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LABORATORY 
TECHNIQUE 

IN 

BIOLOGY  AND  MEDICINE 


BY 

E.  V.  COWDRY 

Professor  of  Anatomy,  Washington  University,  and 

Director  of  Research,  The  Barnard  Free  Skin  and 

Cancer  Hospital,  St.  Louis 


SECOND  EDITION 


BALTIMORE 

THE  WILLIAMS  &  WILKINS  COMPANY 

1948 


Copyright,  1948 
The  Williams  &  Wilkins  Company 


Made  in  the  United  States  of  America 


Published  1943 
Second  Edition  1948 


Formerly  known  as 
Microscopic  Technique  in  Biology  and  Medicine 


Composed  and  Printed  at  the 
WAVERLY  PRESS,  INC. 

FOR 

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


PREFACE  TO  THE  SECOND  EDITION 

Advances  in  technique  have  continued.  Those  Avho  would  learn  about  the 
minute  structure  of  the  body  in  health  and  disease  must  have  many  methods  of 
investigation  at  their  disposal  so  that  they  can  choose  the  one  most  likely  to  be 
useful.  To  be  dynamic,  their  concept  of  structure  must  include  the  organization 
of  living  material  in  space  from  what  can  be  seen  with  the  naked  eye  down 
through  that  which  is  microscopically  visible  to  submicroscopic  groupings  of  the 
smallest  particles  known  to  physicists.  Consequently  brief  descriptions  of, 
and  leading  references  to,  physical  methods  capabb  of  yielding  information 
as  to  the  organization  of  living  material  are  much  in  demand.  The  same  can 
be  said  for  microchemical  techniques,  because  the  more  accurately  investigation 
of  chemical  composition,  and  changes  therein,  can  be  focussed  on  vital  structural 
units  the  more  effective  it  becomes.  Moreover  living  organisms  from  the 
highest  to  the  lowest  have  many  features  in  common.  Techniques  of  proved 
value  at  one  level  in  the  scale  may  well  be  of  use  in  the  investigation  of  higher  or 
more  lowly  organisms  so  that  it  is  well  to  present  them  briefly;  for  otherwise 
the  purpose  of  this  book,  to  expose  in  an  introductory  way  the  technical  oppor- 
tunities for  research,  v/hould  be  limited.  For  these  reasons,  and  also  to  include 
new  methods  discovered  since  the  first  edition  was  published,  as  well  as  im- 
provements in  manj^  standard  techniques  this  book  has  been  considerably 
expanded. 

As  in  the  case  of  the  First  Edition,  I  am  grateful  to  many  of  my  friends  for  the 
frankness  of  their  criticism  and  their  help.  This  assistance  has  been  given  in 
two  principal  ways.  In  numerous  cases  techniques  published  in  the  first  edition 
have  been  submitted  to  those  who  devised  them  for  revision  which  is  acknowl- 
edged in  the  text.  In  other  cases  investigators  have  themselves  written  for 
me  accounts  of  their  own  methods,  or  of  groups  of  techniques  in  the  use  of 
^vhich  they  are  leaders,  which  accounts  are  of  course  credited  to  them.  Mr.  J.  M. 
Albrecht  has  given  very  helpful  technical  advice.  Thanks  arc  due  to  Miss 
Margaret  Goessling  for  help  in  preparing  the  manuscript. 


^^OSi 


PREFACE  TO  THE  FIRST  EDITION 

AVhat  appeared  altogether  impossible  twentj'-five  jTars  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  relying  more  upon  microchemical  and  physical 
procedures  are  not  explored  as  they  should  be.  The  purpose  of  this  book  is  to 
extend  the  horizon  bj^  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  like  Pepsin  and  Phosphatase  and  of  many 
other  components  of  living  material.  It  is  diflScult  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 


VI  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  the}^  will  wish  to  dig  out  what 
they  want  as  directly  and  quickh'  as  possible.  It  is  for  this  reason  that  every- 
thing is  listed  alphabetically.  Obviously  this  book  can  be  nothing  more  than 
a  brief  entr^  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  particularl}^  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,  Chairm.an  of  the 
Biological  Stain  Commission,  who  very  kindly  read  the  manuscript  and  made 
several  useful  suggestions. 


CHOICE  OF  METHODS 

The  selection  uill  depend  upon  what  it  is  desired  to  do.  In  most  cases  a 
particular  kind  of  information  is  sought.  Feasible  methods  of  obtaining  it 
with  the  materials  available  are  needed  and  it  is  important  that  the  information 
secured  be  trustworthy  ha\dng  a  minimum  of  experimental  error.  A  brief  out- 
line of  what  can  be  done  is  presented  in  the  hope  that  some  of  the  techniques 
mentioned  will  be  suitable  or  will  suggest  satisfactory  ways  to  proceed.  Further 
data  are  given  in  the  bodj^  of  this  book  concerning  the  subjects  given  in  bold 
face  type. 

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.  Y/ith  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  seriousl}^  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 
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  underh' ing  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  directl}"  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  readih^  be  seen  without  any  surgical  procedure.  It 
is  even  ]jossible  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, 

1 


Z  CHOICE    OF   METHODS 

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  greatlj'-  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 
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  (Wearn,  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.  For 
collection  of  alveolar  fluid  see  Terry,  R.  J.,  Anat.  Rec,  1926,  32,  223-304;  1936, 
64,  75. 

Other  investigators  have  availed  themselves  of  the  natural  \^indow,  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- 


CHOICE    OF   METHODS  6 

Tnals.  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,  0.,  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  Covell  (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  shght  displacement  of  the  spleen  so  that  it  can  be 
transilluminated  b}^  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. 

The  first  method  of  Reconstruction  from  serial  sections  is  well  known.  Briefly 
stated  the  particular  tissue,  organ  or  sj^stem  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,  formerl}^  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  aw^ay  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  av/ay  the  tissues  which  it  is  desired  to  eliminate  or 
loosen  their  connections  wdth  those  under  investigation,  which,  latter,  can  then 
be  individually  examined.     Techniques  of  this  sort  are  the  only  available  means 


4  CHOICE   OF  METHODS 

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  exammation  of  serial  sections.  The  Epidermis  is  so  tightly  boimd  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  epithelial  sheets  can 
be  similarly  isolated.  However  such  sheets  are  of  little  value  for  chemical 
analysis  because  of  the  action  of  the  acetic  acid.  Fortunately  it  has  been  found 
that  the  epidermis  ma}''  also  be  quickly  loosened  bj''  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  bj''  the  method 
of  Spalteholz  or  Schultze.  These  techniques  give  admirable  results  in  the  study 
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  topograpliic  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 


CHOICE   OF   METHODS  5 

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  wdth;  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  Carnoy'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  wliich 
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. 

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-likc  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  arc  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. 


6  CHOICE   OF  METHODS 

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 
djdng.  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 
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 
poliomyelitis  (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  brilliant  colors  is  obtainable  wrlh.  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 


CHOICE    OF   METHODS  7 

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  thej^  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. 

Thus  cells  can  be  grown  in  Tissue  Cultures  of  chemical  composition  suited  to 
their  requirements.  Mixed  cultures  are  those  containing  se^'eral  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 


8  CHOICE    OF   METHODS 

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  hving  cells  in  tissue  cultures  than  to  cells  viewed  in  other  situations.  In 
tissue  cultures  they  can  be  photographed  at  high  magniiication,  both  by  direct 
illumination  and  in  the  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 
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 
difi"ering  in  vascularity,  fiber,  fat,  tissue  fluid  and  gland  contents  in  various 


CHOICE    OF   JIETHODS  9 

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,  when 
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  fiuid  ^^  hich  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  volatiHzed 
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  stnicturc  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. 

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  enzymatic  properties  with  the  cellular  composition  of  tissues,  the 
localization  of  many  others  can  be  inferred. 

In  the  case  of  these  and  other  microchemical  ir.ethods  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- 


10  CHOICE  OF   METHODS 

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  Altniann-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  fiarts  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  applicable  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  infonna- 
tion  in  the  following  reference  books.  (1)  New  and  Nonofficial  Remedies, 
1946.  Chicago:  Am.  Med.  Assoc,  770  pp.;  (2)  The  National  Formulary. 
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  validity.  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  photometricalh^  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. 

UtiUzation  of  physical  techniques  in  biology  and  medicine  is  now  the  order 


CHOICE   OF   METHODS  11 

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.  Dififraction  methods  are  labor  saving 
and  often  preferable.  Filters  of  djifferent  porosity  are  available  so  that  the 
sizes  of  particles  passing  through  can  be  roughly  gaged.  To  employ  llltra- 
centrifugation  techniques  are  among  several  other  possibilities.  There  are 
now  Microscopes  of  manj^  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  ^  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  knoAvledge  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. 
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  nearlj'  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  mil  depend  to  some  extent  on  the 
propert}'^  under  consideration  and  on  the  number  of  observations  necessary  to 


12  CHOICE    OF   METHODS 

establish  the  percentage  within  the  Hmits  of  probability.  It  would  not  do  to 
compare  the  amount  of  pigment  in  the  specimen,  the  normality  of  wliich  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  wliich  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  Avisely  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 
sj'nonyms,  and  it  is  difficult  to  be  sure  of  their  meaning.  Two  comprehensive 
dye  indexes  have  been  published.  One,  "Schultz'  Farbstofftabellen",  is  now  in 
its  7th  edition  (1928  to  1934)  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  Chemusts 
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  difTerent  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  v/ere  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 
concerning  the  nature  of  dyes  as  related  to  their  use  in  microscopic  technique; 

13 


14  STANDARDIZATION    OF   STAINS 

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  publication  by  the 
Commission  of  a  quarterly,  "Stain  Technology."  The  second  object  is  being 
brought  about  by  the  certifjang  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  would 
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  fourth  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 
office;  (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  (1942)  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,  ahzarin  red  S  (C.C). 

Alizarin  red  S  Carmine 

Anilin  blue,  water  soluble  Chlorazol  black  E 

Auramine  O  Congo  red 

Azocarmine  G  Cresyl  violet 

Azure  A  Crystal  violet 

Azure  B  Eosin,  bluish 

Bismarck  brown  Y  Eosin,  yellowish 

Brilliant  cresyl  blue  Erythrosin  B 

Brilliant  green  Ethyl  eosin 


STANDARDIZATION   OF   STAINS 


15 


Fast  green  FCF 

Fuchsin,  acid 

Fuchsin,  basic 

Giemsa  stain 

Hematoxylin 

Indigo  carmine 

Janus  green  B 

•Tenner's  stain 

Light  green,  S.F.,  yellowish 

Malachite  green 

Martins  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 

Resazurin 

Rose  bengal 

Safranin  O 

Sudan  III 

Sudan  IV 

Tetrachrome  stain  (MacNeal) 

Thionin 

Toluidine  blue  O 

Wright's  stain 


Eight  ccmpanies  in  the  United  States  are  now  submitting  their  stains  to  the 
Commission  for  certification  before  putting  them  on  the  market.  It  must  be 
reahzed,  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 
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  specialize.  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  Executive  Committee  of  the  Com- 
mission. This  interlocking  membership  is  assurance  that  the  work  of  preparing 
staining  formulae  for  the  next  edition  of  the  National  Formulary  is  being  carried 
on  in  close  cooperation  with  the  Stain  Commission.  This  cooperation  has  al- 
ready resulted  in  two  important  steps: 

1.  Specifications  of  the  most  important  stains  now  on  the  certification  basis 
have  been  drawn  up  for  the  National  Formulary  (1942).  These  specifications 
are  partly  chemical  and  spectrophotometric,  but  also  contain  detailed  state- 


IG  STANDARDIZATION   OF   STAINS 

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,"  pubhshed  by  the 
Society  of  American  Bacteriologists,  have  been  compared  and  critically  studied 
with  the  object  of  making  them  identical  in  all  three. 

ABBREVIATIONS 

1  M  (Greek  letter  for  micron)  =  1/lOOOth  part  of  a  millimeter  (mm.)  =  0.001  mm.  =  10"' 

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

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

10-»  mm.  =  0.000,001  m  =  10"^  A. 
1  Kg.  =  approximately  2.2  lbs. 

1  gm.  =  10-3  Kg.^  0.001  K.,  1000  mgm.,  1,000,000  /xg. 
1  mgm.  =  10-«  Kg.,  10-3  gm.,  1000  fig. 
1  pg.  =  l-y  =  10-«  Kg.,  10-6  gru.^  10-3  mgm. 

N  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.). 
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. 
Bourne,  G.,  Cytology  and  Cellular  Physiology,  Oxford:  Clarendon  Press,  1942,  296  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. 
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. 
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.) 
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. 
Simmons,  J.  S.  and  Gentzkow,  C.  J.,  Laboratory  Methods  of  the  United  States  Army, 

Philadelphia:  Lea  &  Febiger,  1944,  823  pp. 
Stitt,  E.  R.,  Clough,  p.  W.  and  M.  C,  Practical  Bacteriology,  Haematology,  and  Animal 

Parasitology,  Philadelphia:  Blakiston,  1938,  961  pp. 


TECHNIQUES 


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

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). 

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). 

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). 

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. 

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  (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  parafiin  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. 
-f-  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- 


17 


ACID  FAST  BACILLI 


18 


ACID  RUBIN 


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.  A  brecht  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  +  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.  Tuberc,  1941, 
44,  255-266.  Efiiciency  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 
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  Phloxine  GR,  see  Chromotrope  2R. 

Acid  Rubin,  see  Acid  Fuchsin. 


ACID  VIOLET 


19 


ADRENAL 


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. 

Acidopliilic,  see  Staining. 

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

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). 

Acriflavine  (CI.  790).  A  yellow  fiuorchrome. 
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  Flhrlich's  aniline  crystal  violet  (see 
Anilin  Crystal  Violet), 5-15  min.  Wash 
in  water  and  treat  with  Gram's  Iodine, 
1  rain.  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. 

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. 

Adenylpyrophosphata.se.  The  technique  of 
localization  of  this  important  enzyme 
in  cytoplasmic  granules  has  been  de- 
scribed and  used  in  extracts  of  chick 
embryos  bv  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  Click  and  Fischer,  Science, 
1945,  102,  429-430. 

Adhesiveness,  or  stickiness  of  cellular  sur- 
faces is  a  phenomenon  of  great  im- 
portance in  connection  with  movement, 
phagoc3'tosis  embryological  develop- 
ment and  other  processes.  There  is  no 
standard  technique  to  iiieasure  it,  ex- 
cept in  special  circumstances  as  when 
it  is  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., 


ADRENAL 


20 


ALCOHOL-FORMALIN 


Am.  J.  Anat.,  1940,  67,  151-227  for  keto- 
steroid  cortical  hormone  said  bj^  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). 

Agar,  as  matrix  for  cutting  plant  material 
with  freezing  microtome  (Evenden,  W. 
and  Schuster,  C.  E.,  Stain  Techn., 
1938,  13,  145-146). 

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  CoHagenic  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. 

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 
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,  I,  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.  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, 
Carnoy'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-Formalin  is  a  fixative  containing  9 
parts  of  absolute  alcohol  and  1  part  of 
formalin.  Since  it  penetrates  quickly 
and  deh3'dration  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'  tiine.  For 
routine  purposes  3-6  hrs.  fixation  will 
suffice  but  as  a  preliminary  to  Micro- 
incineration 24  hrs.  is  recommended. 
Alcohol-formalin  is  recommended  for 
Fibrin,  Glycogen,  Indigo-Carmine 
stains  aud  Peroxidase.  It  is  employed 
with  acetic  acid  in  Bodian's  Method 
for  nerve  fibers. 


ALDEHYDE  GREEN 


21 


ALTMANN'S  METHOD 


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  xjdene  and  sclerenchyma  gives 
rather  brighter  shade  than  Alalachite 
Green  (Emig,  p.  48). 

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  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  bj"  a  mordant  dye.  Additional 
diiections  are  supijlied  (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.  ^-1  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,  sec  Phosphatase 
and  Kidney. 

Alkalinity,  see  Hydrogen  Ion  Concentration. 

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

Allergy,  see  Pollens. 

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- 
distinctlj'-  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)." 

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  4^.  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  yellow   background.     Altmann's 


ALTMANN'S  METHOD 


22 


ALVEOLAR  EPITHELIUM 


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- 
liminarj'  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 
paraffin  without  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. 

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  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 
carrnalum.  Gives  blue  violet  color. 
Very  penetrating  but  not  so  specific  for 
chromatin  as  carmalum   (Lee,  p.  142). 

Alveolar  Epithelium  of  Lungs 

1.  Gold  sodium  thiosulphate  (Bensley, 
R.  D.  and  S.  H.,  Anat.  Ilec,  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  bj"  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.  D.  and  S. 


ALVEOLAR  EPITHELIUM 


23 


AMYLASE 


H.,  Anat.  Rec,  1935,  64,  41-49).  Use 
guinea  pigs.  Silver  Citrate  sol.  (which 
see)  is  injected  intx)  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  Pores  of  the  lung  (Macklin,  C.  C. 
Arch.  Path.,  1936,  21,  202-216).  For- 
malin (10%)  and  Zenker-formalin  are 
among  the  fixatives  suggested.  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  physiological  salt  solu- 
tion. 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;u  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  (Revised  by  C.  C.  Macklin, 
Dept.  of  Histology,  University  of 
Western  Ontario,  London,  Canada, 
1946). 

Alzheimer's  Modification  of  Mann's  eosin- 
methyl  blue  for  neuroglia  and  degenerate 
nerve  fibers  as  given  by  IVIallory  (p.  245) 
is  abbreviated.  Fix  thin  slices,  14  days, 
in  Weigert's  Neuroglia  Mordant  +  10% 
of  formalin.  Wash  8-12  hrs.  in  running 
water.  Mordant  lOyu  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  0,  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,  see  Endamoeba. 

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

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. 

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). 

Ammonium  molybdate,  as  mordant  for 
Mann's  stain  and  Weigert-Pal  (Perdrau, 
J.  R.,  J.  Path.  &  Bact.,  1939,  48,  609- 
610). 

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  and 
the  latter  plastered  on  its  surface. 

Amphophilic,  see  Staining. 

Amyl  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.  Merck  lists  3,  commercial, 
normal  and  tertiary.  It  mixes  with 
95%  alcohol  and  with  xylol.  Hollande 
(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 
stained  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 


AMYLASE 


24 


ANILIN  CRYSTAL  VIOLET 


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 
and  Engel  (H.and  C,  Enzymologia,  1938, 
5,  71-80)  localized  this  enzyme  by 
analj^sis  of  horizontal  sections  through 
the  intestinal  wall.  It  was  found  that  it 
is  present  in  rabbits  in  maximum  amounts 
in  Brunner's  glands.  Holt6r  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. 
Amyloid  (G.  amylon,  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  byMallory 
and  Parker  (IMcClung,  pp.  417-419). 
From  numerous  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  bj'  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  Apdthy'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%  crj^stal  violet  or  methyl 
violet  in  2.5%  aq.  acetic  acid,  washing 
again  in  water  and  mounting  in  Lillie's 
Apathy'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. 

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). 

Angstrom  Unit.  lA  =  0.1  m^  =  0.0001/i  = 
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  CoUagenic  Fibers  and 
is  employed  for  many  other  purposes. 

Anilin  Crystal  Violet  1.  Ehrlich's.  Shake 
up  5  cc.  anilin  oil  with  95  cc.  aq.  dest. 


ANILIN  CRYSTAL  VIOLET 


25 


ANTHRAQUINONE  DYES 


Filter  and  to  84  cc.  of  filtrate  add  16  cc. 
sat.  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  in  alcohol,  clear  in 
xylol,  imbed  in  paraffin  and  cut  sections 
about  4  M  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 
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)  +  95%  ethyl  alco- 
hol, 12  cc.  B :  anilin  oil  2  cc.  +  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. 

Anterior  Chamber  of  Eye.  This  is  in  many 
respects  the  best  site  for  observations 
on  transplanted  tissues.  See  trans- 
plantation of  uterine  mucosa  (Markee, 
J.  E.,  Contrib.  to  Embryol.,  Carnegie 
Inst,  of  Washington,  1940,  28,  219-308) 
and  of  tumors  (Saphir,  O.,  Appel,  M. 
and  Strauss,  H.,  Cancer  Res.,  1941,  1, 
545-547).  Aqueous  humor  is  not  so 
species  specific  as  other  tissue  fluids 
that  have  been  investigated.  Conse- 
quently transplants  from  other  species 
will  often  develop.  There  is  of  course 
the  advantage  of  direct  observation 
through  the  transparent  cornea.  More 
recently  the  technique  of  transplanting 
animal  and  human  tumors  into  this 
favorable  environment  has  been  de- 
veloped mainly  by  Greene  and  his  asso- 
ciates. For  discussion  of  literature 
and  techniques  see  Greene,  H.  S.  N.  and 
Murphy,  E.  D.,  Cancer  Research,  1945, 
269-282. 

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


ANTICOAGULANT  SOLUTIONS 


26 


ARGINASE 


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. 

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. 

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). 

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  Fontana's  sol.  prepared  by 
adding  NH4OH  to  5%  AgNOa  until  ppt. 
is  dissolved,  then  AgNOa  drop  by  drop 
until    fluid    exhibits    slight    presistent 


opalescence.  Wash  in  glass-dist.  water, 
1  min.,  5%  Na2S203,  1  min.  and  tap 
water  10  min.  Counterstain  with  car- 
naalum.  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  LioCOa.  After 
about  1|  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  argentaffin  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  36-40  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 
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 


ARGINASE 


27 


ARSENIC 


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  m.l.  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  a- 
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 
freshly  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  6  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  (See  Fig.  1-3). 

"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." 

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 
(Schweinfurth's  green). 

2.  Fix  pieces  of  tissue  12-24  hrs.  in 
abs.  ale.  50  cc;  chloroform,  50  cc;  pure 
hydrochloric  acid,  3  cc.  saturated  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  has  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 
and  were  unable  to  conclude  that  the 
yellow  crystals  were  in  fact  those  of 
arsenic  trisulphide.  The}'  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  akin. 


ARSENIC 


28 


ARTERIOVENOUS  ANASTOMOSES 


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  bv  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  fi'equently  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  he 
made  translucent  by  treatment  with 
pure  glycerin  for  1-2  hrs.  as  described 
by  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 
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  thickness  of  arteriolar 
wall  to  width  of  lumen  is  1:2. 

Arteriovenous  Anastomoses  are  direct  con- 
nections between  arteries  and  veins 
without  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^) 
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 


ARTERIOVENOUS  ANASTOMOSES         29 


ASTRA  VIOLET 


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. 

Articular  Nerve  Terminals.  Gardner, 
E.  D.,  Anat.  Rec,  1942,  83,  401-419, 
working  in  our  laboratory,  adapted 
silver  methods  to  the  demonstration  of 
nerve  terminals  associated  with  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.  10%  and  20%  formalin 
and  Bouin's  fluid  were  the  fixatives  em- 
ployed. Wash,  dehydrate  to  70%  al- 
cohol. Decalcify  in  2.5%  nitric  acid  in 
70%o  alcohol.  Wash  in  70%  alcohol 
until  neutral  to  blue  litmus  paper. 
Dehydrate  and  infiltrate  with  nitro- 
cellulose dissolved  in  methyl  benzoate. 
Harden  in  chloroform,  clear  in  xylol 
and  leave  in  half  paraffin  and  xylol  over 
night  at  37°C.  Imbed  in  paraffin  and 
cut  lO/n  sections.  Subsequently  the 
Bodian  technique  is  followed,  leaving 
the  sections  in  protargol  (Winthrop) 
12-48  hrs.  Axones  stain  black  against 
a  reddish  gray  background  whose  den- 
sity may  be  varied  according  to  the 
length  of  treatment  with  oxalic  acid. 
To  obtain  consistent  results  chemically 
clean  glassware  and  doubly  distilled 
water  are  helpful.  Most  difficulties, 
however,  are  due  to  improper  fixation. 
Fixatives  with  heavy  metals  should  be 
avoided.  Formalin  should  be  neutral- 
ized, preferably  with  MgCOs-  Bouin's 
fluid  is  especially  good  for  fetal  mate- 
rial and  is  equal  to  formalin  for  young 
and  adult  material.  (Revised  by  E.  D. 
Gardner,  Dept.  of  Anatomy,  Wayne 
University  School  of  Medicine,  De- 
troit, Mich.,  1946.) 

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  e.xisting 
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. 

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.,  1028,  153  pp. 

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. 


ATABRINE 


30 


AURAMIN 


Atabrine,  anti -malarial  agent;  fluorescence 
microscopical  localization  of  atabrine 
in  the  body  (Jailer,  J.  W.,  Science, 
1945,  102,  258-259. 

Atomic  Weights.  The  11th  Report  of  the 
Committee  on  Atomic  Weights  of  the 
International  Union  of  Chemistry. 
Baxter,  G.  P.  (chairman)  et  al.,  J.  Am. 
Chem.  Soc,  1941,  63,  845-850  gives  the 
following  as  International  Atomic 
Weights,  1941. 


Element  Symbol 

Aluminum Al 

Antimony Sb 

Argon A 

Arsenic As 

Barium Ba 

Beryllium Be 

Bismuth Bi 

Boron B 

Bromine Br 

Cadmium Cd 

Calcium Ca 

Carbon C 

Cerium Ce 

Cesium Cs 

Chlorine CI 

Chromium Cr 

Cobalt Co 

Columbium Cb 

Copper Cu 

Dysprosium Dy 

Erbium Er 

Europium Eu 

Fluorine F 

Gadolinium Gd 

Gallium Ga 

Germanium Ge 

Gold Au 

Hafnium Hf 

Helium .' He 

Holmium Ho 

Hydrogen H 

Indium In 

Iodine I 

Iridium Ir 

Iron Fe 

Krypton Kr 

Lanthanum La 

Lead Pb 

Lithium Li 

Lutecium Lu 

Magnesium Mg 

Manganese Mn 

Mercury Hg 

Molybdenum Mo 

Neodymium Nd 

Neon Ne 

Nickel Ni 

Nitrogen N 

Osmium Os 

Oxygen O 

Palladium Pd 

Phosphorus P 

Platinum Pt 


Atomic 

Atomic 

Number 

Weight 

13 

26.97 

51 

121.76 

18 

39.944 

33 

74.91 

56 

137.36 

4 

9.02 

83 

209.00 

5 

10.82 

35 

79.916 

48 

112.41 

20 

40.08 

6 

12.010 

58 

140.13 

55 

132.91 

17 

35.457 

24 

52.01 

27 

58.94 

41 

92.91 

29 

63.57 

66 

162.46 

68 

167.2 

63 

152.0 

9 

19.00 

64 

156.9 

31 

69.72 

32 

72.60 

79 

197.2 

72 

178.6 

2 

4.003 

67 

164.94 

1 

1.0080 

49 

114.76 

53 

126.92 

77 

193.1 

26 

55.85 

36 

83.7 

57 

138.92 

82 

207.21 

3 

6.940 

71 

174.99 

12 

24.32 

25 

54.93 

80 

200.61 

42 

95.95 

60 

144.27 

10 

20.183 

28 

58.69 

7 

14.008 

76 

190.2 

S 

16.0000 

46 

106.7 

15 

30.98 

78 

195.23 

Atomic    Atomic 
Element  Symbol       Number    Weight 

Potassium K  19  39.096 

Praseodymium Pr  59  140.92 

Protactinium Pa  91  231 

Radium Ra  88  226.05 

Radon Rn  86  222 

Rhenium Re  75  186.31 

Rhodium Rh  45  102.91 

Rubidium Rb  37  85.48 

Ruthenium Ru  44  101.7 

Samarium Sm  62  150.43 

Scandium So  21  45.10 

Selenium Se  34  78.96 

Silicon Si  14  28.06 

Silver Ag  47  107.880 

Sodium     Na  11  22.997 

Strontium Sr  38  87.63 

Sulfur S  16  32.06 

Tantalum Ta  73  180.88 

Tellurium Te  62  127.61 

Terbium  Tb  65  159.2 

Thallium TI  81  204.39 

Thorium Th  90  232.12 

Thulium Tm  69  169.4 

Tin Sn  50  118.70 

Titanitim Ti  22  47.90 

Tungsten W  74  183.92 

Uranium U  92  238.07 

Vanadium V  23  50.95 

Xenon Xe  54  131.3 

Ytterbium Yb  70  173.04 

Yttrium Y  39  88.92 

Zinc Zn  30  65.38 

Zirconium Zr  40  91.22 

Auditory  System,  see  Ear. 

Auer  Bodies.  A  technique  whereby  red 
cells  are  overstained  and  leucocytes 
understained  has  proved  helpful  for  the 
demonstration  of  these  rod  like  bodies  in 
leucocytes  (Goodwin,  A.  F.,  Folia Hae- 
mat.,  1933,  51,  359-366).  The  smears 
are  colored  in  the  regular  fashion  by 
Wright's  stain  except  that  water  is  not 
added  to  the  stain. 

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  pj^rocatechine  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. 

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" 


AURAMIN 


31 


AZURE  TOLUIDIN  BLUE 


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  SB,  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 :  amethj^st  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  (Monne,  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,  sec  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,  sec  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. 

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 
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  asj'mmetrical  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 
(Haynes,  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  Toluidin  Blue.— Written  by  Dr.  R.  D. 
Lillie,  Division  of  Pathology,  National 


AZURE  TOLUIDIN  BLUE 


32 


BACTERIA 


Institute  of  Health,  Bethesda,  Md., 
April  22,  1946 — Dissolve  10  gm.  Azure 
A  or  Azure  C  (about  85%  dye  content) 
or  13  gm.  Toluidin  blue  (about  60%  dye 
content)  in  600-800  cc.  aq.  dest.  Or 
polychrome  10  gm.  methylene  blue  in 
600  cc.  aq.  dest.  by  boiling  20  min. 
with  5  gm.  potassium  bichromate  and 
7  cc.  concentrated  sulfuric  acid  (95.5% 
spc.  gf.  1.84),  cooling  to  10°C.  and 
neutralizing  by  adding  gradually  21  gm. 
sodium  bicarbonate.  This  makes  a 
crude  Azure  A.  Dissolve  8  gm.  Eosin 
Y  or  Eosin  B  (a  redder  shade)  in  100  cc. 
aq.  dest.  and  add  to  the  selected  azure 
or  toluidin  blue.  Filter  with  vacuum 
on  hard  filter  paper  on  a  Buchner  fun- 
nel. Just  as  filtration  is  completed, 
add  successively  2  washes  of  50  cc.  aq. 
dest.  and  2  of  25  cc.  95%  alcohol.  Dry 
precipitate  on  filter  paper  at  37°C. 
Make  1%  stock  solution  in  equal  vol- 
umes of  95-98%  C.P.  glycerol  and  C.P. 
methanol  (A.C.S.)  (or  100%  ethyl  alco- 
hol), shaking  at  intervals  for  2  or  3 
days.  The  stock  solution  is  best  kept 
in  a  cool  place,  but  is  quite  stable  at 
room  temperature. 

Bring  paraffin  sections  to  water  as 
usual,  pre -stain  5  min.  in  alum  hema- 
toxylin if  desired,  wash  and  stain  1  hr. 
in  stock  solution  0.5  cc,  Mcllvaine 
buffer  of  desired  pH  level  2  cc,  C.P. 
acetone  5  cc.  and  distilled  water  to 
make  40  cc  Rinse,  dehydrate  in  ace- 
tone, clear  with  50:50  acetone  xylene 
and  2  changes  of  xylene,  mount  in 
clarite. 

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  cj^toplasm 
and  oxyphil  inclusion  bodies.  Further 
details  are  given  in  the  original:  Histo- 
pathologic Technic,  Lillie,  1946  (in 
press). 
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 ,  H  am ,  E Az  "  of  a  great 
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. 

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,  1939),  which 
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. 


BACTERIA 


33 


BACTERIA.    MEDIA 


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 
Simnions  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 persulfate.  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.  H2O2  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.  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. 

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- 
lizer, 1  hr.,  add  5  gm.  sodium  chloride 
and  10  gm.  peptone.  Dissolve  our 
flame,  filter,  add  aq.  dest.  to  1000  cc, 


BACTERIA.    MEDIA 


34 


BACTERIA.    MEDIA 


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  l|-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 
sterility. 

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  lOO^C.  l|-hrs.,  strain 
through  fine  wire  sieve,  filter  through 
clean  glass  wool,  adjust  pH  to  7,  tuiae 
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  -f  5  cc.  in 
NaOH  +  1  cc.  dil.  aq.  CuS04  will  give 
pink  color.  If  not  incubate  again  1  hr. 
and  re-test.  When  complete,  slightly 
acidifj'  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 
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 l|-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. 


BACTERIA.    MEDIA 


35 


BACTERIA.    MEDIA 


Blood  culture  (Kracke).  Add  500 
gm.  finely  ground  fat-free  beef  heart 
muscle  to  lOOO  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.  INIix  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. 
Pilter  through  cotton,  place  in  fer- 
mentation tubes,  sterilize  after  which 
pH  by  potentiometer  (not  colorimeter) 
should  be  7.1-7.4. 

Levine'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  steriUty. 

Endo's  (Standard  for  water  analysis). 
Add  5  gm.  beef  extract,  10  gm.  peptone 
and  30  gra.  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  carefull}^  pour  into  sterile  Petri 
dislics,  let  agar  set  at  room  tempera- 
ture and  harden  over  night  in  incubator. 
Check  sterility. 

Agar,  sodium  desoxy chelate.  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  A'^  sodium  hydroxide  plus  ferric  am- 
monium citrate,  2  gm.  dipotassium 
phosphate  and  1  gm.  sodium  desoxy- 
cholate.  Titrate  with  phenol  red  in- 
dicator to  pli  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.  lyphosa. 

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 
thi'ough  cotton,  tube,  autoclave  15 
lbs.,  15  min.  Slant  with  deep  butt. 
Check  reaction  of  medium  with  known, 
E.  coll.,  A.  aerogenes,  S.  scholtmuelleri 
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  min. 
Check  reaction  of  medium  with  known 
S.  aertrycke,  S.  enteriiidis,  S.  ■paratyphi 
and  S.  schoitmuelleri. 

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  ascptically  and  incubate  to  prove 
sterility.  Check  reaction  of  medium 
with  known  S.  paratyphi  and  S.  schotl- 
muelLeri. 

Dieudonne's  alkaline  blood  agar  (for 
Vibrio  comma).    Make  700  cc.  nutrient 


BACTERIA.    MEDIA 


36 


BACTERIA.    MEDIA 


agar  and  neutralize  to  litmus  about  pH 
6.8.  Mix  150  cc.  defibrinated  beef  blood 
and  150  cc.  in  1  A'^  KOH  and  steam  in 
Ai-nold  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,  de.xtrose  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  -f  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  -f  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, 

Loefiler'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,  paraifinize  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. 

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 


BACTERIA.    MEDIA 


37 


BARIUM 


milk,  place  measured  volumes  in  flasks 
or  tubes  and  autoclave  15  lbs.,  15  min. 
Tellurite  (for  C.  diphtheriae).  Melt 
infusion  a^ar,  or  0.2%  dextrose  agar, 
and  cool  to  50°C.  To  each  10  cc.  add 
1  cc.  citrated,  or  defibrinated,  blood 
+  1  cc.  sterile  2%  aq.  potassium  tellu- 
rite, mix  and  pour  into  Petri  dishes. 

Bismuth  sulfite  agar  (Wilson  and 
Blair  for  E.  typhosa) .  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- 
oughly mix  200  cc.  (B)  and  45  cc.  (C). 
Immediately  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). 

Bacteriostatic  Titration  of  Dyes.  (Reed, 
M.  V.  and  Genung,  E.  F.,  StainT'echn., 
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). 

Balsam  for  mounting  sections  is  usually 
satisfactory  as  purchased.  To  make, 
rnix  equal  parts  dry  balsam  and  sodium 
bicarbonate  and  grind  in  mortar.  Add 
sufficient  xylol  to  make  clear  solution. 
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  Eomp  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.  Exp.  Biol.  &  Med.,  1940,  44,  555- 
556).  Barium  chloride  and  formalin  are 
advised  as  fixative  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 .    Fix  tissues 


BARIUM 


38 


BENDA'S 


by  hypodermic  injection  of  formalin 
and  subsequent  immersion  in  it.  Take 
x-ray  photographs  of  the  radiopaque 
barium. 

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  ivasserlich.  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. 
+  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  +  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. 

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  by  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  fijiative  for 
Tissue  Basophiles. 

Basic  Rubin,  see  Basic  Fuchsin. 

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  H.  L.,  Am.  J. 
Clin.  Path.,  1941,  11,  Tech.  Suppl.  5, 
1-39,  with  beautiful  colored  plates) 
reconamend  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 
Hematology,  1938,  1,  235-372).  See 
Tissue  Basophiles. 

Basophilic,  see  Staining. 

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.  -f  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. 
Dehj^drate,  clear,  imbed  in  paraffin  and 
cut  sections  at  4^t.  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 


39 


BEST'S  CARMINE 


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  O  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  5  min.- 
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,  297-3S8). 

Benzamine  Blue  3B,  see  Trypan  Blue. 

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

Benzo  Blue  3B,  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). 

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. 

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.  Berberiau,  D.  A., 
Arch.  Dermat.  &  Syphil.,  1937, 36,  1171- 


1175,  has  (iv-^veloped  a  method  for  stain- 
ing fun;.';i  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  Marti notti'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.  x\n  alkaloid  used  as  a 
fluorochrome  for  malarial  parasites 
(Metcalf,  R.  L.  and  Patton,  R.  L., 
Stain  Tecbn.,  1944,  19,  11-22). 

Bergamot  Oil  is  sometimes  used  for  clearing 
because  it  wall  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  lOp  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. 

Best's  Carmine.  Griibler's  oarminum  ru- 
brum  optimum,  or  some  other  good 
carmine,  2  gm.;  potassium   carbonate, 


BEST'S  CAHMINE 


40 


BILE  COMPONENTS 


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. 

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). 

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

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

Biebrich  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.  FeCls  in  95  cc.  aq.  dest.  +  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  in  aq.  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  l%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) 
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  ^-f 
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,  h-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 


BILE  COMPONENTS 


41 


BISMUTH  PIGMENTATION 


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  BiliTerdin,  see  Bile 
Pigments,  tFrobilin  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). 

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. 

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  dyes  of  different  shades. 
Quite   widely   employed,   see   Blaydes, 


G.  W.,  Stain  Techn.,  1939,  14,  105-110 

for  use  with  plant  tissue. 
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  slide. 
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  Maechling's  (Am.  J.  Path., 
1934,  10,  577-588)  study  of  nuclear 
inclusions  in  the  kidney. 

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  thoroughly  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  add  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 


BISMUTH  PIGMENTATION 


42 


BLOOD  CELL  VOLUME 


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 
remove  excess  hydrogen  peroxide.  Ap- 
ply modified  Castel  reagent  to  surface 
and  exanaine  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  Keaction.  "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  CuS04  are 
then  added  with  stirring.  A  blue- 
violet  coloration  indicates  the  pi'esence 
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  cj'tological  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." 

Blastomycosis.  The  differentiation  of  Zy- 
monema  (Blastomyces)  dermatitidis,  the 
cause  of  blastomycosis,  from  Crypto- 
coccus  homlnis,  the  cause  of  crypto- 
coccosis or  torulosis,  is  best  accom- 
plished by  wet  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.  Cryptococcus  horninis  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. 

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 
examination  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 
actually  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% 
anunonium  oxalate.  Centrifuge  and 
transfer  1  cc.  plasma  to  tube  containing 
dye.  Remove  0.1  cc.  dyed  plasma  to 
9.9  cc.  saline  in  photoelectric  colorimeter 


BLOOD  CELL  VOLUME 


43 


BLOOD  VESSELS 


tube.  Make  blank  without  plasma. 
Compare  in  Evelyn  or  Klet-Summerson 
colorimeter  using  filter  to  pass  only  light 
of  about  620  m^.  Calculate  as  directed 
for  the  colorimeter  (Shohl,  A.  T.  and 
Hunter,  T.  H.,  J.  Lab.  &  Clin.  Med., 
19-11,  26,  1829-1837).  See  also  earlier 
cell  opacity  method  (Shohl,  A.  T.,  J. 
Lab.  &  Clin.  Med.,  1939-40,  25,  1325- 
1332). 

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  Protein.  Coagulated  blood  protein 
within  the  vascular  lumina  of  stained 
sections  of  fi.xed  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  does  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  Glassv/are).  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  vyill 
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 
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  make 
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  the  inter- 
vening part.  For  stains  see  Giemsa, 
Wright,  Ehrlich,  Oxidase,  Peroxidase 
and  Gordon's  Silver  Method. 

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 


BLOOD  VESSELS 


44 


BONE 


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 
vpater  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. 

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  required.  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  csm 
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 


BONE 


45 


BORAX  CARMINE 


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  modi- 
fied by  Shipley. )  Employ  a  fixative  not 
containing  mercury.  Decalcify  in  Miil- 
ler's  fluid,  wash  in  running  water,  embed 
in  celloidin  and  section  not  over  10  mi- 
crons. Stain  in  thionin  solution  al- 
kalinised  by  2  drops  ammonia.  Trans- 
fer with  glass  needle  to  sat.  aq. 
phosphotungstic  or  phosphomolybdic 
acid.  Leave  until  blue,  gray  or  green. 
Place  in  water  until  sky-blue.  Am- 
monium hydroxide  1  cc.  and  aq.  dest. 
10  cc,  3-5  min.  Several  changes  90% 
alcohol.  95%  a)c.  Clear  in  carbol- 
xylol  and  mount  in  damar  (or  balsam). 
This  method  is  suggested  for  bones  of 
children.  Processes  of  young  osteoblasts 
in  growing  bone.  Shipley  suggests  fol- 
lowing 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  spe- 
cimens in  dark  when  not  in  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 
checking  by  microscopic  e.xamination 
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. 

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.  To  demon- 
strate the  vascular  pattern  special 
methods  are  required  (Doan,  C.  A., 
Johns  Hopkins  Hosp.  Bull.,  1922,  33, 
222-226).  To  reveal  the  nerve  supply 
is  particularly  difficult.  Glaser  (W., 
Ztsch.  f.  Anat.  u.  Entw.,  1928,  87,  741- 
745)  has  described  a  fine  network  accom- 
panying the  vessels  but  Doan  and  Lang- 
worthy  (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,  C.  M.,  Acta  Med.  Scand., 
1941,  107,  11-52.  See  chapters  by  Sabin 
and  Miller  and  by  Doan  in  Downey's 
Handbook  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  Ptuzicka,  A.  Q.,  Anat. 
Rec,  1945,  93.  213-231). 

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  daj's  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, 


BORAX  CARMINE 


46 


BRANDT'S 


transferred  directly  to  acid  ale.  (70% 
ale.  100  ce.,  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  raono-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 
Banks,  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  grn.; 
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  Iseen  used  for  the  Borrel  bodies  in 
fowl  pox. 

Botanical  Technique.  IVIany  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. 
Taylor  in  McClung,  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's  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, 
6-8. 

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. 

Boutons  Terminaux.  For  this  special  type 
of  nerve  ending  the  methods  given 
under  Nerve  Endings  are  useful,  partic- 
ularly that  of  Bodian.  The.se  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  (IMcClung, 
pp.  481-488). 

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  tlie 
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  and  xylol.  This  takes 
about  30  min.  and  should  be  observed 
under  microscope.  Pass  through  several 
changes  of  xjdol,  rinse  in  benzol  and 
mount  in  benzol  balsam.  Pepsinogen  of 
pepsin-forming  cells,  violet ;  and  parietal 
cells,  red.  Bowie  also  makes  a  crj^stal 
violet-orange  G  stain  which  does  not 
differ  materially  from  Bensley's  Neutral 
Gentian. 

Brandt's  glycerin  jelly.  Melted  gelatin,  1 
part;  glycerin  H  parts  plus  few  drops 
carbolic  acid  to  serve  as  a  preservative. 


BRANDT'S 


47 


BUFFERS 


See  Kaiser's  glycerin  jelly  under  gly- 
cerin. 

Brazilin  (CI,  1243)  is  a  subsUxnce  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.  .1.,  Quart.  J.  Micr.  Sci., 
1901,  44,  469-471)  and  O'Leary's  Bra- 
zilin for  myelin  sheaths.  See  also 
Brazilln-Wasserblau  technique  of 
BensleJ^ 

Brazilin-Wa.sserblau  for  secretion  ante- 
cedents of  thyroid  gland  (Bensley,  R. 
R.,  Am.  J.  Anat.,  191G,  19.  37-54)  as 
described  later  by  the  Bensleys  (p.  80) 
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  emplo}''ed  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.  -f-  1.0  gm.  phosphomolyb- 
dic  acid  and  0.2  gm.  Wasscrblau  (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- 
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  braziUvood  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.,  Inn.,  1944,  95  pp.). 

Breast,  see  Mammary  Glands. 


Brilliant  Alizarin  Blue  (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,  sec  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  Rhodamlne  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  paraffin  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. 

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). 

Buffalo  Garnet  R,  see  Erie  Garnet  B. 

Buffers.  For  many  purposes  it  is  essential 
to  use  solutions  buffered  at  a  certain  pH. 


BUFFERS 


48 


CADMIUM 


Details  concerning  numerous  buffers  are 
given  by  Clark,  W.  M.,  The  Determina- 
tion of  Hydrogen  Ions.  Baltimore: 
Williams  &  Wilkins,  1928,  717  pp. 
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  Buffers. 

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  fuchsin,  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- 
toxylin, 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  Mcll- 
vaine  citric  buffers  in  order  to  improve 
Romanowsky  staining  (see  Toluidine 
Blue  Phloxinate)  after  various  fixatives. 
See  McJunkin-Haden  BufiTer.  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.,  Mc Arthur,  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 
quickly.     See  Hydrogen  Ion  Indicators. 


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

Burns.  Methods  of  experimental  produc- 
tion, vital  staining  with  trj'pan  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.  iu  100  cc.  5%  aq. 
chrome  alum  for  10  min.  After  cooling 
make  up  to  100  cc.  with  aq.  dest.,  filter 
and  add  a  little  formalin  to  filtrate.  (2) 
Orcein  (Hollborn,  2466).  Dissolve  1 
gm.  in  100  cc.  acid  alcohol  (70%  alcohol, 
100  cc.  -f  1  cc.  hydrochloric  acid  stand- 
ard). (3)  Acid  alizarin  blue  (Hollborn, 
2559).  Boil  for  10  min.  5  gm.  iu  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  rain.  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 
green;  myelin  sheaths,  rose;  axis  cylin- 
ders, dark  blue;  erythrocytes,  red 
brown. 

Cadmium.  The  chloride  is  employed  in 
fixation  of  Golgi  apparatus  prior  to  silver 
impregnation  (Aoyama,  F.,  Zeit.  wiss. 
mikr.,  1929,  46,  489-491).  See  comment 
by  Baker  (Bourne,  p.  19)  on  this  and 
use  by  Ciaccio  of  cadmium  nitrate  to 
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 


49 


CANARY  YELLOW 


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.  P'ix 
small  fresh  pieces,  3-15  days,  in:  aq. 
dest.,  85  cc;  formalin,  15  cc.;  ammo- 
nium bromide,  2  gm.  Cut  25m  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,  2.5gm. ;  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  parafRn 
and  cut  15-30^  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  role  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). 
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. 

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., 
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. 

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

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 


50 


CAPILLARIES 


Cancer.  Because  cancer  and  other  malig- 
nant tumors  can  develop  in  so  many 
organs  and  tissues  that  contain  inter- 
niitotic  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  bj^  Radioactive  Isotopes. 

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  fairlj-  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- 
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,  54.5-575.  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 
permeabilitj'-  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 


CAPILLARIES 


51 


CARBOHYDRATES 


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  Dichlorfiuorescineate 
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.  Lepehnc-Pickworth 
peroxidase  method  simplified  by  Camp- 
bell 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  200-300m  and  wash 
in  aq.  dest.  1|  hr.  Change  to  above 
described  solution  for  ^  hr.  at  37°C. 
agitating  often.  Wash  in  aq.  dest.  10 
sec.  and  transfer  to  100  cc.  aq.  dest.  -f 
2-3  drops  30%  hydrogen  peroxide  for 
^  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, 
9.3-106). 

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

Capri  Blue  (CI,  876),  a  basic  dye  of  light 
fastness  3.  0.1  gm.  in  95  cc.  aq.  dest. 
-f  5  cc.  5%  aq.  ammonium  alum  -f-  0.5 
cc.  acetic  acid  stains  plant  tissues  blue 
or  black.  Can  be  employed  in  prefer- 
ence to  Cyanine.  Should  stain  well 
after  Erythrosin  (Emig,  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.  tSee 
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.  by  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  GD  (CI,  1113),  Carban- 
threne  Brilliant  Orange  RK,  Carban- 
threne Jade  Green  (CI,  llUl),  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 


52 


CAREY'S 


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  crj^stals,  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. 
Deipolli,  G.  and  Pomerri,  G.,  Mon. 
Zool.  Ital.,  1938,  49,  123-124  have  ad- 
vised its  use  as  follows  for  Niss!  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.  Xylol  saturated  with  car- 
bolic acid  crystals.  After  using  it  for 
clearing  celloidin  sections,  wash  quickly 
in  xylol  before  mounting  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. 

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-i96).  For  data 
on  the  distribution  of  this  enzyme  in 
lower  forms,  see  Blaschko  and  Jacobson 
(Bourne,  p.  200). 

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  E.  J.  Carey,  Dept.  of 
Anatomy,  Marquette  University  School 
of  Medicine,  Milwaukee,  Wis. 

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


CAREY'S 


53 


CARMINE-GELATIN 


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  5  glycerine  and 

I2  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  ^  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  dj^es  are  more 
popular  but  carmine  is  still  of  great  use 
for  staining  small  animals  in  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-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 
iocUde  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 


CARMINE-GELATIN 


54 


CARTILAGE 


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). 

Carnoy-Lebrun  fixative  for  insects  and  ticks. 
Equal  parts  chloroform,  absolute  alcohol 
and  acetic  acid  saturated  with  mercuric 
chloride.     See  Slifer-King  Method. 

Carney'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. 

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  ab- 
sorption 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  micro- 
scope carotin  crystals  appear  dark  blue 
(Steiger,  A.,  Microkosmos,  1941,  8, 
121-122).  Carotin  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  bj'^ 
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). 

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  cliambers  in- 
serted in  the  ears  of  rabbits  (Clark,  E. 
R.,  and  E.  L.,  Am.  J.  Anat.,  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.  tiiick,  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  tecnnique  used  by  Fell,  H. 
B.,  J.  Morph.,  1925,  40,  417-459.  See 
Chondriotin  Sulphuric  Acid  and  Phos- 
phatase   as    components    of    cartilage. 


CARTILAGE 


56 


CELL  CLASSIFICATION 


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. 

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  1-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  (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. 

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). 

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  8Te  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. 

Cedar  Oil,  see  Clearing  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,  415- 
416  as  a  nuclear  stain. 

Cell  Classification  according  to  manner  of 
life.  Intermitotic  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  diflfer- 
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 


CELL  CLASSIFICATION 


56 


CELL  SHAPE 


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,  Ainitosis  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  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  mji  and 
that  the  whole  membrane  is  between  In 
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 
0.25^.  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, Pinocytosis. 
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 
morphology  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 
layer  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-rayed  not  four- 
rayed  as  might  be  the  case  if  the  cross- 
sections  were  squares.  Mechanically 
this  is  a  great  advantage .  When  the  ep- 
ithelium is  stratified  provision  must  be 
made  for  contact  with  cells  on  all  sides. 


CELL  SHAPE 


57 


CELLOIDIN  IMBEDDING 


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. 
Celloidin  Imbedding.  Celloidin  is  a  kind 
of  generic  term  covering  various  cellu- 
lose compounds,  nitrocellulose,  soluble 
gun  cotton,  etc.,  employed  for  imbed- 
ding. The  collodions  are  solutions  of 
pyroxylin  made  as  specified  in  the 
U.S.P.  Pyroxylin  U.S.P.  XI  consists 
chiefly  of  cellulose  tetranitrite  (Merck 
Index,  p.  465).  Obviously  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.,  1934, 
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  ethyl 
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  ^2  hrs. 

Another  so  called  hot  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  Susa.  Wash  in  aq. 
dest.  several  changes  (1  hr.  each)  at 
room  temperature.  Dehydrate  at  50 °C. 
70,  80,  95  and  abs.  ale.  2  changes  J  hr. 
each.  Equal  parts  abs.  ale.  and  ether 
1  hr.  Infiltrate  at  56°C.  in  (1)  10% 
nitrocellulose  (R.S.  ^  second,  viscosity 
A"~25>  Hercules  Powder  Co.)  in  equal 
parts  abs.  ale.  and  ether,  1  hr.  (2)  25% 
in  45  cc.  ale.  -f  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% 


CELLOIDIN  IMBEDDING 


58 


CENTRIFUGATION 


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,  20gms., 
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 
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 /u.  (It  is  possible  to 
arrange  the  sections  serially  but  it  is  a 
tedious  business.  If  serial  sections  are 
needed,  parafl[in  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  ISO  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. 

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  -|-  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  ^  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- 


CENTRIFUGATION 


59 


CENTROSOMES 


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 
roles.  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  microchemistry 
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- 
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  tliat  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.,  ihid,  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  cenlrosphere.  The  centro- 
some, or  centriole  plus  the  clear  area  is 
called  the  cytocentrum.  For  terminology 
see  Wilson,  E.  B.,  The  Cell.  New  York : 
Macmillan  Co.,  1925,  1232  pp.  For 
functional  significance  see  Fry,  H.J., 
Biol.  Bull.,  1929,  57,  131^150.  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 


CENTROSOMES 


60 


CHAMPY'S  FLUID 


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  ceutrosomes  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 
they  are  seldom  present.  Hatai  (S., 
J.  Comp.  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, 
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,  p.  469)  ad- 
vises 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,  formaldehyde 
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. 

Cerasin  Red,  see  Sudan  III, 

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  te.xture.  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.  Fluorescent  material  in  experi- 
mental nutritional  cirrhosis,  technique 
for:  Popper,  H.,  Gyorgy,  P.  and  Gold- 
blatt,  H.,  Arch.  Path.,  1944,  37,  161. 

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). 

Cesares-Gil  flagella  stain  evaluated, 
Thatcher,  L.  M.,  Stain  Techn.,  1926, 
1,  143-144. 

Cesium,  spectrographic  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. 

Champy-KuU'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.; 
mordant  3%  aq.  potassium  bichrom.ate, 

3  days.  Wash  running  water  24  hrs., 
dehydrate,  clear,  imbed  and  section 
at  4/i.  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  in  aq.  dest.,  then  0.5% aurantia  in 
70%  alcohol  20-40  sec.  Differentiate  in 
95%  alcohol,  dehydrate,  clear  and 
mount.  Mitochondria  red,  nuclei  blue 
and  ground  substance  j'^ellow. 

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 


61 


CHLORIDE 


Charcot-Leyden's      Crystals.       Octahedral 

phosphate  crystals  found  in  stools  of 
persons  infected  with  Endameba  his- 
tolytica and  in  a  varietj^  of  other  condi- 
tions. See  description  and  illustration 
hy  Craig,  p.  58. 

Cheese.  Bacteria  in,  see  Hucker,  G.  J., 
N.  y.  Agric.  Exp.  Sta.  Tech.  Bull. 
1921,87  (McClung,  p.  147). 

Chicago  Blue,  see  escape  from  venules  after 
intravenous  injection  (Smith,  F.  and 
Rous,  P.,  J.  Exp.  Med.,  1931,  54,  499- 
514). 

China  Blue,  see  Anilin  Blue. 

Chitin.  1.  Method  for  softening  of  chitin  in 
formalin  fixed  insects  (Murray,  J.  A., 
J.  Roy.  Micr.  Soc,  1937,  57,  15).  Fix 
primarily  in  10%  fornmlin  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,  x3dol  or  carbon  disul- 
phide.     Imbed  in  paraffin. 

2.  Accordinsr  to  Hennings  (sec  Lee, 
p.  597)  fixation  of  insects  in  the  following 
mixture  softens  the  chitin  sufficiently 
to  permit  the  making  of  paraffin  sections : 
nitric  acid,  IC  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.  folio vred  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  fi.xative  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  alimeutar}'  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  hvdrate  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  polj^-azo 
dye.  First  described  as  a  new  biological 
stain  by  Cannan  (H.  G.,  Nature,  1937, 
139,  549).  Review  of  its  uses  (Cannan, 
H.  G.,  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 
aceto-carmine  auxiliary  stain  for  chro- 
mosomes (Nebel,  B.  R.,  Stain  Techn., 
1940,  15,69-72). 

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). 

Chlorazol  Pink  Y,  see  Thiazine  Red  R. 

Chloride.  In  1908  Macallum  reviewed  the 
older  literature  and  described  his  silver 
test  for  chloride  (Macallum,  A.  B., 
Ergeb.  d.  Physiol.,  1908,  7,  552-652). 
The  possibilitj',  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  a-re  mounted  near 
one  edge  on  chemically  clean  large  cover 
slips  by  simply  pi'essing  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 


CHLORIDE 


62 


CHLOROTHYMOLS 


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.  Dichlorjluorescein  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  charged 
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  IVIarsh, 
B.  S.,  Am.  J.  Physiol.,  1934.  110,  261- 
272;  Eggleton,  M.  G.  and  P.  and  Hamil- 
ton, A.  M.,  J.  Physiol.,  1937,  90,  167- 
182). 

(5)  Conclusion  that  in  many  tissues 
for  all  practical  purposes  all  radioactive 
sodium  and  radioactive  chloride  remain 
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 

'  -  X  1000  =  96  gms.  of  extracellular 

fluid  per  kilogram.  When  the  extra- 
cellular fluid  contains  collagenic  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. 

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 
chloropliyll  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). 

Chloroprene,  see  Neoprene. 

Chlorothymols,  as  preservatives  of  gelatin. 


CHLOROTHYMOLS 


63 


CHORIOALLANTOIC  MEMBRANE 


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. 

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 
crystals  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,  H.  and  Chinn, 
H.,  Proc.  Soc.  Exp.  Biol.  &  Med.,  1942, 
49,  202-204). 

Cholinesterase.  Important  since  it  cat- 
alyses hydrolysis  of  acetylcholine  to 
choline  and  acetic  acid.  Histological 
localization  is  difficult  but  Couteaux,R. 
and  Nachmansohn,  D.,  Proc.  Soc.  Exp. 
Biol.  &  Med.,  1940,  43,  177-181  found  it 
present  in  parts  of  guinea  pig's  muscle 
that  contained  motor-end  plates  and 
absent  in  parts  devoid  of  them.  For 
recent  data  see  Blaschko  and  Jacobson 
(Bourne,  pp.  221-224).  Sharp  localiza- 
tion has  been  found  in  the  giant  nerve 
fiber  of  squids  (Nachmansohn,  D.  and 
Steinbach,  H.  B.,  Science,  1942,  95, 
76-77).  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  of  rat  brain  cortex  can  be 
stained  for  microscopic  examination 
with  methyl  violet  and  thereafter  used 
for  enzyme  measurement. 

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  Mucoproteins. 

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 
gl3'cerin  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).  yVfter  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  througli  the  window.  When  the 
inoculated  area  has  shown  marked 
change,  the  shell  is  cut  below  the  window 


CHORIOALLANTOIC  MEMBRANE 


64 


CHROMOLIPOIDS 


and  the  membrane  exposed.  The 
chorioallantois  is  cut  with  a  pair  of  fine 
curved-end  scissors,  removed,  fixed  in 
Zenker's  sohition  (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.  andWoodhouse,  D.  L., 
J.  Path,  and  Bact.,  194.3,  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.,  f  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  gra.;  aq.  dest.,  95  cc; 
formalin,  5  cc.  Dip  in  water  and  trans- 
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.  96%  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/x.  Mount  in  neutral 
balsam.  Nerves,  dark  brown  or  black; 
muscle   and  connective  tissue,   j'ellow. 

Chrom  Blue  GCB,  see  Gallocyanin. 

Chromaffin  Reaction  (chromic  salts  -f  L. 
affinis,  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 
5%  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,  345  and  of  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  injur,y,  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  CG  (CI,  727).  A  carbo.xyl 
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  chromaflin 
reaction.  In  mixtures  with  other  chem- 
icals it  was  more  used  as  a  fixative  50 
years  ago  tlian  today  but  in  Perenyi 
Fluid  it  is  recommended  strongly  by 
Lee  (p.  32)  for  embr3^os,  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 


CHROMOLIPOIDS 


65 


CHROMOSOMES 


adrenal,  and  are  easily  distiuguisliable 
from  carotinoids  because  they  do  not  give 
the  color  reactions  with  sulphuric  acid 
and  iodine-iodide.  From  melanins  they 
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  chromolipoid:;  blue,  the  mela- 
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. 

Chromophii  (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  chromophii, moreover 
chromophii  reaction  is  unwisely  used  to 
designate  the  chromaffin  reaction  of 
epinephrin  producing  tissues. 

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— 
These  are  discrete  bodies  usually  con- 
stant in  number  in  the  cells  of  a  given 
species  and  frequently  having  distinc- 
tive structure  into  which  the  chromatin 
material  of  a  nucleus  resolves  itself 
during  the  mitotic  process  (see  Mitosis). 
From  a  chemical  standpoint  the  chro- 
mosome is  a  protein  fiber  like  silk  or 
hair,  presumabl}''  depending  on  a  pep- 
tide chain  linkage — C — C— N — .  On  this 
structural  framework  the  permanent 
hereditary  units,  the  genes  (which  may 
be  considered  as  the  atoms  of  heredity), 
are  located  at  definite  loci.  In  a  sense, 
therefore,  the  chromosome  may  be  con- 
sidered a  giant  molecule  (Darlington, 
C.  D.,  Nature,  1942, 149,  66-69,  Astbury, 
W.  T.,  Proc.  7th  Int.  Genet.  Congress., 
1939  (Camb.),  1940,  49-51).  It  is  gener- 
ally stated  that  the  chromosomes  of 
sperm  cells  consist  of  basic  proteins, 
such  as  protamines  or  iiistones,  in  com- 
bination with  highly  polymerized  de- 
soxyribose  nucleic  acid  (Mirsky,  A.  E., 
Advances  in  Euzymology,  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  satisfactorj' 
results,  it  is  imperative  to  employ  cyto- 
logical  techniques  calculated  to  bring 
out  iiue  structures  and  details  a.i 
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  ever}'  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.  BoL.,  1940,  27, 
887-895;  Coleman,  L.  C,  Genetics, 
1943,  2S,  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.,  1930,53,  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  maldng  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  e3.sentially  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 


CHROMOSOMES 


66 


CHROMOTROPE  2R 


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  mi.xture  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- 
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,  8.  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,  IL, 
Biol.  Bull.,  1945,  3,  242-257).  Some  of 
the  best  results  have  been  obtained  by 
treatment  with  K  or  NaCN  21-^  to  21-5 
mol.  solutions  for  periods  varjdng  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  cliromosomes 
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  acetocarnnno  or  acetic 
orcein  and  then  coverslip.  Gently  tap 
with  the  rubber  tipped  end  of  a  pencil 
until  the  cells  are  loosened  up  and  ai'e 
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  I  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,  CL,  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-fi.xative  for 
chromosomes  (LaCour,  L.,  Stain 
Techn.,  1941, 16,  1G9-174).  Demonstra- 
tion of  alkaline  phosphatase  in  chromo- 
somes (Krugelis,  E.  J.,  J.  Cell.  &  Comp. 
Physiol.,  1942,  19,  376-379). 
Chromotrope  2R  (CI,  29) — acid  phlo.xine 
GR,  chromotrope  blue  2R,  fast  fuchsin 
G,XLcarmoisine6R — 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 


67 


CLARK  AND  LUBS  BUFFERS 


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). 

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 
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- 
jiolotf,  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.  The  cjuickest  method  is  to  remove  a 
piece  of  fresh  ciliated  epithelium  from 
the  respiratory  nasal  mucosa  of  an  anes- 
thetized or  recently  killed  animal.  Cut 
up  finely  with  scissors,  tease  out  small 
pieces  with  needles,  mount  in  isotonic 
salt  solution  and  examine  at  low  mag- 
nification in  the  dark  field.  A  simple 
technique  of  demonstrating  the  move- 
ment of  cilia  is  to  examine  at  a  mag- 
nification of  about  80  diameters  the 
epithelium  of  the  roof  of  a  frog's  mouth 
by  reflected  light.  The  angle  between 
incident  and  reflected  light  should  be 
about  90°.  A  strong  source  of  illumina- 
tion is  required  with  a  water  screen  to 


remove  the  heat.  A  useful  set  up  is 
described  and  illustrated  by  Lucas  (A. 
M.,  Arch.  Otolaryng.,  1933,  18,  516-524). 
Many  excellent  moving  pictures  have 
been  made  of  ciliary  action  of  which 
one  by  Dr.  Arthur  Proetz  is  recom- 
mended for  teaching  purposes. 

When  cilia  are  present  they  can  be 
seen  in  almost  any  proi)erly  stained 
section  of  well  fixed  material.  A  good 
stiiin  for  cilia  is  iron  hematoxylin  with 
suitable  counterstain,  after  formalin- 
Zenker  fi.xation.  Engelman  (Lee's  Vade 
Mecum  p.  509)  found  that  ciliated  cells 
of  Lamellibi-anchs  could  be  well  iso- 
lated by  maceration  in  4%  aq.  potassium 
bichromate  and  in  0.1%  osmic  acid. 
This  should  be  tried  for  mammalian 
ciliated  cells.  Cilia  and  their  basal 
granules  are  often  sharply  blackened  by 
silver  impregnation.  See  Cowdry's  de- 
scription of  flagellated  thyroid  cells  of 
the  dogfish  (Cowdry,  E.  V.,  Anat.  Rec, 
1921,  22,  289-299).  Centrosomes  and 
diplosomes  are  often  revealed  in  ciliated 
cells  particularly  in  those  undergoing 
differentiation.  See  Centrosomes.  Lit- 
erature on  ciliated  epithelia  is  well 
presented  by  Lucas  in  Cowdry's  Special 
Cytology,  1932,  409-474.  See  his  illus- 
trations. Application  of  technique  of 
microdissection  to  ciliated  cells  is  de- 
scribed by  Worley,  L.  G.,  J.  Cell.  & 
Comp.,  1941,  18,  187-198. 

Cinephotomicrography.  A  convenient  illus- 
trated account  of  apparatus  and  meth- 
ods is  given  bv  Tattle,  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  of 
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- 


CLARK  AND  LUBS  BUFFERS 


68 


CLEARING 


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  H3BO3-KCI  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 

cc.  of  M/5  NaOH 

7.8 

2.61 

8.0 

3.97 

8.2 

5.90 

8.4 

8.50 

8.6 

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 
di-y  with  opening  downward  on  drying 
racks  as  in  biochemical  laboratories  if 
possible  in  a  dust  free  cupboard.  For 
neiv  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  daj^s.  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  worthwiiile  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 
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  poorly  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  sm.all 
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- 


CLEARING 


69 


COCHINEAL 


rectly  from  the  fixative,  Bouin  or  forma- 
lin, without  washing,  to  3  changes  of 
pure  dioxan  within  4  hrs.  and  thence 
into  3  chiinges  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  dioxan,  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,  beechwood  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. 

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  myo- 
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.  Hirsch  (G. 
C,  Form  und  StofTwechsel  der  Gol- 
gi-Korper.  Protoplasma  Monographs, 
Berlin,  1939)  has  likened  the  Golgi 
apparatus  to  a  coacervate.  See  Bensley, 
R.  R.,  Anat.  Rcc,  1937,  G9,  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- 
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  electrolytes.  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. 

Coccidia.  These  sporozoa  include  many 
parasites  of  great  importance  not  only 
to  physicians  and  veterinarians  but  also 
to  cytologists  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. 

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  v/ith  water  and 
treat  frozen  sections  of  normal  cat  kid- 
ney and  human  adrenal  with  resulting 
solution  for  10-20  min.  This  solution 
is  only  usal^le  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 


COCHINEAL 


70 


COLOR  ESTIMATION 


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,  whicli, 
latter  as  a  commercial  dj'^e,  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  toto  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,  sliake  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. 

Collagenic  Fibers.  On  boiling  they  yield 
collagen.  They  are  also  called  white 
fibers  in  contrast  to  the  elastic  fibers 
which  are  distinctly  yellow.  Details 
can  be  seen  in  fresh,  unstained  spreads 
of  Loose  Connective  Tissue.  The  col- 
lagenic fibers  are  usuall}'^  more  numerous 
in  subcutaneous  connective  tissue,  less 
highly  refractile  than  the  elastic  ones 
and  of  greater  girth.  They  do  not 
branch  though  the  finer  fibrils  of  which 
they  are  composed  and  which  confer  a 
faint  longitudinal  stria tion  sometimes 
do.  The  R.C.A.  electron  microscope 
reveals  a  still  finer  system  of  collagenic 


fibrils  (Scott,  G.  H.  and  Anderson,  T.  F., 
Anat.  Rec,  1942,  82, 445 ;  Schmitt,  F.  O., 
Hall,  C.  E.  and  Jakus,  M.  H.,  J.  Cell, 
and  Comp.  Physiol.,  1942,  20,  11-33). 
On  addition  of  dilute  acetic  acid  they 
swell  except  at  certain  places  in  their 
length  where  they  seem  to  be  constricted 
by  circular  bands.  The  fact  that  they 
also  easily  pass  from  the  gel  to  the  sol 
state  on  alkalinization  and  when  sub- 
jected to  slight  heat  is  the  basis  for 
methods  of  separating  Epidermis  from 
dermis. 

The  best  stain  for  collagenic  fibers  in 
sections  after  Zenker  fixation  is  anilin 
blue  in  Mallory's  Connective  Tissue 
Stain  and  in  Masson's  Trichrome 
Stain.  Phosphomolybdic  Acid  Hema- 
toxylin also  gives  a  fine  coloration  of 
collagenic  fibers.  See  Van  Gieson, 
Buzagio,  Biebrich  Scarlet  and  Picro- 
Anilin  Blue  of  Lillie  and  Curtis'  Sub- 
stitute for  Van  Gieson. 

Lillie,  R.  D.,  J.  Tech.  Methods,  1945, 
No.  25,  45  pp.  has  performed  a  very  use- 
ful service  in  testing  the  effectiveness 
of  a  large  series  of  dyes  as  collagenic 
fiber  stains  in  the  Van  Gieson,  Mixed 
Masson-Van  Gieson  and  Masson-Mal- 
lory  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, 
7U7),  Wool  Green  S  (CI,  737)  and  Vol- 
amine  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  Allara,  E.,  Bull.  d'Hist. 
Appl.,  1938,  15,  220-242.     See  Tendons. 

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: 


COLOR  ESTIMATION 


71 


CONCENTRATION 


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  jellowish  green    purplish  pink 

ycllowwh  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  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  gni.  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  I'Qiiserling  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 
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  difhculty  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  Jores  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. 

Mallory  suggests  fixation  in  Jores' 
first  solution  and  preservation  in  Kaiser- 
ling's  third  solution. 

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  sulphate.  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 
al.so  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. Ravold'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 


CONCENTRATION 


72 


CONTRACTION  BANDS 


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 
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. 

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,  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  trj^pan  blue  (Coleman  and  Bell 
Co.)  into  a  mature  wliite  rat  weighing 
about  90  gms.  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  ol^servation  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  da j^s .  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). 


COPPER 


73 


CORNEA 


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).  When 
search  is  necessary  for  traces  of  copper 
without  need  for  microscopic  localiza- 
tion an  emission  spectrograph  may  give 
the  information  quicklj^  see  Histo- 
spectrography.  If  quantitative  deter- 
minations of  copper  in  small  amounts 
of  tissue  are  required  use  the  polaro- 
graphic  technique  elaborated  by  Car- 
ruthers,  C,  Indust.  and  Engin.  Chem., 
1945,  17,  398-399.  Details  for  deter- 
mination of  copper  in  epidermis  are 
given  by  Carruthers,  C.  and  SuntzeiT, 
v.,  J.  Biol.  Chem.,  1945,  159,  647-651_. 

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  x\ltmann*s  Fluid  or  in  Acetic-Osmic- 
Bichromate  fixative  of  Bensley  12-24 
hrs.  Wash,  dehydrate,  clear,  imbed  in 
paraffin  and  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  cc.)  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. 

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  by  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  bodj''  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.  0.8S)  per,  say, 
50  cc.  After  washing  in  2  or  3  changes 
aq.  dest.,  and  pouring  off  last  aq.  dest., 
filter  onto  cornea  through  cotton  v/ool 
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  ciianges  aq.  dest. 
Then  reduce  by  pouring  onto  cornea 
10%  aq.  neutral  formalin,  lo  min.  Cut 
away  "dome"  of  cornea  with  knife  and 
support  its  concave  surface  with  the 
lens,  freeze  and  section  at  about  15  m- 
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 


CORNEA 


74 


GROSSMAN'S 


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. 
Gross  (L.,  The  Blood  Supply  of  the 
Heart  in  its  Anatomical  and  Clinical 
Aspects.  New  York:  Hoeber,  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,  Chapelle  and  Cohn 
(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. 

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.    Sec  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. 

Cover  Glasses,  see  Cleaning. 

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.  -f  4  drops  1% 
aq.  hydrochloric  acid  but  stop  while 
cells  are  a  little  darker  than  desired. 
Neutralize  sections  in  90%  alcohol  -f- 
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  Grubler  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). 

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  b}''  the 
Magnaflux.  An  interesting  elementary 
account  of  Crime  Detection  Techniques 
is  provided  bv  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 
water  10  min.     Stain  for  1  min.  or  more 


GROSSMAN'S 


75 


CURTIS'  SUBSTITUTE 


in:  acid  fuchsin  (C.C.),  1  giu.;  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.  phosphotungstic  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. 

Crj'ostat. — 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). 
Tissues  frozen  in  liquid  air  or  nitrogen 
are  placed  in  a  chamber  which  is  con- 
nected 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  apparatus  the  relative  amount  of 
water  vapor  in  the  system  is  determined 
by  its  flow  past  a  spaced  pair  of  ioniza- 
tion gauges  between  which  is  placed  the 
P2O6  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  in- 
filtrated. This  latter  procedure  is  ac- 
complished without  the  necessity  of 
breaking  the  vacuum.  This  small  but 
important  step  provides  tissues  which 
have  been  frozen-dried  and  prepared  for 
cutting  without  their  having  been  par- 
tially rehydrated  by  e.xposure  to  air  at 
ambient  pressure  and  temperature. 
See  Altmann-Gersh  frozen  dehydration 
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,  394r- 
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  filter 
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  Crystallog- 
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. 

Curettings,  gelatin  method  for  rapid  frozen 
sections  of  (Meeker,  L.  H.,  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  Weigert'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  rnin.  in 
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 


CURTIS'  SUBSTITUTE 


76 


DAVENPORT'S 


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  heavj^ 
staining  with  red  is  less  likely.  In 
original  account  volumes  are  given  in 
ml.  which  are  of  practically  the  same 
value  as  cc. 

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.,  19i2,  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  less  each.  For 
pieces  more  than  3  mm.  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 
Keihn  (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 
is  oxidized  by  cytochrome — oxidase 
which  is  identical  with  indophenol 
oxidase  and  Warburg's  respiratory  en- 
zyme. See  study  of  cytochrome 
oxidase-cytochrome  system  in  kidney 
(Flexner,  L.  B.,  J.  Biol.  Chem.,  1939, 
131,  703-711).    See  Oxidase. 

Cytcphaga  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  Kurloff  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. 

Dahlia,  see  Hofmann's  Violet. 

Dahlia  B,  see  Methyl  Violet. 

Damar  is  gum  damar  dissolved  in  xylol 
and  used  to  mount  sections. 

Dark  Brown  Salt  R,  see  Chrysoidin  Y. 

Darkfleld  Microscope.  This  is  constructed 
on  the  same  principle  as  that  of  the 
uUramicroscope  developed  more  than  a 
generation  ago  by  Siedentopf  and  Zsig- 
mondy  in  so  far  that  it  depends  on  the 
Faraday-Tyndall  phenomenon  of  the 
illumination  of  minute  particles  by  light 
reflected  from  their  surfaces  as  when 
tobacco  smoke  drifts  into  a  beam  of 
light  in  an  otherwise  darkened  room. 
In  the  old  ultramicroscope  (intended 
mainly  for  colloidal  suspensions)  the 
illumination  v.'as  from  one  side  through 
a  slit,  while  in  the  modern  darkfield 
condenser  (designed  for  work  with 
cells)  it  is  from  below  at  the  sides. 
Ordinary  oculars  and  low  power  ob- 
jectives can  be  employed  but  for  oil 
immersion  work  the  best  objective  is  a 
3  mm.  fitted  with  an  iris  diaphram. 
Especially  adapted  and  more  powerful 
objectives  can  usually  be  obtained  and 
are  of  great  value.  Examination  in  the 
darkfield  is  required  for  the  study  of 
Microincineration  preparations,  of 
living  Spirochetes  and  other  small 
microorganisms,  of  Chylomicrons  and  a 
wide  variety  of  cellular  components. 
Ordinarilj'-  the  full  usefulness  of  the 
method  is  not  realized  because  investiga- 
tors content  themselves  with  inade- 
quate light  and  dry,  low  power,  ob- 
jectives. 

Davenport's  2-hour  method  for  staining 
nerve  fibers  in  paraffin  sections  with 


DAVENPORT'S 


77 


DEAD  CELLS 


protargoL  1946  modification  written 
by  Dr.  H.  A.  Davenport  of  original 
(Davenport,  H.  A.,  Mc Arthur,  J.,  and 
Bruesch,  S.  R.,  Stain  Tecbn.,  1939,  14, 
21-26).  Fix  for  1  to  3  days  in:  Form- 
amide  (Eastman  Kodak  Co.),  10  cc; 
paranitroplienol,  5  gm.:  95%  etiiyl  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  58-62°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.  v/ater.  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  o.xalic  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 
been  obtained  with  material  fixed  in 
Bouin's  picric-fornialin-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  Schizosaccharoviyces  pombi  are 
considered  dead  when  tinged  even 
slightly   by   the  sUxin. 

3.  Decolor ization  (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  difl^icult  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  j  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  fiat  and  rounded  cells.  These  were 
pale  grayish  sacs  with  very  thin  walls 
and  fluid  contents  in  which  varying 
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,  So,  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  pieca 


DEAD  CELLS 


78 


DEHYDRATION 


of  the  lower  epidermis  of  the  scale  of 
the  onion  bulb  is  peeled  off  and  placed, 
cutin  side  down,  on  a  slide.  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  fi.xation,  particu- 
larily  so  when  the  agent  is  a  poor  fi.xative. 
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  MuIIer'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  sliglit  bending  of  the  bone  or 
by  the  needle  method.  Over  decal- 
cification is  not  likely. 

For  rapid  decalcification  he  advises 
using  sat.  aq.  phloroglucin  to  which 
5-30%  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 o.xide  and  stir  until  completely 
dissolved.  If  the  magnesium  oxide 
contains  carbonate  it  sliould  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 
decalcifying,  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  dccalcifer  and  imbedded  in 
paraffin  or  celloidin.  Some  investiga- 
tors prefer  the  latter  but  celloidin 
sections  are  not  so  easily  handled.  See 
Bones,  Teeth. 
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  fi.xative.  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 


DEHYDRATION 


79 


DIANIL  BLUE  2R 


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.  Dioxan 
will  not  only  take  the  place  of  the  alcohol 
but  also  that  of  the  clearing  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  expensive, 
inflammable  and  quickly  takes  up  water 
from  the  air.  Whether  it  is  injurious 
when  breathed  remains  to  be  deter- 
mined. On  the  whole  it  appears  that 
little  is  to  be  gained  by  such  substitutes. 
However,  Cyclohexanone  deserves  fur- 
ther trial.  If  alcohol  must  be  avoided 
it  is  always  possible  to  fix  in  formalin 
and  to  use  frozen  sections.  By  the  Alt- 
mann-Gersh  technique  the  tissues  are 
dehydrated  in  vacuo  while  still  frozen. 

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. 

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. 

Desoxyribonucleic  Acid.  Method  for  deter- 
mination in  isolated  nuclei  of  tumor 
cells  (Dounce,  A.  L.,  J.  Biol.  Chem., 
1943,  151,  235-240). 

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  IP.  Schoenhoimer,  R., 
Harvey  Lectures,  1937,  32,  122-144  em- 
ployed deuterium  combined  with  oxy- 
gen as  heavy  water  H^O  to  mark  fatty 
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 Metabolism  of  Fats.  Biological 
Sjanposia  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., 
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. 

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. 

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.  IL,  Am.  J.  Physiol.,  1918, 
46,  483-186  and  Sutter,  M.,  Anat.  Rec, 
1916,  16,  164-165  for  dye  employed  by 
them  in  study  respectively  of  corpora 


DIANIL  BLUE  2R 


80 


DIPHTHERIA  BACILLI 


lutea  and  mammary  glands  but  these 
authors  do  not  employ  the  name :  dianil 
blue. 

'Manil  Red  4B,  see  Benzopurpurin  4B. 
iianthine  B,  see  Erythrosin,  bluish. 

:Jiaphane  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,  dehj^drate,  clear  in  tetralin 
(if  not  available,  benzol)  and  imbed  in 
paraffin.  See  use  of  diaphanol  in 
demonstrating  Melanins. 

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  everj^  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. 

Differential  Leucocyte  Count,  statistical 
study  of  uniformity  in  (Klotz,  L.  F., 
J.  Lab.  &  Clin.  Med.,  1939,  25,  424-434). 

Diffraction  Methods  for  measuring  diameter 
of  red  blood  cells  (Haden,  R.  L.,  J. 
Lab.  &  Clin.  Med.,  1937-38,  23,  508-518). 

Digitonine  reaction  of  Windaus  for  free 
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. 

Di  Nitrosoresorcinol  test  for  iron,  see  Iron. 

Diotrast,  trade  name  for  an  organic  iodine 
preparation  recommended  by  Gross, 
S.  W.,  Proc.  Soc.  Exp.  Biol.  &  Med., 
1939,  42,  258-259  for  injection  into 
common  carotid  with  later  x-ray  photo- 
graphs of  the  vascular  tree. 

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.  Dio.xan 
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  fi.xation  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.  .A.nz.,  1933,  102,  39-44)  suggest: 
dioxan  80%,  methyl  alcohol  20%,  paral- 
dehyde 2%,  and  acetic  acid  5%.  Sec 
Clearing,  Pituitary.  See  as  ingredient 
of  Lison's  glycogen  method;  al?o  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  bj^  Hall, 
W.  E.  B.,  Am.  J.  Clin.  Path.,  1943,  7 
(Technical  Section),  08-100. 

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. 

Diphtheria    Bacilli.  1.  Neisser's    slain 

(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 
stainsmear  pouron  A,30-60sec.    Wash. 


DIPHTHERIA  BACILLI 


81 


DOPA,  OXIDASE  REACTION 


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  (Hocchst)  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  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 
disa.zo  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  (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  mounting  medium 
giving  good  preservation  of  color  in 
microscopic  slides.  See  also  Ivirk- 
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-c/ihy- 
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  5%  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  (NaaHP04 
-f  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  H.  Sharlit  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  pheuoloxidases  but  thus 
far  they  have  not  been  found  in  mam- 
malian tissues. 


DOROTHY  REED  CELLS 


82 


EAR 


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 :  Alake 
1%  and  3%  solutions  of  celloidin  in 
methyl  benzoate  which  take  about  a 
month.  Pour  some  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 
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. 

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. 

Dysentery,  see  Endamoeba. 

Dysprosium,  see  Atomic  Weights. 

Ear.— Written  by  W.  P.  Covell,  Dept.  of 
Anatomy,  Washington  University,  St. 
Louis,  June  5,  1946 — Microscopic  exami- 
nations of  the  ear  are  nearly  always 
made  on  sections.  This  is  understand- 
able, but  it  is  possible  that  the  study  of 
still  living  tissues,  removed  by  careful 
and  minute  dissections,  is  a  field  of  con- 
siderable promise.  The  close  apposi- 
tion of  epithelial  and  nervous  com- 
ponents to  bone  necessitates  decalcifica- 
tion except  in  the  case  of  young  em- 
bryos. The  frequent  use  of  celloidin  in 
place  of  paraffin  for  imbedding  is  oc- 
casioned by  the  wide  range  of  diversity 
in  resistance  of  the  organ  to  the  micro- 
tome knife,  fluid  containing  lumina 
being  surrosnded  by  hard  dense  bone. 
The  histological  techniques  actually  in 
use  for  the  ear  are  fewer  in  number  and 
more  limited  in  range  than  those  era- 
ployed  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,  or  10%  for- 
malin. The  best  results  are  to  be  ob- 
tained by  the  use  of  animal  material  for 
which  prompt  fixation  by  perfusion 
methods  has  been  done.  Isolated  tem- 
poral bones  placed  in  fixative  are  prone 
to  show  autoiytic  changes  in  end  organs 
and  ganglion  cells  in  one-half  hour  fol- 
lowing death.  The  literature  is  filled 
with  autoiytic  changes  described  as 
specific  pathologic  alterations  due  to 
drugs,   toxins,   poisons,   and   so   forth. 


EAR 


83 


EAR 


Actually  luaiiy  of  these  are  the  result 
of  poor  penetration  of  fixatives  and 
elapse  of  time?  between  autopsy  and 
fixation.  In  an  attempt  to  overcome 
the  slow  penetration  of  a  fixative  and 
prevention  of  artefacts  Guild  made  use 
of  water  from  which  gases  had  been  ex- 
hausted as  the  medium  for  fixatives, 
decalcif3ing  solutions   and   alcohols. 

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.  Xitric  acid  in  2  to 
5%  concentration  is  generally  used  for 
human  temporal  bones.  The  lower  con- 
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 (Sr^^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 
daj's.  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  tliis  method. 

In  his  study  of  kittens,  young  rabbits, 
dogs  and  rats.  Van  der  Stricht,  O., 
Contrib.  to  Embryol.,  Carnegie  inst., 
1020,  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  fi.xatives  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  ins  paper  for  the  demonstra- 
tion of  mitochondria  in  the  sustentacu- 
lar  and  hair  cells.  A  difl'erential  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  difiicult  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  chochlea  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  nucra  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  tne  cochlear  nerve  were 
stained  with  1:5000  methylene  blue  in 
isolated  pieces  of  the  fresh  membranous 
cochlea  removed  under  the  dissecting 
microscope;  Co  veil,  W.  P.,  Ann.  Oto., 
Rhino.,  Laryngo.,  1938,  47,  62-67. 
Ross,  E.  L.  and  Hamilton,  J.  W.,  Arch. 
Otol.,  1939,  29,  428-436  allowed  mer- 
curochrome  to  remain  in  the  middle  car 
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.  Trypan 
blue  has  been  utilized  to  ascertain  the 
effects  of  trauma  on  scala  media  cells  of 
the  cochlea;  Lurie,  M.  H.,  Ann.  Oto., 
Rliino.,  Laryngo.,  1942,  51,   712-717. 

A  method  for  graphic  reconstruction 
of  the  organ  of  Corti  was  introduced  by 
Guild,  S.  R.,  Anat.  Rec,  1921,  22,  141- 


EAR 


84 


ELECTRICAL  RESISTANCE 


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.  Ai)at.,  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. 

Various  methods  of  reconstruction 
have  been  employed  particularly  for 
studv  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.  See 
Endolymph. 

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  lien's  eggs,  see  Chorio- 
allantoic Membrane.  Egg  of  helminths, 
see  Floatation  Techniques. 

Ehrlich-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 
CO.  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  Hemaloxylin.  Dissolve  2 
gm.  hemato.xylin  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- 
dimethylamino-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  methj-l  green 
is  a  basic  dj'e  so  that  it  is  not  made  up  of 
three  acid  dj'cs.     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  miu.;  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,  see  Coccidsa. 

Elastic  Fibers.  Viewed  singly  in  fresh 
unstained  spreads  of  Loose  Connective 
Tissue  these  fibers  have  a  faint  yellow 
color,  are  thinner  and  more  highly  re- 
fractile  than  collagenic  fibers.  More- 
over they  are  optically  homogeneous, 
branch  repeatedly  to  form  networks  and 
do  not  swell  when  subjected  to  dilute 
acetic  acid.  To  demonstrate  them  in 
sections  a  choice  can  be  made  from 
several  quite  specific  stains  including 
Weigert's  Resorcin  Fuchsin,  Verhoflf's 
Elastic  Tissue  Stain,  Unna's  Orcein 
Method,  Krajian's  Congo  Stain. 

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 
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 
CO  rain.  Wash  quickly  in  Acid  Alcohol, 
dehydrate  and  differentiate  in  abs.  ale. 
till  section  is  only  faintly  red.  Pass 
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 aad  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. 

Electric  Tissues  of  fishes,  methods  for  are 
given  by  Dahlgren   (McClung,  p.  434). 

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  iiave  an  important 
bearing  on  structure.  In  view  of  the 
wide  variety  of  cells  studied  it  is  inter- 


ELECTRICAL  RESISTANCE 


85 


ELECTRONi'MICROSCOPY 


esting,   as    Danielli    remarks    (Bourne. 

p.  42),  that  a  definite  pattern  slioula 

emerge  of  a  cell  plasma  membrane  only 

10~^-10~''  cm.  in  thickness  corresponding 

to  a  specific  resistance  of  lO'^-lOi^  ohms. 

Electron  Microscopy.     Details  provided  bj' 

Dr.  W.  L.  Simpson  of  The  Barnard  Free 

Skin  and  Cancer  Hospital. 

L  Transmitted    electron    beam    type. 

The    relationship    of    resolving    power 

(R.P.)  to  the  wave  length   (A)  of  light 

employed  and  to  the  numerical  aperture 

(N.A.)  of  a  lens  system  as  expressed  in 

5\ 
the  relation  R.P.  equals  t^  i-  proved  for 

many  years  an  apparently  insurmount- 
able limitation  to  the  biologist's  desire 
to  investigate  directly  minute  structure 
of  cells  and  tissues.  Even  when  ultra- 
violet light  of  2250A  was  employed  the 
limit  of  resolution  was  O.OS/u  in  a  system 
of  N.A.  L40.  With  visible  light  the 
limit  was  approximately  0.2/i.  On  the 
assumption  that  the  angle  of  visual 
acuity  is  1  minute  of  an  arc,  the  greatest 
magnification  that  was  practical  with 
visible  light  ranged  from  1750  to  2100 
times.  There  is,  of  course,  no  limitation 
to  the  actual  magnification  tliat  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  confirma- 
tion 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 
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  0.00 l/i.  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  fifteen  years  old.  Busch  described 
the  first  such  instrument  using  magnetic 
lenses  (Busch,  H.,  Archiv.  f.  Elektro- 
teknik,  1927,  18,  583-594).  Though 
many  improvements  were  m^ade  in  de- 
sign 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  (numer- 
ous papers,  1934-1940)  the  instrument 
was  developed  to  the  state  that  it  is  in 
at  present.  In  this  country  an  excellent 
instrument,     capable    of    giving    high 


resolution  has  been  developed  and  com- 
mercially marketed  by  the  Radio  Cor- 
poration of  America.  The  apparatus  is 
described  by  Zworykin  (V.  K.,  Science, 
1940,  92,  51-53).  In  this  instrument 
electrons  emitted  from  a  hot  wire  fila- 
ment 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  photo- 
graphed on  a  sensitive  plate. 

Much  work  has  already  been  reported 
on  the  use  of  this  instrument.     Though 
it  is  too  early  to  evaluate  these  new 
findirigs,  it  appears  that  some  of  them 
contribute  greatly  to  our  knowledge  of 
the  finer  structure  of  viruses    (Green, 
R.   H.,   Anderson,   T.   F.  and  Smadel, 
J.  E.,  J.  Med.  Research,  1942,  75,  651- 
656)  and  biological  fibers,  e.g.,  studies 
on  collagen  fibers  by  Scott  and  Anderson 
(G.  H.  and  T.  F.,  Anat.  Rec,  1942,  82, 
445)  and  Schmitt,  Hall  and  Jakus   (F. 
O.,  C.  E.,  and  M.  A.,  J.  Cell,  and  Comp. 
Physiol.,  1942,  20,  11-33).     On  the  other 
hand,  much  of  the  work  appears  to  be 
devoted    to   a    simple   attempt    to    see 
things  magnified  more  than  has  hitherto 
been  possible.     Considerable  experience 
will  undoubtedly  be  required  before  the 
full  range  of  usefulness  of  such  an  appa- 
ratus can  be  realized.     By  modifications 
in    standard    microtomes    and    special 
treatment  of  tissues  sections  can  be  cut 
0.1m  thick  (Richards,  A.  G.,  Jr.,  Ander- 
son, T.  F.,  and  Hance,  R.  T.,  J.  Exp. 
Biol.  &  Med.,  194-L  51,  148-152).     For 
study  of  sperm,  see  Baylor,  M.  R.  B., 
Nalbandov,  A.  and  Clark,  G.  L.,  Pro. 
Soc.  Exp.  Biol.  &  Med.,  1944,  54,  229- 
232.     Recent   developments,  see   Wyc- 
koff,  R.  W.  G.,  Science,  1946,  104,  21-26. 
The  R.  C.  A.  and  General  Electric  Ma- 
chines are  compared  bv  Click,  D.,  Ann. 
Rev.  Biochem.  1944,  13,  705-734. 

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.  II.  and  G.  H.,  R.S.I.,  1937,  8,  288- 
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 


ELECTRON  MICROSCOPY 


86 


ELEMENTARY  BODIES 


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 
tliat  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 Vv'hich  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 
411  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  (Grlibler)  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- 
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  cresyl  blue, 
neutral  red,  methylene  blue  and  azur  it 
are  recommended.  Before  staining 
smears,  fixed  in  a  variety  of  ways,  pre- 
treatment  with  2.5%  aq.  potassium  per- 


ELEMENTARY  BODIES 


87 


ENAMEL 


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.  prirauHne 
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. 

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 
tri chrome  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,  H.  A., 
Stain  Techn.,  1934,  8,  143-149). 

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.  +  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,  &-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  alcohols  to  water. 
Stain  in  Iron  Hematoxylin. 

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  +  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, 


ENAMEL 


88 


ENZYMES 


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. 

Endamoeba,  see  Entameba. 

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. 

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., 
J.  Lab.  &  Clin.  Med.,  1939,  25,  543-544). 

Enrichment  techniques,  see  Conceiitration. 

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  maj''  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 
•iskeptwarm.  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 (Mallorj',  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.  imtil  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,  |-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,  H.,  Ztschr.  f.  Mikr.  Anat. 
Forsch.,  1925,  2,  506-535.  See  Small 
Intestine. 

Entomological  Techniques,  see  Mosquito, 
Ticks,  Insects,  Arachnids,  Parasites. 

Enzymes.  Their  name  is  legion.  At  pres- 
ent only  a  few  can  be  localized  histo- 
chemically  within  or  near  their  cells  of 
origin.  There  is  no  better  example  of 
advantageous  association  between  histo- 
logical and  biochemical  methods.  At 
present  four  principal  kinds  of  technique 
are  employed  for  localization:  (1)  By 
spectrographic  identification  in  the 
tissues — especially  the  Cytochrome 
Oxidases,  (2)  By  close  comparison  of 
enzymatic  properties  with  cellular  com- 
position of  the  tissues — Amylase,  Pep- 
sin, Peptidase,  Esterase,  Protease, 
Cholinesterase,  Lipase,  Urease,  Car- 
bonic Anhydrase,  etc.,  (3)  By  separation 
of  nuclei  from  cytoplasms  by  differential 
centrifugation  and  by  estimation  of 
enzyme  in  each — -Arginase,  and  in  sepa- 
rated cytoplasmic  granules  Adenylpyro- 
phosphatase.  (4)  By  the  development 
of  characteristic  products  within  the 
cells  or  tissues — Cytochrome  Oxidase, 
Oxidase,  Phenolase,  Peroxidase,  Phos- 
phatase, Dopa  Oxidase.  See  also  Nu- 
clease, Cathepsin,  Lysozyme  and 
Adenosinase.  The  terms  lyo-  and 
desmoenzymes  are  used  to  indicate  re- 
spectively the  enzymes  which  can  and 
cannot  be  separated  from  cell  proteins. 
Whether  dyes  are  of  any  service  as 
indicators  of  the  presence  of  enzymes 
remains  to  be  determined.  However 
Robertson,  T.  B.,  J.  Biol.  Chem.,  1906, 
2,  317-383,  found  that  a  little  safranin 
added  to  a  solution  of  trypsin  forms  a 
colored  ppt.  and  Holzberg,  H.  L.,  J. 
Biol.  Chem.,  1913,  14,  335-339  observed 
that  the  ppt.  exhibits  proteolytic  ac- 
tivity, and  Marston,  H.  R.,  Biochem. 
J.,   1923,   17,  851-859,  discovered  that 


ENZYMES 


89 


EOS  IN.' y 


azure  dyes,  including  neutral  red,  like- 
wise precipitate  pepsin,  trypsin,  crep- 
sin  and  papain.  The  linkage  of  enzyme 
to  dye  is,  he  thinks,  through  the  basic 
nitrogen  of  the  heterocyclic  ring  of  the 
latter.  In  view  of  these  observations, 
and  the  coloration  of  mitochondria  with 
janus  green,  Marston  suggests  that  the 
mitochondria  contain  proteolytic  en- 
zymes. Methods  for  the  enzymatic 
analysis  of  purified  elementary  bodies 
of  vaccinia  are  described  by  Hoagland, 
C.  L.,  Ward,  S.  M.,  Smadel,  J.  E.,  and 
Rivers,  T.  M.,  J.  Exper.  Med.,  1942,  76, 
163-173.  Two  very  helpful  reviews  are 
recommended:  Gersch,  I.,  Physiol. 
Rev.,  1941,  21,  242-266,  and  Blaschko, 
H.  and  Jacobson,  W.  (Bourne,  pp.  189- 
224). 

Enzymes  are  coming  into  their  own 
as  technical  tools.  Ribonuclease  in 
study  of  the  mechanism  of  the  Gram 
Stain,  Hyaluronidase  as  a  Spreading 
Factor,  Lysozyme  in  detecting  the  pres- 
ence of  acetyl  amino  polysaccharide  in 
bacteria,  Pectinols  in  demonstrating 
chromosomes,  Pancreatin  in  digesting 
away  the  cellular  components  of  the 
spleen  leaving  onlj^  the  framework,  and 
a  great  many  others  in  specifically  hy- 
drolyzing  the  substrates  on  which  they 
act.  The  enzymatic  destruction  of  the 
capsules  of  pneumococci  as  summarized 
by  Dubos,  R.  J.,  The  Bacterial  Cell. 
Harvard  Univ.  Press,  1945,  460  pp.  is  an 
instructive  example.  When  the  capsu- 
lar polysaccharide  is  hydrolysed  by  the 
enzyme  the  pneumococci  are  made  vul- 
nerable to  the  phagocytic  action  of 
leucocytes. 

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. 

Eosin  B  or  bluish  (CI,  771)— eosin  BN,  BW, 
or  DHV,  eosin  scarlet,  eosin  scarlet  B, 
imperial  red,  nopalin  G,  saff  rosin, 
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.  hematoxylin)  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  gm.) 
7  min.  Rinse  quickly  in  aq.  dest. 
Counterstain  in  0.5%  aq.  toluidin  blue 
20-30  sec.  Rinse  again  aq.  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 
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 


EOSIN  Y 


90 


ERIE  GARNET  B 


thionin  as  substitute  for  Wright's  stain 
(Saye,  E.  B.,  Am.  J.  Clin.  Path.,  Tech. 
Suppl.,  1943,  13,  12). 
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  III. 

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.  unsatisfactorj^  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  oft"  with  a  blunt 
instrument.  Separation  is  more  diffi- 
cult when  tempei'ature  is  over  51  °C. 
Epidermises  removed  in  this  way  for  a 
time  continue  to  consume  oxygen  and 
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  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 dj^e  used  for  staining  frozen  sections 
(Geschickter,  C.  F.,  Stain  Techn., 
1930,5,  81-86). 


ERIE  VIOLET  BW 


91 


ERYTHROCYTES 


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). 

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  shape,  size  and  maturity 
of  erythrocytes  in  smears  and  to  measure 
hemoglobin  content  of  blood  by  hemo- 
globinometers.    See  Reticulocytes. 

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  polychromatophilia.  For  resistance 
to  hemolysis  in  hypotonic  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  K^to,  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). 

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., 
Ani.  J.  Med.  Sci.,  1941,  202,  157-162) 
which  open  up  a  new  field  for  study  of 
age  changes  because  the  cells  are  thereby 
dated.  Stratification  of  contents  of 
erythrocytes  by  ultracentrifugation 
(Beams,  H.  W.,  and  Hines,  E.  H.,  Anat. 
Rec,  1944,  89,  531).  Special  methods 
are  given  under  Hemoglobin,  Flagella 
and  Reticulocytes. 
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.  Maximow  and  Bloom  employing 
mainly  permanent  preparations  list: 

Hemocytoblasts:  "...  large  (up  to 
15m)  ameboid,  non-granular  basophil 
cells  of  Ij^mphoid  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  Romano wsky  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." 


ERYTHROCYTES 


92 


EYES 


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 
polychromatophilia,  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. 

Esterase,  see  method  under  Lipase. 

Ethyl  Eosin  (CI,  770).  The  ethyl  ester  of 
eosin  Y.  Sold  often  as  alcohol  soluble 
eosin.    See  Eosins. 

Ethyl  Green  (CI,  685) .  This  is,  like  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  hexaethyl  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  Heidenhain's  modification 
of  Mallory's  ethyl-violet  orange  G  after 
formalin  fi.xation. 

Ethyl  Violet-Biebrich  Scarlet,  see  Bowie's 
stain  for  pepsinogen. 

Ethylene  Glycol  Mono-Ethyl  Ether  = 
Cellosolve. 

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). 

Extracellular  fluid  or  phase,  see  Chloride. 

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  fixative  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    ia 


EYES 


93 


FECES 


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  saved  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.,  Zcit.  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 
Handbuch  der  Ophthalmologie  (Schieck 
and  Briickner,  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  liaa  been  thorough.  Use  glycerin 
jelly  for  mounting. 

Fahrenheit    Temperature    to     Centigrade. 

Use  the  following  relation: 

a  (F°-32)  =c° 

302  °F  ±  i  (302  -  32)  =  a  (270)  =  150*  C. 

.S-F  ±  S  (5  -  32)  =  a  (-  27)  =  -  15°C. 

-  13°F  ±  5  (-  13  -  32)  =  i  (-  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. 

Farrant's  Medium.  Gum  arable,  30  gm.; 
glycerin,  30  cc. ;  arsenous  oxide  (arsenic 
trioxide),  0.1  gm.;  aq.  dest.,  30  cc. 
(McClung,  p.617). 

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  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  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  tri chrome  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 


FECES 


94 


FILTERS 


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  necessarj' 
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. 

Ferments,  see  Enzymes. 

Ferric  Chlorlde-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,  15,37^1). 

Feulgen  Reaction,  see  Thymonucleic  Acid. 

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. 
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  nan- 
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  8-12ju;  N,  pores 
5-7m;  and  W,  pores  3-4^1. 

Mandler.  American  modification  of 
Berkefeld  but  made  of  kieselguhr,  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. 


FILTERS 


95 


FIXATION 


Elt'ord.     Made  of  collodion. 

Fischler's  modification  of  Benda's  stain  for 
Jally  acids  and  soaps  (Fischler,  F., 
Zentralbl  f.  Allg.  Path.  u.  path.  Anat., 
1904, 15,  913-917)  has  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 
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  fatty  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,  (2.5  gms.ferricyanide  and  2  gm. 
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  stain  neutral  fats  inaddition  stain  with 
scarlet  red  after  washing  out  Weigert's 
fluid,  rinse  in  70%  ale.  and  in  water  and 
mount  in  glycerin. 

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  thereby  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. 

P^educe  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 
l^een  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  mom.ent  after  death. 
See  Postmortem  Changes.  If  delay  is 
unavoidable,  keep  the  body,  or  the  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.,  Flem- 
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 


FIXATION 


96 


FIXATIVES 


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,  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  fixative  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  arc  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  supplj'^  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 
lias  been  studied  l)y  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  fixative  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 
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.  Benin'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 
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  ai'e  listed,  further 
data  being  given  under  each  heading. 


Acetic   osmic   bichro- 
mate 
Alcohol  (ethyl) 


Allen 

Barium  chloride  and 
formalin 


FIXATIVES 


97 


FLOATATION  TECHNIQUES 


Basic  lead  acetate 

Bouin 

Cadmium  chloride 

Carnoy 

Carnoy-Lebru  n  n 

Champy 

Chloral  hydrate 

Destin 

Diaphanol 

Dioxan 

Downey 

Erlicki 

Ferric   chloride-osmic 

acid 
Flemming 
Formalin 
Formalin-Zenker 
Giemsa 
Gilson 
Helly 
Hermann 
Hischler 
Ivleinenberg 
Kolatchew 


Lactophenol 
LUlie 

Mann 

March! 

Maximow 

Mercuric  chloride 

Methyl  alcohol 

Muller 

Orth 

Osmic  acid 

Parabenzoquinone 

Perenyi 

Petrunkevitch 

Regaud 

Rabl 

Schandinn 

Silver  nitrate 

Susa 

Tellyesnicky 

Weigert 

Van  Gehuchten 

Zenker 

Zweibaum 


Flagella.  1.  Of  bacteria.  LoeflQer'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  Shunk,  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  (Pijper, 
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  -f-  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  off'ers  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 
sufficient  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. 

Flagellates,  intestinal.  Those  commonly 
found  in  man  are,  according  to  Craig, 
p.  115,  Giardia  lamblia,  Chilomaslix 
mesnili,  and  TricJiomonas  hominis;  less 
frequently  seen  are  Embadomonas  in- 
testinalis  and  Ealeromonis  hominis. 
Stains  much  the  same  as  for  Endameba 
and  Leishmania.  See  Craig  for  choice 
of  suitable  culture  medium. 

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  l%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. 
They  are  floated  out  by  the  use  of  hyper- 
tonic salt  and  other  solutions,  some- 
times with  the  aid  of  centrifugal  force 


FLOATATION  TECHNIQUES 


98 


FLUORESCENCE  MICROSCOPY 


(E.  C.  Faust,  in  Simmons  and  Gentz- 
kow,  p.  684). 

Florence's  Reaction.  The  standard  test  for 
choline  in  seminal  stains.  As  described 
by  PoUak,  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  25/i  long  and  8m  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  thej'  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  gravitj^  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  Synovial 

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 : 
Fluorescence  is  the  property,  pos- 
sessed by  many  substances,  of  convert- 
ing short  wavelengths  of  light  into 
longer  wavelengths.  lu  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  30  years, 
their  use  has  been  limited  until  recently 
by  their  relatively  high  cost  and  by  the 


apparent  failure  of  biologists  to  appre- 
ciate the  possibilities  of  this  type  of 
observation.  Recent  technological  de- 
velopments 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  laboratories.  Evidence 
of  heightened  interest  in  this  field  is 
found  in  the  numerous  papers  concerning 
fluorescence  microscopy  within  the  past 
10  years.  Although  several  reviews  of 
the  subject  already  exist  (Haitinger, 
M.,  Fluorescenz-Mikroscopie,  Akadem- 
ische  Verlagsgesellschift,  Leipzig,  1938; 
Hamperl,  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-called  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. 
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  liaving  a  high  reflecting  power  for 


PLUORESCENCE  MICROSCOPY 


99 


FLUORESCENCE  MICROSCOPY 


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  lias  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  used 
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  be  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 
paraffin  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 
IL,  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  Pia,gins,  A.  B., 
Arch.  Path.,  1941,  32,  258-271).  Simp- 
son and  Cramer  (W.  L.  and  W.,  Cancer 
Research,  1943,  in  press)  have  used  the 
method  to  follow  the  distribution  and 
persistance  of  methylcholanthrene  in 
skin. 

Another  kind  of  fluorescence  is  the 
"secondary"  fluorescence  that  appears 
in  certain  components  of  the  tissue  after 
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 


FLUORESCENCE  MICROSCOPY 


100 


FORMALIN 


in  ultraviolet  light.  Such  substances 
are  called  fluorochromes.  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.  See  Vitamin  A,  Tubercle  Bacil- 
lus, Cell  Injury,  Uranium,  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. 

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.  NajCOs  to  10  cc.  10%  aq. 
AgNOs.  Let  precipitate  settle.  De- 
cant  supernatant   fluid.    Make   up   to 


50  cc.  with  aq.  dest.  Shake,  repeat  sett- 
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.  Tone  2  min. 
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  Carnoy'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  wash  again  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  to  Laidlaw, 
G.  F.,  Am.  J.  Path.,  1929,  5,  239-247. 
See  general  remarks  on  Silver  Methods. 

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 


FORMALIN 


101 


FROZEN  SECTIONS 


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. Unless  neutral  formalin  is  speci- 
fied and  the  manner  of  neutralization, 
it  is  best  simply  to  use  the  fairly  pure 
product  which  now  can  readily  be  ob- 
tained. Experiments  by  Davenport, 
H.  A.,  Stain  Techn.,  1934,  9,  49-52  show 
that  as  a  neurological  fixative  slightly 
acid  formalin  is  somewhat  better  than 
neutral  formalin.  A  few  of  the  many 
demands  for  formalin  as  a  fixative  will 
be  found  under : 


Alizarin  red  S 

Giemsa  staining 

Alveolar  pores 

Glia  staining 

Amyloid 

Gomori 

ArgentafBne  cells 

Gordon 

Arsenic 

Grieves 

Articular    nerve    ter- 

Johnson's neutral  red 

minals 

Kinney 

Bile  pigments 

Krajian 

Bismuth 

Liebermann  -   Burch- 

Bodian 

ardt 

Bone 

Madder  staining 

Buz  agio 

Mallory's    connective 

Cajal's  brom-formol 

tissue  stain 

Cartilage 

Microglia 

Chitin 

Mucus 

Chloride 

Nile  blue  sulphate 

Chorioallantoic  mem- 

Perdrau 

brane 

Pia  mater 

C  hristeller-  Koy  ama 

Romieu 

Chromafifin  reaction 

Schultz  cholesterol 

Color  preservation 

Sebaceous  glands 

Connective  tissue  cells 

Silver  citrate 

Dopa  oxidase 

Smith-Dietrich 

Fatty  acids 

Spirochetes 

Fluorescence    micros- 

Vorhoeff 

copy 

Weigert-Pal 

Foot 

Weil 

Frozen  sections 

Wilder 

In  combination  with  other  reagents 
formalin  is  also  a  good  fi.xative  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  ^lallory    (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., 
Stain  Tech.,  1940, 15,  9-14). 

Formol  is  a  synonym  for  formalin. 

Formol-Miiller.  This  is  1  part  of  formol  to 
10  parts  Mailer'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. 

Fowl  Pox,  see  Borrel  Bodies. 

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,  see  Revival  after. 

Freezing  and  Drying,  see  Altmann-Gersh. 

Frozen  Sections.  These  are  of  great  value 
when  preparations  must  quickly  be 
made  and  when  methods  of  alcoholic 
dehydration  before  sectioning  are  contra- 


FROZEN  SECTIONS 


102 


FUNGI 


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's  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.  +  few  drops  NH4OH,  passed 
up  through  70,  85  and  95%  alcohol 
counterstained  in  absolute  alcohol  and 
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.  Perliaps  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. 

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. 
NH4OH  at  eO^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%  AgNOa  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%  AgNOa.  Then  add 
freshly  prepared  10%  NH4OH  drop  by 
drop  until  almost  entirely  clear.  Dilute 
to  28  cc.  withaq.  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  CO2  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  should  at  once  be 
removed  to  the  stain.)  The  Cresyl 
Violet  method  of  Tress  and  Tress  is 
recommended. 

Fuchsin  NB,  see  New  Fuchsin. 

Fuchsin  S,  SN,  SS,  ST  or  S  III,  see  Acid 
Fuchsin. 

Fungi.     Data    contributed    by   Dr.    Morris 

Moore  of  The  Barnard  Free  Skin  and 

Cancer  Hospital.     Revised  bv  him  July 

17,  1946. 

1.  Skin     scrapings     and     hair.    The 


FUNGI 


103 


FUNGI 


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  shape  and  size. 

Adamson  (H.  G.,  Brit.  J.  Dermat., 
1895,  7,  201-211,  237-244)  has  recom- 
mended clearing  with  5-10%  KOH  and 
staining  bj'  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  rain. ;  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, 
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 


FUNGI 


104 


FUNGI 


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  hydroxide  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. 

4.  Skin:  Unna,  Jr.  (P.,  Dermat. 
Wchnschr.,  1929,  88,  314-321)  advises 
the  following  modification  of  the  Pap- 
penheim-Unna,  Sr.  method  for  staining 
fungi  in  skin.  Fix  in  absolute  alcohol, 
then  run  through  the  alcohols  to  xylol 
and  imbed  in  paraffin.  Cut  sections  at 
\Qn,  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 


FUNGI 


105 


GAMMA 


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%  aq.  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  changes 
of  xylol  and  mount  in  balsam.  The 
branched  organisms  stain  blue  while 
the  hyaline  sheaths  ("clubs")  become 
pink  to  red. 

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  ni chrome  wire  to 
a  clean  slide.  This  should  be  done 
gently  to  avoid  destruction  of  the 
fungous  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,  Actino- 
mycetes. 

Furfural.  Has  een  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  coiinus  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,  Chlowphora  tinclora, 
which  grows  in  the  same  countries.  It 
is  chiefly  employed  for  woolens  giving 
shades  of  lemon  and  old  gold  (Leggctt, 
W.  F.,  Ancient  and  Medieval  Dyes. 
Brooklyn:  Chemical  Publishing  Co. 
Inc.,  1944,  95  pp.). 

Gadolinium   see  Atomic  Weights. 

Gallein  (CI,  781),  a  mordant  dye  of  light 
fastness  1.  Use  as  solution  0.5  gm.  in 
100  cc.  of  either  1%  aq.  ammonium 
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  (CI,  883)— alizarin  blue  RBN, 
chrom  blue  GCB,  fast  violet — A  basic 
oxazin  dye  which  is  an  excellent  stain 
for  nuclei  and  Nissl  substance  (Einar- 
son,  L.,  Am.  J.  Path.,  1932,  8,  295-307). 
This  method  is  for  celloidin  sections 
and  has  achieved  considerable  popu- 
larity. Almost  any  good  fixative  ap- 
pears to  be  satisfactory.  The  author 
used  96% alcohol,  Zenker's  fluid,  neutral 
formalin  1  part  -\-  4  parts  aq.  dest.  and 
several  others.  He  suggests  double 
imbedding  first  in  celloidin  followed  by 
soft  paraffin  (see  Peterfi)  but  in  this 
laboratory  the  usual  method  of  celloidin 
imbedding  is  used.  To  make  the  stain, 
dissolve  10  gms.  chrome  alum  (K2S04- 
Cr2S04-24H20)  in  200  cc.  aq.  dest. 
Add  0.3  gms.  gallocyanin  and  mix 
thoroughly.  Warm  slowly  and  boil  for 
20  min.  Cool  gradually  and  filter. 
First  rinse  50m  sections  in  aq.  dest. 
Stain,  12-24  hrs.  agitating.  Aq.  dest., 
1  change.  80%  alcohol,  agitate  occa- 
sionally. 95%  alcohol,  1  hr.;  absolute 
alcohol,  15  min.  Half  absolute  and 
ether  sufficient  time  to  dissolve 
celloidin.  Absolute  alcohol  enough  to 
remove  ether.  Transfer  to  white  oil 
of  thyme  to  clear,  carrying  over  a 
minimum  of  alcohol.  Toluol  a  few 
minutes.  Mount  in  clarite  X.  Note: 
the  oil  of  thyme  comes  from  Greece  and 
is  not  at  present  obtainable.  Use  cedar 
oil  instead.  The  above  method  gives 
beautifully  stained  Nissl  bodies  in 
thick  sections.  If  thin  sections  are 
wanted  imbed  in  hard  paraffin. 


Gamma  = 


1 
1000 


mg.  or  0.001  mg. 


GASTRIC  ICONTENTS 


106 


GIANT  CELLS 


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. 

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-Muller  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  tliat  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  usu.al  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  fixed,  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 
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. 

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  imder  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  tliiazole 
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 


GIANT  CELLS 


107 


GIEMSA'S  STAIN 


by  various  counler  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  Gienisa's  Stain. 

2.  Foreign  body  giant  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  (polykaryocytes)  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.  H ypertrophied  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  blood  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). 

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,  25  cc. 
commercial  formalin  and  4  cc.  glacial 
acetic  acid)  is  suggested  for  intracellular 

Erotozoa  (East  Coast  fever  parasites) 
y  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- 


GIEMSA'S  STAIN 


108 


GLYCOGEN 


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.  By  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,  p. 
401).  Eosinophile  leucocytes  in  (Or- 
ban,  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). 

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. 


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  inti'amural 
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  Kai- 
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  methods  have  been 
critically  studied  by  C.  M.  Bensley 
(Stain  Techn.,  1939,  14,  47-52).  This 
account  follows  her  presentation.  Since 
glycogen  is  labile,  immediate  fixation 
of  very  small  pieces  of  tissue  (2-3  mm.) 
and  agitation  of  the  fixative  are  neces- 
sary. She  recommends  9  parts  absolute 
ethyl  alcohol  +  1  part  commercial 
formalin  (i.e.  37%  formaldehyde)  neu- 
tralized with  MgCOa.  If  desired  the 
alcohol  in  this  fixative  can  be  saturated 
with  picric  acid.  After  fixation  for  say 
24  hrs.  wash  in  absolute  alcohol,  embed 
in  the  usual  way  in  paraffin  (carefully 
avoiding  overheating)  or  in  celloidin. 
1.  Best's     carmine.    Griibler's     car- 


GLYCOGEN 


109 


GOLD 


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  hemato.xylin 
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-Feulgen.  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 
HCl.  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  1^  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. 

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 
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,  261-2C6.  To  pre- 
pare silver  solution  dissolve  1  gm.  silver 
nitrate  in  10  cc.  aq.  dest.  and  add  11 
drops  40%  aq.  potassium  hydro.xide. 
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- 
P'eulgen  technique.  Excellent  illus- 
trations. 

Glycol  Stearate.  As  an  imbedding  medium 
(Cutler,  O.  I.,  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. 

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  formaldehj'de  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,  microcheraical  detection  of:  1.  Method 


GOLD 


110 


GOLGI  APPARATUS 


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.  Method  of  Okkels,  H.,  C.  rend. 
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 
arabic  in  the  dark  immediately  before 
use.  B.  Add  1  gm.  hydroquinone  pure 
to  100  cc.  10%  gum  arabic  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  intraperitoneal!}'  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.  SnCli -21120 
(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. 
laefore  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%  H2O2  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 bv  Gomori,  G.,  J.  ]\It.  Sinai 
Hosp.,  1944-45,  11,  317-326,  demonstra- 
tion of  gold  salts  by  reduction  to  metal- 
lic gold  with  SnCls.     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. 

Gold  Chloride  for  nerve  endings,  see 
Craven's  and  Carey's  methods. 

Gold  Orange,  see  Orange  II. 

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). 

Golgi  Apparatus  (reticular  material).  The 
following  account  is  partly  based  on 
Cowdry's  description  in  McClung  (pp. 
274-278).  Wliile  there  is  so  little  agree- 
ment as  to  just  what  the  Golgi  apparatus 
is,  it  is  difficult  to  describe  the  technique 
for  its  demonstration.  What  may,  how- 
ever, be  regarded  as  the  "type  struc- 
ture" was  first  revealed  by  Golgi  (Arch, 
di  biol.,  1898,  19,  448-453)  in  nerve  cells 
through  fixation  in  a  mixture  containing 
potassium  bichromate  and  osmic  acid 
followed  by  impregnation  with  silver. 
The  apparatus  appears  jet  black  against 
a  yellowish  background.  It  is  a  conspic- 
uous structure  consistingof  an  intricate 
network  of  anastomosing  strands.  This 
network  may  closely  envelop  the  nu- 
cleus, be  concentrated  to  one  side  of  it, 
or  else  be  scattered  rather  diffusely 
throughout  the  cytoplasm. 


GOLGI  APPARATUS 


111 


GOLGI  APPARATUS 


In  1902  Kopsch  showed  that  the  same 
material  can  be  blackened  by  prolonged 
treatment  with  2%  osmic  acid.  On  this 
affinity  for  both  silver  and  osmium  all 
the  modern  methods  for  revealing  the 
Golgi  apparatus  are  based.  Few  cy to- 
logical  reactions  are  more  fickle  and 
inconstant,  but,  when  after  many  at- 
tempts the  technique  is  successful, 
convincing  and  very  beautiful  prepara- 
tions result.  Unlike  the  mitochondria, 
the  Golgi  apparatus  cannot  be  studied 
unstained  or  supravitally  colored  in  the 
living  cell  with  any  degree  of  satisfaction 
except  perhaps  in  some  plants  (Guil- 
liermond,  A.,  Arch.  d'Anat.  Micro.. 
1927,  23,  1-98;  see  particularly  colored 
plate  1).  There  is  some  evidence  how- 
ever that  droplets  of  material  stainable 
with  neutral  red  may  be  associated 
topographically  with  the  Golgi  apparatus 
(Cowdry,  E.  V.  and  Scott,  G.  H.,  Arch. 
Inst.  Pasteur  de  Tunis,  1928,  233-252; 
Covell,  W.  P.  and  Scott,  G.  H.,  Anat. 
Rec,  1928,38,377-398). 

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 
impregnating  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 
requirements  should  be  carefully  fol- 
lowed. When  either  the  silver  nitrate 
or  osmic  acid  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  pan- 
creas are  also  recommended  but  are 
somewhat  more  difficult  to  handle.  All 
of  the  methods  of  impregnation  outlined 
below  frequently  bring  to  light  the 
mitochondria  also. 

1.  Cajal's  uranium  nitrate  silver 
method,  Carleton,  H.  M.,  J.  Roy.  Micr. 
Soc,  1919,  321-328.  This  is  one  of 
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  24-48  hrs. 
Rinse  in  aq.  dest.  Hydroquinone,2gm. ; 
formalin,  6  cc;  aq.  dest.,  100  cc;  an- 
hydrous sodium  sulphite,  0.15  gm.,  12 
hrs.  Wash  in  aq.  dest.,  dehydrate 
quickly,  clear,  imbed,  and  section. 

2.  Da  Fa7w's  cobalt  nitrate  silver 
method,  Da  Fano,  C.,  J.  Roy.  Micr.  Soc, 
1920, 157-161 .    Here  the  uranium  nitrate 


is  replaced  by  cobalt  nitrate.  In  other 
respects  the  technique  is  similar.  Da 
Fano  has,  however,  so  carefully  at- 
tempted to  control  troublesome  experi- 
mental conditions  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. 
In  the  case  of  embryos  and  delicate  tis- 
sues, when  shrinkage  is  to  be  feared, 
reduce  the  formalin  to  as  little  as  6  cc. 
With  cartilage  and  small  pieces  less  than 
3  mm.  thick,  like  the  organs  of  mice, 
shorten  the  time  of  fi.xation  to  3  to  4  hrs. 
Hollow  organs,  such  as  the  stomach  and 
intestine,  had  better  be  placed  in  the 
fixing  fluid  for  1  hr.  and  then  be  cut  into 
pieces  of  convenient  size  and  shape. 
For  the  spinal  cord,  cerebellum  and 
cerebrum  of  adults,  8-10  hrs.  is  recom- 
mended, but  fixation  should  never  ex- 
ceed 24  hrs.  In  the  case  of  the  testicle, 
he  advises  injection  of  the  fixative 
through  the  aorta  and  then  immersion 
in  it.  Wash  quickly  in  aq.  dest.  and 
impregnate  in  1.5%  aq.  silver  nitrate 
24-48  hrs.  The  concentration  of  silver 
nitrate  should  be  reduced  to  1%  for  very 
small  fragments  easily  permeable,  and 
be  increased  to  2%  for  tissues  containing 
much  fat  and  for  the  spinal  cord.  Im- 
pregnation is  effected  at  room  tempera- 
ture in  a  majority  of  cases.  When 
difficulty  is  experienced  in  impregnation 
the  use  of  an  incubator  at  36°  to  37  °C. 
is  advised.  Wash  rapidly  in  aq.  dest. 
and  cut  down  the  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.  5  hr.  Cut  with  a  freezing  micro- 
tome or  imbed  in  paraffin.  The  Golgi 
apparatus  should  be  colored  dark 
brown  or  black  against  a  yellow  back- 
ground. The  preparations  may  be  made 
more  permanent  by  gold  toning.  Pass 
to  water.     Then  0.1-0.2%  gold  chloride, 

2  hrs.  Counterstaiu  with  Alum  Car- 
mine, dehydrate,  clear  and  mount. 

3.  Kopsch's  method,  Kopsch,  F.,  Sitz.- 
Ber.  d.  K.  Preuss.  Akad.  d.  wiss.  Math. 
KL,  1902,  40,  929-935.  Immersion  of 
small  pieces  of  tissues  in  2%  aq.  osmic 
acid  for  8-16  days  often  brings  to  light 
the  Golgi  apparatus  but  there  is  con- 
siderable shrinkage  and  the  tissues  be- 
come rather  brittle. 

4.  Sjovall's  modification,  Sjovall,  E., 
Anat.  Hefte,  1906,  30,  261-391.  Fix  in 
10%  formalin,  8  hrs.  Wash  in  aq.  dest. 
2%  osmic  acid  at  35°C.,  2  days.  De- 
hydrate, clear,  imbed. 

5.  Hirschler's  modification,  Hirschler, 
J.  Arch.  f.  mikr.  Anat.,  1918,  89,  1-58. 
Fix  in   sat.  aq.   mercuric   chloride,   10 


GOLGI  APPARATUS 


112 


GOLGI  METHODS 


cc;  2%  osmic  acid,  10  cc,  at  room 
temperature  1-3  hrs.  Wash  in  running 
water  then  in  aq.  dest.,  5  hr.  2%  osmic 
acid  at  25 °C.,  12-16  days.  Wash  for 
24  hrs.  in  running  water,  dehydrate, 
clear  in  chloroform  and  imbed. 

6.  Kolatchew's  method,  Nassonov,  D. 
N.,  Arch.  f.  Mikr.  Anat.,  1924,  103, 
437-482.  Fix  in  3%  aq.  potassium 
bichromate,  10  cc;  1%  chromic  acid, 
10  cc. ;  and  2%  osmic  acid,  5  cc,  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  imbed. 

7.  Weigert's  Mann-Kopsch  method  as 
modified  by  Gatenby  (Lee's  Microt- 
omist's  Vade-mecum.  Ed.  9.  Ed.  by 
Gatenby,  J.  B.,  and  Cowdry,  E.  V., 
London,  1928) .  Fix  in  Mann's  corrosive 
osmic  acid  sol.  (sat.  aq.  corrosive  sub- 
limate in  salt  sol.,  10  cc  ;  1%  osmic  acid, 
10  cc)  j-3  hrs.  or  more.  Wash  in  aq. 
dest.,  15-30  min.  2%  osmic  acid,  room 
temperature  10-14  days.  Wash  in  run- 
ning water  2  hrs.  or  more.  Dehydrate, 
clear,  and  imbed.  In  the  sections 
Gatenby  was  able  to  extract  the  black- 
ening step  by  step  with  turpentine  and 
thus  to  considerably  improve  the  prep- 
arations. 

8.  Ludford's  modification,  Ludford, 
R.  J.,  J.  Roy.  Micr.  Soc,  1926,  107-109 
has  experimented  at  length  with  osmic 
acid  methods  and  states  that  his  best 
results  have  been  obtained  as  follows. 
Fix  mammalian  and  avian  tissues  in 
Mann's  corrosive  osmic  sol.  18  hrs. 
Wash  in  aq.  dest.,  30  min.  2%  osmic 
acid  at  30 °C.  for  3  days.  Water  at 
30°C.  1  day,  dehydrate,  clear,  imbed  in 
paraffin  and  section.  A  useful  variant  is 
to  fix  in  same  way  and  wash  in  aq.  dest. 
Then  osmicate  at  35°C.  for  3  days,  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. 

The  writer  prefers  the  Ludford  tech- 
niques. See  Lee  (pp.  313-326)  for  a 
critical  statement  of  the  problem;  also, 
Owens,  H.  B.  and  Bensley,  R.  R.,  Anat. 
Rec,  1929,  44,  79-109  for  a  careful  study 
of  factors  influencing  the  osmic  acid 
changes  and  for  their  ferric  chloride 
osmic  method.  Before  placing  any 
reliance  in  the  Golgi  apparatus  as  an 
indicator  of  cellular  activity  it  is  essen- 
tial (1)  to  make  sure  that  the  technique 
being  used  brings  to  light  all  the  Golgi 
apparatus,  not  only  a  part  of  it  and  (2) 
not  to  mistake  either  mitochondria  or 
droplets  formed  from  them  for  parts  of 
the  Golgi  apparatus  particularly  when 
it  is  in  a  dispersed  condition  spread 
about    in    the    cytoplasm.    But    when 


every  known  precaution  is  taken  the 
surface  and  volume  of  this  peculiar 
structure  can  be  measured  quantita- 
tively by  means  of  a  special  technique 
in  spinal  ganglion  cells  (Covell,  W.  P., 
Anat.  Rec,  35,  149,  1927).  According 
to  Monne,  L.,  Protoplasma,  1939,  42, 
184-192  it  can  be  demonstrated  by 
polarized  light.  Tarao,  S.,  Cytologia, 
1940,  11,  261-281  has  described  a  novel 
method,  which  involves  the  digestion  of 
frozen  sections  with  trypsin  and  colora- 
tion of  the  Golgi  apparatus  with  Nile 
blue  sulphate,  the  value  of  which  re- 
mains to  be  determined.  An  account 
of  alterations  in  hepatomas  is  of  interest 
(Dalton,  A.  J.  and  Edwards,  J.  E.,  J. 
Nat.  Cancer  Inst.,  1942,  2,  565-575). 
Localization  of  alkaline  phosphatase  in 
Golgi  region  of  cytoplasm  (Deane, 
H.  W.,  and  Dempsey,  E.  W.,  Anat. 
Rec,  1945,  93,  401-417). 

Golgi  Cox  Method.  For  adult  nervous 
system  (Dr.  J.  L.  O'Leary,  personal 
communication).  Fix  pieces  3-6  mm. 
thick  in  following  fluid:  add  20  cc.  5% 
aq.  potassium  bichromate  to  20  cc. 
5%  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  verj^ 
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 


GOLGI  METHODS 


113 


GOODPASTURE'S  METHOD 


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.  E.xcept  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.  (Dr.  J.  L.  O'Leary,  personal 
communication.)  It  is  essential  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  entering  fibers  are 
obtained  at  12  to  15  days  after  birth; 
of  short  axon  cells,  at  18  to  21  da,ys; 
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  80-100^. 
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. 

Gomori's  Method.  Silver  impregnation  of 
reticulum,  Gomori,  G.,  Am.  J.  Path., 
1937,  13,993-1001.  Treat  deparaffinized 
sections  of  formalin  fixed  material  with 
0.5-1%  aq.  potassium  permanganate. 
1-2  min.  Rinse  in  tap  water  ana 
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  |  to  |  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  (Revised 
by  G.  Gomori  May  7, 1946).  See  Phos- 
phatase. 

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,  0.69  gr.; 


GOODPASTURE'S  METHOD 


114 


GRAM'S  STAINS 


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. 

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  -|-  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% 
aq.  silver  nitrate  until  ppt.  is  dissolved. 
Add  5  cc.  3.1%  aq.  sodium  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'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  Tcchn.,  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,  0.8gm.; 
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.  Lab.  &  Clin.  Med., 
1940-41,  26,  1518-1519).  Make  thin, 
uniform  smears  and  fix  over  flame. 
Cover  slide  with  3%  gentian  violet  in 
20%  ale,  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,  dry  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.  Gram-Weigert  method 
(McClung,  p.  152).  Fix  in  Zenker's 
fluid.  Stain  paraffin  sections  lightly  in 
alum  hematoxylin  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 


GRAM'S  STAINS 


115 


GROWTH 


in  acetone  until  no  more  color  is  given 
off,  10-15  sec.  Wash  in  aq.  dost. 
Counterstain  in  0.05%  basic  fuchsin 
in  N/500  hydrochloric  acid  (see  Normal 
Solutions).  Drain,  do  not  wash,  apply 
1%  aq.  trinitrophenol,  5-I  min.  Wash 
in  aq.  dest.  Dehydrate  and  differen- 
tiate in  acetone  10-15  sec,  clear  in  xylol 
and  mount  in  balsam.  Gram  +  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-Wix  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-Ncel- 
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  this  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  mechanism  of  the  Gram  staining 
technique  and  the  interpretation  of  the 
findings  has  been  concisely  presented 
by  Dubos,  R.  J.,  The  Bacterial  Cell. 
Harvard  Univ.  Press,  1945,  460  pp. 
The  Gram  +  bacteria  differ  from  the 
Gram  —  ones  in  being  more  acidic  and 
perhaps  in  possession  of  lipids  with 
higher  content  of  unsaturated  acids. 
Their  Gram  positiveness  depends  on 
intactness  of  their  cell  walls,  for  ero- 
sions 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  re- 
stored by  "replating"  the  bacteria  with 
the  extract  of  the  outer  layer.  The 
.  outer  la5''er  apparently  contains  a  pro- 
tein 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  en- 
try and  retention  of  stains  but  the 
whole  manner  of  life  of  the  cells.  See 
Cell  Membrane,  Acid  Fast  Bacilli,  and 
Dead  Cells. 
Craven'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  l^nvier'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  panni cuius 
carnosus  of  hedgehog,  striated  muscle 
of  frog  and  lizard,  extrinsic  eye  muscle 
of  rabbit  and  human  pectoral  muscle. 

Gray,  R,  B,  BB,  see  Nigrosin,  water  soluble. 

Green  PL,  see  Naphthol  Green  B. 

Grenacher,  see  Alum  Carmine,  Borax 
Carmine. 

Grieves'  method  for  undecalcified  dental 
tissues  and  bone  as  outlined  by  Shipley 
(McCIung,  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 
hrs.  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 
rosiii  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 


GROWTH 


116 


HEAVY  WATER 


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  Phospliorus)  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. 

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. 

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  Daniar. 

Hafnium,  see  Atomic  Weights. 

Hairs.— Written  by  Mildred  Trotter,  Dept. 
of  Anatomy,  Washington  University, 
St.  Louis,  May  15, 1946.— The  hair  shaft 
(above  the  surface  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 
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 
permit  a  clear  view  of  its  structure  first 
bleach  with  hydrogen  peroxide.  In- 
dividual cells  of  the  shaft  can  be  iso- 
lated by  maceration  in  40%  aq.  po- 
tassium hydrate. 

Determination  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  pig- 
ment) requires  clearing  by  immersing 
in  some  oil  the  refractive  index  of  which 
is  approximately  the  same  as  that  of  the 
hair  (Hausmann,  L.  A.,  Sci.  Month., 
1944,  59,  195-202). 

Cross  sections  of  a  large  number  of 
hairs  (approximately  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  or  to  mark 
them  in  order  to  follow  their  cyclic 
changes.  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  Trot- 
ter, M.,  chapter  on  Hair  in  Cowdiy's 
Special  Cytology,  1932,  1,  40-65. 
Cleaning  and  mounting  of  individual 
hairs  (Duncan,  F.  W.,  J.  Roy.  Micr. 
Soc,  1943,  63,  85-88.  Microphotog- 
raphy  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). 

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. 

Harris  Alum  Hematoxylin.  Dissolve  1  gm. 
hematoxylin  in  10  cc  absolute  alcohol 
and  20  gms.  ammonium  or  potassium 
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. 

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 


117 


HEMATOXYLIN 


Heidenhain's  Azan  Stain  (Heidenhain,  M., 
Ztschr.  f .  wiss.  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 
p;lacial  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.,  A.m.  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.  H.  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. 

Helianthin,  see  Methyl  Orange, 

Heliotrope  B,  see  Amethyst  Violet. 

Helium,  see  Atomic  Weights. 

Helly'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.,  Phvtopathology,  1944,  34, 
700). 

Hemalum  (Mayer's)  Hematin,  1  gm.;  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  (NalOj),  1  gm.; 
ammonia  alum  (AINH4 (804)2  +  12 
H2O),  50gm.;aq.  dest.,  700  cc,  glycerol, 
300  cc,  glacial  acetic  acid,  20  cc.  No 
ripening  is  necessary.  Stain  sections 
formalin  fi.xed  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 
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  difficulty  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  kidney.  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  Porphyrins. 

Hematoxylin  is  the  most  useful  of  all  dyea 
in  animal  histology  and  pathology  (Gr. 
haimatodec,  blood  like  -f-  Xylon,  wood). 
It  is  an  extract  of  logwood  (Haematoxy- 
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 


HEMATOXYLIN 


118 


HEMOPHILUS  PERTUSSIS 


advantage.  Ripening  is  brought  about 
by  the  formation  of  oxidation  products. 
Consequently  it  is  recommended  that 
solutions  be  exposed  to  light  and  air. 
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.  eth)^  alcohol  should 
alwaj^s  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  hematoxjdin 
with  aq.  dest.  for  the  Iron  Hematoxylin 
technique.  See  also  Delafield's,  Ehr- 
lich's,  Harris'  and  Mayer's  hema- 
toxylin solutions,  likewise  Azure  II 
eosin  and  Hematoxylin. 

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  inaq.  dest. 
Stain  with  Harris'  Hematoxylin  (full 
strength)  for  12-15  min.  Blue  in  tap 
water  or  in  aq.  dest.  -f  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  maj'  be  em- 
ployed. The  Benslej'-s  (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.  erythrosin  can  take  the  place 
of  the  eosin  but  the  advantage  is  ques- 
tionable. 

Kemin  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,  Donogany 
.(Stitt,  p.  698).  Mix  1  drop  of  suspected 
fluid,  of  pyridin  and  of  20%  aq.  NAOH 
on  a  slide  and  allow  to  dr}'.  Radiating 
crystals  appearing  within  several  hours 
indicate    presence   of    hemochromogen. 

Hemocytoblasts,  see  Erythrocytes,  develop- 
mental series. 

Hemofuscin.  Mallory's  fuchsin  stain.  Fix 
in  Zenker's  fluid,  alcohol  or  10%  forma- 
lin. Stain  nuclei  in  paraflin  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,  hemttsiderin  and  melanin  unstained 
(Mallory,  p.  136). 

Hemoglobin,  histochemical  test  (Ralph, 
P.  IL,  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. 

Hemoglobin  Estimation  is  done  by  compar- 
ing blood  with  a  colored  paper  scale  or  bj' 
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 
ihey  hemolyse  become  more  and  more 
translucent.  Techniques  differ  merely 
in  the  v/aj's  of  measuring  the  trans- 
mitted light.  Simple  visual  photom- 
eters and  photoelectric  ones  are  de- 
scribed by  Ponder,  E.  Glasser's  Medical 
Ph5'-sics,  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. 


HEMOPHILUS  PERTUSSIS 


119 


HISTOSPECTROGRAPHY 


phosphomolybodic  acid.  Growth  on  a 
special  medium  is  advised  (Lawson,  G. 
McL.,  J.  Lab.  &  Clin.  Med.,  1939-40, 
25,  435-438). 

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.  Speed 
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,  401^07, 
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 
acetic  acid,  1  cc.  This  resembles 
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. 

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. 

Histiocyte,  a  term  without  value  as  it 
simply  indicates  a  "tissue  cell,"  often 
applied  to  phagocytic  cells  of  connec- 
tive tissue. 

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,  thyroid 
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., 


HISTOSPECTROGRAPHY 


120 


HYALURONIC  ACID 


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  lo 
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 
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. 

Holmium,  see  Atomic  Weights. 

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,  Vuipian  Reaction,  Os- 
mic  Acid. 

Ruber'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  parafl&n 
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  fiat  in  95%  alcohol, 
5-15  min.  Dehydrate  in  absolute,  clear 
in  xylol  and  mount  in  balsam. 

Humus,  see  soil. 

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  hemaloxylin- 
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  Ihionin  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.  This  is  a  polymer 
of  acetyl  glucosamine  and  glucuronic 
acid.  It  occurs  in  a  polydisperse  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  meso- 
thelioma. (For  an  extensive  treatment 
of  the  subject  of  acid  polysaccharides 
and  a  comprehensive  bibliography, 
refer  to  Karl  Meyer's  reviews  on,  "Mu- 
colytic enzymes"  in  Currents  in  Bio- 
chemical Research,  Interscience  Pub- 
lishers, N.  Y.,  1946;  "Mucoids  and 
Glycoproteins"  in  Advances  in  Protein 
Chemistry,  Academic  Press,  N.  Y. 
1945;  "The  Chemistry  and  Biology  of 
Mucopolysaccharides  and  Glycopro- 
teins" in  Cold  Spring  Harbor  Symposia 
on  Quant.  Biol.,  6,  1938,  91-102.)  The 
enzyme,  hyaluronidase,  depolymerizes 
and  hydrolj'ses  hyaluronic  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  polysaccharides  like 
hyaluronic  acid  is  described  by  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 


HYALURONIC  ACID 


121 


HYDROGEN  ION  INDICATORS 


and  for  related  acid  polysaccharides 
which  do  not  stain  metachromatically. 
Fixation  of  material  is  an  important 
factor  in  the  retention  of  hyaluronic 
acid  for  subsequent  staining.  The  or- 
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. 
-f-  chloroform,  3  pts.  +  glacial  acetic 
acid,  1  pt.,  for  |  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  flood  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% 
FejO,.  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. 

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.  Data  contributed 
by  Mr.  Lester  F.  Wicks  of  The  Barnard 
Free  Skin  and  Cancer  Hospital. 

In  1893  Ehrlich  injected  neutral  red 
in  an  attempt  to  determine  the  reaction 
about  phagocytosed  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 
confirmed  the  contention  that  alkaline 
urine  can  be  formed  by  an  acidic  cortex. 
Harvey  and  Bensley  (B.  C.  H.  and  R. 
R.,  Biol.  Bull.,  1912,  23,  225-249)  used 
pH  indicators  to  indicate  tliat  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, 
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  to 
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.  E.xp. 
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  vital  stain.     It  should 


HYDROGEN  ION  INDICATORS 


122 


HYDROGEN  ION  INDICATORS 


exhibit  a  sharp  and  pronounced  color 
change  in  the  proper  pH  range.  It 
should  be  fairly  soluble,  readily  dif- 
fusable,  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  dye  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. 


HYDROGEN  ION  INDICATORS 


Indicator 

Bromphenol  blue 
3.0  N/20  NaOH 

Sodium  alizarin 
Bulphonate 
(Alizarin  red) 
1%  aq.  or  sat. 
in  saline 

Bromcresol  green 
2.9  N/20  NaOH 

Methyl  red 
7.4  N/20  NaOH 

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  saline 

Bromthymol  blue 
3.2  N/20  NaOH 

Phenol  red 
5.7  N/20  NaOH 


pH  Range  and  Colors 
yellow  ♦-  3.0  —  4.6  -►  blue 

yellow  «-  3.8  —  5.0  -►  pink 


yellow  ■<—  4.0  —  5.6  — ►  blue 
red  ♦-  4.2  —  6.3  -+  yellow 

yellow  <—  4.8  —  red  —  6.8  -♦  purple 
yellow  *—  5.4  —  6.6  —»  purple 

yellow  *-  5.4  —  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) 


Remarks 
Very  strong  stain,  too  far  on   acid  side. 

Very  toxic,  weak  stain. 


Strong  stain,   persistent,   well  tolerated. 

Unstable  in  organism,  weak  stain,  fixes  on 
lipoids. 

Powerful  stain,  well  tolerated. 

Strong  stain  but  rapidly  excreted,  is  toxic 
and  exhibits  dichromaticism. 

Very  strong  stain,  well  tolerated. 


Weak  stain,  toxic. 


Weak  stain,  very  toxic  to  mice,  but  not  for 
insects. 

Rapid,  intense  stain,  very  well  tolerated . 


HYDROGEN  ION  INDICATORS 


123 


ILLUMINATION 


Indicator 

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 


HYDROGEN  ION  INDICATORS— Continued 

pH  Range  and  Colors  Remarks 

(approx.)  red  «— 6.0  —  S.O-»blue  Slow  stain,  diffuses  poorly,  usually  de- 

posits in  granules. 


(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 


Very  weak  stain,  precipitates  out  readily 
in  vivo,  not  toxic  if  pure. 


Somewhat  toxic,  not  a  strong  staia. 


Very  weak  stain,  not  very  soluble. 


Toxic,  range  too  alkaline. 


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  acidic  in- 
stead of  basic.  Examples :  aurin  (or 
rosolic  acid);  red  corallin. 

Hydroxybenzene  Compounds  as  cytoplasmic 
fixatives.  Details  of  use  of  pj'rogallol 
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/x  in  thickness  are  cited  as  the 
advantages  special  attention  having 
been  paid  to  mitochondria  and  secretion 
granules  (Huseby,  R.  A.,  Proc.  Soc. 
Exp.  Biol.  &  Med..  1946,  61.   122-125). 

Hydroxyquinoline  test  for  iron,  see  Iron. 

Hypophysis,  see  Pituitary. 

Ice-crysta!  Artefacts  in  normal  and  chroma- 
tolytic  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  be  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 
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 


ILLUMINATION 


124 


INDULIN 


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  ?/Iicr©scope  by  which 
the  structures  giving  off  fluorescence 
can  be  viewed  in  a  dark  background  at 
high  magnification. 

Imbedding,  see  Celioidin,  Paraffin,  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. 

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  vmi- 
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'. 

Indicators,  see  Hydrogen  Ion  and  Oxidation 
Reduction    Potential    indicators. 

Indigo,  a  fine  blue  dye  produced  from  the 


leaves  of  Indigofera  tinctoria,  employed 
as  a  stain  and  a  cosmetic  for  more  than 
4000  years,  and  early  adopted  officiallj^ 
for  the  uniforms  of  American  and 
British  sailors,  its  history  reads  like  a 
romance.  (-See,  Leggett,  W.  F.,  An- 
cient and  Medieval  IDyes.  Brookh'n: 
Chemical  Publishing  Co.,  Inc.,  1944, 
95  pp.) 

Indigo  (CI.  1177)  is  now  produced 
artificially  as  well  as  from  plants. 
Indigo-Carmine  (CI,  IISO) — 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- 
liain  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, 
657-658). 

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  by  Herx- 
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. 


INDULIN 


125 


IODINE 


Lynch,  J.  E.,  Zoit.  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  rubrocj^anine  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.  Rcc,  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. 
Glj'cerol,  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  +  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 
(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  Trdvoux,  G.  Patissier,  1933, 
535  pp..    ■ 

Intermitoiic  Ceils,  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.,  dehj'drate  in  ale,  clear  in  xylol 
and  mount. 

2.  Long  method  of  Ileidenhain  (Q.M. 
Gciman  in  Simmons  and  Gentzkow, 
p.  616).  Recommended  for  Balanii- 
dium  coli  and  for  permanent  mounts. 
This  is  practically  the  same  except  for 
longer  mord.anting  and  staining.  See 
Iron  Hematoxylin. 

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, 
53-50  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. 

lodeosin  B,  sec  Erythrosin,  bluish. 

Iodine,   detection  of:  1.  Ionized  iodine  in 


IODINE 


126 


IRON 


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,  HI.  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 j^ellow  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  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/x.  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  the  chromidial  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. 


IRON 


127 


IRON  HEMATOXYLIN 


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.,  INlarza,  E.,  and 
Chiosa,  L.  Bull,  d'hist.  Appliq.,  1932, 
9,  213.  Scott  (McClung,  p.  758)  warns 
against  confusion  with  carbon. 

4.  Ilydroxyquinoline  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  gm. 
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  w^ash  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 
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  whicli  turns  dark  blue 
in  5  min.  A  positive  reaction  can  even 
be  obtained  after  death.  (Fishback, 
H.  R.,  J.  Lab.  &  Clin.  Med.  19;J9-40, 
25,  98-99). 

In  special  cases,  as  in  the  analysis  of 
small  amounts  of  epidermis,  resort  may 
be  had  to  a  quantitativ^e  polaro;j;rai>hic 
determination  of  iron,  see  Carruthers, 
C.  and  Suntzeff,  V.,  J.  Nat.  Cancer 
Inst.,  1942,  3,  217-220. 
Iron  Hematoxylin  of  Heidenhain  is  one  of 
the  standard  stains.  It  will  give  excel- 
lent results  after  almost  anj^  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.  Alany 
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.  iroii  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. 


IRON  PIGMENTS 


128 


JALOWY 


Iron  Pigments,  see  Berlin  and  Turnbull  blue 

reactions. 
Iron,    Radioactive,    See  Erythrocytes, 

Isamine  Blue  is  described  by  Conn  (p.  137) 
as  a  sulfonated  uaphthyl-rosauilin  or 
napiithyl-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  4/x  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 


statesthat  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 pH'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  these  techniques.  See  re- 
ticulo-endothelial  cells  (Fautrez,  J., 
Bull.  d'Hist.  Appl.,  1936,  13,  202-206). 

Isohematein,  as  a  biological  stain  (Cole, 
E.  C,  Stain  Techn.,  1931,  6,  93-96). 
Greater  tinctorial  power  than  hematox- 
ylin but  less  selective. 

Isopropanal  in  combination  as  a  new  fixative 
for  animal  tissues  which  also  dehydrates 
(Clever don,  M.  A.,  Science,  1943,  37, 
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- 
projjanal  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  hj^droxide  in  95% 
alcohol . 

Isopropyl  Alcoho!,  Has  been  recommended 
as  a  substitute  for  ethjd  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- 
safrole  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  ail  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  Ilortega  method  for 
the  skin  (Jalowy,  B.,  Zeit.  f.  Zellf.  u. 
Mikr.  Anat.,  1937,  27,  667-690).  To 
make  reagent  wash  ppt.,  formed  bj^ 
adding  20  drops  40%  aq.  NaOH  to20_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- 
solves. Add  100  cc  aq.  dest.  and  store 
in  dark.     Deparaffinize  sections  of  tissue 


JALOWY 


129 


JOHNSON'S  NEUTRAL  RED 


fixed  1-2  days  in  neutral  formalin. 
Treat  with  above  reagent  5-30  min.  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, 
hematoxylin  (Lillie,  R.D.  and  Earle 
W.R.  Stain  Teclmol.,  1939,  14,  53-54). 
Janus  E'lue  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  Embrvol.,  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 
safraninfizodimethyl anilinchloride.  This 
likewise    is    useless    for  mitochondria. 

Janus  green  D  (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. 

Dielhylsafranin  is  a  reduction  product 
of  Janus  green  B.  It  is  a  red  dye  which 
colors  mitochondria  specifically  but  not 
very  strongly. 
Janus  Green  B  (Diazingrim)  is  diethjd 
.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 
Iddney  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  m.'xgnification .  When  the  oxygen 
is  further  consumed  by  the  cells  the  aye 
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   (Eniig,  p.  36). 

Jaws,  see  Teeth  and 

Jenner-Giemsa  method  of  Pappenheim  (see 
JNIay-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 
flash.  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  dtirk  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 
(dOfj.)  and  can  be  employed  after  silver 
methods  on  tissues  fixed  in  alcohol  or 
formalin. 

Kirkman,  1.  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, 
2  cc.  10-20  min.  is  sufficient  for  counter- 
staining       Weigert-Pal       preparations. 


JOHNSON'S  NEUTRAL  RED 


130 


KIDNEY 


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). 

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. 

Kallichrom,  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- 
so?Me,  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.  Hyalin-like  granules  found 
in  tlie  stratum  granulosum.  They  can 
be  beautifullj'  stained  with  picro- 
cartnine. 

Kermes.  This  scarlet  dj^e  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  arbor um  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.  Techniques  for  the  sustaining 
tissues  of  the  kidney  (connective  tissue, 
blood  vessels,  nerves  and  lymphatics) 
are  essentially  the  same  as  those  used 
for  the  same  tissues  in  other  organs .  See , 
however,  the  Silver  Citrate  injection  of 
blood  vessels.  The  epithelial  compo- 
nents are  highly  specialized  and  can  be 
investigated  in  a  host  of  different  ways 
of  which  only  a  few  samples  can  be  given. 

A  clear  distinction  between  glomeruli 
and  the  remainders  of  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  pur- 
pose than  Janus  green)  in  0.85%  aq. 
sodium  chloride  by  vascular  Perfusion 
and  to  determine  their  number,  size  and 
distribution  against  a  background  of  un- 
stained or  faintly  rose  tinged  tubules  in 
slices  of  fresh  kidney  (Cowdry,  E.  V., 
Contrib.  to  Embryol.  Carnegie  Inst., 
Washington,   1918,  8,  39-160). 

Individual  renal  tubules  in  their 
entirety  can  be  isolated  by  maceration 
and  teasing  as  described  by  Huber,  G.  C, 
Cowdry's  Special  Cytology,  1932,  2, 
935-977  slightly  amplified.  Partly  wash 
out  blood  by  injecting  physiological 
saline  into  the  renal  artery.  Then  follow 
with  hydrochloric  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.  Iso- 
late individual  tubules  by  teasing  with 
fine  needles.  Wash,  and  mount  in 
glycerin.  With  small  mammals  Huber's 
results  were  excellent  but  he  was  not 
satisfied  with  his  human  preparations. 
The  method  has  however  been  well  ad- 
justed to  the  human  kidney  by  Oliver, 
J.  and  Lund,  E.  M.,  J.  Exp.  Med.,  1933, 
57,  435-483  and  Arch.  Path.,  1934,  18, 
755-774.  Technique  for  the  micro- 
scopic study  in  vivo  of  the  surface  of  the 
guinea  pig's  kidney,  for  the  marking  of 
single  tubules  with  India  ink  and  for 
their  later  isolation  by  maceration  is 
given  by  Walker,  A.  M.  and  Oliver,  J., 


KIDNEY 


131 


KINNEY'S  METHOD 


Am.  J.  Physiol.,  1941, 134,  562-595.  The 
micro  collection  of  fluid  from  single 
tubules  is  as  the  authors  state  a  direct 
continuation  of  the  researches  of  A.N. 
Richards.  See  Oliver,  J.,  Harvej^  Lec- 
tures, 1944-45,  40,  102-155. 

Vital  staining  of  renal  tubules  is  usu- 
ally carried  out  by  techniques  not  re- 
quiring special  adaptation  to  the  kidney, 
see  Vital  Staining.  But  the  procedure 
employed  by  Oliver,  J.,  Bloom,  F.  and 
MacDowell,  M.,  J.  Exper.  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  con- 
centrate trypan  blue  which  consequently 
stains  the  tubule  wall  diffusely.  This  is 
beautifully  illustrated  in  colors.  Micro- 
scopic observations,  having  a  close  rela- 
tion to  function,  are  easily  made  on  the 
kidneysof  lower  forms.  See  the  account 
of  contractility  of  the  ciliated  necks  of 
renal  tubules  in  Necturus  by  Lucas,  A.M. 
and  White,  H.  L.,  Anat.  Rec,  1933, 
57,7-11. 

The  study  of  renal  tubules  present 
in  tissue  cultures  is  useful  up  to  a  certain 
point  in  the  study  of  function.  Thus 
Chambers,  R.  and  Cameron,  G.,  Radiol- 
ogy, 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  variations 
in  normal  mice  and  of  the  influence  of 
testosterone  proprionate  on  castrated 
mice. 

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  depend  on 
topographic  correlation  between  dis- 
tribution of  cell  types  in  15  n  frozen  sec- 
tions and  decomposition  of  substrates. 
The  techniques  are  capable  of  demon- 
strating catheptic,  aminopoly-peptidase 
and  esterase  activities  in  all  of  the  epi- 
thelial components  and  of  showing  that 
the  cells  of  the  proximal  and  distal  con- 
voluted 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  local- 


ized and  expressed  quantitatively  in 
relative  terms. 

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  Graf- 
flin,  A.  L.,  Am.  J.  Anat.,  1942,  70,  1-20. 
They  can  probably  be  emploj'ed  to  ad- 
vantage 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. 

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,  S.  E.,  Science, 
1931,  73, _  529).  It  is  significant  that 
increase  in  acidity  of  the  renal  cortex 
immediately  follows  ligation  of  the  renal 
artery  and  that  the  mitochondria  re- 
spond by  enspherulation  and  fragmenta- 
tion within  6  minutes.  The  kidney  is 
an  organ  in  which  mitochondria  must  be 
examined  with  the  utmost  promptness. 
But  Fuller,  R.  II.,  Arch.  Path.,  1941, 
32,  556-568  could  find  no  relation  in  a 
rather  large  number  of  cases  studied 
between  age,  hours  postmortem  and 
cause  of  death  (except  renal  disease) 
and  quantity  and  distribution  of  stain- 
able  lipoid. 

For  application  to  proximal  convo- 
luted tubules  in  phlorizin  glycuresis  of 
the  Ivabat  and  Furth  procedure  for  al- 
kaline phosphatase  see  Kritzler,  Ti.  A. 
and  Gutmau,  A.  B.,  Am.  J.  Physiol., 

1941,  134,  94-101.     See  Phosphatase. 
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.  dcst.,  differen- 
tiate in  95%  alcohol.  Pass  through 
equal  parts  absolute  alcohol  and  chloro- 
form to  xjdol  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- 
tcrstain  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 


KINNEY'S  METHOD 


132 


LAMPBLACK 


and  pancreas.  Results  arc  sometinaes 
patchy  in  the  spleen. 

Kleinenberg's  fixative.  Saturated  picric 
acid  in  2%  aq.  sulphuric  acid.  Embryos 
and  marine  organisms. 

Kolatchew  Fluid,  see  Golgi  Apparatus. 

Korff'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,  1.5gm. ;  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. 

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  supra  vitally  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). 

Kultsclutzky'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  oljtaincd  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.) 

Lacteals,  see  Lymphatic  Vessels, 

Lactophenol,  a  fixative  for  Bilharzial  Cer- 
carlae.  See  Lactophenol-cotton  blue 
iRchniqao  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  3^-  Re- 
move paraffin  and  pass  down  to  water. 
Weigert's  iron  hematoxylin  (2%)  5  min. 
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  and  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  changes  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  Vv'ater.  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  211. 

Lampblack.  A  colloidal  suspension  of  lamp- 
black is  an  excellent  substance  to  inject 
intravenously    to    demonstrate  phago- 


LAMPBLACK 


133 


LEISHMANLA.  DONOVANI 


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 
d3^e  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). 

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:  5-10  gm. 
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,  G,  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. 
e.xper.  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,  401-422.  The 
authors  describe  techniques  for  direct 
analysis  of  lead  in  the  tissues. 

Leather  Brown,  see  Bismark  Brown  Y. 

Leather  Yellow,  see  Phosphine. 

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). 

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  aq.  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 


LEISHMANIA 


134 


LEUCOCYTES 


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.  iNL  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  I  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  davs  to  maintain. 

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  hemoglobin  solution  take  1  part 
defibrinated  rabbit's  blood  and  3  parts 
aq.  dest.,  centrifuge  and  use  clear  super- 
natant fluid.)  Tube,  prove  sterility  by 
incubation  before  using.  Subculture 
ever}'  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  + 
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. 

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  0.01% 
aq.  solutions  of  methylene  blue,azur  A, 
thionin  toluidine  blue  and  brilliant  cresyl 
blue.  Add  to  100  cc.  1-2.5  cc.  N/10 
NasSaOa  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). 

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 


LEUCOCYTES 


135 


LEUCOCYTE  COUNTS 


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  varial)ili  ty 
in  relation  to  alterations  in  raeteorologic 
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).  Chemotactic  re- 
sponse and  motility  can  be  measured 
both  in  tissue  cultures  (Coman,  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 
analj'^sis  of  the  movements  and  behavior 
of  leucocytes.  Some  excellent  ones, 
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 
satisfactoiy  than  supravitall.y  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  IjTnphocytosis. 


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 

B 

0  1 

E 

2-4 

M 

0 

J 

0-1 

Leucocytes 

St 

3-5 

S 

51-67; 

L 

21-35 

Mon. 

4-5 

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,  1664-1G67  and  for  inter- 
ference by  decreased  fragility  of  eryth- 
rocytes see  Bohrod,  M.  G.,  J.  Lab.  & 
Clin.  Med.,  1940-11,  2G,  1953-1955. 

A  better  method,  unfortunately  not 
widely  employed,  is  the  filament-non- 
filament  count.  Filaments  arc  neutro- 
philes in  which  th(!  nuclear  segments 
are     connected     by     delicate     strands 


LEUCOCYTE  COUNTS 


136 


LIEBERKlJHN'S  GLANDS 


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  normallj''  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 : 

H emocytoblasts :  ".  .  .  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 
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  v/hile  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  Ijonpho- 
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:  Thej'' 
"show  the  earliest  signs  of  the  nuclear 
changes  toward  polymorphism  and  the 
first  sign  of  the  transformation  of  the 
cytoplasm  to  a  phase  sufficientlj'  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  cr  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. 

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 


137 


LIPASE 


Liebermann-Burchardt  reaction  for  choles- 
terol   and     its     esters     {cholesieridcs) . 

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,  l^rain 
and  examine  in  the  reagent.  Cholesterol 
and  its  esters  dark  blue  or  red  purple 
becoming  green. 

2.  Modification  of  Romicu  (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 
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  Tv^'ort,  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's  chrom-osmic-acetic  fixative.  §% 
aq.  chromic  acid,  15  cc;  2%  aq.  osmic 
acid,  3.5  cc. ;  glacial  acetic  acid,  3  drops. 
Used  by  him  for  echinoderm  eggs. 

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.,  el  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  da3's,  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  30;u  thick  and  4.5 
mm.  in  diameter  of  beef  adrenals  are 
extracted  in  30%  glycerol  +  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  bulTer  pH  6.5. 
Nearby  sections,  some  stained  with 
hematoxylin  and  eosin,  and  others,  with 
Sudan  III,  are  examined  histologically. 
The  medulla,  which  exhibits  most 
lipolj'tic  activity,  contains  least  lipid. 
Estimations  of  esterase  are  also  de- 
scribed by  Click  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 leucocvtes  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  bj^  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 


LIPASE 


138 


LIPIODOL 


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  1|  hrs.  each.  Cut  4-8^ 
sections,  float  on  water  (±  3o°C)  and 
mount  on  slides.  Pass  clown  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. 
-f  aq.  dest.  6  cc).  If  maleate  buffer 
is  omitted  mi.xture  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 
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.  dest.) 
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. 

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.  Glj^cerides  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  osmi cation.  Coloration  with  sudan 
and  mounting  in  syrup  of  levulose 
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 — chromolipoids. 
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. — cholesterides . 
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— LipiTies. 
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. 

5.  Lorrain  Smith  reaction  with  nile  blue 
sulphate:  rose — non-saturated  glyc- 
eride. 
5.  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,  Ciioiesterol  (free).  Cholesterol 
Esters,  Myeloidia,  Myelin,  etc 

Lipines,  see  Lipoids. 

Lipiodol,   reactions  in  tissue  to  fat  stains 


LIPIODOL 


139 


LOEFFLER'S  ALKALINE 


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 
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  fatt}^  degeneration,  see 
Lipids. 

Lison's  glycogen  method  (Lison,  p.  227). 
Fix  in  dioxan  saturated  with  picric 
acid,  8.5  parts;  formalin,  1  part;  and 
acetic  acid,  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  maj^  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  +  5  gm.  carmine 
rubrum  (Grubler)  for  j— 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.  Wash  ppt.  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  Schtill  No. 
589  into  stock  bottle.  See  Laidlaw's 
Methods. 

Litmus  as  a  vital  indicator  of  acidity  and 
alkalinity  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.;  CaCl9,  0.2 
gm.;  KCl,  0.4  gm.;  NaHCOj,  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.;  CaCU,  0.025  gm.; 
NaHCOs,  0.02  gm.,  dextrose,  0.01-0.25 
gm. ;  aq.  dest.,  100  cc.  Should  be  freshly 
made.  Owing  to  presence  of  NaHCOs 
must  not  be  sterilized  by  heat. 

Loeffler's   Alkaline   Methylene    Blue.    As 


LOEFFLER'S  ALKALINE 


140 


LYMPHATIC  VESSELS 


emended  Soc.  Am.  Bact.  A.  Methylene 
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  IGth  centurj^ 
logwood  was  banned  in  England  i3y  Act 
of  Parliament  in  1589  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.  Brooklyn:  Chendcal 
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 bj'  the  local  injection  of  fluid 
(salt  solution,  serum,  etc.). 

Sylvia  H.  Bensley  (Anat.  Rec,  1934, 
60,  93-109)  employed  a  graphic  method 
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 
CoUagenic  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  different 
from  Charcot's  and  Spermin  Crystals. 

Lucidol,  a  trade  name  for  benzoyl  peroxide. 

Lucite,  disadvantages  of  as  substitute  for 
Canada  balsam  (Richards,  0.  W.  and 
Smjth,  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. 

Luminoi  (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. 

Observation  of  lung  through  thoracic 
window  in  vivo  (Terry,  R.  J.,  Science, 
1939,  90,  43-44),  see  Celluloid  Corrosion 
preparations.  Alveolar  Pores. 

Lutecium,  see  Atomic  Weights. 

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 


LYMPHATIC  VESSELS 


141 


LYSOZYME 


sight  it  may  be  difficult  or  impossible  to 
see  any  lymphatics  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.  182-4)  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  Lymphoid  Tissue.  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,  443-44G.  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  hydrokoUag  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 
chambers  in  the  ears  of  rabbits  (Clark, 
E.  R.  and  E.  L.,  Am.  J.  Anat.,  1937, 
62,  59-92.  See  India  ink  method  for 
renal  lymphatics  (Pierce,  C.  E.  2nd., 
Anat.  Rec,  1944,  fiO,  315-329). 

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  lymphocytes  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 
polyhydroxylic  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  tliat 
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  enzj^me 
produced  from  egg  white  and  isolated 
as  a  basic  protein  of  small  molecular 
weight  by  Abraham,  E.  P.,  Biochem. 
J.,  1939,  33,  622-030.  It  is  present  in 
many    animal    and    plant    tissues.    A 


LYSOZYME 


142 


MACROPHAGES 


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  cyto- 
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- 
ysaccharide. 
Lyssa  Bodies  are  small  Negri  bodies  which 
look  optically  hyaline,  see  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  dj'e  and 
viewed  as  units  with  parts  of  their 
processes  attached. 

Smooth  muscle  of  the  bladder  is  well 
dissociated  by  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  tubides.  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,J77-816). 

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 
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. 

Bo7ie  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 


MACROPHAGES 


143 


MAGNESIUM 


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  Ruhia 
Tinclorum  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 
difi'erent  part  so  that  the  Persian  leaders 
at  once  concluded  that  all  they  had  to 
cope  vvith  w^as  an  already  well  damaged 
army.  (Leggett,  W.  F.,  Ancient  and 
Medieval  Dyes.  Brooklvn:  Chemical 
Publishing  Co.,  Inc.     944,  95  pp.) 

Alizarin  and  purpurin,  formed  from 
madder,  are  now  made  sj^nthetically. 
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  nuxed  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 
epiphj^seal  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 
wintcrgreen  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. 
IVIadder  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- 
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  the  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  (Mtigenta  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  m.etal  if  it 
is  sound.  Otiierwise,  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  (Haurv,  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  localized 
magnesium  and/or  calcium  in  muscle. 
The  method  can  be  extended  to  other 
tissues  and  perhaps  to  other  minerals. 
Histospectrography  gives  diita  on  the 


MAGNESIUM 


144 


MALLORY'S  CONNECTIVE 


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,  429-440). 

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'. 

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 
extensivel}^  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., 
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  ferri  hemic 
acid,  or  hematin,  which  does  contain 
iron. 

Malaria  Plasmcdta.  Technique  of  examina- 
tion of  process  of  "exflagellation" 
(Anderson,  Ch.  W.  and  Cowdry,  E.  V., 
Arch,  de  I'Inst.  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.  II.,  Ihid.,  233-252). 

For  staining  the  piasmodia  in  smears, 
see    Giemsa,    Jenner,    Marino,    Nocht, 


Plehn,  Wilson  and  Wright's  stains.  A 
simple  method  for  staining  piasmodia 
in  paraffin  sections  is  described  with 
numerous  illustrations  by  Tomlinson, 
W.  J.  and  Grocott,  R.  G.,  Am.  J.  Clin. 
Path.,  1944,  14,  318-326.  The  Barber 
Komp  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  o.xa- 
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  off  all  but  about  i  in.  of  super- 
natant plasma.  Smear  on  2  slides  bj' 
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  (Goosmanu,  C.,  J.  Lab.  & 
Clin.  Med.,  1935-36,  21,  421-424).  See 
Protozoa. 
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 
McCIung,  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 
fi.xed  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. — 


MALLORY'S  CONNECTIVE 


145 


MANN'S  METHYL  BLUE-EOSIN 


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  paraffin 
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. 
Mammary  Glands.  These  can  be  studied 
in  sections  bj'  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  parafhn  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 
oridnally  described  by  Turner,  C.  W. 
and  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  1%  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 
authorsliip  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  Martius   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- 
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%  NaC'l). 
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- 
paraffinized,  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 


MANN'S  METHYL  BLUE-EOSIN 


146 


MASSON'S  TRICHROME 


and  mounted.  See  Alzheimer's  Modi- 
fication of  Mann's  method. 
Marchi  Method.  For  degenerating  nerve 
fibers.  Modification  by  Swank,  R.  L. 
and  Davenport,  H.  A.,  Stain  Teclin., 
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  40^  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. 

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.  Allg.  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  gently.  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  for  connective 
tissue  (Masson,  P.,  Am.  J.  Path.,  1928, 
4,  181-212;  J.  Tech.  Meth.,  1929,  12, 
75-90) .  The  following  is  an  abbreviated 
account  of  the  technique  as  recom- 
mended by  IVIallory  (p.  156).  Use  5n 
paraffin  sections  of  Bouin's  fluid  (3 
days)  or  Regaud's  (1  day)  fixed  tissues. 
Mordant  in  5%  aq.  ammonio-ferric 
alum  previously  warmed  to  45-50  °C. 
for  5  min.  Wash  in  water  and  stain 
for  5  min.  at  45-50°C.  in  Regaud's 
hematoxylin  (hematoxylin,  1  gm.;  95% 
ale,  10  cc;  glycerin,  10  cc;  aq.  dest., 
80  cc).  Rinse  in  aq.  dest.  and  dif- 
ferentiate in  picric  alcohol  (sat.  picric 
acid  in  95%  ale,  2  parts;  95%  alcohol, 
1  part).  Wash  in  running  tap  water. 
Stain  for  5  min.  in:  acid  fuchsin,  0.3 
gm.;  Ponceau  de  xylidine,  0.7  grn.; 
aq.  dest.,  100  cc. ;  glacial  acetic  acid, 
1  cc.  Rinse  in  aq.  dest.  Differentiate 
in  1%  aq.  phosphomolybdic  acid,  5 
min.  Without  rinsing  add  10  drops  sat. 
aniline  blue  in  2%  acetic  acid  and  leave 
for  5  min.  Rinse  in  aq.  dest.  and  place 
again  in  phosphomolybdic  acid.  1% 
acetic  for  5  min.  Dehydrate  in  95% 
alcohol,  then  absolute,  xylol  and  balsam. 
Collagen,  deep  blue;  neuroglia  fibrils, 
red;  nuclei,  black;  argentaffin  granules, 
black  or  red.  See  modifications  by 
Goldner,  J.,  Am.  J.  Path.,  1938,  14, 
237,  and  Larson,  C.  P.  and  Levin,  E.  J., 
Arch.  Path.,  1940,  29,  272-273.  Tech- 
nique for  application  of  Masson's  tri- 
chrome stain  to  tissue  previously 
colored  with  Weigert's  resorcin-fuchsin 
is  given  by  Mendelotf,  J.  and  Blech- 


MASSON'S  TRICHROME 


147 


McILVAINE  BUFFERS 


man,  H.,  Am.  J.  Clin.   Piith.,  Teohn. 
Suppl.,  1943,  7,  Cj5. 

The  difficulty  is  that  the  French  "pon- 
ceau de  X2jlidi7ie"  cannot  be  secured. 
It  appears  to  be  similar  to  ponceau  2R 
(C.l.  79)  but  the  latter  does  not  give 
regularly  good  results.  Lillie  (R.  D., 
Stain  Tech.,  1940,  15,  17-22)  suggests 
the  following  substitutes  for  ponceau 
de  xylidine :  azofuchsin  3B  (C.L,  54), 
nitrazine  yellow  and  biebrich  scarlet 
(C.L,  280).  See  the  Biebrich  Scarlet 
and  Picro-Anilin  Blue  method  of  Lillie. 

Mast  Cells,  see  Basophile  Leucocytes  and 
Tissue  Basophiles. 

Mastoid  Process.  Use  methods  for  Bone. 
Technique  for  measurements  of  size  of 
air  cell  svstem  is  given  by  Diamant,  M., 
Acta  Radiol.,  1940,  31,  543-548. 

Mauveine  (CI,  846),  a  basic  dye  of  light  fast- 
ness 3,  the  first  dj'e  made  from  aniline 
in  1856.  Gives  stain  of  plant  tissues 
like  Methyl  violet  (Emis,  p.  57). 

Maximow  (see  Azure  11  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  method  alcohol  (Griibler  or  IIoll- 
born).  If  methylene  blue  eosinate  is 
not  available  make  it  as  originally  de- 
scribed bv  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.,  193.5-3G,  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).  Dis.solve  1  gra.  hematoxylin 
in  1000  cc.  aq.  dest.  witli  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  quickly, 
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  dj^es. 

Mcllvaine  Buffers  after  Stitt  from  Lillie, 
R.  D.,  Stain  Techn.,  1941,  16,  1-6  who 
employed  them  to  improve  Romanowsky 
staining  after  various  fi.xatives.  See 
Toluidine  Blue  Phloxinate  Method, 
(see  Molecular  Solution)  To  make 
M/15  citric  acid  required  dissolve  14.01 
gm.  mono-hydrated  crj^stalline  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 
1.3 
1.25 
1.2 
1.15 
1.1 
1.05 
1.0 
0.95 
0.9 
0.85 
0.8 
0.75 
0.7 
0.G5 
0.6 
0.55 


Na2HP04 
0.7 
0.75 
0.8 
0.85 
0.9 
0.95 
1.0 
1.05 
1.1 
1.15 
1.2 
1.25 
1.3 
1.35 
1.4 
1.45 


pH 

(3.873) 
(4.034) 
(4.205) 
(4.44) 
(4.653) 
(4. SO) 
(5.042) 
^5.201) 
(5.428) 
(5.096) 
5.85  (5.S3S1 
6.05  (6.036) 
6.3    (6.20) 
6.5    (6.444) 

6.5  (6.522) 

6.6  (6.60) 


3.9 
4.0 
4.2 
4.4 
4.6 
4.8 
5.0 
5.2 
5.4 
5.7 


Since  it  is  difficult  to  measure  out  these 
small  volumes  accurately  at  least  ten 
times  the  volume  in  eacli  case  should  be 
taken   and    the    amount    not    required 


McILVAINE  BUFFERS 


148 


MELANINS 


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.,  ICOO  cc. 
(Haden,  R.  L.,  J.  Lab.  &  Clin.  Med., 
1923,  9,  64-65). 

Meat  Exiract  Brotli  and  other  media  con- 
taining meat,  see  Bacteria  Media. 

Meckel's  Diverticulum.  Literature  on 
(Curd,  II.  H.,  Arch.  Surg.,  1936,  32, 
606-523). 

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  fi.xative  suggested,  Downey  (Folia 
haematol.,  Archiv,  1913,  15,  25)  uses 
commercial  formalin  10  cc.  and  sat. 
mercuric  chloride  in  0.9%  aq.  NaCl 
90  cc.^ 

2.  Kingsley's  method  (Kingsley,  D. 
M.,  Folia  Ilaemat.,  1937,  57,  87-98). 
Fix  in  Downey's   iiuid    (given  above) 

4  parts,  saturated  picric  acid  1  part, 
24  hrs.  Wash  in  running  water,  18-24 
hrs.  Dehydrate  through  alcohols  up 
to 70%,  1-1  hr.  each.  80% ale.  +  iodine, 
overnight.  95%  ale,  45  min.  Repeat 
with  fresh  ale.  N  butyl  alcohol  (techni- 
cal), 1  hr.  Repeat  with  fresh.  Paraf- 
fin (58°C.),  ^  hr.,  then  3  more  changes, 
each  I  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;  CHjOn  (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.  KH2PO4  per  liter  +  60  cc. 
of  B  =  11.876  gm.  Na2HP04 -21120  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  +  4  cc.  1%, 
acetic  acid.  Rinse  in  n  butyl  ale.  -j-  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  bod}^  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 
chara.'ceristic  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.,  Teclm.  Suppl.,  19-t3,  7,  127-128). 
A  method  for  the  collection  of  melanin 
for  analysis  by  differential  Centrifuga- 
iion  is  described  bv  Claude,  A.,  Trans. 
New  York  Acad.  Sci.,  1942,  II,  4,  79-83. 


MELANINS 


149 


METHYL  BLUE 


See  Dopa  Reaction  for  melanogen  in 
n!o];in<il.lasts. 

Melanoblasts,  see  Dopa  Reaction. 

Meldola's  Blue,  son  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  sam(!  amount  of 
acetic  acid.  See  (1)  with  formalin, 
glacial  acetic  and  phj^siological  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  fi.vatives  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,  19-11, 
21,47). 

Mercurochrome  220.  Trade  name  for  di- 
brom-oxy-mercuri-fiuorescein.  Can  be 
used  as  substitute  for  eosin  (Baldwin, 
W.  M.,  Auat.  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  acidl  cc,  saturated 
with  H2S  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  mercurv.  Simonet  (M., 
Arch.  d'Anat.  Micr.,''l929,  25,  372-381) 
uses  instead  fixation  for  10  hrs.  in  equal 
parts  alcohol  and  chloroform,  100  cc, 
-f-  nitric  acid,  2  cc.  the  mixture  satu- 
rated with  HoS  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). 

Intravenous  injections  of  colloidal 
solutions  of  mercury  in  rabbits  are 
described  by  Duliamel,  B.  G.,  C.  rend. 
Soc.  dc  Biol.,  1919,  82,  724-726. 
Mesentery  spreads,  sections  and  cultures. 
Maximow,  A.,  Arch.  f.  exper.  Zellf., 
1927,  4,  1-42  (nice  colored  plates). 


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  polj'chrome  (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  ])y  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  arabic  or  other 
negatively  charged  colloid  (Bank,  O. 
and  Hungenberg  de  Jona,  H.  G.,  Proto- 
plasma,  1939,  32,  489-516). 

MetacrescI  Purple.  See  K)'drogen  Ion  In- 
dicators. 

Metallurgic  Microscope.  Since  the  mate- 
rials routinely  studied  are  opaque  the 
light  is  reflected  verticailj''  down  upon 
them  through  the  objective.  This  in- 
strument is  of  little  use  in  biology  and 
medi'''iRe. 

Metamyelocytes,  see  Leucocytes,  develop- 
mcritfil  SGriGS 

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. 

Methacryiate.  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, 


METHYL  BLUE 


150 


METRIAL  GLAND 


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  (hexa-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  CO.;  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). 

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  hexa-methyl  rosanilins.  R  indi- 
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).  Abasic  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  practicallj^  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 
satisfactorJ^  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  apparentlj'  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  hematoxj'lin  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  bv  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. 

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 
counterstain  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  bj'  recrystallization  but  little 
is  to  be  gained.  The  dye  is  not  much 
used. 

Melrial  Gland.  This  is  a  transitory  struc- 
ture of  unknown  function  in  the  mouse 
appearing  at  approximately  the  8th  day 


METRIAL  GLAND 


151 


MICROINCINERATION 


of  pregnancy.  Failure  of  its  cells  to 
take  up  trypan  blue  seems  to  eliminate 
the  hypothesis  that  it  is  active  in  phago- 
cytosis (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  hj'pothetical  structural 
units  of  the  cell. 

Michiavello  Stain.    See  Rickettsia. 

Michrochemical  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- 
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.  An  excellent  account  of 
the  apparatus  required  and  of  its  proper 
use  is  provided  by  Robert  Chambers 
and  M.  J.  Kopac  in  McClung,  pp.  62-109, 
and  more  recently  by  Chambers  in  J. 
Roy.  Micr.  Soc,  1940,  60,  113-127. 
However  an  attempt  should  not  be  made 
to  learn  the  technique  de  novo  from  the 
printed  word.  Actual  experience  under 
the  supervision  of  a  master  will  save 
valuable  time.     A  helpful  preliminary 


is  to  view  motion  picture  films  of  micro- 
dissections which  can  be  obtained  on 
loan  from  the  Wistar  Institute  of 
Anatomy  in  Philadelphia.  See  Col- 
loquium on  Micrurgj'-  (Microdissec- 
tion), edited  by  j"  C.  Regniers, 
Publisher :  Clmrles  C.  Thomas,  In  Press. 
See  Micromanipulation. 

Muelengracht  Test,  see  Icterus  Index. 

Microglia.  Method  for  impregnating  with 
silver  in  pyro.xylin  (celloidin)  sections 
(Weil,  H.  and  Davenport,  II.  A.,  Trans. 
Chicago  Path.  Soc,  1933,  14,  95-96). 
Wash  15ai  sections  in  aq.  dest.  Treat 
for  15-20  see.  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 
slightly  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  20-40/i  of  formalin  fixed  mate- 
rial. Immediately  place  them  in  aq. 
dest.  +  20  drops  ammonia  per  100  cc. 
Thence  pass  directly  to  5%  aq.  am- 
monium bromide  40-50  °C.  10-15  min. 
Equal  parts  ammonia,  pyridine  and  aq. 
dest.  2  min.  Then  3-5%  aq.  sodium 
sulfite,  2-3  min.  Pa«s  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  bv  Gordon  H. 
Scott,  July  26,  1946.— This  method  is 
one  which  has  been  used  by  plant  and 
animal  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 
variety  of  heating  processes.  Most 
tissues  in  the  body  can  be  treated  by 
the  ashing  process  with  some  success. 
Those  which  contain  large  quantities 
of  phospholipids  ordinarily  do  not  give 
as  good  results  as  tissues  lacking  them. 
The  method  is  one  which  requires 
some  care  and  the  observance  of  certain 
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 


MICROINCINERATIONi 


152 


MICROMANIPULATION 


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  with 
paraffin  in  the  usual  manner. 

The  alternative  method,  that  of 
frozen-dehydration,  is  the  most  suit- 
able for  preparation  of  tissues  for  micro- 
incineration. (See  Altmann-Gersch 
and  Cryostat.)  This  technique  j^ields 
tissues  which,  except  for  the  ice  crystal 
formation,  have  not  been  altered,  to 
any  perceptible  degree,  either  physi- 
cally or  chemically.  Dehydration  at 
sufficiently  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  studj' 
and  for  photography  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  PoUcard.  This  has  some 
advantages  over  the  dark-field  but 
makes  the  use  of  higher  magnifications 
diflacult  if  not  actually  impossible. 
Cellular  details  are,  therefore,  to  be 
observed  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  subse- 
quent fluorescence  of  minerals.  Stu- 
dents should  consult  reference  and  text- 
books on  mineralogy  for  details  of 
identification. 

Calcium  and  magnesium  are  char- 
acterized in  the  dark-field  by  their 
dense  white  ash  residues.  Iron  is  oxi- 
dized during  the  incineration  process 
and  appears  as  varying  tints  of  red. 
The  amount  of  this  element  present  can 
be  correlated  with  the  color  intensity. 
Silicon  is  definitely  crystalline  in  char- 
acter and  is  recognizable  by  its  prop- 
erty 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  ele- 
ments which  yield  black  sulfides  can  be 
detected  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  be- 
cause of  the  utter  relativity  of  the  re- 
sults obtained.  The  most  useful  find- 
ing obtained  from  microincineration, 
therefore,  is  the  appi-eciation  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,  Histospectrog- 
raphy  and  Ultraviolet  Photomicrog- 
raphy. 

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. 

Micromanipulation. — Written  by  Dr.  Robert 
Chambers,  Dept.  of  Biology,  Washing- 
ton Square  College  of  New  York  Uni- 
versity, New  York,  October,  1946 — 
Broadly  speaking,  this  term  covers  two 
sorts  of  operation:  delicate  free-hand 
operations  in  which  the  only  accessory 
is  a  dissecting  microscope,  and,  second, 
operations  conducted  by  means  of 
micrurgical  instruments. 

Considerable  training  is  required  in 
using  a  compound  microscope  for  free- 
hand operation  because  of  the  inversion 
of  the  image.  This,  however,  has  been 
corrected  by  using  the  so-called  erect- 
ing ocular.  A  decided  help  to  relieve 
fatigue  from  too  long  holding  of,  a  dis- 
secting 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  encircles  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.  De- 
scriptions of  excellent  methods  for  in- 
jecting minute  vessels,  such  as  the 
marginal  vein  of  chick  embryos  or  lym- 


MICROMANIPULATION 


153 


MICROMANIPULATION 


phatic  vessels  of  frog  tadpoles,  are  as 
follows:  H.  McE.  Knower,  Chapter  in 
McClung's  Handbook  Microscopical 
Technique,  New  York:  Hoeber,  1937; 
A.  L.  Brown,  Anat.  Rec,  1922,  24,  295. 

Micromanipulation  in  its  more  re- 
stricted sense  applies  to  the  use  of 
special  devices  for  controlling  the  move- 
ments 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  Hand- 
book. 

Several  instruments  are  now  being 
built.  The  ones  in  most  general  use  in 
this  country  are  those  of  Chambers, 
P^terfi  and  Emerson,  although  others 
are  first-class.  The  essential  condition 
of  an  instrument  is  that  the  movements 
be  sufficiently  smooth  and  controllable 
under  the  highest  magnifications  of  the 
compound  microscope.  De  Fonbrune 
of  Paris  has  recently  produced  an  im- 
proved form  of  the  one  already  de- 
scribed in  McClung's  Handbook. 

Micrurgical  instruments  lend  them- 
selves to  several  types  of  operations: 
(1)  Microdissection  and  injection  of 
animal  or  plant  cells  and  tissues  for 
studies  in  cell  anatomy  and  physiology, 
also  cy  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  drop  of  an  inert  oil.  This 
prevents  evaporation  and  the  sphericity 
of  the  droplets  permits  quantitative 
determination.  Application  of  the 
technicjue  to  certain  phases  of  micro- 
chemistry  are  given  by  Benedetti- 
Pichler  in  his  book.  (3)  Isolation 
studies  for  obtaining  pure  line  cultures 
(of  bacteria,  protozoa,  etc.,  breaking  of 
asci  and  isolation  of  the  liberated 
spores,  etc.).  A  good  isolation  tech- 
nique 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 are  best  managed  with  the  de 
Fonbrune  and  Emerson  instruments. 
The  vertical  movement  is  operated  by 
a  separate  controlling  screw.  Since 
the  operations  are  performed  under  a 
single  high-power  objective,  the  only 
criterion  for  ascertaining  different 
levels  is  whether  the  object  being  oper- 
ated on  lies  above,  below  or  in  the  focus 
of  the  objective. 

The  manufactured  instruments  are 
supplied  with  instructions  as  to  their 


use.  Emerson  supplies  two  types,  one 
which  is  cheaper  for  coarser  move- 
ments although  it  is  possible  to  use  the 
cheaper  model  for  remarkably  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 
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 
Tradescautia  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  microflame 
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.  Tne 
shaft  about  2  mm.  from  the  tip,  is  bent 
in  the  microflame  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- 


MICROMANIPULATION 


154 


MICROSOMES 


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. 

Injections  are  performed  by  breaking 
the  tip  of  a  mieropipette  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 
mieropipette,  the  syringe  is  filled  with 
water  and,  by  means  of  the  plunger,  the 
water  is  driven  into  the  brass  tubing 
and  the  pipette  holder  after  which  the 
mieropipette  is  inserted.  Thus,  we 
have  a  water-filled  system  extending 
from  the  syringe  to  the  base  of  the 
mieropipette  which  is  filled  with  air. 
Micro-amqunts  of  any  given  solution 
are  then  drawn  into  or  ejected  from  the 
*  tip  of  the  mieropipette  by  a  delicate 

handling  of  the  plunger  of  the  syringe. 

The  instruments  are  generally  supplied 
in  pairs,  one  part  carrying  a  micro- 
needle for  holding  the  tissue  to  be 
injected,  the  other  carrying  the  miero- 
pipette. For  microdissection,  the  in- 
strument carries  two  needles,  each  of 
which  can  be  operated  independently. 

Wilhelm  PfefTer,  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.  Biologie,  1928, 
1  (4),  5;  and  Schonten,  S.  L.,  Zeit.  f. 
wiss.  Mikr.,  1934,  51,  421.  An  excellent 
book  which  covers  a  broad  range  of  the 
field  of  Micrurgy  is  that  of  J.  A.  Rey- 
niers  Micrurgical  and  Germ-Free  Tech- 
niques, C.  C.  Thomas,  1943. 
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  10^ 
apart. 

Micromicron  (mm)  =  1/1, 000 ,000th  part  of  a 
micron  =  1/1, 000 ,000 ,000th  part  of  a 
mm.„=  10-9  mm.  =  0.000,001  micron  = 
IQ-^A.  Unfortunately  often  used  syn- 
onymously with  ^millimicron  (m/x)  = 
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  sulpnate  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. 

Microrespirometer  to  indicate  production  of 
carbon  dioxide  by  bacteriophages, 
viruses  and  bacteria  (Bronfenbrenner, 
J.,  Proc.  Soc.  Exp.  Biol.  &  Med.,  1924, 
22,  81-82. 

Microscope.  The  ordinary  microscope  usu- 
ally has  darkfield  equipment  and  needs 
no  description.  A  special  illuminator 
to  throw  light  down  on  the  object  has 
been  devised  (Preston,  J.  M.,  J.  Roy. 
Micr.  Soc,  1931,  51,  115-118).  Centri- 
fuge, Fluorescence,  Electron,  Polarizing, 
Ultraviolet,  Metallurgical,  and  Darli- 
field  Microscopes. 

Microsomes  (G.  mikros,  small,  soma,  body). 
Term  introduced  by  Hanstein  in  1880 
originally  to  indicate  tiny  granules — as 
compared  with  ground  substance. 
Claude,  A.  Biological  Symposia,  1943, 
10,  111-129  estimates  their  size  to  be 
50-300  m/x  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- 


MICROSOMES 


155 


MITOCHONDRIA 


teins    and    pho.spholipins    in    definite 
proportions. 

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. 

Miliado  Yellow  G  (CI,  622)— Stilbene  Yel- 
low— a  direct  dye  of  light  fastness  3. 
Similar  to  Sun  Yellow  but  lighter  in 
color  (Eniig,  p.  46). 

Millv,  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  vmtil  d3'^e  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 
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. 

Millimicron  (myu)  =  1 /1000th  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  v/ith  tyrosin 
in  vitro  an  intense  red  color  jdelding  red 
ppt.  not  clianging  to  yellow  within  24 
lars.  They  give  the  following  directions. 
Add  600  cc.  aq.  dest.  to  400  cc.  cone, 
nitric  acid  (sp.  gr.  1.42)  making  4G%  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.  Ma.ximum  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). 

Mingazzini  Phenomenon  in  intestinal  villi 
interpreted  as  an  agonal  or  early  post- 
mortem change  (by  Macklin,  C.  C.  and 
M.  T.,  J.  Anat.,  1926,  Gl,  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  (G.  mitos,  thread  +  chondros, 
grain).  Granules,  rods  and  filaments 
existing  in  the  cytoplasm  of  practically 
all  living  cells  of  plants  and  animals. 
They  can  be  studied  in  living  cells 
unstained,  after  supravital  staining  and 
in  fixed  tissues. 

In  mammals  the  best  place  to  observe 
them  unstained  is  in  pieces  of  pancreas 
cut  so  small  that  when  mounted  in  a 
little  physiological  salt  solution  they 
are  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  zj^mogen  granules. 
The  proximal  poles  are  nearer  the  sur- 
rounding blood  vessels  and  compara- 
tively free  from  zymogen  granules.  In 
them  careful  search,  with  the  aid  of  a 
good  oil  immersion  objective,  will  reveal 
the  mitochondria  as  delicate  but  slightly 
refractile  filaments  oriented  in  general 
with  their  length  parallel  to  the  length  of 
the  acinous  cell.  Even  when  well  flat- 
tened such  preparations  are  too  thick  for 
satisfactory  examination  in  the  dark 
field.  When,  however,  a  mount  of  fresh 
blood  is  studied  in  dark  field  the  mito- 
chondria can  be  distinguished  as  bril- 
liantly illuminated  short  rods  and 
granules  in  the  lymphocytes  in  which 
they  are  not  obscured,  as  in  the  granular 
leucocytes,  by  masses  of  specific  gran- 
ules. Beautiful  illustrations  of  mito- 
chondria seen  in  the  d^irk  field  are 
provided  (Strange ways,  T.  S.  P.  and 
Canti,  R.  G.,  Quart.  J.  Micr.  Sci.,  1927, 
71,1.) 

The  easiest  way  to  demonstrate  mito- 
chondria supravitally  stained  is  to  place 
on  each  of  a  scries  of  say  6  slides  a  small 
drop  of  1:10,000  janus  green  B  (diethyl- 
safraninazo  dimethylanilin  chloride)  in 
0.85%  sodium  chloride  solution.  The 
dye  should  be  added  from  a  1%  stock 
solution  in  distilled  water  because  the 
powder  does  not  dissolve  easily  in  salt 
solution.  Prick  a  finger  and  touch  a 
small  amount  of  blood  to  each  lot  of 


MITOCHONDRIA 


156 


MITOCHONDRIA  AND  BACTERIA 


janus  green  and  cover  eacli  immediately. 
Do  not  wait  to  cover  until  blood  has  been 
added  to  all  of  them.  The  weight  of  the 
cover  glass  is  sufficient  to  spread  the 
mixture.  If  the  right  amounts  of  stain 
and  blood  have  been  used  the  cover  glass 
will  settle  down  on  a  very  thin  film  of 
fluid.  If  too  much  of  either  has  been 
used  it  will  float  on  the  fluid  and  it  will 
not  be  possible  to  see  clearly.  After 
about  5  or  10  minutes  the  mitochondria 
will  be  seen  colored  deep  bluish  green, 
first  in  the  lymphocytes  and  later  among 
the  granules  in  the  other  white  cells. 
To  study  the  preparations  at  leisure  it 
may  be  desirable  to  prevent  evaporation 
by  ringing  with  warm  vaseline.  For 
colored  illustrations  see  Cowdry,  E.  V., 
Internat.  Monatschr.  f.  Anat.  u.  Phy- 
siol., 1912,  29,  1-31. 

Another  satisfactory  method  is  to 
supravitally  stain  the  mitochondria  in 
the  pancreas  by  vascular  injection  as 
described  originally  by  Bensley,  R.  R., 
Am.  J.  Anat.,  1911,  12,  297-388.  About 
1  liter  of  solution  is  put  in  a  bottle,  from 
the  bottom  of  which  a  glass  tube  leads 
off,  or  from  which  the  fluid  is  syphoned 
through  a  bent  glass  tube.  About  6 
feet  of  rubber  tubing  connect  this  with 
a  glass  cannula.  The  rubber  tube  is 
supplied  with  a  clamp.  Artery  forceps 
do  nicely.  A  guinea  pig,  or  other  animal 
of  suitable  size,  is  killed  and  bled  from 
the  throat  because  removal  of  a  good 
deal  of  the  blood  facilitates  the  injection. 
The  cannula  is  inserted  into  the  aorta 
through  the  left  ventricle,  or  into  the 
thoracic  aorta  directly,  and  tied  in 
place.  In  the  former  case  the  branches 
going  up  toward  the  head  and  arms  must 
be  ligated.  When  all  is  ready  hoist  the 
injection  bottle  about  4  or  5  feet  above 
the  animal  and  remove  the  clamp.  Open 
the  right  auricle  so  that  blood  and  solu- 
tion can  flow  out.  In  about  a  minute 
open  the  abdomen  by  a  long  medial 
incision  but  do  not  display  the  pancreas. 
To  make  sure  that  all  the  vessels  in  the 
pancreas  are  being  perfused  by  the  solu- 
tion it  is  desirable  to  momentarily  clamp 
the  superior  vena  cava  and  thus  let  the 
solution  back  up  a  little  under  pressure. 
Now  lay  bare  the  pancreas.  When 
the  optimum  staining  is  obtained, 
usually  about  10  minutes  after  the  be- 
ginning of  the  injection,  it  should  be 
slightly  swollen,  owing  to  separation  of 
lobes  and  lobules  by  increase  in  tissue 
fluid,  and  of  a  uniform  fairly  dark  bluish 
green  color.  Remove  the  pancreas  and 
place  it  in  salt  solution.  For  examina- 
tion it  is  essential  to  take  very  small 
pieces  not  more  than  1  mm.  in  diameter. 
Mount  them  in  a  little  salt  solution  on 
slides  so  that  they  will  flatten  by  the 


pressure  of  the  cover  glasses,  one  piece 
per  slide.  Study  at  low  magnification 
shows  irregular  masses  of  small  deeply 
stained  cells.  These  are  the  islands  of 
Langerhans.  It  is  in  the  acinous  tissue, 
which  is  less  deeply  colored,  that  search 
should  be  made  for  the  mitochondria. 
Identify  first  the  distal  poles  of  the 
cells  cliarged  with  zyomgen  granules. 
Then  look  for  greenish  blue  stained 
mitochondria  in  the  proximal  parts  of 
the  cells.  After  a  time  the  oxygen  in 
the  center  of  the  tissue  is  used  up,  the 
dye  becomes  bleached  to  a  leucobase  and 
then  to  a  pink  colored  base  (diethyl- 
safranin).  This  method  of  supravital 
staining  of  mitochondria  with  janus 
green  can  be  used  for  any  tissue  in  the 
body.  It  is  particularly  recommended 
for  the  pancreas  because  its  lobules  are 
thin,  and  easily  separated  without 
mechanical  injury. 

Other  supravital  stains  for  mito- 
chondria are  Diethylsafranin,  Janus 
Blue,  Janus  Black  1,  Pinacyanol  and 
Rhodamin  B,  which  see.  When  a  very 
dilute  solution  of  methylene  blue  is 
applied  to  mitochondria  in  tissue  cul- 
tures they  can  be  stained  a  brilliant 
blue  (Ludford,  R.  J.,  Arch.  f.  exp. 
Zellf.,  1935,  17,  339-359).  It  is  not 
unlikely  that,  in  conditions  difficult  to 
define,  a  considerable  number  of  dyes 
will  color  mitochondria  supravitally. 
When  fixed  tissues  are  to  be  used  the 
choice  of  method  is  important.  The 
difficulty  with  the  osmic  acid  containing 
fixatives  is  that  they  penetrate  poorly. 
The  best  fixative  is  the  formalin  bichro- 
mate fluid  of  Regaud  followed  by  mor- 
danting with  3%  potassium  bichromate 
and  the  best  stain  is  probably  Anilin- 
Fuchsin  Methyl  Green  as  used  by 
Bensley.  See  methods  of  Altmann, 
Benda,  Champy-Kull,  Regaud  and  Vol- 
konsky,  and  AlcClung  (pp.  265-274). 
Mitochondria  can  now  be  collected  by 
Centrifugation  and  subjected  to  direct 
chemical  analysis  (Bensley,  R.  R.  and 
Hoerr,  N.  L.,  Anat.  Rec,  1934,  60, 
251-266;  449-455).  Valuable  technique 
for  study  of  isolated  mitochondria  by 
Electron  Microscope  has  been  intro- 
duced by  Claude,  A.  and  Fullam,  E.  F., 
J.  Exper.  Med.,  1945,  81,  51-62. 
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 


MITOCHONDRIA  AND  BACTERIA        157 


MOLECULAR  SOLUTION 


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, 7G()-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  deparafnnise  and  stain  sections 
in  aniiin-safranin  (Babes),  wash  quickly 
in  tap  water,  differentiate  in  acid  alcohol 
until  the  resting  nuclei  arc  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  Carney'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 
aniiin-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). 

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.  e.xper.  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 
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. 
Molecular  Film  Technique,  see  Taylor,  H. 
S.,  Lawrence,  E.  0.,  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  ^  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 


158 


MOUNTING  MEDIA 


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,  uarcein,  nitrazine,  oil  red 
O,  orange  G,  orange  I,  orange  II,  orange 
IV,  ponceau  2R,  sudan  R,  thiazine 
redR. 

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.  Alany  of  their  properties  can 
to  great  advantage  be  investigated  in 
Tissue  Cultures.  The  best  way  to 
demonstrate  the  remarkably  close  rela- 
tion that  may  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). 

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  aq.  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 
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.  3  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  nitrste  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. 

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  maldng 
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  ba^sis. 

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  many 
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. 


MOUNTING  MEDIA 


159 


MUCUS 


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 
S  parts  resin  which  becomes  solid  on 
cooling.  Apply  to  edges  in  the  same 
manner  as  the  paraffin.  Kronig'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  and 
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 
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,  0.6  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  paraffin  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). 
Makeup:  (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.  Dehj-drate  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. 

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  tissues  has  been 
worked  out  by  L.  H.  Hempelmann,  Jr., 
Anat.  Rec,  1940,  78,  197-206.  Briefly 
stated  toluidin  blue  in  1:280,000  will 
stain  the  former  vividlj'  and  the  latter 
not  at  all . 

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 


MUCUS 


160 


MUSCLE 


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 
serous  or  zymogenic  cells  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  Schiflf'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  xjdol  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. 

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). 

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  vrave  mechanics,  see 
Carev,  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., 


MUSCLE 


161 


NAPHTHOL  GREEN  B 


1944,  19,  11-27).  See  Myosin  und  Pur- 
kinje  cells  and  fibers. 

Museum  Specimens,  see  Color  Preservation. 

Mycobacteria,  see  Acid  Fast  Bacteria. 

Mycologica!  Tecliniques,  sec  t'ungi. 

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  differentiated  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,  see  Leucocytes,  developmental 
series. 

Myeloidin  is  the  term  applied  to  the  mate- 
rial of  certain  spheroitial  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 
with  muscle  cells  well  demonstrated  by 
Mallory's  Connective  Tissue  Stain. 

Myosin  is  a  protein,  present  in  muscle,  the 
molecules  of  which  are  needle-sliaped. 
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  oxida,tion  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.  II.,  Practical  Handbook  of 
the  Pathology  of  the  Skin.  London : 
H.  K.  Lewis,  1903,  408  pp.  gives  Heller's 
method  which  involves  fixation  of  un- 
gual phalanx  for  a  few  days  in  Muller's 
fluid,  prolonged  washing,  decalcification 
for  4-0  days  in  1  p)art  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  dj'e  (NAC- 
7080  and  DuPont  L  6401)  gives  excellent 
staining  in  combination:  Stain  with 
Weigert's  iron  hematoxylin,  6  min. 
Wash  in  water  and  counterstain  5  min. 
in  3  parts  1%  brilliant  purpurin  R  (CI, 
454)  in  1%  aq.  acetic  acid  and  2  parts 
1%  azofuchsiii  (CI,  153)  likewise  in  1% 
aq.  acetic  acid.  Ptinse  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  Harvev,  B.  C.  II.,  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.  sodium  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 
Mollier,  G.,  Zeit.  f.  Wis.  Mikr.,  1938,  55, 
472-473. 

Lillio,  R.  D.,  J.  Techn.  Methods,  1945, 
No.  25,   47    pp.  recommends  naphthol 


NAPHTHOL  GREEN  B 


162 


NECROSIS 


green  B  for  connective  tissue.  Stain 
sections  6  min.  in  Weigert's  or  other 
iron  hematoxylin.  Wash  thoroughly 
in  water  and  stain  3  nain.  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  xjdeue  or 
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  I. 

Napbtho!  Red  S,  C  or  O,  see  Amaranth. 

Naphthol  Yellow,  see  Martins  Yellow. 

Naphthyi  Red  (CI,  S56),  a  basic  dye  of  light 
fastness  5.  Only  nuclei  of  mature  plant 
cells  colored  fugitive  red  (Emig,  p.  57). 

Naphtliylamine  Brown  (CI,  170),  an  acid 
monoazo  dye  which  stains  plant  tissues 
da,rker  in  presence  of  potassium  bi- 
chromate (Emig,  p.  34). 

Naphthylamine  Pink,  secMagdala  Red. 

Narceln  (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  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,  518-541  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  ioio  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  lyraphiitic  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.  -I-  10  cc.  butyl,  2  hrs.;  80  cc.  62% 
ale.  +  20  cc.  butyl,  2  hrs.;  65  cc.  77% 
ale.  +  35  cc.  butyl,  4  hrs.;  45  cc.  90% 
ale.  -f  55  cc.  butyl,  6  hrs.  to  days;  25 
cc.  abs.  ale.  -f  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  (G.  nekrosis,  a  killing).  The  term 
is  usually  applied  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- 


NECROSIS 


16S 


NEOPRENE 


crease  in  temperature,  confers  renewed 
vitality  (see  Luyet,  B.,  C.  rend.  Soc. 
de  bioL,  193S,  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,  +  rhexis, 
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. 
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  en  a  slide,  dry  and 
examine.  Higgins'  India  Ink  is  usually 
employed;  but  congo  red  (Cumley, 
R.  W.,  Stain  Techn.,  1935,  10,  53-50) 
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 
(Schleifstein,  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- 
cerollOO  cc.  and  methyl  alcohol  100  cc). 
Steam  gently  5  min.  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  quicklj'^  as  possible,  put 
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  outwara 
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  Loeffier'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.  Negri  bodies 
pale  red.  The  red  is  much  more  intense 
if  the  pll  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  bv  Jordan,  J.  H.,  and  Heather, 
H.  H.,  S"tain  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,  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  grosslj'- 
leaking  vessels.  Continue  8-18  hrs. 
until  organ  is  pale  graj'.  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  warm  it  with 
tap  water.  Connect  cannula  with  bottle 
containing  neoprene.  A  special  appara- 
tus for  maintenance  of  15tf-160  mm.  Hg. 
is  advised  by  Lieb  but  it  is  probably 
sufficient   to   provide  gravity   pressure 


NEOPRENE 


164 


NERVE  FIBER  DEGENERATION 


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.  Employed  for  injection  of 
coronary  arterial  system,  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  (5  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,  441-446). 
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. 
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. 


NERVE  FIBER  DEGENERATION  165 


NERVOUS  SYSTEM 


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,  Rao,  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  Mann'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.,  1335,  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,  and 
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  Ternxinaux.  Use  of  quartz  rod 
illuminator  in  study  of  living  nerve 
fibers  is  described  bv  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).  Sec  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 
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 


NERVOUS  SYSTEM 


166 


NEUROFIBRILS 


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  eyidings  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  bj''  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  approximatelj''  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.,  Froc.  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 
microscopicaliy  demonstrable  swelling 
of  the  capsules  of  pneumococci  which  is 
of  distinct  value  in  typing  (L.  W.  Parr, 
in  Simmons  and  Gentzkow,  p.  426). 

Neumann's  Crystals,  sec  Charcot-Leyden. 

Neurofibrils.  These  delicate  fibrils  and 
networks  can  be  demonstrated  with 
difficulty  mainly  by  methods  of  silver 
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 
tiiey  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 


NEUROFIBRILS 


167 


NEUTRAL  RED 


purple  black  color.  5%  aq.  sodium 
hyposulphite  5  inin.  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  teclinique  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 
verj^  useful  synopsis  is  given  by  Addi- 
son (McClung,  pp.  452-466).  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  arc  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- 
matous  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  ModiScation  of  Mann's 
eosin-methyl  blue  method. 

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  arc  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  (Bcnslcy,  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. 
(2)  Potassium  bichromate  2.5  gms.; 
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  ifjL  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  hi  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 
(Benslev,  R.  R.,  Am.  J.'  Anat.,  1911, 
12,  297-388).  Add  2  cc.  of  a  previously 
prepared  1%  aq.  neutral  red  to  300  cc. 


NEUTRAL  RED 


168 


NEUTRAL  STAINS 


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  different  sizes 
in  a  pale  red  background.  After  a  time 
the  dye  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,  ISI.  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  C.C. 
(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  Giemsa,  Wright  and  others.  See 
critical  evaluation  by  Hall  (Downey, 
pp.  643-698).  See  application  in  study 
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 


NEUTRAL  STAINS 


169 


NICKEL 


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  dj^e  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  follov.-ers,  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 
(9-12/i)    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. 
in     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  erythrocj'tes  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  Bo3"d,  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,  chcmotaxis  and  other 
properties,  see  Leucocytes. 

Neutrophilic,  see  Staining. 
Nevillite  V  and  No.  1  have  been  compared 
with  gum  da  mar  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,  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,  and 
ammonium  hydrosulphate  5  drops.  Im- 
merse in  a  solution  of  ammonium 
phosphate  in  order  to  produce  the 
insoluble  double  salt:  NIl4NiP04  + 
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 


170 


NINHYDRIN  REACTION 


Nicotinic  Acid.  Preliminary  detection  of  it 
or  its  amide  by  fluoresence  microscopy 
(Hirt,  A.  and  Wimroer,  K.,  Klin.  Woch- 
nesdir.,  1939,  18,  705-767).  Lasting 
yellow  fluorescence. 

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  fats  {glycer- 
ides)  were  thereby  colored  red  and  the 
fatty  acids  blue,  but  Kaufmann  and 
Lehmann  (C.  and  E.,  Virchow's  Arch, 
f.  Path.  Auat.  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. 

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, 
polypeptidps  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  95%  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. 

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/lo  primary  potassium  phos- 
phate (9.078  gm.  KH2PO4  per  liter). 
The  reaction  is  carried  out  in  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.  coUophonium  and 
20  gms.  heated  lanolin  as  reconxmended 
bj^  Romeis  but  they  must  be  studied  the 
same  day  for  the  color  fades  quickly. 

Serra  cai-efuUy  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 


NINHYDRIN  REACTION 


171 


NITRO  REACTION 


above.  To  be  sure  that  u  secoiidu,ry  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 niuiiydrin  reaction  in  the  same 
structure  does  not  necessarily  demon- 
strate a  proteic  or  amino  acid  nature." 
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- 
noy'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  Ijecause 
of  the  striking  difference  in  appearance 
of  Nissl  bodies  when  intensely  and 
lightly  colored.  There  are  many 
methods  from  which  to  make  a  choice. 
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.,  1943,  18,  77- 
86).  An  apparatus  has  been  devised 
apparently  suitable  for  obtaining  the 
Absorption  Spectra  of  Nissl  bodies. 

Nitrates.  Make  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  crj^stallize 
and  examine  in  polarized  light.  Nitron 
is  diphenyl-endo-anilo-dihydriazole.  It 
precipitates  nitrates  as  insoluble  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-NO;.  All 
strongly  acid.  Aurantia,  martins  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 


NtTRO  REACTION 


172 


NORMAL  SOLUTIONS 


nitrified    (Lison,    p.    162).     See   Lison, 
L.,  J.  PhysioL  et  Path.  Gen.,  1933,  31, 
82-99). 
Nitroprusside    Reaction    for    Glutathione. 

1.  Mattei  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  NH3  vapor. 
Freeze  solidly  with  ice  or  solid  CO2. 
E.xamine  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  ce.  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  Microscopic. 
For  fresh  tissues  (pieces,  smears,  frozen 
sections)  (a)  Glutathione  reduced.  Add 
to  tissue  on  slide  1  drop  5%  sodium 
nitroprusside  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  nitroprusside  (saturated  with 
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). 

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  o.ximes).  Produced 
by  nitrous  acid  acting  on  phenolic  com- 
pounds.    Naphthol  gieen  B  and  Y. 

NNN  Medium,  see  Leisiimania. 

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  (eqviivalent  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  -f  CI  =  35.457 
(see  Atomic  Weights)  =  36.465.  Con- 
sequently make  up  36.465  gms.  of  HCI 
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  appro.ximately  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 
130.136.  But  there  are  2  replaceable 
hydrogen  atoms  so  that  in  makinga 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- 
2HjO   with   molecular   weight   of    126. 


NORMAL  SOLUTIONS 


173 


NORMALS,  GROSS  SIZES 


Owing  to  presence  of  2  hydroxyl  groups 
it  has  2  liydrogen  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  (NaOPI) 
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 
hj^drogen  equivalent  is  h  Na2HP04,  so 
that  the  molecular  weight  is  divided  by 
2.  Similarly  with  the  salt  Na2S04  the 
molecular  weight  is  halved.  For  sodium 
triphosphate,  NaaPO^,  the  hydrogen 
equivalent  is  i  Na3P04,  or  the  molecular 
weight  is  divided  by  3. 

Normality.  Microscopic  study  of  tissues 
will  be  of  little  value  in  medical  research 
unless  their  normal  structure  is  at  least 
approximately  known  as  a  basis  on  which 
to  interpret  the  findings.  Unfortu- 
nately there  is  no  general  agreement  as 
to  what  constitutes  normal  and  abnor- 
mal, but  the  statistical  definition  of 
normality  provides  at  least  a  working 
basis.  According  to  it  the  normal  state 
is  the  usual  one  in  a  homogeneous  group. 
Bj'  usual  we  mean  that  it  is  present  in 
the  majority,  51%  or  more,  of  the  indi- 
viduals. By  homogeneous  we  mean 
that  the  individuals  are  of  the  same  age, 
sex,  race  and  are  living  under  similar 
conditions,  that,  in  other  words,  no 
factor  is  to  the  best  of  our  knowledge 
operative  likely  to  produce  diversity 
among  them  in  the  particular  feature 
the  normality  of  which  is  under  con- 
sideration. Thus,  if  a  certain  measure 
of  calcification  of  the  wall  of  the  aorta  is 
found  in  56%  of  individuals  of  a  homo- 
geneous group  in  St.  Louis,  it  must  be 
regarded  as  normal  for  them.  But  it 
does  not  follow  that  the  same  grade  of 
aortic  calcification  is  normal  for  a  group 
of  Japanese  of  the  same  sex  and  age  in 
Tokj'o.  For  them  an  entirely  different 
grade  may  be  normal  occasioned  by  factors 
of  race,  environment,  etc.  not  operative 
in  the  same  way  for  the  St.  Louis  group. 
In  speaking  of  normality  it  is  necessary 
to  be  very  specific.  An  aorta  may  be 
normal  in  respect  to  degree  of  calcifica- 
tion but  abnormal,  or  unusual,  in  other 
respects.  Consequently'  the  normality 
of  this  or  any  other  tissue  can  only  be 
established  for  the  particular  property 
measured  assuming  tliat  the  technique 
of  observation  is  adequate  and  the  num- 
ber of  individuals  examined  is  sufficiently 
large . 

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  <lcaling 
with  the  organ  in  which  one  is  interested 
in  the  Quart.  Cum.  Index  Med.  The 
older  data  are  summarized  by  Vierordt, 
H.,  Anatomische  Physiologische  und 
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. ;  capacity,  30-45  cc. 

Heart— Weight,  female,  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,  tricu.spid 
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 


NORMALS,  GROSS  SIZES 


174 


NORMALS,  MICROSCOPICAL 


ileum.  Large  intestine,  length,  180- 
195  cm.;  duodenum,  length,  25-28.5 
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 — Weiglit,  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.S  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. 

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— Male,    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    slightlj'     longer     than     the 
anterior. 
Normals,  Microscopical.    Most  tissues  are 
e.xamined    in    but   a    cursory    way.     If 
something  is  encountered  which  looks 
definitely     unusual     the     question     of 
normality  comes  up,  but  there  are  prob- 
ably   numerous    instances    of    tissues 
which  look  enough  like  what  was  ex- 
pected to  be  passed  without  comment 
even  though  they  were  not  in  fact  normal. 
This  will  continue  to  be  the  c^se  for  ap- 
pearances     that      cannot      easily      be 
expressed        quantitatively.     To        be 
specific,  the  normal  range  in  size  of  the 
nuclei  of  human  liver  cells  is  not  known, 
neither  are  the  limits  of  normal  varia- 
tion in  amount  of  interstitial  cells  of  the 
testicle  appreciated.    One  difficulty  is 
that  a  microscopically  complete  exam- 
ination of  any  tissue  is  very  rarely  made 
so  there  is  always  a  chance  that  the 
unseen  part  deviates  from  the  normal. 
Particularly  is  this  so  in  large  organs 
like  the  liver  and  lungs  and  in  small 
ones  whicli  characteristically  are  prone 
to  exhibit  regional  diversity  such  as  the 
prostate,    thyroid    and    the    mammary 
glands.     Data     concerning     the     gross 
examination  of  organs  and  tissues  stud- 
ied in  sections  are  always  desirable  and 
may   provide   a   significant    clue.     One 
must  not  be  led  astray  by  histological 
Artifacts  or  Postmortem   clianges.    In 
experimental    animals    the    problem    is 
less    complicated,    because   the   tissues 
can  always  be  obtained  fresh  and  it  is 
easier  to  prepare  an  adequate  series  of 
controls  for  comparison  with  the  sus- 
pected   specimen.     But    when    we   get 
away  from  sections  to  body  fluids  that 
can  be  readily  and  accurately  sampled 
and  in  which  the  cells  can  be  counted 
per  c.  mm.  both  absolutely  and  differ- 
entially the  verdict  of  normal  or  ab- 


NORMALS,  MICROSCOPICAL 


175 


NUCLEASE 


normal  can  be  returned  with  greater 
assurance.  This  is  particularly  true 
for  the  blood  and  cerebrospinal  fluid. 
Histological  criteria  of  normality  are 
also  of  some  value  in  the  examination  of 
joint  fluids,  serous  fluids  and  vaginal 
smears. 

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,  paraffin  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  acidophilic  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. 
It  is  only  when  stronglj^  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  crj's- 
talline  structure  are  not  so  well  shown 
in  fixed  and  stained  preparations.  The 
inclusions  give  the  usual  protein  reac- 
tions but  arc  Feulgen  negative.  The}' 
apparently  contain  virus. 

An  interesting  and  well  illustrated 
account  of  intranuclear  parasites  is  pro- 
vided bj'  H.  Kirby,  Jr.  in  Calkins,  G.  X. 
and  Summers,  F.  M.,  Protozoa  in  Bio- 
logical Research.  New  York:  Colum- 
bia University  Press,  1941,  1148  pp. 
Nuclease.  This  enzyme  acting  on  nuclcins 
is  very  elusive.  A.  Van  Herwerden  has 
described  it  in  several  publications,  of 
which  the  most  recent  is  Anat.  Anz., 
1914,  47,  312-325.  Lison  (p.  175)  refers 
to  two  other  papers  by  Sachs  and  Oes 
but  does  not  give  references  to  them. 
If  one  could  rely  on  digestion  of  sections 
for  two  days  at  37 °C.  removing  all 
nucleins  to  the  exclusion  of  all  other 
cellular  materials  an  important  path 
for  investigation  would  be  opened  up. 
A  purified   nuclease   is   required.    Sec 


NUCLEASE 


17€ 


NUCLEI 


Barnes,  J.  M.,  Brit.  J.  Exp.  Path.,  1940, 
21,  264-275  for  analysis  of  nuclease  in 
lymphocytes  and  polymorphonuclear 
leucocytes. 
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  polymorphism  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  opportvmities 
for  advance.  The  Vital  Staining  of 
nuclei  without  killing  the  cells  is  diffi- 
cult and  not  particularly  helpful  (Acri- 
flavine);  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 
virus  action  are  usually  acidophdic, 
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  heterogenicity,  of  internal 
structure  which  cannot  be  observed  by 


the  most  careful  examination  of  the 
nuclei  of  living  cells.  In  the  latter  only 
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-osmic  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  (oxy  chromatic  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., 


NUCLEI 


177 


NUCLEOLUS 


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  Mayer's 
Hemalum,  blue  in  tap  water,  counter- 
stain  in  eosin,  dehydrate,  clear  and 
mount.  Perhaps  the  technique  can  be 
so  adjusted  that  it  will  permit  the  sepa- 
ration of  nuclei  from  other  tissues. 
See  Arginase. 

Nucleic  Acids,  see  Desoxyribonucleic,  Ribo- 
nucleic and  Thymonucleic. 

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 
by  dividing  the  nucleic  acid  nitrogen 
by  the  total  coagulable  nitrogen  less 
nucleic  acid  nitrogen. 

Nucieolinus  is  a  term  introduced  by  Ilaeckel 
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  plastosomcs,  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,  easilj^  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 
and  karyospheres.  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.  Amphiniicleoli  (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  necessarilj' 
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 
inaterial  originally  distributed  diffusely 
in  the  nucleoplasm.  Nucleoli  which 
look  bubbly,  or  are  surrounded  by  halos, 
are  to  be  regarded  with  suspicion. 
Fixation  in  Acetic-Osmic-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) 


NUCLEOLUS 


178 


OLIGODENDROGLIA 


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  generallj'^  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  (glioma- 
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  probablj''  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 
review  of  the  literature  (Mendes  Fer- 
reira,  H.  E.,  J.  Lab.  &  Clin.  Med.,  1940- 
41,  26,  1612-1628). 

Nucleotides,  see  Pentose  Nucleotides. 

Nutriles,  growth  promoting  (Williams,  R. 
J.,  Biol.  Rev.,  1941,  16,  49-80). 

Oi!  Ehie  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  Red  IV,  see  Sudan  IV. 

Oil  Red  AS,  O,  B  or  3B,  see  Sudan  IIL 

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  MuUer'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  Anatomy, 
Washington  University,  St.  Louis,  May 
24,  1946.)  For  myelin  sheaths.  Run 
paraffin,  or  celloidin  sections  of  prop- 
erly 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.  for 
4-24  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. 
Differentiate  in  0.25%  aq.  potassium 
permanganate  1—5  min.  Remove  po- 
tassium permanganate  with  Weil's  solu- 
tion (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  myelinated 
fibers.  If  differentiation  not  complete 
after  first  immersion  in  potassium  per- 
manganate followed  by  oxalic  acid- 
bisulphite  mixture,  repeat  the  proce- 
dure.'   Wash,  dehydrate  and  mount. 

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 


OLIGODENDROGLIA 


179 


OSAGE  ORANGE  PIGMENTS 


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). 

Omentum,  spreads  of  (McClung,  p.  336). 
Transplants  of  spleen  into  (Holyoke.E. 
H.,  Am.  J.  Anat.,  1940,  66,  87-132. 

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.,  Fillet,  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.  + 
6%  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.  -f-  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  dye 
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  tho  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,  Federigo,  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 
Dyes.  Brooklvn:  Chemical  Publishing 
Co.,  Inc.,  1944;' 95  pp.). 

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  is  omitted  as  useless. 
Since  the  fluid  docs  not  keep  it  should 
be  made  up  immediatelj'^  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  n.s  brilliant  mordant 
dyes  for  wool  and  silk.  Wolfsom, 
M.  L.,  Harris,  W.  D.,  Johnson,  G.  F., 
Mahan,  J.  E.,  Moffett,  S.  M.  and  Wildi, 
B.,  J.  Am.  Chem.  Soc,  1040,  68.  406- 
418.yiShould  be  tried  on  animal  tissues. 


OSMIC  ACID 


180 


OSSIFICATION 


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,  has  been  im- 
proved by  Palmer  (R.,  J.  Roy.  Micr. 
Soc,  1930,  50,  221-226).  _ 

The  fumes  of  osmic  acid  are  very  in- 
j  urious  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 
Bensley  (II.  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,  Flamming  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  Benslev,  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  IS  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.  KoCO.i  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 


OSSIFICATION 


181 


OXIDASE 


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  Griff  en,   A.   B.,   Stain  Techn., 

14,  7-11.  This  staining  of  ossification 
centers  with  alizarin  red  can  be  com- 
bined with  the  coloration  of  the  cartil- 
aginous skeleton  with  toluidin  blue  to 
make  quite  brilliant  specimens  (Wil- 
liams, T.  \V.,  Stain  Techn.,  1941,  16,  23- 
25).  _ 

Ossification,  intense  glycogenesis  during 
(Gendre,  H.,  Bull.  d'Hist.  Appl.,  1938, 

15,  165-178). 

Otoliths,  technique  for  (Johnston,  M.,  J. 
Roy.  Micr.  Soc,  1938,  58,   112-119). 

Ova,  concentration  of  parasitic  ova  in  Feces. 

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 
IMicrodissection  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- 
per, H.,  Arch.  Path.,  1942,  36,  647-662 
have  indeed  investigated  variations  in 
ovarian  fluorescence  during  cyclical 
changes. 

Owen's  Blue  (British  Drug  Houses  Ltd.),  a 
dis-azo  djx  similar  in  composition  to 
IVIanchester  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 
"oxidases"  all  enzymes  capable  of  cata- 
Ij^sing  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  O2  is  the  same  as  a  per- 
oxidase in  the  presence  of  H2O2.  But 
the  particular  oxidases  are  more  delicate 
and  easil}'  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  (Graff) 
=  oxidase  reaction,  modification  A  (W- 
II.  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.  Agitate  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. 
4-  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  Graff  (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. 


OXIDASE 


182 


OXIDATION-REDUCTION 


Frozen  sections  of  formalin  fixed  tissues 
treated  with  this  reagent  show  violet  or 
black  granules,  which  quickly  disappear. 
Oxidation-Reduction  Potential.  Details 
supplied  by  Dr.  Christopher  Carruthers 
of  The  Barnard  Free  Skin  and  Cancer 
Hospital. 

This  very  important  measurement  is 
particularly  well  explained  by  Seifriz, 
W.,  Protoplasm,  New  York:  McGraw- 
Hill  Book  Co.,  1936,  584  pp.  For  a 
comprehensive  developmenta.1  treat- 
ment of  the  subject  see  Clark,  W.  M. 
and  coworkers.  Hygienic  Laboratory 
Bull.,  1928,  151,  1-352. 

O.xidation  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  FeCla  gains  an  electron  it  is 
reduced  to  FeCU,  or 

Fe'"'*^  -h  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''"'^  -|-  electron  ;;zi  Fe"*^. 

When  an  acid  mixture  of  ferrous  and 
ferric  chloride  is  placed  in  an  electrode 
vessel  it  will  j'ield  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 
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++) 
(Fe+++)  • 

Fe+-^   ^  (Reductant) 
(Oxidant) 


Eh^Eo  - 


Fe+ 


Eh  is  the  observed  difference  in  electro- 
motive force  between  the  electrode  and 
the  normal  hydrogen  electrode;  £'<,  is  a 
constant  characteristic  for  the  ferrous- 
ferric  system  (the  so-called  normal  po- 
tential); R,  T,  and  F  liave  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  : 

R.,  _  p        liT  ,     (Rod) 

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  ;z±  Red  (2) 

For  equation  (1),  the  relation  would  be 
p,  _  r.       ff^  ,„  (Red-) 

E,  -  So  -  —  In  -^Q^ 

The  active  reductant  of  equation  (1) 
is  the  anion  of  au  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 

,  _  RT        (Red-) 

nF  (Ox) 

at  any  constant  pH  (For  development 
see  Cohen,  B.,  Symposia  Quant.  Biol., 
1933,  1,  195-20-1). 

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  ReznikofT,  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  made  with 
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 
upon  the  relative  activity  of  the  proces- 
ses of  o.xidation  and  reduction. 

Although  it  is  difficult  accurately  to 
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 
genei"ate.  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 

ni-Bromophenol  indophenol 0.24S 

o-Chlorophenol  indophenol 0.233 

Phenol  blue  chloride 0.227 

Phenol  indo-2,6  dichlorophenol 0.217 

o  Cre<5ol  indophenol 0.195 

o  Cresol  indo-2,6  dichlorophenol 0.1  SI 

i-Naplithol-2-sulfonate   indophenol  ©-sul- 
fonate     0.135 

l-Naphthol-2-gulfonate  indophenol 0.123 

Toluylene  blue  chloride 0.115 

Brilliant  cresyl  blue  chloride 0.047 

Methylene  blue  chloride -t-0.011 

K4  indigo  tetrasulfonate —0.046 

Ethyl  capri  blue  nitrate —0.072 

K«  indigo  trisulphonate —0.081 

Kj  indigo  disulphonate —0.125 

Cresyl  violet -0.167 


OXIDATION -REDUCTION 


183 


OXIDATION-REDUCTION 


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.  Needham, 
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- 
plasm, reoxidation  of  the  reductant  can 
be  accomplished  by  injecting  dilute  po- 
tassium ferri cyanide  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  dye  assumed  a  distinct 
blue  color.  This  indicated  that  the  sub- 
cutaneous tissue  maintained  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. 


Eh  =  E'o-  0.062  log 


100 -a 


(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 


OXIDATION -REDUCTION 


184 


PALITZSCH'S  BORAX-BORIC 


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  4-0.100  volts,  and  the  stroma 
—0.130  volts.  Anaerobically  both  had 
estimated  potentials  of  —0.290  volts. 

Lewis,  M.  R.,  Barron,  E.  S.  G.  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 
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  and 
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. 
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  -f  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   Method,    see    Weigert-Pal. 

Palitzsch's  Borax-Boric  Acid  Buffers 
(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.  Na2B407  10  H2O  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 

Boras 

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 

5.6 

4.5 

8.51 

5.0 

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 

PALITZSCH'S  BORAX-BORIC 


185 


PARAFFIN  EMBEDDING 


French,  R.  W.  Stain  Techn.,  1930,  5 
87-90;  1932,  7,  107-108  recommended 
the  use  of  these  buffers  for  the  range  pH 
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  (G rubier)  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). 

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 
purposes  a  paraffin  with  melting  point 
of  56-58°C.  is  employed;  but  60-62°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- 


PARAFFIN  IMBEDDING 


186 


PARAFFIN  SECTIONS 


ter.  First  smear  a  little  glycerin  evenly 
over  the  bottom  and  sides  of  the  dish. 
Then  pour  in  a  little  paraffin,  a  tliin 
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  v/ater.  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 
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).  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 


187 


PASTEURELLA 


Farafuchsin,  see  Pararosanilin  (Magenta  O). 
Paraganglion,  see  Aortic. 

Paraldehyde  is  paracetaldehyde,  a  polymer 
of  acetaldehyde  employed  in  Dioxan 
fixative  and  in  other  \va3's. 

Paraloidin,  see  Celloidin. 

Paramagenta,  see  Pararosanilin  (Magenta 
O). 

Paramylum,  a  form  of  carbohydrate  store  in 
lower  plants  (Taylor  in  McClung,  p. 
221). 

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  lesa  doreiventrally  flattened 3 

Body  in  cross  section  ordinarily  round 2 

2.  Body  never  annulated,  without  legs  or  jaws. . . .  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, 
intastine  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  passages  or  externally  (leeches,  blood 
suckers) Hirudinea 

4.  Intestine  absent,  armed  rosteUum  present,  very 


rare  in  human  intestine,  thorn  headed  worms 

Acanthocephala 
Intestine  present,  but  no  armed  rostcllum 

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  moat  species,  larvae 

annulated,  breathe  by  trachea,  adults  ecto- 
parasites, occasionally  under  skin,  in  wounda, 

intestine  or  bladder  (insects) Insecta 

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,  adults  occasionally  in  nasal  passages 
(tongue  worms) Linguatulidae 

Numerous  legs,  occasionally  in  nasal  passages  and 
intestine    (thousand   leggers) Myriapoda 

See  Tapeworm  Proglottids,  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  (Hollborn) 
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  -f  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  J  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 


188 


PERFUSION 


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. 

Pentose  Nucleotides,  identifiable  by  ultra- 
violet absorjation  spectra  maximum 
about  2600  A.  Their  high  concentra- 
tion appears  to  be  correlated  with  the 
generally  noted  basophilia  of  young 
tissues  (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  CO2.  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  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 
material  (Holter,  H.,  J.  Cell,  and  Comp. 
Physiol.,  1936,  8,  179-199).  Similar 
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.  Lab. 
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. 

Perdrau's  Modification.  Bielschowsky's 
silver  method  for  reticulum  as  detailed 
by  Bailey,  P.  and  Hiller,  G.,  J.  Nerv. 
&  Ment.  Dis.,  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  4§-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  ia  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 


PERFUSION 


189 


PERMEABILITY 


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  arterj'  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  |  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 
janus  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  pericardium  (Popa,  J.  T. 
and  Lucinescu,  E.,  J.  Anat.,  67,  78-107) . 


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  potassium  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-S  cc.  30%  aq. 
sodium  chloride  during  10  min.  and 
3-4  cc  particulate  suspension  of  India 
ink  or  mercury  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  n  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  macropliages  and  can- 
cer cells  W.  H.  Lewis  (Am.  J.  Cancer, 
1937,  29,  666-679)  has  enabled  us  to  see 
tliat  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. 


PERMEABILITY 


190 


PEROXIDASE 


Covell  (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),  \-l  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  tliem. 

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 
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  pero.xide 
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%  H2O2,  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  paraflRn;  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-dichlor-phenol-mdo- 
phenol  (Jacoby,  F.,  J.  Physiol.,  1944, 
103,  Proc  Physiol.  Soc  July  29).  Fix 
air  dried  bloocl  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  H2O2 


PEROXIDASE 


191 


PHLOXINE-METHYLENE  BLUE 


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. 

Peterfi,  see  Double  Imbedding,  and  Osmic 
Acid  Method  for  nerve  fibers. 

Petrunkevitch's  Fixatives:  Cupric-phcnol. 
Stock  solution  A  =  aq.  dest.,  100  cc; 
nitric  acid  (c.p.  sp.  gr.  1.41-1.42),  12 
cc;  Cu(N03)2-3  H2O,  8  gm.  Stock 
solution  B  =  80%  alcohol,  100  cc. ; 
phenol  crystals,  c.p.  4  gm.;  ether  6  cc. 
Employ  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,  c.p.  crystals,  5  gm. 
Time  unspecified.  Wash  in  70%  alco- 
hol. Said  not  to  harden  tissues  like 
ordinary  fixatives.  IVIay  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. 
II.  Stockard,  in  Cowdry's  Special  Cy- 
tology, 1932,  3,  1611-1629,  has  described 
this  remarkable  process  as  seen  in  the 
living  state.  "A  leucocj^te  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)  phagocyto- 
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 
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. 

Phenol  Compounds,  see  Azo  Reaction,  Indo 
Reaction. 

Phenolase,  see  Oxidase. 

Phenoloxidase,  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  cre.solphthalein. 

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. 

Phcnosulfonphthalein,  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 
methanes,  and  hydroxy  tri-phenyl 
methanes. 

Phenylene  Blue,  see  Naphthol  Blue  R. 

Phenylene  Brown,  see  Bismark  Brown  Y. 

Phloroglucin  is  1,3,5-trihydroxybenzene. 
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 
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.  sodiiun  thiosulfate 


PHLOXINE-METHYLENE  BLUE 


192 


PHOSPHOLIPID  CONTENT 


(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. 
Phosphatases.  The  following  accounts 
have  been  contributed  by  Dr.  G.  Go- 
mori  (May  7,  1946) : 

For  Acid,  phosphatase  based  on  his  paper 
in  Arch.  Path.,  1941,  32,  189: 

1.  Fix  thin  slices  of  tissues  in  ice  cold 
acetone  for  24  horns. 

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  above  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 
for  U  to  24  hours  at  37°C.: 

Molar  acetate  buffer  pH  5* 3  jjarts 

6%  lead  nitrate 1  part 

Dist.  water  add  slowly,  under  stir- 
ring   6  parte 

2%  Na  glycerophosphate  t 3  parts 

*  100  cc.  of  13.6%  CHsCOONa-SHaO  plus  50  cc.  6% 

acetic  acid, 
t  Commercial  grade  (mixture  of  alpha  and  beta  salts). 

Shake  well,  let  stand  for  a  few  hours. 
Keep  in  the  ice  box.  Before  use,  filter 
a  small  amount  and  dilute  with  2  to  3 
parts  of  dist.  water. 

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  (10  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  2h  at  37 °C.  in  the  following  mixture : 

2%  sodium  gl:,'cerophosphate .  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 (5  to  6  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  Kabat,  E.  A.  and 
Furth,J.,Am.J.  Path.,  1941, 17,  303-318. 
For  phosphatase  in  elementary  bodies 
of  vaccinia  virus,  see  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  Kidney. 

For  "localization  of  different  phos- 
phatases in  duodenal  epithelium",  see 
Deane,  H.  W.  and  Dempsey,  E.  W., 
Anat.  Rec,  1946,  94,  12-13;  "effects  of 
KCN  on  alkaline  phosphatase  activity 
in  the  kidney  and  intestine",  see  Em- 
mel,  V.  M.,  Anat.  Rec,  1946,  94,  15; 
and  for  "intracellular  distribution  of 
alkaline  phosphatase  activity  following 
various  methods  of  histologic  fixation", 
see  Emmel,  V.  M.,  Anat.  Rec,  1946, 
94,  92. 

Phosphate  Solutions.  A  method  for  the 
direct  observation  of  the  effect  of 
buffered  phospliate  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 


193 


PHYSIOLOGICAL 


Phosphomolybdic  Acid  Hematoxylin  (Mal- 

lory's,  see  McClung,  p.  406).  Fix  in 
Zenker's  fluid,  imbed  in  paraffin  and 
remove  mercury  with  iodine.  Rinse  in 
water.  Phosphomolybdic  acid  henaa- 
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 
molybdate,  3gm.;  aq.  dest.,  20  cc;  30% 
aq.  hydrochloric  acid,  20  cc;  reduces  in 
N/50  stanonus  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)  Winter  and  Smith  (L.  G., 
and  W.,  J.  Physiol.,  1922,  56,  227-231). 
See  Radiophosphorus. 

Phosphotungstic  Acid  Hematoxylin.  (Alal- 
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  C.l  gm.  hematoxylin  by  heat  in 
50  cc.  aq.  dest.,  when  cool  add  2.0  gm. 
phosphotungstic  acid  dis.solved  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 


;x»d  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- 
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- 
photungstic acid  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.  This  ap- 
paratus has  been  developed  in  The  Bar- 
nard Free  Skin  and  Cancer  Hospital  by 
Stowell,  R.  E.,  J.  Nat.  Cancer  Inst., 
1942,  3,  111-121  to  measure  the  light  ab- 
sorbed as  a  result  of  the  specific  colora- 
tion of  tissue  components.  It  consists 
of  a  lamp,  microscope,  photocell  and 
equipment  for  amplification  and  record- 
ing. The  particular  component  inves- 
tigated has  been  Thymonucleic  Acid 
as  demonstrated  in  the  epidermal  cells 
of  mice  by  the  Feulgen  reaction.  The 
method  is  one  of  wide  usefulness  and 
will  probably  be  employed  as  a  means  of 
securing  quantitative  data  from  many 
microchemical  reactions.  For  tech- 
nique of  determining  many  other  com- 
ponents 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 


194 


PIROPLASMA 


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  about  100  cc.  Transfer 
directly  to  ammoniated  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  as  mounting  media  (Wicks, 
L.  F.,  Carruthers,  C.  and  Ritchey, 
M.  G.,  Stain  Techn.,  1946,  21,  121-126. 

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, 
Vir chow'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  j 
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  tor  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  tis.sue  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. 
nigrosiue  in  sat.  aq.  picric  acid.  Muscle 
yellow,   connective   tissue   black. 

Picro-Suiphuric  fixative,  see  Kleinenberg's. 

Pigments,  general  reviews:  Bergmann,  E. 
Ergeb.  d.  Phvsiol.,  1933,  35,  158-300; 
Lederer,  E.,  Biol.  Rev.,  1940,  15,  273- 
306  (invertebrates).  See  Bacterial, 
Bile  Pigment,  Bilirubin,  Biliverdin, 
Carotin,  Carotinalbumins,  Carotinoids, 
Chromolipoids,  Cytochrome,  Hematin, 
Hematoidin,  Hematoporphyrin,  Hemo- 
fuscin.  Hemoglobin,  Hemosiderin,  Tri- 
chosiderin.  Iron  Pigments,  Lipoclirome, 
Lipofuscin  (wear  and  tear  pigment), 
Malarial,  Melanins,  Parhemoglobin, 
Porphyrins,  Rhodopsin,  Sulfmethemo- 
globin. 

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. 
Hetheringtou,  D.  C,  Stain  Techn., 
1936,  11,,  153-154,  used  pinacyanol  as  a 
supravital  stain  for  mitochondria  in 
blood  cells. 

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,  666-879)  microscopi- 
cally visible  drinking,  not  submicro- 
scopic  "sipping"  v/hich  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  ruffle  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  +  G.  plasma,  a 
formed    thing).     Piroplasmas  are   pear 


PIROPLASMA 


195 


PLASTIDS 


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 
proposed  by  Dawson ,  A .  B .  and  Fri  edgood , 
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.  H.,  Stain  Techn., 
193S,  13,  111-114  give  following  direc- 
tions to  demonstrate  2  types  of  granular 
cells  in  the  pars  nervosa.  Fix  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  gm.;  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- 
hypophj^sis  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.  Those  proposing  to  investigate 
the  placenta  would  do  well  to  consult 
the  most  recent  paper  in  a  series  dir- 
ected by  Dr.  George  B.  Wislocki  at 
Harvard  (Wislocki,  G.  B.,  Deane,  H.  W. 
and  Dempsey,  E.  W  ,  Am.  J.  Anat., 
1946,  78,  281-345).  Many  techniques, 
mostly  histochemical,  have  been 
brought  to  bear  on  this  organ  in  a  well 
developed,  long  term,  program  of  re- 
search and  the  results  obtained  thereby 
are  well  illustrated,  frequently  by  ex- 
cellent colored  figures. 

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. 

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  important  to  use  a  technique 
which  reveals  basophilia.  In  practice 
Giemsa 's  stain,  or  a  good  coloration  with 
hematoxjdin  and  eosin,  is  generally 
sufficient.  Unna  used  the  term  "plasma 
cell"  for  almost  any  kind  of  cell  with 
much  plasma  including  macrophages  so 
that  the  designation  Unna's  plasma  cell 
is  almost  meaningless. 

Plasma  Membrane,  see  Cell  Membranes. 

Plasma!  Reaction,  see  SchiflF's  Reaction. 

Plasmalogen.  A  component  of  the  cyto- 
plasm which  gives  a  positive  Feulgen 
test  (Bourne,  p.  22). 

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,  chemistry  and  physics  of  (Bartoe, 
W.  F.,  J.  Tech.  Meth.,  1940,  20,  6-11). 
In  museum  work  (Kramer,  F.  M.,  J. 
Tech.  Meth.,  1940,  20,  14-23).  As  sub- 
stitute for  cover  glasses  (Suntzeff,  V. 
and  Smith,  I.,  Science,  1941,  93,  158- 
159;  Russell,  W.  O.,  J.  Tech.  Meth., 
1942,  22,  65-70).  As  mounting  media 
(Hutner,  S.  H.,  Stain  Techn.,  1941,  16, 
177).  As  substitute  for  quartz  for  light 
conduction  (Williams,  R.  G.,  Anat. 
Rec,  1941,  79,  263-270).  As  cover 
glasses.  DeAngelis,  E.,  J.  Lab.  & 
Clin.  Med.,  1945,  30,  469-471  recom- 
mends 2,  Acryloid  B-77  and  Vinylseal. 
After  preparation  is  stained  and  passed 
through  alcohols  to  xylol,  dip  it  slowly 
into  plastic  solution,  allow  to  remain 
30  sec.  slowly  lift  out  and  drain  oft'  ex- 
cess plastic  by  placing  at  an  angle  in  a 
rack.  Repeat  if  thicker  covering  is  de- 
sired, covering  can  be  made  very  hard 
by  baking  in  oven  at  140°C.  for  1  hr. 
Many  advantages  and  some  disadvan- 
tages are  discussed  by  DeAngelis. 

Plastids  are  by  definition  simply  formed 
bodies.  The  term  is  usually  applied  to 
certain  cytoplasmic  bodies  in  plants. 
They  may   be   colorless   leucoplastids, 


PLASTIDS 


196 


PLEUROPNEUMONIA 


chloroplastids  containing  chlorophyll 
or  chromoplastids  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 
techniques  are  only  required  to  reveal 
the  organization  of  the  ground  sub- 
stance, holding  the  chlorophyll,  and 
their  rdles  in  photosj^nthesis.  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.  Total  counts  can  be  made 
in  plasma.  Walker  and  Sweeney  (T.  F. 
and  P.  A.,  J.  Lab.  &  Clin.  Med.,  1939, 
25,  103-104)  proceed  as  follows  :  Moisten 
inside  white  blood  cell  pipette  by  draw- 
ing in  and  expelling  1.1%  aq.  sodium 
oxalate.  Immediately  draw  in  fresh 
blood  to  0.5  mark,  then  oxalate  solution 
to  mark  11.  Shake  vigorously.  Place 
heavy  rubber  band  around  pipette  to 
close  ends.  Centrifuge  pipette  §  min. 
at  1,600  revolutions,  or  the  shortest  time 
to  draw  red  blood  cells  into  its  stem. 
Stand  pipette  vertically  to  permit  red 
blood  cells  to  settle  into  stem  (about  2 
hrs.).  Gently  expell  red  blood  cells  by 
blowing  and  count  platelets  in  clear 
supernatant  solution. 

Another  method  (Buckman,  T.  E. 
and  Hallisey,  J.  E.,  J.A.M.A.,  1921, 
76,  427-429)  is  to  prick  the  finger,  or 
ear,  through  a  drop  of  0.1%  brilliant 
cresyl  blue  in  physiological  saline.  The 
fluid,  plus  blood  is  mounted  and  the  num- 
ber per  red  cell  is  counted.  If  there 
is  one  platelet  per  20  reds  and  there 
are  6  million  reds  per  c.  mm.,  then  there 
are  approximately  300,000  platelets  per 
c.  mm.  of  blood  which  is  a  normal  count. 
The  number  may  exceed  1  million  in 
myelogenous  leukemia. 

A  choice  can  be  made  from  many 
platelet  staining  solutions:  (1)  Buck- 
man,  T.  E.,  and  Hallisey,  J.  E.,  J.A.M. 
A.,  1921,  76,  427:  Glucose,  6.0  gm.; 
sodium  citrate,  0.4  gm.;  aq.  dest.,  100 
cc.  Filter,  add  0.02  gm.  toluene  red 
(dimethyldiamidotoluphenazin )  then 
0.1  gm.  crystal  violet.  Heat  gently  to 
60°C.  5  min.;  cool  and  centrifuge  at 
2000  revolutions  per  min.  for  10  min. 
Filter  supernant  fluid  twice  through  2 
thicknesses  fdter  paper.  Preserve  solu- 
tion by  adding  0.2  cc.  formaldehyde. 
(2)  Kristenson,  A.,  Acta  Med.  Scan- 
dinav.,  1922,  57,  301:  Urea,  10  gm.; 
sodium  citrate,  2.5  gm.;  corrosive  sub- 
limate, 0.005  gm. ;  brilliant  cresyl  blue, 
0.5  gm.;  and  aq.  dest.,  500  cc.  (3) 
Ottenberg,  R,  and  Rosenthal,  N.,  J.A. 


M.A.,  1917,  69,  999:  3%  aq.  sodium 
citrate  to  which  1:500  cresyl  blue  or 
1 :500  methyl  violet  is  added  and  filtered 
before  staining.  (4)  Pratt,  J.  H.,  J.A. 
M.A.,  1905,  45,  1999:  Sodium  citrate, 
3.8 gm. ;  aq.  dest.,  100  cc. ;  formaldehyde, 
0.2  cc;  brilliant  cresyl  blue  0.1  gm. 
(5)  Wright,  J.  H.  and  Kinnicutt,  R., 
J.A.M.A.,  1911,  56,  1457:  A.  Brilliant 
cresyl  blue,  1  gm.;  aq.  dest.,  300  cc. 
B.  Potassium  cyanide,  1  gm.;  aq.  dest., 
1400  cc.  Keep  A  in  ice  box.  For  use  2 
parts  of  A  and  3  parts  of  B  are  mixed  and 
filtered. 

A  differential  platelet  count,  in  which 
4  classes  are  recognized,  is  described  by 
Olef  (I.,  Arch.  Int.  Med.,  1936,  57, 
1163). 

Platelets.  These  can  best  be  seen  in  the 
dark  field  in  mounts  of  fresh  blood  and 
of  fresh  blood  first  treated  with  Anti- 
coagulants." The  contrast  between  the 
two  is  instructive.  It  is  important  to 
remember  that  when  held  under  ob- 
servation in  preparations  sealed  with 
vaseline  for  a  considerable  time,  plate- 
lets may  become  elongated  and  exhibit 
a  superficial  resemblance  to  parasites. 
Data  on  the  rate  of  disintegration  of 
platelets  are  provided  by  Olef,  I.,  J. 
Lab.  &  Clin.  Med.,  1936-37,  22,  128- 
146.  In  blood  regeneration  atypical 
platelets  may  be  encountered  measur- 
ing as  much  as  25-50  m  in  length  (Tocan- 
tins,  M.,  Medicine,  1938,  17,  175-258). 
The  contained  granules  are  easily 
stained  supravitally  and  in  smears. 
Excellent  coloration  of  platelets  in  sec- 
tions are  given  by  Wrights  and  Kings- 
ley's   methods    (see  Megakaryocytes). 

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. 

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. 


PLEUROPNEUMONIA 


197 


POLiVRIZATION  OPTICAL 


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. 

Polarization  Optical  Method. — Written  by 
Francis  O.  Schmitt,  Dept.  of  Biology, 
Massachusetts  Institute  of  Technology, 
Cambridge,  Mass.,  May  28,  1946.— 
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  (1),  Frey-Wyssling 
(2)  and  Schmitt  (3,  4). 

The  polarizing  microscope  's  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,  A^e  and  A^o- 
It  is  usually  determined  by  the  use  of 
a  compensator  which  measures  the 
phase  difference  expressed  as  fractional 
wavelength,  6,  or  retardation,  r,  ex- 
pressed in  mju.  Thickness  of  the  speci- 
men, d,  is  also  expressed  in  m/x.    Then 

u-    i-  •  ^'         AT         o\        r 

birefringence  =   rMo  —  No  =  -t-=j. 

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  diagf 
nosing  the  ultrastructure  of  biologica- 


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 
or  muscle  will  appear  blue  in  one  diag- 
onal position  and  yellow  in  the  diagonal 
perpendicular  thereto;  this  is  because 
these  fibers  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 v/hich  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.,DicDoppelbrechungvon 
Karyoplasma,  Zytoplasma  und  Meta- 
plasma,  Berlin  Geb.  Borntrager,  1937. 


POLARIZATION  OPTICAL 


198 


PONTACYL  CARMINE  6B 


Frey-Wyssling,  A.,  Submikroskopische 
Morphologie  des  Protoplasmas  und 
seiner  Derivate,  Berlin  Geb.  Borntra- 
ger,  1938.  Schmitt,  F.  O.,  The  ultra- 
structure  of  protoplasmic  constituents. 
Physiol.  Rev.,  1939,  19,  270,  Schmitt, 
F.  O.,  Tissue  ultrastructure  analysis: 
polarized  light  method.  In  Glasser's 
Medical  Physics,  1944,  p.  1586.  See 
Nerve  Fibers,   Muscle  Fibers. 

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  (Monne,  L.,  Pro  to - 
plasma,  1939,  32,  184-192). 

Polarizing  Microscope.  Any  ordinary 
microscope  can  be  adjusted  for  crude 
polarization  studies  by  use  of  a  polarizer 
and  analyser.  See  Polarization  Optical 
Methods. 

Polaroid.  This  is  a  polarizing  material 
made  up  of  extremely  minute  crystals 
of  quinine  sulphate  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  ig  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  by 
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.  Literallj' 
many  colored,  but  actually  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  polychrome  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. 

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  used  by  Leach,  E.  H., 
Stain  Techn.,  1946,  21,  107-119  in  Cur- 
tis' Substitute  for  Van  Gieson  Stain. 

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.  40j. 

Pontacyl  Carmine  6B  (CI,  57),  DuPont,  is  an 
acid,  monoazo  dye  which  colors  sections 


PONTACYL  CARMINE  6B 


199 


PORPHYRINS 


bluish  fuchsia  darkened  by  mordanting 
with  potassium  bichromate.  Not  im- 
portant in  microtechnique  (Emig, 
p.  oG). 

Pontacyi  Carmine  2  G  (CI,  31)— Made  by 
DuPont.  Light  fastness  3.  More  in- 
tense color  than  azofuchsin.  Action  on 
fungous  mycelia    (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, 
()5,  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,  University  of  Mary- 
land Medical  ISchool,  Baltimore,  Md. 
1916— 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  be- 
lieve that  it  is  the  protoporphyrin  in 
reticulocytes  that  stains  with  brilliant 
cresyl  blue.  They  have  demonstrated 
that  this  dye  and  protoporphyrin  are 
mutual  precipitants  (see  Reticulocytes) . 
Minute  quantities  of  porphyrins  may  be 
detected  in  tissues  or  solutions  by  vir- 
tue 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  Chemic  des  Pyrrols,  1937, 
press  of  Paul  Dunhaupt,  Kothen.  It  is 
also  available  in  Lithoprint  form:  Ed- 
wards Bros.,  Ann  Arbor,  Mich.).  At- 
tem^pts  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  porphyrins  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  substanecs 


which  quench  the  fluorescence  of  the 
porphyrin,  i.e.,  protoporphyrin  and 
coproporphyrin  are  abundant  in  bone 
marrow,  but  the  fluorescnce  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  chein- 
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- 
phyrin III,  and  mesoporphyrin  may  all 
be  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"). 


PORPHYRINS 


200 


POTASSIUM 


Normally  20-100  micrograms  of  co- 
proporphyrin  I  are  excreted  daily  in  the 
urine.  Coproporphyrin,  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 
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- 
12S).  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  Pillet,  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  pai-affin  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  parafl[in  sections  as  for  Chloride, 
which  see.  Transfer  these  to  a  fairly 
large  cool  room  (—1°  to  -fl°C.)  and  re- 
move paraffin  and  petroleum  ether  as 
for  chloride.    Cover  with  12%  sodium 


POTASSIUM 


201 


phostate 


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.  SI).  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 
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  37°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. 

Preputial  Gland  of  rats.  Useful  histochemi- 
cal  methods  of  investigation  and 
changes  following  thyroidectomy  (Mon- 
tagna,  W.,  Anat.  Rec,  1948,  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. 

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. 

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). 

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- 
factorjf  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., 
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). 


PROSTATE 


202 


PROTOZOA 


Examination  of  corpora  amylacea  by 
various  methods  is  described  by  Moore, 
R.  A.,  Arch.  Path.,  1936,  22,  24-40. 

Protactinium,  see  Atomic  Weights. 

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,  "l,  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. 

Protease.  A  proteolytic  leucocytic  enzyme 
which  can  be  demonstrated  by  a  special 
method  in  very  small  amounts  of  blood 
(Cooke,  J.  v.,  1932,  49,  836-845).  A 
micromethod  for  protease  is  described 
by  Pickford  and  Dorris  (G.  E.and  F., 
Science,  1934,  80,  317-319). 

Protein,  see  following  reactions :  Alloxan, 
Axenfeld,  Azo,  Indo,  Ninhydrin,  Nitro, 
Nitroprusside,  Nitrosamino,  Romieu, 
Xanthroproteic. 

Proteinase,  determinations  (Maver,  M.  E., 
Mider,  G.  B.,  Johnson,  J.  M.  and 
Thompson,  J.  W.,  J.  Nat.  Cancer  Inst., 
1941,  2,  278). 

Prothrombin,  rapid  micro  test  (Abramson, 
D.  J.  and  Weinstein,  J.  J.,  Ajq.  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  thi"omboplas- 
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). 

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  50  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- 
von,  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 


PROTOZOA 


203 


PULP  OF  TEETH 


by  Wenrich,  D.  H.,  J.  Parasitol.,  1911, 
27,  1-28  of  alterations  in  the  form  of 
protozoa  resulting  from  variations  in 
microtechnique. 

Protozoa  can  be  beautifully  demon- 
strated by  fiuorochromes  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. 
Protozoa.  Media.  The  following  are  rec- 
ommended for  intestinal  protozoa  by  Q. 
M.  Geiman  (Simmons  and  Gentzkow, 
617-619) : 

1.  Modification  of  Cleveland's  and 
Sanders'  (for  E.  histolytica).  (1)  Dis- 
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  18  X  150  mm.  culture  tube  and  steril- 
ize with  tube  horizontal  in  hot  air  oven 
160-1S0°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.  Na.HPOi  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  -f  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 
I  to  \  with  20:1  sterile  Ringer-horse 
serum  mixture.  Smaller  quantity  for 
intestinal  flagellates,  larger  quantity  for 
TricJiomonas  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  fcrrocy- 
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  witliin  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- 


l^tJLP  OF  TEETH 


204 


PYRROL  COMPOUNDS 


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.  Silver  methods  for  histochemical 
detection  are  according  to  Lison  (p. 
185)  absolutely  useless. 

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 
cannulas,  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  tri chrome 
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  madder-stained 
bones  is  due  to  purpurin  carboxylic  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  otliers 
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. 

Pyoktanin  Yellow,  see  Auramin. 

Pyoktaninum   Aureum,   see   Auramin. 

Pyoktaninum  Coeruleum,  see  Methyl  Vio- 
let. 

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  diamino  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  red  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  (collodion  cotton,  colloxylin, 
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. 


"QUAX)"  STAIN 


205 


QUARTZ  ROD  TECHNIQUE 


"Quad"  Stain.  A  recent  modification  of 
this  excellent  orcein-aiizarine-Orange 
Gphosphotungstic  and  phosphormolyb- 
dic  acid  technique  is  given  in  detail 
by  Kornhauser,  S.  I.,  Stain  Techn., 
1945,  20,  33-35. 

Quartz  Rod  Technique  for  illuminating  liv- 
ing organs. — Written  by  Dr.  M.  H. 
Knisely,  Department  of  Anatomy,  Uni- 
versity of  Chicago,  September  6,  1946 — 
The  general  purpose  of  this  technique  is 
to  permit  direct  microscopic  study  of 
living  internal  organ  in  situ  while  main- 
taining experimental  conditions  which 
disturb  the  structures  and  processes  to 
be  observed  as  little  as  possible.  Like 
all  techniques  it  has  advantages  and 
limitations;  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  magnifica- 
tion those  living  structures  whose 
colors  and/or  indices  or  refraction  differ 
from  those  of  adjacent  structures. 
With  quartz  rods  we  can  illuminate  for 
examination  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 
fieut  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  sinmltaneously  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  bods'"  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- 
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 


QUARTZ  ROD  TECHNIQUE 


206 


QUARTZ  ROD  TECHNIQUE 


was  maintained  normal  by  a  continu- 
ously flowing  solution.  In  the  best 
experiments  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  Kni.seiy,  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  w'here- 
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., 
Stratrnan-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  iis  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  j'et  unknown,  or  one  or  more 
phases  of  the  particular  functions  one 
is  trying  to  stud3^  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  Idnds 
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- 
tions. For  some  detailed  descriptions 
of  complex  integrated  vascular 
reactions  see  Knisely,  M.  H.,  Bloch, 
E.  H.,  and  Warner  L.,  K.  Danske-viden- 
skabernesselskab.  Biologiske  skrifter. 
1947,   4.-    (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 


QUARTZ  ROD  TECHNIQUE 


207 


QUARTZ  ROD  TECHNIQUE 


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  sj'stem  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 
woridng  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,  tha  higher  the  mag- 
nifications used  the  more  closely  are 
observations  restricted  toward  surface 
struct ui'es.    The  natural  transparency 


or  translucency  of  the  tissues  also  limits 
the  depth  of  observations.  Some  curi- 
ous eifects  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  600J, 
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.  11.,  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- 
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  manj'  problems  including:  How 
are  the  bodies  of  the  adults  of  each 
species  constructed?  How  does  each 
body  develop?  How  does  it  change 
witli  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- 


QUARTZ  ROD  TECHNIQUE 


208 


QUARTZ  ROD  TECHNIQUE 


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  ortjanized  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,  289-274,  Stain  Techno!. 
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 
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  (Kniseiy,  M.  H.,  Eliot, 
T.  S.,  and  Bloch,  E.  H.,  1945;  Kniseiy, 
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 


QUARTZ  ROD  TECHNIQUE 


209 


QUARTZ  ROD  TECHNIQUE 


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  100  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  possible  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  yet  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.,  Ztschr.  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 
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.  II.,  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 


QUARTZ  ROD  TECHNIQUE 


210 


RADIOACTIVE  ISOTOPES 


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  belittle  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  accuracy  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  as  tracer  substances 
(from  Dr.  W.  L.  Simpson  of  The  Bar- 
nard Free  Skin  and  Cancer  Hospital). 

In  the  20  years  that  have  elapsed  since 
Hevesy  first  used  a  radioactive  isotope 
of  lead  to  trace  the  lead  metabolism  of 
plants,  advances  in  nuclear  physics  have 
made  available  to  biologists  materials 
that  appear  to  open  up  new  approaches 
to  a  variety  of  problems  limited  only  by 
the  ingenuity  of  the  investigator  and 
the  availability  of  the  tracer  substances 
he  desires.  Discovery  of  the  phe- 
nomenon of  artificial  radioactivity  in 
1934  by  I.  Curie  and  F.  Joliot  and  the 
development  of  the  cyclotron  by  E.  O. 
Lawrence  and  his  associates  at  the  Uni- 
versity of  California  are  acknowledged 
generally  to  be  the  chief  factors  that 
have  produced  these  important  ad- 
vances. 

The  assumption  is  made  that  an  iso- 
tope is  accepted  by  tissues  without 
discrimination,  and  that  its  distribu- 
tion, metabolism,  and  elimination  will 
be  the  same  as  that  of  the  non-radioac- 
tive form  of  the  element.  This  appears 
valid  except  perhaps  for  the  lightest 
elements  in  which  relatively  great  dif- 
ferences of  atomic  weight  exist  between 
the  radioactive  and  the  stable  isotopes. 
Although  radiations  from  large  (thera- 
peutic) doses  of  some  isotopes  do  exert 
profound  effects  on  tissues,  the  concen- 
tration of  those  employed  as  tracer  sub- 
stances is  usually  so  low  (often  less  than 
one  part  to  several  billion  of  the  stable 
isotope)  that  no  tissue  changes  can  rea- 
sonably be  attributed  to  the  radiation 
accompanying   their  decay. 

Less  than  5%  of  the  cyclotron  pro- 
duced radioactive  isotopes  have  been 
employed  in  biological  studies.  Among 
the  limitations  to  their  use  are  the  fol- 
lowing :  (1 )  They  are  sometimes  difficult 
to  obtain.  Isotopes  that  decay  rapidly 
are  available  only  to  experimenters 
near  the  cyclotron.  (2)  The  rate  of 
decay  of  unstable  isotopes  must  be  slow 
enough  to  permit  the  measurement  of 
their  radiation  at  the  end  of  an  experi- 
ment. While  larger  quantities  can  be 
employed  to  offset  rapid  decay,  a  limit 
is  soon  reached  beyond  which  further 
increases  in  concentration  is  either  not 
possible  because  of  difficulties  in  pre- 
paring them  or  is  not  desirable  because 
of  the  effects  produced  by  radiation  of 
tissues.  The  length  of  experiment 
should  not  be  longer  than  5  or  6  times  the 
half  life  of  the  element  used.  (3)  The 
form  in  which  the  radioelement  is  de- 
sired places  a  limit  on  some  investiga- 
tions.    Since    they    are    usually    pre- 


RADIOACTIVE  ISOTOPES 


211 


RADIOCALCIUM 


pared  from  pure  elements  or  simple 
compounds,  the  use  of  elements  in  com- 
plex forms  is  limited  by  the  amount  of 
sj'nthesis  that  can  be  accomplished.  In 
some  cases  synthesis  of  complex  organic 
compounds  can  be  carried  out  most 
readily  by  the  introduction  of  simple 
radioactive  salts  into  an  animal  or  plant 
and  the  subsequent  recovery  from  the 
organism  of  complex  substances  that 
contain  the  radioelements  incorporated 
in  their  structure. 

Three  methods  of  detection  of  the 
radioactive  isotopes  are  commonly  used  : 

1.  In  vitro  method:  Most  common  is 
the  measurement  of  the  radiation  from 
the  isotope  with  either  a  Geiger-Miiller 
counter  or  an  electroscope.  Tissues  to 
be  examined  are  either  ashed  arid 
measured  or  extracted  and  measured  in 
solution.  The  Geiger-Muller  counter 
is  extremely  sensitive  but  only  gross 
tissue  localization  is  possible,  since  rela- 
tively large  amounts  of  tissue  must  be 
extracted. 

2.  In  vivo  method:  Detection  and  lo- 
calization of  some  isotopes  that  emit 
penetrating  Gamma  rays  are  feasible 
within  the  living  body  by  placing  a 
shielded  Geiger-Muller  counter  against 
the  body  so  that  it  will  receive  rays  from 
restricted  areas.  Thus  Hamilton  has 
studied  the  accumulation  of  radioiodine 
in  the  thyroid  gland. 

3.  Autoradiography  (or  radioautog- 
raphy):  Known  since  1924  (Lacassagne, 
A.  and  Lattes,  J.  S.,  C.  rend.  soc.  d. 
biol.,  1924,  90,  352-353;  C.  rend.  d. 
I'Acad.  d.  sc,  1924,  178,  488-490)  this 
technique  secures  on  photographic  emul- 
sions images  representing  the  location  of 
radioactive  elements  in  tissue  and  organ 
slices  that  have  been  held  in  contact 
with  photographic  films.  Photographic 
records  of  sections  of  fixed  tissues  con- 
taining radioelements  can  be  made  by 
simply  laying  the  mounted  unstained 
sections  on  a  photographic  plate  and 
leaving  them  until  adequate  exposures 
are  obtained.  Subsequently  sections 
are  stained  for  comparison  with  the 
silver  deposit  on  the  developed  plate. 
See  distribution  of  thorium  B  (a  lead 
isotope)  in  animal  tissues  by  B.  Behrens 
and  A.  Baumann  (Zeits.  f.  d.  ges. 
exper.  med.,  1933,  92,  241-250).  In- 
teresting studies  ha,ve  been  carried  out 
also  by  J.  G.Hamilton  on  the  localization 
of  radioiodine  in  normal  and  enlarged 
thyroid  glands.  The  deposition  of 
radiophosphorus  and  radiostrontium  in 
bones  and  osteogenic  tumors  has  been 
autoradiographed  by  Treadwell,  Low- 
Beer,  Friedell  and  Lawrence.  Radio- 
phosphorus  distribution  in  leaves  and 
fruit  of  plants  has  been  studied  by 
Aruon,  D.  J.,Stout,  P.  R.,andSipos,  F., 


Am.  J.  Botany,  1940,  27,  791.  Accord- 
ing to  Hamilton  J.  G.  (Radiology,  1942, 
39,  541-572),  Lindsey  and  Craig  have 
proved  that  the  method  is  valuable  in 
the  study  of  phosphorus  distribution  in 
insect  larvae.  Gorl)inan,  A.  and  Evans, 
H.  L.,  Proc.  Soc.  Exper.  Biol.  &  Med., 
1941,  47,  103  have  similarly  determined 
the  time  in  embryonic  development 
when  the  thyroid  first  accumulates 
iodine. 

Space  does  not  allow  further  review 
of  the  many  problems  tliat  can  be  in- 
vestigated using  radioactive  tracer  sub- 
stances. See  Hevesy,  G.,  Ann.  Rev. 
Biochera.,  1940, 9,  641-662 and  Hamilton, 
J.  G.,  J.  Appl.  Physics,  1941,  12,  440- 
460  and  Radiology,  1942,  39,  541-572. 
Theoretical  considerations  are  discussed 
by  Hevesy,  G.  and  Paneth,  F.  A.,  "A 
Manual  of  Radiology,"  2nd  edition, 
London :  Oxford  Univ.  Press.,  1938, 
and  in  a  popular  review  of  the  develop- 
ment of  the  cyclotron  by  Abersold, 
Paul  C,  Radiology,  1942,  39,  513-540. 
For  literature  on  nearly  400  radioactive 
isotopes  see  Seaborg,  G.  T.,  Chem.  Rec, 
1940,  27,  199-285.  The  effects  of  radio- 
elements  on  growth  of  cells,  tissues, 
and  organisms  have  been  considered 
by  Haven,  F.  L.,  and  Hodge,  H.  C, 
Growth,  1941,  5,  257^266.  The  Annual 
Reviews  of  Biochemistry,  volumes  8  to 
11  contain  many  data  on  the  use  of  tracer 
substances.  See  review  of  mineral 
metabolism  by  Greenberg,  D.  M.,  Ann' 
Rev.  Biochem.,  1939,  8,  269-300. 

Radioarsenic  (As™)  half  life  26.8  hrs. 
Used  as  a  tracer  for  the  distribution  of 
sodium  dihydrogen  arsenate  in  rabbit 
tissues  by  duPont,  O.,  Ariel,  I.  and 
Warren,  S.  L.,  Am.  J.  Syph.,  Conor, 
and  Ven.  Dis.,  1942,  26,  96-118.  Highest 
concentrations  appear  in  liver,  kidney, 
and  lungs.  In  lower  concentration  it 
is  found  in  muscle,  bone,  and  skin. 
Browne-Pearce  tumor  tissue  takes  sig- 
nificant amounts  but  loses  them  within 
4  days.  Elimination  is  chiefly  by 
kidneys. 

Radiobromire  (Br^z)  half  life  34  hrs.  Perl- 
man,  I.,  Morton,  M.  E.  and  Chaikoff, 
I.  L.,  Am.  J.  Physiol.,  1941,  134,  107-113 
followed  the  uptake  of  very  small  doses 
of  radiobromine  by  various  tissues  of 
rat  and  guinea  pig.  Highest  concen- 
trations appear  in  thyroids  in  both 
normal  animals  and  in  animals  with 
thyroids  made  hyperplastic  by  the 
pituitary  thyrotropic  hormone. 

Radiocalcium  (Ca^^)  half  life  180  days. 
Stored  almost  entirely  in  bone.  Only 
small  traces  are  found  in  other  tissues 
(Campbell,  W.  W.  and  Greenberg,  D. 
M.,  Proc.  Nat.  Acad.  Sci.,  1940,  26, 
176-180  and  Pecher,  C,  Proc.  Soc. 
E.xper.    Biol.    Med.,    1941,    46,  86-91). 


RADIOCALCIUM 


212 


RADIOIODINE 


Pecher  also  predicted  that  strontium  is 
handled  in  the  body  in  the  same  fashion 
as  shown  in  his  experiments.  Long  half 
life  makes  this  element  rather  difficult 
to  work  with. 

Radiocarbon  (C")  half  life  21  min._  Short 
life  makes  use  difficult  in  many  investi- 
gations. In  spite  of  this  handicap,  S. 
Ruben,  M.  D.  Kamen  and  their  co- 
workers have  used  radiocarbon  to  study 
CO2  metabolism  and  photosynthesis  in 
a  wide  variety  of  lower  animals  and 
plants.  Their  findings  on  the  nature  of 
photosynthesis,  at  variance  with  the  long 
accepted  view,  afford  a  nice  illustration 
of  the  manner  in  which  well  planned 
experiments  with  the  radioisotopes  can 
support  or  dispel  classical  assumptions. 
They  showed,  for  instance,  that  chloro- 
phyll containing  plants  can  assimilate 
radiocarbon  dioxide  in  the  absence  of 
light  and  convert  it  to  a  carboxylicacid 
radical  attached  to  a  particle  of  high 
(approximately  1090)  molecular  weight. 
The  process  is  limited  in  the  absence  of 
light,  but  in  the  presence  of  light  as- 
similation continues  with  a  photosyn- 
thetic  reduction  of  the  carboxylic  acid 
radical  to  an  alcohol  group  with  the 
liberation  of  oxygen .  This  newly  formed 
alcohol  radical  accepts  CO2  in  another 
non-photosynthetic  reaction.  Succes- 
sive alternate  photosynthetic  and  non- 
photosynthetic  reductions  lead  to  the 
building  of  longer  carbon  chain  radicals 
on  the  large  enzyme  molecule.  Pre- 
sumably these  chains  eventually  split 
off  as  simple  sugars,  etc.  See  Ruben 
S.,  Hassid,  W.  Z.  and  Kamen,  M.  D 
(J.  Am.  Chem.  Soc,  1939,  61,  661 
1940,  62,  34-13),  Ruben,  S.,  Kamen,  M 
D.,  Perry,  L.  H.,  ibid,  1940,  62,  3450 
Ruben,  S.  and  Kamen,  M.  D.,  ibid,  p 
3451.  Kamen,  M.  D.  and  Ruben,  S. 
J.  Appl.  Physics,  1941,  12,  310A  suggest 
the  possibility  of  i?i  vivo  synthesis  of 
sugars, acetic  acid,  etc.  from  radiocarbon 
as  a  means  of  obtaining  radioactive  sub- 
stances that  are  too  complex  to  be 
synthesized  in  the  laboratory  in  the 
time  available  during  the  useful  life  of 
radiocarbon. 

Radiocarbon  (C^^)  half  life  estimated  to  be 
over  1000  years.  Very  small  quantities 
are  produced  but  specific  activity  is 
high.  It  may  be  useful  for  stud}'-  of 
some  biological  problems. 

Radiochlorine  (Cps)  half  life  37  min.  Used 
chiefly  to  investigate  rate  of  chloride  ion 
penetration  into  various  tissues  (Man- 
ery,  J.  F.  and  Haege,  L.  F.,  Am.  J. 
Physiol.,  1941,  134,  83-93).  The  per- 
meability of  human  erythrocytes  to 
radiochloride  ion  lias  been  determined 
and  compared  with  permeability  toother 
non-radioactive  ions  by  Smith,  P.  K., 
Eisenmann,  A.  J.  and  Winkler,  A.  W., 


J.  Biol.  Chem.,  1941,  141,  555-561.  A 
complete  exchange  between  radio - 
chloride  ions  of  serum  and  erythrocytes 
was  found  within  less  than  10  min. 

Radiocobalt  (Co")  half  life— 270  days. 
Little  work  has  been  done  with  this 
isotope.  Copp,  D.  H.  and  Greenberg, 
D.  M.,  Proc.  Nat.  Acad.  Sci.,  1941,  27, 
153-157  report  on  the  distribution  of 
minute  doses.  The  bulk  of  ingested 
radiocobalt  is  rapidly  excreted.  Less 
than  5%  is  retained  after  4  days.  This 
fraction  is  found  chiefly  in  pancreas, 
kidney,  spleen,  and  liver.  The  inter- 
esting question  is  raised  of  a  possible 
relation  between  cobalt  retention  in  the 
pancreas  and  the  association  of  cobalt 
with  insulin. 

Radiocopper  (Cu^'»)  half  life  13  hrs.  Dis- 
tribution in  blood  serum  and  red  cells 
has  been  briefly  reported  by  Yoshikawa, 
H.,  Hahn,  P.  F.  and  Bale,  W.  F.,  Proc. 
Soc.  Exper.  Biol.  Med.,  1942,  49,  285- 
289,  and  J.  Exper.  Med.,  1942,  75, 
489-494.  A  peak  concentration  is 
reached  in  plasma  2  to  5  hrs.  after  inges- 
tion when  it  falls  off  rapidly.  The  con- 
centration in  red  cells  continues  to 
increase  over  2  days. 

Radioelement  85  (ekaiodine,  85-")  half  life 
71  hrs.  This  element,  which  does  not 
occur  naturally  in  anj'^  known  form,  has 
been  used  by  Hamilton,  J.  G.  and  Soley, 
M.  H.,  Proc.  Nat.  Acad.  Sci.,  1940, 
26,  483-489)  as  a  heavy  homologue  of 
iodine  in  their  studies  on  thyroid. 
General  behavior  resembles  iodine  in 
thyroid. 

Radiofluorine  (Pi^)  half  life  112  min. 
Volker,  J.  F.,  Sagnnaec,  R.  F.  and 
Bibby,  B.  G.,  Am.  J.  Physiol.,  1941, 132, 
707-712  studied  distribution  in  rats  and 
cats  after  intravenous  and  intra- 
peritoneal injection  of  radiofluorine  salts. 
Blood  concentration  falls  rapidly  as  the 
concentration  in  calcium  containing 
tissues  rises.  Ultimate  concentration 
in  calcified  tissues  is  in  proportion  to 
their  vascularity. 

Radioiodine  (I"0  half  life  8  days.  A  com- 
parison of  the  rate  of  uptake  of  radio - 
iodine,  radiosodium,  radiopotassium, 
radiochloride,  and  radiobromide  has 
been  made  in  normal  human  subjects 
by  Hamilton,  J.  G.,  Am.  J.  Physiol., 
1938,  124,  667-678.  The  rateof  gamma 
ray  emission  from  the  hand  is  followed 
after  the  subjects  receive  the  salts  by 
mouth.  This  indicates  the  rate  at 
which  the  radiosalt  enters  the  circula- 
tion. Peak  absorption  is  reached  within 
1^-2  hrs.  for  all  except  radiopotassium. 
Its  absorption  continues  to  rise  slowly 
for  4 J  hrs. 

The  metabolism  of  iodine  by  the 
thyroid  gland  in  various  physiological 
and  pathological   conditions   has   natu- 


RADIOIODINE 


213 


RADIOPHOSPHORUS 


rally  attracted  much  attention.  Hertz, 
S.,  Roberts,  A.  and  Evans,  R.D.,Proc. 
Soc.  Exper.  Biol.  Med.,  1938,  38,  510- 
513  first  demonstrated  the  rapid  uptake 
of  radioiodine  in  the  thyroid  gland. 
Increase  of  the  radiosalt  is  even  more 
rapid  in  hyperplastic  thyroids.  Nu- 
merous studies  on  thj'roids  of  humans 
have  been  reported  by  Hamilton,  J.  G. 
and  Soley,  M.  H.,  Am.  J.  Physiol.,  1939, 
127,  557-572  and  ibid,  1940,  131,  135- 
143.  Much  of  this  work  was  done  on 
the  living  glands  by  measuring  the 
gamma  radiation  from  the  neck  region 
over  the  thyroids  by  a  Geiger-Miiiler 
coimter.  These  same  workers,  with  the 
cooperation  of  Eichoru,  K.  B.,  Univ. 
Calif.  Publ.  Pharmacology,  1940,  1, 
339-367  demonstrated,  by  means  of  the 
technique  of  autoradiography,  that  radio- 
iodine  is  deposited  in  the  colloid  of 
normal  and  hyperplastic  thj^roids.  The 
storage  is  markedly  lower  in  the  colloid 
of  nontoxic  goiter,  and  almost  no  radio- 
iodine  enters  cancerous  thyroid  tissue. 
Mention  has  already  been  made  of  the 
work  of  Gorbman  and  Evans  on  deter- 
mination by  autoradiography  of  time  of 
first  storage  of  radioiodine  in  the  thy- 
roids of  embryonic  frogs. 
Radiciron  (Fe^s)  half  life  47  days.  Yield 
very  low.  (Fe^^)  with  a  half  life  of  4 
yrs.  has  not  been  much  used  as  yet  in 
biological  problems.  What  follows  re- 
lates   to   Fe^^ 

Whipple  and  his  associates  have 
availed  themselves  of  radioiron  to  show 
that  iron  metabolism  is  controlled  by 
the  rate  of  absorption  of  the  iron  salts 
from  the  intestine  and  not  by  the  rate 
of  elimination  of  the  iron  already  in  the 
tissues.  A  higher  proportion  of  ab- 
sorbed iron  goes  into  the  formation  of 
hemoglobin  in  anemic  animals  than 
in  normal  ones.  The  rate  of  absorp- 
tion from  the  intestine  seems  to  be 
controlled  bj^  the  iron  content  of  the 
tissues,  especially  of  the  intestinal 
mucosa,  and  not  by  degree  of  anemia 
per  se.  See  numerous  papers  by  Whip- 
ple, G.  H., Bale,  W.  F.,  Lawrence,  E.  0., 
Hahn,  P.  F.  et  al.,  chieflv  in  the  J. 
Exper.  Med.,  1938-1941,  much  of  which 
is  confirmed  by  Austoni,  M.  E.  and 
Greenberg,  D.  M.,  J.  Biol.  Chem., 
1940,  134,  27-41  who  also  demonstrate 
that  the  muscles  serve  as  an  important 
storehouse  for  iron  in  anemia. 

Use  of  erythrocytes  "tagged"  with 
radioactive  iron  opens  up  a  new  approach 
to  many  baffling  problems  in  hematology. 
One  recent  investigation  employing 
such  labeled  red  cells  is  that  of  Chapin, 
M.  A.  and  Ross,  J.  F.,  Am.  J.  Physiol., 
1942.  137,  447-155  who  checked  the 
values  for  true  red  cell  volume  using 
"tagged"    erythrocytes    in    comparison 


with  results  from  dye  dilution,  protein 
dilution,  and  the  hematocrit.  The 
technique  of  measuring  the  activity  of 
such  red  cells  is  described  by  Ross,  J. 
F.  and  Chapin,  M.  A.,  Rev.  Sci.  Instr., 
1942,  13,  77-80.  Erythrocytes  can  also 
be  labeled  with  radiophosphorous. 

Radiomanganese  (Mn^<)  half  life  310  days. 
Not  much  use  has  been  made  of  this 
isotope  to  date  but  Greenberg,  D.  M. 
and  Campbell,  W.  W.,  Proc.  Nat.  Acad. 
Sci.,  1940,  26,  448-452  have  observed 
that  90%  of  ingested  radiomanganese 
is  eliminated  in  the  feces  within 75  hrs. 
The  highest  retention  is  in  the  liver, 
bones,  and  muscles. 

Radionitrogen  (N^s)  half  life  9.93  min. 
Short  useful  life  limits  study  to  experi- 
mental procedures  that  are  completed 
within  approximately  1  hr.  Ruben,  S., 
Hassid,  W.  Z.  and  Kamen,  M.  D., 
Science,  1940,  91,  578  have  found  that 
it  enters  into  the  complex  compounds 
within  barley  plants  which  live  in  air 
containing  it.  Whether  this  represents 
nitrogen  fixation  by  a  non-leguminous 
plant,  or  a  simple  exchange  between 
the  radionitrogen  and  ordinary  nitro- 
gen within  the  plant,  is  not  established 
as  yet. 

Radiophosphorus  (P^^)  j^^jf  jjfg  14  3  days. 
Employed  more  extensively  than  any 
other  isotope,  radiophosphorus  is  rather 
easy  to  prepare  and  its  useful  life  is 
long  enough  to  permit  most  experi- 
mental procedures,  and  short  enough 
to  allow  ready  detection  with  the  Geigcr- 
Miiller  counter  or  photographic  plate  of 
radiation  from  its  decay.  Since  no 
gamma  rays  arc  given  off  during  its 
decay  radiophosphorus  can  not  be  de- 
tected in  the  intact  organism,  except 
in  the  skin.  Also  encoui'aging  its  use 
is  the  fact  that  phosphorus  plaj's  such 
an  extensive  role  in  the  compounds 
found  in  living  organisms.  In  deciding 
whether  to  employ  radiophosphorus  it 
IS  helpful  to  bear  in  mind  the  kinds  of 
work  in  which  it  has  already  proved 
useful. 

Chiewitz,  O.  and  Hevesy,  G.  (Nature, 
1935,  136,  754)  were  the  first  to  use 
artificially  produced  radioactive  isotopes 
in  biological  research.  Initial  studies 
were  devoted  to  investigation  of  metabo- 
lism of  P^2  in  rats.  Absorption  and 
excretion  of  P'^  in  experimental  animals 
and  humans  have  been  studied  by  Hevesy 
and  coworkers,  Lawrence  and  associates, 
and  Greenberg  and  Cohn.  At  least 
70%  of  ingested  P^^  (as  inorganic  phos- 
pliates)  is  absorbed  from  intestine  when 
fed  to  a  fasting  subject.  The  balance 
is  excreted  in  feces.  Glucose  and 
neutral  fat  enhance  absorption.  In- 
travenous disodium  phosphate  con- 
taining tracer  quantities  of  P'^  in  the 


RADIOPHOSPHORUS 


214 


RADIOSODIUM 


phosphate  ion:  4-23%  is  eliminated  in 
24  hrs.  in  urine  and  feces.  Rate  falls 
to  less  than  1%  per  day  after  3d  day. 
The  retention  of  radiophosphorus  varies 
in  different  tissues.  In  decreasing 
order  the  activity  of  the  element  ap- 
peared in  bone,  muscle,  liver,  stomach 
and  small  intestine,  blood,  kidneys, 
heart,  lungs  and  brain.  The  turnover 
of  radiophosphorus  in  brain  is  much 
slower  than  in  other  tissues.  On  basis 
of  weight  retention  it  is  highest  in  bone, 
liver,  intestinal  tract,  heart,  kidneys, 
lungs,  blood,  muscle,  skin,  and  brain 
(in  decreasing  order).  Scott,  K.  G. 
and  Cook,  S.  F.,  Proc.  Nat.  Acad.  Sci., 
1937,  23,  285-272  found  that  large  doses 
of  P^2  cause  decrease  in  polymorpho- 
nuclear leukocytes  in  circulating  blood 
of  chicks,  presumably  due  to  selective 
beta  ray  irradiation  of  the  bone  mar- 
row in  consequence  of  higher  absorption 
and  retention  in  bone.  Lawrence  and 
his  group  (Lawrence,  J.  H.  and  Scott, 
K.  G.,  Proc.  Soc.  Exp.  Biol.  &  Med., 
1939,  40,  694-696;  and  Lawrence,  J.  H., 
Tuttle,  L.  W.,  Scott,  K.  G.,  Conner, 
C.  L.,  J.  Clin.  Invest.,  1940,  19,  267- 
271),  as  result  of  this  finding,  com- 
pared phosphorus  metabolism  of  normal 
and  leukemic  mice.  Although  the  total 
phosphorus  content  of  lymph  nodes, 
spleen,  and  liver  was  about  the  same  in 
normal  and  leukemic  animals,  the  pro- 
portion of  P^2  in  the  leukemic  animal 
was  distinctly  higher,  indicating  a 
higher  rate  of  phosphorus  metabolism 
in  these  animals.  Lawrence,  J.  H., 
Radiology,  1940,  35,  51-60  reported  the 
use  of  radiophosphorus  on  a  group  of 
patients  suffering  from  leukemia  and 
polycythemia.  Hevesy,  G.  and  Lunds- 
gaard,  E.,  Nature,  1927,  140,  275-276 
and  Arton,  C,  Sarzana,  G.,  Perrier,  C, 
Santangelo,  M.  and  Segr6,  E.,  Nature, 
1937,  139,  836-837  have  studied  conver- 
sion of  inorganic  phosphates  to  phos- 
pholipids. They  observed  different 
rates  of  synthesis  and  storage  in  various 
organs.  Studies  on  phospholipids  using 
P^2  as  tracer  are  reviewed  by  Sinclair, 
R.  G.,  Biol.  Symposium,  1941,  5,  82-98. 
Chaikoff  and  his  colleagues  (numerous 
papers  in  J.  Biol.  Chem.,  1937  and  fol- 
lowing years)  confirmed  these  results 
on  different  animals  and  extended 
studies  to  isolated  tissue  slices  in  vitro. 
Jones,  H.  B.,  Chaikoff,  I.  L.  and 
Lawrence,  J.  H.  (J.  Biol.  Chem.,  1939, 
128,  631-634)  found  different  types  of 
malignant  tumors  had  characteristic 
patterns  of  phospholipid  metabolism 
not  related  to  cell  types.  Marshak 
separated  cell  nuclei  from  cytoplasm 
and  observed  malignant  cell  nuclei 
accumulated  more  P'^  than  normal 
nuclei  and  that  relative  to  cytoplasm 


malignant  nuclei  took  up  more  than 
normal  cells,  comparing  lymphoma  cells 
with  normal  liver  cells  (Marshak,  A., 
Science,  1910,  92,  460-461  and  J.  Gen. 
Physiol.,  1941,  25,  275-291).  This  com- 
bination of  the  techniques  using  tracers 
and  methods  of  separating  components 
of  cells  offers  great  promise  for  further 
investigation.  Numerous  reports  on 
plant  tissues  and  on  insects  are  also 
available.  Manly,  M.  L.  and  Bale, 
W.  F.  (J.  Biol.  Chem.,  1939,  129,  125- 
134)  have  described  P^-  distribution  in 
rat  bones  and  teeth.  Sognnaes,  R.  F. 
and  Volker,  J.  F.,  Am.  J.  Physiol.,  1941, 
133,  112-120  have  studied  distribution 
of  P^^  in  parts  of  the  teeth  of  cats,  dogs, 
and  monkeys.  Most  P^^  is  found  in 
dentin,  and  little  in  enamel.  Of  that  in 
enamel,  highest  concentration  is  in 
outermost  layer,  suggesting  that  some 
minerals  reach  the  teeth  by  diffusion 
from  saliva.  Radiophosphorus  may  be 
used  to  "label"  erj^throcvtes  in  much 
the  same  manner  as  radioiron  is  used. 
Recently  Brown,  Jr.,  F.  A.,  Hempel- 
man,  Jr.,  L.  H.  and  Elman,  R.  have 
used  such  "tagged"  erythrocytes  to 
determine  true  blood  volume  (Science, 
1942,  96,  323-324). 

Radiopotassium  (K«)  half  life  12.4  hrs. 
Rate  of  absorption  of  radiopotassium 
from  the  gut  was  investigated  in  con- 
junction with  the  study  of  radioiodine 
by  Hamilton.  The  distribution  of  in- 
jected radiopotassium  in  tissues  of  the 
rat  has  been  studied  by  Noonan,  T.  R., 
Fenn,  W.  O.  and  Haege,  L.  (Am.  J. 
Physiol.,  1941,  132,  474-488).  An  early 
concentration  of  the  ion  occurs  in  liver, 
heart,  kidney,  lung,  diaphragm,  and 
gastrointestinal  tract.  After  equilib- 
rium is  reached,  most  of  the  radio- 
activity is  present  in  tissues  normally 
high  in  potassium,  i.  e.  muscle,  skin  and 
viscera.  Recently  Lyman,  C.  P.,  Am. 
J.  Physiol.,  1942,  137,  393-395  employing 
this  isotope  has  demonstrated  an  in- 
creased permeability  of  clenervated 
skeletal  muscle  to  potassium  ion. 

Radiorhubidium  (Rb^^)  half  life  IS  days. 
It  is  possible  that  radiorhubidium  can 
be  used  in  place  of  radiopotassium  which 
is  difficult  to  prepare  but  no  work  ap- 
pears to  have  been  reported  as  yet. 

Radioselenium  (Se'^)  half  life  48  days. 
McConnell,  K.  P.,  J.  Biol.  Chem.,  1941, 
141,  427-437  has  reported  on  the  reten- 
tion of  radioselenium  in  various  tissues. 
19%  is  found  in  the  liver;  considerable 
amounts  in  muscle,  intestine,  and  blood ; 
less  in  the  testis.  None  is  present  in 
the  skin,  fur,  teeth,  and  long  bones. 
The  element  is  chiefly  excreted  by  the 
kidneys. 

Radiosodium  (Na^^)  half  life  14.8  hrs. 
Easiest  element  to  prepare.    The  yield 


R.\DIOSODIUM 


215 


REGAUD'S  METHOD 


is  very  high.  Another  isotope,  Na" 
has  a  half  life  of  3yrs.,  and  may  eventu- 
ally prove  quite  useful.  To  date  little 
use  has  been  made  of  this  longer  lived 
isotope  in  biology.  All  references  below 
are  to  the  Na^^  isotope. 

This  lias  been  extensively  employed 
by  Flexner,  Gellhorn,  and  Pohl  to  deter- 
mine rates  of  placental  transfer  in 
mammals.  See:  Flexner,  L.  B.  and 
Pohl,  H.  A.,  J.  Cell,  and  Comp.  Physiol., 

1941,  18,  49-60;  Am.  J.  Physiol.,  1941, 
134,  344-349;  Gellhorn,  A.,  Flexner,  L. 
B.  and  Pohl,  H.  A.,  J.  Cell,  and  Comp. 
Physiol.,  1941,18,  385-392;  and  Flexner, 
L.  B.  and  Gellhorn,  A.,  Am.  J.  Obst. 
and  Gynec,  1942,  43,  965-974. 

Radiostrontium  (Sr^s)  half  life  55  days. 
Distribution  of  this  isotope  in  the  body 
is  much  like  radiocalcium.  The  more 
intense  radiation  from  this  element 
than  from  radiocalcium  makes  it  a  possi- 
ble choice  for  localized  radiation  therapy 
of  bones  and  osteogenic  tissues.  Auto- 
radiographic and  other  evidence  for 
accumulation  in  a  human  osteogenic 
sarcoma  is  presented  by  Treadwell,  A. 
deG.,  Low-Beer,  B.  V.  A.,  Friedell,  H.  L. 
and  Lawrence,  J.  H.,  Am.  J.  Med.  Sci., 

1942,  204,  521-530. 

Radiosulphur  (S^^)  half  life  88  days.  The 
metabolism  of  radiosulphur  in  inorganic 
salts  and  in  synthetically  prepared 
thiamine  chloride  (vitamin  Bi)  in  nor- 
mal and  vitamin  deficient  human  sub- 
jects lias  been  studied  by  Borsook  and 
his  coworkers  (Borsook,  H.,  Hatcher, 
J.  B.  and  Yost,  D.  M.,  J.  Appl.  Phys., 
1941,  12,  325A  and  earlier  papers). 

Ranson  Pyridine  method  for  unmyelinated 
nerve  fibers  (Ranson,  S.  W.,  Rev. 
Neurol.  &  Psychiat.,  1914,  12,  467-474). 
Fix  in  absolute  alcohol  +  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,  265-2G6) ;  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). 

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  employed  in  reduc- 
tion-oxidation potential  determina- 
tions, see  Oxidation-Reduction  Po- 
tential. 

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 
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  description  in  1878  will  probably 
not  be  followed. 

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 


REGAUD'S  METHOD 


216 


RESTAINING  FADED  SECTIONS 


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 
(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.  Exp.  Biol.  &  Med.,  1938,  39,  323- 
324  have  discovered  that  the  produc- 
tion of  new  cells  is  rhythmic  being 
greatest  at  night  and  least  by  daJ^  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. 
Physico-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 
thefatty  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  bodj^  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. 

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  oft'ered.  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. 

Restaining  Faded  Sections.  This  is  some- 
times very  desirable.  Trj--  technique 
outlined  by  Small,  C.  S.,  (Amer.  J.  Clin. 
Path.,  Techn.  Suppl.,  1943,  7,  66-67). 


RETICULAR  FIBERS 


217 


REVIVAL  OF  VINEGAR  EELS 


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 
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-lOfj.  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  just  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  naake  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  with  vaseline  and  observe.  To 
make  relatively  permanent  specimens, 
remove  the  coverglass  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.,  19.30,  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  cliiefly  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  Cool- 
ing.— Written  by  B.  Luyet,  St.  Louis 
University,  June  1,  1946) — A  drop  of  a 
concentrated  vinegar  eel  suspension, 
obtained  by  centrifugation,  is  deposited 
on  a  glass  slide  and  most  of  the  remain- 


REVIVAL  OF  VINEGAR  EELS 


218 


RICKETTSIA 


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  ha,nd,  the  droplet 
of  liquid  air  which  may  adhere  to  the 
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. 

Rhodamine  B  (CI,  749)— brilliant  pink  B, 
rhodamine  0 — 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). 

Rhodium,  see  Atomic  Weights. 

Rhodopsin  (G.  rhodon,  rose  +  ops,  eye). 
Visual  purple  present  in  external  seg- 
ment of  the  rod  cells  of  retina  (See 
Arey  in  Cowdry's  Special  Cytology,  1932, 
3,  1211-1291). 

Riboflavin  (lactoflavin)  shows  typical  green 
fluorescence  in  living  liver  and  kidney 
observed  under  fluorescence  micro- 
scope (Ellinger,  P.,  and  Koschara,  \V., 
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. 


Ribonuclease  (Yeast  thymonucleic  acid), 
occurrence  (Greenstein,  J.  P.  and  Jen- 
rette,  W.  V.,  J.  Nat.  Cancer  Inst.,  1941, 
2,  301).  Used  in  investigation  of 
chromatolysis  of  Nissl  Bodies  of  Nerve 
Cells  by  Gersh,  I.,  and  Bodian,  D., 
Biological  Symposia,  1943,  10,  163-184. 

Ribonuclease,  see  Gram  Staining. 

Ribonucleic  Acid.  Biesele,  J.  J.,  Cancer 
Research,  1944,  4,  737-750. _ 

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  bufl'ered  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 
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 


RICKETTSIA 


219 


RUTHENIUM 


lightly  in  aq.  dest.  after  the  citric  acid 
treatment  and  by  staining  again  with 
methylene  blue,  before  ditferentiation 
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,  4i>-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.;  KCl, 
0.025  gm.;  CaCb,  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  NaHCOj  is 
present  it  must  not  be  sterilized  by 
heat. 

Ringer-Locke  solution.  NaCl,  0.85  gm.; 
KCl,  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  NaHCOs  must  not  be  steri- 
lized by  heat. 

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  {ibid,  730-752).  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.;  CaCh,  0.01  gm.;  5%  aq. 
phenol,  100  cc.  Wash  quickly  in  water. 
Dry  and  examine.    See  Eosins. 

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. 

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 


220 


SANDISON'S  TECHNIQUE 


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,  lM-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,  see  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  fiuid  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.  (Revised  by  C.  H. 
Sawyer,  May  14,  1946.)  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- 
clusions 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  N  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. 

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 


SANDISON'S  TECHNIQUE 


221 


SEMEN  STAINS 


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. 

Sarcolemma.  Special  technique  for,  see 
Dahlgren  in  McClung  (p.  132). 

Scandium,  see  Atomic  Weights. 

Scarlet  B  or  EC,  see  Biebrich  Scarlet, 
water  soluble. 

Scarlet  B  Fat  Soluble,  see  Sudan  III. 

Scarlet  J,  J  J,  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. 

Schiflf's  Reaction  for  aldehydes  (Bourne, 
p.  22)  is  basis  of  Feulgen  reaction  for 
Thymonucleic  Acid. 

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. 

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 
25  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. 

Sebaceous  Glands.  Method  for  staining 
intoto  (Badertscher,  J.  A.,  Stain  Techn., 
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. 

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 


SEMEN  STAINS 


222 


SHADOW-CASTING 


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)  +  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  5  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. 

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). 

Sharpening,  see  Microtome  Knife. 

Shrinkage  caused  by  fixation,  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. 

Shadow-casting. — Written  by  Dr.  W.  T. 
Dempster,  Dept.  of  Anatomy,  Univer- 
sity of  Michigan,  Ann  Arbor,  Mich., 
May  28,  1946 — This  is  a  technique  for 
revealing  the  surface  form  and  texture 
of  microscopical  material  in  either  light 
or  electron  microscopy.  It  is  an  out- 
growth 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. 
It  was  found  that  the  particles  travelled 
in  straight  line  paths  from  the  filament 
and  that  obstructions,  however  small, 
cast  a  metal-free  shadow.  With  R.  W. 
G.  Wyckoff  (J.  Appl.  Phys.,  1944,  15, 
712-716),  a  successful  technique  of 
oblique  casting  of  metal  films  was  ap- 
plied to  electron  microscopy  for  measur- 
ing shadow  lengths  and  calculating 
heights  of  minute  objects.    Bacteria, 


viruses  and  minute  chemical  aggregates 
have  been  studied  (Williams,  R.  C.  and 
Wyckoff,  R.  W.  G.,  Proc.  Soc.  Exp. 
Biol.  &  Med.,  1945,  58,  265-270;  59,  265- 
270;  Science,  1945,  101,  594-596;  102, 
277-278;  Nature,  156,  68).  Unusual 
contrast,  surface  texture  and  the  possi- 
bility of  measuring  heights  are  positive 
advantages  of  the  technique.  Further 
applications  to  both  electron  and  visual 
microscopy  involved  a  method  of  study- 
ing opaque  surfaces  by  coUoidin  replicas 
that  are  shadowed  (Williams,  R.  C, 
and  Wyckoff,  R.  W.  G.,  J.  Appl.  Piiys., 
1946,  17,  23-33).  Applications  of  the 
method  to  biological  material  viewed 
with  the  light  microscope  and  an  ac- 
count of  the  casting  apparatus  (W.  T. 
Dempster  and  R.  C.  Williams)  are 
forthcoming. 

Material  is  affixed  to  cover  slips; 
smears  are  thoroughly  dried;  paraffin  is 
dissolved  from  sections  with  solvents. 
With  no  further  preparation,  other  than 
thorough  drying,  the  slips  are  shadowed 
with  a  metal  deposit  in  a  vacuum  cham- 
ber. After  this,  they  are  mounted  face 
down  on  slides  with  clarite  or  balsam. 
For  electron  microscopy,  regular  screen 
grids  with  a  thin  coUoidin  film  over  the 
mesh  are  used  as  substrates  for  suspen- 
sions; replicas  are  placed  directly  on  the 
mesh.  Metallic  chromium  is  about  the 
best  metal  for  general  shadowing  but, 
for  finest  detail  with  the  electron  micro- 
scope, gold  or  uranium  is  preferable. 
The  casting  technique  is  similar  for  the 
different  metals.  Shadow-casting  pro- 
duces a  visually  structureless  deposit 
which  sticks  to  all  surfaces  save  those 
directed  away  from  the  hot  filament  and 
shadow  areas  due  to  obstructions.  Sur- 
faces perpendicular  to  straight  line 
paths  from  the  filament  get  the  heaviest 
deposit;  oblique  surfaces  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  preparations  transmit 
light  and  appear  bright;  highlights  are 
dark.  The  eye,  however,  readily 
adapts  to  this  reversal  of  tone.  Photo- 
graphic negatives  or  prints  made  from 
glass  positives  reverse  the  microscope 
appearance;  highlights  then  are  bright, 
shadows  are  dark,  and  variations  of 
surface  texture  are  shown  by  gradations 
of  tone. 

Electron-microscope  negatives  show 
the  same  natural  appearance  of  light 
and  dark.  The  observer  looks  at  sur- 
faces rather  than  through  specimens  as 
in  ordinary  microscopy.  The  appa- 
ratus consists  of  a  bell  jar  and  a  base 


SHADOW-CASTING 


223 


SILVER  CITRATE 


plate  with  vacuum  tight  electrical  con- 
nections. Electrodes  raised  above  the 
level  of  the  base  plate  carry  a  tungsten 
filament  on  which  metallic  chromium  is 
placed  for  vaporizing.  Cover  slips 
with  affixed  material  (or  the  grid 
screens)  are  arranged  in  a  semicircle  at  a 
predetermined  distance  from  the  fila- 
ment and  the  metal  thereon  to  be 
vaporized.  The  height  of  the  filament 
and  the  distance  from  filament  to  speci- 
mens determine  the  casting  angle. 
Both  an  oil-diffusion  pump  and  a  me- 
chanical pump  must  be  used  to  produce 
the  degree  of  vacuum  required  (at  least 
10"'*  mm.  Hg.).  With  a  suitable  vac- 
uum provided,  the  filament  is  heated 
electrically  and  a  measured  weight  of 
metal  is  vaporized.  Preparations  are 
then  ready  for  mounting  or  examina- 
tion. A  figure  of  the  apparatus  and  the 
formula  for  calculating  the  appropriate 
mass  of  metal  for  the  conditions  of 
shadowing  are  presented  in  the  Demp- 
ster and  Williams  paper. 

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  aiscussed  by  Poli- 
card.  A.,  ana  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). 


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  andp- 
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). 

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.  dichlorfiuorescein  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  gms.  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 
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 


SILVER  CITRATE 


224 


SILVER'S 


make  frozen  sections  to  determine  re- 
sults and  fix  ottier  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  Gray,  see  Nigrosin,  water  soluble. 

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,  401^28.  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  nvclei,  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  10-40ai  or  dehy- 
drate slowly,  imbed  in  paraffin  or  cel- 
loidin  and  cut  2-20/i.  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 
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 


SILVER'S 


225 


SKIN 


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  ana  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  collagenic  tissue.  Weigert 's  re- 
eorcin  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., 
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. 


SKIN 


226 


SMEARS 


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  gm.,  5%  aq.  phenol 
100  cc,  1%  aq.  CaCh,  1  cc;  then  wash 
quickly  and  dry   (McClung,  p.  146). 

Small  Intestine.  Many  conditions  influence 
the  appearance  seen  in  sections.  If 
fixed  while  distended  with  food  rnate- 
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  e.xamine  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 
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, 


SMEARS 


227 


SOLID  GREEN  JJO 


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  fixed  in 
Helly's  fluid  (i.e.  formalin  Zenker)  as 
advised  by  Maximow  (see  Downey,  p. 
2001).  McClung  (p.  262)  recommends 
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. 
Smith-Dietrich  method  for  lipoids  (Die- 
trich, A.,  Verh.  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  chlor- 
ides 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- 
dios odium. 

An  ultramicromethod  for  sodium 
employing  the  polarograph  has  been 
devised  by  Carruthers,  C.,  Indust.  and 
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. 

Solanylin,  a  dye  extracted  from  the  egg- 
plant (Solanum  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 


228 


SPHINGOMYELIN 


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  differ  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). 

Sorensen's  Buffers.  Sorenson's  phosphate 
buflfers  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: 

NatHPO*  anhydrous 9.47  gm. 

KH2PO4 9.08  gm. 

To   obtain   a  solution  of  the  pH  re- 
quired, mix  them  in  following  amounts : 


cc.  M/15 

cc.M/16 

pH 

NaiHPOi 

KHtPOi 

5.4 

3.0 

97.0 

6.6 

5.0 

95.0 

5.8 

7.8 

92.2 

6.0 

12.0 

88.0 

6.2 

18.6 

81.5 

6.4 

26.6 

73.5 

6.6 

37.6 

62.5 

6.8 

60.0 

60.0 

7.0 

61.1 

38. 

7.2 

71.6 

28.5 

7.4 

80.4 

19.6 

7.6 

86.8 

13.2 

7.8 

91.4 

8.6 

8.0 

94.6 

6.5 

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 
pressure  in  vacuum  pump)  until  cleared. 
Mount  or  store  in  this  clearing  fluid.  In 
practice  it  is  possible  to  get  good  results 
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  Histospectro- 
graphy  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  by  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. 
IVIake  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.  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 


229 


SPREADING  FACTORS 


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 
glands  of  humans  (Doenges,  J.  L.,  Arch. 
Path.,  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   Pallida. 

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;  IVIacKenzie,  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  by  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 instantaneously  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. 
subiilis  (Snyder,  M.  A.,  Stain  Techn., 
1934,  9,  71-72). 

Stain  heat  fixed  film  with  carbol- 
fuchsin  (see  Acid  Fast  Bacilli).  Rinse 
quickly  and  differentiate  in  95%  alcohol. 
Wash  in  hot  tap  water  and  again  rinse 
in  alcohol.     Counterstain  for  2-5  min. 


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 
fi.xed  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.  The  recognition  of 
these  factors  constitutes  a  major  ad- 
vance in  biology  and  medicine.  In  this 
Duran  Reynals  and  his  associates  have 
taken  the  lead.  Those  seeking  informa- 
tion as  to  techniques  for  the  investiga- 
tion of  spreading  factors  should  begin 
with  a  detailed  statement  by  Duran 
Reynals,  F.,  Bact.  Rev.,  1942,  6,  197- 
252,  on  which  the  following  account  is 
mainly  based. 

Included  under  this  heading  are  "sub- 
stances present  in  animal  tissues  which 
have  the  common  property  of  increas- 
ing the  permeability  of  connective 
tissue."  By  so  doing  they  promote 
spread  through  connective  tissue  of  a 
wide  variety  of  substances,  viruses  and 
microorganisms.  Duran  Reynals  di- 
vides them  tentatively  into  3  groups: 

1.  "Factors  with  spreading  power  in 
vivo  and  an  enzymatic  effect  on  hyal- 
uronic acid  in  vitro  as  shown  by  reduc- 
tion of  viscosity  and  by  hydrolysis,"  in 
extracts  of  testicle,  spleen,  skin,  etc. 

2.  "  Factors  showing  spreading  power, 
but  no  enzymatic  activity  in  vitro,  on 
hyaluronic  acid"  in  other  tissues  and  in 
some  bacteria. 

3.  Ascorbic  acid,  some  other  reducing 
substances,  possibly  azoproteins. 

The  factor  acting  on  Hyaluronic  Acid 
is  the  enzyme  hyaluronidase.  The  sub- 
stance now  known  as  hyaluronic  acid 
was  demonstrated  in  the  ground  sub- 
stance of  connective  tissue  by  staining 
with  toluidine  blue  by  Bensley,  S.  H., 
Anat.  Rec,  1934,  60,  93-108.  The  same 
investigator  studied  the  consistency  of 
the  ground  substance  by  the  ingenious 
device  of  first  adapting  paramoecia  to 
tissue  fluid  and  then  of  observing  that 
their  movements  in  the  intercellular 
and  interfibrous  spaces  of  connective 
tissue  are  restrained  by  invisible  mate- 
rial. See  Loose  Connective  Tissue.  A 
specific  microchemical  test  is  greatly 


SPREADING  FACTORS 


230 


STARCH  GRAINS 


needed   for   compounds   of   hyaluronic 
acid. 

Spreading  factors  can  be  measured  by 
comparing  the  spread  of  a  colored  fluid 
injected  intradermally  in  rabbits  to- 
gether with  the  factor  and  in  its  ab- 
sence. 
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- 
adelphia: Blakiston  Co.,  1940,  676  pp. 
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)  possess  granules  which  are  homo- 
logous for  the  several  species  but  differ 
in  staining  reaction  for  the  species 
(Maximow — 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  fiuids.  Stains  and  smell  of 
putrefaction  caused  by  them  can  be 
removed,  as  above,  by  permanganate 
and  oxalic  acid. 
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- 


STARCH  GRAINS 


231 


SUBMAXILLARY  GLANDS 


nin,  20min.  Whas  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.). 

Starch  Paste,  as  substitute  for  albumin- 
glycerin  mixture  in  mounting  paraffin 
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, 
432-434. 

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. 

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. 

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  glana  virus  when 
this  plays  an  inapparent  role.  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 


232 


SUDAN  BLACK  B. 


Submicroscopic  Fibrils.  These  by  close 
association  may  constitute  the  neuro- 
fibrils, spindle  and  astral  fibers,  myo- 
fibrils, and  so  on.  Use  of  polarization 
optical  methods  suggests  the  orienta- 
tion of  submicroscopic  rodlets  parallel 
to  the  length  of  the  fibers.  The  elec- 
tron microscope  is  capable  of  demon- 
strating the  component  submicroscopic 
fibrils  of  collagenic  fibrils  (Schmitt, 
F.  O.,  Hall,  C.  E.  and  Jakus,  M.  A., 
Biol.  Symposia,  1943,  10,  261-276). 

Submicroscopic  Particles.  In  summarizing 
work  in  R.  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/x.  (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). 

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- 
methane).  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 
Govan,  A.D.T.,  J.  Path.  &  Bact.,  1944, 
56,  262-264 .  See  Bell's  Method  for  stain- 
ing 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  If  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  than  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/a<.  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- 
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  5%  formalin  24  hrs. 
Cut  frozen  sections,  whether  first  em- 


SUDAN  BLACK  B. 


SULFHYDRYL  GROUPS 


bedded  in  gelatin  or  not.  6-10  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, 
30  min.  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  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. 

Sulfhydryl  Groups.  1.  Prussian  blue  histo- 
chemical  reaction  for  (Ch^vremont,  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  mixture  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 
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  away. 

"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  ma.xi- 
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 


SULFHYDRYL  GROUPS 


234 


SULPHUR 


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  as 
well  localized,  but  in  case  of  doubt  a 
test  of  secondary  impregnation  can  be 
made  in  the  way  described  for  the 
ninhydrin." 
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- 
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 


SULPHUR 


235 


SURFACE  MEASUREMENTS 


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. 

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  (Emig,  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  diethylsa.ha,nm-azodi- 
methylanilin,  that  the  dimethyl  com- 
pound will  not  work.  The  supravital 
staining  of  blood  cells  began  with  the 
demonstration  by  Cowdry  at  Hopkins 
(Internat.  Monatschr.  f.  Anat.  u. 
Physiol.,  1914,  31,  267-286),  that  this 
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). 
Suramin,  a  drug  purchasable  under  term 
of  Naphuride  (Winthrop),  is  only  a 
feeble  inhibitor  of  growth  of  lympho- 
sarcoma transplants.  Its  cytotoxic 
effect  is  rather  similar  to  that  of  colchi- 
cine on  lymphoid  tumors  (Williams, 
W.  L.,  Cancer  Research,  1946,  6,  344- 
353). 
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  intestine — 


SURFACE  MEASUREMENTS 


236 


TANTALUM 


5  sq.  meters — Policard,  A.,  Precis 
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  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,  1,  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 
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.  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. 

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 


237 


TEETH 


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. 

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 
dye  is  useful  in  coloring  foodstuffs,  light 
filters,  etc.  (Emig,  p.  46). 

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 
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  ana  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. 

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%,  J  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 
evapxjrate  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  Physiologi cal  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.  ^  hr.  each  in  following  paraffins 
(1)  42-46°C.,  (2)  52-56°C.  ancl  (3)  58- 
60°C.  within  oven.  Imbed  in  a  mix- 
ture of  235  cc.  52-56 °C.  paraflan  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  -f,  abs.  ale.  (exposed  to 
anhydrous  copper  sulphate,  see  Alco- 
hol) 2  weeks  -f .    Equal  parts  abs.  and 


TEETH 


238 


TEETH,  DECALCIFICATION 


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%alc.48hrs.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.  -f.  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- 
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  inlO% 
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  in  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,  Washmgton  University 
(School  of  Dentistry) .  Revised  by  him 
July  27,  1946. 

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- 


TEETH,  DECALCIFICATION 


239 


TEETH,  DECALCIFICATION 


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. 

Recently,  excellent  results  have  been 
obtained  with  the  use  of  formic  acid 
according  to  the  technique  of  Morse, 
J.  Dent.  Res.,  1945,  24,  143-153.  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  c.c.  aq. 
dest.  At  the  time  of  use  combine 
equal  parts  of  A  and  B.  Change  solu- 
tion daily  until  decalcification  is  com- 
plete as  shown  by  chemical  test.  (See 
below.) 

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  and  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.  Boling  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  fixative.  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  squeez- 
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- 


TEETH,  DECALCIFICATION 


240 


TEETH,  INNERVATION 


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  paraffin  block,  cut 
surface  down,  on  acid  if  the  dentin  is 
exposed. 
Teeth,  Developing.  1.  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  Cultivation  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 
et  al.  (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 
rats  1-5  days  old  in  a  variety  of  fluids. 
They  employed  the  Nassonov  technique 
for  the  Golgi  apparatus  and  Regaud'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.  Bol- 
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  n  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 

fiven  by  Lee  (p.  494)  will  serve.  Treat 
rozen  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 


TEETH,  INNERVATION 


241 


TESTIS 


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;  Bodecker,  C. 
F.,  and  Lefkowitz,  W.,  J.  Dent.  Res., 
1937,  16,  463-475  and  others  relating  to 
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.  593.  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., 
1935-36,  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. 

Terpineol  (or  terpinol),  a  mixture  of  sub- 
stances of  composition  CioHu  and 
CioHnO  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,  165- 
166)  is  partly  to  substitute  for  ethyl  al- 
cohol by  passing  through  the  following 
series  of  mixtures :  (1)  Aq.  dest.,  50  cc. ; 
95%  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.  R.  and  Stowell, 
R.  E.,  in  press).  Do  not  confuse  with 
n  Butyl  alcohol. 

Testis.  Methods  described  elsewhere  for 
the  Connective  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,  175-198,  using 
the  Benda  Method,  obtained  prepara- 
tions  of  opossums   which   he   thought 


TESTIS 


242 


THYMONUCLEIC  ACID 


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's  Special  Cytology,  1932, 
3,  1674-1725. 

Testosterone,  Pollock,  Anat.  Rec,  1942,  84, 
23-27. 

Tetrachrome  Blood  Stain,  see  MacNeal's. 

Tetralin  is  tetrahydronaphthalene  used  as  a 
clearing  agent  after  Diaphanol. 

Thallium.  Barbaglia's  Method.  Fi.x  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  and  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 
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. 

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  (Leathern,  J.  H., 
Anat.  Rec,  1944,  89,  540). 

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  {Feidgen  or  nucleal  reac- 
tion for).  Pass  paraffin  sections,  fixed 
in  equal  parts  sat.  aq.  corrosive  subli- 
mate and  absolute  alcohol ,  through  xylol 
and  alcohols  to  water.  Place  in  a  stain- 
ing jar  containing  normal  HCl  (82.5  cc. 
HCl,  sp.  gr.  1.17-1.185  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  ^1  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  (NaHSOs)  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 
Barnard  Free  Skin  and  Cancer  Hospital 
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  N  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. 


THYMONUCLEIC  ACID 


243 


TISSUE  BASOPHILES 


Three  types  of  organisms  were  tested: 
(1)  Vai'ious  small,  enclosome-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.  N  HCl  room  temperature, 
2  min.  iV  HCl  at  60°C.,  8-10  min. 
Rinse  in  iV  HCl  at  room  temperature. 
Rinse  in  aq.  dest.,  Sulphurous  acid, 
2  min.  Leucobasic  fuchsrn,  1^-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  Trielhyl  Phos- 
phate technique. 
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,  7597772.  A  fine 
beginning  has  been  made  in  the  direct 
study  of  vacuoles  within  the  follicles  in 
living  mice  by  transillumination  after 
the  fashion  of  Kniselv  (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).  Paraffinabout  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  lias  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 
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  extra  vascular  origin. 
They    can    easily   be   studied    in  fresh 


TISSUE  BASOPHILES 


244 


TISSUE  CULTURE 


spreads  of  Loose  Connective  Tissue  or 
omentum.  Their  granules  are  readily 
colored  supra  vitally  with  brilliant  cresyl 
blue,  methylene  blue  and  other  stains. 
Tissue  basophiles  disintegrate  quickly. 
Ivlaximow,  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%  NaoCOs  to  20  cc. 
thionin  sol.  and  filtering  before  use. 
IVIaximow  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., 
Ztsciir.  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. 

Sylven,  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  J%  aq.  toluidin  blue  and 
other  dyes,  and  reached  the  conclusion 
that  the  radiation  brings  about  liberation 
of  organic  sulphuric  acids  of  high  molec- 
I  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  SO  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,  Md. — By  these  methods  a  small 
clump  of  cells  can  be  removed  from  an 
organism  and  maintained  in  a  condition 
of  survival  or  growth  for  periods  rang- 
ing from  a  few  hours  for  some  cells  to 
an  indefinite  number  of  years  for  others. 
While  so  maintained  they  can  be  ex- 
amined microscopically  at  various  mag- 
nifications. The  differentiation  of  em- 
bryonic tissues  can  be  followed  (Fell, 
H.  B.,  J.  Roy.  Micr.  Soc,  1940,  60,  95). 
Malignant    cells    may    be    grown    and 


studied  for  an  extended  interval  and 
their  characteristics  compared  with 
those  of  normal  cells  or  with  malignant 
cells  in  vivo  (Earle,  W.  R.,  J.  Nat. 
Cancer  Inst.,  1943,  4,  165).  Cell  form, 
size,  internal  motion,  locomotion  and 
rate  and  manner  of  cell  proliferation  can 
be  routinely  studied  either  visually,  or 
by  means  of  photographic  records,  in- 
cluding time-lapse  cinematography 
(Earle,  W.  R.,  J.  Nat.  Cancer  Inst., 
1943,  4,  147).  The  tissue  cultures  can 
be  killed,  fixed  in  situ  and  stained,  or 
even  examined  unstained  by  means  of 
the  electron  microscope  (Porter,  K.  R., 
Anat.  Rec,  1946,94.  490). 

In  addition  nutritional  and  physio- 
logical studies  are  possible.  The  cul- 
ture medium  can  be  modified  by  the 
addition  or  omission  of  various  nutri- 
tional elements  or  other  physiologically 
active  substances  and  the  influence  of 
this  altered  medium  on  the  cells  grown 
in  it  studied.  Tissue  cultures  can  also 
be  used  for  investigation  of  both  pro- 
toxoan  and  bacterial  parasites  of  tissues 
(Fischer,  A.,  Gewebezuchtung.  Munich: 
R.  Muller,  1930),  and  in  the  production 
of  vaccines. 

Culture  Support.  For  the  satisfac- 
tory routine  maintenance  of  cultures 
of  most  tissue  cells  a  solid  support  for 
their  growth  and  migration  is  required. 
Various  types  of  supports  have  been 
employed,  such  as  silk  thread,  spider 
web,  glass  wool,  lens  tissue,  cellophane, 
gelatin,  and  agar,  and  for  simple  cul- 
tures the  glass  surface  of  the  culture 
dish.  By  far  the  most  satisfactory 
support  is  provided  by  placing  the  cell 
clump  in  a  thin  layer  of  fluid  plasma,  by 
clotting  the  plasma  into  a  solid  gel  and 
by  addition  of  a  little  tissue  extract  or 
thrombin.  When  of  correct  consistency 
the  clot  is  a  solid,  somewhat  elastic, 
optically  clear  gel,  of  a  fibrillar  struc- 
ture which  enables  the  cells  to  migrate, 
through  it,  although  they  exhibit  a 
tendency  to  collect  at  its  surfaces. 
With  such  a  matrix,  culture  main- 
tenance and  growth  are  usually  much 
more  satisfactory  than  with  any  other 
type  of  solid  support.  (1)  There  is 
better  contact  of  the  explant  surface 
with  the  threads  of  the  fibrillar  matrix. 
(2)  Adhesion  of  cells  to  a  solid  support 
facilitates  cleavage  since  the  ceils 
finally  pull  apart  by  adhering  to  the 
solid  medium  and  by  migrating  in  op- 
posite directions.  (3)  The  freshly 
prepared  fibrin  clot  contains  some 
serum  which  enhances  the  growth  of 
many  cell  types.  (4)  The  fibrin  matrix 
reduces  loss  of  cells  by  their  being 
washed  out  of  the  culture  in  changing 
the  culture  fluid.     (5)  It  is  also  possible 


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245 


TISSUE  CULTURE 


that  the  fibrin  clot  contributes  other 
chemical  or  physical  factors  favorable 
to  cell  growth  and  migration. 

Chicken  plasma  is  usually  employed 
for  the  preparation  of  the  clot  since  it 
is  less  likely  than  others  to  clot  spon- 
taneously and  since  a  gel  of  satisfactory 
consistency  can  be  more  routinely  pre- 
pared from  it.  Plasma  homologous 
with  the  cells  is  often  used,  however,  as 
is  also  plasma  from  other  convenient 
animals.  Premature  clotting  of  the 
plasma  can  be  prevented  by  addition  of 
a  small  amount  of  purified  heparin. 

While  the  fibrin  clot  seems  the  best 
available  culture  matrix  for  general 
work,  it  is  far  from  perfect.  For  in- 
stance, with  certain  combinations  of 
cells  and  media  the  solid  matrix  may 
completely  dissolve  in  the  area  of  the 
cells  and  ruin  the  culture ;  or,  in  the  case 
of  very  slow-growing  cultures,  the  clot 
may  gradually  become  so  opaque  as  to 
handicap  optical  examination.  In 
studies  of  cell  nutrition  the  chemically 
undefined  nature  of  the  plasma  clot  and 
the  difficulties  of  making  a  routine 
quantitative  separation  of  cells  and  clot 
interfere  with  chemical  analysis. 

Isotonic  Saline.  A  satisfactory  iso- 
tonic saline  solution  is  necessary  for 
washing  cultures  and  for  suitable  dilu- 
tion of  plasma  and  nutrient  media. 
Often  only  minor  differences  exist  in 
their  formulae.  Mammalian  Ringer, 
Drew,  Locke,  Tyrode,  or  Earle's  Solu- 
tion are  all  satisfactory  for  mammalian 
cells,  while  such  solutions  as  amphibian 
Ringer  are  advised  for  amphibian  cells. 
Any  solution  simulating  roughly  the  in- 
organic salt  content  of  serum,  and  hav- 
ing a  comparable  osmotic  pressure  can 
be  used  for  routine  tissue  cultures. 
About  1%  of  glucose  is  usually  included 
as  a  source  of  carbohydrate.  For  much 
tissue  culture  work  the  solution  used 
by  Earle,  W.  B.,  J.  Nat.  Cancer  Inst., 
1943,  4,  165  has  the  advantage  of  an 
alkali  reserve,  in  the  form  of  sodium 
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  a  sealed  container  with  an  ade- 
quate tension  of  CO2  in  the  air  over- 
lying the  fluid.  The  heat  of  steriliza- 
tion changes  the  bicarbonate  to  car- 
bonate, the  pH  rises  to  a  value  in  excess 
of  8.0  and  this  frequently  causes  sec- 
ondary changes  like  precipitation  of  the 
calcium  or  magnesium.  Even  steri- 
lization by  filtration  through  a  bac- 
teriological   filter    under    vacuum    can 


cause  such  an  alkaline  shift,  and  there 
is  a  slight  shift  even  when  filtered  under 
pressure.  Probably  the  most  satisfac- 
tory procedure  for  sterilizing  such  a 
solution  is  to  bring  it  to  a  pH  somewhat 
acid  to  that  desired  (to  compensate  for 
loss  of  CO2  during  handling),  to  filter 
by  pressure  and  to  store  in  sealed  con- 
tainers. For  routine  culture  work  an 
initial  pH  of  about  7.8  in  the  culture  is 
desirable  because  elaboration  of  acid 
by  the  cells  of  the  culture  will  carry  the 
pH  to  somewhat  more  acid  levels. 
For  further  control  of  the  pH  of  the 
media  see  Parker,  R.  C,  Methods  of 
Tissue  Culture.  New  York:  Hoeber, 
1938. 

Nutrient  Media.  When  survival  or 
growth  is  desired  for  longer  than  a  very 
limited  time  adequate  nutrient  factors 
(embryonic  extracts  and  sera)  must  be 
incorporated  into  the  culture  medium. 
Optimum  proportions  of  extract  and 
serum  must  be  determined  for  different 
types  of  cells.  20%  embryo  extract, 
40%  horse  serum  and  40%  saline  solu- 
tion is  satisfactory  for  growing  mouse, 
rat  and  human  fibroblasts,  rat  mam- 
mary carcinoma  and  mouse  sarcomas, 
but  Carrel  and  Ebeling  obtained  their 
best  results  with  chicken  macrophages 
by  using  a  medium  composed  chiefly  of 
serum. 

Numerous  sera  have  been  mixed  with 
tissue  culture  media.  In  some  hospital 
centers  human  cord  serum  has  been 
available  and  has  proved  satisfactory 
(Gey,  G.  O.  and  M.  K.,  Am.  J.  Cancer, 
1936,  27,  45).  Horse  serum  has  been 
found  extremely  satisfactory  in  my 
laboratory  for  it  can  be  obtained  in 
large  amounts  with  little  trouble  from 
hemolj^sis,  and  it  is  relatively  stable 
over  periods  of  at  least  a  year.  Such 
sera  can  be  sterilized  by  pressure  filtra- 
tions  but  before  sterilization  by  filtra- 
tion care  should  be  taken  to  prevent 
bacterial  growth  and  resultant  produc- 
tion of  toxic  substance  in  the  serum. 

The  tissue  extract  now ,  commonly 
used  for  routine  tissue  cultures  of  cells 
from  many  species  is  made  by  extract- 
ing briefly  minced  embryonic  tissue 
with  an  equal  volume  of  isotonic  saline 
and  by  decanting  the  supernatant  solu- 
tion after  centrifuging.  To  eliminate 
living  tissue  cells  freeze  in  CO2  snow 
and  recentrifuge.  The  extract  loses 
potency  rapidly  and  should  therefore  be 
used  within  a  few  days  after  prepara- 
tion. 

A  current  source  of  embryo  extract  is 
chick  embryos  of  9  days  incubation. 
Where  facilities  of  a  local  slaughter 
house  are  available  some  workers  (Gey), 
find  it  convenient  to  employ  beef  em- 


TISSUE  CULTURE 


246 


TISSUE  CULTURE 


bryos  removed  from  the  uteri  by  aseptic 
methods.  Whatever  the  source  of  tis- 
sue, the  extract  must  be  prepared  with 
rigid  asepsis  because  no  means  of  steri- 
lization has  proved  satisfactory.  Fil- 
tration through  a  bacteriological  candle 
results  in  great  reduction  in  potency. 

Extracts  of  malignant  tissues  are  very 
effective  in  stimulating  growth  of  some 
types  of  cells.  Earle,  W.  R.,  Arch.  f. 
exp.  Zellforsch.,  1937,  20,  140  for  ex- 
ample, using  horse  serum  and  extracts 
of  Walker  256  rat  mammary  carcinoma, 
in  certain  instances  obtained  media 
which  had  no  stimulative  action  on  rat 
subcutaneous  fibroblasts  and  on  the  epi- 
thelium of  normal  rat  mammary,  but 
which  had  a  very  great  stimulative 
effect  on  the  growth  of  cells  of  car- 
cinoma from  the  rat  mammary  epi- 
thelium. 

In  exploring  the  possibilities  of  grow- 
ing any  cell  type  various  percentage 
combinations  of  embryonic  extract  and 
serum  are  among  the  first  media  to  be 
tried;  but,  in  attempting  to  make  syn- 
thetic culture  media  for  particular  kinds 
of  cells,  tissue  and  protein  hydrolysates, 
amino  acids,  vitamin  and  hormone 
preparations  and  nucleic  acids,  have 
been  used  (Fischer  and  Parker). 

Because  of  limited  knowledge  of  the 
nutritional  requirements  of  cells  it  is 
not  possible  to  obtain  survival  or 
growth  for  any  considerable  period. 
As  knowledge  increases  we  can  reason- 
ably expect  greater  facility  in  the 
growth  of  many  kinds  of  cells,  and  an 
increased  ease  in  obtaining  an  optimal 
selective  growth  of  any  one  cell  type  in 
a  mixed  tissue  with  the  suppression  of 
other  unwanted  types. 

Slide  Cultures.  The  tissue  clump  is 
planted  in  a  drop  of  plasma  and  nutrient 
culture  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 
large  coverslip  until  contact  of  the 
coverslip  with  the  vaseline  ring  on  the 
slide  seals  the  preparation:  For  cover- 
slips  of  the  size  cited  a  rectangular  hol- 
low ground  slide  55  x  80  nun.  by  6  nmi. 
thick  and  with  a  polished  concavity 
40  nxm.  in  diameter  and  about  4.5  mm. 
deep  at  its  deepest  point  is  convenient 
(these  Pj're.x  slides  may  be  obtained  on 
special  order  from  Bausch  and  Lomb 
Optical  Company). 

Such  preparation  can  then  be  given 
an  outer  edge -seal  of  paraffin.    By  us- 


ing very  thin  coverslips,  and  if  neces 
sary,  by  even  omitting  the  small  innei 
slip,  the  cells  can  be  critically  studiec 
with  high  numerical  aperture  lenses. 
In  fact  this  type  of  preparation  is  prob- 
ably the  best  for  work  with  short  work- 
ing 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  prepa- 
ration is  onlj'  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  iso- 
tonic saline,  fresh  nutrient  fluid  added 
and  the  whole  resealed  onto  a  new  outer 
coverslip  and  hollow  ground  slide.  By 
this  partial  renewal  of  the  culture 
medium  everj^  2  or  3  days  such  a  culture 
may  be  carried  for  a  long  time.  Pogo- 
geff,  I.  A.,  and  Murray,  M.  R.,  Anat. 
Rec,  1946,  94,  321-335,  report  carrying 
such  cultures  of  muscle  cells  for  over 
a  year.  When  the  cell  clump  gets  too 
large  a  small  fragment  of  it  may  be  re- 
explanted  to  a  new  culture. 

Such  slide  cultures  may  be  killed  and 
fi.xed  and  stained  in  tolo.  For  even 
more  e.xacting  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  extremely 
thin  sheets.  These  are  suitable  for 
critical  microscopic  study  of  chromo- 
somes, mitochondria,  Golgi  apparatus 
and  other  cellular  components.  If 
grown  on  thin  plastic  sheets  they  can 
even  be  fixed  and  examined  with  the 
electron  microscope  (Porter,  K.  R., 
Anat.  Rec,  1946,  94,  490). 

Slide  culture  technique  is  recom- 
mended for  beginners;  but,  when  it  is 
necessary  to  carry  slide  cultures 
through  consecutive  changes  of  media, 
it  is  difficult  to  maintain  sterility. 
When  dangerous  infectious  agents  are 
employed,  slide  cultures  should  be 
handled  with  great  care  to  avoid  hazard 
to  the  operator,  as  the  preparations 
frequently  develop  leaky  seals,  and  be- 
cause the  coverslip  used  for  very  high 
resolution  microscopic  study  is  ex- 
tremely fragile.  Accurate  control  of 
conditions  over  long  periods  of  time  is 
more  difficult  in  slide  culture  than  in 
Carrel  flask  cultures  containing  more 
media  which  can  be  changed  with  less 
disturbance  of  cells  embedded  in  the 
fibrin  clot. 

Roller  Tube  Cultures  introduced  by 
Gey,  G.  O.  and  M.  K.,  Am.  J.  Cancer, 
1936,  27,  45.  The  culture  vessel  is  a 
round  bottle,  or  a  pyrex  test  tube  about 


TISSUE  CULTURE 


247 


TISSUE  CULTURE 


15  X  180  mm.  Using  the  tube  as  an 
example,  a  thin  laj'er  of  plasma  and 
nutrient  medium  is  placed  over  the 
inner  surface  to  within  5  cm.  of  the 
mouth,  and  while  this  plasma  layer  is 
still  liquid  numerous  small  cell  dumps 
to  be  grown  are  embedded  in  it.  After 
the  plasma  has  clotted  about  1  cc.  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  end  of  a  slowly  rotating  drum  so 
that  as  the  drum  rotates  about  its  axis 
the  supernatant  culture  fluid  is  slowly 
washed  over  the  clumps  of  cells  em- 
bedded 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  are  separated  from  the  plasma 
mass  lining  the  culture  tube  by  pushing 
them  loose  with  a  pipette  tip,  removed 
from  the  culture  tube  by  means  of  the 
pipette,  cut  to  convenient  size,  and  sub- 
planted  to  new  cultures. 

This  type  of  culture  is  better  adapted 
than  the  slide  culture  for  routine  grow- 
ing of  large  numbers  of  cell  clumps, 
since  every  test  tube  can  accommodate 
20  or  more  clumps,  each  of  them  at  least 
as  large  as  that  in  a  slide  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  probably  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  preparation.  Moreover  the 
rotating  mechanism  for  the  roller-tube 
unit  is  cheaply  and  easily  constructed 
while  the  cost  of  routine  culture  tubes 
(pyrex  test  tubes)  is  only  a  few  cents. 

But  the  use  of  "roller-tube"  cultures 
is  not  without  its  limitations.  The  thin 
layer  of  plasma  clot  used  is  often  eroded 
through  by  the  cells  so  that  frequent 
patching  of  the  clot  by  fresh  addition 
of  plasma  becomes  necessary.  This 
patching  interferes  with  accuracy  in 
control  of  conditions  of  the  culture  and 
the  cultures  themselves  are  not  infre- 
quently lost  by  eroding  entirely  out  of 
the  clot.  The  curved  tube  surface,  the 
thick  tube  wall,  and  the  distance  of 
separation  v/hich  they  make  necessary 
between  microscopic  objective  and  con- 
denser complicate  examination  and  limit 
it  to  low  magnifications.  Thishandicap 
can  be  only  partiallj'  overcome  by  sub- 
culturing  to  slide  cultures  to  facilitate 
detailed  microscopic  study.  The  sub- 
culturing  is  objectionable  because  the 
orientation  of  cells  is  interfered  with 
and  new  conditions  require  control. 
Though    numerous    explants    are    pro- 


vided in  roller  tube  cultures  each  of 
them  is  usually  small  so  that  the  total 
volume  of  explanted  tissue  is  not  large. 
The  necessity  of  handling  many  cell 
clumps  makes  the  initial  planting  of  the 
cultures  relatively  slow. 

Carrel  Flask  Cultures,  Carrel,  A.,  J. 
Exper.  Med.,  1923, 38,  407.  These  flasks 
are  made  in  many  sizes  of  which  the 
"D"  3.5  flask  will  be  described.  It  is 
disc  shaped,  3.5  cm.  in  diameter,  with 
topand  bottom  blown  plane  and  parallel, 
each  about  §  mm.  in  thickness.  The 
sides  of  the  flask  are  vertical,  about  10 
mm.  high,  so  that  the  total  separation 
from  top  to  bottom  of  the  flask  is  about 
10  mm.  A  side  neck  of  10  mm.  internal 
diameter,  and  25  to  35  mm.  long  pro- 
jects out  from  the  side  wall  of  the  flask 
and  slopes  upward  at  about  35°  to  the 
bottom  of  tlie  flask. 

The  cell  clump  is  planted  on  the  bot- 
tom of  the  flask  in  a  layer  of  solid  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).  Sometime  after  this  has 
clotted  1  to  2  cc.  of  the  same  fluid  cul- 
ture medium  is  added,  the  flask  sealed 
with  a  rubber  stopper  and  incubated  as 
usual .  About  3  times  weekly  the  prepa- 
ration is  unsealed,  the  old  culture  me- 
dium removed,  the  solid  clot  with  its 
contained  cells  soaked  for  about  15  min. 
in  isotonic  saline,  this  saline  removed, 
fresh  nutrient  fluid  added  and  the  flask 
resealed.  At  intervals  of,  say,  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 
and  these  reinoculated  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  car- 
ried out  at  the  National  Cancer  In- 
stitute, the  actual  time  required  for  2 
relatively  new  operators  to  wash  and 
renew  the  nutrient  medium  on  142  cul- 
tures is  70  min.  from  the  time  they  enter 
the  sterile  room  to  the  sealing  of  the 
last  culture  a  substantial  part  of  which 
time  is  spent  in  setting  up  of  apparatus 
and  preparation  of  solutions.  Each 
culture  flask  receives  one  explant  of 
about  4  to  4.5  mm.  width  and  15  mm. 
length,  while  the  thickness  of  the  ex- 
plant  is  only  the  thickness  of  the  culture 
sheet  of  the  previous  culture  generation. 
Since  only  one  culture  is  made  in  each 
culture   flask,  transplantation  is  rapid 


TISSUE  CULTURE 


248 


TISSUE  CULTURE 


and  growth  from  this  explant  will  often 
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.  "Patching" 
of  the  clot,  with  fresh  plasma  is  rarely 
necessary.  The  clot  is  of  such  thick- 
ness and  texture  that  it  can  be  slipped 
loose  from  the  flask  as  a  sheet,  and 
poured  out  onto  a  sterile  glass  plate, 
where  the  culture  can  be  easily  and 
accurately  cut  up  for  subinoculation 
by  means  of  a  Von  Graefe  No.  5  cataract 
knife.  Because  a  single  very  thin  strip- 
shaped  explant  is  placed  in  each  flask, 
actual  sub-planting  of  cultures  is  much 
more  rapid  than  with  the  roller  tube  prep- 
arations in  general  use  and  the  actual 
amount  of  tissue  probably  greater. 

If  desired  these  cultures  may  be  in- 
cubated on  slowly  rocking  shelves  but 
this  is  required  only  in  more  exacting 
studies  in  which  it  is  necessary  to  have 
the  whole  surface  of  the  culture  verj' 
uniformly  washed  with  the  fluid.  Such 
cultures  can  be  routinely  photographed 
at  magnifications  in  excess  of  200  to  400 
diameters  and  can  be  examined  regu- 
larly with  up  to  a  4  mm.  .65  KA.  achro- 
matic objective.  A  satisfactory  lens 
is  the  Bausch  and  Lomb  5.5  mm.  .65  NA. 
objective.  For  higher  numerical  aper- 
ture photographs,  subinoculation  must 
be  made  to  slide  cultures. 

Among  the  objections  to  employing 
Carrel  flasks  is  their  initial  cost.  (D 
3.5  flasks  are  now  quoted  at  about  $1.75 
each).  They  must  be  made  accurately 
and  can  only  be  obtained  from  a  few 
manufacturers.  (Satisfactory  flasks  of 
3.5  and  5.0  cm.  diameter  can  be  pur- 
chased on  specification  from  Otto  Hopf , 
Glassblower,  Upper  Black  Eddy,  Pa. 
and  from  E.  Machlett  and  Son,  220  E. 
23rd  St.,  New  York.)  When  the  re- 
search is  such  that  a  rocking  shelf  unit 
for  the  cultures  is  necessary  this  device 
is  mechanically  slightly  more  complex 
than  is  the  mechanism  of  the  roller 
tube  unit. 

Limitations  of  Tissue  Culture.  1. 
All  tissue  cultures  more  of  than  just  a 
few  hours  duration  must  be  planned  so 
as  to  establish  and  maintain  the  steril- 
ity of  the  cultures  and  of  the  media  used 
on  them.  This  often  greatly  increases 
the  difficulty  of  otherwise  simple  tech- 
nical operations.  Recent  introduction 
of  antibiotics  offers  help  to  the  worker 
to  obtain  sterile  cultures  from  infected 
tissues  such  as  skin.  The  new  "Selas" 
porcelain  filters  (Selas  Corporation  of 
America,  Philadelphia  34,  Pa.)  on  pre- 


liminary study,  give  promise  of  being 
useful  in  sterilization. 

2.  It  is  not  possible  at  present  to  es- 
tablish a  tissue  cell  strain  from  a  single 
cell.  Instances  have  been  described  in 
which  a  single  tissue  cell  grown  in  a 
culture  dish  with  other  cells  has  pro- 
liferated and  developed  into  a  colony, 
but  such  cultures  are  always  suspect  as 
having  possibly  received  cells  from  the 
other  colonies  in  the  dish. 

3.  Culture  conditions  cannot  be  con- 
sidered as  "normal"  to  any  cell  or  group 
of  cells,  nor  can  they  be  made  so.  Any 
extrapolation  of  cell  or  tissue  behavior 
from  in  vitro  to  normal  in  vivo  condi- 
tions must  therefore  be  made  with 
reserve. 

4.  While  practically  any  tissue  cell 
can  probably  be  kept  viable  in  culture 
for  a  limited  time,  our  present  knowl- 
edge of  the  metabolic  and  other  cultural 
requirements  of  various  cell  types  is  so 
incomplete  that  only  a  limited  number 
of  cell  types  have  as  yet  been  kept 
growing  in  culture  for  a  year.  Cells 
from  normal  adult  tissue  are  generally 
more  difficult  to  grow  than  embryonic 
cells,  particularly  when  the  adult  cells 
are  highly  specialized  and  are  under 
complex  endocrine  control,  as  in  the 
case  of  normal  mammary  epithelium. 
Even  in  the  adult  animal,  however,  the 
relatively  undifferentiated  mesoderma,! 
cells,  loosely  termed  "fibroblasts"  can 
be  readily  grown  from  many  species  and 
are  well  suited  to  experiments.  In  any 
study  when  an  easily  grown  cell  tj'pe 
(the  "fibroblast")  can  be  used  with 
equal  value  to  a  cell  type  which  has  not 
been  satisfactorily  grown,  the  more 
easily  grown  cell  type  is  obviously  the 
one  of  choice  leaving  the  more  difficult 
cell  types  until  special  studies  can  de- 
fine and  remove  difficulties  complicating 
their  gi'owth  in  culture.  Incomplete 
cells,  such  as  adult  erythrocytes,  which 
lack  a  nucleus,  or  certain  nerve  cells 
which  lack  a  centriole,  of  course  cannot 
be  expected  to  proliferate  in  vitro. 
Malignant  cells  can  frequently  be  grown 
for  indefinite  intervals  in  cases  where 
attempts  to  grow  normal  tissues  of  the 
same  sort  have  not  yet  succeeded.  For 
instance,  growth  of  the  normal  mam- 
mary gland  epithelium  has  been  possible 
as  yet  for  only  limited  periods,  but  cer- 
tain mammary  carcinomas  have  been 
grown  for  a  year  or  longer  (Earle,  W.  R., 
Am.  J.  Cancer,  1935,  24,  566). 

5.  The  media  now  used  for  growing 
cultures  for  extended  intervals  of  time 
are  made  up  of  such  complex  sub- 
stances as  serum,  chick  embryo  extract, 
liver  digest,  etc.,  which  canuot  be  de- 
fined chemically.    There    s  at   3    sent 


TISSUE  CULTURE 


249 


TISSUE  CULTURE 


apparently  no  satisfactory  chemically 
defined  culture  medium  for  the  healthy 
survival  and  active  growth  of  any  mam- 
malian tissue  in  vitro  for  an  extended 
period  of  time,  although  there  is  a  great 
(.leal  of  research  in  progress  with  the  aim 
of  obtaining  such  a  medium  (White, 
P.  R.,  Anat.  Rec,  1946,  94,  61). 

6.  Only  small  amounts  of  tissue  can 
be  cultured  by  present  methods.  How- 
ever, by  using  thin,  strip-shaped  ex- 
plants,  cultures  of  25  or  30  mm.  diam- 
eter may  be  more  or  less  routinely 
grown  from  at  least  several  cell  types 
(Earle,  W.  R.,  Arch.  Path.,  1939,  27, 
88).  Workers  with  roller  tube  and 
Carrel  flask  cultures  frequently  have 
been  impressed  with  the  extensive 
sheets  of  cells  arising  from  proliferation 
of  isolated  cells  which  have  scattered 
over  the  surface  of  the  medium  by  lique- 
faction of  the  plasma  around  the  parent 
culture.  Probably  more  extended 
study  of  the  explant,  the  cell  support 
and  the  nutrient  medium  will  permit 
growing  routine  cultures  of  far  larger 
tissue  masses,  and  allow  further  uti- 
lization of  the  use  of  tissue  culture  tech- 
niques in  research  on  vaccines  and  endo- 
crine products. 

7.  When  a  fragment  of  animal  tissue 
is  first  explanted  to  culture  the  proc- 
esses of  adaptation  to  the  new  condi- 
tions are  often  very  complicated. 
Some  cell  types  die  rapidly,  others  more 
slowly  while  still  others  are  finally 
eliminated  apparently  by  overgrowth. 
Meanwhile  surviving  cells  are  being 
affected  by  products  of  necrosis  and  by 
residual  tissue  substances  brought  over 
from  the  parent  organism  by  the  ex- 
planted  tissue.  The  cycle  of  adapta- 
tion and  final  dominance  of  a  cell  strain 
able  to  survive  under  the  culture  condi- 
tions may  last  for  months. 

For  many  purposes  short-term  cul- 
tures of  freshly  e.xplanted  tissues  are 
entirely  adequate  and  satisfactory. 
For  other  purooses,  however,  the  study 
of  such  cultures  carried  only  a  few  hrs., 
or  a  few  days,  may  be  ineffective  and 
misleading  in  reflecting  only  the  initial 
reaction  of  the  cells  to  the  new  culture 
conditions.  Where  this  is  the  case  re- 
sort must  be  made  to  the  use  of  more 
exacting  culture  methods  which  permit 
longer  experimental  periods  and  the 
use  of  relatively  stable  cell  strains  iso- 
lated and  maintained  in  vitro. 

8.  The  technique  for  long-term  tissue 
cultures  under  even  incompletely  con- 
trolled experimental  conditions  requires 
consideraole  equipment  and  technical 
assistance  and  an  extensive  period  of 
training  both  in  operation  of  equip- 
ment and  in  the  maintenance  of  rigidly 


aseptic  techniques.  It  is  necessary  to 
establish  a  fundamental  working  knowl- 
edge of  the  behavior  of  cells  iu  vitro. 
Therefore  much  valuable  time  and  dis- 
appointment will  be  saved  if,  in  the 
initiation  of  any  extensive  tissue  culture 
program,  adequate  training  of  the 
worker  in  an  established  tissue  culture 
laboratory  is  first  insured. 

Measurement.  1.  The  most  direct 
method  is  to  measure  growth  by  in- 
crease in  weight  of  the  culture;  but 
without  a  fibrin  matrix  growth  of  the 
culture  is  often  erratic,  while  if  the 
cells  are  grown  in  such  a  matrix  extreme 
difficulty  is  experienced  in  routinely 
separating  the  cells  from  the  matrix. 
The  data  relate  to  cell  mass,  not  to  cell 
number. 

2.  Another  way  is  to  estimate  change 
in  area  of  culture,  the  width  being  used 
as  an  index.  This  is  satisfactory  for 
experts  observing  long-term  cultures, 
but  interpretation  is  difficult.  An  in- 
crease in  culture  diameter  may  be  due 
to  increase  in  mitosis  or  in  cell  migra- 
tion. In  some  cases  measurement  of 
growth  by  determination  of  surface 
area  is  not  feasible.  The  action  of  20- 
methylcholanthrene  on  mouse  sub- 
cutaneous fibroblasts  so  alters  the  archi- 
tecture and  cell  density  of  the  cultures, 
the  rate  of  cell  migration  that  use  of  the 
culture  width  or  area  as  an  index  of  cell 
proliferation  is  grossly  misleading. 

3.  By  determining  the  number  of  cells 
seen  in  mitosis  in  a  culture  area  relative 
to  the  total  number  of  cells  seen  in  that 
area  an  estimate  may  be  made  of  the 
relative  frequency  of  cell  mitotic  pro- 
liferation. This  method  is  valid  only 
if  it  is  clearly  shown  that  the  experi- 
mental conditions  do  not  disturb  the 
duration  of  mitosis,  or  if  correction  is 
made  for  such  disturbance.  Possible 
complication  from  a  diurnal  variation 
in  the  rate  of  cell  mitosis  must  also  be 
eliminated.  The  value  of  the  method 
may  be  increased  by  development  of 
comparative  microcinematographic 
techniques  and  also  by  use  of  Chalkey's 
methods  for  statistical  analysis  of  tissue 
elements  (Chalkey,  H.  W.,  J.  Nat. 
Cancer  Inst.,  1943,4,  47). 

4.  Chemical  methods  estimating  cul- 
ture growth  by  determination  of  in- 
crease in  ash  content,  aerobic  or  an- 
aerobic oxygen  consumption,  glycolysis, 
etc.  are  at  present  not  entirely  satis- 
factory but  measurement  of  intake  of 
radioactive  isotopes  may  prove  helpful. 
See  in  this  connection  Cohn,  W.  E.,  and 
Bones,  A.  M.,  J.  Gen.  Physiol.,  1945, 
28,  449. 

Many  factors,  often  unknown,  influ- 
ence    results.     Sometimes     these     are 


TISSUE  CULTURE 


250 


TRACHEA 


recognized  only  after  data  have  been 
collected  over  a  considerable  time. 
Consequently  experimental  conditions 
should  be  systematically  and  com- 
pletely recorded  in  a  uniform  way,  so 
that  their  influence  can  be  compared  in 
many  investigations.  Descriptions  of 
cells  should  be  supplemented  by  photo- 
graphs and  cinematographic  records, 
carefully  fixed  and  stained  slides,  or 
other  objective  permanent  records,  pre- 
pared from  materials  as  accurately 
standardized  in  handling  as  possible. 

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.  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  (Auat.  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. 


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 
particles  can  easily  be  recognized  as  a 
black  accumulation  in  the  phagocytes 
and  its  amount  can  be  determined 
chemically  in  fairly  large  bone  samples 
by  a  method  detailed  by  the  author. 

Toluene  Red.  Dimethyldiamidotoluphen- 
azin.     See   Platelet   staining   solutions. 

Toluidin  Blue  O  (CI,  925)— methylene  blue 
T  50  or  T  extra — Employed  very  widely. 

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. 

Tolyi  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  IIL 

Torulosis,  see  Blastomycosis. 

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 
Srnears).  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  mth  standard 
preparations  of  Sarcocystis  and  En- 
cephalitozoa. 

Trachea.     Excellent  experimental  methods 


TRACHEA 


251 


TREPONEMA  PALLIDUM 


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-Grimwald,  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  Tooth  Germs,  Anterior 
Chamber  of  Eye. 

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  signifj' 
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 
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- 
onemas,  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.  m-ed.  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- 
nicacid,  5gm.;  liquid  carbolicacid,  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.  i\Iix 
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 


TREPONEMA  PALLIDUM 


252 


TRICHINELLA  SPIRALIS 


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  + 
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  5m  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^  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 beforehand  by  pipetting  into 
a  beaker:  (1)  15  cc.  5%  aq.  gelatin  at 
56°C.;  (2)  3  cc.  2%  aq.  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 
paraffin  with  xylol.  Pass  through  2 
changes  abs.  ale  Treat  \-\^  min.  in 
4%  uranium  nitrate  in  abs.  ale,  20  cc. ; 
25%  gum  mastic  in  abs.  ale,  40-50  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. 

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 


TRICHINELLA  SPIRALIS 


253 


TRYPANOSOMES 


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., 
Techn.  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). 

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.; 
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,  Roche  ter,  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 
(C2H5)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. 

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. 

Trypan  Blue  (CI,  477)— azidine  blue  3B, 
benzamine  blue  3B,  benzo  blue  3B, 
chlorazol  blue  3B,  Congo  blue  3B, 
dianil  blue  II3G,  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.  Plaarmac.  and 
Expcr.  Therap.,  1941,  73,  405-411). 

Trypan  Red  (CI,  438).  So  named  because 
of  influence  on  Trypanosome  infections 
(G.  trypanon,  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 


TRYPANOSOMES 


254 


TUNGSTIC  ACID 


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.  Kelser's 
medium,  described  fully  by  Craig,  seems 
to  be  the  best.  See  references  supplied 
by  him  (p.  199)  to  culture  in  chick 
embryoes. 

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.  CaClc,  0.1 
gm.  MgCla,  0.05  gm.  NaUPjOj,  1  gm.. 
NatlCOa,  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  appro.xi- 
mately  1  week.  Subculture  at  6-8 
week  intervals. 

Trypsin,  a  gelatin  plate  method  as  described 
under  Pepsin  but  slightly  modified  is 
recammended. 


Tryptagar,  see  Bacteria  Media. 
Tryptophane    Reaction.     The    procedure   of 

Serra  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 
(d  =  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  NaNOo.  The  reagent 
is  prepared  freshly  every  day  and  the 
nitrite  solution  must  also  be  freshly 
made. 

"4.  Mount  directlj^  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  ISO).  For  visible  and 
red  rays  employ  3.5  mm.  "Uvet"  lens 
and  2%  aq.  copper  sulphate.  Bacilli, 
golden  yellow  rods  in  violet  fluorescent 
background.  See  Sputum. 
Tungstic  Acid,  a  stable  solution  (Abraham- 
son,  E.  M.,  Tech.   Bull.,  1940,  1,  75). 


TURNBULL  BLUE 


255 


ULTRAVIOLET 


Turnbull  Blue  reaction  for  iron.  Same  as 
Berlin  blue  except  use  K  ferri cyanide 
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  3'^ellow  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  v\-as  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  "roj^al  color"  em- 
ployed bj^  royalty  in  Persia,  Babylon, 
Media  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 
Tj'rian  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 
IMedieval  Dyes.  Brooklyn:  Chemical 
Publishing  Co.,  Inc.,  1944,  95  pp.). 

Tyrode  solution.  NaCl,  0.8  gm.;  KCl, 
0.02  gm.;  CaClj,  0.02  gm.;  MgCh,  0.01 
gm.;  NaHjPO^,  0.005  gm.;  NaHCOs, 
0.1  gm.  (giving  pli  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:  MgS04,  7.5  g.; 
MgClz,  5.5  g. ;  NazSOi,  7.0  g. ;— dissolved 


in  85  ml.  of  distilled  water  to  which 
12.5  g.  of  concentrated  H2SO4  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  squashed,  if  necessary. 

"The  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  tj'^ro- 
sine  in  the  proteins." 
Ultracentrifuge,  see  Centrifugation. 
Ultramicroscope,  see  Darkfield. 
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.  Because  the  wave 
length  of  ultraviolet  light  is  much 
shorter  than  that  of  visible  light  greater 
resolution  is  possible  by  its  use  (ap- 
proximately 0.1m)-  The  lenses  must  be 
of  quartz  and  the  image  must  be  located 
and  photographed  which  is  cumber- 
some. It  was  employed  chiefly  for 
localization  of  substances  like  nucleic 
acid  which  strongly  absorb  ultraviolet 
light.  Since  greater  resolution  and  a 
visible  image  on  a  fluorescent  screen 
can  be  secured  by  employing  an  Elec- 
tron Microscope  the  ultraviolet  instru- 
ment is  seldom  used. 
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 


ULTRAVIOLET 


256 


UREASE 


(R.  W.  G.  and  A.  L.,  J.  Exper.  Med., 
1931,  54,  449-451),  because  some  pro- 
teins absorb  ultraviolet  more  strongly 
than  others,  details  can  be  brought  out 
with  it  not  revealed  by  visible  light. 
This  they  demonstrate  by  experiments 
with  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. 

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  cc;  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;  collagenic 
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-Andre'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 
^-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. 
Employed  by  HoUande  for  bacteriocytes 
of  Periplaneta  orientalis  L  (Hollande, 
A.  C.,  Bull.  d'Histol.  AppL,  1931,  8, 
176-178). 

Urea.  IVIany  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. 
methjd  alcohol  and  20  cc  glacial  acetic 
acid.  Lison  (p.  169)  criticizes  these 
methods  severely. 

Urease.  A  method  for  determining  the 
distribution  of  urease  in  the  gastric 
mucous  membrane  (pylorus  and  fundus) 
of  the  dog  has  been  described  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 


UREASE 


257 


UROBILIN 


think  it  very  unlikely  that  the  parietal 
cells  contain  urease. 

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^  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. 

Urinary  Casts,  staining  with  methyl  blue 
picric  acid.  To  sediment  from  centri- 
fuged  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  methyl  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,  erythrocj'tes, 
casts,  crystalline  materials,  bacteria 
and  so  forth. 

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 
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     crystals     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  eas}'.     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  bloody.     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  II.  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    an> 


UROBILIN 


258 


VAN  GIESON'S  CONNECTIVE 


urobilinogen  not  converted  into  uro- 
bilin bj'  adding  1-2  drops  of  Lugol's 
Iodine.  Then  H,dd  10  cc.  Schleslnger's 
Reagent,  filter  let  stand  1-2  lirs.  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. 

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. 

Vaginal  Smears.  On  the  basis  of  large 
experience  Papanicolaou  G.  N.,  J.  Lab. 
&  Clin.  Med.,  1940-41,  26,  1200-1205 
has    described    techniques    in    detail. 

1.  Fix  immediately  (before  drying) 
in  equal  parts  95%  alcohol  and  ether 
1-2  min.  Rinse  in  70%,  50%  alcohol 
and  in  aq.  dest.  Ehrlich's  hematoxylin 
(or  other  hematoxylin),  1-2  min.  Rinse 
in  aq.  dest.  Rinse  few  times  in  1% 
hydrochloric  acid  (may  be  omitted). 
Running  water,  5  min.,  or  aq.  dest.  100 
cc.  +  3  drops  cone.  aq.  lithium  car- 
bonate, 1  min.  Do  not  leave  slides  too 
long  in  running  water.  Rinse  in  aq. 
dest.  and  stain  for  2  min.  in  any  of  6 
combinations  of  stains  recommended. 
One  of  these  is  made  up  of  National 
Aniline  and  Chemical  Co.  dyes  in  0.5% 
aq.  sol.  as  follows:  Light  green  S.  F. 
yellowish,  12  cc. ;  orange  G,  24  cc,  acid 
fuchsin,  20  cc,  eosin  yellowish,  40  cc.  -f 
phosphomolybdic  acid  (Merck)  0.45 
gm.  Rinse  in  water.  Rinse  in  dioxan 
10-15  times  until  smears  are  clear. 
Pass  through  absolute  alcohol  to  xylol. 
Mount  in  clarite,  balsam  or  dammar. 
See  a  newer  procedure  by  Papanicolau, 
G.  N.,  Science,  1942,  95_,  438-439.  _ 

2.  A  shorter  method  is,  after  similar 
fixation  of  smears  brought  down  to  aq. 
dest.,  to  stain  them  2-3  min.  in  anilin 
blue,  water  soluble,  16  cc. ;  acid  fuchsin, 
23  cc. ;  orange  G,  17  cc. ;  eosin  yellowish, 
44  cc,  (all  of  5%  aq.  solutions)  +  phos- 
phomolybdic  acid  0.2  gm.,  and  phos- 
photungstic  acid,  0.2  gm.  Rinse  in 
water.  Rinse  in  dioxan  until  clear, 
then  through  absolute  alcohol  and 
xylol  to  clarite.  Nuclei,  red;  erythro- 
cytes, orange ;  cornified  cells,  red,  pink 
or  orange;  basophile  cells,  green  or  blue. 
Nuclei  not  as  dark  and  cell  outlines  as 
sharp  as  after  hematoxylin,  but  corni- 


fied cells  are  more  prominent  and  basal 
cells  more  transparent. 

3.  See  detailed  method  advised  by 
George  Svihla  for  the  rat  (Hartman, 
C.  G.,  Yale  J.  Biol.  &  Med.,  1944,  17, 
99-112). 

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.  + 
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, 
next  layer  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  cc. 

Van    Gehuchten's    mixture,    see    Carnoy'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 


VAN  GIESON'S  CONNECTIVE 


259 


VERHOEFF'S  ELASTIC  TISSUE 


95%  ale,  dehydrate,  dear  and  mount. 
Muscle  yellow,  collagcnic  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  grapliic  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). 

Verhoeflf'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 
mj^elin  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  stiiin :  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.  Counters  tain  in  0.2%  water 
sol.  eosin  in  80%  alcohol.  Dehydrate, 
clear  in  origanum  and  mount  in  balsam. 
Elastic  tissue,  black;  fibroglia,  myoglia, 
neuroglia,  mj'^elin  and  fibrin,  pink. 
Degenerated  elastic  tissue  (elacin) 
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  Alarchi'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 


VERHOEFF'S  ELASTIC  TISSUE 


260 


VIRUSES 


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. 

Vestibular  Apparatus,   see  Ear. 

Vesuvln,  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  difi"erent  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 
arid  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  (King, 
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.  Auat.,  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  extra vasated  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- 
ban and  Schwarzenberg,  1939,  384  pp. 
The  "ultra virus"  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 


261 


VITAL  STAINING 


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  cytoplasm 
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. 
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- 


VITAL  STAINING 


262 


VITAMINS 


tribution  of  the  dye  in  the  tissues. 
The  skin,  Icidneys,  adrenals,  liver, 
spleen  and  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 
d}^e  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.  Exper.  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.  IVIake  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,  Iridjgo-Carmine, 
Manganese  Dioxide,  Higgins'  Ink, 
Protargol  (silver).  Lampblack,  Leuco- 
Dyes,  Nuclei,  Titanium  Dioxide,  Tho- 
rium Dioxide,  Copper,  Platinum,  Iron, 
Mercury,  Lymphatic  Vessels. 
Vitamins.  (Revised  by  C.  Carruthers, 
Barnard  Free  Skin  &  Cancer  Hospital, 
St.  Louis,  May  29,  1946.)  Some  vita- 
mins are  susceptible  of  microscopic 
localization.  Deficiencies  in  most  of 
them  leave  structural  imprints  in  the 
tissues.  A  list  may  therefore  be  useful. 
Up-to-date  information  is  usually  given 
in  Annual  Review  of  Biochemistry. 
See  papers  by  P.  Gj^orgy  and  R.  A. 
Morton  in  the  re\dew  for  1942,  11,  309- 
364  and  365-390.  A  useful  background 
is  provided  by  Sherman,  H.  C,  Chemis- 
try of  Food  and  Nutrition,  New  York: 
Macmillan,  1941,  611  pp.  For  a  sum- 
mary of  tissue  changes  in  vitamin 
deficiencies  see  Wolbach,  S.  B.  and 
Bessey,  O.  A.,  Physiol.  Rev.,  1942,  22, 
233-289. 
A.  Growth  promoting,  anti -infective 
and  anti-xerophthalmic  vitamin. 
C20H29OH,  mol.  wt.  286.4.  The  term 
vitamin  A  is  also  applied  to  its  provita- 
mins: alpha-,  beta-  and  gamma,  caro- 
tene and  cryptoxanthin.  There  are 
two  tests  for  this  vitamin.  (1)  The 
antimony  trichloride  test  is  the  basis  of 
the  Carr-Price  reaction,  which  see,  as 
applied  to  mitochondria  of  hepatic  cells. 
When  the  mitochondrial  fraction  is 
separated  and  collected  by  Centrifuga- 
tion  the  vitamin  A  can  be  easily 
measured  in  it  as  the  Goerner's  have 
done  in  their  several  investigations 
(A.  and  M.  M.,  J.  Biol.  Chem.,  1937-38, 
122,  529-538;  ibid,  1939,  128,  559-565). 
This  test  also  has  been  employed  for 
vitamin  A  in  serum  the  colors  being 
checked  against  alizarin  solutions 
(Parker,  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  antimony  trichloride  test. 
(2)  Green  fluorescence.  Much  work  has 
been  done  on  the  identification  of  vita- 
min A  within  cells  bj''  its  characteristic 
but  short  lived  fluorescence  in  ultra- 
violet light.  Popper,  H.,  Proc.  Soc. 
Exp.  Biol.  &  Med.,  1940,  43,  133-136, 
234-236  advises  fixation  of  liver  in  10% 


VITAMINS 


263 


VITAMINS 


formalin  and  examination  of  frozen 
sections  with  Fluorescence  Microscope 

wittiin  24  hrs.  The  green  fluorescence 
fades  during  irradiation  especially  in 
the  Kupffer  cells.  The  same  fluo- 
rescence is  found  in  epithelial  cells  of 
fascicular  and  glomerular  zone  of 
adrenals  (but  it  is  absent  in  adrenals  of 
newborn  infant),  in  the  cells  of  the 
corpus  luteum,  interstitial  cells  of  the 
testis  and  several  others.  Greenberg, 
R.  and  Popper,  H.,  J.  Cell.  &  Comp. 
Physiol.,  1941,  18,  269-272  report  that 
vitamin  A,  gives  "striking  green  and 
quickly  fading"  fluorescence  and  A2 
"faint  yellow-brown  and  slowly  fading." 

B.  Complex  contains  many  factors. 

Nicotinic  acid  and  nicotin  amide. 
An  excellent  chemical  method  for 
quantitative  determination  of  nicotinic 
acid  has  been  advocated  by  Dam,  W.  J. 
and  Handler,  P.,  J.  Biol.  Chem.,  1941, 
140,  _201-213_,  755-762.  As  to  tissue 
localization  it  is  reported  by  the  same 
authors  that  nicotinic  acid  exists  as 
part  of  nucleotide  molecules  (see 
Pentose  Nucleotides)  but  in  muscle 
or  renal  cortex  most  of  it  occurs  in  some 
other  form.  The  status  of  the  fluo- 
rescent substances  present  in  the  urine 
of  pellagrins  is  not  clear  (Najjar,  V.  A. 
and  Cleckley,  H.  M.,  Proc.  Soc.  Exp. 
Biol.  &  Med.,  1941,  48,  413-414). 

Pantothenic  acid.  Filtrate  factor. 
Factor  W.,  Anti-grey  hair  factor. 
Localization  as  between  blood  plasma 
and  cells  apparently  is  possible  (Pear- 
son, P.  B.,  J.  Biol.  Chem.,  1941,  140, 
423-426. 

Choline.    No    histochemical   method. 

Bi.  Thiamine  hydrochloride;  thiamin 
chloride,  anti-BeriBeri  or  antineuritic 
vitamin;  Aneurine,  Betabion,  Beta- 
toxin,  Oryzaniu,  Torulin.  C12H18, 
ON4SCI2,  mol.  wt.  337.26.  There  is  no 
microchemical  test  for  thiamin  but 
tissue  analysis  reveals  the  curious  fact 
that  the  amount  in  the  adrenal  cortex 
of  the  bull  is  more  than  7  times  that  in 
the  medulla  while  in  the  cow  the  medulla 
contains  1.9  times  as  much  as  the  cortex 
(Wright,  L.  D.,  et  al.,  Univ.  Texas 
Publ.,  1941,  4137,  38-60). 

B2.  Vitamin  G,  Riboflavin;  Lactoflavin. 
C17H20O6N4,  mol.  wt.  376.19.  No  micro- 
scopic methods  are  available  but  a 
microbiological  technique  for  riboflavin 
has  been  described  by  Snell,  E.  E.  and 
Strong,  F.  M.,  Univ.  Texas  Pub.,  1941, 
4137,  11-13  which  Gyorgy  says  results 
in  satisfactory  agreement  with  those 
secured  in  other  ways.  Riboflavin  can 
now  be  determined  by  polarographic 
analysis  (Lingane,  J.  J.,  and  Davis, 
O.  L.,  J.  Biol.  Chem.,  1941,  137,  567- 
574.     Sherman   (p.  373)  states  that  the 


respiratory  enzyme  (Warburg's  yellow 
enzyme)  is  a  combination  of  riboflavin 
phosphate  and  protein  (see  Cyto- 
chrome). 

B3,  4,  6.  Data  insufficient.  See  Sher- 
man   (p.  390). 

Be.  Pyridoxin,  adermin,  antidermatitis 
vitamin.  CgHuNOs,  mol.  wt.  169.18. 
This  essential  nutrilite  apparently  oc- 
curs in  tissues  to  a  large  extent  in  bound 
form.  The  trouble  with  microbiological 
methods  of  analysis  is  that  it  may  be 
only  incompletely  extracted  as  noted 
by  Gyorgy. 

C.  Antiscorbutic  vitamin,  Cebione,  Re- 
doxon.  CeHgOe,  mol.  wt.  176.06. 
Bourne  (Anat.  Rec,  1936,  G6,  369-385) 
has  made  a  critical  study  of  cytological 
methods  for  the  detection  of  vitamin  C. 
The  technique  recommended  is  based 
on  the  assumption  that  the  only  sub- 
stance other  than  vitamin  C  capable 
of  reducing  an  acid  silver  nitrate  solu- 
tion 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.  sol.  of  silver  nitrate 
to  which  5  cc.  acetic  acid  is  added  for 
each  100  cc.  for  a  few  minutes.  The 
vitamin  C  granules  blacken.  After 
w'ashing  in  aq.  dest.  fat  may  be  stained 
in  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 
advises:  Fresh  tissue  is  subjected  to 
glacial  acetic  acid  vapor  for  several 
minutes.  Cut  into  ver}^  thin  slices 
and  put  in  atmosphere  of  hydrogen 
sulphide  for  15  min.  All  vitamin  C 
is  thereby  converted  to  reduced  form. 
Remove  hydrogen  sulphide  by  keeping 
in  partial  vacuum  for  10  to  30  min. 
followed  by  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  hydrogen  sulphide  treat- 
ment, to  momentarily  wash  the  section, 
then  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--U,  75,  251-264  for  methods  of 
demonstrating  vitamin  C  in  chick 
embryos. 

Modification  of  Giroud  and  Leblanc 
silver  method  (Tonutti,  E.,  Proto- 
plasma,  1938,  31  (1),  151-158).  Briefly 
wash  tissue  in  5.4%  aq.  levulose.  10% 
aq.  AgNOj  -f-  2  drops  glacial  acetic  per 


VITAMINS 


264 


WALKER'S  METHOD 


cc,  up  to  30  min.  Rinse  in  aq.  dest. 
30  15-min.  3%  aq.  NajSjOs,  15-30  min. 
Rinse  in  aq.  dest.  15-30  min.  All  this 
in  dark  room  with  red  light.  Change 
to  70%  alcohol  and  imbed  in  paraffin. 
Counterstain  with  "Kernechtrot"  and 
light  green. 

A  photometric  method  for  quantita- 
tive determination  of  vitamin  C  in  very 
small  amounts  of  epidermis  has  been 
worked  out  by  Carruthers,  C,  Indust. 
and  Engin.  Chem.,  1942,  14,  826-828. 

D.  According  to  Sherman  (p.  430)  there 
are  probably  at  least  10  such  substances 
having  antirachitic  potency  of  which  5 
are  (in  1940)  recognized  fairly  clearly 
as  chemical  individuals,  D2  and  D3 
being  of  great  importance.  Action  is 
measured  histologically  by  the  Line 
Test. 

Di.  is  molecular  compound  of  D2  and 
luminsterol  which  is  first  product  of 
irradiation  of  ergosterol  with  ultra- 
violet light. 

Dj.  Calciferol,  C28n440  produced  by  ir- 
radiation or  ergosterol. 

D3.  Antirachitic  vitamin.  C27H44O,  mol. 
wt.  384.6.  This  is  activated  7-dehydro- 
cholesterol. 

E.  Antisterility  vitamin,  a  Tocopherol, 
C29H5PO2,  mol.  wt.  430.4.  The  2  other 
vitamin  E  factors  are  /?  and  7  tocopherol. 

F.  The  designation  of  vitamin  F  was 
originally  applied  to  essential  fatty  acid 
but  it  has  not  been  officially  accepted. 

G  =  B2. 

H.  This  term  as  Sherman  (p.  393)  points 
out  has  been  used  in  at  least  3  ways. 
It  is  considered  to  be  Biotin. 

Ki  is  the  first  form  of  the  antihemor- 
rhagic  vitamin  to  be  isolated  by  Dam 
(see  Dam,  H.,  Helv.  chim.  Acta,  1939, 
22,  310-313).  It  is  2  methyl-3-phytyl- 
1 ,4-naphthoquinone. 

K2  is  the  second  isolated  by  Doisy,  et  al. 
(see  Brinkley,  S.  B.,  MacCorquodale, 
D.  W.,  Thayer,  S.  A.  and  Doisy,  E.  A., 
J.  Biol.  Chem.,  1939,  130,  219-234). 
It  is  the  same  except  that  there  is  a 
longer  more  unsaturated  side  chain. 
Neither  of  the  two  can  be  localized 
histologically  but  we  may  expect  histo- 
logical studies  of  their  action. 

M.  An  unknown  factor  said  to  be  essential 
to  the  nutrition  of  monkeys  (Day,  P.  L., 
et  al.,  J.  Exp.  Med.,  1940,  72,  463-^77). 

P.  Permeability  vitamin,  citrin,  said  to 
be  essential  for  maintenance  of  walls  of 
small  blood  vessels.  For  a  discussion 
of  vitamin  P  as  measured  by  capillary 
fragility  see  Rapaport,  H.  G.,  and  Klein, 
S.,  J.  Pediatr.,  1941,  18,  321-327. 
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, 
p.  39),  cell  volume  is  a  function  of  the 
number  of  contained  osmotically  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  (Jacobs,  M.  H.,  Biol.  Bull., 
1930,  58,  104).  The  simplest  way  to 
obtain  ratio  for  cytoplasmic  and  nuclear 
volumes  is  to  outline  nuclei  and  cyto- 
plasms on  kodaloid  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.  =  16?  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. 
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." 
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 


WALKER'S  METHOD 


2G5 


WEIGERT  METHOD 


technique  for  routine  diagnostic  work 
by  Craig,  p.  55.  To  make  the  stain 
dissolve  1  gni.  hematoxylin  crystals  in 
300  CO.  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  xjdol  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. 
Ptecommended  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 
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 


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- 
tails about  washing  see  the  individual 
fixatives. 

Wasserblau,   see   Brazilin-Wasserblau. 

Water  Absorption  by  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 
cc.  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  chromatc  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  m3'elin  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 


WEIGERT  METHOD 


266 


WEIL'S  METHOD 


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  directlj^  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  100  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  35|  ounces 

1  gram  =  155  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- 
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. 


WEIL'S  METHOD 


267 


WO.AJD 


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  forri- 
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 
bv  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  responsibilitj^  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. 

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 
tliat  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  lias  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  Clmmberd,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.    Sec  Cell  Membranes. 

Whole  Mounts  of  tissues  which  are  fairly 
thick  are  often  very  useful.  See  Elood 
Vessels,  Cartilaginous  Skeleton,  Cor- 
rosion Preparations,  Epidermis,  In- 
sects, Mammary  Glands,  Nerve  End- 
ings, Ossification,  etc. 

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  min. 
This  can  be  omitted  after  phosphomolyb- 
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  S.1%  aq.  NH4OH  drop  by 
drop  to  5  cc.  10.2%  aq.  AgNOs  until 
brown  ppt.  is  just  dissolved.  Then  add 
5  cc.  3.1%  aq.  NaOH  and  sufficient 
NH4OH  to  just  dissolve  ppt.  Make  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  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. 

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 


WOAD 


268 


YEASTS 


flints  in  many  patterns  and  had  rubbed 
into  them  anil  of  the  vvoad  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  b}^ 
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  B  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  fairly  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  Mdunkin-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  directlj^  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. 

Xanthin,  see  Phosphine. 

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  e.xpose  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 
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." 

Xenon,  see  Atomic  Weights. 

XL  Carmoisine  6R,  see  Chromotrope  2R. 

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 


269 


ZYMONEMA  DERMATITIDIS 


Yellow M,  seeMetanil  Yellow. 

X-ray  Diffraction  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  ju  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  liarm  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  12hrs. 
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  that 
produces  an  enzyme  (G.  zj^me,  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 
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. 

Zymonema  Dermatitidis,  see  Blastomyco- 
sis.