  of  virus  to  inclusion 
and  a  critical  review  of  the  whole  prob- 
lem of  plant  viruses,  see  Bawden,  F.  C. 
Plant  Virus  Diseases,  Waltham: 
Chronica  Botanica  Co.,   1943,  294  pp. 

Cytosiderin  Pigment,  Lillie,  p.  127. 

Dahlia,  see  Hofmann's  Violet. 

Dahlia  B,  see  Methyl  Violet. 

Damar  is  gum  damar  dissolved  in  xylol 
and  used  to  mount  sections. 

D'Antoni's  Iodine  solution.  First  make 
standardized  10%  aq.  potassium  iodide. 
Adjust  by  specific  gravity  method  to 
exact  10%  concentration.  To  100  cc. 
of  1%  aq.  potassium  iodide  made  from 
it  add  1.5  gm.  powdered  iodine  crystals. 
Allow  to  stand  for  4  days  before  using. 
Recommended  for  staining  intestinal 
protozoa  (D'Antoni,  J.  S.,  Am.  J.  Trop. 
Med.,  1937,  17,  79-84).  See  McClung, 
1950,  p.  450. 

Dark  Brown  Salt  R,  see  Chrysoidin  Y. 

Dark-Field  Microscope  (From  account  in 
Cowdry's  Histology,  1950).  In  dark- 
field  microscopy  the  light  comes  in  at 
an  angle,  and  does  not  enter  the  ob- 
jective lens  at  all.  It  may  be  thought 
of  as  oblique  illumination  carried  to  the 
limit  of  obliquity.  If  there  is  nothing 
in  the  field  of  view  the  background  will 
of  course  be  perfectly  dark;  but  the 
presence  of  objects  will  result  in  the 
reflection  of  some  light  laterally  into 
the  objective  lens.  Since  only  a  minute 
fraction  of- the   illumieation  beam   of 


DARK-FIELD  MICROSCOPE 


93 


DAVENPORT'S 


light  will  go  toward  the  formation  of 
an  image  this  system  will  require  a  very 
strong  light  source  as  well  as  a  dark- 
adapted  eye  for  observation.  If  a  spe- 
cial dark-field  condenser  is  not  avail- 
able, one  can  be  easily  improvised  by 
adding  a  central  opaque  stop  just  be- 
low the  regular  condenser  in  the  fol- 
lowing manner: 

Select  an  objective  of  not  over  0.66  N. 
A.  and  a  condenser  of  at  least  1.25  N. 
A.  and  set  up  the  illumination  as  for 
bright  field,  with  the  slide  oiled  to  the 
top  of  the  condenser.  Stop  down  the 
iris  diaphragm  until  it  just  encircles  the 
bright  central  disk  which  is  seen  through 
the  tube  with  ocular  removed.  Now 
cut  from  black  paper  or  thin  metal  a 
disk  of  diameter  equal  to  the  diameter 
of  this  opening  in  the  diaphragm  and 
cement  it  to  a  piece  of  glass  that  fits 
the  insertion  slot  under  the  condenser. 
This  central  stop  should  block  all  light 
from  entering  the  objective  if  it  is 
properly  centered  below  the  iris  open- 
ing. Next  open  the  diaphragm  com- 
pletely, thus  permitting  a  hollow  cone 
of  light  to  focus  on  the  specimen  but 
not  to  enter  the  objective.  If  the 
microscope  is  already  supplied  with  a 
dark-field  stop  it  should  be  tested  in 
this  manner. 

The  great  advantage  of  dark-field 
microscopy  is  that  it  enables  one  to  per- 
ceive the  presence  of  particles  far  be- 
low the  theoretical  limit  of  resolution. 
Even  though  a  particle  may  be  much 
smaller  than  the  wave  length  of  light  it 
does  scatter  incident  light  laterally  into 
the  objective  and  is  imaged  as  a  rather 
fuzzy  point  of  light.  We  have  here  an 
exact  analogy  of  the  situation  whereby 
we  can  "see"  dust  particles  dancing  in 
a  strong  beam  of  light  when  it  enters  a 
darkened  room.  Most  of  these  particles 
are  of  a  size  far  below  the  limit  of  visual 
resolution.  Another  advantage  is  that 
small,  transparent  objects,  like  chylo- 
microns, can  be  seen  in  dark-field 
whereas  they  are  invisible  in  the  glare 
of  bright-field  illumination. 

For  the  examination  of  ordinary  his- 
tological sections  little  is  to  be  gained 
by  dark-field  observation.  Neverthe- 
less Ralph,  P.H.  (Stain  Tech.,  1942,  17, 
7-10)  recommends  dark-field  examina- 
tion in  the  study  of  stained  blood 
smears.  It  is  most  helpful  however  in 
the  search  for  very  small,  scattered 
bodies  in  a  more  or  less  empty  medium, 
such  as  chylomicrons  or  spirochetes  in 
blood  plasma.  Dark  field  examination 
is  the  standard  technique  for  the  study 
of  microincineration  preparations. 
The  cytological  results  of  20  years  of 
of  microincineration  have  been  summa- 


rized by  Policard,  A.    (J.   Roy.  Micr. 
Soc.,1942,  42,  25-35). 

The  source  of  the  light  must  be  very, 
very  strong.  To  obtain  the  best  results 
do  not  rely  on  improvised,  or  home 
made,  equipment.  A  special  condenser 
is  needed.  Probably  the  highest  ob- 
jective that  can  be  usefully  employed 
18  a  3  mm.  oil  immersion  objective  with 
iris  diaphragm.  See  Microincineration, 
Spirochetes,  Chylomicrons,  etc. 
Davenport's  2-hour  method  for  staining 
nerve  fibers  in  paraffin  sections  with 
protargol.  1946  modification  written 
by  Dr.  H.  A.  Davenport  of  original 
(Davenport,  H.  A.,  McArthur,  J.,  and 
Bruesch,  S.  R.,  Stain  Techn.,  1939,  14, 
21-26).  Fix  for  1  to  3  days  in:  Form- 
amide  (Eastman  Kodak  Co.),  10  cc; 
paranitrophenol,  5  gm.;  95%  ethyl  alco- 
hol, 45  cc;  aq.  dest.,  45  cc.  Transfer 
thru  graded  alcohols  to  absolute,  then 
either  n-butyl  alcohol  or  xylene  and 
embed  in  paraffin.  Sections  are  cut  and 
mounted  in  the  usual  manner,  paraffin 
removed  and  the  slides  run  thru  graded 
alcohols  to  dist.  water.  Impregnate 
for  1  hr.  at  58-^2 °C.  in  a  5%  aq.  silver 
nitrate.  Rinse  in  3  changes  of  aq. 
dest.  with  20-30  sec.  allowed  for  each 
change.  The  rinse  water  should  cover 
the  slides  completely,  each  slide  sepa- 
rate (not  back  to  back)  and  the  water 
discarded  with  each  change  to  prevent 
carry-over  of  silver  nitrate  into  the 
protargol.  Place  the  slides  in  0.2% 
protargol  (Winthrop  Chemical  Co.)  for 
1  hour  at  room  temperature.  Rinse 
quickly  (2  sec.)  in  aq.  dest.  and  reduce 
for  1  to  2  min.  in  the  following  mixture : 
Sodium  sulfite,  5  gm. ;  Kodalk  (Eastman 
K.  Co.),  0.5  gm.;  hydroquinone,  1  gm.; 
aq.  dest.,  100  cc.  Wash  in  running  tap 
water  several  minutes  and  rinse  once  in 
dist.  water.  Tone  in  0.1%  aq.  gold 
chloride  for  5  to  10  min.  Wash  again 
for  about  1  min.  and  reduce  in  1%  aq. 
oxalic  acid  for  10-20  sec.  Rinse  and 
place  in  hypo  (10%  aq.  sodium  thio- 
sulfate)  for  about  1  min.  Wash  in  run- 
ning water,  dehydrate  and  cover. 

Notes:  If  the  stain  is  too  dark,  try 
any  or  all  of  the  following  modifications : 
rinse  longer  after  the  protargol,  use 
0.1%  protargol,  omit  the  oxalic  acid  re- 
duction after  gold  toning.  If  too  pale: 
double  the  concentration  of  the  pro- 
targol, double  the  time  of  either  or  both 
silver  impregnations,  omit  rinsing  after 
protargol,  double  the  concentration  of 
kodalk  in  the  reducer,  lengthen  the 
time  of  reduction  in  oxalic  acid.  The 
technic  is  suitable  for  mammalian  cen- 
tral or  peripheral  nervous  tissue,  but 
for  sympathetic  fibers  in  intestine  and 
uterus  a  moderate  degree  of  success  has 


DEAD  BACTERIA 


94 


DECALCIFICATION 


been  obtained  with  material   fixed  in 

Bouin's     picric-formalin-acetic     (aq.)- 

Use  clean  glassware  and  fresh  solutions! 

Dead  Bacteria.    To  distinguish  from  living 

try: 

1.  Proca-Kayser  stain  (Gay,  F.  P. 
and  Clark,  A.  R.,  J.  Bact.,  1934,  27,  175- 
189).  Fix  bacterial  smear  by  drying 
and  flaming.  Stain  3-5  min.  in  Loef- 
fler's  alkaline  methylene  blue.  Wash 
quickly  and  stain  in  Ziehl-Neelsen's 
carbol  fuchsin  only  5-10  sec.  Wash  and 
dry.  Living  bacteria  blue,  dead  ones 
purple  to  red. 

2.  Neutral  red  (Knaysi,  G.,  J.  Bact., 
1935,  30,  193-206).  Add  a  little  neutral 
red  to  the  medium.  Escherichia  coli 
and  Schizosaccharomyces  pombi  are 
considered  dead  when  tinged  even 
slightly   by   the  stain. 

3.  Decolorization  (Prudhomme,  R.  O., 
Ann.  Institut  Pasteur,  1938,  61,  512- 
518).  Living  bacilli  separated  from  all 
tissue  decolorize  solutions  of  1-naphthol- 
2-sodium  sulphonate-indo-2-6-dibrom- 
phenol,  O-cresol-2-6  dichlorophenol 
and  0-chlorophenol-indo-2-6-dichloro- 
phenol.  Bacilli  killed  by  100  °C.  for  15 
min.  do  not  decolorize  them. 

The  value  of  these  methods  is 
questionable. 
Dead  Cells.  Often  it  is  very  difficult  to 
say  whether  a  particular  cell  was  dead 
or  alive  when  the  preparation  was  made. 
The  appearance  of  nuclei  in  Postmortem 
Degeneration  may  be  a  clue.  Evans 
and  Schulemann  (H.  M.  and  W. 
Science,  1914,  39,  443-454)  remarked 
upon  the  extraordinary  rapidity  with 
which  dead  cells  take  in  vital  benzidine 
dyes  and  the  diffuse,  uniform  coloration 
that  ensues. 

In  cells  supravitally  stained  with 
neutral  red  Lewis  and  McCoy  (W.  H. 
and  C.  C,  Johns  Hopkins  Hosp.  Bull., 

1922,  33,  284-293)  employed  the  follow- 
ing criteria  for  death:  "  (1)  loss  of  color 
from  the  granules  and  vacuoles;  (2) 
diffuse  pink  staining  of  the  cytoplasm 
and  nucleus;  (3)  the  appearance  of  a 
sharp  and  distinct  nuclear  membrane 
and  a  change  in  texture  of  the  cyto- 
plasm and  nucleus."  Using  dark-field 
illumination  W.  H.  Lewis  (Anat.  Rec, 

1923,  26,  15-29)  observed  the  appear- 
ance in  dying  cells  of  certain  very  small 
brightly  shining  (white)  bodies  which 
he  called  d  or  "death  granules."  These 
were  first  in  Brownian  movement  which 
soon  ceased.  To  quote  Lewis:  "During 
the  period  when  the  cells  were  dying, 
spherical  blebs  often  appeared  on  both 
the  flat  and  rounded  cells.  These  were 
pale  grayish  sacs  with  very  thin  walls 
and  fluid  contents  in  which  va,rying 
numbers  of  small  white  granules  in  ac- 


tive Brownian  motion  were  seen.  The 
blebs  varied  in  size  and  were  occasion- 
ally as  large  as  a  contracted  cell. 
Sometimes  the  blebs  were  so  crowded 
with  granules  that  they  were  milky  in 
appearance.  Frequently  one  would 
burst,  freeing  its  granular  contents  into 
the  surrounding  fluid  medium  where 
they  showed  Brownian  motion  until 
they  settled  down  on  the  slide." 

Luyet's  (B.,  Science,  1937,  85,  106) 
method  for  the  differential  staining  of 
living  and  dead  plant  cells  may  prove 
of  value  for  animal  cells  also.  He  has 
written  the  following  account:  A  piece 
of  the  lower  epidermis  of  the  scale  of 
the  onion  bulb  is  peeled  off  and  placed, 
cutin  side  down,  on  a  sHde.  A  drop  of 
a  .5  per  cent,  slightly  alkaline,  aqueous 
solution  of  neutral  red  is  deposited  on 
the  piece  of  epidermis  and  left  there  for 
2  minutes;  then  it  is  blotted  off  and  re- 
placed by  a  drop  of  a  .4  per  cent  potas- 
sium hydroxide  solution,  which  is  imme- 
diately removed  (also  with  a  blotter) ; 
then  the  preparation  is  washed  with 
tap  water.  The  living  cells  take  with 
that  treatment  a  bright  cerise  red  color, 
while  the  dead  cells  are  of  an  intense 
orange  yellow.  The  contrasts  are  vio- 
lent. There  are  intermediate  tints 
which  correspond  to  the  dying  cells. 
See  Necrosis,  Necrobiosis,  Survival 
of  tissue. 
Decalcification.  The  removal  of  calcium  so 
that  bony  tissues  can  be  cut  in  sections. 
There  are  many  methods  almost  all  of 
which  involve  acid  treatment.  It  is 
generally  better  to  apply  the  de- 
calcifying agent  after  fixation,  particu- 
larily  so  when  the  agent  is  a  poor  fixative. 
The  volume  of  decalcifier  should  be 
about  100  times  that  of  the  tissue.  The 
usual,  crude,  way  of  testing  the  progress 
of  decalcification  is  to  stick  a  fine  needle 
into  the  bone  being  careful  to  avoid  the 
area  that  will  be  cut  in  sections;  but 
less  objectionable  methods  can  be  used, 
see  Teeth,  Decalcification. 

Saturated  aq.  sulphurous,  5%  tri- 
chlorlactic,  5%  hydrochloric  and  equal 
parts  of  1%  hydrochloric  and  1% 
chromic  acids  are  all  fairly  good  de- 
calcifiers.  Lactic,  acetic,  phosphoric 
and  picric  acids  are  usually  unsatis- 
factory. Shipley  (McClung,  p.  347) 
recommends  slow  decalcification  by 
long  immersion  in  Muller's  Fluid 
through  liberation  of  small  amounts  of 
chromic  acid  from  the  bichromate. 
The  bones  of  an  adult  rat  require  21-30 
days.  The  process  can  be  hurried 
somewhat  by  using  an  incubator  at 
37°C.  Adequate  decalcification  is  de- 
tected by  slight  bending  of  the  bone  or 


DEGENERATION 


95 


DEHYDRATION 


by    the    needle    method.     Over    decal- 
cification is  not  likely. 

For  rapid  decalcification  he  advises 
using  sat.  aq.  phloroglucin  to  which 
6-30%  Nitric  Acid  is  added.  A  some- 
what slower  formula  is :  nitric  acid,  5 
cc;  phloroglucin,  70  cc;  95%  ale,  1 
cc;  and  aq.  dest.,  30  cc.  The  phloro- 
glucin allows  use  of  stronger  acids. 
1-2%  aq.  hydrochloric  acid  decalcifies 
quickly  but  it  causes  the  tissue  to  swell. 
Formic  Acid  1-5%  in  70%  alcohol  is, 
according  to  Shipley,  the  best  decal- 
cifying agent  for  large  masses  of  bone. 
With  5%,  the  decalcification  is  com- 
pleted in  4-5  days.  Use  70%  ale.  not 
water,  to  wash  out  the  acid. 

Kramer  and  Shipley  devised  a  Magne- 
sium Citrate  method  of  decalcification 
in  neutral  solutions.  To  make  the  de- 
calcifier  dissolve  80  gms.  citric  acid  in 
100  cc.  hot  aq.  dest.,  add  4  gms.  magne- 
sium oxide  and  stir  until  completely 
dissolved.  If  the  magnesium  oxide 
contains  carbonate  it  should  be  freshly 
ignited.  Cool  and  add  100  cc.  ammonium 
hydroxide  (density  0.90)  and  aq.  dest. 
to  make  300  cc.  Allow  to  stand  24  hrs. 
and  filter.  Titrate  filtrate  with 
hydrochloric  acid  to  about  pH  7.0- 
7.6  and  add  equal  volume  aq.  dest.  In 
decalcifjdng,  this  reagent  should  be 
changed  every  3  days.  A  dog's  rib  is 
decalcified   in  approximately    15   days. 

After  decalcification,  by  whatever 
method,  the  bone,  or  the  area  of  calcifica- 
tion, must  be  thoroughly  washed  to 
remove  the  decalcifer  and  imbedded  in 
paraffin  or  celloidin.  Some  investiga- 
tors prefer  the  latter  but  celloidin 
sections  are  not  so  easily  handled.  See 
Bones,  Teeth. 

The  ion  exchange  resin  technique  for 
bone  is  new  and  interesting  (Dotti,  L. 
B.,  Paparo,  G.  P.  and  Clarke,  B.  E., 
Am.  J.  Clin.  Path.,  1951,  21,  475-479). 
Try  keeping  tissue  in  cork  stoppered 
bottle  containing  10  gms.  of  the  resin 
(Win-3000  supplied  by  Winthrop- 
Stearns,  Inc.  1450  Broadway,  New  York 
18)  in  80  cc  10-20%  formic  acid  for  1-4 
days.  The  optimum  time  should  be 
determined  for  the  particular  kind  of 
bone  and  the  size  of  piece.  The  authors 
supply  helpful  figures  illustrating  the 
dependence  of  intranuclear  details  on 
time  of  decalcification. 
Degeneration.  Because  the  structural  or- 
ganization of  various  sorts  of  cells  is, 
like  their  function,  so  very  different 
the  types  of  degeneration  leading  to 
death  are  also  different  at  least  in  many 
of  their  aspects.  See  Nerve  Fiber 
Degeneration,  Cloudy  Swelling,  Necro- 
sis, Caseation,  Parenchymatous  Degen- 
eration, Postmortem  Changes. 


Dehydration  is  the  removal  of  water  from  a 
tissue  preliminary  to  clearing  and  paraf- 
fin or  celloidin  imbedding.  This  is 
routinely  done  by  treating  the  tissue 
after  Fixation  and  Washing  by  passing 
it  through  a  series  of  ethyl  alcohols  of 
increasing  concentration.  Usually  the 
percentages  are  30,  50,  70,  80,  95  and 
absolute.  The  time  depends  upon  the 
size  and  kind  of  the  tissue  and  the  sort 
of  fixative.  For  slices  of  tissue  less 
than  3  mm.  thick  the  dehydration  can 
be  accomplished  in  6-12  hours.  The 
alcohols  for  large  slices  fixed  say  in 
Zenker's  fluid  are  ordinarily  changed 
every  morning  and  evening,  but  it  is  not 
desirable  to  leave  them  in  absolute 
alcohol  very  long  because  it  makes  them 
brittle.  Three  to  6  hours  should  be 
sufficient.  Tissues  fixed  in  alcoholic 
solutions  take  a  shorter  time  to  de- 
hydrate. After  fixation  in  alcohol- 
formalin  or  in  Carnoy's  fluid  the  tissue 
can  be  dehydrated  and  partly  washed  in 
several  changes  of  absolute  alcohol 
skipping  the  lower  grades  of  alcohol 
entirely. 

When,  for  some  reason,  it  is  desired 
to  eliminate  treatment  with  absolute 
alcohol  the  tissues  can  be  passed  directly 
from  95%  alcohol  into  Aniline  Oil  (say 
30  min.)  which  is  itself  later  removed, 
at  least  partly,  in  5-10  minutes  by 
washing  in  2  changes  of  chloroform. 
Clearing  is  continued  in  chloroform  for 
imbedding  in  paraffin,  or  the  tissue  may 
be  passed  from  95%  alcohol,  even  from 
80%,  into  Terpineol  and  cleared  in  half 
terpineol  and  xylol.  Still  another  way 
to  avoid  absolute  alcohol  is  to  transfer 
from  95%  alcohol  to  Bergamot  Oil 
which  serves  as  a  clearing  agent. 

Several  substitutes  for  ethyl  alcohol 
as  a  dehydrating  agent  are  available. 
Acetone  is  the  best  known.  This  is 
strongljr  recommended  by  R.  D.  Lillie, 
p.  43.  Four  changes  of  acetone  each 
lasting  40  minutes  are  suggested  for 
routine  work  but  this  can  be  reduced 
to  4  changes  each  20  min.  Thereafter 
pass,  the  blocks  of  tissue  to  a  paraffin 
solvent  such  as  benzene,  toluene  or 
xylene  before  placing  in  melted  para- 
ffin. The  schedules  which  he  provides 
(p.  46)  for  dehydration,  clearing  and 
infiltrating  with  paraffin  are  useful. 
See  Cellosolve. 

Dioxan  will  not  only  take  the  place 
of  the  alcohol  but  also  that  of  the  clear- 
ing agent  so  that  it  is  possible  to  greatly' 
simplify  the  technique  and  make  the 
sequence:  fixative  to  dioxan  to  paraffin. 
See  Dioxan  and  note  as  to  possible 
danger  to  those  using  it.  Cellosolve 
has  also  been  proposed  as  a  dehydrating 
agent.     Lee  (p.  64)  says  that  it  is  ex- 


DEHYDROGENASE 


96 


DESOXYRIBONUCLEIC  ACID 


pensive,  inflammable  and  quickly  takes 
up  water  from  the  air.  Wtiether  it  is 
injurious  when  breathed  remains  to 
be  determined.  On  the  whole  it  ap- 
pears that  little  is  to  be  gained  by  such 
substitutes.  However,  Cyclohexanone 
deserves  further  trial.  If  alcohol  must 
be  avoided  it  is  always  possible  to  fix 
in  formalin  and  to  use  frozen  sections. 
By  the  Altmann-Gersh  technique  the 
tissues  are  dehydrated  in  vacuo  while 
still  frozen. 

Dehydrogenase — Written  by  E.  W.  Demp- 
sey,  Dept.  of  Anatomy,  Washington 
University,  St.  Louis.  February  26, 
1951 — Sometimes  used  synonomously 
with  Oxidase,  since  it  is  an  enzyme 
which  catalyzes  the  transfer  of  hydro- 
gen from  one  substance  to  another,  and 
therefore  operates  to  reduce  one  ma- 
terial and  simultaneously  to  oxidise 
another.  A  variety  of  dehydrogenases 
are  recognized,  depending  upon  the  sub- 
strate which  acts  as  the  hydrogen 
donor  (e.g.  succinic,  malic,  lactic 
dehydrogenase).  Dehydrogenases  have 
been  measured  in  slices  or  homogenates 
of  tissues,  particularly  with  reference 
to  malignancy  (Greenstein,  J.  P.,  Bio- 
chemistry of  Cancer,  New  York:  Aca- 
demic Press,  1947, 389  pp. )  and  to  changes 
in  the  reproductive  cycle,  a  review  of 
which  is  presented  in  Chapter  4  of 
The  Enzymes,  New  York:  Academic 
Press,  1950,  Vol.  1,  Part  1,  724  pp. 

Methylene  blue  and  other  dyes  have 
been  used  as  hydrogen  acceptors  in 
the  dehydrogenase  systems.  The  rate 
of  decolorization  of  the  dye  serves 
as  an  index  of  activity.  Recently,  tet- 
razolium  salts,  which  form  colorless 
solutions  but  which  upon  reduction 
are  converted  into  colored,  insoluble 
formazans,  have  been  used  to  localize 
the  enzymes  in  tissue  sections  or  blocks. 
Rutenberg,  A.  M.,  R.  Gofstein  and 
A.  M.  Seligman,  Cancer  Research,  1950, 
10,  113-121  present  a  review  of  the 
use  of  tetrazolium  salts  and  methods 
for  studying  both  non-specific  and  spe- 
cific dehydrogenase  systems  in  living 
tissue  and  in  frozen  sections.  These 
methods  are  still  somewhat  lacking  in 
histological  precision,  but  are  provid- 
ing interesting  data  showing  altered 
concentrations  of  the  enzyme  in  states 
of  different  physiological  activity 
(Zweifach,  B.  W.,'M.  M.  Black  and  E. 
Shorr,  Proc.  Soc.  Exp.  Biol,  and  Med., 
1950,  74,  848-854).  See  Succinic  De- 
hydrogenase and  Triphenyltetrazolium 
Chloride. 

Dehydropyridines.  Warburg  noted  a  marked 
whitish  fluorescence  in  ultraviolet  light. 
Blaschko  and  Jacobson  (Bourne,  p.  196) 
report  that  the  pyridines  do  not  show 


this  fluorescence  and  that  the  small  gran- 
ules that  exhibit  it  in  sections  of  living 
liver  tissue  may  well  be  dehydropyri- 
dines. Their  brilliant  white  fluores- 
cence quickly  fades. 

Delafield's  Alum  Hematoxylin.  To  400  cc. 
sat.  aq.  ammonia  alum  add  4  gms. 
hematoxylin  dissolved  in  25  cc.  95% 
ale.  Leave  exposed  to  air  and  light  4 
days.  Add  100  cc.  methyl  ale.  and  10 
cc.  glycerin;  filter.  Filtrate  will  slowly 
ripen.  To  hasten  ripening  add  10  cc. 
hydrogen  peroxide. 

Delta  Dye  Indicator,  see  Nitrazine. 

Dempsey,  see  Phosphatases,  Esterases,  En- 
zymes,  Dehydrogenase,  Nucleases. 

Dempster,  see  Shaddow  Casting. 

Density  Determinations.  Technique  de- 
scribed for  amebae  is  by  use  Starch 
Density  Gradient  (L0vtrup,  S.,  C.  rend. 
Lab.  Carlsberg,  S6r.  Chim.,  1950,  27, 
137-144). 

Dental  Enamel,  see  Enamel. 

Dentin.  Can  be  studied  in  ground  sections 
of  undecalcified  teeth  as  well  as  in 
paraffin  and  celloidin  sections  of  de- 
calcified ones  (see  Teeth).  For  the 
latter  Hematoxylin  and  Eosin,  Mal- 
lory's  Connective  Tissue  stain  and 
many  others  can  be  applied  as  in  the 
case  of  decalcified  bone.  Hanazawa's 
(Dent.  Cosmos,  1917,  59,  125)  methods 
for  the  minute  structure  of  dentin  are 
given  in  detail  by  Wellings,  A.  W., 
Practical  Microscopy  of  the  Teeth  and 
Associated  Parts,  London:  John  Bale, 
Sons  &  Curnow.  Ltd.,  1938,  281  pp. 
Dentin  can  be  advantageously  ex- 
amined after  vital  staining  with  Alizarin 
Red  S.  Its  pH  can  be  estimated 
(Grossman,  L.  I.,  J.  Dent.  Res.,  1940, 
19,  171-172).  For  determination  of 
rate  of  mineral  replacement  see  Radio- 
active Phosphorus;  for  Korff's  fibers, 
see  Teeth,  Developing;  and  for  nerve 
endings,  see  Teeth,  Innervation. 

Desmoglycogen,  see  Glycogen. 

Desoxyribonuclease  is  an  enzyme  for  de- 
grading desoxyribonucleic  acid.  It  has 
been  used  for  histochemical  observa- 
tions (Catheside,  D.  G.,  and  Holmes 
B.,  Symp.  Soc.  Exp.  Biol.,  No.  1, 
Nucleic  Acids,  1947,  225-231,  Cambridge 
University  Press)  to  remove  this  type 
of  nucleic  acid  from  cells. 

Desoxyribonucleic  acid.  Method  for  deter- 
mination in  Isolated  Hepatic  Nuclei — 
Written  by  Joseph  A.  Falzone,  Dept. 
of  Anatomy,  Washington  University, 
St.  Louis  10,  Mo.  October  26,  1951— 
This  technique  may  be  applied,  with 
slight  modifications  to  a  wide  variety  of 
tissues  and  organisms,  but  is  here  de- 
scribed for  use  with  rat  liver.  In  prin- 
ciple, the  nuclei  of  a  tissue  homogenate 
are  isolated    and  washed    by  centrifu- 


DESOXYRIBONUCLEIC  ACID 


97 


DESOXYRIBONUCLEIC  ACID 


gation,  counted  in  an  hemocytometer 
chamber,    and    the    Desoxyribonucleic 

Acid  (DNA)  extracted  with  hot  per- 
chloric acid  and  determined  by  the 
Dische  (diphenylamine)  reaction.  By 
this  means,  the  DNA  content  of  a 
known  number  of  nuclei  is  determined, 
making  it  possible  to  calculate  the  av- 
erage DNA  content  per  nucleus  (Boivin, 
A.,  Vendrely,  R.,  and  Vendrely,  C, 
Compt.  rend.  Acad,  sci.,  1948,  226, 
1061-1063. 

Isolation,  Counting,  and  Washing  of 
Nuclei:  Anesthetize  the  rat,  expose  ab- 
dominal and  thoracic  viscera,  clamp 
the  inferior  vena  cava  just  below  the 
liver,  sever  the  portal  vein,  and  perfuse 
the  liver  through  the  hepatic  vein  or 
thoracic  inferior  vena  cava,  using  about 
50  ml.  of  cold  normal  saline,  followed 
by  30-50  ml.  of  cold  2%  citric  acid. 
Remove  the  liver,  which  will  weigh 
about  10  gm.,  and  dissect  away  from 
it  any  adhering  diaphragmatic  and  con- 
nective tissues.  Frozen  liver  may  be 
used,  but  nuclear  yield  is  reduced,  and 
increased  cellular  debris  makes  counting 
difficult. 

The  following  steps  are  to  be  carried 
out  in  a  cold  room  at  5°C.  After  taking 
a  specimen  for  histological  control,  ho- 
mogenize remainder  of  the  liver  in  about 
50  ml.  cold  2%  citric  acid  (Cunningham, 
L.,  GrifRn,  C.,  and  Luck,  J.  M.,  Gen. 
Physiol.  1950,  34,  59-63)  using  a  Waring 
Blendor  with  metal  head  equipped  with 
an  ice-brine  cooling  jacket.  The  cold 
room  alone  does  not  prevent  heating, 
but  with  a  cooling  jacket,  the  tempera- 
ture should  not  exceed  5°C.  Homogenize 
for  5  minutes,  adding  a  few  drops  of 
capryl  alcohol  to  prevent  foaming. 

The  yield  is  50-70  ml.  of  a  20%  ho- 
mogenate,  which  is  filtered  through  four 
layers  of  cheesecloth.  Microscopic  ex- 
amination at  this  stage  reveals  a  uni- 
form suspension  of  relatively  undis- 
torted  nuclei,  without  any  lumped  or 
intact  cells. 

Nuclei  which  are  spun  down  for  analy- 
sis cannot  be  counted  directly,  due  to 
clumping,  but  are  estimated  by  first 
counting  the  whole  homogenate  then 
subtracting  from  this  value  the  numbers 
remaining  in  the  various  supernates 
after  centrifugation,  as  described  below. 
Thoroughly  shake  the  homogenate, 
withdraw  four  5  ml.  aliquots,  and  dilute 
each  with  10  ml.  2%  citric  acid.  These 
are  the  counting  dilutions,  each  of 
which  is  counted  twice  in  the  red  cell 
space  (0.1  mm.')  of  an  hemocytometer 
chamber.  Eight  to  ten  individual 
counts,  totaling  3000-5000  nuclei,  should 
keep  the  error  within  5%. 

Fifty  ml.  graduated  conical  centrifuge 


tubes  are  used  in  the  following  steps. 
Remove  four  10  ml.  homogenate  ali- 
quots (each  containing  about 
200,000,000  nuclei  as  determined  above) 
and  carefully  layer  each  over  20  ml.  of 
a  solution  containing  2%  citric  acid 
and  10%  sucrose.  Centrifuge  for  20 
min.  at  about  500  x  G.  By  use  of  this 
layering  technique  nuclei  are  both 
washed  and  isolated  in  a  single  step 
(Wilbur,  K.  M.,  and  Anderson,  N.  G.. 
Exp.  Cell  Res.  1951,  2,  47-57). 

Decant  and  keep  this  supernate.  Re- 
wash  sediment  (containing  nuclei)  in 
35  ml.  2%  citric  acid,  centrifuging  as 
above.  Combine  supernates  1  +  2  from 
each  aliquot,  measure  volumes,  and 
count  nuclei  without  diluting,  if  more 
than  10  per  chamber  (0.9  mm.^)  are 
present. 

Extraction  of  DNA:  A  recent  modi- 
fication of  the  Schneider  procedure  is 
suggested  (Schneider,  W.  C,  Hoge- 
boom,  G.  H.,  and  Ross,  H.  E.,  J.  Nat. 
Cancer  Inst.,  1950,  10,  977-982).  Ex- 
tract sediment  once  with  cold  12% 
perchloric  acid  and  twice  with  cold  6% 
perchloric  acid,  centrifuging  for  15  min. 
at  500  X  G  each  time.  This  removes 
nucleotides  and  sucrose.  Combine 
these  supernates  (3,  4,  +  5),  measure 
volumes,  and  count  nuclei  as  above, 
adding  this  value  to  that  of  supernates 
1-1-2,  and  subtracting  the  total  from 
that  for  the  whole  homogenate.  Super- 
nates 3,  4  +  5  should  not  be  mixed  with 
1  +  2  because  cytoplasmic  proteins 
in  the  latter  would  be  precipitated,  with 
clumping  of  nuclei. 

Extraction  of  phospholipids  with  ab- 
solute alcohol  may  be  omitted  in  work- 
ing with  liver  and  some  other  tissues. 
It  is  necessary  for  brain. 

Extract  sediment  with  30  ml.  6% 
perchloric  acid  at  90°C.  for  15  minutes, 
cool,  and  centrifuge.  Wash  sediment 
with  10-15  ml.  cold  6%  perchloric  acid 
and  combine  these  supernates  (6  + 
7)  as  the  DNA  extract.  This  may  be 
stored  for  24  hrs.  in  a  deep  freeze  with 
little  change  but  longer  storage  (several 
weeks)  produces  unreliable  results. 

Determination  of  DNA:  The  Dische 
reagent  should  be  prepared  with  di- 
phenylamine that  is  colorless  or  nearly 
so,  with  a  well  defined  melting  point 
at  53°C.  Otherwise  this  compound 
must  be  recrystallized  before  use. 

Dische  Blank 

Reagent  Dische 

Glacial  acetic  acid 100  ml.  100  ml. 

Concentrated  H2SO1 2.75  ml.  2.75  ml. 

Diphenylamine 1.0  gm.  — 

Following  the  Seibert  procedure  (J. 


DESOXYRIBONUCLEIC  ACID 


98 


DEUTERIUM 


Biol.  Chem.  1940,  133,  593-604)  set  up 
10  8"  X  1,   pyrex  tubes  as  below. 

1  3  ml.  DNA  extract  from  aliquot  1  +  6  ml.  blank 

Dische 

2  3  ml.  DNA  extract  from  aliquot  2  +  6  ml.  blank 

Dische 

3  3  ml.  DNA  extract  from  aliquot  3  +  6  ml.  blank 

Dische 

4  4  ml.  DNA  extract  from  aliquot  4  +  6  ml.  blank 

Dische 

5  3  ml.  DNA  extract  from  aliquot  1  +  6  ml.  Dische 

reagent 

6  3  ml.  DNA  extract  from  aliquot  2  +  6  ml.  Dische 

reagent 

7  3  ml.  DNA  extract  from  aliquot  3  +  6  ml.  Dische 

reagent 

8  3  ml.  DNA  extract  from  aliquot  4  +  6  ml.  Dische 

reagent 

9  3  ml.  6%  perchloric  acid  +  6  ml.  blank  Dische 
10    3  ml.  6%  perchloric  acid  +  6  ml.  Dische  reagent 

Heat  tubes  10  minutes  at  100°C.,  cool 
to  room  temperature  and  read  optical 
density  (D)  at  GOO  m/i.  Read  unknowns 
against  corresponding  blanks  set  at  zero, 
as  below: 


5  10 

C  D C  D —  =  Corrected  DNA  concentration  of 

1  ®  tube  5 

a  in 

Q  J) Q  J) =  Corrected  DNA  concentration  of 


tube    5  against  tube  1  as  a  blank 
tube    6  against  tube  2  as  a  blank 


tube    7  against  tube  3  as  a  blat 


(°t) 

tube    8  against  tube  4  as  a  blank  (  D —  I 

/     10\ 
tube  10  against  tube  9  as  a  blank  1  D —  I 


The  nucleic  acid  concentrations  (C) 
corresponding  to  the  five  D  readings 
are  then  obtained  from  a  standard 
curve.  The  latter  is  constructed  in 
the  usual  manner  from  at  least  20  points, 
by  subjecting  various  concentrations  of 
a  standard  DNA  to  the  Dische  pro- 
cedure and  plotting  the  resulting  D 
readings  against  these  concentrations. 
The  solvent  (O.OIN  NaOH  or  hot  6% 
perchloric  acid)  plus  Dische  reagent 
is  used  as  a  blank.  Beer's  law  should 
be  closely  obeyed  in  the  useful  range. 

The  DNA  concentration  correspond- 
ing to  the  D  reading  of  tube  10  against 

tube  9  (  C  D^  )   is  the  correction  for 

color  developed  by  the  reagents  alone 
and  is  subtracted  from  the  concentra- 
tions found  for  the  unknowns. 


tube  6 


etc. 


The  volumes  of  DNA  extracts  are 
measured  and  the  total  DNA  per  aliquot 
thus  found.  This  value  is  divided  by 
the  number  of  nuclei  per  aliquot  (ho- 
mogenate  nuclei  minus  supernate  nu- 
clei) to  obtain  the  average  DNA  content 
per  nucleus,  most  conveniently  ex- 
pressed in  fifig  (mgm.  X  10  —  9).  This 
will  usually  range  from  5-10  nixg.,  de- 
pending upon  the  purity  of  DNA  em- 
ployed in  constructing  the  standard 
curve.  The  values  of  the  four  aliquots 
should  agree  within  5%. 

If  an  efficient  means  could  be  found 
of  separating  the  various  nuclear  types 
of  a  given  tissue,  the  value  of  this 
technique  would  be  greatly  enhanced. 
A  method  for  determination  of  De- 
soxyribonucleic  Acid  in  isolated  nuclei 
of  tumor  cells  (Dounce,  A.  L.,  J. 
Biol.  Chem.,  1943,  151,  235-240). 
Same  as  DNA  and  Thymonucleic 
Acid.  The  structure  of  this  acid  in 
relation  to  the  cytochemical  significance 
of  methyl  green-pyronin  staining  is 
carefully  considered  by  Vercauteren, 
R. ,  Enzymologia,  1950, 14, 134-140.  See 
Nucleic  Acid. 

Destin's  fixative.  1%  aq.  chromic  acid,  99 
cc;  formalin,  6  cc;  glacial  acetic  acid, 
2  cc.  After  standing  for  a  few  days  it 
becomes  green  when  it  can  be  used. 

Detergents,  see  discussion  of  cutaneous 
detergents  by  Lane,  C.  G.  and  Blank, 
I.  H.,  J.A.M.A.,  1942,  118,  807-817. 
See  Aerosol. 

Deuterium  is  heavy  hydrogen.  It  is  an  iso- 
tope having  atomic  weight  of  2.0135  and 
the  symbol  H^.  Schoenheimer,  R., 
Harvey  Lectures,  1937,  32,  122-144  em- 
ployed deuterium  combined  with  oxy- 
gen as  heavy  water  H|0  to  mark  fatt}'- 
acids.  In  his  experiments  on  mice, 
held  on  a  carbohydrate  diet  plus  heavy 
water  the  fatty  acids  of  the  body  are 
replaced  by  new  fatty  acids  containing 
deuterium.  The  rate  of  replacement  of 
fatty  acids  can  therefore  be  deter- 
mined. For  further  experiments  along 
this  line  see  Symposium  on  Interme- 
diate MetaboUsm  of  Fats.  Biological 
Sj'mposia  Lancaster:  Jaques  Cattell 
Press,  1941.  Leading  references  on 
deuterium:  Cope,  O.,  Blatt,  H.  and 
Ball,  M.  R.,  J.  Clin.  Invest.,  1943,  22. 
111-115;  Flexner,  L.  B.,  Gellhorn,  A. 
and  Merrell,  M.,  J.  Biol.  Chem.,  1942, 
144,  35-40;  Stern,  K.  and  Dancey,  T.  E., 


DEUTROPLASM 


99 


CENTRIFUGATION  OF 
PARTICULATES 


Proc.  Soc.  Exp.  Biol.  &  Med.,  1941,  48, 
619-620. 

Deutoplasm,  see  Paraplasm. 

Diacetin  (glycerol  diacetate)  use  in  flatten- 
ing paraffin  sections  (Carleton,  H.  M. 
and  Leach,   E.  H.,  J.   Path.   &  Bact., 

1939,  49,  572-576). 

Diamin    Red    4B,    see    Benzopurpurin    4B. 

Diamine  Bordeaux  CGN,  see  Erie  Garnet  B. 

Diaminoacridines  have  marked  affinity  for 
nuclei  in  vivo.  They  can  be  visualized 
by  their  fluorescence  in  near  ultraviolet 
light.  Their  localization  resembles  the 
chromatin  pattern  as  revealed  by  "nu- 
clear" dyes.  These  compound  are  ap- 
parently not  toxic  because  regeneration 
of  rat  liver  cells  while  they  are  still 
within  the  nuclei  takes  place  at  the 
same  rate  as  in  controls  (DeBruyn,  R. 
S.,  Anat.  Rec,  1950,  108,  279-307). 

Di-Amino  Tri-Phenyl  Methane  Dyes.  Ex- 
amples :  brilliant  green,  fast  green  FCF, 
light  green  SF  yellowish  and  malachite 
green. 

Diamond  Green,  see  Brilliant  Green. 

Diamond  Green  B,  BX  or  P  Extra,  see 
Malachite  Green. 

Diamond  Fuchsin,  see  Basic  Fuchsin. 

Dianil  Blue  H3G,  see  Trypan  Blue. 

Dianil  Blue  2R  (CI,  265)— benzo  new  blue 
2B,  direct  steel  blue  BB,  naphthamine 
brilliant  blue  2R — Conn  (p.  63)  gives 
the  same  formula  for  this  acid  dis-azo 
dye  as  that  supplied  by  Corner,  G.  W. 
and  Hurni,  F.  H.,  Am.  J.  Physiol.,  1918, 
46,  483-486  and  Sutter,  M.,  Anat.  Rec, 
1916,  16,  164-165  for  dye  employed  by 
them  in  study  respectively  of  corpora 
lutea  and  mammary  glands  but  these 
authors  do  not  employ  the  name :  dianil 
blue. 

Dianil  Red  4B,  see  Benzopurpurin  4B. 

Dianthine  B,  see  Erythrosin,  bluish. 

Diaphane  for  mounting  Giemsa  preparations 
(Coulston,  F.,  J.  Lab.  &  Clin.  Med., 

1940,  26,  869-873). 

Diaphanol  is  according  to  Lee  (p.  598)  the 
trade  name  for  a  mixture,  formerly 
obtainable  from  Leitz,  produced  by 
passing  chlorine  dioxide  vapor  into  ice 
cold  70%  acetic  acid.  It  should  be 
fresh.  He  advises  against  attempts  to 
make  it  and  outlines  its  use  in  the  soft- 
ening of  Chitin.  Rinse  well  fixed  tissues 
in  63%  alcohol  and  transfer  them  to 
diaphanol  until  they  are  softened  and 
bleached.  If  the  diaphanol  becomes 
discolored,  repeat.  Transfer  to  63% 
alcohol,  dehydrate,  clear  in  tetralin 
(if  not  available,  benzol)  and  imbed  in 
paraffin.  See  use  of  diaphanol  in 
demonstrating  Melanins. 

Diazin  Black,  see  Janus  Black. 

Diazin  Green,  see  Janus  Green  B. 

Diazo  Reaction.  Serra,  J.  A.,  Stain  Techn., 
1946,  21,  5-18  gives  the  technique  as 


follows :  Prepare  tissue  as  described 
under  Ninhydrin  Reaction.  "Treat  the 
pieces  for  2-3  minutes  with  a  saturated 
aqueous  solution  of  sodium  carbonate; 
afterwards  add  some  drops  of  the  diazo 
reagent  and  stir  the  liquid  well.  Ob- 
serve in  glycerin.  (The  coloration  de- 
velops rapidly  and  lasts  for  some  days.) 
Preparation  of  the  diazo-reagent:  into 
a  50  ml.  flask  immersed  in  an  ice  bath, 
pour  1.5  ml.  of  a  sulphanilic  acid  solu- 
tion (dissolve  0.9  g.  of  pure  sulphanilic 
acid  in  9  ml.  of  concentrated  HCl  and 
add  water  to  100  ml.);  add  1.5  ml.  of  a 
5%  aqueous  solution  of  NaN02,  shaking 
the  flask  meanwhile.  After  5  minutes 
in  the  ice  bath  add,  also  while  shaking, 
another  6  ml.  of  nitrite.  After  5  min- 
utes fill  up  to  50  ml.  with  cooled  dis- 
tilled water.  The  reagent  must  be 
prepared  every  day  and  kept  in  the  ice 
chest. 

"The  reaction  gives  an  orange  or  yel- 
low color  with  the  histidine  and  the 
tyrosine  of  the  proteins." 

Dichlorofluorescein.  Structure  of,  Milligan, 
R.  F.  and  Hope,  F.  J.,  J.  Am.  Chem. 
Soc,  1945,  67,  1507-1508. 

Dientamoeba  fragilis.  Technique  of  stain- 
ing and  points  to  be  considered  in  diag- 
nosis (Hood,  M.,  J.  Lab.  &  Clin.  Med., 
1939-40,  25,  914-918). 

Diethylene  Dioxide  =  Dioxan. 

Diflferential  Centrifugation  of  Cell  Particu- 
lates— Written  by  Joseph  A.  Falzone, 
Department  of  Anatomy,  Washington 
University  Medical  School,  St.  Louis. 
November  27,  1951 — Since  the  pioneer- 
ing work  of  R.  R.  Bensley  and  N.  Hoerr 
(Anat.  Rec,  1934,  60,  449-455),  this 
technique  has  proven  one  of  the  most 
versatile  and  direct  in  the  armamen- 
tarium of  cytology.  In  essence  it  con- 
sists of  a  rather  drastic  mechanical 
disruption  of  large  numbers  of  cells  in 
various  media,  separation  of  the  result 
ing  particulates  by  centrifugation,  and 
determination  of  chemical  components 
and  enzymatic  activities  in  these  frac- 
tions. There  are  almost  as  many  varia- 
tions in  the  technique  as  investigators, 
each  variation  having  its  peculiar  merits 
or  limitations,  the  one  chosen  depend- 
ing upon  the  particular  cell  organ- 
elle or  chemical  data  desired. 

The  obvious  and  inherent  weakness 
of  these  methods  is  their  tendency  to 
produce  morphological  and  chemical 
artefacts.  For  example,  when  a  tissue 
is  homogenized  in  an  aqueous  medium, 
enzymes  and  other  substances  may  be 
lost  by  denaturation  or  solution,  or 
what  is  worse,  adsorbed  to  particulates 
that  never  contained  them  in  life. 
Large  particles  may  fragment  and  sedi- 
ment with  an  unrelated  small  granule 


CENTRIFUGATION  OF 
PARTICULATES 


100 


CENTRIFUGATION  OF 
PARTICULATES 


fraction.  With  nuclei,  at  least,  these  ob- 
stacles are  in  part  overcome  by  the  M. 
Behrens  technique  (Ztschr.  f.  physiol. 
Chem.,  1932,  209,  59)  by  which  tissue 
is  frozen  and  dehydrated  at  the  outset, 
fractionation  being  accomplished  in  non- 
polar  organic  media. 

The  factor  of  tonicity  is  poorly  under- 
stood. A  solution  isotonic  to  eryth- 
rocytes may  not  be  so  to  mitochondria 
or  nuclei.  Mitochondria  eventually 
swell  and  burst  in  distilled  water,  but 
appear  well  preserved  in  almost  syrupy 
sucrose  solutions.  The  review  of  W. 
C.  Schneider  and  G.  H.  Hogeboom 
(Cancer  Res.,  1951,  11,  1-22)  should  be 
consulted  in  this  respect.  Nuclei,  on 
the  other  hand,  do  not  appear  to  behave 
as  osmometers. 

Hydrogen  ion  concentration,  of 
course,  is  an  important  variable  and 
should  be  adjusted  according  to  experi- 
mental aims.  In  using  citric  acid 
media,  for  example,  we  find  a  number  of 

EH  dependent  effects.  At  pH  4  or 
elow  (2%  citric  acid)  nuclei  are  readily 
isolated  in  bulk,  morphologically  free 
of  cytoplasm,  with  no  tendency  to 
clumping,  and  with  an  apparently  com- 
plete complement  of  desoxyribonucleic 
acid  (DNA) .  At  pH  4-6,  agglutination 
of  cytoplasmic  particles  produces  nu- 
clear clumping.  At  pH  6,  nuclei  are 
again  dispersed,  and  contain,  in  addi- 
tion to  DNA,  some  enzymatic  activity. 
Above  pH  6.5,  they  disintegrate  com- 
pletely, in  contrast  to  cytoplasmic  com- 
ponents, which  are  better  preserved  in 
nearly  neutral  media  (Bounce,  A.  L., 
Ann.  N.  Y.  Acad.  Sci.,  1950,  50,  982- 
999). 

It  might  appear  that  ideal  media 
would  duplicate  the  tonicity,  pH,  and 
electrolyte  pattern  of  cytoplasm,  and 
some  attempts  in  that  direction  have 
been  made  (Wilbur,  K.  M.  and  Andre- 
son,  N.  G.,  Exp.  Cell  Res.,  1951,  2, 
47-57).  However,  the  most  versatile 
media  employed  to  date  have  been  simple 
iso-  or  hypertonic  sucrose  solutions, 
without  buffers  or  electrolytes.  These 
media,  especially  if  hypertonic,  yield 
excellent  morphological  preservation  of 
all  components,  including  nuclei,  which 
resemble  those  of  living  cells  in  their 
optical  homogeneity  (Schneider  and 
Hogeboom,  Ibid.).  One  wonders  if  this 
is  really  an  advantage  in  the  determina- 
tion of  nuclear  nucleic  acids,  where 
acid  precipitation  may  be  desirable, 
but  the  question  is  not  fully  answered. 
Bearing  on  this  problem  is  the  fact  that 
many  such  unbuffered  homogenates  are 
slightly  acid,  due  probably  to  glycoly- 
sis, but  that  liver  appears  to  be  an 


exception  in  this  respect  (Wilbur  and 
Anderson,  Ibid.). 

The  separation  of  cell  components 
by  centrifugation  has  relied  more  upon 
difference  in  particle  size,  with  resulting 
difference  in  velocity  of  fall,  than  upon 
any  variation  in  specific  gravity.  This 
would  be  expected  from  a  consideration 
of  Stokes'  Law,  which  states  that  the 
velocity  of  particle  fall  is  proportional 
to  the  square  of  its  radius,  but  only 
directly  proportional  to  the  density 
difference  between  particle  and  medium. 
Moreover,  we  are  dealing  with  semi- 
permeable or  frankly  porous  cell  parti- 
cles, whose  density  is  altered  with  that 
of  the  medium,  so  that  very  dense  (and 
correspondingly  viscous)  fluids  must 
be  employed  to  effect  separation  by 
flotation.  In  these  cases  we  may  be 
roughly  measuring  'dry  weight  densi- 
ties', as  in  the  Behrens  procedure.  An 
obvious  exception  to  these  considera- 
tions is  that  of  lipid-rich  constituents, 
which  migrate  centripetally. 

As  a  result,  whole  cells  are  commonly 
sedimented  at  very  low  speeds,  nuclei 
at  approximately  5(X)  x  g  mitochondria 
at  2000-20,000  x  g,  and  microsomes  at 
20,000  to  over  100,000  x  g.  The  "super- 
nate"  is  the  remaining  non-sediment- 
able  fraction,  and  with  the  preceding 
particulates,  completes  the  list  of 
usually  studied  components.  Others, 
such  as  chromosomes  and  melanin  gran- 
ules, will  not  be  considered  here.  The 
time  of  centrifugation  or  field  required 
varies  with  the  viscosity  of  the  medium, 
the  time  ranging  from  a  few  minutes  to 
several  hours. 

A  layering  technique,  as  emphasized 
by  Wilbur  and  Anderson  (Ibid.),  is  the 
most  efficient  means  of  centrifugation, 
as  the  mean  distance  of  particle  fall  is 
both  increased  and  nearly  equalized. 
This  naturally  has  the  effect  of  both 
isolating  and  washing  the  most  rapidly 
sedimenting  fraction  at  a  single  step. 
From  this  and  the  above  considerations 
it  is  obvious  that  a  more  efficient  centri- 
fuge would  be  one  constructed  to  hold 
longer  tubes.  With  such  an  instru- 
ment, employing  tubes  only  double  the 
length  of  standard  models,  an  even 
further  fractionation  of  particulates 
might  be  achieved,  with  diminished  loss 
due  to  washing.  Separation  of  nuclear 
types  suggests  itself,  and  some  en- 
deavors in  that  direction  are  promising 
(Marshak,  A.,  Cancer  Res.,  1950,  10, 
232). 

Below  is  a  brief  outline  of  some  of 
the  more  popular  and  useful  techniques, 
listed  according  to  homogenizing  fluids 
employed.  Centrifuging  media  are  us- 
ually similar  to  these,  or  made  slightly 


CENTRIFUGATION  OF 
PARTICULATES 


101 


DIGITONINE 


denser  by  addition  of  sucrose,  if  layer- 
ing is  employed.  Temperature  should 
be  kept  at  5°C.  or  below  throughout. 
No  attempt  at  completeness  has  been 
made,  and  some  interesting  methods, 
including  ultrasonic  and  enzymatic  cell 
disruption,  are  omitted. 

1)  Citric  acid,  low  pH  (Bounce,  Ibid; 
Cunningham,  L.,  Griffin,  A.  C.  and 
Luck,  J.  M.,  J.  Gen.  Physiol.,  1950, 
34,  59-63). 

Procedure:  Tissue  homogenized  sev- 
eral minutes  in  cold  2%  citric  acid  in 
a  Waring  Blendor.  The  latter  should 
be  equipped  with  a  cooling  jacket. 

Particulates  obtained:  Nuclei. 

Morphology:  Reasonably  good. 
Nuclei  plump  and  unwrinkled,  but 
with  fine  internal  granulation.  No 
clumping  or  cytoplasmic  tags. 

Value:  Simplicity  and  use  of  large 
volumes  of  tissue,  if  desired.  Nuclei 
readily  counted  and  DNA  per  nucleus 
determined  by  the  Dische  or  other 
suitable  analytical  technique.  The 
method  of  choice  for  this  purpose, 
but  of  little  other  value. 

Limitations:  Water  soluble  sub- 
stances, including  much  protein  and 
probably  RNA,  are  lost;  enzymes 
inactivated. 

2)  Citric  acid,  controlled  pH 
(Bounce,  Ibid.). 

Procedure:  Enough  O.IM  citric  acid 
is  added  dropwise  to  partially  homog- 
enized tissue  in  ice  water  to  give  a 
pH  of  6.0.  Used  with  a  low  speed 
Waring  Blendor  or  glass  homogenizer. 

Particulates  obtained:  Nuclei. 

Morphology:  Similar  to  procedure  1. 

Value:  Suitable  for  BNA  and  many 
enzyme  studies. 

Limitations:  Similar  to  1,  except 
that  apoenzj'mes  are  retained.  Co- 
enzymes must  often  be  added  in  de- 
terminations of  their  activities,  how- 
ever. Adsorption  effects  must  be 
controlled  by  repeated  washing  of  nu- 
clei. 

3)  Sucrose,  near  pH  7  (Schneider  and 
Hogeboom,  Ibid.). 

Procedure:  Small  amounts  of  tissue 
homogenized  by  ground  glass  appara- 
tus or  other  "gentle"  mechanical 
means,  in  a  medium  of  neutral  but 
unbuffered  iso-  or  hypertonic  (usually 
0.88  M)  sucrose.  iProbably  best  to 
avoid  NaCl  and  other  electrolytes  for 
general  purposes. 

Particulates  obtained:  Nuclei,  mito- 
chondria, microsomes,  supernate. 
Also  melanin  granules  and  particulate 
glycogen. 

Morphology:  Excellent,  with  pres- 
ervation of  all  particulates  in  a 
form  resembling  that  in  living  cells. 


Mitochondria  filamentous,  at  least 
in  hypertonic  sucrose,  and  stainable 
with  Janus  Green  B;  nuclei  homo- 
geneous with  several  nucleoli. 

Value:  Versatility,  due  to  the  large 
number  of  fractions  obtained.  Suit- 
able for  determinations  of  enzymes, 
nucleic  acids,  proteins  and  lipids  of 
these  fractions.  The  method  of 
choice  for  microspectrophotometry  of 
stained  nuclei,  as  optical  homogeneity 
is  a  definite  prerequisite  for  valid 
results  with  this  method. 

Limitations:  Solution  and  adsorp- 
tion effects  again  come  into  play,  but 
many  artefacts  can  be  partially  ruled 
out.  Nuclei  probably  not  so  readily 
obtained  in  bulk  as  with  methods  1) 
and  2). 

4)  Behrens  technique  (Behrens, 
Bounce;  Schneider  and  Hogeboom, 
Ibid.). 

Procedure:  Tissue  frozen,  dried  in 
vacuo,  ground  in  ball  mill  to  disrupt 
cells,  and  centrifuged  in  non-aqueous 
media,  usually  hydrocarbons. 
Particulates  obtained:  Nuclei. 
Morphology:  More  seriously  dis- 
torted than  with  any  other  proce- 
dure; nuclear  membranes  wrinkled. 
Contamination  with  cytoplasm  must 
be  appraised  from  smears  stained 
with  both  acid  and  basic  dyes. 

Value:  Probably  the  most  widely 
applicable  method  for  nuclei.  Pro- 
cedure of  choice  for  water  soluble 
substances,  i.e.,  coenzymes,  vitamins, 
free  amino  acids,  etc.  Results  with 
total  protein  and  RNA  also  appear 
the  most  reliable  of  any  method. 
Suitable  also  for  BNA  and  many 
enzyme  studies. 

Limitations:  Procedure  is  laborious. 
Lipids,  lipases,  and  many  other  en- 
zymes removed  or  destroyed. 
This  brief  summary  can  only  suggest 
the  variety  of  applications  of  cell  frac- 
tionation to  cytology,  as  new  ones  are 
continauUy  uncovered.     For  example, 
B.  L.  Opdyke  has  recently  described  a 
valuable  technique  for  the  isolation  of 
keratohyaline    granules    of    epidermis, 
using    isotonic    saline    and    a    Waring 
Blendor  for  homogenization.     The  par- 
ticles are  sedimented  at  25,000  x  g  and 
appear  to  have  all  the  morphological 
and   staining   characteristics    of    these 
granules  (See  Keratohyaline  Granules, 
procedure  for  isolation). 
Differential     Leucocyte    Count,    statistical 
study  of  uniformity  in    (Klotz,  L.  F., 
J.  Lab.  &  Clin.  Med.,  1939,  25,  424^34). 
Diffraction  Methods  for  measuring  diameter 
of   red   blood   cells    (Haden,   R.   L.,  J. 
Lab.  &  Clin.  Med.,  1937-38,  23,  508-518). 
Digitonine    reaction   of    Winaaus    for  free 


DILATOMETRIC  TECHNIQUES 


102 


DIRECT  GARNET  R 


cholesterol.  This  has  been  adapted  to 
histochemical  use  by  Brunswick  and 
by  Leulier  and  Noel  (A.,  and  R.,  Bull. 
d'Hist.  Appl.,  1926,  3,  316-319).  Lison 
(p.  211)  recommends  a  slight  change. 
Immerse  frozen  sections  of  formalin 
fixed  tissue  in  0.5%  digitonine  in  50% 
ale.  for  several  hrs.  Rinse  in  50%  ale, 
then  in  water  and  mount  in  Apathy's 
syrup    or    glycerin     gelatin.  With 

crossed  nicols  (polarizing  microscope) 
one  observes  appearance  of  needles  or 
rosettes  of  the  complex  cholesterol- 
digitonide.  To  resolve  this  complex 
stain  with  sudan.  The  esters  will  color 
and  lose  their  birefringence  while  the 
cholesterol  will  remain  uncolored  and 
retain  birefringence. 

Dilatometric  Techniques.  These  have  been 
adjusted  so  that  they  will  give  the  fine 
quantitative  measurements  needed  in 
histochemistry  by  Linderstr0m-Lang,  K 
Nature,  1937,  139,  713.  He  reported 
especially  ultra-microestimations  of 
peptidase  activity. 

Di-Nitrosoresorcinol  test  for  iron,  see  Iron. 

Diodrast,  trade  name  for  an  organic  iodine 
preparation  recommended  by  Gross,  S. 
W.  (Proc.  Soc.  Exp.  Biol.  &  Med., 
1939,  42,  258-259)  for  injection  into  com- 
mon carotid  with  later  x-ray  photo- 
graphs of  the  vascular  tree.  For  visual- 
ization of  ventricular  chambers  and 
large  arteries  see  method  of  Ponsdome- 
nech,  E.  R.  and  Nunez,  V.  B.,  Am. 
Heart  J.    1951,  41,  643-651. 

Dioxan  is  diethylene  dioxide.  It  mixes 
with  water,  ethyl  alcohol,  many  clearing 
agents  and  paraffin  (slightly).  McClung 
(p.  39)  recommends  its  use  to  replace 
ordinary  agents  like  xylol.  Dioxan 
fumes  are  said  to  be  dangerous  to 
laboratory  workers  so  that  it  should  be 
used  under  a  hood  or  in  a  well  ventilated 
room  with  container  covered  when  not 
in  use  (Magruder,  S.  R.,  J.  Lab.  &  Clin. 
Med.,  1937-38,  23,  405-411). 

For  fixation  the  following  mixtures 
are  recommended  (McClung,  p.  39) : 
(1)  Sat.  aq.  picric  acid,  5  parts;  glacial 
acetic,  1  part;  dioxan,  4  parts.  (2) 
Sat.  picric  acid  in  dioxan ,  4  parts ;  glacial 
acetic,  1  part;  absolute  alcohol,  4  parts. 
Graupner  and  Weissberger  (von  H.  and 
A.,  Zool.  Anz.,  1933,  102," 39-44)  suggest: 
dioxan  80%,  methyl  alcohol  20%,  paral- 
dehyde 2%,  and  acetic  acid  5%.  See 
Clearing,  Pituitary.  See  as  ingredient 
of  Lison's  gh'cogen  method;  also  dioxan 
imbedding  of  Pituitary. 

A  method  for  the  dehydration,  puri- 
fication and  clarification  of  dioxan  so 
that  its  use  in  tissue  technique  can  be 
continued  has  been  described  by  Hall, 
W.  E.  B.,  Am.  J.  Clin.  Path.,  1943,  7 
(Technical  Section),  98-100. 


Dipeptidase  can  be  localized  in  chief  cells 
of  stomach.  See  review  of  methods 
(Gersh,  I.,  Physiol.  Rev.,  1941,  21, 
242-266). 

Di-Phenyl  Methane  Dyes.  Of  these  only 
auramin  need  be  referred  to. 

Diphosphopyridine  Nucleotide,  see  tech- 
nique of  Anfinsen,  C.  B.,  J.  Biol.  Chem., 
1944,  152,  285-291. 

Diphtheria    Bacilli.  1.  Neisser's    stain 

(Stitt,  p.  863).  A  =  methylene  blue, 
0.1  gm.;  95%  ale,  2  cc;  glacial  acetic 
acid,  5  cc;  aq.  dest.,  95  cc.  B  = 
Bismark  brown,  0.2  gm. ;  aq.  dest.  (boil- 
ing) 100  cc.  Dissolve  and  filter.  To 
stain  smear  pour  on  A ,  30-60  sec.  Wash. 
Then  B,  30  sec.  Wash  in  water,  dry 
and  mount.  Bacilli  brown  with  dark 
blue  dots  at  either  end.  Better  results 
can  be  secured  by  adding  1  part  of 
crystal  violet  (Hoechst)  1  gm.;  95% 
ale,  10  cc;  aq.  dest.,  300  cc.  to  2  parts 
of  A  before  using.  Chrysoidin  1  gm. 
in  hot  aq.  dest.  300  cc.  is  more  satis- 
factory counterstain  than  Bismark 
brown.  Most  American  brands  of  crys- 
tal violet  are  satisfactory. 

2.  Pander's  stain  (Stitt,  p.  863). 
Toluidin  blue  (Grubler)  0.02  gm.;  aq. 
dest.,  100  cc;  glacial  acetic  acid,  1  cc ; 
abs.  ale,  2  cc  Add  small  amount  to 
fixed  film  on  cover  glass.  Invert  and 
mount  on  slide.  Diphtheria  bacilli 
recognizable  by  metachromatic  granules 
intensely  stained,  diphtheroids  by  their 
strong  color  in  contrast  with  ordinary 
cocci  and  bacilli  the  bodies  of  which 
are  only  faintly  blue. 

3.  Laybourn's  modification  of  Albert's 
stain  (Stitt,  p.  863).  A  =  toluidin 
blue,  0.15  gm. ;  malachite  green,  0.2  gm. ; 
glacial  acetic  acid,  1  cc. ;  95%  ale,  2 
cc,  aq.  dest.,  100  cc.  B  =  iodine 
crystals,  2  gm.;  potassium  iodide,  3 
gm.;  aq.  dest.,  300  cc.  Let  both  stand 
24  hrs.  and  use  filtrate.  Apply  A  to 
heat  fixed  smears  3-5  min.  Wash  in 
water.  Apply  B  for  1  min.  Wash, 
blot  and  dry.  Granules  of  diphtheria 
bacilli,  black;  bars,  dark  green;  inter- 
mediate parts,  light  green  and  all  three 
in  sharp  contrast. 

Diplosome,  a  double  centrosome. 

Direct  Black,  see  Chlorazol  Black  E. 

Direct  Fast  Orange  (CI,  326)~Erie  Fast 
Orange  (NAC),  Erie  Fast  Scarlet  YA 
(NAC) — a  direct  disazo  dye  of  light 
fastness  3  (Emig,  p.  38). 

Direct  Fast  Scarlet  4  BS  (CI,  327)— Pont- 
amine  Fast  Scarlet  4  BS  of  DuPont — ,  a 
disazo  direct  dye  of  light  fastness  3,  can 
be  employed  instead  of  carmine  as  a 
general  stain.  Details  of  use  in  study 
of  plant  and  animal  tissues  are  de- 
scribed (Emig,  p.  38}. 

Direct  Garnet  R,  see  Erie  Garnet  B. 


DIRECT  GREEN  B 


103 


DUCTS 


Direct  Green  B  (CI,  593)— Diazine  Green 
B — a  direct  disazo  dye  of  light  fastness 
3  to  4.  Recommended  as  counterstain 
for  Crocein  Scarlet  7  B  of  invertebrates 
or  paraffin  sections,  time  5  min.  (Emig, 
p.  43). 

Direct  Green  G  (CI,  594)— Alkali  Green  D— 
a  direct  disazo  dye  of  light  fastness  3  to 
4.  Formula  for  blue  green  algae  and 
whole  mounts  is  given  (Emig,  p.  43). 

Direct  Red  4B,  see  Benzopurpurin  4B. 

Direct  Red,  C,  R,  or  Y,  see  Congo  Red. 

Direct  Sky  Blue,  see  Niagara  Blue  4B. 

Direct  Steel  Blue  BB,  see  Dianil  Blue  2R. 

Direct  Violet  B,  see  Azo  Blue. 

Direct  Violet  C,  see  Erie  Garnet  B. 

Dis-Azo  Dyes.  Azo  blue,  benzopurpurin 
4B,  Biebrich  scarlet,  Bismark  brown 
Y  and  R,  brilliant  purpurin  R,  Congo 
red,  dianil  blue  2R,  Erie  garnet  B, 
Niagara  blue  4B,  orseillin,  trypan  blue, 
trypan  red,  sudan  III,  sudan  IV, 
vital  new  red,  vital  red,  etc. 

Dissociation,  see  Maceration. 

Distrene  80  is  a  polysterene  which  forms  a 
water  clear  solution  in  xylol.  It  is 
recommended  by  Kirkpatrick  and  Len- 
drum  (J.  and  A.  C,  J.  Path,  and  Bact., 
1939,  49,  592-594)  as  a  naounting  medium 
giving  good  preservation  of  color  in 
microscopic  slides.  See  also  Kirk- 
patrick, J.  and  Lendrum,  A.  C,  J.  Path. 
&  Bact.,  1941,  53,  441. 

Dominici's  Stain,  see  Eosin-Orange  G  and 
Toluidin  blue. 

Donaldson's  lodine-Eosin  Method,  see  lo- 
dine-Eosin. 

Dopa,  Oxidase  Reaction  for  Melanoblasts 
(Laidlaw,  G.  F.,  Anat.  Rec,  1932,  53, 
399-407).  Dopa  is  short  for  3.4-dihy- 
droxyphenylalanin,  a  substance  which 
when  applied  in  a  certain  way  picks  out 
the  melanoblasts  by  blackening  them. 
Use  frozen  sections  of  fresh  material  or 
of  tissues  fixed  2  to  3  hours  but  not 
longer  in  6%  formalin.  Rinse  4  or  5 
seconds  in  aq.  dest.  and  immerse  in 
buffered  dopa.  (To  make  dopa  stock 
solution  dissolve  0.3  gm.  dopa  powder- 
manufactured  by  Hoffmann-La  Roche, 
Nutley,  New  Jersey— in  300  cc.  cold 
aq.  dest.  Keep  in  refrigerator  and  dis- 
card when  solution  becomes  dark  red. 
To  make  buffers  dissolve  11.87  gms.  di- 
sodium  hydrogen  phosphate  (Na2HP04 
+  2H2O) — or  what  would  be  better 
9.47  gm.  anhydrous  Na2HP04— in  1000 
cc.  aq.  dest.  and  9.08  gms.  anhydrous 
potassium  dihydrogen  phosphate 
(KH2PO4)  in  an  equal  amount  aq.  dest. 
Immediately  before  use  buffer  to  pH  7.4 
by  adding  2  cc.  potassium  phosphate 
solution,  and  6  cc.  sodium  phosphate 
solution  to  25  cc.  dopa  solution).  The 
reaction  is  slow  for  3-4  hours  at  room 
temperature.       If     solution     becomes 


sepia  brown  it  is  likely  to  overstain. 
Observe  under  microscope.  Wash  in 
aq.  dest.,  dehydrate  and  counterstain 
if  desired  with  alcoholic  crystal  violet, 
clear  and  mount  in  balsam.  Melan- 
oblasts should  be  black. 

This  much  used  method  has  been 
criticized  by  11.  Sliarlit  et  al.  (Arch. 
Dermat.  and  Syph.,  1942,  45,  103-111) 
chiefly  on  the  ground  that  the  incuba- 
tion for  3  hrs.  at  room  temperature  may 
itself  increase  the  amount  of  melanin 
present  which  happened  in  their  ex- 
perience at  37  °C.  See  also  remarks 
by  Blaschko  and  Jacobson  (Bourne, 
p.  198)  on  specificity  of  the  reaction. 
It  is  given  by  phenoloxidases  but  thus 
far  they  have  not  been  found  in  mam- 
malian tissues. 

Dorothy  Reed  Cells,  see  Reed-Sternberg 
Cells. 

Double  Green,  see  Methyl  Green. 

Double  Imbedding.  To  facilitate  section 
cutting  by  making  a  celloidin  block 
firmer,  harden  first  in  chloroform  vapor, 
then  in  chloroform,  transfer  to  benzol 
until  it  becomes  transparent  and  in- 
filtrate with  38°C.  paraffin  (Lee,  p. 
104).     See  Fleas, 

Another  method  of  double  imbedding 
is  that  of  Peterfi  (T.,  Zeit.  f.  wiss. 
mikr.,  1921,  38,  342-345).  As  employed 
in  this  laboratory  it  is  as  follows  :  Make 
1%  and  3%  solutions  of  celloidin  in 
methyl  benzoate  which  take  about  a 
month.  Foursome  1%  into  a  dish.  Add 
absolute  alcohol  containing  the  tissue 
which  gradually  sinks  down  into  the 
celloidin.  Transfer  tissue  to  3%  solu- 
tion, 48-96  hrs.  Drop  tissue  directly 
into  benzol  for  a  few  hrs.  Then  infiltrate 
and  imbed  in  40  °C.  paraffin  about 
12-24  hrs. 

Double  Scarlet  BSF,  see  Biebrich  Scarlet, 
water  soluble. 

Downey's  Fluid,  see  Megakaryocytes. 

Ducts.  These  structures  lead  (L.  ducere) 
the  products  of  glands  to  the  site  of 
discharge.  They  are  of  considerable 
variety.  Ordinarily  they  are  easily 
identified  by  their  morphology  in  hema- 
toxylin and  eosin  preparations.  But 
special  techniques  are  required  for  their 
visualization  in  whole  mounts  of  some 
glands. 

In  the  pancreas  for  example  the 
system  of  small  ducts  (ductules)  can 
easily  be  demonstrated  by  perfusion  of 
the  pancreas  with  pyronin — one  of  the 
many  methods  discovered  by  R.  R. 
Bensley.  Proceed  as  described  under 
Perfusion  using  a  solution  made  up  by 
adding  10  cc.  of  1%  aq.  pyronin  to  1000 
cc.  0.85%  aq.  sodium  chloride.  When 
the  pancreas  has  assumed  a  rose  red 
color  the  optimum  intensity  of  which 


DUGGINS 


104 


DUST  CELLS 


must  be  determined  by  trials,  remove  a 
piece  of  it,  tease  out  a  small  lobule  and 
examine  under  low  power  mounted  in 
0.85%  aq.  sodium  chloride.  The  com- 
plicated system  of  ducts  should  be 
sharply  delineated  by  their  deep  rose 
red  color  in  an  almost  colorless 
background.  If  there  is  any  question  of 
their  identification  examine  the  original 
figures  of  Bensley,  R.  R.,  Am.  J.  Anat., 
1911,  12,  297-388.  A  double  staining  of 
ducts  and  Islets  of  Langerhans  can  be 
obtained  by  perfusing  in  the  same  way 
with  pyronin  solution  to  1000  cc.  of 
which  6  cc.  1%  aq.  neutral  red  has  been 
added.  The  islets  appear  yellow  red  in 
contrast  to  the  rose  red  ductules.  See, 
in  addition,  ducts  in  whole  mounts  of 
Mammary  Glands  and  in  sections  of 
Submaxillary  Glands  which  are  of  par- 
ticular interest  in  detecting  the  action  of 
salivary  gland  viruses. 

Duggins,  see  Hairs 

Duodenal  Fluid.  Microscopic  study  must 
be  prompt  because  of  the  presence  of 
cytolytic  engymes.  Examine  sediment 
after  centrifugal  concentration  as  in  the 
case  of  urinary  sediments.  Epithelial 
cells  from  the  entire  alimentary  tract 
leading  to  and  including  the  duodenum 
may  be  present,  generally  bile  stained, 
also  a  few  neutrophiles.  A  great  in- 
crease in  both  or  either  may  indicate 
inflammatory  lesions.  A  polarizing  mi- 
croscope is  helpful,  but  not  essential, 
in  recognizing  cholesterol  crystals  as 
thin,  flat,  colorless  fragments  with 
chipped  edges.  The  more  irregular  the 
crystals  the  more  significant  they  are 
of  calculi  formation.  Bilirubin  is  easily 
detected  as  amorphous  amber,  brown  or 
black  material  and  calcium  bilirubinate 
as  bright  yellow  granular  deposits. 
See  Gentzkow  and  Van  Auken  in  Sim- 
mons and  Gentzkow,  p.  63. 

Duodenal  Drainage  Smears,  see  Papani- 
colaou Techniques. 

Duran-Reynals,  see  Spreading  Factors. 

Dust  Cells  of  the  Lungs— Written  by  C.  C. 
Macklin,  Dept.  of  Histological  Re- 
search, The  University  of  Western  On- 
tario, London,  Canada.  November  28, 
1951 — These  phagocytes  develop  in  the 
alveolar  walls  of  the  lungs  of  man  and 
other  mammals  from  the  primitive 
pneumonocy tes  to  arrest  particles  of  car- 
bon and  other  particulate  matter  which 
elude  the  apprehensional  mechanism  of 
the  respiratory  tract;  and  to  dispose  of 
them  by  conveying  them  to  the  ciliary 
escalator  which  evacuates  them.  Col- 
lapse of  the  lungs  dislodges  them  from 
their  normal  position  in  the  alveolar 
wall  (Macklin,  C.  C,  Lancet,  Feb.  24, 
1951,  432-435)  where  they  are  wedged 
in    between    capillaries,    whence    they 


derive  their  nutriment.  Thus  the  ordi- 
nary human  lung  material  available 
to  pathologists  is  unfavorable  in  that 
it  shows  phagocytes  lying  loose  which 
normally  are  tethered.  If  the  skin  and 
outer  musculature  be  removed  from  a 
fresh  intact  thorax  of  a  small  animal 
such  as  the  mouse  and  it  be  at  once  im- 
mersed in  a  fixing  fluid  such  as  Regaud 
or  Bouin's,  the  lung  is  quickly  fixed  in 
its  normal  uncollapsed  condition.  Pen- 
etration is  facilitated  by  exposing  the 
lower  surface  of  the  diaphragm,  but  this 
must  not  be  breached.  Sections  from 
such  material  show  as  many  as  99%  or 
more  of  the  dust  cells  morphologically 
affixed  to  the  alveolar  walls,  while  only 
1%  or  less  are  free,  and  these  are  re- 
garded as  spent  and  on  their  way  to 
the  exterior.  If,  however,  fresh  normal 
mouse  lungs  are  collapsed  and  promptly 
filled  with  fixing  fluid  via  the  trachea, 
and  cut  into  sections,  it  is  found  that 
only  about  5%  of  the  dust  cells  remains 
fixed  in  the  alveolar  walls,  the  re- 
mainder now  appearing  free.  An  er- 
roneous picture  of  the  relation  of  the 
dust  cells  to  the  alveolar  walls  is  thus 
presented.  That  collapse  alone  dis- 
lodges many  of  the  phagocytes  formerly 
affixed  in  the  alveolar  walls  is  shown  by 
finding  undue  numbers  of  free  dust  cells 
in  sections  from  pieces  of  lung  tissue 
which  have  been  fixed  by  immersion. 
For  larger  lungs  the  method  of  perfusion 
of  fixing  solution  through  the  pulmo- 
narvvasculature  of  the  unopened  thorax 
(Hartroft,  W.  S.,  Anat.  Rec,  1942,  82, 
419)  also  showed  the  majority  of  alveo- 
lar phagocytes  affixed  to  the  walls  rather 
than  free.  Thus  dust  cells  are  funda- 
mentally mural  phagocytes,  and  are 
regarded  as  originated  ultimately  from 
endodermal  epithelium  (Macklin,  C.  C, 
Trans.  Roy  Soc.  of  Canada,  Sect.  V, 
1946,  40,  93-111) .  The  presence  of  fluid 
in  the  alveoli  favors  a  shedding  of  mural 
phagocytes  from  their  walls.  No  ev- 
dence  of  dust  cell  entry  into  the  con- 
nective tissue  or  lymphatic  channels 
of  the  lung  was  found,  contrary  to  the 
opinion  of  many  pathologists.  On  the 
other  hand,  dust  cells  are  often  found 
stuck  in  the  mucus  overlying  the  epi- 
thelium of  the  bronchioli  and  bronchi, 
and  these  are  probably  wornout  cells 
being  carried  to  the  glottis  by  ciliary 
movement  (Macklin,  C.  C,  Can.  J. 
of  Res.,  D,  1949,  27,  50-58;  Macklin, 
C.  C,  Proc.  Instit.  of  Med.  of  Chicago, 
1950,  18,  78-95).  They  are  recoverable 
from  sputum,  and  then  have  been  re- 
ferred to  by  the  unsuitable  term  "heart 
failure  cells"  (which  see). 

In  the  mouse  most  alveolar  phago- 
cytes are  in  alveolar  angles  and  have 


DYES 


105 


EAR 


processes  inserted  into  tunnels  encircled 
by  capillary  loops;  but  a  lesser  number, 
resting  on  connective  tissue,  occupy 
sockets.  After  brief  treatment  of  fresh 
lungs  with  ammoniacal  silver  solution, 
many  of  the  dust  cells  are  blackened 
and  their  mode  of  insertion  into  mural 
vaginae  are  made  clear  (Macklin,  C.  C, 
Anat.  Rec,  194S,  100,  693).  After  with- 
drawal of  such  processes  these  vaginae 
or  tunnels,  now  vacated,  have  become 
pores  (Macklin,  C.  C,  J.  of  Thor. 
Surg.,  1938,  7,  536-551).  These  mural 
phagocytes  may  have  as  many  as  four 
functional  air  faces  or  particle  receptors 
of  varying  area.  These  often  conform 
to  the  contour  of  the  alveolar  wall, 
but,  in  presumably  active  cells,  project 
into  the  air  space.  The  edges  may 
extend  upon  adjacent  capillaries  and 
have  been  mistaken  for  respiratory 
squames  by  some  workers.  They  are 
sister  cells  of  the  alveolar  granular 
cells  (septal  cells,  niche  cells,  etc.)  and 
the  term  "pneumonocyte"  (which  see) 
includes  both  types.  Dust  cells  may  be 
recovered  from  fresh  lungs  by  the 
"gash-irrigation"  and  "wash-out" 
methods  (which  see),  and  studied  in 
fresh  mounts  or  after  being  dried  and 
stained  like  a  blood  smear.  Mito- 
chondria are  scarce  or  absent  in  them. 
One  hour  after  e.xposure  to  carmine 
particles  in  the  inspired  air  they  con- 
tain much  carmine,  which  is  almost 
completely  absent  on  the  remaining 
alveolar  wall  surface,  and  almost  100% 
are  inserted  into  the  walls.  One  week 
after  such  dusting,  however,  only  35% 
are  mural  phagocytes,  the  rest  being 
free.  Since  dust  cells  are  constantly 
being  lost  to  the  body  they  must  be 
replaced  correspondingly  by  the  mul- 
tiplication of  sister  cells  remaining  in 
the  walls.  Dust  cells  often  show  bi- 
zarre shapes  and  a  common  one  is  that 
of  a  dumb-bell  in  which  the  middle 
narrow  bridge  occupies  a  pore.  Such 
cells  are  probably  not  in  transit  from 
one  avleolus  to  another. 

Histocj^tes  of  the  lung  connective 
tissue  often  pick  up  dust  particles  which 
have  passed  the  inefficient  surface  guards 
of  the  broncho-alveolar  system.  These 
are  regarded  as  quite  different  from  the 
endodermal  alveolar  phagocytes. 
Great  amounts  of  such  aberrant  dust 
may  accumulate  and  may  give  rise  to 
disease  processes. 

Dyes,  see  "standardization  of  Stains"  pp. 
xxvii-xxx  of  this  book  and  Staining. 

Dysentery,  see  Endamoeba. 

Dysprosium,  see  Atomic  Weights. 

Ear— Written  by  W.  P.  Covell,  Dept.  of 
Anatomy,  Washington  University,  St. 
Louis  10,  Mo.  October  26,  1951— Micro- 


scopic examinations  of  the  ear  are 
nearly  always  made  on  sections.  This 
is  understandable,  but  it  is  possible  that 
the  study  of  still  living  tissues,  removed 
by  careful  and  minute  dissections,  is  a 
field  of  considerable  promise.  The  close 
apposition  of  epithelial  and  nervous 
components  to  bone  necessitates  de- 
calcification except  in  the  case  of  young 
embryos.  The  frequent  use  of  celloidin 
in  place  of  paraffin  for  imbedding  is 
occasioned  bj^  the  wide  range  of  diver- 
sity in  resistance  of  the  organ  to  the 
microtome  knife,  fluid  containing 
lumina  being  surrounded  bj'  hard  dense 
bone.  The  histological  techniques 
actually  in  use  for  the  ear  are  fewer  in 
number  and  more  limited  in  range  than 
those  employed  for  most  other  parts  of 
the  body.  The  difficulty  experienced 
in  obtaining  fresh  and  normal  adult 
specimens  has  turned  investigation 
toward  human  fetuses  and  the  ears  of 
experimental  animals. 

The  commonly  used  fixatives  are 
either  Zenker's  fluid,  with  or  without 
acetic  acid,  Zenker-formol,  10%  forma- 
lin, Wittmaack's  solution  and  Heiden- 
hain-Susa.  Mygind,  S.  H.  and  cowork- 
ers (Acta  oto-laryng.,  1945,  33,  273- 
280)  prefer  the  Wittmaack's  for  fixation 
of  hair  cells  and  use  the  latter  for 
study  of  such  structures  as  the  stria 
vascularis.  The  best  results  are  to  be 
obtained  by  the  use  of  animal  material 
for  which  prompt  fixation  by  perfusion 
methods  has  been  done.  Kristensen, 
H.  K.  (Acta  oto-laryng.,  1945,  33.  225- 
244)  recommends  the  use  of  a  6%  gum 
arable  in  physiologic  saline  for  intravital 
perfusion  to  precede  the  fixative.  Iso- 
lated temporal  bones  placed  in  fixative 
are  prone  to  show  autolytic  changes 
in  end  organs  and  ganglion  cells  in 
one-half  hour  following  death.  The  lit- 
erature is  filled  with  autolytic  changes 
described  as  specific  pathologic  altera- 
tions due  to  drugs,  toxins,  poisons,  and 
so  forth.  Actually  many  of  these  are 
the  result  of  poor  penetration  of  fixa- 
tives and  elapse  of  time  between  au- 
topsy and  fixation.  In  an  attempt  to 
overcome  the  slow  penetration  of  a  fixa- 
tive and  prevention  of  artefacts  Guild 
made  use  of  water  from  which  gases 
had  been  exhausted  as  the  medium  for 
fixatives,  decalcifying  solutions  and  al- 
cohols. 

A  variety  of  decalcificants  have  been 
used  with  the  view  to  preserving  finer 
cell  structures  of  the  soft  tissues,  viz: 
Formic,  trichloracetic,  and  trichlora- 
lactic  acids,  long  immersion  in  MuUers' 
fluid  and  so  forth.  Nitric  acid  in  2  to 
5%  concentration  is  generally  used  for 
human  temporal  bones.     The  lower  con- 


EAR 


106 


EAR 


centration  while  it  takes  longer  to  act 
is  less  likely  to  overdecalcify.  A  few 
investigators  recommend  the  use  of  3% 
nitric  acid  in  water  and  a  constant  tem- 
perature (37^°C.)  to  hasten  the  proce- 
dure. Degree  of  decalcification  is  us- 
ually judged  by  probing  with  a  needle, 
or  a  simple  test  with  an  indicator  such 
as  phenol  red.  Most  small  animal 
bones  are  decalcified  in  about  4  to  10 
days.  However,  human  temporal 
bones  vary  considerably  and  may  take 
as  long  as  6  to  8  weeks  with  weekly 
change  of  the  solution.  After  decalci- 
fication, thorough  washing  for  24  hours 
in  running  water  is  necessary  following 
which  neutralization  in  5%  sodium  sul- 
fate may  be  used  and  washing  in  run- 
ning water  repeated. 

In  order  to  preserve  cytologic  detail 
attempts  have  been  made  to  circumvent 
decalcification  in  strong  acids.  Small 
laboratory  animals  may  be  perfused 
with  Regaud's  solution  and  following 
fixation  mordanted  in  potassium  bi- 
chromate for  a  considerable  length  of 
time.  The  blocks  can  be  embedded  in 
paraffin  and  sections  made  although  de- 
calcification is  usually  incomplete. 
Mitochondria  in  hair  cells,  stria  vas- 
cularis and  spiral  ganglion  cells  can  be 
studied  by  this  method. 

In  his  study  of  kittens,  young  rabbits, 
dogs  and  rats,  Van  der  Stricht,  O., 
Contrib.  to  Embryol.,  Carnegie  Inst., 
1920,  9,  109-142  fixed  isolated  cochleas 
in  5%  aq.  trichlorlactic  acid,  Bouin's 
and  Zenker's  fiuids,  mordanted  for 
"many  weeks"  in  70%  alcohol  +  a  few 
drops  of  iodine  solution.  After  the  last 
2  fixatives  he  completed  decalcification 
in  2%  nitric  acid  in  70%  alcohol.  Be- 
fore imbedding  in  paraffin  he  stained 
with  Borax  Carmine  and  he  colored  the 
sections  with  Iron  Hematoxylin,  Congo 
Red  and  Light  Green.  Directions  will 
be  found  in  his  paper  for  the  demonstra- 
tion of  mitochondria  in  the  sustentacu- 
lar  and  hair  cells.  A  differential  stain 
for  hair  cells  is  described  by  MacNaugh- 
ton,  I.  P.  J.,  and  Peet,  E.  W.,  J.  Laryng. 
and  Otol.,  1940,  55,  113-114  with  a  fine 
colored  figure  of  the  results. 

Celloidin  is  generally  used  for  im- 
bedding animal  and  human  material. 
It  is  not  ideal  since  it  is  difficult  to 
handle,  takes  considerable  length  of 
time  to  infiltrate  and  is  expensive. 
Various  nitrocellulose  samples  have 
been  tried  for  small  blocks  of  bone  with 
success  but  usually  centers  of  large 
blocks,  particularly  human  temporal 
bones,  do  not  become  sufficiently  hard. 
Concentrations  of  celloidin  usually 
ranging  up  to  15%  are  employed  in  suc- 
cessive steps.    While  the  material  is  in 


8%,  10%  and  15%  celloidin  negative 
pressure  is  used  in  moderate  amounts 
to  insure  infiltration  of  celloidin  into 
chambers  of  the  inner  ear.  This  should 
be  done  with  considerable  caution  as 
rupture  of  the  delicate  membranes  may 
result.  When  the  specimen  is  ready  for 
embedding  it  is  amply  covered  with  15% 
celloidin  and  allowed  to  remain  in  the 
refrigerator  (the  lower  temperature  pre- 
vents excessive  bubble  formation)  until 
it  can  be  blocked.  Blocking  of  the  ma- 
terial is  important  and  depends  upon 
whether  vertical  or  horizontal  sections 
through  the  cochlea  are  desired.  This 
is  readily  determined  by  such  land- 
marks as  the  eminentia  arcuata,  and 
external  and  internal  auditory  meati. 

Sections  of  large  blocks  are  usually 
cut  at  10  to  15  micra  in  thickness  on  a 
sliding  microtome.  Every  section  is 
numbered  and  kept  for  further  study 
if  necessary,  while  every  tenth  or  every 
twentieth  section  is  put  aside  for  stain- 
ing as  a  "tracer"  series. 

The  nerves  of  the  tympanic  mem- 
brane were  successfully  stained  intra- 
vitally  by  the  use  of  methylene  blue; 
Wilson,  J.  G.,  J.  Comp.  Neurol,  and 
Psychol.,  1907,  17,  459-468.  Peripheral 
endings  of  the  cochlear  nerve  were 
stained  with  1:5000  methylene  blue  in 
isolated  pieces  of  the  fresh  membranous 
cochlea  removed  under  the  dissecting 
microscope;  Covell,  W.  P.,  Ann.  Oto., 
Rhino.,     Laryngo.,     1938,     47,     62-67. 

The  peripheral  fibers  of  the  cochlear 
nerve  have  recently  been  demonstrated 
by  Fernandez,  C.  (Laryngoscope,  1951, 
59,  (in  press))  using  Bodian  and  Cajal 
silver  Methods.  The  course  of  the 
fibers  were  traced  in  pieces  removed 
by  dissection  and  mounted  on  slides. 
A  method  for  study  of  Wallerian  de- 
generation in  the  cochlear  nerve  by 
use  of  the  Swank-Davenport  osmic  mix- 
ture is  described  by  Rasmussen,  G.  L. 
(Abstr.,  Anat.  Rec,  1950,  106,  120). 
Ross,  E.  L.  and  Hamilton,  J.  W.  (Arch. 
Otol.,  1939,  29,  428-436)  allowed  mer- 
curochrome  to  remain  in  the  middle 
ear  cavities  of  dogs  for  20  minutes  to 
2  hours,  fixed  the  mercurochrome  in 
acid  solution,  decalcified  the  bones,  and 
studied  the  distribution  of  the  dye  in 
frozen  sections  of  the  cochlea.  The  pas- 
sage of  fluorescein  after  arterial  injec- 
tion was  observed  by  Gisselsson,  L. 
(Acta  oto-laryng.,  1949,  37,  268-275)  in 
the  cochlea.  Trypan  blue  has  been 
utilized  to  ascertain  the  effects  of 
trauma  on  scala  media  cells  of  the 
cochlea  (Lurie,  M.  H.,  Ann.  Otol.,  Rhin., 
Laryng.,  1942,  51,  712-717).  The  capil- 
lary areas  of  the  cochlea  have  been 
further  studied  in  animals  by  Smith, 


EAR  SMEARS 


107 


EAR  SMEARS 


C.  (Laryngoscope,  1951,  59,  (in  press)). 
The  precipitation  of  Prussian  Blue  in 
the  small  vessels  or  the  lead  chromate 
method  of  Williams,  T.  W.  (Anat.  Rec, 
1948,  100,  115-125)  gave  satisfactory 
preparations. 

A  method  for  graphic  reconstruction 
of  the  organ  of  Corti  was  introduced  by 
Guild,  S.  R.,  Anat.  Rec,  1921,  22.  141- 
157.  This  method  has  been  used  for 
ascertaining  damage  to  the  organ  of 
Corti  and  for  measurements  of  the 
length  of  the  organ  of  Corti  in  man  by 
Hardy,  M.,  Am.  J.  Anat.,  1938,  62,  291- 
311.  A  simple  technique  for  measuring 
the  length  of  the  basilar  membrane  is 
reported  by  Keen,  J.  A.,  J.  Anat.,  1939- 
40,  74,  524-527.  To  study  the  mode  of 
vibration  of  the  basilar  membrane 
B^k^sy,  G.  v.,  J.  Acous.  Soc.  Am.,  1948, 
20,  227-241,  used  fresh  human  temporal 
bones  and  suspended  fine  silver  crystals 
in  the  cochlear  duct  to  visualize  the 
transparent  memarane. 

Various  methods  of  reconstruction 
have  been  employed  particularly  for 
study  of  development  of  the  ear.  See 
Bast,  T.  H.,  Arch.  Otol.,  1932,  16,  19-38 
and  others.  Casts  of  the  labyrinth 
have  been  made  of  a  number  of  different 
materials  including  Wood's  metal,  wax, 
rubber  and  so  forth.  Cummins,  H., 
J.  Comp.  Neurol.,  1924-25,  38,  399-459 
used  mercury  for  this  purpose.  With 
monomeric  methyl  methacrylate  (Pera- 
pe.x)  Gray,  0.,  J.  Laryng.  and  Otol.,  1948, 
62,  308,  developed  a  method  for  making 
an  accurate  anatomic  cast  of  the  laby- 
rinth. See  Endolymph. 
Ear  Smears — Written  by  Marian  Pfingsten 
Bryan,  Dept.  of  Otolaryngology,  Wash- 
ington University,  St.  Louis  10,  Mo. 
September  20, 1951 — Cytological  studies 
of  aural  exudates  are  valuable  in  help- 
ing to  understand  the  patterns  of  disease 
pertaining  to  the  middle  ear,  mastoid 
and  external  canal  of  the  ear.  The 
smear  technique,  more  fully  appreci- 
ated now,  although  stressed  by  Dean, 
L.  W.  (J.  A.  M.  A.,  1932,  99,  543-546), 
yields  reliable  information.  When  this 
is  accurately  correlated  with  clinical 
symptoms  it  is  of  diagnostic  value. 
With  repeated  consecutive  smears  the 
course  of  an  inflammatory  reaction  in 
the  middle  ear  can  be  charted  and  the 
bacteria  generally  observed.  The  se- 
verity and  type  of  infection  is  often 
indicated  by  the  number  and  variety 
of  inflammatory  cells.  The  response  of 
the  individual,  as  evidenced  by  the 
number  and  type  of  phagocytes  is  im- 
portant. The  phagocytic  power  of  the 
polymorphonuclear  neutrophilic  leuco- 
cytes along  with  the  monocytes  and 
other  cellular  forms  can  be  evaluated. 


These,  with  other  distinguishing  cy- 
tological features,  are  evident  in  the 
following-categories  of  aural  disease: 
chronic  and  acute  suppurative  otitis 
media,  cholesteatoma  of  the  middle  ear 
and  mastoid,  acute  and  chronic  mas- 
toiditis, external  otitis  and  carcinoma. 

Studies  of  these  aural  secretions  have 
been  sparsely  scattered  through  the 
literature.  Ts'en,  Shih-Ping  (China 
Med.  J.,  1926,  40,  136)  reported  seven 
aural  smears  with  case  histories  and 
suggested  that  the  differences  in 
cytology  were  due  to  the  duration  of 
the  discharge.  He  noted  that  in 
chronic  cases  the  neutrophiles  were  more 
degenerated  than  in  acute  ones.  Since 
then  it  has  been  found  that  chronic 
suppurative  otitis  media  can  readily 
be  differentiated  from  acute  suppurative 
otitis  media  by  smears.  The  amount 
of  fatty  squamous  epithelial  degenera- 
tion is  of  importance.  This  type  of 
degeneration  is  rarely  found  in  acute 
otitis  media.  The  cytology  in  these 
cases  was  studied  by  Dean,  L.  W.  Jr.  and 
Pfingsten,  M.  G.  (Ann.  Oto.,  Rhinol. 
and  Laryngol.,  1933,  42,  484-496). 
Chronic  suppurative  otitis  media  is 
also  characterized  by  excessive  numbers 
of  mixed  bacteria  and  the  neutrophiles 
usually  exhibit  marked  degeneration. 
If  there  is  an  acute  exacerbation  of  a 
chronic  condition,  the  typically  chronic 
picture  is  seen  plus  the  acute  one,  in 
which  the  neutrophiles  are  many,  well 
preserved,  and  often  exhibit  phagocytic 
activity  as  evidenced  by  containing 
bacteria.  In  acute  otitis  media  all  gra- 
dations of  monocytes  are  found  from 
those  characteristic  of  the  circulating 
blood  to  other  larger  mononuclears, 
but  in  chronic  otitis  media  the  larger 
ones  are  rare. 

In  some  cases  of  chronic  otitis  media 
an  increase  of  eosinophiles  has  been 
observed  in  the  exudates.  Dohlman, 
F.  G.  (Nord.  Med.  Tidskr.,  1943,  17, 
224)  worked  on  this  problem.  In  178 
cases  of  varying  types  of  otitis  he  found 
in  99  of  them  some  increase  in  eosino- 
philes. Koch,  Hjalmar  (Acta  Otolaryn- 
gol., 1947,  Supp.  62)  has  also  made  a 
complete  and  detailed  study  concerned 
with  the  finding  of  eosinophiles  in  the 
ear  discharges  from  cases  of  chronic 
otitis  media.  He  stated  that  in  the 
210  chronic  ears  examined,  there  were 
52  cases  which  exhibited  an  increase 
in  eosinophiles.  He  concluded  that  in 
the  eosinophile  cases  the  healing  time 
of  the  lesion  was  lengthened  and  that 
they  were  characterized  by  a  very  vis- 
cous secretion  varying  from  clear  glass 
to  serum-like  in  appearance.  Hansel, 
F.  K.,  Allergy  of  the  Nose  and  Para- 


EAR  SMEARS 


108 


EAR  SMEARS 


nasal  Sinuses;  St.  Louis:  C.  V.  Mosby 
Co.  1936,  383  pp.)  believes  that  increase 
of  eosinophiles  in  affections  of  the 
middle  ear  is  due  to  the  chronicity  of 
the  infection  rather  than  to  an  allergic 
condition  comparable  to  that  which 
appears  in  the  nose.  Eosinophiles  are 
frequently  noted  where  there  is  a  con- 
tinual irritation  factor.  In  aural  polyps 
there  may  be  eosinophilic  infiltration 
in  the  tissue  and  if  discharge  is  present 
eosinophiles  may  be  seen.  Proetz,  A.  W. 
(Ann.  Otol.,  Rhin.  and  Laryngol.,  1931, 
40,  67)  reported  in  detail  an  asthmatic 
infant  who  had  attacks  of  otitis  media 
accompanied  by  asthma.  The  secre- 
tion from  the  ear  did  not  contain  eo- 
sinophiles. The  problem  merits  careful 
consideration  and  further  investigation. 

Considerable  significance  in  the  past 
has  been  attached  to  the  microscopic 
findings  of  cholesterol  crystals  and  fatty 
degenerated  epithelium  in  chronic  mid- 
dle ear  suppurations.  Some  have  re- 
garded these  cytological  findings  as 
diagnostic  of  cholesteatoma  of  the  mid- 
dle ear  and  mastoid.  Particular  care 
must  be  taken  in  these  conditions  when 
studying  the  cytology  of  the  ear,  as  it 
has  been  shown  by  Dean,  L.  W.,  Jr.  and 
Pfingsten,  M.  G.  (Ann.  Otol.,  Rhinol. 
and  Laryngol.,  1933,  42,  484-496)  that 
characteristic  cholesterol  crystals  and 
positive  chemical  tests  for  cholesterol 
can  be  found  in  acute  otitis  media  and 
in  external  otitis  as  well  as  in  chronic 
otitis  media.  Normal  cerumen  from 
the  ear  may  contain  more  amorphous 
and  crystalline  cholesterol  than  choles- 
teatoma. It  is  thus  important  that 
the  ear  canal  be  thoroughly  cleansed 
and  that  the  exudate  studied,  be  taken 
from  the  point  of  perforation  of  the 
drum  membrane.  It  would  seem  that 
the  finding  of  cholesterol  crystals  along 
with  fatty  degenerated  epithelium  in 
aural  secretions  is  not  alone  diagnostic 
of  cholesteatoma,  but  may  be  sugges- 
tive in  the  light  of  the  clinical  symp- 
toms. 

Cholesterol  crystals  show  double  re- 
fraction of  polarized  light,  so  are  best 
observed  in  unstained  direct  smears  of 
secretions  with  a  microscope  equipped 
with  polarizer  and  analyzer.  Since 
lipoid  substances  are  the  only  ones,  so 
far  as  is  known,  that  doubly  refract 
polarized  light,  this  method  is  simple 
and  convenient  for  detecting  cholesterol 
in  its  crystalline  state.  When  the  Nicol 
prisms  of  the  polarizing  apparatus  are 
crossed  the  field  appears  dark,  but 
cholesterol  crystals,  when  present,  are 
brightly  illuminated  against  the  dark 
background.  They  are  seen  in  the  char- 
acteristic  flat  rhomboid  plates,   often 


with  irregular  edges.  The  crystals,  be- 
ing very  fragile,  may  be  broken  into 
fragments  lacking  true  crystal  forma- 
tion. What  the  actual  association  of 
the  crystal  formation  may  be  to  the 
fatty  degenerated  epithelial  cells  is  not 
known. 

Cholesterol  is  also  evident  in  the 
secretions  of  infected  ears  and  in  ceru- 
men in  the  form  of  liquid  crystals  ob- 
served only  with  a  polarizing  micro- 
scope. They  appear  as  small  luminous 
spheres  with  a  black  maltese  cross 
superimposed  on  each  one.  The  phe- 
nomenon is  caused  by  a  molecular  for- 
mation of  cholesterol  esters  that  takes 
place  in  a  liquid  medium.  The  maltese 
crosses  are  the  result  of  interference 
lines  of  light  rays  passing  through  the 
crystals.  They  are  sometimes  seen 
within  large  phagocytic  cells.  Nothing 
is  known  of  their  significance  except 
that  their  presence  indicates  choles- 
terol. 

In  mastoiditis  the  smear  is  equally 
useful  in  helping  to  evaluate  whether 
the  condition  is  acute  or  chronic.  Some 
consider  the  clinical  signs  and  symp- 
toms of  the  patient  sufficient,  but  in 
these  serious  diseases  every  laboratory 
aid  available  is  needed  to  facilitate 
diagnosis.  In  acute  suppurative  mas- 
toiditis, the  secretion,  if  there  is  ample 
opportunity  for  it  to  drain  through  the 
tympanic  membrane,  may  contain  large 
lipoid  bone  phagocytes,  which  hint  at 
bone  destruction.  These  same  cells  are 
usually  found  at  mastoidectomy  in  cell 
pockets  of  diseased  bone  containing 
purulent  material  where  the  bone  is 
actively  being  destroyed.  They  were 
first  observed  in  the  ear  and  mastoid 
by  Pfingsten,  M.  G.  in  1934  by  using 
the  supravital  staining  technique  as 
developed  by  Sabin,  F.  R.  (Bull.  Johns 
Hopkins  Hosp.,  1923,  34,  277-288). 
Their  identification  was  reported  at 
that  time  to  the  clinical  conference  for 
Otolaryngology,  Barnes  Hospital.  A 
complete  report  concerning  these  cells, 
with  case  histories,  before  and  after 
the  use  of  antibiotics,  is  to  be  published 
this  year  by  Bryan,  M.  P.  and  Bryan, 
W.  T.  K. 

Such  cells  are  best  studied  in  the  liv- 
ing condition  since  stains  and  fixations 
dissolve  the  lipoid  substance  within 
the  cell  leaving  large  empty  vacuoles 
in  the  cytoplasm  giving  it  a  foamy 
appearance.  The  cell  membrane  in  ei- 
ther the  fixed  or  living  condition  may 
be  indistinct  and  irregular.  The  nu- 
cleus is  usually  eccentric  and  sometimes 
double.  The  cells  are  for  the  most 
part  spherical  varying  in  size  from  20- 
42  microns  in  diameter.     In  comparison 


EAR  SMEARS 


109 


EAR  SMEARS 


to  the  size  of  the  cell  the  nucleus  is 
small  and  ovoid,  often  obscured  by 
the  lipoid  globules  which  completely 
fill  the  cytoplasm.  Pseudopodia  may  be 
present  but  active  motility  has  never 
been  observed.  They  stain  positively 
with  Sudan  III  in  the  fresh  condition. 
The  stain  dissolves  the  globules  and  the 
cytoplasm  fills  with  the  orange  dye. 
These  large  phagocytes,  when  not  filled 
with  lipoid  material,  may  be  seen  to 
phagocytose  cellular  debris,  red  blood 
cells  and  entire  neutrophiles  as  well  as 
bacteria. 

All  of  the  cellular  elements  should 
be  taken  into  consideration  in  order 
to  evaluate  the  inflammatory  response. 
The  display  of  polymorphonuclear  neu- 
trophilic leucocytes  is  important.  It 
can  be  demonstrated  that  45-65%  of 
these  cells  will  engulf  carmine  particles 
suspended  in  tyrode  or  neutral  saline 
solution.  If  the  same  preparation  is 
counterstained  with  dilute  neutral  red 
the  identical  vacuoles  containing  car- 
bon will  react  to  neutral  red  dye,  in- 
dicating that  neutral  red  staining  is  a 
fairly  accurate  criterion  for  judging 
phagocytic  activity.  Hesse,  Herr 
(Ztschr.  f.  Hals,  Nasen  u.  Ohrenh., 
1927-1928,  18,  377-382)  studied  fresh 
exudates  from  middle  ear  suppurations 
and  distinguished  phagocytic  and  non- 
phagocytic  leucocytes,  but  he  stated 
that  ceil  death  occurred  very  rapidly. 
For  staining  he  used  the  Synderhel- 
mishe  dye,  a  preparation  of  trypan  blue 
and  Congo  red.  The  dye  has  been  used 
here  and  found  to  be  very  toxic  for  the 
cells.  It  does  not  give  sufficient  time 
for  observation  in  the  living  state.  He 
made  no  mention  of  the  giant  bone 
phagocytes. 

Other  cells  encountered  in  acute  mas- 
toiditis include  monocj^tes,  lympho- 
cytes, eosinophiles  and  basophiles. 
Many  variations  in  the  mononuclear 
cells  are  found.  Besides  the  typical 
blood  monocytes  there  are  gradual  tran- 
sitions between  these  cells  and  the 
giant  phagocytes.  Often  it  is  advisable 
to  distinguish  between  these  cells  when 
they  are  not  engorged  with  lipoid  ma- 
terial, but  yet  are  larger  than  the 
typical  monocyte  with  many  varying 
shades  of  neutral  red  vacuoles  and  a 
smaller  ovoid  eccentric  nucleus,  rather 
than  the  typical  horse-shoe  nucleus 
of  the  monocyte.  These  are  termed 
"transitional  mononuclears",  or  cor- 
respond in  appearance  to  the  "clasmato- 
cytes"  in  the  classification  of  Sabin, 
F.  R.,  Doan,  C.  A.  and  Cunningham, 
R.  S.  (Contrib.  to  Embryol.,  1925,  16, 
127-162).  The  lymphocytes  may  show 
slight    motility   in    fresh   preparations 


and  sometimes  a  few  neutral  red  in- 
clusions. If  Janus  Green  is  used  the 
mitochondria  may  be  observed.  Eosin- 
ophiles, if  seen  in  these  secretions,  may 
exhibit  motility  in  the  living  state  but 
are  never  seen  to  store  particulate 
matter.  In  acute  mastoiditis  there  are 
rarely  more  than  a  few  seen.  Baso- 
phile  cells  are  also  noted  in  many  cases. 
They  are  usually  few  in  number  and 
their  significance  is  not  known. 

In  contrast  to  the  various  cells  as- 
sociated with  active  mastoiditis,  there 
are  those  observed  in  chronic  mas- 
toiditis, namely,  large  numbers  of  de- 
generated neutrophiles,  fattj^  degen- 
erated squamous  epithelial  cells  and 
masses  of  mixed  bacteria.  Bacteria  are 
numerous  along  with  cellular  debris. 
Giant  phagocytes  are  rarely  found  in 
the  chronic  condition  unless  there  is  an 
acute  exacerbation  of  the  infection. 
Many  other  cellular  details  require  to 
be  correlated  with  the  symptoms  of  the 
patient  in  these  diseases. 

Cytological  studies  aid  in  differentiat- 
ing the  many  types  of  external  otitis. 
Senturia,  B.  H.,  Matthews,  J.  I.  and 
Adler,  B.  C.  (The  Laryngoscope,  1950, 
60,  543-550)  have  made  important  con- 
tributions showing  that  the  smear  gives 
information  as  to  the  causative  agents 
in  external  otitis,  whether  they  are 
bacilli,  cocci  or  fungi.  Examination  of 
the  smear  is  helpful  in  distinguishing 
between  suppurative  otitis  media  and 
external  otitis  (except  the  circum- 
scribed type).  Senturia  states  that  the 
ear  with  a  hidden  tympanic  perforation 
and  a  secondary  diffuse  external  otitis 
may  be  difficult  to  differentiate  from  a 
diffuse  external  otitis  with  exudate  cov- 
ering the  tympanic  membrane.  Cyto- 
logical examination  usually  makes  the 
difference  clear.  Otitis  media  with  per- 
foration shows  neutrophiles,  lympho- 
cytes, mucus  strands,  with  a  few  bac- 
teria and  epithelial  cells.  Diffuse 
external  otitis  shows  a  predominance  of 
epithelial  cells  and  bacteria,  with  per- 
haps an  occasional  leucocyte  or  mucus 
strand  in  the  secretion.  The  most 
striking  finding  in  the  exudates  of  acute 
and  diffuse  external  otitis  is  the  absence 
of  neutrophiles. 

In  regard  to  the  early  diagnosis  of 
malignant  and  benign  tumors  of  the 
middle  ear  and  external  canal,  the 
cytology  of  the  discharge  should  be 
studied  carefully  with  the  staining  tech- 
nique developed  by  Papanicolaou,  G. 
N.  (Science,  1942,  95,  438-439).  This 
is  a  reliable  aid  in  conjunction  with 
biopsy  and  it  is  especially  important  if 
biopsy  is  not  possible.  Fortunately 
carcinoma  of  the  middle  ear  is  somewhat 


EAR  SMEARS 


110 


EHRLICH'S  TRIACID 


rare .  House ,  H .  P .  (Ann .  Otol . ,  Rhinol . 
and  Laryngol.,  1949,  58,  789-797)  re- 
viewed the  literature  and  reported  that 
201  authentic  cases  of  the  disease  have 
been  reported.  He  discussed  two  cases 
in  which  the  technique  of  Papanicolaou 
aided  in  diagnosis  and  indicated,  that 
since  the  majority  of  malignancies  of 
the  middle  ear  are  superimposed  on 
chronically  discharging  ears,  the  method 
should  prove  of  value  in  the  early 
diagnosis  of  carcinoma  of  the  middle 
ear.  Diamont,  M.  (Acta  Otolaryng., 
1941,  29,  77-79)  pointed  out  the  diffi- 
culty even  with  the  Papanicolaou  stain 
of  differentiating  clinically  between 
granulations  in  chronic  otitis  and  early 
malignancy.  Smears,  however  are  very 
useful  in  these  cases  in  efforts  to  follow 
the  cytology  of  the  lesions  after  opera- 
tive procedures.  Subsequent  biopsies 
are  not  always  feasible.  The  smear 
technique  causes  no  inconvenience  to 
the  patient.  It  is  reliable  in  the  evalua- 
tion of  x-ray  and  radium  therapy  since 
the  effects  of  such  treatments  are  re- 
flected in  the  cellular  response. 

It  seems  important,  in  order  to  ob- 
serve all  of  the  cellular  details  possible, 
to  use  the  method  of  Papanicolaou  as 
well  as  a  good  polychrome  stain,  such 
as  Wright's  or  Hansel's,  especially  in 
the  chronic  ear  conditions,  so  that  the 
exudate  can  be  studied  in  both  stains. 
Details  of  the  light  staining  with 
Wright's  and  of  the  use  of  buffer  solu- 
tion are  described  in  the  staining  of 
Nasal  Smears.  Short  drying  of  the 
slide  in  air,  rather  than  flaming,  is 
recommended  for  maximum  cellular  de- 
tail. Whenever  possible,  in  order  to 
complete  the  cytological  picture,  it  is 
interesting  to  use  the  supra-vital  tech- 
nique on  living  fresh  material.  There 
are  vast  differences  in  appearance  be- 
tween living  and  stained  cells  so  that 
accurate  cellular  differentiation  be- 
comes a  complicated  problem.  In  order 
to  insure  good  results  from  any  of  the 
staining  techniques,  a  fresh  sampling 
of  the  discharge  is  imporatnt.  It  has 
often  been  found  satisfactory  to  use  a 
frontal  sinus  silver  cannula  with  rubber 
bulb  to  obtain  the  secretion  as  close  as 
possible  to  the  point  of  perforation  of 
the  drum  membrane.  The  discharge 
may  then  be  released  and  spread  evenly 
and  thinly  on  a  clean  glass  slide.  At 
mj^ringotomy  it  is  taken  directly  follow- 
ing the  incision  of  the  drum  membrane. 

Since  there  are  a  number  of  reliable 
staining  methods  that  are  adaptable 
to  the  study  of  aural  exudates,  the 
knowledge  gained  thereby  can  be  cor- 
related with  the  clinical  symptoms  of 


the  patient.  This  laboratory  aid  is 
important  in  otologic  examination  and 
stimulates  an  awareness  of  the  ever 
changing  pathological  processes. 

Earle,  see  Tissue  Culture. 

Ectoplasm.  Cytoplasm  lying  immediately 
internal  to  the  plasma  membrane.  It 
is  usually  gelled,  and,  being  free  from 
various  formed  bodies  present  in  the 
endoplasm,  has  a  clear  hyaline  appear- 
ance. 

Egg,  inoculation  of  hen's  eggs,  see  Chorio- 
allantoic Membrane.  Egg  of  hel- 
minths, see  Floatation  Techniques. 
Transplantation  of  living  fertilized 
eggs,  see  account  of  Placenta. 

Ehrllch-Biondi  Stain,  known  also  as  the 
Ehrlich-Biondi-Heidenhain  mixture,  is 
one  of  the  classical  stains. 

Add  20  cc.  sat.  aq.  acid  fuchsin  and  50 
cc.  sat.  aq.  methyl  green  to  100  cc.  sat. 
aq.  orange  G  agitating  the  fluid  while 
doing  so.  Add  60-100  cc.  aq.  dest.  The 
diluted  mixture  should  redden  slightly 
if  a  little  acetic  acid  is  added.  A  drop 
placed  on  filter  paper  should  be  bluish 
green  at  the  center  and  orange  at  the 
periphery.  If  there  is  an  outside  red 
zone  too  much  fuchsin  has  been  used. 
Stain  sections  of  sublimate  fixed  tissues 
12-24  hrs.  Do  not  wash  in  water  but 
dehydrate  quickly.  Clear  and  mount. 
This  stain  gives  beautiful  results  when 
properly  employed  but  it  is  fickle. 
Many  helpful  suggestions  are  given  in 
Lee,  p.  179. 

Ehrlich's  Acid  Hematoxylin.  Dissolve  2 
gm.  hematoxylin  in  100  cc.  95%  alcohol 
and  add;  aq.  dest.,  100  c;  glycerin,  100 
cc;  ammonium  (or  potassium)  alum,  3 
gm.,  glacial  acetic  acid,  10  cc.  Ripen 
by  exposure  to  air  (but  not  dust)  2  or 
3  weeks,  or  immediately  by  addition  of 
0.4  gm.  sodium  iodate. 

Ehrlich's  Aldehyde  Reagent.  2  gms.  para- 
dimethj^lamino-benzaldehyde  in  100  cc. 
20%  aq.  hydrochloric  acid.  See  Uro- 
bilin. 

Ehrlich's  Triacid  blood  stain.  This,  also, 
is  one  of  the  classic  stains,  now  seldom 
used.  It  contains  methyl  green,  orange 
G  and  acid  fuchsin;  but  methyl  green 
is  a  basic  dye  so  that  it  is  not  made  up  of 
three  acid  dyes.  Ehrlich  explained  that 
it  is  so  called  "because  in  it  all  the  three 
basic  groups  of  the  methyl  green  are 
combined  with  acid  dye-stuffs"  (Lee, 
p.  167)  with  which  modern  chemists  do 
not  agree.  Air  dried  smears  are  fixed 
by  heat  (110°C)  about  2  min.;  stained 
in  triacid  (Griibler)  5  min. ;  washed  in 
aq.  dest.  until  no  more  color  is  extracted 
and  dried  with  smooth  filter  paper. 
Said  to  color  neutrophile  granules  and 
leave  azur  granules  unstained. 


EIMERIA 


111 


ELASTICA-TRICHROME  STAIN 


Eimeria,  see  Coccidia. 

Einarson,  see  Gallocyanin-Chromalum 
Staining  of  Basophilic  Cell  Structures. 

Elacin,  see  Elastic  Fibers. 

Elastase.  An  elastolytic  enzyme  from  pan- 
creas has  been  reported  by  Bal6,  J.  and 
I.  Banga,  Biochem.  J.,  1950,  46,  384- 
387.  This  enzyme  renders  elastic  tis- 
sue soluble,  without  the  formation  of 
amino  acid  residues.  Consequently,  it 
apparently  depolymerizes  elastin. 
Since  elastic  tissue  is  highly  insoluble, 
this  enzyme  should  prove  most  useful 
both  for  chemical  and  histological  in- 
vestigations. 

Elastic  Fibers — Written  by  A.  I.  Lansing, 
Dept.  of  Anatomy,  Washington  Uni- 
versity, St.  Louis  10,  Mo.  October  5, 
1951 — Viewed  in  fresh  unstained  spreads 
of  Loose  Connective  Tissue  these  fibers 
are  generally  yellow  and  are  more  highly 
refractile  than  coUagenic  fibers.  They 
are  optically  homogeneous,  branch  re- 
peatedly to  form  networks,  are  of  vari- 
able thickness  and  do  not  swell  in  dilute 
acids.  Elastic  fibers  are  resistant  to 
both  pepsin  and  pure  trypsin  and  are 
slowly  digested  in  commercial  grade 
trypsin.  The  latter  may  be  due  to 
contamination  by  elastase,  a  new,  rela- 
tively specific  enzyme  which  solubilizes 
elastic  fibers  (Bal6,  J.  and  Banga,  I., 
Biochem  J.,  1950,  46,  384).  Elastic 
fibers  are  also  solubilized  by  prolonged 
boiling  in  0.25  N  oxalic  acid  (Adair,  G. 
S.,  Nature,  1951,  167,  605). 

Elastin  may  also  be  distinguished 
from  collagen  by  its  amino  acid  compo- 
sition. As  indicated  in  the  accompany- 
ing table,  collagen  is  very  rich  in  hy- 
droxyproline  while  elastin  has  only 
small  amounts.  Not  all  elastins  have 
the  same  amino  acid  compositions. 
Ligamentum  nuchae  (cow)  differs  sig- 
nificantly from  elastin  of  human  arteries 
which  differ  from  one  another.  Indeed 
age  influences  the  amino  acid  composi- 
tion of  elastin  from  human  aortas. 

Age  influences  many  of  the  properties 
of  elastic  tissue.  Senile  elastic  tissue 
of  skin  is  often  called  elacin.  This 
material  is  not  unlike  elastic  tissue  in 
old  arteries.  Age  differences  in  elastic 
tissue  may  be  summarized  as  follows: 
Elastic  fibers  are  readily  demon- 
strated in  sections  by  the  use  of  one  of 
several  fairly  specific  stains  including 
Weigert's  Resorcin-Fuchsin,  Verhoeff's 
Hematoxylin  Stain,  Unna's  Orcein 
Method,  Krajian's  Congo  Stain.  After 
oxidation  elastic  fibers  are  colored  red 
by  the  Schiff  Reagent.  Victoria  blue 
and  Basic  Fuchsin  also  effectively  stain 
elastic  fibers. 
When   viewed   with   polarized   light 


elastic  fibers  are  not  birefringent  but 
become  so  when  stretched.  This  would 
suggest  that,  although  the  fibers  are 
optically  homogeneous,  they  possess 
internal  structural  orientation.     Elec- 


Amino  Acid  Composition  of  Collagen  and  Elaslin* 


Amino  Acid 


Total  N 

Glycine 

Alanine 

Leucine 

Isoleucine 

Valine 

Phenylalanine. .. 

Tyrosine 

Tryptophan 

Serine 

Threonine 

Cystine 

Methionine 

Proline 

Hydroxyproline . 

Lysine 

Hydroxylysine. . 

Arginine 

Histidine 

Aspartic  acid .... 
Glutamic  acid. . . 
Amide  N 


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0.0 
0.8 
15.1 
14.0 
4.5 
1.3 
8.8 
0.8 
6.3 
11.3 
0.66 


Total  found 119.00 


17.1 

15.71 

29.9 

26.10 

18.9 

23.18 

8.7 

4.52 

4.0 

2.10 

17.4 

13.00 

5.0 

1.73 

1.61 

1.45 

0.01 

0.06 

.82 

0.29 

.96 

0.65 

0.15 

0.06 

0.03 

0.06 

17.0 

10.10 

2.0 

— 

0.39 

0.49 

0.89 

1.78 

0.07 

0.15 

0.63 

0.38 

2.1 

1.83 

0.04 

2.90 

— 

90.83 

15.56 

21.30 

21.58 

4.76 

2.33 

11.50 

1.97 

1.76 

0.24 

0.70 

1.13 

0.10 

0.35| 

9.20i 

1.17 

4.35 
0.75 
l.Il 
3.01 
2.86 


16.00 
32.00 

4.78 
2.28 
12.9 


90.17 


11.0 


0.62 
1.75 
1.51 


•  Adapted  in  part  from  Gross:  J.  Gerontol.,  1950,  5, 
343. 

t  Bowes,  J.  H.,  and  Kenton,  R.  H.:  The  amino  acid 
composition  and  titration  curve  of  collagen.  Biochem. 
J.,  1948,  43,  358-365. 

t  Neuman,  R.  E.:  Amino  acid  composition  of  gela- 
tins, coUagens  and  elastins  from  different  sources.  Arch. 
Biochem.,  1949,  24,  289-298. 

§  Lan.sing,  A.  I.,  Roberts,  Eugene,  Ramasarma,  G. 
B.,  Rosenthal,  Theodore  B.,  and  Alex,  Morris:  Changes 
with  age  in  amino  acid  composition  of  arterial  elastin. 
Soc.  Exp.  Biol.  Med.,  1951,  76,  714-717. 

tron  microscopy  has  so  far  failed  to 
reveal  internal  structure.  Elastic  fibers 
partially  digested  with  elastase  do  re- 
veal the  presence  of  intertwined  fibrils. 

Elastic    Properties,    see    Surface    Tension. 

Elastica-Trichrome  Stain.  In  order  to 
demonstrate  elastic  fibers  with  equal 
clearness  to  the  smooth  muscular  and 
collagenic  fibers,  especially  in  the  walls 


ELASTIN 


112 


ELECTRON  MICROSCOPY 


of  blood  vessels,  a  useful  combination 
of  Weigert's  elastic  tissue  stain  and 
Masson's  trichrome  stain  has  been 
worked  out  by  Mendeloff,  J.,  Am.  J. 
Clin.  Path.,  1943,  Tech.  Suppl.  7,  65. 
Deparaffinize  sections  in  usual  way, 
wash  thoroughly  in  water  and  stain  in 
Weigert's  Resorcin  Fuchsin  mixture  for 
60  min.  Wash  quickly  in  Acid  Alcohol, 
dehydrate  and  differentiate  in  abs.  ale. 
till  section  is  only  faintly  red.     Pass 


Some 

Properties  of  Human 

Arterial  Elastin 

Young 

Senile 

Physical 

Straight,  anasto- 

Frayed, fragmented, 

appear- 

mosing threads  or 

thin    strands    and 

ance 

ribbons 

granules 

Glistening,  refrac- 

Tend  to  clump,  yel- 

tile, water-clear 

low,  dull 

Tinctorial 

1.  Not  stained  by 

1.  Take     up     hema- 

hematoxylin 

toxylin 

2.  Red  color  with 

2.  Yellow    -    orange 

Congo  Red 

with  Congo  Red 

3.  Resorcin-fuchsin, 

3    Stain               more 

orcein  "Van 

densely    with    re- 

Giesen" positive 

sorcin,  etc. 

Chemical 

1.  Mineral -free 

1.  Severely  mineral- 

2. Phosphorus-free 

ized.     Contains  as 

3.  Amino  acids: 

much  as  14%  cal- 

a) trace  of  as- 

cium 

partic  acid 

2.  Large  amounts  of 

b)  1%  glutamic 

phosphorus 

acid 

3.  Amino  acids: 

a)  appreciable 
amounts  of  as- 
partic  acid 

b)  4%       glutamic 
acid 

Physical 

Dry  isolated  elas- 

Dry  isolated  elastin. 

tin,  has  specific 

has  specific  gravity 

gravity  less  than 

greater  than  1.30 

1.30 

through  70%  ale.  to  aq.  dest.  and  stain 
in  Harris'  Alum  Hematoxylin  8  min. 
Differentiate  in  water  5  min.  Stain  in 
Ponceau  acid  fuchsin  mixture  (see  Mas- 
son's  Trichrome)  5  min.  Wash  thor- 
oughly and  place  in  3%  aq.  phospho- 
tungstic  acid,  10  min.  Wash  again 
thoroughly  in  water  and  stain  with 
light  green.  Transfer  directly  to  1% 
acetic  acid,  3  min.  Do  not  wash  but 
dehydrate,  clear  and  mount  in  Gum 
Damar.  Elastic  tissue,  blue -black; 
smooth  muscle,  red;  collagen,  green. 

Elastin,  see  Elastic  Fibers. 

Electric  Tissues  of  fishes,  methods  for  are 
given  by  Dahlgren  (McClung,  1950, 
p.  343). 


Electrical  Resistance  and  capacity  or 
Impedence.  By  employing  alter- 
nating currents  of  varying  frequencies 
figures  for  apparent  resistance  and 
capacity  can  be  obtained.  Red  cells, 
yeast  cells,  ova  etc.  have  been  investi- 
gated. The  technique  is  not  micro- 
scopic but  the  data  have  an  important 
bearing  on  structure.  In  view  of  the 
wide  variety  of  cells  studied  it  is  inter- 
esting, as  Danielli  remarks  (Bourne, 
p.  42),  that  a  definite  pattern  should 
emerge  of  a  cell  plasma  membrane  only 
10~*-10~'  cm.  in  thickness  corresponding 
to  a  specific  resistance  of  lO^^-lO"  ohms. 

Electrodes.  See  the  several  varieties  de- 
scribed in  full  with  literature  references 
and   diagrams  by   Click,   pp.    183-188. 

Electromagnet  Technique  to  determine  elas- 
ticity (Heilbroun,  A.,  Jahrb.  wiss.  Bot., 
1922,  61,  284),  employed  by  Seifriz,  W. 
and  Hock,  C.  W.  (Paper  Trade  J., 
1936,  102,  36)  and  described  by  Cham- 
bers, R.  W.  and  Kopac,  M.  J.  in  Mc- 
Clung's  Microscopical  Technique,  1950, 
p.  542. 

Electron     Microscopy — Details     originally 

Erovided  by  Dr.  W.  L.  Simpson;  revised 
y  Dr.  T.  B.  Rosenthal,  Dept.  of  Anat- 
omy, Washington  University,  St.  Louis. 
June  6,  1951. 

1.  Transmitted  electron  beam  type. 
The  relationship  of  the  wave  length 
(X)  of  light  employed  and  to  the  nu- 
merical aperture  (N.A.)  of  a  lens  system 
as  expressed  in  the  relation  R.P.  equals 

T;^—r-    proved  for  many  years   an   ap- 

parently  insurmountable  limitation  to 
the  biologist's  desire  to  investigate  di- 
rectly minute  structure  of  cells  and 
tissues.  Even  when  ultraviolet  light 
of  2250A  was  employed  the  limit  of 
resolution  was  0.08  y.  in  &  system  of 
N.A.  1.40.  With  visible  light  the  limit 
was  approximately  0.2  y..  On  the  as- 
sumption that  the  angle  of  visual  acuity 
is  1  minute  of  arc,  the  greatest  mag- 
nification that  was  practical  with  visi- 
ble light  ranged  from  1750  to  2100  times. 
There  is,  of  course,  no  limitation  to  the 
actual  magnification  that  may  be 
achieved.  Increases  beyond  the  limit 
mentioned,  however,  do  not  reveal  new 
structures.  As  long  as  this  was  true 
there  seemed  no  hope  of  direct  confir- 
mation of  the  amazing  findings  made 
possible  by  such  new  methods  as  x-ray 
diffraction,  ultracentrifugation  studies, 
chemical  studies  of  virus  structure,  and 
polarized  light  methods. 

Small  wonder  is  it  then  that  the 
biologist  has  grasped  with  enthusiasm 
at  the  possibilities  of  visualizing  ultra- 
microscopic  structure  by  means  of 
devices  that  have  grown  from  the  fertile 


ELECTRON  MICROSCOPY 


113 


ELECTRON  MICROSCOPY 


field  of  electron  optics.  Of  most  general 
interest  is  the  electron  microscope. 
With  this  instrument,  using  the  same 
equation  for  resolving  power,  it  should 
be  possible  to  reach  a  resolution  of  at 
least  O.OOl/x-  Thus,  an  improvement  of 
at  least  200  times  over  the  limit  with 
visible  light  might  be  achieved.  The 
practical  limit  on  magnification  has  been 
placed  at  from  70,000  to  100,000  times. 

Historically  the  electron  microscope 
is  now  twenty-four  years  old.  Busch 
described  the  first  such  instrument  us- 
ing magnetic  lenses  (Busch,  H.,  Arch, 
f.  Elektroteknik,  1927,  18,  583-594). 
Though  many  improvements  were  made 
in  design  it  was  not  until  10  years  later 
that  the  instrument  reached  the  point 
of  being  of  practical  use.  Chiefly 
through  the  work  of  Ruska  and  Borries 
(numerous  papers,  1934-1940)  the  in- 
strument was  developed  to  the  state 
that  it  is  in  at  present.  In  this  country 
an  e.xcellent  instrument,  capable  of 
giving  high  resolution  has  been  de- 
veloped and  commercially  marketed  by 
the  Radio  Corporation  of  America. 
The  apparatus  is  described  by  Zwory- 
kin,  V.  K.  (Science,  1940,  92,  51-53). 
In  this  instrument  electrons  emitted 
from  a  hot  wire  filament  are  accelerated 
by  a  potential  of  30,000  or  more  volts. 
This  beam  is  condensed  and  passed 
through  the  object  which  is  carried  on 
a  wire  screen  supported  nitro-cellulose 
film.  The  transmitted  electron  beam  is 
focussed  in  a  greatly  enlarged  image  by 
means  of  two  magnetic  lenses.  The 
image  can  be  seen  on  a  fluorescent  screen 
or  photographed  on  a  sensitive  plate. 

The  conditions  under  which  good  re- 
sults are  obtainable  are  rather  stringent. 
The  ideal  object  must  be  very  small,  or 
capable  of  being  minutely  divided  with- 
out losing  its  identity;  it  must  maintain 
its  form  on  drying  in  vacuum;  and  it 
must  be  rigid  enough  to  resist  the  heat 
and  disruption  caused  by  exposure  to 
the  electronic  bombardment.  Rela- 
tively few  biological  structures  fall  into 
this  category;  hence  early  studies  were 
confined  to  bacteria,  viruses,  connective 
tissue  fibers,  blood  cells,  spermatozoa, 
etc. 

The  great  need  in  histological  in- 
vestigation with  the  electron  micro- 
scope has  been  a  method  for  uniformly 
cutting  tissues  no  thicker  than  0.1  ^ 
and  about  1  mm*  or  larger  in  area. 
With  these  dimensions  a  section  will 
yield  acceptable  electron  micrographs. 
Such  a  method  seems  to  have  been 
found  in  a  modification  of  the  conven- 
tional microtome  whereby  the  advance 
of  the  block  is  reduced  about  tenfold, 
either  mechanically  (Baker  and  Pease, 


J.  Appl.  Phys.,  1949,  20,  480)  or  by 
means  of  a  thermal  expansion  device 
(Newman,  Borysko,  and  Swerdlow, 
Anat.  Rec,  1949,  105,  267).  The  tissue 
must  be  embedded  in  celloidin  or  plas- 
tic, but  a  special  blade  is  probably 
necessary  for  the  best  results.  Since 
1950  publications  have  appeared  with 
electron  micrographs  of  nearly  all  tis- 
sues of  importance.  See  Excerpta  Med- 
ica,  Sect.  I,  for  listings  and  abstracts. 

Histologists  have  also  borrowed  spe- 
cial methods  from  metallurgists  for 
investigation  of  hard,  opaque  structures 
like  bone,  tooth,  hair,  keratinized  epi- 
thelium, etc.  These  methods  involve 
the  casting  of  very  thin  replicas  of 
surface  details.  Additional  contrast  is 
gained  by  the  ingenious  process  of 
"shadow  casting",  either  on  the  original 
specimen  or  on  its  replica.  A  very  thin 
layer  of  metal  is  deposited  over  the 
object  in  a  vacuum  by  evaporation  from 
a  hot  filament.  When  done  at  an  acute 
angle,  the  elevated  portions  of  the 
object  shadow  the  depressed  areas,  thus 
giving  a  striking  three-dimensional  pic- 
ture (Williams  and  Backus,  J.  Appl. 
Phys.,  1949,  20,  98).  Methods  have 
also  been  developed  for  true  stereoscopic 
images  (Heidenreich  and  Matheson,  J. 
Appl.  Phys.,  1944,  15,  423).  See  Shad- 
dow  Casting. 

The  old  technique  of  selective  stain- 
ing has  been  combined  with  this  newest 
of  procedures  in  histology.  It  has  been 
shown  that  the  salts  of  heavy  metals, 
such  as  osmium  and  phosphotungstate, 
are  deposited  in  a  highly  localized  way 
on  certain  protein  structures,  e.g.  mus- 
cle fibrils,  thus  enabling  visualization 
of  practically  molecular  details  (Hall, 
Jakus,  and  Schmitt,  J.  Appl.  Phys., 
1945,  16,  459). 

With  the  proper  selection  and  com- 
bination of  histological  methods  now 
on  hand  the  electron  microscopist 
should  have  little  difficulty  in  studying 
any  problem  in  morphology.  A  practi- 
cal handbook  for  the  guidance  of  novice 
and  expert  has  been  prepared  by  the 
Royal  Microscopical  Society:  "The 
Practice  of  Electron  Microscopy", 
edited  by  D.  G.  Drummond  and  pub- 
lished as'Part  I  of  vol.  70,  1-141,  1950  of 
the  J.  Roy.  Micro.  Soc.  Papers  dealing 
with  details  of  manipulation  are  to  be 
found  regularly  in  the  Journal  of  Ap- 
plied Physics. 

2.  Emission  electron  type.  The  earli- 
est description  of  an  electron  micro- 
scope in  this  country  was  of  an  entirely 
different  type  from  the  new  R.C.A.  mi- 
croscopes that  give  such  prodigious 
magnifications.  McMillan  and  Scott 
(J.  H.  and  G.  H.,  R.S.I. ,  1937,  8,  288- 


ELECTROPHORESIS 


114 


ELEMENTARY  BODIES 


290)  published  an  account  of  an  electron 
microscope  of  simple  design  that  used 
as  a  source  the  electron  emission  of 
heated  sections  of  tissues.  These  were 
accelerated  by  a  potential  of  1000  to  2000 
volts,  focussed  by  a  magnetic  lens  onto 
a  fluorescent  screen.  An  improved 
design  (Scott,  G.  H.  and  Packer,  D.  M., 
Anat.  Rec,  1939,  74,  17-29)  makes  pos- 
sible magnifications  of  at  least  150 
times.  By  certain  modifications  the 
magnification  can  be  increased  con- 
siderably. It  is  feasible  with  this  in- 
strument to  obtain  photographs  that 
record  the  precise  localization  of  cal- 
cium and  magnesium  salts  in  tissues. 
Scott  and  Packer  {ibid,  31-45)  showed 
that  the  calcium  and  magnesium  of 
skeletal  muscle  was  confined  almost  en- 
tirely to  the  muscle  fibers  themselves, 
and  that  in  contracted  fibers  a  great 
concentration  of  magnesium  appeared 
in  the  contraction  nodes. 

Tissues  to  be  studied  with  this  tech- 
nique must  be  preserved  in  a  manner 
that  permits  no  redistribution  of  min- 
erals. The  satisfactory  method  is  that 
of  Altmann-Gersh. 

Electron  microscopic  technique  sup- 
plements histospectrography  by  pre- 
cisely locating  certain  elements  within 
tissues  and  is  very  useful  in  conjunction 
with  the  technique  of  microincineration 
as  a  means  of  identifying  certain  com- 
ponents of  the  ash  seen  in  sections. 

Electrophoresis.  Most  particles  suspended 
in  water  carry  electricity.  If  placed 
in  an  electric  field  those  possessed  of 
positive  charge  move  toward  the  cath- 
ode and  those  with  a  negative  charge 
toward  the  anode.  Obviously  there- 
fore the  nature  of  the  charge  and  the 
speed  of  movement  can  be  determined 
by  microscopic  study  of  particles  sus- 
pended in  fluid  in  what  is  known  as  a 
micro-electrophoresis  cell.  Types  of 
cell  and  precautions  to  be  observed  in 
their  use  are  described  by  Moore,  D.  H. 
and  Abramson,  H.  A.  Glasser's  Medical 
Physics,  403-407.  Their  account  of  the 
"moving  boundary"  method  of  electro- 
phoresis and  of  the  Tiselius  apparatus 
is  clear  and  to  the  point.  This  latter 
method,  in  contrast  with  the  micro- 
scopic one,  affords  a  technique  of  great 
accuracy  and  sensitivity  for  separating 
concentrations  and  purifying  submicro- 
scopic  components  in  blood  serum  and 
other  complete  liquids. 

Eleidin  (G.  elaia,  oil)  gives  to  the  stratum 
lucidum  its  clear,  glassy  appearance. 
It  may  be  a  dissociation  product  of 
keratohyalin.  There  has  been  no  great 
improvement  on  the  specificity  of  the 
older  methods.  Mallory  (p.  260)  gives 
the  method  of  Buzzi   (1889),  first  cau- 


tioning that  fixation  must  be  in  formalin, 
Orth's  or  Bouin's  fluid.  Stain  frozen 
sections  of  10%  formalin  fixed  tissue  in 
sat.  aq.  picric  acid  (approximately 
1.2%)  5  min.  Rinse  in  aq.  dest.  and 
counterstain  for  1  min.  in  1%  aq.  nigro- 
sin.  Wash  in  water  and  then  in  95% 
ale.  (Skip  absolute)  Clear  in  ter- 
pineol  or  origanum  oil.  Mount  in  bal- 
sam: keratin,  bright  yellow;  eleidin, 
blue  black.  Ranvier's  Picro-Carmine 
gives  a  fine  red  staining  of  eleidin.  See 
finger  Nails. 
Elementary  Bodies  are  the  smallest  particles 
of  viruses.  Those  of  certain  viruses  are 
large  enough  for  direct  microscopic 
examination  in  suitably  stained  prepara- 
tions which  usually  show  also  the  larger 
Inclusion  Bodies  if  these  are  present. 
Various  methods  designed  for  Rickettsia 
are  usually  satisfactory.  Many  special 
techniques  have  been  proposed  of  which 
2  follow : 

1.  Methyl  violet  or  Victoria  blue 
for  smears  (Gutstein,  M.,  J.  Path.  & 
Bact.,  1937,  45,  313-314).  Dry  smears 
on  perfectly  clean  slides  in  air  or  incu- 
bator. If  necessary  remove  excess 
protein  by  rinsing  in  physiological  saline 
solution  followed  by  aq.  dest.  Fix  in 
methyl  alcohol  1  hr.  Stain  in  either  of 
2  ways:  (1)  Place  slide  in  Petri  dish. 
Mix  equal  parts  1%  aq.  methyl  violet 
and  2%  aq.  NaHCOs.  Filter  imme- 
diately onto  the  slide,  cover  dish  and 
incubate  at  37 °C.  20-30  min.  Rinse 
in  aq.  dest.,  dry  and  mount  in  cedar  oil 
or  liquid  paraffin.  Elementary  bodies 
light  violet.  (2)  Same  except  filter 
onto  slide  equal  parts  (a)  Victoria  blue 
4R  1  gm.,  ale.  (abs.)  10  cc.  and  aq.  dest. 
90  cc.  and  (b)  0.02%  aq.  KOH  and  leave 
at  room  temperature  over  night.  Ele- 
mentary bodies  of  vaccinia  and  other 
viruses  dark  blue. 

2.  Methyl  blue  acid  fuchsin  for  sec- 
tions (Nicolau,  S.  and  Kopciowska,  L., 
C.  r.  Acad.  d.  Sci.,  1937,  204,  1276-1278). 
Fix  in  alcoholic  Bouin's  fluid.  Stain 
4-5  micron  paraffin  sections  30-60  min. 
in:  methyl  blue  (Griibler)  1.5  gm.,  aq. 
dest.  65  cc,  methyl  alcohol  35  cc,  glyc- 
erin 5  cc,  3%  aq.  oxalic  acid  5  cc. 
Wash  well  in  aq.  dest.  and  change  to 
absolute  alcohol.  Stain  20  min.  in: 
acid  fuchsin  1.5  gm.,  aq.  dest.  100  cc, 
3%  aq.  oxalic  acid  2  cc  Wash  directly 
in  absolute  alcohol  and  mount  in  the 
usual  way.  Small  particles  in  cells 
associated  with  following  viruses : 
herpes,  Borna,  Zoster,  rabies  and 
pseudo-rabies  are  stained  bright  red. 

A  summary  of  methods  for  demon- 
strating elementary  bodies  is  given  by 
Seiffert,  G.,  Virus  Diseases  in  Man,  Ani- 
mal and  Plant.     New  York :  Philosophi- 


ELLIPSIN 


115 


ENAMEL 


cal  Library,  Inc.,  1944,  332  pp.  Under 
favorable  conditions  some  kinds  of 
of  elementary  bodies  are  visible  at  high 
magnification  unstained  by  both  direct 
and  dark  field  illumination.  Supra- 
vital stains  such  as  brilliant  crcsyl  blue, 
neutral  red,  methylene  blue  and  azur  II 
are  recommended.  Before  staining 
smears,  fixed  in  a  variety  of  ways,  pre- 
treatment  with  2.5%  aq.  potassium  per- 
manganate or  2%  aq.  chromic  acid  is 
advised.  Giemsa  stain  gives  good  re- 
sults but  the  methods  of  Paschen,  Moro- 
sow  and  Herzberg  are  suggested  by 
Seiffert.  The  fluorescence  technique 
of  Hagemann  consists  of  staining  thin 
air  dried  smears  with  1  gm.  primuline 
dissolved  in  1000  cc.  aq.  dest.  +  20  cc. 
pure  phenol  for  15  sec.  washing  in  aq. 
dest.  and  observation  in  ultraviolet 
light  by  fluorescence  microscope. 

New  methods  for  the  collection  and 
purification  of  elementary  bodies  permit 
their  direct  examination  at  very  high 
magnifications  with  the  electron  micro- 
scope (von  Borries,  E.  G.,  Ruska,  E. 
and H.,  Klin.  Woch.,  1938, 17, 921 ;  Green, 
R.  H.,  Anderson,  T.  F.,  and  Smadel, 
J.  E.,  J.  Exp.  Med.,  1942,  75,  651-656) 
and  their  chemical  analysis  for  vitamin 
catalysts,  copper  and  enzymes  (Hoag- 
land,  C.  L.,  Ward,  S.  M.,  Smadel,  J.  E., 
and  Rivers,  T.  M.,  J.  Exp.  Med.,  1942, 
76,  163-173).  See  fluorescence  of  ele- 
mentary bodies  (Turevich,  E.  I.  ab- 
stracted in  Stain  Techn.,  1941,  16,  182.) 

Ellipsin  is  structure  protein  of  cells.  Meth- 
ods for  its  isolation  from  liver  cells  of 
rabbit  and  guinea  pig  by  grinding  fresh 
tissue,  washing,  centrifugation  and  so 
on  are  fully  described  by  Bensley,  R.  R. 
and  Hoerr,  N.  L.,  Anat.  Rec,  1934,  60, 
251-266. 

Embedding,  see  Imbedding. 

Embryo  Chick,  see  Chorioallantoic  Mem- 
brane. 

Embryological  Methods.  In  general  the 
techniques  which  give  good  results  with 
adult  tissues  are  also  satisfactory  for 
embryos ;  but  there  are  differences  as  for 
example  in  silver  impregnations.  More- 
over greater  care  is  necessary  to  avoid 
too  sudden  changes  in  the  fluids  used. 
Helpful  suggestions  are  given  in  Mc- 
Clung,  pp.  279-286.  Application  of 
trichrome  staining  methods  to  embryos 
(Baxter,  J.  S.,  J.  Anat.,  1940-41,  75, 
137-140).  See  demonstration  of  Car- 
tilaginous Skeleton,  Ossification  and 
Spalteholz  method.  Technique  for 
handling  chick  embryos  (Adamstone, 
F.  B.,  Stain  Techn.,  1931,  6,  41-42). 
Block  staining  of  nervous  tissue  of  em- 
bryos with  silver  (Davenport,  II.  A., 
Stain  Techn.,  1934,  8,  143-149). 


Emission  Electron  Microscopy,  see  Electron 
Microscopy. 

Enamel  (dental).  This  can  best  be  studied 
in  ground  sections  of  Teeth.  1.  Cuticle. 
Wash  and  brush  tooth  in  tap  water.  4% 
neutral  formalin,  24  hrs.  Wash  tap 
water,  24  hrs.  Mallory's  anilin  blue 
(0.5%  aq.)  24  hrs.  Again  wash  and 
brush  in  tap  water.  10%  aq.  hydro- 
chloric acid,  10  min.  As  enamel  is  dis- 
solved delicate  opaque  white  membrane 
appears.  Tease  membrane  off  onto  slide 
coated  with  egg  albumen  (Albumen- 
Glycerin).  Blot  with  filter  paper.  5% 
aq.  sodium  thiosulphate  or  bicarbonate 
10  min.  Wash  in  tap  water  10  min. 
Dehydrate  in  alcohols,  clear  in  xylol 
and  mount  in  gum  damar  (McClung, 
p.  371). 

2.  Rods.  Macerate  tooth  in  5-10% 
aq.  hydrochloric  acid  for  24  hrs.  Re- 
move a  little  softened  enamel  and 
examine  (McClung,  p.  372).  See  Chase, 
S.  W.,  Anat.  Rec,  1927,  36,  239-258. 

3.  Organic  Matrix.  Boedeker's 
method  abbreviated  from  McClung  (p. 
372).  Dehydrate  small  piece  (0.5- 
1  mm.  thick),  free  from  dentin,  through 
alcohols  10  min.  each.  Methyl  alcohol 
1-2  hrs.  Decalcify  in  celloidin  mixture 
(parlodion,  DuPont)  made  by  dissolving 
sufficient  in  methyl  ale.  C.P.  to  give 
thick  syrupy  solution.  To  150  cc.  of 
this  add  drop  by  drop  constantly  stir- 
ring nitric  acid  C.P.,  10  cc.  -f  methyl 
ale.  40  cc.  Keep  tissue  in  this  mixture 
in  glass  dish  with  air  tight  cover.  Or- 
ganic matrix  appears  as  brown,  spongy 
material  in  10-12  hrs.  care  being  taken 
to  leave  the  dish  stationary.  After 
decalcification  is  completed,  2-7  days, 
uncover  and  permit  celloidin  to  harden. 
Cut  out  specimen  with  narrow  margin  of 
celloidin.  70  and  40%  ale.  1-2  hrs.  each. 
Aq.  sol.  alum,  24  hrs.  Running  water, 
6-12  hrs.  Ascending  alcohols  to  95% 
1-2  hrs.  each.  Anilin  oil,  6-12  hrs. 
(becomes  brown  and  transparent). 
Equal  parts  anilin  oil  and  chloroform, 
6-12  hrs.  Imbed  in  paraffin  not  over 
52°C.  Mount  3-10^  sections,  dry  and 
treat  with  xylol  3  min.  Dissolve  cel- 
loidin in  ether-alcohol.  Abs.  ale.  1 
min.  Descending  alcoliols  to  water. 
Stain  in  Iron  Hematoxylin. 

Improved  results  may  be  obtained  by 
slow  decalcification  and  concurrent  fixa- 
tion as  described  b.y  Sognnaes,  J.  Dental 
Res.,  1949,  28,  558-564.  Teeth  are 
placed  in  celloidin  bags  filled  with  10% 
formalin  and  immersed  in  a  solution  of 
5  gm.  trichloroacetic  acid,  2^  gm.  po- 
tassium bichromate  in  100  cc.  distilled 
water.  Ten  centimeter  of  formalde- 
hyde is  added  to  the  solution.  Decalci- 
fication of  enamel  is  completed  after 


ENDAMOEBA 


116 


ENTEROCHROMAFFIN  CELLS 


one  week  in  a  refrigerator  at  a  tempera- 
ture of  1°  C.  Decalcification  of  dentin 
is  completed  by  placing  the  bag  in 
5%  trichloroacetic  acid.  Not  more 
than  two  weeks  should  be  required. 
Washing  and  dehydration  in  ethyl  alco- 
hol and  butyl  alcohol  is  carefully  ac- 
complished with  tooth  still  in  the  bag. 
Imbed  in  paraffin,  section  and  stain  by 
ordinary  methods.  Constant  pressure 
during  decalcification  may  further  im- 
prove results  (Sognnaes,  R.  F.,  J.  Den- 
tal Res.,  1948,  27,  609-622). 

4.  Cape-Kitchin  celloidin  decalcifica- 
tion method.  Cut  DuPont's  parlodion 
into  small  cubes  and  dissolve  in  acetone 
free  methyl  alcohol  making  thick  solu- 
tion. To  200  cc.  add  90  cc.  methyl 
alcohol  constantly  stirring  and  9  cc. 
nitric  acid,  sp.  gr.  1.42.  Follow  decalci- 
fication of  enamel  in  this  mixture  be- 
tween crossed  nicols  of  polarizing  micro- 
scope with  24  mm.  objective.  Double 
refraction  disappears  with  decalcifica- 
tion (Bodecker,  C.  F.,  J.  Dent.  Res., 
1937,  16,  143-150). 

5.  Permeability.  When  the  apex  of  a 
tooth  is  immersed  in  strong  alcoholic 
solution  of  fuchsin  -f  NaCl  the  enamel 
becomes  stained  (v.  Beust,  T.,  Dental 
Cosmos,  1912,  54,  659).  Another  way 
is  to  test  for  penetration  of  lead,  boron 
and  other  easily  recognizable  chemicals 
(Howe,  P.  R.,  Dental  Cosmos,  1926,  68, 
1021-1033).  After  intraperitoneal  in- 
jections of  trypan  blue  blue  coloration 
can  be  observed  in  developing  enamel 
only  (not  adult)  as  well  as  in  dentin  of 
dogs  (Gies,  W.  J.,  J.  Nat.  Dent.  Assoc, 
1918,  5,  529-531).  Marshall  (J.  S.,  J. 
Dent.  Res.,  1921,  3,  241-255)  employed 
Naphthamine  brilliant  blue  similarly 
as  a  vital  stain.  See  Dentin,  vital 
staining.  A  "Triple  Embedding"  tech- 
nique is  described  by  Brain,  E.  B.,  J. 
Roy.  Micr.  Soc,  1950,  70,  313-316. 

Endamoeba,  see  Entameba. 

Endocervical  Smears,  see  Papanicolaou 
Techniques. 

Endolymph.  To  demonstrate  its  circulation 
employ  method  used  by  Guild,  S.  R., 
Am.  J.  Anat.,  1927,39,  57-81.  Introduce 
solution  of  potassium  ferrocyanide  and 
iron  ammonium  citrate  into  cochlear 
ducts  of  living  guinea  pigs  under  anes- 
thesia. Kill  at  intervals  up  to  48  hrs. 
Excise  tissue  and  fix  in  acid  fluid  which 
precipitates  Prussian  Blue  wherever 
the  solution  has  circulated. 

Endometrial  Smears,  see  Papanicolaou 
Techniques. 

Endospore  stain  for  bacteria  in  blood  smears. 
Smear,  air  dry  and  fix  by  flaming  3  times. 
5%  aq.  malachite  green  5  min.,  wash  in 
tap  water  10-20  sec.  0.5%  aq.  safranin, 
10  sec,  wash  quickly,  dry  and  examine 


(Bruner,  D.  W.  and  Edwards,  P.  R., 
3.  Lab.  &  Clin.  Med.,  1939,  25,  543-544). 

Enrichment  techniques,  see  Concentration. 

Entameba.  Craig  (p.  35)  gives  a  useful 
table  of  diagnostic  features  of  intestinal 
amebae  in  man;  also,  on  p.  55,  a  list  of 
objects  that  may  be  mistaken  for 
amebae  in  unstained  and  stained  prepa- 
rations; and  details  as  to  media  for  cul- 
tivation of  which  the  Boeck  and  Doboh- 
lav  media  and  the  simpler  Craig  media 
are  the  most  helpful. 

This  genus  includes  E.  histolytica, 
the  cause  of  amebic  dysentery  and  E. 
coli  and  E.  gingivalis,  two  apparently 
harmless  commensals.  The  technique 
is  essentially  the  same  for  all  three. 
In  searching  for  E.  histolytica  or  E.  coli 
take  a  small  amount  of  fresh  feces,  mix 
with  physiological  saline  solution  and 
examine  directly.  Recognize  amebae 
by  large  size  and  movements  if  slide 
is  kept  warm.  E.  histolytica  frequently 
contains  erythrocytes.  Mallory  (p. 
296)  advises  mixture  with  Gram's 
Iodine  solution  to  demonstrate  glycogen 
if  present,  or  mixing  with  drop  1-2% 
formalin,  then  treatment  with  drop  2% 
acetic  acid  and  coloration  with  1  drop 
1%  aq.  neutral  red.  E.  gingivalis  is  to 
be  found  in  decayed  teeth.  Only  E. 
histolytica  extensively  invades  tissues. 

1.  To  make  permanent  smear  prepara- 
tions (Mallory,  p.  296)  fix  thin  smear 
while  moist  in  95%  alcohol,  1  part,  and 
sat.  aq.  corrosive  sublimate,  2  parts, 
for  15  min.  Wash  for  few  sec.  in  water 
and  cover  with  1%  alcoholic  iodine  for 
3  min.  Wash  in  aq.  dest.  until  iodine 
color  is  extracted.  Wash  again  and  stain 
with  Phosphotungstic  Acid  Hema- 
toxylin, 30  min.  Wash  in  water,  dehy- 
drate in  95  and  abs.  alcohol,  clear  in  xylol 
and  mount  in  balsam.  Nuclei  and  ecto- 
sarc,  deep  blue;  cytoplasm,  bluish. 

2.  To  stain  differentially  in  sections 
(Mallory,  p.  297).  Fix  in  95%  or  abs. 
ale,  and  make  paraffin  or  celloidin  sec- 
tions. Stain  in  0.25%  aq.  thionin  3-5 
min.  Differentiate  in  2%  aq.  oxalic 
acid,  i-1  min.  After  washing  in  water, 
dehydrate  in  95%  and  abs.  ale  Clear 
in  xylol  and  mount  in  balsam,  except  for 
celloidin  sections  which  require  clearing 
in  terpineol,  or  origanum  oil,  after  95% 
ale.  Nuclei  of  amebae  brownish  red, 
those  of  all  other  cells,  blue.  See 
lodine-Eosin  stain  and  Walker's 
Method. 

Enterochromaffin  Cells.  Perhaps  the  best 
technique  is  Bodian's  protargol  method 
as  described  by  Dawson,  A.  B.  and 
Barnett,  Julia,  Stain  Techn.,  1944,  19, 
115-118.  For  the  influence  of  pilo- 
carpin  on  enterochromaffin  cells  see 
Hamperl,   II.,   Ztschr.   f.   Mikr.   Anat. 


ENTOMOLOGICAL  TECHNIQUES 


117 


EOSIN -ORANGE  G 


Forsch.,  1925,  2,  506-535.  See  Small 
Intestine. 

Entomological  Techniques,  see  Mosquito, 
Ticks,  Insects,  Arachnids,  Parasites. 

Enzymes — Written  by  E.  W.  Dempsey, 
Dept.  of  Anatomy,  Washington  Uni- 
versity, St.  Louis.  February  26,  1951 — 
Their  name  is  legion.  At  present  only 
a  few  can  be  localized  with  any  degree 
of  histological  precision,  but  the  number 
of  histochemical  methods  available  is 
increasing  rapidly.  There  are  no  bet- 
ter examples  of  felicitous  association 
between  histological  and  biochemical 
methods.  Four  principal  kinds  of  tech- 
niques are  employed  for  localization: 

(1)  By  spectrographic  identification  in 
the  tissues — especially  the  metal-con- 
taining Cytochromes  and  Peroxidase. 

(2)  By  close  comparison  of  enzymic 
activity  with  cellular  composition  of 
tissues.  See  Glick  for  detailed  meth- 
ods, derived  from  the  Linderstr0m- 
Lang  procedures — Amylase,  Pepsin, 
Peptidase,  Esterase,  Protease,  Cho- 
linesterase,  Lipase,  Urease,  Carbonic 
Anhydrase,  etc.  (3)  By  separation  of 
cellular  components,  such  as  nuclei, 
granules  and  mitochondria,  from  homo- 
genates  of  tissue.  Such  separation  is 
accomplished  by  differential  flotation  or 
centrifugation,  and  is  followed  by  esti- 
mation of  the  enzymic  activity  of  the 
purified  component  fractions.  Argi- 
nase,  Adenylpyrophosphatase,  Phos- 
phatase, etc.  have  been  investigated  by 
such  methods.  (4)  By  the  develop- 
ment of  characteristic  insoluble  prod- 
ucts within  tissues  or  cells — Aldolase, 
Acid  and  Alkaline  Phosphatase,  Cholin- 
esterase.  Cytochrome  Oxidase,  Dehy- 
drogenase, Dopa  Oxidase,  Esterase 
Glucuronidase,  Lipase,  Peroxidase, 
Phosphamidase,  Sulfatase. 

Enzymes  are  coming  into  their  own 
as  technical  tools.  Purified  enzymes 
may  be  used  to  destroy  certain  tissue 
components.  Ribonuclease  selectively 
destroys  basophilic  substances  in  cyto- 
plasm and  nucleus,  Desoxyribonuclease 
similarly  attacks  nuclear  chromatin. 
Hyaluronidase  solubilizes  certain  meta- 
chromatic ground  substances,  whereas 
other  mucoid  materials  react  only  with 
more  potent  Mucinases.  The  connec- 
tive tissue  fibers  have  long  been  charac- 
terized by  their  digestibility  in  Pepsin 
and  trypsin,  and  recently  an  Elas- 
tase  has  been  prepared.  A  purified 
Collagenase  has  been  reported.  The 
solubility  of  glycogen  in  solutions  of 
salivary  Amylase  is  an  old  histochemical 
procedure.  The  effects  of  Lysozyme 
and  other  enzymes  on  the  capsules  of 
pneumococci  and  upon  the  Gram  stain 
has  been  summarized  by  DuBos  (The 


Bacterial  Cell,  Harvard  Univ.  Press, 
1945). 

Eosin  B  or  bluish  (CI,  771)— eosin  BN,  BW, 
or  DHV,  eosin  scarlet,  eosin  scarlet  B, 
imperial  red,  nopalin  G,  saffrosin, 
scarlet  J,  JJ,  V — Dibrom  derivative  of 
dinitro-fluorescein.  Chemistry      of 

(Holmes,  W.  C,  Melin,  C.  G.  and 
Paterson,  H.  R.,  Stain  Techn.,  1932, 
7,  121-127). 

There  are  several  fluorescein  dyes 
and  guidance  may  be  needed  in  the 
choice  of  the  one  best  suited  for  a  par- 
ticular purpose.  Conn,  H.  J.  and 
Holmes,  W.  C,  Stain  Tech..  1926,  1, 
87-95 ;  1928,  3,  94-104  have  made  a  study 
of  color,  acidity  and  chemical  structure 
and  Conn  (p.  145)  gives  further  data. 
Their  color  increases  in  depth  in  this 
order:  eosin  Y,  ethyl  eosin,  eosin  B, 
erythrosin  B,  phloxine  and  rose  bengal. 
This  increase  in  color  is  proportional  to 
increase  in  number  of  hologen  atoms. 
Their  acidity  increases  in  a  different 
order:  rose  bengal,  phloxine,  erythrosin, 
eosin  Y  and  eosin  B.  (1)  When  the 
eosin  is  to  follow  in  alcoholic  solution  a 
basic  dye  always  in  aqueous  solution 
(cf.  hemato.xylin)  the  more  acid  and 
lighter  colors  are  recommended  (eosin 
Y,  ethyl  eosin  and  eosin  B.  (2)  When 
it  is  to  precede  in  aq.  solution  a  basic 
dye  (cf.  methylene  blue)  also  in  aq. 
solution,  use  phloxin  or  erythrosin  (see 
phloxine-methylene  blue). 

Eosin  lOB,  see  Phloxine  B. 

Eosin  BN,  BW,  or  DHV,  see  Eosin  B  or 
bluish. 

Eosin  J,  see  Erythrosin,  bluish. 

Eosin-Methyl  Blue,  see  Mann's. 

Eosin-Methylene  Blue  has  been  employed 
in  many  combinations  for  years.  But 
when  the  acid  dye  is  applied  first, 
phloxine  is  preferred  to  eosin.  See 
therefore    Phloxine    Methylene    Blue. 

Eosin-Orange  G — Toluidine  Blue  for  bone 
marrow,  spleen  and  connective  tissue 
(Dominici,  M.  C.  rend.  Soc.  biol.,  1902, 
54,  221-223).  Stain  eosin-orange  G 
(eosin  B.  A.  of  Hollborn  or  eosin  yellow- 
ish of  American  manufacturers  0.5  gm. ; 
aq.  dest.,  100  cc;  orange  G.  0.5  em.) 
7  min.  Rinse  quickly  in  aq.  dest. 
Counterstain  in  0.5%  aq.  toluidin  blue 
20-30  sec.  Rinse  again  aci.  dest.  Dif- 
ferentiate in  95%  ale,  dehydrate  in 
abs.,  clear  in  xylol  and  mount  in  balsam. 
Instead  of  eosin,  0.5%  aq.  acid  fuchsin 
gives  a  little  sharper  contrast.  In 
place  of  toluidin  blue  0.1%  Azur  A  can 
be  employed  to  advantage.  Phloxine- 
orange  G  can  be  tried  as  a  substitute  for 
eosin-orange  G.  (phloxine  0.12  gm.,  aq. 
dest.  100  cc,  orange  G,  0.3  gm.).  The 
crucial  point  is  the  differentiation  in  95% 
ale.     This  should  be  quickly   checked 


EOSIN  SCARLET 


118 


EPIDERMIS 


under  the  microscope  until  the  time  has 
been  determined. 

Eosin  Scarlet,  see  Eosin  B  or  bluish. 

Eosin  Scarlet  B,  see  Eosin  B  or  bluish. 

Eosin  Y  or  yellowish  (CI,  768).  Tetrabrom 
fluorescein  with  some  mono-  and  di- 
brom  compounds.  This  is  the  usual 
kind  of  eosin  employed.  Eosin  Y  and 
thionin  as  substitute  for  Wright's  stain 
(Saye,  E.  B.,  Am.  J.  Clin.  Path.,  Tech. 
Suppl.,  1943,  13,  12). 

Eosinophile  Leucocyte  (acidophilic  or 
coarsely  granular  leucocyte).  Can 
easily  be  examined  while  still  living  in 
mounts  of  fresh  blood.  The  dark  field 
is  useful.  Most  frequently  studied  in 
Blood  Smears,  which  see.  Mitochon- 
dria are  readily  stainable  with  Janus 
Green.  For  occasional  presence  of 
basophile  granules  and  pigment  see 
Downey,  H.,  Folia  Haemat.,  1915,  19, 
148-206.  Techniques  for  rapid  experi- 
mental increase  of  eosinophiles  in 
circulating  blood  are  described  by 
Banerji,  N.,  Am.  J.  Med.  Sci.,  1933, 
186,  689-693;  Chillingworth,  F.  P., 
Healy,  J.  C.  and  Haskins,  F.  E.,  J.  Lab. 
and  Clin.  Med.,  1933-34,  19,  486-494; 
Hajos,  K.,  Nemeth,  I.,  and  Enyedy,  Z., 
Zeit.  f.  d.  ges.  Exper.  Med.,  1926, 
48,  590-592.  Variations  and  errors  in 
eosinophile  counts  of  blood  and  bone 
marrow  are  described  by  Best,  W.  R. 
and  Samter,  M.,  Blood,  1951,  6,  61-74. 

Epidermis.  This  can  be  studied  in  situ 
with  the  dermis,  see  Skin,  or  it  can  be 
examined  in  3  ways  apart  from  the 
dermis. 

1.  Isolated  pieces.  Examination  of 
scrapings  of  the  epidermal  surface  is  of 
limited  usefulness  in  special  cases.  To 
cut  away  a  few  of  the  deeper  cells,  sepa- 
rate them  by  teasing  and  to  study  them 
in  the  still  living  state  with  or  without 
supravital  stains  is  not  particularly 
helpful.  But  their  microdissection  is 
capable  of  giving  important  data  on 
cellular  consistency  and  connections 
(Chambers,  R.  and  deRenyi,  G.,  Am. 
J.  Anat.,  1925,  35,  385-402  and  Than- 
hoffer,  L.,  Zeit.  f.  Anat.  u.  Entw.,  1933, 
100,  559-562).  Their  cultivation  is 
possible,  see  Tissue  Culture. 

2.  Whole  mounts  for  microscopic  study 
(Cowdry's  Histology,  p.  530).  Place 
excised  fresh  skin  in  1%  acetic  acid  in 
ice  box  for  12-36  hrs.  depending  upon 
size,  age  and  region.  Wash  in  tap  water, 
5  min.  Pin  skin  down  with  epidermis 
up  and  cover  with  water.  Strip  off 
epidermis  as  a  compete  sheet.  Wash  in 
aq.  dest.,  5  min.  Stain  in  Harris' 
hematoxylin,  20  min.  Wash  in  aq.  dest., 
1  min.  Differentiate  in  50  cc.  70% 
alcohol  plus  3  drops  hydrochloric  acid 
until  epidermis  becomes  light  pink  color. 


Treat  with  50  cc.  aq.  dest.  plus  6  drops 
ammonia  until  it  becomes  blue.  Wash 
in  aq.  dest.  5  min.  several  changes. 
Dehydrate  in  50,  70,  95  and  2  changes  of 
absolute  alcohol,  10  min.  each.  Clear 
in  2  changes  xylol,  1  hr.  each  and  mount 
in  balsam  inner  side  up. 

If  the  skin  is  hairy,  before  excising  it, 
remove  hair  with  scissors  and  electric 
razor  or  depilatory  solution.  Hair  fol- 
licles and  sebaceous  glands,  unless  par- 
ticularly large,  generally  remain  at- 
tached to  the  epidermis,  but  the  coiled 
bodies  of  the  sweat  glands  are  too  deeply 
situated  to  come  off  with  it.  Conse- 
quently only  their  straight  ducts  are  to 
be  seen.  Before  dehydration,  in  the 
above  technique  the  sebaceous  glands 
can  be  sharply  counterstained  with 
Sudan  IIL 

Such  whole  mounts  of  epidermal 
sheets  are  of  value  insofar  that  their 
study  gives  a  concept  of  the  morphology 
of  the  epidermal  covering  of  the  body 
which  can  be  obtained  in  no  other  way. 
For  the  counting  of  mitoses  they  are  far 
better  than  sections  and  have  been 
extensively  employed  for  this  purpose 
by  Dr.  Cooper  and  her  associates  in  The 
Barnard  Free  Skin  and  Cancer  Hospital. 
See  her  latest  paper  (Cooper,  Z.  K.  and 
Reller,  H.  C,  J.  Nat.  Cancer  Inst., 
1942,  2,  335-344).  Since  the  mucous 
membrane  covering  the  nasal  septum 
can  be  similarly  prepared  as  a  whole 
mount  it  is  likely  that  the  method  may 
be  of  service  in  the  study  of  other  sheets 
of  epithelial  cells. 

3.  Sheets  of  epidermis  for  chemical 
analysis.  Until  very  recently  the 
handicap  experienced  in  chemical  analy- 
sis of  the  skin  has  been  the  difficulty  of 
separating  epidermis  and  dermis  by 
themselves  for  analysis .  All  data  on  the 
epidermis  are  of  doubtful  value  because 
variable  amounts  of  dermis  have  been 
included.  The  method  of  obtaining 
pure  epidermis  by  dilute  acetic  acid 
separation  is  unsatisfactory  for  numer- 
ous reasons.  Baumberger,  J.  P.,  Sunt- 
zeff,  V.  and  Cowdry,  E.  V.,  J.  Nat. 
Cancer  Inst.,  1942,  2,  413-424  have 
discovered  that  dilute  alkali  will  serve 
as  well  as  dilute  acetic  but  this  also 
is  objectionable  from  the  chemical 
point  of  view.  They  therefore  advance 
a  heat  method.  Place  excised  skin  with 
dermis  down  on  warm  plate  such  as  is 
used  for  mounting  paraffin  sections. 
Apply  temperature  of  50°C.  for  2  min. 
which  loosens  the  epidermis  so  that  it 
can  be  easily  pushed  off  with  a  blunt 
instrument.  Separation  is  more  diffi- 
cult when  temperature  is  over  51  °C. 
Epidermises  removed  in  this  way  for  a 
time  continue  to  consume  oxygen  and 


EPINEPHRIN 


119 


ERYTHROCYTES 


are  very  suitable  for  chemical  analysis. 
They  have  been  used  for  epidermal  iron 
and  ascorbic  acid  by  Carruthers,  C.  and 
Suntzeff,  v.,  J.  Nat.  Cancer  Inst.,  1942, 
3,  217-220,  and  for  total  lipid-protein 
nitrogen  ration  by  Wicks,  L.  F.  and 
Suntzeff,  v.,  3,  221-226.  _ 

Epinephrin  (adrenin,  adrenalin),  hormone 
of  adrenal  medulla. 

Erbium,  see  Atomic  Weights. 

Erhlicki's  Solution.  Potassium  bichromate, 
2.5  gm.;  copper  sulphate,  1  gm.;  aq. 
dest.,  100  cc.  Used  for  hardening 
nervous  tissues. 

Erie  Fast  Red  F  D  (CI,  419)  of  NAC  is  a 
direct  disazo  dye  of  light  fastness  3  to  4. 
Resembles  Congo  red  insofar  that  wash- 
ing in  water,  or  in  95%  alcohol,  takes 
all  color  out  of  paraffin  sections.  In 
alkaline  solutions  it  colors  blue-green 
algae  deep  red  to  reddish  brown  (Emig, 
p.  40). 

Erie  Fast  Rubine  B  cone.  A  sulfonated  azo 
dye.  For  formula  nad  influence  on 
mouse  tumors,  see  Stern,  K.,  Cancer 
Res.,  1950,  10,  565-570. 

Erie  Fast  Yellow  WB,  see  Titan  Yellow. 

Erie  Garnet  B  (CI,  375). —  amanil  garnet 
H,  Buffalo  garnet  R,  Congo  corinth  G  or 
GW,  corinth  brown  G,  cotton  corinth 
G,  diamine  Bordeaux  CGN,  direct 
garnet  R,  direct  violet  C  —  an  acid  dis- 
azo dye  used  for  staining  frozen  sections 
(Geschickter,  C.  F.,  Stain  Techn., 
1930,  5,  81-86). 

Erie  Violet  BW  (CI,  387)  of  NAC  is  an  acid 
disazo  dye  of  light  fastness  2  to  3. 
Directions  for  use  in  making  prepara- 
tions of  animal  and  plant  tissues  are 
described  (Emig,  p.  40). 

Erie  Violet  3R  (CI,  394)  of  NAC  is  a  direct 
disazo  dye  of  light  fastness  3  not  as 
satisfactory  for  microscopic  work  as 
Erie  Violet  BW  (Emig,  p.  40). 

Eriochrome  Azurol  V  (CI,  720),  a  mordant 
dye  of  acid  fastness  3  to  4.  Gives  color 
like  that  of  Niagara  Sky  Blue.  Direc- 
tions for  use   (Emig,  p.  52). 

Eriometer,  apparatus  advocated  by  Em- 
mons, W.  F.,  Quart.  J.  Med.,  1927, 
21,  83  to  measure  mean  diameter  of 
erythrocytes.     See     Erythrocytometer. 

Erythroblasts,  see  Erythrocytes,  Develop- 
mental Series. 

Erythrocyte  Counts  do  not  fall  in  the  scope 
of  this  book.  It  is  sufficient  to  state 
that  they  are  going  out  of  fashion  be- 
cause of  the  large  experimental  error 
involved  and  since  it  is  so  easy  to  detect 
variations  in  sliapc,  size  and  maturity 
of  erythrocytes  in  smears  and  to  measure 
hemoglobin  content  of  blood  by  hemo- 
globinometers.  Blum,  L.  L.,  Am.  J.  Clin. 
Path.,  1945, 15,  85  has  introduced  a  rabid 
photoelectric  technique  for  estimating 


the  number  of  erythrocytes.  See  Retic- 
ulocytes, 
Erythrocytes.  For  chemical  and  physical 
studies  erythrocytes  are  particularly 
adapted,  because  they  can  be  collected 
in  enormous  numbers  free  from  other 
kinds  of  cells  and  from  intercellular 
substances.  In  order  to  determine 
marked  differences  in  size  and  shape 
and  hemoglobin  content  examination  of 
fresh  blood  with  direct  illumination,  or 
in  the  dark  field,  is  probably  the  best 
procedure.  An  interesting  photographic 
method  for  the  stereoscopic  visualiza- 
tion of  the  shape  of  erythrocytes  has 
been  described  and  illustrated  by 
Haden,  R.  L.,  J.  Lab.  &  Clin.  Med., 
1936-37,  22,  1262-1263.  For  more  accu- 
rate techniques  see  Wintrobe,  M.  M., 
Clinical  Hematology,  Philadelphia :  Lea 
&  Febiger,  1942,  792  pp.  A  new  aniso- 
cytosis  index  is  proposed  by  van  den 
Berghe,  L.,  and  Weinberger,  E.,  Am.  J. 
Med.  Sci.,  1940,  199,  478-481.  The 
refractile  body  of  Isaacs  (R.,  Anat.  Rec, 
1925,  29,  299-313)  can  also  be  well 
studied  in  fresh  blood.  See  Flagella. 

Smears,  colored  by  Giemsa  or  Wright's 
stain,  are  satisfactory  for  Howell-Jolly 
bodies,  Cabot  rings,  basophilic  stippling 
and  polychromalophilia.  For  resistance 
to  hemolysis  in  hy  potoni  c  sodium  chloride 
solutions,  see  Daland,  G.  A.,  and  Worth- 
ley,  K.,  J.  Lab.  &  Clin.  Med.,  1934-35, 
20,  1122-1136.  A  lysolecithin  fragility 
test  is  described  by  Singer,  K.,  Am. 
J.  Med.  Sci.,  1940,  199,  466-477.  For 
microfragility  tests  see  Kato,  K.,  J.  Lab. 
&  Clin.  Med.,  1940,  26,  703-713  and  for 
basophilic  erythrocytes  of  the  newborn 
see  McCord,  C.  P.,  and  Bradley,  W.  R., 
Am.  J.  Clin.  Path.,  1939,  Tech.  Suppl., 
2,  329-338.  A  thorough  investigation  of 
erythrocytes  in  fetus  and  newborn  has 
been  made  by  Wintrobe,  M.  M.  and 
Schumacker,  H.  B.,  Jr.,  Am.  J.  Anat., 
1936,  58,  313-328.  A  simple  method  for 
determination  of  specific  gravity  of 
erythrocytes  is  described  by  Reznikoff, 
P.,  J.  Exper.  Med.,  1923,  38,  441-444. 
After  hemolysis  the  stroma  remains  and 
can  be  studied  microscopically  or  chemi- 
cally. Lipid  analyses  are  particularly 
significant  (Erickson,  B.  N.,  et  al.,  J. 
Biol.  Chem.,  1937-38,  122,  515-528). 

Isolation  and  collection  en  masse  of 
nuclei  of  chicken  erythrocytes  by 
Dounce,  A.  L.,  and  Lan,  T.  H.,  Science, 
1943,  97,  584-585. 

Experiments  have  been  made  with 
radioactive  iron  as  a  means  of  tagging 
red  blood  cells  (Cruz,  W.  O.,  Hahn, 
R.  F.,  Bale,  W.  F.  and  Balfour,  W.  M., 
Am.  J.  Med.  Sci.,  1941,  202,  157-162) 
which  open  up  a  new  field  for  study  of 
age    changes    because    the    cells    are 


ERYTHROCYTES,  DEVELOPMENTAL  120 
SERIES 


ESTERASES 


thereby  dated.  Stratification  of  con- 
tents of  erythrocytes  by  ultracentrifu- 
gation  (Beams,  H.  W.,  and  Hines,  E.  H., 
Anat.  Rec,  1944,  89,  531).  Special  meth- 
ods are  given  under  Hemoglobin, 
Flagella,  Reticulocytes,  Cabot  Rings, 
Jolly  Bodies,  Pencil  Red  Cells,  and 
Target  Cells.  For  red  fluorescent 
erythrocytes  in  anemia,  see  Seggel, 
K.  A.,  Ergeb.  d.  inn,  Med.  u.  Kinderh., 
1940,  58,  582. 
Erythrocytes,  Developmental  Series.  The 
technique  employed  apparently  makes 
a  great  deal  of  difference  in  the  conclu- 
sions reached.  See  Cowdry's  His- 
tology,  1938  p.  99. 

1.  Maximo w  and  Bloom  employing 
mainly  permanent  preparations  list: 

Hemocytoblasts:  "...  large  (up  to 
15m)  ameboid,  non-granular  basophil 
cells  of  lymphoid  nature."  Occur  ex- 
tra vascularly. 

Basophil  erythroblasts :  The  youngest 
erythroblasts,  characterized  by  the 
intense  basophilia  of  their  cytoplasm. 
Also  called  megaloblasts ,  but  "this  term 
is  misleading  because  it  was  first  used 
for  the  erythroblasts  of  pernicious 
anemia  which  are  cells  of  quite  different 
nature." 

Polychromatic  erythroblasts:  So-called 
because  after  "fixation  and  staining  with 
the  Romanowsky  mixture,  especially  in 
dry  smears,  the  protoplasm  has  a  mixed 
color  varying  from  purplish-blue  to  lilac 
or  gray."  This  is  due  to  the  presence 
of  two  substances,  a  basophile  material 
and  hemoglobin. 

Orthochromatic  erythroblasts  or  normo- 
blasts: These  are  smaller  "and  only 
slightly  larger  than  the  mature,  non- 
nucleated  erythrocytes."  Since  the 
basophile  substance  diminishes  and  the 
hemoglobin  increases,  the  protoplasm 
becomes  acidophilic  "and  stains  a  bright 
pink  with  the  Romanowsky  mixture." 
They  continue  to  divide  mitotically  for 
an  unknown  number  of  generations  until 
the  nucleus  disappears. 

2.  Sabin  and  associates  relying  chiefly 
on  supravital  stains  list : 

Endothelial  cells:  Occur  in  special 
"erythrogenic  capillaries." 

Megaloblasts:  "...  a  daughter  endo- 
thelial cell  which  starts  to  synthesize 
hemoglobin."  "The  megaloblast  has 
maximum  basophilia,  a  moderate  num- 
ber of  rod-shaped  mitochondria,  a  trace 
of  hemoglobin,  and  a  nucleus  with  a 
minimum  of  chromatin  and  conspicuous 
nucleoli." 

Early  erythroblasts:  "The  young  ery- 
throblast  represents  a  growth  phase, 
with  less  rapid  division,  for  the  cell  is 
much  larger  than  the  megaloblast;  it 
contains  the  maximum  number  of  mito- 


chondria. The  amount  of  hemoglobin 
is  still  small,  but  sufficient  to  give  a 
poly  chroma  tophilia ,  predominately 

basophilic  in  methylene  blue-azur.  The 
nucleus  has  a  marked  increase  in 
chromatin." 

Late  erythroblasts:  This  cell  "is  inter- 
mediate in  size  between  the  early 
erythroblast  and  the  definitive  red  cell. 
The  nucleus  has  lost  the  nucleoli  but 
still  has  massive  chromatin.  .  .  .  The 
increase  in  hemoglobin  is  marked  and  in 
fixed  films  the  cytoplasm  is  more 
acidophilic." 

Normoblasts:  "The  stage  of  the  nor- 
moblast is  defined  as  a  nucleated  red 
cell  after  its  last  cell  division.  It  has 
a  small  pyknotic  nucleus  ready  for 
extrusion  or  fragmentation." 

Erythrocytometer  for  measuring  the  diam- 
eter of  red  blood  cells.  Pijper,  A.,  Med. 
J.  South  Africa,  1919,  14,  472  and  Lan- 
cet, 1935,  1,  1152,  deserves  great  credit 
for  the  discovery  independently  of 
Thomas  Young  (1813)  of  a  technique 
for  the  measurement  of  small  objects 
utilizing  the  principle  of  diffraction  and 
Zeiss  has  manufactured  an  instrument 
on  his  specifications.  Another,  the 
Haden-Hausser  erythrocytometer,  is 
made  by  C.  A.  Hausser  and  Son  and  is 
sold  by  Arthur  H.  Thomas  Co.,  Phil- 
adelphia (Haden,  R.  L.  J.  Lab.  &  Clin. 
Med.,  1939-40,  25,  399-403). 

Erythrosin  B,  see  Erythrosin,  bluish. 

Erythrosin  BB  or  B  extra,  see  Phloxine. 

Erythrosin,  bluish  (CI,  773)— dianthine  B, 
eosin  B,  erythrosin  B,  iodeosin  B,  pyro- 
sin  B — Fluorescein  with  2  iodine  atoms. 
See  Eosins. 

Escherichia  Coli,  see  Triphenyltetrazolium 
Chloride. 

Ester  Wax.  An  embedding  medium  de- 
signed especially  to  promote  ribboning 
of  sections.  It  is  made  up  of  diethylene 
glycol  distearate,  73  gm.;  ethyl  cellu- 
lose, 4  gm.;  stearic  acid,  5  gm.;  castor 
oil,  8  gm.;  and  diethylene  glycol  mono- 
stearate,  10  gm.  (Steedman,  H.  F., 
Quart.  J.  Micr.  Sci.,  1947,  88,  123-133). 

Esterases. — Written  by  E.  W.  Dempsey, 
Dept.  of  Anatomy,  Washington  Uni- 
versity, St.  Louis.  February  26,  1951^ 
Strictly  speaking,  these  are  enzymes 
which  hydrolyze  the  ester  linkage  de- 
rived from  any  acid,  organic  or  inor- 
ganic. Thus,  the  phosphatases,  cho- 
linesterases,  glucuronidase,  lipase,  and 
sulfatase  properly  belong  among  the 
esterases.  However,  ordinary  usage 
restricts  the  term  to  the  enzymes 
hydrolyzing  carboxy  esters,  particu- 
larly those  of  fatty  acids.  These  es- 
terases apparently  exhibit  some  sub- 
strate specificity;  at  least,  the  enzymes 
hydrolyzing  esters  of  short-chain  fatty 


ETHYL  EOSIN 


121 


EYES 


acids  appear  to  differ  from  the  true 
lipases,  which  act  upon  long-chain 
substrates,  and  from  cholinesterase. 
Gomori,  G.  (Proc.  Soc.  Exp.  Biol,  and 
Med.,  1945,  58,  362-364,  and  ihid, 
1949,  72,  697-700)  has  devised  a  method 
for  localizing  lipase.  Nachlas,  M.  M. 
and  A.  IM.  Seiigman  (J.  Nat.  Cancer 
Inst.,  1949,  9,  415-425)  believe  the 
histochemical  methods  do  not  differen- 
tiate a  true  lipase,  but  only  a  nonspe- 
cific esterase.  They  describe  a  new  and 
vivid  method  for  this  enzyme,  based 
upon  the  hydrolysis  of  the  acetyl  ester 
of  naphthol  and  staining  the  liberated 
naphthol  by  diazotization.  Seiigman, 
A.  M.,  M.  M.  Nachlas,  L.  H.  Man- 
heimer,  O.  M.  Friedman  and  G.  Wolf. 
(Ann.  Surg.,  1949,  130,  333-341)  describe 
the  development  of  specific  methods 
for  a  number  of  hydrolytic  enzymes. 

Ethyl  Eosin  (CI,  770).  The  ethyl  ester  of 
eosin  Y.  Sold  often  as  alcohol  soluble 
eosin.     See  Eosins. 

Ethyl  Green  (CI,  685) .  This  is,  Hke  methyl 
green,  prepared  from  crystal  violet  but 
differs  from  it  insofar  that  an  ethyl  group 
is  added  instead  of  a  methyl  one.  For 
most  purposes  it  is  a  satisfactory  sub- 
stitute for  methyl  green. 

Ethyl  Purple  6B,  see  Ethyl  Violet. 

Ethyl  Violet  (CI,  682)— ethyl  purple  6B— 
It  is  he.xaethyl  pararosanilin,  a  basic  dye 
employed  by  Bowie,  D.  J.,  Anat.  Rec, 
1924,  29,  57  to  make  a  neutral  stain  with 
biebrich  scarlet  for  staining  islets  of 
Langerhans  of  fish.  Kernohan,  J.  W., 
Am.  J.  Clin.  Path.,  1931,  1,  399-403 
has  used  in  Heidenliain's  modification 
of  Mallory's  ethyl-violet  orange  G  after 
formalin  fixation. 

Ethyl  Violet-Biebrich  Scarlet,  see  Bowie's 
stain  for  pepsinogen. 

Ethylene  Glycol  Mono-Ethyl  Ether  = 
Cellosolve. 

Euchrisine,  see  Acridine  Orange. 

Eunematoda,  see  Parasites. 

Euperal  is,  according  to  Lee  (p.  227),  a  mix- 
ture of  camsal,  eucalyptol,  paraldehyde 
and  sandrac,  n  =  1.483  of  two  sorts 
colorless  and  green.  Since  the  green 
one  contains  a  copper  salt  it  strengthens 
hematoxylin  stains. 

Euporium,  see  Atomic  Weights. 

Evans  Blue  (T.  1824  Eastman  Kodak  Co.). 
Used  clinically  in  man  for  estimation  of 
blood  volume.  Vital  staining  of  malig- 
nant tumors  in  man  (Brunschwig,  A., 
Schmitz,  R.  L.,  and  Clarke,  T.  H., 
Arch.  Path.,  1940,  30,  902-910).  It  is 
not  taken  in  by  red  cells  and  hence  is 
valuable  for  the  determination  of  plasma 
volume  (Gregersen,  M.  I.,  and  Schiro, 
H.,  Am.  J.  Physiol.,  1938,  121,  284-292. 
See  Blood  Cell  Volume. 


Excelsior  Brown,  see  Bismark  Brown  Y. 

Excretion  contrasted  with  secretion  (Cow- 
dry's  Histology,  p.  259). 

Exfoliative  Cytology,  see  Papanicolaou 
Techniques. 

Exogenous  Pigments,  classified  by  color, 
Lillie,  p.  134 

Extracellular  fluid  or  phase,  see  Chloride. 

Exudates,  see  Agar  infiltration  to  hold 
materials  in  place,  also  Papanicolaou 
Techniques. 

Eyes.  Techniques  easily  used  for  other 
parts  of  the  body  require  special  care  in 
the  case  of  the  eye.  When  sections 
through  the  entire  eye  are  required  it  is 
important  to  see  that  the  fixative  chosen 
penetrates  properly  and  that  the  normal 
shape  of  the  organ  is  retained.  Fixation 
by  vascular  injection  may  be  helpful  but 
it  is  not  sufficient  because  so  much  of  the 
eye  is  avascular.  After  removal  of  the 
eye  from  the  orbit,  whether  previously 
injected  or  not,  and  after  the  dissecting 
away  of  unwanted  muscular  and  other 
tissues,  it  should  be  immersed  in  the 
fixative.  This  will  harden  the  outer 
coats  somewhat.  After  a  few  minutes 
small  amounts  of  the  fi.xative  should  be 
injected  by  a  hypodermic  syringe  into 
both  chambers  choosing  locations  not  in 
the  plane  of  the  proposed  sections  and 
providing  opportunity  for  fluid  also  to 
leave.  Then,  with  a  sharp  razor  blade, 
a  deep  cut  should  be  made  to  permit  free 
entrance  of  the  fixative.  After  several 
hours,  more  of  the  tissue  on  either  side 
of  the  plane  should  be  cut  away.  Im- 
bedding in  celloidin  by  the  rapid  method 
is  preferable  to  paraffin  since  it  affords 
much  needed  support  to  the  less  dense 
parts.  Orientation  for  sectioning  is 
also  easier  in  celloidin  because  one  can 
see  through  it  fairly  well. 

If,  on  the  other  hand,  preparations  are 
needed  of  small  parts  of  the  eye  these 
parts  should  be  carefully  dissected  out 
and  the  paraffin  technique  employed. 
Much  time  will  be  saveu  by  following 
the  excellent  suggestions  made  by  S.  L. 
Polyak,  The  Retina.  Univ.  of  Chicago 
Press,  1941,  607  pp.  and  by  G.  L.  Walls 
(Stain  Techn.,  1938,  13,  69-76). 

Dr.  Polyak  in  a  letter  dated  April  19, 
1946  calls  attention  to  the  advisability 
of  soaking  celloidin  blocks  in  oil  as  first 
described  by  Apdthy,  S.,  Zeit.  f.  wis. 
Mikr.,  1912,  29,  464.  The  same  method 
is  well  presented  by  Kranse,  R.,  Enzyk. 
d.  Mikr.  Technik.,  3rd  edit.,  1926,  1, 
281.  For  the  investigation  of  perme- 
ability, oxidation-reduction  potential, 
enzyme  sj'stems,  and  such  properties, 
see  Friedenwald,  J.  S.  and  Stiehler, 
R.  D.,  Arch.  Ophth.,  1938,  20,  761-786. 
Useful  data  are  to  be  found  in  Kurzes 


FAHRENHEIT  TEMPERATURE 


122 


FECES 


Handbuch  der  Ophthalmologie  (Schieck 
and  Bruckner,  Berlin:  Julius  Springer, 
1930,  1,  882  pp.)  The  Anterior  Chamber 
is  a  favourite  site  for  tissue  trans- 
plantation. 

Frozen  sections  of  bird's  eyes.  (Oak- 
ley, C.  L.,  J.  Path.  &  Bact.,  1937,  44, 
365-368).  Fix  in  10%  formol  saline  4 
days,  in  Miiller's  fluid,  6  weeks  in  incu- 
bator, or,  in  case  speed  is  necessary,  in 
Perdrau's  fluid  4  days.  Incise  large 
eyes  to  aid  penetration.  Wash  in  run- 
ning water  at  least  24  hrs.  because 
formalin  and  bichromate  should  be  com- 
pletely removed.  Cut  eye  in  half  being 
careful  not  to  disturb  various  structures. 
12.5%  gelatin  +  1%  phenol  over  night, 
25%  24  hrs.  at  37 °C.  Employ  at  least 
25  cc.  for  each  half  eye.  Mount  with 
cut  surface  down  in  dish  containing  25% 
melted  gelatin.  Set  overnight  in  run- 
ning water  or  in  icebox  (not  refrigera- 
tor). Cut  out  block,  trim  away  excess 
gelatin.  Harden  in  large  amount  10% 
formalin,  2-3  days,  store  in  4%  formalin. 
Before  freezing  soak  15  min.  in  tap 
water.  Freeze  slowly,  over-freeze  and 
then  stain  usual  methods  but  carefully 
avoid  strong  alcohols.  They  will  stand 
70%  and  1%  HCl  provided  washing  in 
water  has  been  thorough.  Use  glycerin 
jelly  for  mounting.  Fluorescence  Mi- 
croscopy of  the  eye  is  very  revealing, 
see  Evans,  J.  N.  and  Singer,  E.,  Arch. 
Ophthal.,  1941,  25,  1007. 

Fahrenheit  Temperature  to  Centigrade. 
Use  the  following  relation: 

8  (F°-  32)  =0° 
302  °F  ±  I  (302  -  32)  =  a  (270)  =  160°  C. 
.5°F  ±  a  (5  -  32)  =  J  (-  27)  =  -  15°C. 
-  13°F  ±  §  (-  13  -  32)  =  S  (-  45)  =  -  25°C. 

Fallopian  Tubes  (oviducts,  uterine  tubes). 
References  to  many  techniques  will  be 
found  in  C.  G.  Hartman's  chapter  in 
Allen,  Danforth  and  Doisy's  Sex  and 
Internal  Secretions.  Baltimore:  Wil- 
liams and  Wilkins,  1939,  1346  pp. 

Falzone,  see  Desoxyribose  Nucleic  Acid. 

Farrant's  Medium.  Gum  arabic,  30  gm.; 
glycerin,  30  cc;  arsenous  oxide  (arsenic 
trioxide),  0.1  gm.;  aq.  dest.,  30  cc. 
There  are  many  types  of  this  medium 
differing  slightly  in  composition,  see 
Gray,  P.  and  Wess,  G.,  J.  Roy.  Micr. 
Sci.,  1950,70,287-291. 

Fast  Acid  Blue  R  (CI,  760).  An  acid  xan- 
thene  dye.  Conn  (p.  143)  says  that  it 
is  almost  the  same  as  violamine  3B  which 
contains  small  amount  of  a  red  dye.  See 
Romell,  L.  G.,  Stain  Techn.,  1934,  9, 
141-145  under  Soil,  bacteria. 

Fast  Acid  Green  N,  see  Light  Green  SF 
yellowish. 


Fast   Blue   B,    OB,    R,    etc.,   see   Indulin, 

water  soluble. 

Fast  Blue  3R,  see  Naphthol  Blue  R. 

Fast  Crimson  GR,  see  Azophloxine  GA. 

Fast  Fuchsin  G,  see  Chromotrope  2R. 

Fast  Green  FCF.  Commission  Certified. 
Closely  related  to  Light  Green  SF 
yellowish  and  recommended  as  a  sub- 
stitute because  it  fades  less. 

Fast  Oil  Orange  II,  see  Oil  Red  O. 

Fast  Phosphine  NAL,  see  Rheonine  A. 

Fast  Red,  see  Amaranth. 

Fast  Red  B,  BN  or  P,  see  Bordeaux  Red. 

Fast  Violet,  see  Gallocyanin. 

Fast  Yellow  (CI,  16)— acid  yellow,  fast 
yellow  FY,  G,  S,  BG,  etc.— An  acid 
mono-azo  dye.  Employed  by  several 
investigators,  see  use  by  Wallart,  J.  and 
Houette,  C,  Bull.  d'Hist.  Appl.,  1934, 
11,  404-407  in  rapid  trichrome  hematox- 
ylin, acid  fuchsin  fast  yellow  method. 
They  used  "Jaune  solide  G  or  GG 
(Ciba). 

Fasting.  Structural  changes  in  human  di- 
gestive tract  (Cowdry's  Histology, 
p.  305). 

Fat  Blue  B,  see  Victoria  Blue  B. 

Fat  Blue  4R,  see  Victoria  Blue  4R. 

Fat  Ponceau,  see  Oil  Red  O. 

Fat  Ponceau,  see  Sudan  IV. 

Fat  Ponceau  G,  see  Sudan  III. 

Fat  Ponceau  R  or  LB,  see  Sudan  IV. 

Fats,  see  Lipids. 

Fatty  Acids,  see  Lipids,  examination  with 
polarized  light,  also  lack  of  specificity 
of  blue  color  with  Nile  Blue  Sulphate. 
A  review  of  the  method  of  tagging  fatty 
acids  with  radioactive  isotopes  is  given 
by  Bloor  (W.  R.,  Physiol.  Rev.,  1939, 
19,  557-577). 

Feathers,  see  Ceresin  imbedding. 

Feces.  1.  To  demonstrate  ova  of  parasites 
(Mallory,  p.  301).  If  they  cannot  be 
seen  when  a  small  bit  of  feces  is  mixed 
with  water  on  a  slide  attempt  to  concen- 
trate them.  To  a  small  amount  of  feces 
add  sufficient  sugar  solution  (common 
granulated  sugar,  500 gm.,  water,  360  cc, 
phenol,  1%)  to  almost  fill  tube.  Cover 
and  gently  mix  contents.  Centrifuge 
at  1000  r.p.m.  5-6  min.  Remove  ma- 
terial from  surface  in  wire  loop  and 
examine  microscopically  for  ova. 
Another  method  is  to  use  hypertonic 
salt  solution  in  proportion  to  feces  of  not 
more  than  20:1  in  the  same  way,  remov- 
ing large  particles  as  may  be  necessary 
before  centrifuging. 

2.  To  find  segments  and  whole  adult 
worms.  Wash  feces  in  small  amount 
water  through  medium  mesh  screen, 
collect  and  examine  at  low  magnification. 
For  identification  consult  a  text  book  of 
parasitology. 

See  Floatation  Techniques,  Intestinal 
Protozoa. 


FELL 


123 


FISCHLER'S 


Fell,  see  Organ  Culture  in  Vitro. 

Ferments,  see  Enzymes. 

Ferric  Chloride-Osmic  Acid  for  demonstra- 
tion of  Golgi  apparatus  (Owens,  H.  B. 
and  Bensley,  R.  R.,  Am.  J.  Anat.,  1929, 
44,  79-100).  Fix  and  impregnate  each 
piece  of  tissue  7-10  days  at  37 °C.  in 
ferric  chloride,  0.05  gm. ;  2%  osmic  acid, 
10  cc. 

Ferrihemate,  see  Hematin. 

Fettblau  -braun,  -griin,  -orange,  -rot  and 
-Schwartz.  These  are  lipid  stains  of 
Hollborn.  For  use  of  hydrotropes 
(Hadjioloff,  A.,  Bull.  d'Hist.  Appl., 
1938,  15,  37-41). 

Feulgen  Reaction,  see  Thymonucleic  Acid. 

Fiberglass,  see  Glass  Cloth. 

Fibers.  Many  are  recognized.  See  Nerve, 
Collagenic,  Reticular,  Elastic,  Neu- 
roglia. Muscle  fibers  are  given  under 
Muscle. 

Fibrils.  These  are  really  small  fibers  many 
of  which  are  intracellular.  See  Neuro- 
fibrils, Myofibrils,  Epidermal  Fibrils, 
Fibroglia,  Myoglia. 

Fibrin.  Usually  easily  identifiable  in  Hem- 
atoxylin and  Eosin  preparations.  Wei- 
gert's  (1887)  standard  differential  stain 
for  fibrin  may  be  used  as  advised  by 
Mallory  (p.  193).  Paraffin  sections  of 
material  fixed  in  abs.  alcohol,  Carnoy 
or  Alcohol-Formalin  can  be  employed. 
If  the  fixative  contains  chrome  salts 
(Zenker,  Helly)  treat  first  with  0.25% 
aq.  potassium  permanganate,  10  min., 
then  5%  aq.  oxalic  acid,  20  min.  and 
wash  in  aq.  dest.  Stain  nuclei  with 
Lithium  Carmine.  Mix  3  cc.  of  A:  abs. 
ale,  33  cc;  anilin  oil,  9  cc.  saturated 
with  methyl  violet  (crystal  violet)  with 
27  cc.  of  B:  sat.  aq.  methyl  violet. 
Stain  5-10  min.  Drain  and  blot.  Treat 
sections  with  Gram's  Iodine,  5-10  min. 
Drain  and  blot.  Differentiate  in  equal 
parts  anilin  and  xylol  drop  by  drop  until 
purple  ceases  to  be  removed.  Blot  and 
remove  anilin  with  xylol.  Mount  in 
balsam.     Fibrin  blue-black,  nuclei  red. 

Fibroblasts.  There  is  no  specific  stain  for 
fibroblasts.  In  fresh  spreads  of  Loose 
Connective  Tissue  they  are  fairly  con- 
spicuous elements  identifiable  by  their 
large  usually  slightly  kidney  shaped 
nuclei  (possessed  generally  of  a  single 
nucleolus)  and  tapering  cytoplasmic 
processes  devoid  of  specific  granulations. 
In  sections  less  cytoplasm  is  seen  and 
it  may  be  impossible  in  some  cases  to 
identify  the  nuclei  with  assurance. 
Recognition  is  mainly  by  position  and 
the  exclusion  of  other  possibilities. 
View  the  beautiful  colored  plates  of 
Evans,  H.  M.  and  Scott,  K.  T.,  Contrib. 
to  Embryol.,  Carnegie  Inst.,  1922,  47, 
1-55  for  a  comprehensive  picture  of  the 
responses  of  fibroblasts  to  vital  stains. 


Pure  strains  of  fibroblasts  can  easily  be 
cultured,  their  behavior  watched  and 
their  nutritional  and  other  requirements 
investigated.     See  Tissue  Culture. 

Fibroglia  Fibrils.  Mallory's  Phosphotungs- 
tic  Acid  Hematoxylin  stain  for. 

Fibrous  Connective  Tissue.  Since  this  is 
much  denser  than  Loose  Connective 
Tissue  the  method  of  making  spreads 
is  not  feasible.  It  can  best  be  examined 
in  sections  of  Zenker  fixed  material 
colored  by  Mallory's  Connective  Tissue 
Stain  supplemented  by  specific  stains 
for  Elastic  Fibers. 

Figge,  see  Porphyrins,  Hematoporphyrins. 

Filament-Nonfilament  Count.  Neutro- 
philic leucocytes  are  divided  into  two 
classes:  filament,  in  which  nuclear  seg- 
ments are  connected  by  delicate  strands 
consisting  apparently  of  nuclear  mem- 
brane only  and  nonfilament  in  which 
there  are  no  filaments  the  strand  being 
so  coarse  that  it  may  be  resolved  into 
nuclear  membrane  plus  nuclear  con- 
tents. The  former  are  mature  and  the 
latter  are  less  differentiated  cells.  Ac- 
cording to  Pepper,  O.  H.  and  Farley, 
D.L., Practical  Hematological  Diagnosis, 
Philadelphia,  Saunders,  1933,  562  pp., 
8-16%of  neutrophiles  are  normally  non- 
filament  cells.  A  shift  to  the  right  is  a 
decrease  in  this  percentage.  The  count 
is  easier  to  make  than  the  Arnett  or 
Schilling  count  and  is  probably  of  equal 
value.  See  also  Nonfilament-Filament 
Ratio. 

Filterable  Viruses,  see  Victoria  Blue  4B 
and  Virus. 

Filters.  Choice  and  use  of  the  various  types 
of  filters  employed  in  the  study  of 
viruses  and  bacteria  are  well  described 
by  J.  R.  Paul  (Simmons  and  Gentzkow, 
584-586).  There  are  4  principal  kinds. 
Berkefeld.  German,  from  diatoma- 
ceous  earth.  V.  pores  8-l2fi;  N,  pores 
5-7 fi;  and  W,  pores  3-4^. 

Mandler.  American  modification  of 
Berkefeld  but  made  of  kioselguhr,  as- 
bestos and  plaster  of  Paris.  Corre- 
sponding grades  of  porosity  are  styled 
"preliminary",  "regular"  and  "fine." 
Chamberland,  French,  from  unglazed 
porcelain,  in  9  grades  of  porosity. 

Seitz.     Made  of  asbestos,  in  2  grades 
K  (coarse)  and  E.  K.  which  filters  out 
ordinary  bacteria. 
Elford.     Made  of  collodion. 

Firminger,  see  Carbowax  Embedding. 

Fischler's  modification  of  Benda's  stain  for 
fatty  acids  and  soaps  (Fischler,  F., 
Zentralbl  f.  Allg.  Path.  u.  path.  Anat., 
1904, 15,  913-917)  lias  been  severely  criti- 
cized by  Lison  (p.  203)  who  concludes 
that  it  is  of  no  microchemical  value. 

Mallory  (p.  120)  has,  however,  given 
a  somewhat  different  description  of  the 


FITE 


124 


FIXATION 


technique.  He  explains  that  since  the 
Na  and  K  fatty  acid  salts  (soaps)  are 
soluble  in  formalin,  it  is  necessary  to 
change  them  into  insoluble  Ca  soaps  by 
saturating  the  10%  formalin  fixative  with 
calcium  salicylate.  Comparison  of 
stained  sections  of  such  material  with 
others  fixed  simply  in  formalin  shows  the 
presence  and  absence  of  the  fatty  acid 
salts  (soaps) .  Calcium  soaps  can  be  dis- 
tinguished from  falty  acids  because  they 
resist  solution  in  a  mixture  of  equal  parts 
abs.  ale.  and  ether  or  in  hydrochloric 
acid  whereas  the  fatty  acids  are  soluble 
in  this  mixture  and  calcium  in  hydro- 
chloric acid.  The  method,  as  detailed 
by  Mallory,  is :  Mordant  frozen  sections 
of  10%  formalin  fixed  material  in  sat. 
aq.  copper  acetate  (12.5%),  2-24  hrs.  at 
room  temperature.  Wash  in  aq.  dest. 
Stain  20  min.  or  more  in  Weigert's  he- 
matoxylin made  up  by  mixing  1  gm. 
hematoxylin  dissolved  in  10  cc.  abs.  ale. 
with  1  cc.  sat.  aq.  lithium  carbonate 
(about  1.25%)  plus  90  cc.  aq.  dest. 
several  days  before  use.  Differentiate 
in  Weigert's  borax-potassium  ferri- 
cyanide,  (2.5gms.ferricyanideand  2gm. 
borax  plus  100  cc.  aq.  dest.)  much 
diluted  until  red  blood  cells  become 
decolorized.  Wash  thoroughly  in  aq. 
dest.  Mount  in  glycerin  jelly  or  glyc- 
erin. Fatty  acids  deep  blue  black.  Fe, 
Ca  and  hemoglobin  may  also  be  stained. 
To  stainneutralfats  inaddition  stain  with 
scarlet  red  after  washing  out  Weigert's 
fluid,  rinse  in  70%  ale.  and  in  water  and 
mount  in  glycerin. 

Fite,  see  his  method  for  Acid  Fast  Bacilli. 

Fixation  by  immersion  is  usually  the  first 
step  in  making  permanent  preparations. 
Compared  with  the  direct  microscopic 
examination  of  still  living  cells  removed 
from  the  body  and  placed  in  approxi- 
mately isotonic  media,  it  has  both  ad- 
vantages and  disadvantages.  Among 
the  first  is  the  fact  that  the  normal  form 
relations  of  the  tissue  components  are 
more  faithfully  preserved  in  large  pieces 
by  fixation;  because  it  is  not  necessary 
to  separate  the  tissue  by  teasing,  or  in 
some  other  way,  into  sufficiently  small 
or  thin  pieces  for  microscopic  study. 
Moreover,  by  fixation,  the  cells  are 
suddenly  and  uniformly  killed,  so  that 
the  changes  resulting  from  unfavorable 
fluid  environment  outside  the  body, 
leading  slowly  or  quickly  to  injury  and 
death,  are  not  encountered.  The  chief 
objection  to  fixation  is  that  the  structure 
is  very  definitely  modified  therebj*  and 
care  must  be  exercised  in  reaching  con- 
clusions as  to  living  tissues  from  the 
study  of  fixed  ones.  It  is  important  to 
restrict  these  structural  changes  to 
those  inseparable  from  the  action  of  the 


fixative  itself,  and  of  the  subsequent 
technique  under  the  most  favorable 
conditions. 

Reduce  to  a  minimum  the  time  in 
which  these  complicating  alterations  can 
occur  by  prompt  fixation.  Remove  the 
tissue  from  an  animal  under  general 
anesthesia,  or  immediately  after  it  has 
been  killed,  by  a  method  unlikely  to 
injure  the  tissues.  In  the  case  of  human 
tissues  removed  at  operation  one  should 
be  on  the  look  out  for  complicating 
factors.  If  the  tissue  is  collected  at 
autopsy  the  autopsy  should  be  made  at 
the  earliest  possible  moment  after  death. 
See  Postmortem  Changes.  If  delay  is 
unavoidable, keep  the  body,orthe  tissue, 
inaniceboxto  reduce  the  speed  of  chemi- 
cal change.  In  case  an  excised  tissue 
cannot  be  immediately  fixed,  place  it  in 
a  covered  glass  container  with  some  cot- 
ton moistened  with  physiological  saline 
solution.  Do  not  put  it  in  the  solution. 
Keep  the  container  likewise  at  a  low 
temperature. 

Carefully  avoid  injury  to  the  tissue 
from  any  cause.  Letting  its  surface  dry 
during  removal  from  the  body,  or  at  any 
time  before  fixation,  produces  Artifacts. 
So  also  does  mechanical  manipulation. 
If  forceps  must  be  used,  do  not  pinch  the 
part  of  which  the  preparation  is  to  be 
made.  It  is  better  to  lift  the  tissues. 
Scissors  tend  to  squeeze  the  tissue,  but 
it  is  necessary  to  cut  with  them  in  some 
cases.  The  ideal  way  is  to  cut  with  a 
sharp  razor  blade.  This  is  easy  with  the 
liver,  kidney,  brain  and  other  more  or 
less  compact  organs,  but  the  sweep  of  a 
razor  blade  tends  to  draw  the  tissue  and 
cause  displacement,  especially  when  the 
specimen  is  heterogeneous,  some  parts 
being  loose  connective  tissue,  others 
muscle,  others  gland,  etc.  When  feas- 
ible, cut  the  tissues  into  slices  and  lift 
them  into  the  fixative.  For  fixatives  that 
penetrate  easily  (formalin,  Zenker's  and 
Bouin's  fluids,  etc.)  make  the  slices  4-6 
mm.  thick.  For  the  poor  penetrators, 
in  which  osmic  acid  is  the  principal 
ingredient  (Bensley's  A.O.B.,  Flera- 
ming's  fluid,  etc.),  the  slices  must  be 
not  more  than  2  mm.  thick.  In  the  case 
of  surface  tissues  (skin,  gastric  mucous 
membrane,  bladder  wall,  etc.)  fix  a  strip, 
flattened  on  the  surface  of  a  piece  of 
wooden  tongue  depressor  or  stiff  paper 
card.  A  volume  of  fixative  at  least  20 
times  that  of  the  tissue  fixed  is  required. 
Agitate  the  bottle  slightly  to  prevent  the 
tissue  from  sticking  to  the  bottom  and 
to  ensure  penetration  from  all  sides. 

It  may  be  desirable  to  inject  the  fixa- 
tive via  a  large  artery  supplying  the 
tissue  to  be  examined.  This  eliminates 
mechanical  injury  to  the  tissue  before 


FIXATION 


125 


FIXATIVES 


fixation,  preserves  gross  form  relations 
better  and  is  suggested  when  sections  are 
required  of  large  specimens.  Before  in- 
jecting the  fixative  wash  out  some  of 
the  blood  by  Perfusion  with  physio- 
logical salt  solution,  or  at  least  let  the 
blood  drain  out  from  the  veins,  because, 
if  all  is  left  in,  it  may  clog  the  arteries 
and  block  the  entry  of  the  fixative. 
After  fixation  by  vascular  injection  it  is 
customary  to  cut,  with  a  razor  blade, 
suitable  slices  and  to  continue  the  fixa- 
tion by  immersion.  Obviously  such 
tissues  should  not  be  employed  for  micro- 
chemical  analyses  because  there  is  a 
danger  of  washing  out  chemical  sub- 
stances. Clearly,  also,  the  speed  of 
fixation  depends  upon  the  degree  of  vas- 
cularity. For  avascular  tissues  such  as 
epidermis,  cornea  and  cartilage  fixation 
by  injection  is  not  recommended. 

After  the  tissues  have  hardened  a 
little  by  immersion  in  or  injection  with 
the  fixative,  it  may  be  helpful  to  remove 
them  from  the  fi.xative  and  trim  them 
with  a  razor  blade  so  that  their  size  and 
shape  will  be  almost  what  is  needed  when 
they  are  finally  cut  into  sections.  The 
slices  should  have  smooth  upper  and 
lower  surfaces  including  an  area  which 
will  yield  sections  that  will  fit  nicely 
under  a  22  x  22  mm.  cover  glass  unless 
larger  covers  are  to  be  used.  The  shape 
should  be  rectangular  with  opposite  edges 
parallel.  In  general  it  is  well  to  have  two 
longer  parallel  edges  and  two  shorter 
ones,  because  a  square  surface  is  not  so 
convenient  to  section  as  an  oblong 
one.  However  one  must  bear  in  mind 
exactly  what  one  wishes  to  demonstrate. 
This  making  of  uneven  surfaces  smooth 
does  however  introduce  an  experimental 
error;  because,  where  much  is  shaved 
off,  the  fixation  has  penetrated  less  than 
where  little  or  no  tissue  has  been  re- 
moved. After  trimming  return  tissues 
to  a  fresh  supply  of  fixative.  Tissues 
fixed  in  poor  penetrators  should  not  be 
trimmed. 

The  time  of  fixation  depends  upon  the 
tissue,  the  fixative  and  the  purpose  in 
mind.  In  general,  24  hrs.  is  suitable. 
Some  fixatives,  particularly  those  con- 
taining potassium  bichromate  and/or 
osmic  acid,  are  not  very  stable  and  for 
this  reason  should  be  renewed.  The 
fixative  deteriorates  less  quickly  if  the 
fixation  is  carried  out  at  a  low  tempera- 
ture in  an  ice  box.  The  speed  of  fixation 
is  probably  also  somewhat  diminished. 
The  effect  of  pH  on  chromium  fixatives 
has  been  studied  by  Zirkle  (C,  Proto- 
plasma,  1928,  4,  201-227).  See  results 
obtained  by  adding  Wetting  Agents  and 
Hydroxybenzene  Compounds  to  fixa- 
tives.    Fixation  involving  Decalcifica- 


tion and  Mordanting  are  special  cases 
described  under  these  headings.  For 
choice  of  fi.xative  see   Fixatives. 

After  fixation  Washing  may  be  neces- 
sary, or  Mordanting.  The  tissue  may 
be  prepared  as  a  Whole  Mount,  or  Frozen 
Sections  may  be  made,  or  it  may  be 
dehydrated,  cleared  and  imbedded  in 
Paraffin  or  dehydrated  and  imbedded  in 
Celloidin  for  Sectioning. 
Fixatives.  The  number  from  which  to 
choose  is  enormous  but  the  number 
actually  employed  is  comparatively 
small.  Formalin  unquestionably  heads 
the  list  as  being  used  for  a  far  greater 


Acetic   osmic   bichro- 

Hischler 

mate 

Kleinenberg 

Alcohol  (ethyl) 

Kolatohew 

Allen 

Lactophenol 

Barium  chloride  and 

LUlie 

formalin 

Mann 

Basic  lead  acetate 

Marchi 

Bouin 

Maiimow 

Cadmium  chloride 

Mercuric  chloride 

Carnoy 

Methyl  alcohol 

Carnoy-Lebrunn 

MuUer 

Champy 

Orth 

Chloral  hydrate 

Osmic  acid 

Destin 

Parabenzoquinone 

Diaphanol 

Perenyi 

Dioxan 

Petrunkevitch 

Downey 

Regaud 

Erlicki 

Rabl 

Ferric    chloride-osmio 

Schandian 

acid 

Silver  nitrate 

Flemming 
Formalin 

Susa 

Formalin-Zenker 

Tellyesnicky 

Giemsa 

Weigert 

Gilaon 

Van  Gehuchten 

HeUy 

Zenker 

Hermann 

Zweibaum 

variety  of  purposes  than  any  other  fixa- 
tive. It  penetrates  well  and  is  an  ex- 
cellent preservative.  It  is  the  only 
satisfactory  fixative  for  use  before  the 
cutting  of  frozen  sections  and  as  a  pre- 
liminary to  certain  microchemical  re- 
actions. Alcohol  comes  next  in  variety 
of  services  performed  but  unfortunately 
it  brings  about  considerable  shrinkage. 
Both  formalin  and  alcohol  are  frequently 
combined  with  other  ingredients. 

For  routine  purposes  Zenker's  Fluid, 
either  alone  or  with  formalin,  is  perhaps 
the  most  popular  fixative.  Tissues  so 
fixed  give  better  contrasts  of  acidophilic 
and  basophilic  components  than  are 
obtained  after  fixation  in  formalin  or 
alcohol  by  themselves.  Bouin's  Fluid 
is  also  an  excellent  fixative  for  general 
use  and  is  being  employed  with  increasing 
frequency.  It  is  particularly  advocated 
by  dermatologists.     Regaud's    Fluid  is 


FLAGELLA 


126 


FLOATATION  TECHNIQUES 


the  fixative  of  choice  for  mitochondria 
because  it  penetrates  so  much  better 
than  Osmic  Acid  containing  fixatives. 
No  important  new  fixatives  have  recently 
been  devised. 

In  making  the  selection  one  is  natu- 
rally guided  by  data  concerning  the 
structures  which  it  is  desired  to  demon- 
strate (see  Nerve  Endings,  Mitochon- 
dria, etc.)  or  the  substances  to  be  re- 
vealed (Lead,  Copper,  Oxidases,  Lipids, 
etc.)  or  the  techniques  that  seem  best 
adapted  to  the  purpose  in  mind  (Mal- 
lory's  Connective  Tissue  stain,  Wei- 
gert's  Method,  etc.).  Some  of  the  more 
important  fixatives  are  listed,  further 
data  being  given  under  each  heading. 
Flagella.  1.  Of  bacteria.  Loeffler's  stain. 
Mordant  in  fresh  20%  aq.  tannic  acid, 
10  cc;  sat.  aq.  ferrous  sulphate,  5  cc; 
3-5%  basic  fuchsin  in  95%  ale,  1  cc. 
gently  heated,  1  min.  Rinse  in  water 
stain  with  slight  heat  in  Carbol  Fuchsin 
1  min.  wash  and  dry.  For  other  flagella 
stains  see  discussion  in  McClung  (pp. 
143-145)  and  Shuuk,  I.  V.,  J.  Bact., 
1920,  5, 181 ;  Galli-Valerio,  B.,  Centralbl. 
f.  Bakt.  Orig.,  1915,  76,  233;  Gray,  P.  H., 
J.  Bact.,  1926,  12,  273.  See  technique 
for  darkfield  study  of  flagella  (Pi j per, 
A.,  J.  Path.  &  Bact.,  1938,  47,  1-17). 

2.  Of  erythrocytes  (Oliver,  W.  W.,  J. 
Inf.  Dis.,  1934,  55,  266-270).  Add  1  mg. 
hirudin  to  2-3  cc.  sterile  Ringer's  solu- 
tion in  small,  sterile  test  tube.  Draw 
up  about  0.5  cc.  into  a  sterile  Pasteur 
pipette  fitted  with  rubber  bulb.  Apply 
to  drop  fresh  normal  blood  from  finger. 
Suck  up  quickly  into  pipette  and  expel 
into  test  tube.  Incubate  at  37 °C.  40-50 
min.  which  promotes  flagella  production. 
Add  small  drop  to  clean  slide  held  at 
40°  angle.  After  the  drop  has  rundown 
slide,  let  dry  completely  in  horizontal 
position  at  room  temperature.  Mor- 
dant in  fresh  10%  aq.  tannic  acid,  50  cc. ; 
sat.  aq.  ferrous  sulphate,  25  cc.  and  sat. 
ale.  basic  fuchsin,  5  cc.  which  is  poured 
on  slide  and  warmed  slightly  20  min. 
Wash  thoroughly  in  running  tap  water 
and  dry.  Flood  with  fresh  Ziehl-Neelsen 
(1  gm.  fuchsin,  10  cc.  alcohol  -j-  90  cc. 
5%aq.  phenol  acid)  20  min.  not  warmed. 
Wash  carefully  in  running  water,  blot 
dry  and  examine  with  oil  immersion. 
It  will  be  helpful  to  examine  Oliver's 
illustrations.  (Revised  by  Wade  Oli- 
ver, Dept.  of  Bacteriology,  Long  Island 
Medical  College,  Brooklyn,  N.  Y., 
1946). 

The  interpretation  of  observations  on 
bacterial  flagella  offers  many  pitfalls. 
Dubos,  R.  J.,  The  Bacterial  Cell. 
Harvard  Univ.  Press,  1945,  460  pp. 
calls  attention  to  their  fineness,  the 
slight   affinity   of  their  substance   for 


stains,  the  use  of  mordants  which  ad- 
here to  their  surface  increasing  their 
apparent  diameter  when  stained,  and 
the  fact  that  mechanical  agitation 
alone  is  sufficient  to  detach  them  from 
the  cells.  By  thus  releasing  flagella 
sufiicient  flagellar  material  can  be  col- 
lected for  immunological  study  and  the 
action  of  flagellar  antibody  on  mobile 
flagella  can  be  followed  microscopically. 
Dubos  remarks  that  the  amounts  of 
flagellar  material  available  are  too 
small  to  permit  chemical  analysis  but 
we  may  hope  that  techniques  both  of 
collecting  material  and  of  analysis  will 
be  so  improved  as  to  make  this  feasible. 
He  refers  to  numerous  papers  on  elec- 
tron microscopic  examination  of  flagella 
as  revealing  structural  details  pre- 
viously unknown. 

Details  in  the  structure  of  flagellae 
are  revealed  by  Electronmicroscopy. 
See  Brown,  H.  P.,  Ohio  J.  Sci.,  1945, 
45,  247;  and  DeRobertis,  E.,  and 
Franchi,  C.  M.,  Exp.  Cell  Res.,  1951, 
2,  295-298.  See  also  Cilia  and  Polysac- 
charides. 

Flagellates,  intestinal.  Those  commonly 
found  in  man  are,  according  to  Craig, 
p.  115,  Giardia  lamblia,  Chilomastix 
mesnili,  and  Trichomonas  hominis;  less 
frequently  seen  are  Embadomonas  in- 
testinalis  and  Enteromonis  hominis. 
Stains  much  the  same  as  for  Endameba 
and  Leishmania.  See  Craig  for  choice 
of  suitable  culture  medium. 

Flame  Photometer.  Use  of  this  instrument 
in  the  analysis  of  biological  materials 
is  critically  presented  by  Wallace, 
W.  M.  et  al.,  J.  Lab.  &  Clin.  Med., 
1951,  37,  621-629. 

Flavins  under  fluorescence  microscope  show 
green  fluorescence  in  liver  tissue.  See 
Riboflavin. 

Fleas,  see  method  of  double  imbedding  for 
(Lee,  p.  598). 

Flemming's  Fluid.  Weak:  0.25%  chromic 
acid,  0.1%  osmic  acid  and  0.1%  glacial 
acetic  acid  in  aq.  dest.  Strong:  1% 
chromic  acid,  15  cc;  2%  osmic  acid, 
4  cc. ;  glacial  acetic  acid,  1  cc.  These  are 
classic  fixatives  now  not  much  used. 
The  Bensleys  (p.  45)  advocate  same 
ingredients  differently  made  up.  A: 
1%  aq.  chromic  acid,  11  parts;  glacial 
acetic  acid,  1  part;  and  aq.  dest.,  4  parts. 
B  :  2%  osmic  acid  in  1%  aq.  chromic  acid. 
Immediately  before  use,  mix  4  parts  of  A 
with  1  part  of  B  and  employ  a  volume 
ten  times  that  of  the  tissue.  Fix  2-72 
hrs.  and  wash  in  water  24  hrs.  See 
Safranin-Gentian  Violet  and  Orange  G 
method.  Mitosis,  Benda's  Method. 

Floatation  Techniques.  Many  methods  are 
available  for  separating  helminth  eggs 
from  feces  for  microscopic  examination. 


FLORENCE'S  REACTION 


127 


FLUORESCENCE  MICROSCOPY 


They  are  floated  out  by  the  use  of  hyper- 
tonic salt  and  other  solutions,  some- 
times with  the  aid  of  centrifugal  force 
(E.  C.  Faust,  in  Simmons  and  Gentz- 
kow,  p.  684). 

Florence's  Reaction.  The  standard  test  for 
choline  in  seminal  stains.  As  described 
by  Pollak,  O.  J.  Arch.  Path.,  1943,  35, 
140-196:  Place  one  drop  semen,  or  of 
aqueous  extract  of  seminal  stain,  on 
slide.  Add  drop  of  reagent  (Pot. 
iodide,  1.65  gm.;  iodine,  2.54  gm.;  aq. 
dest.,  30  cc),  cover  and  examine  micro- 
scopically. Dark  brown,  rhombic  crys- 
tals appear,  about  25m  long  and  Sm  wide 
with  bifurcated  ends  resembling  swal- 
low tails  and  Teichmann's  hemin  crys- 
tals. In  polarized  light  these  show 
double  contours. 

Fluids.  Samples  of  body  fluids  are  often 
presented  for  microscopic  examination. 
In  a  human  being  containing,  say,  100 
lbs.  of  water  the}^  are  naturally  of  great 
variety  even  under  normal  conditions. 
Abnormal  fluids  are  usually  described 
as  transudates  or  exudates.  The  for- 
mer compared  with  the  latter  are 
mainly  filtrates,  are  more  watery,  have 
lower  specific  gravity,  less  albumin,  no 
bacteria  and  are  the  result  of  mechani- 
cal forces  rather  than  inflammation. 
See: 

Aqueous  humor  Intracellular  phase 

Cerebrospinal  Pericardial 

Duodenal  Peritoneal 

Endolymph  Pleural 

Extracellular  phase  Sjiiovial 

Tissue 

Fluoran  Derivatives.  As  explained  by  Conn 
(p.  144)  fluoran  is  not  a  dye  but  a  prod- 
uct of  phthalic  anhydride  containing  a 
xanthene  ring  and  a  lactone  ring  with 
introduced  hydroxyl  groups  and  halogen 
atoms  in  particular  positions.  Ex- 
amples :  eosin  B  and  Y,  erythrosin 
bluish  and  yellowish,  ethyl  eosin, 
fluorescein,  mercurochrome  220,  methyl 
eosin,  phloxine,  phloxine  B,  rose  bengal. 

Fluorescein  (CI,  766)  is  simplest  fluoran 
dye.  It  stains  very  poorly  but  is  highly 
fluorescent.  Its  sodium  salt  is  called 
uranin. 

Fluorescence  Microscopy.  Details  pro- 
vided by  Dr.  W.  L.  Simpson  of  The 
Barnard  Free  Skin  and  Cancer  Hospital. 
Supplemented  by  Dr.  T.  B.  Rosenthal, 
Dept.  of  Anatomy,  Washington  Univer- 
sity, St.  Louis.  June  6,  1951. 

Fluorescence  is  the  property,  pos- 
sessed by  many  substances,  of  convert- 
ing short  wavelengths  of  light  into 
longer  wavelengths.  In  the  field  of 
microscopy  those  structures  and  sub- 
stances are  of  most  interest  that  convert 
ultraviolet  light  into  light  of  the  visible 
spectrum,  since  it  is  only  these  sub- 


stances that  can  be  observed  directly. 
Though  fluorescence  microscopes  de- 
signed for  this  type  of  observation  have 
been  available  commercially  for  many 
years,  their  use  has  been  limited  until 
recently  by  their  relatively  high  cost 
and  by  the  apparent  failure  of  biolo- 
gists to  appreciate  the  possibilities  of 
this  type  of  observation.  Recent  tech- 
nological developments  in  the  glass 
and  electric  lamp  industries  now  make 
it  possible  to  assemble  an  apparatus 
for  fluorescence  microscopy  at  a  cost 
well  within  the  budget  of  most  labora- 
tories. Evidence  of  heightened  interest 
in  this  field  is  found  in  the  numerous 
papers  concerning  fluorescence  micros- 
copy within  the  past  10  years.  Al- 
though several  reviews  of  the  subject 
already  exist  (Haitinger,  M.,  Flu- 
orescenz-Mikroscopie,  Akademische 
Verlagsgesellschift,  Leipzig,  1938;  Ham- 
perl,  H.,  Virchows  Arch.  f.  path.  Anat., 
1934,  292,  1-51;  Sutro,  C.  J.,  Arch. 
Path.,  1936,  22,  109-112;  and  McClung's 
Handbook  of  Microscopical  Technique, 
New  York,  Paul  B.  Hoeber  Inc.,  1937), 
the  technique  will  be  described  as  it 
can  be  used  with  an  assembly  of  low 
cost  apparatus  available  in  the  United 
States  at  the  present  time. 
Apparatus  required: 

1.  An  intense  source  of  ultraviolet 
light  that  is  rich  especially  in  the  region 
from  300  to  400  millimicrons.  Certain 
electric  arcs  using  electrodes  of  special 
metal  alloys  (the  Haitinger  Arc,  C. 
Reichert — Vienna)  have  been  developed 
for  this  purpose.  More  easily  avail- 
able, low  in  cost,  and  having  an  intense 
output  in  the  desired  region,  are  the 
medium  pressure  mercury  vapor  arcs  in 
capillary  quartz  tubes  (the  A  H  4  lamp 
of  the  General  Electric  Company  or 
Westinghouse  Electric  Co.  and  lamps 
made  by  Hanovia  Chemical  Co.,  etc.). 

2.  Filters  that  remove  all  or  nearly 
all  of  the  visible  light.  A  considerable 
selection  of  glass  and  liquid  filters  may 
be  used  for  this  purpose.  Since  most 
of  the  so-ca,lled  ultraviolet  filters  pass 
also  a  certain  amount  of  red  light, 
supplemental  blue  filters  must  be  used 
with  them.  A  solution  of  copper  sulfate 
in  a  cell  or  tube  of  quartz,  or  of  ultra- 
violet transmitting  glass,  is  satisfactory 
and  readily  available.  A  combination 
of  Shott  glass  filters  U  G  2  and  B  G  14 
are  recommended  by  Jenkins  (R.,  Stain 
Techn.,  1937,  12,  167-173).  Corning 
Filters  ^5840,  5860,  or  5874  used  with  a 
copper  sulfate  solution  are  satisfactory 
in  our  experience.  An  entirely  liquid 
filter,  using  solutions  of  cobalt  sulfate 
and  nickel  sulfate,  is  described  by 
Backstrom  (H.  L.  J.,  Arkiv.  for  Kemi. 


FLUORESCENCE  MICROSCOPY 


128 


FLUORESCENCE  MICROSCOPY 


Mineralogi    Och    Geologi,    1940,    13A, 
1-16). 

3.  Condensing  lenses,  if  used  at  all, 
must  be  of  quartz  or  ultraviolet  trans- 
mitting glass. 

4.  A  quartz  prism  or  mirror  of  polished 
metal  having  a  high  reflecting  power  for 
ultraviolet.  Aluminum  and  magne- 
sium-aluminum alloys  are  best  for  this. 
By  mounting  the  microscope  and  light 
source  horizontally  this  item  can  be 
eliminated. 

5.  An  ordinary  microscope  that  is 
fitted  with  a  substage  condenser  of 
quartz  or  ultraviolet  transmitting  glass. 
Since  the  ultraviolet  light  has  served  its 
purpose  when  it  has  reached  the  tissue, 
ordinary  glass  objectives  and  eyepieces 
are  used.  With  some  older  objectives 
the  balsam  of  the  lenses  fluoresces  in 
ultraviolet  and  causes  an  unpleasant 
diffuse  light  to  appear  in  the  microscope 
that  masks  the  fluorescence  of  the  tissue. 
This  may  be  eliminated  with  a  darkfield 
stop  that  prevents  direct  rays  of  ultra- 
violet light  from  entering  the  objective. 
Newer  lenses  are  free  from  this  fluores- 
cence and  may  be  used  without  a  dark- 
field  stop.  This  is  desirable  since  it 
permits  the  utilization  of  a  greater  por- 
tion of  the  light  that  strikes  the  con- 
denser. Popper  has  reported  that  the 
fluorescence  of  Vitamin  A  can  be  ob- 
served with  an  ordinary  microscope  with 
glass  condenser.  Ordinary  optical  glass 
transmits  sufficiently  far  into  the  near 
ultraviolet  that  this  type  of  apparatus 
might  be  successfully  used  for  strongly 
fluorescent  substances. 

6.  Slides  for  the  specimens  of  ultra- 
violet transmitting  glass.  (Corex  D 
glass  slides,  obtainable  from  Corning 
Glass  Co.  are  suitable.) 

7.  An  eyepiece  filter  that  excludes 
ultraviolet  light  with  a  minimum  ab- 
sorption of  visible  light.  This  may  be 
of  glass  (Leitz  ultraviolet  protecting 
filter  no.  8574  A,  Corning  Glass  Works 
filters  no.  3389  or  3060)  or,  simplest  and 
cheapest,  a  circle  of  Wratten  2A  gelatin 
filter  cut  to  fit  within  the  eyepiece  (the 
Wratten  2  filter  is  not  suitable  since  it 
fluoresces  itself  in  ultraviolet  light). 

8.  Non-fluorescent  media  for  mount- 
ing the  section  to  be  examined.  Me- 
dicinal mineral  oil,  or  glycerin  is  suit- 
able. If  immersion  lenses  are  to  be  usep 
a  non-fluorescing  immersion  medium 
must  be  employed.  Sandlewood  oil  has 
been  recommended  for  this  purpose. 

Preparation  of  tissues:  Hamperl  (loc. 
cit.)  recommends  that  tissues  for  fluores- 
cence examinations  be  fixed  only  in  a 
dilute  solution  of  formalin,  since  metal 
containing  fixatives  destroy  the  fluores- 
cence  of    some    substances.     A   5-10% 


solution  of  U.S. P.  formalin  in  aq.  dest. 
is  ordinarily  employed.  Tissues  should 
should  not  be  fixed  longer  than  24  hrs.; 
certain  components  of  tissue  acquire 
abnormal  fluorescence  if  the  time  of 
fixation  is  prolonged.  If  fats  and  other 
alcohol  soluble  substances  are  to  bo  ex- 
amined, i.e.,  vitamin  A,  polycyclic 
organic  carcinogens,  etc.,  frozen  sections 
must  be  made.  If  these  substances  are 
not  of  interest,  the  tissue  may  be  de- 
hydrated, cleared,  and  imbedded  in 
paraflan  in  the  usual  manner.  High 
quality  reagents  are  required,  because 
the  impurities  found  in  many  organic 
substances  themselves  fluoresce.  All 
paraffin  must  be  removed  since  this  too 
fluoresces.  The  section  can  be  cleared 
in  anhydrous  glycerin  or  pure  medicinal 
mineral  oil.  Gelatin  and  celloidin  are 
not  recommended  for  imbedding  because 
of  their  fluorescence. 

Two  types  of  fluorescence  may  be  pro- 
duced in  tissues  with  this  type  of  appa- 
ratus. The  first  is  that  seen  in  tissues 
that  have  been  subjected  to  no  special 
treatment  and  is  due  to  the  presence  of 
fluorescent  substances  in  the  tissues 
themselves.  This  is  termed  "primary" 
fluorescence  or  natural  fluorescence  and 
is  exhibited  by  many  substances  found 
in  animal  organisms.  In  most  tissues 
there  are  present  sufficient  quantities  of 
these  materials  to  permit  the  observer  to 
recognize  the  general  structure  of  the 
tissue  without  recourse  to  stained  con- 
trol sections  studied  with  transmitted 
visible  light.  Hamperl  {loc.  cit.)  de- 
scribes, in  considerable  detail,  the 
natural  fluorescence  of  many  human 
tissues.  Jenkins  (loc.  cit.)  summarizes 
the  findings  in  the  most  common  animal 
tissues.  Cornbleet  and  Popper  (T.and 
H.,  Arch.  Dermat.  &  Syph.,  1942,  46, 
59-65)  review  the  natural  fluorescence 
of  human  skin.  Popper  and  his  co- 
workers have  contributed  a  series  of 
papers  on  the  fluorescence  of  vitamin  A 
in  animal  tissues  (Popper,  H.,  J.  Mt. 
Sinai  Hosp.,  1940,  7,  119-132.  Arch. 
Path.,  1941,  31,  766-802;  Popper,  H.  and 
Brenner,  S.,  J.  Nutrition,  1942,  23,  431- 
443;  Popper,  H.  and  Ragins,  A.  B., 
Arch.  Path.,  1941,  32,  258-271).  Simp- 
son and  Cramer  (W.  L.  and  W.,  Cancer 
Research,  1943,  3,  362,  515,  604)  have 
used  the  method  to  follow  the  distri- 
bution and  persistence  of  methylcholan- 
threne  in  skin.  Fluorescence  color  of 
various  tissues  and  of  drugs  in  tissue 
sections  is  described  by  Helander,  S., 
Acta  Physiol.  Scand.,  1945,  10,  Suppl. 
29,  103  pp. 

Another  kind  of  fluorescence  is  the 
"secondary"  fluorescence  that  appears 
in  certain  components  of  the  tissue  after 


FLUORESCENCE  MICROSCOPY 


129 


FOOT'S  METHODS 


sensitization  with  dyes  and  plant  ex- 
tracts. This  extends  considerably  the 
range  of  fluorescence  microscopy  and  has 
been  developed  chiefly  by  Haitinger 
(loc.  cit.)  in  conjunction  with  Hamperl 
and  Linsbauer.  Various  fluorescent  al- 
kaloids, azo  dyes,  primulins,  auramine, 
berberine  sulfate,  chelidonium,  rhubarb 
extracts,  etc.,  are  selectively  absorbed 
by  certain  parts  of  the  cell  and  cause 
them  to  show  characteristic  fluorescences 
in  ultraviolet  light.  Such  substances 
are  called  fluorochroraes.  Sections  of 
tissue  are  immersed  in  such  substances 
for  a  short  period  of  time  before  being 
examined.  Examples  of  the  use  of  these 
fluorochromes  are  found  in  papers  by 
Haitlinger  (loc.  cit.),. Jenkins  (loc.  cit.), 
Clark  and  Perkins  (W.  M.  and  M.E.,  J. 
Am.  Chem.  Soc,  1932,  54,  1228-1248), 
Lewis  (M.  R.,  Arch.  f.  exp.  Zellf.,  1935, 
17,  96-105)  and  Popper  (H.,  J.  Mt. 
Sinai  Hosp.,  1940,  7,  119-132).  A  good 
account  of  fluorescence  microscopy  of 
insects  is  given  by  Metcalf,  R.  L..  and 
Patton,  R.  L.,  Stain  Techn.,  1944,  19, 
11-27. 

Noteworthy  applications  have  been 
made  in  the  use  of  acridine  derivative 
dyes.  Krebs  and  Gerlach  (Am.  J. 
Roentgenol.  Rad.  Therap.,  1951,  65, 
93-98)  have  demonstrated  that  acridine 
orange,  C.I.  788,  is  a  sensitive  indicator 
of  the  viability  of  cells  following  ther- 
mal and  radiational  damage.  The 
same  dye  was  used  by  Zeiger  and  Har- 
ders  (Z.  Zellforsch.,  1951,  36,  62-78) 
for  flurochroming  of  nerve  tissues.  In 
a  comprehensive  investigation  of  24 
diaminoacridine  stains  by  DeBruyn, 
Robertson  and  Farr  (Anat.  Rec,  1950, 
108,  279-307)  it  was  found  that  nuclei 
of  many  tissues  and  organs  were  vitally 
fluorochromed  without  perceptible  toxic 
or  degenerative  effects. 

Primulin  is  recommended  by 
Schmidt-LaBaume  and  Jager  (Arch.  f. 
Dermat.  Syph.,  1939,  179,  531)  in  an 
ingenious  method  for  visualizing  sur- 
face detail  of  the  epidermis. 

A  most  interesting  method  of  demon- 
strating the  distribution  of  the  right 
and  left  coronary  arteries  is  given  by 
Peterson  and  Gibson  (Med.  Radiogr. 
Photogr.,  1951,  27,  14-17),  who  inject 
the  vessels  with  plastics  containing 
red  and  green  fluorescing  pigments. 
The  heart  is  then  encased  in  a  plastic 
shell,  following  which  the  tissues  are 
digested  by  KOH.  Finally,  the  shell 
holding  the  arterial  cast  is  filled  with 
the  transparent  plastic.  Under  ultra- 
violet illumination  the  arterial  system 
is  revealed  with  extraordinary  clarity. 
The  use  of  fluorescein  dyes  in  the  diag- 
nosis of  malignancy  with  special  refer- 


ence to  tumors  of  the  central  nervous 
system  is  described  by  Hubbard,  T.  B. 
and  Moore,  C.  E.,  J.  Nat.  Cancer 
Inst.,  1949,  10,  303-314  (good  bibliog- 
raphy). The  Diaminoacridines  are  im- 
portant vital  stains  for  nuclei  in  the 
sense  that  they  accumulate  within 
nuclei  without  toxic  action  and  can 
there  be  revealed  b}'  fluorescence 
microscopy  (de  Bruyn,P.  P.  H.,  Robert- 
son, R.  C.  and  Farr,  R.  S.,  Anat.  Rec, 
1950,  108,  279-307).  See  Vitamin  A, 
Tubercle  Bacillus,  Cell  Injury,  Ura- 
nium, Porphyrins,  etc. 

Fluorescence  Spectra.  The  technique  in 
some  detail  is  described  for  3:4-Benz- 
pyrene  by  Hieger,  I.,  Am.  J.  Cancer, 
1937,  29,  705-714  who  thinks  that  the 
photographs  of  the  spectra  can  well  be 
studied  by  simple  visual  examination. 

Fluorescent  Blue,  see  Resorcin  Blue. 

Fluorescent  X.  A  special  type  of  reduced 
neutral  red  (Clark,  W.  M.  and  Perkins, 
M.  E.,  J.  Am.  Chem.  Soc,  1932,  54, 
1228-1248)  employed  for  tissue  cultures 
(Lewis,  M.  R.,  Arch.  f.  exp.  Zelf., 
1935,  17,  96-105). 

Fluorine,  see  Atomic  Weights. 

Fluorochromes.  See  Fluorescence  micros- 
copy. 

de  Fonbrune  pneumatic  micromanipulator 
can  be  obtained  from  Aloe  Scientific, 
5655  Kingsbury,  St.  Louis  12,  Mo. 

Foods.  The  examination  of  foods  to  ascer- 
tain their  suitability  for  human  con- 
sumption involves  not  only  organolep- 
tic tests  (use  of  unaided  senses,  sight, 
smell,  taste,  etc.),  but  direct  micro- 
scopic examination  and  certain  cul- 
tural, experimental  feeding,  and  other 
tests.  The  techniques  for  adultera- 
tions, bacteria,  fungi,  crystals,  spores, 
parasites  and  so  on  are  usually  the 
routine  ones.  However,  much  time 
will  be  saved  by  knowledge  as  to  what 
to  look  for  in  each  case,  how  to  look  and 
the  best  means  of  making  the  observa- 
tions accurately  quantitative  (Schnei- 
der, A.,  The  Microbiology  and  Micro- 
analysis of  Foods.  Philadelphia:  P. 
Blakiston's  Son  &  Co.,  1920,  262  pp.). 

Foot's  Methods.  1.  Rapid  silver  impreg- 
nation of  reticular  fibers  (Foot,  W.  C, 
J.  Tech.  Meth.,  1929,  12,  117-119). 
Fix  in  10%  formalin  (not  necessarily 
neutral),  Zenker's,  Bouin's  or  Helly's 
fluids,  24  hrs.  Make  paraffin  sections. 
Remove  mercury,  if  present,  with  iodine. 
Treat  with  0.25%  potassium  permanga- 
nate, 5  min.  and  5%  oxalic  acid,  10  min. 
Wash  in  aq.  dest.  Impregnate  15  min. 
in  following  silver  solution  at  50°C. :  Add 
40  cc  5%,  aq.  NajCOa  to  10  cc  10%  aq. 
AgNOa.  Let  precipitate  settle.  De- 
rant  supernatant  fluid.  Make  up  to 
50  cc.  with  aq.  dest.  Shake,  repeat  sett- 


FORAMINIFERA 


130 


FORMALIN 


ling  and  decanting.  Dissolve  ppt.  in 
just  sufficient  NH4OH,  added  drop  by 
drop,  to  almost  completely  dissolve  it 
leaving  a  few  gray  granules.  Heat  to 
steaming  to  drive  off  excess  NH3  and 
cool  to  50 °C.  Reduce  in  1%  formalin 
2  min.  Wash  in  tap  water.  Tone2min. 
in  0.2%  aq.  gold  chloride.  Wash.  Tone 
in  5%  aq.  sodium  thiosulphate.  Coun- 
terstain  with  hematoxylin-Van  Gieson. 
Reticulum,  black;  collagenic  fibers, 
Vermillion;  cytoplasm,  yellow;  and 
nuclei,  brown. 

(2).  Silver  method  for  nerve  fibers  in 
paraffin  sections  (Foot,  N.  C,  Am.  J. 
Path.,  1932,  8,  769-775).  This  is  a 
modification  of  Cajal's  technique.  Fix 
in  fresh  Carney's  Fluid  for  24  hrs. 
Transfer  to  absolute  alcohol  for  1-2  hrs., 
clear  in  chloroform  and  imbed  in  par- 
affin. Remove  paraffin  from  sections  in 
usual  way.  Treat  with  2  parts  pyridine 
and  1  part  glycerol  for  1-12  hrs.  Wash 
in  95%  alcohol  and  then  in  aq.  dest.  to 
remove  most  of  pyridine.  A  trace  re- 
maining does  no  harm.  Immerse  in 
10%  aq.  silver  nitrate  at  37 °C.  for  12  hrs. 
or  so  covering  container  to  avoid  evapo- 
ration. Wash  in  2  changes  aq.  dest. 
Place  in  5%  aq.  neutral  formalin  con- 
taining 0.5%  pyrogallol  in  which  sections 
become  yellow-brown,  20  min.  Wash 
under  tap.  Tone  in  1:500  aq.  gold 
chloride,  5  min.  (Nuclear  precision  is 
improved  and  glacial  impregnation  is 
made  less  intense  if  2%  glacial  acetic  is 
added  to  gold  solution).  Then  place  in 
2%  oxalic  acid  containing  1%  neutral 
formalin  for  5  min.  Wash  at  the  tap  and 
transfer  to  5%  aq.  sodium  thiosulphate 
for  5  min.  Finally  washagain  in  running 
water,  dehydrate,  clear  and  mount. 
Foot  also  recommends  Rogers'  technique 
practically  as  given  by  him  (Rogers, 
W.  M.,  Anat.  Rec,  1931,  49,  81-85) 
The  idea  of  intensifying  the  gold  toning 
with  oxalic  acid  he  credits  toLaidlaw, 
G.  F.,  Am.  J.  Path.,  1929,  5,  239-247. 
See  general  remarks  on  Silver  Methods. 

Foraminifera,  see  Carpenter,  W.  B.  The 
Microscope  and  its  Revelations,  Lon- 
don, 1901. 

Formaldehyde  is  a  gas  (HCOH)  soluble  to 
40%  in  water  producing  a  solution  which 
is  termed  commercial  formalin  or  for- 
mol.     See  Formalin. 

Formalin  (Formol)  is  a  37%  aq.  solution  of 
the  gas,  formaldehyde.  Solutions  em- 
ployed as  fixatives  and  preservatives 
are  made  in  terms  of  the  percentage  of 
formalin,  not  of  formaldehyde.  Thus, 
a  10%  solution  of  formalin  (formol)  is  by 
convention  10  cc.  of  formalin  plus  90  cc. 
of  water.  It  is  not  however  10%  for- 
maldehyde. (Obviously  to  dilute  10  cc. 
cone,  hydrochloric  acid  with  90  cc.  of 


water  would  not  give  10%  hydrochloric 
acid  because  cone,  hydrochloric  acid  is 
not  100%,  so  that  this  practice  cannot  be 
extended.)  Formerly  it  was  necessary 
to  neutralize  commercial  formalin  in 
various  ways,  and  it  still  is  for  special 
purposes.  The  best  way  to  obtain 
neutral  formalin  is  to  distil.  Atkins 
(Lee,  p.  61)  advises  addition  of  borax 
to  the  diluted  formalin  until  it  shows  a 
good  red  color  with  phenolphthalein  or 
slaty  blue  with  thymol  blue.  Others 
simply  add  a  little  calcium,  sodium, 
magnesium  or  even  lithium  carbonate. 
Obviously  the  addition  of  such  minerals 
greatly  complicates  the  problem  when 
formalin  is  employed  with  alcohol  as  a 
fixative  preliminary  to  microincinera- 
tion. Lillie  (p.  28)  specifies  a  neutral 
buffered  formaldehyde  solution  (pH 
70)  as  follows:  37-40%  formaldehyde 
solution,  100  cc;  water,  900  cc;  acid 
sodium  phosphate,  monohydrate,  4  gm. ; 
anhydrous  disodium  phosphate,  6.5 
gm.  Constant  pH  is  desired.  Unless 
neutral  formalin  is  specified  and  the 
manner  of  neutralization,  it  is  best 
simply  to  use  the  fairly  pure  product 
which  now  can  readily  be  obtained. 
Experiments  by  Davenport,  H.  A., 
Stain  Techn.,  1934,  9,  49-52  show  that 
as  a  neurological  fixative  slightly  acid 
formalin  is  somewhat  better  than  neu- 
tral formalin.  A  few  of  the  manj^  de- 
mands for  formalin  as  a  fixative  will 
be  found  under: 


Alizarin  red  S 

Alveolar  pores 

Amyloid 

Argentaffine  cells 

Arsenic 

Articular  nerve  ter- 
minals 

Bile  pigments 

Bismuth 

Bodian 

Bone 

Buzaglo 

Cajal's  brom-formol 

Cartilage 

Chitin 

Chloride 

Chorioallantoic  mem- 
brane 

Christeller-Koyama 

Chromaffin  reaction 

Color  preservation 

Connective  tissue  cells 

Dopa  oxidase 

Fatty  acids 

Fluorescence  micros- 
copy 

Foot 

Frozen  sections 


Giemsa  staining 

GUa  staining 

Gomori 

Gordon 

Grieves 

Johnson's  neutral  red 

Kinney 

Krajian 

Liebermann  -   Burch- 

ardt 
Madder  staining 
Mallory's     connective 

tissue  stain 
Microglia 
Mucus 

Nile  blue  sulphate 
Perdrau 
Pia  mater 
Romieu 

Schultz  cholesterol 
Sebaceous  glands 
Silver  citrate 
Smith- Dietrich 
Spirochetes 
Vorhoeff 
Weigert-Pal 
Weil 
Wilder 


FORMALIN-ALCOHOL 


131 


FROZEN  SECTIONS 


In  combination  with  other  reagents 
formalin  is  also  a  good  fixative  cf.  For- 
malin-Zenker  in  which  formalin  is  sub- 
stituted for  acetic  acid,  Bouin,  Regaud's 
Fluid  and  many  others.  Since,  how- 
ever, formalin  is  a  strong  reducing  agent 
mixtures  of  which  it  is  a  part  are  un- 
stable so  that  it  must  be  added  immedi- 
ately before  use.  As  Mallory  (p.  40) 
points  out,  formalin  also  has  certain 
disadvantages.  It  is  inferior  to  alcohol 
as  a  preservative  for  iron  and  other 
pigments.  It  often  changes  the  color  of 
bile  concretions  from  yellow  to  green  and 
it  may  produce  in  the  tissues  a  trouble- 
some brown-black  finely  divided  crystal- 
line precipitate  from  laked hemoglobin. 
He  advises  removal  of  this  precipitate 
by  treating  sections  for  30  min.  in  75% 
alcohol,  200  cc;  plus  25-28%  ammonia 
water,  1  cc.  (Schridde's  method),  or  for 
10  min.  in  80%  alcohol,  100  cc.  plus  1% 
aq.  potassium  hydroxide,  1  cc.  (Vero- 
cay's  method)  after  either  of  which  they 
are  washed  thoroughly  in  water  before 
placing  in  80%  alcohol  and  staining. 
When  employed  as  a  preservative  con- 
centration of  formalin  should  be  4%. 

Formalin-Alcohol,     see    Alcohol-Formalin. 

Formalin-Zenker.  Zenker's  fluid  modified 
by  substituting  5%  formalin  in  place  of 
the  5%  acetic  acid.  It  is  also  known 
as  Helly's  fluid  and  Zenker-formol. 
This  is  one  of  the  three  major  routine 
fixatives  the  others  being  Zenker  and 
Bouin.  See  Acid  Fast  Bacilli,  Alveolar 
Pores,  Arteries,  Basal  Bodies,  Brazilin- 
Wasserblau,  Mucus,  Goodpasture's 
Method,  Methyl-Green  Pyronin.  In 
some  cases  10%  formalin  is  inserted 
instead  of  5%. 

Formalose  see  Formalin. 

Formamide  of  Eastman  Kodak  Co.  is  a  sub- 
stance, called  a  "modifier",  which  when 
added  in  10%  to  50%  alcohol  improves 
fixation  and  staining  of  peripheral  nerve 
(Bank,  E.  W.  and  Davenport,  H.  A., 
StainTech.,1940,  15,  9-14). 

Formazan,  see  Triphenyltetrazolium  Chlo- 
ride. 

Formic  Acid,  see  Decalcification. 

Formol  is  a  synonym  for  formalin. 

Formol-Miiller,  This  is  1  part  of  formol  to 
10  parts  MuUer's  fluid. 

Formol-Nitric  fixative.  3  parts  10%  for- 
malin and  1  part  10%  nitric  acid.  This 
has,  according  to  McClung,  proved  very 
valuable  for  chick  embryos. 

Formol-saline  is  the  fluid  resulting  when 
formalin  is  diluted  with  isotonic  salt 
solution  (presumably  0.85%  aq.  sodium 
chloride)  instead  of  with  aq.  dest.  It  is 
not  advised  as  a  fixative. 

Formvar,  a  plastic  occasionally  employed 
to  make  supporting  films  in  electron 


microscopy.     It  is  made  by  Shawinigan 
Products  Corporation,  New  York,  N.  Y. 

Fowl  Pox,  see  Borrel  Bodies. 

Fractionation,  see  collection  of  cell  com- 
ponents under  Centrifugation. 

Fractures.  Vital  staining  with  Alizarin 
Red  S  (Schour,  et  al.,  J.  Dent.  Res., 
1941,20,411-418). 

Fragility    Tests.    Micro    for     erythrocytes 
(Kato,  K.,  J.  Lab.  &  Clin.  Med. ,1940, 
26,  703-713.  See  Capillary  Fragility  Tests. 

Freezing.  Details  on  the  formation  of 
ice  within  cells  are  given  by  Chambers, 
R.  and  Hale,  H.  P.,  Proc.  Roy.  Soc, 
B,  1932,  110,  336.  See  Revival  after. 

Freezing  and  Drying,  see  Altmann-Gersh. 

Freifeld's  Blood  Stain.  To  20  cc.  tap  water 
add  7  drops  Ziehl's  Carbol  Fuchsin 
and  5  drops  1%  aq.  methylene  blue. 
Stain  methyl  alcohol  fixed  blood  smears 
1  hr.  Shows  clearly  basophilic  granules 
of  toxic  neutrophiles  (Naegeli,  O., 
Zlutkrankheiten  und  Blutdiagnostik, 
Berlin,  1931,  p.  19,  quoted  from  Mc- 
Clung, 1950,  p.  225). 

Frozen  Sections.  These  are  of  great  value 
when  preparations  must  quickly  be 
made  and  when  methods  of  alcoholic 
dehydration  before  sectioning  are  contra- 
indicated.  They  are  specified  elsewhere 
in  this  book  under  several  headings 
including : 

Amyloid  Lipase 

Cholesterol  Lipids 

Digitonine  reaction  Microglia 

Dopa  oxidase  Millon's  reaction 

Gold  Oxidase 

Indigo-carmine  Pepsin 

Krajian'a  Congo  stain  Spirocheta  pallida 

Liebermann-Burchardt  Urease 

To  make  the  sections  take  recently 
excised  still  living  tissue,  or  better  fresh 
tissue  fixed  for  about  30  min.  in  10% 
formalin.  First  freeze  a  little  water  on 
the  block  of  a  freezing  microtome. 
Then  add  the  tissue  and  freeze  it  too 
plus  a  drop  more  of  water.  Allow  block 
to  thaw  to  optimum  consistency,  cut 
sections  15-50m  thick  as  desired,  and 
remove  them  with  a  camel's  hair  brush 
from  the  microtome  knife  to  formalin, 
water  or  physiological  saline.  When 
many  are  required,  it  may  be  necessary 
to  freeze  several  times  as  the  tissue  be- 
comes too  soft.  If  thinner  sections  are 
wanted  resort  to  Gelatin  Imbedding 
before  sectioning. 

For  quick  staining  Thibaudeau,  A.  A., 
J.  Lab.  &  Clin.  Med.,  1933,  19,  204-209 
advises  that  sections  of  formalin  fixed 
tissue  be  stained  in  Harris'  hematoxylin 
5-15  sec,  rinsed  in  aq.  dest.,  blued  in 
aq.  dest.  -f  few  drops  NH4OH,  passed 
up  through  70,  85  and  95%  alcohol 
counterstained  in  absolute  alcohol  and 


FROZEN  SECTIONS 


132       FROZEN-DEHYDRATION  METHOD 


eosin  (5  sec),  cleared  in  carbol  xylol, 
blotted  with  filter  paper  and  mounted 
in  balsam.  Proescher,  F.,  Proc.  Soc. 
Exp.  Biol,  and  Med.,  1933,  31,  79-81 
recommends  pinacyanol  as  giving  ex- 
cellent color  contrasts.  Perhaps  the 
simplest  method  advised  by  the  Bens- 
leys  (p.  138)  is  to  stain  the  sections  in 
Goodpasture's  Acid  Polychrome  Meth- 
ylene Blue  (which  see)  1  min.  or  longer, 
wash  and  mount  in  aq.  dest.  This 
colors  nuclei  dark  purple  and  connective 
tissue  bright  rose  red.  But  methylene 
blue  is  less  permanent  than  hema- 
toxylin. Adamstone  and  Taylor  (Stain 
Techn.  1948,  23,  109-116)  have  devel- 
oped a  modified  technique  useful  for 
histochemical  and  routine  studies. 

For  reticular  and  collagenic  fibers  in 
frozen  sections  proceed  as  follows 
(Krajian,  A.  A.,  Arch.  Path.,  1933,  16, 
376^378) :  After  fixation  in  10%  for- 
malin, cut  sections  5-10  microns  and 
wash  in  aq.  dest.  Then  10%  aq. 
NH«OH  at  60°C.,  15  min.  Wash  in  3 
changes  aq.  dest.  and  place  in  0.3% 
KMn04  for  5  min.  Rinse  in  aq.  dest., 
decolorize  in  1.5%  oxalic  acid  until 
brown  color  has  entirely  disappeared. 
Wash  4-5  times  in  aq.  dest.  and  soak 
in  5%  AgNOj  at  60°C.  for  1  hr.  Wash 
twice  in  aq.  dest.  Transfer  to  ammoni- 
acal  silver  sol.  (to  make  add  6  drops  10% 
NaOH  to  8  cc.  10%  AgNO,.  Then  add 
freshly  prepared  10%  NH4OH  drop  by 
drop  until  almost  entirely  clear.  Dilute 
to  28  cc.  with  aq.  dest.)  16  min.  at  60°C. 
Wash  3  times  quickly  in  aq.  dest. 
Change  to  30  cc.  formaldehyde  +  70  cc. 
aq.  dest.  1-3  min.  at  60 °C.  Wash  in 
tap  water.  Mount  on  slide.  Dehydrate 
with  a  little  absolute  alcohol  and  blot 
into  position.  Dehydrate  more,  blot, 
3  changes  equal  parts  anilin  oil  and  xylol, 
xylol,  balsam.  Reticular  fibers  jet 
black,  collagenic  ones  dark  brown. 

For  serial  sections  of  brain  (Marshall, 
W.  H.,  Stain  Tech.,  1940,  15,  133-138) 
fix  slices  24  hours  or  longer  in  10  or 
15%  formalin  and  then  treat  them  with 
a  20-30%  alcohol  or  in  15%  formalin  in 
20%  alcohol.  The  object  of  the  alcohol 
treatment  is  to  avoid  formation  of  hard 
and  brittle  ice  crystals  which  fracture 
the  sections  as  they  are  made.  Cut 
tissue  into  blocks  about  1.0  cm.  thick. 
Place  on  a  CO2  ice  freezing  disc  which 
has  been  covered  by  a  piece  of  wet  blot- 
ting paper.  (In  our  laboratory  we  use  a 
regular  COj  gas  freezing  disc  which  has 
been  adapted  to  a  precision  sliding 
microtome.)  Freeze  the  block  of  tissue 
slowly  throughout.  The  proper  degree 
of  freezing  depends  on  the  thickness  of 
the  sections  to  be  cut.  Marshall  recom- 
mends a  paraffin  knife,  20-30°  angle  with 


block,  knife  set  in  a  line  perpendicular 
to  the  direction  of  motion.  Remove  cut 
sections  by  a  camel's  hair  brush  to  50% 
alcohol  and  keep  them  in  serial  order. 
Mount  sections  serially  on  slides  coated 
with  Albumen-Glycerin.  Smooth  out 
wrinkles  and  flatten  sections  by  gentle 
pressure  with  blotting  paper  moistened 
with  50%  alcohol.  Remove  slides  to  a 
38  "C.  oven  for  4-6  hrs.  when  they  are 
ready  for  staining.  (In  some  cases  it 
may  be  inadvisable  to  press  the  sections 
flat  upon  the  slide.  Thin  sections  re- 
quire less  drying  than  thick  ones.  In 
any  case  until  one  has  gained  confidence 
in  the  use  of  the  technique,  the  sections 
should  be  observed  at  intervals  in  the 
38 °C.  oven.  At  the  least  sign  of  exces- 
sive drying  (whitening  of  parts  of  the 
section)  the  sections  shoula  at  once  be 
removed  to  the  stain.)  The  Cresyl 
Violet  method  of  Tress  and  Tress  is 
recommended. 
Frozen-Dehydration  Method  for  histologi- 
cal fixation — Written  by  Normand  L. 
Hoerr,  Department  of  Anatomy,  West- 
ern Reserve  University  School  of  Medi- 
cine, Cleveland,  Ohio.  November  28, 
1951 — This  Method  has  distinct  ad- 
vantages over  the  usual  methods  of 
preparation  of  tissues  for  microscopy, 
particularly  for  histochemical  studies 
and  for  microincineration.  Tissues  are 
frozen  as  rapidly  as  possible  and  then 
maintained  below  the  freezing  point 
while  being  dried  in  vacuo.  The 
method  changes  tissues  little  or  not  at 
all  chemically,  and  physically  only  by 
the  removal  of  water.  If  the  freezing 
of  a  block  of  tissue  can  be  effected 
rapidly  enough  the  ice  crystals  which 
form  during  the  freezing  out  of  free 
water  will  be  below  microscopic  size. 
The  constituents  of  the  tissue  are  then 
not  displaced  to  an  extent  appreciable 
by  microscopic  examination.  To  pre- 
vent displacement  of  any  constituent 
of  the  tissue  after  freezing,  it  is  neces- 
sary that  the  dehydration  be  carried 
out  below  the  eutectic  point  of  the 
tissue.  There  is  no  way  of  knowing 
what  the  eutectic  point  of  such  a  com- 
plex mixture  of  substances  as  obtains 
in  animal  tissues  may  be  but,  since  in 
tertiary  and  quaternary  systems  the 
eutectic  point  is  usually  lower  than  the 
eutectic  points  of  mixtures  of  any  two 
of  the  substances,  it  is  reasonable  to 
assume  that  the  absolute  depression  of 
the  freezing  point  in  tissues  may  be 
well  below  — 54.9°C.  (the  eutectic 
point  of  aqueous  calcium  chloride  solu- 
tion). From  our  experience,  we  would 
say  that  better  cytologic  appearance 
of  the  tissues — i.e.,  an  appearance  com- 
paring most  favorably  with  the  appear- 


FUCHSIN  NB 


133 


FUNGI 


ance  of  living  tissues — is  obtained  by 
dehydrating  at  temperatures  below 
—  3(J°C.  If  the  freezing  can  be  effected 
rapidly  enough,  the  constituents  of  a 
tissue  are  not  appreciably  changed 
morphologically. 

Die  Elementarorganismen  und  ihre 
Beziehungen  zu  den  Zellen,  Altmann, 
R., /Leipzig,  Veit  and  Co.,  1890,  intro- 
duced the  method  of  freezing  tissues 
and  dehydrating  them  over  sulfuric 
acid  in  vacuo  at  temperatures  below 
-15°C.  Gersh,  I.  (Anat.  Rec,  1932, 
53,  309-337)  overcame  many  early  diffi- 
culties and  elaborated  the  technic  so 
that  good  histologic  fixation  could  be 
procured  on  a  number  of  tissues. 
Packer,  D.  M.  and  Scott,  G.  H.  (J. 
Tech.  Methods,  1942,  22,  85-96)  have 
overcome,  by  a  newly  designed  cryo- 
stat,  many  of  the  dehydration  difficul- 
ties. The  frozen  dehydration  method 
has  been  of  value  in  the  preliminary 
fixation  of  tissues  for  a  study  with  the 
electron  microscope  of  the  localization 
of  their  contained  salts  by  microincin- 
eration. It  has  been  used  for  the  pre- 
liminary preparation  of  tissues  in  the 
study  of  mitochondria  and  Nissl  sub- 
stance, of  secretion  in  the  stomach  and 
the  thyroid  gland,  and  in  a  histochemi- 
cal  study  of  the  Golgi  apparatus.  More 
extended  discussions  of  the  process  of 
freezing  and  of  the  subsequent  process 
of  dehydration,  with  considerations  of 
the  advantages  and  limitations  of  the 
method  are  given  by  Hoerr,  N.  L. 
(Anat.  Rec,  1936,  65,  293-295)  and 
Simpson,  W.  L.  (Anat.  Rec.  1941,  80, 
173-189). 

A  number  of  cryostats  have  been 
designed  recently,  including  those  by 
Stowell,  R.  E.  (Stain  Technology,  1951, 
26,  105-108)  Emmel,  V.  M.  (Anat.  Rec, 
1946,  95,  159-175)  Scott,  G.  H.,  and 
Hoerr,  N.  L.  (Medical  Physics,  1950, 
2,  292-296)  Wang,  K.  J.,  and  Grossman, 
M.  I.  (J.  Lab.  and  Clin.  Med.,  1949, 
34,  292-296)  and  Mendelow,  H.,  and 
Hamilton,  J.  B.  (Anat.  Rec,  1950,  107, 
443-451).  A  good  commercial  design 
is  available  from  Scientific  Specialties 
Corporation,  Cambridge,  Mass.,  or 
Euclid  Glass  Engineering  Laboratory, 
11310  Wade  Park  Avenue,  Clevelend, 
Ohio. 

Fuchsin  NB,  see  New  Fuchsin. 

Fuchsin  S,  SN,  SS,  ST  or  S  III,  see  Acid 
Fuchsin. 

Fungi. — Written  by  Morris  Moore,  Barnard 
Free  Skin  and  Cancer  Hospital,  St. 
Louis,  Mo.  November  10,  1951. 

1.  Skin  scrapings  and  hair.  The 
usual  method  is  to  mount  the  material 
in  an  alkali — either  sodium  hydroxide 
(NaOH)      or      potassium      hydroxide 


(KOH).  The  latter  is  preferable  and 
should  be  used  in  a  10-30%  solution. 
For  rapid  work  40%  is  employed  but  this 
tends  to  swell  and  disintegrate  the 
fungi.  A  weak  solution  takes  longer  to 
clear  the  skin.  The  skin  usually  clears 
in  5  min.  to  2  hrs.  in  concentrations  of 
10-30%.  A  little  heat  helps.  Use  sub- 
dued light  in  order  to  avoid  high  lights. 
The  fungus  is  clearly  discernible  against 
the  irregular  nondescript  background 
of  skin  which  is  usually  clear.  Dip 
infected  hairs  taken  from  scalps,  par- 
ticularly those  that  are  oily,  in  ether  or 
in  alcohol  (absolute  alcohol  is  preferable 
to  95%)  for  a  moment  in  order  to  get  rid 
of  the  oil  which  often  simulates  spores 
in  sliape  and  size. 

Adamson  (H.  G.,  Brit.  J.  Dermat., 
1895,  7,  201-211,  237-244)  has  recom- 
mended clearing  with  5-10%  KOH  and 
staining  by  the  Gram  method.  Chal- 
mers and  Marshall  (A.  J.  and  A.,  J.  Trop. 
Med.  Hyg.,  1914,  17,  256-265,  289-291) 
suggest  soaking  scales  in  40%  KOH  for 
some  hours  in  a  watch  glass  in  an  in- 
cubator at  40  °C.  Transfer  specimens 
to  watch  glass  containing  15%  alcohol  for 
30  min.,  remove  to  slide,  allow  alcohol 
to  evaporate  and  dry  over  flame ;  stain 
with  Anilin-Gentian  Violet  for  20  min. 
Treat  with  Gram's  Iodine  for  3  min.; 
decolorize  with  anilin  oil,  30  min.;  stain 
in  concentrated  alcoholic  eosin,  1  min.; 
wash  off  eosin  with  anilin  oil  or  clove  oil ; 
treat  with  xylol  and  mount  in  balsam. 

Priestley  (H.,  Med.  J.  Australia, 
1917,  2,  471-475)  recommends  lacto- 
phenol  (lactic  acid,  1  part;  phenol,  1 
part;  glycerol,  2  parts,  aq.  dest.,  1  part) 
for  clearing  instead  of  40%  KOH ;  or 
chloral  hydrate  crystals,  2  parts;  lactic 
acid,  1  part;  phenol  crystals,  1  part, 
may  be  used.  For  thick  material 
Langeron  suggests :  chloral  hydrate 
crystals,  40  gm. ;  phenol  crystals,  40 
gm.;  lactic  acid  (U.S.P.),  20  gm.;  and 
sodium  salicylate,  10  gm.  Slight  heat 
facilitates  clearing.  To  stain,  Priestley 
recommends  treatment  with  chloroform 
to  remove  the  fat;  boiling,  2-3  min., 
with  formic  acid ;  washing  for  a  few 
minutes  in  water  and  staining  with 
Sahli's  methylene  blue :  after  which  the 
tissue  is  to  be  washed,  differentiated 
with  alcohol  if  necessary,  dehydrated, 
cleared  and  mounted  in  balsam. 

Bachman  (R.  W.,  Arch.  Dermat.  & 
Syph.,  1920,  1,  50-54)  recommends  the 
following  procedure :  Place  scrapings  in 
a  drop  of  water  on  a  cover  slip,  tease 
thoroughly  with  a  dissecting  needle, 
dry  over  a  flame  but  do  not  scorch. 
Stain  for  2  min.;  decolorize  in  95%  al- 
cohol, 15-30  sec;  immerse  in  aq.  dest., 
15-30  sec. ;  pour  off  excess,  dry  by  heat, 


FUNGI 


134 


FUNGI 


and  mount  in  balsam.  Spores  and 
mycelium,  blue;  scrapings,  yellow. 
The  dye  is  sat.  ale.  gentian  violet,  2.5 
parts;  aq.  dest.,  17.5  parts;  orange  G 
solution,  9  parts;  acetic  acid,  1  part; 
95%  ale,  5  parts.  The  orange  G  solu- 
tion is  orange  G,  2  parts;  95%  ale,  20 
parts;  water,  80  parts.  Decolorize  with 
10-20%  KOH. 

The  hydroxide  method  of  examination 
is  simple  and  often  rapid,  but  unless 
used  by  one  familiar  with  it  the  results 
may  be  misleading.  There  is  danger  of 
confusion  with  structures  which  Becker 
and  Ritchie  (J.  W.  and  E.  B.,  Arch. 
Dermat.  &  Syph.,  1940, 22,  790-802)  have 
indicated  as  resembling  yeast  cells. 
These  artifacts  may  be  removed  by 
treating  the  material  progressively  with 
absolute  alcohol,  ether,  absolute  and 
95%  alcohol.  They  have  been  termed 
'mosaic  fungus'  and  have  been  reported 
by  Greenwood  and  Rockwood  (A.M.  and 
E.  M.,  Arch.  Dermat.  &  Syph.,  1930, 
21,  96-107)  as  degenerate  fungi.  In 
fact  they  are  cholesterol  crystals.  The 
use  of  dyes  eliminates  in  great  measure 
such  artifacts.  However,  the  use  of 
dyes  is  not  practical  with  thick  sections 
for  which  recourse  must  be  had  to  the 
hydroxide  method. 

When  the  scrapings  or  scales  are  thin, 
or  when  sputum,  pus  or  exudate  are  ex- 
amined, a  1%  aq.  methylene  blue  and 
glycerin  can  be  used  as  follows :  One 
drop  of  the  1%  solution  of  methylene 
blue  is  placed  on  a  clean  slide  and  the 
material  is  stirred  within  it,  allowed  to 
stand  for  approximately  2  min.  when  a 
clean  cover  slip  is  placed  over  the  mix- 
ture and  pressed  down  to  flatten  out  the 
material  and  to  express  the  excess  solu- 
tion. The  superfluous  stain  is  taken  up 
by  filter  paper.  A  drop  of  glycerin  is 
then  placed  along  one  edge  of  the  cover 
slip  and  allowed  to  seep  under,  dis- 
placing the  stain  and  giving  a  clear  back- 
ground to  the  stained  material.  The 
fungus  appears  bright  blue. 

The  lactophenol-cotton  blue  technique 
was  developed  in  the  French  labora- 
tories using  the  formula  of  Amann  (J., 
Zeit.  Wiss.  Mikr.,  1896,  13,18-21).  Lac- 
tophenol  consists  of  phenol  crystals, 
20  gm.;  glycerin,  40  gm.;  lactic  acid, 
20  gm.  andaq.  dest.,  20 gm.  Cotton  blue 
(anilin  blue,  China  blue)  is  a  mixture  of 
the  trisulphonates  of  tri-phenyl  para- 
rosanilin  (C.I.  706)  and  of  di-phenyl 
rosanilin.  Place  a  drop  of  the  cotton 
blue  (0.5%  aq.)  on  the  slide ;  stir  up  the 
material  within  it  and  allow  to  stand  for 
about  2  min.  Add  cover  slip  and  press 
down  to  squeeze  out  any  excess  dye, 
which  is  taken  up  by  filter  paper.  Add 
a  drop  of  lactophenol  to  the  edge  of  cover 


slip  and  allow  it  to  replace  the  cotton 
blue  which  dries  out.  The  stain  may  be 
rapidly  replaced  by  holding  a  bit  of  filter 
paper  at  the  edge  of  the  cover  slip  op- 
posite the  lactophenol.  The  cell  wall 
stains  lightly  as  compared  with  the 
darkly  colored  central  portion  of  the 
fungus.  The  tissue  elements  also  stain 
light  blue. 

Swartz  and  Conant  (J.  H.  and  N.  F., 
Arch.  Dermat.  &  Syph.,  1936,  33, 
291-305)  have  modified  the  lactophenol 
and  cotton  blue  procedure.  First  put 
a  few  scrapings  in  5%  aq.  potassium 
hydroxide,  heat  somewhat  and  wash  in 
water.  Place  material  in  a  drop  of  the 
combined  cotton  blue  (0.5%)  and  lacto- 
phenol. The  fungi  stain  a  darker  blue 
than  the  tissue  cells. 

Schubert  M.,  Dermat.  Wchnschr., 
1937,  105,  1025-1029)  has  modified  the 
Swartz-Conant  technique.  Soak  the 
scales  in  2%  KOH  for  30  min.  or  until 
they  appear  glassy  and  then  wash  in 
aq.  dest.  2-10  hrs.  Transfer  small 
particles  to  a  slide  and  add  1  or  2  drops 
of  following  stain :  cotton  blue,  0.25  gm. ; 
lactic  acid,  10  gm.;  phenol  crystals,  10 
gm.;  and  aq.  dest.,  20  gm.  The  fungi 
appear  dark  blue  while  the  epidermal 
cells  stain  lightly.  See  also  Berberian's 
Method. 

2.  Sputum,  pus  and  exudates:  Exam- 
ine for  fungi  after  mounting  directly 
on  a  slide  after  mixing  in  20%  KOH  or 
on  stained  smears.  The  latter  are  not 
very  satisfactory  because  smearing 
tends  to  disturb  the  arrangement  of  the 
cells  but  they  are  useful  for  detection  of 
mycelium.  Many  contaminating  or- 
ganisms are  generally  present  in  these 
exudates  unless  material  is  secured  from 
fresh  lesions  opened  aseptically.  Sev- 
eral examinations  may  be  necessary  since 
the  organisms  in  exudates  are  seldom 
numerous.  The  hydro .xide  usually  dis- 
solves most  of  the  tissue  elements  and 
the  fungi  stand  out  as  refractile  bodies. 
Several  of  the  staining  methods  em- 
ployed in  the  study  of  hair  and  scrapings 
may  be  used.  Of  these,  the  methylene 
blue  and  glycerin  method  is  best  but  the 
lactophenol-cotton  blue  technique  is 
likewise  advised. 

3.  Vesicles,  blister  fluid,  spinal  fluid 
or  urine:  These  can  also  be  directly 
examined.  But  vesicle,  or  blister,  fluid 
yields  only  a  small  amount  of  material 
and  for  best  results,  the  methylene 
blue-glycerin  method  or  the  lactophenol- 
cotton  blue  technique  is  advised. 
Urine,  or  spinal  fluid,  should  be  con- 
centrated by  centrifugation  before 
examination.  The  same  staining  pro- 
cedures are  advocated.  See  Blasto- 
mycosis. 


FUNGI 


135 


FUNGI 


4.  Skin:  Unna,  Jr.  (P.,  Dermat. 
Wchnschr.,  1929,  88,  314-321)  advises 
the  following  modification  of  the  Pap- 
penheim-Unna,  Sr.  method  for  stainins 
fungi  in  skin.  Fix  in  absolute  alcohol, 
then  run  through  the  alcohols  to  xylol 
and  imbed  in  paraffin.  Cut  sections  at 
10/x,  stain  with  pyronine-methyl  green 
(pyronine,  9  parts;  methyl  green,  1 
part;  96%  alcohol,  90  parts;  glycerol, 
100  cc;  0.5%  phenol  to  make  1000  cc), 
5-10  sec;  rinse  in  water;  dry  with 
absolute  alcohol;  and  mount  in  balsam. 
Fungi,  rubin  red;  leukocytes,  green  to 
blue  green ;  nuclei  of  cells  of  basal  horny 
layer  of  the  epidermis,  red. 

Fungi  in  tissue  can  be  easily  stained 
by  Iron-Hematoxylin  and  eosin.  The 
fungous  elements  take  the  hematoxylin 
stain  nicely,  although  some  difficulty 
may  be  encountered  in  distinguishing 
spherical  cells  or  spores  from  tissue 
elements.  The  Gram  method  of  stain- 
ing for  bacteria  has  been  used  with  a 
measurable  amount  of  success  since 
fungi  are,  in  general,  gram -positive. 

Unna's  Alkaline  Methylene  Blue 
(Unna,  P.,  Monatsh.  f.  prakt.  Dermat., 
1891,  13,  225-237,  286-311),  although 
recommended  for  staining  plasma  cells 
and  as  a  general  stain  in  combination 
with  phloxine  or  eosin,  has  been  espe- 
cially adapted  for  staining  organisms  in 
the  stratum  corneum.  It  consists  of 
methylene  blue,  1  gm.;  potassium 
carbonate,  1  gm.;  and  aq.  dest.,  100 
cc.  The  solution  stains  better  after 
ripening  for  a  week  or  two  and  should 
be  diluted  1  to  10  or  1  to  5  before  use. 

Malcolm  Morris  (Mallory,  F.  B.  and 
Wright,  J.  H.,  Pathological  Technique, 
Philadelphia  and  London,  1924,  p.  175) 
in  staining  various  parasites  of  the  skin, 
avoids  the  use  of  potassium  hydrate. 
Place  skin  in  ether,  or  in  1:1  alcohol- 
ether;  stain  for  5-30  min.  in  5%  gentian 
violet  in  70%  alcohol.  Then  pass 
through  iodine  solution,  1  min.;  anilin, 
or  anilin  plus  2-4  drops  of  nitric  acid; 
anilin;  and  xylol  (2  changes)  to  balsam. 

5.  Other  tissues:  A  number  of  methods 
listed  for  staining  Bacteria  in  tissue 
can  be  used  successfully  for  fungi. 
Mallory's  Connective  Tissue  stain  is 
good  for  Cryptococcus  hominis  in  brain 
tissue.  Fungus  cells,  red ;  thick  mucoid 
capsules,  light  blue.  TheGram-Weigert 
staining  method  is  also  excellent. 
Organisms,  deep  violet;  nuclei,  blue  to 
violet;  connective  tissue,  red. 

Actinomyces  in  sections  may  be 
stained  successfully  with  Alum-Hema- 
toxylin  followed  by  strong  eosin.  Mal- 
lory (p.  279)  lists  2  methods  of  which 
the  following  gives  good  results  with 
paraffin  sections  of  formalin  or  Zenker 


fixed  tissue.  Stain  in  alum-hematoxy- 
lin,  3-5  min.;  wash  in  water;  stain  in 
a  2.5%  a,q.  phloxine  or  5%  aq.  eosin, 
15  min.  in  the  paraffin  oven;  wash  in 
water;  stain  in  Anilin  Crystal  Violet 
(try  Stirling's),  5-15  min.;  wash  in 
water;  treat  with  Gram's  Iodine  solu- 
tion, 1  min.;  wash  in  water  and  blot 
with  filter  paper;  differentiate  in 
several  changes  of  anilin  until  no  more 
color  comes  off;  rinse  in  several  cliangea 
of  xylol  and  mount  in  balsam.  The 
branched  organisms  stain  blue  while 
the  hyaline  sheaths  ("clubs")  become 
pink  to  red. 

The  Hotchkiss-McManus  or  Periodic 
Acid-Schiff  Stain  (Kligman,  A.  L.  and 
Mescon,  H.,  J.  Bact.,  1950,  60,  415-421) 
may  be  used  to  find  fungi  in  tissue  or 
sections.  Tissue  fixed,  embedded  in 
paraffin  and  sectioned  in  usual  manner. 
After  deparaffinizing,  sections  rinsed 
in  absolute  alcohol;  washed  in  distilled 
water;  immersed  in  1%  aq.  periodic 
acid  C.P.  (Eimer  &  Amend)  5  min.; 
washed  in  tap  water,  10  min.;  placed  in 
Schiff  reagent,  10  to  15  min. ;  transferred 
to  1  normal  HCl,  5  min.;  washed  in 
tap  water,  10  min.;  counterstained  with 
1%  aq.  light  green,  for  sec. ;  dehydrated, 
cleared  and  mounted.  Fungi  stain  red 
to  purple.  Tissue  cell  nuclei  do  not 
stain. 

Schiff  reagent  prepared  as  follows: 
Dissolve  0.5  gm.  basic  fuchsin  (Cole- 
man &  Bell)  by  passing  over  it  100  cc. 
boiling  water;  cool  to  50°C.;  filter  and 
add  10  cc,  1  normal  HCl  and  0.5  gm. 
potassium  metabisulfite  dry  reagent 
(Eimer  &  Amend)  to  filtrate.  Solu- 
tion becomes  colorless  to  pale  straw 
colored  by  standing  in  dark  overnight. 
Add  0.25  to  0.5  gm.  activated  charcoal, 
shake  well  and  filter  immediately  to 
completely  decolorize.  Refrigerate  in 
tightl}^  stoppered  bottle. 

For  smears  or  nails:  Use  thin  smears 
and  fine  nail  scrapings.  Material  made 
to  adhere  to  slide  with  egg  albumen. 
Coplin  jar  or  drop  bottle  technique 
employed.  Immerse  material  in  95% 
alcohol,  1  min.;  cover  with  5%  aq. 
periodic  acid,  3  minutes;  wash  in  run- 
ning water.  2  min.;  stain  in  Schiff  re- 
agent, 5  min.;  rinse  in  running  tap 
water,  1  min.;  dehydrate  in  95%  then 
absolute  alcohol  and  two  changes 
xylol,  1  min.  each.  Mount  in  clarite. 
See  Polysaccharides. 

After  the  fungi  have  been  successfully 
cultivated  on  the  various  mediums 
recommended  (Moore,  M.,  Arch. 
Dermat.  &  Syph.,  1936,  34,  880-886) 
they  can  be  examined  microscopically 
by  transferring  part  of  the  growth  with 
a  sterile  platinum  or  nichrome  wire  to 


FURFURAL 


136 


G  ALLOC  YAN  IN -CHROMALUM 
STAINING 


a    clean    slide.    This   should    be    done 

fently  to  avoid  destruction  of  the 
ungous  growth.  The  fungus  is  teased 
apart  gently  in  one  of  several  fluids 
such  as  water,  alcohol,  alcohol  and 
glycerine  (equal  parts)  or  other  mount- 
ing fluids.  Water  has  a  high  surface 
tension  and  causes  disruption  of  the 
growth;  while  alcohol  evaporates  rap- 
idly and  must  be  replaced.  The 
following  solution  serves  well :  2% 
potassium  acetate,  50  cc;  glycerin,  20 
cc;  and  95%  alcohol,  30  cc.  The 
preparation  is  examined  with  reduced 
light.  The  preparations  may  be  stained 
using  one  of  the  several  methods  listed : 
lactophenol-cotton  blue ;  methylene 
blue-glycerin;  or  Giemsa-glycerin.  See 
Chorioallantoic  Membrane,  Actlno- 
mycetes. 

Furfural.  Has  been  suggested  but  is  not 
recommended  as  substitute  for  formal- 
dehyde (Stowell,  R.  E.  and  Stokes, 
J.  M.,  J.  Tech.  Meth.  and  Bull.  In- 
ternal. Assoc.  Med.  Museums,  1944,  24, 
25-30). 

Fuscin  (L.  fuscus,  dusky),  a  dusky  pigment 
of  retinal  epithelium  usually  present 
in  crystalline  formations  made  up  of 
albuminous  cores,  which  determine 
their  shape,  plus  the  adsorbed  fuscin 
material.  A  relationship  to  melanin 
is  debated  but  the  pigment  is  very 
resistant  to  chemical  attack.  It  can, 
however,  be  bleached  completely  when 
exposed  to  light  in  vitro.  For  details 
see  Arey,  L.  B.  in  Cowdry's  Special 
Cytology,  1932,  3,  1218. 

Fustics.  "Young"  fustic  is  a  stain  obtained 
from  the  smoke  tree,  Rhus  cotinus  of 
West  Indies  and  South  America  giving 
colors  from  bright  yellow  to  dark  olive 
now  seldom  used  by  dyers.  "Old"  fus- 
tic is  obtained  from  a  tree  of  the  mul- 
berry family,  Chlorophora  tinctora, 
which  grows  in  the  same  countries.  It 
is  chiefly  employed  for  woolens  giving 
shades  of  lemon  and  old  gold  (Leggett, 
W.  F.,  Ancient  and  Medieval  Dyes. 
Brooklyn:  Chemical  Publishing  Co. 
Inc.,  1944,  95  pp.). 

Gadolinium   see  Atomic  Weights. 

Galliamine  Blue  (CI.  894)  can  be  employed 
in  place  of  hematoxylin  as  an  iron  lake 
stain  for  nuclei. 

Gallein  (CI,  781),  a  mordant  dye  of  light 
fastness  1.  Use  as  solution  0.5  gm.  in 
100  cc.  of  either  1%  aq.  anmionium 
acetate  or  0.1%  sulphuric  acid.  Small 
invertebrates  should  be  previously 
mordanted,  30  min.  in  1%  aq.  ferric 
ammonium  sulpha  e  and  rinse  in  aq. 
dest.  before  staining  for  1  to  2  min.  in 
the  solution  at  50°C.  Color  blue  black. 
If  copper  sulphate  is  employed  for  mor- 
dant   color    is    hematein    purple.     In 


paraffin  sections  of  animal  tissues  nuclei 
color  blue  black  in  15  to  20  sec.  at  50°C. 
Directions  are  also  given  for  plant  tis- 
sues and  Blue-green  algae  (Emig,  p. 
54-55). 
Gallium,  see  Atomic  Weights. 
Gallocyanin-Chromalum  Staining  of  Baso- 
philic Cell  Structures — Written  by 
Ldrus  Einarson.  Normal-Anatomisk 
Institut.  Aarhus  Universitet,  Aarhus, 
Denmark.  February  27,  1951. — Baso- 
philia is  an  essential  property  common 
to  some  most  important  cell  structures, 
and  the  study  of  the  nature  as  well  as 
the  attainment  of  a  method  for  quanti- 
tative estimation  of  basophily  have 
been  among  the  outstanding  general 
problems  in  histology  and  cytology. 
Here,  the  nerve  cells  make  an  especially 
favourable  object  on  account  of  their 
high  degree  of  cytoplasmic  basophily 
due  to  the  Nissl  substance,  which  con- 
tains nuclein,  acid  and  basic  proteins 
respectively  (Einarson  L.  J.  comp. 
Neur.,  1935,  61,  101-133). 

An  accurate  estimation  of  basophily 
can  be  achieved  by  staining  with  dis- 
solved colour  lakes  of  some  synthetic 
dye-stuffs  (Ranvier,  1875;  Grenacher, 
1879;  Rawitz,  1896;  Becher,  1921),  and 
the  staining  by  gallocyanin-chromalum 
affords  the  most  accurate  representative 
of  this  principle  of  staining  by  inner- 
complex  dye-metal  salts  (Einarson,  L., 
Acta  path.  &  Microbiol.  Scand.,  195, 
28,82-102). 

The  staining  solution  is  made  as  fol- 
lows: 5  gm.  chromalum  puriss.  is  dis- 
solved in  100  cc  redistilled  water,  then 
0.15  gm.  gallocyanin  (Griibler-HoU- 
born  or  Nat.  Aniline  Division,  New 
York)  is  added  and  the  whole  is  mixed 
by  shaking  the  bottle.  The  mixture  is 
warmed  up  gradually  and  boiled  for  5 
min.  After  cooling  at  room  tempera- 
ture, filtration  through  filter  paper  and 
addition  of  redistilled  water  through 
the  filter  until  the  volume  again  is  100 
cc,  the  solution  is  ready  for  use;  its 
pH  is  1.64.  Staining  time  is  48  hrs  at 
room  temperature.  After  staining 
washing  in  aq.  dest.,  alcohols,  xylol 
balsam. 

The  process  by  which  the  lake  is 
formed  consists  in  the  formation  of  a 
soluble  lake-cation  (reddish),  a  slightly 
soluble  lake-sulphate  (blue)  and  a  non- 
soluble  lake-hydroxide;  the  latter  is 
formed  by  an  inmiediate  hydrolysis  of 
some  of  the  lake  under  formation. 
After  the  solution  has  been  prepared 
no  further  hydrolysis  occurs  until  4-5 
weeks  later  (late  hydrolysis),  and  a 
gradual  sedimentation  of  lake-hydrox- 
ide takes  place;  at  the  same  time  the 
staining    power    of    the    solution    be- 


GALLOCYANIN-CHROMALUM  137 

STAINING 

comes  weaker.  Thns,  for  safety  the 
solution  should  not  be  used  for  more 
than  3  weeks.  Lake-sulphate  and  lake- 
hydroxide  are  the  main  constituents  of 
the  precipitate  filtered  off  the  solution 
before  use.  Although  such  inner-com- 
plex salts  all  have  very  low  solubilities 
in  water,  the  lake-sulphate,  however, 
reamins  dissolved  in  a  sufficient  amount 
to  become  dissociated  into  the  mono- 
valent lake-cation  and  an  anion  ac- 
cording to  the  equation: 

(gallocyanin  Cr(H20)4)2  S04^  2  galloc 
^  Cr(H20)4+  +  SO4 


In  the  lake-cation,  Cr(H20)6+++  is 
attached  to  one  gallocyanin  molecule 
by  two  valency  bonds  to  its  OH  and 
COOH,  and  by  two  coordinating  bonds 
to  its  =0  and/0\  through  substitu- 
tion of  two  HoO-molecules  from  the 
internal  sphere  of  the  Cr-atom.  The 
designations  =0  and  /0\  are  used 
for  the  sake  of  brevity.  =0  means 
the  2-carbonvl-O,  and  /0\  means  the 
oxazin-0  of  the  chromophor-group). 

The  reddish  lake-cations,  the  actual 
staining  compound,  unite  progressively 
and  selectively  with  the  nucleoproteins 
of  the  fixed  cell  structures  to  form  a  blue 
salt  of  the  lake-cation  and  the  tissue. 
This  proceeds  until  maximum  occupa- 
tion has  taken  place;  then  no  further 
attachment  of  the  stain  to  the  baso- 
philic cell  structures  is  possible.     When 
the  staining  has  attained  a  certain  in- 
tensity it  will  not  be  further  increased, 
even  if  the  tissue  be  left  in  the  staining 
solution   for    a    considerable    length    of 
time;    in    this    respect    the    staining   is 
independent    of   the    staining    time    {ex- 
quisite  progressivity) ;   no   other   histo- 
logical staining  known  to  me  possesses 
this  quality  in  the  same  degree.     Owing 
to  the  great  stability  of  the  staining  it 
is  also  completely  unaffected  by  alcohol 
and  xylol  during  the  procedure  of  de- 
hydration  and   mounting.     In   consid- 
eration of  these  qualities  the  staining 
intensity  depends  directly  on  the  in- 
herent capacity  to  bind  the  stain,  pos- 
sessed by  the  living  cell  at  the  moment 
of  fixation. 

Briefly  the  staining  consists  of : 

1.  A  specific  binding  of  the  lake- 
cation  to  the  nucleic  acids,  in  which  it 
reacts  with  the  phosphoric  acid  groups 
to  form  a  lake-salt  of  nucleic  acid; 
within  the  range  from  pH  0.83  to  4.35 
this  specific  binding  always  takes  place 
by  means  of  the  free  -f-valency  of  the 
lake-cation,  regardless  of  the  type  of 
nucleic  acid. 

2.  An  adsorption  of  lake-sulphate 
to  the  tissue,  i.e.,  an  unspecific  co- 
staining  of  non-basophilic  structures, 


G  ALLOC  Y  AN  IN  -CHROMA  LUM 
STAINING 

which  increases  directly  with  the  pH 
of  the  dye-solution;  it  mainly  takes 
place  by  the  basic  7-(CHj)2N-group  of 
the  dye  molecule;  by  staining  at  a  suffi- 
ciently low  pH  the  co-staining  is  re- 
duced to  a  minimum. 

3.  A  binding  of  the  lake-cation  to  the 
proteins  of  the  basophilic  structures, 
which,  however,  only  takes  place  in  a 
definite  range  of  pH  on  the  alkaline 
side  of  the  iso-electric  point  of  the  pro- 
teins and  in  fact  is  unimportant;  on  the 
acid  side  of  the  iso-electric  point  the 
lake-cation  combines  with  the  nucleic 
acids  alone. 

As    the    lake    compounds    stabilize 
(buffer)  the  staining  solution  towards 
acids  and  alkalies  its  pH  can  be  varied 
simply  bv  adding  1   to  10  cc  of  N/1 
HCl    and'  N/1    NaOH   respectively   to 
each  40  cc.  of  stock  solution.     In  the 
range  from  pH  1.50  to  1.80  the  specific 
staining  reaches  its  maximum  intensity 
at  the  same  time  as  the  co-staining  re- 
mains completely  negligible;   pH   1 .64 
represents   the   optimum  acidity  for  the 
specific  attachment  of  the  lake-cations  to 
the   nucleic  acids,   the   relative   quantity 
of  which  can  be  estimated  by  photometric 
or  densitometric  measurements.    The  in- 
crease of  the  co-staining  first  sets  in  at 
pH  1.80  to  reach  its  maximum  between 
pH  3.4  and  3.5  whereupon  every  stain- 
ing fades  away;  at  pH  4.27  it  is  barely 
visible.     The  co-staining  at  pH  2.90- 
3.42   may   be   used   for   demonstrating 
axons     and     neuroglia     (Einarson     & 
Ringsted  1938,  p.  43).     After  the  addi- 
tion of  alkali  the  solution  should  not  be 
used  longer  than  4-8  days. 

The  usual  basic  blues  preferably  stain 
proteins  and  other  amphoteric  electro- 
lytes which  display  ''facultative  baso- 
phily",\.e.,  under  another  set  of  condi- 
tions they  are  less  basophilic  or  even 
acidophilic;  they  are  amphophilic  sub- 
stances, and  the  staining  is  a  mere  ad- 
sorption of  the  dye. 

Gallocyanin-chromalum  indicates  the 
degree  of  "genuine  basophily"  which 
depends  directly  on  the  quantity  of 
nucleic  acids  present;  genuine  baso- 
phily  is  not  in  the  same  way  dependent 
on  the  external  conditions  as  the 
facultative  one,  and  its  changes  reflect 
the  decomposition  during  neuronal 
function  or  regeneration  and  the  sub- 
sequent repair  of  cytoplasmic  nucleic 
acid  from  the  nucleus  (Einarson,  L., 
Am.  J.  Anat.,  1933,  53,  141-176). 

Gallocyanin  (CI.  883)-alizarin  blue 
RBN,  chrom  blue  GCB  and  fast  violet. 
It  is  a  basic  oxazin  dye. 


Gamma  = 


1000 


mg.  or  0.001  mg. 


GARDNER 


138 


GELATIN  E^IBEDDING 


Gardner,  see  Articular  Nerve  Terminals. 

Garven's  Gold  Chloride  method  for  nerve 
endings  in  muscle  (Garven,  H.S.D., 
Brain,  1925,  48,  380-441).  This  is  Fis- 
cher's modification  of  Ranvier's  tech- 
nique as  used  in  Golgi's  Laboratory. 
Immerse  small  pieces  of  tissue  in  25% 
aq.  pure  formic  acid  and  tease  a  little 
to  assure  penetration  10-15  min.  Blot 
with  clean  cloth.  Place  in  1%  aq.  gold 
chloride  just  sufficient  to  completely 
cover  tissue  and  shake.  Avoid  all  iron 
instruments.  Cover  dish  with  blue  or 
yellow  glass.  Leave  20  min.  Blot 
with  clean  cloth  and  repeat  above  treat- 
ment with  formic  acid  and  gold  leaving 
this  time  in  latter  24  hrs.  in  absolute 
darkness.  Repeat  still  again.  Pass  to 
glycerin  and  leave  in  closed  vessel  in 
ordinary  light.  The  sharpness  of  the 
intensely  purple  black  nerves  in  a 
lightly  colored  background  increases 
with  time.  Small  pieces  can  then  be 
transferred  to  aq.  dest.  and  the  indi- 
vidual fibers  separated.  This  is  facili- 
tated by  dissociation  in  dilute  nitric 
acid.  Wash  and  make  final  mounts  in 
glycerin.  The  author  used  panniculus 
carnosus  of  hedgehog,  striated  muscle 
of  frog  and  lizard,  extrinsic  eye  muscle 
of  rabbit  and  human  pectoral  muscle. 

Gas  Analysis. — Techniques  are  given  in  de- 
tail by  Glick,  pp.  313-412. 

Gash-Irrigation  recovery  method  for  lung 
cells  (GI).— Written  by  C.  C.  Macklin, 
Dept.  of  Histological  Research,  The 
University  of  Western  Ontario,  London, 
Canada.  November  28,  1951. — Fresh 
collapsed  mouse  or  other  mammalian 
lung  is  cut  through  a  drop  of  physiologi- 
cal salt  solution,  blood  serum  or  other 
suitable  liquid,  inverted  and  drained 
onto  a  slide.  It  is  then  covered  and  ex- 
amined as  a  fresh  mount,  or  spread, 
dried,  stained  and  mounted  like  a  blood 
smear.  Liberated  phagocytic  and  gran- 
ular pneumonocytes  (dust  and  foam 
cells — which  see)  are  thus  obtained 
(Macklin,  C.  C,  Proc.  6th  Intern.  Con- 
gress of  exper.  Cytol.,  Stockholm,  1947; 
published  1949,  383-387;  Macklin,  C.  C, 
The  Lancet,  Feb.  24,  1951,  432-435). 

Gastric  Contents.  Examine  microscopically 
material  obtained  by  stomach  tube  after 
test  meal  as  described  by  Stitt  (p.  753). 
Look  for  mucus,  epithelial  cells,  leu- 
cocytes, Gram  positive  bacilli  in 
smears. 

Gastrointestinal  Tract.  Immediate  fixation 
is  desirable  because  postmortem  changes 
occur  especially  quickly.  Do  not  wash 
first  with  water  but  with  physiological 
saline  or  with  the  fixative  itself.  It  may 
be  desirable  to  place  the  excised  pieces, 
with  peritoneal  surface  down,  on  wooden 
tongue  depressor  or  stiff  paper.    Some 


flattening  is  required.  The  mucous 
surface  must  not  be  allowed  to  dry. 
See  Small  and  Large  Intestine.  See 
Papanicolaou  Techniques. 

Gautheria  Oil  used  to  be  employed  as  a 
clearing  agent.     It  has  been  displaced 
_  by  the  artificial  oil,  methyl  salicylate. 

Geiger  Counters  are  instruments  for  the 
counting  of  electrons  which  provide 
quantitative  data  of  great  importance 
in  this  electron  age.  A  concise  descrip- 
tion of  the  history  of  counter  develop- 
ment and  of  the  Geiger-Miiller  type  is 
supplied  by  Rovner,  L.  in  Glasser's 
Medical  Physics,  487-495. 

Gelatin-Carmine  injections,  see  Carmine 
Gelatin  injections. 

Gelatin  Glue,  method  of  mounting  sections, 
see  Masson's. 

Gelatin  Imbedding  and  Sectioning.  This  is 
used  when  sections  are  required  of  loose, 
friable  tissues  which  easily  fall  apart. 
Since  the  imbedding  is  directly  from 
water,  no  alcoholic  or  other  dehydration 
is  required.  Probably  the  best  method 
is  that  of  Zwemer  (R.  L.,  Anat.  Rec, 
1933,  57,  41-44),  devised  primarily  for 
the  study  of  adrenal  lipoids.  Wash 
material  fixed  in  formalin  or  other  fluid 
in  water,  4  hrs.  5%  gelatin  in  incubator 
at  35-37 °C.  24  hrs.  10%  gelatin  at  same 
temperature,  12-16  hrs.  Imbed  by 
placing  in  10%  gelatin  in  Petri  dish  in 
refrigerator.  Cut  out  blocks  of  tissue 
and  fix  in  10%  formalin  several  hours 
to  make  gelatin  insoluble  in  water. 
In  this  formalin  solution  tissues  can  be 
preserved  indefinitely.  Before  section- 
ing rinse  block  in  water  and  trim. 
Freeze  with  CO2  until  block  is  uniformly 
white.  Allow  to  thaw  until  knife  cuts 
easily.  Sections  as  thin  as  5  microns 
can  be  obtained.  Float  onto  slide  in 
aq.  dest.  Drain  off  excess  water  and 
run  a  drop  or  two  of  1%  gelatin  under 
setion.  Again  drain  off  excess.  After 
heating  in  drying  oven  at  33-37°C.  place 
slide  in  10%  formalin  for  10  min.  to  fix 
gelatin.  In  this  formalin  solution  the 
mounted  sections  can  be  stored.  Stain 
sections  in  usual  way  with  Sudan,  Nile 
Blue  Sulphate,  Osmic  Acid,  Laidlaw's 
Silver  Method,  and  mount  in  Gly- 
chrogel. 

Wright's  method  as  described  by 
Mallory  (p. 34)  is  much  quicker  and  is 
recommended  for  fragmented  tissues 
such  as  those  from  curettings.  Make  a 
10%  solution  of  gelatin  in  warm  aq. 
dest.  and  while  still  fluid  add  0.5% 
carbolic  acid.  Do  not  overheat.  The 
tissue,  unfixed  or  fi.xed,  preferably  in 
10%  formalin,  is  "dried"  and  placed  in 
a  small  "pool"  of  gelatin  liquified  by 
heat  on  a  or  slide  in  a  glass  vessel.  This 
is  allowed  to  solidify  in  the  ice  box  for 


GELATIN  MEDIA 


139 


GIEMSA'S  STAIN 


2  hrs.  or  more.  If  necessary,  store 
gelatin  blocks  in  10%  formalin.  Cut 
out  block  containing  the  tissue,  freeze 
and  section.  Float  sections  from  water 
onto  slide  well  coated  with  albumen- 
glycerin  and  spread.  Remove  excess  of 
fluid  and  cover  with  piece  of  thin 
cigarette  paper.  Blot  with  fine  filter 
paper  till  cigarette  paper  is  partly  dry. 
Cover  cigarette  paper  with  equal  parts 
anilin  oil  and  oil  of  cloves  for  few 
seconds.  Drain  and  peel  off  cigarette 
paper.  Remove  oil  by  washing  in  95% 
alcohol  and  pass  to  water  when  sections 
are  ready  for  staining.  Mallory  suggests 
methods  for  Amyloid,  Fat  and  staining 
with  Hematoxylin  and  Phloxine  for 
general  purposes. 

Gelatin  Media,  see  Bacteria,  Media. 

Gelatin-Ringer  electrode  vessel,  modifica- 
tion by  Kriest,  A.  C,  Acta  Physiol,  et 
Pharmacol.  Neerland.,  1950,  1,  32-34. 

Gentian  Blue  6B,  see  Spirit  Blue. 

Gentian  Violet.  The  problem  afforded  by 
this  dye,  like  many  others,  has  been 
attacked  by  the  Stain  Commission. 
The  stain  thus  referred  to  has  no  con- 
stancy. Originally  it  was  a  mixture 
in  about  equal  parts  of  dextrin  and 
methyl  violet,  the  latter  itself  a  mixture 
in  widely  varying  proportions  of  tetra-, 
penta-  and  hexa-methyl  pararosanilins. 
Later  were  placed  on  the  market  methyl 
violets  with  and  without  dextrin  and 
crystal  violet  (the  hexa  methyl  com- 
pound) all  under  the  label  of  Gentian 
violet.  As  Conn  (p.  124)  advises  the 
term  Gentian  violet  should  be  elim- 
inated and  crystal  violet  used 
wherever  in  the  past  the  former  has 
been  specified.  See  Neutral  Gentian, 
Methyl  Violet,  Crystal  Violet. 

Geranine  G  (CI,  127).  An  acid  thiazole 
dye  employed  in  fluorescence  studies 
on  account  of  color  imparted  by  it 
under  ultraviolet  illumination  (Conn, 
p.  70). 

Germanium,  see  Atomic  Weights. 

Giant  Cells.  There  is  no  special  technique 
for  their  demonstration.  Since  the 
features  usually  employed  in  classifica- 
tion are  size  and  nuclear  detail  and 
arrangement,  Hematoxylin  and  Eosin, 
or  Iron  Hematoxylin  the  latter  followed 
by  various  counter  stains  as  for  Acid 
Fast  Bacilli  are  recommended.  The 
following  is  a  much  abbreviated  classi- 
fication of  Giant  Cells  from  Cowdry's 
Histology  1938  Edition : 

1.  Megakaryocytes  of  bone  marrow, 
granules  in  cytoplasm,  best  demon- 
strated by  Giemfsa's  Stain. 

2.  Foreign  body  gianl  cells  formed 
probably  by  a  fusion  of  cells  of  mesen- 
chymatous  origin,  perhaps  of  non- 
granular   leucocytes,    in    response    to 


foreign  materials  of  many  kinds — 
tubercular  giant  cells,  foam  cells  in 
leprosy,  lympsocystic  giant  cells  of  fish 
(Weissenberg),  and  possibly  Reed- 
Sternberg  cells  in  Hodgkin's  disease. 

3.  Osteoclasts  (polj^karyocytes)  of 
bone  marrow  and  Langhans'  giant  cells 
of  placenta  are  normal  inhabitants  of 
these  organs.  Myeloplague  and  Myelo- 
plax  are  other  terms  for  osteoclast. 
Chorioplague  is  a  plate  like  giant  cell 
of  the  chorion.  See  original  account 
for  lack  of  specific  properties  of  so- 
called  Langhans'  cells  which  designa- 
tion should  be  abandoned. 

4.  Epithelial  giant  cells  are  clearly  of 
epithelial  origin.  Found  in  epidermis 
in  chicken-pox  and  other  diseases,  oc- 
casionally in  the  liver  and  in  kidney  in 
many  conditions.  Often  show  nuclear 
irregularity  and  evidence  of  nuclear 
budding. 

5.  Hypertrophied  cells  can  be  either 
normal  to  meet  physiological  demands, 
as  enormously  enlarged  smooth  muscle 
cells  of  pregnant  uterus,  or  due  to  vari- 
ous pathological  conditions.  Mauth- 
ner's  Giant  Cell  in  the  fish  brain  is  al- 
ways of  tremendous  size  in  adults. 

Giemsa's  corrosive  sublimate  fixative.  Sat. 
aq.  corrosive  sublimate  2  parts,  absolute 
alcohol  1  part. 

Giemsa's  Stain.  1.  For  hlood  or  bacteria 
in  smears.  Fix  air  dried  smears  in 
methyl  alcohol  in  a  covered  dish  3-4 
minutes.  Remove  and  blot  dry.  Di- 
lute stock  solution  of  Giemsa  in  propor- 
tion of  1  drop  to  1  cc.  aq.  dest.  and  stain 
for  15  minutes.  Then  wash  in  aq.  dest., 
blot  and  dry.  If  a  precipitate  is  formed 
in  the  smear  by  the  stain,  invert  the 
slide,  support  both  ends,  and  the  stain 
will  adhere  like  a  hanging  drop,  kept 
away  from  the  ends  by  lines  ruled  in 
wax  or  paraffin.  The  pH  of  the  aq.  dest. 
used  to  dilute  the  stain  may  be  altered 
by  adding  very  dilute  acid  or  alkali. 
Optimum  pH  of  6.4  is  given  by  the 
McJunkin-Haden  buffer.  This  may  be 
used  as  diluting  medium  in  place  of  aq. 
dest.  Usually  the  azurophile  are 
stained  more  distinctly  and  the  neutro- 
phile  granules  less  sharply  than  by 
Wright's  stain.  Bacteria  and  intra- 
cellular protozoa  are  better  colored  than 
by  Wright's  stain.  The  May-Giemsa, 
and  Jenner-Giemsa  and  the  panchrome 
stains  of  Pappenheim  are  important 
modifications.  They  are  listed  sepa- 
rately. Present  situation  concerning 
Giemsa's  stain  is  that  American 
products  give  equally  good  results  with 
thin  films  but  the  German  product 
appears  to  be  better  for  thick  ones 
(Conn,  H.  J.,  Stain  Techn.,  1940,  15, 
41-43). 


GIEMSA'S  STAIN 


140  GLIA  STAINING  WITH  ANILIN  DYES 


2.  For  sections.  Much  depends  upon 
the  choice  of  fixative.  Formalin,  gener- 
ally employed  in  10%  solution,  acts  as 
a  sort  of  mordant  for  the  blue  component 
so  that  the  blue  coloration  is  particularly 
strong.  Fixation  in  Regaud's  gives  good 
results  particularly  with  Rickettsia, 
Zenker's  fluid  is  recommended  by 
Wolbach.  When  this  is  used  it  is  neces- 
sary to  remove  the  mercuric  chloride  by 
treating  the  sections  with  Lugol's  solu- 
tion. They  are  then  washed  in  95% 
alcohol  and  the  last  traces  of  iodine  are 
extracted  by  0.5%  aqueous  sodium 
hyposulphite  for  10-15  min.  The  hypo- 
sulphite in  turn  is  washed  out  in  run- 
ning water  about  5  min.  and  rinsing 
in  aq.  dest.  See  Cowdry's  colored 
figures  of  Rickettsia,  J.  Exper.  Med., 
1925,  42,  231-252.  Bouin's  fluid  (75 
cc.  saturated  aq.  picric  acid,  26  cc. 
commercial  formalin  and  4  cc.  glacial 
acetic  acid)  is  suggested  for  intracellular 
protozoa  (East  Coast  fever  parasites) 
by  Cowdry  and  Danks  (Parasitology, 
1933,  25,  1-63)  because  after  Giemsa 
staining  it  gives  the  chromatin  a 
desirable  purple  color  (see  colored 
plate).  Stain  sections  placed  vertically 
in  staining  jars  in  1.5  cc.  Giemsa 's 
solution  plus  50  cc.  aq.  dest.,  changed 
during  the  first  hour,  overnight.  Dif- 
ferentiate in  95%  alcohol,  dehydrate 
quickly  in  absolute  alcohol,  clear  in 
xylol  and  mount  in  balsam. 

If  the  sections  are  not  blue  enough  add 
1-2  drops  0.5%  sodium  bicarbonate  and 
1.5  cc.  methyl  alcohol  to  the  stain;  or 
remove  excess  of  mordanting  potassium 
bichromate  from  Zenker  fixation  by 
rinsing  1  min.  in  1%  potassium  per- 
manganate followed  by  5%  oxalic  acid 
4  min.  and  thorough  washing  in  aq. 
dest.,  or  do  both.  If  on  the  contrary 
they  are  too  blue  mordant  in  5%  potas- 
sium bichromate  15  min.,  rinse  in  aq. 
dest.  until  no  more  yellow  is  removed 
and  stain;  or  add  a  little  colophonium 
to  the  alcohol  used  in  differentiating 
and  dehydrating  of  the  sections,  as 
advised  by  Wolbach,  or  again  do  both. 

Usually  Giemsa's  stain  gives  satis- 
factory results  without  any  special  pre- 
cautions. The  difficulty  is  that  the 
colors  fade  quite  rapidly  particularly 
when  the  balsam  is  noticeably  acid  and 
when  the  sections  are  left  in  direct 
sunlight.  Their  period  of  usefulness 
can  be  extended  by  mounting  in  cedar 
oil,  used  for  oil  immersion  objectives, 
instead  of  in  balsam.  Try  Clarite. 
If  a  variety  of  fixatives  is  employed 
it  may  be  necessary  to  suit  the  stain  to 
the  fixative  by  use  of  buffers,  in  which 
case  see  Lillie,  R.  D.,  Stain  Techn., 
1941,  16,1-6. 


To  demonstrate  the  "nucleoids"  of 
bacteria  in  smears  the  technique  of 
C.  F.  Robinow  published  as  Addendum 
to  Dubos,  R.  J.,  The  Bacterial  Cell. 
Harvard  Univ.  Press,  1945,  460  pp.  is 
suggested.  Fix  smears  in  osmium 
tetroxide  vapor,  treat  7-10  min.  with 
N/1  HCl  at  60°C.  and  color  with 
Giemsa's  solution.  Bj'  this  method  nu- 
cleoids are  stained  whereas  similar 
bacteria  not  treated  with  the  acid  are 
uniformly  colored  by  Giemsa.  Robi- 
now prefers  this  staining  of  nucleoids 
by  Giemsa  after  hydrolysis  to  the  Feul- 
gen  technique. 

Gilson's  Fluid.  Nitric  acid  (sp.  gr.  1.456), 
15  cc;  acetic  acid,  4  cc;  mercuric 
chloride,  20  gm.;  60%  ale,  100  cc;  aq. 
dest.,  880  cc.  Used  mostly  for  inverte- 
brates. 

Gilson's  Mixture  is  equal  parts  chloroform 
and  cedar  oil. 

Gingiva.  Capillaroscopy  of  (McClung,  Mi- 
croscopical Technique,  1950,  328);  Eo- 
sinophile  leucocytes  in  (Orban,  B.,  J. 
Dent.  Res.,  1940,  19,  537-543.) 

Glacial  Acetic  Acid,  see  Acetic  Acid. 

Gland  Cells  contrasted.  Endocrine,  exo- 
crine, apocrine,  merocrine,  holocrine, 
serous,  zymogenic  and  mucous  (Cow- 
dry's  Histology,  p.  257). 

Glass  Cloth,  as  a  substrate  for  tissue  cul- 
ture, Warner,  D.,  Hanawalt,  C.  and 
Bischoff,  F.,  J.  Nat.  Cancer  Inst.,  1949, 
10,  67-74. 

Glass  Electrode.  Sisco,  R.  C,  Cunning- 
ham, B.  and  Kirk,  P.  L.,  J.  Biol.  Chem., 
1941,  139,  1-10  have  devised  an  open 
cup  variety  described  with  diagrams  by 
Click,  pp.  183-184.  See  Claff  and  Swen- 
son  glass  capillary  electrode  and  the 
Pickford  sealed-in  capaillary  glass 
electrode  also  described  by  Click. 

Glia  Staining  with  Anilin  Dyes  (Proescher, 
Fr.,  Stain  Techn.,  1934,  9,  33-38). 
Fix  in  10%  formalin  or  in  90%  alcohol 
followed  by  formalin.  Wash  frozen 
sections,  10-15  microns  thick,  in  aq. 
dest.  Stain  in  sat.  aq.  victoria  blue  B 
(not  filtered  but  poured  off  from  the 
undissolved  dye),  14-24  hrs.  Wash 
quickly  in  aq.  dest.,  mount  with  glyc- 
erin-albumen, blot  and  dry  in  air. 
Treat  with  ultraviolet  light  30  min. 
Pass  to  N/20  iodine  few  sec.  Remove 
iodine,  blot,  dry,  destain  in  xylol- 
anilin,  clear  first  in  clove  oil,  then 
xylol,  mount  in  balsam.  Glia  blue, 
nerve  cells  lightly  stained,  connective 
tissue  metachromatic  violet  or  colorless. 
Instead  of  ultraviolet  light  stained 
sections  can  be  treated  with  0.5% 
potassium  bichromatic  for  30  min.  In 
place  of  victoria  blue,  methyl  violet  2B, 
ethyl  violet  or  crystal  violet  can  be 
employed. 


GLICK 


141 


GLYCOGEN 


Glick,  see  Linderstr0m-Lang,  Kaj,  u.,  and 
Holter,  Heinz,  Histochemical  Advan- 
ces. 

Glomus.  Aortic  and  carotid,  see  Aortic 
Paraganglion. 

Glucose  Agar,  see  Bacteria  Media. 

Glutathione.  Demonstrated  by  Nitro- 
prusside  Reaction.  Inhibiting  factor 
in  Vitamin  C  silver  test. 

Glycerides,  see  Neutral  Fats. 

Glycerine.  Much  used  in  histological  tech- 
nique in  the  making  up  of  stock  solu- 
tions of  hematoxylin,  like  Delafield's, 
in  Albumen-Glycerin  used  for  mounting 
paraffin  sections,  etc.  It  serves  as  an 
excellent  clearing  agent  for  the  walls 
of  large  Arteries  so  that  the  intramural 
vessels  can  easily  be  distinguished  by 
the  blood  in  them.  With  potassium 
hydrate  it  is  employed  to  clear  speci- 
mens in  the  demonstration  of  Ossifica- 
tion centers.  As  a  mounting  medium 
for  frozen  sections  glycerin  is  invaluable. 
In  the  form  of  Brandt's  glycerin  jelly 
(which  see)  glycerin  is  specified  in  the 
technique  for  Sebaceous  Glands  and 
many  other  structures.  To  make  Ilai- 
ser's  glycerin  jelly  (Mallory,  p.  100) 
soak  40  gms.  gelatin  in  210  cc.  aq.  dest. 
for  2  hrs.  Add  250  cc.  glycerin,  stir 
and  heat  gently  10-15  min.  Keep  in 
ice  box  and  melt  before  use.  The  5 
gms.  carbolic  acid  crystals  specified  in 
Kaiser's  formula  has  unfortunately, 
according  to  Mallory,  a  deleterious 
influence  on  alum  hematoxylin  prepara- 
tions. See  also  Glychrogel  and 
Lactophenol. 

Glychrogel,  as  a  mounting  medium  for  teased 
preparations,  Marchi  stained  sections, 
gelatin  sections,  etc.  To  make  100  cc. 
dissolve  0.2  gm.  chrome  alum  (potas- 
sium chromium  sulphate)  in  30  cc.  aq. 
dest.  with  aid  of  heat.  Add  3  gm. 
Knox  granulated  gelatin  in  50  cc.  hot 
aq.  dest.  Add  20  cc.  glycerin  with 
constant  stirring  and  warm.  When 
thoroughly  mixed  add  crystal  of  camphor 
(Wotton,  R.  M.  and  Zwemer,  R.  L., 
Stain  Techn.,  1935,  10,  21-22).  For 
use  in  mounting  nematodes  (Wotton, 
R.  M.,  Stain  Techn.,  1937,  12,  145-146). 

Glycogen,  the  3  chief  microchemical  meth- 
ods have  been  critically  studied  by 
C.  M.  Bensley  (Stain  Techn.,  1939,  14, 
47-52).  This  account  follows  her  pre- 
sentation. Since  glycogen  is  labile, 
immediate  fixation  of  very  small  pieces 
of  tissue  (2-3  mm.)  and  agitation  of  the 
fixative  are  necessry.  She  recom- 
mends 9  parts  absolute  ethyl  alcohol  -f  1 
part  commercial  formalin  (i.e.  37% 
formaldehyde)  neutralized  with  MgCOj. 
If  desired  the  alcohol  in  this  fixative  can 
be  saturated  with  picric  acid.  After 
fixation  for  say  24  hrs.  wash  in  aboslute 


alcohol,  embed  in  the  usual  way  in  par- 
affin (carefully  avoiding  overheating) 
or  in  celloidin. 

1.  Best's  carmine.  Griibler's  car- 
minum  rubrum  optimum  or  some 
other  good  carmine  2  gm.,  potassium 
carbonate  1  gm.,  potassium  chloride  5 
gm.,  aq.  dest.  60  cc.  Boil  gently  until 
color  darkens,  cool  and  add  20  cc.  con- 
centrated ammonia.  Allow  to  ripen  24 
hrs.  This  is  stock  solution.  Mount 
paraffin  sections,  bring  down  to  aq. 
dest.  Stain  nuclei  with  hematoxylin 
as  in  the  H.  and  E.  technique.  Transfer 
to  fresh  stain  (stock  solution  10  cc,  15 
cc.  cone,  ammonia  and  30  cc.  pure 
methyl  alcohol)  for  20  min.  Rinse  in 
3  changes  methyl  alcohol,  dehydrate  in 
acetone,  clear  in  toluol  and  mount 
in  balsam.     Glycogen  brilliant  red. 

2.  Iodine  (Gage).  Mount  paraffin 
sections  as  before,  being  again  careful 
to  avoid  unnecessary  heat,  and  bring 
down  to  water.  Lugol's  aq.  iodine 
10-15  min.  Blot  with  filter  paper  and 
dry  in  air.  Mount  in  yellow  vaseline 
as  advised  by  S.  H.  Gage  (J.  Comp. 
Neur.,  1917,  27,  451-465)  with  minimum 
of  heat.     Glycogen  reddish  brown. 

3.  Bauer -Feidgen.  To  make  Feulgen 
reagent  dissolve  1  gm.  basic  fuchsin  in 
100  cc.  aq.  dest.  by  heat.  Filter  while 
warm  and  add  when  cool  20  cc.  normal 
HCI.  Add  1  gm.  NaHSOs.  Allow  to 
rest  24  hrs.,  when  it  should  be  of  pale 
straw  yellow  color.  Treat  deparaffinized 
sections  with  4%  chromic  acid  for  1  hr. 
or  with  1%  chromic  acid  over  night. 
After  washing  in  running  water  5  min., 
place  in  Feulgen  reagent  10-15  min. 
Rinse  IJ  min.  in  each  of  3  changes  of 
molecular  sol.  NaHSOs  1  part  and  tap 
water  19  parts.  Wash  in  running 
water  10 min.  Counterstain  nuclei  with 
hematoxylin  if  desired.  Dehydrate, 
clear  and  mount  in  balsam.  Glycogen 
deep  reddish  violet,  nuclei  lavender. 
See  Polysaccharides. 

Control.  Prepare  at  same  time  some 
sections  of  liver  rich  in  glycogen.  Be- 
cause glycogen  is  quickly  removed  by 
salivary  digestion,  when  sample  sections 
are  brought  down  to  aq.  dest.,  spit  on 
them  and  allow  to  rest  15-30  min.  chang- 
ing saliva  several  times.  Wash  thor- 
oughly in  water  at  body  temperature 
to  remove  mucus  and  stain  by  either  of 
the  3  above  mentioned  techniques.  If 
the  material  is  then  absent  in  such 
sections  and  present  in  other  similarly 
stained  and  not  digested,  it  is  evidently 
glycogen.  Fixation  by  the  freezing  and 
drying  method  is  even  better  than  with 
the  alcohol,  picric,  formalin  mixture 
because  it  is  quicker  and  there  is  less 


GLYCOGEN 


142 


GOLD 


chance  for  displacement  of  glycogen  in 
the  cells. 

See  also  for  glycogen  staining  of 
Trachoma  inclusions  Thygeson,  P.,  Am. 
J.  Path.,  1938,  14,  455-462.  Glycogen  is 
immobilized  in  its  natural  position 
within  the  cells  by  the  Freezing  and 
Drying  technique  (Altmann-Gersh). 
Compare  figures  3  and  4  of  Bensley  and 
Gersch  (R.  R.  and  I.,  Anat.  Rec,  1933, 
57,  205-215)  showing  results  by  this 
and  other  methods. 

A  new  ammoniacal  silver  nitrate 
method  for  glycogen  is  described  by 
Mitchell,  A.  J.,  and  Wislocki,  G.  B., 
Anat.  Rec,  1944,  90,  261-266.  To  pre- 
pare silver  solution  dissolve  1  gm.  silver 
nitrate  in  10  cc.  aq.  dest.  and  add  11 
drops  40%  aq.  potassium  hydroxide. 
Dissolve  ppt.  by  adding  26%  ammonia 
drop  by  drop  and  make  up  with  abs. 
ale.  to  100  cc.  Allow  to  stand  over 
night  before  use. 

Fix  livers  of  guinea  pigs  and  placentas 
of  same  and  other  animals  for  6-12hrs. 
in  sat.  picric  acid  in  abs.  ale,  90  cc. 
and  neutral  formaldehyde,  10  cc. 
Wash  in  abs.  ale.  several  times  likewise 
in  chloroform  and  abs.  ale.  Transfer 
to  chloroform  and  embed  in  paraffin. 

Place  sections  in  0.25%  aq.  potassium 
permanganate,  5-10  min.;  rinse  in  aq. 
dest.  1-2  min.,  decolorize  in  5%  aq. 
oxalic  acid,  5  min.  and  rinse  again  in  aq. 
dest.  Place  in  2%  aq.  silver  nitrate, 
12-24  hrs.,  transfer  to  ammoniacal  sil- 
ver nitrate,  15-30  min.,  rinse  in  4% 
neutral  formalin,  5-20  sec.  and  in  run- 
ning water,  1  min.  Fix  in  5%  aq. 
sodium  thiosulphate,  5-10  min.  After 
washing  in  running  water,  1  min., 
counterstain  in  paracarmine  (Mayer), 
dehydrate,  clear  in  xylol  and  mount  in 
balsam.  Glycogen,  dense  black  corre- 
sponds with  that  shown  by  Bauer- 
Feulgen  technique.  Excellent  illus- 
trations. 

The  recently  developed  techniques 
of  Gomori  (Am.  J.  Clin.  Path.,  1946,  16, 
177)    and  Hotchkiss    (Arch.   Biochem., 

1948,  31,  131)  can  be  used  on  tissue  sec- 
tions or  blood  smears  (Gibb,  R.  P.,  and 
Stowell,  R.  E.,  Blood,  J.  Hematology, 

1949,  4,  569-579).  The  evaluation  of 
methods  for  the  histochemical  study  of 
glycogen  by  Carpenter,  A.M.,  Polon- 
sky,  B.  and  Mesiten,  M.  U.  (Arch. 
Path.,  1951,  51,  480-485)  may  be  help- 
ful. 

The  best  way  to  separate  out  glycogen 
en  masse  is  by  centrifugal  isolation  as 
employed  by  Lazarow,  A.  (Anat.  Rec, 
1942,  84,  31-50;  Biol.  Symposia,  1943, 
10,  9-26)  for  suspensions  of  fragmented 
liver  cells. 

Colorimetric    methods    for    glycogen 


may  afford  valuable  evidence.  See 
Boettiger,  E.  G.  (J.  Cell.  Comp. 
Physiol.,  1946, 27, 1-8)  and  Van  Wagten- 
donk,  W.  J.,  Simonsen,  D.  H.  and  Hack- 
ett,  P.  L.  (J.  Biol.  Chem.,  1946,  163, 
301-306)  and  the  critical  discussion  by 
Click,  p.  247.  See  Heatley,  N.  G.  and 
Lindahl,  P.  E.  (Proc.  Roy.  Soc,  B, 
1937,  122,  395-402)  for  separation  of 
desmo-  and  lyoglycogen. 

Glycol  Stearate.  As  an  imbedding  medium 
(Cutler,  O.  L,  Arch.  Path.,  1935,  20, 
445-446).  Pass  up  through  alcohols  to 
equal  parts  95%  ale.  and  glycol  stearate 
in  incubator  at  56''C.  12-24  hrs.  Pure 
glycol  stearate  at  56°C.  24  hrs.  Imbed 
as  in  paraffin. 

Glucuronidase.  An  enzyme,  widespread  in 
occurrence  in  the  mammalian  organism, 
which  hydrolyzes  esters  of  glucuronic 
acid.  Glucuronides  are  important  de- 
toxification products.  Chemical  meth- 
ods for  identification  of  glucuronidase 
are  available  (see  Fishman,  W.  H., 
Chapter  18,  The  Enzymes  New  York: 
Academic  Press).  1950  Vol.  1,  part  1, 
pp.  635-652,  Friedenwald,  J.  S.  and  B. 
Becker   (J.  Cell,  and  Comp.  Physiol., 

1948,  31,  303-309)  have  described  a 
method  for  localizing  glucuronidase  on 
tissue  sections,  and  Seligman,  A.M., 
M.  M.  Nachlas,  L.  H.  Manheimer,  O. 
M.  Friedman  and  G.  Wolf  (Ann.  Surg., 

1949,  130,  333-341)  describe  a  method 
involving  the  hydrolysis  of  the  beta 
glucuronide  of  beta  naphthol.  The 
liberated  naphthol  is  converted  to  a 
dye  by  diazotization. 

Glyoxal.  As  substitute  for  formaldehyde 
in  tissue  fixation  (Wicks,  L.  F.  and  Sunt- 
zeff,  v..  Science,  1943,  98,  204;  Stowell, 
R.  E.  and  Stokes,  J.  M.  J.  Tech.  Meth. 
and  Bull.  Internat.  Assoc.  Med.  Mu- 
seums, 1944,  24,  25-30).  Concentra- 
tions 2-6%  produce  less  shrinkage  and 
give  better  cytoplasmic  preservation 
than  4%  formaldehyde.  Glyoxal  is 
only  recommended  as  general  substi- 
tute for  formaldehyde  when  latter  is 
not  available. 

Gmelin's  test  for  bile  pigments.  On  addi- 
tion of  nitric  acid  containing  a  little 
nitrous  acid,  color  changes  to  green, 
then  red  and  finally  blue  observable 
under  microscope. 

Gold,  microchemical  detection  of:  1.  Method 
of  Borchardt.  Modified  by  Michaelis, 
O.,  Biochem.  Zeit.,  1930,  225,  478-488. 
Treat  sections  of  formalin  or  alcohol 
fixed  tissues  for  15  min.  in  a  boiling 
water  bath  or  for  12-24  hrs.  at  40 °C. 
with  5%  aq.  silver  nitrate.  Remove 
ppt.  from  section  with  20%  aq.  nitric 
acid.  Gold  appears  as  black  granules 
(Lison,  p.  100). 
2.  M.ethod  of  Okkels,   H.,   C.   rend. 


GOLD 


143 


GOLGI  APPARATUS 


Soc.  Biol.,  1929,  102,  1089-1091.  Simply 
produce  gold  salt  in  sections  by  exposing 
for  at  least  12  hrs.  to  sunlight  or  to 
ultraviolet  lamp  for  same  time  (Gau- 
thier-Villars,  P.,  C.  rend.  Soc.  de  Biol., 
1932,  109,  197-198).  Lison  (p.  100) 
explains  that  whatever  the  technique 
used  it  is  necessary  to  prove  that  the 
black  granules  are  gold  by  their  insolu- 
bility in  concentrated  acids,  solubility 
in  aqua  regia  (equal  parts  nitric  and 
hydrochloric  acids)  and  solubility  in 
solutions  of  potassium  or  sodium  cya- 
nide. 

3.  Method  of  Roberts,  W.  J.,  Bull. 
d'Hist.  Appl.,  1935,  12,  344-361.  Fix 
tissues  in  20%  neutral  formalin  or  in 
Bouin's  fluid.  Avoid  fixatives  contain- 
ing a  metal.  Wash  thoroughly  in  water. 
Make  paraffin  or  frozen  sections.  The 
latter  has  the  advantage  of  speed.  Make 
2  solutions  :  A.  Add  2  gm.  silver  nitrate 
pure  for  analysis  to  100  cc.  10%  gum 
arable  in  the  dark  immediately  before 
use.  B.  Add  1  gm.  hydroquinone  pure 
to  100  cc.  10%  gum  arable  the  day  before 
use.  Take  off  the  frozen  sections  in  aq. 
dest.  Mix  2  cc.  A  and  2  cc.  B,  add  1-3 
drops  5%  citric  acid,  shake  30  sec. 
Leave  sections  in  this  mixture  5-10 
min.  Then  without  first  washing  plunge 
into  5%  aq.  sodium  hyposulphite  for  a 
few  minutes.  Wash  thoroughly  and 
mount.  Gold  in  cells  is  covered  with 
black  deposit  of  reduced  silver.  Said 
to  be  more  sensitive  method  than 
spectrographic  analysis.  See  author's 
illustrations. 

4.  A  technique  for  demonstration 
of  gold  in  abs.  ale.  or  neutral  formalin 
fixed  tissues,  based  upon  reaction  with 
p  -  Dimethylaminobenzylidenrhodanin 
is  described  by  Okamoto,  K.,  Akagi, 
T.  and  Mikami,  G.,  Acta.  Scholae 
Med.  Univ.  Imp.  in  Kioto,  1939,  22, 
373-381. 

5.  Tin  chloride  method  (Elftman,  H. 
and  Alice  G.  Stain  Techn.,  1945,  20, 
59-62.  After  rats  and  guinea  pigs  are 
injected  intraperitoneally  with  aqueous 
yellow  gold  chloride  fix  by  injection  of 
neutral  formalin  through  heart.  Make 
paraffin  sections.  Pass  down  to  water 
in  usual  way.  Place  slides  in  mixture 
of  10  parts  stock  5%  aq.  SnCl2-2H20 
(with  some  pieces  metallic  tin  added  to 
retard  oxidation)  and  1  part  cone.  HCl 
(mixture  prepared  and  filtered  just  be- 
fore use)  in  incubator  at  56°C.  for  24 
hrs.  Wash  several  changes  aq.  dest. 
before  dehydrating  clearing  and  mount- 
ing in  damar.  Presence  of  gold  indi- 
cated by  particles  exhibiting  purple  of 
Cassius  grading  into  brown.  Colloidal 
gold  in  red,  blue  and  black  may  likewise 
occur.    To  eliminate  disadvantages  of 


occasional  precipitates  of  tin  unrelated 
to  gold  and  possible  confusion  with  bile 
pigments  and  others  the  following  tech- 
nique is  proposed  by  these  authors. 

6.  Fix  in  neutral  formalin,  bring  down 
mounted  sections  to  water.  Place  in 
3%  HjOs  in  incubator  at  37°C.  for  at 
least  24  hrs.  better  3  days.  Wash  in  aq. 
dest.  Run  up  and  mount  in  damar. 
Gold  thus  reduced  to  metallic  form 
shows  range  of  colors,  rose  chiefly  grad- 
ing into  purple,  blue  and  black. 

7.  Christeller,  E.,  Verh.  deutsch. 
Path.  Ges.,  1927,  22,  173  reports,  as  de- 
scribed by  Gomori,  G.,  J.  Mt.  Sinai 
Hosp.,  1944-45,  11,  317-326,  demonstra- 
tion of  gold  salts  by  reduction  to  metal- 
lic gold  with  SnCh.     Similar  to  No.  5. 

8.  For  micro-determination  of  gold 
in  biological  fluids  and  tissues,  see 
Block,  W.  D.,  Ann.  Rheumatic  Dis., 
1944-45,  4,  39-42.  Use  of  this  tech- 
nique provides  a  good  check  on  above 
described  microchemical  methods. 

Radioactive  gold,  distribution  of 
within  cartilage  of  knee-joint,  Ekholm, 
R.,  Acta  Anat.,  1951,  Supp.  15  to  II, 
75  pp. 

Gold  Chloride  for  nerve  endings,  see 
Craven's  and  Carey's  methods. 

Gold  Orange,  see  Orange  IL 

Gold  Orange  MP,  see  Methyl  Orange. 

Gold  Particles.  The  particles  of  gold  are 
held  in  colloidal  state  by  the  protective 
colloid,  sodium  lysalbinate,  and  are 
employed  to  stimulate  macrophage  pro- 
duction by  intravenous  injections  in 
animals  (Simpson,  M.  J.,  J.  Med.  Res., 
1922,43,77-144). 

Goldman,  see  Iron  Hematoxylin  Single 
Stain. 

Golgi  Apparatus — Written  by  Geoffrey 
Bourne,  London  Hospital  Medical  Col- 
lege, London,  England.  November  5, 
1951 — -Most  recent  books  which  give 
details  of  techniques  for  demonstration 
of  the  Golgi  apparatus  preface  their 
descriptions  with  the  statement  that,  as 
some  doubt  exists  as  to  the  nature  of  the 
apparatus,  it  is  difficult  to  describe 
techniques  for  demonstrating  it.  This 
doubt  is  still  unresolved  and  various 
authors  hold  widely  divergent  views 
as  to  the  structure  of  the  apparatus. 
These  may  be  summarized  as  follows: 
Gatenby  (IVIicrotomist's  Vade  Me- 
cum,  nth  Ed.  1950)  believes  that  the 
original  conception  of  the  Golgi  appara- 
tus in  vertebrate  cells  as  a  juxta-nuclear 
argentophil  network  is  still  correct. 
Baker's  view  (Quart.  J.  Micr.  Sci. 
1944,  85,  1-71 — a  modification  of  that 
expressed  by  Parat,  M.  Arch.  d'Anat. 
Micr.,  1928,  24,  73)  is  that  the  apparatus 
is  composed  of  a  series  of  neutral  red 
staining    vacuoles    more    or    less    sur- 


GOLGI APPARATUS 


144 


GOLGI APPARATUS 


rounded  by  a  zone  of  dense  phospholipid 
material  and  in  some  cases  embedded 
in  a  zone  of  diffuse  phospholipid. 
Palade  and  Claude  (J.  Morph.  1949,  85, 
71)  claim  that  the  Golgi  apparatus  is 
composed  solely  of  neutral  red  staining 
vacuoles  which  possess  a  membrane  con- 
taining an  appreciable  proportion  of 
lecithin  and  that  the  classical  Golgi 
apparatus  is  formed  by  myelin  figures 
produced  by  the  action  of  fixatives  on 
these  vesicles.  Bensley  (Exp.  Cell. 
Res.  1951,  2,  1-19)  contends  that  the 
Golgi  apparatus  is  composed  of  vesicles 
or  canals  containing  a  watery  solution 
in  which  various  substances,  e.g., 
polysaccharides  are  dissolved  (Gersh, 
I.,  Arch.  Path.  1949,  47,  99-109).  The 
canalicular  conception  of  the  Golgi  ap- 
paratus is  also  claimed  by  Gatenby  and 
Moussa  for  the  mammalian   neurone. 

Against  these  views  we  must  put  the 
fact  that  no  canals  have  been  seen  in 
cells  by  numerous  workers  using  the 
phase  contrast  microscope  and  as 
Hibbard,  H.  (Quart.  Rev.  Biol.  1945, 
20,  1-19)  has  pointed  out  Lewis  has 
never  seen  anything  resembling  the 
classical  apparatus  in  a  life-time  of 
study  of  tissue  culture  cells.  Most 
workers  on  the  Golgi  apparatus  have 
tended  to  range  themselves  behind  one 
or  other  of  these  views.  It  should  be 
realized,  however,  that  all  these  points 
of  view  about  the  structure  of  the  ap- 
paratus simply  continue  the  con- 
troversy which  has  gone  on  almost  since 
its  discovery.  The  canalicular  concep- 
tion of  the  apparatus  originated  in  1902 
with  Holmgren's  Trophospongium,  the 
neutral  red  vacuole  theory  found  its 
origin  in  Parat's  1928  work  and  has 
seen  some  extension  in  Cowdry's  labora- 
tory by  the  work  of  Cowdry,  E.  V.  and 
Scott,  G.  H.  (Arch.  Inst.  Pasteur  de 
Tunis,  1928,  233),  Covell,  W.  P.  and 
Scott,  G.  H.  (Anat.  Rec,  1928,  38,  377), 
and  of  course  the  argentophil  network 
theory  originated  with  Golgi  himself 
in  1898  (Arch.  Ital.  de  Biol.,  30,  60)  and 
in  more  recent  years  has  been  supported 
bj'^  Ludford  and  particularly  by 
Gatenby.  So,  it  is  apparent  that  our 
knowledge  of  the  nature  of  the  Golgi 
apparatus  has  remained  more  or  less  in 
the  same  state  as  it  was  30  or  40 
years  ago. 

In  view  of  this  discrepancy  of  views 
techniques  wall  be  given  for  demon- 
strating the  classical  Golgi  net  and  also 
for  demonstrating  the  apparatus  as 
discrete  bodies. 

The  Classical  Golgi  Apparatus:  Even 
if  the  Golgi  network  is  an  artefact  as 
some  workers  believe  the  conventional 


Golgi  preparation  does  give  us  informa- 
tion about  its  position  in  the  cell  and 
whether  the  Golgi  material  is  present  in 
small  or  in  large  amount. 

Golgi's  original  technique  involved 
the  fixation  of  tissue  in  a  mixture  con- 
taining potassium  bichromate  and 
osmic  acid  followed  by  impregnation 
with  silver.  The  apparatus  with  this 
technique  appears  jet  black  against  a 
yellowish  background.  It  is  a  con- 
spicuous structure  consisting  of  an 
intricate  network  of  anastomosing 
strands.  This  network  may  closely  en- 
velop the  nucleus,  be  concentrated  to 
one  side  of  it  or  else  be  scattered  rather 
diffusely  throughout  the  cytoplasm. 
In  glandular  cells  the  apparatus  grows 
in  size  with  the  development  of  secre- 
tory granules  and  strands  from  it  ramify 
between  the  various  granules. 

Kopsch,  F.  (Sitzungsber.  K.  Akad. 
Wissensch,  1902,  40,  929)  showed  that 
the  Golgi  apparatus  can  be  blackened 
by  prolonged  treatment  with  2%  osmic 
acid.  On  this  affinity  for  both  silver 
and  osmium  all  the  conventional  meth- 
ods of  demonstrating  the  Golgi  appara- 
tus are  based.  Few  cytological  re- 
actions are  more  fickle  and  incon- 
stant; but  when,  after  many  attempts, 
the  technique  is  successful,  convincing 
and  very  beautiful  preparations  result. 
Mitochondria  can  be  stained  supra- 
vitally  by  some  vital  dyes  but  no  vital 
dyes  will  show  up  the  reticular  Golgi 
apparatus,  a  fact  which  is  taken  by 
some  authors  to  indicate  the  non-exist- 
ence in  vivo  of  such  networks.  How- 
ever, neutral  red  will  stain  spheres  in 
the  region  of  the  apparatus. 

With  both  silver  and  osmium  methods 
considerable  experimentation  is  neces- 
sary in  order  to  obtain  the  best  results. 
The  factors  to  be  varied  are  principally 
the  composition  of  the  fixative  and  im- 
pregnating substance  and  the  time 
during  which  they  are  allowed  to  act. 
During  impregnation  it  is  always  ad- 
visable to  keep  the  tissues  in  the  dark 
and  instructions  as  to  temperature  re- 
quirements should  be  carefully  fol- 
lowed. When  either  the  silver  or 
osmium  solution  becomes  blackened 
it  should  be  renewed.  It  is  important 
for  the  beginner  to  start  with  the  most 
favorable  material.  The  spinal 
ganglion  cells  of  young  mammals  such 
as  the  rabbit  are  perhaps  the  best  for 
this  purpose.  The  acinous  cells  of  the 
pancreas  are  also  recommended  but  are 
somewhat  more  difficult  to  handle.  All 
the  methods  of  impregnation  outlined 
below  frequently  bring  to  light  the 
mitochondria  also. 


GOLGI APPARATUS 


145 


GOLGI APPARATUS 


Osmic  Acid  Technique: 

1.  Mann-Kopsch  method  (Lee's  Mi- 
crotomists  Vade  Mecum,  Uth  Ed. 
1950,  edited  by  J.  Bronte  Gatenby  and 
H.  W.  Beams).  Fix  in  Mann's  fluid 
(equal  parts  of  1%  osmic  acid  and  sat. 
sublimate  in  normal  saline),  for  J-3  hrs. 
Wash  in  aq.  dest.  15-30  min.  2%  osmic 
acid  room  temperature  10-14  days. 
Wash  in  ruiming  water  3  hrs.  or  more. 
Dehydrate  clear  and  embed.  In  the 
sections  Gatenby  was  able  to  extract  the 
blackening  step  "by  step  with  turpentine 
and  thus  to  improve  considerably  the 
preparations. 

2.  Liidford's  modification  (Ludford, 
R.  J.,  J.  Roy.  Micr.  Soc,  1926,  107). 
Fix  mammalian  and  avian  tissues  in 
Mann's  corrosive  osmic  solution  18  hrs. 
Wash  aq.  dest.  30  min.,  2%  osmic  at 
30°C.  for  3  days.  Water  at  30°C.  for  1 
day,  dehydrate,  clear,  embed  in  paraffin. 
A  useful  variant  is  to  fix  in  the  same 
way,  and  wash  in  aq.  dest.  Then 
osmicate  at  35°C.  for  3  daj'S,  first  day  in 
2%  osmic,  second  in  1%  and  third  in 
0.5%.  Leave  in  water  for  1  day  at 
35°C.  He  recommends  various  counter 
stains.  See  Lee's  11th  Ed.  (pp.  404- 
410)  for  a  discussion  of  osmication 
methods;  also  Owens,  H.  B.  and 
Bensley,  R.  R.  (Anat.  Rec,  1929,  44, 
79)  for  a  careful  study  of  factors  in- 
fluencing the  osmic  acid  changes  and  for 
their  ferric   chloride   osmic  method. 

3.  Sjovall's  method  (Sjovall,  E.,  Anat. 
Hefte,  1906,  30,  261-291).  Fix  in  10% 
formalin  8  hrs.  Wash  in  aq.  dest.  2% 
osmic  acid  at  35°C.,  2  days.  Dehy- 
drate, clear  and  embed. 

4.  Kolatchew's  method  (Nassonov,  D. 
N.,  Arch.  f.  Mikr.  Anat.,  1924,  103, 
437).  Fix  in  3%  aq.  potassium  bi- 
chromate, 10  cc;  1%  chromic  acid  10 
cc;  and  2%  osmic  acid,  5  cc.  for  24  hrs. 
Wash  in  running  water  24  hrs.  2%  osmic 
acid  40°C.  8  hrs.  3-5  days  at  35°C. 
Wash  in  aq.  dest.,  dehydrate,  clear  and 
embed.  Osmic  methods  sometimes  im- 
pregnate mitochondria  as  well  as  the 
Golgi  material  particularly  if  the  period 
in  osmic  acid  is  prolonged,  care  must  be 
taken  therefore  in  interpreting  results. 

Silver  Nitrate  Methods:  Tissues  from 
young  animals  usually  respond  best  to 
silver  methods  but  ganglia  from  older 
animals  respond  very  well. 

1.  Aoyaina's  method  (Baker,  J.  R., 
Cytological  Technique,  3rd  Ed.,  1950, 
p.  194).  Fix  tissues  in  Aoyama's  fixa- 
tive (cadmium  chloride  1  gm.,  neutral 
formalin  4%,  15  cc,  aq.  dest.  85  cc.) 
for  4  hrs.  Wash  with  aq.  dest.  and  then 
place  in  silver  nitrate  solution  for  12-14 
hrs.    Wash    with   aq.    dest.    and    then 


place  in  silver  nitrate  solution  for  12-14 
hrs.  After  8-12  hrs.  (kept  in  dark  or  in 
diffuse  light).  Wash  with  aq.  dest.  and 
place  in  Aoyama's  reducer  (Hj'dro- 
quinone  1  gm..  Neutral  Formaldehyde 
40%  15  cc,  aq.  dest.  80  cc,  anhydrous 
sodium  sulphite  0.15  gm.)  for  5  hrs. 
Leave  for  I  hr.  in  running  water  and 
then  place  in  50%  alcohol  overnight. 
Dehydrate  and  embed. 

2.  Cajal's  method  (Carleton,  H.  M.,  J. 
Roy.  Micr.  Soc,  1919,  321-329).  This 
is  one  of  the  many  methods  devised  by 
Cajal.  It  is  recommended  for  embryos 
and  young  animals.  Fix  in  uranium 
nitrate,  1  gm.,  formalin  15  cc,  and  aq. 
dest.  100  cc,  8-24  hrs.  Wash  quickly 
in  aq.  dest.  1.5%  aq.  silver  nitrate  24^8 
hrs.  Rinse  in  aq.  dest.,  hydroquinone 
2  gm.,  formalin  6  cc,  aq.  dest.  100  cc, 
anhydrous  sodium  sulphite  0.15  gm.  12 
hrs.  Wash  in  aq.  dest.,  dehydrate 
quickly,  clear,  embed  and  section. 

3.  Da  Fano's  method  (Da  Fano,  C.,  J. 
Roy.  Micr.  Soc,  1920,  157-161).  Here 
the  uranium  nitrate  is  replaced  by  co- 
balt nitrate.  In  other  respects  the 
technique  is  similar.  Da  Fano  has, 
however,  so  carefully  attempted  to  con- 
trol troublesome  experimental  condi- 
tions that  the  various  steps  are  given  in 
detail.  Fix  in  cobalt  nitrate  1  gm.,  aq. 
dest.  100  cc,  formalin  15  cc  6-8  hrs. 
The  formalin  need  not  be  neutralized 
unless  it  is  strongly  acid.  For  embryos 
and  delicate  tissues  where  there  is  dan- 
ger of  shrinkage  reduce  formalin  to  6 
cc.  Cartilage  and  small  pieces  of 
tissue  (not  more  than  3  mm.)  fix  half 
fixation  time.  Hollow  organs  (e.g. 
stomach  and  intestine)  fix  1  hr.  then 
cut  into  smaller  pieces.  Spinal  cord, 
cerebellum  and  cerebrum  of  adults  fix 
8-10  hrs.  (fixation  should  never  go 
beyond  24  hrs.).  Testicle,  inject  fixa- 
tive through  aorta  then  immerse  testicle 
in  fixative.  Wash  quickly  in  aq.  dest. 
and  impregnate  in  15%  aq.  AgNOs 
24-48  hrs.  Very  small  fragments  im- 
pregnate in  1%  AgNOs,  2%  for  tissues 
containing  much  fat  and  for  spinal 
cord.  Impregnation  normally  satis- 
factory at  room  temperature.  If  un- 
satisfactory at  35°-37°C.  Wash  rapidly 
in  aq.  dest.  and  cut  down  tissues  again 
to  a  thickness  of  2  mm.  or  less.  Reduce 
in  Cajal's  hydroquinone  mixture,  above 
mentioned,  12-24  hrs.  Wash  in  aq. 
dest.  i  hr.  Cut  with  freezing  micro- 
tome or  embed  in  wax.  Golgi  ap- 
paratus dark  brown  or  black  against  a 
yellow  background.  Tone  sections 
with  gold  to  clear  preparation.  Pass  to 
water.    Then  0.1-0.2%  gold  chloride  2 


GOLGI APPARATUS 


146 


GOLGI  METHOD,  QUICK 


hrs.  Counterstain,  dehydrate,  clear 
and  mount. 

It  is  of  interest  that  all  silver  prepara- 
tions depend  upon  the  treatment  of  the 
tissue  by  fixatives  which  contain  the 
salt  of  a  heavy  metal.  Silver,  M.  L. 
(Anat.  Rec,  1942,  82,  507-529)  has 
pointed  out  that  silver  micelles  are  not 
adsorbed  on  to  the  Golgi  material  un- 
less the  cells  have  been  treated  with 
salts  of  heavy  metals — hence  the  pres- 
ence of  metals  like  cobalt  and  ura- 
nium in  the  fixatives  used  for  silver 
techniques. 

Stains: 

1.  Baker's  Sudan  Black  Method  (Baker, 
J.  R.,  Quart.  J.  Micr.  Sci.,  1944,  85, 
1-71).  Fix  small  pieces  of  tissue  in 
formol  calcium  (formalin  40%,  10  cc, 
calcium  chloride,  anhydrous  10%  aq.  10 
cc,  aq.  dest.  80  cc.)  for  3  days,  embed  in 
gelatine.  Harden  block  in  formalin-cal- 
cium-cadmium (formalin  10  cc,  calcium 
chloride,  anhydrous  10%  aq.  10  cc, 
cadmium  chloride,  10%  aq.  10  cc,  aq. 
dest.  70  cc.)  and  then  wash  3-4  hrs.  in 
running  water.  Section  on  freezing 
microtome  at  15m.  Sections  when  fixed 
to  the  slide  are  placed  in  filtered  for- 
malin-calcium-cadmium solution  until 
ready  for  staining.  Then  wash  slides 
for  3  min.  in  running  water,  pass 
through  50%  and  70%  ale  to  a  sat.  solu- 
tion of  Sudan  black  in  70%  ale,  leave  for 
7  min.  Pass  through  3  lots  of  50%  ale, 
rinse  in  aq.  dest.,  counterstain,  mount 
in  glycerine  jelly  or  Apathy's  medium. 
This  method  does  not  demonstrate  the 
Golgi  apparatus  as  a  network  but  as  a 
series  of  discrete  bodies,  a  form  which  is 
claimed  by  some  authors  to  represent 
more  nearly  the  true  form  of  the  Golgi 
apparatus  in  living  cells.  Many  of  the 
vesicles  which  are  demonstrated  by 
this  method  also  stain  with  neutral  red. 

2.  Baker's  Neutral  Red  Method  (Baker, 
J.  R.,  Quart.  J.  Micr.  Sci.,  1944,  85, 
1-71).  This  is  a  vital  method.  Cells 
are  separated  by  teasing  in  a  salt  mix- 
ture (sodium  Chloride,  0.7%  aq.  100 
cc,  calcium  chloride,  anhydrous,  10% 
aq.,  0.2  cc).  To  3  drops  of  solution 
containing  a  suspension  of  cells  add  3 
drops  of  a  solution  made  up  of  Neutral 
red  0.1%  aq.,  1  cc.  and  sodium  and 
calcium  chloride  solution  (as  above) 
9  cc.  Final  concentration  of  dye  is 
0.005%.  Mix  cell  suspension  with  dj^e 
by  sucking  up  with  pipette  and  pass  out 
again.  Leave  preparation  (covered) 
for  20  min.  Put  2  drops  of  mixture  on 
the  glass  of  a  compressorium  in  such  a 
way  as  to  include  some  air.  Cover  edge 
of  compressorium  with  soft  paraffin 
(e.g.  Vaseline).  Examine  under  im- 
mersion lens. 


Golgi  Cox  Method  for  adult  nervous  system. 
— Written  by  J.  L.  O'Leary,  Dept.  of 
Neuropsychiatry,  Washington  Uni- 
versity School  of  Medicine,  St.  Louis 
10,  Mo.  May  8,  1950.— Fix  pieces  3-6 
mm.  thick  in  following  fluid :  add  20  cc 
5%  aq.  potassium  bichromate  to  20  cc 
6%  aq.  mercuric  chloride.  Dilute  16 
cc.  5%  aq.  potassium  chromate  with  40 
cc.  aq.  dest.  and  add  this  to  the  first 
two.  Do  not  agitate  but  leave  in 
fixative  until  scum  forms  on  surface, 
usually  after  1^-2  months.  When  im- 
pregnation is  nearly  complete,  wash 
rapidly,  dehydrate  through  graded  alco- 
hols and  imbed  in  low  viscosity  celloidin 
(see  Celloidin  Imbedding).  Cut  cel- 
loidin sections  serially  at  80  to  120 
microns.  Arrange  in  serial  order  on 
slides  (80%  alcohol).  Blot  sections 
dry  and  cover  immediately  with  1% 
celloidin.  When  somewhat  dry,  bring 
slides  with  sections  to  water.  The 
sections  on  each  slide  may  thereafter 
be  treated  as  a  unit.  Run  sections 
from  water  into  a  saturated  solution 
of  sodium  sulfite.  They  rapidly  turn  a 
yellow  gray.  Wash  over  night  and  de- 
hydrate through  graded  alcohols  to  ab- 
solute. Coat  with  the  following  var- 
nish, applying  it  repeatedly  in  thin  even 
layers,  and  allowing  each  to  dry  par- 
tially before  applying  the  next  (san- 
darac,  75  gm.;  camphor,  15  gm.; 
turpentine  C.P.,  30  cc;  oil  of  lavender, 
22.5  cc;  abs.  ale,  75  cc;  add  castor  oil, 
7  drops.  Mixture  dissolves  very 
slowly).  Since  sections  are  somewhat 
opaque,  the  varnish  must  dry  for  several 
days  until  abs.  ale  has  evaporated. 

Golgi  Methods.  Fundamentally  these  are 
different  from  both  the  Cajal  and  Biel- 
chowsky  techniques  which  were  later 
developments.  They  depend  upon  a 
preliminary  fixation  in  a  potassium 
bichromate  solution  often  containing 
formalin  and  sometimes  other  sub- 
stances such  as  osmic  acid.  The  silver 
is  selective  tending  to  impregnate  a  few 
cells  completely  which  become  black- 
ened when  it  is  reduced.  Except  for  the 
occasional  demonstration  of  the  Golgi 
Apparatus  these  methods  do  not  reveal 
details  of  the  inner  structure  of  nerve 
cells  like  Neurofibrils  and  Nissl  Bodies. 
They  are  of  great  service  in  the  demon- 
stration of  many  non-nervous  tissue 
components,  the  parietal  cells  of  the 
stomach,  bile  canaliculi  of  the  liver, 
Rouget  or  perivascular  cells,  etc. 

Golgi  Method,  Quick.  For  brains  of  new- 
born animals,  and  of  those  1  day  to  30 
days  old. — Written  by  J.  L.  O'Leary, 
Dept.  of  Neuropsychiatry,  Washing- 
ton University  School  of  Medicine,  St. 
Louis  10,  Mo.  May  8,  1950.— It  is  es- 


GOLGI,  METHOD 


147 


GOMORI'S  METHODS 


sential  to  determine  the  age  of  the 
animal  at  which  the  cell  or  fiber  selected 
for  study  is  reaching  maturity.  For 
example,  if  new  born  kittens  are  chosen, 
and  the  area  striata  is  the  object  of 
study,  the  best  impregnations  of  enter- 
ing fibers  are  obtained  at  12  to  15  days 
after  birth ;  of  short  axon  cells,  at  18  to  21 
days;  and  of  pyramids  at  21  to  24  days. 
Cut  slices  of  brains  3-4  mm.  in  thick- 
ness by  quick  cuts  of  a  sharp  scissors. 
Fix  in:  potassium  bichromate,  10  gm.; 
osmic  acid,  1  gm.;  aq.  dest.,  330  cc. 
Time  of  fixation  must  be  determined 
for  each  part  of  the  CNS  studied.  In 
general  the  older  the  animal,  the  longer 
it  is.  After  fixation,  blot  blocks  of 
tissue  on  filter  paper  and  transfer  to  a 
bottle  containing  f%  aq.  silver  nitrate. 
After  24  hrs.  the  reaction  is  complete. 
Imbed  in  celloidin.  Subsequent  treat- 
ment is  very  important.  Place  block  in 
95%  ale.  for  about  5  min.,  remove  and 
blot  dry.  Place  block  on  paraffin  disc 
mounted  on  a  block  holder  in  the  orienta- 
tion desired  for  cutting.  Using  a  hot 
teasing  needle,  melt  paraffin  around  the 
block  so  as  to  fasten  block  to  paraffin. 
Be  sure  that  melted  paraffin  does  not 
creep  up  on  the  block.  Use  knife  at  45° 
angle  to  the  block.  Cut  serially  80-100ai. 
Place  each  section  as  cut  in  order  in  95% 
ale.  using  Petri  dishes.  Be  sure  not  to 
miss  first  and  last  section  of  the  block 
for  these  are  often  more  valuable  than 
the  entire  remainder  of  the  block. 
Using  a  spatula,  transfer  to  another  95% 
ale.  after  5  min.  After  another  5  min. 
transfer  to  oil  of  cloves,  arranging  in 
serial  order,  by  placing  each  section  as 
it  enters  oil  of  cloves  near  the  edge  of 
the  Petri  dish  so  that  it  adheres  to  the 
edge.  When  all  sections  are  transferred 
the  group  will  be  placed  around  the 
circumference  of  the  Petri  dish.  As  the 
sections  start  to  retract  from  the  edge, 
begin  to  arrange  them  in  the  usual  order 
for  serial  sections.  After  clearing  (clove 
oil  5  to  10  min.)  transfer  in  serial  order 
to  slides.  Blot  off  excess  of  clove  oil 
and  apply  xylol,  blot  off  xylol  similarly 
and  apply  a  thin  layer  of  Damar,  using 
the  drop  method.  Let  the  slide  dry  on 
an  even  surface  adding  more  Damar  as 
necessary  to  keep  sections  protected. 
Golgi,  Method  (Porter,  R.  W.  and  Daven- 
port, H.  A.,  Anat.  Rec,  1949,  103,  583). 
Radical  departure  from  prior  methods 
because  silver  impregnation  precedes 
the  potassium  bichromate  solution. 
Fix  48  hours  at  25°C.  in  90  cc.  0.5  aq. 
AgNOj,  10  cc.  formalin,  0.5  cc.  Pyridine. 
Mix  in  order  given,  disregarding  slight 
turbidity.  Colorimetric  test  of  fixing 
solution  with  bromcresol  purple  should 
show  5.5  to  6.0.    Fixation  can  be  done 


by  perfusion,  or  only  by  immersion. 
Cut  slices  to  thickness  of  0.5  to  1.0  cm. 
After  fixation  rinse  blocks  with  dis- 
tilled water  and  place  in  2.5%  (aq)  po- 
tassium bichromate  to  which  1  cc.  of 
1%  osmic  acid  is  added  for  each  100  cc. 
Leave  there  3  to  5  days.  Dehydrate 
quickly  through  alcohols  and  xylol  to 
soft  paraffin.  Sections  should  be  cut 
50  to  100  microns  thick. 
Gomori's  Methods  For  Reticulum  and  Acid 
Phosphatase. 

1.  Silver  impregnation  of  reticulum 
(Gomori,  G.,  Am.  J.  Path.,  1947,  13, 
993-1001).  Treat  deparaffinized  sec- 
tions of  formalin  fixed  material  with 
0.5-1%  aq.  potassium  permanganate. 
1-2  min.  Rinse  in  tap  water  and 
decolorize  in  1-3%  aq.  potassium  meta- 
bisulphite,  1  min.  Wash  for  several 
minutes  in  running  tap  water.  2% 
aq.  iron  ammonium  sulphate  (violet 
crystals),  1  min.  Wash  in  tap  water 
few  minutes  and  then  pass  through  2 
changes  aq.  dest.  Impregnate  for  1 
min.  in  following  solution:  To  10% 
aq.  silver  nitrate  add  g  to  J  of  its  volume 
of  10%  aq.  potassium  hydroxide.  While 
shaking  add  strong  ammonia  drop  by 
drop  until  ppt.  is  completely  dissolved. 
Add  carefully  silver  solution  drop  by 
drop  as  long  as  resulting  ppt.  easily 
disappears  on  shaking.  Finally  add 
equal  vol.  aq.  dest.  Can  be  kept  2  days 
in  stoppered  bottle.  Rinse  in  aq.  dest., 
5-10  sec.  Reduce  in  commercial  forma- 
lin diluted  5-10  times  with  tap  water. 
Wash  under  tap  few  min.  Tone  in 
0.1-0.2%  aq.  gold  chloride,  10  min. 
1-3%  aq.  potassium  metasulphite  for  1 
min.  Fix  in  1-2%  aq.  sodium  thio- 
sulphate  (hyposulphite)  for  1  min. 
Wash  under  tap,  dehydrate,  clear 
and  mount.  Reticulum  black.  Note 
author's  figures  of  sarcomata  (Revisde 
by  G.  Gomori  May  7, 1946). 

2.  For  Acid  phosphatase — Written 
by  G.  Gomori  (University  of  Chicago. 
May  7,  1950— see  Stain  Techn.,  1950, 
25,  81. 

1.  Fix  thin  slices  of  tissues  in  ice  cold 
acetone  for  24  hours. 

2.  Change  acetone  at  room  tempera- 
ture twice  for  the  next  24  hours. 

3.  Two  changes  of  benzene,  45  min. 
each. 

4.  Embed  in  paraffin  (not  abo\'e  56°C. 
and  preferably  below),  2  changes,  30  to 
45  min.  each. 

5.  Cut  sections.  Float  them  on  luke- 
warm (30°C.)  water. 

6.  Carry  sections  through  xylene  and 
2  alcohols  to  dist.  water. 

7.  Incubate  in  the  following  solutions 


GOMORI'S  METHODS 


148 


GORDON'S  SILVER  METHOD 


for  U  to  24  hours  at  37°C.: 

Molar  acetate  buffer  pH  5* 2.5  ml. 

5%  lead  nitrate 1.5  ml. 

Dist.  water 40.0  ml. 

2%  Na  glycerophosphatet 7.5  ml. 

•  100  cc.  of  13.6%  CHiCOONa-3HiO  plus  60  cc.  6% 

acetic  acid, 
t  Commercial  grade  (mixture  of  alpha  and  beta  salts)  • 

Shake  well,  heat  to  ±60°C.  for  about 
10  min.;  filter. 

8.  Rinse  sections  thoroughly  first  in 
dist.  water  and  afterwards  in  2  to  3% 
acetic  acid,  followed  again  by  dist. 
water. 

9.  Immerse  sections  in  a  solution  of 
yellow  ammonium  sulfide  (1-12  drops  to 
a  Coplin  jar)  for  1  minute. 

10.  Wash.  Counterstain  as  desired. 
For  Alkaline  phosphatase: 

1.  Fix  thin  slices  of  tissues  in  80%  al- 
cohol (or  absolute  acetone).  Dehy- 
drate in  95%  and  absolute  alcohol  (or  2 
changes  of  absolute  acetone),  embed 
through  benzene  or  xylene  in  paraffin. 
Cut  sections  around  6  micra  thick. 

2.  Run  slide  through  xylene  and  2  al- 
cohols to  distilled  water.  Incubate  for 
1  to  2  hr.  at  37°C.  in  the  following  mix- 
ture: 

2%  sodium  glycerophosphate.  25  cc. 
2%  sodium  barbital 25  cc. 

Distilled  water 50  cc. 

2%  calcium  chloride 5  cc. 

2%  magnesium  sulfate 2  cc. 

Chloroform a  few  drops 

This  solution  will  keep  in  the  ice  box 
for  months. 

3.  Rinse  slide  thoroughly  in  repeated 
changes  of  distilled  water. 

4.  Immerse  slide  for  3  minutes  in  a 
1  to  2%  solution  of  some  cobalt  salt 
(chloride,  acetate,  sulfate). 

5.  Wash  thoroughly  under  the  tap. 

6.  Immerse  slide  for  2  minutes  in  a 
dilute  solution  of  yellow  ammonium  sul- 
fide (1-12  drops  to  a  Coplin  jarful  of 
distilled  water).     Wash  under  the  tap. 

7.  Counterstain  as  desired;  dehy- 
drate, clear  and  mount. 

Attention  is  called  to  the  earlier 
demonstration  of  phosphatase  in  bone 
by  Robison  (R.,  Biochem.  J.,  1923,  17, 
286-293)  and  to  recent  discussion  by 
Blaschko  and  Jacobson  (Bourne,  pp. 
217-221).  The  distribution  of  phos- 
phatase in  some  normal  tissues  is  indi- 
cated in  colors  by  ICabat,  E.  A.  and 
Furth,  J.,  Am.J.  Path.,  1941, 17, 303-318. 
For  phosphatase  in  elementary  bodies 
of  vaccinia  virus,  see  Macfarlane, 
M.  0.,  and  Salaman,  M.  H.,  Brit.  J. 
Exp.   Path.,   1938,   19,    184;   Hoagland, 


C.  L.  et  al.,  J.  Exp.  Med.,  1942,  76. 
163-173.    See  Kidney. 
Gonococcus,    methyl   green-pyronin   stain. 
To  10  cc.  absolute  methyl  alcohol  add 
1  gm.  methyl  green  (dye  content_60%) 
and  0.2  gm.  pyronin  (bluish  certified). 
Add  100  cc.  2%  aq.  phenol  and  shake  2 
hrs.  per  day  for  2  days  in  a  mechanical 
shaker.     Filter  and  add  20  cc.  glycerin, 
C.P.  to  filtrate.     Fix  smears  by  passing 
slides  lengthwise  through  flame  4  or  5 
times.    Add  stain  immediately  and  warm 
to  slight  steaming.    Wash  off  stain  20-50 
sec.     Dry     and     examine.     Gonococci, 
deep  red ;  other  bacteria  except  these  of 
Neisseria  group  pale  purplish  or  barely 
noticeable;  nuclei  of  pus  cells  green  in 
soft  pink  or  rose   cytoplasm    (Walton, 
S.  T.,  J.  Lab.  &  Clin.  Med.,  1938-39, 
24,  1308-1309). 
Goodpasture's  Method  as  modified  by  Mac- 
Callum      for      bacteria      in      sections 
(McClung,   p.    152).    Fix   in   Zenker's 
fluid  or  in  formalin  Zenker.     Stain  thin 
paraffin    sections    10-30    min.    in:  30% 
ale,    100   cc;   basic   fuchsin,  0.59  gr.; 
anilin  oil,  1  cc;  phenol  crystals,  1  gm. 
Wash  in  water.     Differentiate  in  forma- 
lin (37%  solution  of  formaldehyde)  few 
seconds  until  bright  red  color  changes 
to  rose.    Wash  in  water.     Counterstain 
in  sat.  aq.  picric  acid  3-5  min.  until 
sections  become  purplish  yellow.    Wash 
again  in  water.    Differentiate  in  95% 
ale.  until  red  reappears  and  some  of  it 
as  well  as  of  the  yellow  is  washed  out. 
Wash    in     water.    Stain    in    Stirling's 
gentian  violet   (gentian  violet,  5  gms.; 
95%  ale,  10  cc;  aniline  oil,  2  cc;  aq. 
dest.,  88  cc.)  5  min.  or  more.    Wash  in 
water.     Gram's  iodine  solution  (iodine, 
1  gm.;   potassium  iodide,  2  gms.;  aq. 
dest.,  300  cc.)  1  min.     Blot  dry.     Clear 
in  equal  parts  aniline  oil  and  xylol  until 
no  color  is  removed.     Clear  in  2  changes 
xylol    and    mount    in    balsam.     Gram- 
negative    bacteria,    red;    gram-positive 
ones,  blue;  tissue  red  and  blue;  fibrin 
deep  blue.    See  his  Polychrome  Methyl- 
ene  Blue   and   Carbol-Anilin   Fuchsin 
Methylene  Blue. 

Gopal-Ayengar,  see  Chromosomes,  Hyal- 
uronic Acid. 

Gordiacea,  see  Parasites. 

Gordon's  SilverMethod.  For  blood  smears, 
also  shows  parasites,  Gordon,  H.,  J. 
Lab.  &  Clin.  Med.,  1936-37,  22,  294- 
298.  Dry  smears  of  blood  or  bone 
marrow  in  air  and  fix  in  10%  formalin. 
Wash  in  water.  2.5%  aq.  iron  alum 
10  min.  or  more.  4  changes  aq.  dest. 
Dip  in  1%  aq.  gelatin  -f  1  or  2  drops 
2%  sodium  carbonate  and  drain.  Wash 
quickly  in  aq.  dest.  Impregnate  5-15 
min.  in  silver  solution  (Add  strong 
ammonia  drop  by  drop  to  5  cc.  of  10.2% 


GOSSYPIMINE 


149 


GRAM'S  STAINS 


aq.  silver  nitrate  until  ppt.  is  dissolved. 
Add  5  cc.  3.1%  aq.  soaium  hydroxide 
and  redissolve  ppt.  with  strong 
ammonia.  With  aq.  dest.  dilute  to  100 
cc).  Wash  in  aq.  dest.  at  60 °C.  Re- 
duce in:  10%  formalin  90  cc.  +  2.5% 
iron  alum  10  cc.  Wash  in  tap  water, 
dehydrate  in  alcohol,  clear  in  xylol 
and  mount  in  balsam. 

Gossypimine,  see  Safranin  O, 

Grafts.  Intracoelomic  of  eye  primordium, 
Joy,  E.  A.,  Anat.  Rec,  1939,  74,  461- 
486.    See  Transplantation. 

Gram,  see  Weight  measurements. 

Gram's  Iodine  Solution.  Iodine,  1  gm.; 
potassium  iodide,  2  gm.;  aq.  dest., 
300  cc.  A  stronger  solution  may  be 
desirable  with  only   100  cc.  aq.   dest. 

Gram-Pappenheim  stain  as  modified  for 
smears  and  paraffin  sections  (Scudder, 
S.  A.,  Stain  Techn.,  1944,  19,  39-44). 

Gram's  Stains  for  bacteria: 

1.  In  smears.  Hucker  modification 
(McClung,  p.  138).  Stain  1  min.  in 
equal  parts  A  and  B  :  A  =  crystal  violet 
(85%  dye  content,  4  gm.;  95%  ale,  20 
cc.)  B  =  ammonium  oxalate,  0.8 gm.; 
and  aq.  dest.  80  cc.  After  washing  in 
water  immerse  in:  iodine,  1  gm.  potas- 
sium iodide,  2  gm.,  aq.  dest.,  300  cc. 
1  min.  Then  wash  in  water  and  dry 
by  blotting.  Decolorize  30  sec.  in  95% 
ale.  gently  moving.  Blot  and  counter- 
stain  in:  10  cc.  sat.  safranin  in  95% 
ale.  and  aq.  dest.  100  cc.  Wash  and  dry. 
Kopeloff-Beerman  Modification  (Mc- 
Clung, p.  139).  Stain  5  min.  or  more 
in:  1%  aq.  gentian  or  crystal  violet,  1.5 
cc.  mixed  before  use  with  0.4  cc.  5% 
aq.  sodium  bicarbonate.  Rinse  in  iodine 
solution  made  by  dissolving  2  gm. 
iodine  in  10  cc.  normal  sol.  sodium 
hydroxide  and  adding  90  cc.  aq.  dest. 
and  stand  2  min.  or  more.  Blot  dry. 
Add  100%  acetone  drop  by  drop  with 
specimen  tilted  till  no  more  color  is 
removed,  less  than  10  sec.  Dry  in  air. 
0.1%  aq.  basic  fuchsin,  10-30  sec. 
Wash  in  water  and  dry.  Weiss  Modifica- 
tion (Weiss,  E.,  J.  I^ab.  &  Clin.  Med., 
1940-41,  26,  1518-1519).  Make  thin, 
uniform  smears  and  fix  over  flame. 
Cover  slide  with  3%  gentian  violet  in 
20%  alc^  3-5  min.  Wash  in  warm 
water.  Cover  3-5  min.  with  iodine,  20 
gm. ;  potassium  iodide,  40  gm.,  aq.  dest. 
300  cc.  Wash  with  warm  water.  De- 
colorize in  acetone  and  wash  imme- 
diately in  water.  Counterstain  quickly 
in  2%  basic  fuchsin  in  95%  ale.  Wash 
in  water,  drj-^  and  examine. 

The  use  of  colloidal  iodine  has  been 
suggested  to  improve  the  reaction  be- 
tween bacteria  and  stain  (Lyons,  D.  C, 
J.  Lab.  &  Clin.  Med.,  1936^37,  22, 
523-524).    Methods   for   preparing   col- 


loidal iodine  are  described  by  Chandler 
and  Miller  (W.  L.  and  E.  J.,  J.  Phys. 
Chem.,  1927,  31,  1091-1096). 

2.  In  sections.  Grarn-Weigert  method 
(McClung,  p.  152).  Fix  in  Zenker's 
fluid.  Stain  paraffin  sections  lightly  in 
alum  hemato.xylin  and  wash  in  running 
water.  1%  aq.  eosin,  1-5 min.,  followed 
by  washing  in  water.  Stain  ^-1  hr.  in 
anilin  methyl  violet  made  by  mixing  1 
part  of  A  with  9  of  B  :  A.  abs.  ale.  33  cc. ; 
aniline  oil,  9  cc. ;  methyl  violet  in  excess. 
B.  Saturated  aq.  methyl  violet  and  wash 
in  water.  Lugol's  iodine  1-2  min.  and 
wash  in  water.  Blot;  dehydrate  and 
clear  in  equal  parts  aniline  oil  and 
xylol  several  changes.  Wash  with  xylol 
and  mount  in  balsam.  Glynn's  method. 
(Glynn,  J.  H.,  Arch.  Path.,  1935,  20, 
896-899).  To  make  stain  triturate  1 
gm.  crystal  violet  and  1  gm.  phenol 
crystals  in  mortar  and  add  10  cc. 
absolute  alcohol.  Before  using  dilute 
10  times  with  aq.  dest.,  allow  to  stand 

2  days  and  filter.  Stain  deparaffinized 
sections  of  Zenker  (less  acetic),  Bouin, 
Helly  or  10%  formalin  fixed  material 
for  2  min.  Drain  off  but  do  not  wash. 
Add  Gram's  iodine,  1  min.  Differentiate 
in  acetone  until  no  more  color  is  given 
off,  10-15  sec.  Wash  in  aq.  dest. 
Counterstain  in  0.05%  basic  fuchsin 
in  N/500  hydrochloric  acid  (see  Normal 
Solutions).  Drain,  do  not  wash,  apply 
1%  aq.  trinitrophenol,  j-l  min.  Wash 
in  aq.  dest.  Dehydrate  and  differen- 
tiate in  acetone  10-15  sec,  clear  in  xylol 
and  mount  in  balsam.  Gram  -f  bac- 
teria, violet;  Gram—,  red;  nuclei,  light 
red;  cytoplasm,  yellow. 

3.  For  organisms  in  frozen  sections 
by  Krajian,  A.  A.,  Arch.  Path.,  1941, 
32,  825-827.  Stain  7-10m  frozen  sec- 
tions for  2  min.  in  Harris'  alum  hema- 
toxylin. Wash  in  tap  water  till  blue 
and  destain  quickly  by  dipping  5  to  7 
times  in  acid  alcohol.  Rinse  in  tap 
water  and  apply  following  solution  for 

3  min. — copper  sulfate,  7  gm.;  zinc  sul- 
fate, 4  gm.  dissolved  in  100  cc.  aq.  with 
aid  of  heat.  Pour  off  and  apply  0.3  gm. 
brilliant  green  in  10  cc.  above  copper 
zinc  mixture  for  5  min.  Rinse  in  water 
and  fortify  with  5%  aq.  ammonium  ni- 
trate for  1  min.  Rinse  in  tap  water  and 
stain  with  carbol  fuchsin  (Ziehl-Neel- 
sen)  for  2  min.  Rinse  in  tap  water,  blot 
and  apply  dioxane  for  2  min.  Pour  off 
and  add  equal  parts  creosote  and  .xylol, 
changing  tliis  mixture  and  agitating  to 
promote  even  differentiation  until  back- 
ground appears  clear  red.  Clear  in 
pure  xylol  (2  min.)  and  mount  in  damar. 
Gram  positive  organisms  bluish  green; 
gram  negative  ones  red. 

The   Gram   staining   technique    and 


GRAM  STAINS  MECHANISM 


150 


GRAM  STAINS  MECHANISM 


the  interpretation  of  the  findings  has 
been  concisely  presented  by  Dubos,  R. 
J.,  The  Bacterial  Cell.  Harvard  Univ. 
Press,  1945,  460  pp.  The  Gram  +  bac- 
teria differ  from  the  Gram  —  ones  in 
being  more  acidic  and  perhaps  in  pos- 
session of  lipids  with  higher  content  of 
unsaturated  acids.  Their  Gram  posi- 
tiveness  depends  on  intactness  of  their 
cell  walls,  for  erosions  of  the  walls  make 
them  Gram  negative.  When  the  outer 
layer  of  the  cell  walls  is  removed  by 
extraction  with  bile  salts  they  become 
Gram  negative.  The  Gram  positive 
property  can  be  restored  by  "replating" 
the  bacteria  with  the  extract  of  the 
outer  layer.  The  outer  layer  appar- 
ently contains  a  protein  ribonucleate 
complex,  for  Gram  positive  organisms 
can  be  made  Gram  negative  by  action 
of  the  enzyme,  ribonuclease.  The 
quality  of  the  cell  membrane  conditions 
not  only  the  entry  and  retention  of 
stains  but  the  whole  manner  of  life  of 
the  cells.  See  Cell  Membrane,  Acid 
Fast  Bacilli,  and  Dead  Cells. 
Gram  Stains  Mechanism — Written  by 
James  W.  Bartholomew,  Dept.  of 
Bacteriology,  University  of  Southern 
California,  Los  Angeles  7,  Calif.  July 
9,  1951 — The  literature  concerning  the 
mechanism  of  the  Gram  stain  is 
voluminous  and  makes  very  interesting 
reading  although  the  exact  mechanism 
is  still  unknown.  The  laboratory  tech- 
nician need  not  be  too  concerned  with 
the  mechanism.  It  is  important  for 
the  technician  to  realize  that  the  tech- 
nique involved  is  differential  due  to 
relative  rather  than  absolute  differences 
in  the  organisms  studied.  Thus,  re- 
gardless of  whether  one  uses  the 
Hucker,  Kopeloff-Beerman,  Weiss,  or 
other  modification  of  the  Gram  pro- 
cedure, the  dependability  of  the  results 
obtained  are  greatly  influenced  by  the 
experience  and  skill  of  the  operator. 
One  method  should  be  adopted,  and 
practiced,  until  proficiency  and  self 
assurance  are  obtained. 

Gram  differentiation  is  mostly  due  to 
one  fact.  Gram-positive  cells  resist 
decoloration  after  treatment  with  dye 
and  iodine  to  a  much  greater  extent 
then  Gram-negative  cells.  The  cause 
of  this  resistance  has  been  attributed  to 
1)  cell  membrane  permeability  char- 
acteristics, 2)  the  presence  of  certain 
unique  chemical  compounds,  3)  a  low 
isoelectric  point  of  the  cell  protein,  and 
4)  the  presence  of  a  morphological  struc- 
ture, the  Gram-positive  cortex.  Each 
of  these  theories,  by  itself,  fails  to  ex- 
plain certain  experimental  data.  It  is 
certain  that  the  mechanism  of  the  Gram 
stain  is  very  complex  and  several  of  the 


above  factors  probably  combine  to  give 
the  differentiation  effect.  Cell  mem- 
brane permeability  is  hard  to  ignore 
since  it  is  well  known  that  rupturing 
the  cell  wall  immediately  results  in  a 
Gram-positive  cell  staining  Gram-nega- 
tively.  The  importance  of  a  chemical 
factor  has  been  well  established  through 
the  easily  demostrable  dependence  of 
the  Gram-positive  state  on  the  pres- 
ence of  magnesium  ribonucleate  in  the 
cell.     The  isoelectric  point  theory  ap- 

Elies  to  the  general  staining  phenomena, 
ut  on  close  inspection  looses  its  sig- 
nificance as  a  prime  factor  in  the  mecha- 
nism of  the  Gram  stain.  The  morpho- 
logical concepts  must  await  further 
development  in  our  knowledge  of  bac- 
terial cell  morphology,  and  better  cell 
sectioning  methods,  before  the  con- 
tribution of  morphological  factors  to 
the  Gram  staining  mechanism  can  be 
determined. 

Currently,  an  appealing  explanation 
based  on  published  literature  could  be 
written  as  follows.  Cell  membrane 
permeability  to  iodine  in  alcoholic  solu- 
tion is  of  first  importance.  Magnesium 
ribonucleate  and  possibly  other  com- 
pounds such  as  lipoproteins  are  im- 
portant in  that  they  influence  the  cell 
membrane  permeability.  Gram-posi- 
tive cells  are  less  permeable  to  iodine 
in  alcoholic  solution  than  are  Gram- 
negative  cells.  On  performing  the 
Gram  stain  a  reaction  between  the  dye 
and  iodine,  and  also  possibly  the  cell 
protein,  takes  place  within  the  cell. 
The  alcohol,  which  is  added  next,  enters 
the  cells  and  dissociates  the  dj'^e-iodine 
precipitate,  or  dye-iodine-cell  protein 
complex,  which  has  been  formed.  Since 
the  alcoholic  iodine  permeates  fastest 
through  the  cell  membrane  of  the  Gram- 
negative  cells  the  precipitate  or  complex 
are  dissociated  more  rapidly  in  them 
than  in  the  Gram-positive  cells. 
Hence,  the  Gram-negative  cells  are  the 
first  to  declorize.  The  counter  stain 
also  has  a  certain  decolorization  action 
and  it  must  be  employed  if  a  true  Gram 
stain  differentiation  is  desired. 

While  this  concept  of  the  Gram  stain 
mechanism  has  not  been  proven,  it  has 
the  happy  quality  of  combining  chemi- 
cal concepts  with  cell  membrane  perme- 
ability and  it  does  not  rule  out  possible 
morphological  factors.  In  the  case  of 
the  often  observed  Gram-positive 
bodies  within  the  cell,  a  more  strictly 
chemical  concept  would  have  to  be  used 
to  explain  them. 

As  is  often  the  case,  the  segregation 
of  cells  into  two  distinct  groups,  the 
Gram-negative  and  the  Gram-positive, 
does  not  coincide  with  the  state  actually 


GRAM-TWORT  STAIN 


151 


HAIRS 


existing  in  nature.  There  are  many 
intermediates  between  the  two,  and 
this  fact  should  be  kept  in  mind  by  the 
technician.  Attempts  to  create  termi- 
nology for  intermediate  groups  have 
not  been  well  received  and  the  classifi- 
cation as  it  now  stands  is  extremely 
useful  and  comparitively  simple.  Fur- 
ther information  is  supplied  by 
Bartholomew,  J.  W.  and  Umbreit,  W. 
W.,  J.  Bact.,  1944,  48,  567;  Bartholo- 
mew, J.  W.  and  Mittwer,  T.,  Stain 
Tech.,  1949,  25,  103-110;  Dubos,  R.  J., 
The  Bacterial  Cell.  Cambridge:  Har- 
vard Univ.  Press,  1946,  pp.  72-85; 
Knaysi,  G.,  Elements  of  Bacterial 
Cytology.  Ithaca:  Comstock  Pub.  Co., 
1951,  pp.  267-278;  Mittwer,  T.,  Bar- 
tholomew, J.  W.,  and  Kallman,  B.  J., 
Stain  Tech.,  1950,  25,  169-179. 

Gram-Twort  Stain.  For  study  of  Gram- 
positive  and  Gram-negative  bacteria 
in  sections  (Ollett,  W.  S.,  J.  Path.  & 
Bact.,  1947,  59,  357).  Twort  originally 
used  a  neutral  red-light  green  mixture. 
Ollett,  W.  S.,  J.  Path.  &  Bact.,  1951, 
63,  166  supplies  further  details.  The 
stain  advised  is  approximately  as  fol- 
lows depending  on  dye  content  of  the 
samples  employed.  For  stock  solution 
0.2%  ale.  neutral  red,  (CI  no.  825  9  ml. 
+  0.2%  ale.  fast  green  FCF  10  ml. 
For  use  dilute  1  vol.  stock  solution  with 
3  vols.  aq.  dest.  Employ  as  counter- 
stain  in  Gram's  method  as  described 
(Ollett,  1947)  and  mount  sections  in 
L.  P.  M.  which  is  Lustron  (L  2020), 
apparently  the  same  as  Distreme  80 
crystal  clear  100  gm.  dissolved  in  Di- 
butyl  phthalate  50  ml.  +  monochlor- 
benzene  300  ml. 

Gray,  R,  B,  BB,  see  Nigrosin,  water  soluble. 

Green  PL,  see  Naphthol  Green  B, 

Greene,  see  Anterior  Chamber  Transplanta- 
tion. 

Gregarines.  Technique  given  by  McCIung, 
Microscopical  Technique,  1950  p.  455. 
Fix  in  picric  acid  mixtures.  Stain 
smaller  ones  in  Heidenhain's  Iron 
Hematoxylin  and  larger  ones  with 
Hemalum.  For  Golgi  bodies  see  Joyet- 
Lavergne,  P.,  C.  R.  Soc.  de  Biol.,  1926, 
94,  830. 

Grenacher,  see  Alum  Carmine,  Borax 
Carmine. 

Grieves'  method  for  undecalcified  dental 
tissues  and  bone  as  outlined  by  Shipley 
(McClung,  p.  345)  is:  Fix  small  pieces 
in  10%  formalin  24-36  hrs.  or  any  other 
desired  fixative.  Wash  in  running  water 
24  hrs.  Then  pass  through  2  changes 
of  aq.  dest.  1  hr.  each.  Dehydrate 
through  ascending  alcohols  beginning 
with  50%  ale.  Equal  parts  abs.  ale. 
and  chloroform,  2  hrs.  Chloroform,  2 
hrs.    5%  sol.  of  rosin  in  chloroform,  2 


hra.  10%  sol.  rosin,  2  hrs.  Sat.  sol. 
rosin  until  it  becomes  transparent. 
Imbed  in  melted  rosin  using  one  after 
another  the  rosins  in  3  small  glass  dishes 
on  a  heated  copper  bar,  1  min.  each. 
The  chloroform  carried  over  evaporates. 
The  rosin  containing  the  tissue  is  al- 
lowed to  cool.  The  block  is  ground 
very  thin  by  hand  on  a  carboriundum 
stone  and  polished  on  a  fine  hone  all 
grinding  being  done  under  luke  warm 
water.  The  smooth  surface  is  then 
mounted  on  a  slide  with  a  little  melted 
rosin  after  which  the  surface  is  ground 
and  polished  in  the  same  waj'  and  the 
section  is  ready  for  mounting  or  for 
staining. 

Gross  Specimens,  see  Color  Preservation. 

Ground  Substance  (intercellular),  see  Tis- 
sue Fluid. 

Growth.  Many  techniques  are  now  avail- 
able for  the  measurement  of  growth  of 
tissues.  Increase  in  number  of  cells 
can  be  revealed  by  mitotic  counts 
(Mitosis).  The  amount  of  bone  or  of 
dentine  laid  down  while  Alizarin  S 
or  Madder  is  in  the  circulation  can  be 
estimated.  The  amount  of  radioactive 
isotropes  accumulated  is  a  third  method 
(see  Radioactive  Phosphorus)  if  the 
amount  increases  per  unit  of  time  while 
elimination  of  the  nonradioactive  ele- 
ment in  question  remains  the  same. 
Valuable  histochemical  methods  are 
given  by  Lowry,  O.  H.  and  Hastings, 
A.  B.,  in  Cowdry's  Problems  of  Ageing, 
Baltimore  :  Williams  and  Wilkins,  1942, 
936  pp. 

Gout,  see  Urates. 

Guanin  appears  as  white  granules  in  retinal 
tapetum  of  certain  animals  including 
nocturnal  ones.  Decreases  in  amount 
in  regions  containing  more  fuscin. 
For  details  see  Arey,  L.  B.  in  Cowdry's 
Special  Cytology,  1932,  3,   1218. 

Guanine,  see  microxide  test  under  Purines. 

Guarnieri  Bodies,  cytoplasmic  inclusions  in 
smallpox  and  vaccinia.  See  Inclusion 
Bodies  and  Cowdry,  E.  V.,  J.  Exper. 
Med.,  1922,  36,  667-684  for  supravital 
staining  with  brilliant  cresyl  blue.  For 
sections    Giemsa's   stain    is   excellent. 

Gum  Damar,  see  Damar. 

Gypsum  test,  see  Calcium  7. 

Habermann,  see  Anethole  Clearing  Agent. 

Hafnium,  see  Atomic  Weights. 

Hairs — Written  by  Mildred  Trotter  and 
Oliver  H.  Duggins,  Dept.  of  Anatomy, 
Washington  University,  St.  Louis,  May 
8,  1951 — The  hair  shaft  (above  the  sur- 
face of  the  skin),  the  hair  root  (below 
it)  and  the  hair  follicle  (encasing  the 
root)  call  for  somewhat  different 
techniques. 

The  shaft  may  be  examined  in  a  dry 
mount  after  first  washing  thoroughly 


HAIRS 


152 


HARRIS  ALUM  HEMATOXYLIN 


and  repeatedly  in  ether-alcohol,  or  the 
shaft  and  root  can  be  cleared  and 
mounted  in  balsam  for  repeated  study. 
In  case  it  is  too  highly  pigmented  to 

Eermit  a  clear  view  of  its  structure  first 
leach  with  hydrogen  peroxide.  Indi- 
vidual cells  of  the  shaft  can  be  isolated 
by  maceration  in  40%  aq.  potassium 
hydrate. 

Scale  counts  are  made  readily  after 
a  dry  mount  has  had  applied  a  drop  or 
two  of  a  glycerine-alcohol  mixture  at 
the  ends  of  the  specimen.  This  mix- 
ture progresses  along  the  shaft  by 
capillary  attraction  thus  bringing  into 
relief  the  free  borders  of  the  scales 
(Trotter,  M.  and  Duggins,  O.  H.,  Am. 
J.  Phys.  Anthrop.,  in  press).  Deter- 
mination of  the  cuticular  scale  pattern 
may  be  made  after  partially  embedding 
the  hair  in  a  glycerine  jelly  (Eddy,  M. 
W.  and  Raring,  J.  C,  Proc.  Acad.  Sci., 
1941,  15,  164-168).  Study  of  the  cortex 
(fusi  and  pigment  granules  under  very 
high  power)  and  medulla  (when  present 
with  its  clumps  of  pigment)  requires 
clearing  by  immersing  in  some  oil  the 
refractive  index  of  which  is  approxi- 
mately the  same  as  that  of  the  hair 
(Hausman,  L.  A.,  Sci.  Month.,  1944, 
59,   195-202). 

The  refractive  index  of  hair  may  be 
determined  to  the  greatest  accuracy  by 
the  double-variation  method  using  both 
the  Becke  line  and  half  shadow  tech- 
niques (Gibb.  T.  R.  P.,  Jr.,  Optical 
Methods  of  Chemical  Analysis,  pp. 
249-250,  McGraw-Hill,  1942).  Cut 
hairs  into  a  number  of  1  mm.  lengths, 
agitate  for  30  minutes  in  ether-alcohol 
and  mount  in  oil  of  a  similar  refractive 
index  on  a  temperature  cell  under  a 
polarizing  microscope.  True  index  is 
found  only  at  crossed  Nicols.  It  is 
suggested  that  the  index  be  taken  both 
perpendicular  to  the  lower  Nicol  (90°) 
and  parallel  to  the  lower  Nicol  (0°) 
and  that  the  lower  index  (90°)  be  sub- 
tracted from  the  higher  index  (0°)  in 
order  to  determine  the  birefringence. 
The  Becke  line  method  may  be  followed 
by  the  half  shadow  method  for  greater 
accuracy.  The  limit  of  accuracy  will 
be  approximately  +3  in  the  third  place. 
In  addition,  it  is  suggested  that  the  use 
of  the  phase  microscope  for  such  deter- 
minations may  increase  the  accuracy  to 
the  fourth  decimal  place. 

Cross  sections  of  a  large  number  of 
hairs  (appro.ximately  150)  may  be  made 
at  one  time  with  very  little  preliminary 
preparations  by  using  the  "Dr.  J.  I. 
Hardy  Thin  Cross-Section  Device" 
(Gosnell  Mfg.  Co.,  Washington,  D.  C). 

The  root  and  the  follicle  are  to  be 
seen  in  most  sections  of  hairy  skin  and 


require  no  special  technique  unless  one 
wishes  to  study  the  follicles  attached  to 
whole  mounts  of  epidermis.  In  order 
to  study  the  growth  of  a  given  hair  or 
to  determine  the  cyclic  activity  of  its 
follicle  it  is  convenient  to  place  a  small 
tattoo  mark  with  India  ink  in  the  skin 
near  the  mouth  of  the  follicle  (Trotter, 
M.,  Am.  J.  Phys.  Anthrop.,  1924,  7, 
427-437). 

Distribution  of  alkaline  phosphatase 
in  growth  of  hair  follicle  (Johnson, 
P.  L.,  Butcher,  E.  O.  and  Bevelander, 
G.,  Anat.  Rec,  1945,  93,  355-361).  For 
further  details  see  Trotter,  M.,  Chapter 
on  Hair  in  Cowdry's  Special  Cytology, 
1932,  1,  40-65.  Cleaning  and  mounting 
of  individual  hairs  (Duncan,  F.  W., 
J.  Roy.  Micr.  Soc,  1943,  63,  85-88. 
Microphotography  of  keratin  fibers  of 
hairs  (Stoves,  J.  L.,  J.  Roy.  Micr.  Soc, 
1943,  63,  89-90).  The  less  pigment  in 
the  hair,  the  greater  the  fluorescence,  so 
that  gray  hair  is  clear  white.  Hair 
containing  tricophyton  or  microsporon 
fluoresces  bright  green.  See  Kinnear, 
J.,  Brit.  Med.,  J.,  1931,  1,  791-793  on 
diagnosis  of  ringworm. 

Halides,  microscopic  localization  in  tissues 
by  precipitation  methods  (Gersh,  I. 
and  Stieglitz,  E.  J.,  Anat.  Rec,  1933, 
56,  185). 

Hal  ©meter,  apparatus  designed  by  Eve, 
F.  C,  Lancet,  1928,  1,  1070  to  measure 
mean  diameter  of  erythrocytes,  see 
Erythrocytometer. 

Halowax,  see  Paraffin  Sections. 

Hanging  Drop  preparations  are  mostly  em- 
ployed in  the  examination  of  living 
bacteria  and  protozoa.  A  drop  of  the 
fluid  is  simply  attached  to  the  under 
surface  of  a  cover  glass  which  is  mounted 
over  a  depression  in  a  slide.  Equally 
satisfactory  results  can  usually  be 
obtained  by  simply  mounting  under  a 
cover  glass  on  an  ordinary  slide  unless 
the  greater  depth  of  the  hanging  drop  is 
required.  When  in  Microdissection  it 
is  necessary  to  get  at  the  cells  from  the 
under  surface  of  the  cover  glass  special 
chambers  and  hanging  drops  are  em- 
ployed. 

Harderian  Glands,  fluorescence  in  mice 
(Strong,  L.  C.  and  Figge,  F.  H.  J., 
Science,  1941,  93,  331).  Technique  for 
rat  is  given  by  Grafflin,  A.  L.,  Am.  J. 
Anat.,  1942,  71,  43-64. 

Harleco  Synthetic  Resin  is  recommended 
by  McClung,  Microscopical  Technique, 
1950,  p.  24  as  a  mounting  medium  some- 
what similar  to  Clarite.  It  can  be  ob- 
tained from  Hartman-Liddon  Co.,  5821 
Market  Street,  Philadelphia  39,  Pa. 

Harris  Alum  Hematoxylin.  Dissolve  1  gm. 
hematoxylin  in  10  cc  absolute  alcohol 
and  20  gms.  ammonium  or  jrotassium 


HEART 


153 


HEMATOCRIT 


alum  in  200  cc.  aq.  dest.  the  latter  with 
the  aid  of  heat.  Mix  the  2  solutions, 
bring  quickly  to  boiling  and  add  0.5  gm. 
mercuric  oxide.  Solution  turns  purple. 
Cool  quickly  in  cold  water  bath.  Mal- 
lory  (p.  72)  recommends  adding  5%  of 
acetic  acid. 

Heart,  see  Coronary  Arteries,  Myocardium. 
Pericardium,  Purkinje  Cells  and 
Fibers.  Technique  and  results  of 
"electron  histology"  of  the  heart  are 
presented  by  Kisch,  B.  and  Bardet, 
J.  M.,  E.xp.  Med.  &  Surg.,  1951,  9,  1-47. 

Heart  Failure  Cells— Written  by  C.  C. 
Macklin,  Dept.  of  Histological  Re- 
search, The  University  of  Western 
Ontario,  Loudon,  Canada.  November 
28,  1951. — This  inapt  designation,  used 
by  pathologists,  applies  usually  to 
modified  human  pneumonocytes  which 
have  become  free  in  the  lung  alveoli,  or 
which  are  affixed  to  the  moving  mucous 
sheet  of  the  air  tract  in  transit  to  the 
mouth  region,  or  which  have  been 
hawked  up  in  the  sputum.  The  cells 
may  thus  be  examined  in  fresh  mounts 
with  or  without  vital  or  supravital 
staining;  or  in  stained  dried  smears. 
They  may  also  be  fixed,  embedded  and 
cut  into  thin  sections  (Macklin,  C.  C, 
The  Lancet,  Feb.  24,  1951,  432-435). 
See  Dust  Cells. 

Heavy  Water  is  water  in  which  deuterium, 
the  heavy  hydrogen  isotope  H^,  has 
taken  the  place  of  ordinary  hydrogen. 
See  Deuterium  which  is  used  as  a  tracer 
substance. 

Heidenhain's  Azan  Stain  (Heidenhain,  M., 
Ztschr.  f.  vyiss.  Mikr.,  1915, 32,  361-372). 
The  following  details  are  from  Lee  (1928, 
p.  279) :  Color  sections  1  hr.  at  55°C. 
in  2%  aq.  azocarmine  plus  10  drops 
glacial  acetic  acid  in  small  staining  jar. 
Wash  in  water.  Differentiate  in  96% 
ale.  100  cc.  plus  anilin  oil  0.1  cc.  until 
cytoplasm  becomes  pale  pink  and  nuclei 
clear  red.  To  hurry  differentiation  add 
2  drops  anilin  oil.  Rinse  in  96%  ale. 
containing  few  drops  acetic.  Put  in 
5%  aq.  phosphotungstic  acid  about  2 
hrs.  until  connective  tissue  is  com- 
pletely decolorized.  Wash  rapidly  in 
water.  Stain  ^-3  hrs.  in  following  solu- 
tion diluted  with  equal  or  double  parts 
aq.  dest.:  anilin  blue  (water  sol.  Griib- 
ler)  0.5  gm.;  orange  G,  2  gm.;  acetic 
acid,  8  cc;  aq.  dest.  100  cc.  Examine 
staining  under  microscope.  Wash  in 
water,  dehydrate  in  abs.  ale,  clear  in 
xylol  and  mount  in  balsam.  This  is  a 
very  useful  stain.  See  also  McGregor, 
L.,  Ajn.  J.  Path.,  1929,  5,  545-557  for  use 
of  this  technique  particularly  as  applied 
to  normal  renal  glomerules.  Under 
Islets  of  Langerhans  is  given  use  of  a 


slightly  modified  azan  method  by 
Gomori. 

Heidenhain's  Iron  Hematoxylin,  see  Iron 
Hematoxylin. 

Heinz  Bodies.  These  spherical  bodies  are 
sometimes  seen  in  erythrocytes  espe- 
cially when  examined  in  the  dark  field  or 
when  colored  with  Azur  1.  They  have 
been  referred  to  as  Substantia  Meta- 
chromatica  Granularis  and  B-substance. 
The  best  way  to  demonstrate  them  is 
to  use  the  technique  of  Figge,  F.  11.  J., 
Anat.  Rec,  1946,  94,  17.  Give  0.3%  aq. 
sulfanilamide  to  mice  as  drinking  water. 
Within  4-6  days  these  bodies  will  appear 
in  at  least  90%  of  erythrocytes  whence 
they  are  cast  out  into  the  plasma. 
They  are  most  readily  seen  in  unstained, 
unmounted  blood  smears.  They  dis- 
appear when  studied  in  oil,  balsam  or 
other  mounting  media.  Heinz  bodies 
are  granules  of  heme-containing  pro- 
tein denatured  by  this  drug  within  the 
cells.  They  are  not  produced  by  sod- 
ium sulfathiazole.  The  Heinz  Body 
phenomenon  in  erythrocytes  is  dis- 
cussed in  detail  by  Webster,  S.  H., 
Blood,  1949,  4,  479-504. 

Helianthin,  see  Methyl  Orange. 

Heliozoa,  techniques  for,  see  McClung, 
Microscopical  Technique,  1950,  p.  468, 
also  Rumjantzew,  A.,  and  Wermel,  E. 
Arch.  f.  Protistenk.,  1925,  52,  217. 

Heliotrope  B,  see  Amethyst  Violet. 

Helium,  see  Atomic  Weights. 

Kelly's  Fluid  is  Zenker's  fluid  in  which  5% 
formalin  is  substituted  for  5%  acetic 
acid. 

Helminthosporia.  Stain  for  nuclei  in  (Par- 
ris.  G.  K.,  Phytopathology,  1944,  34, 
700). 

Helvetia  Blue,  see  Methyl  Blue. 

Hemalum  (Mayer's)  Hematin,  1  gra.;  90% 
ale,  50  cc. ;  aq.  dest.,  1000  cc. ;  ammonia 
alum,  50  gms.;  thymol,  1  crystal. 
Keeps  better  after  adding  20  cc.  glacial 
acetic  acid  and  making  Acid  Hemalum. 
A  good  nuclear  stain  when  diluted  with 
aq.  dest.  1:20.  The  above  formula  has 
been  modified  by  Lillie  (R.  D.,  Stain 
Techn.,  1942,  17,  89-90):  hematoxylin, 
5  gm.;  sodium  iodate  (NalOs),  1  gm.; 
ammonia  alum  (A1NH4(S04)2  +  12 
IIjO),  50gm.;aq.  dest.,  700  cc,  glycerol, 
300  cc,  glacial  acetic  acid,  20  cc.  No 
ripening  is  necessary.  Stain  sections 
formalin  fixed  material,  2-5  min.  Blue 
2-10  min.  in  tap  water.  Counterstain 
in  0.2%  aq.  eosin  Y.  Dehydrate  clear 
and  mount  as  usual.  This  method  is 
quick  and  gives  a  sharp  stain. 

Hematin,  identified  by  luminescence  with 
Luminol.  Do  not  confuse  with  hema- 
tein,  see  Hematoxylin. 

Hematocrit,  a  tube  used  to  concentrate  red 


HEMATOIDIN 


154    HEMOCHROMOGEN  CRYSTAL  TEST 


blood  cells  by  centrifugation  and  to 
measure  their  volume,  see  Ponder,  E. 
in  Glasser's  Medical   Physics,  597-600. 

Hematoidin  (hematin  +  G.  eidos,  appear- 
ance). An  iron  free  pigment  produced 
by  phagocytic  digestion  of  erythrocytes 
or  in  clots  and  old  hemorrhages,  chemi- 
cal composition  similar  or  identical  with 
Bilirubin.  Seen  as  red  or  orange  rhombic 
plates  or  radiating  yellow  needles, 
insoluble  in  ether,  water  and  soluble  only 
with  difliculty  in  alcohol,  easily  soluble 
in  chloroform.  Gives  positive  Gmelin's 
test. 

Hematoporphyrin  (G.  haima,  blood  -{- 
porphyra,  purple).— Written  by  Frank 
H.  J.  Figge,  Dept.  of  Anatomy  Univer- 
sity of  Maryland  Medical  School, 
Baltimore,  Md.  Contrary  to  a  deeply 
rooted  misconception,  this  substance 
is  not  the  pigment  as  it  occurs  in  hemo- 
globin, but  is  artificially  produced  by 
the  drastic  decomposition  of  hemo- 
globin in  concentrated  strong  acids. 
Since  it  does  not  occur  in  nature,  such 
terms  as  "hematoporphyrinuria"  are 
obsolete.  In  addition,  protoporphyrin, 
which  is  the  true,  unaltered,  pigment 
found  in  heme  compounds,  is  not  ex- 
creted as  such  by  the  Iddney.  Proto- 
porphyrin is  heme  minus  iron  and  has 
two  vinyl  group  side-chains.  Hemato- 
porphyrin is  heme  minus  iron,  plus  two 
hydrogen  and  two  hydroxy!  groups. 
Hematoporphyrin  is  soluble  in  water, 
ether,  alcohols,  dilute  alkalies,  and  acids. 
For  references  and  additional  informa- 
tion, see  PorpJiyrins. 

Hematoxylin  is  the  most  useful  of  all  dyes 
in  animal  histology  and  pathology  (Gr. 
haimatodec,  blood  like  +  Xylon,  wood). 
It  is  an  extract  of  logwood  {Haernatoxy- 
lon  campechianum)  and  is  marketed  in 
crystalline  form.  When  the  crystals 
are  first  dissolved  in  water  or  alcohol  it 
is  not  an  energetic  stain;  but  requires 
to  be  "ripened"  before  it  can  be  used  to 
advantage.  Ripening  is  brought  about 
by  the  formation  of  oxidation  products. 
Consequently  it  is  recommended  that 
solutions  be  exposed  to  light  and  ajr. 
Hematein  (not  hematin — a  blood  pig- 
ment) is  the  oxidation  product  which 
yields  a  fine  deep  blue  coloration  and  is 
the  one  most  desired.  It  can  be  pur- 
chased. To  make  up  solutions  of 
hematein  instead  of  hematoxylin  is 
logically  sound  but  there  is  no  way  to 
prevent  further  ripening  (oxidation) 
with  the  development  of  other  browner 
unwanted  products  and  precipitation  of 
dyes.  Therefore  it  is  good  practice  to 
begin  with  hematoxylin,  to  let  it  ripen 
naturally  over  a  fairly  long  period  of  time 
or  to  ripen  almost  immediately  by 
adding  about  5%  hydrogen  peroxide,  or 


5%  of  1%  aq.  potassium  permanganate. 
10%  solution  of  hematoxylin  in 
96%  or  abs.  ethyl  alcohol  should 
always  be  kept  on  hand.  It  attains 
maximum  ripening  in  about  one  year, 
but  must  be  kept  in  a  stoppered  bottle 
for  otherwise  the  alcohol  will  evaporate. 
It  is  diluted  to  0.5%  of  hematoxylin 
with  aq.  dest.  for  the  Iron  Hematoxylin 
technique.  See  also  Delafield's,  Ehr- 
lich's,  Harris'  and  Mayer's  hema- 
toxylin solutions,  likewise  Azure  11 
eosin  and  Hematoxylin.  Addition  of 
a  drop  of  Tergitol  No.  7  to  hematoxylin 
solution  will  greatly  increase  speed  of 
staining  but  has  no  other  advantage 
(McClung  1950  p.  136). 

Hematoxylin  and  Eosin  is  rightly  the  most 
used  of  all  staining  methods.  If  the 
tissues  have  been  fixed  in  a  fluid  con- 
taining mercuric  chloride  such  as  Zen- 
ker's fluid  deparaffinize  sections  and 
treat  with  dilute  iodine  in  70%  alcohol 
for  1-2  min.  Wash  in  aq.  dest.,  bleach 
in  5-10%  aq.  sodium  hyposulphite  to 
remove  iodine  and  wash  again  in  aq.  dest. 
Stain  with  Harris'  Hematoxylin  (full 
strength)  for  12-15  min.  Blue  in  tap 
water  or  in  aq.  dest.  +  few  drops  sat. 
aq.  lithium  carbonate,  5-10  min.  Stain 
in  0.2%  aq.  eosin,  1  min.  Rinse  in  aq. 
dest.  and  95%  alcohol.  Dehydrate  in 
absolute  alcohol,  clear  in  xylol  and 
mount  in  balsam.  Nuclei,  deep  blue; 
cytoplasm,  pink.  In  place  of  Harris' 
alum  hematoxylin,  which  we  use, 
Delafield's  Alum  Hematoxylin  or  Ehr- 
lich's  Acid  Hematoxylin  may  be  em- 
ployed. The  Bensleys  (p.  73)  dilute 
1  part  of  the  last  named  with  2  parts 
cold  sat.  aq.  ammonium  alum  and  4 
parts  aq.  dest.  Nuclei,  dark  blue; 
cytoplasm,  collagenic  fibers,  erythro- 
cytes, pink;  smooth  muscle,  lavender. 
0.2%  aq.  erj'throsin  can  take  the  place 
of  the  eosin  but  the  advantage  is  ques- 
tionable. 

Hemin  Crystal  Test  for  blood  pigment, 
Teichmann  (Stitt,  p.  698).  Dissolve  in 
100  cc.  glacial  acetic  acid,  0.1  gm.  of 
KI,  of  K  Br  and  of  K  CI.  Add  few 
drops  to  suspected  material  on  a  slide 
and  cover.  Gently  warm  until  bubbles 
begin,  then  slowly  cool  and  examine  for 
typical  dark  brown  crystals.  The  test 
is  not  very  sensitive  but  positive  result 
is  conclusive. 

Hemochromatosis,  clinical  test  for,  see 
Iron. 

Hemochromogen  Crystal  Test.  Donogdny 
(Stitt,  p.  698).  Mix  1  drop  of  suspected 
fluid,  of  pyridin  and  of  20%  aq.  NAOH 
on  a  slide  and  allow  to  dry.  Radiating 
crystals  appearing  within  several  hours 
indicate    presence    of    hemochromogen. 


HEMOCYTOBLASTS 


155 


HERMANN'S  FLUID 


Hemocytoblasts,  see  Erythrocytes,  develop- 
mental series. 

Hemoflagellates  on  tissues  may  be  demon- 
strated by  many  methods.  (McClung, 
Microscopical  Technique,  1950,  p.  464). 

Hemofuscin.  Mallory's  fuchsin  stain.  Fix 
in  Zenker's  fluid,  alcohol  or  10%  forma- 
lin. Stain  nuclei  in  paraffin  or  celloidin 
sections  with  Iron  Hematoxylin.  Wash 
thoroughly  in  water.  Stain  5-20  min. 
in  :  basic  fuchsin  0.5  gm.,  95%  ale.  50  cc. 
and  aq.  dest.  50  cc.  Wash  in  water. 
Differentiate  in  95%  alcohol,  dehydrate 
in  abs.  ale,  clear  in  xylol  and  mount  in 
balsam  in  the  case  of  paraffin  sections. 
Celloidin  sections  are  to  be  cleared  in 
terpineol  or  origanum  oil  after  95%  ale. 
Nuclei  blue,  hemofuscin  granules  bright 
red,  hemosiderin  and  melanin  unstained 
(Mallory,  p.  136). 

Hemoglobin,  histochemical  test  (Ralph, 
P.  H.,  Stain  Techn.,  1941,  16,  105-106). 
Flood  dried  blood  smear  with  1% 
benzidine  in  absolute  methyl  ale,  1 
min.  Pour  off  and  replace  with  25% 
superoxol  in  70%  ethyl  ale,  90  sec. 
Wash  in  aq.  dest.,  15  sec.  Dry  and 
mount  in  neutral  balsam.  Hemoglobin 
dark  brown. 

Goulliart,  M.  C.  rend.  Soc.  Biol., 
1941,  135,  1260-1262  adds  to  frozen  sec- 
tion or  dried  smear  a  drop  from  a  bottle 
containing  glacial  acetic  acid  to  which 
has  been  added  less  than  a  week  ago  a 
few  crystals  of  potassium  iodide.  After 
about  30  min.  examination  with  a 
polarizing  microscope  shows  tiny  boat 
shaped  birefringent  crystals  of  proto- 
iodoheme  which  later  change  into 
Teichmann  crystals. 

Dunn,  R.  6.,  Arch.  Path.,  1946,  41, 
676,  677.  Dissolve  1  gm.  cyanol  (Na- 
tional Aniline  Division,  Allied  Chemical 
and  Dye  Corporation,  40  Rector  Street, 
New  York)  in  100  cc.  aq.  dest.  Add 
2  cc.  glacial  acetic  acid.  Boil  gently 
and  blue  color  will  disappear.  Keep  for 
several  weeks.  Immediately  before 
using  filter  10  cc.  and  add  2  cc.  glacial 
acetic  acid  and  1  cc.  commercial  3% 
hydrogen  peroxide.  Treat  frozen  or 
paraffin  sections  of  tissue  fixed  in  4% 
formaldehyde  buffered  to  pH  7.0  after 
rinsing  in  water  with  this  fresh  cyanol 
mixture,  3-5  min.  Rinse  in  water, 
counterstain  with  0.1%  safranin  in  1% 
glacial  acetic  acid.  Wash  in  water, 
dehydrate,  clear  and  mount  in  Clarite. 
Hemoglobin,  blue;  nuclei,  red  and  cyto- 
plasm, pink  (from  Click,  p.  63). 

Hemoglobin  Estimation  is  done  by  compar- 
ing blood  with  a  colored  paper  scale  or  by 
a  more  accurate  scale  in  a  hemoglobinom- 
eter.  The  experimental  error  is  at 
least  5%.    Staining  reactions  for  hemo- 


globin within  cytoplasm    (Kindred,  J. 
E.,  Stain  Techn.,  1935,  10,  7-20). 

Hemolysis.  Methods  for  measuring  the 
velocity  of  hemolysis  depend  on  the 
fact  that  red  blood  cell  suspensions  as 
they  hemolyse  become  more  and  more 
translucent.  Techniques  differ  merely 
in  the  ways  of  measuring  the  trans- 
mitted light.  Simple  visual  photom- 
eters and  photoelectric  ones  are  de- 
scribed by  Ponder,  E.  Glasser's  Medical 
Physics,  605-612.  The  same  authority 
explains  the  "equilibrium  methods"  for 
measuring  the  amount  of  hemolysis 
which  has  taken  place  if  the  process  has 
been  arrested.  One  of  these  is  to  count 
the  cells  remaining,  another  to  deter- 
mine the  amount  of  hemoglobin  set 
free,  etc. 

Hemophilus  Pertussis.  Staining  of  cap- 
sules in  air  dried  smears  with  5%  aq. 
phosphomolybodic  acid.  Growth  on  a 
special  medium  is  advised  (Lawson,  G. 
McL.,  J.  Lab.  &  Clin.  Med.,  1939-40, 
25,  435-43S). 

Hemosiderin,  soluble  in  acids  and  other 
reagents  used  in  histological  technique. 
After  formalin  fixation  the  order  of 
decreasing  removal  is  oxalic,  sulphuric, 
nitric,  formic  and  hydrochloric.  Speeu 
of  solution  is  but  little  influenced  by  age 
of  pigment  (Lillie,  R.  D.,  Am.  J.  Path., 
1939,  15,  225-239).  See  Iron,  Di- 
nitrosoresorcinol  method. 

To  demonstrate  hemosiderin  micro- 
scopically pour  on  deparaffinized  sec- 
tions of  freshly  fixed  tissue  1  part  of 
fresh  2%  aq.  potassium  ferrocyanide 
and  3  parts  1%  aq.  hydrochloric  acid 
heated  to  60°-80°C.  Thoroughly  wash 
in  several  changes  of  water.  Counter- 
stain  in  0.1-0.5%  basic  fuchsin  in  50% 
alcohol,  5-20  min.  Wash  in  water. 
Pass  through  95%  and  abs.  alcohol  and 
xylol  and  mount  in  balsam.  Nuclei 
and  hemofuscin,  red;  hemosiderin,  blue 
(J.  E.  Ash  in  Simmons  and  Gentzkow, 
p.  744).     See  Iron  and  Hemofuscin. 

Heparin.  A  method  for  the  histological 
demonstration  of  heparin  has  been  de- 
scribed by  Jorpes,  E.,  Holmgren,  H.  and 
Wilander,  O.,  Ztsch.  f.  mikr.  anat. 
Forsch.,  1937,  42,  279-301.  It  is  based 
on  evidence  that  Tissue  Basophiles 
contain  this  substance.  See  also  Anti- 
coagulants. 

Heptaldehyde.  An  agent  said  by  Strong, 
L.  C,  Am.  J.  Cancer,  1939,  35,  401-407, 
to  produce  liquefaction  of  spontaneous 
mammary  tumors  of  mice.  It  was  not 
helpful  when  injected  into  rat  lepro- 
mata  (Cowdry,  E.  V.  and  Ruangsiri,  C, 
Arch.  Path.,  1941,  32,  632-640). 

Hermann's  Fluid.  2%  osmic  acid,  4  cc; 
1%  platinum  chloride,  15  cc;  glacial 
aceticjflkacid.     1     cc.    This     resembles 


HERRING  BODIES 


156 


HOFMANN'S  VIOLET 


Flemming's  fluid  and  is  a  good  cyto- 
logical  fixative. 

Herring  Bodies,  see  Gushing,  H.,  Proc.  Soc. 
Exp.  Biol.  &  Med.,  1932-33,  30,  1424- 
1425. 

Hertzberg's  Victoria  Blue  stain  for  elemen- 
tary bodies  is  described  by  Seiffert,  G., 
Virus  Diseases  in  Man,  Animal  and 
Plant.  New  York:  Philosophical  Li- 
rary,  Inc.,  1944,  332  pp.  It  is  rather 
like  Gutstein's  technique  (see  Ele- 
mentary Bodies)  except  that  Hertzberg 
does  not  make  up  the  stain  with  po- 
tassium hydrate  and  Gutstein  does  not " 
destain  in  1%  citric  acid. 

Herxheimer's  solution  for  staining  fat : 
scarlet  red  (scharlach  R,  sudan  IV), 
1  gm. ;  70%  alcohol,  50  cc. ;  acetone  C.P., 
50  cc.     See  Sudan  IV. 

Heterophile,  see  Staining. 

Hexamethyl  violet,  see  Crystal  violet. 

Hexuronic  Acid  as  antiscorbutic  factor 
(Harris,  L.  J.,  and  Ray,  S.  N.,  Biochem. 
J.  1933,  27,  58-589). 

Hickson  Purple,  a  disazo  dye,  giving  in  aq. 
sol.  a  purple  color  to  leucocytes  and  a 
red  color  to  erythrocytes  introduced  by 
H.  G.  Cannan  (J.  Roy.  Micr.  Soc,  1941, 
61,88-94). 

Higgins'  Ink.  This  was  apparently  first 
used  as  a  vital  stain  by  George  Wislocki, 
see  Foot  (McClung,  p.  114).  Dilute 
with  equal  volume  sterile  aq.  dest. 
Warm  and  inject  into  marginal  vein  of 
rabbit's  ear  5  cc.  daily  for  3-4  days,  then 
every  3  days  as  long  as  desired.  Since 
the  carbon  is  relatively  insoluble  it  is  a 
simple  matter  to  fix,  imbed,  section  and 
counterstain.  Smaller  amounts  are  to 
be  used  for  smaller  animals,  see  Vital 
Stains. 

Hirudinea,  see  Parasites. 

Hischler's  Fluid,  see  Golgi  Apparatus. 

Hiss's  Method  for  capsule  staining,  see 
Capsule. 

Histamine,  improved  colorimetric  method 
for  estimating  (Rosenthal,  S.  M.  and 
Tabor,  H.,  J.  Pharmacol.  &  Exp. 
Therap.,  1948,  92,  425).  For  use  in  de- 
termination of  the  histamine  content  of 
experimental  tumors,  see  Rosenthal, 
S.  M.,  J.  Nat.  Cancer  Inst.,  1949,  10, 
89-91. 

Histiocyte,  a  term  without  value  as  it 
simply  indicates  a  "tissue  cell,"  often 
applied  to  phagocytic  cells  of  connec- 
tive tissue. 

Histones,  see  discussion  of  Saint-Hilaire's 
method  under  Purines. 

Histoplasma  Capsulatum.  The  halos  about 
this  organism  and  Sporotrichum 
schencku  as  seen  in  H  &  E  stained  tissue 
sections  suggest  that  they  are  encapsu- 
lated. Technique  for  search  for  such 
capsules  and  its  failure  to  reveal  them 
is   described  by  Kligman,   A.   M.   and 


Baldridge,  G.  D.,  A.  M.  A.  Arch.  Path., 
1951,  51,  567-574.  The  Hotchkiss- 
McManus  stain,  as  employed  by  Klig- 
man, A.  M.  and  Mescon,  H.,  J.  Bact., 
1950,  60,  415  is  considered  by  Kligman 
and  Baldridge  to  be  the  best  suited  one 
for  the  demonstration  of  capsules. 

Historadiography  is  the  x-ray  photography 
of  tissues.  By  a  special  technique 
Larmaque,  P.,  Bull.  d'Hist.  Appl., 
1937,  14,  1-16)  rays  emitted  at  a  tension 
of  50-100  KV  having  a  length  of  0, 12-0,2 
A°  are  directed  upon  a  section  closely 
applied  to  a  particularly  finely  grained 
emulsion.  The  absorption  of  the  rays 
by  the  section  depends  upon  the  density 
of  its  parts.  Total  opacity  of  the  tissue 
to  the  rays  is  marked  on  the  photo- 
graphic negative  by  white,  permeability 
by  black,  and  there  are  usually  all  grades 
between  the  two.  Subsequent  magnifi- 
cation of  about  500  times  is  possible,  but 
is  not  advisable.  Sections,  not  more 
than  4  microns  thick,  of  formalin  fixed 
tissues,  are  recommended.  An  illus- 
trated description  of  the  appearance  of 
epidermis,  cartilage,  artery  wall,  thjToid 
and  other  tissues  is  provided  by  Tur- 
chini  (J.  Bull.  d'Hist.  Appl.,  1937,  14, 
17-28).  Historadiography  may  have 
many  uses  in  the  measurement  of 
densities  in  different  physiological  states 
and  in  study  of  the  distribution  of  sub- 
stances opaque  to  x-rays  experimentally 
introduced.  In  some  cases  great  den- 
sity may  accompany  high  Viscosity. 

Histospectrography.  This  is  a  very  valu- 
able survey  method  for  minerals  in 
tissues.  See  Policard,  A.,  Protoplasma, 
1933,  19,  602-629;  Scott,  G.  H.  and 
Williams,  P.  S.,  Anat.  Rec,  1935,  64, 
107-127;  Cowdry,  E.  V.,  Heimburger, 
L.  F.,  and  Williams,  P.  S.,  Am.  J.  Path., 
1936,  12,  13-29.  Optic  lens  and  cata- 
racts have  been  analysed  particularly 
for  iron,  copper  and  zinc  (Busnel,  R.  G., 
Pillet,  P.  and  Tillie,  H.,  Bull.  d'Hist. 
Appl.,  1938,  15,  99-109).  MacCardle, 
R.  C.,  Engman,  M.  F.,  Jr.  &  Sr.,  Arch. 
Dermat.  and  Syph.,  1941,  44,  429-440 
have  employed  histospectrography  to 
advantage  in  determination  of  skin 
magnesium.    See    Absorption    Spectra. 

Hodgkin's  Disease,  see  Reed-Sternberg 
Cells. 

Hofmann's  Violet  (CI,  679)— dahlia,  iodine 
violet,  primula  R  water  soluble,  red 
violet,  violet  R,  RR  or  4RN — Conn 
(p.  120)  says  above  names  are  applied 
rather  indiscriminately  to  stains  varying 
in  shade  from  methyl  violet  to  basic 
fuchsin  which  are  mixtures  of  methyl- 
ated and  ethylated  rosanilins  and 
pararosanilins  having  less  than  5  methyl 
or  ethyl  groups.  He  further  remarks 
that  a  mixture  of   basic   fuchsin  and 


HOLLANDE'S  FIXATIVE 


157 


HYALURONIC  ACID 


methyl  violet  of  the  color  desired  may 
perhaps  be  made  by  the  worker  himself 
as  a  substitute  for  Hofmann's  violet 
which  is  in  fact  the  composition  of  some 
samples  sold  as  Dahlia  and  Hofmann's 
violet. 

Hollande's  Fixative.  Picric  acid,  4.0  gm.; 
copper  acetate,  2.5  gm.;  formol,  10  cc; 
glacial  acetic  acid,  1.5  cc;  aq.  dest. 
100  cc.  Recommended  for  flagellate 
protozoa  (McClung,  1950,  p.  445). 

Holmium,  see  Atomic  Weights. 

Holtfreter's  Solution,  for  use  in  examina- 
tion of  fresh  tissues;  NacCl,  0.35  gm.; 
KCl,  0.005  gm.;  CaClj,  0.01  gm., 
NaHCOa-HsO,  0.02  gm.,  aq.  dest.  100 
cc.  (Holtfreter,  J.,  Arch.  Entio.-Mech., 
1931,  124). 

Hookworms.  To  eliminate  opacity  in 
mounts  of,  see  Tahmisian,  T.  N.,  Stain 
Techn.,  1945,  20,  26. 

Hormones.  Consult  volume  entitled  New 
and  Nonofficial  Remedies  published 
each  year  by  the  American  Medical 
Association.  See  Testosterone,  Chro- 
maffin Reaction,  Vulpian  Reaction,  Os- 
mic  Acid. 

Hotchkiss'Method,  see  Polysaccharides. 

Howell-Jolly  Bodies,  see  Jolly  Bodies. 

Huber's  Toluidin  Blue  stain  for  Nissl  bodies 
(Addison  in  McClung,  p.  150).  This 
much  used  method  is  suggested  for 
autopsy  material.  Fix  in  95%  alcohol, 
100  cc;  trichloracetic  acid  (Mallinck- 
rodt),  1.5  gm.;  mercuric  chloride  (Mal- 
linckrodt),  3  gm.  2-10  days  depending 
upon  size  of  piece  of  tissue.  Change 
fixative  every  2  days  for  larger  speci- 
mens. Pour  off  fluid  and  store  in  95% 
alcohol  until  used.  Do  not  take  out 
mercury  with  iodine.  Stain  paraffin 
sections  in  toluidin  blue  15-18  hrs. 
(Make  up  solution  by  adding  1  gm.  to 
500  cc.  aq.  dest.  Heat  gently  and  when 
it  is  dissolved  add  500  cc.  aq.  dest.). 
Pour  off  stain.  Wash  in  aq.  dest. 
Leave  2  hrs.  in  lithium  carbonate. 
(Make  this  by  adding  5  gm.  to  1000  cc. 
aq.  dest.  Boil  several  minutes.  Cool. 
Filter.  To  100  cc.  filtrate  add  900  cc. 
aq.  dest.) .  Differentiate  in  70%  alcohol 
5-30  min.  Leave  flat  in  95%  alcohol, 
5-15  min.  Dehydrate  in  absolute,  clear 
in  xylol  and  mount  in  balsam. 

Humus,  see  soli. 

Huntoon's  Hormone  Medium,  see  Bacteria, 
Media. 

Hyalin.  This  is  usually  easily  recognizable 
in  sections  stained  with  Hematoxylin 
and  Eosin  or  by  Phloxin  and  Methylene 
Blue,  by  its  affinity  for  eosin  or  phloxin. 
Phosphotungstic  Acid  Hematoxylin 
colors  it  deep  blue.  A  hematoxylin- 
phloxin  method  is  also  recommended 
by  Mallory  (p.  207).  Fix  in  alcohol  or 
10%  formalin  and  imbed  in  paraffin  or 


celloidin.  Stain  in  alum  hematoxylin, 
1-5  min.  or  more.  Wash  in  tap  water 
and  stain  with  0.5%  phloxin  in  20% 
alcohol,  10-30  min.  or  longer.  Wash  in 
tap  water  and  treat  for  |-1  min.  with 
0.1%  aq.  lithium  carbonate.  Wash  in 
tap  water,  dehydrate,  clear  and  mount. 
In  case  of  celloidin  sections,  clear  in 
terpineol  or  origanum  oil  from  95% 
ale  Nuclei,  blue;  fresh  hyalin,  in- 
tensely red;  older  hyalin,  pink  to 
colorless.  A  simple  thionin  stain  is  also 
given  by  Mallory.  It  is  to  stain  similar 
sections  for  5-10  min.  in  0.5%  thionin 
in  20%  ale  Differentiate  and  dehy- 
drate in  80%  alcohol.  Then  95%  alco- 
hol, terpineol  and  terpineol  balsam. 
Nuclei  and  old  hyalin,  blue. 
Hyaluronic  Acid. — Written  by  A.  R.  Gopal- 
Ayengar,  Barnard  Free  Skin  &  Cancer 
Hospital,  St.  Louis.  (Now  at  Tata 
Memorial  Hospital,  Bombay.)  This  is 
a  polymer  of  acetyl  glucosamine  and 
glucuronic  acid.  It  occurs  in  a  poly- 
disperse  form  in  a  variety  of  tissues  such 
as  umbilical  cord,  synovial  fluid, 
vitreous  humor,  skin,  tumors  due  to 
virus  of  leucosis  and  sarcoma  of  fowls, 
and  in  pleural  fluid  associated  with 
human  mesothelioma.  (For  an  exten- 
sive treatment  of  the  subject  of  acid 
polysaccharides  and  a  comprehensive 
bibliography,  refer  to  Karl  Meyer's  re- 
views on,  "Mucolytic  enzymes"  in 
Currents  in  Biochemical  Research,  In- 
terscience  Publishers,  N.  Y.,  1946; 
"Mucoids  and  Glycoproteins"  in  Ad- 
vances in  Protein  Chemistry,  Academic 
Press,  N.  Y.  1945;  "The  Chemistry  and 
Biology  of  Mucopolysaccharides  and 
Glycoproteins"  in  Cold  Spring  Harbor 
Symposia  on  Quant.  Biol.,  6,  1938,  91- 
102.)  The  enzyme,  hyaluronidase, 
depolymerizes  and  hydrolyses  hyal- 
uronic acid.  It  is  a  Spreading  Factor 
and  has  been  ably  presented,  along  with 
other  spreading  factors,  by  Duran- 
Reynals,  F.,  Bact.  Rev.,  1942,  6,  197- 
252;  Meyer,  K.  and  Chaffee,  E.,  Proc. 
Soc  Exp.  Biol.  &  Med.,  1940,  43,  487- 
489;  Meyer,  K.  et  al.,  Proc  Soc  Exp. 
Biol.  &  Med.,  1940,  44,  294-296,  and 
others. 

A  histochemical  method  for  the  dem- 
onstration of  acid  polj'saocharides  like 
hyaluronic  acid  is  described  bv  Hale, 
C.  W.,  Nature,  1946,  157,  802.  the  use 
of  metachromatic  stains  such  as  tolui- 
dine  blue  while  satisfactory  for  sul- 
phated  polysaccharides  like  chondroitin 
sulphate  is  valueless  for  hyaluronic  acid 
and  for  related  acid  polysaccharides 
which  do  not  stain  metachromatically. 
Fixation  of  material  is  an  important 
factor  in  the  retention  of  hj'aluronic 
acid  for  subsequent  staining.     The  or- 


HYALURONIDASE 


158 


HYDROGEN  ION  INDICATORS 


dinary  aqueous  fixatives  containing 
formalin,  while  eminently  suitable  for 
fixing  protein  components,  tend  to  dis- 
solve the  hyaluronic  acid.  To  preserve 
intact  hyaluronic  acid  it  is  therefore 
imperative  to  employ  dehydrating  fix- 
ing agents  like  Carnoy.  The  material 
after  fixation,  dehydration  and  embed- 
ding is  sectioned  in  the  usual  manner 
and  treated  with  an  acid  solution  of 
ferric  hydroxide.  The  iron  combines 
with  hyaluronic  acid  but  not  with  the 
neutral  polysaccharides  or  proteins. 
The  combined  iron  is  then  characterized 
as  Prussian  blue  by  treatment  with 
hydrochloric  acid  and  potassium  ferro- 
cyanide.  A  counter  stain  like  fuchsin 
is  recommended  in  order  to  bring  out 
sharply  the  blue  stained  acid  polysac- 
charides against  a  background  of  red 
stained  cells. 

The  detailed  outline  of  the  Hale 
technique  is  as  follows:  Fix  small  pieces 
of  tissue  in  Carnoy  (Abs.  alcohol,  6  pts. 
-H  chloroform,  3  pts.  +  glacial  acetic 
acid,  1  pt.,  for  j  hr.  Dehydrate  in  abs. 
alcohol,  clear,  embed  in  paraffin  and 
section  in  the  usual  manner.  Mount 
sections  on  clean  slides  without  albu- 
men. Bring  sections  rapidly  to  water 
and  fiood  with  a  mixture  of  dialysed 
iron,  1  vol.  and  acetic  acid  (2M),  1  vol., 
10  min.  (Dialyzed  iron  may  be  pre- 
pared by  adding  ammonia  water  to  a 
concentrated  solution  of  ferric  chloride 
and  dialysing  the  resulting  solution  un- 
til free  or  nearly  free  of  ammonium 
salts.  It  is  a  dark  red  liquid  easily 
miscible  with  water  and  contains  ap- 
proximately 3.5  per  cent  Fe,  or  5% 
Fe20,.  M  =  Molecular  Solution,  which 
see.)  Wash  well  with  aq.  dest.  Flood 
with  a  solution  containing  potassium 
ferrocyanide  (0.02M)  and  hydrochloric 
acid  (0.14M)— 10  min.  Wash  with  wa- 
ter and  counterstain  with  appropriate 
contrasting  dye.  Dehydrate  rapidly, 
clear  in  xylol  and  mount  in  Canada 
balsam. 

In  order  to  distinguish  hyaluronic 
acid  from  other  blue  staining  structures 
Hale  recommends  interpolation  of 
another  step  during  the  staining  proc- 
ess. The  procedure  suggested  involves 
use  of  the  specific  enzyme-hyaluroni- 
dase — soon  after  fixation.  The  enzyme 
hydrolyses  the  hyaluronic  acid  and 
prevents  the  combination  of  the  pol- 
ysaccharide with  iron.  Since  hyal- 
uronidase  is  specific,  it  has  no  similar 
action  on  other  polysaccharides. 
Hyaluronidase  is  the  spreading  factor  which 
increases  the  permeability  of  connec- 
tive tissue  by  reduction  in  viscosity  and 
by  hydrolysis  of  Hyaluronic  Acid. 
Commercial      preparations     of     hyal- 


uronidase from  bull  testes  are  available 
from  the  Schering  Corp.,  Bloomfield, 
N.  J.  Enzyme  prepared  from  certain 
bacteria  apparently  have  hydrolytic 
powers  different  from  those  of  the 
testicular  preparations. 

Hydrax  is  a  synthetic  resin  used  as  a  mount- 
ing medium  (Hanna,  D.,  J.  Roy.  Micr. 
Soc.,1930,  50,  424-426). 

Hydrogen  Acceptors.  These  are  substances 
like  p-amidophenol,  p-phenylenedia- 
mine  and  resorcin,  recommended  to 
strengthen  supravital  staining  of  nerve 
fibers  with  methylene  blue,  see  Auer- 
bach's  Plexus. 

Hydrogen  Ion  Indicators — Written  by  L.  F. 
Wicks,  Veterans  Administration  Hos- 
pital, Jefferson  Barracks,  Missouri. 
February  1, 1951. — These  are  also  called 
acid-base  indicators  and  pH  indicators. 
They  are  dye  compounds  which  are 
themselves  weak  acids  or  weak  bases, 
more  usually  the  latter,  and  have  defi- 
nite ionization  constants.  According 
to  the  old  theory  of  Ostwald,  the  color 
of  the  indicator  in  solution  depends 
upon  the  degree  of  dissociation  and  the 
relative  ratio  of  dissociated  and  un- 
dissociated  forms.  This  ratio,  and  the 
corresponding  shift  in  color,  varies 
with  the  concentration  of  hydrogen  ions 
present,  the  effect  being  a  composite 
one.  The  color  change  interval  will 
span  a  certain  range,  usually  of  two  pH 
units  or  less,  and  it  does  not  require  a 
shift  which  crosses  neutrality.  Of  the 
very  many  dyes  and  plant  coloring 
matters  which  alter  color  with  pH, 
only  a  few  change  sufficiently  sharply 
to  be  of  analytical  value. 

Acid-base  indicators  may,  of  course, 
be  employed  for  adjusting  the  pH  of  a 
solution.  If  direct  addition  is  not  de- 
sired, small  portions  of  both  liquids  may 
be  transferred  to  a  spot  plate,  or  the 
indicator  may  be  applied  in  the  form 
of  test  papers.  (Some  indicators  such 
as  litmus  are  now  rarely  employed 
otherwise.  This  is  partly  true  also  for 
Congo  red  and  nitrazine.) 

Indicators  may  also  be  used  to  esti- 
mate the  reaction  of  a  solution  by  the 
application  of  a  series  with  different  pH 
ranges.  Once  roughly  determined, 
there  is  a  procedure  ("Gillespie's  drop- 
ratio  method")  by  which  a  fairly  ac- 
curate pH  measurement  may  be  easily 
made  with  a  single  indicator  of  proper 
range. 

Perhaps  the  commonest  use  for  hy- 
drogen ion  indicators  by  the  analytical 
chemist  (who  usually  prefers  the  glass 
electrode  for  pH  measurement  and  ad- 
justment), is  as  an  end  point  device  in 
acid-base  titrations.  When  titrating  a 
weak  acid  or  a  weak  base,  the  choice  of 


HYDROGEN  ION  INDICATORS 


159 


HYDROGEN  ION  INDICATORS 


indicator  is  very  important.  That 
chosen  should  be  one  which  has  its 
sharpest  color  transition  near  the  true 
equivalence  point.  For  example,  when 
titrating  the  weak  base  ammonium 
hydroxide  with  hydrochloric  acid,  the 
resulting  ammonium  chloride  is  an 
acidic  salt,  and  the  indicator  selected 
should  be  one  that  shifts  below  neu- 
trality, methyl  red  for  example. 
Again,  when  titrating  a  weak  acid  such 
as  lactic  acid  with  sodium  hydroxide, 
the  salt  formed  is  alkaline,  and  the 
indicator  of  choice  should  shift  above 
neutrality,  bromthymol  blue,  for  ex- 
ample. In  titrating  strong  acids  and 
strong  bases  against  each  other,  the 
selection  of  indicator  is  not  critical,  as 
here  the  pH  change  near  the  end  point 
is  very  great  for  only  a  small  increment 
of  the  added  reagent.  One  should 
never  titrate  weak  acids  and  weak  bases 
together,  for  the  results  cannot  be 
accurate. 

There  are  several  possible  sources  of 
error  in  the  use  of  hydrogen  ion  indi- 
cators. Some  are  present  at  all  times 
and  others  especially  so  in  biological 
fluids.  An  occasional  indicator,  with 
two  groups  sensitive  to  acid  or  alkali, 
has  two  ranges  of  color  transition  at 
perhaps  widely  separately  pH  values. 
For  example,  th3^mol  blue  shifts  from 
red  to  yellow  at  a  low  range  (1.2-2.8) 
and  from  yellow  to  blue  at  a  much  higher 
one  (8.0-9.6) .  A  very  few  indicators  ex- 
hibit "dichroism"  (or  "dichromatism") 
in  which  the  color  varies  with  the  depth 
and  concentration  of  the  solution. 
Bromcresol  purple  and  bromphenol  blue 
are  examples. 

In  attempting  to  determine  the  pH 
of  a  very  dilute  solution,  a  false  result 
may  be  obtained  by  the  use  of  indicators 
as  they  are  themselves  acids  or  bases. 
(Recall,  for  example,  that  water  in  con- 
tact with  the  carbon  dioxide  of  the 
normal  atmosphere  has  a  pH  of  about 
5.7.)  For  such  cases,  a  very  small 
amount  of  buffer  should  be  present  to 
offset  this  effect. 

Indicators  are  intended  for  aqueous 
sj^stems,  and  the  presence  of  other 
solvents  such  as  alcohol  decreases  the 
dissociation  constant.  Acidic  indi- 
cators then  become  more  sensitive  to 
hydrogen  ions,  and  basic  ones  less  sensi- 
tive. A  control  solution  of  the  same 
solvent  composition  may  be  used  for 
comparison,  however. 

Temperature  errors  are  slight  over 
the  usual  ranges. 

Indicators  may  be  altered  or  de- 
stroyed by  the  presence  of  oxidizing  and 
reducing  agents,  and  thej^  niay  unite 
with  heavy  metal  ions.     Fortunately, 


these  are  negligible  considerations  in 
biological  fluids,  but  greater  potential 
errors  exist. 

Proteins  and  their  hydrolysis  prod- 
ucts are  usually  amphoteric  and  may 
combine  with  the  indicator.  Congo 
red,  for  example,  is  almost  worthless  in 
protein  solutions. 

The  presence  of  much  neutral  salt 
will  affect  the  color  of  indicator  solu- 
tions, partly  by  influencing  the  light 
absorption  and  partly  by  shifting  the 
ratio  between  dissociated  and  non- 
dissociated  forms  of  the  indicator. 

The  use  of  mixed  indicators  deserves 
greater  attention  than  it  has  yet  re- 
ceived. As  employed  for  titrations, 
they  are  of  two  general  types.  One 
sort  consists  of  two  acid-base  indicators 
which  have  color  transitions  in  op- 
posite directions,  resulting  in  a  very 
sharp  change  at  a  narrow  pH  zone. 
The  other  kind  utilizes  for  contrast 
color  a  dye  which  itself  is  not  influenced 
by  hydrogen  ion  concentration.  The 
composite  color  change  resulting  is 
usually  much  sharper  than  that  of  the 
indicator  alone.  In  recent  years,  there 
have  appeared  "universal  indicators" 
consisting  of  a  mixture  of  half  a  dozen 
compounds  with  a  "spectrum"  of  colors 
which  may  vary  over  the  entire  pH 
range.  Such  indicators  are  not  very 
accurate  and  should  be  used  only  as  a 
first  rough  test  on  an  unknown  solu- 
tion. These  mixtures  are  more  com- 
mon as  test  papers. 

It  is  not  surprising  that  hj^drogen  ion 
indicators  have  been  employed  as  a 
sort  of  vital  stain  to  determine  the  re- 
action of  various  living  components. 

In  1893  Ehrlich  injected  neutral  red 
in  an  attempt  to  determine  the  reaction 
about  phagocj'tosed  granules.  Since 
then,  other  workers  have  applied  other 
dyes,  striving  to  estimate  the  approxi- 
mate pH  of  tissues,  of  the  fluids  bathing 
them,  and  even  of  individual  cells. 
Alizarin  red  and  litmus  have  been  much 
used,  the  later  especially  with  lower 
organisms.  Thus,  Steiglitz  applied  all 
three  dyes  mentioned  above  to  estimate 
the  reaction  of  living  kidney  (E.  J., 
Arch.  Int.  Med.,  1924,  33,  483-496)  and 
confi.rmed  the  contention  that  alkaline 
urine  can  be  formed  by  an  acidic  cortex. 
Harvey  and  Benslev  (B.  C.  H.  and  R. 
R.,  Biol.  Bull.,  1912,  23,  225-249)  used 
pH  indicators  to  indicate  that  gastric 
fluid  does  not  arise  directly  within  the 
cells  of  the  mucosa.  Margaria  (R.,  J. 
Physiol.,  1934,  82,  496-497)  injected 
bromcresol  purple  and  bromphenol  blue, 
and  claimed  to  have  measured  pH 
changes  upon  stretching  a  muscle. 
Orr  (J.  W.,  J.  Path.  &  Bact.,  1937,  44, 


HYDROGExX  ION  INDICATORS 


160 


HYDROGEN  ION  INDICATORS 


19-27)  employed  phenol  red  to  estimate 
alterations  in  pH  in  the  skin  of  tarred 
mice  during  carcinogenesis.  Chambers 
and  his  colleagues  have  added  pH  indi- 
cators to  tissue  cultures  (R.,  Proc.  Roy. 
Soc,  B,  1932,  110,  120-124)  and  have 
injected  them  directly  into  individual 
living  cells  (McClung,  pp.  62-109). 
The  most  enthusiastic  investigator  tx) 
employ  the  phthalein  and  sulphon- 
phthalein  indicators  is  Rous  (P.,  Sci- 
ence, 1924,  60,  363:  J.A.M.A.,  1925,  85, 
33-35,  and  many  articles  in  J.  Exp. 
Med.,  1925  to  1927).  The  literature  is 
extensive  but  scattered.  There  are 
brief  reviews  by  Rous  (P.,  J.  Exp.  Med., 
1925,  41,  379-411)  and  von  Mollendorf 
(W.,  Ergebn.  Physiol.,  1920,  18,  141- 
306).  See  W.  M.  Clark  in  Simmons  and 
Centzkow  161-171. 

It  is  well  to  question  the  dependa- 
bility of  data  upon  pH  of  living  material 
as  apparently  indicated  by  vital  staining 
methods.  Consider  the  ideal  require- 
ments for  such  a  vita,l  stain.  It  should 
exhibit  a  sharp  and  pronounced  color 
change  in  the  proper  pH  range.  It 
should  be  fairly  soluble,  readily  dif- 
f usable,  strongly  colored,  of  low  toxicity 
and  stable  in  the  organism  (not  readily 
oxidized  or  reduced  or  precipitated  by 
tissue  electrolytes).  Of  the  many  indi- 
cators employed  in  analytical  chemistry, 
only  a  few  meet  these  requirements. 
Certain  errors  are  to  be  guarded  against 
in  their  use.  The  "salt  error"  and 
"protein  error"  are  unavoidably  pres- 
ent. In  the  application  of  these  vital 
stains  changes  may  take  place  that  will 
themselves  cause  a  pH  change.  Among 
them  anesthesia,  trauma,  loss  of  carbon 
dioxide  from  exposed  tissues,  interfer- 
ence with  blood  supply,  and  postmortem 
change  deserve  special  mention.  How- 
ever crude  though  the  methods  may  be, 
these  dye  indicators  are  of  value  in  pre- 
liminary experiments  or  where  no  better 
procedure  is  applicable. 


The  indicator  dyes  of  most  promise  are 
certain  of  the  phthalein  and  sulphon- 
phthalein  compounds.  They  are  gen- 
erally quite  soluble,  highly  diffusable, 
show  marked  color  shifts  and  are  fairly 
constant  in  composition.  The  dye 
solutions  diffuse  quickly  when  injected, 
and  quickly  appear  in  the  urine  and 
stools.  For  these  reasons,  fairly  large 
doses  given  intraperitoneally  are  more 
suitable  than  subcutaneous  injections. 
But  it  is  doubtful,  according  to  Cham- 
bers (personal  communication),  whether 
the  more  soluble  dyes  actually  penetrate 
the  walls  of  most  cells. 

The  following  selection  of  indicators 
is  based  upon  the  reports  of  Rous  and 
others,  and  upon  experiments  with  mice 
carried  out  at  The  Barnard  Free  Skin 
and  Cancer  Hospital.  Their  chemical 
names  can  be  found  in  The  Merck  Index 
or  in  any  good  textbook  of  chemistry. 
Some  are  to  be  used  in  1%  aq.  solutions, 
others  in  sat.  solutions  in  physiological 
saline,  litmus  in  either  aqueous  or  agar 
solution  (Rous,  P.,  J.  Exp.  Med.,  1925, 
41,  379),  while  the  remainder,  which 
are  acidic  (the  sulphonphthaleins  and 
methyl  red),  require  to  be  converted  to 
their  corresponding  sodium  salts  be- 
cause the  latter  are  more  soluble  in 
water.  Consequently  the  proper  equiv- 
alent of  sodium  hydroxide  must  be 
reacted  with  each  compound.  Rub  up 
0.1  gm.  of  the  dry  dye  in  a  mortar 
(agate,  preferably)  with  the  volume  of 
N/20  sodium  hydroxide  solution  given 
in  cc.  below  the  dj'^e  in  the  table.  Filter, 
wash  out  the  mortar  with  several  small 
portions  of  saline  (0.9%  NaCl)  and  make 
all  to  a  volume  of  10  cc.  For  a  mouse, 
0.5-2.0  cc.  of  the  dye  solution  should 
be  injected  intraperitoneally. 

It  should  be  emphasized  that  wher- 
ever possible,  the  glass  electrode  should 
be  employed  for  direct  measurement  of 
pH  in  biological  fluids  rather  than  indi- 
cators. Micro  electrodes,  including 
injection  types,  have  been  developed. 


Indicator 

Bromphenol  blue* 
3.0  N/20  NaOH 

Sodium  alizarin 
sulphonate 
(Alizarin  red) 
1%  aq.  or  sat. 
in  aaline 

Bromcresol  green* 
2.9  N/20  NaOH 

Methyl  red* 
7.4  N/20  NaOH 


HYDROGEN  ION  INDICATORS 

pH  Range  and  Colors  Value  as  Vital  Stain 

yeUow*— 3.0  —  4.6— »  blue  Very  strong  stain,  too  far  on   acid  side. 


yellow  «-  3.8  —  5.0  -►  pink 

yellow  <—  4.0  —  5.6  — ►  blue 
red  «-  4.2  —  6.3  -♦  yellow 


Very  toxic,  weak  stain. 


Strong  stain,   persistent,   well  tolerated. 

Unstable  in  organism,  weak  stain,  fixes  on 
lipoids. 


HYDROKOLLAG 


161 


ICTERUS  INDEX 


HYDROGEN  ION  INDICATORS— Conitnued 


Indicator 

Chlorphenol  red* 
4.7  N/20  NaOH 

Bromcresol  purple* 
3.7  N/20  NaOH 

Bromphenol  red 
3.9  N/20  NaOH 

Methyl  violet 
1%  aq.  or  sat. 
in  ealine 

Bromthymol  blue* 
3.2  N/20  NaOH 

Phenol  red 
6.7  N/20  NaOH 

Litmus,  purified 
(Azolitmin) 
1%  aq.  or 
in  agar  sol. 

Neutral  red 
(Toluylene  red) 
1-2%  aq.  or  sat. 
in  saline 

Cresol  red 
5.3  N/20  NaOH 

Metacresol  purple 
5.3  N/20  NaOH 

Thymol  blue* 
4.3  N/20  NaOH 


pH  Range  and  Colors 
yellow  ♦-  4.8  —  red  —  6.8  -»  purple 

yellow  «—  5.4  —  6.6  -♦  purple 
yellow  <—  6.4  —  red  —  7.0  — ►  purple 
blue- violet  <-  6.0  —  7.0  -►  violet 

yellow  «-  6.0  —  7.4  -*  blue 

yellow  ♦-  6.6  —  7.8  -»  red 
(6.8  —  8.4) 

(approx.)  red  ♦-  6.0  —  8.0  -«  blue 

(approx.)  red  «—  6.8  —  8.0  — »  yellow 

yellow  *-  7.2  —  8.4  —>  purple-red 
yellow  ♦-7.4  —  9.0  —»  purple 
yellow  ♦-  8.2  —  9.4  -»  blue 


Value  as  Vital  Stain 
Powerful  stain,  well  tolerated. 

Strong  stain  but  rapidly  excreted,  is  toxic 
and  exhibits  dichromatism. 

Very  strong  stain,  well  tolerated. 
Weak  stain,  toxic. 


Weak  stain,  very  toxic  to  mice,  but  not  for 
insecte. 

Rapid,  intense  stain,  very  well  tolerated  . 


Slow  stain,  diffuses  poorly,  usually  de- 
posits in  granules. 


Very  weak  stain,  precipitates  out  readily 
in  vivo,  not  toxic  if  pure. 


Somewhat  toxic,  not  a  strong  stain. 


Very  weak  stain,  not  very  soluble. 


Toxic,  range  too  alkaline. 


The  indicators  starred  are  perhaps  of 
of  widest  ordinary  laboratory  use.  To 
these  may  also  be  added  cresol  purple 
(yellow  <—  7.4-9.4  — >•  purple)  and  phenol- 
phthalein  (colorless  <—  8.0-9.8  — »•  red- 
violet).  The  latter  is  usually  made  up 
in  70-90%  alcohol.  It  should  not  be 
used  to  titrate  ammonia. 

Hydrokollag,  a  particulate  material  em- 
ployed for  injection  of  Lymphatic  Ves- 
sels which  see. 

Hydrotropes,  see  Sudan  Stains. 

Hydroxy  Tri-Phenyl  Methanes.  These  are 
the  rosolic  acids.  Amino  groups  of  tri- 
amino  tri-phenyl  methanes  are  replaced 
by  hydroxyls  making  them  aciuic  in- 
stead of  basic.  Examples :  aurin  (or 
rosolic  acid);  red  corallin. 

Hydroxybenzene  Compounds  as  cytoplasmic 
fixatives.  Details  of  use  ofpyrogallol 
and  resorcinol  in  neutralized  formal- 
dehyde solutions  are  given.  The  sim- 
plicity and  rapidity  of  the  procedures 
and  the  ease  of  thereafter  cutting  sec- 
tions 1-2^1  in  thickness  are  cited  as  the 
advantages  special  attention  having 
been  paid  to  mitochondria  and  secretion 
granules  (Huseby,  R.  A.,  Proc.  Soc. 
Exp.  Biol.  &  Mod.,  1946,  61.  122-125). 

Hydroxyquinoline  test  for  iron,  see  Iron. 


Hypophysis,  see  Pituitary. 

Hypoxanthine,  see  Murexide  test  under 
Purines. 

Ice-crystal  Artefacts  in  normal  and  chroma- 
tolj'tic  anterior  horn  cells  (Gersh,  I., 
and  Bodian,  D.,  Biological  Symposia, 
1943,  10,  163-184). 

Icterus  Index  is  a  simple  measure  of  the 
degree  of  yellow  color  of  blood  plasma, 
or  serum,  in  comparison  with  standard 
potassium  bichromate  solutions.  Make 
up  in  tubes  of  same  thickness  and  bore 
as  hematocrit  tubes  a  series  of  unit 
dilutions  of  the  bichromate  solution 
Unit  1  =  1  gm.  potassium  bichromate 
in  10,000  cc.  aq.  dest..  Unit  3  =  3  gm. 
in  10,000  cc.  Unit  5  =  5  gm.  in  10,000 
cc.  etc.  The  plasma  of  centrifuged 
blood  in  hematocrit  is  compared  with 
these.  If  it  has  a  color  corresponding 
to,  say,  Unit  5  of  the  bichromate  solu- 
tion the  icterus  index  is  considered  to 
be  5.  The  normal  value  of  the  icterus 
index  is  usually  given  as  4-7  units. 
The  measure  being  that  of  color,  and, 
since  increase  in  color  can  bo  caused  by 
substances  other  than  bilirubin,  the  in- 
dex is  not  a  specific  measure  of  bili- 
rubinemia.  Lipochromes  can  increase 
the  index.     If  the  blood  is  unusually 


IDIOCHROMATIN 


162 


INDIGO 


concentrated  the  index  is  higher  al- 
though the  total  amount  of  bilirubin  in 
the  circulation  may  not  be  elevated. 
See  much  more  adequate  description 
by  Wintrobe,  M.  M.,  Clinical  Hematol- 
ogy. Philadelphia:  Lea  &  Febiger, 
1942,  703  pp. 

Idiochromatin  (G.  idios,  one's  own,  pe- 
culiar). The  chromatin  concerned  par- 
ticularly with  reproductive  functions 
such  as  chromosome  formation  con- 
trasted with  nutritive  trophochromatin 
(G.  trophe,  food,  nourishment).  There 
is  no  special  technique  for  it. 

Illumination.  For  microscopic  work  the 
lighting  is  of  great  importance.  Direct 
visible  light  can  best  be  obtained  from 
various  electric  microscopic  lamps  on  the 
market.  Only  when  the  light  is  more 
intense  than  that  required  for  routine 
purposes  can  it  be  properly  employed 
for  dark  field  examination  or  for  polari- 
zation. Therefore  an  intense  source 
should  be  available.  The  intensity  can 
be  reduced  to  optimum  by  using  an 
iris  diaphragm.  When  it  is  desired  to 
deliver  light  into  the  body  to  a  position 
behind  living  tissues  or  organs  for 
transillumination  the  Quartz  Rod  tech- 
nique is  suggested. 

Even  to  make  the  light  equivalent  in 
quality  to  that  from  the  white  cloud  on 
a  bright  day,  that  microscopists  used  to 
search  for,  is  quite  unnecessary.  If  the 
light  is  too  much  screened  by  "day- 
light" or  other  glass  its  intensity  will 
be  impaired.  Green  light  was  recom- 
mended quite  enthusiastically  about  20 
years  ago.  But  it  is  difficult  to  secure 
green  light  of  the  necessary  intensity 
and  it  is  unpleasant  to  work  with. 
Ultraviolet  light,  which  permits  higher 
resolution  and  is  selectively  absorbed 
especially  by  nucleoproteins,  is  used 
occasionally  for  Ultraviolet  Photomicro- 
graphy. The  objects,  however,  can  of 
course  not  be  seen  directly  so  that  to 
photograph  them  is  a  hit  and  often  miss 
experience,  though  it  is  possible  to 
focus  on  a  fluorescent  screen.  The 
principal  use  of  ultraviolet  light  is  in 
the  Fluorescence  Microscope  by  which 
the  structures  giving  off  fluorescence 
can  be  viewed  in  a  dark  background  at 
high  magnification. 

Imbedding,  see  Celloidin,  ParafiBn,  Glycol- 
Stearate,  Rubber  Paraffin,  Ceresin, 
Double  and  Gelatin  for  imbedding 
preparatory  to  sectioning.  The  Mount- 
ing of  sections  and  whole  tissues  is  a 
kind  of  imbedding. 

Immersion  Oils.  A  special  grade  of  cedar 
oil  is  usually  emploj'ed  for  oil  immersion 
objectives.  Mineral  oil  is  also  sold  for 
this  purpose.  If  a  refractometer  is  not 
available  Lillie  (p.  6)  recommends  ad- 


justment of  the  index  of  refraction  of 
the  mineral  oil  by  the  addition  to  it  of 
alphabromonaphthalene  gradually  to 
the  point  where  a  white  glass  slide  im- 
mersed in  the  oil  can  no  longer  be  seen 
through  the  oil.  It  should  be  N  A  1.515 
to  1.520.  For  darkfield  work  he  uses  a 
mixture  of  4  parts  of  "heavy"  mineral 
oil  with  one  part  of  alphabromonaph- 
thalene. Cedar  oil  as  supplied  for  this 
purpose  may  easily  get  too  thick  by 
evaporation.  It  should  never  be  left  to 
harden  on  the  objective  but  should 
always  be  brushed  oft"  with  lens  paper 
immediately  after  use.  If  it  does 
harden  on  the  objective,  condenser  or 
slide  wet  the  lens  paper  with  xylol 
which  will  dissolve  the  oil  when  it  is 
gently  applied.     See  Lens  Paper. 

Immunization  of  monocytes  against  foreign 
erythrocytes  with  phagocytosis  of  the 
latter  (Bloom,  W.,  Arch.  Path,  and  Lab. 
Med.,  1927,  3,  608-628). 

Impedence,  see  Electrical  Resistance. 

Imperial  Red,  see  Eosin  B  or  bluish. 

Imperial  Yellow,  see  Aurantia. 

Impression  Preparations,  see  Smears. 

Inanition,  see  Fasting. 

Inclusion  Bodies  are  any  substances  in- 
cluded in  a  cell,  tissue  or  organ.  There 
is  the  implication  that  the  substance  is 
included  from  without,  that  is  to  say, 
it  is  of  extraneous  origin.  But  the 
designation  is  so  loosely  used  as  to  be 
almost  meaningless.  It  is  applied  to 
droplets  of  fat,  ingested  pigments, 
remnants  of  phagocytosed  materials, 
bodies  developed  in  cells  as  a  result  of 
virus  action  and  so  forth.  The  virolo- 
gists have  taken  over  the  designation 
from  normal  cytology  in  which  it  is 
used  less  and  less.  In  certain  virus 
diseases  inclusions  form  in  the  nucleus, 
in  the  cytoplasm  or  in  both  (Cowdry, 
E.  V.  in  Rivers'  book  on  Virus  Diseases, 
Baltimore,  Williams  &  Wilkins,  1928, 
pp. 113-154). 

Since  the  nucleus  is  shielded  from  the 
environment  by  the  cytoplasm  its  reac- 
tivity is  restricted  and  the  materials 
available  for  the  formation  of  nuclear 
inclusions  are  also  limited  as  compared 
with  those  in  the  cytoplasm.  Conse- 
quently the  composition  of  nuclear  in- 
clusions in  virus  diseases  is  more  uni- 
form than  that  of  cytoplasmic  inclusions. 
See  Nuclear  and  Cytoplasmic  Inclusions 

Indamin  Dyes.  Methylated  amino  deriva- 
tives of  indamin.  Bindschedler's  green 
and  toluylene  blue. 

India  Ink,  see  Higgins'. 

Indian  Blue  3  RD,  see  Naphthol  Blue. 

Indicators,  see  Hydrogen  Ion  and  Oxidation 
Reduction    Potential    indicators. 

Indigo,  a  fine  blue  dye  produced  from  the 
leaves  of  Indigofera  tinctoria,  employed 


INDIGO-CARMINE 


163 


INSECTS 


as  a  stain  and  a  cosmetic  for  more  than 
4000  years,  and  early  adopted  officially 
for  the  uniforms  of  American  and 
British  sailors,  its  history  reads  like  a 
romance.  (iSee,  Leggett,  W.  F.,  An- 
cient and  Medieval  Dyes.  Brooklyn: 
Chemical  Publishing  Co.,  Inc.,  1944, 
95  pp.) 

Indigo  (CI.  1177)  is  now  produced 
artificially  as  well  as  from  plants. 
Indigo-Carmine  (CI,  1180) — indigotine  la — 
This  sodium  salt  of  indigosulfonic  acid 
is  blue  with  acid  characteristics  so  that 
it  is  a  good  counterstain  for  carmine. 
It  has  been  employed  with  fuchsin  by 
Shumway,  W.,  Stain  Techn.,  1926,  1, 
37-38.  See  renal  excretion  of  (Kemp- 
ton,  R.  T.,  Bott,  P.  A.  and  Richards, 

A.  N.,  Am.  J.  Anat.,  1937,  61,  505-521). 
It  was  used  as  a  vital  stain  by  Heiden- 
hain  who  employed  35-60  cc.  of  0.4% 
suspension  for  rabbits  and  150-1500  cc. 
for  dogs  (see  Foot,  McClung,  p.  113). 
The  Bensleys  (p.  151)  advise  intra- 
venous injection  of  4  cc.  sat.  filtered 
aq.  indigo-carmine  per  kilogram  of  body 
weight.  Fix  by  vascular  perfusion  with 
formalin  alcohol  (neutral  formalin,  10 
cc;  absolute  alcohol,  90  cc.)  or  by  im- 
mersion in  it.  Counterstain  frozen  sec- 
tions with  Mayer's  Acid  Carmine  or 
with  1%  acridine  red.  Another  way  is 
to  imbed  (in  paraffin),  section,  clear  and 
examine  with  or  without  this  counter- 
staining. 

Indigotine  la,  see  Indigo-Carmine. 

Indin  Blue  2rd,  see  Naphthol  Blue  R. 

Indium,  see  Atomic  Weights. 

Indo  Reaction  for  phenols.  Formation  by 
oxidation  of  an  aromatic  paradiamine  in 
presence  of  tissue  phenol  of  a  blue  or 
green  indamine.  A  difficult  reaction 
(Lison,  p.  142).  See  Lison's  study  of 
the  venom  gland  of  toads  (Lison,  L., 
C.  Rend.  Soc.  de  Biol.,  1932,  111, 
657-^58). 

Indol  Compounds,  see  Nitro  Reaction, 
Nitrosamino  Reaction. 

Indophenol  Blue  (CI,  821).  This  is  formed 
by  oxidation  of  a  mixture  p-amino- 
dimethylaniline  and  a  naphthol.  Conn 
(p.  73)  says  that  this  is  probably  the 
dye  employed  for  staining  fat  byHerx- 
heimer,  G.,  Deut.  Med.  Wochenschr., 
1901,27,607-609. 

Indophenol   1.     See  Oxidation-Reduction. 

Indophenol  Oxidase,  see  Nadi  Reagent, 
Cytochrome,  Oxidase. 

Indophenols.  Dyes  closely  related  to  inda- 
mines.     Example:  indophenol  blue. 

Indulin.     1.  Spirit  soluble  (CI,860)— spirit 
indulin  and  spirit  nigrosin  R. 
2.  Water  soluble  (CI,  861)— fast  blue 

B,  OB,  R,  etc.,  soluble  indulin  3B — 
An  infrequently  used  acid  azin  dye. 
Lynch,  J.  E.,  Zeit.  f.  wis.  mikr.,  1930, 


46,  465-469;  Cumley,  R.  W.,  Stain 
Techn.,  1935,  10,  53-56. 

Indulin  Black,  see  Nigrosin,  water  soluble. 

Infra  Red  photography  shows  split  appear- 
ance of  chromosomes  (Ganesan,  D.,  J. 
Roy.  Micr.  Soc,  1939,  59,  75-78)  and 
gives  better  definition  of  epiphyseal 
layers  of  normal  and  rachitic  bone 
(Siegel,  L.,  Allen,  R.  M.,  McGuire,  G. 
and  Falk,  K.  G.,  Am.  J.  Path.,  1939,  15, 
273-277).  Guardabassi,  M.,  C.  rend. 
Soc.  de  Biol.,  1935,  118,  559-561  has 
used  this  technique  for  alcohol  fixed 
sections  of  brain  of  rabid  dog  sensitized 
with  rubrocyanine  to  demonstrate  struc- 
ture of  Negri  bodies.  Transmission  of 
infra  red  light  through  the  skin  facili- 
tates photography  of  superficial  veins 
in  the  living  state.  Resolution  with  this 
light  of  relatively  long  wave  length  is 
inferior  to  that  with  visible  light. 

Injection,  see  Microinjection.  Perfusion 
of  blood  vessels  and  Neutral  Red 
method  of  staining  pancreas  by  vascular 
injection. 

Innervation,  determination  by  dissection 
(Wharton,  L.  R.,  Anat.  Rec,  1937,  67, 
467-475).  Place  tissue  sheets  or  thin 
organs  on  writing  paper.  Allow  to 
adhere  5-10  min.  Place  in  1  part  gly- 
cerol, 1  part  glacial  acetic  acid  and  6 
parts  1%  aq.  chloroal  hydrate,  18  hrs. 
Glycerol,  1  part ;  Ehrlich's  hematoxylin, 
1  part;  and  1%  aq.  chloral  hydrate,  6 
parts,  24  hrs.  or  more.  If  overstained 
decolorize  in  first  solution  or  in  1% 
hydrochloric  acid  in  70%  alcohol. 
Transfer  to  glycerol  10  days.  Dissect 
under  binocular  microscope  in  fresh 
glycerol.  To  make  permanent  prepara- 
tions, pass  up  to  95%  alcohol,  then 
through  bergamot  oil,  2  parts;  cedar 
oil,  1  part;  and  pure  carbolic  acid  liq- 
uefied by  heat,  1  part,  to  xylol.  Mount 
in  balsam.     See  Nerve  Endings. 

Inoculation  is  to  introduce  materials  into 
the  body  usually  disease  producing  or 
antigenic.  They  are  in  reality  injected 
and  we  speak  of  injecting  a  host  of 
different  substances,  see  in  this  connec- 
tion Microinjection,  Perfusion  and 
Transplantation. 

Insects.  For  whole  mounts  of  large  insects 
Stapp,  P.  and  Cumley,  R.  W.,  Stain 
Techn.,  1936,  11,  105-106,  specify  abs. 
ale,  5-15  days;  95,  85,  70,  and  50%  each 
15  min.  Ale  35%,  30  min.  Equal 
parts  H2O  and  H2O2  -f  trace  NH4OH, 
12-24  hrs.  Ale.  35,  50,  85,  and  95%, 
15  min.  each.  Abs.  ale.  2-3  changes, 
3  days  or  more.  Toluol,  10-21  days. 
Pass  from  thin  to  thick  dammar  and 
mount.  Perhaps  the  simplest  method 
for  small  insects  (fleas,  etc.)  is  simply  to 
drop  them  in  creosote,  U.S. P.  and  after 
24  hrs.  to  mount  them  directly  in  balsam 


INTERFERENCE  COLORS 


164 


IODINE  NUMBER 


(Fox,  1.,  Science,  1942,  96,  478).  Sec- 
tioning is  facilitated  by  methods  de- 
signed to  soften  Chitin,  see  also  Fleas, 
Ticks.  Use  of  fluorescence  microscopy 
in  entomology  (Metcalf,  R.  L.  and  Pat- 
ton,  R.  L.,  Stain  Techn.,  1944,  19,  11- 
27).  In  making  preparations  of  insect 
tissues  one  must  of  course  be  on  the 
lookout  for  infecting  organisms.  A 
well  illustrated  volume,  giving  many 
technical  details,  is  that  of  Paillot,  A. 
L'Infection  Chez  Les  Insectes.  Im- 
primerie  de  Tr^voux,  G.  Patissier,  1933, 
535  pp. 

Interference  Colors  for  daylight,  Newton's 
scale,  see  McClung's  Microscopical 
Technique,  1950,  p.  633. 

Intermitotic  Cells,  see  Cell  Classification. 

Intestinal  Protozoa.  1.  Johnson's  rapid 
iron  hematoxylin  method  (Johnson, 
C.  M.,  Am.  J.  Trop.  Med.,  1935, 15,  551). 
Fix  thin  smears  10  min.  in  Schaudinn's 
fixative  containing  5-10%  glacial  acetic 
acid  (37°-45°C).  Treat  for  5  min.  with 
iodine  in  95%  alcohol  (port  wine  color). 
After  placing  in  70%  alcohol  for  5  min. 
rinse  in  tap  water  1-3  min.  Mordant 
in  4%  aq.  iron  alum  (purple  crystals) 
for  15  min.  Rinse  in  tap  water  1-2 
min.  and  stain  for  10  min.  in  0.5%  aq. 
hematoxylin  (10  cc.  5%  hematoxylin  in 
95%  ale.  plus  90  cc.  aq.  dest.).  Differ- 
entiate in  0.25%  aq.  iron  alum  6-10  min. 
for  flagellates  and  12  min.  for  amoebae. 
After  washing  in  running  water  for  3- 
30  min.,  dehydrate  in  ale,  clear  in  xylol 
and  mount. 

2.  Long  method  of  Heidenhain  (Q.M. 
Geiman  in  Simmons  and  Gentzkow, 
p.  616).  Recommended  for  Balanti- 
dium  coll  and  for  permanent  mounts. 
This  is  practically  the  same  except  for 
longer  mordanting  and   staining.     See 

Iron  Hematoxylin  and  Iron  Hematoxylin 
Single  Stain. 

Intestine.  Difference  in  appearance  of  wall 
when  contracted  and  normally  distended 
(Johnson,  F.  P.,  Am.  J.  Anat.,  1912-13, 
14,  235-250).  Alterations  in  human 
mucosa  from  absorption  of  fat  and  from 
fasting  (Cowdry's  Histology,  pp.  302- 
305).  Effect  of  different  dehydration 
and  clearing  agents  on  intestine  (Ralph, 
P.,  Stain  Techn.,  1938,  13,  9-15).  Ros- 
enberg, L.  E.,  Stain  Techn.,  1940,  15, 
53-56  has  given  an  interesting  account 
of  postmortem  autodigestion.  Mingaz- 
zini  phenomenon  (Macklin,  C.  C.  and 
M.  T.,J.  Anat.,1926,  61,  144-150).  See 
Large  and  Small  Intestines. 

Intracellular  Phase,  see  Chloride. 

Intranuclear  crystals.  Hepatic  cells  of 
dogs.  Determination  of  properties 
(Weatherford,  H.  L.,  and  Trimble, 
H.  C,  Anat.  Rec,  1940,  77,  487-502). 


Intranuclear  Inclusions,  see  Nuclear  In- 
clusions. 

Intravitam  Staining,  the  same  as  Vital 
Staining  but  different  from  Supravital 
Staining,  which  is  upon  or  after,  the 
death  of  the  animal  whose  tissues  are 
stained. 

lodeosin  B,  see  Erythrosin,  bluish. 

Iodides.  Histochemical  localization  not 
feasible  (Gersh,  I.  and  Stielglitz,  E.  J., 
Anat.  Rec,  1933,  56,  185-193).  As  ex- 
plained by  Glick,  p.  34,  a  precipitating 
agent  to  fix  iodide  will  also  precipitate 
protein  and  thus  prevents  its  own  pene- 
tration into  the  tissue. 

Iodine,  detection  of:  1.  Ionized  iodine  in 
the  form  of  iodides.  Stieglitz  (E.,  J. 
Pharm.  and  Exp.  Therap.,  1924,  22, 
89-98)  injects  20  cc.  5%  aq.  lead  nitrate 
intravenously  into  an  animal  to  be  killed 
and  fixes  the  tissue  in  formalin.  In  the 
sections,  iodine  is  found  in  the  form  of 
yellow  crystals  of  lead  iodide.  Methods 
have  been  reviewed  by  Gersh  and  Stie- 
glitz (I.  and  E.  J.,  Anat.  Rec,  1933,  56, 
185-193). 

2.  Methods  for  iodine  in  organic  com- 
bination appear  to  be  unsatisfactory. 
The  whole  subject  of  iodine  has  been 
critically  considered  by  Lison  (p.  111- 
113).  See  Gram's  and  Lugol's  solu- 
tions. 

Iodine,  as  a  stain  is  one  of  the  stains  used 
for  Glycogen  and  Starch  Grains.  It  is 
also  advised  in  the  form  of  Lugol's  solu- 
tion to  bring  out  in  frozen  sections  of 
nervous  tissue  certain  extremely  minute 
bodies  in  the  cytoplasm  and  along  the 
processes  of  nerve  cells  bv  Adamstone, 
F.  B.  and  Taylor,  A.B.,  Science,  1946, 
104,  111.  See  Gram-Pappenheim  stain 
and  Gram  Stain  for  bacteria. 

lodine-Eosin  stain  of  Donaldson,  R.,  Lan- 
cet, 1917,  1,  571  is  highly  recommended 
by  Craig,  p.  45  for  intestinal  amebae 
and  flagellates.  Saturate  one  volume  of 
5%  aq.  potassium  iodide  with  iodine 
crystals  and  mix  with  equal  volume  of 
sat.  aq.  eosin  (yellow  aqueous  eosin). 
Mix  small  drop  with  a  little  feces  on 
slide,  cover  and  examine.  Cysts  of 
amebae  and  flagellates,  yellow  to  green- 
ish yellow  in  red  background;  glycogen 
bodies  within  cysts,  brown. 

Iodine  Green  (CI,  686),  closely  related  to 
methyl  green,  only  used  occasionally. 

Iodine-Iodide  Solution.  This  term  is  em- 
ployed for  almost  any  solution  contain- 
ing iodine  and  iodide  as  Lugol's  and 
Grams. 

Iodine  Number  of  lipids,  see  Schmidt- 
Nielsen,  K.,  C.  rend.  trav.  lab.  Carls- 
berg,  S^r.  Chim.,  1944,  22,  87-96  and 
Kretchmer,  N.,  Holman,  R.  T.  and 
Burr,  G.  O.,  Arch.  Biochem.,  1946,  10, 
101-105. 


IODINE  VIOLET 


165 


IRON 


Iodine  Violet,  see  Hofmann's  Violet. 

Iris  Blue,  see  Resorcin  Blue. 

Iris  Violet,  see  Amethyst  Violet. 

Iron  occurs  in  tissues  "masked"  in  organic 
compounds  which  are  not  ionisable  and 
free  in  inorganic  compounds  which  are 
ionisable  into  ferric  and  ferrous  salts. 

1.  Macallum's  hematoxylin  method 
depends  upon  the  formation  of  a  blue 
black  iron  hematoxylinate.  The  tissue 
is  fixed  in  95%  alcohol  24-48  hours, 
dehydrated,  cleared,  imbedded  in  paraf- 
fin and  the  sections  are  passed  down  to 
distilled  water.  Contact  with  iron  is 
reduced  to  a  minimum.  The  microtome 
knife  must  be  free  of  rust.  Treat  sec- 
tions with  a  freshly  prepared  straw 
yellow  0.5%  aqueous  solution  of  hema- 
toxylin which  must  be  of  the  highest 
purity.  Inorganic  iron  produces  the 
blue-black  compound  which  is  rela- 
tively insoluble.  Dehydrate,  clear  and 
mount  in  balsam  in  the  usual  way. 

The  technique  for  organic  iron  is 
more  difficult  because  it  must  be  un- 
masked before  it  will  react  in  this  way. 
The  best  account  is  Nicholson,  F.  M., 
J.  Comp.  Neurol.,  1923,  36,  37-87.  In 
studying  the  cytoplasmic  iron  contain- 
ing proteins  of  nerve  cells  of  the  medulla 
of  rats,  he  fixed  in  95%  alcohol  48  hours ; 
dehydrated  in  absolute  alcohol  2-5 
hours;  cleared  in  cedarwood  oil  until 
transparent;  imbedded  in  paraffin  (2 
changes)  and  cut  sections  7/1.  After 
being  deparaffinized,  the  sections  were 
passed  through  alcohols  to  4%  pure 
sulphuric  acid  in  95%  alcohol  held  at 
60 °C.  for  5-60  minutes.  This  liberated 
the  iron.  The  sections  were  washed  in 
95%  alcohol ;  passed  down  through 
graded  alcohols  to  aq.  dest.,  and  placed 
in  freshly  prepared  0.5%  aqueous 
hematoxylin,  1-5  minutes  in  which  the 
blue-black  hematoxylinate  forms .  Then 
wash  in  aq.  dest.  (not  tap  water). 
Counterstain  in  dilute  alcohol  erythro- 
sin  and  mount  as  usual.  As  a  check  the 
nuclear  chromatin  of  sections  not  treated 
with  the  acid  alcohol  should  not  be 
colored  black  by  this  hematoxylin 
solution .  Difficulty  may  be  experienced 
because  the  color  of  the  unmasked  iron 
is  faint.  The  reaction  is  a  chemical  one 
of  great  delicacy  and  requires  practice. 
Pancreatic  acinous  cells  also  afford 
favorable  material.  Look  for  cyto- 
plasmic iron  in  the  poles  distant  from 
the  lumen  where  thechromidial  material, 
which  resembles  the  Nissl  bodies,  is 
most  concentrated. 

2.  Prussian  blue  reaction.  Prepare 
sections  in  the  same  way,  deparaffinize 
and  test  as  described  in  Lee  (p.  291). 
For  ferric  salts  of  inorganic  iron  wash  in 
aq.  dest.,  2%  aqueous  potassium  ferro- 


cyanide,  3-15  minutes;  Prussian  blue 
is  formed,  wash,  dehydrate,  clear  and 
mount.  For  ferrous  salts  substitute 
ferricyanide  for  ferrocyanide  in  the  test. 
For  both  use  equal  parts  of  ferrocyanide 
and  ferricyanide.  When  the  iron  is 
organic  it  is  unmasked  by  treating  the 
sections  with  3%  pure  nitric  acid  in  95% 
alcohol  for  24-36  hours  at  room  tem- 
perature or  at  35 °C.  if  necessary.  Wash 
in  pure  90%  alcohol  and  in  aq.  dest. 
Place  in  equal  parts  freshly  made  of 
1.5%  aqueous  potassium  ferrocyanide 
and  0.5%  aqueous  hydrochloric  acid  for 
not  more  than  5  minutes.  Wash  well 
in  aq.  dest.,  colored  with  eosin  or 
safranin,  dehydrate,  clear  and  mount. 
Hemosiderin  gives  Prussian  blue 
reaction  for  inorganic  iron.  The  iron  in 
hemoglobin  is  not  unmasked  by  these 
acid  alcohols.  Brown,  W.  H.,  J.  Exper. 
Med.,  1911,  13,  477-485,  devised  special 
methods  for  its  demonstration.  Test- 
ing for  iron  in  association  with  calcium 
particularly  in  bone  is  critically  de- 
scribed by  Cameron,  G.  R.,  J.  Path, 
and  Bact.,  1930,  33,  929-955.  He  em- 
phasizes the  fact  that  exposure  of  tissues 
and  fluids  to  dust  in  a  city  like  London 
is  an  important  source  of  error. 

3.  Microincineration  yields  a  mineral 
residue  that  contains  iron  originally 
both  organic  and  inorganic.  Color  of 
the  iron  oxides,  viewed  in  the  dark  field, 
varies  according  to  Policard  (C.  rend. 
Acad.  d.  sc,  1923, 176,  1187)  from  yellow 
to  deep  red.  He  suggests  that  perhaps 
the  yellow  to  brown  ash  is  of  organic 
iron  and  the  red  ash  is  of  free  iron.  See 
also  Marza,  V.  D.,  Marza,  E.,  and 
Chiosa,  L.  Bull,  d'hist.  Appliq.,  1932, 
9,  213.  Scott  (McClung,  p.  758)  warns 
against  confusion  with  carbon. 

4.  Hydroxyquinoline  test  (Thomas, 
J.  A.  and  Lavollay,  J.,  Bull.  d'Hist. 
Appl.,  1935,  12,  400^402).  Fix  in  alco- 
hol, trichloracetic  acid  or  neutral  forma- 
lin. Avoid  formol  with  alkaline  water 
and  fixatives  containing  chromium. 
Make  up  reagent  by  dissolving  2.5  gra. 
8-hydroxyquinoline  in  4  cc.  pure  acetic 
acid  warming  gently.  Add  quickly  aq. 
dest.  to  make  100  cc.  Filter.  Wash 
sections  (or  smears  or  cultures)  well  in 
neutral  aq.  dest.  Then  add  few  drops 
of  reagent  5-15  min.  Pour  off  reagent. 
Add  to  preparation  1  drop  25%  aq. 
ammonia  which  produces  a  ppt.  Wash 
in  a  stream  of  neutral  aq.  dest.  If 
large  crystals  remain  wash  more  ener- 
getically. Stain  nuclei  with  lithium 
carmine.  Examine  in  neutral  aq.  dest. 
or  dehydrate  in  terpinol  and  mount  in 
vaseline  oil.  Iron,  green  black;  nuclei, 
red.  Recommended  for  localization  of 
iron  in  granules  of  vitellus,  in  red  blood 


IRON  HEMATOXYLIN 


166 


ISAMINE  BLUE 


cells,  and  in  connection  with  micro- 
incineration. Said  to  be  better  than 
Prussian  Blue  reaction  for  iron. 

5.  Dinitrosoresorcinol  (Humphrey, 
H.  A.,  Arch.  Path.,  1935,  20,  256-258). 
Treat  paraffin  sections  of  formalin  fixed 
tissue  with  30%  aq.  ammonium  sulphide, 
1  min.  Rinse  in  water  and  immerse  in 
sat.  aq.  dinitrosoresorcinol  (Eastman) 
6-20  hrs.  A  counterstain  can  be  em- 
ployed. Humphrey  does  not  say  which. 
1%  eosin  in  50%  alcohol  should  be  satis- 
factory because  the  iron  containing  com- 
pounds such  as  hemosiderin  are  colored 
green.  Wash,  dehydrate,  clear  and 
mount. 

Intravenous  injections  of  colloidal 
solutions  of  iron  in  rabbits  are  described 
by  Duhamel,  B.  G.,  C.  rend.  Soc.  de 
Biol.,  1919,  82,  724-726. 

6.  A  clinical  demonstration  of  iron  in 
the  skin  in  hemochromatosis  involves 
intradermal  injection  of  equal  parts  of 
sterile  0  5%  aq.  potassium  ferrocyanide 
and  1/100  N  hydrochloric  acid.  This 
produces  a  wheal  which  turns  dark  blue 
in  5  min.  A  positive  reaction  can  even 
be  obtained  after  death.  (Fishback, 
H.  R.,  J.  Lab.  &  Clin.  Med.  1939-40, 
25,  98-99). 

In  special  cases,  as  in  the  analysis  of 
small  amounts  of  epidermis,  resort  may 
be  had  to  a  quantitative  polarographic 
determination  of  iron,  see  Carruthers, 
C.  and  SuntzefT,  V.,  J.  Nat.  Cancer 
Inst.,  1942,3,  217-220. 

Kirk,  P.  L.  and  Bentley,  J.  T., 
Mikrochemie,  1936,  21,  250^259  advo- 
cate a  titrimetric  method  for  iron.  The 
difficulty  of  interference  by  small 
amounts  of  copper  has  been  overcome 
by  Ramsav,  W.  N.  M.,  Biochem.  J., 
1944,  38,  467-469.  Click  (p.  277)  is  of 
the  opinion  that  such  methods  may  be 
adapted  for  use  with  the  quantities  of 
material  employed  in  histochemical 
work. 
Iron  Hematoxylin  of  Heidenhain  is  one  of 
the  standard  stains.  It  will  give  excel- 
lent results  after  almost  any  good  fixa- 
tion. Zenker's  fluid  and  formalin- 
Zenker  are  suggested.  Bring  paraffin 
sections  down  to  aq.  dest.  Mordant  in 
5%  aq.  iron  ammonium  sulphate  (iron 
alum,  light  violet  colored  crystals,  dis- 
card the  brownish  material  accompany- 
ing them)  12-24  hrs.  Rinse  quickly  in 
aq.  dest.  Transfer  to  1%  aq.  hema- 
toxylin (made  up  by  diluting  1  cc.  sat. 
sol.  hematoxylin  in  abs.  ale.  with  99  cc. 
aq.  dest.)  for  12-24  hrs.  Differentiate 
under  microscope  in  1%  aq.  iron  alum. 
Wash  thoroughly  in  tap  water.  Many 
counterstains  can  then  be  used  such  as 
1%  aq.  Bordeaux  red,  orange  G.,  acid 
fuchsin,  acridine  red,  or  Mucicarmine. 


Dehydrate,  clear  and  mount.  Nuclei 
dense  blue-black  in  background  of  color 
selected.  See  Centrosomes,  Nuclei, 
Regaud's  Method  for  mitochondria. 

1.  Koneff,  A.  A.,  Anat.  Rec,  1936, 
66,  173-179  advises  use  with  anilin  blue. 
Mordant  sections  5-10  min.  in  5% 
aq.  iron  ammonium  sulphate.  Rinse 
quickly  in  aq.  dest.,  stain  3-15  min.  in 
Harris'  hematoxylin.  Rinse  again  in 
aq.  dest.  and  stain  in:  anilin  blue 
(Griibler)  0.1  gm. ;  oxalic  acid,  2  gm. ; 
phosphomolybdic  acid,  15  gm.  and  aq. 
dest.  300  cc.  Wash  in  aq.  dest.,  differ- 
entiate in  alcohol,  dehydrate  (2  changes 
of  absolute),  clear  in  xylol  and  mount  in 
balsam.  If  euperal  is  used  for  mounting 
omit  the  xylol.  Nuclei,  violet-brown; 
cytoplasm,  light  brown;  erythrocytes, 
dark  violet;  myelin  and  muscle  brown; 
elastic    fibers,    reddish    brown    to    red. 

2.  Lillie,  R.  D.  and  Earle,  W.  R., 
Am.  J.  Path.,  1939,  15,  765-770  recom- 
mend employment  of  a  hematoxylin 
containing  ferric  and  ferrous  iron:  (A). 
Ferric  ammonium  sulphate,  violet  crys- 
tals, 15  gm.;  ferrous  sulphate,  15  gm.; 
aq.  dest.,  100  cc.  (B).  Hematoxylin, 
1  gm.;  95%  alcohol,  50  cc,  glycerin, 
C.P.,  50  cc.  Mix  A  and  B  in  equal 
quantities  before  using.  For  best 
general  discussion  of  iron  hematoxylin, 
see  Lillie,  p.  58. 

Iron  Hematoxylin  Single  Stain — Written  by 
Morris  Goldman,  Dept.  of  Parasitology, 
School  of  Hygiene  and  Public  Health, 
Johns  Hopkins  University,  Baltimore. 
January  29,  1951 — This  stain  is  useful 
for  the  purpose  of  diagnosing  intestinal 
protozoa  occurring  in  fecal  smears.  It 
is  not  intended  to  replace  the  longer  and 
more  precise  iron  hematoxylin  methods. 
The  stain  is  prepared  from  the  following 
relatively  stable  solutions:  Solution  A: 
1%  hematoxylin  in  95%  ale.  (best  pre- 
pared from  a  stock  10%  ale.  solution  of 
hematoxylin).  Solution  B:  NH^Fe- 
(804)2- I2H2O,  4.0  gms.,  glacial  acetic 
acid,  1.0  ml.,  concentrated  H2SO4,  0.12 
ml.,  and  aq.  dest.  to  make  100  ml.  Mix 
equal  parts  of  Solution  A  and  B,  filter 
after  several  hours  and  use.  Staining 
time  varies  from  30  sec.  when  stain  is 
fresh  to  3  min.  after  24  hrs.  Discard 
stain  after  3  days.  Fecal  smears  are 
fixed  in  Schaudinn's,  passed  through 
iodine  alcohol  to  50%  alcohol,  stained, 
washed  in  running  tap  water  5  minutes, 
dehydrated  and  mounted.  The  stock 
solutions  used  in  this  technique  may 
also  be  used  in  the  Heidenhain  iron 
hematoxylin  procedure. 

Iron  Pigments,  see  Berlin  and  Turnbull  blue 
reactions. 

Iron,    Radioactive.    See   Erythrocytes. 

Isamine  Blue  is  described  by  Conn  (p.  137) 


ISLETS  OF  LANGERHANS 


167 


JALOWY 


as  a  sulfonated  naphthyl-rosanilin  or 
naphthyl-pararosanilin.  He  questions 
the  synonym  (alkali  blue  XG)  given  in 
the  Colour  Index.  This  acid  has  been 
much  used  as  a  Vital  Stain  in  European 
laboratories.  It  is  not  made  in  the 
United  States. 
Islets  of  Langerhans  of  the  pancreas .  There 
are  many  techniques  for  the  study  of 
these  cellular  masses. 

1.  To  study  in  the  living  state  the 
method  employed  by  O'Leary,  J.  L., 
Anat.  Rec,  1930,  45,  27-58  is  recom- 
mended. It  consists  essentially  of 
partly  withdrawing  the  pancreas  from 
a  mouse  and  of  mounting  it  in  such  a 
way  that  a  thin  film  of  tissue  can  be 
closely  examined  with  circulation  still 
active.  The  islet  cells  can  be  studied 
with  oil  immersion  lenses  and  the 
changes  in  them  on  the  injection  of 
insulin  noted. 

2.  To  obtain  an  idea  of  the  distribu- 
tion, number  and  size  of  the  islets 
supravital  staining  with  Neutral  Red 
or  Janus  Green  is  indicated,  which  see. 

3.  To  stain  the  cell  types  specifically 
Neutral  Gentian  and  other  stains 
advised  by  Lane,  Bensley  and  their 
followers  are  available.  The  Azan  Stain 
suggested  by  Bloom,  W.,  Anat.  Rec, 
1931,  49,  363-371  (see  his  beautifully 
colored  plate),  has  been  further  investi- 
gated by  Gomori,  G.,  Anat.  Rec,  1939, 
74,  439-459  whose  technique  abbreviated 
is  as  follows :  Fix  thin  slices  of  pancreas 
in  Bouin's  fluid  8-10  hrs.  Wash  in  aq. 
dest.  Imbed  in  paraffin  and  cut  ifi  sec- 
tions. Stain  45-60  min.  at  56°C.  in 
azocarmine.  (To  make  dissolve  0.1% 
azocarmine  in  aq.  dest.  Boil  about  5 
min.  Cool  and  add  1.0  cc  glacial  acetic 
acid  to  each  50  cc.  solution.  Before  use 
filter  at  60 °C.  Stain  will  keep  for 
months.)  Rinse  quickly  inaq.  dest.  and 
blot.  Destain  in  90%  alcohol  containing 
1%  aniline  oil  until  acinous  tissue  is  al- 
most wholly  decolorized  and  B  cells 
show  red  against  pink  background  of  A 
cells.  Rinse  briefly  and  treat  with  5% 
aq.  iron  alum  for  5  min.  or  more.  Rinse 
again  and  stain  2-20  min.  in  the  usual 
mixture  (anilin  blue,  0.5  gm.;  orange 
G,  2.0 gm.;  +  aq.  dest.  to  make  100  cc.) 
diluted  with  2-3  times  its  volume  of  aq. 
dest.  until  under  the  microscope  colla- 
genic  tissue  becomes  deep  blue.  Rinse 
and  blot.  Differentiate  and  dehydrate 
in  absolute  alcohol,  clear  in  xylol  and 
mount  in  balsam.  Cytoplasm  of  A  cells 
rich  orange  yellow,  of  B  cells  fiery  red 
and  of  D  cells  sky  blue.  The  author 
states  that  by  first  staining  with  Bens- 
ley's  neutral  gentian,  decolorizing  and 
restaining  by  above  Azan  method  it  can 


be  seen  that  there  is  no  gradation  be- 
tween A  and  B  cells. 

Isoelectric  Points  of  cellular  structures. 
Methods  for  their  determination  at  con- 
trolled pll's  by  intensity  of  staining 
have  been  critically  evaluated  by  Levine, 
N.  D.,  Stain  Techn.,  1940,  15,  91-112. 
His  conclusion  is  that  no  true  isoelectric 
points  have  yet  been  established  for 
nucleus,  cytoplasm  or  other  tissue  ele- 
ments by  tliese  techniques.  See  re- 
ticulo-endothelial  cells  (Fautrez,  J., 
Bull.  d'Hist.  Appl.,  1936,  13,  202-200). 

Isohematein,  as  a  biological  stain  (Cole, 
E.  C,  Stain  Techn.,  1931,  6,  93-96). 
Greater  tinctorial  power  than  hematox- 
ylin but  less  selective. 

Isomerase  with  Aldolase  are  known  as 
Zymohexase. 

Isopentane  recommended  by  Hoerr,  N.  L., 
Anat.  Rec,  1936,  65,  293  for  freezing  in 
the  Altmann-Gersh  technique. 

Isopropanal  in  combination  as  a  new  fixative 
for  animal  tissues  which  also  dehydrates 
(Cleverdon,  M.  A.,  Science,  1943,  97, 
168).  Isopropanal,  55  cc;  picric  acid, 
5  gms.,  acetone,  30  cc;  glacial  acetic 
acid,  55  cc;  formalin  (40%  formalde- 
hyde CP),  5  cc  Fix  2  hrs.— 4  days  de- 
pending on  size.  Store  in  70%  iso- 
propanal or  imbed  in  paraffin  after  first 
washing  in  2  changes  nearly  absolute 
isopropanal.  Remove  picric  acid  from 
mounted  sections  just  before  staining 
with  1.5%  ammonium  hydroxide  in  95% 
alcohol. 

Isopropyl  Alcohol.  Has  been  recommended 
as  a  substitute  for  ethyl  alcohol  since  it 
mixes  with  water  and  xylol.  It  is  said 
to  be  less  hardening  than  ethyl  alcohol 
(Bradbury,  O.  C,  Science,  1931,74,225) 
but  it  is  more  expensive.  See  Herman, 
C.  M.,  J.  Lab.  &  Clin.  Med.,  1941,26, 
1788. 

Isorubin,  see  New  Fuchsin, 

Iso-Safrol  is  obviously  an  isomer  of  safrol 
which  is  given  as  3,4-methylene-dioxy- 
allylbenzene  in  the  Merck  Index.  Iso- 
saf  role  is  listed  among  Eastman's  organic 
chemicals.  It  is  sometimes  recom- 
mended as  a  partly  dehydrating  and 
clearing  agent  (Silver  Citrate  injection 
of  blood  vessels,  etc)  but  in  all  likeli- 
hood other  clearing  agents  can  be  used 
as  substitutes. 

Isospora,  see  Coccidia. 

Jacobson's  Organ,  innervation,  Bellairs, 
A.,  J.  Anat.,  1942,  76,  167-177. 

Jalowy  modification  of  Hortega  method  for 
the  skin  (Jalowy,  B.,  Zeit.  f.  Zellf.  u. 
Mikr.  Anat.,  1937,  27,  667-690).  To 
make  reagent  wash  ppt.,  formed  by 
adding  20  drops  40%  aq.  NaOH  to 20  cc. 
10%  aq.  silver  nitrate,  10  times  with 
aq.  dest.  Suspend  ppt.  in  20  cc.  aq.  dest. 
Add  ammonia  drop  by  drop  till  it  dis- 


JANSSEX'S  IRON  HEMATOXYLIN 


168 


JOHNSON'S  NEUTRAL  RED 


solves.  Add  100  cc.  aq.  dest.  and  store 
in  dark.  Deparaffinize  sections  of  tissue 
fixed  1-2  days  in  neutral  formalin. 
Treat  with  above  reagent  5-30  miu.  at 
30°C.  Rinse  in  aq.  dest.  and  in  ammonia 
water.  After  treating  with  1  part  neu- 
tral formalin  to  4  of  aq.  dest.  wash  in 
running  water,  dehydrate,  clear  and 
mount  in  balsam.  Collagen,  yellow  to 
brownish  yellow;  reticular  fibers,  black. 
Janssen's  Iron  Hematoxylin  recommended 
in  place  of  Weigert's  acid  iron  chloride, 
hemato.xylin  (Lillie,  R.D.  and  Earle 
W.R.  Stain  Technol.,  1939,  14,  53-54). 
Janus  Blue  can  be  used  in  exactly  the  same 
ways  as  Janus  green  and  with  equal 
success. 
Janus  Dyes.  Named  after  the  God,  Janus 
with  two  faces  since  they  often  exhibit 
two  colors.  Their  chemistry  and  use  in 
histology  is  described  by  Cowdry,  E.  V. 
Contrib.  to  Embryol.,  Carnegie  Inst. 
Washington,  1918,  No.  25,  pp.  39-148. 

Janus  green  (formerly  made  by  Grub- 
ler)  is  safraninazodimethylanilinchlo- 
ride.  This  is  useless  for  staining 
mitochondria. 

Janus  green  C  (Hoechst)  is  dimethyl 
safraninazodimethyl  anilinchloride.  This 
likewise    is    useless    for  mitochondria. 

Janus  green  B  (Hoechst)  is  diethyl- 
safraninazodimethylanilinchloride.  This 
is  the  most  specific  stain  for  mito- 
chondria and  is  now  supplied  by  many 
companies  both  as  Janus  Green  B  and 
simply  as  Janus  Green. 

Janus  blue  G  and  R  (Hoechst)  is 
diethylsafranin-B-naphthol  and  stains 
mitochondria  as  well  as  Janus  Green  B. 
The  marks  G  and  R  indicate  differences 
in  method  of  manufacture  not  different 
dyes. 

Janus  black  D,  I,  II  and  0  (Hoechst), 
of  these  Janus  Black  I  is  a  mixture  of  two 
substances  Janus  green  B  and  a  brown 
dye.  It  colors  mitochondria  by  virtue 
of  the  former. 

Janus  gray  B,  BB  (Hoechst)  are  also 
safranin  derivatives  but  useless  for 
mitochondria. 

Janus  yellow  G,  R,  (Hoechst)  likewise 
safranin  derivatives  and  no  good  for 
mitochondria. 

Diethylsafranin  is  a  reduction  product 
of  Janus  green  B.  It  is  a  red  dye  which 
colors  mitochondria  specifically  but  not 
very  strongly. 
Janus  Green  B  (Diazingriin)  is  diethyl 
safraninazodimethylanilinchloride.  Ja- 
nus green  now  sold  without  the  qualifica- 
tion B  is  usually  the  same  substance 
because  it  has  become  well  known  that 
the  dye  required  must  have  the  composi- 
tion indicated.  Owing  to  its  toxicity 
Janus  green  cannot  be  injected  into 
living  animals  like  trypan  blue  and  other 


"vital"  stains.  It  is  employed  as  a 
supravital  stain  by  simply  immersing 
tissues  in  it  or  better  by  its  injection 
into  the  vessels  of  a  freshly  killed  animal 
the  individual  cells  of  which  remain  for 
some  time  alive.  Janus  green  is  the 
best  supravital  stain  for  mitochondria. 
Janus  green  is  also  very  useful  for  stain- 
ing the  islets  of  Langerhans  of  the 
pancreas  and  the  renal  glomeruli  of  the 
kidney  when  injected  intravascularly, 
see  Neutral  Red.  Both  islets  and 
glomeruli  are  colored  deep  bluish  green 
against  a  background  at  first  colorless, 
or  faintly  green,  and  changing  to  pink 
by  reduction  of  the  dye  to  diethyl- 
safranin. This  permits  the  counting  of 
islets  and  glomeruli  in  pieces  of  tissue 
mounted  in  salt  solution  and  observed 
at  low  magnification.  When  the  oxygen 
is  further  consumed  by  the  cells  the  dye 
is  reduced  to  a  second  colorless  leucobase. 
It  is  therefore  an  oxidation -reduction 
indicator  as  well  as  a  specific  stain  for 
mitochondria.  See  Neutral  Red-Janus 
Green  stain. 

Janus  Red  B  (CI,  266),  a  basic  disazo  dye 
of  light  fastness  4.  Action  on  paren- 
chyma described   (Emig,  p.  36). 

Jaws,  see  Teeth  and 

Jenner-Giemsa  method  of  Pappenheim  (see 
May-Giemsa). 

Jenner's  Stain  for  Leishmania  as  described 
by  Craig,  p.  146:  To  make,  mix  equal 
parts  1.2%  water  soluble  eosin  (Grub- 
ler  or  NAC)  in  acid  free  aq.  dest.  and 
1%  aq.  medicinal  methylene  blue  in  a 
flask.  Shake  thoroughly  and  let  stand 
at  room  temperature  24  hrs.  Collect 
ppt.  on  small  filter  paper  and  wash  with 
aq.  dest.  till  filtrate  is  almost  colorless. 
Dry  ppt.  and  store  in  dark  at  room 
temperature.  Dissolve  0.5  gm.  ppt.  in 
100  cc.  pure  methyl  alcohol  (Merck's 
Reagent).  Cover  smears  with  this  1-2 
min.  Then  add  aq.  dest.  drop  by  drop 
till  metallic  sheen  forms  on  surface. 
Leave  5-15  min.  longer  as  desired  for 
intensity.  Method  said  by  Craig  to  be 
less  reliable  than  Giemsa,  Leishman  or 
Wright  techniques. 

Johnson's  Neutral  Red  stain  for  Nissl 
bodies  (Addison  in  McClung,  p.  450). 
Ripen  1%  aq.  neutral  red  1-4  years. 
Dilute  to  0.25-0.5%  before  using.  Differ- 
entiate and  dehydrate  in  the  usual  way. 
Clear  in  1  part  xylol  +  2-3  parts  castor 
oil.  Gives  good  results  in  thick  sections 
(50m)  and  can  be  employed  after  silver 
methods  on  tissues  fixed  in  alcohol  or 
formalin. 

Kirkman,  I.  J.,  Anat.  Rec,  1932,  51, 
323-326  used  the  following  unripened 
stain  after  Bouin  and  formalin  fixatives : 
neutral  red  (Coleman  &  Bell),  1  gm. ;  aq. 
dest.,  500  cc,  1%  aq.  glacial  acetic  acid, 


JOINTS 


169 


KERATOHYALIN  GRANULES 


2  cc.  10-20  min.  is  sufficient  for  counter- 
staining  Weigert-Pal  preparations. 
Then  rinse  in  aq.  dest.,  differentiate  in 
95%  alcohol,  dehydrate  in  absolute, clear 
and  mount. 

Joints.  Meniscus  (Raszela,  F.,  Bull. 
d'Hist.  Appl.,  1938,  15,  186-210). 

Jolly  Bodies.  These  structures  are  nuclear 
remnants  in  erythrocytes  that  are 
strongly  colored  by  Giemsa's  Stain. 

Jores'  Solution,  see  under  Color  Preserva- 
tion of  gross    specimens. 

Kabunylin,  a  dye  extracted  from  beetroot. 
Said  to  be  good  for  use  with  picrofuchsin 
(Fuse  and  Hino,  Arb.  Anat.  Inst,  zu 
Sendai,  1937,20,   111-113). 

Kaiserling's  Solution,  see  under  Color 
Preservation  of  gross  specimens. 

Kaliichrom,  a  combination  of  cresyl  violet 
and  auramin  recommended  for  both 
plant  and  animal  tissues  (Kisser,  J., 
Mikr.  f.  Naturfreunde,  1931,  9,  95). 

Kardos-Pappenheim  modification  of  Giem- 
sa's stain  (Kardos,  E.,  Folia  haematol., 
Archiv.,  1911,  12,  39).  To  make  the 
methyl  green-orange  stain  mix  2%  aq. 
orange  G.  with  concentrated  aq.  methyl 
green.  Filter,  dry  the  ppt.  and  dis- 
solve in  methyl  alcohol.  Shake  well 
together  5  drops  methyl  green-orange,  10 
drops  of  Giemsa's  stock  solution  and  15 
cc.  aq.  dest.  The  fluid  under  the  foam 
is  used  for  staining.  First  fix  and  stain 
the  blood  smear  with  May-Griinwald 
mixture  3  minutes ;  add  equal  volume  aq. 
dest.,  1  minute;  pour  off  and  add  the 
methyl  green-orange  15  minutes;  wash 
quickly  in  water  and  blot  dry. 

Karo,  white  corn  syrup  (Corn  Products  Co.) 
is  a  useful  medium  for  mounting  whole 
insects  because  they  can  be  transferred 
to  it  directly  from  water  or  weak  alcohol 
and  clearing  is  unnecessary  (Patrick, 
R.,  Science,  1936,  33,  85-86). 

Karotin,  see  Carotin. 

Karyosome  (G.  Karyon  nut,  nucleus  + 
soma,  body).  A  basic  staining  or  chro- 
matin-nucleolus, in  contrast  to  a  plasmo- 
some,  generally  more  numerous,  smaller 
and  of  less  regular  shape  often  called  a 
net-knot. 

Kerasin  is  a  Cerebroside. 

Keratin,  a  scleroprotein  contained  in  hair, 
nails,  horns,  epidermis,  etc.  There  are 
apparently  two  sorts.  Their  chemistry 
is  discussed  by  Giroud,  A.,  Bulliard,  H. 
and  Lebond,  C.  P.,  Bull.  d'Hist.  Appl., 
1934,  11,  365-373.  See  Orange  II,  Oral 
Mucosa. 

Keratohyalin  Granules,  separation  and 
analysis — Written  by  Donald  L. 
Opdyke,  Dept.  of  Anatomy,  Washing- 
ton University  Medical  School,  St. 
Louis,  Mo.  November  8,  1951 — Highly 
refractile  cytoplasmic  granules  of  vary- 
ing size  found  in  the  cells  of  the  stratum 


granulosum  of  stratified  squamous 
epithelia.  They  were  first  described  by 
Langerhans  in  1882  and  were  called 
"keratohyalin"  by  Waldeyer  who  as- 
cribed to  them  a  constant  relation  to  the 
process  of  cornification. 

The  tinctorial  properties  of  these 
granules  may  be  summarized  as  follows: 
They  stain  beautifully  with  picro- 
carmine,  have  an  intense  affinity  for  all 
of  the  hematoxylin  stains  and  stain  as 
well  with  unoxidized  hematoxylin  as 
with  the  aged  stain.  They  stain  meta- 
chromatically  with  toluidine  blue,  and 
after  staining  with  crystal  violet  and 
iodine  they  resist  destaining  by  acetone. 
They  do  not  stain  by  the  Feulgen  pro- 
cedure but  give  beautifully  intense 
reactions  to  the  Bauer,  Iodine,  and 
Best's  Carmine  technics  for  glycogen. 
The  reactions  with  the  P.A.S.  method  of 
McManus  give  varying  results.  The 
granules  are  not  tannophil;  do  not 
osmicate  or  stain  with  the  Sudan  dyes. 
They  are  argentophil. 

Microincineration  studies  reveal  large 
concentrations  of  mineral  in  the 
granules  as  demonstrated  by  quantities 
of  the  white  ash  typical  of  calcium 
and/or  magnesium.  These  ashed  sec- 
tions, as  well  as  unashed  sections  show 
intense  reactions  of  the  granules  to  the 
gallamine  blue  staining  method  of 
Stock,  A.,  J.  Roy.  Micr.  Soc,  Series 
III,  1949,  69,  20-24. 

Enzyme  studies  have  resulted  in  dis- 
agreement in  the  literature  with  respect 
to  ribonuclease  digestibility,  but  the 
granules  are  definitely  not  totally  digest- 
ible in  ribonuclease,  hyaluronidase, 
crystalline  trypsin  or  salivary  enzymes. 
They  are  readily  dissolved  by  elastase. 

The  most  striking  features  of  these 
granules,  both  tinctorially  and  histo- 
chemically,  are  their  similarities  to 
elastic  fibers:  These  common  features 
include  their  tinctorial  reactions  to 
Verhoeff's  procedure,  congo  red,  and 
orcein;  their  affinity  for  mineralization 
with  calcium  salts;  their  solubility  in 
the  elastolytic  enzyme  of  Bal6  and 
Banga   (See  Elastic  Fibers). 

References  for  the  above  data  are: 
Lansing,  A.  I.,  and  Opdvke,  D.  L., 
Anat.  Rec,  1950,  107,  379  398;  Smith, 
C,  and  Parkhurst,  H.  T.,  Anat.  Rec, 
1949,  103,  649-674;  and  unpublished 
work  of  D.  L.  Opdyke. 

The  origin  of  these  granules  is  much 
debated.  Stam,  Von  F.  C,  Acta 
Dermato-Venereol.,  1951,  31,  407-411 
believes  them  to  be  extruded  nucleoli. 
Favre  of  the  Bacteriological  Institute  of 
Lyon  is  of  the  opinion  that  they  are 
formed  by  the  spiral  shaped  mito- 
chondria of  the  basal  layer  of  the  epi- 


KERMES 


170 


KIDNEY 


dermis  (Favre,  M.  Ann.  de  Dermat.  et 
Syph.,  1950, 10  (3) ,  241-262) .  The  litera- 
ture 1886  to  date  includes  reports  of  the 
origin  of  keratohyalin  granules  from 
nuclear  substance,  intercellular  fibrils, 
and  cytoplasm. 

The  particulate  nature  of  the  granules 
has  been  demonstrated  recently  by  their 
isolation  from  epidermis  (D.  L. 
Opdyke).  Epidermis  is  separated  by 
the  heat  method  of  Baumberger,  J.  P., 
Suntzeff,  v.,  and  Cowdry,  E.  V.,  J. 
Nat.  Cancer  Inst.,  1942, 2,  413-423.  The 
sole  of  the  foot  offers  an  ideal  place  to 
obtain  keratohyalin  granules. 

After  separating  the  epidermis  from 
the  dermal  layers,  the  proximal  side  of 
the  epidermal  sheet  is  scraped  with  a 
knife,  removing  all  of  the  epidermis  in 
fine  shavings  down  to  the  highly  trans- 
parent, thick  stratum  corneum.  The 
shavings  are  then  homogenized  in  a 
Waring  Blendor  until  no  cells  remain 
intact.  This  homogenate  is  made  in  a 
0.85%  NaCl  solution  of  pH  7.3  to  which 
a  crystal  of  thymol  is  added  to  minimize 
bacterial  contamination.  All  proce- 
dures are  carried  out  in  a  cold  room. 
The  process  of  cell  fractionation  in  the 
micro  cup  of  the  Waring  Blendor  re- 
quires about  45  min.  If  2%  citric  acid 
is  employed  as  the  medium,  this  time 
may  be  reduced  to  15  min. 

The  components  of  the  suspension  are 
then  separated  by  differential  centrif- 
ugation.  Nuclei  and  whole  cells  are 
found  to  sediment  at  2.5  to  3  Kg/g. 
The  keratohyalin  granules  sediment  at 
25.2  Kg/g.  after  12  min.  of  centrifuga- 
tion.  These  can  be  resuspended  in 
fresh  saline,  washed,  and  reprecipi- 
tated.  They  can  be  distinguished  from 
mitochondria  by  their  tinctorial 
properties. 
Kermes.  This  scarlet  dye  was  known  in 
Egypt  and  farther  East  at  a  very  early 
date.  Kermes  is  the  Armenian  term 
for  a  "little  worm",  variously  identi- 
fied as  Coccus  arborum  and  Coccus  ilicis. 
Moses  referred  to  it  as  "Fola"  and 
"Fola  shami".  Remember  the  promise 
of  Jehovah:  "Though  your  sins  be  as 
scarlet  (Fola)  they  shall  be  as  white  as 
snow;  though  they  be  red  as  crimson 
(Fola  shami),  they  shall  be  as  wool". 
So  valuable  was  Kermes  that  after  the 
subjugation  of  Spain  by  the  Romans 
the  people  were  made  to  pay  half  of  the 
tribute  in  Kermes.  At  about  1640  a 
Dutch  chemist  discovered  the  similarity 
of  this  dye  to  cochineal.  Its  history 
affords  interesting  reading  (Leggett, 
W.  F.  Ancient  and  Medieval  Dyes. 
Brooklyn:  Chemical  Publishing  Co. 
Inc.,  1944,  95  pp.). 
Kidney — Written  by  Jean  Oliver,  Dept.  of 


Pathology,  State  University  of  New 
York,  Brooklyn  2,  N.  Y.,  September  4, 
1951 — Techniques  for  the  general 
demonstration  of  the  elements  of  the 
renal  tissue,  epithelial  cells,  sustaining 
tissues,  blood  vessels  and  nerves  are 
essentially  the  same  as  those  used  for 
other  organs.  Masson's  Trichrome 
stain  has  the  advantage  of  affording  a 
particularly  colorful  differentiation  of 
the  various  elements  in  a  single  section. 

The  individual  renal  organs  that  make 
up  the  kindey,  the  nephrons,  can  be 
isolated  in  their  entirety  by  maceration 
and  teasing  as  described  by  Huber,  G. 
C,  Cowdry's  Special  Cytology,  1932,2, 
935-977.  Partly  wash  out  blood  by  in- 
jecting physiological  saline  into  the 
renal  artery.  Then  follow  with  hydro- 
chloric acid  (cone.  HCl,  3  parts  and  aq. 
dest.  1  part)  using  care  to  protect  the 
eyes.  Remove  and  immerse  the  organ 
in  the  same  fluid.  After  a  suitable  time, 
determined  by  excising  pieces,  wash  a 
block  of  tissue  with  aq.  dest.  and  stain 
in  Hemalum.  Wash  in  very  dilute  aq. 
sodium  hydrate.  Isolate  individual 
tubules  by  teasing  with  fine  needles. 
Wash,  and  mount  in  glycerin.  With 
small  mammals  Ruber's  results  were 
excellent  but  he  was  not  satisfied  with 
his  human  preparations.  The  method 
has  however  been  well  adjusted  to  the 
human  kidney  by  Oliver,  J.,  Architec- 
ture of  the  Kidney  in  Chronic  Bright's 
Disease,  New  York:  Paul  B.  Hoeber, 
1939,  by  a  simpler  procedure  and  dis- 
sected nephrons  may  be  mounted, 
stained  and  photographed.  A  method 
of  montage  then  affords  a  demonstra- 
tion in  their  natural  continuity  of  the 
cellular  elements  of  the  nephron  at 
high  magnification  (J.  Clin.  Invest. 
1951,    in    press). 

A  clear  distinction  between  glomeruli 
and  the  renal  tubules  is  important.  It 
is  a  simple  matter  to  color  the  former 
with  1:5000  Janus  blue  (which  is  more 
satisfactory  for  this  purpose  than  Janus 
green)  in  0.85%  aq.  sodium  chloride  by 
vascular  Perfusion  and  to  determine 
their  number,  size  and  distribution 
against  a  background  of  unstained  or 
faintly  rose  tinged  tubules  in  slices  of 
fresh  kidney  (Cowdry,  E.  V.,  Contrib. 
to  Embryo! .  Carnegie  Inst.,  Washing- 
ton, 1918,  8,  39-160). 

Perhaps  in  no  other  organ  is  it  pos- 
sible to  correlate  morphological  struc- 
ture with  functional  activity  so  closely 
and  by  so  many  methods  as  in  the  kid- 
ney. See  Oliver,  J.,  Am.  J.  Med., 
1950,  9,  88  for  a  general  statement  of 
the  problem  with  various  examples  of 
such  procedures.  As  an  example,  the 
technique  for  the  microscopic  study  in 


KIDNEY 


171 


KIDNEY 


vivo  of  the  surface  of  the  guinea  pig'a 
kidney,  and  for  the  marking  of  single 
tubules  with  India  ink  for  their  later 
isolation  by  maceration,  is  given  by 
Walker,  A.  M.  and  Oliver,  J.,  Am.  J. 
Physiol.,  1941,  134,  562-595.  The  micro 
collection  of  fluid  from  single  tubules 
is  as  the  authors  state  a  direct  continu- 
ation of  the  researches  of  A.  N. 
Richards.  See  Oliver,  J.,  Harvey  Lec- 
tures, 1944^5,  40,  102-155. 

Vital  staining  of  kidney  tubules  is  a 
valuable  method  for  "seeing"  func- 
tional activity  in  morphological  form. 
It  is  usually  carried  out  by  techniques 
not  requiring  special  adaptation.  See 
Vital  Staining.  The  procedure  em- 
ployed by  Oliver,  J.,  Bloom,  F.  and 
MacDowell,  M.,  J.  Exp.  Med.,  1941, 
73,  141-160  deserves  mention  because 
it  gives  a  clear  demonstration  that  the 
cells  of  abnormal  proximal  convoluted 
tubules  can  be  marked  by  their  inability 
to  concentrate  trypan  blue  which  con- 
sequently stains  the  tubule  wall 
diffusely.  This  is  illustrated  in  colors. 
Microscopic  observations,  having  a 
close  relation  to  function,  are  easily 
made  on  the  kidneys  of  lower  forms. 
See  the  account  of  contractility  of  the 
ciliated  necks  of  renal  tubules  in  Nec- 
turus  by  Lucas,  A.  M.  and  White,  H. 
L.,  Anat.  Rec,  1933,  57,  7-11.  The 
functional  handling  of  fluorescent  dj^es 
by  the  amphibian  kidney  may  be  ob- 
served microscopically  in  vivo  by  means 
of  ultraviolet  light  (Ellinger,  P.,  Quart. 
J.  Exp.  Physiol.,  1940,  30,  255,  and 
Singer,  E.,  Anat.  Rec,  1936,  66,  343). 
The  amphibian  kidney  also  lends  itself 
to  the  application  of  the  "extra-vital" 
method  where  the  functional  activities 
(secretion  of  dyes)  of  the  isolated  per- 
fused kidney  are  correlated  with  the 
structural  aspect  of  the  process  by 
subsequent  histological  examination 
(Oliver,  J.  and  Smith,  P.,  J.  Exp.  Med., 
1931,  53,  785;  Oliver,  J.  and  Lund,  E.  A., 
Ibid,  1933,  57,  435). 

The  study  of  the  renal  tubules  present 
in  tissue  cultures  is  useful  in  the  study 
of  function.  Thus  Chambers,  R.  and 
Cameron,  G.,  Radiology,  1941,  37, 
186-193  have  found  that  susceptibility 
to  x-rays  is  increased  when  a  secretory 
stimulant  is  added  but  that  in  cultures 
it  is  distinctly  less  than  in  vivo.  See 
references    accompanying    this    paper. 

A  method  has  been  devised  by  Crab- 
tree,  C.  E.,  Endocrinology,  1941,  29, 
197-203  of  measuring  by  a  differential 
count  the  number  of  Bowman's  capsules 
made  of  cuboidal  as  contrasted  with 
squamous  cells.  The  count  appears  to 
provide  an  index  of  age  and  sex  varia- 
tions in  normal  mice  and  of  the  in- 


fluence of  testosterone  propionate  on 
castrated  mice.  The  glomeruli  may 
also  be  counted  in  aliquot  portions  of 
macerated  renal  cortex  and  decreases 
in  their  number  correlated  with  the 
abnormal  conditions  of  disease  or 
senility  (Aratabi,  M.,  Am.  J.  Anat., 
1926,  36,  399). 

Methods  for  estimating  the  distribu- 
tion of  enzymes  in  the  tissue  components 
of  the  rabbit's  kidney  are  given  by 
Weil,  L.,  and  Jennings,  R.  K.,  J.  Biol. 
Chem.,  1941,  139,  421-432.  They  de- 
pend on  topographic  correlation  be- 
tween distribution  of  cell  types  in 
15  ft  frozen  sections  and  decomposition 
of  substrates.  The  techniques  are  ca- 
pable of  demonstrating  catheptic,  ami- 
nopolypeptidase  and  esterase  activities 
in  all  of  the  epithelial  components  and 
of  showing  that  the  cells  of  the  proximal 
and  distal  convoluted  tubules  are  about 
twice  as  active  enzymatically  as  those 
of  the  ascending  and  descending  loops 
of  Henle  and  about  4  times  as  active  as 
the  cells  of  the  collecting  tubules. 
Amylase  and  dipeptidase  activities  can 
also  be  localized  and  expressed  quanti- 
tatively in  relative  terms. 

For  the  application  to  proximal  con- 
voluted tubules  in  phlorizin  glycuresis 
of  the  Kabat  and  Furth  procedure  for 
alkaline  phosphatase  see  Kritzler,  R.  A. 
and  Gutman,  A.  B.,  Am.  J.  Physiol., 
1941,  134,  94-101.  See  Phosphatase. 
Lipase  may  also  be  shown  by  the  pro- 
cedure of  Gomori,  G.,  Arch.  Path., 
1946,  41,  121. 

Techniques  capable  of  revealing  very 
interesting  data  on  the  shape  of  cells  of 
the  proximal  tubule  have  been  devised 
and  employed  by  Foote,  J.  J.,  and 
Grafflin,  A.  L.,  Am.  J.  Anat.,  1942,  70, 
1-20.  They  can  probably  be  employed 
to  advantage  in  different  functional 
states  and  to  other  than  renal  cells. 

Methods  have  been  elaborated  for 
measurement  of  the  renal  filtration  sur- 
face and  data  have  been  supplied  for 
the  albino  rat  (Kirkman,  H.  and 
Stowell,  R.  E.,  Anat.  Rec,  1942,  82, 
373-389).  The  original  paper  should 
be  consulted.  See  also  the  measure- 
ments of  glomerular  number  and  size 
in  various  species  by  Rytand,  D.,  Am.  J. 
Anat.,  1938,  62,  507.  A  valuable  collec- 
tion of  measurements  of  renal  struc- 
tures will  be  found  in  v.  Mollendorf- 
Handbuch  d.  mikr.  Anat.,  1930,  VII-1, 
20-34. 

pH  determinations  can  be  made  as 
described  by  Emmel,  V.  M.,  Anat.  Rec, 
1940,  78,  361-377  by  means  of  a  capillary 
glass  electrode  (Voegtlin,  C.  and 
Kahler,  H.,  Science,  1932,  75,  362)  and 
a   vacuum   tube   potentiometer    (Hill, 


KING'S  CARBOL-THIONIN 


172 


KURLOFF  BODIES 


S.  E.,  Science,  1931,  73,  529).  It  is 
significant  that  increase  in  activity  of 
the  renal  cortex  immediately  follows 
ligation  of  the  renal  artery  and  that  the 
mitochondria  respond  by  enspherula- 
tion  and  fragmentation  within  6  min- 
utes. The  kidney  is  an  organ  in  which 
mitochondria  must  be  examined  with 
the  utmost  promptness.  A  delay  in 
fixation  of  15  minutes  at  room  tempera- 
ture is  sufficient  to  cause  disturbances 
in  the  mitochondrial  rodlets  of  the 
proximal  convolution.  Material  from 
human  autopsies  is  therefore  of  ques- 
tionable value.  Fuller,  R.  H.,  Arch. 
Path.,  1941,  32,  556-568  could  find  no 
relation  in  a  rather  large  number  of 
cases  studied  between  age,  hours  post- 
mortem and  cause  of  death  (except  renal 
disease)  and  quantity  and  distribution 
of  stainable  lipoid. 

The  interstitial  framework  of  the 
kidney  in  both  normal  and  pathological 
conditions  is  well  shown  by  the  silver 
methods  that  impregnate  "reticular" 
fibers  (q.v.).  The  "polysaccharide" 
content  of  the  interstitial  tissue  may 
be  examined  by  the  use  of  periodic  acid 
(McManus,  J.  F.  A.,  Amer.  J.  Path., 
1948,  24,  643.  See  also,  Ritter,  H.  B. 
and  Oleson,  J.  J.,  Amer.  J.  Path.,  1950, 
26,  639. 

The  blood  vessels  of  kidney  may  be 
injected  with  Neoprene  (q.v.)  and  also 
with  radio-opaque  suspensions  for  a 
radiographic  demonstration  of  micro- 
arteriography (Barclay,  A.  J.,  Amer.  J. 
Roent.,  1948,  60,  1).  For  a  demonstra- 
tion of  the  functional  status  of  the 
renal  circulation,  the  fluorescent  dye 
Vasoflavine  may  be  injected  into  the 
living  animal  and  the  kidney  removed 
and  viewed  with  ultraviolet  light 
(Moses,  J.  B.,  Emery,  A.  J.,  and 
Schlegel,  J.  V.,  Proc.  Soc.  E.xp.  Biol, 
and  Med.,  1951,  77,  233). 

The  cytoplasmic  particulates  of  the 
renal  epithelium,  (mitochondria,  micro- 
somes and  droplets  of  absorbed  protein) 
can  be  isolated  in  sucrose  suspensions 
and  examined  by  standard  biochemical 
procedures.  Cf.  Oliver,  J.,  J.  Mt. 
Sinai  Hospital,  1948,  15,  175. 

King's  Carbol-Thionin  stain  for  Nissl  bodies 
(Addison  in  McClung,  p.  450).  Stain 
paraffin  or  celloidin  sections,  2-3  min., 
in  sat.  thionin  in  1%  aq.  carbolic  acid. 
Then  wash  quickly  in  aq.  dest.,  differen- 
tiate in  95%  alcohol.  Pass  through 
equal  parts  absolute  alcohol  and  chloro- 
form to  xylol  and  mount  in  balsam. 

Kinney's  Method  for  staining  reticulum 
(Kinney,  E.  M.,  Arch.  Path.,  1928,  5, 
283).  Fix  18  hrs.  in  1  gm.  sodium  sul- 
phantimonate  dissolved  in  100  cc.  4% 
formalin     immediately     before     using. 


Imbed  in  paraffin,  but  more  than  1  or  2 
hrs.  in  xylol  or  cedar  oil  will  remove  the 
dark  brown  stain  from  the  reticulum. 
Hematoxylin  is  contraindicated  as  coun- 
terstain  because  it  obscures  the  color  of 
the  reticulum.     Other  ordinary  counter- 
stains  can  be  used.     This  method  works 
well  even  with  autopsy  material.     It  is 
recommended    particularly    for    kidney 
and  pancreas.    Results  are  sometimes 
patchy  in  the  spleen. 
Kleinenberg's    fixative.    Saturated    picric 
acid  in  2%  aq.  sulphuric  acid.     Embryos 
and  marine  organisms. 
Knee-Joint,      method      for      investigation 
therein   of   radioactive   gold    (Ekholm, 
R.,   Acta  Anat.,   1951,   Suppl.    15,    11, 
75  pp.). 
Knisely,  see  Quartz  Rod  Technique. 
Kolatchew  Fluid,  see  Golgi  Apparatus. 
Korfif's  Fibers  of  dentin,  see  Teeth,  De- 
veloping. 
Kossa,  see  his  test  for  Calcium. 
Krajian's  Congo  Stain.     Elastic  fibers  (Kra- 
jian,  A.  A.,  Arch.  Path.,  1934,  18,  378- 
380).     Fix  in  10%  formalin,  24  hrs.  or 
more.     Cut     frozen     sections.       Wash 
them  in   tap  water.     Place   in  2%  aq. 
aluminum  chloride  5-10  min.    Wash  and 
stain  10  min.  in  8  cc.  4%  Congo  red  in 
5%  aq.  sodium  citrate  -|-  2  cc.  glycerin 
C.P.     After  washing  in  tap  water  trans- 
fer to  1%  aq.  KI  for  10  sec.  agitate. 
After  again  washing  in  tap  water,  stain 
5-10  min.  in:anilin  blue,  1.5  gm. ;  orange 
G,  2.5  gm.;  resorcinol,  3  gm.;  phospho- 
molybdic  acid,   1  gm.;  aq.  dest.,   100. 
Wash  carefully  in  tap  water.     Blot  sec- 
tions on  slides.     Dehydrate  in  absolute 
alcohol  2  min.;  clear  in  origanum  oil; 
pass  through  xylol  to  balsam.     Elastic 
fibers  bright  red,  fibrin  dark  blue. 
Krause's  End-Bulbs.    Methylene  blue  dem- 
onstration of  in  skin  of  forearm   (Wed- 
dell,  G.,  J.  Anat.,  1940-41,  75,  346-367). 
See  Skin. 
Krause's    Membrane.     Special    technique 
for,  see  Dahlgren    (McClung,  p.  427). 
Kronig's  Cement  is  recommended  by  Bens- 
leys    (p.   41)    for   ringing   preparations 
mounted  in  glycerin  jelly  or  glycerin : 
7-9  parts  colophonium    (resin)   melted 
and  stirred  with  2  parts  beeswax. 
Kuff,  see  Nucleic  Acid  Dye   Interactions. 
Kurloff  Bodies  are  cytoplasmic  inclusions 
which  frequently  occur  in  the  non-gran- 
ular leucocytes  of  guinea  pigs.     They 
show  particularly  well  in  smears  of  the 
spleen,  may  attain  a  size  equal  to  that 
of  the  nucleus  and  can  be  brilliantly 
colored  supravitally  by  1:2000  brilliant 
cresyl  blue  in  physiological  salt  solution 
(Cowdry,  E.  V.  chapter  in  Rivers 'book 
on  Viruses,  Baltimore,  Williams  &  Wil- 
kins,  1928,  p.  141). 


KULTSCHITZKY'S  HEMATOXYLIN       173 


LEAD 


Kultschitzky's  Hematoxylin  is  1  gm.  hema- 
toxylin dissolved  in  a  little  alcohol  made 
up  to  100  cc.  with  2%  aq.  acetic  acid 
(Lee,  p.  526). 

Lac,  a  crimson  dye  obtained  from  resinous 
incrustation  caused  by  the  insect,  Coc- 
cus lacca,  of  Siam,  Indo-China  and 
Southern  India.  This  dye,  introduced 
into  England  about  1790  A.D.,  became 
an  important  article  of  commerce  in 
competition  with  cochineal  of  Mexican 
origin,  but  before  long  proved  inferior 
to  cochineal  and  was  no  longer  im- 
ported. The  crimson  dyes,  Kermes, 
cochineal  and  lac  have  played  im- 
portant parts  in  the  history  of  civiliza- 
tion (Leggett,  W.  F.,  Ancient  and 
Medieval  Dyes.  Brooklyn:  Chemical 
Publishing  Co.,  Inc.,  1944,  95  pp.) 

Lacmoid,  an  indicator  similar  to  Resorcin 
Blue. 

Lacteals,  see  Lymphatic  Vessels. 

Lactoflavin,  see  Vitamin  Bj. 

Lactophenol,  a  fixative  for  Bilharzial  Cer- 
cariae.  See  Lactophenol-cotton  blue 
technique  under  Fungi. 

Laidlaw's  Methods.  1.  For  inclusion  bodies 
(quoted  from  Pappenheimer,  A.  W.  and 
Hawthorne,  J.  J.,  Am.  J.  Path.,  1936, 
12,  625-633,  see  colored  figure,  who  used 
it  for  cytoplasmic  inclusions  in  liver 
cells).  Fix  in  sat.  aq.  corrosive  sub- 
limate 100  cc.  -f  5%  glacial  acetic  acid 
or  in  Zenker's  fluid  without  acetic. 
Imbed  in  paraffin,  cut  sections  3m.  Re- 
move paraffin  and  pass  down  to  water. 
Weigert's  iron  hematoxylin  (2%)  5min. 
Differentiate  in  0.5%  acid  alcohol. 
Rinse  in  tap  water,  then  aq.  dest.  1% 
aq.  acid  fuchsin  5-15  min.  Rinse  in 
aq.  dest.  Mordant  in  1%  phospho- 
molybdic  acid  30  sec.  Rinse  in  aq.  dest. 
Differentiate  in  0.25%  orange  G  in  70% 
ale.  Dehydrate,  clear  ana  mount  in 
balsam. 

2.  For  silver  staining  of  skin  and  tu- 
mors (Laidlaw,  G.  F.,  Am.  J.  Path.,  1929, 
5,  239-247).  Fix  in  Bouin's  fluid  or  in 
10%  neutral  formalin  for  3  days.  (To 
make  the  Bouin's  fluid  he  uses,  add  100 
cc.  commercial  formalin  and  20  cc.  glacial 
acetic  acid  to  300  cc.  tap  water  and  satu- 
rate with  picric  acid).  Fix  paraffin 
sections  to  slides  by  Masson's  Gelatin 
Glue.  Wash  Bouin  sections  for  20  min. 
in  running  water,  and  formalin  ones  for 
5  min.  1%  ale.  iodine,  3min.,  rinse  in  tap 
water.  5%  aq.  hypo  (sodium  thiosul- 
phate),  3  min.,  rinse  in  tap  water. 
^%  aq.  potassium  permanganate  3  min., 
rinse  in  tap  water,  5%  oxalic  acid,  5  min. 
Wash  in  running  water,  10  min.  Aq. 
dest.  3  clianges  in  5-10  min.  to  clean 
before  adding  silver.  Heat  stock  Lith- 
ium Silver  solution  to  50°C.  and  stain 
in  oven  for  5  min.     Pour  aq.  dest.  over 


both  sides  of  slides.  Flood  sections  fre- 
quently for  3  min.  with  1%  formalin  in 
tap  water.  Again  rinse  both  sides  of 
slides  with  aq.  dest.  1:500  yellow  gold 
chloride  in  aq.  dest.  in  Coplin  jar  at 
room  temperature,  10  min.  Rinse  both 
sides  with  aq.  dest.  Pour  on  5%  oxalic 
acid  10  min.  Rinse  in  aq.  dest.  Pour 
on  5%  hypo  changing  as  often  as  it  be- 
comes turbid,  10  min.  Wash  in  running 
water.  Counterstain  if  desired.  De- 
hydrate, clear  and  mount  in  usual  way. 
Reticulum,  black  threads;  collagen  red- 
dish purple. 

Lake  Ponceau,  see  Ponceau  2R. 

Lampblack.  A  colloidal  suspension  of  lamp- 
black is  an  excellent  substance  to  inject 
intravenously  to  demonstrate  phago- 
cytosis, especially  by  monocytes.  Mc- 
Junkin,  F.  A.,  Arch.  Int.  Med.,  1918, 
21,  59-64,  advised  adding  0.4  gm.  of 
carefully  pulverized  lampblack  to  100  cc. 
2%  gelatin  in  aq.  dest.  Inject  intra- 
venously with  5-9  cc.  10%  aq.  sodium 
citrate,  as  in  the  case  of  Higgins'Ink. 
The  method  has  been  slightly  modified 
by  Simpson,  M.  J.,  J.  Med.  Res. ,1922, 
43,  77-144;  Wislocki,  G.  B.,  Am.  J. 
Anat.,  1924,32,  423-445;  and  Lang,  F.J., 
Arch.  Path.,  1926,  1,  41-63. 

Lanacyl  Blue  BB  (CI,  210),  an  acid  monoazo 
dye  which  colors  cell  walls  and  paren- 
chymatous cells  light  blue  but  less  well 
than  other  blue  acid  dyes  (Emig,  p.  35). 

Lanacyl  Violet  B  (CI,  207),  an  acid  monoazo 
dye  of  light  fastness  3.  Directions  for 
staining  plant  tissue  and  fungous  my- 
celia  (Emig,  p.  35). 

Langerhans,  see  Islets  of. 

Lard,  reactions  in  tissue  to  fat  stains  after 
various  fixations  (Black,  C.  E.,  J.  Lab. 
&  Clin.  Med.,  1937-38,  23,  1027-1036). 

Large  Intestine.  The  conditions  that  in- 
fluence the  appearance  of  sections  are 
easier  to  guard  against  than  in  the  Small 
Intestine  because  of  the  absence  of  villi 
and  greater  uniformity  of  contents. 
The  pronounced  influence  of  degree  of 
distention  is  described  and  well  illus- 
trated by  Johnson  (F.  P.,  Am.  J.  Anat., 
1912-13,  14,235-250). 

Lansing,  see  Collagen,  Elastin. 

Latex-Cast  Techniques  for  study  of  the 
circulation  have  been  applied  to  the 
spleen  by  Gall,  D.  and  Maegraith, 
M.  G.,  Ann.  Trop.  Med.  &  Parasit., 
1950,  44,  331-338,  who  give  good  illus- 
trations of  results  and  references  to 
previous  work. 

Lauth's  Violet,  see  Thionin. 

Lead,  histological  demonstration. 

1.  Mallory  and  Parker's  method  (Mal- 
lory,  F.  B.  and  Parker,  F.  J.,  Am.  J. 
Path.,  1939,  15,  517-522) :  Fix  tissues  in 
95  or  abs.  alcohol  (not  formalin).  Stain 
celloidin  sections  at  54°C.  in:  5-lOgm. 


LEATHER  BROWN 


174 


LEISHMANIA.    MEDIA 


hematoxylin  dissolved  in  few  drops  abs. 
or  95%  alcohol  +  10  cc.  freshly  filtered 
2%  aq.  K2HPO4  for  2-3  hrs.  Wash 
changing  tap  water  10-60  min.,  dehy- 
drate in  95%  ale,  clear  in  terpineol  and 
mount  in  terpineol  balsam.  Lead  light 
to  grayish  blue,  nuclei  deep  blue. 
Another  method  applicable  to  paraffin 
sections  of  Zenker  fixed  material  is  to 
stain  in  0.1%  methylene  blue  in  20% 
ale.  10-20  min.  Differentiate  10-20 
min.  in  95%  ale,  dehydrate,  clear  and 
mount.  Phloxine  is  recommended  as  a 
contrast  stain  before  the  methylene 
blue. 

2.  Chromate  method  (Frankenberger, 
Cretin).  By  simply  fixing  in  Regaud's 
Fluid  lead  is  precipitated  as  insoluble 
yellow  lead  chromate  easily  identifiable 
microscopically.  This  method  is 
strongly  advised  by  Lison  (p.  101). 
It  has  been  used  by  True  (E.,  Bull. 
d'Hist.  Appl.,  1929,  6,  393-399).  See 
Sieber  (E.,  Arch.  f.  exper.  path.  u. 
pharmak.,  1936, 181,  273-280)  for  demon- 
stration  of   lead   in    bones. 

3.  Attempts  have  been  made  to 
identify  lead  after  microincineration  by 
exposure  to  hydrogen  sulphide,  because 
lead  sulphide  is  black,  but  Gordon  H. 
Scott  emphasizes  difficulty  in  dis- 
tinguishing it  from  other  sulphides  and 
from  carbon  in  imperfectly  incinerated 
specimens  (McClung,  p.  660). 

4.  The  method  of  Sieber,  E.,  Arch.  f. 
exper.  Path.  u.  Pharmak.,  1939,  181, 
273  depending  on  production  of  acid 
resistant  brown-black  lead  sulfide  when 
tissue  is  treated  with  acidulated  H2S 
solution  is  said  to  be  satisfactory  by 
Gomori,  G.,  J.  Mt.  Sinai  Hosp.,  1944- 
45,  11,  317-326  when  presence  of  other 
heavy  metals  is  ruled  out. 

Methods  for  chemical  determination 
of  lead  in  biological  materials  are 
important  as  checks  on  above.  Consult 
Smith,  F.  L.  2nd.,  Rathmell,  T.  K.  and 
Williams,  T.  L.,  Am.  J.  Clin.  Path., 
1941,  11,  Suppl.  5,  653-668. 

For  a  convenient  method  of  giving 
colloidal  lead  intravenously  to  rabbits 
see  Crawford,  B.  L.,  Stewart,  H.L., 
Willoughby,  C.  E.  and  Smith,  F.  L., 
Am.  J.  Cancer,  1938,  33,  401^22.  The 
authors  describe  techniques  for  direct 
analysis  of  lead  in  the  tissues. 

Leather  Brown,  see  Bismark  Brown  Y. 

Leather  Yellow,  see  Phosphine. 

Leblond,  see  Radioantographic  Technique. 

Lebowich's  soap-wax  technique  eliminates 
use  of  alcohol ,  xylol  and  overnight  drying 
of  paraffin  sections.  Takes  only  6-8  hrs. 
(Moritz,  C.  E.,  Stain  Techn.,  1939,  14, 
17-20). 

L.  E.  Cells.  The  discovery  of  these  cells 
in    acute    disseminated    lupus   erythe- 


matosus by  Hargraves,  M.  M.,  Rich- 
mond, M.  and  Morton,  R.,  Proc.  Staff 
Meet.,  Mayo  Clin.,  1948,  23,  25-28,  was 
a  definite  advance  in  diagnostic  pro- 
cedure. The  simplest  test  for  L.  E. 
cells  is  that  of  Lee,  Stanley  L.,  Am.  J. 
Clin.  Path.,  1951,  21,  492-496.  With- 
draw 1-2  cc.  venous  blood  into  a  clear 
dry  test  tube.  Let  clot  and  remain  at 
room  temperature  for  2  hrs.;  with 
wooden  applicator  "fish  out"  the  clot. 

Lecithin,  a  compound  of  phosphoric  acid, 
glycerol,  choline  and  2  fatty  acid  mole- 
cules. It  is  a  phosphatide  soluble  in 
alcohol,  chloroform,  ether  and  benzene, 
see  Lipoids. 

Lee-Brown.  Modification  of  Mallory's  ani- 
line blue  connective  tissue  stain  (Lee- 
Brown,  R.  K.,  and  Laidley,  J.  W.  S., 
J.  Urol.,  1929,  21,  259-274).  Mallory 
(p.  155)  states  that  the  following  tech- 
nique is  particularly  valuable  for  the 
kidney.  Treat  paraffin  sections  of  Zen- 
ker fixed  material  with  iodine  to  remove 
mercury.  Wash.  1%  aq.  phosphomolyb- 
dic  acid,  30  sec.  Wash  in  aq.  dest.  1-2 
min.  Stain  in:  aniline  blue,  0.5  gm.; 
orange  G.,  2  gm. ;  phosphomolybdic  acid, 
2  gm.;  aq.  dest.,  100  cc.  for  30  min.  at 
55 °C.  Wash  in  an.  dest  2-5  min.  1% 
aq.  phosphomolybdic  acid,  30  sec.  95% 
ale . ,  abs .  ale . ,  xylol ,  balsam .  Glomerular 
basement  membrane  and  collagen,  deep 
blue;  nuclei,  orange. 

Leishmania  Donovani,  a  search  for  stains 
that  will  color  more  rapidly  than  Giemsa 
revealed  Astra  violet  F.  F.  Extra, 
Himmelblau,  Magenta  Lermont  and 
Navy  blue  shade,  each  to  be  used  in 
fresh  10%  aq.  solution  (Takasaki,  S., 
Lues,  Tokyo,  1938, 16,  127). 

Leishmania.  Media.  Direct  microscopic 
examination  of  peripheral  blood  may 
be  negative  while  detection  in  culture 
is  feasible.  Q.  M.  Geiman  (Simmons 
and  Gentzkow)  recommends  addition 
of  10  cc.  blood  to  sodium  citrate  in 
physiological  saline,  centrifuge  and  in- 
oculate few  drops  buffey  coat  into  tubes 
of  NNN  medium,  incubate  22-28°C.  and 
examine  microscopically  10-20th  day 
for  motile  forms.  The  following  media 
are  abbreviated  from  Geiman 's  ac- 
count. 

1.  Blood  agar  or  NNN  (Novy,  Mac- 
Neal  and  Nicolle,  1908).  Agar,  14  gm., 
sodium  chloride,  6  gm.,  aq.  dest.  1000 
cc.  Add  5  vol.  sterile  defibrinated 
rabbit's  blood  cooled  to  45°-50°C.  Mix, 
tube  long  slant.  After  agar  sets,  cap 
with  sterile  rubber  stoppers.  Prove 
sterility  by  incubation  37°C.,  24  hrs. 
Inoculate  material  to  be  cultivated  on 
slant  and  in  water  of  condensation. 
Incubate  20°-25°C.  Transfer  every  20- 
30  days  to  maintain. 


LENGTH 


175 


LEUCOCYTES 


2.  Leptospira  (Noguchi,  1924).  0.9% 
aq.  sodium  chloride,  800  parts;  fresh 
rabbit  serum,  100  parts;  2%  nutrient 
agar  pH  7.2,  100  parts,  rabbit  hemo- 
globin solution  10-20  parts.  (To  make 
this  hemo'^^lobin  solution  take  1  part 
defibrinated  rab!)it's  blood  and  3  parts 
aq.  dest.,  centrifuge  and  use  clear  super- 
natant fluid.)  Tube,  prove  sterility  by 
incubation  before  using.  Subculture 
every  30  days.  An  increase  in  hemo- 
globin solution  improves  growth  of 
Leishmania. 

3.  Adler's  modification  of  above. 
Agar,  1  part;  Locke's  solution  contain- 
ing 0.2%  dextrose,  8  parts;  fresh  rabbit 
serum,  1  part.  For  species  of  Leish- 
mania and  Trypanosoma  cruzi. 

4.  Modified,  Salle  and  Schmidt 
(Cleveland  and  Collier,  1930).  Veal 
infusion  (50  gm.  Bacto-veal,  Difco  -t- 
1000  cc.  aq.  dest.),  250  cc;  proteose, 
peptone  (Difco),  10  gm.;  sodium  chlo- 
ride, 5  gm.;  aq.  dest.,  550  cc.  Dissolve 
make  pH  7.4  and  autoclave.  Add  20  cc. 
50%  aq.  glucose  (sterilized  by  filtration 
or  in  autoclave  10  lbs.,  10  min.)  and 
60  cc.  horse  red  cells  laked  with  2  parts 
aq.  dest.  Pour  in  medium  flasks  or 
tubes.     Vigorous   long   lived    cultures. 

Length  measurements : 

Millimeters  to  inches  X  0.0394.    Inches 
tomm.  X25.4.     SeeMicron. 

Lens  Paper,  a  specially  prepared  soft  paper 
indispensible  for  cleaning  immersion 
oil  from  objectives. 

Leprosy  Bacilli.  Stain  by  carbol-fuchsin  in 
smears.  See  Concentration  method  for 
collecting  bacilli  from  lesions.  For 
study  in  sections,  see  Acid  Fast  Bacilli. 

Leptospira  Medium,  Noguchi's,  see  Leish- 
mania. 

Leptospiras,  method  for  isolation  from  water 
(Bauer,  J.  H.,  Am.  J.  Trop.  Med.,  1927, 
7,  177-179.    See  Spirochetes. 

Leuco  Basic  Fuchsin.  To  make  add  to  200 
cc.  aq.  sol.  fuchsin,  2  gm.  potassium 
metabisulphite  and  10  cc.  N  hydro- 
chloric acid.  After  bleaching  24  hrs. 
add  0.5 gm.  Novit,  shake  1  min.  and  filter 
through  coarse  paper.  Resulting  clear 
solution  works  nicely  in  Feulgen  tech- 
nique (Coleman,  L.  C,  Stain  Techn., 
1938,13,  123-124). 

Leuco-Dyes  as  vital  stains.  Make  001% 
aq.  solutions  of  methylene  blue,azurA, 
thionin  toluidine  blue  and  brilliant  cresyl 
blue.  Add  to  100  cc.  1-2.5  cc.  N/10 
NajSaO,  and  1-4  cc.  N/10  HCl.  Mix 
and  store  at  room  temperature  in  dark. 
To  stain,  add  1-2  drops  of  leucobase  to 
the  protozoa,  blood  cells,  etc.  in  physio- 
logical saline.  Said  to  give  good  contrast 
staining  of  nucleus  and  cytoplasm  and 
to  be  useful  in  oxidation-reduction 
determinations  (Roskin,  G.,  Arch.  Russ. 


Anat.  Hist.  Embr.,   1937,   16,107-109). 

Leuco-Patent  Blue  V,  see  Lillie,  p.  285. 

Leucocytes.  In  the  broad  sense  they  in- 
clude all  white  blood  cells  but  the  term 
is  generally  restricted  to  the  "granular" 
leucocytes  as  compared  with  the  "non- 
granular" ones  (Lymphocytes  and  Mon- 
ocytes). In  a  still  narrower  sense  the 
leucocytes  include  only  polymorphonu- 
clear neutrophiles,  eosinophiles  and 
basophiles  which  are  easily  found  in 
circulating  blood  as  contrasted  with  less 
differentiated  leucocytes  called  Myelo- 
cytes and  Myeloblasts  generally  con- 
fined to  the  bone  marrow. 

For  mitochondria  within  leucocytes 
supravital  staining  with  Janus  green  is 
indicated.  In  smears  Giemsa's  stain 
has  a  little  advantage  over  Wright's  in 
the  fact  that  it  better  demonstrates  any 
bacteria  that  may  be  present.  The 
May-Giemsa  technique  is  most  used  in 
Europe.  It  is,  in  effect, a  double  staining 
because  the  air  dried  smears  are  first 
treated  with  the  May-Grunwald  com- 
bined fixative  and  stain  and  are  later 
colored  by  Giemsa's  stain.  It  gives 
satisfying  deep  colors.  TheKardos- 
Pappenheim  modification  is  suggested 
when  a  particularly  intense  coloration  of 
neutrophilic  granules  is  desired.  Ehr- 
lich's  triacid  stain  may  likewise  be  use- 
ful because  it  is  said  to  stain  the  neutro- 
philic granules  leaving  the  azur  granules 
untouched. 

Leucocytes  give  strong  Peroxidase 
and  Oxidase  reactions,  which  are,  how- 
ever, not  specific  for  them.  The  Golgi 
Apparatus  (reticular  material)  can  be 
demonstrated  by  long  treatment  with 
osmic  acid  or  by  the  Cajal  uranium  ni- 
trate and  silver  method  (Cowdry,  E.  V., 
J.  Exper.  Med.,  1921,  33,  1-11).  The 
demonstration  of  degenerative  leucocytic 
changes  associated  with  ageing  is  de- 
scribed by  Lowell  (A.  L.,  J.  Lab.  &  Clin. 
Med.,  1937-38, 23, 791-796),  of  variability 
in  relation  to  alterations  in  meteorologic 
conditions  by  Berg  (M.,  J.  Lab.  &  Clin. 
Med.,  1937-38,  23, 797-803)  and  of  lipoid 
components  by  Bacsich  (P.,  J.  Anat., 
1935-36,  70,  267-272).  Chemotaclic  re- 
sponse and  motility  can  be  measured 
both  in  tissue  cultures  (Comau,  D.  R. 
Arch.  Path.,  1940,  30,  896-901)  and 
directly  by  observing  the  behavior  of 
leucocytes  with  relation  to  bacteria  and 
in  temporary  mounts  (Mallery,  O.  T. 
and  McCutcheon,  M.,  Am.  J.  Med.  Sci., 
1940, 200, 394-399 ) .  By  the  latter  method 
differences  in  behavior  of  neutrophiles 
from  seriously  ill  and  normal  persons 
have  been  reported .  Motion  pictures  are 
of  great  assistance  in  making  a  thorough 
analysis  of  the  movements  and  behavior 
of    leucocytes.    Some    excellent    ones, 


LEUCOCYTE  COUNTS 


176 


LEUCOCYTES 


taken  by  Dr.  W.  H.  Lewis,  are  available 
for  distribution  by  the  Wistar  Institute 
of  Anatomy  in  Philadelphia.  To  in- 
vestigate their  behavior  after  they  have 
left  the  blood  vessels  and  entered  the 
surrounding  tissues  is  immensely  more 
difficult.  The  only  method  that  gives 
promise  of  important  results  is  to  employ 
for  this  purpose  special  chambers  in- 
serted in  the  ears  of  rabbits  (Clark, E.R. 
and  E.  L.,  Am.  J.  Anat.,  1936,  59,  123- 
173) .  See  Neutrophile,  Eosinophile  and 
Basophile  Leucocytes. 
Leucocyte  Counts,  1.  Total  number  white 
blood  cells  per  c.  mm.  Over  12,000  a 
leucocytosis,  less  than  5000,  a  leucopenia. 
Average  about  7,500. 

2.  Differential.  Smears  colored  by 
Giemsa's  or  Wright's  stains  are  more 
satisfactory  than  supravi tally  stained 
preparations  because  the  latter  are  more 
difficult  to  handle  and  the  cells  are 
slowly  dying  and  showing  more  and  more 
deviations  from  normal.  Relative  num- 
ber of  different  white  cells  is  expressed 
in  percentages,  i.e.  neutrophiles 55-75, 
eosinophiles,  2-4,  basophiles,  0-1,  lym- 
phocytes 21-31,  and  monocytes  4-5. 
Both  total  and  differential  counts  should 
be  correlated  to  avoid  misconceptions. 
60%  neutrophiles  in  total  count  of  8,000 
amounts  to  4,800  neutrophiles  per  c.  mm. 
80%  neutrophiles  in  total  count  of  4,800 
is  the  same,  namely  4,800  neutrophiles 
per  c.  mm.  although  a  relative  neutro- 
philic leucocytosis  exists.  60%  neutro- 
philes in  a  total  count  of  16,000  makes 
on  the  other  hand  9,600  neutrophiles 
per  c.  mm.  which  is  an  actual  neutro- 
philic leucocytosis.  20%  lymphocytes 
of  9,000  is  the  same  number  per  c.  mm. 
as  60%  of  3,000 ;  while  30%  of  11,000  is  an 
actual  lymphocytosis. 

3.  Age.  Since  young  neutrophiles 
have  fewer  nuclear  lobes  than  older  ones 
counts  of  the  number  with  from  1-5 
lobes  were  made  by  Arneth.  Today 
simpler  methods  are  used. 

The  Schilling  is  the  usual  one.  It  is 
both  a  total,  a  differential  and  an  age 
count  combined.     The  normal  is  given 


Total 

5,000  to  10,000 

£ 

d-l 

E 

2-4 

M 

0 

J 

0-1 

Leucocytes 

St 

3-5 

S 

51-67 

L 

21-35 

Mon. 

4-6 

above.  B  =  basophile.  E  =  eosino- 
phile. M  =  myelocyte  (Nucleus  large, 
occupying  about  half  cytoplasmic  area. 


spherical  to  oval  or  kidney-shaped,  pale 
staining,  chromatin  reticulated,  nu- 
cleoli present.  Cytoplasm  faintly 
basophilic  with  few  specific  granules 
which  are  small,  often  difficult  to  stain 
and  irregularly  distributed).  J  = 
juvenile  (A  little  larger  than  mature 
neutrophiles.  Nucleus  saucer  to  bean 
shaped.     Stains  poorly.     Circum- 

scribed basophilic  nucleoli).  St  = 
stab  nuclear  (Slightly  smaller  than 
juveniles.  Nucleus  T  V  or  U  shaped 
but  not  divided  into  segments  by  fila- 
ments and  without  nucleoli).  S  =  seg- 
ment nuclear  (Fully  differentiated  neu- 
trophiles having  2-5  or  more  segments 
often  joined  only  by  filaments.  Nuclei 
stain  intensely.)  L  =  lymphocyte. 
Mon  =  monocyte. 

When  the  numbers  of  M.  J.  St.  are 
increased  relative  to  S.,  it  is  called  a 
"shift  to  the  left",  meaning  that  im- 
mature leucocytes  are  called  into  the 
circulation ,  which  is  an  unfavorable  sign. 
When  the  relative  number  of  S  is  in- 
creased over  the  others,  it  is  termed  a 
"shift  to  the  right",  meaning  that  only 
mature  leucocytes  are  called  out,  which 
is  a  favorable  sign  if  it  follows  a  previous 
shift  to  the  left.  Details  are  given  by 
Wintrobe,  M.  M.,  Clinical  Hematology, 
Philadelphia,  Lea  &  Febiger,  1942,  792 
pp.  For  blood  containing  gum  acacia, 
see  Monke,  J.  V.,  J.  Lab.  &  Clin.  Med., 
1940-41,  26,  1664-1667  and  for  inter- 
ference by  decreased  fragility  of  eryth- 
rocytes see  Bohrod,  M.  G.,  J.  Lab.  & 
Clin.  Med.,  1940-41,  26,  1953-1955. 

A  better  method,  unfortunately  not 
widely  employed,  is  the  filament-non- 
filament  count.  Filaments  are  neutro- 
philes in  which  the  nuclear  segments 
are  connected  by  delicate  strands 
apparently  made  up  of  nuclear  membrane 
only  and  nonfilaments  are  those  in  which 
the  connections  are  so  wide  that  they 
can  be  resolved  into  nuclear  membrane 
plus  nuclear  contents.  In  100  neutro- 
philes there  are  normally  8-16  nonfila- 
ment  cells.  A  greater  per  cent  is  a  shift 
to  the  left.  For  counts  see  Krusen, 
F.  H.,  Am.  J.  Med.  Sci.,  1937,  193.  470- 
474. 
Leucocytes.  Developmental  series.  The 
technique  employed  apparently  makes 
a  great  deal  of  difference  in  the  conclu- 
sions reached.  See  Cowdry's  His- 
tology, p.  99. 

1.  Maximow  and  Bloom  employing 
mainly  permanent  preparations  list: 

Hemocytoblasts:  "...  large  (up  to  15) 
ameboid,  non-granular  basophil  cells  of 
lymphoid  nature."  Occur  extra vascu- 
larly. 

Promyelocytes:  "The  oval  or  kidney- 
shaped,  clear  nucleus  contains  a  loose 


LEUCOCYTIC  INDEX 


177 


LIEBERMANN-BURCIIARDT 


chromatin  network  and  several  nucleoli. 
At  the  indentation  of  the  nucleus  there 
is  a  distinct  cytocentrum .  The  ame- 
boid protoplasm  is  slightly  basophil, 
although  it  often  shows  acidophil  areas." 
Specific  granules  "are  scarce  and  usual- 
ly confined  to  the  periphery  of  the  cyto- 
centrum and  to  the  acidophil  spots  in 
the  cell  body."  Azurophil  granules  are 
present  but  later  disappear.  They  often 
show  mitosis. 

Myelocytes:  "The  protoplasm  becomes 
diffusely  acidophil  while  the  specific 
granules  increase  in  number  and  fill  the 
whole  cell  body,  except  for  the  cyto- 
centrum. The  nucleus  keeps  its  com- 
pact form  while  its  previously  loose 
chromatin  network  becomes  coarser  and 
stains  darker.  The  nucleoli  are  indis- 
tinct.   Mitoses  are  common." 

Metamyelocytes:  After  an  unknown 
number  of  mitoses  a  generation  appears. 
The  nucleus  "as  soon  as  it  is  recon- 
structed after  the  last  mitosis,  shows  a 
beginning  polymorphism  and  has  the 
shape  of  a  horse-shoe."  The  mature 
leucocyte  is  formed  from  these  cells  by 
individual  maturation  without  division. 

2.  Sabin  and  associates  relying  chiefly 
on  supravital  stains  list : 

Reticular  cells:  They  "are  small,  their 
cytoplasm  is  faintly  basophilic,  as  seen 
in  fixed  films,  and  in  supravital  prepa- 
rations they  show  no  differentiation  of 
specific  substances."  Reticular  cells 
"lack  the  striking  rod-shaped  mito- 
chondria which  characterize  the  lympho- 
cytic strain.  .  .  .  The  nuclei  have  less 
sharp  contours  and  less  chromatin  than 
those  of  lymphocytes." 

Myeloblasts:  These  differ  "through 
the  elaboration  of  a  marked  basophilia 
and  of  great  numbers  of  small  mito- 
chondria. ...  In  supravital  technique, 
the  myeloblast  has  usually  no  stainable 
substance  except  mitochondria  .  .  ."but 
occasionally  a  few  vacuoles  reacting  to 
neutral  red  are  present  as  well  as  some 
which  are  not  colored  by  it. 

Myelocytes  A:  The  earliest  stage  with 
the  specific  granules  up  to  10  "reacts 
with  a  single  blue  granule  in  the  oxydase 
test." 

Myelocytes  B:  "May  be  conveniently 
divided  into  those  with  less  than  half 
and  those  with  more  than  half  the  full 
quota  of  granules." 

Myelocytes  C:  These  cells  contain 
the  full  quota.  Metamyelocytes:  They 
"show  the  earliest  signs  of  the  nuclear 
changes  toward  polymorphism  and  the 
first  sign  of  the  transformation  of  the 
cytoplasm  to  a  phase  sufficiently  fluid 
to  allow  the  flowing  of  granules  which  is 
essential  for  ameboid  movement.  In 
passing  through  these  stages,  there  is  a 


gradual  decrease  of  basophilia  of  the 
cytoplasm  and  in  the  numbers  of  mito- 
chondria. The  basophilia  disappears 
entirely  in  the  early  leucocytes,  while 
the  mitochondria  persist  in  small  num- 
bers until  the  stage  of  senility  in  the 
leucocytes." 

Leucocytic  Index,  ratio  of  number  of  pol- 
ymorphs to  number  of  lymphocytes, 
considered  by  Turley,  L.  A.  and  Mc- 
Clellan,  J.  T.,  Am.  J.  Clin.  Path.,  1943, 
7,  87-95  to  be  valuable  indicator  of 
condition  of  the  patient,  a  high  or  rising 
index  being  a  bad  sign  and  a  low  or  fall- 
ing one,  a  favorable  sign. 

Leucocytic  Infiltrations.  A  convenient  way 
to  produce  an  intense  local  neutrophilic 
infiltration  is  to  inject  starch  as  de- 
scribed by  Chambers,  R.  and  Grand, 
C.  G.,  Am.  J.  Cancer,  1937,  29,  111-115. 
Cowdry,  E.  V.  and  Ruangsiri,  C,  Arch. 
Path.,  1941,  32,  632-640  made  repeated 
injections  of  1%  corn  starch  suspensions 
in  physiological  saline  in  amounts  of 
0.1-0.2  cc.  into  leprous  nodules  of  rats. 

Leucocytozoa,  Protozoa,  belonging  to  the 
Hepatozoidae,  which  inhabit  the  mono- 
cytes of  dogs,  rats,  and  other  animals 
particularly  in  the  tropics.  See,  Wen- 
yon,  C.  M.,  Protozoology.  New  York: 
William  Wood  &  Co.,  1926,  2,1053-1563. 

Leucosin,  a  stored  reserve  in  lower  plants 
(Taylor  in  McClung,  p.  221). 

Levitation  Method,  see  Floatation  Method. 

Levulose  Syrup  for  fluid  mounts.  Mallory 
(p.  99)  specifies  30  gms.  levulose  dis- 
solved in  20  cc.  water  by  warming  at  37  °C. 
for  24  hrs. 

Lewis-Locke    solution,    see    Locke-Lewis. 

Leydig  Cells  are  usually  known  as  inter- 
stitial cells  of  the  Testis  which  see. 

Liang  uses  Schiff  reagent  for  nerve  endings 
(Liang,  H.  M.,  Anat.  Rec,  1947,  97, 
419). 

Lieberkiihn's  Glands,  data  on  size,  surface 
area,  number  of  cells  etc.  in  human 
large  intestine  (Policard,  A.,  Bull. 
d'Hist.  Appl.,  1939,  16,  261-262). 

Liebermann-Burchardt  reaction  for  choles- 
terol   and     its     esters     {cholesterides) . 

1.  Modification  of  A.  Schultz.  Ex- 
pose frozen  sections  of  formalin  fixed 
tissue  at  least  4  days  (more  in  winter) 
to  strong  light,  if  possible  sunlight. 
Mount.  Dry  carefully  with  blotting 
paper.  Cover  with  few  drops  equal 
parts  acetic  and  sulphuric  acids.  Drain 
and  examine  in  the  reagent.  Cholesterol 
and  its  esters  dark  blue  or  red  purple 
becoming  green. 

2.  Modification  of  Romieu  (M.,  C. 
rend.  Acad.  d.  Sci.,  1927,  184,  1206-1208) 
Mount  frozen  sections  of  formol  or  Bouin 
(less  acetic)  fixed  tissues  and  dry. 
Cover  with  1  drop  cone,  sulphuric  acid, 
3-15  sec.    Stop  reaction  by  adding  2-3 


LIGHT  BLUE 


178 


LINDERSTR0M-LANG,  ET  AL 


drops  acetic  anhydride.  Wash  with 
several  drops  of  same.  Cover  and 
examine  immediately.  Cholesterol  and 
its  esters  violet  lilac  or  red  purple,  be- 
coming green.  The  above  two  methods 
abbreviated  from  Lison  (p.  210)  are  in 
his  excellent  judgment  specific  for 
cholesterol  and  its  esters  if  positive. 
A  negative  reaction  does  not  definitely 
prove  their  absence.  See  Swyer,  G.  I. 
M.,  Cancer  Research,  1942,  2,  372-375 
for  quantitative  measurement  of  the 
color. 

Light  Blue,  see  Spirit  Blue. 

Light  Green,  see  Methyl  Green. 

Light  Green  N,  see  Malachite  Green. 

Light  Green  SF  yellowish  (CI,  670)  S— acid 
green,  fast  acid  green  N — Commission 
Certified.  This  acid  di-amino  tri- 
phenyl  methane  dye  is  a  sulfonated 
derivative  of  brilliant  green  and  a 
valuable  counterstain  for  safranin.  It 
is  used  by  Twort,  F.  W.,  Brit.  J.  Exp. 
Path.,  1924,  5,  350-351  as  a  double  stain 
with  neutral  red  for  animal  parasites 
and  microorganisms  in  tissues.  Un- 
fortunately light  green  fades  quickly. 
Conn  (p.  110)  recommends  fast  green 
FCF  as  a  substitute. 

Lighting,  see  Illumination. 

Lignin  Pink,  a  monazo  acid  dye  (British 
Drug  Houses  Ltd.).  Advised  0.5% aq. 
solution  as  a  chitin  stain  and  a  contrast 
stain  with  chlorazol  Black  E  (Cannan, 
H.  G.,  J.  Roy.  Micr.  Soc,  1941,  61, 
88-94). 

Lilienfeld-Monti  test  for  phosphorus  is  not 
a  satisfactory  microchemical  method. 
See  Bensley's  method  (R.  R.,  Biol. 
Bull.,  1906,  10,  49-65)  and  criticism  by 
Lison  (p.  118). 

Lillie,  see  Azure  or  Toluidin   Blue   Eosin. 

Lillie's  chrom-osmic-acetic  fixative.  J% 
aq.  chromic  acid,  15  cc;  2%  aq.  osmic 
acid,  3.5  cc. ;  glacial  acetic  acid,  3  drops. 
Used  by  him  for  echinoderm  eggs. 

Lime,  see  Calcium. 

Linderstr0m-Lang,  Kaj.  U.,  and  Holier, 
Heinz,  Histochetnical  Advances — 
Written  by  David  Glick,  Dept.  of 
Physiological  Chemistry,  University  of 
Minnesota,  Minneapolis  14-,  Minn.  Octo- 
ber 17,  1951 — Prof.  Linderstr0m-Lang, 
since  1938  the  head  of  the  Department 
of  Chemistry  of  the  Carlsberg  Labora- 
tory, Copenhagen,  Denmark,  and  Dr. 
Heinz  Holter,  his  associate  and  head  of 
the  Cytochemical  Department,  are 
responsible  for  some  of  the  most  sig- 
nificant advances  in  the  field  of  quanti- 
tative histochemistry  as  well  as  of 
protein  and  proteolytic  enzyme  chemis- 
try. Their  histochemical  work  began 
in  1930  when  Dr.  Holter  came  to  the 
Carlsberg  Laboratory.  Their  first  ef- 
forts were  directed  to  the  development 


of  micro  titration  techniques  that 
would  enable  a  thousandfold  refinement 
of  common  macro  methods  of  biochemi- 
cal analysis  without  loss  of  precision. 
This  degree  of  refinement  constituted  a 
temporary  compromise  since  it  was 
their  goal  to  bring  reliable  quantitative 
chemical  techniques  to  bear  on  the 
study  of  the  single  cell,  and  a  hundred 
thousandfold  refinement  would  be  re- 
quired to  achieve  this  end  in  the  case 
of  mammalian  cells.  However  the  one 
thousandfold  refinement  did  permit 
work  on  single  microtome  sections, 
protozoa,  and  ova  of  certain  marine 
invertebrates. 

The  first  investigation  of  the  series 
(Linderstr0m-Lang,  K.  and  Holter,  H., 
Compt.  rend.  trav.  lab.  Carlsberg,  Ser. 
Chim.,  1931,  19,  No.  4)  described  the 
micro  titration  apparatus  that  was  de- 
vised, (the  burettes  used  were  graduated 
in  0.0002  ml.  divisions  and  readings  were 
taken  to  0.00002  ml.)  and  subsequently 
Linderstr0m-Lang  and  Holter  adapted 
the  equipment  to  the  measurement  of 
proteolytic  enzyme  activity.  They  em- 
ployed this  method  for  a  study  of  the 
distribution  of  peptidase  along  the  roots 
and  sprouts  of  barley  (Linderstr0m- 
Lang,  K.  and  Holter,  H.,  Ibid.,  1932, 
19,  No.  6).  When  one  has  developed 
a  new  tool  it  is  natural  that  he  employ 
it  for  diverse  purposes  to  test  its  range 
of  usefulness.  And  accordingly,  in  the 
next  application  of  their  technique, 
Holter  and  Linderstr0m-Lang  (Zeit.  f. 
Physiol.  Chem.,  1932,  219,  223-240)  in- 
vestigated the  proteinases  of  Drosera 
Rotundi folia,  an  insectivorous  plant, 
and  then  Linderstr0m-Lang  (Ibid,  1933, 
215,  167-178)  made  a  study  of  the  pepti- 
dase content  of  single  eggs  of  two  marine 
invertebrates,  the  California  sand  dollar 
and  Urechis  caupo,  before  and  after  fer- 
tilization. These  eggs  have  a  wet 
weight  of  only  about  5  X  10~'  g. 

Linderstr0m-Lang  and  Holter  next 
extended  their  technique  to  a  method 
for  the  iodometric  determination  of 
reducing  sugars  with  a  precision  corre- 
sponding to  25  X  10~*  g.  of  glucose 
(Compt.  rend.  trav.  lab.  Carlsberg, 
1933,  19,  No.  14),  and  this  was  followed 
by  the  development  of  a  diffusion 
method  for  ammonia  sensitive  to  14  X 
10~'  g.  of  nitrogen  (Compt.  rend.  trav. 
lab.  Carlsberg,  Ser.  Chim.,  1933,  19, 
No.  20.) 

The  elegance  and  general  applica- 
bility to  diverse  problems  of  the  tech- 
niques and  methods  being  developed 
began  to  attract  research  workers  to 
the  Carlsberg  Laboratory  from  other 
countries,  and  the  series  of  publications 
that  had  evolved  under  the  heading  of 


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Studies  in  Enzymatic  Histochemistry 
soon  had  contributions  from  T. 
Philipson,  who  investigated  the 
peptidase  activity  of  single  centri- 
fugally  separated  parts  of  eggs  of 
Psammechinus  miliaris  {Ibid,  1934,  20, 
No.  4),  and  D.  Glick,  who  elaborated 
an  acidimetric  method  for  lipolytic  en- 
zymes {Ibid,  No.  5)  and  applied  it  to  a 
study  of  the  distribution  of  esterase  in 
the  gastric  and  duodenal  mucosa  of  the 
hog  (Glick,  D.  76id,  No.  11). 

The  latter  was  undertaken  since  Lin- 
derstr0m-Lang  and  Holter  had  already 
underway  similar  investigations  of  the 
distribution  of  pepsin  {Ibid,  1935,  20, 
No.  11),  acid  {Ibid,  20,  No.  11),  and 
peptidase  {Ibid,  20,  No.  11).  A.  S0e- 
borg-Ohlsen  joined  in  a  collaborative 
study  of  the  enzj'me  distribution  in  the 
hog  stomach  as  a  function  of  its  histo- 
logical structure  {Ibid,  20,  No.  11)  and 
the  enzyme  pattern  in  the  mucosa  was 
revealed  to  a  significant  degree. 

The  need  of  methods  for  the  chemical 
determination  of  additional  cellular 
constituents  diverted  the  Carlsberg 
Laboratory  workers  and  visiting  scien- 
tists to  the  task  of  elaborating  them, 
and  for  the  visitors  this  served  the  ad- 
ditional purpose  of  providing  excellent 
first  hand  training  and  experience  in 
the  use  of  the  techniques.  With 
Palmer,  A.  H.  {Ibid,  1935,  21,  No.  1), 
an  electrometric  chloride  titration  was 
developed  with  a  precision  of  about 
14  X  10-'  g.  of  chloride;  Levy,  M.  {Ibid, 
1936,  21,  101-110)  refined  the  Kjeldahl 
nitrogen  analysis  to  3  X  10""®  g.  of 
nitrogen,  Linderstr0m-Lang  (Compt. 
rend.  trav.  lab.  Carlsberg,  Ser.  Chim., 
1936,  21,  111-122)  worked  out  an  electro- 
metric  titration  method  for  sodium  plus 
potassium  sensitive  to  1  X  10"^  m. 
equiv.,  Norberg,  B.  {Ibid,  1937,  21, 
233-241)  evolved  a  titrimetric  estima- 
tion of  potassium  isolated  as  the 
iodoplatinate  that  was  accurate  to  1-2 
X  10— '  m.  equiv.,  and  with  Weil,  L. 
{Ibid,  1935,  21,  7-14)  micro  methods  for 
arginase  and  urease  were  established. 

Linderstr0m-Lang,  K.  and  Engel,  C. 
{Ibid,  1938,  21,  243-258)  employed  the 
method  that  had  been  previously  de- 
veloped for  reducing  sugars  for  the 
measurement  of  amylase  activity,  the 
distribution  of  which  they  studied  in 
barley,  and  Holter  and  Doyle,  W.  L. 
{Ibid,  1938,  22,  219-225)  subsequently 
modified  the  technique  to  gain  greater 
precision.  Meanwhile  micro  methods 
for  other  enzymes  were  being  developed. 
Holter  and  Doyle  (J.  Cell  Compt. 
Physiol.,  1935,  12,  295-308)  adapted  the 
iodometric  method  of  Stern  to  the  esti- 
mation   of    catalase    with    a    precision 


equivalent  to  the  decomposition  of  2  X 
10"*  g.  of  hydrogen  peroxide,  and  Glick, 
D.  (Compt.  rend.  trav.  lab.  Carlsberg, 
Ser.  Chim.,  1938,  21,  2G3-268)  extended 
his  esterase  method  to  include  the  meas- 
urement of  cholinesteraso  with  a  sensi- 
tivity equivalent  to  the  hydrolysis  of 
1  X  10~*  mole  of  ester. 

As  the  new  methods  were  made  avail- 
able their  application  to  specific  prob- 
lems followed  at  once.  Thus,  Linder- 
str0m-Lang,  K.,  and  Duspiva,  F.,  (Ibid, 
1936,  21,  53-84)  studied  the  digestion  of 
keratin  by  larvae  of  the  clothes  moth, 
and  Duspiva  followed  this  work  by  in- 
vestigations of  various  proteolytic  en- 
zymes of  clothes-  and  wax-moth  larvae 
{Ibid,  1936,  21,  177-202),  as  well  as  of 
pH  of  the  intestinal  juice  of  these  organ- 
isms for  which  a  micro  glass  electrode 
was  used  {Ibid,  21,  167-176).  Holter  (J. 
CellComp.  Physiol.,  1936,  8,  179-200) 
studied  peptidase  localization  in  marine 
ova,  with  Kopac  {Ibid,  1937,  10,  423- 
437)  the  localization  of  this  enzyme 
in  amoeba,  and  with  Doyle,  W.  L. 
(Compt.  rend.  trav.  lab.  Carlsberg,  Ser. 
Chim.,  1938,  22,  219-225)  the  amylase 
localization  in  amoeba.  Doyle  {Ibid, 
1938,  21,  291-299)  also  investigated  the 
catalase  and  peptidase  activity  in  ma- 
rine ova.  The  activation  of  leucylpep- 
tidase  of  single  Tubifcx  eggs  by  magnes- 
ium salts  was  reported  by  Holter,  H., 
Lehmann-Bern,  F.  E.  and  Linderstr0m- 
Lang  (Compt.  rend.  trav.  lab.  Carls- 
berg, Ser.  Chim.,  1938,  21,  259-262). 

Other  applications  in  this  period  in- 
clude a  study  of  the  distribution  of 
urease  in  the  dog  stomach  by  Linder- 
str0m-Lang  and  S0eborg-Ohlsen,  A. 
(Enzymologia,  1936,  1,  92-95),  and  an 
investigation  of  the  cholinesterase  dis- 
tribution in  the  hog  stomach  before  and 
after  administration  of  drugs  that  affect 
gastric  secretion  by  Glick  {Ibid,  1938, 
21,  269-281).  Using  a  titrimetric  dye 
method  previously  worked  out  (J.  Biol. 
Chem.,  1935,  109,  433-436)  Glick  fol- 
lowed the  changes  in  ascorbic  acid  in 
different  parts  of  the  developing  barley 
embryo  up  to  the  ten-day  sprout  stage 
{Ibid,  1937,  21,  203-209). 

Titrimetric  procedures  alone  had 
been  exploited  up  to  this  time.  But  in 
the  attempt  to  extend  the  applicability 
of  this  approach  to  quantitative  histo- 
chemistry, other  analytical  principles 
were  tested.  Colorimetry,  if  conducted 
on  the  required  micro  scale,  would  ob- 
viously open  a  vast  new  region  for 
exploitation,  but  the  limiting  factor  in 
the  middle  nineteen  thirties  was  the 
availability  of  the  necessary  equipment. 
At  that  time,  the  Zeiss  Pulfrich  step 
photometer,  that  employed  cuvettes  re- 


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180 


LINDERSTR0M-LANG  ET  AL 


quiring  0.2  ml.  of  liquid,  was  used  by 
Lundsteen,  E.  and  Vermehren,  E. 
(Ibid,  1936,  21,  147-166)  at  the  Carlsberg 
Laboratory  for  the  determination  of 
phosphorus  and  phosphatase  in  0.05 
ml.  blood  samples.  But  for  many  histo- 
chemical  purposes  the  liquid  volume 
needed  for  the  colorimetric  measure- 
ment was  still  too  great.  This  problem 
was  largely  solved  subsequently  as  will 
be  mentioned  later. 

In  1937  two  new  analytical  techniques 
were  ingeniously  developed  by  Linder- 
str0m-Lang  for  micro-biochemical  re- 
search. One  of  these  was  based  on  the 
dilatometric  principle  that  in  certain 
reactions  a  volume  change  of  the  total 
solution  occurs  which  results  in  a 
change  in  specific  gravity  great  enough 
for  measurement.  As  applied  by  Lin- 
derstr0m-Lang  (Nature,  1937,  139,  713- 
714),  a  density  gradient  is  set  up  by 
half -filling  a  glass  tube  with  a  kerosene- 
bromobenzene  mixture  of  appropriate 
specific  gravity,  and  then  filling  the  re- 
mainder of  the  tube  with  a  similar  mix- 
ture having  a  greater  proportion  of 
kerosene  to  give  a  lower  specific  gravity. 
If  the  liquid  in  the  middle  of  the  tube 
is  stirred,  and  the  tube  mounted  in  a 
thermostat,  a  stable  vertical  density 
gradient  is  produced.  If  into  this 
gradient  an  aqueous  drop  of  a  reaction 
mixture  is  placed,  it  will  fall  until  it 
reaches  the  level  at  which  its  specific 
gravity  equals  that  of  the  surrounding 
medium.  As  the  reaction  within  the 
drop  proceeds,  the  resultant  volume 
change  of  the  drop  will  alter  its  specific 
gravity  and  the  drop  will  then  move  to 
a  new  level.  In  some  cases  the  distance 
the  drop  will  move  has  been  found  to 
be  directly  proportional  to  the  extent 
that  the  chemical  reaction  has  occurred. 
By  employing  a  cathetometer  the  move- 
ment of  the  drop  can  be  followed  to  0.01 
mm.  and  finer  movements  can  be  ob- 
served with  an  ocular  micrometer  in  the 
telescope  of  the  instrument.  After  cali- 
bration of  the  gradient  with  drops  of 
salt  solution  of  known  specific  gravity, 
measurements  of  the  excursion  of  the 
drop  can  be  used  to  calculate  the 
amount  of  reaction  products  formed  in 
certain  instances. 

The  dilatometric  method  was  em- 
ployed by  Linderstr0m-Lang  and  H. 
Lanz,  Jr.  (Ibid,  1938,  21,  315-338),  for 
peptidase  measurements,  and  a  study 
was  made  by  Holter,  Lanz,  and  Linder- 
str0m-Lang  {Ibid,  1938,  23,  1^9)  of  this 
enzyme  during  the  first  cell  divisions  in 
the  egg  of  Psammechinis  miliaris. 
Linderstr0m-Lang,  Jacobsen,  O.  and 
Johansen,  G.  {Ibid,  17-25)  also  em- 
ployed the  density  gradient  tube  for 


the  measurement  of  the  deuterium  con- 
tent of  heavy  water  mixtures. 

The  other  new  technique  that  was 
evolved  at  this  time  was  based  on  the 
principle  of  the  Cartesian  diver  (Lind- 
erstr0m-Lang,  K.  Nature,  1937,  140, 
108-108).  The  diver  is  a  small  vessel, 
open  at  one  end,  that  will  rise  or  fall, 
while  submerged  in  a  salt  solution,  as  a 
result  of  a  change  in  its  gas  volume. 
The  volume  of  the  first  divers  used  was 
about  0.01  ml.,  and  a  reaction  mixture 
of  about  0.001  ml.  placed  in  the  bottom 
of  the  diver  was  employed  for  gaso- 
metric  measurements  of  enzyme  activity 
or  respiration.  The  measurement  was 
carried  out  by  applying  a  known  posi- 
tive or  negative  pressure  to  the  air  over 
the  tube  containing  the  submerged 
diver  to  bring  it  to  a  marked  level  in  the 
tube.  At  this  level  the  gas  volume 
in  the  diver  has  a  fi.xed  value,  and  by 
determining  the  external  pressure 
necessary  to  bring  the  gas  volume  back 
to  this  value  after  expansion  (in  case  of 
a  reaction  evolving  gas)  or  contraction 
(in  case  of  reaction  using  up  gas),  the 
actual  amount  of  gas  evolved  or  con- 
sumed can  be  calculated.  In  other 
words  the  apparatus  is  a  constant 
volume,  variable  pressure,  unit  similar 
in  this  respect  to  the  ordinary  Warburg 
apparatus  but  capable  of  measuring  gas 
changes  of  a  magnitude  a  thousand 
times  smaller.  Further  refinements 
were  developed  later  and  will  be 
mentioned. 

The  first  applications  of  the  Cartesian 
diver  method  were  to  the  gasometric 
measurement  of  cholinesterase  activity 
in  ganglia,  Linderstr0m-Lang  and  Glick 
(Compt.  rend.  trav.  lab.  Carlsberg, 
1938,  22,  300-306),  the  determination  of 
thiamine  and  cocarboxylase  in  yeast, 
Westenbrink,  H.  G.  l<i.\lbid,  1940,  23, 
195-212),  and  the  respiration  of  sepa- 
rated parts  of  the  embryos  of  Para- 
centrotus  lividus,  Lindahl,  P.  E.  and 
Holter  {Ibid,  249-256),  and  of  cells 
grown  in  tissue  culture,  Zamecnik  {Ibid, 
1941,24,59-67). 

The  following  period  in  the  work  of 
Linderstr0m-Lang  and  Holter,  although 
beclouded  by  the  German  occupation  of 
Denmark  and  the  accompanying  diffi- 
culties and  distractions,  was  devoted  to 
the  consolidation  of  the  advances 
already  made  by  further  refinements  of 
technique  and  the  extension  of  the 
methods  to  new  uses.  Meanwhile, 
Linderstr0m-Lang  and  Mogensen,  K.  R. 
{Ibid,  1938,  23,  27-35)  had  already 
described  a  cryostat  for  the  accurate 
sectioning  of  fresh  frozen  tissue  at  a 
constant  low  temperature  and  the  tech- 
nique of  handling  the  cut  sections  to 


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LINDERSTRPIM-LAXG  ET  AL 


prevent  their  curling  etc.,  and  Holter 
and  Lindahl  (Ibid,  1940,  23,  249-256)  had 
already  studied  the  distribution  of  pep- 
tidase in  the  Paracentrotus  embryo  in 
continuation  of  the  line  of  cytochemical 
work  on  marine  invertebrates  that  had 
been  instituted  earlier. 

Considering  the  disruptive  influence 
of  the  occupation  of  Denmark  by  the 
Nazis,  it  is  remarkable  that  the  Carls- 
berg  Laboratory  workers  continued  to 
be  productive,  but  perhaps  the  very 
need  to  have  a  refuge  from  the  wild 
events  outside  served  to  hold  them  to 
their  laboratory  benches  for  periods  of 
sweet  reasonableness.  The  record 
shows  that  during  these  years  the  Car- 
tesian diver  technique  was  subjected  to 
intensive  theoretical  analysis,  Linder- 
str0m-Lang  and  Holter  {Ibid,  1942,  24, 
105-138)  and  Linderstr0m-Lang  {Ibid, 
1942,  24,  249-280;  Ibid,  1943,  24,  333- 
398),  and  laboratory  refinement,  Holter 
{Ibid,  1943,  24,  399-478).  Zeuthen,  E. 
{Ibid,  479-518)  devised  a  diver  with  a 
gas  volume  a  hundred  times  smaller 
than  the  first  ones  used,  and  this  opened 
many  new  possibilities. 

Applications  of  Cartesian  diver  gas- 
ometry  at  this  time  included  studies  on 
the  respiration  of  the  egg  and  enibryo 
of  the  ascidian,  Ciona  intestinalis  L., 
Holter  and  Zeuthen  {Ibid,  1944,  25, 
33-65),  the  respiration  of  syncytia 
formed  by  abnormal  development  of 
Ciona  eggs,  Andresen,  Holter,  and 
Zeuthen  {Ibid,  1944,  25,  67-85),  and  ox- 
ygen uptake  during  mitosis  of  frog  eggs, 
Zeuthen  {Ibid,  1946,  25,  191-228). 
Holter  {Ibid,  1944,  25,  156-167)  also  de- 
veloped a  colorimetric  method  for 
measuring  the  volume  of  large  amoebae. 

Parallel  with  these  developments 
during  the  war  years  were  others. 
Schmidt-Nielsen  {Ibid,  1942,  24,  233- 
247)  worked  out  a  titrimetric  method 
for  analysis  of  fat  in  quantities  of  10"* 
g.  and  later  he  extended  this  investi- 
gation to  methods  for  the  determination 
of  the  iodine  number  in  amounts  of  fat 
of  this  order  {Ibid,  1944,  25,  87-96),  and 
he  described  the  extraction  and  frac- 
tionation of  fats  in  1  mg.  of  tissue 
{Ibid,  97-105).  Previous  work  on  the 
stomach  was  also  continued  by  Rask- 
Nielsen  {Ibid,  1944,  25,  1-32)  who 
studied  the  peptidase  content  of  the 
pyloric  portion  of  the  hog  stomach,  and 
Bottelier,  H.  P.,  Holter,  H.,  and  Linder- 
str0m-Lang,  K.  {Ibid,  1943,  24,  289-314) 
investigated  the  peptidase  in  the  roots 
of  barley. 

Further  extension  to  new  uses  of  the 
microtechniques  that  had  been  de- 
veloped marked  the  post-war  years. 
Bruel,  D.,  Holter,  H.,  Linder8tr0m-Lang, 


K.  and  Rozits.  K.  {Ibid,  1946,  25,  289- 
324)  elaborated  a  titrimetric  method  for 
the  determination  of  total  nitrogen 
with  an  accuracy  of  5  X  10~*  g.  nitrogen, 
Levi  and  Zeuthen  {Ibid,  273-288) 
adapted  the  density  gradient  tube  to 
micro  weighing,  and  later  Zeuthen,  E. 
{Ibid,  1948,  26,  243-266)  constructed  a 
Cartesian  diver  balance  that  gave  re- 
duced weights  to  1  X  10~*  g.  The  re- 
duced weight  is  the  weight  of  an  object 
minus  the  weight  of  an  equal  volume  of 
water.  This  quantity  is  convenient  for 
expressing  the  amount  of  a  small  bio- 
logical sample  since  it  is  a  measure  of 
the  quantity  of  the  substance  independ- 
ent of  its  water  content.  Zeuthen 
{Ibid,  1948,  26,  267-276)  followed  the 
reduced  weight  and  volume  of  amoebae 
during  starvation,  and  Holter  and 
Zeuthen  {Ibid,  277-296)  studied  metabo- 
lism and  reduced  weight  of  amoebae. 
A  theoretical  treatment  of  the  use  of  the 
Cartesian  diver  for  respiration  measure- 
ments on  single  cell  organisms  was 
given  by  Linderstr0m-Lang  {Ibid,  1946, 
25,  229-272). 

The  main  program  of  the  cytochemi- 
cal department  in  recent  years  has  been 
the  continuation  of  Holter's  previous 
work  on  the  biochemistry  of  unicellular 
organisms  and  the  developing  inverte- 
brate and  amphibian  embryo.  The 
demand  for  microanalysis  of  different 
constituents  during  the  course  of  these 
studies  has  led  to  further  expansion  of 
method  development. 

Apparatus  that  will  permit  colori- 
metric analysis  of  substances  at  the  level 
required  for  work  on  microtome  sec- 
tions, single  early  embryos,  or  proto- 
zoa was  developed  more  recently  by 
Holter  and  his  coworkers.  The  vol- 
umes of  liquid  needed  for  the  color- 
imetry  are  of  the  order  of  0.010  ml.  The 
cuvettes  employed  to  hold  these  small 
volumes  are  pieces  of  capillary  tubing 
having  a  lumen  of  about  1  mm.  in 
diameter  and  about  10  mm.  long.  The 
cuvette  is  sealed  to  a  microscopic  slide 
with  stopcock  grease,  filled  with  the 
colored  liquid,  and  a  cover  slip  is  placed 
on  top.  This  unit  is  set  on  the  stage  of  a 
microscope  and  a  fine  beam  of  filtered 
light  is  passed  through  it.  The  trans- 
mitted light  is  collected  by  a  low-power 
objective  and  sent  on  to  a  photocell 
fixed  over  the  ocular.  Galvanometer 
readings  are  taken  as  for  any  other 
photoelectric  colorimeter.  With  this 
equipment  Holter  and  L0vtrup  {Ibid, 
1949,  27,  27)  studied  the  proteolytic 
enzymes  in  amoebae  and  Krugelis 
{Ibid,  1950, 27,  273-290)  investigated  the 
properties    and    changes    of    alkaline 


LINE  TEST 


182 


LIPASE 


phosphatase  activity  during  amphibian 
development. 

Other  recent  work  that  has  come  from 
the  Carlsberg  Laboratory  includes  a 
micro  gasometric  method  for  sulfur 
compounds  such  as  cystine,  for  which  it 
is  accurate  to  1  X  10~'  g.  within  2.5%, 
Holter  and  L0vtrup  {Ibid,  1949,  27, 
72-78).  Further  development  of  the 
Cartesian  diver  technique  was  effected 
by  using  silicone  coated  divers, 
Schwartz  {Ihid,  1949,  27,  79-92),  and 
L0vtrup  {Ibid,  1950,  27,  125-136)  modi- 
fied the  diver  balance  of  Zeuthen.  In 
the  course  of  the  latter  study  L0vtrup 
designed  a  microbalance  with  a  sensi- 
tivity of  1  X  10~*  g.  To  conduct  studies 
on  the  density  of  microorganisms  such 
as  amoebae,  a  starch  density  gradient 
tube  was  developed  by  L0vtrup  {Ibid, 
1950,  27,  137-144).  From  a  combina- 
tion of  density  and  reduced  weight 
measurements  the  volume  of  amoebae 
could  be  determined  with  an  accuracy  of 

1  X  10-9  ml. 

Aside  from  the  classical  applications 
of  the  quantitative  techniques  and 
methods  that  Linderstr0m-Lang  and 
Holter  instituted,  their  procedures  are 
being  employed  ever  more  extensively 
by  an  increasing  number  of  scientists 
in  the  diverse  fields  to  which  quantita- 
tive histo-  and  cytochemistry  is  of 
great  importance.  In  fact,  the  develop- 
ment of  the  fundamental  aspects  of  all 
of  the  life  sciences  can  be  expected  to  be 
significantly  enhanced  by  their  con- 
tributions. 
Line  Test  for  vitamin  D.  This  is  the  basis 
for  calculating  the  U.S. P.  unit  of  vita- 
min D  potency.  The  line  test  was 
apparently  first  introduced  by  McCol- 
lum,  E.  v.,  et  al.,  J.  Biol.  Chem.,  1922, 
51,  41-49.  A  critique  of  the  test_  is 
given  by  Bills,  C.  E.,  et  al.,  J.  Biol. 
Chem.,  1931,  90,  619-636.  See  also 
Sherman,  H.  C.,  The  Chemistry  of  Food 
and  Nutrition,  New  York:  MacMillan, 
1941,  611  pp.  A  slightly  modified  tech- 
nique is  proposed  and  given  in  detail 
by  Martin,  G.  J.,  J.  Lab.  &  Clin.  Med., 
1940,  26,  714-719.  Inject  rats  intra- 
peritoneally  with  1  cc.  1%  aq.  sodium 
alizarin  sulfonate  at  pH  8.0  and  give 
supplements  of  measured  amounts  of 
vitamin  D  orally.  Animals  similarly 
stained  but  not  given  the  vitamin  serve 
as  controls.     After  test  periods  of  1  or 

2  days,  kill  the  animals,  remove  radii 
and  ulnae  and  examine  grossly  and  mi- 
croscopically for  alizarin  stained^  lines 
at  epiphysis.  See  also  use  of  Alizarin 
Red  S.  Both  this  and  the  sulfonate  are 
better  than  Madder  because  they  pro- 
vide quicker  and  more  intense  colora- 
tion of  bony  calcium  laid  down  during 


the  period  that  they  are  available  in  the 
circulation  as  accelerated  by  vitamin  D. 

Linguatulidae,  see  Parasites. 

Linin  (L.  linum,  flax).  The  acidophilic, 
thread-like  framework  of  nucleoplasm 
seen  in  sections  but  not  in  the  living 
nucleus. 

Lipase.  Frozen  sections  30ju  thick  and  4.5 
mm.  in  diameter  of  beef  adrenals  are 
extracted  in  30%  glycerol  -f  equal 
volume  1%  methyl  butyrate  in  glycine 
—  NaOH  buffer  at  pH  8.7;  digested  at 
40°C. ;  enzyme  action  arrested  by  addi- 
tion of  2%  phenol  (10  parts)  and  0.04% 
brom-thymol  blue  (1.5  parts)  to  3.5 
times  total  volume;  and  end  point  ti- 
trated at  pH  6.5  with  0.05  N  HCl. 
This  point  is  determined  by  comparing 
color  with  standard  color  of  brom-thy- 
mol blue  in  phosphate  buffer  pH  6.5. 
Nearby  sections,  some  stained  with 
hematoxylin  and  eosin,  and  others,  with 
Sudan  III,  are  examined  histologically. 
The  medulla,  which  exhibits  most 
lipolytic  activity,  contains  least  lipid. 
Estimations  of  esterase  are  also  de- 
scribed by  Glick  and  Biskind  (D.and 
G.  R.,  J.  Biol.  Chem.,  1935,  110,  575- 
582).  See  Barnes,  J.  M.,  Brit.  J.  Exp. 
Path.,  1940,  21,  264-275  for  analysis  of 
lipase  in  lymphocytes  and  polymor- 
phonuclear leucocytes  and  Hoagland, 
C.  L.,  et  al.,  J.  Exper.  Med.,  1942,  76, 
163-173  for  lipase  determinations  in 
elementary  bodies  of  vaccine  virus. 

An  important  new  technique  is  de- 
scribed and  well  illustrated  by  Gomori, 
G.,  Arch.  Path.,  1946,  41,  121-129: 

1.  Fix  thin  slices  of  fresh  tissue  in 
chilled  acetone  12-24  hrs.  in  ice  box. 

2.  Dehydrate  in  2  changes  absolute 
acetone,  12-24  hrs.  each,  room  tempera- 
ture. 

3.  Impregnate  in  5%  acetylcellulose 
(Eastman's  cellulose  acetate  "high 
acetyl,  low  viscosity,  no.  4644")  for 
24  hrs. 

4.  Drain  off  fluid,  transfer  to  2 
changes  benzene,  1  hr.  each. 

5.  Embed  in  paraffin  (56-62°C),  2 
changes,  1  to  \\  hrs.  each.  Cut  4-8m 
sections,  float  on  water  (db  35°C)  and 
mount  on  slides.  Pass  down  through 
xylol  and  alcohols  to  aq.  dest. 

6.  Incubate  at  37°C  6-12  hrs.  in  50  cc. 
Solution  I  +  2  cc.     Solution  II. 

Solution  I:  Glycerin  150  cc,  10%  aq. 
calcium  chloride,  50  cc;  half-molar 
maleate  buffer  pH  7  to  7.4  (maleic  acid, 
5.8  gm. ;  4%  aq.  sodium  hydroxide  94  cc. 
+  aq.  dest.  6  cc).  If  maleate  buffer 
is  omitted  mixture  should  be  adjusted 
to  pH  indicated. 

Solution  II:  5%  aq.  Tween  40,  or 
Tween  60  (Atlas  Powder  Co.,  Wilming- 
ton, Del.)   or  Product  81  with  about 


LIPIDS 


183 


LIPOCHRIN 


0.02%  merthiolate  added.     Keep  both 
stock  solutions  in  ice  box. 

7.  Rinse  in  aq.  dest.  and  transfer  to 
1-2%  aq.  lead  nitrate,  10-15  min. 

8.  Rinse  thoroughly  in  repeated 
changes  aq.  dest.  and  transfer  to  dilute 
solution  of  light  yellow  ammonium  sul- 
fide (few  drops  to  Coplin  jar  of  aq.  dost.) 
whereupon  sites  of  lipase  activity  be- 
come dark  brown. 

9.  Wash  under  tap  and  counterstain 
with  hematoxylin  and  very  lightly  with 
eosin. 

10.  Dehydrate  in  alcohols;  clear  in 
gasoline  or  tetrachloroethylene  (per- 
chloroethylene)  and  mount  in  clarite  in 
these  solvents.     Avoid  toluol  and  xylol. 

Modifications  have  been  suggested  bv 
Stowell  and  Lee  (Arch.  Path.,  1950,  50, 
519-537).  Consult  also  the  newer  tech- 
nique of  Nachlas  and  Seligman  (Anat. 
Rec.  1949,  105,  677-695)  See  Esterase.  _ 
Lipids.  Identification  of  various  kinds  in 
microscopic  preparations  is  extremely 
difficult.  As  Lison  (p.  192)  has  shown, 
reliance  cannot  be  placed  in  solubility 
tests.  Some  bodies,  soluble  in  alcohol, 
ether,  chloroform,  carbon  tetrachloride 
and  so  on,  are  not  fats  while  some  fats 
show  considerable  resistance  to  such  sol- 
vents. Formalin  fixation  itself  causes 
marked  changes  in  solubility  of  fatty 
bodies  (Ivaufmann,  C.  and  Lehmann, 
E.,  Virchow's  Archiv.  f.  Path.  Anat. 
und  Physiol.,  1926,  261,  623-648).  It  is 
not  unusual  to  find  fats  slightly  soluble 
or  insoluble  in  microscopic  preparations 
which  on  chemical  extraction  are  soluble. 
Results  of  examination  in  polarized  light 
must,  he  states,  be  interpreted  with 
caution.  Glycerides  and  fatty  acids 
examined  in  vivo  are  never  birefringent 
in  the  dissolved  condition.  After  freez- 
ing or  treatment  with  formalin  they  can 
become  crystalline  and  birefringent. 
Cholesterol,  in  the  form  of  birefringent, 
rhombic  plates,  is  of  rare  occurrence 
in  vivo,  but  easily  recognizable.  Choles- 
terides  appear  sometimes  as  droplets 
presenting  the  black  cross  of  polarization 
when  viewed  at  low  temperature. 
When  temperature  is  increased  they  lose 
birefringence  and  look  like  droplets  of 
fat.  Birefringence  is  lost  as  a  result 
of  osmication.  Coloration  with  sudan 
and  mounting  in  syrup  of  levulos 
decreases  birefringence.  Lison  gives 
following  tabular  method  of  analysis 
(abbreviated). 

1.  In  frozen  sections,  mounted  in  levulose  syrup, 
without  artificial  coloration,  generally  yellow 
orange  or  brown. 

2.  Iodine    -    iodide   solution    (like    Gram's    or 
Lugol's)  gives  black -green  or  brown.  Chromic 


acid  solution  decolorizes  quickly  or  slowly— 

carotinoids. 
2.  Above    reactions    negative.    Sulphuric    acid 
sometimes    gives    red    color — chTomolipoida. 
1.  In  frozen  sections  show  no  natural  color. 

2  Liebermann  reaction  (Schultze  or  Romieu 
technique)  positive:  color  blue,  purple  or  violet, 
becoming  green. 

3.  Digitonine  reaction  (Brunswick  or  Leulier- 
Noel  technique)  gives  crystals  strongly  illu- 
minated between  crossed  nicols,  unstainable 
by    histological    methods — free    cholesterol. 
3.  Digitonine   reaction   gives    no   crystalline 
ppt. — cholesteridea . 
2.  Liebermann  reaction  negative  after  repeated 
attempts,  no  coloration  or  brown  or  red  color. 
3.  Mounted  in  levulose  syrup,  without  arti- 
ficial  coloration,    examined    with    crossed 
nicols,  brightly  illuminated  and  showing 
cross  of  polarization — Lipines. 
3.  Mounted  in  same  way,  without  artificial 
coloration,  examined  with  crossed  nicols, 
not  illuminated  or  illuminated  but  without 
showing  cross  of  polarization. 
4.  Smith-Dietrich  reaction  at  50°C.   posi- 
tive, color  black — Lipines. 
4.  Above    reaction    negative.    Coloration 
gray  or  absent. 

6.  Lorrain  Smith  reaction  with  nile  blue 
sulphate:  rose — non-saturated  glyc- 
eride. 
6.  Above  reaction  absent  or  blue — Sat- 
urated or  non-saturated  glyceride,  or 
fatty  acid  or  Lipine. 

The  much  used  Osmic  Acid  and  Sudan 

staining  methods  are  helpful  when 
other  evidence  is  available  as  to 
chemical  constitution  of  substances 
demonstrated.  Fluorochromes  are  use- 
ful for  fluorescent  visualization  of  fats 
(Metcalf,  R.  L.  and  Patton,  R.  L.,  Stain 
Techn.,  1944,  19,  11-27).  See  Fatty 
Acids,  Soaps,  Neutral  Fats  (Glycerides), 
Lipoids,  Cholesterol  (free),  Cholesterol 
Esters,  Myeloldin,  Myelin,  etc. 

In  following  up  almost  any  problem 
in  biology  and  medicine  one  has  to  re- 
sort to  chemical  methods  and  seek  the 
help  of  chemists.  A  microtitration 
technique  for  lipid  in  amounts  of  ap- 
proximately 10  M  gm.  in  say  1  mg.  of 
tissue  has  been  elaborated  by  Schmidt- 
Nielsen,  K.  C.  rend.  trav.  lab.  Carls- 
berg,  Ser.  Chim.,  1942,  24,  233-247.  In 
compiling  a  book  like  this  it  is  interest- 
ing to  note  how  many  advances  have 
come  from  the  Carlsberg  laboratories. 

Lipines,  see  Lipoids. 

Lipiodol,  reactions  in  tissue  to  fat  stains 
after  various  fixations  (Black,  C.  E., 
J.  Lab.  &  Clin.  Med.,  1937-38,  23, 
1027-1036). 

Lipochrin  is  the  term  applied  to  certain 
usually  solitary  fatty  droplets  present 
in  retinal   cells  of  several  vertebrates 


LIPOCHROME 


184 


LOEFFLER'S  METHYLENE  BLUE 


but  absent  in  guinea  pigs  and  man. 
For  literature  see  Arey,  L.  B.  in 
Cowdry's  Special  Cytology,  1932,  3, 
1219. 

Lipochrome.  Defined  by  Lison  (p.  244) 
as  a  solution  of  a  carotinoid  in  a  fatty 
body,  the  latter  by  itself  uncolored, 
often  found  in  nerve,  hepatic,  cardiac 
muscle  cells  and  elsewhere.  See 
Carotinoids. 

Lipofuscins  are  fats  colored  by  the  carotene 
dissolved  in  them  found  in  nerve,  hepa- 
tic and  cardiac  muscle  cells  (Mallory, 
p.  125). 

Lipoids  (G.  lipos,  fat  +  eidos,  appearance). 
This  term  is  taken  to  mean  almost 
anything  even  remotely  looking  like 
fat.  Generally  included  under  it  are 
lecithin,  cephalin,  sphingomyelin,  kera- 
sin,  phrenosin,  etc.  which  cannot  be 
identified  microchemically  in  sections. 
They  are  referred  to  as  Lipines  by  Lison . 
See  his  tabular  analysis  under  Lipids. 
See  methods  of  Ciaccio  and  Smith- 
Dietrich. 

Lipolytic  Enzymes,  see  Lipase. 

Lipomicrons,  small  droplets  of  lipid  in 
circulating   blood.    See    Chylomicrons. 

Lipophanerosis  is  fatty  degeneration,  see 
Lipids. 

Lipoprotein.  Method  for  separation  and 
isolation  from  liver  cells  by  differential 
centrifugation,  see  Lazarow,  A.,  Anat. 
Rec,  1942,  84,  31-50;  Biological  Sym- 
posia, 1943,  10,  9-26. 

Lison's  glycogen  method  (Lison,  p.  227). 
Fix  in  dioxan  saturated  with  picric 
acid,  8.5  parts;  formalin,  1  part;  and 
acetic  acia,  0.5  cc.  Pass  direct  through 
dioxan,  dioxan-paraffin,  paraffin,  im- 
bed, section  and  stain  in  the  usual  way. 

Lithium  Carmine  1.  To  make  Orth's 
lithium  carmine  dissolve  2.5-5  gms. 
carmine  in  100  cc.  sat.  aq.  lithium 
carbonate.  Boil  for  10-15  min.  and, 
when  cool,  add  a  crystal  of  thymol  as 
an  antiseptic.  Stain  sections  about  3 
min.  Differentiate  in  Acid  Alcohol. 
Wash  in  water,  dehydrate  in  alcohol, 
clear  in  xylol,  or  toluol,  and  mount  in 
balsam.  Gives  sharp  bright  red  stain 
of  nuclei  often  useful  in  place  of  the 
blue  of  hematoxylin,  of  methylene  blue, 
etc.  It  may  be  used  after  almost  any 
good  fixative. 

2.  Lithium  carmine  has  also  been 
employed  in  many  classical  experiments 
as  a  vital  stain  (Aschoff,  L.  and  Kiyono, 
K.,  Folia  Haemat.,  1913,  6,  213;  Suzuki, 
T.,  Nierensekretion,  Jena,  1912 ;  Kiyono, 
K.,  Die  Vitale  Karminspeicherung, 
Jena,  1914,  etc.).  Filter  a  sterilized 
concentrated  suspension  of  carmine 
rubrum  optimum  (5  gm.)  in  cold  sat. 
aq.  lithium  carbonate  and  slowly  in- 
ject 5-10  cc.  intravenously  in  rabbits 


(Foot,  McClung,  p.  115) .  The  Bensleys 
(p.  151)  give  the  following  directions. 
Cook  on  water  bath  100  cc.  sat.  aq. 
lithium  carbonate  -f-  5  gm.  carmine 
rubrum  (Grubler)  for  ^1  hr.  Filter 
hot.  Allow  to  settle  and  cool.  Filter 
cold.  Sterilize  in  autoclave  and  filter 
again  through  sterile  filter.  Inject 
intravenously  once  or  more.  Kill  the 
animal  and  fix  tissues  in  alcohol,  forma- 
lin or  formalin-Zenker. 

Lithium  Silver  of  Hortega  as  described  by 
Laidlaw  (G.  F.,  Am.  J.  Path.,  1929,  5, 
239-247) :  In  250  cc.  glass  stoppered 
bottle  dissolve  12  gms.  silver  nitrate, 
C.P.  in  20  cc.  aq.  dest.  Add  230  cc. 
sat.  lithium  carbonate,  C.P.  in  aq.  dest. 
Shake  well.  Let  settle  to  about  70  cc. 
ppt.  Decant.  Washppt.  with  aq.  dest. 
3  or  4  times.  Decant  all  except  70  cc. 
ppt.  Add  ammonia  water  (26-28%) 
shaking  until  fluid  is  nearly  clear.  Add 
aq.  dest.  to  total  vol.  of  120  cc.  Filter 
through  Whatman  filter  paper  No.  42 
or  44  or  Schleicher  and  Schiill  No. 
589  into  stock  bottle.  See  Laidlaw's 
Methods. 

Litmus  as  a  vital  indicator  of  acidity  and 
allcalinity  in  rats  and  mice  (Rous,  P., 
J.  Exper.  Med.  1925,  41.  379-397).  See 
Hydrogen  Ion  Indicators. 

Liver.  In  this  very  large  organ,  as  in  the 
lungs,  it  is  necessary  to  carefully  select 
the  specimens  excised  for  study.  It  is 
bad  practice  to  take  only  slices  vertical 
to  the  surface  including  the  capsule. 
The  deeper  parts  should  be  included. 
How  the  weight  and  structure  of  the 
human  liver  varies  with  phases  of  as- 
similation and  secretion  as  in  rabbits 
(Forsgren,  E.,  Act.  med.  Scandin., 
1931,  76,  285^-315)  and  in  rats  (Higgins, 
G.  M.,  Berkson,  J.  and  Flock,  E., 
Am.  J.  Physiol.,  1933,  105,  177-186) 
remains  to  be  determined.  Effect  of 
different  dehydration  and  clearing 
agents  on  liver  (Ralph,  P.,  Stain  Techn., 
1938,  13,  9-15).  A  well  illustrated  ac- 
count of  the  influence  of  fixatives  on 
liver  cells  is  given  by  Schiller,  W.,  Zeit. 
f.  Zellf.  u.  Mikr.  Anat.,  1930,  11,  63-178. 

Locke  Solution.  As  given  by  Craig,  p.  69 
as  a  component  of  culture  medium  for 
amebae  it  is:  NaCl,  9.0  gm.;  CaCU,  0.2 
gm.;  KCl,  0.4  gm.;  NaHCOs,  0.2  gm.; 
glucose,  2.5  gm.;  aq.  dest.  1000.0  cc. 
sterilized  in  Arnold  sterilizer  or  in  auto- 
clave. 

Locke-Lewis  solution.  NaCl,  0.85  gm.; 
KCl,  0.042  gm.;  CaClj,  0.025  gm.; 
NaHCOa,  0.02  gm.,  dextrose,  0.01-0.25 
gm. ;  aq.  dest.,  100  cc.  Should  be  freshly 
made.  Owing  to  presence  of  NaHCOa 
must  not  be  sterilized  by  heat. 

Loeffler's  Alkaline  Methylene  Blue.  As 
emended  Soc.  Am.  Bact.     A.  Methylene 


LOGWOOD 


185      LUNGS,  UNCOLLAPSED,  FIXATION 


blue  (90%  dye  content)  0.3  gm.  +  95% 
ethyl  alcohol,  30  cc.  B.  0.01%  aq.  KOH 
by  weight  100  cc.  Mix  A  and  B 
(McClung,  p.  137). 

Logwood.  This  source  of  hematoxylin,  the 
most  important  of  the  older  dyes,  was 
discovered  by  the  Spaniards  at  the 
Bay  of  Campeachy  in  Mexico  and  was 
introduced  by  them  into  Europe. 
Much  used  in  Spain  in  the  16th  century 
logwood  was  banned  in  England  by  Act 
of  Parliament  in  1580  and  punishment 
provided  for  its  use.  A  hundred  years 
later  this  Act  was  repealed  and  since 
1715  the  tree  has  been  cultivated  in 
Jamaica  (Leggett,  W.  F.,  Ancient  and 
Medieval  Dyes.  Brooklvn:  Chemical 
Publishing  Co.,  Inc.,  1944,  95  pp.). 

Loose  Connective  Tissue.  Subcutaneous 
tissue  of  this  sort  is  often  chosen  for 
investigation.  It  may  be  dissected  out 
and  spread  on  slides.  A  good  way, 
demanding  practice,  is  to  tease  the  tis- 
sue apart,  without  the  addition  of  any 
saline  solution,  so  that  one  edge  is  paral- 
lel to  the  end  of  the  slide  and  about  4 
cm.  from  it.  This  edge  is  allowed  to 
dry  and  become  affixed  to  the  slide, 
while  the  remainder  of  the  tissue  is 
kept  moist  and  is  stretched  with  needles 
evenly  along  the  length  of  the  slide  into 
a  fairly  thin  film.  This  spread  is  then 
examined  in  the  fresh  state,  with  various 
solutions  added,  or  it  is  fixed  and  stained 
like  a  blood  smear.  Separation  of 
components  into  a  sufficiently  thin 
spread  is  facilitated  by  first  making  a 
bulla  (L.  for  bubble)  under  the  epi- 
dermis by  the  local  injection  of  fluid 
(salt  solution,  serum,  etc.). 

Sylvia  H.  Bensley  (Anat.  Rec,  1934 
60,  93-109)  employed  a  graphic  methoa 
for  demonstration  of  ground  substance. 
She  adapted  a  culture  of  paramoecia  to 
0.6-0.8%  salt  solution,  injected  sub- 
cutaneously  into  a  guinea  pig,  excised 
the  bulla  and  examined  it  as  a  whole 
mount  with  cover  glass  supported  at 
edges.  Actively  motile  organisms  sud- 
denly rebounded  without  coming  into 
contact  with  microscopically  visible 
structure  and  none  escaped  into  the 
surrounding  fluid  from  the  bulla.  This 
is  evidence  of  the  existence  in  loose 
connective  tissue  of  an  amorphous 
ground  substance  in  the  physical  condi- 
tion of  a  gel.  She  described,  and  used 
to  advantage,  methods  for  determina- 
tion of  the  refractive  index,  consistency, 
digestability  and  tinctorial  properties 
of  this  substance  in  several  parts  of  the 
body. 

Methods  for  the  identification  of 
Collagenic  and  Elastic  Fibers,  Fibro- 
blasts, Tissue  Basophiles  and  other 
constituents  are   described   under   the 


respective   headings.    See  also  Tissue 
Fluid. 

Lorrain  Smith,  see  Nile  Blue  Sulphate. 

Lubarsch  Crystals  are  tiny  formations  occa- 
sionally seen  post-mortem  intracellu- 
larily  in  testis  and  said  to  be  differont 
from  Charcot's  and  Spermin  Crystals. 

Lucas,  see  Clia,  Plastics. 

Lucidol,  a  trade  name  for  benzoyl  peroxide. 

Lucite,  disadvantages  of  as  substitute  for 
Canada  balsam  (Richards,  O.  W.  and 
Smith,  J.  A.,  Science,  1938,  87,  374). 
It  is  used  in  place  of  Quartz  for  transil- 
lumination by  Williams,  R.  G.,  Anat. 
Rec,  1941,  79,  263-270,  and  in  making 
containers  for  museum  specimens  by 
Snitman,  M.  F.,  Arch.  Otolaryng., 
1942,  36,  220-225. 

Lugol's  Iodine.  Potassium  iodide,  6  gm.; 
iodine,  4  gm.;  aq.  dest.,  100  cc. 

Luminescence,  Bacterial.  Technique  for 
inhibition  and  leads  to  literature  (John- 
son, F.  H.,  Flagler,  E.  A.,  Simpson,  R. 
and  McGreer,  K.,  J.  Cells  and  Comp. 
Physiol.,  1951,  37,  1-14). 

Luminol  (3-aminophthalhydrazide)  made 
by  Eastman  Kodak  Co.  has  a  marked 
affinity  for  hematin  yielding  brilliant 
luminescence  in  ultraviolet  light. 
Hematin  in  a  dilution  of  1:100,000,000 
can  be  detected  thereby.  This  is_  a 
medicological  test  of  great  sensitivity 
but  is  not  limited  to  human  blood 
(Proescher,  F.  and  Moody,  A.  M.,  J. 
Lab.  &  Clin.  Med.,  1938-39,  24,  1183- 
1189). 

Lungs.  To  excise  properly  pieces  for  fixa- 
tion requires  great  skill  especially  if 
lesions  are  present.  The  slices  should 
be  cut  with  the  sweep  of  a  particularly 
sharp  knife  to  minimize  squeezing  and 
the  resultant  distortion  and  displace- 
ment of  fluids  when  these  are  present. 
The  contents  of  small  cavities  and 
bronchi  may  escape  unless  care  is  taken 
to  retain  them  by  immediate  coagula- 
tion by  fixation.  Owing  to  regional 
differences  it  is  important  to  select 
representative  areas.  To  demonstrate 
the  fibrin  often  present  in  lesions, 
Weigert's  stain  is  recommended. 

Illumination  of  circulation  in  lung  by 
quartz  rod  (Weaver,  J.  T.  et  al.  Am.  J. 
Physiol.,  1934,  109,  23(>-256).  Observa- 
tion of  lung  through  thoracic  window 
in  vivo  (Terry,  R.  J.,  Science,  1939,  90, 
43-44),  See  Alveolar  Epithelium,  Alveo- 
lar Fluid,  Alveolar  Foam  Cells,  Alveolar 
Pores,  Alveolus,  Ammoniacal  Silver, 
Bronchiolar  Epithelium,  Carmine  Dust- 
ing, Celluloid  Corrosion,  Dust  Cells, 
Gash  Irrigation,  Heart  Failure  Cells, 
Pneumonocytes,  Silver  Lineation,  Tis- 
sue Phagocytes,  Vacuoloids,  Wash-out 
Recovery  Method. 

Lungs,  Uncollapsed,   Fixation — Written  by 


LUTECIUM 


186 


LYMPHATIC  VESSELS 


C.  C.  Macklin,  Dept.  of  Histological 
Research,  The  University  of  Western 
Ontario,  London,  Canada.  November 
28,  1951— This  may  be  done  (1)  by 
prompt  immersion  of  the  fresh  flayed 
intact  thorax  (IIT)  in  any  good  fixative 
which  is  adaptable  to  small  animals, 
such  as  mice;  (2)  by  perfusion  of  the 
blood  vessels  of  the  intact  thorax  of  the 
recently  exsanguinated  animal  (PIT); 
(3)  by  injection  of  the  fixative  into  the 
trachea  with  the  thorax  unopened 
(BF).  This  is  known  as  bronchial 
filling.  For  technique  of  methods  Nos. 
1  and  2  see  Dust  Cells.  For  method 
No.  3  a  cannula  is  tied  into  the  trachea 
and  fixing  fluid  injected  in  amount  equal 
to  one-third  to  one-half  of  the  volume 
of  the  lung  in  full  expiration.  Presence 
of  air  does  not  prevent  spread.  Fix- 
ation is  rapid.  After  tying  the  trachea 
the  preparation  is  allowed  to  stand  one 
to  twenty-four  hours.  This  method 
may  be  combined  with  No.  1.  Methods 
1  and  3  may  be  used  without  previous 
exsanguination.  In  methods  1  and  2 
the  capillaries  of  the  alveolar  walls  are 
fully  opened,  but  in  3  they  are  only 
partly  so.  Methods  1  and  2  possess  the 
advantage  of  not  having  loose  particles 
washed  into  the  lower  part  of  the  air 
tract.  Other  advantages  are  con- 
sidered under  "Dust  Cells".  Methods 
1  and  2  demonstrate  the  close  and  ex- 
tensive relation  of  the  capillaries  of  the 
alveolar  wall  to  the  pneumonocytes 
(Macklin,  C.  C,  Trans.  Roy.  Soc.  of 
Can.,  Sect.  V,  1946,  40,  93-111). 

Lutecium,  see  Atomic  Weights. 

Lutein,  see  Lillie,  p.  129. 

Luyet,  see  Revival  after  Ultra  Rapid  Cooling. 

Lymphatic  Vessels.  There  are  many  ways 
of  demonstrating  lymphatic  vessels. 
The  most  convenient  is  to  sit  in  an  easy 
chair  and  view  the  splendid  moving 
picture  prepared  by  Dr.  Richard  L. 
Webb  of  the  Department  of  Anatomy  of 
the  University  of  Illinois  College  of 
Medicine  entitled:  "Mesenteric  lym- 
phatics, their  conduct  and  the  behavior 
of  their  valves  in  the  living  rat". 

Another  easy  method  is  to  watch 
absorption  of  cream  in  a  cat.  A  fasting 
animal  is  fed  |  pint  of  cream  and  the 
abdominal  cavity  is  opened  under  ether 
anesthesia  a  few  minutes  later.  At  first 
sight  it  may  be  difficult  or  impossible  to 
see  any  lyroplmtics  in  the  mesentery 
although  a  few  bean  shaped  lymph  nodes 
are  visible  near  its  base  and  can  be 
easily  felt.  Keep  the  abdominal  con- 
tents moist  with  saline.  Close  the 
opening.  In  a  little  while,  when  again 
examined,  the  lymphatic  vessels  will  be 
clearly  marked  in  white  by  the  milk  fat 


which  has  been  absorbed  by  the  lacteals 
and  is  being  transported  in  them. 

A  simple  method  to  visualize  the 
pathways  of  lymphatic  drainage  from 
the  nasal  mucous  membrane  has  been 
described  by  Yoffey,  J.  M.,  Lancet, 
1941,  1,  529-530.  Anesthetize  a  cat. 
Drop  into  each  nostril  1  cc.  5%  trypan 
blue  (T.  1824)  in  physiological  saline 
(0.85%  aq.  NaCl).  T.  1824  is  specified 
because  it  is  a  trypan  blue  isomer  which 
is  deeply  colored  even  in  high  dilutions 
but  any  good  trypan  blue  will  do.  Dis- 
sect away  the  side  of  the  neck. 
Lymphatic  vessels,  deeply  stained,  will 
be  seen  from  the  nose  and  pharynx 
converging  to  the  deep  cervical  node 
and  from  the  posterior  border  of  this 
node  a  single  deep  cervical  vessel  takes 
origin  and  proceeds  downward  in  the 
neck.  The  technique  delineates  a  func- 
tioning system  of  vessels  actually  at 
work. 

Lymphatic  vessels  and  capillaries 
constitute  a  drainage  system  provided 
in  largest  measure  beneath  the  external 
surface  of  the  body  and  the  invagina- 
tions of  this  surface  into  it  in  the  respira- 
tory, alimentary  and  urinogenital 
systems.  They  are  absent  in  the 
brain  and  bone  marrow  and  rare  or 
absent  in  skeletal  muscle.  See  detailed 
information  concerning  the  organ  or 
tissue,  in  which  it  is  desired  to  demon- 
strate them,  to  be  found  in  Drinker, 
C.  K.  and  Yoffey,  J.  M.,  Lymphatics, 
Lymph  and  LymphoidTissue.  Harvard 
Univ.  Press,  1941,  406  pp. 

Methods  for  the  injection  of  lympha- 
tics involve  forcing  fluid  containing 
particulate  matter  into  areas  where 
there  are  many  lymphatic  capillaries. 
A  technique  for  the  observation  in  vivo 
of  the  superficial  lymphatics  of  human 
eyelids  is  described  by  Burch,  G.  E., 
Anat.  Rec,  1939,  73,  443-446.  0.02  cc. 
of  a  dilute  solution  of  patent  blue  V  is 
injected  intradermally  5-10  mm.  beyond 
the  middle  of  the  lid  margin.  The 
lymphatics  are  apparent  in  about  5 
min.  and  may  be  observed  as  long  as 
75  min.  Consult  earlier  experiments 
with  this  dye  by  McMaster,  P.  D., 
J.  Exp.  Med.,  1937,  65,  347-372. 

A  good  way  is  to  utilize  the  trans- 
parent ears  of  white  mice  to  inject  the 
lymphatics  with  hydrokollag  by  means 
of  a  microdissection  apparatus  (Pul- 
linger,  B.  D.  and  Florey,  W.  H.,  Brit.  J. 
Exp.  Path.,  1935,  16,  49-61).  But  the 
best  available  technique  is  closely  to 
examine  over  long  periods  of  time  living 
non-injected  lymphatics  in  Sandison 
cliambers  in  the  ears  of  rabbits  (Clark, 
E.  R.  and  E.  L.,  Am.  J.  Anat.,  1937, 
62,   59-92.     See   India  ink  method  for 


LYOGLYCOGEN 


187 


MACERATION 


renal  lymphatics  (Pierce,  C.  E.  2nd., 
Anat.  Rec,  1944,  90,  315-329). 

Lyoglycogen,  see  Glycogen. 

Lyons  Blue,  see  Spirit  Blue. 

Lymphocytes.  There  is  no  specific  stain 
for  lymphocytes,  but  identification  is 
usually  easy  at  least  for  small  lympho- 
cytes. To  observe  motility,  mount 
fresh  blood  and  ring  with  vaseline  to 
prevent       evaporation.  Movements 

usually  begin  after  the  neutrophiles 
have  become  active.  Examination  in 
the  darkfield  may  be  helpful.  Mito- 
chondria can  be  demonstrated  easier 
in  lymphocytes  by  supravital  staining 
with  Janus  Green  than  in  polymorpho- 
nuclear leucocytes  because  they  are  not 
obscured  by  the  specific  granulations. 
In  the  study  of  smears  the  characteristic 
cytoplasmic  basophilia  of  lymphocytes 
can  be  brought  out  by  most  of  the  usual 
stains  (Giemsa's,  Wright's).  The 
Peroxidase  Reaction  of  Ijonphocytes  is 
negative,  or  very  strictly  limited. 
Methods  demonstrating  Cathepsin,  Nu- 
clease, Amylase,  Lipase,  Lysozyme  and 
Adenosinase  in  lymphocytes  are  de- 
scribed by  Barnes,  J.  M.,  Brit.  J.  Exp. 
Path.,  1940,  21,  264-275.  To  determine 
the  age  of  lymphocytes  is  extraordinarily 
difficult.  Perhaps  the  nearest  approach 
to  this  goal  is  the  work  of  Wiseman, 
B.  K.,  J.  Exper.  Med.,  1931,  54,  270-294. 

Lysis.  In  histology  this  term  means  the 
solution  of  a  cell  resulting  from  injury 
to  the  cell  membrane.  A  choice  may 
be  made  of  several  agents  productive 
of  this  change.  As  classified  by  Danielli 
(Bourne,  pp.  74-75)  antibodies  and 
px)lyhydroxylic  phenols  probably  act 
almost  wholly  on  the  protein  component 
of  the  membrane;  lipoid  solvents, 
lecithinase,  digitonin,  sodium  or  potas- 
sium salts  of  fatty  acids  and  paraffin 
sulphonates  mainly  on  the  lipoid  part ; 
and  the  heavy  metals  probably  on  both. 
He  suggests  the  probable  modes  of 
action.  It  is  therefore  possible  that 
these  lytic  agents  may  in  their  action 
provide  clues  as  to  the  nature  of  the 
plasma  membrane.  See  Cell  Mem- 
branes. 

Lysozyme  a  heat  and  acid  resistant  enzyme 
produced  from  egg  white  and  isolated 
as  a  basic  protein  of  small  molecular 
weight  by  Abraham,  E.  P.,  Biochem. 
J.,  1939,  33,  622-630.  It  is  present  in 
many  animal  and  plant  tissues.  A 
method  for  its  determination  in  lympho- 
cytes and  polymorphonuclear  leuco- 
cytes (neutrophiles)  is  given  by  Barnes, 
J.  M.,  Brit.  J.  Exp.  Path.,  1940,  21,  264- 
275).  The  use  of  lysozyme  as  a  cy to- 
logical  agent  in  bacteriology  is  de- 
scribed by  Dubos,  R.  J.,  The  Bacterial 
Cell.  Harvard  Univ.  Press,  1945,  460  pp. 


Observation  that  a  bacterium  is  sus- 
ceptible to  lysozyme  is  an  indication 
that  it  contains  as  an  essential  part  of 
its  structure  a  substrate  for  this  en- 
zyme, probably  an  acetyl  amino  pol- 
j'saccharide. 
Lyssa  Bodies  are  small  Negri  bodies  which 
look  optically  hyaline,  sec  Negri  Bodies. 
Maceration  (L.  macerare,  to  soak)  is  a  very 
important  technique  by  which  tissues 
are  soaked  for  considerable  periods  of 
time  in  various  fluids  which  loosen  the 
connections  between  the  cells  and  allow 
them  to  be  easily  separated  for  micro- 
scopic study.  This  is  a  method  em- 
ployed by  the  great  masters  in  histology 
which  is  unfortunately  not  sufficiently 
used  now-a-days. 

For  nervous  tissue  Addison  (McClung, 
p.  439)  recommends  Gage's  dissociator 
which  is  2  cc.  formalin  in  1000  cc. 
physiological  salt  solution  for  2  or  3 
days.  After  this  treatment  large  ven- 
tral horn  nerve  cells  can  easily  be  dis- 
sected out  with  the  aid  of  a  binocular 
microscope,  stained  with  carmine,  picro- 
carmine  or  a  dilute  anilin  dye  and 
viewed  as  units  with  parts  of  their 
processes  attached. 

Smooth  muscle  of  the  bladder  is  well 
dissociated  bv  10-20%  nitric  acid 
(Dahlgren,  in  McClung,  p.  423).  The 
resulting  fibers  are  suitable  for  class  use. 

Thyroid  follicles  are  freed  from  the 
surrounding  tissue  and  can  be  examined 
individually  after  maceration  in  cone, 
hydrochloric  acid  3  parts  and  aq.  dest. 
1  part  for  about  24  hrs.  and  thorough 
washing  in  at  least  10  changes  of  tap 
water  (Jackson,  J.  L.,  Anat.  Rec,  1931, 
48,  219-239). 

Epidermis  can  be  separated  from 
dermis  by  maceration  in  1%  acetic  acid, 
see  epidermis. 

Kidney  tubules.  Pieces  of  kidney 
fixed  in  10%  formalin  or  in  Kaiserling's 
solution  are  placed  in  cone,  hydrochloric 
acid  at  room  temperature  until  they 
become  sufficiently  softened  after  2-7 
days.  The  time  depends  upon  size  of 
piece,  degree  of  fibrosis  and  other  factors. 
There  is  no  advantage  in  using  fresh 
tissue.  When  adequately  macerated 
the  almost  diffluent  tissue  is  washed  in 
repeated  changes  of  aq.  dest.  in  which 
it  may  be  kept  for  several  days.  Dis- 
sect out  individual  tubules  with  the 
aid  of  a  binocular  microscope  (Oliver, 
J.  and  Luey,  A.  S.,  Arch.  Path.,  1934, 
18,  777-816). 

Seminiferous  tubules.  Whole  human 
testicles  are  fixed  in  formalin.  They 
are  then  cut  into  segments  1  cm.  thick 
parallel  to  direction  of  the  lobules.  The 
tunica  vaginalis  is  not  removed  but  is 
slit  through  in  one  or  two  places  with  a 


MACNEAL'S  TETRACHROME 


188 


MAGDALA  RED 


razor.  Each  segment  is  placed  in  cone, 
hydrochloric  acid,  75  cc,  aq.  dest.  25  cc. 
1-7  days.  Heat  just  below  boiling 
20-30  min.  Tissue  shrinks,  turns  dark 
brown  and  softens.  A  sediment  collects 
in  the  dish.  Part  of  acid  is  drawn  off 
with  a  pipette,  boiled  water  is  added 
and  the  process  is  repeated  until  practi- 
cally all  of  the  acid  is  removed.  The 
water  is  boiled  to  prevent  formation  of 
air  bubbles  along  the  tubules.  It  turns 
the  tubules  a  yellowish  white  color  in 
which  condition  they  should  be  isolated 
by  careful  teasing.  When  the  tubules 
cannot  be  easily  lifted  away  from  one 
another,  the  maceration  is  insufficient. 
When,  on  the  other  hand,  they  break 
it  is  a  sign  of  over  maceration  (Johnson, 
F.  P.,  Anat.  Rec,  1934,  59,  187-199). 
A  similar  method  was  used  by  Johnson 
in  1916  to  separate  the  lobules  of  the 
pig's  liver. 

Bone  cells  and  lamellae.  Treat  a 
thin  bone  section  with  cone,  nitric 
acid  as  long  as  24  hrs.  Mount  on  a 
slide  and  squeeze  out  bone  cells  by  pres- 
sure on  cover  glass.  The  lamellae  can 
be  pealed  off  easily  from  a  piece  of 
decalcified  bone  which  has  been  gently 
boiled  in  water  (Shipley,  in  McClung, 
p.  348). 

Enamel  rods.  A  piece  of  dental  ena- 
mel is  dissociated  with  5-10%  hydro- 
chloric acid  for  24  hrs.  When  it  has 
become  soft,  remove  a  little  with  a 
needle  to  a  slide  and  tease  out.  Mount 
in  physiological  salt  solution  under  a 
cover  glass.  Draw  through  a  little 
carmine  stain  with  a  blotter  and  wash 
it  out  with  10%  acetic  acid.  The 
specimen  can  be  ringed  with  paraffin 
(Churchill,  and  Appleton,  in  McClung, 
p.  372). 

Nerve  cells.  Pieces  of  gray  matter 
of  ventral  horn  are  soaked  for  2  or  3 
days  in  0.02  formalin.  The  tissue 
softens,  the  cells  are  dissected  out  and 
stained  with  carmine  or  picro-carmine 
(Addison,  in  McClung,  p.  439). 

MacNeal's  Tetrachrome  is  a  blood  stain 
containing  eosin,  methylene  azure  A, 
methylene  blue  and  methylene  violet. 
It  is  employed  like  Wright's  stain. 
For  details  see  MacNeal,  W.  J.,  J.  A. 
M.  A.,  1922,  78,  1122,  and  Conn,  H.  J., 
Stain  Technology,  1927,  2,  31. 

Macrophages.  These  are  the  free  cells  of 
the  reticulo-endothelial  system.  Al- 
most any  method  of  exposure  to  rela- 
tively non-toxic,  finely  particulate 
matter  is  sufficient  to  bring  them  out. 
The  simplest  way  is  to  inject  mice  with 
trypan  blue  as  described  under  Vital 
Staining  and  to  look  for  the  macro- 
phages in  spreads  of  Loose  Connective 
Tissue.    Another     method,     used     by 


Maximow,  is  to  give  rabbits  intra- 
venous injections  of  saccharated  iron 
oxide  or  India  ink  and  to  examine  blood 
from  right  ventricle  in  smears  (see 
Cowdry's  Histology,  p.  69).  Lines  of 
division  between  macrophages  and 
monocytes,  if  they  exist,  are  difficult 
to  establish.  Supravital  staining  with 
Neutral  Red  and  Janus  Green  is  useful 
to  demonstrate  neutral  red  granules 
and  mitochondria  respectively. 

Madder  Staining  of  bone.  Madder  is  a  red 
dye,  prepared  from  the  plant  Rubia 
Tinctorum  which  has  been  used  for 
thousands  of  years.  It  is  perhaps  the 
first  dye  to  be  used  in  camouflage  in  war. 
With  its  help  Alexander  defeated  the 
Persians  by  staining  the  clothing  of  his 
Greek  soldiers  red,  each  garment  in  a 
different  part  so  that  the  Persian  leaders 
at  once  concluded  that  all  they  had  to 
cope  with  was  an  already  well  damaged 
army.  (Leggett,  W.  F.,  Ancient  and 
Medieval  Dyes.  Brooklyn:  Chemical 
Publishing  Co.,  Inc.     944,  95  pp.) 

Alizarin  and  purpurin,  formed  from 
madder,  are  now  made  syntheticall3\ 
Madder  should  be  employed  for  the 
vital  staining  of  growing  bone  as  de- 
scribed by  Macklin  (C.  C,  Anat.  Rec, 
1917,  12,  403-405;  J.  Med.  Res.,  1917, 
36,  493-507).  Young  rats  are  suggested 
as  material.  Each  should  eat  1-5  gms. 
of  madder,  thoroughly  mixed  with  its 
food,  daily.  The  calcium  deposited  in 
the  growing  bone  while  madder  is  thus 
made  available  in  the  circulation  is 
colored  red.  Staining  is  noticeable 
after  1  day  but  the  feeding  should  be 
continued  for  a  week  or  more. 

The  ventral  ends  of  the  ribs  and  the 
epiphyseal  lines  of  long  bones  are  most 
intensely  colored.  The  bones  selected 
are  fixed  in  10%  neutral  formalin, 
washed  and  cleaned  in  water,  dehy- 
drated thoroughly  in  alcohol,  placed  in 
benzene  for  24  hrs.,  cleared  in  oil  of 
wintergreen  by  the  method  of  Spalteholz 
and  examined  with  binocular  microscope 
as  whole  objects. 

Chemistry  of  madder  staining  is  dis- 
cussed by  Dr.  Richter  (Biochem.  J., 
1937,  31,  591-595).  The  substance  giv- 
ing the  intense  carmine  red  color  is 
apparently  purpurin  carboxylic  acid. 
Madder  is  one  of  the  most  classical  of 
stains.  Its  history  extends  back  through 
the  centuries  and  has  been  well  reviewed 
by  F.  T.  Lewis  (Anat.  Rec,  1942, 
83,  229-253).     See  Line  Test. 

Magdala  Red  (CI,  857) — naphthalene  pink, 
naphthalene  red,  naphthylamine  pink, 
Sudan  red — According  to  Conn  (p.  102) 
this  basic  naphtho-safranin  differs  from 
commercial  magdala  red  which  is  an 
acid  dye  belonging  to  an  entirely  dif- 


MAGENTA 


189 


MALARIAL  PIGMENT 


ferent  group.  He  calls  attention  to  its 
use  by  Kultschitzky,  N.,  Arch.  f. 
Mikr.  Anat.,  1895,  46,  673-695)  in  stain- 
ing elastic  tissue  of  tlie  spleen.  Used 
as  a  fluorochrome  for  Lipids. 

Magenta,  see  Basic  Fuchsin. 

Magenta  II  is  triamino  ditolyl-phenyl- 
methane  chloride  probably  present  in 
most  samples  of  Basic  Fuchsin.  See 
Pararosanilin  (Magenta  O),  Rosanilin 
(Magenta  I)  and  New  Fuchsin  (Magenta 
III). 

Magnafiux  is  a  useful  instrument  employed 
in  the  FBI  Laboratory  to  detect  the 
occurrence  of  small  cracks  and  defects 
in  the  surface  of  metallic  objects. 
When,  for  example,  a  magnetizable 
object  is  placed  in  a  magnetic  field, 
created  by  the  magnafiux,  the  field  is 
distributed  throughout  the  metal  if  it 
is  sound.  Otherwise,  magnetizable  pig- 
ments become  oriented  around  the 
breaks  in  the  surface  indicating  their 
location  (Hoover,  J.  E.,  Scientific 
Monthly,  1945,  60,  18-24).  Obviously 
metallic  laboratory  equipment  can  be 
tested  in  this  way. 

Magnesium,  Titan  yellow  method  for  de- 
termination of  small  amounts  in  body 
fluids  (Haury,  V.  G.,  J.  Lab.  &  Clin. 
Med.,  1938,  23,  1079-1084). 

Methods  for  detection  in  plant  cells 
(Broda,  B.,  Mikrokosmos,  1939,  32, 
184).  (1)  Triturate  1  part  quinalizarin 
with  5  parts  sodium  acetate  crystals. 
Make  to  fresh  0.5%  solution  in  5%  aq. 
NaOH.  Addition  of  1-2  drops  to  paraf- 
fin section,  then  1-2  drops  10%  NaOH 
results  after  some  hours  in  blue  stain. 
(2)  Add  to  paraffin  section  1-2  drops 
0.2%  aq.  Titan  yellow,  then  1-2  drops 
10%  NaOH  gives  rise  to  brick  red  stain 
of  magnesium.  (3)  Add  to  paraffin  sec- 
tion 0.1%  aq.  azo  blue.  Gives,  without 
the  NaOH,  a  violet  stain  of  magnesium. 
An  attempt  should  be  made  to  adjust 
these  techniques  to  human  tissues  in 
which  a  magnesium  salt  has  been 
injected. 

By  means  of  a  specially  constructed 
electron  microscope  Scott  and  Packer 
(G.  H.  and  D.  M.,  Anat.  Rec,  1939 
74,  17-45)  have  accurately  localizea 
magnesium  and/or  calcium  in  muscle. 
The  method  can  be  extended  to  other 
tissues  and  perhaps  to  other  minerals. 
Histospectrography  gives  data  on  the 
amount  of  magnesium  relative  to  the 
other  minerals  in  the  skin  of  normal  and 
neurodermatitis  patients.  In  the  latter 
there  is  a  magnesium  deficiency  (Mac- 
Cardle,  R.  C.,  Engman,  M.  F.,  Jr.  and 
Sr.,  Arch.  Dermat.  and  Syph.,  1941, 
44,  429^40). 

If  it  is  desired  to  supplement  micro- 
scopic and  spectrographic  detection  of 


magnesium  by  quantitative  analysis  of 
very  small  amounts  of  tissue  a  tech- 
nique of  microdermination  with  the 
polarograph  devised  by  Carruthers,  C., 
Indust.  and  Engin.  Chem.,  1943,  15, 
412-414  will  be  useful.  It  has  been 
employed  for  analysis  of  pure  epidermis 
by  Carruthers,  C,  and  Suntzeff,  V., 
Cancer  Research,  1943,  3,  744-748.  See 
Calcium  5. 

The  Spectrophotometric  determina- 
tion of  magnesium  in  human  serum  has 
been  advanced  by  Craig,  P.,  Zak,  B., 
Iseri,  L.  T.,  Boyle,  A.  J.  and  Myers,  G. 
B.  (Am.  J.  Clin.  Path.,  1951,  21,  394-398) 
through  the  making  of  several  innova- 
tions: (1)  destruction  of  protein  by 
nitric  acid  digestion,  (2)  use  for  stabi- 
lizer of  a  sodium  lauryl  sulfate-poly- 
vinyl alcohol  reagent,  (3)  choice  of  a  550 
m/x  wave  length  for  quantitation  and 
(4)  the  preparation  of  calibration  curves 
from  standards  containing  urea  and 
electrolytes  normally  occurring  in 
plasma.  It  would  seem  feasible  by 
different  calibration  and  other  adjust- 
ments to  utilize  this  technique  for  the 
estimation  of  magnesium  in  fluids  other 
than  serum  and  perhaps  in  tissue 
homogenates. 

Magnetic  Particle  Technique  to  investigate 
the  phj'sical  properties  of  the  cytoplasm 
of  living  cells  as  determined  by  move- 
ments of  phagocytized  particles  of 
various  sorts  in  a  magnetic  field  (Crick, 
F.  H.  C.  and  Hughes,  A.  F.  W.,  Exp. 
Cell  Res.,  1950,  1,  37-80).  This  appears 
to  be  a  very  promising  method. 

Malachite,  a  mineral  mined  by  the  Egyp- 
tians, and  applied  as  a  powder  gave  a 
green  pigmentation  about  the  eyes. 
It  is  said  to  be  the  oldest  coloring  mat- 
ter known  to  them  (Leggett,  W.  F., 
Ancient  and  Medieval  Dyes.  Brook- 
lyn: Chemical  Publishing  Co.  Inc., 
1944,  99  pp.). 

Malachite  Green  (CI,  657) — diamond  green 
B,  BX  or  P  extra,  light  green  N,  new 
Victoria  green  extra,  O,  I  or  II,  solid 
green  O,  Victoria  green  B  or  WB — 
Commission  Certified.  A  feebly  basic 
di-amino  tri-phenyl  methane  dye  quite 
extensively  employed  as  a  counterstain 
for  safranin  or  carmine. 

Malachite  Green  G,  see  Brilliant  Green. 

Malarial  Pigment.  Produced  in  erythro- 
cytes by  action  of  the  parasites,  black 
and  distinguishable  from  carbon  by  its 
solubility  in  concentrated  sulphuric 
acid.  Among  distinguishing  character- 
istics given  by  Lison  (p.  254)  are 
solubility  in  dilute  alkalis,  argentaffine 
reaction  negative,  specific  stains  for 
lipids  negative,  likewise  reactions  for 
iron.  But  Morrison  and  Anderson  (D. 
B.  and  W.  A.  D.,  Public  Health  Rep., 


MALARIA  PLASMODIA 


190 


MALLORY-HEIDENHAIN  STAIN 


1942,  57,  90-94)  find  that  when  the 
pigment  within  the  parasites  (Plas- 
modium Knowlesi)  is  extracted  in 
such  a  way  as  not  to  influence  the 
spectra  of  hemoglobin  it  can  be  identified 
spectrophotometrically  as  ferrihemic 
acid,  or  hematin,  which  does  contain 
iron. 
Malaria  Plasmodia.  Technique  of  examina- 
tion of  process  of  "exflagellation" 
(Anderson,  Ch.  W.  and  Cowdry,  E.  V., 
Arch,  de  I'lnst.  Pasteur  de  Tunis,  1928, 
17,  46-72),  of  quantitative  determina- 
tions of  gametocytes  (Cowdry,  E.  V. 
and  Covell,  W.  P.,  Ibid.,  147-456)  and 
of  demonstrating  neutral  red  granules 
and  Golgi  apparatus  (Cowdry,  E.  V. 
and  Scott,  G.  H.,  Ibid.,  233-252). 

For  staining  the  plasmodia  in  smears, 
see  Giemsa,  Jenner,  Marino,  Nocht, 
Plehn,  Wilson  and  Wright's  stains.  A 
simple  method  for  staining  plasmodia 
in  paraffin  sections  is  described  with 
numerous  illustrations  by  Tomlinson, 
W.  J.  and  Grocott,  R.  G.,  Am.  J.  Clin. 
Path.,  1944,  14,  316-326.  The  Barber 
Eomp  thick  film  method  is  strongly 
recommended  for  surveys. 

Serlin,  N.  J.  and  Lissa,  J.  R.,  Am.  J. 
Clin.  Path.,  1942,  6,  8  advise  the  follow- 
ing method  when  diagnosis  depends  on 
finding  gametocytes,  or  malarial  pig- 
ment, in  peripheral  blood.  Completely 
evaporate  1  cc.  1%  aq.  potassium  oxa- 
late in  a  15  cc.  centrifuge  tube.  Add  10 
cc.  venipuncture  blood.  Mix  carefully 
and  centrifuge  30  min.  at  2,500  R.P.M. 
Pipette  oil  all  but  about  J  in.  of  super- 
natant plasma.  Smear  on  2  slides  by 
wiping  buffer  layer  with  stick  applicator 
having  non-absorbent  cotton  tip. 
Stain  by  Wright's  method.  Study  of 
Giemsa  stained  smears  by  dark  field  is 
suggested  (Goosmann,  C.,  J.  Lab.  & 
Clin.  Med.,  1935-36,  21,  421-424).  See 
Protozoa. 

Taylor,  D.  J.,  Greenberg,  J.  and 
Josephson,  E.  S.  (J.  Lab.  Dis.,  1951,  88, 
158-162)  describe  a  useful  method  for 
the  maintenance  of  intraerythrocytic 
forms  of  Plasmodium  gallinaceum  in  a 
whole  medium  on  vitro. 
Mallory's  Connective  Tissue  Stain.  This 
is  name  usually  given  to  his  anilin 
blue-acid  fuchsin-orange  G  stain.  See 
also  his  Phosphomolybdic  and  Phospho- 
tungstic  Acid  Hematoxylin  Stains. 
(Mallory,  p.  155).  Fix  in  Zenker's 
fluid.  Imbed  in  paraffin  or  celloidin. 
Remove  mercury  from  sections  with 
iodine  or  0.5%  sodium  hyposulphite. 
Stain  in  0.5%  aq.  acid  fuchsin,  1-5  min. 
Drain  off  stain  and  put  in :  anilin  blue, 
water  soluble,  0.5  gm. ;  orange  G,  2  gm. ; 
1%  aq.  phosphotungstic  acid,  100  cc, 
20  min.  or  longer.    Rinse  in  95%  ale. 


2  or  3  changes  until  no  more  stain  is 
removed.  Dehydrate  in  abs.  ale,  clear 
in  xylol,  mount  in  neutral  balsam.  For 
celloidin  sections,  reduce  staining  time 
and  pass  from  95%  ale.  to  terpineol  and 
mount  in  balsam.  This  is  one  of  the 
most  beautiful  of  all  stains  and  is  very 
widely  used.  Collagenic  fibrils  blue, 
fibroglia,  neuroglia  and  myoglia  fibrils 
red,  elastic  fibrils  pink  or  yellow.  In 
McClung,  p.  405,  Mallory  and  Parker 
advise  0.25%  aq.  acid  fuchsin  and 
staining  in  the  anilin  blue  mixture  for 
1-24  hrs.  or  for  1  hr.  in  paraffin  oven  at 
60  °C.  The  modifications  of  this  stain 
are  almost  endless. 

Adaptation  to  formalin  fixed  material 
is  often  desirable.  Kernohan  (J.  W., 
J.  Tech.  Meth.,  1934,  13,  82-84)  has 
outlined  the  following  method  of  doing 
this  by  mordanting.  Wash  formalin 
fixed  tissue  in  running  water  or  in 
ammonia  water  for  short  time.  Place 
in  Weigert's  primary  mordant — potas- 
sium bichromate,  5  gm.;  chromium 
fluoride,  2  gm.  and  aq.  dest.  100  cc. — 
for  4  days  and  in  his  secondary  mordant 
— copper  acetate,  5  gm.;  chromium 
fluoride,  2.5  gm.;  acetic  acid  (36%), 
5  cc;  aq.  dest.,  100  cc.  and  formol, 
10  cc. — for  2  days.  Imbed  in  paraflin 
in  the  usual  way. 

Rexed,  B.,  and  Wohlfart,  G.,  Zeit. 
wiss.  Mikr.,  1939,  56,  212-215  suggest 
control  of  pH  of  the  acid  fuchsin.  It  is 
stated  that  fresh  0.1%  acid  fuchsin  has 
pH  4.49  and  that  increase  in  alkalinity 
makes  it  defective.  To  prepare  one  at 
pH  3.29  ±  0.01,  which  is  recommended, 
take  acid  fuchsin  1  gm.;  N/10  HCl,  60 
cc. ;  aq.  dest.  900  cc. ;  Storensen's  citrate 
(citric  acid  crystals,  21  gm.;  N/1 
NaOH,  200  cc;  +  aq.  dest.  to  make 
1000  cc),  40  cc.  Most  tissues  stain  in 
range  pH  3-4,  red  blood  cells  alone  at 
pH5-7. 

In  1936,  Mallory  considered  (Stain 
Tech.,  11,  101-102)  the  most  important 
modifications  of  his  stain  to  be  Heiden- 
hain's  Azocarmine  (Azan),  the  Lee- 
Brown  and  Masson  Trichrome  methods. 
See  Grossman's  modification  and  Pitui- 
tary for  special  adaptations. 
Mallory-Heidenhain  Rapid  One-Step  Stain 
for  Connective  Tissue — Written  by 
Jane  E.  Cason,  Dept.  of  Pathology, 
Medical  College  of  Alabama,  Birming- 
ham, Ala.  January  27,  1951— Although 
innumerable  modifications  of  Mallory's 
aniline  blue  collagen  stain  for  tissue 
sections  prevail,  this  procedure  appears 
to  be  an  improvement  over  the  original 
and  subsequent  modifications. 

The  necessity  for  adjusting  the  in- 
tensity of  the  aniline  blue  and  the  acid 
fuchsin  in  routine  staining  suggested 


MAMMARY  GLANDS 


191 


MANGANESE  DIOXIDE 


the  possibility  of  combining  the  two 
original  solutions  at  a  modified  ratio. 
The  results  proved  quite  successful,  and 
three  definite  advantages  of  this  pro- 
cedure were  apparent:  1)  Only  one  step 
is  involved.  2)  sufficient  and  desirable 
coloration  is  accomplished  in  five  min. 
3)  The  intensity  of  the  color  is  con- 
sistent. 

The  coloration  appears  the  same  as 
that  listed  by  Mallory:  Collagenous 
fibrils  stain  intense  blue;  ground  sub- 
stances of  cartilage  and  bone,  mucus, 
amyloid  and  certain  other  hyaline  sub- 
stances stain  varying  shades  of  blue; 
nuclei,  fibroglia,  myoglia  and  neuroglia 
fibrils,  axis  cylinders  and  fibrin  stain 
red;  nucleoli,  red  blood  corpuscles  and 
myelin  stain  yellow;  elastic  fibrils  stain 
pale  pink  or  yellow.  This  technique  is 
suggested  for  routine  pathologic  stain- 
ing because  it  is  simple  to  follow,  rapid 
and  consistent. 

To  make  stain  add  to  200  cc.  aq.  dest. 

Phosphotungstic  acid  crystals  (Merck). . .  1  gm. 

Orange  G  (C.  I.  No.  27) 2  gm. 

Aniline  blue,  W.  S.  (C.  I.  No.  707) 1  gm. 

Acid  fuchsin  (C.  I.  No.  692 3  gm. 

Formalin-Zenker  is  the  preferred  fixa- 
tive, but  Bouin's,  formalin,  and  alcohol 
have  been  used  with  success.  Embed 
tissue  in  paraffin,  and  cut  sections  at  6^. 

1.  Deparaffinize  in  xylene,  pass 
through  graded  alcohols  to  tap  water. 
(If  formalin-Zenker  is  the  fixative,  treat 
with  iodine  and  sodium  thiosulfate.) 

2.  Stain  sections  5  min.  in  staining 
solution. 

3.  Wash  in  running  tap  water  3  to 
5  sec. 

4.  Dehydrate  rapidly  through  95% 
and  100%  ale,  clear  in  xylene,  and 
mount  in  balsam. 

Mammary  Glands.  These  can  be  studied 
in  sections  by  methods  intended  to 
reveal  the  particular  data  sought.  For 
general  purposes  Hematoxylin  and 
Eosin,  Mallory's  Connective  Tissue 
Stain,  or  Phloxine-Methylene  Blue  is 
recommended  after  Zenker  fixation. 
For  fat  use  Sudan  Black  and  Oil  Red  O 
on  frozen  sections  after  fixation  in  10% 
formalin  or  examine  in  paraffin  sections 
after  fixation  in  Flemming's  fluid  or 
some  other  osmic  acid  containing  mix- 
ture. 

In  the  case  of  the  small  glands  of  mice, 
rats,  rabbits  and  other  mammals  the 
method  of  making  whole  mounts  is 
invaluable  in  investigations  of  the 
responses  of  mammary  glands  to  endo- 
crine stimulation.  The  following  is 
essentially  the  same  technique  as  that 
originally  described  by  Turner,  C.  W. 
ana  Gardner,  W.  U.,  Agri.  Exp.  Res. 


Stat.  Bull.,  Univ.  of  Mo.,  1931,  158, 
1-57  :  Remove  skin  and  mammary  gland. 
Stretch  out  and  fasten  on  a  cork  block 
with  the  external  surface  of  the  skin 
down.  Fix  in  Bouin's  fluid  24  hrs. 
Wash  in  tap  water.  Dissect  away  all 
tissue  over  the  gland  which  has  been 
tinged  light  yellow  by  the  picric  acid 
in  the  fixative.  Remove  the  gland  from 
the  skin.  Stain  in  Mayer's  Hemalum. 
Wash  in  l%aq.  potassium  alum  and  then 
in  water.  Differentiate  in  70%  ale.  + 
2%  of  hydrochloric  acid  until  the  color 
has  been  removed  from  the  connective 
tissue  and  the  acini  and  ducts  of  the 
glands  show  up  in  sharp  contrast  in  a 
light  background.  Wash  in  tap  water. 
Dehydrate  in  alcohol,  clear  in  xylol, 
mount  in  balsam  between  glass  plates 
and  close  the  edges  with  sealing  wax. 
Much  can  be  made  out  when  magnified 
only  2-5  times.  Small  pieces  can  be 
mounted  on  slides,  with  edges  of  cover 
glasses  supported  as  may  be  necessary, 
for  examination  at  higher  magnifications. 
There  are  many  excellent  pictures  in 
the  paper  cited. 

For  examination  of  fetal  mice,  see 
Turner,  C.  W.  and  Gomez,  E.  T.,  ibid, 
1933,  182,  1-43.  Valuable  data  are 
given  in  Turner's  chapter  on  mammary 
glands  in  Allen's  Sex  and  Internal 
Secretions,  Baltimore:  Williams  &Wil- 
kins,  1939,  1346  pp.  For  techniques  to 
reveal  secretory  phenomena  in  mam- 
mary glands,  see  Weatherford,  H.  L., 
Am.  J.  Anat.,  1929,  44,  199-281 ;  Jeffers, 
K.  R.,  Am.  J.  Anat.,  1935,  56,  257-277, 
279-303.  Technique  for  localizing  site 
of  fat  formation  in  mammary  glands  is 
given  by  Kelly  and  Petersen,  J.  Dairy 
Sci.,  1939,  22,  7.  The  differential  stain- 
ing of  sections  of  unpreserved  bovine 
udder  tissue  is  to  be  found  in  U.  S. 
Dept.  of  Agri.  Circular  No.  514,  under 
authorship  of  W.  T.  Miller  and  H.  W. 
Johnson.  A  method  for  obtaining 
serial  slices  of  whole  human  breasts  is 
described  by  Ingleby,  H.  and  Holly,  C, 
J.  Tech.  Meth.,  1939,  19,  93-96. 

Manchester  Blue  (British  Drug  Houses 
Ltd),  a  dis-azo  dye  of  the  benzidine 
series.  In  either  alcoholic  or  aqueous 
solution  it  gives  a  sharp  deep  blue 
effect  (H.  G.  Cannan,  J.  Roy.  Micr. 
Soc,  1941,61,88-94). 

Manchester  Brown,  see  Bismark  Brown  Y. 

Manchester   Yellow,   see  Martins   Yellow. 

Mandarin  G,  see  Orange  II. 

Manganese.  Histochemical  detection  un- 
certain (Lison,  p.  98). 

Manganese  Dioxide.  Drinker,  C.  K.  and 
Shaw,  L.  H.,  J.  Exper.  Med.,  1921, 
33,  77-98  employed  a  suspension  of  fine 
particles  in  acacia  water  to  investigate 
phagocytic   power  of   endothelium   be- 


MANN'S  FIXATIVE 


192 


MASSON'S  TRICHROME  STAIN 


cause  the  particles  can  be  seen  within 
the  cells  and  the  amounts  of  manganese 
in  the  tissues  can  be  determined  by 
chemical  analysis. 

Mann's  Fixative  is  equal  parts  1%  aq.  osmic 
acid  and  sat.  corrosive  sublimate  in  phys- 
iological salt  solution  (0.85%  NaCl). 
It  is  a  good  way  to  apply  osmic  acid  for 
the  blackening  of  fat. 

Mann's  Methyl  Blue-Eosin  Stain.  This 
is  used  for  protozoa  and  for  inclusions 
caused  by  viruses.  Sections  are  de- 
parafhnized,  stained  12  hrs.  in  1%  aq. 
methyl  blue  35  cc,  1%  aq.  eosin  45  cc. 
and  aq.  dest.  100  cc.  They  are  then 
rinsed  in  95%  ale,  dehydrated  cleared 
and  mounted.  See  Alzheimer's  Modi- 
fication of  Mann's  method. 

Manometer  for  capillary  blood  pressure, 
see  Landis,  E.  M.,  Am.  J.  Physiol., 
1926,  75,  548. 

Marchi  Method.  For  degenerating  nerve 
fibers.  Modification  by  Swank,  R.  L. 
and  Davenport,  H.  A.,  Stain  Techn., 
1935,  10,  87-90.  Details  provided  by 
Dr.  J.  L.  O'Leary.  Degeneration  time 
of  approximately  14  to  20  days.  Kill 
animal  by  overdose  of  nembutal  or  some 
other  barbiturate  given  intraperi- 
toneally.  Open  left  ventricle,  insert 
cannula  into  aorta  and  perfuse  with 
2.5-5%  anhydrous  (10%  crystalline) 
magnesium  sulfate  solution  containing 
2-3%  potassium  bichromate.  Imme- 
diately afterwards  remove  the  brain 
and  spinal  cord  and  put  into  10% 
formalin  for  48  hrs.  Place  slices  3  mm. 
thick  directly,  without  washing,  in : 
1%  aq.  potassium  chlorate,  60  cc. ; 
1%  aq.  osmic  acid,  20  cc. ;  glacial  acetic 
acid,  1  cc. ;  37%  formaldehyde  (Merck's 
reagent),  12  cc.  Use  about  15  volumes 
of  this  fluid  to  1  of  tissue.  Agitate  and 
turn  over  daily.  After  staining  for  7-10 
days,  wash  in  running  water,  12-24  hrs., 
dehydrate  in  70%  and  95%  and  absolute 
alcohol  and  imbed  in  low  viscosity  nitro- 
cellulose as  described  by  Davenport, 
H.  A.  and  Swank,  R.  L.,  Stain  Tech., 
1934,  9,  134-139.  See  Celloidin  Im- 
bedding. Cut  40m  sections  serially, 
mount  on  slides,  dehydrate  to  toluol, 
placing  chloroform  in  absolute  alcohol 
since  low  viscosity  nitrocellulose  is 
soluble  in  absolute  alcohol.  Clear  in 
toluol.  Mount  in  clarite  X  dissolved 
in  toluol.  See  these  authors  (Stain 
Techn.,  1935,  10,  45-52)  for  artifacts 
and  effects  of  perfusion  in  Marchi 
technique.  Rasmussen,  G.  L.,  Anat. 
Rec,  1944,  89,  331-338  has  elaborated  a 
very  useful  cellophane  strip  method  for 
preparation  and  study  of  Marchi  serial 
sections. 

Marchi's  Fluid.  Miiller's  Fluid,  2  parts; 
1%  osmic  acid,  1  part.     Fix  5-8  days; 


wash  in  running  water.  Employed  to 
blacken  degenerated  nerve  fibers.  See 
Nerve  Fibers. 

Method,  underlying  mechanisms  in- 
volved (Swank,  R.  L.  and  Davenport, 
H.  A,  Stain  Techn.,  1934,  9,  11-19; 
1935,  12,  45-52). 

Marine  Blue  V,  see  Anilin  Blue. 

Marino's  Stain  for  malaria  plasmodia  is  de- 
scribed in  detail  by  Craig,  p.  286  who 
states  that  it  gives  excellent  results; 
but,  owing  to  its  complexity,  is  little 
used  for  routine  blood  examinations. 

Marrow,  see  Bone  Marrow. 

Marshall  Red  (British  Drug  Houses  Ltd), 
a  disazo  dye.  Stain  sections  in  sat. 
aq.  solution  20  min.  Rinse  in  aq. 
dest.  Stain  in  sat.  Victoria  Green  G 
in  70%  alcohol  30  min.  Rinse  in  95% 
alcohol,  dehydrate,  clear  and  mount 
in  usual  way.  Myofibrils  sage  green, 
nuclei  crimson.  Advised  also  for  retina 
(H.  G.  Cannan,  J.  Roy.  Micr.  Soc, 
1941,  61,88-94). 

Martius  Yellow  (CI,  9) — Manchester  yellow, 
naphthol  yellow — An  acid  nitro  dye 
employed  by  Pianese  (G.,  Beitr.  z. 
Path.  Anat.  u.  AUg.  Path.,  1896,  Suppl. 
I,  193  pp.)  for  investigating  cancer 
tissue  in  association  with  acid  fuchsin. 
Conn  (p.  44)  reports  good  results  in 
staining  of  plant  tissue  with  CC  product. 

Masson's  Gelatin  Glue.  Method  for  mak- 
ing sections  stick  to  slides  (Masson, 
P.,  Am.  J.  Path.,  1928,  4,  181-212). 
Dissolve  0.05  gm.  sheet  gelatin  in  20 
cc.  aq.  dest.,  warming  gentlj'.  Filter  a 
large  drop  on  each  slide  on  warm  plate. 
Float  paraffin  sections  on  drops.  When 
drops  spread  place  slides  upright  to 
drain  but  do  not  permit  drying.  Blot 
and  transfer  to  dish  containing  formalin 
(so  arranged  that  vapor  only  will  act 
on  slides)  in  oven  45-50 °C.  For  sub- 
sequent staining  20  minutes  in  hot  vapor 
is  enough.  For  silver  treatment  over- 
night is  suggested. 

Masson's  Trichrome  Stain — Written  by 
Pierre  Masson,  Dept.  of  Pathology, 
University  of  Montreal,  Montreal, 
Canada.  October  24,  1951— The  prin- 
ciple established  by  F.  B.  Mallory  in  his 
famous  method  of  employing  acid 
fuchsin,  phosphomolybdic  acid,  anilin 
blue,  orange  G.  can  be  advantageously 
applied  to  other  acid  dyes  yielding  a 
more  specific  staining  of  the  chromatin. 
It  then  gives  a  very  instructive  com- 
bination of  tints. 

Due  to  the  intensity  of  the  staining, 
the  sections  must  be  thin;  5^  is  opti- 
mum. In  order  to  prevent  swelling  of 
the  collagen  and  its  deformation  during 
the  desiccation,  the  sections  must  not 
be  left  too  long  (20-30  sec.)  on  the 
warmed    water,    or   gelatin,    especially 


MASSON'S  TRICHROME  STAIN 


193 


MASSON'S  TRICHROME  STAIN 


after  fixation  by  picro-formol  (Bouin) 
or  formalin.  So  altered,  the  collagen 
does  not  properly  absorb  the  dyes  and 
differentiates  badly.  Trichrome  stains 
with  iron  haematoxylin.  All  trichromic 
methods  are  based  upon  a  common  first 
step:  the  staining  of  nuclei  by  iron  alum 
haematoxylin,  followed  by  differen- 
tiation with  picric  alcohol. 

Three  solutions  are  required:  A.  Iron 
alum,  violet  crystals,  5  gm.,  aq.  dest. 
100  cc.  B.  Regaud's  hematoxylin  solu- 
tiin  made  up  by  dissolving  1  gm.  hema- 
toxylin in  60  cc.  hot  aq.  dest.  Cool  and 
add  10  cc.  glycerin  and  10  cc.  of  95% 
alcohol.  This  stain  is  ready  for  use  at 
once.  C.  Differentiating  mixture  con- 
sisting of  2  parts  sat.  picric  acid  in  95% 
alcohol  and  1  part  of  95%  alcohol. 

Step  1.  Place  iron  alum  solution  and 
the  hematoxylin  solution  in  staining 
jars  in  a  water  bath  heated  to  40-45°C. 
Mordant  deparaflined  sections  in  heated 
iron  alum,  15  min.  Rinse  with  aq. 
dest.  Stain  in  heated  hematoxylin,  15 
min.  or  more.  Rinse  the  uniformly 
black  sections  with  95%  alcohol  and 
immerse  in  picric  alcohol.  Control  the 
progress  of  differentiation  under  the 
microscope.  As  soon  as  the  nuclei  alone 
remain  colored,  wash  with  running 
water  for  15  min. 

If  after  washing,  the  background  of 
the  preparation,  particularly  the  col- 
lagen remains  gray,  rinse  with  alcohol 
and  complete  picric  differentiation. 
The  chromatin  is  electively  black  and 
opaque. 

Various  authors  have  proposed  to 
"simplify"  this  method  by  staining  the 
nuclei  with  Weigert's  iron  perchloride 
hematoxylin  or  with  alum  hematoxylin. 
I  must  say  that  I  have  long  ago  tried 
such  modifications  before  adopting  the 
above  method  and  that  I  have  aban- 
doned them  entirely.  The  red  of  the 
ponceau-fuchsin  solution  superposes 
itself  on  the  gray  or  blue  color  imparted 
by  hematoxylin  so  that  chromatin  takes 
a  dull  color  lacking  in  specificity. 
Rather  than  to  use  such  modifications 
it  would  be  better  to  omit  all  nuclear 
staining  and  start  with  the  next  step 
(ponceau-fuchsin  and  so  forth) :  the 
results  are  thus  comparable  to  those  ob- 
tained by  Mallory's  original  method. 

Step  2.  Stain  in  a  mixture  of  acid 
fuchsin  and  Ponceau  ofxylidin  BS  (J.  R. 
Geigy,  S.  A.  Basel).  No  other  Ponceaux 
I  have  used,  French,  German  or  Amer- 
ican give  results  comparable  to  that  of 
Geigy's. 

Three  solutions  are  required:  A. 
Ponceau,  BS  (Geigy),  1  gm.,  aq.  dest. 
100  cc,  glacial  acetic  acid  1  cc.  B.  Acid 
fuchsin     (National     Anilin    Co.,    New 


York),  1  gm.,  aq.  dest.  100  cc,  glacial 
acetic  acid  1  cc.  1  part  of  A  with  2 
parts  of  B.  C.  Aq.  dest.  100  cc,  glacial 
acetic  acid  1  cc.  D.  Aq.  dest.  100  cc, 
phosphomolybdic  acid  1  gm. 

Place  sections  stained  with  the  iron 
hematoxylin  in  the  A  B  mixture,  5  min. 
Rinse  them  with  C.  Transfer  them  to 
Z>  in  a  Copeland  or  Borrel  jar  at  40°C. 
5  min.  or  more.  The  collagen  should 
remain  perfectly  colorless. 

Step  3.  Stain  the  collagen  with 
anilin  blue  or  Fast  green.  To  make  the 
former  dissolve  2  gms.  anilin  blue  (Na- 
tional Anilin  Co.,  New  York)  in  100  cc. 
warmed  aq.  dest.  Cool  and  add  1  cc. 
glacial  acetic  acid.  To  make  the  latter 
dissolve  1  gm.  fast  green  (National 
Anilin  Co.,  New  York)  in  100  cc.  aq. 
dest.  and  add  1  cc.  glacial  acetic  acid. 
After  the  phosphomolybdic  differenti- 
ation (Step  2  jD)  rinse  the  sections  in 
acetic  water  C  Pour  on  them  8  to  10 
drops  of  the  anilin  blue  or  fast  green 
solutions  2  to  3  min.  Wash  in  acetic 
water  C  2  to  3  min.  Dehydrate  with 
absolute  alcohol,  clear  in  toluol  or  xylol, 
mount  in  balsam  or  permount.  Total 
length  of  this  technique  is  approxi- 
mately 60  minutes.  Chromatin  is 
black,  cytoplasms  stain  in  various 
shades  of  red,  granulations  of  eosino- 
philes  and  mast  cells  stain  ruby  red, 
erythrocytes  are  black,  elastic  fibers 
stain  red,  collagenic  fibers  and  mucus 
stain  dark  blue  (with  anilin  blue)  or 
green  (if  Fast  green  is  used). 

To  this  relatively  fast  technique,  I 
prefer  a  slow  one,  based  on  the  use  of 
diluted  solutions  of  Ponceau-fuchsin, 
anilin  blue  or  fast  green. 

After  nuclear  staining  with  iron  alum 
hematoxylin  (Step  1)  wash  and  immerse 
the  slides  in  1  part  A  B  mixture  and  9 
parts  1%  acetic  acid,  30  min.  Rinse  in 
acetic  water  C,  1  min.  Transfer  the 
sections  into  D  at  40°C.  5  min.  or  more. 
Rinse  in  acetic  water  C.  Stain  Anilin 
blue  solution  1  part  {or  Fast  green  solu- 
tion 1  part)  with  acetic  water  C  9  parts, 
15  to  30  minutes.  Rinse  in  acetic  water. 
Dehydrate  with  absolute  alcohol,  clear 
in  toluol  or  xylol,  mount  in  balsam  or 
permount. 

The  results  are  grossly  the  same  as 
after  the  rapid  method,  but  more  deli- 
cate and  precise.  Moreover,  the  stain- 
ing of  the  collagen  is  slow  and  progres- 
sive and  can  be  stopped  at  the  most 
favorable  step;  in  many  circumstances 
an  excessive  staining  of  collagen  masks 
some  fine  details,  for  example  the  fine 
prolongations  of  the  connective  tissue 
cells. 

N.B.  After  staining  of  collagen  with 
Fast  green,  it  is  preferable  to  mount  in 


MAST  CELLS 


194 


MCILVAINE  BUFFERS 


salicylic  balsam  or  salicylic  permount. 
Spread  a  drop  of  saturated  solution  of 
salicylic  acid  in  toluol  upon  a  cover 
slip.  Dry.  The  glass  is  then  covered 
with  a  thin  crystalline  layer  of  salicylic 
acid.  Put  on  it  a  drop  of  balsam  or 
permount  and  cover  the  section  taken 
out  of  the  toluol  (or  xylol).  Salicylic 
acid  instantaneously  redissolves  in  the 
mounting  medium.  This  addition  of 
salicylic  acid  is  not  advantageous  after 
staining  with  anilin  blue. 

Mast  Cells,  see  Basophile  Leucocytes  and 
Tissue  Basophiles. 

Mastigophora,  Techniques  for  given  by  Mc- 
Clung,  Microscopical  Technique,  1950, 
p.  469. 

Mastoid  Process.  Use  methods  for  Bone. 
Technique  for  measurements  of  size  of 
air  cell  system  is  given  by  Diamant,  M., 
Acta  Radiol.,  1940,  31,  543-548. 

Mauveine  ((T,  846),  a  basic  dj^e  of  light  fast- 
ness 3,  the  first  dye  made  from  aniline 
in  1856.  Gives  stain  of  plant  tissues 
like  Methyl  violet  (Emi_g,  p.  57). 

Maximow  (see  Azure  II  Eosin  Hematoxylin 
method).  He  has  advised  as  a  fixative 
90  cc.  Zenker's  fluid  less  acetic  acid  + 
10  cc.  formalin.  This  is  essentially 
Formalin  Zenker.  See  Buzaglo's  con- 
nective tissue  stain. 

May-Giemsa  stain  of  Pappenheim  (Folia 
Haematol.,  Arch.,  1917,  22,  15).  This 
is  the  same  as  Jenner-Giemsa.  Fix 
and  stain  air  dried  blood  smears  about 
3  min.  in  May-Griinwald  mixture  (sat. 
sol.  methylene  blue  eosinate  in  methyl 
alcohol).  Add  same  amount  aq.  dest. 
and  leave  1  min.  Pour  off  (but  do  not 
wash)  and  add  diluted  Giemsa's  solu- 
tion. Stain  in  this  15-30  min.  Rinse 
aq.  dest.  1  min.  or  until  desired  color 
is  reached.  Blot  dry.  This  is  a  good 
modification  of  the  ordinary  Giemsa's 
stain  because  it  gives  slightly  more 
intense  colors. 

May-Griinwald  combined  fixative  and  stain 
is  a  sat.  sol.  of  methylene  blue  eosinate 
in  methyl  alcohol  (Grtibler  or  Holl- 
born).  If  methylene  blue  eosinate  is 
not  available  make  it  as  originally  de- 
scribed by  Jenner  (Lancet,  1899,  No.  6, 
370).  Mix  equal  parts  1.25%  water  sol. 
eosin  and  1%  methylene  blue;  after  24 
hrs.  filter;  wash  ppt.  on  filter_  with 
water;  dry  and  dissolve  powder  in  200 
cc.  pure  methyl  alcohol.  It  is  employed 
in  the  May-Giemsa  and  Kardos-Pap- 
penheim  methods  for  staining  blood 
smears. 
May-Griinwald-Giemsa  stain  in  one  solu- 
tion. Strumia  (M.  M.,  J.  Lab.  &  Clin. 
Med.,  1935-36,  21,  930-934)  gives  di- 
rections for  combining  the  stains  and 
for  use  and  notes  that  a  standardized 
product  is   prepared   by   Coleman  and 


Bell    Co.     Intensity    of    coloration    is 
enhanced  by  the  combination. 
Mayer's    Acid    Alum    Hematoxylin.     The 

following  formula  is  given  by  Mallory 
(p.  73).  Dissolve  1  gm.  hematoxylin 
in  1000  cc.  aq.  dest.  with  a  little  heat  if 
required.  Add  0.2  gm.  sodium  iodate 
and  50  gm.  ammonium  or  potassium 
alum.  When  latter  is  dissolved  add  1 
gm.  citric  acid  and  50  gm.  chloral  hy- 
drate. Color  turns  reddish  violet. 
Does  not  easily  over-ripen. 

Mayer's  Acid  Carmine.  The  Bensleys 
(p.  131)  advise  its  preparation  as 
follows.  Add  4  gm.  carmine  to  15  cc. 
aq.  dest.  +  30  drops  hydrochloric  acid. 
Boil  until  it  is  dissolved.  Add  95  cc. 
85%  ethyl  alcohol.  Neutralize  with 
ammonia  until  the  carmine  begins  to 
precipitate  as  seen  in  a  graduate  against 
white  paper  background.  Add  4  more 
drops  ammonia  after  first  precipitation. 
If  this  acid  carmine  stains  too  quicklj^ 
slow  it  down  by  dilution  with  80-90% 
alcohol.  This  gives  a  fine  red  nuclear 
counterstain  for  tissues  vitally  stained 
with  Indigo-Carmine,  Trypan  Blue 
and  similar  dyes. 

Mayer's  Albumen,  see  Albumen  Glycerin. 

Mcllvaine  Buffers  after  Stitt  from  Lillie, 
R.  D.,  Stain  Techn.,  1941,  16,  1-6  who 
employed  them  to  improve  Romanowsky 
staining  after  various  fixatives.  See 
Toluidine  Blue  Phloxinate  Method, 
(see  Molecular  Solution)  To  make 
M/15  citric  acid  required  dissolve  14.01 
gm.  mono-hydrateu  crystalline  citric 
acid  in  500  cc.  aq.  dest.  and  add  enough 
neutral  methyl  alcohol  C.P.  to  make 
total  volume  1,000  cc.  after  careful 
mixing.  To  make  M/15  Na2HP04  dis- 
solve 9.47  gm.  anhydrous  Na2HP04 
in  500  cc.  aq.  dest.  and  make  up  to 
1,000  cc.  with  methyl  alcohol.  These, 
in  following  proportions  listed  in  cc, 
give  pH  values  indicated. 


cc.  Citric  Acid 

cc.  NajHPOi 

pH 

1.3 

0.7 

3.9 

(3.873) 

1.25 

0.75 

4.0 

(4.034) 

1.2 

0.8 

4.2 

(4.205) 

1.15 

0.85 

4.4 

(4.44) 

1.1 

0.9 

4.6 

(4.653) 

1.05 

0.95 

4.8 

(4.80) 

1.0 

1.0 

5.0 

(5.042) 

0.95 

1.05 

5.2 

(5.201) 

0.9 

1.1 

5.4 

(5.428) 

0.85 

1.15 

5.7 

(5.696) 

0.8 

1.2 

5.85  (5.838) 

0.75 

1.25 

6.05  (6.036) 

0.7 

1.3 

6.3 

(6.29) 

0.65 

1.35 

6.5 

(6.444) 

0.6 

1.4 

6.5 

(6.522) 

0.55 

1.45 

6.6 

(6.60) 

Since  it  is  difficult  to  measure  out  these 


MCJUNKIN-HADEN  BUFFER 


195 


MELANINS 


small  volumes  accurately  at  least  ten 
times  the  volume  in  each  case  should  be 
taken  and  the  amount  not  required 
simply  be  discarded.  For  ordinary 
purposes  employ  aq.  dest.  in  place  of 
methyl  alcohol. 

McJunkin-Haden  Buffer  has  pH  6.4  and  is 
useful  in  place  of  aq.  dest.  for  diluting 
Giemsa,  Wright  and  other  blood  stains. 
Monobasic  potassium-phosphate,  6.63 
gm.;  anhydrous  dibasic  sodium  phos- 
phate, 2.56  gm.;  aq.  dest^  1000  cc. 
(Haden,  R.  L.,  J.  Lab.  &  Clin.  Med., 
1923,  9,  64-65). 

Meat  Extract  Broth  and  other  media  con- 
taining meat,  see  Bacteria  Media. 

Mechanical  Stages,  see  Lillie  p.  287. 

Meckel's  Diverticulum.  Literature  on 
(Curd,  H.  H.,  Arch.  Surg.,  1936,  32, 
506-523). 

Media,  see  Bacteria,  Leishmania,  Protozoa, 
Trypanosomes. 

Megakaryocytes.  These  can,  like  blood 
cells,  be  examined  in  fresh  and  stained 
smears  of  bone  marrow.  For  a  deter- 
mination of  their  role  in  platelet  forma- 
tion it  is  essential  to  clearly  show  the 
granules  typical  of  both.  This  can 
best  be  done  in  sections  of  bone  marrow 
prepared  by : 

1.  Wright's  method  (Wright,  J.  H., 
J.  Morph.,  1910,  21,  263-277).  After 
fixation  in  sat.  mercuric  chloride  in 
0.9%  aq.  NaCl,  dehydrate  in  alcohol, 
follow  with  acetone,  clear  first  in  thick 
cedar  oil  and  then  in  xylol,  embed  in 
paraffin.  Sections  deparaffinized  are 
covered  with  equal  parts  stain  (poly- 
chrome methylene  blue  solution  3  parts 
and  0.2%  eosin  yellowish  in  methyl 
alcohol  10  parts)  10  min.  A  metallic 
looking  scum  forms  but  the  stain  should 
not  be  allowed  to  precipitate.  Stop 
staining  when  cytoplasm  looks  bright 
red  and  reticular  fibers  light  red.  Wash 
in  water,  dehydrate  in  acetone,  clear 
in  turpentine  and  mount  in  thick 
colophonium  in  pure  turpentine  oil. 
See  Wright's  colored  plates.  In  place 
of  the  fixative  suggested,  Downey  (Folia 
haematol.,  Archiv,  1913,  15,  25)  uses 
commercial  formalin  10  cc.  and  sat. 
mercuric  chloride  in  0.9%  aq.  NaCI 
90  cc. 

2.  Kingsley's  method  (Kingsley,  D. 
M.,  Folia  Haemat.,  1937,  57,  87-98). 
Fix  in  Downey's  fluid  (given  above) 
4  parts,  saturated  picric  acid  1  part, 
24  hrs.  Wash  in  running  water,  18-24 
hrs.  Dehydrate  through  alcohols  up 
to70%,i-lhr.each.  80%  ale.  +  iodine, 
overnight.  95%  ale,  45  min.  Repeat 
with  fresh  ale.  A^  butyl  alcohol  (techni- 
cal), 1  hr.  Repeat  with  fresh.  Paraf- 
fin (58°C.),  ^  hr.,  then  3  more  changes, 
each     5     hr.     Imbed.     Prepare     stock 


solutions  A :  methylene  blue  (U.S. P. 
med.  88%),  0.065  gm.;  methylene  azure 
A  (80%),  0.01  gm.;  glycerin,  C.P., 
5  cc;  CH3OH  (C.P.),  5  cc;  aq.  dest., 
25  cc;  buffer  (pH,  6.9),  15  cc  B: 
methylene  violet  (Beruthsen  85%), 
0.013  gm.;  eosin,  yel.  (92%),  0.45  gm.; 
glycerine, 5  cc;  CH3OH,  10  cc  ; acetone, 
C.P.,  35  cc.  The  buffer  is  40  cc.  of  A 
=  9.078  gm.  KHjPOi  per  liter  +  60  cc. 
of  B  =  11.876  gm.  Na2HPO«-2H20  per 
liter  of  aq.  dest.  Immediately  before 
use  mix  equal  parts  of  stock  stains  A 
and  B.  After  washing  deparaffinized 
sections  in  aq.  dest.  stain  8-10  min. 
Wash  off  in  current  of  aq.  dest.  Wash 
in  aq.  dest.  100  cc.  +  1%  acetic  acid, 
0.8  cc.  Wash  again  in  aq.  dest.  to  re- 
move acid.  Blot.  Rinse  in  acetone, 
100  cc  +  0.001  gm.  eosin  -f  4  cc.  1% 
acetic  acid.  Rinse  in  n  butyl  ale  -\-  a 
little  eosin.  Neutral  xylol  several 
changes.  Mount  in  neutral  xylol  dam- 
mar. See  Kingsley's  plate  for  colors. 
Granules  dark  red.  It  is  important  to 
fix  the  bone  marrow  promptly  after 
death  or  to  obtain  it  by  biopsy. 

Megaloblasts,  see  Erythrocytes,  develop- 
mental series. 

Meibomian  Glands.  Whole  mounts  of  the 
glands  stained  with  Sudan  IV  in  a  trans- 
parent background  by  a  method  de- 
scribed for  Sebaceous  Glands. 

Meissner's  Corpuscles.  To  investigate  by 
supravital  staining  with  methylene 
blue  in  skin  of  amputated  fingers,  see 
Weddell,  G.,  J.  Anat.,  1940-41,  75, 
441-446.  Skin  from  general  body  sur- 
face will  not  do  because  of  rarety  of 
the  corpuscles. 

Meissner's  Plexus,  see  Auerbach's. 

Melanins.  Lison  (p.  248)  gives  many  dif- 
ferential microchemical  properties  from 
which  the  following  are  selected.  Ex- 
treme resistance  to  most  chemicals,  not 
modified  by  concentrated  acids  but 
soluble  in  concentrated  alkalis.  They 
are  depigmented  by  oxydants.  Thus, 
Schultze  treats  them  with  diaphanol 
(chlordioxyacetic  acid)  for  24  hrs.  in 
hermetically  sealed  container  in  dark- 
ness ;  and  Alfiere  treats  sections  with 
0.1%  potassium  permanganate  2-24 
hrs. ;  washes  with  much  water,  treats 
with  0.3%  oxalic  acid  and  again  washes. 
Their  power  of  reducing  ammoniacal 
silver  nitrate,  Lison  regards  as  very 
characteristic.  Melanins  occur  nor- 
mally' in  epidermis,  hair,  choroid  of  eyes. 
Greatly  increased  in  Addison's  disease. 
Contain  no  iron  or  fat.  Difficulties  in 
histological  identification  (Jacobsen, 
V.  C.  and  Klinck,  G.  H.,  Arch.  Path., 
1943,  17,  141-151).  Use  of  Bodian 
method  (Dublin,  W.  B.,  Am.  J.  Clin. 
Path.,  Techn.  Suppl.,  1943,  7,  127-128). 


MELANOBLASTS 


196 


MESONEPHRIC  TUBULES 


A  method  for  the  collection  of  melanin 
for  analysis  by  differential  Centrifuga- 
tion  is  described  by  Claude,  A.,  Trans. 
New  York  Acad.  Sci.,  1942,  II,  4,  79-83. 
A  very  complete  account  of  the 
melanins  has  been  presented  by  Gordon, 
H.  (Organizing  Chairman) :  The  Bi- 
ology of  Melanomas — Special  Publica- 
tions, New  York  Acad.  Sci.,  1948, 
466  pp. 

Lillie  (p.  131)  cites  Alfieri  from 
Romeis  as  advising  for  the  bleaching  of 
melanin  in  sections  treatment  with 
0.05%  aq.  potassium  permanganate 
until  thoroughly  brown  followed  by  de- 
colorization  in  0.33  aq.  oxalic  acid  and 
repeating  the  process  if  necessary. 
Obviously  stronger  solutions  could  be 
employed.  This  removal  of  melanin 
might  be  advantageous  in  some  cases  to 
reveal  more  sharply  other  properties  of 
the  cells  like  mitochondrial  content. 
Treatment  with  10%  hydrogen  peroxide, 
as  suggested  by  Lillie,  is  perhaps  a 
better  method. 

See   Dopa  Reaction   for   melanogen   in 
melanoblasts. 

Melanoblasts,  see  Dopa  Reaction. 

Meldola's  Blue,  see  Naphthol  Blue  R. 

Mercuric  Chloride  (corrosive  sublimate) 
in  various  combinations  is  an  excellent 
fixative.  It  can  be  used  in  saturated 
aq.  sol.  plus  5%  acetic  acid  or  in  satu- 
rated ale.  sol.  with  the  same  amount  of 
acetic  acid.  See  (1)  with  formalin, 
glacial  acetic  and  physiological  saline 
for  Centrosomes,  (2)  sat.  in  0.9%  aq. 
sodium  chloride  for  Megakaryocytes, 
(3)  sat.  in  70%  alcohol  +  5%  acetic 
for  Mitosis,  (4)  sat.  aq.  +  equal  parts 
2.5%  aq.  potassium  bichromate  for 
Neutral  Gentian,  (5)  sat.  aq.  with  equal 
parts  abs.  alcohol  for  Thymonucleic 
Acid,  and  (6)  with  nitric  acid  for  Urea. 
The  mercuric  chloride  is  removed  from 
the  sections  by  Lugol's  iodine  solution. 
See  also  fixatives  of  Zenker,  Gilson, 
Rabl  and  Petrunkewitsch.  Zinc  chlo- 
ride is  suggested  as  substitute  for 
mercuric  chloride  in  Zenker's  fluid 
(Russell,  W.  O.,  J.  Techn.  Methods  & 
Bull.  Int.  Asso.  Med.  Museums,  1941, 
21,47). 

Mercurochrome  220.  Trade  name  for  di- 
brora-oxy-mercuri-fluorescein.  Can  be 
used  as  substitute  for  eosin  (Baldwin, 
W.  M.,  Anat.  Rec,  1928,  39,  229)  but 
it  has  little  to  commend  it. 

Mercury,  microchemical  tests  for. 

1.  Method  of  Almkvist-Christeller. 
Fix  tissues  2  days  in  sat.  aq.  picric  acid, 
100  cc. ;  25%  nitric  acid  1  cc,  saturated 
with  HjS  gas,  filtered  after  1  day.  After 
fixation  wash  in  running  water  for  24 
hrs.  Imbed  in  paraffin.  Mercury  ap- 
pears as  black  ppt.  of  sulphide.    Lison 


(p.  102)  explains  that  it  is  necessary  to 
make  parallel  tests  for  iron  because  this 
method  changes  iron  into  the  black  sul- 
phide which  could  be  mistaken  for  the 
sulphide  of  mercury.  Simonet  (M., 
Arch.  d'Anat.  Micr.,  1929,  25,  372-381) 
uses  instead  fixation  for  10  hrs.  in  equal 
parts  alcohol  and  chloroform,  100  cc, 
+  nitric  acid,  2  cc.  the  mixture  satu- 
rated with  HjS  by  bubbling. 

2.  Method  of  Brandino  (G.,  Studi 
Sassari,  1927,  5,  85).  Fix  in  formalin 
or  in  alcohol.  Treatment  of  sections 
with  1%  sol.  of  diphenylcarbazide  which 
forms  with  mercury  a  violet  ppt.  Gives 
results  with  organs  of  persons  killed  by 
mercury  poisoning  kept  in  formalin 
17  years  (Lison,  p.  102). 

3.  Method  of  Hand  et  al.,  J.  Lab.  & 
clin.  Med.,  1943,  28,  1835-1841.  Re- 
agents: (A)  Mercurous.  1  cc.  thioglycol- 
lic  acid  +9  cc.  glycerol.  (B)  Mercuric. 
Heat  until  clear  100  cc.  glycerol.,  +5 
gm.  tartaric  acid,  +5  gm.  stannous 
chloride.  Add  few  gms.  metallic  tin 
and   store   in   glass   stoppered   bottle. 

(C)  Iodine.  Dissolve  50  gm.  potassium 
iodide  in  50  cc.  aq.  dest.,  add  70  gm. 
iodine  and  95%  ale.  to  make  1,000  cc. 

(D)  Chloroauric  acid  1%  aq.  (E)  Con- 
trol. Let  5  gm.  tartaric  acid  stand  over 
night  in  100  cc.  glycerol.  Cut  15  m 
frozen  sections  of  tissue.  Place  on 
slides  and  dry.  To  section  on  each  of  4 
slides  add  1  drop  of  one  reagent.  Add 
cover  glasses.  Remove  with  filter 
paper  excess  of  reagents.  Seal  edges  of 
cover  glass  with  commercial  gold  size, 
an  adhesion  intended  to  hold  gold  foil 
on  glass.  Melted  paraffin  is  less  satis- 
factory but  will  serve  for  a  short  time. 
After  10  min.  examine  slides.  Metallic 
mercury  visible  as  small  black  spherules. 
These  are  soluble  in  C  and  form  gold 
amalgam  losing  glossy  surface  when 
treated  with  D.  Reagent  A  gives  typi- 
cal yellow  crystals  with  mercurous 
mercury  in  addition  to  globules  of 
mercury  when  mercuric  mercury  also  is 
present.  Sections  treated  with  B  and  E 
are  unchanged  (adapted  from  Click 
p.  25). 

Intravenous  injections  of  colloidal 
solutions  of  mercury  in  rabbits  are 
described  by  Duhamel,  B.  G.,  C.  rend. 
Soc.  de  Biol..  1919,  82,  724-726. 

Mesentery  spreads,  sections  and  cultures. 
Maximow,  A.,  Arch.  f.  exper.  Zellf., 
1927,  4,  1-42  (nice  colored  plates).  For 
microinjection  of  small  vessels  of  the 
mesentery  see  Florey,  H.,  Proc.  Roy. 
Soc.  B,  1926  100,  269. 

Mesonephric  Tubules,  cultivation  in  vitro 
and  method  for  collection  of  fluid  there- 
from (Keosian,  J.,  J.  Cell  &  Comp. 
Physiol.,  1938,  12,  23). 


METACHROMASIA 


197 


METHYL  VIOLET 


Metachromasia,  see  Metachromatism. 

Metachromatism  (metachromasia)  is  the 
property  of  certain  dyes  to  stain  (G., 
meta,  beyond)  the  usual  color  (G. 
chroma).  The  action  of  some  impure 
methylene  blues  is  sometimes  cited  as 
an  example.  Thus  polychrome  (many 
colored)  methylene  blue  stains  some 
objects  blue  and  certain  granules  red- 
dish. This  methylene  blue  is  however 
a  mixture  of  methylene  blue  and  methyl- 
ene red.  The  latter  dye  accounts  for 
the  staining  beyond.  Orcein  colors 
nuclei  blue  and  cytoplasm  pink.  Safra- 
nin  stains  nuclei  in  its  ordinary  solution 
color  (red)  and  the  ground  substance  of 
cartilage  that  of  its  free  color  base 
(orange).  Michaelis  (Lee,  p.  136) 
thinks  that  the  appearance  of  the  color 
base  is  not  occasioned  by  the  alkalinity 
of  the  objects  stained.  The  red  stain 
of  mucin  by  thionin  can  be  altered  to 
blue  by  alcohol  and  be  shifted  back  to 
red  by  water.  For  colored  plates  show- 
ing metachromatic  staining  of  mast 
cells,  see  Maximow,  A.,  Arch.  f.  mikr. 
Anat.,  1913,  83  (1),  247-289.  Meta- 
chromasia of  acid  dyes  is  increased  by 
adding  strychnine,  quinine  or  clupein 
and  of  basic  dyes  by  gum  arable  or  other 
negatively  charged  colloid  (Bank,  O. 
and  Hungenberg  de  Jona,  H.  G.,  Proto- 
plasma,  1939,  32,  489-516).  The  dis- 
tinction between  "true"  and  "false" 
metachromatic  staining  with  toluidine 
blue  is  fully  described  by  Sylvan,  B., 
Acta  Radiol.,  1945,  Suppl.  59,  100  pp. 

Metacresol  Purple.  See  Hydrogen  Ion  In- 
dicators. 

Metallurgic  Microscope.  Since  the  mate- 
rials routinely  studied  are  opaque  the 
light  is  reflected  vertically  down  upon 
them  through  the  objective.  This  in- 
strument is  of  little  use  in  biology  and 
medicine. 

Metamyelocytes,  see  Leucocytes,  develop- 
mental series. 

Metanil  Yellow  (CI,  138)— acid  yellow  R, 
orange  MNO  or  MN,  soluble  yellow  OL, 
tropaeolin  G,  yellow  M — An  acid  mono- 
azo  dye  employed  in  the  Masson  tech- 
nique, see  Foot,  N.  C.,  Stain  Techn., 
1933,  8,  101-110. 

Methacrylate.  Plastic  for  mounting  ali- 
zarin-red-S  preparations.  (Holcomb, 
R.  C.  and  Apterman,  P.  M.,  J.  Tech. 
Methods,  1944,  24,  21-24). 

Methyl  Alcohol,  see  Elementary  Bodies. 
It  is  much  used  in  many  techniques. 

Methyl  Benzoate.  Refractive  index  close 
to  that  of  cedar  wood  oil.  It  can  be 
used  in  place  of  immersion  oil.  In 
addition  it  is  a  substitute  for  absolute 
alcohol  and  an  excellent  clearing  agent 
but  it  is  expensive.  See  Ceresin 
imbedding. 


Methyl  Blue  (CI,  706)— cotton  blue,  Hel- 
vetia blue — Widely  used.  Recom- 
mended for  connective  tissue  by  Lillie, 
R.  D.,  J.  Tech.  Methods,  1945,  No.  25, 
47  pp.  See  Mann's  Methyl-Blue  Eosin 
and  staining  of  Elementary  Bodies. 

Methyl  Blue-Eosin,  see  Mann's. 

Methyl  Eosin  (CI,  769).  The  methyl  ester 
of  eosin  Y,  see  Eosins,  choice  of. 

Methyl  Green  (CI,  684) — double  green, 
light  green — This  basic  triphenyl  meth- 
ane dye  is  crystal  violet  (he.xa-methyl 
pararosanilin)  into  which  a  seventh 
methyl  group  has  been  incorporated. 
Conn  (p.  130)  points  out  that  this  is 
loosely  bound  so  that  some  methyl  or 
crystal  violet  is  always  present  with  the 
methyl  green  to  which  circumstance  the 
metachromatic  properties  of  the  dye  are 
partly  due.  Methyl  green  is  not  as 
stable  as  most  dyes  and  cannot  therefore 
be  kept  too  long  in  the  powdered  state. 
It  is  very  similar  to  Ethyl  Green. 

Methyl  Green-Pyronin  (Pappenheim).  Sec- 
tions of  formalin-Zenker  fixed  tissues 
are  stained  about  6  min.  in :  methyl 
green  0.5  gm.;  pyronin  Y,  0.5  gm.;  ale. 
2.5  cc;  glycerin  20  cc;  aq.  dest.  0.5% 
carbolized  100  cc.  Rinse  in  aq.  dest.; 
dehydrate  in  acetone;  clear  in  cedar 
oil  followed  by  xylol  and  mount.  Opti- 
mum time  of  staining  must  be  deter- 
mined experimentally.  A  brilliant 
stain  particularly  for  lymphocytes  and 
plasma  cells.  Very  useful  for  spleen 
and  lymph  nodes.  (Slider  and  Downey 
in  McClung's  Microscopical  Technique, 
p.  342). 

Modification  of  Scudder  (Stain 
Techn.,  1944,  19,  39-44)  gives  good 
results  on  tissue  sections  and  bacteria 
and  has  been  used  for  identification  of 
2  types  of  nucleic  acids  (Taft,  E.  G., 
Exper.  Cell  Research,  1951,  in  press). 
Dehydration  of  stained  material  with 
tertiary  hiilyl  alcohol  gives  better  results 
than  with  ethyl  alcohol. 

Methyl  Orange  (CI,  142)— gold  orange  MP, 
helianthin,  orange  III,  tropaeolin  D — 
A  slightly  acid  mono-azo  dye  widely 
employed  as  an  Indicator. 

Methyl  Red  (CI,  211).  A  slightly  acid 
mono-azo  dye  widely  used  as  an  Indica- 
tor. See  also  Carter,  J.  S.,  J.  Exp. 
Zool.,  1933,  65,  159-179  for  vital  staining 
of  rabdites  of  Stenostomum  with 
methyl  red. 

Methyl  Salicylate  (oil  of  Wintergreen)  is 
employed  in  Spalteholz  Method  of 
clearing. 

Methyl  Violet  (CI,  680)— dahlia  B,  gentian 
violet,  Paris  violet,  pyoktaninum  coeru- 
leum — Exists  in  various  shades  2R,  R, 
B,  2B,  3B,  etc.,  depending  upon  propor- 
tions of  the  mixture  of  tetra-,  penta- 
and  he.xa-methyl  rosanilins.       R  indi- 


METHYLENE  AZURE 


198 


MICRODISSECTION 


cates  reddish  and  B  bluish.  2B  is  the 
one  which  Conn  (p.  123)  regards  as  most 
satisfactory  whenever  methyl  violet, 
or  one  of  the  redder  types  of  gentian 
violet,  is  requested.  (It  is  Commission 
Certified.)  The  pure  hexamethyl  com- 
pound is  called  crystal  violet — a  dye 
much  in  demand.  See  Hydrogen  Ion 
Indicators. 

Methylene  Azure  (CI,  923) .  A  basic  thiazin 
dye  long  recognized  as  a  component  of 
Polychrome  Methylene  Blue.  Conn 
(p.  76)  says  that  the  term,  methylene 
azure,  should  be  discarded  because  it 
is  composed  of  three  components  Azure 
A,  B,  and  C  which  see. 

Methylene  Blue  (CI,  922)— Swiss  blue- 
Conn  (p.  80)  says  that  this  basic  thiazin 
dye  is  theoretically  tetra-methyl  thio- 
nin  but  the  homologues  of  lower 
methylation  are  practically  always 
present ;  he  lists  the  following  grades : 
methylene  blue  BX,  B,  BG,  BB,  and 
methylene  blue  chloride.  The  last 
named  is  Commission  Certified  and  least 
toxic.  Methylene  blue  Med.  U.S.P. 
is  required  to  be  zinc  free  and  is  also 
satisfactory.  New  methylene  blue  N 
(methylene  blue  NN)  is  a  basic  dye  of 
the  same  type  but  of  a  slightly  greener 
shade.  Conn  (McClung,  p.  595)  states 
that  it  was  apparently  in  certain  lots 
of  prewar  methylene  blue.  Methylene 
blue  O  is  the  same  as  toluidin  blue  O 
which  resembles  azure  A,  a  component 
of  methylene  azure  produced  by  poly- 
chromizing  methylene  blue.  Another 
of  the  series  is  methylene  blue  GG  but 
it  has  no  particular  advantage.  Prob- 
ably no  dye,  other  than  hematoxylin  and 
eosin,  is  more  widely  used.  The  oxida- 
tion products  of  methylene  blue  are 
described  by  Holmes,  W.  C,  Stain 
Techn.,  1926,  1,  17-26  and  the  influence 
of  pH  on  staining  of  plasma  cells  and 
lymphocytes  by  Kindred,  J.  E.,  Stain 
Techn.,  1935,  10,  7-20.  Its  cytological 
action  has  been  fully  studied  by  Lud- 
ford,  R.  J.,  Arch.  f".  exp.  Zellf.,  1935, 
17,  339-359.  It  is  an  excellent  counter- 
stain  for  Acid  Fast  Bacilli.  See  Poly- 
chrome Methylene  Blue,  Loeffler's 
Alkaline  Methylene  Blue,  Nerve  End- 
ings, Phloxine  Methylene  Blue,  Mac- 
Neal's  Tetrachrome,  Pancreas,  Pro- 
tozoa, etc.  For  use  of  methylene  blue 
as  a  supravital  stain  fixed  with  ammo- 
nium molybdate,  see  Lillie,  p.  245. 

Methylene  Blue  NN,  see   New  Methylene 
Blue  N. 

Methylene  Blue  T  50  or  T  Extra,  see  Toluidin 
Blue  O. 

Methylene  Blue  Eosinate,  see  May-Griin- 
wald  fixative  and  stain. 

Methylene   Green    (CI,   924).    This   basic 
thiazin    dye    is    mono-nitro    methylene 


blue.  Conn  (p.  86)  says  that  it  is  oc- 
casionally employed  as  a  substitute  for 
methyl  green  and  gives  good  results  as 
counters  tain  for  eosin. 

Methylene  Violet.  Commission  Certified. 
This  feebly  basic  thiazin  dye  is,  as 
Conn  (p.  86)  explains,  formed  whenever 
methylene  blue  is  heated  with  a  fixed 
alkali  or  alkali  carbonate.  It  may  be 
purified  by  recrystallization  but  little 
is  to  be  gained.  The  dye  is  not  much 
used. 

Metrial  Gland.  This  is  a  transitory  struc- 
ture of  unknown  function  in  the  mouse 
appearing  at  approximately  the  8th  d.n}' 
of  pregnancy.  Failure  of  its  cells  to 
take  up  trypan  blue  seems  to  eliminate 
the  hypothesis  that  it  is  active  in  phago- 
cvtosis  (Lobo,  B.  A.,  and  Atkinson, 
W.  B.,  Anat.  Rec,  1946,  94,  77). 

Micelle  (dim.  of  L.  Mica  a  crumb,  micella, 
micellae).  Term  introduced  by  Nageli 
in  1884  for  then  hypothetical  structural 
units  of  the  cell. 

Michiavello  Stain.     See  Rickettsia. 

Microchemical  Reactions.  For  the 
microscopic  identification  of  particular 
elements  or  substances  some  micro- 
chemical  reactions  are  available  but  it 
is  difficult  to  sharply  distinguish  them 
from  other  techniques  not  usually  styled 
microchemical.  An  attempt  is  made  to 
list  them  under  the  objects  demon- 
strated :  Lead,  Iron,  Vitamin  C,  Peroxi- 
dase, etc.  Many  are  generally  known 
under  personal  names.  See  for  exam- 
ple: Axenfeld  (proteins),  Burchardt 
(gold) ,  Carr-Price  (vitamin  A) ,  Feulgen 
(thymonucleic  acid),  Gmelin  (bile  pig- 
ments), Lilienfeld-Monti  (phosphorus), 
Millon  (tyrosin),  Romieu  (proteins), 
Schiff  (aldehydes),  Vulpian  (epineph- 
rine), etc. 

Microdissection.  In  the  selection  of  this 
method  for  use  in  any  particular  problem 
it  is  well  to  bear  in  mind  several  con- 
siderations. It  is  of  particular  value 
in  the  direct  examination  of  large  cells 
easily  isolated,  like  sea  urchin  eggs, 
and  of  tissues  that  exist  in  thin  sheets, 
like  highly  vascularized  membranes 
which  can  be  easily  approached  in  the 
living  state  without  serious  injury. 
The  data  to  be  secured  relate  chiefly 
to  the  responses  of  the  cells  to  the 
mechanical  stimulus  of  the  microneedle, 
to  the  character  of  the  connections  be- 
tween fibers,  cells  and  parts  of  cells  as 
determined  by  their  resistance  to  at- 
tempts to  separate  them  and  to  the 
physical  consistency  of  cellular  and 
nuclear  membranes  and  of  cytoplasm 
and  nucleoplasm.  Moreover  individual 
cells  can  be  isolated  by  microdissection 
just  as  Barber  was  able  to  isolate  single 
bacteria  by  the  pipette  which  he  intro- 


MICROELECTRODES 


199 


MICROINCINERATION 


duced  and  which  was  in  fact  the  inspira- 
tion of  G.  L.  Kite's  first  microdissection 
apparatus.  Today  this  has  been  very 
greatly  improved  chiefly  by  Chambers 
and  Peterfi.  See  detailed  account  by 
Chambers  under  Micromanipulation. 

Microelectrodes,  see  full  discussion,  Mc- 
Clung,  Microscopical  Technique,  1950, 
p.  532. 

Microglia.  Method  for  impregnating  with 
silver  in  pyroxylin  (celloidin)  sections 
(Weil,  H.  and  Davenport,  11.  A.,  Trans. 
Chicago  Path.  Soc,  1933,  14,  95-96). 
Wash  15yu  sections  in  aq.  dest.  Treat 
for  15-20  sec.  with  silver  solution  (made 
by  adding  10% aq.  silver  nitrate  drop  by 
drop  from  a  burette  to  2  cc.  cone,  am- 
monia (28%)  shaking  to  prevent  ppt. 
formation  until  about  18  cc.  have  been 
added  and  the  solution  has  become 
slightlj'  opalescent).  Transfer  to  15% 
formalin,  moving  section  rapidly  until 
coffee-brown  in  color.  Pass  through 
3  changes  aq.  dest.  Dehydrate  in 
alcohol,  clear  in  xylol  and  mount  in 
balsam. 

Microglia  and  Oligodendroglia.  In  frozen 
sections  20-40m  of  formalin  fixed  mate- 
rial. Immediately  place  them  in  aq. 
dest.  +  20  drops  ammonia  per  lOO  cc. 
Thence  pass  directly  to  5%  aq.  am- 
monium bromide  4O^50°C.  10-15  min. 
Equal  parts  ammonia,  pyridine  and  aq. 
dest.  2  min.  Then  3-5%  aq.  sodium 
sulfite,  2-3  min.  Pass  through  and 
shake  in  3  changes  1  min.  each  of  follow- 
ing: 8  parts  5%  aq.  sodium  carbonate, 
2  parts,  10%  aq.  silver  nitrate  +  am- 
monia till  ppt.  Reduce  in  1%  formalin 
less  than  1  min.  Wash,  dehydrate  clear 
and  mount  (King,  L.  S.,  Arch.  Neurol, 
and  Psychiat.,  1937,  38,  362-364). 

Microincineration — Written  by  Gordon  H. 
Scott,  July  26,  1946  and  revised  by  him 
January  16,  1951 — This  method  is  one 
which  has  been  used  by  plant  and  ani- 
mal histologists  intermittently  for  over 
a  hundred  years.  In  concept  it  is 
simple  in  that  it  consists  primarily  of 
ashing  tissue  sections  carefully  so  as 
to  retain  the  minerals  in  their  position 
in  the  fixed  tissue.  The  ashing  can  be 
done  on  glass  or  quartz  slides  by  a  vari- 
ety of  heating  processes.  Most  tissues 
in  the  body  can  be  treated  by  the  ash- 
ing process  with  some  success.  Those 
which  contain  large  quantities  of  phos- 
pholipids ordinarily  do  not  give  as  good 
results  as  tissues  lacking  them. 

The   method   is   one   which   requires 
some  care  and  the  observance  of  cer- 


tain very  definite  precautions  if  good 
results  are  to  be  had. 

Fixation:  There  are  two  methods  of 
fixation  which  can  be  used.  These  are 
the  chemical  and  the  frozen-dehydra- 
tion. If  the  cryostat  or  other  suitable 
devices  for  frozen-dehydration  are  not 
available,  fixation  by  absolute  alcohol 
plus  10  per  cent  formalin  yields  reason- 
ably good  pictures.  This  particular 
fixative  is  one  of  the  few  chemical  mix- 
tures which  dissolves  the  minimum  of 
mineral  from  fresh  tissue  and  adds  none 
to  it.  Tissues  are  passed  through  re- 
peated changes  of  absolute  alcohol  to 
dry  them  and  are  then  infiltrated  wdth 
paraffin  in  the  usual  manner. 

The  alternative  method,  that  of 
frozen-dehydration,  is  the  most  suitable 
for  preparation  of  tissues  for  micro- 
incineration. (See  Altmann-Gersh  and 
Cryostat.)  This  technique  yields  tissues 
which,  except  for  the  ice  crystal  forma- 
tion, have  not  been  altered,  to  any 
perceptible  degree,  either  ph3^sically  or 
chemicallJ^  Dehydration  at  suffi- 
ciently low  temperatures  maintains  an 
ice-salt  equilibrium  and  no  shifting  of 
minerals  in  the  cell  results.  If  the 
paraffin  infiltration  is  done  with  care, 
shrinkage  and  consequent  cellular  dis- 
tortion is  avoided. 

Methods  of  examination  of  the  in- 
cinerated preparations  are  several. 
One  of  the  simplest  and  best  for  study 
and  for  photograph}'  is  the  dark-field. 
Of  the  several  types  of  dark-field,  the 
cardioid  condenser  probably  gives  the 
most  uniform  results.  Illumination 
from  above  with  the  incident  light  fall- 
ing on  the  slide  at  an  angle  of  30°  is 
advised  by  Policard.  This  has  some 
advantages  over  the  dark-field  but 
makes  the  use  of  higher  magnifications 
difficult  if  not  actually  impossible.  Cel- 
lular details  are,  therefore,  to  be  ob- 
served best  by  using  the  cardioid  dark- 
field. 

Identification  of  minerals.  Some 
good  results  can  be  achieved  by  the  use 
of  ultraviolet  light  and  with  the  sub- 
sequent fluorescence  of  minerals.  Stu- 
dents should  consult  reference  and  text- 
books on  mineralogy  for  details  of 
identification. 

Calcium  and  magnesium  are  charac- 
terized in  the  dark-field  by  their  dense 
white  ash  residues.  Iron  is  oxidized 
during  the  incineration  process  and 
appears  as  varying  tints  of  red.  The 
amount  of  this  element  present  can  be 


MICROINJECTION 


200 


MICROMANIPULATION 


correlated  with  the  color  intensity. 
Silicon  is  definitely  crystalline  in  char- 
acter and  is  recognizable  by  its  property 
of  double  refraction  in  polarized  light. 
This  may  at  times  be  confusing  since 
all  minerals  blend  to  some  extent  with 
the  glass.  Lead  and  other  elements 
which  yield  black  sulfides  can  be  de- 
tected by  treating  the  section  with 
gaseous  hydrogen  sulfide.  Uranium 
in  pathological  tissues  fluoresces  with  a 
unique  color  under  ultraviolet  radia- 
tion. 

Attempts  have  been  made  to  quanti- 
tate  the  ash  residue  by  photographic 
means  and  by  the  use  of  a  photoelectric 
cell  whose  output  current  is  properly 
amplified.  Both  methods  leave  much 
to  be  desired  both  in  accuracy  and 
because  of  the  utter  relativity  of  the 
results  obtained.  The  most  useful 
finding  obtained  from  microincinera- 
tion, therefore,  is  the  appreciation  of  the 
distribution  of  the  total  minerals  in 
the  cell.  Experimental  alterations  in 
them  can  be  detected  by  the  technique. 
See  account  by  Scott  in  McClung's 
book  and  Electron  Microscope,  Histo- 
spectrography  and  Ultraviolet  Photo- 
micrography. 

Microinjection.  This  is  an  important  exten- 
sion of  microdissection  whereby  various 
fluids  are  injected  directly  into  the 
cytoplasm  or  nuclei  of  living  cells.  It 
is  capable  of  yielding  information  on 
Permeability,  Hydrogen  Ion  Concen- 
tration, Oxidation-Reduction  Poten- 
tial which  cannot  be  secured  in  any 
other  way,  but  in  reaching  conclusions 
due  allowance  must  be  made  for  the  fact 
that  cells  thus  treated  are  of  necessity 
severely  injured.  Microinjection  with 
glass  pipettes  but  without  an  expensive 
micromanipulator  can  yield  worthwhile 
results  as  described  by  Knower  (Mc- 
Clung,  pp.  51-61)  but  for  direct  work  on 
cells  the  micromanipulator  is  essential. 

Microliter  Burettes  are  essential  in  some 
histochemical  techniques.  They  are  of 
two  sorts.  In  the  first  a  capillary  glass 
tube  is  calibrated  so  that  volume  is 
indicated  by  the  level  of  the  meniscus. 
In  the  second  the  tube  is  not  calibrated 
but  instead  a  screw  determining  the 
level  is  provided  as  a  micrometer.  The 
best  micrometer  burette  was  designed 
by  Scholander  and  has  been  improved 
by  Scholander,  P.  F.,  Edwards,  G.  A. 
and  Irving,  L.,  J.  Biol.  Chem.,  1943, 
148,  495-500.  In  selecting  a  microliter 
burette  consult  Click,  pp.  255-264. 

Micromanipulation — Revised     by     Robert 


Chambers,  Dept.  of  Biology,  Washing- 
ton Square  College  of  New  York 
University,  New  York.  May,  1950 — 
Broadly  speaking,  this  term  covers  two 
types  of  operations:  delicate  free-hand 
operations  in  which  the  only  accessory 
may  be  a  dissecting  microscope,  and, 
second,  operations  conducted  by  means 
of  micrurgical  instruments  under  high 
magnifications. 

For  freehand  operations  considerable 
training  is  required  in  using  a  com- 
pound microscope  because  of  the  in- 
version of  the  image.  This,  however, 
can  be  corrected  by  using  the  so-called 
erecting  ocular.  A  decided  help  to 
relieve  fatigue  from  too  long  holding 
of,  a  dissecting  needle,  for  instance, 
is  to  have  the  shaft  of  the  needle  held 
in  the  apex  of  a  pyramid  of  plastic 
clay,  the  base  of  which  has  been  pressed 
down  on  the  stage  to  one  side  of  the 
microscope.  The  operator's  hand  en- 
circles the  mound  of  clay  which  bends 
as  his  fingers  guide  the  needle.  The 
tiring  fingers  can  be  released  at  any 
time  while  the  needle  tip  remains 
in  position.  Descriptions  of  excellent 
methods  for  injecting  minute  vessels, 
such  as  the  marginal  vein  of  chick 
embryos  or  lymphatic  vessels  of  frog 
tadpoles,  are  as  follows:  H.  McE. 
Knower,  Chapter  in  McClung's  Hand- 
book Microscopical  Technique,  3rd  ed., 
New  York:  Hoeber,  1950;  A.  L.  Brown, 
Anat.  Rec,  1922,  24,  295. 

Micromanipulation  in  its  more  re- 
stricted sense  applies  to  the  use  of 
mechanical  devices  for  controlling  the 
movements  of  the  tips  of  microneedles 
and  micropipettes  in  the  field  of  high 
powers  of  the  compound  microscope. 
A  full  account  is  given  in  McClung's 
Handbook. 

Several  instruments  are  now  being 
built.  The  ones  in  most  general  use  in 
this  country  are  those  of  Chambers, 
P6terfi  and  Emerson.  The  micro- 
needles or  pipettes  extend  into  a  moist 
chamber  on  the  stage  of  the  microscope 
so  that  their  tips  can  be  inserted  into 
hanging  drops  of  fluid  suspended  from 
the  undersurface  of  a  coverslip  which 
roofs  the  chamber.  The  essential  con- 
dition of  an  instrument  is  that  the 
movements  be  sufficiently  smooth  and 
controllable  under  the  highest  magni- 
fications of  the  compound  microscope. 

P^terfi's  instrument  was  manufac- 
tured by  Carl  Zeiss  Co.  and  is  now 
difficult  to  procure.  Chambers'  was 
manufactured    by    E.    Leitz    and    has 


MICROMAN IPULATION 


201 


MICROMANIPULATION 


undergone  many  modifications  as  to 
its  accessory  parts.  It  is  now  being 
manufactured  by  the  Gamma  Instru- 
ment Company,  Great  Neck,  New  York. 
Both  instruments  are  supplied  with 
two  main  holders  each  independent 
of  the  other  for  carrying  a  microneedle 
or  a  micropipette.  The  advantage  of 
Chambers'  is  that  the  two  holders  are 
so  adjusted  as  to  permit  the  needles 
and  pipettes  to  extend  parallel  to  one 
another  on  the  microscope  stage.  This 
permits  the  insertion  of  the  needles  into 
the  moist  chamber  through  one  opening, 
thus  increasing  the  chances  of  main- 
taining moisture  conditions  in  the 
chamber. 

More  recently  an  instrument  devised 
by  de  Fonbrune  of  Paris  is  being  manu- 
factured by  A.  S.  Aloe  Co.,  St.  Louis, 
Mo.  An  early  form  of  it  is  described 
in  L'lllustration,  February  15  and  22, 
1941.  It  depends  for  its  fine  move- 
ments on  hydraulic  pressure  conveyed 
through  flexible  tubing  from  a  hand 
operated  lever.  Circus  movements  in 
the  horizontal  plane  are  performed  by 
rotating  the  lever,  the  vertical  move- 
ment being  accomplished  by  a  thrusting 
action  of  a  plunger  in  the  shaft  of  the 
lever.  Two  such  instruments  mounted 
on  opposite  sides  of  the  microscope  per- 
mit the  use  of  two  microneedles.  The 
instrument  has  great  possibilities  but 
its  specific  usefulness  for  the  perform- 
ance of  circus  movements  is  fully 
covered  by  the  Emerson  instrument 
which,  incidentally,  is  sturdily  con- 
structed. 

The  Emerson  is  a  first  class  machine 
with  a  mechanically  controlled  lever 
using  circus  movements  in  a  horizontal 
plane,  also  a  separate  fine  adjustment 
device  for  the  vertical  ones.  Emerson 
has  two  models,  the  lower  priced  one 
having  a  lever  control  for  both  hori- 
zontal and  vertical  fine  adjustments. 
These  are  being  supplied  by  the  J.  H. 
Emerson  Company,  Cambridge,  Massa- 
chusetts. 

The  Chambers  instrument  is  the  only 
one  supplied  with  a  microinjection  ap- 
paratus a  description  of  which  is  given 
in  the  latter  part  of  this  article. 

Micrurgical  instruments  lend  them- 
selves to  several  tj^pes  of  operations : 
(1)  Microdissection  and  injection  of 
animal  or  plant  cells  and  tis.sues  for 
studies  in  cell  anatomy  and  physiology, 
also  cj'to-chemistry  in  which  chemical 
reactions  can  be  obtained  by  applying 
chemical  agents  not  only  to  individual 
cells  but  to  localized  regions  within  a 
given  cell.  (2)  Chemical  reactions  in 
micro-drops.  A  very  useful  method  is 
to  deposit  the  droplets  with  a  micro- 


pipette in  a  hanging  drop  of  an  inert 
oil.  This  prevents  evaporation  and 
the  sphericity  of  the  droplets  in  the 
oil  permits  quantitative  determina- 
tions. Application  of  the  technique  to 
certain  phases  of  microchemistry  are 
given  by  Benedetti-Pichler  in  his  book 
Introduction  to  the  Microtechnique  of 
Inorganic  Analysis,  New  York:  Wiley, 
1942.  (3)  Isolation  studies  for  obtain- 
ing pure  line  cultures  (of  bacteria,  pro- 
tozoa, etc.,  breaking  of  asci  and  isola- 
tion of  the  liberated  spores,  etc.). 
A  good  isolation  technique  is  given  by 
Reyniers,  J.  A.,  J.  Bact.,  1933,  26,  251. 

The  movements  of  the  instruments 
can  be  controlled  in  any  of  three  dimen- 
sions; the  horizontal  permits  circus 
movements  in  one  plane.  Circus  move- 
ments in  the  horizontal  plane  are  best 
managed  with  the  de  Fonbrune  and 
Emerson  instruments.  The  vertical 
movement  is  operated  by  a  separate 
controlling  screw.  Micro  operations 
also  can  be  performed  under  relatively 
low  powers  of  the  microscope.  How- 
ever, the  operator  should  realize  that 
the  compound  microscope,  even  though 
it  be  binocular,  is  monobjective.  This 
means  that  the  position  of  an  object 
in  the  vertical  plane  can  be  deduced 
only  by  observing  whether  the  object 
is  in  or  out  of  focus.  The  lower  the 
magnifying  power  of  the  objective  the 
greater  is  the  depth  of  its  focus. 
Hence,  there  may  be  occasions  when 
the  tip  of  the  microneedle  and  the  ob- 
ject to  be  operated  upon  are  at  different 
levels  although  both  are  in  focus  to  the 
eye. 

The  mechanical  stage  of  the  micro- 
scope is  a  useful  adjunct  for  micrurgy. 
Particularly  for  injections,  the  most 
satisfactory  way  is  to  keep  the  tip  of 
the  micropipette  in  the  center  of  the 
field  and  to  perform  the  operation  by 
raising  the  tip  into  the  object  to  be 
injected  after  having  brought  the  ob- 
ject into  position  by  means  of  the 
mechanical  stage. 

The  manufactured  instruments  are 
supplied  with  instructions  as  to  their 
use.  Emerson  supplies  two  types,  one 
for  coarser  movements  although  it  is 
possible  to  use  this  model  for  remark- 
ably fine  operations.  The  only  way  to 
select  an  instrument  is  to  know  what  is 
wanted  and  then  to  decide  after  having 
the  instrument  demonstrated  to  him. 
All  require  the  use  of  a  good  mechanical 
stage  to  move  the  moist  chamber  which 
carries  the  drops  containing  the  tissue 
to  be  operated  on.  All  in  all,  micro- 
manipulation requires  not  only  ability 
but  mechanical  aptitude  on  the  part  of 
the  would-be  operator.     It  is  one  thing 


MICROMANIPULATION 


202 


MICROMANIPULATION 


to  have  an  instrument  and  a  good  micro- 
scope. It  is  another  matter  to  build 
the  many  accessories,  with  cement,  out 
of  wood,  glass  or  plastic,  which  the 
operator  may  need  for  his  special  pur- 
poses. Any  gadget  built  may  well 
mean  a  new  discovery. 

Tissues  and  cells  to  be  operated  on 
often  require  special  means  for  holding 
them  in  place.  Actively  moving  pro- 
tozoa can  be  kept  quiet  by  immersing 
them  in  egg  albumen  or  a  solution  of 
hemi-cellulose.  Strips  of  the  epidermis 
of  onion  or  tulip  bulbs,  immersed  in 
varying  concentrations  of  cane  sugar, 
offer  good  objects  for  operation  on  pro- 
toplasts under  different  degrees  of 
plasmolysis,  likewise  stamen  hairs  of 
Tradescantia  which  show  mitotic 
figures.  Similar  studies  may  also  be 
made  on  the  epidermis  of  the  tails  of 
tadpoles.  For  these,  the  operator 
should  use  frogs'  Ringer  solution  to 
maintain  the  proper  balance  of  elec- 
trolytes in  the  medium.  Muscle  fibers 
stripped  from  the  semitendinosus  of  the 
frog  are  good  material.  Urodeles  fur- 
nish excellent  material.  An  effective 
means  of  obtaining  red  cells  undergoing 
mitosis  is  to  bleed  a  Necturus  or  other 
member  of  the  same  order  and  take  a 
sample  of  blood  after  a  week  or  so. 

The  microneedles  and  micropipettes 
are  usually  made  from  glass  capillary 
rods  or  tubes.  Serviceable  sizes  with 
an  outside  diameter  of  1-2  mm.  can  be 
drawn  out  in  a  bunsen  flame.  The 
needle  tips  are  made  over  a  microfiame 
by  heating  and  pulling  the  shaft  of  a 
capillary  held  at  both  ends  with  the 
two  hands.  A  serviceable  gas  micro- 
burner  for  this  purpose  is  a  hypodermic 
needle.  When  successful,  the  drawn- 
out  tips  taper  to  a  point  rapidly  enough 
so  that  the  invisibly,  fine  tip  is  sup- 
ported on  a  relatively  rigid  shaft.  The 
shaft  about  2  mm.  from  the  tip,  is  bent 
in  the  microfiame  to  about  a  right  angle. 
The  other  end  of  the  capillary  is  then 
inserted  into  a  specially  constructed 
needle-holder  and  mounted  in  a  micro- 
manipulator so  that  the  tip  extends 
over  the  microscope  stage  into  a  moist 
chamber.  The  bent-up  tip  is  adjusted 
with  the  screws  of  the  instrument  until 
the  tip  lies  in  a  hanging  drop  of  fluid 
suspended  from  a  glass  cover-slip  serv- 
ing as  the  roof  of  the  moist  chamber 
and  in  the  field  of  the  microscope. 

Mechanical  contrivances  for  drawing 
out  the  end  of  a  glass  capillary  into  a 
tapering  tip  are  available.  The  most 
elaborate  one  is  that  of  de  Fonbrune, 
the  microforge.  This  is  a  highly  am- 
plified mechanism  with  a  built  in  micro- 
scope and  coarse  adjustment  parts  for 


holding,  one,  a  glass  capillary  and  the 
other  a  platinum  loop.  The  microscope 
is  adjusted  to  view  the  loop  of  an  elec- 
trically incandescing  fine  platinum  wire 
to  which  one  end  of  the  glass  capillary 
is  approached  and  then  withdrawn  as 
the  glass  begins  to  melt.  Then  prop- 
erly done  the  tip  of  the  capillary  is 
drawn  out  to  a  tapering  tip. 

The  principle  of  the  microforge  is 
based  on  a  much  simpler  device  long 
ago  devised  by  S.  L.  Schouten  and  de- 
scribed by  him  in  1934.  This  can  be 
readily  built  in  any  laboratory  equipped 
with  an  ordinary  microscope  and  micro- 
manipulator. 

A  fairly  good  mechanical  device  for 
drawing  microtips  is  being  supplied  by 
the  Gamma  Instrument  Company.  It 
depends  upon  springs  to  which  the  two 
ends  of  a  glass  capillary  (1-2  mm.  diam.) 
are  fastened  while  the  middle  of  the 
capillary  passes  through  several  coils 
of  a  fine  platinum  or  nichrome  wire. 
When  the  wire  is  caused  to  incandesce 
electrically  the  glass  softens  and  the 
taut  springs  pull  the  capillary  in  two, 
each  part  with  a  tapering  tip.  A  more 
precise  needle  puller  based  on  the  same 
principle  is  the  Livingston  Micro  Pi- 
pette Puller  made  by  Otto  K.  Hebel, 
Swarthmore    College,    Pennsylvania. 

There  are  several  possible  ways  by 
means  of  which  a  person  with  some 
ingenuity  should  be  able  to  devise  from 
the  usual  laboratory  equipment  a  simple 
mechanical  device  for  drawing  needle 
tips.  If  the  glass  capillary  is  of  tubing 
the  microtips  can  be  used  for  micro- 
capillaries. 

Injections  are  performed  bj^  breaking 
the  tip  of  a  micropipette  against  the 
undersurface  of  the  coverslip  while  the 
tip  is  in  view  under  the  microscope. 
Capillarity  draws  fluid  into  the  shaft 
of  the  pipette  when  the  open  tip  is  in- 
serted into  a  hanging  drop  of  fluid,  be 
it  oil  or  any  given  solution.  For  micro- 
injection, the  pipette  holder,  mounted 
on  the  instrument,  is  attached  to  a 
looped,  capillary  brass  tube  of  which 
the  other  end  is  attached  to  the  nozzle 
of  a  syringe.  Before  mounting  the 
micropipette,  the  syringe  is  filled  with 
water  previously  boiled  to  be  air-free 
and,  by  means  of  the  plunger,  the  water 
is  driven  into  the  brass  tubing  and  the 
pipette  holder  after  which  the  micro- 
pipette is  inserted.  Thus,  we  have  a 
water-filled  system  extending  from  the 
syringe  to  the  base  of  the  micropipette 
the  shaft  of  which  may  contain  air. 
Micro-amounts  of  any  given  solution 
are  then  drawn  into  or  ejected  from  the 
tip  of  the  micropipette  by  a  delicate 
handling  of  the  plunger  of  the  syringe. 


MICROMETRY 


203 


MICROSCOPES 


Considerable  deviations  are  possible 
in  the  matter  of  the  microinjection 
technique.  For  example,  if  it  is  deemed 
desirable  to  have  no  air  in  the  system, 
the  shafts  of  the  capillary  tubing,  on 
which  the  microtips  are  drawn,  may 
previously  be  filled  with  either  oil  or 
water.  By  using  some  ingenuity  the 
entire  microinjection  apparatus  can  be 
built  in  the  laboratory,  the  parts  re- 
quired being  a  Luer  syringe,  hypodermic 
needles  the  shafts  of  which  can  be  cut 
off,  a  strip  of  flexible  brass  or  copper 
tubing,  glass  tubing  and  of  course  the 
operator's  constant  companion:  a  stick 
of  deKhotinsky  cement  or  an  analo- 
gous superior  sealing  wax. 

The  instruments  are  generally  supplied 
in  pairs,  one  part  carrying  a  micro- 
needle for  holding  the  tissue  to  be 
injected,  the  other  carrying  the  micro- 
pipette.  For  microdissection,  the  in- 
strument carries  two  needles,  each  of 
which  can  be  operated  independently. 
Wilhelm  Pfeffer,  to  whom  we  owe  the 
term  "plasma  membrane"  for  the  limit- 
ing boundary  of  protoplasm,  stated,  in 
one  of  his  papers  in  1887,  that  an  instru- 
ment with  which  one  could  operate 
delicate  needles  and  pipettes  in  the 
field  of  a  compound  microscope  would 
go  far  toward  the  elucidation  of  the 
nature  of  living  cells.  Pfeffer's  dream 
has  been  realized  in  the  development  of 
the  special  field  of  science  called  today 
Micromanipulation  or  Micrurgy. 

Of  general  interest,  and  also  for  many 
details  not  described  elsewhere,  are  the 
following:  Barber,  M.  A.,  Philippine  J. 
Science,  B,  1914,  9,  307;  Chambers,  R., 
Anat.  Rec,  1922,  24,  1;  P^terfi,  T.,  in 
methodik  der  wissensch.  Biologic,  1928, 
1  (4),  5;  and  Schouten,  S.  L.,  Zeit.  f. 
wiss.  Mikr.,  1934,  51,  421.  An  excellent 
book  which  covers  a  broad  range  of  the 
field  of  Micrurgy  is  that  edited  by 
J.  A.  Reyniers  on  Micrurgical  and 
Germ-Free  Techniques,  C.  C.  Thomas, 
1943,  an  article  on  micromanipulation 
by  Chambers,  R.  and  C.  G.  Grand, 
Encyclopaedia  Britannica,  1948. 

Micrometry  is  the  measurement  of  an  object 
observed  microscopically.  This  can  be 
done  either  by  using  an  ocular  microm- 
eter in  which  there  are  lines  which  can 
be  accurately  moved  the  length  of  the 
structure  to  be  measured  or  by  inserting 
a  ruled  disc  in  an  ordinary  ocular  with 
which  it  can  be  compared.  Both  must 
be  standardized  in  relation  to  a  microm- 
eter slide  generally  ruled  with  lines  10m 
apart.     See  Cell  Measurements. 

Micromicron  (nn)  =  1/1 ,000,000th  part  of  a 
micron  =  1/1, 000 ,000 ,000th  part  of  a 
mm.  =  10- »  mm.  =  0.000,001  micron  = 
10~^A.    Unfortunately  often  used  syn- 


onymously   with  o  millimicron  (m/i)  = 
0.001  micron  =  lOA. 

Micron  (Gr.  Mikros,  small)  expressed  by 
Gr.  letter  n  =  approximately  1/25,000 
inch  =  1/1000  part  of  a  mm.  =  0.001 
mm.  =  10-3  mm.  =  10,000  A  (see 
Millimicron  and  Micromicron). 

Microphotometer,  see  Photoelectric. 

Microradiographic  examination.  This  con- 
sists of  magnification  of  a  Roentgen  ray 
image  after  it  has  been  registered  pho- 
tographically. The  essential  point  is 
to  use  film  of  very  fine  grain  emulsions. 
Thus  the  Gevaert  Lipmann  emulsion 
permits  enlargement  300  times  without 
much  loss  of  detail.  In  some  cases  it 
is  helpful  before  microradiographic 
examination  to  increase  the  absorption 
of  Roentgen  rays  by  "absorption  stain- 
ing" through  adding  radio-opaque  mate- 
rials such  as  barium  sulp'aate  and  thoro- 
trast.  The  application  of  this  technique 
in  the  study  of  biologic  materials 
is  described  and  illustrated  by  Clark, 
G.  L.  and  Bick,  E.  J.,  in  Glasser's  Medi- 
cal Physics,  730-733. 

The  importance  of  extremely  soft 
Roentgen  rays  and  the  properties  of 
fine-grained  emulsions  are  described 
by  Engstrom,  A.  and  Lindstrom,  B., 
Acta  Radiol.,  1951,  35,  33-44.  See  also 
their  illustrations  of  striated  muscle 
fibers  and  of  bone  by  microradiography. 

Microrespirometer  to  indicate  production  of 
carbon  dioxide  by  bacteriophages, 
viruses  and  bacteria  (Bronfenbrenner, 
J.,  Proc.  Soc.  Exp.  Biol.  &  Med.,  1924, 
22,  81-82.     See  Capillary  Respirometry. 

Microscopes  (From  Cowdry's  Histology, 
1950).  While  excellence  in  histological 
technique  is  important,  knowledge  of 
microscopes  and  of  how  to  get  the  best 
service  out  of  them  is  also  important. 
There  are  several  kinds  from  which  a 
choice  must  be  made  of  the  one  capable 
of  yielding  the  information  required. 
The  following  account  of  what  these 
instruments  are,  what  their  particular 
use  is,  what  their  limitations  are,  sup- 
plemented by  leading  references  to 
literature  on  the  subject  is  offered  for 
guidance.  It  was  written  for  Cowdry's 
Histology  by  Dr.  T.  B.  Rosenthal. 

The  ordinary  compound  microscope  is 
a  precision  optical  instrument  designed 
to  give  magnified  images  of  50  to  1200X. 
Daylight,  or  strong  artificial  light,  is 
used  to  illuminate  by  transmission  a 
more  or  less  thin,  flat,  transparent 
object.  The  image  appears  in  its  nat- 
ural form  and  color,  but  inverted  and 
reversed  in  position,  at  a  distance  of 
about  10  inches  from  the  eye.  Height 
and  depth  are  not  reversed,  however, 
so  it  is  possible  to  judge  or  measure 


MICROSCOPES 


204 


MICROSCOPES 


the  elevations  on  an  object  by  differen- 
tial focussing. 

Three  lens  systems,  condenser,  ob- 
jective, and  ocular  cooperate  to  form 
the  final  image.  The  purpose  of  the 
condenser  is  two-fold:  to  gather  light 
for  illumination,  and  to  focus  this  light 
on  the  object  in  a  cone  of  the  proper 
dimensions  so  that  the  full  resolving 
power  of  the  objective  lens  can  be 
gained.  By  means  of  the  objective 
lens  a  real,  magnified,  inverted,  re- 
versed image  of  the  object  is  formed  at 
the  upper  end  of  the  tube.  The  ocular 
further  magnifies  this  primary  image  to 
yield  a  virtual,  inverted,  reversed  sec- 
ondary image.  Total  magnification  is 
calculated  by  multiplying  the  magnify- 
ing powers  of  the  objective  and  the 
ocular. 

Inspection  of  the  markings  on  a  set 
of  objectives  will  show  their  magnifying 
powers  and  another  optical  property 
called  the  numerical  aperture  (N.  A.). 
Usually  the  10 X  (also  called  16  mm) 
objective  is  marked  with  the  number 
"0.25;"  the  44X  (4  mm,  "high  dry"), 
"0.66;"  and  the  oil  immersion,  95X 
(1.8  or  2.0  mm),  "1.25."  Numerical 
aperture,  calculated  from  a  geometrical 
property  of  the  front  lens  element,  is 
proportional  to  the  theoretical  resolving 
power  of  the  objective  and  is  related 
to  the  maximum  power  of  the  ocular 
that  can  be  profitably  used  with  that 
objective.  In  order  to  understand 
this  relation  between  magnification, 
resolution,  and  N.  A.  it  is  necessary  to 
consider  certain  fundamentals. 

As  successively  higher  powers  of  mag- 
nification are  brought  to  bear  on  a  tis- 
sue section  more  and  more  fine  detail 
becomes  visible,  which  is,  after  all,  the 
only  purpose  of  microscopy.  But  to 
fineness  of  detail  a  limit  is  set,  not  by 
magnification,  but  by  the  nature  of 
light  and  by  the  optical  properties  of 
lenses.  As  long  as  the  object  to  be 
seen  is  large  compared  to  the  wave 
length  of  the  light  which  illuminates  it 
the  microscopic  image  will  be  sharp. 
If  structural  details  are  so  small  that 
their  size  nearly  approaches  the  wave 
length  of  light,  the  image  becomes 
fuzzy.  Although  magnification  may 
be  secondarily  raised  by  employing 
stronger  eyepieces  no  further  resolu- 
tion of  detail  takes  place;  the  image 
remains  fuzzy,  and  we  have  what  is 
called  "empty  magnification." 

The  formula  R  =  X/2  N.  A.  gives  a 
general  relationship,  where  X  =  wave 
length  of  light  used,  N.  A.  is  a  given 
constant  of  the  objective  lens,  and  R  is 
the  size  of  minimum  resolvable  detail 
(given  in  /i  if  X  is  given  in  fx).    Taking 


an  average  value  for  X  in  white  light  to 
be  0.5  fi  and  substituting  the  N.  A. 
values  mentioned  above,  we  have  for 
the  lOX  objective,  R  =  1.0  m;  for  the 
44X,  R  =  0.4  m;  and  for  oil  immersion, 
R  =  0.2  /x.  Thus,  with  the  best  lenses, 
details  lying  less  than  0.2  m  apart  are 
not  discerned. 

Another  general  rule  in  microscopy 
states  that  the  maximum  total  magnifi- 
cation should  not  be  pushed  higher  than 
about  one  thousand  times  the  N.  A.; 
otherwise  empty  magnification  results. 
With  the  lOX  objective,  1000  X  0.25  = 
250.  Therefore  a  25X  ocular  is  the 
maximum.  For  the  oil  immersion,  a 
13X  ocular  gives  the  limiting  useful 
magnification. 

It  is  thus  evident  that  the  objective 
lens  is  the  heart  of  the  microscope. 
The  ocular  brings  out  only  the  details 
which  already  have  been  resolved  by 
the  objective. 

The  role  of  the  condenser  may  be 
reviewed  in  the  light  of  these  interpre- 
tations. Historically  the  condenser 
received  its  name  from  its  original  pur- 
pose: it  was  simply  a  lens  used  to  con- 
centrate light  on  the  subject.  E. 
Abbe  (1840-1905),  who  contributed  a 
great  deal  to  theoretical  and  practical 
microscopy,  including  the  concept  of 
numerical  aperture,  came  to  the  con- 
clusion that  an  objective  lens  could  not 
work  at  its  maximum  N.  A.  unless  it 
were  matched  with  a  condenser  lens 
system  of  equal  N.  A.  Hence  the 
modern  condenser  is  also  rated  in  terms 
of  N.  A.  and  is  provided  with  a  variable 
iris  diaphragm  to  alter  its  N.  A.  in  order 
to  match  with  that  of  the  objective. 

Since  the  standard  microscope  pro- 
vides all  the  convenient  magnifications 
and  even  more  resolution  than  is  usually 
required  in  routine  histology,  it  is  not 
necessary  to  take  extraordinary  pains 
to  coUimate  (parallel)  the  microscope 
with  the  light  source  or  to  worry  over 
the  fine  points  of  diaphragm  control. 
However,  good  photomicrography  de- 
mands such  attention  to  details,  be- 
cause the  plate  is  much  more  sensitive 
to  inequalities  of  lighting  than  the  ob- 
server's eye.  On  the  other  hand  the 
eye  is  subject  to  fatigue  unless  the 
proper  illumination  is  employed.  In 
student  classes  one  commonly  finds  that 
the  lighting  with  monocular  micro- 
scopes is  too  bright  and  with  binoculars 
too  dim.  To  minimize  visual  fatigue 
the  brightness  of  the  microscopic  field 
of  view  should  about  equal  the  bright- 
ness of  the  table-top  and  be  in  keeping 
with  the  general  level  of  illumination 
in  the  room. 

The  following  suggestions  for  setting 


MICROSOMES 


205 


MICROSOMES 


up  the  microscope  apply  to  the  oil 
immersion  lens.  When  dry  lenses  are 
to  be  used  omit  step  2. 

1.  Clean  the  slide  with  alcohol. 
Put  a  drop  of  xj-lol  on  the  oil  immersion 
lens  and  polish  it  with  lens  paper. 
Do  the  same  for  the  eye  piece. 

2.  Raise  the  condenser  so  that  its 
upper  surface  is  on  a  level  with  the 
stage,  or  slightly  below  it.  Put  a  drop 
of  immersion  oil  on  the  condenser  and 
lay  the  slide  down.  Put  a  drop  of  oil 
on  the  slide,  lower  the  lens,  and  focus 
as  usual. 

3.  Open  the  diaphragm  wide,  and 
removing  the  eye  piece,  look  down  the 
tube.  Adjust  the  position  of  the  light 
source  and  mirror  so  that  the  aperture 
appears    sj^mmetrically    illuminated. 

4.  At  this  point  the  aperture  should 
look  like  a  bright  disk  surrounded  by  a 
rim  of  dim  illumination.  Now,  close 
down  the  diaphragm  until  about  nine- 
tenths  of  the  area  of  bright  central 
field  remains  visible.  Replace  the 
eye  piece.  The  N.  A.  of  condenser 
and  objective  are  now  approximately 
equal. 

5.  Modify  the  brightness  of  the  image 
for  visual  comfort  by  altering  the  in- 
tensity of  illumination;  not  by  altering 
the  diaphragm.  If  the  image  is  too 
bright  put  tissue  paper  over  the  lamp 
or  pull  the  lamp  away  and  readjust  by 
repetition  of  Steps  3  and  4.  If  the 
image  is  not  bright  enough  move  the 
lamp  in,  and  if  necessary  dispense  with 
the  substage  mirror.  As  the  final  step, 
try  a  slight  change  in  the  position  of 
the  condenser,  but  avoid  breaking  the 
oil  pool  between  the  condenser  and  the 
slide. 

Resolving  power  may  be  improved 
somewhat  for  striated,  or  periodic, 
structures  by  using  oblique  illumina- 
tion. Light  is  sent  diagonally  from 
below  so  that  only  part  of  it  enters 
the  objective,  with  the  striations  (as  in 
muscle)  lying  across  the  direction  of 
the  light.  If  the  condenser  is  not 
laterally  movable  it  should  be  masked 
below  so  that  light  enters  only  from  one 
side.  By  trial  and  error  a  favorable 
orientation  of  condenser,  slide  and 
mask  can  be  found. 

The  limitations  imposed  on  the  re- 
solving power  of  the  conventional 
microscope  cause  no  inconviences  in 
general  histology.  It  is  with  the  finer 
details  of  cytology  that  need  is  felt 
for  greater  resolution.  As  is  obvious 
from  the  formula  R  =  X/2  N.  A.,  either 
an  increase  of  N.  A.  or  a  decrease  of  X 
will  reduce  the  value  of  R.  The  best 
available  oil  immersion  objectives  and 
condensers    are    rated    at    1.40   N.    A. 


A  special  lens  working  at  1.60  has  been 
designed,  but  the  improvement  in  re- 
solving power  over  one  at  1.40  is  not 
important.  Fortunately  we  can  ob- 
tain the  shorter  values  of  X  by  em- 
ploying ultra-violet  light  and  photog- 
raphy   (see    ultraviolet-microscope). 

A  word  should  be  said  in  favor  of 
water-immersion  objectives,  items  for- 
merly found  in  every  laboratory  of 
microscopy  but  now  no  longer  listed  in 
the  catalogues  of  American  manufac- 
turers. These  are  made  in  a  series  of 
magnifications,  and  in  resolving  power 
are  intermediate  between  air  and  oil- 
immersion  objectives.  For  study  of 
living  aquatic  organisms  and  tissue  cul- 
tures the  lOX  and  44X  objectives  are 
very  useful  since  they  are  made  to  be 
lowered  directly  into  the  medium. 
For  histological  slide  material  the  high- 
power  water-immersion  objective  is 
probably  not  inferior  to  the  oil-immer- 
sion, besides  being  more  convenient 
to  use. 

It  is  commonly  believed  that  a  bin- 
ocular, i.  e.,  two  eyepiece,  microscope 
is  superior  to  the  monocular  because  it 
affords  stereoscopic  vision.  Actually, 
it  cannot  be  so  since  a  single  objective 
forms  only  one  primary  image  regard- 
less of  whether  it  be  viewed  by  one  or 
two  eyes.  Nevertheless  our  habit  of 
seeing  with  two  eyes  probably  creates 
an  illusion  of  stereoscopic  vision  with 
the  binocular  microscope.  On  the 
other  hand  a  binocular  microscope  em- 
ploying two  objectives  on  a  pair  of 
converging  tubes  does  provide  stereo- 
scopic views.  The  dissecting  micro- 
scope is  built  on  this  pattern,  having 
in  addition  a  set  of  reversing  and  erect- 
ing prisms  so  that  the  final  image  is 
normally  orientated.  Magnifications 
above  30X  become  rather  useless  for 
dissection  purposes  because  the  depth 
of  focus  is  greatly  reduced.  Manipula- 
tion is  usually  by  hand. 

See  Centrifuge,  Color  Translation, 
Darkfield,  Electron,  Phase  Contrast, 
Metallurgical,  Polarizing,  Reflecting 
and  Ultraviolet  Microscopes.  Many 
excellent  books  on  microscopes  and 
photomicrography  are  available.  The 
following  are  suggested:  Gage,  S.  H., 
The  Microscope.  Ithaca:  Cornstock, 
1941,  616  pp.  Shillaber,  C.  P.,  Pho- 
tomicrography in  Theory  and  Prac- 
tice. New  York:  John  Wiley  and  Sons, 
1944,  773  pp.  Wredden,  J.  H.,  The 
Microscope,  Its  Theory  and  Applica- 
tion. New  York:  Grune  and  Stratton, 
1948,  296  pp. 
Microsomes  (G.  mikros,  small,  soma,  body). 
Term  introduced  by  Hanstein  in  1880 
originally  to  indicate  tiny  granules — as 


MICROSPECTROPHOTOMETRY 


206 


MICROSPECTROPHOTOMETRY 


compared  with  ground  substance. 
Claude,  A.  Biological  Symposia,  1943, 
10,  111-129  estimates  their  size  to  be 
50-300  ran  and  therefore  beyond  limits 
of  ordinary  microscopic  visibility. 
These  microsomes  of  Claude  are  ob- 
viously not  the  ones  which  Hanstein 
had  in  mind.  According  to  Claude 
they  are  essentially  ribose  nucleopro- 
teins  and  phospholipins  in  definite 
proportions. 
Microspectrophotometry — Barry  Commo- 
ner, The  Henry  Shaw  School  of  Botany, 
Washington  University,  St.  Louis  5. 
November  28,  1951 — Microspectropho- 
tometry is  a  technique  for  examination 
of  cells  and  cell  structures  designed  to 
yield  data  on  the  chemical  composition 
of  these  objects.  The  method  is 
based  on  the  fact  that  given  molecular 
configurations  absorb  specific  wave- 
lengths of  ultraviolet,  visible  or  infra- 
red radiation.  Under  ideal  conditions, 
as  in  dilute  solutions,  the  molecular 
group  and  therefore  the  substance  in 
which  it  occurs  may  be  identified  from 
the  shape  of  the  absorption  spectrum, 
and  its  concentration  in  the  sample 
determined  from  the  amount  of  absorp- 
tion at  a  characteristic  wavelength. 
Unfortunately,  cytological  objects 
never  offer  such  simple,  readily  analyz- 
able  situations.  Consequently,  special 
steps  must  be  taken  to  evaluate  the 
absorption  spectra  of  cytological 
objects,  and  in  most  instances,  data  com- 
parable with  those  obtained  from  solu- 
tions are  not  yet  attainable.  Never- 
theless, the  technique  has  thus  far 
proved  to  be  a  valuable  source  of  in- 
formation on  cell  composition,  and  if 
used  with  care  can  be  advantageously 
applied  to  a  number  of  biological  prob- 
lems. 

The  essential  measurement  in  micro- 
spectrophotometry is  determination 
of  the  reduction  in  intensity  of  a  light 
beam  after  passing  through  a  cytologi- 
cal object.  This  measurement,  made 
at  a  series  of  specific  wavelengths,  gives 
the  absorption  spectrum  of  the  object. 
The  measurements  may  be  made  by 
passing  a  monochromatic  beam  through 
the  object;  or  by  passing  a  heterogene- 
ous beam  through  the  object  and  then 
dispersing  it  into  a  spectrum.  Equip- 
ment employing  the  second  of  these 
methods  has  been  described  by  (Mellors, 
R.  C,  Science,  1951,  112,  381-389). 
Since  most  microspectrophotometry 
has  employed  the  first  principle  of 
operation,  details  for  this  type  of  ap- 
paratus are  given  below. 

1.  General.  The  fundamental  parts 
of  a  microspectrophotometer  consist 
of  a  suitable  light  source,  a  monochrom- 


ator,  microscope  optics,  a  photoelec- 
tric tube  which  receives  the  projected 
image  of  the  object,  and  appropriate 
means  of  measuring  the  phototube 
response. 

2.  Light  sources.  Ordinary  automo- 
bile headlight  tungsten  lamps  operated 
on  a  storage  battery  are  suitable  for 
work  in  the  visible  spectrum.  For  work 
in  the  ultraviolet  ranges  mercury  dis- 
charge tubes  provide  adequate  light 
intensity.  High  pressure  mercury 
lamps  (such  as  General  Electric  AH6) 
emit  a  continuous  spectrum  in  the  vis- 
ible and  ultraviolet  superimposed  on  a 
number  of  bright  and  dark  lines.  In 
the  infra-red  ranges  Nernst  glowers  are 
suitable. 

3.  The  monochromator.  The  light 
beam  from  source  is  directed  into  the 
entrance  slit  of  a  monochromator, 
thus  giving  an  emergent  beam  of  a 
determined  mean  wavelength.  The 
monochromator  should  supply  the  con- 
denser of  the  microscope  with  a  beam 
sufficient  to  fill  the  aperture  of  the 
latter. 

4.  The  microscope.  The  optical  ar- 
rangement of  the  microscope  must  be 
such  as  to  give  a  true  light  image  of  the 
object  at  the  plane  of  the  photocell. 
The  requirements  for  this  condition 
have  been  presented  by  Caspersson, 
T.  (Cell  growth  and  cell  function,  New 
York:  Norton,  1949).  For  visible  work 
any  good  apochromatic  system  of  suffi- 
cient numerical  aperture  is  adequate. 
In  the  ultraviolet  range  quartz  lenses 
or  reflecting  objectives  must  be  used. 
The  latter  have  the  advantage  of  being 
achromatic  in  the  ultraviolet  spectrum. 

5.  The  light  detector.  The  most 
suitable  detector  for  this  type  of  work 
is  the  photomultiplier  tube.  The  tube 
is  supplied  with  a  suitable  power  source 
and  its  output  amplified  and  read  off 
a  microammeter.  Less  conveniently 
the  direct  output  may  be  detected  with 
a  galvanometer. 

The  construction  of  microspectro- 
photometric  equipment  has  been  de- 
scribed in  the  following  papers:  Norris, 
K.P.  and  Wilkins,M.H.F.,  Discussions 
of  the  Faraday  Society,  1950,  No.  9, 
360-363,  Barer,  R.,  Discussions  of  the 
Faraday  Society,  1950,  No.  9,  369- 
378,  Mellors,  R.  C.,  Discussions  of  the 
Faraday  Society,  1950,  No.  9,  398-406, 
Thorell,  B.,  Discussions  of  the  Faraday 
Society,  1950,  No.  9,  432-436,  Walker, 
P.  M.  B.  and  Davies,  H.  G.,  Discus- 
sions of  the  Faraday  Society,  1950, 
No.  9,  461-470,  Commoner,  B.,  Ann. 
Mo.  Bot.  Card.,  1948,  35,  239-254, 
Burch,  C.  R.,  Proc.  Physic.  Soc,  1947, 
59,  41-46,   Grey,  D.   S.,  J.   Opt.   Soc. 


MICROSPECTROPHOTOMETRY 


207 


MICROSPECTROPHOTOMETRY 


Amer.,  1950,  40,  283-290,  Mellors,  R.  C, 
Science,  1951,  112,  381-389.  The  prob- 
lem of  instrumental  accuracy  is  dis- 
cussed by  Click,  D.,  Engstrom,  A.  and 
Malmstrom,  B.  G.  (Science,  1951,  114, 
253-258)  and  Caspersson,  T.  (Cell 
growth  and  cell  function,  New  York: 
Norton,  1949). 

The  light  absorption  due  to  the  ob- 
ject may  be  determined  in  the  following 

ways. 

1.  Direct  method.  The  microscope 
is  focused  on  an  area  of  the  object 
slide  adjacent  to  the  object  itself. 
The  light  impinging  on  the  photo-cell 
is  then  adjusted  to  give  a  fixed  response 
in  the  detector  circuit.  Without  alter- 
ing the  optical  conditions  the  slide  is 
then  moved  so  that  the  object  is  now 
centered  in  the  field  and  a  reading  of 
the  detector  response  is  taken.  This 
measurement  compared  with  the  first 
one  yields  the  per  cent  of  the  incident 
light  which  is  transmitted  by  the  ob- 
ject. 

2.  Split-beam  method.  In  this 
method  the  light  beam  is  split  before 
entering  the  microscope  and  a  small 
fraction  directed  toward  an  accessory 
phototube  and  detector  circuit.  It  is 
then  possible  to  evaluate  the  intensity 
of  the  light  reaching  the  main  photo- 
tube in  terms  of  the  intensity  of  the 
beam  directed  toward  the  seconday 
phototube.  This  method  has  the  ad- 
vantage of  being  independent  of  random 
fluctuations  in  the  intensity  of  the  light 
source.  As  in  the  direct  method  the 
absorption  of  the  object  is  determined 
by  comparison  with  a  blank  area  in  the 
slide. 

Interpretation  of  data:  The  derivation 
of  valid  conclusions  from  intracellular 
absorption  data  is  a  considerably  more 
difficult  task  than  the  experimental 
work  itself;  in  fact,  it  is  frequently  the 
case  that  data  are  obtained  for  which 
there  are  as  yet  no  valid  interpretations. 
The  difficulties  in  interpretation  arise 
from  the  fact  that  little  is  known  con- 
cerning the  physical  state  of  intracel- 
lular objects  and  its  effect  on  their 
optical  properties.  Since  the  rules  for 
analysis  of  absorption  data  are  based 
exclusively  on  the  optical  properties  of 
homogeneous  systems  such  as  gases  and 
dilute  solutions,  entirely  new  methods 
need  to  be  developed  for  the  hetero- 
geneous structures  of  the  cell. 

For  homogeneous  solutions  of  ab- 
sorbing materials,  the  following  rela- 
tions (the  Beer-Lambert  Laws)  are 
found,  within  limits,  to  hold: 

D  =  kcd 
where  c  is  the  concentration  of  absorb- 
ing material,  d  is  the  length  of  the  opti- 


cal path  through  the  absorbing  layer, 
and  k  is  the  extinction  coefficient  of  the 
substance  in  question.  D,  the  optical 
density,  is  defined  by  the  expression 

D  =  logio  y,  where  lo  is  the  intensity 

of  the  incident  beam  and  I  the  intensity 
of  the  transmitted  beam.  The  Beer- 
Lambert  relationship  holds  only  where 
all  light  absorption  is  due  to  the  trap- 
ping of  photons  by  the  substance  in 
question,  and  where  each  molecule  in 
the  light  path  contributes  equally  to 
the  absorption. 

In  microspectrophotometric  work, 
the  value  of  D  for  a  given  optical  sec- 
tion through  the  object  is  determined 
experimentally  at  various  wavelengths. 
This  gives  the  absorption  spectrum  of 
the  entire  object.  Since  the  aim  of  the 
measurement  is  usually  the  identifica- 
tion and  estimation  of  a  specific  sub- 
stance present  in  the  object,  the  initial 
spectrum  needs  to  be  evaluated  in  the 
light  of  the  above  relationships. 

In  order  to  identify  a  specific  sub- 
stance from  the   absorption   spectrum 
of  an  object  believed  to  contain  this 
substance,     the     following     conditions 
must   be   met.     1)  It   must   be   known 
that  the  object  contains  no  absorbing 
material   other  than  the  substance  in 
question;  or  the  relative  contents  and 
absorption  spectra  of  extraneous  sub- 
stances must  be  determined.    Without 
this  information  one  cannot  conclude 
that  a  given  absorption  maximum  in  the 
spectrum  of  the  object  is  due  to  a  spe- 
cific substance ;  such  a  maximum  may  re- 
sult   from    the    superposition    of    the 
spectra  of  two  or  more  different  sub- 
stances.    2)  It  must  be  known  that  the 
absorption  of  light  at  any  given  wave- 
length is  due  only  to  the  trapping  of 
photons  by  the  substance  in  question 
and  is  not  a  consequence  of  scattering 
and  similar  non-specific  processes.     In 
general,  light  scattering  rises  with  in- 
crease   in    frequency    but    since    sharp 
changes  in  refractive  index  occur  near 
the    absorption    maximum    of    a    sub- 
stance,   scattering   effects    may    some- 
times pass  through  a  maximum  with 
variation  in  wavelength. 

If  in  addition  to  identification  an 
estimation  of  the  relative  arnount  of 
the  substance  present  in  the  intracel- 
lular object  is  to  be  made,  the  follow- 
ing further  conditions  must  be  fulfilled. 
3)  The  length  of  the  optical  path 
through  the  object  must  be  known. 
This  is  not  necessarily  identical  with 
the  thickness  of  the  object  for  in  hetero- 
geneous systems  internal  reflections 
may  cause  a  significant  fraction  of  the 
entering  beam  to  be  reflected  through 


MICROSPECTROPHOTOMETRY 


208 


MICROSPECTROPHOTOMETRY 


a  total  path  length  considerably  longer 
than  the  dimensions  of  the  object. 
This  effect  will  enhance  the  absorption 
of  the  object  and  may  be  variable  with 
wavelength.  4)  The  relationship  be- 
tween the  optical  density  of  an  object 
and  the  concentration  of  the  specific 
substance  must  be  demonstrated  since 
it  cannot  be  assumed  that  the  linearity 
predicted  by  Beer's  Law  will  hold. 
5)  The  effect  of  inhomogeneous  distri- 
bution of  the  absorbing  material  in  the 
planes  perpendicular  to  the  optical 
axis  must  be  estimated  since  relatively 
small  departures  from  homogeneity 
will  lead  to  significant  alterations  in  the 
absorption  due  to  a  given  amount  of 
material. 

If  the  absolute  quantity  of  a  given 
substance  in  an  intracellular  object  is 
to  be  determined,  two  further  condi- 
tions must  be  met.  6)  The  specific 
extinction  of  the  substance  in  question, 
that  is,  the  absorption  per  mole  per 
liter  must  be  known.  7)  The  influences 
of  the  conditions  noted  above  on  the 
extinction  must  be  determined  quan- 
titatively. If  accepted  analytical  prac- 
tices are  to  be  followed,  this  would  mean 
that  the  absorption  due  to  a  known 
amount  of  the  specific  substance  when 
added  to  the  intracellular  object  be 
determined  or  that  the  amount  of  the 
substance  found  in  the  object  by  inde- 
pendent analytical  means  be  correlated 
with  its  absorption  characteristics. 

Unfortunately,  presently  available 
techniques  can  meet  the  above  condi- 
tions only  in  part.  The  procedures 
thus  far  worked  out  or  suggested  are 
noted  below. 

/ .  The  problem  of  complex  composition 
of  the  object.  If  it  can  be  shown  that 
the  composition  of  the  object  is  uniform 
throughout  its  optical  depth,  then  the 
relative  contribution  of  individual  sub- 
stances to  the  overall  absorption  spec- 
trum may  be  worked  out  by  comparison 
of  the  latter  with  individually  deter- 
mined spectra  of  the  separate  compo- 
nents. However,  large  differences  in  the 
height  of  the  maxima  frequently  make 
this  procedure  a  difficult  one.  To 
demonstrate  that  this  optical  uni- 
formity exists,  it  must  be  shown  that 
the  absorption  spectrum  of  the  object 
is  constant  for  all  thickness  (obtained, 
for  example,  by  sectioning).  Where 
this  is  not  possible  as  in  the  case  of 
structures  enclosed  within  intact  liv- 
ing cells  an  optical  method  may  be  used 
(Commoner,  B.,  Discussions  of  the 
Faraday  Society,  1950,  No.  9,  449-460). 
This  procedure  may  be  applied  to  ob- 
jects in  which  one  component  is  dis- 
tributed in  an  invariant  layer  in  a  cell 


which  varies  considerably  in  thickness. 
In  this  case  it  is  possible  to  determine 
the  absorption  due  to  the  separate  com- 
ponents in  situ  by  measuring  the  ab- 
sorption spectra  of  two  regions  in  the 
cell  which  differ  in  thickness  by  a  known 
amount.  In  favorable  instances  this 
method  may  be  used  to  determine  the 
absorption  spectrum  of  the  nucleus  con- 
tained within  a  living  cell.  Thus  far 
the  procedure  has  been  applied  only  to 
certain  types  of  plant  cells.  Failure 
to  conform  with  this  condition  casts 
doubt  on  the  meaning  ascribed  to  the 
absorption  spectra  of  structures  such 
as  the  nucleolus  which  have  been  ob- 
tained without  detailed  analysis  of  the 
contribution  made  by  over-lying  and 
under-lying  material. 

2.  Non-specific  light  losses.  It  has 
been  frequently  assumed  (Caspersson, 
T.,  Cell  growth  and  cell  function.  New 
York:  Norton,  1949)  that  light  lost 
due  to  scattering  is  related  to  wave- 
length according  to  the  Rayleigh  equa- 
tion. Using  this  assumption  and  the 
further  assumption  that  at  some  spe- 
cific wavelength  range  such  as  300-350 
mM  no  specific  absorption  occurs,  the 
scattering  losses  are  calculated  by  ex- 
trapolation from  the  readings  obtained 
in  this  limited  range.  The  usefulness 
of  this  method  is  considerably  weakened 
by  the  fact  that  the  first  assumption 
has  never  been  demonstrated  to  be  true 
for  intracellular  objects  and  that  the 
second  assumption  can  be  true  only  very 
rarely.  An  experimental  determina- 
tion of  losses  due  to  scattering  may  be 
made  by  an  apparatus  described  by 
Caspersson,  T.  (Cell  growth  and  cell 
function,  New  York:  Norton,  1949) 
which  measures  the  light  emerging 
from  the  object  at  various  angles  from 
the  optical  axis.  Scattering  losses 
may  vary  considerably  with  the  physi- 
cal state  of  the  object  and  are,  there- 
fore, very  sensitive  to  fixation  proce- 
dure, etc. 

3.  The  length  of  the  optical  path. 
Thus  far  no  method  for  determining  this 
value  has  been  proposed  and  in  prac- 
tice the  only  approach  to  the  value  of 
this  dimension  is  the  observed  thickness 
of  the  object.  Thus  in  making  this 
assumption,  calculations  are  exposed  to 
an  error  of  unknown  magnitude. 

4-  The  validity  of  Beer's  law.  This 
essential  determination  has  thus  far 
been  carried  out  in  only  one  instance 
(Commoner,  B.,  Discussions  of  the 
Faraday  Society,  1950,  No.  9,  449-460) 
in  which  the  cellular  material  occurred 
in  solution  in  the  vacuole  of  a  mature 
plant  cell.  In  this  case  it  was  possible 
to  alter  the  concentration  of  the  dis- 


MICROTOME 


209 


MILK 


solved  material  by  plasmolysing  the 
cell  to  various  degrees  and  thereby  al- 
tering the  water  content  of  the  solution. 
This  method  permits  comparison  of  the 
optical  density  of  the  vacuole  at  various 
volumes  and  provides  a  direct  test  of 
Beer's  law.  In  the  case  of  solid  struc- 
tures such  as  the  nucleus  the  osmotic 
method  is  probably  invalidated  by  the 
fact  that  the  absorbing  material  is  not 
freely  dissolved  throughout  the  struc- 
ture. 

When  the  cytological  object  contains 
oriented  absorbing  material  in  signifi- 
cant amounts,  considerable  departure 
from  the  Beer-Lambert  relationship  is 
to  be  expected  (Commoner,  B.,  Science, 
1949,  110,  31-40).  It  may  be  possible 
to  avoid  this  difficulty  by  the  use  of  a 
polarized  incident  light  beam. 

6.  The  effect  of  lateral  inhomogeneity. 
This  problem  has  been  discussed  from 
a  theoretical  point  of  view  (Glick,  D., 
Engstrom,  A.  and  Malmstrom,  B.  G., 
Science,  1951,  114,  253-258;  Danielli, 
J.  F.,  Cold  Spring  Harbor  Symp.,  1949, 
14,  32-39).  It  is  apparent  from  these 
discussions  that  large  errors  may  arise 
from  this  effect  but  at  present  there  is 
no  experimental  way  of  determining 
their  magnitude.  In  general  it  would 
seem  essential  that  objects  studied  be 
homogeneous  at  least  with  respect  to 
their  microscopic  appearance. 

6.  The  determination  of  absolute  quan- 
tities. In  some  instances  (Caspersson, 
T.  and  Schultz,  J.,  Proc.  Nat.  Acad. 
Sci.,  1940,  26,  507-515)  investigators 
have  calculated  the  absolute  content  of, 
say,  nucleic  acid  in  a  nucleus  from  meas- 
urements of  the  optical  density  of  the 
nucleus  and  the  specific  extinction  of 
nucleic  acid  solutions.  Such  a  proce- 
dure has  never  been  validated  by  the 
customary  tests  of  the  analyst. 

7.  The  effect  of  ultraviolet  light  on  the 
optical  properties  of  the  cell.  One  of  the 
most  serious  difficulties  encountered  in 
the  microspectrophotometric  work  is 
the  fact  that  exposure  to  ultraviolet 
light  is  damaging  and  frequently  lethal 
to  the  object.  At  the  same  time  irra- 
diation seriously  alters  the  absorption 
properties  of  the  cell.  It  was  first 
shown  by  Brumberg,  E.  M.  and 
Larionov,  L.  P.  (Nature,  1946,  158, 
663-664)  that  in  living  cells  undamaged 
by  ultraviolet  the  optical  density  of  the 
nucleus  does  not  exceed  that  of  the 
cytoplasm.  When  the  cell  has  been 
killed  by  ultraviolet  radiation  the  opti- 
cal density  of  the  nucleus  rises  sharply 
whereas  the  absorption  of  the  cytoplasm 
drops.  Similar  changes  have  now  been 
observed  by  a  number  of  other  workers 
(Bradfield,  J.  R.  G.,  Discussions  of  the 


Faraday  Society,  1950,  No.  9,  481-490; 
Walker,  P.  M.  B.  and  Davies,  H.  G., 
Discussions  of  the  Faraday  Society, 
1950,  No.  9,  461-470).  These  observa- 
tions show  that  1)  absorption  measure- 
ments of  living  cells  must  be  made  on 
cells  which  actually  survive  the  ex- 
perimental procedure,  2)  fixation  pro- 
cedures may  seriously  alter  the  apparent 
distribution  of  ultraviolet  absorbing 
material  and  3)  the  structural  ar- 
rangement peculiar  to  the  living  nu- 
cleus probably  has  a  significant  effect 
on  its  absorption  properties. 

Microtome.  The  freezing,  rotatory  and 
sliding  microtomes  are  well  known  and 
advertised.  The  high  speed  micro- 
tome required  for  cutting  especially 
thin  sections  for  Electron  Microscopy 
is  essential  for  research.  See  account 
by  G.  H.  Scott  in  McClung's  Micro- 
scopical Technique,  1950,  p.  720;  also 
FuUam,  E.  F.  and  Gessler,  A.  E.,  Rev. 
Sci.  Inst.,  1946,  17,  23  and  Gessler,  A.  E. 
and  Fullam,  E.  F.,  Am.  J.  Anat.,  1946, 
78,  245.  Geren,  B.  B.  and  McCuUoch, 
D.  (Exp.  Cell  Research,  1951,  2,  97-102) 
have  made  adjustments  in  the  Minot 
rotary  microtome  by  which  sections  of 
tissues,  prepared  by  the  methacrylate 
embedding  technique  of  Newman,  S. 
B.,  Borysko,  E.  and  Swerdlow,  M. 
(Science,  1949,  110,  66)  can  be  cut  1/20 
n  in  thickness.  The  edges  of  some  sec- 
tions are  much  thinner.  Glass  knives 
with  water  trough  (Latta,  H.  and  Hart- 
mann,  J.  F.,  Proc.  Soc,  Exp.  Biol,  and 
Med.,  1950, 74,  436)  are  a  great  improve- 
ment over  steel  ones. 

Microtome  Knife,  sharpening.  There  is  no 
easy  method.  Care  and  long  practice 
are  essential.  (See  Bensleys,  p.  57.) 
For  the  usual  oil  and  water  stones  a 
ground  glass  is  now  sometimes  substi- 
tuted (Uber,  F.  M.,  Stain  Techn.,  1936, 
11,93-98). 

Micrurgical  Technique  (Gr.  micros,  small 
+  ergon,  work)  is  referred  to  under  the 
heading  of  microdissection. 

Mikado  Yellow  G  (CI,  622)— Stilbene  Yel- 
low— a  direct  dye  of  light  fastness  3. 
Similar  to  Sun  Yellow  but  lighter  in 
color  (Emig,  p.  46). 

Milk,  bacteria  in,  a  modification  of  Newman 
technic  (Broadhurst,  J.  and  Paley,  C, 
J.  Am.  Vet.  Med.  Assoc,  1939,  94, 
525-526).  To  prepare  stain  add  0.4  cc. 
cone.  H2SO4  to  54  cc.  95%  alcohol. 
Mix  with  40  cc.  technical  tetrachlor- 
ethane  in  flask  and  heat  to  55  °C.  but  no 
higher.  Add  about  1.0-1.2  gm.  methy- 
lene blue  while  mixture  is  still  hot. 
Shake  until  dye  goes  into  solution. 
Then  add  8.0  cc.  1%  basic  fuchsin  in 
95%  alcohol.  Mix,  cool,  filter  and  put 
up  in  glass  stoppered  bottle.    Spread 


MILLIMICRON 


210 


MITOCHONDRIA 


0.01  cc.  milk  over  area  of  1-2  sq.  cm.  on 
slide.  Dry  on  flat  warm  surface  5  min. 
Flood  with  stain  15  sec.  Drain  off  ex- 
cess and  dry  while  flat  with  gentle  heat. 
Wash  in  cold  water  till  all  blue  is  re- 
moved and  a  faint  pink  color  appears. 
Dry  and  examine. 

Technique  for  the  rapid  detection  of 
Mycobacterium  tuberculosis  in  cows, 
milk  is  described  by  Maitland,  M.  L.  C, 
J.  Hyg.,  1950,  48,  397-401. 

Millimicron  (m^)  =  1/lOOOth  part  of  a 
micron  =  1/1, 000,000th  part  of  a  mm.  = 
10~'  mm.  =  0.001  micron  =  10  A  (see 
Micromicron). 

Millon's  Reaction.  For  microchemical  pur- 
poses it  is  necessary,  as  Bensley  and 
Gersh  (R.  R.,  and  I.,  Anat.  Rec, 
1933,  57,  217-233)  point  out,  for  the 
reagent  to  act  without  the  aid  of  heat, 
to  give  almost  immediately  with  tyrosin 
in  vitro  an  intense  red  color  yielding  red 
ppt.  not  changing  to  yellow  within  24 
hrs.  They  give  the  following  directions. 
Add  600  cc.  aq.  dest.  to  400  cc.  cone, 
nitric  acid  (sp.  gr.  1.42)  making  40%  by 
volume.  After  48  hrs.  add  1  part  to  9 
parts  aq.  dest.  Saturate  with  mercuric 
nitrate  crystals  frequently  shaking  sev- 
eral days.  To  make  the  reagent  take 
400  cc.  filtrate,  add  3  cc.  original  40% 
solution  plus  1.4  gm.  sodium  nitrite. 
Mount  sections  (preferably  after  freez- 
ing and  drying  technique)  to  slides 
without  using  water.  Immerse  in  rea- 
gent in  cold.  Maximum  reaction  should 
be  within  3  hrs.  when  sections  show 
noticeable  rose  color.  However  use 
several  slides,  remove  them  from  reagent 
in  a  Coplin  jar  at  intervals,  dip  imme- 
diately in  1%  aq.  nitric  acid,  dehydrate 
quickly  in  absolute  alcohol,  clear  in 
xylol  and  mount  in  balsam.  Bensley 
and  Gersh  found  that  mitochondria  are 
positive  to  Million's  reagent. 

Mineral  Oil,  reactions  in  tissue  to  fat  stains 
after  various  fixations  (Black,  C.  E., 
J.  Lab.  &  Clin.  Med.,  1937-38,  23, 
1027-1036).     See  Immersion  Oils. 

Mingazzini  Phenomenon  in  intestinal  villi 
interpreted  as  an  agonal  or  early  post- 
mortem change  (by  Macklin,  C.  C.  and 
M.  T.,  J.  Anat.,  1926,  61,  144-150). 

Mites.  The  techniques  given  for  Ticks  and 
Insects  are  applicable  for  making  whole 
mounts.  The  simple  creosote  method 
(see  Insects)  is  recommended. 

Mitochondria — Written  by  Geoffrey  Bourne, 
London  Hospital  Medical  College,  Lon- 
don, England.  November  5,  1951 — 
Granules,  rods  and  filaments  existing 
in  the  cj'toplasm  of  practically  all  liv- 
ing cells  of  plants  and  animals.  They 
can  be  studied  in  living  cells  unstained 
and  after  supravital  staining,  and  in 
fixed  tissues. 


They  can  be  seen  in  living  cells  even 
with  direct  illumination  if  it  is  critical. 
In  mammals  they  probably  are  best 
seen  unstained  in  very  small  pieces  of 
pancreas  mounted  in  normal  saline  and 
flattened  out  by  the  pressure  of  the 
cover  glass.  The  distal  poles  of  the 
acinous  cells,  facing  the  glandular 
lumen  may  be  identified  by  densely 
packed,  highly  refractile  zymogen  gran- 
ules. The  proximal  poles  are  nearer 
the  surrounding  blood  vessels  and  com- 
paratively free  from  zymogen  granules. 
With  direct  illumination  a  careful 
search  with  an  oil  immersion  objective 
will  distinguish  the  mitochondria  as 
delicate,  slightly  refractile  filaments 
oriented  in  general  with  their  long  axes 
parallel  with  the  length  of  the  cell. 
Wernicke  illumination  (phase  contrast) 
will  permit  immediate  recognition  of 
these  bodies  which  are  shown  up  clearly 
by  this  method.  Phase  contrast  en- 
ables mitochondria  to  be  studied  with 
ease  in  a  variety  of  living  unstained 
cells  (H.  U.  Zollinger,  Rev.  d'Hematol., 
1950,5,696). 

The  clarity  with  which  mitochondria 
may  be  observed  with  phase  contrast 
in  living  unstained  cells  has  rendered 
their  supravital  staining  less  necessary. 
However,  on  occasions,  this  may  be 
desirable.  When  very  dilute  methylene 
blue  is  added  to  tissue  culture  cells  the 
mitochondria  become  stained  a  bril- 
liant blue  (Ludford,  R.  J.,  Arch.  f.  Exp. 
Zellf.,  1935,  17,  339-359).  Other  vital 
dyes  are  diethylsafranin,  Janus  blue, 
Janus  black  1,  Pinacyanol,  Rhodamin 
B.  and  Janus  green  B.  (diethylsafra- 
ninazo-dimethyl-analin  chloride) . 

The  latter  is  used,  as  an  example,  as 
follows: 

Place  a  small  drop  of  1:10,000  Janus 
Green  B  in  0.85%  aq.  sodium  chloride, 
dj^e  should  be  added  from  a  1%  stock 
The  solution  in  aq.  dest.  because  the 
power  does  not  dissolve  easily  in  salt  so- 
lution. In  this  drop  a  variety  of  small 
pieces  of  tissue,  may  be  teased,  and  then 
covered  with  a  cover  glass,  and  mito- 
chondria may  be  demonstrated  very 
beautifully  in  blood  leucocytes  by  add- 
ing a  drop  of  freshly  drawn  blood  to  a 
drop  of  1  in  10,000  solution  of  Janus 
Green  B  and  examining  after  adding  a 
coverslip.  Mitochondria  become  col- 
ored a  dark  bluish-green  after  5-10  min. 
They  first  appear  in  the  lymphocytes 
and  then  between  the  granules  of  the 
granular  cells.  R.  R.  Bensley  (Am.  J. 
Anat.,  1911,  12,  297-388)  has  described 
a  method  of  intravascular  staining  of 
mitochondria  using  Janus  Green  B. 
Janus  Green  preparations  of  mito- 
chondria    are     not     permanent — they 


MITOCHONDRIA 


211 


MITOCHONDRIA 


bleach  in  from  30-60  min.  to  a  colorless 
leucobase.  Mitochondria  show  up  bril- 
liantly in  living,  particularly  tissue 
culture,  cells  using  dark-ground  il- 
lumination (see  Strangeways,  T.  S.  P. 
and  Canti,  R.  G.,  Quart.  J.  Micr.  Sci., 
1927,  71,  1-14).  They  may  be  also 
photographed  at  high  magnification 
with  the  electron  microscope  (R. 
Claude  and  E.  F.  Fullam,  J.  Exp.  Med., 

1945,  81,  51-62;  H.  U.  Zollinger,  E.x- 
perientia,  1950,  6,  16-17). 

Mitochondria  can  be  isolated  from 
tissues  by  the  process  of  differential 
centrifugation  (R.  R.  Bensley  and  N. 
Hoerr,  Anat.  Rec,  1934,  50,  251,  499). 
This  technique  has  been  developed  by 
Porter,  K.  and  his  colleagues  (J.  Exp. 
Med.,  1945,  81,  233-246);  Claude,  A, 
(Science,  1943,  97,  451-456;  J.  Exp. 
Med.,  1944,  80,  19);  Hogeboom,  G.  H. 
and    his    colleagues    (J.    Biol.    Chem., 

1946,  165,  615-630).  These  authors  not 
only  developed  the  technique  of  isola- 
tion of  mitochondria  by  differential 
centrifugation  and  were  able  to  obtain 
mitochondria  which  were  morphologi- 
cally identical  with  those  in  the  living 
cell,  but  by  a  series  of  chemical  studies 
were  able  to  show  that  such  isolated 
mitochondria  contain  the  greater  part 
of  the  respiratory  enzymes  of  the  cell, 
a  fact  which  suggests,  not  only  that  the 
mitochondria  may  be  the  main  respira- 
tory centers  of  the  cell  but  that  they 
can  function  as  synthetic  centers  as 
well.  The  conception  of  the  respira- 
tor}^ function  of  mitochondria  was  sug- 
gested as  long  ago  as  1912  by  Kingsbury 
A.  (Anat.  Rec,  1912,  6,  39). 

Isolation  of  Mitochondria.  Hoge- 
boom, G.  H.,  Schneider,  W.  C.  and 
Pallade,  G.  E.  (Proc.  Soc.  Exp.  Biol. 
Med.,  1947,  65,  320-321)  have  described 
a  method  of  obtaining  morphologically 
intact  mitochondria  from  rat  liver. 
Rat  liver  is  homogenized  by  the  method 
of  Potter,  V.  R.  and  Elvehjem,  C.  A. 
(J.  Biol.  Chem.,  1936,  114,  495-504)  in 
0.88  M.  sucrose.  The  homogenate  is 
centrifuged  3  times  at  600  g.  for  10  min. 
This  removes  nuclei  and  intact  cells. 
The  supernatant  is  then  centrifuged  at 
24,000  g.  for  20  min.  This  brings  down 
the  mitochondria  together  with  a  few 
microsomes.  Mitochondria  obtained  in 
this  way  remain  stable  in  form  for 
several  days  at  4°C.  There  are  a 
variety  of  methods  for  making  perma- 
nent preparations  of  mitochondria  in 
tissue  sections. 

1.  Altmann's  method.  See  Carleton, 
H.  M.  and  Leach,  E.  H.,  Histological 
Technique,  Oxford,  1949.  Fix  in 
Champy's  fluid  or  in  Flemming  without 
acetic.     Postchrome  for  3  days  (trans- 


fer tissue  direct  to  2§-3%  aq.  potassium 
dichromate).  Wash  12-24  hrs.  in  run- 
ning water,  dehydrate,  embed  and  sec- 
tion. Bring  sections  to  water,  then: 
(1)  flood  slide  with  aniline  fuchsin 
(aniline  water,  made  by  adding  10 
cc.  of  aniline  to  one  half  or  one  litre 
of  hot  aq.  dest.  in  a  flask  and  ashing, 
cooling  and  filtering,  100  cc,  acid 
fuchsin  12  gm.).  Warm  slide  with 
bunsen  flame  till  the  stain  steams  (but 
does  not  boil).  Leave  for  5  min.  (2) 
rinse  rapidly  in  aq.  dest.  (3)  differen- 
tiate in  slide  jar  of  picric  acid,  sat.  sol. 
in  ab.  ale.  20  cc,  30%  ale  80  cc.  Dif- 
ferentiation should  be  stopped  when 
the  red  dye  has  diffused  out  of  the  nuclei 
and  cytoplasm  and  the  mitochondria 
are  bright  red.  (4)  rinse  in  aq.  dest. 
(5)  dehydrate  rapidly,  mount  in  bal- 
sam. 

2.  Heidenhain's  iron  hematoxylin 
method.  Tissues  may  be  fixed  in 
formaldehyde,  Helly's  fluid,  Zenker  for- 
maldehyde or  Flemming-without-ace- 
tic,  for  24  hrs.  Sections  are  mor- 
danted in  a  5%  aq.  iron  alum  for  5  to 
24  hrs.  according  to  the  nature  of  the 
tissue.  Rinse  in  water.  Stain  in  0.5% 
aq.  hematoxylin  for  a  period  of  time 
equal  to  that  of  the  iron  alum  treat- 
ment. Rinse  in  water,  differentiate 
in  iron  alum  solution  and  control  dif- 
ferentiation with  microscope  until  only 
nuclei  and  mitochondria  are  black. 
Counterstain  if  necessary,  dehydrate, 
mount  in  balsam. 

3.  Cain's  method  (Quart.  J.  Micr.  Sci., 
1948,  89,  229-231).  Fix  in  Helly's  fluid, 
6  hrs.  postchrome  48  hrs.  at  37°C.  in 
sat.  aq.  potassium  dichromate,  wash 
overnight  in  running  water,  embed  in 
parafRn  wax  and  cut  sections  about 
3  n.  Bring  sections  to  water  treating 
with  iodine  (i  in  70%  ale)  and  then 
5%  aq.  sodium  thiosulphate  on  the  way. 
Dry  slide,  except  sections,  flood  with 
aniline  fuchsin,  (see  Altmann's  method) 
and  heat  until  steaming  as  for  Alt- 
mann's technique.  Wash  off  acid  fuch- 
sin with  aq.  dest.  Irrigate  with  alka- 
line solution  (one  drop  of  aq.  sodium 
carbonate  in  10°  cc.  of  aq.  dest.).  Dif- 
ferentiate 30  sec.  to  I5  min.  To  stop 
differentiation  dip  slide  into  1%  HCl. 
Wash  in  aq.  dest.,  counterstain  1%  aq. 
water-soluble  methjd  blue.  Wash  aq. 
dest.,  dip  into  1%  acid  3  sec.  only. 
Wash  aq.  dest.,  dehydrate  and  mount 
in  balsam. 

The  Bensley-Cowdry  acid  fuchsin  and 
methyl  green  method  (Cowdry,  E.  V., 
Contrib.  Carnegie  Inst.,  Wash.,  VIII, 
1918)  gives  beautiful  results.  In  it  the 
methyl  green  is  used  both  as  differen- 
tiator and  counterstain. 


MITOCHONDRIA  AND  BACTERIA         212 


MITOSIS 


Schridde's  method  (Ergn.  Anat.  u. 
Entw.,  Bonnet,  XX,  1911)  can  also  be 
recommended  as  giving  beautiful  prep- 
arations of  mitochondria. 

4.  Pritchard's  silver  method  (J.  Anat., 
1951 ,  85,  in  press) .  This  method,  which 
is  dependent  upon  reduction  of  silver 
on  the  slide,  gives  very  beautiful  and 
precise  preparations  of  mitochondria 
which  show  up  black.  Small  pieces  of 
tissue  should  be  fixed  in  Regaud's  or 
Helly's  fluid  for  3  da3's  and  post- 
chromed  for  4  days  in  3%  aq.  potassium 
bichromate.  Prepare  paraffin  sections 
in  the  usual  way,  avoiding  excessive 
heat  in  flattening  and  drying.  Remove 
paraffin  and  proceed  to  aq.  dest.  Slides 
placed  for  20  sec.  with  agitation  into  a 
dilute  solution  of  silver  diamino  hy- 
droxide (Wilder's  Solution  diluted  with 
an  equal  volume  of  aq.  dest.  to  which 
is  added  2  drops  of  8%  NH4OH  per  50 
cc).  Drain  quickly  and  without  rins- 
ing immerse  in  a  large  volume  (e.g.  200 
cc.)  of  very  dilute  formalin  (1/1000 
commercial  formalin  in  aq.  dest.)  agi- 
tating for  10-20  sec,  not  more.  Fresh 
formalin  solution  for  each  section  is 
preferable.  Wash  in  aq.  dest.  Dif- 
ferentiate carefully  under  the  micro- 
scope with  1-2%  aq.  potassium  ferri- 
cyanide  until  mitochondria  show  up 
black  against  a  clear  background. 
Wash  in  aq.  dest.,  counterstain  with  1% 
Safranin  or  Ponceau  fuchsin.  Regaud's 
fixative  gives  the  best  results  but  after 
Helly's  fluid  the  Golgi  element  is  often 
sharply  impregnated  as  well  as  the  mito- 
chondria. 

Mitochondria  and  Bacteria.  Demonstration 
in  the  same  cells.  See  Cowdry,  E.  V. 
and  Olitsky,  P.  K.,  J.  Exper.  Med., 
1922,  36,  521-533,  Cowdry,  E.  V.,  Am.  J. 
Anat.,  1923,  31,  339-343.  Stain  as  for 
mitochondria  with  Anilin  Fuchsin  and 
Methyl  Green.  Mitochondria  are  col- 
ored crimson.  When  the  bacilli  are  acid 
fast  as  in  leprosy  they  are  colored  a  dark 
reddish  purple;  but  when  they  are  not 
acid  resistant  they  are  stained  bluish 
green. 

Mitogenic  Radiations.  It  is  questionable 
whether  these  rays,  said  to  generate 
mitosis,  really  exist.  A  critical  and 
well  balanced  statement  is  afforded  by 
Glasser,  O.,  in  Glasser's  Medical  Phy- 
sics, 760-763. 

Mitosis  (G.  Mitos,  thread).  Indirect  nu- 
clear division  in  which  the  chromatin 
forms  a  thread  which  breaks  up  into 
chromosomes. 

Material  should  be  freshly  fixed,  less 
than  half  hour  after  removal.  But  mito- 
sis can  be  seen  in  some  tissues  24  hrs.  or 
longer  after  death,  especially  if  the  body 
is  kept  at  a  low  temperature  but  the 


number  is  less  and  the  details  not  so 
clear  as  after  quick  fixation  (Mallory, 
p.  108).  Sat.  mercuric  chloride  in  70% 
ale.  plus  5%  acetic  acid,  Zenker's  fluid, 
formalin-Zenker ,  Bouin's  fluid  and  Flem- 
ming's  strong  fluid  are  satisfactory 
fixatives  but  the  last  named  penetrates 
very  badly. 

The  most  beautiful  stain  for  mitotic 
figures  is  safranin  light  green  but  the 
mitoses  can  be  more  clearly  distin- 
guished without  the  green  counterstain. 
Simply  deparaffinise  and  stain  sections 
in  anilin-safranin  (Babes),  wash  quickly 
in  tap  water,  differentiate  in  acid  alcohol 
until  the  resting  nuclei  are  less  intensely 
colored  than  the  dividing  ones,  wash  in 
95%,  dehydrate  in  abs.  clear  in  xylol 
and  mount  in  balsam. 

Another  excellent  method  is  to  apply 
the  Feulgen  reaction  for  Thymonucleic 
Acid  to  sections  of  tissues  preferably 
fixed  in  Carnoy's  fluid  or  acetic  subli- 
mate. This  demonstrates  thymonucleic 
acid  in  the  chromatin,  and  the  dividing 
nuclei,  as  with  safranin,  are  more  deeply 
stained  than  the  others.  This  method 
is  displacing  the  older  safranin  tech- 
nique. 

To  demonstrate  mitosis  in  whole 
mounts  of  epidermis  place  freshly  ex- 
cised skin  (circumcision  specimen  pre- 
ferred) in  0.1%  aq.  acetic  acid  in  the 
icebox  over  night.  Wash  quickly  in 
aq.  dest.  Strip  off  the  epidermis  with 
needles,  stain  it  like  a  section  with 
anilin-safranin  or  with  Harris'  hema- 
toxylin and  mount  with  the  outer  sur- 
face uppermost.  This  technique  could 
probably  be  adapted  to  relatively  flat 
epithelia  of  the  respiratory  digestive, 
urinary  and  genital  systems. 

In  order  to  reveal  the  maximum  num- 
ber of  mitotic  figures  it  is  important  to 
study  the  mitotic  rhythm  of  the  par- 
ticular tissue  or  organ  and  take  tissues 
at  the  peak  which  in  the  case  of  the 
human  foreskin  is  probably  between 
9  p.m.  and  midnight  (Cooper,  Z.  K.  and 
Schiff,  A.,  Proc.  Soc.  Exp.  Biol.  &  Med., 
1938,  39,  323-324).  The  relation  of 
alimentation  and  nutrition  to  cyclic 
variations  in  mitotic  activity  is  pre- 
sented by  Blumenthal,  H.  T.,  Growth, 
1950,  14,  231-250. 

To  experimentally  increase  the  num- 
ber of  mitosis  use  colchicine  which  ar- 
rests the  process  chiefly  in  the  meta- 
phase  by  causing  failure  of  the  mitotic 
spindle  to  form  and  function  (Ludford, 
R.  J.,  Arch.  f.  exper.  Zellf.,  1936,  18, 
411-441).  Consequently  as  long  as  the 
cells  are  under  the  influence  of  colchi- 
cine— a  matter  of  a  few  hours  only — 
mitosis  begins  as  usual;  but,  since  it  is 
not  completed,  the  proportion  of  mitotic 


iMOIST  CHAMBERS 


213 


MOROSOW'S  METHOD 


figures  to  resting  nuclei  is  temporarily 
greatly  increased.     Sodium  cacodylate, 
auramine  and  other  substances  listed  by 
Ludford     likewise     influence     mitosis. 
For  checks  on  the  method  of  estimating 
growth   by   counting  arrested  mitoses, 
see   Paletta  and   Cowdry    (F.    X.   and 
E.  v.,  Am.  J.  Path.,  1942,  18,  291-311). 
Aisenberg  (E.  J.,  Bull.  d'Hist.  Appl., 
1935,  12,  100-122)  has  found  that  mitosis 
of  epidermal   cells   is   arrested    in   the 
metaphase  simply  by  passing  a  ligature 
around  a  frog's  leg  and  keeping  the  foot 
in    distilled    water.     The    mitoses    ac- 
cumulate in  large  numbers  but  continue 
when     released     from     the     hypotonic 
environment.     Aisenberg     {ibid.     1936, 
13,  265-286)  also  discovered  low  concen- 
tration  of   ethyl   alcohol   to   stimulate 
mitosis,  0.4-0.8  M  to  arrest  in  meta- 
phase, 1.2-1.5  M.  to  cause  gelatinization 
of  mitosis  and  higher  concentrations  to 
kill  the  cells.     Comprehensive  data  on 
the  influence  of  Colchicine  are  supplied 
by  Levine,  M.,  Ann.  N.  Y.  Acad.  Sci., 
1951,  51,  1365-1408. 
Moist  Chambers,  Small  for  stage  of  micro- 
scope (Chambers,  R.  and  Kopac,  M.  J. 
in  McClung's  Microscopic  Technique, 
1950).     Large  in  which   hands  can   be 
used   (Holter,  H.,  C.   rend.   trav.  lab. 
Carlsberg,   S6r.   Chim.,    1945,  25,    15fr- 
167).     See  diagrams  for  air-condition- 
ing (Click,  p.  182). 
Molecular  Film  Technique,  see  Taylor,  H. 
S.,  Lawrence,  E.  O.,  and  Langmuir,  I., 
Molecular  Films,  the  Cyclotron  and  the 
New  Biology,  Rutger's  University  Press, 
1942,  95  pp. 
Molecular  Solution  is  the  molecular  weight 
of  the  substance  in  grams  made  up  to  1 
liter  with  aq.  dest.     Thus  M  oxalic  acid 
(COOH)2-2H20   is    126  gms.    with  aq. 
dest.  added  to  1  liter;  but  A-^  oxalic  acid 
is     half     of     this     concentration.     See 
Normal  Solutions. 

The  molecular  weight  expressed  in 
grams  is  called  the  gram-molecular 
weight  or  mole. 

Millimole  is  1/1000  of  a  mole. 

Milligram  equivalent  (milliequiva- 
lent).  The  equivalent  weight,  the 
gram-equivalent,  or  the  equivalent  of  a 
substance  is  the  weight  in  grams  which 
in  its  reaction  corresponds  to  a  gram 
atom  of  hydrogen,  or  of  hydroxyl,  or  half 
a  gram  atom  of  oxygen,  or  gram 
atom  of  a  univalent  ion.  Milliequiva- 
lent  is  1/1000  of  the  equivalent  weight, 
i.e.,  the  equivalent  weight  of  sodium 
carbonate  is  j  the  molecular  weight,  or 
53.0.  Therefore,  the  milliequivalent 
(m.e.)  or  the  weight  in  1  ml.  of  normal 
solution  is  0.0530  gm. 


Molybdenum,  see  Atomic  Weights. 

Mono-Azo  Dyes.  Amarnth,  azo  fuchsin, 
benzene-azo-a-naphthylamine,  bordeaux 
red,  brilliant  yellow  S,  chromotrope  2R, 
chrysoidin  Y,  fast  yellow,  janus  green 
B,  metanil  yellow,  methyl  orange, 
methyl  red,  narcein,  nitrazine,  oil  red 
O,  orange  G,  orange  I,  orange  II,  orange 
IV,  ponceau  2R,  sudan  R,  thiazine 
red  R. 

Monocytes.  When  "typical"  these  are 
easily  recognized  in  stained  blood  smears 
and  in  supravital  preparations  but  there 
is  no  technique  by  which  they  may 
always  be  distinguished  from  all  Lym- 
phocytes and  Macrophages.  That  is, 
they  possess  no  single  feature,  like  the 
eosinophile  granules  of  eosinophile  leuco- 
cytes, for  their  certain  identification 
(see  Cowdry,  p.  66-71).  They  ingest 
particulate  matter  including  Trypan 
Blue  and  similar  vital  stains  and  are 
therefore  to  be  considered  as  com- 
ponents of  the  Reticulo-Endothelial 
System.  Many  of  their  properties  can 
to  great  advantage  be  investigated  in 
Tissue  Cultures.  The  best  way  to 
demonstrate  the  remarkably  close  rela- 
tion that  paay  exist  between  monocytes 
and  contained  bacilli  is  to  stain  leprous 
tissue_  for  acid  fast  bacilli  (see  Leprosy 
Bacilli).  See  Bacterium  Monocyto- 
genes. 

Monolayer  technique  is  a  physico-chemical 
line  of  investigation  that  gives  valuable 
data  on  the  structure  of  protein  and 
lipoprotein  films  and  consequently  on 
the  plasma  membrane  of  cells.  See 
Schulmann  (Bourne,  pp.  51-67). 

Moore,  see  Fungi. 

Mordant  (L.  mordere,  to  bite),  a  substance, 
like  alum,  employed  to  make  a  dye  bite 
into  the  tissue  and  hold  on.  The  dye 
combines  with  the  mordant  which  is 
itself  in  high  concentration  in  the 
structures  to  be  stained.  In  the  Iron 
Hematoxylin  technique  the  sections 
are  mordanted  with  iron  alum.  They 
are  briefly  washed  in  aa.  dest.  to  remove 
some  of  the  excess  mordant.  Then  they 
are  stained  with  a  dilute  aqueous  solu- 
tion of  hematoxylin  and  differentiated 
in  the  mordant  which  draws  out  most  of 
the  hematoxylin  until  it  remains  only 
in  the  structures  which  took  up  the 
mordant  most  energetically  in  the  first 
place  and  which  therefore  alone  remain 
colored.  Copper  salts  are  also  good 
mordants.    See     Weigert's     mordants. 

Morosow's  Method  for  elementary  bodies  as 
modified  by  Fonta  and  Triboudeau  and 
given  by  Seiffert,  G.,  Virus  Diseases  of 
Man,  Animal  and  Plant.  New  York: 
Philosophical  Library,  Inc.,  1944,  332 
pp.  Dry  thin  smear  in  air.  Place 
vertically  in  aq.  dest,,  10-15  min.  and 


MOSKOWITZ 


214 


MOUNTING  MEDIA 


dry  again.  Cover  with  mixture  of 
acetic  acid,  1  cc;  40%  formalin  (com- 
mercial formaldehyde),  2  cc;  aq.  dest., 
100  cc.  Rinse  well  in  aq.  dest.  and  heat 
till  steam  rises  in  mixture  of  carbolic 
acid,  1  cc;  tannin,  50  gm.;  aq.  dest., 
100  cc  Rinse  in  aq.  dest.  j  min.  and 
heat  slightly  1-2  min.  till  smear  becomes 
brown  or  slightly  black  in  silver  solu- 
tion made  up  as  follows:  To  20  cc.  aq. 
dest.  add  "platinum  loop"  of  25%  am- 
monia and  then  drop  by  drop  from 
pipette  of  10%  aq.  silver  nitrate  until 
an  opalescent  ppt.  appears.  About 
0.5  cc.  of  silver  solution  will  be  needed. 
After  silvering  smear  rinse  well  in  aq. 
dest.,  mount  and  seal  edges  with 
paraffin. 

Moskowitz,  see  Protein  Silver  for  Staining 
Protozoa. 

Mosquito  larvae,  technique  of  raising  anoph- 
eline  (Bates,  M.,  Am.  J.  Trop.  Med., 
1941,  21,  103-122).  Bodian  technique 
for  mosquito  nervous  system  (Rogoff, 
W.  M.,  Stain  Techn.,  1946,  21,  59-61). 

Motion  Pictures.  The  technique  of  making 
motion  pictures  of  living  cells  and  or- 
gans has  proved  its  worth.  The  movies 
can  be  projected  again  and  again  and 
the  sequence  of  events  made  very  clear. 
It  is  important  to  remember  that  mo- 
tile cells  do  not  run  around  at  the  speed 
indicated,  because  the  actual  distance 
travelled  is  far  less  than  on  the  screen 
and  the  time  much  greater.  The  Wis- 
tar  Institute  of  Anatomy  in  Philadel- 
phia is  distributor  of  a  comprehensive 
series  of  motion  picture  films  on  either 
a  purchase  or  rental  basis. 

Motor  End  Plates.  The  particular  morpho- 
logical type  of  nerve  ending  in  muscle 
does  not  concern  us  here ;  but  reference 
can  be  made  if  desired  to  the  classifica- 
tion by  Hines,  M.,  Am.  J.  Anat.,  1931, 
47,  1-55.  The  methods  advocated  for 
histological  demonstration  are  legion. 
Reference  is  made  to  2  gold  techniques 
(Craven's  and  Carey's)  and  to  1  silver 
method  (Chor's).  The  former  can  be 
ultimately  traced  back  to  Ranvier  and 
the  latter  to  Cajal.  See  also  techniques 
described  under  Nerve  Endings. 

Mounting  Media.  The  refractive  index  of 
the  medium  is  important  and  a  table 
giving  the  indices  for  many  substances 
used  is  supplied  by  Lee  (p.  218).  As 
pointed  out,  the  greatest  transparency  is 
secured  when  the  refractive  indices  of 
media  and  tissues  are  equal  and  media  of 
lower  index  than  the  tissues  give  some- 
what greater  visibility  of  tissue  com- 
ponents, while  those  of  higher  index 
provide  less  visibility.  There  are  nmny 
media  to  choose  from,  the  refractive 
indices  of  which  are  more  or  less  satis- 
factory.   The    selection    will    depend 


more  upon  whether  the  medium  can  be 
employed  for  the  particular  tissue  and 
its  relative  permanence. 

For  frozen  sections  and  tissues  to  be 
mounted  from  water  and  aqueous  solu- 
tions various  glycerin  mixtures  are 
popular :  Lactophenol,  Glychrogel, 
Brandt's  and  Kaiser's  glycerin  jellies. 
Having  taken  the  easiest  one  to  prepare, 
for  their  merits  are  about  equal,  the 
tissue  is  mounted  and  covered  and  it  is 
necessary  to  seal  the  edges.  In  the  case 
of  temporary  mounts  a  little  paraffin 
applied  with  a  heated  scalpel,  or  wire, 
will  suffice.  Lee  (p.  230)  advocates 
Peter  Gray's  sealing  medium  made  up 
by  melting  together  4  parts  anhydrous 
lanolin,  1  part  Canada  balsam  (dry)  and 
8  parts  resin  which  becomes  solid  on 
cooling.  Apply  to  edges  in  the  same 
manner  as  the  paraffin.  Eronig's  ce- 
ment is  employed  in  Bensley's  labora- 
tory. Duco  cement  is  very  worthwhile 
because  it  is  insoluble  in  xylol,  alcohol 
and  other  chemicals  used  to  clean  micro- 
scopic preparations.  Mallory  (p.  99) 
dilutes  it  with  an  equal  volume  of  ace- 
tone.   See   Karo. 

For  sections  and  tissues  which  are  first 
dehydrated  and  cleared  the  investiga- 
tor must  choose  the  mounting  medium 
best  adapted  to  his  purpose  from  a  con- 
siderable number  proposed  of  which  the 
following  are  given  elsewhere  in  this 
book :  Balsam,  Cedar  Oil,  Clarite, 
Colophonium,  Damar,  Diaphane,  Eupe- 
ral,  Nevillite,  Sandarac,  Terpineol 
Balsam. 

The  chief  desiderata  are  a  medium 
which  will  harden  fairly  quickly,  which 
will  not  become  acid  and  bring  about  the 
fading  of  anilin  dyes  and  which  will  not 
crack  or  develop  granules.  Clarite  is 
competing  for  first  place  with  balsam* 
because,  to  make  the  balsam  neutral  ana 
keep  it  so,  is  a  troublesome  job.  Direc- 
tions for  its  preparation  are  given  by  the 
Bensleys  (p.  39).  But  the  balsam  ordi- 
narily purchased  is  satisfactory  for 
hematoxylin  and  eosin  and  iron  hema- 
toxylin preparations  except  when  the 
latter  are  counterstained  with  an  anilin 
dye.  The  writer  used  to  employ  cedar 
oil  (for  immersion  objectives),  in  mount- 
ing sections  stained  by  Giemsa's 
method,  which  is  superior  to  balsam, 
but  it  drys  slowly  and  is  not  better  than 
clarite.  Damar  has  been  recommended 
for  stains  likely  to  fade  and  colophonium 
for  thick  sections  of  the  nervous  system 
for  which  covers  are  not  used;  both 
however  appear  to  be  less  valuable  than 
clarite. 

Museum  specimens  require  an  aque- 
ous mounting  medium  which  preserves 


MUCICARMINE 


215 


MUCUS 


colors.    See    Color    Preservation.     See 

Plastics  for  museum  work. 

Mucicarmine  for  mucus,  Mayer's  (Mallory 
and  Parker  in  McClung,  p.  417).  To 
make  up  stain,  mix  carmine,  1  gm.; 
aluminum  chloride,  0.5  gm. ;  and  aq. 
dest.,  2  cc.  Heat  over  flame  for  2  min. 
Color  of  solution  darkens.  Add  grad- 
ually 100  cc.  50%  ale.  stirring  constantly 
until  dissolved.  After  24  hrs.  filter. 
Filtrate  keeps  well.  Stain  parafTin  sec- 
tions of  absolute  alcohol  fixed  tissue  in 
carmine  sol.  5-10  min.  Wash  in  water, 
dehydrate,  clear  and  mount.  Mucus 
is  red.  When  nuclei  also  are  colored  red, 
add  few  drops  1%  aq.  sodium  bicarbon- 
ate to  the  stain.  It  is  customary  to 
stain  cells  and  nuclei  before  hand  with 
alum  hematoxylin.  Bensley  (Cowdry's 
Special  Cytology,  1932,  p.  203)  uses 
alcoholic  chrome  sublimate  (sat.  mer- 
curic chloride  and  potassium  bichromate 
in  95%  ale.)  and  increases  the  content  of 
carmine  5  times. 

Mucigen,  intracellular  antecedent  of  Mucin. 

Mucihematein  for  mucus,  Mayer's,  Mal- 
lory and  Parker  in  McClung,  p.  416). 
Alake  up  :  (A)  hematein,  0.2  gm. ;  alumi- 
num chloride,  0.1  gm.;  glycerin,  40  cc; 
aq.  dest.,  60  cc.  and  (B)  hematein,  0.2 
gm.;  aluminum  chloride,  0.1  gm.;  70% 
alcohol,  70  cc;  nitric  acid,  1-2  drops. 
A  is  advised  except  when  the  mucus 
swells  much  in  which  case  use  B. 
Stain  paraffin  sections  of  absolute  alco- 
hol fixed  tissue  5-10  min.  Wash  in 
water.  Dehydrate  in  95%  ale.  and  in 
abs.  Clear  in  xylol  and  mount  in  bal- 
sam. Mucus  stains  blue.  The  other 
materials  are  colorless.  _  Preliminary 
coloration  with  carmine  is  suggested. 
Bensley  (Cowdry's  Special  Cytology, 
1932,  p.  203)  used  alcoholic  chrome  sub- 
limate fixation  (sat.  mercuric  chloride 
and  potassium  bichromate  in  95%  ale.) 
and  increased  the  content  of  hematein 
five  times. 

Mucin,  one  of  several  glycoproteins  found  in 
mucus.  See  Mucus  and  Mucicarmine, 
Mucihematein  and  Mucisudan  stains, 
also  Polysaccharides. 

Mucinase.  Enzyme  (s)  which  hydrolyze 
mucus  or  mucoproteins.  They  are  not 
very  well  understood,  and  specific 
preparations  are  not  yet  available. 
Purified  enzymes,  capable  of  differenti- 
ating the  various  mucopolysaccharides 
one  from  another  would  be  very  valu- 
able. 

Mucisudan  is  a  dye  of  undetermined  compo- 
sition made  by  hydrolysis  of  sudan 
black  B  with  acetic  acid  and  recom- 
mended as  a  new  stain  for  mucin  (Leach, 
E.  H.,  J.  Path,  and  Bact.,  1938,  47, 
637-639). 
Mucoproteins.     A  method   for   histological 


distinction  between  the  chondroitin 
sulphuric  acid  protein  of  connective 
tissue  mucus  and  the  mucotin  sulphuric 
acid  protein  of  epithelial  ti-ssues  has  been 
worked  out  by  L.  H.  Hempelmaiin,  Jr., 
Anat.  Rec,  1940,  78,  197-206.  Briefly 
stated  toluidin  blue  in  1:280,000  will 
stain  the  former  vividly  and  the  latter 
not  at  all.  Click,  p.  46,  notes  that  con- 
firmation of  Hempelmann's  work  is  still 
lacking. 

Metachromatic  staining  with  toluid 
in  blue  is  specific  for  mucoid  compounds 
containing  polysaccharide  esters  of 
sulfuric  acid  provided  that  Lison's  tech- 
nique is  followed  closely  in  the  opin- 
ion of  Sylvan,  B.,  Acta  Radiol.,  1945, 
suppl.  59,  100  pp.  Fix  tissue  (12-24 
hrs.)  in  equal  parts  8%  aq.  basic  lead 
acetate  and  14-16%  formalin.  Stain 
some  paraffin  sections  (30  min.)  in  0.1% 
toluidin  blue  in  1%  ale.  and  others  in 
0.1%  toluidin  blue  in  30%  ale  (first 
allowing  these  stains  to  age  for  several 
days).  Wash  in  ale  briefly,  mount  in 
natural  cedar  oil.  See  Michaelis,  L. 
and  Granick,  S.,  J.  Am.  Chem.  Soc, 
1945,  67,  1212-1219  and  Hess,  M.  and 
Hollander,  F.,  J.  Lab.  &  Clin.  Med., 
1947,  32,  905-909  for  data  about  meta- 
chromasia.  Leach,  E.  H.,  Stain 
Techn.,  1947,  22,  73-76  has  advocated 
Bismark  brown  as  a  stain  for  muco- 
proteins. 
Mucus  means  slime.  It  is  a  viscid,  stringy 
material  which  ordinarily  stains  with 
basic  dyes  and  is  found  in  many  parts  of 
the  body.  The  chemical  composition 
of  mucus  is  not  uniform.  It  may  consist 
of  one  of  several  glycoproteins,  called 
mucins,  which  are  by  contrast  definite 
chemical  substances.  The  term  mu- 
cous is  an  adjective  describing  a  cell  or 
tissue  which  produces  or  contains 
mucus.  Mucigen  is  the  intracellular 
antecedent  of  a  mucin.  Since  there  are 
several  mucins  there  are  several  corre- 
sponding mucigens. 

Pathologists  sometimes  divide  mucins 
into  two  categories,  epithelial  and  con- 
nective. The  connective  tissue  type  is 
found  in  the  ground  substance  of  bone, 
synovial  fluid  and  in  other  locations. 
It  increases  in  amount  in  the  myxedema 
(G.  myxa,  mucus  -|-  oidema,  swelling) 
of  certain  thyroid  deficiencies  as  well 
as  in  arteriosclerosis  and  various  tumors. 
The  ubiquitous  fibroblast  is  said  to  be  a 
great  former  of  mucins.  Epithelial 
mucins  are  produced  by  epithelial 
secretory  cells.  The  goblet  cells  are 
easily  recognized  by  the  fact  that  the 
material  to  be  discharged  is  held  in  a 
goblet  like  expansion  of  the  cell.  Other 
mucous  cells  can  be  distinguished  from 


MUELENGRATH  TEST 


216 


MYELOBLASTS 


serous  or   zymogenic   cella   by   several 
criteria : 

1 .  The  nuclei  instead  of  being  roughly 
spherical  are  often,  but  not  always, 
pressed  against  the  cell  membrane  re- 
mote from  the  lumen. 

2.  The  mitochondria  are  usually  of 
smaller  diameter  and  shorter  than  in 
zymogenic  cells. 

3.  The  secretion  antecedents  (Muci- 
gens)  of  mucous  cells  are  more  difficult 
to  see  in  the  fresh  state,  more  labile, 
and  in  fixed  tissues  are  metachromatic 
and  can  be  stained  almost  specifically 
with  mucicarmine  and  mucihematein. 

See  Mucicarmine  and  Mucihematein 
of  Mayer. 

A  simple  method  for  mucus  has  been 
described  by  Lillie  (R.  D.,  J.  Tech. 
Methods,  1929,  12,  120-121).  Sections 
of  tissue  fixed  in  formalin  or  in  Zenker- 
formol  (Helly)  are  passed  to  water.  In 
the  case  of  the  latter  remove  mercury 
with  iodine  and  sodium  thiosulphate  as 
usual.  Stain  1  min.  in  0.2%  aq.  toluidin 
blue.  Wash  in  water.  Dehydrate  in 
pure  acetone,  clear  in  xylol  and  mount  in 
balsam.  Mucus,  reddish  violet;  nuclei, 
blue ;  red  cells,  yellow  or  greenish  yellow. 
In  the  case  of  old  formalin  material 
rinse  in  95%  alcohol  before  the  acetone. 

McManus,  J.  F.  A.,  Nature,  1946,  158, 
202,  recommends  the  use  of  Schiff's 
Reagent  followed  by  periodic  acid. 
Material  fixed  in  Zenker-formal  is  de- 
hydrated and  embedded  in  the  usual 
manner  and  the  sections  transferred  to 
water  after  treatment  with  iodine  and 
hypo  and  placed  in  a  0.5%  aq.  periodic 
acid  2  min.  The  slides  are  washed  in 
tap  water  and  aq.  dest.  and  kept  in 
Schiff's  reagent  for  15  minutes;  rinsed 
in  Sulphurous  Acid,  dehydrated  and 
cleared  in  the  alcohol  and  xylol  series 
respectively  and  mounted  in  balsam. 
According  to  McManus,  the  mucus  of 
the  goblet  cells  of  the  human  intestine 
and  bronchus,  mucus  salivary  glands, 
certain  pituitary  cells,  the  colloid  of  the 
pituitary  stalk  and  thyroid,  granules 
in  some  nerve  cells  in  the  medulla  of  the 
rat  and  in  the  human  intestine,  the 
basement  membranes  of  the  tubular 
epithelium  and  of  the  glomerulus  in  the 
kidney  were  tested  by  this  method  and 
an  intense  coloration  detected  in  all 
instances. 

Hotchkiss,  R.  D.  deserves  credit  for 
reporting  in  a  personal  communication 
to  Glick  (p.  43)  in  1946  the  independent 
discovery  of  a  similar  technique  to  that 
of  McManus  employing  periodic  acid. 
This  is  given  in  detail  by  Glick. 
Muelengrath  Test,  see  Icterus  Index. 
Miiller's  Fluid.  Potassium  bichromate,  2- 
2.5  gm.;  sodium  sulphate,   1  gm.;  aq. 


dest.,  1  gm.  This  was  formerly  much 
used  for  long  fixation  and  mordanting  of 
nervous  tissue.  See  Chromaffin  Reac- 
tion, Decalcification,  O'Leary's  Bra- 
zilian Method,  Weigert  Method.  It  is 
now  largely  replaced  by  Orth's  Fluid 
which  is  really  formalin -Miiller. 

Mumps.  Refractile,  eosinophilic  bodies  in 
red  blood  cells  are  very  small  first  5-6 
days.  Increase  in  size  and  elongate 
7-14  days.  (Parsons,  H.  H.,  Military 
Surgeon,  1938,  83,  541-543). 

Murexide  Test,  see  Purines. 

Muscle,  to  distinguish  in  sections  from  con- 
nective tissue,  Dahlgren  (McClung,  p. 
306)  suggests  Retterer's  and  Van 
Gieson's  stains,  picronigrosine  and 
Unna's  orcein  to  which  may  be  added 
Mallory's  stain.  Demonstration  of 
chloride  in  muscle  fibers  (Heilbrunn, 
L.  V.  and  Hamilton,  P.  G.,  Physiol. 
Zool.,  1942,  15,  363-374).  For  contrac- 
tion bands  and  wave  mechanics,  see 
Carey,  E.  J.,  Arch.  Path.,  1940,  30, 
881-892,  1041-1072.  A  technique  for 
separating  nuclei  from  cytoplasm  for 
analysis  is  given  under  Nuclei.  If 
microdissection  is  contemplated  the 
pioneer  paper  by  Kite,  G.  L.,  Am.  J. 
Physiol.,  1913,  32,  146-164  should  be 
consulted.  The  experimental  produc- 
tion of  myocardial  segmentation  is 
described  by  Saphir,  O.  and  Karsner, 
H.  T.,  J.  Med.  Res.,  1923-24,  44,  539- 
556.  Methods  of  Maceration  are  often 
useful  in  the  isolation  of  single  fibers. 
Mitoses  can  only  be  induced  in  excep- 
tional cases  (Allen,  E.,  Smith,  G.  M. 
and  Gardner,  W.  U.,  Am.  J.  Anat., 
1937,  61,  321).  An  electron  microscopic 
technique  for  localization  of  magnesium 
and  calcium  is  described  by  Scott,  G. 
H.  and  Packer,  D.  M.,  Anat.  Rec, 
1939,  74,  31-45.  Muscle  gives  beautiful 
fluorescent  colors  in  ultraviolet  light 
with  many  fluorochromes  (Metcalf, 
R.  L.  and  Patton,  R.  L.,  Stain  Techn., 
1944,  19,  11-27).  See  Myosin  and  Fur- 
kinje  cells  and  fibers. 

Museum  Specimens,  see  Color  Preservation. 

Mycelia,  see  Lillie,  p.  289. 

Mycobacteria,  see  Acid  Fast  Bacteria. 

Mycological  Techniques,  see  Fungi. 

Myelin,  see  various  methods  for  demonstra- 
tion of  Nerve  Fibers. 

Myeloblasts.  The  recognition  of  these  cells 
is  a  fine  art ;  because,  by  definition,  they 
are  so  little  dififerentiated  that  the 
granules  characteristic  of  the  3  types  of 
leucocytes  are  absent.  For  contrasting 
views,  dependent  largely  on  whether 
supravital  staining  or  fixed  and  stained 
preparations  are  used,  see  Cowdry's 
Histology,  p.  100,  also  Leucocytes,  de- 
velopmental series. 


MYELOCYTES 


217 


NAPHTHOL  GREEN  B 


Myelocytes,  see  Leucocytes,  developmental 
series. 

Myeloidin  is  the  term  applied  to  the  mate- 
rial of  certain  spheroidal  or  cuboidal 
bodies  of  wax-like  luster  present  in  the 
bases  of  retinal  pigment  cells  of  monkeys 
and  some  other  animals  but  reported  as 
absent  in  man  and  said  to  resemble 
myelin.  For  literature  see  Arey,  L.  B. 
in  Cowdry's  Special  Cytology,  1932, 
3,  1218. 

Myocardium.  Method  for  separation  of 
fiber  bundles  (Mall,  F.  P.,  Am.  J.  Anat. 
11,211-266). 

Myofibrils.  The  best  method  is  to  fix  in 
Zenker's  fluid  or  strong  Flemming's 
mixture  and  to  stain  with  iron  hema- 
toxylin (see  Dahlgren  in  McClung 
p.  425).  Microincineration  is  useful 
for  the  demonstration  of  minerals. 

Myoglia  is  a  fine  network  of  fibers  associated 
wuth  muscle  cells  well  demonstrated  by 
Mallory's  Connective  Tissue  Stain. 

Myonemes  of  trypanosomes,  gold  chloride 
method  for  (Ogawa,  M.,  Arch.  f. 
Protistenk.,  1913,  29,  248).  See  Wen- 
rich,  D.  H.  and  Diller,  W.  F.  in  Mc- 
Clung's  Microscopical  Technique,  1950, 
p.  464. 

Myosin  is  a  protein,  present  in  muscle,  the 
molecules  of  which  are  needle-shaped. 
Cross  striations  of  muscle  are  thought 
to  depend  on  their  arrangement.  In 
the  isotropic  (non-birefringent)  bands 
the  myosin  molecules  are  believed  to  be 
disposed  at  random  and  in  the  aniso- 
tropic (birefringent)  bands  parallel  to 
the  length  of  the  fiber  (see  Bourne,  p.  30). 

Myriapoda,  see  Parasites. 

Nadi  Reagent  is  dimethyl-paraphenylene- 
diamin  -|-  a  naphthol.  Indophenol 
oxidase  catalyses  oxidation  of  nadi  to 
indophenol  blue  and  that  of  parapheny- 
lene  diamin  to  diamin. 

Nails.  These  very  interesting  structures 
are  seldom  examined  microscopically 
despite  the  fact  that  changes  in  them 
may  provide  significant  clues  to  the  con- 
dition of  other  tissues.  They  are  chiefly 
made  up  of  stratum  lucidum  thickened 
by  much  eleidin.  It  is  a  simple  matter 
to  macerate  cut  finger  or  toe  nails  in 
40%  aq.  potassium  hydroxide  or  in  con- 
centrated sulphuric  acid  for  a  few  days 
and  then  to  isolate  the  individual  cells 
some  of  which  are  nucleated.  Mac- 
Leod, J.  M.  H.,  Practical  Handbook  of 
the  Pathology  of  the  Skin.  London: 
H.  K.  Lewis,  1903,  408  pp.  gives  Heller's 
method  which  involves  fi.xation  of  un- 
gual phalanx  for  a  few  days  in  Muller's 
fluid,  prolonged  washing^  decalcification 
for  4-6  days  in  1  part  nitric  acid  and  3 
parts  of  water  followed  by  thorough 
imbedding  in  celloidin.  The  sections 
can  then  be  stained  with  hematoxylin. 


gentian  violet,  safranin  or  any  other  of 
a  number  of  dyes. 

Naphthalene  Pink,  see  Magdala  Red. 

Naphthalene  Red,  see  Magdala  Red. 

Naphthamine  Blue  3BX,  see  Trypan  Blue. 

Naphthamine  Brilliant  Blue  2R,  see  Dianil 
Blue  2r. 

Naphthol  Blue  Black  (CI,  246).  Lillie, 
R.  D.,  J.  Tech.  Methods,  1945,  No.  25, 
47  pp.  has  reported  that  this  dye  (NAC- 
7080  and  DuFont  L  6401)  gives  excellent 
staining  in  combination:  Stain  with 
Weigert's  iron  hematoxylin,  6  min. 
Wash  in  water  and  counterstain  5  niin. 
in  3  parts  1%  brilliant  purpurin  R  (CI, 
454)  in  1%  aq.  acetic  acid  and  2  parts 
1%  azofuchsin  (CI,  153)  likewise  in  1% 
aq.  acetic  acid.  Rinse  in  1%  aq.  acetic 
acid  and  stain  5  min.  in  1%  naphthol 
blue  black  (CI,  246)  in  sat.  aq.  picric 
acid.  Rinse  in  1%  aq.  acetic  acid, 
2  min.  Dehydrate  and  clear  in  alcohol, 
alcohol  and  xylol,  xylol  and  mount  in 
clarite.  Collagen,  reticulum  and  base- 
ment membranes,  dark  green;  smooth 
muscle,  brown;  nuclei  brownish-black. 

Naphthol  Blue  R  (CI,  909)— fast  blue  3R, 
Indian  blue  2RD,  Meldola's  blue,  new 
blue  R,  phenylene  blue — An  oxazin  dye 
used  by  Harvey,  B.  C.  H.,  and  Bensley, 
R.  R.,  Biol.  Bull.,  1912,  23,  225-249  as  a 
supravital  stain  for  gastric  mucosa. 
The  Bensleys'  report  that  this  dye  has 
proved  useful  in  the  localization  of  un- 
suspected parathyroid  and  thyroid  tis- 
sue in  experimental  animals.  After 
vascular  perfusion  in  a  concentration  of 
1 :  40 ,000  of  0 .85%  aq .  sodi um  chloride  the 
thyroid,  parathyroid  and  lymph  nodes 
become  colored  intensely  blue ;  whereas 
other  tissues,  muscles,  salivary  glands 
etc.,  are  colored  pale  greenish  blue. 

Naphthol  Green,  see  Naphthol  Green  B. 

Naphthol  Green  B  (CI,  5) — acid  green  O, 
green  PL,  naphthol  green — An  acid 
nitroso  dyefor  whicha  probable  formula 
is  given  by  Conn  (p.  42)  and  which  he 
thinks  was  the  naphthol  green  used  by  v. 
Volkmann,  R.  and  Strauss,  F.,  Zeit.  f. 
Wis.  Mikr.,  1934,  51,  244-249,  and  by 
MoUier,  G.,  Zeit.  f.  Wis.  Mikr.,  1938,  55, 
472-473. 

Lillie,  R.  D.,  J.  Techn.  Methods,  1945, 
No.  25,  47  pp.  recommends  naphthol 
green  B  for  connective  tissue.  Stain 
sections  6  min.  in  Weigert's  or  other 
iron  hematoxylin.  Wash  thoroughly 
in  water  and  stain  3  min.  in  1%  aq. 
eosin  Y  (CI,  768  .  Rinse  in  water  and 
mordant  4  min.  in  10%  dilution  of 
U.S. P.  ferric  chloride  solution.  Rinse 
in  water  and  stain  5  min.  in  1%  naph- 
thol green  B.  Differentiate  2  min.  in 
1%  aq.  acetic  acid.  Dehydrate  in 
aceton,  clear  in  acetone-xylene  and  in 
.xylene  and  mount  in  clarite  xylene  or 


NAPIITHOL  ORANGE 


218 


NASAL  CELL  SMEARS 


in  salicylic  acid  balsam.  Connective 
tissue,  green;  muscle  and  cytoplasm, 
pink. 

Y  (CI,  2) — fast  printing  green,  Gam- 
bine — An  acid  nitroso  dye  apparently 
not  used  in  histology. 

Naphthol  Orange,  see  Orange  L 

Naphthol  Red  S,  C  or  O,  see  Amaranth. 

Naphthol  Yellow,  see  Martins  Yellow. 

Naphthyl  Red  (CI,  856),  a  basic  dye  of  light 
fastness  5.  Only  nuclei  of  mature  plant 
cells  colored  fugitive  red  (Emig,  p.  57). 

Naphthylamine  Brown  (CI,  170),  an  acid 
monoazo  dye  which  stains  plant  tissues 
darker  in  presence  of  potassium  bi- 
chromate (Emig,  p.  34). 

Naphthylamine  Pink,  see  Magdala  Red. 

Naples  Yellow,  an  exogenous  pigment — 
Pb3(Sb04)-.— Lillie,  p.  134. 

Narcein  (CI,  152).  An  acid  mono-azo  dye. 
Was  used  by  Ehrlich  in  combination 
with  pyronin  and  methylene  blue  or 
methyl  green  to  produce  a  neutral  dye 
(Conn,  p.  54).     No  longer  available. 

Nasal  Cell  Smears — Written  by  Marian 
Pfingsten  Bryan,  Dept.  of  Otolaryngol- 
ogy, Washington  University,  St.  Louis 
10,  Mo.— September  14,  1951— Since 
1927  the  study  of  the  cytology  of  nasal 
secretions  has  been  recognized  as  an 
important  aid  in  the  clinical  diagnosis 
and  differentiation  of  certain  nasal  and 
sinus  conditions.  Eyerman,  C.  H., 
one  of  the  early  investigators  (Ann. 
Otol.,  Rhinol.  and  Laryngol.,  1927,  36, 
808-815),  identified  the  main  tj^pes  of 
cells  found  in  nasal  secretion.  Tillot- 
son,  R.  S.  (Arch.  Otolaryng.,  1929,  10, 
262-265)  reported  on  the  value  of  cyto- 
logic studies  in  the  diagnosis  of  sphen- 
oiditis.  Sewall,  E.  C.  and  Hunnicut, 
L.  (Arch.  Otolaryng.,  1929,  10,  1-15)  dis- 
cussed cytologic  examination  of  the 
antrum.  Dean,  L.  W.  (J.  A.  M.  A., 
1932,  99,  543-546)  emphasized  the  sig- 
nificance of  the  nasal  smear  in  helping 
to  diagnose  allergy  of  the  nose  and  si- 
nuses. Hansel,  F.  K.  (J.  Allergy,  1934, 
5,  357)  contributed  many  observations 
on  the  cytology  of  these  secretions,  es- 
pecially in  regard  to  allergy  of  the  nose 
and  paranasal  sinuses.  There  is  a  good 
review  of  the  numerous  articles  in  the 
literature  dealing  with  nasal  cytology 
by  Hansel,  F.  K.,  Allergy  of  the  Nose 
and  Paranasal  Sinuses,  St.  Louis:  C.  V. 
Mosby  Co.,  1936,  p.  383. 

The  particular  emphasis  has  been  on 
the  number  of  eosinophilic  cells  in  rela- 
tion to  neutrophiles;  but,  as  knowledge 
has  progressed  in  nasal  cytology,  the 
value  of  the  study  of  smears  has  been 
extended.  The  objectives  of  nasal  and 
sinus  cytology  include  the  following: 
First,  the  demonstration  of  eosinophiles 
in  the  secretions  in  relation  to  neutro- 


philes may  be  helpful  in  distinguishing 
allergic  conditions  from  infections,  or 
in  establishing  the  coexistence  of  both. 
In  the  second  place  the  method  makes 
possible  detailed  observations  of  the 
ciliated  columnar  epithelial  cells,  ex- 
foliated in  the  early  stages  of  acute 
upper  respiratory  infections.  These 
studies  may  aid  further  in  differentiat- 
ing the  common  cold  from  allergic 
rhinitis.  Thirdly  the  smear  technique 
proves  to  be  a  reliable  help,  along  with 
biopsy,  in  the  diagnosis  of  carcinoma, 
other  tumors  and  lesions  of  the  upper 
respiratory  tract.  Fourth,  for  the 
microscopic  study  of  radiation  changes 
produced  in  cells  after  treatment  with 
x-ray  and  radiation,  it  is  invaluable. 
Fifth,  it  is  a  useful  tool  for  working  out 
unknown  cellular  patterns  which  may 
support  clinical  diagnoses  of  other 
nasal  and  sinus  conditions. 

For  cytological  studies,  nasal  secre- 
tion is  collected,  after  blowing  the  nose, 
on  wax  paper  or  on  a  cellophane  hand- 
kerchief. Specimens  are  preferably 
taken  separately  from  each  side  of  the 
nose.  The  material  may  also  be  blown 
directly  on  to  a  clean  glass  slide,  then 
gently  smeared  with  a  probe  (not  a  dry 
cotton  applicator).  Thin  smears  are 
more  suitable  for  microscopic  study 
than  thick  ones.  In  order  to  obtain  a 
sampling  from  a  normal  nose,  the  naso- 
pharynx is  wiped  with  a  small  cotton 
applicator,  slightly  moistened  with 
saline.  For  abnormal  dry  noses,  the 
raping  technique  may  also  be  used  be- 
fore application  of  any  drugs.  A  saline 
tampon,  inserted  into  the  nostril,  may 
stimulate  the  flow  of  secretion  in  order 
to  obtain  a  specimen.  Secretions  from 
the  sinuses  may  be  collected  by  aspira- 
tion of  the  nose,  displacement  or  irri- 
gation of  sinuses,  or  be  taken  directly 
from  the  area  during  an  operation. 
These  secretions,  direct  from  the  re- 
spective paranasal  sinuses,  are  often  of 
value  in  locating  the  source  of  the  in- 
fection and  in  determining  the  type 
of  cellular  response.  They  thus  help 
to  complete  the  diagnosis.  The  cyto- 
logical picture  presented  in  the  nose 
may  be  somewhat  different  from  the 
cellular  pattern  observed  when  the  ma- 
terial is  obtained  directly  from  a  par- 
ticular sinus.  Both  should  be  studied 
and  correlated  whenever  possible. 

In  the  differential  diagnosis  of  acute 
and  chronic  infections  from  allergic 
ones,  or  in  demonstrating  the  joint  ex- 
istence of  both,  the  numerical  propor- 
tions of  eosinophilic  to  neutrophilic 
cells  in  the  nasal  smear  should  be  eval- 
uated. The  ratio  is  usually  an  expres- 
sion of  the  nature  and  stage  of  the  nasal 


NASAL  CELL  SMEARS 


219 


NASAL  CELL  SMEARS 


condition.  Hansel,  F.  K.,  St.  Louis: 
C.  V.  Mosby  Co.,  1936,  gives  a  useful 
scheme  on  page  380  for  recording  the 
cytology  of  nasal  secretions  similar  to 
that  used  in  the  recording  of  skin  tests. 
In  view  of  the  irregularitj-  of  distribu- 
tion of  the  cells  and  the  amount  and 
type  of  secretions,  it  is  difficult  to  de- 
termine the  cells  in  percentages.  Long 
personal  experience  in  the  observation 
of  smears  and  in  the  correlation  of  them 
with  clinical  symptoms  is  of  the  utmost 
value.  The  smears  should  be  repeated 
in  the  same  patient  in  order  to  obtain 
a  complete  picture  of  the  sequence  of 
events.  In  estimating  the  numbers  of 
neutrophiles  in  the  secretion,  it  must  be 
realized  that  when  acute  or  chronic  in- 
fections complicate  allergic  responses, 
the  neutrophiles  outnumber  the  eosino- 
philes  in  the  secretion  about  10  to  1. 
In  seasonal  hay  fever,  however,  un- 
complicated by  infection,  there  may  be 
a  few  neutrophiles  along  with  squamous 
epithelial  cells,  but  the  major  cellular 
response  will  be  eosinophilic.  Some 
cases  of  hay  fever  show  a  response  of 
mononuclears  with  coarse  blue  granules 
filling  the  cell  and  nearly  obscuring  the 
nucleus.  Their  actual  significance  is 
still  unknown.  A  criticism  of  Hansel's 
scheme  is  that  it  does  not  take  into  con- 
sideration the  other  cellular  elements 
besides  eosinophiles  and  neutrophiles. 
The  type,  color,  quantity  and  consist- 
ency of  the  secretion  should  be  included 
and  correlated  with  clinical  symptoms, 
but  the  macroscopic  appearance  cannot 
be  relied  upon  as  an  indication  of  the 
microscopic  cellular  contents  of  the  se- 
cretion. The  smears  must  also  be  ex- 
amined for  the  presence  of  microorgan- 
isms and  the  tj'pes  of  epithelial  cells 
should  be  carefully  studied. 

Smears  from  normal  noses  may  or 
may  not  show  a  few  scattered  neutro- 
philes. A  small  amount  of  thin  mucus 
is  usualljr  present  with  some  squamous 
epithelial  cells.  Ciliated  cells  are  seen 
if  they  have  been  wiped  from  the  nose 
with  the  applicator.  In  the  normal 
nose  they  are  rarely  exfoliated. 

In  cases  of  nasal  polyps,  eosinophiles 
usually  are  predominant;  but  repeated 
studies  of  the  secretions  are  necessary 
to  determine  whether  or  not  complicat- 
ing acute  or  chronic  infection  is  present. 
If  a  marked  predominance  of  neutro- 
philes persists,  a  chronic  infection  is 
present.  The  cytology  of  nasal  polypi 
is  fully  discussed  by  Walsh,  T.  E.,  and 
Lindsay,  J.  R.  (Arch.  Otolaryng.,  1934, 
20,  649).  Stagnation  and  secondary 
infection  are  frequently  present  inthese 
cases,  since  polypi  cause  obstruction  in 
the  nose  and  a  resultant  neutrophilic 


response.  A  single  failure  to  find  eosin- 
ophiles in  a  smear  does  not  rule  out  the 
existence  of  an  allergic  condition,  but 
repeated  failures  to  demonstrate  eosin- 
ophiles usually  means  that  an  active 
nasal  allergy  is  not  present. 

The  differentiation  of  an  allergic 
rhinitis  from  a  common  cold  may  at 
times  be  difficult  clinicall3^  In  these 
cases  microscopic  examination  of  the 
secretions  is  helpful;  but  nevertheless, 
even  with  cases  of  acute  upper  respira- 
tory infections  in  non-allergic  patients, 
the  eosinophilic  response  is  so  marked 
that  it  may  closely  approach  the  num- 
ber found  in  allergic  individuals  during 
an  infection.  In  the  non-allergic  per- 
sons the  eosinophiles  may  disappear 
before  the  neutrophiles  but  not  in  many 
of  them.  In  allergic  persons,  the  eosin- 
ophiles often  increase  as  the  neutro- 
philes disappear,  but  these  differences 
are  uncertain.  Acute  allergic  exacer- 
bations may  so  closely  resemble  com- 
mon colds  that  in  numerous  intances, 
even  microscopically,  it  is  difficult  to 
distinguish  between  them  unless  re- 
peated consecutive  smears  are  examined 
throughout  the  course  of  the  condition. 
Hilding,  A.  C.  (Tran.  Amer.  Laryngol. 
Assoc,  1934,  253-271)  carefully  studied 
fresh  unstained  nasal  secretions  from 
common  colds  in  adults  and  noted  that 
ciliated  columnar  cells  were  shed  in  the 
exudates  during  the  first  few  days  of  the 
infection.  More  recently  Bryan,  W. 
T.  K.  and  Brvan,  M.  P.  (The  Larvngo- 
scope,  1950,  60,  523-531)  applied  the 
staining  technique  of  Papanicolaou  to 
nasal  secretions.  In  a  series  of  upper 
respiratory  infections,  in  which  the 
secretions  were  examined  at  timed 
intervals  during  the  course  of  the  in- 
fection, it  was  possible  to  chart  a  de- 
generative pattern  of  change  exhibited 
by  the  ciliated  columnar  epithelial  cells. 
These  changes  suggest  virus  etiology. 
In  seasonal  allergic  hay  fever  the  ex- 
foliation of  the  ciliated  columnar  cells 
and  their  degenerative  patterns  of 
change  have  thus  far  not  been  observed. 
Smears  from  these  cases  reveal  that 
large  numbers  of  clumped  or  grouped 
eosinophiles  are  significant  when  they 
are  not  complicated  bj^  infection. 
Ciliated  epithelial  cells  are  of  rare 
occurrence,  but  some  basal  and  squa- 
mous epithelial  cells  are  frequently 
encountered.  Consequently  exfoliation 
of  the  ciliated  columnar  epithelial  cells 
in  acute  upper  respiratory  infections 
may  have  diagnostic  value. 

Within  the  last  few  years  cytological 
studies  of  nasal  diseases  have  been  ex- 
tended by  use  of  the  smear  technique  to 
include  diagnostic  information  in  regard 


NASAL  CELL  SMEARS 


220 


NASAL  CELL  SMEARS 


to  malignancies,  other  tumors  and  le- 
sions occurring  in  this  area.  Morrison, 
L.  F.,  Hopp,  E.  S.  and  Wu,  R.  (Ann. 
Otol.,  Rhinol.  and  Laryngol.,  1949,  58, 
18-31)  have  employed  the  smear  tech- 
nique as  an  adjunct  in  the  diagnosis  of 
exfoliating  neoplasms  of  the  naso- 
pharynx. This  proved  so  reliable  that 
a  positive  smear  demanded  discovery 
of  the  source  of  the  malignant  cells. 
Sooy,  F.  A.  (The  Laryngoscope,  1950, 
60,  964-992)  in  a  study  of  primary  tu- 
mors of  the  nasal  septum,  has  also  used 
the  smear  technique  to  advantage. 
Early  diagnosis  of  carcinoma  of  the 
maxillary  sinus  in  a  series  of  cases  re- 
ported by  Fitz-Hugh,  G.  S.,  Moon,  C. 
N.  Jr.  and  Luptom,  C.  H.  Jr.  (The 
Laryngoscope,  1950,  60,  376-387)  was 
thus  facilitated. 

This  cell  smear  method  is  not  only  an 
aid  in  diagnosis,  but  also  is  a  convenient 
means  for  studying  the  microscopic 
course  of  a  lesion  during  and  after  treat- 
ment. Biopsies  are  not  always  possi- 
ble, but  smears  are  easily  obtained  with- 
out discomfort  to  the  patients.  The 
effects  on  the  cells  of  x-ray  or  radium 
therapy  comprise  a  whole  new  field 
of  cytological  research.  New  knowl- 
edge thus  gained  is  of  great  importance 
when  closely  correlated  with  clinical 
symptoms. 

Satisfactory  techniques  for  staining 
nasal  secretions  include  the  following: 

1.  Wright's  Stain.  Slides  are  dried 
in  air  (avoid  flaming).  Many  direc- 
tions advise  staining  as  a  blood  film; 
but  two  points  will  improve  this  tech- 
nique for  nasal  work,  namely  the  use 
of  a  buffer  solution  for  the  diluent  and 
the  shortening  of  the  time  recommended 
for  blood  smears  to  only  15  or  20  sec. 
of  staining.  After  marking  off  the 
ends  of  the  slides  with  a  wax  pencil, 
they  are  flooded  with  the  dye  for  15 
sec.  The  buffer  diluent  is  added  to  the 
stain  and  allowed  to  mix  well  for  15 
sec.  more.  The  slides  are  then  washed 
in  buffer  solution  and  placed  on  end 
on  a  blotter  to  drain  and  thus  to  dry 
more  rapidly.  This  light  rapid  stain- 
ing shows  cellular  detail  better  in  the 
nasal  smear  than  the  usual  technique. 
The  slides  can  be  kept  and  stored  for 
many  years  without  coverslips  and 
thereafter  show  no  signs  of  deteriora- 
tion. The  cellular  details  are  even 
better  if  the  slides  are  stained  within 
30  min.  after  being  made  and  dried. 
The  stain  is  especially  good  for  eosino- 
philes,  neutrophiles  and  mononuclears. 
It  is  not  very  suitable  for  differentiating 
epithelial  cells. 

2.  HanseVs  Stain.  Color  slide  30 
sec.   with  his  dye   which  is   an  eosin- 


methylene  blue  combination  and  can 
be  obtained  directly  from  him  (Dr  F 
K.  Hansel,  634  N.  Grand  Blvd.,  St. 
Louis  3,  Mo.).  Then  add  alkaline 
water  which  is  made  by  adding  one 
drop  of  1%  potassium  carbonate  to  60 
cc.  aq.  dest.,  for  30  sec.  Wash  in  alkaline 
water  followed  by  washing  in  acid  water 
which  is  made  by  adding  one  drop  of 
1%  hydrochloric  acid  in  60  cc.  aq.  dest. 
Wash  again  in  alkaline  water  and  finally 
rinse  in  95%  ethyl  alcohol.  This  stain 
is  especially  good  for  eosinophiles. 
The  granules  are  very  brilliant  and  re- 
fractile.  Other  cellular  detail  may  be 
somewhat  dark  and  indefinite. 

5.  Gtemsa  Stain.  The  preparation 
from  Gradwohl  is  very  satisfactory. 
The  dilution  is  one  drop  of  stain  to  1  cc. 
ac[.  dest.  The  slide  is  flooded  with 
diluted  stain  for  one  minute  then 
washed  with  aq.  dest.  If  overstaining 
occurs,  this  may  be  decolorized  with 
ethyl  alcohol.  The  restulting  colora- 
tion is  excellent  for  eosinophiles  but  is 
not  especially  recommended  for  the 
other  cells. 

4.  Supra-Vital  Staining.  This  tech- 
nique is  particularly  useful  for  study- 
ing secretions  in  the  fresh  condition 
when  one  wishes  to  observe  motility 
of  eosinophiles  and  neutrophiles,  the 
phagocytic  activity  of  neutrophiles  and 
of  mononuclears,  as  well  as  the  ciliary 
activity  of  the  exfoliated  columnar 
epithelial  cells.  One  drop  of  exudate 
may  be  mi.xed  with  a  drop  of  1:15,000 
aqueous  Neutral  Red  or  Janus  Green, 
or  of  both  in  combination.  The  tech- 
nique is  fully  described  by  Sabin,  F. 
R.  (Bull.  Johns  Hopkins  Hosp.,  1923, 
34,  277-288)  who  used  it  to  study  living 
human  blood  cells.  The  stock  solution 
of  Neutral  Red  contains  100  mg.  of 
dye  to  10  cc.  of  absolute  alcohol.  The 
dilute  solution  contains  0.4  cc.  of  stock 
Neutral  Red  in  10  cc.  of  absolute  al- 
cohol. An  even  dye  film  is  obtained 
by  flaming  the  slide,  then  flooding  it 
with  the  dilute  Neutral  Red  solution  or 
with  a  mixture  of  Neutral  Red  and 
Janus  Green,  which  is  2  cc.  of  dilute 
Neutral  Red  to  3  drops  of  saturated 
solution  of  Janus  Green  in  absolute  al- 
cohol. The  slide  is  quickly  drained  and 
placed  upright  to  dry.  Fresh  exudate 
IS  mounted  on  the  slide  with  coverslip 
and  ringed  wth  vaseline.  The  cells 
may  last  for  2  to  3  hrs.  if  examined  un- 
der a  warm  stage.  The  mitochondria 
of  the  cells  may  be  studied  when  Janus 
Green  is  used.  This  dye  is  more  toxic 
to  the  cells  than  Neutral  Red.  The 
ciliated  epithelial  cells  are  of  particular 
interest  and  the  motility  of  the  cilia  is 
not  impaired.     Nuclear  staining  of  the 


NASAL  PASSAGES 


221 


NECROBIOSIS 


cells  with  either  of  the  dyea  is  indica- 
tive of  cell  death.  The  supra-vital 
staining  technique  was  applied  to  the 
study  of  nasal  secretion  by  Pfiugsten, 
M.  G.,  in  1933  and  reported  to  the 
E.  N.  T.  clinical  conference,  Barnes 
Hospital  (unpublished).  It  is  possible 
to  distinguish  cells  which  are  stimu- 
lated to  activity  from  degenerating 
cells  with  this  technique.  A  good  azure 
stain  may  be  obtained  by  using  10-12 
drops  of  Wright's  stain  in  absolute  al- 
cohol, but  this  is  more  toxic  than  Neu- 
tral Red. 

5.  Papanicolaou  Stain.  This  stain, 
as  developed  by  Papanicolaou,  G.  N. 
(Science,  1942,  95,  438-439)  is  the  first 
satisfactory  stain  and  fixation  found 
which  permits  accurate  identification 
of  the  different  types  of  epithelial  cells 
found  in  nasal  exudates.  Ciliated 
columnar  cells  are  well  preserved  and 
can  be  readily  identified  from  the 
squamous  and  basal  types.  The  nu- 
clear detail  of  all  cells  in  this  stain  is 
very  sharp  and  clear.  The  eosinophilic 
granules,  however,  are  not  as  distinct 
as  in  the  other  stains.  Slight  modifica- 
tions can  be  made  to  bring  out  certain 
details  in  nasal  smears.  The  chromatin 
masses  may  be  more  distinct  when  the 
time  in  hematoxylin  is  shortened  to 
2  to  3  min.  The  granules  of  the  eosin- 
ophilic cells  and  other  acidophilic  bod- 
ies are  brighter  red  if  the  time  in  eosin 
is  increased  to  3  min.,  or,  if  a  trace  of 
phlo.xine  is  added  (0.5%  solution  in  95% 
alcohol).  All  cellular  details  disclosed 
by  the  Papanicolaou  stain  are  revealed 
by  Wright's  stain,  even  the  patterns  of 
epithelial  degeneration,  but  they  are 
noted  with  greater  difficulty. 

It  follows  that  one  stain  is  not  suffi- 
cient to  see  all  cellular  details  to  the 
best  advantage.  It  seems  important 
to  use  several  staining  methods  on  the 
same  material  to  obtain  more  complete 
knowledge  of  cellular  responses.  The 
smears,  with  their  cellular  patterns, 
may  be  considered  to  be  an  approximate 
index  of  the  pathological  processes  oc- 
curring in  the  tissues. 
Nasal  Passages.  The  fluid,  when  present 
in  unusual  amounts  can  obviously  be 
studied  in  Smears.  Nasal  clearance 
depends  upon  the  movement  by  the 
cilia  toward  the  pharynx  of  a  mucous 
sheet  (to  which  foreign  materials  be- 
come attached)  over  a  layer  of  fluid  in 
which  the  cilia  act  as  can  be  demon- 
strated by  the  techniques  of  Lucas, 
A.  M.  and  Douglas,  L.  C,  Arch.  Oto- 
laryng..  1934,  20,  518-641  and  others. 
Methods  for  Mucus  and  Cilia  are  given 
under  their  respective  headings.  The 
wall    of    the    nasal    passages    exhibits 


marked  regional  diversity  (Hilding,  A., 
Arch.  Otolaryng.,  1932,  16,  9-18).  The 
nasal  mucous  membrane  covering  the 
septum  can  be  removed  in  toto  by  the 
dilute  acetic  acid  method  (see  Epider- 
mis) and  examined  as  a  whole  mount 
which  gives  valuable  data  impossible  to 
secure  from  the  study  of  sections. 
Those  interested  in  wound  healing  would 
do  well  to  consult  a  paper  by  Boling, 
L.  R.,  Arch.  Otolaryng.,  1935,  22,  689- 
724.  An  easy  and  graphic  method  for 
visualization  of  lymphatic  drainage  is 
described  under  Lymphatic  Vessels. 
For  numerous  suggestions  as  to  tech- 
nique see  Proetz,  A.  Applied  Physi- 
ology of  the  Nose.  St.  Louis:  Annals 
Publishing  Co.,  1941,  395  pp. 

Nasal  Sinuses.  The  mechanism  of  clear- 
ance is  similar.  To  make  sections  of 
the  nasal  sinuses,  especially  the  smaller 
ones,  fixation  in  Formalin  Zenker  is 
suggested  followed  by  Decalcification 
and  Celloidin  Imbedding.  The  sec- 
tions can  be  stained  by  the  method  best 
adapted  to  the  purpose  in  mind. 

Nasmyth's  Membrane,  see  Enamel  cuticle. 

n-Butyl  Alcohol  (prophylcarbinol).  Rec- 
ommended by  Stiles  (K.  A.,  Stain 
Techn.,  1934,  9,  97-100)  to  replace 
higher  concentrations  of  alcohol  in  histo- 
logical technique  especially  for  lightly 
chitinized  insects  but  also  as  a  routine 
for  vertebrates.  After  fixation  in  Gil- 
son's  Fluid  pass  the  tissues  through 
35%  (ethyl)  alcohol  ^1  hr.;  90  cc.  45% 
ale.  +  10  cc.  butyl,  2  hrs.;  80  cc.  62% 
ale.  -f  20  cc.  butyl,  2  hrs.;  65  cc.  77% 
ale.  +  35  cc.  butyl,  4  hrs.;  45  cc.  90% 
ale.  +  55  cc.  butyl,  6  hrs.  to  days;  25 
cc.  abs.  ale.  +  75  cc.  butyl,  6  hrs.  to 
over  night;  butyl  2  changes  several 
hrs.  (or  store  in  butyl  if  desired).  To 
imbed  transfer  to  mixture  of  butyl  and 
paraffin  and  to  paraffin,  n  Butyl  alcohol 
is  helpful  in  making  permanent  prepara- 
tions of  tissues  freshly  stained  with 
Methylene  Blue,  which  see.  It  should 
not  be  confused  with  Tertiary  Butyl 
Alcohol. 

Necrobiosis  was  for  Minot  (C.  S.,  The 
Problem  of  Age,  Growth  and  Death. 
New  York,  G.  P.  Putnam's  Sons,  1908, 
280  pp.)  a  condition  in  which  the  cells 
continue  to  live  but  change  their  chemi- 
cal organization  so  that  their  substance 
passes  from  a  living  to  a  dead  state. 
"Here  (he  says)  life  and  death  play 
together  and  go  hand  in  hand."  The 
term  is  current  but  is  of  little  use  be- 
cause it  has  no  advantage  over  the  word 
Necrosis  for  the  disorganization  of 
death  seldom  if  ever  takes  place  simul- 
taneously throughout  the  substance  of 
any   living   thing.    See   Dead   Cells. 


NECROSIS 


222 


NEODYMIUM 


Necrosis  (G.  ne/crosis,  a  killing).  The  term 
is  usually  appliea  to  indicate  the  local 
death  of  a  cell  or  of  group  of  cells,  not 
that  of  the  body  as  a  whole.  Death  is 
defined  by  Webster  and  others  as  the 
"cessation  of  life"  which  merely  poses 
the  question  of  what  life  is.  Perhaps 
the  most  fundamental  vital  phenomenon 
is  the  oxygen  consumption  involved  in 
respiration.  This  may  persist  in  eryth- 
rocytes even  after  the  loss  of  their 
nuclei  (Harrop,  G.  A.,  Arch.  Int.  Med., 
1919,  23,  745-752).  But  cells  frozen 
by  special  techniques  do  not  respire 
while  frozen.  They  endure  in  a  state 
of  suspended  animation  (called  vitrifica- 
tion) indefinitely.  They  are  not  dead 
since  they  retain  the  structural  organi- 
zation, which,  when  unlocked  by  in- 
crease in  temperature,  confers  renewed 
vitality  (see  Luyet,  B.,  C.  rend.  Soc. 
de  biol.,  1938,  127,  788-789  and  many 
others).  Death  can  therefore  be  better 
defined  as  the  disorganization  of  living 
matter  which  makes  permanently  im- 
possible all  vital  phenomena.  Since 
the  organization  of  different  sorts  of 
living  cells  is  fundamentally  different 
the  loss  of  organization  in  them  is  likely 
also  to  be  different.  See  various  forms 
of  Degeneration.  In  general  necrosis 
of  tissue  is  often  evidenced  by  a  break- 
ing up  of  the  nucleus  known  as  caryor- 
rhexis  (G.  Karyon,  nucleus,  -f-  rhcxis, 
rupture)  or  by  its  solution,  caryolysis 
(G.  lysis,  solution).  Consequently  any 
good  nuclear  strain  such  as  hematoxylin 
or  methylene  blue  is  satisfactory.  See 
techniques  for  Dead  Cells,  Necrobiosis. 

Neelsen,  see  Carbol-Fuchsin. 

Negative  Stains  are  used  to  show  the  back- 
ground in  which  bacteria  and  other 
organisms  are  present  in  smears  and  by 
contrast  thus  to  reveal  them  unstained, 
that  is  in  a  negative  way.  The  tech- 
nique is  very  simple.  Simply  mix  the 
fluid  containing  the  organisms  with  the 
"stain",  smear  on  a  slide,  dry  and 
examine.  Higgins'  India  Ink  is  usually 
employed;  but  congo  red  (Cumley, 
R.  W.,  Stain  Techn.,  1935,  10,  53-56) 
and  azo  blue  (Butt,  E.  M.,  Boynge, 
C.  W.  and  Joyce,  R.  L.,  J.  Inf.  Dis., 

1936,  58,  5-9)  are  among  many  other 
materials  used.     See  Azo  Blue. 

Negri  Bodies.  1.  Rapid  section  method 
(Schleif stein,  J.,  Am.  J.  Pub.  Health, 

1937,  27,  1283-1285).  Fix  in  Zenker's 
fluid,  wash,  dehydrate  in  dioxan,  embed 
in  paraffin,  cut  at  4  microns,  mount, 
deparaffinize.  Flood  slides  with  1  drop 
1:40,000  aq.  KOH  in  2  cc.  stock  solution 
of  stain  (Rosanilin  of  Grubler  1.8  gm., 
methylene  blue,  Nat.  Col.,  1  gm.,  gly- 
cerol 100  cc.  and  methyl  alcohol  100  cc). 
Steamgentlv5min.    Rinse  in  tap  water. 


Decolorize  by  gently  moving  in  90% 
ethyl  alcohol  until  color  is  faintly  violet. 
Pass  quickly  through  95%  alcohol, 
absolute,  xylol  and  mount  in  balsam. 
Negri  bodies  deep  magenta  with  dark 
blue  inclusions. 

2.  Rapid  smear  method  (Dawson, 
J.  R.,  J.  Lab.  &  Clin.  Med.,  1934-35, 
20,  659-663).  Remove  brain  to  be 
examined  as  quickly  as  possible.  Cut 
several  small  segments  (3-4  mm.  thick) 
from  Ammon's  horn  perpendicular  to 
its  long  axis  and  place  in  Petri  dish. 
Cut  away  adjacent  tissue  leaving  only 
the  horn.  Place  a  segment,  cut  surface 
down,  on  small  end  of  a  new  1  in.  cork. 
With  wooden  applicator,  or  match, 
gently  wipe  peripheral  tissue  outward 
and  downward.  The  segment  is  thus 
more  firmly  attached  to  the  cork  and 
the  gray  matter  containing  the  pyra- 
midal cells  bulges  upward.  Press  this 
gently  against  a  slide  (clean  and  entirely 
free  from  grease)  held  at  one  end  be- 
tween thumb  and  forefinger.  Repeat 
3  or  4  times,  starting  at  end  away  from 
fingers,  quickly  so  tissue  does  not  dry. 
Immediately  immerse  in  abs.  methyl 
alcohol  5  min.  or  more.  Rinse  in  run- 
ning water  and  stain  in  2%  aq.  phloxine 
2-5  min.  Wash  off  excess  stain  in  run- 
ning water  and  color  in  Loeffler's  alka- 
line methylene  blue,  10-20  sec.  De- 
colorize in  80%  ethyl  ale,  dehydrate  in 
95%  and  2  changes  of  absolute,  clear  in 
xylol  and  mount  in  balsam.  Handle 
slides  with  forceps  and  avoid  danger 
from  contact  with  tissue  throughout 
process.  Pyramidal  cells  blue,  Negri 
bodies  bright  red  to  reddish  brown. 
Time  including  examination  25  min. 
Stovall,  W.  D.  and  Black,  C.  E., 
Am.  J.  Clin.  Path.,  Tech.  Suppl.,  1940, 
4,  8  recommend  control  of  pH  in  staining 
with  eosin  methylene  blue  (see  Buffers) . 
Stain  with  1%  eosin  in  95%  alcohol  at 
pH  6.0  or  more  alkaline.  ISIegri  bodies 
pale  red.  The  red  is  much  more  intense 
if  the  pH  is  3.0.  Loeffler's  methylene 
blue  is  best  as  counterstain  at  pH  5.3. 
At  pH  6.0  it  removes  eosin. 

Azur  B  is  advised  for  staining  of  Negri 
bodies  by  Jordan,  J.  H.,  and  Heather, 
H.  H.,  Stain  Techn.,  1929,  4,  121-126; 
see  also  Carbol-Anilin  Fuchsin  methyl- 
ene blue. 

Neisserian  Infection.  A  differential  stain 
favorable  for  diagnosis  (Scudder,  S.  A., 
StainTechn.,  1931,  6,  99-105). 

Neisser's  Stain  for  Diphtheria  Bacilli, 
which  see. 

Nemathelminthes  is  the  phylum  of  round 
worms.     See  Parasites. 

Nematodes.  See  Glychrogel  for  mounting. 
See  Parasites. 

Neodymium,   see   Atomic  Weights. 


NEON 


223 


NERVE  FIBER  DEGENERATION 


Neon,  see  Atomic  Weights. 

Neoprene,  injection  of  blood  vessels  (Lieb, 
E.,  J.  Tech.  Methods,  1940,  20,  50-51). 
Neoprene  is  a  colloidal,  finely  divided 
suspension  of  synthetic  chloroprene  in 
an  alkaline  aqueous  medium.  Instruc- 
tions for  the  human  kidney.  Cannulate 
renal  artery  and  wash  with  tap  water 
at  slow  but  constant  rate.  Ligate  grossly 
leaking  vessels.  Continue  8-18  hrs. 
until  organ  is  pale  gray.  Cover  and 
keep  in  ice  box  6-7  hrs.  or  until  the 
next  day.  Keep  specimen  at  room 
temperature  about  one  hour  before  in- 
jection. If  it  feels  cold  warrn  it  with 
tap  water.  Connect  cannula  with  bottle 
containing  neoprene.  A  special  appara- 
tus for  maintenance  of  150^160  mm.  Hg. 
is  advised  by  Lieb  but  it  is  probably 
sufficient  to  provide  gravity  pressure 
by  raising  the  bottle  5  ft.  or  more. 
Close  vessels  ejecting  the  neoprene 
with  hemostats  and  tie  them  when  ves- 
sels are  completely  filled.  Rinse  in 
warm  water.  If  a  corrosion  specimen 
is  wanted  leave  kidney  in  cone,  com- 
mercial HCl  in  tightly  covered  vessel 
at  56 °C.  over  night.  Next  morning 
pour  off  acid  and  allow  stream  of  water 
to  flow  over  the  cast  itself  in  the  bottom 
of  the  container.  When  all  debris  is 
removed  examine  under  water  with 
dissecting  microscope.  Store  in  0.3% 
Dowicide  sol.  (American  Anode  Inc., 
60  Cherry  St.,  Akron)  to  avoid  mold. 
Lieb  gives  more  details  and  describes 
combined  corrosion,  histological  and 
roentgenological  methods.  Technique 
should  be  adapted  to  other  organs. 
(Revised  by  Ethel  Lieb,  May  16,  1946). 
Lieb's  method  has  been  modified  in 
several  respects  by  Duff,  G.  L.  and 
More,  R.  H.,  J.  Tech.  Methods,  1944, 
24,  1-11.  The  technique  for  mounting 
separately  for  detailed  microscopic 
examination  small  sprigs  of  the  renal 
cortical  arteries  greatly  increases  its 
usefulness. 

Neoprene  Latex.  Emplo3^ed  for  injection  of 
coronary  arterial  sj'stem,  well  illus- 
trated and  with  a  list  of  earlier  papers 
(Smith,  J.  R.  and  Henry,  M.  J.,  J.  Lab. 
&  Clin.  Med.,  1945,  30,  462-466). 

Nerve  Endings.  These  may  be  demon- 
strated in  many  ways.  Nothing  will 
adequately  take  the  place  of  their  study 
in  vivo  (Speidel,  C.  C.,  J.  Comp.  Neur., 
1942,  76,  57-73) ;  but  no  method  should 
be  used  with  expectation  of  satisfactory 
results  the  first  time.  Experimentation 
is  required.  Most  of  the  silver  methods 
for  neurofibrils  show  nerve  endings. 
The  writer  has  obtained  good  results 
by  Bodian's  Method  applied  to  paraffin 
sections  of  experimental  tumors.  Cra- 
ven's  Gold   Chloride  method  may   be 


tried.  For  silver  impregnation  of  intra- 
cellular nerve  endings  in  pars  inter- 
media of  pituitary,  see  Tello,  F.,  Trab. 
d.  Lab.  Rech.  Biol.  Univ.  Madrid,  1912, 
10,  145-183.  Methylene  blue  is,  since 
the  time  of  Ehrlich,  a  very  popular  stain 
for  nerve  endings.  Addison  (McClung, 
pp.  477-480)  has  given  a  full  account  of 
the  technique.  Commission  Certified 
zinc-free  methylene  blue  is  suggested. 
Dye  can  be  applied  locally  or  by  vascular 
perfusion. 

1.  Local  application.  Place  tissue  in 
shallow  dish  on  thin  layer  of  glass-wool 
moistened  with  0.1-0.05%  methylene 
blue  in  physiological  salt  solution.  Add 
enough  stain  every  few  minutes  to  keep 
tissue  moist  and  covered  by  film  of 
stain.  Beginning  after  15  min.  examine 
frequently  at  low  magnification  until 
nerves  are  colored  blue.  Fix  stain  by 
immersion  in  cold  8%  ammonium  molyb- 
date  in  physiological  salt  solution  or 
Ringer's  (^  hr.).  Wash  in  cold  water. 
Dehydrate  in  alcohols  in  refrigerator 
a  little  above  32 °C.  Either  clear  in 
xylol  and  mount  in  balsam  or  imbed  in 
paraffin  and  section.  Cole  (E.  C,  J. 
Comp.  Neurol.,  1925,  38,  375-387) 
proceeded  much  in  this  way.  He 
immersed  whole  alimentary  tract  of 
frog  in  1:10,000  methylene  blue  solution 
for  1  hr.  and  cut  it  in  pieces. 

2.  Vascular  perfusion.  Insert  can- 
nula in  main  artery  leading  to  the  tissue. 
Inject  1:10,000  methylene  blue  in 
physiological  saline  until  tissue  becomes 
light  blue.  Leave  15  min.  Remove 
thin  pieces  or  slices.  Place  in  dish 
and  moisten  with  methylene  blue  solu- 
tion. Examine  uncovered  at  low  magni- 
fication at  intervals  until  nerve  fibers 
and  endings  are  stained.  It  is  essential 
as  in  local  application  not  to  exclude  air 
from  tissue  by  covering  with  too  much 
fluid.  Fix  in  ammonium  molybdate  and 
continue  as  described  above.  For  large 
fetuses  use  Langworthy's  method  (O. 
R.,  J.  Comp.  Neurol.,  1924,  36,  273-297), 
for  the  lungs  of  rabbits  that  of  Larsell 
(O.,  J.  Comp.  Neurol.,  1921,  33, 
105-131),  for  arteriovenous  anasto- 
moses Brown's  (M.  E.,  Anat.  Rec, 
1937,  69,  287-295),  and  for  skin  Weddell's 
(G.,  J.  Anat.,  1940-41,  75,  441-416). 
Staining  may  perhaps  be  accentuated 
by  hydrogen  acceptors,  see  Auerbach's 
Plexus.  See  Pacinian  Corpuscles, 
Meissner's  Corpuscles,  Krause's  End 
Bulbs,  Motor  End  Plates,  Boutons 
Terminaux  and  Synapses. 

Nerve  Fiber  Degeneration.  The  standard 
techniques  are  the  Marchi  Method  by 
which  the  lipids  produced  by  degenera- 
tion are  blackened  with  osmic  acid  and 
the  staining  of  lipoids  by  Sudan  III. 


NERVE  FIBERS 


224 


NERVOUS  SYSTEM 


In    addition    3    other    much    quicker 
methods  are  recommended: 

1.  To  stain  vitally  with  neutral  red 
(Covell,  W.  P.  and  O'Leary,  J.  L., 
J.  Tech.  Meth.,  1934,  13,  92-93).  In- 
tensity of  staining  of  degenerating 
myelin  depends  upon  amount  and  con- 
centration of  the  dye.  It  can  be  applied 
in  3  ways:  (1)  Inject  4  cc.  4%  neutral 
red  in  physiological  salt  solution  into 
marginal  ear  vein  of  a  rabbit  over 
period  of  1  hr.;  (2)  Perfuse  through 
aorta  with  large  volume  of  1:1,000 
solution;  (3)  Immerse  finely  teased 
piece  of  degenerated  nerve  in  1:10,000 
solution  for  about  12  min.  Vital  stain- 
ing permits  immediate  determination 
of  extent  and  degree  of  degeneration. 
See  the  author's  excellent  colored 
figures. 

2.  To  examine  by  polarized  light 
(Weaver,  H.  M.,  J.  Lab.  &  Clin.  Med., 
1940-41,  26,  1295-1304).  Lay  excised 
nerves  without  stretching  on  piece  of 
wooden  tongue  depressor  and  fix  24 
hrs.  or  more  in  10%  neutral  formalin. 
Cut  longitudinal  frozen  sections  10 
microns  thick.  Float  them  onto  slides 
from  water,  mount  in  neutral  glycerin 
and  examine.  Weaver  gives  diagrams 
to  aid  in  interpretation  of  findings.  See 
also  Pritchett,  C.  O.  and  Stevens,  C, 
Am.  J.  Path.,  1939,  15,  241-250;  Rad- 
hakrishana,  BLao,  M.  V.,  Ind.  J.  Med. 
Res.,  1938,  26,  103-106. 

3.  To  demonstrate  early  changes  in 
the  axis  cylinders  (cores  of  the  fibers) 
Alzheimer's  modification  of  IVIann's 
eosin-methyl  blue  method  is  strongly 
recommended  by  Mallory  as  showing 
normal  axis  cylinders  deep  blue  and 
degenerated  ones,  red. 

Nerve  Fibers.  Many  excellent  methods 
present  themselves :  the  continuous 
direct  observation  of  the  growth  of 
individual  fibers  in  living  tissues  of 
lower  animals  (Speidel,  C.  S.,  Biol. 
Bull.,  1935,  68,  140-161);  the  micro- 
dissection of  living  fibers  (De  Renyi, 
G.  S.,  Cowdry's  Special  Cytology,  1932, 
3,  1370-1402);  x-ray  diffraction  studies 
of  the  sheaths  (Schmitt,  F.  O.,  Bear, 
R.  S.  and  Palmer,  K.  J.,  J.  Cell,  ana 
Comp.  Physiol.,  1941,  18,  31-42)  and 
microincineration  (Scott,  G.  H.,  Proc. 
Soc.  Exp.  Biol.  &  Med.,  1940,  44,  397- 
398).  For  their  demonstration  in  fixed 
tissues  consult  methods  of  Bodian, 
Davenport,  Golgi,  O'Leary,  Osmic 
Acid,  Weigert  and  WeiL  The  methylene 
blue  technique  of  staining  nerve  fibers 
is  given  under  Auerbach's  Plexus. 
See  Nerve  Endings,  Motor  End  Plates, 
Bouton  Terminaux.  Use  of  quartz  io<l 
illuminator    in    study    of    living   nerve 


fibers  is  described  by  Speidel,  C.  C, 
J.  Comp.  Neurol.,  1935,  61,  1-80  and  by 
Bensley,  S.  H.,  Anat.  Rec,  1944,  90, 
1-11. 

Nerve  Grafts,  methods,  histological  and 
otherwise  (Sanders,  F.  K.,  and  Young, 
J.  Z.,  J.  Anat.,  1942,  76,  143-166). 

Nerve  Plexuses,  see  Auerbach's. 

Nerves.  A  red  lead  and  carpenter's  glue 
method  for  injection  and  visualization 
of  blood  vessels  of  nerves  (Epstein,  J., 
Anat.  Rec.,  1944,  89,  65-69).  See  Pia 
Mater  perivascular  nerves. 

Nervous  System.  This,  the  most  compli- 
cated of  bodily  parts,  can  be  investi- 
gated microscopically  in  a  great  many 
different  ways.  It  is  however  shielded 
from  the  environment  so  that  there  are 
great  obstacles  in  the  way  of  direct 
observation  in  vivo.  In  mammals  the 
best  that  can  be  done  is  to  insert  win- 
dows in  the  wall  of  the  skull.  A 
technique  for  this  purpose,  designed  by 
Forbes  (H.  S.,  Arch.  Neurol,  and 
Psychiat.,  1928,  19,  75),  permits  direct 
study  at  low  magnification  of  blood 
vessels  with  so  little  injury  that  their 
behavior  in  various  experimental  condi- 
tions can  be  investigated.  It  is  likely 
that  by  the  Sandison  Technique  very 
significant  observations  can  be  made 
on  living,  growing  nerve  fibers  of  the 
rabbit.  In  amphibia  Speidel  (C.  S., 
Biol.  Bull.,  1935,  68,  140-161)  has  been 
particularly  successful  in  devising 
methods  for  study  of  nerve  fibers 
in  vivo. 

Another  group  of  techniques  is  avail- 
able for  marking  in  vivo  and  examination 
of  the  tissues  after  removal.  Vital 
Staining  has  been  much  used.  Some 
factors  that  condition  the  coloration  of 
nerve  cells  with  trypan  blue  have  been 
described  by  King,  L.  S.,  J.  Anat., 
1934-35,  69,  177-180.  The  pathways 
of  drainage  of  cerebrospinal  fluid  can 
be  marked  with  Prussian  Blue  (Weed, 
L.  H.,  J.  Med.  Res.,  1914,  26,  21-117). 
Nerve  fibers  and  cells  can  of  course  be 
marked  by  the  in  vivo  creation  of  in- 
juries and  subsequently  examined.  To 
determine  the  distribution  of  Radio- 
phosphorus  may  prove  helpful. 

For  the  examination  of  excised  tissues 
a  host  of  methods  present  themselves. 
Consider  first  the  classical  techniques 
from  which  several  others  spring. 

1.  The  original  Nissl  method  for 
internal  structure  of  the  nerve  cell 
consisted  of  fixing  in  alcohol  and  of 
staining  sections  with  methylene  blue. 
It  revealed  a  basophilic  material  called 
Nissl  Substance.  The  unfortunate  ten- 
dency now-a-days  is  to  loosely  designate 
all   methods   intended    to   demonstrate 


NEUFELD'S  QUELLING  REACTION       225 


NEUROFIBRILS 


this  substance  as  Nissl  techniques  even 
though  resemblance  to  the  original 
method  is  lacking. 

2.  The  original  Golgi  method  for  the 
external  form  of  nerve  cells  depends 
upon  preliminary  mordanting  of  tissue 
in  potassium  bichromate  solutions,  fol- 
lowed by  immersion  in  weak  aqueous 
silver  nitrate,  and  the  cutting  of  thick 
sections  in  which  occasional  nerve  cells 
and  processes  are  outlined  with  startling 
clarity  by  the  black  deposit  of  silver 
chromate.  Cajal  modified  and  speeded 
up  the  technique  by  addition  of  osmic 
acid  to  the  bichromate  solution  (see 
Golgi  Method,  quick).  But  the  most 
used  modification  is  the  Golgi  Cox 
technique. 

3.  The  original  Weigert  method  for 
myelin  sheaths  of  nerve  fibers  depended 
likewise  upon  preliminary  mordanting 
in  bichromate  and  the  formation  of 
hematoxylin  "lakes"  when  the  sections 
were  later  stained  with  hematoxylin. 
Its  most  important  modification  is 
known  as  Weigert-Pal.  The  Marchi 
method,  as  modified  by  Swank  and 
Davenport  is  based  on  similar  mordant- 
ing with  bichromate  after  which  they 
are  treated  with  osmic  acid  and  was 
designed  to  reveal  degenerated  myelin 
sheaths  the  lipids  of  which  are  unaf- 
fected by  the  mordanting  and  are 
blackened  while  those  of  the  normal 
sheaths  are  not. 

4.  Cajal  and  Bielchowsky  introduced 
valuable  methods  for  axones,  neuro- 
fibrils, and  nerve  endings  including 
synapses.  Both  techniques  as  applied 
to  blocks  of  tissue  depend  on  preliminary 
"silvering"  with  weak  silver  nitrate 
solution  but  in  those  of  the  former  the 
silver  is  reduced  by  a  photographic 
developer  generally  hydroquinone  or 
pyrogallic  acid;  while  in  those  of  the 
latter  the  tissues  are  first  brought  into 
an  ammoniacal  silver  solution  and  then 
reduced  in  formalin.  The  most  useful 
modification  is  the  Bodian  Method 
of  activated  protargol.  See  its  evolu- 
tion under  Silver  Methods  which  are 
of  assistance  in  the  study  of  many 
other  tissues  of  the  body  as  well  as  the 
nervous  system. 

5.  Weigert 's  neuroglia  stain  was  also 
a  classic,  likewise  Cajal's  gold  chloride 
and  sublimate  method  (1913)  which 
was  soon  followed  by  Hortega's  car- 
bonate silver  method  (1917).  See  recent 
techniques  under  Neuroglia. 

There  are  still  other  techniques  to 
choose  from  which  are  not  so  directly 
developments  of  the  neurological 
classics.  Nerve  cells  are  closely  mixed 
with  fibers .    To  isolate  them  sufficiently 


for  direct  study  at  high  magnification 
in  approximately  isotonic  media  in- 
volves considerable  injury  and  they 
cannot  be  held  under  observation  for 
long  periods  because  their  death  ensues 
fairly  quickly.  Spinal  ganglion  cells 
are  the  easiest  studied.  The  Macera- 
tion technique  is  not  much  used  for  the 
nervous  system  but  Addison  (McClung, 
p.  439)  states  that,  if  pieces  of  the 
anterior  horn  of  the  spinal  cord  are 
treated  with  Gage's  dissociator  (0.2% 
formalin  in  physiological  saline)  for 
2-3  days,  the  nerve  cells  can  easily  be 
dissected  out  under  a  binocular  micro- 
scope, stained  and  examined  more  or 
less  as  units.  Tissue  Culture  of  nerve 
cells  of  the  adult  is  not  feasible  because 
they  are  fixed  postmitotics  (having 
permanently  lost  the  power  of  multi- 
plication) ;  but  culture  of  young  tissues 
provides  interesting  results  (Levi,  G., 
Arch,  de  Biol.,  1941,  52,  1-278,  profusely 
illustrated).  Nerve  Fibers  are  more 
easily  isolated  and  their  investigation 
in  the  fresh  state  is  very  profitable. 
The  histological  localization  of  Cho- 
linesterase  is  now  feasible.  The  meas- 
urement of  oxidative  metabolism  in  dif- 
ferent parts  of  the  nerve  cell  by 
reduction  of  ferric  chloride  (Gerard, 
R.  W.,  Assoc,  for  Res.  in  Nerv.  &  Ment. 
Dis.,  Baltimore,  Williams  &  Wilkins, 
1938,  18,  316-345)  can  probably  be  tied 
up  with  localization  of  Oxidases  and 
Peroxidases.  Marinesco  (G.,  Arch. 
Suisse  de  Neurol,  et  de  Psych.,  1924, 
15,  1-24)  has  published  repeatedly  on 
these  enzymes  in  nerve  cells.  Methods 
for  Pigments  and  Lipids  can  easily  be 
applied  to  the  nervous  system.  For 
microincineration  of  nerve  cells  and 
fibers  see  Scott,  G.  H.,  Proc.  Soc.  Exp. 
Biol.  &  Med.,  1940,  44,  397-398.  If  it  is 
desired  to  demonstrate  mitochondria 
the  Anilin-Fuchsin  Methyl  Green 
method  is  suggested  after  fixation  by 
vascular  perfusion  plus  immersion.  See 
in  addition  to  above  headings :  Auer- 
bach's  Plexus,  Axis  Cylinders,  Boutons 
Terminaux,  Centrosomes,  Cresyl  Violet, 
Golgi  Apparatus,  Microglia,  Motor 
End  Plates,  Nerve  Endings,  Neuro- 
fibrils, Neurosecretory  Cells,  Oligo- 
dendroglia. 

Neufeld's  Quelling  Reaction.  This  is  a 
microscopically  demonstrable  swelling 
of  the  capsules  of  pneumococei  which  is 
of  distinct  value  in  typing  (L.  W.  Parr, 
in  Simmons  and  Gentzkow,  p.  42fi). 

Neumann's  Crystals,  see  Charcot-Leyden. 

Neurites,  see  Nerve  Fibers. 

Neurofibrils.  These  delicate  fibrils  and 
networks  can  be  demonstrated  with 
difficulty  mainly  by  methods  of  silver 


NEUROGLIA 


226 


NEUTRAL  GENTIAN 


impregnation  in  the  cytoplasm  of  nerve 
cells.  In  the  living  nerve  cells  of 
selected  invertebrates  they  can  also  be 
seen  but  opinion  is  divided  as  to  whether 
they  can  be  detected  in  the  living  nerve 
cells  of  vertebrates   (Cowdry,  p.  393). 

None  of  the  techniques  for  neuro- 
fibrils are  really  satisfactory,  but,  with 
patience,  fairly  good  results  can  be 
secured  of  adult  nerve  cells  by  the 
following  modification  (Cowdry,  E.  V. 
Internat.  Monatssch.  f.  Anat.  u  Phy- 
siol., 1912,  29, 1-32)  of  Cajal's  technique. 
Fix  pieces  not  more  than  2  mm.  thick 
in  Carnoy's  6:3:1  fluid  2-6  hrs.  Wash 
in  aq.  dest.  24  hrs.  1.5%  aq.  silver 
nitrate  at  39 °C.  for  3  days  with  one 
change.  Rinse  in  aq.  dest.  and  reduce 
in  pyrogallic  acid  1  gm.;  aq.  dest.,  100 
cc;  formalin  5  cc.  in  the  dark,  24  hrs. 
Wash  in  aq.  dest.  1  hr.  Dehydrate  1 
hr.  in  95%;  2-4  hrs.  in  abs.  changed 
twice;  clear  in  cedar  oil,  2  hrs.;  imbed 
in  paraffin  2  hrs.  Rinse  deparaffinised 
sections  in  aq.  dest.  0.1%  aq.  gold 
chloride  neutralized  with  lithium  car- 
bonate 2  hrs.  The  sections  take  a  dark 
purple  black  color.  5%  aq.  sodium 
hyposulphite  5  min.  to  bleach  out 
excess  of  silver.  Rinse  in  aq.  dest. 
dehydrate,  clear  in  toluol  and  mount  in 
balsam. 

The  neurofibrils  are  exaggerated  op- 
tically by  their  sharp  blue  black  stain 
in  a  colorless  background.  Moreover 
they  form  centers  for  the  deposit  of 
silver  which  probably  increases  their 
bulk.  The  Nissl  bodies  can  be  brought 
out  by  staining  in  the  usual  way  with 
toluidin  blue  after  washing  in  aq.  dest. 
following  treatment  of  the  sections  with 
sodium  hyposulphite.  The  essential 
step  in  this  technique  is  the  impregna- 
tion with  silver.  Consequently  the 
time  in  the  silver  solution  should  be 
varied  and  perhaps  its  concentration 
likewise.  To  obtain  a  good  preparation 
without  many  trials  is  not  to  be  ex- 
pected. 

Silver  techniques  for  neurofibrils  are 
legion.  A  book  has  been  written  on 
the  subject  (Cajal,  S.  R.  and  deCastro, 
F.,  Elementos  de  Tecnica  micrografica 
del  sistema  nerviosa.  Madrid,  1933). 
Special  methods  are  advised  for  different 
parts  of  the  nervous  system  and  for 
animals  of  different  sorts  and  ages.  A 
very  useful  synopsis  is  given  by  Addi- 
son (McClung,  1950,  p.  364).  See  also 
Seki,  M.,  Ztschr.  f.  Zellf.  u.  Mikr. 
Anat.,  1939-40,  30,  548-566. 
Neuroglia.  This  is  the  connective  tissue 
of  the  nervous  system.  Like  that  of  the 
rest  of  the  body  it  consists  of  cells,  fibers 
(or  fibrils  as  they  are  called)  and  inter- 
cellular substance.     The  last  named  is 


inconspicuous  and  little  known.  The 
Neuroglia  Fibrils  are  considered  sepa- 
rately. The  cells  are  of  three  principal 
sorts:  (1)  microgliocytes  of  mesenchy- 
raatous  origin.  These  may  be  resting 
and  extend  long,  delicate  processes  or 
they  may  be  ameboid  in  which  case 
they  look  something  like  lymphocytes 
being  usually  identifiable  by  intensely 
staining  nuclei.  (2)  astrocytes  (star 
cells)  and  (3)  oligodendrocytes  (little 
tree  cells)  both  of  ectodermal  origin. 
A  tabular  comparison  of  the  three  is 
given  in  Cowdry's  Histology,  p.  406. 
No  neuroglial  cells  possess  Nissl  bodies. 
See  Cajal's  Brom-Formol-Silver 
Method,  the  Phosphotungstic  Acid 
Hematoxylin  method  of  Mallory,  Weil 
and  Davenport's  silver  methods  given 
under  Microglia  and  Oligodendroglia 
and  Alzheimer's  Modification  of  Mann's 
eosin-methyl  blue  method.  See,  also 
Silver  Diaminohydroxide  after  sensitiz- 
ing with  sodium  sulfite. 

Neurones,  see  Nerve  Cells. 

Neurosecretory  Cells.  A  good  deal  has  been 
written  on  the  subject.  The  most 
recent  data  on  location  in  nervous 
system  and  methods  are  provided  by 
Scharrer,  E.,  J.  Comp.  Neurol.,  1941, 
74,   87-92;   Scharrer,    B.,   ibid,   93-130. 

Neutral  Fats.  These  are  glycerides  of 
fatty  acids.  See  Lipids,  examination 
of  with  polarized  light.  Colored  rose 
red  by  Nile  Blue  Sulphate.  See  Sudan 
Stains,    Osmic   Acid  and   Oil   Red   O. 

Neutral  Gentian  (Bensley,  R.  R.  Am.  J. 
Anat.,  1911,  12,  297-388).  This  gives  a 
very  fine  deep  violet  coloration  of  secre- 
tion antecedents  of  serous  (or  zymo- 
genic) cells.  It  has  been  used  particu- 
larly for  the  pancreas  and  the  stomach. 
Neutral  gentian  is  the  neutral  dye 
obtained  when  aq.  gentian  violet 
(crystal  violet)  is  precipitated  by  its 
equivalent  of  aq.  orange  G  which  is 
added  slowly  and  the  mixture  agitated. 
Use  solutions  almost  but  not  quite  satu- 
rated. If  the  right  amount  of  orange  G 
solution  is  added  almost  complete 
precipitation  is  obtained.  If  too  much 
is  added  the  precipitate  is  dissolved  in 
which  case  add  more  gentian  violet. 
Excess  of  orange  G  can  be  detected  by 
the  production  of  a  yellow  ring  of  stain 
about  a  violet  center  when  a  drop  of  the 
solution  with  the  precipitate  is  touched 
to  a  piece  of  filter  paper.  When  satisfied 
that  ppt.  is  maximal,  filter;  and  dissolve 
dried  ppt.  in  20%  ale.  until  "color  of  a 
good  haemalum  solution  is  obtained". 
Allow  the  solution  to  stand  24  hrs. 
before  use. 

Fixatives:  Several  are  advised.  (1) 
Equal  parts  sat.  ale.  mercuric  chloride 
and   2.5%   aq.    potassium   bichromate. 


NEUTRAL  RED 


227  NEUTRAL  RED  AND  JANUS  GREEN 


(2)  Potassium  bichromate  2.5  ems.; 
mercuric  chloride,  5  gms.;  aq.  dest., 
100  cc.  (3)  Zenker's  fluid  less  acetic 
90  cc,  neutral  formalin  10  cc.  or  (4) 
2%  osmic  acid  2  cc;  2.5%  potassium 
bichromate  8  cc. ;  glacial  acetic  acid  1 
drop.  In  the  case  of  the  last  the  paraf- 
fin sections  are  treated  with  1%  aq. 
potassium  permanganate  1  min.;  5% 
aq.  oxalic  acid  1  min.  and  are  washed 
thoroughly  in  water  before  staining. 
Stain  4/i  sections  24  hrs.  Blot  with 
several  layers  filter  paper.  Dehydrate 
in  acetone.  Place  in  toluol.  Dif- 
ferentiate in  1  part  abs.  ale.  and  3  parts 
oil  of  cloves.  Wash  in  toluol  and  mount 
in  balsam.  Zymogen  granules,  purple; 
cytoplasm  and  nucleus,  yellow;  chromo- 
phile  material,  lavender. 
Neutral  Red  (CI,  825)— toluylene  red— This 
weakly  basic  amino-azin  dye  is  used  for 
many  purposes .  It  is  a  chloride.  Some 
advocate  the  iodide  as  more  easily 
purified  but  neutral  red  sold  by  any 
reliable  manufacturer  is  satisfactory. 
Vital  neutral  red  is  recommended  by 
Conn.  The  principal  uses  of  neutral 
red  are  to  stain: 

1.  Islets  of  Langerhans  of  the  pancreas 
(Bensley,  R.  R.,  Am.  J.  Anat.,  1911, 
12,  297-388).  Ad.d  2  cc.  of  a  previously 
prepared  1%  aq.  neutral  red  to  300  cc. 
physiological  salt  solution  (0.85%  NaCl) 
thus  making  a  concentration  of  neutral 
red  of  1:15,000.  Place  this,  and  as 
much  more  as  may  be  required  in  a 
bottle  from  the  bottom  of  which  a  glass 
tube  leads  off,  or  in  an  ordinary  bottle 
with  a  bent  glass  tube  to  serve  as  a 
siphon.  The  tube  is  connected  with  a 
glass  cannula  by  about  5  feet  of  rubber 
tubing.  A  freshly  killed  guinea  pig  is 
bled  from  the  throat.  Insert  the  can- 
nula in  the  thoracic  aorta  and  inject 
the  solution  by  raising  the  bottle  to 
a  height  of  4  or  5  feet.  Expose  the 
pancreas.  Cut  the  inferior  vena  cava 
near  the  heart  so  that  the  blood,  followed 
by  the  solution,  can  easily  escape.  The 
pancreas  will  take  on  a  deep  rose  red 
color.  Remove  pieces,  mount  in  phys- 
iological salt  solution  under  cover  glasses 
and  examine  at  low  magnification.  The 
optimum  depth  of  staining  must  be 
determined  experimentally.  The  islets 
of  Langerhans  appear  as  deep  yellow 
red  irregular  masses  of  difi'erent  sizes 
in  a  pale  red  background.  After  a  time 
the  aye  is  bleached  from  the  background 
and  the  islets  become  more  sharply 
stained. 

A  wonderfully  fine  color  contrast  can 
be  secured  when  methylene  blue  is 
added  to  the  neutral  red  solution  in  a 
concentration  of  1:10,000  and  both  are 
injected  in  the  same  way.    The  islets 


are  stained  yellow  red  and  the  ducts 
blue.  But  it  is  desirable  first  to  obtain 
satisfactory  results  with  the  methylene 
blue  alone. 

2.  Parietal  cells  in  the  stomach, 
(Harvey,  B.  C.  H.  and  Bensley,  R.  R., 
Biol.  Bull.,  1912,  23,  225-249).  These 
are  beautifully  stained  by  injection 
with  neutral   red  as  described  above. 

3.  Granules  in  blood  cells.  Touch  a 
drop  of  fresh  blood  to  a  little  1:15,000 
neutral  red  on  a  slide  and  cover  imme- 
diately without  attempting  to  mix. 
When  the  size  of  the  drop  of  blood  and 
the  amount  of  stain  are  properly 
estimated  the  cover  glass  will  press  out 
the  fluid  into  a  thin  film  suitable  for 
examination.  The  specific  granules  of 
leucocytes  are  stained  red.  In  the 
monocytes  red  stained  granules  appear 
and  sometimes  increase  in  size.  When 
the  staining  is  fairly  intense,  or  after 
a  sufficient  interval  the  nuclei  of  the 
leucocytes  become  colored  and  also  a 
basophilic  material  in  young  reticulated 
red  blood  cells.  Simultaneous  colora- 
tion with  Neutral  Red  and  Janus  Green 
is  frequently  carried  out  by  hema- 
tologists. 

Fluorescent  X  is  a  special  type  of 
reduced  neutral  red  (Lewis,  M.  R., 
1935,  17,  96-105).  See  Nerve  Fiber 
Degeneration  and  Nissl  Bodies. 
Neutral  Red  and  Janus  Green.  These  are 
often  employed  together  as  a  supravital 
stain  for  blood  cells.  A  recent  com- 
prehensive statement  of  the  technique 
is  given  by  Cunningham  and  Tompkins 
(Downey,  pp.  555-579).  They  add  3 
drops  cone  janus  green  in  absolute 
alcohol  to  1  cc  dilute  neutral  red,  which, 
latter,  is  20-30  drops  cone  neutral  red 
in  absolute  alcohol.  This  mixture  is 
spread  evenly  on  slides  and  evaporated. 
They  caution  that  for  exudates,  tissue 
scrapings,  leucemic  blood,  bone  marrow 
and  lymph  nodes  it  is  necessary  to  use 
stronger  solutions.  Neutral  red  CC. 
(Commission  Certified)  is  satisfactory 
in  place  of  the  neutral  red-iodide  advised 
by  Sabin.  Fresh  blood  is  mounted  on 
the  dye  deposit,  and  is  ringed  with 
vaseline  to  prevent  evaporation.  This 
technique  has  had  a  profound  influence 
on  cytology.  Obviously  it  must  be 
cautiously  used  and  observations  dis- 
continued as  soon  as  evidences  are  seen 
of  experimental  modifications  in  the 
cells.  It  affords  valuable  information 
on  the  mitochondria  and  neutral  red 
granules  not  stainable  together  by  other 
methods,  but  it  will  not  supplant  the 
staining  of  blood  smears  by  the  methods 
of  Gierasa,  Wright  and  others.  See 
critical  evaluation  by  Hall  (Downey, 
pp.  643-698).    See  application  in  studiy 


NEUTRAL  RED  IODIDE 


228 


NEW  METHYLENE  BLUE 


of  lymphosarcoma ta  (Hu,  C.  H.  and 
Pai,  H.  C,  Arch.  Path.,  1942,  34,  106- 
116). 

Neutral  Red  Iodide.  This  is  a  special  form 
of  neutral  red  prepared  by  Phillips, 
M.  and  Cohen,  B.,  Stain  Techn.,  1927, 
2,  17-18  and  recommended  by  Sabin 
for  the  Neutral  Red  Janus  Green 
method. 

Neutral  Safranin,  or  Safranin-acid  violet 
(Bensley,  R.  R.,  Am.  J.  Anat.,  1911, 
12,  297-388).  Make  the  neutral  dye 
by  precipitating  sat.  aq.  safranin  O 
with  sat.  aq.  acid  violet.  The  latter  is 
added  slowly  and  the  mixture  is  agitated 
gently.  The  precipitation  should  be 
complete  so  that  when  it  settles  the 
supernatant  fluid  is  of  a  faintly  violet 
color.  Filter  and  dissolve  dried  ppt. 
in  abs.  ale.  Dilute  this  stock  solution 
with  equal  vol.  aq.  dest.  allow  to  stain 
30  min.  before  use.  Stain  sections, 
fixed  as  described  under  Neutral  Gen- 
tian, in  the  same  way  as  with  neutral 
gentian.  Nuclei  are  colored  with  safra- 
nin and  secretion  antecedents  with  the 
acid  violet.  The  method  has  been  used 
chiefly  for  the  pancreas  but  it  gives  fine 
coloration  of  nerve  as  well  as  gland  cells. 
Unfortunately  the  colors  are  not  very 
permanent. 

Neutral  Stains.  As  explained  by  the 
Bensleys  (p.  65)  acid  and  basic  dyes 
are  mutually  antagonistic.  One  will 
extract  the  other  from  a  section.  This 
can  be  overcome  by  having  them  react 
on  each  other  to  form  a  molecularly 
balanced  neutral  compound  insoluble 
in  pure  water  and  which  must  therefore 
be  employed  in  alcoholic  solution. 
Because  the  staining  depends  upon  the 
hydrolytic  splitting  of  the  compound 
they  must  be  applied  at  maximum  con- 
centration of  water  consistent  with 
retaining  the  dye  in  solution.  It  is  on 
account  of  the  necessity  for  dilution 
with  water  to  promote  dissociation  that 
water  is  added  to  Wright's  blood  stain 
on  the  slide.  These  neutral  dyes  are 
of  particular  value  in  the  staining  of 
secretion  antecedents  by  R.  R.  Bensley 
and  his  followers,  see  Neutral  Gentian 
(gentian  violet-orange  G),  Neutral 
Safranin  (safranin-acid  violet).  Crystal 
Violet-Acid  Fuchsin  and  Bowie's  Stain. 

Neutrophile  Leucocyte  (finely  granular 
leucocyte,  polymorphonuclear  leuco- 
cyte). Most  numerous  granular  leuco- 
cyte, percentage  55-75;  slightly  smaller 
(9-12m)  than  eosinophile;  nucleus  lo- 
bated,  usually  also  filamented,  stains 
deeply;  specific  granules,  refractile, 
neutrophilic,  small,  uniform  and 
numerous;  highly  motile  and  phago- 
cytic. Special  methods  for  their  study 
are    far    too    numerous    even    to    list. 


The  so-called  toxic  neutrophiles  in 
certain  pathological  states  differ  from 
normal  ones  in  the  staining  of  nuclei 
and  specific  granules  (Mommsen,  H., 
Ztschr.  exper.  Med.,  1929,  65,  299). 
A  comprehensive  account  of  neutro- 
philes is  provided  by  Bunting,  C.  H. 
m  Downey's  Hematology,  1938,  1, 
160-177.  Because  these  cells  normally 
constitute  by  far  the  majority  of  leuco- 
cytes in  the  circulating  blood,  chemical 
analyses  of  total  leucocytes  separated 
from  the  erythrocytes  relate  chiefly  to 
them.  The  most  convenient  way  is  to 
mix  fresh  blood  with  Anticoagulant, 
centrifuge  and  take  the  so-called  buffy 
layer.  For  lipid  analysis  of  such 
material,  see  Boyd,  E.  M.,  Arch.  Path., 
1936,  21,  739-748.  Another  useful 
method,  described  by  Haan  and  em- 
ployed by  Barnes,  J.  M.,  Brit.  J.  Exp. 
Path.,  1940,  21,  264-275,  which  works 
nicely  with  the  rabbit  but  poorly  with 
the  cat,  is  to  inject  intraperitoneally 
200-300  cc.  warm  sterile  saline  solution 
and  4  hrs.  later  to  withdraw  fluid  with  a 
cannula  into  5  cc.  4%  sodium  citrate. 
This  fluid  contains  95-98%  neutrophiles. 
Barnes  has  outlined  methods  for  de- 
termination of  Cathepsin,  Nuclease, 
Amylase,  Lipase,  Lysozyme  and  Adeno- 
inase.  Since  it  is  possible  now  to  break 
up  cells  and  to  collect  by  centrifugation 
masses  of  Mitochondria  and  Nuclei, 
it  should  be  feasible  to  collect  and 
similarly  to  analyse  the  neutrophilic 
granulations.  For  technique  of  meas- 
uring motility,  chemotaxis  and  other 
properties,  see  Leucocytes. 

Neutrophilic,  see  Staining. 

Nevillite  V  and  No.  1  have  been  compared 
with  gum  damar  and  Canada  balsam  as 
mounting  media  by  Groat  (R.  H., 
Anat.  Rec,  1939,  74,  1-6).  Both  are 
clean,  colorless,  inert  and  neutral. 
He  recommends  a  60%  solution  of 
either  V  or  No.  1  in  toluol. 

New  Blue  R,  see  Naphthol  Blue  R. 

New  Fuchsin  (Magenta  III)  (CI,  678)— 
fuchsin  NB,  isorubin — It  is  triamino- 
tritolyl-methane  chloride.  This  new 
fuchsin  is  sometimes  specified  for 
staining  of  acid  fast  bacilli. 

New  Methylene  Blue.  The  Colour  Index 
lists  several  dyes  by  this  name  of  which 
2  deserve  mention:  (1)  GG  (CI,  911) 
is  recommended  by  the  Bensleys  (p. 
16)  as  a  supravital  stain  for  mast  cells 
and  for  the  thyroid  because  of  its  meta- 
chromatic capacity.  (2)  N  (CI,  927)— 
methylene  blue  NN— Conn  (p.  88) 
says  that  it  may  be  of  some  value  though 
it  is  practically  never  used  in  micro- 
scopical work.  Cowdry  tried  it  and 
found  that  it  had  no  particular  ad- 
vantages. 


NEW  PINK 


229 


NINHYDRIN  REACTION 


New  Pink,  see  Phloxine. 

New  Ponceau  4R,  see  Ponceau  2R. 

New  Victoria  Blue  B  or  R,  see  Victoria 
Blue  R. 

New  Victoria  Green  Extra  O,  I  or  II,  see 
Malachite  Green. 

Niagara  Blue  3B,  see  Trypan  Blue. 

Niagara  Blue  4B  (CI,  520) — benzo  sky  blue, 
direct  sky  blue,  pontamine  sky  blue 
5BX — A  disazo  dye,  see  Varrelman, 
F.  A.,  Stain  Techn.,  1938,  13,  115-119. 
Niagara  blue  2B  (N.A.C.)  is  the  Ameri- 
can prototype  of  trypan  blue  for  which 
it  can  be  substituted  (Foot,  McClung, 
p.  115). 

Niagara  Sky  Blue  6  B  (CI,  518),  a  direct  di- 
sazo dye  of  light  fastness  3.  Instruc- 
tions for  employing  this  useful  stain  in 
the  examination  of  plant  and  animal 
tissues  are  given  (Emig,  p.  41). 

Nickel.  The  microchemical  technique  of 
Cretin  and  Pouyanne  (A.,  and  L., 
Bordeaux  chirurgical,  1933,  4,  321-364) 
employed  in  a  study  of  the  influence  of 
metals  on  bone  deposition,  as  given  by 
Lison  (p.  102),  is:  Fix  in  formol,  30  cc, 
"s6rum  physiologique",  100  cc,  ana 
ammonium  hydrosulphate  5  drops.  Im- 
merse in  a  solution  of  ammonium 
phosphate  in  order  to  produce  the 
insoluble  double  salt:  NHiNiPO^  + 
6H2O.  Decalcify.  In  the  sections  stain 
the  nickel  by  an  alcoholic  solution  of 
pure  hematoxylin  which  forms  a  lilac 
colored  nickel  lake  appearing  blue  when 
very  thick  (Lison,  p.  102). 

Nicotinic  Acid.  Preliminary  detection  of  it 
or  its  amide  by  fluoresence  microscopy 
(Hirt,  A.  and  Wimmer,  K.,  Klin.  Woch- 
nesdir.,  1939,  18,  765-767).  Lasting 
yellow  fluorescence.  See  Vitamin  B 
complex. 

Night  Blue  (CI,  731),  a  basic  dye  of  light 
fastness  4  gives  beautiful  blue-violet 
coloration  of  plant  tissues  but  fades 
(Emig,  p.  52). 

Nigrosin,  water  soluble  (CI,  865) — gray 
R,  B,  BB,  indulin  black,  silver  gray, 
steel  gray — Commission  Certified.  This 
is  a  mixture.  It  has  been  used  as  a 
counterstain  for  neutral  red  in  colora- 
tion of  Nissl  bodies  by  Bean,  R.  J., 
Stain  Techn.,  1927,  2,  56-59,  as  a  nega- 
tive stain  for  bacteria,  Treponema,  etc. 
See  Picro-Nigrosin. 

Nile  Blue  A,  see  Nile  Blue  Sulphate. 

Nile  Blue  Sulphate  (C  1.  913)— Nile  Blue  A 
— This  is  an  important  oxazin  dye  for 
which  purity  tests  have  been  estab- 
lished (Conn,  p.  270).  It  was  intro- 
duced by  Lorrain  Smith  as  a  fat  stain. 
Briefly  the  method  is  to  stain  fresh 
tissues,  or  frozen  sections  of  formalin 
fixed  tissues,  for  10-20  min.  in  a  cone, 
aq.  solution  of  Nile  blue  sulphate,  to 
differentiate    in    1%    aq.    acetic    acid, 


wash  in  water  and  mount  in  glycerin. 
He  thought  that  the  neutral  fais  {glycer- 
ides)  were  thereby  colored  red  and  the 
fatty  acids  blue,  but  Kaufmann  and 
Lehmann  (C.  and  E.,  Virchow's  Arch, 
f.  Path.  Anat.  und  Physiol.,  1926,  261, 
623-648)  came  to  the  conclusion  that  the 
method  was  valueless.  However  Lisson 
(p.  202)  was  unimpressed  by  their 
evidence.  In  his  opinion  the  rose  (or 
red)  color  does  signify  the  presence  of 
a  nonsaturated  glyceride  whereas  the 
blue  color  is  of  no  significance  because  of 
its  lack  of  specificity.  He  reported 
that  some  mixtures  of  free  fatty  acids 
remain  uncolored;  for  those  containing 
saturated  fatty  acids  non -coloration  is 
the  rule;  while  some  others,  not  con- 
taining fatty  acids,  are  colored.  See 
Lipids,  tabular  analysis. 

Stone,  L.  S.,  Anat.  Rec,  1931,  51, 
267-273  has  advanced  a  technique  for 
the  preservation  of  supravital  staining 
with  Nile  blue  sulphate  the  essential 
feature  of  which  is  repeated  treatment 
with  phosphomolybdic  acid.  Zenker's 
fluid  with  acetic,  2  hrs.  Running  tap 
water,  1  hr.  1%  aq.  phosphomolybdic 
acid,  2  hrs.  Dehydrate  in  50,  70,  80,  95 
and  abs.  ale.  each  containing  0.1% 
phosphomolybdic  acid,  30  min.  each. 
Clear  in  equal  parts  0.1%  phospho- 
molybdic acid  in  abs.  ale.  and  cedar 
wood  oil,  30  min.  Then  pure  cedar  wood 
oil  over  night.  Embed  in  paraffin  3 
changes  15-20  min.  each.  Counterstain 
sections  in  stain  desired  applied  in  abs. 
ale.  containing  0.1%  phosphomolybdic 
acid.  Mount  in  damar.  Balsam  will  do. 

Nile  Pink,  fat  stain  prepared  from  nile 
blue  sulphate  by  boiling  with  dilute 
sulphuric  acid  (Rettie,  T.,  J.  Path.  & 
Bact.,  1931,  34,  595-596). 

Ninhydrin  Reaction.  Berg's  (W.,  Pfluger's 
Arch.,  1926,  214,  243-249)  directions: 
Fix  tissues  in  10%  formalin,  wash  in 
water.  Boil  section  for  1  min.  in  2  cc. 
0.2%  ninhydrin.  Wash,  mount  in  glyc- 
erin or  glycerin  jelly.  Amino  acids, 
polypeptides  and  proteins  blue  or  violet. 
Romieu  (M.,  Bull.  d'Hist.  AppL,  1925, 
2,  185-191)  employs  a  strong  solution 
heated  less.  See  Giroud  (A.,  Proto- 
plasma,  1929,  7,  72-98). 

Details  are  given  by  Serra,  J.  A., 
Stain  Techn.,  1946,  21.  5-18.  He  ad- 
vises that  the  tissue  first  be  hardened 
by  fixation  for  an  unspecified  time  in  2 
parts  96%  alcohol  and  1  part  commercial 
formalin  (40%  formaldehyde)  plus 
"some  drops"  of  glacial  acetic  acid  in 
10  cc.  of  the  mixture.  After  this  it  is 
well  washed  in  running  water  and  in  aq. 
dest.  before  the  frozen  sections  are 
made.  He  also  gives  a  method  for 
paraffin  sections. 


NIPPLE  SECRETION  SMEARS 


230 


NISSL  BODIES 


The  reaction  consists  of  immersing 
the  sections  or  fresh  materials  in  equal 
volumes  of  0.4%  aq.  triketo-hydrinden- 
hydrate  (ninhydrin)  and  phosphate 
buffer  pH  6.98.  The  ninhydrin  solution 
must  be  freshly  prepared  and  the  phos- 
phate buffer  not  too  concentrated.  For 
the  latter  he  suggests  6  cc.  M/15  solu- 
tion secondary  sodium  phosphate 
(11.1876  gm.  Na2HP04-2H20  per  liter) 
and  4  cc.  M/15  primary  potassium  phos- 
phate (9.078  gm.  KH2PO4  per  liter). 
The  reaction  is  carried  outin  a  covered 
glass  container  placed  on  a  boiling 
water  bath.  This  is  allowed  to  stand 
1-2  min.  in  the  vapor  after  it  has 
reached  the  boiling  point.  A  blue,  or 
violet,  color  developing  while  hot  or 
after  cooling  indicates  the  presence  of 
amino  acids,  fre,  or  bound  in  peptides, 
or  proteins. 

For  microscopic  examination  mount 
in  pure  glycerin  squeezing  if  necessary. 
The  edges  can  be  cemented  by  using  a 
mixture  of  80  gm.  collophonium  and 
20  gms.  heated  lanolin  as  recommended 
by  Romeis  but  they  must  be  studied  the 
same  day  for  the  color  fades  quickly. 

Serra  carefully  states  that  the  reac- 
tion is  given,  not  only  by  all  amino 
acids  except  proline  and  hydroxypro- 
line,  by  peptides  and  proteins  but  also 
by  other  compounds  such  as  amines, 
aldehydes,  sugars  with  free  aldehyde  or 
keto  groups  and  by  ammonia  and  am- 
monium salts.  "However,  with  com- 
pounds other  than  amino  acids  and 
proteides,  the  reaction  is  much  less 
sensitive  and  sometimes  it  gives  a  more 
reddish  color.  In  general  it  is  easy  to 
exclude  the  possibility  of  these  com- 
pounds being  present,  by  their  solubil- 
ity and  localization.  It  must  also  be 
remembered  that  the  intensity  of  the 
ninhydrin  reaction  varies  according  to 
the  nature  of  the  amino  acid  and  the 
binding  of  this  in  the  peptides. 

"The  coloring  formed  during  the 
reaction  can  diffuse  and  be  absorbed 
by  several  cell  structures.  This  com- 
monly happens  when  the  heating  is 
exaggerated  and  when  compounds  easily 
soluble  are  present,  for  instance  after  a 
weak  fixation.  It  is,  therefore,  recom- 
mended to  employ  fixatives  which 
harden  the  tissues,  as  we  have  said 
above.  To  be  sure  that  a  secondary  im- 
pregnation or  adsorption  of  the  coloring 
has  not  taken  place,  the  following  test 
may  be  executed :  A  small  weight  (some 
milligrams)  of  a  pure  amino  acid,  such 
as  glycine,  is  dissolved  in  distilled 
water;  an  equal  volume  of  phosphate 
buffer  of  pH  6.98  and  a  few  drops  of  0.4% 
ninhydrin  solution  are  added;  it  is 
boiled    slowly    and    cooled    for    20-30 


minutes.  The  ninhydrin  employed 
must  be  completely  consumed — by  addi- 
tion of  more  amino  acid  solution.  The 
colored  liquid  of  this  reaction  is  now 
used  to  immerse  the  pieces,  with  boiling, 
etc.,  as  for  a  ninhydrin  reaction.  If 
then  a  certain  structure  shows  a  colora- 
tion, this  means  that  an  absorption  or 
adsorption  has  taken  place  and  a  posi- 
tive ninhydrin  reaction  in  the  same 
structure  does  not  necessarily  demon- 
strate a  proteic  or  amino  acid  nature." 

Nipple  Secretion  Smears,  see  Papanicolaou 
Techniques. 

Nissl  Bodies  (Tigroid  bodies,  chromophile 
granules,  chromidia,  etc.)  are  masses 
of  basophilic  material  easily  demon- 
strable in  the  cytoplasm  of  most  nerve 
cells  after  a  wide  variety  of  fixations. 
Certain  types  of  nerve  cells  are  char- 
acterized by  the  shape,  number,  size 
and  distribution  of  their  Nissl  bodies. 
Since,  moreover,  the  Nissl  bodies  ap- 
pear at  a  definite  stage  in  the  develop- 
ment of  the  cells  and  undergo  distinctive 
modifications  in  physiological  and  path- 
ological conditions  there  can  be  no 
question  that  they  represent  material 
present  in  vivo  although  they  cannot 
be  distinguished  as  such  in  living  nerve 
cells.  Bensley,  R.  R.  and  Gersh,  I., 
Anat.  Rec,  1933,  47,  217-237  claim  that 
their  discovery  of  well-formed  Nissl 
bodies,  stainable  with  toluidin  blue,  in 
sections  of  tissues  frozen  in  liquid  air 
and  dehydrated  in  vacuo  while  still 
frozen  is  proof  of  the  presence  of  Nissl 
bodies  in  the  living  state.  Wiemann, 
W.,  Zeit.  f.  d.  ges.  Neurol,  u.  Psychiat., 
1925,  98,  347-404  appears  to  have  made 
ultraviolet  photomicrographs  of  Nissl 
bodies,  and  a  dense  ash,  revealed  by 
microincineration  (Scott,  G.  H.,  Proc. 
Soc.  Exp.  Biol.  &  Med.,  1940,  44,  397- 
398),  corresponds  with  them  topo- 
graphically. 

The  influence  of  fixation  on  the  shape 
(and  perhaps  to  a  slight  degree  on  the 
distribution)  of  Nissl  bodies  in  nerve 
cells  has  never  been  clearly  defined. 
It  is  known  that  the  Nissl  bodies  are 
much  more  pronounced  after  fixation  in 
95%  alcohol,  Zenker's  fluid  and  Car- 
ney's fluid  than  they  are  after  fixation 
in  osmic  acid,  Altmann's  fluid  and 
Regaud's  fluid.  Fixatives  of  the  first 
group  also  result  in  more  stainable 
particles  in  the  nucleoplasm  than  those 
of  the  second.  For  other  details  see 
Hopkins,  A.  E.,  Anat.  Rec,  1924,  28, 
157-163.  Influence  of  staining  is  also 
a  factor  to  be  reckoned  with  because 
of  the  striking  difference  in  appearance 
of  Nissl  bodies  when  intensely  and 
lightly  colored.  There  are  many 
methods  from  which  to  make  a  choice. 


NITRATES 


231 


NITROPRUSSIDE  REACTION 


Some  of  these  are  given  under  Gallo- 
cyanin,  Gallamin  Blue  and  Carbol- 
Fuchsin.  See  also  the  methods  of 
Huber,  Johnson  and  King  and  buffered 
thionin  (Windle,  W.  V.,  Rhines,  R.  and 
Rankin,  J.,  Stain  Techn.,  19-13,  18,  77- 
86).  An  apparatus  has  been  devised 
apparently  suitable  for  obtaining  the 
Absorption  Spectra  of  Nissl  bodies. 

Nitrates.  Atake  frozen  sections  of  fresh 
tissues.  Cover  section  on  a  slide  with 
1-2  drops  hot  10%  "Nitron"  in  5%  aq. 
acetic  acid.  Place  in  refrigerator  30 
min.  to  permit  nitrates  to  crystallize 
and  e.xamine  in  polarized  light.  Nitron 
is  1 , 4-Diphenyl-3 , 5-endanilo-dihydro- 
triazol.  It  precipitates  nitrates  as  in- 
soluble salts  (Cramer,  G.,  Zbl.  allg. 
Path.,    1940,  74,   241-244). 

Nitrazine — nitrazine  yellow,  delta  dye  in- 
dicator— An  acid  mono-azo  dye  sug- 
gested as  substitute  for  ponceau  de 
xylidine  in  Masson's  Trichrome  Stain. 

Nitrazine  Yellow,  see  Nitrazine. 

Nitrocellulose  for  imbedding.  Low 
viscosity  nitrocellulose  ("Hercules  Pow- 
der Co.'s  R.S.  0.5  second  nitro- 
cellulose") does  not  require  to  be  washed 
as  in  the  case  of  celloidin.  First  add 
absolute  alcohol,  break  up  lumps  and 
add  ether.  Use  100  gms.  nitrocellulose, 
100  cc.  absolute  alcohol  and  140  cc. 
anhydrous  ether.  For  evaporation  a 
large  surface  is  required  in  proportion 
to  depth.  A  precision  microtome  is 
needed  for  sectioning  blocks  after  first 
hardening  in  70-80%  alcohol.  Blocks 
are  cut  both  dry  and  wet.  Serial 
sections  4  microns  thick  are  obtainable 
whereas  in  celloidin  the  minimum  is 
about  12  microns.  Since  low  viscosity 
nitrocellulose  (L.V.N.)  is  more  readily 
dissolved  than  celloidin  by  absolute 
alcohol  the  use  of  butyl  alcohol  between 
95%  alcohol  and  xylol  is  suggested 
(Davenport,  H.  H.  and  Swank,  R.  L., 
Stain  Techn.,  1934,  9,  137-140). 

Nitro  Dyes.  Chromophore-N02.  All 
strongly  acid.  Aurantia,  martius  yel- 
low, picric  acid. 

Nitro  Reaction  to  distinguish  between  pyr- 
rols and  indols.  Treat  preparation  with 
a  mixture  of  sulphuric  and  nitric  acids 
(equal  parts).  Substances  containing 
the  benzene  ring  (and  among  them 
indol  compounds)  are  nitrified  and 
recognizable  by  their  canary  yellow 
color  whereas  the  pyrrols  are  not 
nitrified  (Lison,  p.  162).  See  Lison, 
L.,  J.  Physiol,  et  Path.  G6n.,  1933,  31, 
82-99). 

Nitrogen.  The  titrimetric  method  of 
Bruel,  D.,  Holter,  H.,  Linderstr0m- 
Lang,  K.  and  Rozlts,  K.,  C.  rend.  trav. 
lab.  Carlsberg,  S^r.  chim.,  1946,  25, 
289-324  is  recommended. 


Nltroprusside    Reaction    for    Glutathione. 

1.  IVIattei  and  Dulzetto  (Atti.  e.  rend, 
della  Accad.  dei  Lincei,  1928,  8,  317). 
Fix  in  20%  trichloracetic  acid.  Treat 
frozen  sections  3-4  min.  with  a  fresh 
solution  of  sodium  nitroprussiate.  After 
quickly  drying  expose  to  NII3  vapor. 
Freeze  solidly  with  ice  or  solid  CO2. 
Examine  frozen  on  slide  at  5°C.  The 
violet  color  of  sulphydryl  rapidly  disap- 
pears. 

2.  Joyet-Lavergne  (Ph.,  Bull.  d'Hist., 
1928,  5,  331-349)  Method  1 :  apply  to 
tissue  1  drop  5%  aq.  sodium  nitroprus- 
siate, then  1  drop  ammonia  and  examine 
immediately.  Method  2:  before  apply- 
ing reagent  as  above  he  uses  a  stimulant 
10%  aq.  potassium  cyanide,  5  min.;  or 
2%  aq.  sodium  sulphite,  10  min.,  or  sat. 
ammonium  sulphate,  15  min.,  or  tri- 
chloracetic acid,  2-5  min.  Method  3  for 
fixed  tissues:  fix  several  hours  in  abs. 
ale.  or  in  formol  15  cc.  +  physiological 
saline  sol.  75  cc.  Tease  tissue  or  make 
frozen  sections.  Stimulate  with  potas- 
sium cyanide  or  ammonium  sulphate. 
Then  apply  reagent. 

3.  Giroud  and  Bulliard  (A.  and  H., 
Protoplasma,  1933,  19,  381-384).  Apply 
to  fresh  teased  tissues  or  frozen  sections 
10%  aq.  sodium  nitroprussiate  alka- 
linized  by  about  2%  ammonia.  Fix  the 
color  by  treatment  for  several  seconds 
with  5%  aq.  zinc  acetate.  Dehydrate, 
clear  and  mount  in  balsam  in  the  usual 
way.  The  violet  color  becomes  red  but 
lasts  some  time  especially  if  kept  in  ice 
box.  The  same  technique  is  possible 
after  alcohol  fixation. 

Lison  (p.  135)  has  considered  the  spec- 
ificity of  these  reactions  and  recom- 
mends analysis  given  in  an  article  by 
Rapkine  contained  in  the  last  edition 
of  Langeron's  Precis  de  Microscopie. 
For  fresh  tissues  (pieces,  smears,  frozen 
sections)  (a)  Glutathione  reduced.  Add 
to  tissue  on  slide  1  drop  5%  sodium 
nltroprusside  for  plants,  2%  for  animals. 
Add_  a  reinforcer  such  as  sat.  aq.  am- 
monium sulphate  or  crystals,  then  drop 
of  ammonia.  Red  or  violet  color,  (b) 
Glutathione  total.  Treat  tissue  with  10% 
cyanide  of  potassium,  5-10  min.  Then 
(a),  (c)  SH  radicals  fixed  to  proteins. 
10%  trichloracetic  acid  15  min.  Wash 
in  much  water.  Repeat  several  times. 
For  fixed  tissues  avoid  employing 
absolute  alcohol  or  trichloracetic  acid. 
Use  instead  formol-saline  (above). 
Then  follow  as  for  fresh  tissues.  Fix 
colors  with  zinc  acetate  as  described. 

Bourne  (G.,  Austral.  J.  Exp.  Biol.  & 
Med.  Sci.,  1935,  13,  238-249)  puts  frozen 
sections  into  hot  5%  aq.  acetic  acid 
30-90  sec. ;  drains  off  the  acid ;  adds  5% 
sodium    nltroprusside    (saturated    with 


NITROSAMINO  REACTION 


232 


NORMALITY,  MICROSCOPIC 


ammonium  sulphate)  2  min.,  then  few- 
drops  cone,  ammonium  hydroxide  which 
turns  them  purplish  blue.     For  quanti- 
tative unreliability  of  the  test  for  -SH 
and  -S.S-  see  Hammett  and  Chapman, 
(F.  S.  and  S.  S.,  J.  Lab.  &  Clin.  Med. 
1938-39,  24,  293-298).     See  Sulfhydryl 
Groups. 
Nitrosamino   Reaction   of   Lison    (p.    161) 
consists  in  transforming  the  amino  group 
present  in  pyrrol  and  indol  into  nitrosa- 
mine  by  action  of  nitric  acid;  then  by 
demonstrating  the  nitrosamine  by  the 
reaction  of  Liebermann. 
Nitroso  Dyes  (quinone  oximes).     Produced 
by  nitrous  acid  acting  on  phenolic  com- 
pounds.   Naphthol  green  B  and  Y. 
NNN  Medium,  see  Leishmania, 
Nocht's  Stain  for  malaria  plasmodia  is  de- 
scribed by  Craig,  p.  287  as  less  satis- 
factory and  more  time  consuming  than 
Wright's. 
Nonfilament-Filament  Ratio.    This  is  de- 
rived from   the   Filament-Nonfilament 
Count,    the    number   of    nonfilamented 
neutrophiles   being   multiplied   by    100 
and  divided  by  the  number  of  filamented 
ones.    See   Stiles,   M.   H.,   J.    Lab.    & 
Clin.  Med.,  1940-41,  26,  1453-1460. 
Nopalin  G,  see  Eosin  B  or  bluish. 
Normal    Solutions.     The    equivalent    of    a 
substance  (equivalent  weight,  the  gram 
equivalent)  is  the  weight  in  grams  which 
in  its  reaction  corresponds  to :  a  gram 
atom  of  hydrogen,  or  of  hydroxyl,  or  a 
univalent  ion,  or  to  half  a  gram  atom  of 
oxygen.     A    normal    solution    contains 
one  equivalent  per  liter,  a  0.05  normal 
contains  0.05  equivalent. 

Hydrochloric  acid  (HCl),  the  molecu- 
lar weight  is  H  =  1.008  -|-  CI  =  35.457 
(see  Atomic  Weights)  =  36.465.  Con- 
sequently make  up  36.465  gms.  of  HCl 
to  1  liter  with  aq.  dest.  But  it  can  not 
be  weighed  out  in  this  way.  Since 
cone,  hydrochloric  acid  (sp.  gr.  1.19) 
is  approximately  12  N,  to  make  a  normal 
solution  (approximate)  dilute  83.3  cc. 
to  1  liter  with  aq.  dest.  The  normality 
can  be  accurately  determined  by  stand- 
ardizing with  sodium  carbonate,  or  by 
titration  with  a  solution  of  sodium 
hydroxide  of  known  normality. 

Sulphuric  acid  is  H2SO4.  Molecular 
weight  calculated  in  the  same  way  is 
98.08.  But  there  are  2  replaceable 
hydrogen  atoms  so  that  in  making  a  nor- 
mal solution  the  molecular  weight  is 
divided  by  2  which  means  that  65.068 
gms.  of  H2SO4  is  to  be  made  up  to  1  liter 
with  aq.  dest.  A  cone.  sol.  (sp.  gr. 
1.84)  is  approximately  36  N.  To  make 
approximately  1  N  dilute  27.8  cc.  to 
1  liter. 

Oxalic  acid  has  the  formula  (C00H)2- 
2H2O    with    molecular    weight    of    126. 


Owing  to  presence  of  2  hydroxyl  groups 
it  has  2  hydrogen  equivalents  and  it  is 
necessary  to  divide  the  molecular  weight 
by  2  so  that  63  gms.  is  made  up  to  1  liter 
with  aq.  dest. 

The  alkali  sodium  hydroxide  (NaOH) 
has  1  hydroxyl  group,  so  that  the  molecu- 
lar weight  is  taken  without  division. 
But  with  disodium  phosphate,  the 
formula  of  which  is  Na2HP04,  the 
hydrogen  equivalent  is  ^  NajHPOi,  so 
that  the  molecular  weight  is  divided  by 
2.  Similarly  with  the  salt  Na2S04  the 
molecular  weight  is  halved .  For  sodium 
triphosphate,  Na3P04,  the  hydrogen 
equivalent  is  3  NajPO*,  or  the  molecular 
weight  is  divided  by  3. 
Normality,  Microscopic  (From  Cowdry's 
Histology,  1950).  Knowledge  of  the 
microscopic  structure  of  normal  tissues 
is  prerequisite  to  an  understanding  of 
their  microscopic  structure  in  abnormal 
conditions.  These  latter  conditions  in- 
deed are  defined  in  terms  of  the  former 
by  merely  adding  a  prefix  "differing 
from,"  the  Latin  ab.  Yet  it  is  extra- 
ordinarily difficult  to  reach  agreement 
on  what  constitutes  justification  for  the 
use  of  the  adjective  "normal."  Dis- 
cussion tends  to  drag  out  almost  end- 
lessly. 

Definition:  We  choose  here  to  advo- 
cate the  statistical  definition  of  normal- 
ity. According  to  this :  a  normal  condi- 
tion is  the  usual  one  in  members  of  a 
homogenous  group .  By  usual  is  intended 
in  the  majority,  that  is  in  any  number 
over  50  per  cent.  By  homogeneous  is 
meant  in  individuals  who  are  alike  at 
least  insofar  that  they  are  of  the  same 
race,  sex  and  age. 

Exam-pies:  The  existence  of  races, 
characterized  by  structural  differences, 
is  a  fact  to  be  reckoned  with.  What  is 
normal,  or  usual,  in  one  may  or  may  not 
be  normal  or  usual  in  another.  Physi- 
cal anthropologists  deal  with  such 
matters  but  histologists  are  also  in- 
volved because  it  is  desirable  to  explore 
microscopically  differences  that  are  de- 
tectable by  naked-eye  inspection. 
These  racial  differences  are  grafted  on 
a  basic  and  fundamental  similarity, 
for  all  races  of  mankind  belong  to  one 
and  the  same  species.  Conspicuous 
among  them  are  certain  features  of 
hair,  skin  and  body  build,  which  are 
normal  for  one  race,  but  are  not  normal 
for  others.  The  list  on  careful  study, 
may  prove  to  be  a  long  one. 

Between  the  two  sexes  there  is  like- 
wise a  fundamental  similarity.  Many 
tissues  of  males  and  females  are  indis- 
tinguishable. Some  others  show  quan- 
titative differences.  The  normal  num- 
ber of  erythrocytes  per  cubic  millimeter 


NORMALITY,  MICROSCOPIC 


233 


NORMALITY,  MICROSCOPIC 


of  blood  in  males  differs  from  that  in 
females.  And  there  are  qualitative 
differences  in  the  primary  and  second- 
ary sex  organs.  Consequently,  as  be- 
tween races,  so  also  as  between  the 
sexes  what  is  normal  for  one  is  not 
necessarily  normal  for  the  other.  One 
must  alwaj's  bear  in  mind  many  known 
differences  and  the  likelihood  of  almost 
innumerable  others. 

Though  a  basic  similarity  exists  in 
the  structure  and  function  of  the  body 
at  all  ages  in  the  life  of  a  given  indi- 
vidual, some  very  profound  differences 
clearly  obtain,  as  between  different 
ages.  What  is  normal  for  individuals 
of  the  same  race  and  sex,  at,  say,  age 
10  is  clearly  not  normal  at  age  twentj', 
or  at  age  fifty.  It  has  been  said  that 
the  boy  is  not  a  little  man  and  the 
senile  is  not  an  old  boy;  he  is  a  different 
individual  but  he  is  constructed  in  the 
same  general  pattern. 

No  assessment  of  normality  is  there- 
fore of  any  value  unless  the  group  is 
homogeneous  in  race,  sex  and  age. 
Even  this  qualification  is  only  sufficient 
for  a  few  properties  relating  to  the  body 
as  a  whole  such  as  weight,  height,  basal 
metabolism,  urinary  excretion  and  so 
on. 

The  body  is  made  up  of  so  many  parts 
that  it  is  frequently  essential  to  con- 
sider the  normality  of  each  separately 
maintaining  this  homogeneity  in  race, 
sex  and  age  of  the  persons  having  the 
parts  compared.  In  the  vascular  sys- 
tem the  coronary  artery  ages  much  more 
rapidly  than  the  radial  artery.  Struc- 
ture normal,  or  usual,  for  the  coronary 
artery  at  thirty  is  normal  for  the  radial 
at  about  sixty.  The  skin  of  the  face 
and  hands  ages  more  rapidly  than  that 
in  less  exposed  situations  as  for  example 
the  back.  What  is  normal  for  one  at 
age  sixty  is  definitely  not  normal  for 
the  other. 

To  be  on  the  safe  side  still  other  pre- 
cautions are  indicated.  The  method 
employed  to  determine  the  normality 
of  the  particular  property  in  question 
should  be  given,  because  some  other 
technique  might  yield  higher  or  lower 
values.  One  must  also  be  on  the  look- 
out for  modifjnng  conditions  which 
should  be  identified  and  specified. 
Thus  the  normal  secretory  activity  of 
the  sweat  glands  is  modified  consider- 
ably by  changes  in  the  weather.  When 
temperature  and  humidity  are  high 
during  the  summer,  the  level  of  normal 
activity  is  different  from  that  in  the 
winter.  What  is  normal  at  high  alti- 
tudes is  frequently  not  normal  for  parts, 
or  functional  mechanisms,  of  indi- 
viduals of  the  same  race,  sex  and  age 


measured  by  the  same  methods  at  sea 
level.  To  further  multiply  examples 
would  be  wearisome. 

Errors  in  Estimation:  Histologists, 
who  would  provide  a  sound  basis  for 
work  in  pathology,  should  bear  in  mind 
the  considerations  already  mentioned 
and  others  incident  on  the  very  in- 
adequate techniques  they  employ. 

Before  attempting  to  catalogue  them, 
however,  it  should  be  freely  admitted 
that,  even  in  the  best  textbooks,  there 
is  a  deplorable  looseness  in  descriptions 
intended  to  convey  information  on  the 
normal  microscopic  structure  of  the 
human  body.  Illustrations  of  human 
tissues  are  often  presented  without 
any  qualifying  data  concerning  them 
and  others  of  the  tissues  of  a  wide  range 
of  lower  animals  are  supplied,  likewise 
without  qualifying  data,  the  assump- 
tion being  made,  all  too  frequently, 
that  they  are  truly  representative  of 
normal  human  tissues.  In  many  cases 
they  are  representative  and  serve  the 
purpose.  And  it  hardly  needs  to  be 
said,  that  for  accurate  and  controlled 
experiments  animal  tissues  are  essen- 
tial. But  it  is  not  difficult  to  cite  cases 
in  which  examination  of  normal  animal 
tissues  could  convey  a  misleading  idea 
of  normal  human  tissues  of  the  same 
kind.  Kurloff  bodies  are  a  normal 
component  of  the  spleens  of  guinea 
pigs,  but  not  of  human  beings.  The 
demarkation  of  hepatic  lobules  by  con- 
nective tissue  is  notably  characteristic 
of  certain  species.  What  is  normal  in 
this  respect  for  the  domestic  pig  is  not 
normal  for  man. 

Appearance  in  microscopic  prepara- 
tions of  any  kind  may  be  technical 
artifacts  and  may  not  indicate  any  de- 
viation from  normal  in  the  tissue  or 
organ  from  which  the  preparation  has 
been  made.  The  term  artifact  is  de- 
rived from  L.  ars,  art  and/aciw.'?,  made. 
It  is  something  made  artificially. 
Webster  defines  an  artifact  as  being 
"in  histology,  a  structure  or  appearance 
in  a  tissue  or  cell  due  to  death  or  to 
the  use  of  reagents  and  not  present 
during  life."  The  degree  of  artifact 
is  proportional  to  the  difference  be- 
tween the  structure  existing  normally 
in  the  living  body  and  the  structure  ob- 
served in  the  preparation.  Details 
are  given  in  Laboratory  Technique. 

In  the  case  of  living  tissues,  observed 
with  blood  and  nerve  supply  intact, 
there  is  a  possibility  of  artifact.  It  is 
at  a  minimum  in  the  rabbit  ear  cham- 
bers, in  which  the  ingrowing  tissue  is 
very  thin  and  can  be  studied  with  high 
magnifications,  and  rather  more  to  be 
reckoned   with   when   tissues   must   be 


NORMALITY,  MICROSCOPIC 


234 


NORMALITY,  MICROSCOPIC 


displaced  in  order  to  supply  the  neces- 
sary illumination.  But  it  is  small  with 
relatively  thick  organs  like  the  spleen 
and  liver  transilluminated  by  Knisely's 
quartz  rod  technique  and  examined 
at  lower  magnification.  With  increase 
in  time  modifications  due  to  changes  in 
light,  temperature,  hydrogen  ion  con- 
centration, etc.  are  likely  also  to  in- 
crease. 

In  living  cells  removed  from  the  body 
and  examined  in  Tissue  Cultures  the 
possibility  of  artifact  is  again  at  a 
minimum;  but,  though  the  cells  in  suc- 
cessive generations  in  suitable  media  go 
on  living  indefinitely,  their  environ- 
ments are  different  from  those  existing 
within  the  body.  When  after  vital 
staining,  or  supravital  staining,  still 
living  cells  are  examined  in  approx- 
imately isotonic  media,  there  is  a  grave 
danger  of  artifact  if  the  study  is  pro- 
longed because  the  cells  are  slowly 
dying. 

In  fixed  tissiies  the  degree  of  diver- 
gence from  the  living  condition  is  ob- 
viously much  greater  than  in  the  case 
of  still  living  ones.  However  death 
has  been  sudden  so  that  artifacts  due 
to  gradual  death  are  eliminated.  If 
the  technique  has  been  carefully 
standardized  the  same  fixative  applied 
to  the  same  type  of  cell  in  the  same 
physiological  state  is  likely  to  yield 
similar  results. 

1.  The  folds  seen  in  stained  sections 
of  skin  are  often  at  least  partly  due  to 
shrinkage  of  the  collagenic  and  elastic 
tissue  of  the  dermis  after  excision  and 
before  fixation.  The  fixative  serves 
further  to  exaggerate  them  (Evans  et 
al.,  1943).  Some  ballooning  of  the  epi- 
thelial caps  of  intestinal  villi  occurs 
in  vivo.  This  phenomenon  is  likewise 
exaggerated  by  ligating  the  arteries  of 
supply  in  the  living  animal  and  by  em- 
ploying fixatives  which  induce  forcible 
contraction  of  smooth  muscle  (Mack- 
lins,  1932). 

2.  Alterations  occurring  postmortem 
in  tissues  before  fixation  can  be  con- 
fusing. The  most  frequent  one  is  a 
breaking  up  of  the  tips  of  intestinal 
villi,  sometimes  also  caused  by  mechan- 
ical injury  during  excision  or  washing 
out  the  contents  of  the  gut.  In  all 
tissues  autolytic  and  osmotic  changes 
occur  after  death  which  profoundly 
alter  their  structure  after  fixation. 
The  rate  of  autolysis  is  very  rapid  in 
organs  like  the  pancreas  rich  in  enzymes 
let  loose  when  the  action  of  said  en- 
zymes ceases  with  death  to  be  properly 
regulated.  The  rate  is  slower  in  the 
nervous  system  and  especially  in  the 
walls  of  elastic  arteries  in  which  the 


proportion  of  inanimate  components 
(elastic  and  collagenic  fibers)  is  high 
and  that  of  living  cells  is  low. 

3.  Shrinkage,  and  increased  affinity 
of  surface  cells  for  stains,  occasioned  by 
letting  the  surface  dry  before  fixation 
should  be  recognized  as  such. 

4.  Differences  in  the  appearance  of 
the  peripheral  and  central  parts  of 
tissues,  owing  to  the  unequal  penetra- 
tion of  the  fixative  as  a  whole,  or  of  its 
components,  are  sometimes  encount- 
ered. 

5.  The  displacement  of  glycogen  in 
the  direction  of  entrance  and  passage 
of  the  fixative,  so  well  illustrated  in 
a  book  by  de  Robertis  and  his  associates 
(1948),  suggests  the  likelihood  of  similar 
artifacts  in  the  cytoplasmic  localization 
of  other  substances. 

6.  The  glassy  appearance  of  nuclei 
and  cytoplasm,  sometimes  occasioned 
by  overheating  in  paraffin  imbedding, 
or  in  spreading  out  sections,  is  easily 
detected. 

7.  The  presence  of  formed  material 
within  blood  vessels,  faintly  resembling 
organisms,  caused  by  the  coagulation 
of  blood  proteins,  has  led  some  people 
astray. 

8.  The  introduction  of  extraneous 
substances  present  either  in  the  al- 
bumin fixative  used  to  mount  the  sec- 
tions or  deposited  as  dust  from  the  air 
(bacteria,  spores,  mineral  matter,  etc.) 
is  a  possibility  to  be  borne  in  mind. 
Careful  focussing  is  required  to  deter- 
mine whether  particulate  matter  is 
located  below,  within  or  upon  the  sec- 
tion. In  stained  smears  of  blood  and 
other  cells  precautions  against  ex- 
traneous substances  are  also  indicated. 

Interpretation:  One  has  to  be  almost 
constantly  on  guard  because  the  fore- 
going list  of  artifacts  is  by  no  means 
complete.  It  is  simply  illustrative  of 
some  of  the  conditions  that  may  be  met 
with  and  must  be  excluded.  Even  when 
appearances,  that  do  not  seem  to  be 
artifacts,  are  observed  it  is  not  safe  to 
conclude  in  an  unqualified  fashion  that 
the  tissue,  or  organ,  from  which  the 
preparations  have  been  made  was  in 
fact  normal.  On  the  contrary  several 
conditions  require  fulfillment  before 
an  opinion  should  be  offered. 

1.  The  appearance  noted  must  be 
present  in  more  than  50  per  cent  of  in- 
dividual tissues  comprising  a  homo- 
geneous series  in  the  sense  already  ex- 
plained. The  advice  of  a  colleague 
versed  in  statistics  is  desirable  as  to 
the  number  required  in  the  said  series. 

2.  In  the  event  that  the  appearance 
in  question  is  observed  in  but  small 
pieces   excised    from    large    organs    or 


NORMALITY,  MICROSCOPIC 


235 


NORiMA]  JTY,  MICROSCOPIC 


tissues  care  is  indicated  that  the 
samples  are  truly  representative  of  the 
whole.  Examination  of  samples  all 
from  the  tail  of  the  pancreas  well  might 
fail  to  reveal  conditions  prevalent  else- 
where in  the  pancreas  and  convey  a  false 
idea  of  the  normality  of  the  whole. 
Inadequate  preliminary  gross  examina- 
tion might  lead  to  the  overlooking  of 
small  localized  modifications,  as  in 
beginning  cancers,  and  might  result 
in  a  verdict  of  normality  based  on  ap- 
pearances in  poorly  selected  samples. 

3.  If  the  samples  have  been  taken 
from  organs  or  tissues  which  are  larger 
(hypertrophied)  or  smaller  (atrophied) 
than  normal  then  their  examination 
may  likewise  result  in  a  misconception 
of  normality.  The  alterations  in 
volume,  which  have  to  be  taken  into 
consideration,  may  be  abnormal,  or 
within  the  range  of  normal  volumetric 
changes  in  physiological  activity. 
Among  the  latter  are  alterations  in 
the  volume  of  the  liver  consequent  upon 
the  different  phases  in  intestinal  ab- 
sorption. We  must  face  not  only 
changes  in  volume  but  also  changes 
in  microscopic  appearance.  The  nor- 
mality of  microscopic  structure  there- 
fore must  be  specified  in  relation  to 
physiologic  state.  What  is  normal  for 
one  stage  in  the  menstrual  cycle  is  not 
normal  for  another.What  is  normal  for 
the  gastric  mucous  membrane  during 
active  digestion  is  not  normal  while 
fasting. 

4.  Since  the  appearance  depends  upon 
the  technique  employed  the  qualifica- 
tion is  essential  that  it  is  normal  for, 
say,  intracellular  lipids  demonstrable 
by  Sudan  III,  or  for  whatever  is  in 
question.  Not  all  lipids  are  detectable 
by  this  method.  It  is  quite  possible 
that  the  lipid  contents  not  so  revealed 
are  in  abnormal  quantities. 

5.  In  returning  a  verdict  that  tissues 
examined  microscopically  are  normal, 
histologists  should  still  further  qualify 
their  conclusions.  It  is  the  part  of 
wisdom  for  them  to  remember,  es- 
pecially when  employing  microchemical 
reactions,  that  most  of  them  are  ig- 
noramuses as  to  chemistry.  A  smatter- 
ing of  chemistry  is  insufficient.  It  is 
a  kind  of  wine  that  goes  to  the  head 
releasing  inhibitions.  Not  to  obtain 
expert  advice  from  real  chemists  is 
foolish. 

Consider  only  the  sections  of  tissues 
fixed  in  formalin,  or  in  Zenker's  fluid, 
and  stained  with  hematoxylin  and  eosin 
which  are  examined  routinely  in  so 
many  laboratories.  Reflect  upon  the 
functional  attributes  of  these  tissues 
while  they  were  alive  and  to  changes 


to  which  these  specimens  give  no  clues 
whatsoever.  Among  these  are  respon- 
siveness to  stimuli,  permeability,  the 
entry  and  outgo  of  water  and  of  in- 
numerable substances,  the  integration 
of  thousands  of  enzymatic  activities, 
the  basic  submicroscopic  structural  ar- 
chitecture that  makes  this  possible, 
and  so  on  almost  ad  infinitum.  It  is 
no  wonder  that  tissues  examined  by 
such  a  crude  technique,  as  well  as  by 
other  more  delicate  ones,  may  look 
normal,  since  so  much  is  overlooked, 
while  in  fact  they  are  far  from  normal. 
Neuroanatomists  are  among  the  first 
to  recognize  the  inadequency  of  micro- 
scopic examinations.  They  designate 
abnormalities  without  structural  traces, 
"functional."  Parts  of  the  brain  that 
look  normal  actually  may  be  far  from 
normal.  This  qualification  probably 
holds  for  all  parts  of  the  body. 

Standards  for  the  comparison  are 
few  and  disappointing.  Some  gross 
weights  and  measurements  are  given 
in  Laboratory  Technique  but  these  are 
only  for  adults  and  corrections  for  race 
are  not  supplied.  For  a  summary  of 
measurements  for  children  see  R.  E. 
Scammon's  account  in  Abt's  Pediatrics, 
Philadelphia:  Saunders,  1923,  1,  257- 
444.  Anatomische,  Physiologische  und 
Physikalische  Daten  by  H.  Vierordt, 
Jena:  Fischer,  1906,  616  pp.  is  still  use- 
ful. R.  Pearl  and  his  associates  have 
published  quantitative  data  on  the 
endocrines  in  Human  Biology,  1935,  7, 
350-391;  555-607.  1936,  8,  92-125. 
1937,  9,  245-250.  For  a  spleen  and 
thymus  see  E.  B.  Krumbhaar  in 
Cowdry's  Problems  of  Ageing,  1942. 
The  Tabulae  Biologicae  (Amsterdam: 
Vitgevery,  Dr.  W.  Jurik)  is  often  use- 
ful. The  last  part  on  the  eye  (oculus) 
constitutes  vol.  22  of  408  pp.  and  was 
published  in  1947.  The  most  recent 
publication  on  normal  values  in  clinical 
medicine  is  that  of  Sunderman  and 
Boerner  (1949).  A  "Handbook  of  Bi- 
ological Data"  is  in  course  of  organiza- 
tion by  the  American  Institute  of  Bi- 
ological Sciences  under  the  direction 
of  E.  C.  Albritton  at  the  National  Re- 
search Council,  Washington,  D.  C. 

Summary:  Histologists  must  be  alert 
for  artifacts,  for  otherwise  there  is  a 
danger  of  describing  appearances  not 
present  in  vivo,  or  exaggerated  or  min- 
imized by  the  procedure  employed. 
They  must  be  good  physiologists  always 
mindful  of  the  fact  that  only  a  minority 
of  functional  states  are  reflected  by 
microscopic  structure.  They  must  re- 
member that  their  techniques  just  skim 
the  surface.  As  experimentalists  it  is 
essential  for  them  to  gain  a  knowledge 


NORMALS,  GROSS  SIZES 


236 


NORMALS,  GROSS  SIZES 


of  the  normal  structure  of  the  cells, 
tissues  and  organs  of  the  body  for  other- 
wise their  conclusions  based  on  well 
controlled  experiments  on  animals  and 
the  microscopic  observation  of  parts 
of  the  human  body  in  health  and  disease 
will  be  in  jeopardy.  And  they  must 
have  a  good  idea  where  in  the  literature 
they  can  quickly  find  helpful  accounts 
of  what  little  has  been  determined  ac- 
curately of  gross  and  microscopic 
normality.  It  is  safe  to  say  that  no 
human  bodj'  is  at  any  time  normal,  or 
usual,  throughout  its  extent,  because 
is  is  a  far  too  complex  organization  of 
multifunctional  interlocking  mecha- 
nisms for  uniformity  in  this  respect. 
Health  is  not  synonymous  with  an  all 
pervading  normality.  It  is  merely  a 
state  in  which  no  particular  normality 
is  lost  to  a  conspicuously  detrimental 
degree. 
Normals,  Gross  Sizes.  What  these  are  is 
only  known  in  a  very  hazy  way.  Yet 
if  the  size  of  an  organ  is  distinctly  ab- 
normal this  fact  must  clearly  be  taken 
into  consideration  in  evaluating  the 
results  of  its  microscopic  study.  The 
best  way  is  to  search  for  papers  dealing 
with  the  organ  in  which  one  is  interested 
in  the  Quart.  Cum.  Index  Med.  The 
older  data  are  summarized  by  Vierordt, 
H.,  Anatomische  Physiologische  una 
Physikalische  Daten  und  Tabellen. 
Jena:  Fischer,  1906,  616  pp.  A  sum- 
mary of  measurements  on  infants  and 
children  is  provided  by  R.  E.  Scammon 
in  Abt's  Pediatrics,  Philadelphia: 
Saunders,  1923,  1,  257-444.  See  also 
Coppoletta,  J.  M.  and  Wolbach,  S.  B., 
Am.  J.  Path.,  1933,  9,  55-70.  Useful 
quantitative  data  on  the  endocrines  are 
supplied  by  R.  Pearl  and  his  associates 
in  Human  Biology,  1935,  7,  350-391,  555- 
607;  1936,  8,  92-125;  1937,  9,  245-250. 
For  spleen  and  thymus  see  Krumbhaar, 
E.  B.,  Cowdry's  Problems  of  Ageing. 
Baltimore:  Williams  &  Wilkins,  1942, 
139-184.  There  is  a  wide  range  in  indi- 
vidual variation.  Size  may  be  greater 
or  smaller  than  the  normal  or  usual 
without  being  indicative  of  disease. 
Stitt,  E.  R.,  Clough,  P.  W.  and  M.  C., 
Practical  Bacteriology,  Haematology 
and  Animal  Parasitology.  Phila- 
delphia: Blakiston,  1938,  961  pp.  give 
these  approximate  measurements  (ab- 
breviated) : 

Adrenals— Length,  6-7  cm.;  breadth, 
3-3.5  cm. ;  weight,  5-6  gms.  each. 

Aorta — Length,  42.5-50  cm.:  thick- 
ness of  wall,  1.5-2  mm.;  diameter, 
1.7-3  cm. ;  weight,  35-45  gms. 

Appendix— Length,  9-10  cm.;  diameter, 
6  mm.;  weight,  7-14  gm.,  quite 
variable. 


Bladder — Capacity,  500  cc.  when  nor- 
mally distended;  thickness  of  wall, 
2.5  mm. ;  weight,  30-60  grams. 

Brain— Weight,  female,  1250-1275  gms.; 
male,  1365-1450  gms.;  length,  16.5 
cm.;  transverse  diameter,  14  cm.; 
vertical  diameter,  12.7  cm.;  dimen- 
sions in  female  being  1  cm.  less. 

Fallopian  tubes — Length,  7.6-12.6  cm., 
the  right  usually  the  longer ;  diameter 
of  lumen  averages  2.5  mm. 

Gall  bladder — Length,  7.5-10  cm. ;  diam- 
eter, 2.5-3  cm.;  thickness  of  wall, 
1-2  mm. ;  capacitv,  30-45  cc. 

Heart— Weight,  feLoale,  250-280  gms., 
male,  270-360  gms.;  length,  11.5-14 
cm.;  breadth,  7.5-10  cm.;  thickness, 
5-8  cm. ;  thickness,  wall  left  ventricle, 
9-12  mm.,  right  ventricle,  2.5-3  mm. ; 
circumference,  mitral  orifice,  10.4- 
10.9  cm. ;  circumference,  tricuspid 
orifice,  12-12.7  cm. ;  circumference, 
aortic  orifice,  7.7-8  cm. ;  circumference, 
pulmonary  orifice,  8.5-9  cm. 

Intestines — Small  intestine,  length, 
6.75  meters,  2/5  jejunum  and  3/5 
ileum;  diameter  from  47  mm.  in 
duodenum  to  27  mm.  at  the  end  of 
ileum.  Large  intestine,  length,  180- 
195  cm.;  duodenum,  length,  26-28.5 
cm. 

Kidneys — Weight,  left,  150  gms.,  right, 
140  gms.;  thickness  of  cortex,  1  cm.; 
length,  11.5  cm.;  breadth,  6.2  cm.; 
thickness,  3.2  cm. ;  the  left  longer  and 
the  right  thicker. 

Liver — Weight,  1440-1680  gms. ;  greatest 
transverse  diameter,  20-24  cm.,  great- 
est antero-posterior  diameter,  10-15 
cm.,  vertical  diameter,  12.7-15  cm. 

Lungs— Weight,  combined,  1020-1290 
gms.;  weight,  male,  right  lung,  680 
gms.,  left  lung,  600  gms.;  weight, 
female,  right  lung,  480  gms.,  left  lung, 
420  gms.;  length,  26-30  cm.;  antero- 
posterior diameter  at  base,  17.5-20 
cm.;  transverse  diameter  at  base, 
10-12.7  cm.;  right  lung  is  shorter, 
broader  and  thicker  than  the  left; 
dimensions  in  female  average  2.5  cm. 
less. 

Mammary  gland — Weight  in  adult,  150- 
200  gms.;  weight  during  lactation, 
400-900  gms. 

Oesophagus — Length,  25-30  cm.;  diam- 
eter of  lumen,  3  cm.;  thickness  of 
wall,  8  mm. ;  weight,  40  gms. 

Ovaries — Weight  (each).  4-8  gms., 
length,  3.8  cm.;  breadth,  1.9  cm.; 
thickness,  1.2  cm. 

Pancreas — Weight,  quite  variable,  60- 
135  gms. ;  length  varies,  average  15- 
20  cm. 

Parathyroids — Length,  6-7  mm.; 
breadth,  3-4  mm.;  thickness,  1.5-2 
mm. 


NORMOBLASTS 


237 


NUCLEAR  INCLUSIONS 


Pineal  gland — Length,  1  cm.;  breadth, 
5   mm.;    thickness,   5   mm.;   weight, 
0.2  gm. 
Pituitary      body — Length,      8      mm.; 
breadth,  1.2  cm.;  weight,  0.3-0.6  gm. 
Prostate — Weight,  22  gms.;  length  3.1- 
3.8  cm.;  breadth,  3.8—4.5  cm.;  thick- 
ness, 2.5  cm. 
Salivary   glands — Parotid,    weight,    25- 
30  gms.;  sublingual,  weight,  2-3  gm.; 
submaxillary,  weight,  8-9  gms. 
Seminal  vesicles — Length,  5  cm. 
Spinal   cord — Length,  45  cm.;  weight, 
27-30  gms. ;  transverse  diameter  aver- 
ages  1.2  cm.;  antero-posterior  diam- 
eter averages  9  mm. 
Spleen — Weight,  155-195  gms.;  length, 
10-12.5  cm.;  breadth,  7.7  cm.;  thick- 
ness, 2.5-3.7  cm. 
Stomach — Capacity,  1-2  liters.;  thick- 
ness of  wall,  6  mm.;  weight,  125-175 
gms. 
Testes — Weight,      20-25     gms.      each; 
length,    3.8    cm.;    breadth,   2.5    cm.; 
thickness,  2  cm. 
Thoracic  duct — Length,  37-5-45  cm. 
Thymus  gland— Weight  at  birth,   13.7 
gms.  and  increases  to  26.2  gms.  at  end 
of  second  year  when  it  gradually  de- 
creases until  gland  disappears ;  dimen- 
sions at  birth,  length,  6  cm. ;  breadth, 
3.7  cm. ;  thickness,  6  mm. 
Thyroid — Transverse  diameter,  6-7  cm. ; 

height,  3  cm.;  weight,  30-40  gm. 
Ureters — Length,    28-30    cm.,    slightly 
longer  on  left  side  and  longer  in  male, 
diameter  of   lumen   varies,  averages 
2.5  mm. 
Urethra— IVIale,    length,    16-20.6    cm.; 
prostatic,  2.5-3.1   cm.,  membranous, 
1.5-2.5  cm.,  and  the  anterior,  12-15 
cm. ;  female,  length,  3.8  cm. ;  diameter 
of  lumen  averages  7-10  mm. 
Uterus — (Virginal)      length,      7      cm.; 
breadth,  4  cm. ;  thickness,  2.5  cm. ; 
weight,  40-50  gm. ;  the  dimensions  of 
a    multiparous    uterus   are    each    in- 
creased 1  cm.  or  more  and  the  weight 
is  increased  20  gms. ;  length  of  cavity 
in  virgin,  5  cm.,  in  multiparae,  5.7  cm. 
Vagina — Length,  7.6-8.9  cm.;  posterior 
wall     is     slightly    longer     than     the 
anterior. 
Normoblasts       (orthochromatic       erythro- 
blasts).     Stage  in  formation  of  erythro- 
cyte between  erythroblast  and  reticulo- 
cyte ;  nucleus  spherical  or  oval,  picnotic, 
often  excentrically  placed.     Cytoplasm 
contains    much    hemoglobin,    not    nor- 
mally     present     in      circulation.     See 
Erythrocytes,     Developmental     series. 
Nucleal   Reaction  is  a  microchemical   test 
for  Thymonucleic  Acid  which  see,  also 
Feulgen  Reaction. 
Nuclear    Inclusions    are    characteristic    of 
some  virus  diseases  but  in  many  such 


diseases  they  are  not  found.  Only 
when  they  are  present  in  large  numbers 
as  in  yellow  fever  is  it  feasible  to  in- 
vestigate them  in  fresh  tissues.  Stain- 
ing reactions,  solubility  tests  and  other 
properties  of  fresh  inclusions  are  de- 
scribed by  Cowdry,  E.  V.  and  Kitchen, 
S.  F.,  Am.  J.  Hygiene,  1930,  11,  227-299. 
Methods  for  their  identification  in  fixed 
tissues  are  summarized  by  Cowdry, 
E.  v..  Am.  J.  Clin.  Path..  1940,  10,  133- 
148.  For  general  purposes  fixation  in 
Zenker's  fluid,  parafhn  imbedding  and 
coloration  with  Hematoxylin  and  Eosin 
is  the  most  satisfactory.  Coloration 
with  Phloxine  or  Eosin  Methylene  blue 
gives  more  brilliant  colors  but  they  fade 
more  rapidly.  The  nuclear  inclusions 
are  typically  acidophilic  and  therefore 
take  eosin  and  phloxine  energetically. 
When  it  is  desired  to  reverse  the  colors 
use  Safranin-Light  Green  which  gives 
green  inclusions  and  red  chromatin. 
For  microchemical  methods  see  Cowdry, 
E.  v..  Science,  1928,  68,  40-41,  see  also 
Specific  Gravity  determinations.  Paper 
by  Lucas,  A.  M.,  Am.  J.  Path.,  1940, 
16,  739-760. 

When  the  following  features  are  noted 
in  a  section  it  is  likely  that  a  virus  has 
been  at  work : 

1.  A  considerable  number  of  inclu- 
sion-laden nuclei  which  can  be  arranged 
in  series  representing  stages  in  develop- 
ment. This  indicates  an  active  process 
in  which  the  nuclei  exhibiting  the  most 
advanced  alterations  were  affected  first 
and  the  others  in  succession. 

2.  A  change  in  which  the  accumula- 
tion of  aciaophilic  material,  forming 
the  inclusion,  is  accompanied  by  mar- 
gination  of  basophilic  chromatin  on  the 
nuclear  membrane,  a  disappearance  of 
nucleoli  and  ultimate  death  and  disin- 
tegration of  the  cells.  This  suggests 
that  the  inclusion  formation  is  not 
merely  an  intranuclear  heaping  up  of 
material  effected  without  injury. 

3.  A  cellular  reaction  characterized 
by  hyperplasia,  hypertrophy  or  necrosis. 

Nuclear  inclusions  are  of  two  general 
sorts — A  and  B  (Cowdry,  E.  V.,Arch. 
Path.,  1934,  18,  527-542).  Type  A  are 
the  most  definite  and  exhibit  the  proper- 
ties noted  above  under  2.  When  the 
basophilic  chromatin  does  not  marginate 
on  the  nuclear  membrane  and  the 
nuclear  structure  does  not  disintegrate 
— we  have  to  proceed  warily.  Such 
inclusions  (type  B)  are  droplet-like 
masses  of  acidophilic  material  sur- 
rounded by  clear  halos.  They  have 
been  reported  in  Borna  disease,  in  polio- 
myelitis and  in  several  other  conditions. 
When  observed  in  routine  preparations 
they  are  seldom  conspicuous  structures. 


NUCLEASES 


238 


NUCLEI 


It  is  only  when  strongly  stained  with 
fuchsin,  for  instance,  that  they  catch 
the  eye.  Perhaps  careful  search  of 
tissues  not  subjected  to  virus  action 
might  reveal  similar  bodies.  Therefore 
in  the  case  of  type  B  inclusions,  insist- 
ence on  criteria  1  and  3  is  desirable. 
The  nuclear  inclusions  in  the  liver 
following  severe  burns  look  very  much 
like  those  caused  by  viruses  (Belt,  T. 
H.,  J.  Path,  and  Bact.,  1939,  48,  493- 
498). 

In  plants,  as  in  animals,  some  nuclear 
inclusions  are  indicative  of  the  action 
of  certain  viruses,  see  excellent  sum- 
marizing account  by  Bawden,  F.  C, 
Plant  Viruses  and  Virus  Diseases. 
Waltham:  Chronica  Botanica  Co.,  1943, 
294  pp.  Since  the  inclusions  usually 
occur  in  the  form  of  "thin  flat  crystal- 
line plates"  they  should  be  examined  in 
living  cells  in  the  dark  field  and  in 
polarized  light  because  details  of  crys- 
talline structure  are  not  so  well  shown 
in  fixed  and  stained  preparations.  The 
inclusions  give  the  usual  protein  reac- 
tions but  are  Feulgen  negative.  They 
apparentl}'  contain  virus. 

An  interesting  and  well  illustrated 
account  of  intranuclear  parasites  is  pro- 
vided by  H.  Kirby,  Jr.  in  Calkins,  G.  N. 
and  Summers,  F.  M.,  Protozoa  in  Bio- 
logical Research.  New  York:  Colum- 
bia University  Press,  1941,  1148  pp. 
Nucleases — Written  by  E.  W.  Dempsey, 
Dept.  of  Anatomy,  Washington  Uni- 
versity, St.  Louis.  February  26,  1951 — 
For  many  j-ears,  cytologists  have  at- 
tempted to  apply  enzjane  preparations 
which  chemically  degrade  or  destroy 
nucleic  acids.  Van  Herwerden,  A., 
Anat.  Anz.,  1914,  47,  312-325,  and  Lison 
(p.  175)  describe  the  older  experiments. 
Until  recently,  however,  the  enzyme 
preparations  were  contaminated  with 
trypsin  and  other  proteolytic  enzj'mes, 
so  that  the  results  after  digestion  of 
sections  with  nucleases  were  difficult  to 
interpret.  Kunitz,  M.,  J.  Gen.  Phys- 
iol., 1940,  24, 15-32  described  the  prepa- 
ration of  crystalline  ribonuclease,  an 
enzyme  which  depolymerizes  and  there- 
fore solubilizes  pentose-nucleic  acid. 
McDonald,  M.  R.,  J.  Gen.  Physiol., 
1948,  32,  39-42,  reported  that  the  last 
traces  of  proteolytic  activity  in  this 
preparation  could  be  removed  by  heat 
treatment.  Ribonuclease  has  been 
widely  used  to  identify  many  basophilic 
cytoplasmic  components  (Nissl  sub- 
stance, ergastoplasm  of  pancreatic  cells, 
basophilic  inclusions  in  placenta,  cyto- 
plasmic basophilia  in  general),  and 
nucleoli  in  some  but  not  all  cells  fail  to 
stain  after  digestion  in  the  enzyme. 
Desoxyribonuclease,  an  enzyme  which 


depolymerizes  desoxypentosenucleic 
acids,  has  also  been  crystallized  by 
Kunitz,  M.,  J.  Gen.  Physiol.,  1950, 
33,  349-362.  This  preparation  de- 
stroys the  stainability  of  chromatin, 
but  has  not  yet  had  as  wide  an  applica- 
tion to  cytology  as  had  had  ribo- 
nuclease. These  preparations  are 
available  through  the  Worthington 
Biochemical  Laboratories,  Freehold, 
N.J. 
Nuclei.  To  look  into  the  body  and  study 
the  nuclei  of  living  cells  is  feasible 
only  up  to  a  certain  point.  The  ob- 
servation of  the  Clarks'  (E.  R.  and  E. 
L.,  Am.  J.  Anat.,  1936,  59,  123-173) 
that  in  transparent  chambers  inserted 
into  the  ears  of  rabbits  (Sandison's 
Technique)  the  finely  granular  leuco- 
cytes may  be  followed  about  and  seen 
to  lose  their  nuclear  poljonorphism  is 
significant  of  what  can  be  done.  In 
Tissue  Cultures  the  cells  are  living 
under  less  natural  conditions  but  they 
grow  in  thin  films  and  can  therefore  be 
observed  at  high  magnification.  Care- 
ful analysis  of  moving  pictures,  show- 
ing nuclear  form  and  structure,  like 
those  of  Dr.  W.  H.  Lewis  distributed 
by  the  Wistar  Institute,  can  prove  very 
fruitful.  By  ultracentrifugation  data 
can  be  obtained  bearing  on  intranuclear 
Viscosity  and  the  relative  Specific 
Gravity  of  nuclear  components.  The 
techniques  of  Microdissection  and 
microinjection  also  offer  opportunities 
for  advance.  The  Vital  Staining  of 
nuclei  without  killing  the  cells  is  diffi- 
cult and  not  particularly  helpful  (Acri- 
fiavine);  but  it  appears  to  be  feasible 
in  a  variety  of  vertebrate  cells  with 
dilute  solutions  of  methylene  blue 
(Russel,  D.  G.,  J.  Exp.  Med.,  1914,  20, 
545-553),  in  amebae  by  microinjection 
(Monne,  L.,  Proc.  Soc.  Exp.  Biol.  R. 
Med.,  1934-35,  32,  1197-1199),  and  in 
the  fibroblasts  of  tissue  cultures  with 
crystal  violet  (Bank,  O.  and  Kleinzeller, 
H.,  Arch.  f.  exp.  Zellf.,  1938,  21,  394- 
399).  The  same  can  be  said  for  Ultra- 
violet Photomicrography. 

The  choice  of  fixative  is  important. 
It  is  difficult  to  secure  after  formalin 
fixation  a  brilliant  color  contrast  of 
basophilic  and  acidophilic  nuclear  mate- 
rials by  staining  with  Giemsa,  Eosin- 
Methylene  Blue  and  other  mixtures  of 
"basic"  and  "acid"  dyes,  because  the 
former  take  very  intensely  and  the 
latter,  lightly.  But  following  Zenker's 
fluid  and  other  mixtures  containing 
potassium  bichromate,  which  acts  as  a 
sort  of  mordant,  these  stains  color  the 
acidophilic  as  well  as  the  basophilic 
components.  It  is  for  this  reason,  and 
because  nuclear  inclusions   caused  by 


NUCLEI 


239      NUCLEIC  ACID-DYE  INTRACTIONS 


virus  action  are  usually  acidophilic, 
that  formalin  used  alone  is  contraindi- 
cated  as  a  fixative. 

On  the  morphological  side  it  is  known 
that  nuclei  stained  in  sections  after 
fixation  in  the  usual  ways  show  a  di- 
versity, or  heterogenic! ty,  of  internal 
structure  which  cannot  be  observed  by 
the  most  careful  examination  of  the 
nuclei  of  living  cells.  In  thelatteronly 
the  nucleolus  can  generally  be  distin- 
guished. The  so-called  linin  network, 
and  small  irregular  particles  staining 
with  acid  and  basic  dyes,  are  not  ob- 
served. These  probably  result  from  the 
coagulating  action  of  the  fixative  upon 
materials  present  in  solution  or  fairly 
uniformly  distributed  in  the  nucleo- 
plasm. Stained  sections  of  tissues  fixed 
in  fluids  containing  fair  amounts  of 
osmic  acid  (Altmann's  Mixture  and 
Bensley's  Acetic-Osmic-Bichromate) 
exhibit,  on  the  contrary,  nuclei  with 
quite  homogeneous  looking  nucleoplasm, 
containing  nucleoli,  which  portray  the 
condition  in  vivo  more  accurately. 
Colored  illustrations  of  the  nuclei  of 
liver  cells  containing  inclusions  after 
osmic  and  non-osraic  fixation  (Figs.  47 
and  20)  are  provided  by  Cowdry,  E.  V. 
and  Kitchen,  S.  F.,  Am.  J.  Hyg.,  1930, 
11,  227-299.  This  does  not  mean, 
however,  that  the  ground  substance  is 
always  optically  homogeneous   in  vivo. 

The  shrinkage  of  nuclei  when  exam- 
ined in  stained  sections  is  generally 
more  than  10%  of  their  size  in  vivo. 
In  post-mortem  autolysis,  particularly 
of  the  kidney,  one  of  the  first  nu- 
clear modifications  is  shrinkage.  The 
shrunken  nuclei  may  stain  intensely 
with  both  basic  and  acid  dyes.  The 
acidophilic  material  in  them  may  even 
appear  to  be  increased;  for  it  is  more 
concentrated,  owing  to  decrease  in 
volume  (oxychromatic  degeneration). 
They  are  also  more  spherical  and  less 
oval  in  shape.  In  early  stages  this 
modification  can  easily  be  identified 
by  its  occurrence  in  some  tubules  and 
not  in  others.  A  comparable  hyper- 
chromatism  of  nuclei  at  the  edge  of  a 
section  accompanied  by  a  flattening  of 
them  may  indicate  that  a  surface  film 
of  tissue  was  permitted  to  dry  before 
fixation. 

Among  the  stains  Iron  Hematoxylin 
is  a  favourite  because  of  its  sharpness 
and  permanence.  Phloxin-Methylene 
Blue  is  also  recommended.  If  one  de- 
sires to  reverse  the  colors  and  get  red 
nuclei  and  green  cytoplasm  Safranin 
Light  Green  is  suggested.  The 
Safranin-Gentian  Violet-Orange  G 
technique  gives  several  beautiful  color 
tones.    Recently  the  Feulgen  reaction 


by   which  Thymonucleic   Acid   can   be 

demonstrated  has  become  very  popular 
as  the  most  sharply  discriminating 
nuclear  stain.  Microchemical  studies 
are  now  possible  which  a  few  years  ago 
were  undreamed  of.  The  method  of 
Microincineration  reveals  some  of  the 
mineral  constituents  (Scott,  G.  H., 
Proc.  Soc.  Exp.  Biol.  &  Med.,  1935, 
32,  1428-1429). 

The  collection  of  nuclei  in  bulk  for 
chemical  analysis  is  now  feasible  (see 
Centrifugation).  Thus  nuclei  of  liver 
cells  can  be  separated  from  cytoplasms 
by  centrifugation  after  treatment  with 
dilute  citric  acid.  Normal  liver  nuclei 
do  not  accumulate  P32  while  tumor 
nuclei  and  regenerating  nuclei  do 
(Marshak,  A.,  Federation  Proceedings, 
Baltimore,  1942,  1,  (2)  57).  A  method 
for  separating  nuclei  from  rest  of  thy- 
mus is  described  by  Williamson,  M.B 
and  Gulick,  A.,  J.  Cell.  &  Comp.  Phys- 
iol., 1942,  20,  116-118.  The  authors 
analysed  the  mass  of  nuclei  for  calcium, 
magnesium  and  phosphorus.  Another 
method  for  separating  from  cytoplasm 
(Crossmon,  G.,  Science,  1937,  85,  250) 
is  to  place  drop  5%  aq.  citric  acid  in 
center  of  a  slide  smeared  with  Mayer's 
Albumin  Glycerin.  Add  piece  fresh 
muscle.  This  slowly  becomes  trans- 
parent and  infiltrated.  The  cloudiness 
of  the  citric  acid  is  caused  by  released 
nuclei.  Remove  muscle  and  allow  fluid 
containing  nuclei  to  dry  completely. 
Hold  nuclei  in  place  by  treating  with 
95%  ethyl  alcohol.  Wash  in  tap  water, 
then  in  aq.  dest.,  stain  with  A-Iayer's 
Hemalum,  blue  in  tap  water,  counter- 
stain  in  eosin,  dehydrate,  clear  and 
mount. 

For  a  technique  to  demonstrate  sex 
differences  in  neuroglial  and  nerve 
cell  nuclei  consult  Barr,  M.  L.  (Exp. 
Cell  Res.,  1951,  2,  288-290).  See  Argin- 
ase  and  Diaminoacridines  for  visual- 
ization of  nuclei  in  vivo  by  their  fluores- 
cence. 

Nucleic  Acids,  see  Ribonucleic,  Thymo- 
nucleic and  Desoxyribonucleic.  Micro- 
spectrophotometry. 

Nucleic  Acid-Dye  Interactions — Written  by 
Edward  L.  Kuff,  Dept.  of  Anatomj'^, 
Washington  University,  St.  Louis  10, 
Missouri.  October  18,  1951— Pure 
nucleic  acids  are  not  known  to  react 
with  acid  dyes.  The  staining  of  nuclei 
with  acid  dyes  which  can  be  made  to 
occur  in  tissue  sections  is  due  pre- 
sumably to  interaction  of  the  dye  with 
protein  constituents  of  the  nuclei, 
although  this  phenomenon  has  not  been 
adequately  studied. 

Nucleic  acids  of  both  cytoplasmic  and 
nuclear   origin    react   strongly    with   a 


NUCLEOCYTOPLASMIC  RATIO 


240 


NUCLEOCYTOPLASMIC  RATIO 


wide  variety  of  basic  dyes,  both  in  the 
test  tube  and  in  tissue  sections.  In 
a  rougli  way,  it  can  be  stated  that  the 
amount  of  a  basic  dye  taken  up  by  a 
given  nucleic  acid-containing  structure 
in  the  cell  is  proportional  to  the  amount 
of  nucleic  acid  present.  Attempts  at 
a  precise  quantitative  estimation  of 
nucleic  acid  content  by  means  of  basic 
dye  uptake  are  in  the  early  stages  of 
development. 

The  physico-chemical  nature  of  the 
interaction,  in  buffered  aqueous  solu- 
tion, of  rosaniline  with  yeast  and 
pancreatic  pentose  nucleic  acids  (PNA) 
and  with  thymus  desoxypentose  nucleic 
acid  (DNA)  has  recently  been  studied 
(Cavalieri,  L.  F.,  Kerr,  S.  E.,  and 
Angelos,  A.  J.  Am.  Chem.  Soc.  1951, 
73,  2567-2578;  Cavalieri,  L.  F.  and 
Angelos,  A.,  ibid.,  1951,72,  4686-4693). 
The  reaction  was  found  to  be  reversible 
and  stoichiometric .  The  curves  relating 
amount  of  dye  taken  up  by  the  nucleic 
acid  to  the  concentration  of  free  dye 
were  different  for  each  type  of  nucleic 
acid.  However,  in  all  cases  the  data 
could  be  explained  satisfactorily  on  the 
basis  of  a  salt-like  linkage  between  the 
dj^e  cation  and  the  negatively  charged 
phosphoric  acid  groups  of  the  nucleic 
acid,  without  the  assumption  of  more 
complicated  adsorptive  forces.  Speci- 
fic dissociation  constants  for  the  dye- 
nucleic  acid  complexes  could  thus  be 
calculated.  Similar  studies  using  other 
common  histologic  dyes  are  needed 
before  the  staining  of  nucleic  acids  in 
tissue  sections  can  be  quantitatively 
evaluated.  It  should  be  noted  that 
the  dye  concentrations  used  in  the 
above  experiments  were  far  below  those 
ordinarily  employed  in  staining.  There 
is  some  evidence,  however,  that  the  dye- 
nucleic  acid  interaction  is  stoichio- 
metric at  high  dye  concentrations  also. 

Mixtures  of  methyl  green  and  pyronin 
stain  nuclei  green  and  cytoplasmic 
nucleoproteins  red  (see  Methyl  Green- 
Pyronin).  This  differential  staining  of 
the  two  types  of  nucleic  acid  by  two 
different  basic  dyes  has  been  investi- 
gated by  N.  B.  Kurnick  (J.  Gen.  Phys- 
iol., 1950,  33,  243-264),  who  concluded 
the  DNA  of  the  nucleus  owed  its  specific 
staining  by  methyl  green  to  its  high 
degree  of  polymerization.  DNA  which 
had  been  depolymerized  by  treatment 
with  heat  or  acid  failed  to  stain  well 
with  methyl  green  but  did  stain  strongly 
with  pyronin  or  other  basic  dyes.  DNA 
combined  with  histone  stained  less 
strongly  with  methyl  green  than  did  the 
free  DNA.  Under  conditions  of  high 
dye  concentrations,  the  reaction  be- 
tween DNA  and  methyl  green  was  said 


to  be  stoichiometric  (Kurnick,  N.  B. 
and  Mirsky,  A.  E.,  J.  Gen.  Physiol., 
1950,  33,  265-274),  and  has  been  made 
the  basis  of  a  quantitative  histochem- 
ical  test  for  DNA  (Leuchtenberger,  C, 
Vendreley,  R.,  and  Vendreley,  C,  Proc. 
Nat'l.  Acad.  Sci.  1951,  37,  33-38). 

The  interaction  of  nucleic  acids  with 
basic  dyes,  while  apparently  primarily 
of  an  ionic  character,  can  be  modified 
by  many  factors.  Among  these  are: 
1)  availability  of  the  phosphate  groups 
of  the  nucleic  acid  and  their  spatial 
arrangements  (polymerization) ;  2)  com- 
petition for  available  phosphate  groups 
by  substances  other  than  dye,  such  as 
the  histones  of  the  nucleus,  heavy  metal 
cations  of  the  fixing  fluids,  or  impurities 
in  the  dye  solution;  3)  ionic  strength, 
pH,  and  temperature  of  the  staining 
bath;  and  4)  the  dye  concentration  used 
for  staining.  To  approach  any  degree 
of  stoichiometry  in  dye-nucleic  acid 
interactions,  all  of  these  factors  must 
be  controlled  as  rigidly  as  possible. 

The  absorption  spectra  of  all  basic 
dyes  which  have  been  investigated  show 
changes  when  the  dyes  are  bound  to 
nucleic  acids.  This  fact  is  of  im- 
portance if  the  measured  absorption  at 
some  particular  wave  length  is  to  be 
used  to  measure  the  amount  of  dye  pres- 
ent in  a  cellular  structure.  It  may 
also  provide  information  as  to  the 
structure  of  the  nucleic  acids  them- 
selves. Such  spectral  shifts  have  been 
studied  by  Michaelis  and  Granick 
(Michaelis,  L.,  J.  Phys.  and  Colloid 
Chem.  1950,  54,  1-17),  who  found  that 
basic  dyes  bound  to  nucleic  acids  almost 
invariably  show  a  shift  in  their  absorp- 
tion maxima  towards  the  longer  wave 
lengths.  Binding  to  nucleic  acid  was 
also  found  to  inhibit  the  development 
of  metachromasia  shown  by  such  dyes 
as  thionin  and  toluidin  blue  either  in 
concentrated  solutions  or  when  bound 
to  other  strongly  acid  substrates  such 
as  the  acid  mucopolysaccharides. 
Under  conditions  of  excess  nucleic  acid 
with  regard  to  the  dye,  the  bound  dye 
was  shown  to  follow  the  Beer-Lambert 
Law.  Under  conditions  of  high  dye 
concentration  with  regard  to  nucleic 
acid,  such  as  obtain  in  the  usual  stain- 
ing reactions,  it  has  not  been  shown 
that  the  bound  dye  follows  Beer's  law. 
Nucleocytoplasmic  Ratio.  A  histological 
method  for  computing  this  ratio  is  fully 
described  by  Cowdry,  E.  V.and  Paletta, 
F.  X.,  J.  Nat.  Cancer  Inst.,  1941,  1, 
745-759 ;  but  there  are  many  such  tech- 
niques. A  chemical  method  has  been 
used  to  advantage  by  Dawbarn,  M.  C, 
Australian  J.  Exp.  Biol.  &  Med.  Sci., 
1932,  9,  213-226.     Her  ratio  is  obtained 


NUCLEOLINUS 


241 


NUCLEONUCLEOLAR  RATIO 


by  dividing  the  nucleic  acid  nitrogen 
by  the  total  coagulable  nitrogen  less 
nucleic  acid  nitrogen. 

Nucleolinus  is  a  term  introduced  by  Haeckel 
to  indicate  a  deeply  staining  granule 
within  a  nucleolus.  For  details  see 
Champy,  C.and  Carleton,  H.M.,  Quart. 
J.  Micr.  Sci.,  1921,  65,  589-610. 

Nucleolus  (L.  dim.  of  nucleus)  is  a  body 
within  a  nucleus.  There  are  at  least 
three  sorts. 

1.  Plasmosomes.  These  can  be  de- 
fined as  roughly  spherical  bodies,  which 
can  easily  be  seen  in  the  nuclei  of  some 
living  cells  without  the  aid  of  any  stains, 
which  stain  after  appropriate  fixation, 
namely,  with  plasma  or  "acid"  stains 
like  eosin,  (hence  the  name)  and  which 
do  not  directly  contribute  material 
to  the  formation  of  chromosomes. 

Plasmosomes  are  not  to  be  confused 
with  cytoplasmic  granules  called  plas- 
mosomes by  Arnold  many  years  ago  or 
with  plastosomes,  a  term  given  by 
Meves  to  mitochondria  and  now  fortu- 
nately being  discarded.  They  can  be 
referred  to  as  acidophilic  or  oxyphilic 
nucleoli,  but  sometimes  they  are  tinged 
quite  strongly  with  basic  dyes.  They 
are  of  dense  consistency,  easily^  shifted 
by  centrifugal  action  and  are  in  some 
cases  more  resistant  to  the  digestive 
action  of  pepsin  and  hydrochloric  acid 
than  karyosomes. 

2.  Karyosomes,  are  by  contrast  in- 
tensely basophilic  and  do  contribute 
material  to  the  making  of  chromosomes 
during  mitosis.  But  they  are  resistant 
to  peptic  hydrochloric  acid  digestion. 
Wilson  (E.  B.,  Heredity,  New  York: 
Macmillan  1925,  p.  93)  recognizes  3 
types,  net-knots,  chromosome-nucleoli 
andkaryospheres.  There  is  doubt  about 
the  existence  in  vivo  of  the  net-knots  in 
the  same  shape,  size  and  position  as 
observed  in  stained  sections. 

3.  Amphinucleoli  (G.  amphi  on  both 
sides)  are  nucleoli  consisting  of  both 
plasmosome  and  karyosome  material. 
Often  the  acidophilic  substance  acts  as 
a  kind  of  core  and  the  basophilic  sub- 
stance is  close  to  it  or  appears  to  be 
plastered  on  its  surface.  The  latter  may 
not  occur  in  the  same  form  in  the  living 
nucleus. 

The  fixation  which  shows,  when  the 
sections  are  stained,  the  highest  degree 
of  nucleolar  detail  is  not  necessarily 
the  best  (see  remarks  about  Nuclei,). 
The  Linin  network,  net-knots  and 
basophilic  material  marginated  on  plas- 
mosomes may  result  in  part  from  the 
coagulating  action  of  the  fixative  on 
material  originally  distributed  diffusely 
in  the  nucleoplasm.  Nucleoli  which 
look  bubbly,  or  are  surrounded  by  halos, 


are  to  be  regarded  with  suspicion. 
Fixation     in   Acetic-Gsmic-Bichromate 

and  in  other  fluids  containing  osmic 
acid  is  indicated  but  they  penetrate 
poorly.  Staining  by  almost  any  tech- 
nique which  gives  a  good  color  contrast 
between  acidophilic  and  basophilic 
materials  is  satisfactory.  The  classical 
stain  is  with  safranin  and  light  green. 
Eosin  and  methylene  blue,  hematoxylin 
and  eosin  are  recommended,  likewise  all 
methods  advised  for  Nuclear  Inclusions 
caused  by  viruses. 

Usually  no  difficulty  is  experienced 
in  the  identification  of  nucleoli.  How- 
ever with  the  plasmosomes  there  may 
be  some  question.  In  the  first  place 
nuclear  inclusions  type  B  (Cowdry 
E.  v..  Arch.  Path.,  1934,  18,  527-542) 
look  something  like  plasmosomes.  For 
example,  the  nuclear  inclusions  in  Borna 
disease  are  acidophilic  and  may  be  of 
the  same  size  as  plasmosomes ;  but,  they 
like  others  of  type  B  are  strongly 
acidophilic,  are  seldom  tinged  with 
basic  stains  and  are  generally  surrounded 
by  halos  of  unstained  nucleoplasm. 
Moreover  they  are  not  present  in  normal 
animals. 

Secondly  cells  are  sometimes  encoun- 
tered in  which  there  is  an  increase  in 
acidophilic  nuclear  material  often  ac- 
companied by  nuclear  hypertrophy. 
The  material  may  occur  in  the  form 
of  dense  spherules  or  of  masses  which 
are  bluntly  angular  and  without  halos. 
Colored  illustrations  of  liver  cell  nuclei 
are  given  by  Cowdry,  E.  V.  and  Kitchen, 
S.  F.,  Am.  J.  Hyg.,  1930,  11,  227-299, 
figs.  43  and  44.  These  bodies  may  be 
true  nucleoli  which  have  undergone 
hypertrophy  or  they  may  be  simply 
accumulations  in  the  nuclei  of  aci- 
dophilic material.  The  only  sure  way 
to  tell  would  be  to  ascertain  whether 
they  comport  themselves  like  true 
plasmosomes  during  mitosis  but  the 
cells  involved  have  not  been  seen  in 
division.  In  other  conditions  (^lioma- 
tous  tumors,  etc.)  cells  are  found  whose 
nuclei  are  enlarged  and  possess  roughly 
spherical,  vacuole-like  masses  of  granu- 
lar acidophilic  material.  The  granules 
have  the  appearance  of  coagula  produced 
by  the  fixative  in  a  rather  thin  fluid 
medium.  There  is  no  halo.  Such 
bodies  are  probably  not  altered  plas- 
mosomes.   Their  density  is  much  less. 

Differential  staining.  Nucleoli  are 
colored  brown  after  fixation  in  equal 
parts  of  1%  aq.  chromic  acid  and  10% 
formalin  and  staining  of  chromosomes 
by  Feulgen  Reaction  (Bhaduri,  P.  N., 
J.  Roy.  Micr.  Sci.,  1938,  58,  120-124). 
Nucleonucleolar  Ratio  recommended  as  an 
aid  in  the  grading  of  malignancy  with 


NUCLEOTIDES 


242 


ONCOCYTES 


review  of  the  literature  (Mendes  Fer- 
reira,  H.  E.,  J.  Lab.  &  Clin.  Med.,  1940- 
41,  26,  1612-1628). 

Nucleotides,  see  Diphosphopyridine  Nucleo- 
tide and  Pentose  Nucleotides. 

Nutriles,  growth  promoting  (Williams,  R. 
J.,  Biol.  Rev.,  1941,  16,  49-80). 

Oil  Blue  NA  (Calco)  a  stain  which  colors 
rubber  bright  blue  in  various  plant 
species  (Whittenberger,  R.  T.,  Stain 
Techn.,  1944,  19,  93-102).  This  dye  is 
also  a  good  stain  for  fat  in  animal  cells 
(Lillie,  R.  D.,  Stain  Techn.,  1945,  20, 
7-9). 

Oil  Immersion,  see  Immersion  Oils. 

Oil  Red  IV,  see  Sudan  IV. 

Oil  Red  AS,  O,  B  or  3B,  see  Sudan  III. 

Oil  Red  O  (CI,  73).— fast  oil  orange  II,  fat 
ponceau,  oil  scarlet,  orange  RR,  red  B, 
Sudan  II — an  acid  mono-azo  dye  sug- 
gested as  fat  stain  by  French,  R.  W., 
Stain  Techn.,  1926,  1,  79.  Proescher's 
(F.,  Stain  Techn.,  1927,  2,  60-61)  oil  red 
pyridine  stain  for  fat  is  to  immerse 
frozen  sections  of  formalin,  Muller- 
formalin  (see  Muller's  fluid)  and  5  cc. 
10%  formalin  in  100  cc.  sat.  aq.  picric 
acid  fixed  tissues  in  50%  aq.  pyridine, 
3-5  min.  Stain  3-5  min  in  3-5  gms.  oil 
red  O  dissolved  in  100  cc.  70%  aq. 
pyridine  C.P.  Differentiate  in  50% 
pyridine  several  minutes  and  counter- 
stain  for  2-3  min.  in  Delafield's  Hema- 
toxylin. Mount  in  levulose  syrup.  For 
central  nervous  system  differentiate 
30  min.  in  pyridine  and  use  16  cc.  Dela- 
field's +  2  cc.  glacial  acetic  acid.  Ac- 
cording to  Proescher,  oil  red  O  stains 
fats  and  lipids  more  intensely  and 
quickly  than  Sudan  III  or  IV. 

Oil  Scarlet,  see  Oil  Red  O. 

Oil  Soluble  Dyes.  List  with  physical  prop- 
erties of  each  and  use  as  fat  stains. 
Very  comprehensive  (Lillie,  R.  D.,  J. 
Tech.  Methods,  1944,  24,  37-45). 

Oil  Vermillion,  see  Sudan  R. 

Okajima's  "omnichrom"  stain  (Ito,  T., 
Folia   Anat.   Jap.,    1937,    15,    357-359). 

O'Leary's  Brazilin  Method — Revised  by 
James  L.  O'Leary,  Dept.  of  Neuro- 
psychiatry, Washington  University,  St. 
Louis,  May  8, 1950 — For  myelin  sheaths. 
Run  paraffin,  or  celloidin  sections  of 
properly  fixed  and  mordanted  (Muller's 
Fluid)  tissue  to  water.  After  rinsing 
transfer  to  3%  aq.  potassium  bichro- 
mate or  in  Muller's  fluid,  12-24  hrs. 
Stain  in:  10%  Grubler's  Brazilin  in 
abs.  ale.  (1-6  months  old),  10  cc;  aq. 
dest.,  100  cc;  acetic  acid,  glacial,  5 
drops.  Wash  in  aq.  dest.  Differ- 
entiate in  0.25%  aq.  potassium  per- 
manganate 1-5  min.  Remove  potassium 
permanganate  with  Weil's  solution 
(oxalic  acid,  2.5  gm.;  sodium  bisulphite, 
2.5  gm.;  aq.  dest.  1,000  cc.)     Sections 


should  show  gray  matter  light  pink, 
white  matter  brilliant  red.  Cell  bodies 
stain  in  addition  to  mj^elinated  fibers. 
If  differentiation  not  complete  after 
first  immersion  in  potassium  perman- 
ganate followed  by  oxalic  acid-bisul- 
phite mixture,  repeat  the  procedure. 
Wash,  dehydrate  and  mount.  See 
Golgi-Cox  Method  and  Golgi  Method, 
Quick. 

Oligodendroglia.  Method  for  impregna- 
tion with  silver  in  pyroxylin  (celloidin) 
sections  (Weil,  H.  and  Davenport,  H. 
A.,  Trans.  Chicago  Path.  Soc,  1933,  14, 
95-96).  This  resembles  their  Microglia 
method.  Wash  sections  in  aq.  dest. 
and  transfer  to  aq.  dest.  containing  1 
drop  cone,  ammonia  per  10  cc.  Treat 
for  15-20  sec.  with  silver  solution  made 
up  as  for  microglia  except  that  15% 
aq.  silver  nitrate  is  used  and  the  end 
point  of  the  titration  is  reached  when 
about  12  cc.  of  it  have  been  added  to  the 
2  cc.  cone,  ammonia.  Transfer  to  10% 
formalin  and  allow  section  to  drop  to 
bottom  without  moving  dish.  After 
the  pyroxylin  has  become  deeply  stained 
and  the  tissue  begins  to  take  a  brown 
color,  move  it  with  glass  rods  until  it  is 
stained  coffee-brown.  Use  fresh  forma- 
lin for  each  section.  Pass  section 
through  3  changes  aq.  dest.  Dehydrate 
in  alcohol,  clear  in  xylol  and  mount  in 
balsam. 

Olive  Oil,  reactions  in  tissue  to  fat  stains 
after  various  fixations  (Black,  C.  E., 
J.  Lab.  &  Clin.  Med.,  1937-38,  23, 
1027-1036). 

Oliver,  see  Kidney. 

Omentum,  spreads  of  (McCIung,  p.  336). 
Transplants  of  spleen  into  (Holyoke,E. 
H.,  Am.  J.  Anat.,  1940,  66,  87-132. 

Oncocytes  (G.  onkos,  tumor,  swelling  -f- 
kytos,  cell).  These  cells  are  recogniz- 
able in  ordinary  hematoxylin  and  eosin 
preparations  by  their  (1)  large  size,  (2) 
usually  single  centrally  placed  picnotic 
nuclei  and  (3)  the  large  volume  of  finely 
granular  eosin  staining  cytoplasm  in 
proportion  to  nuclear  volume.  They 
are  most  frequently  encountered  in  the 
parotid  and  submaxillary  glands  but 
they  have  been  reported  in  many  other 
epithelia  including  those  of  the  thyroid, 
parathyroid,  pituitary,  pancreas,  liver, 
stomach,  Fallopian  tube,  uvula,  nose, 
pharynx,  trachea  and  esophagus. 
Nohteri,  H.  (Acta  Path,  et  Micr.  Scand., 
1946,  23,  473-483)  found  them  only  in 
individuals  over  52  years  of  age.  Ham- 
perl,  H.  (Virchow's  Arch.,  1937,  298, 
327-375)  reported  that  they  are  very 
rarely  seen  under  50.  An  excellent 
cytological  account  of  oncocytes  in  the 
salivary  glands  of  a  large  series  of 
animals  of  known  age  is  provided  by 


OPDYKE 


243 


ORGAN  CULTURE  IN  VITRO 


Andrew,  W.  (J.  Gerontol.,  1949,  4,  95- 
103). 
Opdyke,  see  Keratohyalin  Granules,  Sepa- 
ration and  Analysis. 
Opsonocytophagic  Index,  method  for  rapid 
staining  of  blood  smears  in  (Bondi,  A. 
Jr.,  J.  Lab.  &  Clin.  Med.,  1941,  26,  1811). 
Derivation  of  index  number  in  (Foshay, 
L.,  LeBlanc,  T.  J.,  J.  Lab.  &  Clin.  Med., 
1936-37,  22,  1297-1300). 
Opal  Blue  (CI,  689)— Aniline  Blue,  alcohol 
soluble,  Bleu  Lumiere,  Gentiana  Blue 
6B,  Spirit  Blue— a  basic  dye  of  light 
fastness  3,  to  be  employed  in  contrast 
staining  with  Biebrich  Scarlet,  Crocein 
Scarlet  and  other  dyes  (Emig,  p.  50)_. 
Optic  Lens,  methods  of  microincineration 
and  histospectrography  as  applied  to 
cataracts  of  various  sorts  and  normal 
lenses  with  special  attention  to  copper, 
zinc  and  iron  (Busnel,  R.  G.,  Pillet,  P. 
and  Tille,  H.,  Bull.  d'Hist.  AppL,  1938, 
15,  99-109). 

Oral  Mucosa.  Smear  method  for  study  of 
keratinization  (Weinmann,  J.,  J.  Dent. 
Res.,  1940,  19,  57-71).  With  end  of 
agate  spatula  gently  scrape  area  about 
1.5  sq.  cm.  Smear  on  slide,  dry  in  air 
and  stain  for  30  sec.  in  :  sat.  ale.  gentian 
violet  (or  better  crystal  violet)  10  cc.  + 
5%  aq.  phenol,  90  cc.  Lugol's  Iodine, 
30  sec.  Wash  in  water  until  no  more 
color  is  extracted.  Counterstain  for 
2  min.  in  sat.  safranin  O  in  95%  alcohol, 
10  cc.  +  aq.  dest.,  100  cc.  Wash  in 
water  2-3  sec,  dry  and  mount  in  balsam. 

Orange  I  (CI,  150).  Synonyms:  naphthol 
orange,  tropaeolin  G  or  000  No.  1.  An 
acid  mono-azo  dye  used  as  an  Indicator. 

Orange  II  (CI,  151).  Synonyms:  acid 
orange  II,  Y  or  A,  gold  orange,  mandarin 
G,  orange  A,  P,  or  R,  orange  extra, 
tropaeolin  000  No.  2.  An  acid  mono- 
azo  dye.  Ebbinghaus,  H.,  Centralbl. 
f.  allg.  Path.  u.  Path.  Anat.,  1902,  13, 
422-425  employed  gold  orange  with 
hematoxylin  as  a  special  stain  for  keratin. 

Orange  III,  see  Methyl  Orange. 

Orange  A,  P,  or  R,  see  Orange  II. 

Orange  Extra,  see  Orange  II. 

Orange  G  (CI,  27).  Synonym;  wool  orange 
2G.  Of  slightly  different  grade  ac- 
cording to  Conn  (p.  47)  are  orange  GG 
and  GMP.  An  acid  mono-azo  dj'e 
widely  used. 

Orange  MNO  or  MN,  see  Metanil  Yellow. 

Orange  R  (CI,  161),  an  acid  monoazo  dye  of 
light  fastness  3-4  action  of  which  on 
plant  and  animal  tissue  is  described 
(Emig,  p.  33). 

Orange  RR,  see  Oil  Red  O. 

Orcein  (CI,  1242)  is  a  natural  dye  produced 
from  lecanora  parella  (a  lichen)  and 
should  not  be  confused  with  orcin  pro- 
duced from  the  same  plant.  It  is  now 
prepared  synthetically.     Its  precise  for- 


mula remains  to  be  determined  but  it 
is  a  most  valuable  stain  for  Elastic 
Fibers.  Mollier,  G.,  Zeit.  f.  wis.  mikr., 
1938,  55,  472-473  employed  it  with  iron 
hematoxylin,  naphthol  green  B  and 
azocarmine  G.  Acetic-orcein  is  advo- 
cated as  a  new  stain-fixative  for  chromo- 
somes (LaCour,  L.,  Stain  Techn.,  1941 
16,  169-174).  An  acid  orcein  Giemsa 
is  described  for  use  in  dermatology  by 
Pinkus,  H.,  Arch.  Dermat.  and  Syph., 
1944,  49,  35.5-356. 

Orceille,  a  purple  dye,  derived  from  Lichens 
growing  on  the  rocks  of  the  Near  East 
and  Mediterranean  areas,  achieved 
great  favor  among  the  ancients  being 
said  by  Theophrastus  and  Dioscorides 
to  even  excel  Tyrian  Purple.  A  Floren- 
tine dye  trader,  P^ederigo,  promoted 
this  dye,  built  up  a  thriving  business  and 
calling  himself  Orcelli,  founded  a  large, 
distinguished  and  prolific  family  (Leg- 
gett,  W.  F.,  Ancient  and  Medieval 
I)3^es.  Brooklyn:  Chemical  Publishing 
Co.,  Inc.,  1944,  95  pp.). 

Organ  Culture  in  Vitro — Written  by  Honor 
B.  Fell,  Strangeways  Research  Labora- 
tory, Cambridge,  England.  June  8, 
1951 — In  most  forms  of  tissue  culture, 
the  investigator  is  not  concerned  with 
the  original  tissue  fragment  but  with 
the  cells  which  migrate  from  it  and 
multiply  in  the  medium  to  form  a  zone 
of  new  growth.  The  object  of  "organ 
culture"  is  to  grow  tissue  in  a  differen- 
tiated state  as  an  independent  organ- 
ism. 

There  are  several  variations  of  the 
method,  but  in  general  the  e.\plants 
are  grown  on  the  surface  of  a  fairly 
large  volume  of  culture  medium  with 
an  abundant  air  supply.  Tissue  ex- 
tracts used  in  the  preparation  of  the 
medium  are  never  made  from  young 
embryos,  as  such  extracts  have  been 
shown  to  inhibit  differentiation 
(Gaillard,  P.  J.,  Hormones  regulating 
growth  and  differentiation  in  embryonic 
explants.  Act.  Sci.  et  Industr.,  Paris: 
Hermann  et  Cie,  1942).  The  tissue 
fragments  are  transplanted  at  frequent 
intervals,  partly  to  restrict  outgrowth 
which  disintegrates  the  histological 
structure  of  the  explants,  and  partly 
because  the  compact  mass  of  tissue  soon 
exhausts  the  food  material  in  its  im- 
mediate neighborhood. 

Although  as  j^et  the  method  is  prac- 
ticed in  a  few  laboratories  only,  many 
different  types  of  differentiated  tissues 
have  successfully  been  grown  (Fell, 
H.  B.,  J.  Roy.  Micr.  Soc,  1940,  60,  95- 
112;  Chapter  on  "Histogenesis  in  Tissue 
Culture"  in  Bourne,  G.  H.,  Cytology 
and  Cell  Physiology,  Oxford:  Clarendon 
Press,  1951).     Brachet,  A.  (C.  R.  Acad. 


ORGAN  CULTURE  IN  VITRO 


244 


ORGAN  CULTURE  IN  VITRO 


Sci.,  1912,  155,  1191),  Thomson,  G.  D. 
(Proc.  Roy.  Soc.  Med.,  1914,  7  and 
Marcus  Beck  Lab.  Reports,  21) ,  Chlopin, 
N.  (Arch.  f.  Mikr.  Anat.,  1922,  96, 
435-493)  and  Maximow,  A.  (Contrib. 
Embry.  Carnegie  Inst.,  1925,  16,  49- 
115),  showed  that  small  fragments  of 
chicken  and  mammalian  embryos  would 
continue  to  develop  when  cultivated 
in  vitro.  It  has  since  been  found  that 
isolated  organ  rudiments  have  a  sur- 
prising capacity  for  growth  and  differ- 
entiation in  culture.  Thus  the  eye 
rudiment  of  a  3-day  check  embryo  forms 
a  retina  of  almost  adult  type  during 
cultivation  (Strangeways,  T.  S.  P.,  and 
Fell,  H.  B.,  Proc.  Roy.  Soc,  B,  1926, 
100,  273-283);  the  rudiments  of  the 
long-bones  may  increase  to  four  times 
their  original  length  in  11  days,  ossify 
and  even  undergo  considerable  anatom- 
ical development  (Fell,  H.  B.,  and  Robi- 
son,  R.,  Biochem.  J.,  1929,  23,  767-784, 
and  Fell,  unpublished  results) ;  the  early 
stages  of  joint-formation  take  place 
in  vitro  (Fell,  H.  B.  and  Canti,  R., 
Proc.  Roy.  Soc,  B,  1934,  116,  316-351); 
the  thyroid  rudiment  forms  vesicles 
and  secretion  (Carpenter,  E.,  J.  Exper. 
Zool.,  1942,  89,  407-431);  embryonic 
ovaries  produce  sex  cords  and  ova 
(Martinovitch,  P.  N.,  Proc.  Roy.  Soc, 
B,  1938,  125,  232-249,  Ibid,  1939,  128, 
138-143;  Gaillard,  P.  J.,  Proc.  Roy. 
Neth.  Acad,  of  Sci.,  1950,  53).  Some 
organs  which  are  already  largely  or 
completely  differentiated  when  ex- 
planted,  can  be  maintained  in  a  healthy 
state  for  various  periods  in  vitro,  e.g. 
the  prostate  gland  of  infant  rats  (Price, 
D.,  Ann.  Rep.  Strangeways  Res.  Lab., 

1949,  13)  and  mice  (Lasnitzki,  Z.,  Brit. 
J.  Cancer,  in  press),  late  foetal  mouse 
bones  (Fell,  H.  B.  and  Mellanby,  E., 
Brit.  Med.  J.,  1950,  2,  535-539),  rabbit 
lymph  glands  (Trowell,  A.  O.,  Exp. 
Cell  Research,  in  press),  parathyroid 
(Kooreman,  P.  J.,  and  Gaillard,  P.  J., 
Arch.  Chir.  Nederl.,  1950,  2,  326), 
pituitary  (Martinovitch,  P.  N.,  Nature, 

1950,  165,  33-34). 

It  is  possible  to  practice  organ  culture 
under  very  simple  conditions.  The 
writer  works  on  an  open  bench  in  a 
small,  clean  culture  room  with  a  sealed 
window  and  no  outside  ventilation; 
the  door  of  the  culture  room  which 
opens  into  a  larger  laboratory,  is  left 
open.  A  separate  culture  room,  though 
an  advantage,  is  not  necessary,  how- 
ever. No  mask  or  cap  is  worn.  The 
culture  bench  is  covered  with  a  large 
sheet  of  plate  glass;  a  beaker  of  distilled 
water  is  kept  boiling  on  a  tripod  beside 
the  operator  and  instruments,  pipettes, 
etc.  are  frequently  rinsed  in  it.     Direct 


sunlight  is  excluded  by  a  green,  trans- 
lucent plastic  curtain  which  makes  a 
very  pleasant  diffused  light.  Under 
these  conditions  the  writer  often  works 
for  weeks  without  losing  a  single  ex- 
plant  from  contamination.  If  occa- 
sional colonies  should  appear,  the 
culture  room  is  thoroughly  steamed  by 
boiling  a  bucket  of  water  over  a  gas 
ring  for  some  hours  and  the  walls, 
ceiling,  floor  and  benches  are  then 
wiped  with  a  cloth  impregnated  with 
lysol.  This  procedure  usually  abolishes 
any  airborne  infection. 

It  may  well  be  that  in  a  city,  or  in 
a  hot  dusty  atmosphere,  much  more 
stringent  precautions  may  be  necessary 
than  these  simple  arrangements,  but 
it  is  advisable  to  try  the  simple  organ- 
isation first  and  only  elaborate  when 
strictly  necessary. 

1.  Preparation  of  tissue.  In  experi- 
ments with  early  organ  rudiments,  the 
most  difficult  and  also  the  most  im- 
portant part  of  the  technique  is  to 
dissect  and  handle  the  delicate  tissues 
without  damaging  the  cells.  For  this 
it  is  essential  to  prepare  adequately 
fine  instruments.  The  writer  uses  a 
narrow  Graefe's  cataract  knife  and  an 
ordinary  sewing  needle  broken  in  half 
and  mounted  in  a  thin  glass  rod. 

A  No.  1  knife  is  best  but  it  is  much 
too  thick  and  coarse  as  it  comes  from 
the  makers,  and  for  the  dissection  of 
early  embryos  it  must  be  ground  to  a 
suitable  shape  by  the  worker  himself. 
It  is  first  rubbed  on  a  fairly  coarse  car- 
borundum stone  to  render  the  distal 
third  of  the  blade  as  thin  and  flexible 
as  possible,  and  is  then  smoothed  and 
shaped  on  an  Arkansas  stone.  Through- 
out the  sharpening  process  the  knife  is 
repeatedly  examined  under  a  dissect- 
ing binocular  microscope  and  when 
finished,  the  end  third  of  the  blade 
should  be  narrow,  very  flexible  and  have 
a  perfect  point;  the  flexibility  should 
be  carefully  graded  or  the  end  will  snap. 
In  the  writer's  experience  beginners 
have  great  difficulty  in  learning  to  pre- 
pare knives  properly. 

The  needle  is  mounted  by  melting 
the  tip  of  a  glass  rod  and  pushing  the 
broken  end  of  the  needle  into  it.  The 
needle  should  not  be  stuck  too  far  into 
the  rod  or  the  glass  may  crack  during 
sterilisation.  To  sharpen  the  point, 
the  needle  is  rotated  with  the  finger  and 
thumb  and,  at  the  same  time  it  is  rubbed 
on  the  end  of  an  Arkansas  stone.  It 
should  be  remembered  that  where 
needles  are  sharpened  the  stone  is 
spoilt  for  knives. 

When  the  knives  and  needles  are 
ready,  they  are  carefully  wiped  with 


ORGAN  CULTURE  IN  VITRO 


245 


ORGAN  CULTURE  IN  VITRO 


a  clean  duster,  then  rinsed  in  two 
changes  of  fresh  absolute  alcohol  and 
thoroughly  dried  on  a  glass  cloth.  It 
is  convenient  to  sterilize  a  knife  and 
a  needle  together  in  a  single  tube.  A 
very  firm,  deep  wad  of  cotton  wool  is 
ranimed  into  the  bottom  of  a  test  tube, 
the  instruments  are  slid  gently  into  the 
tube  which  is  corked  with  a  cotton  wool 
bung  and  sterilized  by  dry  heat.  It 
is  advisable  to  sterilize  the  knives  and 
needles  point  downwards,  otherwise 
moisture  may  condense  on  the  steel 
and  corrode  it. 

After  use,  the  instruments  should  be 
very  carefully  wiped  to  avoid  rusting 
and  re-sharpened  before  being  sterilized 
again.  When  once  the  knives  have 
been  properly  ground,  it  takes  only  a 
few  minutes  to  clean  and  sharpen  them 
on  the  Arkansas  stone. 

The  explants  are  prepared  under  a 
dissecting  binocular,  the  stage  of  which 
is  fitted  with  a  small  glass  shade  to  pre- 
vent airborne  contamination  during 
the  manipulation.  It  is  important  to 
select  the  right  magnification  and  it  is 
often  convenient  to  make  the  gross  dis- 
section (e.g.  the  removal  of  a  limb  or 
eye  from  the  body)  under  a  low  power 
which  provides  ample  depth  of  focus, 
and  to  complete  the  process  under  a 
higher  magnification.  The  writer  uses 
transillumination  for  the  dissection 
which  is  done  in  a  large  hollow  ground 
slide  containing  Tyrode;  the  tissue 
should  not  be  left  in  Tyrode  for  longer 
than  is  absolutely  necessary. 

It  is  important  to  remove  from  the 
rudiments  as  much  of  the  epidermis 
as  possible,  otherwise  the  epithelium 
envelopes  the  explant,  keratinizes  and 
the  imprisoned  cells  degenerate.  To 
detach  the  epidermis  the  very  thin, 
sharp  knife  blade  is  slid  beneath  it  and 
the  tissue  is  then  cut  by  gently  stroking 
the  edge  of  the  knife  with  the  point  of 
the  needle.  It  is  essential  to  reduce 
trauma  to  a  minimum,  or  the  explants 
will  not  prosper  during  subsequent 
cultivation. 

There  is  an  optimum  size  of  explant. 
If  it  is  very  minute  it  may  not  thrive 
in  vitro,  though  sometimes  this  difficulty 
may  be  overcome  by  placing  it  in  con- 
tact with  some  other  tissue  (Borghese, 
E.,  J.  Anat.,  1950,  84,  303).  If  the 
explant  is  too  large  the  interior  be- 
comes necrotic.  There  may  also  be 
an  optimum  stage  of  development  at 
which  a  given  tissue  should  be  ex- 
planted.  If  taken  too  early  it  may  not 
differentiate  in  complete  isolation, 
while  if  removed  from  the  embryo  a 
few  hours  later  it  may  attain  an  almost 
adult  structure.     On  the   other  hand, 


if  explanted  when  differentiation  is 
already  well  advanced,  the  tissue  may 
be  unable  to  adapt  itself  to  life  in  vitro 
and  either  degenerate  or  lose  its  charac- 
teristic structure. 

2.  Watch-glass  method.  In  the 
Strangeways  Research  Laboratory  a 
simple  form  of  moist  chamber  is  em- 
ployed for  organ  culture  (Fell,  H.  B. 
and  Robison,  R.,  Biochem.  J.,  1929, 
23,  767-784).  It  consists  of  a  Petri 
dish  80  mm.  in  diameter  and  10  mm. 
deep,  carpeted  with  a  thin  layer  of  ab- 
sorbent cotton  wool  in  the  center  of 
which  a  round  hole  is  cut.  A  watch- 
glass  40  mm.  in  diameter  is  laid  over  the 
hole  and  the  dish  is  sterilized  by  dry 
heat.  Ten  cubic  centimeters  of  sterile 
aq.  dest.  is  pipetted  into  the  Petri 
dish.  It  is  not  essential  to  use  exactly 
the  size  of  watch-glass  and  Petri  dish 
mentioned;  but  it  is  important  that  the 
diameter  of  the  Petri  dish  should  be 
fairly  large  relative  to  that  of  the  watch- 
glass  for  otherwise  the  culture  medium 
will  tend  to  dry. 

The  culture  medium  is  dropped  into 
the  watch-glass  with  a  pipette;  the 
first  drop  is  always  deposited  on  the 
cotton  wool  as  this  is  found  to  reduce 
the  risk  of  infection.  The  type  of 
medium  depends  on  the  tissue  to  be 
cultivated.  Many  avian  and  mam- 
malian rudiments  grow  well  in  a  mixture 
of  fowl  plasma  and  the  extract  of  an  old 
(12-14  day)  chick  embryo.  Bone  rudi- 
ments grow  very  well  in  a  mixture  of 
3  pts.  of  plasma:  1  pt.  of  concentrated 
extract  of  a  14-day  chick  embryo  made 
with  Tyrode  containing  1%  glucose, 
so  that  the  final  medium  contains  0.25% 
glucose.  It  is  important  to  see  that 
the  tissue  has  a  suitable  depth  of  me- 
dium beneath  it,  as  large  explants  soon 
exhaust  and  partly  liquefy  the  clot  in 
their  neighborhood.  For  chick  bone 
rudiments  of  about  1.25  mm.  in  length 
the  writer  uses  12  drops  of  medium, 
but  by  the  time  they  have  attained  a 
length  of  about  5  mm.  she  adds  20  drops 
to  each  watch-glass.  Organ  cultures 
are  often  more  fastidious  about  their 
culture  medium  than  unorganised  tis- 
sues. It  is  therefore  advisable  to  use 
freshly  made  embrj-o  extract  and 
plasma  which  are  not  more  than  a  week 
old.     Antibiotics  are  unnecessary. 

For  many  mammalian  tissues  an  ad- 
mixture of  homologous  plasma  is  desir- 
able. Martinovitch,  P.  N.  (Nature,  1950, 
165,  33-34)  grows  the  anterior  pituitary 
of  young  rats  in  a  mixture  of  6  drops  of 
heparinized  rat  plasma,  3  drops  of 
chicken  plasma  and  3  drops  of  concen- 
trated chick  embryo  extract. 

When  the  organ  rudiments  are  ready 


ORGAN  CULTURE  IN  VITRO 


246 


ORGAN  CULTURE  IN  VITRO 


for  explantation,  the  writer  washes 
them  in  fresh  Tyrode  which  is  then 
removed  and  replaced  by  embryo  ex- 
tract of  about  half  the  concentration 
of  that  used  for  the  culture  medium 
and  lacking  the  extra  glucose.  They 
are  sucked  into  a  pipette  and  deposited 
on  the  surface  of  the  clot  in  the  watch- 
glass.  The  Petri  dish  is  then  placed 
under  the  shade  on  the  dissecting 
binocular  microscope,  the  lid  removed 
and  the  surplus  extract  carefully  sucked 
off  with  an  extremely  fine  pipette,  care 
being  taken  not  to  damage  the  clot. 
Organ  cultures  do  not  grow  well  under 
a  fluid  phase,  but  they  quickly  become 
surrounded  by  a  shallow  pool  of  fluid 
exuded  from  the  clot. 

The  Petri  dishes  are  not  sealed,  but 
under  the  conditions  described,  the 
medium  does  not  dry.  If  the  dishes 
are  laid  directly  on  the  incubator  shelf, 
drops  of  moisture  form  on  the  inner 
surface  of  the  lid,  but  this  can  easily 
be  prevented  by  placing  the  vessels  on 
planks  of  wood  which  are  left  perma- 
nently in  the  incubator.  Should  drops 
form  in  spite  of  this  precaution,  it  is 
probable  that  the  temperature  of  the 
incubator  is  fluctuating  unduly. 

Many  tissues  grow  and  develop  well 
at  normal  body  temperature,  but  others 
do  better  if  the  temperature  is  lowered 
to  34°C.  Martinovitch  has  kept  rat 
and  mouse  ovarian  explants  and  rat 
pituitaries  in  a  healthy  differentiated 
state  for  several  weeks  at  34°C. 

The  writer  transfers  explants  to  fresh 
medium  three,  or  in  the  case  of  large, 
rapidly  growing  avian  rudiments,  four 
times  a  week.  To  make  the  fourth 
transplantation,  she  prepares  a  double 
set  of  watch-glasses  on  the  Friday  of 
each  week;  one  set  is  employed  the  same 
day  and  the  other  placed  in  the  refriger- 
ator for  use  on  the  Saturday  when 
the  explants  are  again  subcultivated. 
They  are  then  left  undisturbed  until 
the  Monday.  This  routine  gives  excel- 
lent results  for  cultures  of  avian  long 
bone  rudiments. 

During  incubation,  some  types  of 
explant  become  firmly  anchored  to  the 
clot  by  migrating  cells.  To  detach  the 
tissue  without  damage,  the  clot  is 
pulled  away  on  one  side  so  as  to  rupture 
the  zone  of  outgrowth;  the  explant  is 
then  loosened  from  the  underlying 
clot  with  the  knife  and  needle,  after 
which  the  zone  of  growth  on  the  other 
side  is  ruptured,  so  that  the  explant 
now  lies  freely  in  the  pool  of  exuded 
fluid.  It  is  advisable  not  to  rupture 
both  sides  of  the  zone  of  growth  before 
detaching  the  explant  from  the  clot; 
none  of  the  old  clot  should  be  left  at- 


tached to  the  tissue.  The  explant  is 
sucked  into  a  pipette  containing  a  little 
Tyrode  and  deposited  in  a  hollow- 
ground  slide  in  a  Petri  dish.  The 
Tyrode  is  replaced  by  the  more  dilute 
extract  mentioned  above  and  the  tissue 
is  placed  on  a  fresh  clot  in  the  manner 
already  described. 

3.  Gaillard's  method.  Professor  P.  J. 
Gaillard  (personal  communication)  uses 
a  rather  different  technique  which  has 
given  excellent  results  in  the  cultiva- 
tion of  the  parathyroid  from  human 
infants  and  of  the  human  fetal  ovary. 

The  explants  are  cultivated  in  em- 
bryological  watch  glasses  sealed  with 
a  small  glass  plate.  They  are  grown 
on  a  very  soft  clot  composed  of  a  mix- 
ture of  (a)  5-15%  adult  human  blood 
plasma  containing  0.5  cc.  of  a  0.1% 
solution  of  heparin  for  10  cc.  of  blood, 
(b)  10%  placental  vein  serum,  (c) 
65-75%  of  Gey's  balanced  saline  solu- 
tion, (d)  human  fetal  brain  press  juice. 
To  make  the  brain  press  juice,  frag- 
ments of  fetal  brain  are  placed  in  a 
Petri  dish  at  4°C.  for  24  hrs.  which 
facilitates  the  separation  of  the  tissue 
fluid;  the  tissue  is  then  minced  with  a 
tissue  press,  an  equal  quantity  of  Gey's 
saline  solution  is  added  and  the  mix- 
ture is  centrifuged  for  15  min.  at  6,000- 
8,000  r.p.m.  The  supernatant  fluid  is 
decanted  into  Pyrex  glass  tubes  and 
stored  at  —  20°C.  The  explants  are 
transferred  to  fresh  medium  twice  a 
week. 

Parathyroid  explants  completely 
liquefy  the  clot  in  3  days.  During  the 
first  few  days  in  vitro  the  peripheral 
zone  degenerates  and  is  washed  away 
during  transplantation,  leaving  a 
healthy  ball  of  tissue  which  enlarges 
during  subsequent  cultivation  and  may 
survive  for  as  long  as  60  days.  The 
central  part  of  the  ovarian  cultures 
degenerates,  but  the  necrotic  matter 
is  later  resorbed  and  replaced  by 
healthy  tissue  growing  in  from  the 
periphery. 

4.  Other  methods.  Recently  Trowell 
has  devised  a  modification  of  the  watch- 
glass  method  which  can  be  used  for 
metabolic  studies  and  which  enables 
organized  tissue  to  be  grown  in  a  fluid 
medium  in  any  type  of  gaseous  at- 
mosphere required.  He  has  success- 
fully grown  rabbit  lymph  glands  by 
this  technique  which  should  be  ap- 
plicable to  many  physiological  prob- 
lems. 

Some  rudiments  develop  well  in 
tubes  (Strangeways,  T.  S.  P.  and  Fell, 
H.  B.,  Proc.  Roy.  Soc,  B,  1926,  100, 
273-283).  The  medium  is  placed  in  a 
small  test  tube  (2"  x  ^")  and  allowed  to 


ORGANOIDS 


247 


OSMIC  ACID 


clot.  The  explant  is  deposited  on  the 
surface  of  the  clot  and  the  tube  is 
corked  and  incubated  in  a  vertical 
position.  At  2-3  day  intervals  the 
explant  is  removed  from  the  clot  with 
a  pipette,  washed  and  re-planted  in  a 
fresh  tube. 

Although  large  explants  thrive  best 
in  some  form  of  watch-glass  culture, 
ver}'  small  rudiments  may  do  better 
in  large  hanging  drop  preparations 
(li  inch  square  coverslips  on  3  x  I5 
inch  hollow  ground  slides)  (Jacobson 
W.  and  Fell,  H.  B.,  Quart.  J.  Micr. 
Sci.,  1941,  82,  563-586;  Borghese,  E., 
J.  Anat.,  1950,  84,  287-302).  When  the 
tissue  is  transplanted  most  of  the  zone 
of  outgrowth  is  cut  away  and  the  ex- 
plant  is  preserved  intact. 

Application  of  Organ  Culture:  The 
cultivation  of  organized  tissue  has  an 
almost  unlimited  number  of  possible 
applications.  So  far  it  has  been  used 
mainly  by  embryologists  for  investiga- 
tions in  developmental  mechanics, 
because  explants  of  embryonic  tissue 
are  so  readily  accessible  to  manipula- 
tion and  observation.  An  important 
and  almost  unexploited  field,  however, 
is  the  study  of  tissue  metabolism  by 
this  method.  Much  work  has  been 
done  on  the  growth  requirements  and 
ph3^siology  of  unorganised  tissue  cul- 
tures, but  very  little  on  the  metabolism 
of  the  many  types  of  differentiated 
tissue  which  can  be  grown  in  vitro  and 
which  might  provide  information  which 
would  be  impossible  to  obtain  from  the 
intact  animal  or  from  short-lived  tissue 
slices. 

It  will  be  seen  from  the  foregoing 
description  of  technique  that  the  neces- 
sary procedure  and  equipment  for  this 
kind  of  tissue  culture  are  not  elaborate. 
All  that  is  needed  for  success  is  skill 
in  fine  dissection  and  a  sound  knowledge 
of  histology  and  embryology. 

Organoids  (G.  organon,  organ  -+-  cidos, 
appearance).  The  term  organoid  is 
not  a  happy  one.  It  is  used  to  denote 
the  organ-like  appearance  of  some 
structure  that  the  user  fails  accurately 
to  describe  as  in  the  case  of  some 
tumors.  Also,  certain  bodies,  such  as 
the  mitochondria,  are  occasionally 
listed  as  organoids,  or  organelles,  con- 
veying an  unwarranted  impression  of 
similarity  to  the  complex  organs  of 
the  body. 

Origanum  Oil.  With  it  tissues  can  be 
cleared  from  95%  alcohol,  but  care  must 
be  taken  to  obtain  a  pure  product. 
The  kind  required  consists  of  carvacrol 
and  cymene  terpenes.  Ordinary 
origanum  oil  is  oil  of  thyme. 

Orseillin  BB  (CI,  284).    A  little  used  acid 


dis-azo  dye.  See  Cohen,  I.,  and  Doak, 
K.  D.,  Stain  Techn.,  1935,  10,  25-32. 
For  staining  fungi  (Alcorn,  G.  D.  and 
Yeager,  C.  C,  Stain  Techn.,  1937,  12, 
157-158). 

Orthochromatic  Erythroblasts,  see  Ery- 
throcytes, developmental  series. 

Orth's  Fluid.  Potassium  bichromate,  2.5 
gm.;  aq.  dest.,  100  cc,  formalin,  10  cc. 
The  1  gm.  sodium  sulphate  originally 
advised  by  Orth  i.s  omitted  as  useless. 
Since  the  fluid  does  not  keep  it  should 
be  made  up  immediately  before  use. 
Regaud's  fluid,  the  best  fixative  for 
mitochondria,  is  the  same  except  that 
the  amount  of  formalin  is  increased 
See  Lithium  Carmine  (Orth). 

Osage  Orange  Pigments  as  brilliant  mordant 
dyes  for  wool  and  silk.  Wolfsom, 
M.  L.,  Harris,  W.  D.,  Johnson,  G.  F., 
Mahan,  J.  E.,  Moffett,  S.  M.  and  Wild!, 
B.,  J.  Am.  Chem.  Soc,  1940,  68,  406- 
418.     Should  be  tried  on  animal  tissues. 

Osmic  Acid.  This  is  the  tetroxide  of 
osmium  and  has  no  acid  properties. 
It  comes  in  sealed  glass  tubes  usually 
each  containing  1  gm.  To  make  the  2% 
aq.  sol.of  osmic  acid  generally  employed, 
wash  the  label  off  the  tube  with  soap 
and  water.  After  washing  repeatedly 
in  aq.  dest.  rinse  in  absolute  alcohol  and 
dry.  Carefully  clean  the  inside  of  a 
glass  stoppered  bottle  and  of  a  graduate 
in  the  same  way.  With  clean  forceps  put 
the  tube  in  the  bottle.  If  it  is  not  easily 
broken  by  vigorous  shaking  it  will  be 
necessary  to  take  it  out,  file  one  side, 
break  and  return  to  the  bottle.  Finally 
add  50  cc.  of  aq.  dest,  measured  in  the 
graduate.  The  osmic  acid  slowly  dis- 
solves forming  a  clear  light  yellow  solu- 
tion. Do  not  hasten  solution  by  heat. 
Keep  in  dark  or  subdued  light.  To  use 
a  bottle  made  of  colored  glass  or  the  out- 
side of  which  has  been  blackened  is  a 
bad  practice  because  it  hides  the  con- 
dition of  the  solution  from  the  person 
using  it.  If  there  is  a  blackening  of  the 
solution  its  potency  is  probably  reduced. 
An  indicator  of  concentration,  dis- 
covered by  Tschngaeff,  lias  been  im- 
proved by  Palmer  (R.,  J.  Roy.  Micr. 
Soc,  1930,50,  221-226). 

The  fumes  of  osmic  acid  are  very  in- 
jurious to  the  eyes.  They  are  a  good  fixa- 
tive for  well  separated  cells  as  in  smears. 
They  blacken  the  chromaffin  cells  of  the 
adrenal  charged  with  epinephrine  or  its 
precursor  (Cramer,  W.,  Fever,  Heat 
Regulation,  Climate  and  Thyroid- 
Adrenal  Apparatus.  London:  Long- 
mans, Green  &  Co.,  1928,  153  pp.) 
Alone,  a  solution  of  osmic  acid  is  a  fair 
fixative  for  mitochondria  and  by  pro- 
longed action  may  reveal  the  Golgi 
apparatus.     See  critique  by  Owens  and 


OSMIC  ACID  METHOD 


248 


OVARY 


Bensley  (H.  S.  and  R.  R.,  Am.  J.  Anat.. 
1929,  44,  79-109).  But  osmic  acid 
penetrates  very  badly  indeed  and  is  best 
employed  in  mixtures  with  other  chem- 
icals as  in  the  fixatives  of  Altmann, 
Mann,  Bensley,  Flemming  and  others. 
Its  chief  value  is  that  it  blackens  many 
but  not  all  fatty  droplets.  However  it 
also  blackens  some  materials  which  are 
not  fatty.  Osmic  acid  plays  an  impor- 
tant part  in  the  Marchi  method  for 
nerve  fiber  degeneration. 

Osmic  Acid  Method  for  fat.  When  reduced 
to  osmium  dioxide  in  the  presence  of 
some  fats  it  blackens  them  as  may  be 
seen  by  the  examination  of  tissues  fixed 
in  fluids  containing  osmic  acid  (Alt- 
mann's,  Flemming's  etc.)  but  unless 
rigidly  controlled  other  substances  may 
be  blackened  as  well  or  not  all  of  the  fats 
may  be  shown.  See  remarks  by  Owens, 
H.  B.  and  Bensley,  R.  R.,  Anat.  Rec, 
1929,  44,  79-109.  It  is  best  to  proceed 
as  advised  by  Mallory  (p.  119).  Place 
frozen  sections  of  tissue  fixed  in  10% 
formalin  for  24  hrs.  in  aq.  dest.  1%  osmic 
acid  24  hrs.  (or  Flemming's  or  Marchi 's 
solution).  Wash  thoroughly  in  running 
water  6-12  hrs.  Abs.  ale.  for  several 
hours  in  order  to  get  secondary  stain- 
ing of  palmitic  and  stearic  compounds  as 
well  as  of  oleic.  Wash  in  water  and 
mount  in  glycerin  jelly  (glycerin  alone 
will  do).  Fat  is  black  against  a  yellow- 
ish brown  background.  Non-fatty  sub- 
stances like  tannic  acid  and  eleidin  of 
epidermis  are  also  blackened. 

For  nerve  fibers  (Dr.  J.  L.  O'Leary, 
personal  communication).  Use  fresh  or 
10%  formalin  fixed  material.  Tie  a 
stretch  of  freshly  isolated  nerve  to  short 
length  of  glass  rod  and  immerse  in  2% 
aq.  osmic  acid.  Leave  for  24  hrs.  Wash 
4-6  hrs.  in  running  water.  Dehydrate 
in  ascending  alcohols  and  doubly  imbed 
by  the  Peterfi  method  as  follows :  Pour 
1%  celloidin  in  methyl  benzoate  (which 
takes  about  1  month  to  dissolve)  into  a 
dish.  Add  absolute  alcohol  and  the  tis- 
sue. The  latter  gradually  sinks  into  the 
celloidin.  Transfer  to  2-3%  celloidin 
in  methyl  benzoate.  Leave  2-4  days. 
Drop  tissue  directly  into  benzol.  After 
a  few  hours  in  benzol  begin  infiltration 
in  paraffin  at  40°C.  This  takes  12-24 
hrs.  Change  paraffin  several  times  and 
imbed. 

Ossicles,  see  Ear. 

Ossification.  Demonstration  of  in  embryos 
and  fetuses  up  to  18  weeks  by  staining 
with  alizarin  red  S  (Richmond,  G.  W. 
and  Bennett,  L.,  Stain  Techn.,  1938, 
13,  77-79).  Eviscerate.  Fix  in  95% 
alcohol  2  weeks  or  more.  Rinse  in  tap 
water  and  put  in  1%  aq.  KjCOj  for 
month  or  longer.     Clear  soft  parts  and 


make  bones  clearly  visible  by  placing  in 
1%  aq.  KOH  for  10  days  or  more.  (Spec- 
imens fixed  in  formalin  instead  of  alco- 
hol require  about  1  month  in  10%  KOH) 
If  tissues  become  too  soft  harden  in 
equal  parts  glycerin,  95%  alcohol  and 
water  12-24  hrs.  and  continue  KOH  if 
necessary.  In  last  few  days  reduce 
KOH  to  0.5%.  Wash  in  running  tap 
water  12  hrs.  Immerse  in  0.1%  aq. 
alizarin  red  S  to  which  few  drops  1% 
aq.  KOH  has  been  added  for  30-60 min. 
Wash  for  30  min.  in  running  tap  water. 
Remove  deep  purple  color  from  soft 
parts  by  immersing  in  20%  aq.  glycerin 
containing  1%  KOH.  For  small  speci- 
mens reduce  KOH  to  0.5%.  This  de- 
colorization  may  require  1-2  weeks  be- 
fore ossified  skeleton  remains  deep  red 
in  transparent  background.  Dehydrate 
by  passing  slowly  through  95%  ale, 
glycerin  and  aq.  dest.  in  following  pro- 
portions 10 :  20 :  70—20 :  20 :  60—30 :  30 :  40— 
40:40:20—50:50:0.  Seal  in  specimen 
jar  in  the  final  mixture  of  alcohol  and 
glycerin. 

A  rather  similar  technique  leading  up 
to  dehydration  in  absolute  alcohol, 
clearing  in  toluol  and  final  storage  in 
anise  oil  saturated  with  naphthalene  is 
presented  by  Cumley,  R.  W.,  Crow,  J. 
F.  and  Griffen,   A.   B.,   Stain  Techn., 

14,  7-11.  This  staining  of  ossification 
centers  with  alizarin  red  can  be  com- 
bined with  the  coloration  of  the  carti- 
laginous skeleton  with  toluidin  blue  to 
make  quite  brilliant  specimens  (Wil- 
liams, T.  W.,  Stain  Techn.,  1941,  16,  23- 
25). 

Ossification,  intense  glycogenesis  during 
(Gendre,  H.,  Bull.  d'Hist.  AppL,  193S, 

15,  165-178). 

Otoliths,  technique  for  (Johnston,  M.,  J. 
Roy.  Micr.  Soc,  1938,  58,   112-119). 

Ova,  concentration  of  parasitic  ova  in  Feces. 

Ovalocytosis,  see  Pencil  Red  Cells. 

Ovary.  For  routine  purposes  fixation  in 
Zenker's  Fluid  and  coloration  by  Mal- 
lory's  Connective  Tissue  stain  or  by 
Masson's  Trichrome  technique  is  in- 
dicated. Follicular  atresia  can  be  beau- 
tifully demonstrated  by  Vital  Staining 
with  trypan  blue  or  by  other  similar 
dyes,  see  Evans,  H.  M.  and  Swezy,  D. 
R.,  Memoirs  Univ.  California,  1931, 
9,  119-224.  For_  the  utilization  of 
Microdissection  in  determination  of 
the  physical  properties  of  the  follicular 
wall  see  Thanhoffer,  L.,  Zeit.  f.  Anat. 
u.  Entw.,  1933,  100,  559-562.  The  in- 
teresting fluorescence  studies  on  the 
ovary  by  Policard,  A.,  C.  rend.  Acad, 
d.  Sci.,  1924,  179,  1287  are  likely  to  be 
extended  now  that  the  possibilities  of 
Fluorescence  Microscopy  are  better 
appreciated.     Ragins,  A.  R.  and  Pop- 


OWEN'S  BLUE 


249 


OX  ID ATION  -REDUCTION 
POTENTIAL 


per,  H.,  Arch.  Path.,  1942,  36,  647-662 
nave  indeed  investigated  variations  in 
ovarian  fluorescence  during  cj^clical 
changes. 

Owen's  Blue  (British  Drug  Houses  Ltd.),  a 
dis-azo  dye  similar  in  composition  to 
Manchester  blue.  Used  best  in  alco- 
holic solution  (H.  G.  Cannan,  J.  Roy. 
Micr.Soc,  1941,61,88-94). 

Oxalate  Solutions,  see  Anticoagulant  Solu- 
tions. 

Oxazins.  Dyes  resembling  the  thiazins  but 
in  which  sulphur  atom  is  replaced  by 
oxygen.  Examples:  brilliant  cresyl 
blue,  celestin  blue  B,  cresyl  violet,  gal- 
lamin  blue,  gallocyanin,  Nile  blue  sul- 
phate, resorcin  blue. 

Oxidase.  Unfortunately,  as  Lison  (p.  263) 
points  out,  histologists  and  biochemists 
are  not  always  agreed  as  to  terms.  The 
latter  include  under  the  designation 
"o.xidases"  all  enzymes  capable  of  cata- 
lysing a  reaction  of  oxidation,  for  in- 
stance the  phenolases,  purinoxidases, 
succinoxidase,  tyrosinase,  etc.;  whereas 
what  the  former  describe  as  "oxidases" 
are  in  reality  phenolases  and  thus  only  a 
part  of  the  whole  group  of  oxidases. 
The  action  of  oxidase  (or  phenolase)  in 
the  presence  of  Oj  is  the  same  as  a  per- 
o.xidase  in  the  presence  of  H2O2.  But 
the  particular  oxidases  are  more  delicate 
and  easily  modified  in  their  action  by 
variations  in  temperature,  pH  and  other 
factors.  The  following  methods  are 
from  Lison,  much  abbreviated. 

1.  M.  nadi  oxidase  reaction  (Gratf) 
=  oxidase  reaction,  modification  A  (W. 
H.  Schultze)  and  stabile  oxidase  reac- 
tion (V.  Gierke).  Make  2  solutions  :  A. 
Boil  1  gm.  anaphthol  in  100  cc.  aq.  dest. 
Add  drop  by  drop  25%  aq.  potassium 
hydroxide  until  melted  a  naphthol  is 
dissolved.  Cool.  Can  be  kept  in  dark 
at  least  1  month.  B.  Obtain  good 
sample  dimethyl  -  p  -  phenylenediamine 
furnished  in  sealed  tubes.  It  blackens 
quickly  when  secured  in  bulk.  Graff 
advised,  as  more  stable,  dimethyl-p- 
phenylenediamine  hydrochloride.  Make 
1%  solution  of  either  in  aq.  dest.  Boil 
and  cool.  Keeps  2-3  weeks  in  dark. 
Immediately  before  using  take  equal 
parts  A  and  B,  filter  and  employ  filtrate. 
Place  frozen  sections  of  formalin  fixed 
tissues  or  smears  (after  fixing  for  2  hrs. 
in  formalin  vapor  or  in  formol,  10  cc. 
+  96%  alcohol,  40  cc.)  in  above  mixture 
of  A  and  B  in  a  thin  layer  at  the  bottom 
of  a  Petri  dish.  .\gitJite  a  little  to  per- 
mit oxygenation  of  the  fluid.  Blue 
granules  quickly  appear  (1-5  min.). 
Rinse  in  water  and  examine.  To  make 
more  permanent  treat  with  Lugol's 
iodine  diluted  one  third,  2-3  min., 
which  makes  the  blue  granules  brown. 


Restore  blue  by  washing  in  aq.  dest. 
-f  few  drops  sat.  aq.  lithium  carbonate. 
Counterstain  with  hemalum  or  .safranin. 
mount  in  glycerin.  Schmorl  advised 
instead  of  Lugol's  a  cone.  aq.  sol.  am- 
monium molybdate. 

2.  G.    nadi   oxidase    reaction    (Graff) 
=  labile  oxidase  reaction   (V.  Gierke). 

This  more  difficult  method  is  for  fresh 
tissues.  The  nadi  reagent  is  prepared 
without  addition  of  alkali.  The  re- 
quired pH  depends  on  the  cells  investi- 
gated. For  animal  tissues  Lison  recom- 
mends about  8.2,  8.1  and  7.8  and  for 
plants  3.4-5.9.  Directions  are  given 
by  Grjiff  (S.,  Die  Mikromorphologischen 
Methoden  der  Fermentforschung,  Ab- 
derhalden's  Handb.,  1936,  4  (1),  93-142). 

3.  Naphthol  reaction  of  Loele.  This 
is  not,  in  the  opinion  of  Lison,  strictly 
speaking  a  microchemical  reaction,  but 
it  is  as  simple.  Place  small  amount 
a  naphthol  in  a  test  tube.  Add  drop  by 
drop  10%  aq.  potassium  hydroxide  until 
naphthol  is  completely  dissolved.  Add 
200  cc.  aq.  dest.  Solution  may  be  used 
after  24  hrs.  It  will  last  about  3  weeks. 
Frozen  sections  of  formalin  fixed  tissues 
treated  with  this  reagent  show  violet  or 
black  granules,  which  quickly  disappear. 

Oxidation-Reduction  Potential.  Written 
by  Christopher  Carruthers,  Division 
of  Cancer  Research,  Washington  Uni- 
versity, St.   Louis    10.    May   12,    1950. 

This  very  important  measurement  is 
particularly  well  explained  by  Seifriz, 
W.,  Protoplasm,  New  York:  McGraw- 
Hill  Book  Co.,  1936,  584  pp.  For  a 
comprehensive  developmental  treat- 
ment of  the  subject  see  Clark,  W.  M. 
and  coworkers,  Hygienic  Laboratory 
Bull.,  1928,  151,  1-352. 

Oxidation  is  the  process  in  which  a 
substance  loses  electrons,  and  reduction 
is  the  process  in  which  a  substance  takes 
on  electrons.  For  example  when  ferric 
chloride  FeCU  gains  an  electron  it  is 
reduced  to  FeCU,  or 

Fe"''"'^  +  electron  — ►  Fe'^ 

Because  the  ion,  Fe"*^,  can  lose  an  elec- 
tron it  is  a  reducing  agent  or  reductant, 
and  since  Fe"'"++  can  gain  an  electron  it 
is  an  oxidizing  agent  or  oxidant.  The 
change  is  reversible 

Fe-'-^  -t-  electron  :^  Fe++. 

When  an  acid  mixture  of  ferrous  and 
ferric  chloride  is  placed  in  an  electrode 
vessel  it  will  yield  a  potential— the  oxi- 
dation potential.  This  potential  can 
be  measured  by  placing  a  noble  metal, 
such  as  a  bright  platinum  wire  in  the 
solution,  and  measuring  the  potential 
against  the  normal  calomel  electrode 
with  a  potentiometer.  The  intensity 
of  the  oxidizing  or  reducing  action  of  a 


OXIDATION-REDUCTION 
POTENTIAL 


250 


system  is  determined  by  its  oxidation 
potential.  The  potential  produced  is 
determined  by  the  ratio  of  ferrous  to 
ferric  ions,  and  is  given  by  the  relation : 


RT         (Fe^)  . 
£a  -  E.  -  —  In  ^-p^^j  . 

Fe-^^   ^  (Reductant) 
Fe*++  (Oxidant) 

Eh  is  the  observed  difference  in  electro- 
motive force  between  the  electrode  and 
the  normal  hydrogen  electrode;  Eo  is  a 
constant  characteristic  for  the  ferrous- 
ferric  system  (the  so-called  normal  po- 
tential); R,  T,  and  F  have  their  cus- 
tomary significances.  The  parentheses 
represent  concentrations  of  the  two  com- 
ponents. 

Certain  groups  of  organic  dyes  are 
likewise  able  to  induce  upon  electrodes 
reversible  potentials.  These  organic 
dyes  can  be  used  as  indicators  of  oxida- 
tion-reduction, and  the  following  rela- 
tion holds : 

RT        (Red) 

^^  -  ^o  -  -^ '°  loir 

If  the  reductant  is  identified  as  an  ion, 
or  the  oxidant  as  a  cation,  for  two  simple 
cases  there  would  be 

Ox  +  electron  ^::±  Red"  (1) 

Ox+  +  electron  ;=±  Red  (2) 

For  equation  (1),  the  relation  would  be 

The  active  reductant  of  equation  (1) 
is  the  anion  of  an  acid,  and  its  concen- 
tration depends  not  only  upon  the 
amount  of  reductant,  but  also  upon  the 
hydrogen  ion  concentration.  The  rela- 
tion then  becomes 

at  any  constant  pH  (For  development 
see  Cohen,  B.,  Symposia  Quant.  Biol., 
1933,  1,  195-204). 

The  use  and  interpretation  of  indica- 
tor dyes  in  biological  systems  is  given 
by  Cohen,  B.,  ibid,  214-223,  and  Cham- 
bers, R.,  ibid,  205-213.  Sources  of  error 
are  also  indicated  by  Cohen,  B.,  Cham- 
bers, R.  and  Reznikoff,  P.,  J.  Gen. 
Physiol.,  1928,  11,  585-612.  Most  of  the 
following  material  is  taken  from  the 
above  papers. 

On  a  microscopic  basis,  the  measure- 
ments, like  those  of  pH,are  madejwith 
indicators  in  which  the  cells  are  bathed 
or  which  are  injected  with  them.  They 
are  applied  in  sequence  and  their  reac- 
tions observed.  Methylene  blue,  for 
instance,  will  be  oxidized  (retain  color) 
or  be  reduced    (lose  color)   depending 


OXIDATION-REDUCTION 
POTENTIAL 

upon  the  relative  activity  of  the  proces- 
ses of  oxidation  and  reduction. 

Although  it  is  difficult  to  accurately 
measure  the  amount  of  indicator  in- 
jected into  cells,  it  is  imperative  that 
the  quantity  be  small.  Otherwise  too 
much  indicator  may  be  more  than  the 
cell  can  reduce,  or  be  greater  than  the 
reducing  intensity  which  the  cell  can 
generate.  The  following  indicators  from 
Cohen  provide  a  useful  range  in  potential 
values : 

Name  of  Oxidant  E    at  pH  7.0 

Phenol    m-sulfonate    indo-2,6    dibromo- 

phenol 0.273 

m-Bromophenol  indophenol 0.248 

o-Chlorophenol  indophenol 0.233 

Phenol  blue  chloride 0.227 

Phenol  indo-2, 6  dichlorophenol 0.217 

o  Cresol  indophenol 0. 195 

o  Cresol  indo-2, 6  dichlorophenol 0. 181 

l-Naphthol-2-sulfonate  indophenol  o-sul- 

fonate 0.135 

l-Naphthol-2-8ulfonate  indophenol 0.123 

Toluylene  blue  chloride 0.115 

Brilliant  creayl  blue  chloride 0.047 

Methylene  blue  chloride H-0.011 

Ki  indigo  tetrasulfonate —0.046 

Ethyl  capri  blue  nitrate —0.072 

Ki  indigo  trisulphonate —0.081 

Ki  indigo  disulphonate ...—0.125 

Cresyl  violet —0.167 

E'o  represents  the  potential  at  any  given 
pH  of  a  system  in  which  the  ratio  of  oxi- 
dant to  reductant  is  unity. 

In  order  to  get  the  indicator  dyes  into 
single  cells  the  microinjection  technique 
of  Chambers  is  used.  Chambers  recom- 
mends dilute  aqueous  solutions  of  the 
basic  dyes,  i.e.,  0.05%  to  0.1%,  and  in- 
jects successive  small  doses.  Needhara, 
J.  and  D.  M.,  Proc.  Roy.  Soc.  B,  1926, 
99,  173-199;  383-397  used  1%  solution 
since  weaker  solutions  of  particular 
dyes  could  not  be  seen  under  the  micro- 
scope when  injected  into  cells. 

The  determinations  are  carried  out 
aerobically  (cells  maintained  in  a  micro 
drop  in  water-saturated  air  at  atmos- 
pheric pressure)  andanaerobically  (cells 
held  in  an  atmosphere  of  purified  process 
nitrogen  saturated  with  water). 

For  example,  under  aerobiosis,  if  all 
the  indicators  down  to  and  including 
methylene  blue  are  reduced  at  pH  7.0 
by  cells  of  a  particular  type;  and  if 
ethyl  capri  blue  is  only  partially  re- 
duced (and  the  rest  of  the  indicators  not 
reduced),  the  reducing  intensity  of  the 
aerobic  cell  is  approximately  —0.072 
volts  at  pH  7.0.  The  same  procedure  is 
followed  for  cells  anaerobically. 

To  detect  the  presence  of  the  indicator 
after  decolorization  by  the  cell  proto- 


OXIDATION-REDUCTION 
POTENTIAL 


251 


OXIDATION-REDUCTION 
POTENTIAL 


plasm,  reoxidation  of  the  reductent  can 
be  accomplished  by  injecting  dilute  po- 
tassium ferricyanide  or  of  potassium 
dichromate  in  the  anaerobic  state,  or  by 
exposure  to  air  in  the  anaerobic  state. 
The  recovery  of  color  on  oxidation  is  a 
necessary  control  demonstrating  that 
the  indicator  has  been  reversibly  re- 
duced and  not  reversibly  destroyed. 

It  is  also  essential  to  bring  the  cell 
interior  into  contact  both  with  oxidant 
and  reductant  of  the  indicator.  This 
is  necessary  to  determine  whether  the 
indicator,  which  would  shift  to  the 
potential  of  the  electromotive  system 
present,  is  behaving  in  a  truly  rever- 
sible manner. 

The  aqueous  solutions  of  the  acid 
dyes,  e.g.  the  various  indophenols  give 
the  most  clear  cut  results.  Upon  in- 
jection they  rapidly  diffuse  throughout 
the  cell  before  being  reduced.  The 
experimental  evidence  indicates  that 
the  speed  of  reduction  of  the  indicator 
dyes  decreases  as  the  potential  of  the  in- 
dicator approaches  that  of  the  cell. 

In  the  immersion  method  slices  of 
tissue  are  bathed  in  solutions  of  the  in- 
dicator dyes.  Here  it  is  not  only  neces- 
sary to  distinguish  between  penetrating 
and  nonpenetrating  indicators  but  also 
to  watch  for  differences  in  the  rapidity 
with  which  cells  and  certain  cell  inclu- 
sions are  stained  by  the  various  in- 
dicators. For  example,  indicators  con- 
taining the  sulfonated  radicals  do  not 
readily  penetrate  cells,  while  the  non- 
sulfonated  more  or  less  rapidly  pene- 

Fildes,  P.,  Brit.  J.  Exp.  Path.  1929, 
10,  151-175  measured  the  oxidation- 
reduction  potential  of  the  subcutaneous 
tissue  fluid  of  the  guinea  pig,  and  also 
its  effect  on  infection.  Guinea  pigs 
were  inoculated  with  indicator  dyes 
(0.01%)  in  both  the  reduced  and  oxi- 
dized states  and  he  observed  whether 
change  had  occurred.  The  injections 
were  made  superficially  so  that  the  im- 
mediate effect  could  be  seen  through  the 
shaved  skin.  The  oxidized  form  of 
methylene  blue  remained  a  strong  blue, 
and  the  reduced  d\'e  assumed  a  distinct 
blue  color.  This  indicated  that  the  sub- 
cutaneous tissue  mainta-ined  an  oxida- 
tion-reduction potential  on  the  positive 
side  of  reduced  methylene  blue. 

Then  "indophenol  1"  (naphthol-2  so- 
dium sulphonate  indo  2,  6  dibromo- 
phenol)  in  both  states  was  injected  and 
the  animals  examined.  After  40  min- 
utes the  oxidized  and  reduced  forms  of 
the  dye  were  at  about  the  same  intensity 
of  blue.  Therefore  it  was  concluded 
that  the  Eh*'  of  the  subcutaneous  tissue 
was  positive  to  that  of  reduced  indo- 


phenol 1.  The  rate  of  oxidation  was 
slower  here  than  in  the  case  of  methy- 
lene blue,  because  the  difference  in  Eh 
of  the  tissues  and  the  reduction  point 
of  the  dye  was  less. 


Ek'=  Ei  —  0.062  log 


100 -o 


(at    37 °C.) 


where  Eo'  is  a  constant  characteristic 
of  the  particular  system  and  a  =  % 
reduction. 

Finally  the  dye  indicator,  "indophe- 
nol 2"  (phenolindophenol  2,  6  dibromo- 
phenol)  was  injected.  The  reduced 
form  of  the  dye  remained  colorless  while 
the  oxidized  form  faded  from  20  to  80 
minutes.  Addition  of  ferricyanide 
failed  to  restore  all  the  reduced  dye,  so 
the  results  were  complicated  by  decom- 
position of  the  dye  in  the  tissues.  It 
was  concluded  that  the  Eh  of  the  tissue 
fluids  is  positive  to  the  zone  of  complete 
reduction  of  indophenol  1. 

The  oxidation-reduction  potential  of 
the  ciliary  body  was  determined  (Frie- 
denwald,  J.  S.  and  Stieher,  R.  D.,  Arch. 
Ophth.,  1938,  20,  761-786)  by  introduc- 
ing indicator  dyes  into  the  stroma  or 
epithelium  of  ciliary  body  under  aerobic 
and  anaerobic  conditions.  After  equi- 
librium had  been  reached,  the  degree 
of  bleaching  was  observed  microscopi- 
cally. Then  an  oxidizing  agent  was 
added,  such  as  ferricyanide,  and  re- 
covery of  the  color  was  noted.  The 
ratio  of  intensity  of  color  before  and 
after  oxidation  with  ferricyanide  gave 
the  potential  in  the  system  since  it 
afforded  a  measure  of  the  ratio  of  oxi- 
dant to  reductant  of  the  indicator  in 
equilibrium  in  the  tissue.  Aerobically 
the  epithelium  had  an  estimated  poten- 
tial of  -fO.lOO  volts,  and  the  stroma 
—0.130  volts.  Anaerobically  both  had 
estimated  potentials  of  —0.290  volts. 

Lewis,  M.  R.,  Barron,  E.  S.  0.  and 
Gardner,  R.  E.,  Proc.  Soc.  Exp.  Biol.  & 
Med.,  1930-31,  28,  684-685  compared  the 
power  of  cancer  tumors,  tumors  pro- 
duced by  viruses  and  normal  tissue  to 
reduce  methylene  blue.  The  tissues 
were  cut  in  a  manner  similar  for  tissue 
respiration,  and  the  pieces  were  placed 
in  M/15  Sorensen's  phosphate  buffers 
at  pH  7.38.  Anaerobiosis  was  main- 
tained by  a  stream  of  nitrogen.  The 
time  of  reduction  of  the  dye  by  tumors 
was  the  same  as  that  of  normal  tissues. 

Voegtlin,  C,  Johnson,  J.  M.  and  Dyer, 
H.  A.,  J.  Pharm.  &  Exp.  Therap.,  1925, 
24,  305-337  have  quantitatively  esti- 
mated the  reducing  power  of  normal  and 
cancerous  tissue.  For  the  anaerobic 
experiments  tissues  were  sliced  about  2 
mm.  thick  and  weighed  about  0.5  gm. 
Samples  of  tissue  were  placed  in  sterile 


OXYME 


252 


PALITZSCH'S  BUFFERS 


vacuum  tubes,  and  5  cc.  of  a  sterile  solu- 
tion of  the  indicator  in  a  phosphate  buf- 
fer solution  (M/15  Na2HP04,  KH2PO4 
Sorensen)  of  pH  7.6  were  added  to  each 
tube  by  means  of  a  sterile  pipette. 
After  evacuation  of  the  tubes  by  a 
vacuum  pump,  they  were  rapidly  fixed 
in  a  constant  temperature  bath  at  38 °C. 
on  a  revolving  rack. 

The  indicator  .solutions  were  prepared 
by  adding  phosphate  buffer  to  an  ac- 
curately weighed  amount  of  the  dye  in 
a  mortar  and  grinding.  The  solutions 
were  made  up  to  volume  and  boiled  to 
sterilize. 

The  reducing  power  of  tissues  was 
based  upon  the  time  needed  to  reduce 
anaerobically  equimolar  amounts  of  the 
indicators  used  (the  dye  content  of  each 
indicator  was  determined  on  a  moisture 
free  basis).  For  the  indicators  used  it 
was  found  that  the  optimum  concentra- 
tion for  comparative  purposes  was  ap- 
proximately M/42,533.  A  more  useful 
concentration  of  M/40,000  was  suggested 
for  future  work. 

All  the  tissues  (brain,  carcinoma — 
peripheral  portion,  heart  muscle,  spleen, 
kidney,  liver,  lung,  skeletal  muscle  ana 
testis)  had  a  reducing  power  which 
varied  according  to  the  type  of  tissue 
having  the  highest  reducing  power  (with 
the  exception  of  the  necrotic  portion  of 
carcinoma).  The  latter  was  devoid  of 
reducing  power  while  the  viable  portion 
reduced  the  indicators  as  rapidly  as 
did  some  of  the  normal  tissues. 

See  Recording  Autotitrator  as  de- 
scribed bj^  Jacobsen,  C.  F.  and  L^onis, 
J.  C,  C.  rend.  trav.  Lab.,  Carlsberg, 
Ser.  China.,  1951,  27,  333-339. 

Oxyme.  Estimation  in  plant  and  animal 
tissues  (Yamafuji,  K.,  Kondo,  H.  and 
Omura,  H.,  Enzymologia,  1950,  14, 
153-156. 

Oxychromatic  Degeneration.  A  kind  of 
degeneration  in  which  oxychromatic 
(acidophilic)  material  appears  in  the 
nuclei.  See  Luger,  A.  and  Lauda,  E., 
Med.  Klin.,  Berlin,  1926,  22,  415,  456, 
493. 

Oxydase,  see  Oxidase. 

Oxygen  Consumption.  A  method  is  de- 
scribed for  epidermis  separated  from 
dermis  by  heat  (Baumberger,  J.  P., 
Suntzeff,  V.  and  Cowdry,  E.  V.,  J.  Nat. 
Cancer  Inst.,  1942,  2,  413-423. 

Oxyntic  Cells  (G.  Oxyntos,  making  acid), 
an  unsatisfactory  term  for  the  parietal 
cells  of  the  stomach  because  it  implies 
actual  manufacture  of  acid. 

Oxyphil  (G.  oxys.,  acid  -1-  philos,  fond)  same 
as  acidophilic.  The  term  is  commonly 
applied  to  the  colloid  cells  of  the  para- 
thyroid and  thyroid  which  are  colored 
with  "acid"  dyes  such  as  eosin. 


Ozokerite,  see  Ceresin  Imbedding. 

Pacinian  Corpuscles  can  best  be  located  by 
naked  eye  inspection  of  the  abdominal 
viscera  of  a  freshly  killed  cat  as  small 
elongated,  cigar  shaped  bodies  situated 
just  within  the  tunica  serosa  which 
appear  china  white  because  they  have  a 
very  poor  blood  supply.  Fix  in  Zen- 
ker's Fluid  and  color  with  Mallory's 
Connective  Tissue  stain  for  general 
purposes  or  employ  Bodian's  method  for 
nerve  fibers. 

Pal-Weigert  Stain  for  myelin  sheaths  as 
modified  by  Erhart,  E.  A.,  Zeit.  f.  wis. 
Mikr.,  1951,  60,  155-156  recommended 
as  fast,  simple  and  giving  a  blue  black 
stain  of  myelin  sheaths  in  an  almost 
uncolored  background.  For  paraffin, 
celloidin  or  frozen  sections  of  material 
fixed  in  formalin.  Wash  sections  in 
water  in  4%  aq.  iron  alum,  2-3  min. 
Rinse  rapidly  in  water.  Stain  in  aq. 
dest  80  cc,  lithium  carbonate  1  gm., 
10%  ale.  hematoxylin  for  5-20  min.  at 
room  temperature.  Wash  in  water, 
counterstain  if  desired  in  1%  aq.  neu- 
tral red,  carmin  or  cresyl  violet.  De- 
hydrate, clear  and  mount  in  balsam. 
See  Weigert-Pal. 

Palitzsch's  Borax-Boric  Acid  Buflfers 
(Clark,  W.  M.  The  Determination  of 
Hydrogen  Ions.  Baltimore:  Williams 
and  Wilkins,  1928,  717  pp.)  Prepare: 
(1)  M/20  borax  solution  by  dissolving 
19.0715  gms.  NasBiOv  10  HjO  in  1  liter 
aq.  dest.  (2)  A  solution  containing 
M/5  boric  acid  and  M/20  NaCl  by  dis- 
solving 12.368  gms.  H3BO3  and  2.925 
gms.  NaCl  in  1  liter  aq.  dest.  To  make 
buffer  of  the  desired  pH  mix  1  and  2  in 
the  proportions  indicated. 


(2)  M/5  Boric 

(1)  M/20 

Acid.  M/20 

pH 

Borax 

NaCl 

9.24 

10.0 

0.0 

9.11 

9.0 

1.0 

8.98 

8.0 

2.0 

8.84 

7.0 

3.0 

8.69 

6.0 

4.0 

8.60 

5.5 

4.5 

8.51 

6.0 

5.0 

8.41 

4.5 

5.5 

8.31 

4.0 

6.0 

8.20 

3.5 

6.5 

8.08 

3.0 

7.0 

7.94 

2.5 

7.5 

7.88 

2.3 

7.7 

7.78 

2.0 

8.0 

7.60 

1.5 

8.5 

7.36 

1.0 

9.0 

7.09 

0.6 

9.4 

6.77 

0.3 

9.7 

French,  R.  W.  Stain  Techn.,  1930,  5 
87-90;  1932,  7,  107-108  recommended 
the  use  of  these  buffers  for  the  range  pH 


PALLADIUM 


253 


PAPANICOLAOU  TECHNIQUES 


9.2-8.2  but  he  made  them  up  in  a  dif- 
ferent way. 

Palladium.  Histochemical  detection  based 
on  reaction  between  palladium  and  p- 
Dimethylaminobenzyl-idenrhodanin  in 
neutral  formalin  or  alcohol  fixed  tissues 
(Okamoto,  K.,  Mikami,  G.  and  Nishida, 
M.,  Acta  Scholae  Med.  Univ.  Imp.  in 
Kioto,  1939,  22,  382-387). 

Panchrome  is  a  modification  by  Pappenheim 
(Folia  haematol.,  Arch.,  1911,  11,  194) 
of  the  Giemsa  stain.  Add  0.75  gm.  of 
the  panchrome  powder  (Griibler)  to  75 
cc.  pure  methyl  alcohol  and  25  cc.  acid 
free  glycerin  at  60°C.  After  filtering 
keep  in  glass  stoppered  bottle.  Use 
after  May-Griinwald  fixation  as  de- 
scribed for  Giemsa  after  methyl  alcohol 
fixation.  According  to  Slider  and 
Downey  (McClung's  Microscopical 
Technique,  p.  329)  it  gives  better 
coloration  of  neutrophilic  granules  and 
metachromasia  of  mast  granules  than 
the  plain  Giemsa's  stain  but  "some 
delicacy  is  lost,  and  the  cells  are  more 
likely  to  be  muddy." 

Pancreas.  This  organ  lends  itself  very  well 
to  microscopic  examination  in  the  fresh 
state.  The  classic  which  everyone  seek- 
ing technical  details  should  consult  is 
Bensley,  R.  R.,  Am.  J.  Anat.,  1911,  12, 
297-388.  The  techniques  for  Blood 
Vessels  and  Nerve  Endings  are  those 
employed  generally  and  are  described 
under  these  headings.  No  particular 
difficulties  will  be  encountered  in  their 
adaptation  to  the  pancreas.  It  may  be 
helpful  however  to  consult  Beck,  J.  S. 
P.  and  Berg,  B.  N.,  Am.  J.  Path.,  1931, 
7,  31-35  on  the  blood  vessels.  The 
same  holds  for  the  Connective  Tissue 
components.  Epithelial  parts  of  the 
pancreas  can  routinely  be  examined  in  a 
preliminary  way  with  the  other  parts  in 
tissues  fixed  in  Formalin-Zenker  and 
stained  with  Hematoxylin  and  Eosin. 
For  details  see  Zymogen,  Ducts  and 
Islets  of  Langerhans. 

Pancreatin  digestion  method  for  spleen 
(Kyes,  P.,  Am.  J.  Anat.,  1901,  1,  37- 
43). 

Paneth  Cells.  Influence  of  fasting  on 
(Klein,  S.,  Am.  J.  Anat.,  1905-1906,  5, 
315-330).  To  observe  storage  and  dis- 
charge phases  examine  in  guinea  pigs  24 
and  6  hrs.  after  feeding  (Klein,  S.,  Am. 
J.  Anat.,  1905-06,  5,  315-330) .  By  com- 
bining DeGalantha's  amyloid  stain  with 
mucicarmine,  Paneth  granules  are  col- 
ored green  and  mucous  granules  red 
(Hertzog,  A.  J.,  Am.  J.  Path.,  1937, 
13,  351-360). 

Pantothenic  Acid.  Detection  by  fluores- 
cence microscopy  in  tomato  plants 
(Bonner,  J.  and  Dorland,  R.,  Am.  J. 
Bot.,  1943,  30,  414-418). 


Papanicolaou    Techniques    in    Exfoliative 

Cytology— written  by  Charlotte  M. 
Street,  Department  of  Anatomy,  Cor- 
nell University  Medical  College,  New 
York.  May  21,  1951— Exfoliative  cy- 
tology, i.e.  the  study  of  cells  which 
have  been  exfoliated  from  the  epithelial 
lining  of  an  organ  or  cavity  of  the  body, 
is  gradually  becoming  an  independent 
branch  of  the  morphological  sciences. 
Its  usefulness  in  hormonal  studies  and 
in  cancer  diagnosis  has  been  largely 
responsible  for  its  great  expansion  in 
the  last  two  decades.  The  progress 
achieved  in  this  field  has  been  primarily 
due  to  the  introduction  of  more  ade- 
quate technical  methods  for  the  preser- 
vation and  staining  of  cells  in  smears 
prepared  from  various  physiological 
secretions.  The  techniques  described 
herein  will  be  limited  chiefly  to  those 
which  have  been  developed  by  Dr. 
George  N.  Papanicolaou  in  the  course 
of  his  cytologic  studies,  and  which  are 
now  in  use  in  his  laboratory  in  the  De- 
partment of  Anatomy  of  Cornell  Uni- 
versity Medical  College. 

These  technical  procedures  are  based 
on  the  use  of  smears  which  are  fixed 
without  drying,  in  a  solution  of  equal 
parts  of  95'%  ethyl  alcohol  and  ether, 
by  which  good  preservation  of  e.xfoli- 
ated  cells  is  attained.  In  such  fixed 
smears  the  cells  are  well  preserved  and 
when  stained,  their  cytoplasmic  and 
nuclear  details  are  clearly  defined.  On 
the  contrary,  in  dried,  unfixed  smears 
many  of  the  structural  details  are  lost, 
there  is  an  apparent  increase  in  the 
size  of  the  cells  and  their  nuclei,  and 
their  affinity  for  stains  is  much  im- 
paired. Since  cells  subjected  to  drying 
prior  to  fixation  show  a  preference  for 
acidophilic  stains,  it  is  impossible  in 
dried  smears  to  make  the  proper  evalua- 
tion of  basophilic  and  acidophilic  cells 
which  is  so  necessary  in  endocrine 
studies.  Furthermore,  in  the  examina- 
tion of  smears  for  malignant  cells, 
good  nuclear  preservation  and  staining 
are  of  utmost  importance.  For  this 
reason  thorough  fixation  of  wet  smears 
is  essential.  The  relative  size  of  cells 
and  the  nuclear-cytoplasmic  ratio  also 
play  an  important  role  in  the  diagnosis 
of  malignancy.  Therefore,  drj^ing  of 
smears  which  results  in  apparent  en- 
largement of  cells  and  nuclei  may  give 
a  false  impression  of  malignancy. 

The  development  of  Papanicolaou's 
staining  procedures  may  be  divided  into 
two  periods.  The  first  includes  his 
animal  experimentation  and  his  early 
work  in  the  human,  during  which  vagi- 
nal smears  were  used  as  an  index  for 
evaluating  cytologic  changes  occurring 


PAPANICOLAOU  TECHNIQUES 


254 


PAPANICOLAOU  TECHNIQUES 


during  the  normal  estrous  or  menstrual 
cycle,  as  well  as  changes  due  to  various 
hormonal  factors.  For  his  studies  in 
both  guinea  pigs  and  the  human,  he 
used  alcohol-ether  fixed  smears  which 
were  stained  first  with  hematoxylin 
and  then  counterstained  with  aqueous 
solutions  of  eosin  and  water  blue  (see 
staining  technique  I).  This  method 
was  a  simple  one  technically  and  re- 
sulted in  good  differentiation  between 
basophilia  and  acidophilia.  It  is  still 
considered  useful  in  hormonal  studies. 

During  the  second  period,  modifica- 
tions in  technique  were  investigated 
which  would  be  better  adapted  to  the 
diagnosis  of  malignant  neoplasms.  In 
thick  smears  where  cells  are  found  in 
compact  clusters,  overljang  one  an- 
other, or  mixed  with  blood  or  mucus, 
a  light,  transparent,  cytoplasmic  stain 
is  necessary  to  give  a  clearer  view  of 
individual  cells  and  their  nuclei. 
Papanicolaou  found  that  this  could  be 
achieved  by  using  95%  alcohol  as  the 
solvent  for  the  counterstains.  Stains 
006  and  EA36  (formulae  2  &  3  follow- 
ing the  staining  procedures)  were  intro- 
duced in  1942;  EA  65  (formula  4)  is  a 
later  modification  of  EA36.  When 
used  in  combination  with  a  good  nuclear 
stain,  these  counterstains  are  particu- 
larly well  adapted  to  cancer  diagnosis 
but  are  also  excellent  for  endocrine 
studies  because  of  their  delicate  differ- 
ential staining  qualities.  They  can 
also  be  used  in  staining  tissue  which 
is  advantageous  in  the  correlation  of 
smears  and  sections.  (For  use  of  alco- 
holic counterstains,  see  staining  tech- 
niques II,  III,  IV,  V.) 

Female  Genital  Tract.  Vaginal,  endo- 
cervical  and  endometrial  aspiration 
smears;  cervical  swab  and  spatula 
smears. 

Equipment:  For  vaginal  smears:  a 
glass  pipette,  five  inches  in  length  and 
one  quarter  inch  in  diameter,  slightly 
curved  about  two  inches  from  its  blunt 
tip,  and  equipped  with  a  tightly  fitting 
strong  rubber  bulb.  The  pipette  should 
be  made  of  hard  pyrex  glass  to  with- 
stand sterilization.  For  endocervical 
and  endometrial  aspiration  smears:  a 
Becton-Dickenson  laryngeal  cannula 
equipped  with  a  strong  rubber  bulb. 
For  cervical  smears:  a  non-absorbent 
cotton  swab  or  the  wooden  spatula 
designed  by  Ayre  (Avre,  J.  E.,  Am.  J. 
Obst.  &  Gynec,  1947,  53,  609).  For 
all  types  of  smears:  a  jar  of  fixing  solu- 
tion, i.e.  equal  parts  of  95%  ethyl  alco- 
hol and  ether,  and  clean  slides.  When 
more  than  one  slide  is  placed  in  a  bottle 
of  fixative,  a  paper  clip  is  attached  to 
one  end  of  each  slide  in  order  to  prevent 


contact  between  the  smeared  surfaces. 
The  slides  are  marked  in  advanced 
with  the  patient's  name  or  case  num- 
ber, type  of  smear  and  date.  This  can 
be  done  either  with  a  diamond  pencil, 
or  by  clipping  a  small  piece  of  paper  or 
card  to  the  slide.  In  the  latter  case, 
the  label  should  be  written  with  lead 
pencil,  not  ink. 

Preparation  of  smears:  Smears  should 
not  be  prepared  after  a  douche,  bath, 
or  use  of  a  lubricant.  They  should  be 
prepared  prior  to  the  pelvic  examina- 
tion, if  possible. 

Vaginal  smears:  The  dry  pipette, 
with  bulb  compressed,  is  introduced 
into  the  posterior  fornix  of  the  vagina 
and  the  bulb  released.  The  pipette  is 
rotated  carefully  during  aspiration  to 
obtain  samples  from  different  parts  of 
the  vagina.  The  fluid  thus  obtained 
in  the  pipette  is  expelled  on  to  a  slide, 
and  spread  in  a  uniform  smear  with 
the  side  of  the  pipette.  The  slide  is 
immersed  immediately,  before  the 
smear  dries,  in  the  alcohol-ether  fixative 
and  allowed  to  remain  for  at  least  15 
minutes  for  thorough  fixation  of  cells 
before  staining.  Smears  may  be  left 
in  alcohol-ether  for  a  week  or  even 
longer  without  harmful  effect. 

Endocervical  or  endometrial  smears: 
After  a  speculum  is  in  place,  endocervi- 
cal or  endometrial  secretion  is  aspirated 
by  means  of  the  laryngeal  cannula, 
expelled  on  to  a  slide,  spread  uniformly 
with  the  cannula,  and  the  slide  im- 
mersed immediately  in  alcohol-ether. 

Cervical  swab  smears:  Secretion  ob- 
tained by  means  of  a  swab  or  spatula 
from  the  external  os,  or  from  any  suspi- 
cious appearing  lesion  of  the  cervix, 
is  quickly  spread  on  a  slide  and  fixed 
in  alcohol-ether. 

The  vaginal  smear,  because  it  is 
composed  chiefly  of  squamous  cells 
which  reflect  hormonal  activity  is  the 
best  type  for  hormonal  evaluation,  and 
for  a  study  of  the  menstrual  cycle. 
For  the  latter,  smears  may  be  prepared 
daily  throughout  the  complete  cycle 
by  the  patient.  Slides,  either  dated 
or  numbered  serially  in  advance,  with 
clips  attached,  a  pipette  and  jar  of 
fixative  are  given  to  the  patient  and 
she  is  instructed  how  to  prepare  them. 

For  diagnosis  of  malignancy,  a  vagi- 
nal smear  gives  a  more  complete  overall 
picture  of  the  cytology  of  the  genital 
tract  since  cells  from  all  parts  accumu- 
late in  the  vaginal  pool.  Endocervical 
smears  reveal  the  presence  of  a  cervical 
carcinoma,  endometrial  smears  are 
better  for  diagnosis  of  fundal,  and  occa- 
sionally, tubal  carcinomas.  Cervical 
swab  or  spatula  smears  give  informa- 


PAPANICOLAOU  TECHNIQUES 


255 


PAPANICOLAOU  TECHNIQUES 


tion  of  more  specific  nature  but,  unless 
prepared  and  fixed  very  quickly,  swab 
smears  are  apt  to  be  drier  than  aspira- 
tion smears  and  therefore  diSicult  to 
interpret. 

Smears  of  the  female  genital  tract 
are  stained  by  either  method  I  or  II. 

Urinary  Tract. 

Collection  of  specimens:  Urine  from 
females  must  be  catheterized  to  avoid 
contamination  from  vaginal  secretion. 
Ureteral  specimens  are  advisable  if 
carcinoma  of  the  kidney  pelvis  is  sus- 
pected. In  cases  of  suspected  carci- 
noma of  the  prostate,  separate  voided 
urine  specimens  are  obtained  before 
and  after  massage.  In  such  cases, 
prostatic  smears  can  also  be  prepared 
by  spreading  prostatic  secretion  di- 
rectly on  slides  and  fixing  them  in  alco- 
hol-ether. 

Preparation  of  smears:  Upon   collec- 
tion, the  urine  specimen  (approximately 
50  cc.  of  a  bladder  specimen;  as  much 
of  a  ureteral  specimen  as  can  be  ob- 
tained)   must    be    mixed    immediately 
with  an  equal  volume  of  95%  alcohol 
for   fixation    and    preservation    of    the 
cells.     The  mixture  of  urine  and  alcohol 
is  centrifuged  in  50  cc.  conical  tubes  at 
medium  speed  for  15-30  minutes  (until 
sediment  is  well  packed),  after  which 
the    supernatant    liquid    is    decanted. 
Clean  slides,  marked  in  advance  with 
a  diamond  pencil  or  water-proof  lab- 
oratory ink,  are  coated  with  a  thin  film 
of  Mayer's  albumin  fixative  in  order  to 
make    the    cells    adhere    to    the    slide. 
Sediment    can    be    removed    from    the 
tube  by  means  of  a  small  spoon-shaped 
instrument   such  as   a  nasal   or  small 
bone    curette    or,    if   very   scanty,    by 
pipetting.     It  is  spread  on  the  slide, 
using  a  second  clean  slide  to  distribute 
it  in  a  uniform  smear.     When  the  smear 
starts  to  dry  at  its  periphery,  it  is  im- 
mersed   in    95%    alcohol    and    ether, 
equal    parts.     Care    must    be    taken, 
however,   not   to   allow   the   smear   to 
become  too  dry  before  fixation  for  the 
reasons      discussed      previously.     The 
smears  should  be  left  in  the  alcohol- 
ether    fixative    for   at    least    one    hour 
before  staining  or  they  tend  to  wash 
off    during    the    staining    process.     All 
instruments  used  in  transferring  sedi- 
ment from  tube  to  slide  must  be  care- 
fully cleaned  to  avoid  contamination 
of  the  next  specimen.     If  there  is  suffi- 
cient sediment,   three   or  four  smears 
are  prepared  from  each  specimen.     If 
there  is  an  abundance  of  sediment,  it 
should   be   stirred   up   in   the   tube   in 
order  to  insure  representative  sampling. 
If  smears  cannot  be   prepared   imme- 
diately after  centrifugation,  the  super- 


natant liquid  is  decanted,  the  sediment 
covered  with  a  few  cubic  centimeters 
of  absolute  alcohol  and  placed  in  the 
refrigerator. 

Graham  et  al.  (Vincent  Memorial 
Hospital,  The  Cytologic  Diagnosis  of 
Cancer.  W.  B.  Saunders  Co.,  1950) 
suggest  mixing  two  or  three  drops  of 
albumin  fixative  with  the  sediment  in 
the  centrifuge  tube  before  making 
smears  so  that  the  cells  will  stick  better 
to  the  slides.  This  has  been  found  to 
be  very  helpful  in  urine  as  well  as 
other  sediments. 

Urine  smears  are  stained  by  method 
IV. 

Respiratory  Tract. 

Sputum:  This  must  be  a  deep  cough 
specimen  as  a  salivary  specimen  is 
obviously  of  no  value  in  the  diag- 
nosis of  carcinoma  of  the  lung.  It 
is  ordinarily  collected  directly  in  70% 
alcohol  to  retard  the  growth  of  bacteria 
and  fungi  as  well  as  to  prevent  cellular 
degeneration.  However,  if  smears  can 
be  made  within  four  hours  after  the 
specimen  is  obtained,  it  need  not  be 
fixed  in  alcohol.  Since  alcohol  hardens 
sputum,  smears  are  more  easily  pre- 
pared from  a  fresh,  unfixed  specimen. 
When  alcohol  is  used  for  fixation,  70% 
is  preferable  to  95%  because  it  causes 
less  hardening  of  the  sputum.  Before 
preparing  the  smears,  the  specimen  is 
examined  for  blood-tinged  portions. 
If  any  are  present,  they  are  transferred 
to  slides  which  have  been  marked  pre- 
viously and  coated  with  a  thin  film  of 
Mayer's  albumin.  If  no  blood  tinged 
particles  are  found,  samples  from  differ- 
ent parts  of  the  specimen  are  to  be 
transferred  to  the  slides.  A  fairly 
thin  uniform  smear  is  prepared  by 
spreading  the  material  with  another 
clean  slide,  gently  rubbing  it  between 
the  two  slides  in  order  to  crush  any 
hard  rubbery  masses  and  distribute 
them  evenly.  The  smear  is  fi.xed,  with- 
out drying,  in  alcohol-ether  fixative  for 
a  minimum  of  one  hour.  Three  or 
four  smears  should  be  prepared.  They 
are  stained  by  method  III. 

Bronchial  aspirates  or  saline  washings 
of  the  bronchus  are  mi.xed  immediately 
with  an  equal  volume  of  95%  alcohol. 
Because  of  the  scantiness  of  material 
obtained  by  bronchial  aspiration,  the 
collection  tube  should  always  be  rin.sed 
well  with  alcohol  and  the  rinse  solution 
added  to  the  specimen.  Specimens  are 
centrifuged  and  smears  prepared  from 
the  sediment  by  the  method  given  for 
smears  of  urine  sediment.  They  are 
stained  by  method  III. 


PAPANICOLAOU  TECHNIQUES 


256 


PAPANICOLAOU  TECHNIQUES 


Gastro- Intestinal  Tract. 

Esophageal  aspirates  are  obtained  by 
means  of  a  rubber  suction  tube  passed 
through  the  esophagoscope.  Due  to 
the  scantiness  of  material  usually  ob- 
tained, the  tube  should  be  rinsed  with 
saline  and  this  rinse  added  to  the  as- 
pirate. The  entire  specimen  is  then 
mixed  immediately  with  95%  alcohol. 

Gastric  specimens  should  be  obtained 
after  at  least  eight  hours  of  fasting. 
Because  of  the  action  of  free  acid  and 
digestive  juices  in  the  stomach,  best 
results  are  obtained  if  the  residual 
gastric  contents  are  first  aspirated  and 
discarded.  Freshly  exfoliated  material 
can  then  be  obtained  by  either  of  the 
following  methods.  (1)  Using  a  Reh- 
fuss  tube  with  a  bucket  tip,  the  stomach 
is  washed  repeatedly  with  Ringer's 
solution  by  aspirating  and  re-injecting 
the  same  solution.  (2)  A  gastric  bal- 
loon can  be  used  which  has  been  spe- 
cially designed  to  cause  gentle  friction 
of  the  gastric  mucosa,  thereby  increas- 
ing exfoliation.  The  balloon  and  the 
technique  for  its  use  in  obtaining  gas- 
tric specimens  have  been  described  by 
Panico,  Papanicolaou  and  Cooper 
(Abrasive  balloon  for  exfoliation  of 
gastric  cancer  cells,  J.  A.  M.  A.,  1950, 
143,  1308-11).  Gastric  specimens  must 
be  fixed  immediately  in  an  equal  volume 
of  95%  alcohol  to  prevent  further  de- 
generation and  digestion  of  cells. 

Duodenal  drainage  specimens  are  ob- 
tained through  a  Rehfuss  tube,  allow- 
ing the  tip  of  it  to  pass  from  three  to 
six  inches  beyond  the  pjdorus.  The 
specimen  is  immediately  mixed  with 
an  equal  volume  of  95%  alcohol. 

Rectal  or  colonic  washings:  In  order 
to  obtain  specimens  which  are  as  free 
as  possible  of  fecal  material,  the  patient 
is  given  a  laxative  at  night,  and  a  hot 
soapsuds  enema  the  next  morning. 
Saline  washings  of  the  rectum  or  colon 
are  collected  two  to  five  hours  later  and 
mixed  immediately  with  95%  alcohol. 
A  special  suction  apparatus  for  rectal 
washings  is  described  by  Loeb  and 
Scapier  (Rectal  washings,  technic  for 
cytologic  study  of  rectosigmoid.  Am. 
J.  Surg.,  1951,  81,  298-302).  Colonic 
washings  can  be  obrained  by  high 
colonic  irrigation,  using  normal  saline. 

Preparation  of  smears:  All  specimens 
from  the  gastro-intestinal  tract  must 
be  processed  with  the  least  possible 
delay  because  of  deterioration  of  the 
cells.  This  is  particularly  important 
in  gastric  and  duodenal  specimens 
due  to  the  action  of  the  digestive  juices. 
The  specimens  are  centrifuged  as  soon 
as  received  and  the  supernatant  fluid 
promptly   decanted.     The   addition   of 


ten  drops  of  Mayer's  albumin  per  50 
cc.  of  specimen  before  centrifugation 
will  facilitate  sedimentation.  If  smears 
cannot  be  prepared  immediately,  the 
sediment  can  be  covered  with  absolute 
alcohol  and  kept  temporarily  in  the 
refrigerator.  Smears  are  prepared  in 
the  same  manner  as  urine  smears  but 
stained  by  method  III. 

Other  Sources. 

Exudates,  pleural,  peritoneal,  and 
pericardial.  Fifty  cc.  of  50%  alcohol 
is  added  immediately  to  50  cc.  of  the 
exudate  and  smears  prepared  from  the 
sediment  in  the  same  way  as  urine  sedi- 
ment smears,  but  stained  by  method 
III.  Fifty  percent  alcohol  is  used 
rather  than  95%  so  that  there  will  be 
less  precipitation  of  protein  material. 

Cystic  and  other  fluids  or  washings. 
An  equal  volume  of  95%  alcohol  is 
added  immediately  to  the  specimen  and 
smears  prepared  from  sediment  and 
stained  by  method  III. 

Nipple  secretion  smears.  If  secre- 
tion can  be  obtained  easily  from  the 
nipple  by  very  gentle  pressure,  a  clean 
slide  is  brought  in  contact  with  the 
secretion  expressed.  Care  should  be 
taken  not  to  bring  the  slide  in  contact 
with  the  nipple  itself  in  order  to  prevent 
the  collection  of  an  excess  of  epidermal 
cells.  The  material  is  spread  uniformly 
with  another  slide,  the  smear  is  fixed 
in  alcohol-ether  and  stained  by  method 
III. 

Method  I.  (Papanicolaou,  G.  N., 
Am.  J.  Anat.,  1933,  52:  No.  13,  supple- 
ment). This  is  the  original  Papan- 
icolaou technique  for  staining  vaginal 
smears  in  rodents  and  the  human;  it 
can  be  used  to  advantage  in  endocrine 
work  and  in  cytologic  studies  of  lower 
mammals. 

1.  Transfer  slides  from  alcohol -ether 
directly  to  80%  alcohol  and  run  down 
through  70%  and  50%  alcohols  to 
aq.  dest. 

2.  Stain  in  Ehrlich's  hematoxylin  5  min. 

3.  Rinse  in  aq.  dest.  and  place  in  run- 
ning tap  water  15  min. 

4.  Stain  in  0.5%  aq.  eosin  3-4  min. 

5.  Rinse  well  in  tap  water. 

6.  Stain  in  0.5%  aq.  waterblue  1  min. 

7.  Rinse  in  tap  water  and  run  up 
through  50%,  70%,  80%,  95%  and 
absolute  alcohols,  absolute  alcohol 
and  xylol  (equal  parts),  to  xylol, 
leaving  in  each  solution  long  enough 
to  clear. 

8.  Mount,  without  drying,  with  a  cover 
slip. 

Nuclei — dark  purple;  cytoplasm  of 
non-cornified  cells — pale  blue;  cyto- 
plasm of  cornified  cells — pink. 


PAPANICOLAOU  TECHNIQUES 


257 


PAPANICOLAOU  TECHNIQUES 


Method    II.     This    is    the    technique 
described  by  Papanicolaou  in  Science, 
1942,  95,  438-9,  with  a  few  minor  modifi- 
cations.    It  is  recommended  for  stain- 
ing smears  of  the  female  genital  tract 
which  are  to  be  used  for  either  cancer 
diagnosis  or  endocrine  studies. 
L  Transfer    slides,    without    drying, 
from  alcohol-ether  fixative  to  80% 
alcohol  and  run  down  through  70% 
and  50%  alcohols  to  aq.  dest.,  leaving 
in    each    solution   long    enough   to 
clear. 

2.  Stain  in  Harris  Hematoxylin  (1) 
6  min. 

3.  Rinse  in  aq.  dest. 

4.  Dip  in  0.25%  aq.  hydrochloric  acid 
6  times. 

5.  Place  in  running  tap  water  6  min. 

6.  Rinse  in  aq.  dest.  and  run  up 
through  50%,  70%,  80%,  and  95% 
alcohols,  leaving  in  each  long 
enough  to  clear. 

7.  Stain  in  0G6  (2)  for  U  minutes. 

8.  Rinse  in  95%  ale,  two  changes. 

9.  Stain  in  EA36  (3)  for  H  min. 

10.  Rinse  in  95%  alcohol,  3   changes. 

11.  Dehydrate  and  clear  by  running 
through  absolute  ale,  absolute 
alcohol  and  xylol  (equal  parts)  to 
xylol,  leaving  in  each  solution  long 
enough  for  the  smears  to  be  thor- 
oughly penetrated  and  dehydrated. 

12.  Mount,  without  drying,  with  a 
coverslip,  using  permount,  gum 
damar,  Canada  balsam  or  another 
neutral  mounting  medium. 

Nuclei — dark  purple;  non-cornified 
cells — pale  greenish-blue;  cornified  cells 
— variations  from  pink  to  orange,  de- 
pending on  the  degree  of  cornification; 
keratinized  cells — intense  orange. 

Method  III.  A  modification  of 
method  II.  Recommended  for  smears 
of  sputum,  prostatic  secretion  and  all 
sediments  except  urine. 

1-8.  Same  as  steps  1-8  or  method  II. 
9.  Stain  in  EA65  (4)  1|  min. 
10-12.  Same  as  steps  10-12  of  method  II. 

Note:  Rectal  and  colonic  washings 
are  washed  in  running  tap  water  (step 
5)  15  min.  instead  of  6  min. 

Method  IV.  A  modification  of 
method  III.  In  this  procedure  blueing 
of  the  nuclei  is  accomplished  by  the 
use  of  ammonium  hydroxide  rather 
than  running  tap  water,  as  the  latter 
tends  to  loosen  cells  from  the  slide. 
It  is  therefore  recommended  for  smears 
of  urine  sediment  which  is  the  least 
adhesive  of  the  sediments.  It  is  also 
shorter  than  method  II  and  may  be 
used  for  other  types  of  smears  for  that 
reason.  However,  in  thick  smears,  it 
has  a  disadvantage  in  that  the  cyto- 
plasm  retains   some   hematoxylin   and 


therefore  does  not  take  a  clear  cyto- 
plasmic stain. 

1.  Transfer  slides  from  alcohol-ether 
to  80%  alcohol  and  run  down  through 
70%  and  50%  alcohols  to  aq.  dest. 

2.  Stain  in  Harris  hematoxylin  (1)  2 
min. 

3.  Rinse  in  distilled  water,  then  50% 
ale. 

4.  Place  in  a  solution  of  1.5%  ammo- 
nium hydroxide  made  up  in  70% 
alcohol  for  1  min. 

5.  Rinse  in  70%  alcohol,  2  changes. 

6.  Run  up  through  80%  and  95%  ale. 

7.  Stain  in  0G6  (2)  for  U  min. 

8.  Rinse  in  95%  alcohol,  2  changes. 

9.  Stain  in  EA65  (4)  for  U  min. 
10-12.  Same  as  steps  10  to  12  in  method 

II. 
Method  V.     For  sections. 

1.  Remove  paraffin  in  xylol  and  run 
down  through  descending  alcohols 
to  aq.  dest.  in  the  usual  way. 

2.  Stain  in  Harris  hemato.xylin  (1)  2^ 
min. 

3.  Rinse  in  aq.  dest.  (2  changes),  and 
50%  and  70%  alcohols. 

4.  Place  in  3%  ammonium  hydroxide 
made  up  in  70%  ale.  for  1  min.  (or 
until  sections  turn  blue). 

5.  Rinse  in  70%  alcohol  (2  changes), 
80%  and  95%  ale. 

6.  Stain  in  0G6  (2)  for  If  min. 

7.  Rinse  in  95%  alcohol  (2  changes). 

8.  Stain  in  EA65  (4)  or  EA36  (3)  for 
1^  min. 

9.  Rinse  in  95%  alcohol  (3  changes), 
dehydrate,  clear  and  mount  in  the 
usual  way. 

Formulae  for  Stains. 

(1)  Harris  hematoxylin  is  prepared  from  the 
standard  formula,  using  ammonium  alum- 
inum sulphate,  but  omitting  the  glacial  ace- 
tic acid.  It  is  diluted  with  an  equal  volume 
of  distilled  water  before  using,  and  filtered 
into  a  dark  bottle  for  storage  when  not  in  use. 
It  should  be  reinforced  by  the  addition  of  a 
small  amount  of  fresh  undiluted  stock  solu- 
tion fairly  often  in  order  to  maintain  uniform 
staining  results. 

(2)  0G6: 

Orange  G— 0. 5%  in  95%  alcohol .  100  cc. 
Acid  phosphotungstic 0.015  gm. 

(3)  EA36: 

Light  Green  SF  (yellowish) — 
0. 1%  in  95%  alcohol* 45  cc. 

Bismarck  Brown  Y— 0.5%  in 
alcohol 10  cc. 

Eosin  yellowish— 0.5%  in  95% 
alcohol 45  cc. 

•Original  formula  for  EA36  calls 
for  0.5%  light  green  solution 
but  0. 1%  is  now  used 


PAPANICOLAOU  TECHNIQUES 


258 


PAPANICOLAOU  TECHNIQUES 


Acid  phosphotungstic 0.200  gm. 

Lithium  carbonate,  saturated 
aqueous  solution 1  drop 

(4)  EA6S: 

Light  Green  SF  (yellowish)  — 
0.05%  in  95%  alcohol 45  cc. 

Bismarck  Brown  Y — 0.5%  in 
95%  alcohol 10  cc. 

Eosin  yellomsh  (water  and  alco- 
hol soluble)— 0.5%  in  95%  al- 
cohol      45  cc. 

Acid  phosphotungstic,  C.P 0.2  gm. 

Lithium  carbonate,  saturated 
solution 1  drop 

National  Aniline  and  Chemical  Com- 
pany certified  stains  are  used  for  pre- 
paring the  above  stains.  If  other  dyes 
are  used,  the  amounts  may  have  to  be 
adjusted. 

For  the  preparation  of  stains  0G6, 
EA36  and  EA65,  it  is  advisable,  because 
of  the  low  solubility  in  alcohol  of  orange 
G,  Bismarck  brown  and  light  green,  to 
make  the  individual  alcoholic  stock 
solutions  from  aqueous  solutions  as 
follows: 

Orange  G,  0.5%  in  95%  alcohol— to  95 
cc.   95%  alcohol,   add  5  cc.   of   10% 
aqueous  solution  of  orange  G. 
Bismarck  Brown,  0.5%  in  95%  alcohol— 
to  95  cc.  alcohol,  add  5  cc.  of  10% 
aqueous  solution  of  Bismarck  Brown. 
Light  Green,  0.1%  in  95%  alcohol— to 
95  cc.  95%  alcohol,  add  5  cc.  of  2% 
aqueous  solution  of  light  green. 
Light  Green,  0.05%  in  95%  alcohol— to 
95  cc.  95%  alcohol,  add  5  cc.  of  1% 
aqueous   solution    of   light    green    or 
dilute  the  0.1%  alcohol  solution  with 
an    equal    volume    of    95%    alcohol. 
By  this  method,  the  alcoholic  content 
of  the  stock  stains  is  less  than  95% 
but  it  makes  no  appreciable  differ- 
ence in  the  staining  results.     Some 
of   the   stain   may   precipitate   when 
added  to  the  alcohol,  but  the  solubil- 
ity   may    be    increased    by    heating 
slightly  (not  over  an  open  flame). 
The  strength  of  the  light  green  solu- 
tion is  less  in  the  EA65  than  in  EA36 
because     smears    of    sputum,     gastric 
aspirates,  exudates,  etc.  are  apt  to  be 
thicker  than  those  of  the  female  genital 
tract    and    therefore    require    a    paler 
basophilic    stain.     In    smears    stained 
with  either  EA36  or  EA65,  as  soon  as 
the    green    begins    to    predominate    at 
the  expense  of  the  acidophilic  stains, 
the  counterstain  should  be  discarded. 
All  stains  should  be  stored  in  dark 
bottles  when  not  in  use.     If  used  con- 
stantly, they  should  be  filtered  daily 
and  frequently  reinforced  by  the  addi- 
tion of  fresh  stain.     Because  of  oxida- 
tion,   hematoxylin    should    always    be 


filtered  daily  regardless  of  the  volume 
of  staining. 

Vaginal,  cervical  and  endometrial 
smears  may  be  stained  after  fixation 
of  only  15  minutes,  but  other  smears 
will  adhere  better  if  left  in  alcohol- 
ether  for  an  hour  or  more.  In  the 
staining  process,  when  slides  are  trans- 
ferred from  one  solution  to  another, 
they  should  be  drained  as  much  as 
possible,  particularly  during  alcohol 
rinses  following  the  counterstains,  and 
during  dehydration.  It  is  also  im- 
portant that  slides  be  passed  through 
solutions  slowly  and  carefully,  with 
minimal  agitation  of  the  solutions,  so 
that  cells  will  not  be  washed  from  the 
slides.  Besides  the  loss  of  cells,  there 
is  also  the  danger  of  contamination  of 
other  smears  with  "floaters"  (cells  or 
clumps  of  cells  which  have  fallen  into 
one  of  the  solutions).  However,  these 
floaters  can  usually  be  recognized 
microscopically  since  they  are  on  a 
higher  focusing  level  than  the  fixed 
smear.  There  is  also  danger  of  con- 
tamination while  mounting  the  smears 
if  the  glass  rod  used  for  applying  the 
mounting  medium  touches  the  smear 
and  picks  up  loose  cells. 

Smears  which  have  been  fixed  but 
not  stained  may  be  mailed  to  a  labora- 
tory for  staining  by  the  method  sug- 
gested by  Ayre  and  Dakin.  (Ayre,  J. 
E.  and  Dakin,  E.,  Canad.  M.  A.  J., 
1946,  54,  489-91.)  After  the  smear  has 
been  fixed  in  alcohol -ether  for  one  hour, 
it  is  removed  from  the  fixative  and, 
without  drying  it,  two  or  three  drops  of 
glycerine  are  placed  on  it.  A  clean 
coverslip  or  slide  is  placed  on  top  of 
the  smear  thereby  sealing  it  from  the 
air.  When  the  slides  are  received  in 
the  laboratory,  thej'  are  placed  in  a 
jar  of  alcohol -ether  until  the  covering 
slide  or  coverslip  becomes  loose  and 
slips  off  without  disturbing  the  smear. 

It  should  be  stressed  that  the  Papa- 
nicolaou smear  technique  as  applied  to 
cancer  diagnosis  is  not  a  substitute  for 
biopsy  or  other  established  methods  of 
diagnosis  and  that  positive  smears 
should  be  corroborated  whenever  pos- 
sible by  biopsy  or  x-raj^  before  an}'' 
major  surgery  is  performed.  How- 
ever, it  can  be  extremely  useful  in  the 
discovery  of  earlj^  or  hidden  lesions. 
This  is  particularly  true  in  cervical 
and  bronchogenic  carcinomas,  where 
the  degree  of  accuracy  of  the  test  is 
high.  It  has  an  additional  advantage 
in  that  it  is  a  short  and  relatively  simple 
laboratory  procedure  and,  in  most 
instances,  the  specimens  can  be  ob- 
tained with  little  discomfort  to  the 
patient. 


PAPER  CHROMATOGRAPHY 


259 


PARAFFIN  IMBEDDING 


Too  much  emphasis  cannot  be  placed 
on  the  importance  of  good  technique 
in  the  preparation  and  staining  of 
smears  which  is  absolutely  essential 
for  their  correct  evaluation.  Regard- 
less of  the  amount  of  training  and  ex- 
perience one  has  had  in  cytology,  it  is 
most  difficult  and  often  impossible  to 
interpret  cells  which  have  been  poorly 
preserved  or  improperly  stained. 
Paper  Chromatography — Written  by  Eugene 
Roberts,  Division  of  Cancer  Research, 
Washington  University,  St.  Louis  10. 
July  15,  1951 — This  technique  makes 
possible  the  identification  and,  in  some 
cases,  the  quantitative  determination 
of  minute  amounts  of  numerous  sub- 
stances of  biological  interest.  Impetus 
was  given  to  the  development  of  these 
methods  by  their  classical  application 
to  the  separation  of  amino  acids  (Cons- 
den,  R.,  Gordon,  A.  H.  and  Martin, 
A.  J.  P.,  Biochem.  J.,  1944,  38,  224- 
232).  Subsequent  studies  using  paper 
chromatographic  procedures  have  been 
made  of  the  chemistry  and  metabolism 
of  such  diverse  materials  as  inorganic 
ions,  amino  acids,  proteins  and  en- 
zymes, carbohydrates,  fats,  vitamins, 
purines  and  pyrimidines,  pigments, 
growth  factors,  and  antibiotics.  The 
extensive  literature  dealing  with  the 
theoretical  and  practical  aspects  of 
this  rapidly  expanding  subject  has  been 
summarized  in  recent  reviews  (Clegg, 
D.  L.,  Analytical  Chemistry,  1950, 
22,  48-59;  Strain,  H.  H.,  Analytical 
Chemistry,  1951,  23,  2^38)  and  in  an 
excellent  symposium  (Biochemical  So- 
ciety Symposia  No.  3,  Partition  Chro- 
matography, Cambridge  University 
Press). 

Basically,  the  technique  consists  of 
applying  a  small  amount  of  solution 
containing  the  sample  near  the  edge  of 
a  strip  or  square  of  filter  paper  and  al- 
lowing a  suitable  solvent  to  flow  along 
the  paper  past  the  sample  either  by 
downward  or  upward  migration.  The 
paper  is  removed  and  dried.  When 
conditions  are  properly  chosen,  the 
components  of  the  mixture  are  sepa- 
rated in  such  a  manner  that  each  one 
occupies  a  discrete  spot  on  the  paper. 
When  the  substances  of  interest  are 
colorless,  numerous  optical  and  chemi- 
cal methods  may  be  applied  in  localizing 
them.  Radioautography  has  proven 
to  be  an  extremely  useful  adjunct  in 
work  with  isotopically  labeled  com- 
pounds. The  separation  of  components 
in  mixtures  containing  substances  of 
similar  chemical  properties  is  often 
facilitated  by  the  use  of  two  solvents 
(two  dimensional  chromatography). 
The  sample  is  placed  6  cm.  from  one 


corner  of  a  large  sheet  of  filter  paper 
(i.e.,  18  X  22").  It  is  developed  with 
one  solvent,  taken  out,  and  dried.  The 
sheet  is  then  rotated  90°  and  placed 
into  a  solvent  of  different  properties. 
After  drying,  the  spots  are  visualized 
by  an  appropriate  method.  For  any 
given  pair  of  solvent  systems  the  sub- 
stances will  occujJy  characteristic  posi- 
tions on  the  chromatograms.  The 
preparation  of  reference  chromatograms 
with  known  compounds  of  a  high  degree 
of  purity  is  made  under  the  same  condi- 
tions. The  spots  on  the  unknown  can 
then  be  identified  by  comparison  with 
the  reference  "map".  Paper  chro- 
matography can  be  performed  success- 
fully in  equipment  usually  found  in 
biological  laboratories  (test  tubes,  bell 
jars,  crocks,  petri  dishes)  and  does  not 
require  training  in  complicated  chemi- 
cal methodology. 

Results  of  interest  to  the  cytologist 
have  been  obtained  in  the  examination 
of  the  free  (or  loosely  bound)  amino 
acids  in  normal  rat  tissues  (Awapara, 
J.,  J.  Biol.  Chem.,  1949,  178,  113-116), 
in  normal  and  neoplastic  mouse  tissues 
(Roberts,  E.  and  Frankel,  S.,  Cancer 
Res.,  1949,  9,  645-648),  and  in  isolated 
hepatic  cell  nuclei  of  the  rat  (Dounce, 
A.  L.,  Tishkoff,  G.  H.,  Barnett,  S.  R. 
and  Freer,  R.  M.,  J.  Gen.  Physiol., 
1950,  33,  629-642).  Amino  acids  in 
acid  hydrolysates  of  mitochondria  iso- 
lated from  various  tissues  have  also 
been  studied  by  this  method  (Li,  C. 
and  Roberts,  E.,  Science,  1949,  110, 
559-560). 

Pappenheim,  see  Panchrome,  Kardos-Pap- 
penheim,  Methyl  Green-Pyronin  and 
May-Giemsa  Stains. 

Para  Red  (CI,  44)  is  useless  as  a  stain  (Emig, 

P-30).  ,       .       ,  . 

Parabenzoquinone,  as  a  fixative  for  mito- 
chondria (Baker,  J.  R.,  Nature,  1932, 
130,  134;  Sircar,  S.  M.,  J.  Roy.  Micr. 
Soc,  1935,55,  238-244). 

Paracarmine  (Mayer).  Dissolve  1  gm.  car- 
minic  acid,  0.5  gm.  aluminium  chloride 
and  4  gms.  calcium  chloride  in  100  cc. 
70%  alcohol.  Warm  slightly,  if  required. 
Allow  to  settle  and  filter.  Tissues  to  be 
stained  should  not  be  alkaline  or  con- 
tain much  lime  (Lee,  p.  147). 

Paraffin  Imbedding.  For  routine  it  is  more 
convenient  than  celloidin  imbedding. 
Thinner  sections  can  be  cut  and  it  is 
easier  to  make  them  in  series.  Paraffin 
imbedding  is  quicker  and  the  blocks 
being  dry  are  easily  stored  in  a  smaller 
space. 

After  the  specimen  has  been  cleared 
(see  Clearing)  it  is  placed  in  paraffin 
held  at  a  temperature  just  sufficiently 
high  to  keep  it  melted.     For  ordinary 


PARAFFIN  SECTIONS 


260 


PARAFFIN  SECTIONS 


purposes  a  paraffin  with  melting  point 
of  56-58°C.  is  employed;  but  6(>-62°C. 
is  sometimes  selected  for  very  thin  sec- 
tions and  52-54''C.  for  thick  ones. 
Paraffins  of  low  melting  points  are  de- 
scribed by  Waterman,  H.  C,  Stain 
Techn.,  1939,  14,  55-62.  When  it  is 
desired  to  give  the  imbedding  medium 
more  firmness  than  60-62 °C.  paraffin, 
use  is  occasionally  made  of  Rubber 
Paraffins  or  Ceresin.  Under  Clarite 
is  described  a  mixture  of  paraffin  and 
clarite  for  use  in  hot  weather  when  thin 
sections  are  demanded.  Routine  paraf- 
fin infiltration  is  best  done  in  wide 
mouthed  glass  bottles  or  jars  in  an  in- 
cubator held  at  the  proper  temperature. 
Excessive  temperatures  harden  and 
shrink  the  tissues.  The  paraffin  over 
each  specimen  should  be  changed  at 
least  once  to  insure  removal  of  the  xylol 
or  other  clearing  agent.  If  this  removal 
is  incomplete  difficulties  will  be  later 
encountered  in  crystallization  of  the 
paraffin  block  and  in  sectioning.  The 
time  necessary  for  infiltration  will  de- 
pend on  the  size  of  the  tissue  and  its 
penetrability.  Five  to  6  hours  is  about 
the  average  with  limits  of  2  to  24  hours 
in  special  cases.  See  special  treatment 
for  Teeth  and  Bone. 

For  actual  imbedding,  folded  paper 
containers  have  now  been  rather  gen- 
erally replaced  by  glass  dishes.  Watch 
glasses  (Syracuse  preferred)  are  satis- 
factory ;  but  Petri  dishes,  the  inner  sides 
of  which  are  not  quite  vertical  but  slope 
outward  slightly  from  the  base,  are  bet- 
ter. First  smear  a  little  glycerin  evenly 
over  the  bottom  and  sides  of  the  dish. 
Then  pour  in  a  little  paraffin,  a  thin 
layer  of  which  will  harden  so  that  when 
the  tissue  is  placed  in  the  dish,  it  will 
not  come  in  contact  with  the  bottom. 
It  is  customary  to  orient  the  tissue  so 
that  the  surface  to  be  cut  first  is  next 
to  the  bottom  of  the  dish.  Quickly 
pour  in  more  paraffin  until  the  tissue  is 
covered  to  a  depth  of  say  6  mm.  Hold 
the  dish  in  ice  water  until  the  surface 
of  the  paraffin  has  hardened  just  to  the 
point  when  on  immersion  in  the  iced 
water  the  surface  will  hold  its  shape  and 
not  run.  However  too  rapid  cooling 
of  paraffins  of  high  melting  point  may 
cause  cracks  in  the  surface  and  even  in 
the  depth  of  the  blocks.  After  a  few 
minutes  the  paraffin  block  slips  out 
easily  because  the  glycerin  prevented  it 
from  sticking.  When  several  different 
specimens  are  imbedded  in  the  same 
dish  identify  each  by  partly  imbedding 
near  it  a  small  strip  of  paper  bearing  its 
number.  Finally  some  of  the  paraffin 
is  cut  away  from  each  tissue  so  that  it 
can  be  conveniently  filed  away  but  it  is 


important  not  to  remove  too  much 
paraffin . 
Paraffin  Sections.  1.  Blocking.  If  the 
specimen  is  a  slice  of  tissue  it  was 
trimmed  at  the  time  of  fixation  into  a 
quadrangular  form  with  each  edge  and 
surface  parallel  to  the  opposite  one.  If 
the  specimen  is  a  cross  section  of  a 
tubular  structure  the  cutting  will  be 
more  difficult.  Heat  the  metal  holder 
of  the  microtome,  gently  press  the  sur- 
face of  the  paraffin  block  against  it  and 
harden  in  iced  water.  The  surface  of 
tissue,  protected  by  the  most  paraffin 
(which  is  the  upper  surface,  remote 
from  bottom  of  the  dish,  as  it  was  im- 
bedded), should  be  next  to  the  holder 
and  as  far  as  possible  evenly  equidistant 
from  the  surface  of  the  holder.  Unless 
there  is  plenty  of  paraffin  between  the 
tissue  and  the  holder,  difficulties  will 
be  encountered  if  it  becomes  necessary 
to  remount  the  block  on  subsequent  oc- 
casions to  cut  more  sections.  Since  the 
slice  of  tissue  is  of  even  thickness  its 
outer  surface  will  be  evenly  parallel 
to  the  sweep  of  the  knife  so  that  the 
tissue  included  in  a  given  section  will 
be  approximately  the  same  distance 
from  the  surface  of  the  block  and  equally 
subjected  to  fixation  and  subsequent 
technique. 

2.  Cutting.  The  knife  should  cut 
from  long  side  to  opposite  long  side. 
Trim  the  edges  of  the  paraffin  block  so 
that  it  will  have  to  pass  through  an  even 
layer  of  paraffin  at  least  5  mm.  wide  both 
before  and  after  it  enters  and  leaves  the 
tissue.  When  more  paraffin  is  cut  away 
it  may  be  later  needed  if  more  trimming 
is  required  to  make  the  sections  into 
straight  ribbons.  The  sides  of  the 
tissue  should  also  be  protected  by  layers 
of  paraffin  which  are  parallel  and  of  even 
thickness.  The  object  of  all  this  is  for 
the  knife  to  cut  through  the  paraffin  and 
tissue  squarely  and  for  it  to  encounter 
as  nearly  as  possible  equal  resistance. 
The  resistance  of  the  paraffin  at  the  sides 
will,  however,  always  be  less  than  that 
of  the  paraffin  plus  the  tissue  at  the  cen- 
ter. For  this  reason  it  may  be  necessary 
to  cut  away  most  of  the  paraffin  from  the 
sides. 

But  all  specimens  are  not  rectangular 
slices  of  tissue  of  uniform  thickness. 
Spherical  bodies  are  easy  to  cut  but  the 
sections  obtained  are  very  difficult  to 
flatten.  Specimens  containing  large 
cavities  are  troublesome  because  the 
paraffin  in  the  cavities  offers  so  little 
resistance.  In  such  cases  celloidin  im- 
bedding is  advised.  When  a  part  of  the 
tissue  is  brittle  and  the  rest  soft  it  is 
best  to  orient  the  tissue  so  that  the  knife 
passes  through  the  soft  part  first.     In 


PARAFUCHSIN 


261 


PARASITES 


orientation  of  fairly  large  objects  a 
beam  of  light  passed  through  the  paraffin 
block  from  an  arc  lamp  or  other  powerful 
source  is  of  great  assistance.  For  very- 
minute  objects  a  method  described  by 
Fry  (H.  J.,  Anat.  Rec,  1927,  34,  245- 
252)  is  suggested.  For  refractory  tis- 
sues, like  yolk  laden  eggs,  McClung  (p. 
40)  suggests  hydration.  The  block  is 
trimmed  until  the  imbedded  tissue  is 
exposed  when  it  is  soaked  in  water  for 
several  hours.  This  reduces  friability 
and  brittleness  and  good  sections  may 
often  be  obtained. 

Temperature  and  humidity  are  factors 
in  securing  a  good  ribbon  by  making  one 
section  stick  evenly  to  the  next  in  series. 
Sometimes  a  little  boiling  water  near  at 
hand  will  help  but  it  should  not  be 
necessary  if  the  tissue  has  been  properly 
infiltrated  with  paraffin  of  the  right 
melting  point  which  set  firmly  when 
cooled.  Static  electricity,  causing  the 
ribbon  to  adhere  in  a  troublesome  way 
to  surfaces,  is  partly  dependent  upon 
difference  in  density  of  tissue  and  paraf- 
fin. But  the  most  important  factor  in 
obtaining  excellent  sections  is  have  the 
microtome  in  good  working  order  and 
the  knife  sharp  (see  Sharpening). 
Lillie  (p.  44)  says  that  ribboning  con- 
sistency of  paraffin  is  sometimes  im- 
proved by  adding  to  the  paraffin  before 
infiltration  10-20%  of  beeswax  or  3-5% 
of  halowax.  For  ordinary  purposes 
sections  should  be  cut  6  microns  thick. 
To  mount  them  on  slides  first  smear 
carefully  cleaned  slides  (see  Slides)  with 
Albumen-Glycerin,  cover  with  aq.  dest. 
and  gently  heat  over  an  alcohol  lamp 
if  a  slide  warmer  is  not  available.  Then 
mark  the  slides  with  a  diamond  point 
pencil  and  leave  for  about  6  hrs.  in  a 
drying  oven  at  40-45°C. 

Parafuchsin,  see  Pararosanilin  (Magenta  O). 

Paraganglion,  see  Aortic. 

Paraldehyde  is  paracetaldehyde,  a  polymer 
of  acetaldehyde  employed  in  Dioxan 
fixative  and  in  other  ways. 

Paraloidin,  see  Celloidin. 

Paramagenta,  see  Pararosanilin  (Magenta 
O). 

Paramylum,  a  form  of  carbohydrate  store  in 
lower  plants  (Taylor  in  McClung,  p. 
221). 

Paramoecia.  Directions  for  using  dyes  for 
intravitam  staining  of  food  vacuoles, 
contractile  vacuoles  and  various  other 
structures,  as  well  as  minimum  concen- 
tration for  effective  staining  and  toxic- 
ity, are  supplied  by  McClung,  1950, 
pp.  437-439. 

Paraplasm  is  a  term  supposed  to  include  non- 
living cellular  components  such  as  gly- 
cogen and  lipid  granules.  It  is  mis- 
leading because  all  cellular  components 


contribute  in  one  way  or  another  to 
vital  phenomena.  Deutoplasm  is  syn- 
onymous. 

Pararosanilin  (Magenta  O)  (CI,  676) — basic 
rubin,  parafuchsin  and  paramagenta — 
This  is  triamino  -  triphenyl  -  methane 
chloride,  the  chief  component  of  most 
Basic  Fuchsins. 

Parasites.  These  range  all  the  way  from 
ultramicroscopic  viruses  to  organisms  a 
yard  or  more  long.  Microscopic  tech- 
niques for  viruses  are  given  under  Cyto- 
plasmic Inclusions,  Elementary  Bodies, 
and  Nuclear  Inclusions.  Certain  Gram 
negative  intracellular  insect  or  arachnid 
transmitted  bacteria  -  like  microor- 
ganisms are  called  Rickettsia  and  re- 
quire special  methods  for  their  demon- 
stration. See  also  Bacteria  and  Spiro- 
chaetales,  Fungi,  Piroplasma  and  Pro- 
tozoa. A  search  for  such  small  para- 
sites involves  not  only  an  examination 
of  tissues  but  also  of  body  fluids  includ- 
ing Blood,  Feces,  Gastric  Contents, 
Urinary  Sediment,  etc.  When  the  para- 
sites are  scarce  resort  is  made  to  methods 
of  Concentration.  Elementary  orienta- 
tion in  respect  to  the  larger  animal  para- 
sites (metazoa)  is  provided  by  the  fol- 
lowing classification  (according  to 
Stiles)  from  Stitt  (p.  387)  which  has 
been  slightly  modified. 

1 .  Body  more  or  less  dorsiventrally  flattened 3 

Body  in  cross  section  ordinarily  round 2 

2.  Body  never  annulated,  without  legs  or  jawB.. . .  4 
Body  annulated  (at  least  possesses  mouth  parts), 

breathes  usually  through  tracheal  system, 
adults  with  jointed  legs  or  other  appendages..  6 

3.  Intestine  present  without  anus,  1  or  2  suckers, 

body  not  segmented.  (In  liver,  lungs,  blood, 
intestine  rarely  elsewhere — flukes)  Trematoda 

Intestine  absent,  2  or  4  suckers  on  head,  body 
of  adults  segmented,  tissue  usually  contains 
calcareous  bodies,  adults  (tapeworms)  in  in- 
testine,    larvae   (bladder     worms)    elsewhere 

Cestoda 

Intestine  and  anus  present,  sucker  on  posterior 
end,  body  annulated  like  earthworm,  in  upper 
air  passagei  or  externally  (leeches,  blood 
suckers) Hirudinea 

4.  Intestine  absent,  armed  rostellum  present,  very 

rare  in  human  intestine,  thorn  headed  worm* 

Acanthocephala 
Intestine  present,  but  no  armed  rostellum 

Nematoda  5 

5.  Intestine   rudimentary    in    adults,    no    lateral 

chords,  rare  in  human  intestine  (hair  snakes  or 

horse   hair  worms) Gordiacea 

Intestine  present  with  lateral  chords,  common 
in  intestine,  muscles,  lymphatics,  etc.  (round 
worms) Eunematoda 

6.  Six  legs  in  adult,  wings  in  most  species,  larvae 

annulated,  breathe  by  trachea,  adults  ecto- 
parasites, occasionally  under  skin,  in  wounds, 
intestine  or  bladder  (insects) Insecta 


PARENCHYMATOUS 
DEGENERATION 


262 


PEPTIDASE 


Eight  legs  in  adult,  6  in  larva,  head  and  ab- 
domen coalesced,  ectoparasites,  may  burrow 
under  skin  or  live  in  hair  follicles  (ticks,  mites, 
etc.) Acarina 

Four  claws  about  mouth,  larvae  encysted  in  various 
tissues,  adulta  occasionally  in  nasal  passages 
(tongue  worms) Linguatulidae 

Numeroiis  legs,  occasionally  in  nasal  passages  and 
intestine    (thousand   leggera) Myriapoda 

See  Tapeworm  Progloltids,  Trema- 
todes,  Taenia,  Ticks,  Insects,  Enda- 
moeba,  Trichinella,  Glychrogel. 

Parenchymatous  Degeneration,  see  Cloudy 
Swelling. 

Parhemoglobin,  a  kind  of  hemoglobin  which 
crystallizes  in  same  fashion  but  is  in- 
soluble   in   alcohol    (Mallory,    p.    135). 

Paris  Blue,  see  Spirit  Blue. 

Paris  Violet,  see  Methyl  Violet. 

Parlodion,  a  derivative  of  pyroxylin  (see 
Celloidin). 

Paschen's  Method  for  elementary  bodies  as 
given  by  Seiffert,  G.,  Virus  Diseases  in 
Man,  Animal  and  Plant.  New  York: 
Philosophical  Library,  Inc.,  1944,  332 
pp.  Dry  very  thin  smears  in  air. 
Place  slides  perpendicular  in  aq.  dest. 
Ringer  or  physiological  saline,  3-10 
min.,  longer  for  older  specimens.  Then 
dry  and  place  in  abs.  ale.  1-24  hours,  or 
in  methyl  alcohol,  10  min.  Dry,  cover 
with  filtered  Loeffler  caustic  (HoUborn) 
and  heat.  Rinse  in  aq.  dest.  and  color 
with  well  filtered  Carbol  Fuchsin. 
Rinse  in  aq.  dest.  (To  destain  if  neces- 
sary dip  in  abs.  ale,  then  rinse  in  aq. 
dest.),  blot  dry. 

Pasteurella,  capsules  of.  A  modification  of 
Hiss's  method  advocated  by  Jasmin, 
A.  M.,  J.  Bact.,  1945,  50,  361-363. 
Transfer  amount  of  surface  culture  ad- 
hering to  a  fine,  straight  platinum  wire 
to  loopful  physiological  saline  4-0.5-1% 
phenol  and  10%  blood  serum.  Spread 
thin  film  on  clean  polished  slide;  fix 
dried  film  by  quickly  dipping  in  methyl 
alcohol.  Drain  and  flame  to  remove 
excess  alcohol.  Finally  color  i  to  1  min. 
in  any  regular  bacterial  stain,  wash  in 
water  and  dry.  Capsules  appear  as 
clear  areas  about  strongly  stained  bac- 
teria in  lightly  colored  background. 

Patent  Blue  A  (CI,  714)— Brilliant  Acid 
Blue  A — an  acid  dye  of  light  fastness  4, 
stains  parenchyma  blue  green  with  poor 
definition  (Emig,  p.  52). 

Pectinols  are  enzyme  preparations  of  4 
grades  supplied  by  Rohm  and  Haas  Co. 
of  Philadelphia.  Their  primary  action 
is  on  pectins.  McKay,  H.  H.  and 
Clarke,  A.  E.,  Stain  Techn.,  1946,  21, 
111-114  recommend  their  use  to  demon- 
strate chromosomes  of  root  tip  smears 
after  colchicine  and  before  staining 
with  carmine. 


Pectins,  macromolecular  properties,  test  for 
(Hueper,  W.  C,  Arch.  Path.,  1942,  33. 
267-290).    See  Ruthenium  Red. 

Pencil  Red  Cells  are  oval  or  elongated 
erythrocytes  in  the  condition  of  ovalo- 
cytosis 4  or  5  times  as  long  as  they  are 
broad. 

Pentose  Nucleic  Acid  is  present  in  cyto- 
plasm, nucleoli  and  possibl}^  in  the 
chromatin  of  cells.  With  proper  con- 
trols the  substance  may  be  identified 
in  cells  by  use  of  ultraviolet  micro- 
spectrophotometry  or  by  Pyronin- 
methyl  Green  and  ribonuclease.  It  is 
found  in  high  concentration  in  cell, 
actively  producing  protein  (Stowells 
R.  E.,  Cancer,  1948,  2,  121-131)  and  is 
generally  associated  with  basophilia 
(Caspersson,  T.  and  Schultz,  J.,  Nature, 
1939,143,602-603). 

Pepsin,    microchemical    determination : 

1.  Freeze  gastric  mucous  membrane 
of  freshly  killed  pig.  Keep  at  —  10°C. 
Cut  cylinders  of  tissue  (2.5  mm.  in  diam- 
eter) with  sharp  cylindrical  borer  ver- 
tical to  surface.  Mount  cylinders  with 
muscle  down  on  a  piece  of  cardiac  mu- 
cosa or  on  stiffened  gelatin.  Freeze 
with  COj.  Cut  sections  at  25  microns. 
Make  enzyme  determinations  on  section 
and  correlate  with  structure  in  adjacent 
sections  and  with  known  distribution  of 
cell  types  at  different  distances  from 
lumen.  This  shows  that  chief  cells  are 
the  source  of  the  pepsin  (Holter,  H.  and 
Linderstr0m-Lang,  K.,  C.  rend.  Trav. 
Lab.  Carlsberg,  1935,  20  (11)  1-32). 

2.  Make  extract  of  tissue,  mix  with 
buffers  at  suitable  pH,  apply  to  gelatin 
surface  of  Eastman  lantern  slide  plate, 
incubate,  wash  gelatin  surface,  fix  in 
20%  formalin,  stain  with  acid  fuchsin 
or  Delafield's  hematoxylin  and  observe 
sites  of  proteolytic  activity  evidenced 
by  clear  spots.  Test  is  positive  for  0.0001 
-0.0002  gm.  stomach  of  young  ambly- 
stoma  weighed  wet.  Details  of  this  in- 
genious technique,  also  applicable  with 
slight  modification  for  trypsin,  are  given 
by  Dorris  (F.,  J.  Exp.  Zool.,  1935, 
70,  491-527).  See  also  Peptidase,  Pep- 
sin and  Dipeptidase. 

Pepsinogen,  antecedent  of  pepsin  in  body 
chief  cells  of  stomach.  For  staining 
reaction  and  discharge  by  vagal  stimu- 
lation, see  Bowie,  D.  J.  and  Vineberg, 
A.  M.,  Quart.  J.  Exper.  Physiol.,  1935, 
25,   247-257. 

Peptidase  can  be  localized  in  centrifuged 
marine  eggs  by  direct  enzymatic  analysis 
of  fragments  containing  different  cyto- 
plasmic components  using  a  procedure 
essentially  the  same  as  that  advocated 
by  Linderstr0m-Lang  and  his  associates. 
It  occurs  in  the  hyaline  ground  sub- 
stance and  is  not  bound  to  the  granular 


PERDRAU'S  MODIFICATION 


263 


PERICARDIUM 


material  (Holter,  H.,  J.  Cell,  and  Comp. 
Physiol.,  1936,  8,  ITQ^IOQ).  Simittr 
studies  with  amebae  indicate,  likewise, 
association  with  cytoplasmic  matrix 
(Holter,  H.  and  Kopac,  M.  J.,  J.  Cell, 
and  Comp.  Physiol.,  1937,  10,  423-427). 
These  techniques  are  likely  to  be  of  wide 
usefulness.  Peptidase  has  been  loca- 
lized in  gastric  and  duodenal  mucosa  of 
the  pig  by  Linderstr0m-Lang  and  Hol- 
ter (K.  and  H.,  C.  rend  Trav.  I^b. 
Carlsberg,  1935,  20  (11),  42-56).  See 
also  Mauer  et  al.  (J.  Nat.  Cancer  Inst., 
1941,  2,  278).  An  excellent  critical 
discussion  of  the  histological  distribu- 
tion of  peptidase  is  provided  by 
Blaschko  and  Jacobson  (Bourne,  pp. 
207-216). 

Anfinsen,  C.  B.,  Lowry,  O.  H.  and 
Hastings,  A.  B.,  J.  Cell,  and  Comp. 
Physiol.,  1942,  20,  231-237  have  de- 
veloped a  method  whereby  the  same 
section  can  be  stained  for  microscopic 
examination  and  thereafter  used  for 
enzyme  analysis.  It  works  also  for  di- 
phosphopyridine  nucleotide  and  choli- 
nesterase.     See  Protease. 

Perdrau's  Modification.  Bielschowsky's 
silver  method  for  reticulum  as  detailed 
by  Bailey,  P.  and  Hiller,  0.,  J.  Nerv. 
&  Ment.  bis.,  1924,  59,  337-361.  Fix 
in  10%  formalin.  Wash  in  running  tap 
water  12-24  hrs.,  then  in  several  changes 
aq.  dest.,  24  hrs.  more.  Cut  frozen 
sections,  15-25  m,  and  leave  in  aq.  dest. 
24  hrs.  0.25%  aq.  potassium  perman- 
ganate, 10  min.  Wash  in  aq.  dest. 
Decolorize  in  equal  parts  1%  oxalic  acid 
and  1%  acid  potassium  sulphite.  Wash 
in  several  changes  aq.  dest.  over  night. 
Treat  with  following  solution  40-60 
min. :  Add  2  drops  40%  sodium  hydrox- 
ide to  5  cc.  20%  silver  nitrate.  Just 
dissolve  ppt.  with  ammonia.  Dilute  to 
50  cc.  with  aq.  dest.  and  filter.  Wash 
sections  rapidly  with  aq.  dest.  Reduce 
in  20%  formalin  in  tap  water,  30  min. 
Wash  in  aq.  dest.  Tone  with  gold 
chloride  and  continue  as  in  Laidlaw's 
Method.  Reticulum,  black;  collagen 
reddish.  This  is  intended  primarily 
for  nervous  system,  see  Bailey  and  Hil- 
ler's,  Fig.  3. 

Perenyi's  Fluid.  3  parts  95%  alcohol,  4 
parts  10%  aq.  nitric  acid,  3  parts  0.5% 
chromic  acid  is  according  to  Lee  (p.  32) 
an  important  fixative  for  embryos,  seg- 
menting eggs,  etc. 

Perfusion.  The  technique  of  washing 
through  the  blood  vessels  with  a  fluid  is 
one  of  wide  usefulness.  It  is  in  general 
the  same  but  varies  somewhat  depend- 
ing upon  what  is  to  be  perfused.  The 
apparatus  consists  of  a  bottle  capable 
of  holding  at  least  1000  cc.  equipped  with 
an  outlet  near  the  bottom  or  a  bent  glass 


tube  siphon  connected  by  a  rubber  tube 
about  6  feet  long  with  a  glass  Cannula. 
An  artery  clamp  placed  about  1  foot  from 
the  cannula  will  serve  as  a  shut  off. 

If  one  wishes  to  perfuse  a  mouse  the 
best  way  is  to  tie  a  small  cannula  into 
the  ventricle,  if  it  is  the  abdominal 
organs  of  a  guinea  pig  the  following  pro- 
cedure is  advised:  Kill  the  animal  with 
chloroform  if  this  anesthetic  will  not 
interfere  with  the  results  as  is  seldom 
the  case.  Cut  carotids  and  jugular 
veins  to  partly  exsanguinate  the  animal. 
Clip  away  sternum  and  most  of  the  ribs. 
Displace  left  lung,  expose  thoracic  aorta 
and  free  a  portion  of  it  from  surrounding 
tissue.  Pass  moistened  ligature  thread 
behind  aorta.  Make  with  scissors  a 
small  slit  in  wall  of  aorta  not  at  right 
angles  to  it  but  directed  into  it  and 
downward  (toward  tail)  being  careful 
not  to  cut  more  than  3  through  it.  In- 
sert wet  cannula  into  the  slit  with  slight 
rotatory  motion  until  the  constriction 
in  the  cannula  is  about  1  cm.  within  the 
aorta.  Then  bring  the  two  ends  of  the 
thread  together  and  tie  the  cannula  in 
place.  Remove  clamp  from  rubber  tube 
and  allow  fluid  to  flow  in  from  bottle 
suspended  about  4  feet  above  cannula, 
open  right  auricle  to  permit  free  exit  of 
fluid.  It  may  be  necessary  to  clamp  in- 
ferior vena  cava  just  above  diaphragm 
and  increase  pressure  somewhat.  Some- 
times it  is  helpful  to  vary  pressure  by 
opening  and  closing  clamp.  After  4  or 
5  minutes  open  abdomen  and  examine 
organ  which  it  is  desired  to  perfuse. 
The  absence  of  blood  color  in  it  and  the 
color  of  the  perfusate  (if  colored)  are 
indicators  of  completeness  of  the  oper- 
ation. The  pancreas  and  the  liver  will 
swell  considerably  but  this  may  not  be  a 
disadvantage. 

Pericapillary  Cells,  or  pericytes,  are  closely 
applied  to,  or  wrapped  about,  the  endo- 
thelium of  blood  capillaries.  The  desig- 
nation relates  to  position  only  and  it 
includes  cells  of  several  sorts  from 
much  branched  Rouget  cells  to  simple 
fusiform  muscle  cells  and  connective 
tissue  cells.  Methods  of  silver  im- 
pregnation and  beautiful  illustrations 
are  provided  by  Zimmermann,  K.  W., 
Zeit.  f.  Anat.,  1923,  68,  29-109.  The 
myofibrils  in  contractile  pericapillary 
cells  can  be  stained  supravitally  with 
jauus  green,  (Bensley,  R.  R.  and  Vim- 
trup,  R.,  Anat.  Rec,  1928,  39,  37-55). 
Valuable  data  can  be  obtained  by  micro- 
dissection of  the  living  tissues  (Zwei- 
fach,  B.  W.,  Am.  J.  Anat.,  1937,  60, 
473-657). 

Pericardium.  Special  dissections  of  bands 
of  fibers  in  periciirdium  (Popa,  J.  T. 
and  Lucinescu,  E.,  J.  Anat.,  67,  78-107). 


PERITONEAL  FLUID 


264 


PEROXIDASE 


Methods  for  study  of  absorption  of  sub- 
stances placed  in  pericardial  sac  (Drin- 
ker, C.  K.  and  Field,  M.  E.,  J.  Exper, 
Med.,  1931,  53,  143-150). 

Peritoneal  Fluid.  Cells  present  (Webb, 
R.  L.,  Am.  J.  Anat.,  1931-32,  49,  283- 
334;  Folia  Haemat.,  1933,  51,  445-451). 

Periodontium,  see  method  for  Teeth  and 
Jaws. 

Peritoneum.  Outlines  of  mesothelial  cells 
blackened  with  silver  nitrate  (Pumala, 
R.  H.,  Anat.  Rec,  1937,  68,  327-338, 
good  illustrations).  Exudate  cells 
stained  vitally  with  lithium  carmine 
(Maximow,  A.  A.,  Cowdry's  Special 
Cytology). 

Perivascular  Spaces  of  the  brain.  The  Weed 
McKibben  method  (Weed,  L.  H.,  Am. 
J.  Anat.,  1923,  31.  191-221),  based  on 
dehydrating  the  brain  by  increasing 
osmotic  pressure  of  the  blood  and  draw- 
ing into  these  perivascular  spaces  solu- 
tions of  pota.ssium  ferrocyanide  and 
iron  ammonium  citrate,  after  injection 
into  the  subarachnoid  space,  and  their 
later  precipitation  as  Prussian  blue  by 
fixing  tissue  in  acid  formalin,  has  been 
modified  by  Patek,  P.  E.,  Anat.  Rec, 
1944,  88,  1-24.  In  rabbits  and  cats  he 
injects  intravenously  6-8  cc.  30%  aq. 
sodium  chloride  during  10  min.  and 
3-4  cc.  particulate  suspension  of  india 
ink  or  mercur}'^  sulfide  in  the  cisterna 
magna  under  atmospheric  pressure  dur- 
ing 15-20  min.  The  animal  is  then 
killed  by  bleeding  and  perfused  via 
the  aorta  with  10%  formalin.  After 
further  fixation  of  brain  by  immersion 
1  mm.  slices  are  cut  and  mounted  un- 
stained or  the  tissue  maybe  imbedded 
in  paraffin  in  celloidin  and  10-50  /x  sec- 
tions colored  with  gallocyanin  or  some 
other  appropriate  stain.  Dogs  can  also 
be  used  as  he  directs. 

Permeability.  This  is  a  fundamental  prop- 
erty for  the  study  of  which  there  are 
many  microscopic  techniques.  The 
idea  that  what  goes  in  and  what  comes 
out  through  the  plasma  membrane  (see 
Cell  Membranes)  always  depends  upon 
the  character  of  the  particular  substance 
and  of  the  membrane  is  fallacious.  By 
his  method  of  observing  in  vivo  the  ruffle 
Pseudopodia  of  macrophages  and  can- 
cer cells  W.  H.  Lewis  (Am.  J.  Cancer, 
1937,  29,  666-679)  has  enabled  us  to  see 
that  materials  can  be  drawn  into  the  cy- 
toplasm in  invaginations  of  the  plasma 
membrane  which  lose  connection  with 
the  outside  so  that  when  the  isolated 
membranous  investments  disintegrate 
the  materials  are  liberated  in  the  cyto- 
plasm without  ever  traversing  the  intact 
surface  plasma  membrane.  This  is  the 
converse  of  observations  made  possible 
by  the  direct  examination  of  secreting 


acinous  cells  of  the  pancreas  by  W.  P. 
Coyell  (Anat.  Rec,  1928,  40,  213-223) 
which  show  secretory  products  leaving 
the  cell  in  protrusions  of  the  plasma 
membrane.  These  later  become 
pinched  off,  the  membranes  disintegrate 
and  the  product  is  set  free  in  the  lumen. 
See  literature  review  (Blinks,  L.  R., 
Ann.  Rev.  Physiol.,  1942,  4,  1-24).  See 
Spreading  Factors. 
Peroxidase.  This  enzyme  catalyses  oxida- 
tion of  several  oxidizable  substrates  in 
presence  of  peroxide.  It  is  most  abun- 
dant in  plants  being  usually  prepared 
from  horse-radish.  In  mammals  it 
occurs  in  mammary  glands  and  in  milk. 
In  the  peroxidase  reaction,  so  commonly 
employed  in  the  study  of  leucocytes,  a 
colored  product  is  formed  in  the  pres- 
ence of  peroxide  from  a  suitable  sub- 
strate, benzidine  or  alpha  naphthol. 
Blaschko  and  Jacobson  (Bourne,  p.  197) 
remind  us  that  it  is  still  uncertain  that 
this  reaction  in  leucocytes  demonstrates 
a  true  peroxidase  because  it  is  relatively 
stable  to  heat. 

1.  Alpha  naphthol -pyronin  (Gra- 
ham, G.  S.,  J.  Med.  Res.,  1916,  30,  231- 
242).  Fix  blood  smears  in  9  parts  95% 
alcohol  and  1  part  formalin  freshly  pre- 
pared, 1-2  min.  Wash  in  water  and 
flood  with :  alpha  naphthol  (Merck's 
"recrystallized"  or  "Reagent"),  1  gm.; 
40% alcohol,  100  cc. ;  hydrogen  peroxide, 
0.2  cc  for  4-5  min.  Wash  in  dish  of 
running  water,  15  min.  Stain  in: 
pyronin  0.1  gm.;  anilin  oil,  4  cc;  40% 
alcohol  96  cc,  2  min.  Wash  in  water. 
Stain  in  0.5%  aq.  methylene  blue 
(Griibler's  BX),  ^-;1  min.  Wash  in 
water,  blot,  mount  in  neutral  balsam. 
Fresh  smears  should  be  used.  When 
used  by  a  class  of  students  tie  droppers 
to  bottles  to  avoid  spoiling  solutions  by 
mixing  them. 

2.  Benzidine-methylene  blue  (Gra- 
ham. G.  S.,  J.  Med.  Res.,  1918,  39,  15- 
24).  Fix  as  above.  Wash  in  water. 
Treat  5-10  min.  in  0.2%  hydrogen 
peroxide  in  40%  alcohol  saturated  before 
using  with  benzidine,  5-10  min.  Wash 
and  stain  with  methylene  blue. 

3.  Benzidine-safranin  (Sato,  A.  and 
Shoji,  K.,  J.  Lab.  and  Clin.  Med.,  1927- 
28,  13,  1058-1060).  Dry  blood  smear 
in  air.  Flood  the  slides  with  solution 
A  (0.5%  copper  sulphate).  After  1 
minute  pour  off  solution  but  do  not 
wash  or  dry  slides.  Apply  solution  B 
(rub  up  in  a  mortar  0.2  gms.  benzidine 
with  a  few  drops  distilled  water.  Then 
add  200  cc.  aq.  dest.  and  filter.  To 
filtrate  add  4  drops  3%  hydrogen 
peroxide)  for  2  min.  Then  wash  in  tap 
water.  Stain  with  solution  C  (1% 
safranin  in  aq.  dest),  1  min.    Wash  in 


PEROXYDASE 


265 


PHAGOCYTOSIS 


tap  water  and  dry.  Peroxidase  granules 
are  colored  blue  in  granular  leucocytes 
and  the  nuclei  orange  red. 

4.  Nitroprusside-benzidine  (Goodpas- 
ture, E.  W.,  J.  Lab.  &  Clin.  Med., 
1919,  4,  442-444).  To  make  the  stain 
dissolve  0.05  gm.  sodium  nitroprusside 
in  2  cc.  aq.  dest. ;  add  100  cc.  95%  alco- 
hol;  0.05  cc.  benzidine  C.P.;  0.05  gm. 
basic  fuchsin  and  0.5  cc.  hydrogen 
peroxide.  Cover  well  dried  blood  smear 
with  known  amount  of  stain,  1  min.;add 
equal  volume  aq.  dest.  plus  hydrogen 
peroxide,  3-4  min. ;  rinse  thoroughly  in 
water  and  blot  dry.  Shows  many  blue 
granules  in  granular  leucocytes  and  few 
in  monocytes.  Nuclei  are  colored  red. 
To  increase  intensity  of  stain  dilute 
with  a  little  less  aq.  dest.  and  stain 
longer.  Method  can  be  used  for  frozen 
sections  of  material  fixed  in  formalin 
and  preserved  in  80%  ale.  A  modifica- 
tion of  this  stain  has  been  proposed  by 
Beacom  (J.  Lab.  &  Clin.  Med.,  1925-26, 
11,  1092-1093)  with  hydrogen  peroxide 
omitted  and   basic  fuchsin  doubled. 

5.  Benzidine-Giemsa  (Armitage,  F. 
L.,  J.  Path.,  1939,  49,  579-580).  Fix 
smears  in  96%  alcohol  containing  10% 
formol  freshly  made  up.  Flood  with 
benzidine  mixture  (0.75  gm.  benzidine 
in  500  cc.  40%  ethyl  alcohol.  Filter. 
Add  7  cc.  3%  HjOi,  mix  by  shaking  im- 
mediately before  using)  2  min.  for  fresh 
films,  longer  for  older  ones.  Wash  in 
40%  alcohol  until  definite  yellow  gran- 
ules are  seen  in  granular  leucocytes. 
Absolute  alcohol  and  dry  in  incubator. 
Counterstain  with  dilute  Giemsa,  wash 
in  water,  blot  and  dry. 

6.  Benzidine  for  paraffin  sections 
(McJunkin,  F.  A.,  Anat.  Rec,  1922-23, 
24,  67-76).  After  fixation  of  small 
pieces  in  10%  formalin  imbed  quickly 
in  paraffin;  70%  alcohol,  1  hr. ;  acetone, 
30  min.;  benzol,  20  min.;  paraffin,  20 
min.  Mount  thin  sections  in  usual 
fashion.  Deparaffinize  in  benzol  20 
sec,  acetone,  10  sec.  Water,  few  sec- 
onds. Drain  off  water,  apply  mixture 
(80%  alcohol,  25  cc. ;  benzidine,  0.1  gm. ; 
hydrogen  peroxide,  2  drops)  diluted  with 
1  or  2  parts  aq.  dest.,  5  min.  Water,  5 
min.;  hematoxylin,  2  min.;  water,  1 
min.,  0.1%  aq.  eosin,  20  sec;  95% 
alcohol,  30 sec. ;  abs.  alcohol,  5 sec.  Clear 
in  xylol  and  mount  in  balsam. 

Note :  In  above  methods  a  blue 
counterstain  tends  to  obscure  the  blue 
peroxidase  reaction. 

7.  DCPIP-2,  6-(iichlor-phenol-indo- 
phenol  (Jacoby,  F.,  J.  Physiol.,  1944, 
103,  Proc.  Physiol.  Soc.  July  29).  Fix 
air  dried  blood  smear  in  9  parts  abs.  ale. 
and  1  part  formol  for  2-3  min.  Wash  in 
water.     Treat  smear  for  3-5  min.  with 


0.5%  aq.  2.6-dichlor-phenol-indophenol 
to  every  5  cc.  of  which  4  drops  IIjOi 
is  added  prior  to  use.  Wash  in  water, 
blot  dry  and  examine.  "Peroxidase- 
positive"  granules,  deep  purple-violet. 
No  precipitation  of  crystals  and  gran- 
ules on  smear.  Author  suggests  0.5% 
aq.  neutral  red  as  a  counterstain  to  be 
applied  after  treatment  with  DCPIP. 
If  smear  is  to  be  mounted  use  neutral 
balsam.  Solution  of  DCPIP  can  be 
stored  in  ice  box  for  few  months. 

Peroxydase,  see  Peroxidase. 

P6t6rfi,  see  Double  Imbedding,  and  Osmic 
Acid  Method  for  nerve  fibers. 

Petrunkevitch's  Fixatives:  Cupric-phenol. 
Stock  solution  A  =  aq.  dest.,  100  cc; 
nitric  acid  (cp.  sp.  gr.  1.41-1.42),  12 
cc;  Cu(N03)2-3  H^O,  8  gm.  Stock 
solution  B  =  80%  alcohol,  100  cc; 
phenol  crystals,  cp.  4  gm.;  ether  6  cc. 
iSmploy  1  part  A  with  3  parts  B.  Fix 
12-24  hrs.  Wash  in  70%  alcohol. 
Cupric-paranitrophenol.  60%  alcohol, 
100  cc;  nitric  acid  (same),  3  cc;  ether 
5  cc. ;  cupric  nitrate  (same),  2  gm.; 
paranitrophenol,  cp.  crystals,  5  gm. 
Time  unspecified.  Wash  in  70%  alco- 
hol. Said  not  to  harden  tissues  like 
ordinary  fixatives.  May  be  followed 
by  all  common  stains.  (Petrunkevitch, 
A.,  Science,  1933,  77,  117-118). 

Petrunkevitch's  Fluid  is  sat.  mercuric 
chloride  in  aq.  dest.,  300  cc,  abs.  ale, 
200  cc;  acetic  acid,  90  cc;  and  nitric 
acid,  10  cc. 

pH,    see   Hydrogen    Ion  Indicators. 

Phagocytosis.  There  are  numerous  methods 
for  the  demonstration  of  this  phenome- 
non from  which  to  choose. 

1.  In  Vaginal  Smears  (which  see), 
made  after  intercourse,  neutrophilic 
leucocytes  can  be  observed  in  the  act  of 
engulfing  individual  spermatozoa.  C. 
R.  Stockard,  in  Cowdry's  Special  Cy- 
tology, 1932,  3,  1611-1629,  has  described 
this  remarkable  process  as  seen  in  the 
living  state.  "A  leucocyte  comes  in 
contact  with  a  spermatozoon  which  with 
its  tail  is  longer  than  the  leucocyte. 
The  leucocyte  by  stretching  and  con- 
tracting finally  takes  into  itself  the 
entire  spermatozoon,  the  tail  being 
wound  in  a  circular  fashion  within  the 
cell   body." 

2.  In  temporary  mounts  of  bacteria 
and  Leucocytes  (which  see)  pliagocyto- 
sis  can  be  followed  in  detail.  Differ- 
ences in  the  behavior  of  neutrophiles 
from  seriously  ill  and  normal  persons 
have  been  described. 

3.  Under  Vital  Staining  will  be  found 
many  techniques  which  permit  the 
observation  of  the  phagocytosis  of 
inanimate  particulate  materials  by 
macrophages.     A  graphic  demonstration 


PHASE  CONTRAST  MICROSCOPE 


266 


PHLOXINE-METHYLENE  BLUE 


of  the  immunologic  control  of  phagocy- 
tosis of  erythrocytes  by  these  cells  can 
be  provided  by  using  a  method  de- 
scribed by  Bloom,  W.,  Arch.  Path.  & 
Lab.  Med.,  1927,  3,  608-628. 
Phase  Contrast  Microscope.  By  phase 
contrast  one  generally  means  a  system 
of  illumination  and  phase  retardation 
by  means  of  which  phase  differences 
of  light  waves  are  converted  into  ampli- 
tude differences.  Its  purpose  is  to 
create  contrast,  thus  rendering  objects 
visible  which  would  otherwise  be  im- 
possible to  see  in  a  bright-field  because 
of  e.xcessive  transparency. 

The  equipment  consists  of  an  annular 
diaphragm  in  the  front  focal  plane  of 
the  condenser  and  a  diffraction  plate 
of  special  design  placed  between  the 
components  of  the  objective  lens. 
Phase  contrast  microscopes,  developed 
in  Europe  just  before  the  war,  have 
been  commercially  available  in  the 
United  States  only  recently  but  already 
an  impressive  amount  of  investigation 
has  been  reported  on  biological  material 
(Bennett,  A.  H.,  Sci.  Monthly,  1946,  43, 
191-193;  Richards,  O.  W.,  Cold  Spring 
Harbor  Svmposium  Quant.  Biol.,  1947, 
11,  208-214).  Their  great  usefulness 
lies  in  the  fact  that  with  their  help  one 
can  see  more  clearly  than  by  other 
means  at  high  magnification  and  resolu- 
tion such  external  and  internal  cellular 
structures  as  surfaces  (Ralph,  P.  H., 
Anat.  Rec,  1947,  98,  219-223,  489-507); 
vaginal  smears  (Culiver,  A.,  and  Gluck- 
man,  J.,  J.  Obst.  and  Gynaec,  Brit. 
Empire,  1948,  55,  261-267);  centrioles 
(Buchsbaum,  R.,  Anat.  Rec,  1948,  102, 
19-27) ;  mitochondria  (Ludford  et  al., 
J.  Roy.  Micr.  Soc,  1948,  68,  1-9;  Zollin- 
ger, H.  v.,  Am.  J.  Path.,  1948,  24,  569- 
589);  viral  inclusion  bodies  (Angulo, 
J.  J.  et  al,  J.  Bact.,  1949,  57,  297-303) 
etc.  in  the  living  stage,  or  in  fixed  and 
unstained  material. 

Lack  of  contrast  has  always  been  a 
problem  peculiar  to  histology  and 
cytology  because  the  refractive  indices 
of  cytoplasm  and  its  inclusions  are  so 
nearly  the  same.  The  classical  method 
of  creating  color  contrast  by  selective 
or  differential  staining  is  subject  to 
well-known  limitations.  Phase  micros- 
copy now  provides  for  the  first  time 
an  effective,  reliable  method  of  creating 
contrast  by  purely  optical  means. 
The  chief  deterrent  to  its  universal 
adoption  for  research  is  its  present 
high  cost,  ranging  from  $500  to  $1,000 
for  a  microscope  and  accessories. 
Moreover  special  adjustments  have  to 
be  made  for  the  particular  tissue  to  be 
examined.  You  cannot  simply  take 
the    microscope   out    of   the    cupboard 


and  get  busy  in  the  examination  of 
any  old  tissue  as  is  customary  with 
the  standard  bright  light  microscope. 
The  phase  contrast  microscope  is  not  of 
any  particular  assistance  in  the  study 
of  fixed  and  stained  preparations  in 
which  differential  tissue  transparency 
is  not  a  factor  of  primary  importance. 

Phenol  Compounds,  see  Azo  Reaction,  Indo 
Reaction. 

Phenolase,  see  Oxidase. 

Phenoloxida  ^,  see  criticism  of  Dopa  Oxi- 
dase reaction. 

Phenolphthalein.  This  compound  of 
phthalic  acid  with  phenol  and  sulfuric 
acid  is  an  important  indicator.  Closely 
related  to  it  is  cresolphthalein. 

Phenosafranin  (CI,  840) — safranin  B  extra — 
This  is  the  simplest  of  the  safranins. 
It  has  been  used  by  Moore,  E.  J., 
Science,  1933,  77,  23-24_  for  staining 
fungi  on  culture  media  or  in  host  tissue. 

Phenosulfonphthalein,  use  in  renal  function 
tests  (Shaw,  E.  C,  in  Glasser's  Medical 
Physics,  1628-1630). 

Phenyl  Methane  Dyes.  The  hydrogen 
atoms  of  methane  can  be  replaced  by 
phenyl  groups  and  it  is  possible  to  add 
amino  groups  to  the  benzene  rings. 
See  di-phenyl  methanes,  di-amino  tri- 
phenyl  methanes,  tri -amino  tri -phenyl 
metlmnes,  and  hydroxy  tri -phenyl 
methanes. 

Phenylene  Blue,  see  Naphthol  Blue  R. 

Phenylene  Brown,  see  Bismark  Brown  Y. 

Phloroglucin  is  1,3,5-trihj'^droxybenzene. 
It  is  obtained  in  the  form  of  a  yellowish 
white  crystalline  powder.  It  protects 
the  organic  components  of  tissues  so 
that  acids  can  be  used  in  higher  con- 
centrations for  decalcification.  Make 
sat.  aq.  sol.  phloroglucin  and  add 
5-25%  of  the  acid. 

Phloxine    (CI,   774)— erythrosin  BB  or  B 
extra,  new  pink. 

Phloxine  B  (CI,  778) — cyanosine,  eosin 
lOB,  phloxine  TA,  N  or  BB— Conn 
(p.  154)  explains  that  this  differs  from 
phloxine  in  possessing  4  in  place  of  2 
chlorine  atoms  in  phthalic  acid  residue 
of  molecule.  This  phloxine  B  is  the 
one  ordinarily  used.     See  Eosins. 

Phloxine  Ta,  N  or  BB,  see  Phloxine  B. 

Phloxine-Azure.  This  resembles  Mallory's 
phloxine-methylene  blue.  Stain  sec- 
tions after  Bouin  or  Zenker  fixation 
in  2.5%  aq.  phloxine,  15  min.;  wash  in 
water  and  stain  in  0.1%  aq.  azure  A, 
30  min.;  wash  in  water,  differentiate  in 
95%  ale.  plus  few  drops  xylene  colo- 
phonium;  dehydrate  in  abs.,  clear  in 
xylol  and  mount.  Particularly  good 
for  bone  marrow.  (Haynes,  R.,  Stain 
Technology,  1926,  1,  68). 

Phloxine-Methylene  Blue.  Mallory  (p.  86) 
recommends  that  phloxine  be  employed 


PHOSPHAMIDASE 


207 


PHOSPHATE  ION 


in  place  of  eosin  in  the  following  method 
because  it  gives  (as  Conn  suggested)  a 
more  brilliant  color.  Deparaffinize  sec- 
tions of  Zenker  fixed  material  in  usual 
way.  Remove  mercury  with  0.5% 
iodine  in  95%  alcohol  5-10  min.  and  the 
iodine  with  0.5%  aq.  sodium  thiosulfate 
(hypo)  5  min.  Wash  thoroughly  in 
water.  2.5%  aq.  phloxine  in  paraffin 
over  1  hr.  or  more.  Cool  stain,  drain 
and  rinse  in  water.  Take  5  cc.  1%  methy- 
lene blue  on  1%  borax,  5  cc.  1%  aq. 
azure  II,  add  90  cc.  aq.  dest.,  filter  onto 
the  sections.  Pour  on  and  off  several 
times.  After  required  time  differentiate 
in  100  cc.  95%  alcohol  plus  2-5  cc.  10% 
colophony  (rosin)  in  absolute  alcohol. 
Control  differentiation  under  micro- 
scope. Dehydrate  in  several  changes 
abs.  ale.  Clear  in  xylol  and  mount  in 
balsam.  Nuclei  and  bacteria,  blue: 
collagen,  etc.  bright  rose.  The  method 
yields  beautiful  preparations  of  intra- 
nuclear inclusions  in  yellow  fever  and 
is  extensively  used  for  many  purposes. 

Phosphamidase  an  enzyme  capable  of 
hydrolyzing  para-chloranilido  phos- 
phonic  acid,  at  acid  pH  (5.6).  Gomori, 
G.,  Proc.  Soc.  Exp.  Biol,  and  Med., 
1948,  69,  407-409,  describes  the  method. 
Phosphamidase  is  reported  to  be  espe- 
cially intense  in  the  grey  matter  of  the 
central  nervous  system  and  in  malig- 
nant tumors. 

Phosphatases — Written  by  E.  W.  Dempsey, 
Dept.  of  Anatomy,  Washington  Uni- 
versity, St.  Louis.  February  26,  1951 — 
Since  the  original  papers  of  Gomori,  G. 
(Proc.  Soc.  Exp.  Biol,  and  Med.,  1939, 
42,  23-26),  and  Takamatsu,  H.  (Trans. 
Jap.  Path.  Soc,  1939,  29,  492-498),  a 
host  of  papers  on  the  localization  of 
these  enzymes  has  appeared.  The 
Gomori  and  Takamatsu  methods  in- 
volve incubating  sections  in  a  solution 
of  organic  phosphate,  during  which 
free  phosphate  radicals  are  liberated. 
These  are  instantly  precipitated  as  the 
calcium  or  the  lead  salt,  for  the  alkaline 
and  acid  phosphatase  reactions,  respec- 
tively. The  insoluble  metal  phosphate 
is  then  visualized  by  staining  with 
alizarin  or  silver  solutions  (Kabat, 
E.  A.  and  J.  Furth,  Am.  J.  Path.,  1941, 
17,  303-318)  or  by  transformation  to 
black  cobaltous  sulfide.  For  technical 
procedures,  see  Gomori's  Method. 

The  question  whether  alkaline  phos- 
phatase is  a  single  enzyme,  or  whether 
several,  substrate-specific  enzymes  ex- 
ist, is  an  actively  debated  topic. 
Dempsey,  E.  W.  and  H.  W.  Deane  (J. 
Cell,  and  Comp.  Physiol.,  1946,  27, 
159-179)  suggested  that  there  are  several 
different  alkaline  phosphatases,  and 
Emmel,  V.   E.    (Anat.   Rec,  1946,  96, 


423-438)  has  demonstrated  that  the 
enzyme  of  the  intestine  is  easily  poi- 
soned by  KCN  whereas  that  of  the  kid- 
ney is  not.  Contrariwise,  Gomori,  G. 
(Proc.  Soc.  Exp.  Biol,  and  Med.,  1949, 
70,  7-11)  reports  no  difference  in  locali- 
zation of  phosphatase  when  19  different 
substrates  were  employed,  although  a 
phosphonate  substrate  gave  different 
results  in  the  acid  range.  Later, 
Gomori,  G.  (Proc.  Soc.  Exp.  Biol,  and 
Med.,  1949,  72,  449-150)  found  that 
5-nucleotidase  was  localized  differently 
than  is  glycerophosphatase.  The  pos- 
sible multiplicity  of  the  alkaline  phos- 
phatases is  therefore  unsettled,  but  the 
weight  of  evidence  suggests  there  is 
more  than  one  enzyme  for  dephospho- 
rylating  mechanisms. 

A  new  chemical  approach  to  the 
phosphatases  appeared  when  Menten, 
M.  L.,  J.  Junge  and  M.  H.  Green  (J. 
Biol.  Chem.,  1944,  153,  471-477)  em- 
ployed beta  naphthyl  phosphate  as  a 
substrate.  After  enzymatic  hydrolysis, 
beta  naphthol  was  visualized  by  cou- 
pling with  a  diazonium  salt.  This  pro- 
cedure has  been  greatly  improved  by 
Mannheimer,  L.  H.  and  A.  M.  Selig- 
man,  J.  (Nat.  Cancer  Inst.,  1948,  9, 
181-200)  and  by  Seligman  and  Mann- 
heimer {ibid.,  1949,  9,  427-434)  for 
alkaline  and  acid  phosphatase  respec- 
tively. The  present  methods  give 
results  fully  comparable  in  precision 
with  the  Gomori  procedures. 

The  possible  occurrence  of  artifacts 
of  various  sorts  in  the  phosphatase 
methods  has  been  actively  debated. 
Most  observers  agree  that  histochemi- 
cal  preparations  are  of  considerable 
value,  but  many  warn  of  the  possible 
migration  of  enzyme  or  of  its  products 
during  the  technical  procedures.  The 
problem  therefore  becomes  one  of  re- 
solving power — -What  are  the  spatial 
limits  of  dependability  of  the  phos- 
phatase procedures?  Two  references 
on  this  debated  topic  are:  Martin,  B. 
F.  and  Jacoby,  F.,  J.  Anat.,  1949,  83, 
351-363,  and  Leduc,  E.  H.  and  Demp- 
sey, E.  W.,  J.  Anat.,  1951,  in  press. 

Finally  attention  should  be  called  to 
the  excellent  review  on  phosphatases 
by  Lison,  L.,  Bull.  d'HistoL,  1949,  25, 
23-41. 
Phosphate  Ion.  A  capillary  colorimetric 
technique  has  been  described  by 
Walker,  A.  M.,  J.  Biol.  Chem.,  1933, 
101,  239-254.  He  employed  it  for 
glomerular  urine.  See  discussion  by 
Sumner,  J.  B.,  Science,  100,  413-414. 
The  technique  is  suitable  for  even 
0.08  jttl  containing  less  than  1  nm  gm. 
phosphate  phosphorus  with  a  mean 
error    approximately   0.1%    (Glick,    p. 


PHOSPHATE  SOLUTIONS 


268 


PHOSPHOTUNGSTIC  ACID 
HEMATOXYLIN 


208).  See  method  of  Lowry,  O.  H.  and 
Lopez,  J.  Biol.  Chem.,  1946,  162,  421- 
428  for  determination  of  inorganic 
phosphate  in  presence  of  labile  phos- 
phate esters.     See  Phosphorus. 

Phosphate  Solutions.  A  method  for  the 
direct  observation  of  the  effect  of 
buffered  phosphate  solutions  on  a  thin 
layer  of  living,  vascular  tissue  in  moat 
chambers  introduced  into  the  rabbit's 
ear  is  described  by  Abell,  R.  G.,  Anat. 
Rec,  1935-36,  64,  51-73. 

Phosphine  (CI,  793)— leather  yellow,  xan- 
thin — a  basic  xanthene  dye  used  as  a 
microchemical  test  for  nucleoproteins 
by  Schumacher,  J.,  Zentralbl.  Bakt., 
Abt.  I.  Orig.,  1922,  88,  362-366.  Phos- 
phine 3  R  is  fluorchrome  for  lipids. 

Phospholipid  Content  of  white  blood  cells 
(Boyd,  E.  M.,  J.  Lab.  &  Clin.  Med., 
1935-36,  21,  957-962). 

Phosphomolybdic  Acid  Hematoxylin  (Mal- 
lory's,  see  McClung,  p.  406).  Fix  in 
Zenker's  fluid,  imbed  in  parafhn  and 
remove  mercury  with  iodine.  Rinse  in 
water.  Phosphomolybdic  acid  hema- 
toxylin at  room  temperature  12-24  hrs. 
or  at  about  54°C.  2-3  hrs.  (That  is 
hematoxylin  1  gm.,  phosphomolybdic 
acid  crystals  2  gm.,  aq.  dest.  100  cc. 
Requires  several  weeks  to  ripen  or  ripen- 
ing may  be  immediate  after  addition  of 
5  cc.  1%  aq.  potassium  permanganate.) 
Wash  in  water.  Decolorize  in  95%  ale. ; 
dehydrate  in  abs.  Clear  in  xylol  and 
mount  in  balsam.  Collagenic  fibers 
deep  blue.  To  counterstain  first  color 
5-10  min.  in  0.5%  aq.  acid  fuchsin,  drain 
and  stain  directly  in  the  hematoxylin. 

Phosphorescence  Microscope,  Science 
(News),  1943,  98,  8  (No.  2547). 

Phosphorus.  The  histochemical  detection 
of  phosphorus  is  a  matter  of  great  im- 
portance but  the  techniques  are  open  to 
much  criticism.  Lison  (pp.  113-120) 
has  reviewed  the  whole  question  and 
advises  two  techniques  as  vigorously 
specific  for  phosphorus  in  the  ionic 
form: 

1.  Angeli,  A.,  (Riv.  di  Biol.,  1933,  10, 
702)  using  plant  material  treats  sections 
for  20  min.  with:  ammonium  molyb- 
date,  3  gm.;  aq.  dest.,  20  cc;  30%  aq. 
hydrochloric  acid,  20  cc;  reduces  in 
N/50  stannous  chloride,  rinses  quickly 
in  aq.  dest.,  washes  longer  in  2.5%  aq. 
ammonia  which  results  in  elements  con- 
taining phosphorus  being  colored  blue 
green. 

2.  Serra,  J.  A.  and  Queiros  Lopes,  A., 
Portugaliae  Acta  Biol.,  1945,  1,  111- 
122.  Reagents:  (A)  Fixative.  Add  few 
drops  glacial  acetic  acid  to  mixture  2 
parts  96%  ale  and  1  part  formalin. 
(B)  Molybdate.  0.5  gra.  ammonium 
molybdate    dissolved    in    20    cc.    aq. 


dest.  -f  10  cc.  cone.  (30%)  hydrochloric 
acid  diluted  to  50  cc.  with  aq.  dest. 
(C)  Benzidine.  25  mg.  dissolved  in  5 
cc.  glacial  acetic  acid  diluted  to  50  cc. 
with  aq.  dest.  Fix  tissue  in  "A"  and 
wash  well  in  water.  Treat  frozen  sec- 
tions, or  small  pieces,  with  "B"  at 
10-12°  for  2-3  weeks  and  then  at  20-25° 
for  2-3  days.  Cover  with  drop  of  "C" 
for  3  min.  Add  2  drops  sat.  aq.  sodium 
acetate.  Mount  in  glycerol  from  a 
supply  containing  crystals  of  sodium 
acetate.  Phosphate  is  revealed  by 
intense  blue  color.  When  these  authors 
digest  tissue  with  nuclease  to  liberate 
phosphate  from  nucleic  acid  this  visual- 
ization of  phosphate  indicates  localiza- 
tion of  the  nucleic  acid  (partially  quoted 
from  Glick,  p.  35). 

By  the  titrimetric  method  of  Lind- 
ner, R.  and  Kirk,  P.  L.,  Microchemie, 
1937,  22,  300-305  phosphorus  can  be 
detected  quantitatively  in  the  range 
0.5-10.0  fjL  gm.  The  whole  subject  has 
been  reviewed  by  Glick,  D.,  J.  Chem. 
Education,  1935,  12,  253-259. 
Phosphotungstic  Acid  Hematoxylin.  (Mal- 
lory's,  see  McClung,  p.  403)  Fix  in 
Zenker's  fluid  and  remove  mercury  from 
sections  with  iodine  or  0.5%  sodium 
hyposulphite.  Rinse  in  water.  0.25% 
aq.  potassium  permanganate,  5-10  min. 
Wash  in  water.  5%  aq.  oxalic  acid, 
10-20  min.  Wash  carefully  in  several 
changes  of  water.  Phosphotungstic  acid 
hematoxylin,  12-24  hrs.  (To  make  this 
dissolve  0.1  gm.  hematoxylin  by  heat  in 
50  cc.  aq.  dest.,  when  cool  add  2.0  gm. 
phosphotungstic  acid  dissolved  in  50  cc. 
aq.  dest.  Requires  a  few  weeks  to 
ripen.  Ripening  can  be  done  at  once  by 
addition  of  10  cc.  0.25%  aq.  potassium 
permanganate).  95%  ale,  30  sec; 
dehydrate  quickly  in  abs.  Clear  in 
xylol  and  mount  in  balsam.  Fibroglia, 
myoglia,  neuroglia  and  fibrin,  deep 
blue;  ground  substance,  cartilage  and 
bone,  yellowish  to  brownish  red; 
coarse  elastic  fibers,  purple. 

Mullen,  J.  P.  and  McCarker,  J.  C, 
Am.  J.  Path.,  1941,  17,  289-291  suggest 
the  following  procedure  for  nervous  tis- 
sues fixed  in  formalin.  Tissues  stored 
in  4%  aq.  formalin  for  several  years  give 
good  results.  After  fixation  in  4%,  cut 
blocks  5  mm.  or  less  in  thickness.  Wash 
for  6-12  hrs.  in  running  water.  Dehy- 
drate to  include  95%  alcohol  as  usual. 
Complete  dehydration  in  2  changes  n 
butyl  alcohol,  4  hrs.  each  (but  absolute 
alcohol  xylol  is  satisfactory).  Imbed  in 
paraffin  directly  from  n  Butyl  Alcohol 
(which  see). 

Treat  sections  for  2  hrs.  or  longer  in 
following  mordant:  Dissolve  5  gms. 
chromium  chloride   (green  crystals  ob- 


PHOTODYNAMIC  ACTION 


269 


PICCOLYTE  RESINS 


tainable  from  General  Chemical  Co., 
New  York)  in  100  cc.  aq.  dest.  and  add  5 
cc.  glacial  acetic  acid.  This  dark  green 
solution  soon  becomes  purple  black  but 
is  usable  after  many  weeks.  Rinse  in 
aq.  dest.  Stain,  as  above,  with  phos- 
photungsticacid  hematoxylin. 

Photodynamic  Action  of  thiazine  dyes  on 
vaccine  virus  may  be  due  to  red  or  infra 
red  rays  (Hirano,  N.  and  Sayama,  K., 
Arch.  exp.  Med.,  1936,  13,  324-332). 

Photoelectric  Colorimeter,  construction  and 
use  (Hanselman,  R.  C,  Am.  J.  Clin. 
Path.,  1943,  13,  108-116). 

Photoelectric  Microphotometer — Written 
by  R.  E.  Stowell,  Dept.  of  Oncology, 
University  of  Kansas  Medical  Center, 
Kansas  City  2,  Kan.  January  19, 
1951 — The  original  type  of  apparatus 
developed  by  Stowell,  R.  E.  (J.  Nat. 
Cancer  Inst.,  1942,3,  111-121)  was  used 
to  measure  the  light  absorbed  as  a 
result  of  the  specific  coloration  of  tissue 
components.  Such  equipment  consists 
of  a  light  source,  microscope,  and  photo- 
electric equipment  with  amplification 
and  recording  of  light  absorption. 
Modified  equipment  is  discussed  by 
Pollister,  A.  W.  and  Moses,  M.  J.  (J. 
Gen.  Physiol.,  1949,  32,  567-577). 
Among  substances  measured  by  these 
techniques  are  Feulgen  reaction  for 
desoxypentose  nucleic  acid,  pyronin 
methyl-green  stain  for  nucleic  acids 
and  Millon's  reaction  for  tyrosine.  For 
other  possible  uses  of  this  type  of  equip- 
ment see  Stowell,  R.  E.  (J.  Invest. 
Derm.,  1945,  6,  183-189). 

Photoxylin,  see  Celloidin. 

Phrenosin  is  a  Cerebroside. 

Phthalein  Indicators.  Table  giving  rela- 
tive reactions  of  the  several  organs  and 
tissues  after  vital  staining  (Rous,  P., 
J.  Exper.  Med.,  1925,  41,  739-759). 
See  Indicators  of  pH. 

Physiological  solutions.  These  are  in- 
tended for  the  examination  of  living 
cells  with  a  minimum  of  change.  Blood 
serum,  or  plasma,  is  an  unnatural  me- 
dium for  any  living  cells  except  those 
naturally  intravascular  as  shown  by  the 
fact  that  alone  and  undiluted  it  is  a  poor 
medium  for  tissue  culture.  Physio- 
logical saline  is  for  mammals  0.85-0.9% 
aqueous  NaCl  and  for  amphibians  about 
0.65%  aqueous  NaCl.  For  others  see 
Ringer,  Ringer  -  Locke,  Locke  -  Lewis 
and  Tyrode.  Normal  solutions  (which 
see)  are  different. 

Pia  Mater.  Perivascular  nerves.  Washout 
blood  by  vascular  perfusion  with  saline 
solution  or  by  rinsing  nonperfused  tis- 
sue with  saline.  Fix  with  10.5%  citric 
acid  in  20%  formalin  preferably  by  in- 
jection. Dissect  out  blood  vessels  of 
pia  under  binocular  microscope.     Wash 


in  aq.  dest.  twice  and  place  in  20%  aq. 
silver  nitrate  2  hrs.  Pass  through  4 
changes  20%  formalin  in  Petri  dishes 
each  containing  al)out  100  cc.  Transfer 
directly  to  ammoiiiated  silver  nitrate 
made  by  adding  cone,  ammonia  (28%) 
drop  by  drop  to  20%  aq.  silver  nitrate 
using  3  drops  more  than  amount  re- 
quired to  dissolve  ppt.  Observed  under 
the  microscope  the  nerves  "come  up" 
slowly  and  when  they  are  dark  enough 
transfer  to  20%  ammonia  water  for  1-2 
min.  Wash  in  aq.  dest.  plus  few  drops 
glacial  acetic  acid.  Tone  in  0.2%  aq. 
yellow  gold  chloride  30-60  min.  Wash 
in  water,  dehydrate  in  3  changes  95% 
alcohol,  clear  in  carbol-creosote-xylol 
mixture  and  mount  in  balsam  (Penfield, 
W.,  Am.  J.  Path.,  1935,  11,  1007-1010); 
revised  by  W.  Penfield,  Montreal  Neu- 
rological Institute,  Montreal,  Canada, 
May  1,  1946. 

Pianese  Method.  Much  used  a  generation 
ago  for  study  of  cancerous  tissue. 
Pianese,  G.,  Beitr.  z.  Path.  Anat.,  u. 
Allg.  Path.,  1896,  Suppl.  I,  193  pp. 

Piccolyte  Resins — Written  by  L.  F.  Wicks, 
Veterans  Administration  Hospital,  Jef- 
ferson Barracks,  Mo.  February  1, 
1951 — These  synthetic  terpene  resins 
(/3  pinene  polymers)  have  been  recom- 
mended for  permanent  mounting  media 
(Wicks,  L.  F.,  Carruthers,  C.  and 
Ritchey,  M.  G.,  Stain  Techn.,  1946, 
21,  121-126).  Natural  resins  are  quite 
variable  in  quality,  and  with  age  the 
poorer  samples  may  turn  yellow,  crack 
or  develop  acidity  which  fades  basic 
stains.  Synthetic  resins,  being  of  con- 
trolled manufacture,  are  much  more 
uniform  in  composition  and  predicta- 
ble in  behavior.  An  examination  of 
many  such  resins  was  stimulated  by 
the  war-time  scarcity  of  Canada  bal- 
sam. 

The  requirements  for  a  good  mount- 
ing medium  are  abbreviated  from  the 
original  article.  It  should  possess: 
1.  Correct  refractive  index,  2.  Freedom 
from  acidity,  3.  Clarity,  present  and 
future,  4.  Solubility  in  the  proper  or- 
ganic solvents,  5.  Good  adhesion  for 
glass,  6.  Reasonable  hardening  time 
(and  not  craze  or  granulate  later),  7. 
An  approximately  right  softening  tem- 
perature, 8.  Constant  composition, 
stability,  inertness,  availability  and 
moderate  price. 

The  Piccolytes  meet  all  the  above 
stipulations,  being  of  correct  refractive 
indices,  very  low  acid  numbers,  pale, 
non-yellowing,  adherent  to  glass,  and 
soluble  in  .xylol  and  toluol.  They  are 
also  available  in  a  variety  of  softening 
points.  Unfortunately,  despite  con- 
siderable interest  shown  in  the  descrip- 


PICRIC  ACID 


270 


PINEAL 


^'^  tion  of  these  resins,  the  manufacturer 

•-,  fe.  (Pennsylvania      Industrial      Chemical 

;^'j:  Corp.,  Clairton,  Penn.)  is  not  interested 

,,  ^in  packaging  for  such  small  specialty 

,^.  sale.  However,  one  of  the  Piccolytes 
("S-115")  is  on  the  market  under  the 
name  of  "Harleco  Mountant"  (Hart- 
man-Leddon  Co.,  Philadelphia).  Pos- 
sibly, one  of  lower  softening  point  than 
S-115  would  have  been  a  better  choice 
for  general  use. 

Picric  Acid  is  a  very  important  ingredient  of 
several  fixatives.  It  penetrates  rapidly 
and  serves  to  some  extent  as  a  mordant 
like  potassium  bichromate.  See  Bouin's 
fluid.  Picric  acid  staining  of  chromo- 
phobe bodies  of  Lipschutz  (Schiller, 
Virchow's  Arch.,  1930,  278,  663-689). 

Picro-Carmine  (Ranvier) .  There  are  many 
sorts  most  of  them  based  on  Ranvier 's 
original  formula:  Add  carmine  (dis- 
solved in  ammonia)  to  sat.  aq.  picric 
acid  to  saturation.  Evaporate  to  ^ 
original  volume,  cool,  filter  out  ppt.  and 
evaporate  filtrate  to  dryness.  The 
resultant  red  crystalline  powder  is 
picro-carmine.  Make  a  1%  aq.  sol.  for 
staining.  If  overstained  decolorize  with 
0.2%  hydrochloric  acid.  This  is  an 
excellent  and  very  popular  stain.  It 
colors  keratohyalin  very  brightly  (Lee, 
p.  146). 

Picroformaldehyde  Formic  Acid  for  fixation 
(Lillie,  R.  D.,  J.  Tech.  Methods,  1944, 
24,  35-36).  Formaldehyde  (37%  solu- 
tion), 10  cc,  formic  acid,  5  cc.  and  sat. 
aq.  picric  acid,  85  cc.  is  recommended 
as  a  substitute  for  Bouin's  Fluid.  It 
decalcifies  femurs  of  mice  well  in  48 
hrs.,  provides  sections  adapted  to 
Romanovsky  staining,  and  in  general 
acts  like  Bouin's  fluid. 

Picro-Formol,  see  Bouin's  Fluid. 

Picro-Indigo-Carmine  is  a  much  used  stain. 
Lee  (p.  433)  advises  3  parts  sat.  indigo- 
carmine  in  70%  ale.  and  1  part  sat.  picric 
acid  also  in  70%  ale. 

Picro-Mallory.  Several  fine  modifications 
of  Mallory's  connective  tissue  stain 
using  picric  acid  (McFarlane,  D.,  Stain 
Techn.,  1944,  19,  29-37). 

Picro-Nigrosine  for  muscle.  After  alcohol 
or  Bouin  fixation,  stain  sections  in  sat. 
nigrosine  in  sat.  aq.  picric  acid.  Muscle 
yellow,   connective   tissue  black. 

Picro-Sulphuric  fixative,  see  Kleinenberg's. 

Pigments,  general  reviews:  Bergmann,  E. 
Ergeb.  d.  Physiol.,  1933,  35,  158-300; 
Lederer,  E.,  Biol.  Rev.,  1940,  15,  273- 
306  (invertebrates).  See  Aposiderin, 
Bacterial,  Bile  Pigment,  Bilirubin, 
Biliverdin,  Carotin,  Carotinalbumins, 
Carotinoids,  Ceroid,  Chromolipoids, 
Cytochrome,  Cytolipochrome,  Cytosid- 
erin,  Exogenous,  Hematin,  Hematoi- 
din,     Hematoporphyrin,     Hemofuscin, 


Hemoglobin,  Hemosiderin,  Trichosid- 
erin,  Iron  Pigments,  Lipochrome,  Lipo- 
fuscin  (wear  and  tear  pigment), 
Malarial  Melanins,  Parhemoglobin, 
Porphyrins,  Rhodopsin,  Scheele's 
Green,  Sulfmethemoglobin,  Schwein- 
furt  Green,  Ultramarine  Green  and 
Verdigris. 

Pinacyanol  (CI,  808) — sensitol  red — A  basic 
xanthene  dye  of  the  cyanine  group. 
Proescher,  F.,  Proc.  Soc.  Exp.  Biol.  & 
Med.,  employed  the  Eastman  Kodak  Co. 
product  of  which  a  0.1-0.5%  solution  in 
absolute  ethyl  or  methyl  alcohol  for  5- 
10  sec.  stains  frozen  sections  brightly. 
Wash  in  water,  mount  in  glycerine. 
Chromatin,  blue  violet;  protoplasm, 
purple;  connective  tissue,  red;  elastic 
tissue,  black  violet;  muscle,  bluish 
violet  to  purple;  amyloid,  red;  etc. 
Hetherington,  D.  C,  Stain  Techn., 
1936,  11,,  153-154,  used  pinacyanol  as  a 
supravital  stain  for  mitochondria  in 
blood  cells. 

Pineal.  Staining  pineal  parenchyma  by  a 
modified  Hortega  method  after  paraffin 
embedding — Written  by  W.  M.  Shank- 
lin,  American  University  of  Beirut, 
Beirut.  March  30,  1951— Fix  the  gland 
as  soon  as  it  is  removed  in  10%  formalin 
(Merck  blue  label  40%)  3  days  at  room 
temperature  of  about  24°C.     Wash  for 

2  hrs.  in  aq.  dest.  to  which  6  drops  of 
ammonium  hydroxide  are  added  for 
each  100  ml.  Wash  again  in  aq.  dest. 
for  a  few  minutes  and  dehydrate  the 
gland  in  70%,  80%,  95%  and  absolute 
alcohol,  leaving  3  hrs.  in  each.  Clear 
in  cedar  oil  over  night,  followed  by 
pure  xylene  for  half  an  hr.  Infiltrate 
with  paraffin  (melting  point  48°C.)  for 

3  hrs.  Embed  in  pure  paraffin.  Cut 
sections  7-10  m,  fix  to  slides  by  the 
albumen-water  method  and  dry  thor- 
oughly. Heat  the  slide  gently  to  soften 
the  paraffin  and  pass  through  2  changes 
of  xylene.  Pass  slides  through  abso- 
lute alcohol,  95%  and  80%  alcohol  1-2 
min.  in  each.  Wash  in  3  changes  aq. 
dest.  for  3  min.  each.  Place  slides  in 
10%  aq.  silver  nitrate  to  which  3  drops 
of  pyridine  for  each  10  ml.  are  added 
and  keep  in  the  dark  at  room  tempera- 
ture 24°C.  for  24  hrs.  Wash  in  aq. 
dest.  and  sensitize  by  placing  in  5% 
sodium  sulfite  1  hr.  This  step  may  be 
omitted,  but  sometimes  this  is  neces- 
sary as  it  acts  as  a  rejuvenator.  Wash 
in  aq.  dest.  to  which  2  drops  of  pyridine 
for  each  10  ml.  are  added  for  1  to  2 
min.  followed  by  2  changes  aq.  dest. 
Impregnate  in  strong  silver  carbonate 
at  room  temperature  for  2  to  5  min. 
Formula:  10%  aq.  Silver  nitrate  (pure 
crystals)  100  ml.,  5%  aq.  Sodium  car- 
bonate    (pure)    300    ml.,    Ammonium 


PINOCYTOSIS 


271 


PLACENTA 


hydroxide,  just  sufficient  to  dissolve 
the  precipitate,  Distilled  water  100 
ml.  Dip  in  aq.  dest.  for  a  few  seconds, 
without  agitating.  Reduce  in  10% 
formalin  (Merck  blue  label),  to  which 
4  drops  of  pyridine  are  added  for  each 
10  ml.,  1  miu.  agitating  gently.  Wash 
in  aq.  dest.  Tone  in  yellow  gold  chlo- 
ride (1  g.  to  500  ml.  aq.  dest.)  for  a 
few  seconds  to  1  min.  Time  should  be 
checked  carefully.  Fi.x  in  5%  hypo  for 
1  to  2  min.  Wash  and  counterstain 
lightlv  with  1%  erythrosin  or  safranin. 
Dehydrate  in  80%,  95%  and  absolute 
alcohol.  Clear  in  two  changes  of 
xylene,  mount  in  neutral  Canada  bal- 
sam and  cover  with  cover  slips  (see 
Nassar,  T.  and  Shanklin,  W.  M.,  Stain 
Techn.,  1950,  25,  35-38). 

Pinocytosis,  a  term  introduced  by  Lewis 
(W.H.,  Bull.  Johns  Hopkins  Hosp.,  1931, 
49,  17-26)  to  indicate  drinking  by  cells 
as  opposed  to  phagocytosis,  or  eating  by 
cells.  It  means  (Lewis,  W.  H.,  Am.  J. 
Cancer,  1937,  29,  666-679)  microscopi- 
cally visible  drinking,  not  submicro- 
scopic  "sipping"  which  Meltzer  termed 
"Potocytosis".  By  this  process  in  tis- 
sue cultures  proteins  and  other  sub- 
stances that  do  not  diffuse  into  the  cells 
are  engulfed  by  wavy  rufBe  pseudo- 
podia.  The  cell  membrane,  which  first 
invests  the  globulus  of  fluid  taken  into 
the  cytoplasm,  later  disappears  and  the 
fluid  becomes  part  of  the  cytoplasm. 
Can  be  best  seen  in  cultures  of  cancer 
cells  of  which  an  excellent  moving  pic- 
ture is  available  for  distribution  by  the 
Wistar  Institute  of  Anatomy  at  Phila- 
delphia. 

Piroplasma  (L.  pirum,  pea  -f  G.  plasma,  a 
formed  thing).  Piroplasmas  are  pear 
shaped  parasites  of  red  blood  cells  caus- 
ing diseases  of  great  importance  in 
domestic  and  other  animals  but  not  as 
yet  found  in  man.  They  can  be  colored 
by  any  good  blood  stain.  Giemsa  and 
May-Giemsa  are  recommended. 

Pituitary.  The  microscopic  techniques  for 
this  conductor  of  the  endocrine  sym- 
phony are  obviously  too  numerous  to 
mention.  Consult  each  issue  of  the 
Quart.  Cum.  Index  Med. 

To  differentiate  2  classes  of  acido- 
philes  in  the  cat  a  modification  of 
Heidenhain's  "azan"  modification  of 
Mallory's  connective  tissue  stain  is 
proposedby  Dawson,  A.  B.  and  Friedgood, 
H.  B.,  Stain  Tech.,  1938,  13,  17-21.  T. 
Maxwell,  Jr.  (ibid,  93-96)  proposes  a 
modification  especially  designed  for  the 
basophiles  and  Koneff,  H.  H.  {ibid,  49- 
52)  one  for  the  rat.  In  addition  all 
within  the  space  of  a  few  months,  Lewis, 
M.  R.,  and  Miller,  C.  II.,  Stain  Techn., 
1938,  13,   111-114  give  following  direc- 


tions to  demonstrate  2  types  of  granular 
cells  in  the  pars  nervosa.     Vix  in  3% 
aq.  potassium  bichromate  2  parts  and 
half   sat.    corrosive   sublimate   in   95% 
ale.  1  part,  12-24  hrs.  with  1  change. 
Dehydrate  to  70%  alcohol  to  which  add 
few    drops    iodine.     Change    each    day 
until    solution    retains    color.     Dioxan, 
8-24  hrs.,  3  changes.     Dioxan  -|-  little 
paraffin.     Paraffin  4  changes.     Cut  sec- 
tions 4  microns  and  deparaffinize.  Stain 
0.25%  aq.   acid   fuchsin   30  min.  Then 
1-24  hrs.  in  Mallory's  stain  (aq.  dest., 
100  cc;  anilin  blue,  0.5  ^m.;  orange  G, 
2  gm.  and  phosphotungstic  acid,  1  gm.). 
Differentiate  in  95%  alcohol  until  no 
more  color  comes  out.     Abs.  ale,  xylol, 
balsam.     To  identify  microglia  in  neuro- 
hypophysis see  Vazquez -Lopez,  E.,  J. 
Anat.,    1942,  76,    178-186.     Differential 
stain  for  mouse  pituitary  (Dickie,  M. 
M.,  Science,  1944,  100,  297-298).     Pitui- 
cytes  by  Hortega  silver  carbonate  tech- 
nique (Shanklin,  W.  M.,  Stain  Techn., 
1943,  18,87-89). 
Placenta — Written     by     Dr.     George     B. 
Wislocki,  Dept.  of  Anatomy,  Harvard 
Medical  School,  Boston  15.     March  8, 
1951 — Of  all  the  organs  of  the  body  the 
placenta  has  been  the  least  investigated 
by  histological,  cytological  and  cyto- 
chemical  methods.     Compared  to  most 
other  tissues,  its  study  is  greatly  com- 
plicated by  the  marked  and  perplexing 
differences  in  its  structure  in  various 
mammals,  as  well  as  by  the  fact  that 
from  the  moment  of  implantation  up  to 
the  time  of  parturition,  it  goes  through 
a    complex    succession     of    structural 
changes.     The    latter    are    doubtlessly 
related  to  differing  physiological   and 
nutritional  needs,  first  of  the  implant- 
ing egg,  then  of  the  differentiating  em- 
bryo  before    the    heart    has   begun   to 
beat,  and  finally  of  the  growing  and 
maturing     fetus.     Besides     subserving 
the  immediate  needs  of  fetal  nutrition 
and  of  excretion  of  fetal  waste  products, 
the  placenta  is  also  an  endocrine  organ 
which  produces  both  steroid  hormones 
and     chorionic     gonadotropin.     Rela- 
tively little  is  known  concerning  the 
functions  of  these  hormones,  but  the 
placental  steroids  apparently  play  an 
important  part  in  the  growth,  regula- 
tion and  maintenance  of  the  pregnant 
uterus  by  exerting  their  influence  prin- 
cipally upon  the  uterine  musculature 
and  blood  vessels  and  thereby  influenc- 
ing also  the  length  of  gestation  and  the 
onset  of  labor.     The  role  of  chorionic 
gonadotropin  is  more  obscure,  but  it 
seems  to  interplay  with  the  pituitary, 
as  well  as  with  the  steroid-producing 
glands    including    the    placenta   itself. 


PLACENTA 


272 


PLACENTA 


in  maintaining  gestation  and  regulating 
maternal  metabolism. 

In  view  of  the  manifold  functions  of 
the  placenta,  which  surpass  in  number 
those  of  any  other  organ,  it  is  not 
surprising  to  find  that  it  gives  a  great 
many  cytological  and  histochemical 
reactions.  Despite  the  variety  of  these, 
none  of  them  is  specific  for  the  placenta 
alone.  The  placenta  in  this  respect 
differs,  for  example,  from  brain  where 
for  various  neuronal  and  glial  elements 
a  variety  of  special  cytological  tech- 
niques has  been  developed. 

For  a  general  understanding  of  the 
comparative  anatomy  and  phylogeny 
of  the  placenta,  consult  Grosser  O. 
(Frlihentwicklung  Eihautbildung  und 
Placentation  des  Menschen  und  der 
Saugetiere,  1927)  Bergmann,  Munich, 
and  Mossman,  H.  W.  (Carnegie  Contrib. 
to  Embryol.,  1937,  26,  129-246).  See 
also  the  chapter  by  E.  C.  Amoroso  on 
Placentation  in  the  forthcoming  3rd 
edition  of  Marshall's  Physiologj^  of 
Reproduction.  For  the  placentation 
of  the  Primates  consult  Hill,  J.  P. 
(Phil.  Trans.  Roy.  Soc.  Lond.  Ser.  B., 
1932,  221,  45-78),  Wislocki,  G.  B.,  and 
Streeter,  G.  L.  (Carnegie  Contrib.  to 
Embryol.,  1938,  27,  1-66)  and  Stieve, 
H.  (Ztschr.  f.  mikro-anat.  Forsch.,  1944, 
54,  480-543).  Various  aspects  of  the 
topography,  growth  and  vasculariza- 
tion of  the  human  placenta  are  de- 
scribed by  Spanner,  R.  (Morph.  Jahrb., 
1935,  75,  374-392)  and  Ztschr.  f.  Anat. 
u.  Entwicklungsgesch.,  1936,  105,  163- 
242)  and  Stieve,  H.  (Ztschr.  f.  mikro- 
anat.  Forsch.,  1940,  48,  287-449). 

Interest  in  placental  cytology  and 
histochemistry  was  initiated  by  Hof- 
bauer  (Biologie  d.  menschlichen  Pla- 
zenta,  1905,  Braumiiller,  Vienna  and 
Leipzig)  who  investigated  the  placental 
assimilation  of  iron,  protein  and  fats 
in  man,  and  by  Edwin  Goldmann 
(Beitrage  z.  klin.  Chir.,  1912,  78,  1-108) 
who  studied  extensively  the  distribu- 
tion and  significance  of  glycogen,  fat, 
iron  and  hemoglobin  in  the  placentas 
and  fetuses  of  mice  and  rats. 

In  the  past  two  decades  with  the  de- 
velopment of  many  new  cytological 
and  histochemical  methods,  interest 
in  the  placenta  has  been  rekindled. 
The  human  placenta  has  been  investi- 
gated by  a  variety  of  histochemical 
techniques  for  the  demonstration  of 
various  proteins,  carbohydrates,  lipids, 
enzymes  and  inorganic  compounds. 
Wislocki,  G.  B.  and  Bennett,  H.  S. 
(Am.  J.  Anat.,  1943,  73,  335-449)  have 
described  the  presence  in  the  tropho- 
blastic syncytium  of  birefringent,  su- 
danophilic   droplets   which  react   with 


phenylhydrazine  and  which  are  soluble 
in  acetone,  and  they  concluded  that 
these  reactions  indicate  the  site  of 
formation  of  placental  steroid  hor- 
mones. The  Liebermann-Burchardt  re- 
action, the  Schiff  reaction  and  the 
property  of  autofluorescence  have  been 
introduced  as  further  means  of  charac- 
terizing placental  steroid  hormones  in 
histological  sections  (Dempsey,  E.  W. 
and  Wislocki,  G.  B.,  Endocrin.,  1944, 
35,  409-428).  These  investigators  also 
describe  glycogen,  revealed  by  Best's 
carmine  and  Bauer's  method,  and  iron, 
demonstrated  by  both  the  Turnbull 
blue  reaction  and  microincineration. 
The  distribution  of  acid  and  alkaline 
phosphatases  has  been  explored  (Demp- 
sey, E.  W.  and  Wislocki,  G.  B.,  Am.  J. 
Anat.,  1947,  80,  1-33)  by  using  Gomori's 
methods  with  a  variety  of  substrates 
(glycerophosphate,  fructose  diphos- 
phate, nucleic  acid,  adenylic  acid  and 
lecithin) .  Mucopolysaccharides  as  well 
as  glycogen  have  been  investigated  by 
McManus'  periodic  acid-Schiff  reaction, 
metachromatic  substances  by  their 
responses  to  toluidin  blue  after  basic 
lead  acetate  fixation  and  lipids  with 
sudan  black  B  (Wislocki,  G.  B.  and 
Dempsey,  E.  W.,  Am.  J.  Anat.,  1948, 
83,  1-30).  The  affinity  of  syncytium, 
fibrin  and  fibrinoid  of  the  human 
placenta  for  acid  and  basic  dyes  has 
been  studied  (Singer,  M.  and  Wislocki, 
G.  B.,  Anat.  Rec,  1948,  102,  175-193). 
A  basophilic  substance  abundantly 
present  in  syncytium  and  cytotropho- 
blast  and  digested  by  crystalline  ribo- 
nuclease  has  been  identified  as  ribo- 
nucleoprotein  (Dempsey,  E.  W.  and 
Wislocki,  G.  B.,  Am.  J.  Anat.,  1945, 
76,  277-301).  Some  of  the  functional 
activities  of  the  placental  trophoblast, 
as  revealed  by  histochemical  studies, 
have  been  summarized  (Wislocki,  G. 
B.,  Dempsey,  E.  W.  and  Fawcett,  D. 
W.,  Obstet.  and  G}^.  Survev,  1948,  3, 
604-614). 

Our  knowledge  of  the  trophoblastic 
cell  columns  and  shell  and  of  the  basal 
plate,  as  well  as  of  the  relationships  of 
of  these  structures  to  the  underlying 
basal  decidua  in  the  human  placenta 
has  been  materially  advanced  by  the 
use  of  cytological  and  histochemical 
methods  (Wislocki,  G.  B.  and  Bennett, 
H.  S.,  1943,  I.e.;  Wislocki,  G.  B.  and 
Dempsey,  E.  W.,  1948,  I.e.).  By  these 
means  two  types  of  cytotrophoblast 
can  be  distinguished  from  the  elements 
of  the  maternal  decidua  (Wislocki, 
G.  B.,  Proc.  Am.  Assn.  Anat.,  1951). 

Tissue  cultures  of  young  human 
placentas  containing  actively  growing 
cytotrophoblast  produce  gonadotropic 


PLANT  CELL  WALLS 


273 


PLASMA  MEMBRANE 


hormone,  even  after  repeated  trans- 
plantation (Jones,  G.  E.  S.,  Gey,  G.  O. 
and  Gey,  M.  K.,  1943,  Bull.  J.  H.  Hosp., 
72,  26-38). 

The  placentas  of  various  mammals 
other  than  man  have  been  investigated 
by  multiple  cytological  and  histochemi- 
cal  methods,  for  example,  pig's  placenta 
(Wislocki,  G.  B.  and  Dempsev,  E.  W., 
Am.  J.  Anat.,  1946,  78,  181-225),  cat's 
placenta  (Wislocki,  G.  B.  and  Dempsey, 
E.  W.,  Am.  J.  Anat.,  1946,  78,  1-45), 
rodent's  placenta  (VVislocki,  G.  B., 
Dempsey,  E.  W.  and  Deane,  H.  W., 
Am.  J.  Anat.,  1946, 78,  281-345),  shrew's 
placenta  (Blarina  brevicauda  and  Sore.x 
fumeus)  (Wislocki,  G.  B.  and  Wirasatt, 
W.  A.,  Am.  J.  Anat.,  1947,  81,  269-307), 
and  bat's  placenta  (Myotis  lucifugus 
lucifugus)  (Wimsatt,  W.  A.,  Am.  J. 
Anat.,  1949,  84,  63-141).  The  distribu- 
tion of  acid  and  alkaline  phosphatases 
in  the  placentas  of  pig,  cat,  rodents  and 
man  has  been  the  subject  of  a  separate 
report  (Dempsey,  E.  W.  and  Wislocki, 
G.  B.,  Am.  J.  Anat.,  1947,  80,  1-33). 
By  his  method  for  lipase,  Gomori  (Men- 
struation and  its  Disorders,  edited  by 
E.  T.  Engle,  1950,  C.  C.  Thomas) 
depicts  lipase  activity  in  the  allantoic 
and  vitelline  placentas  of  mouse  and 
rat.  The  distribution  of  saliva-insolu- 
ble glycoproteins,  stained  y)y  the  peri- 
odic acid-Schiff  procedure,  is  briefly 
described  in  the  placentas  of  pig,  cat, 
mouse,  rat  and  man  (Wislocki,  G.  B., 
J.  Natl.  Cancer  Inst.,  1950,  10,  1341). 

Goldmann  (Beitrage  z.  klin.  Chir., 
1909,  64,  192-265),  in  his  classic  account 
of  vital  staining,  described  the  intra- 
vitam  deposition  of  pyrrhol  blue  in 
the  placenta  of  the  mouse.  Wislocki 
(Carnegie  Contrib.  to  Embryol.,  1920, 
11,  45-60  and  1921,  13,  89-lOi),  by  em- 
ploying the  technique  of  vital  staining, 
demonstrated  the  storage  of  trypan 
blue  in  the  trophoblast  of  the  placentas 
of  cats,  rabbits  and  guinea  pigs,  while 
Gerard  (Arch.  d.  biol.,  1925,  35,  269- 
293)  and  Everett  (J.  Exp.  Zool.,  1933, 
70,  243-284)  carried  out  further  similar 
experiments  in  mice  and  rats. 

By  using  solutions  of  iron  ammonium 
citrate  and  sodium  ferrocyanide  which 
could  be  traced  in  the  placenta  by 
converting  them  into  the  Prussian 
blue  reaction,  Cunningham  (Am.  J. 
Physiol.,  1920,  53,  439-456  and  1922, 
60,  448-460;  Proc.  Soc.  Exper.  Biol,  and 
Med.,  1923,  20,  343-345)  investigated 
the  relative  permeabilities  of  the 
placentas  of  cats  and  rabbits  to  these 
two  substances. 

Everett  (J.  Exp.  Zool.,  1935,  70,  243- 
280)  has  made  a  series  of  important 
observations  concerning  the  activities 


of  the  yolk-sac  epithelium  in  the  rat 
by  devising  a  technique  for  perfusing 
the  uterine  circulation  with  thionin, 
toluidin  blue,  methylene  blue,  brilliant 
blue,  Nile  blue  sulfate,  Janus  green, 
neutral  red  and  trypan  blue.  This 
important  study  included  direct  micro- 
scopic observation  of  the  vitelline 
epithelium  in  hanging  drops  in  a  con- 
stant temperature  chamber. 

An  ingenious  technique  of  tying  off 
the  vitelline  blood  vessels  in  living 
rabbit  fetuses  in  utero,  for  the  purpose 
of  ascertaining  the  route  followed  by 
antibodies  in  their  passage  across  the 
placental  barrier,  has  been  devised 
bv  Brambell,  Hemmings  and  Rowlands 
(Proc.  Roy.  Soc,  London,  B,  1949,  136, 
131-144). 

Transplantation  of  living  fertilized 
eggs  and  pieces  of  placenta,  for  purposes 
of  observing  their  growth,  has  also  been 
successfully  carried  out.  Fertilized 
mouse  eggs,  introduced  into  the  ante- 
rior chamber  of  the  ej'e,  into  the  ova- 
rian bursa,  or  under  the  capsule  of  the 
kidney,  undergo  development,  produc- 
ing large  amounts  of  seemingly  normal 
placental  trophoblast  (Runner,  M.  N., 
Anat.  Rec,  1947,  98,  1-18;  Fawcett, 
D.  W.,  Wislocki,  G.  B.  and  Waldo,  C. 
M.,  1947,  Am.  J.  Anat.,  81,  413-444; 
Fawcett,  Anat.  Rec,  1950,  108,  71-91). 
Implants  of  trophoblast  from  9-11 
days  of  development  also  establish 
themselves  in  the  eye,  although  their 
capacity  for  growth  declines  in  relation- 
ship to  their  age  at  the  time  of  implanta- 
tion (Grobstein,  C,  J.  Exp.  Zool., 
1950,  114,  359-373). 

Plant  Cell  Walls,  see  Polysaccharides. 

Plants.  Except  for  pathogenic  Bacteria 
and  Fungi,  technique  for  plants  does  not 
come  within  the  scope  of  this  book. 
However  much  is  to  be  learned,  es- 
pecially in  microchemistry,  from  many 
methods  employed  by  botanists  and  the 
reader  is  advised  to  consult  Johansen, 
D.  A.  Plant  Microtechnique.  New 
York:  McGraw-Hill,  1940,  523  pp.,  also 
McClung,  1950. 

Plasma  Cells.  Since  plasma  cells  (of  the 
Marschalko  type)  are  mainly  identified 
by  recognition  of  a  small  area  near  the 
nucleus  which  does  not  stain  as  intensely 
as  the  rest  of  the  cytoplasm  with  basic 
dyes,  it  is  imjx)rtant  to  use  a  technique 
which  reveals  basophilia.  In  practice 
Giemsa's  stain,  or  a  good  coloration  with 
hematoxylin  and  eosin,  is  generally 
sufficient.  Unna  used  the  term  "plasma 
cell"  for  almost  any  kind  of  cell  with 
much  plasma  incluaing  macrophages  so 
that  the  designation  Unna's  plasma  cell 
is  almost  meaningless. 

Plasma  Membrane,  see  Cell  Membranes. 


PLASMA  STAINS 


274 


PLASTICS 


Plasma  Stains  are  too  numerous  to  cata- 
logue here.  Lillie  (p.  66-68)  states 
that  they  functionally  are  divisible 
into  two  groups:  plasma  stains  proper 
and  those  selective  for  extracellular 
materials  such  as  collagenic  and  elastic 
fibers,  bone  and  cartilage  matrix  and 
so  on. 

Plasma  Reaction,  see  Aldehydes  and  SchifT 
Reaction. 

Plasmalogen.  A  component  of  the  cyto- 
plasm which  gives  a  positive  Feulgen 
test   (Bourne,  p.  22).     See  Aldehydes. 

Plasmodesmata,  dehydrogenase  activity 
in,  see  Triphenyltetrazolium  Chloride. 

Plasmosin,  the  gel  and  fiber  forming  con- 
stituent of  the  hepatic  cell.  Method  of 
isolation  and  properties  (Bensley,  R.  R., 
Anat.  Rec,  1938,  72,  351-369). 

Plasmosome.  The  true  nucleolus  staining 
with  "plasma"  or  "acid"  dyes,  that  is 
to  say,  red  with  eosin  in  the  hematoxylin 
and  eosin  combination.  The  plasmo- 
some apparently  does  not  make  any 
direct  contribution  to  chromosome  for- 
mation. Acidophilic  nucleoli  are  quite 
different  from  certain  cytoplasmic  gran- 
ules which  Arnold  called  "plasmo- 
somes"  and  mitochondria  termed  "plas- 
tosomes"  by  Meves. 

Plastics — Written  by  M.  S.  Lucas,  Dept. 
Biological  Science,  Michigan  State 
College,  East  Lansing.  August  10, 
1951 — Acrylic  plastics  have  been  de- 
scribed very  early  for  use  as  embedding 
media  in  biological  fields  whereas  poly- 
ester  resin  plastics,  the  so-called  "cold- 
setting"  plastics,  have  more  recently 
come  into  use.  Acrylics  require  very 
careful  handling  and  the  fumes  are  more 
toxic  than  those  of  the  polyester  resins. 
These  features  have  somewhat  deterred 
the  general  use  of  acrylics.  Finished 
blocks  of  both  types  of  plastic  are  clear 
and  resistant  to  damage;  acrylics  are 
a  little  clearer  but  the  difference  is  so 
slight  that  it  is  not  noticeable  except 
when  a  block  of  acrylic  plastic  is  com- 
pared directly  with  a  block  of  polyester 
resin.  The  plastics  can  be  tinted  or 
even  rendered  opaque  on  the  back  by 
addition  of  dyes  and  chemicals.  The 
specimens  to  be  embedded  can  be 
opaque   or   cleared. 

Techniques  involved  in  successful 
embedding  of  biological  and  medical 
specimens  are  not  difficult.  However, 
use  of  plastics  is  not  as  simple  as  some 
pamphlets  indicate.  It  is  as  precise  a 
technique  as  any  used  in  histology  and 
for  this  reason  many  people  have 
mediocre  success.  Most  successful 
users  of  plastics  feel  that  the  possible 
uses  for  them  have  only  begun  to  be 
described.  It  is  a  good  idea  for  those 
interested,  to  secure  current  pamphlets 


concerning  new  methods  and  also  about 
the  development  of  new  and  simpler- 
to-use  plastics  from  the  several  firms 
listed  below  as  well  as  others  not  listed 
here. 
Acrylics 

1.  The  Polychemicals  Dept.,  E.  I.  du 
Pont  de  Nemours  and  Co.,  Wilming- 
ton, Del.  "Du  Pont  Plastics  in 
Medicine  and  Surgery",  Mimeo. 
Pp.  9,  references. 

2.  The  Plastics  Dept.,  Rohm  and  Haas 
Co.,  Washington  Sq.,  Phila.  5,  Pa. 
"Embedding  Biological  Specimens 
in  Acrylic  Plastic",  Mimeo.  Pp.  6, 
references. 

3.  Description  of  and  Methods  for 
Acrylics  in  general:  "Preservation 
of  Agricultural  Specimens  in  Plas- 
tics". Miscell.  Pub.  No.  679 
U.S.D.A.,  G.  R.  Fessenden,  1949. 
Pp.  78,  111.,  Lit.  Cited,  bibliography. 

Polyester  Resin  Plastics. 

Selectron  No.  5026,  manufactured  by 
the  Pittsburgh  Plate  Glass  Co.  The 
following  are  some  trade  names  under 
which  it  is  retailed. 

1.  "Bio-plastic" — Wards  Natural  Sci- 
ence Establishment,  Inc.,  3000  Ridge 
Rd.,  E.,  Rochester  9,  N.  Y.  "How 
to  Embed  in  Bio-plastic",  1950, 
Pp.  20,  25^,  references,  111.  "Nat- 
ural Science  Bulletin"  Published 
monthly. 

2.  "Castolite"— The  Castolite  Co., 
Woodstock,  111.  "Preserving  Speci- 
mens in  Castolite",  1950,  Mimeo. 
Pp.  23,  references.  Instruction  Man- 
ual. 

3.  "Turtox  Embedding  Plastic"— The 
General  Biological  Supply  House, 
761  E.  69th  PL,  Chicago,  ill.  "Em- 
bedding Specimens  in  Transparent 
Plastic",  Turtox  Service  Leaflet  No. 
33,  1951. 

4.  "Carolina     Embedding     Plastic" — 
Carolina  Biological  Co.,  Elon  Col- 
lege,   N.    Carolina.     Instruction   Book- 
let 50?f. 

The  pamphlets  listed  above  contain 
detailed  procedures.  They  also  give 
the  history  and  specifications  of  the 
plastic  and  indicate  biological  materials 
that  are  suitable  for  embedding.  Most 
of  them  contain  large  bibliographies. 
In  addition  to  the  references  listed  in 
these  pamphlets,  certain  additional 
work,  can  be  mentioned. 

Albert  Jehle  (University  Museum, 
Univ.  of  Pennsylvania,  Phila.,  Pa.)  is 
using  dental  acrylics  as  reconstruction 
material  in  restoration  of  skeletons 
(report  in  preparation).  In  some  in- 
stances he  has  replaced  an  entire  bone 
by  preparing  a  plastic  cast. 

A  new  technique  for  light  and  electron 


PLASTICS 


275 


PLASTIDS 


microscop3'^  (Newman,  S.  B.,  Borysko, 
E.  B.,  and  Swerdlow,  M.,  U.  S.  National 
Bur.  Standards,  J.  of  Res.,  1949,  43, 
183-199)  uses  n -butyl  methacrylate  for 
mounting  specimens  which  can  be 
sectioned  for  studj'  under  a  microscope. 

Plastics  in  the  form  of  sheets  find 
interesting  uses.  Microscopy  with 
plastic  substitutes  for  cover  glasses  is 
discussed  by  Richards,  O.  W.,  Small, 
J.  H.  and  Collyer,  P.  W.,  Stain  Tech., 
1944,  19,  59-62.  Natural  color  plant 
specimens  have  been  mounted  between 
plastic  plates  by  Fessenden  (G.  R., 
loc.  cit.  under  Acrylics  3).  Preserved 
flowers  can  be  placed  in  transparent 
plastic  containers  with  a  desiccant  and 
kept  for  long  periods  of  time  (Specht, 
R.  C,  Engineering  Progress,  Univ. 
Florida,  1950  IV  No.  12,  Gainesville). 

Polyester  resins:  Gross  anatomical 
and  embryological  specimens  have 
been  mounted  and  are  described  in  the 
following  articles: 

1.  "Embedding  Gross  Sections  in 
Ward's  Bio-plastic",  T.  H.  Ro- 
maniak.  Ward's  Nat.  Sci.  Bull., 
1948,  22,  34. 

2.  "The  Mounting  of  Human  Sections 
in  Bio-plastic,"  Ward's  Nat.  Sci. 
Bull.,  1950,  24,  27. 

3.  "On  the  Mounting  of  Anatomical 
Museum  Specimens  in  Transparent 
Plastic",  Kampmeier,  O.  T.  and 
Haviland,  R.  N.,  Anat.  Record,  1948, 
100,  201-231  (Used  Castolite  on  100 
specimens) . 

4.  "The  Relationships  of  Herniated 
Intervertebral  Discs  to  the  Spinal 
Cord  and  Spinal  Nerves",  (Used 
Castolite).  A.  F.  Reed  and  H.  D 
Kirgis,  Abstract  D2,  Anat.  Rec, 
1951,  109,  146. 

5.  "The  Blood  Vessels  of  the  Jejunum 
and  Ileum  in  Man  and  Certain  Lab- 
oratory Animals",  (Used  Polysty- 
rene). Abstract  D  16,  Anat.  Rec, 
1951,  109,  144. 

6.  Charles  H.  Glines  (with  Alexander 
Barry  and  Bradley  M.  Patten  at  the 
Anatomy  Dept.,  School  of  Medicine, 
University  of  Michigan,  Ann  Arbor) 
has  prepared  both  cleared  and 
opaque  embryos  following  techniques 
set  out  in  Ward's,  "How  to  Embed 
in  Bio-Plastic".  Glines  and  his 
co-workers  have  devised  some  modi- 
fications over  methods  described  by 
Kampmeier.  These  data  are  avail- 
able in  mimeograph  form,  and  were 
also  presented  at  the  1951  meeting 
of  the  Am.  Assoc,  of  Anatomists 
(Barry,  A.,  Patten,  B.  M.  and  Glines, 
C.  H.,  Abstract  D  31,  Anat.  Rec,  1951, 
109,133). 

Demonstration    sets     composed     of 


animal  types  have  been  prepared  in 
plastic  for  class  use  (Lucas,  M.  S. 
report  in  preparation)  in  connection 
with  a  study  of  "unknown"  specimens 
(Study  1,  1951,  Guide  for  Laboratory 
Studies  Biological  Science,  Mich.  State 
College  Press,  E.  Lansing).  Each 
animal  is  prepared  individually  in  a 
block  and  later  is  set  with  others  into 
an  illuminated  box  or  into  a  large  block 
of  plastic.  This  provides  an  attrac- 
tive variety  of  unknown  animal  types 
to  supplement  conventional  identifica- 
tion studies.  Plastic-corrosion  speci- 
mens prepared  by  injecting  blood  ves- 
sels of  animals  with  Vinyl  Acetate 
(Vinylite)  can  be  embedded  in  blocks 
of  polyester  resins  (Service  Bulletin 
No.  5,  1950,  and  Natural  Science  Bul- 
letin 1950,  24,  3,  Ward's  Natural  Science 
Est.).  Vinylite  of  the  type  carried  by 
this  firm  contains  a  pigment  which  will 
not  bleed  out  of  the  preparation  into 
the  plastic  block,  whereas,  latex-in- 
jected vessels  will  permit  dye  diffusion. 
The  use  of  plastic  in  reconstructions 
from  serial  sections  is  advocated  by 
Boyer,  C.  C,  Anat.  Rec,  1948,  100, 
191-197. 

In  soils  studies,  vinvlite  resin  (The 
Bakelite  Corp.,  230  N.  Mich.  Ave., 
Chicago  1,  111.)  and  cellulose  acetate 
(Hercules  Powder  Co.,  Cellulose  Prod- 
ucts Dept.,  Parlin,  N.  Y.)  are  used  to 
make  soil  profiles  (Berger,  K.  C.  and 
R.  J.  Muckenhirn,  Soil  Sci.  Soc  of 
Amer.  Proc,  1945,  10,  368-370;  Smith, 
H.  W.  and  C.  D.  Moodie,  Soil  Science, 
1947,  64,  61-69).  Permanent  plastic 
color  standards  for  rapid  soil  and  plant 
tissue  testing  are  emploved  bj^  Lynd, 
J.  Q.,  and  L.  M.  Turk,  J.  Soc.  Agron- 
omy, 1948,  40,  940-941.  Plant  ma- 
terials showing  deficiency  symptoms 
can  be  prepared  in  plastics  as  described 
by  Lynd,  J.  Q.,  Quart.  Bull.  Mich.  State 
Coll.  Agric.  Exper.  Station,  In  press. 
Art.  No.  1238. 

A  process  for  embedding  flowers  in 
plastic  so  as  to  preserve  their  natural 
shape  and  color  has  been  described  by 
Specht,  R.  C,  Engineering  Progress 
at  the  U.  of  Fla.,  1950,  IV  No.  12, 
Gainesville,  $1. 
Plastids  are  by  definition  simply  formed 
bodies.  The  term  is  usually  ap])lied  to 
certain  cytoplasmic  bodies  in  plants. 
They  may  be  colorless  leucoi)l.astids, 
chloroplastids  containing  chlorophyll 
or  chrornoplastids  containing  other  pig- 
ments. Chlorophyll  thus  segregated 
in  these  bodies  acted  on  by  light  plays 
its  part  in  starch  production,  as  hemo- 
globin (erythroplastids)  acts  in  trans- 
port of  oxygen.  The  chloroplastids  are 
easily  visible  microscopically.     Special 


PLATELET  COUNTS 


276 


PNEUMONOCYTES 


techniques  are  only  required  to  reveal 
the  organization  of  the  ground  sub- 
stance, holding  the  chlorophyll,  and 
their  r61es  in  photosynthesis.  A  full 
account  is  provided  by  Guilliermond, 
A.,  The  Cytoplasm  of  the  Plant  Cell. 
Waltham:  Chronica  Botanica  Co.,  1941, 
247  pp.  (translated  from  the  French  by 
L.  R.  Atkinson). 

Platelet  Counts,  see  Blood  Platelets. 

Plastosomes,  see  Mitochondria. 

Platino-Acetic-Osmic  mixture,  see  Her- 
mann's Fluid. 

Platinum.  Intravenous  injections  of  col- 
loidal solutions  of  platinum  in  rabbits 
are  described  by  Duhamel,  B.  G.,  C. 
rend.  Soc.  de  Biol.,  1919,  82,  724-726. 
For  microchemical  detection  of  plat- 
inum reference  is  made  to  the  method 
of  Okamoto  and  Associates  as  described 
by  Click  (p.  26). 

Platinum  Chloride  is  the  name  usually  given 
to  hydro-chloroplatinic  acid.  It  is  used 
occasionally  as  an  ingredient  of  fixa- 
tives. 

Platyhelminthes  is  the  phylum  of  flatworms. 
The  two  classes  of  important  parasites 
are  the  Cestodes  and  Trematodes. 
See  Parasites. 

Pleuropneumonia.  Staining  of  organisms. 
Stain  paraffin  sections  4  microns  thick 
of  tissue  fixed  in  Zenker,  Bouin,  abso- 
lute alcohol  or  Carnoy's  fluid  brought 
down  to  water  directly  in  Mallory's 
phosphotungstic  acid  hematoxylin  (18- 
24  hrs.)  without  preliminary  treatment 
with  permanganate  and  oxalic  acid. 
Do  not  wash  but  blot  nearly  dry  and 
dehydrate  rapidly  in  absolute  alcohol, 
clear  in  xylol  and  mount  in  balsam. 
Organisms  in  lungs  appear  as  deep  blue 
masses  of  mycelial  threads  (Turner,  H. 
W.,  Austral.  J.  Exp.  Biol.  &  Med.  Sci., 
1935,13,149-155). 

Plehn's  Stain  for  malaria  plasmodia  is  de- 
scribed by  Craig,  p.  289  as  uncertain  in 
its  action  and  is  not  recommended  if 
other  modifications  of  Romanowsky 
stain  are  available. 

Plimmer's  Bodies,  see  Bird's  Eye  Inclu- 
sions. 

Plutonium.  Use  of  micrurgy  in  study  of 
chemical  properties  of  first  micro- 
quantities  of  plutonium,  Seaborg,  G.  T., 
Science,  1946,  104,  379  also  Chambers, 
R.  W.  and  Kopac,  M.  J.  in  McClung's 
Microscopical  Technique,  1950,  p.  543. 
Pneumonocytes — Written  by  C.  C.  Macklin, 
Dept.  of  Histological  Research,  The 
University  of  Western  Ontario,  London, 
Canada.  November  28,  1951— These 
elements  are  also  known  as  pulmonary 
cells  (la  cellule  pulmonaire),  epicytes, 
septal  cells,  niche  cells,  alveolar  epi- 
cytes, alveolar  cells,  alveolar  wall  cells, 
alveolar   granular   cells,   alveolar   epi- 


thelial cells,  residual  epithelial  cells  on 
the  pulmonary  alveolar  walls,  little 
granular  cells,  etc.,  and  are  demon- 
strable in  mammalian  lungs  by  the 
usual  methods  of  fixation  and  staining. 
Their  most  prominent  feature  is  an 
array  of  Vacuoloids  (which  see)  in  the 
cytoplasm  (Macklin,  C.  C,  Anat.  Rec. 
1947,  97,  397).  This  gives  to  them  in 
ordinary  sections  a  frothy  appearance. 
Many  contain  phagocytized  particles. 
The  latter  are  the  mural  phagocytes  or 
dust  cells  (which  see).  They  are  found 
inserted  into  canals  in  partitional  al- 
veolar walls,  which,  when  vacated,  are 
natural  pores.  In  the  marginal  alveolar 
walls  they  are  inserted  into  sockets. 
Most  are  lodged  in  crotches  of  adjoin- 
ing alveolar  walls.  Normally  they  are 
5m  to  15/x  in  maximum  diameter,  but 
when  stimulated  exceed  this  size. 

The  form  is  altered  by  the  fixation 
technique  employed  to  demonstrate 
them.  After  perfusion  of  the  pul- 
monary blood  vessels  of  the  unopened 
thorax  with  physiological  saline  solu- 
tion followed  by  a  fixative  such  as 
Bouin's  fluid,  the  pneumonocytes  of 
partitional  alveolar  walls  often  in  pro- 
file have  the  shape  of  a  carafe  with 
larger  head  and  attenuated  foot,  and 
a  connecting  stalk  which  is  indented 
by  the  environing  capillary.  In  ma- 
terial fixed  by  immersion  of  a  block  of 
lung  tissue  the  cell  often  appears 
rounded  with  little  or  no  indication 
that  it  extends  from  one  alveolus  to 
another.  When  fixation  was  by  filling 
of  the  air  spaces  with  preservative  the 
cell  is  often  extended  laterally,  that 
is  flattened,  and  appears  wider  and 
shorter  than  normal.  Probably  the 
best  preservation  of  normal  form  is 
attained  in  the  mouse  by  immersion 
of  the  unopened  thorax,  cleared  of  skin 
and  muscle,  in  fixing  fluid.  In  such 
preparations  the  cell,  when  situated  in 
an  alveolar  "corner"  may  show  as 
many  as  four  separate  air  faces,  and  a 
cytosome  often  forked  in  longitudinal 
section  (Macklin,  C.  C,  Trans.  Roy. 
Soc.  of  Canada,  Sect.  V,  1946,  40,  93- 
1 1 1 — bibliography) . 

H.  von  Hayek,  Anat.  Anz.,  1942,  93, 
149-155,  estimated  their  combined 
volume  in  both  human  lungs  to  be  150 
cubic  centimeters;  and  they  would  thus, 
in  his  view,  compose  a  diffuse  intra- 
pulmonic  alveolar  epithelial  organ  as 
large  as  the  spleen  of  man.  Sjostrand 
and  Sjostrand  (Zeitsch.  f.  mik.-anat. 
Forsch.,  1938,  44,  370-411)  give  their 
combined  volume  as  10%  of  the  lung 
tissue. 

From  them  tumors  may  originate  in 
man  and  the  lower  mammals.     Mostofi 


POLARIZATION  OPTICAL  METHOD      277      POLARIZATION  OPTICAL  METHOD 


and  Larsen  (J.  Nat.  Cancer  Inst.,  1951, 
11, 1187-1222)  have  shown  that  in  Strain 
A  mice,  adenomata  may  arise  from  them 
during  continuous  administration  of 
urethane,  0.1%  in  the  drinking  water. 
Macklin  (J.  Thor.  Surg.,  1938,  7,  536- 
551;  Trans.  Roy.  Soc.  of  Can.,  1946, 
Sect.  V,  40,  93-111;  and  BioL  BulL, 
1949,  96,  173-178)  has  admitted  a  high 
degree  of  mitotic  potency  in  the  pneu- 
monocyte  (epicyte)  and  has  accepted 
it  as  a  primary  center  for  lung  cancer. 
Pneumonocytes  may  become  phago- 
c>i;ic  and  appear  with  more  or  less 
foreign  material  within  the  cytoplasm. 
When  such  cells  become  free  they  take 
a  rounded  or  oval  form  and  are  known 
as  epithelial  phagocytes,  alveolar 
phagocytes  or  Dust  Cells  (which  see), 
and  are  quite  different  from  the  histo- 
cytes  of  the  pulmonary  connective 
tissues.  Free  pneumonocytes  without 
particulate  matter  within  them  are 
known  as  Foam  Cells  (which  see).  In 
silver-wash  preparations  (see  Silver 
Lineation)  these  alveolar  mural  cells 
are  encircled  each  by  a  heavy  golden- 
brown  line,  and  the  air-faces  are  sprin- 
kled with  particles  of  the  same  hue. 

Large  rounded  granules  of  different 
sizes  are  seen  in  them  in  frozen  sections 
from  Regaud-fixed  lungs  which  have 
been  well  mordanted  with  potassium 
bichromate  and  strongly  stained  with 
Heidenhain's  iron  hematoxylin  (Mack- 
lin, C.  C,  Can.  J.  Res.,  D,  1950, 28, 5-15). 
Similar  granules,  but  gray  or  black,  may 
be  seen  in  Aquax  (which  see)  sections 
from  lungs  that  have  been  filled  while 
fresh  with  fixative  containing  from  jg 
to  1%  of  osmium  tetroxide.  Similar 
granules,  but  of  dark  brown  color,  have 
been  described  in  them  by  Sjostrand  and 
Sjostrand  (Zeitsch.  f.  mik.-anat.  Forsch., 
1938,  44,  370-411)  where  the  living  cells 
have  come  into  contact  with  blood  and 
an  aldehyde,  as  pure  formalin.  They 
find  that  this  colored  substance  has 
properties  of  hemin. 
Polarization  Optical  Method. — Written  by 
Francis  O.  Schmitt,  Dept.  of  Biology, 
Massachusetts  Institute  of  Technology, 
Cambridge,  Mass.,  May  19,  1950.— 
The  examination  of  tissues  and  cells 
with  the  polarizing  microscope  gives 
information  about  the  presence  of 
preferentially  oriented  constituents, 
the  direction  of  their  orientation,  their 
shape,  regularity  of  internal  construc- 
tion, partial  volume  and  refractive  in- 
dex. Details  of  the  theory  and  methods 
by  which  such  information  may  be  ob- 
tained are  contained  in  the  books  and 
papers  of  Schmidt,  Frey-Wyssling  and 
Schmitt  listed  at  the  end  of  this  sec- 
tion. 


The  polarizing  microscope  is  equipped 
with  a  polarizer  (nicol  prism  or  polaroid 
disc)  below  the  condenser  and  an  ana- 
lyzer in  the  draw  tube  above  the  objec- 
tive. Between  the  analyzer  and  objec- 
tive is  a  slot  into  which  may  be  inserted 
a  compensator  or  gypsum  plate.  When 
the  planes  of  polarization  of  polarizer 
and  analyzer  are  perpendicular  no  light 
passes  through  the  ocular.  If  a  speci- 
men is  now  placed  on  the  stage,  oriented 
constituents  may  become  visible  on  a 
dark  field.  The  intensity  will  be  maxi- 
mum when  the  distinguishing  direction 
of  the  object,  such  as  a  fiber,  is  oriented 
at  45°  to  the  planes  of  polarization  of 
polarizer  and  analyzer.  Objects  hav- 
ing internal  regularity  of  structure  may 
have  two  descriptive  refractive  indices, 
hence  show  double  refraction  or  bire- 
fringence. It  is  the  object  of  polarized 
light  microscopy  to  detect,  measure  and 
interpret  this  birefringence. 

Birefringence  is  numerically  equal 
to  the  difference  between  the  two  de- 
scriptive refractive  indices,  iV,  and  No- 
It  is  usually  determined  by  the  use  of 
a  compensator  which  measures  the 
phase  difference  expressed  as  fractional 
wavelength,  0,  or  retardation,  r,  ex- 
pressed in  ra/i.  Thickness  of  the  speci- 
men, d,  is  also  expressed  in  m/x.     Then 

birefringence  =   N,  —  No  =  -y  =   -3. 

Commonly  used  are  the  Berek,  quar- 
ter-wave (S^narmont)  and  Kohler  ro- 
tating mica-plate  compensators,  in  or- 
der of  increasing  sensitivity. 

Besides  the  magnitude  of  birefring- 
ence its  sign  is  of  importance  in  diag- 
nosing the  ultrastructure  of  biological 
constituents.  If  the  refractive  index 
for  vibrations  paralleling  the  distinc- 
tive direction,  e.g.  the  long  axis  of  a 
fiber,  is  greater  than  that  for  vibrations 
perpendicular  to  this  direction,  the 
birefringence  is  positive  with  respect  to 
this  direction.  If  the  refractive  index 
relations  are  reversed  the  birefringence 
is  negative.  Most  protein  and  carbo- 
hydrate fibers  show  positive  birefring- 
ence while  nucleic  acid  and  nucleo- 
proteins  usually  show  negative 
birefringence.  While  the  sign  of  bire- 
fringence may  be  determined  with 
compensators,  the  gypsum  Red  I  plate 
may  be  very  useful.  When  this  plate 
is  inserted  into  the  compensator  slot, 
the  field  appears  red  if  the  nicols  are 
crossed.  Birefringent  objects  show 
addition  or  subtraction  colors,  such  as 
blue  or  yellow,  respectively,  depending 
on  the  orientation  of  the  object  with 
respect  to  the  planes  of  polarizer  and 
analyzer  and  on  the  sign  of  birefring- 
ence.   Thus  a  fiber  of  connective  tissue 


POLARIZED  LIGHT 


278 


POLAROGRAPHIC  TECHNIQUE 


or  muscle  will  appear  blue  in  one  diag- 
onal position  and  yellow  in  the  diagonal 
perpendicular  thereto;  this  is  because 
these  j&bers  manifest  birefringence 
which  is  positive  with  respect  to  the 
fiber  axis.  A  nerve  fiber  shows  the 
same  colors  in  its  myelin  sheath  except 
that  the  diagonal  positions  in  which  it 
shows  these  colors  are  reversed  from 
those  of  the  above  case;  this  is  because 
the  myelin  sheath  manifests  birefrin- 
gence which  is  negative  with  respect  to 
the  fiber  axis. 

The  birefringence  of  most  biological 
objects  is  due  to  regularity  of  structure 
of  components  considerably  smaller 
than  the  wavelength  of  light.  To  get 
at  the  nature  of  these  components,  one 
studies  the  relation  of  the  birefringence 
to  the  refractive  index  of  the  medium 
in  which  the  object  is  immersed,  using 
consecutively  a  number  of  media  (us- 
ually organic  solvents)  of  varying  re- 
fractive index.  Application  of  Wie- 
ner's theory  then  makes  it  possible  to 
deduce  the  orientation  of  the  submicro- 
scopic  particles  as  well  as  their  internal 
regularity  of  structure,  refractive  in- 
dices and  approximate  partial  volumes. 
Electron  microscope  observations 
have  confirmed  many  of  the  deductions 
based  on  the  polarization  optical  anal- 
ysis of  tissue  ultrastructure.  This 
method  will  continue  to  be  of  impor- 
tance biologically  despite  the  great 
possibilities  of  the  electron  microscopy, 
for  the  polarized  light  method  is  appli- 
cable to  tissues  in  the  fresh  state.  See 
Schmidt,W.J.,DieDoppelbrechungvon 
Karyoplasma,  Zytoplasma  und  Meta- 
plasma,  Berlin  Geb.  Borntrager,  1937. 
Frey-Wyssling,  A.,  Submicroscopic 
morphology  of  protoplasm  and  its 
derivatives,  Elsevier  Publishing  Co., 
Inc.,  1948.  Schmitt,  F.  O.,  The  ultra- 
structure  of  protoplasmic  constituents. 
Physiol.  Rev.,  1939,  19,  270.  Schmitt, 
F.  O.,  Tissue  structure:  polarization 
optical  analysis.  In  Glasser's  Medical 
Physics,  Vol.  II,  1950,  p.  1128. 
Bennett,  H.  S.,  The  microscopical  in- 
vestigation of  biological  materials  with 
polarized  light.  In  McClung's  Micro- 
scopical Technique,  in  press. 

Polarized  Light  is  said  to  be  better  than 
Marchi  and  Sudan  III  methods  for 
study  of  myelin  degeneration  of  periph- 
eral nerves  (Prickett,  C.  O.  and  Stevens, 
C,  Am.  J.  Path.,  1939,  15,  241-250). 
Used  in  study  of  mitochondria  and 
Golgi  apparatus  (Monn6,  L.,  Pro  to - 
plasma,  1939,  32,  184-192). 

Polarizing  Microscope.  The  polarizing  mi- 
croscope in  its  simplest  form  is  a  con- 
ventional microscope  used  with  plane- 
polarized  light  for  illumination  and  with 


a  polarizing  ocular  (called  the  analyzer) 
to  detect  the  presence  of  birefringence. 
Or  it  may  be  thought  of  as  a  polariscope 
equipped  with  magnifying  lenses.  This 
instrument  has  been  borrowed  from 
the  minerologists,  who  employ  it  in 
the  study  of  crystalline  materials.  Its 
purpose  in  histology  is  to  seek  sub- 
stances characterized  by  their  ability 
to  rotate  the  plane  of  polarized  light 
(Schmitt,  1939). 

A  Nicol  prism,  or  a  disk  of  Polaroid, 
is  placed  below  the  condenser.  Ordi- 
nary light  is  thereby  polarized,  but  goes 
on  to  form  an  image,  exactly  as  in  the 
bright-field  microscope.  With  an  ana- 
lyzer in  the  ocular,  oriented  so  that  its 
polarizing  direction  is  parallel  to  that 
of  the  polarizer  below,  one  sees  the 
regular  image.  With  a  90°  rotation  of 
the  analyzer,  however,  the  field  be- 
comes completely  dark,  except  in  those 
places  on  the  slide  where  birefringent 
crystals  or  crystalloidal  materials  are 
found.  Since  this  image  in  general  will 
not  be  very  bright  it  is  best  to  observe 
with  the  dark-adapted  eye  and  to  have 
a  very  strong  source  of  light  for  so  much 
light  is  lost.  Starch  grains,  cholesterol 
droplets,  certain  salt  crystals,  any  or- 
ganic structures  with  preferentially 
oriented  molecular  aggregates,  such  as 
nerve  fibers  (Prickett,  C.  O.  and 
Stevens,  C,  Am.  J.  Path.,  1939,  15, 
241-250),  striated  muscle,  bone  (Klee- 
men  1945)  plant  cell  walls,  etc.  stand  out 
well  (Johnson,  B.  K.,  Endeavour,  1948, 
7,  57-65).  With  a  more  elaborate 
polarizing  microscope  one  may  make 
quantitative  measurements  leading  to 
the  identification  of  these  materials 
and  to  certain  conclusions  regarding 
the  fine  structure  of  organic  forms. 
See  Polarization  Optical  Methods. 
Polarographic  Technique — Written  by 
Christopher  Carruthers,  Division  of 
Cancer  Research,  Washington  Uni- 
versity, St.  Louis,  10,  Mo.  October  5, 
1951 — The  polarographic  method  of 
analysis  is  ideally  suited  for  the  quanti- 
tative and  qualitative  measurement  of 
minute  amounts  of  reducible  sub- 
stances. It  is  especially  suitable  for 
concentrations  of  10"'^  to  10"^  molar, 
and  since  the  analysis  can  be  performed 
with  a  very  small  volume  of  solution, 
as  little  as  a  drop  to  a  few  tenths  of  a 
cc,  traces  of  reducible  substances  can 
be  detected.  This  method  was  dis- 
covered by  Professor  J.  Heyrovsky  of 
Charles  University,  Prague  (Rev.  trav. 
chim.,  1925,  44,  488-498).  It  is  based 
upon  the  reducibility,  or  oxidizability, 
of  a  substance.  For  example,  when 
electrons  are  removed  from  the  ferrous 
ion,  Fe*"*^,  it  is  oxidized  to  the  ferric 


POLAROGRAPHIC  TECHNIQUE 


279 


POLAROGRAPHIC  TECHNIQUE 


ion  Fe"*'++,  or  Fe"*"*"  z^  Fe"^'"  +  e  (elec- 
tron). Conversely,  when  an  electron 
is  added  to  the  ferric  ion,  the  latter 
is  reduced  to  the  ferrous  state.  In  the 
polarographic  method  the  electrolysis 
is  carried  out  with  a  small  easily  polariz- 
able  electrode,  the  dropping  mercury 
electrode,  and  one  large  non-polarizable 
electrode. 

In  the  reduction  of  cadmium  ion  to 
metallic  cadmium,  for  e.xample,  the 
applied  potential  is  gradually  changed 
from  0.0  volts  and  the  current  flowing 
through  the  solution  is  measured  by 
means  of  a  galvanometer.  From  0.0 
volt  to  about  —0.5  volt  very  little 
current  flows  through  the  solution  be- 
cause the  potential  at  which  the  cad- 
mium ion  is  reduced  has  not  been 
reached.  At  about  —0.53  volt  the 
current  begins  to  increase  as  the  voltage 
is  decreased  (negative)  and  at  —0.7 
volt  the  current  becomes  steady  again. 
Between  —0.52  volt  and  —0.7  volt  an 
S  shaped  curve  is  produced,  and  a 
measurement  of  the  mid-point  of  this 
curve  gives  the  half -wave  potential, 
a  characteristic  constant  of  any  sub- 
stance under  controlled  conditions  while 
the  current  obtained  gives  the  amount 
of  cadmium  in  solution. 

In  other  words,  the  half-wave  poten- 
tial indicates  the  nature  of  the  reducible 
substance  while  the  current  observed 
is  a  function  of  its  concentration. 
When  the  current  in  microamperes  is 
plotted  against  the  applied  voltage, 
the  resulting  figure  is  called  a  polaro- 
gram.  The  small  current  flowing 
through  the  cell  prior  to  reduction  is 
called  the  residual  current.  This  is 
due  either  to  the  reduction  of  traces  of 
oxjrgen,  impurities  or  to  the  solution 
of  minute  amounts  of  mercurj^  from  the 
dropping  mercury  electrode.  The  cur- 
rent, which  is  obtained  at  about  —0.7 
volt  for  the  reduction  of  cadmium  ions, 
is  termed  the  diffusion  current  and  it 
is  a  function  of  the  concentration.  The 
diffusion  current  is  so  named  since  the 
ions  in  solution  are  reduced  as  fast  as 
they  reach  the  dropping  mercury  elec- 
trode— that  is,  the  reduction  is  diffusion 
controlled.  Now  if  the  concentration 
of  the  cadmium  ions  is  increased,  the 
diffusion  rate  will  increase  in  propor- 
tion to  the  concentration  and  this  will 
be  reflected  bj^  an  increase  in  the  diffu- 
sion current.  A  plot  of  the  diffusion 
current  minus  the  residual  current 
against  the  concentration  can  be  used 
for  analytical  purposes. 

The  half-wave  potential  can  be  em- 
ploj'ed  to  determine  the  nature  of  un- 
known substances.  For  example,  when 
the  electrolysis  of  a  mixture  of  Fe^ 


Pb++,  Zn+*-  and  Co++  is  carried  out  in 
a  solution  of  0.1  N  KCl,  the  supporting 
electrolyte,  the  half-wave  potentials 
in  volts  v.s.  the  saturated  calomel 
electrode  of  these  ions  are  respectively 
-0.396,  -0.995,  -1.20  and  -1.3.  The 
half-wave  potentials  are  separated 
sufTiciently  to  permit  the  determination 
of  each  one  in  a  mixture  of  the  four 
ions,  and  they  enable  one  to  determine 
any  of  these  ions  in  an  unknown  solu- 
tion. 

The  principal  objectives,  therefore, 
of  the  polarographic  method  are  two. 
In  the  first  place  the  determination  of 
the  nature  of  a  substance  from  the  half- 
wave  potential.  This  aspect  of  the 
procedure  is  very  important  since  it 
enables  one  to  get  at  structural  altera- 
tions in  reducible  compounds  from  a 
single  measurement.  An  application 
of  this  aspect  of  the  method  in  biology 
(carcinogenesis)  is  given  by  (Car- 
ruthers,  C,  and  Suntzeff,  V.  Science 
1950,  114,  103-107).  Secondly,  minute 
amounts  of  several  reducible  substances 
can  be  quantitatively  determined  in 
a  mixture  without  previous  separation 
by    the    classical    chemical    methods. 

The  equation  for  polarographic  analy- 
sis was  developed  by  Ilkovik  (Collec- 
tion Czechoslov  Chem.  Commun,  1934, 
6,  498-513).  This  equation  is  'd  =  605 
nD'/^Cm^'H"*  in  which  'd  is  the  mean 
diffusion  current  (in  microamperes),  n 
is  the  number  of  electrons  involved  in 
the  reduction  of  one  molecule  of  re- 
ducible substance,  F  is  the  Faraday 
(96,000  coulombs),  D  is  the  diffusion 
coefficient  (in  cm"  8ec~i),  C  is  its  con- 
centration (millimoles  per  liter),  m  is 
the  weight  of  mercury  flowing  from  the 
capillary  per  second  in  (mg.)  and  t  (in 
sec.)  is  the  time  necessary  for  the  forma- 
tion of  one  drop  of  mercury.  This 
equation  can  be  reduced  to  'd  =  KC 
when  all  the  other  factors  are  constant. 
Hence  the  concentration  of  a  reducible 
substance  is  given  by  the  equation 
C  =  'd/K.  Since  K  can  be  determined 
experimentally  from  the  electrolysis  of 
a  reducible  substance  at  known  con- 
centrations, the  amount  in  an  unknown 
can  be  measured  from  the  diffusion 
current. 

For  a  general  and  detailed  description 
of  the  polarographic  method  of  analysis, 
the  following  books  are  recommended: 
Kolthoff,  I.  M.,  and  Lingane,  J.  J. 
Polarography,  New  York:  Interscience 
Publishers,  Inc.  1941,  215-254;  Muller, 
O.  H.,  The  Polarographic  Method  of 
Analysis,  Easton,  Pa.,  Chemical  Educa- 
tion Publishing  Co.,  1951,  32-80,  141- 
193;    ilohn,    H.,    Chemische   Analysen 


POLORAGRAPHIC  TECHNIQUE 


280 


POLAROGRAPHIC  TECHNIQUE 


mit  dem  Polarographen,  Berlin:  Verlag 
von   Julius   Springer,   1937,  34-47. 

There  are  several  American  made 
polarographs  which  automatically  re- 
cord current-voltage  curves  (polaro- 
grams).  These  instruments  have  stim- 
ulated research  in  the  field  and  have 
simplified  the  measurements  consider- 
ably. The  following  companies  make 
self-recording  polarographs:  E.  H. 
Sargent  and  Co.,  155-165  East  Superior 
Street,  Chicago  11,  111.;  Leeds  and 
Northup,  4912  Stenton  Avenue,  Phila- 
delphia, Pa.;  Rutherford  Instrument 
Co.,  8616  Georgia  Avenue,  Silver 
Springs,  Md.  Manual  recording 
polarographs  are  also  made  by  Fischer 
Scientific  Co.,  2850  S.  Jefferson  Ave., 
St.  Louis,  Mo.  and  E.  H.  Sargent  and 
Co.,  155-165  East  Superior  St.,  Chicago, 
11,  111. 

Training  in  the  polargraphic  method 
of  analysis  is  best  achieved  by  working 
with  the  instrument  under  supervision 
of  an  expert,  or  by  taking  a  course  in 
analytical  chemistry  in  which  the  bases 
of  the  method  are  taught.  The  latter 
training  can  be  obtained  at  the  Chem- 
istry Department  of  the  University  of 
Minnesota,  Minneapolis,  Minn.,  or  at 
Harvard  University,  Cambridge,  Mass. 

Polarographic  analysis  has  been  found 
useful  in  many  fields  of  endeavor: 

Vitamin  B  (Thiamin).  Lingane,  J. 
J.  and  Davis,  O.  L.,  J.  Biol.  Chem., 

1941,  137,  567-574  found  that  this  vita- 
min gave  a  well  defined  wave  at  —0.25 
volt  (v.s.  the  saturated  calomel  elec- 
trode in  0.1  N  KCl).  The  method  has 
not  been  applied  to  natural  products. 
Vitamin  B2  (Riboflavin)  has  been  found 
to  be  reducible  by  the  same  investiga- 
tors   (Lingane    and    Davis,    Ibid). 

Nicotinic  Acid  and  Nicotinamid. 
Nicotinic  acid  is  reducible  in  0.1  N 
NaHCOs,  Shitkata,  M.  and  Tachi,  I., 
Bull.  Agr.  Chem.  Soc.  (Japan)  1927, 
3,  95-96.  A  detailed  investigation  of 
the  polarographic  behavior  of  nicotinic 
acid  and  related  compounds  has  been 
published  by  Tompkins,  B.  C.  and 
Schmidt,  C.  L.  A.  (Univ.  Calif.  Pub. 
Physiol.  1943,  8.  229-247). 

Pantothenic  acid  is  also  reducible 
(Lingane,  J.  J.  and  Davis,  O.  L.,  Ibid.). 

Vitamin  C  has  been  determined  in 
fruits  and  vegetables  polarographically 
by  Gillam,  W.  S.  (Indus,  and  Eng. 
Chem.   Anal.   Ed.,   1945,    17;  217-220). 

Vitamin  E  and  related  compounds 
have  been  extensively  investigated  by 
Kolthoff  and  coworkers,  see  Smith, 
L.  I.,  Kolthoff,  I.  M.,  Wawzonek,  S. 
and  Ruoff,  P.  M.,  J.  Am.  Chem.  Soc, 

1942,  64,  447-451;  644-648.  This  class 
of  compounds  was  oxidized  having  half- 


wave  potentials  of  approximately  0.1 
to  0.2  volt. 

Vitamin  K-Vitamin  Kb  is  also  re- 
ducible at  the  dropping  mercury  elec- 
trode, see  Knoblock,  E.,  Collection 
Czechoslov  Chem.  Commun.,  1949,  14, 
508-531. 

Folic  acid  can  be  determined  polaro- 
graphically in  small  amounts  in  tablets, 
see  Mader,  W.  J.  and  Frediani,  H., 
Amal.  Chem.,  1948,  20,  1199-1201. 

Steroids  and  Related  Compounds. 
Steroids  such  as  testosterone,  pro- 
gesterone, pregnenol-17-one-3,  desoxy- 
corticosterone,  etc.  which  have  a  car- 
boxyl  group  conjugated  with  a  double 
bond  are  reducible  (Eisenbrand,  J. 
and  Picher,  H.,  Zeit.  Physiol.  Chem., 
1939,  260,  83-99).  The  reaction  prod- 
ucts of  17-ketosteroids  with  Girard's 
Reagent  T  (trimethyl  acethydrazide 
ammonium  chloride)  make  the  method 
more  general  in  scope  (Wolfe,  J.  K., 
Hershberg,  E.  C.  and  Fieser,  L.  F., 
J.  Biol.  Chem.,  1940,  136,  653-687). 

Minerals.  The  polarograph  is  ideally 
suited  for  the  determination  of  minute 
amounts  of  most  of  the  metals  with  the 
exception  of  the  alkali  and  alkali-earth 
metals  (Kolthoff,  I.  M.,  and  Lingane, 
J.  J.,  Ibid.). 

Proteins.  Very  few  pure  proteins 
have  been  studied  polarographically. 
Proteins  containing  sulphydryl  groups 
or  — S — S — groups  can  be  determined 
and  their  purification  followed  by  the 
shape  of  the  catalytic  reduction  waves 
(Carruthers,  C,  H.  Biol.  Chem.,  1947, 
171,  641-651).  Proteins  and  polypep- 
tides containing  cystine  or  cysteine, 
when  dissolved  in  suitably  buffered 
cobalt  solutions,  produce  a  catalytic 
reaction  during  electrolysis  at  the 
dropping  mercury  electrode  (Brdicka, 
R.  Collection  Czechoslov.  Chem. 
Commun.,  1936,  8,  366-376).  This  ob- 
servation was  used  by  some  investiga- 
tors as  a  test  for  cancer  since  the  blood 
sera  of  normal  individuals  and  of 
persons  suffering  from  cancer  revealed 
significant  differences  in  the  height  of 
the  catalytic  waves  (Brdicka,  R., 
Nature,  1937,  139,  330;  1020-1021). 
However,  this  test  proved  to  be  non- 
specific (Rusch,  H.  P.,  Klatt,  T., 
Meloche,  V.  W.,  and  Dirksen,  A.  J., 
Proc.  Soc.  Exptl.  Biol.  Med.,  1940,  44, 
362-365). 

Amino  acids.  Amino  acids,  with  the 
exception  of  cysteine  and  cystine  which 
give  catalytic  waves  in  buffered  cobalt 
solutions,  are  not  reducible. 

Many  other  types  of  organic  com- 
pounds are  also  reducible.  For  ex- 
ample, carcinogenic  hydrocarbons  such 
as  3-methylcholanthrene,  3-4,benzypy- 


POLAROID 


281 


POLYSACCHARIDES 


rene  and 9, 10-dimethyl-l ,2-benzanthra- 
cene  can  be  determined  polarograph- 
ically  (Wawzouek,  S.,  and  Laitinen, 
H.  A.,  J.  Am.  Chem.  Soc,  1942,  &4, 
2365-2368). 

Many  other  types  of  organic  com- 
pounds (aldehydes,  ketones,  acids, 
nitro  nitroso  and  azo  compounds,  per- 
oxides, sugars  (ketases)  and  heterocylic 
compounds),  which  can  be  qualitatively 
and  quantativel}^  determined  polaro- 
graphically  are  given  in  a  review  by 
Wawzonek  (Anal.  Chem.,  1949,  21,  61- 
66),  and  in  the  books  listed  above. 

Polaroid.  This  is  a  polarizing  material 
made  up  of  extremely  minute  crystals 
of  quinine  sulpliate  periodide.  A  nitro- 
cellulose film  containing  the  crystals 
all  oriented  in  the  same  direction  can 
be  mounted  between  sheets  of  glass 
with  a  total  thickness  fo  about  3  mm. 
See  Bourne,  p.  26. 

Pollens.  The  microscopic  identification  of 
the  different  sorts  of  pollen,  especially 
the  allergens,  does  not  involve  any 
complicated  technique.  From  a  good 
textbook,  Feinberg,  S.  M.,  Allergy  in 
Practice.  Chicago,  The  Year  Book 
Publishing  Co.,  1944,  798  pp.,  one  is  first 
guided  by  data  on  pollens  likely  to  be 
in  the  atmosphere  at  the  particular 
season  and  in  the  special  locality.  The 
next  step  is  to  spread  on  microscopic 
slides  very  thin  films  of  white  petrola- 
tum. Then  expose,  for  measured  time, 
these  in  a  horizontal  position  coated 
side  up  protected  by  a  suitable  covering 
from  rain  but  not  so  as  to  interfere  with 
access  of  air.  Examine  directly  bj' 
direct  illumination  or  in  dark  field.  If 
staining  is  necessary  apply  Calberla's 
solution  as  described  by  Gay,  L.  N., 
Curtis,  H.  and  Norris,  T.,  Bull.  Johns 
Hopkins  Hosp.,  1941,68,  179-189  (glyc- 
erin 5  cc;  95%  ale,  10  cc;  aq.  dest., 
15  cc;  sat.  aq.  basic  fuchsin,  2  drops). 
Most  important  is  detailed  microscopic 
comparison  of  the  grains  observed  with 
the  illustrations  in  the  following  mono- 
graph: Wodehouse,  R.  P.,  Pollen 
Grains.  New  York :  McGraw-Hill  Book 
Co.,  1935. 

Poly-Azo  Dyes.  Chlorazol  black  E,  sudan 
black  B. 

Polychromatic  Erythroblasts,  see  Erythro- 
cytes, developmental  series. 
^  Polychrome  Methylene  Blue.  Literally 
many  colored,  but  actuallj'^  in  this  case 
two  colored.  It  is  a  methylene  blue 
which  contains,  in  addition  to  the  blue 
itself,  large  amounts  of  azures  especially 
A  and  B .  These  are  redder  than  methy- 
lene blue  and  are  partly  responsible  for 
the  metachromatic  staining  (G.  meta, 
beyond  +  chroma,  color)  given  by 
polychrome  methylene  blue.    The  color 


is  beyond  and  different  from  the  simple 
blue  by  reason  of  its  marked  reddish 
tint.  It  is  usually  better  to  purchase 
the  fKjlychrome  methylene  blue  rather 
than  to  make  it.  If  it  has  to  be  made 
dissolve  1  gm.  methylene  blue  in  100 
cc.  0.5%  aq.  NaHCOj;  place  in  steam 
sterilizer  1^  hrs.;  cool  and  filter  (Mc- 
Clung,  p.  334).  It  should  be  a  good 
methylene  blue.  Goodpasture's  (E. 
W.,  J.A.M.A.,  1917,  69,  998)  recipe  for 
polychrome  methylene  blue  is :  Boil  400 
cc.  aq.  dest.  +  1  gm.  methylene  blue  and 
1  gm.  potassium  carbonate  for  30  min. 
Cool  and  add  3  cc.  acetic  acid  and  shake 
dissolving  ppt.  Boil  gently  down  to 
200  cc.  volume  (5  min.).  Cool.  Eosi- 
nates  spectra  and  staining  potency 
(Lillie,  R.  D.  and  Roe,  M.  A.,  Stain 
Techn.,  1942,  17,  57-63).  See  also 
Lillie,  R.  D.,  Stain  Techn.,  1942,  17, 
97-110  for  acid  oxidation  methods  of 
polychroming. 

Polyethylene  glycols  are  also  known  as 
Carbowaxes.  See  Carbo  Wax  embed- 
ding. Rinehart,  J.  F.  and  Suleiman 
Abul-Haj,  A.  M.  A.  Archiv.  Path., 
1951,  51,  666-669  advise  the  dehydration 
and  embedding  of  formation  fixed 
tissues  and  the  staining  therein  of 
lipids  by  Sudan  and  other  dyes.  This 
is  a  great  improvement  over  the  frozen 
section  technique. 

Polysaccharides — Written  by  R.  D.  Hotch- 
kiss.  The  Rockefeller  Institute  for 
Medical  Research,  New  York  21,  N.  Y. 
November  14,  1951 — Polysaccharides 
are  oxidized  by  periodic  acid  with  pro- 
duction of  polyaldehydes  which  are 
stained  with  Schiff's  fuchsin-sulfite 
reagent.  Only  substances  with  1,2- 
glycol  groupings  (almost  exclusively 
carbohydrates,  which  in  fixed  tissue 
preparations  are  chiefly  present  as 
polysaccharides,  mucins  or  mucopro- 
teins)  are  supposed  to  react  in  this  way. 
In  the  Schiff-aldehyde  reactions,  it  is 
important  to  note  that  the  fuchsin 
combines  chemically  with  the  alde- 
hydic  substances  (Wieland  and  Scheu- 
ing,  Ber.,  1921,  54,  2527-2555);  the  dye 
is  presumably  therefore  fixed  at  the 
site  of  the  original  polysaccharide. 
J.  F.  A.  McManus  (Nature  1946,  158, 
202),  R.  D.  Lillie  (Bull.  Internat. 
Assoc,  Med.  Museums,  1947,  27,  23-61), 
and  R.  D.  Hotchkiss  (Arch.  Biochem. 
1948,  16,  131-141)  independently  dis- 
covered and  described  (he  periodic  acid- 
Schiff  method;  the  description  below 
is    that    of    Hotchkiss'    procedure. 

Solutions:  Alcoholic  periodic  acid: 
0.4  gm.  periodic  acid,  dissolved  in  15 
cc.  aq.  dest.,  add  0.14  gm.  cryst.  Na 
acetate,  then  add  35  cc.  alcohol  95%. 
Reducing    rinse:    Dissolve    1    gm.    KI, 


POLYSACCHARIDES 


282 


POLYSACCHARIDES 


1  gm.  Na  thiosulfate  hydrate  in  20  cc. 
aq.  dest.  Add,  with  stirring,  30  cc.  95% 
alcohol,  and  then  0.5  cc.  2  N  hydro- 
chloric acid.  Precipitated  sulfur  is 
allowed  to  settle  out,  but  the  mixture 
can  also  be  used  immediately.  Schiff's 
reagent:  fuchsin-sulfite  as  used  in  the 
reaction  for  Thymonucleic  Acid,  or  the 
Bauer-Feulgen  reaction  for  Glycogen; 
maximum  sensitivity  and  stability  are 
obtained  by  adjusting  the  acidity  to 
the  minimum  amount  which  allows  the 
reagent  to  dry  in  a  thin  film  on  a  glass 
slide  without  becoming  spontaneously 
colored.  Preserved  in  the  cold.  Sul- 
fite wash:  Aqueous  bisulfite,  e.g.  0.2 
gm.  K  metabisulfite  and  0.5  cc.  cone, 
hydrochloric  acid  to  50  cc.  water. 

Procedure:  Fix  in  usual  fixatives; 
alcoholic  or  picric  acid  fixatives  are 
suitable  if  water  soluble  polysaccharides 
such  as  glycogen  are  to  be  demon- 
strated. Bring  to  70%  alcohol.  Leave 
5  min.  in  periodic  acid,  rinse  with  70% 
alcohol,  leave  in  reducing  rinse  5  min., 
rinse  with  70%  alcohol,  leave  in  Schiff's 
reagent  15  to  45  min.,  wash  2  to  3  times 
in  sulfite  water.  Examine  or  dehydrate 
and  mount  as  desired.  Counterstain- 
ing  with  a  basic  dye  such  as  malachite 
green  (2  mg.  per  100  cc.  aq.  dest.)  is 
satisfactory.  Control  sections  treated 
same  except  not  exposed  to  periodic 
acid. 

Modifications:  Aqueous,  instead  of 
alcoholic,  periodic  acid  and  reducing 
rinse  may  be  used,  with  increased  likeli- 
hood of  removing  soluble  polysaccha- 
ride such  as  glycogen  or  dextrins  from 
the  section.  McManus  uses  0.5% 
aqueous  periodic  acid  without  acetate 
buffering  (Stain  Technology,  1948,  23, 
99-108) ;  Lillie  uses  aqueous  sodium 
periodate  containing  nitric  acid  (ibid., 
1951,  26,  123-136).  Periodate  or  iodate 
entrapped,  or  bound  to  such  com- 
ponents as  calcium  in  the  sections,  will 
color  the  Schiff  reagent;  the  reducing 
rinse  is  intended  to  destroy  these 
iodates  chemically.  Ordinary  washing 
is  used  in  the  McManus  and  Lillie  pro- 
cedures, and  is  adequate  whenever 
iodate  retention  does  not  prove  to  be 
a  difficulty  in  the  particular  tissue  under 
investigation.  The  periodate-Schiff  re- 
action may  be  used  subsequent  to  treat- 
ment with  enzymes  such  as  diastase 
(Lillie)  or  hyaluronidase  to  further 
identify  the  material  being  stained. 
Isolated  substances  can  be  tested  for 
stainability  by  "spot  tests"  with  the 
same  reagents  to  help  predict  their  be- 
havior in  tissue  sections  (see  Hotchkiss, 
loc.  cit.). 

Mechanism  of  reaction  and  specificity: 
Aldehyde     groups    newly    formed     by 


periodic  acid  and  reacting  with  Schiff's 
reagent  are  almost  certainly  produced 
by  the  breakage  of  1,2-glycols  or  equiv- 
alent 1,2-amino  alcohols  or  1,2-dia- 
mines.  Biologically  occurring  sub- 
stances containing  such  groups  are  the 
simple  sugars,  polj'saccharides,  cere- 
brosides,  inositol  compounds,  certain 
hydroxy-amino  acids,  adrenaline.  The 
specificity  of  the  stain  further  depends 
in  part  upon  the  removal  of  low  mole- 
cular compounds  in  fixation  and  wash- 
ing, leaving  the  polysaccharides  (and 
mucins,  glycoproteins)  as  probably  the 
only  known  naturally  occurring  sub- 
stances which  remain  to  be  stained. 
The  sugar  residues  contained  in  nucleic 
acids  and  the  hydroxyamino  acids  of  the 
proteins  (except  perhaps  terminal  resi- 
dues or  hydroxj'lysine)  are  chemically 
so  substituted  that  they  do  not  react 
with  the  periodic  acid.  A  number  of 
other  substances  which  reduce  the  oxi- 
dant (such  as  tryptophan)  do  not  yield 
aldehyde  groups. 

Not  all  sugars  or  polysaccharides 
give  equallj'  intense  coloration  (Hotch- 
kiss, loc.  cit.:  Jeanloz,  R.,  Science 
1950,  111,  289)  and  the  presence  or 
amount  of  color  cannot  in  every  single 
case  be  predicted  from  the  known  struc- 
ture. A  further  difficulty  is  that  in 
some  cases  the  structure  of  a  complex 
biological  material  (e.g.  hyaluronic 
acid)  is  inferred  from  the  chemical 
study  of  a  highly  purified  component 
isolated  from  only  one  or  two  special 
sources.  In  such  cases  the  spot  tests 
mentioned  maj-  be  used  to  investigate 
empirically  the  behavior  of  crude,  or 
purified,  fractions  from  the  tissue  under 
consideration. 

A  method  of  acetjdating  tissue  sec- 
tions has  been  described  (McManus, 
J.  F.  A.  and  Cason,  J.  E.,  J.  Exp.  Med., 
1950,  91,  651-654)  by  which  the  1,2- 
glycol  structures  are  blocked  with 
acetyl  groups  and  no  longer  are  able 
to  be  oxidized  by  periodic  acid.  De- 
acetylation  with  cold  alkali  introduces 
an  element  of  greater  specificity,  since 
glycol  substances  such  as  polj'saccha- 
rides  can  now  again  be  stained,  but  the 
amino  derivatives  of  glycols  are  prob- 
ably not  released. 

The  mechanism  of  the  Bauer-Feulgen 
and  Casella  reactions,  in  which  chromic 
acid  and  permanganate  are  the  oxidants 
is  probably  similar,  but  these  oxidants 
tend  to  destro}'-  the  aldehyde  groups 
which  they  first  produce.  Lillie  (loc. 
cit.  1951)  has  made  a  critical  study  of 
the  various  Schiff  reactions  for  carbo- 
hydrates and  should  be  consulted  for 
a  review  of  this  subject  (see  also  review 
by   McManus,   loc.    cit.    1948).     C.    P. 


POLYVINYL  ALCOHOL 


283 


PORPHYRINS 


Leblond  (Amer.  J.  Anat.  1950,  86,  1-50) 
has  surveyed  the  staining  of  rat  tissues 
rather  thoroughly.  A  discussion  of  the 
chemical  mechanism  and  tests  of  many 
isolated  substances  were  provided  by 
Hotchkiss  (loc.  cit.). 

Uses  and  appUcations:  The  following 
substances  have  been  stained  with  the 
periodic-acid-Schiff  methods:  glycogen, 
starch,  mucins,  hyaluronic  acid,  cell- 
ulose, chitin,  agar,  amyloid,  reticulin, 
kerasin,  colloid  of  the  thj'roid  and  hypo- 
physis, collagen.  Structures  or  regions 
revealed  have  included  basement  mem- 
branes, cartilage  matrix,  ground  sub- 
stance of  connective  tissue,  hyaline 
casts,  hyaline  of  arteriosclerosis,  ovar- 
ian cj'st  and  follicle  fluid,  lens  capsule, 
Descemet's  membrane,  collagenous  re- 
ticulum, chromaffin,  brush  border  of 
the  renal  tubules,  elements  in  the  mi- 
totic figures  of  tumor  tissue,  zymogen 
granules,  mold  mycelium,  flagella  in 
certain  algae,  plant  cell  walls,  sperm 
acrosomes.  The  stain  is  particularly 
convenient  for  demonstrating  vividly 
the  pathogenic  fungi  infecting  the  less 
intensely  staining  animal  tissues  (Klig- 
man,  A.  M.  and  Mescon,  H.,  J.  Bact., 
1950,  60,  415-421).  In  a  few  cases,  ma- 
terial stained  in  tissues  has  been  corre- 
lated, with  a  view  to  tentative  identifi- 
cation, with  such  specific  glycoproteins 
as  gonadotrophic  hormone  in  the  pitui- 
tary (Catchpole,  H.  R.,  Fed.  Proc. 
1947,  6,  88),  renin  in  the  kidney 
(Marshall,  J.  and  Wakerlin,  G.  E., 
Fed.  Proc.  1949,  8,  106-7),  accessory 
reproductive  gland  secretion  in  beetles 
(Anderson,  J.  M.,  Biol.  Bull.,  1950, 
99,  49-64),  and  hyaluronidase  in  the 
sperm  acrosome  (Leutchtenberger,  C. 
and  Schrader,  F.,  Proc.  Natl.  Acad. 
Sci.,  1950,  36,  677-683). 

Polyvinyl  Alcohol,  macromolecular  proper- 
ties (Heuper,  W.  C,  Arch.  Path., 
1942,  33,  271).  Use  in  preparing  tissues 
for  staining  with  Sudan  III  (Lubkin, 
V.  and  Carsten,  M.,  Science,  1942,  95, 
634). 

Ponceau  B,  see  Biebrich  Scarlet,  water 
soluble. 

Ponceau  R,  RG,  G,  4R,  2RE,  NR,  J,  FR, 
GR,  see  Ponceau  2R. 

Ponceau  2R  (CI,  79).— Brilliant  ponceau  G, 
lake  ponceau,  new  ponceau  4R,  ponceau 
R,  RG,  G,  4R,  2RE,  NR,  J,  FR,  GR, 
scarlet  R,  xylidine  ponceau  3RS. — 
An  acid  mono-azo  dye  which  may  be  the 
ponceau  de  xylidine  called  for  in 
Masson's   Trichrome   Stain. 

Ponceau  S  (CI,  282)  of  National  Aniline 
Division  of  Allied  Chemical  and  Dye 
Corporation  is  u.sed  by  Leach,  E.  II., 
Stain  Techn.,  1946,  21.  107-110  in  Cur- 
tis' Substitute  for  Van  Gieson  Stain. 


Ponceau  de  Xylidine.  The  difficulty  is 
that  the  Fench  "ponceau  de  xylidine" 
cannot  be  secured.  It  appears  to  be 
similar  to  ponceau  2R  (C.I.  79)  but  the 
latter  does  not  give  regularly  good  re- 
sults. Lillie  (R.  D.,  Stain  Tech.,  1940, 
15,  17-22)  suggests  the  following  sub- 
stitutes for  ponceau  de  xylidine:  azo- 
fuchsin  3B  (C.I.,  54),  nitrazine  yellow 
and  biebrich  scarlet  (C.I.,  280)".  See 
the  Biebrich  Scarlet  and  Picro-Anilin 
Blue  method  of  Lillie.  Sec  Masson's 
Trichrome. 

Ponder's  Stain  for  Diphtheria  Bacilli, 
which  see. 

Ponsol  Red  5  GK  (CI,  1131)  and  Ponsol  Red 
AFF,  both  of  DuPont  are  referred  to  by 
Emig,  p.  64. 

Pontachrome  Brown  MW  (CI,  101)  of  Du- 
Pont, a  monoazo  mordant  dye,  light 
fastness  4,  action  of  which  on  blue  green 
algae  is  described  (Emig,  p.  31). 

Pontachrome  Orange  R  (CI,  415)  of  DuPont, 
a  direct  disazo  dye  of  color  fastness  5. 
Gives  fugitive  colors  only  (Emig,  p.  40). 

Pontacyl  Carmine  6B  (CI,  57),  DuPont,  is  an 
acid,  monoazo  dye  which  colors  sections 
bluish  fuchsia  darkened  by  mordanting 
with  potassium  bichromate.  Not  im- 
portant in  microtechnique  (Emig, 
p.  30). 

Pontacyl  Carmine  2  G  (CI,  31)— Made  by 
DuPont.  Light  fastness  3.  More  in- 
tense color  than  azofuchsin.  Action  on 
fungous   myoelia    (Emig,   p.   29). 

Pontamine  Fast  Pink  BL  (CI,  353),  a  disazo 
direct  dye  of  light  fastness  3  to  4.  Use 
in  acid  and  alkaline  solutions  as  stain 
for  plant  tissues  and  algae  are  described 
(Emig,  p.  39). 

Pontamine  Sky  Blue  5BX,  see  Niagara  Blue 
4B.  Use  in  measurement  of  lymph  flow 
(McMaster,  P.  D.,  J.  Exper.  Med.,  1937, 
65,  373-392) . 

Poppy  Seed  Oil,  reactions  in  tissue  to  fat 
stains  after  various  fixations  (Black, 
C.  E.,  J.  Lab.  &  Clin.  Med.,  1937-38, 
23,  1027-1036). 

Porphyrins. — Written  by  Frank  H.  J.  Figge, 
Dept.  of  Anatomy,  Universitj'  of  Marj'- 
land  Medical  School,  Baltimore,  Md. 
October  10,  1951 — There  is  no  specific 
histo-chemical  reaction  for  porphyrins, 
but  Watson,  C.  J.  and  Clark,  W.  O., 
Proc.  Soc.  Exp.  Biol.  &  Med.,  1937, 
36,  65-70  believe  that  it  is  the  proto- 
porphyrin in  reticulocytes  that  stains 
with  brilliant  cresyl  blue.  See  Reticu- 
locytes. 

Watson  and  his  workers  and  other  in- 
vestigators have  more  recently  modified 
their  views  regarding  this  point  (Wat- 
son, C.  J.,  Blood,  1946,  1,  99-120) 
Keller  and  Seggel  had  demonstrated 
that  the  porphyrin  containing  cells 
were   not   identical   with   reticulocytes 


PORPHYRINS 


284 


PORPHYRINS 


but  could  be  recognized  by  their  fluores- 
cence. These  fluorescytes  normally 
constitute  only  0.1%  of  the  red  cells 
(Keller,  Ch.  J.  and  K.  A.  Seggel,  Folia 
Haematol,  52,  241,  1934).  No  paral- 
lellism  was  found  to  exist  between  the 
reticulocytosis  in  cases  of  remission  of 
pernicious  anemia  and  the  concentra- 
tion of  porphyrin  in  the  erythrocytes, 
which  reaches  a  ma.ximum  after  the 
reticulocyte  peak.  Watson,  Grinstein, 
and  Hawkinson  have  confirmed  this 
latter  observation  (J.  Clin.  Invest., 
1944,  23,  69-80).  A  very  comprehen- 
sive review  describing  the  distribution 
of  porphyrins  in  the  human  body  ap- 
peared in  a  book  by  Lemberg  and 
Legge,  "Hematin  Compounds  and  Bile 
Pigments"  (Interscience  Publishers, 
1949). 

Minute  quantities  of  porphyrins  may 
be  detected  in  tissues  or  solutions  by 
virtue  of  the  red  fluorescence  of  these 
substances  when  they  are  examined  in 
near  ultraviolet  light  (Wood's  light). 
Konigsdorfer,  Borst,  and  Fischer  em- 
ployed a  spectral  analysis  microscope 
to  detect  and  identify  porphyrins  in 
histological  material  (See  Fischer  and 
Orth's  Die  Chemie  des  Pyrrols,  1937, 
press  of  Paul  Dunhaupt,  Kothen.  It  is 
also  available  in  Lithoprint  form:  Ed- 
wards Bros.,  Ann  Arbor,  Mich.).  At- 
tempts have  been  made,  Kliiver,  H., 
Science,  1944,  99,  482-484,  to  identify 
the  type  of  porphyrin  present  in  tissues 
and  in  nervous  tissue  by  means  of 
fluorescence  spectra  determination. 
The  precise  identification  and  deter- 
mination of  porphyrins  involves  deter- 
mination of  relative  solubility  in  ether 
and  in  acid  solutions  of  various  concen- 
trations, absorption  spectra,  and  melt- 
ing points  of  the  methylesters. 

The  detection  of  porphj^rins  in  tissues 
by  means  of  the  visually  observed  red 
fluorescence  is  beset  with  several  pit- 
falls. Red  fluorescence  is  not  a  specific 
test,  because  occasionally  other  nat- 
urally occurring  red  fluorescent  sub- 
stances are  encountered.  The  red 
fluorescence  of  porphyrins  may  also  be 
masked  in  at  least  two  ways: 

1.  The  presence  of  certain  substances 
which  quench  the  fluorescence  of  the 
porphyrin,  i.e.,  protoporphyrin  and 
coproporphyrin  are  abundant  in  bone 
marrow,  but  the  fluoresence  is  not  ap- 
parent because  of  the  high  concentra- 
tion of  heme  compounds  and  other 
forms  of  iron. 

2.  The  presence  of  a  substance  or  sub- 
stances with  a  blue-green  or  in  other 
words,  a  complimentary  fluorescence 
spectrum.  As  one  would  expect,  por- 
phyrin in  such  a  combination  gives  rise 


to  a  white  fluorescence,  i.e.,  urine  us- 
ually contains  substances  which  flu- 
oresce blue-green.  The  addition  of 
porphyrin  changes  this  to  white  fluores- 
cent urine.  Urine  fluoresces  red  only 
when  the  concentration  of  porphyrin  is 
very  high. 

For  an  excellent  account  of  the  chem- 
istry and  distribution  of  porphyrins  in 
tissues  and  organs,  the  reader  is  referred 
to  the  review  of  Dobriner,  K.,  and 
Rhoads,  C.  P.,  Physiol.  Rev.,  1940,  20, 
416-468.  Everett's  Medical  Biochem- 
istry (1942,  Paul  B.  Hoeber,  New  York) 
also  contains  a  good  summary  of  this 
field.  In  the  following  discussion,  some 
of  the  original  references  to  statements 
regarding  porphyrins  have  been 
omitted.  These  may  be  found  in  one 
of  the  above  reviews  or  in  Fischer  and 
Orth.  Most  of  the  porphyrins  en- 
countered in  nature  may  be  classified 
as  type  III  or  type  I  of  the  four  series 
of  isomers.  This  is  because  proto- 
porphyrin, which  belongs  to  the  type 
III  series,  is  involved  in  the  formation 
of  such  important  substances  as  chloro- 
phyl,  hemoglobin,  myoglobin,  cyto- 
chromes, catalase,  peroxidase,  and 
cytochrome  oxidase.  Protoporphyrin 
(and  a  small  amount  of  coproporphyrin) 
are  usually  formed  during  the  synthesis, 
but  as  a  general  rule,  porphyrin  is  not 
formed  during  the  breakdown  of  these 
compounds  in  the  liver. 

Intestinal  bacteria  convert  many  of 
these  heme  compounds  to  protopor- 
phyrin. Deuteroporphyrin,  copropor- 
Ehyrin  III,  and  mesoporphyrin  may  all 
e  derived  from  this.  These  same  por- 
phyrins may  also  result  from  the  sterile 
autolysis  of  hemoglobin  or  myoglobin 
(Hoagland,  R.,  J.  Agr.  Res.,  1916,  7, 
41-45).  It  is,  therefore,  probable  that 
these  pigments  would  be  present  in 
thrombotic  areas,  severely  damaged 
tissues,  and  necrotic  tissues  in  general. 
Hematoporphyrin  is  an  artificial  por- 
phyrin resulting  from  the  treatment  of 
reduced  hemoglobin  with  strong  acids. 
Since  it  does  not  occur  in  nature,  the 
name  is  unfortunate  and  has  given  rise 
to  much  confusion  (see  "Hematopor- 
phyrin"). 

Normally  20-100  micrograms  of  co- 
proporphyrin I  are  excreted  daily  in  the 
urine.  Coproporphj'rin,  as  its  name 
implies,  is  present  in  large  amounts  in 
the  feces,  but  is  also  found  in  the  am- 
niotic fluid,  meconium,  and  in  the 
sebaceous  glands  in  certain  areas  of  the 
skin  of  the  human  subject  (Fischer- 
Orth;  Figge,  Symposium  on  Cancer, 
A.  A.  A.  S.,  1945,  117-128).  In  certain 
pathological  states,  large  amounts  of 
the  ether  insoluble  uroporphyrins  are 


POSTMITOTIC  CELLS 


285 


POTASSIUM 


excreted  in  the  urine.  Protoporphyrin, 
which  is  now  known  to  be  the  same  as 
ooporphyrin,  is  excreted  in  relatively 
large  amounts  by  female  birds.  A 
porphyrin-secreting  gland  deposits  this 
on  the  egg  shell  as  it  passes  through  the 
oviduct.  The  purpose  of  this  is  not 
known.  Protoporphyrin  and  copropor- 
phyrin  develop  in  abundance  in  eggs  as 
they  are  incubated  and  embryonic  tis- 
sues and  fluids  in  general  have  a  rela- 
tively high  porphyrin  content.  Graf- 
lin,  A.  L.,  Am.  J.  Anat.,  1942,  71,  43-64 
gives  the  technic  for  histochemical 
studies  of  the  protoporphyrin  in  rat 
harderian  glands.  This  includes  sev- 
eral good  illustrations.  These  glands 
excrete  porphyrins  which  pass  via  the 
naso -lachrymal  duct  and  larynx  to  the 
gastro-intestinal  tract  (Figge  and  Salo- 
mon, J.  Lab.  &  Clin.  Med.,  1942,  27, 
1495-1501).  Most  of  the  porphyrin  in 
the  feces  of  rats  is  derived  from  the 
harderian  gland  excretions.  In  addi- 
tion to  rats,  mice  also  excrete  relatively 
large  amounts  of  protoporphyrin  via 
the  harderian  glands.  The  variability 
with  respect  to  the  red  fluorescence  of 
the  harderian  glands  of  mice  of  strains 
with  different  susceptibility  to  spon- 
taneous mammary  carcinoma  gave  rise 
to  the  hypothesis  that  porphyrins  were 
involved  in  the  regulation  of  suscepti- 
bility to  mammary  carcinoma  (Figge, 
Strong,  Strong,  Jr.,  and  Shanbrom, 
Cancer  Res.  1942,  2,  335-342).  Ham- 
sters, which  are  very  susceptible  to 
chemically-induced  tumors,  were  also 
found  to  have  brilliant  red  fluorescent 
harderian  glands.  The  occurrence  of 
porphyrins  in  certain  organs  and  tissues 
of  the  human  subject  which  exhibit  a 
high  cancer  incidence  (cervix  of  uterus, 
skin,  etc.)  led  to  the  concept  that  these 
substances  may  act  as  co-carcinogens  in 
a  more  general  manner  than  postulated 
at  first  (Figge,  A.  A.  A.  S.,  1945,  117- 
128).  Jones,  E.  G.,  Shaw,  H.  N.,  and 
Figge,  F.  H.  J.,  Am.  J.  Obs.  &  Gyn., 
1946,  51,  467-479  give  technics  for 
demonstrating  porphyrin  on  the  cervix 
of  the  uterus  in  the  human  subject. 
See  Hematoporphyrin. 

Postmitotic  Cells,  see  Cell  Classification. 

Postmortem  Change.  These  are  alterations 
in  structure  due  to  autolytic  and  os- 
motic changes.  The  rate  of  autolysis 
is  very  rapid  in  some  organs  such  as  the 
pancreas  which  are  enzyme  producers. 
It  is  relatively  slow  in  the  walls  of  elas- 
tic arteries  in  which  the  proportion  of 
inanimate  components  (elastic  and  col- 
lagenic  fibers)  is  high.  In  the  case  of 
tissues  which  cannot  be  immediately 
fixed  certain  precautions  should  be 
taken  to  minimize  postmortem  change. 


See  Agonal  Changes,  Artifacts,  Fixa- 
tion, and  Small  Intestine,  Necrosis, 
Necrobiosis. 

Potocytosis,  a  term  introduced  by  Meltzer 
to  designate  submicroscopic  "sipping" 
of  fluid  by  cells.     See  Pinocytosis. 

Pottenger's  Dilution  Flotation  method,  see 
Concentration  of  bacteria. 

Potassium,  Histochemical  methods. 

1.  Policard,  A.  and  Fillet,  D.,  BuU- 
d'Hist.  Appl.,  1926, 3,  230-235  have  sug- 
gested that  potassium  and  sodium  prob- 
ably occur  as  chlorides  and  that  their 
conversion  to  sulphates  by  treating  the 
sections  with  sulphuric  anhydride  fumes 
makes  them  more  stable  and  better  able 
to  withstand  the  high  temperature  of 
Microincineration  which  see. 

2.  Marza,  V.  D.,  Bull.  d'Hist.  Appl., 
1935,  13,  62-71  has  modified  Macallum's 
well  known  technique.  Fix  small  pieces 
of  tissue  in  96%  pure  ale.  in  the  ice  box. 
Make  paraffin  sections.  To  eliminate 
the  possibility  of  the  presence  of  iron 
leave  {control  sections  5  min.in  freshly 
prepared  sol .  yellow  ammonium  sulphate . 
Wash  in  aq.  dest.,  dehydrate,  clear  and 
mount  in  neutral  balsam.  There  should 
be  no  ppt.  Make  up  following  solu- 
tions: A.  Cobalt  nitrate,  5  gm.;  aq. 
dest.,  10  cc;  glacial  acetic  acid,  2.5  cc. 
B.  Sodium  nitrite,  25  gm.;  aq.  dest.,  36 
gm.  To  A  add  41  cc.  of  B  and  use 
immediately.  If  delay  is  necessary 
keep  in  ice  box  and  filter  before  using. 
Cover  test  sections  with  this  for  2 
hrs.  in  a  closed  Petri  dish  to  avoid 
evaporation.  Wash  slowly  in  50%  ale. 
to  remove  every  trace  of  reagent. 
Plunge  in  ammonium  sulphate  solution 
3  min.  Wash  in  aq.  dest.  to  remove 
ammonium  sulphate.  Dehydrate,  clear 
and  mount.  Examine  illustrated  paper 
by  Marza  and  Chiosa  (V.  D.  and  L.  T., 
Bull.  d'Hist.  Appl.,  1935,  13,  153-177) 
on  application  of  this  method  to  the 
problem  of  ovogenesis. 

3.  Gersh,  I.,  Anat.  Rec,  1938,  70, 
311-329  has  also  modified  Macallum's 
method.  It  involves  the  making  of 
similar  paraffin  sections  as  for  Chloride, 
which  see.  Transfer  these  to  a  fairly 
large  cool  room  (—1°  to  -f  1°C.)  and  re- 
move paraffin  and  petroleum  ether  as 
for  chloride.  Cover  with  12%  sodium 
cobalti-nitrite  solution  of  Biilman 
(Treadwell,  F.  P.,  Analytical  Chemis- 
try, vol.  1,  4th  English  Ed.  translated 
by  W.  T.  Hall,  New  York,  John  Wiley 
&  Sons,  Inc.,  1916,  p.  81).  Decant 
fluid,  mount  in  glycerin  in  same  way  and 
examine.  Crystals  of  sodium  potassium 
cobalti-nitrite  are  just  visible  with  oil 
immersion  lens.  They  are  short  yellow 
rods  with  rounded  ends  in  a  diffuse  pale 


PRATT 


286 


PROSTATE 


yellow  background  soluble  at  room  tem- 
perature. 

4.  Carer-Comes,  O.,  Zeit.  f.  wis. 
Mikr.,  1938,  55,  1-6  has  advised  histo- 
chemical  demonstration  of  potassium 
by  Siena  orange  (K.  Hollborn),  which  is 
sodium  paradipicrylamine.  Deparaf- 
finize  sections  of  neutral  formalin  fixed 
tissue.  Place  in  Siena  orange  solution, 
as  received  ready  for  use  from  Kollborn, 
2  min.  Then  10%  HCl  3  min.  Wash 
twice  in  aq.  dest.  10  min.  Blot  with 
filter  paper  and  dry  at  ST^C.  Mount  in 
thickened  cedar  oil.  Tissues  contain- 
ing potassium,  orange;  others,  pale  yel- 
low or  unstained. 

5.  Radioactive  potassium  can  be 
easily  measured  in  tissues  and  cells. 
There  is  40%  penetration  of  red  blood 
cells  in  vivo  (Mullins,  L.  J.,  Noonan, 
W.  O.  and  T.  R.  and  Halge,  L.,  Am.  J. 
Physiol.,  (1941,  135,  93-101).  See 
Radiopotassium. 

Titrimetric  methods  for  potassium. 
The  one  of  Norberg,  B.  (C.  rend.  trav. 
lab.  Carlsberg,  S6r.  Chim.,  1937,  2], 
233-241)  is  given  by  Click,  p.  268.  Ap- 
parently the  presence  of  sodium  in 
amounts  150  times  that  of  the  potassium 
did  not  interfere.  The  one  of  Cunning- 
ham, B.,  Kirk,  P.  L.  and  Brooks,  S.  C. 
(J.  Biol.  Chem.,  1941,  139,  21-28)  is 
effective  when  amount  of  sodium  is  not 
more  than  20  times  that  of  potassium 
and  the  quantity  of  potassium  is  2  /x 
gm.  +. 

Pratt,   see   Triphenyltetrazolium   Chloride. 

Preputial  Gland  of  rats.  Useful  histochemi- 
cal  methods  of  investigation  and 
changes  following  thyroidectomy  (Mon- 
tagna,  W.,  Anat.  Rec,  1946,  94,  38). 

Pressure.  Increase  in  pressure  beyond  a 
certain  limit,  somewhat  characteristic 
for  particular  cells  (300-1000  atmos- 
pheres), brings  about  a  liquefaction  of 
the  plasmagel  which  can  be  directly 
observed  microscopically  or  determined 
by  certain  measurements  like  action 
potential  for  nerve  fibers.  Danielli 
(Bourne,  p.  38)  has  expressed  the 
opinion  that  the  factor  causing  in- 
hibition of  movement  may,  in  all  cases, 
be  increased  hydration  of  protein 
molecules  and  that  the  method  of  in- 
creased pressure  may  be  of  great  value 
to  large  scale  and  micro-biologists. 

New  technique  for  differential  pres- 
sure measurements  employing  con- 
denser manometers  as  given  by  Hansen, 
A.  T.  and  Warburg,  E.,  Acta  Physiol. 
Scand.,  22,  211-215. 

Price-Carr  Reaction,  see  Carr-Price  Reac- 
tion. 

Primula  R  Water  Soluble,  see  Hofmann's 
Violet. 

Primulin  (CI,  812) — primuline  yellow — An 


acid  thiazole  dye  used  in  fluorescence 
microscopy  (Pick,  J.,  Zeit.  Wis.  Mikr., 
1935,  51,  338-351). 

Praseodymium,  see  Atomic  Weights. 

Primuline  Yellow,  see  Primulin. 

Proflavine,  a  dye  similar  to  Acrlflavine. 

Prolactan.  Methods  for  assay  (Bates,  R. 
W.,  Cold  Spring  Harbor  Symposium  on 
Quantitative  Biol.,  1937,  5,  191-197). 

Promyelocytes,  see  Leucocytes,  develop- 
mental series. 

Prontosil  as  a  vital  dye  (Carter,  W.,  Science, 
1939,   90,   394).     It   is   fluorescent. 

Propylcarbinol,  see  n-Butyl  Alcohol. 

Prostate.  This  organ  cannot  be  examined 
microscopically  in  vivo  and  supravital 
staining  has  not  proved  very  fruitful. 
The  cutting  and  staining  of  sections  is 
the  conventional  method.  It  is  impor- 
tant that  the  blocks  of  tissue  fixed  be 
oriented  with  great  care,  and  that 
microscopic  and  gross  observations  be 
correlated.  For  normal  size  and  weight 
see  Moore,  R.  A.,  Am.  J.  Path.,  1936, 
12,  599-624  and  for  age  changes  a  chap- 
ter by  the  same  author  in  Cowdry's 
Problems  of  Ageing,  Baltimore :  Wil- 
liams &  Wilkins,  1942,  936  pp.  Since 
the  structure  of  the  prostate  exhibits 
so  many  local  differences  there  is  a 
danger  of  erroneous  conclusions  from 
incomplete  examination.  In  their  clas- 
sic paper  on  the  rat-prostate  cytology 
as  testis  hormone  indicator  Moore,  C. 
R.,  Price,  D.  and  Gallagher,  T.  F.,  Am. 
J.  Anat.,  1930,  45,  71-107  secured  best 
results  after  fixation  in  Bouin's  Fluid 
and  staining  with  Harris'  Hematoxylin 
and  Eosin. 

Swyer,  G.  I.  M.,  Cancer  Research, 
1942,  2,  372-375  has  checked  with  satis- 
factory results  the  Schultz  test  for  cho- 
lesterol by  chemical  analyses.  He  has 
also  outlined  a  method  for  measuring 
the  color  in  the  Liebermann-Burchardt 
reaction.  For  singly  refractile  fat  in 
the  epithelial  cells  see  Gylling,  P., 
Acta  Path,  et  Microb.  Scan.,  1941,  18, 
247-258. 

To  demonstrate  the  ducts  (Le  Due, 
I.  E.,  J.  Urol.,  1939,  42,  1217-1241)  in 
autopsy  material  lay  open  prostate  by 
incising  length  of  anterior  commissure 
and  express  secretion  from  ducts  by 
gentle  massage  and  careful  sponging. 
Locate  orifices  of  ducts  with  aid  of  a 
dissecting  microscope.  Inject  celloidin 
solution  into  them  through  No.  26  or  27 
gauge  hypodermic  needle  fitted  with 
tapering  solder  tip.  Then  macerate 
with  hydrochloric  acid  and  remove  all 
except  casts  of  the  ducts.  See  his 
illustrations. 

A  method  for  demonstrating  arterial 
supply  is  described  and  illustrated  in 
some  detail  by  Flocks,  R.  H.,  J.  Urol., 


PROTACTINIUM 


287 


PROTOSIDERIN 


37,  524-548.  Inject  internal  iliac  ar- 
teries of  a  fresh  cadaver  with  equal 
parts  barium  sulphate  and  water  at  200- 
250  mm.  mercury  pressure.  But  be- 
forehand cut  small  branch  of  superior 
vesical  artery  to  relieve  pressure  in 
prostatic  vessels.  Remove  prostate 
with  sufficient  surrounding  tissue.  Cut 
gland  into  5-6  sections  each  about  1  cm. 
thick.  Dehydrate  in  ascending  alco- 
hols and  clear  in  oil  of  wintergreen 
(methyl  salicylate). 

Examination  of  corpora  amylacea  by 
various  methods  is  described  by  Moore, 
R.  A.,  Arch.  Path.,  1936,  22,  24-40. 

Protactinium,  see  Atomic  Weights. 

Protamines,  see  Saint  Hilaires  Method 
discussed  under  Purines. 

Protargol.  This  is  a  light  brown  protein 
silver  compound  containing  approxi- 
mately 8%  silver.  To  demonstrate 
phagocytosis  by  the  reticulo-endothelial 
system  fine  suspensions  may  be  injected 
intravenously  (Askanazy,  M.,  Aschoff 
Path.  Anat.,  Jena,  1923,  1,  183)  but  the 
method  is  not  recommended  by  Foot 
(McClung,  p.  115).  Protargol  is  also 
used  for  staining  of  paraffin  sections 
(Bank,  E.  W.  and  Davenport,  H.  A., 
Stain  Techn.,  1940,  15,  9-14).  See 
Silver  Methods,  Bodian  Method,  Pro- 
tein Silver. 

Protease.  An  enzyme  located  in  leucocytes 
which  can  be  demonstrated  in  small 
quantities  of  blood  has  been  described 
(Cooke,  J.  v..  Arch.  Int.  Med.,  1932, 
49,  836-845).  Pickford,  G.  E.  and  F. 
Dorris  (Science,  1934,  80,  317-319)  have 
reported  a  micromethod  for  protease. 
DeRobertis,  E.  (Ann.  N.  Y.  Acad.  Sci., 
1949,  50, 317-335)  devised  micro  methods 
for  the  analysis  of  proteolytic  activity 
in  thyroid  colloid.  Sections  were  in- 
cubated on  plates  covered  with  gelatin, 
which  was  subsequently  stained.  Pro- 
teolytic activity  digested  the  gelatin, 
(pausing  the  film  to  stain  less  intensely 
than  neighboring  undigested  areas. 
Similar  experiments  for  localizing  pro- 
tease have  been  carried  out  using  fibrin 
film  as  a  substrate. 

Protein,  see  following  reactions:  Alloxan, 
Axenfeld,  Azo,  Indo,  Ninhydrin,  Nitro, 
Nitroprusside,  Nitrosamino,  Romieu, 
Xanthroproteic. 

Proteolytic  enzymes.  These  include  pro- 
tease and  many  others.  See  Click, 
pp.  302-306. 

Protein  Silver  for  Staining  Protozoa — 
Written  by  Norman  Moskowitz,  Dept. 
of  Zoology,  University  of  Pennsylvania, 
Philadelphia.  January  24,  1951 — When 
commercially  prepared  silver  products 
suitable  for  staining  Protozoa  by  the 
Bodian  silver  technic  apparently  be- 
came   unavailable,     a    substitute    for 


Protargol  was  prepared  as  follows:  0.9 
g.  of  granular  gelatin  is  dissolved  by 
heat  in  100  ml.  aq.  dcst.;  to  this  0.1  g. 
of  silver  nitrate  is  added  at  60°  C. ;  this 
solution  is  poured  into  Columbia  stain- 
ing dishes  (10  ml.)  in  which  one  or  two 
drops  of  M/10  sodium  hydroxide  have 
been  added.  Copper  is  not  used  in  the 
impregnating  bath.  Smears  fixed  in 
Hollande's  or  Schaudinn's  fixatives  are 
bleached  and  impregnated  for  36  hours 
or  more  at  35°C.  Impregnated  smears 
are  reduced  with  a  mixture  of  hydro- 
quinone  and  sodium  sulfite,  and  toned 
with  gold  chloride. 

1.  Fix  smears  in  the  recommended 
fixative. 

2.  Wash  in  50%  ale.  to  remove  fixative. 

3.  Harden  the  smears  in  70%  ale.  in 
which  they  may  be  stored  for  not 
more  than  a  few  days. 

4.  Hydrate  smears. 

5.  Bleach:  Treat  preparations  in  0.5% 
potassium  permanganate  2  min. 
Wash  in  at  least  two  changes  of  aq. 
dest.,  1  min.  each.  Treat  with  5% 
oxalic  acid  2  min.  Wash  in  three 
changes  aq.  dest. 

6.  Impregnate  smears  in  protein  silver 
solution  for  36  hours  at  approx- 
imately 35°C.  Results  in  staining 
can  be  varied  by  omitting  the  M/10 
sodium  hydroxide  or  by  adding  one 
or  two  drops  of  M/10  nitric  acid 
instead  of  the  recommended  alkali. 

7.  Reduction  of  silver.  Rinse  im- 
pregnated smears  in  aq.  dest.  water 
and  treat  for  5  minutes  in  a  reducing 
bath  prepared  by  dissolving  1  g. 
of  hydroquinone  and  5  g.  of  sodium 
sulfite  in  100  ml.  aq.  dest. 

8.  Wash  in  aq.  dest.  1  min. 

9.  Toning.  Immerse  preparations  for 
4  min.  in  0.2%  (1%  may  be  used) 
aq.  yellow  gold  chloride.  Staining 
is  affected  by  the  duration  of  the 
gold  toning. 

10.  Rinse  in  aq.  dest. 

11.  Treat  with  2%  oxalic  acid  for  3 
min.  Variation  of  time  in  oxalic 
acid  produces  staining  differences. 

12.  Wash  1  min.  each  in  of  3  changes 
aq.  dest. 

13.  Treat  with  5%  sodium  thiosulfate 
for  7-8  min. 

14.  Wash  in  running  tap  water  for  ^  hr. 
or  more. 

15.  Dehydrate  and  mount.  The  im- 
pregnating medium  should  not  be 
exposed  to  intense  daylight  at  any 
time. 

Proteinase,  determinations  (Maver,  M.  E., 
Mider,  G.  B.,  Johnson,  J.  M.  ana 
Thomp.son,  J.  W.,  J.  Nat.  Cancer  Inst., 
1941,  2,  278). 

Protoslderin,   see   Lillie,  p.    127. 


PROTHROMBIN 


288 


PROTOZOA.    MEDIA 


Prothrombin,  rapid  micro  test  (Abramson, 
D.  J.  and  Weinstein,  J.  J.,  AJn.  J.  Clin. 
Path.  Technical  Suppl.,  1942,  6,  1-7) : 

1.  Make  M/40  calcium  chloride  by 
dissolving  1.11  gms.  anhydrous  calcium 
chloride  C.P.  in  400  cc.  aq.  dest. 

2.  Make  thromboplastin  suspension 
from  brain  freshly  killed  rabbit  as  de- 
scribed by  Quick,  A.  J.  Am.  J.  Clin. 
Path.,  1940,  10,  222.  Dehydrate  macer- 
ated brain  in  acetone,  dry  completely, 
mix  with  normal  saline  (0.3  gm.  to  5 
cc.)  and  incubate  at  50°C.  15  min.  The 
supernatant  turbid  fluid  is  thromboplas- 
tin. It  must  be  kept  in  ice  box  when 
not  in  use. 

3.  Measure  separately  in  micro- 
hemopipettes  10  cc.  of  calcium  chloride 
sol.,   of   thromboplastin   and  of  blood. 

4.  After  adding  blood,  mix  thor- 
oughly with  fine  glass  rod,  tilt  gently 
from  side  to  side  until  gelation  begins, 
then  time  end  point  by  passing  rod 
through  mass. 

Prothrombin  time  (Sherber,  D.  A., 
J.  Lab.  &  Clin.  Med.,  1940,  26,  1058- 
1061;  and  Isenberg,  H.  D.,  J.  Lab.  & 
Clin.  Med.,  1951,  37,  807-809). 

Protoporphyrin  in  Harderian  glands,  see 
Porphyrins. 

Protozoa,  staining  in  bulk.  (Stone,  W.  S., 
J.  Lab.  &  Clin.  Med.,  1935-36,  21,  839- 
842) :  Suggested  for  mucous  surface 
protozoa  of  man  and  used  at  Army 
Medical  School.  Thoroughly  emulsify 
20  cc.  feces  in  200  cc.  37°C.  physiological 
saline  solution.  Allow  to  stand  for  5 
min.  and  pour  supernatant  fluid  into 
two  50  cc.  centrifuge  tubes.  Centri- 
fuge at  1,850  r.p.m.  5  min.  Decant 
supernatant  fluids.  Examine  residue 
from  one,  fresh,  and  to  other  add  25  cc. 
Schaudinn's  Fixative.  Mix  and  leave 
24  hrs.  Protozoa  in  cultures  and  other 
fluids  are  to  be  concentrated  by  centri- 
fugation  and  fixed  in  the  same  way. 
Between  each  of  following  steps  centri- 
fuge organisms  and  discard  supernatant 
fluid  before  adding  the  next.  Wash 
twice  in  aq.  dest.  Wash  with  70%  al- 
cohol plus  sufficient  Gram's  iodine  to 
make  it  light  brown  color,  10  min. 
Wash  70%  alcohol  10  min.  Stain 
Harris'  Hematoxylin  1-24  hrs.  Wash 
tap  water.  Destain  by  adding  20  cc. 
acid  alcohol  (1%  HCl  in  70%)  controlled 
by  microscope.  When  desired  defini- 
tion is  reached  add  sufficient  ammonia 
water  (6  drops  NH4OH  to  60  cc.  aq. 
dest.)  to  neutralize  acid  and  give 
bright  blue  solution.  Wash  in  tap 
water.  Dehydrate  10  mins.  in  each  of 
5  alcohols:  70,  95,  95,  abs.,  and  abs. 
Clear  in  xylol.  Mount  in  balsam.  See 
author's  figures. 

Perhaps  the  best  method  for  concen- 


trating and  sectioning  protozoa  is  that 
of  Lucas,  M.  S.,  Science,  1929,  70,  482- 
483.  Use  a  round  bottom  vial.  Let 
protozoa  settle  to  bottom,  pipette  off 
fluid  to  within  4  mm.  of  level  of  top  of 
protozoan  mass,  then  add  dilute  alco- 
hol. Next  change,  pipette  off,  and  add 
stronger  alcohol.  Alcohol,  xylol,  pure 
xylol,  melted  paraffin  (the  vial  being 
held  under  an  electric  bulb,  etc.)  sev- 
eral changes  of  each.  Finally  lower 
protozoa  with  as  little  paraffin  as 
possible  into  a  specially  prepared  paper 
tray  and  harden. 

Levine  W.  D.,  Stain  Techn.,  1939, 
14,  29-30  suggests  following  method  to 
make  Methylene  Blue  stains  perma- 
nent :  Wash  methylene  blue  stained 
smears  of  protozoa  repeatedly  in  aq. 
dest.  15  min.  to  1  hr.  Place  in  tertiary 
butyl  alcohol  1-2  min.  then  in  3  or  more 
changes  15  min.  each.  Pass  through 
xylol  to  balsam  or  mount  directly  in 
balsam.  Other  dyes  like  toluidin  blue 
0,  nile  blue  sulfate,  eosin  Y,  ponceau  2R 
can  likewise  be  retained. 

The  protargol  method  of  Bodian  has 
been  adjusted  to  protozoa  by  Cole,  R.  M. 
and  Day,  M.  F.,  J.  Parasitology,  1940, 
26  Suppl.  29.  See  also  Parasites, 
Endamoeba  Leishmania,  Leucocyto- 
zoa,  Malaria,  Intestinal  Protozoa.  Wen- 
yon,  C.  M.,  Protozoology.  New  York: 
William  Wood,  1926,  1563  pp.  is  a  con- 
venient book  of  reference.  It  gives  a 
fine  list  of  blood  protozoa.  No  investi- 
gator can  afford  to  ignore  the  discussion 
by  Wenrich,  D.  H.,  J.  Parasitol.,  1941, 
27,  1-28  of  alterations  in  the  form  of 
protozoa  resulting  from  variations  in 
microtechnique. 

Protozoa  can  be  beautifully  demon- 
strated by  fluorochromes  showing  in 
ultraviolet  light  various  fluorescent 
colors  (Metcalf,  R.  L.  and  Patton,  R. 
L.,  Stain  Techn.,  1944,  19,  11-27). 

Obviously  the  investigation  of  proto- 
zoa extends  far  beyond  their  identifica- 
tion in  preparations  made  by  various 
methods.  Those  dealing  with  patho- 
genic protozoa  will  greatly  extend  their 
horizon  by  consideration  of  the  form 
and  function  of  these  organisms  and  the 
ingenious  techniques  of  investigation 
ably  presented  in  a  volume  entitled 
Protozoa  in  Biological  Research  edited 
by  Calkins,  G.  N.  and  Summers,  F.  M., 
New  York:  Columbia  University  Press, 
1941,  1148  pp.  See  Intestinal  Protozoa, 
Protein  Silver. 
Protozoa.  Media.  The  following  are  rec- 
ommended for  intestinal  protozoa  b}'  Q. 
M.  Geiman  (Simmons  and  Gentzkow, 
617-619): 

1.  Modification  of  Cleveland's  and 
Sanders'  (for  E.  histolytica).     (1)  Dis- 


PRUSSIAN  BLUE 


289 


PURINES 


solve  33  gm.  Bacto-Entamoeba  medium 
(Difco)  in  1000  cc.  aq.  dest.  Pour  in 
test  tubes  in  amounts  sufficient  to  make 
medium  length  slants  with  no  butts. 
Autoclave,  slant,  harden  at  room  tem- 
perature several  days.  (2)  Place  few 
gm.  Bacto-Rice-Starch  powder  (Difco) 
in  IS  X  150  mm.  culture  tube  and  steril- 
ize with  tube  horizontal  in  hot  air  oven 
160-180°C.  1  hr.  Repeat  twice  at  daily 
intervals  being  careful  to  avoid  chemi- 
cal changes  in  the  starch  occasioned 
by  higher  temperatures.  (3)  Dissolve 
11.23  gm.  NasHPO*  I2H2O  +  0.269  gm. 
KH2PO4  +  8.0  gm.  NaCl  in  aq.  dest. 
to  make  1000  cc,  autoclave  15  lbs.  20 
min.  and  cool.  Add  10  parts  above 
solution  to  1  part  sterile  horse  serum. 
Cover  f  of  each  slant  with  this  mixture, 
add  2-3  loopfuls  of  the  sterile  starch, 
incubate  37°C.  24  hrs.  to  prove  sterility. 
Final  pH  should  be  7-7.2.  Store  in 
refrigerator  till  used. 

2.  Boeck  and  Drbohlav's.  Wash  6 
eggs  in  70%  alcohol  and  emulsify  con- 
tents in  75  cc.  sterile  Locke  or  Ringer. 
Distribute  in  4  cc.  lots  in  15  X  150  mm. 
culture  tubes,  slant  in  inspissator  and 
heat  70°C.  till  mixture  solidifies,  then 
autoclave  15  lbs.,  20  min.  Slant  tubes 
in  autoclave,  close  doors  and  ports,  turn 
in  steam  increasing  quickly  to  15  lbs., 
for  10  min.  Through  lower  port  run  in 
live  steam  in  place  of  steam-air  mixture 
maintaining  constant  15  lbs.  pressure. 
After  replacement  by  steam  close  lower 
port  and  hold  15  lbs.  another  15  min. 
Cut  off  steam  and  let  cool  slowly. 
Cover  each  slant  with  4  cc.  10: 1  Ringer- 
horse  serum  mixture  +  2  or  3  loopful 
sterile  rice  starch.  Incubate  37°C., 
24  hrs.  to  prove  sterility. 

3.  Nutrient  agar  serum-saline. 
Cover  long  slants  of  nutrient  agar 
(Difco.  1.5%)  in  standard  test  tubes 
^  to  ^  with  20:1  sterile  Ringer-horse 
serum  mixture.  Smaller  quantity  for 
intestinal  flagellates,  larger  quantity  for 
Trichomonas  vaginalis. 

4.  Trussell  and  Plass  (for  Tricho- 
monas vaginalis).  Overlay  slants  of 
liver  infusion  agar  (Difco)  with  a  se- 
lected mixture  as  for  nutrient  agar 
medium.  Adjustment  of  agar  and  solu- 
tion by  1  A'^  HCl  and  0.25%  aq.  sodium 
phosphate  is  suggested,  likewise  addi- 
tion of  0.2%  aq.  dextrose.  Incubate 
37°C.,  24  hrs.  to  prove  sterility;  store  in 
refrigerator. 

The  technique  of  obtaining  cultures 
of  protozoa  free  from  bacteria  has  been 
described  in  a  comprehensive  fashion 
by  G.  W.  Kidder  in  Calkins,  G.  N.  and 
Summers,  F.  M.,  Protozoa  in  Biological 
Research.  New  York:  Columbia  Uni- 
versity Press,  1941,  1148  pp.     He  was 


concerned  mainly  with  protozoa  from 
natural  waters,  soil  and  so  forth,  closely 
associated  with  bacteria  throughout 
their  existence.  The  techniques  advo- 
cated are  of  3  types:  (1)  to  get  rid  of  the 
bacteria  by  simply  washing  the  pro- 
tozoa in  sterile  fluid;  (2)  to  scrape  off 
the  adhering  bacteria  by  causing  the 
protozoa  to  migrate  through  semi-solid 
media  and  (3)  to  kill  off  the  bacteria  by 
agents  non-toxic  for  the  protozoa.  The 
establishment  of  sterilized  protozoa  in 
culture  is  an  essential  prerequisite  to 
investigation  of  their  behavior  in  re- 
sponse to  accessory  food  factors  and 
nutritional  supplements. 

Prussian  Blue  (CI,  1288)  is  ferric  ferrocy- 
anide,  a  colored  salt.  It  is  also  known 
in  commerce  as  Berlin  blue,  Chinese 
blue,  Paris  blue,  Milori  blue  and  Steel 
blue.  An  aqueous  solution  of  Prussian 
blue  is  a  good  medium  for  the  injection 
of  blood  vessels.  It  contrasts  nicely 
with  carmine.  The  particles  of  both  are 
sufficiently  large  to  be  held  within  the 
endothelium.  Deposition  of  Prussian 
blue  is  useful  in  the  localization  of  drain- 
age of  Cerebrospinal  Fluid  (Weed,  L. 
H.,  J.  Med.  Res.,  1914,  26,  21-117)  and 
in  the  microchemical  demonstration  of 
Iron  (Gomori,  G.,  Am.  J.  Path.,  1936, 
12,655-663).     See  Berlin  Blue. 

Pulp  of  Teeth.  This  can  be  studied  in  situ 
in  undecalcified  teeth  or  in  paraffin  or 
celloidin  sections  of  decalcified  ones. 
See  Teeth.  If  it  is  to  be  examined  by 
itself  after  removal  from  the  teeth  and 
fixation,  attempt  to  preserve  its  natural 
elongated  shape.  Almost  all  methods 
available  for  other  soft  tissues  are  ap- 
plicable. Wellings,  A.  W.,  Practical 
Microscopy  of  Teeth  and  Associated 
Parts.  London:  John  Bale,  Sons  & 
Curnow,  Ltd.  1938,  281  pp.  gives  many 
of  them.     See  Teeth,  Innervation. 

Purines.  See  critical  evaluation  of  micro- 
chemical  techniques  for  purines  by 
Click,  p.  72-73.  The  difficulty  is  that 
the  tests  are  positive  for  all  the  purines 
and  specificity  is  lacking.  Saint- 
Hilaire's  method  of  precipitating  them 
as  insoluble  copper  salts  and  the  forma- 
tion therefrom  of  red  ferrocyanide 
gives  positive  reactions  for  protamines, 
histones  and  other  protein  products. 
Detection  by  reduction  of  silver  salts 
is  worthless. 

The  murex  test  is  positive  with  xan- 
thine, guanine  and  uric  acid  but  nega- 
tive with  adenine  and  hypoxanthine 
(Lison,  1936,  p.  186).  However,  it  in- 
volves the  use  of  strong  acid  and  alkalis 
and  is  thus  very  drastic.  It  is  included 
by  Click  since  it  may  be  useful  in  some 
cases  though  it  is  no  different  from  the 
Xanthroproteic  Reaction.    To  a  section 


PSITTACOSIS 


290 


PYROSIN  B 


prepared  by  standard  methods  add  1 
drop  cone,  nitric  acid  and  warm  gently 
for  30  sec.  Drain  off  acid  with  blotting 
paper.  Add  drop  of  aq.  dest.  and  re- 
move in  same  fashion.  Expose  section 
to  ammonia  vapor.  Uric  acid,  guanine, 
xanthine  and  its  methyl  derivatives, 
purple  violet;  protein  material  often 
yellow  orange. 

Psittacosis,  method  for  staining  elementary 
bodies  (Hornus,  G.  J.  P.,  Ann.  Inst. 
Pasteur,  1940,  64,  97-116).  See  other 
kinds  of  Elementary  Bodies. 

Purkinje  Cells  of  heart.  Distend  entire 
heart  by  injecting  fixative  through  4 
cannulae,  in  aorta,  in  pulmonary  artery, 
in  superior  vena  cava,  in  one  pulmonary 
vein  and  ligating  other  vessels.  Fix 
in  Zenker's  or  Bouin's  fluid.  Sino- 
auricular  node  is  at  junction  of  superior 
vena  cava  and  right  auricle.  Cut  blocks 
perpendicular  to  the  node.  Color  paraf- 
fin sections  with  Masson's  trichrome 
stain  or  with  hematoxylin  and  eosin  for 
transitions  between  Purkinje  and  car- 
diac muscle  cells.  The  sharpest  differ- 
ential stain  for  the  former  is  Best's 
carmine  stain  for  glycogen  (Taussig, 
H.  B.,  J.  Tech.  Methods,  1934,  13,  85- 
87). 

Purkinje  Fibers.  In  excising  the  specimen 
the  presence  of  Purkinje  fibers  is  lo- 
calized by  the  dimpling  in  a  cross  section 
because  in  the  fresh  state  the  Purkinje 
fibers  contract  more  than  the  cardiac 
fibers  (Todd,  T.  W.  in  Cowdry's  Special 
Cytology,  1932,  2,  1179).  Todd  recom- 
mends for  general  purposes  Bouin's 
fixative  and  Mallory's  stain.  Safranin 
light  green  is  good  for  the  intercalated 
discs  (Jordan,  H.  E.,  and  Banks,  J.  B., 
Am.  J.  Anat.,  1917,  22,  285-338).  Tech- 
niques for  bringing  out  the  Purkinje 
system  particularly  of  mammalian  ven- 
tricles are  described  by  Abramson, 
D.  I.  and  Margolin,  S.,  J.  Anat.,  1935- 
36,  70,  250-259. 

Purpurin  (CI,  1037) — alizarin  No.  6,  alizarin 
purpurin — An  acid  anthraquinon  dye. 
The  bright  red  color  of  mauder-stained 
bones  is  due  to  purpurin  carbo.xylic  acid 
(Richter,  D.,  Biochem.  J.,  1937,  31, 
591-595). 

Pycnosis  (G.  pyknos,  dense)  When  the  sub- 
stance of  a  cell,  as  seen  in  stained  sec- 
tions is  unusually  dense  it  is  sometimes 
said  to  be  pycnotic.  The  increase  in 
density  is  usually  accompanied  by  a 
decrease  in  size  of  cytoplasm  and/or 
nucleus  and  the  nucleus  may  be  hyper- 
chromatic,  that  is  have  an  increased 
affinity  for  stains  like  hematoxylin  and 
methylene  blue.  Sometimes  pycnotic 
cells  occur  singly  surrounded  by  others 
not  in  the  same  condition  but  they  may 
be  present  in  group.     Those  in  the  cen- 


tral nervous  system  have  been  called 
chromophile  cells  (Cowdry,  E.  V., 
Contrib.  to  Embry.,  Carnegie  Inst., 
1917,  11,  29-41).  Information  is  needed 
on  the  cause  or  causes  of  pycnosis  and 
on  the  fate  of  cells  in  this  condition. 
Technique  for  the  microspectrophoto- 
metric  study  of  pyknosis)  of  red  blood 
cell  nuclei  is  given  by  Korson,  R.,  J. 
Exp.  Med.,  1951,  93,  121-128. 

Pyoktanin  Yellow,  see  Auramin. 

Pyoktaninum   Aureum,   see   Auramin. 

Pyoktaninum  Coeruleum,  see  Methyl  Vio- 
let. 

Pyridoxine,  see  Vitamin  Bj. 

Pyronin.  There  are  2  pyronins :  B  (CI, 
741)  and  Y  (CI,  739)  also  known  as  G. 
Conn  (p.  140)  describes  them  as 
closely  related  to  diphenyl  methanes 
since  they  have  one  carbon  atom  at- 
tached to  2  benzene  rings  and  exhibit 
similar  tendency  to  quinone  structure. 
Their  formula  also  resembles  that  of 
oxazins  except  that  nitrogen  of  central 
ring  is  replaced  by  CH  radical.  Pyro- 
nin B  is  tetra-ethyl  diamine  xanthene 
and  Y  is  the  tetra-methyl  compound. 
Conn  (McClung  p.  599)  advises  Y  with 
methyl  green  in  Pappenheim's  stain, 
for  the  granules  of  mast  cells  and  the 
gonococcus  in  smears  of  pus.  B  is  satis- 
factory for  most  purposes.  Only  re- 
cently has  the  distinction  been  made 
so  that  most  formulae  call  simply  for 
pyronin.  American  pyronins  are  now 
more  concentrated  than  those  imported 
before  1914.  Conn  says  that  allowance 
should  be  made  for  this  difference  in  the 
proportions  of  pyronin  and  methyl 
green. 

Pyronin  G  is  the  best  supravital  stain 
for  the  duct  system  of  the  pancreas 
(Bensley,  R.  R.,  1911,  12,  297-388). 
It  is  applied  by  Perfusion  a  solution  of 
1:1000  in  0.85%  aq.  NaCl  being  used 
until  the  pancreas  takes  a  light  rose 
color.  Small  pieces  are  then  mounted 
in  salt  solution  and  examined.  The 
ducts  from  the  main  ones  to  the  centro- 
acinous  cells  are  sharply  stained  in  red 
against  an  almost  colorless  background. 
The  ducts  may  be  similarly  stained  by 
methylene  blue  in  a  concentration  of 
1:10,000.  To  obtain  a  beautiful  contrast 
coloration  Bensley  injects  with  a  salt 
solution  containing  1 :  100  pyronin  and 
1:15,000  janus  green.  This  stains  the 
ducts  rea  and  the  islets  bluish  green. 
The  combination  of  1:1000  pyronin  and 
1:15,000  neutral  red  also  demonstrates 
ducts  and  islets  but  without  an  equally 
distinct  color  contrast.  The  pyronin 
method  for  ducts  is  one  of  the  most  use- 
ful techniques  both  for  investigation  and 
for  class  room  demonstration. 

Pyrosin  B,  see  Erythrosin,  bluish. 


PYROXYLIN 


291 


QUARTZ  ROD  TECHNIQUE 


Pyroxylin  (collodion  cotton.  coUoxylin, 
soluble  gun  cotton,  xyloidin,  cellodion 
wood).  It  is  chiefly  cellulose  tetra- 
nitrite.  Mainly  used  in  manufacture  of 
Collodions,  Celloidin,  Paraloidin,  Pho- 
toxylin,  etc. 
Pyrrol    Compounds,    see    Nitro    Reaction, 

Nitrosamino  Reaction. 
"Quad"  Stain.  A  recent  modification  of 
this  excellent  orcein-alizarine-Orange 
G  phosphotungstic  and  phosphormolyb- 
dic  acid  technique  is  given  in  detail 
by  Kornhauser,  S.  I.,  Stain  Techn., 
1945,  20,  33-35. 
Quartz  Fiber  Balance  and  quartz  torsion 

balances,  see  Balances. 
Quartz  Rod  Technique  for  Illuminating  Liv- 
ing Organs. — Written  by  Dr.  M.  H. 
Knisely,  Department  of  Anatomy,  Uni- 
versity of  South  Carolina,  Charleston, 
S.  C.  June  27,  1950— The  general 
purpose  of  this  technique  is  to  perrnit 
direct  microscopic  study  of  living  in- 
ternal organ  in  situ  while  maintaining 
experimental  conditions  which  disturb 
the  structures  and  processes  to  be  ob- 
served as  little  as  possible.  Like  all 
techniques  it  has  advantages  and  limita- 
tions; there  are  specific  purposes  for 
which  it  works  well,  and  purposes  for 
which  it  has  not  yet  worked  at  all.  The 
method  makes  it  possible  to  study  at  32 
to  about  600  diameters  magnification 
those  living  structures  whose  colors 
and/or  indices  or  refraction  differ  from 
those  of  adjacent  structures.  With 
quartz  rods  we  can  illuminate  for  ex- 
amination under  nearly  normal  condi- 
tions many  living  tissues  and  organs 
which  heretofore  have  been  inacces- 
sible. The  method  depends  upon  two 
physical  principles: 

1.  Conducting  light  from  a  suitably 
intense  source  directly  to  the  structures 
to  be  studied  by  way  of  a  fused  quartz 
rod.  Clean,  smooth  transparent  rods 
conduct  light  around  bends  and  turns 
by  internal  reflection  almost  like  a  hose 
conducts  water.  With  suitably  shaped 
rods  brilliant  illumination  of  relatively 
inaccessible  structures  is  relatively 
easy.  As  evidence  of  intensity,  with  a 
750  watt  T-12  tungsten  filament  bulb 
and  a  two  foot  length  of  7  millimeter 
rod,  so  much  light  can  be  sent  into  a 
microscope  objective  that  one  can 
scarcely  look  into  the  ocular.  Lesser 
degrees  of  intensity  are  of  course  easily 
obtainable.  Substitutes  for  quartz 
rods  have  been  suggested  and  occasion- 
ally used.  (Cole,  E.  C,  Science,  1938, 
87,  396-398.  Williams,  R.  G.,  Anat. 
Rec,  1941,  79,  263-270).  We  have 
tested  several.  No  substitute  has  yet 
proven  as  effective  for  illuminating 
living  tissues  as  fused  quartz  itself. 


2.  Maintaining  the  normal  tempera- 
tures  of   intensely    illuminated    living 
structures  with  a  slowly  flowing  isotonic 
isothermal    wash    solution.     It    is    im- 
possible to  illuminate  a  non-transparent 
structure  without  heating  it  at  the  same 
time.     The  color  of  an  object,  even  a 
translucent  object,   as  seen  by  either 
transmitted  or  reflected  light  is  due  to 
the  patterns  of  the  wave  lengths  which 
reach  the  eye  after  parts  of  the  incident 
light  are  "absorbed",  and  the  word  ab- 
sorbed   here    means    transformed   into 
heat  by  and  within  the  substance  of  the 
object  seen.     Light  filters  as  commonly 
used  between  light  source  and  illumi- 
nated  object   can   shelter   a   specimen 
from  the  wave  lengths  which  the  filters 
absorb,  but  they  do  not  alter  the  fact 
that  a  part  of  the  light  energy  which 
passes  the  filters  and  falls  on  the  speci- 
men is  always  transformed  into  heat 
within  the  specimen  by  the  materials  of 
the    specimen    itself.     Hence,    in    con- 
tinuously illuminating  a  living  object 
heat  is  simultaneously  developed  in  it 
at  a  constant  rate.     If  the  specimen  is 
small,  thin,  and  very  nearly  transparent 
and  if  its  illumination  is  dim,  the  small 
amount  of  continuously  produced  heat 
may  be  transferred  to  adjacent  objects 
so  rapidly  that  the  temperature  of  the 
specimen  never  rises  enough  to  interfere 
with  its  normal  functioning.     However, 
in  illuminating  relatively  thick  trans- 
lucent structures  such  as  frog  kidney  or 
liver,  or  mammalian  spleens,  brightly 
enough  for  microscopic  study,  heat  is 
developed  in  the  illuminated  structures 
faster  than  it  can  be  removed  without 
assistance.     To  remove  this  heat  a,  flow- 
ing solution  at  constant  temperature  is 
applied  to  the  illuminated  tissue,  either 
through  sets  of  glass  tubes,  or  more 
recently  through  hollow  tipped  quartz 
rods  which  deliver  both  light  and  flow- 
ing solution  precisely  to  the  selected 
portions   of   the   specimen.     The   fluid 
delivered  to  the  tissue  must  of  course 
be    isothermal    and   isotonic    with   the 
fluid  which  normally  bathes  it,  i.e.  plain 
water  at  room  temperature  is  used  to 
carry  heat  from  frog  skin  or  tongue, 
amphibian  Ringer's  solution   at   room 
temperature  to   carry  heat   from  frog 
kidney,    and    mammalian    Ringer's    at 
mammalian  body  temperature  to  carry 
heat  from  monkey  omentum.     On  ac- 
count of  the  high  specific  heat  of  water 
the  flowing  solution  can  take  up  the 
heat  as  fast  as  it  is  produced  with  but 
little   change  in  its  own  temperature; 
each  small  portion  of  flowing  solution 
is  warmed  but  little  as  it  passes  through, 
then  leaves  the  illuminated  field.     By 
these  physical  mechanisms  the  heat  in- 


QUARTS  ROD  TECHNIQUE 


292 


QUARTZ  ROD  TECHNIQUE 


escapably  developed  by  transformation 
of  light  energy  is  removed  as  fast  as  it 
is  produced  and  in  consequence  the 
temperature  of  the  illuminated  tissue 
does  not  rise. 

Thus  far  in  a  series  of  careful  tests  we 
have  found  no  visible  change  in  any 
structure  and/or  process  within  any 
living  tissue  or  organ  in  response  either 
to  a  sudden  change  from  dim  to  intense 
illumination  or  to  hours  of  continuous 
intense  illumination,  provided  the  tem- 
perature of  the  illuminated  specimen 
was  maintained  normal  by  a  continu- 
ously flowing  solution.  In  the  best 
e.xperiments  the  tissue  being  studied 
floats  on  a  thin  film  of  slowly  moving 
fluid  but  does  not  itself  touch  the  rod 
which  conducts  light  to  it. 

For  more  detailed  descriptions  of  the 
method  see  Knisely,  M.  H.,  Anat.  Rec, 

1936,  64,  499-524;  McClung,  C.  E., 
Handbook  of  Microscopical  Techniques 
for  Workers  in  Animal  and  Plant  Tis- 
sues, New  York:  Paul  B.  Hoeber,  Inc., 

1937,  p.  632-642;  Knisely,  M.  H.,  Anat. 
Rec,  1938,  71,  503-508;  Hoerr,  N.  L., 
1944,  see,  Glasser,  O.,  Medical  Physics, 
Chicago:  Year  Book  Publishers,  Inc., 

1944,  625-626. 

The  limitations  and  range  of  applica- 
bility and  usefulness  of  this  technique 
may  be  roughly  indicated  by  a  few  notes 
describing  some  of  its  current  and  pro- 
jected uses.  As  the  method  depends 
upon  seeing,  its  usefulness  is  continu- 
ously limited  by  the  mechanisms  where- 
by we  see.  As  a  brief  rough  statement 
we  "see"  by  recognizing  patterns  of 
color  and/or  intensity  of  the  light 
"rays"  coming  to  the  retina.  The  vas- 
cular system  with  its  refractile  (brightly 
transparent)  vessel  walls,  plasma  and 
white  cells,  and  its  brightly  colored 
erythrocytes  is  one  of  the  most  con- 
spicuous features  of  living  tissues  and 
has  thus  far  in  our  laboratory  received 
more  attention  than  other  living  struc- 
tures. Further,  the  vascular  system  is 
worth  intensive  study,  because  from 
moment  to  moment  continuously  under 
all  conditions  of  health  and  disease  it 
sets  the  maximum  rates  at  which  oxy- 
gen, glucose  and  other  anabolites  are 
carried  to  and  metabolites  are  removed 
from,  almost  every  cell,  tissue,  and 
organ  of  the  body.  For  an  elaboration 
of  this  theme  see:  Knisely,  M.  H., 
Stratman-Thomas,  W.  K.,  Eliot,  T.  S. 
and  Bloch,  E.  H.,  J.  Nat.  Malaria  Soc, 

1945,  4,  285-300. 

For  microscopic  study  of  the  periph- 
eral vascular  beds  of  internal  organs, 
the  method  is  limited  by  the  necessity 
of  an  anesthetic,  an  operation,  and  the 
exposure    of    the    surfaces    of   internal 


organs  to  the  outer  air,  an  unusual 
gaseous  environment. 

The  method  is  most  successful  when 
employed  to  examine  structures  just 
below  normal  anatomical  surfaces, 
rather  than  just  under  cut  surfaces  of 
tissues.  Thus  studies  have  been  carried 
out  in  frog  skin,  brain,  peripheral  nerves, 
smooth  muscles  of  the  gastrointestinal 
tract,  stomach  mucosa,  mesentery, 
striated  muscles,  lung,  suprarenal, 
kidney,  and  liver,  and  in  mam- 
malian spleen,  stomach  and  intestinal 
wall,  intestinal  villi,  omentum,  mesen- 
teries, liver,  and  brain  surfaces.  All 
these  have  natural  anatomical  surfaces 
which  can  be  exposed  without  damaging 
the  underlying  microscopic  structures. 
In  contrast,  much  as  we  would  like  to 
study  mammalian  bone  marrow,  we 
have  not  yet  found  a  way  to  expose  a 
portion  of  it  while  preserving  its  struc- 
tures and  their  functioning  well  enough 
so  that  the  specimen  was  worth  any 
serious  attention. 

The  conditions  of  an  experiment  limit 
the  phenomena  which  occur  during  that 
experiment.  An  anesthetized  animal 
obviously  does  not  run  or  swim  about; 
it  cannot  perform  many  obvious  well- 
known  functions  of  normal  unanes- 
thetized  animals.  By  extension,  there 
is  no  reason  to  assume  that  a  particular 
set  of  experimental  conditions  do  not 
inhibit,  retard,  alter,  or  prevent  func- 
tions as  yet  unknown,  or  one  or  more 
phases  of  the  particular  functions  one 
is  trying  to  study.  When  one  selects 
an  anesthetic,  gives  an  animal  a  specific 
quantity  of  it,  ties  the  animal  down, 
and  operates  upon  it,  he  thereby  puts 
that  animal's  circulatory  system  into 
one  of  its  reaction  states,  and  all  tests 
made  on  the  animal  from  that  time  on 
can  show  only  various  factors  of  that 
reaction  state  or  those  deviations  from 
it  which  are  possible  under  those  par- 
ticular experimental  conditions.  For 
example,  the  circulatory  responses  to 
exercise  are  not  occurring  in  an  anes- 
thetized animal  whose  muscles  have 
been  and  are  in  a  prolonged  state  of  rest. 
It  cannot  be  too  strongly  emphasized 
that  within  our  experience  each  experi- 
ment, or  class  of  experiments,  always 
acts  toward  minimizing  or  preventing 
known  and  probably  unknown  func- 
tions. Each  time  that  a  new  type  of 
experiment  has  been  devised,  new  kinds 
or  degrees  of  responses  of  peripheral 
vascular  beds  have  been  encountered. 
Each  time  we  have  learned  how  to  main- 
tain lesser  degrees  of  anesthesia  and/or 
to  do  less  damaging  operations  the  pe- 
ripheral vascular  beds  have  exhibited 
increasingly  complex  integrated    reac- 


QUARTZ  ROD  TECHNIQUE 


293 


QUARTZ  ROD  TECHNIQUE 


tions.  For  some  detailed  descriptions 
of  complex  integrated  vascular 
reactions  see  Knisely,  M.  H.,  Bloch, 
E.  H.,  and  Warner  L.,  K.  Danske-viden- 
skabernes  selskab.  Biologiske  skrifter. 
1947,    h-    (No.    7). 

By  careful  operative  techniques  some 
of  the  common  deleterious  effects  of 
operations  can  be  prevented.  Blood- 
less sludgeless  operations  can  be  per- 
formed on  animals  from  those  as  small 
as  frogs  and  mice  up  to  those  at  least 
as  large  as  rhesus  monkeys.  Sufficient 
care  can  be  taken  so  that  almost  no 
blood  is  lost;  simultaneously  care  can 
be  taken  to  traumatize  but  very  little 
tissue,  thus  minimizing  the  amounts  of 
precipitated-agglutinated  blood  pour- 
ing from  traumatized  tissues  into  the 
general  circulation  (Knisely,  M.  H., 
Eliot,  T.  S.,  and  Bloch,  E.  H.,  "Sludged 
Blood  in  Traumatic  Shock",  Archives 
of  Surgery,  1945,  51,  220-236).  As  (a) 
hemorrhage  and  (b)  precipitation- 
agglutination  of  the  circulating  blood 
are  two  separate  factors  which  can  act 
alone  or  in  combination  in  initiating 
some  of  the  pathologic  processes  which 
are  commonly  included  under  the  term 
"shock",  it  cannot  be  too  strongly  em- 
phasized that  bloodless  sludgeless  oper- 
ations must  be  performed  if  one  wishes  to 
study  the  circulatory  system  when  its 
parts  are  not  participating  in  shock 
reactions. 

Living  tissues  move,  and  the  move- 
ments tend  to  limit  the  microscopic 
study  of  living  structures.  When  an 
object  moves  under  a  microscope,  each 
point  of  its  microscopic  image  moves  as 
many  times  as  far  as  the  object  moves 
as  the  magnifying  power  of  the  lenses 
employed.  Thus,  at  100  diameters 
magnification  each  point  of  an  image 
moves  100  times  as  far  as  the  correspond- 
ing part  of  the  object.  Further,  the 
image  moves  during  the  same  time  in- 
terval that  the  object  moves,  so  in  each 
small  interval  of  time  the  image  goes 
100  times  as  far  as  the  object :  thus  at  all 
times  during  the  movement  the  image 
is  going  100  times  as  fast  as  the  object. 
From  this  example  it  is  obvious  that  when 
an  object  moves  under  a  microscope  each 
point  of  the  image  moves  as  many  times 
as  far  and  as  many  times  as  fast  as  the 
object  moves,  as  the  magnifying  power 
of  the  lens  system  employed.  These 
factors  rapidly  increase  the  difficulty  of 
observing  moving  structures  as  higher 
magnifications  are  used.  However,  the 
movements  of  most  tissues  do  not  pre- 
sent as  formidable  an  obstacle  as  the 
bare  statement  of  the  problem  might 
imply.  For  as  one  gains  experience  in 
working  with  living  tissues,  many  small 


methods  are  developed  for  holding  tis- 
sues still,  and  for  observing  between 
movements,  and  one  learns  to  swing  his 
eyes  with  the  image  and  observe  many 
details  sharply  even  while  the  tissues 
are  in  moderately  rapid  motion. 

The  depth  in  the  transilluminated 
tissue  to  which  one  can  observe  is 
limited  by  a  number  of  factors.  Most 
important  is  the  focal  length  of  the 
lenses  employed,  the  higher  the  mag- 
nifications used  the  more  closely  are 
observations  restricted  toward  surface 
structures.  The  natural  transparency 
or  translucency  of  the  tissues  also  limits 
the  depth  of  observations.  Some  curi- 
ous effects  result  from  this,  for  instance : 
when  smooth  muscle  is  relaxed  it  is  on 
the  transparent  side  of  translucent,  but 
when  it  contracts  it  becomes  quite 
opaque,  hence,  in  this  tissue,  the  maxi- 
mum possible  depths  of  observations 
are  a  function  of  the  physiological  state 
of  the  tissue.  For  similar  and  other 
reasons,  such  as  the  amount  of  blood 
present  at  any  moment  in  very  vascular 
tissues,  the  depth  to  which  one  can  see 
in  many  tissues  is  partly  dependent  on 
the  particular  set  of  physiologic  proc- 
esses going  on  at  the  time  the  tissue  is 
studied. 

The  maximum  duration  of  the  obser- 
vations made  in  any  one  animal  depends 
upon  the  species,  the  care  in  maintain- 
ing light  anesthesia,  the  care  exercised 
in  the  initial  operation,  and  the  purpose 
of  the  study  itself.  Individual  frog 
kidney  glomeruli  have  often  been  kept 
under  continuous  observation  at  mag- 
nifications up  to  400  (sometimes  600), 
up  to  as  long  as  12  hrs.,  without  injuring 
the  tissues  enough  so  that  the  blood 
began  to  agglutinate  or  so  that  passing 
white  cells  ever  began  to  stick  to  the 
inner  surfaces  of  the  brilliantly  illumi- 
nated glomerular  endothelium.  (Clark, 
E.  R.  and  E.  L.,  Am.  J.  Anat.,  1935,  57, 
385-438).  For  a  record  of  prolonged 
observations  see  Knisely,  M.  H.,  Strat- 
man-Thomas,  W.  K.,  Eliot,  T.  S.  and 
Bloch,  E.  H.,  J.  Nat.  Malaria  Soc, 
1945,  4,  285-300. 

Thus  far  the  limitations  of  the  method 
have  been  more  considered  than  the 
range  of  its  usefulness.  The  limitations 
are  important  and  must  be  clearly 
recognized  and  understood  by  all  who 
plan  either  to  use  it  or  to  evaluate  re- 
ports of  work  done  by  means  of  it. 
However,  as  one  purpose  of  this  book 
is  to  help  experimenters  select  methods 
which  may  be  useful  to  them,  the  range 
of  usefulness  of  the  method  will  now 
be  roughly  outlined. 

The  fused  quartz  method,  like  all 
others  does  not  have  uses  which  are  in- 


QUARTZ  ROD  TECHNIQUE 


294 


QUARTZ  ROD  TECHNIQUE 


dependent  of  the  purposes  of  those  who 
use  it.  Methods  are  always  dependent 
upon  purposes.  Analytical  mecha- 
nistic biologists  are  working  on  the  solu- 
tions of  many  problems  including:  How 
are  the  bodies  of  the  adults  of  each 
species  constructed?  How  does  each 
body  develop?  How  does  it  change 
with  time?  How  is  it  constructed  while 
it  is  alive?  How  is  it  constructed  so 
that  it  can  function?  What  physical 
and  chemical  functions  does  each  small 
part  have?  During  each  phase  of 
physiology  how  does  each  small  part 
behave?  How  do  the  coordinated  func- 
tions of  the  small  parts  summate?  How 
does  the  function  or  functions  of  each 
small  part  contribute  at  each  moment 
to  the  integrated  symphony  of  the 
functioning  of  the  whole?  Further, 
what  can  go  wrong  with  each  part? 
And  in  addition  the  clinical  sciences 
continually  ask,  "What  can  we  do  to 
prevent  or  help  repair  whatever  can  go 
wrong  with  each  part,  with  each  group 
of  parts,  with  the  integrated  function- 
ing of  the  body  as  a  whole?" 

Histological  studies  are  made  for  a 
definite  purpose,  to  help  collect  evi- 
dences from  which  to  develop  accurate 
concepts  of  the  structure,  functioning 
and  responses  of  the  small  parts  of  liv- 
ing bodies.  When  we  have  accurate 
concepts  of  the  structure  and  behavior 
of  small  parts  then  we  can  deal  induc- 
tively with  this  information  and  so 
build  up  concepts  of  the  functioning  of 
whole  organs.  Our  current  more  trust- 
worthy concepts  of  the  structure  and 
function  of  the  kidney  were  built  up  by 
this  inductive  approach  (Vimtrup,  Bj., 
Am.  J.  Anat.,  1928,  41,  123-151;  Rich- 
ards, A.  N.,  Proc.  Royal  Soc.  London 
B.  1938,  126,  398-432),  which  is  exactly 
opposite  from  trying  to  deduce  the 
function  of  microscopic  parts  from  the 
results  of  gross  experiments  performed 
on  whole  organs  or  systems. 

Each  living  animal  lives  in  four  di- 
mensions, three  of  space  and  one  of 
time.  At  any  moment  each  feature  of 
an  animal's  structure  exists  in  the  three 
space  dimensions.  But  many  features 
of  the  spacial  architecture  undergo 
rapid  or  slow  cyclical,  intermittent,  or 
progressive  changes  with  time.  The 
chemical  and  physical  characteristics, 
the  shapes,  the  magnitudes  and  the 
positions  of  structures  change  as  parts 
of  development,  of  physiology  and  of 
pathology.  New  structures  appear  and 
old  ones  disappear.  These  are  changes 
along  the  time  dimension.  The  rates 
at  which  changes  occur  are  most  impor- 
tant parts  of  our  concepts  of  the  struc- 


ture and  functioning  of  the  small  parts 
of  living  bodies. 

The  usefulness  of  microscopic  studies 
of  living  organized  tissues  (as  opposed 
to  tissue  cultures)  becomes  most  appar- 
ent when  one  recognizes  the  limitations 
of  histological  sections.  A  histological 
section  is  not  the  original  living  mate- 
rial. It  is  only  a  two  dimensisnal  slice 
out  of  a  four  dimensional  system,  minus 
what  had  been  lost  and  plus  what  has 
been  added  in  its  preservation-prepa- 
ration. No  one  can  possibly  begin  to 
appreciate  "what  has  been  lost"  in  the 
preparation  of  histological  sections  un- 
less and  until  he  studies  tissues  by 
methods  which  do  not  involve  any  of  the 
steps  commonly  used  in  preparing 
sections. 

The  spacial  dimensions  of  living 
tissues  are  invariably  altered  in  the 
preparation  of  histological  sections. 
The  alterations  in  dimensions  fre- 
quently or  usually  are  as  great  or 
greater  than  the  changes  in  dimension 
which  microscopic  structures  undergo 
as  parts  of  their  own  physiologic  proc- 
esses. Hollow  structures,  for  example, 
liver  sinusoids,  collapse  during  death 
and  fixation,  their  fixed  tissue  dimen- 
sions becoming  less  than  meaningless. 
Knowledge  of  the  exact  dimensions  of 
structures,  the  surface  areas  of  vascular 
networks,  the  surface  areas  of  glands 
etc.,  are  urgently  needed  as  a  basis  for 
quantitative  physiological  work. 
(Krogh,  A.,  Anatomy  and  Physiology 
of  Capillaries,  New  Haven:  Yale  Uni- 
versity Press,  1929,  p.  46.)  It  cannot 
be  too  strongly  emphasized  that  for 
strict,  mathematical  treatment  of  phys- 
iological problems  (Bloch,  I.,  Bull. 
Math.  Biophysics,  1941,  3,  121-126, 
ibid.,  1943,  5,  1-14)  measurements  of  the 
dimensions  of  microscopic  structures 
taken  from  fixed  tissues,  untempered 
by  knowledge  obtained  from  the  living, 
cannot  be  used.  For  after  the  abuse 
which  the  tissues  undergo  in  death  and 
fixation,  shrinking  and  swelling  in  vari- 
ous reagents,  and  the  mechanical  dis- 
tortions caused  by  the  cutting  processes 
(Dempster,  W.  T.,  Anat.  Rec,  1942,  84, 
241-267,  ibid,  269-274,  Stain  Technol. 
1943,  18,  13-24),  the  dimensions  of  the 
microscopic  parts  of  a  section  bear  no 
known  or  at  present  knowable  relation- 
ship to  any  of  the  size  or  sizes  which 
these  parts  had  in  life. 

In  the  light  of  the  above  paragraphs 
it  becomes  apparent  that  microscopic 
observations  of  living  organized  tissues, 
illuminated  by  quartz  rods  or  other 
techniques,  makes  available  certain 
classes  of  information  not  obtainable 


QUARTZ  ROD  TECHNIQUE 


295 


QUARTZ  ROD  TECHNIQUE 


by  other  histological  techniques.     This 
method  permits  study  of  the  following: 

1.  The  true  dimensions  of  visible 
microscopic  structures.  Further,  it 
permits  direct  study  of  changes  of  di- 
mensions of  structures  during  physio- 
logic and/or  pathologic  processes.  The 
dimensions  of  visible  structures  can  be 
measured  by  ocular  micrometers,  or  by 
taking  motion  pictures  of  the  structures 
and  making  "cine  tracings"  of  their 
projected  images  (Knisely,  M.  H.,  Eliot, 
T.  S.,  and  Bloch,  E.  H.,  1945;  Knisely, 
M.  H.,  Bloch,  E.  H.,  and  Warner,  L., 
cited  above) .  When  a  set  of  physiologic 
processes  have  been  studied  throughout 
their  course,  the  method  then  permits 
study  of  the  dimensions  of  living  micro- 
scopic structures  during  defined  phases 
of  physiologic  processes,  or  during  de- 
fined physiologic  states.  (The  same 
can  be  said  of  pathologic  processes.) 
The  results  of  this  kind  of  study  are 
quite  different  from  summations  of  the 
records  of  dimensions  of  tissues  taken 
at  unknown  phases  of  physiologic  proc- 
esses and  studied  and  measured  after 
unknown  amounts  of  distortion.  For 
an  example  which  demonstrates  this  see 
Knisely,  M.  H.,  Bloch,  E.  H.,  and 
Warner,  L.,  cited  above. 

2.  The  rates  and  changes  in  rate  of 
visible  processes,  most  of  which  are 
quite  unknown  today.  Histological 
sections  reveal  steps  in  processes  which 
have  long  cycles,  such  as  the  endome- 
trial changes  during  the  menstrual 
cycle.  They  frequently  fail  to  record 
as  sequences  changes  which  are  parts  of 
short  cycles,  the  reasons  being  (a)  that 
the  stages  of  short-cycle  phenomena 
appear  in  a  collection  of  sections  simply 
as  a  frequency  distribution  of  the  states 
of  the  observed  structures  and  (b)  that 
the  dimensions  are  so  altered  during 
death,  fixation  and  sectioning  that 
functional  differences  are  quite  obliter- 
ated, jumbled,  and  obscured.  Further, 
all  too  frequently  the  series  of  sections 
present  no  real  indicator  valid  for  de- 
termining the  sequence  of  the  steps  in 
short-cycle  phenomena.  When  motion 
pictures  are  taken  through  the  micro- 
scope the  method  permits  accurate 
recording  and  measuring  of  the  rates 
of  very  rapid  processes.  For  example, 
Knisely,  M.  H.,  Eliot,  T.  S.,  and  Bloch, 
E.  H.,  1945,  cited  above,  measured  the 
rate  of  formation  of  precipitates  in 
blood  flowing  through  crushed  tissues, 
finding  that  the  precipitates  formed  in 
from  l/8th  to  l/4th  of  a  second  while 
the  blood  flowed  from  lUO  to  150  micra. 
In  the  future  this  method  should  make 
it  possible  to  measure,  in  organized 
tissues,  the  rates  of  many  visible  phys- 


iologic, pathologic,  pharmacologic  and/ 
or  therapeutic  processes  or  responses. 
It  should  make  it  possible  to  measure 
the  rate  of  formation  of  any  visible  end 
product  of  in  vivo  chemical  reactions. 
Further,  and  most  important,  the 
study  of  processes  as  they  occur  fre- 
quently makes  it  possil)le  to  determine 
steps  in  chains  of  causation.  If  one 
assumes  that  an  effect  cannot  precede 
its  cause  in  time,  then  it  is  possible  to  be 
certain  that  some  phenomena  do  not 
cause,  but  rather  may  be  caused  by, 
others. 

3.  The  method  should  make  it  pos- 
sible to  obtain  small  samples  of  tissues 
and/or  fluids  from  defined  micro- 
anatomical  regions,  during  defined 
phases  of  physiologic  and/or  pathologic 
processes.  Wearn,  J.  T.  and  Richards, 
A.  N.,  Am.  J.  Physiol.,  1924,  71,  209- 
227,  used  micro-pipettes  to  remove 
glomerular  filtrate  from  the  Bowman 
spaces  of  frog  Malphigian  corpuscles. 
This  was  a  triumph  of  imagination,  in- 
sight, and  technique.  It  initiated  and 
provided  a  firm  foundation  for  the  whole 
modern  series  of  studies  of  kidney  func- 
tion. The  example  set  by  Richards 
and  Wearn  should  not  be  lost  or  ignored. 
Similar  studies  of  samples  from  defined 
micro-anatomical  structures,  taken  dur- 
ing defined  phases  of  physiologic  and 
pathologic  processes  will  undoubtedly 
go  a  long  way  toward  unravelling  many 
current  and  future  problems.  This 
must  be  kept  in  mind  as  increasingly 
sensitive  and  accurate  methods  are  de- 
vised for  measuring  the  concentrations 
of  substances  in  very  small  samples  of 
rather  dilute  solutions.  The  use  of 
special  isotopes  (initiated  by  Hevesy) 
is  greatly  increasing  the  abilities  of 
analysts  to  detect  and  measure  sub- 
stances in  extremely  small  biological 
samples.  One  next  necessary  step  in 
this  growing  branch  of  knowledge  must 
consist  in  defining  and  knowing  the 
micro-anatomical  regions  from  which 
each  sample  comes  and  the  physiologic 
or  pathologic  states  under  which  each, 
sample  is  collected,  as  accurately  as  the 
composition  of  the  sample  can  now  be 
determined.  This  seems  obvious;  ob- 
vious also  is  the  fact  that  in  many 
quarters  it  seems  not  j^et  to  be  appre- 
ciated. 

4.  The  method  plus  suitable  and  ade- 
quate micro-dissection  and  micro-in- 
jection techniques  (Chambers,  R.  and 
Kopac,  M.  J.,  in  McClung,  2nd  ed.,  pp. 
62-109;  Buchtal,  F.,  Ztsclir.  f.  Wis- 
sensch.  Mikr.,  1942,  58,  126-133)  should 
make  it  possible  to  place  samples  of 
various  substances  in  defined  micro- 
anatomical  areas,  during  defined  phases 


QUARTZ  ROD  TECHNIQUE 


296 


RADIOACTIVE  ISOTOPES 


of  physiologic  or  pathologic  processes 
and  watch  or  otherwise  determine  the 
responses  of  parts  of  living  systems  to 
the  newly  arrived  material.  For  an 
extensive  example  of  one  such  set  of 
studies,  see  Knisely,  M.  H.,  Bloch, 
E.  H.,  and  Warner,  L.,  cited  above. 

5.  The  method  permits  the  study  of 
some  kinds  of  pathologic  processes  while 
they  are  still  in  reversible  stages,  that 
is,  in  controllable  stages.  Autopsies 
and  autopsy  sections  show  the  final 
cumulative  results  of  all  of  the  simul- 
taneous and  consecutive  pathologic  and 
reparative  processes  which  had  been 
going  on.  That  is,  they  show  the  pre- 
servable,  visible  part  of  the  accumu- 
lated results  after  some  one  or  more  sets 
of  pathologic  processes  have  become  ir- 
reversible. The  microscopic  studies  of 
living  tissues  allow  examination  of  some 
pathologic  processes  (a)  as  they  de- 
velop, (b)  as  they  proceed  at  sublethal 
degrees  of  intensity,  and  (c)  as  they 
accumulate  toward  lethal  combinations 
of  factors,  but  are  still  reversible,  that 
is  while  the  animal's  life  can  still  be 
saved,  and  (d)  as  they  accumulate  into 
non-reversible  stages.  Further,  the 
method  permits  study  of  the  results  of 
experimental  therapeutics  on  visible 
pathologic  processes.  For  demonstra- 
tions and  elaboration  of  this  theme  see 
Knisely,  M.  H.,  Stratman -Thomas, 
W.  K.,  Eliot,  T.  S.,  and  Bloch,  E.  H., 
1945,  cited  above. 

It  may  seem  to  some  that  the  above 
discussion  is  too  critical  or  unjustly 
critical  of  the  histological  sectioning 
techniques,  or  that  the  author  is  trying 
to  laelittle  their  use.  This  I  do  not  be- 
lieve to  be  so.  The  best  histologists 
have  always  studied  sections  not  for  the 
structure  of  the  dead  sections  them- 
selves, but  rather  to  determine  as  closely 
as  possible  the  structure  and  functions 
the  tissues  had  had  when  last  alive.  Pre- 
cision and  accurac}'^  in  developing  con- 
cepts from  the  evidences  gathered  by  a 
technique  can  never  be  greater  than  the 
user's  understanding  of  the  inherent 
limitations  of  that  technique.  The  ac- 
curacy of  a  technique  cannot  be  deter- 
mined simply  by  repeating  its  steps  an 
infinite  number  of  times;  its  limitations 
and  degrees  of  accuracy  must  also  be 
cross-checked  by  other  and,  if  possible, 
quite  different  techniques.  Each  useful 
technique  delineates  one  or  more  aspects 
of  the  original  tissue  more  accurately 
than  do  other  techniques.  Obviously 
the  most  accurate  and  comprehensive 
concepts  of  micro-anatomy,  microscopic 
physiology  and  microscopic  pathologic 
physiology  can  be  developed  only  by 
synthesis;   by  putting  together  in  the 


mind  of  the  student  the  most  accurate 
of  the  available  individual  aspects.  For 
this  purpose  each  technique  has  special 
values  of  its  own;  for  this  purpose  not 
enough  different  techniques  are  yet 
available. 

Quinoline  Dyes.  Only  pinacyanol  is  of  ap- 
parent value  to  histologists. 

Quinone-Imine  Dyes.  Possess  2  chromo- 
phores  :  indamin-N=  and  quinoid  ben- 
zene ring.  They  are  divisible  into 
Azins,  Indamins,  Indophenols,  Ox- 
azins,  Thiazins. 

Quinone  Oximes,  see  Nitroso  Dyes. 

Rabbit  Ears,  see  Sandison's  Technique  for 
inserting    transparent    chambers    in. 

Rabies,  see  Negri  Bodies. 

Rabl's  Fluid  is  sat.  aq.  mercuric  chloride, 
1  part;  sat.  aq.  picric  acid,  1  part;  aq. 
dest.,  2  parts. 

Radiation.  Methods  and  results  of  radia- 
tion of  normal  tissues  reviewed  (Warren, 
S.  and  Dunlap,  C.  E.,  Arch.  Path., 
1942,  34,   562-608  and  earlier  papers). 

Radioactive  Isotopes— Written  by  T.  B. 
Rosenthal,  Dept.  of  Anatomy,  Washing- 
ton Universit}-,  St.  Louis.  June  6, 
1951. — The  release  of  radioactive  iso- 
topes in  large  quantities  for  civilian 
use  (1946)  has  resulted  in  a  voluminous 
literature  concerned  with  fundamental 
concepts  of  metabolism  and  with  the 
diagnosis  and  treatment  of  malignant 
disease.  Although  only  a  few  notable 
discoveries  may  be  directly  attributed 
to  the  use  of  these  isotopes  (since  the 
basic  ideas  were  derived  from  earlier 
work  with  stable  isotopes  and  conven- 
tional biochemical  methods),  it  must 
be  admitted  that  the  relative  ease  and 
speed  of  operations  with  radioactive 
isotopes  have  thrown  open  a  hitherto 
difficult  field.  By  these  means  the 
validity  of  many  old  concepts  has  been 
confirmed,  while  others  have  been 
conclusively  demonstrated  to  be  false. 
It  must  be  emphasized  however  that 
research  with  isotopes  has  not  dis- 
placed the  traditional  methods  in  biol- 
ogy and  chemistry.  Reliable  tech- 
niques are  still  requisite,  and  there  is 
more  need  for  good  analytical  chemistry 
than  ever  before.  The  radioactive  iso- 
tope now  takes  its  proper  place  among 
the  resources  of  the  laboratory  as  a  tool 
indispensable  for  certain  problems,  use- 
ful for  others,  and  merely  optional  for 
still  others. 

Mass  production  of  reliable  instru- 
ments for  measurement  of  radio-activ- 
ity has  extended  the  use  of  isotopes  to 
those  laboratories  previously  deterred 
by  expense  or  lack  of  specially  trained 
personnel.  The  choice  of  equipment, 
i.e.,  Geiger-MuUer  counters,  propor- 
tional counters,  scintillation  counters, 


RAD  10  AUTOGRAPHY 


297 


RADIOAUTOGRAPHY 


ionization  chambers,  electroscopes,  etc. 
will  depend  on  the  nature  of  the  prob- 
lem and  the  degree  of  versatility  de- 
sired. For  complete  details  see  the 
monographs  listed  below  and  the  latest 
manufacturers'  catalogs. 

Renewed  interest  in  the  pioneering 
technique  of  the  autoradiograph  has 
resulted  from  the  development  of  new 
procedures  and  specially'  prepared  emul- 
sions. Under  favorable  circumstances 
it  is  now  possible  to  achieve  intra- 
cellular localization  of  tracer  elements. 
See  Gross,  Bogoroch,  Nadler,  and 
Leblond,  Am.  J.  Roentgenol.  Rad. 
Therap.,  1951,  65,  420-458  for  a  review 
of   the    potentialities    and    limitations. 

The  first  step  in  a  biological  investiga- 
tion would  naturally  be  to  decide 
whether  the  use  of  isotopes  is  war- 
ranted, on  the  basis  of  results  expected 
in  relation  to  the  economic  investment. 
The  next  step  is  a  survey  of  the  liter- 
ature to  ascertain  whether  isotopes 
suitable  for  the  purpose  are  available 
and  what  measure  of  success  could  be 
expected  on  the  basis  of  similar  experi- 
ments. From  these  considerations  a 
choice  of  instruments  and  procedures 
would  be  made.  On  the  other  hand, 
a  certain  project  may  be  warranted 
and  feasible,  but  rendered  impracticable 
by  technical  complications  of  various 
kinds. 

For  a  source  of  materials  one  would 
first  consult  the  catalog  issued  by  the 
Oak  Ridge  Laboratory  of  the  Atomic 
Energy  Commission.  Herein  are  listed 
the  available  stable  and  radioactive 
isotopes  together  with  prices,  shipping 
information,  and  official  regulations  on 
clinical  applications,  health  protection, 
etc.  Of  the  96  elements,  only  He,  Li, 
B,  Ne,  N,  O,  Mg,  Al,  F,  Si,  and  Fa  lack 
radioactive  isotopes  of  suitable  half- 
life,  leaving  85  elements  whose  employ- 
ment is  limited  only  by  the  ingenuity 
and  resources  of  the  investigator.  In 
many  cases  the  form  of  the  material 
may  be  specified:  pure  metal,  inorganic 
compound,  labelled  organic  compound, 
colloid,  solution,  etc. 

In  addition  a  number  of  commercial 
firms  are  engaged  in  supplying  on  order 
and  from  stock  an  ever-increasing  vari- 
ety of  organic  intermediates  and  com- 
pounds of  metabolic  importance  syn- 
thesized with  tagged  atoms  in  various 
positions.  If  only  small  quantities  are 
needed,  the  investigator  may  conduct 
a  biological  synthesis  from  plant  or 
animal  sources  in  his  own  laboratory. 
Although  the  nuclear  reaction  pile 
now  supplies  the  great  bulk  of  radio- 
isotopes, those  who  have  access  to 
cvclotrons  or  similar  machines  should 


inquire  about  the  possibility  of  getting 
small  amounts  of  the  shorter-lived 
isotopes. 

Studies  involving  radiation  by  alpha- 
particles    require    the    naturally-occur- 
ring radioactive  elements.     The  meta- 
bolism   of    Pb,    for   example,    may    be 
traced  by  means  of  those  elements  iso- 
topic  with  Pb. 
Radioactive  Tracers  in  Biology.     2nd 
Ed.     M.     D.     Kamen,     Academic 
Press,  New  York,  1951,  429  pp. 
Radioactive  Indicators.     G.  Hevesy, 
Interscience  Publishers,  Inc.,  New 
York,  1948,  556  pp. 
Isotopic  Tracers  and  Nuclear  Radi- 
ations.    W.  E.  Siri,  Ed.,  McGraw- 
Hill,  New  York,  1949,  653  pp. 
The  latter  features  a  selected  bibliog- 
raphy to  1947  of  papers  on  biological 
and  medical   applications  arranged  by 
elements,  together  with  extensive  tables 
which  aid  in  reviewing  previous  find- 
ings  and  suggesting  new  possibilities. 
The   current  literature  is  recorded  in 
the  abstract  journals  and  in  Nuclear 
Science     Abstracts,     published     twice 
monthly  by  the  A. E.G.     The  "Annual 
Reviews   of   Biochemistry"   should   be 
consulted    for    comprehensive    reviews 
of  special  topics. 
Radioautography — Written       by       C.       P. 
Leblond  and  R.  Bogoroch,  Department 
of  Anatomy,  McGill  University,  Mon- 
treal.   September  12, 1951 — Radioautog- 
raphy is  a  technique  devised  to  define 
the    localization    of    radioactive    sub- 
stances   within    biological    specimens. 
The  usual  practice  is  to  place  a  histo- 
logical section  as  closelj^  as  possible  to 
a  phtographic  emulsion,  expose  for  a 
suitable  time  interval  and  subsequently 
develop   as   in   ordinary   photograph}'. 
In  place  of  light,  the  rays  emitted  by 
the    radio-isotope    furnish    the    energy 
necessary  to  transform  the  silver  bro- 
mide of  the  emulsion  into  photolj'tic 
silver,  which  may  then  be  developed 
by  photographic  reagents  into  visible 
black  silver  grains.     Such  grains  overlie 
the   sites   of   deposition   of   the   radio- 
active   substance    in    the    histological 
sections,    thus    giving    a    permanent, 
visible  record  of  the  localization  of  this 
substance. 

The  use  of  the  silver  bromide  grain 
as  a  detector  of  radioactivity  has  four 
advantages  over  other  techniques:  1) 
the  effect  of  the  radiation  is  cumulative, 
and,  therefore,  minute  quantities  of 
radioactivity  may  be  detected  if  ex- 
posure is  sufficiently  prolonged;  2)  the 
radioautographic  image  is  a  permanent 
record;  3)  the  record  is  two-dimensional 
and  may  be  three-dimensional  in  case 
of  tracks;  and  4)  the  smallness  of  the 


RADIOAUTOGRAPHY 


298 


RADIOAUTOGRAPHY 


silver  bromide  granules  makes  it  pos- 
sible to  localize  the  radioactivity  to 
a  comparablj'^  small  area  in  the  tissue. 

It  IS,  therefore,  possible  by  this 
method  to  virtually  "see"  a  "labeled" 
element  in  an  animal  tissue.  Further- 
more, the  fate  of  that  element  in  the 
animal  body  may  be  detected  by  follow- 
ing the  element  through  from  structure 
to  structure.  The  metabolism  of  any 
labeled  physiological  or  pharmacologi- 
cal compound  can  thus  theoretically  be 
traced  throughout  the  animal  body 
using  the  radioautographic  method, 
providing  the  labeled  material,  be  it 
a  product  of  synthesis  or  degradation, 
is  retained  in  its  original  site  in  a  tissue 
during  processing  for  radioautography. 
The  advantage  of  this  method  for  the 
progress  of  histology  is  apparent,  since 
the  significance  of  known  structures  will 
often  be  revealed  by  the  substance 
which  they  incorporate.  From  the 
medical  point  of  view  the  sites  of  ac- 
cumulation of  the  isotopes  as  well  as 
their  length  of  stay  in  the  body  provide 
useful  information  as  to  the  radio- 
toxicity  of  known  amounts  of  these  iso- 
topes. The  applications  of  radioautog- 
raphy to  biology  and  medicine  have 
been  fully  reviewed  most  recently  bji- 
Gross,  J.,  Bogoroch,  R.,  Nadler,  N.  J., 
and  Leblond,  C.  P.,  Am.  J.  Roentg. 
and  Radium  Ther.,  1951,  65,  420-458. 
The  use  of  a-ray-producing  isotopes  was 
examined  by  Yagoda,  H.,  Radioactive 
Measurements  with  Nuclear  Emulsions. 
New  York:  John  Wiley  and  Sons,  Inc., 
1949. 

As  with  other  methods  involving  the 
use  of  radioisotopes,  it  is  assumed  that 
the  chemical  behavior  of  a  labeled  sub- 
stance is  identical  with  that  of  its  stable 
counterpart.  This  will  also  be  true  of 
its  biological  behavior  providing  that 

1)  the  amount  of  radioactivity  admin- 
istered be  small  enough  not  to  have  a 
significant    radio-chemical    effect,    and 

2)  the  amount  or  weight  of  material  in- 
jected be  sufficiently  small  not  to  pro- 
duce a  significant  increase  in  the  amovmt 
of  this  substance  present  in  the  circula- 
tion. The  labeled  substance  will  then 
truly  act  as  a  "tracer"  of  the  normal 
metabolism. 

The  success  of  radioautography  de- 
pends on  1)  the  geometrical  relationship 
between  specimen  and  emulsion,  the 
best  conditions  being  a  minimal  dis- 
tance between  source  and  emulsion,  a 
thin  section  (5ai  or  less),  and  a  thin 
emulsion  coating  (10;u  or  less) ;  2)  the 
features  of  the  emulsion — the  silver 
bromide  grains  should  be  uniform  (for 
contrast),  of  a  small  size,  and  in  high 
concentration;  and  3)  the  characteristics 


of  radiations  (i.e.,  energy  and  intensity) 
— particles  of  low  energy  such  as  soft 
/3-rays  give  a  better  resolution.  For 
a  more  detailed  discussion  of  each  of 
these  factors  see  Nadler,  N.  J.,  Cana- 
dian J.  Med.  Sci.,  1951, 29, 182-194;  Gross 
et  al.,  referred  to  above;  Doniach, 
I.  and  Pelc,  S.  R.,  Brit.  J.  Radiol., 
1950,  23,    184-192. 

Exposure  time  for  a  radioautograph 
is  influenced  by  these  three  groups  of 
factors:  1)  Geometrical  relations.  Thus, 
minimizing  the  distance  between  emul- 
sion and  specimen  will  tend  to  reduce 
exposure  time,  while  decreasing  the 
thickness  of  section  and  emulsion  will 
tend  to  prolong  it.  2)  Properties  of 
photographic  emulsions.  Emulsions  vary 
in  sensitivity  to  /3-rays.  Also,  the  more 
contrasty  the  emulsion  is,  the  narrower 
is  the  range  of  satisfactory  exposure 
time.  3)  Character  of  the  radiation. 
The  radiation  influences  the  exposure 
time  not  only  by  its  concentration  per 
unit  area,  but  also  according  to  the 
half  life  and  energy  of  the  isotopes  used. 
With  soft  radiation,  the  absorption  in 
the  tissue  will  also  increase  the  exposure 
time. 

It  is  important  to  keep  the  exposure 
to  a  minimum,  as  overexposure  results 
in  a  more  diffuse  image  with  correspond- 
ing loss  in  resolution.  Unstained 
"test"  slides  developed  periodically 
will  indicate  the  optimum  exposure 
time. 

Preparation  of  Tissues. 

In  the  case  of  soft  tissues,  paraffin 
or  celloidin  sections  are  prepared  in 
the  routine  histological  manner  and  de- 
hydrated with  or  without  staining. 
For  practical  purposes,  5^t  paraffin  sec- 
tions and  10/i  celloidin  sections  seem 
to  be  most  convenient.  Carbowax 
(Blank,  H.,  McCarthy,  P.  L.,  and  De- 
Lamater,  E.  D.,  Stain  Techn.,  1951, 
26,  193-197)  and  frozen  dried  (Holt, 
M.  W.,  Cowing,  R.  F.,  and  Warren,  S., 
Science,  1949,  110,  328-330)  sections 
have  also  been  used. 

Bones  and  teeth  may  be  embedded 
and  cut  according  to  Bloom's  method 
(see  Leblond,  C.  P.,  Wilkinson,  G.  W., 
B(51anger,  L.  F.,  and  Robichon,  J.,  Am. 
J.  Anat.,  1950,  86,  289-341)  for  bones  of 
young  animals  or  according  to  Arnold, 
J.  (Science,  1951,  114,  178-180)  for  hard 
bones  of  older  animals.  Methods  em- 
ploying grinding  machines  have  to  be 
used  for  teeth  as  described  by  Hoecker, 
F.  E.  and  Roofe,  P.  G.  (Radiology, 
1949,  52,  856-865),  Laude,  P.  P.,  Janes, 
R.  G.  and  Boyd,  J.  D.  (Anat.  Rec, 
1949, 104, 11-15),  Sognnaes,  R.  F.,  Shaw, 
J.  H.,  Solomon,  A.  K.  and  Harvold,  E. 
(Anat.    Rec,    1949,    104,    319-330). 


RADIOAUTOGRAPHY 


299 


RADIOAUTOGRAPHY 


Large  specimens  of  soft  tissues  may 
be  frozen,  ground  in  the  same  way  as 
above,  or  sectioned  with  a  chilled  blade 
or  fine  electric  saw  while  being  kept 
in  the  frozen  state,  and  thus  applied 
to  the  photographic  emulsion. 

1.  Contact  method:  This  technique, 
the  oldest  and  crudest  method,  has  been 
used  extensively  especially  in  the  in- 
vestigations of  the  sites  of  localization 
of  isotopes  in  hard  tissues  such  as  bones 
and  teeth,  and  in  frozen  sections  of  soft 
tissues.  (See  reviews:  Gross,  J.  and 
Leblond,  C.  P.,  Canadian  Med.  Assoc. 
J.,  1947, 57, 102;  Gross,  J.,  Bogoroch,  R., 
Nadler,  N.  J.  and  Leblond,  C.  P.,  Am. 
J.  Roentg.  and  Radium  Therap.,  1951, 
65,  420-458.)  The  method  consists  es- 
sentially of  placing  a  microscopic  slide 
bearing  a  radioactive  tissue  in  close  con- 
tact with  a  photographic  plate;  and 
of  keeping  these  in  close  contact  by 
pressure. 

1.  A  slide  containing  the  histological 
section  is  dipped  twice  into  an  ether- 
alcohol  solution  of  1%  celloidin  fol- 
lowing staining  and  passage  through 
95%  and  absolute  alcohol.  The  sec- 
tion is  then  dried  overnight  to  in- 
sure hardening  of  the  celloidin. 

2.  In  the  darkroom,  the  histological 
slide,  which  should  be  free  from  dust 
particles  or  granules  which  may 
make  for  uneven  contact,  is  placed 
gently  against  a  photographic  plate 
or  film  (x-ray,  Eastman  Kodak 
Medium  Lantern  Slide  plate,  etc.). 
Various  methods  may  be  used  to 
insure  an  intimate  uniform  contact 
between  slide  and  plate  or  film. 
They  may  be  held  in  a  roentgen-ray 
type  pressure  cassette  or  in  a  print- 
ing frame.  Naturally,  these  holders 
should  be  light-tight. 

3.  After  exposure,  the  photographic 
plate  or  film  is  processed  according  to 
the  routine  photographic  procedures, 
i.e.,  developed  in  D-72  for  2  min., 
rinsed  in  water,  fixed  in  acid  fixer 
for  10  min.,  washed  for  30  min.  and 
dried.  To  protect  the  radioauto- 
graphic  image  in  the  emulsion  from 
scratches  or  abrasions,  it  is  useful 
to  cover  the  image  with  a  drop  of 
Canada  balsam  and  a  histological 
coverslip.  The  histological  section 
and  the  radioautographic  image  may 
then  be  simultaneously  examined 
under  a  dissection  or  ordinary  micro- 
scope. 

This  method  has  the  advantage  of 
simplicity  but  the  localization  at  best 
is  rather  crude  due  to  low  resolution. 

2.  Mounting  method:  The  one  most 
usually  followed  is  that  of  floating  sec- 
tions   onto    a   photographic   emulsion. 


Recent  attempts  at  doing  this  under 

dry  conditions  will  be  mentioned  later. 

In  the  Wet  Mounting  method  (Evans, 

T.  C,  Proc.  Soc.  Exp.  Biol,  and  Med., 

1947,  64,  313-315;  Endicott,  K.  M.,  and 
Yagoda,  H.,  Thid.j  p.  170-172;  Boyd, 
G.     A.,    and    Williams,    A.    I.,    Ibid., 

1948,  69,  225-232)  the  tissue  section  is 
floated  directly  onto  a  photographic 
film  or  plate.  This  insures  a  more 
intimate  contact  between  tissue  and 
emulsion  and  results  in  a  better  resolu- 
tion, i.e.,  permits  a  finer  localization. 
This  method,  however,  cannot  be  used 
if  the  radioactive  substance  in  the 
tissue  is  water  soluble. 

1.  Unstained  strips  of  paraffin  sections 
of  radioactive  tissues  are  floated  in 
a  40°C.  water  bath  to  remove  any 
wrinkles  from  the  tissue.  The  water 
is  cooled  to  18°C.  by  the  addition  of 
ice  cubes  at  18°C.  and  all  subsequent 
steps  are  carried  out  in  the  darkroom 
using  a  Wratten  No.  1  safelight. 

2.  A  photographic  plate  or  film  (East- 
man Kodak  Medium  Lantern  Slide 
Plate,  NTA,  NTB  1,  2,  or  3  plates, 
etc.)  is  slipped  under  the  tissue  sec- 
tion, the  corner  of  the  tissue  being 
held  against  the  photographic  plate 
with  a  needle,  and  the  plate  with  ad- 
herent tissue  is  lifted  out  of  the 
water.  After  the  excess  water  has 
drained  off  the  plate  and  the  emul- 
sion has  dried  completely,  the  tissue 
adheres  intimately  and  permanently 
to  the  photographic  emulsion. 

3.  After  proper  exposure  at  refrigerated 
temperatures  (4°C.  or  lower),  the 
section  is  deparaffinated  in  two 
changes  of  xylol  and  hydrated 
through  graded  alcohols. 

4.  The  preparations  may  be  developed 
in  D-72  developer  for  2  minutes  or  in 
D-19  for  10  minutes,  rinsed  in  water, 
fixed  in  acid  fixer  F5  or  in  30%  thio- 
sulphate,  and  washed  in  running 
water  for  30  minutes. 

5.  Unstained  sections  may  be  dehy- 
drated, cleared  and  mounted  im- 
mediately or  they  may  be  stained 
with  either  dilute  hematoxylin  over- 
night, or  metanil  yellow  (Gross,  J. 
and  Leblond,  C.  P.,  Canadian  Med. 
Assoc.  J.,  1947,  57,  102-122;  Simmel, 
E.  B.,  Fitzgerald,  P.  J.  and  Godwin, 
J.  T.,  Stain  Techn.,  1951,  26,  25-28). 
For  staining  prior  to  development, 
Doniach  and  Pelc  have  used  hot 
carbol  fuchsin-neutral  red  which  re- 
sists the  destaining  action  of  the 
developing  reagents. 

Because  the  mounting  method  allows 
for  a  very  intimate  contact  between 
tissue  section  and  emulsion,  a  very 
very     fine     localization     is     possible. 


RADIOAUTOGRAPHY 


300 


RADIOAUTOGRAPHY 


This  method  has  been  used  extensively 
especially  in  hospitals  where  a  minimum 
of  equipment  was  desirable.  The 
method,  however,  does  not  always  give 
reproducible  results  because  of  1)  un- 
even penetration  of  developer  through 
the  tissue  leaving  some  areas  with  latent 
images  in  the  emulsion  beneath  un- 
developed, 2)  staining  of  the  emulsion 
gelatin  which  obscures  histological  and 
radioautographic  detail  to  some  extent, 
and  3)  chemical  action  of  the  tissue 
on  the  emulsion  causing  in  some  cases 
blackening  of  the  emulsion  or  in  other 
cases  insensitization  of  the  emulsion. 
These  possibilities  are  eliminated  in 
the  "coating"  methods.  It  must  be 
emphasized,  however,  that  the  prepara- 
tions obtained  by  the  mounting  tech- 
nique are  often  more  satisfactory  under 
high  than  under  low  magnification. 

Several  "Dry"  Mounting  methods 
have  been  described  (Hoecker,  F.  E. 
and  Roofe,  P.  G.,  Radiology,  1949,  52, 
856-865;  Williams,  A.  I.,  Oak  Ridge, 
Tenn.,  Los  Alamos  Scientific  Labora- 
tory, AECU,  1950,  930;  Gallimore,  J., 
Oak  Ridge,  Tenn.,  1951).  These  elim- 
inate the  use  of  large  quantities  of 
water  on  the  tissue  prior  to  exposure, 
thus  preventing  loss  or  displacement  of 
water  soluble  material.  The  published 
results  indicate  that  the  method  is  still 
in  the  experimental  stage. 

3.  Coating  method:  A  slide  bearing  a 
histological  tissue  section  is  covered 
with  either  a  fluid  emulsion  or  a  strip- 
ping emulsion  removed  from  its  backing. 
The  method  as  used  with  fluid  emulsion 
is  very  satisfactory  for  research  pur- 
poses, because  of  the  possibility  of  con- 
trolling the  thickness  of  the  emulsion. 
In  "fluid  coating": 

1.  The  sections,  stained  or  unstained, 
are  coated  with  celloidin  as  in  the 
contact  method.  The  area  on  the 
slide  to  be  coated  is  outlined  with  a 
diamond  pencil. 

2.  Using  a  Wratten  No.  1  safelight  at 
a  distance  of  three  feet,  photographic 
emulsion  is  prepared  for  coating, 
a)  The   most   convenient   emulsions 

with  which  to  work  are  the  bulk 
emulsions,  several  of  which  have 
been  kindly  given  the  authors  by 
Ansco  Corporation  and  British 
Kodak  Limited.  These  emul- 
sions are  scooped  directly  into 
a  50  cc.  beaker  which  is  placed  in 
a  37°C.  water  bath.  Ansco 
Radioautographic  Emulsion  A 
has  a  large  grain  size  and  a  very 
high  sensitivity  but  gives  a  poor 
resolution,  while  British  Kodak 
NT4  bulk  emulsion  has  a  small 
grain  size   and  medium  to  high 


sensitivity  but  gives  a  good 
resolution.  Unfortunately,  none 
of  the  bulk  emulsions  is  available 
on  a  commercial  bais. 

b)  The  next  best  possibility  is  to 
remove  emulsion  from  photo- 
graphic plates  or  films  such  as 
Eastman  Kodak  NH  Special 
Spectroscopic  plates  or  Eastman 
Kodak  "Special"  Medium  Lan- 
tern Slide  films.  Both  these 
emulsions  have  a  medium  grain 
size  and  sensitivity.  The  emul- 
sion is  hydrated  in  distilled  water 
(18-20°C.)  for  10  minutes  and 
scraped  with  a  clean  edged  object 
(such  as  a  glass  slide)  into  the 
50  cc.  beaker. 

c)  "Stripping"  emulsions,  such  as 
Ilford  Special  Half  Tone  Strip- 
ping Plate,  that  can  be  stripped 
away  from  a  glass  or  film  support 
have  also  been  used  successfully. 
These  may  be  stripped,  hydrated, 
and  melted,  or  may  be  treated  as 
above. 

d)  Another  form  in  which  emulsion 
is  available  is  the  "pellicle"  which 
consists  of  a  fairly  thick  (250/x) 
layer  of  concentrated  silver  bro- 
mide grains  in  a  matrix  of  low 
gelatin  content.  Although  these 
pellicles  have  no  support,  they 
must  be  presoaked  in  distilled 
water  and  dupanol  C  solution 
(10  cc  of  water  and  10  cc  of  1% 
dupanol  per  1"  x  3"  pellicle)  for 
24  hours  at  18°C.  The  beaker 
containing  the  solution  and  the 
hydrated  pellicle  is  then  trans- 
ferred to  a  37°C.  water  bath. 
Eastman  Kodak  NTB2  and  NTBj 
pellicles  have  a  small  grain  size 
and  high  sensitivity  to  /3  radia- 
tions, NTBs  being  the  more 
sensitive  of  the  two. 

All  these  emulsions  are  ready  to  be 
applied  after  15  minutes  in  the  water 
bath. 

To  further  improve  resolution,  these 
emulsions  may  be  diluted  with  distilled 
water  and  dupanol  (Gross  et  al.,  re- 
ferred to  above). 

3.  Using  a  medicine  dropper,  two  drops 
of  melted  emulsion  are  applied  per 
square  inch  of  slide  to  be  coated. 
These  are  spread  quickly  and  evenly 
with  a  camel's  hair  brush  (kept  at 
37°C.)  over  the  outlined  area  and 
the  emulsion  is  allowed  to  gel  and 
dry  completely  on  a  leveling  table 
at  room  temperature. 

4.  The  dry  preparations  are  stored 
horizontally  in  light-tight  slide  boxes 
(plastic  are  recommended)  which 
are  kept  at  refrigerated  temperatures 


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301 


RADIOAUTOGRAPHY 


for  the  duration  of  exposure.  A 
drying  agent  (Drierite  or  CaClj)  in 
the  slide  box  prevents  the  hydration 
of  the  emulsion  during  exposure, 
thus  reducing  the  possibility  of 
fogging  due  to  the  solution  of  gases 
in  the  emulsion  and  also  causes  a 
sharper  image  to  be  produced. 

5.  After  a  suitable  exposure,  the  prepa- 
rations are  developed  in  D-72  de- 
veloper for  one  and  a  half  minutes, 
rinsed,  fixed  in  acid  fi.xer,  washed  in 
running  water  (18°C.)  for  15  min- 
utes, dehydrated  for  2  minutes  in 
each  of  the  following  solutions: 
95%  alcohol,  absolute  alcohol,  alco- 
hol-xylol,  and  cleared  in  three 
changes  of  xylol.  To  prevent  arte- 
facts, the  preparations  are  immersed 
in  a  1%  solution  of  balsam  or  a  50% 
solution  of  permount  for  at  least 
one  hour.  Longer  periods  of  time 
are  recommended  for  hard  tissues. 
Slides  should  at  all  times  be  kept 
horizontal. 

6.  The  sections  are  mounted  in  balsam 
or  permount  under  a  coverslip  and 
the  preparations  allowed  to  dry  at 
room  temperature. 

For  a  more  detailed  description  of  this 
method  see  Gross  et  al.,  referred  to 
above. 

In  "Strip  Coating":  Instead  of  using 
fluid  emulsion,  the  sections  may  be 
coated  with  a  Stripping  emulsion,  such 
as  Ilford  Special  half  tone  stripping 
plate  or  British  Kodak  stripping  plates 
for  autoradiograph}'.  Eastman  Kodak 
NTB  stripping  films  have  also  been 
used. 

1.  The  emulsion  strip  is  removed  from 
its  glass  support  by  cutting  the  emul- 
sion along  the  three  edges  of  the 
glass  plate. 

2.  The  emulsion  is  then  slowly  peeled 
off  to  about  1  inch  from  the  edge 
and,  with  the  side  that  formerly 
adhered  to  the  glass  facing  upwards, 
kept  for  5  minutes  in  a  shallow  dish 
containing  a  1%  solution  of  dupanol 
C  at  18-20°C. 

3.  The  microscope  slide  bearing  the 
section  is  slipped  underneath  the 
emulsion  and  lifted  together  with 
the  emulsion  out  of  the  water. 

4.  Three  edges  of  the  emulsion  are 
folded  underneath  the  slide  to  insure 
adherence.  (Pelc  dips  the  glass 
slides  in  gelatin  prior  to  mounting 
the  section  on  them.  This  also 
insures  adherence.) 

5.  The  slides  are  then  stored  dry  at 
1-2°C.  as  above. 

For  more  detailed  report  see  Berriman, 
R.  W.,  Hertz,  R.  H.,  and  Stevens,  G. 
W.  W.,  Brit.  J.  Radiol.,  1950,  23,  472- 


477;  Bogoroch,  R.,  Stain  Techn.,  1951, 
26,  43-50. 

The  difficult}^  with  the  coating  meth- 
ods as  described  is  that  the  prepara- 
tions must  be  stained  before  they  are 
coated.  This  procedure  in  some  cases 
such  as  P"  in  bone  removes  radio- 
active material,  thus  making  staining 
of  sections  before  radioautography  in- 
advisable. The  removal  of  P'^  is  negli- 
gible, however,  when  prestaining  is 
done  with  safranin  or  acid  fuchsin. 
By  using  both  stained  and  unstained 
preparations  at  the  same  time,  it  is 
possible  to  make  sure  of  the  extent 
of  the  loss.  It  is  also  possible  to 
completely  prevent  loss  by  using 
the  original  coating  method  first  de- 
scribed by  Belanger,  L.  F.,  and  Leblond, 
C.  P.,  Endocrinology,  1946,  39,  8-13, 
in  which  the  sections  were  stained 
through  the  developed  and  fi.xed  emul- 
sion. This  method  has  recently  been 
successfully  used  by  Arnold,  1951,  in 
the  study  of  calcium^  in  bone.  In 
this  case,  however,  success  may  be  ob- 
tained only  if  very  thin  emulsion  coats 
are  used  and  celloidin  is  not  deposited 
on  the  sections  prior  to  emulsion  coat- 
ing. The  danger  of  artefacts  produc- 
tion must  be  kept  in  mind. 

3.  Inverting  Method:  In  this  the  sec- 
tions are  coated  with  liquid  emulsions 
without  prestaining  of  the  sections 
(Belanger,  L.  F.,  Anat.  Rec,  1950, 
107,  149.  Kodak  matrix  emulsion  is 
used  for  best  results)  or  with  a  strip 
emulsion  (MacDonald,  A.  M.,  Cobb,  J., 
and  Solomon,  A.  K.,  Science,  1948,  107, 
550-552).  After  e.xposure  and  develop- 
ment the  preparation  is  placed  under 
water  and  the  emulsion,  with  the  sec- 
tion adhering  to  its  undersurface,  is 
lifted  from  the  slide  using  a  razor  blade. 

The  complex  is  then  inverted  and 
fixed  on  a  clean  slide,  section  side  up. 
The  free  edges  are  sealed  with  1% 
celloidin,  which  is  allowed  to  dry  com- 
pletely. The  section  may  now  be 
stained  with  ease  since  the  celloidin 
protects  the  emulsion  from  the  action 
of  the  dyes. 

The  preparation  is  mounted  under  a 
coverslip  using  Canada  balsam. 

4.  Wet  collodion  method:  The  Wet 
Collodion  method  (Gomberg,  H.  J., 
Nucleonics  (in  press)) — an  adaptation 
of  the  old  wet  collodion  process  of 
photography — involves  the  deposition 
of  a  very  thin  and  dense  layer  of  sensi- 
tized silver  bromide  in  a  collodion 
matrix  formed  by  chemical  reaction 
directly  on  the  surface  to  be  studied. 
After  a  suitable  exposure  time  in  a 
silver  nitrate  bath,  the  affected  silver 
grains  are  physically  developed  using 


RADIOAUTOGRAPHY 


302 


RADIOAUTOGRAPHY 


a  ferrous  sulfate  solution.  By  using 
a  physical  rather  than  a  chemical  de- 
velopment, it  is  theoretically  possible 
to  obtain  developed  grains  ranging 
from  10~V  to  1m  in  diameter.  This 
method,  however,  is  still  in  the  experi- 
mental stage  and  there  are  several 
serious  technical  difficulties  that  must 
be  overcome  before  the  method  can  be 
used  in  biological  research,  e.g.,  expo- 
sures for  more  than  two  days  have  not 
been  successful  due  to  the  great  accumu- 
lation of  background  fog. 

Quantitative  Determinations. 

Quantitative  results  have  been  ob- 
tained using  1)  densitometric  measure- 
ments, 2)  grain  counts,  and  3)  track 
counts. 

The  measurement  of  photographic 
densities  using  a  photometer  is  satis- 
factory for  work  at  low  magnifications. 
The  magnified  radioautographic  image 
is  projected  onto  a  frosted  glass,  and 
estimated  as  "densities"  (which  are 
measured  as  the  log  of  the  intensity  of 
incident  over  transmitted  light).  This 
method  has  been  used  by  Dobyns, 
B.  M.,  Skanse,  B.,  and  Maloof,  F. 
(J.  Clin.  Endocrinol.,  1949,  9,  1171- 
1184)  and  by  Axelrod,  D.  J.  and  Hamil- 
ton, J.  G.  (Am.  J.  Path.,  1947,  23,  389- 
412).  Much  work  remains  to  be  done 
before  densities  may  be  translated  in- 
to concentrations  of  radio-elements. 

The  counting  of  photographic  grains 
in  an  area  outlined  by  a  micrometer 
placed  in  the  ocular  is  the  most  satis- 
factory method  to  estimate  densities. 
It  may  be  used  with  small  or  large  speci- 
mens. All  the  grains  in  the  area  of  the 
emulsion  overlying  a  reactive  zone  may 
be  counted  (Pelc)  or  only  the  grains  at 
definite  levels  are  recorded.  Results 
have  been  obtained  with  the  latter 
method  in  this  Department  in  collabo- 
ration with  N.  Nadler  and  a  good  agree- 
ment with  theoretical  calculations  was 
found. 

The  results  may  be  expressed  in 
several  ways.  The  density  or  grain 
counts  in  one  structure  may  be  com- 
pared with  that  of  another  structure  in 
the  same  tissue  or  animal  or  they  may 
be  expressed  in  more  absolute  terms  by 
comparison  with  densities  or  grain 
counts  of  standards  radioautographed 
at  the  same  time  as  the  test  material 
and  containing  known  amounts  of 
radioactivity  and  geometry  (Dudley, 
R.  A.,  and  Dobyns,  B.  M.,  Science, 
1949,  109,  327-328;  Nadler,  N.  J.  and 
Bogoroch,  R.,  Anat.  Rec,  Supplement, 
1951,   109,  69). 

When  attempting  quantitative  com- 
parisons, the  geometry  of  the  source, 
the  distance  between  source  and  emul- 


sion, i.e.,  interspace,  the  emulsion  and 
section  thickness  must  all  be  taken  into 
consideration.  It  must  also  be  re- 
membered that  the  density  remains 
proportional  to  the  intensity  of  the 
source,  and  thus  to  the  concentration 
of  the  isotope,  only  when  the  exposure 
is  kept  to  a  minimum.  Otherwise  the 
relationship  between  density  and  con- 
centration is  more  complex. 

It  is  necessary  to  have  uniform  emul- 
sions of  absolute  thicknesses  (such  as 
stripping  films)  for  densitometric  meas- 
urements since  all  the  grains  in  every 
level  of  the  emulsion  are  measured. 
On  the  other  hand,  when  only  definite 
levels  in  the  emulsion  are  examined 
for  grain  counts,  small  variations  in 
emulsion  thickness  are  unimportant. 
The  liquid  coating  method  has  made 
it  possible  to  obtain  reproducible 
quantitative  results  under  these  condi- 
tions. 

The  counting  of  tracks  per  unit  area 
using  nuclear  emulsions  for  o  and  0  rays 
may  also  be  used  as  a  quantitative 
method  (Boyd,  G.  A.  and  Levi,  H., 
Science,  1950,  111,  58-59). 

Qualitative  Results. 

Space  does  not  permit  a  review  of 
the  interpretation  of  the  many  results 
that  have  been  obtained  using  the  vari- 
ous techniques  described  above.  Such 
a  review  with  bibliography  as  previ- 
ously mentioned  has  been  given  by 
Gross  et  al.  Only  a  few  examples  of 
the  more  extensive  work  with  this 
technique  will  therefore  be  cited. 

Foreign  substances  such  as  heavy 
metals  or  fission  products  have  been 
investigated  radioautographically  es- 
pecially by  the  Berkeley  group  under 
J.  G.  Hamilton.  The  actinide  and 
lanthanide  groups  of  elements,  adminis- 
tered as  their  ions,  are  found  to  be 
localized  primarily  in  bone  where  they 
remain  for  considerable  periods  of 
time.  The  elements  related  to  calcium 
localize  mostly  in  the  growing  ends  of 
bone. 

The  dynamics  of  phosphate  deposi- 
tion were  used  to  study  the  growth  of 
bone  by  Leblond,  C.  P.,  Wilkinson, 
G.  W.,  B^langer,  L.  F.,  and  Robichon, 
J.,  (Am.  J.  Anat.,  1950,  86,  289-341). 
By  studying  coated  and  inverted 
radioautographs  of  serial  sections  of 
bones  obtained  from  young  rats  sacri- 
ficed at  various  time  intervals  after  in- 
jection of  P^'^,  these  authors  found  that 
two  types  of  reactions  occurred  soon 
after  injection,  diffuse  and  localized. 
The  diffuse  reaction,  extending  through- 
out the  bone,  was  due  to  exchange 
between  labeled  circulating  phosphates 
and  the  surface  phosphates  of  crystals 


R ANSON  PYRIDINE 


303 


REED-STERNBERG  CELLS 


of  bone  salts.  These  reactions  dimin- 
ished rapidly  in  intensity  within  a  short 
interval  of  time  (one  day).  The  local- 
ized reactions,  on  the  other  hand,  did 
not  decrease  significantly  with  time 
and  were  due  to  a  deposition  of  labeled 
phosphate  salts  in  the  formation  of 
new  bone.  The  displacement  of  the 
new  bone  with  time  revealed  the  mecha- 
nism of  bone  formation. 

Similarly,  using  I"',  Leblond,  C.  P. 
and  Gross,  J.  (Endocrinol.,  1948,  43, 
306-320)  followed  the  synthesis  and 
degradation  of  the  protein,  thyroglobu- 
lin,  in  the  thyroid  gland  of  the  rat. 
They  found  that  labeled  iodide  verj' 
shortly  after  injection  into  normal  ani- 
mals was  incorporated  into  thyroglobu- 
lin  at  the  apical  portion  of  the  thyroid 
follicle  cells.  Later,  the  labeled  thyro- 
globulin  was  deposited  into  the  colloid 
where  it  was  eventually  broken  down 
into  amino  acids,  one  of  which  was  the 
hormone,  thyroxine  (which  then  diffuses 
out  of  the  follicle).  This  study  re- 
vealed that  regardless  of  the  physio- 
logical state  of  the  animals,  all  the  cells 
of  the  thyroid  gland  are  always  func- 
tioning in  one  direction.  The  rate  at 
which  the  cells  of  any  one  follicle  is 
functioning,  however,  changes  with 
the  physiological  state  of  the  animal. 

The  pathological  physiology  of  the 
thyroid  gland,  especially  in  reference 
to  the  detection  of  thyroglobulin  in 
neoplasms,  has  also  been  studied  radio- 
autographically  by  Marinelli,  L.  D., 
Foote,  F.  W.,  Hill,  R.  F.  and  Hocker, 
A.  F.,  (Am.  J.  Roentgen,  and  Radium 
Therap.,  1947,  58,  17-32),  Franz,  V.  K., 
Quimby,  E.  H.  and  Evans,  T.  C.  (Radi- 
ology, 1948,  51,  532-552),  Dobyns,  B. 
M.  and  Lennon,  B.,  (J.  Clin.  Endo- 
crinol., 1948,  8,  732-748),  and  more  e.x- 
tensively  by  Fitzgerald,  P.  J.  and  Foote, 
F.  W.,  (J.  Clin.  Endocrinol.,  1949,  9, 
1153-1170). 

Still  another  application  of  the  tech- 
nique was  used  by  Leblond,  C.  P., 
Stevens,  C.  E.  and  Bogoroch,  R.  (Sci- 
ence, 1948,  108,  531)  in  which  they  fol- 
lowed the  turnover  of  desoxyribonucleic 
acid  labeled  with  P^''  in  newly  formed 
cells.  This  method  not  only  gives  an 
indication  of  the  rate  of  formation  of 
cells  in  different  tissues  in  the  rat  body 
but  also  is  a  means  to  demonstrate  the 
displacement  and  fate  of  these  newly 
formed  cells. 

The  examples  cited  are  but  a  few  of 
the  important  contributions  to  biology 
and  medicine  that  have  been  made 
possible  through  the  use  of  the  radio- 
autographic  techniques  as  they  exist 
todaJ^  Many  more  contributions  can 
and  most  likely  will  be  made  with  these 


techniques — at  present  the  only  means 
available  to  obtain  minute  localiza- 
tion of  radioactive  substances. 

Ranson  Pyridine  method  for  unmyelinated 
nerve  fibers  (Ranson,  S.  W.,  Rev. 
Neurol.  &  Psychiat.,  1914,  12,  467-474). 
Fix  in  absolute  alcohol  -f  1%  ammonia, 
48  hrs.  Rinse  in  aq.  dest.  and  treat 
with  pyridine,  24  hrs.  Wash  repeatedly 
in  aq.  dest.,  24  hrs.  2%  aq.  silver  nitrate 
at  35°C.  in  dark,  3  days.  Rinse  in  aq. 
dest.  Reduce  in:  pyrogallol,  4  gm.; 
5%  formalin  in  aq.  dest.,  100  cc.  Wash 
and  imbed  in  paraffin.  This  much  used 
technique  gives  a  fine  blackening  of 
unmyelinated  fibers.  See  also  Ranson, 
S.  W.  and  Billingsley,  P.  R.,  J.  Comp. 
Neurol.,  1918,  29,  313-358;  Johnson,  S. 
E  ibid,  1928,  38,  299-314).  The  latter 
believes  the  essential  features  of  the 
technique  to  be  vascular  perfusion  with 
physiological  saline  solution  followed 
by  1%  ammonia  in  absolute  alcohol. 

Ranvier's  Gold  Chloride  method  for  nerve 
endings  in  muscle,  see  Craven's  and 
Carey's  methods.  See  also  Ammonia 
Carmine  and  Picrocarmine  of  Ranvier. 

Reconstruction.  Stereoscopic  x-ray  method 
(Morton,  W.  R.  M.,  J.  Anat.,  1940-41, 
75,  265-266);  wax  plate  method  as  ap- 
plied to  the  stapes  (Anson,  B.  J.,  Kara- 
bin,  J.  E.  and  Martin,  J.,  Arch.  Oto- 
laryng.,  1939,  29,  939-973). 

Rectal  Washings,  see  Papanicolaou  Tech- 
nique. 

Red  B,  see  Oil  Red  O. 

Red  Blood  Cell,  see  Erythrocyte. 

Red  Corallin,  (CI,  726).  Look  up  in  Colour 
Index. 

Red  Violet,  see  Hofmann's  Violet. 

Redox  dyes  are  those  emploj'^ed  in  reduc- 
tion-oxidation potential  determina- 
tions, see  Oxidation-Reduction  Po- 
tential. 

Reducing  Sugars,  Titrimetric  methods  for: 
Linderstr0m-Lang,  K.  and  Holter,  H., 
C.  rend.  trav.  lab.  Carlsberg,  S6r. 
Chim.,  1933,  19,  1-12;  and  Heck,  K., 
Brown,  W.  H.  and  Kirk,  P.  L.,  Mikro- 
chemie,  1937,  22,  306-314. 

Reed-Sternberg  Cells.  Recognition  of 
these  cells  is  helpful  in  reaching  a  diag- 
nosis of  Hodgkin's  disease.  Special 
technique  other  than  Hematoxylin  and 
Eosin  is  ordinarily  not  necessary. 
Comparison  by  Jackson,  H.,  Jr.  and 
Parker,  F.,  Jr.,  New  England  J.  Med., 
1944,  231,  35-44  of  Reed-Sternberg  Cells 
with  certain  other  multinucleated  cells 
will  be  helpful.  The  use  of  tissue  cul- 
ture technique  in  their  investigation 
opens  many  promising  leads  (Grand, 
C.  G.,  Proc.  Soc.  Exp.  Biol.  &  Med., 
1944,  56.  229-230).  Thus,  it  should  be 
possible  to  determine  their  life  history 
and  check  on  the  suggestion  that  the 


REFLECTING  MICROSCOPE 


304 


REISSNER'S  FIBER 


hyperchromatic  Sternberg  Cells  are  a 
later  development  of  Reed  cells  (Ber- 
sack,  S.  R.,  Am.  J.  Clin.  Path.,  1943, 
13,  253-259).  The  cytoplasmic  inclu- 
sions, reported  by  Grand,  are  sugges- 
tive of  virus  action.  The  claim  of  Sym- 
mers,  D.,  J.  A.  M.  A.,  1945,  128,  1248- 
1249,  that  these  cells  should  be  called 
Greenfield  Cells  in  honor  of  Greenfield's 
first  de.scription  in  1878  will  probably 
not  be  followed.  The  Phase  Micro- 
scope can  be  helpful  in  the  study  of 
Reed-Sternberg  cells  (Hoffmann,  J.  T. 
and  Rottino,  A.,  Blood,  1950,  5,  74-78). 
Reflecting  Microscope.  As  is  well  known, 
magnified  images  can  be  produced  by 
mirrors  as  well  as  by  lenses.  Proposals 
have  been  made  many  times  to  take 
advantage  of  the  special  properties  of 
mirror  systems  to  create  a  microscope 
of  a  pattern  analogous  to  the  reflecting 
telescope.  Such  an  instrument  would 
be  completely  achromatic,  and  thus 
superior  to  the  best  refracting  micro- 
scope objective,  the  apochromatic, 
which  is  corrected  for  only  three  colors 
of  chromatic  aberration  and  two  colors 
of  spherical  aberration.  There  would 
be  no  great  loss  of  light  by  reflection 
compared  to  the  considerable  loss  en- 
countered in  lenses  by  absorption. 
Furthermore,  a  reflecting  microscope 
focused  by  visible  light  would  also  be 
in  focus  for  ultraviolet  and  infra-red 
light,  thus  simplifying  the  process  of 
photography.  The  construction  of  sev- 
eral such  microscopes  has  been  de- 
scribed in  the  literature,  but  serious 
consideration  was  not  given  to  them 
because  their  numerical  apertures 
were  low  and  they  had  but  little  to 
offer  over  the  lens  type  of  microscope. 

The  rising  tide  of  interest  in  ultra- 
violet microscopy  has  stimulated  recent 
developments  in  this  field.  Burch  in 
England  (Burch,  C.  R.,  Proc.  Phys. 
Soc,  London,  1947,  59,  41-49)  has  de- 
signed a  long  focus,  reflecting  objective 
with  magnification  and  resolving  power 
equivalent  to  that  of  the  average 
"high-dry"  lens.  This  permits  one  to 
employ  a  micro-manipulator  and  to 
observe  objects  behind  thick  glass 
walls,  such  as  tissue  cultures. 

Another  design,  originating  in  Hol- 
land (Bouwers,  A.,  Achievements  in 
Optics,  New  York:  Elsevier,  1946,  135 
pp.)  has  been  put  into  commercial 
production  recently  by  Van  Leer  of 
Pittsfield,  Mass.  It  can  be  mass  pro- 
duced because,  unlike  Burch's  model, 
it  uses  only  spherical  reflecting  sur- 
faces. 

Bausch  and  Lomb  have  also  brought 
out  a  special  reflecting  objective  (Grey, 
D.  S.  and  Lee,  P.  H.,  J.  Opt.  Soc.  Am., 


1949,  39,  719-728).  This  instrument 
combines  both  lenses  and  mirrors  in 
order  to  gain  even  higher  magnification 
and  resolving  power.  It  is  apochro- 
matic from  220  to  800  m/i,  and  has  a 
working  distance  of  about  1  mm.  A 
reflecting  condenser  has  been  designed 
to  match  this  objective.  The  whole 
outfit  costs  about  $1,000.  Use  of  a 
reflecting  microscope  for  the  study  of 
cells  is  described  by  Mellors,  R.  C, 
J.  Nat.  Cancer  Inst.,  1950, 10,  1358-1361. 

Refractive  Index.  Microscopical  deter- 
mination bystandard  liquids.  See  paper 
by  Kunz,  A.  H.  and  Spulnik,J.,  Re- 
viewed in  J.  Roy.  Micr.  Soc,  1937,57, 
55. 

Regaud's  Fluid.  3%  aq.  potassium  bi- 
chromate, 20  cc. ;  formalin,  5  cc.  When 
this  is  used  for  mitochondria  fix  tissue 
for  4  days  changing  every  day  and  then 
mordant  in  3%  aq.  potassium  bichro- 
mate for  7  days  changing  every  second 
day.  It  is  a  fluid  that  can  be  profitably 
employed  for  many  other  purposes. 
Of  these  see  Giemsa's  Stain,  Lead, 
Masson's  Trichrome,  Romieu  Reac- 
tion and  Starch  Grains. 

Regaud's  Method  of  iron  hematoxylin  for 
mitochondria.  Fix  tissues  in  Regaud's 
fluid,  mordant,  imbed  and  section  as 
described  under  Anilin  Fuchsin  Methyl 
Green  Method.  Run  mounted  sec- 
tions down  to  water  and  mordant  for 
24  hrs.  in  5%  aq.  iron  alum.  Rinse 
quickly  in  aq.  dest.  (not  tap  water) 
and  transfer  to  hematoxylin  (made  by 
dissolving  1  gm.  hematoxylin  crystals 
in  10  cc.  abs.  ale.  adding  10  cc.  glycerin, 
80  cc.  aq.  dest.  and  allowing  to  ripen 
3  weeks).  If  traces  of  iron  alum  are 
carried  to  the  stain  they  will  do  no  harm, 
but  if  too  much  enters  the  hematoxylin 
a  dense  black  precipitate  will  form  and 
ruin  the  hematoxylin.  On  the  other 
hand,  if  the  sections  are  washed  ex- 
cessively in  aq.  dest.  too  much  of  the 
alum  will  be  removed  and  the  hema- 
toxylin will  not  stain  as  intensely  as  it 
should.  The  happy  mean  must  be  de- 
termined. The  hematoxylin  should  be 
used  over  again  about  10  times.  Differ- 
entiate in  5%  aq.  iron  alum  under  low 
magnification.  Wash  in  running  tap 
water  (not  aq.  dest.)  1  hr.  This  should 
bring  out  the  blue-black  color  of  the 
hematoxylin  stain.  Dehydrate,  clear  and 
mount.  Various  counterstains  can  be 
used  if  desired.  Consult  Meves'  beauti- 
ful figures  of  collagenic  fibers  stained 
with  fuchsin  (Meves,  F.,  Arch.  f.  Mikr. 
Anat.,  1910,  75,  149^208).  This  is  the 
most  permanent  stain  for  mitochondria 
but  lacks  the  color  contrast  afforded  by 
anilin  fuchsin  methyl  green. 

Reissner's    Fiber,     staining    reactions    of 


RELIEF  METHODS 


305 


RETICULAR  FIBERS 


(Jordan,  H.,   Am.  J.   Anat.,   1925,  34, 
427-443). 

Relief  Methods,  see  Negative  Stains. 

Replacement  of  Tissue  to  take  the  place  of 
that  worn  out  or  lost  can  now  be 
measured  more  accurately.  Though 
some  signs  of  youth  and  age  of  cells 
can  be  detected  (Chapter  24  in  Cowdry, 
E.  v.,  Problems  of  Ageing.  Baltimore: 
Williams  &  Wilkins,  1942,  936  pp.), 
it  is  not  so  easy  to  determine  the  per- 
centage actually  dying  as  the  per- 
centage of  new  cells  produced  to  replace 
them  by  counting  mitoses.  Using  whole 
mounts  of  separated  human  Epidermis 
from  foreskins  removed  by  circumcision 
Cooper,  Z.  K.,  and  Schiff,  A.,  Proc. 
Soc.  E.xp.  Biol.  &  Med.,  1938,  39,  323- 
324  have  discovered  that  the  produc- 
tion of  new  cells  is  rhythmic  being 
greatest  at  night  and  least  by  day.  To 
obtain  material,  as  they  did  every  hour 
of  the  day  and  night,  of  other  human 
tissues  seems  impossible.  If  one  wishes 
to  investigate  rate  of  cellular  replace- 
ment in  internal  less  accessible  tissues 
that  are  replaceable,  take  advantage  of 
the  fact  that  the  drug,  colchicine,  per- 
mits cells  to  enter  mitosis  but  arrests 
the  process  usually  in  themetaphase. 
In  consequence  of  this  experimental 
summation  many  more  mitoses  can  be 
counted  in  a  given  specimen  than  would 
be  found  if  cell  division  had  been 
completed  as  usual  (See  Mitosis  for 
the  necessary  controls).  There  are  no 
special  means  for  the  study  of  replace- 
ment of  Fibers  but  careful  use  of  avail- 
able techniques  will  probably  yield 
data  as  to  whether  the  fibers  are  newly 
formed  or  old  and  practically  useless. 
Phj'sico-chemical  methods  are  how- 
ever promising  when  backed  by  histo- 
logical researches.  Thus  the  new  bone 
formed,  during  the  time  that  Madder, 
or  better  Alizarin  Red  S,  is  made  avail- 
able in  the  circulation  can  be  measured. 
In  adult  animals,  assuming  that  the 
amount  of  bone  remains  approximately 
constant,  it  can  be  concluded  that  the 
breakdown  is  at  the  same  rate  and  in 
this  round  about  way  arrive  at  a  figure 
for  replacement. 

Some  fats  can  be  conveniently  colored 
with  fat  soluble  dyes  which  they  retain 
on  ingestion  and  after  incorporation  in 
the  fatty  depots  of  the  body.  It  should, 
therefore,  be  possible  to  keep  animals  at 
a  fairly  constant  weight  on  a  diet  con- 
taining a  certain  amount  of  fat,  to  sub- 
stitute for  this  fat  stained  fat  of  the 
same  sort  without  increasing  their 
weight  and  to  estimate  the  ratio  of  stained 
to  unstained  fat  after  a  definite  interval 
of  time — in  other  words  the  replace- 
ment.   Other  possibilities  are  to  employ 


for  the  test  a  fat  of  melting  point  quite 
different  from  the  native  body  fat  of  the 
animals;  and  fatty  acids  tagged  with 
radioactive  isotopes,  see  Fatty  Acids. 
The  radioactive  isotopes,  particu- 
larly those  of  Phosphorus  and  Iron  give 
somewhat  similar  clues.  The  amount  of 
radiophosphorus,  for  example,  accumu- 
lating in  any  particular  tissue  can  be 
accurately  determined.  If  the  supposi- 
tion is  justified  that  the  total  amount  of 
phosphorus  (radioactive  and  non -radio- 
active) remains  about  the  same,  then 
non -radioactive  phosphorus  must  be  lost 
at  the  rate  that  the  radiophosphorus 
enters.  It  is  too  soon  however  to  pre- 
dict what  this  possible  line  of  investiga- 
tion with  the  isotopes  will  show.  See 
Radiophosphorus. 

Resazurin,  a  compound  used  as  an  o.xida- 
tion-reduction  indicator. 

Resorcin  Blue  (CI,  908) — fluorescent  blue, 
iris  blue — Often  called  Lacmoid.  See 
Nebel,  B.  R.,  Stain  Techn.,  1931,  6, 
27-29. 

Resorcin-Fuchsin,  see  Weigert's  resorcin- 
fuchsin  method  for  elastic  fibers. 

Respiratory  System.  This  contains  very 
diversified  structural  components  for 
which  no  single  technique  or  group  of 
techniques  can  be  offered.  But  the 
interpretation  of  the  preparations  de- 
pends, as  in  all  systems  of  the  body, 
on  the  age.  A  chapter  by  Macklin,  C. 
C.  and  M.  T.,  in  Cowdry 's  Problem  of 
Ageing,  Baltimore :  Williams  and  Wil- 
kins, 1942,  936  pp.,  gives  the  necessary 
background  and  numerous  hints  and 
references  to  technique.  See  Lungs, 
Trachea,  Nasal  Passages  and  Nasal 
Sinuses. 

Respiratory  Tract  Smears,  see  Papanicolaou 
Techniques. 

Respirometry,  see  Capillary  Respirometry. 

Restaining  Faded  Sections.  This  is  some- 
times very  desirable.  Try  technique 
outlined  by  Small,  C.  S.,  (Amer.  J.  Clin. 
Path.,  Techn.  Suppl.,  1943,  7,  66-67). 

Reticular  Fibers.  These  are  more  finely 
divided  and  tend  more  to  form  a  reticu- 
lum than  the  collagenic  fibers.  Yet 
there  may  be  anatomical  continuity  be- 
tween collagenic  and  reticular  fibers  and 
there  is  reason  to  believe  that  the  two 
are  fundamentally  similar.  They  are 
not  so  conveniently  viewed  in  the  fresh 
condition  because  to  make  thin  spreads 
is  more  difficult.  For  details  see 
Maximow,  A.  A.,  von  Mollendorf's 
Handbuch  der  Mikroskopischen  Anato- 
mic des  Menschen,  1927,  2  (1),  232-583. 
The  principal  methods  for  reticular 
fibers  in  sections  involve  silver  impreg- 
nation (Perdrau,  Foot,  Wilder,  Gomori 
and  Laidlaw),  the  choice  of  which  will 
to  some  extent  depend  on  the  kind  of 


RETICULAR  AND  COLLAGEN  IC  306 

FIBERS 


REVIVAL  OF  VINEGAR  EELS 


tissue  studied.  There  are,  however, 
several  which  are  stains  (Kinney's 
Method  and  Biebrich  Scarlet  and 
Picro-Anilin   Blue). 

Reticular  and  Collagenic  Fibers  in  frozen 
sections  (Krajian,  A.  A.,  Arch.  Path., 
1933,  16,  376-378).  Cut  frozen  sections 
5-lOii  thick  of  tissue  fixed  in  10%  forma- 
lin and  wash  in  3  changes  aq.  dest. 
After  treating  with  10%  aq.  ammonium 
hydroxide  at  60°C.  for  15  min.  (in  par- 
affin oven)  wash  them  again  in  3  changes 
aq.  dest.  Place  in  0.3%  aq.  potassium 
permanganate  for  5  min.,  wash  in  aq. 
dest.  for  a  few  seconds  and  decolorize 
in  1.5%  aq.  oxalic  acid  till  brown  color 
has  j  ust  disappeared .  Wash  thoroughly 
in  aq.  dest.  and  place  in  5%  aq.  silver 
nitrate  at  60°C.  for  1  hr.  Wash  in  2 
changes  aq.  dest.  and  place  in  ammonia- 
cal  silver  nitrate  solution  at  60°C.  for 
15  min.  (To  make  this  solution  add 
6  drops  10%  aq.  sodium  hydroxide  to 
8  cc.  10%  aq.  silver  nitrate  which  gives 
a  brownish  black  precipitate.  Add 
fresh  10%  aq.  ammonium  hydroxide 
drop  by  drop  until  only  a  few  small 
particles  of  the  precipitate  remain. 
Dilute  to  28  cc.  with  aq.  dest.).  Wash 
sections  quickly  in  3  changes  aq.  dest. 
Treat  them  with  30%  formalin  at  60°C. 
1-3  min.,  wash  in  large  amount  of  tap 
water  and  mount  on  slides.  Cover 
sections  with  a  little  absolute  alcohol 
and  blot  into  position.  Then  complete 
dehydration  with  absolute,  blot,  clear 
in  equal  parts  aniline  oil  and  xylol. 
Wash  in  xylol  and  mount  in  gum  dammar 
or  Canada  balsam.  Reticulum  black; 
collagen,  brown. 

Reticulocytes.  These  are  the  stages  recog- 
nized in  the  red  series  before  the  as- 
sumption of  properties  of  Erythrocytes. 
An  excellent  review  of  the  properties  of 
reticulocytes  is  supplied  by  Orten,  J. 
M.,  Yale  J.  Biol.  &  Med.,  1933-34,6, 
519-539.  Reticulocytes  can  easily  be 
identified  by  supravital  staining  with 
brilliant  cresyl  blue.  First  make  a  thin 
film  of  the  dye  on  slide  by  allowing  a  1% 
solution  in  absolute  alcohol,  spread 
evenly,  to  evaporate.  Then  mount  fresh 
blood,  ring  withvaseline  and  observe.  To 
make  relatively  permanent  specimens, 
remove  the  cover  glass  after  2  min . ,  smear 
dry  and  color  by  Wright's  Stain.  The 
supravital  staining  with  cresyl  blue  is 
inhibited  by  certain  substances  (Heath, 
C.  W.  and  Daland,  G.  A.,  Arch.  Int. 
Med. ,  1931 ,  48,  133-145) .  For  a  calcula- 
tion of  experimental  error  in  reticulo- 
cyte counts,  see  Marcussen,  P.  V., 
Folia  Haemat.,  1938-39,  61,  49-64  and 
for  fragility  tests,  see  Mermod,  C. 
and  Dock,  W.,  Arch.  Int.  Med.,  1935, 
55,     52-60.    Resistance    to     hypotonic 


sodium  chloride  solutions  is  described 
by  Daland,  G.  A.,  and  Zetzel,  L.,  Am. 
J.  Med.  Sci.,  1936,  191,  467-474.  The 
protoporphyrin  content  of  reticulocytes 
can  be  estimated  by  the  fluorescence 
technique.  Watson  and  Clarke  (C.  J. 
and  W.  O.,  Proc.  Soc.  Exp.  Biol.  & 
Med.,  1937,  36,  65-70)  have  discovered 
that  it  is  greater  than  in  erythrocytes 
and  that  brilliant  cresyl  blue  is  pre- 
cipitated by  protoporphyrin  which  may 
explain  the  characteristic  staining  of 
reticulocytes  by  this  dye. 

Reticulo-Endothelial  Blockade.  Supposed 
to  be  a  method  whereby  R.  E.  cells  are 
so  blocked  by  the  ingestion  of  one 
foreign  material  that  they  are  unable  to 
take  in  another.  For  experiments  with 
India  ink  and  brilliant  vital  red  and 
critical  statement,  see  Victor,  J.,  Van 
Buren,  J.  R.,  and  Smith,  H.  P.,  J. 
Exper.  Med.,  1930,  51,  531-548. 

Reticulo-Endothelial  System.  This  is  by 
definition  made  up  of  the  reticular  cells 
of  the  connective  tissues  plus  certain 
special  endothelial  cells  chiefly  located 
in  the  spleen,  liver,  bone  marrow, 
adrenals  and  lymph  nodes.  All  have 
the  common  property  of  phagocytosing 
particulate  matter  such  as  trypan  blue, 
carbon,  etc.  These,  and  possibly 
others,  may  leave  their  moorings  and 
become  free  cells  when  they  become 
known  as  Monocytes  or  Macrophages. 
A  better  term  is  the  "system  of  macro- 
phages" (or  big  eaters)  in  which  empha- 
sis is  placed  on  function  not  origin.  See 
Vital  Staining. 

Retina,  see  Eyes. 

Retterer's  Stain  for  muscle.  Fix  in  10 
parts  80%  alcohol  plus  1  part  formic 
acid.  Stain  paraffin  sections  with  alum 
carmine.  Muscle  light  red,  all  connec- 
tive tissue  unstained. 

Revival  of  Vinegar  Eels  after  Ultrarapid 
Cooling. — Written  by  B.  J.  Luyet, 
Dept.  of  Biology,  St.  Louis  University. 
January  15,  1951 — A  drop  of  a  con- 
centrated vinegar  eel  suspension,  ob- 
tained by  centrifugation,  is  deposited 
on  a  glass  slide  and  most  of  the  remain- 
ing vinegar  is  blotted  off.  Then  a  drop 
of  30%  ethylene  glycol  is  added  to  the 
squirming  mass  in  order  to  reduce  some- 
what the  water  content  of  the  worms. 
After  about  5  minutes  the  excess  ethyl- 
ene glycol  is  blotted  off,  and  the  eels, 
still  moving  actively,  are  "wiped  up," 
in  a  thin  layer,  on  very  thin  pieces  of 
mica  (about  5  mm.  on  a  side  and  about 
35  micra  thick).  The  eels,  supported 
on  this  mica  slip,  are  then  immersed  in 
liquid  air.  After  about  one  minute 
they  are  removed,  and,  by  means  of  a 
vigorous  shake  of  the  hand,  the  droplet 
of  liquid  air  which  may  adhere  to  the 


RHENIUM 


307 


RICKETTSIA 


mica  support  is  dislodged,  whereupon 
the  preparation  is  abruptly  swished  in 
a  little  water  (about  2  cc.)  in  a  watch 
glass,  at  room  temperature  or  prefer- 
ably at  30°C.  (The  purpose  of  the 
immersion  in  water  is  rapid  rewarming.) 
After  about  5  minutes  one  sees,  under  a 
low  power  microscope,  several  eels  be- 
gin to  move  and,  after  about  ten  min- 
utes, some  50  out  of  200  in  the  drop 
become  quite  active,  though  they  are 
never  entirely  normal.  See  Luyet,  B., 
C.  rend.  Soc.  Biol.,  1938,  127,  788-789. 

Rhenium,  see  Atomic  Weights. 

Rheonine,  A,  Synonymous  with  Rheonine 
AL,  G  or  N  and  Fast  Phosphine  NAL, 
an  acridine  dye  occasionally  employed 
in  fluorescence  microscopy  (Conn,  H. 
J.  in  McClung's  Microscopical  Tech- 
nique, 1950,  p.  98). 

Rhodamine  B  (CI,  749)— brilliant  pink  B, 
rhodamine  O — A  basic  xanthene  dye. 
It  gives  a  good  color  contrast  with 
methylene  blue  in  coloration  of  the 
spleen  (Houcke,  E.,  C.  Rend.  Soc.  de 
Biol.,  1928,  99,  788-789). 

Rhodamine  O,  see  Rhodamine  B. 

Rhodamines.  Similar  in  some  respects  to 
pyronins  but  there  is  a  third  benzene 
ring  affixed  to  central  carbon  atom  and 
to  this  in  turn  is  attached  a  carboxyl 
in  ortho  position.  Examples:  Rhoda- 
mine B  and  fast  acid  blue  R.  Rhoda- 
min  B  (Merck)  and  6G  IG.  have  been 
employed  as  vital  stains.  When  used 
with  plant  cells  mitochondria  become 
fluorescent  (Strugger,  S.,  Protoplasma, 
1938,  30,  85-100). 

Rhodanid.     Ammonium  thiocyanate. 

Rhodium,  see  Atomic  Weights. 

Rhodopsin  (G.  rhodon,  rose  +  ops,  eye)  is 
a  photosensitive  visual  purple  pigment 
of  the  rods  of  the  retina  easily  seen  in 
ordinary  microscopic  preparations.  It 
is  a  protein  vitamin  A  combination. 
Night  blindness  is  an  indication  of 
vitamin  A  deficiency. 

Riboflavin  (lactoflavin)  shows  typical  green 
fluorescence  in  living  liver  and  kidney 
observed  under  fluorescence  micro- 
scope (Ellinger,  P.,  and  Koschara,  W., 
Ber.  deutsch.  Chem.  Ges.,  1933,  66, 
315-317,  808-813,  1411-1414).  Detected 
also  in  Malpighian  tubules  of  American 
roach  (Metcalf,  R.  L.  and  Patton,  R.  L., 
J.  Cell  and  Comp.  Physiol.,  1942,  19, 
373-376)  and  in  tomato  plants  (Bonner, 
J.  and  Borland,  R.,  Am.  J.  Bot.,  1943, 
30,  1008-1009).  See  Hirt,  A.  and  Wim- 
mer,  K.,  Klin.  Wochnschr.,  1939,  18, 
733-740.     See  Vitamine  B2 

Rinbonuclease  is  an  enzyme  which  degre- 
dates  ribonucleic  acid.  When  used  to 
remove  ribonucleic  acid  from  sections 
for  histochemical  observations,  (Sto- 
well,  R.  E.  and  Zorzoli,  A.,  Stain  Tech. 


1947,  22,  51-61)  pure  preparation 
(McDonald,   M.    R.,   J.   Gen.   Physiol. 

1948,  32,  39-42)  is  desirable.  See 
Gram  Staining,  Nuclei  Acids  and 
Nuclease. 

Ribonucleic  Acid.  A  type  of  Pentose 
Nucleic  Acid  containing  the  ribose 
type  of  sugar  present  in  yeast.  Also 
known  as  UNA.  (Ribonucleic  Acid) 
Use  of  electrotitration  technique  shows 
that  yeast  ribose  nuclei  acid  under  the 
influence  of  ribonuclease  yields  a  sec- 
ondary phosphate  and  a  hydroxyl  group 
(of  a  purine  or  pyrimidine).  See 
Vandendriessche,  L.,  C.  rend,  trave. 
Lab.  Carlsberg,  S^r.  Chim.,  1951,  27, 
341-391. 

Rickettsia  are  small,  gram  negative,  bacteria- 
like  organisms  which  are  insect  trans- 
mitted and  typically  inhabit  endothelial 
cells  of  vertebrate  hosts  named  after 
H.  T.  Ricketts  who  died  of  typhus  fever 
while  investigating  them.  They  are 
best  stained  by  Giemsa's  method 
after  fixation  in  Zenker's,  Bouin's  or 
Regaud's  fluids. 

1.  Rapid  staining  with  thionin.  Make 
sat.  sol.  of  thionin  in  aq.  dest.  Pre- 
cipitate by  adding  10%  NaOH.  Collect 
ppt.  on  filter  and  wash  until  filtrate 
becomes  neutral.  Dissolve  ppt.  in  2% 
phenol.  Stain  absolute  alcohol  fixed 
smears  only  30-50  sec.  Drain,  wash 
quickly  in  absolute  alcohol,  clear  in 
xylol  and  mount  in  cedar  oil.  Rick- 
ettsia, deep  violet;  cytoplasm,  light 
violet ;  red  cells  bluish  green  (Laigret, 
J.  and  Auburtin,  P.,  Bull.  Soc.  Path. 

-exat.,  1938,  31,  790-791). 

2.  Fuchsin  staining  method.  Smear 
tissue  culture  on  slide.  Dry  in  air, 
then  by  heat.  Filter  directly  on  to 
smear  0.25%  basic  fuchsin  in  phosphate 
solution  buffered  to  pH  7.4  or  in  aq. 
dest.  made  pH  7.2-7.4  by  adding  sodium 
hydrate  or  carbonate  (see  Buffers) . 
Stain  4  min.  Wash  quickly  with  0.5% 
aq.  citric  acid.  Pour  off  citric  and  wash 
rapidly  in  tap  water.  Counterstain  in 
1%  aq.  methylene  blue,  10  sec.  Rick- 
ettsia, red;  cells,  blue;  not  recom- 
mended for  tissue  sections  (Zinsser,  H., 
Fitzpatrick,  F.  and  Hsi  Wei,  J.  Exp. 
Med.,  1939,  69,  179-190).  This  is  very 
similar  to  Michiavello's  method  de- 
scribed by  Cox  (H.  R.,  Publ.  Health 
Rep.,  1939,  53,  2241-2247)  as  superior 
to  Giemsa's  stain  for  Rickettsiae  of 
Rocky  Mt.  Spotted  Fever  and  Typhus 
groups. 

The  Michiavello  technique  has  been 
adapted  for  sections  by  Pinkerton  (see 
Harry  Plotz,  in  Simmons  and  Gentz- 
kow,  p.  572).  Stain  paraffin  sections 
after  Regaud  fixation  overnight  in  1% 
aq.  methylene  blue  and  decolorize  in 


RICKETTSIA  ORIENTALIS 


308 


ROULEAUX  FORMATION 


95%  alcohol.  Counterstain  with  0.25% 
aq.  basic  fuchsin  for  30  min.  Decolor- 
ize quickly  (say  3  sec.)  in  0.5%  aq. 
citric  acid.  Differentiate  rapidly  in 
abs.  ale.  clear  in  xylol  and  mount  in 
dammar.  Rickettsiae,  deep  red;  sur- 
rounding tissue,  partly  red.  Back- 
ground can  be  made  bluer  by  washing 
lightly  in  aq.  dest.  after  the  citric  acid 
treatment  and  by  staining  again  with 
methylene  blue,  before  differentiation 
in  95%  alcohol  dehydration,  clearing 
and  mounting  as  advised  by  Plotz. 
Plotz  gives  details  of  use  of  Michia- 
vello's  stain  in  demonstration  of  Rick- 
ettsiae in  yolk  sac  cultures. 

A  fuchsin  and  methyl  violet  combina- 
tion is  recommended  for  typhus  fever 
Rickettsiae  by  Nyka,  W.,  J.  Path.  & 
Bact.,  1944,  56,  264. 

See  cultivation  of  Rickettsiae  in  eggs 
(Fitzpatrick,  F.  K.,  J.  Lab.  &  Clin. 
Med.,  1946,  31,  45-55),  Typhus  Fever 
rickettsiae,  and  Rickettsia  orientalis. 
A  convenient  list  of  pathogenic  Rick- 
ettsia is  provided  by  Pinkerton,  H., 
Bact.  Rev.,  1942,  6,  37-78. 

Rickettsia  orientalis.  Rapid  method  for 
staining  in  smears  by  Clancy,  C.  F.  and 
Wolfe,  D.  M.,  Science,  1945,  102,  483. 
Air  dry  smears  of  infected  yolk  sac 
membranes,  or  of  other  tissues,  and  fix 
by  heat.  Flood  slide  with  xylol,  dry 
in  air  current,  immerse  in  1:5,000 
methylene  blue  and  basic  fuchsin  in  aq. 
dest.  for  5  min.  Wash,  dry  and  ex- 
amine. Organisms  blue,  background 
pinkish  purple.  Grams  should  be  di- 
luted from  1%  stock  solutions  on  the 
day  used. 

Ringer  solution.  NaCl,  0.85  gm.;  KCI, 
0.025  gm.;  CaCU,  0.03  gm.;  aq.  dest., 
100  cc.  Lee  (p.  731)  advises  for  am- 
phibians same  except  that  NaCl  is  0.65 
gm.  and  NaHCOs,  0.02  gm.  is  added  to 
make  pH  about  7.0-7.4.  If  NaHCOs  is 
present  it  must  not  be  sterilized  by 
heat. 

Ringer-Locke  solution.  NaCl,  0.85  gm.; 
KCI,  0.042  gm.;  CaCh,  0.025  gm.; 
NaHCOs,  0.02 gm.;aq.  dest.,  100  cc.  for 
cold  blooded  animals.  Lee  (p.  73)  ad- 
vises same  except  that  NaCl  is  0.65  gm. 
Should  be  freshly  made.  Owing  to 
presence  of  NaHCOa  must  not  be  steri- 
lized by  heat. 

Rivanol,  a  dye  sometimes  employed  in 
fluorescence  microscopy. 

Roberts,  see  Paper  Chromatography. 

Rocky  Mountain  Spotted  Fever,  see  Rick- 
ettsia. 

Roller  Tube  Cultures.  Control  of  pH  in,  see 
paper  by  Paff,  G.  H.,  Proc.  Soc.  Exp. 
Biol.  &  Med.,  1946,  62,  184-187.  See 
Tissue  Culture. 

Romanowsky    Stains    contain    polychrome 


methylene  blue  eosinates.  Those  of 
Wright,  Leishman  and  Wilson  are  well- 
known.  The  Romanowsky  effect  is 
the  lavender-red  coloration  by  them  of 
the  nuclei  of  lymphocytes,  monocytes, 
protozoa  and  other  materials.  Acetone 
solvents  for  Romanowsky  stains  (Kings- 
ley,  D.  M.,  J.  Lab.  &  Clin.  Med., 
1936-37,  22,  524-531).  Polychroming 
process  (i6 id,  736-752).  Dyes  for  (ibid, 
1264-1273).     Large     bibliographies. 

Romieu  Reaction  for  proteins.  Fix  in 
formalin,  in  alcohol  or  in  Bouin's  fluid. 
Make  rather  thick  sections  in  paraffin 
or  preferably  in  celloidin.  Cover  sec- 
tion with  a  drop  of  syrupy  phosphoric 
acid.  After  few  minutes  in  oven  at 
56°C.  examine  directly.  A  red  or 
violet  color  develops  in  location  of  pro- 
teins. According  to  Blauchetiere  and 
Romieu  (A.  and  B.,  C.  Rend.  Soc.  de 
Biol.,  1931,  107,  1127)  it  is  due  to  the 
tryptophane  grouping.  See  Lison,  p. 
129. 

Rongalite  White,  said  to  stain  normal  but 
not  cancerous  cells  (Roskin,  G.,  Bull. 
d'Hist.  appl.,  1938,  15,  20-23). 

Rosanilin  (Magenta  I)  is  triamino-tolyl- 
diphenyl-methane  chloride,  a  compo- 
nent of  most  Basic  Fuchsins.  Rosan- 
ilin with  methylene  blue  for  Negri 
bodies  (Schleifstein,  J.,  Am.  J.  Pub. 
Health.,  1937,  27,  1283-1285). 

Rosazine,  see  Azocarmine  G. 

Rose  Bengal  (CI,  779).  A  xanthene  dye  of 
fine  color  used  for  several  purposes 
including  the  staining  of  Soil  Bacteria 
by  Conn  (p.  157).  Make  suspension 
of  soil  in  9  times  its  weight  of  0.015% 
aq.  gelatin.  Spread  drop  on  clean  slide 
and  dry  over  boiling  water  bath.  Cover, 
while  still  on  bath  for  1  min.,  with  rose 
bengal  1  gm.;  CaClj,  0.01  gm.;  5%  aq. 
phenol,  100  cc.  Wash  quickly  in  water. 
Dry  and  examine.  Used  to  demon- 
strate injured  liver  cells  by  vital  stain- 
ing (Williams,  W.  U.,  Yale  J.  Biol,  and 
Med.,   1950,   23,  177-183).     See  Eosins. 

Rosenthal,  see  Fluorescence,  Microscopy, 
Radioactive  Isotopes,  Electron  Micros- 
copy. 

Rosin  U.S. P.  XI  (Colophony,  yellow  resin, 
abietic  anhydride)  used  in  Grieves' 
method  for  undecalcified  dental  tissues 
and  bone. 

Rosinduline  GXF,  see  Azocarmine  G. 

Rosophenine  lOB,  see  Thiazine  Red  R. 

Rouget  Cells,  see  Pericapillary  cells. 

Rouleaux  Formation  of  red  cells.  They 
are  stacked  like  coins,  outlines  distinct, 
usually  break  up  on  shaking,  diminished 
on  washing  with  saline  solution; 
whereas  agglutinated  reds  stick  to- 
gether irregularly,  outlines  indistinct, 
usually  become  more  compact  on  shak- 
ing, not  affected  by  washing. 


RUBBER 


309 


SAFRANIN-LIGHT  GREEN 


Rubber.  To  stain  rubber  in  tissues  many 
techniques  have  been  reported  by 
Haasis,  F.  W.,  Stain  Techn.,  1945,  20, 
37-38.  The  work  was  done  in  Guayule 
studies  under  project  of  Bureau  of 
Plant  Industry.  Micromanipulation  in 
study  of  latex  particle  of  rubber 
(Hauser,  E.  A.,  Ind.  Eng.  Chem.,  1926, 
18,  1146-1147). 

Rubber.  To  stain  rubber  in  tissues  many 
techniques  have  been  reported  by 
Haasis,  F.  W.,  Stain  Techn.,  1945,  20, 
37-38.  The  work  was  done  in  Guayule 
studies  under  project  of  Bureau  of  Plant 
Industry. 

Rubber  Paraffin.  Johnson,  J.  (Applied 
Micr.,  1903,  6,  2662)  has  recommended 
1%  crude  India  rubber  in  paraffin  col- 
ored amber  yellow  by  addition  of  asphalt 
heated  to  100°C.  1-2  days.  The  super- 
natant fluid  is  poured  off  and  used  as 
ordinary  paraffin.  Double  Imbedding 
in  celloidin  and  paraffin  has  been  sug- 
gested. See  Beyer,  E.  M.  (Am.  J. 
Clin.  Path.,  Tech.  Suppl.,  1938,  2, 
173-175). 

Rubidium,  see  Atomic  Weights. 

Russell-Body  Cells,  Russell  bodies  and  the 
cytoplasm  of  plasma  cells  are  probably 
not  hemoglobiniferous  because  they  do 
not  react  as  do  the  substances  in  known 
hemoglobiniferous  cells  with  reference 
to  isoelectric  point  of  hemoglobin 
(Kindred,  J.  E.,  Stain  Techn.,  1935,  10, 
7-20). 

Ruthenium,  see  Atomic  Weights. 

Ruthenium  Red  is  ammoniated  ruthenium 
oxychloride,  a  mineral  pigment.  Conn 
(p.  187)  says  that  it  is  used  microscopi- 
cally as  a  test  for  Pectin  for  which  some 
consider  it  to  be  specific. 

Ruthenium  Tetroxide,  as  a  fixative  said  to 
be  superior  in  some  ways  to  osmium 
tetroxide;  but  it  decomposes  readily 
and  penetrates  poorly.  To  prevent 
decomposition  make  1%  sol.  in  sat. 
chlorine  water  (Carpenter,  D.  C.  and 
Nebel,  B.  R.,  Science,  1931,  74,  154-155). 

Saffron,  a  yellow  dye  obtained  from  the 
plant.  Crocus  sativus.  Long  cultivated 
in  Persia  this  plant  was  introduced  into 
China  by  the  Mongols  and  throughout 
the  Orient.  In  the  early  days  of  Greece 
saffron  was  the  official  color.  Saffron 
was  spread  on  the  streets  of  Rome  to 
welcome  the  Emperor  and  his  army. 
Some  monks  discovered  that  by  use  of 
an  iron  mordant  and  saffron  manu- 
scripts could  be  cheaply  made  to  appear 
golden.  The  City  of  Florence  for  a 
time  incorporated  the  saffron  blossom 
in  its  coat  of  arms.  Later  the  City  of 
Basle,  Switzerland,  followed  suit  and 
the  "Saffron  war"  resulted  in  1374  A.D. 
This  acknowledged  imperial  color  has 
come  down  through  the  ages;  witness 


the  yellow  roofs  of  the  Imperial  and 
Forbidden  Cities  in  Peking.  For  a 
valuable  account  read  Leggett,  W.  F., 
Ancient  and  Medieval  Dyes.  Brook- 
lyn: Chemical  Publishing  Co.,  Inc., 
1944,  95  pp.  See  also  saffron  as  em- 
ployed by  Vieussens  and  Leeuwenhoek 
(Lewis,  F.  T.,  Anat.  Rec,  1942,  83,  229). 

Saffrosin,  see  Eosin  B  or  bluish. 

Safranin.  In  the  safranins  one  nitrogen 
of  the  azin  group  is  pentavalent  and  to 
this  a  benzene  ring  is  attached.  All 
are  strongly  basic.  Amethyst  violet, 
azocarmine  G,  Magdala  red,  pheno- 
safranin  and  safranin  O  are  mentioned. 

Safranin  Acid  Violet,  see  Neutral  Safranin. 

Safranin  B  Extra,  sec  Phenosafranin. 

Safranin  O  (CI,  841) — cotton  red,  Gos- 
sypimine,  safranin  Y  or  A — Commission 
Certified.  A  basic  azin  dye  of  great 
usefulness  which  is  sold  as  a  mixture 
of  di-methyl  and  tri-methyl  pheno- 
safranins.  Conn  (p.  97)  explains  that 
the  shade  depends  upon  their  relative 
proportion.  The  red  is  deeper  when 
there  is  more  of  the  latter.  Safranin  O 
can  be  employed  irrespective  of  whether 
safranin  0  wasserloslich,  or  safranin 
spiritloslich  or  safranin  gelb  is  called 
for.  The  safranin  pur,  likewise  of 
Grubler  and  Co.,  is  in  his  opinion 
methylene  violet  (CI,  842).  Safranin 
O  is  one  of  the  finest  nuclear  stains 
especially  in  the  Safranin  Light  Green 
method.  It  is  also  useful  in  making 
certain  neutral  stains  (Neutral  Safra- 
nin). Standardized  technique  for 
safranin  O  employing  buffered  solutions 
is  given  by  Sawyer,  C.  H.,  Stain  Techn., 
1940,  15,  3-7. 

Safranin  Y  or  A,  see  Safranin  O. 

Safranin-Gentian  Violet-Orange  G.  This 
is  Flemming's  tricolor  stain  for  nuclei. 
As  described  by  the  Bensleys  (p.  88). 
Fix  in  Flemming's  fluid  and  bring 
paraffin  sections  down  to  95%  alcohol. 
Stain  in  equal  parts  sat.  safranin  in 
95%  alcohol  and  filtered  sat.  anilin  oil 
in  aq.  dest.,  2-24  hrs.  Rinse  in  aq. 
dest.  and  stain  in  sat.  aq.  gentian  violet 
(crystal  violet),  ^2  hrs.  Drop  on  sat. 
aq.  orange  G,  30-60  sec.  Drop  95% 
alcohol  on  slide  until  clouds  of  color 
cease  coming  off.  Drop  on  clove  oil 
and  differentiate  under  microscope. 
Clear  in  benzol  and  mount  in  balsam. 
Violet  should  color  diffused  chromatin 
strand ;  safranin  denser  part ;  and  orange 
G,  the  background. 

Safranin-Light  Green. — Written  by  C.  H. 
Sawyer,  Duke  Hospital,  Durham,  North 
Carolina,  Dec.  16,  1950. — Stain  sections 
24  hrs.  in  2%  aq.  safranin  O  and  wash 
out  the  excess  safranin  in  0.25%  aq. 
light  green  (acid  violet).  Chromatin 
appears  red  and  acidophilic  nuclear  in- 


SALMONELLA 


310 


SCHULTZE'S  METHOD 


elusions  caused  by  viruses  green.  A 
very  brilliant  stain  but  the  green  fades 
in  the  course  of  a  month  or  two.  Stand- 
ardized safranin  O  technique  advised 
by  C.  H.  Sawyer  (Stain  Techn.,  1940, 
15,  3-7)  is:  overstain  deparaffinized  sec- 
tions in  0.1%  light  green  S.F.  or  fast 
green  FCF  in  50%  alcohol  adjusted  to 
pH  2.4  with  0.1  A^  HCl  (100  cc.  =  20  cc. 
0.5%  stain  +  50  cc.  100%  alcohol  + 
8  cc.  0.1  A^  HCl  +  22  cc.  aq.  dest.) 
for  4  hrs.  or  more.  Destain  in  Soren- 
sen's  buffer  pH  8,  30  minutes  or  more. 
Overstain  in  0.1%  aq.  safranin  O  at 
least  4  hrs.  Rinse  in  aq.  dest.  De- 
stain  in  0.01  A^  HCl  (pH  2)  or  in  0.001 
N  HCl  (pH  3)  for  light  green  and  fast 
green  respectively,  15  min.  After  rins- 
ing in  aq.  dest.  dehydrate  in  2  changes 
dioxan,  pass  through  xylol  and  mount 
in  balsam.  As  fixatives  Sawyer  finds 
Petrunkevitch's  paranitrophenol-cu- 
pric-nitrate-nitric  and  picro-formol-ace- 
tic  better  than  Bouin's  fluid.  Zenker's 
fluid  can  be  employed. 

Salmonella,  Flagella  of  non-motile,  Ed- 
wards, P.  R.,  Moran,  A.  B.  and  Bruner, 
D.  W.,  Proc.  Soc.  Exp.  Biol.  &  Med., 
1946,  62,  296-298.  See  Triphenyltetra- 
zolium  chloride. 

Samarium,  see  Atomic  Weights. 

Sandarac  mixed  with  dioxan,  camphor  and 
salol  is  recommended  by  McClung 
(p.  40)  as  a  mounting  medium  in  place 
of  balsam. 

Sandison's  Technique  for  inserting  trans- 
parent chambers  in  rabbit  ears  (Sandi- 
son,  J.  C,  Anat.  Rec,  1924,  28,  281). 
This  has  been  improved  by  Clark,  E.  R. 
et  al.,  Anat.  Rec,  1930,  47,  187-211  and 
by  Abell,  R.  G.  and  Clark,  E.  R.,  Anat. 
Rec,  1932,  53,  121-140.  See  modifica- 
tions by  Williams,  R.  G.,  Anat.  Rec, 
1934,  60,  487-491  and  by  the  same 
author  (ibid,  493-499)  the  latter  for 
insertion  into  skin.  Moore,  R.  L., 
Anat.  Rec,  1935-36,  64,  387-403)  has 
adapted  the  chamber  for  insertion  into 
dog's  ear. 

Saponin,  for  hemolysis  in  centrifugal  isola- 
tion of  nuclei  from  chicken  erythro- 
cytes (Dounce,  A.  L.  and  Lau,  T.  H., 
Science,  1943,  97,  584-585). 

Sarcolemma.  Special  technique  for,  see 
Dahlgren  in  McClung's  Microscopical 
Technique,  1950,  p.  341. 

Sawyer,  see  Safranin-Iight  green. 

Scandium,  see  Atomic  Weights. 

Scarlet  B  or  EC,  see  Biebrich  Scarlet, 
water  soluble. 

Scarlet  B  Fat  Soluble,  see  Sudan  III. 

Scarlet  J,  JJ,  V,  see  Eosin  B  or  bluish. 

Scarlet  R,  see  Ponceau  2R. 

Scarlet  Red,  see  Sudan  IV. 

Schaudinn's  Fixative.  Sat.  mercuric  chlo- 
ride  in   0.85%  aq.   sodium    chloride   2 


parts.  Add  1  part  95%  ethyl  alcohol 
and  enough  glacial  acetic  to  make  1% 
solution  immediately  before  use.  For 
Protozoa,  staining  in  bulk. 

Scheele's  Green,  an  exogenous  pigment, 
copper  arsenite. 

SchiflF's  Reaction  for  aldehydes  (Bourne, 
p.  22)  is  basis  of  Feulgen  reaction  for 
Thymonucleic  Acid. 

Schistosomes.  When  it  is  necessary  to 
collect  at  autopsy  all  parasites  irrespec- 
tive of  the  stage  of  development  or 
location,  a  modification  of  previous 
techniques  described  by  Pan,  C,  and 
Hunter,  III.  G.  W.  (J.  Lab.  &  Clin. 
Med.,  195,  37,  815-816)  is  suggested. 
To  infect  small  mammals  with  Schisto- 
soma Japonicum  see  method  of  Pan, 
C,  Kaufman,  E.  H.,  and  Hunter,  III, 
G.  W.,  Ibid.,  817-819. 

Schlesinger's  Reagent.  Add  to  4  gms.  zinc 
acetate  in  a  bottle  95%  ethyl  alcohol  to 
make  up  100  cc.  Shake  occasionally 
and  use  supernatant  fluid.  See  Uro- 
bilin. 

Schmitt,  see  Polarization  Optical  Method. 

Schneider's  Aceto-Carmine,  see  Aceto- 
Carmine. 

Schultz,  H.  Cholesterol  Test.  Cut  frozen 
sections  of  formol  fixed  material.  Place 
sections  in  a  2.5%  solution  of  iron  alum 
mordanting  for  3  days  in  low  tempera- 
ture (37°)  oven.  Rinse  the  sections 
after  removal  from  the  alum  solution 
in  aq.  dest.  to  which  are  added  a  few 
drops  of  nitric  acid  (2  to  3  drops  per 
26  cc).  This  removes  alum  precipitate 
in  the  sections.  They  are  then  trans- 
ferred to  2-3%  gelatin  solution  and 
mounted  in  dilute  gelatin  on  the  slide. 
After  the  mounted  sections  have  com- 
pletely dried  add  a  few  drops  of  a  mix- 
ture of  equal  parts  of  concentrated 
sulphuric  acid  and  glacial  acetic  acid. 
The  appearance  of  a  blue -green  color 
indicates  that  cholesterol,  either  in  free 
or  ester  form,  was  present  in  the  sec- 
tions before  treatment.  Both  acids 
must  be  of  analytical  reagent  standard 
and  the  sulphuric  acid  at  least  98% 
pure.  The  appearance  of  bubbles  in 
large  numbers  indicates  impure  re- 
agents. See  Knouff,  R.  A.,  Brown, 
J.  B.  and  Schneider,  B.  M.,  Anat.  Rec, 
1941,  79,  17-38.  Revised  by  R.  A. 
Knouff,  Dept.  of  Anatomy,  Ohio  State 
University,  Columbus,  Ohio,  April  24, 
1946.  Swyer,  G.  I.  M.,  Cancer  Re- 
search, 1942,  2,  372-375  has  checked  in  a 
satisfactory  way  the  Schultz  test  with 
quantitative  determinations  of  cho- 
lesterol in  normal  and  enlarged  pros- 
tates. 

Schultze's  Method  for  clearing  embryos 
has  been  modified  by  Miller.  See 
Cartilaginous  Skeleton. 


SCHWEINFURT  GREEN 


311 


SEPARATION  OF  CELL 
COMPONENTS 


Schweinfurt  Green,  an  exogenous  pigment, 
cupric  acetoarsenite. 

Scott,  see  Altmann-Gersh  Frozen  Dehydra- 
tion Method. 

Sebaceous  Glands.  Method  for  staining 
intoto  (Badertscher,  J.  A.,  StainTechn., 
1940,  15,  29-30).  Fix  fresh  skin  for  24 
hrs.  in  10%  formalin,  or  take  skin  from 
dissecting  room  cadaver  and  fix  in  the 
same  way.  Make  free  hand  vertical 
sections  1-2  mm.  thick  from  region  pos- 
sessing the  glands.  Whole  pieces  of 
skin  12  mm.  square  or  larger  (without 
subcutaneous  fat)  can  be  used  in  place 
of  the  sections.  Pass  through  50  to 
70%  alcohol.  Stain  for  12-24  hrs.  in  a 
mixture  of  70  parts  absolute  ethyl 
alcohol,  20  parts  10%  aq.  sodium  hy- 
droxide and  10  parts  of  aq.  dest.  satu- 
rated with  Sudan  IV.  Wash  away  excess 
stain  by  repeated  changes  of  70%  alcohol 
until  glands  become  sharply  outlined. 
Clear  in  glycerin.  Mount  in  Brandt's 
glycerin  jelly  (melted  gelatin,  1  part; 
glycerin,  1^  parts  -{-  few  drops  carbolic 
acid).  Glands  scarlet  in  transparent 
background.  This  method  may  prove 
useful  to  bring  out  the  distribution, 
number,  size  and  other  features  of 
sebaceous  glands  in  different  conditions 
as  well  as  at  different  ages.  The  same 
method  can  be  used  for  Meibomian 
(tarsal)  glands  after  a  little  preliminary 
dissection  described  by  the  author. 

Another  method  of  staining  sebaceous 
glands  in  toto  employed  in  the  Barnard 
Free  Skin  and  Cancer  Hospital  is  to 
separate  epidermis  from  dermis  by  the 
dilute  acetic  acid  method  (see  Epi- 
dermis) and  stain  the  epidermal  sheet, 
with  sebaceous  glands  attached,  with 
Sudan  III  or  IV  as  one  would  a  section 
for  fat.  A  hematoxylin  counterstain  is 
useful. 

The  technique  of  Fluorescence  Mi- 
croscopy is  useful.  Figge,  F.  H.  J., 
Bull.  School  of  Med.  Univ.  Maryland, 
1942,  26,  165-176  has  described  the  re- 
markable red,  white  or  yellow  fluo- 
rescence of  blackheads  which  is  charac- 
teristic of  different   individuals. 

Secretion  contrasted  with  excretion  (Cow- 
dry's  Histology,  p.  259). 

Sectioning,  see  Celloidin,  Frozen,  Gelatin 
and  Paraffin  Sections.  Also  Bone 
grinding  and  Teeth  cutting  with  power 
lathe. 

Selectron,  a  synthetic  resin  recommended 
by  R.  McClung  Jones  in  McClung's 
Microscopial  Technique,  1950,  p.  152 
for  embedding  embryos.  May  be  pur- 
chased from  Pittsburgh  Plate  Glass 
Co.,  Grant  Building,  Pittsburgh,  Pa. 

Selenium.  Intravenous  injections  of  col- 
loidal solutions  of  selenium  in  rabbits 


are  described  by  Duhamel,  B.  G.,  C. 
rend.  Soc.  de  Biol.,  1919,  82,  724-726. 
See  Radioselenium. 

Semen  Stains,  examination  of  for  sperma- 
tozoa. Place  piece  of  soiled  cloth  not 
more  than  \  inch  in  diameter  on  a  slide. 
Add  few  drops  saline  solution  and 
scrape  surface  of  cloth  with  blunt  edge 
of  a  scalpel.  Carry  scrapings  off  with 
fluid  anci  spread  on  a  slide.  Dry  and 
fix  with  heat.  Cover  with  4  cc.  1%  aq. 
Wollschwartz  (Grubler)  -f  0.05  cc.  2% 
aq.  sulphuric  acid,  5  min.  Wash  in 
water.  Counterstain  6-8  sec.  with  Loef- 
fler's  methylene  blue  diluted  with  15 
parts  aq.  dest.  Wash  in  aq.  dest.,  dry 
and  examine.  Heads  of  spermatozoa 
bright  golden  or  yellowish  color,  all  else 
gray.  Useful  in  legal  medicine  (Wil- 
liams, W.  W.,  J.  Lab.  &  Clin.  Med., 
1936-37,  22,  1173-1175).  See  author's 
figures.  See  Pollak,  O.  J.,  Arch.  Path., 
1943,  35,  140-196. 

Seminal  Fluid.  To  study  in  sections 
centrifuge  fluid  3  to  1  hr.  after  ejacula- 
tion for  20  min.  at  3000  r.p.m.  Fix 
centrifugate  in  4%  formalin,  48  hrs. 
2  changes.  Take  sediment  into  abs. 
ale,  then  9  parts  abs.  and  1  part  xylol. 
Gradually  increase  xylol  to  9  parts  to 
1  part  ale.  Xylol  paraffin  30  min. 
Then  54°C.  melting  paraffin  for  3  hrs. 
in  incubator  at  58°C.  After  3  hrs.  in 
60°C.  melting  paraffin  embed  and  sec- 
tion 2-3  microns  thick  (Joel,  K.,  J. 
Lab.  &  Clin.  Med.,  1939,  24,  970-972). 

Sense  Organs,  see  Eyes,  Ear,  Pacinian 
Corpuscles,  Meissner's  Corpuscles, 
Krause's  End  Bulbs,  Nerve  Endings. 

Sensitol  Red,  see  Pinacyanol. 

Separation  of  Cell  Components  by  Differ- 
ential Centrifugation— Written  by  A. 
Lazarow,  Department  of  Anatomy, 
Western  Reserve  University  School  of 
Medicine,  Cleveland,  Ohio.  Novem- 
ber 28,  1951— When  R.  R.  Bensley  and 
N.  Hoerr  (Anat.  Rec,  1934,  60,  449- 
455)  successfully  separated  mitochon- 
dria from  the  cell,  they  initiated  a  new 
era  in  cytochemistry.  The  cells  are 
disintegrated  by  passing  the  tissue 
through  bolting  silk  (mild  homogeniza- 
tion  in  a  glass  homogenizer  may  also 
be  used).  This  procedure  ruptures  the 
cytoplasmic  membranes  but  leaves 
most  of  the  cell  components  relatively 
unaltered.  The  resulting  suspension 
of  cell  fragments  is  subsequently 
fractionated  by  differential  centrifuga- 
tion. 

The  sedimentation  of  a  particle  in  a 
centrifugal  field  is  dependent  upon  its 
size,  its  shape,  and  its  density  (relative 
to  the  suspending  media)  and  upon  the 
centrifugal  force.  The  sedimentation 
of  non-spherical  particles  is  slower  than 


SERUM  AGAR 


312 


SHADOW-CASTING 


that  of  corresponding  spherical  parti- 
cles; this  difference  is  attributable  to  a 
viscosity  factor  and  to  Brownian  bom- 
bardment (the  larger  surface  area  of 
the  non-spherical  particles  results  in 
an  increased  Brownian  bombardment 
and  a  greater  tendency  to  keep  the 
particles  in  suspension).  If  the  density 
of  the  particle  is  equal  to  that  of  the 
media  no  sedimentation  will  take  place 
regardless  of  the  centrifugal  force 
applied.  If  the  particle  is  less  dense 
than  the  suspending  media  (as  lipid 
particles)  it  will  be  subjected  to  a  flota- 
tion  during   centrifugation. 

The  centrifugal  force  is  defined  as 
the  force  in  dynes  acting  on  a  mass  of 
one  gram.  Since  it  is  customary  to 
express  the  centrifugal  force  in  terms  of 
the  earth's  gravitational  force,  and 
since  the  gravitational  force  acting 
upon  a  mass  of  one  gram  is  980  dynes, 
the  centrifugal  force  is  given  by  the 
equation 

47r^rn^ 
Centrifugal  force  =     „„„     where  n  is 

yoU 

equal  to  the  number  of  revolutions  per 
second  and  r  is  equal  to  the  radius  of 
centrifuge.  Thus  the  centrifugal  force 
is  proportional  to  the  square  of  the 
number  of  revolutions  and  directly 
proportional  to  the  radius  of  the  centri- 
fuge. [Thus  for  an  angle  centrifuge 
with  a  radius  of  8  cm.,  the  centrifugal 
force  is  12,400  X  g.  at  a  speed  of  12,000 
RPM.  For  the  International  centri- 
fuge with  a  radius  of  20  cm.  the  cen- 
trifugal force  is  1,400  X  g.  at  a  speed  of 
2500  RPM]. 

In  carrying  out  the  fractionation  of 
the  cytoplasmic  constituents,  a  short 
preliminary  centrifugation  at  low  speed 
removes  the  intact  cell  nuclei  and  larger 
cell  fragments.  The  mitochondria  are 
subsequently  separated  from  the  su- 
pernatant by  centrifuging  at  about 
1,000-1,500  X  gravity  for  10-30  minutes. 
Less  contaminated  preparations  can  be 
obtained  by  successively  centrifuging 
the  supernatant  for  several  10-minute 
periods.  The  purity  of  the  prepara- 
tions is  checked  by  microscopic  ob- 
servation of  the  separated  fractions. 
After  the  mitochondria  have  been  re- 
moved, the  supernatant  is  recentrifuged 
at  about  10,000  X  g.  for  30-60  minutes. 
This  removes  the  submicroscopic  par- 
ticulate components  of  the  cell  (micro- 
somes, or  ultramicrosomes).  There  are 
at  least  two  types  of  submicroscopic 
particulate  components,  a  lipo  protein 
nucleic  acid  complex  and  a  hydrated 
glycogen-particle  (Lazarow,  A.,  Biol. 
Symposium,  1943,  10,  9,  edited  by  N.  L. 
Hoerr).     The  supernatant  of  this  high 


speed  centrifugation  contains  the  solu- 
ble proteins,  metabolites,  electrolytes, 
etc. 

When  sucrose  is  used  as  a  suspending 
media  (Hogeboom,  Schneider,  and 
Palade,  J.  Biol.  Chem.,  1946,  177,  610) 
the  morphology  of  the  mitochondria  is 
better  preserved  (in  salt  solution  the 
mitochondria  tend  to  swell  and  they 
lose  their  rod -like  shape).  By  using 
various  buffered  citric  acid  solutions 
it  is  possible  to  make  a  clean  separa- 
tion of  the  nuclei  (Dounce,  A.  L., 
Ann.  N.  Y.  Acad.  Sci.,  1950,  50,  982- 
999). 

In  the  past  decade  considerable 
progress  has  been  made  with  regard  to 
the  localization  of  enzymes  with  the 
component  parts  of  the  cell.  In  de- 
termining the  localization  of  enzymes 
(etc.)  it  is  necessary  to  consider  both 
the  concentration  [i.e.,  the  amount  of 
enzyme  per  unit  of  weight  or  per  unit 
of  protein  nitrogen]  and  the  relative 
fraction  [i.e.,  what  fraction  of  the  total 
enzyme  of  the  cell  is  present  in  a  given 
fraction].     The  cytochrome-cyto- 

chrome oxidase  system  as  well  as  many 
of  the  components  of  the  Kreb's  tri- 
carboxylic acid  cycle  are  concentrated 
within  the  mitochondria  (Schneider, 
W.  C,  and  Hogeboom,  G.  H.,  Cancer 
Res.,  1951,11,  1-22).  The  submicro- 
scopic particles  (ultramicrosomes)  are 
thought  to  be  concerned  with  protein 
synthesis. 

Serum  Agar,  see  Bacteria,  Media. 

Setoglaucine  O  (CI,  658),  a  basic  dye  less 
light  fast  than  Malachite  green  (CI, 
657),  a  constituent  of  some  bacterio- 
logical media  (Emig,  p.  47). 

Shadow-Casting — Written  by  W.  T.  Demp- 
ster, Dept.  of  Anatomy,  University  of 
Michigan,  Ann  Arbor,  Mich.,  and  R. 
C.  Williams,  Dept.  of  Biochemistry, 
University  of  California,  Berkeley, 
Calif.  June  9,  1950— This  is  a  tech- 
nique for  revealing  the  surface  form 
and  texture  of  microscopical  material 
in  either  light  or  electron  microscopy. 
It  is  an  outgrowth  of  R.  C.  Williams' 
experience  with  vacuum  deposited 
metal  films  on  astronomical  mirrors 
and  of  studies  on  the  physics  of  metallic 
films.  Metal  evaporated  from  a  hot 
filament  in  a  high  vacuum  is  of  atomic 
dimensions,  and  upon  condensation  on 
a  perfectly  smooth  surface  it  forms  a 
continuous  film  of  uniform  thickness. 
The  atomic  particles  of  metal  travel 
in  straight  line  paths  from  the  filament. 
When  these  are  directed  to  an  obliquely 
placed  surface,  obstructions,  however 
small,  cast  a  "shadow"  that  is  metal- 
free.  The  technique  of  condensing 
metal  films  obliquely  upon  specimens 


SHADOW-CASTING 


313 


SHADOW-CASTING 


was  first  applied  successfully  to  elec- 
tron microscopy  by  R.  C.  Williams  and 
R.  W.  G.  Wyckoff  (J.  Appl.  Phys.,  1944, 
15,  712-716)  who  used  the  technique 
for  determining  the  heights  of  minute 
objects  by  measuring  the  lengths  of 
their  shadows.  It  was  found  by  them, 
however,  that  the  process  greatly  en- 
hanced the  contrast  in  images  of  very 
small  objects,  or  of  very  small  irregular- 
ities on  larger  objects,  and  this  advan- 
tage of  the  technique  has  come  to  out- 
grow in  importance  the  original  purpose 
of  measuring  heights. 

Bacteria,  viruses  and  the  larger  pro- 
tein molecules  have  been  studied  by 
the  use  of  the  shadowing  technique 
(for  complete  bibliography,  see  R.  W. 
G.  Wyckoff,  Electron  Microscopy,  1949, 
Interscience  Publishers  Inc.,  New  York) 
as  applied  to  electron  microscopy.  The 
advantages  of  improved  contrast  are 
so  great  that,  in  the  case  of  the  observa- 
tion of  small  biological  objects  and 
minute  chemical  aggregates,  the  tech- 
nique has  improved  the  practical  use- 
fulness of  the  electron  microscope 
almost  ten-fold.  Further  applications 
to  both  electron  and  visual  microscopy 
involved  a  method  of  studj'ing  opaque 
surfaces  by  colloidin  replicas  that  are 
shadowed  (Williams,  R.  C,  and  Wyck- 
off, R.  W.  G.,  J.  Appl.  Phys.,  1946,  17, 
23-33).  Applications  of  the  method 
to  biological  material  viewed  with  the 
light  microscope  and  an  account  of  the 
casting  apparatus  have  been  presented 
by  W.  T.  Dempster  and  R.  C.  Williams 
(Anat.Rec,  1946,  96,  27-38). 

For  the  shadowing  of  non-opaque 
objects,  materials  to  be  observed  with 
the  light  microscope,  the  following 
procedure  is  followed:  Material  is  af- 
fixed to  cover  slips;  smears  are  thor- 
oughly dried;  paraffin  is  dissolved  from 
sections  with  solvents.  With  no  fur- 
ther preparation,  other  than  thorough 
drying,  the  slips  are  shadowed  with  a 
metal  deposit  in  a  vacuum  chamber. 
After  this,  they  are  mounted  face  down 
on  slides  with  clarite  or  balsam. 

If  large,  opaque  materials  are  to  be 
examined  under  the  light  microscope, 
a  surface  replica  can  be  taken  by  flow- 
ing a  dilute  solution  of  celloidin  over 
the  surface,  allowing  it  to  harden,  and 
stripping  the  celloidin  film  off  after 
drying.  The  film  is  then  shadowed  with 
its  replicating  surface  uppermost. 

For  electron  microscopy,  regular 
screen  grids  with  a  thin  colloidin  film 
over  the  mesh  are  used  as  substrates 
for  suspensions;  replicas  are  placed 
directly  on  the  mesh.  A  droplet  of 
suspension  containing  small  biological 
objects  is  placed  on  the  substrate  and 


allowed  to  dry.  Salts  are  then  rinsed 
off  with  distilled  water  and  the  speci- 
men is  ready  for  shadowing;  then  the 
specimen  may  be  studied  with  the  elec- 
tron microscope. 

A  metal  to  be  used  in  shadowing  for 
light  microscopy  should  be  readily 
evaporated  and  it  should  be  relatively 
opaque  when  jjresent  in  a  very  thin 
film.  Metallic  chromium  appears  to 
be  the  best  metal  to  use  for  light  micros- 
copy since  it  transmits  less  than  50% 
of  the  incident  light  in  a  thickness  less 
than  0.03  micra.  For  electron  micros- 
copy chromium  is  generally  satisfac- 
tory for  objects  as  large  as  bacteria, 
but,  for  finer  detail,  the  metals  palla- 
dium and  uranium  are  superior,  as  they 
can  be  applied  to  yield  satisfactory 
contrast  in  thicknesses  of  only  0.001 
micra. 

The  casting  technique  is  similar  for 
the  different  metals.  Shadow-casting 
produces  a  visually  structureless  de- 
posit which  sticks  to  all  surfaces  save 
those  directed  away  from  the  hot  fila- 
ment and  shadow  areas  due  to  obstruc- 
tions. Surfaces  perpendicular  to 
straight  line  paths  from  the  filament 
get  the  heaviest  deposit;  oblique  sur- 
faces get  less  and  shadows  none.  Metal 
deposited  at  a  rather  oblique  angle  has 
a  distribution  much  like  light  from  a 
point  source  shining  obliquely  on  three- 
dimensional  objects.  Highlights  and 
shadows  are  produced.  Through  the 
microscope,  shadows  in  the  prepara- 
tions transmit  light  and  appear  bright; 
highlights  are  dark.  The  eye,  however, 
readily  adapts  to  this  reversal  of  tone. 
Photographic  negatives  or  prints  made 
from  glass  positives  reverse  the  micro- 
scope appearance;  highlights  then  are 
bright,  shadows  are  dark,  and  varia- 
tions of  surface  texture  are  shown  by 
gradations  of  tone. 

Electron-microscopic  negatives  show 
the  same  natural  appearance  of  light 
and  dark.  Although  the  electron  mi- 
crographs are  taken  by  transmitted 
electrons,  in  complete  analogy  with 
photomicrographs,  the  negative  prints 
give  one  the  impression  that  one  is 
looking  down  on  the  surface  of  the  ma- 
terial being  examined. 

The  apparatus  for  shadowing  con- 
sists of  a  bell  jar  and  a  base  plate  with 
vacuum  tight  electrical  connections. 
Electrodes  raised  above  the  level  of 
the  base  plate  carry  a  tungsten  filament 
on  which  the  metal  is  placed  for  vapor- 
izing. Cover  slips  with  affixed  ma- 
terial (or  the  grid  screens)  are  arranged 
in  a  semicircle  at  a  predetermined 
distance  from  the  filament  and  the 
metal   thereon   to  be   vaporized.     The 


SHANKLIN 


314 


SILVER  CITRATE 


height  of  the  filament  and  the  distance 
from  filament  to  specimens  determine 
the  casting  angle.  Both  an  oil-diffu- 
sion pump  and  a  mechanical  pump  must 
be  used  to  produce  the  degree  of  vacuum 
required  (at  least  ICT*  mm.  Hg.).  With 
a  suitable  vacuum  provided,  the  fila- 
ment is  heated  electrically  and  a  meas- 
ured weight  of  metal  is  vaporized. 
Preparations  are  then  ready  for  mount- 
ing or  examination.  A  figure  of  the 
apparatus  and  the  formula  for  calcu- 
lating the  appropriate  mass  of  metal  for 
the  conditions  of  shadowing  are  pre- 
sented in  the  Dempster  and  Williams 
paper.  Wyckoff's  book  (above)  covers 
completelj'  applications  to  electron 
microscopy. 

Shanklin,  see  Pineal,  Silver  Diaminohy- 
droxide  after  sensitizing  with  sodium 
sulfite. 

Sharpening,  see  Microtome  Knife. 

Shrinkage  caused  by  fi.xation,  dehydration 
and  clearing  of  nervous  tissues  has  been 
measured  by  King,  H.  D.,  Anat.  Rec, 
1910,  4,  213-244  and  by  Allen,  Ezra, 
Anat.  Rec,  1916,  10,  565-589. 

Sickle-Cell  Trait.  A  critical  study  of 
methods  for  detection  by  Diggs  and 
Pettit  (L.  W.  and  V.  D.,  J.  Lab.  &  Clin. 
Med.,  1939,  25,  1106-1111)  gives  first 
place  to  the  Moist  Stasis  technique  of 
Scriver  and  Waugh.  Place  a  rubber 
band  about  proximal  part  of  a  finger. 
Leave  5  min.  Puncture  and  examine 
fresh  blood  for  sickle  cells.  According 
to  Hansen-Pruss  (O.  C,  J.  Lab.  &  Clin. 
Med.,  1936-37,  22,  311-315)  the  maxi- 
mum percentage  of  sickle  cells  is 
produced  in  4-5  hrs.  by  supravital 
staining  with  brilliant  cresyl  blue  or 
janus  green,  while  it  takes  24  hrs.  in 
unstained  moist  preparations. 

The  following  rapid  method  of  diag- 
nosis is  reported  by  Neuda,  P.  M.  and 
Rosen,  M.  S.,  J.  Lab.  &  Clin.  Med., 
1945,  30,  456-458.  Mix  "cherry -size" 
piece  of  feces  with  5  cc.  isotonic  sodium 
chloride  solution.  Add  0.1  cc.  of  fil- 
trate to  tube  of  nutrient  broth,  incubate 
24  hrs.  at  37°C.  To  top  of  suspected 
blood  on  slide  add  drop  of  culture. 
Something  in  broth  makes  susceptible 
cells  quickly  assume  sickle  form. 

Siena  Orange  (K.  Hollborn,  Leipsig)  = 
sodium  paradipicrylamine,  an  alleged 
stain  for  potassium  (Carere-Comes,  O., 
Zeit.  wiss.  Mikr.,  1938,  55,  1-6). 

Silicon.  Easily  recognizable  in  sections 
viewed  in  polarized  light.  It  often 
occurs  as  sericite  in  combination  with 
magnesium,  iron  and  other  minerals,  see 
Jones, W.  R.,J.  Hyg  1933,  33,  307-329. 
Microtechnique  is  discussed  by  Poli- 
card,  A.,  and  Mastin,  E.,  Bull.  d'Hist. 
Appl.,  1933,  10,  22-36.    Microincinera- 


tion is  useful  but  Scott  says  that  an 
exaggerated  idea  of  amount  may  be 
obtained  (McClung,  p.  659). 

Sintered-glass  filters,  see  Cunningham, 
B.,  Kirk,  P.  L.  and  Brooks,  S.  C,  J. 
Biol.  Chem.,  1944,  139,  21-28. 

Silver  is  occasionally  found  in  the  tissues 
particularly  after  treatment  with  silver 
nitrate  or  argyrol.  It  appears  as  brown 
to  black  granules  or  masses,  is  definitely 
blackened  by  ammonium  sulphide  and 
may  be  removed  by  a  mixture  of  sodium 
thiosulphate  and  potassium  ferri- 
cyanide  solutions.  Recently  a  method 
based  on  reaction  between  silver  and  p- 
dimethylaminobenzylidenrhodanin  has 
been  described  and  illustrated  in  colors 
(Okamoto,  K.,  Utamura,  M.  and  Akagi, 
T.,  Acta  Scholae  Med.  Univ.  Imp.  in 
Kioto,  1939,  22,  361-372).  Details  are 
supplied  by  Glick,  p.  26. 

Silver  Chloride  Dichlorfluoresceinate 
coloration  of  vascular  endothelial  cells 
(Bensley,  R.  D.  and  S.  H.,  Anat.  Rec, 
1935,  64,  46-49).  Inject  intravenously 
0.8%  aq.  dichlorfluorescein  until  animal 
becomes  quite  yellow.  Kill  animal: 
remove  tissues  and  immerse  in  10% 
aq.  silver  nitrate  or  in  Bensley's  Silver 
Citrate  solution  until  salmon  pink  color 
develops.  Fix  in  10%  neutral  formalin. 
Dehydrate  in  alcohol  and  Iso-Safrol, 
clear  in  iso-safrol  and  mount  in  balsam. 
Endothelial  cells  outlined  in  pink.  On 
exposure  to  light  color  changes  in  time 
the  silver  becoming  brown  and  black. 
See  demonstration  of  Chlorides  in  lungs 
by  this  method. 

Silver  Citrate  injection  of  blood  vessels 
(Bensley,  R.  D.,  Am.  J.  Anat.,  1929, 
40,  146-169).  This  method  has  proved 
of  great  value  in  the  investigation  of 
efferent  vessels  of  renal  glomeruli.  It 
can  be  employed  to  advantage  in  other 
situations  particularly  in  association 
with  supravital  staining  of  Pericapillary 
Cells  with  janus  green.  To  make  up 
the  solution  dissolve  4  gm.  silver  nitrate 
in  100  cc.  aq.  dest.  and  remove  to  dark 
room.  Completely  precipitate  silver 
as  silver  phosphate  by  addition  of 
sodium  phosphate  solution.  Wash  ppt. 
repeatedly  with  aq.  dest.  decanting 
supernatant  fluid.  Make  up  to  volume 
approximately  30  cc.  Dissolve  ppt.  by 
adding  28  cms.  pure  citric  acid  (or 
tartaric  acid)  in  crystals.  Dilute  with 
aq.  dest.  to  1000  cc.  and  keep  in  dark. 

For  use,  dilute  this  stock  solution 
with  3  times  its  volume  1%  aq.  sodium 
citrate.  Kill  the  animal  by  bleeding. 
For  kidneys  and  other  abdominal 
viscera  insert  into  aorta  cannula  con- 
nected by  rubber  tubing  with  pressure 
bottle.  First  perfuse  with  1%  aq. 
sodium  citrate  with  the  pressure  bottle 


SILVER  DEPOSITS 


315 


SILVER  LINEATION 


about  60  cm.  above  cannula.  When 
clear  fluid,  free  from  blood,  appears  in 
inferior  vena  cava,  clamp  tube  and 
replace  citrate  solution  with  silver 
solution.  Raise  bottle  about  150  cm. 
above  cannula  and  release  clamp.  De- 
termine length  of  time  of  perfusion  by 
trials.  When  complete,  immediately 
make  frozen  sections  to  determine  re- 
sults and  fix  other  pieces  in  10% 
formalin  for  24  hrs.  Cut  paraffin  sec- 
tions desired  thickness.  Mount  them 
in  usual  way,  run  down  to  water  and 
develop  in  light  in  diluted  photographic 
developer  or  simply  by  direct  exposure 
to  sunlight  or  arc  light.  Counterstain 
in  Mayer's  Acid  Carmine,  hematoxylin, 
acridine  red  or  some  other  suitable  dye. 
Dehydrate,    clear,    mount    in    balsam. 

Silver  Deposits,  methods  for  removal, 
Lillie,  p.  135. 

Silver  Diaminohydroxide  after  Sensitizing 
with  Sodium  Sulfite  for  Neuroglia — 
Written  by  William  M.  Shanklin, 
American  University  of  Beirut,  Beirut, 
Lebanon.  March  30,  1951 — Fix  small 
fresh  pieces  of  the  central  nervous  sys- 
tem in  formalin  ammonium  bromide  for 
4  days  at  room  temperature  (Del  Rio 
Hortega,  P.,  Arch.  Hist.  Normal  Y 
Path.,  1942,  1,  165-205,  329-361;  1943, 
2,  231-244,  577-604) :  Formalin  (Merck, 
blue  label  40%)  70  ml..  Ammonium 
bromide  14  gm.  and  aq.  dest.  680  cc. 
Wash  10  hrs.  in  aq.  dest.  to  which  30 
drops  of  strong  ammonia  water  are 
added  for  each  100  cc.  of  water,  and 
cover  the  jar.  Wash  in  two  changes  of 
aq.  dest.  for  1-2  hrs.  in  each;  dehydrate 
with  alcohol.  Clear  in  cedar  oil  fol- 
lowed by  xylene  30  min.  Infiltrate 
with  paraffin  for  3-4  hrs.,  embed,  sec- 
tion at  10-15  M  and  fix  to  slide  by  the 
albumen  water  method.  Remove  the 
paraffin  with  xylene,  dehydrate  with 
alcohol  and  pass  through  three  changes 
of  aq.  dest.  1-2  min.  each.  Sensitize 
by  placing  the  slides  in  5%  aq.  sodium 
sulfite,  2  hrs.  Pass  quickly  through  3 
changes  of  aq.  dest.  To  prepare  silver 
diaminohj^droxide  solution  place  1 
ml.  28%  ammonia  water  in  a  small 
flask  and  add  7  or  8  ml.  10%  silver 
nitrate  rapidly.  Continue  to  add  10% 
silver  nitrate  drop  by  drop  shaking 
between  each  addition  to  clear  the 
solution  until  a  faint  permanent  turbid- 
ity remains  after  the  last  drop  is  added. 
This  takes  a  total  of  9  to  10  ml.  silver 
solution.  Then  dilute  the  resultant 
solution  with  an  equal  volume  of  aq. 
dest.  (Lillie,  R.  D.,  Stain  Techn.,  1946, 
21,  69-72;  Histopathologic  Technic, 
Philadelphia:  Blakiston  Co.  1948). 
Impregnate  by  immersion  in  the  silver 
solution  at  room  temperature  for  2  to  5 


min.;  the  time  is  varied  until  the  opti- 
rnum  is  determined.  The  silver  solu- 
tion will  keep  for  several  daj'^s  but 
should  be  changed  frequently  after 
use.  Dip  in  aq.  dest.  1  or  2  sec.  Re- 
duce for  1  min.  in  2%  neutral  formalin 
(Merck,  blue  label)  agitating  gently. 
The  formalin  should  be  changed  fre- 
quently. Wash  in  aq.  dest.  for  1  min. 
Tone  in  yellow  gold  chloride  (1  g.  to 
500  ml.  aq.  dest.)  a  few  seconds  to  one 
minute.  This  step  must  not  be  pro- 
longed beyond  the  exact  time  needed. 
Fix  in  5%  hypo  for  1-2  min.  Wash  in 
tapwater  and  counterstain  lightly  with 
1%  picric  acid.  Dehydrate  in  alcohol, 
clear  in  xylene,  mount  in  neutral  bal- 
sam and  cover  with  cover  slips. 

This  method  successfully  stains 
fibrous  and  protoplasmic  astrocytes, 
microglia  and  oligodendroglia.  By  em- 
bedding the  tissue  in  paraffin  the  prob- 
lem of  overformalinization  is  avoided 
and  the  tissues  are  still  suitable  for 
staining  years  later  (See  Nassar,  T.  K. 
and  Shanklin,  W.  M.,  Stain  Techn., 
1951,26,  13-18). 
Silver  Electrode  of  Linderstrom-Lang, 
Palmer  and  Holter  described  by  Glick, 
p.  183. 
Silver  Gray,  see  Nigrosin,  water  soluble 
Silver  Lineation  on  pulmonary  alveolar 
walls — Written  by  C.  C.  Macklin,  Dept. 
of  Histological  Research,  The  Univer- 
sity of  Western  Ontario,  London,  Can- 
ada. November  28, 1951 — The  following 
modernization  of  a  very  old  tech- 
nique is  recommended.  Immediately 
after  cessation  of  the  circulation  the 
collapsed  lungs  of  healthy  mammals  are 
filled,  via  the  trachea  or  a  large  bron- 
chus, with  0.2  per  cent  aqueous  silver 
nitrate  for  not  more  than  one  minute; 
they  are  at  once  drained.  They  are 
promptly  refilled  with  distilled  water 
and  evacuated,  and  this  last  process  is 
repeated.  They  are  then  filled  with 
10%  neutral  formalin  in  water;  the 
trachea  is  tied  and  the  preparation  im- 
mersed in  the  same  fixative  for  24  hours 
or  more.  The  degree  of  distention 
should  be  approximately  that  of  full 
inspiration  (See  C.  C.  Macklin,  J.  Thor. 
Surg.,  1938,  7,  536-551,  for  further 
details  and  bibliographj')-  Silver  cit- 
rate may  be  used  instead  of  silver 
nitrate  (see  R.  D.  Bensley,  Am.  J. 
Anat.,  1929,  40,  146-169).  Blocks  are 
cut  out  and  sectioned  while  frozen,  or 
after  having  been  embedded  in  paraffin 
or  celloidin.  Flattened  frozen  sections 
are  best  for  en  face  examination  of  the 
silver  lines  on  an  expanse  of  alveolar 
wall.  The  silver  lines  are  darkened  by 
exposure  of  the  sections  to  direct  sun- 
light; or,  as  an  alternative,  the  lungs 


SILVER  METHODS 


316 


SILVER'S 


may  be  briefly  filled  with  y*5  strength 
hydroquinone  solution  after  the  silver 
has  been  thoroughly  washed  out  and 
before  fixation.  Stains  such  as  Mayer's 
paracarmine  may  be  used.  When  the 
silver  is  thus  applied  for  but  an  instant 
it  does  not  involve  the  underlying 
endothelium  or  reticulin  fibers.  In 
thick  sections  the  silver-line  network 
of  the  bronchial  epithelium  may  often 
be  followed  directly  into  that  of  the 
alveolar  walls,  and  the  two  are  then 
seen  to  form  one  system.  On  the 
alveolar  walls  the  areas  circumscribed 
by  the  meshes  of  the  net  are  not  uni- 
form. The  smaller,  more  heavily 
marked,  meshes  enclose  alveolar  wall 
cells  (pneumonocytes,  septal  cells,  etc., 
which  see)  while  the  larger  meshes, 
known  as  bare  areas,  contain  no  surface 
cells.  Around  them  the  lines  in  some 
animals,  as  rabbit,  are  often  incomplete. 
Silver  Methods.  General  statement.  A 
brief  historical  review  by  Silver,  M.  L., 
Anat.  Rec,  1942,  82,  507-529  shows  that 
progress  has  been  made  in  the  control 
of  these  techniques  to  the  point  where 
they  yield  reliable  results  with  con- 
siderable uniformity.  Impregnation  of 
blocks  of  tissue  and  reduction  of  the 
silver  in  various  ways  were  and  still  are 
the  bases  of  the  methods  of  Golgi, 
Cajal  and  Bielchowsky  which  have 
contributed  so  much  to  our  knowledge 
of  the  Nervous  System,  which  see. 
But  one  had  to  wait  until  the  sections 
were  cut  and  examined  to  ascertain  the 
results.  Sometimes  they  were  all  that 
heart  could  desire;  at  other  times  the 
worker  faced  repeated  disappointments. 
Having  labored  with  the  silver  impreg- 
nation of  neurofibrils  I  have  always 
avoided  silver  methods  whenever  others 
can  be  employed  in  their  place.  Now 
however  with  the  successful  application 
of  reduced  silver  to  sections  mounted 
on  slides  the  technique  is  brought 
from  the  insides  of  the  blocks  of  tissue 
which  one  cannot  see  into  the  open, 
thanks  to  Rogers,  W.  M.,  Pappenheimer, 
A.  M.,and  Goettsch,  M.,  J.  Exp.  Med., 
1931,  54,  167-169.  Another  advance 
was  the  introduction  of  protargol  as  the 
silver  salt  for  treating  sections  of  the 
central  nervous  system  by  Bartelmez, 
G.  W.  and  Hoerr,  N.  L.,  J.  Comp. 
Neurol.,  1933,  57,  401-428.  Then,  like- 
wise in  Bensley's  laboratory,  Bodian, 
D.,  Anat.  Rec,  1936,  65,  89-97  employed 
protargol  with  hydroquinone  as  reducer 
and  speeding  up  results  by  copper, 
mercury  and  acid.  Finally  Davenport, 
H.  A.,  McArthur,  J.,  and  Bruesch,  S.  P., 
Stain  Techn.,  1939,  14,  21-26  dispense 
with  copper,  and,  by  combining  pro- 
targol and  silver  nitrate  at  optimum  pH, 


reduce  staining  time  of  sections  of  pe- 
ripheral nerves  to  2  hrs.  In  addition, 
Silver  (loc.  cit.)  by  well  planned  experi- 
ments has  shown  that  staining  with 
silver  is  brought  about  through  adsorp- 
tion and  flocculation  of  electrically 
charged  silver  micelles  by  suitably 
charged  surfaces.  When  these  newer 
methods  are  widely  brought  to  bear 
upon  tissues  of  the  body  in  normal  and 
pathological  conditions  a  significant 
service  will  be  performed.  Suffice  it 
here  to  give  a  few  details  under  Nervous 
System,  Spirochetes,  tests  for  Calcium, 
Chloride,  Vitamin  C,  Reticular  Fibers, 
Melanin,  etc. 

It  is  in  some  cases  desirable  to  destain 
silver  slides.  To  do  this  pass  down  to 
running  water  for  5  min.  and  treat  sec- 
tions with  0.25%  aq.  potassium  per- 
manganate to  which  1%  of  cone,  sul- 
phuric acid  is  added  for  15  min.  Wash 
in  running  water  2  min.  Bleach  in  5% 
aq.  oxalic  acid  2-5  min.  Wash.  Re- 
peat the  stain  omitting  preliminary 
oxidation-reduction,  or  apply  some 
other  technique  (Wilson,  R.  A.  J.,  Am. 
J.  Clin.  Path.,  1943,  Tech.  Suppl.  7,  39). 

Silver  Nitrate  is  employed  in  many  tech- 
niques. It  is  important  to  remember 
that  ammoniacal  silver  nitrate  solutions 
on  evaporation  yield  an  explosive  com- 
pound. Consequently  solutions  of  this 
sort  should  never  be  allowed  to  dry  but 
should  be  washed  down  the  sink  with 
plenty  of  water. 

Silver  Staining  of  bone  (McCollum,  E.  V., 
Simmonds,  N.,  Shipley,  P.  G.  and 
Park,  E.  A.,  J.  Biol.  Chem.,  1922,  51, 
41-49). 

Silver's  rapid  silver-on-the-slide  method 
for  nervous  tissue  (Silver,  M.  L.,  Stain 
Techn.,  1942,  17,  123-127).  A  new 
feature  of  this  technique  is  the  reducing 
solution. 

1.  For  nuclei,  fine  fibers  and  nerve 
terminals,  fix  with  10%  neutral  or 
commercial  formalin  in  1%  aq.  sodium 
chloride  with  Bouin's  fluid  or  with  some 
other  fixatives  which  he  specifies  prefer- 
ably by  Perfusion. 

Cut  frozen  sections  10-40/x  or  dehy- 
drate slowly,  imbed  in  paraffin  or  cel- 
loidin  and  cut  2-20^.  Mount  paraffin 
sections  on  slides  and  deparaffinize  in 
the  usual  way.  In  the  case  of  celloidin 
sections  remove  celloidin  with  several 
changes  acetone  and  of  equal  parts 
absolute  alcohol  and  ether  and  pass  down 
through  alcohols  to  water. 

To  make  reducing  solution  dissolve 
64  gm.  Rochelle  salts  (potassium  sodium 
tartrate)  in  500  cc.  aq.  dest.  Boil  vigor- 
ously. Add  10  cc.  10%  aq.  silver  nitrate 
and  boil  again  at  least  5  min.  Remove 
from    flame.     Add   0.3  gm.    crystalline 


SINUSOIDS 


317 


SKIN 


magnesium  sulphate  and  while  simmer- 
ing 0.2  gm.  KjS  (U.S. P.)  employing 
only  the  brown  unoxidized  part  of  1 
piece.  Filter  while  hot  and  make  up 
filtrate  with  aq.  dest.  to  4  liters.  This 
reducer  improves  slightly  with  age. 

Place  mounted  paraffin  sections  or 
frozen  or  celloidin  sections  in  equal 
parts  above  reducer  and  0.5%  aq.  pro- 
targol  (Winthrop  Chemical  Co.,  Inc., 
New  York)  at  45-55°C.  Staining  is 
progressive  and  ordinarily  takes  2-3 
hrs.  Remove  and  examine.  When  com- 
plete, generally  before  a  grossly  visible 
reduction  of  silver  is  evident  in  the 
solution,  remove,  wash  in  2  changes  aq. 
dest.,  dehydrate,  clear  and  mount. 
More  finely  myelinated  fibers  are  re- 
vealed than  are  demonstrated  by  the 
standard  Weigert  technique. 

2.  For  myelin  sheaths  and  mito- 
chondria fix  with  10%  formalin  in  1% 
aq.  potassium  bichromate  or  with  10% 
formalin  in  1%  aq.  NaCl  again  prefer- 
ably by  perfusion,  and  mordant  small 
blocks  of  the  tissue  in  3%  aq.  potassium 
bichromate  for  7  days  (This  mordanting 
can  be  dispensed  with  if  tissue  is  in 
fixative  for  more  than  1  week.).  Wash, 
dehydrate,  imbed  (in  paraffin),  cut 
4-20m  and  mount  on  slide.  Remove 
imbedding  medium  and  proceed  as 
described  above. 

Sinusoids  are  capillaries  of  large  diameter 
through  which  the  circulation  is  slower. 
The  endothelial  cells  of  their  walls 
ingest  some  forms  of  particulate  matter 
in  the  blood  stream.  The  best  place  to 
demonstrate  them  is  in  carmine  stained 
sections  of  formalin  fixed  liver  of  an 
animal  injected  intravenously  with 
India  ink  as  described  under  Vital 
Staining. 

Sizes  of  Organs.    See  Normals. 

Skin.  No  other  part  of  the  body  is  simi- 
larly spread  out  for  examination  in  vivo. 
Much  is  to  be  gained  by  correlation  of 
gross  and  microscopic  study.  Altera- 
tions in  color,  moisture,  consistency  and 
thickness  can  easily  be  detected. 
Changes  in  sensitivity  and  in  the  num- 
ber and  activity  of  sweat  glands  can  be 
determined  by  appropriate  methods. 
Simple  techniques  are  available  for  the 
visualization  of  Lymphatic  Vessels, 
and  the  Capillaries  in  the  dermal  papil- 
lae can  be  demonstrated  microscopically 
and  their  behavior  recorded  in  moving 
pictures.  See  Thomas  Lewis'  classic, 
The  Vessels  of  the  Human  Skin  and 
their  Responses.  London :  Shaw  & 
Sons,  1927,  322  pp.  Very  important  is 
direct  study  of  the  skin  with  hand 
lens  or  binocular  microscope. 

But  examination  in  sections  will 
always    remain    the    basic    method    of 


study.  Hair,  where  present,  should  be 
cut  short  with  scissors  and  removed 
with  an  electric  razor,  an  instrument 
which  does  not  require  the  use  of  any 
soap  and  does  not  scrape  away  the  sur- 
face. Samples  of  skin  removed  at 
autopsy  are  satisfactory  for  some  pur- 
poses up  to  about  24  hrs.  if  the  body  has 
been  kept  cool  because  autolytic  changes 
take  place  comparatively  slowly  in  the 
skin.  But  biopsy  specimens  are  much 
better.  The  local  anesthetic  should  be 
injected  in  a  circle  about  the  skin  to  be 
excised  and  the  observer  should  be  on 
the  lookout  for  slight  modifications  if  the 
sections  include  the  actual  area  into 
which  it  is  forced.  Obviously  the 
specimen  should  be  lifted,  never 
pinched  with  forceps. 

Because  the  skin  is  made  up  of  2  tis- 
sues, avascular  epidermis  and  vascular 
dermis,  closely  bound  together,  differ- 
ential shrinkage  is  a  troublesome  factor. 
Evans,  R.,  Cowdry,  E.  V.  and  Nielson, 
P.  E.,  have  found  in  this  laboratory 
that,  owing  to  shrinkage  or  drawing  to- 
gether of  the  dermis,  the  folds  in  the 
epidermis  are  accentuated  to  an  extent 
much  greater  than  is  generally  realized. 
This  is  more  marked  in  young  skins  than 
in  those  of  old  people  and  in  living  skin 
than  in  skin  excised  after  long  delayed 
autopsy.  It  is  apparently  not  feasible 
to  entirely  side  step  this  kind  of  artefact 
but  the  tendency  of  the  whole  specimen 
to  curl  up  can  be  obviated  by  spreading 
it  out  with  dermis  down  on  a  piece  of 
wooden  tongue  depressor  or  stiff  card- 
board for  the  first  few  minutes  of  fixa- 
tion. If  interest  definitely  centers  in 
the  dermis  it  should  be  mounted  with 
epidermis  down.  But  it  should  not  be 
kept  in  either  position  too  long  because 
the  complete  entry  of  fixative  will  there- 
by be  prevented.  After  3  or  4  hrs.  the 
specimen  should  be  trimmed  with  a 
new  wet  razor  blade. 

Frozen  sections  are  essential  for  rapid 
diagnosis,  for  staining  with  Sudan  and 
for  many  other  purposes.  The  tech- 
nique most  used  by  dermatologists  is  to 
fix  in  Bouin's  Fluid  and  to  stain  paraffin 
sections  with  Hematoxylin  and  Eosin. 
After  Zenker  Fixation,  Mallory's  Con- 
nective Tissue  Stain,  or  Masson's 
Trichrome  Stain,  is  suitable  for  muscle 
and  coUagenic  tissue.  Weigert's  re- 
sorcin  fuchsin  is  recommended  for  elas- 
tic fibers.  The  Dopa  Reaction  is  re- 
quired for  melanin  precursors.  For 
nerve  fibers  the  Bodian  method  is  prob- 
ably the  best.  Another  silver  tech- 
nique advised  for  the  skin  is  that  of 
Jalowy. 

MacCardle,  R.  C,  Engman,  M.  F., 
Jr.  and  Sr.,   Arch.  Dermat.   &  Syph., 


SKUNK'S  STAIN 


318 


SMALL  INTESTINE 


1941,  44,  429-440  give  details  of  spectro- 
graphic  analysis  of  skin  lesions.  See 
also  Microincineration.  Ultracentrif- 
ugation  method  for  determination  of 
intranuclear  viscosity  (Cowdry,  E.  V. 
and  Paletta,  F.  X.,  Am.  J.  Path.,  1941, 
17,  335-357).  Methods  of  transplanta- 
tion are  described  by  Kelly,  R.  W.  and 
Loeb,  L.,  Anat.  Rec,  1939,  74,  487-509 
and  of  fluorescence  examination  by 
Cornbleet,  T.  and  Popper,  H.,  Arch. 
Dermat.  and  Syph.,  1942,  46,  59-65" 
An  adaptation  of  the  Sandison  tech- 
nique is  recommended  by  Williams,  R. 
G.,  Anat.  Rec,  1934,  60,  493-499.  See 
Sebaceous  and  Tarsal  glands.  Hairs, 
Nails,    Feathers. 

If  it  is  not  desired  to  investigate  a 
particular  area,  to  which  attention  has 
been  called  by  its  unusual  gross  appear- 
ance; but,  instead,  to  demonstrate  some 
special  component,  or  response,  of  the 
skin  one  should  be  guided  in  selection 
of  the  specimen  by  the  location  where 
the  component  or  response  is  most  likely 
to  be  found.  Thus  Meissner's  corpuscles 
are  best  seen  in  sections  of  skin  of 
palmar  surface  of  finger  tips.  Weddel, 
G.,  J.  Anat.,  1941,  75  (3),  346-367  reports 
that  multiple  groups  of  Krause's  end- 
bulbs  occur  beneath  each  cold  spot  in  the 
forearm  about  1  mm.  inward  from  the 
skin  surface.  Many  helpful  clues  are 
supplied  by  Lewis,  T.,  Pain.  New 
York:  MacMillan,  1942,  192  pp.  He 
quotes  Strughold  as  stating  that  pain 
spots  are  aggregated  as  closely  as  200 
per  sq.  cm.  in  supraclavicular,  ante- 
cubital,  inguinal  and  popliteal  fossae 
while  they  are  rare  (40-70  per  sq.  cm.) 
on  tip  of  nose  and  ear,  soles  and  palms 
(see  Nerve  Endings).  The  skin  of 
axillary,  pubic  and  nipple  areas  is  more 
likely  than  that  of  the  rest  of  the  body 
to  respond  to  sex  hormones.  Adjust- 
ments to  external  environment  are  to  be 
expected  in  exposed  parts.  To  search 
for  sweat  glands  in  those  mammals  which 
do  not  possess  any  is  futile.  To  expect 
all  epidermal  layers  in  thin  epidermis  is 
likewise  contraindicated. 

Fluorescence  Microscopy  is  capable 
of  yielding  interesting  results  in  dis- 
tinction between  psoriasis  and  hyper- 
keratosis scales  (Radley,  J.  A.  and 
Grant,  J.,  Fluorescence  Analysis  in  Ul- 
traviolet Light.  New  York:  Van  Nos- 
trand,  1935).  Further  indications  on 
fluorescence  are  given  under  Hair  and 
Sebaceous  Glands. 

Now  that  epidermis  can  be  conven- 
iently separated  from  dermis  it  is  desir- 
able to  give  details  of  technique  relating 
to  it  under  a  separate  heading.  See 
Epidermis. 
Skunk's  Stain,  see  Flagella. 


Skyblue  (CI,  1286)— coelestin  blue,  coeline, 
coeruleum — a  mineral  pigment,  cobal- 
tous  stannate,  seldom  used  in  medical 
research. 

Slides,  see  Cleaning. 

Slifer-King  method,  see  Ticks. 

Slime  Forming  Bacteria,  Conn's  method. 
Stain  for  about  1  min.  with  a  little  heat 
in  Rose  bengal  1  gni.,  5%  aq.  phenol 
100  cc,  1%  aq.  CaClj,  1  cc;  then  wash 
quickly  and  dry   (McClung,  p.  146). 

Small  Intestine.  JVIany  conditions  influence 
the  appearance  seen  in  sections.  If 
fixed  while  distended  with  food  mate- 
rial, the  spaces  between  the  villi  are 
more  noticeable,  the  villi  shorter  and 
the  muscular  layers  thinner  than  when 
fixed  while  strongly  contracted.  See 
illustrations  provided  by  Johnson,  F. 
P.,  Am.  J.  Anat.,  1912-13,  14,  235-250 
and  Contraction  Bands.  The  time  after 
feeding  and  the  character  of  the  food  has 
a  marked  influence  on  structure.  The 
cytoplasmic  granules  of  the  Paneth 
Cells  are  almost  all  discharged  in  guinea 
pigs  6  hrs.  after  feeding.  They  are  pres- 
ent in  large  numbers  after  fasting  for  24 
hrs.  (Klein,  S.,  Am.  J.  Anat.,  1905-06, 
5,315-330).  Even  vitamin  B  deficiency 
alters  the  distribution  of  intraepithelial 
fat  (Mottram,  J.  C,  Cramer,  W.,  and 
Drew,  A.  H.,  Brit.  J.  Exp.  Path.,  1922, 
3,  179-181).  According  to  Hamperl 
(H.,  Ztschr.  f.  Mikr.-Anat.  Forsch., 
1925,  2,  506-535)  Enterochromaffin  Cells 
can  no  longer  be  found  in  humans  autop- 
sied  as  late  as  4-5  hrs.  after  death.  The 
incidence  of  Contraction  Bands  in 
muscle  is  increased  by  exposure  to  air 
and  mechanical  manipulation  before 
fixation.  Villi  are  very  prone  to  ex- 
hibit Agonal  Changes.  If  the  indi- 
vidual has  fasted  for  a  long  time  before 
death  a  marked  invasion  of  the  mucous 
membrane  by  lymphocytes  is  to  be  ex- 
pected. See  Fig.  158,  Cowdry's  His- 
tology. It  may  extend  throughout  the 
gastrointestinal  tract  being  greatest  in 
the  stomach  and  least  in  the  large  in- 
testine. 

A  good  way  to  examine  the  wall  of  the 
small  intestine  is  to  push  a  test  tube  of 
appropriate  size  into  the  lumen  of  a 
segment.  This  will  hold  it  open  and 
facilitate  dissection.  Strip  off  the 
serosa,  then  the  tunica  muscularis,  not- 
ing the  direction  of  the  fibers  and  leaving 
only  the  mucosa.  Take  small  pieces  of 
mucosa  and  mount  in  physiological 
saline  inside  up  and  examine  at  low 
magnification.  Finally  with  dissecting 
needles  pick  out  separate  villi  and  study 
with  oil  immersion  objective.  To  ob- 
tain a  clearer  concept  of  individual 
muscle  fibers  first  macerate  the  intestine 
on  the  tube  in  15%  aq.  nitric  acid  for 


SMEARS 


319 


SODIUM 


2-3  days.  Consult  Carey,  E.  J.,  Anat. 
Rec,  1921,  21,  189-215  and  Goerttler, 
K.,  Morph.  Jahrb.,  1932,  69,  329.  See 
Chloralhydrate  Maceration. 
Smears.  To  examine  fluids  and  tissues  as 
thin  films  so  that  the  components  are 
individually  clearly  visible  is  often  nec- 
essary. Careful  preliminary  cleaning 
of  the  slides  is  necessary.  Touch  the 
surface  of  a  slide  about  2  cm.  distant 
from  the  end  to  a  drop  of  blood  imme- 
diately on  the  appearance  of  the  latter 
from  a  puncture  in  the  skin.  Quickly 
apply  the  smooth  end  of  another  slide 
to  the  drop  and  the  surface  of  the  first 
slide  so  that  the  drop  spreads  along  the 
line  of  contact.  Then  evenly  push  the 
second  slide,  with  the  blood  following  it, 
along  the  surface  of  the  first  slide.  The 
angle  at  which  the  pusher  is  held  plus 
the  speed  of  smearing  and  the  amount 
of  blood  will  determine  the  thickness  of 
the  film.  Ordinarily  it  should  be  so 
thin  that  the  reds  are  smeared  in  a  single 
layer.  But  for  certain  purposes  as  in 
the  search  for  some  parasites  thick 
smears  are  the  best  (see  Blood  Smears). 

In  the  case  of  cells  in  cerebrospinal 
and  other  fluids  and  of  some  bacteria 
and  parasites  it  may  be  desirable  to 
concentrate  the  objects  by  centrifuga- 
tion  because  otherwise  smears  would 
show  too  few  of  them.  See  Concentra- 
tion Methods.  The  precautions  de- 
tailed above  to  obtain  evenness  are  sel- 
dom required.  The  material  simply  is 
transferred  to  the  slide  in  a  platinum 
loop  or  glass  pipette  and  spread  on  it. 
Smears  of  lymph  nodes  and  spleen  are 
generally  made  by  drawing  "streaking" 
the  freshly  cut,  moist  surfaces  along 
slides.  Impression  preparations  of 
these  tissues  are  not  smears  but  they 
serve  the  same  purpose.  In  making 
them  the  surface  of  slide  is  quickly 
pressed  against  the  surface  of  the  tissue 
and  a  considerable  number  of  the  easily 
detachable  cells  adhere  to  the  slide 
where  they  are  quickly  dried,  or,  while 
still  wet  the  impression  can  be  fi.xed  in 
Helly's  fluid  (i.e.  formalin  Zenker)  as 
advised  by  Maximow  (see  Downey,  p. 
2001).  McClung  (p.  262)  reconrimends 
smears  on  cover  glasses  for  certain  germ 
cells. 

The  smears  can  be  fixed  by  gentle 
heat,  or  by  methyl  alcohol  or  in  special 
cases  in  formalin  or  osmic  vapor.  Giem- 
sa's  stain  is  the  most  popular  but  a 
great  many  others  are  available  es- 
pecially for  Bacteria. 

Smears  cannot  be  made  of  fixed  cells 
isolated  by  Maceration  in  the  same  way 
because  they  are  not  present  in  body 
fluids  which  when  they  dry  facilitate 
sticking  of  the  cells  to  the  slides.     It  is 


therefore  necessary  to  spread  them  on 
slides  previously  moistened  with  a  very 
small  amount  of  Albumen-Glycerin 
before  drying.  Sec  Papanicolaou  Tech- 
niques, Ear  and  Nasal  Cell  Smears. 

Smith-Dietrich  method  for  lipoids  (Die- 
trich, A.,  Verb.  d.  Deut.  Path.  Ges., 
1910,  14,  263-268).  Treat  frozen  sec- 
tions of  formalin  fixed  tissues  1-3  days 
in  5%  aq.  potassium  bichromate  at  37°C. 
After  washing  in  aq.  dest.  stain  4-5  hrs. 
in  Kultschitzky's  hematoxylin  (stock 
solution  10%  in  abs.  ale.  ripened  at 
least  6  months,  10  cc.  +  2%  acetic  acid, 
90  cc).  Wash.  Differentiate  over 
night  in  Weigert's  borax  ferricyanide 
(borax,  2  gm.;  potassium  ferricyanide, 
2.5  gm. ;  aq.  dest.,  100  cc).  Wash  care- 
fully. Mount  in  syrup  of  levulose. 
Lipoids  dark  blue.  Lison  (204)  consid- 
ers the  positive  staining  as  characteris- 
tic for  a  lipine  (lipoid)  if  the  possible 
presence  of  cholesterides  and  cholesterol 
is  excluded. 

Smooth  Muscle,  see  Contraction  Bands. 

Soap-Wax  technique  for  paraffin  imbedding, 
see  Lebowich. 

Soaps.  Sodium  and  potassium  salts  of  fatty 
acids,  see  Fischler's  modification  of 
Benda   method. 

Sodium.  A  method  for  the  retention  of 
sodium  and  potassium  in  microinciner- 
ated  tissue  has  been  proposed  by  Poli- 
card,  A.,  and  Fillet,  D.,  Bull.  d'Hist. 
Appl.,  1926,  230-235.  In  their  opinion 
these  two  elements  are  present  as  chlo- 
rides in  the  tissue  and  their  conversion 
to  sulphates  by  treating  the  sections 
with  sulphuric  anhydride  fumes  makes 
them  more  stable  and  better  able  to 
withstand  the  high  temperature  of  in- 
cineration. See  Microincineration  Ra- 
dioisotopes. 

A  good  colorimetric  method  for  so- 
dium is  reported  by  Bott,  P.  A.  (J.  Biol. 
Chem.,  1943,  147,  653-661).  He  used 
it  for  determinations  of  sodium  in 
glomerular  urine.  It  works  even  with 
0.3  M  gm.  of  urine  in  0.2  ^l  with  error  of 
about  3%.  Such  techniques  are  not 
advised  for  people  untrained  in  chem- 
istry. 

Probably  the  best  titrimetric  method 
for  sodium  is  that  of  Linder,  R.  and 
Kirk,  P.  L.  (Mikrochemie.  1938,  23, 
269-279)  for  small  amounts  of  tissue 
having  0.13-4.13  m  gm-  of  sodium.  Ac- 
cording to  Glick,  p.  270  the  technique 
of  Clark,  W.  G.,  Levitan,  N.  I.,  Gleason, 
D.  F.  and  Greenberg,  G.  (J.  Biol. 
Chem.,  1942,  145,  85-100)  might  be 
adapted  to  the  required  micro  level  for 
histochemical  investigation. 

An  ultramicromethod  for  sodium 
employing  the  polarograph  has  been 
devised  by  Carruthers,  C,  Indust.  and 


SODIUM  ALIZARIN  SULPHONATE 


320 


SPALTEHOLZ  METHOD 


Engin.  Chem.,  1943,  15,  70-71.  It  has 
been  used  for  analysis  of  small  amounts 
of  epidermis  by  Suntzeff,  V.  and  Car- 
ruthers,  C,  Cancer  Research,  1943,  3, 
431-433.  If  it  is  only  necessary  to 
prove  presence  or  absence  of  traces  of 
sodium  try  Histospectrography. 

Sodium  Alizarin  Sulphonate.  See  Hydrogen 
Ion  Indicators. 

Sodium  Fluoride  effect  on  teeth  (Cowdry's 
Histology,  p.  267). 

Sodium  Paradipicrylamine,  see  Siena  Or- 
ange. 

Soil.  Bacteria.  1.  Conn's  Rose  Bengal 
method  (McClung,  p.  146).  To  1  gm. 
soil  add  gelatin  fixative  (0.015%  gelatin 
in  boiling  water  used  after  it  has  cooled) 
to  make  10  cc.  Place  about  0.01  cc.  on 
slide  to  cover  1  sq.  cm.  Dry  on  boiling 
water  bath.  Stain  with  Rose  bengal  as 
for  Slime  Bacteria.  Unless  counts  are 
to  be  made  the  amount  smeared  on  the 
slide  is  not  important. 

2.  Fast  acid  blue  (C.I.  760)  is 
strongly  recommended  (Romell,  L.  G., 
Stain  techn.,  1934,  9,  141-145)  but  it  is 
doubtful  whether  any  manufacturer 
other  than  I.  G.  Garbenindustrie  makes 
the  dye.  According  to  the  General 
Dyestuffs  Corporation  it  is  contained  in 
violamin  3B.  Dry  suspension  of  soil 
on  slide  which  has  been  fixed  in  alcohol 
with  0.05%  dye  in  4%  aq.  phenol. 
Washing  is  unnecessary.  Examine 
smears  in  water.  Details  are  given  by 
Romell. 

Solantine  Red  8  BLN.  A  sulfonated  'azo 
dye.  For  formula  and  influence  on 
mouse  tumors,  see  Stern,  K.,  Cancer 
Res.,  1950,  10,  565-570. 

Solanylin,  a  dye  extracted  from  the  egg- 
plant (Solarium  melongena,  var.  escu- 
lenta)  proposed  as  a  substitute  for 
hematoxylin.  It  will  stain  nuclei  and 
mucus  (Fuse  and  Suzuki,  Arb.  Anat. 
Inst,   zu  Sendai,   1935,   17,   175-181). 

Solid  Green  JJO,  see  Brilliant  Green. 

Solid  Green  O,  see  Malachite  Green. 

Soluble  Blue  3M  or  2R,  see  Anilin  Blue. 

Soluble  Indulin  3B,  see  Indulin,  water 
soluble. 

Soluble  Yellow  OL,  see  Metanil  Yellow. 

Solutions.  In  technique  several  kinds  are 
employed. 

1.  Physiological  solutions  are  in- 
tended to  approximate  as  closely  as 
possible  to  the  tissue  fluid  environments 
of  cells  so  that  cells  examined  in  them 
will  not  be  greatly  altered  thereby. 
See  Physiological  Solutions. 

2.  Normal  solutions  are,  on  the  other 
hand,  chemical  standards  made  by  dis- 
solving definite  amounts  of  substance 
(easily  calculated)  in  sufficient  aq.  dest. 
to  make  1  liter.     See  Normal  Solutions. 

3.  Molar,  molecular  and  grammolecu- 


lar  solutions  contain  the  molecular 
weight  of  the  substance  in  grams  per 
liter.  They  are  of  the  same  concentra- 
tion as  normal  solutions  of  substances 
possessed  of  one  hydrogen  or  other 
equivalent  and  difTer  from  those  of  sub- 
stances containing  more  than  1  such 
equivalent.  See  Molecular  Solutions. 
4.  Molal  solutions  contain  the  molec- 
ular weight  of  the  substance  in  grams 
+  1000  grams  aq.  dest.  The  designa- 
tion molal  is  rarely  used,  molecular  is 
common  and  normal  most  frequent. 

Sonic  Vibrations.  Employed  as  a  means  for 
fractionating  spermatozoa  so  that  their 
several  parts  can  later  be  collected  by 
centrifugation  (Zittle,  C.  A.  and  O'Dell, 
R.  A.,  J.  Biol.  Chem.,  1941,  140,  899- 
907).     See    Ultrasonic    Vibrator. 

Sorensen's  BuflFers.  Sorenson's  phosphate 
bufi'ers  are  prepared  from  Merck's 
special  reagents.  Dry  salts  at  105°C. 
overnight  and  store  in  a  dessicator  over 
CaCh.  M/15  solutions  are  used.  To 
make  them  dissolve  the  following 
amounts  in  aq.  dist.  and  make  each  so- 
lution up  to  one  liter: 

NaiHPOi  anhydrous 9.47  gm. 

KH2P04 9.08  gm. 

To    obtain    a   solution   of   the   pH   re- 
quired, mix  them  in  following  amounts: 


ec.  M/15 

cc.  M/15 

pH 

NajHPOi 

KH,PO. 

5.4 

3.0 

97.0 

5.6 

5.0 

15.0 

5.8 

7.8 

92.2 

6.0 

12.0 

88.0 

6.2 

18.5 

81.5 

6.4 

26.5 

73.5 

6.6 

37.5 

62.5 

6.8 

50.0 

60.0 

7.0 

61.1 

38. 

7.2 

71.5 

28.5 

7.4 

80.4 

19.6 

7.6 

86.8 

13.2 

7.8 

91.4 

8.6 

8.0 

94.5 

5.5 

For  range  pH  8.2-9.2  see  Palitzsh  Buf- 
fers.    See  affect  of  Phosphate  Solutions 

on  living  cells. 
Spalteholz  Method  for  clearing  small  em- 
bryos as  suggested  by  the  Bensleys. 
After  appropriate  fixation  80  and  95% 
alcohol  1  day  each.  Two  changes  ab- 
solute alcohol,  2  days.  Equal  parts 
benzol  and  absolute  alcohol,  1  day. 
Two  changes  pure  benzol,  1  day.  Then 
Wintergreen  oil  (methyl  salicylate)  and 
benzyl  benzoate  by  weight  5:1,  3:1  and 
2:1  for  very  young,  young  and  older 
embryos   respectively    (under  negative 

Kressure  in  vacuum  pump)  until  cleared, 
lount  or  store  in  this  clearing  fluid.     In 
practice  it  is  possible  to  get  good  results 


SPECIFIC  GRAVITY 


321 


SPORE  STAIN 


without  the  negative  pressure.  This 
method  can  be  used  for  many  tissues 
besides  embryos.  For  author's  account 
see  Spalteholz,  W.,  Ueber  das  Durch- 
sichkigmachen  von  menschlichen  und 
Tierischen  Praparaten.  Leipzig,  2nd 
Edition,   1914. 

Specific  Gravity.  It  is  often  desirable  to 
ascertain  the  relative  specific  gravities 
of  tissues,  cells  and  parts  of  cells.  See 
Centrifugation. 

Spectrographic  Analysis,  see  Histospectrog- 
raphy  and  Absorption  Spectra. 

Spectrophotometric  Analysis  of  tissue  stain- 
ing has  been  greatly  advanced  by 
Stowell,  R.  E.  and  Albers,  V.  M.,  Stain 
Techn.,  1943,  18,  57-71.  Comparison 
of  spectral  absorption  curves  of  stains 
and  substances  colored  bj^  them  has 
demonstrated  that  data  can  thereby  be 
obtained  on  the  chemical  processes  in- 
volved. No  evidence  was  found  of  sig- 
nificant chemical  alterations  in  the 
chromophox  radicals  of  the  stains  asso- 
ciated with  the  tissue  staining  under  the 
conditions  of  the  experiments. 

Spectrophotometric  Evaluation  of  blood 
stains  (Lillie,  R.  D.  and  Roe,  M.  A., 
Stain  Techn.,  1942,  17,  57-63). 

Spermatozoa,  simple  method  for  staining. 
Make  smears  of  fresh  spermatic  fluid  on 
slides  and  dry  in  air.  Fix  3  minutes  in 
10%  formalin.  Stain  in  Harris'  hema- 
toxylin 1  minute,  wash  in  water  and  dry 
(Fetterman,  G.  H.,  Am.  J.  Clin.  Path., 
1942,  6,  9).  Microincineration  (Poli- 
card,  A.,  Bull.  d'Hist.  Appl.,  1933,  10, 
313-320).  Helpful  histochemical  meth- 
ods are  detailed  by  Marza,  V.  B., 
Bull,  d'hist.  appl.,  1931,  8,  85-102. 

Sperms  are  excellent  material  for 
Electron  Microscopy.  See  Schmitt,  F. 
O.  (Biological  Symposia,  1943,  10,  261) 
and  Scott,  G.  H.  in  McClung's  Micro- 
scopical Technique,  1950,  p.  723.  The 
Keilin,  D.  and  Hartres,  E.  F.  (Nature, 
1950,  165,  504)  device  of  manifold  in- 
tensification of  absorption  spectra  in 
liquid  air  has  been  employed  by  Mann, 
T.  (Biochem.  J.,  1951,  48,  386-388)  for 
cytochrome  determinations  in  sperm. 
See  Semen. 

Spermin  Crystals  are  long  prism-like  forma- 
tions produced  in  dried  semen  colored 
brown  or  violet  with  iodine  or  potas- 
sium iodide,  also  known  as  Boettcher's 
crystals. 

Sphingomyelin,  a  compound  of  phosphoric 
acid,  a  fatty  acid,  choline  and  sphingo- 
sine  without  glycerol,  soluble  in  ben- 
zene, pyridine  and  hot  alcohol  and  al- 
most insoluble  in  ether,  see  Lipoids. 

Spirit  Blue  (CI,  689)— anilin  blue  alcohol 
soluble,  gentian  blue  6B,  light,  Lyon 
and  Paris  blues— A  mixture  of  di-  and 
tri-phenyl    rosanilin     chlorides.     Conn 


(p.  133)  reports  that  it  is  a  good  stain 

for  growing  nerve  fibers. 
Spirit  Indulin,  see  Indulin,  spirit  soluble. 
Spirit  Nigrosin  R,  see  Indulin,  spirit  soluble. 
Spirochaetales.    The     organisms     of     this 

group  often  require  special  methods  for 

demonstration ;  but  within  the  gastric 

f lands  of  humans  (Doenges,  J.  L.,  Arch, 
'ath.,  1939,  27,  469-477)  dogs,  cats,  rats 
and  Macacus  rhesus  monkeys  (Cowdry, 
E.  V.  and  Scott,  G.  H.,  Arch.  Path., 
1936,  22,  1-23)  they  can  frequently  be 
seen  in  ordinary  hematoxylin  and  eosin 
preparations.  Preparations  of  these  be- 
nign organisms  are  therefore  easily 
made  and  useful  as  showing  intracellular 
forms  within  parietal  cells.  For  special 
techniques  see  Treponema  Pallidum, 
Warthin-Starry  method  and  Vincent's 
Angina. 
Spleen.  Fixatives  penetrate  the  spleen 
poorly  on  account  of  the  large  amount 
of  blood  in  it.  Consequently  it  is  desir- 
able to  fix  only  thin  slices  of  it,  say  3-4 
mm.  thick.  If  the  spleen  is  particularly 
soft  to  begin  with  the  slices  will  not  hold 
their  shape  and  it  may  be  necessary  to 
cut  parallel  to  the  surface  and  include 
the  capsule  as  a  support.  Direct  ob- 
servation of  splenic  venous  sinuses 
in  vivo  (Knisely,  M.  H.,  Anat.  Rec, 
64,  499-524;  65,  23-50;  MacKenzie,  D. 
W.,  Whipple,  A.  O.  and  Wintersteiner, 
M.  P.,  Am.  J.  Anat.,  1941,  68,  397-456). 
Transplants   into   omentum    (Holyoke, 

E.  A.,  Am.  J.  Anat.,  1940,  66,  87-132). 
For  vascular  injections  of  Malpighian 
bodies,  see  Nisimaru,  Y.  and  Staggerda, 

F.  R.,  J.  Physiol.,  1932,  74,  327-337. 
See  Kurlof  Bodies. 

Spodogramme,  term  used  bj'  French  his- 
tologists  for  the  mineral  skeleton  of 
tissue  revealed  by  Microincineration. 

Spore  Stain,  a  modification  of  Dorner's. 
Make  thin  film  on  slide.  Cover  with 
blotting  paper  and  add  freshly  filtered 
Ziehl's  carbol  fuchsin.  Steam  5-10  min. 
on  hot  plate,  the  blotting  paper  being 
moistened  with  the  fuchsin.  Decolor- 
ize instantaneouslj'  with  95%  alcohol 
and  wash  in  water.  Add  drop  of  sat. 
aq.  nigrosine  and  spread  thinly.  Dry 
quickly  and  examine.  Spores  red,  other 
parts  of  bacilli  almost  colorless  against 
dark  background.  Said  to  be  simpler, 
quicker  than  the  unmodified  Dorner's 
method.  It  is  recommended  for  Bacil- 
lus megatherium,  B.  niger,  B.  cereus, 
B.  mycoides  and  some  cultures  of  B. 
subtilis  (Snyder,  M.  A.,  Stain  Techn., 
1934,  9,  71-72). 

Stain  heat  fixed  film  with  carbol - 
fuchsin  (see  Acid  Fast  Bacilli).  Rinse 
quickly  and  difforentiate  in  95%  alcohol. 
Wash  in  hot  tap  water  and  again  rin.se 
in  alcohol.     Counterstain  for  2-5  min. 


SPREADING  FACTORS 


322 


SPREADING  FACTORS 


with  Loeffer's  methylene  blue.  In  case 
of  thick  films  pour  off  and  add  more 
blue.  Rinse  in  tap  water  and  blot  dry 
(S.  Bayne-Jones  in  Simmons  and  Gentz- 
kow,  p.  386). 

A  modification  of  Schaeffer's  spore 
stain.  Support  a  small  metal  tray  over 
asbestos  centered  wire  gauze.  Add 
water  and  heat  to  steaming.  Slides  with 
ends  resting  on  either  side  of  the  tray 
should  have  droplets  of  water  condense 
on  their  under  surface.  Flood  properly 
fixed  smear  on  slide  with  5%  aq.  mala- 
chite green  and  leave  in  this  way  on 
steam  bath  1  min.  Drop  in  cold  water, 
rinse  thoroughly  and  while  wet  add  0.5% 
aq.  safranin  30  sec.  Rinse  again  in  cold 
water.  Spores,  green;  vegetative  cells, 
red  (Ashby,  G.  K.,  Science,  1938,  87, 
443). 
Spreading  Factors  and  ground  substance  of 
Mesenchyme — Written  by  F.  Duran- 
Reynals,  Dept.  of  Microbiology,  Yale 
University,  New  Haven,  Conn.  Octo- 
ber 8,  1951 — A  discussion  on  the  nature 
and  function  of  the  spreading  factors 
(S.F.)  must  be  indispensably  preceded 
by  a  survey  on  the  newer  knowledge  of 
the  ground  substance  (G.S.)  of  the 
mesenchyme,  a  knowledge  to  which, 
precisely,  the  discovery  of  the  S.F. 
has  largely  contributed. 

The  importance  of  the  G.S.  is  easily 
emphasized:  it  is  impossible  to  have  a 
complete  concept  of  cell  function  if  this 
cell  is  not  considered  together  with  its 
immediate  environment,  namely,  the 
G.S.  or  other  intercellular  matrices 
which,  it  is  to  be  hoped,  some  day  will 
be  known.  One  cannot  have  a  clear 
idea  of  the  effects  on  cells  of  poisons  or 
therapeutic  agents;  of  the  portal  of 
entry  and  of  so  many  other  phenomena 
in  infection  and  defense  against  infec- 
tion without  a  previous  knowledge  of 
the  initial  effect  of  the  invading  agent 
on  the  G.S.  or,  conversely,  of  the  effect 
of   the   G.S.   on   the   infectious   agent. 

The  G.S.  of  the  mesenchyme  is,  of 
course,  present  in  all  mesodermic  struc- 
tures, and  consequently  it  pervades  the 
whole  animal  body.  It  is  present  in 
the  skin  where  it  spreads  as  a  continu- 
ous sheet  underneath  the  epidermis; 
and  in  between  the  follicular  cells  sur- 
rounding the  mammalian  ovum.  The 
synovial  fluid  is  a  modified  G.S.,  and 
the  joint  cavities  can  be  considered  as 
giant  intercellular  formations.  The 
G.S.  is  also  abundantly  in  the  vitreous 
humor,  in  the  umbilical  cord,  and  still 
other  structures. 

From  direct  studies  by  a  group  of 
physiologists,  the  following  has  been 
learned  about  the  G.S. : 

(a)  It    is    a    jelly    placed    between 


vessels,  cells  and  fibers,  and  does  not 
contain  spaces; 

(b)  It  does  not  contain  free  water 
under  physiological  conditions.  Water 
is  associated  with  components  of  the 
jelly,  which  is  capable  of  hydration 
in  different  degrees; 

(c)  Dyes,  and  presumably  metabo- 
lites, do  not  seem  to  progress  through 
the  jelly  proper,  but  following  the  path- 
ways   of   the    fibrillar   structures;    and 

(d)  It  shows  a  resistance  to  penetra- 
tion by  inoculated  fluids,  and  this  re- 
sistance is  not  overcome  until  a  pressure 
of  8.5  cm  of  water  has  been  reached. 
Therefore,  the  G.S.  is  a  barrier. 

The  main,  or  best  known,  compo- 
nents of  the  G.S.  are  mucopolysaccha- 
rides— -linked  with  protein,  to  which 
the  G.S.  owes  its  viscid  consistency. 
The  most  significant  of  the  polysac- 
charides   is    hyaluronic    acid    (H.A.) 

H.A.  is  a  member  of  a  very  important 
group  of  substances  such  as  heparin, 
mucoitin,  and  chondroitin  sulphuric 
acids,  and  still  others,  all  of  which 
play  preponderant  physiological  roles. 

The  G.S.  can  be  identified  by  means  of 
staining  reactions,  more  or  less  selec- 
tive for  polysaccharides,  such  as  peri- 
odic acid,  leuco  fuchsin,  toluidine  blue, 
Prussian  blue,  and  still  others. 

One  may  suppose  that  since  H.A.  is 
so  widely  present  in  normal  and  patho- 
logical tissues,  its  secretion  is  the  work 
of  many  mesenchymal  cells,  including 
plain  fibroblasts,  in  a  manner  perhaps 
comparable  to  the  formation  of  the  acid 
in  the  capsule  of  some  bacteria,  e.g. 
streptococcus  group  A  and  C.  However, 
a  variety  of  contributions  seem  to 
incriminate  specialized  granule  -  con- 
taining cells,  identified  or  not  to  mast 
cells  as  the  elements  responsible  for  the 
formation  of  the  G.S.  in  general;  or  of 
synovial  fluid;  or  hyaluronic  acid  in 
particular.  One  may  wonder  whether 
H.A.,  so  rapidly  and  abundantly  ac- 
cumulated in  some  normal  or  patho- 
logical tissues,  such  as  in  the  sex  skin 
of  monkeys  and  some  tumors,  is  origi- 
nated by  the  same  cells  that  secrete  the 
acid  present  in  other  tissues.  Be  it  as 
it  may,  H.A.  formation  is  controlled 
by  endocrine  factors  which  will  be  re- 
viewed later. 

The  S.F.  are  the  agents  which  act 
selectively  or  exclusively  on  the  G.S., 
changing  its  physical  and  chemical 
characteristics.  They  were  discovered 
in  1928  by  the  enhancing  effect  that 
extracts  from  mammalian  testes  proved 
to  have  on  infection.  The  most  im- 
portant, or  at  least  the  best  known,  of 
the  S.F.  is  hyaluronidase  (H)  which  is 
present,  in  mammalian  testes,  in  cer- 


SPREADING  FACTORS 


323 


SPREADING  FACTORS 


tain  invasive  bacteria  such  as  staphy- 
lococcus, streptococcus,  pneumococcus, 
in  animal  poisons  such  as  that  of  rattle- 
snakes, in  secretions  of  insects  such 
as  mosquitoes,  etc.  Often  H.  is  asso- 
ciated with  other  spreading  factors  in 
the  animal  secretions. 

The  in  vivo  effect  of  H.  is  shown  by  the 
spreading  reaction.  In  this  reaction 
any  material  injected  in  the  G.S.,  to- 
gether with  H.,  spreads  rapidly  as  if 
ink  were  dropped  on  a  blotter.  The 
reaction  is  best  shown  when  a  mixture 
of  any  H. -containing  material  and  any 
colored  matter  is  injected  intradermally 
into  a  rabbit.  A  control  inoculum  of 
the  colored  material  alone  remains 
largely  localized  where  the  presence  of 
the   injection    has   left   it. 

H.  was  identified  to  a  mucolytic  en- 
zyme in  1939.  The  action  of  the  en- 
zyme, or  rather  of  the  group  of  enzymes, 
has  been  and  still  is  subject  to  extensive 
biochemical  study  into  which  we  cannot 
enter.  Essentially,  H.A.  attacked  by 
the  specific  enzyme  contained,  for  in- 
stance, in  a  simple  rabbit  testicle  ex- 
tract, is  first  depolymerized  and  then 
split  into  its  components,  glucuronic 
acid  and  acetyl  glucosamine.  The 
first  effect  manifests  itself  by  a  sudden 
drop  in  the  viscosity  of  the  solution 
containing  the  polysaccharide,  for  in- 
stance, a  simple  extract  of  umbilical 
cord. 

This  reaction  in  the  test  tube  is  the 
counterpart  of  the  spreading  reaction 
in  the  animal.  In  the  latter  case,  there- 
fore, the  H.A.  of  the  ground  substance 
is  similarly  attacked  by  the  injected 
H.  and  the  jelly  is  quickly  liquefied. 
What  used  to  be  a  barrier  is  now  a 
pathway,  and  consequently,  any  mate- 
rial injected  together  with  the  enzyme 
will  easily  spread  throughout  the  inter- 
cellular atmosphere.  Mechanical  pres- 
sure increases  the  diffusion  consider- 
ably. 

The  spreading  reaction  also  takes 
place,  although  far  less  conspicuously 
than  in  the  skin,  in  tissues  in  which  con- 
nective tissue  exists  in  formations  sus- 
ceptible of  being  injected.  Whether  H. 
attacks  components  of  the  blood  capil- 
laries resulting  in  increased  capillary 
permeability  is  still  a  debatable  point. 
At  any  rate,  the  permeability  of  the 
blood  capillaries  is  considerably  in- 
creased by  some  factor  which  is  regu- 
larly present  in  the  materials  contain- 
ing H. 

There  are  other  S.F.  which  act  on  the 
G.S.  of  the  mesenchyme,  but  their 
mode  of  action  is  far  less  known  than 
in  the  case  of  H.  The  effect  of  azopro- 
teins  and  ascorbic  acid  seems  to  be  of 


only  a  physical,  depolymerizing  nature. 
Others,  found  in  several  tissue  extracts, 
bacterial  cultures  and  animal  secre- 
tions, and  also  simple  chemicals,  do 
not  affect  H.A.  in  vitro,  and  their 
spreading  effect  may  largely  be  due  to 
edema  formation,  as  shown  by  the  lack 
of  spreading  in  the  skin  of  the  dead 
rabbit.  It  may  be  quite  important 
that,  as  shown  recently,  derivations  of 
H.A.  have  a  spreading  effect  even  in 
the  dead  rabbit.  All  these  facts  em- 
phasize the  complexity  of  the  spreading 
reaction,  and  suggest  that  more  atten- 
tion should  be  paid  to  S.F.  other  than 
H.  Such  study  could  prove  to  be  ex- 
tremely fruitful. 

With  the  above  reviewed  as  a  back- 
ground, we  are  on  a  solid  basis  to 
explain  phenomena  of  fundamental 
importance  in  pathology,  such  as  the  in- 
vasion of  the  lung  by  the  pneumococ- 
cus; the  progressive  march  of  erj^sip- 
elas;  the  brutal  invasion  of  tissues 
following  snake  bite;  the  efficacy  in 
the  inoculation  of  infectious  agents 
carried  by  insect  vectors;  and  so  many 
others. 

In  the  domain  of  physiology,  besides 
other  possible  effects  we  can  under- 
stand the  liberation  of  the  ovum  from 
its  surrounding  granulosa  cells  as  an 
event  preceding  fertilization.  H.A. 
is  largely  present  in  the  cement  holding 
these  cells  together,  and  from  the  at- 
tack of  the  acid  by  the  enzyme  secreted 
by  the  spermatozoa,  this  cement  is 
liquefied  and  the  granulosa  cells  be- 
come dispersed. 

In  the  field  of  pharmacology,  we  can 
also  understand  the  powerful  enhance- 
ment of  the  effect  of  therapeutic  agents 
such  as  the  various  solutions  adminis- 
tered in  clysis,  of  local  anesthetics,  of 
antibiotics,  etc.  when  the  agent  is  in- 
jected together  with  H.  which  will  pro- 
mote its  rapid  spreading  and  absorp- 
tion. Also,  the  injection  of  H.  in 
edematous  areas  subsequent  to  injury 
greatly  accelerates  the  absorption  of 
the  extravasated  fluid,  possibly  the 
effect  of  the  enzyme  being  here  of  a 
more  complex  nature. 

If  a  dye  solution  is  injected  intra- 
dermally into  animals  it  will  sometimes 
diffuse  (although  far  less  than  if  H. 
is  added)  depending  on  different  fac- 
tors such  as  age,  sex,  genetic  constitu- 
tion, etc.  of  the  host.  This  simple 
experiment  indicates  that  the  G.S.  has  a 
physiological  permeability  or  tonus, 
subject  to  changes.  When  the  per- 
meability is  decreased  the  effect  is 
manifest  by  a  suppression  of  the  spread- 
ing effect  of  H.,  the  opposite  being  prob- 


SPREADING  FACTORS 


324 


SPUTUM 


ably  true  when  the  permeability  of  the 
G.S.  is  increased. 

Hormonal  effects  play  a  very  impor- 
tant part  in  controlling  the  permeabil- 
ity of  the  G.S.  Thus,  estrogenic,  and 
some  adrenal  cortical,  hormones  de- 
crease the  permeability  of  the  G.S. — 
that  is,  increase  its  barrier  value — 
whereas  chorionic  gonadotropin  hor- 
mone has  the  opposite  effect.  Other 
hormones  have  been  studied  in  this 
respect,  sometimes  with  contradictory 
results. 

The  mechanism  of  action  of  these 
hormones — a  crucial  point  in  our  sub- 
ject— is  a  matter  of  conjecture.  We 
have  three  clear  cases  in  which  the 
hormone  has  promoted  the  accumula- 
tion of  vast  amounts  of  hyaluronic 
and  chondroitin  sulphuric  acid  in  tis- 
sues of  election:  estrogens  causing  the 
development  of  the  sex  skin  of  monkeys; 
testosterone  inducing  the  develop- 
ment of  the  cock's  comb;  and  thyroid 
stimulating  hormone  provoking  the 
formation  of  periorbital  tissue  in  the 
guinea  pig  and  pretibial  myxedematous 
tissue  in  man.  In  all  these  cases  H. 
injected  into  the  tissue  causes  their 
quick  dissolution  or  collapse  through 
an  effect  on  H.A. 

In  the  case  of  the  sex  skin  of  mon- 
keys, at  least,  the  accumulation  of  H.A. 
increases  very  much  the  barrier  value 
of  the  tissue  to  the  penetration  of 
foreign  matter.  However,  we  do  not 
have  any  evidence  that,  in  the  other 
cases  where  the  permeability  of  the 
G.S.  has  been  diminished  by  endocrine 
effects,  the  effect  is  due  to  quantita- 
tive changes  in  the  tissue  polysaccha- 
rides. This  statement  holds  specially 
true  in  the  case  of  the  adrenal  cortical 
hormones  because  their  effect  is  very 
rapid,  being  most  marked  one  hour 
after  intravenous  injection.  There- 
fore, it  is  logical  to  think  that  qualita- 
tive besides  quantitative  changes  in  the 
polysaccharides  are  effective  in  alter- 
ing the  permeability  of  the  G.S. 

It  would  also  seem  logical  to  suppose 
that  there  is  a  common  denominator 
to  the  effects  of  at  least  some  of  the 
hormones  effective  on  the  G.S.,  and, 
in  view  of  the  newer  knowledge  on  the 
physiology  of  the  adrenal  cortex,  one 
could  suspect  that  some  of  the  adrenal 
cortical  hormones  are  this  common 
denominator.  In  favor  of  this  sup- 
position one  could  quote  the  fact  that 
a  great  variety  of  stimuli  which  de- 
crease the  permeability  of  the  G.S. 
are  also  known  to  act  on  the  adrenal 
in  the  alarm  reaction  syndrome. 

Of  special  interest  in  this  respect  is 
the  case  of  infection,  notably  that  in- 


duced by  rapidly  invading  bacteria. 
Here,  shortly  after  the  experimental 
inoculation  of  e.g.  staphylococcus  or 
streptococcus,  the  permeability  of  the 
G.S.  is  considerably  diminished,  and 
this  phenomenon  is,  in  itself,  a  power- 
ful element  in  the  defense  of  the  or- 
ganism against  infection,  specially  in 
its  first  phases. 

Whatever  hormones  are  responsible 
for  the  diminished  permeability  or  in- 
creased barrier  value  of  the  G.S.,  we 
do  not  know  how  the  effect  is  brought 
out,  although  one  could  theorize  on  a 
direct  effect  on  the  polysaccharides  or 
on  whatever  cells  of  the  mesenchyme 
responsible  for  the  elaboration  of  these 
polysaccharides. 

These  problems  are  of  far  reaching 
importance.  For,  fluctuations  in  the 
permeability  of  the  G.S.  of  the  mesen- 
chyme manifest  themselves  by  parallel 
alterations  in  susceptibility  or  resist- 
ance to  infection,  and  to  a  certain 
extent  it  is  possible  to  protect  against 
e.g.  vaccinia  or  tuberculosis  by  treat- 
ment of  the  host  with  estrogenic  hor- 
mones. True,  we  still  do  not  know  the 
precise  mechanisms  of  the  effect  above 
reviewed,  but  we  do  know  that  the 
harmonious  integration  of  these  effects 
reflects  on  the  G.S.,  which  will  main- 
tain its  perfectly  physiological  tonus. 

If  we  know  so  much  about  acquired 
immunity,  it  is,  to  a  large  extent,  be- 
cause the  natural  immunity  has  failed. 
Maintaining  this  tonus  of  the  G.S. 
would  mean  controlling  of  complex 
mechanisms  leading  to  natural  resist- 
ance, that  is  to  health.  (See  Duran- 
Reynals,  F.,  Bact.  Rev.,  1942,  6,  197- 
252.  Also  Symposium  on  "The  ground 
substance  of  the  Mesenchyme  and  hyal- 
uronidase"  in:  Ann.  N.  Y.  Acad.  Sci., 
1950,  52,  943-1196.) 
Sputum.  Amount,  gross  appearance,  color 
and  odor  (if  present)  are  important. 
Microscopic  examination  should  first 
be  made  mounted  but  unstained.  Look 
for  pus,  elastic  tissue,  pigmented  heart 
failure  cells,  amebae,  fungi,  ova  of  ani- 
mal parasites,  colorless,  hexagonal 
pointed  Charcot-Leyden  crystals,  other 
crystalline  material,  etc.  Stain  smears 
by  methods  of  Giemsa,  Gram  and  for 
Acid  Fast  bacilli.  It  may  be  necessary 
to  use  Concentration  methods.  Inter- 
pretation of  findings  requires  much 
experience .  Comparison  of  chlorox  and 
sodium-hydroxide-alum  techniques  for 
tubercle  bacilli  in  sputum  (Cameron, 
G.  M.  and  Castles,  R.,  J.  Lab.  &  Clin. 
Med.,  1946,  31,  361-368).  See  also  Sec- 
tion on  Sputum  Examination  in  Osgood, 
E.    S.,    Laboratory    Diagnosis.     Phil- 


STAINING 


325 


STARCH  PASTE 


adelphia:  Blakiston  Co.,  1940,  676  pp. 
See  Papanicolaou  Techniques. 

Staining  is  the  act  of  giving  color  to  some- 
thing. It  is  said  to  be  progressive  when 
the  structures  colored  take  up  the  stain 
progressively  to  a  greater  degree  than 
do  others  which  by  contrast  are  not 
colored.  Thus,  in  testing  for  iron  by 
the  Macallum  method  the  iron  is  stained 
progressively  with  hematoxylin.  Stain- 
ing is  called  regressive  when  many 
structures  are  over  stained  and  by 
decolorization,  or  differentiation,  the 
color  regresses  and  is  retained  only  by 
those  which  hold  it  most  tightly  in  con- 
trast with  which  the  others  are  not 
stained.  To  demonstrate  Nissl  bodies 
in  nerve  cells  the  cells  are  over  stained 
with  toluidin  blue.  By  decolorization 
in  alcohol  the  color  is  made  to  regress  to 
the  point  where  the  Nissl  bodies  stand 
out  colored  in  a  cytoplasm  no  longer 
blue.  See,  also  vital  and  supravital 
staining  and  acid  and  basic  dyes. 

Acid  stains  are  often  contrasted  with 
basic  ones  though  the  dyes  are  usually 
neutral  salts.  In  "acid"  dyes  it  is  the 
acid  part,  or  anion,  that  is  colored  and 
does  the  staining;  while  in  "basic"  dye 
the  reverse  holds  and  it  is  the  basic  por- 
tion, or  cation,  that  is  the  coloring  agent. 
For  instance,  acid  fuchsin  is  a  sodium 
salt  of  sulphonic  acid  of  fuchsin  and  it 
is  the  acid  part  which  gives  the  color. 
Basic  fuchsin,  on  the  other  hand,  is  a 
hydrochloride  of  rosanilin  and  it  is  the 
base,  rosanilin,  which  stains.  A  "neu- 
tral" dye  is  a  more  complex  association 
between  a  color  acid  and  a  color  base. 

Basic  materials  may  be  colored  by 
acid  dyes  and  acid  ones  by  basic  dyes, 
but  this  does  not  by  any  means  always 
hold.  A  substance  staining  by  an 
"acid"  dye  is  said  to  be  acidophilic,  as 
for  example  the  specific  granules  of 
eosinophile  leucocytes  which  take  the 
"acid"  dye  eosin.  Similarly  another 
material,  such  as  nuclear  chromatin  is 
termed  basophilic  because  it  colors  with 
toluidin  blue  which  is  a  "basic"  stain. 
A  neutrophilic  granule  is  colored  by 
both  the  color  acid  and  the  color  base  of 
a  neutral  dye.  An  amphophilic  one 
(G.  ampho,  both;  philos,  fond)  will 
stain  with  either  acid  or  basic  dyes  or 
with  a  neutral  dye  for  it  likes  both  color 
acids  and  color  bases.  Heterophile 
leucocytes  (G.  heteros,  other,  and  philos, 
fond)  px)ssess  granules  which  are  homo- 
logous for  the  several  species  but  dififer 
in  staining  reaction  for  the  species 
(Ma.ximow — Bloom,  Histology,  2nd  Edit. 
1934).  See  Supravital  and  Vital  Stains. 

Stains.  The  laboratory  worker  desiring  to 
keep  clean  can  use  the  methods  advised 


by  W.  C.  Tobie  (Simmons,  and  Gentz- 
kow,  p.  358). 

Bacteriological  stains  on  hands. 
Wash  in  2  or  3%  cone,  hydrochloric  acid 
in  95%  alcohol  (by  vol.)  and  then  in 
soap  and  water.  For  fabrics,  wash  in 
10%  acetic  acid  in  95%  alcohol  (by  vol.) 
and  rinse  repeatedly  in  much  water;  in 
case  stain  remains  wash  with  dilute 
chlorine,  or  bromine  water,  or  with  fil- 
tered chlorinated  lime  solution  (as 
"HTH"  high  test  hypochlorite)  and 
rinse  again  in  water. 

Iodine  stains.  Remove  with  aq. 
sodium  thiosulphate  and  wash  in  water. 

Blood  stains.  Wash  away  with  3% 
aq.  hydrogen  peroxide,  and  rinse  in 
water. 

Silver  stains  occasioned  by  silver 
nitrate,  argyrol  and  the  like.  Treat 
with  hot  solution  of  5  gm.  mercuric 
chloride  +  5  gm.  ammonium  chloride  in 
100  cc.  water. 

Mercurochrome  stains.  Wash  out 
fresh  ones  with  dilute  bromine  water  or 
chlorine  water  or  fresh  aq.  filtered 
chlorinated  lime  (HTH).  Old  ones 
should  be  treated  with  2%  aq.  potas- 
sium permanganate  followed  by  5%  aq. 
oxalic  acid  and  washing  in  water. 

Biological  fluids.  Stains  and  smell  of 
putrefaction  caused  by  them  can  be 
removed,  as  above,  by  permanganate 
and  oxalic  acid. 
Standards.  See  Biological  Standards,  Nor- 
mality, Normals. 
Starch  Grains.  The  usual  microchemical 
test  is  to  color  blue  with  dilute  iodine. 
Starch  grains  can  also  be  stained  side 
by  side  with  mitochondria  in  plant  cells 
(Pea  roots,  Elodea,  etc.).  After  Re- 
gaud  fixation  stain  sections  with  warmed 
anilin  fuchsin  about  5  min.  Differen- 
tiate in  5%  alcoholic  aurantia.  Wash 
in  aq.  dest.  Mordant  in  2%  aq.  Tan- 
nin, 20  min.  Wash  in  aq.  dest.  and  stain 
in  1%  aq.  toluidin  blue,  gentian  violet 
or  methyl  green,  5-10  min.  Milovidov, 
(P.  F.,  Arch.  d'Anat.  Micr.,  1928,  24, 
8-18).  Differentiate  in  95%  ale.  dehy- 
drate in  abs.  ale,  clear  in  xylol  and 
mount.  Mitochondria  red,  starch  blue, 
violet  or  green.  Well  shown  in  an 
excellent  colored  plate.  Armed  with 
illustrations  showing  the  distinctive 
structural  features  of  starch  granules 
from  many  species  of  plants  it  is  ordi- 
narily a  simple  matter  by  direct  micro- 
scopic examination  to  identify  a  given 
sample  of  starch  (Schneider,  A.,  The 
Microbiology  and  Microanalysis  of 
Foods.  Philadelphia:  P.  Blakiston's 
Son  &  Co.,  1920,  262  pp.).  See  Poly- 
saccharides. 
Starch  Paste,  as  substitute  for  albumin- 
glycerin  mixture  in  mounting  paraffin 


STATISTICAL  CONTROL 


326 


SUBMICROSCOPIC  FIBRILS 


sections.  Mix  thoroughly  1  gm.  pow- 
dered starch  in  10  cc.  cold  water.  Pour 
into  20  cc.  boiling  water.  Add  2  drops 
dilute  HCl  and  boil  5  min.  constantly 
stirring  to  free  opalescent  sol.  from 
lumps  of  starch.  Add  crystal  of  thymol 
after  paste  has  cooled.  Use  as  the  albu- 
min mixture.  Has  advantages  in  stain- 
ing techniques  as  it  is  unaffected  by 
dyes,  gives  a  very  light  background 
especially  in  silver  preparations;  it  is 
easily  made,  and  sections  adhere  firmly 
to  slides.  R.  Spoerri,  Science,  1939, 
90,  260,  see  also  McDowell,  A.  M.,  and 
Vassos,  A.  A.  Jr.,  Arch.  Path.,  1940,  29, 
432^34.  See  account  by  Bates,  J.  C, 
Stain  Techn.,  1942,  17,  49-56  on  the 
structure  and  staining  of  starch  grains 
in    the    potato    tuber. 

Statistical  Control  in  hematology  (Lancas- 
ter, H.  O.,  J.  Hyg.,  1950,  48,  402-417). 

Steel  Gray,  see  nigrosine,  water  soluble. 

Stereocilia  of  ductus  epididymis  are  not  true 
cilia.  For  technique  and  discussion, 
see  Lucas  A.  ,M.,  in  Cowdry's  Special 
Cytology,  1932  1,  409-474. 

Sternberg  Cells,  see  Reed-Sternberg  Cells. 

Stieve's  Fluid.  Saturated  aqueous  mer- 
curic chloride,  76  cc;  formaldehyde 
solution,  20  cc;  and  glacial  acetic  acid, 
4  cc.  Fix  for  18  hours  and  wash  in 
several  changes  95  per  cent  alcohol. 
A  good  general  fixative  recommended 
by  R.  E.  Stovvell. 

Stomach,  secretory  cells  of.  Use  Mucicar- 
mine  or  Mucihematein  for  surface 
epithelial  cells  and  neck  chief  cells ; 
Bensley's  Neutral  Gentian  for  body 
chief  cells  and  any  combination  of  dyes 
including  a  strongly  "acid"  stain  like 
eosin  for  the  parietal  cells,  all  after  Ben- 
sley's alcoholic  chrome  neblimate  fixa- 
tion. The  parietal  cells  can  be  sharply 
stained  by  supravital  intravascular  in- 
jection with  Neutral  red  or  Naphthol 
Blue  R.  The  canaliculi  of  the  parietal 
cells  can  be  impregnated  with  silver  by  a 
modified  Golgi  method  (Plenk,  H.,  von 
Mollendor'f  Handb.  d.  Mikr.  Anat.  d. 
Menschen.  1932,  5,  (2),  235-402).  To 
observe  the  cytological  changes  after 
discharge  of  strongly  acid  gastric  juice 
and  of  juice  rich  in  pepsin  inject  hist- 
amine and  stimulate  the  vagus  respec- 
tively (Bowie,  D.  J.,  and  Voneberg,  A. 
M.,  Quart.  J.  Exper.  Physiol.,  1935 
25,  247-257).  For  mitochondria  inject 
Janus  Green  intravascularly  or  fix  in 
Regaud's  fluid,  mordant  in  potassium 
bichromate  and  stain  with  Anilin- 
Fuchsin  Methyl  Green.  See  localiza- 
tion of  Pepsin. 

Stools,  see  Feces. 

Storage  of  specimens  whether  microscopic 
slides,  paraffin  or  celloidin  blocks  or 
simply  in  preservative  fluids  should  be 


systematic  in  all  laboratories.  Every 
specimen  coming  in  for  examination 
should  be  given  an  accession  number 
and  the  data  about  it  should  be  inscribed 
in  a  book.  A  book  is  better  than  a  series 
of  cards  because  cards  can  be  removed 
by  irresponsible  persons  and  lost.  The 
number,  and  other  necessary  informa- 
tion, should  be  written  on  the  slide  with 
a  diamond  pencil.  This  is  usually  done 
in  pathological  laboratories  where  there 
is  much  routine  to  be  attended  to.  It  is 
equally  important  in  other  laboratories 
devoted  primarily  to  teaching  and  re- 
search even  when  a  number  of  inde- 
pendent investigators  are  involved. 
System  pays ;  lack  of  a  unified  system 
serving  several  people  means  loss  and 
waste  of  valuable  material. 

Street,  see  Papanicolaou  Techniques  in 
Exfoliative  Cytology. 

Strength,  see  Tensile. 

Striated  Cuticular  Border  of  intestinal  epi- 
thelial cells  is  frequently  confused  with 
cilia,  see  Lucas,  A.  M.,  in  Cowdry's 
Special  Cytology,  1932,  1,  409-474. 

Striated  Muscle,  glycogen  distribution 
(Gendre,  H.,  Bull.  d'Hist.  AppL,  1938, 
15,  265-276).  Effect  of  different  dehy- 
dration and  clearing  agents  (Ralph,  P., 
Stain  Techn.,  1938,  13,  8-15).  Methods 
for  study  of  wave  mechanics  in  living 
state  (Carey,  E.  J.,  Zeit,  W.  and  Masso- 
pust,  L.,  Am.  J.  Anat.,  1942, 70, 119-133. 

Styrax,  a  very  highly  refractile  mounting 
medium  seldom  employed  in  histology 
(Lee,  p.  228). 

Subcutaneous  Tissue  spreads.  Making 
(McClung's  Microscopical  Technique 
p.  336). 

Sublimate  Acetic  is  a  fixative  of  which  the 
usual  composition  is  95  parts  sat.  aq. 
mercuric  chloride  plus  5  parts  glacial 
acetic  acid.  See  Laidlaw's  method  for 
inclusion  bodies.  When  the  saturated 
solution  of  mercuric  chloride  is  made  in 
95%  alcohol  the  fixative  should  be  called 
Sublimate  Alcohol  Acetic.  See  Mer- 
curic Chloride. 

Submaxillary  Glands.  These  can  be  nicely 
stained  by  the  supravital  methods  em- 
ployed for  the  Pancreas.  Stains  for 
Zymogen  and  for  Mucus  are  useful .  The 
duct  cells  are  the  principal  sites  of 
action  of  the  salivary  gland  virus  when 
this  plays  an  inapparent  r61e.  The 
tremendously  enlarged  duct  cells  pro- 
vided with  Nuclear  Inclusions  are  often 
seen  in  the  guinea  pig's  submaxillary 
and  in  several  other  species,  see  Cowdry , 
E.  V.  and  Scott,  G.  H.,  Am.  J.  Path., 
1935,  11,  647-657. 

Submicroscopic  Fibrils.  These  by  close 
association  may  constitute  the  neuro- 
fibrils, spindle  and  astral  fibers,  myo- 
fibrils, and  so  on.     Use  of  polarization 


SUBMICROSCOPIC  PARTICLES 


327 


SUDAN  BLACK  B 


optical  methods  suggests  the  orienta- 
tion of  submicroscopic  rodlcts  parallel 
to  the  length  of  the  fibers.  The  elec- 
tron microscope  is  capable  of  demon- 
strating the  component  submicroscopic 
fibrils  of  coUagenic  fibrils  (Schmitt, 
F.  O.,  Hall,  C.  E.  and  Jakus,  M.  A., 
Biol.  Symposia,  1943,  10,  261-276). 

Submicroscopic  Particles.  In  summarizing 
work  in  II.  R.  Bensley's  laboratory, 
Lazarow,  A.,  Biol.  Symposia,  1943,  10, 
9-26  mentions  two  of  these  barely 
visible  as  shimmering  points  of  light  in 
the  dark  field:  (1)  Lipoprotein  complex 
discovered  by  Claude  at  the  Rockefeller 
Institute  containing  fats,  proteins  and 
nucleo-protein  and  when  concentrated 
en  masse  by  centrifugation  of  cherry 
red  color.  Particle  size  0.06-0. 2^.  (2) 
Particulate  glycogen  discovered  by 
Lazarow  containing  a  little  protein  but 
no  fat.  Water  content  75%.  See  Mi- 
crosomes. 

Submicrosopicc  Structure  of  cytoplasm, 
methods  and  results  (Frey-Wyssling,  A., 
J.  Roy.  Micr.  Soc,  1940,  60,   128-139). 

Succinic  Dehydrogenase.  Semenoif,  W.  E., 
Zeit,  f.  Zellforsch.  Micr.  Anat.,  1935, 
22:  305-309  as  detailed  by  Click,  p.  96. 
Treat  frozen  sections  of  fresh  tissue 
with  2  cc.  0.05  methylene  blue  plus  2 
cc.  10%  sodium  succinate  made  up  to 
10  cc.  with  M/15  phosphate  buffer, 
pH  7.6-8.0  for  10-15  min.  under  cover 
slip  with  air  bubbles  excluded  and 
edges  sealed  with  paraffin.  Compare 
with  section  in  control  medium  made 
up  without  sodium  succinate.  Fading 
of  dj^e  indicates  enzymatic  activities. 
Click,    p.    96.     See    Dehydrogenase. 

Sudan,  II  (CI.  73)— Oil  red  O.  Physical 
properties,  Lillie,  R.  D.,  J.  Tech. 
Methods,  1944,  24,  37-45. 

Sudan  III  (CI,  248) — cerasin  red,  fat  pon- 
ceau G,  oil  red  AS,  O,  B  or  3B,  scarlet 
B  fat  soluble,  Sudan  G,  Tony  red — A 
weakly  acid  dis-azo  dye,  the  most 
popular  of  fat  stains  in  alcoholic  solu- 
tion. A  sat.  sol.  in  70%  alcohol  is  used 
in  the  same  manner  as  Sudan  IV  in 
Herxheimer's  solution  (see  below). 
Variations  in  action  of  Sudan  stains 
depending  on  character  of  fat  and  kind 
of  fixation  (Black,  C.  E.,  J.  Lab.  & 
Clin.  Med.,   1937-38,  23,   1027-1036). 

Staining  in  aqueous  phase  (Dufrenoy, 
J.,  Stain  Techn.,  1937,  12,  71-72). 
Make  concentrated  solution  of  Sudan 
III  in  5  cc.  methylal  (dimethyloxy- 
metliane).  Add  10-20  cc.  aq.  dest. 
The  mixture  separates  into  2  layers :  the 
lower  made  up  of  water,  methylal  and 
Sudan  III  and  the  upper  of  methylal, 
Sudan  III  and  water.  Whether  sections 
float  or  sink  they  take  up  Sudan  III. 
Another  method  of  staining  with  Sudan 


III  in  gelatin  solution  is  given  by 
Telford  Govan,  A.  D.,  J.  Path.  &  Bact., 
1944,   56,   262-264.     See   Bell's  Method 

for  staining  fats  mobilized  by  heat. 
A  promising  acetic-carbol-sudan  tech- 
nique for  lipids  is  described  by  Jackson, 
C,  Onderstepoort,  J.  Vet.  Sci.  &  Animal 
Industry,  1944,  19,  169-177.  To  prepare 
stock  solution  heat  to  simmering  2  gms. 
finely  powdered  Sudan  III  in  450  cc. 
95%  ale.  Filter  hot.  Stopper,  leave 
in  refrigerator  over  night  and  filter  cold. 
Add  to  any  desired  amount  stock  solu- 
tion 5%  aq.  carbolic  drop  by  drop  agi- 
tating vigorously  till  alcohol  content 
is  reduced  to  60%.  About  2  cc.  carbolic 
to  6  cc.  stock  solution  is  required.  Let 
stand  few  hours  well  corked.  Add 
glacial  acetic  drop  by  drop  2.5  drops  per 
cc.  of  carbol  sudan,  or  20  drops  to  the 
8  cc.  in  above  instance. 

Cut  frozen  sections  of  formol  or 
formol-saline  fixed  tissue.  Place  in 
50%  ale.  1  min.  Stain  in  acid-carbol- 
sudan  mixture  l^  hrs.  in  well  stoppered 
container.  Differentiate  in  50%  alco- 
hol, containing  5%  acetic  acid,  10-60, 
sec.  Wash  in  aq.  dest.,  1  min.  Coun- 
terstain  in  filtered  Delafield's  hema- 
toxylin diluted  1:2  with  aq.  dest. 
Differentiate  in  acid  water,  10-20  sec, 
blue  in  ammonia  water  (5  min.)  and 
wash  in  aq.  dest.  Finally  mount  in 
glycerin-jelly.  Method  is  particularly 
recommended  when  existence  of  so- 
called  "Sudanophobe"  lipids  is  sus- 
pected. 

Sudan  IV  (CI,  258) — cerotine  ponceau  3B, 
fat  ponceau,  fat  ponceau  R  or  LB,  oil 
red  IV,  scarlet  red — A  weakly  acid  dis- 
azo  dye  also  widely  used  as  fat  stain 
sometimes  under  heading  of  Scharlach 
R,  especially  in  Herxheimer's  Solution. 
Place  frozen  sections  of  formalin  fixed 
tissue  in  70%  alcohol  for  a  few  sec. 
Transfer  to  Herxheimer's  solution  for 
2-5  min.  in  a  covered  container  to  re- 
duce evaporation  and  precipitation. 
Rinse  in  70%  alcohol.  Wash  quickly  in 
aq.  dest.  Counterstain  with  Harris' 
hematoxylin.  Wash  in  tap  water. 
Mount  in  Glycerin.  Seal  with  paraffin, 
or,  if  permanency  is  desired,  with  Duco 
or  Kronig's  cement.  As  a  rule  these  fat 
stains  do  not  last  more  tlmn  a  few  months. 
Physical  properties  of  Sudan  IV  (Lillie, 
R.D.,  J.  Tech.Methods,  1944,24,  37-45). 

Sudan  Black  B.  This  dye  is  of  English 
manufacture  and  is  not  available  in  U.S. 
during  the  war.  Its  identity  is  still 
uncertain. 

1.  For  fat.  Fix  tissues  24  hrs.  in  5% 
formalin  in  0.9%  saline  or  in  Zweibaum's 
fluid.  The  latter  is  made  by  adding 
1  part  of  2%  aq.  osmic  acid  to  7  parts 
of  a  mixture  consisting  of  3%  potas- 


SUDAN  BLACK  Bj 


328 


SULFHYDRYL  GROUPS 


sium  bichromate  6  cc;  2%  chromic 
acid,  3  cc. ;  and  aq.  dest.  5  cc.  Wash  in 
running  water  24  hrs.  In  case  tissue 
is  delicate  and  requires  support  embed 
in  gelatin  before  cutting  frozen  sections  : 
12.5%  gelatin  in  1%  aq.  phenol  filtered, 
37°C.,  24  hrs.  25%  solution,  same. 
Embed  in  fresh  25%  aq.  gelatin,  cool, 
trim,  harden  in  6%  formalin  24  hrs. 
Cut  frozen  sections,  whether  first  em- 
bedded in  gelatin  or  not,  5-10  microns 
thick.  Transfer  to  aq.  dest.  and  then 
into  50%  diacetin  agitated  30  sec.  To 
make  stain,  add  excess  Sudan  Black  B 
(I.G.F.)  to  equal  volumes  of  diacetin 
and  aq.  dest.,  incubate  at  55°C.  for  2 
days.  Cool.  Before  use  filter  off 
amount  required.  Stain  15  micron  sec- 
tions 2  hrs.  If  speed  is  necessary  warm 
in  paraffin  oven.  50%  diacetin  30  sec. 
Counterstain  with  carmalum.  Place 
in  dish  of  water  with  care  making  sec- 
tions "spin  on  surface  and  flatten." 
Float  on  to  slide  and  mount  in  Apathy's 
medium.  Nuclei  red,  lipids  including 
myelin  black  (Leach,  E.  H.,  J.  Path.  & 
Bact.,  1938,  47,  635-637).  Diacetin  is 
glycerol  diacetate  introduced  as  solvent 
for  scharlach  R  by  Gross  (W.,  Zeit., 
wiss.  Mikr.,  1930,  47,  64).  Since  Leach 
does  not  specify  what  Apathy's  medium 
is,  it  is  suggested  that  temporary 
mounts  be  made  in  glycerin. 

2.  For  myelin  (Lison,  L.  and  Dag- 
nelie,  J.,  Bull.  d'Histol.  Appl.,  1935, 
12,  85-91).  To  stain  lipoid  granules  in 
leucocytes.  Dry  blood  smear  and  fix 
in  methyl  alcohol,  30  sec.  Stain  in  a  jar 
with  sat.  Sudan  black  B  in  70%  alcohol, 
30min.  Rinse  in  water  and  wash  1  min. 
in  70%  alcohol  to  remove  deposit. 
Counterstain  with  sat.  alcoholic  eosin  in 
70%  alcohol,  30  sec.  Wash  and  stain 
in  sat.  aq.  methylene  blue  3  min.  Rinse, 
blot  dry  and  examine  with  oil  immersion. 
Lipoid  granules,  deep  black;  nuclei, 
blue;  and  erythrocytes,  red.  (Sheehan, 
H.L  ,J.  Path.  &  Bact.,  1939,  49, 580-581). 

Sudan  Black  Bi  as  a  bacterial  fat  stain. 
Sat.  sol.  of  Sudan  black  B  (Nat.  Aniline 
and  Chemical  Co.)  in  70%  alcohol,  or 
in  ethylene  glycol  stains  fat  bodies  in 
bacteria  deep  blue  black  (Hartman,  T. 
L.,  Stain  Techn.,  1940,  15,  23-28). 

Sudan  Blue  G,  Brown  5  B,  Corinth  B,  as  fat 
stains  (Lillie,  R.  D.,  J.  Lab.  &  Clin. 
Methods,  1944,  24,  35-42).  This  gives 
good  account  of  all  oil  soluble  dyes  as 
fat  stains. 

Sudan  Dyes  suspended  in  watery  media 
for  use  in  the  staining  of  fat  are  de- 
scribed by  Telford  Govan,  A.  D.,  J. 
Path.  &  Bact.,  1944,  56,  262-264.  While 
stirring  add  sat.  Sudan  dye  in  acetone 
drop  by  drop  from  capillary  pipette  to 
1%   aq.   gelatin   containing   1%   acetic 


acid  to  development  of  a  deep  brick-red 
color  and  a  milk  like  consistency. 
Evaporate  acetone  for  2  hrs.  at  37°. 
Remove  sediment  by  filtration.  Cut 
frozen  sections.  Transfer  them  from 
water  to  1%  aq.  gelatin  for  2-3  min. 
Stain  for  30  min.  at  37°  in  above  de- 
scribed dye  suspension.  Wash  in  1% 
aq.  gelatin  2-3  min.  and  thoroughly  in 
water.  Mount  in  glycerin  jelly,  see 
Glycerine   Jelly   or   in   Karo   Syrup. 

Sudan  G,  see  Sudan  III. 

Sudan  Hydrotropes.  Sudan  stains  are  rela- 
tively insoluble  in  water.  They  can  be 
changed  to  hydrotropes  (Neuberg)  which 
are  water  soluble.  The  hydrotropes  of 
red  lipid  stains  are  of  a  blue  color 
which  changes  to  red  when  the  dye 
passes  into  a  lipid  or  a  lipid  solvent. 
This  is  the  basis  of  a  useful  technique 
for  lipids  (Hadjioloff,  A.,  Bull.  d'Hist. 
Appl.,  1938,  15,  37-41). 

Sudan  R  (CI,  113)— brilliant  fat  scarlet  B, 
oil  Vermillion — A  weakly  acid  mono-azo 
dye. 

Sudan  Red,  see  Magdala  Red. 

Sugars,  see  Reducing  Sugars. 

Sulfatase.  An  enzyme  capable  of  hydrolyz- 
ing  sulfuric  acid  from  its  ester  linkages. 
Since  cartilage,  mucus  and  many  de- 
toxification products  contain  esterified 
sulfuric  acid,  an  understanding  of  the 
localization  of  this  enzyme  would  be 
most  interesting.  Seligman,  A.  M., 
M.  M.  Nachlas,  L.  H.  Maunheimer,  O. 
H.  Friedman  and  G.  Wolf,  Ann.  Surg., 
1949,  130,  333-341,  describe  a  method 
involving  the  hydrolysis  of  beta  naph- 
thyl  sulfate  and  subsequent  diazotiza- 
tion  of  the  enzymatically  liberated 
naphthol. 

Sulfhydryl  Groups.  1.  Prussian  blue  histo- 
chemical  reaction  for  (Chevremont,  M. 
and  Frederic,  J.  Arch,  de  Biol.,  1943, 
54,  589-605).  Fresh  or  fixed  tissue  sec- 
tions or  smears  can  be  used.  Formol, 
formol  Ringer  (saline)  and  Bouin  are 
suitable  fixatives;  but  fluids  containing 
sublimate,  such  as  those  of  Zenker  and 
Helly  are  contraindicated.  The  opti- 
mum time  of  fixation  is  from  a  few  hours 
to  one  day.  Time  of  heating  during 
paraffin  embedding  should  be  reduced 
to  a  minimum.  Wash  sections  care- 
fully in  aq.  dest.  to  remove  formalin. 
Plunge  sections  or  smears  in  3  succes- 
sive baths  of  the  following  mixture: 
1  part  fresh  0.1%  aq.  ferricyanide  of 
potassium  (For  Analysis,  C.P.)  and  3 
parts  1%  aq.  ferric  sulphate  (For  Anal- 
ysis, C.P.).  The  mi.xture  thus  pre- 
pared has  a  pH  of  2.4  and,  in  ordinary 
light,  it  is  stable  for  2  hrs.;  in  darkness 
it  lasts  longer.  The  time  in  the  baths 
is  approximately  10-20  min.  for  frozen 
sections,  20-25  min.  for  paraffin  sections 


SULFHYDRYL  GROUPS 


329 


SULFONPHTHALEINS 


and  for  blood  smears  and  1  hr.  for 
smears  of  yeast.  If  desired,  stain  the 
background  with  Azo  carmin.  No 
metal  instruments  must  enter  the  baths. 
A  positive  result  is  indicated  by  appear- 
ance in  cells  of  blue  granules  or  of  a  blue 
colloidal  precipitate  which  gives  the  im- 
pression of  being  diffuse.  After  long 
washing  in  water  preparations  can  be 
mounted  in  Canada  balsam  after  dehy- 
dration or  in  syrup  of  levulose  without 
dehydration.  They  last  as  long  as  7 
months.  Consult  original  article  for 
histochemical  controls  and  for  illustra- 
tions of  epidermis  and  other  tissues. 

2.  Another  reaction  is  given  as  fol- 
lows by  Serra,  J.  A.,  Stain  Techn.,  1946, 
21,  5-18:  "This  reaction  has  been  exten- 
sively used  for  the  study  of  the  dis- 
tribution of  the  tripetide  glutathione. 
One  of  the  better  methods  of  accom- 
plishing the  reaction  is  that  of  Giroud 
and  Bulliard  (see  Lison,  1936),  which 
gives  a  stable  red  coloration,  while 
other  methods  produce  a  violet  color 
rapidly  fading  awaJ^ 

"The  pieces  are  immersed  for  some 
seconds  (in  general  an  excess  of  time 
does  no  harm)  in  a  5%  aqueous  solution 
of  zinc  acetate.  Directly  afterwards 
they  are  treated  with  a  10%  aqueous 
solution  of  sodium  nitroprusside,  con- 
taining about  2%  concentrated  am- 
monia. The  pieces  acquire  a  bright 
red  coloration,  which  attains  its  maxi- 
mum in  3-5  minutes.  Afterwards  they 
are  mounted  in  pure  glycerin  for  micro- 
scopic observation,  if  necessary  with  a 
preliminary  washing  in  distilled  water. 

"The  materials  may  be  studied 
freshly  or  after  fixation.  It  must  be 
noted,  however,  that  the  majority  of 
the  fixatives  hinder  the  reaction.  We 
obtained  good  results  with  a  fixation  in 
10%  neutral  formaldehyde  during  2-15 
hours  at  room  temperature.  A  more 
prolonged  action  of  this  fixative  also 
hinders  the  reaction;  it  is  recommended, 
therefore,  that  if  possible  2-4  hours  of 
fixation  be  used. 

"The  results  of  the  reaction  have 
different  meanings  according  to  the 
fixation,  washings,  etc.,  because  the 
glutathione  is  partly  soluble.  When 
the  tissues  are  treated  several  times 
with  a  10%  solution  of  trichloroacetic 
acid  for  15  minutes,  the  glutathione  is 
dissolved  and  only  "fixed",  that  is,  pro- 
teic  sulfhydryl  groups  remain  in  the 
preparation.  It  is  still  possible  not 
only  to  demonstrate  the  existing  SH 
groups  but  also  to  reduce  SS  groups  to 
SH  groups,  by  means  of  a  pre-treatment 
of  the  materials  with  a  solution  of  10% 
KCN  for  10  minutes  in  a  small  stoppered 
bottle    (the    cyanide    solution    can    be 


weakly  alkalinized  with  potassium  hy- 
droxide, to  make  its  use  safe). 

"The  reaction  has  been  recognized  aa 
well  localized,  but  in  case  of  doubt  a 
test  of  secondary  impregnation  can  be 
made  in  the  way  described  for  the 
ninhydrin."  See  Nitroprusside  Reac- 
tion. 
Sulfmethemoglobin,  a  greenish  compound 
of  methemoglobin  and  sulphur  often 
encountered  in  abdominal  walls  of 
cadavers,  but  it  may  be  present  in 
blood  where  it  can  be  diagnosed  by 
spectroscopic  examination  (Mallory, 
p.  135). 
Sulfonamides.  Great  importance  of  sulfa 
drugs  makes  their  demonstration  in 
tissues  useful.  Mackee,  G.  M.,  Herr- 
mann, F.,  Baer,  R.  L.  and  Sulzberger, 
M.  B.,  Science,  1943,  98,  66-68;  J.  Lab. 
&  Clin.  Med.,  1943,  28,  1642-1649. 

Fix  fresh  tissue  with  dry  formalde- 
hyde gas  and  visualize  sulfa  compounds 
as  orange  precipitates  in  frozen  sections 
treated  with  p-dimethylaminobenzalde- 
hyde  in  acid  alcohol  solution.  In  at- 
tempting to  identify  sulfonamides 
microscopically  in  urinary  sediments 
the  descriptions  and  diagrams  of  the 
various  crystals  given  by  C.  J.  Gentz- 
kow  and  H.  A.  Van  Auken  in  Simmons 
and  Gentzkow  will  be  helpful,  viz. 

Sulfadiazine:  (1)  free  drug,  "dark 
greenish  irregularity  striated  spheres 
with  either  fuzzy  or  clean  edges";  (2) 
acetyl  crystals  like  "sheaves  of  wheat 
with  eccentric  bindings". 

Sulfaguanidine :  (1)  free  drug,  rare; 
(2)  acetyl  crystals  as  "thin  oblong 
plates,  clear  or  with  fine  mesh  pattern, 
often  aggregated  into  cross  or  star-like 
clusters". 

Sulfanilamide:  (1)  free  drug  as  large 
needles  with  angle  of  106°  at  ends 
often  in  sheaves;  (2)  acetyl  crystals 
similar  needles  with  square  ends. 

Sulfapyridine :  (1)  free  drug  as  stubby 
prisms;  (2)  acetyl  crystals  as  "boat- 
or  petal -shaped  forms  with  rounded 
edges;  start  angled  crystals;  bow  ties  or 
burrs;  and  occasionally  as  large  ro- 
settes." 

Sulphathiazole:  (1)  free  drug  rare  as 
flattened  or  6  sided  crystals  with  angle 
at  end  of  84°;  (2)  acetyl  crystals  may 
resemble  those  of  free  drug  but  with 
end  angles  of  136°  when  they  look  like 
wheat  sheaves  with  central  binding. 
These  may  be  swollen  suggesting  2  half 
circles  fused  at  center;  striated  spheru- 
lites  frequently  occur. 

Sulfasuccidine    crystals    absent     be- 
cause of  but  slight  absorption  of   this 
drug  from  intestine. 
Sulfonphthaleins.    These  are  compounds  of 
phthalic    anhydride    and    ortho-sulfo- 


SULFUR  BORDEAUX 


330 


SUPRAVITAL  STAINING 


benzoic  acid.  They  are  most  valuable 
indicators.  Examples:  brom  chlor  phe- 
nol blue,  brom  cresol  green,  brom  cresol 
purple,  brom  phenol  blue,  brom  phenol 
red,  brom  thymol  blue,  chlor  cresol 
green,  chlor  phenol  red,  cresol  red, 
metacresol  purple,  phenol  red,  thymol 
blue. 

Sulfur  Bordeaux  (CI,  1012),  Sulfogene  Bor- 
deaux BRN  (DuPont)  and  Sulfur  Bor- 
deaux BCF  (NAC)  are  direct  dyes  of 
light  fastness  2.  Specifications  for 
staining  invertebrates  and  plant  tissues 
are  given  (Emig,  p.  62). 

Sulfur  Brilliant  Blue  (CI,  957),  Sulfindone 
Brilliant  Blue  CG  (NAC),  Sulfogene 
Brilliant  Blue  6BS  (DuPont),  and  Sulfo- 
gene Brilliant  Blue  3  GCF  (DuPont) 
are  the  best  blue  direct  sulfur  dyes  of 
color  fastness  2,  the  use  of  which  for 
staining  algae  and  invertebrates  is  de- 
scribed (Emig,  p.  61). 

Sulfur  Direct  Blue  (CI,  956),  Sulfogene 
Direct  Blue  BRS  (DuPont),  a  direct 
dye  of  light  fastness  2  which  does  not 
color  blue  green  algae  as  intensely  or 
brightly  as  Sulfur  Brilliant  Blue,  but 
does  present  details  of  cell  structure 
clearly  (Emig,  p.  61). 

Sulfur  Green  (CI,  1006),  Sulfogene  Green 

2  B  (DuPont),  Sulfogene  Brilliant 
Green  2  G  (DuPont)  and  Sulfur  Green 

3  G  cone.  (NAC),  direct  dyes  of  light 
fastness  2  action  of  which  on  plant  tis- 
sues and  invertebrates  is  described 
(Emig,  p.  62). 

Sulfur  Orange  (CI,  949)  and  Sulfur  Yellow 
(CI,  948)  resemble  Sulfur  Bordeaux 
(Emig,  p.  61). 

Sulphonal  Poisoning.  Effect  on  liver  cell 
mitochondria  (Grynfeltt,  E.,  and  La- 
font,  R.,  C.  rend.  Soc.  de  Biol.,  1921, 
85,  406-408). 

Sulphur.  In  inorganic  form  sulphur  is  not 
found  in  living  things  except  in  the 
thiobacteria.  Histochemically  one  has 
to  consider  sulphates  and  masked  sul- 
phur. Macallum  has  devised  a  method 
for  sulphates  but  Lison  (p.  121)  says 
that  it  only  gives  very  rough  localization 
in  tissues  because  the  salt  is  diffusible. 
For  organic,  masked  sulphur  see  Sulf- 
methemoglobin,  Glutathione,  Radio- 
sulphur. 

Sulphur  Compounds.  Microdetermination 
by  means  of  the  iodine-azide  reaction 
(Holter,  H.  and  L0vtrup,  S.,  C.  rend. 
Lab.  Carlsberg,  S6r.  Chim.,  1949,  27, 
72-78).  They  worked  with  tetrathio- 
nate  and  cystine.  1  X  10~'  ng  could  be 
determined  with  an  accuracy  of  2.5% 
(standard  deviation). 

Sulphurous  Acid.  This  is  used  for  rinsing 
sections  which  have  been  stained  with 
Feulgen  or  Schiff's  reagent.  Prepare 
by  dissolving  1  gm.  potassium  or  sodium 


meta  bisulphite  in  200  cc.  of  tap  water 
to  which  10  cc.  of  N  HCl  are  added. 

Sultan  Red  4B,  see  Benzopurpurin  4B. 

Sun  Yellow  (CI,  620),  a  direct  stilbene  dye, 
light  fastness  3.  Serves  as  a  mordant 
to  produce  green  in  combinations  with 
blue  counterstains.  Many  combina- 
tions of  Sun  Yellow  with  blue  and  red 
dyes  in  double,  triple  and  quadruple 
stains  are  described  (P^mig,  p.  44-45). 

Superchrome  Black  PV  (CI,  170)  of  NAC, 
an  acid  monoazo  mordant  dye  action  of 
which  on  plant  sections  and  blue  green 
algae  is  described  (Emig,  p.  34). 

Superchrome  Violet  B  (CI,  169)  of  NAC,  an 
acid  monoazo  mordant  dye  of  light  fast- 
ness 3  of  which  action  on  blue  green 
algae  is  described  (Emig,  p.  34). 

Superchrome  Garnet  Y  (CI,  168)  of  NAC,  an 
acid  monoazo  mordant  dye  of  light  fast- 
ness 3  of  which  action  on  blue  green 
algae  is  described  (Emig,  p.  34). 

Supravital  Staining.  By  this  is  meant 
staining  upon  the  living  state.  In  other 
words  stains  are  applied  to  cells  re- 
moved from  a  living  animal,  or  to  cells 
within  a  recently  killed  animal.  Thus 
blood  cells  are  removed  from  the  body 
and,  while  still  living,  are  stained  supra- 
vitally  or  the  stains  are  applied  to  still 
living  cells  of,  say,  the  stomach  within 
the  body  of  a  recently  killed  animal  by 
vascular  injection.  The  essential  point 
is  that  the  stains  act  upon  living  cells 
but  the  cells  do  not  go  on  living,  neither 
does  an  animal  injected  intra vascularly 
with  a  supravital  stain.  Janus  green 
is  our  most  useful  supravital  stain. 
Cells  supravitally  stained  by  it  die  and 
when  it  is  injected  in  sufficient  quantity 
into  a  living  animal,  the  animal  dies 
likewise  for  it  is  toxic.  Vital  stains, 
on  the  contrary,  do  not  kill  cells  and  can 
be  safely  injected  into  living  animals 
since  they  are  nontoxic  in  the  concen- 
trations necessary  to  obtain  the  desired 
results.  This  kind  of  staining  used  to 
be  called  intravital  in  contrast  to  supra- 
vital.   See  Vital  Stains. 

Supravital  stains  have  been  known 
for  a  long  time  but  their  introduction  as 
essential  means  of  investigation  is  due 
primarily  to  Professor  R.  R.  Bensleyof 
the  University  of  Chicago  (Am.  J.  Anat., 
1911,  12,  297-388).  He_  showed  their 
usefulness  in  demonstrating  specifically 
by  vascular  injection  the  different  epi- 
thelial components  of  the  pancreas  and 
he  called  attention  to  the  fact  that  to 
stain  mitochondria  specifically  it  is 
essential  to  use  janus  green  having  the 
composition  of  die^/ij/Zsafranin-azodi- 
methylanilin,  that  the  dimethyl  com- 
pound will  not  work.  The  supravital 
staining  of  blood  cells  began  with  the 
demonstration  by  Cowdry  at  Hopkins 


SURAMIN 


331 


SURFACE  TENSION 


(Internat.  Monatschr.  f.  Anat.  u. 
Physiol.,  1914,  31,  267-286),  that  thia 
particular  janus  green  B  as  used  in  Ben- 
sley's  laboratory  stains  the  mitochon- 
dria in  human  white  blood  cells  specifi- 
cally. The  method  was  later  further 
developed  by  Sabin  and  her  associates. 
Details  of  techniques  are  given  under 
janus  green,  neutral  red,  brilliant  cresyl 
blue,  pyronin,  methylene  blue,  naph- 
thol  blue  and  cyanamin.  Useful  table 
giving  reactions  of  types  of  blood  cells 
(Gall,  E.  A.,  J.  Lab.  &  Clin.  Med., 
1934-35,  20,  1276-1293).  A  critique  of 
supravital  staining  of  blood  cells  is 
provided  by  Schwind,  J.  L.,  Blood, 
1950,  5,  597-622. 
Suramin,  a  drug  purchasable  under  term 
of  Naphuride  (Winthrop),  is  only  a 
feeble  inhibitor  of  growth  of  lympho- 
sarcoma transplants.  Its  cytotoxic 
efTect  is  rather  similar  to  that  of  colchi- 
cine on  lymphoid  tumors  (Williams, 
W.  L.,  Cancer  Research,  1946,  6,  344- 
353). 
Surface  Films,  study  of  by  micrurgical 
technique,  Chambers,  R.  W.  and  Kopac, 
M.  J.  on  McClung's  Microscopical 
Technique,  1950,  p.  542. 
Surface  Measurements.  To  determine  the 
surface  area  of  structures  of  microscopic 
size  involves  many  techniques  some  of 
which  are  rather  complicated.  The 
following  references  are  given  to  methods 
and  results  in  a  wide  variety  of  in- 
stances. Perhaps  the  particular  surface 
to  be  measured  will  be  sufficiently  simi- 
lar to  one  of  these  to  justify  employ- 
ment of  the  same  technique  or  a  modi- 
fication of  it. 

Endothelium  of  vascular  capillaries — 
6300  sq.  meters — Krogh,  A.,  Anatomy 
and  Physiology  of  Capillaries,  Yale 
Press,  1929,  422  pp. 

Erythrocytes  combined — 3500  sq.  me- 
ters— Evans,  C.  L.,  Recent  Advances  in 
Physiology.  Philadelphia:  Blakiston, 
1926,  383  pp. 

Filtration  surface  of  both  kidneys 
combined — 1.56  sq.  meters — Vimtrup, 
B.  J.,  Am.  J.  Anat.,  1928,  41,  132-151. 
See  also  recent  measurements  for  al- 
bino rat  by  Kirkman,  H.  and  Stowell, 
R.  E.,  Anat.  Rec,  1942,  82,  373-389. 

Gastric  glands  secreting  surface — 
2.7  sq.  meters — Scott,  G.  H.  (personal 
communication),  see  Cowdry's  Histol- 
ogy  (p.  282).  ,  „   .  . 

Lacteal  surface  in  small  intestme-;- 
5  sq.  meters — Policard,  A.,  Pr6cis 
d'Histologie  Physiologique.  Collection 
Testut,  Paris:  G.  Doin,  923  pp.,  after 
Potter. 

Large  intestinal  crypts — 4.2  meters — 
Policard,  ibid. 

Mitochondrial,  zymogenic  and  nuclear 


surfaces  in  pancreatic  acinous  cells  of 
guinea  pig — duNouy,  P.  L.  and  Cowdry, 
E.  v.,  Anat.  Rec,  1927,  34,  313-329. 

Respiratory  surface  plus  nonrespira- 
tory epithelial  surface  of  airways  of 
lungs — 70  sq.  meters — Wilson,  H.  G., 
Am.  J.  Anat.,  1922,  30,  267-295. 

Surface  Replica  Method,  see  Shadow  Cast- 
ing in  Electron  Microscopy. 

Surface  Tension.  This,  or  more  correctly 
interfacial  tension,  is  tension  at  the 
surface  of  a  fluid  tending  to  produce 
a  sphere.  Surface  tension  is  high  for 
water  and  low  for  alcohol.  Soap  de- 
creases surface  tension  of  water  because 
it  concentrates  at  surfaces.  Bile  acids 
lower  surface  tension  of  blood  serum. 
According  to  Gibbs  any  substance 
lowering  interfacial  tension  will  con- 
centrate at  the  interfaces.  Surface 
tension  is  best  determined  by  a  Cenco- 
du  Nouy  tensiometer  capable  of  meas- 
uring the  force  required  in  lifting  a 
standard  platinum  ring  out  of  the 
surface  of  the  liquid.  The  ring  must 
obviously  be  held  absolutely  horizontal 
and  be  pulled  away  slowly  (Holmes, 
H.  N.,  Glasser's  Medical  Physics,  257- 
263). 

Much  has  been  written  about  surface 
tension  (Reviews:  Harvey,  E.  N.,  and 
Danielli,  J.  F.,  Biol.  Rev.,  1938,  13, 
319-341  and  Danielli,  J.  F.  in  Bourne, 
pp.  69-98).  Before  measurements  can 
be  made  on  cells  it  is  obviously  neces- 
sary to  isolate  them  and  this  entails 
risk  of  injury  which  is  much  greater 
in  the  case  of  mammalian  cells  than  of 
the  sea  urchin  eggs  usually  employed. 
The  following  techniques  are  given  as 
examples : 

1.  By  centrifuging  marine  eggs  elon- 
gation can  be  produced  and,  when  the 
length  exceeds  a  certain  ratio  of  diam- 
eter, the  egg  divides.  Knowing  the 
minimum  force  required,  the  difference 
in  density  between  the  2  halves  and  the 
circumference  of  the  cylinder,  it  is 
apparently  possible  to  calculate  the 
tension  at  the  surface  (Harvey,  E.  N., 
J.  Franklin  Inst.,  1932,  214,  1-23). 

2.  By  compressing  sea  urchin  eggs 
by  a  minute  gold  beam  the  internal  pres- 
sure can  be  calculated  and  from  this  the 
surface  tension  (Cole,  K.  S.,  J.  Cell  & 
Comp.  Physiol.,  1932,  I,  1-9). 

3.  By  stretching  a  cell  between  the 
two  needles  of  a  microdissection  ap- 
paratus the  force  required  to  secure  a 
given  degree  of  elongation  can  be  deter- 
mined and  thence  the  surface  tension 
(Norris,  C.  H.,  J.  Cell  &  Comp.  Physiol., 
1939,  14,  117-133). 

4.  Surface  tension  is  probably  to 
some  extent  at  least  conditioned  by  the 
elasticity  of  the  superficial  plasma  gel 


SURVIVAL  OF  TISSUES 


332 


TEETH 


layer  which  brings  in  the  methods  and 
observations  of  Lewis,  W.  H.,  Arch.  f. 
exp.  Zellf.,  1939,  28,  1-7;  Am.  J.  Cancer, 
1939,  35,  408-415  who  refers  to  previous 
work  along  this  line. 

Survival  of  Tissues  after  death  of  the  body 
(Alvarez,  W.  C,  Quart.  Rev.  Biol., 
1937,  12,  152-164).  Often  determined 
by  measuring  how  long  the  tissue  con- 
tinues to  respire.  Data  for  whole  skin, 
kidney  and  liver  (Walter,  E.  M.,  Shar- 
lit,  H.  and  Amersbach,  J.  C,  J.  Invest. 
Dermat.,  1945,  6,  235-238).  Schrek,  R., 
Radiology,  1946,  46,  395-410  has  made 
much  use  of  a  method  for  measuring  the 
survival  of  cells  in  terms  of  the  per- 
centage which  do  not  stain  with  eosin 
(and  are  presumably'  alive)  in  emulsions 
of  cells  in  a  special  fluid  held  at  definite 
pH  and  temperature  for  various  lengths 
of  time.  See  Dead  cells.  Revival  after 
freezing. 

Susa  fixative  of  Heidenhain.  Corrosive 
sublimate,  4.5  gm.;  common  salt,  0.5 
gm.;  aq.  dest.,  80  cc;  formalin,  20  cc; 
and  trichloracetic  acid,  4  cc.  Fix  about 
12  hrs.,  wash  in  95%  alcohol.  Has  the 
advantage  over  most  sublimate  mix- 
tures that  treatment  with  iodine  is  not 
usually  required  to  remove  black  mer- 
cury deposit  in  the  tissues.  It  has 
been  modified  by  several  people.  See 
Buzaglo. 

Swiss  Blue,  see  Methylene  Blue. 

Synapses,  see  methods  employed  by  Bartel- 
mez,  G.  W.  and  Hoerr,  N.  L.,  J.  Comp. 
Neurol.,  1933,  57,  401-428. 

Synovial  Fluid  of  normal  knee  joint.  Method 
of  examination  and  results  (Coggeshall, 
H.  C,  Warren,  C.  F.  and  Bauer,  W., 
Anat.  Rec,  1940,  77,  129-144). 

Syphilis,   see  Treponema  pallidum. 

Syrup,  see  Apathy. 

Taenia  Echinococcus,  a  parasite  of  dogs 
which  produces  hydatic  cysts  in  human 
liver  and  other  tissues.  The  laminated 
cyst  wall  is  typical  and  the  heads  have 
double  circle  of  hooks  and  4  suckers. 

Taenia  Saginata.  In  examination  of  fresh 
Feces  identify  by  head  with  4  suckers 
but  without  hooks. 

Taenia  Solium.  Look  in  Feces  for  head 
with  4  suckers  and  a  circle  of  small 
hooks  best  seen  in  fresh  mounts.  The 
genital  system  opens  at  the  side  and 
the  uterus  is  only  slightly  branched. 

Tagged  Atoms,  see  Radioactive  Isotopes, 
Deuterium. 

Tannic  acid  iron  technique  is  described  by 
Salazar,  A.  L.,  Stain  Techn.,  1944,  19, 
131-135.  He  advocates  it  for  study  of 
Golgi  apparatus  and  with  Giemsa's 
stain  to  give  sharper  differentiation 
between  agranulocytes  and  granulo- 
cytes. 

Tantalum,  see  Atomic  Weights. 


Tapeworm  Proglottids.  Orient  pieces  4-5 
cm.  long  containing  gravid  proglottids 
between  glass  slides  held  together  by 
elastic  bands.  Fix  in  Bouin's  fluid  (sat. 
aq.  picric  acid,  7  parts;  glacial  acetic 
acid,  20  parts;  and  formalin,  10  parts 
10-12  hrs.  Wash  in  running  water  2-3 
min.  Flood  with  10%  aq.  NaOH  (out- 
lines of  uterus  become  visible  deep 
orange).  Rinse  in  tap  water.  Flood 
with  5%  HCl  1-2  min.  Tap  water  10- 
15  min.  Dehydrate  in  alcohol,  clear  in 
xylol  and  mount  in  balsam  (Dammin, 
G.  J.,  J.  Lab.  &  Clin.  Med.,  1937-38, 
23,  192-194).  An  oxidation  reduction 
method  for  stain  differentiation  is  pro- 
vided by  Tapmisian,  T.  N.,  Stain 
Techn.,  1945,  20,  11-12.     See  Parasites. 

Target  Cells.  Er3'throcytes  looking  some- 
what like  the  "bull's  eyes"  of  targets 
because  a  central  hemoglobin  rich  area 
is  surrounded  by  a  clear  area  enclosed 
by  a  peripheral  ring  likewise  rich  in 
hemoglobin.  Target  cells  are  well 
demonstrated  by  Wright's  Blood  Stain. 
They  are  often  more  numerous  when  the 
blood  plasma  concentration  is  increased 
and  they  show  greater  resistance  than 
normal  erythrocytes  to  hypotonic  salt 
solutions.  Target  tissues  are  those 
especially  influenced  by  certain  hor- 
mones which  seem  to  reach  them  as  if 
aimed  at  targets. 

Tarsal  Glands.  Whole  mounts  can  be  made 
by  the  method  described  for  Sebaceous 
Glands.  They  are  also  known  as 
Meibomian  glands. 

Taste  Buds.  To  demonstrate,  choose  cir- 
cumvallate  papillae,  fix  in  Bouin's 
Fluid  and  stain  with  Hematoxylin  and 
Eosin.  See  Arey,  L.  B.  et  al.,  Anat. 
Rec,  1935-36,  64,  9-25. 

Tartrazine  (CI,  640),  a  pyrazolone  acid  dye 
of  light  fastness  4.  This  bright  yellow 
dj^e  is  useful  in  coloring  foodstuffs,  light 
filters,  etc.  (Emig,  p.  46).  Recom- 
mended as  a  substitute  for  Orange  G 
in  Mallory's  Stain. 

Tatooing  Pigments,  see  Exogenous  Pig- 
ments. 

Teeth.  The  most  comprehensive  statement 
of  microscopical  technique  is  contained 
in  A.  W.  Wellings'  "Practical  Micros- 
copy of  the  Teeth  and  Associated 
Parts."  London:  John  Bale  Sons  & 
Curnow  Ltd.,  1938,  281  pp.  A  chapter 
by  Churchill  and  Appleton  in  McClung's 
Technique  is  also  useful.  Teeth  can 
be  studied  from  so  many  different  angles 
that  to  outline  the  techniques  in  a  few 
words  is  extraordinarily  difficult.  Their 
composition  of  (1)  enamel,  the  hardest 
tissue  in  the  whole  body,  with  (2)  dentin 
which  is  highly  mineralized  and  contains 
the  processes  of  cells  but  not  their  nu- 
cleated bodies  plus    (3)   richly  cellular 


TEETH 


333 


TEETH 


pulp  confers  numerous  obstacles.  The 
wise  histologist  or  pathologist  will  save 
valuable  time  by  at  once  seeking  advice 
from  experts  in  some  dental  research 
laboratory.  They  possess  experience 
and  instruments  for  grinding  and  sawing 
both  of  which  he  lacks.  Teeth  of  adults 
can  be  prepared  for  examination  in  2 
principal  ways : 

1.  Without  decalcification.  Church- 
ill and  Appleton  (McClung,  p.  253) 
recommend,  in  place  of  the  usual  grind- 
ing method,  a  cutting  technique  used  by 
Johnston  at  Yale.  After  extraction  fix 
the  tooth  immediately  in  formalin.  Then 
dry  and  fix  to  wooden  block  by  modelling 
compound.  Sections  are  then  made  by 
the  cutting  wheels  of  a  power  lathe.  If 
necessary  they  are  polished  on  a  Belgian 
stone,  dehydrated  in  alcohol,  cleared  in 
xylol  and  mounted  in  balsam. 

Ground  sections  of  very  brittle  teeth 
or  teeth  with  supporting  structures 
intact  may  be  ground  after  imbedding 
in  methyl  methacrylate  according  to 
Sognnaes,  R.  F.,  Anat.  Rec,  1947,  99, 
133-144.  This  method  permits  sawing 
of  such  thin  slices  before  grinding  that 
serial    sections   may   be    prepared. 

When  one  wishes  to  include  the  soft 
as  well  as  the  hard  parts  Chase's  tech- 
nique of  petrifaction  is  advised  by 
them.  Fix  as  desired  (say  10%  forma- 
lin) and  wash  as  required.  Transfer  to 
aq.  gum  arable  or  dextrin  of  syrupy 
consistency.  Freeze  on  freezing  micro- 
tome and  cut  slices  with  very  fine  saw 
(jeweler's).  Remove  gum  arable  by 
washing  in  water  and  stain  with  carmine 
or  hematoxylin.  Dehydrate  through 
alcohols  to  95%,  |  to  several  hours  each 
depending  on  size  of  slice.  Acetone  ^ 
hr.  or  more.  Cover  with  thin  celloidin 
in  a  container  to  depth  twice  or  more 
thickness  of  slice.  Leave  container 
top  open  very  slightly  permitting  evap- 
oration until  celloidin  will  scarcely  flow 
when  container  is  steeply  tilted.  Trans- 
fer with  considerable  celloidin  to  con- 
tainer of  heavy  lead  foil  and  further 
evaporate  until  completely  hardened. 
Grind  and  polish  both  sides  of  slice  in 
presence  of  water.  Remove  celloidin 
with  acetone  and  acetone  with  xylol. 
Mount  in  balsam.  Sections  obtained 
by  this  and  the  Johnston  technique  can 
be  examined  by  direct  illumination, 
in  the  dark  field,  in  ultraviolet  light 
(Walkhoff,  O.,  Dental  Cosmos,  1923, 
65,  160-176),  in  polarized  light  (Andre- 
sen,  V.  The  Physiological  and  Artificial 
Mineralization  of  Enamel.  Oslo.  Dancke, 
1926)  and  by  x-ray  for  which  many 
references  are  given  (McClung,  381- 
385). 

2.  With  decalcification.     In  the  par- 


affin technique,  advised  by  Churchill 
and  Appleton,  clip  ends  of  roots  of  a 
freshly  extracted  tooth  or  drill  hole. 
Fix  in  4%  formalin.  Dry  with  towel 
and  seal  openings  to  pulp  with  celloidin. 
Quickly  dry.  Decalcify  in  10%  hydro- 
chloric acid  C.P.  10  days  or  more  testing 
with  needle.  Running  water,  24  hrs. 
95% ale. ,24  hrs.  Abs.  ale,  5  hrs.  Chlor- 
oform, 1  hr.  Equal  parts  chloroform 
and  45°C.  paraffin  in  glass  stoppered 
bottle  on  top  of  oven  (oven  58°C.)  over 
night.  5  hr.  each  in  following  paraffins 
(1)  42-46°C.,  (2)  52-56°C.and  (3)58- 
60°C.  within  oven.  Imbed  in  a  mix- 
ture of  235  cc.  52-56°C.  paraffin  and  15 
cc.  beeswax.    See  Paraffin  Sections. 

In  the  celloidin  technique  (Churchill 
and  Appleton)  cut  off  apex  of  tooth  or 
drill  a  hole  to  pulp  through  crown. 
Fix  in  4%  formalin,  buffered  to  counter- 
act acid,  45  hrs.  for  single  teeth.  (Wash 
in  water)  change  to  80%  ale.  95%  ale. 
2  weeks  -\-  depending  on  size.  Abs. 
ale.  2  weeks  +,  abs.  ale.  (exposed  to 
anhydrous  copper  sulphate,  see  Alco- 
hol) 2  weeks  +.  Equal  parts  abs.  and 
ether,  2  weeks  +.  Then  1  month  or 
more  in  |,  1,  2,  5,  7,  10,  12%  celloidin 
(parlodion).  Orient  and  imbed  in  12% 
in  stender  dish.  Make  depth  of  cel- 
loidin twice  height  of  tissue.  Place  lid 
of  stender  dish  on  tightly.  Allow 
bubbles  to  rise  24  hrs.  If  bubbles  still 
present  move  tissue  gently  so  they  can 
escape.  Put  piece  of  paper  between 
lid  and  dish,  24  hrs.  +.  Evaporate  to 
consistency  hard  rubber,  7  days  +. 
80% ale.  48  hrs. or  until  beginning  decal- 
cification. Trim  block  leaving  sufficient 
celloidin  about  tissue  to  facilitate  cut- 
ting. 10%  acetic  or  hydrochloric  acid 
in  70%  ale.  changing  daily  3  weeks  + 
until  needle  penetrates  easily.  When 
spaces  appear  in  the  celloidin  drill  holes 
to  reach  them.  Wash  24  hrs.  in  running 
water;  then  same  time  in  weak  sol. 
sodium  bicarbonate.  Wash  24  hrs.  + 
in  water.  50,  70  and  80%  ale.  each  24 
hrs.  +.  95%  and  abs.  ale,  §  hr.  each. 
Ale.  ether,  0.5%  and  12%  celloidin  5-20 
min.  each.  Harden  in  chloroform,  24 
hrs.  Leave  in  80%  until  sections  are 
made,  see  Celloidin  Sections. 

For  small  and  developing  teeth  a  wider 
variety  of  methods  is  possible  see  Teeth 
Developing.  To  classify  examples  of 
all  the  methods  available  for  old  and 
young  teeth  and  associated  structures 
in  a  manner  expected  by  the  reader  is 
not  feasible.  In  general  however  there 
are  methods  that  involve  whole  teeth 
which  come  under  Teeth  (Blood  Ves- 
sels, Innervation,  Lymphatics)  and 
their  response  to  Alizarin  Red  staining 
and    exposure    to    Radioactive    Phos- 


TEETH,  BLOOD  VESSELS 


334 


TEETH,  DECALCIFICATION 


phorus.  Some  techniques  are  also  pro- 
vided under  Teeth  and  Jaws  and  parts 
of  teeth :  Enamel,  Dentin,  and  Pulp. 
Teeth,  Blood  Vessels  (Boling,  L.  R.,  Anat. 
Rec,  1942,  82,  25-32).  Revised  by  L.  R. 
Boling,  July  27,  1946.  Two  suspensions 
are  recommended:  (1)  cinnabar,  120 
gms. ;  gum  arable,  40  gms. ;  water,  160 
cc.  (2)  cinnabar  (red  mercuric  sul- 
phide), 80  gms.;  corn  starch,  40  gms.; 
10%  formalin  in  physiological  saline, 
125  cc.  Grind  up  the  mixtures  slowly 
in  a  glass  ball  mill  for  2  or  3  days,  strain 
through  gauze,  and  use  immediately. 
Anesthetize  a  cat  or  dog  with  sodium 
pentobarbital.  Expose  and  ligate  both 
common  carotid  arteries.  Perfuse  the 
head  with  physiological  saline  through 
a  glass  cannula  inserted  in  one  carotid. 
Incise  the  carotid  of  the  opposite  side 
distal  to  the  ligature  and  allow  it  to 
bleed  until  clear  saline  appears  when  it 
should  be  clamped.  Open  the  jugular 
veins  and  allow  them  to  drain.  As 
soon  as  all  blood  has  been  washed  from 
the  vessels  of  the  head  direct  the  sus- 
pension through  the  same  cannula  by 
means  of  a  two  way  stop  cock.  Main- 
tain a  pressure  of  120  mm.  of  mercury  by 
air  pressure.  Aid  penetration  by  gentle 
rhythmic  pressure  on  a  hand  bulb  in- 
serted in  the  conducting  system.  When 
injection  of  the  mass  is  begun  remove 
the  clamp  momentarily  from  the  op- 
posite carotid  to  allow  free  flow  of  the 
mass  in  all  large  arteries.  This  pro- 
motes good  injections  of  both  right  and 
left  sides  from  the  single  cannula. 
After  completion  of  the  injection  remove 
the  head  and  place  in  strong  formalin 
over  night,  then  cut  away  the  soft  tissue 
from  the  jaws  and  place  the  jaws  inl0% 
formalin  in  saline  solution  for  several 
days,  wash,  and  decalcify  in  5%  nitric 
acid.  After  decalcification  dehydrate 
thoroughly  in  graded  series  of  alcohol  and 
clear  in  two  changes  of  methyl  salicylate. 
Dissect  away  any  bone  interfering  with 
observation  of  teeth.  This  is  best  done 
with  a  dental  engine  and  round  bur  while 
the  specimen  is  immersed  in  clearing 
fluid.  Moisture  or  heat  will  cause 
clouding  of  the  specimen  and  must  be 
avoided.  In  addition  to  the  desirable 
color  of  cinnabar,  is  the  radiopacity  of 
these  injections;  the  course  of  all 
macroscopically  visible  vessels  may  be 
followed  m  roentgenograms  before  decal- 
cification. The  method  also  works  well 
on  soft  tissues.  The  first  mass  will  pass 
through  all  capillaries  in  a  tooth  and 
fill  both  arteries  and  veins.  Better 
demonstration  of  arteries  is  obtained 
with  the  second  which  has  not  been 
found  to  pass  through  capillaries.  The 
use  of  formalin  seems  to  aid  in  the  reten- 


tion of  the  mass  in  the  blood  vessels  and 
to  prevent  the  formation  of  gas  bubbles 
in  the  pulp  cavity  during  decalcification. 
Teeth,  Decalcification:  Details  from  Dr. 
L.  R.  Boling,  Washington  University 
(School  of  Dentistry).  Revised  by  him 
Dec.  16,  1950. 

Decalcification  of  teeth  for  the  prep- 
aration of  histological  sections  presents 
several  problems  not  encountered  with 
other  tissues  especially  if  the  surround- 
ing bone  and  soft  tissues  are  also  pre- 
served. The  great  difference  in  salt 
content  and  organic  matrix  of  enamel, 
dentin,  cementum,  bone  and  soft  tis- 
sues makes  difficult  the  preservation  of 
one  while  the  others  are  being  decal- 
cified. 

Enamel,  except  in  the  most  immature 
portions  of  developing  teeth,  is  entirely 
destroyed  by  ordinary  decalcification 
methods.  The  organic  portion  of  adult 
enamel  may  be  observed  by  the  slow 
decalcification  of  thin  ground  sections 
under  a  cover  slip  (Chase,  S.  W.,  Anat. 
Rec.  36,  239-258,  1927).  The  acid,  one 
per  cent  nitric,  hydrochloric  or  sul- 
phuric, or  five  per  cent  chromic,  acetic 
or  citric,  is  run  under  a  propped  cover 
slip  over  the  section.  Action  may  be 
stopped  at  any  point  by  substituting 
water  for  acid  and  the  remaining  mate- 
rial stained  and  mounted  as  de- 
sired without  disturbance.  Boedeker's 
method  of  "celloidin-decalcifying"  is 
also  said  to  give  good  results  (Funda- 
mentals of  Dental  Histology  and  Embry- 
ology, New  York,  The  MacMillan  Co., 
1926,  p.  223)  and  allows  sectioning  of  the 
organic  remainder  in  any  plane.  See 
Enamel. 

For  the  examination  of  sections  of 
whole  teeth  without  enamel  or  for  teeth 
in  relation  to  the  bone  of  the  jaws  five 
per  cent  nitric  acid  in  water  has  been 
found  by  most  investigators  to  give  con- 
sistent results.  Hydrochloric  acid  may 
be  used  but  causes  too  much  swelling. 
For  delicate  objects  one  to  five  per  cent 
nitric  acid  in  70  per  cent  alcohol  may 
prove  superior. 

Excellent  results  have  been  ob- 
tained with  the  use  of  formic  acid  ac- 
cording to  the  technique  of  Morse, 
J.  Dent.  Res.,  1945,  24,  143-153  in  the 
decalcification  of  single  human  teeth 
or  teeth  and  jaws  of  small  animals. 
Two  solutions  are  made  as  follows: 
Solution  A:  1  part  90%  formic  acid 
C.P.  and  1  part  aq.  dest.,  Solution  B: 
20  grams  sodium  citrate  C.P.  and  100 
cc.  aq.  dest.  At  the  time  of  use  com- 
bine equal  parts  of  A  and  B.  Change 
solution  daily  until  decalcification  is 
complete  as  shown  by  chemical  test. 
(See    below.)     When    this    method    is 


TEETH,  DEVELOPING 


335 


TEETH,  DEVELOPING 


used  with  very  large  teeth  or  large 
blocks  of  tissue  containing  bone  and 
teeth,  objectionable  precipitates  may- 
be formed  in  the  depth  of  the  block. 

Celloidin  imbedding  before  decal- 
cification helps  preserve  tissue  relation- 
ships (See  Teeth,  celloidin  technique). 
Arnim  has  perfected  a  technique  of 
double  imbedding  for  rat  jaws  ana  teeth 
which,  though  tedious,  yields  beautiful 
results.  Enamel  matrix  is  frequently 
preserved.  (Anat.  Rec,  62,  pp.  321- 
330,  1935.)  This  method  has  been 
modified  by  Burket  for  larger  teeth 
(McClung,  p.  366). 

Tooth  buds  may  be  decalcified  after 
paraffin  imbedding  by  the  following 
method  given  by  Dr.  L.  R.  Doling  in  a 
personal  communication.  Carefully  re- 
move from  the  tooth  bud  all  surround- 
ing bone.  Fix,  dehydrate,  clear  and 
imbed  in  paraffin  in  the  usual  way. 
Shave  away  paraffin  and  soft  tissue  from 
one  surface  of  the  specimen  so  that 
enamel  is  exposed.  Immerse  block  in  5 
per  cent  aq.  nitric  acid  until  decalci- 
fication is  complete.  Place  in  5  per  cent 
aq.  sodium  sulphate  for  a  few  hours. 
Wash  over  night  in  running  water  and 
reimbed,  handling  the  tissue  as  gently 
as  possible  in  order  not  to  disturb  rela- 
tionship of  hard  and  soft  tissues.  This 
method  permits  demonstration  of  Golgi 
apparatus  and  mitochondria  in  amelo- 
blasts  and  odontoblasts  in  situ.  It 
works  best  with  teeth  of  small  animals 
easily  penetrated  by  fi.xative.  The 
paraffin  protects  the  soft  tissues  but  does 
not  interfere  with  action  of  acid  on 
enamel  and  dentin.  (See  also  Teeth, 
Developing.) 

Successful  preparation  of  decalcified 
tooth  sections  depends  as  much  or  more 
on  the  care  of  the  tissues  before  and 
after  decalcification  than  on  the  actual 
process.  Good  fixation  of  the  pulp 
tissue  is  difficult  but  essential  to  pre- 
vent shrinkage.  Ten  per  cent  formalin 
in  physiological  salt  solution  may  be 
used  for  several  days  or  weeks  without 
injury  to  the  soft  tissue  and  allow 
thorough  penetration.  Better  results 
are  obtained  in  a  short  time  if  the  fixa- 
tive can  be  perfused  through  the  blood 
vessels.  In  the  preparation  of  human 
or  other  large  teeth,  fixation  artifacts 
are  minimized  if  the  tooth  is  ground 
longitudinally  on  a  flat  stone  until  the 
pulp  is  just  exposed.  Two  opposite 
surfaces  may  be  ground.  Grinding 
should  be  done  on  a  sharp  stone  under 
running  water  to  prevent  heating. 
Cutting  of  holes  through  the  dentin  to 
the  pulp  or  the  amputation  of  the  tips 
of  teeth  is  often  resorted  to  in  order  to 
get  better  penetration  but  these  meth- 


ods are  apt  to  disturb  the  position  of 
the  pulp  and  should  be  avoided  if  pos- 
sible. After  decalcification  the  teeth 
should  be  carefully  handled  and  the  de- 
hydration process  should  be  slow  to 
prevent  separation  of  tissues  of  different 
densities.  The  substitution  of  n-butyl 
alcohol  for  ethyl  alcohol  and  xylol  in 
dehydration  and  clearing  processes  has 
proven  advantageous  (Morse,  loc.  cit.). 
By  this  method  dehydration  may  be 
prolonged  with  less  hardening. 

Over  decalcification  should  be  care- 
fully avoided  because  it  will  partially 
destroy  the  dentin  matrix,  cause  sepa- 
ration of  tissues  of  differing  consistency 
and  disturb  staining  reactions.  Testing 
for  completion  of  decalcification  by  prob- 
ing with  needles  or  bending  and  saueez- 
ing  in  the  fingers  should  be  avoided  at 
all  costs  if  tissue  relationships  are  de- 
sired. The  progress  of  decalcification 
can  be  followed  radiographically  but  the 
end  point  can  not  be  accurately  deter- 
mined. The  best  method  of  testing  is 
that  described  by  Arnim  (loc.  cit.). 
Five  cc.  of  the  acid  used  in  decalcifica- 
tion is  placed  in  a  clean  test  tube  and 
neutralized  with  ammonium  hydroxide, 
and  .1  cc.  of  a  saturated  solution  of 
ammonium  oxalate  added.  If  no  pre- 
cipitate forms  additional  .1  cc.  portions 
of  oxalate  are  added  at  15  minute  inter- 
vals until  .4  cc.  have  been  added.  If  a 
precipitate  is  formed  the  tissue  is  placed 
in  fresh  acid  and  retested  in  24  hours. 
Formation  of  no  precipitate  with  .4  cc. 
oxalate  solution  after  24  hours  in  fresh 
acid  is  indicative  of  complete  decalcifica- 
tion. 

When  tissues  are  found  to  be  not  suffi- 
ciently decalcified  after  imbedding  the 
process  can  be  completed  by  immersing 
the  celloidin  block  in  acid  70  per  cent 
alcohol  or  floating  the  paraflfin  block,  cut 
surface  down,  on  acid  if  the  dentin  is 
exposed. 
Teeth,  Developing.  L  Tooth  germs.  Glas- 
stone  (S.,  J.  Anat.,  1935-36,  70,  260- 
266)  has  described  a  method  for  the 
excision  of  tooth  germs  from  18-21  day 
rat  embryos  and  their  CultiTation  in 
fowl  plasma  and  embryo  extract.  The 
technique  of  Transplantation  of  tooth 
germs  of  young  pups  into  the  abdominal 
wall  has  been  reported  by  C.  H.  Huggins 
etal.  (J.  Med.,  1934,  60,  199).  Bevelan- 
der,  G.,  Anat.  Rec,  1941,  31,  79-97  ob- 
tained fine  preparations  of  Korff 's  fibers 
in  pig's  tooth  beginning  with  110  mm. 
stage  by  fixation  in  Formalin-Zenker 
and  silver  impregnation  by  Foot's 
Method. 

2.  Young  teeth.  Beams,  H.  W.  and 
King,  R.  L.,  Anat.  Rec,  1933,  57,  29-40 
fixed  the  developing  molar  teeth  of  white 


TEETH,  INNERVATION 


336 


TEETH,  LYMPHATICS 


rats  1-5  days  old  in  a  variety  of  fluids. 
They  employed  the  Nassonov  technique 
for  the  Golgi  apparatus  and  Regand's 
for  mitochondria  without  any  special 
provision  for  decalcification.  In  some 
cases  Boling's  Decalcification  (Teeth, 
Decalcification)  method  after  paraffin 
imbedding  may  prove  useful.  Dr.  Dol- 
ing states  in  a  personal  communication 
that  a  modification  of  Bouin's  picro- 
formol  fixative  may  be  used  for  fixing 
and  decalcifying  very  young  tooth  buds 
or  teeth  and  jaws  of  rats.  A  mixture 
of  75  parts  saturated  aqueous  solution 
of  picric  acid,  25  parts  formalin  and  10 
to  20  parts  glacial  acetic  acid  will  de- 
calcify a  mature  rat  jaw  and  teeth  in 
less  than  a  week.  Ordinary  Bouin's 
picro-formol  is  sufficiently  acid  to  de- 
calcify very  young  tooth  buds  in  a  few 
days.  After  decalcification  the  tissues 
are  handled  in  the  same  manner  as  soft 
tissues  after  Bouin  fixation  except  that 
a  longer  period  is  allowed  for  removal  of 
picric  acid.  This  procedure  allows 
better  than  average  staining  of  decal- 
cified tissues.  Nuclear  structure  is 
especially  well  preserved  and  little 
separation  of  hard  and  soft  tissues  is 
found.  The  method  of  microincinera- 
tion has  been  adjusted  to  developing 
teeth  by  Hampp,  E.  G.,  Anat.  Rec, 
1940,  77,  273-286. 
Teeth,  Innervation.  Methods  described 
under  Nerve  Endings  require  consider- 
able adaptation  before  they  can  be  em- 
ployed for  the  teeth.  For  obvious 
reasons  methylene  blue  is  particularly 
difficult  to  use.  From  a  great  many 
techniques  2  are  selected. 

1.  Van  der  Sprenkel,  H.  B.,  J. 
Anat.,  1935-36,  70,  233-241.  Grind  den- 
tinal wall  of  normal  human  canine  tooth 
down  to  a  thickness  of  300-500  microns 
leaving  the  cavity  closed  and  the  pulp 
untouched.  Saw  remainder  of  tooth 
into  rings  (not  decalcified) .  From  them 
cut  on  freezing  microtome  cross  sections 
about  40  fi  thick  and  impregnate  accord- 
ing to  the  Gros  method.  Van  der  Spren- 
kel does  not  give  a  reference  to  this 
method.  Perhaps  the  Gros  method,  as 
given  by  Lee  (p.  494)  will  serve.  Treat 
frozen  sections  with  pyridine.  Wash 
with  aq.  dest.  to  remove  odor  of  pyri- 
dine. 20%  aq.  silver  nitrate,  in  dark,  1 
hr.  Transfer  without  washing  to  20% 
formalin  neutralized  with  magnesium 
carbonate.  Change  twice  until  no  more 
white  ppt.  is  formed.  Reduce  under 
microscope  in  following  solution :  Add 
ammonia  to  15  cc.  20%  silver  nitrate 
until  ppt.  formed  just  disappears. 
Then  add  1  drop  ammonia  per  each  cc. 
silver  nitrate  solution.  After  this  20% 
aq.  ammonia  1  min.  or  more.     1%  acetic 


acid,  same.  Tone  in  0.2%  aq.  gold 
chloride  treat  with  sodium  hyposul- 
phite, wash,  dehydrate,  clear  and 
mount.  Counterstain  with  Van  Gieson 
or  toluidin  blue,  if  desired  before  dehy- 
dration. See  Van  der  Sprenkel's  illus- 
trations. 

2.  Christensen,  K.,  J.  Dent.  Res., 
1940,  19,  227-242  was  concerned  pri- 
marily with  determination  of  the  source 
of  the  large  proportion  of  unmyelinated 
and  small  myelinated  fibers  in  the  pulp. 
His  technique  is  a  combination  of  dis- 
section and  the  making  of  histological 
preparations  of  cats.  First  inject  ar- 
teries with  a  yellow  corn  starch  mass 
(composition  not  specified)  and  harden 
tissues  in  formalin.  Expose  cervical 
sympathetic,  common  carotid  and  its 
chief  branches,  mandibular  canal  and 
floor  of  orbit.  Wash  dissected  areas 
with  aq.  dest.,  and  brown  nerves  with 
dilute  aq.  silver  nitrate  so  that  they  can 
be  easily  followed  along  the  walls  of  the 
yellow  colored  vessels.  To  trace  their 
final  path  to  lower  teeth  serial  sections 
of  inferior  alveolar  nerve  and  artery  are 
required  and  to  upper  teeth  similar  ones 
of  internal  maxillary  plexus  and  superior 
alveolar  nerves.  Wrap  canine  teeth  in 
cotton,  carefully  crack  with  vise  and 
remove  pulps.  Slightly  stretch  each 
pulp  along  surface  of  short  glass  tube 
attaching  the  ends  to  the  tube  by  silk 
threads  to  prevent  tortuosity  of  nerve 
fibers  in  the  final  preparations  made  by 
the  Bodian-Method.  Examine  the  cer- 
vical sympathetic  ganglia  by  techniques 
for  Nissl  Bodies  as  well  as  for  nerve 
fibers  before  and  after  degeneration  re- 
sulting from  experimental  destruction 
of  dental  pulp. 

Teeth  and  Jaws.  Sections  through  (Will- 
man,  M.,  J.  Dental  Res.,  1937,  16,  183- 
190).  Fix  in  10%,  formalin,  10-30  days. 
Transfer  to  95%  alcohol  for  same  time. 
After  decalcification  in  5%  aq.  nitric 
acid,  change  to  5%  aq.  sodium  sulphate 
for  24  hrs.,  then  wash  in  running  water 
24  hrs.  Dehydrate  through  ascending 
alcohols  to  95%,  then  2  changes  of  ab- 
solute, 6%,  12%  and  25%  celloidin  solu- 
tion, 7  days  each.  Cut  sections  with 
heavy,  sledge  type  of  microtome.  Re- 
move celloidin  from  sections  with  alco- 
hol-ether and  pass  down  to  aq.  dest. 
Stain  with  Harris'  hematoxylin  and  acid 
alcohol  eosin.  Mount  in  dammar. 
Control  decalcification  either  by  testing 
a  second  tooth  with  a  needle  or  by 
polariscope.    See   Dental    Enamel. 

Teeth,  Lymphatics.  Obviously  the  work 
of  Fish,  E.  W.,  Proc.  Roy.  Soc.  Med., 
1926-27,  20  (3),  225-236;  Bddecker,  C. 
F.,  and  Lefkowitz,  W.,  J.  Dent.  Res., 
1937,  16,  463-475  and  others  relating  to 


TEICHMANN 


337 


THIAMINE 


the  "lymph  supply"  of  dentin  and 
enamel  does  not  refer  to  lymph  but  to 
tissue  fluid  for  the  spaces  are  not  lined 
with  lymphatic  endothelium.  For  tis- 
sue fluid  in  these  situations  see  Cowdry, 
E.  V.  Problems  of  Ageing.  Baltimore: 
Williams  &  Wilkins,  1942,  p.  693.  An 
excellent  account  of  techniques  designed 
for  investigation  of  the  lymphatic  sys- 
tem of  teeth  and  jaws  is  provided  by 
MacGregor,  A.,  Proc.  Roy.  Soc.  Med., 
1936-36,  29  (2),  1237-1272.  His  favorite 
injection  masses  were  strong  solutions 
of  basic  lead  acetate  and  acid  suspen- 
sions of  carmine.  Before  killing  and 
injecting  the  animals  (cats,  dogs,  guinea 
pigs  and  monkeys)  he  caused  them  to 
inhale  large  doses  of  amyl  nitrite  with 
the  idea  of  dilating  the  peripheral  blood 
vessels. 

Teichmann,  see  Hemin  Crystal  Test,  Flor- 
ence Reaction. 

Tellurium,  see  Atomic  Weights. 

Tellyesniczky's  fixative.  5  parts  of  formol, 
100  of  70%  alcohol  and  5  of  acetic  acid. 

Tendons.  These  are  dense  bands  of  col- 
lagenic  fibers  interspersed  by  a  few 
flattened  fibroblasts  (lamellar  cells). 
Fixatives  penetrate  the  larger  ones 
poorly.  Zenker's  Fluid  and  Hematoxy- 
lin and  Eosin  are  fairly  satisfactory. 
For  mechanical  factors  in  structure  see 
Carey,  E.  J.,  Am.  J.  Anat.,  1936,  59, 
89-122;  Anat.  Rec,  1936,  64,  327-341. 

Tensile  Strength.  An  ingenious  method  has 
been  worked  out  to  measure  this  prop- 
erty of  skin  (Herrick,  E.  H.,  Anat.  Rec, 
1945,  93,  145-149). 

Terbium,  see  Atomic  Weights. 

Tergitol,  see  Wetting  Agents. 

Terpineol  (or  terpinol),  a  mixture  of  sub- 
stances of  composition  CioHu  and 
CioHuO  formed  by  action  of  dil.  HCl 
on  terpin  hydrate.  Used  as  a  clearing 
agent.  Can  clear  tissues  from  90%, 
even  from  80%  ale.  A  good  mixture  is 
4  parts  terpineol  +  1  part  xylol. 

Tertiary  Butyl  Alcohol  (trimethyl  carbinol). 
Has  been  recommended  as  a  substitute 
for  ethyl  alcohol  and  clearing  agents  like 
xylol  in  the  paraffin  technique  because 
it  mixes  easily  both  with  water  and 
paraffin.  It  causes  but  little  shrinkage 
and  hardening  of  tissue.  One  method 
(Stowell,  R.  E.,  Science,  1942,  96,  166- 
166)  is  partly  to  substitute  for  ethyl  al- 
cohol by  passing  through  the  following 
series  of  mixtures:  (1)  Aq.  dest.,  60  cc. ; 
96%  ethyl,  40  cc. ;  butyl,  10  cc. ;  1-2  hrs. 
(2)  Aq.  dest.,  30  cc. ;  95%  ethyl,  50  cc. ; 
butyl,  20  cc,  2  hrs.  to  several  days.  (3) 
Aq.  dest.,  15  cc. ;  95%  ethyl,  50  cc. ; 
butyl,  35  cc;  1-2  hrs.  (4)  95%  ethyl, 
45  cc. ;  butyl,  55  cc. ;  1-2  hrs.  (5)  Butyl, 
75  cc;  abs.  ethyl,  25  cc ;  1-3  hrs.  (6) 
Pure  butyl,  3  changes  4  hrs.  to  over- 


night. (7)  Equal  parts  pure  butyl  and 
paraffin  oil,  1-2  hrs.  Infiltrate  in  paraf- 
fin. Another  method  (Stowell,  R.  E., 
J.  Tech.  Methods,  1942,  22,  71-74)  is  to 
entirely  substitute  50%,  70%,  85%  and 
pure  butyl  alcohol  for  the  corresponding 
ethyl  alcohols.  Stowell  provides  useful 
suggestions  as  to  the  details  of  paraffin 
imbedding.  Tertiary  butyl  alcohol  has 
been  recommended  for  dehydrating 
material  stained  with  methylene  blue 
and  other  dyes  readily  extracted  during 
ethyl  alcohol  dehydration  (Levine,  N. 
D.,  Stain  Techn.,  1939,  14,  29-30).  It 
may  be  used  as  a  substitute  for  ethyl 
alcohol  in  the  acid  fast  and  Gram  stains 
for  bacteria  (Beamer,  P.  B.  and  Stow- 
ell, R.  E.,  J.  Lab.  &  Clin.  Med.,  1943, 
28,  1599-1602).  Do  not  confuse  with 
n  Butyl  alcohol. 

Testis,  M[ethods  described  elsewhere  for 
the  ConnectiTe  System,  Blood  Vessels, 
Nerve  Fibers  and  so  on  are  available. 
Technique  for  isolation  of  seminiferous 
tubules  is  given  under  Maceration. 
See  also  Chromosomes.  Wagner,  K., 
Biologia  Generalis,  1925,  1,  22-51  has 
employed  a  method  of  vital  staining  with 
trypan  blue  which  he  claims  differen- 
tiates between  interstitial  cells  and 
histiocytes  or  macrophages.  Duesberg, 
J.,  Biol.  Bull.,  1918,  35,  176-198,  using 
the  Benda  Method,  obtained  prepara- 
tions of  opossums  which  he  thought 
indicated  discharge  of  material  from  the 
interstitial  cells  into  the  blood  stream. 
Wagner  (loc.  cit.)  has  observed  some- 
what similar  phenomena  in  other  ani- 
mals, but  there  has  been  no  satisfactory 
follow  up.  For  detailed  information 
about  interstitial  cells  see  Rasmussen, 
A.  T.,  Cowdry '8  Special  Cytology,  1932, 
3,  1674-1726. 

Testosterone,  Pollock,  Anat.  Rec,  1942,  84, 
23-27. 

Tetrachrome  Blood  Stain,  see  MacNeal's. 

Tetralin  is  tetrahydronaphthalene  used  as  a 
clearing  agent  after  Diaphanol. 

Tetrazolium  Salt.  Smith,  F.  Y..,  Science, 
1951,  113,  751-754  gives  uses  and  limita- 
tions. Neotetrazolium  gives  deep  pur- 
ple to  black  on  reduction  and  is  thus 
superior  to  the  tetrazolium  salt.  Blue 
tetrazolium  also  has  certain  special 
advantages.  See  Triphenyltetrazolium 
Chloride. 

Thallium.  Barbaglia's  Method.  Fix  in 
95%  alcohol  iodized.  This  precipitates 
thallium  in  the  form  of  insoluble  crystals 
of  thallium  iodide  recognizable  by  their 
yellow  color  (Lison,  p.  66). 

Thiamine.  Blaschko  ana  Jacobson  (H.  and 
W.  in  Bourne's  Cytology,  1942,  p.  196) 
refer  to  the  work  of  Ellinger  and  Kos- 
chara  in  the  observation  under  the  fluo- 
rescence microscope  of  green  fluorescence 


THIAZIN  DYES 


338 


THYMONUCLEIC  ACID 


due  to  flavin  and  that  on  alkalinization 
this  is  replaced  by  a  bluish  fluorescence 
which  is  known  to  be  occasioned  by  the 
presence  of  thiamine,  itself  identical 
with  vitamin  B,  or  aneurin. 

See  Cartesian  diver  technique  of 
Westenbrink,  H.  G.  K.,  Enzymologia, 
1940,  8,  97-107.  Click,  p.  395  thinks 
that  the  technique  of  Schultz,  A.,  At- 
kin,  L.  and  Frey,  C.  N.,  Ind.  Eng. 
Chem.,  Anal.  Ed.,  1942,  14,  35-39  based 
on  stimulation  of  yeast  fermentation 
by  thiamine. 

Thiazin  Dyes.  A  very  useful  group  of  dyes 
for  the  histologist.  The  two  benzene 
rings  are  joined  by  =N—  and  — S— . 
Examples :  azure  A,  B  and  C,  methylene 
azure,  methylene  blue,  methylene  green, 
methylene  violet,  new  methylene  blue 
N,  thionin,  toluidin  blue  O. 

Thiazine  Red  R  (CI,  225)— chlorazol  pink 
Y,  rosophenine  lOB — An  acid  mono-azo 
dye  employed  especially  as  counterstain 
for  iron  hematoxylin. 

Thiazole  Dyes  contain  thiazole  ring  with 
indamine  as  chroma tophore.  Geranine 
G,  primalin,  thioflavine  S,  and  titan 
yellow.  All  of  these  dyes  appear  to  be 
useful  in  fluorescence  microscopy.  Pick, 
J.,  Zeit.  f.  wis.  Mikr.,  1935,  51,  338-351 
refers  to  three  of  them. 

Thiazole  Yellow,  see  Titan  Yellow. 

Thioflavine  S  (CI,  816).  An  acid  thiazole 
dye  used  in  fluorescence  microscopy. 

Thionin  (CI,  920)— Lauth's  violet— Com- 
mission Certified.  An  extremely  useful 
basic  thiazin  dye.  See  Tissue  Baso- 
philes,  King's  Carbol  Thionin,  etc. 

Thiourea.  A  derivative  of  urea  with  sul- 
phur replacing  oxygen.  As  means  of 
activating  thyroid  gland  (Thomas,  O. 
L.,  Anat.  Rec,  1944,  89,  461-469). 
Effect  on  organ  weights  and  plasma 
proteins  of  the  rat  (Leathem,  J.  H., 
Anat.  Rec,  1944,  89,  540). 

Thomas,  see  Arginine  Reaction. 

Thorium  Dioxide  is  occasionally  employed 
as  a  vital  stain  for  reticulo-endothelium. 
Angermann,  M.  and  Oberhof,  K.,  Zeit. 
f.  Ges.  Exp.  Med.,  1934,  94,  121-126  give 
directions  for  its  administration  to  rab- 
bits and  for  determination  of  its  dis- 
tribution chemically,  radiologically  and 
histologically.     (Thorotrast) 

Thulium,  see  Atomic  Weights. 

Thyme  Oil  N.F.  VI.  Sometimes  misnamed 
oil  of  origanum.  Contains  thymol,  car- 
vacrol,  cymene,  pinene,  linalool  and 
bornyl  acetate.  It  is  said  to  be  useful 
for  clearing  celloidin  sections. 

Thymol  Blue.     See  Hydrogen  Ion  Indicators. 

Thymonucleic  Acid  is  a  type  of  desoxypen- 
tose  nucleic  acid  isolated  from  nuclei 
of  the  thymus.  Also  called  desoxyribo- 
nucleic  acid  on  DNA  for  short.  {Feul- 
gen     or     nucleal     reaction    for).     Pass 


paraffin  sections,  fixed  in  equal  parts 
sat.  aq.  corrosive  sublimate  and  ab- 
solute alcohol,  through  xylol  and  al- 
cohols to  water.  Place  in  a  staining 
jar  containing  normal  HCl  (82.5  cc. 
HCl,  sp.  gr.  1.17-1.85  per  liter  of  water) 
at  room  temperature  for  1  min.  Trans- 
fer to  normal  HCl,  at  60°C.  and  there 
hydrolyze  for  4  min.  Treat  with  the 
fuchsin  sulphurous  acid  reagent  in  a 
staining  jar  for  3-I  hr.  (This  reagent 
is :  One  gram  of  basic  fuchsin  is  dis- 
solved in  100  cc.  of  distilled  water  with 
the  aid  of  a  little  heat.  The  solution  is 
filtered  while  still  warm  and  20  cc.  of 
normal  HCl  is  added  to  the  filtrate.  The 
resulting  fluid  is  then  cooled  and  1 
gm.  dry  sodium  bisulfite  (NaHSOj)  is 
added.  Then,  after  standing  for  about 
24  hrs.,  the  reagent  is  ready  for  use  and 
should  have  a  pale  straw  color.)  Pass 
through  a  series  of  3  jars,  each  contain- 
ing a  solution  made  by  adding  10  cc.  of  a 
molecular  solution  of  sodium  bisulfite 
(i.e.,  104  grams  per  liter)  to  200  cc.  of 
tap-water,  allowing  1^  min.  in  each  and 
agitating  frequently.  Wash  in  tap 
water  for  5  min.,  dehydrate,  clear  and 
mount  in  balsam.  Thymonucleic  acid 
is  colored  purple  or  violet  and  color 
holds  (Cowdry,  E.  V.,  Science,  1928, 
68,40-41).  Collected  references  (Milo- 
vidov,  P.,  Protoplasma,  1938,  31  (2), 
246-266) ;  technique  for  plant  tissues 
(Whitaker,  T.  W.,  Stain  Techn.,  1939, 
14,  13-16).  A  more  recent  account  is 
given  by  Stowell,  R.  E.,  Stain  Techn., 
1945,  20,  45-58.  Specificity  has  been 
considered  by  Dodson,  E.  O.,  Stain 
Techn.,  1946,  21,  103-105.  See  Bauer- 
Feulgen  stain  for  Glycogen. 

Dr.  A.  R.  Gopal-Ayengar  of  the 
Tata  Memorial  Hospital,  Bombay  has 
supplied  details  of  a  modification  of 
the  Feulgen  technique  by  Rafalko, 
J.  S.,  Stain  Techn.,  1946  21,  91-93.  In- 
stead of  using  HCl  and  sulphites,  as  in 
the  usual  method,  Rafalko  directly 
charges  both  basic  fuchsin  and  the 
bath  water  with  SO2  gas,  using  A'^  HCl 
only  for  the  necessary  process  of  hy- 
drolysis. By  this  method,  he  claims  to 
have  been  able  to  stain  diffuse  and  small 
chromosomes,  which  give  negative 
results  with  conventional  procedure. 
Three  types  of  organisms  were  tested: 
(1)  Various  small,  endosome -containing 
amoebae;  (2)  oocytes  of  parasitic  wasps, 
Habrobracon;  and  (3)  the  yeasts  Sac- 
charomyces  cerevisiae  and  S.  carlsber- 
gensis.  Fix  smears  for  2-20  min. 
Wash  in  water  20  min.  and  in  aq.  dest. 
20  min.  A'^  HCl  room  temperature, 
2  min.  N  HCl  at  60°C.,  8-10  min. 
Rinse  in  A'"  HCl  at  room  temperature. 
Rinse   in    aq.   dest..    Sulphurous   acid, 


THYMONUCLEOHISTONE 


339 


TICKS 


2  min.  Leucobasic  fuchsin,  lJ-2  hrs. 
Sulphurous  acid  bath,  for  sufficient 
time  to  remove  the  free  untreated 
leucobasic  fuchsin  (2-3  changes).  Tap 
water,  10-15  min.  Counterstain,  if 
necessary,  with  aq.  or  ale.  fast  green. 
Dehydrate,  clear  and  mount  in  the 
usual  manner,  or  follow  Triethyl  Phos- 
phate technique. 

See  Aldehydes  for  discussion  of  spe- 
cificity of  Feulgen  reaction  and  Lessler, 
M.  A.,  Arch.  Biochem.  and  Bioph., 
1951,  32,  42-54  who  thinks  that  it 
should  be  feasible  to  correlate  deter- 
minations of  color  intensity  of  gelatin 
DNA  preparations  of  known  DNA  con- 
centration with  that  of  Feulgen  stained 
nuclei  and  thus  measure  approximately 
the  nuclear  DNA  content.  Lessler 
specifies  certain  sources  of  e.xperimental 
error  to  be  guarded  against. 

The  histophotometric  measurement 
of  DNA  in  the  course  of  embryonic 
development  is  described  by  Lison, 
L.  and  Pasteels,  J.,  Arch,  de  Biol., 
1951,  62,  1-43.  A  biometric  investiga- 
tion of  their  technique  is  provided  by 
Martin,  L.,  Arch,  de  Biol.,  1951,  62, 
45-64. 
Thymonucleohistone.  Technique  for  di- 
electric properties,  Lars-Goran,  All- 
g^n.  Acta  Phj^siol.  Scand.,  Suppl.  76, 
22,  140  pp. 
Thymus.  Isolation  en  7nasse  of  nuclei  from 
Behrens'  method  as  modified  by  Mayer, 
D.  T.  and  Gulick,  A.,  J.  Biol.  Chem., 
1942,  146,  433-440. 
Thyroid.  For  routine  purposes  Zenker  fixa- 
tion and  hematoxylin  and  eosin  staining 
of  paraffin  sections  is  suggested.  If  one 
is  interested  in  the  colloid,  its  appear- 
ance after  various  fixations,  its  shrinkage 
patterns  and  the  significance  of  its 
acidophilic  and  basophilic  staining  are 
described  by  Bucher,  D.,  Zeit.  f.  Zellf. 
u.  Mikr.  Anat.,  1938,  28,  359-381.  The 
effect  on  colloid  of  different  agents  for 
dehydration  and  clearing  is  described 
by  Ralph,  P.,  Stain  Techn.,  1938,  13,  9- 
15.  A  method  for  determination  of  the 
volume  of  colloid  is  given  by  Stein,  H. 
B.,  Am.  J.  Anat.,  1940,  66,  197-211. 

The  shape  of  thyroid  follicles  can  be 
distinguished  but  imperfectly  in  sec- 
tions unless  reconstructions  are  made 
from  serial  sections.  For  an  excellent 
method  of  viewing  entire,  isolated 
follicles  see  Maceration.  The  localiza- 
tion of  unsuspected  masses  of  follicles, 
not  present  in  the  gland,  in  the  neck 
tissues  of  experimental  animals  can  be 
accomplished,  by  supravital  staining 
with  Naphthol  Blue. 

Many  methods  are  available  for  the 
detailed  examination  of  the  secretory 
epithelial     cells     not     requiring     their 


special  adjustment  to  the  thyroid  gland. 
See       Mitochondria,       Microchemical 

methods,  etc.  The  Brazilin-Wasser- 
blau  technique  is  recommended  for  in- 
cellular  secretion  antecedents.  If  the 
Golgi  apparatus  is  to  be  investigated 
consult  Welch,  C.  S.  and  Broders,  A., 
Arch.  Path.,  1940,  29,  759-772.  A  fine 
beginning  has  been  made  in  the  direct 
study  of  vacuoles  within  the  follicles  in 
living  mice  by  transillumination  after 
the  fashion  of  Knisely  (Williams,  R.  G., 
Anat.  Rec,  1941,  79,  263-270).  Minute 
instructions  for  demonstration  of  blood 
vessels  and  lymphatics  and  results 
which  are  to  be  expected  are  given  by 
Rienhoff,  W.  F.,  Arch.  Surg.,  1931,  23, 
783-804.  For  fluorescence  see  Grafflin, 
A.  L.,  J.  Morph.  and  Physiol.,  1940,  67, 
455-470.  Effect  of  Thiourea  on  thyroid 
secretion  (Thomas,  O.  L.,  Anat.  Rec, 
1944,89,461-469). 
Ticks.  The  following  method  for  softening 
and  sectioning  is  an  adaptation  by  Miss 
Slifer  of  the  Slifer-King  technique  for 
grasshopper  eggs  (Slifer,  E.  H.,  and 
King,  R.  L.,  Science  1933,  78,  366-367). 
Drop  animal  into  dish  of  Carnoy-Le- 
brun.  After  5  min.  place  under  binocu- 
lar and  puncture  with  a  glass  needle. 
Allow  fixative  to  act  for  at  least  20  min. 
longer.  (Variations  in  the  size  of  the 
puncture  and  in  the  length  of  time  for 
fixation  should  be  tried.)  Transfer  to 
70%  alcohol  colored  a  light  yellow  with 
iodine  over  night.  If  alcohol  is  colorless 
next  morning  let  stand  a  few  hours 
longer.  Repeat  if  necessary.  At  this 
point  (or  somewhat  earlier)  it  is  well  to 
make  a  larger  incision  in  the  animal 
with  a  scalpel.  The  viscera  should  now 
be  well-hardened  and  should  not  ooze  out 
through  the  hole.  70%  alcohol,  several 
hrs.  70%  alcohol  containing  4%  phenol, 
2  or  3  days.  95%  alcohol  2  hrs.  Anilin 
oil,  several  hrs.  Chloroform  (2  changes 
of  5  min.  each).  Paraffin  about  an  hour. 
Imbed  and  block.  Trim  block  away  so 
that  viscera  are  just  exposed,  at  the 
point  where  sectioning  is  to  begin. 
Place  block  in  water  containing  4% 
phenol.  Be  sure  that  the  cut  surface  is 
under  water  and  examine  occasionally  to 
see  that  air  bubbles  do  not  form  on  it. 
After  3  days  a  swelling  of  the  tissues 
should  be  noticeable  so  that  they  pro- 
trude a  little  beyond  the  cut  surface  of 
the  paraffin.  If  this  has  not  occurred, 
cut  away  a  little  more  and  soak  several 
days  longer.  Trim  block,  place  on 
microtome  and  section  5-7  microns. 
Work  rapidly  once  you  have  begun.  A 
slight  delay  between  sections  will  allow 
the  cut  surface  to  dry.  If,  for  any 
reason,  it  is  necessary  to  stop  wet  a 
scrap  of  paper  and  stick  it  to  the  cut 


TIGROID  BODIES 


340 


TISSUE  CULTURE 


surface.  In  case  of  difficulty  in  making 
sections  stick  to  slides  try  Haupt's 
gelatine  fixative  (Stain  Techn.,  1930, 
5,  97-98).  After  the  sections  have 
been  spread,  arranged  on  the  slide  and 
albumen  (Webb,  R.  L.,  Am.  J.  Anat., 
1931-32,  49,  283-334). 
Tigroid  Bodies  (G.  tigris,  tiger  and  eidos, 
appearance).  A  term  applied  to  Nissl 
bodies  since  they  sometimes  look 
streaked  and  spotted  like  a  tiger.  See 
Nissl  Bodies. 
Tissue  Basophiles  (tissue  mast  cells). 
Some  think  that  these  cells  are  emi- 
grated Basophile  Leucocytes  and  others 
that  they  are  of  extravascular  origin. 
They  can  easily  be  studied  in  fresh 
spreads  of  Loose  Connective  Tissue  or 
omentum.  Their  granules  are  readily 
colored  supravi tally  with  brilliant  cresyl 
blue,  methylene  blue  and  other  stains. 
Tissue  basophiles  disintegrate  quickly. 
Maximow,  A.,  Arch,  f .  mikr.  Anat.,  1913, 
83  (1),  247^289  gives  the  following 
metachromatic  stain  for  mast  cells. 
Sections  of  abs.  ale.  fixed  tissues  are 
stained  24-48  hrs.  in  sat.  thionin  in  50% 
ale.  Staining  can  be  reduced  to  20  min. 
by  adding  4  drops  3%  Na2C03  to  20  cc. 
thionin  sol.  and  filtering  before  use. 
Maximow  gives  technique  for  smears 
and  spreads  fixed  in  formalin  Zenker. 
See  his  beautiful  colored  plates.  See 
Toluidine  Blue  Phloxinate. 

Holmgren  and  Wilander  (H.  and  O., 
Ztschr.  f.  mikr.  Anat.  Forsch.,  1937, 
42,  242-278)  recommend  fixation  in  10% 
aq.  basic  lead  acetate  and  staining  with 
1%  ale.  Toluidin  blue.  They  show 
that  fixation  in  formalin-alcohol  gives 
very  inferior  results.  In  their  opinion 
the  metachromatic  substance  colored  is 
identical  with  Heparin. 

Sylvan,  B.,  Acta  Radiol.,  1940,  21, 
206-212  has  followed  this  matter  up  by 
subjecting  rats  and  guinea  pigs  (in  which 
the  basophilic  granules  are  said  to  be 
less  soluble  in  water  than  in  most  other 
animals)  to  Gamma  rays.  He  fixed  the 
tissues  in  weaker  aq.  basic  lead  acetate 
(4%)  for  24  hrs.,  stained  paraffin  sec- 
tions with  5%  aq.  toluidin  blue  and 
other  dyes,  and  reached  the  conclusion 
that  the  radiation  brings  about  liberation 
of  organic  sulphuric  acids  of  high  molec- 
ular weight.  It  would  be  natural  to 
investigate  the  relation  if  any  between 
heparin  and  the  basophilic  granules  in 
buffy  coat  of  centrifuged  human  blood 
containing  say  0.5%  basophiles  and  in 
that  of  certain  turtles  in  which  the  per- 
centage is  as  high  as  80  as  well  as  in 
livers. 

Another  method  of  study  is  to  investi- 
gate heparin  in  relation  to  the  charac- 
teristic dissolution  of  basophiles  2  days 


after  the  intraperitoneal  injection  of  egg 
albumen  (Webb,  R.  U.,  Am.  J.  Anab., 
1931-32,49,283-334). 
Tissue  Culture— Written  by  Wilton  R. 
Earle,  National  Cancer  Institute,  Be- 
thesda.  July  10, 1951 — This  technique  is 
obviously  of  great  value  in  biology  and 
medicine.  For  orientation  reference 
should  be  made  to  two  recent  books: 
Parker,  R.  C,  Methods  of  Tissue  Cul- 
ture, 2nd.  ed.,  New  York:  Hoeber, 
1950,  Cameron,  Gladys,  Tissue  Culture 
Technique,  New  York:  Academic  Press, 
1950.  At  present  (March  1951)  the 
Tissue  Culture  Association  (%  Dr.  Mar- 
garet Murray,  College  Physicians  & 
Surgeons,  W.  168  St.,  New  York)  has 
nearly  ready  for  publication  an  elab- 
orately cross  indexed  bibliography 
containing  over  16,000  primary  refer- 
ences to  papers  involving  tissue  culture. 
This  association  also  serves  as  a  coor- 
dinating organization  for  tissue  culture 
workers. 

By  the  methods  of  tissue  culture  a 
small  clump  of  cells  can  be  removed 
from  an  organism  and  maintained  in  a 
condition  of  survival  or  growth  for 
periods  ranging  from  a  few  hours  for 
some  cells  to  an  indefinite  number  of 
years  for  the  descendants  of  others. 
While  so  maintained  they  can  be  exam- 
ined microscopically  at  various  mag- 
nifications. The  differentiation  of  em- 
bryonic organs  can  be  followed  (see 
account  in  this  book  written  by  Honor 
B .  Fell :  Organ  Culture  in  vitro) .  Malig- 
nant cells  may  be  grown  and  studied  for 
an  extended  interval  and  their  charac- 
teristics compared  with  those  of  nor- 
mal cells  or  with  malignant  cells  in 
vivo  (Lewis,  W.  H.,  Arch.  f.  Exp. 
Zellf.,  1939,  23,  8;  Earle,  W.  R.,  J. 
Nat.  Cancer  Inst.,  1943,  4,  165).  Cell 
form,  size,  internal  motion,  locomotion 
and  rate  and  manner  of  cell  prolifera- 
tion can  be  routinely  studied  either 
visually,  or  by  means  of  phase  inter- 
ference photography,  or  by  time-lapse 
cinematography  (Fell,  H.  B.,  and 
Hughes,  A.  F.,  Quart.  J.  Micr.  Sci., 
1949,  90,  355).  The  tissue  can  be 
vitally  stained  (Ludford,  R.  J.,  Uth 
Scientific  Report,  Imper.  Cancer  Re- 
search Fund,  1934,  169)  or  fixed  in  situ, 
and  stained  for  microscopic  examina- 
tion, (Cameron,  above  cited),  for  micro- 
chemical  test,  or  for  electron  microg- 
raphy. Physiological  and  nutritional 
studies  are  possible.  The  culture  me- 
dium can  be  modified  by  the  addition 
or  omission  of  various  nutritional  ele- 
ments or  other  physiologically  active 
substances  (Pogogeff,  I.  A.,  and  Mur- 
ray, M.  R.,  Anat.  Rec,  1946,  95,  321), 
and  the  influence  of  the  altered  medium 


TISSUE^CULTURE 


341 


TISSUE  CULTURE 


on  the  cells  may  be  studied.  The  cul- 
ture medium  may  be  removed  and 
tested  to  determine  what  changes  the 
cells  have  induced  in  it  (Brues,  A.  M., 
Rathbun,  E.  N.,  Cohn,  W.  E.,  J.  Cell, 
and  Comp.  Physiol.,  194-i,  24,  155). 
The  tissue  growing  in  culture  may  be 
used  as  a  host  for  the  growth  and 
study  of  bacteria  or  viruses  (Fo.x,  J. 
P.,  Amer.  J.  Hygiene,  1947,  46,  1). 
Rapidly  increasing  facility  in  the  han- 
dling of  manj^  types  of  cells  in  culture 
suggests  a  constantly  increasing  useful- 
ness of  tissue  culture  methods  in  many 
fields  of  future  scientific  work. 

1.  The  Culture  Medium  and  Sub- 
strate. A  satisfactory  isotonic  balanced 
saline  solution  is  necessary  for  washing 
cultures  and  for  dilution  of  plasma  and 
nutrient  media.  This  balanced  salt 
solution  can  be  prepared  best  from 
glass  distilled  water  free  from  fungus 
or  bacterial  growth  which  might  give 
rise  to  toxic  substances.  Reagent 
quality  chemicals  should  be  used  and 
heavy  metal  contaminants  in  partic- 
ular should  be  avoided.  Only  minor 
differences  exist  in  the  formulae  of 
many  of  the  solutions  in  current  use: 
Mammalian  Ringer  (Ringer,  S.,  J. 
Physiol.,  1895,  18,  425),  Tyrode  (Arch. 
Intern,  de  Pharmacodyn.  et  de  Thdr., 
1910,  20,  205;  Gey,  G.  O.  and  Gey,  M. 
K.,  Am.  J.  Cancer,  1936,  27,  45).  Hanks' 
(Hanks,  J.  H.,  J.  Cell,  and  Comp. 
Physiol.,  1948,  31,  235),  Simms'  (Simms, 
H.  S.,  and  Sanders,  M.,  Arch.  Pathol., 
1942,  33,  619),  Earle's  solution  (J.  Nat. 
Cancer  Inst.,  1943,  4,  165).  Solutions 
as  amphibian  Ringer  are  used  with 
amphibian  cells.  Any  solution  ap- 
proximating the  inorganic  salt  content 
of  serum,  and  having  a  comparable 
osmotic  pressure,  can  be  employed  as  a 
diluting  and  wash  solution  for  routine 
tissue  cultures.  For  skin  epithelium 
Parshley  and  Simms  (Am.  J.  Anat., 
1950,  86,  163)  have  used  a  diluting 
solution  containing  no  calcium  or  bi- 
carbonate, but  with  increased  phos- 
phate, and  with  aspartic  acid  added. 
This,  however,  apparently  has  not  been 
tested  for  longer  culture  intervals  than 
about  a  week.  About  0.1%  glucose  is 
usually  included  in  balanced  saline 
solutions  as  a  source  of  carbohydrate. 
For  much  tissue  culture  work  the  solu- 
tion used  by  Earle  has  the  advantage 
of  an  alkali  reserve,  in  the  form  of  so- 
dium bicarbonate,  comparable  to  that 
of  serum. 

All  physiological  solutions,  such  as 
serum,  depending  chiefly  on  sodium 
bicarbonate  for  their  alkali  reserve,  can 
be  maintained  at  a  stable  pH  within 
workable     physiological     limits     only 


when  kept  in  sealed  containers  with  an 
adequate  tension  of  CO2  in  the  air  over- 
lying the  fluid.  Sterilization  by  heat, 
or  by  vacuum  filtration  through  a  bac- 
teriological filter,  causes  an  alkaline 
shift,  due  to  loss  of  CO2,  and  this 
causes  secondary  changes  in  the  solu- 
tion such  as  precipitation  of  the  cal- 
cium and  magnesium  salts.  Probably 
the  most  satisfactory  procedure  for 
sterilizing  such  a  solution  is  to  filter  by 
pressure  (2-10  p.s.i.)  and  to  store  in 
sealed  containers.  Before  the  filtra- 
tion CO2  should  be  passed  through  the 
solution  to  bring  it  to  a  pH  somewhat 
more  acid  than  that  desired.  For 
routine  culture  work  an  initial  pH  of 
about  7.6  in  the  culture  is  desirable 
because  elaboration  of  acid  by  cells  in 
the  culture  will  carry  the  pH  to  some- 
what  more    acid    levels. 

While  the  balanced  salt  solution  may 
be  used  for  rinsing  cultures,  and  while 
tissue  fragments  may  be  left  in  it  for 
short  periods,  these  times  should  be 
limited  as  continued  action  of  the  bal- 
anced saline  on  the  cells  is  injurious. 
It  is  probably  wiser  to  consider  the 
balanced  salt  solution  as  having  its 
major  use  as  a  diluent  for  other  media 
such  as  serum. 

For  the  satisfactory  routine  main- 
tenance of  cultures  of  most  tissue 
cells  a  solid  support  or  substrate  for 
their  growth  and  migration  is  neces- 
sary. Various  types  of  substrates  have 
been  employed,  such  as  silk  thread, 
spider  web,  glass  wool,  lens  tissue, 
cellophane,  gelatin  and  agar,  and  for 
simple  cultures,  the  glass  surface  of 
the  culture  dish. 

By  far  the  most  widely  used  sub- 
strate, and  the  one  which  has  proven 
most  satisfactory  until  recently  has 
been  a  thin  layer  of  plasma  or  fibrin 
clot.  To  prepare  the  culture  with 
this  substrate,  the  clump  of  cells  to  be 
planted  (the  implant)  is  placed  in  a 
thin  layer  (0.1  to  1.0  mm.  thick)  of 
plasma  or  fibrinogen  solution,  and  this 
is  clotted  into  a  solid  gel  by  addition 
of  a  little  tissue  extract  or  thrombin 
solution.  When  of  correct  consistency 
the  clot  is  a  solid,  somewhat  elastic, 
optically  clear  gel.  Its  fibrillar  struc- 
ture enables  the  cells  to  migrate 
through  it,  although  they  tend  to  col- 
lect at  its  surfaces. 

This  type  of  culture  substrate  has 
certain  advantages:  1.  The  fibrin  ma- 
trix anchors  the  cells  so  that  as  long  as 
the  matrix  is  intact  few  cells  wash 
loose  and  are  lost  when  the  culture  fluid 
is  renewed.  2.  The  fibrils  of  the  sub- 
strate present  effective  surfaces  for 
adhesion   and   migration   of   the   cells. 


TISSUE  CULTURE 


342 


TISSUE  CULTURE 


3.  Consequently,  with  some  cell  types 
at  least  the  fibrillar  structure  of  the 
clot  probably  facilitates  the  final 
stages  of  cell  division,  since  final 
separation  of  the  cells  is  accomplished 
by  the  daughter  cells  migrating  in 
opposite  directions.  4.  The  freshly 
prepared  fibrin  clot  contains  some 
serum  and  possibly  other  factors  which 
enhance  the  growth  of  many  cell  types. 

Chicken  plasma  is  most  often  used 
for  the  preparation  of  the  clot  because 
it  is  less  likely  to  clot  spontaneously 
and  since  a  gel  of  good  consistency  can 
be  more  routinely  prepared  from  it; 
but  plasma  homologous  with  the  cells 
is  frequently  employed,  as  is  also 
plasma  from  other  animals.  Premature 
clotting  of  the  plasma  is  usually  pre- 
vented by  addition  of  a  small  amount 
of  purified  heparin. 

Although  the  plasma  or  fibrin  clot  is 
widely  used,  it  is  in  many  ways  un- 
satisfactory. 1.  With  certain  combina- 
tions of  cells  and  media  the  clot  rapidly 
dissolves  and  ruins  the  culture.  2. 
In  the  case  of  very  slow-growing  cul- 
tures the  clot  may  gradually  become  so 
opaque  as  to  make  optical  examina- 
tion impossible.  3.  The  chemically  un- 
defined nature  of  the  plasma  or  fibrin 
clot  and  the  variability  of  its  physical 
structure  interfere  with  many  types  of 
work,  such  as  studies  in  cell  nutrition. 

4.  Since  the  cells  are  embedded  in  the 
clot  and  cannot  be  separated  from  it, 
differential  staining,  weighing  of  the 
cells,  or  chemical  analyses  of  the  cells 
alone  in  the  absence  of  the  clot,  are 
difficult  or  impossible. 

Perforated  cellophane  (Evans,  V.  J., 
and  Earle,  W.  R.,  J.  Nat.  Cancer  Inst., 
1947,  8,  103)  offers  a  substrate  superior 
to  plasma  for  many  kinds  of  work.  In 
the  cellophane  substrate  culture  the 
cell  clump  is  placed  on  the  floor  of  the 
culture  container,  is  inamersed  in  a 
nutrient  fluid  medium,  and  is  covered 
with  a  perforated  cellophane  sheet  which 
holds  it  in  position  against  the  glass 
floor  of  the  flask.  This  type  substrate 
is  cheap,  easily  handled,  may  be  steri- 
lized by  autoclaving,  is  relatively  inert, 
and  is  chemically  defined  (regenerated 
cellulose,  Earle, 'W.  R.,  Evans,  V.  J., 
Edward,  M.  F.,  and  Duchesne,  E.,  J. 
Nat.  Cancer  Inst.,  1949,  10,  291). 
Experience  to  date  indicates  that  when 
grown  on  this  substrate  in  an  adequate 
nutrient  culture  fluid,  many  cell  types, 
both  normal  and  malignant,  continue 
to  proliferate  with  far  greater  luxuri- 
ance than  when  embedded  in  the 
plasma  clot.  Loose  cells  from  the 
implant  float  loose,  reattach  to  the 
cellophane  or  the  glass  floor  of  the  cul- 


ture vessel,  and  rapidly  extend  the 
area  covered  by  the  cells  (Earle,  W.  R., 
Evans,  V.  J.  and  Schilling,  E.  L.,  J. 
Nat.  Cancer  Inst.,  1950,  10,  943).  The 
luxuriance  of  growth  is  such  that  cul- 
ture flasks  of  60  square  cm.  floor  area 
may  often  be  used  for  established  stock 
cultures.  Frequently  this  area  is  cov- 
ered bj^  cells  so  rapidly  that  trans- 
planting is  necessary  in  as  short  time 
as  5-10  days.  Transplanting  of  cello- 
phane substrate  cultures  is  most  easily 
accomplished  by  scraping  or  shaking 
the  culture.  Many  cells  come  loose 
from  the  substrate  and  are  trans- 
ferred as  a  cell  suspension  to  a  new  flask. 
Once  a  luxuriant  cell  growth  is  ob- 
tained on  cellophane  and  a  heavy 
cell  suspension  is  prepared  from  it,  the 
cellophane  sheet  may  in  instances  be 
discarded  and  the  cells  may  be  allowed 
to  settle  on  the  glass  floor  of  the  flask 
on  which  they  attach  and  proliferate 
(Earle,  W.  R.,  Evans,  V.  J.,  Sanford, 
K.  K.,  Shannon,  J.  E.,  Jr.  and  Waltz, 
H.  K.,  J.  Nat.  Cancer  Inst.,  in  press; 
Shannon,  J.  E.,  Jr.  and  Earle,  W.  R., 
J.  Nat.  Cancer  Inst.,  in  press). 

When  survival  or  growth  is  desired 
for  longer  than  a  few  hours,  nutrients 
must  be  included  in  the  culture  medium. 
In  successful  instances  of  cell  prolif- 
eration over  an  extended  period  of  time 
the  nutrient  materials  used  have  been 
a  tissue  extract,  most  often  from  em- 
bryonic tissue,  a  serum,  or  usually  a 
combination  of  the  two.  In  many  in- 
stances, particularly  in  the  older  litera- 
ture, where  a  tissue  extract  alone  has 
been  found  satisfactory  as  a  nutrient, 
serum  was  actually  also  present  in  sub- 
stantial amounts  owing  to  use  of  a 
plasma  substrate   for   the   culture. 

The  tissue  extract  now  commonly 
employed  for  routine  tissue  cultures  of 
cells  from  many  species  is  made  by 
briefly  extracting  minced  embryonic 
tissue  with  an  equal  volume  of  balanced 
salt  solution,  and  by  decanting  the 
supernatant  fluid  after  centrifuging. 
This  concentration  is  known  as  1:1 
embryo  extract.  An  attempt  is  gener- 
ally made  to  eliminate  living  tissue 
cells  from  the  solution  by  freezing  in 
CO2  snow  and  recentrifuging.  The 
extract  loses  potency  rapidly  and 
should  therefore  be  used  within  a  few 
days  after  preparation,  but  if  lyophil- 
ized  it  can  be  kept  for  much  longer 
periods  (Hetherington,  D.  C,  and 
Craig,  J.  S.,  Proc.  Soc.  Exp.  Biol,  and 
Med.,  1939,  42,  831). 

One  current  source  of  embryo  extract 
is  chick  embryos  of  9-11  days  incuba- 
tion. Where  facilities  of  a  local  slaugh- 
ter house  are  available  some  workers 


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343 


TISSUE  CULTURE 


find  it  convenient  to  employ  beef  em- 
bryos removed  from  the  uteri  by  asep- 
tic methods  (Gey  and  Gey,  cited 
above). 

At  present,  whatever  the  source  of 
tissue,  the  extract  is  routinely  pre- 
pared with  rigid  asepsis  because  no 
means  of  sterilization  has  yet  proven 
satisfactory.  Filtration  through  a  bac- 
teriological filter  results  in  rapid  clog- 
ging of  the  filter  and  in  great  reduction 
in  potency.  However,  it  has  been 
recently  reported  (Bryant,  Jay  C, 
Earle,  "W.  R.  and  Peppers,  E.  V.,  at 
the  1951  Annual  Meeting  of  the  Tissue 
Culture  Association,  Detroit)  that  by 
treating  the  extract  first  with  hyalu- 
ronidase,  and  then  by  centrifuging  at 
approximately  40,000  G,  extract  from 
9-day  chicks  may  be  rapidly  filtered 
by  low  pressure  through  a  ^03  porosity, 
4  mm.  wall  thickness  Selas  filter  (Selas 
Filter  Co.,  Philadelphia,  Penn.).  The 
resulting  extract,  used  with  serum, 
caused  rapid  proliferation  of  mouse 
strain  L  fibroblasts  for  the  17  day 
interval  studied.  If  these  results  are 
confirmed  for  other  cell  types  and  for 
longer  intervals  of  culture,  this  method 
of  preparation  of  embryo  extract  should 
substantially  facilitate  tissue  culture 
work. 

Extracts  of  malignant  tissues  have 
often  been  extremely  effective  in 
stimulating  growth  of  some  types  of 
cells.  A  balanced  saline  extract  of 
Walker  256  rat  mammary  carcinoma, 
used  with  horse  serum,  has  caused 
rapid  proliferation  of  the  Walker 
256  carcinoma  cells  in  culture,  but 
no  stimulative  action  was  noted  on 
either  rat  normal  mammary  epithelium 
or  subcutaneous  fibroblasts  (Earle, 
W.  R.,  Arch.  f.  Exp.  Zellf.,  1937,  20, 
140). 

Horse  serum  has  been  routinely  used 
in  this  laboratory  over  a  period  of 
years  as  a  serum  component  of  nutrient 
fluid.  The  mixture  consisted  of  40% 
horse  serum,  20%  1 : 1  extract  from  9-day 
chick  embryos  and  40%  balanced  saline 
solution.  This  has  given  luxuriant 
long-term  growth  with  such  different 
cells  as  Walker  256  rat  mammary  car- 
cinoma (Earle,  W.  R.,  Arch,  of  Path., 
1939,  27,  80),  fibroblasts  from  rats, 
mice,  and  humans,  and  a  number  of 
mouse  fibrosarcoma  strains  (Earle, 
W.  R.,  J.  Nat.  Cancer  Inst.,  1943,  4, 
165).  Not  all  cells  do  well  in  this  me- 
dium. A  high  concentration  of  chicken 
serum  with  low  embryo  extract  gave 
superior  growth  of  chicken  monocvtes. 
Parker,  R.  C.  (J.  Exp.  Med.,  1932, 
55,  713;  1933,  58,  97;  1933,  58,  401)  has 
found  that  various  strains  of  fibroblasts 


required  very  different  concentrations 
of  embryo  extract  and  serum  to  attain 
their  optimal  growth. 

Where  it  can  be  used  horse  serum 
offers  certain  technical  advantages. 
Eight  to  10  liters  of  horse  blood  can 
be  obtained  from  one  bleeding  without 
injury  to  the  horse,  and  after  clotting 
and  separation  of  the  blood  cells  by 
centrifugation  the  serum  may  be 
sterilized  by  pressure  filtration  through 
a  #03  Selas  filter  at  5  p.s.i.,  and  stored 
under  refrigeration  for  a  year  or  longer. 
Before  filtration  extreme  care  should  be 
taken  to  prevent  any  fungus  or  bac- 
terial growth  in  the  serum;  to.xic  sul)- 
stances  produced  can  pass  the  filter 
and   injure   or   kill    the   culture. 

Numerous  other  types  of  sera  have 
been  used  in  tissue  culture.  Beef 
serum  would  probably  be  as  satisfac- 
tory as  horse.  If  local  facilities  make 
it  available  sheep  serum  could  be 
tried.  In  hospital  centers  human  cord 
serum  has  been  available  and  has  been 
found  extremely  satisfactory  bj^  many 
workers  (Gey,  G.  O.  and  M.  K.  cited 
above).  Dr.  Margaret  Murray  finds 
that  for  human  tumor  material  it  is 
superior  to  horse  serum  (personal 
communication) . 

In  exploring  the  possibilities  of  grow- 
ing any  cell  type,  various  percentage 
combinations  of  embryo  extract  and 
serum  are  among  the  first  media  to  be 
tried,  after  which  these  combinations 
may  be  supplemented  by  addition  of 
other  physiologically  active  substances. 

There  has  been  a  great  deal  of  work 
toward  preparing  a  chemically  defined 
or  synthetic  culture  medium  for  cells 
growing  in  culture  (Vogelaar,  J.  P. 
M.,  and  Erlichman,  E.,  Am.  J.  Cancer, 
1933,  18,  28;  Baker,  L.  E.,  Science, 
1936,  83,  605;  Fischer,  A.,  Astrup,  T., 
Ehrensvard,  G.  and  Oehlenschlager, 
v.,  Proc.  Soc.  Exp.  Biol.  &  Med.,  1948, 
67,  40;  White,  P.  R.,  Growth,  1946, 
10,  231;  Davidson,  J.  N.,  Leslie,  I. 
and  Waymouth,  C,  Biochem.  J.,  1949, 
44,  5;  Morgan,  J.  F.,  Morton,  H.  J. 
and  Parker,  R.  C,  Proc.  Soc.  Exp. 
Biol.  &  Med.,  1950,  73,  1).  Substan- 
tial progress  has  been  and  is  being 
made  in  defining  the  nutritional  re- 
quirements of  cells  in  culture,  but  at  the 
present  time  no  chemically  defined 
medium  now  available  appears  to  be 
satisfactory  for  the  continued  pro- 
liferation of  any  type  of  tissue  cell 
in  vitro.  This  study  of  cell  nutrition 
of  both  the  normal  and  the  malignant 
cell  will  undoubtedly  continue  as  one  of 
the  most  interesting  and  active  fields 
of  tissue  culture  research. 

At    present    our    knowledge    of    the 


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344 


TISSUE  CULTURE 


media  and  other  conditions  prevailing 
in  culture,  and  of  the  nutrients  and 
other  materials  influencing  the  cells 
in  vitro  are  all  too  inadequate.  Con- 
sequently any  extrapolation  of  cell  or 
tissue  behavior  from  in  vitro  to  normal 
in  vivo  conditions  must  be  made  with 
great  reserve.  Great  caution  should 
be  observed  in  undertaking  any  tissue 
culture  study  the  significance  of  which 
is  dependent  on  in  vitro  conditions 
being  identical  or  closely  comparable 
with  those  in  vivo. 

2.  Types  of  Cultures.  In  cover  slip 
preparations  the  tissue  clump  is  planted 
in  a  drop  of  plasma  and  nutrient  cul- 
ture medium  on  a  round  coverslip  of 
24  mm.  diameter.  This  coverslip  is 
laid,  culture  side  up,  on  a  coverslip 
48  mm.  square,  and  is  attached  to  the 
larger  coverslip  through  capillarity  by 
allowing  a  small  drop  of  culture  medium 
to  run  between  them. 

A  hollow  ground  slide,  charged  with 
a  vaseline  ring,  is  then  lowered  onto  the 
the  large  coverslip  until  contact  of 
the  coverslip  with  the  vaseline  ring  on 
the  slide  seals  the  preparation:  For 
coverslips  of  the  size  cited  a  rectangular 
hollow  ground  slide  55  x  80  mm.  by  6 
mim.  thick  and  with  a  polished  concav- 
ity 40  mm.  in  diameter  and  about  4.5 
mm.  deep  at  its  deepest  point  is  excel- 
lent. (The  more  usual  Maximow  slide  is 
75  X  45  X  8  mm.,  with  a  cavity  36  mm. 
in  diameter,  and  requires  a  40  x  40  mm 
coverslip.) 

Coverslip  preparations  can  be  given 
a  final  outer  edge-seal  of  paraffin.  By 
using  very  thin  coverslips,  and  if 
necessary,  by  even  omitting  the  small 
inner  slip,  the  cells  can  be  critically 
studied  with  high  numerical  aperture 
lenses.  This  type  of  preparation  is 
probably  the  best  for  routine  work  with 
short  working  distance  high  resolution 
objectives. 

Since  the  total  amount  of  culture 
medium  is  only  1-3  drops,  a  tissue  clump 
of  very  limited  size  must  be  used  and 
the  reasonably  healthy  life  of  the 
preparation  is  only  a  few  days.  At  the 
end  of  that  time  however,  the  culture 
may  be  opened,  the  inner  coverslip 
with  the  actual  culture  lifted  out,  rinsed 
in  balanced  saline;  fresh  nutrient 
fluid  is  added  and  the  whole  resealed 
onto  a  new  outer  coverslip  and  hollow 
ground  slide.  By  this  partial  renewal 
of  the  culture  medium  every  2  or  3 
days  the  culture  may  be  carried  for  ex- 
tended periods.  Pogogeff"  and  Murray 
(Anat.  Rec,  1946,  95,  321)  report  carry- 
ing such  cultures  of  muscle  cells  for 
more  than  a  year  and  a  half.  When  the 
cell  clump  gets  too  large  a  small  frag- 


ment of  it  may  be  re-explanted  to  a  new 
culture. 

Instead  of  using  a  plasma  substrate 
for  the  culture,  the  cell  clump  may  be 
placed  on  the  coverslip,  a  few  drops  of 
nutrient  fluid  added,  and  a  disc  of 
perforated  cellophane  dropped  on  the 
cell  clump  (Schilling,  E.  L.,  Earle, 
W.  R.  and  Evans,  V.  J.,  J.  Nat.  Cancer 
Inst.,  1950,  10,  883).  Or  the  cell  clump 
may  be  placed  on  an  inner  coverslip 
in  nutrient  fluid,  and  covered  with  a 
disc  of  perforated  cellophane.  Trans- 
fer of  the  culture  to  a  fresh  slide  is 
similar  to  transfer  of  the  usual  double 
coverslip  culture  preparation. 

Coverslip  cultures  may  be  killed  and 
fixed  and  stained  in  toto.  For  even 
more  exacting  visual  or  photographic 
work  the  plasma  may  be  omitted  and 
the  cells  grown  or  allowed  to  migrate 
out  directly  on  the  glass  coverslip. 
In  migrations  under  these  conditions 
the  cells  spread  on  the  glass  in  ex- 
tremely thin  sheets.  These  are  suit- 
able for  critical  microscopic  study  of 
chromosomes,  mitochondria,  Golgi  ap- 
paratus and  other  cellular  components. 
If  grown  on  thin  plastic  sheets  they 
can  even  be  fixed  and  examined  with 
the  electron  microscope. 

Coverslip  cultures  for  short  periods 
of  time  are  recommended  in  beginning 
tissue  culture  work,  but  when  it  is 
necessary  to  carry  them  through  con- 
secutive changes  of  media,  sterility  is 
difficult  to  maintain.  When  dangerous 
infectious  agents  are  employed,  cover- 
slip  preparations  should  be  handled 
with  great  care  to  avoid  hazard  to  the 
operator,  as  they  frequently  develop 
leaky  seals  and  because  the  thin  cover- 
slips  are  easily  broken.  Accurate  con- 
trol of  culture  conditions  over  long 
periods  of  time  is  more  difficult  in 
coverslip  preparations  than  in  tube  or 
Carrel  flask  cultures. 

For  preparations  of  high  optical  per- 
fection, such  as  are  necessary  in  high 
resolution  microcinematography,  the 
culture  is  often  prepared  on  a  large 
coverslip  as  described,  and  over  this  a 
thin  glass  or  metal  slide,  having  a  hole 
through  it  the  diameter  of  the  usual 
hollow-slide  concavity,  is  placed  and 
sealed.  The  open  top  of  the  prepara- 
tion is  then  sealed  by  means  of  another 
coverslip.  In  this  type  of  mount  the 
nutrient  fluid  of  the  culture  can  form 
a  continuous  film  joining  a  small 
central  area  of  the  upper  and  lower 
coverslips.  While  the  optical  perfec- 
tion of  such  a  preparation  is  high,  the 
cell  clump  used  must  be  small  and  its 
life  is  short  due  to  limited  volume  of 


TISSUE  CULTURE 


Mi 


TISSUE  CULTURE 


culture  fluid  and  limited  access  of  the 
cells  to  adequate  oxygenation. 

In  Roller  Tube  Cultures  (Gey.G.O.and 
M.  K.,  Am.  J.  Cancer,  1936,  27,  45) 
the  culture  vessel  is  a  test  tube  usually 
about  15  X  150  mm.,  or  in  some  cases  a 
larger  container  is  used.  A  thin  layer 
of  plasma  and  nutrient  medium  is 
coated  over  the  inner  surface  of  the 
tube  to  within  5  cm.  of  the  mouth,  and 
while  this  plasma  layer  is  still  liquid 
numerous  small  tissue  fragments  are 
embedded  in  it.  After  the  plasma  has 
clotted,  about  1  ml.  of  nutrient  solution 
is  added  and  the  tube  sealed  with  a 
rubber  stopper.  In  the  incubator  the 
tube  is  slipped  into  a  hole  in  the  front 
end  of  a  drum-shaped  frame  rotating 
at  about  7-10  r.p.h.,  so  that  as  the 
drum  rotates  about  its  axis  the  super- 
natant culture  fluid  slowly  washes  over 
the  clumps  of  cells  embedded  in  the 
plasma  lining  the  tube.  The  fluid  is 
changed  every  2  to  4  days.  At  periods 
of  9  to  15  days,  colonies  of  cells  may  be 
dissected  loose  from  the  plasma  film 
lining  the  culture  tube.  They  are  then 
removed  from  the  culture  tube  by 
means  of  the  pipette,  cut  to  convenient 
size,  and  subplanted  to  new  cultures. 

This  type  of  culture  is  better  adapted 
than  is  the  coverslip  preparation  for 
routine  growing  of  large  numbers  of 
cell  clumps  since  each  test  tube  can 
accommodate  5  to  20  cell  clumps,  each 
of  them  as  large  as  that  in  a  coverslip 
culture.  The  fluid  can  be  readily 
changed  with  only  minimal  disturbance 
of  the  embedded  cultures.  Where  an 
extensive  series  of  cultures  is  carried 
bacterial  infection  is  usually  less 
troublesome  than  with  slide  cultures. 
Since  the  tube  may  be  sealed  with  a 
rubber  stopper,  there  is  less  gas  (CO2 
and  O2)  leakage  than  in  the  slide  prepa- 
ration (Earle,  W.  R.,  U.  S.  Pub.  Health 
Reports,  1931,  46,  199S).  Moreover  the 
rotating  mechanism  for  the  roller-tube 
unit  is  cheaply  and  easily  constructed 
and  the  cost  of  routine  culture  tubes 
(pyrex  test  tubes)  is  only  a  few  cents. 

But  the  use  of  "roller-tube"  cultures 
is  not  without  limitations.  The  thin 
layer  of  plasma  clot  used  is  often  eroded 
by  the  cells  so  that  frequent  patching 
of  the  clot  by  fresh  additions  of  plasma 
becomes  necessary.  This  patching  in- 
terferes with  accuracy  in  control  of 
conditions  of  the  culture  and  the  cul- 
tures themselves  are  not  infrequently 
lost  by  eroding  entirely  out  of  the  clot. 
The  curved  tube  surface,  the  thick  tube 
wall,  and  the  separation  which  the 
tube  makes  necessary  between  micro- 
scope objective  and  condenser  all  com- 
plicate   microscopic    examination    and 


limit  it  to  low  magnifications.  This 
handicap  can  be  partially  overcome  by 
subculturing  to  coverslip  preparations 
for  detailed  microscopic  study,  but  this 
is  objectionable  because  of  disturbance 
of  the  culture  and  because  of  the  poor 
control  of  culture  conditions  in  the 
coverslip  preparations.  Although  each 
roller  tube  may  contain  a  number  of 
implants,  each  of  them  is  usually  small 
so  that  the  total  volume  of  explanted 
tissue  is  not  large.  The  consequent 
necessity  of  handling  many  cell  clumps 
makes  the  initial  planting  of  the  cul- 
tures relatively  slow. 

With  some  cell  types,  following  lique- 
faction of  the  plasma  film  in  the  roller 
tube,  loose  cells  scatter  over  the  inner 
surface  of  the  tube  and  proliferate 
luxuriantly.  This  gives  roller  tube 
cultures  which  can  be  handled  and 
transferred  by  scraping  and  shaking 
cells  loose,  as  described  below  for  the 
cellophane  substrate  cultures. 

Attempts  have  been  made  to  adapt 
the  cellophane  substrate  to  roller  tubes 
by  inserting  a  loose  sleeve  of  perforated 
cellophane  in  the  roller  tube  on  top  of 
the  cell  clumps.  Some  workers  have 
reported  that  the  cellophane  sleeve  has 
a  tendency  to  rotate  within  the  tube 
and  so  scrape  cells  off.  Some  method 
must  be  used  to  prevent  this.  Other 
workers  have  apparently  had  extremely 
satisfactory  results  with  cellophane 
substrate  roller  tube  cultures.  Too 
little  information  is  at  present  available 
to  evaluate  the  method. 

Flasks  for  Carrel  Flask  Cultures 
(Carrel,  A.,  J.  Exp.  Med.,  1923,  38,  407) 
are  made  in  several  sizes.  A  well  made 
"D"  3.5  type  flask,  as  currently  used  is 
disc  shaped,  3.5  cm.  in  diameter,  with 
top  and  bottom  blown  plane  and  paral- 
lel, each  about  ^  mm.  in  thickness. 
The  sides  of  the  flask  are  vertical,  so 
that  the  total  separation  from  top  to 
bottom  of  the  flask  is  10.0  to  11.0  mm. 
A  side  neck  of  10.3-10.8  mm.  internal 
diameter,  1.0-1.3  mm.  wall  thickness, 
32-34  mm.  length,  projects  from  the 
side  wall  of  the  flask  and  slopes  upward 
to  form  an  angle  of  140°  with  the  top 
surface  of  the  flask.  The  end  of  the 
throat  is  fire-polished  and  free  from 
bead  or  overhang.  The  entire  flask  is 
made  of  Pyrex  glass,  and  is  oven  an- 
nealed to  be  strain-free  under  polarized 
light. 

The  cell  clump  is  planted  on  the  bot- 
tom of  the  flask  in  a  layer  of  medium 
which  consists  of  0.6  cc.  of  chicken 
plasma  and  0.7  cc.  of  some  fluid  culture 
medium  (20%  chick  embryo  extract, 
40%  horse  serum  and  40%  physiological 
saline).     After  this  has  clotted,  1  to  2 


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346 


TISSUE  CULTURE 


ml.  of  the  same  fluid  culture  medium 
is  added;  the  flask  sealed  with  a  rubber 
stopper,  and  incubated  as  usual. 
About  3  times  weekly  the  preparation 
is  unsealed,  the  old  culture  medium  re- 
moved, the  solid  clot  with  its  contained 
cells  soaked  for  about  15  min.  in  iso- 
tonic balanced  saline,  this  saline  re- 
moved, fresh  nutrient  fluid  added,  and 
the  flask  resealed.  At  intervals  of 
for  instance  28  days,  the  whole  sheet 
of  plasma  may  be  slipped  loose  from  the 
floor  of  the  flask,  poured  out  of  the 
flask  and  the  cell  sheet  cut  into  explants 
of  suitable  size.  These  may  be  reinocu- 
lated  to  make  new  cultures  in  other 
flasks. 

This  type  of  culture,  like  the  "roller 
tube"  culture  is  well  suited  for  carrying 
relatively  large  numbers  of  cultures 
over  extended  periods.  Washing  of  the 
culture  and  renewal  of  the  culture  fluid 
can  be  done  quickly.  As  routinely 
carried  out  at  the  National  Cancer  In- 
stitute, the  actual  time  required  for  2 
operators  to  set  up  apparatus  and  solu- 
tions, wash  and  renew  the  nutrient 
medium  on  200  plasma  substrate  cul- 
tures is  about  90-110  min.  In  plant- 
ing, each  culture  flask  receives  one  ex- 
plant  of  about  3.0  to  4.5  mm.  width 
and  15  mm.  length,  while  the  thickness 
of  the  explant  is  only  the  thickness  of 
the  culture  sheet  of  the  previous  cul- 
ture generation.  Since  only  one  cul- 
ture is  used  in  each  culture  flask,  trans- 
plantation is  rapid.  Growth  from  this 
type  explant  of  a  rapidly  growing  cell 
strain  will  often  routinely  cover  the 
floor  of  the  flask  at  28  days. 

There  are  several  advantages  over 
the  roller  tube  culture.  The  plasma 
clot  is  usually  thicker  (though  a  thin 
clot  can  be  used)  so  that  there  is  less 
trouble  from  clot  erosion;  with  many 
cell  strains  "patching"  of  the  clot  with 
fresh  plasma  is  not  necessary.  The 
clot  is  of  such  thickness  and  texture  that 
it  can  be  slipped  loose  from  the  flask 
as  a  sheet,  and  slid  out  onto  a  sterile 
glass  plate,  where  the  culture  can  be 
easily  and  accurately  cut  up  for  sub- 
inoculation  by  means  of  a  Bard-Parker 
^11  blade  attached  to  a  j^7  handle. 
Because  a  single  very  thin  strip-shaped 
explant  is  placed  in  each  flask,  actual 
subplanting  of  cultures  is  much  more 
rapid  than  with  the  roller  tube  prepara- 
tions in  general  use  and  the  actual 
amount  of  tissue  is  probably  greater. 
If  desired,  flask  cultures  may  be  in- 
cubated on  slowly  rocking  shelves  but 
this  is  necessary  only  in  studies  in 
which  the  whole  surface  of  the  culture 
must  be  washed  with  a  moving  film  of 
fluid.     Cultures  can  be  routinely  photo- 


graphed at  magnifications  of  200  to  400 
diameters  and  can  be  examined  regu- 
larly with  up  to  a  4  nmi.,  0.65  N.A. 
achromatic  objective.  The  5.5  mm. 
Bausch  and  Lomb  objective  of  0.65 
N.A.  is  extremely  useful.  For  higher 
numerical  aperture  photographs  sub- 
inoculation  must  be  made  to  slide 
cultures. 

The  general  use  of  the  Carrel  flask  has 
been  limited.  Since  the  flasks  require 
a  high  quality  of  precision  glassblowing, 
thej^  are  expensive.  D3.5  flasks  cur- 
rently sell  for  $2.25  each.  Apparently 
the  dimensional  and  fabrication  speci- 
fications of  the  flask  were  never  pub- 
lished so  that  many  of  the  flasks  sold 
have  been  unsatisfactory  in  use,  or 
have  been  so  fragile  as  to  make  their 
use  prohibitively  expensive  due  to 
breakage.  (Satisfactory  flasks  are  now 
available  from  Mr.  Otto  Hopf,  Upper 
Black  Eddy,  Penn.)  In  many  points 
their  manner  of  use  and  accessory  ap- 
paratus used  with  them  has  been  in- 
adequately described  in  text  and  papers 
on  tissue  culture.  The  writer  considers 
the  Carrel  flask  as  the  most  satisfactory 
of  the  three  culture  methods  described 
for  routine  qualitative  work,  and  as 
a  method  which  warrants  greater  at- 
tention than  it  has  received. 

The  preceding  types  of  tissue  culture 
preparations  have  all  allowed  the 
growth  of  a  very  small  mass  of  tissue, 
or  of  a  very  thin  sheet  of  cells  adherent 
to  a  more  or  less  plane  substrate.  An- 
other type  of  three  dimensional  substrate 
culture  has  recently  been  reported. 
(Earle,  W.  R.,  SchilUng,  E.  L.,  Shan- 
non, J.  E.,  Jr.,  1951  Annual  Meeting 
of  the  Tissue  Culture  Association,  De- 
troit; J.  Nat.  Cancer  Inst.,  in  press). 
In  some  instances  this  was  built  from 
folded  perforated  cellophane  sheets; 
in  others  it  consisted  of  a  mass  of  com- 
mercially available  Pyrex  glass  chem- 
ical absorption  tower  packing  helices 
of  I  inch  lumen,  |  mm.  rod  size.  The 
mouse  strain  L  cells  studied  were  im- 
planted as  a  cell  suspension  and  ad- 
hered to  the  surfaces  of  the  matrix, 
while  nutrient  fluid  was  periodically 
circulated  through  the  interstices  of 
the  substrate  mass.  Cell  proliferation 
of  4  X  to  8.5  X  the  original  inoculum 
was  obtained  with  both  types  of  sub- 
strates. In  one  instance  an  estimated 
1145  mg.  (wet  weight)  of  cells  was  ob- 
tained. In  a  number  of  instances  the 
weight  exceeded  500  mg.   per  culture. 

It  appears  that  the  general  methods 
and  principles  involved  in  this  type 
culture  may  already  be  extrapolated  to 
make  practical  the  growth  of  far  larger 
masses  of  tissue  cells.     Even  this  type 


TISSUE  CULTURE 


347 


TISSUE  CULTURE 


of  culture,  however,  must  at  the  present 
time  be  considered  as  merely  a  step  in 
the  development  of  other  and  still 
more  useful  culture  types.  Klein 
(Klein,  G.,  Cancer,  1950,  3,  1052)  and 
others  have  been  able  to  grow  numerous 
types  of  malignant  tumors  intraperi- 
toneally  in  animals,  and  by  repeated 
subinoculation  in  this  site  have  caused 
the  tumor  cells  to  form  a  dense  cell  sus- 
pension in  the  ascitic  fluid  of  the  animal. 
It  appears  quite  probable  that  with 
further  increase  of  our  knowledge  of 
the  factors  which  control  the  prolifera- 
tion of  cells  on  these  various  types  of 
substrates,  it  may  be  practical  to  en- 
tirely eliminate  the  necessity  of  the 
substrate,  as  we  now  know  it.  By 
control  of  culture  conditions  it  may  be 
possible  to  routinely  grow  many  types 
of  cells  free-floating,  or  virtually  free 
floating,  as  a  suspension  in  a  nutrient 
fluid.  Already  these  possibilities  are 
being  explored. 

Numerous  other  types  of  tissue  cul- 
ture preparations  are  employed  for 
special  purposes.  A  watchglass  culture 
is  often  used  in  embrvological  studies 
(Fell,  H.  B.,  above  cfted).  Porter,  K. 
R.,  Claude,  A.  and  Fullam,  E.  F.,  (J. 
Exp.  Med.,  1945,  81,  233)  have  intro- 
duced a  special  flask,  for  use  in  a  roller- 
tube  unit,  designed  particularly  for 
electron  microscopy.  Special  flasks 
designated  T-12  and  T-60  flasks  (Earle, 
W.  R.  and  Highhouse,  F.,  unpublished) 
have  been  designed  to  handle  quan- 
titatively cultures  planted  from  cell 
suspensions  obtained  from  cellophane 
substrate  cultures.  These  will  be  con- 
sidered in  more  detail  below. 

3.  The  Cells  in  Cultures.  With  respect 
to  the  types  of  cells  which  may  he  grown 
in  tissue  culture  at  the  present  time, 
certain  rough  but  possibly  useful  gener- 
alizations may  be  made.  1.  Nearly 
any  type  of  cell  can  be  kept  alive  or  in 
a  state  of  survival,  from  a  few  hours 
to  a  few  days.  2.  Cell  proliferation  is 
probably  not  to  be  expected  at  all  of 
anatomically  incomplete  cells,  such  as 
the  erythrocyte,  which  lacks  a  nucleus. 

3.  Embryonic  cells  which  have  not 
yet  assumed  a  high  degree  of  func- 
tional specialization  or  differentiation, 
are  in  general  easier  to  grow  than  adult, 
highly  specialized  or  differentiated  cells. 

4.  Manj'  malignant  cells  are  more 
easily  grown  than  are  the  normal  cells 
from  which  they  arise.  For  instance, 
there  has  been  no  satisfactory  long- 
term  culture  of  the  normal  mammary 
gland  epithelium,  although  there  have 
been  frequent  instances  of  culture  of 
epithelium  from  carcinoma  of  the  mam- 
mary   gland.     5.  That    group    of    cells 


which  we  loosely  designate  as  "fibro- 
blasts", and  closely  related  cells  which 
arise  from  the  mesenchyme  or  meso- 
derm are  in  general  reasonably  easy  to 
grow,  particularly  from  very  young 
animals.  The  epithelial  tissues,  how- 
ever, especially  the  highly  differen- 
tiated secretory  epithelia,  from  the 
liver  or  the  thyroid,  are  often  far  more 
difficult.  6.  It  appears  quite  probable 
that  as  our  knowledge  of  the  nutrition 
and  endocrine  control  of  specific  cell 
types  increases  we  shall  become  in- 
creasingly able  to  grow  these  more 
highly  specialized  cells.  7.  At  present 
relatively  few  cell  strains,  normal  or 
malignant,  have  been  maintained  in  a 
state  of  rapid  proliferation  for  as  long 
as  one  year.  9.  If  an  easilj'  grown  cell 
type  can  be  used  with  equal  value 
to  one  which  has  never  been  satisfac- 
torily grown,  the  easily  grown  cell  is 
obviously  the  one  of  choice.  The  study 
necessary  to  grow  luxuriantly  a  cell 
type  which  has  never  been  grown  may 
well  take  years.  Any  problem  which 
depends  for  its  success  on  cultivation 
of  a  cell  type  which  has  never  been 
satisfactorily  grown  should  be  entered 
into  with  caution  and  only  after  a  careful 
evaluation  of  whether  the  results  to  be 
obtained  justify  the  expenditure  of 
effort  involved. 

Until  recently  it  has  never  been  pos- 
sible to  grow  a  single  isolated  tissue 
cell  of  any  type.  Consequently  it  has 
never  been  possible  to  establish  a  pure 
culture  or  strain  of  cells  which  could  be 
considered  with  assurance  as  made  up 
of  only  one  single  cell  type.  Cultures 
of  such  cells  as  the  chick-heart  fibro- 
blasts or  the  malignant  mammary  epi- 
thelium have  in  instances  been  cultured 
and  have  appeared  stabilized  for  ex- 
tended periods  of  years;  but  there  has 
been  no  assurance  that  all  cells  within 
such  cultures  were  identical  in  type  or 
in  origin.  At  best  they  could  be  con- 
sidered as  made  up  of  similarly  ap- 
pearing, or  comparably  reacting  cells, 
as  judged  by  the  tests  used.  Recently, 
however,  methods  have  been  worked 
out  by  which  a  single  isolated  cell 
from  the  subcutaneous  connective  tis- 
sue of  a  CsH  strain  mouse  was  success- 
fully isolated  and  grown  (Sanford,  K. 
K.,  Earle,  W.  R.  and  Likely,  G.  D., 
J.  Nat.  Cancer  Inst.,  1948,  9,  229). 
This  cell  strain  has  continued  to  pro- 
liferate luxuriantly'-  in  horse  serum  and 
chick  embryo  extract  for  a  number  of 
years.  (The  strain  has  now  been  made 
available  to  laboratories  having  facili- 
ties for  carrying  it.)  The  methods  used 
have  allowed  proliferation  of  a  number 
of  other  types   of  isolated  cells   both 


TISSUE  CULTURE 


348 


TISSUE  CULTURE 


normal  and  malignant,  and  should 
ultimately  allow  the  growth  of  large 
cultures  from  them.  To  date,  however, 
(March  1951)  technical  difficulties  have 
interfered  with  carrying  the  proliferat- 
ing isolated  cell  of  most  of  these  other 
types  through  to  large  cultures. 

For  accurate  cell  descriptions  which 
are  to  be  compared  with  descriptions 
from  other  cultures  at  other  times, 
culture  conditions  and  media  should 
be  standardized  as  accurately  as  pos- 
sible. Cultures  should  be  well  estab- 
lished yet  relatively  young,  and  of 
comparable  size  (Earle,  W.  R.  and 
Thompson,  J.  W.,  U.  S.  Pub.  Health 
Rep.,  1930,  45,  2672).  Chilling  of  the 
cultures,  inadequate  frequency  of 
change  of  culture  media,  severe  vibra- 
tion, all  tend  to  produce  aberrant  cell 
shapes.  Prolonged  exposure  to  light 
in  the  presence  of  erythrocytes  can 
injure  or  kill  otherwise  relatively  in- 
sensitive cells  (Earle,  W.  R.,  J.  Exp. 
Med.,  1928,  48,  457;  48,  683).  The  cen- 
tral part  of  a  dense  culture  is  often 
degenerating  or  necrotic.  Cells  which 
have  migrated  out  from  an  explant 
frequently  exhibit  aberrant  and  "giant 
cell"  forms  at  the  extreme  periphery  of 
the  culture.  Cells  located  in  a  plasma 
substrate  at  its  interface  with  the 
supernatant  fluid  often  show  different 
structural  features  from  cells  at  the 
interface  of  the  plasma  with  the  glass 
surface  of  the  culture  dish  (Earle, 
W.  R.,  Schilling,  E.  L.  and  Shelton, 
E.,  J.  Nat.  Cancer  Inst.,  1950,  10, 
865  and  1067). 

These  variations  should  be  recognized 
by  the  worker  and  every  effort  made  to 
standardize  conditions  to  eliminate  them 
as  complicating  factors  in  the  records. 
Photographic  records  of  the  culture 
and  of  its  living  cells  are  extremely  sat- 
isfactory as  records  if  correctly  made. 
Phase  contrast  optics  are  often  useful 
or  essential.  For  following  changes 
in  cell  activities  the  time-lapse  cinemat- 
ograph is  extremely  useful. 

4.  Tissue  Culture  and  Quantitative 
Research.  When  an  attempt  is  made 
to  prepare  substantial  numbers  of  repli- 
cate cultures  for  quantitative  studies 
involving  changes  of  cell  proliferation 
rate  in  culture,  numerous  difficulties  are 
encountered.  If  fresh  tissue  is  used, 
the  amount  of  residual  tissue  material 
brought  over  from  the  host,  and  the 
variability  among  the  explants  of  an 
extensive  series  of  cultures  often  raise 
serious  questions  as  to  the  significance 
of  results  obtained.  By  using  plasma 
substrate  tissue  colonies  of  a  well  estab- 
lished cell  strain,  and  by  bisecting 
each  colony,  one  half  of  each  colony 


may  be  used  as  a  control  on  the  other, 
but  even  this  type  preparation  contains 
the  residual  materials  of  the  plasma 
matrix,  while  the  number  of  control 
cultures  necessary  to  achieve  reason- 
ably accurate  conclusions  is  often  pro- 
hibitive. 

If  the  cell  type  can  be  grown  satis- 
factorily on  a  surface  substrate  (e.g. 
cellophane  or  glass),  and  if  a  cell  sus- 
pension can  be  prepared  from  it,  this 
suspension  may  be  handled  by  special 
burettes  and  other  accessory  equip- 
ment, and  from  the  suspension  large 
numbers  of  replicate  cultures  having  a 
high  degree  of  accuracy  may  be  rapidly 
planted  (Evans,  V.  J.,  Earle,  W.  R., 
Sanford,  K.  K.,  Shannon,  J.  E.  Jr.  and 
Waltz,  H.  K.,  J.  Nat.  Cancer  Inst., 
1951,  11,  907). 

The  term  growth  has  been  so  loosely 
used  in  the  tissue  culture  literature, 
and  particularly  in  the  early  literature, 
as  to  be  confusing  and  often  mislead- 
ing (Essentials  of  Tissue  Culture, 
Parker,  R.  C,  Cunningham,  B.  and 
Kirk,  P.  L.,  J.  Cell,  and  Comp.  Physiol., 
1942,  20,  343).  Estimation  of  change 
in  culture  area  or  diameter  has  prob- 
ably been  the  most  widely  used  and 
most  easily  applied  index  of  culture 
"Growth".  Substantial  cell  prolifera- 
tion for  an  extended  period  of  time  can 
be  easily  recognized,  but  in  the  early 
stages  of  the  life  of  the  culture  the 
method  may  be  grossly  inaccurate  due 
to  increase  in  the  culture  area  resultant 
from  cell  migration  rather  than  from 
cell  proliferation.  Even  with  older 
cultures  the  method  is  accurate  only 
where  cell  density  per  uuit  area  is 
relatively  constant  and  if  necrosis  has 
not  supervened. 

Actual  observation  of  cell  division  is 
the  ultimate  evidence  of  cell  prolifera- 
tion. By  determining  the  number  of 
cells  which  undergo  mitosis  in  a  cul- 
ture area  per  unit  time  relative  to  the 
total  number  of  cells  in  that  area,  an 
estimate  may  be  made  of  the  relative 
frequency  of  cell  proliferation.  But 
such  observations  are  arduous  and  are 
subject  to  a  number  of  possible  errors. 
Cell  proliferation  rate  may  be  very 
different  in  different  parts  of  the  same 
culture,  so  that  the  method  is  valid 
only  for  a  constant  zone  or  for  com- 
parable areas  of  cultures.  If  the 
enumerations  are  made  visually  on 
living  cells,  the  accuracy  of  enumera- 
tion is  often  very  poor  while  the  con- 
tinuous exposure  to  light  may  be  in- 
jurious. When  fixed  preparations  are 
counted  the  method  is  valid  only  if 
the  average  interval  of  duration  of 
mitosis   is    known   and   if   the   experi- 


TISSUE  CULTURE  OF  PLANTS 


349 


TISSUE  CULTURE  OF  PLANTS 


mental  conditions  do  not  disturb  the 
duration  of  mitosis.  If  such  disturb- 
ance occurs  it  must  be  compensated 
for,  as  must  also  variation  in  the  pro- 
liferation rate  with  different  periods  in 
the  diurnal  cycle. 

By  time-lapse  cinematography  at  low 
magnifications  for  a  period  of  hours,  it 
is  possible  to  include  100  or  more  cells 
in  the  field,  and  at  the  same  time  to 
obtain  detail  adequate  to  recognize  any 
cell  undergoing  division.  It  is  conse- 
quently practical  to  determine  the  per- 
centage of  cells  which  undergo  mitosis 
per  unit  interval  of  time  with  a  high 
degree  of  accuracy.  If  at  least  two  or 
more  optical  systems  are  available  a 
control  culture  and  one  or  more  under 
experimental  treatment  may  be  simul- 
taneously recorded.  While  the  method 
has  the  disadvantage  of  requiring  ex- 
pensive equipment,  such  multiple  op- 
tical system  time-lapse  cinematographs 
promise  to  be  powerful  instruments  in 
the  quantitative  study  and  comparison 
of  such  phenomena  as  rates  of  cell 
proliferation,  migration  and  necrosis 
in  tissue  culture  preparations. 

Where  the  cells  can  be  grown  from  a 
cell  suspension  planted  on  the  surface 
of  a  substrate  such  as  cellophane  or 
glass,  the  cells  may  be  treated  with  a 
special  citric  acid  diluting  solution  and 
the  cell  nuclei  may  be  enumerated  in 
a  hemocytometer  (Sanford,  K.  K., 
Earle,  W.  R.,  Evans,  V.  J.,  Waltz,  H. 
K.  and  Shannon,  J.  E.,  Jr.,  J.  Nat. 
Cancer  Inst.,  1951,  11,  773).  While  as 
yet  tested  for  only  a  few  cell  types,  of 
which  the  L  strain  of  mouse  cells  of 
single  cell  origin  is  the  chief  one,  the 
method  should  be  applicable  to  other 
cell  types  and  should  be  tried.  From 
the  results  so  far  obtained  the  method  is 
slow,  but  is  both  practical  and  accurate 
for  measuring  any  change  in  the  total 
number  of  nuclei  in  the  culture  planted 
from  the  cell  suspension.  It  has  the 
advantage  that  it  enumerates  only  ap- 
parently healthy,  living  nuclei,  and  it 
also  allows  distinguishing  and  dif- 
ferentially enumerating  cells  actually 
in  mitosis  if  this  is  desired. 

Measurement  of  growth  by  increase 
of  weight  of  the  culture  is  usually  im- 
practical with  cultures  of  sizes  and 
types  now  available.  If  the  cells  are 
within  a  plasma  matrix  they  cannot  be 
separated  from  it  for  weighing.  Even 
when  the  cells  are  grown  on  cellophane 
or  glass  substrates,  difficulties  of  getting 
the  cells  and  substrate  freed  of  media 
and  of  fluid  without  changing  the 
weight  of  the  cells  makes  weight  deter- 
minations difficult.  Determinations  of 
dry  weight  are  similarly  complicated. 


While  optical  methods  for  determin- 
ing the  "growth"  of  a  culture  have  been 
tried,  optical  quality  of  cultures  and  of 
culture  media  may  vary  so  greatly  due 
to  other  changes  (e.g.,  clouding  or 
precipitation  of  the  culture  media, 
cell  granulation,  accumulation  of  fat 
or  granules;  occurrence  of  necrosis) 
that  various  methods  suggested  have 
not  proven  practical  under  the  widely 
varied  experimental  conditions  which 
may  prevail. 

Many  chemical  indices  of  "growth" 
have  been  suggested  and  tried.  Some 
of  these  have  been:  rate  of  utilization 
of  glucose,  lactic  acid  production, 
change  of  pH  of  the  medium,  rates  of 
aerobic  and  anaerobic  glycolysis,  oxy- 
gen consumption,  increase  in  (Kjel- 
dahl)  nitrogen  content  of  the  culture, 
increase  in  desoxyribose  nucleic  acid 
or  ribose  nucleic  acid,  increase  in 
organic  phosphorus  and  in  lipid-free 
organic  phosphorus  and  the  accumula- 
tion of  radioactive  isotopes  of  some 
metabolized  substance.  To  a  greater 
or  lesser  degree  these  are  useful  as 
indices  of  change  of  one  or  more  com- 
pounds or  groups  of  compounds  by  the 
cell  or  its  enzyme  systems.  Such 
chemical  indices  of  change  are  par- 
ticularly valuable  when  a  number  of 
them  can  be  simultaneously  provided 
in  a  single  study  so  that  results  may  be 
correlated.  In  future  work  such  chem- 
ical studies  will  probably  become  in- 
creasingly significant.  However,  under 
the  wide  range  of  experimental  condi- 
tions which  may  be  encountered  in 
tissue  culture  studies  and  with  our 
present  limited  knowledge,  it  is  unwise 
to  rely  on  any  one  of  these  chemical 
indices  as  an  accurate,  quantitative 
measure  of  the  proliferation  of  tissue 
cells  or  nuclei. 
Tissue  Culture  of  Plants  is  also  a  fine  art. 
Fortunately  an  excellent  account  is 
available  in  book  form:  White,  P.  R., 
A  Handbook  of  Plant  Tissue  Culture. 
Lancaster:  Jaques  Cattell  Press,  1943, 
277  pp.  The  nutrient  fluids  used  are 
chiefly  composed  of  pure  chemicals, 
blood  plasma,  embryo  juice  and  so  forth 
are  lacking.  The  temperature  of  incu- 
bation ranges  from  about  30°C.  down 
to  5°C.  The  tissues  are  easily  killed 
by  high  temperatures.  The  special 
techniques  required  in  physiology, 
pathology  and  morphogenesis  are  de- 
scribed by  White  who  also  reviews  the 
literature.  The  technique  of  tissue 
culture  has  proved  useful  in  researches 
on  the  disorderly  growth  of  cells  from 
Crown-galls  (White,  P.  R.  and  Braun, 
A.  C,  Cancer  Research,  1942,  2,  597- 
617). 


TISSUE  EOSINOPHILES 


350 


TOXOPLASMA 


Tissue  Eosinophiles.  Demonstration  is 
easy  by  the  same  techniques  as  for 
Eosinophile  Leucocytes.  In  rabbits  a 
marked  increase  of  tissue  eosinophiles 
can  be  produced  in  maxillary  sinus 
mucosa  by  pilocarpinization.  This  at- 
tains a  maximum  in  5  min.  and  disap- 
pears after  24  hrs.  (Nemours,  P.  R., 
Arch.    Otolaryng.,    1933,    17,    38-42). 

Tissue  Fluid.  All  living  cells  of  the  body 
are  aquatic.  There  is  reason  to  think 
that  the  tissue  fluids,  which  they  in- 
habit, are  not  of  uniform  composition 
throughout  the  body  but  exhibit  regional 
differences  (Cowdry,  E.  V.,  Problems  of 
Ageing,  Baltimore:  Williams  &Wilkins, 
1942, 583-625) .  Except  when  present  in 
large  amounts,  these  tissue  fluids  can- 
not be  collected  for  chemical  analysis. 
Consequently  microchemical  means  are 
important  in  determination  of  their 
nature.  They  are  often  described  in 
the  literature  as  intercellular  ground 
substance.  Many  methods  have  been 
described  by  S.  H.  Bensley  (Anat.  Rec, 
1934,  60,  93-109)  for  the  ground  sub- 
stance of  Loose  Connective  Tissue. 
See  Spreading  Factors.  A  method  for 
quantitative  evaluation  of  tissue  fluid- 
lymph  cellular  ratios  has  been  reported 
by  Allen,  L.,  Anat.  Rec,  1945,  92,  279- 
287.    See  also  Cartilage  and  Bone. 

Tissue  phagocytes  of  the  lungs  (histocytes, 
histiocytes,  etc.) — Written  by  C.  C. 
Macklin,  Dept.  of  Histological  Re- 
search, The  University  of  Western 
Ontario,  London,  Canada.  November 
28,  1951 — These  cells  are  of  mesodermal 
origin  and  akin  to  the  phagocytic  cells 
of  the  general  connective  tissue.  They 
are  made  conspicuous  by  the  grains  of 
carbon  or  other  particulate  matter 
which  they  ingest,  which  have  escaped 
the  phagocytic  clearance  mechanism 
of  the  pulmonary  alveolar  surfaces, 
and  which  have  worked  their  way  into 
the  environments  of  the  phagocytes. 
They  are  demonstrable  by  any  good 
fixation  and  staining  technique,  and 
may  be  made  outstanding  by  the  Vital 
Staining  method  (which  see).  They  are 
not  to  be  confused  with  the  Dust  Cells 
(which  see)  which  are  of  endodermal 
origin  and  do  not  enter  the  connective 

Titan  Yellow  (CI,  813)— Erie  fast  yellow 
WB,  thiazole  yellow — An  acid  thiazole 
dye  used  in  fluorescence  microscopy. 
See  method  for  Magnesium. 

Titanium  Dioxide.  Huggins,  C,  Anat.  Rec, 
1939,  74,  231-253  used  this  compound 
in  a  suspension  as  a  vital  stain  for  bone 
marrow  because  the  amounts  taken  in 
by  reticuloendothelial  cells  can  be 
measured.  He  employed  specially  puri- 
fied  titanium    chloride   obtained   from 


Dr.  J.  L.  Turner  and  the  Titanium  Pig- 
ment Corporation,  111  Broadway,  New 
York.  The  method  is  to  make  a  fine 
5%  suspension  in  2%  aq.  gum  acacia 
by  mixing  with  an  electrical  mixer  for 
1  hr.  After  keeping  this  at  4°C.  for  2 
days  siphon  off  the  supernatant  fluid  for 
use  to  avoid  aggregates  which  settle  to 
the  bottom.  Keep  this  likewise  on  ice 
but  warm  to  body  temperature  before 
intravenous  injection.  Inject  slowly 
into  ear  veins  of  rabbits,  each  animal  to 
receive  3-6  injections  of  10  cc  on  con- 
secutive days.  The  titanium  dioxide 
E articles  can  easily  be  recognized  as  a 
lack  accumulation  in  the  phagocytes 
and  its  amount  can  be  determined 
chemically  in  fairly  large  bone  samples 
by  a  method  detailed  by  the  author. 

Tocopherol,  see  Vitamin  E. 

Toisson  Solution  for  diluting  blood;  aq. 
dest.  160  cc;  neutral  glycerin,  30  cc; 
sodium  sulphate,  8  gm.;  sodium  chlo- 
ride, 1  gm.;  methyl  violet,  0.025  gm. 

Toluene  Red.  Dimethyldiamidotoluphen- 
azin.     See  Platelet  staining  solutions. 

Toluidin  Blue  O  (CI,  925)— methylene  blue 
T  50  or  T  extra — Employed  very  widely. 
Metachromatic  staining  with  this  dye 
is  specific  for  certain  mucoproteins. 
See  Sylvto,  B.,  Acta  Radio.,  1945, 
Suppl.  59,  100  pp. 

Toluidine  Blue  Phloxinate.  Instructions 
for  preparation  (Lillie,  R.  D.,  Stain 
Techn.,  1941,  16,  1-6).  Lillie  now 
recommends  Azure  Toluidine  blue. 

Toluylene  Blue  (CI,  820).  A  basic  indamin 
dye,  homologue  of  Bindschelder's  Green 
which  see. 

Toluylene  Red,  see  Neutral  Red. 

Tolyl  Blue  5  R  (CI,  289),  a  disazo  mordant 
dye  of  light  fastness  3  preparation  and 
use  of  which  for  plant  and  animal  tissues 
is  described   (Emig,  p.  37). 

Tony  Red,  see  Sudan  III. 

Torsion  Balances,  see  Balances. 

Torulosis,  see  Blastomycosis. 

Tourmaline,  as  a  polarizer,  see  Bennett, 
H.  S.  in  McCIung's  Microscopical 
Technique,  1950,  p.  614. 

Toxic  Neutrophiles  (see  Neutrophiles, 
toxic). 

Toxoplasma.  These  protozoa  can  be  identi- 
fied microscopically.  They  can  be 
colored  with  Wright's  or  Giemsa's 
stain  in  impression  preparations  (see 
Smears).  To  demonstrate  them  in  sec- 
tions use  Giemsa's  stain  after  Regaud's 
fixative,  eosin-methylene  blue  after 
Zenker-acetic  or  hematoxylin  and 
phloxin  after  formalin  (Pinkerton,  H, 
and  Weinman,  D.,  Arch.  Path.,  1940, 
30,  374;  Sabin,  A.  B.,  Advances  in  Pe- 
diatrics, 1942,  1,  1).  It  is  helpful  in 
diagnosis    to    compare    with    standard 


TRACHEA 


351 


TRANSPARENT  CHAMBER 
TECHNIQUE 


preparations   of   Sarcocystis   and    En- 
cephalitozoa. 

Trachea.  Excellent  experimental  methods 
to  demonstrate  secretion  of  Mucus 
are  detailed  by  Florey,  H.,  Carleton, 
H.  M.  and  Wells,  A.  Q.,  Brit.  J.  Exper. 
Path.,  1932,  13,  269-284.  Techniques 
for  Nerve  Endings  are  given  under  this 
heading  but  it  would  be  helpful  to  con- 
sult Larsell,  O.  and  Dow,  R.  S.,  Am.  J. 
Anat.,  1933,  52,  125-146  who  illustrates 
what  one  may  expect  to  find.  Tech- 
niques for  Cilia  require  no  special 
adaptation.  Celloidin  sections  are 
smoother  than  paraffin  ones. 

Tracer  Techniques,  see  Radioactive  Isotopes. 

Trachoma  Bodies.  These  are  easily  colored 
by  Giemsa's  stain.  For  demonstration 
of  glycogen  in  them  and  other  pertinent 
data  see  Thygeson,  P.,  Am.  J.  Path., 
1938,  14,  455-462. 

Evolution  forms  of  Rickettsia  tra- 
chomatis. Fix  smears  in  iodine  alcohol, 
4-5  min.  Stain  in  May-Griinwald,  1 
part;  Giemsa,  1  part;  neutral  aq.  dest. 
10  parts  for  1  hr.  Differentiate  in  95% 
alcohol  (Foley,  H.  and  Parrot,  L.,  Arch. 
Inst.  Pasteur  d'Alg^rie,  1938,  16,  283- 
292) .     See  colored  plates  by  the  authors. 

Transplantation.  This  technique  provides 
opportunities  for  important  microscopic 
studies.  See  Anterior  Chamber  of 
Eye,  Chorioallantoic  Membrane,  Or- 
gan Culture,  Tissue  Culture,  and  Tooth 
Germs. 

Transparent  Chamber  Technique — Writ- 
ten hj  Eliot  R.  Clark,  Department  of 
Anatomy,  University  of  Pennsylvania, 
and  Wistar  Institute  of  Anatomy  and 
Biology,  Philadelphia,  Pa.  Novem- 
ber 28,  1951 — Several  types  of  trans- 
parent chambers  and  windows  have 
been  developed,  that  have  been  inserted 
in  the  outer  ears,  and  in  other  locations, 
of  rabbits  and  other  animals,  with  the 
aid  of  which  it  is  possible  to  watch 
through  the  microscope — ^in  some  types 
of  chambers  with  the  oil  immersion  lens — 
the  growth  and  behavior  of  cells  and  tis- 
sues within  the  living  mammal.  The 
stimulus  for  this  study  came  from  the 
desire  to  extend  to  the  mammal  the  type 
of  prolonged  microscopic  studies  of  cells 
and  tissues  within  the  living  animal  that 
had  been  carried  out  in  the  tails  of 
living  amphibian  larvae  (cf.  E.  R. 
Clark,  Anat.  Rec,  1909,  3,  183;  Am.  J. 
Anat.,  1912,  13,  351,  and  1918,  23,  37). 
The  first  chambers  were  developed  by 
Sandison,  J.  C.  (Anat.  Rec,  1924,  28, 
281;  Am.  J.  Anat.,  1928,  41,  447),  at  the 
instigation  of  E.  R.  Clark.  His  original 
attempts  were  with  mica  chaml)ers, 
but  his  successful  ones  were  constructed 
of  celluloid  (cellulose  nitrate).  The 
latter  provided  beautiful  preparations, 


in  which  details  of  growth  and  behavior 
of  blood  vessels  and  of  other  tissues 
could  be  seen  with  highest  microscopic 
magnifications.  They  remained  in  the 
ear  a  maximum  of  4|  months. 

After  Sandison  transferred  from 
Anatomy  to  Surgery  (1928)  a  group  at 
the  University  of  Pennsylvania  under- 
took to  develop,  improve  and  modify 
the  chamber,  and  succeeded  in  develop- 
ing several  types  of  chambers  designed 
for  attacking  a  variety  of  problems. 

One  of  the  most  useful  types,  which 
is  a  modification  of  Sandison's  chamber, 
is  a  type  named  the  "round-table" 
chamber  (E.  R.  Clark,  Kirby-Smith, 
Rex,  and  R.  G.  Williams,  Anat.  Rec, 
1930,  47,  187).  The  "round-table" 
chamber,  as  described  in  1930,  has  been 
modified  as  follows:  the  washers,  both 
celluloid  and  rubber,  have  been  found 
unnecessary  in  "round-table"  cham- 
bers, though  essential  in  "preformed- 
tissue"  chambers,  and  in  "round-table" 
chambers  installed  in  dogs'  ears;  the 
transplant  hole  in  the  base  of  "round- 
table"  chambers  has  been  omitted, 
since  the  Williams  removable-top  cham- 
ber has  proven  a  more  satisfactory 
transplant  type;  the  protective  discs 
have  been  replaced  by  splints  and 
shields  unattached  to  the  chamber; 
the  celluloid  rings  of  both  base  and  top 
have  been  stiffened  by  gluing  on  an 
extra  0.66  mm.  thick  celluloid,  over 
their  medial  two-thirds;  the  edges  of 
the  table  have  been  bevelled  outward, 
from  above  down.  This  type  is  es- 
pecially useful  for  observation  of  the 
growth  and  behavior  of  blood  vessels, 
lymphatic  vessels,  connective  tissue, 
nerves,  bone,  cartilage,  epidermis  and 
blood  cells.  To  date  it  has  been  the 
most  used  and  most  imitated  of  all 
the  chamber  types.  Published  results 
have  appeared  largely  in  the  Anatomi- 
cal Record  and  the  American  Journal 
of  Anatomy,  from  1930  to  the  present. 
In  nearly  all  of  them,  Eleanor  Linton 
Clark  has  been  joint  author. 

Construction,  installation  and  meth- 
ods of  study  are  briefly  as  follows. 
The  chamber  consists  of  two  parts,  a 
base  and  a  top.  The  base  is  made  of 
celluloid,  1.8  mm.  thick,  3.2  cm.  in 
diameter,  at  the  center  of  which  is  a 
raised  round  table,  1.3  mm.  high,  6.3 
mm.  in  diameter.  The  top  consists  of 
a  celluloid  ring,  also  1.8  mm.  thick  and 
3.2  cm.  in  diameter,  having  a  central 
hole  1.4  cm.  across.  To  the  ring  is 
glued  a  mica  disc,  75  micra  thick  using 
Varian's  glue:  gum  copal,  Venice  tur- 
pentine and  xylol  (Science,  1931,  73, 
678).  Three  holes  are  bored  at  points 
equidistant  from  each  other,  near  the 


TRANSPARENT  CHAMBER 
TECHNIQUE 


352 


TRANSPARENT  CHAMBER 
TECHNIQUE 


outer  rim  of  both  the  base  and  the  top 
ring,  large  enough  to  take  bolts  0.040" 
in  diameter.  The  chamber  fits  the  ear 
best  if  trinmied  to  a  pear  shape.  The 
edges  of  both  base  and  top  are  rounded. 
To  the  table,  close  to  its  outer  rim,  are 
glued  4  "buffers"  (or  separators) — 
squares  or  octagons  of  celluloid,  5  mm. 
wide  and  either  42  or  75  micra  thick, 
depending  upon  the  thickness  of  grow- 
ing space  desired.  The  purpose  of  the 
"buffers"  is  to  leave  a  non-compressible 
open  space,  into  which  tissues  may  grow 
when  the  top  of  the  chamber  is  bolted 
to  the  base.  The  bolts  are  of  brass, 
and  may  be  either  headed  (1  cm.  long) 
or  headless  (1.5  cm.  long).  They  are 
made  from  brass  wire  0.040"  (1.0  mm.) 
in  diameter,  threaded  120  turns  to  the 
inch  (47  to  the  cm.).  The  brass  nuts 
are  he.xagonal.  The  central  3  mm.  of 
the  bolts  is  coated  with  celluloid  by 
dipping  in  celluloid  dissolved  in  equal 
parts  of  absolute  alcohol  and  ether 
and  removing  the  celluloid  from  the 
end  sections. 

Rabbits  used  for  installation  of  ear 
chambers  should  have  ears  at  least  5 
inches  long,  should  be  free  from  "snuf- 
fles" and  from  "ear-mite,  and  should 
have  no  scars  in  the  chamber  region. 
The  chamber  is  inserted  in  the  ear  at 
a  place  about  two-thirds  of  the  distance 
from  base  to  tip,  in  the  flat  portion  be- 
low the  central  artery  and  nerve,  with 
the  round-table  close  to  the  central 
artery.  With  a  nearly-sharp,  heart- 
shaped  dissector,  the  skin  is  raised  on 
both  sides  of  the  ear,  over  an  area  about 
two  mm.  beyond  the  limits  of  the 
chamber — care  being  taken  to  remove 
all  the  dermis,  but  to  leave  the  vascular 
subcutaneous  layer  behind.  The  cen- 
tral portion  of  the  raised  skin  is  re- 
moved to  a  distance  about  4  mm.  from 
the  outer  edge.  A  round  hole  is  cut 
clear  through  the  ear,  slightly  larger 
than  the  top  of  the  table,  and  the  base 
of  the  table  is  placed  in  position  with 
the  table  projecting  through  the  hole. 
The  three  bolts  are  forced  through  the 
ear  following  a  needle  and  the  end  of  a 
probe,  the  table  top  is  brought  down 
over  the  three  bolts,  and  the  loosened 
skin  is  lifted  up  over  the  edges  of  the 
table  on  both  surfaces  of  the  ear.  The 
nuts  are  screwed  down  on  the  bolts, 
until  the  three  buffers  are  clearly  seen, 
but  not  enough  to  cause  the  mica  to 
bulge.  The  splints  and  shields  (Clark, 
E.  R.  and  E.  L.  Clark,  Anat.  Rec, 
1932,  51,  55) — the  former  a  flat  ring  of 
.66  mm.  thick  celluloid,  the  latter  a 
pie-plate  shaped  piece  of  0.24  mm.  thick 
celluloid,  shaped  by  heating  in  a 
mould — are  installed  as  follows.     Two 


holes  are  made  in  the  ear  with  a  leather 
punch.  Through  each  hole  is  passed  a 
headless  bolt  outside  of  which  is  a  glass 
sleeve  about  4  mm.  long.  This  sleeve 
protects  the  ear  from  the  bolt.  The 
hole  in  the  outer  splint  is  large  enough 
so  that  the  splint  clears  the  chamber. 
The  inner  splint  is  trimmed  down  to  a 
half  ring,  to  fit  the  ear  fold,  and  the 
edge  rounded.  Bits  of  sterile  gauze  are 
pushed  into  the  space  around  the 
sleeves.  The  operations  are  performed 
under  strict  asepsis-aqueous  solution 
of  metaphen,  1:500,  being  the  disin- 
fectant of  choice,  both  for  preparing 
the  ear  and  for  sterilizing  the  chambers. 
Local  anesthesia  is  used — injections  of 
novocaine,  2%,  being  made  across  the 
base  of  the  ear. 

Microscopic  observations  are  made 
with  the  rabbit  lying  on  its  back,  held 
by  a  special  rabbit  board  (E.  R.  Clark, 
Sandison  and  Hou,  Anat.  Rec,  1931, 
50,  169),  while  the  ear  is  clamped  to  a 
wooden  or  bakelite  holder,  which  is 
held  and  moved  about  by  the  mechan- 
ical stage.  Camera  ludida  drawings 
are  made  using  the  Leitz  "Zeichen- 
ocular  #4",  which  allows  the  micro- 
scope to  be  tilted  at  an  angle  of  45 
degrees.  The  microscope  rests  on  a 
sheet  of  heavy  plate-glass  which  is 
raised  at  the  sides  sufficiently  to  permit 
a  drawing  board  to  move  underneath. 
Illumination  is  provided  by  a  6-volt 
concentrated  or  ribbon  filament  lamp, 
the  light  passing  through  water  filter 
or  heat-resisting  glass.  Photographs 
of  whole  chamber  are  made  with  a 
bellows  camera,  using  16  or  32  mm. 
lenses;  photomicrographs  with  the 
Leitz  "Leica,"  or  similar  type  camera, 
that  concentrate  the  light,  permitting 
oil  immersion  pictures  of  ^q"  to  -^■^"  ex- 
posure; and  motion  pictures  including 
'time-lapse"  motion  pictures.  While 
oil  immersion  studies  may  be  made, 
a  most  useful  lens  is  the  8  mm.,  20X 
apochromatic  objective  long  working- 
distance  type  with  small-tipped  nose- 
piece),  since  e.xcellent  resolution  is 
obtained  with  even  a  25X  compensat- 
ing ocular.  Since  the  object  is  about 
1  cm.  above  the  top  of  the  microscope 
stage,  illumination  is  improved  by 
either  removing  the  top  lens  of  the 
condensor,  or  by  using  a  supplementary 
lens  placed  between  the  regular  con- 
densor and  the  mirror — as  designed  by 
Dr.  Poser,  for  Bausch  and  Lomb,  which 
sends  a  beam  of  parallel  rays,  instead 
of  rays  converging  at  the  thickness  of 
a  glass  slide  and  then  diverging. 

A  second  type  of  chamber  has  been 
named  the  "preformed  tissue"  chamber 
(E.   R.   Clark,  Kirby-Smith,  Rex  and 


TRANSPARENT  CHAMBER 
TECHNIQUE 

Williams,  Anat.  Rec,  1930,  47,  187). 
It  makes  possible  a  microscopic  study 
of  the  original  tissues  of  the  ear,  with 
their  original  nerve,  blood  vessel  and 
lymphatic  vessel  supply. 

A  third  type,  termed  the  "moat  cham- 
ber", developed  by  Abell  and  Clark 
(Anat.  Rec,  1932,  53,  121;  and  Abell, 
Anat.  Rec,  1937,  69,  14)  contains 
a  small  space,  or  "moat",  accessible 
to  the  outside,  in  which  may  be  placed 
any  fluid  substance,  in  order  to  ob- 
serve, through  the  micrscope,  the  effect 
upon  the  tissues.  The  fluid  may  be 
withdrawn  later,  for  chemical  analysis. 
This  chamber,  in  Abell's  hands,  has 
been  used  for  studies  on  the  absorption 
of  methylene  blue,  reactions  to  a  phos- 
phate buffer,  rate  of  absorption  of  urea, 
rate  of  diffusion  from  blood  vessels  of 
nitrogenous  substances,  reaction  of 
blood  vessels  to  foreign  protein  in  a 
sensitized  animal  (Abel  and  Schenck), 
and  behavior  of  vessels  in  hypertension 
(Abel  and  Page). 

A  fourth  type,  named  the  "removable 
top"  chamber  has  been  developed  by 
R.  G.  Williams,  for  the  purpose,  origi- 
nally, of  obtaining  easy  access  to  the 
living  tissue  of  the  chamber,  in  order 
that  transplants  of  organs  or  tissues, 
or  implantation  of  granular  substances, 
might  be  made,  without  seriously  dis- 
turbing the  delicate  tissues.  It  con- 
tains a  device  by  which  the  top  cover 
may  be  temporarily  lifted  off  merely  by 
unscrewing  two  or  three  nuts.  This 
chamber  has  undergone  steady  improve- 
ment. The  original  model  (R.  G. 
Williams,  J.  Morph.,  1939,  65,  17)  was  a 
modification  of  the  "moat"  chamber- 
constructed  of  glass  and  mica.  This 
was  improved  (R.  G.  Williams,  Anat. 
Rec,  1948,  101,  291)  by  the  substitution, 
for  glass,  of  tantalum — the  metal  which 
is  practically  inert  in  contact  with 
living  tissues — and  the  replacement  of 
a  major  part  of  the  relatively  large, 
flat  surface  that  comes  in  contact  with 
living  tissue,  by  a  tantalum  gauze, 
which  becomes  virtually  a  part  of  the 
ear.  Finally  Williams  and  Roberts 
(Anat.  Rec,  1950,  107,  359)  have  intro- 
duced a  fifth  type  of  chamber,  a  radi- 
cally modified  tantalum  and  mica 
chamber,  which  still  further  reduces  the 
flat  surfaces  next  living  tissue,  has  a 
longer  life  than  any  other  type  of 
chamber  (although  many  "round-table" 
and  "moat"  chambers  have  survived 
well  over  a  year),  is  so  little  irritating 
to  the  ear  that  outer  splints  and  shields 
have  been  found  unnecessary,  can  be 
installed  in  much  less  time  than  is  re- 
quired for  other  chambers,  gives  beau- 
tiful pictures  of  blood  capillary  growth 


353  TRANSPARENT  CHAMBER 

TECHNIQUE 

and  behavior,  has  a  removable  top  for 
transplanting,  could  be  modified  to 
study  preformed  tissue,  and  has  been 
adapted  for  the  introduction  or  removal 
of  fluids,  for  which  the  "moat"  cham- 
ber was  devised.  Epidermis,  which 
forms  an  annoying  temporary  invader 
of  "round-table"  chambers  in  about 
one-fourth  of  installed  chambers,  never 
invades  the  latest  tantalum  chambers. 
On  the  other  hand,  up  to  the  present, 
neither  nerves  nor  lymphatics,  which 
have  been  studied  in  "round-table" 
chambers,  have  been  seen  in  the  new 
type  chambers,  although  these  tissues 
could  probably  be  brought  in  by  slight 
modifications  of  the  chamber.  Success- 
ful autogenous  transplants  have  been 
made  by  Williams  in  one  or  the  other 
variety  of  his  ingenious  chambers,  of 
thyroid,  adrenal  cortex,  spleen,  lymph 
node  and  testis,  many  of  which  have 
survived  and  have  been  available  for 
microscopic  study  for  months,  and  even 
for  3-ears  (cf.  Am.  J.  Anat.,  vols.  62, 
77,  81,  and  Anat.  Rec,  vols.  73,  104, 
179). 

In  other  laboratories,  Ebert,  Florey 
and  Pullinger  (J.  Path,  and  Bact., 
1939,  48,  379)  have  described  a  "round- 
table"  chamber,  modified  chiefly  by  the 
substitution  of  another  plastic,  "per- 
spex",  for  celluloid,  and  the  use  of  a 
vertical  instead  of  an  oblique  access 
hole  in  the  base.  They  give  an  excel- 
lent description  of  the  entire  method  of 
construction  and  installation  of  the 
chambers.  Ebert,  Ahern  and  Block 
(Science,  1949,  110,  No.  2868,  p.  665) 
describe  further  modifications,  using 
the  same  or  a  similar  plastic,  namely 
"plexiglas"  (acrylic  and  methacrylate 
resin),  which  include  a  reduction  in 
outside  diameter  from  32  to  25  mm.  (a 
dimension  also  used  by  Essex);  replace- 
ment of  "buffers"  by  remote  supports 
(as  in  Sandison's  chambers);  replace- 
ment of  metal  bolts  and  nuts  by  plastic 
rods;  elimination  of  splints  and  shields; 
and  a  quadruple  punch,  guided  by  a 
double,  transparent  template,  which  is 
very  helpful  in  installing  chambers. 
Essex,  H.  E.  (Methods  in  Medical 
Research,  Year  Book  Publishers,  1948, 
1,  139)  gives  again  a  complete  descrip- 
tion of  the  construction  and  installa- 
tion of  a  chamber  made  of  "lucite" 
(methyl  methacrylate  resin),  modelled 
after  the  Ebert,  Florey,  Pullinger  style 
of  "round-table"  chaml)er.  He  studies 
the  ear  with  the  rabbit  in  its  normal 
position,  rather  than  on  its  back. 

On  account  of  the  greater  ease  of 
construction,  greater  clearness  and 
freedom  from  warping,  we  have  tried 
lucite  in  our  laboratory,  but  it  has  been 


TRANSPARENT  CHAMBER 
TECHEIQUES 


354 


TRANSPARENT  CHAMBER 
TECHNIQUES 


found  to  incite  what  seemed  to  us  to 
be  definite  abnormal  or  pathological 
reactions  in  the  living  tissue,  in  the 
form  of  excessive  extravasations  of  red 
and  emigrations  of  white  blood  cells  in 
the  early  stages,  and  later,  of  large  ab- 
normal accumulations  of  cells  next  the 
Incite.  Consequently,  for  "round- 
table"  chambers  we  have  preferred 
celluloid,  to  which  the  tissues  do  not 
appear  to  react  unfavorably.  How- 
ever, beautiful  preparations  may  be 
obtained  with  chambers  made  of  metha- 
crylate  resin  plastics. 

Moore,  R.  L.  (Anat.  Rec,  1936,  64, 
387)  (in  E.  R.  Clark's  laboratory),  has 
adapted  the  "round-table"  chamber  to 
the  ear  of  the  dog.  The  chamber  used 
was  similar  in  construction  to  the  one 
used  in  the  rabbit,  except  that  the  table 
was  about  2.00  mm.,  instead  of  1.3  mm. 
high.  He  also  found  that,  in  order  to 
obtain  chambers  in  which  the  ingrowths 
from  the  periphery  filled  the  table  area 
completely,  it  was  necessary  to  have  the 
two  parts  of  the  chamber  held  more 
rigidly  together  than  in  the  rabbits' 
chambers.  This  was  accomplished  by 
using,  in  addition  to  the  buffers,  the 
celluloid  and  rubber  washers  as  advo- 
cated in  the  original  "round-table" 
description  (Clark  et  al.  Anat.  Rec, 
1930,  47,  187).  Of  4  "round-table" 
chambers  installed  in  ears  of  dogs,  2 
without  washers  failed  to  fill  the  table 
area,  while  in  both  chambers  provided 
with  washers,  the  growth  was  complete. 
Quiet,  large-eared  beagle  (or  rabbit) 
hounds  were  used.  Moore  also  success- 
fully installed  a  "preformed-tissue" 
chamber  in  the  dog's  ear. 

Williams,  R.  G.  (Anat.  Rec,  1934, 
60,  493)  has  installed  "round-table" 
chambers  in  skin  flaps,  made  in  the 
lateral  body-wall  of  rabbits,  and  has 
pointed  out  that,  by  the  use  of  the 
"preformed-tissue"  type  of  chamber, 
striated  muscle  (the  panniculus  carno- 
sus)  could  be  brought  under  microscopic 
observation  there. 

Algire,  G.  H.  (J.  Nat'l.  Cancer  Inst., 
1943,  4,  p.  1),  at  the  National  Cancer 
Institute,  Bethesda,  Maryland,  has 
developed  methods  for  the  installation 
and  microscopic  study  of  double-walled, 
transparent  chambers  in  the  dorsal 
skin  of  mice.  The  chambers  are  an 
adaptation  of  the  "preformed-tissue" 
chambers  of  Clark  et  al.  (1930).  Re- 
cently Algire  and  Legallais  (J.  Nat'l. 
Cancer  Inst.,  1949,  4,  225)  have  de- 
scribed a  "round-table"  modification, 
for  the  study  of  new  forming  tissue, 
and  also  a  method  of  obtaining  access 
to  the  chambers.  The  maximum  time 
of  survival   of   Algire 's   chambers   has 


been  two  months.  Several  cancer  stud- 
ies have  been  published  from  Algire 's 
laboratory. 

Other  parts  of  the  mammal  have 
been  brought  under  long-continued 
(weeks  or  months)  microscopic  observa- 
tion by  the  extension  of  the  chamber  or 
window  methods.  Zintel,  H.  A.  (Anat. 
Rec,  1936,  66,  437)  studied,  in  rabbits, 
a  loop  of  small  intestine  with  its  at- 
tached mesentery,  drawn  outside  the 
body  into  a  semicircular  celluloid  con- 
tainer, and  maintained  there  for  several 
weeks.  Microscopic  studies  with  trans- 
mitted light  were  possible  on  the  mesen- 
tery. Similar  chambers  have  been 
used  on  dogs  (Abell,  R.  G.  and  I.  H. 
Page,  Surg.,  Gyn.,  and  Obst.,  1943, 
77,  348).  Wentsler,  N.  E.  (Anat.  Rec, 
1936,  66,  423)  has  successfully  installed 
single-paned  windows,  constructed  of 
celluloid,  in  the  skulls  of  rabbits, 
through  which  it  was  possible  to  watch, 
through  the  microscope,  the  pial  cir- 
culation, over  a  period  of  8  months. 
In  Montevideo,  Estabile,  C.  (Proc 
Am.  Sci.  Cong,  [of  1940],  1942,  6,  147) 
has  used  a  window  similar  to  the  Wents- 
ler cranial  window,  to  observe  the 
contractions  of  the  musculature  of  the 
right  atrium,  with  transmitted  light 
provided  by  an  electric  light  bulb 
passed  through  the  internal  jugular 
vein  into  the  atrium.  In  a  personal 
communication  Estabile  described  to 
the  author  the  use  of  the  same  window 
to  observe  the  stomach  wall  in  action, 
with  light  furnished  by  a  light  bulb 
inside  the  stomach. 

Transparent  chambers  and  windows 
have  provided  new  methods  for  micro- 
scopic observation  of,  and  for  experi- 
ment upon  cells,  tissues  and  organs  in 
the  living  mammal.  They  are  of 
value,  in  both  research  and  teaching 
to  the  physiologist,  pathologist,  phar- 
macologist, bacteriologist  and  surgeon, 
but  especially  to  the  living  anatomist, 
whose  field  is  fundamental  to,  and  in  a 
way  encompasses  all  the  others. 
Transparent  Chamber  Techniqnes — Writ- 
ten by  Glenn  H.  Algire,  U.  S.  Public 
Health  Service,  Bethesda,  Md.  June 
15,  1950 — Transparent-chamber  tech- 
niques seek  to  make  attainable  a 
dynamic,  functional  approach  to  prob- 
lems of  histophysiology  and  cellular 
biology  through  microscopic  observa- 
tion of  tissues  and  cells  in  unanesthe- 
tized  animals  over  periods  of  from 
several  weeks  to  many  months.  This 
technique  was  first  reported  by  Sandi- 
son,  J.  C.  (Anat.  Rec.  1924,  28,  281-287; 
1928,  41,  447-474),  working  under  Dr. 
E.  L.  Clark,  in  whose  laboratory  the 
rabbit    ear    chambers    were    later    on 


TRANSPARENT  CHAMBER 
TECHNIQUES 


355 


TRANSPARENT  CHAMBER 
TECHNIQUES 


greatly  improved  (Clark,  E.  R.,  et.  al., 
Anat.  Rec,  1930,  47,  187-211). 

The  cartilage  of  the  rabbit  ear  serves 
as  a  natural  support  for  chambers 
which  were  constructed  first  of  cellu- 
loid and  mica  and  later  on  of  glass  and 
mica,  or  methyl  methacrylate  resin  (lu- 
cite,  perspex).  An  area  of  skin  the  size 
of  the  chamber  is  dissected  away  from 
the  cartilage  on  both  sides  of  the  ear, 
leaving  the  central  artery  intact.  The 
chamber  units  are  inserted  close  to  the 
main  artery,  usually  with  the  observa- 
tion area  at  the  inner  surface  of  the 
ear,  and  the  skin  is  drawn  over  the 
chamber  edges  and  trimmed  to  expose 
the  observation  area.  The  chamber  is 
held  in  position  by  the  use  of  bolts  or 
pegs  and  by  fitting  the  cartilage  into 
a  space  between  the  upper  and  lower 
pieces.  Protective  covers  of  celluloid 
or  vinylite  are  used  to  prevent  injury. 

Various  types  of  chambers  have  been 
designed  for  special  purposes.  The 
preformed-tissue  design  used  in  studies 
of  the  original  tissues  of  the  ear  is 
restricted  to  low  magnification  observa- 
tions of  blood  vessels  within  a  rela- 
tively thick  layer  of  skin.  Introduc- 
tion of  the  preformed-tissue  chamber 
into  the  rabbit  ear  arouses  cellular  and 
circulatory  disturbances  which  require 
10-14  days  to  subside  before  satisfac- 
tory studies  can  be  made. 

Greatly  improved  resolution  of  cells 
and  tissues  at  high  magnifications  was 
achieved  by  the  round-table  designs 
(Clark,  E.  R.,  et  al.,  cited  above),  in 
which  a  central  hole  is  punched  through 
the  cartilage  and  a  narrow  space  of 
from  40-75  micra  thickness  is  provided 
for  the  ingrowth  of  newly  formed  blood 
vessels  and  connective  tissue  from  the 
edges  of  the  cartilage.  The  thickness 
of  the  space  is  controlled  by  spacers 
placed  between  the  central  round  table 
and  the  coverslip.  Clark,  E.  R.  and 
Clark,  E.  L.,  have  applied  these  tech- 
niques to  detailed  descriptive  accounts 
of  the  growth,  differentiation,  and  be- 
havior of  blood  vessels,  growth  of 
lymphatics  and  nerves,  development  of 
fat  tissues,  migration  of  leukocytes  and 
macrophages,  and  inflammatory  reac- 
tions. 

Abell  and  Clark,  in  devising  the  moat 
chamber  (Anat.  Rec,  1932,53,  121-140) 
modified  the  round-table  design  to 
include  a  well,  provided  with  silver 
cannulae,  for  the  introduction  of  chem- 
ical solutions  into  the  chamber  in  con- 
tact with  the  living  tissue.  This  design 
was  used  in  studies  of  diffusion  of  dyes 
and  of  effects  of  chemical  agents  on 
tissues.  The  round-table  designs  re- 
quired 3  to  4  weeks  for  complete  vas- 


cularization of  a  table  area  having  a 
diameter  of  approximately  7  mm. 

In  the  removable  top  chamber,  Will- 
iams improved  the  method  for  the  in- 
troduction of  cell  transplants.  These 
are  implanted  several  weeks  after 
insertion  of  the  chamber,  when  vascu- 
larization of  the  round  table  is  com- 
plete. Recently  Williams  has  suc- 
ceeded in  prolonging  the  duration  of 
the  transplant  chamber  through  the 
use  of  tantalum  gauze  (Williams,  R. 
G.,  Anat.  Rec,  1948,  101,  291-298). 
Growth  of  blood  vessels  and  connective 
tissue  through  the  meshes  serves  to 
reinforce  the  attachments  between 
tissue  and  chamber.  The  useful  dura- 
tion of  some  of  the  tantalum  chambers 
has  been  approximately  3  years  (per- 
sonal communication). 

Ebert,  Florey,  and  Pullinger  (Ebert, 
R.  H.,  Florey,  H.  W.  and  Pullinger, 
B.  D.,  J.  Path,  and  Bact.,  1939,  48, 
79-94),  introduced  the  use  of  methyl 
methacrylate  resin  (Incite,  perspex)  in 
the  construction  of  the  chamber  and 
modified  an  earlier  design  of  the  trans- 
plant chamber  by  Clark  (cited  above), 
to  give  access  through  a  removable 
plug  introduced  into  the  central  table. 
This  design  has  been  used  in  studies  of 
tissue  reactions  in  tuberculous  infec- 
tion (Ebert,  R.  H.,  Ahern,  J.  J.  and 
Bloch,  R.  G.  (Proc  Soc  Exp.  Biol. 
Med.,  1948,  68,  625-^33).  Further  mod- 
ifications of  this  design  were  published 
recently  which  simplify  the  procedures 
(Ahern,  J.  J.,  Barclay,  W.  R.  and  Ebert, 
R.  H.,  Science,  1949,  110,  665). 

Essex  has  made  use  of  a  Incite  cham- 
ber in  studies  of  peripheral  nerve  in- 
jury and  repair  (Essex,  H.  E.  and  de 
Rezende,  N.,  Am.  J.  Physiol.,  1943, 
140,  107-114),  and  in  studies  of  leuko- 
cytes in  leukopenia  (Essex,  H.  E.  and 
Grana,  A.,  Am.  J.  Physiol.,  1949,  159, 
396-400).  Construction  details  of  this 
chamber  have  been  given  (Essex,  H, 
E.  in  Methods  in  Medical  Research, 
Year  Book  Publishers,  1948  1,  139-145). 

Chambers  have  been  described  for 
other  sites,  organs,  and  species.  Per- 
manent cranial  windows  have  been  de- 
signed, (Wentsler,  N.  E.,  Anat.  Rec, 
1936,  66,  423^435),  in  which  a  small  glass 
window  set  into  a  celluloid  frame  is  se- 
cured to  the  skull  with  small  silver  pegs. 
A  disadvantage  of  this  type  of  window, 
as  of  the  Incite  disks  made  by  Sohler, 
Lothrop,  and  Forbes  (J.  Pharmacol., 
1941,  71,  325-330),  is  its  small  size, 
having  a  diameter  of  12-16  ram. 

Sheldon    et    al.     (Sheldon,     C.     H. 
Pudenz,   R.   H.,   Restarski,   J.   S.   and 
Craig,    W.    McK.,    J.    Neuro-phj-siol., 
1944,  1,  67-75)  describe  a  technique  for 


TRANSPARENT  CHAMBER 
TECHNIQUES 


356 


TREPONEMA  PALLIDUM 


the  preparation  of  a  lucite  calvarium, 
in  which  the  convex  portion  of  the  skull 
of  a  monkey  is  permanently  replaced 
by  a  transparent  lucite  plate,  thus  ex- 
posing the  surface  of  both  cerebral 
hemispheres.  These  preparations  have 
been  used  in  studies  of  superficial 
cerebral  vessels  in  head  injury,  drug 
administration,  and  oxygen  poisoning. 

Several  attempts  have  been  made  to 
install  permanent  type  chambers  for 
the  study  of  internal  organs.  Zintel 
(Anat.  Rec,  1936,  66,  437-447)  intro- 
duced one  of  the  earliest  of  these  for 
exteriorizing  a  loop  of  intestine  and  its 
mesentery.  A  chamber  for  the 
pancreas  of  the  mouse  has  been  de- 
scribed (Flory,  C.  M.  and  Thai,  A., 
Anat.  Rec,  1947,  97,  33-40).  Estable 
(Proc.  Soc.  Exp.  Biol,  and  Med.,  1948, 
67,  445-447)  has  described  a  technique 
for  biomicroscopic  study  of  the  ovary 
and  the  Fallopian  tube  in  rabbits. 
The  ovary  is  transferred  to  a  subcu- 
taneous space,  retaining  intact  its 
vascular  and  nerve  supply.  A  trans- 
parent capsule  is  applied  for  protection 
during  repeated  microscopical  examina- 
tion. 

Adaptation  of  the  transparent  cham- 
ber technique  to  mice  (Algire,  G.  H., 
J.  Nat.  Cancer  Inst.,  1943,  4,  1-11); 
Algire,  G.  H.  and  Legallais,  F.  Y.,  J. 
Nat.  Cancer  Inst.,  1949,  10,  225-243) 
makes  the  procedure  especially  useful 
in  the  many  phases  of  cancer  research 
involving  inbred  strains  of  mice.  In 
this  modification  tantalum  sutures  are 
used  to  support  a  dorsal  fold  of  two 
skin  layers  between  two  plastic  (vinyl- 
ite)  splints.  One  skin  layer  is  incised 
and  retracted  to  allow  the  introduction 
of  a  lucite  ring  and  attached  coverslip 
which  is  in  contact  with  the  inner  sur- 
face of  the  second  layer  of  skin.  Both 
the  lucite  ring  and  the  supporting 
splints  are  held  in  position  by  tantalum 
bolts.  This  procedure  makes  acces- 
sible to  study  by  transmitted  light  a 
layer  of  tissue  approximately  500  micra 
thick,  consisting  of  peripheral  nerves, 
striated  muscle,  peripheral  blood  ves- 
sels and  lymphatics,  subcutaneous 
connective  tissue  and  fat,  hair  follicles, 
and  epidermis. 

Implantation  of  normal  and  neoplas- 
tic cells,  of  embryonic  organs,  or  of 
carcinogenic  chemicals  is  readily  per- 
formed during  the  operation  for  in- 
sertion of  the  chamber.  Experimental 
studies  of  the  tissue  may  be  under- 
taken immediately  after  the  operation, 
and  carried  on  for  from  30  to  60  daj^s. 
Response  of  the  host  and  the  implanted 
tissues  to  physical  and  chemical  agents 
may  be  studied  in  terms  of  cellular  and 


circulatory  reactions,  including  quanti- 
tative measurements  of  blood  pressure, 
dye  diffusion,  and  arterial  oxygen 
saturation.  Microscopic  resolution  is 
sufficiently  good  to  observe  cross-stria- 
tions  in  muscle,  platelets  in  circulating 
blood,  and  cytoplasmic  detail  in  cells 
adhering  to  the  coverslip.  Additional 
resolution  of  cytologic  detail  has  been 
obtained  through  use  of  a  round-table 
design.  The  access-type  consists  of  a 
thick  (1  mm.)  coverslip  of  lucite 
through  which  a  1  mm.  diameter  hole 
provides  for  local  application  of  chem- 
icals at  any  time  after  introduction 
of  the  chamber.  The  operative  pro- 
cedure requires  less  than  one  hour  and 
two  workers  can  readily  handle  10  to 
15  animals  in  daily  observations, 
measurements,  and  photographic  rec- 
ords. 

The  dynamic,  functional  aspects  of 
the  transparent-chamber  approach  in- 
dicates future  increased  development 
and  correlations  with  other  methods 
of  biological  research. 

Trematodes.  Make  up  stain  by  mixing 
1  gm.  of  dried  residue  on  filter  paper 
from  Schneider's  aceto-carmine  with 
10  gm.  ammonia  alum  in  200  cc.  aq. 
dest.  with  aid  of  heat.  When  dissolved, 
cool,  filter  and  to  filtrate  add  crystal 
of  thymol.  After  fixation  bring  worms 
to  water  or  to  20%  alcohol.  Stain  12-36 
hrs.  depending  on  size.  Remove  to 
water  2  changes.  Dehydrate  through 
20,  35,  and  50  to  70%  alcohol.  Place 
few  crystals  potassium  chlorate  in  small 
glass  covered  dish;  add  few  drops  cone. 
HCl.  When  chlorine  is  given  off  fill 
dish  with  70%  alcohol.  If  deeply 
stained  differentiate  in  this  chlorinated 
alcohol.  If  not  or  the  specimens  are 
small  ones  add  it  to  the  alcohol  covering 
them  and  agitate.  When  sufficiently 
destained  remove  to  fresh  80%  alcohol. 
Dehydrate  in  alcohol.  Add  cedar  wood 
oil  to  the  absolute  until  mixture  is  one 
half  oil.  Clear  in  cedar  oil  and  mount 
in  balsam  (Gower,  W.  Carl,  Stain 
Techn.,  1939,  14,31-32). 

Treponema  Pallidum.  The  organisms  can 
best  be  seen  in  the  primary  lesions  by 
Darkfield  examination.  The  same 
method  is  useful  for  skin  and  lymph 
nodes  in  the  secondary  stage  but  for 
the  tertiary  lesions  in  deep  lying  tissues 
sections  are  desirable  supplemented 
by  smears.  A  negative  finding  is  com- 
forting but  does  not  necessarily  signify 
absence  of  parasites  unless  confirmed 
serologically. 

1.  Low  surface  tension  stain  for 
smears  (Haire,  R.  D.,  J.  Lab.  &  Clin. 
Med.,  1938,  23,  1215-1216).  Mix  1  gm. 
Gentian   violet    (or   crystal   violet)   in 


TREPONEMA  PALLIDUM 


357 


TREPONEMA  PALLIDUM 


mortar  slowly  adding  100  cc.  hexylre- 
sorcinol.  Filter  and  store  filtrate  in 
stock  bottle.  Stain  smears  30  min. 
Wash  in  water,  dry  and  examine.  Stain 
on  slide  must  not  be  heated.  Trep- 
onenias,  light  purple. 

2.  Wright's  stain  for  smears  (Mallory, 
p.  289).  To  make  stain  add  1  cc. 
Wright's  stain  and  1  cc.  1%  aq.  potas- 
sium carbonate  to  10  cc.  aq.  dest.  in 
test  tube  and  heat  to  boiling.  Spread 
material  thinly  on  cover  glass  (not  slide) 
and  hold  level  with  forceps.  Cover 
with  hot  stain  3-4  min.  After  fluid  has 
turned  violet,  and  a  yellow  metallic 
scum  has  formed  over  it,  pour  off  and 
repeat  process  twice  with  hot  stain. 
Wash  in  water,  dry  and  mount  in  balsam. 
Treponemas,    intensely    violet. 

3.  Giemsa's  stain  for  smears  (Giemsa, 
G.,  Deut.  med.  Wochn.,  1909,  35,  1751- 
1752)  after  Mallory  (p.  290).  Fix 
smears  for  15  min.  in  absolute  alcohol 
or  pass  them  through  flame  thrice. 
Pour  on  freshly  diluted  stain  (1  cc.  aq. 
dest.  +  1  drop  stock  Giemsa).  Steam 
gently  and  leave  15  sec.  Decant  and 
add  immediately  fresh  diluted  stain, 
warm  and  let  cool  15  sec.  Repeat  4 
times  leaving  1  min.  last  time.  Rinse 
quickly  in  running  water.  Blot. 
Mount  in  balsam.  Treponemas,  dark 
red. 

4.  Fontana-Tribondeau  silver  method 
for  serum  (Fontana,  A.,  Dermat.  Zeits., 
1925-26,  46,  291-293)  after  Mallory 
(p.  291).  To  make  silver  solution  add 
ammonia  water  (diluted  1:20)  drop  by 
drop  to  50-100  cc.  1%  aq.  silver  nitrate 
until  a  coffee  colored  clouding  takes 
place.  Air  dry  thin  smears  of  serum. 
Pour  on  few  drops  Ruge's  sol.  (aq.  dest., 
100  CO.;  glacial  acetic,  1  cc;  formalin, 
2  cc.)  and  change  several  times  during 
1  min.  Rinse  in  running  water.  Mor- 
dant witha  little  aq.  dest.,  100  cc;  tan- 
nic acid,  5  gm. ;  liquid  carbolic  acid,  1  cc. 
for  20  sec.  warming  to  steaming.  Rinse 
in  aq.  dest.  Treat  with  silver  solution 
30  sec.  heating  slightly.  Wash  in  tap 
water.  Dry  in  air.  Mount  in  balsam. 
Treponemas,  brown  to  deep  black. 

5.  Burri's  India  Ink  method  for  lesion 
fluid  (Mallory,  p.  291).  Make  1:4 
suspension  of  India  ink  in  aq.  dest. 
Sterilize  in  autoclave,  15  min.  Mix 
this  in  equal  parts  with  fluid  from  lesion 
on  slide  with  platinum  loop.  Spread 
thinly.  Dry  and  examine.  Trep- 
onema (and  bacteria  if  present),  white 
in  brown  to  black  background. 

6.  Quick  method  for  demonstration 
in  fresh  autopsy  tissues.  This  is 
Krajian's  modification  of  Dieterle's 
method  (Am.  J.  Syphilis,  1933,  17,  127) 
as  amplified  in  Stain  Techn.,  1935,  10, 


68.  Fix  tissue  5  mm.  thick  10  min.  in 
10%  formalin,  70°C.  Cut  frozen  sec- 
tions 5-7  microns.  Place  in  2%  aq. 
sodium  cobalti  nitrite  5  min.  Wash  2 
changes  aq.  dest.  Mordant  for  15  min. 
at  70°C.  in  uranium  nitrate  1  gm. ;  85% 
formic  acid,  3  cc;  glycerin,  5  cc; 
acetone,  10  cc;  95%  alcohol,  10  cc. 
Wash  quickly  in  aq.  dest.  Develop 
5  min.  in  10  cc.  of  following  mixture  -f- 
1  drop  albumin-glycerin  before  use 
(hydroquinone,  0.62  gm. ;  sodium  sulfite, 
0.12  gm.;  acetone,  5  cc. ;  40%  neutral 
formaldehyde,  5  cc;  pyridine,  5cc.; 
sat.  gum  mastic  in  95%  alcohol,  5  cc, 
aq.  dest.,  30  cc).  Wash  few  sec.  aq. 
dest.  Then  warm  silver  solution  15-25 
sec.  and  wash  in  aq.  dest.  Keep  all 
solutions  in  cool  place.  (Original  gives 
treatment  with  0.75%  aq.  silver  nitrate 
at  70°C.  for  1  hr.  upon  the  development 
in  hydroquinone  mixture.) 

7.  Levaditi's  block  silver  method 
(Mallory,  p.  293).  Fix  tissue  pieces 
(1  mm.  thick)  in  10%  formalin,  24  hrs. 
Rinse  in  aq.  dest.  95%  alcohol,  24  hrs. 
Transfer  to  aq.  dest.  and  leave  until 
tissue  sinks  to  bottom.  Fresh  1.5-3% 
aq.  silver  nitrate  at  37°C.  in  dark  3-5 
days  changing  3  times.  (The  stronger 
silver  is  advised  for  tissues  excised 
during  life.)  Wash  in  aq.  dest.  Re- 
duce 24-72  hrs.  in  dark  at  room  tempera- 
ture in:aq.  dest.,  100  cc. ;  formalin, 
5  cc;  pyrogallic  acid,  2-4  gms.  Wash 
in  aq.  dest.  Dehydrate  in  80,  95  and 
absolute  alcohol.  Clear  in  oil  of  cedar 
wood,  imbed  in  paraffin,  mount  5fx  sec- 
tions on  slides,  remove  paraffin  and 
mount  in  balsam.     Treponemas,  black. 

8.  Heitzman's  modification  of  the 
Warthin-Starry  and  Nieto's  methods  as 
given  by  Mallory  (p.  293).  Cut  frozen 
sections  15/n  or  less  of  10%  formalin  fixed 
tissue.  Place  directly  in  pyridine, 
10  min.  Wash  in  aq.  dest.,  3  changes. 
1%  aq.  uranium  nitrate  at  37°C.,  15 
min.  Wash  quickly  in  aq.  dest.,  2 
changes.  0.25%  aq.  silver  nitrate  at 
56°C.,  15-30  min.  Develop  until  dark 
brown  in  following  mixture  made  im- 
mediately beforeliand  by  pipetting  into 
a  beaker:  (1)  15  cc.  5%  aq.  gelatin  at 
56°C.;  (2)  3  cc.  2%  an.  silver  nitrate; 
(3)  0.5  cc.  1%  aq.  hydroquinone.  Re- 
move and  thoroughly  wash  in  warm  aq. 
dest.  Dehydrate  on  slide  adding  by 
pipette  increasing  alcohols  to  absolute. 
Clear  in  benzol  and  mount  in  balsam. 
A  heavy  black  ppt.  indicates  too  long 
development.  Treponenmas,  black. 
See  Warthin-Starry  method. 

9.  For     routine      paraffin      sections, 
Steiner,  G.,  J.  Lab.  &  Clin.  Med.,  1939 
25,  204-210.     Fix  in  10%  formalin  and 
make  sections   9-10   microns.    Remove 


TRIACID  BLOOD  STAIN 


358 


TRICHROME-STAINS 


paraffin  with  xylol.  Pass  through  2 
changes  abs.  ale.  Treat  1-1^  min.  in 
4%  uranium  nitrate  in  abs.  ale,  20  cc; 
25%  gum  mastic  in  abs.  ale,  4(>-50  cc; 
abs.  ale,  20-30  cc.  Wash  in  at  least  3 
changes  aq.  dest.  until  streaks  of  gum 
mastic  are  removed.  0.1%  aq.  silver 
nitrate  in  water  bath  at  100°C.,  1-1§ 
hrs.  Wash  in  aq.  dest.  Then  through 
80%  and  95%  to  abs.  ale.  10-12.5%  gum 
mastic  in  abs.  ale.  5  min.  Repeat  3 
washings  described  in  aq.  dest.  Re- 
duce 20-30  min.  in:  hydroquinone,  10 
gm.;  12.5%  gum  mastic  in  abs.  ale, 
1  cc.;  aq.  dest.,  200  cc.  (with  tempera- 
ture gradually  raised  to  100°C.).  Wash 
thoroughly  in  aq.  dest.  Counterstain 
with  hematoxylin  and  eosin  if  desired. 
Dehydrate  in  abs.  Clear  in  xylol  and 
mount  in  balsam.  The  advantages  are 
speed  and  decrease  in  confusing  silver 
deposits.  See  Steiner's  illustrations. 
A  technician's  experience  with  Steiner's 
method  has  been  published  (Wilson, 
R.  A.  J.,  Am.  J.  Clin.  Path.,  1946,  16, 
21-24). 

10.  Nigrosine  has  been  proposed  as  a 
negative  stain  for  treponema  (Dienst, 
R.  B.  and  Sanderson,  E.  S.,  Am.  J. 
Public  Health,  1936,  26,  910).  Com- 
parison of  dark  field,  nigrosine  stain 
and  Kahn  test  in  diagnosis  (Nagle,  N., 
J.  Lab.  &  Clin.  Med.,  1939-40,  25,  660- 
661). 

11.  Ziehl's  fuchsin  stain  (Perrin,  T. 
G.,  Am.  J.  Clin.  Path.,  1943,  Tech. 
Suppl.,  7,  28).  Make  smears  on  slides 
of  exudate  secured  by  compressing  base 
of  chancre  or  by  scraping  surface  of 
ulcer.  Dry  in  air  and  fix  by  heat  in 
flame,  if  desired.  Stain  2  min.  while 
heating,  or  for  6  min.  at  room  tempera- 
ture, being  careful  not  to  let  the  stain 
dry.  The  stain  is  aq.  dest.,  10  cc; 
commercial  formalin,  1  cc;  acetic  acid, 
1  cc;  Ziehl's  fuchsin  (Ziehl's  Carbol- 
Fuchsin)  4  cc.  Wash  in  water,  moving 
gently,  and  dry  in  air.  See  Vincent's 
Angina. 

Triacid  Blood  Stain,  see  Ehrlich's. 

Tri-Amino  Tri-Phenyl  Methane  Dyes. 
These  are  the  rosanilins.  Examples: 
acid  fuchsin,  acid  violet,  anilin  blue  WS, 
basic  fuchsin,  benzyl  violet,  crystal 
violet,  ethyl  green,  ethyl  violet,  Hof- 
mann's  violet,  iodine  green,  isamine 
blue,  magenta  II,  methyl  blue,  methyl 
green,  methyl  violet,  new  fuchsin  (ma- 
genta III),  pararosanilin  (magenta  O), 
rosanilin  (magenta  I),  spirit  blue,  vic- 
toria blue  B  and  R  and  victoria  blue  4R. 

Trichinella  Spiralis.  Mallory  (p.  304)  gives 
as  a  rapid  method  of  diagnosis  the 
squeezing  of  small  pieces  of  jaw  muscle 
or  of  muscle  near  tendon  of  diaphragm 
between  two  slides  and  direct  examina- 


tion at  low  magnification.  A  useful 
device  for  squeezing  the  muscle,  called 
a  "trichinoscope"  has  been  constructed 
by  Gould,  S.  E.,  Am.  J.  Clin.  Path., 
Teehn.  Suppl.,  1944,  8,  98-100.  If 
trichinellae  are  calcified  or  encapsu- 
lated specimens  can  be  cleared  with 
acid.  In  permanent  preparations  of 
Zenker  or  formalin  fixed  material 
stained  with  hematoxylin  and  phloxine 
or  eosin  the  parasites  are  best  seen  in 
longitudinal  sections  of  muscle  fibers. 
To  demonstrate  in  migratory  phase 
withdraw  blood  from  vein  in  arm  into 
syringe  containing  3%  aq.  acetic  acid, 
centrifuge  and  examine. 

Rapid  iodine-silver  technique  (Kal- 
waryjski,  M.  B.  E.,  Wojsk.  Przegl. 
Weteryn.,  1938,  9,  123-136).  Place  thin 
slices  of  muscle  for  10  min.  in  iodine, 
potassium  iodide,  aq.  dest.  sol.  in  fol- 
lowing proportions  2:4:100  or  0.5:1:100 
or  0.1:0.2:100  Wash  in  aq.  dest. 
Destain  in  2.5%  aq.  sodium  thiosulphate 
until  muscle  is  clear.  Wash  in  aq.  dest. 
Equal  parts  10%  aq.  silver  nitrate  and 
strong  ammonia  until  iodine  leaves  para- 
sites. Wash  in  aq.  dest.  Decolorize 
in  5%  aq.  sodium  thiosulphate.  Wash 
in  aq.  dest.  and  mount  in  glycerin. 
Parasites  stained  dark  brown  owing  to 
conversion  of  iodine  to  silver  iodide. 

See  investigation  of  larvae  with  radio- 
active phosphorus  (McCoy,  O.  R., 
Downing,  V.  F.  and  Voorhis,  S.  N.,  J. 
Parasit.,  1941,  27,  53-58). 

Trichomonas  Vaginalis,  technique  for,  Van 
Someren,  V.  D.,  Vet.  J.,  1946,  102, 
73;  detailed  by  Wenrich,  D.  H.  and 
Diller,  W.  F.  on  McClung's  Microscop- 
ical Technique,  1950,  p.  465. 

Trichloracetic  Acid  employed  with  mercuric 
chloride  and  acetic  acid  as  a  fixative 
(Heidenhain,  Zeit.  wiss.  Mikr.,  1909, 
25,  405)  also  used  in  4  or  5%  aq.  sol.  as 
decalcifying  agent. 

Trichlorethylene,  as  a  solvent  in  histo- 
logical technique  in  place  of  xylol 
(Oltman,  R.  E.,  Stain  Techn.,  1935,  10, 
23-24). 

Trichlorlactic  Acid  used  as  fixative  fol- 
lowed by  staining  with  resorcin  fuchsin 
for  cytoplasmic  canalicular  apparatus 
(Holmgren,  E.,  Ergeb.  d.  Anat.,  1901, 
11,  274-329;  Cowdry,  E.  V.,  Internat. 
Monatsschr.  f.  Anat.  u.  Physiol.,  1912, 
29,  1-32). 

Trichosiderm  name  suggested  for  iron  pig- 
ment from  red  hair  (Flesch,  P.  and 
Rothman,  S.,  J.  Invest.  Dermat.,  1945, 
6,257-270). 

Trichrome-Stains.  There  are  many  such 
stains.  See  Mallory's  and  Masson's. 
A  rapid  one  is  described  by  Pollak, 
O.  J.,  Arch.  Path.,  1944,  37,  294.  Com- 
position of  stain:  acid  fuchsin,  0.5  gm.; 


TRIETHYL  PHOSPHATE 


359 


TRIPHEXYLTKTRAZOLIUM 
CHLORIDE 


ponceau  2  R,  1.0  gm.;  light  green  S  F, 
yellowish,  0.45  gm.;  orange  G,  0.75  gm.; 
phosphotungstic  acid  C.P.,  1.5  gm.; 
phosphomolybdic  acid,  C.P.,  1.5  gm.; 
glacial  acetic  acid,  3.0  cc;  ethyl  ale, 
50%  up  to  300  cc.  Add  acetic  to  alcohol 
and  put  50  cc.  in  each  of  4  beakers.  In 
first  dissolve  acid  fuchsin  and  ponceau, 
in  second  light  green,  in  third  orange 
and  phosphotungstic  acid,  and  in  fourth 
phosphomolybdic  acid  (the  last  named 
by  slight  warming).  Mix  and  use  bal- 
ance of  alcohol  to  wash  out  contents  of 
beakers  adding  them  to  mixture.  Stain 
keeps  well;  can  be  obtained  from  Will 
Corporation,  Rochester,  N.  Y.  See 
colored  plate  by  the  author. 

Triethyl  Phosphate  in  dehydration.  Nelsen, 
O.  E.,  Stain  Tech.,  1945,  20.  131-132. 
recommends  the  use  of  this  compound 
(C2H6)3P04)  in  histological  technique, 
as  it  displaces  water  in  tissues  readily 
without  shrinkage  or  distortion.  Since 
tissues  may  be  transferred  directly  into 
it  from  water,  the  tedious  alcohol  dis- 
placement series  in  the  paraffin  tech- 
nique is  unnecessary.  It  is  soluble  in 
the  alcohols,  benzene,  ether,  chloroform 
and  xylol.  Nelsen  reports  excellent 
results  with  smears  following  the  tri- 
ethyl phosphate  method.  Following 
fixation  and  subsequent  staining  with 
Feulgen,  the  smears  are  first  transferred 
to  equal  parts  of  water  and  triethyl 
phosphate,  then  to  triethyl  phosphate 
and  finally  into  xylene  before  mounting. 
Fast  green  may  be  dissolved  in  it  if 
counterstaining  is  desired. 

Trimethylcarbinol,  see  Tertiary  Butyl 
Alcohol. 

Triphenyltetrazolium  Chloride,  A  Valuable 
Reagent  for  Studies  of  Reducing  Ac- 
tivity in  Living  Organisms — Written 
by  Dr.  Robertson  Pratt,  University 
of  California  College  of  Pharmacy, 
San  Francisco.  January  29,  1951 — In 
aqueous  solutions,  2,3,5-triphenyltet- 
razolium  chloride  (TPTC)  is  colorless, 
diffusible,  and  readily  transported 
through  plant  and  animal  tissues;  but 
in  the  presence  of  living  tissue  or  of 
some  other  reducing  agents,  an  in- 
soluble red  formazan  is  precipitated 
from  solutions  of  TPTC.  This  is  the 
basis  for  use  of  this  compound  in  histo- 
physiology  to  determine  sites  of  de- 
hydrogenase or  other  reducing  activity 
in  living  organisms,  including  cultures 
of  bacteria  (Pratt  and  Dufrenoy,  Stain 
Technol.,  1948,23,137).  The  compound 
has  practical  applications  also  outside 
of  the  field  of  research  (see  below). 

TPTC  has  been  used  successfully  in 
concentrations  ranging  from  0.05  to  1.0% 
in  distilled  water  or  in  buffers  within 
the  biologic  range  of  pH  values.     The 


optimum  concentration  depends  largely 
on  the  kind  and  location  of  tissue  under 
investigation.  Studying  sites  of  re- 
ducing activity  in  stalks  of  sugar  cane, 
where  the  compound  had  to  be  trans- 
ported through  a  considerable  distance 
of  vascular  tissue  and  some  adsorption 
undoubtedly  occurred  on  the  way, 
Dufrenov  and  Pratt  (Am.  J.  Bot., 
1948,  35',  333)  found  a  0.5%  solution 
satisfactory  for  demonstrating  that 
dehydrogenase  activity  was  most  pro- 
nounced in  the  plasmodesmata  and  in 
lipidic  parts  of  the  cytoplasm. 

In  other  investigations  Pratt  and 
Dufrenoy  (Antibiotics,  1949,  Phila- 
delphia, Lippincott;  J.  Bact.,  1949,  57, 
9)  found  0.1%  solutions  of  TPTC 
ideal  for  studying  the  efTects  of  peni- 
cillin and  other  antibiotics  on  the 
dehydrogenase  systems  of  bacteria 
(Huddleson  and  Baltzer,  Science,  1950, 
112,651). 

Reduction  of  colorless  TPTC  to  the 
red,  insoluble,  formazan  occurs  over  a 
wide  range  of  pH  values.  Reducing 
sugars  do  not  interfere  with  the  reac- 
tion in  the  normal  biologic  range  of 
hydrogen  ion  concentrations,  since 
they  do  not  reduce  TPTC  below  pH 
11.0  (Mattson  et  al.,  Science,  1947, 
106,  294).  The  apparent  redox  poten- 
tial of  2,3,5-triphenyltetrazolium  chlo- 
ride is  about  —0.08  volt  (Jerchel  and 
Mohle,  Ber.  deutsch.  chem.  Ges.,  1944, 
77-B,  591).  Thus  the  compound  can 
act  as  an  electron  acceptor  in  reactions 
involving  dehydrogenases  of  pyridine 
nucleotides  (Jensen  et  al..  Science, 
1951,113,65). 

Other  tetrazolium  compounds  and 
several  derivatives  of  2,3,5-triphenyl- 
tetrazolium chloride  have  been  used 
in  the  same  way  as  TPTC.  One  of  the 
most  useful  of  these  is  4,4'-bis(3,5- 
diphenyl-2-tetrazolium)-biphenyl  di- 
chloride  which  is  colorless  in  aqueous 
solution  and  upon  reduction  by  living 
cells  yields  an  insoluble,  violet  or 
mauve  formazan.  Other  agents  which 
may  be  used  similarly  are  the  2,5- 
diphenyl-3(p-iodophenyl),  the  2,3-di- 
(p-iodophenyl)-5  phenj'l,  and  the 
2-  (p-iodophenyl)  -3- (p-nitrophenyl)  -5- 
phenyltetrazolium  compounds. 

Applications  of  TPTC  and  similar 
compounds  outside  the  fields  of  histo- 
physiologic  and  bacteriologic  research 
are  in  the  practical  testing  of  viability 
of  seeds,  grains  and  yeasts;  in  the  dairy 
industry,  for  performing  the  Brucella 
ring  test  (Wood,  Science,  1950,  112, 
86);  and  in  the  field  of  antibiotics,  for 
rapid  microbiologic  assaying  of  prepa- 
rations of  penicillin  and  other  similar 
agents.     Triphenyltetrazolium        chlo- 


TROPAEOLIN  D 


360 


TRYPANOSOMES 


ride  and  some  of  its  derivatives  may 
find  application  also  in  plant  pathology 
as  diagnostic  agents  or  for  detection  of 
various  bacterial  or  fungus  infections 
in  the  tissues  of  plants  (Atkinson  et  al., 
Science,  1950,  111,  385). 

Stock  solutions,  prepared  in  a  satu- 
rated solution  of  NaHCOs  are  stable 
in  the  dark  and  may  be  kept  for  some 
time,  although  freshly  prepared  solu- 
tions are  preferable.  Solutions  should 
be  protected  from  direct  sunlight  or 
other  strong  radiations,  especially  of 
the  shorter  wave-lengths,  (Gierlach 
and  Krebs,  Am.  J.  Roentgen.  &  Radium 
Therap.,  1949,  62,  559).  Similarly 
TPTC  solutions  should  be  shielded 
from  the  radiations  of  radio-active 
substances  (ibid). 

Solutions  of  TPTC  may  be  flooded 
over  living  tissues  of  plants  or  animals 
or  over  cultures  of  bacteria  on  solid 
or  semi-solid  media  for  a  few  minutes 
and  then  removed  (by  decanting  or 
pipetting)  whereupon  the  sites  of  active 
reduction  are  clearly  defined  by  the 
red  deposits  of  insoluble  formazan 
which  is  not  diffusible.  Alternatively, 
the  roots  of  intact  plants  or  the  bases 
of  plant  stems  may  be  immersed  in 
solutions  of  TPTC.  The  compound  is 
readily  absorbed  and  translocated  up- 
ward. Subsequent  longitudinal  sec- 
tioning of  the  plant  reveals  the  sites 
where  the  compound  has  been  reduced 
to  the  formazan.  Stained  material 
can  be  dehydrated  in  alcohol  and  ace- 
tone and  preserved  for  cyto-histologic 
examination.  Bacterial  cultures  on 
agar  or  other  suitable  media  can  be 
dehydrated  similarly  and  used  for 
cytologic  study. 

In  bacteriology,  TPTC  may  be  in- 
corporated in  either  liquid  or  semi- 
solid culture  media.  Relative  reducing 
activity  of  different  organisms  under 
different  conditions  may  be  determined 
by  noting  relative  intensities  of  color 
developed  in  different  tubes.  In  agar 
media,  TPTC  has  been  used  to  dis- 
tinguish variants  of  Salmonella  typhi- 
niurium,  of  S.  sonnei,  of  Escherichia 
coli,  and  of  several  species  of  Brucella 
on  the  basis  of  reducing  activity  as 
evidenced  by  the  color  of  different 
colonies. 

Triphenyltetrazolium  chloride  has 
proved  of  value  in  cancer  studies  also 
and  has  helped  in  elucidation  of  some 
problems  in  cancer  physiologv  (H61- 
scher,  Agnew.  Chem.,  1950,  62,^174). 

The  reaction  involved  in  reduction 
of  the  water-soluble,  diffusible,  color- 
less tetrazolium  salts  to  the  water- 
insoluble,  non-diffusible,  colored  forma- 
zans  is  as  follows: 


N— N- 


\ 


y    \ 


y    \ 


-i-2e+2H+ 


N=N- 

ci- 

2,3,5-Triphenyltetrazolium  Chloride 


"N=N— /^ 
Triphenyl  Formazan 

The  details  of  the  chemistry  of  tetra- 
zolium salts  and  of  their  insoluble 
formazans  have  been  reviewed  by  Ben- 
son (Chem.  Rev.,  1947,  41,  1). 

Tropaeolin  D,  see  Methyl  Orange. 

Tropaeolin  G,  see  Metanil  Yellow. 

Tropaeolin  G  or  OOO  No.  1,  see  Orange  I. 

Tropaeolin  OOO  No.  2,  see  Orange  II. 

Trotter,  see  Hair. 

Trypan  Blue  (CI,  477)— azidine  blue  3B, 
benzamine  blue  3B,  benzo  blue  3B, 
chlorazol  blue  3B,  Congo  blue  3B, 
dianil  blue  H3G,  naphthamine  blue 
3BX,  Niagara  blue  3B — This  acid  dis- 
azo  dye  is  the  most  popular  of  all  Vital 
Stains.  See  also  trypan  blue  capillary 
permeability  test  (e  Silva,  M.  R.,  and 
Dragstedt,  C.  A.,  J.  Pharmac.  and 
Exper.  Therap.,  1941,  73,  405-411). 

Trypan  Red  (CI,  438).  So  named  because 
of  influence  on  Trypanosome  infections 
(G.  irypanon,  anger  +  soma,  body). 
An  acid  dis-azo  dye  much  used  as  a 
vital  stain  but  less  satisfactory  than 
trypan  blue. 

Trypanosomes.  The  following  is  based  upon 
Craig's  account.  Before  examining 
peripheral  blood,  or  cerebrospinal  fluid, 
for  trypanosomes  it  is  advisable  to  con- 
centrate them  by  centrifugation.  They 
can  be  well  seen  in  the  darkfield. 
Smears  of  blood  should  be  made  a  little 
thicker  than  for  malaria  plasmodia  and 
after  being  air  dried  should  be  stained 
immediately.  The  methods  of  Giemsa 
and  Wright  are  preferred  giving  a  little 
more  time  for  the  stains  to  work.  For 
details  of  structure  use  iron  hematoxy- 
lin after  Schaudinn's  fluid  (Craig  p.  49). 
The  South  American  trypanosome, 
T.  cruzi,  is  more  easily  cultured  than 
either  of  the  African  forms,  T.  gam- 
biense  or  T.  rhodesiense.  Reiser's 
medium,  described  fully  by  Craig,  seems 
to  be  the  best.  See  references  supplied 
by  him  (p.  199)  to  culture  in  chick 
embryoes. 

Trypanosomes.  Inclusion  Bodies  are  pro- 
duced in  Trypanosomes  in  vivo  by 
effective  doses  of  antrycide,  dimidium 
bromide  and  Suramin.  Their  forma- 
tion can  also  be  observed  in  response  to 


TRYPANOSOMES 


361 


TUMOR  CELLS 


vital  stains  under  the  microscope  but 
in  this  case  they  are  not  permanent. 
The  determination  of  the  chemical 
nature  of  these  inclusions  by  Ormerod, 
W.  E.,  Brit.  J.  Pharm.  and  Chemo- 
therap.,  1951,  6,  334-341  is  a  fine  example 
of  the  application  of  histochemical 
methods  to  protozoa.  They  contain  ri- 
bonucleic acid  and  protein  and  resemble 
Volutin  granules. 

Trypanosomes.  Media.  Summarized  from 
Q.  M.  Geiman  (Simmons  and  Gentzkow, 
658,  661). 

Brutsaert  and  Henrard's  (A)  6.50  gm. 
NaCl.,  0.14  gm.  KCl,  0.12  gm.  CaCU  + 
aq.  dest.  to  make  1000  cc.  (B)  8.0  gm. 
NaCl,  0.2  gm.  KCl,  0.2  gm.  CaCls,  0.1 
gm.  MgClj,  0.05  gm.  NaHP204,  1  gm. 
NaHCOs,  1  gm.  glucose  +  aq.  dest.  to 
make  1000  cc.  Sterilize  both  by  filtra- 
tion and  distribute  in  culture  tubes 
2  cc.  A  +  2.5  cc.  B.  Add  2  cc.  citrated 
human  blood  (1%  citrate)  and  incubate 
at  37°C.  24  hrs.  to  prove  sterility. 
Keep  in  refrigerator  useful  up  to  2 
weeks.  Into  a  syringe  containing  1  cc. 
1%  aq.  sodium  polyanethol  sulfonate 
draw  up  5  cc.  patient's  blood.  Dis- 
tribute 0.5  cc.  to  each  of  10  culture 
tubes,  incubate  25-28°C.  Examine  mi- 
croscopically for  trypanosomes  after 
10-20  days. 

Kelser's.  Dissolve  2.5  gm.  Bacto- 
beef  (Difco)  in  500  cc.  aq.  dest.  on 
water  bath  55°C.,  1  hr.  Add  12.5  gm. 
Bacto  peptone  (Difco)  and  3.5  gm. 
sodium  chloride  by  placing  flask  in  boil- 
ing water  5  min.  Clarify  by  filtering 
through  cotton  and  make  pH  7  with 
IN  sodium  hydroxide.  Determine  vol- 
ume and  add  1%  Bacto-agar.  Dissolve 
and  distribute  5  cc.  per  test  tube  or 
10  cc.  per  small  flask.  Autoclave  12 
lbs.,  30  min.  Store  for  latter  addition 
dextrose  and  blood  or  for  immediate 
use  add  5%  of  1%  aq.  dextrose  (0.25  cc. 
per  tube  or  0.5  cc.  per  flask)  and  5% 
fresh  sterile  defibrinated  guinea  pig 
blood.  After  thorough  mixing  slant 
with  short  slant  or  deep  butt.  Use 
sterile  rubber  corks  to  prevent  evapora- 
tion. Prove  sterility  by  incubation. 
Inoculate  by  adding  organisms  to  slant 
or  water  of  condensation.  On  incuba- 
tion at  room  temperature  (22-25°C.) 
growth  becomes  apparent  in  approxi- 
mately 1  week.  Subculture  at  6-8 
week  intervals. 

Trypsin,  a  gelatin  plate  method  as  described 
under  Pepsin  but  slightly  modified  is 
recommended. 

Tryptagar,  see  Bacteria  Media. 

Tryptophane  Reaction.  The  procedure  of 
Scrra  and  Lopes  is  specified  as  follows 
by  Serra,  J.  A.,  Stain  Techn.,  1946,  21, 


5-18:  Prepare  tissue  as  described  under 
Ninhydrin  Reaction. 

"1.  Harden  the  fixed  pieces  in  10% 
formaldehyde  for  at  least  1-5  hours  (an 
unnecessary  step  if  a  fixative  with  for- 
malin has  been  employed);  then  wash 
well. 

"2.  Immerse  for  3-5  seconds  in  an 
aqueous  solution  of  sodium  silicate 
(a  =  1.1).  When  the  materials  are 
sufficiently  hardened  this  step  may  also 
be  omitted;  it  is  recommended,  how- 
ever, that  the  coloration  should  be  tried 
both  with  and  without  it. 

"3.  Immediately  afterwards,  immerse 
the  pieces  in  the  Voisenet  reagent  for 
10-15  minutes,  in  a  small  glass  stoppered 
bottle.  This  reagent  is  composed  of 
10  ml.  concentrated  HCl  to  which  is 
added,  with  a  thorough  stirring,  one 
drop  of  2%  aqueous  formol  and  one  drop 
of  0.5%  aqueous  NaNOj.  The  reagent 
is  prepared  freshly  every  day  and  the 
nitrite  solution  must  also  be  freshly 
made . 

"4.  Mount  directly  in  glycerin  and 
observe,  with  squeezing,  if  necessary. 
As  the  coloration  fades,  it  is  necessary 
to  observe  the  preparations  on  the  same 
day. 

"The  reaction  is  given  by  indolic 
compounds,  and  in  proteins  it  is  specific 
for  tryptophane,  which  reacts  even 
when  bound.  The  localization  of  the 
reaction  seems  to  be  satisfactory  and 
the  sensitivity  is  sufficient  for  it  to  be 
used  in  cytophysiological  work."  See 
Romieu  Reaction. 
Tubercle  Bacilli.  Stain  by  Carbol  Fuchsin, 
see  Acid  Fast  Bacilli.  See  Concentra- 
tion method  for  sputum.  Fluorescence 
with  auramine  has  been  described 
(Hagemann,  P.  K.  H.,  Miinch.  med. 
Woch.,  1938,  85,  1066).  Fix  smears  by 
flame  and  stain  15  min.  in  1:1000  aq. 
auramine  (Bayer)  containing  5%  pheno- 
lum  liquefactum  (liquid  carbolic  acid). 
Wash  in  tap  water.  Decolorize  in 
ethanol  100  cc. ;  HCl  cone,  4  cc. ;  sodium 
chloride,  4  gm.  renewing  solution  after 
li  min.  Wash  thoroughly  in  tap  water. 
Examine  without  cover  glass  under 
fluorescence  microscope  using  apochro- 
matic  dry  objective  and  3  compensating 
ocular  (X  about  180).  For  visible  and 
red  rays  employ  3.5  mm.  "Uvet"  lens 
and  2%  aq.  copper  sulphate.  Bacilli, 
golden  yellow  rods  in  violet  fluorescent 
background.  Kaiserling,  C.  Deutsche 
Med.  Wochenschr.,  1939,  64,  1354,  has 
described  differences  in  fluorescence  of 
human  bovine  tubercle  bacilli.  See 
Coproporphyrin  and  Sputum. 
Tumor  Cells.  All  of  these  are  not  cancer 
cells  but  see  Cancer  for  technique. 


TUNGSTIC  ACID 


362 


ULTRACENTRIFUGES 


Tungstic  Acid,  a  stable  soltiuon  (Abraham- 
son,  E.  M.,  Tech.   Bull.,  1940,  1,  75). 

Turnbull  Blue  reaction  for  iron.  Same  as 
Berlin  blue  except  use  K  ferricyanide 
and  HCl. 

Turpentine.  Not  advised  as  clearing  agent. 
See  test  for  Alcohol  absolute. 

Typhus  Fever  rickettsiae  in  lungs  of  mice. 
(Nyka,  W.,  J.  Path.  &  Bact.,  1945,  52, 
317-324).  Fix  in  10%  neutral  formalin. 
Stain  sections  in  1:10,000  aq.  methyl 
violet  30  min.  to  1  hr.  Differentiate  in 
acetic  acid  (2  drops  glacial  acetic  in 
100  cc.  aq.  dest.)  till  cytoplasm  is  de- 
colorized. Counterstain  in  1 :  10,000  aq. 
metanil  yellow  for  few  seconds.  Dehy- 
drate in  acetone,  clear  in  xylol  and 
mount  in  neutral  medium  (say  immer- 
sion oil).    Rickettsiae,  violet. 

Tyrian  Purple.  The  ancients  prized  this 
dye  very  highly.  Said  to  have  been 
discovered  when  a  sheep  dog  of  Hercules 
bit  into  a  shellfish  and  stained  his  mouth 
bright  red,  this  wonderful  dye  was  first 
produced  for  local  use  in  Crete  about 
B.C.,  1600,  and  was  later  distributed  by 
the  Phoenicians  bringing  business  to 
Tyre;  hence  the  name  Tyrian  purple. 
Pliny  has  given  a  detailed  description 
of  its  preparation.  Factories  for  ex- 
traction of  the  dye  from  Murex  trunclus 
were  established  by  the  Phoenicians  at 
many  points  in  the  Mediterranean 
basin,  chiefly  at  Tyre,  Tarentum  and 
Palermo,  and  trading  points  at  Cadiz, 
and  in  present  day  Morocco.  Tyrian 
purple  became  the  "royal  color"  em- 

Eloyed  by  royalty  in  Persia,  Babylon, 
ledia  and  Syria.  The  robes  of  Greek 
generals  were  purple,  likewise  those  of 
their  Gods.  Jewish  tabernacle  decora- 
tions were  colored  by  a  bluish  type  of 
Tyrian  purple.  The  sails  of  Cleo- 
patra's barge  were  colored  purple.  Ac- 
cording to  a  decree  by  Caesar  Augustus 
none  in  the  Roman  Empire  but  the  Em- 
peror and  his  household  could  wear 
purple  (Leggett,  W.  F.,  Ancient  and 
Medieval  Dyes.  Brooklyn:  Chemical 
Publishing  Co.,  Inc.,  1944,  95  pp.). 

Tyrode  solution.  NaCl,  0.8  gm.;  KCl, 
0.02  gm.;  CaClo,  0.02  gm.;  MgCh,  0.01 
gm.;  NaHjPOi,  0.005  gm.;  NaHCO,, 
0.1  gm.  (giving  pH  about  7.5-7.8) ; 
dextrose,  0.1  gm.;  aq.  dest.,  100  cc. 
Solution  cannot  be  boiled  but  can  be 
passed  through  a  Berkfeld  filter. 

Tyrosine  Reaction.  The  procedure  of  Serra 
and  Lopes  which  gives  better  results 
than  the  Millon  Reaction  is  specified  as 
follows  by  Serra,  J.  A.,  Stain  Techn., 
1946,  21,  5-18:  Prepare  tissue  as  de- 
scribed under  Ninhydrin  Reaction. 

"1.  Immerse  the  objects  for  30  min- 
utes in  a  few  milliliters  of  the  mercuric 
solution    (composition:  HgS04,  7.5  g.; 


HgClz,  5.5  g. ;  NajSOi,  7.0  g. ;— dissolved 
in  85  ml.  of  distilled  water  to  which 
12.5  g.  of  concentrated  H2S04  is  added; 
after  dissolving  dilute  to  100  ml.  with 
distilled  water).  Perform  the  treat- 
ment in  a  small  glass  stoppered  bottle, 
placed  in  a  water  bath  which  is  main- 
tained at  60°C. 

"2.  After  the  30-minute  treatment, 
cool  the  bottle  in  running  water  and 
allow  to  stand  at  room  temperature  for 
10  minutes. 

"3.  Dilute  the  mercuric  solution  in 
the  bottle,  by  addition  of  an  equal  vol- 
ume of  distilled  water. 

"4.  Develop  the  color,  adding  now 
some  drops  of  a  freshly-prepared  1  M 
solution  of  sodium  nitrite  (6.9  g.  NaN02 
in  100  ml.  of  water). 

"The  coloration  attains  its  maximum 
in  3  minutes  and  lasts  for  some  months, 
though  it  fades  gradually  with  time. 
The  materials  are  mounted  and  ob- 
served in  pure  glycerin,  where  they  can 
be  squeezed  or  sc^uashed,  if  necessary. 

"Tne  reaction  is  principally  due  to 
the  presence  of  tyrosine  in  the  protein 
molecule,  and  is  also  produced  by  other 
phenolic  compounds.  The  method  here 
described  gives  with  tryptophane  only 
a  transient  coloration,  which  lasts  no 
more  than  a  few  minutes;  it  is  hoped, 
therefore,  that  by  this  procedure  this 
histochemical  test  reveals  only  the  tyro- 
sine in  the  proteins." 
Turnick,  see  Aceto-Orcein-Fast  Green. 
Ultracentrifuges — Written  by  H.  W.  Beans, 
Dept.  of  Zoology,  State  University  of 
Iowa,  Iowa  City.  September  27,  1951 — 
Few  instruments  are  more  essential  to 
certain  phases  of  biological  and  medical 
laboratory  analj^sis  and  research  than 
the  centrifuge.  However,  because  of 
the  relatively  low  centrifugal  force 
generated  by  this  instrument,  its  use- 
fulness is  limited  to  the  displacement 
of  materials  within  living  cells  of  rela- 
tively low  viscosity  and  to  biological 
materials  within  the  test  tube  that 
differ  greatly  in  their  relative  densi- 
ties. Recent  improvement  of  the  elec- 
trically driven  laboratory  type  centri- 
fuge has  however,  resulted  in  machines 
capable  of  generating  forces  of  the  order 
of  10,000  to  30,000  times  gravity.  It 
was  this  type  centrifuge  that  Bensley 
and  Hoerr  used  to  separate  and  analyze 
mitochondria  (Anat.  Rec,  1934,  60, 
251),  a  technique  which  paved  the  way 
for  the  important  discovery  that  mito- 
chondria are  the  carriers  of  all  the  im- 
portant oxidative  enzymes  (Green,  D. 
E.,  Scientific  American,  1949,  181,  48). 

The  development  of  the  ultracentri- 
fuge  has  provided  a  tool  for  the  study 
of   the   components   of   living   cells   of 


ULTRACENTRIFUGES 


363 


ULTRACENTRIFUGES 


high  viscosity,  as  well  as  certain  of  their 
molecular  components  after  disruption 
of  the  cell.  In  fact  it  was  the  latter 
that  led  Svedberg  and  his  associates 
to  experiment  with  machines  to  de- 
velop higher  centrifugal  force,  optical 
methods  of  recording  the  behavior  of 
mixtures  during  rotation,  and  suitable 
mathematical  formula  for  interpreting 
the  molecular  weights  of  proteins  (Sved- 
berg, T.  and  K.  C.  Pedersen,  The 
Ultracentrifuge.  Oxford  Univ.  Press 
1940).  Svedberg's  many  years  of  ex- 
perimentation culminated  in  an  oil- 
turbine  driven  rotor  surrounded  by  an 
atmosphere  of  hydrogen  to  reduce 
heating  and  hence,  convection  which 
is  always  a  troublesome  problem  in  the 
high  speed  centrifuging  of  mixtures. 
The  safe  operating  speed  of  the  Sved- 
berg ultracentrifuge  is  said  to  be  about 
67,000  R.P.M.,  producing  about  350,000 
times  gravity.  By  means  of  this  instru- 
ment Svedberg  and  his  associates  have 
demonstrated  that  the  protein  molecule 
is  relatively  large.  In  addition,  they 
have  made  an  important  contribution 
to  biology  by  determining  the  molecular 
weights  of  many  of  them. 

Because  the  oil-driven  ultracentri- 
fuge is  relatively  complicated  and 
costly,  only  9  or  10  of  them  have  been 
made  (Gay,  G.  W.,  Scientific  American 
1951,  184,  43).  In  1930,  J.  W.  Beams 
developed  the  air-turbine  ultracentri- 
fuge of  Henriot  and  Huguenard  to  use 
in  connection  with  some  experiments 
in  physics  (J.  Appl.  Phys.,  1937,  8, 
795).  This  instrument  consists  of  a 
cone  shaped  rotor  which  is  supported 
and  driven  bj'^  air  under  pressure  from 
properly  directed  jets.  It  is  relatively 
simple  (costs  less  than  $100.00)  and  has 
proven  most  useful  for  histological 
and  cytological  studies.  Displacement 
of  materials  such  as  Golgi  apparatus, 
mitochondria,  Nissl  bodies,  neuro- 
fibrillae,  centrosomes,  chromosomes, 
spindles,  acrosomes,  plastids,  nuclei 
and  nuclear  components,  intracellular 
virus  bodies,  membrane  materials, 
erythrocyte  components,  secretion 
products,  vitamine  C  granules,  organizer 
substances,  enzymes,  bacteriophage, 
and  sarcoma  virus  have  been  observed. 
In  addition,  it  has  been  successfully 
used  to  study  polarity  in  both  plants 
and  animals  as  well  as  the  relative 
viscosity  of  certain  cancer  cells.  Thus, 
by  use  of  this  method,  information  has 
been  gained  concerning  both  the  struc- 
ture and  function  of  many  cellular  com- 
ponents and  inclusions  both  inside  and 
outside  the  cell. 

E.  N.  Harvey  has  adapted  his  centri- 
fuge-microscope  principle   to   the   air- 


turbine  rotor  thus  making  possible  the 
direct  observation  of  cells  in  a  centrif- 
ugal field  of  100,000  to  250,000  times 
gravity  (Biol.  Bull.    1934,  66,  48). 

While  the  air-turbine  ultracentrifuge 
above  described  has  proved  satisfac- 
tory for  the  study  of  many  materials 
within  cells,  it  is  not  so  suitable  for 
the  separation  of  colloidal  solutions 
within  a  test  tube.  This  is  because  of 
the  convection  induced  by  the  slight 
heating  of  the  rotor  as  it  spins  in  air  at 
atmospheric  pressure.  This  difficulty 
has  been  overcome  by  the  development 
of  the  air-turbine  vacuum-tj^pe  ultra- 
centrifuge. This  machine  consists  of 
a  large  rotor  (4  to  7  inch)  situated  in- 
side a  vacuum  tight  chamber  which 
is  driven  and  supported  by  an  air- 
turbine  of  the  type  described  above, 
located  outside  and  vertically  above  the 
vacuum  tight  chamber.  The  turbine 
and  rotor  are  connected  by  a  flexible 
shaft  which  enters  the  vacuum  chamber 
through  a  vacuum  tight  oil  gland.  By 
this  means  a  convection  free  centrifugal 
field  is  established,  the  intensity  of 
which  is  limited  only  by  the  bursting 
strength  of  the  rotor  spining  the  vac- 
uum chamber.  (Beams,  J.  W.,  J. 
Appl.  Phys.,  1937,  8,  795;  Rev.  Mod. 
Phys.,  1938,  10,  245;  Bisco,  Pickels  and 
Wyckoff,  J.  Exp.  Med.,   1936,  64,  39). 

The  development  of  this  compara- 
tively inexpensive  centrifuge  has  made 
available  to  many  biophysical  and  bio- 
chemical laboratories  an  instrument 
comparable  to,  if  not  superior  to,  the 
oil-turbine  ultracentrifuge  of  Sved- 
berg. With  it  investigators  have  con- 
centrated, separated,  and  determined 
the  molecular  weights  of  substances 
such  as  many  different  types  of  virus, 
antibodies,  bacteriophage,  cancer 
agents,  hormones,  and  various  proto- 
plasmic constituents. 

New  uses  for  the  ultracentrifuge  in 
biological  research  are  continually  ap- 
pearing. For  example,  press  reports 
state  that  Gofman  and  associates  have 
separated  cholesterol  from  human  blood 
by  use  of  this  machine.  Their  studies 
indicate  that  it  may  be  possible  to  antici- 
pate certain  circulatory  diseases,  such  as 
high  blood  pressure  and  arteriosclerosis, 
by  this  technique.  In  other  words, 
eventually  we  may  have  available 
"diagnostic  ultracentrifuges". 

The  air-turbine  tubular-type  ultra- 
centrifuge has  been  used  successfully 
to  separate  uranium  235  from  uranium 
238  (Smyth,  H.  D.,  Atomic  Energy  for 
Military  Purpo.ses,  Princeton  Univ. 
Press,  1945).  This  machine  was  first 
described  by  J.  W.  Beams,  and  has 
become  a  subject  of  classified  military 


ULTRAFILTER 


364 


ULTRAVIOLET  MICROSCOPE 


research.  Efforts  to  improve  the  ultra- 
centrifuge  are  continuing.  Beams  J. 
Wash.  Acad.  Sci.,  1947,  37,  221  has  de- 
scribed a  magnetically  supported  and 
magnetically  driven  ultracentrifuge. 
This  machine  in  experimental  tests 
obtained  rotational  speeds  of  48  million 
R.  P.  M.,  thus  producing  a  centrifugal 
force  of  500  million  times  gravity. 
Further  development  of  the  ultra- 
centrifuge  will  undoubtedly  lead  to  a 
wider  application  and  more  refined  tech- 
niques for  researchers  interested  in 
problems  involving  sedimentation  and 
fractionation  of  mixtures.  See  Cen- 
trifugation. 

Ultrafilter.  For  filtering  small  volumes  of 
fluid  the  ultrafilter  of  Johnson,  H.  C. 
and  Kirk,  P.  L.,  Mikrochemie  ver. 
Mikrochim,  Acta,  1940,  28,  254-257  is 
recommended  by  Glick,  p.  487.  See 
easily  constructed  apparatus  described 
by  Clark,  L.  C,  J.  Lab.  &  Clin.  Med., 
1951,37,481-484. 

Ultramarine  Green,  an  exogenous  pigment, 
a  sodium  aluminum  silicate  and  sulfide 
(Lillie,  p.  138). 

Ultramicroscope,  see  Darkfield. 

Ultrasonic  Vibrator.  Type  used  to  test 
effect  of  ultrasonics  on  blood  elements 
by  Morrow,  P.  L.,  Bierman,  H.  R.  and 
Jenkins,  R.,  J.  Nat.  Cancer  Inst.,  1950, 
10,  843-859. 

Ultrasonics.  The  division  of  acoustics  com- 
prising sound  frequencies  beyond  the 
limits  of  perception  by  the  human  ear. 
Radiation  of  this  sort  can  be  very  de- 
structive to  living  cells.  The  tech- 
nique and  results  are  well  described  by 
Gregg,  E.  C,  Jr.  in  Glasser's  Medical 
Physics,  1591-1596. 

Ultraviolet  Microscope  and  Color  Transla- 
tion Process.  A  microscope  using 
ultraviolet  radiation  instead  of  or- 
dinary light  to  form  an  image  is  some- 
what of  a  misnomer,  for  there  is  nothing 
to  be  seen.  One  must  expose  a  photo- 
graphic plate  sensitive  to  the  ultra- 
violet rays  to  record  the  image.  If  the 
ultraviolet  is  used  only  to  excite 
fluorescence  then  of  course  a  visible 
image  is  produced  as  has  been  described. 
We  are  here  concerned  with  images 
which  are  not  visible. 

In  accordance  with  the  law,  already 
mentioned,  R  =  X/2  N.  A.  increased 
resolving  power  can  be  achieved  by 
employing  the  shorter  wave  lengths 
of  the  spectrum.  Considerable  im- 
provement of  the  image  is  obtained  by 
using  monochromatic  blue  light  with 
the  ordinary  "achromatic"  type  of 
objective;  but  with  highly  color  cor- 
rected "apochromatic"  lenses  there  is 
little  to  be  gained  by  so  doing.    The 


ultraviolet  region  of  the  spectrum  of- 
fers wave  lengths  as  short  as  0.15  m, 
most  of  which  are  produced  very  con- 
veniently by  modern  mercury  and 
hydrogen  arcs.  Therefore,  photog- 
raphy in  the  ultraviolet  should  double 
or  even  triple  the  resolving  power  of 
a  lens  if  everything  else  is  held  constant. 

Since  ordinary  optical  glass  is  nearly 
opaque  to  ultraviolet  light  lens  makers 
must  use  natural  quartz,  or  fluorite, 
components  throughout  the  system. 
This  puts  rather  severe  limitations  on 
the  lens  designer  as  it  narrows  his 
range  of  possible  corrections.  For 
example,  an  all-quartz  objective  must 
be  used  with  ultraviolet  radiation  of  a 
specified  wave  length  in  order  to  get  a 
good  image  (Lavin,  G.  I.,  Rev.  Sci. 
Inst.,  1943,  14,  375-376).  Focus  is 
obtained  by  trial  and  error.  This 
makes  observation  somewhat  labori- 
ous and  almost  impracticable  for  living 
material.  Ultraviolet  microscopy  is, 
however,  used  with  considerable  success 
in  the  study  of  fixed,  unstained  cells 
because  proteins  and  nucleic  acids 
show  specific  absorbtion  at  0.280  m 
and  0.260  ^  respectively  (Caspersson, 
L,  J.  Roy.  Micr.  Soc.,  1940,  60,  8-25). 
Thus  chromosomes,  rich  in  nucleic  acid, 
reveal  themselves  in  strong  contrast 
and  high  resolution  (Ludford  et  al., 
J.  Roy.  Micr.  Soc,  1948,  68,  1-9). 
After  proper  calibration  one  can  use 
the  blackening  of  a  plate,  or  ultraviolet 
photocell  currents,  to  measure  the 
concentrations  of  absorbing  materials 
in  various  structures  under  examina- 
tion. 

A  remarkable  advance  in  ultraviolet 
microscopy  has  recently  come  about 
with  the  development  of  the  color 
translation  rnicroscope  (Land,  E.  H.  et 
al.,  Science,  1949,  109,  371-374),  for 
which  an  objective  lens  corrected  for 
three  wave  lengths  in  the  ultraviolet 
has  been  designed.  The  operator  takes 
a  photograph  at  each  wave  length. 
These  negatives  show  somewhat  dif- 
ferent details  because  the  materila 
being  studied  has  different  ultraviolet 
absorbing  powers  at  these  three  wave 
lengths.  The  negatives  are  then 
treated,  as  in  the  conventional  process 
for  visual  color  photography,  by  as- 
signing to  each  negative  one  of  the 
primary  colors.  The  result  is  a  color 
print  representing  in  an  arbitrary  way 
something  which  is  invisible  to  the  eye. 
Thus,  changes  in  the  ultraviolet  ab- 
sorption spectra  of  the  materials  are 
revealed  by  changes  in  the  apparent 
colors  of  details  in  the  print;  hence  the 
term  "color  translation." 


ULTRAVIOLET  MICROSPECTRO- 
PHOTOMETRY 


365 


URATES  AND  URIC  ACID 


Ultraviolet     Microspectrophotometry      has 

been  developed  by  T.  Caspersson  (Cell 
Growth  and  Cell  Function.  A  Cyto- 
chemical  Study.  W.  W.  Norton  and 
Company,  New  York,  1950)  and  per- 
mits the  determination  of  absorption 
curves  of  light  from  240  to  350  milli- 
microns w^avelength  in  minute  parts 
of  cells.  Information  on  steroids,  nu- 
cleic acids,  proteins  and  iodides  can 
be  obtained.  Equipment  is  being  de- 
veloped now  in  several  laboratories 
to  extend  the  range  of  measurements 
from  230  niM  to  the  infra  red  and  the 
usefulness  of  these  types  of  technics. 

Ultraviolet  Photomicrography  has  certain 
advantages  over  visible  light  photo- 
micrography because  the  resolving 
power  of  the  former  is  greater  in  conse- 
quence of  its  shorter  wave  length,  and 
as  pointed  out  by  Wyckoff  and  Louw 
(R.  \V.  G.  and  A.  L.,  J.  Exper.  Med., 
1931,  54,  449-451),  because  some  pro- 
teins absorb  ultraviolet  more  strongly 
tlian  others,  details  can  be  brought  out 
with  it  not  revealed  by  visible  light. 
This  they  demonstrate  by  experiments 
w^ith  B.  subtilis.  It  was  then  found 
that  the  substances  that  strongly  ab- 
sorb ultraviolet  light  give  a  positive 
Feulgen  reaction  (Wyckoff,  R.  W.  G., 
Ebeling,  H.  H.,  and  Ter  Louw,  A.  L., 
J.  Morph.,  1932,  53,  189-199)  and  that 
they  also  yield  conspicuous  mineral 
ash  on  microincineration  (Scott,  G.  H., 
Science,  1932,  76,  148-150)— an  inter- 
esting superposition  of  three  technical 
methods.  The  work  of  the  Swedish 
group  is  summarized  in  Caspersson's 
book  (Cell  Growth  and  Cell  Function. 
A  Cytochemical  Study,  W.  W.  Norton 
and  Co.,  New  York,  1950).  Two  avail- 
able articles  in  the  American  literature 
are  by  Stowell  (Arch.  Path.  1948,  46, 
164-178  and  Cancer,  1949,  2,  121-131). 
The  use  of  other  newer  types  of  lenses 
should  be  mentioned.  See  (Mellors, 
R.  C,  Berger,  R.  E.,  and  Streim,  H. 
G.,  Science,  1950,  111,  627-632  for  refer- 
ences). 

Union  Green  B,  see  Janus  Green  B.  Ultro- 
pak  illuminator  of  Leitz  is  helpful  for 
micrological  work.  The  Epi  Condenser 
W  of  Zeiss  is  similar.  See  Chambers, 
R.  W.  and  Kopac,  M.  J.  in  McClung's 
Microscopical  Technique,  1950,  p.  508. 

Unna's  Orcein  method  for  elastic  fibers. 
This  is  simple  and  direct.  Stain  paraf- 
fin sections,  after  almost  any  fixation, 
in:  orcein,  1  gm.;  absolute  alcohol, 
100  CO. ;  and  hydrochloric  acid,  1  cc.  for 
several  hours.  Wash  in  70%  alcohol 
and  sharpen  the  deep  brown  coloration 
of  the  elastic  fibers  by  removing  excess 
stain   from   background   by   destaining 


under  the  microscope  in  95%  alcohol 
plus  a  trace  of  hydrochloric  acid. 
Wash  in  95%,  dehydrate,  clear  and 
mount.  If  desired  counterstain  with 
methylene  blue. 

Dahlgren  (McClung,  p.  425)  advises 
a  modification  of  this  stain  for  Muscle. 
After  sublimate  fixation  stain  sections 
24  hrs.  in  Wasserblau,  0.25  gm.;  abso- 
lute alcohol,  60  cc;  orcein,  1  gm.; 
glycerin,  10  cc;  water,  30  cc.  Wash 
in  70%  alcohol,  dehydrate,  clear  and 
mount.  Muscle,  purple;  coUagenic 
fibers,  blue;  elastic  fibers,  red.  It  is 
important  in  doubtful  cases  to  compare 
with  similar  tissue  colored  by  other 
specific  stains  before  identification  of 
muscle  is  assured. 

Uranin,  sodium  salt  of  Fluorescein. 

Uranium.  Salts  injected  into  tissues  can 
be  demonstrated  by  (1)  a  method  of 
Schneider  (G.,  Skand.  Arch.  Physiol., 
1903,  14,  383-389).  Fix  in :  5%  aq. 
potassium  ferrocyanide,  50  cc,  sat.  aq. 
picric  acid,  50  cc. ;  hydrochloric  acid, 
10  cc.  Wash  in  4%  aq.  hydrochloric 
acid  and  then  in  80%  alcohol  acidified 
with  hydrochloric  acid.  Imbed  and 
cut.  The  uranium  ferrocyanide  of 
potassium  is  detected  by  its  dark  brown 
color  (Lison,  p.  103).  (2)  the  Prussian 
blue  reaction  for  iron  as  employed  by 
Gerard  and  Cordier  (P.  and  R.,  Arch. 
Biol.,  1932,  43,  367-413).  According  to 
Lison  this  method  is  highly  specific. 
The  possibility  of  detecting  uranium 
salts  in  incinerated  sections  by  their 
fluorescent  properties  in  ultraviolet 
light  has  been  described  (Policard,  A. 
and  Okkels,  H.,  Abderhalden's  Handb. 
d.  biol.  Arbeitsmethoden,  1931,  5,  1815). 
Gordon  H.  Scott  has  been  successful 
when  large  amounts  are  present  but 
has  called  attention  to  complicating 
factors  (McClung's  Microscopical  Tech- 
nique, p.  660). 

Urates  and  Uric  Acid.  A  modification  of 
Courmont-Andr^'s  method  is  suggested. 
Neutralize  some  formalin  with  calcium 
carbonate.  Fix  tissue  in  equal  parts 
1%  aq.  silver  nitrate  and  4.4%  neutral 
formalin  in  darkness,  12-24  hrs.  Wash 
in  several  changes  aq.  dest.,  24  hrs. 
Imbed  in  paraffin.  Stain  sections 
hematum  10  min.;  running  tap  water 
i-1  hr. ;  1%  aq.  orange  G  or  eosin  ^1 
hr.  Wash  quickly  in  aq.  dest.  Place 
in  0.5%  aq.  phosphomolybdic  acid,  rinse 
in  aq.  dest.  and  color  in  0.12%  aq.  light 
green,  1-10  min.  Differentiate  quickly 
in  96%  alcohol,  dehydrate  in  iso-amyl- 
alcohol,  clear  in  xylol  and  mount  in 
balsam.  Urates,  black;  chromatin, 
blue;  protoplasmic  inclusions  red  to 
orange    and    collagenic    fibers,    green. 


UREA 


366 


URINE 


Employed  by  HoUande  for  bacteriocytes 
of  Periplaneta  orientalis  L  (HoUande, 
A.  C,  Bull.  d'Histol.  Appl.,  1931,  8, 
176-178). 

Urea.  Many  histochemical  techniques  have 
been  proposed.  Leschke  (E.,  Zeit. 
Klin.  Med.,  1915,  81,  14-35)  fixes  in 
half  sat.  sol.  mercuric  nitrate  in  1% 
nitric  acid  for  1  day,  then  washes  in 
frequently  changed  aq.  dest.,  imbeds 
in  paraffin  and  treats  the  sections  with 
sat.  aq.  hydrogen  sulphide  staining 
nuclei  with  hemalum.  Stiibel  (H., 
Anat.  Anz.,  1921,  54,  237-239)  fixes  small 
pieces  in  6%  xanthydrol  in  glacial  acetic 
acid  6-12  hrs.,  imbeds  in  paraffin,  stains 
sections  by  ordinary  methods  and 
examines  by  polarizing  microscope. 
Oliver  (J.,  J.  Exper.  Med.,  1921,  33, 
177-186)  employs  instead  a  solution 
containing  2  gm.  xanthydrol,  10  cc. 
methyl  alcohol  and  20  cc.  glacial  acetic 
acid.  Lison  (p.  169)  criticizes  these 
methods  severely. 

It  may  be  necessary  to  resort  to  the 
capillary  tube  colorimetric  technique 
of  Walker,  A.  M.  and  Hudson,  C.  L., 
Am.  J.  Physiol.,  1937,  118,  153-166, 
or  to  the  titrimetric  method  of  Kinsey, 
V.  E.,  and  Robison,  P.,  J.  Biol.  Chem., 
1946,   162,  325-331. 

Urease.  A  method  for  determining  the 
distribution  of  urease  in  the  gastric 
mucous  membrane  (pylorus  and  fundus) 
of  the  dog  has  been  d.escribed  and  used 
by  Linderstr0m-Lang  and  Ohlsen  (K. 
and  A.  S.,  Enzymologia,  1936-37,  1, 
92-95).  Cylinders  of  tissue  (2.5  mm. 
in  diameter)  are  cut  vertical  to  the 
surface  from  frozen  mucosa.  Cross 
frozen  sections  (25  microns  thick) 
of  the  cylinders  are  then  tested  for 
urease.  This  is  concentrated  in  the 
surface  layers  containing  cells  stainable 
with  mucicarmine.  Chief  cells  in  the 
bases  of  the  glands  are  inactive  in  both 
fundus  and  pylorus  and  the  authors 
think  it  very  unlikely  that  the  parietal 
cells  contain  urease. 

For  help  in  the  problem  of  adapting 
methods  for  urea  to  urease  see  Glick, 
p.  287. 

Uremia.  Microscopic  demonstration  of 
uremia  by  precipitation  of  xanthydrol 
urea  in  tissue.  A  modification  of 
Oestreicher's  original  method  is  pro- 
vided by  Brown,  A.  F.  and  Krajian, 
A.  A.,  Arch.  Path.,  1936,  21,  96-99. 

Cut  blocks  of  tissue  2-3  mm.  thick. 
Immerse  in  fresh  xanthydrol  solution 
(xanthydrol,  5  gm.,  glacial  acetic  acid 
100  cc.)  at  80°C.  for  2  hrs.  Wash  in 
running  water,  5  mm.  Fix  in  1  part 
formaldehyde  U.S. P.  and  10  parts  aq. 
dest.  at  70°C.  for  15  min.     Wash  in  tap 


water  and  cut  5-10/x  frozen  sections. 
Transfer  them  to  slide  and  pour  on 
several  drops  "dehydrated  alcohol" 
(presumably  abs.  ethyl  ale.)  from  a  drop 
bottle  and  blot.  Repeat.  Cover  by 
dipping  in  thin  pyroxylin  (celloidin) 
contained  in  wide  mouthed  bottle. 
Fix  film  of  pyroxylin  to  slide  by  blowing 
breath  over  section  and  stain  in  1%  aq. 
eosin  for  several  minutes.  Wash  in 
water,  dehydrate  in  3  changes  dehy- 
drated alcohol,  place  in  carbol-xylene, 
clear  in  2  changes  pure  xylene  1  min. 
each  and  mount  in  dammar.  Xanthy- 
drol urea  crystals  appear  as  closely 
packed  clusters  of  yellow-green  needles. 

Uric  Acid.  Micro  colorimetric  method, 
Borsook,  H.,  J.  Biol.  Chem.,  1935, 
110,  481-493.     See  Urates. 

Urinary  Casts,  staining  with  methyl  blue 
picric  acid.  To  sediment  from  centri- 
luged  urine  add  1  drop  0.5%  aq.  eosin. 
Mix  by  side  to  side  shaking.  After  1-2 
min.  add  2  drops  from  1  cc.  1%  aq. 
methyl  blue  +  10  cc.  sat.  aq.  picric  acid 
and  again  mix.  Color  of  sediment 
should  be  distinctly  bluish  green.  If  it 
is  reddish  brown  add  more  methjd  blue- 
picric  acid.  Transfer  to  slide  cover 
and  examine.  The  casts  should  be  dis- 
tinct blue  but  not  too  dark.  Numerous 
details  are  brought  out  (Behre,  J.  A. 
and  Muhlberg,  W.,  J.  Lab.  &  Clin.  Med., 
1936-37,  22,  853-856).  See  the  author's 
figures. 

Urinary  Sediments.  The  following  outline 
is  from  Stitt  (pp.  707-713)  much  ab- 
breviated. Concentrate  sediment  by 
centrifuging  15  cc.  fresh  urine  1500 
r.p.m.  5  min.  but  not  longer.  Decant 
supernatant  urine.  Suspend  sediment 
in  2  cc.  urine  as  is  the  practice  in  the 
Naval  Medical  School.  By  always  using 
these  amounts  quantitative  differences 
from  normal  in  individual  sediments 
become  apparent.  Examine  for  epi- 
thelial cells,  leucocytes,  erythrocytes, 
casts,  crystalline  materials,  bacteria 
and  so  forth. 

Urinary  Tract  Smears,  see  Papanicolaou 
Techniques. 

Urine.  For  microscopic  study  sediments 
are  divided  into  classes. 

Details  with  helpful  diagrams  are  sup- 
plied by  C.  J.  Gentzkow  and  H.  A.  Van 
Auken  in  Simmons  and  Gentzkow, 
26-33. 

Unorganized   components   depending 
chiefly    on    metabolic    activities    and 
changes  in  content  of  bladder  before 
urination.     See  also  Sulfonamides. 
Examine  for: 
In  acid  urines 
Urates,  as  pink  amorphous  mate- 
rials 


UROBILIN 


367 


VACUOLOIDS 


Uric  acid,  as  yellow  brown,  wedge- 
like "whetstones",  dumb-bell  and 
rosette  crystals 
Calcium    oxalate     as    "envelope" 

crystals 
Cystine    as    colorless    refractile    6 

sided  plates 
Leucine    (yellow  spheroids) 
Tyrosine  (fine  needles) 
Hippuric    acid    (brownish    needles 
or  prisms) 
In  neutral  urines 
Above  components  plus 
Neutral  calcium  phosphate  (slender 
pyramidal     ci-ystals     united     at 
apices  forming  rosettes) 
In  alkaline  urines 
Phosphate   deposits    (white    amor- 
phous) 
Ammonium      calcium      phosphate 

(coffin  lid  or  feathery  crystals) 
So-called  triple  phosphate  crystals 
Calcium  carbonate  (spheres,  dumb- 
bells or  amorphous  masses) 
Ammonium     urate     (dark     yellow 
brown  cockle  burr  crystals) 
Organized  components  consisting  of 
cells  and  their  products  as  well  as  of 
casts.     Microscopically  to  identify  leu- 
cocytes,  red   blood   cells   and   sperms, 
when  present,  is  easy.     It  is  necessary 
to  distinguish  between  cells  from  renal 
tubules,  transitional  cells  from  bladder 
and    squamous    epithelial    cells.     The 
casts  are  of  4  sorts,  hyaline,  granular, 
waxy  and  blood3^     See  Addis  Count. 

Detection  of  acid  fast  bacilli  in  urine 
(Kelso,  R.  E.  and  Galbraith,  T.  W., 
Am.  J.  Clin.  Path.,  1943,  Techn.  Suppl., 
7,  8-11). 
Urobilin  is  a  derivative  of  bilirubin. 
Schmidt's  test  for  urobilin  in  feces  con- 
sists of  rubbing  up  small  amount  of 
feces  in  white  dish  in  sat.  aq.  mercuric 
chloride  whereupon  particles  containing 
this  pigment  take  on  a  deep  red  color 
(C.  J.  Gentzkow  and  H.  A.  Van  Auken 
in  Simmons  and  Gentzkow,  p.  82). 
Wintrobe,  M.  M.,  Clinical  Hematology. 
Philadelphia:  Lea  &  Febiger,  1942,  703 
pp.  gives  several  tests  for  urobilinogen 
and  urobilin. 

1.  Remove  bile  pigments,  if  present 
from  10  cc.  urine  (or  aq.  suspension 
feces)  by  addition  of  2  cc.  10%  calcium 
chloride  and  filtration.  Oxidize  any 
urobilinogen  not  converted  into  uro- 
bilin by  adding  1-2  drops  of  Lugol's 
Iodine.  Then  add  10  cc.  Schlesinger's 
Reagent,  filter  let  stand  1-2  hrs.  Uro- 
bilin is  indicated  by  green  fluorescence 
when  examined  against  dark  back- 
ground in  bright  light. 

2.  Make  dilutions  of  urine  by  adding 
1  cc.  to  10,  20,  30,  40  etc.  cc.  of  aq.  dest. 


To  10  cc.  of  each  dilution  in  test  tubes 
add  1  cc.  Ehrlick's  Aldehyde  Reagent. 

Urobilinogen  is  indicated  by  pink  color 
within  5  min.  seen  by  looking  down 
through  mouths  of  tubes. 

Urography,  a  new  technique  for  study  of 
individual  metabolic  spectra  (Beer- 
stecher,  Jr.  E.,  and  Sutton,  II.  1'^, 
Texas  Reports,  Biol.  &  Med.,  1951, 
9,  8-12. 

Vaccinia,  Cytoplasmic  inclusions  in,  see 
Cowdry,  E.  V.,  J.  Exper.  Med.  1922, 
36,  667-684.  Summary  of  methods  used 
in  the  investigation  of  elementary 
bodies  of  vaccine  virus  (Smadel,  J.  E. 
and  Hoagland,  C.  L.,  Rev.  Bact.,  1942, 
6,  79-110. 

Vaccinia,  see  Guarnieri  Bodies. 

Vacuoles,  food  and  contractile,  see  Para- 
mecia. 

Vacuoloids — Written  by  C.  C.  Macklin, 
Dept.  of  Histological  Research,  The 
University  of  Western  Ontario,  London, 
Canada.  November  28,  1951 — This  is 
a  provisional  name  for  numerous  clear 
round  regions,  often  containing  a  mi- 
nute central  granule,  which  appear  in 
the  pneumonocytes  and  foam  cells 
(which  see)  after  the  usual  methods  of 
fixation  and  staining.  They  superfi- 
cially resemble  vacuoles  and  give  to  the 
cells  a  frothy  appearance.  They  are 
the  dominant  formations  of  the  cyto- 
somes  of  these  cells.  They  are  often 
referred  to  as  granules,  for  after  certain 
techniques  many  of  them  are  found  to 
have  an  optically  substantial  nature, 
though  they  ordinarily  do  not  take 
stains  (Brodersen,  J.,  Zeitschr.  f. 
mikros.-anat.  Forsch.,  1933,  32,  73-83; 
Macklin,  C.  C,  Trans.  Roy.  Soc.  of 
Can.,  1946,  Sect.  V,  40,  93-111).  They 
are  relatively  stable,  indenting  the 
nucleus  (Macklin,  C.  C,  Canadian 
J.  of  Research,  D,  1950,  28,  5^15)  and 
are  from  0.5m  to  1.5^  or  more  in  diam- 
eter. In  fresh  mounts  from  (jash-irriga- 
tion  and  wash-out  recoveries  (which 
see)  they  appeared  as  clear  balls 
(Macklin,  C.  C,  Proc.  6th  Internat. 
Cong.  exp.  Cytol.,  Stockholm,  1947; 
published  1949,  383-387) .  They  seemed 
to  be  in  inverse  ratio  to  the  amount  of 
ingested  dust.  In  the  pure  foam  cells 
they  were  fairly  uniform  in  size,  sphe- 
roidal and  refractile,  gleaming  like 
transparent  glass  beads;  they  were  not 
so  sharply  marked  as  air  bubbles,  and 
had  an  extremely  delicate  greenish- 
yellow  cast.  The  contour  was  evenly 
curved  and  sharply  set  off  from  the 
environing  cj'toplasm.  In  the  fresh 
condition,  with  conventional  lighting, 
no  structural  detail  could  be  made  out 
in  the  pellucid  interior.     They  were  not 


VAGINAL  SMEARS 


368 


VAN  DEN  BERGH  TEST 


birefringent;  and  in  the  dark  field  the 
surface  was  brilliantly  illuminated. 
They  did  not  take  colors  dissolved 
weakly  in  the  mounting  fluid.  When 
Janus  Green  B  was  offered  in  weak  di- 
lution, coccoid  mitochondria  were  ob- 
served between  the  vacuoloids.  Fine 
red-stained  granules  were  found  be- 
tween the  vacuoloids  in  foam  cells  of 
frozen  sections  which  had  been  stained 
with  Sudan  IV.  Vacuoloids  seem  to  be 
composed  largely  of  water,  but  should 
not  be  regarded  as  simple  capsules. 
They  should  certainly  not  be  confused 
with  myelin  figures.  When  foreign 
particles  are  taken  into  the  cell  they 
lodge  between,  never  within,  the  vacuo- 
loids, and  this  is  true  for  vital  dye  accu- 
mulations. The  possibility  of  differen- 
tiation in  vacuoloids  is  admitted. 

In  material  fixed  in  Regaud's  fluid, 
mordanted  in  potassium  bichromate, 
cut  into  frozen  sections  and  well  stained 
with  Heidenhain's  iron  hematoxylin, 
some  of  them  appear  as  dense  blue- 
black  balls.  Some  of  them  appear  as 
large  dense  darkbrown  spheroids  or 
ovals  within  pneumonocytes  which 
have  been  in  contact  wath  blood  and 
have  been  fixed  with  pure  formalin  or 
other  aldehyde  (Sjostrand  and  Sjos- 
trand,  Zeitsch.  f.  mikros.-anat.  Forsch., 
1938,  44,  370-411).  These  workers  find 
that  the  colored  material  has  properties 
of  hemin.  The  same  coloration  of 
vacuoloids  may  occur  in  mice  suffering 
from  a  virus  infection  after  fixation 
with  Bouin's  fluid  by  immersion  of  the 
skinned  intact  thorax  (which  see). 
Some  of  them,  in  apparently  normal 
pneumonocytes  and  foam  cells  appear 
black  or  gray-black  after  exposure  to 
osmic  acid  fixation  intrabronchially 
after  Aquax  (which  see)  embedding  and 
mounting  in  levulose  (which  see).  In 
apparently  degenerate  pneumonocytes, 
all  of  the  vacuoloids  are  impregnated 
with  osmium. 

The  presence  of  vacuoloids  brings 
about  a  lacelike  picture  in  the  mito- 
chondria of  pneumonocytes,  for  mito- 
chondria are  not  admitted  to  the  in- 
teriors of  the  vacuoloids  but  occupy  the 
cytoplasm  between  them  (Macklin, 
C.  C.,  Biol.  Bull.,  1949,  96,  173-178). 
With  Nile  Blue  Sulphate  (Lorrain 
Smith,  which  see)  the  vacuoloids  in 
foam  cells  which  are  not  stained  dark 
after  osmic  acid  fixation  are  some- 
times colored  light  red  as  seen  in 
Aquax   sections   mounted   in   levulose. 

Macklin  spoke  in  the  following 
words  at  the  1947  International  Cyto- 
logical  Congress:  "The  function  of  the 
foam  cell  is  obscure,  and  may  be  con- 
cerned  with    the    conditioning   of   the 


alveolar  wall;  as  for  instance  in  the 
maintenance  of  a  proper  surface  ten- 
sion. However,  the  finding  of  these 
cells  in  augmented  quantities  under 
pathological  conditions  in  man  and 
rat  suggests  that  they  may  be  impli- 
cated in  the  local  body  reactions  to 
chronic  infection.  Amoeboidism  seems 
to  be  a  property.  ...  It  looks  as  if  the 
antecedent  entodermal  cell  has  a  dual 
differential  potency,  directing  it  to  the 
left,  so  to  speak,  where  it  may  become 
an  irreversible  pure  foam  cell,  or  to  the 
right,  where  it  may  end  up  as  an  irre- 
versible pure  dust  cell.  The  in-be- 
tween stages,  presumably  very  useful, 
may  be  termed  "bireactives."  (Mack- 
lin, C.  C.,  Proc.  6th  Internat.  Cong, 
exp.  Cytol.,  Stockholm,  1947;  published 
1949,  383-387). 

Vaginal  Smears,  see  Papanicolaou  Tech- 
niques. 

Valves.  Aortic,  staining  of  elastic  tissue 
in  (Wilens,  S.  L.,  Arch.  Path.,  1940, 
29,  200-211).  X-ray  demonstration  of 
valves  of  veins  (Edwards,  E.  A.,  Anat. 
Rec,  1936,  64,  369-385). 

Vanadium,  see  Atomic  Weights. 

Van  den  Bergh  Test  for  bilirubin  as  de- 
scribed by  Wintrobe,  M.  M.,  Clinical 
hematology.  Philadelphia:  Lea  & 
Febiger,   1942,  703  pp.   abbreviated: 

1.  Qualitative: 

(a)  Add  1.5  cc.  cone,  hydrochloric 
acid  C.P.  to  30  or  40  cc.  aq.  dest.  -f 
0.1  gm.  sulphanilic  acid  which  keeps 
well. 

(b)  Dissolve  0.5  gm.  sodium  nitrite 
C.P.  in  100  cc.  aq.  dest.  making  up  fresh 
every  3-4  weeks. 

Make  diazo  reagent  by  mixing  5  cc. 
of  (a)  with  0.15  cc.  of  (b)  freshly  each 
day. 

Mix  0.25  cc.  reagent  with  0.2  cc.  clear 
plasma  or  serum  (2  cm.  column  in  hema- 
tocrit). Immediate  purplish  color  at- 
taining maximum  in  30  sec.  is  direct 
reaction.  Color  appearing  at  once  but 
reaching  maximum  later  is  biphasic  re- 
action. If  no  color  in  1  min.  but  on 
addition  of  5  cc.  alcohol  reddish  violet 
color  appears  reaction  is  indirect. 

2.  Quantitative. 

(a)  Stir  and  shake  80-90  gms.  am- 
monium sulphate  C.P.  in  100  cc.  aq. 
dest.  until  saturated  and  filter. 

(b)  Make  standard  color  by  dissolving 
3.92  gm.  cobalt  sulphate  (7H2O)  in  100 
cc.  aq.  dest.  over  night. 

Mix  0.5  cc.  diazo  reagent  with  1  cc. 
serum  or  plasma  in  centrifuge  tube. 
After  standing  few  minutes  add  2.5  cc. 
95%  ethyl  alcohol  and  1  cc.  of  (a).  Mix 
and  centrifuge.  In  positive  reaction 
uppermost  layer  is  reddish  violet  alco- 
holic  extract   of   diazotized    bilirubin, 


VAN  GEHUCHTEN'S  MIXTURE 


369 


VERHOEFF'S  ELASTIC  TISSUE 

METHOD 


next  laj^er  is  flocculated  protein  and 
residue  is  ammonium  sulphate.  Com- 
pare supernatant  fluid  with  the  stand- 
ard (b)  in  colorimeter.     Then: 

mm.  standard 

; X  4  X  0.5 

mm.  unknown 

=  mg.  bilirubin  per  100  CO. 

Van    Gehuchten's    Mixture,    see    Carney's 

Fluid. 
Van    Gieson's    Connective    Tissue    Stain. 

Paraffin  sections  of  Zenker  fixed  mate- 
rial are  stained  with  Harris'  hema- 
toxylin. Rinse  in  water.  Stain  in  1% 
aq.  acid  fuchsin  7.5  cc.  and  sat.  aq. 
picric  acid  50  cc,  10  min.  Wash  in 
95%  ale,  dehydrate,  clear  and  mount. 
Muscle  yellow,  collagenic  fibers  red, 
nuclei  blue  black.  A  brilliant  stain. 
But  it  fades  quickly  and  is  not  so  much 
employed  at  present  as  Mallory's  con- 
nective tissue  stain.  See  Buzaglo's 
Method,  Curtis'  Substitute  for  Van 
Gieson,  Collagenic  Fibers,  Connective 
System. 

Van  Wijhe's  method  for  staining  cartilage 
in  whole  tissues  with  methylene  blue. 
See  Cartilage. 

Vasa  Vasorum.  Injection  with  India  ink 
(Winternitz,  M.  C,  Thomas,  R.  M. 
and  LeCompte,  P.  M.,  The  Biology  of 
Arteriosclerosis.  Springfield:  Thomas, 
1938,  142  pp.).  Filter  Higgins  Engros- 
sing ink  through  coarse  filter  paper  and 
dilute  filtrate  with  8  times  volume  of  aq. 
dest.  Obtain  pressure  apparatus  con- 
sisting of  2  liter  metal  tank  with  top 
and  bottom  outlets  and  air  pressure 
gauge.  Connect  upper  outlet  with 
escape  valve  and  high  pressure  air  line 
and  the  lower  one  with  rubber  tube  and 
cannulae.  To  inject  vasa  of  coronary 
arteries  place  fresh  human  heart  un- 
opened in  0.9%  aq.  sodium  chloride 
containing  0.1%  sodium  nitrite  and  a 
little  thymol  for  24  hrs.  at  3-4°C.  Just 
before  injection  warm  heart  to  37 °C., 
tie  cannulae  in  openings  of  coronary 
arteries  and  clamp  or  ligate  all  openings 
of  heart  except  the  aorta.  By  opening 
and  closing  the  escape  valve  the  ink  in 
the  tank  is  driven  into  the  coronaries 
by  a  pulsating  pressure.  During  first 
10  min.  maintain  the  minimum  pressure 
at  about  100  mm.  of  mercury  with 
maximum  pressure  of  pulsations  not 
more  than  200.  Then  increase  slowly 
so  that  during  next  20  min.  the  mini- 
mum pressures  vary  500-800  mm.  and 
the  maximum  800-1000.  After  injec- 
tion put  heart  in  10%  formalin  for  24 
hrs.  Dissect  out  main  coronaries. 
Clear  by  Spalteholz  Method  for  whole 
mounts  or  imbed  in  paraffin  section  and 
color  by  Masson's  Trichrome  stain. 
The  authors  give  special  directions  for 


injecting  the  aorta  and  vessels  of  kid- 
neys and  amputated  legs.  Their  illus- 
trations afford  useful  guides  to  the 
results  expected. 

Vaseline  in  tissues  can  be  distinguished 
from  the  normal  fats  by  the  fact  that 
the  former  is  colored  clear  violet  and 
the  latter  intense  blue  black  by  stain- 
ing for  15  min.  with  Sudan  Black  B. 
Terebenthine,  turpineol  and  methyl 
benzoate  are  colored  blue  black  (Gerard, 
P.,  Bull.  d'Hist.  Appl.,  1935,  12,  92-93). 

Vegetative  Intermitotics,  see  Cell  Classifica- 
tion. 

Veins,  see  Blood  Vessels  and  a  very  fine 
presentation  by  Franklin,  K.  J.,  A 
Monograph  on  Veins.  Springfield : 
Thomas,  1937,  410  pp.  with  hundreds 
of  references  to  techniques  and  results. 

Venous  Sinuses,  splenic,  direct  observa- 
tion in  vivo  (Knisely,  M.  H.  Anat. 
Rec,  1936,  64,  499-524;  65,  23-50).  See 
Spleen. 

Venules.  A  graphic  demonstration  of 
venules  in  the  ears  of  white  mice  can 
be  obtained  by  intravenous  injection 
of  Chicago  blue  because  this  dye  escapes 
into  the  surrounding  tissue  fluid  more 
easily  from  venules  than  from  capil- 
laries (Smith,  P.  and  Rous,  P.,  J.  Ex- 
per.  Med.,  1931,  54,  499-514). 

Verdigris.  A  green  progeneous  pigment 
made   up   of   basic    copper   acetates. 

VerhoefiF's  Elastic  Tissue  Method  (Ver- 
hoeff,  F.  H.,  J.  A.  M.  A.,  1908,  50, 
876-877).  Gives  good  results  after 
fixation  in  Zenker's  fluid,  formalin 
alone  or  after  Weigert's  mordant  for 
myelin  sheaths  or  Marchi's  fluid.  It 
is  fairly  satisfactory  for  tissues  decalci- 
fied with  nitric  acid.  Mercury  deposits 
resulting  from  Zenker's  fixation  are 
removed  by  the  stain :  Hematoxylin 
crystals,  1  gm.;  Abs.  ale,  20  cc;  Dis- 
solve in  test  tube  with  aid  of  heat, 
filter  and  add  in  order  given:  10%  aq. 
ferric  chloride,  8  cc. ;  Cone  Lugol's 
solution  (iodine,  2;  potassium  iodide,  4; 
water,  100),  8  cc  Stain  sections  in 
above  sol.  5  min.  or  more.  Differenti- 
ate in  2%  aq.  ferric  chloride  for  a  few 
sec.  until  the  connective  tissue  takes 
the  color  of  Lugol's  solution.  Keep 
sections  in  motion  during  differentia- 
tion. They  can  be  examined  at  low 
magnification  in  water  and  if  over 
differentiated  can  be  restained  at  this 
stage.  Wash  in  water  followed  by  95% 
ale  to  remove  the  stain  of  Lugol's  solu- 
tion. Then  leave  in  water  5  min.  or 
more.  Counterstain  in  0.2%  water 
sol.  eosin  in  80%  alcohol.  Dehydrate, 
clear  in  origanum  and  mount  in  balsam. 
Elastic  tissue,  black;  fibroglia,  myoglia, 
neuroglia,  myelin  and  fibrin,  pink. 
Degenerated     elastic     tissue      (elacin) 


VERONAL-ACETATE 


370 


VIRUSES 


can  be  distinguished  by  less  intensity 
of  staining  and  by  diffuse  outlines. 
To  differentially  stain  myelin  sheaths 
fair  results  are  obtained  after  Zenker's 
fixative  or  formalin  followed  by  Marchi's 
fluid.  For  best  results  fix  in  formalin 
4  days,  or  longer,  and  mordant  in 
Weigert's  potassium  bichromate  and 
chrome  alum  for  4  days.  Again  it  is  not 
necessary  before  hand  to  remove  mer- 
curial precipitates.  Place  sections  in 
3%  aq.  potassium  permanganate,  30 
min.  Wash  in  water  and  color  for  30 
min.  in  the  hematoxylin  stain  described. 
Wash  in  water  and  differentiate  in  10% 
aq.  ferric  chloride  until  the  internal 
elastic  membranes  of  blood  vessels  are 
decolorized  as  determined  by  examina- 
tion in  water  at  low  magnification. 
1-2  min.  are  required.  Wash  in  water 
for  5  min.,  counterstain  with  eosin  and 
mount  in  usual  way. 

Veronal-Acetate  buffers,  see  Michaelis,  L., 
J.  Biol .  Chem. ,  1930,  87,  34.  Employed 
for  thionin  staining  of  Nissl  substance 
by  Windle,  W.  F.,  Rhines,  R.  and  Ran- 
kin, J.,  Stain  Tech.,  1943,  18,  77. 

Vestibular  Apparatus,  see  Ear. 

Vesuvin,  see  Bismark  Brown  Y. 

Victoria  Blue  (1)  B  (CI,  729)— corn  blue 
BN,  fat  blue  B— (2)  R  (CI,  728)— corn 
blue  B,  new  Victoria  blue  B  or  R — (3) 
4R  (CI,  690)— fat  blue  4R— A  useful 
basic  tri -phenyl  methane  dye.  4R  is 
quite  extensively  discussed  with  other 
vital  stains  by  Gutstein,  M.,  Zeit.  f.  d. 
Ges.  Exp.  Med.,  1932,  82,  479-524. 
Herzberg,  K.,  Zentralbl.  Bakt.  I  Abt. 
Orig.  1934,  131,  358-366  employed  4B 
highly  concentrated  (Bayer  standards, 
Hollborn),  as  a  stain  for  filterable 
viruses  (Kikuth,  variola,  varicella, 
ectromelia  and  possibly  herpes).  Dry 
smears  in  air  24  hrs.  Stain  5-20  min. 
in  3%  aq.  Victoria  blue.  This  dye 
solution  should  have  been  heated  to 
60°C.  for  half  an  hour,  allowed  to  stand 
2  weeks  and  filtered  before  use.  To 
increase  intensity  of  stain  add  0.3  cc. 
10%  aq.  tartaric  acid  to  10  cc.  of  stain. 
Response  of  different  viruses  to  stain 
is  not  uniform.  Various  counterstains 
are  suggested.  The  various  Victoria 
blues  are  not  easily  disentangled.  Vic- 
toria blue  (variety  unspecified)  has, 
according  to  Lee  (p.  187),  a  special 
affinity  for  elastic  fibers  and  mucous 
cells. 

Victoria  Green  B  or  WB,  see  Malachite 
Green. 

Victoria  Green  G  (British  Drug  Houses 
Ltd),  a  triazo  dye  of  benzidine  series. 
In  alcoholic  solution  gives  blue  green 
and  yellow  green  colors.  Can  be  used 
with  Marshall  red  or  Hickson  purple  (H. 


G.  Cannan,  J.  Roy.  Micr.  Soc,  1941,  61, 

88-94). 

Victoria  Rubin  O,  see  Amaranth. 

Villi,  method  for  study  of  movements  (ffing, 
C.  E.  and  Arnold,  L.,  Am.  J.  Physiol., 
1922,  59,  97-131;  King,  Arnold  and 
Church,  J.  G.,  ibid,  61,  80-92).  See 
Agonal  Changes.  Changes  in  shape 
when  intestine  is  distended  (Johnson, 
E.  P.,  Am.  J.  Anat.,  1912-13,  14,  235- 
250). 

Vincent's  Angina,  staining  of  spirochete. 
Spread  ulcerative  material  on  clean 
slide.  Dry  in  air  and  fix  with  heat. 
N/20  HCl,  10  sec.  Running  water,  5 
sec.  Cover  with  Gram's  iodine  solu- 
tion, 5-10  sec.  Wash.  Cover  with 
anilin  gentian  violet,  5-10  sec.  Wash. 
Gram's  iodine,  5-10  sec.  Wash.  Anilin 
gentian  violet,  5-10  sec.  Wash,  blot 
and  examine.  Spirochetes  deep  violet 
color.  Also  good  for  T.  pallidum 
(Bailey,  H.  D.,  J.  Lab.  &  Clin.  Med., 
1937-38,  23,  960). 

Violamin  3B,  possibly  related  to  fast  acid 
blue. 

Violamin  R  (CI,  758).  Lillie,  R.  D.,  J. 
Tech.  Methods,  1945,  No.  25,  47  pp.  has 
reported  that  this  dye  is  a  good  stain 
for  collagen  and  more  light  fast  than 
acid  fuchsin.  Pass  sections  down  to 
water  and  stain  for  6  min.  in  Hemalum 
(Mayer-Lillie) .  Wash  in  tap  water  and 
stain  4  min.  in  0.1%  fast  green  FCF  or 
in  0.3%  Wool  Green  S  (CI,  737)  both  in 
1%  aq.  acetic  acid.  Wash  in  1%  aq. 
acetic  acid  and  stain  10-15  min.  in  0.2% 
acid  fuchsin,  or  in  0.2%  violamine  R, 
both  in  sat.  aq.  picric  acid.  Wash  2 
min.  in  1%  aq.  acetic  acid.  Dehydrate 
in  alcohol  and  alcohol -xylol,  clear  in 
xylol  and  mount  in  clarite.  Connective 
tissue,  red;  erythrocytes,  green;  cyto- 
plasm and  muscle,  gray-green;  and 
nuclei,  brown. 

Violet  R,  RR  or  4RN,  see  Hofmann's  Violet. 

Virchow's  Crystals  are  orange  or  bright 
yellow  crystals  of  hematoidin  occasion- 
ally met  with  in  extravasated  blood. 

Viruses  may  now  be  studied  microscopically 
in  several  different  ways.  There  is  a 
general  but  not  very  satisfactory  dis- 
tinction made  between  Elementary 
Bodies  of  the  viruses  which  may  be 
extracellular  and  the  Inclusion  Bodies 
which  may  be  larger,  are  intracellular 
and  may  contain  cellular  material 
perhaps  combined  with  virus.  The 
Chorioallantoic  Membrane  has  proved 
to  be  an  excellent  tissue  in  which  to 
examine  virus  action.  See  further  data 
under  above  headings.  A  very  compre- 
hensive description  is :  Rocha-Lima,  H., 
Reis,  J.,  and  Silberschmidt,  K.,  Metho- 
den   der   Virusforschung.     Berlin:    Ur- 


VISCOSITY 


371 


VITAL'STAINING 


ban  and  Schwarzenberg,  1939,  384  pp. 
The  "ultravirus"  diseases  of  insects  re- 
quire special  techniques  and  they 
should  not  so  often  be  ignored  in  ob- 
taining a  clear  view  of  the  viruses  as  a 
whole.  The  following  book  is  a  mine 
of  useful  information  Paillot,  A.,  L'ln- 
fection  Chez  Les  Insectes.  Imprimerie 
de  Tr^voux,  G.  Patissier,  1933,  535  pp. 
The  Electron  Microscope  is  of  great 
service  in  study  of  viruses. 

Botanists  have  greatly  advanced 
knowledge  of  the  chemical  composition 
of  viruses.  Discussion  by  Bawden, 
F.  C,  Plant  Viruses  and  Virus  Diseases. 
Waltham:  Chronica  Botanica  Co.,  1943, 
294  pp.  of  data  bearing  on  the  purity  of 
virus  crystals,  paracrystals  and  liquid 
crystals  shows  the  use  and  limitations 
of  present  day  techniques.  His  photo- 
micrographs of  the  virus  crystals  are 
interesting.  The  earlier  literature  is 
well  summarized. 
Viscosity.  According  to  Heilbrunn  (L.  V., 
An  Outline  of  General  Physiology. 
Philadelphia:  Saunders,  1937),  "Vis- 
cosity can  be  roughly  defined  as  the 
force  which  tends  to  hold  the  particles 
of  a  substance  together  when  a  shearing 
force  acting  on  the  substance  tends  to 
pull  it  apart."  Viscosity  is  the  in- 
verse of  fluidity.  It  is  of  great  im- 
portance to  histologists  to  be  able  to 
detect  and  if  possible  to  measure  changes 
in  viscosity.  When  a  living  cell  is 
examined  in  approximately  an  isotonic 
medium  and  tiny  particles  in  it  begin 
Brownian  Movement  a  decrease  in 
viscosity  is  indicated  and  when  the 
movement  ceases  an  increase  is  to  be 
expected.  Thus  Lewis  (W.  H.,  Bull. 
J.  Hopkins  Hosp.,  1923,  34,  373-379) 
took  cessation  of  Brownian  movement 
of  particles  in  the  nucleus  viewed  in  the 
dark  field  to  mean  gelation  which  is 
increase  in  viscosity.  A  Microdissec- 
tion method  is  to  insert  2  microneedles 
into  a  cell.  If  they  can  be  pulled  apart 
easily  the  viscosity  is  low;  if  with  diffi- 
culty, it  is  high.  The  idea  back  of  the 
Ultracentrifuge  method  is  that  if  two 
cells  of  the  same  sort  are  subjected  to 
equal  centrifugal  force  and  a  component, 
say  the  nucleus,  is  displaced  more  in 
one  than  in  the  other  the  viscosity  of 
the  cytoplasm  is  greater  in  the  cell 
showing  the  least  nuclear  displacement. 
But  this  is  not  necessarily  true.  One 
has  to  be  sure  that  the  nuclei  are  of 
equal  Specific  Gravity.  If  the  more 
displaced  nucleus  is  of  higher  specific 
gravity  than  the  other  it  will  be  more 
subjected  than  the  other  to  the  centrifu- 
gal force  and  its  greater  displacement 
will  not  signify  a  lower  viscosity  of  the 


surrounding  cytoplasm.  Similarly  if 
the  specific  gravity  of  the  cvtoplasm 
surrounding  the  more  displaced  nucleus 
is  less  than  that  in  the  other  cell  the 
greater  displacement  subjected  to  the 
centrifugal  force  of  the  nucleus  through 
it  will  not  indicate  a  lower  cytoplasmic 
viscosity.  When  a  material  changes 
from  a  sol  to  a  gel  its  viscosity  increases 
without  a  change  in  specific  gravity. 
Consequently  in  the  interpretation  of 
alterations  in  displaceability  of  cellular 
components  subjected  to  centrifugal 
force  one  has  to  be  on  the  lookout  for 
changes  in  specific  gravity  and  col- 
loidal state.  For  details  in  respect  to 
intranuclear  viscosity,  see  Cowdry,  E. 
V.  and  Paletta,  F.  X.,  Am.  J.  Path., 
1941,    17,   335-357;   1942,    18,   291-311). 

Vital  New  Red.  This  is  an  acid  dis-azo 
dye  not  listed  in  indexes  but  Conn  (p. 
64)  calls  attention  to  chlorazol  fast 
pink  4BL  (CI,  353)  as  most  nearly 
resembling  it.  Vital  new  red  is  one  of 
the  many  dis-azo  dyes  employed  by 
Evans,  H.  M.,  and  Scott,  K.  J.,  Car- 
negie Inst.  Wash.,  Contrib.  to  Embryol., 
1921,  10,  1-56  to  bring  out  a  difference 
in  reaction  of  the  two  great  groups  of 
connective  tissue  cells. 

Vital  Red  (CI,  456)— acid  Congo  R,  azidine 
scarlet  R,  brilliant  Congo  R,  brilliant 
Congo  red  R,  brilliant  dianil  red  R. 
brilliant  vital  red — An  important  acid 
dis-azo  dye  frequently  used  in  standard 
method  for  determination  of  blood 
volume. 

Vital  Staining.  This  technique  has  been 
contrasted  with  Supravital  Staining. 
It  must  be  viewed  broadly.  Any 
nontoxic  coloration  of  the  living  body  is 
vital  staining.  It  is  not  restricted  to 
particulate  materials  or  to  colloidal 
suspensions  which  are  phagocytosed  by 
certain  cells.  The  fat  depots  of  an 
animal  become  vitally  stained  red 
when  the  said  animal  is  fed  fat  colored 
with  alcohol  soluble  sudan  III.  Bone 
formed  while  madder  is  available  in 
the  circulation  is  stained  red  and  dentin 
is  vitally  stained  violet  by  intravenous 
injections  of  1%  sodium  alizarin  sul- 
phonate  (Gottlieb,  B.,  Ztschr.  f.  Somat., 
1913,  11,  452).  The  phthalein  indi- 
cators tint  the  tissues  of  living  animals 
faintly  but  almost  all  the  colors  of  the 
rainbow.  Bile  capillaries  of  the  liver 
can  easily  be  stained  by  intravenous 
injection  of  sodium  sulphindigotate. 
Many  other  examples  of  similar  phe- 
nomena could  be  cited.  But  it  is 
customary  to  think  of  vital  stains  as 
substances  which  are  regularly  taken 
in  by  cells  of  the  Reticulo-Endothelial 
System  and  by  a  few  others  on  occasion. 


VITAMINS 


372 


VITAMINS 


These  include  colloidal  suspensions  of 
various  benzidine  dyes  (trypan  blue, 
isamin  blue,  pyrrhol  blue,  trypan  red, 
etc.),  of  silver,  Higgins  ink,  lamp  black 
etc.;  and  of  simple  suspensions  of  India 
ink,  carmine,  graphite  and  so  on.  They 
are  injected  intravenously,  intraperi- 
toneally  or  subcutaneously.  The  litera- 
ture is  enormous.  Consult  latest  issue 
of  the  Quarterly  Cumulative  Index 
Medicus.  For  chemistry  of  Benzidine 
dyes  see  Evans,  H.  M.  and  Schulemann, 
W.,  Science,  1914,  39,  443. 

The  following  experiment  is  suggested. 
Give  each  of  a  dozen  or  more  white 
mice  1  cc.  of  0.5%  trypan  blue  in  sterile 
aq.  dest.  intraperitoneally  and  in  the 
course  of  a  few  minutes  the  beginning  of 
deposition  of  the  dye  in  the  ears  will  be 
noted.  Give  similar  doses  every  sec- 
ond day  for  8  days.  A  few  hours  after 
the  last  draw  a  little  blood  from  the 
tail  and  observe  that  some  of  the  mono- 
cytes have  taken  up  the  dye.  Then 
autopsy  the  mice  and  study  the  dis- 
tribution of  the  dye  in  the  tissues. 
The  skin,  kidneys,  adrenals,  liver, 
spleen  ana  bone  marrow  will  be  found 
quite  deeply  colored  while  the  nervous 
system  has  escaped.  The  heaviest 
accumulation  will  be  in  the  peritoneal 
cavity  near  the  sites  of  injection  and  in 
the  loose  connective  tissue  everywhere. 
Examination  of  fresh  mounts  in  physio- 
logical salt  solution  will  reveal  that  the 
dye  is  concentrated  within  (1)  the 
epithelial  cells  of  the  convoluted  tubules 
of  the  kidney,  of  the  adrenal  and  choroid 
plexus;  (2)  certain  cells  of  the  ovary 
and  testicle;  (3)  the  macrophages  of 
loose  connective  tissue  and  especially 
of  the  spleen,  liver,  bone  marrow,  ad- 
renals and  lymph  nodes — fibroblasts 
are  colored  less  deeply;  and  (4)  the 
"specific  endothelia"  of  the  five  organs 
mentioned.  If  permanent  preparations 
are  desired  fix  in  10%  formalin  and  im- 
bed in  paraffin. 

Vital  staining  in  the  narrow  sense  is 
used  for  many  purposes.  (1)  To  iden- 
tify phagocytic  cells  of  the  reticulo- 
endothelial system  and  to  see  how  they 
behave  in  normal  and  pathological 
conditions.  (2)  To  locate  injured  cells 
because  some  cells  that  do  not  ordi- 
narily stain  take  up  the  dye  when 
injured.  (3)  To  influence  the  activity 
of  R.  E.  cells  by  blocking  them  with 
particulate  matter.  This  has  not  been 
very  successful.  See  R.  E.  Blockade 
(Victor,  J.,  Van  Buren,  J.  R.  and 
Smith,  H.  P.,  J.  Exper.  Med.,  1930,  51, 
531-548).  (4)  To  measure  the  absorp- 
tion by  membranes  of  particulate  matter 
(Wislocki,  G.  B.,  Anat.  Rec,  1921,  21, 
29-33).     (5)  To     distinguish     between 


malignant  and  non-malignant  cells  (Lud- 
ford,  R.  J.,  Arch.  f.  exp.  Zellf.,  1933, 
14,  42-55).  (6)  To  determine  pH  of 
different  organs  and  tissues  by  injec- 
tion with  phthalein  indicators  (Rous, 
P.,  J.  Kxper.  Med.,  1925,  41,  739-759). 
(7)  To  identify  calcium  salts  laid  down 
(Alizarin  Red  S  and  Madder).  See 
method  for  Reticulo-endothelial  system. 
It  is  sometimes  very  worthwhile  to 
inject  simultaneously  three  materials, 
for  example  Higgins'  Ink  intravenously, 
trypan  blue  or  Niagara  blue  intraperi- 
toneally, and  lithium  carmine  intra- 
pleurally  (Foot,  McClung,  p.  116). 
An  interesting  experiment  is  to  feed 
Sudan  III  or  Scharlach  (scarlet  = 
Sudan  IV)  colored  lipids.  Make  solu- 
tion in  olive  oil  (about  20%).  Intro- 
duce by  stomach  tube  into  a  cat.  There 
is  slight  staining  of  fatty  tissue  within 
24  hrs.  and  maximum  in  3-7  days 
(Hadjioloff,  A.,  Bull.  d'Hist.  AppL, 
1938,  15,  81-98).  Try  also  inducing 
cat  to  drink  large  amount  of  milk  or 
cream  colored  with  Sudan  III  or  Sudan 
black,  see  colored  illustrations  of  Gage 
and  Fish  (S.  H.  and  P.  A.,  Am.  J.  Anat., 
1924-25,  34,  1-81).  History  of  vital 
staining  (Conn,  H.  J.  and  Cunningham, 
R.  S.,  Stain  Techn.,  1932,  7,  81-90, 
115-119).  See  Chorioallantoic  Mem- 
brane, Carmine,  Indigo-Carmine, 
Manganese  Dioxide,  Higgins'  Ink, 
Protargol  (silver),  Lampblack,  Leuco- 
Dyes,  Nuclei,  Titanium  Dioxide,  Tho- 
rium Dioxide,  Copper,  Platinum,  Iron, 
Mercury,  Lymphatic  Vessels. 
Vitamins — Written  bj^  C.  Carruthers,  Wash- 
ington University  School  of  Medicine, 
St.  Louis.  February  15,  1951 — Only  a 
few  vitamins  are  susceptible  of  micro- 
scopic localization.  Deficiencies  in  most 
of  them  leave  structural  imprints 
in  the  tissues.  A  list  may  be  therefore 
useful  giving  briefly  required  tech- 
niques. Current  information  is  sup- 
plied in  Annual  Reviews  of  Biochem- 
istry and  Physiology. 

A  useful  background  is  provided  by 
Sherman,  H.  C.  (Chemistry  of  Food  and 
Nutrition,  New  York:  Macmillan,  1941, 
611  pp.).  For  a  summary  of  tissue 
changes  in  vitamin  deficiencies  see 
Wollach,  S.  B.  and  Bessev,  O.  H.  (Phvs- 
iol.  Rev.,  1942,  22,  233-^290).  For  the 
biochemistry  and  chemical  determina- 
tion of  the  vitamins  the  following  are 
recommended:  Gyorgy,  P.,  Vitamin 
Methods,  New  York,  Academic  Press. 
1950,  571  pp.;  Williams,  R.  J.,  Eakerj 
R.  E.,  Beerstecker,  E.,  and  Shive,  W., 
The  Biochemistry  of  B.  Vitamins,  New 
York,  Reinhold  Publishing  Co.,  1950, 
741  pp.;  Harrow,  B.,  One  Family. 
Vitamins,  Enzymes,  Hormones,  Minne- 


VITAMINS 


373 


VITAMINS 


apolis,  Burgess  Publishing  Co.,  1950, 
115  pp.;  The  Association  of  Vitamin 
Chemists,  Inc.,  Methods  of  Vitamin 
Assay,  New  York;  Interscience  Pub- 
lishers, Inc.,  1947,  189  pp.;  Johnson, 
D.  C,  Methods  of  Vitamin  Determina- 
tion, Minneapolis,  Burgess  Publishing 
Co.,  1949,  109  pp. 
A.  Growth  promoting,  anti-infective  and 
anti-xerophthalmic  vitamin,  a  polyene 
alcohol.  There  are  also  vitamins  A2, 
A3  and  the  so-called  Sub-vitamin  A, 
and  neo-vitamin  A,  which  is  a  sterioiso- 
mer  of  vitamin  A.  The  members  of 
the  vitamin  A  group  differ  in  their 
maximum  absorption  in  the  ultraviolet. 
Provitamins  A  are  alpha,  beta  and 
delta  carotene  which  vary  in  their 
vitamin  A  activity.  Vitamin  A  can 
be  determined  by  the  following  methods : 

1.  Its  absorption  at  326  m^  measured 
by  a  spectrophotometer,  or  photoelec- 
tric spectrophotometer,  under  con- 
trolled   conditions    of    analysis. 

2.  The  glycerol  dichlorohydrin 
method  of  Sobel,  A.  E.  and  Werbin, 
H.,  Ind.  Eng.  Chem.,  Anal.  Ed.,  1946, 
18,  570-573;  1947,  19,  107-112. 

3.  The  Carr-Price  reaction  which 
employs  the  antimony  trichloride  re- 
agent. This  reaction  has  been  applied 
to  determine  the  vitamin  A  content 
of  hepatic  mitochondria  of  rats  after 
separation  by  differential  centrifuga- 
tion.  The  mitochondria  contain  249 
to  910  U.S. P.  units  of  vitamin  A  per 
100  mg.  mitochondrial  lipid  (Goerner, 
A.  and  M.  M.,  J.  Biol.  Chem.,  1937- 
38,  122,  529-538;  1939,  128,  559-565). 
This  test  has  also  been  employed  for 
vitamin  A  in  serum,  the  colors  being 
checked  against  alizarin  solutions  (Par- 
ker, R.  C,  Methods  of  Tissue  Culture, 
New  York:  Hoeber,  1938,  292  pp.). 
According  to  Joyet-Lavergne,  P.,  C. 
rend.  Acad.  d.  Sci.,  1935,  201,  1219-1221, 
vitamin  A  can  be  demonstrated  in  the 
red  blood  cells  of  rays  (marine  fish)  by 
the  Carr-Price  test.  This  same  investi- 
gator has  also  applied  the  Carr-Price 
reaction  to  a  variety  of  tissues  of  sev- 
eral animals,  and  has  found  that  in 
every  case  the  mitochondria  appear  as 
bright  blue  bodies  (Ann.  physiol.  physi- 
cochem.  biol.  1937,  13,  1019-1021),  see 
also  Bourne,  G.  Austral.  J.  Exp.  Biol. 
Med.  Sci.  1934,  13,  239-249.  The  Carr- 
Price  reaction  can  localize  vitamin  A 
precisely  in  individual  mitochondria 
according  to  Jones,  J.,  Lab.  Clin. 
Med.,  1947,   12,  700. 

4.  Fluorescence  microscopy.  The 
fading  green  fluorescence  of  vitamin  A 
is  used  by  Popper  for  the  identification 
and  localization  of  vitamin  A  in  cells 
(Gyorgy,    1950,    p.    89).     Tissues    are 


fixed  in  5%  formalin  and  examined  as 
soon  as  possible.  Human  tissues  are 
kept  in  the  fixitive  not  longer  than  18 
hrs.  and  animal  tissues  not  longer  than 
8  hrs.  Why  this  difference  should  be 
is  not  clear.  Frozen  sections  are  made, 
mounted  in  water  and  examined  at 
once  with  a  fluorescence  microscope. 
Any  green  fading  fluorescence  is  con- 
sidered to  be  vitamin  A  and  is  usually 
located  in  fatty  material.  For  a  more 
exact  localization  of  this  vitamin, 
sections  may  be  stained  with  1%  aq. 
methylene  blue  for  30  sec.  This  stain 
interferes  only  slightly  with  the  vita- 
min A  fluorescence.  By  Popper's 
method  vitamin  A  has  been  found  in 
the  liver,  adrenal  cortex,  testicle,  and 
in  the  ovary  where  it  undergoes  charac- 
teristic changes  during  the  mentrual 
cycle  and  during  pregnancy.  It  is  also 
present  in  the  intestinal  tract  during 
absorption,  in  lactating  breasts,  in 
the  retina  and  in  certain  pathologic 
conditions  of  the  kidney.  For  a  re- 
view of  its  distribution  in  tissues,  see 
Popper,  H.,  Physiol.  Rev.,  1944,  24, 
205-224.  According  to  Popper  caro- 
tene can  be  differentiated  from  vitamin 
A  by  its  very  slowly  fading  green 
fluorescence,  and  anhydro-  or  "cy- 
clized",  vitamin  A  may  be  recognized 
by  its  dark  brown  fluorescence,  which 
gradually  becomes  dull  green  and  fi- 
nally fades  out  entirely. 

B.     Complex    contains    inany   factors. 

Bi.  Thiamine  hydrochloride  (anti-neuritic 
factor,  aneurin).  Thiamine  can  be  de- 
termined colorimetrically  by  the 
method  of  Hochberg,  M.,  Melnick,  D. 
and  Oser,  B.  L.,  Cereal  Chem.,  1945, 
22,  83-90.  This  vitamin  can  also  be 
determined  microbiologically  using 
Streptococcus  salwarius,  or  Lactobacillus 
fermenti  36  (Gyorgy,  1950,  pp.  372-376). 
The  coenzyme  derived  from  thiamine 
is  thiamine  pyrophosphate.  It  is  in- 
volved in  the  decarboxylation  of  alpha 
keto  acids,  particularly  pyruvic  acid, 
and  consequently  in  the  tissue  oxida- 
tion of  carbohydrates.  In  thiamine 
deficiency  the  pyruvate  and  lactate 
blood  levels  are  increased,  the  increase 
in  the  former  being  due  to  a  lack  of 
ability  to  change  pyruvate  to  lactate 
without  thiamine.  No  histochemical 
method  is  available  for  this  vitamin. 

B2.  Vitamin  G  (Riboflavin,  Lactoflavin). 
Riboflavin  can  be  determined  chemi- 
cally by  measuring  its  fluorescence  in 
light  of  wave  length  440  to  500  mn 
(Gyorgy,  1950,  pp.  102-144).  Ribo- 
flavin can  also  be  determined  polaro- 
graphically  in  pure  solutions.  The 
flavoproteins  catalyze  the  metabolism 
of  such  substances  as  D-amino  acids, 


VITAMINS 


374 


VITAMINS 


L-amino  acids,  L-hydroxy  acids,  alde- 
hydes and  purines.  In  Warburg's  "old 
yellow  enzyme",  the  respiratory  en- 
zyme, riboflavin  exists  as  the  mono- 
nucleotide. The  j^ellowish-green  fluo- 
rescence of  riboflavin  has  been  used  for 
its  detection  in  tissues  by  Ellinger, 
P.,  and  Koschara,  W.,  von  Euler,  H., 
et  al.,  Hert,  A.  and  Wimmer,  K.  and 
Metcalf,  R.  L.  and  Patton,  R.  L.  See 
Glick,  D.,  Techniques  of  Histo-  and 
Cyto-Chemistry,  New  York,  Inter- 
science  Publishers,  Inc.,  1950,  pp.  531 
Metcalf,  R.  L.  and  Patton,  R.  L.  claim 
that  another  form  of  riboflavin  exists 
which  gives  a  yellow-orange  fluores- 
cence. 

B3.  Pantothenic  acid,  first  designated 
B3.  filtrate  factor,  factor  II,  anti- 
grey-hair-factor.  This  vitamin  is  a 
component  of  coenzyme  A  involved 
in  the  acetylation  of  aromatic  amines 
and  choline,  and  in  the  metabolism  of 
fats  and  carbohydrates.  Pantothenic 
acid  is  determined  microbiologically 
using  Lactobacillus  arabinosus  17-5 
(Johnson,  1949,  pp.  79-80). 

Be.  Group  (Pyridoxal,  Pyridoxine,  and 
Pyridoxamine).  Pyridoxamine  is  basi- 
cally as  important  as  pyridoxine, 
and  pyridoxal  is  even  more  so.  Pyri- 
doxal phosphate  is  the  coenzyme  of 
this  vitamin  group  and  is  involved  in 
the  decarboxylation  of  amino  acids  and 
in  transamination.  Pyridoxine  can  be 
determined  chemically  by  techniques 
based  upon  the  indophenol  test  (Gy- 
orgy,  1950,  p.  239).  This  method, 
however,  is  only  suitable  for  pyridoxine 
and  not  for  the  other  two  forms  so 
that  it  is  not  adaptable  for  tissue  analy- 
sis. Saccharomyces  Carlsbergensis  and 
Neurospora  sitophila  are  employed  for 
the  microbiological  determination  of 
the  Be  group  of  vitamins.  Pyridoxal, 
pyridoxamine  and  pyridoxine  have  ap- 
proximately the  same  activity  in  stim- 
ulating growth  (Gyorgy,  1950,  pp.  406- 
414). 

B12.  Aniipernicious  anemia  factor  (ani- 
mal protein  factor).  This  vitamin  con- 
tains 4  per  cent  of  cobalt.  It  produces 
a  positive  hematological  response  in 
pernicious  anemia  in  quantities  as  low 
as  3  micrograms.  This  vitamin  also 
improves  the  hatchability  of  hen's  eggs, 
the  growth  and  survival  of  chicks,  and 
the  growth  of  rats.  Vitamin  B12 
has  been  implicated  in  the  following 
processes:  (1)  the  synthesis  of  purines 
and  pyrimidines  and  their  derivatives, 
(2)  the  synthesis  of  methionine  and 
folic  acid,  and  (3)  the  utilization  of 
p-aminobenzoic  acid  and  folic  acid. 
It  is  related  metabolically  to  folic  acid 
(see    The    Nutritional    and    Chemical 


Significance    of    Folic    Acid,    Lederle, 
American  Cyanamid  Co.  1950,  121  pp. 

Choline,  a  constituent  of  phospho- 
lipids, lecithins  and  sphingomyelins, 
acts  as  a  donator  of  labile  methyl 
groups  and  prevents  hemorrhagic  de- 
generation of  kidneys  of  j^oung  rats 
deficient  in  choline.  Choline  can  be 
determined  chemically  or  microbio- 
logically (Gyorgy,  1950,  pp.  243  and 
464). 
C.  Antiscorbutic  vitamin  (Cebione,  Re- 
doxon).  Bourne,  G.,  Anat.  Rec,  1936, 
66,  369-385,  has  made  a  critical  study 
of  cytological  methods  for  the  deter- 
mination of  vitamin  C.  The  technique 
reconomended  is  based  on  the  assump- 
tion that  the  only  substance,  other 
than  vitamin  C,  capable  of  reducing 
an  acid  silver  nitrate  solution  in  the 
dark  is  hydrogen  sulphide  "which  is 
not  by  any  means  a  common  constituent 
of  living  tissue,  if  it  occurs   at  all." 

To  demonstrate  reduced  vitamin  C, 
frozen  sections  of  fresh  tissue  are 
treated  with  5%  aq.  silver  nitrate,  to 
which  5  cc.  acetic  acid  is  added  for 
each  100  cc,  for  a  few  minutes.  The 
vitamin  C  granules  blacken.  After 
washing  in  aq.  dest.  fat  may  be  stained 
by  a  solution  of  Sudan  III  or  Scharlach 
R  in  90%  ale.  and  the  section  cleared 
and  mounted  in  glycerin. 

To  reveal  both  reduced  and  oxidized 
vitamin  C  is  more  difficult.  Bourne 
advised:  Subject  fresh  tissue  to  glacial 
acetic  acid  vapor  for  several  minutes. 
Cut  into  very  thin  slices  and  put  in 
atmosphere  of  hydrogen  sulphide  for 
15  min.  All  vitamin  C  is  thereby  con- 
verted to  reduced  form.  Remove  hy- 
drogen sulphide  by  keeping  in  partial 
vacuum  for  10  to  30  min.,  follow  by 
exposure  to  a  strong  stream  of  nitrogen 
gas  for  15  min.  Treat  with  acid  silver 
nitrate    solution     as    described. 

If  there  is  reason  to  believe  that 
glutathione  inhibits  the  reaction, 
Bourne  suggests,  after  the  hydrogen 
sulphide  treatment,  to  momentarily 
wash  the  section,  then  to  plunge  into 
mercuric  acetate  solution  for  a  few 
minutes,  wash  and  apply  acid  silver 
nitrate  solution.  See  Barnett,  S.  A. 
and  Bourne,  G.,  J.  Anat.,  1940-41,  75, 
251-264  for  methods  of  demonstration 
of  vitamin  C  in  chick  embryos. 

Modification  of  Giroud  and  Leblanc 
silver  method  (Tonutti,  E.,  Proto- 
plasma,  1938,  1,  151-158).  Briefly  wash 
tissue  in  5.4%  aq.  levulose  and  10%  aq. 
AgNOs  plus  2  drops  glacial  acetic  per 
cc,  up  to  30  min.  Rinse  in  aq.  dest. 
30  15-min.  and  in  3%  aq.  NajSoOj, 
15-30  min.  Rinse  in  aq.  dest.  15-30, 
min.    All  this  is  to  be  done  in  the  dark 


VITAMINS 


375 


VITAMINS 


room  with  red  light.  Change  to  70% 
ale.  and  imbed  in  paraffin.  Counter- 
stain  with  "Kernechtrot"  and  light 
green. 

For  a  description  of  the  localization 
of  vitamin  C  in  fibroblasts,  during  the 
histogenesis  of  chick  embryos,  goblet 
cells,  Golgi  apparatus,  mitochondria, 
etc.  see  Bourne,  G.,  Cytology  and  Cell 
Physiology,  London:  The  Clarendon 
Press,  1951,  p.  262  et  seq. 

D.  The  group  of  "vitamins  D"  consists 
of  a  number  of  compounds  (Rosenberg, 
H.  P.,  Chemistry  and  Physiology  of  the 
Vitamins,  New  York:  Interscience 
Publishers,  Inc.,  New  York:  1942,  341 
pp.)  Di  is  a  molecular  compound  con- 
sisting of  vitamin  D2  and  lumesterol. 
D2-activated  ergosterol,  calciferol,  or 
viosterol.  D3  is  activated  7-dehydro- 
cholesterol  and  D4  is  activated  22- 
dihydrocholesterol.  D5  is  activated  7- 
dehydrositosterol . 

There  are  at  least  10  provitamins  D. 
The  vitamins  D  can  be  determined 
chemically;  but,  since  the  methods  are 
based  on  conjugated  unsaturation  of 
the  vitamin  D  molecule,  no  distinction 
between  them  can  be  made  (Rosenberg, 
1942,  pp.  412-413).  The  biological 
methods  for  the  determination  of  vita- 
mins D  are  reliable  when  properly 
conducted  and  they  are  superior  to 
chemical  and  physical  techniques.  See 
the  Line  Test. 

E.  Antisterility  vitamins  of  which  there 
are  four:  alpha,  beta,  delta  and  gamma 
tocopherol.  This  vitamin  can  be  de- 
termined by  chemical  and  biological 
methods  (Rosenberg,  1952,  p.  452). 
Vitamins  E  are  also  antioxidants,  the 
gamma  isomer  being  more  effective  than 
the  beta  isomer  which,  in  turn,  is  more 
effective  than  the  alpha  isomer  which  is 
most  active  as  the  antisterility  factor. 

A  pigment,  classified  as  a  lipofuscin, 
develops  in  the  uterus  of  vitamin  E 
deficient  rats  (Elftman,  H.,  Kaunitz, 
H.  and  Slanetz,  C.  A.,  Annals  N.  Y. 
Academy  of  Sciences,  1949,  52,  72-79). 
This  pigment  probably  arises  from 
the  peroxidation  and  polymerization 
of  unsaturated  fatty  acids.  Its  ap- 
pearance in  the  uterus  can  be  pre- 
vented by  ovariectomy  and  by  main- 
taining the  rats  on  a  vitamin  E-deficient 
diet  low  in  unsaturated  fat  (Atkinson, 
W.  B.,  Kaunitz,  H.  and  Slanetz,  C.  A., 
Ann.,  N.  Y.  Acad.  Sci.,  1949,  52,  68-71). 

Folic  acid  (pteroylglutamic  acid, 
vitamin  Bo,  factor  U,  L  casei  factor, 
Norite  eluate  factor).  A  deficiency  in 
folic  acids  results  in  megaloblastic 
arrest  in  the  bone  marrow  and  the  de- 
velopment of  a  macrocytic  anemia. 
In   chicks  it  is   necessary   for  normal 


growth,  feathering,  and  egg  hatchabil- 
ity.  For  relationships  between  folic 
acid  and  vitamin  B^  see  The  Nutri- 
tional and  Chemical  Significance  of 
Folic  Acid,  Lederle:  American  Cyana- 
mid  Co.  1950. 

F.  A  Vitagen,  Essential  fatty  acids 
consisting  of  linoleic,  linolenic  and 
arachidonic.  Arachidonic  acid  is  phys- 
iologically the  most  important  com- 
pound. 

H.  Biotin  (vitamin  H,  anti-egg-white 
injury  factor).  Biotin  can  be  assayed 
microbiologically  (Gyorgy,  1950,  p. 
61).  It  is  probably  a  coenzyme  for 
oxalacetate  decarboxylase  involved  in 
the  mechanisms  of  growth  since  its 
content  in  embryonic  and  tumor  tissue 
is  high. 

I.  Inositol  (mouse  anti-alopecia  factor). 
This  vitamin  is  hexahydroxy  cyclohex- 
ane.  Its  significance  in  human  nutri- 
tion has  not  yet  been  established.  It 
is  present  in  relatively  large  amounts 
in  tissues  and  its  occurrence  as  a  unit 
structure  in  lipids  may  later  remove  its 
inclusion  as  a  vitamin.  Definite  proof 
that  inositol  acts  catalytically  has  not 
been    obtained. 

K.  The  antihemorrhagic  factor  (phyllo- 
quinone). 

Ki.  is  the  first  form  of  the  antihemor- 
rhagic vitamin  isolated  bv  Dam,  H., 
Helv.  Chem.  Acta,  1939,  22,  310-313. 
It  is  2-methyl-3-phytyl-l,4-naphtho- 
quinone. 

K2.  is  the  second  form  isolated  by 
Brinkly,  S.  B.,  MacCorquodale,  D.  W., 
Toyer,  S.  A.,  and  Doisy,  E.  A.,  J. 
Biol.  Chem.,  1939,  130,  219-234.  It  is 
2  -  methyl  -  3  -  difarnesyl  - 1,4  -  naptho- 
quinone.  Since  the  various  K  vitamins 
are  quinones,  or  easily  oxidized  to 
quinones,  most  chemical  methods  pro- 
posed for  their  determination  are  based 
on  oxidation-reduction  titrations  or 
on  color  reactions  utilizing  the  quinone 
character  of  vitamin  K  (Gyorgy, 
1950,  p.  207,  et  seq.). 

Nicotinic  acid  (niacin,  nicotin  amide 
and  niacin  amide).  Microbiological 
methods  employing  Lactobacillus  arabi- 
nosus  17-5,  a  yeast,  Torula  crernoris, 
and  a  nonpathogenic  bacterium,  Pro- 
teus H  X  19,  are  more  sensitive  than 
chemical  methods  (Williams,  pp.  55- 
56.  The  colorimetric  methods  which 
have  been  widely  used  in  nicotinic  acid 
assays  all  involve  the  interaction  of 
this  vitamin  with  cyanogen  bromide 
and  an  aromatic  amine  (Williams,  1950, 
p.  54).  The  coenzymes,  diphosphopy- 
ridine  nucleotide  and  triphosphopyri- 
dine  nucleotide,  contain  nicotinic  acid. 
About  forty  different  enzyme  reactions 
have  been  reported  to  be  catalyzed  by 


VALKONSKY  METHOD 


376 


WALKER'S  METHOD 


one  or  the  other  of  these  enzymes. 
The  chief  metabolites  of  nicotinic  acid 
which  are  most  abundant  in  human, 
swine  and  dog  urine,  are  N'-methyl- 
nicotinamide  and  6-pyridone.  These 
can  be  determined  in  a  fluorometer 
having  a  maximum  transmission  at  365 
m^t  in  the  ultraviolet  (Johnson,  1949, 
pp.  68-69). 

Paraminobenzoic  acid.  This  vitamin 
is  unique  among  vitamins  in  that  it 
makes  up  an  integral  part  of  one  of 
the  B  vitamins,  folic  acid.  Its  nutri- 
tional significance  may  depend  on  its 
action  as  a  building  block  of  the  folic 
acid  molecule.  The  catalytic  action 
of  paraminobenzoic  acid  probably  de- 
pends upon  this  latter  property. 
P.  Permeability  factor  (citrin),  consid- 
ered to  be  a  minor  water-soluble  vita- 
min. The  active  fraction  of  vitamin  P, 
extracted  from  lemon  peels,  is  a  mixture 
of  glucosides.  Rutin  is  another  flavone 
glucoside  isolated  from  tobacco  leaves 
and  buckwheat  which  resembles  vita- 
min P  in  structure  and  activity.  It 
was  first  used  clinically  by  Griffith, 
J.  Q.,  Couch,  J.  F.  and  Lindauer,  M. 
A.,  Proc.  Soc.  Exp.  Biol.  &  Med.,  1944, 
55,  228-229  and  since  has  been  exten- 
sively employed  for  the  control  of  capil- 
lary fragility.  This  vitamin  may  act 
as  a  biological  antioxidant  for  ascorbic 
acid,  or  epinephrine,  or  it  may  inhibit 
hyaluronidase  activity. 

Volkonsky  Method  for  mitochondria.  This 
is  a  complicated  technique  involving 
staining  with  anilin  fuchsin,  aurantia, 
methylene  violet  and  azure  II  but  can 
give  splendid  results.  See  original  ac- 
count (Volkonsky,  M.,  Bull,  d'hist. 
AppL,  1928,5,  220-222). 

Volume.  As  explained  by  Danielli  (Bourne, 
1951  p.  102),  cell  volume  is  a  function 
of  the  number  of  contained  osmoti- 
cally  active  particles  unless  change  is 
restricted  by  rigidity  of  the  enveloping 
membrane.  A  satisfactory  technique 
for  measuring  the  volume  of  red  blood 
cells  is  to  determine  photoelectrically 
light  absorption  of  a  suspension  (Ja- 
cobs, M.  H.,  Biol.  Bull.,  1930,  58,  104). 
The  simplest  way  to  obtain  ratio  for 
cytoplasmic  and  nuclear  volumes  is  to 
outline  nuclei  and  cytoplasms  on  koda- 
loid  and  determine  the  weights  as  has 
been  recently  done  in  carcinogenesis 
(Cowdry,  E.  V.  and  Paletta,  F.  X.,  J. 
Nat.  Cancer  Inst.,  1941,  1,  745-759). 
The  technique,  of  course,  varies  with 
structure  involved,  for  example  thyroid 
colloid  (Stein,  H.  B.,  Am.  J.  Anat., 
1940,  66,  197-211),  fresh  endocrine 
glands  (Swinvard,  C.  A.,  Anat.,  Rec, 
1939,  74,  71-78).  To  determine  volume 
and    cell     numbers    in    small     organs 


(Dornfeld,  E.  J.,  et  al.,  Anat.  Rec, 
1942,  82,  255-259).  For  influence  on 
tissue  volume  of  various  methods  of 
fixation,  dehydration  and  imbedding, 
see  Stowell,  R.  E.,  Stain  Techn., 
1941,  16,  67-83. 
Volume  measurements 

1  liter  =  2.1  U.  S.  pints   (1.76  Imperial 
pints) 

1   cc.  =  16i  minims    (17  minims   B.P.) 

1  gallon  =  3.79  liters  (1  Imperial  gallon  = 
3.79  liters) 

1  pint  =  473  cc.  (1  Imperial  pint  =  568  cc.) 

1  fluid  ounce  =  29.5  cc.    (1  fluid  ounce 
B.P.  =  28.4  cc.) 

1  fluid  drachm  =  3.7  cc.  (1  fluid  drachm 
B.P.  =  3.5  cc.) 

1  minim  =  0.065  cc. 

For  accurate  measurements  in  capil- 
lary tube  colorimetry  and  titrametric 
techniques  capillary  tubes,  pipettes, 
microliter  burettes  and  other  instru- 
mentation is  required.  These  are  de- 
scribed in  detail  by  Click  1949.  To 
measure  the  volume  of  irregular  aspects 
(0.01  to  1.0  M  1)  a  method  of  Holter, 
H.,  C.  rend.  trav.  lab.  Carlsberg,  S6r. 
chem.,  1945,  25,  156-167  is  suggested. 
This  is  a  colorimetric  technique,  the 
amount  of  color  displaced  by  the  ob- 
ject being  measured. 
Volutin.  Spherical  bodies  in  fungi,  bacteria 
and  other  organisms  (Taylor  in  Mc- 
Clung's  Microscopical  Technique,  p. 
221).  According  to  R.  F.  MacLennan, 
in  Calkins,  G.  N.  and  Summers,  F.  M., 
Protozoa  in  Biological  Research.  New 
York:  Columbia  University  Press. 
1941,  1148  pp.,  the  term  "volutin  should 
either  be  dropped  or  definitely  re- 
stricted to  metachromatic  granules 
which  respond  to  Feulgen's  stain  when 
used  without  hydrolysis." 
Volocidae,   technique  for,   Hartmann,  M., 

Arch.  f.  Protistenk.,  1918,  39,  1. 
Von  Kossa,  see  Calcium. 
Vulpian  Reaction  named  after  a  Parisian 
physician.  Fresh  slices  of  the  adrenal 
immersed  in  dil.  aq.  ferric  chloride  show 
a  green  coloration  of  the  chromaffin  cells 
of  the  medulla.  It  is  a  test  for  tissues 
producing  epinephrine.  See  :  chromaffin 
reaction  and  osmic  acid. 
Walker's  Method  for  intestinal  protozoa  is 
recommended  as  an  excellent  rapid 
stream.  However,  almost  equally  satis- 
factory results  can  be  obtained  by  the 
more  tedious  method  of  frequently 
changing  the  water.  Osmic  acid  con- 
taining fixatives  are  to  be  washed  in  aq. 
dest.  for  about  an  hour.  After  Regaud's 
fixative  the  tissue  is  transferred  to  3% 
aq.  potassium  bichromate  without  wash- 
ing in  water.  Tissues  fixed  in  alcoholic 
mixtures  are  to  be  briefly  washed  in 
alcohol    before    dehydration.     For    de- 


WASH-OUT  RECOVERY  METHOD 


377 


WASHING 


tails  about  washing  see  the  individual 
fixatives. 

Wash-out  Recovery  Method  (WO)— Written 
by  C.  C.  Macklin,  Dept.  of  Histological 
Research,  The  University  of  Western 
Ontario,  London,  Canada.  November 
28,  1951 — A  cannula  loaded  with  physio- 
logical saline,  serum  or  other  medium 
is  tied  into  the  trachea  of  a  mouse  or 
other  mammal  and  the  contents  in- 
jected into  the  fresh  lung,  withdrawn, 
and  examined  as  fresh  or  dried  and 
stained  mounts.  For  most  animals  it 
is  convenient  to  wash  out  a  part  of  a 
lung  through  a  cannulated  bronchus. 
Granular  alveolar  and  phagocytic  al- 
veolar cells  (pneumonocytes)  are  so 
recovered.  (Macklin,  C.  C,  Proc.  6th 
Intern.  Cong.  exp.  C.ytology,  Stock- 
holm, 1947;  published' 1949,"  383-387). 
See  Dust  Cells,  Foam  Cells,  Vacuoloids. 

Wasserblau,   see   Brazilin-Wasserblau. 

Water  Absorption  bj'  slices  of  liver.  The 
method  has  been  standardized  by  Sperry 
and  Brand  (W.  M.  and  F.  C,  Proc.  Soc. 
Exp.  Biol.  &  Med.,  1939,  42,  147-150) 
and  may  prove  useful  in  the  investiga- 
tion of  other  tissues. 

Water  Blue  (Wasserblau),  see  Anilin  Blue. 

Wear  and  Tear  pigment,  see  Lipofuscin. 

Weigert  Method.  For  myelin  sheaths. 
Kultschitzky  modification  (Romeis,  B. 
Taschenbuch  der  mikroskopischen  tech- 
nik,  ii  Auflage  Section  999,  p.  332).  Fix 
in  10%  formalin  and  mordant  in  Miil- 
ler's  Fluid,  or  in  Formalin  Miiller  or  in 
Weigert's  Quick  Mordant.  Bring  par- 
affin or  celloidin  sections  to  water.  Im- 
merse in  3%  aq.  potassium  bichromate  or 
in  Miiller's  fluid  12  hrs.  Stain  for  12- 
24  hrs.  in  :  10%  hematoxylin  in  abs.  ale. 
(1-6  months  old),  10  cc;  aq.  dest.,  100 
CO.  Wash  and  destain  in:  aq.  lithium 
carbonate,  100  cc. ;  1%  aq.  potassium 
ferricyanide,  10  cc.  until  clear  differen- 
tiation appears  between  gray  and  white 
matter.  Wash,  dehydrate  and  mount. 
The  following  is  provided  by  Dr.  J. 
L.  O'Leary :  Mordanting  in  the  Weigert 
procedure  serves  two  purposes :  (1 )  It 
'renders  the  myelin  sheath  components 
insoluble  in  the  fat  solvents  necessary 
to  secure  dehydration  and  imbedding. 
(2)  It  distributes  the  chromate  ion  in 
sufficient  concentration  in  the  myelin 
sheaths  to  ensure  the  formation  of  an 
adequate  lake  with  hematoxylin  in  the 
subsequent  staining  procedure.  If  par- 
affin imbedding  is  to  be  used,  it  is  abso- 
lutely necessary  to  carry  block  mordant- 
ing to  the  point  where  the  myelin  of  all 
fibers  has  been  rendered  insoluble.  For 
this  reason  paraffin  imbedding  of  mate- 
rial to  be  used  for  Weigert  staining 
should  be  restricted  to  small  nerves  and 
thin   pieces   of   spinal    cord,   otherwise 


overhardening  results.  Here  excellent 
results  are  to  be  achieved,  the  smallest 
fibers  staining  as  completely  as  by  the 
osmic  acid  method.  Two  methods  are 
applicable  to  paraffin  imbedded  sections, 
the  procedures  for  which  are  given  sub- 
sequently. These  are  :  the  Kultschitzky 
modification  of  the  Weigert  method  and 
technique  for  routine  diagnostic  work 
by  Craig,  p.  55.  To  make  the  stain 
dissolve  1  gm.  hematoxylin  crystals  in 
300  cc.  aq.  dest.  with  aid  of  a  little  heat 
and  add  100  cc.  sat.  aq.  ammonium  alum 
with  a  crystal  of  thymol.  Allow  to 
ripen  10  days  in  flask  stoppered  with 
cotton;  then  keep  in  dark.  Fix  smears 
in  Schaudinn's  Fixative  5-10  min. 
Wash  thoroughly  in  several  changes 
aq.  dest.  Immerse  in  above  hematoxy- 
lin solution  3-5  min.  Then  pass 
through  50,  60,  70,  90  and  95%  alcohol, 
at  least  5  min.  each.  After  immersing 
in  absolute  10  min.  clear  in  xylol  and 
mount  in  xylol  balsam. 

Warburg's  Respiratory  Enzyme,  see  Cyto- 
chrome-Oxidase. 

Warthin-Starry  method  for  spirochaetes  in 
sections  has  been  modified  by  Faulkner, 
R.  R.  and  Lillie,  R.  D.  Stain  Techn., 
1945,  20,  81-82  by  the  use  of  a  buffered 
solution.  Use  Walpole's  buffer :  18.5  cc. 
of  solution  of  11.8  cc.  acetic  acid  in 
1000  cc.  aq.  dest.  +  1.5  cc.  of  solution 
of  16.4  gm.  sodium  acetate  in  1000  cc. 
aq.  dest.  which  gives  pH  of  3.6.  1. 
Pass  paraffin  sections  through  xylol  and 
alcohols  to  aq.  dest.  buffered  to  pH  3.6 
by  addition  of  20  cc.  of  above  buffer  to 
480  cc.  aq.  dest.  2.  Impregnate  45  min. 
at  55-60°C.  in  paraffin  oven  in  1%  aq. 
silver  nitrate  similarily  buffered.  3. 
Place  slides  sections  up  on  glass  rods 
pour  on  developer  previously  warmed 
to  55-60°C.  This  developer  is  made  by 
heating  15  cc.  5%  aq.  gelatin  in  above 
buffered  aq.  dest.  and  just  before  using 
add  3  cc.  2%  aq.  silver  nitrate  and  1  cc. 
3%  aq.  hydrochinone  also  made  up  in 
the  same  buffered  solution.  While  de- 
veloping avoid  direct  sunlight  and  cold 
drafts.  Continue  3-5  min.  until  sec- 
tions become  golden  brown  or  grayish 
yellow  and  developer  starts  to  turn 
black.  Pour  off,  rinse  with  warm  55- 
60°C.  tap  water  and  then  with  aq.  dest. 
4.  Dehydrate,  clear  and  mount  in  xylene 
clarite  or  balsam.  Spirochaetes  black. 
Recommended  for  syphilitic  lesions, 
yaws  and  Vincent's  stomatitis. 

Washing.  The  surplus  of  most  aqueous 
fixatives  is  removed  by  washing  the  tis- 
sue in  water.  In  the  case  of  Zenker's 
fluid,  for  example,  wash  for  12-24  hrs. 
in  running  tap  water.  A  convenient 
way  is  to  cover  the  wide  mouth  of  a 
bottle  containing  the  tissue  with  gauze 


WEIGERT'S  MORDANTS 


378 


WEIL'S  METHOD 


secured  by  an  elastic  band .  Water  from 
the  tap  is  allowed  to  drop  onto  the  gauze 
or  better  is  led  into  the  bottle  through 
the  gauze  in  a  small  glass  tube.  Most 
laboratories  are  provided  with  many 
such  water  carrying  tubes.  The  water 
pressure  should  be  so  regulated  that  the 
tissue  is  not  bumped  about  by  the 
O'Leary's  Brazilin  method.  All  large 
blocks  of  brain  or  spinal  cord  should  be 
imbedded  in  celloidin,  the  length  of  time 
in  celloidin  and  the  type  of  celloidin  to 
be  used  being  determined  by  the  thick- 
ness of  the  sections  desired.  The  fol- 
lowing general  rules  apply  to  the  block 
mordanting  of  material  to  be  stained  by 
the  Weigert  method : 

1.  If  it  is  advisable  to  stain  nerve  cells 
and  myelinated  fibers  in  alternate  sec- 
tions, it  is  best  to  forego  block  mordant- 
ing in  Miiller's  fluid.  Formalin  fixed 
blocks  are  imbedded  directly  in  celloidin 
and  alternate  sections  are  stained  by 
Weil's  Method  and  the  Gallocyanin 
Technique. 

2.  If  only  staining  by  a  Weigert  pro- 
cedure is  contemplated,  the  blocks  may 
be  mordanted  in  Miiller's  fluid  for  sev- 
eral weeks  to  several  months  depending 
upon  the  size  of  the  block,  imbedded  in 
celloidin  and  stained  by  the  Weigert-Pal 
method. 

3.  In  special  cases  (cerebral  cortex) 
the  small  myelinated  fibers  are  stained 
completely  with  great  difficulty.  Blocks, 
premordanted  or  not,  are  sectioned  in 
celloidin  and  the  sections  given  long 
mordanting  (one  week  to  one  month)  in 
Miiller's  fluid.  Stain  by  Kultschitzky 
modification  of  Weigert  or  Weigert-Pal. 

Weigert's  Mordants.  (1)  Primary,  or  rapid 
mordant :  potassium  bichromate,  5  gm. ; 
Fluorchrome,  2  gm.;  boiling  aq.  dest., 
100  cc.  (2)  Secondary,  or  copper,  or 
neuroglia  mordant:  boil  2.5  gm.  Fluor- 
chrome with  1(X)  cc.  aq.  dest.  Take 
away  flame.  When  boiling  has  stopped, 
add  5  cc.  glacial  acetic  acid,  then  5  gm. 
finely  powdered  copper  acetate.  Stir 
vigorously  until  dissolved  and  cool. 

Weigert  Pal  Method.  For  myelin  sheaths 
(Dr.  J.  L.  O'Leary,  personal  communi- 
cation). Fix  in  10%  formalin,  1-2  wks. 
Wash  in  running  tap  water,  3  hrs.  Mor- 
dant in  Miiller's  fluid  1  wk.  to  3  mo. 
depending  on  the  size  of  block.  Change 
Miiller's  thrice  weekly  at  first,  later  once 
weekly.  Wash  in  running  tap  water,  6- 
12  hrs.  Imbed  in  celloidin.  Cut  sec- 
tions 20-100  M  depending  upon  size  of 
block  and  detail  desired.  0.25%  aq. 
chromic  acid,  3-5  hrs.  3  changes  aq. 
dest.  10%  hematoxylin  in  abs.  ale. 
ripened  and  diluted  to  1%  with  aq.  dest. 
prior  to  use,  12-24  hrs.  3  changes  of  aq. 
dest.    Differentiate  in  Pal's  fluid  (ox- 


alic acid,  1  gm.;  potassium  sulphite,  1 
gm. ;  aq.  dest.,  200  cc),  alternating  with 
0.25%  aq.  potassium  permanganate  if 
differentiation  is  too  slow.  Wash  in  3 
changes  aq.  dest.  Dehydrate  in  2 
changes  95%  ale.  Clear  in  carbol- 
creosol-xylol  followed  by  pure  toluol. 
Mount  in  balsam.  Myelin  sheaths,  deep 
black;  background,  unstained.  Ano- 
ther variation  of  the  Pal -Weigert  method 
is  given  by  Clark,  S.  L.  and  Ward,  J.  W., 
Stain  Tech.,  1935,  10,  53-55.  See  John- 
son's Neutral  red  for  counterstain. 
Weigert's  Borax  Ferricyanide.  Borax,  1 
gm.;  potassium  ferricyanide,  1.25  gm.; 
aq.  dest.,  100  cc.  A  fluid  for  differentia- 
tion of  hematoxylin  stain  in  Weigert's 
method.  Employed  also  in  copper 
chrome  hematoxylin  method  of  Bensley. 
Weigert's  Resorcin-Fuchsin.  Stain  for  elas- 
tic fibers.  Given  by  Mallory,  p.  168. 
Add  2  gm.  basic  fuchsin  and  4  gm.  resor- 
cin  to  200  cc.  aq.  dest.  Boil  in  enamel 
dish  and  while  boiling,  add  25  cc.  29%  aq. 
ferric  chloride.  Stir  and  boil  2-5  min. 
Cool.  Collect  ppt.  and  discard  filtrate. 
Dry  ppt.  on  filter  paper  and  return  both 
to  the  enamel  dish  which  has  also  been 
dried.  Add  200  cc.  95%  alcohol,  warm 
carefully,  stir  and  discard  filter  paper 
when  ppt.  is  dissolved  out.  Cool,  add 
95%  alcohol  to  200  cc.  and  4  cc.  hydro- 
chloric acid.  Mixture  keeps  well.  For- 
malin fixed  material  is  preferred,  but 
most  other  fixatives  are  satisfactory. 
Stain  paraffin  sections,  after  removing 
paraffin,  for  20  min.  or  more  in  above  mix- 
ture. Wash  off  excess  in  95%  alcohol  and 
differentiate  in  Acid  Alcohol  if  required. 
Wash  thoroughly  in  tap  water.  Dehy- 
drate, clear  and  mount.  Elastic  fibers 
dark  blue  black.  It  is  well  to  stain  nu- 
clei with  Lithium  Carmine  (Orth's) 
before  coloring  the  elastic  tissue.  Wei- 
gert's resorcin  fuchsin  for  elastic  tissue 
has  been  supplemented  by  Masson's 
trichrome  for  other  connective  tissue 
components  in  a  helpful  way  by  Mende- 
loff,  J.  and  Blechman,  H.,  Am.  J.  Clin. 
Path.,  Techn.  Suppl.,  1943,  7,  65. 
Weight  measurements 

1  kilogram  =  2.2  lbs.,  or  35j  ounces 

1  gram  =  15^  grains 

1  pound  =  453.6  gms. 

1  ounce  =  28.4  gms. 

1  drachm  ==  3.89  gms. 

1  grain  =  0.065  gms. 

The  Troy  pounds  and  ounces  are  dif- 
ferent but  seldom  used.  For  weights 
of  organs,  see  Normals. 
Weil's  Method.  For  myelin  sheaths  (Weil, 
A.,  Arch.  Neurol,  a.  Psychiat.  1928,  20, 
392  and  Weil,  A.,  Textbook  of  Neuro- 
pathology, 2nd.  ed.  p.  328.  New  York 
1945.  Place  celloidin  sections  of  for- 
malin   fixed    material    (not    yet    mor- 


WELD 


379 


WILDER'S  METHOD 


danted)  in  aq.  dest.  Stain  for  15  min. 
at  45-50°C.  in  equal  parts  of  4%  aq. 
iron  alum  and  1%  aq.  hematoxylin  pre- 
pared from  10%  sol.  in  abs.  ale.  at  least 
6  months  old.  (Note :  do  not  filter  this 
stain;  do  not  use  it  twice;  mix  fluids 
just  before  using.)  Wash  2  times  in 
tap  water.  Differentiate  in  4%  aq. 
iron  alum  until  gray  matter  or  degener- 
ated areas  become  recognizable.  Wash 
3  times  in  tap  water.  Differentiate 
over  white  background  to  desired  de- 
gree in:  borax,  2.5  gm.;  potassium  ferri- 
cyanide,  12.5  gm.;  aq.  dest.,  1000  cc. 
(For  paraffin  sections,  differentiate  just 
long  enough  in  4%  aq.  iron  alum  to 
remove  stain  from  back  of  slide.  Care 
should  be  taken  not  to  over-differen- 
tiate, for  in  so  doing  fine  fibers  are  lost). 
Wash  2  times  in  tap  water  and  next  in 
aq.  dest.,  to  which  28%  ammonia  had 
been  added  (6  drops  to  100  cc.  of  water) . 
Dehydrate  in  95%  ale,  abs.  ale.  and 
ether  (equal  parts),  clear  in  xylol  and 
mount  in  balsam  or  claritex.  Revised 
by  A.  Weil,  Northwestern  University 
Medical  School,  Chicago,  111.  May  14, 
1946. 

Weld,  a  plant,  Reseda  luteola  which  yields  a 
yellow  dye.  The  use  of  this  source  of 
yellow  coloring  matter  is  said  to  be  of 
greater  antiquity  than  any  other  source 
of  yellow  dye.  It  was  employed  to  dye 
the  clothes  of  the  six  vestal  virgins 
whose  responsibility  it  was  to  keep  the 
fire  burning  in  the  temple  of  Vesta  in 
Rome  (Leggett,  W.  F.,  Ancient  and 
Medieval  Dyes.  Brooklyn:  Chemical 
Publishing  Co.,  Inc.  1944,  95  pp.). 

Wetting  Agents.  These  have  been  used  in 
experiments  designed  to  increase  the 
rapidity  of  penetration  of  fixatives  by 
Chermock,  R.  L.  and  Muller,  H.  E. 
Science,  1946, 103,  731-732.  They  found 
that  Tergitol-4  when  added  to  10%  for- 
malin, Zenker's  fluid  and  some  others 
improved  fixation  and  staining.  Tergi- 
tol-08  was  also  an  advantage  when 
employed  in  Zenker's  fluid.  The 
authors  give  the  literature  on  the 
subject.  Tergitol-7  is  recommended 
in  McClung's  Microscopical  Technique, 
1950,  p.  136  to  accelerate  the  staining 
with  hematoxylin.  One  merely  adds 
one  drop  to  the  jar  containing  the 
aqueous  hematoxylin.  To  add  the 
tergitol  to  the  mordant  before  staining 
with   hematoxylin   is   not   helpful. 

Wetting  Properties.  An  interesting  method 
for  investigating  the  cell  membrane  is 
to  measure  its  wetting  properties.  The 
Mudds  (S.,  and  E.  B.  H.,  J.  Exp.Med., 
1926,  43,  127-142;  J.  Gen.  Physiol.,  1931, 
14,  733-751)  have  noticed  the  responses 
of  cells  to  a  film  of  oil  advancing  between 
slide    and    cover    glass.    Erythrocytes 


are  easily  wetted  by  the  oil; whereas, 
when  leucocytes  are  surrounded  by  the 
film  of  oil,  the  oil  does  not  wet  their  sur- 
faces but  remains  separated  from  them 
by  thin  films  of  saline  solution.  The 
Mudd's  thought  that  this  indicated  that 
the  surface  of  erythrocytes  is  lipoid  and 
that  of  leucocytes  protein.  Danielli 
(Bourne,  p.  78)  has  expressed  the  view 
that  the  surfaces  of  both  cells  are  prob- 
ably coated  with  protein,  the  erythro- 
cytes with  serum  albumen  and  the  leu- 
cocytes with  serum  globulin.  The  wet- 
ting technique  has  been  employed  in  a 
considerable  number  of  experiments. 
Dawson  and  Belkin,  J.  A.  and  M.,  Biol. 
Bull.,  1929,  56,  80-86  and  Marsland,  D., 
J.  Cell.  &  Comp.  Physiol.,  1933,  4,  9-33 
worked  with  amebae  and  Chambers,  R., 
Biol.  Bull.,  1935, 69,  331,  and  Kopac,  M. 
J.  and  Chambers,  R.,  J.  Cell.  &  Comp. 
Physiol.,  1937,  9,  331-361  with  naked 
arbacia  eggs.     See  Cell  Membranes. 

Whole  Mounts  of  tissues  which  are  fairly 
thick  are  often  very  useful.  See  Blood 
Vessels,  Cartilaginous  Skeleton,  Cor- 
rosion Preparations,  Epidermis,  In- 
sects, Mammary  Glands,  Nerve  End- 
ings, Ossification,  etc. 

Wicks,  see  Hydrogen  Ion  Indicators,  Pic- 
colyte  Resins. 

Wilder's  Method  of  silver  impregnation  for 
reticular  fibers  (Wilder,  H.  C,  Am.  J. 
Path.,  1935,  11,  817-819).  Fix  in  10% 
formalin,  Zenker  or  formalin-Zenker. 
Treat  paraffin,  celloidin  or  frozen  section 
in  0.25%  aq.  potassium  permanganate  or 
in  10%  aq.  phosphomolybdic  acid  for  1 
min.  Rinse  in  aq.  dest.  and  transfer  to 
hydrobromic  acid  (Merck's  cone.  34%, 
1  part;  aq.  dest.,  3  parts)  for  1  rain. 
This  can  be  omitted  after  phosphornolyb- 
dic  acid.  Wash  in  tap  water  and  in  aq. 
dest.,  then  dip  in  1%  aq.  uranium  nitrate 
(sodium  free)  5  sec.  or  less.  Wash  in 
aq.  dest.  10-20  sec.  and  place  in  Foot's 
silver  diamino  hydroxide  for  1  min.  To 
make  this:  add  8.1%  aq.  NH4OH  drop  by 
drop  to  5  cc.  10.2%  aq.  AgNOj  until 
brown  ppt.  is  just  dissolved.  Then  add 
5  cc.  3.1%  aq.  NaOH  and  sufficient 
NH4OH  to  just  dissolve  ppt.  IVlake  up 
to  50  cc.  with  aq.  dest.  Dip  quickly  in 
95%  ale.  and  reduce  for  1  min.  in:  aq. 
dest.,  50  cc.;  40%  neutral  formalin 
(neutralized  with  magnesium  carbon- 
ate), 0.5  cc;  1%  aq.  uranium  nitrate, 
1.5  cc.  Wash  in  aq.  dest.  Tone  in 
1:500  gold  chloride  (Merck's  reagent), 
1  min.  Rinse  in  aq.  dest.  and  treat  with 
5%  aq.  sodium  thiosulphate  (hyposul- 
phite), 1-2  min.  Wash  in  tap  water. 
Counterstain  as  desired,  dehydrate, 
clear  and  mount  in  balsam.  Reticular 
fibers  black.  Note  author's  figures  of 
lymph  nodes. 


WILSON'S  STAIN 


380 


XANTHOPROTEIC  REACTION 


Wilson's  Stain  for  Leishmania  is  compli- 
cated. Details  are  provided  by  Craig, 
p.  147  in  whose  opinion  it  gives  no  better 
results  than  Wright  or  Leishman  stains. 

Windaus,  see  Digitonine  Reaction. 

Wintergreen  Oil  (methyl  salicylate)  is  used 
in  the  Spalteholz  Method  of  clearing. 

Wislocki,  see  Placentas. 

Woad  is  a  blue  dye  derived  from  the  plant 
Isatis  tinctoria,  now  only  of  historic 
interest,  as  it  was  replaced  by  indigo 
after  over  a  1,000  years  of  supremacy  in 
Europe.  When,  nearly  2,000  years  ago, 
Julius  Caesar's  Roman  legions  crossed 
the  English  Channel  they  encountered 
a  race  of  Celtic  origin  which  they  called 
"Picts",  or  painted  people,  because 
they  had  punctured  their  skins  with 
flints  in  many  patterns  and  had  rubbed 
into  them  anil  of  the  woad  plant.  The 
account  of  this  dye  by  Leggett  is  in- 
teresting reading  (Leggett,  W.  F.,  An- 
cient and  Medieval  Dyes.  Brooklyn: 
Chemical  Publishing  Co.,  Inc.,  1944, 
95  pp.).  Leggett  quotes  opinion  of 
Guest  that  the  word  "Britain"  is  the 
Latinized  form  of  Brythen,  a  Celtic 
term,  indicating  "painted  men". 

Woods  Metal  is  now  largely  replaced  by 
celluloid  in  the  making  of  corrosion  prep- 
arations. 

Wool  Black  B  (CI,  315),  an  acid  disazo  dye 
of  light  fastness  3  to  4  staining  action 
of  which  is  briefly  reported  (Emig, 
p.  38). 

Wool  Green  S  (CI,  737)  Lillie,  R.  D.,  J. 
Tech.  Methods,  1945,  No.  25,  47  pp.  has 
reported  this  dye  in  a  good  combination 
for  connective  tissue.  Mordant  sec- 
tions 2  min.  in  sat.  ale.  picric  acid. 
Wash  3-5  times  in  running  water  and 
stain  6  min.  in  Weigert's  or  other  iron 
hematoxylin.  Wash  in  water  and  stain 
4  min.  in  1%  Biebrich  scarlet  in  1%  aq. 
acetic  acid.  Wash  in  water  and  mor- 
dant 4  min.  in  10%  dilution  of  U.S. P. 
ferric  chloride  solution.  Wash  in  water 
and  stain  4  min.  in  1%  aniline  blue, 
methyl  blue,  or  wool  green  S  in  1%  aq. 
acetic  acid.  Destain  2  min.  in  1%  aq. 
acetic  acid.  Dehydrate  and  clear  in 
acetone,  acetone  and  xylene  and  in 
xylene.  Mount  in  clarite  in  xylene  or 
in  salicylic  acid  balsam.  Connective 
tissue  and  basement  membranes,  dark 
blue  or  green;  muscle  and  cytoplasm, 
red. 

A  substitute  for  Wright's  stain  is  pro- 
posed by  Saye,  E.  B.,  Am.  J.  Clin. 
Path.,  1943,  Tech.  Suppl.  7,  12-13,  made 
up  of  Eosin  Y  and  Thionin.  It  is 
recommended  for  blood  cells  and  mala- 
rial parasites. 

Wool  Orange  2G,  see  Orange  G. 

Wool  Red,  see  Amaranth. 

Wound  Healing,  method  for  study  in  vitro 


(Bentley,  F.  H.,  J.  Anat.,  1935-36,  70, 
498-506). 

Wright's  Blood  Stain.  This  is  a  compound 
stain  of  the  Romanowsky  type.  The 
Commission  Certified  (C.C.)  product  is 
available.  Dry  the  smear  in  air.  Cover 
the  area  between  the  wax  lines  with 
stain  measured  by  drops  from  a  medicine 
dropper.  After  1  min.  add  same  volume 
aq.  dest.,  shifting  the  slide  a  little  from 
side  to  side  so  that  it  mixes  fairlj-^  well. 
A  green  metallic  looking  scum  forms  on 
the  surface.  Leave  2  or  3  min.  Too  long 
staining  produces  a  precipitate.  It  may 
be  necessary  to  use  for  dilution  instead 
of  aq.  dest.  the  McJunkin-Haden  buffer. 
Wash  in  tap  water  30  sec.  or  more  until 
thin  parts  of  smear  become  pink  or  yel- 
low. Dry  by  blotting  with  smooth  filter 
paper  and  examine  directly  without 
mounting  in  balsam  and  adding  a  cover 
glass.  Usually  excellent  results  are  ob- 
tained. If  however  it  is  desired  to  em- 
ploy buffered  solutions  especially  for 
sections  consult  Petrunkevitch,  A., 
Anat.  Rec,  1937,  68,  267-280  and  Lillie, 
R.  D.,  Stain  Techn.,  1941,  16,  1-6.  The 
other  most  used  blood  stain  is  that  of 
Giemsa  with  its  several  modifications. 
Ehrlich's  triacid  stain  is  less  used 
nowadays . 

X  Bodies,  see  Cytoplasmic  Inclusions  in 
plants. 

Xanthene  Dyes.  As  the  name  implies  they 
are  derivatives  of  xanthene.  They  com- 
prise many  indicators  and  are  classified 
as  acridines,  fluoran  derivatives,  phe- 
nolphthalein,  pyronins,  quinolines,  rho- 
damines,  and  sulfonphthaleins. 

Xanthene     Dye,     see     Phosphine. 

Xanthine,  see  Purines. 

Xanthoproteic  Reaction.  Treat  section 
with  cold  fuming  nitric  acid.  After  a 
few  minutes  the  proteins  become  colored 
yellow.  Then  rinse  and  expose  to  am- 
monia vapor  which  changes  the  color  to 
orange.  Not  specific  for  proteins  be- 
cause there  is  also  a  nitration  of  aromatic 
radicals  of  phenols,  alkaloids,  etc.  The 
color  is  often  faint  but  fairly  sharp 
(Lison,  p.  127).  See  also  Bensleys 
(p.  126). 

The  reaction  is  described  as  follows 
by  Serra,  J.  A.,  Stain  Techn.,  1946,  21, 
5-18:  Fix  tissue  as  given  under  Nin- 
hydrin  Reaction.  "The  pieces  are 
treated  for  some  minutes  with  concen- 
trated HNO3  until  they  become  in- 
tensely yellow.  After  a  washing  in 
distilled  water,  immerse  in  a  diluted 
ammonia  solution,  or  expose  the  pieces 
to  ammonia  vapors.  The  color  changes 
to  orange.  The  observation  can  be 
made  by  mounting  directly  in  pure 
glycerin. 
"The  reaction  is  due  to  the  presence 


XENON 


381 


ZYMOGEN 


of  tyrosine,  phenylalanine  or  trypto- 
phane in  the  protein  molecule,  and  is 
also  given  by  all  phenolic  compounds. 
Among  the  peptides,  only  the  prota- 
mines do  not  show  a  positive  reaction. 
To  withstand  the  treatments,  a  strong 
fixation  is  recommended,  though  the 
reaction  can  also  be  performed  on  fresh 
materials."     See  Purines. 

Xenon,  see  Atomic  Weights. 

XL  Carmoisine  6R,  see  Chromotrope  2R. 

Xylene  Cyanol  FF  (cyanol  FF).  Related 
to  Aniline  blue,  an  acid  dye  of  the 
phenylmethane  series  employed  as 
ultracellular  indicator  (Conn,  H.  J., 
in  McClung's  Microscopical  Technique, 
1950,  p.  103). 

Xylidine   Ponceau  3RS,   see   Ponceau  2R. 

Xyloidin,  see  Pyroxylin. 

Yeasts,  vital  staining  of,  see  Brilliant  Pur- 
purin  R.  Malachite  green-safranin 
technique  for  staining  spores  (McClung, 
L.  S.,  Science,  1943,  98,  159-160). 

Yellow  M,  seeMetanil  Yellow. 

X-Ray  Absorption  of  tissues  provides 
method  of  quantitative  study  of  tissue 
elements  and  their  localization  (Eng- 
strom.  A.,  Acta  Radiologica,  suppl.  53, 
1946).  This  method  has  been  ad- 
vanced by  Engstrom  over  that  de- 
scribed under  Historadiography.  It 
has  been  much  used  in  the  quantitative 
determination  of  the  density  of  enamel 
and  dentin,  see  Boedeker,  C.  F.  and 
Appelbaum,  E.,  Dental  Cosmos.,  1933, 
75,  21. 

X-ray  DifiFraction  method  for  investigating 
structure  of  nerve  myelin  sheath 
(Schmitt,  F.  O.,  Bear,  R.  S.  and  Palmer, 
K.  J.,  J.  Cell.  &  Comp.  Physiol.,  1941, 
18,  31-42.    See,  also,  Historadiography. 

Yaws.  Treponema  pertenue,  18-20  m  long, 
6-20  uniform  spirals.  Same  technique 
as  for  Treponema  Pallida. 

Ytterbium,  see  Atomic  Weights. 

Yttrium,  see  Atomic  Weights. 

Zenker's  Fluid.  Potassium  bichromate, 
2.5  gms.;  mercuric  chloride,  (corrosive 
sublimate)  5  gms.;  aq.  dest.,  100  cc; 
glacial  acetic  acid,  5  cc.  Because  this 
mixture  does  not  keep  well  make  a  stock 
fluid  of  say  2  liters  by  adding  mercuric 
chloride  to  saturation  in  5%  potassium 
bichromate.  It  will  do  no  harm  if  more 
than  sufficient  mercuric  chloride  is  used 
and  remains  undissolved  at  the  bottom 
of  the  bottle.  The  main  point  is  to  reach 
saturation.  This  will  require  several 
hours  unless  the  mercuric  chloride  is 
dissolved  in  the  aq.  dest.  with  the  aid  of 
gentle  heat  before  adding  the  bichromate 
which  has  been  pulverized  in  a  mortar 
to  facilitate  solution. 

Immediately   before   use  add   5%  of 


glacial  acetic  acid.  Fix  tissues  24hrs. 
and  wash  in  running  water  about  12  hrs. 
Dehydrate  and  imbed  in  the  usual  way. 
Remove  mercuric  chloride  from  sections 
by  Lugol's  iodine  solution  5-10  min.  and 
wash  out  the  iodine  in  alcohol  before 
staining.  This  fluid  is  employed  in 
techniques  too  numerous  to  mention. 
It  is  called  for  in  case  of  Mallory's  Con- 
nective Tissue  stain  and  for  demonstra- 
tion of  Tendons,  Purkinje  Cells,  Muscle, 
Fibrin,  Hemofuscin,  etc. 

Zenker  Less  Acetic  is  the  stock  solu- 
tion without  addition  of  acetic  acid. 
This  will  serve  as  a  fixative  for  mitochon- 
dria; because,  since  it  does  not  contain 
acetic  acid,  they  are  not  dissolved.  It 
is,  however,  not  recommended  for  mito- 
chondria. 

Formalin-Zenker  or  Zenker-Formol 
is  a  very  useful  fixative  indeed.  Helly's 
fluid  is  Zenker  with  5%  formalin  in  place 
of  the  5%  acetic  acid.  Maximow  has 
used  10%  formalin  instead  of  5%.  It 
is  added,  like  the  acetic  acid,  just  before 
use.  The  time  of  fixation,  washing,  etc. 
is  the  same  as  for  Zenker's  fluid. 

Ziehl's  Carbol-Fuchsin  (as  emended  Soc. 
Am.  Bact.):  A.  Basic  fuchsin,  0.3  gm.; 
95%  ethyl  alcohol,  10  cc. :  B.  Phenol,  5 
gm.;  aq.  dest.,  95  cc.  Mix  A  and  B. 
Much  used  for  the  staining  of  Acid  Fast 
Bacilli. 

Zinc.  Mendel  and  Bradley's  Method  (L. 
B.  and  H.  C,  Am.  J.  Physiol.,  1905, 
14,  313-327).  Treat  paraffin  sections 
with  10%  aq.  sodium  nitroprussate  for 
15  min.  at  50°C.  Wash  carefully  in 
running  water.  Add  cover  glass.  In- 
troduce under  it  one  drop  potassium 
sulphide  solution  which  causes  an  in- 
tense purple  color  (Lison,  p.  98). 

Zinc  Chloride,  as  substitute  for  mercuric 
chloride  in  Zenker's  fluid  (Russell,  W. 
O.,  J.  Techn.  Meth.  &  Bull.  Int.  Assoc. 
Med.  Museums,  1941,  21,  47). 

Zirconium,  see  Atomic  Weights. 

Zweibaum's  Fixative.  Add  1  part  2%  aq. 
osmic  acid  to  7  parts  3%  aq.  potassium 
bichromate,  6  cc,  2%  chromic  acid,  3  cc. 
and  aq.  dest.,  5  cc.     See  Sudan  Black  B. 

Zymogen  is  substance  within  cells  tliat 
produces  an  enzyme  (G.  zyme,  leaven 
+  gennao,  I  produce).  It  is  usually 
seen  in  the  form  of  granules.  These 
zymogen  granules  as  they  occur  in  the 
acinous  cells  of  the  pancreas,  in  the 
chief  cells  of  the  stomach,  in  the  serous 
(or  zymogenic  cells  of  the  salivary 
glands  and  in  other  situations  can  be  well 
stained  with  Bensley's  Neutral  Gentian 
or  Bowie's  Ethyl  Violet-Biebrich  Scar- 
let. They  can  also  be  readily  studied 
in  living  cells  and  their  behavior  noted 


ZYMOHEXASE 


382 


ZYMONEMA  DERMATITIDIS 


as  material  is  discharged  from  the  cells 
into  the  lumina  of  the  acini  by  a  method 
elaborated  by  Covell,  W.  P.,  Anat.  Rec, 
1928,  40,  213-223.  The  technique  con- 
sists of  carefully  mounting  the  pan- 
creas of  a  living  mouse  in  such  a  way 
that  the  circulation  continues  and  the 
influence  of  pilocarpine  can  be  observed. 
Zymohexase.  This  enzyme  system  is  made 
up  of  aldolase  and  isomerase.  The 
former  converts  hexosediphosphate  into 


dihydrocyacetone  phosphate  and  phos- 
phoglyceraldehyde.  The  latter  cataly- 
ses equilibrium  between  these  two 
products.  Allen,  R.  J.  L.  and  Bourne, 
G.  H.,  J.  Exper.  Biol.,  1943,  20,  61-64 
adapted  methods  for  phosphatase  to 
the  demonstration  of  zymohexase  ac- 
tivity. See  technique  and  comments 
by  Glick,  p.  86-88. 
Zymonema  Dermatitidis,  see  Blastomyco- 
sis.