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':..A.  FBy-Wyssiing 

SUBMIGROSCOPIC 

MORPHOLOGY  OF 

PROTOPLASM 


:^  :-•:■''* 


I 


SUBMICROSCOPIC  MORPHOLOGY  OF  PROTOPLASM 


SOLE   DISTRIBUTORS    FOR   THE   U.S.A.   AND    CANADA: 
ELSEVIER    PRESS,    INC.,    402    LOVETT   BOULEVARD,    HOUSTON    (tEXAs) 
FOR   THE   BRITISH    COMMONWEALTH    EXCEPT    CANADA: 
CLEAVER-HUME    PRESS,   LTD.,   42A    SOUTH   AUDLEY    STREET,    LONDON,    W.I 


vy  >   I — '    #    ■ 


SUBMICROSCOPIC  MORPHOLOGY 

OF  PROTOPLASM 


xj 


by 


A.  FREY-WYSSLING 

PROFESSOR  OF  GENERAL  BOTANY  AT  THE  FEDERAL  INSTITUTE  OF  TECHNOLOGY 

ZURICH    (SWITZERLAND) 


Second  English  Edition 


No^  a  i^ir 


ELSEVIER  PUBLISHING  COMPANY 

AMSTERDAM  •  HOUSTON  •  LONDON*  NEW  YORK 

1953 


German  Edition  1938 

First  English  Edition  1948 

Second  English  Edition  1953 

English  Translation  by  May  Hollander,  Selborne 


ALL    RIGHTS    RESERVED 
THIS    BOOK   OR   ANY    PART    THEREOF   MAY   NOT    BE    REPRODUCED    IN  ANY    FORM 
(including    PHOTOSTATIC    OR    MICROFILM    FORM) 
WITHOUT   WRITTEN    PERMISSION    FROM    THE    PUBLISHERS 

Library  of  Congress  Catalog  Card  Number:  j2-j6j9 


FOREWORD 


'      '  LIBF?A5t:Y     ^ 

MASS.  ~     ' 


This  monograph  is  the  third  edition  of  my  "Submikroskopische 
Morphologie  des  Protoplasmas  und  seiner  Derivate"  published  in  1938 
by  Gebriider  Borntrager  Berlin.  War  and  post-war  conditions  made  it 
impossible  to  republish  this  book  in  German.  For  that  reason  I  was 
glad  to  accept  the  offer  of  the  Elsevier  Publishing  Company,  Amster- 
dam to  translate  the  manuscript  of  the  second  edition  into  English. 

The  aim  of  the  first  edition  was  to  introduce  Submicroscopic 
Morphology  as  a  new  branch  of  General  Morphology.  As,  in  1938, 
the  electron  microscope  had  not  yet  become  an  instrument  of  biological 
research,  that  introduction  was  based  on  the  results  of  indirect  methods 
of  investigation  (macromolecular  chemistry,  double  refraction,  di- 
chroism,  X-ray  diffraction  etc.),  which  made  it  possible  to  provide 
evidence  of  the  arrangement  of  submicroscopic  elements.  In  general, 
one  indirect  method  alone  will  not  produce  unequivocal  evidence  of 
a  structure  invisible  in  the  ordinary  microscope.  But  a  combination 
of  several  such  methods  made  it  possible  to  exclude  certain  possi- 
bilities. Submicroscopic  Morphology,  therefore,  was  an  exciting  and 
inspiring  field  of  trial  and  error  for  morphologists  interested  in 
Biophysics. 

Since  then,  the  electron  microscope  has  made  it  feasible  to  photo- 
graph submicroscopic  structures  and  to  check  the  results  of  the 
indirect  methods.  It  is  a  great  satisfaction  for  the  pioneers  of  Sub- 
microscopic Morphology  to  know  that  their  postulates  as  to  the  struc- 
tures of  gels,  fibres  etc.  were  right.  On  the  other  hand,  our  science 
has  lost  one  of  its  attractive  charms ;  we  no  longer  have  the  satisfacdon 
of  inventing  new  methods  of  research  and  seeking  the  particular 
structural  arrangement  which  agrees  with  the  results  given  by  all  the 
available  indirect  methods  and  therefore  must  correspond  to  the  real 
invisible  structure.  This  romance  of  discovery  has  given  place  to  the 
technical  problem  of  obtaining  objects  thin  enough  to  get  the  best 
possible  image  in  the  electron  microscope. 


VI  FOREWORD 

By  the  time  the  second  edition  appeared  in  1948,  Submicroscopic 
Morphology  had  become  generally  accepted  as  an  important  branch 
of  the  biological  sciences.  The  morphologists  who  did  not  trust 
indirect  methods,  willingly  accepted  the  results  of  electron  microscopy, 
although  electron  optics  are  even  more  complicated  than  those  of 
polarized  light  or  X-rays.  But  the  objectively  visible  image  has  always 
been  the  foundation  of  Morphology,  and  therefore  research  in  Sub- 
microscopic  Morphology  is  henceforth  governed  by  the  remarkable 
invention  of  the  electron  microscope. 

As  a  consequence,  this  third  edition  is  centred  on  the  results  of  the 
electron  microscope;  the  old  indirect  methods,  however,  are  treated 
as  equally  valid  means  of  research.  The  polarizing  microscope  and 
even  the  X-ray  camera  are  more  accessible  to  the  average  biologist 
who  is  interested  in  iine-structures  than  the  expensive  electron  micro- 
scope. There  are  several  excellent  monographs  on  electron  micro- 
scopy, but  there  is  no  other  synopsis  of  the  value  and  the  results  of  the 
indirect  methods  in  Submicroscopic  Morphology.  In  the  first  rush  of 
publishing  electron  micrographs,  many  micrographs  were  produced 
which  would  have  been  discarded  as  mere  pictures  of  artefacts  if  the 
conclusions  of  indirect  methods  had  been  considered.  Where  there  is 
doubt  as  to  the  accuracy  of  an  electron  micrograph,  the  results 
estabhshed  by  indirect  methods  ought  to  be  taken  into  consideration. 
Any  discrepancies  between  the  interpretation  of  the  results  of  indirect 
methods  and  those  of  the  electron  micrograph  must  be  cleared  up 
before  a  submicroscopic  structure  may  be  regarded  as  definitely 
established. 

This  book  is  written,  not  for  specialists,  but  for  students  who  are 
attracted  to  this  interesting  field  of  research.  It  is  merely  an  outline 
and  does  not  attempt  to  give  full  details,  which  should  be  sought  in 
the  original  publications  quoted.  The  extensive  literature  published  on 
this  subject  since  1948  has  been  taken  into  account  as  far  as  it  was 
possible  in  this  condensed  monograph.  It  shows  the  enormous 
development  of  Submicroscopic  Morphology  during  this  short  period. 

Institut  Fiir  Allgemeine  Botanik 
der  Eidgenossischen  Technischen 
Hochschule,  Zurich. 

November,  1952.  A.  Frey-Wyssling 


CONTENTS 

INTRODUCTION:  THE  DOMAINS  OF  MORPHOLOGY i 

I.  FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY 8 

§  I.  Organization  of  Sols 8 

a.  Invisible  Particles 8 

b.  Homogeneity 12 

c.  Concept  of  Phase  in  Colloids 15 

d.  Coacervation 18 

§  2.  Principles  of  Structure 24 

a.  Crystal  Structure 26 

b.  Structural  Chemistry 33 

c.  Structure  of  Phase  Boundaries 40 

d.  Liquid  Crystals 5^ 

§  3.  Structure  of  Gels 5^ 

a.  Chemistry  of  High  Polymers 58 

b.  Structural  Viscosity 64 

c.  Gel  Structure 66 

d.  Micellar  Theory 76 

§  4.  Studies  in  Gels 82 

a.  Polarization  Microscopy 82 

b.  X-ray  Analysis  of  Gels 96 

c.  Swelling  of  Gels 109 

d.  Electron  Microscopy 115 

e.  Summary 13° 

n.  THE  FINE-STRUCTURE  OF  PROTOPLASM 131 

§  I.  Cytoplasm 132. 

a.  Molecular  Constituents  of  the  Cytoplasm 132 

b.  Physicochemical  Behaviour  of  Proteins 141 

c.  Physical  Properties  of  the  Cytoplasm 163 

d.  Submicroscopic  Structure  of  Cytoplasm 172 

e.  Protoplasmic  Flow  and  Cell  Polarity 186 

f.  Separation  of  the  Cytoplasm  into  Different  Phases 191 

g.  Morphological  Principles  of  the  Permeability  Problem 197 

h.  Molecular  Morphology  of  the  Cytoplasm 207 

§  2.  Nucleus 210 

a.  Molecular  Constituents  of  the  Nucleus 210 

b.  Fine-Structure  of  the  Nucleus 215 

c.  Fine-Structure  of  the  Chromosomes 224 

d.  Submicroscopic  Morphology  of  Hereditary  Processes 230 

§  3.  Chloroplasts 243 

a.  Microscopic  Structure  of  the  Chloroplasts 243 

b.  Molecular  Constituents  of  Chloroplasts 246 

c.  Submicroscopic  Structure  of  the  Chloroplasts 251 


ci^^^-i 


VIII  CONTENTS 

§  4.  Erythrocytes 262 

a.  The  Microscopic  Structure  of  Erythrocytes 262 

b.  Molecular  Constituents  of  the  Erythrocytes 265 

c.  Submicroscopic  Structure  of  Erythrocytes 266 

§  5.  Gametes 274 

a.  Spermatozoa 274 

b.  Eggs 276 

III.  FINE-STRUCTURE  OF  PROTOPLASMIC  DEiaVATIVES 279 

§  I.  Carbohydrates,  Chitin  and  Cutin 279 

a.  Meristematic  Plant  Cell  Walls  (Cellulose) 279 

b.  Cutinized  Cell  Walls  (Cutin) 293 

c.  The  Chitin  Frame  (Chitin) 301 

d.  Starch  Grains  (Amylose  and  Amylopectin) 310 

§  2.  Proteins 326 

a.  Reserve  Protein 326 

b.  Silk  (Silk  Fibroin) 331 

c.  Horny  Substances  (Keratin) 338 

d.  Connective  Tissue  (Collagen) 345 

e.  Muscle  Fibres  (Actomyosin) 352 

f.  Nerves  (Neurokeratin  and  Neuronin) 360 

g.  Fibrillar  Proteins.  Recapitulation 364 

RETROSPECT 371 

LITERATURE 375 

AUTHOR  INDEX 401 

SUBJECT  INDEX '. 407 


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INTRODUCTION 
THE  DOMAINS  OF  MORPHOLOGY 

Dass  ich  erkeniie,  was  die  Welt 
I»!  Imiersten  :^usammenhdlt. 

Goethe's  Faust 

Morphological  biology  comprises  the  study  of  organs^  (anatomy 
in  medicine,  organography  according  to  Goebel),  of  tissues  (histology) 
and  of  cells  (cytology).  Together  these  domains  form  a  hierarchic 
system,  since  they  describe  units  of  diminishing  size  in  the  above 
order.  The  diiferent  domains  defined  by  the  concepts  organ,  tissue, 
and  cell  can  also  be  characterized  by  the  expedients  which  are  used 
to  make  the  units  under  investigation  visible,  since  each  of  the  three 
sciences  makes  use  of  different  instruments  of  observation.  The 
organographer  observes  with  the  naked  eye  or  with  the  magnifying 
glass,  the  histologist  with  the  ordinary  microscope  and  the  cytologist 
with  the  more  refined  immersion,  phasecontrast  (Zernike,  1946)  or 
even  ultraviolet  microscopes.  Accordingly,  the  range  of  research  of 
organography  is  in  general  limited  by  the  resolving  power  of  the  eye, 
the  domain  of  cytology  by  the  resolving  power  of  the  microscope 
(Fig.  i).  In  biology,  all  that  can  be  described  with  the  aid  of  these 
means  of  observation  is  referred  to  as  morpholo^. 

The  hierarchy  of  morphology,  however,  goes  beyond  the  resolving 
power  of  the  microscope.  The  persistent,  I  might  almost  say  the 
heroic,  struggle  with  which  the  resolving  power  of  the  microscope 
has  been  increased  (Abbe,  1879;  Kohler,  1904)  is  the  best  evidence 
of  this.  Fig.  I  shows  how  the  microscopic  domain  was  widened  step 
by  step  by  advances  in  the  theory  and  technique  of  optics  until  at  about 

^  In  this  connection,  "organ"  is  to  be  understood  in  the  morphological  sense  as  part 
of  an  organism,  and  not  in  the  physiological  sense  "organ  =  instrument",  which  is  based 
on  specific  functions;  according  to  that  definition,  single  tissues,  special  cells  or  even 
parts  of  cells  can  also  act  as  "organs". 


2  INTRODUCTION 

O.I  /<  the  absolute  limit  was  reached  for  a  true  image  of  the  object, 
due  to  the  wavelength  of  ultraviolet  light.  Until  recently,  morphology 
was  forced  to  remain  at  this  limit.  We  have  great  admiration  for  the 
numerous  cytologists  who  have  worked  in  the  limiting  regions  of  the 
optical  resolving  power  of  the  immersion  microscope,  pursuing  ever 
finer  structures,  with  ineffable  devotion  and  utter  disregard  for  their 
eyesight.  However,  if  one  remembers  their  labour  and  its  limited 
prospects  of  success  (since  the  actual  ultrastructure  of  the  protoplasm 
cannot  be  obtained  with  any  certainty  by  microscopic  means),  it  is 
remarkable  how  few  biologists  have  drawn  the  obvious  conclusion 
from  the  theory  of  the  limit  of  microscopic  images  and  have  turned 
their  attention  to  indirect  methods  of  research. 

The  resolving  limit  of  the  microscope  is  like  the  shore  of  a  mys- 
terious mountain  lake.  On  land  the  geo-morphologist  can  easily  re- 
Fig.  I 

THE  DOMAINS  OF  MORPHOLOGY 


Organography 
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Histology    Cytology 

Micellar  studies 
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cognize  all  details  of  shape  and  size ;  he  can  measure  and  photograph 
them.  If,  however,  he  wants  to  study  the  morphology  of  the  bed  of  the 
lake,  he  will  derive  no  benefit  from  a  stndy  of  the  shoreline,  however 
carefully  this  may  be  done.  Nor  will  it  serve  his  purpose  if  he  tries  to 


THE    DOMAINS  OF  MORPHOLOGY  3 

look  at  the  bottom  through  the  water  above  it.  He  must  not  cUng 
stubbornly  to  the  rocks  on  the  shore  but  must  free  himself  from  the 
land.  He  must  "swim",  and  from  the  surface  of  the  water  must  find 
out  indirectly  with  a  plummet  how  the  bed  is  shaped.  Each  fathoming 
provides  him  with  a  point,  and  the  profile  of  the  bed  can  then  be 
constructed  by  interpolation. 

Until  recent  times,  in  the  submicroscopic  domain  which  lies  beyond 
the  microscopic  limit,  the  situation  was  completely  analogous.  Views 
on  submicroscopic  morphology  could  be  obtained  only  by  sounding, 
i.e.,  by  indirect  means,  and  the  invisible  shapes  and  sizes  could  be 
deduced  only  from  a  combination  of  the  various  methods  of  research. 

The  discovery  of  the  electron  microscope  after  1938  suddenly 
brought  the  submicroscopic  regions  within  reach.  By  means  of  electron 
rays  the  resolving  power  has  been  increased  a  hundredfold  in  one 
sudden  leap.  The  surface  of  the  water  in  the  lake  to  be  studied  has,  so 
to  speak,  been  reduced  to  a  much  lower  level.  The  precipices  and  gullies 
which  had  hitherto  been  hidden  have  become  accessible  to  the  in- 
vestigator, who  is  now  equipped  with  the  means  whereby  he  can  move 
about  in  this  difficult  province.  Submicroscopic  morphology  has 
accordingly  lost  something  of  its  mysterious  charm.  The  unravelling 
of  its  secrets  no  longer  wholly  depends  upon  an  ingenious  com- 
bination of  partial  evidence  obtained  indirectly,  as  it  still  does  in  the 
study  of  the  constitution  of  organic  molecules  in  structural  chemistry. 
There  is  now  a  direct  means  of  checking  the  conceptions  developed 
so  far.  The  objective  micrographs  given  by  the  electron  microscope 
have  made  submicroscopic  morphology  very  popular  in  biology, 
whereas  formerly  it  had  been  left  to  those  few  biologists  with  a  working 
knowledge  of  physics. 

However,  the  electron  microscope  cannot  completely  replace  the 
indirect  methods  which  have  been  so  successful  up  to  now.  All 
specimens  have  to  be  dried  and  this  may  cause  serious  artefacts  in 
structures  like  protoplasm  containing  80  to  90  %  water,  and  there 
exist  many  objects  which,  for  technical  reasons,  cannot  yet  be  imaged 
in  the  electron  microscope;  furthermore,  irradiation  by  electrons 
represents  a  bombardment  which,  compared  with  irradiation  by  light, 
involves  incomparably  greater  energies.  These  are  apt  to  destroy  the 
structures  of  specimens  cut  into  insufficiently  thin  sections.  Amicro- 
scopic  structures,  invisible  in  the  electron  microscope,  may,  moreover. 


4  INTRODUCTION 

occur.  The  electron-optical  images  of  biological  objects  should  therefore 
be  considered  critically.  They  must  be  compared  with  the  results  ob- 
tained from  the  indirect  methods  and,  in  cases  of  contradiction,  it  must 
be  made  clear  on  which  side  the  error  lies.  In  this  way  it  has  been 
possible  in  some  instances  to  show  that  the  electron  microscope  had 
produced  spurious  effects.  Electron  microscopy  should  not,  therefore, 
supersede  the  methods  formerly  appHed,  such  as  polarization  micro- 
scopy or  X-rays  analysis,  but  the  new  direct  method  and  the  valuable 
indirect  methods  must  be  used  jointly,  each  acting  as  a  check  on  the 
other,  in  the  exploration  of  the  submicroscopic  domain. 

The  history  of  this  science  will  soon  be  able  to  celebrate  its  first 
centenary  (Nageli,  1858).  However,  only  in  the  last  thirty  years  has 
there  been  enough  interest  to  produce  a  continuous  development  of 
this  field  of  research.  For  Ambronn,  who  devoted  his  whole  life  to 
this  branch  of  science  and  who  published  his  fundamental  researches 
on  the  rod-Hke  nature  of  the  structural  elements  of  gels  in  I9i6-'i7, 
had  to  carry  out  his  work,  according  to  his  own  statement,  "excluded 
from  publicity",  and  until  his  death  in  1927  he  considered  that  his 
was  the  voice  of  a  biologist  crying  in  the  wilderness.  The  general  lack 
of  interest  in  submicroscopic  problems  was  without  doubt  due  to  the 
following.  Colloid  chemistry  had  developed  into  a  general  doctrine 
of  dispersoids.  The  discovery  of  the  ultramicrbscope  (Siedentopf 
and  ZsiGMONDY,  1903)  had  suddenly  widened  the  range  of  the  sub- 
microscopic morphology  of  sols.  With  great  enthusiasm  biologists 
mastered  the  new  method,  but  discovered  with  disappointment  that 
nearly  all  important  biological  objects:  cytoplasm,  nuclei,  plastids, 
cell  walls,  etc.  are  "optically  empty".  We  know  now  that  this  is  due 
not  only  to  the  close  packing  or  the  hydrophilic  nature  of  the  hypo- 
thetical particles,  but  also,  and  mainly,  to  the  fact  that  we  have  to  deal 
with  anisodiametric  structural  elements,  which  are  invisible  in  the 
ultramicroscope  if  only  one  of  their  dimensions  is  amicroscopic,  even 
if  such  structural  elements  accumulate  in  loose  meshworks  of  sub- 
microscopic or  even  microscopic  dimensions.  This  indicates  that 
biological  gels  do  not  at  all  represent  disperse  systems  in  the  classical 
sense  of  colloid  chemistry  (see  Table  II).  The  failure  of  the  ultra- 
microscope  seemed  to  imply  that  these  objects  do  not  possess  a  sub- 
microscopic structure. 

In  the  meantime,  structural  chemistry  has  developed  amicroscopic 


THE  DOMAINS  OF    MORPHOLOGY  5 

molecular  morphology.  X-ray  analysis  has  provided  us  with  exact 
data  on  the  mutual  position  and  distances  of  the  atoms  and  groups  of 
atoms  in  organic  molecules,  and  this  has  greatly  added  to  our  knowl- 
edge of  stereochemistry.  Nowadays  we  know,  not  only  the  formulae 
of  many  compounds,  but  also,  with  astounding  accuracy,  their  entire 
morphological  structure. 

From  the  molecular  region,  the  elucidation  of  the  constitution  of 
high  polymers  has  already  advanced  into  the  submicroscopic  region 
as  a  new  branch  of  structural  chemistry.  In  the  case  of  polysaccharides 
and  polypeptides,  for  instance,  it  shows  that  thousands  of  similar 
structural  elements  can  be  united  to  gigantic  chain  molecules  which 
sometimes  even  reach  microscopic  lengths.  Staudinger,  to  whom 
we  owe  this  knowledge,  designates  this  new  kind  of  study  as  macro- 
molecular  chemistry. 

This  might  lead  one  to  believe  that  the  link  between  cytological 
and  molecular  morphology  has  been  forged  and  that,  consequently, 
a  special  submicroscopic  morphology  would  become  superfluous. 
This,  however,  is  by  no  means  true,  for,  the  high  polymer  chains  can 
arrange  themselves  in  more  or  less  regular  lattices  which  in  their  turn 
cluster  together  to  form  porous  structures,  interspersed  with  numerous 
capillary  spaces  of  various  sizes.  Or  again,  they  may  form  loose  mesh- 
works  with  a  totally  different  degree  of  order.  Besides  chains,  there 
may  occur  lamellar  high  polymers,  thus  allowing  for  a  great  many 
possible  arrangements  of  the  submicroscopic  elements.  Consequently, 
in  addition  to  the  problems  of  constitution  in  macromolecular  chemis- 
try, there  exist  morphological  problems  of  a  special  kind,  the  de- 
scription of  which  can  best  be  characterized  as  the  morphological  study  of 
fine-structure.  In  biology  this  nomenclature  is  synonymous  with  the 
study  oi  micellar  systems  (Frey  1928b),  provided  the  new  definition  on 
p.  81  be  taken  into  account. 

In  Fig.  I  the  lower  boundary  line  of  the  morphological  domain  of 
the  fine-structures  has  been  drawn  arbitrarily  at  the  limit  of  visibility 
of  the  smallest  gold  particles  in  the  ultramicroscope.  The  resolving 
power  of  the  electron  microscope,  which  may  yet  be  improved,  lies 
for  the  present  within  the  same  range.  This  serves  to  show  that  the 
order  of  magnitude  of  our  field  of  research  coincides  with  that  of 
classical  colloid  chemistry.  In  contrast  with  the  isolated  dispersed 
particles,  however,  the  colloid  dimensions  do  not  refer  to  all  three 


INTRODUCTION 


directions  in  space  but,  in  the  case  of  rod-shaped  elements,  to  two 
dimensions  only,  or  even  to  only  one  in  the  case  of  lamellar  submicro- 
scopic  elements,  which  may  be  clustered  to  form  complicated  systems. 


TABLE  I 
MORPHOLOGY 


Instrument 

Order  of 

Morphological  hierarchy 

of  research 

Scale 

magnitude 

Organs 

Organography 

Eye,  magnif. 
glass 

mm  scale 

>  o.  I  mm 

Tissues 

Histology 

Microscope 
I  Immersion 
^  and  ultraviolet 
(  microscope 

Micrometer 
Wavelengths 

>  I  /x 

Cells 

Cytology 

of  light 

>   O.I   IX 

Fine-structure 

Micellar 

Electron 

Colloid 

>  I  m^u. 

studies 

microscope 

dimensions 

Molecule 

Structural 

X-rays 

Wavelength 

>  lA 

structure 

chemistry 

of  X-rays 

Atom  structure 

Electron 

Electron  rays 

Wavelength  of 

<  O.I  A 

theory 

electron  rays 

According  to  Table  I  the  domain  of  fine  structures  forms  a  link 
between  our  present  knowledge  of  cytological  and  molecular 
morphology.  We  must  therefore  attempt  to  penetrate  into  the  study 
of  micellar  systems  from  these  two  known  sides.  Starting  from  the 
region  of  visible  structures,  we  must  resort  to  our  knowledge  of 
phases,  while  on  the  other,  molecular,  side  we  should  apply  our  knowl- 
edge of  crystal  structure.  Both  these  theories  cover  morphological 
domains  which  fall  outside  the  hierarchy  given  in  Table  I.  There  is  no 
upper  Hmit  to  the  dimensions  of  phases,  although  there  does  exist  a 
lower  hmit  which  we  shall  have  to  consider.  Similarly,  there  exists  no 
upper  limit,  on  theoretical  grounds,  to  the  regular  arrangement  of 
atoms  and  molecules  in  crystal  lattices.  For  this  reason,  we  can  use 
these  abstract  sciences,  which  are  less  sensitive  to  dimensions,  as  an 
introduction  to  the  study  of  fine  structures. 

Morphology  is  not  an  ultimate  goal  of  science,  but  it  represents  one 
of  its  most  important  foundations.  No  physical  problem  can  be  attacked 
without  first  defining  accurately  the  mutual  positions  of  the  various 


THE  DOMAINS  OF  MORPHOLOGY  7 

points  in  the  system  to  be  investigated.  It  is  only  after  this  that  time 
can  be  introduced  as  a  parameter,  to  pass  on  from  static  to  dynamic 
considerations.  Just  so  in  biology.  Every  physiological^  research,  being 
concerned  essentially  with  changes  in  course  of  time,  presupposes  a 
complete  knowledge  of  morphology.  The  relatio  ns  between  the  various 
organs  and  tissues  can  only  be  studied  in  their  dependence  on  time  if 
their  spatial  arrangement  has  been  ascertained  with  accuracy.  This 
explains  the  tremendous  flight  which  the  physiology  of  the  human 
body  has  taken  in  connection  with  the  development  of  anat  omy  and 
histology. 

Passing  from  the  total  organism  to  the  elementary  organism  of  the 
cell,  we  must  expect  similar  relations.  If,  therefore,  we  want  to  study 
the  physiology  of  cells  successfully,  we  must  know  their  morphology 
as  thoroughly  as  that  of  the  total  organism.  The  invisible  texture  of  the 
cell,  however,  which  is  the  object  of  line  structure  or  micellar  morpho- 
logy, is  still  in  its  infancy.  The  difficulties  in  this  field  of  research  are 
great  and  at  present  we  still  do  not  know  how  far  we  shall  be  able  to 
proceed.  Each  new  gain  in  this  direction,  however,  will  not  only 
augment  the  archives  of  the  descriptive  science  of  nature,  but  will 
redound  to  the  benefit  of  physiology,  and  will  in  the  end  satisfy  our 
thirst  for  knowledge. 

1  Physiologji  is  the  science  of  events  and  processes  in  living  organisms.  Both  these  ex- 
pressions clearly  indicate  that  time  is  involved,  i.e.,  they  show  the  dynamic  character  of 
physiology.  Biomorphohgy  and  biochemistry,  on  the  other  hand,  are  not  concerned  with  time; 
the  one  describes  the  spatial  arrangement  and  the  other  the  properties  of  organic  matter. 
It  is  only  when  time  begins  to  play  a  part  that  morphology  hQCome.s  physiolo^  of  development 
and  biochemistry  becomes  physiology  of  metabolism  which,  combined,  give  general  physio- 
logy, taking  into  account  all  variable  quantities,  i.e.  space,  matter  and  time,  which  are 
accessible  to  our  tools  of  research.  In  view  of  this,  we  fail  to  see  why  the  attribute 
"dynamic"  is  nowadays  added  so  readily  to  the  branches  of  knowledge  which  describe 
biology.  A  combination  such  as  "dynamic  morphology"  is  quite  inconsistent  because, 
by  definition,  morphology  can  do  no  more  than  describe  or  explain  given  spatial  arrange- 
ments, whereas,  as  soon  as  changes  in  spatial  arrangement  are  considered,  we  enter  the 
domain  of  physiology. 


I.    FUNDAAEENTALS    OF    SUBAETCROSCOPIC 

MORPHOLOGY 

"Le  cytoplasnie  propremeiit  dit  se  present e  sur  le  vivant 
comme  une  substance  collo'idale  homogene,  translucide,  op- 
tiquement  vide  a  I'ultramicroscope.  .  ." 

GuiLLiF.RMOND,  Mangf.not  et  Plantefol 

(1933,  p.  386) 

§  I.  Organization  of  Sols 

a.  Invisible  Particles 

Ever  since  Graham  (1861)  showed  that  the  pseudo-solutions  which 
impede  filtration  and  which  nowadays  we  call  sols  contain  relatively 
large,  slowly  diffusing  particles,  the  nature  of  these  invisible  particles 
has  been  explored  in  all  directions  by  colloid  chemistry  (Zsigmondy, 
1925;  OsTWALD,  1927). 

Demonstration  and  shape  of  the  particles.  Numerous  methods  have  been 
worked  out  to  distinguish  and  to  separate  the  originally  hypothetical 
submicroscopic  colloid  particles  from  the  amicroscopic  molecules. 
By  means  of  dialysis  the  amicroscopic  particles  can  be  made  to 
permeate  through  a  semi-permeable  membrane  (parchment)  through 
which  the  colloid  particles  cannot  follow  (Graham,  1862).  This 
method  has  since  been  developed  into  ultrafiltration^  by  which  sols  are 
pressed  through  filters  with  submicroscopic  pores  (collodion  films  of 
varying  pore  size)  and  in  this  way  are  split  up  into  fractions  of  different 
particle  sizes.  Further,  since  most  colloid  pa'rticles  carry  an  electric 
charge  or  can  be  charged  by  a  change  in  the  acidity  of  the  sur- 
roundings, they  can  be  made  to  migrate  in  an  electric  field  to  the 
anode  or  to  the  cathode  according  to  their  charge,  and  it  is  possible 
in  this  way  to  concentrate  them  by  electrophoresis. 

None  of  these  methods  of  indirect  particle  identification,  however, 
is  quite  as  convincing  as  ultramicroscopj,  which  makes  the  particles 
visible  (SiEDENTOPF  and  Zsigmondy,  1903).  Admittedly,  the  ultra- 
microscope  does  not  give  a  true  image  of  the  colloid  particles,  for  the 


I 


I  ORGANIZATION  OF  SOLS  9 

reason  that  its  resolving  power  does  not  surpass  that  of  the  ordinary 
microscope.  It  merely  reveals  the  existence  of  submicroscopic  particles. 
The  possibility  of  ultramicroscopic  demonstration  is  based  on  the  fact 
that  hght  incident  upon  small  particles  is  scattered  in  all  directions.  In 
this  way  they  become  radiant  (like  the  dust  particles  in  a  dark  room 
where  sunlight  penetrates  through  some  gap),  so  that  the  path  of  a 
beam  of  light  in  a  sol  is  clearly  traced  (Tyndall  scattering).  The  lighted 
sphere  surrounding  such  a  dust  particle  is  much  larger  than  the 
scattering  particle  itself,  and  an  image  of  it  can  be  obtained  in  the 
microscope  if  the  distance  between  the  colloid  particles  is  not  too 
small.  As  the  objective  of  the  microscope  gives  an  image  of  planes 
only,  optical  cross-sections  of  the  lighted  spheres  are  imaged  in  the 
form  of  deflexion  discs.  Since  the  particles  in  the  sol  take  part  in 
Brownian  movement,  these  scintillating  "deflexion  discs"  oscillate 
vividly  in  an  irregular  manner.  It  is  an  impressive  sight  to  watch  these 
luminous  spots  which,  in  untiring  movement,  stand  out  like  bright 
stars  from  the  pitch-dark  background. 

To  what  extent  the  size  of  the  "deflexion  discs"  exceeds  that  of  the 
particles  we  do  not  know;  nor  can  we  determine  the  exact  shape  of 
the  particles.  All  the  same,  the  ultramicroscope  enables  us  to  draw 
conclusions  as  to  their  circumference  in  cases  of  marked  deviation 
from  the  spherical.  Non-spherical  particles  may  be  oriented  in  a  field 
of  flow.  In  that  case  they  scintillate  to  difterent  extents  according  as  the 
incident  ultramicroscopic  irradiation  is  parallel  or  perpendicular  to  the 
direction  of  flow;  they  show  what  is  calJed  azimuth  effect.  If  the  light 
falls  upon  the  small  endplane  of  submicroscopic  rods,  they  scatter 
much  less  than  with  sideways  irradiation.  From  such  diflferences  in 
intensity  of  the  "deflexion  discs",  depending  on  the  direction  of  the 
incident  beam,  the  rod-hke  shape  of  the  particles  can  be  inferred. 

Anisodiametric  particles  are  usually  birefringent.  As  they  are 
oriented  in  a  field  of  flow,  sols  containing  such  colloid  particles 
become  optically  anisotropic  in  a  velocity  gradient  (Freundlich, 
Stapelfeldt,  and  Zocher,  1924).  Long  rods  are  oriented  at  lower 
rates  of  shear  than  shorter  ones  (Signer  and  Gross,  1935).  From 
measurements  of  the  birefringence  of  flow,  conclusions  can  therefore 
be  drawn  regarding  the  rations  between  length  and  thickness. 

Si^e  of  the  particles.  A  clear  picture  of  the  world  of  submicroscopic 
particles  can  be  obtained  with  the  aid  of  the  methods  mentioned. 


lO  FUNDAMENTALS  OF  SUBMI  C  ROSCOPIC  MORPHOLOGY  I 

But  colloid  chemistry  was  not  content  with  these  qualitative  con- 
clusions ;  it  tried  to  obtain  quantitative  facts  as  to  the  size  of  the  par- 
ticles. Some  information  was  provided  by  ultrafiltration,  but  apart  from 
that,  much  more  accurate  methods  were  available. 

If  the  number  of  particles  per  unit  volume  is  determined  in  the 
ultramicroscope,  the  particle  size  can  be  calculated  from  the  con- 
centration of  the  sol.  Moreover,  there  exist  mathematical  relations 
between  Brownian  movement  (Einstein's  formula),  velocity  of  sedi- 
mentation (Stokes'  formula)  or  diffusion  on  the  one  hand,  and  particle 
size  on  the  other.  These  make  it  possible  to  determine  the  diameter 
of  spherical  colloid  particles.  The  ultramicroscope  plays  an  important 
part  in  these  investigations  (Zsigmondy,  1925),  since  the  particles 
have  to  be  observed  when  counting  or  measuring  the  Brownian 
movement.  In  many  cases,  however,  the  colloid  particles  cannot  be 
observed  ultramicroscopically,  not  only  because  their  dimensions  are 
frequently  too  small  but,  above  all,  because  their  refractive  power  is 
often  only  sUghtly  different  from  that  of  the  dispersing  medium,  so 
that  light  scattering  is  insufficient.  This  usually  applies  to  biological 
sols  with  their  organic  colloid  particles,  which  means  that  the  limit  of 
visibility  of  these  sols  in  the  ultramicroscope  is  reached  long  before 
that  of  inorganic  sols  (compare  Fig.  i,  p.  2). 

The  method  of  sedimentation  is  free  from  this  difficulty,  because  the 
change  in  concentration  of  the  solution  as  a  result  of  sedimentation 
of  the  particles  can  be  determined  by  analytic  means  or,  still  more 
simply,  by  the  change  in  refractive  index.  Moreover,  the  sedimentation 
velocity  can  be  increased  at  will  by  applying  stronger  centrifugal 
forces.  The  ultracentrifuge,  which  was  developed  by  Svedberg  (1938a) 
into  an  instrument  of  the  highest  accuracy  and  great  power  (cen- 
trifugal fields  which  are  750,000  times  that  of  the  gravitational  field!) 
allows  of  the  determination  of  particle  weights  down  to  amicroscopic 
molecules. 

The  various  methods  referred  to  have  revealed  much  of  the 
morphology  (size  and  shape)  of  submicroscopic  particles,  so  that  the 
electron  microscope  has  only  confirmed  by  direct  micrographs  the 
results  obtained  by  indirect  means. 

Fig.  2  represents  a  series  of  submicroscopic  particles  of  biological 
importance,  facilitating  comparison  with  the  microscopic  and  amicro- 
scopic regions.  The  size  and  shape  of  the  particles  were  determined  by 


ORGANIZATION  OF  SOLS 


II 


Fig.  2 

PARTICLE  SIZES   (PARTLY  FROM  STANLEY,   1938a,  b) 


measuring 
scale 


Mol. 

weight 


0  in 


I .  Erythrocyte .... 
z.  Bacterium  coli  .  .  . 
3 .  Bacterium  prodigiosum 


4.  Treponema  pallidum 


5 .  Small-pox  virus 


fj         6.  Chicken  plague  virus . 


7.  Megatherium  bacteriophagus  . 


8  .Yellow  fever  virus 


100 


m/x 


9.  Gene,  calcul.  accord,  to 
Muller(i935) 

10.  Tobacco  mosaic  virus.    .    .    . 

1 1.  Foot  and  mouth  virus.    .     .    . 

12.  Glycogen,  according  to 
HusEMANN  and  Ruska  (1940) 

1 3 .  Haemocyanin  from  Octopus    . 

14.  Smallest  ultramicroscopically 
visible  gold  particles,  accord- 
ingtoZsiGMONDY(i925)  . 

15.  Horse  haemoglobin    .    .    .    . 

16.  Ovalbumin  (Svedberg  1930) 

17.  Saccharose 

18.  Hydrogen  molecule     .    .    .    . 


2300- 10° 


300- lO" 


23- 10° 


4.3-10" 
33-io« 


43- 10" 
0.4-  10® 
1.5- 10^ 


2.8-  10° 


2.7-  10° 


69- 10* 

5-5 

40- 10' 

4-3 

342 

0.5 

2 

0.2 

7500 

3000 

750 


200 


Length 

{mfi) 


175 


90 

38 
22 
20 

12.3 

10 

10 


6000 
1000 


18000 


125 
430 

64 


i.o 


12         FUNDAMENTALS  OF  SURMIC  ROSCOPIC  MORPHOLOGY  I 

the  methods  mentioned  and  in  many  cases  also  by  the  electron  micro- 
scope. It  is  seen  that  there  is  a  continuous  transition  from  the  lifeless 
amicroscopic  molecules  to  the  living  cells  at  the  limit  of  microscopic 
visibility.  The  smallest  particles  which  exhibit  phenomena  of  life  (self- 
multiplication)  are  in  the  submicroscopic  region.  Theoretical  biology^ 
being  concerned  with  the  definition  and  the  essence  of  life,  is  therefore 
called  upon  to  give  serious  attention  to  our  branch  of  morphology. 
On  the  other  hand,  these  colloid  particles  often  give  the  impression  of 
consisting  of  uniform,  chemically  well-defined  substances,  and  the 
biochemist  attributes  molecular  weights  to  them  which,  depending 
on  the  size  of  the  particles,  may  assume  fantastically  large  values. 

b.  Homogeneity 

Real  solutions  containing  amicroscopic  particles  are  designated  as 
uniform  or  homogeneous  from  a  physico-chemical  point  of  view.  Sols, 
however,  are  not  considered  as  uniform;  they  are  heterogeneous.  The 
concept  of  homogeneity  applied  here  is  essentially  different  from  the 
optical  homogeneity  which  plays  such  an  important  part  in  microscopy. 
A  medium  is  optically  homogeneous  when  its  constituent  parts  have 
the  same  refractive  index,  so  that  it  is  impossible  to  establish  their 
boundary  line  by  means  of  light. 

Physico-chemical  homogeneity,  however,  requires  that  two  parts 
taken  from  the  object  shall  be  identical,  not  only  in  their  behaviour 
towards  light,  but  also  in  all  other  properties.  This  will  be  the  case  if 
the  particles  are  similarly  arranged  throughout  the  whole  object 
(Figs.  3-7). 

Several  homogeneous  arrangements  of  particles  are  possible.  The 
structural  elements  can  be  arranged  irregularly,  like  the  molecules  of 
a  liquid  or  gas.  The  distances  between  the  particles  are  not  all  equal, 
but  if  we  proceed  through  the  mass  along  a  straight  line,  the  average 
distance  found  will  be  constant,  and  equal  volume  elements  will  on 
the  average  contain  an  equal  number  of  particles.  Such  arrangements 
are  called  statistically  homogeneous  in  contrast  to  the  distribution  of  the 
atoms  in  a  crystal,  which  are  arranged  in  a  certain  pattern.  As  all 
distances  in  a  given  direction  are  identical,  this  is  called  a  lattice 
arrangement.  The  spacings  can  be  equal  in  three  directions  which 
are  mutually  perpendicular;  in  that  case  the  lattice  arrangement  is 
isotropic  (Fig.  4).  Or  else,  the  spacings  are  different  in  different  di- 


ORGANIZATION  OF  SOLS 


13 


f .    •.  .       •      •  • 

.\            •       •       •    • 

/                           • 

•  ^^* 

•/•     •          •    • 

9 

^,    •       •   •      .  • 

• 

•             .         • 

•            •          « 

•    •      . 

•     • 

•  .  . 

•   ^    ^  • 

•    • 

e          ^      •        \          •         • 

f*              •   \»     . 

•         • 

•     •/              •     •   1        •       •     • 

• 

-            •     •                          • 

•    •   • 

•     ."-     •         •/            •      • 

•      • 
•     • 

•      • 

•      •         •           •         •     • 

•  ••••• 

•  c  •  •  • 

•  •/ "  •  ^  ^«      •      • 

•  /  •      •  ^  •      • 

•  I*         •         •!        •         • 

•  V     •        •     /  •         • 

•  •        •         •         • 

•  o  •         •         •  • 

•  •    •    '/"'-^^ 

•  •      •      •/    •      • 

•  «    •    •  -V _•__ ^ 
•    ••••• 


•  •  •   •!  •  •••••••••• 

•  ••••/*•••*. 

•  •    •    •.•  ^    •    •    •    f^M    •  •    •    ^ 

•  •••!•••    •••'••••9' 

, I  ^_,/ 

I 1     ^ 

•  •    •(•   ••••••••  •!•   • 

I  I 

I  I 


Fig.  3 


Fig.  4 


Fig-  5 


Fis.  6 


Fig-  7 


Homogeneous  arrangements 


Fig.  3.  Statistically  homogeneous  distribution  -  Fig.  4.  Homogeneous  isotropic  lattice  - 

Fig.  5.  Homogeneous  anisotropic  lattice  -  Fig.  6.  Statistically  homogeneous  distribution 

of  polar  particles  -  Fig.  7.  Homogeneous  lattice  arrangement  of  polar  particles. 

rections,  in  which  case  the  lattice  arrangement  is  anisotropic  (Fig.  5). 
The  homogeneous  lattice  arrangement  has  in  common  with  the 
statistically  homogeneous  arrangement  that  equal  volumes  contain 
an  equal  number  of  particles.  With  anisotropic  arrangements  it  is 
not  sufficient  to  compare  volumes  of  equal  size;  they  must  also  have 
the  same  orientation.  For,  if  from  Fig.  5  instead  of  circles  we  draw 
two  congruent  rectangles  with  different  orientations,  the  properties  of 
one  of  these  rectangles  will  be  different  from  those  of  the  other  on 
account  of  the  different  distribution  ot  lattice  points  with  respect  to  the 
length  of  the  rectangle  (the  linear  thermal  expansion  of  the  long  side 
of  the  two  rectangles,  for  instance,  will  be  different).  The  necessity  of 
taking   orientation   into   account   becomes    particularly   apparent    if 


14  FUNDAMENTALS  OF  SUBM  I  C  RO  SC  OPI  C  MORPHOLOGY  I 


Fig.  8 


%^®^ 


at 


•^ 

2 

^ 

a: 

^ 

cA 

Fig.  9 


Fig.  lo 


Fig.  II  Fig.  12  Fig.  13 

Homogeneous  states  of  the  compound  ABj.     QA,  0   B,  ^  E. 
Fig.  8.  Solid  -  Fig.  9.  Liquid  -  Fig.  10.  Gaseous  -  Fig.  11.  Homogeneous  solution  of 

EBj  in  AB2. 

Mixed  crystals  (A,  E)  Bg. 
Fig.  12.  Homogeneous  -  Fig.  13.  Heterogeneous. 


polar  particles  such  as,  for  instance,  water  molecules  are  arranged 
homogeneously.  Fig.  6  shows  such  particles  in  a  statistically  homoge- 
neous distribution  and  Fig.  7  gives  an  example  of  an  arrangement  in  a 
lattice  which  has  identical  spacings  in  three  directions. 

From  these  considerations  we  derive  the  following  definition  of 
homogeneity:  an  object  is  homogeneous  if  equal  and  equally  oriented  parts, 
taken  arbitrarily  from  the  object,  possess  the  same  internal  structure.  This 
implies  that  all  the  parts  thus  compared  have  the  same  physical  and 
chemical  properties. 

An  important  condition  in  these  considerations  is  the  order  of 
magnitude  of  the  volumes  to  be  compared.  Physico-chemical  homo- 


I  ORGANIZATION  OF  SOLS  I5 

o-eneity  requires  that  they  shall  be  oi submicroscopic dh/iens'ions .Y\xQ.vc\X&t- 
nal  structure,  therefore,  refers  to  the  arrangement  ot  atoms,  ions  and 
molecules,  which  in  Figs.  3-7  have  been  indicated  by  points  or  arrows. 

It  follows  from  this  definition  that  sols  cannot  be  homogeneous. 
For,  if  in  a  sol  we  consider  submicroscopic  volumes  of  sufficiently 
small  size,  the  one  may  contain  a  colloid  particle,  while  the  other  may 
merely  contain  the  solvent,  i.e.,  the  dispersing  medium.  In  contrast  to 
sols,  not  only  are  all  pure  substances  homogeneous,  whether  in  the 
solid,  liquid  or  gaseous  state  (Figs.  8-10),  but  so  also  are  real  solutions, 
provided  the  solute  consists  of  amicroscopic  particles  (Fig.  11).  If, 
however,  differences  in  the  concentration,  for  instance  concentration 
gradients,  occur  in  the  solution,  it  is  heterogeneous.  Similarly,  either 
homogeneous  or  heterogeneous  mixed  crystals  can  originate  from  a 
solution  or  melt,  according  as  the  two  components  can  unite  to  a 
crystal  lattice  in  a  regular  or  in  an  irregular  distribution  (Figs,  iz 
and  15). 

Colloid  solution  having  been  recognized  as  heterogeneous,  the 
further  question  arises  whether  the  colloid  particles  themselves  may  be 
considered  as  homogeneous.  To  answer  this  question  we  must  deal 
shortly  with  the  phase  theory,  which  treats  of  relations  between 
homogeneous  states. 

c.  Concept  of  Phase  in  Colloids 

According  to  the  thermodynamical  definition,  any  homogeneous  state 
is  called  a  phase.  Figs.  8-12  thus  picture  the  structure  of  phases.  Fig.  13 
representing  not  a  homogeneous  phase  but  a  heterogeneous  system  of 
AB2  and  EBg. 

The  colloid  particles  were  formerly  believed  to  be  homogeneous 
and  the  dispersed  particles  were  therefore  designated  as  dispersed  phase 
and  the  surrounding  liquid  as  dispersing  medium  (Fig.  14).  Thus  a  sol 
represents  a  two-phase  system.  The  study  of  the  structure  of  colloids 
need  not,  of  course,  be  confined  to  the  liquid  state.  Dispersions  of 
liquid  or  solid  particles  in  liquid  or  solid  media  (emulsions,  suspen- 
sions, etc.)  are  known  in  the  microscopic  domain.  We  may  also  expect 
to  find  them  in  the  submicroscopic  world.  Since,  however,  the  par- 
ticles in  such  dispersions  are  no  longer  visible,  colloid  systems  were 
designated  as  dispersoids.  In  this  way  an  attempt  was  made  to  charac- 
terize, not  only  the  organization  of  sols,  but  in  the  most  general  sense 


l6  FUNDAMENTALS  OF  SUBMICROSCOPI C  MORPHOLOGY  I 

that  of  all  colloids,  as  will  be  clear  from  the  following  system  (Wo. 
OsTWALD,  1909). 

Systematics  of  dispersoids.  According  to  the  theory  of  dispersions, 
each  of  the  three  states  of  matter,  solid,  liquid  or  gas,  can  occur  either 
as  dispersing  medium  or  as  dispersed  particles  (Fig.  14),  so  that  3^  =  9 
combinations  are  possible  (Table  II).  Fig.  14  shows  how  in  these 
systems  the  dispersed  part  I  is  distributed  in  the  dispersing  phase  11. 

TABLE  II 
DISPERSOID  SYSTEMS,  ACCORDING  TO  WO.  OSTWALD,   1909 


Dispersing  medium 

Dispersed  portion 

Dispersoids 

Solid 

Solid 

Grain-structure 

Solid 

Liquid 

Drop-structure 

Solid 

Gas 

Bubble-structure 

Liquid 

Solid 

Suspensoids 

Liquid 

Liquid 

Emulsoids 

Liquid 

Gas 

Foams 

Gas 

Solid 

Smoke 

Gas 

Liquid 

Mist 

Gas 

Gas 

On  the  strenght  of  the  definition  of  phases  it  was  originally  believed 
that  the  dispersed  part  I  was  homogeneous.  In  the  dispersoids,  how- 
ever, this  leads  to  difficulties.  Often  it  was  doubtful  whether  a 
dispersed  phase  was  liquid  or  soHd.  For,  suppose  the  dispersoid 
particles  become  smaller  and  smaller  until  they  contain  only  a  few 
molecules,  then  it  would  be  difficult  to  decide  whether  they  are  solid 
or  liquid.  Liquid  drops  may  be  taken  to  be  homogeneous,  whereas  it  is 
very  difficult  to  prove  this  of  solid  suspended  particles.  It  was  only 
by  the  introduction  of  X-ray  iiiethods  in  colloid  chemistry  that  the 
particles  of  certain  dispersoids,  for  instance  gold  and  silver  sols, 
could  be  proved  to  possess  a  crystal  lattice  and,  therefore,  to  be  really 
homogeneous.  With  increasing  degree  of  dispersion,  however,  the 
homogeneity  of  a  crystal  lattice  also  becomes  questionable.  For,  the 
energy  ot  the  points  lying  at  its  surface  is  different  from  that  of  the 
points  inside  the  lattice,  because  they  are  no  longer  surrounded  on  all 
sides  by  equivalent  fields  of  force  (Fig.  i6).  In  the  case  of  liquids  this 
sives  rise  to  surface  tension.  For  instance,  in  the  smallest  gold  particles 


ORGANIZATION  OF  SOLS 


17 


which  can  be  measured  by  X-ray  methods  (Scherrer,  1920),  200  of 
the  380  Au-atoms,  i.e.,  more  than  half  the  total  number,  lie  at  the  sur- 
face of  the  crystals.  With  decreasing  particle  size,  of  course,  an  even 
higher  percentage  of  atoms  lies  at  the  surface,  until,  with  14  or  still  less, 
all  the  atoms  lie  at  the  surface  (face-centred  cube.  Fig.  25,  p  .27).  Thus 
one  can  no  longer  speak  of  a  homogeneous  phase  in  the  case  of  atoms 


Phase  I 


Phase E 


Fig.  14 


Fig.  15 


Fig.  16 


Fig.  14.  Colloid  chemical  concept  of  phase.  I  Dispersed  phase  (colloid  portion),  11 
dispersing  medium.  The  inhomogeneity  of  boundaries  reigns  throughout  the  system  - 
Fig.  15.  Thermodynamical  concept  of  phase.  The  homogeneity  of  the  phases  reigns 
throughout  the  system.  -  Fig.  i6.  Inbomogeneous  surroundings  of  the  lattice  points  of 
boundary'  planes  (face-centered  cubic  lattice). 


that  are  not  similarly  surrounded  on  all  sides.  It  is  only  by  a  still 
further  increase  in  dispersion  that  finally  a  homogeneous,  molecularly 
dispersed  solution  of  Au-ions  is  obtained. 

On  the  other  hand,  it  has  been  ascertained  (Zsigmondy,  1925,  p.  39) 
that  the  homogeneous  primary  particles  of  suspensoids  can  cluster 
together  to  form  bigger  heterogeneous  secondary  particles  (compare 
Fig.  73,  p.  104)  without  any  fundamental  change  in  the  properties  of 
such  sols.  This  strengthened  the  opinion  that  the  properties  of  sols 
and  other  colloids  were  not  decided  by  the  inner  structure  of  the 
particles.  Since  with  increasing  dispersion  the  surface  of  the  particles 
increases  considerably  in  proportion  to  their  mass,  colloid  chemistry  has 
developed  much  more  into  a  science  of  surfaces.  The  properties  and 
reactions  of  colloids  have  been  elucidated  to  a  great  extent  by  the  study 
of  surface  reactions.  Whereas  the  phase  theory  is  concerned  with  the 
equilibrium  between  different  phases  and  is  able  to  predict  under  what 
conditions  phases  cease  to  exist  (dissolution)  or  new  phases  appear 


l8        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

(separation  into  two  layers),  classical  colloid  chemistry  is  interested  in 
the  first  place  in  phase  boundaries  (capillary  chemistry  according  to 
Freundlich,  1922). 

Thermodynamics  require  that  all  parts  of  a  phase  have  exactly  the 
same  energy  content.  This  is  only  realized,  however,  when  the  phases 
are  so  extended  that  the  irregular  distribution  of  energy  at  their 
surface,  i.e.,  the  inhomogeneity  in  the  immediate  neighbourhood  of 
the  phase  boundary  (Fig.  16)  can  be  neglected  (Fig.  15).  Thus,  the 
classical  phase  theory  has  to  forego  all  considerations  concerning  phase 
boundaries  (compare  Figs.  3-11,  13,  14)  because  of  their  inhomoge- 
neity, and  its  laws  only  apply  to  homogeneous  regions  of  at  least  micro- 
scopic dimensions.  The  properties  of  colloids,  on  the  contrary,  are 
determined  in  the  first  place  by  the  inhomogeneity  of  the  phase  boun- 
daries, the  predominant  effect  of  which  is  due  to  the  very  large  surface. 
For  this  reason  it  has  been  suggested  by  Ostwald  (1938)  that  the 
definition  "dispersed  phase"  should  be  avoided,  and  that  we  should 
speak  of  the  "colloid  portion"  of  the  dispersoid. 

The  phase  theory  once  seemed  to  hold  out  promise  of  explaining 
the  formation  of  new  phases  (separation  into  two  strata,  formation  of 
vacuoles)  or  the  disappearance  of  phases  (melting-in)  in  biological 
systems.  From  the  above,  however,  it  is  clear  that  the  phase  theory 
does  not  hold  good  in  colloid  chemistry,  since  it  has  been  developed 
by  emphasizing  the  homogeneity  of  the  phase  and  neglecting  the 
specific  properties  of  surfaces,  while  conversely,  in  cytological  systems, 
homogeneity  usually  fails  and  the  surfaces  are  of  quite  outstanding 
importance.  Bungenberg  de  Jong  and  his  fellow-workers  have 
elucidated  the  principles  according  to  which  visible  boundary  layers 
can  appear  and  disappear  in  those  heterogeneous  systems  to  which 
the  phase  theory  does  not  apply.  In  his  theory  of  coacervation  Bungen- 
berg DE  Jong  has  summarized  the  rules  which  govern  these  phe- 
nomena. 

d.  Coacervation 

In  the  separation  of  a  sol  into  two  non-miscible  parts,  the  dispersing 
medium  and  the  dispersed  portion  often  do  not  separate  completely. 
Flakes  are  formed  which  still  contain  a  certain  amount  of  dispersing 
medium  and  therefore  remain  suspended.  For  this  reason  the  floccu- 
lation  is  usually  reversible.  If,  however,  such  flakes  collect  into  small 


ORGANIZATION  OF  SOLS 


19 


drops  or  into  a  coherent  liquid  layer,  we  have  to  do  with  a  phenomenon, 
for  which  Bungenberg  de  Jong  (1932)  introduced  the  term 
coacervation  (Fig.  20);  in  English:  piling  up  (acervus  —  pile). 

Hydration.  The  colloid  particles  in  a  sol  are  solvated,  which  means 
that  molecules  of  the  dispersing  medium  adhere  to  the  particle.  In  the 
special  case  of  water,  this  solvation  is  designated  as  hydration,  since  in 
that  case  water  molecules  are  bound  by  the  colloid  particle.    The 


W  H 


\ 


/-  /     1     \   x"  ^^ 


Fig.  17 


Fig.  18 


Fig.  19 


Fig.  17.  Model  of  a  water  molecule  and  scheme  of  dipoles  -  Fig.  i8.  Hy- 
dration of  an  isoelectric  colloid  particle  -  Fig.  19.  Hydration  of  a  charged 
colloid  particle  (from  Pallmann,  193 i). 


attraction  is  brought  about  by  electrostatic  forces,  for,  in  a  water 
molecule  the  electric  charges  are  not  distributed  uniformly,  because 
the  two  positive  hydrogen  atoms  are  separated  in  space  from  the 
doubly  charged  negative  oxygen.  For  that  reason  a  water  molecule  in 
an  electric  field  behaves  like  a  molecular  rod  with  two  different 
electric  poles  and  is  therefore  designated  as  a  dipole  (Fig.  17).  Similarly, 
in  a  colloid  particle  the  electric  charges  are  usually  not  distributed  uni- 
formly, not  even  if  the  particles  are  isoelectric,  i.e.,  if  their  positive 
and  negative  charges  cancel  each  other  so  that  outwardly  they  appear 
neutral.  In  Fig.  18  a  particle  has  been  sketched,  the  negative  charges 
of  which  are  situated  towards  its  surface.  This  has  a  polarizing  effect 
on  the  water  molecules  in  the  immediate  neighbourhood  of  the 
particle.  These  water  molecules  follow  the  particle  in  its  Brownian 
movement  as  the  so-called  solvation  or  hydration  layer.'  If  the  colloid 
particle  is  not  neutral  but  carries  an  excess  negative  or  positive  charge 
as  a  result  of  dissociation  of  H-  or  OH -ions,  the  swarm  of  oriented 


20        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

dipoles  surrounding  the  particle  will  be  correspondingly  larger.  This 
is  why  the  hydration  of  colloids  reaches  its  minimal  value  at  the 
isoelectric  point. 

The  binding  forces  which  attract  the  water  dipoles  decrease  with 
increasing  distance.  Thus  the  swarm  of  water  molecules  which  are 
hampered  in  their  free  movement  becomes  less  dense  in  the  outer 
layers  until  in  the  end  one  reaches  without  noticeable  transition  the 
region  of  the  freely  moving  dipoles  of  the  dispersing  medium.  In  the 
solvation  layer  the  density  of  the  water  therefore  decreases  ex- 
ponentially, in  much  the  same  way  as  the  density  of  the  atmosphere 
with  increasing  distance  from  the  earth.  As  thert  is  no  sudden  transition 
from  the  hydration  layer  to  the  free  water,  such  hydrophilic  colloids 
are  very  stable.  The  particles  show  no  tendency  to  cluster  together; 
in  a  way  they  "have  no  surface  at  all",  their  surface  energy  is  zero 
(Fig.  2oa). 

Dehydration.  If  water  is  withdrawn  from  the  diffuse  solvation  layer, 
the  difference  between  bound  and  freely  moving  dipoles  becomes 
noticeable.  The  water  layer  around  the  particle  now  acquires  a  surface 
(Fig.   2ob)  and  if  two  such  dehydrated  particles  meet,  the  surface 


.    ■    No  surface 

•      z'. 

-■--.V    . 

./•.;. 

."  '■  .   "^  " 

'•   ''.y 

■^•^T"'-*.  *  "\  • 

1  ■•■■% 

sXM***    '  1           ^ 

\--:> 

'^."'•■'.  V  ' 

\-  ■  •  . 

■*/'  .*   */  .  " 

•>^- 

*  .    -    . 

Surface 


Flocculation 
.Coacervafe  droplet 

Coacervafe  layer 


Fig.  20.  Dehydration  of  colloid  particles  (from  Bungenberg  de  Jong,  1932). 
a)  Diffuse  hydration  layer,  b)  definite  hydration  layer,  r)  incipient  coacervation. 

energy  which  tends  towards  a  minimum  value  will  cause  the  surround- 
ing water  layers  to  unite.  The  colloid  particles,  however,  cannot  come 
into  direct  contact  with  each  other  because  of  their  solvation  layers. 
But  they  no  longer  possess  separate  layers,  for  these  have  all  united 
into  a  single  liquid  sphere.  If  the  number  of  particles  united  in  this 
way  becomes  so  large  that  they  form  a  microscopically  visible 
conglomeration,  one  speaks  of  flakes  or  flocculates.  These  can  further 
cluster  into  drops  (microcoacervation)  and  finally  into  a  liquid  layer 
(macrocoacervation).  Thus  coacervates  are  liquids  rich  in  colloid 
which  have  been  separated  by  means  of  dehydration. 


I  ORGANIZATION  OF  SOLS  21 

In  the  coacervate  the  distribution  of  colloid  particles  is  statistically 
uniform,  as  in  the  original  sol,  although  their  concentration  has  been 
increased.  If  the  colloid  particles  are  considered  as  dispersed  phase,  their 
state  has  not  been  changed  in  the  coacervation  process;  and  yet 
clearly  a  new  phase  boundary  is  formed  between  a  layer  rich  in  colloid 
and  one  poor  in  colloid.  This  example  shows  how  vague  is  the  con- 
cept of  phase  in  colloid  chemistry.  For  that  reason  the  hydrophilic 
sols  and  the  coacervates  originating  from  them  are  sometimes  called 
quasi-homogeneous  phases,  since  the  distribution  of  the  particles  is 
completely  uniform  and  the  particle  size  is  liable  to  decrease  to  mole- 
cular dimensions. 

The  dehydration  of  colloid  particles  illustrated  in  Fig.  20  can  be 
brought  about  in  various  ways;  for  instance,  a  rise  in  temperature, 
which  accentuates  the  contrast  between  bound  and  freely  moving 
water,  will  often  suffice.  Usually,  however,  use  is  made  of  dehydrating 
substances  such  as  sahs  (salting  out)  or  aliphatic  alcohols  or  acetone. 
Such  substances,  which  disturb  the  stability  of  the  sol  and  increase 
the  tendency  to  separate,  are  called  sensitizers.  Besides  salts  and  organic 
liquids,  colloid  solutions  may  also  be  used  as  sensitizers  if  they  com- 
pete with  the  particles  of  the  original  sol  to  bind  the  free  water  and 
thus  cause  dehydration. 

The  dispersing  medium  which  is  separated  from  the  coacervate  is 
called  the  equilibrium  liquid  (Fig.  21),  for,  following  changes  in  tempera- 
ture or  composition  in  the  system,  water  is  taken  up  or  given  oflF  by 
the  coacervate.  The  situation  is,  therefore,  analogous  to  the  separation 
in  a  mixture  of  phenol  and  water  (p.  46).  Coacervates  can  be  regarded 
as  a  solution  of  water  in  the  colloid  (swelling)  and  the  equilibrium 
liquid  must  then  represent  a  solution  of  a  small  amount  of  colloid  in 
water.  In  the  example  given  in  Fig.  21,  however,  the  gelatin  is  insoluble 
in  alcohol-water  and  the  concentration  of  the  colloid  in  the  equilibrium 
liquid  is  practically  zero.  Here  the  analogy  therefore  ceases  since,  in- 
stead of  a  reciprocal  solubility,  there  exists  only  a  one-sided  adsorption 
of  water  by  the  colloid.  The  reason  why  gelatin  is  completely  in- 
soluble below  its  melting  point  will  be  made  clear  on  p.  73.  Coacervates 
of  homopolar  substances  have  been  studied  by  Mme  Dobry  (1938, 
1940). 

Discbarge.  In  biological  systems  the  colloid  particles  are  seldom 
neutral;  usually  they  are  electrically  charged.  Particles  carrying  opposite 


22        FUNDAMENTALS  OF  SUBMIC ROSC OPI C  MORPHOLOGY 


charges  tend  to  unite,  but  because  of  their  solvation  layers  can  only- 
approach  each  other  to  a  certain  extent.  The  attraction  is  counteracted 
by  the  hydration  as  by  a  spring  (Fig.  22)  and  thus  no  coagulation 
takes  place  which  would  annul  the  charges,  but  again  a  coacervate 
occurs  which  now  contains  particles  of  opposite  charge.  So,  in  addi- 


Equilibrium   liquid 


Coacervate 


Fig.  21.  Coacervation  of  gelatin  at  41"^  C. 
Isoelectric  gelatin  sol  +  alcohol  as  sensi- 
tizer. Equilibrium  liquid  =  solution  of 
water  and  alcohol.  Coacervate  =  gelatin  + 
small  amounts  of  water  +  alcohol. 


v^AA\AAA 


Fig.  22.  State  of  stability  of  colloid  particles 
(from  BuNGENBERG  DE  JoNG  and  Bonner, 
1935).  Attraction  by  opposite  electrical 
charges  (arrows).  Repulsion  by  solvation 
layer  (spring). 


tion  to  sensitizers,  electric  charges  are  apt  to  cause  coacervation.  For  this 
the  sols  must  have  opposite  charges;  e.g.,  gelatin  (positive)  and  gum 
arable  (negative)  or  lecithin  (positive)  and  nucleic  acid  (negative). 
In  this  case  the  aggregation  is  designated  as  complex  coacervation,  since 
two  oppositely  charged  kinds  of  particles  take  part  in  the  flocculation. 
In  many  cases  colloid  particles  can  be  made  to  reverse  their  charge  by 
adding  neutral  salts,  when  the  familiar  valency  rules  apply,  viz.,  on 
the  addition  of  polyvalent  cations,  negative  particles  change  their  sign 
more  easily  according  as  the  valency  of  the  cation  is  higher,  while 
positive  particles  behave  in  a  similar  way  with  respect  to  polyvalent 
anions.  Negatively  charged  phosphatides,  for  example,  reverse  their 
charge  on  the  addition  of  CaClg.  In  the  sol,  the  phosphatide  particles 
which  have  already  become  positive  and  those  which  have  so  far  re- 
mained negative  attract  each  other,  and  in  this  way  a  separation  occurs 
which  has  been  called  autocomplex  coacervation,  because  in  this  instance 
similar  but  oppositely  charged  particles  attract  each  other. 

Morphologically  the  coacervation  shows  many  features  which  have 
their  counterpart  in  the  phenomena  occurring  in  cells.  In  the  first  place 
the  vacuolization  calls  for  mention.  If,  in  a  system  consisting  of  equili- 


ORGANIZATION  OF  SOLS 


23 


brium  liquid  and  suspended  coacervate  droplets,  the  equilibrium  is 
modified  as  a  result  of  changes  in  temperature  or  composition  in  the 
direction  of  a  further  dehydration  (heating,  addition  of  more  sensiti2er), 
vacuoles  appear  in  the  droplets.  These  represent  separated  equilibrium 
liquid  which  has  remained  inside  the  coacervate  droplets  (Fig.  23). 


Fig.  23.  Vacuolization  by  lowering  the  temperature  of  coacervate  drops 
consisting  of  gelatin  sol  +  resorcin  (from  Bungenberg  de  Jong,  1932). 


Probably  vacuolization  by  dehydration  is  comparable  with  the  forma- 
tion of  vacuoles  in  the  cell,  since,  in  that  case  too,  liquid  is  being 
separated  from  the  plasma  colloids. 

Apart  from  this  striking  analogy,  Bungenberg  de  Jong  (1932) 
mentions  other  models  for  cytological  differentiation  on  the  basis  of 
observations  with  coacervates.  When  mixing  sols  of  gelatin,  gum 
arable  and  nucleic  acid  from  yeast,  two  complex  coacervates  arise,  in 
addition  to  equihbrium  hquid,  one  of  which  consists  mainly  of  gelatin 
and  gum  arable,  the  other  being  composed  chiefly  of  gelatin  and  nucleic 


24        FUNDAMENTALS  OF  S  UBMI  C  RO  S  C  OPIC  MORPHOLOGY  I 

acid.  Their  partition  is  such  that  the  first  always  contains  the  second 
in  the  form  of  enclosed  droplets. 

This  can  easily  be  demonstrated,  as  the  negative  nucleic  acid  coacer- 
vate  can  be  selectively  stained  by  alkaline  dyes  such  as  methyl  green. 
This  is  regarded  by  Bungenberg  de  Jong  as  a  model  for  a  nucleus 
imbedded  in  cytoplasm.  Personally,  however,  I  do  not  believe  that 
such  comparisons  are  admissible,  since  both  nucleus  and  cytoplasm 
possess  a  structure,  whereas  the  liquid  coacervate  droplets  are  com- 
pletely amorphous.  For  that  reason,  the  picture  suggesting  the  resem- 
blance to  the  cell  may  be  incidental  and  should  therefore  not  be  used 
in  analogy  to  cytological  phenomena.  There  would  otherwise  be  too 
great  a  temptation  to  over-simplify  the  relationships  between  cyto- 
plasm and  nucleus.  The  nuclear  changes  in  karyokinesis,  for  instance, 
cannot  possibly  be  attributed  to  changes  in  hydration  or  electric 
charges  alone.  These  phenomena  are  attended  with  complicated  struc- 
tural alterations. 

Whereas  the  early  upholders  of  the  theory  of  coacervation  were 
principally  concerned  with  the  surfaces  of  the  colloid  particles  with 
their  solvation  layers  and  electric  charges,  attempting  to  gain  more 
knowledge  of  the  structure  of  boundary  layers  (see  p.  40  and  267), 
their  studies  were  later  extended  to  include  the  inner  structure  of 
coacervate  systems.  In  biological  objects  we  have  to  assume  that  the 
coacervate  has  a  submicroscopic  gel  structure  (Bank,  i  941).  Therefore, 
apart  from  a  knowledge  of  boundary  structure,  we  are  also  in  need  of 
deeper  insight  into  the  inner  structure  of  colloid  particles  and  coacer- 
vate flocculates.  In  order  to  advance  in  this  direction  we  must  appeal 
to  structural  principles. 

§  2.  Principles  of  Structure 

By  structural  principles  we  mean  the  laws  governing  the  mutual  posi- 
tions of  atoms,  ions,  and  molecules.  The  positions  of  the  atoms  in  the 
molecule  are  studied  by  structural  chemistrj,  which  in  this  respect 
appears  as  a  morphological  science.  For  example,  when  we  represent 
the  carbon  atom  by  its  4  valencies  or  a  benzene  ring  by  the  well- 
known  hexagon  (Fig.  24),  these  are  morphological  illustrations  based 
on  certain  properties  of  these  substances.  The  exact  location  of  the 
valency  bonds  in  space  and  the  distances  between  the  atoms  remained 


2  PRINCIPLES  OF  STRUCTURE  ZJ 

unknown  for  a  long  time,  and  there  was  a  certain  arbitrariness  in  the 
use  of  valency  lines  as  regards  their  direction  and  length  (cf.  Fig.  35  b, 
p.  38).  Today,  however,  the  data  needed  for  an  exact  morphological 
representation  are  known,  and,  if  written  in  a  suitable  way,  at  least  the 
simpler  chemical  formulae  actually  do  represent  molecular  models, 
which  have  been  projected  on  to  a  plane.  We 
owe  our  knowledge  of  the  exact  distances  and  h  x^ 

directions    chiefly  to   X-ray  analysis.   X-rays      ^ j. ^     ii'  m 

enable  us  to  measure  dimensions  of  the  order  |  hc^;;^^      joh 

of  magnitude  of  their  wavelength  (e.g.,  copper  ^ 

radiation:   X  =   1.54  A),  if  identical  distances  Fig.  24 

are  often  repeated  and  act  as  a  lattice,  causing 

interferences  which  can  be  photographed  and  thus  made  macroscopically 
visible.  It  is,  therefore,  the  principle  oi  repetition^hxch.  has  opened  the 
door  to  the  morphology  ofmolecular  structure.  The  more  regularly  the 
given  distances  are  arranged,  the  more  accurately  can  the  absolute  values 
and  directions  be  determined.  From  the  considerations  relating  to  ho- 
mogeneity it  follows,  therefore,  that  in  gases,  liquids  (Fig.  9-11,  p.  14) 
and  solutions  the  morphology  of  the  molecules  cannot  be  determined 
by  means  of  X-rays,  though  an  exception  to  this  rule  is  provided  by 
solutions  of  very  large  molecules  which  in  their  own  construction 
show  a  certain  periodicity  (for  example  carbon  chains).  In  such  cases, 
however,  the  measurements  are  often  ambiguous,  because  the  mole- 
cules are  not  orientated  in  fixed  directions.  The  most  reliable  values 
of  atomic  distances,  often  attaining  almost  incredible  precision  (up  to 
i7oo  of  I  '^)»  have  therefore  been  determined  in  crystal  lattices.  For  a 
quantitative  determination  of  the  arrangement  of  the  atoms  in  a  mol- 
ecule one  must  necessarily  make  use  of  phases  which  possess  a 
structure.  Amorphous  phases  without  structure,  such  as  liquids  and 
real  solutions,  are  not  suitable  for  the  elucidation  of  such  morpholo- 
gical relations. 

In  this  respect,  biological  conditions  are  highly  unfavourable.  Al- 
though the  protoplasm  must  be  presumed  to  have  a  structure,  it  is  not 
governed  by  the  principle  of  repetition  with  sufficient  consistency 
to  permit  of  X-ray  analysis.  Granted  that  periodicity  plays  an  im- 
portant part  in  all  living  matter  as  regards  time  and,  to  some  extent, 
also  spatial  arrangement;  yet  a  strictly  periodic  order  presupposes 
an  equilibrium  of  forces  and  this  is  opposed  to  life,  which  depends  on 


iG        FUNDAMENTALS  OF  SUBMI  C  RO  S  COPI C  MORPHOLOGY  I 

movement  and  the  maintenance  of  non-equilibria.  As  soon,  however, 
as  chemical  substances  are  withdrawn  from  the  metabolic  processes, 
the  ordering  forces  can  intervene  and  form  periodic  structures,  as, 
for  instance,  with  the  skeleton  substances  cellulose,  chitin,  collagen, 
keratin,  etc.  Therefore,  to  study  the  structure  of  protoplasm,  other 
methods  should  be  applied  which,  however,  are  partly  based  on  the 
results  of  the  investigations  on  crystal  structure.  For  this  reason  this 
important  branch  of  morphology  must  be  briefly  touched  upon. 

a.    Crystal  Structure 

Lattice.  The  essential  nature  of  lattices  is  determined  by  the  fact  that 
certain  locations  of  points,  which  in  the  more  simple  cases  are  identical  with 
the  centre  of  gravity  of  the  atoms,  periodically  repeat  themselves  in  three 
given  directions  in  space.  These  directions  coincide  with  the  axes  of  the 
crystallographic  system.  The  distance  from  one  point  to  the  next  identical 
one  is  designated  as  the  identity  period  or  spacing.  Depending  on  the 
crystallographic  system,  the  spacings  are  the  same  in  either  three  (cubic)  or 
only  two  directions  (tetragonal,  hexagonal,  rhombohedral),  or  they  are 
different  in  all  three  dimensions  (rhombic,  monoclinic,  triclinic).  The  reg- 
ularly repeated  points  form  an  array  of  points.  Displacing  such  a  row  by 
constant  amounts  in  a  direction  either  perpendicular  or  obliquely  to  its  own 
direction,  we  obtain  the  lattice  plane,  while  finally  the  crystal  lattice  results 
from  displacing  such  a  plane.  If  a  point  in  the  lattice  is  moved  in  the  three 
principal  directions,  each  time  covering  the  identity  period  involved,  and  if 
the  three  vectors  obtained  are  completed  to  a  three-dimensional  parallel- 
epiped, we  obtain  the  so-called  elementary  or  unit  cell  of  the  crystal  lattice.  In 
analogy  to  a  gas  molecule,  which  represents  the  smallest  unit  with  all  the 
chemical  properties  of  the  gaseous  phase,  the  unit  cell  is  the  smallest  unit 
which  still  shows  all  physical  and  symmetry  properties  of  the  crystal.  It  may 
contain  one  or  several  molecules  (and  in  the  case  of  high  polymers  even 
parts  of  molecules).  We  are,  therefore,  dealing  with  a  geometrical  concept 
and  by  no  means  with  a  chemical  one.  If  the  unit  cell  is  decomposed  into  its 
elements,  the  crystalline  properties  are  lost.  As  the  base  cell  possesses  all  the 
properties  of  the  crystal,  and  this  crystal  can  be  obtained  by  displacing  the 
elementary  unit  in  the  principal  directions,  structure  analysis  aims  at  de- 
termining the  dimensions  and  the  symmetry  of  the  base.  Its  shape  is  de- 
termined by  three  identity  periods  a:b:c  in  Angstrom  units,  to  which  in 
monoclinic  and  triclinic  systems  one  must  add  the  angle  ^,  or  the  angles 
a,  /3,  y  formed  by  the  edges  of  the  unit  cell.  The  macroscopically  determined 
proportions  between  the  axes  of  the  crystals  agree  with  the  proportions 
between  the  dimensions  of  the  unit  cell,  provided  analogous  planes  are 
considered. 

X-ray  analysis  measures  the  distances  between  the  lattice  planes.  In  the 


PRINCIPLES  OF  STRUCTURE 


27 


case  of  crystals  showing  a  high  degree  of  symmetry  (cubic  system),  the 
lattice  points  are  identical  with  the  points  of  intersection  of  symmetry  planes 
and  their  distances  can  therefore  be  calculated  from  the  distances  in  the 
X-ray  diagram.  In  the  case  of  lattices  having  a  lower  degree  of  symmetry, 
however,  the  situation  of  the  points  in  the  lattice  planes  is  not  determined 
unambiguously  by  symmetry  elements;  they  possess  certain  degrees  of 
freedom.  Accordingly,  the  determination  of  the  structure  with  the  aid  of 
the  distances  in  the  X-ray  diagram,  alone,  is  not  possible;  additional  mea- 
surements of  the  intensity  of  the  interferences  are  then  required.  In  this 
case,  however,  the  position  of  all  lattice  points  in  the  unit  cell  can  often  be 
only  approximately  determined.  (Niggli,  1929,  1941/42). 


Fig.  25 


Fig.  26 


Crystal  lattices.  The  encircled  points  belong  to  the  unit  cell 
Fig.  25.  Gold,  a  =  4.07  A,  #  Au  -  Fig.  26.  Sodium  chloride,  a  =  5.60  A,  #  Na,  O  CI 


Figs.  25  and  26  represent  two  of  the  best-known  lattices,  viz.  that  of  the 
element  gold  and  of  the  compound  sodium  chloride.  Both  lattices  are  cubic: 
this  means  that  the  dimensions  and  shape  of  the  unit  cell  are  determined  by 
a  single  identity  period  a  which  is  the  same  in  three  mutually  perpendicular 
directions.  Once  the  spacing  a  has  been  determined  by  means  of  X-rays,  the 
volume  a^  and,  from  the  known  density  of  the  crystalline  substance,  the 
weight  of  the  unit  cell  can  be  calculated.  Dividing  this  weight  by  the  absolute 
weight  of  the  atom  or  molecule  in  question  (  =  atomic  or,  as  the  case  may 
be,  molecular  weight/LoscHMiDT's  number  0.606  •  10-^),  one  finds  the 
number  of  atoms  or  molecules  in  the  unit  cell. 

For  example,  the  elementary  cell  of  gold  contains  4  Au  atoms,  that  of 
sodium  chloride  4  Na-  and  4  Cl-ions.  These  points  have  been  encircled  in 
Figs.  25  and  26;  the  other  points  marked  on  the  planes  of  the  cube  are  to 
be  considered  as  having  originated  from  the  encircled  ones  by  a  simple 
translation,  thus  belonging  to  a  neighbouring  unit  cell.  The  lattice  type  of 
gold  is  termed  face-centred  because  the  points  of  intersection  of  the 
diagonals  of  the  faces  are  all  occupied  by  atoms.  Numerous  elements,  such 
as  Ag,  Cu,  Al,  Pb,  etc.,  crystallize  in  accordance  with  the  same  scheme, 
though  with  different  identity  periods.  In  the  NaCl  type  of  lattice,  which  is 


28        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPh6lOGY  I 

found  in  several  binary  compounds  (NaF,  KCl,  PbS,  etc.)  with  different 
values  of  a,  two  of  such  face-centred  cubic  lattices  overlap. 

If  the  atoms  of  a  crystal  lattice  are  not  represented  by  distinct  points, 
but  by  spheres  touching  each  other,  their  space  requirement  related  to  the 
volume  of  the  unit  cell  can  be  calculated.  It  is  then  found  that  of  all  possible 
crystal  lattices  the  cubic  face-centred  lattice  of  Fig.  25  has  the  closest  possible 
packing.  The  volume  of  the  spheres  amounts  to  0.74  of  the  total  space 
available.  There  is  another  possibiHty  of  closest  packing  where  the  arrange- 
ment of  the  spheres  is  hexagonal  (hexagonal  space-centred  lattice).  The 
ratio  of  the  axis  is  a:c  =  1.63:1  and  the  space  required  exactly  the  same 
as  in  the  cubic  closest  packing  (0.74).  In  other  types  of  close  packing  the 
space  requirement  is  always  smaller  than  0.74.  For  instance,  in  the  space- 
centred  cubic  lattice  the  spheres  fill  only  0.68  of  the  volume  of  the  unit  cell. 

Primary  valency  lattice.  Next  to  the  geometrical  relations  between  the 
points  in  the  crystal  lattices,  the  forces  which  keep  the  atoms  together 
are  of  primary  importance.  The  purely  geom.etrical  consideration  of 
the  lattice  is  quite  independent  of  this.  As  soon,  however,  as  one  is 
interested  in  the  reason  why  certain  distances  in  a  lattice  are  great  and 
others  small,  this  question  must  be  considered.  In  fact,  the  lattice 
forces  are  of  a  varied  nature.  Actually,  in  the  examples  given,  the  forces 
are  different.  In  Fig.  25  similar  atoms,  in  Fig.  26  oppositely  charged 
ions  attract  each  other.  In  both  cases  primary  valencies  act  as  lattice 
forces  which  can  join  together  uncharged  as  well  as  oppositely 
charged  particles.  In  the  first  case  one  speaks  of  a  homopolar  lattice,  in 
the  second  of  a  heteropolar  or  ion  lattice. 

The  morphological  similarity  of  these  two  types  of  lattice  is  due  to 
the  fact  that  in  both  cases  the  construction  of  the  lattice  is  founded 
on  the  rules  of  the  theory  of  co-ordination.  According  to  Werner's 
chemistry  of  complexes,  each  atom  is  surrounded  by  a  fixed  number 
of  neighbouring  particles,  either  4,  6,  8  or  1 2,  depending  on  volume 
conditions.  This  theory,  based  originally  on  the  composition  of  salts 
containing  crystal  water  [e.g.,  Ca(H20)gCl2]  and  other  complex  salts, 
has  also  proved  useful  in  the  elucidation  of  crystal  structures  of  other 
compounds  and  of  the  elements.  In  fact,  in  Fig.  25  each  Au-atom  at  the 
corners  of  the  cube  is  surrounded  by  1 2  neighbouring  atoms  and  in 
Fig.  26  each  Na-ion  by  6  Cl-ions  or,  vice  versa,  each  Cl-ion  by  6  Na- 
ions. 

The  theory  of  co-ordination  has  led  to  another  fundamental  re- 
cognition which  has  become  of  the  greatest  importance  to  the  sub- 


PRINCIPLES  OF  STRUCTURE 


29 


microscopic  morphology  of  organic  compounds.  It  has  been  shown 
that  the  lattice  points  in  Figs.  25  and  26  represent  only  the  centres  of 
gravity  of  the  atoms.  The  range  of  their  electron  orbits,  however, 
extends  over  such  large  volumes,  that  these  can  be  represented  by 
spheres  touching  each  other  in  the  lattice  (Figs.  27-29).  A  crystal 


Fig.  27  Fig.  28  Fig.  29 

Co-ordination  numbers  (fromMAGNUS,  1922) 

Fig.  27.  Number  12;  e.g.,  Au  (Au),2  in  crystallized  gold  -  Fig.  28.  Number  6;  e.g., 
Na(Cl)e  in  sodium  chloride;  Fe(CN)6  as  ion  -  Fig.  29.  Number  4;  e.g.,  CCI4,  C(C)4  in 

diamond. 


lattice,  therefore,  which  is  kept  together  by  main  valencies  is  much 
more  closely  packed  than  the  common  pictures  suggest.  Unfortunately, 
the  representations  in  space  obtained  by  drawing  continuous  spheres  ■ 
instead  of  lattice  points  are  not  very  illuminating,  whereas  in  a  plane 
this  procedure  can  be  applied  with  great  success  (comp.  Fig.  3 1,  p.  34)- 
The  atomic  distances  in  the  lattices  of  elements  correspond,  therefore, 
to  the  atomic  diameters  and  in  binary  compounds  they  represent  the 
sum  of  the  radii  of  the  two  partners  (Goldschmidt).  In  this  way  it 
has  been  possible  to  determine  the  volume  occupied  by  various  atoms 
and  at  the  same  time  to  find  an  explanation  for  the  different  co-ordina- 
tion numbers.  E.g.,  four  Cl-atoms  combined  in  a  tetrahedron  to- 
gether enclose  a  space  which  just  corresponds  to  the  size  of  a  silicon 
atom;  this  accounts  for  the  co-ordination  number  4  in  the  compound 
SiCl4.  Of  the  smaller  fluorine  atoms,  however,  we  need  6  spheres  to 
obtain  the  space  occupied  by  one  Si-atom.  Hence  the  co-ordination 
number  6  (SiFg). 

If  the  lattice  contains  homopolar  valency  bonds,  the  distances  be- 
tween the  atoms,  or  the  diameters  of  their  spheres,  show  a  surprising 


30        SUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

constancy,  not  only  in  simple  compounds,  but  also  in  very  complicated 
ones.  In  the  heteropolar  ion  lattices  a  disturbing  effect  occurs  because 
of  the  opposite  charges  of  the  two  partners.  The  ions  have  a  polarizing 
effect  upon  each  other,  which  may  lead  to  deformations  of  the  electron 
orbits  (Fajans,  1923,  1925)  in  those  cases  where  the  symmetry  of  the 
lattice  does  not  exclude  such  effects,  as,  for  instance,  in  the  lattice  of 
NaCl  (Fig.  26).  The  ions  can  then  no  longer  be  represented  by  spheres; 
they  represent  dipoles  similar  to  the  water  molecules  in  Fig.  17  (p.  19). 
The  result  is  that  ion  lattices  often  possess  Httle  symmetry  and  that 
the  atomic  distances  between  given  partners  are  subject  to  certain 
fluctuations,  depending  on  the  circumstances. 

Fortunately  this  does  not  apply  to  the  molecular  structures  of 
organic  compounds  which  always  have  a  homopolar  character;  the 
distances  found  in  certain  compounds  can  therefore  be  transferred 
with  perfect  confidence  to  other  ones,  so  that  one  can  speak  of 
distance  rules.  In  Table  III  a  number  of  atomic  distances  are  given  as 
determined  in  organic  crystals  by  means  of  X-rays.  In  these  considera- 
tions the  hydrogen  atoms  must  be  neglected,  as  they  do  not  scatter 
X-rays ;  nor  do  they  seem  to  have  a  perceptible  influence  on  the  dis- 
tances between  the  atoms.  Table  III,  for  example,  shows  that  in  single 
bonds  the  atom  radius  of  carbon,  r,;,  amounts  to  0.77  A  and  that  of 
nitrogen,  r^^,  to  0.71  A.  In  spite  of  the  larger  atomic  weight  of  nitrogen, 
its  sphere  of  action  is  smaller  than  that  of  carbon.  It  is  also  seen  that 
the  sphere  of  influence  of  the  carbon  atoms  is  decreased  by  double 
bonds. 

Each  valency  in  an  organic  molecule  corresponds  to  a  definite 
amount  of  energy  (Meyer  and  Mark,  1930).  In  the  combustion  of  the 
homologous  paraffins,  for  instance,  the  heat  of  combustion  per  mole 
increases  by  a  definite  amount  for  each  new  C-atom  introduced;  this 
value  amounts  to  about  70  kcal.  The  energy  equivalents  for  the  other 
compounds  mentioned  in  Table  III  have  been  determined  in  a  similar 
way.  It  will  be  apparent  that  with  decreasing  distance  between  the 
C-atoms  the  energy  content  of  the  different  bonds  increases. 

To  sum  up,  it  can  be  said  that  in  the  main  valency  bonds  which  play 
a  part  in  the  structure  of  protoplasm,  distances  of  1-1.5  A  and  bond 
energies  of  the  order  of  100-200  kcal  occur. 

Molecule  lattice.  In  addition  to  homopolar  main  valency  lattices  and 
heteropolar  ion  lattices  we  must  consider  molecule  lattices.  If  the 


PRINCIPLES  OF  STRUCTURE 

TABLE  III 

DISTANCES  AND  MAIN  VALENCY  FORCES  BETWEEN  THE  ATOMS  IN 

ORGANIC  COMPOUNDS 


31 


Distance  in  A 

Energy-equivalent 

Crystal  lattice 

Bond 

accord,  to 

kcal  (Meyer- 

Stuart,  1954 

Mark,  1930) 

Diamond      .... 

Aliphatic  C— C 

1.54 

71 

Graphite 

Aromatic  C^^C 

1.42-1.4S 

96 

Stilbene 

Double     C=C 

1-35 

125 

Ca-carbide   .... 

Triple       C^C 

1.19 

166 

Carbonic  acid  .    .    . 

Ketone     C=:0 

1.05-1.15 

203 

Polyoxymethylene  . 

Oxygen 

bridge     C — O 

1.49 

Urea ;  hexamethyl- 

ene  tetramine     .    . 

Amino      C — N 

1. 53-1.48 

valency  of  an  atom  species  corresponds  to  the  co-ordination  number 
with  regard  to  another  atom  species  (as,  e.g.,  in  CH4),  the  mutual 
saturation  of  the  valencies  excludes  the  possibility  of  unlimited  lattices 
such  as  those  shown  in  Figs.  25  and  26  (p.  27).  Although  such 
molecules  no  longer  possess  free  valencies,  they  can  still  be  arranged 
in  a  crystal  lattice  (see  Mark  and  Schossberger,  1937).  The  binding 
forces,  however,  are  now  of  a  different  nature;  in  contrast  to  the  pri- 
mary valencies  they  are  called  secondary  valencies.  They  are  explained 
in  theoretical  physics  by  means  of  dipole  moments,  in  much  the  same 
way  as  the  orientation  and  attraction  of  water  molecules  by  an  ion  (see 
Fig.  19,  p.  19).  In  practice  these  forces  between  the  molecules  cause 
the  cohesion.  The  secondary  valence  forces  are,  therefore,  identical  with 
the  Van  der  Waals  cohesive  forces.  In  molecule  lattices  they  are  of 
the  same  nature  as  in  liquids  and  they  can  therefore  be  derived  from 
the  heat  of  sublimation  or  vaporization  of  the  compound.  It  then  be- 
comes apparent  that  each  atom  or  radical  occurring  in  the  structural 
formulae  of  organic  chemistrv  contributes  a  certain  amount  to  the 
cohesion.  At  a  first  approximation  the  cohesion  of  a  molecule  species 
is  composed  additively  of  these  partial  contributions,  and  can  be 
calculated  by  adding  up  the  various  increments,  in  exactly  the  same 
way  as  the  molecular  volume  (according  to  Kopp's  rule),  the  molecular 


32        FUNDAMENTALS  OF  SUBMIC  ROSCOPI  C  MORPHOLOGY 

TABLE  IV 

COHESIVE  FORCES  BETWEEN  ORGANIC  GROUPS,  ACCORDING  TO 
MEYER  AND  MARK  I93O 


Groups 

Molar  cohesion 
kcal/mole 

Aliphatic  C:  methyl  and     ^ 
methylene  groups             S 

Ether  bridge 

Amino  group 

Carbonyl  group 

Aldehyde  group 

Hydroxyl  group 

Carboxyl  group 

—  CHg  and  =  CHj 
CH2     ,  —  CH 
0 

NHo 
CO 
CHO 
OH 
COOH 

1.78 
0.99 
1.63 

5-53 

4.27 

4.70 

7-25 
8.97 

weight  or  the  molecular  refraction.  Accordingly,  the  contribution  of 
the  characteristic  groups  to  the  cohesion  has  been  denoted  as  mo/ar 
cohesion  (Meyer  and  Mark,  1930).  For  example,  the  heat  of  vaporiza- 
tion of  ethyl  alcohol,  which  amounts  to  10  kcal  per  mole,  is  additively 
composed  of  the  molecular  cohesions  of  CH3,  CHg,  and  OH.  The 
values  concerned  can  be  found  in  Table  IV. 

This  table  shows  that,  in  neighbouring  molecules,  methyl  and 
methylene  groups  and  also  oxygen  bridges  attract  each  other  only 
slightly.  The  attraction  between  amino  and  ketone  groups  is  twice  as 
large  and,  in  the  polar  hydroxyl  and  carboxyl  groups,  the  cohesion 
assumes  quite  considerable  values.  None  the  less,  all  the  values  for 
molar  cohesion  are  10  to  100  times  smaller  than  the  energy  equivalents 
of  the  main  valency  bonds,  and  accordingly  the  secondary  valency 
bonds  are  at  least  10  times  weaker.  Consequently,  whenever  secondary 
valencies  play  a  decisive  role  in  the  crystal  lattice,  the  distances  are 
much  greater  than  those  between  atoms  bound  by  primary  valencies. 
In  organic  crystals,  therefore,  in  which  both  bond  types  occur :  primary 
valencies  inside  the  molecule  (intramolecular)  and  secondary  valencies 
between  the  molecules  (intermolecular),  the  lattice  distances  are  essen- 
tially of  two  different  orders  of  magnitude. 


PRINCIPLES  OF  STRUCTURE 


33 


b.  Structural  Chemistry 

After  the  discovery  of  stereoisomery^  structural  chemistry  learnt  to 
distinguish  between  different  positions  of  the  substituents  to  the 
carbon  atom.  At  first,  the  results  of  this  interesting  science  (Werner, 
1904;  Freudenberg,  1933)  were  little  more  than  qualitative  and  re- 
ferred mainly  to  the  directions  radiating  from  the  C-atom.  Quantitative 
determinations  of  distances  along  these  directions  were  not  yet  possible. 
The  results  of  crystal  structure,  however,  determine  not  only  qualita- 
tively but  also  quantitatively  the  relative  positions  of  the  atoms  in  space. 

The  starting  point  for  the  new  development  in  structural  chemistry 
was  the  crystal  lattice  of  diamond,  which  crystallizes  in  the  cubic 


Fig.  30.  Diamond  lattice,  a)  Unit  cell,  h)  projection. 


system.  Its  unit  cell  is  a  cube  containing  8  C-atoms,  4  of  which  belong 
to  a  face-centred  cube  as  in  the  case  of  gold,  while  the  four  remaining 
atoms  are  situated  on  the  body  diagonals  halfway  between  the  corners 
of  the  cube  and  its  centre  (Fig.  50a).  Thus  the  unit  cell  contains,  as  it 
were,  4  central  atoms  surrounded  by  4  neighbouring  atoms  at  the 
corners  of  a  tetrahedron,  in  conformity  with  their  co-ordination  num- 
ber (Fig.  29).  If  this  three-dimensional  lattice  is  projected  on  to  its 
base.  Fig.  50b  is  obtained,  which  shows  the  arrangement  of  valency 
lines  commonly  used  in  organic  chemistry !  Thus  the  usual  scheme  of 
the  quadrivalent  carbon  (Fig.  24,  p.  25)  is  morphologically  correct  if  it 
is  considered  as  the  projection  of  a  tetrahedron. 

According  to  X-ray  analysis,  the  lattice  period  of  diamond,  i.e.,  the  edge 
of  the  cube,  measures  3.55  A.  It  follows  that  the  distance  between  the  lattice 
points  on  the  face  diagonal  is  1-3.55 -Vz  =  2.51  A;  the  shortest  distance 
between  two  C-atoms  on  the  body  diagonal  is  5-3.55  -^3  =  1.54  A.  It  is 
in  this  simple  way  that  the  C — C-distance  corresponding  to  the  sphere  of 
action  of  a  C-atom  in  an  aliphatic  bond  has  been  calculated  (Table  III,  p.  31). 


34        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

If  a  plane  is  drawn  through  two  body  diagonals,  the  arrangement  of 
lattice  points  obtained  is  as  represented  in  Fig.  31a.  In  this  cross- 
section  the  C-atoms  are  joined  by  a  zig-zag  line  whose  links  enclose 
the  so-called  tetrahedron  angle  of  109°.  5.  On  parallel  planes,  further  ar- 
rays of  such  zig-zag  chains  are  found,  one  of  which  has  been  represented 
by  dotted  lines.  It  is  linked  up  with  the  other  two  by  primary  valencies. 


.^ , 


I   / 


7.45^ 


'CH, 

J^ 

/^ 

V 

r'&f 

TH. 

Y 

Tcwp 

\    ' 

y   \ 

V  ^ 

Fig.  31.  «)  Diamond  lattice  (primary  valency  lattice)  as  compared 

with  Fig.  30a  by  45°  inclined,  b)  Paraffin  lattice  (molecule  lattice); 

V  =  valency  angle  =  109°. 5 


Aliphatic  compounds  (chain  lattice).  The  zig-zag  arrangement  described 
is  fundamental  to  the  morphology  of  saturated  carbon  compounds; 
for  it  has  been  found  that  all  aliphatic  molecules  represent  such  kinked 
chains.  In  paraffin  molecules,  for  instance,  the  increase  in  chain  length 
for  each  additional  C-atom  is  1.27  A  instead  of  1.54  A.  It  can  easily 
be  calculated  that  this  is  in  conformity  with  the  zig-zag  chains  show- 
ing the  tetrahedron  angle.  In  this  way  two  carbon  atoms  reach  a 
spacing  of  2.54  A,  which  is  the  intramolecular  period  of  the  paraffins 
(Hengstenberg,  1928;  MiJLLER,  1929;  Halle,  193  i). 

In  Fig.  31b  it  is  shown  how,  by  parallel  alignment,  such  chains 
combine  into  the  rhombic  crystal  lattice  of  the  paraffins.  It  seems 


PRINCIPLES  OF  STRUCTURE 


55 


paradoxical  that  the  soft,  plastic  paraffin  crystals  should  have  a  lattice 
structure  so  similar  to  the  diamond  model  represented  by  Fig.  31a. 
Notwithstanding  the  apparent  analogy,  however,  there  exist  funda- 
mental differences  which  explain  the  differences  in  the  physical  be- 
haviour of  the  two  substances.  In  particular,  the  lattice  of  the  paraffin 
crystals  is  built  much  more  loosely.  This  is  caused  by  the  fact  that 


cleavage< 
plane 


homogeneous 
lattice  region 


<>   ^l  <>   I   S  2 
a  b 

Fig.  32.  Aliphatic  chains,  a)  Molecule  lattice;  b)  chain  lattice. 


these  crystals  possess  not  a  main  valency  lattice,  but  a  molecule 
lattice.  The  chains  are  joined  by  Van  der  Waals  forces  only,  since 
the  CH2  groups  are  able  to  bind  only  two  neighbouring  groups  by 
primary  valencies.  Thus  in  the  paraffin  lattice  we  have  two  types 
of  distances :  molecular  distances  of  the  order  of  magnitude  5  A  and 
atomic  ones  of  the  order  of  magnitude  1.5  A  (Fig.  31b).  The  fact  that 
in  the  diamond  lattice  all  C-atoms  touch  each  other  explains  its  great 
density  and  hardness.  The  paraffin  lattice,  on  the  other  hand,  has  a 
much  lower  density  and  layers  of  molecules  can  be  shifted  with  respect 
to  each  other  with  relative  ease  (Fig.  32).  This  accounts  for  the  soft- 
ness and  plasticity  of  paraffin  crystals. 

As  long  as  the  paraffin  chains  are  short,  they  easily  crystallize  into 
a  molecular  lattice.  This  leads  to  crystals  in  the  form  of  flakes,  which 


2,6        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

can  be  cleft  along  the  base  (Fig.  32a).  When,  however,  the  chains 
grow  to  great  length,  it  becomes  increasingly  difficult  to  arrange  the 
terminal  groups  in  fixed  planes,  and  crystallization  takes  place  as 
pictured  in  Fig.  32b.  Here  no  rigorous  lattice  order  prevails,  since  a 
lengthways  displacement  of  one  chain  with  respect  to  another,  over 
distances  equal  to  some  intramolecular  spacings,  i.e.,  only  a  fraction 
of  the  chain  length,  does  not  affect  the  lattice  structure.  This  is  because, 
owing  to  their  multiplicity,  the  smaller  spacings  inside  the  molecule 
(2.54  A  in  the  case  of  paraffins)  overshadow  the  periodicity  of  the  end 
groups.  These  arrangements  of  long  chains  are  called  c/yam  lattice.  It 
is  significant  that  the  chains  cannot  revolve  around  their  longitudinal 
axis ;  if  they  could,  there  would  be  no  lattice  order.  The  cross-section 
of  the  chain  lattice  is,  therefore,  homogeneous,  but  inhomogeneities, 
which  are  indicated  in  Fig.  32b  by  the  end  groups,  occur  lengthwise, 
leaving  only  small  homogeneous  lattice  regions. 


CH, 


.CH. 


I  \ 

3CH2 


CH2 

CH2      H2C4 


H2C4 


H2/ 


CH2 

CHj 
o) 


\ 

5CH2 


V 

H2 


.H, 


'CHj 


H3C6 


b) 


Fig-  33 


Fig-  34 


Fig.  33.  Graphite  lattice  -  Fig.  34.  Hexane.  a)  Conventional  structural  formula; 
b)  morphologically  correct  formula;  c)  ring  constellation,  supplement  to  the 

valency  angle  v  =  70°. 5. 


Aromatic  compounds  {layer  lattice).  Unlike  the  aliphatic  compounds,  the 
aromatic  ones  cannot  be  derived  from  the  structure  of  diamond.  Their 
structure  is  similar  to  that  oi graphite.  This  modification  of  carbon  crystallizes 
in  the  hexagonal  system  and  possesses  a  crystal  lattice  as  represented  in 
Fig.  33.  The  carbon  atoms  form  rings  containing  6  atoms,  which  are  linked 
together  in  an  uninterrupted  plane.  Thus  at  each  lattice  point  3  primary 
valencies  are  engaged.  The  fourth  valency  is  distributed  among  the  neigh- 
bouring atoms  as  in  the  benzene  ring  (Fig.  24,  p.  25).  Accordingly,  as  a 
result  of  the  larger  bond  energy,  the  C — C-distance  is  reduced  to  1.45  A 
(see  Fig.  33).  As  all  primary  valencies  are  thus  engaged  in  a  plane,  the 


2  PRINCIPLES  OF  STRUCTURE  37 

resulting  main  valency  layers  are  united  into  a  lattice  by  weaker  secondary 
valencies.  The  distance  between  the  layers  (3.41  A)  is  therefore  considerably 
larger  than  that  in  the  rings.  A  structure  in  which  the  lattice  forces  and 
spacings  within  a  plane  are  so  different  from  those  in  a  direction  perpen- 
dicular (or  nearly  perpendicular)  to  this  plane  is  called  a  layer  lattice.  Com- 
pounds of  this  lattice  type  always  crystallize  in  the  form  of  flakes  and  are 
as  a  rule  easily  split  along  the  base  (mica,  serisite).  Many  benzene  derivatives 
and  other  aromatic  compounds  (naphthalene,  anthracene,  etc.)  belong  to  this 
class.  The  division  into  aliphatic  and  aromatic  substances  is  therefore  not 
only  based  upon  their  chemical  behaviour,  but  it  also  has  a  morphological 
background  in  that  the  one  tends  to  crystallize  into  a  chain  lattice,  while  the 
other  shows  a  strong  tendency  towards  the  development  of  a  layer  lattice. 

Cyclic  compounds.  The  structural  formulae  of  aliphatic  chemistry  are 
found  to  be  very  similar  to  molecular  models  if  the  valency  angle 
between  two  successive  C-C  bonds  are  taken  into  account.  A  chain 
such  as  hexane  should  therefore  be  kinked  instead  of  straight  (Fig.  34a, 
and  b).  Molecules  which  do  not  form  part  of  a  crystal  lattice,  but  can 
freely  move  about  in  the  gaseous  or  dissolved  state,  are  subject  to  the 
so-called  free  rotation  of  the  groups  around  the  direction  of  the  valency 
lines.  In  Fig.  34a  rotation  would  not  give  rise  to  a  new  structure. 
In  kinked  chains,  however,  the  free  rotation  means  that,  for  instance, 
group  I  in  Fig.  34b  need  not  necessarily  lie  in  the  plane  of  drawing 
with  2  and  5 ;  it  can  be  located  anywhere  on  the  perimeter  of  a  cone 
which  has  its  apex  in  group  2  and  whose  apical  angle  is  the  supplement 
of  the  valency  angle.  Among  these  possibilities  there  is  one  special 
case  in  which  groups  4  and  5  are  turned  through  1 80°,  thus  resulting 
in  a  ring-shaped  model.  It  is  not  difficult  to  see  that  this  can  easily  lead 
to  cyclic  compounds.  Fig.  34c  shows  why  rings  of  5  or  6  atoms  are 
formed  preferentially:  the  supplement  (70°. 5)  of  the  valency  angle  is 
contained  somewhat  less  than  6  and  somewhat  more  than  5  times  in 
360°  (5 -70°. 5  =  3  5  2°. 5;  6 -70°. 5  =  423°).  The  different  forms  which  a 
molecule  can  assume  are  called  its  constellations;  so  Figs.  34b  and  c 
represent  two  different  constellations  of  the  same  molecule  hexane. 

Other  atoms  besides  carbon  can  also  occur  in  the  ring  (heterocyclic 
rings).  Let  us  here  briefly  discuss  the  example  of  sugar,  which  is  so 
important  in  biology.  The  monosaccharides,  which  formerly  were 
considered  as  "open"  chains  (Fig.  35a),  have  been  shown  to  contain 
a  heterocyclic  ring  with  an  oxygen  bridge.  In  glucose  this  is  usually 
a  T-5  bond,  often  represented  in  the  manner  of  Fig.  35  b.  The  formula, 


38        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

however,  is  not  true  to  reality,  since  the  C-O  distance  in  it  is  unduly 
large.  Haworth  (1925),  therefore,  writes  sugar  as  an  equilateral 
hexagon  or  pentagon,  according  as  to  whether  the  oxygen  bridge  is 
situated  between  the  carbon  atoms  1-5  (derivatives  of  pyranose)  or 


CH=0 
H><OH 
HO^H 
H^OH 
HxOH 
CH2OH 
o) 


C—OH 
\H 


2  HCOH 

I 

3  HOCH 


HCOH 

I 
HC 


CH2OH 
b) 


cj 


dj 


Fig.  35.  Glucose,  a)  Aliphatic,  b)  heterocj^clic  sti-uctural  formula; 
c,  d)  a-  and  /^-configuration  after  Haworth  (1925,  1929). 

1-4  (derivatives  of  furanose).  Figs.  35c  and  d  represent  the  glucose 
pyranoses.  With  the  aid  of  the  distance  rules  (see  Table  III,  p.  31),  the 
dimensions  of  a  glucose  molecule  can  be  calculated.  For  example,  on 
the  assumption  that  the  ring  is  completely  in  one  plane  and  represents 

an  equilateral  hexagon,  the  axis  drawn  through 
the  C-atoms  i  and  4  has  a  length  of  2  •  i .  5  4  + 
2-1.49  =  ^-^^  ^-  '^^^^  value  is  only  approx- 
imate, because,  to  begin  with,  the  hexagon  is 
not  completely  equilateral  on  account  of  the 
somewhat  smaller  diameter  of  the  O-atom, 
and  further,  the  C-atoms,  as  well  as  the  OH- 
groups  represented  by  the  O-atoms,  do  not 
lie  strictly  in  the  plane  in  which  the  distances 
are  measured  so  that,  instead  of  the  distances 
C-C  and  C-O,  only  their  projections  contri- 
bute to  the  length  concerned.  If  all  this  is  ta- 
ken into  account,  the  smaller  value  of  5.15  A 
is  obtained,  which  corresponds  to  that  found 
by  X-ray  analysis.  Fig.  36  shows  the  far-reaching  similarity  between 
the  present  structural  formulae  (Fig.  55c)  and  the  molecular  models. 
The  former  no  longer  represent  arbitrary  schemes,  but  rightly  propor- 
tioned projections  of  the  molecular  structure  on  a  plane. 

According  to  the  aliphatic  manner  of  writing  (Fig.  35a),  glucose 
contains  four  asymmetric  C-atoms  (x),  since  only  the  CHo-  and  the 


Fig.  36.  Molecular  structure 

of  glucose  (from  Meyer  and 

Mark,  1930). C-atomshatch- 

ed,  O-atoms  encircled. 


2  PRINCIPLES  OF  STRUCTURE  39 

C=:0  group  have  a  symmetry  plane.  As  a  result  of  the  ring  forma- 
tion, however,  the  i  C-atom  of  the  carbonyl  group  also  becomes 
asymmetric.  For  that  reason  two  different  configurations  of  the  hetero- 
cyclic ring  are  possible;  they  are  called  a-  and  /3-glucose  (Fig.  35c  and 
d)  and  are  distinguished  by  their  optical  rotation  (/?  shows  the  smaller 
rotation).  It  is  seen  that  the  /S-glucose  shows  a  regularly  alternating 
distribution  of  the  H-  and  OH-groups  on  both  sides  of  the  ring,  while 
in  a-glucose  the  hydroxyl  groups  at  the  i  and  2  C-atoms  are  neighbours. 

With  /3-glucose  it  is  possible  to  lay  a  second  bridge  between  the  i 
and  the  6  C-atoms  by  dehydration  (laevo-glucosan) ;  in  a-glucose  this 
is  impossible.  This  proves  that  in  /5-glucose  the  OH-group  of  the  i 
C-atom  lies  on  the  same  side  of  the  ring  as  the  one  of  the  6  C-atom. 

The  a  and  ^  positions  of  the  OH-groups  at  the  i  C-atom  are 
fundamental  to  an  understanding  of  the  structure  of  disaccharides  and 
high-polymer  carbohydrates.  In  disaccharide  formation  a  1-4-bridge 
between  two  glucose  rings  is  formed  by  loss  of  one  molecule  of  water. 
Now  it  is  easy  to  see  that  in  the  case  of  the  a-position  the  two  rings 
can  simply  be  joined  directly,  whereas  in  the  case  of  /3-position  one 
of  the  rings  must  first  rotate  through  an  angle  of  180°  around  its 
1-4-axis  in  order  to  bring  the  two  OH-groups  which  are  to  react  into 
a  neighbouring  position. 

OH  OH  OH  CHpOH 

CH^OH  CH2OH  CH^OH  OH 

Fig.  37a.  Maltose  Fig.  37b.  Cellobiose 

Disaccharides  from  glucose 

Both  cases  are  realized  in  nature;  in  the  first  case  maltose  is  formed 
and  in  the  second  cellobiose,  the  disaccharide  unit  of  the  cellulose 
chain  (Fig.  37).  In  maltose  the  two  glucose  rings  can  be  made  to 
coincide  by  a  simple  translation,  whereas  in  cellobiose  this  requires 
a  digonal  axis.  The  cellobiose  molecule  therefore  possesses  a  higher 
degree  of  symmetry,  seeing  that  the  coincidence  must  be  achieved  by 
a  combination  of  a  translation  and  a  rotation. 

The  bond  represented  in  Fig.  37a  is  described  as  a-glucosidic  and 
the  one  in  Fig.  37b  as  /5-glucosidic.  Instead  of  sugar  molecules,  all 


40        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

kinds  of  different  molecules  containing  hydroxyl  groups  can  combine 
with  glucose  according  to  both  these  schemes,  which  are  then  dis- 
tinguished as  a-  and  /?-glucosides  respectively.  This  distinction  is 
not  only  interesting  and  important  from  the  point  of  view  of  molecular 
morphology  (structural  chemistry),  but  is  also  of  great  importance  in 
physiology.  In  fact,  the  a-  and  /3-bridges  are  broken  down  by  quite 
different  enzymes.  For  the  hydrolysis  of  maltose  we  need  an  a- 
glucosidase,  which  is  not  capable  of  splitting  cellobiose,  while,  con- 
versely, ^-glucosidases  can  attack  cellobiose  but  are  inactive  with 
respect  to  maltose.  It  seems  that  in  plants  the  reserve  substances, 
which  mus"  be  quickly  mobilized  when  required,  are  more  often  built 
according  to  the  a-type  (saccharose,  starch),  while  glucosides,  which 
cannot  be  used  directly  as  reserves  (e.g.,  amygdalin),  and  cellulose 
are  /5-glucosides.  This  example  shows  that  ultimately  the  problem 
of  enzymes  is  also  of  a  morphological  nature.  To  be  able  to  distinguish 
between  an  a-  and  a  /5-bond,  they  must  possess  a  quite  specific  struc- 
ture. Without  a  knowledge  of  this  structure,  it  is  unlikely  that  the 
riddle  of  organic  catalysis  will  be  solved  (Mittasch,  1936).  The  well- 
known  comparison  of  the  lock  and  the  key  is  not  merely  a  symbol,  but 
substrate  and  enzyme  must  fit  together  in  the  strict  sense  of  the  word 
as  two  parts  which  are  adjusted  morphologically  to  each  other. 

c.  Structure  of  Phase  Boundaries 

Surface  tension.  The  regions  containing  phase  boundaries  are  always 
inhomogeneous.  One  can  only  speak  of  homogeneous  phases  if  in 
comparison  with  their  surface  they  are  so  extended  that  all  surface 
effects  can  be  neglected. 

These  inhomogeneities  are  best  known  in  liquids,  where  they 
manifest  themselves  as  surface  tension;  but  they  also  occur,  although 
less  markedly,  at  the  surface  of  crystal  lattices  or  at  the  boundary  of 
gaseous  phases.  The  surface  tension  of  a  liquid  is  caused  by  the  fact 
that  the  molecules  in  the  bulk  of  the  phase  are  surrounded  on  all  sides 
by  similar  molecules,  whereas  in  the  phase  boundary  this  only  occurs 
on  one  side.  If,  by  way  of  example,  we  consider  a  liquid-gas  boundary 
layer,  the  attractive  forces  of  the  small  number  of  gas  molecules  avail- 
able can  at  a  first  approximation  be  neglected;  therefore,  at  the  surface 
the  molecules  are  subject  to  a  quite  different  field  of  cohesive  forces 
from  that  to  which  those  inside  the  liquid  are  exposed. 


2  PRINCIPLES  OF  STRUCTURE  4I 

As  Fig.  38c  shows,  the  cohesive  forces  acting  on  a  molecule  at  the 
surface  do  not  cancel  each  other.  The  particles  are  therefore  attracted 
by  the  bulk  of  the  liquid.  It  will  yield  to  this  attraction  as  far  as  possible 
and  to  some  extent  decrease  its  distance  from  the  deeper-lying  mole- 
cules. This  results  in  an  increase  in  density,  of  which  a  rough  outline 
is  given  in  Fig.  38d.  In  this  way  a  surface  "skin"  is  formed,  which  on 
its  inner  side  merges  into  the  area  of  the  homogeneous  liquid. 

The  surface  skin  possesses  a  certain  firmness  because  its  molecules 


^-y' •      •   • 

•       •     • 


a 


^J  b)  cj  d) 

Fig.  38.  Inhomogeneity  of  the  phase  boundary  liquid/gas.  Cohesive  forces  a)  symmetrical, 

b)  asymmetrical,  c)  directed  inwards;  ii)  scheme  of  the  inhomogeneous  arrangement  of 

molecules  (greatly  exaggerated,  as  the  compressibility  of  liquids  is  very  small). 


cannot  move  as  freely  as  in  the  ideal  liquid.  This  firmness  can  be  de- 
termined by  stretching  a  lamella  of  the  liquid  suspended  in  a  frame 
by  means  of  a  movable  bar,  and  by  measuring  the  weight  needed  to 
break  the  film.  This  weight  is  independent  of  the  thickness  of  the 
lamella,  but  is  a  Hnear  function  of  the  length  1  of  the  bar,  since  a 
lamella  which  is  twice  as  broad  can  carry  twice  the  weight.  The  firm- 
ness of  the  surface,  therefore,  refers  to  the  unit  of  length,  i  cm,  and 
the  force  which  is  capable  of  rending  a  lamella  surface  i  cm  wide  is 
called  the  surface  tension  a  of  the  liquid.  As  both  the  surface  in  front 
and  that  at  the  back  ol  the  lamella  must  be  broken,  the  force  p  =  2  ct  1 

(Fig-  39)- 

Instead  of  the  more  accurate  methods  of  surface  tension  measure- 
ments with  the  aid  of  capillary  rise  or  stalagmometry  (Hober,  1922, 
p.  154),  the  much  more  primitive  breaking  method  has  been  mentioned 
here,  because  the  definition  of  surface  tension  is  founded  on  it  and  it 
demonstrates  in  a  simple  way  its  dimension  as  force/cm.  Surface 
tension,  therefore,  is  not  tension  in  the  ordinary  sense,  for  otherwise 
its  dimension  would  have  been  force/cm^.  The  difference  between  these 
two  quantities  can  be  seen  from  the  scheme  given  in  Fig.  40.  In  order 


42        FUNDAMENTALS  OF  SUBMIC ROSCOPIC  MORPHOLOGY  I 

to  rend  a  plane,  the  cohesive  forces  have  to  be  overcome  along  a  line 
only,  whereas  in  the  case  of  a  rod  the  force  has  to  be  applied  to  a  plane. 
Hence  Figs.  40a  and  b  are  graphic  representations  of  the  definition 
of  surface  tension  (force/cm)  and  cohesive  tension,  or  pressure  (force/ 
cm^)  respectively. 

To  understand  this  better,  let  us  compare  the  surface  tension  and 
the  cohesive  tension  of  water.  For  water  at  15°  C,  a  amounts  to 
7.30  mg/mm,  which  in  absolute  units  is  71.6  dynes/cm.  In  order  to 


^\~^^\^^^^^^^^^ 

k 

A 

0) 

A— 

rr-7--m 

A            ■          A 



!  —.' 

-y  " 

Fig.  40 

'1 

Fig-  39 

Fig.  39.  Measurement  of  the  surface  tension  of  a  lamella  (from  Lecher, 
1919)  -  Fig.  40.  a)  Dimension  of  surface  tension  (force/cm);  b)  di- 
mension of  cohesive  tension  (force/cm-). 


measure  the  cohesive  pressure  or  inner  pressure  (Freundlich  and 
LiNDAu,  1932),  one  must  tear  apart  planes  of  water  in  which  the  mole- 
cules cannot  change  position  with  respect  to  each  other,  for  example 
a  film  of  water  between  two  hydrophilic  pistons.  Such  experiments, 
however,  do  not  produce  reliable  evidence.  The  cohesive  tension  must 
also  be  overcome  when  water  is  torn  from  the  cell  wall  in  a  desiccating 
cell.  According  to  the  osmotic  measurements  of  Renner  (191 5)  and 
Ursprung  (191 5)  with  fern annulus cells,  thiscohesive  tension  amounts 
to  300  to  350  atm.  From  the  heat  of  vaporization  of  water,  however, 
the  much  larger  value  of  about  10^  atm.  is  derived  (Lecher,  p.  60).  In 
absolute  units  this  corresponds  to  an  order  of  magnitude  of  10^" 
dynes/cm^.  Since  the  surface  layer  of  water  has  a  thickness  of  at  least 
3  A  (the  diameterof  the  water  molecule  is  2.78  A),  about  1/3-10^  of  such 
layers  is  needed  to  account  for  the  cohesive  tension.  Multiplying  the 
surface  tension  of  71.6  dynes/cm  of  a  monolayer  by  1/3  •  10^,  we  obtain 


PRINCIPLES  OF  STRUCTURE 


45 


about  1/4- lo^*^  dynes/cm^,  which  result  corresponds  to  the  order  of 
magnitude  mentioned  above. 

The  product  of  surface  tension  and  area  has  the  dimension  of 
energy :  cm^  •  force/cm  =  force  •  cm  =  energy.  Instead  of  surface  ten- 
sion, the  notion  oi  surface  energy  is  therefore  often  used.  If  much  work 
has  to  be  done  to  increase  the  surface,  as  for  instance  in  water  or 
other  hquids  with  many  OH-groups  in  contact  with  air,  the  surface 
energy  is  large  (see  Table  V). 


TABLE  V 
SURFACE  TENSION  AGAINST  AIR  AT   I  5 

(hober,  1922,  p.  167) 


C 


o.zs  molar  solutions 


o 
dyne  cm 


Relative  o 
(a  H^O  =  i) 


Water 

Cane  sugar 

Urea 

Glycerol 

Acetic  acid 

Ethyl  alcohol     .    .    .    . 
Ethyl  ether  (satur.sol.) 
Ethyl    acetate     .    .    .    . 

i-Valeric  acid 

i-Amyl  alcohol  .    .    .    . 


71.6 
72.1 
71.6 

71-5 
66.8 

66.0 

53-1 
41-^ 

34-9 
29.9 


1.000 
1.007 
1. 000 
0.999 
0.932 
0.922 
0.742 
0.578 
0.487 
0.417 


As  it  is  impossible  to  disperse  water  in  ethyl  alcohol  or  other  liquids 
with  which  it  is  miscible,  in  the  form  of  drops,  obviously  the  water 
molecules  can  be  transferred  to  the  surrounding  dispersing  medium 
without  doing  any  work.  Thus  the  surface  tension  between  two 
mixing  phases  is  2ero  and,  therefore,  no  phase  boundary  is  formed.  By 
analogy,  a  hydrated  solid  colloid  particle  cannot  be  supposed  to  pos- 
sess surface  energy  if  the  water  dipoles  in  the  outer  shell  of  the 
hydration  layer  have  the  same  mobility  as  those  in  the  bulk  of  the 
water.  In  that  case  we  are  dealing  with  the  situation  illustrated  in  Fig. 
20a  (p.  20),  i.e.,  the  particle  loses  its  surface  and  is  in  stable  solution 
in  the  dispersing  medium. 

The  examples  given  show  that  it  is  not  enough  to  speak  merely  of 
the  surface  energy  of  a  Hquid  without  specifying  the  medium  in  con- 


44        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

tact  with  which  the  surface  tension  has  been  measured.  The  data  given 
in  the  literature  usually  refer  to  the  surface  tension  against  air.  In 
cytology,  however,  we  are  concerned  in  the  first  place  with  the  surface 
tension  of  the  protoplasm  against  the  nutrient  solution  or  the  cell 
sap(TableXXI,  p.  i66). 

The  surface  tension  against  air  has  become  of  great  importance  in 
physiology.  As  shown  by  analysis  of  foams,  many  substances  are 
accumulated  at  the  surface,  which  usually  lowers  the  surface  tension 
to  a  considerable  extent  (Table  V),  On  the  basis  of  thermodynamics 
the  Gibbs-Thomson  theorem  renders  account  of  this  phenomenon  by 
the  two  following  rules :  i .  Substances  which  lower  the  surface  tension 
of  water  accumulate  at  the  surface;  2.  a  small  amount  of  a  solute  can 
strongly  reduce  the  surface  tension  but  cannot  appreciably  increase  it. 

Hydrophily  and  Upophily.  To-day  these  relations  can  easily  be  under- 
stood quahtatively  with  the  aid  of  simple  rules  on  the  mutual  mis- 
cibility  of  different  types  of  molecules.  Water  and  ethyl  alcohol, 
for  instance,  are  miscible  in  any  proportions  as  are  also  absolute 
alcohol  and  ethyl  ether.  Water  and  ethyl  ether,  however,  are  only  mis- 
cible to  a  very  small  extent.  The  phase  theory  contents  itself  with 
determining  the  range  of  miscibility,  without  being  concerned  with 
the  cause  of  the  insolublity.  The  theory  of  structure,  however,  tries 
to  form  a  notion  of  the  limited  solubility  of  water  and  ether  and  vice 
versa.  The  reasoning  is  as  follows. 

If  alcohol  and  water  are  mixed,  the  water  molecules  will  be  pre- 
ferably attached  to  the  kindred  hydroxyl  groups,  more  or  less  accord- 
ing to  the  scheme  of  Fig.  41a.  In  the  presence  of  an  excess  of  water 
the  OH-group  is  hydrated  in  much  the  same  way  as  in  Fig.  18  (p.  19) 
by  orienting  and  attracting  the  dipoles,  be  it  only  to  a  small  extent. 
Each  OH-group,  therefore,  is  surrounded  by  a  water  shell  designated 
by  the  dotted  circle  in  Fig.  41a.  The  alkyl  group,  on  the  other  hand, 
tries  to  escape  from  the  water  molecules,  because  it  is  hydrophobic. 
It  therefore  protrudes  from  the  hydration  layer  if  its  size  allows,  as, 
e.g.,  in  butyl  or  amyl  alcohol.  In  ethyl  alcohol,  however,  the  sphere 
of  action  of  the  OH-group  corresponds  approximately  to  the  length 
of  the  alkyl  group,  hence  water  dipoles  can  settle  all  round  the  mol- 
ecule. This  explains  the  unlimited  miscibility  of  ethyl  alcohol  and 
water.  In  the  higher  members  of  the  aliphatic  alcohol  series,  however, 
the  lipophilic  part  of  the  molecular  chain  predominates,  with  the 


PRINCIPLES  OF  STRUCTURE 


45 


result  that  only  a  limited  number  of  water  dipoles  can  be  attached. 
If  very  little  water  is  present,  all  the  hydroxyl  groups  of  the  alcohol 
molecules  accumulate  round  the  few  water  dipoles  available  (Fig.  41b) ; 
in  96%  ethyl  alcohol,  for  instance,  9-10  CH3CH0OH  round  each  HgO 
molecule.  This  water  is  bound  so  strongly,  that  it  can  only  be  separated 


op  o  9 


00  0^0 

^•00.-     'QP^ 


Fig.  41.  Solubility.  Water  molecules  and  OH-groups  hatched,  lipophilic 
groups  (-CH3,  -CHj-,  -O-  bridges)  black.  O.x^'gen  groups  (-OH  and  -0-) 
surrounded.  fl)Ethanol  CHa-CHg-OH  and  water  HgO  (unlimited  miscibil- 
ity).  ^)  bound  water  in  96  °o  ethanol,  c)  ethanol  and  ethyl  ether 
CHg-CHa-O-CHg-CHg  (unlimited  miscibility),  d)  water  in  "moist"  ethyl 
ether  (very  limited  miscibilit}-). 


from  the  hydroxyl  groups  by  chemical  means.  As  is  well-known,  ab- 
solute ethyl  alcohol  cannot  be  obtained  by  distillation,  but  only  by 
chemical  dehydration. 

A  still  more  simple  reasoning  applies  to  the  miscibility  of  alcohol  and 
ether  (Fig.  41c).  Notwithstanding  the  homopolar  character  of  the 
ether  bridge,  i.e.,  the  -O-group,  it  still  has  a  certain  affinity  for  the 
OH-group.  Consequently,  both  the  hydroxyl  group  and  the  alkyl  group 
of  the  alcohol  can  enter  into  some  chemical  relationship  with  the  two 
parts  of  the  ether  molecule.  This  is  not  so  when  we  attempt  to  dissolve 
water  in  ether.  The  -O-bridge  has,  admittedly,  a  certain  affinity  for 
water,  but  this  affinity  is  slight,  so  that  only  a  limited  number  of  water 
molecules  can  be  bound  by  a  given  number  of  ether  molecules  (Fig. 
4 id).  The  circumstances  are  similar  to  those  in  96%  ethyl  alcohol  — 


46 


FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY 


but. 


whereas  in  that  case  the  number  of  HgO  molecules  round  a 


80 
°C 

60 
40 
20 


hydroxyl  group  could  be  increased  ad  libitum,  each  -O-bridge  can 
only  attract  a  fraction  of  a  water  molecule.  For  that  reason,  as  soon  as 
the  amount  of  water  present  exceeds  a  certain  limit,  the  water  mole- 
cules must  cluster  together.  They  accumulate  into  drops  and  form 
their  own  phase.  Conversely,  a  few  ether  molecules  may  be  dispersed 
in  this  phase,  but,  as  will  be  shown,  these  ether  molecules  tend  to 
accumulate  in  the  neighbourhood  of  the  phase  boundary. 

Much  the  same  phenomena  are  observed  in  the  phenol/water  sys- 
tem (Fig.  42).  If  some  phenol  is  added  to 
water,  it  is  dissolved.  Beyond  a  certain 
percentage  of  phenol,  however,  two  co- 
existent phases  are  obtained,  which  do 
not  mix.  Similarly,  traces  of  water  are 
soluble  in  pure  phenol,  but  if  the  amount 
of  water  is  increased,  a  miscibility  limit 
is  reached  beyond  which  the  two  phases 
no  longer  mix.  As  shown  in  Fig.  42,  the 
miscibility  depends  not  only  on  the  con- 
centration of  the  two  components  but  also 
on  the  temperature.  In  the  region  called 
the  Miscibility  gap  the  system  is  hetero- 
geneous. Here  two  phases  are  formed,  one 
consisting  of  phenol  saturated  with  wa- 
ter and  the  other  of  water  saturated  with 
phenol.  Outside  the  miscibility  gap  only 
a  single  phase  exists,  a  homogeneous 
solution  with  a  completely  uniform  distribution  of  intermingled 
phenol  and  water  molecules.  When  heat  is  supplied,  the  miscibility 
of  the  two  components  increases,  until  at  a  certain  temperature  the 
miscibility  gap  disappears.  At  low  temperature  the  hydration  layer  of 
the  phenolic  OH-group  is  smaller  in  si2e  than  the  phenylic  residue, 
so  that  limited  miscibility  results.  With  rising  temperature  the  hydration 
sphere  is  increased  and  at  69°  C  surrounds  the  whole  space  of  the  CgHg- 
group  (comparable  to  Fig.  4 1  a)  causing  in  this  way  unlimited  miscibility. 
To  sum  up,  the  decisive  factor  in  the  solubility  of  organic  substances 
in  water  is  not  only  the  presence  of  hydrophilic  (i.e.,  water-attracting) 
groups,  but  primarily  also  their  nuwber  in  comparison  with  the  number 


Homogeneous 

solution        1 

X 

X, 

/ 

X 

/ 

\ 

X 

\ 

/ 

Miscibility 

\ 

X 

gof. 

> 

\ 

: 

t 

\ 

Water  - 


20 


40 


60 


80  100% 

— ^Phenol 


Fig.  42.  Diagram  of  miscibility  of 
the  water/phenol  system  (from 
Rothmund,  1898).  Abscissa: 
from  left  to  right  content  of 
phenol  in  %  of  weight.  Ordinate: 
Temperature  in  °C. 


PRINCIPLES  OF  STRUCTURE 


47 


of  hydrophobic  (i.e.,  water-repelling)  groups  in  the  molecule.  On  the 
other  hand,  the  latter  groups  determine  the  solubility  in  homopolar 
liquids  (solvents  for  fatty  substances),  such  as  hydrocarbons,  carbon 
tetrachloride,  carbon  disulphide,  ether,  chloroform,  benzene,  olive  oil, 
etc.,  and  for  this  reason  are  designated  as  lipophilic  groups.  Table  VI 
gives   a  survey  of  the  various   hydrophilic  and  lipophilic  groups 


TABLE  VI 
HYDROPHILIC  AND  HYDROPHOBIC  GROUPS 


Hydrophilic  (lipophobic) 
dipole  character  (often 
tendency  to  form  ions) 

Lipophilic  (hydrophobic) 
homopolar 

S            ^\OH        carboxyl 
%        —OH             hydroxyl 
^       — C\tt            aldehyde 

"2            C — O          carbonyl 
^       — NHj            amino 
S            NH             imino 

1           C^NH,       ^^^^« 
^       — C<(qj^         imido 

• — SH               sulphydryl 

^ 

00 

Oh 

C 

•■—* 

'o 

bJO 

G 

<u 
u 
u 

r 

— CH3              methyl 

^Ho       i             1    1 
ChJ       ^       niethylene 

C2H5             ethyl 
— C3H7             propyl 
— QjHan+i        alkyl 
— C5H8 —         isoprene  group 
the  terpenes 

— CgHs              phenyl 

of 

occurring  in  the  organic  compounds  participating  in  the  construction 
of  the  protoplasm.  With  the  aid  of  this  table  it  is  possible  in  many 
cases  to  derive  from  the  chemical  structural  formula  of  a  substance 
its  solubility  in  an  organic  compound. 

Surface  films.  The  lipophilic  nature  of  the  alkyl  radicals  explains  the 
lowering  of  the  surface  tension  reproduced  in  Table  V  (p.  43).  As 
shown  in  Fig.  41a,  the  lipophilic  ends  of  the  alcohol  molecules  pro- 
trude to  a  certain  extent  from  the  hydration  layer.  In  their  attempt  to 
escape  from  the  water  dipoles,  they  tend  to  approach  each  other  and 
to  accumulate  at  a  phase  boundary.  It  is  the  hydrophobic  nature  of 
the  alcohol  molecules,  therefore,  which  causes  their  accumulation  at 
the  surface;  they  are  said  to  be  surface-active.  This  applies,  of  course,. 


48        FUNDAMENTALS  OF  SUBMIC  ROSCOPIC  MORPHOLOGY 


only  to  those  cases  where  the  adjacent  phase  itself  is  not  hydrophilic. 
This  will  almost  always  hold  good  at  the  liquid/gas  phase  boundary. 
Fig.  43a  shows  the  arrangement  of  alcohol  molecules  at  the  surface. 
As  their  molar  cohesion  is  less  than  that  of  water  with  its  OH-groups 
(see  Table  IV,  p.  32),  the  surface  tension  will  decrease.  The  molecules 


fo  ^°  o  1)1 '0% 


o  o  ^^0/ 
o    o 


o    o 


o     o 

o 
o 


o 


o 


o     o 
o 


o 


o  o 


V 


''0^0  o^o 


o=c 


^OH 


o 


0^0   ogooOo 


o°o 


o 


o    o 


d) 


Fig.  43.  Molecular  surface  structure  of  aqueous  solutions.  Accumulation  at  the 
surface  of  a)  ethanol,  h)  ethyl  ether.  Monomolecular  films  of  c)  fatty  acids, 
(i)  di-basic  acids,  o  water;  hydrophilic  groups  white;  lipophilic  groups  black; 

oxygen  encircled. 

of  ether  or  amyl  alcohol,  in  which  the  lipophilic  groups  are  predom- 
inant, will  have  still  less  affinity  for  water  and  will  lower  the  surface 
tension  to  a  greater  extent.  This  explains  the  first  rule  in  the  theory  of 
Gibbs-Thomson,  and  also  explains  why  very  small  amounts  are  suffi- 
cient to  lower  the  surface  tension  appreciably,  since  the  majority  of  the 
molecules  dissolved  accumulate  at  the  surface. 

For  a  substance  to  raise  the  surface  tension  it  must,  so  to  speak,  be 
more  hydrophilic  than  water.  This  applies,  for  example,  to  sugars: 
because,  with  their  numerous  OH-groups,  they  are  able  to  attract 
the  water  strongly.  For  this  reason  they  do  not  enter  the  surface,  but 
remain  in  the  bulk  of  the  phase.  Their  action  on  the  surface  tension 
is  due  to  the  fact  that  the  density  at  the  surface  is  somewhat  increased 
by  the  attractive  forces  acting  on  the  water  molecules.  Clearly,  this 
will  only  be  possible  if  the  concentration  of  the  sugar  is  very  high;  in 
a  0.25  molar  solution  of  cane  sugar  (the  only  substance  in  Table  V 


2  PRINCIPLES  OF  STRUCTURE  49 

which  causes  a  rise  in  surface  tension),  the  surface  properties  of  the 
water  are  almost  unchanged. 

With  increasing  length  of  the  paraffin  chain,  the  hydrophobic 
character  of  the  alcohol  molecules  becomes  more  pronounced,  and 
finally  their  affinity  for  water  is  so  small  that  they  accumulate  in 
quantity  at  the  surface.  The  same  applies  to  the  fatty  acids.  Their 
aliphatic  chains  are  so  hydrophobic,  that  they  float  on  the  surface  of 
the  water.  These  floating  molecules  tend  to  keep  as  far  apart  as  possible, 
in  much  the  same  way  as  the  gas  molecules  in  a  given  volume.  They 
spread  over  the  whole  available  water  surface.  The  expansive  pressure 
which  brings  about  this  spreading  can  be  measured  by  means  of  a 
movable  barrier.  In  the  apparatus,  originally  designed  by  Langmuir 
in  1 91 7  (Langmuir  tray),  the  spreading  pressure  is  transferred  from 
a  movable  barrier  to  a  torsion  balance  and  measured  in  dyn/cm  with 
an  accuracy  of  up  to  o.oi  dynes  per  cm. 

The  surface  law  found  wich  this  measuring  instrument  is  similar  to 
the  gas  law  "volume  X  pressure  =  constant",  in  that  the  product  of 
surface  per  mole  and  surface  pressure  is  constant.  The  floating  mole- 
cules therefore  behave  like  a  gas :  the  surface  density  can  be  increased 
by  reducing  the  surface.  This  "surface  compression",  however,  cannot 
be  carried  too  far ;  if  the  surface  is  reduced  below  a  certain  limit,  the 
surface  pressure  increase  becomes  steeper  than  that  required  by  a 
constant  value  of  the  product.  At  this  Hmit  the  molecules,  which 
hitherto  were  freely  movable,  cluster  into  a  close-packed  monolayer 
(monomolecular  film),  which  has  less  compressibility.  In  these  films 
the  polar  molecules  stand  up,  withdrawing  their  hydrophobic  groups 
from  the  water  and  dipping  their  hydrophilic  groups  into  the  water 
(Fig.  43c). 

The  thickness  of  the  film  can  be  calculated  from  the  amount  of 
substance  spread  on  the  water  and  the  size  of  the  surface  (Adam,  1930). 
This  thickness  corresponds  to  the  length  1  of  the  chain  molecule 
(Fig.  43c),  and  the  values  found  in  this  way  compare  well  with  those 
derived  from  the  X-ray  investigation  of  molecule  lattices.  From  the 
molecular  weight  of  the  substance  under  examination,  i.e.,  from  the 
number  of  molecules  packed  in  the  surface  layer,  the  distance  between 
the  chain  molecules  can  be  computed;  here  again  the  values  obtained 
are  similar  to  those  found  by  X-ray  analysis  for  the  distance  between 
the  chains  in  molecule  lattices  (order  of  magnitude:  4-5  A). 


5° 


FUNDAMENTALS  OF  SUBM  I  C  RO  SC  OPI  C  MORPHOLOGY 


Carrying  out  the  same  experiment  with  a  dibasic  acid,  the  film 
thickness  found  is  half  that  of  the  corresponding  monobasic  acid, 
the  surface  occupied  being  twice  as  large.  For  example,  the  molecular 
surface  of  nonyl  acid  CH3(CH2)7COOH  is  25  A^,  while  sebacic  acid 
COOH(CH2)8COOH  fills  an  area  of  57  A-  (Meyer  and  Mark,  1930). 
This  can  be  explained  by  assuming  that  both  the  carboxyl  groups  of 
the  dicarbon  acid  are  dipping  into  the  water,  which  means  that  the 
molecule  is  bent  (Fig.  43d).  Such  bending  is  made  possible  by  the  free 
rotation  around  C-C-bonds. 

When  a  slide  is  dipped  into  the  liquid  on  which  a  molecular  monolayer  is 
spread,  and  then  withdrawn,  it  is  coated  by  a  double  layer  of  that  compound. 
If  this  procedure  is  repeated,  two,  three,  four  and  more  double  layers  may 
be  deposited  on  the  glass  slide.  Such  experiments  can  be  performed  with 
stearate  films  whose  double  layers  measure  48.8  A;  so  a  slide  can  be  coated 
in  stages  with  layers  of  any  multiple  of  48.8  A. 

Preparations  hke  these  can  be  used  for  the  determination  of  the  sub- 
microscopic  thickness  of  very  thin  objects,  provided  they  have  a  similar 
refractive  index  to  the  stearate  film  for  comparison.  This  method  is  based 
on  the  fact  that  the  intensity  of  the  light  reflected  from  a  glass  surface 
diminishes  when  it  is  covered  by  a  thin  transparent  film.  The  variables 
involved  in  this  phenomenon  are  the  refractive  indices  of  film,  supporting 
material  and  medium  (usually  air)  through  which  they  are  viewed,  and  the 
thickness  of  the  film.  The  reflectivity  depends  further  on  the  angle  of  inci- 
dence and  the  wavelength  of  the  light;  both  are  kept  constant  by  using  an 
appropriate  vertical  illumination.  In  a  comparison  microscope,  called  a /(?/)/(?- 
scope  (Waugh,  1950),  the  density  of  the  biological  object,  e.g.  ghosts  of 
erythrocytes,  can  be  compared  with  the  density  of  stearate  films  of  known 
thickness.  Before  a  measurement  is  possible,  the  refractive  indices  of  the 
object  and  the  comparison  film  must  be  determined,  because  they  must  be 
ahke.  This  is  done  by  using  a  set  of  glass  sUdes  covering  a  range  of  re- 
fractive indices  in  small  increments.  The  slide  on  which  the  object  shows 
the   same   reflectivity   as   the   clean   glass   indicates   its    refractive   index. 
Mixtures  of  barium  stearate  and  stearic  acid  are  used  to  adjust  the  index 
of  the  stearate  film  to  that  of  the  object.  The  eftect  of  reducing  the  intensity 
of  the  reflected  light  is  greatest  when  there  is  considerable  disparity  between 
the  refractivity  of  the  support  and  that  of  the  object;  hence,  the  greater  the 
difl"erence  in  refractive  index  between  object/film  and  glass,  the  better  is 
the  determination  of  the  thicls:ness  of  the  object. 

With  this  method  Waugh  (1950)  has  found  that  the  thickness  of  the  mem- 
brane in  the  red  blood  cells  of  the  rabbit  is  2 1 5  A  ±  1 5  A  at  p^  6  (cf.  p.  264). 

Although  proteins  are  to  a  certain  extent  hydrophilic,  they,  too, 
form  surface  films.  Ovalbumin,  for  instance,  spreads  on  the  surface  of 


2  PRINCIPLES  OF  STRUCTURE  5I 

water  in  the  form  of  solid  skins  (Devaux,  1935;  Gorter  and  co- 
workers, 1955;  JoLY,  1948).  The  structure  of  such  films  is  not  yet 
known  in  all  its  details.  Molecules  of  the  polypeptide  chain  type 
(Fig.  87c,  p.  132)  do  not  stand  erect  but  lie  flat  on  the  surface.  As  a 
result  of  their  amphoteric  nature,  their  spreading  surface  is  not  con- 
stant but  depends  on  p^^.  It  is  important  to  note  that,  judging  from 
their  surface  activity,  not  only  the  skeletal  proteins  but  also  the  re- 
serve proteins  are  hydrophobic  to  a  considerable  degree  (Bull,  1947). 
The  surface  structures  described  in  this  section  are  brought  about, 
not  by  primary  valencies,  but  merely  by  cohesion  forces.  Consequently, 
the  relative  positions  of  the  atoms  are  not  fixed  like  those  in  a  main 
valency  lattice;  a  certain  mobility  exists,  of  which  indications  were 
already  found  in  the  ease  with  which  molecule  lattices  are  split  and 
deformed.  In  surface  films,  however,  the  attractive  forces  are  still 
less  pronounced.  The  molecules  in  a  film  containing  fatty  acids,  for 
instance,  are  free  to  rotate  about  their  axis.  We  might  say  that  surface 
films  are  in  a  state  intermediate  between  the  amorphous  liquids  and 
the  solid  bodies  with  their  well-defined  regular  structure. 

d.  Liquid  Crystals 

Mesophases .  At  one  time  "liquid  crystals"  played  a  great  part  in  the 
discussion  of  protoplasm  structure.  Lehmann  (1917)  went  so  far  as 
to  attribute  life  to  these  remiarkable  structures.  We  know  now, 
however,  that  the  unusual  properties  of  "flowing"  crystals  which, 
on  account  of  their  striking  birefringence,  are  perhaps  better  denoted 
as  anisotropic  liquids^  are  by  no  means  as  enigmatic  as  was  formerly 
believed.  For,  the  structure  of  liquid  crystals  is  similar  to  that  of  the 
surface  films  of  fatty  acids  on  water.  It  is  usually  a  matter  of  chain 
molecules  in  parallel  alignment,  which  are  free  to  move  relatively  to 
each  other  in  the  direction  of  their  axis  and  to  rotate  about  this  axis. 
However,  the  orientation  in  the  surface  films  is  restricted  to  a  small 
number  of  monolayers  or  even  to  a  single  monolayer  only,  whereas 
the  liquid  crystals  contain  oriented  structures  of  microscopic  dimen- 
sions (deformable  crystals,  drops,  etc.) 

The  best  starting  point  for  a  correct  understanding  of  the  structure  of 
crystalline  liquids  is  the  molecule-  or  chain-lattice  represented  in  Fig.  52 
(p.  35).  In  these  lattices  the  molecules  are  immovable;  the  substance  is 
in  the  crystalline  solid  state.  If,  now,  heat  is  applied  to  the  lattice,  the 


52        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY 


molecules  are  released  at  a  certain  temperature  and  finally  the  crystal  melts. 
With  increasing  chain  length,  however,  the  disintegration  of  the  lattice  is 
impeded.  Although  the  mobility  of  the  chain  molecules  is  increased,  their 
parallel  alignment  is  maintained,  in  much  the  same  way  as  in  a  sheaf  of 
pencils  in  which  each  pencil  can  be  turned  about  its  axis  and  shifted  with 
respect  to  its  neighbours,  but  cannot  be  turned  out  of  its  parallel  position. 
This  state  is  evidently  intermediate  between  the  crystalline  solid  and  the 
amorphous  liquid  state,  because  the  mobility  of  the  molecules  does  not 
refer  to  all  directions  in  space  but  is  restricted  to  a  single  one.  We  are, 
hertefore,  dealing  with  a  state  of  matter  which  is  designated  as  tnesophase 

(Friedel,  1922)  or  crystalline  liquid 
(VoRLANDER,  1 93 6).  Since  an  align- 
ment into  loose  sheaves  is  only  pos- 
sible with  rod-shaped  molecules, 
only  chain  molecules  can  occur  as 
mesophases.  If  a  crystal  lattice  of 
isodiametric  molecules  is  dissolved, 
its  molecules  become  at  once  inde- 
pendently mobile.  With  a  chain  lattice 
this  is  not  always  true,  as  the  pattern 
is  often  destroyed  in  two  steps.  The 
first  step  frees  the  crystalline  bonds 
between  the  chain  molecules;  but 
there  remains  some  cohesion,  which 
maintains  a  certain  parallelism  of  the 
individual  chains,  which  can  rotate 
and  shift  along  each  other  as  indicat- 
ed above.  If  the  rod-shaped  molecules 
can  only  rotate  round  their  longi- 
tudinal axis,  their  ends  remaining  in 
definite  planes  (cf.  Fig.  32a),  the  crystalline  mesophase  is  in  the  so-called  j'/^?^^//V 
state;  but  if  rotation  and  shifting  in  the  direction  of  the  molecular  axis  is 
possible,  so  that  the  ends  of  the  molecular  rods  no  longer  correspond  (cf. 
Fig.  32b,  p.  35),  the  mesophase  is  said  to  be  nematic.  In  many  cases  spindle- 
shaped  bundles  are  formed  which  are  strongly  birefringent  (cf.  Fig.  44). 
It  is  only  by  a  second  step  that  the  crystalline  mesophase  can  be  converted 
into  an  isotropic  amorphous  liquid  phase,  where  the  molecules  become  com- 
pletely mobile. 

The  transformation  of  the  chain  lattice  into  a  mesophase  occurs  at  a  well- 
defined  temperature  (melting  point  I),  whereas  the  conversion  into  an 
amorphous  liquid  takes  place  at  a  given  higher  temperature  (melting 
point  II). 

Compared  to  solid  crystals,  the  optics  of  mesophases  is  simple.  As  all 
molecules  in  the  sheaf  can  be  rotated  about  their  axes,  no  order  exists  in 
directions  perpendicular  to  these  axes.  All  directions  perpendicular  to  the 


Fig.  44.   Anisotropic  liquid  aggregates  in 

a  sol  of  benzopurpurin  between  crossed 

nicols  (from  Zocher,  1925). 


PRINCIPLES  OF  STRUCTURE 


53 


axis  are,  therefore,  equivalent  in  all  respects.  Consequently,  mesophases 
are  usually  optically  uniaxial,  and  as  a  rule  no  isotropic  or  biaxial  mesophases 
are  observed  (Zocher,  1925,  1931).  In  a  polarization  microscope  between 
crossed  nicols,  mesophases  will  therefore  appear  completely  dark  if  we 
observe  in  the  direction  of  the  sheaf  axis,  whereas  they  light  up  in  all  other 
directions.  According  as  to  whether  the  refractive  index  parallel  to  the  axis 
is  larger  or  smaller  than  that  perpendicular  to  it,  the  mesophase  is  called 
optically  positive  or  optically  negative  (cf.  p.  87). 

TABLE  VII 

CRYSTALLINE  LIQUID   STATE  (ACCORDING  TO  VORLANDER     1 95  6) 

Compare  Fig.  45 


(1) 
(2) 
(3) 

(4) 


Solid  phase 


Alesophase 


melt.pt.  I 


Liquid  phase 
melt.pt.  II 


Crystalline  solid 


:  amorphous  liquid 


„                       ^  crystalline  liquid                   ^            „         ,, 
^          „            ,,      (supercr.)  ->  decomposed 
,,     (supercr.)  ->  decomposed,  infusible 
rising  temperature 


The  ease  with  which  a  mesophase  is  changed  into  an  isotropic 
liquid  is  a  function  of  the  chain  length.  This  is  apparent,  in  particular, 
from  Vorlander's  researches  (1936).  With  increasing  chain  length  it 
becomes  increasingly  difficult  to  attain  the  amorphous  liquid  state, 
because  finally  the  melting  point  II  is  such  a  high  temperature  that 
the  chain  molecules  are  decomposed  before  the  mesophase  is  converted 
into  a  real  liquid.  With  still  greater  chain  lengths  the  substance  does 
not  fuse  at  all,  because  the  molecules  are  subject  to  degradation  be- 
fore becoming  movable.  In  this  case,  therefore,  the  cohesive  forces  be- 
tween the  very  long  chains  are  stronger  than  the  main  valency  bonds 
in  the  chain  molecule:  the  molecular  structure  breaks  down  before 
the  lattice  disintegrates.  Substances  which  cannot  be  changed  into 
the  amorphous  state,  because  the  ////^r-molecular  forces  in  the  lattice 
or  in  sheaves  (mesophase)  are  larger  than  the  /«/r<2-molecular  binding 
forces,  are  called  super -crystalline  (Vorlander).  A  survey  is  given  in 
Table  VII;  the  substances  (2)  and  (3)  occur  as  mesophases  at  certain 
intervals  of  temperature. 

Fig.  45  shows  a  series  of  molecules  of  increasing  chain  length  which 


54    FUNDAMENTALS  OF  SU  BM  I  C  RO  S  C  OPI  C  MORPHOLOGY      I 

corresponds  to  the  general  plan  of  Table  VII.  The  striking  fact  in  this 
series  is  that  the  addition  of  only  a  single  pair  of  members  to  the  chain 
results  in  such  radical  changes  in  the  physical  properties.  It  is  to  be 
noted  that  this  holds  good  only  for  para-substituents  in  the  benzene 
ring,  leading  to  one-dimensional  chain  molecules. 

i)  p-Azoxybenzene 
36° 
cryst. -solid  Z^  am.  liq. 

o 

2)  p-Azoxyphenetolc 

134"  il'5 

crvst. -solid  5^  cryst.  liq.  ^  am.  liq. 

^-/     II  ^— 

O 

5)  p-Azoxy-azobenzene 
226'' 
crvst. -solid  *^  cryst.  liq.  ^  dccomp. 


-N=N-<        >-N  =  N^        >-N  =  N 

6 


4)  p-Azoxy-disazobenzene 
cryst.-solid  -^  decomp. 

o 

Fig.  45.  Series  of  chain  molecules  which  aggregate  to  mesophases  (cf.  Table  VII). 

Myelin  for //IS.  Cytologists  are  more  famihar  with  the  birefringent 
semi-liquid  tubes,  designated  as  myelin  forms  because  they  were  ob- 
served for  the  first  time  with  myelinated  nerves  (Fig.  179,  p.  362).  When 
water  is  added  to  such  nerve  fibres,  adventitious  threads  issue  from 
their  sheath.  They  bend  and  curl  and  finally  grow  into  irregular 
entanglements.  The  active  substance  causing  these  structures  is  the 
lecithin  in  the  myelin  sheath,  for  exactly  the  same  phenomena  are 
observed  when  water  is  added  to  isolated  lecithin,  especially  if  this  is 
liable  to  decompose.  Although  the  myelin  forms  are  particularly  strik- 
ing  in   organic   phosphoric   acid  compounds,  similar  tubes  emerge 


PRINCIPLES  OF  STRl^CTURK 


55 


from  the  alkali  salts  of  oleic  acid  when  these  are  wetted.  Very  beautiful 
myelin  forms  were  obtained  by  Gicklhorn  (1932a)  in  the  cell  sap  of 
the  well-known  AlUuni  epidermal  cells  by  adding  ammonia  or  sodium 
hydroxide  (Fig.  46).  The  variety  of  shapes  in  these  peculiar  structures 
is  beautifully  demonstrated  in  Nageotte's  microphotograph  atlas 
(1936,  No.  434). 

The   myelin   forms   are   usually   designated   as   liquid   crystals.   It 


Fig.  46.  Myelin  forms  in  the  epidermal  cells  oi  Allium  (from  Gicklhorn,  1932a). 

should  be  pointed  out,  however,  that  there  is  a  fundamental  difference 
between  these  structures  and  the  crystalline  liquid  state  mentioned 
above.  For,  in  the  latter  we  have  to  deal  with  a  special  aggregate 
state  of  a  uniform  substance,  i.e.,  a  system  consisting  of  one  com- 
ponent only,  whereas  at  least  two  components  take  part  in  the  formation 
of  myelin  forms.  In  the  examples  mentioned,  one  of  these  components 
is  water.  It  is  further  essential  that  the  molecules,  which  here  again 
must  have  a  chain-like  structure,  be  not  homopolar  as  in  Fig.  45,  but 
heteropolar,  i.e.,  they  must  contain  a  hydrophilic  and  a  lipophilic 
pole.  The  hydrophilic  group  in  oleic  acid  is  the  carboxyl  group,  that 
in  lecithin  is  the  choline.  If  the  conditions  mentioned  are  realized, 
myelin  forms  may  occur,  provided  the  molecules  are  sufficiently  mobile. 
The  apparent  growth  is  due  to  water  absorption;  it  is,  therefore,  a 
matter  oi  swelling:  the  hydrophilic  groups  are  surrounded  by  water, 
while  the  hydrophobic  groups  are  drawn  away  from  the  surface.  The 
resulting  orientation  in  the  case  of  lecithin  is  represented  in  Fig.  47a; 
the  lecithin  underlying  this  scheme  is  a  /5-lecithin  (see  Fig.  93,  p.  138) 
in  which  the  phosphoric  acid  is  attached  to  the  OH-group  in  the  middle 
of  the  glycerol  molecule.  Obviously,  the  water  penetrating  into  the 


56        FUNDAMENTALS  OF  SUBMI C  ROSCOPI C  MORPHOLOGY  I 

lecithin  causes  the  molecules  to  arrange  themselves  in  layers  which 
are  similar  to  surface  films,  except  that  there  are  no  mono-  or  oligo- 
molecular  layers  but  huge,  microscopically  visible  structures  consisting 
of  bimolecular  lamellae.  If  the  length  of  the  pair  of  overlapping  lecithin 
molecules  is  about  50  A  (Trillat,  1925/27),  a  wall  of  a  myelin  tube  5  (i 
in  thickness  consists  of  some  1000  double  layers  (Fig.  47b).  Water 
continues  to  be  absorbed  until  all  the  hydrophilic  groups  are  saturated. 


T 


o   o  o  „ 

000       o 

.''■■'.'.'.'■','<         „  r.\'.',','.',v 

o  °    °-      ° 


t 


> 


to        o  o   r 
■■■■■■■'  ■!  O    O   .■.'.■.'.■■■.■.■■  I 
o  _  o     00     L 


O  o 


o  o 


o  o 


!f^?^„°  "o^'^tC 


c  O  O  g  "o  O   o 

o  o  o  o  o  o   o  o 
°     O      n  ^  O 

10   o  o   o  o  " 


B ----.1 


^Q     O    O     ^  O        op 

B.'  ■'■'-.''■^.'■.'-'i         V  r.^.-.  ■■-■■■.■■ .  B 

J  o  o  o     o  o  o  L 


o  o 


o  o    o 


00    Q  o    o 

(o   o  o  o  o   o 
■■■.'.v,'.  I    o    O 


o  o 


Lecithin 


°°°H,0°°° 


t 


c.50^ 


Fig.  47.  Myelin  forms  of  lecitiain 

a)  Submicroscopic  structure.  Hatched,  hydrophilic;  black,  lipophilic  part  of  the  fork- 
shaped  lecithin  molecule,  b)  Microscopic  image  and  optics. 

thus  causing  further  growth  of  the  tubes.  In  course  of  time  the  myelin 
forms  therefore  traverse  the  whole  field  of  view  under  the  cover  glass 
of  the  microscopic  preparation. 

It  can  be  proved  by  optical  means  that  the  lecithin  molecules  in  the 
myelin  tubes  are  perpendicular  to  the  surface.  For,  in  a  flowing  solu- 
tion (see  p.  90)  the  lecithin  molecules  appear  to  be  optically  positive. 
The  myelin  tubes,  however,  are  optically  negative  with  respect  to 
their  long  axis.  From  this  it  follows  that  the  lecithin  chains  must  be 
oriented  perpendicular  to  the  tube  axis.  Bear  and  Schmitt  (1956) 
mention  a  formula  (p.  86)  from  which  the  double  refraction  n^ — n^  of 
the  cylindrical  myelin  tube  with  its  optical  axis  in  radial  direction  can 
be  computed.  For  the  myelin  forms  of  lecithin  in  Ringer  solution  the 
authors  fijid  n^  —  n^  =  0.0039  (Schmitt  and  Bear,  1937).  On  further 
absorption  of  water  the  lamellar  structure  of  the  myelin  forms  becomes 


PRINCIPLES  OF  STRUCTURE 


57 


63.5^^-- 


increasingly  pronounced.  Finally,  the  positive  intrinsic  double  refrac- 
tion of  the  molecules  is  overcompensated  by  the  negative  double 
refraction  due  to  the  lamellar  texture  (see  p.  87,)  and  the  sign  of  the 
myelin  birefringence  is  reversed  (Nageotte,  1936). 

The  absorption  of  water  can  be  followed  by  means  of  X-rays.  The 
dry  myehn  substances 
obtained  from  evapor- 
ated benzene  solu- 
tions give  X-ray  in- 
terferences which  cor- 
respond to  twice  the 
chain  length  (lecithin 
and  cephalin  44A,  ster- 
ol 34  A,  sphingomyelin 
and  cerebroside  65- 
67  A;  ScHMiTT  and 
Palmer,  i  940) .  If  water 
is  added  to  these  lipids, 
the  X-ray  periods  are 
enlarged  and  so  allow 
of  an  evaluation  of  the 
thickness  of  the  water 
lamellae  formed.  It  can 
be  seen  from  Fig.  48 
that  the  original  period 
of  63.5  A  of  mixed 
nerve  lipids  has  be- 
come 150  A  at  a  water  content  of  75%.  This  implies  a  water  layer 
of  86  A  between  the  bimolecular  lipid  layers. 

The  myelin  forms  offer  a  good  example  of  the  manner  in  which 
complicated  microscopic  structures  can  result  from  a  simple  arrange- 
ment of  submicroscopic  entities.  They  show,  however,  that  no  co- 
ordinated growth  is  possible  as  a  result  of  such  a  process,  for  the 
myelin  forms  "grow"  at  random  aimlessly  in  the  substrate  and  the 
final  outcome  is  a  chaos  rather  than  an  illustration  of  organized  life 
(Fig.  46). 


Fig.  48.  Water  intercalation  between  bimolecular  lipidic 
films.  Size  of  the  adsorbed  water  layer  with  increasing 
water  content.  The  black  points  correspond  to  o%,  25%, 
50%,  6-j°o  and  75%  of  water  content  (from  Schmitt 
and  Palmer,  1940). 


58         FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

§  3.  Structure  of  Gels 

a.  Chemistry  of  High  Polymers 

Polymerisation  and  condensation.  In  about  1920  Staudinger  drew 
attention  to  the  fact  that  in  the  high-polymer  natural  substances  the  struc- 
tural units  which  can  be  obtained  from  them  by  hydrolysis  are  interlinked 
by  primary  valency  bonds  (Kekule  bonds).  He  first  proved  the  correctness 


Monomer 
H 


H 


Polymer  chain 
H  H  H 


H 


c=o 


-C— O— C— O— C— O— C— O— C— o 


H 

Formaldehyde 
(oxymethylene) 

CH  =  CH., 


H  H  H  H  H 

Polyoxymethylene 

_CH— CH,— CH— CH.,— CH— CH,— CH— CH,— CH— 


Styrene 


/ 


Polystyrene 


CH2=C— CH-CH„ 


— CH,— C  =  CH— CH„— CH,— C  =  CH— CH,— CH^— 


CH3 
Isoprene 


CH3  CH3 

Caoutchouc  (polyprene) 

Fig.  49.  Polvmerization 


of  this  point  of  view  in  synthetic  products.  Fig.  49  shows  some  of  his 
polymerizations.  It  is  seen  that  the  monomer  molecules  always  contain 
double  bonds,  one  of  which  interacts  with  another  molecule  and  thus  links 
two  monomer  molecules  together.  If  this  process  is  repeated,  long  chain 
molecules  are  formed  whose  growth  would  be  theoretically  unlimited  if  the 
possibility  of  further  addition  did  not  diminish  with  increasing  chain  length 
and  the  sensitivity  to  oxygen  (and  the  like)  of  the  giant  molecules  formed 
did  not  become  considerably  enhanced.  For  the  present,  however,  the 
factors  limiting  the  chain  length  will  not  be  considered,  and  the  polymer 
chains  will  simply  be  denoted  by  "open"  formulae.  Polymerization  pro- 
cesses are  particularly  successful  if  the  monomer  contains  a  system  of 
conjugated  double  bonds,  as  e.g.,  in  isoprene,  i.e.,  if  double  bonds  alternate 
with  single  bonds.  The  terminal  double  bonds  may  then  give  rise  to  inter- 
linking with  those  of  neighbouring  monomer  molecules,  while  the  central 
single  bond  is  converted  into  a  double  bond.  In  this  manner  unsaturated 


3  STRUCTURE  OF  GELS  59 

high-polymer  compounds  are  formed,  such  as  rubber  in  the  case  con- 
sidered here. 

Apart  from  this  type  of  chain  formation,  high  molecular  weight  sub- 
stances may  also  be  formed  by  etherification  of  alcohohc  groups  (Fig.  50) 
or  by  a  process  of  esterification  between  carboxylic  and  hydroxylic  groups 
with  elimination  of  water.  This  way  of  interlinking  is  distinguished  as 
condensation  from  the  polymerization  of  unsaturated  compounds.  It  leads  to 
equally  long  molecules;  the  chains  are  then,  however,  no  longer  all-carbon 
chains  like  those  in  polystyrene  or  rubber,  but  always  contain  oxygen  atoms 
as  interconnecting  links.  When  polyvalent  alcohols  react  with  each  other, 
no  chain-like,  but  net-hke  or  even  spatial  giant  molecules  are  formed,  such 
as  probably  occur  in  the  insoluble  huminic  acids  and  in  the  insoluble 
cutins  (see  p.  295).  By  way  of  introduction,  however,  we  shall  confine  the 
discussion  to  the  somewhat  simpler  conditions  in  the  high-polymer  carbo- 
hydrates with  linear  chain  molecules. 

The  high-polymer  molecules  may  become  so  large  as  to  assume  the 
properties  of  colloid  particles.  Staudinger  (1936a)  designates  these  giant 
molecules  as  macromolecules  and  the  branch  of  science  dealing  with  their 
constitution  and  chemical  behaviour  as  niacromokcidar  chemistry. 

Polysaccharides.  The  same  principles  by  which  disaccharides  are 
formed  (see  Fig.  35/37,  p.  39),  govern  the  formation  of  polysaccharides, 
which  are  of  outstanding  importance  in  plant  physiology.  Here  too, 
the  monoses  are  interlinked  by  1-4  oxygen  bridges  with  elimination 
of  water,  and  this  polycondensation  may  embrace  a  large  number  of 
monomer  molecules.  In  cellulose  the  successive  links  of  /3-glucose  are 
rotated  through  180°.  In  starch,  however,  the  a-glucose  residues  can 
interact  without  being  rotated  (Fig.  50).  The  cellulose  chains  have  a 
digonal  screw  axis  as  an  element  of  symmetry,  contrary  to  the  starch 
chains,  which  have  not.  Consequently,  the  cellulose  molecules  are 
more  stable  and  straightened  out,  whereas  the  starch  molecules  tend 
to  become  more  convolute  because  they  are  less  symmetrical.  This 
morphological  difference  is  doubtless  one  of  the  reasons  for  the 
difference  in  behaviour  between  starch  and  cellulose.  Possibly  it  is 
also  responsible  for  the  tendency  of  the  starch  molecule  towards 
branching  (see  Fig.  1 5  2b,  p.  3 1 1).  The  mannans  occurring  in  corozo  nut 
and  in  the  rhizomes  of  Amorphophalhis  konjak  (see  Fig.  160)  can  be 
derived  in  a  similar  way  from  mannose  as  starch  and  cellulose  from  glu- 
cose. The  two  monoses  differ  only  in  the  different  position  of  the  H- 
and  OH-groups  at  the  second  C-atom.  For  the  chain  in  mannan  from 
corozo  nut,  locahzed  in  the  cell  waU,  Meyer  and  Mark  (1930,  p.  168) 


6o        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY 


assume  /5-glucosidic  bonds,  while  it  seems  likely  that  to  Konjak 
mannan,  being  a  reserve  substance,  a  starch-like  structure  with  a- 
glucosidic  bonds  should  be  assigned. 

It  is  highly  significant  that  the  pectic  substances,  which  are  held  to  be 
responsible  for  the  coherence  of  plant  tissues  and  which  contain  poly- 


OH 

> O^OH 

CH20H 

^-Glucose 

OH 
OHy 1  OH 

CH2OH 
cX-G/ucose 

OH. OH 

\VHOhN 

CH^OH 

C(-Monri06e 

OH 

oih-o"^ 

COOR 


CH2OH 


,^ty 


CH2OH 


0— 


CHpOH 


CH2OH 


'^:x°- 


CH2OH         CH2OH 

Starch 


CH2OH 


-0- 


CH20H  CH2OH  CH2OH  CH2OH 

c(-Mannan 


C(_-Galaciuronic  acid 
(R^CHs  or  H) 


COOR 

o—^^o^  \ — A-o 

COOR  COOR 

Polygalocturonic    acid 


^^^' 


O— 


Fig.  50.  Polysaccharides 


galacturonic  acid  as  a  basic  material,  also  have  the  structural  principle 
of  polysaccharides.  Here  the  -CHgOH  side  chain  of  the  monose  ring 
is  replaced  by  the  carboxyl  group  -COOH.  The  pectins  are  therefore 
capable  of  salt  formation.  Polygalacturonic  acid  is  soluble  in  water, 
but  its  Ca-salt  is  not,  so  that  this  polyacid  can  be  precipitated  by  Ca 
ions.  Part  of  the  carboxyl  groups  is  esterified  with  methanol  (Deuel, 
1943).  It  is  interesting  to  note  that  the  methylation  does  not  interfere 
with  the  solubility  in  water,  because  methyl  groups  bound  to  oxonium 
oxygen  obtain  an  induced  polarity  so  that  they  partly  lose  their 
lipophilic  character  and  become  hydrophilic. 

The  monomer  of  the  pectic  acid  is  a-galacturonic  acid.  As  in  a- 
galactose,  the  hydroxyl  groups  of  the  first  and  fourth  C-atoms  are 
not  situated  on  the  same  side  of  the  pyranose  ring  (Fig.  50);  the  a- 
glucosidic  linkage  causes  a  rotation  of  succeeding  chain  members. 


3  STRUCTURE  OF  GELS  6l 

In  crystalline  sodium  pectate  the  screw  axis  is  not  twofold  as  in  cellu- 
lose, but  threefold  (Palmer  and  Hartzog,  1945).  The  crystallizing 
tendency  of  pectic  substances  is  much  smaller  than  that  of  cellulose; 
in  the  plant  it  occurs  in  the  amorphous  state  only  (Wuhrmann  and 
PiLNiK,  1945). 

The  pentosans,  which  come  partly  within  the  hemicellulose  class, 
have  a  similar  structure  to  that  of  the  polysaccharides  already  de- 
scribed, except  for  the  absence  of  the  side  chains,  i.e.,  the  sixth  C- 
atom.  If  in  cellulose  or  polygalacturonic  acid  this  group  is  replaced  by 
H,  we  obtain  the  xylan  chain  or  a  polyarahinan. 

The  polysaccharides  demonstrate  strikingly  how  slight  morpholog- 
ical variations  of  one  and  the  same  structural  principle  may  give  rise 
to  substances  which  behave  quite  differently  from  a  physiological  point 
of  view. 

Chain  length  of  high  polymers.  According  to  Staudinger,  all  high  polymer 
chains  terminate  in  end  groups.  Unfortunately,  so  far  the  terminal  groups 
of  none  of  the  high  molecular  weight  natural  substances  are  known;  the 
chains  are  therefore  preferably  written  in  "open"  formulae  (Fig.  50). 
Contrarily,  in  comparatively  short  synthetic  chains  the  end  groups,  hence 
the  molecular  weights,  of  the  products  can  be  determined.  If  foreign  atoms, 
such  as,  for  instance,  iodine  form  the  terminal  groups,  such  determinations 
can  be  easily  performed.  If,  however,  the  chains  are  terminated  by  OH- 
groups,  the  accuracy  of  this  so-called  end-group  method  diminishes  rapidly 
with  increasing  chain  length.  In  polyoxymethylene  dimethyl  ether  this 
method  can  be  successfully  applied  up  to  a  degree  of  polymerization  of 
about  100.  The  methods  of  freezing  point  depression  and  rise  of  boiUng 
point,  commonly  used  in  molecular  weight  determinations  in  substances 
of  low  molecular  weight,  cannot  be  applied  to  high  polymers,  as  the  effects 
are  too  small. 

On  the  other  hand,  the  molecular  weight,  and  thus  the  chain  length  of 
high  polymers,  can  be  measured  by  osmotic  means, in  which  case  it  must 
be  taken  into  account  that  Van  't  Hoff's  law  does  not  apply  rigorously  to 
molecules  of  so  great  a  volume.  Corrections  similar  to  Van  der  Waals' 
b-correction  in  the  equation  of  state  of  gases  must  therefore ,be  introduced 
(ScHULZ,  1936).  A  method  derived  by  Staudinger  is  based  on  the  fact  that 
the  specific  viscosity  of  a  solution  of  chain  molecules  (i.e.,  the  viscosity 
increase  which  is  imposed  upon  the  solvent  by  the  solute),  within  a  certain 
range  of  molecular  weights,  is  approximately  a  linear  function  of  the  chain 
length.  In  addition  to  osmometry  and  viscometry  we  mention  in  particular 
Svedberg's  ultracentrifuge  for  the  determination  of  the  degree  of  poly- 
merization of  high  polymer  natural  substances.  X-ray  analysis  is  not  suitable 
for  this  purpose  (see  p.  99). 


62        FUNDAMENTALS  OF  SUBMICROSCOPI C  MORPHOLOGY  I 

TiVBLE 
HOMOLOGOUS    POLYMERIC    SERIES    OF    CELLULOSE 


Degree  of 
polymerization 

Chain  length 

Mechanical 
properties 

Oligosaccharides 

y-cellulose 
HemicoUoid  cellulose 

I-IO 
lO-IOO 

-50  A 
^0-500  A 

Pulverizabl^ 

crystal  powder 
Short-fibred 

/5-cellulose 

pulverizable 
powder 

Mesocolloid  cellulose 

100-500 

500-2000  A 

Fibrous,  strong 

a-cellulose  (rayc^n) 
Native  cellulose, 

500-2000 

0.25-1  /x 

Long-fibred, 

a-cellulose 

and  more 

very  strong 

(fibre  cellulose) 

According  to  Staudinger,  the  experimental  data  available  lead  to 
the  following  conclusions  regarding  the  molecule  type  of  cellulose 
(Fig.  50).  If  some  10  glucose  residues  are  linked  together  to  form  a 
chain,  easily  soluble  cellulose  products  are  obtained,  which,  owing  to 
their  particle  length  of  50  A,  already  exhibit  slightly  colloid  properties. 
Compounds  of  this  kind  are  known  as  degradation  products  of  cellu- 
lose, termed  cellodextrines  or  y-celluloses.  If  the  number  of  chain 
links  increases  to  100,  /9-celluloses  are  obtained  which  are  soluble  in 
10%  sodium  hydroxide  without  swelling,  to  form  viscous  sols.  Not 
before  the  degree  of  polymerization  exceeds  100  and  approaches  800  do 
we  obtain  the  so-called  a-celluloses,  which  are  no  longer  attacked  by 
1%  sodium  hydroxide  and  which  find  application  in  the  cellulose 
industry  (rayon,  cellophane).  They  slowly  dissolve  while  swelling  in 
10%  NaOH  and  yield  viscous  "gel  solutions".  Native  cellulose  has  a 
still  higher  degree  of  polymerization;  if  dissolved  in  Schweizer's 
solution  with  complete  exclusion  of  oxygen,  a  degree  of  polymeriza- 
tion of  about  2000  for  the  fibre  cellulose  of  linen,  hemp,  ramie  and 
others  can  be  calculated  from  the  viscosity.  The  values  determined 
from  the  viscosity  can  be  checked  osmometrically  up  to  a  degree  of 
polymerization  of  about  1000  (Staudinger,  1936  a,  b);  beyond  this 
limit  extrapolation  is  carried  out  according  to  the  linear  viscosity 


STRUCTURE  OF  GELS 


65 


VIII 


(ACCORDING   TO   STAUDINGER,    1936b,    1937^) 


Capacity  of  film 
formation 

Solubility  in 
10%  NaOH 

Viscosity  in        \ 

1%     SCHWEIZER 

solution 

Deviation  from 
Hagen-Poiseuille 
law  in  I  %  solution 

1 

None 

Easily  soluble 

Solution  of  low 

None 

Small 

without  swelling 
Soluble  without 
swelling 

viscosity 
Viscous  solution 

None 

Large 

Slowly  dissolved 
wuth  swelling 

Viscous 

"gel  solution" 

Small 

Very  large 

Strong  swelling 
almost  insoluble 

Highly  viscous 
"gel  solution" 

Strong 
structural 

viscosity! 

rule.  Whether  this  applies  to  the  whole  range  from  looo  to  2000  chain 
links  cannot  be  decided.  Furthermore,  it  has  been  questioned  whether 
chain  molecules  of  such  liighly  polymeric  substances  can  be  completely 
dispersed  in  a  micromolecular  solvent  at  all  (Lieser,  1940,  1 941).  On 
the  other  hand,  it  is  possible  that  native  fibres  contain  still  longer  chains 
which  may  be  degraded  on  dissolution  in  cuprammonium.  The  value 
of  2000  for  the  degree  of  polymerization  of  the  fibre  cellulose  is, 
therefore,  not  reliable ;  but  it  is  the  only  value  which  can  be  determined 
at  present  experimentally  and,  for  the  time  being,  we  must  refer  to  it. 
Its  magnitude  is  impressive  enough,  seeing  that  a  degree  of  poly- 
merization of  2000  corresponds  to  a  chain  length  of  i  /(,  each  glucose 
residue  measuring  5  A.  This  means  that  the  cellulose  molecules  have 
microscopic  lengths.  Nevertheless,  they  remain  invisible  because  their 
thickness  is  amicroscopic. 

Chain  molecules  of  a  given  structural  type  but  different  chain  lengths 
are  called  a  homologous  polymeric  series.  The  polyglucosans  mentioned 
represent  the  polymeric  homologues  of  the  celluloses.  In  such  a 
series  the  physical  properties  change  with  increasing  molecular  weight 
according  to  certain  laws.  Table  VIII  gives  data  for  cellulose.  Not  only 
does  the  solubility  decrease  and  the  viscosity  of  the  solutions  increase, 
but  the  fibrous  character  and  the  capacity  for  film  formation,  which 


64        FUNDAMENTALS  OF  S  UBMIC  RO  SCOPIC  MORPHOLOGY  I 

are  of  particular  importance  in  biology,  become  increasingly  pro- 
nounced beyond  a  certain  degree  of  polymerization. 

It  is  only  from  the  low  molecular  weight  members  of  a  homologous 
polymeric  series  that  uniform  substances  of  definite  molecular  weight 
can  be  obtained  by  recrystallization,  fractional  precipitation,  etc.  In 
the  members  of  higher  molecular  weight  this  is  no  longer  possible. 
In  the  series  of  paraffins,  in  particular,  it  has  been  found  that  fraction- 
ation gives  mixtures  of  substances  of  molecular  weights  which  are 
only  approximately  equal.  The  determination  of  the  degree  of  polymeri- 
zation therefore  yields  only  an  average  value ;  the  actual  chain  lengths 
are  spread  more  or  less  around  this  value  according  to  the  method 
of  fractionation.  Such  mixtures  are  called  polymer  uniform  substances 
("'polymer  einheitliche  Stoffe")  by  Staudinger  (1936b).  Whether 
the  high  polymers  occurring  in  nature  are  also  polymer  uniform,  or 
whether  life  always  builds  chains  of  exactly  the  same  length  cannot 
be  decided  at  present. 

Although  the  representatives  of  a  homologous  series  behave  quite 
differently  from  a  physical  point  of  view,  they  show  the  same,  or 
at  least  a  very  similar  chemical  behaviour,  in  conformity  with  their 
uniform  structure.  For  instance,  the  alcoholic  OH-groups  of  all  re- 
presentatives in  Table  VIII  and,  further,  those  of  the  polysaccharide 
molecules  shown  in  Fig.  50  and  even  those  of  the  polygalacturonic 
acid  chains  (Schneider  and  co-workers,  1936;  Deuel,  1947b)  can 
be  etherified  and  esterified  (methylated,  acetylated,  nitrated,  etc.)  with- 
out measurable  change  in  the  degree  of  polymerization.  The  polymer 
mixture  formed  in  this  way  from  the  polymer  uniform  substance  con- 
cerned has  the  same  average  chain  length  as  the  original  material 
(it  is  an  "analogous  polymer",  Staudinger,  1936b).  On  esterifica- 
tion,  the  cellulose  chains  lose  their  polar,  hydrophilic  properties, 
acquire  a  more  homopolar  lipophilic  character  and  on  account  of  their 
solubility  in  organic  liquids  are  then  more  accessible  to  osmotic  ex- 
periments. 

b.  Structural  Viscosity 

Anomalous  flow.  The  four  fractions  of  the  series  of  homologous 
cellulose  polymers  yield  colloid  solutions  of  an  entirely  different 
nature.  Staudinger  divides  them  into  "almost,  meso-,  hemi-  and  eu- 
-colloid"  (Tabic  VIII).  In  the  two  former  cases  the  chain  molecules  in 


2  STRUCTURE  OF  GELS  65 

1%  ScHWEiZER  solution  are  completely  solvated,  i.e.,  completely 
surrounded  by  molecules  of  the  solvent,  and  free  to  move  as  in  real 
solutions.  Their  colloid  character  results  merely  from  the  fact  that,  the 
molecular  length  of  solute  molecules  in  one  dimension  being  almost 
microscopic,  they  attract  a  large  amount  of  solvent  and  thus  increase 
the  viscosity.  Staudinger  denotes  this  state  as  "sol  solution".  From 
a  degree  of  polymerization  of  about  loo  onwards,  however,  a  i% 
ScHWEiZER  solution  can  no  longer  completely  solvate  all  the  chain 
molecules,  and  the  solute  molecules  hamper  each  other's  Brownian 
movement.  They  are  not  completely  dissolved  but  are  in  a  state 
intermediate  between  solid  and  liquid.  At  the  highest  degree  of 
polymerization  detectable,  this  interaction  of  the  giant  chains  with 
2000  links  is  so  intensified,  that  the  fibre  cellulose  dissolves  very 
slowly.  Solutions  in  which  the  chain  molecules  are  hampered  in  their 
Brownian  movement  for  want  of  solvent  were  called  "gel  solutions" 
by  Staudinger  (Staudinger  and  Sorkin,  1957b).  There  exists  a 
reliable  method,  based  on  the  phenomena  of  capillary  flow,  by  which 
the  concentration  or  particle  size  can  be  found  at  which  the  particles 
in  a  colloid  solution  begin  to  disturb  each  other,  viz.,  Hagen- 
Poiseuille's  law 

where  q  is  the  amount  of  Hquid  flowing  through  a  capillary  of  radius 
r  in  a  time  t  under  the  influence  of  a  pressure  gradient  p/1.  In  this  for- 
mula the  viscosity  rj  is  independent  of  the  pressure  gradient  p/1. 

This  no  longer  applies  when  the  colloid  particles  in  the  solution 
influence  each  other's  motion.  In  this  case  the  viscosity  depends  on  the 
pressure  gradient:  r]  =  f(p/l),  in  the  sense  that  the  viscosity  decreases 
with  increasing  pressure  gradient.  This  can  be  explained  by  the  fact 
that  in  these  solutions  the  elastic  properties  of  the  solid  substance  are 
not  completely  eliminated,  since  the  parcicles,  instead  of  being  fully 
dissolved,  enter  into  some  sort  of  relation  with  each  other.  With  in- 
creasing pressure  gradient  in  the  capillary  these  elastic  forces  are 
progressively  counteracted.  For  this  reason,  in  colloid  solutions  with 
long  chain  molecules  the  chains  which  are  originally  present  in  a  ran- 
dom and  disorderly  arrangement  will  be  oriented  parallel  to  the  direc- 
tion of  flow,  and  thus  the  forces  resisting  the  flow  which  are  respon- 
sible for  the  viscosity  will  be  decreased.  According  to  Table  VIII,  such 


66        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY 


deviations  from  Hagen-Poiseuille's  law  are  observed  in  the  case  of 
cellulose  of  polymerization  degrees  exceeding  roo.  Since  the  anomaly 
of  flow  is  caused  by  the  mutual  positions  of  the  colloid  particles,  it 
has  been  designated  as  structural  viscosity  (Ostwald,  1925;  Philip- 
POFF,  1955). 

c.  Gel  Structure 

Gelj'rawe.  If  the  coherence  between  the  individual  colloid  particles 
becomes  still  more  pronounced  than  in  the  "gel  solution",  gels  are 
formed  with  a  more  or  less  fixed  shape  and  distinctly  elastic  properties. 
Of  course  there  exist  all  kinds  of  gradations  from  the  gel  solutions, 
in  which  the  elastic  coherence  of  the  particles  can  only  be  proved  by 
testing  them  for  structural  viscosity,  and  the  real  gels  whose  units  are 
more  or  less  fixed  in  their  mutual  positions.  The  gels  that  become 
liquid  on  shaking  and  soHdify  again  at  rest,  form  a  typical  intermediate 
stage.  This  remarkable  phenomenon  is  due  to  the  same  effect  as  the 
decreasing  viscosity  at  increasing  pressure;  it  is  called  thixotropy  (cf. 
Freundlich,  1942).  Colloid  silicic  acid  and  gelatin,  for  instance,  can 
occur  as  thixotropic  gels  at  suitable  concentrations. 

If  spherical  colloid  particles  cluster  together  to  a  gel,  the  result  is  a 
rather  compact  gel  and  from  Fig.  5 1  a  it  is  obvious  that  such  a  structure 

can  onlv  be  formed  at  a 
relatively  high  concent- 
ration of  the  solute. 
With  sols  containing 
long  chain  molecules, 
however,  a  fixed  mutual 
position,  i.e.,  a  structure 
is  attained  much  more 
easily.  At  concentrations 
as  low  as  a  few  per  cent, 
of  a  long  chain  high 
polymer,  the  chain  molecules  can  combine  into  a  loose  meshwork,  as 
represented  by  Fig.  51.  Such  a  colloid  already  possesses  a  structure, 
although  a  very  loose  one,  which  may  still  easily  undergo  some  plastic 
deformation.  It  also  possesses  a  certain  elasticity,  because  the  places 
where  the  chains  are  interlaced  can  be  regarded  as  fixed  points.  As  will 
be  shown,  these  can  be  due  to  various  kinds  of  forces.  Since  in  biolo- 


Fig.  51.  Submicroscopic  stnacture  formation  a)  with 
spherical,  ^)  with  filiform  particles. 


3  STRUCTURE  OF  GELS  67 

gical  colloids  it  is  often  difficult  to  decide  upon  their  nature,  I  have 
suggested  that  they  should  be  called  by  the  neutral  name  oi  points  of 
attachment  ot  junctions  ("Haftpunkte"),  which  is  non-committal  as  to 
the  kind  of  bonds  involved  (Frey-Wyssling,  1933  b;  1936  a). 

In  Fig.  5 1  the  junctions  are  marked  by  black  dots.  Obviously  in  a  gel 
with  chain  molecules  many  fewer  junctions  are  needed  to  build  up  a 
structure  than  in  the  case  of  spherical  colloid  particles.  A  gel  built  up 
by  high  polymer  chains  can  therefore  contain  up  to  p/%  of  water 
CSeifriz,  1938)  and  yet  possess  a  structure.  This  fact  is  very  important 
to  an  understanding  of  protoplasm  structure,  since  the  water  content 
of  living  matter  is  always  surprisingly  high. 

In  Fig.  5 1  b  further  chains  can  be  interwoven  at  will ;  the  number  of 
junctions  will  then  increase,  and  the  result  is  a  more  solid  gel  structure. 
The  plastic  properties  of  the  structure  become  less  pronounced  at  the 
same  rate,  while  the  elastic  properties  increase.  Thus  the  model  of  a 
gel  structure  projected  here  comprises  all  the  states  ranging  from  gels 
very  rich  in  water  to  those  very  poor  in  water  characteristic  of  active 
and  dormant  protoplasm. 

Limited sivelling.  In  the  swelling  process  the  absorbed  medium  pene- 
trates into  the  interstices  available  in  the  gel  structure  and  widens  the 
framework.  It  is  clear  that  the  permeating  liquid  should  show  chemical 
affinity  for  the  chain  molecules  concerned.  Thus  the  lipophilic  mole- 
cules of  rubber  and  polystyrene  swell  in  benzene,  which  is  lipophilic, 
while  the  hydrophilic  cellulose  swells  in  water.  Whether  in  this  process 
the  framework  of  the  gel  goes  to  pieces,  i.e.,  whether  the  gel  is  dissolved, 
depends  on  whether  the  junctions  present  can  be  disrupted.  If  the 
bonds  are  of  the  type  of  cohesive  forces  and  the  solvent  present  is 
capable  of  completely  solvating  the  chain  molecules,  the  gel  structure 
may  disintegrate  and  change  into  a  gel  solution  in  which  the  particles 
have  greater  mobility.  This  happens,  for  instance,  in  the  swelling  of 
fibre  cellulose  in  cuprammonium.  Limited  swelling,  therefore,  always 
indicates  that  the  chain  molecules  can  only  be  solvated  to  a  limited 
extent. 

Sometimes  main  valency  bonds  may  be  among  the  junctions.  For 
example,  as  shown  by  Staudinger  (1936a),  polystyrene  with  a  degree 
of  polymerization  of  1700  is  soluble  in  benzene,  but  on  the  addition 
of  traces  of  divinylbenzene  (0.002%)  it  is  converted  into  a  product 
showing  limited  swelling  in  benzene  (Fig.  5  2).  In  the  same  way  chains 


68        FUNDAMENTALS  OF  SUBMIC ROSCOPIC  MORPHOLOGY 


CH=CH2 


CH=CH! 

Divinyl- 

benzene 


-CH2—CH-CH2-  CH  -  -CH2-CH 


-CH2—CH-CH2-CH  —  CH2-CH 


-CH2-CH-CH2- 


■CH2-CH-CH2- 


■CH2-CH-CH2- 

-CH2-CH-CH2— 


Fig.  52.  Limited  swelling  of  polystyrene 
(from  Staudinger,  1936a). 


of  methylcellulose  can  be  interlinked  by  dicarbon  acids  (Tavel,  1939) 
or  chains  of  polygalacturonic  acid  (pectic  acid)  by  epioxides  (Deuel, 
1947a).  When  main  valency  bonds  occur  between  the  chain  molecules, 
even  the  most  suitable  solvating  medium  is  no  longer  capable  of 
destroying  the  gel  structure.  Notwithstanding  considerable  swelling 

(e.g.,  a  30-fold  increase 
in  volume)  the  frame- 
work of  the  chains  is 
0^  ^^  preserved.  It  is  possible 

[]  that  cellulose  also  con- 

V  _  _  tains  a  few  of  such  main 

valency  bridges  (see,  for 
instance,  Lieser,  1940; 
Meyer,   1940a;  Pacsu, 
1948).  These  would  li- 
mit  the    swelling    and 
would  have  to  be  de- 
graded chemically  when  cellulose  is  dissolved  in  cuprammonium. 
It  can  be  said  in  general  that  limited  swelling  occurs  when  certain 
junctions  of  the  gel  frame  (cohesive  or  main  valency  bonds)  cannot 
be  loosened. 

Concept  of  phase  in  gels.  In  the  case  of  a  sol  one  can  (if  necessary) 
speak  of  a  "dispersed  phase"  distributed  in  a  dispersing  medium, 
although  difficulties  arise  which  have  already  been  mentioned  on  p. 
16.  With  sols  containing  chain  molecules  instead  of  colloid  particles 
in  the  sense  of  the  classical  theory  of  dispersoids,  to  uphold  the  con- 
cept "phase"  is  decidedly  wrong.  For,  according  to  the  definitions  in 
phase  theory,  separate  molecules  may  not  be  characterized  as  phases. 
With  gels  the  conditions  are  much  the  same.  In  a  chain  framework, 
it  is  incorrect  to  speak  of  a  "dispersed  phase",  because  regions  with 
a  thickness  of  molecular  dimensions  are  not  homogeneous  phases, 
and  the  concept  "dispersing  medium"  also  becomes  questionable. 
Consider  a  gel  consisting  of  equal  percentages  of  chains  and  water; 
a  projection  of  the  structure  then  gives  the  impression  that  the  water 
is  distributed  as  a  "dispersoid"  in  closed  compartments  (Fig.  53a) 
whereas,  conversely,  in  a  cross-section  of  the  gel  the  sections  across 
the  chain  molecules  appear  as  "dispersed"  particles  distributed  in  the 
liquid  (Fig.  5  3b).  In  reality,  however,  neither  of  the  two  partners  is 


STRUCTURE  OF  GELS 


69 


"dispersed"  relatively  to  the  other,  for  they  both  fill  the  available 
space  continuously. 

A  gel  of  chain  molecules  is  therefore  not  a  two-phase  system  but  a 
single  undivided  phase.  It  is  not  only  microscopically  homogeneous 
and  optically  empty,  but  also  homogeneous  from  a  physico-chemical 


I       - 


\ 


^        /        / 


\J 


b) 


Fig.  53.  Gel  framework,  a)  Projection,  b)  section  across  the  frame- 
work. Areas  to  be  compared  encircled. 


point  of  view.  As  in  the  case  of  real  solutions,  if  small  volumes  are 
considered,  one  always  finds  the  same  composition,  with  the  sole 
difference  that  the  volumes  contain  only  parts  of  chains  instead  of 
whole  molecules  (Fig.  53b).  Thus  gels  with  a  framework  consisting 
of  individual  chain  molecules  are  one-phase  systems.  As  in  the  case  of 
mesophases,  this  state  deserves  a  nomenclature  of  its  own.  It  will  be 
designated  as  pseudophase,  especially  in  view  of  the  fact  that  often  not 
all  junction  bonds  are  identical,  so  that  the  condition  of  homogeneity 
is  not  strictly  satisfied. 

In  concentrated  gels  the  chain  molecules  show  a  strong  tendency 
to  orientate  in  parallel  and  to  cluster  in  strands  or  rods.  In  such  cases 
the  parallel  arrangement  may  become  so  pronounced  that  here  and 
there  the  chain  molecules  combine  to  form  a  chain  lattice.  The  length 
of  the  crystal  lattice  in  the  direction  of  the  chain  axis  need  not  be  the 
same  as  the  length  of  these  molecules ;  the  chains  may  protrude  from 
the  end  planes  of  the  crystalline  rods  (Gerngross,  Herrmann  and  co- 
workers, 1930,  1932),  continue  further  and  eventually  enter  again  into 
another  region  of  lattice  order,  as  has  been  indicated  schematically 
in  Fig.  54a.  The  more  complete  the  average  parallel  arrangement  of 
the  chains,  the  greater  the  probability  of  the  occurrence  of  crystals 


70        FUNDAMENTALS  OF  SUBMIC  ROSCOPIC  MORPHOLOGY  I 

(Fig.  54b).  In  this  case  the  gel  is  no  longer  a  one-phase  system:  the 
reo-ions  of  lattice  order  form  a  homogeneous  phase  in  contrast  to  the 
pseudophase  formed  by  the  mixture  of  the  unordered  chains  and  the 
surrounding  liquid. 

Hence,  from  a  structural  point  of  view  there  are  two  kinds  of  gels. 


iJ        L_. 
a)  b) 

Fig.  54.  Ordered  regions  in  a  gel  framework,  a)  Locally 
parallelized  chain  molecules,  b)  local  formation  of  a  crystal 

lattice. 

viz.,  I.  one-phase  gels  whose  framework  consists  of  very  long  chain 
molecules  interlinked  at  the  junctions  (pseudophases)  and  2.  two- 
phase  gels  with  a  crystalline  and  a  non-crystalline  (amorphous)  phase. 
Instead  of  the  fine  chain  framework  of  the  one-phase  gels,  we  then 
have  a  much  coarser  rod  framework. 

Dispersion  series.  Having  derived  the  structure  of  gels  from  the 
special  form  of  the  high  molecular  weight  chain  molecules  —  thus 
starting  from  below,  that  is  from  the  amicroscopic  domain  —  we 
shall  now  try  to  advance  into  the  submicroscopic  domain  of  gels  from 
the  macroscopic  and  microscopic  regions.  In  colloid  chemistry  the 
concept  of  colloid  particles  is  usually  derived  from  macroscopic 
particles  with  the  aid  of  a  dispersion  series.  The  particle  size  in  this  series 
decreases  steadily  to  microscopic  dimensions,  ultimately  declining  to 
invisible  submicroscopic  dimensions.  The  final  step  in  the  direction 
of  progressive  dispersion  leads  from  the  colloid  range  to  the  ami- 
croscopic dispersions  of  true  solutions  (Table  IX). 

When  making  a  similar  dispersion  series  for  gels  one  must  start  from 
re ficu/ar  instead  of  corpuscular  systems.  The  frequency  of  such  systems 
in  biology  is  surprising;  one  comes  to  the  conclusion  that  network 
systems  of  all  dimensions  are  typical  of  living  matter! 


STRUCTURE  OF  GELS 


71 


TABLE  IX 
DISPERSION  SERIES 


Order  of  magnitude  of 

Corpuscular  disperse 
systems 

Reticular  disperse 
systems 

structural  unit 

.••  •  •   • 

•"3-r^— 

K^ll 

^^S 

Macroscopic 

Microscopic 

Submicroscopic 

Amicroscopic 

^  Gravel 
i  Sand 

Dust 

Clay 

Salt  solution 

Liana  undergrowth;  veil 

of  aerial  roots 
Wad  of  threadlike  algae 
VA  gel,  cell  wall 
Methyl  cellulose; 

cytoplasm(?) 

The  entanglement  of  lianas  in  a  virgin  forest  is  a  macroscopic  net- 
work system  (Fig.  5  5).  A  good  example  of  fibre  network  is  the  veil 
of  aerial  roots  of  Cissus  lianas  in  a  tropical  forest :  thin  filiform  roots 
with  a  length  of  several  metres  hang  slackly  from  the  branches.  They 
form,  as  it  were,  a  fabric  in  the  air,  although  none  of  these  aerial  roots 
have  grown  together.  In  moving  air  this  entanglement  of  roots  be- 
haves like  a  coherent  mass  because  neighbouring  filaments  impede 
free  movement.  There  are  many  other,  still  finer  macroscopic  net- 
work systems  viz.,  skeletons  of  vascular  bundles  of  leaves,  succulent 
sprouts  and  fruits  (Fig.  56),  skeletons  of  sponges  (especially  not- 
iceable in  silica  sponges),  spongy  parts  of  bones,  etc.  An  excellent 
example  of  a  meshwork  with  microscopic  elements  is  macerated  skin 
(Fig.  57);  also  latex  tube  systems  of  the  latex  plants.  When  algae 
threads  are  fished  out  of  a  pool,  we  are  astonished  to  find  how  they 
cling  together  in  a  tangled  skein,  although  every  thread  is  an  individual 
in  itself.  Here  the  junction  bonds,  which  are  hypothetic  in  the  case  of 
gels,  can  actually  be  observed  under  the  microscope,  for  at  all  points 
where  two  threads  cross,  they  stick  together  (cf.  Fig.  51b,  p.  66).  The 
number  of  these  junctions  is  so  great  that  a  wad  of  algae  like  this  is 
even  slightly  elastic  when  compressed. 

We  penetrate  into  the  submicroscopic  domain  by  making  the 
threads  so  thin  as  to  become  invisible  under  the  ordinary  microscope, 
thus  obtaining  gels.  Until  recently  their  structural  principles  had  to  be 
found  out  by  indirect  means   (Frey-Wyssling,   1938).   Nowadays, 


72        FUNDAMENTALS  OF  SUBMICROSCOPI  C  MORPHOLOGY 


Fig.  5  5 
500  fJ- 


Fig.  56 


i^ 


-I 


Fig-  57 


Fig.  58 


Reticular   structures   of  different   scales 

Fig.  5  5 .  Coarse  macroscopic  reticular  structure :  liana  brush  in  a  virgin  forest  photographed  skyward. 
Fig.  56.  Macroscopic  reticular  structure:  fascicular  skeleton  of  a  Luffa  fruit  (vegetable  sponge). 
Fig.  57.  Microscopic  reticular  structure:  network  of  collagen  fibres  in  cow's  skin  (Kuntzel,  1941). 
Fig.  58.  Submicroscopic  reticular  structure:  ultra-structure   of  coagulated  blood    fibrin    (from 

WoLPERS  and  Ruska,  1939). 


3  STRUCTURE  OF  GELS  73 

however,  the  reticular  structure  of  gels  can  be  photographed  directly 
in  the  electron  microscope  (Fig.  58).  As  will  be  explained  in  the  next 
paragraph,  the  submicroscopic  strands  or  strings  which  form  the  gel 
frame  will  be  designated  as  micellar  ttrands.  Thus  the  submicroscopic  gel 
structure  is  a  micellar  framework. 

The  transition  into  the  amicroscopic  domain  is  of  particular  im- 
portance. Whereas  corpuscular  disperse  systems  in  this  case  become 
real  solutions  and  are  no  longer  accessible  to  colloid  chemical- 
methods  of  research,  reticular  systems  remain  colloids  even  if  the 
thickness  of  the  strands  of  their  framework  is  reduced  to  amicroscopic 
dimensions,  i.e.,  to  the  cross-section  of  a  single  molecule.  Thus  in  net- 
work systems  there  is  no  lower  Umit  to  the  colloid  domain ;  they  re- 
main gels  irrespective  of  whether  their  network  is  submicroscopic  or 
amicroscopic.  Examples  are  the  polystyrene  gels  mentioned  on  p.  67, 
(Fig.  52,  p.  68),  or  the  methyl  cellulose  gels  prepared  by  Tavel  (1939) 
with  the  aid  of  oxalyl  chloride,  or  pectin  gels  prepared  by  Deuel 
(1947a)  with  ethylene  oxide.  In  these  cases  the  strings  of  the  network 
are  chain  molecules  and  the  gel  structure  is  a  fine  molecular  frameivork. 

Comparison  of  corpuscular  and  reticular  systems.  The  properties  of  net- 
work gels  differ  in  principle  from  those  of  sols  with  their  corpuscular 
dispersed  particles.  This  is  clearly  demonstrated  by  Table  X. 

Whereas  a  liquid  capable  of  solvating  a  substance  will  disperse 
corpuscular  colloids,  reticular  colloids  remain  a  coherent  mass  into 
which  the  solvating  medium  can  penetrate  to  a  certain  extent  only 
(limited  swelling).  In  this  case  the  dispersing  medium  would  be 
better  characterized  as  an  imbibition  medium  (see  p.  81  and  84),  since 
the  colloid  substance  is  not  dispersed  into  separate  particles.  In  the 
coacervation  of  sols  an  equilibrium  liquid  poor  in  colloid  and  the 
coacervate  layer  rich  in  colloid  are  formed  (Fig.  21,  p.  22).  In  reticular 
coacervates,  however,  the  equilibrium  liquid  contains  no  colloid,  be- 
cause the  latter  is  insoluble  in  the  reticular  state.  For  example,  after 
gelation  of  a  gelatin  solution,  no  gelatin  is  found  in  the  supernatant 
liquid  (cf.  p.  21). 

In  reticular  colloids  the  mutual  position  of  their  submicroscopic 
elements  is  fixed,  so  that  a  structure  results.  It  follows  that  gels  possess 
a  certain  elasticity,  although  often  only  slight,  indicating  that  the 
forces  acting  in  the  junction  bonds  are  weak.  Typically  intermediate 
between  gels  and  sols  are  gel  solutions,  whose  particles  impede  each 


74        FUNDAMENTALS  OF  SU  BM  I  C  RO  SC  OPI  C  MORPHOLOGY  I 


TABLE  X 

COMPARISON  OF  CORPUSCULAR  AND  RETICULAR  COLLOIDS 

IN  THE  SOLVATED  STATE 


Corpuscular  colloids 

Intermediate  state 

Reticular  colloids 

Colloid  state 

Sols 

Gel  solution 

Gels 

Colloid  portion 

Individual  particles 

Particles  are  impeding 

Coherent  structure 

(micelles  or  macro- 

each  other's  motion 

(micellar  or  macro- 

U3 

molecules) 

molecular  frame- 

_u 

work) 

Solvating  liquid 

Dispersing  medium 

Imbibition  medium 

Equilibrium  liquid 

Dispersing  medium 

Imbibition  medium 

1— « 

incoacervation 

-f  colloid  portion 

fjee  from  colloid 

Elasticity 

Inelastic 

Structural  viscosity 

Elastic 

Structure 

Structureless 

Short-range  order 

Structured 

Ultramicroscope 

Demonstration  of 

Gel  frame  is  optically 

j: 

particles 

empty 

Ultracentrifuge 

Sedimentation 

Syneresis 

CO 

Ultrafiltration 

Particle  size 

Pore  size 

U4 

Dialysis 

i  With  the  aid  of 

>  Without  membrane 

O 

W3 

Donnan  equilibrium 

^  membranes 

-X3 

C 

Osmotic  laws 

Hold  good 

Disturbed 

Do  not  apply,  because 

-C 

gel  is  insoluble 

Kinetic  migration 

Mutual  diffusion 

Permeation 

Electric  migration 

Electrophoresis 

Electrosmosis 

■|i 

Dilution,  swelling 

Unlimited  dilution 

Unlimited  swelling 

Limited  swelling 

Disturbance  of 

Precipitation 

"Hardening" 

irbanc 
ilibri. 

stability 

(flocculation. 

(tanning,  fixation) 

coagulation) 

■  22   g" 

Separation  into 

Two  coexisting 

Usually  vacuolization 

C   '^ 

different  phases 

layers 

Other's  free  movements.  These  gel  solutions,  therefore,  show  structural 
viscosity,  demonstrating  the  existence  of  some  structure  when  de- 
formed. 

The  difference  between  corpuscular  and  reticular  systems  is  particu- 
larly apparent  when  testing  the  apphcability  of  the  methods  of  research 
developed  by  colloid  chemistry.  As  a  result  of  the  close  packing  of  the 
micellar  strands,  all  gels  are  optically  empty  in  the  ultramicroscope. 
In  the  centrifuge  no  definite  sedimentation  equilibrium  is  established; 


5  STRUCTURE  OF  GELS  75 

some  of  the  imbibing  medium  is  simply  pressed  out  of  the  gel  (syn- 
eresis).  As  the  gels  do  not  contain  individual  particles,  ultrafiltration 
cannot  be  used  as  a  method  to  discover  whether  they  contain  sub- 
microscopic  or  amicroscopic  structural  elements.  It  gives  some  in- 
formation, however,  about  the  approximate  pore  size  of  the  network 
structure,  since  on  account  of  its  structure  each  reticular  colloid  re- 
presents an  ultrafilter,  provided  it  possesses  the  firmness  needed  to 
resist  the  fihration  pressures  applied.  In  all  other  methods  of  research 
mentioned  in  Table  X  the  contrast  between  the  movable  particles  of 
sols  and  the  immovable  frame  of  the  gels  finds  expression.  In  dialysis 
and  in  the  study  of  Donnan  equilibria,  amicroscopic  particles  are 
removed  by  diffusion  through  a  membrane  which  is  impermeable 
to  colloid  particles.  In  the  case  of  insoluble  gels  no  membrane  is 
needed,  because  the  colloid  portion  is  itself  immovable  (see  p.  202). 
For  the  same  reason,  the  osmotic  laws  are  not  applicable  to  gels, 
whereas  in  true  sols,  where  the  individual  particles  are  completely  in- 
dependent, they  allow  of  a  determination  of  the  number  (and  therefore 
the  weight)  of  the  particles.  Finally,  when  concentration  gradients  or 
potential  gradients  are  applied  to  gels,  the  amicroscopic  particles 
diffuse  through  the  gel  frame  (permeation),  or  the  imbibition  liquid 
migrates  through  the  electrically  charged  network  (electrosmosis). 

Similarly  when  the  equilibrium  in  a  colloid  system  is  disturbed, 
the  behaviour  of  gels  and  sols  is  fundamentally  different.  Sols  can  be 
diluted  by  the  solvating  liquid,  whereas  in  true  gels  only  limited  swell- 
ing occurs.  In  sols  the  disturbance  of  stability  factors  (hydration  and 
charge)  may  lead  to  flocculation  or  coagulation.  In  contrast  to  what 
is  commonly  asserted,  gels  do  not  coagulate,  they  are  "hardened". 
In  technology  this  is  denoted  by  tanning  and  in  cytology  by  fixation. 
Separation  of  sols  results  in  two  microscopically  uniform  "phases" 
(Fig.  15,  21,  p.  .17,  22),  whereas  in  gels  the  separated  drops  usually 
cannot  unite  and  give  rise  to  vacuolization  in  the  originally  micro- 
scopically uniform  system  (Fig.  23,  p.  23).  The  concepts  of  limited 
swelling,  fixation  and  vacuolization,  which  are  mentioned  at  the  bot- 
tom of  the  last  column  in  Table  X,  are  familiar  to  all  cytologists  and 
we  need  waste  no  time  on  the  question  as  to  which  colloids  are  of  the 
first  importance  in  microscopic  and  submicroscopic  morphology. 

Indeed,  the  number  of  colloid  systems  in  biology,  whose  nature  has 
been  ascertained  successfully  by  means  of  the  methods  of  research 


76        FUNDAMENTALS  OF  SUBMI C RO  S  COP  I  C  MORPHOLOGY  I 

developed  for  corpuscular  dispersoids,  is  very  small  (blood,  mil  k, 
serum,  suspensions  of  micro-organisms  and  viruses).  No  conclusive 
information  could  be  derived  by  these  methods  on  the  fine  structur  e 
of  the  protoplasm.  The  very  terminology  of  the  theory  of  dispersoid  s, 
which  assumes  dispersed  particles  in  a  dispersing  medium,  is  unsuitable. 
True,  the  introduction  of  dijJorM,  i.e.,  strongly  anisodiametric  par- 
ticles, accounts  to  a  certain  extent  for  the  properties  known  to  modern 
macromolecular  chemistry  (Manegold,  1941).  The  older  technical 
terms  of  Nageli  (Nageli  and  Schwendener,  1877)  are  much  better 
adapted  to  the  needs  of  biologists  working  with  gels.  Nageli's 
ideas  can  be  applied  to  our  present  concept  of  gel  structure.  To  that 
end  let  us  first  give  a  precise  definition  of  the  micellar  concept,  to  which 
unfortunately  various  meanings  have  been  attached  in  colloid  science. 

d.  Micellar  Theory 

The  concept  of  the  micelle.  C.  Nageli  was  the  first  to  develop  a  well-founded 
theory  on  the  structure  of  hydrogels,  which  he  designated  as  organi-:^ed 
substances.  Starting  from  double  refraction,  anisotropy  of  swelling  and  layer 
structure  of  grains  of  starch  (18  5  8,  new  edition  1928)  and  of  cell  membranes, 
he  made  the  assumption  that  these  substances  consist  of  long,  submicro- 
scopic  particles,  supermolecular  in  character  and  of  crystalline  structure.  Such 
a  particle  was  called  a  micelle  (diminutive  of  the  latin  mica  =  a  crumb  or 
little  bit). 

Later,  Nageli  extended  his  theory  to  solutions.  He  stated  that,  when  a 
gel  is  dissolved,  the  micelles  are  maintained  as  units  and  give  a  micellar 
solution.  As  a  result  of  this  transference  of  the  micellar  concept  from  solid 
gels  to  solutions,  this  concept  is  used  in  the  literature  in  various  meanings, 
as  has  been  pointed  out  by  several  authors:  Zsigmondy  (1921),  Ambronn 
and  Frey  (1926,  p.  152).  Whereas  the  biologists,  in  particular  Ambronn's 
school  (Frey,  1926a,  1928a,  b)  and  also  Schmidt  (1934),  adhere  to  the 
original  definition  which  indicates  the:  form  and  crystallinity  of  the  particles, 
the  meaning  attached  to  micelles  by  colloid  chemists  is  as  a  rule  simply 
that  of  dispersed  particles  in  a  colloid  solution,  stressing  in  particular 
their  electrical  charge,  without  heeding  their  form  and  structure.  In  the  latter 
case,  therefore,  it  represents  an  overall  concept  which  may  embrace  all 
possibilities  such  as  primary  particles  (monones),  secondary  particles 
(polyones),  associate^  (^-g-.  i^^  soaps),  etc.,  including  their  charges  and 
solvation  layers.  As  a  result  of  this  situation,  the  origin  of  this  term  is 
scarcely  known  in  colloid  chemistry.  This  led  to  what  Nageli  objected  to  in 
a  discussion  of  Pfeffer's  terminology  in  the  famous  "Osmotische  Unter- 
suchungen"  (1877).  Nageli  says  (new  edit.  1928,  p.  70/71):  "Pfeffer  uses 
the  general  expression  'tagma'  for  molecular  compound,  observing  that  in 
chemistry  one  would  hesitate  to  introduce  the  term  micelle,  which  is  re- 


3 


STRUCTURE  OF  GELS 


77 


miniscent  of  cell.  It  seems,  therefore,  that  the  etymological  error  is  made : 
to  believe  that  we  are  deaUng  with  a  barbaric  composition  of  "cellula"  and 
an  unknown  word  beginning  with  "mi",  in  much  the  same  way  as  the 
word  aldehyde  is  formed." 

By  optical  means,  Ambronn  has  definetely  established  the  existence  of 


o) 


Fig.  59.  Former  conception  of  the  micellar  structure:  a)  from  Nageli  and  Schwendener 
(1877),  b)  from  Seifriz  (1929)  and  K.  H.  Meyer  (1930). 


long,  submicroscopic  particles  in  gels  such  as  celloidin,  denitrated  cellulose, 
celluloid,  gelatin,  aluminium  oxide  fibres  (see  p.  82).  These  particles  often 
showed  an  intrinsic  double  refraction  which  could  only  be  explained  by 
assuming  crystaUine  particles  (Ambronn,  191 6/1 7).  The  existence  of 
crystaUine  micelles  in  chitin  (Mohring,  1922),  in  muscle  fibres  (Stubel, 
1923)  and  in  vegetable  cell  walls  (Frey,  1926b)  was  demonstrated  by  means 
of  the  same  methods. 

At  about  the  same  time  the  crystalline  nature  of  many  colloid  particles, 
for  example  gold  sols,  cellulose  and  many  other  colloids,  was  estabhshed  by 
the  X-ray  method  (Scherrer,  1920).  Nageli's  micellar  theory  was  taken 
up  by  Meyer  and  Mark  (1930)  and  propagated  by  them  among  chemists 
in  an  almost  unaltered  form,  after  having  been  nursed  for  a  long  time  in  its 
original  form  by  a  few  biologists.  This  is  obvious  from  a  comparison  of 
Nageli  and  Schwendener's  scheme  (1877)  and  the  model  of  fibre  struc- 
ture given  by  Seifriz  (1929)  and  K.  H.  Meyer   (1930):    Fig.   59b.    In 


-}%         FUNDAMENTALS  OF  S  U  BM  I  C  RO  S  COPI  C  MORPHOLOGY      I 

Nageli's  scheme  (Fig.  59a),  two  intermicellar  substances  are  drawn 
between  the  micelles;  one  of  these  substances  may  be  eliminated.  What  is 
new  in  Fio-.  59b  is  the  determination  of  the  inner  structure  of  the  micelles; 
for  the  rest,  however,  there  is  complete  agreement  with  Fig.  59a.  The 
micelles  were  considered  as  disperse  phase,  surrounded  by  intermicellar 
spaces  which  are  accessible  to  the  dispersing  medium.  To  account  for  the 
coherence  of  the  crystalline  micelles  in  a  solid  framework,  special  micellar 
forces  had  to  be  assumed.  Meyer  and  Mark  considered  these  to  be  large 
cohesive  forces  which,  in  cellulose  for  instance,  are  additively  composed 
of  the  molar  cohesions  of  the  numerous  OH-groups.  However,  since  these 
same  forces  act  intramicellarly  as  lattice  forces,  it  was  difficult  to  see  what 
the  difference  might  be  between  the  forces  responsible  for  the  />//ramicellar 
coherence  of  the  chain  molecules  in  a  crystal  lattice  and  the  /«/^rmicellar 
"micellar  forces". 

According  to  Nageli,  when  a  gel  is  dissolved,  the  micelles  are  dispersed, 
and  the  sol  contains  independent  crystals.  This  point  of  view  has  often  been 
adopted  by  others,  in  particular  for  cellulose  sols,  although  such  solutions 
do  not  give  X-ray  diagrams  (e.g.,HERZOG,  1927).  According  to  Staudinger 
(1932),  the  high  polymer  natural  substances  are  dissolved  as  separate  chain 
molecules  instead  of  crystalline  particles.  At  present,  therefore,  only  crystal- 
Une  suspensoid  colloids  such  as  gold-,  vanadium  pentoxide-,  ferric  oxide 
sols,  etc.  can  be  claimed  to  be  micellar  solutions  in  Nageli's  sense;  they 
show  mostly  a  strong  birefringence  of  flow  and  partly  also  X-ray  inter- 
ferences. 

In  the  case  of  gels,  our  recent  knowledge  of  the  structure  of  high 
polymers  raises  further  objections  to  Nageli's  concept  of  micelle,  for  it  is 
found  that  the  chain  molecules  are  much  longer  than  the  crystalline  regions 
(Fig,  54,  p.  70).  It  follows  that  the  micelles,  instead  of  possessing  individual 
character  as  assumed  by  Nageli,  have  grown  together  and  are  to  a  certain 
extent  absorbed  in  the  gel  structure.  Nowadays  they  can  no  longer  be 
considered  to  be  substantial  (not  even  conditionally  substantial)  particles 
(Frey-Wyssling,  1936a,  c;  Kratky  and  Mark,  1937).  They  consist  of  well- 
ordered  chain  molecules,  which  protrude  from  the  crystalline  into  the  amor- 
phous regions  and  perhaps  take  part  again  in  other  ordered  lattice  regions. 

We  conclude  that  i .  there  are  sols  containing  chain  molecules  which  are 
more  or  less  independent,  rather  than  micelles  in  Nageli's  sense,  and  2.  that 
the  micelles  in  gels  do  not  represent  independent  crystallites  but  at  best 
can  be  described  as  lattice  regions.  Taking  into  account  the  constant  danger 
of  confusion  with  the  colloid  chemical  concept,  which  by  micelle  means  an 
electrically  charged  instead  of  a  crystalline  particle,  it  would  perhaps  be 
better  in  our  considerations  to  give  up  the  concept  micelle.  If  one  wishes 
to  use  it  nevertheless,  one  should  not  assign  any  special  significance  to  this 
concept,  but  simply  use  it  in  the  sense  of  supermolecular  colloid  particle. 
This  would  exclude  all  possibility  of  confusion.  One  would  then  have  to 
distinguish   between   two   different   kinds   of  colloid   particles:    i.   super- 


5  STRUCTURE  OF  GELS  79 

molecular  micelles  consisting  of  many  molecules,  and  2.  macromolecular 
molecules  of  submicroscopic  dimensions.  However,  since  a  well-founded 
terminology  for  sols  does  already  exist,  the  micellar  theory  will  be  confined 
to  gels,  as  originally  intended  by  Nageli. 

Nomenclature.  Although  the  assumption  of  independent  micelles  in 
gels  has  proved  to  be  erroneous,  Nageli's  work  contains  a  great  many 
other  ideas  on  the  structure  of  gels  which  have  been  shown  to  be  quite 
correct.  I  quote  the  following  paragraph,  for  instance,  (new  edition 
1928,  p.  76/77):  "Die  Micelle  vereinigen  sich  .  .  .  zu  Verbanden  .  .  ., 
indem  sie  sich  beliebig,  bald  baumartig,  bald  mehr  netzartig  anein- 
ander  hangen.  Diese  unregelmiissigen  Verbande  .  .  .  bilden  eine  ste- 
hende  Gallerte".  Elsewhere  he  speaks  of  "Micellar-Reihen,  in  denen 
die  Micelle  miteinander  verwachsen  sind".  Although  at  the  time  the 
existence  of  chain  molecules  was  not  even  suspected,  he  has  given  a 
description  of  gel  structure  which  is  essentially  correct. 

To  current  biology  the  main  concern  is,  not  whether  living  matter 
and  its  derivatives  contain  or  do  not  contain  crystalline  regions,  but 
rather  whether  the  particles  are  independent  of  each  other,  as  presumed 
in  classical  colloid  chemistry  or  in  the  theory  of  dispersions,  or  whether 
they  are  united  in  a  framework  (however  weak),  and  thus  provide 
the  colloid  with  a  structure.  Consequently,  contrary  to  structureless 
dispersoids,  gels  are  in  need  of  an  appropriate  terminology. 

It  is  tempting  to  make  up  for  this  deficiency  by  creating  new  names^ 
However,  one  does  not  always  render  science  a  service  by  doing  so, 
and  it  is  perhaps  preferable  in  this  case  to  use  old  well-tried  expressions 
adapted  to  modern  experimental  results  by  new  definitions.  Following 
Nageli,  the  frame  substance  will  be  designated  as  micellar  portion  and 
the  interstitial  substance  as  intermicellar  portion  of  a  gel.  In  those  cases 
where  the  micellar  structure  consists  of  coarse  beams  or  joists,  which 
are  partly  crystalline  and  therefore  homogeneous,  one  can  also  speak 
of  micellar  phase  and  intermicellar  phase. 

There  is  no  danger  that  this  new  definition  will  again  give  rise  to 
confusion,  for  the  concept  intermicellar  is  used  in  exactly  the  same  sense 
as  hitherto  in  the  literature  of  the  subject,  and  the  concept  micellar  is 
only  changed  so  as  to  apply  not  exclusively  to  the  crystalline  regions 
of  a  framework,  but  to  the  framework  as  a  whole.  This  solves  the 

^  Pfeiffer  (1941b,  i942a)  designates  the  theory  of  fine-structure  as  leptonics  and  the 
invisible  structural  units  as  leptones  (from  Ac-tto'c  =  fine,  small). 


8o        rUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

difficulty  that  gels  whose  framework  units  consist  of  only  a  few 
parallel  chain  molecules  do  not  answer  to  Nageli's  original  definition, 
because  a  small  number  of  chain  molecules  are  not  capable  of  forming 
a  crystal  lattice.  With  still  finer  strands  of  the  gel  structure,  it  is  true, 
it  ultimately  consists  only  of  chain  molecules  and  the  micellar  frame- 
work has  changed  into  a  molecular  frameivork^  as  has  been  pointed  out 
on  page  73.  Just  as  in  the  transition  from  colloid  to  molecular  disper- 
sions, there  also  exist  transitional  forms  between  (a)  gels  with  micellar 
strands  and  (b)  gels  with  chain  molecules  as  structural  units.  In  gels 
with  a  molecular  framework  the  particle  size  of  the  two  components 
of  the  system  are  not  similar  as  in  the  case  of  solvent  and  solute 
molecules  in  a  true  solution.  In  principle  they  remain  different  in  a 
morphological  sense  as  framework  and  interstitial  substance. 

In  the  case  of  micellar  systems  possessing  strands  with  a  thickness 
of  several  molecules,  a  distinction  should  be  made  between  processes 
which  occur  in  the  meshes  of  the  network  (/>^/^r/;//V«'//<«r)  and  those  occur- 
ring inside  the  beams  of  the  frame,  i.e.,  in  the  crystal  lattice  {intra- 
micellar).  In  the  same  sense  the  expression  "intramicellar"  is  used  for 
cation  exchange  inside  layer  lattices  (Wiegner,  1935 ;  Bottini,  1937). 
With  the  aid  of  the  concepts  "micellar",  "intermicellar"  and  "intra- 
micellar", all  processes  occurring  in  gel  structures  can  be  described 
unambiguously.  By  a  relatively  sUght  change  in  concepts  we  thus 
preserve  a  nomenclature  which  has  done  good  service  for  90  years, 
and  renders  honour  toNAGELi,who  laid  the  foundations  of  the  research 
on  biological  gels. 

In  Table  X  we  have  recapitulated  the  most  important  points  which, 
according  to  our  definition,  distinguish  the  reticular  gel  from  its 
counterpart,  the  corpuscular  sol.  As  in  the  case  of  dispersoids  (Table 
II,  p.  16),  the  components  of  a  gel  can  occur  as  gases,  hquids  or 
solids,  with  the  restriction,  however,  that  the  micellar  component  must 
always  be  solid  (Table  XI).  If  the  intermicellar  substance  is  a  gas  or  a 
liquid,  we  have  to  deal  with  network  structures  or  capillary  structures. 
If  it  is  a  solid,  however,  solidified  gels  result,  showing  clearly  in 
contrast  to  dispersoids  that  the  two  components  are  completely 
equivalent  as  regards  the  arrangement  in  space. 

The  micellar  structure  is  determined  by  the  micellar  strands,  by  the 
type  of  bonds  between  them  and  by  the  intermicellar  substance.  For 
a  given  type  of  micellar  units,  however,  the  gels  can  be  built  up  with 


STRUCTURE  OF  GELS 


8i 


TABLE  XI 

(Compare  Table  II) 

RETICULAR  SYSTEMS  (ACCORDING  TO  FREY-WYSSLING,   1 93 yd) 


Imbibition  medium 

Micellar  frame 

Structures 

Solid 

Liquid 

Gas 

Solid 
Solid 
Solid 

i  Composite  solid 

}  Gel  structure 

^  Capillary  structure 

various  different  possibilities  of  orientation.  This  determines  the 
micellar  texture,  which  gives  information  about  the  arrangement  of 
the  structural  elements  in  the  gel,  in  contrast  to  the  micellar  structure, 
which  characterizes  the  fine-structure  in  general. 

Definitions.  To  sum  up,  we  give  the  following  survey: 
By  structure^  we  mean  the  fixed  mutual  positions  of  the  submicro- 
scopic  or  amicroscopic  morphological  units ;  by  texture,  the  special 
arrangement  and  distribution  of  such  structural  units. 


=  colloids  with  freely  moving  particles 

=  colloids  with  a  gel  frame 

=  molecular  colloid  particles 

=  supermolecular  colloid  particles,  most  often 

packets  of  chain  molecules  in  parallel  arrangement 
==  amicroscopic  structure  of  intertwined  cliain 

molecules 
=  submicroscopic  structure  of  coherent  micellar 

strands 
=  substances  in  the  interstices  of  a  molecular 

framework 
=  substances  in  the  interstices  of  a  micellar  frame- 
work 
=  processes  occurring  between  the  strands  of  a  gel 

frame 
=  processes  occurring  inside  the  strands  of  a  gel 

frame 
=  fine-structure  of  gels  in  general 
=  arrangement  of  the  structural  units  in  particular 

^  Not  only  crystalline  but  also  amorphous  solid  phases  possess  a  structure.  For,  in 
amorphous  glasses  (Bussem  and  Weyl,  1936)  and  also  in  isotropic  gels  the  structural 
elements  are  bound  together  elastically  in  fixed  mutual  positions,  notwithstanding  the  lack 
of  order.  We  must  therefore  in  principle  attribute  a  structure  to  ?11  solid  states  of  matter. 


corpuscular  colloids 
reticular  colloids 
macromolecules 
micelles 

molecular  framework 

micellar  framework 

interstitial  substances 

intermicellar  substances 

intermicellar  processes 

intramicellar  processes 

micellar  structure 
micellar  texture 


8i        FUNDAMENTALS  OF  SUBMICROSCOPI C  MORPHOLOGY  E 

§  4.  Studies  in   Gels 

The  colloid  chemical  methods  of  investigation  which  have  proved  so- 
successful  in  the  elucidation  of  the  nature  of  sols  have  only  a  limited 
applicability  to  gels  (compare  the  discussion  of  Table  X,  p.  75).  Gels 
must  therefore  be  investigated  by  different  means.  Of  these  we  shall 
only  discuss  those  which  are  of  special  importance  to  the  investigation 
of  cytological  objects,  leaving  others,  such  as  are  of  interest,  e.g.,  in 
the  technical  testing  of  gels,  out  of  account.  For  lack  of  space  the 
methods  of  investigation  will  not  be  treated  in  great  detail ;  we  shall 
only  deal  with  the  principles  of  these  methods  and  the  problems  which 
they  can  solve. 

a.  Polarisation  Microscopy 

Theory  of  composite  bodies.  The  texture  of  gels  can  be  explored  by 
optical  means  if  two  conditions  are  fulfilled.  In  the  first  place  the 


"//="/ 


i"//="< 


ni-n,f_ 


nj,  =  n^ 


Fig.  60.  a)  Rodlet  composite  body,  ny  big, 

Ha  small  refractive  index,  b)  Layer  or  platelet 

composite  body,  Oy  big,  Hq  small  refractive 

index. 


strands  of  the  framework  must  be  separated  from  the  intermicellar 
space  by  definite  phase  boundaries,  and  secondly  they  may  not  be 
oriented  at  random  but  must  show  a  certain  tendency  to  orientation 
in  a  given  direction  in  space,  Wiener  (191 2)  has  calculated  theoretic- 


4  STUDIES  IN  GELS  85 

ally  the  optical  effects  occurring  in  these  systems.  In  this  calculation 
one  must  assume  greatly  idealized  textures  with,  for  instance,  parallel 
circular  cjdinders  or  parallel  planes  (Fig.  6oa  and  b).  Such  aggregates, 
meeting  the  mathematical  requirements,  are  designated  as  "composite 
bodies"  (German:  Mischkorper).  As  the  structural  units  (cylinders  or 
planes)  are  not  bound  together,  they  do  not  possess  a  micellar  structure 
in  our  sense.  One  can  imagine,  however,  that  a  gel  is  formed  out  of 
such  an  idealized  composite  body  if  the  structtiral  units  are  somehow 
anastomosed  with  each  other.  This  does  not  affect  the  general  character 
of  the  optical  effects,  but  it  is  obvious  that  quantitative  calculations 
according  to  Wiener's  formulae  cannot  give  very  accurate  results  for 
gels  with  a  micellar  structure,  since  the  geometrical  conditions  for  an 
accurate  mathematical  treatment  of  the  problem  are  not  satisfied. 

The  rods  or  layers  of  the  composite  body  must  be  supposed  to  be 
optically  isotropic.  It  then  follows  from  theory  that  the  behaviour 
of  the  composite  body  with  respect  to  polarized  light  depends  on  the 
direction  of  its  vibration,  i.e.,  such  a  body  is  anisotropic,  provided 
the  diameter  of  the  cyHnders  and  the  distances  between  the  cylinders 
or  layers  are  small  compared  with  the  wavelength  of  the  light.  It  should 
be  borne  in  mind  that  by  "small"  we  do  not  mean  arbitrarily  small,  as 
the  structural  units  should  possess  true  phase  boundaries.  Single  chain 
molecules,  for  instance,  cannot  act  as  structural  units  in  a  composite 
body. 

Optical  anisotropy  can  manifest  itself  in  three  different  ways : 

1 .  Birefringence.  The  refractive  power  (n^y)  for  directions  parallel  to 
the  axis  of  the  composite  body  is  different  from  that  perpendicular 
to  it  (nj^)  (Fig.  60a,  b)  so  that,  in  polarized  light,  interference  colours 
occur  as  in  doubly  refracting  crystals. 

2.  Anisotropic  absorption  (dichroism).  In  coloured  composite  bodies, 
absorption  is  different  parallel  (k^^)  and  perpendicular  (kj^)  to  the  axis ; 
they  therefore  show  different  colours  depending  on  their  position  with 
respect  to  the  plane  of  oscillation  of  linearly  polarized  light  (Fig.  6i). 

3.  Anisotropic  diffraction.  Transmitted  light  is  differently  diffracted  in 
different  directions;  the  typical  gloss  of  silk,  for  instance,  must  be 
attributed  to  this  effect. 

The  composite  bodies  possess  a  very  typical  characteristic:  their 
anisotropy  is  not  constant  but  is  a  function  of  the  properties  of  the  sub- 
stance enclosed  between  the  particles,  which  in  microscopy   we  de- 


84   FUNDAMENTALS  OF  S U BM I C ROSC OP  I C  MORPHOLOGY      I 

sign2itc2.smoufitmg\iqmd,  or  better  as  imbibition  liquid.  Hence  the  double 
refraction  changes  with  varying  refractive  index  n  of  the  mounting 
liquid.  For  this  reason  the  double  refraction  of  such  composite  bodies 


s 


Fig.  6i.  Dichroism  of  bast  fibres  (SE  vibration  plane  of  polarizer)  from  Frey  (1927b). 
a)  Stained  with  chlorozinc-iodine :  black/colourless;  b)  stained  with  gold:  green  (marked 

with  little  lines)/claret  (dotted). 

differs  essentially  from  the  double  refraction  of  crystals,  which  re- 
presents a  constant  characteristic  of  the  crystal. 

Fig.  62  shows  the  changes  in  double  refraction  observed  when 
epidermal  hairs  of  incinerated  barley  awns  are  mounted  successively 
in    air    (n  =  i.oo),    water    (n  =  1.33),    alcohol    (n  =  1.36),    xylene 

(n  =  1.49),  benzene  (n  =  1.50),  Canada 
balsam  (n  =  1.54),  mono  bromo  naph- 
thalene (n  =  1.66),  potassium  mercury 
iodide  (n  =  1.72).  The  variation  of  the 
birefringence  with  the  refractive  index 
no  of  the  imbibition  liquid  obeys  a  hyper- 
bolic law.  The  double  refraction  is  zero 
when  n^  =  n^  (n^  =  refractive  index  of 
micellar  component).  Composite  bodies 
with  rodlet  texture  are  optically  positive, 
those  with  layer  texture  are  negative. 
Wiener's  formula  for  the  rodlet  bire- 
fringence runs : 


in  A 


1200 


900 


600 


300 


\ 

\ 

/ 

\ 

/ 

^ 

\ 

/ 

\ 

^^.^ 

10 


1.2 


14 


1.6 


1.B  02 


Fig.  62.  Curve  of  rodlet  birefrin- 
gence of  the  epidermal  hairs  of  in- 
cinerated barley  awns  (from  Frey, 
1926b).  Abscissa:  refractive  index 
ng  of  the  imbibition  liquid.  Or- 
dinate: retardation  yk  in  A  units. 


n 


n 


■1 


(^ 


i)n| 


+    ^2^1^ 


Here  n^^  represents  the  extraordinary 
refractive  index  (parallel  to  the  axis  of  the  composite  body)  and  n_^ 
the  ordinary  index  (perpendicular  to  the  axis),  n^  the  refractive  index 


4  STUDIES  IN  GELS  85 

of  the  isotropic  rods  and  n^  that  of  the  imbibition  hquid;  d^  and  62  are 
the  volume  fractions  of  the  two  components  (^1  +  ^2  =  i)-  Clearly, 
n?,  —  nl  is  a  measure  of  the  double  refraction  n^^  —  n^.  The  formula 
shows  how  this  double  refraction  depends  on  the  refractive  index  n.^  of 
the  imbibition  medium.  It  is  zero  when  n^  =  n^,  and  positive  for  all 
other  values  of  n.,,  because  the  numerator  contains  the  square  of 
nf  — n|.  In  other  words,  the  rodlet  birefringence  is  always  positive : 
n,,  >  nl-  Since  in  birefringent  objects  the  larger  index  is  denoted  by 
ny  and  the  smaller  one  by  na,  it  follows  that  n^^  =  ny  and  n^  =  Ua. 
Conversely,  in  composite  bodies  with  layer  texture  and  negative  bi- 
refringence we  have  nj_  =  riy  and  n^^  =  na. 

It  is  significant  that  besides  the  volume  fractions  6^  and  62  no 
quantities  depending  on  the  dimensions  of  the  rods  occur  in  the  equa- 
tion. The  double  refraction  is  independent  of  the  thickness  of  the  rods. 
This  is  of  particular  importance  to  the  study  of  submicroscopic 
textures,  as  long  as  the  size  of  the  structural  units  is  not  known. 

The  double  refraction  of  the  composite  bodies  has  been  termed 
form  birefringence  (Frey,  1924),  because  its  nature  depends  on  the 
form  of  the  textural  elements  of  the  solid  phase.  The  curves  of  form 
birefringence  are  therefore  used  to  examine  whether  intermicellar 
spaces  occur  in  a  material  and  to  decide  whether  the  micellar  phase 
has  the  form  of  rods  or  platelets.  Usually  one  does  not  measure  the 
birefringence  n^^  —  n^  itself,  since  this  depends  on  the  variable 
thickness  d  of  the  swollen  gel  according  to  the  formula 

njj  —  nj_  =  yA/d, 

but  simply  the  retardation  yX,  where  y  is  the  so-called  phase  difference 
and  A  the  wavelength  of  the  light.  The  introduction  of  this  method 
of  research  into  colloid  optics  is  due  to  Ambronn. 

Measurement  of  tjje  birefringence.  The  basic  formula  for  birefringence 
can  be  simplified  by  introducing  the  notations  Zln  for  n^^  —  nj^  and 
r  for  the  retardation  or  path  difference  yX.  This  gives 

/In  =  r/d, 

which  shows  clearly  the  linear  dependence  of  the  retardation  on  the 
thickness  d  of  the  object,  because  zln  for  a  given  object  in  a  given 
medium  is  constant. 

The  retardation  F  is  measured  by  a  compensator.  This  is  a  crystalline 
lamella  (quartz,  gypsum,  calcite)  with  known  double  refraction  An  which 


86         FUNDAMENTALS  OF  SUBMI C  ROSCOPI C  MORPHOLOGY  I 

is  inserted  into  the  polarizing  microscope.  It  is  in  the  form  of  a  sliding 
wedo-e,  or  a  flat  plate  which  can  be  tilted  so  that  its  thickness  d  is  variable. 
Since  the  light  oscillating  parallel  to  the  direction  of  the  minor  refractive 
index  of  a  double  refracting  specimen  passes  faster  through  the  object  than 
the  beam  oscillating  in  the  perpendicular  direction  parallel  to  the  major 
refractive  index,  a  path  difference  of  these  two  beams  results,  which  causes 
the  interference  colours  observed  in  the  polarizing  microscope.  This 
retardation  can  be  diminished  if  the  direction  of  the  major  refractive  index 
of  the  specimen  is  oriented  parallel  to  the  minor  index  of  the  compensator. 
By  varying  the  thickness  of  the  compensator,  the  retardation  of  the  specimen 
can  be  counterbalanced,  until  the  colours  disappear  completely.  Then  the 
double  refraction  is  compensated  and  the  value  of  F  can  be  read  from  the 
compensator.  For  delicate  measurements  there  are  compensators  which 
permit  determination  of  the  phase  difference  y  of  the  two  beams.  Then  the 
readings  must  be  multiplied  by  the  wavelength  A  of  the  monochromatic 
light  used,  or  by  A  =  550  m/x  for  white  light. 

The  formula  mentioned  above  applies  to  objects  bounded  by  two 
parallel  planes  as,  e.g.,  in  microtome  sections,  where  d  corresponds  to 
the  thickness  of  the  section.  Many  biological  objects,  however, 
(myelin  tubes,  myelin  sheath  of  the  nerves,  fibres  with  narrow  lumen, 
etc.)  occur  in  the  form  of  hollow  cylinders.  In  this  case  the  thickness 
becomes  greater  with  increasing  distance  from  the  edge,  and  according- 
ly the  path  difference  increases.  The  phenomena  are  particularly  com- 
plicated when  the  optical  axis  is  not  parallel  to  the  axis  of  the  cylinders 
as  in  fibres,  but  perpendicular  to  the  cylinder  axis,  as  is  the  case  of 
myelin  objects.  The  birefringence  An.  may  then  be  calculated  from 
a  formula  of  Bear  and  Schmitt  (1936)  if  the  largest  possible  path 
difference  /^(max)  is  measured.  This  formula  runs : 

(d,  +  zda)  arc  cos  [(d^  +  2d2)/3di] 

where  d^  represents  the  diameter  and  d.,  the  inner  diameter  of  the 
hollow  cylinder. 

A  similar  problem  occurs  in  the  determination  of  the  double  re- 
fraction of  objects  with  spherite  texture  and  radially  oriented  optical 
axis  Ce.g.,  grains  of  starch).  In  this  case  the  double  refraction  is 

1  (max) 


An 


1. 122  r 


where  r  is  the  radius  of  the  spherites  (Frey-Wyssling,  1940b).  Bear 
and  Schmitt's  formula  should  yield  this  value  for  a  solid  cvHnder, 


4 


STUDIES  IN  GELS 


87 


where  d.^  =  o.  This  is  not  so,  however,  because  empirical  data  con- 
cerning the  position  of  the  maximum  retardation  /"(max)  in  a  myelin 
tube  have  been  mixed  up  with  the  optical  theory  (Schmitt  and  Bear, 

^937)- 

S2gn  of  the  double  refraction.  The  sign  or  character  of  the  double  re- 
fraction is  a  very  important  datum  for  the  textural  analysis  of  gels.  A 
micellar  texture  is  called  optically  positive  if,  as  mentioned  before, 
n,,  —  nj_  has  a  positive  value.  If,  on  the    other   hand,   n„  —  nj^ 


is 


smaller  than  zero,  the  double  refraction  is  negative.  The  refractive 
index  n,,  always  refers  to  a  direction  which  in  some  way  or  other 
is  of  a  special  character :  direction  of  the  orientation  in  the  composite 
l)odies  mentioned,  direction  of  growth,  direction  of  pressure  or  ten- 
sion, direction  of  flow,  special  morphological  direction,  and  so  on. 
In  fibres  and  threads,  for  example,  the  fibre  axis  is  the  reference  axis, 
in  cross-sections  of  parenchyma  cells  the  tangential  direction,  in 
spherites  the  radial  direction. 

The  character  of  the  birefringence  of  gels  is  indicated  by  the  so- 
■caUed  index  ellipsoid,  the  long  axis  of  which  corresponds  to  the  larger 
index  ny,  while  the  short  axis  corresponds  to  the  smaller  index  na. 
The  direction  of  ny  is  determined  by  comparison  with  a  selenite 


1 

[ 

I 

J    - 

1 

r-" 
-  .  L  J  J-L 

o) 


+ 


b) 


\  I 

ii 

I 

-i- 


CI^ 


d) 


+ 


+ 
f) 


T-ig.  63.  Optical  character  of  gels.  Reference  axis  marked  by  a  dotted  line,  a)  Rodlet 
■composite  body,  b)  layer  composite  body,  c)  thread  of  gum  arable,  d)  thread  of  cherry  gum, 
;)  section  across  a  vegetable  parenchyma  cell  (reference  axis   =  tangential  direction), 
/)  starch  grain  (reference  axis  =  radial  direction). 


plate  (see  Ambronn  and  Frey,  1926).  The  orientation  of  the  index 
■ellipsoid  and  the  direction  to  which  the  double  refraction  refers  have 
heen  drawn  in  Fig.  63,  Many  gels  are  isotropic  when  observed  in  the 
direction  of  the  reference  axis;  they  are  uniaxial  in  the  crystallographic 


88        FUNDAMENTALS  OF  SUBMIC  RO  SCOPIC  MORPHOLOGY  I 

sense,  and  the  definition  of  optically  positive  and  negative  is  in  com- 
plete conformity  with  the  terminology  customary  in  mineralogy.  In 
those  cases,  however,  where  the  object  shows  anisotropic  behaviour 
towards  Hght  incident  along  the  reference  axis,  crystal  optics  use 
other  definitions  to  describe  the  optical  character,  and  the  customary 
terminology  in  gels  is  no  longer  identical  with  that  in  crystal  optics. 
Whenever  there  exists  a  direction  of  isotropy,  this  should  be  chosen  as 
reference  axis. 

Systematics  of  double  refraction.  In  most  cases  the  micellar  texture  itself 
is  birefringent,  because  the  chain  molecules  constituting  the  strands 
of  the  structure  are  themselves  anisotropic.  This  kind  of  optical  aniso- 
tropy  is  called  intrinsic  double  refraction.  In  this  case  the  double  refrac- 
tion of  the  gel  cannot  be  reduced  to  zero  by  changing  the  refractive 
index  n^  of  the  imbibition  Hquid;  there  is  a  residual  double  refraction 
in  the  minimum  of  the  curve  for  form  birefringence :  the  intrinsic 
double  refraction  of  the  substance.  In  all  cases  examined  so  far,  the 
micellar  strands  behave  like  optically  uniaxial  systems,  or  at  any  rate  at 
a  first  approximation.  They  possess,  therefore,  two  principal  refractive 
indices,  designated  by  n^  (extraordinary  index  along  the  fibre  axis)  and 
no  (ordinary  index  perpendicular  to  the  fibre  axis).  The  intrinsic  double 
refraction  is  accordingly  ng  —  no.  As  a  rule  it  is  positive,  but  sometimes 
turns  out  to  be  negative.  In  those  cases  where  the  intrinsic  double 
refraction  is  different  from  zero,  the  refractive  index  n^  in  Wiener's 
formula  is  to  be  replaced  by  the  average  value  \  (ng  +  no)  or,  better 
still,  by  \  (ng  +  2no). 

Both  types  of  form  birefringence  (positive  composite  bodies 
with  rodlet  texture  and  negative  composite  bodies  with  layer  texture) 
may  be  combined  with  the  three  possibilities,  positive,  negative  and 
zero  intrinsic  double  refraction.  On  the  whole  one  can,  therefore, 
distinguish  between  six  types  of  double  refraction  (Frey,  1924). 

Both  the  form  and  the  intrinsic  birefringence  can  be  attributed 
to  the  structure  of  the  object,  but  the  intrinsic  double  refraction  is 
caused  by  the  much  finer  structure  of  the  crystal  lattice,  whereas  the 
form  birefringence  results  from  the  coarser  colloid  structure.  Hence 
the  latter  is  as  a  rule  smaller  than  the  former. 

The  intrinsic  and  the  form  double  refraction  are  both  due  to 
morphological  properties,  in  contrast  to  the  phenomenon  of  incidental 
double  refraction^  which  becomes  apparent  when  solid  objects  are  sub- 


4  STUDIES  IN  GELS  89 

ject  to  tensions  or  pressures;  the  designation  is,  therefore,  double 
refraction  due  to  tension  or  tension  double  refraction.  This  phenomenon 
accompanies  elastic  deformation  (photo-elastic  effect),  and  elastic  de- 
formability  is  a  condition  for  its  occurrence.  Since,  according  to 
definition,  gels  actually  do  possess  this  property  (Table  X,  p.  74), 
effects  of  this  kind  are  to  be  expected  in  gels  exposed  to  stress.  The 
tension  double  refraction  is  usually  positive  with  respect  to  the 
axis  of  deformation,  while  that  due  to  pressure  is  usually  negative.  The 
effect  is  most  pronounced  in  isotropic  gels  (e.g.,  strain-free  gelatin), 
but  is  of  course  also  observed  in  gels  which  are  anisotropic  by  nature 
if  these  are  exposed  to  tensions,  in  which  case  it  is  superposed  on  the 
pre-existing  textural  and  intrinsic  double  refraction.  On  removal  of  the 
stress,  the  tension  double  refraction  must  disappear,  as  with  every 
really  elastic  phenomenon.  If  it  does  not,  the  object  has  been  plastically 
deformed.  The  photo-elastic  effect  is  due  to  the  deformation  of  elec- 
tron orbits  in  the  material  concerned ;  the  distances  between  the  atoms 
in  this  material  are  slightly  increased  or  decreased.  In  cubic  crystal 
lattices  insignificant  changes  in  atomic  distances  cause  considerable 
optical  anisotropy  (Wiener,  1926b). 

Orientation  double  refraction.  The  junction  bonds  in  a  gel  being  seldom 
very  strong,  they  easily  yield  to  the  forces  apphed.  The  elastic  deforma- 
tion is  then  followed  by  a  re-orientation  of  the  micellar  strands,  thus 
intensifying  the  intrinsic  and  textural  birefringence  of  the  gel.  For  this 
reason  the  optical  phenomena  in  gels  exposed  to  stress  are  often  very 
comphcated.  The  difference  between  the  double  refraction  due  to 
tension  and  that  due  to  orientation  is  most  obvious  when  these 
phenomena  are  different  in  sign,  as  for  example  in  the  basic  experi- 
ments of  Ambronn  (1889)  with  cherry  gum.  For,  when  cb^rry  gum 
is  stretched,  the  transient,  weakly  positive  double  refraction  resulting 
from  the  tension  is  followed  by  a  negative  double  refraction  due  to 
the  orientation  of  the  micellar  texture. 

D'stribution  of  orientations.  In  a  stretched  gel,  the  directions  of  the 
micellar  units  are  spread  about  the  reference  axis  according  to  a  com- 
plicated distribution  function  (Kratky,  1933,  1938).  The  majority 
of  micellar  strands  enclose  small  angles  with  the  direction  of  the 
stretch,  and  only  few  of  them  enclose  large  angles  with  this  direction. 
The  distribution  function  depends  on  the  degree  of  stretch.  If  this 
strain  is  unknown,  however,  an  idealized  scheme  of  the  distribution 


90        FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

can  be  made  by  assuming  that  within  a  certain  angle  all  possible 
orientations  about  the  reference  axis  occur  with  equal  frequency.  The 
assembly  of  orientation  then  forms  a  sector  (in  a  plane)  or  a  cone  (in 
space),  whose  vertical  angle  a  can  be  computed  from  the  double  re- 
fraction of  the  gel  when  the  intrinsic  double  refraction  ng  —  n^,  of  the 
micellar  strands  is  known,  provided  that  by  judicious  choice  of  the 
imbibition  liquid  negligible  form  birefringence  is  assured.  The  angle 
of  scattering  a  is  then  given  by  the  following  simple  relation  (Frey- 
Wyssling,  1945): 

r  ■       ■         ,         A  ,  .  sin  2a 

for  scattering  in  a  plane  Zln  =  (ng  —  nj 


for  scattering  in  space  An  =  (n^  —  nj 


2a 
COS  a  +  cos'^a 


For  example,  the  space  angle  in  cellophane  paper,  referred  to  the 
preference  direction,  imposed  by  the  manufacturing  stress,  was  found 
to  be  71°.  5.  The  anisotropy  of  cellophane  is,  therefore,  rather  strong, 
for  the  angle  of  scattering  corresponding  to  the  isotropic  state,  i.e., 
completely  uniform  distribution,  would  have  been  90°.  The  micellar 
strands  with  their  numerous  orientations  in  space  may  be  replaced 
by  a  gel  in  which  only  a  single  orientation  occurs.  This  orientation 
angle  is  called  the  average  orientation  angle  am-  With  the  assumptions 
made  by  us  am  becomes  ^a,  as  shown  by  Fig.  64a. 

The  orientation  of  the  strands  in  a  micellar  texture  can  be  brought 
about  by  a  variety  of  means  other  than  tension  or  pressure,  e.g.,  by 
drying  or  freezing  a  gel  (Ambronn,  1891 ;  Ullrich,  1941);  the  strings 
or  strands  of  the  frame  are  then  shifted  into  more  or  less  parallel 
positions. 

Birefringence  of  flow.  The  best-defined  orientation,  however,  is  that 
in  a  field  of  flow,  if  one  succeeds  in  liquefying  the  gel  to  a  sol  by  re- 
leasing the  junctions.  If  such  a  solution  is  subject  to  flow,  the  colloid 
rodlets  are  turned  parallel  at  all  points  where  a  velocity  gradient 
exists.  A  well-defined  velocity  gradient  can  be  obtained  by  introducing 
the  sol  into  a  narrow  gap  (width  below  14  m^i)  between  a  fixed  hollow 
cylinder  and  a  revolving  inner  cylinder  (Signer,  1930,  1933;  Boehm, 
1939;  Frey-Wyssling  and  Weber,  1941).  When  rotating  the  inner 
cylinder,  the  liquid  in  contact  with  the  surface  of  the  rotor  acquires 
its  velocity,  while  the  liquid  in  contact  with  the  wall  of  the  fixed 


STUDIES  IN  GELS 


91 


cylinder  remains  at  rest.  As  shown  by  Fig.  64b,  this  gives  rise  to  a 
velocity  gradient  in  the  gap  and  thus  to  a  force  couple  on  the  rodlets 
dissolved.  This  force  couple,  however,  is  counteracted  by  the  Brown- 
ian  movement  of  the  particles,  which  tends  to  annihilate  the  orienta- 
tion brought  about  by  the  shear.  As  a  result  of  this  competition 


'Z, 


u   \ 


^ 


\: 


y 


b) 


Fig.  64.  a)  Scattering  of  rodlets  when  oriented;  a  angle  of  scattering,  b)  Orientation  of 

rodlets  by  a  gradient  of  flow.  Zj  revolving  inner  cylinder,  Zg  immobile  outer  cylinder, 

d  gap  between  the  two  cylinders,  u  maximum  velocity  of  flow,  u'  velocity'  of  a  rodlet 

which  is  oriented  by  the  velocity  gradient. 


between  orienting  forces  and  Brownian  movement,  the  rodlets  are 
scattered  with  respect  to  the  axis  of  orientation.  The  distribution 
function  of  the  rodlets  is  very  complicated,  but  can  be  derived  from 
theory  (see,  for  instance,  Peterlin  and  Stuart,  1943)-  It  is  found 
that  the  direction  of  the  axis  of  orientation  depends  on  the  length 
of  the  rods.  With  short  rods  (axis  ratio  a:b  ^  i)  the  orientations  are 
spread  about  an  axis  enclosing  an  angle  of  45°  to  the  direction  of  flow. 
With  increasing  length  of  the  rods  (a:b>  i),  the  axis  tends  to  be 
oriented  in  the  direction  of  flow,  finally  (when  a :  b  ->  00)  becoming 
parallel  to  the  tangent  plane  of  the  cylinder.  The  direction  of  the  axis 
of  orientation  can  be  ascertained  in  the  polarization  microscope  by 
the  direction  of  extinction.  The  extinction  angle  therefore  provides  in- 
formation as  to  the  length  of  the  micelle  rodlets  or  macromolecules 


02        FUNDAMENTALS  OF  SUBMICROSCOPI C  MORPHOLOGY  I 

dissolved,  since  short  particles  give  extinction  angles  of  about  45°, 
whereas  filaments  give  angles  near  0°. 

Having  determined  the  extinction  angle,  one  can  also  measure  the 
retardation  (technical  notes  in  Wissler,  1940,  and  historical  review  in 
PiLNiK,  1946). 

The  birefringence  of  flow  is  not  a  constant  as  is  the  double  refraction 
of  crystals,  because  the  retardation  does  not  only  depend  on  the  thick- 
ness of  the  layer,  but  also  on  the  velocity  gradient  and  the  viscosity, 
and  on  the  concentration  of  the  solution.  All  these  variable  quantities 
are  combined  in  Maxwell's  constant,  by  which  the  anisotropy  of 
flow  of  different  sols  can  be  characterized  and  compared.  With  sols 
in  which  the  particles  of  the  solute  are  chain  molecules  (molecular 
colloids),  the  method  can  be  used  to  obtain  data  on  the  anisotropy 
of  single  macro  molecules. 

In  the  case  of  single  chain  molecules  we  can  no  longer  speak  of 
refractive  indices,  since  the  surface  of  a  molecule  does  not  represent  a 
phase  boundary  where  the  velocity  of  propagation  of  light  is  changed 
by  a  definite  amount.  The  optical  properties  of  the  molecules  are 
therefore  characterized  by  another  quantity,  designated  as  optical 
polan':(ability, -which,  is  a  measure  for  the  influence  of  the  electromagnet- 
ic field  of  a  fight  wave  on  the  orbits  and  oscillations  of  the  electrons 
in  the  molecule.  This  influence  depends  on  the  direction  of  vibration 
of  the  fight,  and  in  a  rod-shaped  molecule  with  rotational  symmetry 
we  must  therefore  distinguish  two  different  principal  polarizabifities, 
the  one  parallel  and  the  other  perpendicular  to  the  mohcule  axis,  in 
the  same  way  as  we  must  distinguish  two  principal  refractive  indices  in 
an  optically  uniaxial  crystal. 

More  than  once  the  question  has  arisen  (e.g.,  Schmidt,  1938)  as  to 
whether  chain  molecules,  like  micellar  strands,  cause  rodlet  bire- 
fringence when  they  are  in  parallel  alignment.  This  problem  has  been 
solved  by  Sadron  (1957).  It  follows  from  the  theory  developed  by 
him  that  the  formula  for  the  double  refraction  of  flow  consists  of  two 
parts.  The  first  part  depends  only  on  the  polarizabifity  of  the  molecule 
(compare  intrinsic  double  refraction),  whereas  the  second  part  contains 
also  the  influence  of  the  shape  of  the  particles  (compare  form  bire- 
fringence). In  contrast  to  the  conditions  prevailing  in  micellar  systems, 
however,  both  terms  depend  on  the  refractive  index  of  the  solvent 
(Snellman  and  BjornstAhl,  1941). 


4  STUDIES  IN  GELS  93 

The  birefringence  of  flow  has  furnished  arguments  in  favour  of  the 
view  that  the  micellar  strands  of  protein  gels  are  beaded  chains  (Fig.  51a, 
p.  66).  A  flowing  solution  of  1.5%  gelatin  is  isotropic  at  40°  C.  This  is  an 
indication  that  this  sol  contains  globular  protein  molecules.  When  cooled 
down  to  20°  C.  the  gelatin  sets  after  some  time.  During  the  incipient 
gelification  the  solution  becomes  birefringent  owing  to  the  formation  of 
micellar  strands.  The  extinction  angle  of  the  double  refraction  of  flow 
permits  calculation  of  the  length  of  the  elongated  particles  in  such  a  gel 
solution.  Whereas  a  diameter  of  only  about  50  A  must  be  attributed  to  the 
globular  protein  molecules,  the  measured  chain  length  is  more  than  1000  A 
and  it  increases  steadily  up  to  over  6000  A  before  the  system  solidifies. 
Particles  of  this  length  could  not  possibly  be  formed  by  unfolding  of  the 
polypeptide  chain,  which  is  somehow  coiled  in  globular  protein  molecules ; 
its  cross-section  measuring  about  46  (A)^  (see  p.  365),  its  length  could  not 
exceed  1500  A  when  coiled  in  a  sphere  of  50  A  diameter.  Joly  (1949) 
therefore  concludes  that  the  micellar  strands  result  from  linear  aggregation 
of  globular  macromolecules  forming  beaded  chains.  When  these  have 
become  sufficiently  long,  they  interact  and  a  three  dimensional  network, 
i.e.  a  gel,  is  formed.  This  gel,  containing  1.5%  gelatin  by  volume  and 
micellar  strands  of  50  A  diameter,  must  have  a  relatively  wide-meshed 
network.  Assuming  that  the  beaded  chains  meet  with  the  tetrahedron  angle 
of  109.5°,  the  edges  of  the  polyhedra  which  compose  the  framework  are 
as  much  as  o.i  /x  long. 

The  force  of  aggregation  in  these  gelatin  chains  is  weak.  By  increasing 
the  velocity  gradient  in  the  apparatus  inducing  birefringence  of  flow,  the 
micellar  chains  of  a  gel  solution  of  gelatin  are  shortened  by  rupture.  The 
applied  force  couple  is  of  the  order  of  Van  der  Waals  forces,  an  indication 
that  no  valency  bonds  have  been  formed  between  the  beads  of  the  chain. 
This  is  the  reason  why  a  gelatin  gel  can  be  melted  and  reduced  to  a  sol  by 
simple  heating. 

According  to  Joly  (1949),  the  same  beaded  chains  are  formed  when 
proteins  with  globular  molecules  are  denatured  (seep.  136),  e.g.  when  a 
solution  of  blood  albumin  is  heated.  At  a  certain  temperature  intra- 
molecular bonds  are  loosened  and  become  free  to  replace  the  Van  der 
Waals  bonds  between  the  aggregated  molecules  by  chemical  bonds,  such 
as  hydrogen-,  salt-  or  ester-bonds  (see  p.  145).  Then  the  protein  has  become 
insoluble  and,  therefore,  denatured. 

Similar  observations  have  been  reported  of  ovalbumin  by  Foster 
and  Samsa  (1950).  This  protein  consists  of  relatively  small  globules 
(Fig.  2,  p.  11)  which  can  be  unfolded  by  a  high  flow  gradient  to 
sinuous  chains  of  600  A  length.  But  this  occurs  only  when  the  con- 
centrations are  low  (<  0.6%).  Particles  of  2000  A  length  have  been 
measured  in  more  highly  concentrated  solutions  (2.4%).  Such  lengths 


94 


FUNDAMENTALS  OF  SUBM IC  RO  S  C  OP  I C  MORPHOLOGY 


are  only  possible  if  several  particles  aggregate.  It  is  unlikely,  however, 
that  the  aggregation  affects  fully  folded  globular  particles;  probably 
they  become  deformed  and  partly  unfolded  by  the  flow  gradient,  so 
that  somewhat  expanded  macromolecules  aggregate. 

Micellar  textures.  Some  examples  will  demonstrate  the  results  ob- 
tained so  far  in  the  optical  structure  analysis  of  gels  (Frey- Wyssling, 
1930).  The  majority  of  gels  to  be  considered  possess  a  micellar  frame- 
work containing  regions  of  lattice  order  with  rod-shaped  crystals.  In 
the  following  schemes  these  are  indicated  by  dashes,  although  it 
should  be  remembered  that  these  lattice  regions  do  not  represent 
isolated  dispersed  particles  but  that  they  are  all  interlinked  and  inter- 
woven by  chain  molecules. 

When  it  has  been  ascertained  by  a  combination  of  optical  results  and 
X-ray  analysis,  or  birefringence  of  flow,  that  the  rod-shaped  lattice 
regions  or  the  chain  molecules  are  optically  positive  with  respect  to  the 
longitudinal  axis,  the  orientation  of  the  lattice  regions  can  be  derived 
from  the  character  of  the  double  refraction  in  various  sections  of  the 
gel.  This  can  be  demonstrated  in  particular  in  the  case  of  all  walls  ot 
anisodiametric  plant  cells.  As  shown  in  Fig.  65,  the  orientation  of 
the  lattice  regions  is  indicated  by  the  arrangement  of  index  ellipsoids 
in  radial,  tangential  and  cross-section. 

In  the  secondary  wall  of  a  bast  fibre  the  lattice  regions  run  almost 
parallel  to  the  axis  {fibre  texture.  Fig.  65a).  If  their  orientations  are 
scattered  with  respect  to  the  cell  axis,  the  cross-section  which  in  the 
first  case  is  almost  isotropic  becomes  birefringent ;  we  obtain  a  fibroid 
texture  (Fig.  65b).  The  counterpart  of  the  fibre  texture  is  the  ring 
texture  (Fig.  65c),  in  which  all  lattice  regions  run  in  tangential  orienta- 
tion. This  texture  occurs  in  the  ring-shaped  reinforcements  of  young 
vascular  cells.  If,  starting  from  this  texture  which  is  optically  negative 
with  respect  to  the  cell  axis,  the  lattice  regions  are  allowed  to  scatter, 
the  widespread  tube  texture  is  obtained  (sieve  tubes,  latex  tubes,  vessels, 
elongated  parenchyma  cells,  etc.).  Here  the  tangential  section  is  optic- 
ally negative;  the  radial  section,  however,  is  positive,  since  all  projec- 
tions of  the  scattered  rod-shaped  lattice  regions  upon  the  radial  section 
are  approximately  parallel  to  the  axis.  As  there  is  a  continuous  change 
from  the  negative  region  to  the  positive  one,  a  front  view  of  these 
cells  will  show  an  isotropic  zone  in  which  the  two  regions  of  opposite 
sign  become  merged  (Fig.  65d). 


STUDIES  IN  GELS 


95 


If  the  lattice  regions  do  not  scatter,  but  deviate  from  the  direction 
of  the  cell  axis  while  remaining  parallel  to  each  other,  a  spiral  texture 
is  obtained,  as  occurs  in  cotton  wool  fibres,  the  tracheids  of  conifers 
(Jaccard  and  Frey,  1928;  Preston,  1934,  1946)  and  the  wood  fibres 
of  deciduous  trees. 


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Fig.  65.  Micellar  textures  of  cell  walls  (from  Frey-Wyssling,  1930). 
a)  Fibre  texture,  b)  fibroid  texture,  c)  ring  texture,  d)  tube  texture,  n^  biggest, 
Hq  smallest  refractive  index  of  cellulose;  n^*  biggest,  n^*  medium,  n^* 
smallest  refractive  index  of  the  cell  wall,  i  isotropic,  -f  optically  positive, 

—  optically  negative. 


In  isodiametric  objects  there  exists  no  morphological  axis  which  may 
serve  as  reference  axis  to  the  double  refraction.  In  spherical  objects 
such  as  starch  grains,  spherites  and  the  like,  the  radial  direction  is 
therefore  chosen  as  reference  axis.  If  the  refractive  power  for  vibrations 
parallel  to  this  axis  is  larger  than  that  for  vibrations  in  a  tangential 
direction,  the  spherite  texture  is  called  positive;  in  the  opposite  case 
it  is  called  negative.  The  determination  of  the  optical  character  of  a 
spherite  built  up  of  chain  molecules  or  rod-shaped  lattice  regions, 
however,  does  not  sufhce  to  derive  its  submicroscopic  texture.  For, 


96    FUNDAMENTALS  OF  S  UBM  I  C  RO  SC  OP  I  C  MORPHOLOGY      I 

as  shown  in  Fig.  66,  spherites  can  be  positive  or  negative  both  with 
radial  and  with  tangential  arrangement  of  the  structural  elements, 
depending  on  whether  the  structural  elements  themselves  are  positive 
or  negative  with  respect  to  their  axis. Hence  the  first  thing  to  ascertain 
is  the  optical  character  of  these  structural  elements.  In  most  cases  the 
texture  is  as  shown  in  Fig.  66a  (starch  grains,  inulin). 


Fig.  66.  Gels  with  spherite  texture,  a)  Positive,  b)  negative  spherite  of  positive 

rodlets;  c)  negative,  d)  positive  spherite  of  negative  rodlets;  e)  positive  myelin 

sphere  (oblate)  of  positive  rodlets. 

In  hollow  spheres,  the  reference  axis  cannot  be  determined  with 
certainty.  In  isodiametric  parenchyma  cells,  for  instance,  the  double 
refraction  of  the  cell  wall  is  referred  to  the  tangential  direction  (Fig. 
63e,  p.  87),  in  analogy  to  the  situation  in  anisodiametric  cells, 
although  they  are  isotropic  in  radial  direction.  This  is  due  to  random 
orientation  of  the  structural  elements  in  the  tangent  plane.  An  arrange- 
ment of  this  kind  is  designated  as  foliate  texture.  For  further  details 
of  optical  texture  analysis  we  must  refer  to  the  literature  concerned 
(Ambronn  and  Frey,  1926;  Frey,  1926b;  Frey-Wyssling,  1930, 
1935a;  Schmidt  1934,  1937a). 

b.  X-ray  Analysis  of  Gels 

Micellar  strands.  A  complete  structural  analysis  by  means  of  X-rays 
is  only  possible  if  crystalline  lattice  regions  are  present.  In  the  case  of 
molecular  colloids  such  as  rubber  solutions,  protein  solutions,  etc., 
irradiation  with  monochromatic  X-rays  furnishes  as  a  rule  no  more 
than  an  "amorphous"  ring,  which  gives  some  information  about  the 
/«/ramolecular  periods  occurring  most  frequently  (for  instance,  in 
rubber:  the  length  of  an  isoprene  unit).  Only  when  the  chain  molecules 
are  arranged  in  a  crystal  lattice  does  X-ray  analysis  yield  interference 
phenomena  rich  in  lines  or  spots,  from  which  far-reaching  morpholog- 
ical conclusions  can  be  derived.  This  will  be  further  illustrated  bv 


4  STUDIES  IN  GELS  97 

means  of  the  cellulose  diagram  of  ramie  fibre  (Fig.  67),  Each  point  on 
the  diagram  corresponds  to  a  set  of  parallel  net-planes  in  the  crystal 
lattice.  The  diagram  of  Fig.  67  enables  us  to  measure  four  quantities : 
I.  the  mutual  distances,  2.  the  density,  3.  the  breadth  and  4.  the 
arrangement  of  interferences,  each  of  which  permits  calculation  of 
a  corresponding  quantity  in  the  undisturbed  lattice  regions. 


Fig.  67.  X-ray  fibre  diagram  from  ramie  showing  lavcr  lines. 

I.  According  to  Bragg's  law  of  reflexion,  the  distance  between  the 
lattice  planes  is  calculated  from  the  distance  between  the  interferences 
and  the  centre  of  the  diagram.  We  learn  from  X-ray  optics  how  the 
unit  cell  (see  p.  26)  in  the  crystal  lattice  of  cellulose  can  be  computed 
from  the  distances  measured  in  the  diagram  of  artificially  oriented 
cellulose  preparations  whose  crystalline  regions  display  an  arrangement 
of  even  higher  orientation  than  in  ramie  fibres.  The  elementary  cell 
found  for  crystalline  cellulose  is  monoclinic;  its  sides  are  a  =  8.35  A, 
b  =  10.3  A,  c  =  7.9  A,  and  the  angle  ^  between  a  and  c  is  84°  (Meyer 
and  Mark,  1930).  Of  these  quantities,  the  most  accurately  determined 
is  the  fibre  period  b  which  corresponds  to  the  length  of  a  cellobiose 
molecule  (Fig.  68).  It  is  calculated  from  the  distances  between  the 
so-called  layer  Hnes  which  are  clearly  visible  in  Fig.  67,  running  parallel 
to  the  equator  of  the  diagram  and  connecting,  as  it  were,  the  inter- 
ference spots.  These  interference  spots  are  broadened  along  the  layer 


q8      fundamentals  of  submicroscopic  morphology  I 

lines  as  a  result  of  cellulose  chains  which  do  not  belong  to  the  crystal 
lattice  (Sauter,  1937). 

2.  From  the  density  of  interferences  the  number  of  atoms  in  the  net- 
planes  can  be  derived,  since  the  lattice  planes  reflect  the  X-rays  more 
intensely  in  proportion  as  they  contain  more  atoms.  The  density  of 
interferences  can  be  estimated,  or  measured  photometrically.  In  Fig. 

67  two  black  spots  can  be  seen  on 
the  equator,  with  a  mutual  distance 
of  261/4  i^rn.  Their  great  density  is 
caused  by  the  family  of  net-planes 
which  contain  the  glucose  rings  of 
the  cellulose  chains  and,  as  both 
points  correspond  to  the  front  plane 
of  Fig.  68,  the  ring  of  the  glucose 
units  must  lie  in  this  plane.  In  this 
way  it  is  possible  from  the  intensity 
of  the  interferences  to  determine 
the  orientation  of  the  molecular 
models  (obtained  on  structural 
chemical  grounds)  in  the  unit  cell 
(derived  from  X-ray  analysis). 

3.  From  the  breadth  of  the  interferences  one  can  calculate  the  width  of 
the  undisturbed  lattice  regions,  using  a  method  developed  by 
Scherrer  (1920)  for  metals,  i.e.,  substances  absorbing  X-rays,  and 
worked  out  by  Laue  (1926)  for  non-absorbing  substances.  To  do  this 
the  density  must  be  measured  photometrically.  The  breadth  at  half- 
maximum  of  the  density  peaks  in  the  photometer  curve  (Fig.  70,  p.  102) 
is  a  measure  of  the  dimension  of  the  crystalline  regions  perpendicular 
to  the  set  of  net-planes  causing  the  interference.  The  broader  the  X- 
ray  interference  in  the  diagram  is,  the  smaller  is  this  dimension.  In 
Fig.  67  the  interference  spots  on  the  equator  are  clearly  broader  than 
those  near  the  poles  of  the  diagram.  It  follows  from  this  that,  in  the 
fibre,  the  dimensions  of  the  lattice  regions  are  considerably  smaller  in 
directions  perpendicular  to  the  fibre  axis  than  in  directions  parallel 
to  this  axis.  They  must,  therefore,  be  rod-shaped,  in  conformity  with 
the  conclusion  drawn  from  the  character  of  their  form  birefringence. 
Hengstenberg  and  Mark  (1928)  find  50-60  A  for  the  thickness  of 
these  rodlets.  Their  length  cannot  be  measured  accurately,  the  for- 


Fig 


68.  Crystal  lattice  of  cellulose  (from 
Meyer  and  Misch,  1937). 


4  STUDIES  IN  GELS  99 

mulae  being  very  insensitive  to  changes  in  lengtii  when  this  length  is 
large  (Frey-Wyssling,  1937a).  The  experiments  admit  of  no  doubt, 
however,  that  the  length  of  the  rodlets  must  be  more  -  probably  much 
more  -  than  10  times  as  long  as  their  thickness. 

4.  From  the  arrangement  of  interferences  can  be  derived  the  arrangement 
of  the  rod-shaped  lattice  regions.  In  the  diagram  considered  all  rodlets 
are  parallel  to  the  fibre  axis  {fibre  diagram) ;  but  if  they  follow  a  screw 
line  within  the  wall,  the  interferences  on  the  equator  are  drawn  out 
into  sickles  {.uckle  diagram).  Finally,  if  they  lack  all  order,  interference 
rings  instead  of  spots  are  obtained  (Debye-Scherrer  or  ring  diagram^ 
see  Fig.  69,  p.  100).  A  comprehensive  and  simple  treatment  of  the 
relation  between  the  arrangement  of  interferences  and  that  of  lattice 
regions  has  been  given  elsewhere  (Frey-Wyssling,  1935a,  p.  11). 
Ring,  sickle  and  fibre  diagrams  are  represented  in  Fig.  75,  p.  106). 

Working  out  the  fibre  diagrams  in  full  detail  from  the  four  points 
of  view  mentioned,  one  arrives  at  the  structural  model  of  the  fibre  wall 
shown  in  Fig,  59b  (p.  77).  This  picture  renders  all  the  facts  which  can 
be  ascertained  by  means  of  X-rays,  though  the  rodlets  are  in  reality 
much  thinner. 

When  drawing  such  a  scheme  it  should  always  be  borne  in  mind 
that  X-ray  analysis  only  gives  information  about  the  regions  of  lattice 
order;  no  information  can  be  obtained  in  this  way  about  the  regions 
without  lattice  structure.  In  particular,  it  cannot  be  decided  by  means  of 
X-rays  whether  the  chain  molecules  in  the  crystal  lattice  are  of  exactly 
the  same  length  as  the  lattice  regions  or  whether  (as  has  already  been 
mentioned)  they  protrude  from  these  regions  without  order  and  invade 
several  other  lattice  regions  (Fig.  54,  p.  70).  X-ray  analysis  therefore 
tells  us  nothing  about  the  manner  in  which  the  crystalline  regions  are 
interlinked  or  about  the  interstices  between  the  regions  of  lattice  order. 
From  a  biological  point  of  view,  however,  these  intermicellar  spaces 
are  of  special  importance.  For,  in  most  substances  possessing  a  frame- 
work, the  micellar  strands  with  their  crystalline  regions  are  to  be  con- 
sidered as  virtually  lifeless,  while  all  perceptible  processes  of  life 
presumably  take  place  in  the  intermicellar  system.  Thus,  the  mechanical 
properties  of  a  gel  are  determined  in  the  first  place  by  the  micellar 
structure,  whereas  for  all  physiological  questions  (such  as  permeability, 
metabolic  processes,  vital  staining,  etc.)  one  should  study  primarily 
the  intermicellar  regions. 


lOO      FUNDAMENTALS  OF  SUBMI  C  RO  SC  OPI  C  MORPHOLOGY  I 

Intermicellar  spaces.  The  regions  between  the  meshes  of  the  micellar 
framework  may  represent  a  homogeneous  phase  if  they  are  filled  with 
a  uniform  liquid  or  gas.  This  only  holds  good  so  long  as  the  gel  frame 
consists  of  strands  which  can  themselves  be  considered  as  a  phase,  so 
that  a  phase  boundary  exists.  If  the  strands  become  so  thin,  however, 
as  to  reach  the  dimensions  of  a  chain  with  the  thickness  of  a  single 
molecule,  the  concept  phase  loses  its  significance. 


(ni)       (002)       (022)       (113)       (222) 


Fig.  69.  X-ray  diffraction  pattern  of  a)  ramie  and  h)  %\W  stained  with  gold.  In  addition  to 
the  fibre  diagram,  Debye-Scherrer  rings  of  gold  (m),  (002)  etc.  are  seen  (from  Frey- 

Wyssling,  1937a). 

Information  as  to  the  dimensions  of  the  intermicellar  spaces  in  the 
gel  frame  can  be  obtained  in  various  ways.  If  one  succeeds  in  filtering 
particles  of  known  size  through  a  gel,  the  inference  is  that  the  pores 
are  bigger  than  the  particles,  as  in  an  ultrafilter.  Unfortunately,  how- 
ever, it  is  not  possible  to  obtain  absolute  values  of  the  pore  size  of  the 
intermicellar  spaces  with  the  aid  of  ultrafiltration  (Czaja,  1950),  since 
differences  in  electric  charge  or  in  chemical  behaviour  (hydrophoby) 
very  strongly  influence  the  ease  with  which  filtration  of  the  particles 
takes  place  (Morton,  1935).  For  this  reason,  only  relative  sizes  can  be 
obtained,  which  cannot  be  compared  with  the  absolute  values  deter- 
mined by  means  of  X-rays. 

Until  now  it  has  not  been  possible  to  obtain  X-ray  diagrams  of  the 
intermicellar  substances ;  for,  even  when  in  the  solid  state,  they  do  not 
usually  show  the  properties  of  crystals.  In  the  plant  cell  wall,  e  g.. 


4  STUDIES  IN  GELS  lOI 

silicic  acid,  lignin,  etc.  are  embedded  in  the  amorphous  state  and  there- 
fore do  not  produce  X-ray  interferences.  For  this  reason  the  amount 
of  space  occupied  by  the  intermicellar  regions  in  frameworks  was 
totally  unknown.  To  obtain  information  in  this  important  field, 
foreign  substances  must  be  introduced  into  these  spaces,  where  they 
crystallize  and  can  then  be  submitted  to  X-ray  analysis  (Frey-Wyss- 
LiNG,  1937a).  We  must  therefore  create  by  artificial  means  an  inter- 
micellar substance  possessing  lattice  order,  which  enables  us  to  derive 
quantitative  data  of  the  dimensions  of  the  unknown  submicroscopic 
regions.  Gold  and  silver  crystals  have  proved  to  be  the  most  suitable 
for  this  purpose.  Following  Ambronn,  the  objects  are  soaked  in 
1-2",',  solutions  of  gold  chloride  or  silver  nitrate,  then  carefully  dried 
with  blotting  paper  and  finally  the  salt  absorbed  is  reduced  by  means 
of  light  or  hydrazin  hydrate  (Frey,  1925).  In  this  way  microscopically 
homogeneous  colourings  are  obtained  displaying  a  beautiful  di- 
chroism  (compare  Ambronn  and  Frey,  1926,  coloured  table;  Wiener 
1926a).  The  X-ray  diagram  of  the  dyed  fibres  shows  Debye-Scherrer 
rings  of  crystalline  silver  or  gold  (Fig.  69)  in  addition  to  the  fibre 
diagram  of  the  framework  substance  (ramie  fibre,  silk  and  wool).  The 
annular  interferences  prove  that  the  metal  crystallites  imbedded  take 
up  all  possible  positions  with  respect  to  the  fibre  axis.  The  size  of  the 
cubic  gold  and  silver  crystals  is  calculated  from  the  breadth  at  half- 
maximum  of  the  interference  rings  (Fig.  70). 

The  investigation  produced  the  surprising  evidence  that  metal 
crystallites  with  a  cross-section  of  about  50  A  are  incrusted  in  silk 
and  wool,  and  particles  even  exceeding  a  diameter  of  100  A  in  ramie 
fibres  (Table  XII).  Since  the  strands  of  the  micellar  framework  in  ramie 
fibres  have  a  thickness  of  only  50  A,  the  artificially  embedded  metal 
crystallites  occupy  an  unexpectedly  large  space.  Notwithstanding  their 
great  strength,  cellulose  fibres  must,  therefore,  be  built  rather  loosely, 
a  fact  which  was  already  known  from  density  measurements  in  the 
bleached  fibres  used  in  these  experiments.  After  removal  of  all  foreign 
substances,  the  density  of  ramie  fibres  amounts  to  only  1.39,  whereas 
the  density  of  cellulose  is  1.59.  There  should  therefore  be  about  12.6% 
of  submicroscopic  empty  space*  (Frey-Wyssling  and  Speich,i942). 

^  The  density  1.39  ~  0.03  is  derived  from  accurate  determinations  of  mass  and  volume. 
If,  instead  of  the  density  of  crystalline  cellulose,  one  uses  the  density  1.55  of  the  incom- 
pletely crystallized  fibre  measured  in  toluene,  one  finds  a  discrepancy  of  10.3%  . 


102      FUNDAMENTALS  OF  SUBM  IC  ROSC  OP  I  C  MORPHOLOGY 

TABLE  XII 

PARTICLE  SIZE  A    OF  GOLD  AND  SILVER  CRYSTALS 

EMBEDDED  IN  FIBRES 


Metal  embedded 

A  in  A 

Ramie  fibres 

Ag 

85 

Ramie  fibres 

Au 

84 

Hemp  fibres 

Au 

90 

Bamboo  fibres 

Au 

85 

Wool 

Au 

58 

Silk 

Au 

50 

It  is  clear  that  not  all  cellulose  rodlets  with  a  cross-section  of  about 
50  A  can  be  surrounded  by  spaces  100  A  wide,  as  otherwise  the  dis- 
crepancy in  density  would  be  much  greater  still.  Furthermore,  the 
phenomena  of  swelling  require  very  narrow  intermicellar  spaces  of  the 


A 


I 


•/- 


Fig.  70.  Photometer  curve  of  hemp  fibres  stained  with  gold  (the  distance  and  breadth  ot 
the  interferences  are  magnified  2.0  times  as  compared  with  Fig.  69).  From  the  breadth  at 
half-maximum  of  the  dcnsitj'  peaks  the  diameter  yl  of  the  embedded  gold  crystals  can  be 
calculated  (from  Frey-Wyssling,  1937a). 

order  of  10  A,  into  which  the  water  can  penetrate,  pushing  the  cellulose 
rodlets  apart.  In  dyed  ramie  fibres  there  must  therefore  be  two  cate- 
gories of  submicroscopic  spaces,  viz.,  i.  narrow  intermicellar  spaces  of 


4  STUDIES  IN  GELS  105 

the  order  of  magnitude  10  A  which  are  responsible  for  the  phenomena 
of  swelling,  and  2.  wider  capillary  spaces  which  are  accessible  to  dyes 
of  much  larger  dimensions  and  to  the  hardening  substances  Hgnin, 
cutin,  etc.  For  this  reason  they  are  of  primary  importance  technically 
in  the  process  of  dyeing  and  physiologically  in  the  hardening  of  the 
cell  wall.  It  must  be  supposed  that  these  larger  spaces  are  widened  by 
the  growth  of  the  substances  embedded. 

The  capillary  shape  of  the  wider  spaces  can  be  proved  in  the  follow- 
ing way:  in  objects  with  a  well-developed  fibre  texture  the  gold  and 
silver  particles  embedded  give  rise  to  a  strong  rodlet  dichroism 
(Frey-Wyssling  and  Walchli,  1946).  This  is  only  possible  if  the 
isodiametric  metal  crystals  are  arranged  in  rows  or  in  rod-shaped 
aggregates;  i.e.,  the  metal  particles  must  lie  in  pre-formed  submicro- 
scopic  canals.  Even  more  can  be  inferred  from  experiments  with 
silver  amalgam.  If  mercury  is  precipitated  in  the  fibre  from  an  alcoholic 
solution  of  sublimate,  dichroic  colouring  is  obtained  which  does  not 
produce  an  X-ray  diagram,  because  the  mercury  is  present  in  the  Uquid 


Fig.  7 1 .  Oriented  embedding  of  silver  amalgam  in  the  fibre.  In  addition 
to  the  fibre  diagram  of  cellulose  (broad  interference  spots)  a  fibre  dia- 
gram of  silver  amalgam  (narrow  interference  spots)appears  (frompREY- 

Wyssling,  1937a). 

state.  Treating  the  fibres  afterwards  with  a  solution  of  silver  nitrate, 
one  obtains  X-ray  diagrams  showing  interferences  of  silver  amalgam 
(Fig.  71)  in  addition  to  the  diagram  of  cellulose.  The  silver  amalgam 


c.lOO^' 


104     FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

crystallizes  in  submicroscopic  hexagonal  needles  which  all  run  parallel 
to  the  fibres  axis,  for,  instead  of  a  Debye-Scherrer  diagram,  3.  fibre 
diagram  of  silver  amalgam  is  obtained.  This  proves  the  presence  of 
submicroscopic  canals  in  the  fibre. 

It  is  much  more  difficult  to  discover  the  dimensions  of  these  pre- 
formed capillaries,  as  the  size  of  the  gold  crystals  embedded  varies 

with  the  speed  at  which 
they  develop  in  the  capil- 
lary system.  Furthermore, 
the  metal  rodlets  causing 
the  dichroism  are  so  large 
that  they  can  easily  be 
shown  in  the  ultramicro- 
scope  (Fig.  72).  Their  rod- 
shape  is  betrayed  by  the 
different  intensity  of  the 
light  scattered  in  lateral 
irradiation  according  as 
the  vibration  of  the  line- 
arly polarized  light  is  par- 
allel or  perpendicular  to 
the  fibre  axis  (Frey-Wyss- 
LiNG,  1937b).  Consequent- 
ly, the  crystals  or  primary 
particles  measured  by 
means  of  X-rays  must  have  clustered  together  to  form  rod-like  aggregates 
or  secondary  particles  (Fig.  73),  widening  the  capillaries  in  so  doing. 
Whether  this  already  takes  place  while  the  crystals  are  growing,  we 
do  not  know.  Presumably,  however,  they  can  develop  fairly  freely, 
since  they  do  not  acquire  the  rodlet  shape  of  the  capillary  system  until 
they  are  collected  in  the  secondary  crystalline  particles.  We  must  there- 
fore take  it  as  proved  that,  apart  from  the  intermicellar  spaces  in  which 
the  water  penetrates  when  the  fibre  swells,  there  exist  even  larger  pre- 
formed inhomogeneities . 

As  a  consequence,  native  fibres  must  possess  long-shaped  submi- 
croscopic regions  containing  intermicellar  spaces  which  are  only 
accessible  to  small  molecules  such  as  water,  salt  ions  and  iodine.  These 
regions  are  designated  as  microfihrils\  they  possess  a  more  or  less  homo- 


Fig.  72 


Fig-  75 


Fig.  72.  Ramie  tibre  stained  with  silver  in  the  ultra- 
microscope   (from   Frey-Wyssling,    1937b). 
Fig.  73.  Ultrastructure  and  shape  of  the  submicro- 
scopic silver  rodlets  in  the  fibre. 


STUDIES  IN  GELS 


105 


capillary  structure  (Fig.  74a).  In  between  these  microfibrils,  however, 
interfibrillar  capillaries  must  occur  in  the  form  of  wider  canals,  in  which 
larger  molecules  such  as  colloid  dyes  and  incrusting  material  are  de- 
posited. The  porous  system  of  ramie  fibres  is,  therefore,  heterocaplllarj ; 
the  smaller  intermicellar  spaces  (of  the  order  of  10  A)  and  the  larger 
interfibrillar  ones  (of  the  order  of  100  A)  communicate  freely.  The 


1000^  =  0.!fi 


i(ca  10  A  } 
mfca  60  J) 

Ifca  100  ^j 


b) 


o) 


Fig.  74.  Miccllar  structure  of  bast  tibres 
(from  Frey-Wyssling,  1936a,  1937a).  a) 
Longitudinal  and  cross-section;  intermicellar 
spaces  all  similar  (homocapillarity).Z')  Cross- 
section  with  coarser  interfibrillar  and  finer 
intermicellar  spaces  (heterocapillarity); 
microfibrils  f  composed  of  micellar  strands 
m ;  i  intermicellar  spaces,  k  interfibrillar 
capillaries. 


microscopically  visible  fibrils  must  still  contain  both  categories  of 
spaces,  because. as  a  rule  they  can  be  dyed  like  the  whole  fibre,  and 
they  thus  represent  aggregate  bundles  of  the  invisible  submicroscopic 
microfibrils. 

Stretching  experiments.  A  subject  which  has  become  of  special  im- 
portance in  the  study  of  gel  structure  is  the  X-ray  analysis  of  the/)ro^(?j-j- 
of  orientation  in  stretching  experiments.  By  way  of  example  we  shall 
briefly  go  into  the  phenomena  observed  in  stretching  regenerated 
cellulose  fibres  obtained  from  viscose. 

It  is  possible  to  make  isotropic  cellulose  fibres  from  viscose 
(Hermans  and  De  Leeuw,  1937).  The  X-ray  diagram  of  these  fibres 


Io6      FUNDAMENTALS  OF  S  U  B.M  I  C  RO  S  COPI  C  MORPHOLOGY  I 

consists  of  Debye-Scherrer  rings.  If  the  orientation  of  the  micellar 
strands  is  completely  random,  photometric  measurements  show  the 
intensity  round  each  ring  to  be  constant.  If,  now,  the  isotropic  fibres 
are  stretched,  the  micellar  strands  are  oriented.  With  increasing  stretch, 
the  X-ray  diagram  changes  into  a  sickle  diagram  and  finally  into  a  fibre 
diagram  (Fig.  67,  p.  97)  when  orientation  is  complete.  If,  at  a  given 


(1    ) 


hO  1.25  1.62 


f»    .| 


1.89 

Fig.  75.  X-ray  diagram  of  Hermans's  threads,  gradually  stretched.  The  numbers  give  the 
degree  of  stretching   (length  of  stretched  gel/original  length).  (From  Kratky,   1940). 


degree  of  stretch,  one  measures  the  intensity  along  the  interference 
sickles  corresponding  to  the  equator  interferences  in  the  fibre  diagram 
(paratropic  interferences),  the/r^^//^;z(7  with  which  the  different  orienta- 
tions of  the  micellar  strands  occur  can  be  derived  from  the  decline  in 
intensity  from  the  equator  towards  the  poles.  In  fact,  the  intensity 
depends  on  the  number  of  lattice  planes  which  take  part  in  the  reflexion 
of  X-rays.  It  is  possible  in  this  way  to  determine  experimentally  the 
distribution  function  of  the  orientations  of  micellar  axes. 


4  STUDIES  IN  GELS  IO7 

If  the  distribution  were  one  which  covers  a  sector  with  uniform 
density  (Fig.  64a,  p.  91),  as  was  assumed  on  p.  90,  the  sickle  inter- 
ferences would  be  circular  arcs  with  sharp  boundaries,  extending  over 
a  sector  angle  dependent  on  the  angle  of  scattering.  As  shown  by 
Fig.  75,  however,  the  density  in  the  sickle  decreases  very  gradually 
towards  the  poles,  and  the  distribution  function  is  a  very  complicated 
one :  the  micellar  strands  which  enclose  a  small  angle  with  the  direc- 
tion of  the  stretch  are  more  frequent  than  'those  which  form  a  large 
angle  with  this  direction,  and  this  distribution  is  a  function  of  the 
degree  of  deformation  (Hermans,  Kratky  and  Treer,  1941).  In 
order  to  explain  the  distribution  curves  found  experimentally  (in- 
tensity depending  on  angular  distance  from  the  equator),  and  their 
change  with  the  degree  of  stretch,  Kratky  (1940)  has  made  two 
different  assumptions  with  regard  to  gel  structure  and  has  calculated 
how  the  distribution  alters  in  the  stretching  process.  Comparing  these 
theoretical  curv^es  with  those  obtained  experimentally,  it  is  possible  to 
decide  which  of  the  two  hypotheses  is  the  more  likely. 

The  first  limiting  case  considered  by  Kratky  (1935,  1940)  conforms 
to  the  older  ideas  about  gel  structure,  assuming  rod-shaped  "freely 
suspended  micelles",  which  are  independent  of  each  other  (Fig.  59b, 
p.  77).  Their  orientation  in  the  stretching  process  is  achieved,  as  it 
were,  by  the  flow  of  liquid  (swelling  medium)  which  turns  the  rodlets 
distributed  at  random  into  positions  which  are  parallel  to  the  direction 
of  the  stretch.  On  this  assumption  the  distribution  of  the  micellar 
orientations  can  be  calculated  for  any  degree  of  stretch  (=  final  length 
divided  by  original  length  of  the  gel).  Advanced  parallel  arrangement 
of  the  rodlets  is  only  reached  at  high  degrees  of  stretch.  A  number  of 
very  swollen  gels  of  cellulose  esters  (cellulose  amyl  oxalate,  trinitro- 
cellulose)  show  a  behaviour  which  is  in  conformity  with  this  theoret- 
ical distribution. 

On  the  other  hand,  it  seemed  surprising  at  first  that,  in  the  case  of 
relatively  low  degrees  of  swelling  (between  1.5  and  2),  neighbouring 
micelles  do  not  disturb  each  other's  movements  and  behave  according 
to  formulae  which  have  been  derived  for  particles  freely  suspended 
in  a  large  amount  of  liquid.  To  explain  this,  Kratky  (1954)  suggested 
that  the  arrangement  of  micellar  rods  is  not  completely  random,  but 
that  there  must  exist  short-range  order  (i.e.,  order  in  small  regions).  This 
means  that  if  small,  submicroscopic  regions  are  considered,  a  certain 


I08      FUNDAMENTALS  OF  SUBMI  C RO SCOP  I  C  MORPHOLOGY  I 

parallel  arrangement  is  founds  At  some  distance,  however,  the  ar- 
rangement becomes  gradually  more  and  more  disturbed,  so  that  all 
possible  orientations  are  found  in  a  gel  volume  of  even  microscopic 
dimensions.  This  is  shown  by  Fig.  76.  Hence,  when  considering 
the  dispersion  of  orientations  in  Fig.  51b  (p.  66)  or  54a  (p.  70),  it 

must  be  borne  in  mind  that  neighbouring 
particles  are  almost  parallel.  An  entirely  dif- 
ferent orientation  is  only  found  at  a  certain 
submicroscopic  distance  as  a  result  of  grad- 
ual changes  in  orientation.  In  the  much 
larger  microscopic  dimensions  this  means. 


>>\nOa\\  i/i,n\l 


Fig.  76.  Short-range  order  of      however,  that  all  anisotropy  effects  are  neu- 

short  rod  molecules   (from      ^ralized  as  if  a  random  criss-cross  arrange- 
Hermans,  1941).  . 

ment  existed. 

The  principle  of  short-range  order  would  explain  why  it  is  that, 
when  stretched,  gels  of  a  low  degree  of  swelling  can  behave  as  if  their 
particles  were  freely  floating  micelles.  In  fact,  the  movement  of  each 
particle  is  very  similar  to  its  neighbour:  there  is  no  steric  hindrance, 
as  would  be  the  case  if  the  arrangement  were  an  irregular  one.  The 
principle  of  short-range  order  does  not  suffice,  however,  to  explain 
altogether  the  behaviour  of  gels  when  stretched ;  for,  the  extensibility 
of  these  gels  would  have  to  be  unlimited,  and  it  should  be  possible 
to  deform  them  to  fibres  of  arbitrary  length,  even  in  those  cases 
where  the  degree  of  swelling  is  low. 

In  the  cellulose  fibres  mentioned,  prepared  by  Hermans,  this  is  im- 
possible. We  are  therefore  forced  to  assume  that  the  micelles  are  not 
freely  movable,  but  that  they  are  interlinked  by  junctions  (Frey- 
Wyssling  1936a,  1936c)  or  hinges  (Fig.  77).  This  assumption  of 
complete  interlinking  of  the  structural  elements  in  the  gel  is  designated 
by  Kratky  as  the  second  limiting  case.  Here  again,  there  exists  short- 
range  order,  and  the  picture  arrived  at  (Hermans,  1941)  corresponds 
more  or  less  to  the  one  given  by  us  (compare  Fig.  54,  p.  70).  In  other 
words,  the  orientation  takes  place  as  if  chains  consisting  of  rigid  links 
and  movable  but  inextensible  hinges  were  stretched  by  pulling  at  the 

^  The  voluntar\'^  parallel  arrangement  of  rod-shaped  particles  is  not  confined  to  colloid 
raatter.lt  occurs  also  in  pure  liquids  and  real  solutions,  where  physicists  speak  of  short- 
range  order  (Zernike,  1939;  Stuart,  1941;  Peterlin  and  Stuart,  1943).  Taking  an 
arbitrary  molecule,  its  immediate  neighbours  are  more  or  less  orderly  as  regards  distance 
and  orientation. 


STUDIES  IN  GELS 


109 


ends.  On  this  assumption  a  distribution  function  for  the  orientation 
in  network  systems  can  be  derived.  A  striking  result  of  this  theory  is, 
that  a  completely  parallel  arrangement  of  all  micellar  strands  would  be 
reached  at  a  degree  of  stretch  2 
(100%  stretch).  This  is  not  in 
keeping  with  the  observed  facts, 
seeing  that  Hermans's  cellulose 
fibres,  especially  when  greatlv 
swollen,  can  undergo  a  stretch 
of  several  times  100",,.  One 
must,  therefore,  assume  that  in 
reality  neither  the  first  nor  the 
second  limiting  case  is  realized. 
The  behaviour  is  intermediate 
between  those  corresponding  to 
the  two  extreme  cases  calculated, 
i.e.,  the  micelles  are  not  freely 
suspended  but  they  are  inter- 
linked to  form  micellar  strands. 
The  junctions  present,  however, 

are  not  fixed  indis  soluble  hinges  which  completely  prevent  the  micellar 
strands  or  parts  of  these  strings  from  gliding  past  each  other.  In  fact,  the 
cohesion  must  be  due  to  forces  which  at  certain  points  can  be  overcome 
by  the  orientating  forces,  so  that  a  "flow  in  small  regions"  takes  place. 

c.  Swelling  of  Gels 

If  isotropic  gels  are  immersed  in  a  swelling  medium,  they  swell 
uniformly  in  all  directions.  If  a  certain  orientation  of  the  micellar 
strands  prevails,  however,  the  swelling  is  anisotropic,  i.e.,  different 
in  different  directions.  The  anisotropy  of  swelling  of  starch  grains 
induced  Nageli  (1858)  to  consider  the  structural  units  (micelles) 
of  the  gel  as  submicroscopic  rodlets. 

Intermicellar  swelling.  According  to  Nageli,  the  swelling  medium 
penetrates  between  the  rodlets  which  we  now  call  the  micellar  strands. 
In  many  cases  X-ray  analysis  has  confirmed  this  view,  as  often  the 
X-ray  diagram  does  not  change  in  the  swelling  process,  so  that  ap- 
parently the  crystalline  regions  remain  unaltered  (e.g.,  plant  cell  walls 
and  cellulose  gels). 


Fig.  77.  Short-range  order  in  a  gel  of  inter- 
linked micelles  (from  Hermans,  1941). 


no      FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

The  swelling  medium  penetrating  between  the  string4ike  structural 
elements  causes  the  system  to  inflate  laterally.  For  this  reason  swelling 
is  always  at  its  greatest  in  directions  perpendicular  to  the  direction 
of  orientation  of  the  micellar  texture,  and  is  almost  zero  along  the 
fibre  axis  if  the  fibre  texture  is  ideal.  The  arrangement  of  the  micellar 
strands  can  therefore  be  derived  from  the  anisotropy  of  swelling,  or 
conversely,  the  anisotropy  of  swelling  or  shrinking  to  be  expected  can 
be  computed  from  the  optical  anisotropy  measured  (Steinbrinck, 
1906;  Ziegenspeck,  1938). 

If  it  is  assumed  that  the  microfibrils  of  native  fibres,  made  up  of 
polyhedral  micellar  strands,  possess  a  more  or  less  circular  cross- 
section  (Fig.  74b,  p.  105),  these  can  be  idealized  as  circular  cylinders. 
It  is  then  found  that  in  the  completely  dry  state  9.5  per  cent,  by  vol.  of 
intermicellar  empty  spaces  must  occur  between  the  strands  (Hermans, 
1938).  This  value  tallies  approximately  with  the  average  empty  space 
(8.5  %)  obtained  from  determinations  of  double  refraction  and  density 
(Frey-Wyssling  and  Speich,  1942),  showing  that  in  well-dried 
fibres  the  microfibrils  are  fairly  closely  packed.  Gels  in  which  the 
colloid  portion  is  crystalHzed  imperfectly,  so  that  a  large  amount  of 
amorphous  substance  is  present,  swell  much  more  than  well-crystallized 
fibres,  the  swelling  medium  being  able  to  penetrate  into  the  unordered 
regions,  causing  them  to  swell.  Nevertheless  it  does  not  succeed  in 
solvating  the  individual  chain  molecules  in  the  ordered  regions. 

Intramicellar  swelling.  If,  however,  the  affinity  between  the  swelling 
medium  and  the  chain  molecules  is  stronger  than  the  binding  forces 
in  the  chain  lattice,  the  swelling  medium  will  penetrate  into  the  lattice 
and  widen  it.  This  widening  can  be  followed  by  means  of  X-rays  and 
is  often  found  to  abolish  the  interferences.  In  that  case  the  chain 
molecules  are  completely  solvated  and  if  they  are  not  kept  together  by 
valence  bridges  (p.  67),  unlimited  swelling  can  take  place  which  will 
gradually  lead  to  the  dissolved  state  of  a  sol. 

In  many  cases,  however,  swelling  media  react  with  the  side  groups 
of  the  macromolecules,  causing  a  change  in  the  chemical  character 
of  the  high  polymer  chains.  This  applies,  for  instance,  to  the  esterifica- 
tion  of  solid  cellulose  (nitration,  acetylation,  Frey-Wyssling,  i936d). 
If  the  changed  chain  molecules  cannot  be  solvated  by  the  penetrating 
swelling  medium,  the  result  is  a  lattice  of  the  newly  formed  substance 
and  no  unlimited  widening  of  the  chain  lattice  takes  place.   This 


4  STUDIES  IN  GELS  III 

phenomenon,  too,  can  be  followed  by  means  of  X-rays,  since  the 
new  chain  lattice  shows  new  interferences,  while  the  orieinal  ones 
disappear.  These  conversions  are  termed  permufoid  or  topochemical 
reactions,  because  the  reacting  groups  undergo  chemical  changes 
within  the  crystal  lattice  itself  without  dissolution  of  the  molecules. 
The  characteristic  feature  of  these  reactions  lies,  therefore,  in  the  fact 
that  chemical  changes  take  place  in  the  solid  state,  in  contrast  to  the 
classical  formula:  corpora  non  agiint  nisi fluida. 

Intramicellar  swelling  clearly  demonstrates  the  great  similarit}- 
between  swelling  and  dissolution.  As  has  been  shown  by  Katz  (1924), 
in  both  cases  the  same  physico-chemical  phenomena  take  place  (heat 
of  swelUng,  volume  contraction  and  swelling  pressure  as  a  result  of 
solvation),  the  only  difference  being  that  swelling  occurs  very  slowly 
because  of  the  slow  Brownian  movement  of  the  macromolecules.  And 
if  in  some  way  or  other  these  form  a  network,  only  limited  swelling 
takes  place  and  the  state  of  a  sol  is  not  reached. 

Shrinkage.  Most  gels  encountered  in  nature  are  liable  to  swell  to 
a  certain  extent.  On  drying,  the  behaviour  depends  on  the  properties 
of  their  gel  frame.  If  this  possesses  meshes  with  fixed  contours,  such 
as,  for  instance,  silica  gels,  the  decrease  in  volume  does  not  correspond 
tothelossof  water.  The  dry  system  is  a  porous  body,  i.e.,  it  has  changed 
into  an  air-containing  aerogel. 

If  the  gel  framework  is  flexible,  however,  the  meshes  will  graduallv 
close  on  continued  shrinkage  till  finally  the  micellar  strands  touch  on 
all  sides.  The  result  is  a  horny,  brittle  xerogel  without  perceptible 
porosity.  The  drying  process  of  these  xerogels  is  very  problematic.  If 
we  assume  the  gel  to  be  isotropic,  it  must  possess  a  gel  frame  of  random 
arrangement.  Were  we  to  apply  this  principle  of  randomness  also  to 
amicroscopic  regions  (Fig.  53a,  p.  69),  the  framework  obtained  when 
the  molecular  or  micellar  strands  approach  each  other  would  be  a 
loose  structure  with  numerous  interstitial  or  intermicellar  spaces.  In 
that  case  the  xerogel  would  possess  a  lower  density  than  the  crystalline 
substance  and  it  would  have  a  white  and  untransparent  appearance 
as  a  result  of  the  light  diffraction  caused  by  the  air-containing  spaces. 
This  only  applies,  however,  to  aerogels,  whereas  xerogels  solidify  to 
completely  transparent  glassy  substances.  If  the  density  of  the  crys- 
talline micellar  strands  is  determined  by  mean?  of  X-rays  and  compared 
with  the  density  of  xerogels,  the  discrepancy  found  is  only  about  10% 


112      FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

(Hermans,  1938),  whereas  a  dried  mass  of  micellar  strands  should 
represent  a  more  airy  structure  with  a  much  lower  density.  Examples 
of  xerogels  are  gelatin  and  celloidin. 

We  are  therefore  compelled  to  assume  the  existence  of  short-range 
order.  Given  this  short-range  order  of  the  micellar  strands,  one  can 
imagine  continuous  strings  intersecting  the  whole  gel  (Fig.  54,  p.  70). 
The  orientation  never  changes  abruptly;  deviations  from  parallel 
alignment  are  only  gradual.  Following  such  a  continuous  string  or 
micellar  strand  in  an  isotropic  gel,  one  finds  a  curve;  neighbouring 
strands  are  approximately  parallel  (Fig.  85,  p.  127).  Shrinkage  causes 
the  strings  to  approach  each  other;  if  the  distance  between  them  re- 
mains the  same  at  all  points,  the  result  must  be  a  decrease  in  the  radius 
of  curvature  (Hermans,  1941).  It  follows  that,  on  the  assumption  of 
short-range  order,  the  gel  is  capable  of  shrinking  uniformly  in  all 
directions  until  the  structural  elements  are  close-packed,  without 
kinks  in  the  micellar  strands  (Fig.  76,  p.  108). 

Discrepancy  in  the  density  of  dry  gels.  The  transparent  brittle  state  of 
dry  xerogels  (dried  glue,  gelatin  foil,  horny  celloidin,  etc.)  has  led 
Hermans  and  Vermaas  (1946)  to  compare  these  substances  with  glass. 
In  the  manufacture  of  glass  the  rapid  cooling  of  melts  gives  the  un- 
wieldy molecules  of  quartz,  silicates,  borates,  etc.  no  time  to  crystallize. 
The  glassy  amorphous  state  is,  therefore,  characterized  by  a  similar 
molecular  framework  to  that  of  the  gels  with  amicroscopic  framework, 
i.e.,  with  chain  molecules  as  structural  units.  Glasses  possess  a  some- 
what lower  density  than  crystals  of  the  same  compound,  since  the 
closest  packing  of  the  molecules  is  attained  in  the  crystal  lattice  only. 
For  instance,  the  difference  between  the  densities  of  butyl  alcohol 
CH3CH2CH2CH2OH  in  the  crystalline  and  in  the  supercooled  glassy 
state  amounts  to  6%.  Gels  with  micellar  structure  contain  ordered 
crystalline  regions  of  micellar  strands  next  to  less  ordered,  more  or 
less  amorphous  regions.  For  the  latter  Hermans  (1946)  assumes  an 
amorphous  glassy  state.  Hence  the  gel  consists  of  crystalline  and  glassy 
amorphous  parts.  If  the  densities  of  the  crystalline  and  the  amorphous 
compounds  are  known,  the  amount  of  crystalline  material  in  the  gel 
can  be  calculated.  Using  the  reciprocal  densities,  i.e.,  the  specific 
volumes,  the  following  holds  good :  X  9?-^^.,  +  (i  — x)  y^^^^j  =  9?,  where 
ff  =  experimentally  determined  specific  volume  of  the  gel,  ^^j^r)  = 
specific    volume    of   the    crystalline    part,   rp^^^^^  =  average    spec.vol. 


4  STUDIES  IN  GELS  II3 

of  the  amorphous  part,  x  =  the  fraction  of  crystalline  material. 
Substituting  1.55  for  the  density  of  the  cellulose  fibre  (determined 
in  toluene),  1.59  for  that  of  crystalline  cellulose  and  6%  less  for 
amorphous  cellulose  (compare  butyl  alcohol),  Hermans  (1946)  cal- 
culated X  =  0.61  for  ramie  fibres  and  0.18  — 0.32  for  regenerated 
cellulose.  In  other  words,  only  1/5  to  1/5  of  the  cellulose  in  rayon  fibres 
is  crystalline.  Whereas  this  result  is  quite  acceptable,  the  amount  of 
crystalline  cellulose  in  ramie  is  likely  to  be  greater  than  60%.  Otherwise 
the  difference  between  the  birefringence  of  ramie  fibres  and  that  of 
crystalline  cellulose  ought  to  be  greater  than  actually  determined 
(Frey-Wyssling  and  Speich,  1942;  according  to  our  measurements  it 
amounts  to  4.4%  and,  based  on  the  double  refraction  0.0705  of 
crystalline  cellulose  determined  by  Hermans  1949,  to  7.3  %). 

The  crystallinity  of  a  gel  can  also  be  determined  by  X-rays.  Since 
amorphous  substances  scatter  the  X-ray  beam,  they  cause  a  diffuse  back- 
ground blackening  of  the  film  in  the  X-ray  camera.  The  photometer  curve 
(cf.  Fig.  70,  p.  102)  taken  from  such  films  permits  computation  of  the  amount 
of  the  amorphous  fraction  in  the  gel  under  investigation.  By  this  method 
Hermans  and  Weidinger  (1949)  find  70%  crystalline  cellulose  in  ramie 
and  39  %  in  regenerated  cellulose.  There  is  a  third  means  of  estimating 
the  amount  of  the  two  fractions.  As  the  hydrolysis  velocity  of  amorphous 
cellulose  is  much  greater  than  that  of  crystallized  cellulose,  the  quantitative 
relation  between  them  can  be  derived  from  a  suitable  hydrolysis/time  curve. 
Philipp,  Nelson  and  Ziifle  (1947)  calculate  by  this  method  95%  crystal- 
linity for  ramie  fibres  and  about  70  %  for  rayon.  As  the  three  methods 
mentioned  (optical.  X-ray  and  chemical)  yield  different  values  for  the 
crystallinity  of  the  same  gel,  we  must  conclude  that  there  is  no  net  difference 
between  crystallized  and  amorphous  cellulose;  hence  the  non-crystallized 
fraction  is  rather  to  be  considered  2lS  paracrystalline  (cf.  Fig.  54,  p.  70). 

Hermans  criticizes  the  opinion  that  dry  xerogels  are  porous  bodies 
on  the  ground  that  no  one  speaks  of  submicroscopic  spaces  in  the  case 
of  glasses  either,  notwithstanding  the  lower  density  than  in  the  crystal- 
line state.  This  comparison,  however,  does  not  seem  quite  justified 
to  me,  since  certain  liquids  (such  as  water,  alcohols  and  aldehydes  in 
the  case  of  cellulose)  are  capable  of  penetrating  into  xerogels,  whereas 
this  does  not  occur  in  glasses.  Thus,  clearly,  there  must  exist  a  differ- 
ence in  the  order  of  magnitude  of  the  "empty  spaces"  present.  In 
the  swollen  state  xerogels  definitely  possess  a  loose  structure,  and  it 
is  not  likely  that  the  micellar  framework  loses  this  structure  completely 


0.80 


0.70 


\j}'     r^  ""  empty   space 
^^o'       A^^  Water 


114     FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  Z 

Upon  drying.  This  is  more  likely  to  occur  in  molecular  frameworks. 
Here  the  empty  spaces  shrink  and  form  interstitial  spaces  which  no. 
longer  possess  the  character  of  submicroscopic  pores.  It  is  therefore 
easy  to  see  why,  in  the  poorly     ^^^^ 
crystallized  rayon  fibres,  por-  | 
osity    will    disappear    to    a  § 
great    extent  in   the   drying  | 
process ;  all  the  same,   even  c^o.90 
these  fibres  contain  about  5  % 
of  empty  space  (Fig.  78).  In 
native  fibres  whose  incrusta- 
tions have  been  removed,  a 
complete  closing  of  the  struc- 
ture would  hardly  be  poss- 
ible. Otherwise  it  would  be 
hard  to  explain  how  the  dens- 
ity  of  ramie  determined  in 
toluene  could  amount  to  i .  5  5 , 
whereas  the  result  of  accurate 
measurements   of  mass   and 
volume  gives  only  1.39.  Fur- 
thermore, an  inner  reserve  of 
space  is  necessary  to  explain 
the  great  flexibility  and  capa- 
city to  twist;  otherwise  these 
fibres  would  be  as  brittle  and 
elastic  as  glass  fibres. 

Do^ible  refraction  of  swollen  gels.  In  the  swelling  process,  isotropic 
imbibition  liquid  penetrates  between  the  anisotropic  micellar  strands. 
In  this  way  the  rodlet  birefringence  of  gels  is  enhanced,  for  it  follows 
from  the  formula  given  on  p.  84  that  if  the  other  conditions  remain 
constant,  this  birefringence  acquires  its  maximum  value  when  the 
relative  volumes  of  rodlets  and  imbibition  medium  are  equal  {b^  =  ^3)- 
The  intrinsic  double  refraction,  however,  is  inversely  proportional  to 
the  volume  so  long  as  it  is  permissible  to  assume  that  no  change  in 
micellar  orientation  occurs  as  a  result  of  swelling.  If  the  intrinsic 
double  refraction  of  the  dry  gel  is  called  i-Do  and  the  degree  of  swell- 
ing is  q  (volume  of  swollen  gel/volume  of  dry  gel),  then,  according; 


%r 
0.60 


' '  ""  Cellulose 
_i •  -•   "    ■  '- 


0.!0 


0.20  0.30 

Regain  g  H2O  per  g  cellulose 


Fig.  78.  Increase  in  volume  of  swelling  isotropic 
(regenerated)  cellulose  threads  (from  Hermans^ 
1946).  Abscissa:  absorption  of  water.  Ordinate: 
specific  volume  (i/density),  q>o  specific  volume 
of  dried  threads  0.66,  ^^cr  specific  volume  of  crys- 
tallized cellulose  0.63.  The  water  absorption^ 
increases  linearly,  but  the  volume  does  not. 


4  STUDIES  IN  GELS  TI5 

to  Kratky  and  Platzek  (1958),  the  total  double  refraction  of  the 
swollen  gel  t-Do  amounts  to : 

t-Do  = t-Do. 

q 

Consequently,  if  the  intrinsic  double  refraction  i-Do  of  the  dry  gel 
is  known  and  the  total  double  refraction  t-Do  of  the  swollen  gel  is 
measured,  the  rodlet  birefringence  r-Do  of  the  swollen  gel  can  be 
calculated.  It  is  therefore  possible  to  measure  rodlet  anisotropy  in  gels 
capable  of  swelling,  provided  the  birefringence  curves  are  only  plotted 
from  points  which  result  from  measurements  in  imbibition  liquids 
giving  rise  to  similar  degrees  of  swelling.  Otherwise  one  would  obtain 
compHcated  kinky  curves  devoid  of  regularity,  instead  of  smooth 
Wiener  curves  (cf.  Fig.  62,  p.  84). 

Apart  from  rodlet  double  refraction,  another  form  of  birefringence 
mav  occur  when  liquids  penetrate  between  the  amorphous  chain 
molecules.  This  is  attributed  by  Vermaas  (1941,  1942)  to  oriented 
adsorption  of  the  penetrating  molecules.  It  might  also  be  due,  how- 
ever, to  a  change  in  the  "intrinsic  anisotropy"  of  the  chain  molecules 
caused  by  the  swelling  medium,  such  as  that  occurring  in  sols  when 
the  refractive  index  of  the  dispersing  medium  is  changed  (Sadron, 

1937)- 

d.  Electron  Microscopy 

Electron  rays.  The  electrons  which  are  emitted  by  a  cathode  are 
electrically  charged  negative  particles  with  a  mass  of  1/1840  of  that 
of  a  hydrogen  atom.  The  range  of  these  electrons  in  air  is  very  short, 
because  they  are  absorbed  or  scattered  by  atoms  or  molecules  which 
they  meet  on  their  path.  All  investigations  with  electron  rays  must 
therefore  be  carried  out  in  vacuo.  On  account  of  their  electric  charge 
they  can  be  made  to  deviate  from  their  straight  trajectory  by  means  of 
electric  or  magnetic  fields.  Bundles  of  electron  rays  can  therefore  be 
focused  by  electric  coils  in  much  the  same  way  as  light  rays  by  lenses. 
This  makes  it  possible  to  form  images  with  electron  rays  according  to 
the  laws  of  geometrical  optics  (Zworykin  and  coll.,  1945;  Burton 
and  Kohl,  1946;  Wyckoff,  1949;  Frey-Wyssling,  195  i). 

In  so  far  as  the  electron  rays  represent  a  stream  of  particles,  they  can 
hardly  be  compared  with  light  rays  or  X-rays.  They  have  the  remark- 


Il6      FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

able  property,  however,  of  possessing  at  the  same  time  the  character 
of  waves.  They  can  be  deflected  by  crystal  lattices  and,  like  X-rays, 
give  rise  to  interferences.  Hence  an  electron  ray  represents  a  corpus- 
cular ray  and  a  wave  train  at  the  same  time!  The  wavelength  X  of 
electron  rays  depends  on  the  voltage  applied  to  the  cathode  tube;  A  is 
inversely  proportional  to  the  square  root  of  the  voltage.  In  the  case 
of  light  waves,  the  velocity  of  propagation  in  vacuo  is  independent  of 
the  wavelength.  This  does  not  apply  to  electron  rays,  for,  besides 
lowering  the  wavelength,  an  increase  in  voltage  also  results  in  a  greater 
velocity  of  the  electrons.  This  velocity  may  become  as  high  as  io^° 
cm/sec,  i.e.,  1/3  of  the  velocity  of  light.  Since  electron  microscopy 
operates  with  very  high  tensions,  the  electrons  are  "rapid",  i.e.,  rich 
in  energy.  At  a  tension  of  57  kV  the  wavelength  amounts  to  about 
5.10"'°  cm  =  0.05  A  (BoRRiES  and  Ruska,  1939a).  This  is  one 
twentieth  of  the  wavelength  of  hard  X-rays  (about  i  A)  and  one  two 
hundredth  of  the  wavelength  of  soft  X-rays  (about  10  A).  In  spite  of 
this  extremely  small  ^^avelength  and  in  contrast  to  X-rays,  electrons 
have  no  penetrating  power,  as  the  electrons  are  already  totally  ab- 
sorbed by  layers  of  solid  substances  of  a  thickness  of  o.i  /x.  When 
passing  through  an  object,  they  lose  part  of  their  energy  and  leave 
it  with  a  somewhat  smaller  velocity,  i.e.,  with  a  changed  wavelength 
depending  on  the  energy  loss  in  the  object.  This  means  that  the  elec- 
tron beam,  originally  monochromatic,  becomes  polychromatic,  and 
images  from  electron  lenses  show  not  only  spherical  but  also  chromatic 
defects  as  light  microscopic  images  do. 

The  electron  microscope.  Since  the  resolving  power  of  the  microscope 
depends  on  the  order  of  magnitude  of  the  wavelength  of  the  light 
used,  one  might  expect  great  improvement  in  the  resolving  power  of 
an  X-ray  microscope  as  compared  to  the  ordinary  microscope.  That 
dream  could  not  be  realized,  because  lenses  for  X-rays  do  not  exist. 
The  possibility  of  focusing  electron  rays  has,  however,  made  the 
construction  of  a  short-wave  microscope  feasible.  (Martin  1938; 
BoRRiES  and  Ruska  1939b;  Ardenne,  1940a,  b;  Zworykin,  1940, 
1941 ;  Zworykin,  Hillier,  and  Vance,  1941 ;  Borries,  1941 ;  Induni, 

I945-) 

The  electron  microscope  operates  according  to  the  same  principle 
as  the  ordinary  microscope.  The  light  source  is  replaced  by  a  source 
of  electrons.  Usually  this  is  a  hot  cathode,  but  Induni  (1945)  has  also 


STUDIES  IN  GELS 


117 


constructed  an  electron  microscope  with  a  cold  cathode.  The  electron 
ravs  emitted  are  focused  bv  a  condenser  coil  and  directed  towards  the 
object  (Fig.  79).  An  object  coil  behind  the  object  projects  a  real  mag- 
nified image  of  the  object, 
in  the  same  way  as  an  object  coid  Cothode~^{~~Z^^^^    Light  Source 


lens  in  the  ordinary  micro- 
scope. In  analogy  to  projec- 
tion  microscopy,   this   real 
image   is    magnified    again  Obiect  Airlock^ 
and  projected  onto  a  screen  ^~ 


\ii 


Illuminating  Optic 


\r:\l^^^^^^J)=p\  Objective 

High   Tension 
Supply 


High   Voltage    f 
Unit  40-60  kV    L» 


TT 


Projective  Lens    I 


Final   Image      ^ 
{Drying    Agents 


A 


by  a  projection  coil,  com- 
parable to  the  ocular.  Since 
electron  rays  are  not  visible, 
a  fluorescent  screen  is  used, 
which  lights  up  in  propor- 
tion to  the  intensity  of  the 
incident  irradiation,  thus 
giving  rise  to  a  visible  image 
Since  photographic  plates 
are  not  only  sensitive  to 
ultraviolet  and  X-rays,  but 
also  to  electron  rays,  the 
fluorescent  screen  may  be 
replaced  by  a  cassette  for 
plates  if  microphotographs 
are  to  be  taken. 

The  whole  path  of  the  rays  must  lie  in  vacuo,  which  is  maintained 
by  means  of  vacuum  pumps.  For  this  reason  the  objects  must  be  in- 
troduced from  the  atmosphere  into  the  evacuated  apparatus  through 
an  air  lock.  The  electron  image  on  the  fluorescent  screen  is  observed 
through  a  window  at  the  side.  As  in  a  projection-drawing  microscope, 
the  source  of  the  rays  is  in  the  upper  part  of  the  apparatus,  the  object 
being  irradiated  from  above,  giving  a  projected  image  at  about  the 
height  of  the  table.  Fig.  79  gives  a  comparison  with  the  ordinary 
microscope  according  to  Induni's  description  (1945). 

So  long  as  the  objects  investigated  have  a  thickness  of  more  than 
0.1  fi,  the  image  in  the  electron  microscope  is  formed  in  the  manner 
of  shadow  images.  Objects  of  considerably  less  thickness  (order  of 


Recording    Device 


Fig.  79.  Comparison  between  light  microscope 

(at  right)  and  electron  microscope  (at  left)  (from 

Induni,  1945). 


Il8      FUNDAMENTALS  OF  SUBMICROSCOPIC  MORPHOLOGY  I 

magnitude  o.oi  /<  =  loo  A)  transmit  electron  rays.  In  this  case  the 
imao-e  formation  is  due  to  the  fact  that  many  of  the  electrons  are  de- 
fleeted  from  their  rectilinear  trajectory  by  the  atoms  in  the  object,  in 
much  the  same  way  as  a  small  celestial  body  which  enters  the  sphere 
of  attraction  of  a  star.  Now  if  the  object  lens  possesses  a  small  aperture, 
the  electrons  which  are  deflected  do  not  reach  the  image  and  the  object 
appears  darker  than  the  background.  Since  heavy  atoms  deflect  elec- 
trons more  strongly  than  do  light  ones,  metallic  colloid  particles  appear 
darker  than  organic  particles,  which  often  furnish  a  very  faint  contrast. 
It  is  possible  to  enhance  the  contrasts  by  introducing  heavy  atoms  such 
as  iodine  (Husemann  and  Ruska,  1940),  osmium  (OsOJ  or  tungsten 
(phosphotungstic  acid;  Hall,  Jakus  and  Schmitt,  1945)  as  "electron 
dyes".  It  must  be  emphasized  that  the  comparison  with  "dyes"  is  not 
strictly  correct  because  the  absorption  of  electrons  is  very  slight.  If 
there  is  appreciable  absorption,  e.g.  in  thick  sections,  organic  objects 
are  instantly  burnt  by  the  high  energy  released  by  the  captured 
electrons.  Therefore,  preparations  for  the  electron  microscope  must 
be  so  thin  that  the  electron  absorption  is  negligible.  As  indicated 
above,  the  contrast  observed  is  due  to  scattering. 

The  electron  scattering  comprises  different  phenomena.  In  the 
lirst  place  there  is  the  coherent  diff'raction  of  the  beam  in  much  the 
same  way  as  in  the  ordinary  microscope.  The  coherent  light  of  the 
diffracted  rays  is  apt  to  interfere  and  to  furnish  a  uniform  image  when 
these  rays  are  collected  by  a  lens.  However,  the  scattering  of  electrons 
which  causes  the  contrast  in  the  electron  microscope  is  incoherent,  i.e. 
the  deflected  rays  are  no  longer  able  to  interfere  with  each  other  and 
to  be  focused  at  the  proper  place  in  the  image  screen.  Most  of  these 
aberrant  electrons  are  scattered  elastically,  when  the  ray  is  deviated 
by  some  atom  nucleus  without  loss  of  energy.  But  there  is  also  inelastic 
scattering  whereby  the  electron  loses  some  of  its  energy,  and  then 
not  only  is  it  deflected  from  its  original  path,  but  its  velocity  is  slowed 
down  at  the  same  time,  so  that  the  wavelength  of  the  ray  is  increased. 
This  corresponds  to  a  chromatic  error.  The  geometric  and  chromatic 
aberrations  of  the  scattered  electron  cause  an  indistinct  blurred  image 
if  they  reach  the  objective.  They  are  therefore  screened  off"  by  a 
narrow  diaphragm  (Fig.  80);  consequently,  the  more  incoherently 
electrons  are  scattered  by  an  object,  the  darker  it  must  appear  on  the 
image  screen  owing  to  this  loss  of  electron  light.  In  order  to  obtain 


4 


STUDIES  IN  GELS 


119 


highly  contrasting  images,  the  aperture  of  the  objective  lens  must  be  as 
small  as  possible.  On  the  other  hand,  a  small  aperture  is  unfavourable 
to  the  resolving  power  of  the  microscope,  for,  according  to  Abbe's 
theory  of  image  formation,  the 
resolving  power  increases  with 
the  aperture  and  reaches  a  maxi- 
mum when  this  becomes  ^-^  i.  It 
would  be  useless,  however,  to 
make  high  aperture  electron  lens- 
es because  their  lack  of  correc- 
tion would  produce  imperfect 
images,  in  the  same  way  as  un- 
•corrected  lig-ht  lenses.  Thev 
•could  only  be  improved  by  cut- 
ting down  the  aperture,  but  this 
would  reduce  the  resolution. 
The  present  quality  of  electron 
lenses  can  be  compared  to  that 
-of  the  optical  lenses  at  the  time 


Fig.  80.  Electron  scattering  by  a  specimen  and 

selective  effect  of  the  objective  Jens  aperture 

(from  HiLLiER,  1946). 


when  Abbe  began  to  eliminate  their  spherical  and  chromatic  defects. 
The  necessary  screening  of  the  scattered  light  and  the  defects  of 
the  lenses  require  very  narrow  bundles  of  electrons  with  apertures 
of  only  0.00 1  to  0.005.  As  a  result  of  the  small  apertures  a  applied,  the 
Tesolving  power  d  is  not  as  large  as  could  have  been  expected  from 
the  exceedingly  small  wavelength.  As  calculated  for  the  ordinary 
microscope  (probably  Abbe's  theory  cannot  be  applied  without  altera- 
tions to  the  electron  microscope,  but,  curiously  enough,  the  results 
are  plausible),  the  resolving  power  \s  l\a  =  0.05  A/0.002  =  25  A. 
This  minimum,  however,  is  only  seldom  reached.  Usually  the  resolving 
power  amounts  to  about  50  A  (Kinsinger  and  co-workers,  1946). 
This  is  near  to  the  smallest  gold  particles  which  have  been  demonstrated 
in  the  ultramicroscope  (60  A).  Instead  of  luminous  points,  however, 
true  images  are  obtained.  Thus  the  electron  microscopy  covers  the 
whole  field  of  particle  sizes  in  colloid  chemistry,  completing  this 
science  by  the  new  branch  of  colloid  morphology.  Considering  the 
hard  work  needed  to  increase  the  resolving  power  of  the  ordinary 
microscope  from  dry  systems  with  d  =  o.^  fxto  quartz  immersion  for 
ultraviolet  light  with  d  =  o.\  /u,  we  cannot  sufficiently  express  our 


I20      FUNDAMENTALS  OF  S  U  BMIC  RO  S  C  O  PI  C  MORPHOLOGY  I 

admiration  on  realizing  how  the  resolving  power  has  been  increased 
by  a  factor  of  about  a  hundred  by  the  discovery  of  the  electron 
microscope ! 

The  small  aperture  of  the  objective  coils  is  responsible  for  the  great 
focal  depth  of  electron  optical  images.  This  depth  determines  the  ratio 
between  the  layer  thickness  in  the  object  imaged  sharply  and  the  re- 
solving power.  In  the  ordinary  microscope  with  large  aperture  the 
focal  depth  is  only  about  i,  which  means  that  a  section  of  several  [x 
thickness  can  be  analyzed  into  successive  optical  sections  at  different 
levels  by  means  of  the  fine  adjustment.  In  the  electron  microscope  this 
ratio  is  about  looo.  This  is  a  drawback  in  the  spatial  analysis  of  the 
object,  but  it  is  a  very  valuable  aid  to  the  sharp  focusing  of  the  image 
and  to  obtaining  stereophotographs  (Ardenne,  1940b;  Muller, 
1942a;  Heidenreich  and  Matheson,  1944). 

The  similarities  and  the  dissimilarities  between  ordinary  and  elec- 
tron microscopy  are  listed  in  Table  XIIL 


TABLE  XIII 
PROPERTIES  OF  ORDINARY  AND  ELECTRON  OPTICAL  IMAGES 


Light  rays 

Electron  rays 

Wavelength 

8000-2000  A 

About  0.05  A 

Penetrating  power 

Fairly  great 

Small 

Contrasts  are  caused  by 

Absorption 

Electron  scattering 

Contrasts  enhanced  by< 

Staining 

Dark  field  illumination 

Impregnation  by  heavy 
atoms  (J,  Wo,  Os) 
Shadowing 

Focal  depth 

Small,  ca.  i 

Large,  ca.  1000 

Most  favourable  magni- 
fication 

Up  to  1500 

Up  to  30,000 

Resolving  power 

3000  A 

30  A 

The  electron  microscope  may  be  changed  into  an  apparatus  produ- 
cing electron  diffraction  spectra  if  the  objective  current  is  turned  oft", 
and  the  projective  lens  is  removed  (E.  Ruska,  1940).  The  electron 
diffraction  diagrams  obtained  have  the  appearance  of  X-ray  diagrams ; 
they  are  only  formed  if  a  crystal  lattice  is  present  in  the  object. 


STUDIES  IN  GELS  121 


Technique  of  making  preparations  (Ruska,  1939;  Wyckoff,  1949).  The 
penetrating  power  of  electron  rays  being  small,  it  is  difficult  to  find 
adequate  specimen  holders.  The  most  suitable  holders  are  nitrocellulose 
films  of  submicroscopic  thickness.  These  can  be  made  by  spreading  a 
drop  of  a  collodion  solution  in  amyl  acetate  on  water,  which  is  saturated 
with  this  solvent.  After  evaporation  of  the  amyl  acetate,  a  nitrocellulose 
film,  which  in  favourable  cases  is  only  lo  m/z,  is  left  on  the  water  surface. 

When  investigating  suspended  objects  (bacteria,  viruses,  colloid  particles), 
a  drop  of  the  suspension  is  left  to  dry  on  the  specimen  holder.  Only  dried 
objects  can  be  placed  in  the  apparatus,  because  the  exposure  has  to  be  made 
in  vacuo.  This  rules  out  the  observation  of  living  organisms  in  the  electron 
microscope,  and  it  is  also  impossible  to  image  cytological  objects  in  their 
natural  swollen  state. 

The  methods  described  are  appropriate  for  the  investigation  of  corpuscular 
colloids.  But  in  general  those  methods  are  unsuitable  for  reticular  colloids 
with  a  coherent  structure  and  a  different  microtechnique  had  to  be  devised 
for  these  objects.  Sometimes  gel  solutions  or  gels  can,  admittedly,  be  dried 
on  a  specimen  holder  to  be  imaged  (Fig.  86a, p.  128).  Before  it  was  possible 
to  prepare  sections  thin  enough  for  the  electron  microscope,  like  those 
obtained  in  ordinary  microscopy  by  means  of  microtomes,  all  kinds 
of  expedients  had  to  be  resorted  to.  In  some  cases  the  thin  edge  of  a  wedge- 
shaped  section  is  thin  enough  for  use  in  electron  microscopy,  but  no  images 
of  suitable  dimensions  can  be  obtained  in  this  manner.  Thick  objects,  such 
as  cell  walls  and  fibres,  can  be  teased  into  small  fragments  after  being 
allowed  to  swell  (Wergin,  1942),  or  else  they  can  be  crushed  into  sub- 
microscopic  splinters  in  a  vibrating  ball  mill.  These  splinters  are  suspended 
and  finally  dried  on  the  specimen  holder  (Hess  and  co-workers,  1941). 
Some  gels  can  be  divided  into  submicroscopic  flocculates  by  means  of  special 
vibrators  (O'Brien,  1945).  Ultrasonic  waves  have  proved  to  be  particularly 
suitable  for  this  purpose;  e.g.,  by  this  method  microscopic  fibres  can  be 
disintegrated  into  submicroscopic  fibrils  (Wuhrmann,  Heuberger,  and 
MiJHLETHALER,  1 946)  without  damage  to  the  structure  such  as  the  vibrating 
mill  inflicts. 

Several  dif^culties  arise  when  these  preparations  are  irradiated.  The 
electrons  absorbed  impart  a  negative  charge  to  the  object,  resulting  in 
repulsive  forces  between  the  structural  elements,  and  may  cause  inflation  of 
the  fibrils  at  the  points  irradiated.  The  changes  brought  about  by  this  effect, 
however,  are  as  a  rule  less  striking  than  those  suffered  by  the  object  as  a 
result  of  the  heat  evolved.  On  absorption,  the  great  energy  content  of  the 
fast  electrons  is  mostly  converted  to  heat.  Silver  and  gold  can  be  fused 
together  or  even  melted  completely  in  the  electron  microscope.  Obviously, 
therefore,  organic  compounds  become  charred  if  exposed  too  long.  Many 
objects,  such  as  bacteria,  appear  brownish  after  exposure  in  the  electron 
microscope,  even  if  precautions  are  taken  to  protect  them.  Naturally,  the 
thicker  the  object,  the  greater  is  the  heat  evolved.  The  object  is  not  easy 


122      FUNDAMENTALS  OF  S UB M I C RO S C OP  I  C  MORPHOLOGY 


Film 


to  cool,  because  in  vacuo  heat  cannot  be  transferred  by  convection.  All  that 
can  be  done  is  to  withdraw  as  much  of  the  heat  evolved  as  possible  by  means 
of  the  metallic  ring  lying  on  the  specimen  holder.  The  best  way  to  do  this 
is  to  place  a  fine  wire  netting  over  the  ring  and  to  irradiate  the  object  through 
the  meshes. 

Only  when  an  organic  preparation  is  thinner  than  o.i  /x,  does  it  become 
sufficiently  transparent  to  electron  rays  and  can  be  irradiated  for  some  time 
without  damage.  Therefore,  the  aim  is  to  produce  sections  lo-ioo  times 
thinner  than  those  used  in  histology.  Various  microtomes  have  been 
employed  for  this  purpose.  Claude  and  Fullam  (1946)  produced  sections 
of  0.3-0.6  IX  thickness  with  a  special  rotating  high  speed  microtome. 
Bretschneider  (1949a)  arrived  at  o.i  /z  with  the  rocking  microtome. 
Similar  results  have  been  obtained  by  Danon  and  Kellenberger  (1950). 
The  fine  0.1  ju.  movement  of  the  specimen  holder  of  the  microtome  is  handi- 
capped by  the  imperfection  of  the  micrometer  screw,  but  it  can  be  achieved 
by  the  thermal  expansion  of  a  massive  metal  block  which  has  previously 
been  cooled  down  by  dry  ice  (Newman  and  co-workers,  1949).  Special 
devices  for  the  block  advance  on  an  inclined  plane  seem  to  be  coming  into 
general  use  (Hillier  and  Gettner,  1950). 

Thin  sections  of  organic  materials  do  not  show  much  contrast  in  the 
electron  microscope,  as  their  constituents  C,  N  and  O  produce  the  same 
electron  scattering  as  the  carrier  film.  Only  cell  components  which  contain 
phosphorus  or  which  are  minerahzed  appear  to  be  darker.  In  certain  cases 
the  contrast  can  be  enhanced  by  osmium  fixation  of  the  cells  and  by  staining 
with  phosphotungstic  or  phosphomolybdic  acid. 

The  best  contrast  is  obtained  by  the  method  oi  metal  shadowing,  developed 
by  Wyckoff  (1949).  In  a  vacuum  bell  jar  a  small  amount  of  metal  is 
vaporized  and  deposited  obliquely  on  the  preparation  (Fig.  81).  As  a  result 

the  faces  of  the  specimen 
turned  to  the  source  of  metal 
vapour  are  coated  with  metal, 
whereas  the  opposite  faces  are 
not.  Behind  the  object  there  is 
a  zone  free  of  metal  which  is 
called  the  shadow  of  the  spe- 
cimen. From  this  shadow  the 
height  of  the  object  can  be  cal- 
culated if  the  shadowing  angle 
is  known  (Muller  1942b). 

When  a  preparation  like  this 
is  irradiated  in  the  electron 
microscope,  the  electrons  are 
greatly  scattered  at  the  places  where  metal  has  accumulated,  passing  freely 
through  the  zones  of  shadow.  As  a  result,  the  picture  on  the  projection 
screen  exhibits  an  astonishing  three-dimensional  effect,  creating  the  impres- 


0.00211 
0.01  u 


O.OOitn 


Specimen 

Fig.  81.  Shadowing  of  a  specimen  by  deposition  of 
metal :  s  length  of  the  shadow,  b  height  of  the  spe- 
cimen, a  shadowing  angle,  h  =  s  tan  a. 


4  STUDIES  IK  GELS  I23 

sion  that  the  objects  are  obHquely  illuminated.  On  the  photographic 
negative  the  shadows  are  black,  comparable  to  the  shadows  in  a  land- 
scape cast  by  the  sun,  which  is  the  reason  why  this  method  has  been  called 
shadowing.  Because  this  effect  is  very  striking,  the  negatives  of  shadowed 
preparations  are  reproduced  and  not  the  positives,  as  in  ordinary  photo- 
graphy. This  means  that  a  positive  film  must  be  made  of  every  photograph 
before  prints  can  be  made. 

Metal  shadowing  permits  even  very  flat  objects  to  be  pictured,  for  the 
shadow  can  be  accentuated  by  lowering  the  shadowing  angle.  A  suitable 
angle  is  ^\^  (1:6),  furnishing  pictures  reminiscent  of  sunset  or  sunrise 
illumination  with  its  very  long  shadows.  As  we  are  accustomed  to  illuminate 
relief  maps  from  the  left-hand  top  corner,  shadowed  electron  micrographs 
ought  to  be  oriented  so  that  their  shadow  points  towards  the  bottom  right- 
hand  corner.  Only  thus  do  we  get  the  natural  impression  of  a  high-relief. 
If  such  a  picture  is  turned  upside-down,  the  impression  received  is  of 
reversed  rehef,  all  elevations  seeming  to  be  depressions. 

Wyckoff  (1949)  has  found  the  most  suitable  metals  for  shadowing  to  be 
chromium  and  palladium.  The  higher  the  atomic  number  of  the  element, 
the  thinner  is  the  metal  film  yielding  the  same  effect  when  deposited  on  the 
preparation.  Whereas  the  thickness  of  a  chromium  film  must  be  40  A, 
a  palladium  film  of  20  A  will  do.  In  this  respect  uranium  would  be  better 
still. 

As  the  scale  of  pubHshed  electron  micrographs  varies  from  i :  looo  up 
to  1:100000,  it  is  as  well  to  mark  the  magnification  on  every  individual 
picture.  This  is  done  by  putting  a  black  line  on  the  micrograph  which 
represents  the  length  of  i  /m;  for  a  magnification  of  10,000,  its  length  is  i  cm. 

Results  of  electron  microscopy.  The  improvement  in  the  resolving 
power  for  structures  invisible  in  the  ordinary  microscope  is  most 
evident  from  the  electron  optical  images  which  have  been  obtained 
from  the  silica  wall  of  the  diatom  Pleurosigma  angtilatum  (Fig.  82a),  the 
well-known  test  object  for  the  immersion  objective  of  the  ordinary 
microscope.  In  the  latter  case  the  best  objectives  show  three  inter- 
secting systems' of  lines  (Fig.  82b),  which  at  the  utmost  give  a  vague 
impression  of  a  perforation  (Fig.  82c;  Ardenne,  1940b),  whereas  in 
the  electron  microscope  Fig.  83a  is  obtained.  The  surmised  pores  are 
clearly  imaged  with  sharp  edges ;  and,  being  so  far  apart,  it  is  evident 
from  this  "coarse"  structure  that  the  electron  microscope  is  able  to 
resolve  exceedingly  minute  details.  It  is  shown  that  the  pores  do  not 
represent  cyUndrical  canals  running  through  the  silica  walls,  but  that 
the  outer  opening  is  in  the  form  of  a  slit,  while  the  inner  one  is  elliptic 
and  closed  by  a  sieve  membrane.  Stereoscopic  pictures  moreover 
betray,  not  canals,  but  spacious  caverns,  whose  outer  openings  re- 


124      FUNDAMENTALS  OF  SUBM I C R O S C O P  I  C  MORPHOLOGY 


.  .^m^^M^umm^ir^s 


c) 


.a)  Pleurosigma  angtdalum^' .  Sm.  contour  (from  Husted,  1930);/?)  light  microscopic 
image  scale  1500:1  (from  Michel,  1940);  c)  light  microscopic  image  with 
numerical  aperture  1.4,  image  scale  10,000:1    (from  Ardenne,  1940b). 


Fig.  83.  a)  Electron  microscopic 
image  of  Pleurosigma,  image  scale 
100,000:1;  l))  sketch  of  the  spatial 
organisation  of  the  silica  wall; 
image  scale  ca.  60,000:1  (from 
MtJLLER  and  Pasew.\ldt,  1942). 


^) 


f 


b) 


4  STUDIES  IN  GELS  I25 

present  the  slits  shown  in  the  image  (Fig.  85  b,  Muller  and  Pase- 
WALDT,  1942).  Hence,  the  diatom  wall  is  not  a  massive  structure, 
but  consists  of  an  outer  and  an  inner  lamella,  separated  by  sub- 
microscopic  spaces  and  connected  by  pillar-shaped  buttresses  (Fig.  85b). 

Fig.  82c  shows  what  was  meant  in  Table  XIII  (p.  120)  by  "most 
favourable  magnification".  A  microscopic  image  or  a  microphoto- 
graph  can  be  magnified  at  will  by  projection,  so  that  the  magnification, 
or  better  the  image  scale,  does  not  provide  an  unambiguous  reference 
by  which  to  compare  different  microscopes.  Nevertheless  there  is  a 
limit  to  the  magnification  of  images,  in  that  the  contours  become 
rague  when  the  image  scale  becomes  too  large.  For  this  reason  there 
exists  a  "profitable"  magnification  which  is  best  maintained  in  micro- 
photography  and  which  is  designated  as  "most  favourable  magnifica- 
tion". Strong  magnifications  of  the  microphotographic  negatives  ob- 
tained result  in  poor  definition  as  shown  in  Fig.  82c,  where  the 
systems  of  lines  are  hazy  as  a  result  of  a  magnification  of  10,000,  which 
is  seven  times  the  "profitable"  one  of  1500. 

The  most  successful  objects  of  research  for  the  electron  microscope 
are  the  submicroscopic  particles  of  suspensoids,  such  as  inorganic 
coUoids,  virus  particles,  bacteriophages,  organic  macromolecules 
which  exceed  50  A  diameter.  Unicellular  objects  such  as  diatoms 
and  bacteria  are  too  thick;  they  furnish  black  shadow  pictures  and 
details  are  only  to  be  seen  if  the  object  is  perforated  or  provided  with 
surface  appendages  (cilia,  flagella).  The  colloid  particles,  however, 
are  thin  enough  to  transmit  electrons,  producing  real  so-called  phase 
images. 

Fig.  84a  shows  shadowed  macromolecules  of  haemocyanin  from 
the  blood  of  a  snail.  This  micrograph  was  the  first  clear-cut  picture 
of  protein  macromolecules  (Williams  and  Wyckoff,  1945).  Ac- 
cording to  SvEDBERG,  the  globulat  proteins  aggregate  by  2,  4,  8  etc. 
to  form  bigger  particles.  This  rule  (see  p.  141)  found  by  experiments 
with  the  ultracentrifuge,  is  now  substantiated  by  electron  micrographs 
such  as  Fig.  84b  (Polson  and  Wyckoff,  1947). 

The  agents  of  virus  diseases  have  been  found  to  be  macromolecules 
of  different  shapes.  The  classical  tobacco  mosaic  virus  is  rod-shaped, 
as  proved  by  indirect  methods  (double  refraction  of  flow.  X-rays). 
The  electron  micrograph  (Fig.  84c)  shows  that  the  length  of  the  rods 
is  not  defined.  Their  mean  length  depends  on  the  p^^  of  the  dispersing 


126   FUNDAMENTALS  OF  S  U  B  M  I  C  RO  S  C  OP  I  C  MORPHOLOGY 


Fig.   84.  Electron  micrographs  of  globular  colloids,  a)  Macromolccules  of  haemocyanin 

(Williams  and  Wyckoff,   1945).  b)   Macromolecules    of  haemocyanin  aggregated  in 

fours    (PoLSON   and  Wyckoff,    1947).     c)   Tobacco   mosaic   virus    (Wyckoff,    1949). 

d)  Cn'stallizcd  tomato  bushy  stunt  virus  (Wyckoff,  1949). 


STUDIES  IN  GELS 


127 


medium  (Takahashi  and  Rawlins,  1948).  There  is  some  indication 
that  the  rods  are  formed  by  linear  aggregation  of"  roundish  particles. 
The  diameter  of  the  straight  rods  is  150  A  (Wyckoff,  1949),  which 
corresponds  to  the  lateral  identity  period  of  1 5  2  A  revealed  by  X-ray 
investigation  (Bernal  and  Fankuchen,  1937)  in  the  hexagonal  crystals 
of  the  virus  protein  (Stanley,  1935,  1936). 

Contrary  to  expectation,  virus  diseases  with  rod-shaped  particles 
are  rare,  globular  virus  macromolecules  occurring  much  more  fre- 
quently.  When  dried,  a  virus  suspension  of  this  kind  crystallizes,  and 
Wyckoff  (1949)  succeeded  in  producing  very  beautiful  pictures  of 
the  lattice  of  those  crystals  (Fig.  84d).  The  arrangement  of  the  molecules 
revealed  by  the  X-ray  diffraction  method  can  now  be  seen,  and  it  is 
most  interesting  to  observe  how  frequently  small  disturbances  within 
the  regular  pattern  of  the  molecule  arrangement  occur. 

Figs.  84a-c  represent  the  dispersed  particles  of  protein  sols  which 
prove  the  applicabihtv  of  the  electron  microscope  in  biochemistry. 
The  biologist  asks,  therefore,  what 
information  the  electron  microscope 
may  give  on  the  structure  of  gels, 
among  which  we  classify  the  shaped 
portion  of  the  protoplasm.  By  way  of 
example  we  reproduce  in  Fig.  8  5  the 
electron  optical  image  of  a  V2O5  gel 
serving  as  ultrafilter  (Ardenne, 
1940b).  One  recognizes  the  reticular 
structure  assumed  on  the  basis  of 
results  obtained  by  indirect  methods. 
The  agreement  with  the  scheme  of 
Fig.  53a  (p.  69),  proposed  before  the 
electron  microscope  had  been  dis- 
covered, is  most  striking. 

Fig.  85  is  a  rather  indistinct  picture 
of  a  dry  gel.  The  first  clear-cut  elec- 
tron micrograph  of  a  very  loose  gel 
which,  previously  to  preparation, 
contained  about  99*^0  water,  is  reproduced  in  Fig.  86a  (Frey-Wyss- 
ling  and  Muhlethaler,  1944).  It  displays  a  beautiful  spatial  frame- 
work with   big  meshes   and   roundish  interstices.    Fig.    86a   seems 


Fig.  85.  Electron  micrograph  of  an 
ultrafilter    of   vanadium    pentoxide, 
image  scale  35,000:  i  (from  Ardenne, 
1940b). 


128   FUNDAMENTALS  OF  S  I' BM  I  C  R  OSCOP  I  C  MORPHOLOGY 


^*} 


If 

ivvS''-  Vi'.'fVV'lAfK 


^mM 
^  ..^ 


wt^^ 


•?/-^ 


Fig.  86.  Electron  micrographs  of  gels,  a)  Gel  of  V2O3,  40,000  : 1  (from  FREY-\X'YSSLiNGand 
MuHLETHALER,  1 944).  Z))  Cellulosc  Cell  wall of  the  alga  ijo/rogyra,  19,000:1  (phot.  A.  Vogel). 
f)  Chitinous  cell  wall  of  the  fungus  Phyconiyces,  12,000:1  (from  Frey-Wyssling  and 
MiJHLETHALER,  1950).  (7)Tunicin  of  the  mantle  of  Cioiia,  16,000:  i  (from  Frey-Wyssling 

and  Frey,  1950). 


4  STUDIES  IN  GELS  I  29 

to  contradict  the  principle  of  short-range  order,  .,s  the  gel  strands 
show  a  criss-cross  random  arrangement,  but  we  have  to  remember 
that  the  picture  represents  a  projection  of  the  reticular  texture,  because 
the  great  focal  depth  of  the  electron  microscope  causes  gel  strands 
separated  in  space  to  be  imaged  in  a  single  plane.  It  is  likely  that  the 
filaments  crossing  each  other  are  not  lying  at  the  same  depth  in  the  gel, 
but  that  the  majority  are  oblique  with  respect  to  the  image  plane,  as  is 
apparent  from  the  faintness  of  outline  of  numerous  strand  "ends". 
A  stereoscopic  view  of  Fig.  86a  justifies  the  comparison  of  a  gel  with 
a  wad  of  cotton  wool.  At  various  points  ramifications  of  the  gel  strings 
are  visible,  showing  that,  notwithstanding  the  apparent  criss-cross 
arrangement  of  the  gel  strands,  there  exists  short-range  order.  The 
figure  further  shows  that,  in  the  case  of  a  gel  thickness  corresponding 
to  an  ultrafilter,  all  possible  orientations  occur  in  spite  of  the  short- 
range  order,  so  that  there  exists  statistical  isotropy,  as  indicated  in 
Fig.  5  3  (p.  69).  The  curved  micellar  strands  which  are  visible  in  Fig. 
86b  are  particularly  interesting  because  they  favour  branching  of  the 
strands  (cf.  Muhlethaler,  1949). 

As  shown  by  Fig.  58  (blood  fibrin,  Wolpers  and  Ruska,  1939),  the 
reticular  structure  postulated  has  also  been  found  in  biological  gels. 
It  can  also  be  observed  in  gels  of  bacterial  cellulose  (Frey-Wyssling 
and  Muhlethaler,  1946),  where  we  found  cellulose  strands  of  about 
250  A  diameter.  Later  the  same  strands  were  discovered  in  cell  walls 
(Frey-Wyssling,  Muhlethaler  and  Wyckoff,  1948).  Fig.  86b 
shows  the  growing  tip  of  the  cellulose  wall  in  the  end  cell  of  a  thread 
of  the  alga  Spirog^ra. 

Whereas  the  gel  strands  of  vanadium  pentoxide  (Fig.  86a),  due 
to  the  atomic  number  23  of  ^^V,  produce  sufficient  contrast  in  the 
electron  microscope,  the  cellulose  strands  with  ^^C  must  be  shadowed 
to  produce  distinct  micrographs.  Figs.  86b-d  show  how  well  high- 
relief  pictures  of  gels  can  be  obtained  if  they  are  properly  prepared  and 
shadowed.  An  important  prerequisite  to  obtaining  such  results  is  the 
complete  removal  of  any  incrusting  material.  In  contrast  to  VgOg, 
biological  gels  are  not  only  full  of  water,  but  also  incrusted  with  all 
kinds  of  amorphous  substances,  such  as  hemicelluloses  and  lignins 
in  plant  cell  walls  or  proteins  in  animal  skeleton  materials.  Thus  Fig. 
86d  represents  tunicin  (Tunicate  cellulose)  from  the  mantle  of  Ciona. 
All  accompanying  substances  have  been  removed,  so  only  the  strands 


130      ir.NDAMEKTALS  OF  SUBMI  C  RO  SCOPI  C  MORPHOLOGY  1 

of  tunicin  are  left.  The  chitin  of  fungi  cell  walls  has  been  prepared  in  a 
similar  way.  Only  after  repeated  boiling  of  the  objects  in  io%  KOH 
is  the  texture  of  the  gel  disclosed  as  in  Fig.  86c,  where  two  different 
textures  (parallel  texture  and  dispersed  texture,  see  p.  95)  are  portrayed 
side  by  side.  If  such  methods  are  not  used,  many  of  the  biological  gels 
furnish  the  picture  of  a  homogeneous  film,  because  the  incrusting 
substances  have  the  same  electron-optical  behaviour  as  those  of  the 
gel  framework.  Clear-cut  micrographs  of  gels  can  only  be  obtained  if 
all  incrusting  substances  are  carefully  removed.  This  is  a  handicap  in 
the  electron  microscopy  of  protoplasm,  as  its  frame  substances  are 
far  less  resistent  to  chemical  agents  used  in  purifying  the  framework 
of  gels  than  are  cellulose  and  chitin. 

e.  Summary 

Gels  with  reticular  structure  are  characterized  by  the  existence  of  a 
framework  whose  constituent  parts  occupy  definite  mutual  positions. 
The  frame  strands  have  either  submicroscopic  or  amicroscopic  dia- 
meters. In  the  first  case  they  can  be  detected  by  electron  microscopy 
and  the  submicroscopic  morphology  of  such  gels  is  thus  accessible  ta 
detailed  direct  investigation  (Fig.  86). 

In  the  second  case  the  framework  is  formed  by  chain  molecules 
which  cannot  be  solvated  completely  and  maintain  c^txAn  junctions. 
If  these  junctions  are  released,  the  network  character  is  lost.  In  this 
case  the  reticular  gel,  which  originally  showed  only  limited  swelling, 
can  change  into  the  sol  state  via  the  gel  solution.  As  will  be  obvious 
from  this  definition,  there  exists  a  transitional  state  between  the 
reticular  and  the  corpuscular  dispersed  state.  It  will  require  further 
studies  to  elucidate  the  morphological  properties  of  such  gel  frames 
and  the  nature  of  the  bonds  in  the  junctions,  which  may  be  quite 
different  in  character  (see  p.  145). 


II.  THE  FINE-STRUCTURE  OF  PROTOPLASM 


The  great  conquests  in  the  field  of  structural  chemistry  have  been  real- 
ized by  means  of  analysis  and  synthesis.  Analysis  provides  information 
about  the  structural  units  and,  with  the  aid  of  synthesis,  their  position 
in  the  molecule  is  determined.  Although  no  inner  relationship  seems 
to  exist  between  chemistry  and  morphology,  i.e.,  between  our  know- 
ledge of  matter  and  that  of  shape,  this  same  procedure  has  been  the 
method  of  research  in  morphology :  detecting  the  structural  units  by 
analysis  and  determining  their  mutual  position.  The  latter  can  be  done 
by  direct  means  both  in  the  macroscopic  and  the  microscopic  domain 
and  thus  has  no  need  of  the  indirect  methods  used  in  organic  chem- 
istry. 

However,  for  the  elucidation  of  the  invisible  submicroscopic  struc- 
ture of  protoplasm,  in  so  far  as  it  is  not  yet  accessible  to  electron 
microscopy,  analysis  must  again  be  combined  with  some  kind  of 
synthesis.  It  is  true  that  this  is  not  a  matter  of  synthesis  in  the  sense 
of  organic  chemistry.  We  can  do  no  more  than  unite  the  structural 
units  obtained  by  analysis  in  a  scheme  which  enables  us  to  explain  the 
optical  and  physico-chemical  properties  of  protoplasm.  Because  of  the 
exceedingly  complicated  state  of  the  inner  morphologic  structure  of 
living  matter,  only  a  very  incomplete  solution  of  the  problem  is 
possible  in  this  way. 

In  this  situation  one  might  be  tempted  to  abandon  the  wearisome 
road  of  analysis  and  synthesis  and  simply  accept  protoplasm  as  a  given 
substance.  This  is,  however,  impossible  for  morphology  as  a  branch 
of  the  exact  sciences.  For,  so  long  as  there  are  possibilities  of  research, 
morphology  must  from  an  inner  necessity  continue  the  analysis  of 
living  matter  —  even  the  sacredness  of  the  human  body  failed 
as  a  taboo  in  former  times.  It  is  only  when  all  the  possibilities  of 
analytic  dissection  which  the  human  mind  places  at  its  disposal 
have  been  exhausted,  that  morphology  will  bow  in  awe  to  the  secrets 
of  nature. 


132 


FINE-STRUCTURE  OF  PROTOPLASM 

§  I.  Cytoplasm 


II 


a.  Molecular  Constituents  of  the  Cytoplasm 

The  chemical  composition  of  the  cytoplasm  is  described  here  only 
from  the  point  of  view  of  the  molecular  shape  of  its  compounds 
(Sponsler  and  Bath,  1942).  The  molecular  structures  concerned  are 
known  in  principle,  but  an  attempt  at  morphological  synthesis  of 
cytoplasm  with  the  aid  of  these  structural  units  is  impossible.  Never- 
theless, this  morphological  point  of  view  enables  us  to  explain  the 
physico-chemical  behaviour  of  cytoplasm  to  a  certain  extent. 

Proteins.  The  basic  substances  of  the  proteins,  isolated  by  means  of 
hydrolysis  and  paper  chromatography,  are  a-amino  acids  which  possess 
the  structure  given  in  Fig.  87a,  where  R  represents  a  group  of  C-atoms. 


a)  R  CHNHz-COOH 
cc 


rRChf- 


To  be  exact,  the  NH2-  and  COOH- 


^NH 


,NH2   basic 


RCH 


•^ 


b) 


'^CQOH  O'^'cf 


/vrt?;. 


\ 


CHR 


NH 


CO 


-RCH 


XO  ^o 


i 


XHR- 


^NH 


".CO 


i 


HOOC 


Fig.    87.    Molecular    structure    of 
amino  acids,  a)   Overall  formula; 
h)    principle    of  chain    formation; 
c)  polypeptide  chain. 


groups  should  be  bound  to  the  C-atom 
as  individual  atom  groups,  as  shown 
in  Fig.  87b.  It  can  easily  be  seen  that 
two  amino  acids  can  form  a  so-called 
dipeptide  by  eliminating  water.  If  this 
process  is  repeated  many  times,  a  long 
polypeptide  chain  is  formed,  the  ends 
of  which  have  been  left  open  in  Fig.  87c. 
Like  the  paraffin  chain,  it  is  kinked.  The 
distance  between  two  equivalent  groups 
is  3 . 5  A,  as  has  been  ascertained  by  means 
of  X-ray  analysis  of  crystalline  fibre 
proteins.  Only  the  >CO  and  >NH 
groups  are  similar  along  the  whole  length  ofthe  chain,  while  R  differs  ac- 
cording to  the  kind  of  protein  and  thus  is  responsible  for  the  great 
variety  in  this  class  of  substances.  The  zig-zag  chain  drawn  in  Fig.  87c 
can  be  considered  as  a  relatively  indifferent  frame,  which  cannot  be 
responsible  for  the  chemical  lability  which  we  know  the  cytoplasm 
to  possess.  Its  unusual  reactivity  is  due  to  the  side  chains  R. 

In  chemical  text  books  the  amphoteric  character  of  the  proteins  is 
often  explained  by  the  fact  that  amino  acids  possess  both  an  acid  and  a 
basic  group  (Fig.  87b).  However,  it  follows  from  the  structural  picture 
of  the  polypeptide  chain  that  these  groups  disappear  in  the  condensa- 
tion process,  thus  losing  their  capacity  for  dissociation.  If  in  spite  of 


CYTOPLASM 


135 


this  the  proteins  clearly  show  acid  or  basic  properties,  this  is  brought 
about  by  the  side  chains  which  in  their  turn  carry  free  CO  OH-  or 
NHg-groups.  This  happens  when  some  members  of  the  polypeptide 
chains   consist  of  dicarbo-amino  acids  or  diamino  acids  (Fig.  88). 


Acid  side  chains 
NH 

^CH-CH^-COOH 
CO 


/ 

NH 
\ 

/' 

CO 


Aspartic    ocid 


CH-CH^-CHp-COOH 


Glutamic   acid 


Lipophilic    side   chains 

,CH-CH,-CH 

/  ^CH- 

CO 

L  Pucine 

/ 

NH 

ycH-cH,^^ 

CO 
^       Phenyl    alanine 

Sulphur   containing 

side  chain 
/ 

NH 

yCH-CH.-SH 

CO 

Cysteine 


Basic   side  chains 

NH 
\ 


^CH-CH2-CH^-CH2-NH2 
CO 

Ornithine 

^LH-CH.-CH.-CHj-NH-C^ 

c'o  ^^^ 

^    Arginine   (Valine  -^  Guanidme) 

Hydrophilic   side  chains 

NH 

.iH-CH^-OH 

CO 

Serine 


\ 

/ 

NH 

\h-chp-(^oh 

CO 

^  Tyrosine 

Possible  chain   end 

CH,^CH2 
NH         \ 
\     ^CH! 

^•P        Proline 


Fig.  88.  Side  chains  R  of  the  polypeptide  chains. 


The  common  amino  acids  (valine,  leucine,  phenyl-alanine,  etc.) 
cannot  exercise  special  influence  on  the  reactivity  of  the  proteins,  but 
they  confer  upon  these  proteins  a  pronounced  lipidic  character,  since 
the  ends  of  the  side  chains  consist  of  methyl  or  phenyl  groups  (Fig. 
88).  In  many  cases,  however,  the  terminal  groups  carry  an  alcoholic 
hydroxyl  group  (serine,  tyrosine),  on  account  of  which  a  certain 
hydrophily  is  maintained. 

A  particularly  important  side  chain  is  cysteine  with  its  very  reactive 
sulphydryl  group.  As  will  be  shown  later,  this  group  very  easily  forms 
bridges  between  neighbouring  polypeptide  chains.  In  contrast  to  such 
constituents  of  protein  chains,  capable  of  bonding  and  thus  favouring 
further  polymerization,  cyclic  amino  acids  such  as  proHne  can  ter- 
minate the  main  valency  chains  and  thus  limit  the  apparently  endless 


134 


FINE-STRUCTURE  OF  PROTOPLASM  II 


polypeptide  chain  molecules.  The  proline  ring  can,  however,  also  be 
built  into  the  peptide  chain  (see  Fig.  173,  p.  546). 

Considering  the  variety  and  the  number  of  20  amino  acids  (besides 
some  rare  amino  acids,  Cohn  and  Edsall,  1943)  which  have  thus  far 
been  isolated  from  proteins,  and  in  view  of  the  fact  that  these  can  occur 
as  side  chains  at  various  points  along  the  polypeptide  chains,  we  realize 
that  the  protein  components  of  the  cytoplasm  represent  a  variegated 
mosaic.  It  follows  from  Fig.  88,  that  the  amino  acid  configuration 
-CH-NHg-COOH  does  ;w/ contribute  to  the  character  of  the  mosaic, 
since  it  is  only  responsible  for  the  peptide  interlinking.  The  chemical 
behaviour  of  the  polypeptides  of  protoplasm  is  determined  by  the 
end  and  side  groups  of  the  amino  acids,  to  which  often  little  attention 
is  paid. 

The  polypeptide  chains  show  a  number  of  properties  which  single 
them  out  from  the  other  substances  of  which  protoplasm  is  built  up. 

1.  The  principle  of  repetition  which  mhlology  we  know  as  segmenta- 
tion or  metamerism.  Most  high  polymer  substances  are  built  according 
to  this  principle.  In  the  majority  of  these  substances,  however,  iden- 
tical monomer  groups  are  repeated,  whereas  in  the  polypeptide  chains 
the  side  groups  R,  which  occur  at  regular  distances  of  3.5  A,  have 
different  constitutions.  Probably  the  typical  side  chains  also  repeat 
themselves  regularly,  but  their  period  is  much  greater  and  is  often  not 
accessible  to  experimental  analysis. 

2.  The  principle  of  specificity.  Owing  to  the  numerous  possible  side 
chains  R  and  the  unlimited  variety  in  their  arrangement  along  the 
polypeptide  chains,  an  almost  infinite  number  of  polypeptides  is  con- 
ceivable, distinguished  only  by  slight  difference  in  construction.  This 
difference  in  construction  may  result  in  a  different  chemical  behaviour 
which  becomes  apparent  in  the  specific  properties  of  the  proteins. 

3.  The  principle  of  contractility.  The  most  striking  property  of  poly- 
peptide chains  is  their  capacity  to  contract,  as  will  be  further  discussed 
on  page  559.  The  origin  of  the  mobihty  of  cells  (protoplasmic  flow, 
cilia,  contractible  fibrils,  etc.)  must  be  sought  in  these  molecular 
structural  units  and  for  this  reason  they  form  undoubtedly  the  most 
important  structural  elements  in  the  fine-structure  of  protoplasm. 

The  number  N  of  amino  acids  in  natural  polypeptides  seems  to 
obey  the  Bergmann-Niemann  rule  (1936/37)  N  =  2" -3™,  which  in- 
dicates that  there  must  be  some  threefold  symmetry  in  protein  mole- 


CYTOPLASM 


155 


:h,  ch. 


NH-, 


SH 


OH 


CH3 


cules.  Established  N-numbers  are  96  =  z%  144  =  z*-f,  288  =  z*-^^, 
^^^  ^   2«-32  etc.  (ScHEiBE,  1948).  These  complicated  proportions 
have  been  derived  from  crystalline  proteins. 
In  this  respect  there  exist  two  different  types : 

a)  Globular  proteins  consisting  of  isodiametric  macromolecules 
which  preferably  crystallize  in  the  system  of  cubic,  hexagonal  or 
•orthorhombic  closed  packing  (Fig.  90a). 

b)  Fibrillar  proteins  formed  by  expanded  polypeptide  chains  aggre- 
gated to  a  chain  lattice  (Fig.  90b).  In  the  chain  lattice  they  may  assume 
a  spiral  configuration  (spiral  chains,  Perutz,  195  i). 

In  the  second  type  the  crystallization  depends  on  the  regularity  of 
the  side  chains  R.  If  these  side  radicals  are  simple  as  in  silk  fibroin 
(Fig.  170),  where  they  consist  mainly  of  H-  and  CHg-groups,  the 
chains  combine  as  easily  as  polysaccharides  to  form  a  crystal  lattice.  As 
will  be  obvious  from  Fig.  89,  however,  this  is  not  possible  if  the  side 
chains  happen  to  be  of 
quite  different  lengths  and 
confio-urations.  These  con- 
ditions  can  be  compared 
with  the  arrangement  of 
bean-  or  peastalks.  Where- 
as there  is  no  difficulty  in 
uniting  a  great  number  of 
smooth  bean  stalks  into  a 
btmdle,  it  is  not  so  easy 

to  obtain  a  parallel  order  in  pea  stalks  with  their  numerous  twigs 
pointing  sidewise;  and  if,  moreover,  the  lengths  of  these  twigs  alter- 
nate in  an  irregular  manner,  the  resulting  structure  becomes  so  spaci- 
ous that  it  is  almost  impossible  to  bundle  them  together.  This  is  the 
case  with  complicated  polypeptide  chains. 

In  general  these  unwieldy  chains  are  folded  up  in  some  complex 
manner  to  form  globular  molecules.  Open  spaces  inside  these  macro- 
molecules  are  occupied  by  bound  hydration  water.  The  protein  part- 
icles crystallize  in  a  molecular  lattice  of  close  packing.  As  their  size 
is  considerable,  some  space  accessible  to  additional  water  or  even  dye- 
stuff  molecules  is  left  between  the  spheres  (Fig.  90a).  Such  crystals 
therefore  swell  or  shrink  and  can  be  stained  in  aqueous  solutions. 

Rigorous  dehydration  removes  not  only  the  water  between  the 


CH. 


COOH 


NH2 


NH 


Fig.  89.  Unequallengths  of  polypeptide  side  chains  R. 


136 


FINE-STRUCTURE  OF  PROTOPLASM 


11 


macromolecular  Spheres,  but  also  the  hydration  water  inside  the  glob- 
ular molecules,  so  that  their  structure  is  destroyed  and  the  solubilitv 
of  the  protein  is  abolished.  This  physico-chemical  transformation  of 
soluble  proteins  is  called  denaturation.  There  are  some  indications  that 
the  denaturation  of  globular  proteins  consists  in  an  unfolding  of  the 


? 


«) 


1/ 


/  \ 


V) 

Fig.  90.  Model  of  the  fine-structure  of  protein  (from  Frey- 
Wyssling,  1944b).  a)  Lattice  of  spherical  macromolecules 
(slightly  anisotropic  or  isotropic;  highly  hydrated);  b)  chain 
lattice  of  thread  molecules  (strongly  anisotropic,  barely  hy- 
drated). The  transformation  a ->  b  is  termed  "denaturation". 


wrapped-up  polypeptide  chains.  In  Fig.  90  the  denaturation  of  globular 
into  fibrillar  proteins  is  indicated  by  an  arrow  a  ->  b.  The  inverse 
reaction,  the  transformation  of  the  denatured  protein  into  globular 
molecules,  is  usually  impossible  in  vitro,  but  it  must  occur  readily 
in  vivo.  Forms  of  protein  molecules  intermediate  between  the  globular 
and  chain  configurations  are  not  well  known.  Such  intermediate 
shapes  do  not  crystallize  out,  but  it  is  probable  that  they  are  involved 
in  protein  metabolism.  Fig.  95c  (p.  144)  shows  the  length  of  the  poly- 
peptide chain  which  is  folded  up  in  a  globular  protein  molecule  of 
100  A  diameter, 

Ranzi  (195  i)  has  devised  a  method  for  distinguishing  globular 
from  fibrillar  proteins  in  dilute  solutions.  The  first  show  an  increase 
in  viscosity  with  KCNS  as  compared  with  a  test  solution  equimolar 


I  CYTOPLASM  137 

in  KCl,  whereas  the  second  show  a  decrease.  With  this  test  Ranzi  has 
shown  that  the  euglobulin  of  frog  embryos  is  fibrillar  between  p^ 
5.5  and  8.5;  but  beyond  this  range  it  is  globular.  Since  the  develop- 
ment of  the  frog  ectoderm  in  tissue  cultures  is  only  possible  within 
the  range  of  pH  5  and  9,  there  is  an  indication  that  fibrillar  proteins 
are  indispensable  for  any  manifestation  of  morphogenesis. 

Lipids.  The  biological  concept  of  lipids  comprises  all  substances 
which  are  hydrophobic.  This  concept  is  therefore  characterized  by  a 
negative  property  (insolubility  in  water)  rather  than  by  a  positive  one 


:y  OH-CH2  y     CH3 

-c                     I  ~^ 

§■  OH-CH "I  CH3 

■0                   I  Q. 

1^  OH-CH2  -■    CH.f 


3  Fatty  acids  +  Glycerol 


0 

Lipophilic      CH3  \y\y\/sy\y\/\/\/\  C-O-CH,  /\/\/vv/VN/s,/\/sy\/v/\/'VN/  CW-,    Lipophilic 

Falty  acid  Higher  alcohol 

Wax 

Fig.  91.  Molecular  structure  of  lipid  chains. 

(solubility  in  organic  liquids).  For  this  reason  it  comprises  different 
families  of  substances  such  as  terpenes,  waxes,  fats,  sterines,  etc.,  of 
which  the  last  two  take  part  in  building  up  protoplasm. 

True  lipids  are  characterized  by  the  fact  that  all  their  free  end  groups 
consist  of  typically  Hpophilic  groups.  This  is  especially  obvious  in  the 
case  of  fats,  which  represent  esters  of  the  three-valent  alcohol  glycerol 
with  fatty  or  oleic  acids.  As  a  result  of  the  esterification,  thehydrophilic 
groups  of  the  original  products  are  screened,  as  shown  in  Fig.  91.  In 
the  same  way  the  hydrophilic  groups  are  masked  in  waxes  which  are 
formed  by  the  esterification  of  higher  alcohols  with  higher  fatty 
acids  (Table  XXVII,  p.  296).  It  is  difficult  to  say  why  they  are  screened 
in  the  course  of  the  metabolic  process,  but  in  any  case  these  lipids 
contrast  strongly  with  the  hydrophilic  compounds  of  living  cytoplasm 
and,  if  they  are  formed  in  excess,  we  observe  the  well-known  phenom- 
enon of  fatty  degeneration  of  protoplasm.  A  correct  balance  between 
hydrophilic  and  Hpophilic  compounds  in  living  matter  is  essential. 

In  contrast  to  fats,  most  lipophilic  compounds  of  the  cytoplasm 
carry  at  least  one  hydrophiUc  group,  which  serves  to  bring  about  the 


138 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


Fie. 


92.  Molecular  structure  of  cholesterol; 
terminal  group  OH. 


contact  with  neighbouring  hydrophilic  groups.  This  applies  in  partic- 
ular to  the  important  group  of  the  sterines  (Windaus,  1923),  from 
among  which  the  formula  of  the  complicated  cholesterol  C27H45OH 
is  reproduced  (Fig.  92).  The  molecule  contains  four  rings  and  a  double 

bond.  According  to  X-ray  ana- 
lysis (Bernal,  1932)  the  length 
of  the  molecule  is  17-20  A  and 
its  cross-section  only  7.2  A.  The 
elongated  form  tallies  well  with 
the  optical  finding  that  dissolved 
cholesterol  molecules  can  be 
easily  oriented  in  a  field  of  flow 
and  like  most  rod-shaped  mole- 
cules show  positive  birefring- 
ence of  flow. 

Phosphatides. 
Because  of  their 
solubility         in 
ether,  phosphat- 
ides are  usually  also  counted  among  the  lipids,  but 
besides  their  lipid  character  they  possess  a  marked 
tendency  toward  hydrophily,  which  is  shown  by  their 
adsorption  of  water  and  the  occurrence  of  myelin  forms. 
Thus,  phosphatides  represent  compounds  which  are 
intermediate  between  hydrophobic  and   hydrophilic 
substances  and  for  this  reason  belong  to  the  most 
important  intermediates  between  the  representatives 
of  these  two  extreme  groups  in  the  cytoplasm.  By 
way  of  example  we  may  mention  lecithin  which,  like  o^ 
the  fats,  consists  partly  of  glycerol  and  fatty  acids.    ^- 
In  this  case,  however,  only  two  OH-groups  are  occu-    "^ 
pied  by  fatty  acids,   the  third   being   esterified    by 
phosphoric  acid  and  the  latter  in  its  turn  by  the  amino 
alcohol  choline  (Fig.  93). 

Choline  HOCH2-CH2-N(CH3)30H  is  a  base  whose 
hydroxy  1  group  is  attached  to  a  methylated  ammonium 
group.  The  three  methyl  groups  might  conceivably 
give  the  end  group  -N(CH3)30H  of  the  molecule  a  lipophilic  character 


O 

HO-k=0 
0 

CH2  CH2 

I  I 

0  0 

1  I 


CHy 


CH2     H^C 


CH2 


H2C 


CH2      H2C 

:  j 

\h2        H2( 


CH2 


H2C 


CH2       H2C 


H.C 


CH2    O 

<\ 
o 

CH2 


CH2    H2C 


HpC 


CH2 


CH2      H2C 


H,C 


CH, 


CH2      H2C 


H.C 


CH, 


\ 


Lecilhin 

Fig-    93- 
Molecular  struc- 
ture of  lecithin. 


I  CYTOPLASM  139 

in  spite  of  the  hydrophilic  OH-group.  This,  however,  is  not  the  case. 
For,  curiously  enough,  alkyl  groups  (-CH3,  -C2H5)  bound  to  am- 
monium nitrogen  are  hydrophilic  in  behaviour  (like  methyl  bound  to 
oxonium  oxygen,  which  makes  pectic  acid  and  methyl  cellulose 
soluble  in  water,  seep.  60).  For  this  reason  the  ammonium  end  group 
tends  to  escape  from  the  neighbourhood  of  the  lipophilic  end  groups 
of  the  fatty  acids.  Consequently,  the  lecithin  molecule  resembles  a 
tuning  fork  (Fig.  94),  in  contrast  to  fats  which  can  be  represented 
schematically  by  a  three-pronged  fork  without  a  handle.  The  prongs 
of  the  fork  represent  the  lipophilic  pole,  the  handle  of  the  fork  the 
opposite  hydrophilic  pole  of  the  lecithin  molecule. 

The  phosphatides  react  with  the  protein  chains  of  the  cytoplasm 
by  combining  with  either  the  lipophiHc  or  the  hydrophilic  end  groups 
of  the  side  chains,  as  indicated  in  Fig.  94.  This  junction  is  not  of  a 


o  °  o 
^oOH- 


o   o 
000 
■OH     OH- 
■  CH-  K  000 

'-"j  >  00 

CHy 

-CH3 


CH, 

CH, 

CH:, 

CH3 

CHs 

Lecithin  Fat 


Water  Lecithin  Polypeptide  chain 

Fig.  94.  Relation  between  polypeptide  side  chains  and  lecithin;  o  =  water  molecule. 

chemical  nature,  for  the  phosphatides  can  be  extracted  from  the 
cytoplasm  with  ether.  Nevertheless  the  phosphatide  molecules  occupy 
quite  definite  places,  according  to  the  character  of  the  side  groups  in 
the  polypeptide  molecules.  Lipids  without  hydrophylic  groups,  such 
as  fats,  can  combine  only  with  the  lipophiHc  side  groups.  For  this 
reason  their  possible  combinations  with  protein  chains  are  limited.  As 
shown  in  Fig.  94,  they  can  only  enter  into  relation  with  hydrophilic 
side  chains  by  interposition  of  phosphatides  or  other  intermediates. 
The  sterines  possess  a  polar  structure  similar  to  that  of  the  phos- 
phatides, but  lecithin  is  more  reactive:  of  its  two  hydroxyl  groups  at 
the  hydrophiUc  tail  one  is  acid  (attached  to  phosphorus)  and  the  other 
basic  (attached  to  nitrogen).  For  this  reason  it  can  form  salts  with 
basic  as  well  as  with  acid  groups  of  the  polypeptide  chain.  Phos- 
phatides can  therefore  react  with  nearly  all  end  groups  occurring  in 
the  side  chains  of  proteins.  Sterines,  on  the  contrary,  are  only  capable 
of  forming  esters.  Finally,  for  fats,  all  side  chains  of  the  polypeptides, 
with  the  exception  of  the  lipophilic  end  groups,  are  blocked.  This 


I40 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


shows  clearly  how  opportunities  of  entering  into  the  protoplasm 
multiply  as  the  lipids  become  more  hydrophilic  in  character. 

Chemical  composition  of  the  cytoplasm.  The  proportions  of  the  com- 
pounds described  above  vary  considerably  in  the  cytoplasm  (Table 
XrV).  This  is  especially  so  for  lipids,  carbohydrates  and  water-soluble 
compounds.  Although  the  two  analyses  in  Table  XIV  represent  ex- 
treme cases,  it  is  evident  that  protein  is  the  main  constituent  of  the 
cytoplasm.  Only  small  amounts  of  the  other  constituents  are  structural 
compounds.  In  the  cytoplasm  of  the  sUme  mould  Reticularia  there  are 
considerable  quantities  of  reserve  substances  such  as  carbohydrates, 
soluble  nitrogenous  compounds  and  probably  most  of  the  lipids. 
Whereas  slime  moulds  can  be  dried  and  analysed  without  difficulty, 
the  cytoplasm  of  tissues  with  solid  cell  walls  can  not.  In  this  case  the 
membranes  have  to  be  broken  in  a  blendor  and  the  cell  contents  sus- 
pended in  an  appropriate  solution,  from  which  the  constituents  of 
the  cell  wall,  the  cytoplasm,  the  plastids  and  the  nucleus  must  be 
separated  by  centrifuging  and  by  fractionated  salting  out,  e.g.  with 
ammonium  sulphate  (Menke,  1938a).  The  fraction  corresponding  to 
the  cytoplasm  yields  the  analysis  recorded  in  the  right  column  of 
Table  XIV.  By  this  method  of  preparation,  all  water-soluble  com- 


TABLE  XIV 

CHEMICAL  COMPOSITION  OF  CYTOPLASM 


Plasmodium 
ReticuLria  Ijcoperdon 

KlESEL   1930,  p.   257 


Leaves 

Spinacia  okracea 

Menke  1938a,  p.  289 


Proteins  

Soluble  N-compounds. 

Lipids 

Phosphatides 

Cholesterol 

Nucleic  acids 

Carbohydrates   .    .    .    . 

Ash 

Unknown 


29.07 
12.00 

19.05 

4.67 

0.58 

5.68 

25.08 

5.87 


100.00 


0/ 

o 

85.0 

0.7 


3-1 
II. 2 


1 00.0 


CYTOPLASM 


141 


pounds,  such  as  sugars,  amino  acids  and  amides,  are  lost;  hence  the 
proportion  of  insoluble  proteins  is  increased.  It  is  noteworthy  that 
the  proportion  of  lipids  is  very  low.  The  considerable  "unknown" 
fraction  probably  comprises  the  nucleic  acids,  which  in  the  present 
case  have  not  been  separately  determined. 

b.  Physicochemical  Behaviour  of  Proteins 

Si^e  of  globular  protein  f/wlecules.  The  molecular  weight  of  globular 
proteins  can  be  determined  with  the  aid  of  the  ultracentrifuge. 
SvEDBERG  has  found  that  the  weights  of  quite  different  proteins  are 
similar.  Thus  the  molecular  weight  of  pepsin,  insuHn  and  egg  albumin 
is  34,500 (SvEDBERG,  i93i).In  many  instances  there  is  an  approximate 
multiple  of  this  figure,  such  as  70,200  for  horse  serum-albumin.  After 
a  systematic  investigation,  Svedberg  (1938b)  came  to  the  conclusion 
that  in  protein  molecules  there  is  a  fundamental  unit  of  molecular 
weight  17,600. 

TABLE  X\' 
WEIGHTS  AND  SIZE  OF  GLOBULAR  PROTEIN  MOLECULES 


Substance 


0 
ci. 

Q 

> 


J3 

EC 


3 


c   ^ 

O     O 

£  'E 
I  S   ° 

k  -^ 


v-l 

^ 

1 

nt 

u 

1 

-C 

J2 

■l-J 

°<: 

3 

0 
u 

u 
£ 

particles 

din  A 

3 

u 

< 

n 

cr 

-a 

Q 

C/3 

1 

1 

Lactalbumin  a,  rnyoglobin i  17600  200 

Lactoglobulin,  ovalbumin,  zein,  pepsin, 

insulin 2  35  200  400 

Serum  albumin,  CO-haemoglobin,  yel- 
low ferment 4  70400  800 

Serum  globulin 8  140800  1600 

Edestin,  excelsin,  phycocyanin,  phyco-  I 

erythrin,  catalase 16  281600  3200 

Haemocyanin    (cleavage    component),  | 

urease 24  422400  4800 

Haemocyanin  (cleavage  component).    .  48  845  000  9  600 

Haemocyanin  (Calocaris) j     96  1690000  19200 

Haemocyanin  (Rossia) I  192  13380000  38400 

Haemocyanin  (Helix  pomatia)   ....  1-384  6760000  j  76800 


c 

c 


34.5        2.87  1.8  I  0.07 
43.5        5-75, 2.o;   0.14 


55 

II. 5 

2.2 

0.28 

69 

23 

2-5 

0.56 

87 

46 

2.8 

1. 12 

100 

69 

3.0 

1.68 

125 

138 

3-4 

3.36 

158 

275 

3.8 

6.72 

200 

550 

4.2 

13.44 

250 

IIOO 

4-7 

26.88 

j^Z  FINE-STRUCTURE  OF  PROTOPLASM  II 

Table  XV  gives  examples  of  this  multiple  series  (cf.  K.  H.  Meyer, 
1940a,  p.  409).  It  shows  how  the  Svedberg  units  combine  in  2's,  4's, 
8's,  i6's  etc.  There  are,  however,  not  only  multiples  of  2,  but  also  of  5 
(e.g.  24),  a  fact  which  recalls  the  Bergmann-Niemann  rule.  Up  to 
384  units  may  be  combined  in  one  molecule.  The  aggregation  or 
dissociation  of  these  large  particles  depends  on  p^  conditions. 

Since  the  nitrogen  content  of  proteins  is  16%,  the  average  mole- 
cular weight  of  the  amino  acids  in  proteins  is  6.25  x  N  =  87.5,  if  no 
allowance  is  made  for  basic  amino  acids  with  more  than  one  N-atom. 
With  this  figure,  the  approximate  number  of  amino  acids  in  globular 
protein  molecules  can  be  calculated.  The  Svedberg  unit  contains 
about  200  (which  is  near  to  the  figures  of  2^  x  3  =  192  or  2^  x  3^ 
=  216)  and  the  largest  particles  mentioned  in  Table  XV  contain  more 
than  75,000. 

Globular  protein  molecules  can  be  photographed  in  the  electron 
microscope  (Fig.  84a,  b,  p.  126).  The  average  space  needed  by  an 
amino  acid  (Fig.  181,  p.  365)  is  3.5  X  4-6  X  10  A^  =  161  A^.  In  the 
electron  microscope  a  sphere  of  diameter  50  A  can  be  readily  recog- 
nized. Its  volume  is  50=^  x  n/G  A^  =  65,500  A^.  This  corresponds  to 
about  400  amino  acids.  Protein  molecules  with  two  Svedberg  units 
must  therefore  be  easily  visible  in  the  electron  microscope,  while  the 
Svedberg  unit  itself  is  just  at  the  limit  of  the  resolving  power. 

A  similar  result  is  obtained  if  we  remember  (Fig.  31b,  p.  34)  that 
in  an  aliphatic  chain  the  carbon  atoms  are  lined  up  at  intervals  of 
1.25  A,  the  distance  between  neighbouring  chains  being  5  A.  Thus 
40  X  10  X  10  =  4000  carbon  atoms  can  be  placed  in  a  cube  of  50^  A=^. 
This  would  yield  a  molecular  weight  of  48,000,  which,  again,  corre- 
sponds roughly  to  2  Svedberg  units. 

A  third  determination  is  possible  based  on  the  average  density  of 
proteins,  which  is  1.33.  Knowing  the  absolute  weight  of  a  Svedberg 
unit  (17,600  divided  by  the  Loschmitt  number  6.06  X  lo^^),  the 
volume  of  the  molecule  can  be  calculated.  Considered  as  a  sphere,  its 
diameter  is  34.5  A.  In  Table  XV  the  size  of  the  macromolecules  in 
the  multiple  series  of  globular  proteins  has  been  calculated  in  this  way 
(Frey-Wyssling,  1949a),  the  dimensions  found  being  as  shown  in 
Fig.  84a,  b  (p.  126).  As  a  result  we  may  note  that  globular  macro- 
molecules  of  protein  with  at  least  400  amino  acids  or  a  molecular 
weight  of  about  40,000,  are  within  the  resolving  power  of  the  electron 


I  CYTOPLASM  145 

microscope.  If  the  diameter  d  is  calculated  from  the  mean  space  of 
161  A^  needed  by  an  amino  acid,  somewhat  largervalues  are  obtained. 
The  length  of  the  completely  unfolded  polypeptide  chain  of  the 
denatured  molecule  is  found  by  multiplying  the  chain  period  of  3.5  A 
(trans-)  or  2.8  A  (cis-configuration,seep.  346)  by  the  number  of  amino 
acid  residues  in  the  molecule.  With  the  period  3.5  A,  the  lengths  L 
indicated  in  Table  XV  are  obtained.  Of  course,  these  figures  are 
maximal  values  which  are  not  realized,  since  the  chains  will  never 
expand  completely,  but  will  assume  a  bent  or  curled  shape. 

Surface  films  of  proteins.  Although  globular  proteins  are  soluble  in 
water  or  salt  solution,  not  all  parts  of  the  molecule  show  an  affinity 
for  water.  The  polypeptide  chains  which  are  coiled  up  in  an  unknown 
manner  within  globular  molecules  carry  hydrophilic  and  lipophilic 
(hydrophobic)  side  groups.  The  former  strive  for  contact  with  water 
but  the  latter  "avoid"  it,  hence  the  proteins  can  be  spread  as  molecular 
films  on  the  surface  of  water  (Gorter  and  co-workers,  1955).  One 
milligram  of  protein  can  cover  a  surface  from  one  to  more  than  two 
and  a  half  square  metres;  assuming  a  density  of  1.53,  this  means  films 
of  7.5  to  3  A  thickness  (Adam,  1941,  p.  87).  These  values  show  that 
the  molecular  film  cannot  consist  of  spherical  macromolecules,  but 
that  these  protein  globules  flatten  and  uncoil  to  form  protein  chains. 
This  spreading  of  the  macromolecule  allows  all  hydrophiUc  groups 
to  make  contact  with  the  water  surface  and  all  hydrophobic  groups 
to  turn  away  from  it  towards  the  air.  If  the  surface  film  is  larger  than 
one  square  metre,  it  is  liquid,  i.e.  the  flattened  molecules  retain  their 
mobility  and  may  change  their  relative  positions  on  the  water  surface. 
However,  as  soon  as  the  film  is  compressed  to  an  area  of  one  square 
metre,  it  becomes  solid,  rigid  and  insoluble;  the  molecules  lose  their 
individuality  and,  because  they  stick  closely  together,  they  can  no 
longer  be  hydrated.  They  assume  the  state  of  fibrous  proteins  and  as 
such  become  insoluble.  This  change  of  solubility  is  known  as  "de- 
naturation",  mentioned  on  p.  136.  Merely  shaking  a  protein  solution 
often  suffices  to  form  a  foam  of  insoluble  denatured  protein. 

If  the  molecular  weight  of  the  protein  is  known,  the  area  per  mole- 
cule in  a  surface  film  of  7,5  A  thickness  can  be  calculated,  as  has  been 
done  in  Table  XV  (p.  141).  If  this  area  is  considered  to  be  circular, 
the  diameter  D  of  the  circle  can  be  compared  with  the  diameter  d  of 
the  globular  molecule.  For  small  protein  molecules  the  ratio  D/d  is 


144 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


about  two,  and  for  larger  ones  four  to  five  (Fig.  95  a,  b).  This  means 
that  the  area  of  the  flattened  molecule  is  four  to  twenty  times  bigger 
than  the  cross-section  or  projection  of  the  spherical  molecule  before 
spreading.  The  polypeptide  chain  may  wind  about  in  this  area.  If  the 
cross-section  of  such  a  chain  measures  4.6  x  10  A  as  in  the  chain 
lattice,  its  length  L  can  be  computed. 


Fig.  95.  Surface  film  of  a  protein   (from  Frey-Wyssling,   1949a).  a)  Globular 

molecule  of  100  A  diameter;  b)  spread  to  a  surface  layer  7.5  A  thick;  c)  denatured 

to  a  polypeptide  chain  11,600  A  long. 


The  chain  length  L  obtained  for  the  globular  particles  is  shown  in 
Table  XV.  For  instance,  a  protein  molecule  of  24  Svedberg  units 
with  a  molecular  diameter  of  100  A  harbours  a  chain  of  11 600  A 
=  1.16  ^  length  (Fig.  95  b,  c).  An  even  greater  length  is  obtained  if 
it  is  assumed  that  this  molecule  consists  of  4800  amino  acids,  each  of 
which  contributes  3.5  A  to  the  chain  length;  this  yields  L  =  1.68  fi. 

Since  globular  proteins  denature  so  easily,  we  may  ask  what  types 
of  force  hold  together  the  inner  architecture  of  these  macromolecules? 
They  must  be  rather  weak,  because  they  are  broken  by  mere  contact 
of  the  globular  molecules  with  a  water  surface.  On  the  other  hand, 
the  expanded  molecules  form  a  solid  film,  which  has  the  character  of 
a  fibrous  protein.  It  must  be  supposed  that  the  individual  molecules 
have  been  fused  to  a  two-dimensional  molecular  aggregate.  Here, 
instead  of  intramolecular  forces  holding  together  the  coiled,  folded  or 
laminated  internal  structure  of  the  globular  molecule,  inter  molecular 
forces  unite  neighbouring  expanded  molecules.  The  same  thing  occurs 
when  globular  protein  molecules  are  connected  to  form  beaded  chains. 


CYTOPLASM 


M5 


These  bonds  between  macromolecules  are  very  important,  because 
they  transform  the  protein  from  the  state  of  a  corpuscular  sol  into 
that  of  a  reticular  gel.  But  in  spite  of  this  fact  it  is  probable  that  the 
intramolecular  and  intermolecular  forces  are  alike,  because  it  makes 
no  difference  whether  distant  parts  of  one  polypeptide  chain  or 
sections  of  two  different  chains  react  with  each  other.  In  both  cases 
attractive  forces  between  side  groups  are  involved.  The  places  where 
the  side  chains  are  mutually  connected  will  again  be  called  "junc- 
tions" (see  p.  67)  and  the  nature  of  these  points  of  attachment  will 
now  be  discussed  in  more  detail. 

The  theory  of  junctions^.  The  attraction  between  the  side  groups  of 
neighbouring  protein  mole- 
cules may  be  of  a  number  of  V 
different  types.  Some  of  these 


I 

CH3 
■cHj      y 
CH3 


CH3- 


o°o°  o 

O  O    n 


.C/" 


^OH 


OH  NH3 


SO4 


0 
-c- 


-0H°  OHf- 

O  O    O    o 

o  o 


0  ^ 

II 

CO-NH3 

Salt    formation 


-COOH* 


o 
'^  o      ° 


-0- 


Ester  bridge 


W 
-S S- 


0  NH 
II  II 
C-NH-C- 

Amide   bridge 


Sulphur  bridge 


-0- 


Ether  bridge 


possibilities  are  shown  in  Fig. 
96.  Both  hpophilic  and  hy- 
drophilic  groups  may  attract 
each  other.  Salt-Uke  or  ester- 
like bonds  can  be  formed  be- 
tween neighbouring  acidic 
and  basic  or  alcoholic  groups, 
and  even  main  valency  bonds 
may  be  operative,  forming 
ether-,  acid  amide- or  sulphur 
bridges.  Not  all  side  chains 
take  part  in  these  reactions, 
but  a  certain  number  with 
free  end  groups  will  combine 
with  lipids,  hydrophiHc  groups  or  water,  as  has  already  been  described 
(Fig,  94,  p.  1 39).  Furthermore,  they  form  points  of  attraction  for  ions 
of  the  inorganic  salts  which,  according  to  their  charge,  will  gather 
round  acidic  or  basic  groups.  It  is  important  that  the  end  groups  of 
many  side  chains  remain  free,  for  if  they  were  all  interlinked,  the 
result  would  be  a  molecular  aggregate  of  very  small  reactivity. 

There  exist  four  kinds  of  junctions  keeping  together  the  molecules 
formed  by  polypeptide  chains.  In  Fig.  96  these  have  been  numbered 
I-IV,  and  thev  can  be  characterized  as  follows : 

^  In  German:  Haftpunkt-Theorie 


Fig.    96.    Schematic   representation  of  junction 

possibilities   between  neighbouring  polypeptide 

chains;  o  =  water  molecule. 


146  FINE-STRUCTURE  OF  PROTOPLASM  II 

I.  Homopolar  cohesive  bonds,   i.e.,   mutual   attraction   of  lipidic 

groups ; 
11.  Heteropolar  cohesive  bonds,  i.e.,  attraction  between  groups  of 
pronounced  dipole  character; 

III.  Heteropolar  valency  bonds,  i.e.,  formation  of  salts  and  esters; 

IV.  Homopolar  valency  bonds  or  bridge  formation. 

We  shall  briefly  discuss  the  characteristics  of  these  types  of  bonds. 

I.  Homopolar  cohesive  bonds  are  of  the  same  kind  as  the  forces  which 
keep  a  paraffin  crystal  together.  Very  little  is  known  about  the  causes 
of  the  attraction  between  lipophilic  groups,  for  the  electric  charges 
in  these  substances  are  distributed  so  regularly  that  the  resulting  field 
of  force  is  negligible,  in  contrast  to  dipole  molecules.  It  has  therefore 
been  suggested  that  weak  dipole  moments  are  induced  in  the  neigh- 
bouring molecules  by  periodic  oscillations  in  the  field  of  force, 
brought  about  by  vibrations  within  the  electronic  configurations 
(Bartholome,  1956).  We  know  more  about  the  energy  of  these 
bonds.  As  follows  from  Table  IV  (p.  32),  the  cohesion  between 
methyl  and  methylene  groups  is  the  weakest  among  the  cohesive 
forces.  This  kind  of  bond  is  loosened  by  small  amounts  of  energy  and 
is  therefore  strongly  sensitive  to  temperature  changes.  For  this  reason, 
paraffins,  fats  and  waxes  melt  at  relatively  low  temperatures  in  spite 
of  their  high  molecular  weight. 

A  similar  behaviour  is  shown  by  the  homopolar  cohesive  bonds 
between  lipidic  side  groups  of  neighbouring  polypeptide  molecules. 
By  a  rise  in  temperature,  this  kind  of  junction  is  easily  loosened. 
Similarly,  lipids  and  phosphatides  which  are  attached  to  these  groups 
become  more  mobile.  This  causes  the  living  matter  to  liquefy  to  a 
certain  extent:  the  rapidly  decreasing  viscosity  of  the  cytoplasm  as  a 
function  of  the  temperature  is  a  well-known  phenomenon  (Heil- 
BRUNN,  1930).  Fig.  97  shows  the  rapid  decrease  in  the  viscosity  of 
amoeba  cytoplasm  between  10  and  20  degrees  C,  which  is  probably 
due  to  the  rupture  of  lipidic  bonds  in  addition  to  the  viscosity  decrease 
of  the  intermicellar  water.  At  temperatures  beyond  20°  C.  another 
process  sets  in,  vi2.,  a  shrinkage  at  those  spots  where  hydrophilic 
chain  ends  come  together,  resulting  in  some  kind  of  solidification.  At 
the  same  time,  however,  the  rupture  of  lipidic  junctions  continues  and 
at  25°  C.  clearly  surpasses  the  solidification  brought  about  by  de- 
hydration. By  raising  the  temperature  still  further,  the  curve  should 


CYTOPLASM 


147 


finally  rise  again,  since  in  that  case  the  cytoplasm  would  solidify  as  a 
result  of  shrinkage.  Death  with  coagulation  occurs  at  about  42°  C. 

Since  in  the  physiological  temperature  range  a  rise  in  temperature 
would  certainly  not  be  able  to  rupture  either  heteropolar  cohesive 
bonds  or  main  valency  bonds,  it  is  permissible  to  attribute  the  change 


v 

Pf) 

p 

A 

0 

> 

20 

A 

"A 

I 

i 

\\ 

I'i 

, 

10 

\ 

^         ° 

r 

\ 

5 

\       J 

\ 

\/ 

\ 

\ 

b-o-^ 

i 

0 

5 

K 

J 

7; 

)            2( 

■)            2t 

)             3 

0 

3 

5 

°C 

Fig.  97.  Viscosity  of  the  cytoplasm  of  the  amoeba  (from  Heil- 

BRUNN,  1930).  Abscissa:  temperature  in  °C.  Ordinate:  viscosity 

(time  in  seconds,  which  a  crystal  enclosed  needs  to  travel  halfway 

through  the  cell  under  the  influence  of  gravity). 


in  viscosity  of  the  cytoplasm  primarily  to  the  abolition  of  homopolar 
cohesive  bonds.  The  weakness  of  the  homopolar  cohesive  bond  is 
demonstrated  by  the  exceedingly  small  surface  tension  of  proto- 
plasmic membranes  (i  dyne/cm  against  nutrient.  Table  XXI,  p.  166),  in 
comparison  with  water  (71.6  dynes/cm  against  air.  Table  V,  p.  43), 
where  the  surface  is  formed  by  heteropolar  HgO  molecules. 

11.  Heteropolar  cohesive  hands  are  of  a  quite  different  character.  The 
underlying  attractive  forces  are  due  to  dipole  moments  (p.  19), 
which  are  mostly  so  strong  that  they  are  designated  as  secondary  or 
residual  valencies. 

Of  recent  years  the  semi-chemical  character  of  heteropolar  cohesive 
bonds  has  come  to  the  fore,  since  they  are  designated  as  hydrogen 
bonds  or  hydrogen  bridges  (Pauling,  1940).  Wherever  dipolar  groups 
with  hydrogen  atoms  situated  in  the  periphery  (OH-,  NHg-groups) 


148  FINE-STRUCTURE  OF  PROTOPLASM  II 

are  present,  the  possibility  exists  of  their  being  attracted  electro- 
statically by  the  local  negative  charges  of  the  dipole  groups  of  neigh- 
bouring molecules.  To  a  certain  extent  the  hydrogen  atom  acts  as  an 
intermediary  between  the  two  molecules  and  connects  them  by  forming 
some  kind  of  bridge.  This  is  represented  in  Fig.  98  for  two  poly- 
peptide molecules  running  in  opposite  di- 

c^o H-  N  rections.  The  hydrogen  atom  is  ifted  some- 

RHC  CHR  what    out  of  its  position  in   the   original 

N-H o^c  molecule  and  it  looks  as  if  part  of  the  hy- 

O'^c  N H-        drogen  valency  is  transferred  to  the  neigh- 

CHR       RHC  bouring  molecule.  Clearly,  this  schematic 

H-N  c-^o  representation  of  the  "secondary  valencies" 

9^'^ >^-i^  gives  only  a  very  incomplete  idea  of  the 

^HC  CHR  interactions  of  the  two  electric  fields  which 

f'"'^' o-^c  attract    the    positively    charged   hydrogen 

°""^\  ^/v- "«•  •        atom  with  different  field  strengths. 

CHR       RHC^  j£^    £qj.   5^g£(-   reasons,   the   heteropolar 

"••■^ "■"\  /'^°  -        groups  (OH,  COOH,  CHO,  NH^  etc.)   of 

„    ^^  ,  ,      ,    ,       neighbouring  molecules  cannot  come  near 

Fig.   98.  Hydrogen  bonds  be-  °  ° 

tween  polypeptide  chains.       enough  together,  their  electric  fields  attract 

water  molecules.  Instead  of  hydrogen 
bridges,  a  hydration  layer  is  formed  between  them  (Fig.  96,  p.  145)  and 
it  is  obvious  that  with  this  kind  of  junction  the  cohesion  depends  on 
the  number  of  water  molecules  between  the  two  end  groups,  i.e.,  on 
their  hydration.  For  this  reason  heteropolar  cohesive  bonds  are  j-^/zj-zV/Vf 
fo  hydration  changes. 

Swelling  depends  largely  on  the  presence  of  inorganic  ions,  in 
which  case  the  so-called  ion  series  of  Hofmeister  holds  good  (see 
HoBER,  1922).  Their  influence  on  swelling  phenomena  can  be  ex- 
plained morphologically  on  the  basis  of  the  diameter  and  hydration 
layers  of  the  ions.  Goldschmidt  has  calculated  the  diameters  of  the 
ions  from  the  distances  between  the  atoms  in  the  crystal  lattice,  and 
the  size  of  the  hydration  layers  can  be  derived  from  the  ion  mobilities. 
For  the  monovalent  cations,  for  instance,  the  following  radii  have 
been  found  (Table  XVI). 

Obviously  the  small  ions  have  thicker  hydration  layers  than  the 
bigger  ones.  This  is  due  to  the  fact  that  the  water  dipoles  are  attracted 
more  strongly  as  the  distance  between  the  centre  of  gravity  and  the 


CYTOPLASM 


[49 


surface  of  the  ion  decreases.  Fig.  99  shows  a  graphical  representation 
of  the  water  layers.  It  demonstrates  how  the  ionic  radii  grow  with 
increasing  atomic  weight  while  the  water  layers  decrease. 

If  a  gel  swollen  in  water  is  imbibed  with  salt  solutions,  the  pene- 
trating ions  will  weaken  the  electric  field  of  the  hydrophilic  dipole 

TABLE  XVI 
ION  RADII 


1  ^^ 

Na 

K 

NH4 

Rb 

Cs 

In  the  crystal  lattice  according  to 

GOLDSCHMIDT 

0.78 

0.98 

1-33 

1.45 

1.46 

1.66A 

Derived  from  the  conductivity  at 

00  dilution 

3.66 

2.81 

1.88 

1.89 

1. 81 

1.80A 

Number  of  H^O  per  ion,  accord- 

ing to  Pallmann  (1937)    .   .    . 

10. 0 

4-3 

0.9 

0.8 

0.5 

0.2 

groups  of  the  gel  frame;  consequently  their  hydration  decreases, 
which  results  in  shrinkage.  In  the  case  of  biogels  this  effect  of  shrinkage 
in  neutral  salts  is  observed  only  in  rather  concentrated  salt  solutions 
(from  about  N/z  upwards)  which  in  most  cases  must  be  considered 
to  be  non-physiological.  Shrinkage  by  means  of  salt  can  therefore  be 
used  for  preserving  purposes  (brining  of  meat)  or  for  the  salting-out 
of  dissolved  proteins. 

The  degree  of  shrinkage  depends  on  the  radius  of  the  hydrated  ions 
as  long  as  other  conditions  remain  constant.  For  instance,  if  dried  agar 
powder  swells  in  Normal  alkali  chloride  solutions  (Brauner,  1932), 
the  degree  of  sweUing  is  less  than  in  water,  and  it  is  found  that  by 
comparison  with  the  other  alkali  ions,  Li  and  Na  ions  result  in  a  higher 
degree  of  swelling,  in  accordance  with  the  series  of  Fig.  99 : 


Fig.  99.  Hydration.  Ions  of  the  alkali  series;  hydration  layer  dotted. 


150  FINE-STRUCTURE  OF  PROTOPLASM  II 

Li  >  Na  >  K  >  Rb  >  Cs 

Using  only  potassium  salts  and  varying  the  anion  in  the  halogen 

series,  one  finds : 

I  >  Br  >  CI, 

i.e.,  the  more  strongly  hydrated  CI  causes  less  swelling  than  the  lesser 
hydrated  I.  This  inversion  of  the  influence  of  ion  hydration  shows 
that  the  influence  of  the  ions  on  swelling  phenomena  is  determined 
primarily  by  their  charge.  Biogels,  such  as  agar  in  the  present  case, 
usually  possess  a  weakly  negatively  charged  gel  frame.  For  this  reason 
the  discharging  effect  of  cations  of  equal  valency  is  inversely  pro- 
portional to  their  hydration.  The  effect  of  the  anions  is  due  to  the 
fact  that  the  discharging  cations  are  accompanied  by  their  gegenions. 
These  lay  greater  claim  to  the  charge  of  the  cations,  in  inverse  ratio 
to  their  hydration.  For  this  reason,  the  discharge  of  the  gel  framework 
by  a  given  cation  accompanied  by  I  ions  is  less  than  if  it  were  ac- 
companied by  CI  ions.  In  other  words,  for  a  given  cation,  the  less 
hydrated  the  anion  of  the  salt  is,  the  greater  will  be  the  water  absorp- 
tion of  the  gel. 

In  many  cases,  however,  gels  swell  not  less  but  more  strongly  in 
salt  solutions  than  in  water.  This  occurs  if  the  gel  framework  possesses 
ionogenic groups,  as  is  the  case  with  proteins.  For  example,  the  gel  frame 

of  gelatin,  when  imbibed  with 
a  neutral  salt  solution,  shows  a 
considerable  negative  charge  as 
a  result  of  the  dissociation  of 
COOH-groups.  For  this  reason 
cations  can  be  retained  by  ad- 
sorption ;  their  hydration  is  great- 
er than  the  dehydration  of  the  gel 
framework,  caused  by  the  adsorp- 
^^-r^'  tion  of  the  cations.  It  is  therefore 

Fig.   100.  Hydration.  Influence  of  ions  on       possible  for  thedegreeof  swelling 

the  hydration  of  polypeptide  chains;  A  =        ^gached    in    salt    solutions  tO  be 
difference  in  swelhng. 

higher  than  that  m  water. 
Fig.   100  indicates  how  ions  of  equal  valency  can  cause  different 
degrees  of  swelling.  Consider  an  anionic  side  chain  and  a  hydrophilic 
OH-group   of  a  neighbouring  polypeptide   chain.    Both  possess    a 


1{0H\^ 


I  CYTOPLASM  151 

hydration  layer.  If  a  Na  ion  surrounded  by  its  hydration  layer  ap- 
proaches this  system,  it  is  held  electrostatically,  and  a  hydration 
equilibrium  between  the  various  groups  is  established.  If  the  Na  ion 
is  replaced  by  a  much  less  hydrated  ion  like  Rb,  the  latter  is  able  to 
approach  the  anionic  group  more  closely  because  of  its  smaller 
hydration  layer.  This  results  in  a  stronger  discharge  than  in  the  case 
of  the  Na  ion;  the  hydration  decreases  and  the  neighbouring  poly- 
peptide chains  approach  each  other. 

An  explanation  along  these  general  lines  becomes  more  difficult  if 
bivalent  ions  such  as  Ca  take  part  in  these  processes.  Since  bivalent 
ions  carry  two  elementary  charges,  they  can  discharge  negative  pro- 
teins more  strongly  than  monovalent  ions.  For  this  reason  they 
usually  cause  shrinkage  of  protoplasm  (decrease  in  permeability,  in- 
crease in  density  and  viscosity;  Cholodny  and  Sankewitsch,  1935). 
In  the  case  of  the  trivalent  ions  Fe  and  Al  these  effects  are  still  more 
pronounced  (tanning).  One  speaks,  therefore,  of  a  valency  rule  of 
shrinkaee,  which  states  that  the  shrinking  effect  of  ions  increases  with 
rising  valency. 

With  increasing  charge  of  the  ions,  however,  the  hydration  layer 
also  increases.  The  Ca  ion,  for  instance,  is  hydrated  more  strongly 
than  the  K  ion  of  the  same  size.  Accordingly,  CaClg  causes  gelatin 
to  swell  to  a  greater  extent,  and  this  can  even  result  in  the  formation 
of  a  sol.  In  the  same  way  the  strongly  hydrated  Zn  ion  in  concentrated 
ZnClg  solutions  causes  unexpectedly  marked  swelling  of  cellulose. 
The  valency  rule  does  not,  therefore,  apply  generally  to  bivalent 
ions. 

The  valency  rule  asserts  itself  more  clearly  in  Hofmeister's  series 
of  the  anions 

SCN  >  I  >  NO3,  Br  >  CI  >  acetate  |  >  SO4  >  tartrate  |  >  citrate. 

The  trivalent  citrate  ion  is  a  weaker  swelling  agent  than  the  bivalent 
tartrate  and  sulphate  ions  and  these  last  two  are  weaker  agents  than 
the  monovalent  ions. 

In  the  case  of  positively  charged  proteins  with  cationic  polypeptide 
chains,  Hofmeister's  ion  series  appears  to  be  reversed,  because  the 
adsorption  now  refers  to  the  anions.  This  inversion  is  particularly 
striking  if  one  succeeds  in  reversing  the  charge  of  a  negative  gel.  For 
instance,  with  gelatin  in  a  neutral  or  basic  medium,  where  the  gel 


IJZ  FINE-STRUCTURE  OF  PROTOPLASM  II 

framework  acts  as  an  anion,  the  order  in  which  ions  furtlier  swelling 
is  as  follows 

Li  >  Na  >  K  >  Rb  >  Cs. 

In  an  acid  medium,  however,  in  which  the  gel  framework  behaves 
like  a  cation: 

Li  <  Na  <  K  <  Rb  <  Cs. 

Now  one  would  expect  that  in  the  isoelectric,  i.e.,  uncharged  state, 
the  gel  frame  would  show  the  same  degree  of  swelling  in  all  neutral 
chlorides,  since  in  that  case  no  electrostatic  attractive  forces  are 
operating.  This  is  not  so,  however;  one  finds  so-called  fransitionarj 
series  which  are  of  special  importance  for  biology : 

Li  >  Na  >  K  <  Rb  <  Cs. 

This  result  is  not  easily  comprehensible  after  what  has  been  said 
before.  For,  if  one  plots  the  degree  of  swelling  against  the  atomic 
weight  of  the  cations,  one  obtains  a  descending  curve  in  alkaline 
solutions  (gel  framework  negative)  and  an  ascending  curve  in  acid 
solutions  (gel  frame  positively  charged;  Fig.  loia).  For  this  reason 
one  would  expect  a  horizontal  hne  if  the  pjj  of  the  swelling  medium 
has  been  adjusted  to  the  isoelectric  point  (L  E.  P.)  of  the  protein. 
However,  the  experiment  yields  a  minimum  curve  in  which  K  holds 
a  special  place. 

By  using  ion  models,  however,  it  is  possible  to  understand  these 
relations,  too.  It  follows  from  the  ion  mobilities  that  the  two  ions  in 
KCl  are  of  equal  size.  For  this  reason  they  are  adsorbed  in  the  same 
way  by  an  isoelectric  framework.  In  LiCl  and  NaCl,  however,  Li  and 
Na  are  adsorbed  to  a  smaller  extent  than  CI  because  of  their  large 
hydration  layer.  Consequently,  the  molecular  framework  assumes  a 
weakly  negative  charge  and  is  more  strongly  hydrated  than  in  KCL 
Conversely,  in  RbCl  and  CsCl  the  cations  are  more  easily  adsorbed 
than  the  CI  ions,  which  again  results  in  a  weak  electric  charge  of  the 
gel,  accompanied  by  increased  hydration  (Fig.  loi  b). 

Since  the  isoelectric  point  of  cytoplasm  usually  lies  in  the  weakly 
acid  region,  cations  have  a  discharging  effect  on  it.  As  a  rule,  therefore, 
cations  cause  less  swelling  than  water.  Anions,  on  the  contrary,  in- 
crease hydration  as  a  result  of  their  similar  electric  charge,  so  that 
cytoplasm  often  swells  considerably  (cap-plasmolysis,  p.  197)  in  par- 


CYTOPLASM 


155 


ticular  with  SCN,  I,  Br,  but  also  with  CI,  which  is  held  to  be  re- 
sponsible for  the  swelling  of  the  cytoplasm  of  haloph^nies  (Stocker, 
1928).  In  the  series  I,Br,Cl,F,  chlorine  often  takes  a  similar  optimal 
place  to  potassium  in  the  alkaline  metals  and  Ca  in  the  alkaline  earths 
(PiRSCHLE,  1930). 


1             1 

1 

! 

Ooj 

Q. 

1           i 

' — Swelling   m/nimum 

1                1                1 

Li           No 

K 
oj 

Rb          Cs 

Li            No 

K            Rb           Cs 
b) 

Fig.  loi.  Change  in  swelling  of  a  gel  frame  consisting  of  polypeptide  chains 

under  the  influence  of  chlorides  of  the  alkali  series  at  various  pn  values ; 

Q  =  degree  of  swelling,  a)  Hofmeister's  series;  dotted  line  =  behaviour 

expected  at  pn  =  I.E. P.;  instead,  one  finds  b)  transitionary  series. 

III.  Heteropolar  valency  bonds.  If  all  acid  and  basic  groups  in  the 
cytoplasm  exactly  cancel  each  other  out,  the  isoelectric  state  (im- 
properly called  "isoelectric  point"  I.E. P.)  is  attained  and  nearly  all 
properties  of  protoplasm  reach  extreme  values :  the  degree  of  swelling 
becomes  a  minimum,  the  danger  of  setting  a  maximum ;  the  stability 
is  low,  the  electric  charge  and  the  migration  in  an  electric  field  become 
zero  by  definition,  etc. 

If  then  positive  and  negative  end  groups  of  the  side  chains  occupy 
suitable  relative  positions,  they  can  enter  into  salt-like  bonds  (Fig. 
96 III,  p.  145).  Their  electric  charges  are  neutralized  and  the  hydration 
of  the  region  in  question  is  reduced  to  a  minimum.  The  salt  bonds 
cannot  be  broken  as  readily  by  neutral  salts  as  the  heteropolar  cohesive 
bonds.  Something  more  drastic  is  required,  viz.,  the  concentration  of 
the  H  ions  (pn),  must  be  changed.  Some  of  the  intermolecular  salt 
bridges  are  then  hydrolyzed  and  a  certain  number  of  the  bound 
carboxyl  and  amino  groups  become  free.  If  hydrolysis  is  achieved  by 
H  ions,  i.e.,  if  the  p^  of  the  liquid  in  which  the  cytoplasm  is  examined 
drops  below  the  I.E. P.,  the  dissociation  of  the  free  COOH  groups 
is  diminished,  that  of  the  amino  groups  (-NH3OH)  is  increased.  Thus 
the  cytoplasm  acquires  a  positive  electric  charge  and  behaves  like  a 
complex  cation.  Conversely,  if  the  p^  of  the  medium  is  greater  than 
the  I.E.  P.  of  the  cytoplasm,  the  dissociation  of  the  COOH  groups  is 


^54 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


increased  and  the  cytoplasm  becomes  negatively  charged,  i.e.,  it  acts 
like  a  weak  anion.  This  occurs  as  a  rule  in  neutral  nutrients,  since  the 
I.E. P.  of  protoplasm  is  usually  lower  than  7  (Table  XVII). 

TABLE  XVII 
ISOELECTRIC  POINT  (i.E.P.)  OF  CERTAIN  PROTOPLASTS 

(according  to  Pfeiffer,  1929) 


PH 


Bacteria: 


Fungi : 
Algae: 
Angiosperms : 


Bacteriutn  coli  .... 
grampositive  bacteria 
gramnegative  bacteria 

Fusarium 

Nitella 

Hyacinthus  (root  tip)  . 
Lupinus,  Pisum    .    .    . 

Rheum 

Solarium 


12-13 
about  5 

2-3 

5-4 

4.4-9.6 

4-3 

4-3 
4.5-4.8 

6.4 


The  isoelectric  state  determines  the  acidity  at  which  the  heteropolar 
junctions  of  the  salt  bonds  are  most  effective.  Any  deviation  of  the  p^ 
from  this  state  results  in  a  loosening  of  this  type  of  bond. 

Up  to  a  certain  point  esterifications,  i.e.,  bridges  formed  between 
alcoholic  OH  and  acid  groups  of  neighbouring  polypeptide  chains 
(Fig.  96III,  p.  145),  can  Hkewise  be  reckoned  among  the  heteropolar 
valency  bonds.  Their  firmness  is  dependent  also  on  the  pjj  of  the 
medium,  since  hydrogen  ions  are  capable  of  hydrolyzing  and  hydroxyl 
ions  of  saponifying  these  ester  bonds  catalytically. 

IV.  Homopolar  valency  bonds  are  formed  either  by  elimination  of 
water  (ether,  glucoside  and  peptide  bridges.  Fig.  96 IV,  p.  145)  or  by 
splitting  off  hydrogen,  i.e.,  dehydrogenation  (methylene  and  sulphur 
bridges.  Fig.  102).  The  former  still  possess  a  certain  polarity  and  can 
be  hydrolyzed  under  suitable  conditions.  Without  the  aid  of  enzymes 
this  can  now  only  be  effected  at  temperatures  above  the  physiological; 
compare,  for  instance,  the  hydrolysis  of  glucosides  and  proteins  by 
boiling  acids.  This  is  of  particular  importance  for  the  stability  of  the 
carbohydrates  and  the  peptide  bonds.  The  purely  homopolar  valency 
bridges  (-CH2-CH2- -S-S-)  can  no  longer  be  hydrolyzed  at  all.  Here 


I  CYTOPLASM  155 

the  loosening  of  the  junctions  is  achieved  according  to  an  entirely 
different  principle,  namely  by  addition  of  elementary  hydrogen 
ihydrogenation). 

cystine  bridge:  CH-R-SH      HS-R-CH  <— — ^  CH-R-S-S-R-CH 


/ 
methylene  bridge:  CH-R-CH3  CH3-R-CH  i===^  CH-R-CH2-CH2-R-CH 


-2H 

TTh 


Fig.  102.  Bridges  dependent  on  r^ 

At  physiological  temperatures  water  in  very  small  amounts  not  only 
dissociates  into  ions  according  to  the  scheme  HgO  ^  H+  +  OH~,  but 
also,  though  admittedly  to  still  less  extent,  into  the  elements  hydrogen 
and  oxygen:  zHgO^  2H2  ^  Og.  These  gases  develop  a  very  low 
gas  pressure,  which  for  hydrogen  we  shall  designate  as  tHg. 

If  the  partial  pressure  of  hydrogen  in  the  cytoplasm  increases,  the 
-S-S-  bridges  tend  to  be  hydrogenated,  which  causes  rupture  of  the 
bonds  (Fig.  102).  The  cystine  bridges  can  therefore  absorb  Hg  and 
for  this  reason  act  in  the  same  way  with  respect  to  the  partial  pressure 
of  Ha  as  a  buffer  with  respect  to  the  concentration  of  H+ions.  These 
conditions  have  been  investigated  in  particular  in  the  case  of  gluta- 
thione (G).  This  is  a  protein  compound  which  can  be  split  into  glutamine, 
cysteine  and  glycine.  It  represents  a  tripeptide  chain  with  the  three 
amino  acids  mentioned  as  side  chains.  However,  whether  it  occurs  in 
the  cytoplasm  as  a  free  molecule  or  only  as  part  of  a  much  larger 
macromolecule  cannot  be  decided  at  present.  In  both  cases  glutathione 
reacts  according  to  the  following  scheme :  2  GSH  ^  GS-SG  +  Hg. 
Thus,  when  sulphydryl  groups  occur  in  the  side  chains  of  proteins 
(Fig.  96 IV,  p.  145),  these  can  give  rise  to  formation  or  dissolution  of 
cross-links. 

Methylene  bridges  cannot  be  formed  with  the  same  ease,  at  any 
rate  in  the  laboratory,  where  methyl  groups  show  a  very  passive 
behaviour.  All  the  same,  it  is  known  that  in  the  metabolism  one 
molecule  of  succinic  acid  can  be  formed  out  of  two  molecules  of  acetic 
acid  by  dehydrogenation  (Mothes,  1933).  This  succinic  acid  is  then 
dehydrogenated  further  to  fumaric  acid,  converted  into  malic  acid, 
dehydrogenated  to  oxalo-acetic  acid  and  finally,  after  decarboxylating 
this  keto-acid,  converted  into  pyruvic  acid.  It  thus  becomes  apparent 


156  FINE-STRUCTURE  OF  PROTOPLASM  II 

that  dehydrogenation  plays  an  important  part  in  the  chemistry  of 
fermentation.  It  is,  therefore,  likely  that  to  a  certain  extent  this  also 
applies  to  the  formation  of  methylene  bridges  between  neighbouring 
polypeptide  chains.  It  is  known  that  in  asphyxia  the  cytoplasm  often 
liquefies;  this  may  be  due  partly  to  hydrogenation  processes,  resulting 
from  increased  partial  pressure  of  hydrogen. 

The  hydrogen  pressure  in  protoplasm  is  characterized  by  its  negative 
logarithm  in  much  the  same  way  as  the  hydrogen  ion  concentration.  The 
Ph  is  derived  from  the  product  of  the  ionic  concentrations  (cH+)  •  (cOR-)  = 
1 0-1*.  Similarly,  the  product  of  the  Hg  and  Og  partial  pressures  in  water 
is  constant.  It  amounts  to  (tH2)'^-t02  =  iQ-^^  in  which  the  pressures  are 
expressed  in  atmospheres.  Thus  the  Hg  and  O2  pressures  are  mutually 
dependent  in  the  same  way  as  the  H+  and  OH"  concentrations.  The  hydro- 
gen and  oxygen  pressure  or  the  so-called  redox  potential  of  a  solution  in 
water  can  therefore  be  characterized  by  a  single  number.  For  this  purpose 
we  choose  the  negative  logarithm  of  the  hydrogen  pressure,  which  is 
designated  as  r^. 

If  hydrogen  is  made  to  flow  through  a  system  under  atmospheric  pressure, 
the  hydrogen  pressure  amounts  to  i  atm.,  or,  written  in  exponential  form : 
10"  atm.,  which  means  that  r^  =  o.  On  the  other  hand,  if  oxygen  flows 
through  the  system,  tOg  =  i,  and  accordingly  (tHg)"  =  iQ-^^  or  ry  =  41. 
Obviously  the  rjj  of  a  system  can  vary  between  o  and  41.  Small  values  of  r^ 
indicate  lack  of  oxygen,  larger  ones  on  the  contrary  are  indicative  of  fav- 
ourable aerobic  conditions,  r^  (like  p^)  can  be  measured  directly  with  the 
aid  of  a  potentiometer  (Fig.  103)  or  indirectly  with  the  help  of  suitable  dyes 
(MiCHAELis,  1933)  which  lose  colour  at  a  certain  r^  as  a  result  of  hydrogena- 
tion (for  instance,  methylene  blue  and  indigo).  The  analogies  between  p^ 
and  Th  are  listed  in  Table  XVIII.  The  characteristic  values  of  the  rjj  scale 
are  apparent  from  the  following  list : 

% 

I  at.  Og  (oxygen  electrode) 41 

air  (1/5  at.  oxygen) 4°^1 

hydrogen  and  oxygen  pressure  in  equilibrium  ....    27.3 

H2  pressure  =2-02  pressure  (middle  of  redox  scale)  .    20.5 

,       1        ^  (  aerobic  life  o 

border  or   ;  1  ■    i-r  ° 

(  anaerobic  lire 

I  at.  H2  (hydrogen  electrode) o 

Table  XIX  gives  a  few  r^  measurements  in  living  cytoplasm  (Needham, 
1925,  RiES,  1938).  The  values  are  not  strictly  comparable,  since  according 
to  the  equation  Hg  —  2  el~  ^  2H+  the  value  of  rjj  is  a  function  of  p^-  This 
dependence  is  apparent  from  Fig.  103  (according  to  Bladergroen,  1945). 
If  the  electric  redox  potential  E  (with  respect  to  the  platinum  hydrogen 


CYTOPLASM 


157 


electrode  E  =  o)  and  the  value  of  p^  in  the  system  are  used  as  rectangular 
coordinates,  the  curves  of  constant  hydrogen  pressure  (rjj)  are  sloping 
lines.  If  two  of  the  three  quantities:  electric  redox  potential  E,  the  exponent 
of  hydrogen  pressure  r^  and  the  exponent  of  hydrogen  ion  concentration 
pjj,  are  known,  the  magnitude  of  the  third  one  can  be  read  from  the  diagram 
in  Fig.  103.  Since  the  redox  system  is  only  determined  by  its  electric  poten- 
tial E,  it  follows  that  in  biological  systems  both  the  t^  value  and  its  corre- 
sponding Pjj  value  should  be  given.  On  this  condition  the  rj^  value  may  be 
identified  with  the  redox  potential,  as  is  usually  done  in  biology. 


TABLE  XVIII 

Pjj  and  rjj  SYMBOLISM 


Actual  acidity 
Ph 


Redox  system 


■■H 


Starting  point.  .  . 
Dissociation  .  .  . 
Law  of  mass  action 

Exponent 

Inter\^al 


hydrogen  ion  cone.  cH+ 
H2O  ^  H+  +  OH- 
cH+-cOH-  =  1 0-1* 

Ph  =  -log  cH+ 
Pjj  varies  from  0-14 


hydrogen  pressure  tHg 
2H2O  ^  2H2  +  O2 

(tH2)2-t02    =   IO-«2 

r„  =  -log  tHo 


■■H 


rjj  varies  from  0-41 


TABLE  XIX 

REDOX  POTENTIAL  (rjj)  OF  CERTAIN  PROTOPLASTS 
(according  TO  NEEDHAM,   1 92 5) 


Sea-urchin  egg 

Amoeha  proteus 

Salivary  gland  of  Chironomus . 


Ph 


6.5 
7.6 

7-2 


^H 


19-21 
17-19 
19-20 


Just  as  the  heteropolar  valency  bonds  are  strongest  at  a  certain  pjj, 
namely  at  the  I.E. P.,  there  is  an  optimum  value  of  Tjj  at  which  the 
homopolar  valency  bonds  are  the  least  endangered.  It  has  already  been 
pointed  out  that  cystine  bridges  are  broken  down  at  high  hydrogen 
pressures,  i.e.,  at  low  rjj-values.  At  high  values  of  rjj  they  are  re- 
established. A  high  rjj  is,  however,  also  capable  of  loosening  bonds 
(oxidation).  As  shown  by  Staudinger  (1957a,  p.  13),  the  glucoside 


158 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


oxygen  bridges  of  cellulose  from  a  certain  degree  of  polymerization 
onwards  are  very  sensitive  to  oxidation,  so  that  the  chains  are  easily 
degraded,  for  instance  according  to  the  scheme:  (C6Hio05)2n  -f  O., 
=  2(QHio05),0. 

Similar  sensitive  ether  bridges  may  be  assumed  to  exist  in  the 
cytoplasm,  so  that  not  only  too  small  a  r^  but  also  too  high  a  r^  may 
interfere  with  the  bonds  inside  a  macromolecule. 

Apart  from  dehydrogenation,  i.e.,  elimination  of  hydrogen,    the 

transfer  of  hydrogen  atoms  from 
one  chain  to  a  neighbouring  chain 
may  also  be  responsible  for  bridge 
formation.  Astbury  (1936)  and 
AsTBURY  and  Wrinch  (1937)  dis- 
cuss two  possibilities  of  bridge  form 
-ation  inside  folded  polypeptide 
chains  of  fibre  proteins,  and  similar 
reactions  may  also  be  considered 
in  protoplasm.  The  hydrogen  can  be 
exchanged  between  neighbouring 
keto  and  imido  groups  following 
the  lactam-lactim  tautomerism  ac- 
cording to  the  abbreviated  equation 
>  CO  +  HN<  ^  >C(OH)-N<  , 
thus  building  a  main  valency 
bridge.  In  the  same  way  bridges 
may  be  formed  between  keto  and 
methylene  groups  by  keto-enol  in- 
version: >  CO  ^  RHC  <  ^  > 
C(OH)-RC  < .    Such     inversions 


Fig.  103.  Relation  between  redox  po- 
tential E,  hydrogen  ion  exponent  pjj  and 
hydrogen  pressure  exponent  r^.  Abscissa : 
PH-value  of  the  system;  ordinate:  electric 
potential  E  volt  of  the  system  with  respect 
to  Pt-Hj-electrode  (from  Bladergroen, 
1943)- 


often  occur  quite  easily,  and  in  many 
cases  it  is  impossible  to  decide  which  of  the  two  forms  is  present.  In 
the  case  of  cytoplasm,  this  would  mean  that  because  of  the  possibilities 
discussed  it  would  remain  doubtful  whether  a  bridge  existed  or  not, 
i.e.,  its  existence  might  be  obvious  at  one  moment  and  fail  at  the  next, 
which  would  be  in  accordance  with  the  great  instability  of  the  bond 
and  with  the  mobility  of  the  cytoplasm. 

V.  Long-range  forces .  Whereas  the  forces  described  under  I-IV  have 
an  extremely  small  radius   of  action,  there  are  reactions  between 


I  CYTOPLASM  159 

protein  macromolecules,  submicroscopic  and  even  microscopic  pro- 
tein particles  which  bridge  submicroscopic  distances.  Such  reactions 
occur  when  rod-shaped  virus  particles  (Fig.  84c,  p.  126)  take  a  parallel 
orientation  in  a  concentrated  sol  (Wyckoff,  i947a-c),  when  protein 
macromolecules  aggregate  according  to  the  rule  of  Svedberg  (Fig. 
84b)  or  when  globular  submicroscopic  particles  crystallize  (Fig.  84 d). 
Similar  attractions  over  considerable  distances  appear  when  antibodies 
(precipitins,  agglutinins)  cause  the  precipitation  of  specific  proteins 
or  even  the  agglutination  of  bacteria  and  blood  corpuscles. 

The  nature  of  long-range  forces  is  difficult  to  understand.  As  their 
radius  of  action  is  greater  than  50  A,  they  play  an  important  role  in 
the  structural  arrangement  of  colloidal  particles.  Oster  (195  i)  shows 
that  long-range  orientation  is  partly  due  to  the  repulsive  effect  of 
electrical  double  layers  in  highly  concentrated  sols,  and  partly  to 
ordinary  Van  der  Waals  attractive  forces  which  are  additive,  so  that 
an  integrating  effect  of  all  the  atoms  of  two  adjacent  macromolecules 
is  involved. 

ROTHEN  (1947)  has  published  experiments  indicating  that  the  action 
of  long-range  forces  is  detectable  at  distances  of  over  200  A.  He 
coated  the  antigen  of  bovine  albumin  on  a  slide  with  a  layer  200  A 
thick  of  barium  stearate  and  was  able  to  observ^e  the  immunological 
reaction  of  the  antibody  applied  to  this  film.  Even  enzymes  such  as 
trypsin  and  pepsin  were  found  to  act  upon  substrate  layers  through 
an  inert  screen.  The  last  experiment  is  in  contradiction  to  the  current 
conception  of  enzyme  action,  which  is  considered  to  be  a  contact 
reaction  with  the  molecules  of  the  substratum.  The  impermeability  of 
the  intervening  stearate  films  has  therefore  been  doubted  (Trurnit, 
1950).  Whatever  the  result  of  this  criticism  may  be,  long-range  forces 
incontestibly  cause  the  aggregation  of  submicroscopic  particles  in  sols 
and  the  formation  of  structures  in  gels. 

There  must  be  a  discrete  number  of  spots  on  the  surface  of  a 
globular  macromolecule  where  junctions  are  possible.  If  this  number 
is  two,  the  protein  globules  have  a  tendency  to  form  beaded  chains 
(Fig.  104),  which  may  yield  a  loose  reticulum.  If  the  number  of  active 
spots  is  three,  they  will  be  the  origin  of  a  two-dimensional  layer  repre- 
senting a  porous  film  (Fig.  104).  Four  junctions  would  cause  a  three- 
dimensional  framework,  since  they  are  arranged  rather  in  a  tetrahedral 
manner  than  in  a  plane.  A  sphere  may  touch  as  many  as  6  neighbours 


[6o 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


in  a  plane.  This  yields  a  dense  film.  When  several  layers  of  the  kind 
are  superposed,  a  close-packed  crystal  lattice  results  (Fig.  84d,  p.  126); 
in  this  case  every  macromolecule  is  fixed  by  1 2  junctions.  This  suggests 
that  the  junctions  are  induced  wherever  the  globules  touch.  Although 
this  seems  true  for  crystallizing  proteins,  it  would  be  difficult  to  under- 


O 


Fig.  104.  Aggregation  of  globular  macromolecules  (dots  =  spots  of  junctions),  a)  Two 
spots  of  junctions  produce  beaded  chains;  h)  three  spots  of  junctions  produce  porous 
layers;  c)  four  spots  of  junctions  produce  tetrahedral  groups;  d)  twelve  spots  of  junctions 

produce  a  close-packed  crystal  lattice. 

stand  the  formation  of  beaded  chains  and  loose  meshworks  without 
assuming  a  restricted  possible  number  of  junctions  per  aggregating 
particle.  In  the  case  of  globules  aggregating  to  beaded  chains,  the 
macromolecules  must  be  endowed  with  a  pronounced  polarity. 

Summary.  The  proteins  are  to  be  considered  as  the  structural  ele- 
ments of  the  cytoplasm.  Their  macromolecules  are  interlinked  to  form 
a  framework,  whose  junctions  can  be  disrupted  by  various  quite 
different  agents.  A  rise  in  temperature  attacks  in  the  first  place  the 
homopolar  cohesive  bonds  or  lipidic  bonds.  Dependent  on  their  state 
of  hydration,  adsorbed  salts  affect  the  heteropolar  cohesive  bonds  or 


I  CYTOPLASM  l6l 

secondary  valency  bonds;  p^  influences  the  heteropolar  or  salt  bonds, 
and  the  redox  potential  is  capable  of  intervening,  either  as  a  con- 
structive or  as  a  destructive  factor,  in  the  homopolar  valency  bonds  or 
bridgelike  bonds.  It  is  therefore  very  difficult  to  explore  the  structure 
of  the  cytoplasm  experimentally,  for  it  is  scarcely  possible  to  vary  only 
a  single  one  amongst  these  four  factors,  keeping  the  three  others 
rigorously  constant.  A  change  in  the  temperature  or  the  salt  concen- 
tration will  often  cause  changes  in  pn  and  tn,  which  in  their  turn  are 
interdependent.  For  this  reason  one  can  never  be  sure  in  an  experiment 
whether  some  measure  has  not  affected  other  tj^pes  of  bonds  besides 
the  group  of  points  of  attachment  which  one  wished  to  investigate. 
In  spite  of  the  fact  that  it  is  practically  impossible  to  keep  the  four 
types  of  junctions  as  neatly  apart  as  in  theory,  this  scheme  gives  an 
idea  of  the  various  kinds  of  bonds  which  by  their  harmonious  col- 
laboration are  responsible  for  the  remarkable  structure  of  protoplasm. 
In  the  case  oi fixation,  the  aim  is  to  preserve  the  molecular  arrange- 
ment as  true  to  life  as  possible.  This  can  never  be  done  perfectly,  since 
the  usual  means  of  fixation  affect  quite  different  categories  of  junctions. 
Alcohol  has  a  dehydrating  and  hardening  effect  on  the  heteropolar 
cohesive  bonds.  In  order  to  counteract  the  accompanying  shrinkage, 
a  swelline  medium  such  as  acetic  acid  has  to  be  added.  Its  H-ions 
lessen  the  contracting  action  of  the  alcohol  by  hydrolysis  of  hetero- 
polar valency  bonds  and  by  maintaining  a  certain  state  of  hydration  of 
the  heteropolar  cohesive  bonds.  Oxidizing  fixatives  like  chromic  acid 
and  osmic  acid  affect  bridges  which  are  sensitive  to  r^^  and  thus  solidify 
the  labile  hompolar  main  valency  bonds.  The  tanning  action  of  formal- 
dehyde may  be  due  to  its  capacity  to  form  bridges  between  neighbour- 
ing polypeptide  chains  according  to  the  same  scheme  as  that  which 
governs  the  polymerization  of  oxymethylene.  It  is  impossible  to  find 
a  fixation  mixture  which  in  no  way  affects  the  structure  of  the 
labile  cytoplasm.  In  spite  of  this,  fixations  which  have  been  carried 
out  correctly  cannot  be  compared  with  precipitations,  since  there  is 
no  separation  of  phases,  but  only  a  coarsening  of  an  existing  structure. 
It  is  shown  by  the  dyeing  experiments  carried  out  by  Drawert  (1957) 
with  varying  p^  of  the  imbibing  liquid,  that  the  molecular  framework 
after  fixation  still  contains  acidic  and  basic  groups  capable  of  dis- 
sociation, although  these  groups  are  no  longer  screened  off  but  are  freely 
accessible  to  dyes.  This  is  why  fixed  cells  can  be  stained  easily,  whereas 


;6z 


FINE-STRUCTURE  OF  PROTOPLASM 


IE 


vital  staining  of  living  cytoplasm  is  almost  impossible.  The  enhanced 
adsorbing  power  of  dead  cytoplasm  allows  of  identifying  dead  cells 
with  the  fluorochrome  acridin-orange  (Strugger,  1949)-  Depending 
on  its  concentration,  this  dye  shows  a  green  (1:50,000)  or  a  red 
(1:100)  fluorescence  in  the  UV  light.  Since  dead  cytoplasm  adsorbs 
a  considerable  amount  of  acridin-orange,  it  displays  a  magnificent  red 
fluorescence,  whereas  living  cells  appear  to  be  green. 

With  the  aid  of  the  diagram  of  Fig.  96  (p.  145)  some  indication 
of  the  sio-nificance  of  the  various  elements  in  the  structure  of  the  proto- 
plasm can  be  given.  In  the  periodic  system  (Table  XX)  all  elements 
which  are  of  importance  to  the  life  of  plants  lie  on  a  line  connecting 
carbon  with  the  inert  gas  argon.  I  have  designated  this  line  as  the 
nutrition  line  (1935c);  only  hydrogen  and  molybdenum  (Arnon  and 
Stout,  1939)  are  an  exception. 

TABLE  XX 
elements  which  are  indispensable  to  plant  nutrition 


Series 

0 

1 

Q 

IQ 

H 

V 

B 

ra 

m. 

0 

Is*    penod 

H 

He 

2nd   penod 

He 

U 

Be 

B 

A^ 

N. 



0^ 

s 

F 

Ne 

3rd   pcnod 

Ne 

Nal 

."» 

-^ 

« 

Is. 

P 

a 

~\ ~ 

^. 

iHi   (»nod 

(A/ 

0. 

(j 

Zn 

Sc 

r, 

6e 

V 
As 

Cr 
5e 

Mn 

Br 

^  Co    Ni 

Kr 

5  Hi   period 

Kr 

Rb 

Sr 

Cd 

Y 
In 

Sn 

Nb 
Sb 

Mo 
Te 

Va 

Ru    Rh    Pd 

X 

Gtti   penod 

X 

Oi 

Ba 

•n 

Ce 
Pb 

la 
Bi 

W 
ft) 

Re 

Os    Ir    P» 

Rn 

7  Hi  period 

Rn 

- 

Ra 

fit 

T>. 

ft 

U 

In  Table  XX  the  indispensable  elements  have  been  framed  by 
squares,  whereas  those  which  are  found  in  nearly  all  plants,  but  whose 
indispensability  remains  to  be  proved,  have  been  framed  in  dotted 
lines.  C  and  N  Ue  in  the  centre.  These  elements  occupy  a  central 
position  in  the  molecular  structure,  too,  since  they  form  the  poly- 
peptide main  chains.  They  may  therefore  be  designated  as  chain- 
building  elements.  The  chains  are  built  according  to  the  scheme 
-C-C-N-C-C-N-.  Notwithstanding  its  close  relation  to  nitrogen, 
phosphorus  does  not  occur  as  a  chain-building  element  in  this  manner, 
but  only  in  combination  with  oxygen  (compare  Fig.  122,  p.  215: 
-C-0-P-0-C-) ;  as  in  the  inorganic  domain,  it  is  always  present  in 
an  oxidized  form  as  phosphoric  acid.  In  the  degradation  of  carbo- 


I  CYTOPLASM  163 

hydrates  it  also  acts  as  a  protector  of  atom  groups  which  should  not 
be  affected  (hexose  diphosphoric  acid,  phosphorus  glyceric  acid,  etc.). 
It  is  possible  that  in  the  cytoplasm  the  phosphatides,  which  can 
combine  with  various  groups  of  the  polypeptide  chains,  render  a 
similar  service.  The  elements  O  and  S  of  the  sixth  row  are  primarily 
bridge-building  elewenis,  since  they  interconnect  the  C-N-polypeptide 
chains.  Apart  from  this,  oxygen  can  act  as  a  chain-building  element 
in  the  high  polymeric  carbohydrates,  and  conversely  N  and  C  are 
capable  of  bridge  formation. 

The  elements  of  the  first  and  second  row:  Na,  K,  Cu,  Mg,  Ca,  Zn, 
and  also  CI  occur  in  cytoplasm  as  ions  and  act  as  hydration  regulators. 
They  do  not  form  stable  bonds  but  only  heteropolar  salt  bonds  with 
the  molecular  framework  (metallic  organic  compounds  like  chloro- 
phyll, haemoglobin,  etc.  are  quantitatively  of  minor  importance).  In 
this  respect  the  most  favourable  ions  in  plants  are  K,  Ca  and  CI  of  the 
so-called  argon  type  (in  animals  Na  takes  the  place  of  K).  Both  in 
mixtures  and  in  pure  solutions  these  ions  are  tolerated  in  concen- 
trations at  which  other  ions  are  detrimental  to  the  cytoplasm  structure. 
This  would  also  explain  why  the  nutrition  line  takes  its  course  towards 
argon.  The  higher  valent  elements  B,  Mn  and  Fe  presumably  enter 
into  some  relation  with  the  protoplasmic  frame.  As  regards  manganese 
and  iron,  it  is  usually  believed  that  their  capacity  to  change  valency 
is  put  to  use  in  metabolism. 

The  most  important  part  is  played  by  the  element  hydrogen,  both 
as  an  ion  and  as  an  element.  It  regulates  p^  and  ry,  thus  preventing 
the  molecular  framework  from  soUdifying,  and  maintaining  the  labile 
changeable  state  which  is  so  characteristic  of  protoplasm. 

c.  Physical  Properties  of  the  Cytoplasm 

Sol  properties.  Many  cytologists  suppose  the  cytoplasm  to  be  a  Hquid 
(Rhumbler,  1898).  Heilbrunn  (1930),  for  example,  writes  about  the 
amoeba:  "it  is  a  tiny  sac  of  fluid  in  motion"  and  Chambers  (1925) 
considers  not  only  the  cytoplasm  but  also  the  nucleus  to  be  a  liquid 
phase. 

The  flow  of  protoplasm,  the  relatively  low  viscosity,  the  large  water 
content,  the  soft  consistency,  the  convex  shape  in  plasmolysis  and 
other  indications  point  to  the  sol  character  of  the  cytoplasm,  i.e.,  to 
a  state  in  which  all  submicroscopic  particles  have  free  relative  move- 


164  FINE-STRUCTURE  OF  PROTOPLASM  II 

ment.  The  most  striking  of  these  effects  \&  protoplasmic  flow,  and  when 
seen  for  the  first  time  this  phenomenon  will  always  convince  the 
observer  of  the  liquid  state  of  the  cytoplasm. 

The  merit  of  having  characterized  the  aggregate  state  of  cytoplasm 
with  the  aid  of  physical  laws  is  due  to  Rhumbler  (1914)-  According 
to  his  observations,  the  cytoplasm  of  the  amoeba  possesses  i.  no 
measurable  elasticity,  2.  no  perceptible  compressibility  at  ordinary 
pressures  and  3.  it  follows  the  capillary  laws  which  are  determined  by 
the  surface  tension  (minimum  surface,  constant  contact  angle,  spread- 
ing on  the  surface  of  liquids,  capillary  rise).  At  the  present  time  our 
picture  will  be  somewhat  different. 

According  to  Newton's  law,  ideal  Hquids  are  completely  free  from 
inner  elasticity:  any  particle  in  the  bulk  of 'the  liquid  can  be  moved 
at  will  without  showing  the  slightest  tendency  to  swing  back  into  its 
original  position.  In  cytoplasm  this  condition  is  not  fulfilled,  for,  as 
will  be  shown  below,  it  possesses  structural  elasticity  or  elasticity  of  flow. 

The  incompressibility  should  not  be  tested  at  "ordinary"  pressures, 
but  at  high  pressures  where  the  low  compressibility  in  comparison 
with  solid  bodies  becomes  apparent.  If  a  living  amoeba  in  its  nutrient 
is  exposed  to  a  uniform  pressure  of  the  order  of  magnitude  required 
to  prove  incompressibility,  its  cytoplasm  is  altered,  whereas  it  is  the 
main  property  of  ideal  liquids  not  to  undergo  any  changes  in  this 
experiment.  Brown  (1934)  and  Marsland  (1942)  show  that  the  cyto- 
plasm of  different  eggs,  of  Amoeba,  Paramaecium,  of  human  erythro- 
cytes and  of  Elodea  leaves  becomes  liquefied  by  high  hydrostatic 
pressure.  It  behaves  therefore  Uke  sols  in  which  the  process  of  gelation 
is  accompanied  by  a  small  increase  of  volume.  According  to  obser- 
vation in  the  centrifuge  microscope  with  a  high  pressure  chamber,  the 
mobility  of  included  particles  increases  by  almost  25%  for  each 
pressure  increment  of  70  atm.  (1000  lbs/in.^).  Under  these  conditions 
protoplasmic  streaming  is  inhibited,  and  within  fairly  broad  limits, 
the  effect  is  reversible.  Pressure  up  to  300  atm.  may  be  maintained 
for  about  an  hour,  and  yet,  when  the  cells  are  returned  to  atmospheric 
pressure,  the  original  structural  characteristics  are  restored  within  a 
minute.  At  700-1000  atm.  even  the  cortical  layer  of  the  cytoplasm  is 
liquefied  and  irreversible  changes  begin  to  appear. 

Rhumbler's  best  arguments  refer  to  the  capillary  properties  of 
naked   cytoplasm,  although  by  no  means  all  cytoplasts  assume  a 


I  CYTOPLASM  165 

Spherical  shape  or  can  be  spread  at  will  on  the  surface  of  another 
liquid.  In  those  cases  where  the  cytoplasm  forms  liquid  drops,  its 
surface  tension  y  can  be  measured  (E.  N.  Harvey,  1936)  by  observing 
the  cell  as  a  sessile  drop  flattened  by  gravity.  The  relation 

y  ==  g  (d  -  d')r-^F 

is  used  to  calculate  y;  g  is  the  acceleration  due  to  gravity,  (d  —  d') 
the  difference  in  density  between  drop  and  medium,  r  the  radius  of 
greatest  flattening  and  F  a  function  of  the  distance  a  in  Fig.  105  a 
representing  the  flattening  of  the  drop.  For  the  egg  of  the  mollusc 
Busjcon  canaliculatum  a  tension  of  0.5  dyne/cm  is  found  by  this  method, 
while  the  egg  of  the  salamander  TriturHS  virescens  gives  only  o.i 
dyne/cm  (Table  XXI). 

The  eggs  of  mackerel  contain  a  large  oil  droplet  which  can  be 
flattened  against  the  rigid  cell  membrane  when  revolving  the  egg  at 
high  speed  in  the  centrifuge  microscope  of  E.  N.  Harvey.  From  its 
shape,  an  oil/cytoplasm  interfacial  tension  of  0.6  dyne/cm  is  calculated: 
if  the  centrifugal  force  is  increased  up  to  450  times  gravity,  this 
tension  does  not  change,  showing  that  the  surface  is  not  elastic.  In 
contact  with  sea  water  this  oil  gives  a  tension  of  7  dyne/cm,  a  high 
value  which  is  explained  by  the  rule  that  the  interfacial  tension  be- 
tween two  immiscible  liquids  is  the  difference  of  the  tensions  of  the 
two  liquids  against  air.  As  the  surface  tension  y  of  water  is  72  dyne/cm 
and  that  of  oils  is  only  about  half  as  much,  it  is  evident  that  the  cell 
surface  cannot  be  formed  of  pure  lipids,  because  this  would  provoke 
a  higher  interfacial  tension  between  the  surface  of  a  cell  and  its  culture 
medium.  A  surface  with  only  o.i  dyne/cm  tension  against  the  medium 
cannot  be  very  lipidic;  besides  the  lipids  it  must  contain  proteins 
with  a  certain  affinity  for  water. 

If  the  cell  does  not  flatten  under  its  own  weight,  the  flattening  can 
be  achieved  by  compression  (E.  N.  Harvey,  1937):  the  spherical  cell 
is  loaded  by  a  thin  beam  of  gold  with  micro  weights.  The  weight  W 
divided  by  the  area  D  of  the  flattened  cell  in  contact  with  the  beam 
gives  the  pressure  P,  from  which  the  surface  tension  is  calculated  by 
the  formula 


l66 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


IV 


oT 


when  Tj  and  fg  are  the  two  radii  ofthe  flattened  cell  indicated  in  Fig.  105b. 

The  unfertilized  egg  of  the 
st2i-\xtchin,  A.rbacia  punctnlata, 
shows  a  surface  tension  of 
0.135  dyne/cm  when  loaded 
with  two  micrograms.  Smaller 
weights  give  lower  values  and 
extrapolation  of  the  tension/ 
compression  curve  yields  0.08 
dyne/cm  for  the  uncompressed  egg.  As  the  surface  tension  is  not  con- 
stant but  depends  on  the  interior  pressure,  the  surface  displays  elasticity : 
this  again  is  evidence  of  the  presence  of  proteins  in  the  cytoplasm 
surface,  since  a  layer  of  pure  lipid  would  not  show  elasticity.  Sols  have 
no  elastic  properties,  so  it  is  evident  that  the  proteins  in  the  surface 
layer  are  in  a  gel-like  state. 


Fig.  105.  Measurement  of  surface  tension  (after 

E.   N.   Harvey,    1936/37);   a)   sessile   drop,   b) 

flattened  drop. 


TABLE  XXI 

SURFACE  TENSION  OF  PROTOPLASM  WITH  RESPECT  TO  SOLUTIONS 
(according  to  E.  N.  HARVEY,   1 93 7) 


Naked  protoplasts 


Leucocytes  (Lepus  caniculus)  .... 

„  {Rana  pi  pi  ens) 

Amoeba  {Amoeba  dnhia) 

Slime  mould  {Physarum  polycephalum) 
Sea-urchin  egg  {Arbacia  punctulatd)  . 
Salamander  egg  ij'riturus  viridescens) 


Medium 


Ringer  sol.  +  serum 

5?  H  " 

„  ,  diluted 

„  ,  250  X  diluted 

seawater 

pond- water  +gum  arable 


It  is  clear  that  the  occurrence  of  capillary  phenomena  gives  no 
conclusive  evidence  of  the  existence  of  a  true  liquid.  On  the  other 
hand,  however,  it  has  not  been  proved  that  Hquid  cytoplasm  possesses 
an  organized  structure;  it  has  only  been  shown  that  the  possibility 
of  such  a  structure  cannot  be  excluded. 

The  same  holds  good  for  the  results  of  viscosity  studies  on  liquid 
cytoplasm,  which  give  valuable  information  on  changes  in  fluidity. 


1  CYTOPLASM  167 

Viscosity  measurements  can  be  performed  by  examining  the  Brownian 
movement  of  granule  inclusions  (Pekarek,  1930)  or  by  observing  the 
speed  of  a  heavy  particle  falling  through  the  cytoplasm  by  its  own 
weight,  or  by  centrifugal  force  (Heilbronn,  1914;  Heilbrunn,  1930). 
The  intensity  of  Brownian  movement  is  given  by 

X2  RT       I 


t  N      ^Ttrrj 

where  X^  represents  the  mean  square  of  the  displacement  of  a  granule 
with  radius  r  during  time  t,  Ris  the  gas  constant,  T  the  absolute  tempera- 
ture, and  N  is  Loschmitt's  number.  It  is  seen  that  the  viscosity  r}  of 
the  medium  is  inversely  proportional  to  the  intensity  of  Brownian 
movement. 

For  the  movement  of  a  particle  through  a  Uquid  (Fig.  1 12a,  p.  192), 
Stokes'  law 

2ng  (d  —  d'y 


T]    = 


9v 


holds  good.  Here  v  is  the  velocity  of  the  moving  spherical  particle, 
(d  —  d')  the  difference  in  density  between  cytoplasm  and  observed 
particle,  g  the  acceleration  due  to  gravity,  and  n  the  number  of  times 
which  the  applied  centrifugal  force  is  stronger  than  gravity. 

With  these  methods  it  is  found  (Table  XXII,  p.  1 69)  that  the  sap 
in  the  vacuole  of  plant  cells  is  often  only  about  twice  as  viscous  as 
water  (Frey,  1926c).  For  the  cytoplasm,  however,  relative  viscosities 
of  six  in  Amoeba  (Pekarek,  1930),  twenty-four  in  parenchyma  cells 
of  the  Viciafaba  seedling  (Heilbronn,  19 14)  or  thirty  in  erythrocytes 
of  man  (Ponder,  1934)  are  found.  Such  values  are  more  reliable  if 
derived  from  Brownian  movement  than  if  determined  by  Stokes'  law, 
since  the  latter  requires  a  uniform  velocity  v  of  the  faUing  particles 
which  is  not  often  realized  in  cytoplasm. 

Once  again  these  measurements  do  not  establish  the  existence  of 
structural  viscosity  in  cytoplasm.  To  solve  this  question  it  is  necessary 
to  carry  out  viscosity  measurements  with  different  pressure  gradients. 
Since  protoplasm  cannot  be  made  to  flow  through  a  narrow  tube  like 
a  liquid,  Pfeiffer  (1936)  sucks  naked  protoplasts  (so-called  gymno- 
plasts  from  the  decomposing  fruit  pulp  of  Pbysalis,  Solanum  or  Juni- 
perus,  of  Allium  epidermic  cells,  etc.)  through  a  capillary  under  a  given 


;68 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


„}00 


^  30 


70 


SO 


^ 

^^^ 

E 

\ 

V 

\ 

X 

V 

/ 


a 


Pressure  p  in  cm  H2O 


pressure  difference  which  can  be  read  from  a  manometer.  At  the  same 
time  he  measures  the  viscosity  by  following  the  Brownian  movement 
of  particles  (dyed  by  means  of  chrysoidin)  which  are  embedded  in  the 
protoplasm  (Pekarek,  1932).  In  Fig.  106  the  viscosity  7]  is  plotted 
against  the  pressure  gradient  p  for  plasmic  drops  from  the  cells  oiCbara 

fragiUs.  The  viscosity  decreases 
rapidly  with  increasing  pressure 
(measured  in  cm  HgO),  where- 
as in  normal  flow  of  glycerin  r\ 
remains  practically  independent 
of  the  pressure.  This  experiment 
shows  clearly  that  protoplasm  is 
not  a  sol-like  liquid,  but  repre- 
sents an  elastic^' gt\  solution".  This 
does  not  yet  imply  a  definite  struc- 
ture, although  once  more  this  pos- 
sibility is  not  ruled  out. 

It  is  otherwise  with  the  devia- 
tions from  Stokes'  law.  Ac- 
cording to  this  law,  microscopic- 
ally visible  particles  or  bubbles  in  a  liquid  either  fall  or  rise  with 
constant  velocity.  Scarth  (1927)  has  ascertained,  however,  that  in 
cytoplasm  the  particles  do  not  move  with  uniform  velocity.  It  looks 
as  though  they  encounter  invisible  obstacles,  and  they  fall  in  a 
hesitant  and  jerky  manner.  According  to  Scarth,  they  give  the 
appearance  of  lead  shot  which  is  run  through  a  brush  heap.  Again 
and  again  the  falling  particles  meet  with  invisible  strands,  lose  speed 
and  change  their  direction.  Accordingly,  the  cytoplasm  cannot  be 
homogeneous  but  must  be  full  of  invisible  fibres  of  a  higher  density. 
It  does  not  possess  a  uniform  viscosity,  and  the  results  derived 
from  the  fall  method  (Heilbronn,  1914)  represent  some  kind  of 
average  value.  In  Pekarek's  viscosity  measurements  (1930,  1952), 
which  are  based  on  the  amplitude  of  oscillation  of  particles  in  Brown- 
ian movement,  the  inhomogeneity  of  the  cytoplasm  is  less  apparent, 
because  the  oscillatory  motion  daes  not  cover  a  long  distance  through 
the  cytoplasm  and  can  be  studied  at  a  fixed  point. 

The  values  reported  for  the  relative  viscosity  of  the  cytoplasm 
do  not  prove  its  true  liquid  state,  even  though  they  are  considerably 


Fig.  106.  Structural  viscosity  of  the  cyto- 
plasm of  Char  a  fragilis  (from  Pfeiffer, 
1936).  Abscissa:  pressure  p  in  cm  HjO.  I 
Cytoplasm  at  21°  C,  II  at  12°  C;  III  glyce- 
rol at  21°  C.  Ordinate:  Viscosity  rj  in 
%  of  the  original  value. 


I 


CYTOPLASM  169 


lower  than  the  values  for  many  true  viscous  liquids  (Table  XXII). 
For  a  true  liquid  should  in  the  first  place  be  homogeneous  in  the 
physical  sense  and  this  certainly  does  not  apply  to  cytoplasm.  The 
following  comparison  may  be  permitted : 

Consider  a  wad  of  thread-like  algae.  The  threads  can  be  moved  at 

TABLE  XXII 
RELATIVE  VISCOSITY  fj 

Water *       i 

Cel/  sap.- 
Stem  parenchyma,  oi  the  Vida  Faba  seedlinp:  ...  1.9  (Heilbronn,  1914) 

Protonemz  of  Lepiobrjum  piriforwe 1.9  (Pekarek,  1933) 

Epidermic  cells  of  the  .^///«w  (7f/>a  bulb 2      (Pekarek,  1930) 

Terminal  vacuole  of  C/i9.r/cr///w  (see  Fig.  1 1 2a,  p.  192)  2.5  (Frey,  1926c) 

Cytoplasm : 

Amoeba 6      (Pekarek,  1930) 

Stem  parenchyma  of  the  Vicia  Faba^eeAhne.   ...        24      (Heilbronn,  19 14) 
Red  cell  of  man 30      (Ponder,  1934,  p.  87) 

Viscous  liquids: 

Glycerol 87  (L.a.ndolt-B6rnstein,  1923) 

Paraffin  oil 92                             ,, 

Castor  oil 1250                              ,, 

wiU  with  respect  to  each  other,  although  they  impede  each  other's 
freedom  of  movements  as  a  result  of  their  extremely  anisodiametric 
shape.  When  transferring  this  microscopic  model  to  the  molecular 
domain,  the  threads  become  long  chain  molecules  in  a  dispersing 
medium  and  a  drop  of  this  macromolecular  sol  would  show  structural 
viscosity  and  all  the  capillary  phenomena  described.  If  the  individu- 
alized algae  threads  of  our  model  were  replaced  by  the  graceful 
reticular  alga  Hydrodictyon  (Oltmanns,  1922,  p.  277),  scarcely  any 
change  in  the  inner  mobility  of  such  a  wad  of  algae  would  be  observed. 
On  a  molecular  scale  this  means  that  a  drop  which  contains  a  coherent 
three-dimensional  molecular  network,  instead  of  free  chain  molecules, 
will  not  only  assume  a  spherical  shape  but  also  show  a  constant 
contact  angle  and  spread  on  the  surface  of  suitable  liquids.  In  spite 
of  this,  the  structural  elements  of  the  network  cannot  move  freely! 
The  network  is  so  flexible,  however,  that  its  shape  within  the  drop 
is  determined  by  the  forces  of  surface  tension.  /Ul  the  same,  we  cannot 


lyo 


FINE-STRUCTURE  OF  PROTOPLASM  II 


Speak  of  a  true  liquid,  for,  when  static  equilibrium  is  established,  the 
drop  is  inhomogeneoiis,  not  only  at  the  surface,  but  also  in  bulk. 

To  sum  up,  it  can  be  said  that  cytoplasm  in  its  Hquid  state  obeys 
neither  the  laws  of  Newton  (Pfeiffer,  1937)  nor  those  of  Poiseuille 
or  Stokes  (Frey-Wyssling,  1940  a).  Although  to  cytologists  it  may 
have  the  appearance  of  a  liquid,  it  certainly  is  no  true  liquid  in  the 
physical  sense.  We  had  better  not  attach  too  much  value  to  this  simi- 
larity, for  we  should  then  be  unable  to  penetrate  its  submicroscopic 
fine-structure,  since  a  liquid  possesses  a  structure  only  in  its  surface. 
On  the  contrary,  it  is  my  aim  to  stress  especially  the  deviations  from 
the  physical  laws  of  liquids,  as  it  is  precisely  these  deviations  which 
offer  us  the  chance  of  elucidating  the  structural  properties  of  cyto- 
plasm. 

Gel  properties.  Often  cytoplasm  does  not  flow  in  Hquid  drops,  but 
shows  plastic  properties.  This  in  itself  would  not  be  sufficient  to 
indicate  a  solid  state ;  but  it  is  also  elastic  and  to  a  certain  extent  pos- 
sesses a  constant  shape.  The  result  of  plasmolysis  is  not  always  separ- 
ation from  the  cell  wall  of  a  definitely  convex  drop.  On  rapid  de- 
hydration with  strongly  hypertonic  solutions  the  shape  in  plasmolysis 
becomes  concave  or  angular,  indicating  a  certain  rigidity  of  the  cyto- 
plasm in  this  state  (Prudhomme  van  Reine,  1955). 

Especially  interesting  is  the  spinning  capacity  of  the  cytoplasm,  which 
is  apparent  from  the  fact  that  long  strands  can  be  drawn  from  it 
(Seifriz  and  Plowe,  193 i).  Often  this  phenomenon  can  also  be  ob- 
served during  plasmolysis  in  the  form  of  the  so-called  strands  of 
Hecht  (Fig.  107a),  although  this  name  is  scarcely  justified,  since  their 
importance  was  pointed  out  by  Chodat  (1907)  many  years  before 
Hecht  (191 2).  From  Fig.  107  a  it  is  apparent  that  spherical  boundaries 
as  claimed  by  Rhumbler  (1898)  occur  only  in  a  few  fibres  in  a  very 
imperfect  form.  A  similar  fact,  which  shows  the  non-liquid  state  of 
the  cytoplasm,  is  the  "angular  plasmolysis"  of  sea-urchin  eggs  (Runn- 
STROM  and  Monne,  1945 ;  Runnstrom,  Monne  and  Wicklund,  1946). 
In  the  plasmoptysis  of  Spirog^ira  cells  the  protoplasm  can  be  drawn 
out  into  a  long  strand  which  contracts  rhythmically  (Fig.  107  b). 
Seifriz  (1929)  has  shown  that  the  cytoplasm  of  amphibian  red  cells 
can  be  drawn  out  to  three  times  its  normal  length  and  the  nucleus 
even  up  to  20  times  its  original  length  without  the  occurrence  of  any 
drops.  All  these  properties  of  the  cytoplasm  are  inconsistent  with  the 


CYTOPLASM 


171 


hypothesis  of  a  true  liquid;  they  point  rather  to  some  fibrous  sub- 
microscopic  structural  element. 

The  inner  elasticity  can  be  demonstrated  by  suspending  iron  filings 
in  the  cytoplasm  and  moving  them  by  means  of  a  magnetic  field.  As 
soon  as  the  field  is  switched  off,  the  particles  swing  back  elastically  to 


Fig.  107.  a)  Plasmic  strands  of  epidermic  cells  from  the  bulb  of  Allium,  plasmolyzed  by 
CaClj,  (according  to  Kuster,  193  5^) ;  h)  plasmic  strand  oiSpirogyra,  extruded  in  plasmoptysis 

(from  Frey-Wyssling,  1940^). 

their  original  positions  (compare  Heilbronn,  1922).  This  method 
has  been  further  developed  by  Crick  and  Hughes  (1950)  to  measure 
the  internal  elasticity  of  cytoplasm  quantitatively.  They  find  the 
modulus  of  rigidity  of  chick  fibroblasts  in  tissue  culture  to  be  of  the 
magnitude  of  100  dynes/cm^.  At  the  same  time  they  give  evidence  of 
the  thixotropic  behaviour  of  the  cytoplasm  which  can  change  its  state 
reversibly  from  solid  to  fluid  when  stirred.  In  this  respect  it  gives 
similar  results  to  elastic  gels  of  sodium  oleate  or  bentonite. 

The  reversible  gel-sol  transition  is  one  of  the  most  important  proper- 
ties of  cytoplasm,  as  it  is  the  basic  phenomenon  in  protoplasmic  flow 


l-jZ  FINE-STRUCTURE  OF  PROTOPLASM  II 

(p.  1 86).  If  a  gel  is  liquefied  under  isothermal  conditions,  the  volume 
can  either  increase  (gelatin,  agar)  or  remain  constant  (Na-oleate  and 
other  thixotropic  gels)  or  decrease  (methyl  cellulose  in  water),  A 
decrease  in  temperature  or  an  increase  in  pressure  favours  gel  form- 
ation in  the  first  case  and  sol  formation  in  the  third  (Freundlich, 
1937).  Cytoplasm  belongs  to  the  third  category  (p.  187).^  In  addition 
and  in  contrast  to  all  other  gels,  it  can  also  change  its  aggregate  state 
by  itself,  even  if  the  external  physical  conditions  remain  unaltered. 

It  seems  that  anaesthetized  cytoplasm  is  more  gelated  than  in  the 
active  state,  as  Seifriz  (1950)  finds  that  any  anaesthetic  causes  a 
reversible  gelation  of  protoplasm. 

We  are  thus  faced  with  the  paradox  that  cytoplasm  simultaneously 
exhibits  the  characteristics  of  hquids  (fluidity)  and  of  solids  (elasticity). 
It  is  noiv  solid,  then  liquid  to  an  extent  rarely  observed  in  any  other 
colloid.  The  task  of  submicroscopic  morphology  consists,  therefore, 
in  drawing  up  a  structural  scheme  which  explains  the  double  nature 
of  cytoplasm  at  the  boundary  of  the  two  classical  aggregate  states.  By 
doing  so,  we  should  gain  more  than  by  adhering  to  the  concept  of 
cytoplasm  either  as  a  liquid  or  as  a  gel,  neither  of  which  can  be  true 
in  a  general  sense. 

d.  Submicroscopic  Structure  of  Cytoplasm 

Particulate  globules.  If  we  disregard  the  microscopic  inclusions  in 
cytoplasm  (plastids,  mitochondria,  lipid  globules,  granules  etc.),  it 
represents  a  microscopically  homogeneous  pseudophase.  This  is  no 
longer  true  when  it  is  observed  in  the  electron  microscope,  where 
submicroscopic  particles  appear  to  be  dispersed  in  a  reticulate,  fiorous 
or  homogeneous  matrix  of  diameters  from  5  00  to  1 5  00  A  (Claude, 
1946;  Faure-Fremiet,  Bessis  and  Thaureaux,  1948;  Lehmann, 
1950).  In  liver  cells  these  particles  are  distinctly  smaller  than  the  mito- 
chondria, which  measure  2000  to  5000  A.  Claude  suggested  calling 
them  "microsomes".  Globules  of  1000  A  diameter  may  lodge  as  many 
as  64  of  the  biggest  macromolecules  listed  in  Table  XV  (p.  141),  so 
the  microsomes  must  contain  a  great  number  of  protein  molecules  and 
other  compounds. 

According  to  Bensley  (1943),  the  submicroscopic  particles,  isolated 
from  homogenized  liver  tissue  by  the  centrifuge,  consist  of  protein, 

^  Brown  (1934)  and  Marslakd  (1942)  have  checked  this  up  to  1000  atm. 


I  CYTOPLASM  173 

nucleoprotein,  flavoprotein,  triglycerides,  lecithin,  sterine,  vitamin  A 
and  80-90%  water.  They  contain  the  ribonucleic  acid  of  the  cytoplasm 
(Jeener,  1948).  According  to  the  view  of  Caspersson  (1941),  they 
are  involved  in  protein  synthesis. 

As  metabolic  centres  they  are  analogous  to  the  mitochondria  or 
chondriosomes,  which,  however,  are  microscopic  particles  and  repre- 
sent a  special  system  in  the  cell  which  is  designated  as  chondriome 
(GuiLLiERMOND,  Mangenot  and  Plantefol,  1933;  Bourne,  1945). 
The  mitochondria  of  guinea  pig  liver  tissue  can  be  isolated  (Hoerr, 
1943)  and  analyzed.  They  are  of  lipidic  nature  (43.6%)  but  contain 
at  the  same  dme  two  proteins  of  different  I.E. P.  They  are  free  of 
lecithin  and  cephalin  (Bensley  and  Hoerr,  1934).  Faure-Fremiet 
(1946)  gives  for  the  same  material  somewhat  different  figures:  Protein 
64.6%,  glycerides  28.8%,  lecithin  and  cephalin  4.2%,  cholesterol 
2.25  %.  At  any  rate  there  is  no  nucleic  acid  present.  This  is  confirmed 
by  the  lack  of  UV  absorption  (Monne,  1948).  According  to  Monne 
(1942  b),  the  mitochondria  may  be  strongly  hydrophilic. 

The  rodlet  shape  of  the  so-called  chondrioconts  and  the  double 
refraction  of  the  filamentous  mitochondria  from  the  intestinal  cells  of 
Ascaris  megalocephala  (Giroud,  1928)  indicate  an  inner  structure 
resembling  a  mesophase.  Originally  Bensley  (1937)  thought  that  the 
chondriosomes  might  be  merely  coacervates.  Claude  and  Fullam's 
(1945)  electron  micrograms  of  fixed  chondriosomes  show  a  lipid 
cortex  and  a  watery,  less  dense  central  zone.  In  addition  Muhle- 
thaler,  Muller  and  Zollinger  (1950)  have  found  that,  in  kidney 
cells,  they  are  coated  with  a  distinct  submicroscopic  membrane. 

In  recent  publications  the  mitochondria  are  considered  as  important 
bodies  with  special  physiological  functions  (Claude,  1944),  as  certain 
enzymes  are  fixed  on  them.  Hogeboom,  Claude  and  Hotchkiss  (1946) 
found  cytochrome  oxidase,  and  Leuthardt  (1949)  was  able  to  localize 
the  enzymes  of  the  tricarboxyHc  acid  cycle  on  the  liver  mitochondria. 
Muller  and  Leuthardt  (1950)  and  Brenner  (1949)  have  demon- 
strated that  the  mitochondria  of  intact  lymphocytes  perform  oxidation 
—  reduction  reactions.  This  means  that  the  respiration  is  assigned  to 
these  bodies.  The  fact  that  the  mitochondria  are  dispersed  throughout 
the  cytoplasm  would  account  for  continuing  respiration  of  parts  dis- 
sected from  a  living  cell. 

It  is  probable  that  new  mitochondria  originate  exclusively  from 


174 


FINE-STRUCTURE  OF  PROTOPLASM  II 


pre-existing  mitochondria,  similar  to  plastids,  chromosomes  and  virus 
particles.  Lehmann  (1947)  has  proposed  the  term  hiosomes  for  such 
bodies  which  are  characterized  by  self-multiplication  and  endowed 
with  specific  functional  tasks. 

Reticulate  ground-cytoplasm.  The  matrix  in  which  the  microsomes  and 
mitochondria  are  suspended  has  quite  a  different  aspect,  depending 
on  the  object  under  investigation  and  on  the  method  of  fixation  used. 

Claude  and  Fullam  (1946)  speak  of  a  fibrous  ground  texture  in 
the  cells  of  the  guinea  pig  liver,  Faure-Fremiet  and  co-workers  (1948) 
of  a  reticulate  ground-plasm  in  the  amoebocytes  of  the  snail.  The 
cytoplasm  of  the  thrombocytes  in  the  blood  is  hyaline,  alveolar  or 
fibrous  depending  on  the  fixation  with  osmic  acid,  formalin  or  alcohol 
(Bessis  and  Bricka,  1948).  Bretschneider  (1950a)  describes  a  three- 
dimensional  network  400  A  wide,  partly  beaded  strands  in  the  cyto- 
plasm of  ciliates  fixed  with  OSO4. 

It  looks  as  though  we  are  about  to  have  a  repetition  of  the  cyto- 
logical  discussions  on  the  structures  of  fixed  cytoplasm  as  seen  in  the 
ordinary  microscope,  this  time  with  reference  to  the  submicroscopic 
aspect.  It  is  obvious  that  only  the  finest  textures  observed  come  any- 
where near  the  natural  situation,  while  the  coarser  textures  are  only 
worth  while  considering  in  relation  to  a  possible  linear  coagulation 
of  previously  filamentous  submicroscopic  structural  elements.  Rozsa 
and  Wyckoff  (1950/5 1)  have  found  that  the  cytoplasm  of  the  dividing 
cells  in  the  onion  root  tip  yields  a  beautiful  dense  reticulate  structure 
with  very  fine  meshes  (smaller  than  0.05  [x  diameter)  when  fixed  in 
neutral  formaHn,  whilst  every  acid  fixative  (especially  OSO4  and  acetic 
acid)  furnishes  a  very  coarse  cytoplasmic  reticulum  with  almost  micro- 
scopic meshes  (0.5  fi  diameter).  Bretschneider  (1950c)  has  made  a 
systematic  study  of  the  influence  of  fixation  on  the  submicroscopic 
structure  of  cytoplasm  as  seen  in  the  electron  microscope,  and  has 
tested  all  the  treatments  used  in  cytology  on  the  same  subject  (root 
tip  of  onion).  The  best  fixation  is  obtained  in  Champy's  and  in  Kopsch- 
Regaud's  fluids  (Fig.  108/1,2),  which  contain  formalin  and  OSO4 
combined  with  chromic  acid  and  potassium  bichromate.  The  hyalo- 
plasm shows  a  fine  network  of  thin  protein  filaments  with  a  diameter 
of  about  160  A  forming  a  regular  hexagonal  pattern.  Pure  solutions 
of  formaHn  (Fig.  108/4, 5),  Bouin's  fluid  (Fig.  108/5)  ^^^  Helly's  fluid 
(Fig.    108/6)    yield   a    slightly    coarser    network.    Substances    which 


CYTOPLASM 


175 


Fig.  108.  Cytoplasm  of  the  meristem  cells  of  the  root  tip  in  onion.  Pictures  after  different 
fixation  fluids  but  at  the  same  magnification  of  12000  X  and  100  kV.    (By  courtesy  of 

L.  H.  Bretschneider,  1950c). 

coagulate  the  proteins  strongly,  such  as  acetic  acid,  trichloracetic 
acid,  phosphotungstic  acid,  alcohol,  sublimate  or  sulphosalicylic 
acid  destroy  the  fine  pattern  of  cytoplasm  by  syneresis.  Contrary  to 
Wyckoff's  statements,  osmic  acid  is  found  to  produce  fairly  good 
fixation  for  animal  cells. 


jj(,  FINE-STRUCTURE  OF  PROTOPLASM  II 

It  is  a  remarkable  fact  that  mixtures  of  fluids  which  fix  different 
types  of  junctions  seem  to  effect  the  best  fixation  (seep.  i6i),  whereas, 
with  the  exception  of  formalin,  pure  compounds  produce  poor 
fixation. 

A  special  feature  of  great  importance  is  the  occurrence  of  beaded 
chains  observed  in  the  electron  microscope.  Bessis  and  Bricka  (1948) 
have  described  such  microfibrils  (of  ^^  500  A  diameter)  in  the  cyto- 
plasm of  thrombocytes,  Matoltsy,  Gross  and  Grignolo  (195  i)  in 
the  vitreous  body  of  cattle  eyes,  and  Lehmann  (195 1)  observes  similar 
chains  in  the  cytoplasm  of  Amoeba.  Sheaves  of  such  beaded  chains 
with  knots  of  600  A  diameter  occur  in  liver  cells;  Bernhard, 
Gautier  and  Oberling  (195  i)  have  shown  that  these  beaded  fibrils 
belong  rather  to  the  ergastoplasm  subject  to  metabolic  changes  than 
to  the  mechanical  cytoplasmic  framework.  In  the  egg  of  Tubifex,  fibrils 
carrying  knots  of  about  0.15  /i  diameter  have  been  found  (Lehmann 
and  Biss,  1949);  these  fibrils  form  the  ground-plasm  in  which  the 
microscopic  yolk  granules  (2  ^t)  are  suspended.  The  knots  (0.15/^) 
reach  microscopic  dimensions  and  are  identical  with  the  chromidia  of 
Hertwig  found  in  the  sea-urchin  and  Tiibifex  eggs.  They  contain 
ribonucleic  acid  (Monne,  1946a),  The  protoplasmic  fibrils  appear  to 
be  segmented  by  the  chromidia  and  display  for  that  reason  a  micro- 
scopical structure  similar  to  the  chromatids  (see  p.  225). 

Monne  (1948)  identifies  these  chromidia  with  the  microsomes,  be- 
cause both  contain  ribonucleic  acid  (Feulgen  negative,  UV  absorption 
at  A  =  260  vafjL,  stainable  with  pyronin),  which  differentiates  them 
from  the  mitochondria.  However,  such  an  identification  must  be  dis- 
carded from  a  morphological  view,  because  the  chromidia  are  im- 
movable bodies  fixed  on  a  beaded  microfibril,  whereas  the  microsomes 
are  corpuscularly  dispersed  free  and  independent  particles. 

In  the  gelated  state  cytoplasm  has  some  continuous  structure  and, 
given  the  chemical  composition  of  the  cytoplasm  (p.  140),  it  must  be 
a  protein  gel.  Protein  molecules  can  aggregate  to  a  framework  in 
different  ways. 

a.  Globular  molecules  or  composite  submicroscopic  particles  may 
associate  to  form  beaded  chains  (Fig.  5  la,  p.  66).  If  these  chains 
become  sufficiently  long  or  branch,  a  framework  is  easily  formed.  The 
gelation  of  gelatin  belongs  to  this  type  (Joly,  1949). 

b.  Expanded  polypeptide  chains  can  aggregate  to  form  fibrils,  such 


I  CYTOPLASM  177 

as  are  found  in  fibrous  proteins,  which  may  give  rise  to  a  meshwork 
or  a  plaitwork  (Fig.  51b,  p.  66). 

The  first  type  of  framework  must  produce  gels  with  a  higher 
percentage  dry  weight  than  the  second,  which  we  can  picture  as  being 
made  up  of  submicroscopic  or  amicroscopic  strands.  If  the  cytoplasm 
appears  homogeneous  in  the  electron  microscope,  the  structural  ele- 
ments (globules  or  threads)  must  be  amicroscopic,  i.e.  they  must  have 
micromolecular  diameters  (<  50  A).  It  is  difficult  to  decide  which 
type  is  really  present,  because  the  structure  easily  changes  in  character 
owing  to  the  denaturation  of  proteins  in  the  fixation  and  drying 
processes.  The  inner  structure  of  the  globules  and  microfibrils  is 
governed  by  the  junction  principles  discussed  on  p.  145.  If  these  sub- 
microscopic  elements  aggregate  to  form  a  gel,  another  type  of  junction 
is  involved,  caused  by  long-range  forces  (p.  158).  The  nature  of  these 
forces  is  not  well  known  but  in  forming  gels  they  act  morphologically 
disjunctions  in  the  submicroscopic  domain  in  very  much  the  same  way 
as  the  chemical  forces  do  in  the  amicroscopic  range.  According  to 
OsTER  (195 1)  there  is  no  real  difference  between  short-range  forces 
and  long-range  forces. 

Assuming  that  there  is  such  a  gel,  all  the  cytoplasmic  properties, 
strange  as  they  may  be,  can  be  accounted  for. 

The  high  water  content  of  the  cytoplasm  (70  to  80%  or  more)  is 
caused  by  the  considerable  width  of  the  meshes  of  the  framework. 
In  addition,  there  is  hydration  water  inside  the  submicroscopic  strands 
and  beaded  chains.  The  water  content  is  liable  to  be  so  great  that 
many  of  the  water  dipoles  are  not  fixed  by  the  framework  and  have 
freedom  of  movement.  In  this  case  excretion  of  water  from  the  cj^to- 
plasts  and  hence  vacuolization  becomes  possible.  As  a  rule,  however, 
all  the  water  is  loosely  bound  by  main  chains  or  side  chains  and  takes 
part  in  establishing  the  maximum  state  of  swelling. 

The  transition  of  protoplasm  to  a  resting  state  is  accompanied  by 
a  gradual  diminution  in  the  amount  of  water  brought  about  by  a 
narrowing  of  the  submicroscopic  interfibrillar  and  intramolecular 
interstitial  meshes.  The  water  is  perhaps  partly  replaced  by  lipids,  as 
hydrophilic  groups  are  screened  off  by  phosphatides,  sterines  and  the 
like.  The  determinant  structure  and  the  organization  of  the  framework 
which  governs  the  processes  of  life  can  thus  sometimes  be  preserved 
for  years  (spores,  seeds).  Evidently  this  natural  deh3"dration  cannot 


178  FINE-STRUCTURE  OF  PROTOPLASM  IE 

be  imitated  by  artificial  drying  at  room  temperature,  since  the  change- 
in  the  framework  structure  has  to  proceed  step  by  step  along  with, 
the  dehydration  caused  by  the  neutralization  or  screening  of  the 
hydrophilic  groups,  without  changing  those  configurations  of  the 
molecular  structure  which  are  necessary  for  the  maintenance  of  life^ 
But  by  the  modern  procedure  oi free^e-drying  a  method  has  been  found 
which  permits  evaporation  of  the  hydration  water  without  altering  am~ 
structure  essential  to  life.  Freeze-dried  bacteria  can  be  preserved  in- 
definitely ;  and  this  method  seems  to  be  very  promising  for  the  preven- 
tion of  denaturation  when  fixing  submicroscopic  protein  structures^ 

The  physical  properties  fluidity^  plasticity  and  elasticity  must  be  at- 
tributed to  the  character  of  the  junctions  between  submicroscopic 
particles.  The  more  these  are  dissolved,  the  more  liquid  the  cytoplasm, 
becomes.  However,  the  junctions  must  never  all  be  weakened  at  the 
same  time.  Jn  other  words,  the  cytoplasm  must  never  become  a  true 
sol  in  which  all  particles  can  move  freely.  Certain  bonds  are  always 
preserved  and  these  cause  the  elastic  properties.  The  dissolution  of" 
all  junctions  would  result  in  the  death  of  the  cytoplasm  by  liquefaction^ 

The  great  marvel  of  the  Uving  framework  is  its  striking  mobility,, 
which  becomes  apparent  in  protoplasmic  flow.  In  this  flow  the  chains, 
are  orientated  not  only  in  small  submicroscopic,  but  even  in  micro- 
scopic regions,  as  indicated  by  the  visible  strand  formation.  The  paral- 
lel alignment  of  the  chains  is  often  so  pronounced  that  birefringence 
of  flow  occurs  (Ullrich,  1936a;  amoeboid  movement  of  the  rhizo- 
podiae,  Schmidt  1937a,  1941b).  The  whole  movement  is  only  intel- 
ligible if  a  great  number  of  junctions  are  continuously  being  formed^, 
only  to  be  broken  down  shortly  afterwards.  The  jimdamental  dijference 
from  dead  gels  lies  in  the  fact  that  in  the  cytoplasm  the  junctions  are  continuously 
reconstructed.  The  pattern  of  junctions  in  living  matter  is  not  rigid  and 
fixed  as,  for  instance,  in  gelatin  or  still  more  in  cellulose  gels ;  its  only 
permanent  feature  is  its  continual  change ! 

The  reconversion  to  the  system  of  junctions  proceeds  according  to- 
some  definite  plan  about  which  we  remain  completely  in  the  dark.. 
A  temporary  change  in  stability  can  also  be  produced  artificially,, 
owing  to  the  thixotropic  properties  of  the  cytoplasm  (see  p.  66).  By 
mechanical  means  (pressure,  shock)  a  reversible  liquefaction  can  be 
brought  about.  Such  drastic  interference  is  always  followed,  however.,^ 
by  a  more  or  less  serious  damage  to  the  cytoplasm  (see  p.  187). 


I  CYTOPLASM  179 

Interrelation  of  the  particulate  globules  and  the  reticulate  ground-cytoplasm. 
While  it  is  fairly  well  established  that  the  submicroscopic  reticulate 
structure  of  the  cytoplasm  is  formed  by  linear  aggregation  or  by 
reversible  denaturation  of  globular  protein  molecules,  there  is  no 
proof  that  all  existing  submicroscopic  protein  particles  participate  in 
these  sol- gel  transformations.  It  is  possible  that  certain  globules, 
as,  e.g.,  the  microsomes  in  the  liver,  may  be  specialized  for  metabolic 
work,  whereas  others  with  the  capacity  of  forming  gels  have  the 
character  of  structural  proteins.  It  seems  unlikely  that  the  two  funda- 
mental tasks  of  the  cytoplasm,  metabolism  and  morphogenesis,  are 
performed  by  the  same  globular  elements.  It  is  true  that  some  investi- 
gators think  of  a  uniform  type  of  cytoplasm;  thus  Virtanen  (1948) 
finds  that  the  number  of  enzymes  in  bacteria  is  so  high,  that  all  protein 
molecules  in  the  cytoplasm  must  be  enzymes.  On  the  other  hand,  we 
find  that  in  the  microscopic  domain  individuaUzed  and  mobile  meta- 
bolic centres,  such  as  erythrocytes  or  chloroplasts,  are  suspended  in 
a  liquid  which  can  gelate  (fibrinogen- fibrin  transformation,  sol-gel 
transformation  of  the  endoplasm).  Similar  specialization  might  there- 
fore conceivably  prevail  in  the  submicroscopic  domain. 

We  may  note  here  that  pieces  of  cytoplasm  separated  from  the  rest 
continue  to  live  independently,  although  they  are  not  capable  of 
restoring  the  original  cell  shape.  Thus,  since  metabolism  is  confined 
to  quite  specific  molecular  configurations,  all  essential  groupings  have 
to  occur  repeatedly  in  each  cytoplast ;  this  is  the  case  if  they  are 
carried  by  submicroscopic  particles. 

The  development  of  the  organism  is  presumably  also  governed  by 
special  specific  groupings  in  the  cytoplasm,  which  can  be  designated 
as  morphogenetic  configurations.  However,  in  contradistinction  to  the 
majority  of  active  groups  regulating  the  metabolic  process,  these 
configurations  do  not  by  any  means  occur  in  every  type  of  cell ;  they 
are  confined  to  the  cells  of  certain  tissues,  probably  located  in  the 
nuclei.  A  tissue  of  this  kind  acts  as  "organizer"  (Spemann,  1936; 
Weiss,  1939;  Baltzer,  1942),  since  the  processes  of  development 
concerned  can  only  take  place  in  its  presence.  This  organizer  can  be 
influenced  by  chemical  means.  Lehmann  (1937a,  b),  for  instance,  has 
succeeded  in  controlling  chorda  formation  by  treating  the  gastrula  of 
Triton  or  Rana  with  lithium  chloride.  This  can  be  explained  by  as- 
suming that  the  essential  morphogenetic  configuration  is  changed 


l8o  FINE-STRUCTURE  OF  PROTOPLASM  II 

either  substantially  by  chemical  compounds  (e.g.,  hydration)  or  only 
in  its  configuration  in  space  (e.g.,  by  changes  in  the  distance  between 
decisive  groups)  in  such  a  way  that  they  can  no  longer  fulfil  their 
task.  These  morphogenetic  groups  often  require  hormones  to  be 
activated  (Hadorn,  1939). 

Since  the  morphogenetic  faculties  are  assigned  to  special  cells, 
whereas  certain  metabolic  phenomena,  such  as  respiration,  are  com- 
mon to  all  cytoplasts,  a  morphological  separation  of  these  manifes- 
tations of  life  in  the  submicroscopic  domain  is  probable. 

As  previously  pointed  out,  the  submicroscopic  microsomes  must 
contain  a  considerable  number  of  protein  macromolecules  and  other 
compounds  such  as  nucleic  acids,  phosphatides,  lipids,  pigments,  etc. 
These  constituents  must  be  united  in  some  very  specific  pattern.  This 
follows  from  the  fact  that  their  arrangement  is  capable  of  specific 
achievements  in  biosynthesis.  Just  as  in  organic  chemistry  an  asym- 
metric synthesis  is  only  possible  if  another  optically  active  compound 
with  asymmetric  carbon  atoms  is  present  which  prevents  the  form- 
ation of  racemic  mixtures,  so,  too,  the  organization  of  biocatalysts 
must  be  adequate  to  the  chemical  structure  of  the  specific  compounds 
synthesized.  For  here,  as  in  the  case  of  asymmetric  synthesis,  the 
theorem  applies:  Specific  structures  can  he  formed  only  hj  the  agency  of 
corresponding  structures. 

The  chemical  compounds  of  the  cytoplasm  would  not  be  capable 
of  accomplishing  any  useful  work  without  definite  positions  in  space. 
The  prosthetic  group  (coenzyme)  of  an  enzyme  is  only  active  when 
attached  to  a  special  protein  carrier  (apoenzyme).  Although  the 
chemical  forces  of  their  linkage  are  not  considerable,  and  the  coenzyme 
can  therefore  be  split  off  and  recombined  with  the  macromolecular 
carrier  with  comparative  ease,  the  system  is  only  effective  when  the 
prosthetic  group  takes  up  its  specific  steric  position. 

When  the  enzymes  are  located  in  individual  particles  such  as  micro- 
somes or  mitochondria,  they  can  be  separated  from  the  other  cell 
constituents  and  examined  in  the  isolated  state.  In  the  case  of  the 
endoenzymes,  however,  which  cannot  be  extracted  from  the  tissues 
(Bersin,  1939),  the  apoenzyme  may  be  a  part  of  the  cytoplasmic 
framework,  in  which  case  there  is,  of  course,  no  possibility  of  dis- 
tinguishing metabolic  from  structural  cytoplasmic  constituents. 

In  connection  with  the  foregoing  it  is  necessary  to  stress  the  fact 


1  CYTOPLASM  l8l 

that  morphogenetic  manifestations  of  the  cytoplasm  are  only  possible 
in  its  gelated  state,  for  this  alone  permits  it  to  assume  shapes  different 
from  those  induced  by  the  surface  laws  of  liquids.  Submicroscopic 
morphology  is  therefore  very  much  concerned  to  know  the  type  of 
junctions  by  which  the  macromolecules  of  the  cytoplasm  lose  their 
individuality  and  aggregate  to  form  a  gel. 

Comparison  with  current  opinions  on  the  structure  of  cytoplasm.  The  views 
on  the  submicroscopic  structure  of  cytoplasm  developed  in  former 
editions  of  this  monograph  have  met  with  some  criticism.  Before 
going  into  this  criticism,  we  shall  briefly  discuss  various  points  which 
make  our  theory  fundamentally  different  from  others. 

It  is  not  permissible  to  draw  a  parallel  between  "protoplasmic  vis- 
cosity" and  the  viscosity  of  liquids  (compare  Table  XXII,  p.  169). 
For  here  it  is  not  merely  a  matter  of  friction  between  freely  moving 
particles,  but  of  an  additional  resistance  offered  by  an  elastic,  sub- 
microscopic framework  as  well.  I  completely  agree  with  Scarth 
(1927)  when  he  writes  that  the  fall  of  a  particle  through  the  cytoplasm 
is  comparable  to  the  zig-zag  path  of  shot  falling  through  a  brush  heap, 
and  that  drastic  methods  like  centrifugation  forcibly  destroy  the  fine 
framework  of  the  plasma  structure.  The  work  of  Scarth  also  contains 
the  essential  points  of  this  monograph  in  those  places  where  he  points 
out  that  the  polarity  and  the  capacity  for  growth  of  cells  are  incom- 
patible with  the  nature  of  a  liquid  such  as  that  which  has  often  been 
attributed  to  the  cytoplasm  and  the  nucleus. 

Often  microscopic  strands  are  visible  in  the  cytoplasm.  As  a  dense, 
tough,  "formed"  protoplasm,  these  are  embedded  in  "unformed" 
protoplasm  of  semi-liquid  consistency.  Such  differentiations  have  been 
distinguished  as  kinoplasm  and  matrix  (Scarth,  1927),  active  plasma 
and  paraplasm  (v.  Mollendorff,  1937)  or  spongioplasm  and  en- 
chylema  (Monne,  1942a).  In  some  cases  the  two  constituents  can  be 
separated  in  the  centrifuge  as  a  gel  rich  in  lipids  and  a  sol,  poor  in 
lipids  but  rich  in  mitochondria,  comparable  to  the  conditions  in  the 
nucleus,  where  the  chromosomal  threads  and  the  karyolymph  can  be 
separated  from  each  other.  The  microscopic  cytoskeleton  (Peters, 
1937)  is  not  to  be  identified  with  the  submicroscopic  structure.  Un- 
doubtedly the  strands  which  are  visible  in  the  ordinary  microscope 
originate  from  far-reaching  bundling  of  the  submicroscopic  stra  nds 
postulated  by  us,  but  they  certainly  are  not  homogeneous  and  poss  ess 


l82  FINE-STRUCTURE  OF  PROTOPLASM  II 

an  invisible  fine-structure,  detailing  of  which  falls  within  the  province 
of  submicroscopic  morphology.  A  further  task  is  to  establish  the 
nature  of  the  plasma  liquor  (enchylema,  paraplasm,  matrix). 

Very  many  of  the  hypotheses  relating  to  the  structure  of  cytoplasm, 
discussed  in  former  times  (Lundegardh,  1922,  p.  242),  are  irre- 
concilable with  our  own  views.  Nowadays  the  emulsion  and  alveolar 
theories  can  no  longer  be  regarded  as  valid.  Taking  clotted  milk  as 
an  example,  Seifriz  (1936)  shows  how  the  droplet  theory  takes  ac- 
count only  of  the  relatively  coarse  units,  whereas  the  fine-structure  is 
caused  by  the  fibre  structure  of  the  casein.  He  applies  this  model  to 
cytoplasm  and  is  thus  led  to  a  scheme  of  protoplasmic  structure  which 
tallies  well  with  ours,  so  long  as  we  bear  in  mind  that,  when  living,  it 
does  not  represent  a  fixed  coagulum  of  protein  particles,  because  the 
particles  may  be  reversibly  released  and  move  freely  and  independently 
of  each  other.  Further  comparison  of  the  proteins  of  protoplasm  with 
a  heap  of  rodlets  seems  less  felicitous  to  me,  since  such  a  heap  has 
a  fortuitous,  statistical  character,  whereas  the  structure  of  protoplasm 
must  be  a  co-ordinated  whole.  Its  framework  cannot  be  a  disorderly 
pile;  it  must  surely  consist  of  an  organized  and  well-defined  structure. 

According  to  our  present  knowledge,  all  hypotheses  of  proto- 
plasmic structure  which  postulate  permanently  individualized  sub- 
microscopic  particles  (granules,  droplets,  alveoles,  ultramicrons)  must 
be  discarded  as  being  corpuscular  theories.  The  framework  structure  of 
gelated  cytoplasm  possesses  no  dispersed  phase  in  the  sense  of  the 
classical  theory  of  colloids:  both  the  framework  and  the  enchylema 
are  continous  throughout  the  whole  space  available.  For  the  same 
reason  Butschli's  foam  structure  or  honeycomb  theory  cannot  be  taken 
into  account,  in  spite  of  its  numerous  merits,  for  a  honeycomb  con- 
sists of  closed  dispersed  regions  in  contradistinction  to  the  open  and 
continuous  system  of  interconnected  strands. 

Flemming's  fibrillar  theory,  on  the  contrary,  conforms  rather  well 
with  the  condition  of  a  complete  intermeshing  of  strands  and  dis- 
persing medium  shown  to  be  likely  in  this  monograph.  Here  again, 
however,  the  fibrillar  structure  has  to  be  transferred  to  submicro- 
scopic regions.  In  fact,  in  a  three-dimensional  network,  both  the 
contours  of  the  meshes  and  the  meshes  themselves  fill  all  space 
continuously.  Monne  (1946  a)  is  of  the  opinion  that  the  protoplasmic 
fibrils  do  not  form  a  network,  but  are  only  plaited  (in  German:  Flecht- 


1  CYTOPLASM  183 

werk).  To  my  mind  this  depends  on  whether  we  have  to  do  with  a 
plasma  gel  or  a  plasma  sol  (p.  1 65).  In  the  first  case  there  must  be  some 
interaction  between  the  invisible  fibrils,  whereas  in  the  second  case 
they  may  be  independent  of  each  other. 

The  fibrillar  theory  has  been  developed  partly  on  the  basis  of  fixed 
■structures.  This  derivation  is  not  as  unreasonable  as  has  often  been 
suggested,  since  on  fixation  the  submicroscopic  or  amicroscopic 
strands  of  the  cytoplasm  combine  into  coarser  strings  by  directed 
^coagulation  and  can  thus  become  microscopically  visible.  It  is  only 
because  the  cytoplasm  actually  possesses  a  thread  structure,  that  the 
good  fixations  obtained  by  cytological  micro-techniques  are  possible. 
In  this  process  the  molecular  framework  may  shrink,  be  coarsened, 
<leformed  and  disturbed,  but  a  clear-cut  separation  of  coagulum  and 
serum  as  in  the  case  oi  protein  solutions  of  like  concentrations  (milk, 
fibrinogen)  does  not  occur. 

The  protoplasmic  framework,  which  proves  to  be  very  stable  with 
respect  to  hydrolyzing  substances,  may  be  identical  with  Reinke's 
plastin  (1881).  The  latter  represents  the  insoluble  and  not  easily  di- 
gestible part  of  the  cytoplasm;  both  these  properties  belong  to  the 
cytoplasmic  protein  framework.  On  drying,  it  becomes  still  less  di- 
gestible, which  may  be  connected  with  the  fact  that  the  strands  of  the 
framework  combine  into  coarser  strings,  as  in  fixation,  and  then  are 
less  accessible  to  the  destructive  enzymes. 

The  introduction  oi  plastin  as  a  collective  concept  for  the  entire 
protein  frame  is  very  convenient  for  describing  these  conditions. 
Although  Reinke  did  not  think  of  a  network,  its  properties  tally  well 
with  the  characteristics  given  by  him.  The  original  concept  "plastin" 
has  no  chemic?l  meaning,  for  it  is  characterized  only  in  the  negative: 
insolubility,  indigestibility,  absence  of  phosphatides  and  lipids;  in 
short,  what  remains  if  everything  sensitive  to  mild  physico-chemical 
intervention  has  been  removed.  Reinke's  expression  plastin  is  there- 
fore a  morphological  concept  Hke  chromatin  in  the  nucleus,  and  as  such 
is  almost  indispensable  for  purely  descriptive  purposes.  For  this  reason 
it  is  regrettable  that  Kiesel  (i93°)'  ^^^^"^  having  isolated  certain 
protein-Hke  skeleton  substances  from  the  plastin  of  slime  moulds  (in 
Reinke's  original  sense),  has  applied  the  name  "plastin"  to  a  well- 
defined  protein  compound.  It  is  better  to  give  a  new  name  to  these 
chemically  defined  substances,  and  to  maintain  the  plastin  concept  in 


l84  FINE-STRUCTURE  OF  PROTOPLASM 


II 


its  original  morphological  meaning  proposed  by  Reinke  (i88r), 
Zacharias  (1883),  Berthold  (1886)  and  others. 

Cytological  morphology  needs  collective  concepts  such  as  lignin, 
chromatin,  lipids  and  plastin,  which  do  not  designate  well-defined 
chemical  compounds  but  classes  of  substances  which  are  defined  in 
a  morphological  sense  as  microscopic  phenomena.  If  these  concepts, 
created  by  the  microscopist,  are  not  satisfactory  from  a  chemical  point 
of  view,  chemistry  should  provide  a  new  and  more  suitable  termi- 
nology. In  fact,  microscopic  microchemistry,  adjusted  to  morphology, 
can  never  satisfy  the  high  demands  of  an  exact  chemical  and  structural 
description. 

Bensley  has  succeeded  in  giving  a  closer  characterization  of  the 
structural  proteins  of  the  liver  (1938,  1943).  The  mobile  proteins  are 
soluble  in  0.8  5  %  NaCl.  On  further  treatment  with  N/200  NH4OH  the 
mitochondria  and  the  nucleochromatin  are  dissolved.  From  the  re- 
mainder a  homogeneous  substance,  plasmosin,  can  be  extracted  with 
NaCl  10%.  This  is  described  as  a  gel-  and  fibre-forming  constituent 
of  the  protoplasm  (Bensley,  1938).  The  protein  ellipsin  is  left,  and 
Bensley  compares  it  with  Reinke's  plastin.  Plasmosin  is  compared 
with  the  muscular  protein  myosin  (Bensley,  1943);  according  to 
Mirsky  and  Pollister  (1945),  however,  it  has  its  origin  in  the 
nucleus  and  should   be   regarded   as   a   nucleoprotein. 

Criticism  of  the  tbeo'j  of  junctions.  The  submicroscopic  reticular 
structure  of  the  cytoplasm  has  been  decidedly  rejected  by  Hofler 
(1940).  In  his  investigations  on  cap-plasmolysis  (compare  Fig.  114, 
p.  197)  he  succeeded  in  making  the  cytoplasm  of  Allium  cells  swell 
up  to  10  and  more  times  its  original  volume  with  the  aid  of  alkali  salts, 
without  causing  the  cells  to  die.  Hofler  concludes  that  no  framework 
can  be  present,  for  the  enormous  swelling  has  pushed  the  structural 
elements  so  far  apart  that  they  must  completely  change  their  mutual 
relations  and  positions.  This  reasoning  would  be  correct  if  only 
granular  particles  were  operative  in  the  cytoplasm.  It  has  been  pointed 
out,  however,  that  a  submicroscopic  or  even  molecular  framework 
can  attain  enormous  degrees  of  swelling  without  breaking  down  its 
structure  (see  p.  67).  It  seems  to  me,  therefore,  that  Hofler's  inter- 
esting observations  are  in  favour  of  the  theory  of  junctions  rather  than 
against  it,  for  what  system  other  than  a  gel  could  be  inflated  ten-fold 
without  losing  its  inner  organization?  That  the  latter  has  been  pre- 


I  CYTOPLASM  185 

served  is  proved  by  the  fact  that  it  is  able  revert  to  the  normal  state 
of  swelling  in  which  protoplasmic  flow  is  resumed.  In  spite  of  its 
magnitude,  cap-plasmolysis  must  be  designated  as  limited  swelling,  and 
in  the  case  of  colloids  with  limited  swelling  we  always  have  some  sort 
of  meshwork. 

It  is  incumbent  upon  us  to  give  the  most  careful  consideration  to 
any  objections  of  a  physico-chemical  nature,  since  these  concern  the 
fundamentals  of  the  postulated  theory  of  junctions.  According  to 
ScHULZ  (1939),  the  Van  der  Waals  cohesive  forces  are  too  small  to 
establish  fixed  bonds  between  molecules,  so  that  a  continual  inter- 
change of  these  junctions  must  be  assumed.  Considering  the  labile 
nature  of  the  invisible  protoplasmic  structures,  it  seems  to  me  that 
this  should  be  valued  as  constructive  rather  than  destructive  criticism. 
The  decisive  point  is,  that  the  cohesive  forces  between  the  macro- 
molecules  of  the  cytoplasm  act  as  struct ure-Joijnmg  elements,  as  is 
clearly  shown  by  the  structure  of  mesophases  (p.  51).  Although  long- 
range  forces  are  even  smaller  than  the  Van  der  Waals  forces,  to 
which  they  are  related,  they  must  also  be  included  among  the  possible 
junctions,  since  they  possess  structure-forming  faculties.  According 
to  Bernal  (1940)  and  Fankuchen  (1941),  they  can  cause  macro- 
molecules  which  are  up  to  1 50  A  apart  to  form  oriented  gel  structures! 

K.  H.  Meyer  (1940  a,  p.  607),  on  the  contrary,  regards  the  cohesive 
bonds  as  true  junctions.  According  to  him,  the  distinction  between 
several  different  types  of  junctions  goes  too  far;  a  division  into 
cohesive  and  valency  bonds  would  amply  suffice.  Against  this  objection 
it  can  be  said  that  chain  molecules  with  homopolar  cohesive  bonds 
(e.g.,  waxes)  or  chiefly  heteropolir  cohesive  bonds  (e.g.,  cellulose) 
show  a  fundamentally  different  behaviour  in  the  physiological  range 
of  temperatures.  Whereas  wax  becomes  plastic  at  37°  as  a  result  of 
the  weakening  of  the  homopolar  cohesive  bonds,  a  separation  of  the 
polysaccharide  chains  in  cellulose  can  only  be  brought,  about  by 
suitable  hydration  of  the  heteropolar  cohesive  bonds.  Admittedly, 
homopolar  cohesive  bonds  can  also  be  solv^ated  by  lipophilic  swelling 
media  (benzene,  etc.).  Under  physiological  conditions,  however, 
solvating  media  of  this  kind  need  not  be  considered,  and  it  would 
seem  that  the  division  suggested  suits  the  purpose  in  the  case  of  living 
hydrogels.  Similarly,  the  reaction  to  chemical  interference  (hydrolysis, 
hydrogenation,   etc.)   of  a   gel  frame   containing   only  heteropolar 


l86  FINE-STRUCTURE  OF  PROTOPLASM  II 

valency  bonds  would  be  fundamentally  different  from  that  of  a  gel 
whose  chain  molecules  are  connected  by  homopolar  valency  bonds. 
While  this  criticism  touches  the  theory  of  junctions  in  the  molecular 
range,  Lehmann  and  Biss  (1949)  raise  objections  to  reticula,  whose 
strands  have  diameters  lying  on  the  borderline  between  submicro- 
scopic  and  microscopic  dimensions.  They  contend  that  the  theory 
considers  only  molecular  or  micellar  frameworks  and  neglects  gel 
structures  with  coarser  strands,  such  as  the  beaded  fibrils  found  in  the 
Tuhifex  egg.  This  argument  disregards  the  basic  principle  of  the  theory 
of  junctions,  which  has  been  advanced  in  opposition  to  the  view  that 
the  cytoplasm  is  a  liquid,  because  to  my  mind  its  capacity  to  gelate 
is  a  vital  necessity.  Since  it  is  not  known  which  forces  cause  the  cyto- 
plasm to  set,  in  1938  I  introduced  the  notion  of  junctions,  a  term  which 
does  not  imply  any  special  type  of  binding  forces.  If  the  possibilities 
of  junctions  in  the  amicroscopic  range  have  been  more  extensively 
described,  it  is  only  because  very  little  is  known  of  other  types  of 
junctions,  such  as  long-range  forces.  But  this  by  no  means  implies 
that  only  molecular  gels  are  involved.  On  the  contrary,  every  possible 
type  of  gel  must  be  taken  into  account;  and  it  is  the  task  of  submicro- 
scopic  cytology  to  establish  the  nature  of  the  junctions  involved. 

e.  Protoplasmic  Flow  and  Cell  Polarity 

Protoplasmic  flow.  The  touchstone  for  the  correctness  of  any  theory 
of  protoplasmic  structure  is  a  self-consistent  explanation  of  proto- 
plasmic flow.  For  this  reason  the  latest  results  of  the  investigations 
"on  this  important  phenomenon  of  life  will  be  briefly  discussed. 

The  cells  and  plasmodia,  in  which  it  has  so  far  been  possible  to 
analyze  flow  in  detail,  all  show  a  sol-like  liquid  inner  protoplasm 
(plasma  sol)  and  a  gel-like,  solidified  outer  skin  (plasma  gel)  (Lewis, 
1942;  Marsland,  1942;  Moyer,  1942;  ScARTH,  1 942;  Seifriz,  1942, 
1943;  Andresen,  1942).  The  difference  in  colloid  state  between  the 
two  types  of  protoplasm  is  demonstrated  by  the  Brownian  movement 
of  microscopic  granules.  These  are  in  lively  movement  in  the  bulk 
protoplasm  (endoplasm)  where  the  viscosity  is  low,  but  in  the  solid 
protoplasmic  skin  (ectoplasm),  they  have  the  appearance  of  being 
frozen.  According  to  Gold  acre  and  Lorch  (1950),  the  protein  mole- 
cules are  in  a  folded  state  in  the  liquid  endoplasm  and  in  an  unfolded 
(denatured)  state  in  the  gelated  ectoplasm. 


I  CYTOPLASM  187 

In  cells  with  amoeboid  movement,  protoplasmic  flow  is  maintained 
by  continuous  gel-sol  transitions.  The  hind  part  of  the  cell  contracts, 
and  simultaneously  part  of  the  gel-like  ectoplasm  is  converted  into 
liquid  endoplasm.  This  can  be  observed  directly,  because  particles 
enclosed  in  the  ectoplasm  become  mobile,  show  increased  Brownian 
movement  and  finally  are  carried  away  by  the  endoplasm.  In  the  front 
part  of  the  amoeba,  inner  pressure  causes  the  skin  to  become  thin 
and  bulge  outward  as  a  pseudopodium.  The  invading  stream  of 
endoplasm  solidifies  into  a  gel  at  the  side  walls  of  the  bulge  and 
thus  rebuilds  the  skin  at  the  same  rate  at  which  the  amoeba  moves 
forward.  To  explain  protoplasmic  flow  we  need,  therefore,  a  deeper 
understanding  both  of  the  contraction  and  of  the  gel-sol  transition  of 
protoplasm. 

In  cytoplasm  liquefied  artificially  (by  high  pressure,  p.  172),  any 
flow  there  may  be  stops;  not  only  does  the  creeping  motion  of 
Amoeba  cells  come  to  an  end,  but  also  the  rotation  in  Elodea  cells. 
The  process  of  cell  division  is  interrupted  also  in  sea-urchin  eggs, 
which  display  incipient  constriction.  If  the  high  pressure  is  not  main- 
tained too  long,  the  cytoplasm  re-solidifies  into  a  gel  on  return  to 
normal  pressure,  and  protoplasmic  flow  and  cell  division  resume  their 
normal  course  again.  These  experiments  show  that  the  plasma  sol  is 
not  capable  of  flowing  and  of  forming  constrictions  such  as  those 
necessary  for  cell  division,  since  no  gel  structure  is  present  to  provide 
the  necessary  forces. 

Lewis  (1942)  has  shown  that  in  sol-gel  transitions  the  solidifying 
protoplasm  can  contract.  In  the  division  of  fibroblasts,  for  instance, 
the  division  of  the  nucleus  is  accompanied  by  the  occurrence  of  a 
thickened  ring  of  plasma  gel,  which  divides  the  cytoplasm  into  two 
parts  by  contraction.  This  explains  how  the  ectoplasm  of  the  Amoeba 
can  exert  pressure  on  the  endoplasm. 

It  is  of  particular  interest  that  these  contractions  take  place  rhyth- 
mically. With  the  aid  of  time  lapse  photography  (80  fold  speeding  up), 
Seifriz  (1937)  has  shown  that  the  flow  is  a  pulsating  movement. 
Kamiya  (1940,  1942)  succeeded  in  analyzing  the  rhythmic  flow  of 
the  cytoplasm  in  ?  plasmodium  strand  of  Physarum  polycephalum  by 
means  of  variable  one-sided  counter-pressures  which  exactly  balance 
the  flow.  He  observed  complicated  oscillatory  changes  in  pressure, 
which  can  be  resolved  into  pure  sine  oscillations  by  Fourier  analysis. 


i88 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


Fig. 
shows 


40      44 
Time  in  minutes 


Fig.  109.  Electrical  record  and  mechanical  record  of  streaming 
Physarum  cytoplasm  (from  Kamiya  and  Abe,  1950). 


This  shows  that  the  plasmic  flow  of  slime  moulds  is  a  polyrhythmic 
movement  caused  by  numerous   sine-like   contractions   of  various 

periods. 

109  (below) 
the  oscilla- 
tions of  the  pressure 
in  a  Plasmodium 
strand  of  Physarum. 
There  are  cyclic 
changes  of  the  amp- 
litudes and  a  sys- 
tematic displacement 
of  the  central  point 
between  maximum 
and  minimum  press- 
ures. This  means 
that  there  is  a  more 
intense  flow  in  one 
direction  of  the 
strand  than  in  the  other,  with  the  result  that  the  cytoplasm  moves 
slowly  in  the  direction  of  lower  pressure. 

Kamiya  and  Abe  (1950)  have  also  measured  the  electric  potential 
difference  between  the  two  poles  of  a  PljsaniM  strand  with  its  oscil- 
lating plasmic  flow.  It  changes  in  a  similar  way  to  the  internal  pressure, 
showing  sine  waves  with  the  same  periods  and  corresponding  am- 
plitudes within  10  mV,  but  there  is  a  small  phase  difference.  The  maxi- 
mum and  minimum  values  of  the  electrical  record  lag  behind  those  of 
the  mechanical  record  by  about  half  a  minute,  indicating  that  the  con- 
traction involving  a  pressure  change  is  not  caused  by  the  measured 
potential  differences.  The  pressure  oscillations  can  be  eliminated  by 
appropriate  counterpressures.  Then  the  rhythmic  potential  changes 
go  on.  This  means  that  the  chemical  processes  causing  contraction 
operate  even  if  the  contraction  is  impeded. 

These  details  of  rhythmic  contraction  are  reminiscent  of  muscle 
activity,  which  is  due  to  the  contractility  of  actomyosin  (see  p.  358). 
It  is  therefore  likely  that  protoplasmic  flow  is  also  maintained  by 
contractile  proteins  in  the  cytoplasm.  These  can  only  develop  their 
full  activity  in  the  gelated  state.  It  would  seem  that  these  statements 


CYTOPLASM 


189 


once  and  for  all  refute  the  idea  that  cytoplasm  is  a  liquid  with  freely 

moving  particles. 

Protoplasmic  flow  in  Atmeha  and  Physarum  seems  to  consist  in  the 

forcing  of  liquid  cytoplasm  through  capillaries  or  other  channels  by 

a  contracting  gel;  but  this  view  cannot  be  generalized.  In  plant  cells, 

such  as  in  the  leaves  of  Elodea,  the  whole 

protoplasm  rotates    along  the   cell    wall 

(cyclosis) ;  or  in  Uving  hairs  cytoplasmic 

strands  even  circulate  across  the  central 

vacuole.  In  these  cases  the  impelling  force 

must  be  sought  in  the  flowing  strand 

itself.  If  we  admit  that  local  contractions 

are  again  involved,  we  may  postulate  the 

following  to  account  for  the  flow  (Frey- 

Wyssling,  1947).  A  submicroscopic  part 

of  the  strand  gelates  and  contracts  for  a 

short  time ;  relaxation  follows  and  an  ad- 
joining spot  contracts,  etc.  When  such 

waves  of  contraction  move  periodically 

along   the   protoplasmic    strand   in    one 

direction,  there  is  flow  either  in  a  perist- 
altic manner  by   transverse    contraction 

of  the  surface  layers,  or  in  pulling  the 

highly    viscous    strand   by   longitudinal 

contraction  (Fig.  no). 

In  the  last  case  the  flow  is  opposite  to  the  direction  of  the  moving 
contraction  and  the  relaxed  part  of  the  strand  must  be  expanded  by 
another  contraction  centre  situated  at  some  distance.  Since  in  the 
same  microscopic  strand,  flow  may  proceed  simultaneously  in  oppo- 
site directions,  diff"erent  waves  of  contraction  with  opposite  polarity 
must  be  admitted.  Loewy  (1949)  stresses  the  fact  that  this  system 
necessitates  a  solid  substratum  (cell  wall,  ectoplasm)  on  which  the 
gelating  centres  of  the  flowing  strand  can  be  temporarily  anchored 
by  some  kind  of  junctions. 

In  any  case  a  contraction  of  submicroscopic  elements  can  only 
produce  a  microscopically  visible  eff"ect,  if  the  system  is  temporarily 
solidified  by  junctions.  This  is  evident  from  Fig.  in.  To  the  left  of 
this  figure  linear  submicroscopic  particles  contract  individually;  the 


Fig.  1 10.  Movement  of  a  protein 
strand  by  a  contraction  wave.  The 
strand  streams  in  the  opposite 
direction  to  the  advancement  of 
the  wave  {a,  b,  c)  (from  Frey- 
Wyssling,   1947). 


190 


FINE-STRUCTURE  OF  PROTOPLASM 


ir 


I  i 
1  \ 


1 


1 


effect  of  this  contraction  is  to  increase  their  distance  apart,  but  no 
external  tension  is  manifest.  Only  if  the  particles  are  joined  by  junc- 
tions (Fig.  1 1 1  b)  is  a  microscopically  visible  shortening  possible  and 
an  external  force  exerted. 

Cell  polarity.  Another  important  fact  which  has  to  be  explained  by 
^  I  a  consistent  theory  of  plasma 

structure  is  the  polarity  of 
cytoplasm.  This  property  is 
especially  evident  with  the 
A  eggs  of  Ecliinodermata  and 
i|  hi  Amphibia.  These  cells  show 
definite  animal  and  vegetative 
poles.  Sometimes  the  animal 
pole  is  characterized  by  a  pa- 
pilla, but  this  is  not  universal. 
There  is  as  well  an  invisible 
physiological  polarity.  Were 
the  structural  elements  of 
cytoplasm  independent  of 
each  other  as  in  a  liquid,  no 
fixed  polar  arrangement  within  the  cytoplasm  would  be  conceivable. 
The  polarity,  therefore,  must  be  inherent  in  the  plasma  gel.  As  the 
cortex  of  the  egg  has  undoubtedly  a  gel-like  character  and  in  this 
state  is  capable  of  considerable  active  transformation  when  the 
fertilization  membrane  is  formed  (Runnstrom,  1944),  one  might  be 
inclined  to  attribute  the  polar  properties  to  this  cortical  layer.  But 
MoNNE  (1946  b)  finds  that  there  is  a  dorsoventral  gradient  also  within 
the  egg,  the  animal  cytoplasm  being  more  solidified  and  the  vegetative 
cytoplasm  more  liquefied.  It  is  admitted  that  the  heteropolar  organi- 
zation of  the  egg  is  predetermined  by  the  foregoing  cell  division 
(Lehmann,  1945).  Cytoplasmic  currents  do  not  destroy  the  hetero- 
polar organization.  From  this  fact  I  suppose  that  important  junctions 
of  the  protoplasmic  framework  are  still  present  throughout  the  moving 
cytoplasm.  As  Monne  points  out,  cytolysis  of  the  sea-urchin  egg  is 
preceded  by  violent  protoplasmic  currents.  This  increased  movement 
is  due  to  a  complete  liquefaction  of  the  cytoplasm,  which  is  followed 
by  the  disorganization  and  the  death  of  the  cell.  Complete  disinte- 
gration of  the  junctions,  therefore,  will  never  occur  in  living  cells. 


Fig.  III.  Contraction  of  protein  molecules;  a) 

without  being  interlinked,  h)  when  interlinked 

by  junctions  (from  Frey-Wyssling,  1947) 


I  CYTOPLASM  191 

On  the  other  hand,  fertilization  of  the  sea-urchin  egg  is  followed  by 
a  solidification  of  the  fluid  endoplasm  into  a  gel  (Mirsky,  1956). 

f .  Separation  of  the  Cytoplasm  into  Different  Phases 

As  long  as  there  exists  a  certain  equilibrium  between  the  cyto- 
plasmic proteins  on  the  one  hand  and  the  amount  of  lipids  and  water 
on  the  other,  the  cytoplasm  remains  microscopically  homogeneous, 
hyaline,  as  clear  as  water  and  optically  empty.  In  the  physico-chemical 
sense  as  well,  the  system  is  a  homogeneous  pseudophase  (p.  69) 
without  inner  surfaces.  This  system  is  bound  to  separate  into  phases 
if  one  of  the  three  components,  protein,  Hpid  or  solvent,  increases  in 
quantity  to  such  an  extent  that  the  state  of  mutual  equilibrium  can 
no  longer  be  maintained,  and  similar  molecules  cluster  together  and 
are  separated  from  the  rest  of  the  cytoplasm  by  a  phase  boundary. 

Formation  of  vacuoles.  Guilliermond  (1933)  attributes  the  origin  of 
vacuoles  to  the  formation  of  hydrophilic  colloids  in  the  cytoplasm. 
These  colloids  attract  water,  are  hydrated  and  thus  cause  a  separation. 
It  is  quite  possible  that  the  vacuoles  are  formed  in  this  manner.  Besides 
colloids,  salts,  which  accumulate  in  the  cell,  may  initiate  the  accumu- 
lation of  water  in  some  of  the  meshes  of  the  submicroscopic  frame- 
work. Then,  according  to  the  laws  of  surface  tension,  the  aqueous 
phase  becomes  spherical  in  shape  and  pushes  the  framework  aside.^  It 
may  therefore  be  assumed  that  the  framework  of  the  cytoplasm  has 
a  higher  density  in  the  neighbourhood  of  a  vacuole.  Thereupon  lipids 
are  accumulated  in  the  boundary  layer  (cf.  Fig.  115,  p.  199). 

The  colloid  content  of  the  vacuolar  liquid  can  be  demonstrated,  or 
at  least  shown  to  be  probable,  in  several  ways.  The  viscosity,  for 
instance,  is  about  twice  that  of  water  (Weber,  1921;  Pekarek,  1933) 
or  of  aqueous  solutions  with  the  same  salt  content  as  the  vacuoles 
(cf.  Table  XXII,  p.  169).  The  large  terminal  vesicle  of  Closterium  algae, 
in  which  the  sedimentation  of  g^'psum  crystals  can  be  measured  accu- 
rately (Frey,  1926  c),  is  particularly  suitable  for  the  application  of  the 
falling  particle  method.  From  Stokes'  law  one  derives  a  relative  vis- 
cosity of  about  2.5  for  the  cell  sap.  The  experiment  shows,  moreover, 
that  the  boundary  of  the  vacuole  is  not  a  smooth  surface,  for  a  number 
of  crystals  do  not  follow  the  shortest  path,  but  glide  down  along  the 

^  Owing  to  the  plasmic  sti-ucture,  the  vacuoles  may  at  first  sight  appear  to  be  rod-like 
in  shape. 


102 


FINE-STRUCTURE  OF  PROTOPLASM 


ir 


wall  (Fig.  112a).  For  this  reason,  when  measuring  the  time  of  fall  of 
crystals  traversing  the  cell  sap,  one  must  always  observe  the  time 
needed  to  detach  the  particle  from  the  phase  boundary  (Weber,  1921). 
In  certain  cases  the  cell  sap  solidifies  on  fixation,  as  shown  in  Fig. 
112  b  in  the  pathological  giant  cells  of  the  fungus  Aspergillus  niger 
(Frey,  1927a).  Here  the  difference  between  the  colloid  systems  of  the 
cell  sap  and  the  protoplasm  is  evident.  In  the  cytoplasm  the  framework 


Fig.  112.  Vacuoles,  a)  Sedimentation  of  gypsum  crystals  in  terminal  vacuoles  of  Closterium 

(from  Frey  1926c);  b)  pathologic  giant  cells  of  Aspergillus  niger  fixed  with  Flemming. 

Cytoplasm  2  and  nucleus  k  have  not  changed  much;  in  the  cell  sap,  however,  a  voluminous 

precipitate  is  formed  (from  Frey,  1927a). 

structure  prevents  a  separation  of  the  different  components,  whereas 
in  the  cell  sap  precipitation  occurs.  The  coagulated  vacuole  of  Fig. 
112b  betrays  a  coarse  structure  of  fibrous,  entangled  bodies.  From 
this  we  may  conclude  that  the  colloids  in  the  cell  sap  do  not  possess 
a  structure  comparable  with  the  cytoplasm,  but  represent  sols  with 
movable  particles  without  definite  mutual  positions.  Here  coagulation 
actually  results  in  an  orderless  "pile",  indicating  an  unordered  state 
before  the  precipitation.  The  end  groups  of  the  organic  compounds 
which  are  the  constituents  of  vacuolar  colloids  are  not  screened  off  as 
in  the  cytoplasm  and  are  consequently  reactive.  This  is  made  use  of 
in  the  vital  staining  of  the  vacuoles.  Their  colloids,  which  evidently 
carry  acid  groups,  are  usually  readily  coloured  by  basic  dyes.  In  the 
cytoplasm,  the  cell  nucleus  (Becker,  1956)  and  the  living,  still  growing 


I  CYTOPLASM  195 

cell  wall,  on  the  contrary,  vital  staining  is  much  less  easily  obtained. 
According  to  Strugger's  investigations  (193  5/1936)  on  vital  staining, 
the  Ph  of  the  surrounding  liquid  is  the  main  factor  in  dyeing;  this  is 
true  not  only  in  the  living  state,  but  according  to  Pischinger  (1937), 
Drawert  (1937)  and  others  also  in  fixed  protoplasts.  According  to 
the  theory  of  junctions  this  means  that  the  acid  and  basic  groups  of 
the  framework,  which  are  screened  off  in  the  I.E. P.,  must  first  be 
liberated  by  slight  hydrolysis  in  order  to  be  capable  of  reacting  with 
the  dyestuff. 

The  vacuoles  owe  their  existence  to  substances  which  are  tempo- 
rarily or  definitively  excluded  from  interaction  with  the  framework 
of  the  cytoplasm.  For  this  reason  these  sap-filled  spaces  represent 
places  in  which  excretory  (definitive  elimination)  or  reserve  substances 
(temporary  elimination)  are  stored.  All  cell  sap  components  like 
anthocyanins,  tannins,  glucosides,  etc.  must  therefore  be  regarded  as 
substances  eliminated  from  the  cytoplasm.  Hence  the  vacuoles  are 
primarily  excretory  organelles  in  which  all  kinds  of  substances  that  are 
inconsistent  with  the  cytoplastic  molecular  structure  are  stored;  their 
function  of  regulating  osmotic  phenomena  is  only  a  secondary  task. 

Lipidic  drops.  As  in  the  case  of  water,  there  is  an  upper  limit  to  the 
amount  of  molecularly  dispersed  lipids  bound  by  the  cytoplasm 
structure.  Beyond  this  limit  the  Hpid  molecules  cluster  together  into 
globules  which  represent  an  analogy  to  the  vacuoles;  they  might  be 
called  lipidic  vacuoles  as  counterpart  to  the  aqueous  vacuoles.  Apart 
from  the  surface  films  at  the  phase  boundaries,  as  a  rule  neither  the 
lipidic  drops  nor  the  vacuoles  possess  a  structure.  Their  content  is 
semi-solid  to  liquid,  optically  isotropic  and  homogeneous  in  the 
physico-chemical  sense. 

These  regions,  which  are  homogeneous  and  therefore  foreign  to 
the  protoplasm,  are  usually  regarded  as  reserves  for  the  metabolic 
process.  In  this  connection  we  think  in  the  first  place  of  oil  and  fat 
containing  seeds,  which  mobilize  their  lipids  during  germination. 
However,  we  also  find  lipidic  secretions  of  an  irreversible  nature, 
which  can  scarcely  be  considered  as  reserve  substance  (fatty  de- 
generation, lipophanerosis). 

A.leurom  grains .  The  accumulation  of  proteins  in  the  cytoplasm  leads 
to  two  types  of  differentiation.  On  the  one  hand,  easily  soluble 
proteins  with  globular  molecules  of  relatively  low  molecular  weight 


IC)4  FINE-STRUCTURE  OF  PROTOPLASM  II 

may  accumulate  in  the  vacuoles  of  storage  ceils,  where  they  crystallize 
or  solidify  into  aleurone  grains.  However,  if  the  amount  of  high  mole- 
cular weight  protein  chains  in  the  cytoplasm  increases  and  these  chains 
cluster  together,  protoplasmic  fibrils  are  formed  (Kuster,  1934a, 
1935  a).  In  other  words,  the  morphological  properties  observed  depend 
upon  whether  reserve  proteins  or  structural  proteins  are  separated. 
Originally  the  aleurone  grains  are  liquid  vacuoles,  which  lose  water 
by  active  dehydration.  In  this  process  the  various  vacuole  components 
precipitate  according  to  their  solubility.  In  the  aleurone  vacuole  of 
Rkinus  seed,  for  instance,  the  almost  insoluble  magnesium-potassium 
salt  of  inositol  phosphoric  acid  (phytin)  is  precipitated  first  as  a  body 
called  "globoid".  Thereupon  the  reserve  proteins  which,  in  contrast 
to  the  insoluble  skeletal  proteins,  are  corpuscularly  dispersed,  begin 
to  arrange  themselves  into  the  lattice  order  of  a  crystalloid  (cf.  p.  136) 
and  to  fill  the  available  space.  Finally  the  last  remnants  of  liquid, 
containing  an  easily  soluble  albumin,  solidify  into  a  homogeneous 
substance  surrounding  both  globoid  and  crystalloid.  On  mobilization 
of  the  reserve  substances,  the  dissolution  proceeds  in  the  reverse 
order:  the  albumin  is  dissolved  first,  thereupon  follows  the  protein 
crystalloid  and  finally  the  mineral  globoid. 

Origin  of  fibrils.  Formerly  the  formation  of  contractile  fibrils  (Proto- 
zoa) and  of  muscular  fibres  (Metazoa)  was  regarded  as  an  extremely 
curious  achievement  of  the  cytoplasm.  Nowadays,  however,  this  kind 
of  differentiation  can  be  understood  from  a  morphological  point  of 
view,  since  the  framework  structure  of  the  cytoplasm  itself  consists 
of  submicroscopic  strands.  These  structural  elements  need  only  be 
accumulated  and  arranged  in  some  order  to  produce  microscopic 
fibrillar  structures.  However,  the  mechanism  of  contraction  of  these 
fibrils  remains  obscure  (cf.  p.  359). 

Phase  separation  by  centrifitging.  The  phases  brought  about  by  sepa- 
ration can  be  stratified  in  the  cell  by  centrifugal  force.  Here  the 
centrifuge  microscope  of  E.  N.  Harvey  and  Loomis  (1930)  renders 
special  service.  Fig.  113  shows  a  centrifuged  sea-urchin  egg  of  A.rbacia 
punctulata.  Centrifuging  has  elongated  the  egg  cell  and  its  various 
components:  pigment  grains,  yolk  globules,  mitochondria  and  oil 
droplets  appear  neatly  separated.  Optically  homogeneous  cytoplasm, 
containing  the  nucleus,  accumulates  in  the  less  dense  part  of  the  cell. 
The  striking  layer  formation  seems  to  indicate  a  stratification  phe- 


CYTOPLASM 


195 


OH 
Nucleus 

Optically 

homogeneous 
zone 

Chondrisomes 


Yolk 


nomenon  in  a  liquid.  This,  however,  is  contradicted  by  the  following 

interesting  and  extremely  remarkable  fact:  by  further  centrifuging, 

the  egg  cell  can  be  separated  into  two  halves,  as  indicated  in  Fig.  1 1 3 

by  a  line.  In  this  process  a  clear  part  containing  the  nucleus  and  a 

pigmented  part  without  nucleus  are  formed.  Both  can  be  inseminated 

and  are  then  capable  of  division  (E.  B.  Harvey, 

1933),  and  the  part  which  does  not  contain  the 

nucleus  may  sometimes  be  induced  to  divide 

without  any  nucleus.  E.  B.  Harvey  (1936) 

concludes  from  this:  "It  must  therefore  be  the 

'ground  substance'  which  is  the  material  for 

development  -  the  matrix  which  is  not  moved 

by  centrifugal  force  and  which,  in  the  living 

egg,  is  optically  empty".   Lehmann  (1945) 

points  out  that  in  the  outer  layers   of  the 

Tuhifex  and  the  sea-urchin  egg,  there  must 

be  a  morphogenetic  pattern,   which  cannot 

be  destroyed  by  centrifugal  forces. 

In  other  words,  the  method  of  centrifuging 
also  leads  to  the  conclusion  that  an  invisible 
ground  framework  must  exist,  which  is  torn 

apart  in  the  centrifuge  by  the  oil  droplets,  yolk  and  pigment  particles 
respectively,  as  a  result  of  their  different  weights.  The  microscopically 
visible  particles  must  move  in  the  opposite  direction  through  the 
meshes  of  this  framework  without  damaging  it  seriously,  seeing  that 
division  and  growth  of  the  plasmic  fragments  separated  by  centrifu- 
gation  still  takes  place  afterwards.  For  this  reason  the  framework  must 
either  possess  very  coarse  meshes,  or  else  it  must  be  possible  for  the 
important  molecular  groupings,  whose  mutual  positions  have  been 
altered  by  centrifugation,  to  be  restored  to  their  original  arrangements. 

By  centrifuging,  the  invisible  cytoplasmic  frame  is  orientated,  for 
the  drawn-out  plasmatic  neck  shows  positive  birefringence  with  respect 
to  the  axis  (Pfeiffer,  1941b).  Its  reticular  structure  must  possess  an 
unexpected  mechanical  stability,  for  A.scaris  eggs  can  stand  centrifugal 
fields  of  950,000  times  gravity  for  10  hours  or  400,000  times  gravity 
for  10  days  (Beams,  1943),  without  dying  or  losing  their  normal 
capacity  for  development,  although,  with  the  exception  of  the  nucleus, 
all  components  of  the  cell  appear  to  be  completely  separated  from  the 


Pigment 


Fig.  113.  Egg  cell  of  Arbacia 
punctulata  after  centrifuging 
(from  E.B.Harvey,  1936). 


[C)6  FINE-STRUCTURE  OF  PROTOPLASM 


II 


cytoplasm.  Nor  can  the  polarity  of  Tithifex  eggs  be  reversed  by 
centrifuging  (Lehmann,  1940). 

We  must  mention  in  particular  that  neither  the  oil  droplets  nor  the 
yolk  and  pigment  combine  into  a  homogeneous  phase,  but  remain 
dispersed.  This  indicates  the  existence  of  surface  layers  which,  either 
by  their  structure  or  by  their  electric  charge,  offer  resistance  to  fusion 
with  the  neighbouring  particles.  It  is  quite  possible  that  the  properties 
of  the  ground  substance  in  which  they  are  still  embedded  prevent  the 
droplets  from  clustering  together  as  might  be  expected  from  the  laws 
of  surface  tension. 

Separation  of  phases  as  a  result  of  freezing.  When  the  cytoplasm  is 
subjected  to  freezing,  ice  crystals  are  formed  which  are  embedded  in 
the  dehydrated  gel.  Thus  we  get  separation  by  crystalHzation.  Ac- 
cording to  LuYET  (1939)  the  dehydration  of  the  living  hydrogel 
proceeds  step  by  step.  As  long  as  the  freezing  is  confined  to  excess 
water,  such  as  that  contained  in  the  vacuoles  of  plant  cells  or  coming 
from  the  metaboHc  process,  the  cell  does  not  die.  It  is  only  when  the 
imbibition  water  which  takes  up  the  plasma  structure  is  withdrawn 
from  the  living  hydrogel,  that  the  structure  breaks  down  and  death 
of  the  cell  sets  in.  The  resistance  of  the  cytoplasm  to  low  temperature 
depends,  therefore,  on  the  persistence  with  which  it  retains  its  hy- 
dration water  and  safeguards  it  against  crystalHzation. 

Thecrystalhzationof  the  imbibition  water,  which  is  enclosed  in  the 
submicroscopic  gel  meshes  and  bound  by  hydration  forces,  can  be 
prevented  if  the  gel  is  cooled  down  to  very  low  temperatures  by  rapid 
removal  of  heat.  This  leads  to  a  state  which  has  been  designated  as 
vitrification  (Luyet,  1937).  The  water  molecules  become  immobile  to 
such  an  extent  that  they  cannot  arrange  themselves  into  a  crystal 
lattice  and  retain  their  original  positions  with  respect  to  the  sub- 
microscopic  gel  strands.  In  this  way  it  is  possible  to  preserve  the  "life 
structure"  of  thin  protoplasmic  films  for  a  considerable  space  of  time, 
for  instance  in  hquid  air.  The  fact,  however,  that  with  rising  tempera- 
ture the  preparation  has  to  pass  through  the  critical  temperature  range 
in  which  the  water  separates  from  the  gel  by  crystallization,  makes  it 
difficult  to  induce  such  a  "vitrified"  protoplasm  to  resume  its  life 
functions.  The  clear  gel  suddenly  becomes  turbid  at  about — 15''^ 
and  then  the  structural  breakdown  sets  in,  which  normally  causes 
death  on  slow  cooling  (cf.  freeze-drying,  p.  178). 


CYTOPLASM 


197 


This  phenomenon  should  not  be  confused  with  the  well-known 
fact  that  frozen  plants  can  often  be  kept  alive  if  thawed  slowly.  In 
these  objects  the  imbibition  water,  indispensable  to  life,  has  not  yet 
crystallized  and  it  is  only  necessary  to  avoid  inundation  of,  and 
damage  to,  the  protoplasmic  structure  by  water  from  ice  melting  too 
suddenly. 

g.  Morphological  Principles  of  the  Permeability  Problem 

Like  all  physiological  questions,  the  problem  of  physiological 
permeability  is  founded  on  morphological  assumptions.  The  lipid 
theory  of  Overton  (1899),  the  :iltrafilter  theory  of  Ruhland  (191 2, 
1950),  the  mosaic  theory  of  Nathanson  (1904)  and  the  modern,  com- 
bined lipid  filter  theory  of  Collander  (1932,  1937a)  are  all  based  on 
certain  morphological  concepts  which,  it  is  true,  have  not  been  gained 
directly,  but  via  physiological  experiments  or  reasoning  (Davson  and 
Danielli;  1943).  Before  going  into  these  questions  of  the  submicro- 
scopic  structure  of  protoplasmic  boundaries,  a  more  accurate  micro- 
scopic description  of  the  cell  boundaries  must  be  given. 

Problem  of  the  boundary  layers.  The  phenomenon  of  the  cap-plasmo- 
lysis  (German:  Kappen-Plasmolyse)  proves  that  certain  plasmolytic 
agents  are  capable  of  penetrating  into  the  cytoplasm,  though  not  into 
the  cell  vacuole.  For  this  reason  Hofler  (193  i)  distinguished  between 
permeability,  i.e.,  the  passage  from  the  outside  through  the  cytoplasm 
into  the  cell  sap,  and  intrability,  in  which  only  the  cytoplasm  is  reached. 
In  addition  one  must  in  certain  cases  take  into  account  a  membrane 


Fig.  114.  Cell  with  cap-plasmolysis  to  demonstrate  the  various  types  of  permeability 

(from  Hofler,  1932).  a)  Membrane  permeability  (p  plasmolysis  forecourt);  Z')  intrability 

(2  cytoplasm);  c)  permeability   (v  vacuole). 

permeability,  i.e.,  a  resistance  of  the  cell  wall  to  penetration  (Fig.  114). 

In  cellulose  cell  walls  the  membrane  permeability  can  be  neglected; 

they  are  permeable  to  all  plasmolytic  agents  and  therefore  also  to 

nutrients.  Cutinized  cell  walls  show  a  different  behaviour  Tmoss  leaves. 


IC)8  FINE-STRUCTURE  OF  PROTOPLASM  II 

fern  anulus,  seed-coats);  they  are  semi-permeable  to  sugars.  Any 
substance  which  has  passed  the  cell  wall  reaches  a  second  permeabilicy 
resistance  at  the  cytoplasmic  surface.  In  former  times  it  was  assumed 
that  all  plasmolytic  agents  were  retained  at  the  plasma  surface  and  there 
exerted  their  plasmolyzing  action.  This  led  to  the  paradox  that  cane 
sugar,  for  instance,  one  of  the  most  important  of  the  nutrients,  could 
not  penetrate  into  the  cell.  The  knowledge,  however,  that  salts  like 
KCNS  cause  the  cytoplasm  to  swell  and  bring  about  cap-plasmolysis 
of  the  cell  (see  Fig.  114)  has  overthrown  rhis  assumption  and  now- 
adays it  is  supposed  with  Hofler  (,1934)  that  the  main  resistance  in 
plasmolysis  should  be  sought  in  the  vacuole  boundary,  the  so-called 
tonoplast,  instead  of  in  the  cytoplasmic  surface.  This  does  away  with 
the  contradiction  inherent  in  the  impHcation  that  important  nutrients 
do  not  penetrate  into  the  cytoplasm  or,  like  KNO3,  can  only  do  so  with 
great  difficulty. 

This  penetration  into  the  cytoplasm  falls  under  the  concept  of 
intrability.  In  the  case  of  substances  which  cause  no  visible  change  in 
the  cell,  their  presence  within  the  cytoplasm  cannot  always  be 
proved  easily.  Yet  the  phenomenon  can  be  very  well  observed,  in  the 
case  of  vital  staining  with  chrysoidin,  which  often  does  not  enter  the 
vacuole.  On  the  strength  of  these  experiments  it  is  supposed  that  the 
outer  boundary  layer  of  the  cytoplasm  is  dilferent  in  nature  from  the 
inner  one  around  the  vacuole. 

In  the  phenomenon  of  deplasmolysis  the  plasmolysing  agent  must  grad- 
ually invade  the  vacuole  also.  For  this  process  Hofler  wants  to  reserve 
the  designation  permeability.  However  suitable  this  distinction  may  be  for 
botanical  objects,  in  which  most  permeability  studies  have  been  carried  out 
with  the  aid  of  deplasmolysis,  it  is  inappropriate  for  animal  cells,  which  do 
not  possess  vacuoles.  I  do  not  believe  that  Hofler's  terminology,  which 
we  want  to  apply  in  this  context,  would  cause  confusion,  since  for  cells 
without  vacuoles  intrability  and  permeability  are,  of  course,  identical.  All 
the  same  we  are  faced  with  a  logical  difficulty,  for  henceforth,by  permeability 
zoologists  will  understand  entrance  into  the  cytoplasm,  whereas  botanists 
will  understand  this  as  traversing  the  cytoplasm,  i.e.  entrance  followed  by 
elimination.  If  this  elimination  represents  a  passive  diffusion,  which  is 
probably  the  case  in  deplasmolysis  experiments,  the  difficulty  is  not  fun- 
damental. In  most  cases,  however,  where  the  elimination  occurs  in  connec- 
tion with  the  natural  intake  of  a  substance,  energy  is  involved,  and  the 
phenomenon  should  then  be  considered  as  active  elimination  [adenoid  activity 
according  to  Overton,  see  CoLLANDERand  Holmstrom,  1937).  This  does 


CYTOPLASM 


199 


not  apply  to  permeability  investigations  which  are  restricted  to  diffusion 
studies  (Barlund,  1929;  Ullrich,  1934;  Hofmeister,  1935;  Marklund, 
1936),  in  which  the  concentration  gradient  applied  is  the  only  potential  and 
no  account  need  be  taken  of  energy  produced  by  the  cell.  Accordingly,  the 
investigations  connected  with  the  respiratory  sorption  of  substances 
(Steward,  1932,  1933;  LundegArdh  and  Burstrom,  1933,  1935;  Hoag- 
LAND  and  Broyer,  1936;  Arisz  and  Van  Dijk,  1939;  Reinders,  1940; 
Brauner,  1943)  are  not  considered  as  permeability  studies. 

All  permeability  theories  have  in  common  that  the  resistance  to 
diffusion  is  located  in  the  so-called  plasmalemma  or  cytoplasmic 
membrane,  which  is  the  outer  boundary  layer  of  the  cytoplasm  and 
which  is  supposed  to  be  either  a 
submicroscopic  Hpidic  layer,  an 
ultrafilter  or  a  combination  of  both 
these  structures.  This  plasma- 
lemma  has  never  been  detected  as 
an  individual  layer  in  the  ordinary 
microscope.  Moreover  the  hyaline 
ectoplasm  of  amoebae  cannot  be 
regarded  as  a  permanent  structure, 
since  in  amoeboid  motion  it  can 
temporarily  change  into  granular 
endoplasm.  Nevertheless  the  hy- 
pothetic skin  must  be  present, 
for  micro-injection  experiments 
(Chambers,  1928)  show  that  dye- 
stuffs,  whose  entrance  is  opposed 
by  the  surface,  readily  spread  into 
the  bulk  of  the.  cytoplasm.  Col- 

lander  (1937  b)  regards  this  outer  skin  as  a  lipid  film  free  of  proteins, 
and  according  to  Danielli  (1936)  and  Tornava  (1939)  it  consists  of 
only  two  to  four  molecular  layers,  since,  on  increasing  the  surface  by 
endosmosis,  semi-permeabilitv  of  certain  cells  suddenly  disappears  at 
a  certain  surface  size,  and  the  cytoplasm  begins  to  "leak".  Curtis 
(1936),  on  the  contrary,  has  found  with  red  blood  cells  that  the  semi- 
permeable skin  does  not  become  "thinner"  when  stretched,  but  is 
continuously  repaired  by  material  supplied  by  inner  layers.  Probably, 
therefore,  the  plasmalemma  does  not  represent  a  definite  skin,  but 
only  a  boundary  layer  in  which  lipids  accumulate.   Sometimes  this 


Fig.  1 1 5.  Scheme  of  submicroscopic  plasma 
boundary  in  vegetable  cells  (from  Scarth, 
1942).  Lipids  dotted,  w  cell  wall;  e  hya- 
line ectoplasm,  coated  with  plasmalem- 
ma ;  pg  plasma  gel  of  endoplasm ;  ps  plasrna 
sol  of  endoplasm;  k  kinoplasm;  t  tono- 
plast;  V  vacuole;  s  transvacuolar  plasmic 
strand;  m  myelin  tube;  p  plastid. 


200  FINE-STRUCTURE  OF  PROTOPLASM  II 

accumulation  comprises  not  only  the  plasmalemma  but  also  visible 
cytoplasmic  layers,  so  that  the  presence  of  lipids  causes  a  distinct 
double  refraction  (Monroy,  1946). 

According  to  E.  N.  Harvey  (1937)  the  cell  surface  is  elastic;  this, 
according  to  model  experiments,  applies  only  to  the  surface  of 
solutions  containing  proteins,  whereas  lipidic  drops  of  lecithin 
(Harvey  and  Danielli,  1936)  or  of  oil  in  living  cells  (E.  N.  Harvey, 
1937)  possess  no  surface  elasticity!  It  follows  from  this  that  proteins 
take  part  in  the  construction  of  the  semi-permeable  plasmalemma, 
as  I  have  already  pointed  out  in  earlier  work  (1935  a,  p.  144).  Un- 
doubtedly the  elastic  properties  of  the  cell  surface  are  determined  by 
the  network  of  proteins.  The  scheme  with  individuaU-:{ed  spherical 
protein  molecules,  which  Danielli  and  Harvey  (1935)  believe  to  be 
the  structure  of  the  phase  boundary  between  oil  inclusions  and  hydro- 
philic  cytoplasm,  can  only  be  valid  for  surfaces  without  elasticity ;  the 
elastic  plasmalemma,  rather,  possesses  a  reticulate  structure.  Lehmann 
(1950/52)  has  produced  electron  micrographs  of  the  plasmalemma  of 
Amoeba  proteus  which  show  a  meshwork  of  globular  macromolecules 
(fig. 104b,  p. 160). This  meshwork  must  be  multilayered, since  Mitchison 
(1950a)  finds  the  plasmalemma  to  show  layer  form   birefringence. 

Probably  this  protein  framework  of  the  cvtoplasm  is  built  more 
densely  into  the  oiater  layers  and  changes  gradually  into  a  much  looser 
structure  towards  the  inside.  Accordingly,  the  cytoplasm  in  the  egg 
of  the  sea-urchin  is  liquid,  and  a  similar  conspicuous  difference  in 
organization  between  ectoplasm  and  endoplasm  seems  to  exist  in 
rhodophyta  (Hofler,  1936b).  At  the  phase  boundary  around  the 
vacuole  the  greater  density  of  the  framework  and  the  accumulation 
of  lipids  must  occur  again,  causing  a  renewed  resistance  to  diffusion 
in  this  region. 

ScARTH  (1942)  has  completed  and  improved  the  scheme  of  the  fine- 
structure  of  the  cytoplasmic  layers  of  plant  cells  suggested  by  me  in 
the  first  edition  of  this  monograph  (Fig.  115).  Underneath  the  cell 
wall  lies  the  hyaline  ectoplasm;  its  outer  boundary  is  formed  by  the 
plasmalemma  rich  in  lipids.  The  endoplasm  consists  at  its  periphery 
of  plasma  gel,  with  a  network  of  protein  filaments,  and  the  central  part 
of  plasma  sol  with  more  or  less  loosened  junctions.  It  is  intersected  by 
strands  of  higher  density  which,  as  kinoplasm,  connect  the  ectoplasm 
with  the  tonoplast. 


CYTOPLASM 


20 1 


Stihmkroscopic  morphology  of  selectively  permeable  membranes.  A  clear 
picture  of  the  permeability  phenomena  in  the  plasmalemma  is  ob- 
tained with  the  aid  of  the  permeability  theory  of  K.  H.  Meyer  (1955) 
and  T.  Teorell  (1955).  This  theory  has  been  developed  for  mem- 
branes with  a  framework  structure  and  for  this  reason  is  also  appli- 


Fig.  116.  Morphological  principle  of  K.  H.  Meyer's  and  T.  Teorell's  permeability  theory 
(1935).  Molecular  frame  a)  anionic,  V)  cationic,  c)  amphoteric. 


cable  to  the  cytoplasm,  which  in  our  opinion  is  built  on  a  similar 
principle.  The  starting  point  of  these  ideas  is  that  a  molecular  frame- 
work represents  a  gigantic,  polyvalent  and  immobile  cation  or  anion. 
In  the  case  of  the  cytoplasm  with  its  amphoteric  character,  the  frame- 
work can  act  either  as  cation  or  as  anion,  according  as  the  p^  changes 
(Fig.  116). 

One  may  imagine  that,  in  the  meshes  of  the  framework,  carboxyl 
groups  or  amino  groups,  or  both,  are  fixed  as  immobile  members  of 
the  main  valency  chains  (Sollner,  1950).  The  first  case  may,  for  in- 
stance, be  realized  in  the  pectin  gel  (Bonner,  1936a;  Deuel,  1943) 
of  polyuronic  acid  chains  (Fig.  116  a),  when  the  framework  acts  as 
an  acid;  the  hydrogen  ions  are  partly  split  off  by  dissociation  and  for 
this  reason  cations  can  diffuse  more  easily  through  this  molecular 
structure  than  anions.  Conversely,  if  the  framework  consists  of  basic 
chains  (e.g.,  of  diamine  acids,  Fig.  ii6b),  the  anion  permeability 
comes  to  the  fore.  Finally,  the  amphoteric  cytoplasm  (Fig.  ii6c)  is 
more  permeable  to  anions  at  low  p^^  and  to  cations  at  higher  pj^  values. 

These  considerations  apply  not  only  to  molecular  frameworks,  but 
to  the  coarser  meshworks  of  submicroscopic  strands  or  globules  as 
well.  This  theory  of  the  submicroscopic  structure  of  the  protoplasmic 
surface  and  the  cytoplasm  may  seem  one-sided,  in  that  it  takes  into 


202 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


account  only  the  ultrafilter  action  (Ullrich,  1936b);  yet  lipid  solu- 
bility is  also  included,  if  one  realizes  that  the  molecular  framework, 
especially  in  its  outer  regions,  contains  lipids  and  phosphatide  mole- 
cules which  are  located  within  the  meshes.  Wilbrandt  (1935)  there- 
fore rightly  remarks  that  no  sharp 
distinction  can  be  made  between  the 
effects  of  filter  action  and  solubility. 

A  colloid  framework  in  the  form  of 
a  polyvalent  immobile  ion,  which  is  in 
contact  with  a  true  solution,  represents 
a  DoNNAN  system,    even    though    no 
semi-permeable  wall  is  present.  For,  as 
required  for  a  Donnan  equilibrium,  the 
migration  of  the  colloid  framework  into 
the  surrounding  solution  is  impossible, 
whereas  its  mobile  ions  can  move  freely 
(Fig.  117).  This  consideration  makes  a 
theory  of  selective  permeability  possible. 
Suppose  an  anionic,  molecular  frame- 
work R  in  the  form  of  a  potassium  salt 
KR  is  in  contact  with  a  KCl-solution. 
Let   A   be  the  number  of  dissociation 
points  of  the  framework  anion,  i.e.,  the  concentration  of  the  potassium 
capable  of  dissociation,  y  the  concentration  of  the  KCl  penetrated 
into  the  meshes  of  the  framework,  and  c  the  KCl-concentration  of 
the  outer  solution.  Then  the  ion  product  [K]  •  [CI]  equals  (y  +  A)y 
inside,   and   c^   outside   the   framework.    Accordingly,  one  obtains 
Donnan's  law^:  (y  +  A)y  =  c^. 

Donnan's  exchange  mechanism  therefore  applies  to  our  framework 
structures,  since  the  immobile  anion  R  expels  the  mobile  anion  CI 
from  the  meshes  of  the  framework.  As  follows  from  Table  XXIII, 
the  CI  concentration,  y,  in  the  framework  decreases  rapidly  with  in- 
creasing A.  Thus,  in  order  to  establish  Donnan  equilibria  in  the  cyto- 
plasm, no  semi-permeable  membranes  are  required:  the  plasma  gel  as  a 
whole  acts  as  a  gigantic,  immobile  and  polyvalent  colloid  ion. 

1  Usually  the  equilibrium  is  formulated  in  a  more  complicated  way  (Hober,  1922,  p.  219) : 
(KCl  —  y)/y  =  (KR  +  KC1)/KC1.  In  this  less  convenient  form  KCl  =  c  -f-  y  and  KR  = 
A,  which  gives  the  above  formula. 


Fig.  117.  Donnan  equilibrium  be- 
tween a  molecular  framework  R 
with  anionic  dissociative  groups 
(A)  and  a  solution  of  KCl ;  (c)  and 
(y)  are  the  outer  and  inner  equili- 
brium concentrations. 


CYTOPLASM 


203 


TABLE  XXIII 
DONNAN  EQUILIBRIUM  IN  THE  MOLECULAR  FRAMEWORK 


KR 

KCl  total 

KCl  inside 

KCl  outside 

(A) 

(c+y) 

(y) 

(c) 

O.OI 

1. 00 

0.497 

0.503 

O.I 

1. 00 

0.476 

0.524 

I 

1. 00 

0-333 

0.667 

10 

1. 00 

0.083 

0.917 

100 

1. 00 

0.0098 

0.990 

K.  H.  Meyer  combines  this  result  with  the  velocity  of  ion  migration  in 
a  membrane  possessing  framework  structure,  in  order  to  arrive  at  a  quan- 
titative expression  for  the  permeability.  Let  U^  be  the  ion  mobility  of  the 
cation  and  U,^  that  of  the  anion  of  the  salt;  further  n^  the  number  of  cations 
and  n^^  the  number  of  anions  of  the  migrated  salt,  defining  these  numbers 
in  such  a  way  that  always  n^  +  n^  =  i  (Meyer  and  Sievers,  1936). 

As  the  number  of  migrating  ions  is  not  only  proportional  to  U  but  also 
proportional  to  the  ion  concentration  in  the  molecular  framework  (com- 
pare Fig.  1 1 7),  we  have : 

Hk       Uk  (y  +  A) 


n, 


UA-y 


Since  n^  +  n^  =  i,  nj^  and  n^  could  be  calculated  if  A  and  y  were 
known.  This,  however,  is  not  the  case  and  for  this  reason  the  known  outer 
concentration,  c,  is  introduced.  We  have 


and  therefore: 


y  =  y'c2  +  A74  —  A/2 

Hk       Uk  (a/4c^  +  A^  +  a)  ^_ 
Ha   ~  Ua  iV4c'  +  A2  -  A)       U, 


U 


K 


X. 


This  relation  is  K.  H.  Meyer's  starting  point  in  his  investigations  on 
permeability.  The  ratio  n-^/ni^  can  be  determined  potentiometrically.  On 
the  other  hand,  the  ratio  Uk/U^  and  the  factor  X  are  unknown. 

By  carrying  out  measurements  at  different  concentrations  c,  one  ob- 
tains several  equations  from  which  both  unknown  quantities  can  be  derived. 
Accordingly,  the  quantity  A  which  Meyer  designates  as  selectivity  constant 
can  be  determined,  and  thus  an  important  property  of  the  framework  can 
be  expressed  numerically. 

For  instance,  from  the  well-known  potential  measurements  of  the  apple 
skin  by  Loeb  and  Beutner  (1912/1913),  a  selectivity  constant  A  =  0.08 


204  FINE-STRUCTURE  OF  PROTOPLASM  II 

is  calculated,  i.e.,  the  normality  of  the  immobile  framework  anion  equals 
0.08  N. 

Meyer  has  proved  the  validity  of  his  theory  in  numerous  synthetic  and 
natural  membranes.  Undoubtedly  it  may  therefore  also  be  applied  to  the 
cytoplasm.  To  this  end,  however,  we  must  take  into  account  not  only  the 
ion  mobility  but  also  the  lipid  solubility.  This  is  done  by  introducing  the 
distribution  coefficients  of  the  migrating  substance  between  membrane 
framework  and  outer  liquid.  If  Ij^  and  1^  are  the  distribution  coefficients 
of  the  cations  and  anions  respectively,  the  Donnan  relation  runs 

(y  +  A)y 


since  the  concentrations  of  the  ions  in  the  framework  are  increased  or 
decreased  according  as  the  distribution  coefficients  are  larger  or  smaller 
than  I.  The  general  permeability  formula  then  takes  the  form 

"K  _  Uk  (a/4cMk  1a  +A^  + A) 
n^       Ua(V4c^1k1a  +  A2-A)' 

Although  this  formula  has  as  yet  hardly  been  applied  to  cytoplasmic 
permeabiHty,  I  think  it  worthy  of  attention,  as  to  a  certain  extent  it 
facilitates  a  synthesis  of  the  theories  of  permeability  in  biology.  Each 
of  the  quantities  occurring  in  it  refers  to  a  different  principle  of  the 
usual  theories  of  permeability.  The  ion  mobility  U  is  a  measure  of 
the  filter  resistance.  In  a  hydrophilic  framework  with  wide  meshes, 
Uj^  and  U^  would  be  equal  to  the  ion  migration  velocities  in  water. 
By  narrowing  of  the  meshes,  however,  larger  organic  ions  are  im- 
peded; and  the  filter  effect  will  influence  the  quantities  U.  The  effect 
of  the  solubihty,  in  the  first  place  the  lipid  solubiHty  in  the  cytoplasm, 
is  accounted  for  by  the  distribution  coefficients  1.  The  concentration 
gradient  applied  is  expressed  by  c  and  the  selectivity  constant  A  is 
related  to  the  electric  phenomena  accompanying  the  permeation.  If 
the  framework  of  a  membrane  has  a  negative  charge,  i.e.,  if  it  behaves 
like  an  anion,  A  becomes  positive;  in  the  reverse  case,  i.e.,  with  a 
positively  charged  framework,  A  is  negative.  For  the  amphoteric 
cytoplasm  the  selectivity  constant  A  must  therefore  be  either  positive 
or  negative,  depending  on  the  p„  of  the  nutrient. 

If  the  p^  value  of  the  imbibing  liquid  lies  above  the  isoelectric  state 
of  the  molecular  framework,  the  cytoplasm  behaves  like  an  anion  and 
thus  is  permeable  to  cations.  In  this  state,  weakly  basic  substances  Hke 
amides  (urea,  methyl  urea,  malonic  amide,  etc.)  will  permeate  more 


I  CYTOPLASM  205 

easily  than  at  a  p^  value  below  the  I.E. P.  Consequently,  if  one  wants 
to  distinguish  amidophiHc  and  amidophobic,  or  urea-permeahle  and 
glycerol-permahle  protoplasts,  the  I.  E.  P.  of  the  cytoplasm  and  the  p^ 
of  the  penetrating  solution  and  the  cell  sap  (Drawert,  1948)  should 
be  known.  Otherwise  it  cannot  be  decided  whether  the  differences 
observed  are  intrinsic  properties  of  the  protoplasm,  as  Hofler  (1936  a, 
1942)  believes,  or  whether  they  have  been  induced  temporarily  by  the 
amphoteric  cytoplasmic  framework  (Bogen,  1938;  Rottenburg, 
1945).  It  may  be  assumed  that  the  relation  between  p^  and  I.E. P. 
plays  a  decisive  part  in  comparative  permeabiHty  experiments,  so  that 
in  the  end,  like  vital  staining,  they  only  represent  new  methods  to 
determine  the  state  of  ionization  of  the  amphoteric  cytoplasmic  frame- 
work. 

In  the  isoelectric  state,  i.e.,  in  the  case  of  a  neutral  framework, 
A  =  o.  Then  the  permeability  formula  reduces  to  n^/n^  —  Uk/U^. 
In  this  state,  therefore,  the  cytoplasm  is  no  longer  selective  in  its 
permeability  to  cations  or  anions. 

Since  K.  H.  Meyer's  theory  is  based  on  potentiometry,  it  allows 
only  of  studying  the  ion  permeability,  which  is  of  greater  importance 
to  metaboHsm  than  the  permeation  of  non-electrolytes  studied  so 
often  in  plant  cells.  For  the  time  being,  however,  its  application  to 
cytoplasmic  permeability  is  difficult  (Meyer  and  Bernfeld,  1946),  as, 
of  the  many  quantities  which  have  to  be  accounted  for,  only  very  few 
are  known  in  the  cytoplasm.  Nevertheless,  the  morphological  prin- 
ciples of  the  considerations  presented  will  doubtless  bear  fruit  in 
future  theories  of  permeability. 

The  tonoplast.  Whereas  the  plasmalemma  in  plant  cells  probably 
differs  from  the  inner  cytoplasm  only  by  a  protein  framework  of 
greater  density  and  a  considerable  lipid  content,  the  vacuole  skin,  or 
the  so-called  tonoplast  membrane,  must  possess  an  essentially  different 
structure.  It  is  this  skin  which  impedes  the  entrance  of  hydrophilic 
substances  into  the  vacuole  and  on  the  contrary  strongly  furthers  the 
passage  of  lipophiUc  substances  (Plowe,  193  i).  It  must  therefore 
contain  large  quantities  of  lipids.  Although  this  statement  should  not 
be  generalized  without  further  criticism,  it  certainly  applies  to  many 
cases  and  especially  to  the  classical  example  of  ^////////epidermal  cells. 
In  an  interesting  controversy  Weber  (1932)  and  Hofler  (1932)  dis- 
cussed the  question  whether  this  lipid  layer  should  be  regarded  as 


2o6 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


belonging  to  the  cytoplasm  or  as  a  membrane  of  the  vacuole.  From 
the  point  of  view  of  molecular  morphology  this  point  of  contention 
can  be  decided  in  the  following  way  (Fig.  ii8). 

As  a  resuh  of  the  accumulation  of  Hpids,  the  latter  are  no  longer 
in  equilibrium  with  the  protein  framework.  Their  molecular  forces 


o  O  O   o      o 
^o  o   o    o     o 
„  °  o  o  o 
o    o      O    o 
0    °  o  o 


}.        Cytoplasm 


'    Tonoplast    skin 


Va  c  u  ole 


°; 


????????????? 
iiUAiiiiiUA 

???????????? 
UiiiiiiiiAl 

O        Oo00o°0 


Fig.  1 1 8.  Scheme  of  the  submicroscopic  structure  of  the  tonoplast  membrane,  consisting 

of  polar  lipid  molecules  (cf.  Fig.  115,  p.   199).  Hydrophylic  groups  white,  lipid  chains 

black,  water  molecules  small  circles,  a)  Bimolecular,  b)  polymolecular  film. 


cause  them  to  arrange  themselves,  turning  their  hydrophilic  poles 
towards  the  hydrophilic  inner  plasm,  the  lipophilic  ones  towards  the 
vacuole.  As  ascertained  in  the  case  of  Allium  (Fig.  46,  p.  55),  the 
inner  part  of  the  vacuole  consists  of  a  hydrophilic  liquid;  the  outer 
boundary,  on  the  contrary,  has  a  more  lipophilic  nature.  In  comparison 
with  the  cytoplasm,  therefore,  the  lipid  molecules  in  the  vacuole  must 
be  arranged  in  exactly  the  reverse  order.  The  result  is  that  the  bound- 
ary region  of  cytoplasm  and  vacuole  consists  of  a  lipid  layer  which  on 
either  side,  without  any  sharp  transition,  gradually  changes  into 
hydrophilic  regions.  The  boundary  membrane  will  therefore  consist 
of  molecular  double  layers. 

It  is  evidently  difficult  to  say  which  part  of  this  lipid  layer  belongs 
to  the  cytoplasm  and  which  to  the  vacuole  surface.  The  only  criterion 
would  be  to  determine  to  what  extent  the  cytoplasmic  protein  frame- 
work penetrates  into  this  layer.  Since,  however,  this  cannot  be  decided 
by  vital  staining,  we  must  content  ourselves  v/ith  the  fact  that  the 
boundary  between  the  two  cytological  parts  cannot  be  accurately 
determined. 


I  CYTOPLASM  207 

After  destroying  the  cytoplasm,  the  tonoplast  can  be  pressed  out 
of  the  cell  as  a  spherical  globule  which  continues  to  exist  for  days. 
Life,  however,  cannot  be  attributed  to  this  sphere,  although  it  may 
manifest  osmotic  changes  in  volume.  Similarly,  in  the  unimpaired  cell 
the  regulation  of  permeability  by  this  layer  in  the  usual  permeability 
experiments  is  not  a  sign  of  life,  but  a  purely  passive  result  of  diffusion 
equilibria. 

h.  Molecular  Morphology  of  the  Cytoplasm 

In  this  monograph  the  explanations  of  molecular  morphology  have 
intentionally  been  kept  very  vague  and  general.  We  have  mentioned 
polypeptide  chains  and  their  junctions,  lipophilic  and  hydrophilic 
groups,  acid  and  basic  side  groups.  These  suffice  for  an  understanding 
of  the  general  properties  of  the  cytoplasm,  but  its  specific  achieve- 
ments cannot  be  approached  in  this  manner  and  require  a  knowledge 
of  the  exact  molecular  constitution.  For  such  an  approach,  however, 
only  one  important  starting  point  is  available,  viz.,  the  asymmetry  of 
the  cytoplasm.  Of  the  stereo-isomeric  amino  acids  only  the  laevo 
forms  occur  in  the  cytoplasm  (Gause,  1936);  accordingly,  the  syn- 
theses and  the  degradations  which  are  carried  out  in  the  cytoplasm 
are  strictly  specific:  of  the  possible  isomers,  only  a  particular  one  is 
formed.  Whereas  artificial  syntheses  of  an  organic  compound  with 
asymmetric  carbon  atoms  lead  to  an  optically  inactive  racemate,  only 
the  dextro  or  the  laevo  form  of  the  same  substance  is  formed  in  the 
cytoplasm. 

This  discovery  of  Pasteur's  is  of  far-reaching  importance  to 
morphology,  for  it  shows  how  new  configurations  result  from  those 
already  present:  in  the  cytoplasm  each  structural  creation  requires  an 
adequate  creator.  This  is  the  principal  reason  why  the  cytoplasm  cannot 
be  a  formless  liquid,  but  must  possess  a  framework  of  well-defined 
molecular  structure. 

In  addition  to  the  asymmetry  of  the  amino  acids,  which  in  the 
scheme  of  Fig.  87  (p.  132)  is  evident  from  the  relative  positions  of 
the  H  and  R  groups,  numerous  other  structural  particulars  must  exist 
in  the  cytoplasm  framework.  All  specific  physiological  reactions  are 
certainly  caused  by  them.  It  has  already  been  pointed  out  that  en^y^»es 
must  carry  such  groups  of  a  specific  structure.  In  Fig.  119  an  example 
is  given  showing  the  dehydrogenase,  which  acts  as  catalytic  carrier  of 


2o8 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


hydrogen  in  respiratory  and  fermentative  processes.  The  active  group 
of  the  molecule  consists  of  a  nucleotide  (adenine,  ribose  and  phos- 
phoric acid,  see  p.  213),  which  is  linked  with  a  second  nucleotide-like 
compound  (nicotinic  acid  amide,  ribose,  phosphoric  acid)  by  a  mole- 
cule of  phosphoric  acid  (Karrer,   1941,   1944).  The  nicotinic  acid 


H2C- 

HOCH-CH 

I      \ 

HOCH       O 


Protein 
carrier 


N  N 

\         I 
C=C 

X      // 

N — QH 


XH 


-0^       OH  HO^      p- 

X     X 

p( 


\ 


-CHz 

HC-HCOH 
I         \ 
0^     HCOH 

HC 


\ 


HC 


<f 


f/Vv 


+  H2^ 

'-H, 


HO^p- 

</\h 


HzNOC^  '^CH 


CH 

w 

CH 


-CH2 

HC—HCOH 
I         \ 
0^     HCOH 
HC^ 

K 

H^C^       CH 


HzNOC 


x^V^" 


Apo-enzyme  Co-enzyme  with  prosthetic  group  (Nicotinic  acid  anriide)  Prosthetic  group  hydrogenized 

Fig.  119.  Structural  formula  of  dehydrogenase  as  an  example  of  a  co-enzyme. 


amide  is  capable  of  taking  up  hydrogen,  and  is  therefore  designated 
as  active  group  or  prosthetic  group.  It  can,  however,  develop  its 
activity  only  together  with  the  whole  molecule  and  only  on  condition 
that  the  latter  be  connected  with  a  colloid  protein  carrier.  The  carrier 
is  designated  as  an  apo-en^yme  and  the  molecule  with  the  prosthetic 
group  as  a  co-en^^^yme  (compare  for  instance  Bersin,  1939).  The  two 
parts  of  the  enzyme  can  be  chemically  separated  and  recombined.  In 
contrast  to  some  co-enzymes,  the  constitution  of  the  apo-enzymes  is 
still  completely  unknown.  In  the  so-called  lyo-enzymes,  which  leave 
the  ceUs  and  are  active  in  solution,  the  apo-enzyme  is  a  corpuscular 
protein  particle  of  colloid  dimensions.  It  must,  however,  be  supposed 
that  in  the  endo-enzymes,  which  are  active  only  in  che  cells  and  can 
be  isolated  only  by  autolysis,  i.e.,  by  breaking  down  the  colloid 
framework  of  the  cytoplasm,  the  apo-enzyme  is  anchored  on  the 
framework  of  the  protoplasm. 

Vitamins  often  contain  specific  structural  units  which  are  necessary 
for  the  formation  of  co-enzymes,  but  cannot  be  formed  by  the  hetero- 
trophic organisms,  since  the  latter  apparently  lack  the  formative 
principle  indispensable  to  the  synthesis  concerned.  Such  molecular 
morphological  particulars  might  likewise  play  a  part  in  the  activity 
of  hormones. 


I  CYTOPLASM  209 

In  this  context  the  group  of  auxins  amongst  the  phytohormones 
will  be  discussed  briefly  as  a  further  example  of  compounds  having 
a  specific  effect  (Went  and  Thimann,  1937).  The  auxins  admittedly 
are  not  very  specific,  as  they  initiate  all  kinds  of  different  reactions  of 
growth :  elongation  growth  of  meristematic  cells,  division  growth  of 


C-CH,-COOH  C^C-CH^-COOH 


f^H  I   Indolyl-^-  acetic  odd  ^^^^CH2         ^     lnden-3-acfiic  acid 

C-CH2-CH2-COOH  |^V~li^ 

iE"    Cumoryl-a- acetic  acid 


^  .CH  \^  X-CH2  -COOH 

/^/^  HI    Indolyl-p- propionic  acid  0 


N—C-CHp-CHNH.-COOH  CH2 

II       II         '  \\ 

CH    CH  CH2 

NH         Y    Histidine  ^     Ethylene 

Fig.  120.  Molecular  structure  of  plant  growth  and  stimulant  substances. 

parenchyma  and  cambium  cells,  epinastic  curvature  of  leaves,  initi- 
ation of  callus  and  root  formation  in  cuttings,  inhibition  of  extension 
of  axillary  buds,  etc.  The  experience  that  chemically  different  com- 
pounds stimulate  the  same,  or  at  least  similar,  growth  created  a  still 
greater  sensation  than  this  diversity  of  positive  or  negative  reactions 
caused  by  the  growth  substances  appUed.  The  nearly  identical,  though 
quantitatively  different  effects  of  indolyl,  inden  and  cumaryl  com- 
pounds (Fig.  120)  are  well-known.  For  this  reason  it  has  often  been 
suggested  that  in  the  case  of  these  auxins  there  is  rather  a  universal 
stimulation  of  the  metaboHsm  than  a  specific  hormonal  effectivity. 

However,  a  comparison  of  the  4  structural  formulae  of  the  com- 
pounds I-IV  in  Fig.  120  (Thimann,  1936),  all  of  which  are  stimulants 
of  growth  (ahhough  the  compounds  II-IV  are  active  to  considerably 
less  extent),  shows  that  they  have  morphological  characteristics  in 
common :  all  of  them  contain  a  five-membered  ring  with  at  least  one 
double  bond.  Six-membered  rings  (naphthyl  derivatives)  are  also 
active  (Thimann  and  Bonner,  1938).  It  appears  to  be  immaterial 
whether  this  ring  is  homo-  or  heterocyclic  and  what  side  chains  are 
substituted  in  it.  A  further  characteristic  is  that  all  four  substances 
are  monobasic  acids,  in  which,  however,  the  COOH-groups  must  be 
separated  from  the  ring  by  at  least  one  C-atom  (Koepfli,  Thimann 
and  Went,  1938;  exception:  2,4,6-trichloro  benzoic  acid).  The  mor- 


210  FINE-STRUCTURE  OF  PROTOPLASM  II 

phological  principle  of  the  unsaturated  five-membered  ring  seems  to 
be  particularly  important.  We  do  not  know  how  this  ring  fits  into 
the  protoplasmic  structure,  but  it  must  possess  a  specific  kind  of 
stimulating  activity,  adapted  to  a  certain  configuration  of  the  cyto- 
plasm frame.  It  cannot  be  accidental  that  histidine  (Fig.  120,  V),  the 
specific  stimulant  to  protoplasmic  flow  (Fitting,  1927,  1936),  should 
also  show  the  unsaturated  five-membered  ring,  although  admittedly 
with  two  double  bonds. 

Even  the  double  bond  alone  is  capable  of  initiating  some  of  the 
reactions  mentioned,  for  traces  of  ethylene  (Fig.  120,  VI)  cause  typical 
epinastic  curvature  of  leaves  (which  are  even  used  as  test  reactions, 
Denny,  1935),  and  give  rise  to  the  formation  of  adventitious  roots 
in  the  presence  of  a  sufficient  amount  of  auxin  (Michener,  1935)- 
For  the  initiation  of  cell  elongation,  however,  the  acid  group  too 
seems  to  be  required.  At  the  moment,  molecular  morphology  is  unable 
to  account  for  the  fact  that  the  combination  of  a  double  bond  and 
an  acidic  group  has  to  be  realized  by  means  of  some  five-  or  six- 
membered  ring. 

§  2.  Nucleus 

a.  Molecular  Constituents  of  the  Nucleus 

The  isolation  of  sufficient  quantities  of  substances  from  the  cell 
nucleus  for  chemical  purposes  meets  with  great  difficulties,  and  so  far 
it  has  been  possible  to  carry  out  a  thorough  chemical  analysis  only 
in  special  cases,  in  particular  in  the  case  of  the  sperm  nuclei  of  fishes, 
where  extremely  interesting  results  have  been  obtained.  The  following 
account  therefore  refers  primarily  to  fish  sperm,  but  a  generalization 
applying  to  the  chemistry  of  other  nuclei  on  the  strength  of  micro- 
chemical  analogies  is  permissible,  howbeit  with  due  caution.  The 
nuclear  substances  designated  as  nucleoproteins  can  be  separated  into 
two  components,  viz.,  into  proteins  on  the  one  hand  and  phosphor- 
containing  nucleic  acids  on  the  other.  Other  compounds  such  as 
lipids  (Hirschler,  1942)  are  present  in  insignificant  quantities. 
ScHMiEDEBERG  (KiESEL,  1930)  finds  fot  the  Sperm  heads  of  salmon: 

nucleic  acid  .    .    .    .    60.50%  by  weight 
protamines    .    .    .    .    35.56%    ,,       ,, 
rest,  with  0.12%  Fe      3.94%   »       » 


2  NUCLEUS  211 

Protein  components.  Not  without  reason,  very  complicated  proteins 
were  presumed  co  be  present  in  the  nucleus  but,  contrary  to  expec- 
tation, only  fairly  simple  polypeptides,  designated  as  protamines,  were 
found  in  the  fish  sperm.  They  are  characterized  by  the  fact  that  on 
hydrolysis  they  produce  a  striking  number  of  basic  amino  acids, 
principally  arginine,  but  also  lysine,  histidine  and  others.  According 
to  KossEL  (1929),  the  proportion  of  the  di-amino  acids  (cf.  Fig.  88, 
p.  133)  to  the  mono-amino  acids,  alanine,  valine,  leucine,  etc.  (ab- 
breviated M),  often  amounts  to  2:1.  For  example,  in  the  case  of  the 
mono-protamines  2  arginine:  i  M;  in  the  di-protamines  2  (arginine, 
histidine):  i  M;  in  the  tri-protamines  2  (arginine,  histidine,  lysine): 
I  M.  Often  the  basic  compounds  preponderate  even  more.  Felix 
(195 1)  finds  that  in  clupeine  from  the  sperm  of  the  herring  80%  of  the 
amino  acids  are  arginine,  and  that  the  molecular  ratio  of  arginine  to 
phosphorus  is  1:1  in  these  nuclei.  This  preponderance  of  the  di- 
amino  acids  results  in  polypeptide  chains  of  strongly  basic  character. 
As  a  further  characteristic  Kossel  mentions  that  the  amino  acids  with 
5  C  members  (ornithine,  proline,  valine)  are  conspicuously  pre- 
dominant over  those  with  6  C  atoms  (for  instance  leucine),  which  are 
typical  for  other  proteins.  Still  more  important  is  the  complete  absence 
of  cystine  and  amino  dicarboxylic  acids  in  the  protamines. 

For  example,  the  formula  given  in  Fig.  121  is  attributed  to  sturine 
from  the  sperm  of  the  sturgeon,  which  represents  a  tri-protamine. 
Where  processes  of  synthesis  and  the  formation  of  organic  substances 
are  concerned,  Kossel  (1905)  is  inclined  to  attribute  special  im- 
portance to  the  alternating  -C-N-C-N-order  of  the  end  groups  of 
the  side  chains  of  arginine  and  histidine,  which  also  occurs  in  the 
nitrogen-containing  bases  of  nucleic  acids.  For  the  time  being, 
however,  these  facts  can  only  be  accepted  as  morphological  statements, 
for  the  functioning  of  such  systems  is  still  unknown.  In  this  context 
it  is  interesting  to  note  that  the  polypeptide  main  chain  represents  a 
-C-C-N-C-C-  N-arrangement. 

The  chains  of  the  protamines  obtained  are  not  very  long.  E.g., 
salmine,  with  a  molecular  weight  between  2000  and  2500,  consists  of 
only  15  to  18  amino  acids  (Kiesel,  1930),  i.e.,  the  polypeptide  chain 
would  measure  only  about  60  A.  Undoubtedly,  however,  the  poly- 
peptide chains  of  the  nucleoproteins  will  be  much  longer  in  the  native 
state  and  will  only  break  off  into  these  short  fragments  as  a  result  of 


212  PINE-STRUCTURE  OF  PROTOPLASM  II 

the  chemical  treatment.  The  protamines  seem  to  be  strictly  limited  to 
fish  sperm.  In  other  nuclei,  proteins  of  less  basic  character  have  been 
found,  the  so-called  histones,  which  have  a  higher  molecular  weight 
and  are  therefore  less  soluble.  They  contain  a  great  variety  of  amino 
acids  and  form  a  transition  to  the  typical  proteins.  Their  I.E. P.  lies 

/ 

NH 
\  A/W 

CH-CH,  -CHi-CHy  -NH-Cf 

CO  Arg/nine 

NH 

NH^-  CHj-  CH^-CH^-CH^-CH 

Lysine  \q 

/ 

NH 

CH-CH. 
rQ       Alanine 

\ 

^N  NH 

CH     \  / 

I  L-CHj-CH 

^^LH  i-iistidine    YO 

y 

NH 

\h-ch,-ch(_'^^^ 

/"     ,  CH, 

QQ       Leucine 

Fig.  121.  Molecular  structure  of  sturine. 

in  the  alkaline  region,  up  to  a  p^  of  about  8.5  (Pischinger,  1937). 

The  ultraviolet  absorption  of  proteins  which  results  from  the 
presence  of  cyclic  amino  acids  (tyrosine,  tryptophan,  histidine)  is 
small.  The  globulins,  for  instance,  whose  I.  E.  P.  lies  on  the  acid  side, 
show  a  weak  absorption  band  at  2800  A,  whereas  in  the  basic  histones 
this  band  occurs  at  2900  A,  which  may  be  used  as  a  means  of  identi- 
fication (Caspersson,  1 941).  The  histones  appear  to  be  concentrated 
in  the  nucleolus. 

The  nucleic  acids  also  possess  a  pronounced  chain  structure.  The 
chemistry  of  the  chain  members,  designated  as  nucleotides,  is  well- 
known.  Hydrolysis  leads  to  three  components:  one  molecule  of 
phosphoric  acid,  one  molecule  of  sugar  from  the  group  of  the  pentoses 
and  one  heterocyclic  basic  ring  from  the  pyrimidine  or  purine  type. 
d-Ribose  or  desoxyribose  is  the  pentose  of  the  majority  of  the  nucleo- 
tides isolated,  while  all  kinds  of  substituted  pyrimidine  rings  (uracil, 


NUCLEUS  213 


cytosine,  thymine)  or  purine  rings  (guanine,  adenine)  can  occur. 
Cytidylic  acid,  a  nucleotide  obtained  from  yeast,  has  the  structural 
formula  shown  in  Fig.  122  c. 

Because  of  the  purine  and  pyrimidine  rings,  the  nucleic  acids  show 
a  strong  ultraviolet  absorption,  having  its  maximum  at  2600  A.  This 


Phosphoric    acid <    '^"a'' 

Ribose- purine  base  {  I     \^    '^\n/ 

0^    ,0H      ■ 


■h\h         Ch\'\  Phosphoric   acid-- -<  ' y^Q       W^ 


\C^- 


0.   ^'"^ 


CH 

N^      .CH  N.       X-^    /  o     .       .        . 

^ru  \u      NH  Pyrimidme  base 

t-"  •'-"                                    ribose      I            A/ -      ^ 

a)  b)                                            \                          \    OH  yO 

■'  ^                                Phosphoric    acid <     ^^P^ 

r 
OH  ^'tbose J  %-0. 


HC=CH 
/          \ 

HCOH 

i,N — C              N— 

-CH      HC — 0 

'            \           / 

\          / 

N — C, 

0 — CH 

\ 

'\5 

^0 

H^COH 

0—P=0 


N   \n 
OH  „/'^0 


0^ 

0.    ^^^ 


Cytosine  .  Ribose         Phosphoric   acid  !\i ' 

0 


Fig.  122.  Molecular  structure  of  the  nucleic  acids,  a)  Pyrimidine  base;  b)  purine  base  (the 

rings  are  usually  represented  in  the  form  of  rectangles,  but  this  might  be  incorrect  from  a 

morphological  point  of  view);  r)  cytidylic  acid  =  nucleotide  cytosine-ribose-phosphoric 

acid  (from  Fischer,  1942);  d)  nucleic  acid  =  polynucleotide. 

property  has  been  very  skilfully  utihzed  in  cytology  by  Caspersson 
(1936). 

In  cells,  the  nucleotides  do  not  occur  in  the  free  state.  A  mutual 
esterification  to  polynucleotide  has  taken  place,  the  latter  representing 
the  actual  nucleic  acids.  The  esterification  takes  place  between  an  OH- 
group  of  the  phosphoric  acid  and  an  alcohoUc  hydroxyl  group  of  the 
ribose.  It  is  possible  that  periodically,  say  after  every  fourth  nucleo- 
tide, other  kinds  of  bonds  also  occur.  For  example,  in  the  nucleic 
acid  of  yeast,  four  nucleotides  (adenine,  uracil,  guanine  and  cytosine 
nucleotides)  are  combined  into  a  tetra-basic  acid.  This  nucleic  acid 
however,  apparently  does  not  occur  in  the  nucleus  but  in  the  cyto- 
plasm. 

The  nucleic  acids  from  the  nucleus  differ  from  the  nucleic  acids  of 


214  FINE-STRUCTURE  OF  PROTOPLASM  II 

the  cytoplasm,  in  that  part  of  their  nucleotides  do  not  contain  d-ribose 
but  d-2-ribodesose.  In  this  desoxypentose  the  OH-group  at  the  2nd 
C-atom  of  ribose  has  been  replaced  by  H.  It  is  likely  that  this  small 
structural  change  causes  the  nucleic  acids  of  the  nucleus  to  be  much 
more  sensitive  to  hydrolysis.  For,  according  to  Feulgen,  after  weak 
acid  hydrolysis  the  desoxyribose  nucleic  acids  of  the  nucleus  show 
Schiff's  aldehyde  reaction  with  fuchsine  in  H.SOa.  Obviously  the 
hydrolysis  of  the  nucleic  acids  of  the  nucleus  hberates  the  aldehyde 
groups  of  ribodesose,  whereas  in  the  case  of  the  nucleic  acids  of  the 
cytoplasm  the  aldehyde  groups  remain  masked.  Accordingly,  this 
specific  staining  has  been  introduced  in  cytological  microchemistry  as 
Feulgen's  nucleal  reaction  to  prove  the  existence  of  desoxyribose 
nucleic  acids  (Feulgen  and  Rossenbeck,  1924). 

It  has  been  possible  to  analyze  macrochemically  a  number  of  nucleic 
acids  showing  a  positive  nucleal  reaction.  Thymonucleic  acid  from  the 
nuclei  of  the  thymus  gland  consists  of  four  nucleotides  with  the  bases 
adenine,  thymine,  guanine  and  cytosine.  A  molecule  of  this  relatively 
small  size,  however,  will  scarcely  show  colloid  properties  Hke  the 
nucleic  acids  in  the  nucleus.  It  must  therefore  be  supposed  that  the 
tetra-basic  acid  of  Fig.  1 22  d  represents  only  a  part  of  the  native  nucleic 
acids  of  high  molecular  weight.  Guanyl  nucleic  acid  from  the  pancreas 
gland,  the  most  complicated  nucleic  acid  known  at  present,  contains, 
in  addition  to  the  tetra-basic  thymonucleic  acid,  a  nucleotide  with 
ribose  as  sugar  and  guanine  as  basic  component.  This  shows  that 
mixed  polymerization  products  of  nucleotides  with  ribose  and  ribode- 
sose groups  can  occur  in  the  nucleic  acids  of  the  nucleus.  For  further 
particulars  we  must  refer  to  che  literature  concerned  (e.g.,  Kiesel, 
1930;  Fischer,  1942).  Specific  differences  of  the  nucleic  acids  in 
different  animals  or  in  different  organs  of  the  same  animal  must  be 
looked  for  in  the  type  of  the  basic  side  groups  and  their  arrangement 
along  the  chain.  A  microanalytical  method  for  the  determination  of 
pyrimidine  and  purine  bases  is  possible  by  paper-chromatography 
(ViscHER  and  Chargaff,  1948;  Chargaff,  1950). 

Whereas  formerly  the  nucleic  acids  were  considered  as  tetra- 
nucleotides,  it  was  suggested  in  the  first  edition  of  this  monograph 
that  they  represent  high  polymer  long  chain  molecules  (Fig.  i22d). 
Signer,  Caspersson  and  Hammarsten  (1938)  confirmed  this  by  means 
of  the  birefringence  of  flow  of  Na-thymonucleate  and  simultaneously 


2  NUCLEUS  215 

AsTBURY  and  Bell  (1938)  proved  the  existence  of  a  chain  lattice  with 
a  fibre  period  of  3.34  A  in  artificial  fibres  of  Na-nucleate.  The  degree 
of  polymerization  is  very  high  and  only  by  taking  special  precautions 
is  it  possible  to  isolate  them  unimpaired  from  the  thymus  gland 
(Knapp,  1946).  According  to  Riley  and  Oster  (195  i),  the  molecules 
of  concentrated  solutions  (gels)  are  arranged  in  a  hexagonal  chain 
lattice. 

The  main  chain  consists  chiefly  of  P-  and  O-bridges;  the  phos- 
phorus carries  a  free  acid  group,  while  the  basic  groups  constitute 
short  side  chains.  Compared  with  the  nitrogen-containing  bases,  the 
dissociation  of  the  phosphoric  acid  preponderates  to  such  an  extent 
that  the  system  represents  a  chain  of  polybasic  acids.  The  isoelectric 
point  lies  below  p^  2  (Pischinger,  1937). 

In  recent  times  ribonucleic  acid,  which  does  not  show  the  Feulgen 
nucleal  reaction  and  which  was  considered  to  be  characteristic  for  the 
cytoplasm  onlv,  has  been  discovered  in  the  nucleus  by  UV  absorption 
analvsis.  After  cell  division  when  the  thvmonucleic  acid  content,  as 
measured  by  Feulgen  colorimetry,  drops  sharply,  the  amount  of 
ribonucleic  acid  increases  in  the  nucleus  and  may  reach  nine  times 
that  of  thymonucleic  acid  (Ogur,  Erickson,  Rosen,  Sax  and  Holden, 
1951). 

b.  Fine-Structure  of  the  Nucleus 

The  active  nucleus.  The  nucleus  possesses  for  the  most  part  a  coarse 
framework.  Its  strands  are  usually  of  microscopic  thickness,  but  as 
they  are  strongly  hydrated  and  insensitive  to  dyes  in  the  living  nucleus, 
they  remain  invisible  in  the  ordinary  microscope;  but  they  can  be 
detected  by  the  phase  contrast  microscope,  which  is  an  invaluable  tool 
for  vital  observations  in  cytology.  These  framework  fibrils  have 
become  of  great  importance,  since  they  could  be  identified  with  the 
uncoiled  chromosomes  (compare  p.  225).  A  sol-like  liquid  is  found 
between  the  strands  of  the  fibrils;  it  is  designated  as  nuclear  sap, 
karyolymph  or  enchylema.  In  other  words,  the  structure  is  analogous 
to  that  of  cytoplasm,  where  the  framework  of  the  plasma  gel  (kino- 
plasm,  spongioplasm)  is  distinguished  from  the  cytoplasmic  sap  (para- 
plasm, enchylema). 

Martens  (1927/29)  and  Pischinger  (1937)  have  elucidated  the 
connection  between  the  invisible  fine-structure  of  living  nuclei  and 


2l6  FINE-STRUCTURE  OF  PROTOPLASM  II 

the  visible  structure  of  fixed  nuclei.  On  fixing,  the  fibrils  of  the  nucleus 
are  dehydrated  and  become  accessible  to  staining.  They  usually 
clot  together  as  a  result  of  the  adhesive  action  of  the  coagulated 
proteins  of  the  enchylema. 

The  chain  molecules,  forming  the  fibrils  in  badly  fixed  nuclei,  may 
be  so  highly  hydrated  that  no  structure  whatsoever  can  be  detected 
in  the  living  nucleus  (Pischinger,  1950).  Such  nuclei  are  homo- 
geneous in  the  electron  microscope  when  properly  fixed  (Rqzsa  and 
Wyckoff,  1950).  These  observations  do  not  negative  an  amicroscopic 
nuclear  structure.  It  is  possible  that  the  chain  molecules,  although 
completely  hydrated,  may  be  paralleli2ed  in  the  same  way  as  they  are 
known  to  be  in  cellulose  solutions.  In  this  state  the  nucleus  is  thixo- 
tropic;  and  it  may  behave  like  a  liquid  drop,  in  which  the  nucleolus 
falls  to  the  bottom  when  observed  in  a  horizontal  microscope  (Harris, 

1939)- 

The  framework  structure  in  the  nucleus  has  received  a  much  more 

appropriate  name  than  in  the  cytoplasm,  where  the  misleading  concept 

of  foam  or  honeycomb  structure  is  often  used.  For  it  is  designated 

as  a  reticulum,  which  clearly  expresses  that  both  framework  substance 

and  karyolymph  are  continuous  structural  components.  In  the  living 

nucleus  the  threads  of  the  reticulum  are  separate,  but  during  fixation 

they  coalesce  and  are  held  together  by  the  coagulating  protein  of  the 

enchylema. 

The  living  reticular  framework  is  not  rigid,  but  is  to  some  extent 
plastic.  By  means  of  centrifugal  forces  Nemec  (1929)  has  displaced 
the  nucleolus  in  the  nucleus,  or  even  removed  it  altogether,  in  which 
case  the  reticulum  was  deformed.  According  to  several  authors  the 
nucleus  has  the  nature  of  a  liquid  (e.g.,  Schaede,  1927)  or  even  no 
structure  at  all.  This  is  derived  from  deformability  and  optical  homo- 
geneity. The  spherical  shape,  the  capacity  to  form  drops  and  the  fact 
that  living  nuclei  are  often  optically  empty  are  put  forward  as  further 
arguments.  For  this  reason  it  is  necessary  to  repeat  that  the  behaviour 
of  a  colloid,  whether  elastic  like  a  gel  or  more,  liquid  like  a  sol,  does 
not  in  itself  prove  or  disprove  the  existence  of  a  submicroscopic 
structure.  To  decide  this,  measurements  of  structural  viscosity  are 
necessary,  a  property  which  the  highly  viscous  nuclear  substance  pos- 
sesses in  a  marked  degree,  as  has  been  shown  by  Harris  (1952). 

Since  the  structural  elements  of  the  nuclei  are  represented  by  the 


2  NUCLEUS  217 

uncoiled  chromosomes,  the  question  arises  whether  they  are  em- 
bedded in  the  karyolymph  as  free  corpuscular  dispersed  particles,  or 
whether  they  occupy  definite  relative  positions  forming  a  structure. 
I  am  convinced  that  the  latter  is  true,  for  in  general  the  nucleolus 
remains  in  contact  with  the  chromosome  fibrils,  on  to  which  it  has 
been  condensed  (Heitz,  193  i;  Geitler,  1940),  and  heterochromatin 
(cf.  p.  220),  if  present,  occupies  a  certain  position  in  the  nucleus  and 
cannot  be  readily  displaced. 

The  karyolytnph  (enchylema),  on  the  contrary,  appears  to  be  a  sol. 
In  Allium  nuclei,  for  instance,  Luyet  and  Ernst  (1934a)  succeeded 
in  separating  it  from  the  framework  substance  of  greater  specific 
weight  by  centrifugal  means.  The  nuclear  sap  of  oocytes  of  Xenopus 
laevis  is  a  solution  of  proteins;  its  hydrolysate  yields  a  paper  chromato- 
gram  with  12  amino  acids  but  no  nucleic  acid  (Brown,  Callan  and 
Leaf,  1950). 

The  nuclear  membrane  varies  greatly  in  thickness.  According  to 
Luyet  and  Ernst  (1934b)  it  is  not  a  self-consistent  skin,  but  only  a 
phase  boundary.  Other  authors,  however,  mention  a  real  envelope,  the 
birefringence  of  which  has  frequently  been  found  to  differ  from  that 
of  the  nucleus  itself.  Schmidt  (1939c)  gives  evidence  of  an  optically 
negative  spherite  texture  in  the  boundary  layer  of  the  nucleus  (lamellar 
birefringence  caused  by  protein  chains  running  in  a  tangential  di- 
rection). According  to  F.  O.  Schmitt  (1938)  the  sign  of  the  spherite 
cross  is  reversed  after  imbibing  with  glycerol,  urea  or  sugar  solutions ; 
this  would  neutralize  the  form  birefringence,  and  the  intrinsic  bire- 
fringence of  the  lipids  would  become  apparent.  Pfeiffer  (1944)  has 
even  published  complete  curves  of  form  birefringence.  Monne 
(1942  c)  believes- the  nuclear  envelope  to  be  a  double  membrane,  con- 
sisting of  a  firm  nuclear  protein  layer  free  of  lipids  and  a  very  tender 
cytoplasmic  protein-lipid  membrane.  The  same  conditions  are  de- 
scribed by  Baud  (1949  a,  b)  for  the  nucleus  of  liver  cells.  He  empha- 
sizes that  in  living  nuclei  there  is  no  optical  anisotropy;  only  after 
fixation  does  a  birefringent  nuclear  membrane  appear  which  is  sur- 
rounded by  a  birefringent  perinuclear  zone.  The  optical  anisotropy 
of  the  nuclear  membrane  is  that  of  a  negative  spherite  indicating  a 
protein  lamellar  texture,  whilst  the  perinuclear  layer  represents  a 
positive  spherite  due  to  radially  oriented  lipid  accumulation  around 
the  nucleus  (cf.  Fig.  118,  p.  206). 


2l8  FINE-STRUCTURE  OF  PROTOPLASM  II 

It  is  curious  that  of  all  these  structures  nothing  is  to  be  seen  in  the 
electron  microscope  after  optimal  fixation  with  4%  neutral  formalin 
(RozsA  and  Wyckoff,  1950).  It  is  true  that  the  birefringent  peri- 
nuclear zone  is  only  visible  after  fixation  with  OSO4,  which  according 
to  RozsA  and  Wyckoff  produces  artefacts.  But  in  the  polarizing 
microscope  the  nuclear  membrane  appears  equally  after  fixation  with 
4%  neutral  formalin,  especially  if  its  double  refraction  is  enhanced  by 
4%  sodium  sulpho-antimoniate.  From  these  facts  it  must  be  concluded 
that  there  are  oriented  arrangements  of  amicroscopic  molecules  in  the 
nuclear  boundary.  Whether  this  structure  should  be  called  a  "mem- 
brane" is  open  to  discussion.  The  actual  evidence  is  rather  in  favour 
of  a  phase  boundary  with  the  structure  of  a  mesophase  (Pischinger, 
1950). 

The  large  nuclei  of  Amphibian  oocytes  seem  to  have  a  composite 
nuclear  membrane  as  revealed  by  the  electron  microscope  (Callan, 
Randall  and  Tomlin,  1949;  Callan  and  Tomlin,  1950).  A  structure- 
less sheet  is  covered  by  a  layer  with  pores  of  300  A  diameter  and 
800  A  distance  in  hexagonal  array.  This  porous  lamella  serves  as  a 
mechanical  support  of  the  homogeneous  nuclear  boundary,  which  must 
have  some  amicroscopic  structure  since  it  shows  semipermeability. 

Nuclear  staining.  The  proteins  isolated  from  the  nucleus  being 
strong  bases,  it  might  be  expected  that  it  would  be  easy  to  dye  the 
structural  elements.  The  living  nucleus,  however,  can  hardly  be 
stained  without  temporary  or  permanent  damage  (Becker,  1936). 
For  this  reason  it  must  be  supposed  that  the  basic  groups  which  ionize 
freely  in  the  isolated  protamines  and  histones  are  screened  off  in  the 
native  state.  If  nevertheless  one  wishes  to  apply  vital  staining,  these 
groups  must  be  liberated  by  slight  hydrolysis.  As  in  the  case  of  cyto- 
plasm, it  can  be  said  that  vital  staining,  which  means  the  formation 
of  coloured  salts  of  the  basic  or  acid  dyestuffs  applied,  always  repre- 
sents a  hydrolytic  intervention;  for  instance,  vital  nuclear  staining  in 
a  dilute  solution  of  erythrosin  is  only  possible  when  acidified  with 
acetic  acid. 

It  is  reasonable  to  assume  that  the  basic  protein  groups  are  screened 
by  nucleic  acids.  Apparently,  however,  the  active  nucleus  contains 
this  component  rather  sparingly,  so  that  other  anionic  substances  must 
also  take  part  in  masking  the  basic  groups.  Active  nuclei  are  less 
intensively  stained  by  the  nucleal  reaction  than  those  which  are 


2  NUCLEUS  219 

dividing.  A  more  convincing  proof  is,  however,  brought  forward  by 
Caspersson's  experiments  (1936),  which  are  based  on  the  absorption 
of  ultraviolet  hght  bv  nucleic  acids.  By  means  of  microphotometric 
measurements  he  shows  that  the  concentration  of  nucleic  acids  in  the 
nucleus  strongly  increases  in  the  preliminary  stage  of  cell  division,  to 
decrease  again  during  the  telophase. 

It  is  perhaps  partly  due  to  changes  in  nucleic  acid  content  that  fixed  nuclei 
are  sometimes  more  easily  stained  with  acid  dyestuffs  (erythrophily),  at 
other  times  with  basic  ones  (cyahophily),  as  has  been  summarized  by 
TisCHLER  (1921/22).  Caspersson,  howcvcr,  has  not  been  able  to  establish 
a  relation  between  nucleic  acid  content  and  basic  or  acidic  nuclear  reaction 
with  respect  to  dyestuffs  in  the  nuclei  of  the  gland  of  the  oesophagus  of 
Helix pomafia.  This  must  probably  be  explained  by  the  fact  that  not  only  the 
number  of  acidic  or  basic  groups  in  the  nuclear  proteins,  but  also  the  pn 
of  the  karyolymph  is  determinative  for  the  anionic  or  cationic  behaviour 
of  the  nucleus  (Keller,  1932;  Becker,  1936).  On  the  other  hand,  the 
nucleic  acid  content  probably  determines  the  I.  E.  P.,  so  that  at  a  constant 
pfj  value  the  adsorptive  power  of  a  nucleus  towards  basic  or  acid  dyestuffs 
may  vary. 

Caspersson's  photometric  determination  of  nucleic  acid  seems  to 
prove  that  the  increasing  chromophily  of  the  fixed  nucleus  is  related 
to  the  accumulation  of  nucleic  acids  in  the  preliminary  stages  of  cell 
division.  It  seems  to  me  that  the  older  cytologists,  who  distinguished 
in  the  nuclear  substance  a  component  Hke  plastin,  linin  or  achromatin 
(difficult  to  stain  and  hardly  digestible)  from  the  easily  stained  "chro- 
matin", already  recognized  the  existence  of  the  two  fundamental 
principles  in  the  nuclear  structure  viz.,  on  the  one  hand  high  poly- 
meric, relativelv  resistant  proteins  and,  on  the  other,  a  compound  very 
sensitive  to  basic  staining,  the  nucleic  acid,  which  predominates  during 
nuclear  division  but  falls  into  the  background  in  the  active  nucleus. 
The  well-known  staining  of  fixed  nuclei  with  basic  dyestuffs  indicates 
the  presence  of  liberated  acid  groups  and  the  nucleal  reaction  points 
to  aldehyde  groups.  Undoubtedly  therefore,  the  chromatic  substance 
consists  mainly  of  nucleic  acids.  In  spite  of  this  it  is  not  possible  to 
designate  these  chemically  well-defined  compounds  as  chromatin.  For, 
in  cytology,  the  term  chromatin  has  become  a  morphological  concept 
for  regions  showing  identical  behaviour  with  respect  to  staining 
(Heitz,  1935).  Those  regions  of  the  active  nucleus  or  parts  of  chro- 
mosomes which  after  division  do  not  lose  their  high  nucleic  acid 


220  FINE-STRUCTURE  OF  PROTOPLASM  II 

content,  are  designated  as  heterochromatic  (positively  heteropycnotic, 
White,  1945).  In  other  words,  heterochromatin  comprises  those  thymo- 
nucleic  acids  which  remain  passive  during  the  changing  phases  of 
mitosis,  whereas  chromatin  or  euchromatin  consists  of  thymonucleic 
acids  which  in  the  process  of  nuclear  division  first  increase  and  after- 
wards decrease  again.  It  has  been  found  that  the  heterochromatic  re- 
gions (chromocentres)  of  a  nucleus  represent  chromosome  parts  which 
locally  have  preserved  their  spiral  structure  (Straub,  1943). 

Birefringence  of  the  nucleus.  As  a  rule  spherical  nuclei  are  isotropic 
aside  from  their  birefringent  boundary.  If  their  shape  is  anisodia- 
metric,  however,  they  often  display  double  refraction. 

The  two  components,  protein  and  nucleic  acid  chains,  do  not  only 
show  opposite  chemical  behaviour,  in  that  the  one  is  positive  (cationic) 
and  the  other  negative  (anionic).  Their  optical  reactions  are  also  op- 
posite. In  the  natural  state  all  fibrillar  proteins  investigated  so  far  are 
optically  positive,  whereas,  according  to  the  interesting  model  experi- 
ments of  Schmidt  (1957a)  and  the  experiments  on  flow  birefringence 
(Signer,  Caspersson  and  Hammarsten,  1938;  Wissler,  1940),  arti- 
ficially prepared  fibres  of  the  sodium  salt  of  a-thymonucleic  acid  are 
optically  negative.  For  this  reason  elongate  nuclei  with  a  high  nucleic 
acid  content,  Hke  certain  sperm  nuclei  (Fig.  125a,  p.  228),  are  optically 
negative.  The  negative  reaction  in  polarized  light  is,  however,  limited 
to  the  chromatic  part  of  the  sperm  head.  Often  the  achromatic  parts 
are  optically  positive.  Any  attempt  to  explain  this  positive  reaction  as 
rodlet  birefringence,  i.e.,  as  positive  textural  double  refraction,  is 
inconclusive  in  the  absence  of  indisputable  Wiener  curves.  Since 
Schmidt  (1937a,  p.  87)  has  proved  that  these  regions  show  positive 
intrinsic  birefringence,  it  seems  to  me  that  the  anisotropy  must  be 
attributed  to  the  submicroscopic  protein  framework.  I  do  not  doubt 
that  it  exists  also  in  the  chromatic  part  of  the  sperm  head,  where  it 
is  over-compensated,  however,  by  the  strongly  negative  nucleic  acid. 
If  it  were  possible  to  eHminate  the  nucleic  acid  components  com- 
pletely without  disturbing  the  structure  and  to  dehydrate  the  protein 
to  a  sufficient  extent,  both  the  positive  rodlet  birefringence  and  the 
positive  intrinsic  double  refraction  of  the  protein  framework  would 
become  apparent.  The  positive  birefringence  of  achromatic  oblong 
nuclei,  such  as  the  fibrous  spindle-shaped  nucleus  of  Aloe  described 
by  Kuster  (1934b),  must  doubtless  be  attributed  tothe  orientated 


2  NUCLEUS  221 

protein  framework.  It  seems  to  me  that  the  chemical  dualism  in  the 
nuclear  structure  is  clearly  demonstrated  by  its  optical  anisotropy,  as 
the  character  of  the  birefringence  is  determined  either  by  the  optically 
positive  proteins  or  the  optically  negative  nucleic  acid  inclusions. 

Nucleolus.  It  has  been  shown  that  the  nucleoli  have  their  origin  in 
the  accumulation  of  proteins  (in  particular  histones,  Caspersson, 
1940a;  Serra  and  Queiroz-Lopes,  1944),  which  can  be  regarded  as 
reserve  substances.  The  reserve  proteins  (e.g.  edestin,  excelsin, 
p.  141)  differ  from  the  framework  proteins  by  a  lower  degree  of 
polymerization  and  the  globular  form  of  their  molecules.  They  may 
be  arranged  into  molecular  crystal  lattices  capable  of  .=. welling.  It  is 
significant  that  the  nucleoli  in  the  nucleus  can  be  substituted  by 
protein  crystalloids  (Kuster,  1955  a,  p.  155)  as  is  the  case  with  repre- 
sentatives of  the  Scrophulariaceae  (Gicklhorn,  1932b)  and  Lenti- 
bulariaceae.  Sometimes  nucleoli  and  crystalloids  occur  simultaneously 
in  the  same  nucleus  (Zimmermann,  1896). 

The  principal  difference  between  the  proteins  of  the  reticulum  and 
those  of  the  nucleoli  is  the  greater  solubiHty  of  the  latter.  In  contrast 
to  the  very  resistant  nuclear  frame,  they  are  readily  dissolved  by 
pepsin  in  hydrochloric  acid.  In  spite  of  its  high  histone  content,  the 
nucleolus  apparently  possesses  weak  anionic  properties,  for  it  vigor- 
ously collects  most  basic  dyestuffs  and  as  a  rule  shows  a  greater  re- 
sistance to  swelling  in  dilute  alkaline  solutions  than  the  reticulum 
(TisCHLER,  1921/22,  p.  45-51).  This  is  due  to  a  certain  content  of 
globuHns  and  ribonucleic  acid  (Caspersson,  1941),  so  that  its  I.E. P. 
does  not  lie  in  the  alkaline  field.  On  the  other  hand,  it  also  stores  acid 
dyestuffs  such  as  eosin,  indicating  an  I.E. P.  near  neutrality.  The 
behaviour  of  the  nucleolus  towards  dyestuffs  depends  very  much  on 
the  process  of  fixing  and  the  method  of  staining  (Romeis,  1943,  p.  323), 
which  may  modify  its  isoelectric  behaviour.  It  is  well  kown  that 
Carnoy  fixation  (alcohol  -j-  acetic  acid)  dissolves  a  good  deal  of  the 
nucleolus,  so  that  it  appears  to  be  surrounded  by  a  broad  areola.  It 
shows  a  specific  affinity  for  the  acid  dyestuff  methyl  green  (Colour 
Index,  I  St  ed.,  No  684,  in  German  Lichtgriin)  which  allows  of  dif- 
ferential staining  in  comparison  with  the  red  Feulgen  reaction  of 
chromatin  (Semmens  and  Bhaduri,  1939).  This  double  staining  has 
become  important  for  the  problem  of  nucleoH  formation  in  the  telo- 
phase of  cell  division. 


222  FINE-STRUCTURE  OF  PROTOPLASM  II 

If  the  nucleoli  represent  reserve  proteins,  their  formation  is  com- 
parable to  that  of  aleurone  grains  in  the  cytoplasm.  In  fact,  it  has  been 
observed  that  the  protein  crystalloids  which  sometimes  replace  the 
nucleoli  grow  in  small  vacuoles  of  the  nucleus.  The  place  where  the 
nucleoli  appear  is  predetermined,  for  they  condense  in  contact  with 
special  chromosomes  provided  with  secondary  constrictions  (Heitz, 
1935;  Hakansson  and  Levan,  1942).  At  first  they  behave  like  real 
vacuoles,  for  in  the  presence  of  several  chromosomes  condensing 
nucleolar  material,  the  several  nucleoli  formed  can  subsequently  unite 
to  form  bigger  ones.  In  the  present  state  of  our  knowledge  the 
nucleolus  formation  must  be  considered  as  an  accumulation  of  the 
karyolymph  proteins  at  a  definite  spot,  which  takes  place  at  the  ex- 
pense of  energy,  until  a  coacervate  droplet  rich  in  proteins  is  formed. 

Nuclear  spindle.  The  microscopic  structure  of  the  spindle  which 
becomes  apparent  in  nuclear  divisions  has  long  remained  an  enigma. 
In  fixed  preparations  spindle-shaped  fibrillae  are  visible,  some  of  which 
stretch  from  the  one  pole  of  the  cell  to  its  equator,  while  others, 
shorter  ones,  coalesce  with  the  chromosomes  at  special  points  of 
attachment  (centromeres).  In  the  living  state,  however,  all  this  re- 
mains invisible;  microscopically  the  spindles  are  homogeneous, 
structureless  and  optically  empty.  Microsurgical  interventions  reveal 
a  relatively  rigid  double  cone  with  distinct  cleavability  but  without 
a  visible  structure  (Belar,  1929).  Accordingly  the  spindle  fibres  have 
been  considered  as  artefacts  of  the  fixing  process. 

In  this  case  it  has  been  possible  to  elucidate  the  true  state  of  affairs 
by  means  of  the  polarizing  microscope.  Schmidt  (1937a)  finds  the 
spindles  to  be  positively  birefringent  in  living  sea-urchin  eggs.  Thus 
the  images  visible  in  the  fixed  material  prove  to  be  real  structures 
existing  in  vivo.  Since  the  poles  of  the  spindle  behave  like  positive 
spherites  whose  rays  can  be  followed  nearly  throughout  the  cell,  they 
must  consist  of  optically  positive  invisible  fibrillae.  Undoubtedly 
the  same  fibrils  stretch  from  each  pole  to  the  chromosomes.  It  was 
thought  that  these  fibrils  were  submicroscopic  and  ought,  therefore, 
to  be  visible  in  the  electron  microscope.  This  is  the  case  when  acid 
fixation  is  used  (e.g.  Bouin's  solution;  Beams,  Evans,  Verne  van 
Bremen  and  Baker,  1950).  But,  when  duly  fixed  with  neutral  formahn, 
the  spindle  region  of  dividing  cells  in  onion  root  dps  appears  to  be 
structureless   (RozsA  and  Wyckoff,   1950).  Therefore,  the  fibrillar 


2  NUCLEUS  223 

elements  of  the  spindle  must  be  amicroscopic,  consisting,  maybe,  of 
polypeptide  chains.  Whether  these  are  individualized  or  in  some  way 
aggregated  cannot  be  decided.  In  any  event  they  must  be  arranged 
in  orderly  array  to  produce  the  observed  birefringence.  As  a  result  of 
the  fixing  process,  the  visible  fibres  arise  by  dehydration  and  by  some 
kind  of  crystalline  aggregation  of  these  birefringent  elements.  Their 
denser  packing  causes  a  marked  increase  in  optical  anisotropy.  As  the 
birefringence  is  positive,  the  assumption  of  expanded  polypeptide 
main  valency  chains  is  not  unreasonable.  At  any  rate  this  hypothesis 
may  be  used  as  long  as  it  is  not  disproved. 

The  spindles  are  formed  primarily  in  the  cytoplasm  when  the 
nucleus  is  still  intact.  In  some  cases  even  cells  devoid  of  nuclei  are 
capable  of  forming  spindles  (E.  B.  Harvey,  1936).  Having  supposed 
the  cytoplasm  to  consist  of  proteins,  there  is  no  difficulty  in  deriving 
the  spindle  structure  from  the  cytoplasmic  frame  structure.  The 
globular  macromolecules  must  simply  denature  to  give  expanded 
chains  which  aggregate  laterally.  In  fixed  preparations  this  fibrillation 
of  the  cytoplasm  can  often  be  observed  in  the  regions  surrounding 
the  poles.  I  fully  realize  that  the  transition  will  not  take  place  according 
to  this  simple  scheme,  but  must  be  connected  with  the  synthesis  of 
additional  protein  chains.  However,  the  principal  condition  is  that  the 
cytoplasm  already  contains  the  structural  elements  required,  i.e.,  the 
polypeptide  chains,  either  as  structural  material  or  as  a  model  for  the 
formation  of  new  chains. 

The  spindle  is  not  always  formed  outside  the  nucleus;  in  special 
cases  it  has  its  origin  inside  the  nuclear  boundary,  or  it  is  observed 
that  cytoplasmatic  and  nuclear  fibrils  together  take  part  in  the  con- 
struction of  the  spindle.  This  once  more  indicates  that  the  submicro- 
scppic  structures  of  cytoplasm  and  nucleus  are  alike.  By  submicro- 
scopic  changes  fibrillar  elements  of  similar  morphological  nature  may 
originate  from  both  of  them.  We  may  conclude  that  the  nucleus  does 
not  separate  chemically  from  the  cytoplasm  as  a  completely  foreign 
substance. 

The  protein  chain  structure  of  the  spindle  fibres  can  be  utilized  in 
the  so-called  "strain  theory"  (Zugfasertheorie),  according  to  which 
the  chromosomes  are  drawn  towards  the  pole  by  shortening  fibres. 
In  fact,  expanded  polypeptide  chains  have  the  property  of  contracting 
considerably  under  certain  circumstances  (see  p.   134).  It  might  be 


224  FINE-STRUCTURE  OF  PROTOPLASM  II 

objected  that  in  this  case  not  only  the  fibrils  connected  with  the  chro- 
mosomes must  be  shortened  but  also  those  seemingly  stretching  from 
pole  to  pole.  Schmidt  (1939a)  has  shown,  however,  that  the  double 
refraction  of  the  spindle  fibrils  is  extinguished  at  the  equator.  In  other 
words,  the  fibres  running  from  pole  to  pole  appear  to  be  interrupted. 
The  intermediate  body  formed  at  the  equator  (phragmoplast)  is  iso- 
tropic. When  the  chromosomes  are  drawn  apart  from  each  other,  the 
birefringence  of  the  fibrils  decreases,  as  is  to  be  expected  in  the  con- 
traction of  protein  fibres. 

The  strain  theory  cannot  explain  why  the  chromosomes  migrate  to 
the  equator  in  the  metaphase.  To  imagine  a  mechanism  which  ac- 
counts for  that  movement,  rather  complicated  assumptions  must  be 
made.  Ostergreen  (1950)  thinks  the  attracting  forces  of  the  poles 
increase  with  increasing  distance.  Therefore,  the  centromere  (kineto- 
chore)  of  the  metaphase  chromosome  is  only  in  equilibrium  when 
located  midway  between  the  two  poles.  After  the  cleavage  of  the 
metaphase  chromosome,  an  additional  hypothesis  is  needed  to  explain 
anaphase,  viz.  that  the  kinetochores  of  the  two  daughter  chromosomes 
have  a  polar  character,  so  that  only  their  side  turned  towards  one  of 
the  poles  is  attracted. 

It  is  obvious  that  a  hypothesis  resting  on  such  uncertain  grounds 
is  no  better  than  the  assumption  that  the  chromosomes  have  active 
locomotion  at  their  disposal  which  allows  them  to  move  to  and  fro. 

c.  Fine-Structure  of  the  Chromosoiues 

The  chromosomes  differentiate  from  the  nuclear  reticulum  in  which 
they  are  preformed.  During  the  prophase  of  cell  division  they  disen- 
tangle, shorten  and  become  independent.  The  membrane  of  the 
nucleus  which  is  the  semi-solidified  peripheral  part  of  the  nuclear  sap 
disappears.  After  the  destruction  of  this  boundary,  the  karyolymph 
readily  mixes  with  the  cytoplasm. 

The  chromosomes  often  possess  two  arms.  The  connecting  part 
between  these  arms  is  somewhat  constricted  (primary  constriction) 
and  cannot  be  stained  by  Feulgen's  reagent;  it  is  anucleal.  The  con- 
striction serves  as  a  point  of  contact  for  the  spindle  fibre.  This  region 
often  possesses  a  clearly  visible  boundary  and  is  then  designated  as 
centromere  or  kinetophore.  In  addition  to  the  primary  constriction, 
so-called  secondary  constrictions  are  sometimes  found,  where  the 


NUCLEUS 


225 


nucleoli  condense  during  the  telophase.  Moreover  Fig.  125  a  shows 
some  heterochromatic  parts  (end  of  left  chromosome  arm  and  satellite). 
An  obvious  hypothesis  relating  to  the  submicroscopic  structure  of 
the  chromosomes,  deriving  support  from  Heitz  (1935)  and  Geitler 
(1934,  1938)  in  their  elaborately  documented  summarizing  studies  on 


.^: 


\ 


% 


^ 


¥1 


»«> 


ft 

*  h 


n 


0 


^-„„-d 


^) 


^) 


Fig.  123.  Microscopic  chromosome  structure  (from  Heitz,  1935).  a)  Idealized  chromo- 
some with  helicoid  chromonema  threads;  heterochromatic  region  hatched;  in  the  upper 
part :  a  primary  (kinetic)  constriction ;  in  the  lower  part  at  the  right :  secondary  constriction 
with  satellite  (corrected  to  satisfy  Geitler's  criticism  1938,  p.  98).  b)  Chromatid  pair  of 
Trillium  erectum.  c)  Spiral  structure  of  the  chromosomes  of  Tradescaiitia  virginica. 
d)  Spiral  structure  of  the  chromosomes  of  Trillium  erectum. 

the  structure  of  chromosomes  can  be  built  up  from  the  chromonema 
theory.  Each  chromosome  contains  two,  according  to  other  investi- 
gators (Nebel  and  Ruttle,  1937;  Nebel,  1939,  1941)  even  four 
spirally  wound  threads,  called  chromonemala  (Fig.  123  a).  In  the  first 
case  they  are  identical  with  the  chromatid  threads  (Fig.  123  b),  well 
known  from  the  prophase  of  meiosis.  It  is  only  in  that  prophase  that 
the  chromonema  spiral  is  completely  uncoiled  and  therefore  survey- 
able  in  its  entire  length,  which  is  many  times  that  of  the  chromosome. 
It  consists  of  a  non-staining  thread  which  at  regular  intervals  is  covered 
with  knots  showing  the  nucleal  reaction  and  designated  as  chromo- 
meres.  In  the  mitosis  chromosomes  these  particulars  can  scarcely  be 
observed,  as  the  chromonemata  are  coiled  (Fig.  123  c)  and  embedded 


220  FINE-STRUCTURE  OF  PROTOPLASM  II 

in  a  ground  mass    (matrix)   which   shows   strong   nucleal   staining. 

It  must  be  mentioned  that  a  spiral  structure  has  been  observed  onl)^ 
in  large  chromosomes.  According  to  Japanese  and  American  cyto- 
logists  (Straub,  1938;  HusKiNS,  1 94 1,  1942)  the  visible  helix  some- 
times possesses  a  spiral  structure  of  its  own,  in  which  case  the  chro- 
mosome would  possess  the  structure  of  a  doubly  wound  helix  with  a 
primary  and  a  secondary  spiral  (large  and  small  spiralling).  With  the 
aid  of  the  phase  contrast  microscope  Ruch  (1945)  has  shown  that  in 
the  case  of  the  much-investigated  chromosomes  of  Tradescantia  the 
chromomeres  occurring  in  pairs  on  the  spirally  wound  chromonema 
fibrils  suggest  the  existence  of  the  small  spiralling;  but  by  judicious 
considerations  of  the  focal  depth  of  the  microscope  objectives  used, 
he  proves  clearly  that  no  doubly  wound  chromosomes  exist  (Ruch, 
1949).  The  question  as  to  how  the  helical  chromonemata  are  separated 
from  each  other  during  mitosis  without  uncoiling  is  a  problem  in 
itself  (Matthey,  1941). 

The  chromonema  theory  has  gained  general  importance  by  the 
discovery  of  the  giant  double  chromosomes  of  the  nuclei  from  the 
salivary  glands  of  the  Diptera.  In  these  marvellous  cytological  objects 
homologous  chromosomes  are  united  into  astonishingly  broad  and 
remarkably  long  ribbons.  These  gigantic  chromosomes  may  be  re- 
garded as  bundles  of  numerous  expanded  chromonemata,  formed  by 
endomitosis  (Heitz,  1935).  They  are  united  into  strings  of  consider- 
able dimensions ;  the  chromomeres  form  transversal  discs  which,  by 
means  of  staining  or  the  nucleal  method,  are  made  visible  as  numerous 
crosshnes  (Fig.  124). 

The  non-stainable,  anucleal  regions  of  the  chromonemata  bundles 
represent  the  protein  components  of  the  chromosome.  It  may  be 
concluded  that  the  protein  thread  is  not  restricted  to  the  colourless 
segments,  but  runs  invisibly  through  the  whole  chromosome.  In  the 
chromomeres  the  nucleic  acid  components  are  localized,  thus  masking 
the  protein  ground  mass.  Their  localization  is  demonstrated  by  the 
nucleal  reaction,  the  ultraviolet  absorption  and  the  X-ray  absorption 
method  of  Engstrom  which  proves  that  the  Feulgen  positive  bands 
contain  2  to  10  times  more  mass  than  the  Feulgen  negative  ones.^ 
Moreover,  the  ultraviolet  microscope  with  its  higher  resolving  power 
furnishes  proof  of  the  existence  of  the  protein  ground  mass  in  the 

^  Engstrom  and  Ruch  (1951). 


NUCLEUS 


227 


chromomeres.  With  the  aid  of  digestion  experiments  in  which  the 
nucleic  acid  was  protected  from  digestion  by  lanthanum  thymo- 
nucleate,  Caspersson  (1936)  finds  that  the  chromomeres  are  resolved 
into  extremely  thin  discs.  Ultraviolet  photography  reveals  a  fine- 
structure  of  lamellae  with  a  thickness 
of  only  0.1  j.1.  Since  at  this  order  of 
magnitude  the  limit  of  the  resolving 
power  in  ultraviolet  hght  is  reached, 
the  question  as  to  whether  these  very 
thin  chromomere  discs  possess  a  still 
finer  submicroscopical  structure  and 
thus  are  subdivided  remains  unsettled. 
Personally  I  do  not  doubt  that  they  are. 

Conversely,  milt  nuclease  digests 
the  nucleic  acids  of  the  chromomeres 
(Mazia  and  Jaeger,  1939)  without 
disturbing  the  ground  structure  of  the 
chromosomes  of  the  salivary  glands. 
The  ability  to  take  the  Feulgen  stain 
disappears;  on  the  other  hand  the 
ninhydrin  test  turns  out  positive  over 
the  entire  length  of  the  chromosome. 
So  the  chromonema  does  not  consist 
of  alternating  protein  and  nucleic  acid 
links,  but  represents  a  continuous 
protein  thread  in  which  at  regular 
intervals  nucleic  acid  knots  are  inter- 
calated. The  nucleic  acids  form  saltlike 
compounds  with  the  protein  ground 
mass,  the  nucleoproteins,  whose  oc- 
currence is  therefore  limited  to  the 
chromomeres  (Fig.  125  b-d). 

Fibrillar  hypothesis.  From  a  morphological  point  of  view  Wrinch 
(1936)  believes  the  molecular  structure  of  the  chromonema  to  be  as 
follows :  the  polypeptide  chains  form  a  system  of  parallel  fibrils  like 
the  warp  of  a  weaving-loom  and  the  nucleic  acids  represents  the  woof 
in  this  system  of  chains.  Every  four  neighbouring  polypeptide  chains 
are  kept  together  by  a  molecule  of  the  tetra-basic  thymonucleic  acid. 


Fig.  124.  Two  incompletely  conjug- 
ated giant  chromosomes  of  the  nuclei 
from  the  salivary  gland  of  a  Drosophila 
hybrid  with  a  chromosome  pattern 
characteristic  of  the  two  parental 
species  (from  Patau,  1935);  mel  from 
Dr.  melaiiogasier,  sim  from  Dr.  simu- 
lans  ;  in  a  a.  structural  difference. 


228 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


As  the  native  nucleic  acids  have  a  much  higher  molecular  weight,  the 
woof  would  not  consist  of  short  chains  with  four  members  but  of 
much  longer  chains.  The  heteropolar  salt  bonds  between  the  acid 
groups  of  the  nucleic  acid  chains  and  the  basic  groups  of  the  poly- 
peptide chains  would  have  to  be  considered  as  the  junctions  of  this 
network  (Fig.  125  b). 


»t  =  =2-t 


Biti?-t4 


h) 


iMm 


d 


«') 


Fig.  125.  Submicroscopic  arrangement  of  nucleic  acid  (shaded);  a)  in  the  head   of  the 
spermatozoon  of  Sepia  (after  Schmidt,   1937a);  b-d  in  the  chromonema:  b)  transversal 
(after  Wrinch,  1936),  c)  lengthwise  (after  Schmidt,  1937c,  1939b),  d)  scattered  orienta- 
tions (from  Frey-Wyssling,  1943b,  1944a). 


This  scheme  is  not  supported  by  the  optical  properties.  The  arti- 
ficial nucleic  acid  threads  obtained  by  Schmidt  (1957a)  are  optically 
negative^ ;  and  since  in  the  spinning  the  molecular  chains  are  arranged 
parallel  to  the  axis,  the  polynucleic  acid  chains  themselves  must  also 
be  optically  negative.  It  follows  from  this  that  the  molecules  of  the 
nucleic  acid  chains  in  the  sperm  nuclei  (Schmidt,  see  Fig.  125  a)  run 
parallel  to  the  morphological  axis  of  the  sperm  head.  But  the  poly- 
peptide frame  of  these  nuclei  also  must  be  orientated  in  the  same 
direction.  This  means  that  the  chain  molecules  of  both  nuclear  com- 
ponents show  parallel  alignment. 

The  chromomere  discs  of  the  chromosomes  of  the  saUvary  glands 
are  optically  negative  (Schmidt,  1937c,  1959b).  For  the  submicro- 

1  Threads  of  sodium  thymonucleate  show  a  reversal  of  their  sign  of  double  refraction 
when  strongly  stretched  (Wilkins,  Gosling  and  Seeds,  195  i). 


Z  NUCLEUS  229 

scopic  fine-structure  of  the  chromomeres  Schmidt  therefore  takes 
into  consideration  a  possible  arrangement  as  given  in  Fig.  125  c.  The 
fact,  ascertained  by  Astbury  and  Bell  (1958),  that  the  fibre  period 
of  3.34  A  of  the  nucleic  acids  is  about  the  same  as  that  in  the  poly- 
peptide chains  (3.5  A,  see  Table  XXXII,  p.  368)  seems  to  support 
this  hypothesis. 

With  the  aid  of  the  ultraviolet  dichroism  of  the  nucleic  acid  chains 
Caspersson  (1940b)  has  checked  the  structure  proposed  in  Fig.  125  c. 
If  the  nucleic  acid  molecules  in  the  protein  fibres  showed  complete 
orientation,  chromomeres,  like  artificial  thymonucleic  acid  fibres, 
would  displav  a  very  pronounced  dichroism  in  polarized  ultraviolet 
light.  Compared  with  these  fibres,  however,  the  chromomeres  of  the 
chromosomes  of  the  salivary  glands  show  only  an  extremely  small 
dichroitic  effect.  Caspersson  therefore  draws  the  conclusion  that  the 
nucleic  acid  chains  are  intercalated  practically  without  orientation. 
Also  the  double  refraction  of  the  chromomeres,  as  derived  from  the 
birefringence  of  flow  of  sodium  nucleate  sols  (Signer,  Caspersson 
and  Hammarsten,  1938),  proves  to  be  very  small.  Meanwhile,  as- 
suming that  nucleic  acids  are  straight  chains,  the  negative  sign  of  the 
chromomere  birefringence  indicates  that  the  chains  have  a  certain 
preferred  orientation.  With  the  aid  of  the  formula  mentioned  on  p.  90 
the  scattering  in  the  orientation  of  the  chain  molecules  can  be  calcu- 
lated (Frey-Wyssling,  1943b),  and  the  scattering  angle  found  in  this 
way  is  86°. 5,  i.e.,  nearly  a  right  angle.  This  means  that  the  scattering 
is  almost  complete,  thus  furnishing  an  important  argument  against 
the  supposition  that  the  nucleic  acid  molecules  are  parallel  to  the 
chromonema  axis.  A  similar  result  is  obtained  if  the  intrinsic  double 
refraction  of  —  0.050  found  by  Schmidt  (1928)  for  the  chromatin  of 
the  Sepia  sperm,  or  even  only  a  fraction  of  this  value,  is  compared 
with  the  birefringence  of  the  chromomeres. 

In  spite  of  the  small  orientation  of  the  nucleic  acids,  Caspersson 
assumes  the  protein  chain  structure  to  be  continuous.  Orientated 
polypeptide  chains  are  supposed  to  cause  the  anucleal  chromosome 
segments  to  appear  positively  birefringent.  This  effect,  however,  can 
only  be  observed  in  stretched  chromosomes  (F.  O.  Schmitt,  1938; 
Pfeiffer,  1 941  a).  Optics  therefore  do  not  provide  sufficiently  reliable 
data  to  assume  orientation  of  the  protein  chains.  There  can  certainly 
be  no  pronounced  fibrillar  texture  of  expanded  polypeptide  chains. 


230 


FINE-STRUCTURE  OF  PROTOPLASM  II 


as  there  is  not  the  slightest  indication  of  the  existence  of  a  chain  lattice. 
All  the  same,  the  anisotropy  of  swelling  and  the  cleavabiHty  of  the 
chromosomes  are  in  favour  of  an  orientation  along  the  long  axis  of 
the  protein  ground  mass.  In  his  microchemical  experiments  with 
chromosomes  of  salivary  glands  Painter  (1941)  is  also  impressed  by 
their  fibrillar  character.  It  must  be  supposed  that  the  isotropy  of  the 
protein  results  from  coiling  and  folding  of  polypeptide  chains,  com- 
bined with  a  corresponding  hydration.  Evidently  the  chromosome 
protein  consists  rather  of  globular  molecules  which  may  be  aggregated 
to  form  beaded  chains. 

The  explanation  of  the  birefringence  of  other  chromosomes 
(Schmidt,  1957a,  1941a)  is  much  impeded  by  our  insufficient 
knowledge  of  the  submicroscopic  orientation  in  the  chromonemata 
bundles  of  the  salivary  glands.  According  to  Becker  and  Kozbial 
(1937),  the  optical  character  of  the  chromosomes  of  the  root  tips  of 
Allium  and  Vicia  depends  on  the  process  of  fixing :  if  treated  directly 
with  alcohol  they  appear  to  be  negative;  after  a  previous  treatment 
with  acetic  acid  vapour  (causing  swelling)  they  are  positive.  On  the 
assumption  of  a  nearly  complete  scattering  of  the  nucleic  acid  chains 
in  the  isotropic  living  chromosome,  these  effects  might  be  explained 
by  tendencies  towards  orientation  as  a  result  of  the  shrinkage  or 
swelling  in  the  fixing  process.  It  seems  to  me  that  considerations  of 
this  kind  open  more  prospects  than  explanations  formerly  attempted 
with  the  aid  of  the  spiral  structure  (Nakamura,  1937).  Kuwada  and 
Nakamura  (1934)  explain  the  positive  double  refraction  of  the  chro- 
mosomes of  Tradescantia  by  a  single  spiral  of  negative  chromonemata ; 
whereas,  in  their  opinion,  optically  negative  chromosomes  are  caused 
by  a  double- wound  spiral. 

d.  Submicroscopic  Morpholo^  of  Hereditary  Processes 

Genes.  The  fibrillar  character  of  the  chromatids  meets  two  important 
morphological  requirements  of  genetics:  i.  the  substrate  is  easilj 
ckavable  in  the  direction  of  the  long  axis,  which  is  not  only  necessary  for 
the  splitting  of  chromosomes  but  also  for  the  phenomena  taking  place 
between  synapsis  and  diakinesis  in  heterotypic  divisions;  2.  the  long 
chromonemata  offer  an  opportunity  for  the  linear  arrangement  and  the 
possibility  of  exchange  of  the  genes. 

AIorgan's  school  has  calculated  that  the  number  of  genes  known 


2  NUCLEUS  231 

in  the  Drosophila  chromosomes  is  so  large  that  for  reasons  of  space 
each  gene  must  be  bound  to  relatively  small  molecules  of  about  the 
same  order  of  magnitude  as  found  in  the  reserve  proteins  (compare 
Fig.  90,  p.  136)  investigated  in  Svedberg's  ultracentrifuge.  It  is 
difficult  to  see,  however,  how  such  freely  moving  particles  are  able 
to  intervene  decisively  in  the  processes  of  development.  To  be  able 
to  do  this  their  carriers  must  have  fixed  mutual  positions  and  it  is 
best  to  imagine  that  they  are  fixed  on  beaded  protein  fibrils.  In  this 
way  we  comply  with  the  requirement  of  Hnear  arrangement  in  a 
manner  which  could  hardly  be  improved. 

In  spite  of  its  great  probability,  however,  irrefutable  proof  of  the 
existence  of  the  submicroscopic  fibrillar  structure  has  not  yet  been 
produced.  As  has  been  shown,  the  quantitative  evaluation  of  the 
optical  results  suggests  that  the  protein  is  not  in  a  pronounced  fibrillar 
state  possessing  the  characteristics  of  a  chain  lattice ;  and  thus  far  the 
electron  microscope  has  failed,  because  even  the  pachytene  and  diplo- 
tene  chromosomes  yield  only  compact  black  shadows  (Elvers,  1943) 
showing  fewer  particulars  than  a  good  light  optical  image.  It  is  of  so 
much  the  greater  value  that  the  experimental  investigation  of  mutation 
or  ray  genetics  (Zimmer  and  Timofeeff-Ressovsky,  1942)  opens  new 
perspectives. 

Target  theory.  Artificial  mutations  are  induced  by  ionizing  rays  (UV- 
rays,  X-rays,  y-rays).  The  dose  of  rays  is  measured  by  the  X-ray  unit 
r  (roentgen),  which  is  defined  as  that  amount  of  rays  which  will  bring 
about  enough  conductivity  under  prescribed  conditions  in  a  chamber 
of  I  cm^  of  air  to  permit  a  charge  of  one  electrostatic  unit  to  be 
measured  at  saturation  current.  It  is  now  established  that  the  mutation 
rate  induced  artificially  by  radiation  is  proportional  to  the  dose  of 
rays  brought  to  bear  (Timofeeff-Ressovsky,  1940).  The  effect  is 
independent  of  the  wavelength  and  the  dose  (intensity  X  time)  can 
be  given  all  at  once,  concentrated  or  diluted,  or  else  given  at  intervals. 
There  appears,  therefore,  to  be  no  recovery.  For  instance,  whereas  the 
sex-linked  mutations  in  the  x-chromosome  of  Drosophila  have  a 
natural  mutation  rate  of  approximately  0.2%,  the  irradiation  of  2500  r' 
produces  a  rate  of  7%  and  5000  r  produces  14^%.  If  the  mutation  rates 
are  plotted  as  a  function  of  the  dose,  a  straight  line  resuhs  which 
intersects  the  zero  point;  thus  there  is  no  threshold  value  and  any 
small  dose  will  give  an  effect. 


232  FINE-STRUCTURE  OF  PROTOPLASM  II 

Ionization  consists  in  the  formation  of  ions  from  neutral  molecules 
by  the  action  of  irradiated  energy.  The  molecules  in  question  are,  as 
it  were,  struck  by  the  energy  quanta  of  the  radiation  and  are  thereby 
modified.  That  is  why  the  occurrence  of  a  single  ionization  is  called 
a  hit.  The  relation  between  mutation  rate  and  dose  of  rays  indicates 
that  a  mutation  is  the  result  of  such  a  hit.  It  can  also  be  demonstrated 
(Timofeeff-Ressovsky,  1940)  that  the  interdependence  of  dose  and 
rate  would  not  produce  a  straight  line  if  several  hits  were  needed  to 
bring  about  one  mutation.  The  conclusion  to  be  drawn  from  the  bio- 
physical analysis  of  chromosome  irradiation  is,  therefore,  that  the 
artificial  mutation  of  genes  is  the  elementary  result  of  a  single  hit. 
There  are,  it  is  true,  other  possible  physical  explanations,  besides  the 
target  theory,  which  may  account  for  the  effects  observed  (Minder 
and  Liechti,  1945). 

The  approximate  number  of  atoms  in  a  cubic  centimetre  of  organic  sub- 
stance being  known,  and  also  the  number  of  single  ionizations  which  one 
r  unit  is  able  to  evoke,  it  is  possible  to  calculate  how  many  atoms  are  needed 
for  one  of  them  to  be  hit  to  produce  the  mutation  in  question,  this  by  means 
of  the  experimentally  ascertained  mutation  constant,  which  indicates  the 
degree  of  probability  to  incite  a  mutation  by  a  given  dose  of  radiation.  The 
volume  occupied  by  these  atoms  altogether  is  called  the  target  area.  It  varies 
with  different  mutations  of  genes  within  the  x-chromosome  of  Drosophila; 
nevertheless  an  average  can  be  calculated,  according  to  which  the  susceptible 
volume  amounts  to  3.20-10-20  cm^.,  from  which  it  follows  that  the  radius 
of  the  target  area  (assumed  to  be  spherical)  is  1.97  m/x  (Timofeeff- 
Ressovsky,  1940). 

There  is  an  alternative  method  by  which  the  target  area  can  be  computed. 
If  very  strong  ionizing  rays  are  used,  of  very  great  density,  such  as  neutron 
rays,  for  example,  more  than  one  ionization  may  take  place  in  one  target  area, 
only  one  of  which,  however,  effects  mutation.  The  other  ionizations  are 
inoperative  and  the  mutation  rate  must  consequently  be  smaller  than  was  to 
be  expected  from  the  irradiated  dose  of  rays  in  r  units.  Indeed,  in  the  case 
of  the  >r-chromosome  of  Drosophila,  the  mutation  rate  actually  is  1.6  times 
smaller  for  neutron  rays  than  for  X-rays,  with  the  same  dose  of  rays.  The 
radius  of  the  spherical  target  area  can  now  be  deduced  from  this  factor  to- 
gether with  the  known  density  of  ionization  for  neutron  rays;  Lea  (1940) 
finds  1.89  m/i,.  Seeing  that  this  figure  so  nearly  agrees  with  the  value  found 
by  Timofeeff-Ressovsky,  it  may  be  taken  as  fairly  certain  that  the  order 
of  magnitude  of  the  target  area  is  roughly  4  m/x  diameter. 

The  target  area  is  not  to  be  identified  with  the  gene,  since  it  only  gives  us 
the  size  of  a  sensitive  area  within  which  something  has  to  happen  favouring 
the  probability  of  a  mutation.  The  gene  may  therefore  be  larger  than  the 


2  NUCLEUS  255 

target  area,  viz.,  if  not  all  parts  of  the  former  are  capable  of  changing  their 
molecular  structure  by  ionization.  It  may,  alternatively,  be  smaller,  if  it  does 
not  attain  the  overall  dimension  of  the  statically  calculated  ionization  area 
which,  according  to  Timofeeff-Ressovsky  (1940),  contains  100-2000  (with 
a  mean  of  roughly  1000)  atoms.  The  latter  possibiHty  is,  however,  discounted 
by  estimations  of  the  size  of  the  gene  made  by  speciaUsts  ingenetics.True.it 
is  often  stated  in  the  literature  that  the  target  area  is  of  the  same  order  of 
magnitude  as  that  of  the  gene  size  found  by  other  methods,  but  we  shall 
show  that  this  is  not  so. 

One  known  estimation  of  the  kind  comes  from  Muller(i93  5).  Assuming 
that  a  single  chromonema  thread  of  the  salivary  gland  chromosomes  had  the 
same  volume  as  in  the  corresponding  metaphase  chromosomes  of  normal 
cells,  the  following  calculation  applies  to  the  x'-chromosome  of  Drosophila. 
In  metaphase  its  volume  is  1/8  /x^,  two-thirds  of  which  fall  to  the  share  of 
the  chromonema,  the  length  of  which  in  the  salivary  gland  chromosome  is 
200  ^.  When  completely  uncoiled,  therefore,  a  single  chromonema  thread 
has  the  submicroscopic  thickness  (cf.  Metz,  1941)  of  0.02  /x.  The  thread 
is  thinner  still  if  it  is  assumed  that  the  chromonema  is  regularly  screwed-up 
in  the  metaphase  chromosome,  the  diameter  of  which  is  \  [x.  The  length  of 
200  fi  gives  us  250  windings;  consequently,  with  the  chromosome  being  2  fx. 
in  length,  the  chromonema  could  not  be  thicker  than  0.004  /x. 

In  calculating  the  length  of  the  chromonema  section  containing  a  gene, 
MuLLER  was  guided  by  the  following  consideration:  By  examining  the 
interchange  of  factors  in  cross-breeds,  four  genes  were  localized  in  a  given 
chromomere  of  0.5  ju.  width  in  the  salivary  gland  chromosome  and  the 
existence  of  further  genes  was  shown  to  be  improbable.  Thus  the  length 
covered  by  a  gene  on  the  chromonema  thread  would  be  about  o.i 25  ^.  This 
is  a  dimension  which  lies  on  the  borderline  of  microscopic  resolving  power. 
The  chromonema  sections  which,  according  to  Muller,  correspond  ap- 
proximately to  one  gene,  are  shown  in  diagram  in  Fig.  1 26d  and,  for  com- 
parison, the  target  area  is  indicated  by  a  black  circle.  It  is  recognized  that  the 
thickness  of  the  chromonema  thread  is  of  the  same  order  of  magnitude 
as  the  diameter  of  the  target  area,  but  never  the  estimated  size  of  the  gene, 
the  volume  of  which  exceeds  that  of  the  target  area  by  two  to  three  orders 
of  magnitude !  It  can  be  shown  that  the  sphere  of  action  within  a  gene  has  a 
similar  size  to  the  target  area. 

Carrier  hypothesis  (Frey-Wyssling,  1944b).  If  the  volume  of  the 
gene  is  liable  to  be  more  than  a  thousand  times  larger  than  the  target 
area,  what,  it  must  be  asked,  are  the  relations  between  these  two 
quantities?  It  will  be  seen  in  Fig.  i26d  how  the  small,  sensitive  region 
is  embedded  in  a  large,  non-mutating  area.  It  is  not  known  where  the 
sensitive  region  lies  and  it  may  therefore,  if  desired,  be  thought  of 
as  placed  anywhere.  The  picture  is  reminiscent  of  that   of  enzymes ^ 


234  FINE-STRUCTURE  OF  PROTOPLASM  II 

where  small  active  groups  are  similarly  carried  by  a  larger  protein 
complex  system  (see  Fig.  119,  p.  208).  I  therefore  propose  to  discuss 
the  picture  of  carrier  and  prosthetic  region  for  the  genes  as  well  and 
to  call  this  view  the  ^''Carrier  hypothesis^'' . 

Since  it  is  not  the  salivary  gland  chromonemata  stretched  to  the 
utmost,  with  their  hypothetical  submicroscopic  thickness,  which 
operate  in  hereditary  processes,  but  the  considerably  shorter  meiotic 
chromosome  threads  (leptonema,  zygonema),  the  size  of  the  gene 
should  be  derived  from  the  conditions  produced  by  reduction  division. 
We  learn  from  genetics  that  the  regions  inciting  mutation  are  placed 
linearly  in  the  conjugating  chromatids;  consequently  the  target  areas 
must  likewise  be  aligned  lengthwise  in  the  leptotenic  chromosomes. 
As  the  x-chromosome  is  supposed  to  contain  about  1800  genes 
(Timofeeff-Ressovsky,  1940),  all  sensitive  regions  of  4  m^^  diameter 
should  together  produce  a  length  of  7.2/^.  Bearing  in  mind  that, 
according  to  Muller  (1935),  the  genetically  active  volume  of  the 
x-chromosome  is  roughly  ^12  Z*^?  one  finds  for  the  thickness  x  of  the 
extended  leptotenic  chromosome  prior  to  conjugation  of  the  chro- 
mosomes, ^l^-K^-Tz-j.z  =  7i2)  from  which  x  =  0.12 /^  is  derived  for 
the  thickness  of  the  so-called  leptonema.  This  value  may  be  of  the 
right  order  of  magnitude,  since  the  diameter  of  the  leptonema  is  in 
the  vicinity  of  microscopic  resolving  power. 

If  the  genetically  active  chromomeres  of  the  leptonema  are  now 
divided  up  into  submicroscopic  slices  of  the  thickness  of  a  target  area, 
the  region  corresponding  to  a  gene  should  be  included.  In  this  view 
and  by  this  calculation,  the  gene  should  be  a  flat  disc  having  an 
estimated  diameter  of  120  mju  and  thickness  of  4  m^.  We  are  com- 
pletely ignorant  as  to  where  the  target  areas  lie  in  these  discs:  the 
arrangement  may  be  any  of  an  almost  unlimited  variety,  as  in  Fig.  1 26  c. 
The  only  certainty  we  have  is  that,  given  the  linear  ahgnment  of  the 
loci  of  the  mutations,  they  must  be  juxtaposed  in  the  axis  of  the 
leptonema.  In  Fig.  i26e  such  a  gene  disc  is  represented  on  the  same 
scale  as  the  dimensions  of  the  gene  calculated  by  Muller  (1935).  The 
position  of  the  target  area  within  the  gene  being  unknown,  it  is  shown 
as  a  globule  placed  arbitrarily  anywhere  in  the  disc.  It  is  interesting  to 
note  that  this  size  of  the  gene  tallies  well  with  that  computed  by  Muller 
(chromonema  cross-section  x  lengthof  gene),  although  found  by  total- 
ly different  means  (leptonema  cross-section  x  diameter  of  target  area) : 


NUCLEUS 


235 


Gene  size  according  to  Muller(i93  5),  Fig.  izGd  (2000)- 50,000  (m/^)^ 
Gene  size  according  to  carrier  hypothesis.  Fig.  126c  45>ooo  (m^)^ 

Target  area  according  to  Timofeeff-Ressovsky  (1940)  32  (m^)^ 


Chromosome 


Gene 


1.7m  u 
lOmu  r 


Fig.  126.  Chromosome  and  genes,  a) -v-Chromosome  of  the  Drosophila;  b)  leptonema  with 
linearly  aligned  chromomeres;  c)  leptonema,  strongly  magnified,  with  interchromomeres 
and  target  areas  indicated  as  points;  d)  size  of  genes  calculated  as  prisms  with  quadratic 
cross-section  according  to  Muller  (1935),  each  containing  a  target  area  in  corresponding 
size,  di  upper  and  dg  lower  limit  of  gene  size;  e)  gene  size  according  to  the  carrier  hypo- 
thesis, at  the  same  magnification  as  d;  e^  front  view  of  the  gene  disc  in  comparison  with 
a  target  area,  e,  ground-plan  of  the  gene  disc,  indicating  the  twofold  construction  out  of  2 
chromatids  with  equivalent  spheres  of  action;/)  yellow  respiratory  enzyme  with  apo-  and 
co-enzvme,  both  drawn  on  the  same  scale  as  d  and  e. 


Scheme  i26e  is  even  more  reminiscent  than  i26d  of  the  structure 
assigned  to  enzymes.  Fig.  i26f  depicts  the  yellow  respiratory  enzymes 
on  the  same  scale.  Both  the  size  of  the  colloidal  carrier  (mol.  wt.  70000) 
of  this  enzyme  and  its  prosthetic  group  are  known.  Presuming  that 
1000  atoms  occupy  a  volume  of  3.2-  io"20cm^  (Timofeeff-Ressovsky, 
1 940)  and  that,  in  accordance  with  the  composition  of  sturine  (Fig. 
121,  p.  212,  27  C  :  II  N  :  5  O  :  47  H),  the  average  weight  of  the  atoms 
of  the  amino  acids  is  6.7,  the  diameter  of  the  apo-enzyme  (thought 
of  as  a  sphere)  is  calculated  to  be  about  10  m/^  and  that  of  the  co- 
enzyme with  81  atoms,  approximately  1.7  m/*. 


236  FINE-STRUCTURE  OF  PROTOPLASM 


II 


It  will  be  clear  from  the  following  that  a  similar  comparison  applies 
to  other  enzymes,  at  any  rate  so  far  as  the  prosthetic  group  is  con- 
cerned : 

Co-enzyme  of  carboxylase  (aneurinopyrophosphoric  acid)      44  atoms 

Co-enzyme  of  the  dehydrogenase  II  (nucleotide  of  nico- 
tinic acid  +  phosphoric  acid  +  nucleotide  of  adenine) 
hydrogenated -^ 7^      ,, 

Co-enzyme  of  the  yellow  respiratory  enzyme  (lactoflavin- 

dinucleotide  of  adenosine) 81       ,, 

Average  target  area  of  the  genes 1000      ,, 

The  apo-enzymes  of  these  desmoenzymes  are  not  freely  moving 
colloidal  particles;  Hke  the  genes  in  the  chromosome,  they  are  em- 
bedded in  the  submicroscopic  cytoplasmic  structure.  Only  by  auto- 
lysis can  they  be  liberated  under  certain  circumstances  and  made 
accessible  to  examination. 

The  comparison  between  gene  and  enzyme  may  not  be  merely  a 
superficial  one ;  one  might  at  least  try  to  probe  further.  It  is  scarcely 
to  be  wondered  at  that  the  gene  and  target  area  should  be  so  much 
larger  than  the  volume  of  the  apo-  and  co-enzymes,  if  it  be  remem- 
bered how  much  more  complicated  than  single  metabolic  reactions 
are  the  processes  of  development  controlled  by  the  genes.  Latterly  it 
has  become  ever  more  evident  that  this  control  is  exercised  chemically. 
When  a  mutation  takes  place,  these  chemical  processes  proceed 
differently.  It  is  therefore  not  wrong  to  assume  that  the  target  area 
acts  like  the  prosthetic  group  of  an  enzyme  and  that  the  controlled 
processes  follow  a  different  course  owing  to  changes  in  this  sensitive 
area.  As  this  area  contains  approximately  1000  atoms,  50  amino  acid 
residues  (with  on  an  average  20  atoms)  are  located  in  it,  allowing 
protein  chemistry  to  come  into  full  play  in  its  almost  unlimited  variety. 

According  to  the  hypothesis  propounded  here,  the  gene  would,  in 
the  terminology  of  Haase-Bessel(i936),  consist  of  a  carrier  (pheron) 
and  chemically  active  regions  (agon),  some  idea  of  the  dimensions  of 
which  can  be  formed  on  the  basis  of  the  target  theory.  Since,  however, 
every  colloidal  particle  of  the  apo-enzyme  carries  only  one  amicro- 
scopic  operative  group,  this  conformity  probably  cannot  be  assigned 
to  the  genes.  There  are,  maybe,  several  chemically  active  regions  in 
the  large  disc  of  Fig.  i26e.  This  would  explain  polyphaeny,  i.e.,  that 


2  NUCLEUS  237 

phenomenon  whereby  often  more  than  a  single  property  is  regulated 
from  one  locus  of  the  chromosome.  This  view  would  also  account  for 
minor  distinctions  in  activity  of  homologous  active  regions  in 
different  individuals  and  would  explain  polyallely.  There  must  be  some 
correlation  between  the  carrier  and  the  active  region,  in  the  same  way 
as  a  co-enzyme  can  only  develop  its  activity  in  close  conjunction  with 
the  apo-enzyme;  to  some  such  reciprocal  action  must  be  attributed 
the  different  ways  in  which  certain  phaena  are  actualized.  To  be  brief, 
the  carrier  model  serves  to  make  intelligible  most  of  the  knowledge 
acquired  by  research  into  heredity. 

There  being  good  grounds  for  assuming  that  the  leptonema  has 
a  double  structure,  falling  into  two  chromatids  after  conjugation,  the 
carrier  discs  may  be  represented  as  two  halves,  each  with  an  operative 
region  of  the  same  value  (Fig.  iz6e).  Of  these  two,  only  one  need 
be  struck  by  the  rays  for  the  origin  of  a  mutated  gamete,  since  the 
chromatids  are  separated  from  each  other  in  the  formation  of  tetra- 
cytes. 

The  advantage  which  the  carrier  hypothesis  possesses,  as  compared 
to  th.Q  fibrillar  hypothesis  developed  in  the  chapter  on  chromosomes,  is 
that  it  disregards  the  disputed  question  of  molecular  protein  structure 
and  nucleic  acid  intercalation ;  the  elementary  units  may  be  conceived 
of  either  as  fibrillar  protein  units  coiled  in  any  way,  or  as  globular 
proteins.  Then,  the  carrier  hypotheses  makes  the  gene  and  operative 
region  of  suhmicroscopic  dimensions,  whereas  the  fibrillar  hypothesis 
allows  the  gene  to  be  of  amicroscopic  size  represented  by  side  chain 
groups  of  polypeptide  chains.  In  the  first  edition  this  picture  un- 
warrantably simplified  the  exceedingly  complicated  facts.  On  the 
other  hand,  the  fibrillar  hypothesis  has  to  its  credit  the  plausibility  it 
confers  upon  the  shape,  cleavability  and  self-duplication  of  the  chro- 
mosomes. It  will  therefore  be  the  aim  of  research  to  reconcile  these 
two  hypotheses  to  a  concordant  theory  by  endeavouring  to  fathom 
the  suhmicroscopic  morphology  of  those  proteins  which  represent 
neither  their  extreme  fibrillar,  nor  an  independently  dispersed  globular 
form. 

Function  of  the  desoxyribose  nucleic  acids.  The  desoxyribose  nucleic 
acids,  which  were  at  first  thought  to  be  the  hereditary  substance  par 
excellence,  are  of  relatively  uniform  chemical  constitution  and,  in 
their  molecular  morphology,  lack  the  diversity  required  by  genetics. 


238  FINE-STRUCTURE  OF  PROTOPLASM  II 

Moreover,  Caspersson's  measurements  show  that  their  appearance  is 
transitory  and  that  they  afterwards  largely  disappear.  For  this  reason 
Kiesel  (1930,  p.  185)  stigmatizes  as  downright  paradoxical  the  fact 
that  cytologists  pay  such  conscientious  attention  to  an  unspecific 
material  like  the  desoxyribose  nucleic  acids,  yet  ignore  the  proteins, 
with  their  specific  structure,  merely  because  constituents  do  not  bind 
the  basic  dyes  used  for  staining  cell  nuclei.  Posternak  (1929)  goes  to 
the  length  of  relegating  the  nucleic  acids  to  the  rank  of  degradation 
products  of  organic  phosphorus  compounds ;  but  this  view  is  invalid- 
ated by  the  morphological  behaviour  of  the  desoxyribose  nucleic 
acids  during  karyokinesis  and  the  interesting  fact  that  many  co- 
enzymes consist  of  nucleotides  (co-dehydrogenase  II  and  others,  see 
p.  208). 

I  have  therefore  suggested  the  following  hypothesis  respecting  the 
function  of  the  desoxyribose  nucleic  acids :  The  genes  play  no  active 
part  during  karyokinesis,  but  2it&  passive  and  in  this  state  are  distributed 
by  some  process  among  the  daughter  nuclei.  Their  operative  groups 
must  therefore  be  reactive  in  the  active  nucleus  to  fulfil  their  task, 
but  they  must  be  screened  off  during  nuclear  division.  This  might  be 
effected  by  a  loose  binding  of  desoxyribose  nucleic  acid  groups.  It  was 
pointed  out  in  the  discussion  of  the  phosphatides  that,  in  the  respira- 
tory combustion  of  carbohydrates,  those  hydroxyl  groups  of  the  sugar 
which  are  not  subject  to  degradation  are  screened  by  phosphorylation 
and  are  thus  temporarily  protected.  Similarly,  the  phosphoric  groups 
of  the  nucleic  acids  might  for  the  time  screen  the  specific  groups  of 
the  genes  during  mitosis.  This  would  account  for  the  localization  of 
the  desoxyribose  nucleic  acids  in  certain  places  only,  viz.,  where  the 
genetically  active  groups  are  to  be  found  in  the  fundamental  protein 
substance.  They  thus  give  a  true  picture  of  the  distribution  of  genes 
as  proved  by  cytology.  There  is,  then,  nothing  "paradoxical"  about 
the  attempts  to  establish  the  distribution  of  the  desoxyribose  nucleic 
acids  in  the  chromonema  down  to  the  finest  detail,  since  these  are  the 
indicators,  as  it  were,  of  the  specific  groups  through  which  the  genes 
operate. 

The  assumption  that  the  desoxyribose  nucleic  acids  accumulate  only 
in  those  parts  which  contain  genes,  and  protect  their  active  groups, 
integrates  the  conflicting  views  championed  by  the  theorists  of  he- 
redity, one  being  founded  on  the  structural  chemical  specificity  of  the 


2  NUCLEUS  259 

proteins,  whereas  the  other  side  upholds  the  micro-morphological 
specificity  of  desoxyribose  nucleic  acid  distribution. 

The  fate  of  the  desoxyribose  nucleic  acids  in  the  cycle  of  nuclear 
division  favours  the  above  hypothesis.  When  the  nucleus  undergoes 
mitosis,  desoxyribose  nucleic  acids  are  built  up  (increasing  chromo- 
philic  tendency,  nucleal  reaction  and  ultraviolet  absorption).  In  pro- 
phase they  appear  to  be  embedded  in  the  chromomeres,  protecting 
the  specific  groups  during  the  cleavage  of  the  chromosomes.  When 
their  task  is  done,  most  of  the  desoxyribose  nucleic  acids  migrate 
from  the  chromomeres  to  the  matrix  of  the  chromosomes.  As  a  result, 
the  latter  absorbs  stain  to  the  full  extent  and  the  chromonemata  thus 
remain  invisible  during  metaphase  and  anaphase;  in  this  stage,  there- 
fore, nothing  at  all  can  be  known  of  their  exact  morphology.  In 
telophase  the  desoxyribose  nucleic  acids  are  for  the  most  part  degraded 
again.  The  chromosomes  become  transparent  and  it  can  be  seen  how 
the  chromonemata,  losing  their  stainability,  uncoil  (Heitz,  1935, 
p.  419)  and  disappear  in  the  nuclear  reticulum. 

There  are  parts  of  certain  chromosomes  which  are  called  hetero- 
chromatic,  where  the  desoxyribose  nucleic  acids  are  not  degraded  after 
cell  division.  When  genes  of  euchromatic  regions  of  the  chromosome 
come  into  the  neighbourhood  of  heterochromatin  by  crossing  over, 
their  manifestation  is  lost  (Prokofyewa  Belgorskaja,  1948;  Lewis, 
1950)  or  changed  from  dominant  to  recessive  (McClintoc,  1950). 
These  facts  indicate  a  screening  effect  of  desoxyribose  nucleic  acids 
on  genes. 

In  the  view  set  forth  here,  the  desoxyribose  nucleic  acids  play  a 
passive  part  in  heredity,  in  that,  although  they  protect  the  genes,  they 
do  not  participate  in  their  spontaneous  propagation.  By  contrast,  on 
the  analogy  of  the  enzymes  with  nucleotides  as  prosthetic  groups,  an 
active  part  may  be  assigned  to  them.  Caspersson  (1941),  applying  his 
ultraviolet  absorption  method,  discovered  that  vigorous  protein 
synthesis  is  initiated  wherever  nucleic  acids  appear;  notably  that 
histones  are  formed  as  the  result  of  the  reaction  of  nucleic  acids  of 
the  ribose  type  (absorption  maximum  at  2900  A)  and  globuhns  from 
that  of  the  nucleic  acids  of  the  desoxyribose  type  (absorption  maxi- 
mum at  2800  A).  Caspersson,  therefore,  declares  nucleic  acids  to  be 
necessary  to  any  and  every  biological  synthesis  of  proteins.  In  this 
case  the  desoxyribose  nucleic  acid  would  be  operative  in  the  redupli- 


240  FINE-STRUCTURE  OF  PROTOPLASM  II 

cation  of  the  chromonema  threads  during  cell  division;  but  then  the 
question  arises  as  to  why  protein  synthesis  is  only  necessary  in  the 
chromomeres  and  how  the  anucleal  parts  of  the  thread  augment  their 
protein  substance.  According  to  this  theory,  nucleic  acids  would  be 
also  temporarily  necessary  in  endomitotic  division  (Geitler,  1940; 
Berger,  1 941),  though  hitherto  this  has  evaded  observation.  In  what- 
ever way  the  function  of  the  nucleic  acids  as  synthesizing  protein 
enzymes  may  be  confirmed  or  modified  in  the  future,  it  will  not  irre- 
concilably contradict  the  propounded  hypothesis  of  screening,  as  in 
both  cases  nucleic  acids  must  be  assumed  to  accumulate  in  the  genet- 
ically active  regions,  as  a  result  of  which  the  chemical  activity  of  the 
genes  is,  for  the  time  of  multiplication,  paralyzed. 

ScHULTZ  (1941)  goes  one  step  further  and  calls  the  genes  nucleo- 
proteins,  that  is  to  say  nucleic  acid  compounds.  He  declares  that  the 
genes  and  nucleoproteins  have  in  common  the  properties  of  speci- 
ficity, auto-reproduction,  similar  distribution  in  the  cell  and  intimate 
relation  to  synthesis  processes.  There  is  this  much  to  be  advanced 
against  this  opinion :  that  the  activity  of  the  genes  only  begins  in  the 
reconstituted  nucleus,  whereas  in  that  state  the  nucleoproteins  dis- 
appear very  much  into  the  background.  Hence,  after  their  duplication 
and  division,  the  genes  must  be  independent,  to  a  large  extent,  of  the 
nucleic  acids,  making  their  influence  felt  in  the  growing  cell,  without 
having  the  character  of  nucleoproteins. 

Identical  auto-reproduction  of  nucleoproteins  (comparison  with  virus  protein). 
Whereas  in  this  monograph  the  genes  have  been  compared  morphologically 
and  chemically  with  enzymes,  the  literature  inclines  rather  to  draw  the 
analogy  with  the  rod-shaped  virus  particles,  notwithstanding  the  fact  that 
important  points  of  comparison  have  lost  cogency  since  the  invalidation  of 
the  classical  fibrillar  hypothesis  of  the  chromosome  structure.  Many  of  the 
varieties  of  virus  isolated  so  far  are  of  similar  chemical  composition  to 
chromatin:  they  are  «»r/i?o/)ro/f/«j-,  i.e.,  proteins  of  polypeptides  and  nucleic 
acids.  They  do,  it  is  true,  still  contain  lipids  and,  under  some  circumstances, 
also  small  amounts  of  polysaccharides.  Minute  amounts  of  lipid  have  also 
been  detected  in  chromosomes  (Hirschler,  1942),  though  as  a  rule  those 
components  are  disregarded  in  discussions  on  the  structure  of  chromatin. 
It  is  the  virus  of  tabacco  mosaic  disease  which  has  been  subjected  to  the  most 
exact  analysis,  as  Stanley's  method  (1958a)  provides  a  suitable  means  (by 
precipitations)  of  obtaining  it  in  a  crystallized  form.  It  contains  1.7  to 
5  %  of  nucleic  acid,  according  to  its  preceding  treatment.  If  the  nucleic 
acid  is  separated  off,  the  virus  protein  loses  its  pathogenic  properties  and 


2  NUCLEUS  241 

its  propagating  power.  This  proves  beyond  doubt  that  the  mysterious  auto- 
reproduction  of  the  crystalhzable  viruses  is  determined  by  nucleoproteins. 

There  is,  however,  a  fundamental  difference  as  compared  w4th  the  nucleo- 
proteins of  the  nuclei  of  the  cell,  the  virus  protein  showing  no  nucleal  reac- 
tion. Thus  the  phosphoric  compounds  in  the  viruses  are  of  the  ribosenucleic 
acid  type,  and  not  the  thymonucleic  acid  found  in  the  nuclei.  The  tobacco 
mosaic  virus  molecules  are  threadlike,  judging  by  their  birefringence  of  flow 
(Takahashi  and  Rawlins,  1933,  1935)  and  as  demonstrated  by  the  electron 
microscope  (Fig.  84c,  p.  126).  The  thread  molecules  unite  into  bundles 
liable  to  grow  to  microscopic  dimensions  and  then  appear  as  crystallized 
virus  protein.  This,  however,  is  not  in  a  true  crystalline,  but  rather  in  a 
mesomorphous  state,  for  the  X-ray  analysis  of  these  "crystals"  produces 
only  intramolecular  interferences  (Bernal,  1939)  and  does  not  reveal  any 
molecular  lattice  arrangement  of  the  virus  molecules  (Wyckoff  and  Corey, 
1936).  Thus,  like  liquid  crystals,  the  parallelized  thread  molecules  are  free 
to  revolve  and  shift  individually. 

The  structure  of  the  mesomorphous  virus  rodlets,  which  is  reminiscent  of 
that  of  the  chromonema,  favours  their  cleavability.  On  the  other  hand,  the 
reduplication  of  the  chromomeres  can  hardly  be  understood  as  a  mere  split- 
ting of  bundles  of  parallelized  molecules.  The  comparison  is  also  prejudiced 
by  our  complete  ignorance  as  to  how  the  nucleic  acids  are  distributed  in  the 
submicroscopically  visible  virus  molecule.  The  analogy  rests  merely  upon 
the  common  filiform  structure. 

It  is  the  mysterious  auto-reproduction  of  the  virus  protein  which 
encourages  comparison  with  the  chromonemata  in  the  chromosomes.  If 
only  a  trace  of  the  thread  molecules  of  tobacco  mosaic  virus  finds  its  way 
into  the  cells  of  the  tobacco  leaf,  they  fill  up  completely,  in  an  astonishingly 
short  time,  with  the  pathogenic  protein,  which  becomes  visible  as  birefrin- 
gent  rodlets,  whereas  the  protein  proper  to  the  cell  diminishes.  Thus,  when 
in  contact  with  virus  molecules,  non-virus  protein  becomes  virus.  This 
phenomenon  has  been  termed  autocataljtic  reproduction.  It  is  known  in  other 
compounds;  for  example,  small  amounts  of  trypsin  are  liable  to  change  a 
larger  amount  of  another  compound,  known  as  "protrypsin",  into  trypsin. 
Energy  is  required  for  the  spontaneous  reproduction  of  the  virus  protein 
and  this  is  supplied  by  the  living  cell.  There  can,  therefore,  be  no  reproduc- 
tion of  virus  outside  the  living  cell. 

It  is  tempting  to  regard  the  duplication  of  the  chromonemata  in  mitosis 
likewise  as  autocatalytic  reproduction;  but  we  should  not  forget  that  we 
have  simply  coined  a  term  for  what  is  at  present  an  inexplicable  process  and 
are  still  quite  in  the  dark  as  to  the  nature  of  the  "first  step"  which,  through 
contact  with  the  specific  nucleoproteins  of  the  chromonema,  has  autocatalyt- 
ically  to  be  transmuted  into  identical  nucleoproteins. 

The  electron  microscope  shows  that  the  rod-like  shape  of  the  tobacco 
mosaic  virus  (Wyckoff,  1947a)  is  an  exception.  The  majority  of  the  virus 
species  photographed  by  Wyckoff  (1947b)  have  a  pronounced  globular 


242  FINE-STRUCTURE  OF  PROTOPLASM  It 

shape  and  agglomerate  in  a  visible  crystal  lattice.  The  morphologicaE 
analogy  of  chromonemata  and  virus,  therefore,  is  no  longer  supported;  the- 
chemical  comparison  of  both  genes  and  viruses  with  enzymes  is  much  more 
convincing. 

Nucleus  and  cytoplasm.  Considered  from  the  morphological  stand- 
point, the  secret  of  karyokinesis  is  evidently  that  the  specific  protein- 
molecules,  which  serve  as  substratum  to  the  genes,  have  to  be  carefully 
transmitted  to  the  daughter  cells,  preferably  without  any  reciprocal! 
changes  of  position  along  the  chromatid.  Their  individuality  and 
specific  spatial  relationships  were  developed  in  the  course  of  phylo- 
genesis and  the  cytoplasm  is  not  capable  of  re-creating  them.  The  great 
riddle  of  heredity  therefore  still  is :  How  can  a  chromonema  of  such 
complicated  submicroscopic  and  amicroscopic  morphology  that  it  can 
never  be  produced  anew,  bring  forth  its  like  from  itself  by  longitudinal 
division?  This  mysterious  process  must  undoubtedly  take  place 
frequently  in  the  giant  chromosomes  of  the  Diptera,  which  are  bundles 
of  similar  chromonemata.  It  is  as  though  the  chromonemata  served^ 
as  it  were,  as  patterns  for  the  creation  of  their  like.  It  is  known  from 
the  evidence  of  the  asymmetrical  C  synthesis  (see  p.  207)  that  certain 
configurations  are  able  to  produce  essentially  the  same  morphological 
forms  in  the  amicroscopic  region,  but  the  refinements  of  this  process, 
and  its  mechanism  are  a  mystery.  For  here,  as  contrasted  with  the 
mode  of  action  of  the  enzymes,  it  is  not  merely  a  question  of  fitting 
a  key  to  a  lock,  but  of  how  the  key  produces  one  identical  to  it,  or 
the  lock  its  exact  like. 

If  we  take  the  specific  structures  to  be  a  given  fact,  we  come  to  an 
important  decision  as  to  the  morphological  signification  of  the 
nucleus.  The  gene-bearing  protein  threads  are  in  a  sense  self-contained 
and  irretrievable  structures  and  it  therefore  becomes  clear  why  they 
are  not  carried  along  by  the  cytoplasmic  stream,  but  are  localized  at 
a  given  spot.  There  they  are  withdrawn  from  the  turbulent  activity 
of  the  cell  and  perform  their  directive  and  formative  task  as  static 
centres. 

It  is  evident  from  the  heredity  of  cytoplasm  (Wettstein,  1957)  that 
specific  groups  must  also  occur  in  it.  These  special  structures,  how- 
ever, are  not  solitary,  for  parts  of  the  cytoplasm  are  similar  in  their 
behaviour  to  the  whole  cytoplast.  Even  fragments  of  the  eggs  of  sea- 
urchin  without  a  nucleus  can  undergo  a  certain  development  involving 


5  CHLOROPLASTS  243 

cell  division  (E.  B.  Harvey,  1936).  If,  on  the  other  hand,  portions  of 
chromosomes  are  removed  from  the  nucleus  while  division  is  going 
on,  the  result  is  a  serious  modification  of  the  hereditary  process. 

Although  the  cytoplasm  is  able  to  build  up  very  complicated 
molecular  systems,  its  architectural  capacities  are  to  some  extent 
limited,  for  it  cannot  produce  from  itself  the  protein  structures  of 
nuclei  and  plastids.  In  heterotrophic  organisms  it  even  lacks  the 
capacity  to  manufacture  relatively  simple  elementary  units,  which  are 
needed  for  protoplasmic  synthesis;  it  is  for  this  reason  that  these 
compounds  have  to  be  added  as  vitawins  to  the  culture  medium 
(ScHOPFER,  1936/37). 

As  a  rule,  all  such  problems  are  studied  in  their  purely  chemical 
aspect.  Yet  the  molecules  should  not  be  considered  only  as  chemical 
supporters  of  reactions,  but  also  morphologically  as  elementary  units 
of  the  high  polymeric  gel  frame.  In  the  cytoplasm,  this  texture  is  very 
finely  spun,  is  labile  and  is  involved  in  permanent  reconstruction. 
In  the  chromosomes  of  the  nucleus,  on  the  contrary,  it  has  far  greater 
density  and  a  certain  stabiHty  and  is  therefore  distinct  from  the  cvto- 
plasm,  not  so  much  on  chemical  as  on  structural  grounds. 


§  3.  Chloroplasts 

a.  Microscopic  Structure  of  the  Chloroplasts 

According  to  the  handbooks  of  Schurhoff  (1924,  p.  57),  Guil- 
LiERMOND,  Mangenot  et  Plantefol  (1933,  p.  1 5  8),  Sharp  (1934)  and 
KiJSTER  (1935a,  p.  288),  the  chloroplasts  are  microscopically  homo- 
geneous. They  are  described  as  hydrogels  and  both  Kuster  (1935a) 
and  HoFMEiSTER  (1940)  even  incline  to  the  view  that  they  are  in  a 
liquid  state  of  aggregation,  though  their  flattened  shape  and  their 
autonomic  transfiguration  (Senn,  1908)  would  discount  this  view.  As 
against  Kuster's  presentation  of  the  matter  (1935  a),  richly  docu- 
mented as  it  is,  publications  have  been  amassing  since  1935  arguing 
in  favour  of  a  microscopic  structure  in  living  chloroplasts  (Hubert, 
1935,  p.  369;  DouTRELiGNE,  1935;  Heitz,  1936a,  b;  Frey- Wyssling, 
1937c;  Geitler,  1937;  Weier,  1938).  All  the  investigators  mentioned 
find  the  chloroplasts  to  be  finely  granulated  and  for  this  reason  appeal 
to  Schimper's  (1885)  and  A.  Meyer's  (1883)  grain  theory.  Schimper's 


244  FINE-STRUCTURE  OF  PROTOPLASM  II 

doctrine  states  that  the  chloroplasts  consist  of  a  colourless  stroma,  in 
which  minute  granules,  lying  on  the  boundary  of  microscopic  visi- 
bility, are  embedded;  and  these  contain  the  green  pigment  (Binz, 
1892).  Colloid  research,  however,  had  utterly  refuted  this  view,  for 
the  methods  employed  by  colloid  optics  seemed  to  show  that  all  living 
components  of  the  cells  are  fluid  (Kuster,  1935  a,  p.  290),  optically 
empty  (Guilliermond,  1930)  and  microscopically  homogeneous. 
Consequently,  any  kind  of  microstructure  made  visible  in  some  way 
or  other  was  said  to  be  a  form  of  precipitation,  structure  of  coagu- 
lation, artificial  product  or  artefact.  The  granular  structure  of  chloro- 
plasts suffered  the  same  fate. 

Photographs  taken  of  living  cells  provided  the  evidence  for  the 
refutation  of  the  theory  that  the  grains  in  chloroplasts  are  a  product 
of  precipitation.  The  first  microphotographic  document  may  be  said 
to  have  come  from  Heitz  (1932),  who  photographed  chlorophyll  grains 
next  to  a  living  nucleus  in  the  leaf  stem  of  Victoria  regia.  Doutreligne 
(1935)  considers  photography  in  red  light  an  especially  suitable  means 
of  proving  beyond  doubt  the  inhomogeneous  distribution  of  chloro- 
phyll in  the  plastids.  Her  objects  are  mosses  {Mniuw),  Vallisneria, 
Cahomha  and  Myriophyllum.  Wieler  (1936)  identifies  the  grains  in  a 
variety  of  Selaginella.  But  the  most  detailed  work  is  undoubtedly  that 
of  Heitz  (1936a, b),  which  contains  microphotographs  of  a  great 
number  and  variety  of  plants.  The  grains  are  decidedly  identified  in 
mosses  {Physcomitrium^  Hypnum,  AInium,  Funaria),  vascular  crypto- 
gams, very  many  Monocotyledons  and  Dicotyledons.  Most  authors 
preferred  single-layer  leaves,  such  as  mosses  and  fern  prothallia,  for 
their  observations  and  Doutreligne  avoids  even  the  source  of  error 
involved  in  the  use  of  an  embedding  medium,  using  transparent 
water-plants.  Heitz  disdains  this  precaution  and  includes  sections  of 
living  tissue  in  his  investigations.  One  of  the  things  he  notices  in  the 
leaf  of  Agapanthus  umhellatus  is  that  certain  chloroplasts  are  liable  to 
be  damaged  (though  the  cause  is  not  known)  and  in  that  state  their 
granular  structure  is  far  more  clearly  apparent  than  in  the  undamaged 
specimens.  Evidently  this  is  a  kindred  case  to  the  fixation  of  the 
nuclei,  where  a  barely  visible  structure  in  the  live  state  is  coarsened 
in  death  and  the  blurred  outlines  of  the  optically  merging  structural 
components  become  more  sharply  defined.  Seeing  that  so  many 
observers  have  described  the  plastids  as  microscopically  homogeneous. 


3  CHLOROPLASTS  245 

we  are  compelled  to  assume  that  the  grana  are  often  submicroscopic 
and  only  become  visible  by  coarsening.  Experience  of  nuclear  struc- 
tures would  seem  to  imply  that,  again,  it  is  not  a  matter  of  artefacts 
in  this  case,  but  rather  of  pre-formed  structures  which,  lying  below 
microscopic  resolving  power,  or  exhibiting  no  optically  demonstrable 
phase  boundaries,  have  become  visible.  The  second  alternative  at  the 
same  time  shows  why  the  chloroplasts  appear  to  be  optically  empty 
in  the  ultramicroscope  (Guilliermond,  1930). 

Heitz  declares  that  the  grana  vary  in  size  from  0.5  to  2  /<  and  that 
the  size  is  specific  to  the  species.  As  against  this,  the  granules  in  light 
plants  are  always  found  to  be  smaller  than  in  shade  plants ;  accordingly, 
the  granular  size  increases  from  the  upper  side  of  the  leaf  (palisades) 
towards  the  underneath  (spongy  tissue).  The  grana  are  especially  large 
and  distinct  in  the  chloroplasts  of  the  green  fruit  of  Polygonatum 
(Menke,  1934a,  who,  however,  calls  them  artificial  products;  Weber, 
1936). 

The  evidence  that  the  grana  are  not  globules,  but  platelets,  is  im- 
portant (Heitz,  1936b).  In  the  side  view  of  the  flat  discs  of  chloro- 
plasts they  look  like  dense  streaks  (cf.  Fig.  130b,  p.  25  5).  The  Heitz 
microphotographs  reveal  no  localization  of  the  grana  in  the  periphery; 
this  conflicts  with  the  observations  made  by  Priestley  and  Irving 
(1907),  ZiRKLE  (1926)  and  Wieler  (1936),  according  to  which  the 
colouring  matter  is  accumulated  in  the  cortex  and  is  lacking  in  the 
centre. 

As  only  the  grana  contain  the  pigment,  they  alone  show  the  fluor- 
escence of  chlorophyll  (Heitz,  1936b;  Metzner,  1937),  appearing 
bright  red,  whereas  the  stroma  remains  dark.  In  this  way  the  hetero- 
geneous distribution  of  the  chlorophyll  can  be  proved  indubitably, 
even  in  what  appears  to  be  optically  homogeneous  chloroplasts. 

MoMMAERTS  (1938)  is  of  Opinion  that  the  minute  green  particles 
occurring  in  infusions  of  ground  leaves  (Noack,  1927)  are  isolated 
grana,  which  he  subjects  to  chemical  analysis.  Gjr.anick  (1938)  and 
Menke  (1938b),  however,  succeeded  in  obtaining  undamaged  chloro- 
plasts from  the  leaves  for  chemical  examination. 

Strugger  (1950)  has  discovered  that  the  small  amoeboid  un- 
diflerentiated  proplastids  which  exist  in  dividing  meristematic  cells 
already  contain  a  single  primary  granum.  This  minute  disc  multiplies 
by  auto-reproduction.  When  two  grana  are  formed  in  this  way,  the 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


246 

proplastid  divides  and  each  part  is  provided  with  one  of  them.  This 
scheme  of  multipUcation  goes  on  as  long  as  there  is  cell  division  and 
the  number  of  proplastids  increases  in  the  young  cells.  Only  when 
their  definite  number  has  been  reached  and  the  cell  differentiates  do 
the  proplastids  evolve  to  mature  plastids.  Then  the  self-reproduction 
of  the  grana  in  the  expanding  plastid  proceeds  in  a  very  characteristic 
way.  After  splitting  parallel  to  the  disc-plane  of  the  granule,  the  two 
new  platelets  remain  juxtaposed,  split  further  and  pile  up,  so  that 
cyHnders  of  grana  result  with  their  axes  perpendicular  to  the  surface 
of  the  flat  plastid.  It  is  due  to  this  arrangement  that  the  green  colour 
of  the  grana  is  visible  in  the  microscope  in  spite  of  their  minute  thick- 
ness; in  fact,  it  is  not  a  single  granule,  but  a  pile  of  grana  that  is 
observed. 

The  grana  produce  chlorophyll  only  in  the  hght.  If  they  contain 
but  a  trace  of  this  pigment,  they  can  easily  be  discovered  in  the 
fluorescence  microscope.  Before  any  chlorophyll  is  synthesized  in  the 
proplastids,  they  must  be  made  visible  by  staining  with  rhodamineB. 

b.  Molecular  Constituents  of  Ch  lor  op  lasts 

Proteins,  lipids  and  the  pigments  chlorophyll  a,  chlorophyll  b,  as 
also  carotene  and  xanthophyll,  which  are  given  the  collective  name 
of  carotenoids,  go  to  the  making  of  the  chloroplasts.  Menke  finds 
47.7%  of  protein  and  37.4%  of  lipids  in  the  chloroplasts  of  spinach 
leaves.  They  are  rich  in  ash  (7.8%)  and  contain  about  7.7%  of  chloro- 
phyll (Menke,  1940b).  Half  the  lipids  consist  of  fats,  20%  of  sterines, 

TABLE  XXIV 
ANALYSIS  OF  CHLOROPLASTIC  MATTER  OF 

Spinacia  ohracea 

IN  %  BY  WEIGHT  (rABINOVITCH,   1 94 5) 


Menke 
(1938b) 

Chibnall 
(1939) 

BOT 

(1939) 

COMAR 
(1942) 

Lipids 

Protein 

Ash 

Residue 

37-4 

47-7 
7.8 

7-1 

25.1 
39.6 
16.9 
18.4 

26-32 

42-54 

16-25 

34 
54 

7 

Chlorophyll  .... 

7-7 

3  CHLOROPLASTS  247 

16%  of  raw  wax  and  2-7%  of  phosphatides  (Menke  and  Jacob, 
T942).  Other  authors  find  similar  values  as  shown  in  Table  XXIV 
(Frey-Wyssling,  1949b). 

There  is  no  intrinsic  chemical  difference  between  the  chloroplastic 
protein  and  cytoplasmic  protein  of  spinach  (Noack  and  Timm,  1942; 
TiMM,  1942) ;  the  former  contains  a  little  more  histidine  and  somewhat 
less  lysine  and  glutamic  acid.  According  to  Noack  (1930),  the  cata- 
lytically  active  iron  (Noack  and  Liebich,  1941;  Liebich,  1941)  is 
bound  by  adsorption  in  the  stroma.  Mommaerts  was  inclined  to  view 
the  grana  as  the  containers  of  the  iron,  but  the  grana  he  used  for  his 
Avork  were  not  perfectly  pure. 

Microchemically,  the  lipid  content  of  the  stroma  has  been  definitely 
proved  both  by  the  myelin  forms  produced  by  Weber  (1933)  and 
Menke  (1934a)  from  chloroplasts,  and  by  the  vital  staining  of  the 
grana  by  the  lipid  dye  rhodamine  B  introduced  by  Strugger  (1936/37). 

The  formation  of  myelin  depends  upon  the  following  two  con- 
ditions: firstly  the  lipid  molecules  must  be  liberated  from  any  loose 
linkage  to  the  protein  frame  so  that  they  can  "coalesce";  secondly, 
they  must  possess  not  only  lipophiHc,  but  also  hydrophiHc  end  groups 
which,  as  seen  on  p.  5  6,  cause  an  infiltration  of  water.  The  presence 
■of  water  alone  does  not  initiate  the  emigration  of  the  plastid  myelin, 
from  which  fact  one  may  infer  that  the  lipids  in  the  chloroplasts  have 
no  free  hydrophilic  groups,  but  that  these  are  screened  ofT,  for  instance, 
by  the  formation  of  a  hpoprotein  complex.  If,  however,  they  are 
liberated  by  saponification  in  a  shghtly  alkahne  medium  (NH4OH), 
myelin  is  formed  at  once. 

We  have  fewer  exact  data  on  the  chemical  constitution  of  the 
grana.  If  they  do  not  serve  merely  as  energy  traps,  but  are  at  the  same 
time  the  loci  of  COo  assimilation,  they  must  contain  proteins  in 
addition  to  pigments.  Euler,  Bergman  and  Hellstrom  are  of 
opinion  that  this  system  is  ten  to  twenty  times  the  size  of  a  chlorophyll 
molecule.  Mestre  (1930)  calls  the  compound  between  chlorophyll, 
lipid  and  protein  the  "phyllochlorine  complex".  Stole,  borrowing 
Willstatter's  nomenclature  (Willstatter  and  Rohdewald,  1934), 
called  the  hypothetic  compound  "chloroplastin  simplex".  (It  should 
be  noted  that  in  this  term  the  word  "plastin"  does  not  cover  the  sense 
in  which  the  older  authors  employed  it;  they  used  it  to  denote  the 
stroma  protein,  whereas  it  is  here  applied  to  the  grana  protein.)  Stole 


248  FINE-STRUCTURE  OF  PROTOPLASM  II 

and  Wiedemann  (1941)  succeeded  in  producing  this  protein  con- 
taining chlorophyll  in  its  pure  state.  They  call  the  resulting  chromo- 
protein  "chloroplastin'".  Its  molecular  weight  in  the  ultracentrifuge  was 
found  to  be  roughly  five  million.  This  compound  was  obtained  from 
thirty  different  plant  species;  it  shows,  as  do  the  haemoglobins  of 
various  vertebrata,  slight  differences,  according  to  the  plant  species. 
The  chloroplastin  of  Aspidistra  contains  about  69%  of  protein 
(plastin),  21%  of  lipids  and  8%  of  pigments,  6%  of  which,  approxi- 
mately, is  chlorophyll.  Menke  (1940b),  finding  7-8%  of  chlorophyll 
in  toto  in  the  chloroplasts,  doubts  whether  the  chloroplastin  contains 
a  pure  chromoprotein.  As,  however,  the  chloroplastin  is  free  from 
iron,  it  may  nevertheless  be  assumed  that  it  does  not  contain  all 
essential  constituents  of  the  stroma. 

We  are  better  informed  as  to  the  structure  of  the  pigments  in  chloro- 
plasts than  on  the  molecular  structure  of  the  protein.  One  reason  for 
this  is  that  the  pigments  are  easier  to  isolate,  another  being  that  they 
are  of  considerable  physiological  interest. 

The  chlorophyll  molecule  C55H7205N4Mg  is  like  a  tadpole  in  appearance, 
having  a  large  head  and  a  long  tail  (Fig.  1 27).  The  head  consists  of  four 
rings  of  pyrrole  linked  together  to  form  one  porphin  ring.  This  harbours 
a  magnesium  atom  in  the  centre  and  at  its  periphery  are,  in  chlorophyll  <2, 
four  methyl,  one  ethyl  and  one  vinyl  groups  and  also  three  oxygenic  side 
chains,  viz.,  one  butyric  acid,  one  acetic  acid  and  one  formaldehyde  residue. 
The  two  latter  are  interconnected  laterally  (shown  by  9  and  10  in  Fig.  127); 
an  isocyclic  ring  is  therefore  formed,  to  which  has  been  ascribed  the  process 
of  assimilation  on  account  of  its  labile  acetic  acid-ester  configuration 
(Fischer,  1935 ;  Stoll,  1936).  The  acid  groups  are  esterified  with  methanol 
and  phytol  (CgoHggOH).  Chlorophyll  b  differs  from  chlorophyll  a  merely 
by  the  substitution  of  the  methyl  group  at  the  3.  C  atom,  shown  by  a  circle 
in  Fig.  127,  by  a  formaldehyde  residue  -CH  =  O. 

There  are  ten  double  bonds  in  the  polycyclic  ring;  they  are  conjugated, 
which  means  to  say  that  they  alternate  regularly  with  simple  bonds.  Systems 
of  conjugated  double  bonds  like  this  cause  absorption  of  light  in  short-wave 
light.  Strong  absorption  in  the  far  red  is  furthermore  induced  by  the  effect 
of  porphin  ring  formation  upon  the  system  of  unsaturated  bonds.  The 
presence  of  magnesium  only  slightly  shifts  the  position  of  the  various 
absorption  bands  of  this  system,  but  it  does  affect  their  intensity.  It  is  there- 
fore responsible  for  the  green  colour  of  chlorophyll.  If  the  magnesium  is 
removed  from  the  porphin  nucleus,  the  brilliant  colouring  fades  and  changes 
to  a  dirty  olive  brown  (phaeophorbids).  The  sUght  morphological  difference 
as  between  chlorophyll  h  and  chlorophyll  a  suffices  to  change  the  bluish 


CHLOROPLASTS 


249 


XH-CH3 

CH2 

\h-CHj 


CH2 


CH, 


CH2 
CH2 


o 

Cm 


CH-CH^ 


CH: 


CH2 
II     ^ 

CH 

I 

CH2 

O 
I 

I        CH2 

-CH         I 

-C<         I  II  '/^CH3 

)C-N^  N-Cs^ 

HCfi  Mg  rCH 

C=N  N—C. 

/        I  \ 

ICH3 


H3C. 


N,/ 


H3C 


HC 


H2  H2 

c-c^ 
j:h2 

-CH, 


>c^c. 


■  C^ 
II 
■CH 


CH3 


o=c- 


A 


HC^ 

M 
HC^f^y 

II 
HC^CH 


CH, 


green  colour  which  distinguishes  chlorophyll  a  by  increased  absorption 
in  the  blue,  to  a  yellowish  green  shade. 

The  head  of  the  chlorophyll  molecule  has  a  hydrophilic  character  owing 
to  the  nitrogen  atoms  of  the  four  pyrrole  rings  and  the  co-ordinately  bound 
magnesium.  Its  long  phytol  tail,  on  the  other  hand,  is  lipophilic;  there  is, 
therefore,  in  this  pigment  a  clavate  molecule  with  a  pronounced  lipophilic 
pole  and  a  lipophobic  pole. 

By    contrast,    the   carotenes, 
which  are  unsaturated  hydro- 
carbons of  the  empiric  formula 
C40H56,   are   completely   lipo- 
phiHc.    The  xanthophylls,  on 
the  contrary,  may  contain  as 
many  as  six  hydroxyl  groups 
and  are  therefore  not  so  decid- 
edly hydrophobic.  Whereas  it 
was    formerly    held    that    the 
carotenoids  are  dissolved  in  the 
chloroplastic     lipids,     Menke 
(1940c)  is  of  opinion  that,  like 
chlorophyll,     they     combine 
with    protein    molecules     to 
form  chromoproteins.  As  far 
as  carotene  is  concerned,   an 
argument  against   this    hypo- 
thesis is  provided  by  the  fact 
that,  unUke  xanthophyll   and 
chlorophyll,  this  pigment  can  ch3Ch.^ 
be  extracted  by  benzene  and 
other  organic    solvents    from 
dry  leaf  powder  without   any 
preliminary  chemical  action. 

The  structural  formula  of 
the  ^-carotene  contained  in  the 
leaves  is  represented  in  Fig. 
127.  It  is  a  chain  of  conjugated 
double  bonds  which  cause  the 

blue  absorption  and,  therefore,  the  yellow-to-orange  colour;  it  has  me- 
thylic  lateral  groups  and  two  terminal  rings  of  six  members.  The  consti- 
tution of  /3-carotene  is  a  matter  of  paramount  importance  to  vitamin 
research,  for  the  break-down  of  the  double  bond  occupying  a  middle 
position  in  the  chain  (shown  by  ->  in  Fig.  127)  and  addition  of  water 
produce  two  molecules  of  vitamin  A  (Karrer,  193  5).  ^-carotene  is  optically 
inactive.  In  the  a-carotene  in  carrot  roots  and  in  palm  oil  the  double  bond 
between  the  C  atoms  marked  in  Fig.  127  5  and  6  in  one  of  the  six-membered 


WCv 


,H 


■CH 
II 


o 


HC 
II 

HC^QI^ 
II 


CH3 


"\/y/^V 


H3CS 


CH3 
AH3 


CH3] 


CH=CH2         " 


HC^ 

hI 

\    yC=C 

H3C^  ^c-c^ 


Chlorophyll     a 


fi- 


H2  H2 


Carotene 


Fig.  127.  Molecular  structure  of  the  pigments  in 
the  chloroplast  (it  should  be  noted  that  it  is  not 
certain  whether  the  carotenes  possess  the  trans- 
configuration or  the  cis-configuration  drawn  here) . 


250  FINE-STRUCTURE  OF  PROTOPLASM  II 

rings  is  shifted  to  a  place  between  atoms  4  and  5 ;  as  a  result,  the  C  atom 
marked  6  becomes  asymmetrical  and  the  molecule  optically  active.  In  the 
case  of  y-carotene  the  six-membered  ring  is  open,  the  bond  between  C 
atoms  I  and  6  lacking.  Small  to  larger  quantities  of  a-  and  y-carotene  are 
often  present  in  leaves,  as,  for  example,  a-carotene  in  the  leaf  of  Daucus 
Carota  (Mackinney  and  Milner,  1933)  and  y-carotene  in  Cuscuta  salina 
(Spoehr,  1935,  p.  193).  To  these  three  carotenes  may  be  added  lycopene 
and  others,  all  of  which  are  distinct  from  each  other  by  virtue  of  their  melting 
points  and  absorption  spectra  (Smith,  1936).  Like  ^-carotene,  a-carotene 
and  y-carotene  are  provitamins  for  the  growth  factor  A,  but  they  produce 
only  half  its  effect.  This  is  because  the  two  symmetrical  halves  of  ^-carotene 
have  exactly  the  same  chemical  constitution  as  vitamin  A,  whereas,  owing 
to  the  slight  morphological  changes  to  one  of  the  terminal  six-membered 
rings  of  a-  and  y-carotene,  only  the  unchanged  half  of  the  structural 
formula  can  produce  vitamin  molecules.  With  lycopene  both  the  terminal 
six-membered  rings  are  open,  which  is  why  this  carotenoid,  known  chiefly 
in  tomato,  has  no  vitamin  A  activity  at  all  (Karrer,  1935;  Kuhn,  1937). 
This  illustrates  most  aptly  the  powerful  influence  of  the  special  morphology 
of  the  molecules  upon  the  specific  reactions  in  the  organism. 

There  are  also  numerous  yellow  xanthophylls  C4  3H56-q(OH)jj.  Except  for 
the  introduction  of  OH  groups  at  certain  places  in  the  structural  formula, 
their  molecules  are  built  up  in  the  same  way  as  the  orange  carotenes.  Crypto- 
xanthin  possesses  one  of  these  hydroxyl  groups  at  the  C  atom  marked  3, 
whereas  in  the  zeaxanthin  from  the  grains  of  maize  both  six-membered 
rings  are  substituted  in  this  way.  There  are  small  amounts  of  both  compounds 
in  leaf  xanthophyll,  though  it  mainly  consists  of  another  xanthophyll  with 
two  OH  groups  viz.,  lutein,  which  has  been  known  for  some  time  from  egg 
yolk.  It  comprises  50-60%  of  the  xanthophyll  (Spoehr,  1935)  in  the  leaves 
of  spinach,  gourd,  sunflower,  lettuce,  barley  and  other  leaves.  The  OH 
groups  cause  the  beginning  of  light  absorption  to  shift  somewhat  towards 
the  shorter  wavelengths  as  compared  to  ^S-carotene.  In  carotenoids  with 
three  and  more  oxygen  atoms,  epioxide-bridges  have  been  discovered 
(Karrer,  1946). 

According  to  the  foregoing  considerations,  the  fundamental  principle  of 
the  molecular  structure  of  all  carotenoids  is  a  relatively  short  chain  of  un- 
saturated hydrocarbon  with  conjugated  double  bonds.  Minor  variations 
in  this  type  of  structure  give  rise  to  the  numerous  carotenoids  and  hydroxyl 
substitution  produces  the  various  xanthophylls  (Smith,  1937). 

As  opposed  to  this  variability  on  the  part  of  the  yellow  pigments,  in 
higher  plants  we  have  the  two  green  pigments,  chlorophyll  a  and  h,  with 
their  strikingly  unvarying  constitution.  Thanks  to  this,  the  percentage  of 
the  two  chlorophyll  pigments  contained  in  leaves  can  be  determined  by  the 
quantitative  method  of  spectral  analysis  (Heierle,  1935 ;  Sprecher,  Heier- 
LE  and  Almasi,  1935).  The  yellow  leaf  pigments  lend  themselves  to  such 
analysis  only  if  they  are  composed  of  ^-carotene  and  lutein  and  nothing 


3  CHLOROPLASTS  25I 

else.  By  this  method  Heierle  (1935)  finds  for  Amersfoort  tobacco  at  the 
end  of  July,  for  instance,  per  square  metre  of  leaf  surface:  chlorophyll  a 
147.5  n^g'  chlorophyll  ^53.8  mg,  carotene  37.2  mg  and  xanthophyll  17.8 
mg.  This  represents  the  famiUar  molecular  ratio  of  3 :  i  for  the  green  pig- 
ments and  roughly  one  molecule  of  carotenoids  to  every  two  chlorophyll 
molecules  (about  1/3  molecule  of  carotene  and  2/3  molecule  of  xanthophyll). 
By  means  of  chromatographic  adsorption  Seybold  (1941)  made  comparative 
measurements  and  found  that  the  molar  ratios  just  given  do  not  invariably 
exist  between  the  pigments.  Chlorophyll  h,  for  instance,  may  be  present  in  far 
smaller  quantities,  or  may  not  occur  at  ail,  this  applying  notably  to  certain 
algae  (Seybold,  Egle  and  Hulsbruch,  1941).  Instead,  those  groups  of  algae 
may  contain  other  varieties  of  the  green  pigment,  such  as  chlorophyll  c  or 
chlorophyll  (/(Aronoff,  1950). 

c.  Suhmicroscopic  Structure  of  the  Chloroplasts 

State  of  chlorophyll  in  the  chloroplast.  Granular  chlorophyll  and  mole- 
cular chlorophyll  solutions  (in  acetone,  alcohol,  etc.;  Fig.  128a)  show 
red  fluorescence  when  exposed  to  light  rays ;  the  fluorescence  is  pro- 


AL  (OhJs 

c 

Fig.  128.  Chlorophvll  molecule,  a)  Molecular  dispersion;   b)  colloid  particle;  <r)  adsorbed 

on  a  monomolccular  lecithin  layer. 

portional  to  the  intensity  but  independent  of  the  wavelength  of  the 
incident  light  (Wassink,  Vermeulen,  Reman  and  Katz,  1938).  On 
the  other  hand,  colloidal  chlorophyll  solutions  do  not  fluoresce;  they 
can  be  obtained  by  the  dilution  of  molecular  solutions  with  water.  The 
chlorophyll  molecules  then  assemble,   on  account  of  their  partial 


252  FINE-STRUCTURE  OF  PROTOPLASM  II 

hydrophobic  bias,  to  form  submicroscopic  droplets  (Fig.  128b).  Then 
they  lose  their  fluorescence,  obviously  because,  owing  to  their  as- 
sociation, the  molecules  reciprocally  influence  each  other.  Noack 
(1927)  was  thus  able  to  show  that,  contrary  to  earlier  ideas,  the  chloro- 
phyll cannot  be  present  in  a  colloidal  state  in  the  plastids.  The  fluor- 
escence persists,  however,  if  the  chlorophyll  is  adsorbed  in  a  mono- 
molecular  layer  on  aluminium  hydroxide  or  globulin.  With  Noack, 
therefore,  we  may  conclude  that,  in  the  molecular  state,  the  chloro- 
phyll is  present  in  the  plastids  as  monon?olecular  films.  Fluorescence  is 
heightened  if  a  monomolecular  layer  of  lecithin  is  interposed  between 
the  chlorophyll  and  the  adsorbant.  The  assumption  must  be  that  this 
makes  the  chlorophyll  molecules  yet  more  independent  of  each  other 
so  that  there  is  much  less  mutual  interference  in  their  fluorescence. 
Hubert  (1935)  devised  a  scheme  by  which  the  molecular  morphology 
of  this  phenomenon  is  clarified  (Fig.  128c).  The  hydrophilic  pole  of  the 
lecithin  is  orientated  with  respect  to  the  hydrophilic  adsorbant, 
whereas  the  hydropobic  phytol  tail  stands  parallel  to  its  hydrophobic 
chains,  making  the  porphin  system  in  Fig.  128  visible  in  profile.  In 
this  state  the  chlorophyll  molecule  may  best  be  compared  to  a  signet, 
the  phytol  chain  being  the  stem  or  handle,  and  the  porphin  ring  the 
seal. 

As  a  counter  to  these  established  facts,  K.  P.  Meyer  (1939)  states 
that  his  colloidal  chlorophyll  solutions  do  fluoresce;  but  his  method 
of  extraction  is  such  that  the  chlorophyll,  instead  of  being  isolated, 
is  dispersed  in  its  natural  association  with  protein  and  lipids.  In  at- 
tempts to  produce  multimolecular  films  from  chlorophyll,  globulin 
and  lecithin,  Nicolai  and  Weurman  (1958)  obtained  non-fluorescing 
systems  of  layers. 

The  state  of  the  chlorophyll  in  the  living  plastids  may  further  be 
revealed  by  the  position  of  its  absorption  bands  (Seybold  and  Egle, 
1940).  For  this  work  the  Baas  Becking  school  favoured  light  ab- 
sorption in  red.  Living  foliage  exhibits  an  absorption  maximum  at 
6810  A  (Baas  Becking  and  Koning,  1934;  Hubert,  1935).  But  in 
chlorophyll  isolated  from  the  plant,  this  absorption  band  shifts  in 
a  varying  degree  towards  the  region  of  the  shorter  wavelengths.  The 
effect  is  most  marked  in  hexane,  where  the  displacement  amounts  to 
nearly  200  A,  for  Wakkie  (1935)  finds  the  absorption  maximum  in 
this  solvent  at  6620  A.  This  faces  us  with  the  task  of  seeking  states 


CHLOROPLASTS  253 


of  the  chlorophyll  in  which  its  absorption  comes  nearest  to  that  of 
the  living  plastids,  which  would  permit  us  to  predict  how  it  will 
behave  in  the  chloroplast. 

According  to  Kundt's  law,  the  position  of  the  absorption  bands 
is  governed  by  the  refractive  index  of  the  solvent,  in  the  sense  that 
the  hio-her  the  refractive  index,  the  more  will  the  bands  shift  towards 
the  long-wave  region.  This,  however,  applies  only  to  a  limited  extent 
to  chlorophyll,  viz.,  only  in  so  far  as  solvents  of  equal  chemical  value 
are  compared.  Thus  Wakkie  finds  four  different  series  of  substances 
to  which  Kundt's  law  applies ;  they  are :  i .  purely  lipidic  liquids  like 
heptane,  carbon  tetrachloride,  benzene;  2.  ethyl  ether  and  ketones; 
3.  alcohols;  4.  water,  glycerol.  In  the  lipidic  solvents  the  red  ab- 
sorption band  is  shifted  farthest  from  its  natural  position  towards 
yellowy  in  the  ketones  somewhat  less  so  (e.g.,  acetone  6640  A)  and  in 
the  alcohols  sdll  less  so  (ethyl  alcohol  6665  A,  benzyl  alcohol  6720  A). 
Hence,  the  more  hydrophilic  the  solvents,  the  closer  is  the  approach 
to  natural  conditions  in  the  leaf.  The  position  of  the  absorption  bands 
cannot,  therefore,  be  improved  by  adding  lipids  (Na  oleate)  to 
alcoholic  solutions;  on  the  contrary,  it  is  worsened  by  20  A.  Solutions 
in  water  most  nearly  approximate  the  natural  green  of  leaves  (6720  A); 
despite  the  fact  that  the  chlorophyll  is  dissolved  coUoidally,  and  not 
molecularly,  in  this  lipophobic  solvent,  the  effect  of  the  increased 
hydrophilic  bias  is  to  strengthen  the  resemblance  to  the  conditions 
existing  in  the  living  chloroplast.  Since  it  does  not  seem  possible  to 
find  solvents  in  which  chlorophyll  displays  the  same  absorption 
maximum  as  in  the  leaf,  it  must  be  assumed  that  the  chlorophyll  is 
not  dissolved,  but  chemically  combined  in  the  chloroplast. 

Birefringence.  Very  important  criteria  are  supplied  by  the  bire- 
fringence of  the  chloroplasts  and  phaeoplasts.  It  was  discovered  by 
ScARTH  (1924)  and  was  found  to  be  widespread  by  Kuster  (1933, 
1935b),  Menke  (1934a,  b,  1943),  Ullrich  (1936a)  and  Weber  (1937)- 
The  Weber  school  rightly  ascribes  the  optical  anisotropy  to  the  lipidic 
substances,  which  can  be  made  to  emigrate;  they  then  produce 
striking  birefringent  myelin  forms  (Weber,  1933;  Menke,  1934a). 

KiJSTER  (1933,  1937)  and  Menke  (1934b)  discovered  the  lamelli- 
form  chloroplasts  of  Mougeotia,  Mesocarpus,  Spirotaenia,  Spirog^fra  and 
other  algae  to  be  clearly  birefringent  in  profile  and  in  cross-section, 
viz.,  negative  with  reference  to  the  thickness  of  the  plastids;  the  top 


2$4 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


view  is,  on  the  contrary,  isotropic.  Given  these  facts,  either  the  entire 
chloroplasts,  or  the  single  grana  must  be  optically  uniaxial  with 
negative  birefringence. 

It  can  be  shown  that  the  birefringence  of  the  big  chloroplasts  in 


Fig.  129.  Layer  birefringence  of  chloroplasts  of  Mougeotia  (from  Frey-Wyssling  and 
Steinmann,  1948).    Abscissa:  refractive  index  nj  of  the  imbibition   liquid.    Ordinate: 

retardation  T  in  m/<. 

Conjugatae  algae  is  a  form  birefringence  (Frey-Wyssling  and  Stein- 
mann, 1948).  The  flat  chloroplasts  of  the  2Xg2.MoMgeotia  were  used  for 
this  study.  They  have  the  shape  of  a  rather  thick  plate  which  is  as 
long  and  wide  as  the  whole  cell  in  which  it  is  located.  Further,  they 
appear  homogeneous  in  the  light  microscope,  no  grana  having  been 


3  CHLOROPLASTS  255 

detected  as  yet.  When  such  a  chloroplast  is  fixed  in  Zenker's  solution, 
picric  acid/HgCL,  or  Flemming's  solution  and  is  then  observed  in 
mixtures  of  acetone  and  methylene  iodide  with  refractive  indices  // 
increasing  from  1.36  to  1.74,  the  birefringence  changes  following  a 
hyperbolic  curve.  According  to  Wiener's  theory  of  the  anisotropy  of 
composite  bodies,  this  behaviour  discloses  a  layered  structure  (Fig. 
60b,  p.  82),  the  lamellae  of  which  are  thin  compared  with  the  wave- 
lengths of  light.  When  the  imbibition  is  made  with  a  mixture  of 
fj  =  1.58,  the  chloroplast  becomes  isotropic.  This  is  the  point  where 
the  lamellae  have  the  same  refractive  index  as  the  imbibing  medium. 
As  acetone  removes  the  lipids,  the  disclosed  lamellae  must  consist  of 
protein.  It  is  of  interest  to  note  that  muscle  protein  and  neurokeratin 
from  nerve  sheaths  also  have  a  refractive  index  as  high  as  i .  5  8  (Schmidt 
1937b). 

If  the  chloroplasts  are  fixed  with  OsO^,  the  lipids  become  partly 
insoluble.  Then  we  find,  in  addition  to  the  variable  layer  birefringence 
mentioned  above,  a  constant  intrinsic  anisotropy,  independent  of  the 
refractive  index  of  the  imbibition  medium,  which  is  due  to  orientated 
adsorbed  lipids  (Fig.  129).  Thus,  the  chloroplast  of  Mougeotia  has  a 
submicroscopic  layered  structure  of  protein  and  interposed  orientated 
lipid  lamellae.  In  Menke's  experiments  (1934b)  with  chloroplasts  of 
Closterium  the  lipids  produced  myelin  forms  which,  like  lecithin, 
sodium  oleate,  etc.,  are  optically  positive  with  reference  to  the  radius 
of  the  tubes.  From  this  it  follows  that  the  orientation  of  the  lipids 
in  the  plastids  must  be  as  shown  in  Fig.  130a  (L). 

At  first,  Menke  (1938c)  regarded  the  proposed  scheme  (Frey- 
Wyssling,  1937c)  of  the  lamellar  fine-structure  of  chloroplasts  with 


p .■.•  ■  ■.■.■.-■■/..'•• 

tmmiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii 

p  .-'.■..-'..■■.■..•.-:-■■■.■:  ■■■ 

f  Tnnriiii iiiiiiiiiiiiiiiiiiiiiiiiiiiiiin':;iiiiiiii 

p  ' ■'  .•.■.•..•. 

L  Mnn iiiii iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii  l>) 

p  //!■.■'■-•.■.■-■.■.•.•.■■."■■■■ 

^) 

Fig.  130.  Structure  of  chloroplasts.  a)  Submicroscopic  layer  structur  neglecting  the  grana 
structure.  P  protein  layer,  L  lipid  layer,  with  indication  of  the  optical  character  (from  Frey- 
Wyssling,  1937O;  ^)  scheme  of  a  cross-section  of  a  chloroplast    in    ultraviolet    light 

(from  Menke,  1940^). 


256  FINE-STRUCTURE  OF  PROTOPLASM  II 

some  scepticism.  However,  gold-stained  chloroplasts  in  profile  clearly 
exhibited  dichroism  (cf.  p.  84,  loi),  which  is  indicative  of  a  laminar 
texture  (Menke  and  Kuster,  1938). 

Further  proof  of  the  lamellar  texture  was  provided  by  the  large 
chloroplasts  oi  Anthoceros,  that  classical  object  which,  at  the  instigation 
of  Ernst,  had  already  been  appealed  to  so  fruitfully  in  the  dispute 
over  the  relationship  between  plastids  and  chondriosomes  (Scherrer, 
1 9 14).  Menke  and  Koydl  (1939)  identified  layers  at  the  limit  of 
microscopic  resolution  in  cross-sections  through  the  chloroplasts  of 
Anthoceros  using  the  enhanced  resolving  power  of  the  UV  microscope. 
Not  only  do  the  big  chloroplasts  without  grana  of  Anthoceros  and 
the  Conjugatae  algae  appear  to  be  laminated,  but  also  the  granulated 
chloroplasts  {Selaginella,  Phaseolus).  The  grana  are  united  by  thin 
lamellae,  which  induced  Menke  (i94od)  to  devise  the  plan  of  Fig. 
130b  of  a  section  through  the  discoid  chloroplasts  of  the  higher  plants. 
The  pile-like  arrangement  of  the  grana  (Strugger,  1950,  195 1)  is 
clearly  visible. 

Electron  microscopy  (Kausche  and  Rusk  a,  1940;  Menke,  1940  a; 
Algera,  Beyer,  v.  Iterson,  Karstens  and  Thung,  1947;  Granick 
and  Porter,  1947).  Besides  stroma  and  grana,  a  distinct  boundary 
layer  has  been  disclosed  (Frey-Wyssling  and  Muhlethaler,  1949a) 
as  a  third  morphological  element  of  the  chloroplast  (Fig.  1 3  la,  p.  259). 
This  layer  must  consist  essentially  of  proteins,  as  it  displays  the 
properties  of  a  solid  and  does  not  show  any  sign  of  the  liquid  or  semi- 
liquid  state  characteristic  of  lipid  matter.  It  is  probable  that  the  living 
boundary  contains  lipids,  but  their  amount  must  be  small  as  compared 
with  the  total  lipid  mass  in  the  chloroplast.  Obviously  they  join  the 
emigrating  myelin.  The  proteins  of  this  plastid  layer  must  be  of 
the  fibrous  type ;  otherwise  the  formation  of  a  membrane  would  not 
be  possible  when  dried.  The  formation  of  strands  of  stretched  chloro- 
plasts (Kuster,  1935c)  is  probably  due  to  this  protein.  How  much 
the  dried  membrane  differs  from  its  natural  state  in  the  living  chloro- 
plast is  not  known. 

Under  the  membrane,  the  grana  are  visible  as  discs.  The  stroma,  on 
the  other  hand,  does  not  show  any  conspicuous  structure.  Wyckoff 
(1949)  has  given  evidence  of  globular  macromolecules  about  250- 
300  A  in  diameter,  which  lie  on  and  between  the  grana.  If  the  plastid 
membrane  has  burst,  as  usually  occurs  during  the  preparation  of  the 


3  CHLOROPLASTS  257 

chloroplasts,  the  whole  carrier  film  is  sprinkled  with  these  globular 
bodies  (Fig.  131c,  d).  This  behaviour  would  indicate  that  the  stroma 
is  a  corpuscular  dispersion  of  macromolecules,  i.e.,  a  sol.  Since  a  sol 
has  no  framework,  the  characteristic  shape  of  the  chloroplast  must  be 
due  to  its  membrane,  much  the  same  as  in  erythrocytes,  and  to  its 
internal  lamination  (Fig.  130b).  The  chloroplast  can  change  its  shape 
(Senn,  1908),  or  even  form  processes  (Heitz,  1952);  this  faculty 
must  also  be  ascribed  to  the  membrane,  which  may  be  compared  with 
the  ectoplasm  of  creeping  protozoa.  This  again  argues  in  favour  of 
a  protein  rather  than  of  a  lipid  ground  mass  of  the  plastid  membrane. 
The  grana  supporting  lamellae  suggested  by  Menke  (Fig.  130b) 
and  Strugger  (195  i)  have  not  yet  been  found  in  the  electron  micro- 
scope. 

We  may  ask  whether  the  macromolecules  found  by  Wyckoff 
(1949)  represent  lipo-proteins  or  only  proteins.  It  is  almost  certain 
that  the  latter  is  the  case.  The  preparations  show  very  thin  flat  discs 
(Kausche  and  Ruska,  1940)  of  various  diameters  up  to  5  //  and  only 
100-200  A  thick.  It  can  be  shown  that  before  desiccation  these  discs 
wxre  in  a  semi-liquid  state.  They  never  have  folds,  as  the  plastid 
membrane  has,  and  dry  perfectly  smoothly  on  the  carrier  film,  even 
if  they  include  isolated  grana  (Fig.  131c).  There  has  been  much  dis- 
cussion on  the  nature  of  these  discs.  They  have  been  looked  upon  as 
protein  lamellae  (Menke,  1940a)  or  phosphatide  bladders  (Algera 
and  co-workers,  1947)  (which  is  unlikely,  as  the  chloroplast  contains 
only  0.5-2.5%  of  phosphatides),  but  there  is  no  doubt  that  they 
represent  the  total  lipid  matter  of  the  chloroplast  and  must  be  con- 
sidered as  myelin  forms.  Fig.  131c  shows  how  this  myelin  flows  out 
of  a  fraction  of  a  disintegrated  chloroplast. 

It  is  likely  that  the  grana  lipids  have  also  emigrated,  because,  as 
seen  in  the  electron  microscope,  the  grana  consist  of  proteins  only. 
Washing  with  lipo-solvents  does  not  alter  them  (Menke,  1940  a; 
Granick  and  Porter,  1947).  They  seem  to  be  layered  Hke  a  low  pile 
of  coins.  Occasionally  such  a  pile  appears  to  be  overturned  (Fig. 
13  id),  when  a  number  of  very  thin  lamellae,  all  of  the  same  diameter, 
are  visible.  The  question  is  justified,  whether  these  lamellae  are  really 
lamellar  parts  of  grana  or  perhaps  ghosts  of  whole  grana.  However, 
Steinmann  has  disclosed  in  Aspidistra  chloroplasts  as  many  as  30  of 
these  lamellae  in  the  same  pile  (unpublished).  This  rules  out  any 


258  FINE-STRUCTURE  OF  PROTOPLASM  11 

errors  in  interpretation,  since  a  pile  of  grana  consists  of  only  about 
8  microscopic  discs  (Strugger,  195  i). 

The  submicroscopic  lamellae  must  consist  of  protein.  In  the  living^ 
state,  the  lipids  in  the  grana  were  probably  located  between  these 
protein  layers.  If  this  picture  can  be  substantiated  by  further  research^ 
the  grana  of  the  chloroplast  would  represent  a  layered  composite  body 
with  alternating  protein  and  hpid  lamellae.  The  chlorophyll  is  closely 
associated  with  the  grana  lipids,  because  it  emigrates  together  with 
them;  on  the  other  hand,  Menke  (1938c,  1943)  points  out  that  chloro- 
phyll migrating  with  the  Hpids  imparts  conspicuous  dichroism  to  the 
myelin  tubes,  lacking  in  the  profile  of  the  chloroplast.  Hence  a  simple 
combination  of  hpids  and  chlorophyll  is  excluded,  which  is  a  further 
argument  in  favour  of  the  existence  of  a  chromoprotein. 

From  Fig.  13 id  it  may  be  concluded  that  this  chromoprotein  is 
arranged  in  layers.  If  this  conception  of  the  arrangement  of  the 
chromoprotein  be  correct,  the  principle  of  laminar  surface  develop- 
ment can  be  consistently  pursued  from  the  molecular  to  the  macro- 
scopical  region.  The  molecular  layers  compose  the  discoid,  sub- 
microscopic  to  microscopic  grana  (Fig.  130a,  p.  255);  these,  again, 
lie  in  layers  in  discoid  or  laminar  chloroplasts  and  finally  the  chloro- 
phyll grana  are  exposed  to  the  light,  again  in  foliar  laminae. 

Tracing  thus  a  given  morphological  principle  through  several 
orders  of  magnitude,  we  are  provided  with  an  interesting  counterpart 
to  fibre  structure,  in  which  linear  development  plays  a  similar  part.  The 
laminar  series:  molecular  layer/grana/chloroplast/foliar  laminae  may 
be  compared  with  the  linear  series :  chain  molecule/microfibril/fibre/ 
fibre  bundles  of  the  pericycle.  It  should  be  emphasized  that  in  both 
cases  the  form  birefringence  has  been  the  key  to  the  submicro- 
scopic structure,  viz.,  the  discovery  of  the  rodlet  birefringence  in 
fibres  and  of  the  platelet  birefringence  in  chloroplasts. 

Chloroplastin  and  the  unit  of  assimilation.  The  definite  estabHshment  of 
the  grana  as  the  only  loci  in  the  chloroplast  containing  chlorophyll, 
calls  for  a  discussion  concerning  the  biochemical  concept  of  chloro- 
plastin. There  is  no  doubt  that  the  grana  represent  a  high  concen- 
tration of  chlorophyll.  According  to  Granick  (1949),  the  chloroplast 
of  spinach  contains  only  40-60  grana,  0.6  ^  in  diameter  and  0.08  fjt 
thick.  Since  in  some  instances  it  has  been  possible  to  photograph  the 
grana  in  profile  with  the  fight  microscope  (Heitz,  1932),  this  sub- 


CHLOROPLASTS 


259 


Fig. :  3 1 .  Chloruplasts  ot  tobacctj  leaves  in  the  electron  microscope  (t rom  !■  rey-\\  yssling 
and  MunLETHALER,  1949a).  a)  Chloroplast  membrane;  b)  grana;  c)  myelin  covering  grana 
and  globular  macromolecules  of  disintegrated  stroma;  d)  intact  granum  and  layers  of  an 

overturned  granum. 


26o  FINE-STRUCTURE  OF  PROTOPLASM  II 

microscopic  thickness  of  0.08  fx  may  be  due  to  desiccation  during  the 
preparation  for  the  electron  microscope,  and  we  may  estimate  the 
thickness  of  the  fresh  grana  to  be  about  o.i  5  [x.  The  whole  chloroplast 
has  a  diameter  of  5  fx  and  its  thickness  in  the  fresh  state  is  about  half 
of  this.  If  we  calculate  the  volume  of  the  chloroplast  as  an  ellipsoid, 
4/3  X  2.5^  X  1.25  X  n,  and  that  of  the  50  grana  as  cylindrical  discs, 
50  X  0.3^  X  0.15  X  n,  wt  obtain  a  volume  ratio  of  15/1.  Thus,  the 
total  volume  of  the  grana  would  be  only  1/ 1 5  or  7  %  of  that  of  the 
whole  chloroplast.  Since  there  is  7.7%  of  chlorophyll  in  the  chloro- 
plast (Menke,  1940b),  this  would  mean  that  the  grana  consist  entirely 
of  chlorophyll.  This  is  obviously  impossible,  for  the  grana  are  still 
visible  in  the  electron  microscope  when  the  pigments  are  extracted. 

We  must  conclude,  therefore,  that  the  discs  visible  in  Fig.  131b  do 
not  represent  individual  grana,  but  piles  of  grana.  The  number  of 
piles  in  the  chloroplast  of  tobacco  leaves  is  about  50,  thus  the  same 
as  in  spinach  leaves,  and  their  diameter  0.4  fx.  The  chloroplast  of  about 
2.5  //  thickness  can  lodge  not  more  than  12  layers  of  grana.  With  these 
figures  the  volume  ratio  chloroplast/total  number  of  grana  is  3,  i.e. 
the  grana  occupy  1/3  and  the  stroma  2/3  of  the  plastid  volume^.  This 
ratio  enables  us  to  calculate  the  chlorophyll  content  of  the  grana 
protein. 

According  to  Table  XXIV  (p.  246),  half  the  weight  of  the  chloroplast 
is  protein  and  7.7%  chlorophyll  (mol.wt.  893).  This  yields  a  molar 
xatio  of  3  chlorophyll  to  i  Svedberg  unit  (mol.wt.  17600).  Since  the 
chlorophyll  is  restricted  to  the  grana  and  their  volume  being  only  one 
third  of  the  chloroplast,  this  ratio  must  be  9/1  in  the  grana,  if  the 
protein  concentration  is  the  same  as  in  the  stroma.  This  result  seems 
to  prove  that  chlorophyll  cannot  be  a  prosthetic  group  of  an  enzyme, 
for,  considered  as  a  co-enzyme,  its  carrier  would  have  a  molecular 
weight  as  low  as  2000,  which  has  never  been  found  for  apo-enzymes. 

Stoll's  chloroplastin  (1936)  has  a  molecular  weight  of  roughly 
5  millions.  If  it  is  really  the  chromoprotein  of  the  chloroplast,  it  must 
come  from  the  grana  alone  and  cannot  be  contaminated  by  stroma 
protein.  It  is  doubtful  whether  these  two  proteins  can  be  separated 
quantitatively  by  fractionated  precipitation.  Supposing  the  chloro- 

1  Thus  about  25  %  by  weight  of  the  grana  consists  of  chlorophyll;  this  is  astonishingly 
high,  as  compared  with  the  haematin  (mol.  wt.  592)  content  of  the  erythrocytes  (p.  265) 
which  is  only  ca.  3  %  of  the  cell  interior. 


3  CHLOROPLASTS  261 

plastin  with  the  molecular  weight  5  millions  to  be  really  the  chromo- 
protein  of  the  chloroplast,  it  ought  to  hold  2500  chlorophyll  molecules, 
but  in  reality  it  contains  only  about  420.  This  indicates  that  the  chloro- 
plastin  is  a  mixture  with  stroma  constituents  rather  than  a  pure  com- 
pound from  the  grana. 

On  the  other  hand,  physiologists  find  that  a  number  of  chlorophyll 
molecules  as  large  as  calculated  above  is  necessary  for  the  assimilation 
of  one  molecule  of  COg.  That  number  is  called  unit  of  assimilation. 

Whereas  chemists  think  of  the  photosynthetic  process  as  associated 
with  the  chlorophyll  molecule  (Stoll,  1936),  physiologists  tend  rather 
to  regard  the  pigment  merely  as  an  energy  trap  ajid  to  attribute  the 
actual  chemical  action  of  the  gradual  hydrogenation  to  the  proteins 
in  the  chloroplast  (Rabinowitch,  1945).  This  is  inferred  partly  from 
Blackman's  dark  reaction  (1905),  but  mainly  from  facts  established 
by  Emerson  and  Arnold  (1932),  according  to  which  a.  unit  of  assimi- 
lation of  roughly  2500  chlorophyll  molecules  is  needed  for  the  re- 
duction of  one  CO2  molecule.  Gaffron  and  Wohl  (1956)  calculate 
about  1000  molecules  for  this  same  unit.  This  observed  fact  calls  into 
question  all  attempts  to  deduce  the  mechanism  of  assimilation  from 
the  chemical  constitution  of  the  chlorophyll  molecule  (Willstatter, 
1933;  Franck,  19.35;  Stoll,  1936).  Gaffron  and  Wohl  state  that 
the  pigment  acts  merely  as  a  specific  energy  transmitter  and  that  a  very 
large  number  of  chlorophyll  molecules  would  be  required  to  capture 
the  necessary  quanta  of  light  for  the  assimilation  of  one  CO2  molecule 
(Warburg  and  Negelein,  1923;  Scplmucker,  1930;  Eymers  and 
Wassink,  1938;  Emerson  and  Lewis,  1939).  It  is  to  be  expected  that 
the  occurrence  of  these  units  of  assimilation  will  be  expressed  morpho- 
logically in  some  way.  Heitz  (1936a)  presumes  that  the  grana  may  be 
involved.  This,  however,  cannot  be  so,  for  if,  as  Euler,  Bergman 
and  Hellstrom  (1934)  state,  a  chloroplast  contains  1.65-10^  chloro- 
phyll molecules,  there  would  have  to  be  something  like  10^  or  a 
million  grana,  whereas  the  actual  number  is  about  600.  In  a  bi- 
molecular  layer,  2000  chlorophyll  molecules  would  occupy  a  surface 
of  1000  X  225  A^  =^  z.i^  X  io~^  fj,^.  As  a  square,  this  surface  has  a 
side  of  only  0.05  /u.  Therefore  the  unit  of  assimilation  is  certainly 
amicroscopical. 

Seeing  that  a  chloroplastin  macromolecule  in  the  grana  ought  to 
contain  about   2500   chlorophvll   molecules,  the   question   naturally 


262  FINE-STRUCTURE  OF  PROTOPLASM  II 

arises  whether  the  unit  of  assimilation  is  identical  with  the  chloro- 
plastin  unit.  This  would  simplify  our  terminology.  But  as  long  as  it 
cannot  be  proved  that  chloroplastin  derives  from  the  grana  alone,  the 
coincidence  of  the  number  of  chlorophyll  molecules  in  the  unit  of 
assimilation  and  in  the  chloroplastin  molecule  seems  to  be  only 
incidental. 

§  4.  Erythrocytes 

a.  The  Microscopic  Structure  of  Erythrocytes 

It  is  not  only  their  lack  of  a  nucleus  which  makes  the  red  blood 
corpuscles  of  mammals  a  cytological  curiosity,  but  it  is  also  the 
peculiar  shape  of  the  cell.  Seen  from  the  top  in  the  microscope,  they 

look  like  round  discs,  the  bound- 
ary of  the  cross-section  of  which 
is  curiously  sinuate,  instead  of 
being  planoparallel.  Thus  the 
erythrocytes  are  biconcave   discs. 

This  remarkable  shape  of  the 
Fig.  1^2.  Cross-section  of  the  red  cell  of  man.  .  .  •  j    j.      i        j 

,^    „  , .  ,        1/  /  J  ,    rv  cross-section  is  said  to  be  due 

ab  =  8.55/i;  thickness  li  (cd  4-  ef)  =  2.40//; 

thickness  gh  =  1.02  /i  (from  Ponder,  1934)-      to  the  function  of  the  red  blood 

corpuscles,  since  from  a  surface 
thus  shaped  the  interior  of  the  cell  can  be  easily  supplied  with  oxygen 
by  diffusion,  whereas  a  globular  shape  would  entail  greater  poverty 
of  oxygen  in  the  centre  than  in  the  surface  layers  and,  with  a  piano- 
parallel  disc,  the  edge  would  be  richer  in  oxygen  than  the  centre. 

The  discs  remain  biconvex  in  shape  as  long  as  the  erythrocytes  are 
suspended  in  the  blood  plasma  or  in  serum,  but  they  round  up 
directly  if  the  medium  is  changed  by  the  addition  of  lecithin  to  the 
blood  plasma.  It  is  a  remarkable  fact  that  the  same  thing  happens 
when  a  thin  layer  of  them  is  covered  with  a  cover  glass.  Ponder 
(1934),  discussing  many  possible  causes  of  this  phenomenon,  omits 
to  mention  the  change  in  r^  of  the  medium  and  asphyxiation,  which 
all  living  cells  undergo  after  some  time  in  the  thin  layer  under  the 
cover  glass.  Under  certain  circumstances  rounded  blood  corpuscles 
can  be  restored  to  their  initial  biconcave  disc  shape  by  the  addition 
of  serum. 

As  any  experiment  with  erythrocytes  involves  possible  transfor- 
mation, it  is  not  an  easy  task  to  establish  their  true  cross-sectional 


4  ERYTHROCYTES  263 

shape.  Ponder  (1954)  obtained  the  image  shown  in  Fig.  132  by  a 
series  of  microphotographs  with  an  objective  of  the  least  possible 
focal  depth.  With  retention  of  the  volume,  the  transformation  to 
spheres  is  effected  by  surface  changes  only.  For  instance,  the  biconcave 
erythrocyte  of  the  rabbit  has  a  surface  of  no  fi^,  whereas  that  of  the 
globular  form  is  only  70  fi^  (reduction  in  surface  of  36%). 

A  further  indication  of  surface  changeability  is  provided  by  the 
dented  blood  corpuscles,  which  are  transitions  between  the  biconcave 
discs  and  the  globules,  or  the  curious  thorn-apple  forms  which  arise 
vmder  certain  conditison.  These  facts  make  it  plain  that  surface  forces 
are  responsible  for  the  shape  of  the  erythrocytes.  Gough  (1924)  points 
out  that  surface-enlarging  forces  must  be  active  in  the  erythrocytes, 
conducive  to  expansion  of  the  surface  of  contact  with  the  suspension 
liquid,  as  in  the  case  of  the  myelin  forms.  The  largest  surface  would 
be  obtained  if  the  blood  corpuscle  were  flattened  to  the  thinnest 
possible  disc.  On  the  other  hand,  there  is  some  shght  surface  tension 
in  the  blood  corpuscles  (presumably  of  the  order  of  i  dyne/cm,  cf. 
Table  XXI,  p.  166),  tending  to  reduce  the  surface  and  to  round  off 
the  erythrocytes  if  other  factors  do  not  interfere. 

Now  it  mav  be  that  the  exceptional  shape  of  the  erythrocytes  of 
mammals  represents  some  kind  of  equilibrium  between  the  surface- 
enlarging  and  surface-reducing  forces.  If  that  be  so,  the  membrane  of 
the  erythrocytes  should  have  the  properties  of  a  mesophase.  No  form 
of  equilibrium  can,  however,  be  mathematically  computed  from  the 
cross-section  in  Fig.  132  and  Ponder  (1934,  p.  89)  therefore  inclines 
to  the  belief  that  there  must  be  a  certain  amount  of  internal  solidity. 
The  micrurgical  investigations  of  Seifriz  (1927,  1929)  tend  to  endorse 
this,  for  they  show  that  deformed  and  elongated  erythrocytes  have 
some  slight  elasticity.  Dervichian  does  not  agree  with  this  view 
(Dervichian,  Fournet  and  Guinier;  1947). 

Inner  structure.  The  various  theories  as  to  the  internal  structure  of 
the  red  blood  corpuscles  are  expressed  in  the  following  two  views. 
One  school  regards  the  anucleate  erythrocytes  as  enclosed  in  a  mem- 
brane which  gradually  changes  towards  the  interior  into  a  very  loosely 
knit  spongy  structure,  in  which  the  red  blood  pigment  is  embedded. 
Some  support  for  this  view  is  afforded  by  the  network  structure  which 
can  be  made  visible  in  young  erythrocytes  by  suitable  fixation  and 
staining.  Representatives  of  the  other  school  of  thought,  however. 


264  FINE-STRUCTURE  OF  PROTOPLASM  II 

dismiss  this  network  as  mere  artefact.  To  their  way  of  thinking,  the 
erythrocyte  consists  merely  of  a  balloon-like  membrane,  a  view  which 
has  some  backing  through  the  absence  of  any  microscopic  structure 
in  the  Hving  cell  interior  as  seen  in  the  ultra  microscope  or  illuminated 
by  ultraviolet  rays.  This  view  is  also  shared  by  most  of  the  research 
workers  who  have  studied  haemolysis.  For,  if  the  erythrocytes  are 
damaged  mechanically,  either  by  heat  or  freezing,  or  by  immersion  in 
sufficiently  hypotonic  or  hypertonic  solutions,  the  contents  of  the  cell 
extravasate  with  the  red  blood  pigment  and  a  colourless  sheath  re- 
mains, which  is  called  ghost,  or  the  stroma. 

These  facts  notwithstanding,  the  contents  of  the  erythrocytes  are 
not  to  be  considered  as  a  sol-like  liquid  of  no  organized  intrinsic 
structure,  an  error  committed  by  the  older  investigators  and,  more 
recently,  by  Gough  (1924').  The  relative  viscosity  of  the  cell  contents 
is  30  (see  Table XXII,  p.  169)  and  Ponder  (1934)  states  that  the  interior 
of  the  cell  shows  respiration  like  other  cells.  Although  the  erythrocyte 
membrane  has  been  proved  to  contain  all  the  chemically  identifiable 
substances  of  the  blood  corpuscles  with  the  exception  of  the  blood 
pigment  and  the  salt  content,  the  assumption  clearly  must  be  that  the 
contents  of  the  cell,  far  from  being  an  unorganized  liquid,  is  a  partially 
gelated  cytoplasm,  the  organization  of  which  is  easily  destroyed  when 
damage  is  inflicted. 

The  thickness  of  the  ghost  membrane  has  been  measured  by 
numerous  investigators  with  a  wide  variety  of  results  ranging  from 
15  to  700  vafx  (Jung,  1950).  This  seems  rather  embarrassing.  But  when 
the  methods  used  for  the  measurements  are  considered  the  results  can 
be  classified  into  two  groups,  viz.  those  obtained  from  dried  ghosts 
(electron  microscope.  Fig.  135,  p.  272,  Wolpers,  1941;  leptoscope^ 
Waugh,  1950),  yielding  15-25  m^,  and  those  from  hydrated  ghosts 
(dark  field  observation,  Lepeschkin,  1927;  micrurgy,  Seifriz,  1927) 
with  about  500  m/<.  The  last  figure  has  also  been  found  by  Mitchison 
(1950b),  who  has  thrown  down  the  ghosts  by  a  centrifugal  force  of 
1 10,000  g  to  a  compact  mass  which  is  still  5  5  %  of  the  volume  of  the 
intact  red  cell.  From  this  result  it  follows  that  the  swollen  membrane 
is  as  thick  as  half  the  depth  of  the  erythrocyte  (diameter  c-d  of  Fig. 
132)  and  that  it  shrinks  when  dried  to  1/25  of  this  size!  The  inner 
part  of  the  membrane,  therefore,  represents  in  vivo  a  very  loose  gel 
with  only  4%  protein,  which  fills  almost  the  whole  erythrocyte. 


4  •  ERYTHROCYTES  265 

b.  Molecular  Constituents  of  the  Erythrocytes 

Erythrocytes  consist  approximately  of  two-thirds  water  and  one- 
third  dry  residue,  which  is  mainly  composed  of  the  red  blood  pig- 
ment, haemoglobin,  and  salts.  It  is  interesting  to  note  that  potassium 
predominates  over  sodium  as  cation  of  the  salts.  Small  amounts  of 
protein  foreign  to  haemoglobin  and  of  lipids  constitute  the  ery- 
throcyte membrane. 

Haemoglobin.  The  red  blood  pigment  is  a  chromoprotein,  Hke 
chloroplastin  in  green  leaves;  yet  the  Hnk  between  chromogen  and 
protein  is  closer  than  in  chlorophyll  and  the  blood  pigment  therefore 
emerges  as  protein  from  the  stroma  in  haemolysis. 

Haemochromogen  is  a  labile  porphyrin  compound  which,  outside 
the  organism,  is  transformed  into  the  more  stable  haematin.  The 
composition  of  this  compound  is  C34H3204N4FeCl  and  it  is  closely 
akin  to  chlorophyll  (Gr.\nick,  1948).  The  main  differences  are  that 
in  the  centre  of  the  porphin  ring  there  is,  instead  of  magnesium,  tri- 
valent  iron,  the  third  valency  of  which  imparts  a  saHne  nature  to  the 
compound  usually  neutralized  by  the  anion  chlorine;  and  the  absence 
both  of  the  phytol  chain  and  the  iso-cyclic  ring  of  the  C  atoms  6-9-10 
(see  Fig.  127,  p.  249).  As  a  result  of  the  missing  phytol  chain  the 
haematin  appears  to  be  morphologically  more  compressed  and  less 
markedly  polar  than  chlorophyll.  The  protein  carrier,  to  which  the 
haemochromogen  is  attached  is  called  "globin". 

The  haemoglobin  molecule  is  of  a  thickset  rod-like  shape  with 
57  A  diameter  and  34  A  height  (Perutz,  1948).  On  the  basis  of  the 
iron  content  its  molecular  weight  is  computed  at  16,000  to  17,000 
(K.A.RRER,  1941),  while  the  reading  in  the  uhracentrifuge  is  69,000,  i.e., 
about  four  times  the  value  (Svedberg's  law  of  multiples,  see  p.  141). 
Stromatin.  Jorpes  (1932)  states  that  approximately  4%  of  the  total 
protein  content  of  the  erythrocytes  consists  of  a  protein  foreign  to 
haemoglobin,  which  is  contained  in  the  erythrocyte  sheath  and  is 
therefore  described  as  stromatin.  According  to  Winkler  and  Bungen- 
BERG  DE  Jong  (1941),  its  I.E.P.  is  at  p^  5.2.  Analysis  of  the  hae- 
molyzed  membrane  of  erythrocytes  shows  that  there  is  80  °o  of 
stromatin  and  20 °o  of  lipids. 

Phospholipids.  The  bulk  of  the  hpids  consist  of  phosphatides,  notably 
lecithin  (Fig.  93,  p.  138),  besides  which  there  are  insignificant 
amounts  of  cephaiin  and  sphingomyelin.  The  I.E.P.  of  the  phospho- 


266  FINE-STRUCTURE  OF  PROTOPLASM  II 

lipids  is  at  p^  2.7.  They  are  thought  to  play  a  decisive  part  in  the 
permeability  phenomena  of  the  red  blood  corpuscles. 

Cholesterol.  Approximately  one  molecule  of  cholesterol  is  found 
for  every  four  phosphatide  molecules  in  the  stroma  (exact  ratio  3.5:1, 
Winkler  and  Bungenberg  de  Jong,  1941).  As  may  be  seen  in 
Fig.  92  (p.  138),  cholesterol,  unlike  the  phosphatides,  possesses 
no  ionogenic  groups.  Bungenberg  de  Jong  therefore  assigns  to  it 
an  important  part  in  the  formation  and  build-up  of  lipid  structures, 
for,-  in  a  lecithin  solution,  the  individual  lipid  molecules  remain 
separated  from  each  other  as  the  result  of  their  negative  charge. 
Although  the  fatty  acid  chains  have  a  tendency  to  agglomerate,  the 
repellent  effect  of  the  ionized  phosphoric  acid  groups  predominates 
and  the  molecules  are  therefore  kept  at  a  distance  from  each  other. 
If  cholesterol  is  added  to  a  solution  of  this  kind,  these  neutral  mole- 
cules are  able  to  penetrate  in  between  the  lecithin  molecules  and 
association  follows  as  the  result  of  Van  der  Waals  cohesive  forces, 
as  the  repelling  action  of  the  charges  does  not  span  the  width  of  the 
cholesterol  molecule.  Cholesterol  therefore  acts  as  a  sensitizer  in  the 
precipitation  of  lipid  solutions  with  ionogenic  groups.  Conversely,  in 
lipid  films  of  phosphatides,  cholesterol  acts  as  a  stabiliser,  as  it  counter- 
acts solution  of  the  film  by  ionogenic  influences. 

Nucleic  acids  are  only  present  during  the  development  of  the  ery- 
throcytes in  the  bone  marrow.  The  stem  cells  contain  5  %  cytoplasmic 
nucleic  acid,  but  during  differentiation  and  maturation  of  the  red  cell, 
its  concentration  drops  to  below  0.5%  (Thorell,  1948). 

c.  Submicroscopic  Structure  of  Erythrocytes 

Stromatin  as  tricompkx  system.  Winkler  and  Bungenberg  de  Jong 
(1941)  have  pubhshed  an  instructive  design  of  the  structure  of  the 
erythrocyte  sheath  (Fig.  133).  By  exact  measurement  of  the  electric 
migration  velocity  of  the  red  blood  corpuscles  in  the  most  various 
salt  solutions,  these  investigators  find  quantitatively  the  same  be- 
haviour as  in  phosphatides,  from  which  they  conclude  that  the  surface 
of  the  erythrocytes  is  covered  by  a  phosphatide  film  (layer  I  in  Fig. 
133),  which  is  stabilized  by  cholesterol.  The  I.E. P.  of  the  stroma  with 
Ph  5.2  being  between  that  of  the  phospholipids  (2.7)  and  of  the 
stromatin  (5.8),  it  is  assumed  that  the  phosphoHpids  form  a  complex 
system  with  the  stromatin  (layer  IV),  their  positive  choline  groups 


ERYTHROCYTES 


267 


entering  into  relationship  with  the  anionic  end  groups  of  the  protein 
(layer  III).  Haemolysis  experiments  have  further  shown  how  calcium 
ions  consolidate  the  erythrocyte  membrane  and  stabiHze  it.  In  layer  II 
the  calcium  ions,  with  their  strong  positive  charge,  are  therefore 
allocated  between  the  negative  phosphoric  acid  groups  of  the  lecithin 
and  a  more  powerful  ionogenic 


I  t 


YL 


280i 


i)^i^i^i^»C  c 


qF 


M 
E 


mn-i^-rom^i^Hi 


/ 


20& 


cohesion  is  thereby  attained.  Thus 
the  stroma  is  regarded  as  a  com- 
plex system  consisting  of  phos- 
phatide-calcium  ions,  stromatin 
protein,  and  the  regular  distri- 
bution of  charge  brings  with  it  a 
definite  arrangement  and  orien- 
tation of  the  various  components  m  ^2o^\ 
of  the  system.  The  tricomplex  sys- 
tem is  completed  by  an  assumed 
complex  linkage  of  the  haemo- 
globin (layer  VI)  with  anionic 
end  groups  in  layer  V  to  cationic 
groups  of  the  stromatin. 

The  design  of  Fig.  1 3  3  is  further 
complicated  by  layer  A.  This  re- 
presents   an    incomplete    film  of 
polar  lipids,  which  turn  their  lipo- 
philic side  towards  the  monomo- 
lecular    phosphatide  layer  I  and 
their  hydrophilic  pole   outwards 
(fat,  fatty  acids,  possibly  chole- 
sterol). It  is  necessary  to  assume  this,  for,  without  the  layer  A,  the 
erythrocytes    would    agglutinate    in    aqueous    solutions    and,    when 
shaken  out  with  paraffin  oil,  would  pass  over  into  the  lipid  phase, 
neither  of  which  they  do. 

The  scheme  devised  by  Winkler  and  Bungenberg  de  Jong  (1941) 
explains  manv  properties  of  erythrocytes,  e.g.,  it  makes  allowance  for 
the  lipid  filter  theory  of  permeability,  there  being  a  fipid  film  with 
molecular  pores  (where  the  cholesterin  covering  is  lacking).  It  ex- 
plains the  permeability  to  anions  which  is  characteristic  of  erythro- 
cvtes,  as  the  calcium  ion  layer  III  debars  the  cations.  The  same  layer 


Fig.  133.  Molecular  structure  of  the  en- 
velope of  the  red  cell  from  Winkler  and 
Bungenberg  DE  Jong  (i940-'4i);  •  anionic 
groups ;  o  cationic  groups  or  cations  (Ca) ; 
shaded:  cholesterin;  ^  phospholipid  acid; 
ch  cholesterin  ester;   • fatty  acid. 


268  FINE-STRUCTURE  OF  PROTOPLASM  II 

of  ions,  with  its  water  of  hydration,  is  responsible  for  the  effect  of 
hydrating  and  dehydrating  ions  upon  the  properties  of  the  erythro- 
cytes. According  to  Fricke  (1925),  the  electric  properties  of  the  wall 
of  the  erythrocytes  are  such  that  the  existence  must  be  assumed  of 
a  non-conductive  layer  3  3  A  thick.  This  thickness  corresponds  to  the 
lipidic  part  of  the  phosphatide  layer  I.  Gorter  and  Grendel  (1925) 
assume  that  there  is  a  bimolecular  lipid  film  on  the  basis  of  the  lipid 
content  of  the  erythrocytes;  and  this  claim  is  likewise  partly  met. 

Finally,  Winkler  and  Bungenberg  de  Jong  calculate  from  the 
stromatin  and  lipid  contents  of  the  erythrocytes  of  pigs  (19.2,  or 
3.5  mg  per  ml  of  blood)  that  the  orientated  lipid  molecules  just  cover 
the  surface  of  the  blood  corpuscles  in  the  manner  indicated  (Fig.  133) 
and  that  the  layer  of  stromatin  below  is  1 20  A  thick.  From  this  we 
get  1 50  A  as  the  thickness  of  the  total  erythrocyte  membfane  (without 
layer  A)  which,  surprisingly,  is  of  about  the  same  order  of  magnitude 
as  the  data  obtained  by  Wolpers  (1941)  by  means  of  electron  optics. 
However,  this  is  only  incidental,  since  Fig.  133  does  not  refer  to  the 
dried,  but  to  the  hydrated  envelope. 

Although  this  explanation  of  many  interesting  phenomena  as- 
sociated with  the  morphology  and  physiology  of  erythrocytes  is  un- 
disputed, the  model  of  Fig.  133  still  raises  a  number  of  difficulties. 
One  of  the  first  points  to  be  noted  is  that  analysis  of  the  erythrocytes 
has  not  revealed  the  presence  of  calcium.  True,  Winkler  and 
Bungenberg  de  Jong  have  calculated  that  the  quantity  of  Ca  present 
is  so  small  that  it  would  escape  detection  in  analysis,  but  they  never- 
theless consider  it  improbable  that,  given  the  percentage  of  calcium 
in  the  blood  serum,  no  Ca  ions  should  be  adsorbed  on  the  erythrocyte 
membrane.  In  the  transition  from  the  biconcave  disc  shape  to  the 
globular,  the  surface  must  shrink  by  37%.  It  is  not  clear  how  this 
could  take  place  without  causing  change  of  structure  since,  compared 
to  their  normal  distances,  the  molecules  are  already  densely  packed. 

An  argument  against  the  parallel  radial  orientation  of  all  the 
molecules  is  the  slight  optical  anisotropy  of  the  erythrocytes.  Stro- 
matin and  haemoglobin  can  scarcely  be  said  to  represent  chain  mole- 
cules; on  the  contrary,  haemoglobin  is  known  to  be  a  globular 
molecule.  Should  stromatin  be  filamentous,  it  would  seem  to  me  that 
the  orientation  of  those  threads,  c^iven  their  great  length,  is  more 
likely  to  be  parallel  to  the  surface  than  a  radial  one,  as  suggested. 


ERYTHROCYTES 


269 


Winkler  and  Bungenberg  de  Jong  discuss  this  possibility;  but, 
iinding  that  the  number  of  anionic  COOH  groups  of  the  side  chains 
is  not  large  enough  for  their  tricomplex  system,  they  place  the  main 
chains  perpendicular  to  the  surface  of  the  cell. 

Haemoglobin  as  a  solute  in  close  packing.  Although  the  concentration 
of  haemoglobin  reaches  34%  in  the  red  cell,  it  does  not  crystallize; 


Fig.  134.  Close  packing  of  haemoglobin  in  the  erythrocyte  (from  Jung,  1950).  t  diameter, 

h  height,  d  body  diagonal  of  the  haemoglobin  molecule,  a  distance  of  molecular  layers. 

The  size  of  a  hydrated  Li  +  and  K+  ion  is  given  for  comparison. 


it  iills  the  erythrocyte  as  an  isotropic  solute.  On  the  other  hand,  an 
X-ray  period  of  62  A  is  furnished  by  living  cells  (Dervichian, 
FouBusiET  and  Guinier,  1947).  This  period  can  be  explained  as  follows 
(Jung,  1950):  The  haemoglobin  molecules  are  covered  by  a  hydration 
layer  of  3  A,  so  that  the  dimensions  of  its  thickset  cylinder  are  63  A 
for  the  diameter  /,  40  A  for  the  height  h  and  74. 5  A  for  the  body 
diagonal  d.  If  these  molecules  are  allowed  free  rotation,  every  one 
requires  a  spherical  space  of  74.5  A  diameter  (Fig.  134).  Further,  if 
these  spheres  are  arranged  in  hexagonal  closest  packing,  a  layer 
distance  of  61  A  results,  which  is  consistent  with  the  X-ray  period 
found.  Therefore,  the  state  of  the  haemoglobin  in  the  erythrocyte  is 
that  of  the  densest  solution  possible,  whose  concentration  has  been 
calculated  to  be  34*^0. 

It  is  evident  that  such  an  arrangement  is  most  favourable  for  the 
gas  exchange  of  O,  and  COo.  But  why  is  it  that  such  a  saturated 


ZyO  FINE-STRUCTURE  OF  PROTOPLASM  II 

solution  does  not  crystallize?  As  a  matter  of  fact,  every  disturbance 
of  the  existing  equilibrium,  say  by  a  hypertonic  salt  solution  or  by 
formation  of  sickle-shaped  cells  in  anaemic  venous  blood  (Perutz 
and  MiTCHisoN,  1950),  provokes  the  crystallization  which  is  re- 
cognized by  the  birefringence  of  the  hitherto  isotropic  haemoglobin. 
The  possibility  exists  that  in  the  swollen  erythrocyte  traces  of  stro- 
matin  between  groups  of  haemoglobin  molecules  prevent  the  crystal- 
lization which  occurs  as  soon  as  this  stabiHzing  system  is  destroyed. 

Birefringence.  Fresh  problems  arise  as  soon  the  optics  of  erythrocytes 
is  taken  into  consideration.  Rabbit's  red  cells,  carefully  haemolyzed 
by  freezing  and  thawing,  are  birefringent  (Schmitt,Bear  and  Ponder 
1936,  1938),  exhibiting  a  very  faint  polarization  cross.  With  respect 
to  the  cell  radius,  the  birefringence  is  slightly  negative  in  isotonic  salt 
solution,  but  positive  polarization  crosses  are  clearly  visible  in 
glycerol  mixtures.  The  inference  from  imbibition  tests  of  this  kind  is 
that,  as  in  the  case  of  the  chloroplasts,  in  the  sheaths  of  the  erythrocytes 
there  is  positive  intrinsic  birefringence  of  the  embedded  lipids,  upon 
which  is  imposed  a  negative  form  birefringence  of  the  protein  frame- 
work. Lipid  solvents,  such  as  butyl  and  amyl  alcohol,  produce 
distinctly  negative  polarization  crosses,  abolishing  the  intrinsic  bire- 
fringence of  the  lipids  and  bringing  the  negative  form  birefringence 
out  clearly. 

ScHMiTT,  Bear  and  Ponder  come  to  the  conclusion  that  there  must 
be  a  composite  body  with  alternating  protein  and  lipid  lamellae.  The 
lipid  layers,  they  think,  must  be  bimolecular  on  account  of  the 
hydrophiHc  bias  of  the  stromatin.  This  view  conflicts  with  the 
calculations  made  by  Gorter  and  Grendel  (i92  5)>  according  to 
which  the  lipid  content  of  the  erythrocytes  would  be  just  sufficient 
for  a  single  bimolecular  covering.  The  possible  layering  throughout 
the  stroma  would  only  be  lipid-protein-cavity-protein-lipid.  Conse- 
quently, unless  those  authors'  statements  are  incorrect,  it  is  difficult 
to  see  how  there  can  be  a  composite  body  of  protein  and  lipid,  like 
that  proved  for  the  chloroplasts. 

Another  possible  explanation,  taking  the  observed  facts  into  ac- 
count, is  that  the  stromatin  is  loosely  layered  and  is  in  itself  a  Wiener 
composite  body.  In  this  case,  too,  the  positive  intrinsic  birefringence 
of  the  hpid  skin  overlays  the  negative  form  birefringence,  the  problem, 
however,  still  being  whether  the  lipid  birefringence  would  then  be 


4  ERYTHROCYTES  27I 

perceptible  at  all.  The  probable  retardation  F  can  be  calculated  with 
the  aid  of  the  formula  on  p.  86  by  inserting  the  value  o.oi  i  for  the 
birefringence  An,  which  Bear  and  Schmitt  (1936)  set  down  for 
orientated  lipid  in  the  nerve  sheath.  In  rabbits,  the  diameter  d^  of  the, 
supposedly,  hollow  cylindrical  rim  of  erythrocytes  is  i.j  /n  (cf.  c-d  in 
Fig.  132,  p.  262),  and  d,  is  shorter  by  twice  the  thickness  of  the  bi- 
molecular  Hpid  layer  (4  X  3  m//),  i.e.,  1.688  //.  The  value  for  the 
retardation  F  is  then  a  httle  above  1.8  m^.  This  is  a  value  which, 
though  at  the  lower  limit  of  quantitative  mensurability  with  sensitive 
compensators,  may,  by  suitable  polarizing  optics,  be  revealed  quali- 
tatively. This  shows  that  a  single  bimolecular  lipid  layer  suffices  to 
produce  the  faint  positive  intrinsic  birefringence  detected  by  Schmitt, 
Bear  and  Ponder. 

Both  the  quantity  of  lipid  present  and  the  slight  intrinsic  bire- 
frino-ence  witness  to  the  fact  that  there  can  hardly  be  more  than  a 
double  film  of  orientated  lipid  molecules  in  the  erythrocyte.  This 
eliminates  the  possibility  of  a  protein-hpid  layer  composite  body, 
such  as  demonstrated  in  chloroplasts.  To  account  for  the  lamellar 
birefringence,  therefore,  one  is  forced  to  assume  that  the  stromatin 
protein  is  lamellar  with,  maybe,  layers  of  water  in  between.  These 
need  not  necessarily  be  continuous;  indeed,  they  are  more  probably 
cavities  shaped  somewhat  Hke  lenses  (Fig.  136,  p.  272).  On  this  as- 
sumption the  direction  in  which  the  stromatin  molecules  of  Fig.  135 
(p.  267)  (layer  IV)  are  orientated  must  undoubtedly  be  turned  through 
an  angle  of  90°  and  lie  parallel  to  the  erythrocyte  surface. 

MiTCHisoN  (1950b)  is  of  opinion  that  the  small  amount  of  hpids 
cannot  contribute  anything  to  the  birefringence  of  the  erythrocyte. 
According  to  him,  the  birefringence  of  a  bimolecular  lipid  layer  6  m/t 
thick  is  not  measurable,  due  to  diffraction  errors.  He  attributes  both 
the  negative  form  and  the  positive  intrinsic  double  refraction  to  the 
stromatin  by  assuming  that  radially  oriented  looped  polypeptide 
chains  are  lodged  in  the  submicroscopic  stromatin  layers.  Such  an 
arrangement  seems  to  be  rather  unlikely. 

Electron  microscopy.  Apart  from  fibres  and  diatoms,  erythrocytes 
were  the  first  cytological  object  to  produce  good  and  impressive 
images  in  the  electron  microscope  (Wolpers,  1941).  This  is  due  to 
their  ability  to  withstand  complete  drying  without  any  essential  change 
in  structure. 


272 


FINE-STRUCTURE  OF  PROTOPLASM 


11 


The  photographs  of  the  residue  of  haemolysis  (Fig.  155)  merely 
show  a  folded  membrane.  No  inner  structure  is  visible,  which,  ac- 
cording to  WoLPERS,  proves  the  balloon  theory  of  the  structure  of 
erythrocytes.  The  average  thickness  of  the  membrane  is  25  m/j,. 
However,  this  measurement  by  Wolpers  refers  to  the  dried  envelope 
which  has  been  reduced  to  1/25  of  its  thickness  in  vivo. 

After  suitable  extraction  of  the  lipid,  Wolpers  found  the  erythro- 


1 -.  ■»  "■ 


Fig.  135  Fig.  136 

Fig.  135.   Membrane  of  red    cells,  osmotic  tixation.  Electron  microscope  9500:1  (from 
Wolpers,    1941).  Fig.    136.   Stretched  membrane  of  red  cell  in  electron   microscope. 
Image  scale  51,000:1   (from  Wolpers,  1941). 


cyte  membrane  to  be  porous.  He  therefore  discards  as  improbable  the 
layer  structure  inferred  from  observations  in  the  polarization  micro- 
scope. He  also  rejects  the  idea  of  a  mosaic  structure,  which  his  electron 
micrographs  would  at  first  sight  seem  to  suggest;  for  he  detected  a 
network  structure  in  stretched  erythrocyte  membranes  which  had  been 
fixed  with  osmium  tetroxide  after  extraction  of  the  lipids  (Fig.  136). 
This  induces  him  to  believe  that  the  stromatin  has  ^i  frame  structure, 
in  the  meshes  of  which  he  imagines  the  lipids  to  be  embedded. 
Whether  this  opinion  is  shared,  or  the  meshes  are  thought  to  be  free 
from  lipids  and  filled  with  an  aqueous  phase,  depends  upon  the  re- 
jection or  acceptance  of  a  superficial  double  film  of  lipid.  However 
this  may  be,  the  optically  proved  lamellar  structure  must  not  be 
ignored;  rather  should  an  attempt  be  made  to  reconcile  the  two 
findings. 


^  ERYTHROCYTES  273 

A  consistent  picture  is  obtained  if  the  filamentous  protein  frame  is 
thought  of  as  stratified  parallel  to  the  surface  and  the  meshes  as 
shallow,  tangentially  extended  lenses,  when  the  body  of  the  frame- 
work will  exhibit  layer  birefringence.  Under  these  circumstances, 
certainly,  no  pores  would  be  visible  in  the  top  view  of  the  skin.  The 
impression  received  is  that  sieve-like  images  are  artefacts  and  not 
natural  structures.  This  suspicion  is  strengthened  when  one  examines 
Jung's  photographs  (1942)  of  erythrocyte  membranes  denatured  by 
heat  haemolysis.  There  are  similar  sieve  images,  with  even  larger  pores. 

More  recent  electron  micrographs  of  ghosts  by  Bessis  and  Bricka 
(1949)  and  2aCek  and  Rosenberg  (1950)  do  not  show  any  sieve  pores, 
but  a  coherent  fine  granulated  structure.  Without  doubt  the  surface 
of  these  membranes  is  formed  by  aggregated  globular  protein  mole- 
cules, which  leave  only  small  capillaries  between  each  other.  It  is  open 
to  discussion  how  the  haemoglobin  molecules  with  a  diameter  of 
action  of  74. 5  A  can  diffuse  across  such  a  membrane  with  the  velocity 
characteristic  for  haemolysis.  Probably  the  texture  of  the  hydrated 
membrane  is  much  looser  in  vivo  than  in  the  completely  dried  state 
necessary  for  the  electron  microscopic  observation.  If  the  capillaries 
in  the  membrane  appear  to  be  too  narrow  for  haemoglobin,  haemo- 
lysis must  locally  destroy  submicroscopic  parts  of  the  membrane 
where  the  haemoglobin  can  freely  escape.  The  electron  microscopy 
does  not  give  evidence  of  any  such  mosaic  structure  of  the  erythrocyte 
membrane,  which  has  often  been  postulated  for  the  understanding  of 
the  complicated  permeability  phenomena  (Ponder,  1948). 

Putting  together  what  we  know  with  fair  certainty  of  the  sub- 
microscopic  intrinsic  structure  of  the  erythrocyte  membrane,  we  must 
come  to  the  conclusion  that  the  stromatin  has  a  coherent  texture  which 
appears  to  be  laminated,  on  account  of  the  form  birefringence.  In  the 
dry  state  there  are  lens-shaped  or  flat  submicroscopic  spaces.  The 
lipids  envelop  the  whole  surface  of  the  erythrocytes  in  a  continuous 
film.  The  quantity  of  lipid  is  too  small  for  a  protein-lipid  layer  body. 
In  the  hydrated  state  the  stromatin  is  considerably  swollen  and  it  is 
likely  that,  in  vivo,  the  spacious  meshes  of  this  dilute  gel  are  filled 
with  haemoglobin,  which  assumes  the  special  state  of  a  solute  in  close 
packing. 

This  expose  of  the  microstructure  of  erythrocytes  demonstrates 
impressively  the   fact  that  submicroscopic   morphology  cannot  be 


274  FINE-STRUCTURE  OF  PROTOPLASM  II 

inferred  from  either  the  indirect  methods,  or  from  direct  electron 
microscopy,  alone,  but  that  the  two  modes  of  enquiry  should  be 
complementary  and  the  results  obtained  with  the  one  should  be 
scrutinized  in  the  light  of  the  data  produced  by  the  other. 


§  5 .  Gametes 

Gametes  are  very  highly  differentiated  cells  with  the  faculty  of 
transmitting  to  the  zygote  the  capacity  of  developing  all  the  pro- 
spective properties  of  the  future  organism.  For  that  reason,  their  sub- 
microscopic  structure  is  of  particular  interest.  The  results  attained  in 
this  direction  are  still  rather  scanty;  but  there  are  already  some 
interesting  electron  microscopic  investigations  on  the  fine-structure  of 
gametes  which  are  reviewed  below. 

a.  Spermatozoa 

The  tails  of  certain  spermatozoa  are  positively  birefringent,  whereas 
their  heads  are  negative  (Schmidt,  1937a).  The  inference  is  that  the 
anisotropy  of  the  tail  is  due  to  protein  fibrils,  that  of  the  head  to  the 
inclusion  of  orientated  nucleic  acid  (Fig.  125a,  p.  228). 

The  head  of  the  sperm  being  too  thick  for  the  transmission  of 
electrons  in  the  electron  microscope,  only  details  of  its  surface  can  be 
explored;  but  the  thinner  tail  offers  excellent  conditions  for  such  an 
investigation,  and  the  submicroscopic  structure  of  this  part  of 
spermatozoa  is  now  thoroughly  known. 

Using  the  microscopic  information  available,  Bretschneider 
(1949b)  has  drawn  the  diagram  of  Fig.  137  as  a  result  of  his  electron 
microscopic  investigations.  A  strong  nuclear  membrane  of  protein 
fibrils  causes  the  characteristic  shape  of  the  head,  which  contains  the 
chromosomes.  It  is  enveloped  by  a  thin  layer  of  cortical  cytoplasm. 
The  apex  is  covered  by  the  so-called  head  cap  consisting  of  a  highly 
hydrated  gel  that  plays  an  important  role  in  fertilization  (Bret- 
schneider, 1950b).  Its  distal  end  is  marked  by  a  sharp  line  in  the 
cortical  plasm.  The  basal  part  of  the  head  is  covered  by  a  very  thin 
sheath,  the  head  tunica.  There  is  a  collar  formed  by  a  ring-shaped 
membrane  around  the  base  of  the  head,  where  the  tail  is  fastened. 

The  tail  consists  of  9  microfibrils   into  which  the   axon  of  the 


GAMETES 


275 


flagellum  can  be  split.  Eight  of  these  microfibrils  are  arranged  in  a 
tube  and  their  ends  are  connected  to  the  base  of 
the  head.  They  surround  the  ninth  microfibril.  This 
central  fibril  is  fastened  to  the  centrosome  which  is 
situated  in  the  crater-shaped  base  of  the  head. 
These  9  microfibrils  are  enveloped  by  a  helical 
sheath  consisting  of  a  double  spiral,  each  band  of 
which  is  about  1 70  m// thick.  The  spiral  body  origin- 
ates from  mitochondria;  it  is  rich  in  lipids.  It 
ends  with  the  so-called  ring  of  Jensen  who  had 
discovered  the  spiral  body  in  the  ordinary  micros- 
cope (1887).  Further  on  the  axon  is  covered  by  a 
thin  cortical  membrane,  which  again  has  a  helical 
texture  (tail  spirals).  It  consists  of  microfibrils  about 
50  m//  thick  with  a  low  pitch  making  about  150 
spiral  windings  around  the  axon. 

The  terminal  part  of  the  tail  protrudes  from  the 
cortical  membrane  showing  the  uncovered  axon. 
Usually  this  part  is  slightly  curved  or  sharply  bent 
at  the  end  (Fig.  137).  When  bull  sperm  is  dried, 
the  microfibrils  of  the  axon  fall  apart,  forming  a 
tiny  brush  which  is  an  artefact.  In  human  sperm 
this  is  not  the  case. 

It  is  remarkable  that  the  number  of  9  microfibrils 
is  not  only  characteristic  for  the  sperm  tail  of  many 
vertebrates  (e.g.  Corregonus;  Rotheli,  Roth  and 
Medem,  1950),  but  also  for  some  invertebrates 
investigated,  such  as  sea-urchins  and  coleoptera 
(Bretschneider  1948).  In  ram  spermatozoa  12 
microfibrils  have  been  found  (Randall  and  Fried- 
lander,  1950),  6  of  which  form  a  tube  surrounding 
a  sixfold  central  fibril.  Minute  details  of  the  connec- 
tions of  the  tail  fibrils  to  the  head  and  the  compli- 
cated helical  textures  of  the  spiral  body  and  the  corti- 
cal membrane  are  also  described  by  these  authors. 

In  algae  there  are  spermatozoa  with  hairy  flagella 
(in  German  "Flimmergeisseln").  With  Euglena  and  Monas  the  hairs 
have  been  discovered  in  the  ordinary  microscope  (Fischer,  1894). 


Fig.  137.  Fine- 
structure  of  the 
sperm  (from  Bret- 
schneider, 1949). 
I  head  cap ;  2  chro- 
mosomes ;  3  head 
tunica  (external 
layer) ;  4  ring- 
shaped  membrane ; 
5  centrosome;  6 
articular     strands ; 

7  axial    filament; 

8  double  helix 
(Jensen's  spiral 
body);  9  Jensen's 
ring;  10  cortical 
helix;  11  terminal 

piece. 


276 


FINE-STRUCTURE  OF  PROTOPLASM 


II 


They  are  of  the  same  order  of  size  as  bacterial  cilia  and,  like  them, 
can  only  be  made  visible  under  the  light  microscope  by  methods 
which  increase  their  width,  for  example,  by  the  use  of  an  apposition 
stain.  They  are  also  described  for  aquatic  fungi,  certain  brown  flagel- 
lates (Chrysophyceae)  and  the  zoospores  of  the  Heterocontae  among 
algae. 

In  the  electron  microscope  these  hairs  are  very  conspicuous  (Brown, 
1945;  Foster  and  co-workers,  1947;  Houwink,  195 i).  Manton  and 
Clarke  (1950)  have  discovered  that  the  longer  one  of  the  two  flagella 
of  the  spermatozoa  of  Fucus  is  also  hairy. 

It  will  be  an  interesting  task  to  find  out  whether  these  hairs  are 
active,  like  the  bacterial  cilia,  or  whether  they  are  passive  microfibrils 
split  off  the  fibrous  flagellum  in  order  to  increase  its  propulsive  power. 

b.  Eggs 

There  is  a  wealth  of  information  on  the  birefringence  of  the  cortex 
(Fig.  138)  of  the  sea-urchin  egg  (Monroy,  1945;  Monroy  and  Mon- 
talenti,    1947),  indicating  that  this  layer  is  a  lipo-protein  system 


Fig.  138.  Tubifex  egg  (from  Lehmann,  1947). 


(Ohman,  1945).  After  fertilization,  the  double  refraction  disappears 
for  15-20  minutes,  indicating  an  activation  of  the  cortical  layer  by 
temporary  hydration  and  disorientation.   Similar  structural  changes 


5  GAMETES  277 

have  been  observed  in  the  dark  iield  microscope  (Runnstrom,  1928/29). 

The  contents  of  the  egg  are  liquid.  They  can  be  stratified  by  cen- 
trifugation  into  layers  of  yolk,  fibrillar  cytoplasm  and  enchylema  with 
mitochondria  (Fig.  113,  p.  195).  The  cytoplasmic  fibrils  are  double 
refracting;  they  carry  the  ribonucleic  chromidia  (Monne,  1946a). 

The  egg  of  Tubifex  has  a  much  thinner  cortical  layer,  which  is  easily 
destroyed  by  lipid  solvents.  The  ground  cytoplasm  consists  of  fibrils 
which  are  beaded  by  chromidia.  In  the  electron  microscope  the 
chromidia  measure  0.15 /^  (Lehmann  and  Biss,  1949).  The  fibrils, 
whose  diameter  is  smaller  than  o.  i  fx,  form  a  coarse  meshwork  which 
harbours  the  microscopic  yolk  granules  (ca,  2  fi  diameter).  This  gel 
can  loosen  its  junctions,  so  that  the  fibrils  display  protoplasmic  flow, 
which  is  the  case  during  anaphase  and  telophase  of  mitosis.  In  this 
state  the  egg  content  is  liquid  and  can  be  stratified  by  centrifugation 
in  the  same  way  as  described  for  the  sea-urchin  egg. 

At  the  two  poles  the  protoplasm  of  the  Tubifex  egg  is  clearly 
differentiated  into  regions  of  animal  and  of  vegetal  cytoplasm.  These 
differentiations  are  microscopically  visible  because  they  contain 
strongly  basophilic  granules  ^Fig-  158)-  Prior  to  fertilization  the  cyto- 
plasm of  either  pole  can  be  forced  across  the  cell  by  centrifugation 
and  united  with  the  cytoplasmic  opposite  pole  (Lehmann,  1948).  There 
is  no  mixture  with  the  central  fibrillar  cytoplasm.  The  stratification 
produced  is  stable  in  Tubifex  eggs,  whereas  in  other  cases,  as  e.g.  in 
Limnaea  eggs  {Molhisca),  the  original  arrangement  is  restored  by 
protoplasmic  flow  (Raven  and  Bretschneider,  1942). 

Tubifex  eggs  with  displaced  polar  cytoplasm  can  develop  normal 
embryos.  But  they  do  not  do  so  when  the  centrifugation  has  been 
applied  too  early.  Lehmann  (1948)  thinks  that  the  polar  regions 
differentiate  at  the  expense  of  the  yolk  and  that  their  development  is 
interrupted  after  the  centrifugal  displacement.  Since,  during  the 
cleavage  of  the  egg,  the  polar  cytoplasm  can  be  traced  into  definite 
somatic  cells,  it  can  be  shown  that  in  germs  with  abnormal  develop- 
ment those  somatic  cells  contain  too  small  a  portion  of  polar  cyto- 
plasm. This  shows  how  local  regions  of  the  egg  are  capable  of  inducing 
the  development  of  definite  parts  of  the  germ.  For  that  reason,  there 
are  not  only  multicellular  organizers  which  control  the  organo- 
genesis during  the  development  of  the  embryo,  but  there  are  already 
regulating  systems  on  a  lower  scale  inside  the  egg  cell.  In  this  way 


278  FINE-STRUCTURE  OF  PROTOPLASM  II 

it  is  shown  that  the  morphologically  differentiated  parts  of  the  cyto- 
plasm fulfil  different  physiological  tasks. 

Lehmann  (1948)  distinguishes  the  following  systems  in  the  cyto- 
plasm of  the  egg  of  Tubifex  which,  during  the  development  of  the 
germ,  play  a  definite  role  of  their  own:  the  cortical  layer,  the  animal 
pole  cytoplasm,  the  vegetal  pole  cytoplasm,  the  fibrillar  central  cyto- 
plasm and  the  cytoplasm  round  the  nucleus  (Fig.  138). 

It  would  be  of  great  interest  to  know  the  submicroscopic  structure 
of  these  different  types  of  cytoplasm.  Lehmann  (1950)  has  started  this 
important  electron  microscope  investigation  with  the  following  pre- 
liminary results.  The  fibrillar  cytoplasm  consists  of  coarse  beaded 
fibrils  carrying  the  chromidia  and  enclosing  the  yolk  granules  as 
mendoned  above.  The  polar  cytoplasm  has  quite  a  different  character; 
it  is  a  dense  mass  of  globular  elements  of  30-100  m^t  diameter.  These 
globules  can  associate  and  form  a  gel.  As  the  polar  cytoplasm  of  the 
egg  is  later  transferred  to  ectodermal  and  mesodermal  cells,  they 
have  been  investigated  individually.  The  ectodermal  cells  contain 
similar  globules  (30-100  m^<),  but  the  mesodermal  cells  produce  large 
ellipsoidal  globules  of  the  dimensions  600  x  300  m// or  300  X  200  m^. 
It  is  open  to  discussion  how  these  large  particles  evolve  from  the 
smaller  globules  in  the  polar  cytoplasm.  From  their  density  in  the 
electron  micrograph  they  are  thought  to  contain  phosphorus.  This 
together  with  their  microscopical  size  (ca.  0.3  fj)  makes  it  look  as 
though  they  are  related  to  the  basophilic  granules  which  characterize 
the  polar  cytoplasm  in  the  ordinary  microscope  (Fig.  138,  p.  276).  It 
is  strange  that  they  should  not  have  appeared  on  the  electron  micro- 
graph of  the  polar  cytoplasm.  All  these  large  basophilic  granules  with 
a  high  phosphorus  content  are  probably  not  structural  elements  at  all, 
but  the  seat  of  important  metabolic  processes. 


III.  FINE-STRUCTURE 
OF  PROTOPLASMIC  DERIVATIVES 

The  distinctive  feature  in  the  structure  of  living  protoplasm  is  the 
absence  of  homogeneous  lattice  regions,  whereas  the  intrinsic  struc- 
ture of  protoplasmic  derivatives  is  as  a  rule  conditioned  by  the  ar- 
rangement of  the  molecular  elementary  units  in  some  lattice  order. 

o 

This  is  due  to  the  fact  that  protoplasm  is  made  up  of  many  varying 
kinds  of  molecules  (including  specifically  different  polypeptides), 
whereas  the  high-polymer  constituent  of  a  protoplasmic  derivative 
generally  consists  of  one  particular  kind  of  macromolecules  which 
combine  to  form  an  orderly  pattern  with  comparative  ease  and  thus 
lends  itself  to  X-ray  analysis.  For  this  reason  we  are  much  better 
informed  on  the  submicroscopic  structure  of  these  mesoplasmatic, 
metaplasmatic  and  alloplasmatic  cell  constituents  than  on  the  intrinsic 
structure  of  the  living  substance.  Thus,  while  we  can  only  trace  the 
intrinsic  structure  of  protoplasm  in  general  outline,  we  have  abundant 
quantitative  data  concerning  the  ultrastructure  of  highly  diiferentiated 
cytoplasm,  frame  and  reserve  substances.  This  part  of  the  monograph 
will  deal  with  those  structures  disclosed  up  to  date  and  will  be  con- 
cerned less  with  the  physico-chemical  than  with  the  biological 
questions  inherent  in  the  theory  of  microstructure.  The  macro- 
molecular  substances  making  up  the  bulk  of  the  structures  concerned 
are  mentioned  within  brackets  after  the  sub-titles. 

§  I.  Carbohydrates,  Chitin  and  Cutin 

a.  Meristen/atic  Plant  Cell  Walls  (Cellulose) 

The  primary  cell  wall.  There  is  a  physiological  and  histochemical 
difference  between  the  primary  cell  wall  of  vegetable  meristems  and 
the  secondary  membranes  of  fully  grown  tissues.  It  is  mainly  in  their 
surface  growth  that  this  difference  stands  out.  the  secondary  wall 
layers  being,  on  the  contrary,  deposited  by  apposition  against  the  ex- 
panded primary  wall  during  the  corresponding  growth  of  the  mem- 
brane in  thickness.  In  many  respects,  therefore,  the  primary  mem- 


28o         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

branes  behave  very  differently  from  the  strong  secondary  walls.  They 
lack  microscopic  lamination  and  iibrilization.  Since  they  represent  the 
intermediate  membrane  between  neighbouring  cells,  they  consist  of 
three  lamellae,  viz.,  the  original  middle  lamella  produced  from  the 
cell  plate  during  cell  division,  and  the  two  primary  walls  added  on 
to  it.  Another  important  point  about  meristematic  cell  walls  is  that 
no  cellulose  can  be  identified  microchemically  in  them  (Tupper-Carey 
and  Priestley,  1923).  Gundermann,  Wergin,  and  Hess  (1937) 
nevertheless  detected  by  X-rays  the  fibre  period  of  cellulose  in  the 
elongating  cells  oi  Avena  coleoptiles  (after  removal  of  the  epidermis). 
As  their  photographs  show  only  the  interferences  of  the  lattice  planes 
perpendicular  to  the  chain  axis,  evidently  the  cellulose  strands  present 
are  either  poorly  crystallized  or  the  X-ray  pattern  is  disturbed  by  the 
large  amount  of  pectic,  hemicellulosic  and  other  non-cellulosic  wall 
substances.  Thimann  and  Bonner  (1933)  found  by  analysis  42% 
of  cellulose  in  dried  A.vena  coleoptiles  but,  just  as  in  Heyn's  X-ray 
investigations  (1933,  1934),  this  percentage  includes  the  epidermis 
with  thickened  walls  (Fig.  140b,  e,  p.  284).  Although  unthickened 
meristem  walls  contain  less  cellulose,  they  certainly  contain  an  already 
cohesive,  fine  framework  of  cellulose  strands.  Seeing  that  the  cellulose 
is  masked  by  other  constituents  of  the  membrane  (see  p.  287),  it  is 
particularly  fortunate  that  its  presence  can  be  betrayed  by  its  bire- 
fringence. Pectins,  which  accompany  cellulose,  have  only  very  rarely 
been  found  to  show  birefringence  in  plants  (Roelofsen  and  Kreger^ 
1951). 

The  view  I  advanced  (1935  b)  at  the  International  Botanical  Con- 
gress held  at  Amsterdam,  to  the  effect  that  quite  young  meristematic 
cell  walls  already  contain  a  submicroscopic  cellulose  framework,  was 
at  first  disputed  by  Hess  and  his  co-workers,  though  they  overlooked 
the  birefringence  of  these  cell  walls  (Hess,  Trogus  and  Wergin, 
1936).  Later,  however,  they  admitted  that  cellulose  can  be  identified 
by  X-ray  after  cold  water  extraction,  since,  after  the  removal  of  water- 
soluble  intermicellar  substances,  collective  crystallization  of  ex- 
ceedingly thin  strands  of  cellulose  takes  place  (Hess,  Kiessig,  Wergin 
and  Engel,  1939). 

Birefringence  enables  the  investigator  to  detect  when,  during  the 
formation  of  the  young  membrane  after  the  division  of  the  cellj 
cellulose  first  makes  its  appearance.  Becker  states  (1934)  that  the  so- 


I  CARBOHYDRATES,  CHITIN  AND  CUTIN  281 

called  cell  plate  in  the  phragmoplast  of  the  staminal  hairs  of  Trades- 
cantia  first  becomes  visible  as  droplets  exhibiting  a  Brownian  move- 
ment. They  do  not,  he  says,  move  along  the  spindle  filaments,  as  is 
assumed  by  others,  but  are  formed,  just  where  they  are,  by  dissoci- 
ation from  the  dense  plasm  (Becker,  1935).  The  drops  adhere  laterally 
and  form  a  grained  isotropic  membrane  which,  however,  does  not  at 
first  touch  the  side  walls  and  shows  a  pectic  reaction  (coloration  with 
ruthenium  red).  Plasmolysis  reveals  its  independence.  From  the 
moment  when  this  system  has  grown  completely  through  the  phrag- 
moplast and  has  reached  the  wall  of  the  mother  cell,  this  diaphragm 
becomes  visible  between  crossed  nicols.  Apparently  the  phragmoplast, 
split  into  two  halves,  immediately  generates  cellulose  on  its  surface 
where  it  is  in  contact  with  the  new  membrane.  It  seems  to  me  im- 
probable that  a  cellulose  frame  would  develop  from  the  droplets 
described  by  Becker.  It  is  also  difficult  to  understand  how  proto- 
pectin  could  be  formed  from  liquid  drops.  I  therefore  suspect  that 
the  drops  are  water  of  hydration  liberated  when  high-polymeric  chain 
molecules  are  built  up  in  the  cell  plate  from  sugars  of  low  molecular 
weight.  The  fact  that  the  microvacuoles  are  dyed  vitally  with  basic 
dyes  (neutral  red)  does  not  invalidate  this  view,  since  they  may  quite 
conceivably  contain  water-soluble  components,  though  they  can 
scarcely  harbour  insoluble  high-polymeric  material  such  as  proto- 
pectin  or  cellulose.  These  wall  substances  must  be  formed  submicro- 
scopically  in  the  phragmoplast  and  do  not  become  visible  until  a 
microscopic  system  of  protopectin  has  been  built  up,  against  which 
cellulose  mixed  up  with  protopectin  is  then  immediately  deposited  on 
both  sides.  Hence  the  original  middle  lamella  and  both  primary  walls 
are  already  present  in  this  very  young  state,  but  presumably  all  three 
membranes  increase  in  thickness  before  surface  growth  begins. 

Cell  elongation.  The  submicroscopic  morphology  of  elongating  cell 
walls  is  familiar.  All  meristematic  cells  capable  of  elongation  are  of 
tubular  texture,  as  has  been  demonstrated  in  the  case  of  Avena  cole- 
optiles  (SoDiNG,  1934;  Bonner,  1935),  of  the  staminal  filaments 
(ScHOCH-BoDMER,  1936;  Frey-Wyssling,  1936c),  thc  rapidly  growing 
sporogonous  stem  of  the  moss  Pellia  (Overbeck,  1934;  Van  Iterson, 
1935),  to  mention  only  a  few.  Likewise  cotton  hairs  (Wergin,  1937), 
bast  fibres  and  all  derivatives  of  cambium  (Meeuse,  1938,  i  941)  possess 
extremely  thin,  scarcely  visible  primary  walls  of  tubular  texture.  The 


282 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


cellulose  framework  of  a  wall  of  this  kind  is  illustrated  in  diagram 
by  Fig.  139,  as  derived  from  the  birefringence  and  iodine  dichroism 
o^  Euphorbia  latex  tubes  (Frey-Wyssling,  1942). 

It  should  be  borne  in  mind  that  with  tubular  texture  the  cell  wall 

is  negative  with  respect  to  the  cell  axis.  When 
elongating  tissues  are  stretched  by  mechanical 
means,  the  birefringence  of  their  cells  changes 
and  becomes  positive  (Bonner,  1935);  but  if 
they  extend  through  growth  they  remain  nega- 
tively birefringent.  We  have  to  ask  ourselves, 
therefore,  why  the  cell  wall  optics  of  artificial 
and  natural  extension  should  be  opposite. 

The  electron  microscopy  of  primary  cell  walls 
has  disclosed  a  texture  of  cellulose  strands  al- 
most identical  with  the  diagram  of  Fig.  139 
drawn  on  the  basis  of  indirect  methods  (Frey- 
Wyssling,  Muhlethaler  and  Wyckoff,  1948; 
MiJHLETHALER,  1950a).  The  Strands  are  the  same 
as  the  microfibrils  observed  in  secondary  cell 
walls  (p.  105);  their  diameters  are  almost  iden- 
tical. This  had  not  been  expected,  because  the 
fraction  of  cellulose  is  only  a  very  small  portion  of  the  total  amount 
of  wall  substances  in  primary  walls  (Table  XXV,  p.  287).  The  microfi- 
brils form  systems  which  cross  at  different  angles,  but  mostly  so  that 
an  angle  smaller  than  90°  points  in  the  transverse  direction  of  the  cell. 
This  causes  the  optical  negative  reaction  in  the  polarizing  microscope. 
A  new  fact,  however,  was  also  found,  viz.,  that  the  microfibrils  are  not 
stratified  in  superposed  planes  but  are  interwoven,  just  as  in  a  textile 
fabric.  This  is  the  reason  why  primary  walls  do  not  show  any  lami- 
nation and  cannot  be  broken  down  into  fibrils.  On  the  other  hand, 
the  question  arises  as  to  how  such  a  woven  texture  can  grow  in  area. 
An  investigation  into  the  surface  growth  of  these  membranes  has 
therefore  been  started. 

Plant  cytology  distinguishes  two  different  types  of  cell  elongation, 

termed  tip  growth  and  cell  extension.  Tip  growth  is  considered  to  consist 

in  the  addition  of  new  areas  to  the  existing  wall  at  the  distal  cell  end, 

such  as  in  elongating  root  hairs,  cotton  hairs,  pollen  tubes,  fungal 

^  k  absorption  coefficient,  n  refrative  index. 


Fig.  139.  Tubular  texture 
of  latex  tubes  (from 
Frey-Wyssling,  1942)^. 


I  CARBOHYDRATES,  CHITIN  AND  CUTIN  283 

hyphae  etc.  On  the  other  hand,  very  rapidly  expanding  cells  in  the 
tissues  of  coleoptiles,  hypocotyls,  radicles,  staminal  jfilaments  etc.  were 
thought  to  elongate  by  increasing  their  cell  surface  along  its  total 
length  owing  to  passive  extension  accompanied  by  active  intus- 
susception. 

The  process  of  the  addition  of  new  microfibrils  to  the  existing 
texture  in  tip  growth  is  difficult  to  observe  in  the  electron  microscope. 
In  growing  root  hairs  the  apex  appears  to  be  covered  by  a  felt  of 
cellulose  microfibrils  which  stiffen  the  slime  around  these  cells  (Frey- 
Wyssling  and  Muhlethaler,  1949  b),  and  those  of  the  pollen  tubes 
(VoGEL,  1950)  or  of  sprouting  sporangiophores  (Frey-Wyssling  and 
Muhlethaler,  1950)  are  so  intensely  cutinized  that  the  cellulose 
texture  is  obscured.  Cells  which  grow  in  water  do  not  present  these 
difficulties.  In  the  end  cell  of  a  Sp'iro^'ra  thread  the  microfibrils  are 
not  intermeshed  (Fig.  86b,  p.  128).  The  tip  consists  of  loose  longi- 
tudinal microfibrils  which  represent  a  kind  of  warp.  At  their  distal 
end  these  microfibrils  seem  to  be  free,  whilst  at  their  base  they  are 
tied  together  by  transverse  microfibrils  which  function  as  a  weft.  In 
this  way  a  woven  texture  results.  Soon  the  number  of  transverse 
microfibrils  exceeds  that  of  longitudinal  fibrils,  thus  producing  the 
optical  negative  reaction  of  the  fully  grown  primary  wall. 

In  order  to  investigate  the  so-called  extension  growth,  elongating 
coleoptiles  were  macerated  and  the  isolated  cells  duly  prepared  and 
observed  in  the  polarizing  and  the  electron  microscope  (Muhle- 
thaler, 1950b).  The  result  of  this  research  is  very  surprising.  It 
transpired  that  there  is  no  extension  of  the  wall  in  its  total  length, 
but  the  cell  elongation  is  due  to  a  rapid  bipolar  tip  growth.  This  is 
illustrated  by  Fig.  140.  Picture  a)  shows  an  expanding  parenchyma 
cell  of  the  oat  coleoptile  stained  with  benzoazurin  in  the  polarizing 
microscope.  The  dichroism  of  this  dyestuff  produces  deep  coloration 
when  the  direction  of  the  bulk  of  the  microfibrils  coincides  with  the 
vibration  plane  of  the  polarizer.  It  is  seen  from  Fig.  140a  that  there 
is  a  heavily  stained  cell  body  with  pits  from  which  a  long  thin-walled 
outgrowth  protrudes.  In  the  cell  body  longitudinal  ribs  are  visible 
which  correspond  to  the  cell  edges.  Fig.  140c  gives  a  detail  of  such 
a  rib  with  the  adjacent  pitted  primary  wall  in  the  electron  microscope. 
It  is  evident  that  a  wall  fortified  by  numerous  parallel  textured  ribs 
cannot  be  extended  in  the  longitudinal  direction.  Therefore,  an  ex- 


284         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


Fig.  140.  Elongating  cells  iii  Arena  coleoptilcs  {c-e  electron  micrographs),  a)  Elongating 

parenchyma    cell,    200:1;    b)   elongating   epidermal  cell,  630:1;  c)  face  and  edge  of  a 

parenchyma  cell,  8000:  i;  d)  tip  of  an  elongating  parenchyma  cell,  8000:  i ;  e)  tip  of  an 

elongating  epidermal  cell,  8000:  i  (from  Muhlethaler,  1950b). 


I  CARBOHYDRATES,  CHITIN  AND  CUTIN  285 

tension  growth  in  the  classical  sense  of  such  a  cell  is  not  possible. 
Growth  in  area  is  only  realized  in  the  two  polar  outgrowths,  of  which 
only  one  is  visible  in  Fig.  140a.  The  tip  of  such  a  process  seen  in 
the  electron  microscope  is  shown  in  Fig.  i4od.  It  is  open,  and  evi- 
dently the  same  weaving  of  a  transverse  weft  into  a  longitudinal  warp 
takes  place  as  was  described  above. 

RoELOFSEN  (1951b)  finds  an  axial  orientation  of  the  microfibrils  on 
the  outer  surface  of  the  primary  wall  of  cotton  hairs  and  a  tangential 
orientation  on  the  inner  surface.  He  thinks  that  the  outer  fibrils  have 
been  oriented  by  cell  extension.  It  is  more  likely,  however,  that  these 
longitudinal  microfibrils  represent  the  "warp"  as  seen  in  Fig.  86b 
(p.  128). 

The  impossibility  of  wall  extension  is  even  better  illustrated  by  the 
epidermal  cells  (Figs.  140b,  e).  They  elongate  in  the  oat  coleoptile 
about  150  times,  (Frey-Wyssling,  1945a),  but  during  the  whole  time 
of  this  rapid  growth,  which  lasts  four  days,  there  is  the  compact  outer 
wall,  several  //  thick,  characteristic  of  the  epidermal  cells  of  plants. 
The  electron  microscope  discloses  tip  growth,  not  only  for  the  thin- 
walled  interior  part  of  the  epidermal  cell,  but  also  for  that  very  thick 
exterior  wall.  It  is  an  amazing  thing  that,  simultaneously,  in  one  and 
the  same  cell,  a  tubular  texture  should  be  laid  down  for  the  interior 
faces  of  the  cell  w-all  and  a  parallel  one  for  the  thick  exterior  faces. 
This  fact  argues  against  any  simple  physico-chemical  origin  of  cell 
wall  textures  comparable  to  that  of  molecular  surface  films.  There  are 
unknown  morphogenetical  principles  inherent  in  the  cytoplasm 
building  the  wall.  From  Figs.  i4od  and  e  it  w^ould  seem  that  the 
cytoplasm  oozes  out  of  the  cell  in  order  to  weave  its  wall,  not  only 
from  inside,  but  also  from  outside. 

The  discovery  of  bipolar  growth  raises  the  question  whether  there 
is  any  intercalation  of  microfibrils  by  intussusception.  Hitherto  the 
growth  in  area  was  considered  to  consist  in  local  expansions  of  the 
wall  and  concomitant  insertion  of  new  cell  wall  substances  into  the 
loosened  area.  The  bipolar  growth  does  not  favour  such  a  view, 
because  it  consists  essentially  of  an  addition  of  a  new  area  to  the 
existing  wall  and  not  in  a  general  enlarging  of  the  cell  faces  by  internal 
growth.  However,  there  are  growth  phenomena,  such  as  the  enlarge- 
ment of  the  cross-section  of  plant  cells,  which  cannot  occur  by  the 
simple  additon  of  new  wall  areas.  This  growth  in  area  consists  in 


-86 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


pushing  the  cellulosic  microfibrils  apart  by  local  plasmatic  growth 
(mosaic  growth;  Frey-Wyssling  and  Stecher,  195  i;  Bosshard, 
1952). 

Actually  the  insertion  of  additional  cellulose  microfibrils  into  the 
existing  fabric  is  not  quite  as  difficult  as  it  seems  from  the  electron 


0.03(1 


Fig.  141.  Cellulose  frame  in  living  cell  walls  (from  Frey-Wyssling,  195  i). 

micrographs.  The  cellulose  texture  observed  represents  only  2.5  %  by 
weight  of  the  growing  cell  wall;  in  the  living  state  it  contains  92.5  % 
of  water  of  hydration  and  only  7. 5  %  of  wall  substances,  of  which  2/5 
are  pectins  and  hemicelluloses  which  are  removed  when  the  cells  are 
prepared  for  examination  in  the  electron  microscope.  On  the  basis  of 
these  figures  and  the  known  diameter  of  the  cellulose  microfibrils  the 
diagram  of  Fig.  141  has  been  drawn  (Frey-Wyssling,  195  i),  which 
shows  how  much  space  is  available  for  living  cytoplasm  [Christian- 
sen and  Thimann  (1950)  find  12  ^%  protein  in  the  primary  wall  of 
pea  seedlings]    and  highly  hydrated  accompanying  substances  in  a 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


287 


primary  cell  wall.  It  also  rules  out  the  possibility  of  direct  interference 
by  auxin  with  the  cellulose  frame. 

The  increased  plasticity  of  elongating  tissues  (Heyn,  193 1 ;  Soding, 
193 1 ;  ZoLLiKOFER,  1955)  is  probably  due  to  the  bipolar  protrusions 
of  the  cells, and  the  effect  of  different  ions  on  the  cell  elongation  (Wuhr- 
MANN,  1937)  must  be  sought  in  the  influence  on  the  cellulose-synthe- 
sizing cytoplasm. 

TABLE  XXV 

CHEMICAL  COMPOSITION  OF  MAIZE  COLEOPTILES  IN  mg/COLEOPTILE 

(blank  AND  FREY-WYSSLING,   I941;  WIRTH,   I946) 


Length  of 

coleoptile  in  mm 

9 

52 

55 

55/6 

Lipids 

Sugar 

Hemicelluloses    .    . 

Cellulose 

Pectin 

Protein 

Ash 

0.040 
1. 016 
0.251 
0.191 

0.052 
0.5 10 
0.160 

0.701 
2.65 1 
0.975 
0.950 
0.272 
1.018 
0.500 

0.975 
5.704 
X.571 
1.616 
0.580 
1. 651 
0.444 

0.162 
0.951 
0.228 
0.269 
0.095 
0.272 
0.078 

Sum 

Total  dry  weight    . 

2.200              6.845 

2.545              6.755 
1 

12.521 

12.400 

1 

2.055 
2.067 

Forces  of  growth.  The  classical  cytologists  considered  the  turgor 
pressure  to  be  the  driving  force  of  cell  elongation  in  plants.  The  cell 
expansion  was  ascribed  to  water  absorption  only.  This  is  not  the  case, 
however,  as  seen  from  Table  XXV,  where  the  chemical  composition 
of  expanding  maize  coleoptiles  is  summarized.  Since  there  are  no  cell 
divisions  when  the  coleoptiles  elongate  from  9  to  55  mm,  each  cell 
must  increase  all  its  constituents,  in  the  same  proportion  as  indicated 
by  Table  XXV.  The  increment  of  cell  substances  appears  to  be  very 
considerable  (Blank  and  Frey-Wyssling,  1941,  1944),  being  almost 
proportional  to  the  cell  elongation.  If  the  figures  relating  to  the  5  5  mm 
coleoptile  are  divided  by  6,  the  values  for  a  coleoptile  section  of  9  mm 
length   are   obtained    (Table   XXV,   last   column),    which   compare 


288 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


c 
o 


50 


-  40 


-  30 


-  20 


-   W 


■c 

c 
J' 


400 


300 


200 


o 
S 

o 

e 

CO 

o 
40 


30 


20 


favourably  with  those  of  the  9  mm  coleoptile;  a  real  increase  per  mm 
occurs  for  lipids,  pectins  and  above  all  for  cellulose,  which  is  compen- 
sated by  a  loss  in  proteins  and  ash.  This  investigation  proves  that  cell 
elongation  is  accompanied  by  a  most  intense  metabolism^.  Osmotic 
phenomena  are  only  accessory  manifestations  of  that  metabolism;  they 
are  never  the  cause  of  any  growth. 

BuRSTROM   (1942)  has   carefully  studied  the   osmotic  conditions 

during  cell  elongation  in  wheat 
QOnjAt  root.  It  is  seen  from  Fig.  142  that 
the  turgor  pressure  temporarily 
decreases  during  the  lengthening 
of  the  cell.  To  raise  it  to  its  initial 
level,  osmotic  material  has  to  be 
brought  into  the  cell.  Since  ener- 
gy is  required  to  transport  ma- 
terial (Arisz,  1943),  there  must 
be  considerable  respiration  dur- 
ing the  elongation  of  the  cell 
(Bonner,  1936b).  This  proceeds, 
therefore,  not  only  by  means  of 
osmotically  accumulated  poten- 
tial energy,  but  chemical  respira- 
tory energy  is  needed  as  well. 
Turgor  extension  is  at  its  greatest 
at  the  moment  when  turgor 
pressure  is  at  its  lowest,  from 
which  it  follows  that  the  wall 
then  has  its  maximum  elasticity 
(Frey-Wyssling,  1948  a,  b).  Af- 
terwards elasticity  is  obviously 
reduced  by  the  stiffening  of  the 
new  wall  areas  (Fig.  140a,  p.  284). 
It  is  curious  that,  despite  the 
turgor,  the  stretchable  bipolar 
cell  outgrowths  show  no  tendency  to  become  spherical  during  the  ex- 
tension. This  is  due  to  the  submicroscopic  tubular  texture  of  the  cells, 

1  BuRSTROM  (195 1)  produces  evidence  showing  that  cell  elongation  and  increase  of  dry 
matter  are  nevertheless  physiologically  separated  processes. 


m  -10 


0 


10 


20 

Time  units 


Fig.  142.  Osmotic  conditions  during  the 
elongation  growth  of  single  cells  in  wheat 
root  (compounded  from  various  illustrations 
in  BuRSTROM,  1942).  Abscissa:  Time  (time 
unit  is  duration  of  mitosis  in  the  tip  of  the 
root).  Ordinates:  a)  Length  of  cell  in^; 
b)  turgor  extension  in  yu ;  c)  turgor  pressure 
in  at.;  d)  osmotic  material  per  cell  in  (lo//)^ 
times  at.  (From  Frey-Wyssling,  1945  a). 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


289 


which  resists  any  such  tendency.  The  microfibrils  of  the  cellulose 
frame,  which  encircle  the  cell  horizontally  to  obliquely,  have  con- 
siderable tensile  strength  which  is  comparable  to  that  of  bast  fibres 
and  is  due  to  primary  valency  bonds.  In  the  axial  direction,  however, 
these  fibrils  are  held  together  only  by  interfibrillar  substances  of  much 


-2r- 


fa  tit.*  itit' 


Pa 


O) 


b) 


Fig.  143.  Wall  tension  in  cylindrical  cells,  a)  Anisotropy  of  the  strength  F  and  of  the 

wall  tension  p  axially  (index  a)  and  tangentially  (index  t) ;  b)  derivation  of  longitudinal 

(Pa)  and  lateral  stress  (pj).  1  length,  r  radius  of  the  cell,  d  thickness  of  the  cell  wall. 


weaker  solidity.  Consequently,  a  cylindrical  cell  of  tubular  texture  has 
less  strength  axially  than  tangentially  (Fig.  143^)-  It  is  therefore  not 
difficult  to  understand  that  the  elastic  extension  by  the  turgor  occurs 
preferentially  in  the  axial  direction. 

The  turgor  tension  in  the  cell  wall  likewise  differs  according  to  the 
direction,  and  in  the  same  sense  as  the  strength  of  the  wall.  As  the 
equation  (Castle,  1937b)  wall  tension  p  X  cross  section  of  wall  = 
turgor  pressure  T  x    liquid  cross  section  applies,  we  have 

p3-(2  7rrd)  =  T-TiT- 
pr(2ld)      =T-2rl 

for  the  axial  (pj  and  tangential  (p,)  wall  tension,  where  d  is  the  wall 
thickness,  r  the  radius  and  1  the  length  of  the  cylindrical  cells  (Fig. 
143b).  The  resultant  ratio  of  p^  to  p^  is  2:1,  i.e.,  the  tangential 
wall  tension  is  double  the  axial  wall  tension.  Although  the  lateral 
stress  in  the  extending  cell  is  twice  the  longitudinal  stress,  it  grows 
in  length  only.  This  is  possible  if  the  F^:  F^  strength  ratio  is  above  2, 
as  there  is  every  reason  to  think  it  will  be,  since  primary  valence  bonds 
are  chiefly  responsible  for  Ft,  whereas  cohesive  forces,  which  are  ten 
times  smaller,  determine  F^^   (see  Tables  III,  p.  31,  and  FV,  p.  32). 


290         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  HE 

This  means  to  say  that  elastic  cell  extension,  instead  of  giving  wa^r 
to  the  lateral  tension,  follows  the  weaker  longitudinal  stress. 

Castle  (1937b)  thinks  that  the  larger  tangential  stress  favours  the 
transverse  orientation  of  the  cellulose  strand  and  thus  causes  the 
tubular  texture.  Careful  examination  of  Fig.  140  (p.  284),  however^ 
leads  one  to  conclude  that  no  such  mechanistic  process  can  explain 
the  very  complicated  facts  of  the  submicroscopic  morphogenesis 
which  is  observed. 

CoRRENS,  who  noted  the  predominance  of  lateral  stress  in  cylindri- 
cal cells  as  far  back  as  1893,  came  to  the  conclusion  that  "the  existing 
stressing  effects"  in  the  micellar  texture  of  laminated  membranes  of 
filiform  algae  "cannot  be  responsible  for  their  orientation"  (1893,. 
p.  284),  since  laterally  and  longitudinally  orientated  systems  occur 
alternately. 

A  further  argument  which  proves  the  relative  unimportance  of  the 
turgor  pressure  in  growth  problems  is  the  study  of  energetics  during 
cell  elongation.  Assuming  the  elongation  to  be  really  an  elastic  stretch, 
which  is  later  fixed  by  intussusception,  the  work  involved  in  wait 
extension  can  be  calculated  (Frey-Wyssling,  1948a,  b).  It  turns  out 
that  this  work  is  only  i/iooo  to  i/ioo  of  the  total  energy  produced  b}~ 
the  cell  when  the  sugar  content  of  its  vacuolar  sap  is  respited.  For 
this  reason,  there  must  be  other  fundamental  processes,  such  as- 
transport  of  substances  and  biosynthesis,  which  cause  growth,  and  the 
problem  of  morphogenesis  remains  as  enigmatic  for  submicroscopic 
morphologists  as  it  was  for  microscopic  cytologists. 

The  secondary  cell  wall.  According  to  Van  Iterson  (1927)  the  sub- 
microscopical  texture  of  the  secondary  cell  wall  depends  on  the 
direction  of  flow  of  the  protoplasm  depositing  the  laminae  of  ap- 
position. Currents  of  protoplasm  can,  in  fact,  be  observed  to  circulate,, 
depositing  rings  or  bars  during  vascular  formation.  Van  Iterson 
(1937)  furthermore  tries  to  explain  the  direction  of  flow  causally.  It 
is,  he  says,  principally  axial  in  the  staminal  hairs  of  Tradescantia,  for 
example,  since,  owing  to  the  tubular  texture  of  the  cellulose  mem- 
brane, the  cells  tend  to  elongate.  However,  the  outer  cuticular  layer 
with  fibrous  texture  impedes  extension,  but  there  is  pronounced  elon- 
gation the  moment  the  cuticularized  outer  layer  of  withered  flowers 
bursts.  On  the  basis  of  these  observations  it  was  inferred  that,  owing 
to  the  tubular  texture  of  the  primary  wall  of  embryonic  fibres,  the 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


2QI 


protoplasm  likewise  circulates  in  an  axial  direction  and  the  nascence 
of  the  fibrous  texture  of  the  secondary  wall  could  be  explained  as 
being  causally  mechanistic.  Van  Iterson  now  goes  so  far  as  to  suggest 
as  an  explanation  for  the  crosswise  layers  of  the  Valonia  cell  wall 
(brought  into  prominence  by  X-ray  investigation)  that  the  proto- 


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Fig.  144.  Diagram  of  intercalation  of  wax  in  meristematic  cell  walls  of  tubular  texture. 
a)  Radial  section;  U)  tangential  and  cross-sections. 

plasm  is  forced  to  change  its  direction  of  flow  by  about  90°  after  the 
deposition  of  every  layer;  for  the  tendency  of  the  cell  to  expand  is 
always  perpendicular  to  the  direction  of  the  iibrillae  of  the  newly 
formed  layer,  for  which  reason  the  flow  of  plasm  is  supposed  to  be 
passively  directed  cross-wise  over  the  youngest  lamella. 

With  all  due  admiration  for  Van  Iterson's  reasoning,  and  con- 
ceding a  certain  contributory  role  to  the  forces  he  has  discovered,  it 
can  scarcely  be  said  that  mechanistic  theories  of  this  kind  are  at 
present  adequate  to  resolve  the  mysteries  of  morphogenesis.  For  there 
are  several  facts  of  observation  which  do  not  come  within  the  compass 
of  causation.  For  instance,  out  of  similar  cells  near  the  cambium,  to 
the  primary  wall  of  which  tubular  texture  is  ascribed,  are  differentiated 
on  the  one  hand  fibres  with  spiral  texture,  which  may  have  been 
generated  as  suggested  above,  but  on  the  other  hand,  vessels  with 
tubular  texture.  This  might  be  due  to  the  fact  that  the  fibres  have  tip 
growth,  whereas  the  vascular  members  have  not;  it  would  then, 


292  FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

however,  have  to  be  explained  why  in  one  case  the  extensibility  of 
the  primary  wall  of  tubular  texture  is  utilized,  while  the  cylindrical 
cells  of  the  vascular  members  follow  the  unconventional  course  of 
growing  in  girth  instead  of  in  length.  It  is  difficult  to  avoid  assuming 
the  existence  of  internal  formative  forces  when  the  purposiveness 
inherent  in  each  individual  cell  development  becomes  apparent  again 
and  again. 

Intercalation  oj  wax.  The  discovery  by  X-ray  of  the  intercalation  of 
wax  has  enriched  our  knowledge  of  the  structure  of  the  primary  cell 
walls.  In  young  cotton  hairs,  A.vena  coleoptiles  without  epidermis  and 
many  meristematic  tissues,  Hess  and  co-workers  (1936)  found  X-ray 
interferences  corresponding  to  periods  of  60  and  83  A.  By  extraction 
these  substances  were  isolated  and  identified  as  vegetable  waxes 
(GuNDERMANN,  Wergin,  and  Hess,  1937).  They  are  comparatively 
short  chains  of  the  type  QHgn+jCO-O-C^^Hoj^  +  j,  n  and  m  amounting 
to  about  24  or  32,  as  established  for  other  vegetable  waxes  by  Chib- 
NALL,  Piper,  and  co-workers  (1934). 

As  these  waxes  produce  far  clearer  interferences  than  cellulose,  of 
which  often  only  the  fibre  period  appears,  they  must  be  assumed  to 
be  better  crystallized  than  the  cellulose  chain  molecules.  The  possi- 
bility therefore  exists  that  waxes  of  this  kind  are  in  part  the  source 
of  the  birefringence  of  the  primary  cell  walls.  Pursuing  this  problem 
as  presented  by  the  meristematic  cell  walls  of  A.vena  coleoptile,  K.  and 
M.  Wuhrmann-Meyer  (1939)  established  that  the  birefringence  is 
affected  by  the  fatty  wax  component  susceptible  of  extraction  by 
pyridine.  Though  this  effect  is,  admittedly,  lacking  in  the  radial 
sections  through  the  cells,  it  appears  in  the  tangential  and  cross- 
sections.  From  this  it  may  be  inferred  that  the  rod-shaped  wax  mole- 
cules are  orientated  at  right  angles  to  the  microfibrils  of  the  tubular 
texture;  then  there  is  isotropy  on  the  radial  section,  whereas  on  the 
tangential  and  cross-sections  we  have  a  birefringence  which  is  the 
reverse  in  character  of  that  of  cellulose,  as  will  be  clear  from  Fig.  144. 

The  waxes  being  extremely  hydrophobic  and  the  cellulose  chains 
very  hydrophilic,  there  can  be  no  direct  contact  between  these  two 
cell  wall  substances,  so  that  an  intermediate,  polar  substance  is  in- 
terposed (Frey-Wyssling,  1 93 yd).  Possible  molecules  with  hydro- 
philic and  hydrophobic  end  groups  are  phosphatides  (Hansteen- 
Cranner,  1926).  Seeing  that  Thimann  and  Bonner  (1933)  found  no 


I  CARBOHYDRATES,  CHITIN  AND  CUTIN  295 

phosphatides  in  the  membranes  of  Avena  coleoptile,  the  question 
arises  as  to  whether  the  wax  alcohols  and  fatty  acids  in  the  primary 
walls  occur  in  the  unesterihed  state,  in  which  case  their  hydrophilic 
pole  would  be  connected  with  the  cellulose  threads.  It  will  be  evident 
from  Fig.  144  why  the  primary  cell  walls  can  be  stained  with  fatty 
acid  dyes,  whereas  the  individual  cellulose  strands  seem  to  be  "masked". 
Physiologically  this  intercalation  of  wax  results  in  the  impaired 
permeability  ot  the  wall  to  water,  ions  and  lipophobic  molecules,  as 
these  substances  are  admitted,  not  through  the  entire  meshes  of  the 
intermicellar  spaces  but  only  through  the  hydrophilic  regions  in  the 
vicinity  of  the  cellulose  strands. 

b.  Cutini':(ed  Cell  Walls  (Cut in) 

Mkrochemistry  and  optics  of  CMtini':(ed  epidermises.  The  morphology  of 
the  thick  cuticular  layers  of  the  leaf  epidermises  of  xerophytes  (Fritz, 
1935,  1937)  is  particularly  interesting,  in  that,  although  optically  often 
appearing  to  be  homogeneous,  they  contain  at  least  four  different 
membranous  substances,  the  submicroscopic  arrangement  of  which  is 
known.  Our  starting  point  will  be  the  optics,  investigated  by  Am- 
BRONN  (1888),  of  the  cuticular  layers  which,  in  the  polarizing  micro- 
scope, behave  in  a  reverse  sense  to  the  cellulose  layers  lying  beneath 
them.  The  cellulose  component  appears  optically  positive  with 
reference  to  the  tangential  direction  of  the  cell  wall,  while  on  the 
contrary  the  cuticular  layer  is  optically  negative  (Fig.  145a).  Extern- 
ally, the  epidermis  is  bounded  by  the  almost  isotropic  cuticle  and 
between  the  cellulose  and  cuticular  layers  is  interposed  a  fairly  wide 
isotropic  layer  of  pectins  (Anderson,  1928).  Ambronn  had  already 
suspected  that  the  optically  negative  reaction  of  the  cuticular  layers 
was  caused  by  intercalated  waxes,  but  this  property  was  later  attributed 
to  the  cutin.  Madeleine  Meyer  (1938),  however,  demonstrated  by 
careful  micromelting  tests  (Fig.  145  b)  that  the  negative  birefringence 
derives  from  a  fusible  wax,  while  the  residual  cutin  proves  to  be  almost 
isotropic.  In  many  cases,  of  which  Gasteria  is  an  example,  a  slightly 
positive  birefringence,  due  to  cellulose,  makes  its  appearance  after  the 
waxes  have  melted  out.  Hence,  besides  the  cutin,  the  cuticular  layer 
must  also  contain  cellulose  and  even  pectins,  which  can  be  identified 
by  ruthenium  red.  The  optics  of  the  longitudinal  section  discloses  the 
fact   that   these   four   cell   wall    substances    (Table    XXVI)    are    not 


294         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

TABLE  XXVI 
CELL  WALL  SUBSTANCES  OF  THE  CUTICULAR  LAYERS 


Optical  behaviour 

TTV 

referred  to  tan- 

Coloured by 

Solubility 

Disintegration 

Absorption 

gential  direction 

by 

Cutin 

Isotropic 

Basic  lipid  dyes 

Insoluble 

NaOH 

saponification 

Strong 

Cutih  waxes 

Opt.  negative 

Lipid  dyes 

Pyridine 

Melting 
above  220° C 

Lacking 

Cellulose 

Opt.  positive 

Iodine-zinc 
chloride  sol. 
(dichroism) 

SCHWEIZER 

reagent 

Hydrolysis 

Lacking 

Pectins 

Isotropic 

Ruthenium  red 

Picric  acid 
followed 
by  H2O2 

Hydrolysis 

Lacking 

Clivia 


Gasieria 


Yucca 


Dasylirion 


A 


20^ 
12° 


4 


(\ 


12    3     4 


[\ 


'-^yr* 


12    3    4 


2(f        40° 


Fig.  145.  Cuticular  layers  of  vegetable  epidermises 
(from  M.Meyer,  1938).  a)  Optics  of  longitudinal 
section.  Ordinate:  Relative  strength  of  bire- 
fringence. Abscissa:  i  cellulose  layer,  pos.  bire- 
fringent;  2  pectin  layer,  isotropic;  3  cutinized 
''•'  wall,  neg.  birefringent;  4  cuticle,  isotropic,  h) 
Hysteresis-melting  curve  of  the  cutin  wax  of 
Clivia,  measured  by  reduction  in  birefringence 
of  the  section.  Ordinate:  Retardation  in  degrees 
of  the  Senarmont  compensator.  Abscissa: 
Temperature  T  in  °C. 


60'' 


evenly  distributed  over  the  thickness  of  the  cuticular  layer.  In  Clivia, 
for  instance,  only  an  inner  zone  — which  iodine-zinc  chloride  solution 
tints  dark  brown — clearly  contains  cellulose.  The  waxes  are  in 
greatest  evidence  in  the  middle  of  the  layer,  so  that  it  is  there  that 


1  CARBOHYDRATES,  CHITIN  AND  CUTIN  295 

the  retardation  is  at  its  most  negative  (Frey,  1926b).  The  wax  content 
diminishes  outside  and  the  cuticle  contains  no  wax  at  all,  consisting 
•of  pure  cutin  (Fig.  145a). 

In  hydrophytes  the  cutinization  of  the  epidermis  is  confined  to  a 
thin,  optically  isotropic  cuticle.  It  is  probable  that  all  cell  walls  that 
are  in  contact  with  air  are  superficially  cutinized,  since  Elsa  Hauser- 
MANN  states  (1944)  that  the  cells  of  mesophylls,  which  serve  to 
ventilate  the  leaf,  are  covered  with  a  submicroscopical  film  of  cutin. 

Molecular  stridcture  of  lipophilic  cell  wall  substances.  To  understand  the 
submicroscopical  arrangement  of  the  four  cell  wall  substances  in 
cuticular  layers  it  is  necessary  to  know  the  morphology  of  their  mole- 
cules. We  shall  therefore  have  to  consider  briefly  the  chemistry  of  the 
waxes  and  of  the  very  imperfectly  known  cutin.  Unlike  the  enormously 
long  cellulose  chain  molecules  and  the  very  long  pectin  chains,  the 
waxes  are,  as  already  mentioned,  short  rod  molecules  of  less  than 
100  A  length.  In  the  simplest  case  they  consist  of  higher  aliphatic 
alcohols,  the  corresponding  fatty  acids  and  higher  paraffins.  According 
to  Kreger  (1949),  there  is  no  stoichiometrical  relation  between 
alcohols  and  acids.  Therefore,  the  plant  waxes  are  only  partly  esters, 
the  rest  being  mixtures  of  higher  alcohols,  paraffins  and  fatty  acids, 
with  a  predominance  of  the  first  two.  The  alcohols  and  fatty  acids 
have  even-numbered  chains  between  C24  and  C34  (Chibnall,  Piper 
and  their  collaborators,  1934);  for  instance,  myricyl  alcohol  CgoHgjOH 
or  cerotic  acid  C25H51COOH.  If  esters  occur  they  have  the  same 
overall  formula  as  fatty  acids  C^HanOg.  On  the  other  hand,  the  paraf- 
fins have  odd-numbered  chains  between  C27  and  C31  (Kreger,  1949); 
e.g.,  n-noneicosane  CogHgo.  As  indicated  in  the  last  formula,  the  mole- 
cules of  plant  waxes  are  always  unbranched  chains. 

Besides  the  aliphatic  waxes  the  cutinized,  and  especially  the 
suberized,  membranes  contain  the  waxes  cerin  and  friedelin,  which 
have  a  substantially  lower  hydrogen  content.  The  inference  therefore 
is  that  they  contain  aromatic  rings  and  thus  approximate  the  sterols, 
which  represent  the  cycHc  alcohols.  Luscher  (1956)  states  that 
friedelin  and  cerin  contain  an  alcoholic  OH  group  which  can  be 
acetylated  or  otherwise  esterified,  while  the  second  constituent  O 
atom  is  masked,  presumably  as  a  cyclic  ether  bridge.  Thus  friedelin 
and  cerin  are  alcohols,  not  esters.  On  the  other  hand,  they  may 
possibly  be  esterified  with  other  molecules  in  the  membrane.  Other- 


'.^6 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


wise  nothing  is  known  of  their  constitution.  On  saponifying  the 
waxes  of  pine  needles,  Bougault  and  Bourdier  (1908)  obtained  co~ 
hydroxyfatty  acids  (such  as  hydroxylauric  acid  and  hydroxypalmitic 
acid)  instead  of  simple  acids  and  alcohols.  Molecules  of  this  kind 
possess  two  reactive  groups;  thus  they  can  together  form  esters  and 
grow  to  high-polymeric  chains,  as  shown  in  Table  XXVII.  Their 
discoverers  call  these  waxes  "estolids".  Their  degree  of  polymeri- 
zation cannot  be  very  high,  as  they  are  still  soluble  and  fusible. 

TABLE  XXVII 
lipophilic  cell  wall  substances 


Aliphatic  Waxes: 

Wax  Acids: 

CH3.  (CH,)„.  C-O^  (CH,)^.  CH3 

'    Palmitic  acid 

C15H31COOH 

Stearic  acid 

Ci^Ha^COOH 

0 

Oleic  acid 

Ci,H33COOH 

Linoleic  acid 

Ci,H3iCOOH 

Arachic  acid 

Q,H3,COOH 

(Chibnall  and  Piper,  1934; 

Cerotic  acid 

Q5H51COOH 

LiJscHER,  1936) 

Higher  fatty  acids  up  to 
Wax  Alcohols: 

QjHejCOOH 

Cetyl  alcohol 

QsHsaOH 

Octadecyl  alcohol 

QSH37OH 

Cer}l  alcohol 

QeHssOH 

Myricyl  alcohol 

QoHsiOH 

\ 


Higher  alcohols  up  to       C34H^90H 


Cyclic  Waxes: 

Molecular  structure : 
(LiJscHER,  1936) 

Estolids: 

-O-CCHa)  -C-0-(CH,)  -C-O- 

i!  "     !l 

o  o 

(Bougault  and  Bourdier,  1908J 


Cerin 

QqH 

5oO. 

Friedelin 

Q«H 

7  60, 

Hydroxyacids : 

Sabinic  acid 

(hydroxylauric 

acid) 

OH. 

^11^22 

COOH 

funiperic  acid 

(hydroxypalmitic  acid) 

OH- 

^isHso 

COOH 

Suberin,  Cutin  and  Sporopollenin: 

Molecular  structure : 

Spatial  network  through  ester 

and  ether  bridges 

(Zetzsche,  1932;  LiJscHER,  1936) 


Suberin 

Cutin 

Sporopollenin 


Saponification 
becomes  more  and 
more  difficult 


Decomposition  Products  of  Sub er in: 

Suberic  acid  COOH-  (CH,)6-  COOH 

Phloionolic  acid  Ci,H3,,(OH)3-COOH 

Phloionic  acid  COOH-  Ci6H3o(OH),-  COOH 

Phellonicacid  C2iH4.(OH).COOH 

Eicosancdicarboxylic  acid  COOH-  (CH2)3„-COOH 


I  CARBOHYDRATES,  CHITIN  AND  CUTIN  297 

The  polymerization  plan  of  the  high-polymeric  cell  wall  substances 
cutin  and  suberin  must  be  similar  to  that  of  the  estolids,  since  their 
hydrolytic  and  decomposition  products  ordinarily  exhibit  two  or  more 
reactive  groups  capable  of  esterifying  or  etherifying  (dicarboxylic 
acids,  hydroxycarboxylic  acids,  Table  XXVII).  This  is  the  distin- 
guishing feature  between  the  monomeric  molecular  residues  of  cutin 
and  suberin,  on  the  one  hand,  and  the  molecules  of  waxes  on  the 
other  (LuscHER,  1936).  Seeing  that  suberin  is  more  readily  decom- 
posed than  the  cutins  (Zetzsche,  1932),  it  is  probable  that  the  degree 
of  polymerization  or  of  interlinking  attained  within  it  is  lower  than 
in  the  latter.  It  is  presumably  at  its  highest  in  sporopollenin,  as  this 
wall  substance  is  exceedingly  resistant  to  saponification  and  decay,  so 
that  the  cell  walls  of  fungus  spores  and  grains  of  pollen  are  preserved 
for  thousands  of  years  in  peat  deposits. 

The  isolated  dicarboxylic  acids  (Table  XXVII)  may  possibly  be 
oxidized  degradation  products  of  higher  hydroxyacids ;  suberic  acid, 
COOH- (CH2)6-COOH,  for  instance,  results  from  the  oxidative  de- 
gradation of  suberin.  Probably  not  all  the  carboxyl  groups  of  the 
carboxylic  acids  in  the  membrane  are  esterified,  for  cutin  has  some 
of  the  characteristics  of  an  acid,  or  a  high-polymeric  anion  (pro- 
nounced negative  charge  Brauner,  1930,  selective  cation  perme- 
ability, staining  by  basic  dyes).  Since  its  behaviour  is  almost  iso- 
tropic, it  must  be  presumed  that  the  linkage  of  the  carboxyl  and 
hydroxyl  groups  is  not  that  of  a  linear  chain  scheme,  but  reticular  in 
aU  spatial  directions  as  in  lignin. 

Suhmicroscopic  structure  of  the  cuticular  layers.  It  now  remains  to  build 
up  a  picture  of  the  mutual  spatial  relationship  between  the  cell  wall 
substances  in  the  cuticular  layers.  A  possible  clue  is  afforded  by  the 
optical  anisotropy  of  the  suhmicroscopic  particles  of  wax.  If  their 
form  and  optics  were  known,  the  orientation  of  the  intercalated  wax 
could  be  inferred  from  the  nature  of  the  wall  birefringence. 

The  wax  molecules  are  rod-shaped  and  therefore,  when  spread  on 
a  slide,  might  be  expected  to  be  orientated  and  reveal  something  as 
to  their  intrinsic  birefringence.  Many  waxes,  like  paraffin,  fats,  phos- 
phatides and  other  lipids,  produce  w^hat  is  known  as  a  "negative 
streak"  (Fig.  146  b),  which  might  incline  one  to  conclude  that  the 
wax  molecules  are  optically  negative  with  reference  to  their  longi- 
tudinal axis.  Such  a  conclusion  is,  however,  inadmissible,  since  short- 


298 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


redded  molecules  have  a  tendency  to  crystallize  as  thin  platelets  or 
lamellae  (Fig.  146a)  and,  when  spread  out,  these  submicroscopic, 
often  plastic,  crystal  lamellae  are  orientated.  Longitudinally,  they  fall 
in  with  the  direction  of  the  stroke  and  the  molecules  then  run  perpen- 
dicular to  the  streak.  In  this  wav  the  streaks  of  paraffin  and  beeswax 


^^ 


a)       + 


b) 


=1 

c) 


W 


^ 


=-c 


/".  /\  /^ 


d) 


Fig.  146.  Submicroscopic  textures  from  optically  positive  lipid  chains.  P  paraflfin  mole- 
cules, E  estolid  molecules,  W  wax  molecules,  C  cellulose  chains,  Cu  interlinked  cutin 
chains,  a)  Paraffin  lamella  optically  positive;  b)  optically  negative  streak  of  paraffin; 
c)  optically  positive  streak  of  pine-needle  wax ;  d)  intercalation  of  cutin  wax  in  the  epidermis. 

are  negative,  but  the  molecules  themselves  are  optically  positive^.  By 
analogy  it  might  therefore  be  supposed  that  the  molecules  of  the 
vegetable  waxes  which  yield  a  negative  streak  are  positive;  but  there 
are  some  waxes  with  a  positive  streak,  as  I  found  with  estolids  from 
pine  needles  (Fig.  146c).  The  streak  test,  therefore,  tells  us  nothing 
definite  and  another  method  has  to  be  resorted  to,  which  consists 
in  dissolving  the  waxes,  in  order  that  their  molecules  may  be  rendered 
independent  of  each  other,  and  then  testing  their  intrinsic  bire- 
fringence in  a  flow  gradient. 

Ambronn  and  Frey  pointed  out  in  "Polarisationsmikroskop" 
(1926,  p.  167)  that  the  only  certain  way  of  establishing  the  intrinsic 
birefringence  of  disperse  particles  is  by  using  a  rotary  drum  in  ac- 
cordance with  Kundt's  system.  Signer  (1930,  1933)  built  a  flow- 


^  On  p.  92  it  is  explained  that  double  refraction  cannot  be  attributed  to  a  single 
molecule.  So  if  we  speak  here  of  optically  positive  molecules,  this  means  that  the  sign 
of  the  double  refraction  of  a  large  number  of  molecules,  made  parallel  by  flow  or  crystal- 
lization, is  positive. 


I  CARBOHYDRATES,  CHITIN  AND  CUTIN  299 

birefringence  apparatus  of  the  greatest  precision,  in  which,  in  spite 
of  their  Brownian  movement,  comparatively  short  rod-molecules  can 
be  orientated.  It  was  with  the  aid  of  this  apparatus  that  Weber  (1942) 
determined  the  optical  nature  of  wax  molecules.  The  experimental 
evidence  points  to  optically  positive  rod-molecules.  Thus  the  molecules 
of  the  membrane  waxes,  like  those  of  paraffin,  fats  and  other  lipids,  are 
optically  positive  rodlets. 

Since  the  waxes,  referred  to  the  tangents  of  the  cuticular  layers, 
produce  negative  birefringence,  their  molecules  must  stand  perpen- 
dicular to  the  surface  of  the  membrane.  So  perfect  is  the  orientation 
of  the  rod-molecules,  that  the  outside  layer  of  the  epidermis  of  Clivia, 
seen  from  above  after  the  removal  of  the  cellulose  layer  underneath 
it,  appears  optically  isotropic.  Hence  the  cuticular  layer  possesses  a 
radial  optical  axis. 

,  After  extraction  of  the  wax,  form  birefringence  is  exhibited  (form 
birefringence  curves  in  M.  Meyer,  1938),  this,  referred  to  the 
optical  axis  of  the  cuticular  layer,  being  negative.  This  means  that  we 
have  to  do  with  lamellar  birefringence;  hence  the  wall  layer  consists 
of  submicroscopic  lamellae,  in  the  texture  of  which,  judging  by  all 
previous  experience,  the  cellulose  of  the  cutin  layer  must  be  involved. 
The  optical  analysis  therefore  suggests  the  presence  of  submicroscopic 
cellulose  lamellae  with  exceedingly  thin  platelets  of  wax  interposed, 
the  wax  molecules  being  orientated  perpendicular  to  the  cellulose 
chains  (see  Fig.  146  d). 

Now,  in  the  presence  of  the  water,  present  not  only  in  cellulose, 
but  also  in  cutinized  cell  walls,  the  hydrophobic  wax  molecules  cannot 
come  into  contact  with  the  hydrophilic  cellulose  chains.  Thus  there 
must  be  some  intermediate  polar  substance,  and  that  is  the  cutin.  This 
wall  material  contains  both  hydrophilic  (-OH,  -COOH)  and  hydro- 
phobic (-CH3)  groups  and  it  may  be  assumed  that  the  former  incline 
more  towards  the  cellulose,  whereas  the  latter  tend  more  towards  the 
wax.  We  then  have  a  scheme  such  as  that  represented  in  Fig.  i46d. 

It  can  be  seen  in  this  model  how  the  cell  wall  substances  in  the 
cuticular  layers  are  placed  one  relatively  to  another:  hydrophilic 
lamellae  consisting  of  cellulose  and  probably  also  of  pectins,  layers  of 
wax  molecules  in  radial  arrangement  and,  in  between  them,  amorphous 
cutin  in  random  orientation.  Apart  from  the  interposition  of  the  wax, 
the  morphological  conditions  are  similar  to  those  in  lignification,  where 


500  FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

amorphous  lignin  is  intercalated  between  cellulose  rodlets  or  lamellae. 
In  both  cases  the  cellulose  is  masked  by  the  incrustation.  For  example, 
it  is  only  with  difficulty  that  the  cellulose  can  be  dissolved  out  of  wood 
with  Schweizer's  reagent,  and  hitherto  could  not  be  eliminated  at 
all  in  this  way  from  the  cutin  layers.  It  is  easier  to  saponify  the  cutin, 
or  the  suberin  (Karrer,  Peyer  and  Zegar,  1923;  M.  Meyer,  1958) 
and  to  hberate  the  cellulose. 

The  scheme  shows  the  relative  positions  of  the  four  cell  wall 
substances,  not  their  quantitative  proportions,  these  being  very 
variable.  Small  or  larger  amounts  of  the  carbohydrate  wall  substances, 
cellulose  and  pectins  can  always  be  identified  in  the  inner  regions  of 
the  cuticular  layer;  they  are,  indeed,  often  quite  prominent.  Further 
out,  it  is  the  waxes  which  are  in  greater  prominence,  with  marked 
and  sometimes  complete  decUne  of  cellulose  and  pectins.  The  outer 
layers  probably  consist  of  cutin  and  wax  only.  This  is  noteworthy  as 
compared  with  lignin  deposition,  since  cutin  can  obviously  occur  as 
an  independent  wall  substance,  whereas  lignin  is  always  found  in 
company  with  cellulose.  Finally,  there  are  no  waxes  in  the  isotropic 
cuticle  (Priestley,  1943),  which,  therefore,  comprises  only  a  thin 
pellicle  of  almost  amorphous  cutin. 

It  would  be  interesting  to  discover  the  still  quite  unknown  history 
of  the  development  of  this  complicated  submicroscopic  system 
originating  in  a  region  remote  from  the  protoplasm.  Martens  (1934) 
states  that  the  cuticle  is  secreted  in  the  fluid  state  and  then  coagulates 
in  the  air.  This  may  also  safely  be  said  to  apply  to  the  cuticular  layers. 
The  cutinic  acids  would  then  be  dissolved  in  a  low  molecular  state, 
migrate  into  the  wall  and  there  polymerize.  It  is  less  difficult  to  under- 
stand the  deposition  of  the  low-molecular  waxes,  though  even  in  this 
case  it  is  necessary  to  assume  that  there  is  some  special  solvent,  or 
that  unesterified  wax  acids  and  alcohols  migrate.  This  process  is 
similar  in  nature  to  the  excretion  of  waxes  through  the  epidermis, 
where  they  form  a  granular,  rod-shaped  or  scaly  coating  (Weber, 
1942). 

Each  component  of  the  wall  in  the  full-grown  cuticular  layer  has 
its  particular  physiological  function.  By  reason  of  its  hydrophobic 
nature,  the  primary  duty  of  the  wax  is  to  make  these  layers  watertight. 
The  cutin  has  a  similar  purpose,  though  in  a  less  extreme  degree,  since 
its  hydrophilic  groups  make  it  less  hydrophobic  and,  therefore,  it  has 


I  CARBOHYDRATES,  CHITIN  AND  CUTIN  3OI 

a  slio-ht  tendency  to  swell.  As  the  cutin  layer  strongly  absorbs  ultra- 
violet  lio-ht  (Frey,  1926  b)  and  retains  this  property  even  after  the 
waxes  have  been  extracted,  it  impedes  any  intensive  ultraviolet  ir- 
radiation of  the  mesophyll  of  xerophytes.  As  aliphatic  compounds  in 
general  do  not  absorb  ultraviolet  light,  there  must  be  some  unknown 
cyclic  compound  (cyclic  waxes)  in  the  cuticular  layer.  The  hydrophilic 
quality  of  the  lamellae  of  cellulose  and  pectins  is  responsible  for  the 
cuticular  transpiration  (Gaumann  and  Jaag,  1936)  of  the  leaves,  which 
occurs  not  only  in  hydrophytes,  but  also  in  xerophytic  evergreens. 
The  loss  of  water  is  a  sign  that  the  submicroscopic  wax  lamellae  are 
not  continuous,  but  that  the  hydrophilic  (cellulose)  and  semi-hydro- 
philic  (cutin)  regions  cohere  and  thus  offer  the  water  an  outlet. 

c.  The  Chitin  Frame  (Cbitin) 

Chitin  is  a  nitrogenous  frame  substance,  primarily  characteristic  of 
the  animal  phylum  of  Arthropoda  (Crustacea,  insects).  It  also  forms 
the  membranous  frame  oi fungi  (Harder,  1937;  R.  Frey,  1950).  The 
behaviour  of  vegetable  and  animal  chitin  is  identical,  as  has  been 
proved  for  the  sporangiophores  of  Phycomyces  chemically,  optically 
and  by  X-rays  (Diehl  and  Van  Iterson,  1935 ;  Van  Iterson,  Meyer 
and  LoTMAR,  1936).  In  the  same  way  as  the  cellulose  characteristic  of 
autotrophic  plants  may  be  built  by  both  bacteria  {Bacterium  xylinum) 
and  by  the  animal  class  of  the  Tunicata  (Fig.  86d,  p.  \z%),  fungi  are, 
inversely,  able  to  synthesize  an  animal  frame  substance.  One  cannot 
go  very  far  wrong  by  assuming  that  this  similarity  is  connected  with 
the  heterotrophic  life  of  fungi,  which,  like  animals,  have  so  much 
nitrogen  to  draw  upon  that  some  of  it  is  deposited  in  the  cell  walls 
and  is  there  immobilized.  As  there  is,  on  the  contrary,  only  a  minimum 
of  nitrogen  in  autotrophic  plants,  it  cannot  contribute  to  the  form- 
ation of  their  frame  substances;  otherwise  chitin,  which  is  more 
resistant  than  cellulose  in  many  respects,  would  certainly  also  occur 
elsewhere  in  the  vegetable  kingdom.  Morphologically,  the  two  frame 
substances  are  very  similar  in  behaviour,  as  will  be  shown  in  what 
follows,  the  micellar  frame  of  each  being  composed  of  very  long  chain 
molecules. 

Molecular  structure  of  chitin.  The  structural  unit  of  chitin  is  glucos- 
amine, i.e.,  a  pyranose  ring  in  which  an  OH  group  has  been  substi- 
tuted by  an  NHg  group  (Fig.  147a).  It  is  not  known  whether  the 


302         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

position  of  this  amino  group  at  the  z"'^  C  atom  corresponds  to  that  of 
the  OH  group  of  the  glucose  or  of  the  mannose  ring  (Itschner,  1935). 
An  acetyl  residue  is  linked  with  the  NH2  group;  thus,  contrary  to 
cellulose,  there  are  here  two  side  chains,  viz.,  an  OHCHo-group  and 
a  CHgCO-group. 

H        NH^  H        H 

OH /oh    h\h  oh /oh  nh^ 

H\i_yOH  ^\h_^/0H 

CH2OH  CH2OH 

Glucose  configuration  Mannose  configuration 

a)  Glucosamine 
NHCOCH3   CH2OH  NHCOCH3   CH2OH  NHCOCH3 

CH2OH  NHCOCH3    CHpOH  NHCOCH3    CH2OH 

h)  Chain  of  chit  in 
Fig.  147.  Molecular  structure  of  chitin. 

The  acetylglucosamine  molecules  are  linked  glucosidically  and 
form  long  chain  molecules,  each  member  of  which  is,  according  to 
Meyer  and  Mark  (1930),  twisted  with  respect  to  its  preceding  and 
succeeding  neighbour  by  180°  (Fig.  147  b).  X-ray  photographs  of  the 
sinews  of  the  spiny  lobster  and  of  the  sporangiophores  of  Phycomyces 
show  that  the  crystallographic  elementary  cell  is  rhombic,  its  di- 
mensions are  9.4 :  10.46  (10.26) :  19.25  A  and  it  contains  eight  acetyl- 
glucosamine  residues,  viz.,  two  to  every  four  main  valence  chains, 
which  traverse  the  crystal  lattice  (Meyer  and  Pankow,  1935).  A 
different  modification  of  chitin  with  the  crystal  lattice  9.32:  10.17: 
22.15  has  been  found  in  Polychaeta  and  Mollusca.  It  has  been  termed 
i5-chitin,  in  contrast  to  the  a-chitin  of  insects,  Crustaceae  and  fungi 
(Lotmar  and  Picken,  1950).  The  fibre  period  10.3  A  is  important, 
because  it  corresponds  to  the  length  of  two  pyranose  rings  and  is 
identical  to  that  of  cellulose.  This  warrants  the  belief  that  the  glucosan 
rings,  hke  the  glucose  residues  of  cellulose,  are  linked  together  by 
/5-glucosidic  1-4  bonds  (see  Fig.  147  b). 

Stihm'icroscopic  texture  of  the  chitin  frame.  Microscopically,  the  chitin 
sheath  of  the  Arthropoda  and  the  membranes  oi fungi  show  lamellation 
and  fibrillation,  as  is  known  to  be  the  case  in  the  cell  walls  of  cellulose. 
By  analogy,  therefore,  it  may  be  assumed  that  fibrillation  is  realized 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


303 


in  the  submicroscopic  region.  The  interfibrillar  spaces  in  crustacea 
are  filled  partly  with  mineral  substances,  especially  with  calcium 
carbonate,  while  the  membranes  of  fungi  are  encrusted  with  sub- 
stances rather  of  a  carbohydrate  or  pectinous  nature  (which  can  be 
extracted  by  boiling  for  several  hours  with  a  ten  per  cent,  solution  of 
caustic  potash). 


r; 


b?) 


e) 


Fig.  148.  Types  of  submicroscopic  texture  with  chitin  as  the  frame  substance,  a)  Crab 
sinew:  fibrous  texture,  b)  Interior  of  lobster  shell:  submicroscopic  lamellar  texture,  with 
direction  of  fibrillae  changing  from  lamella  to  lamella  (i,  2,  3,  4,  etc.);  bj)  cross-section, 
bg)  plan,  /r)  Eggshell  of  Ascaris:  foliate  texture  (Schmidt,  1936b).  d)  Conidiophores  of 
Aspergillus:  fibroid  texture  (Frey,  1927a).  e)  Conidiophores  of  Phycomyces:  spiral  texture 

(OoRT  and  Roelofsen,  1932). 

As  with  cellulose,  the  orientation  of  the  rods  of  the  frame  is 
demonstrable  by  optical  means,  since  the  larger  axis  of  the  index 
ellipse  of  sections  immersed  in  water  or  gh^cerol  runs  parallel  to  the 
submicroscopic  chitin  rodlets.  This  method  reveals  the  same  potential 
orientation  as  that  actualized  in  cellulosic  cell  walls  (Fig.  148). 

Chitinous  tendons  of  crabs,  lobsters,  beetles,  etc.  are  of  an  un- 
mistakable fibrous  texture.  Of  all  chitinous  objects,  therefore,  they 
produce  the  most  richly  pointed  X-ray  diagrams  and  are  thus  the  most 
informative  as  to  the  lattice  structure  of  chitin.  Optically,  the  fibrous 
texture  is  disclosed  by  the  fact  that  the  refractive  power  is  considerably 
more  pronounced  parallel  to  the  axis  of  the  tendon  than  perpendicular 
to  it,  while  something  like  isotropy  prevails  in  the  cross-sections  of 
the  tendon.  This  fibrous  texture  is  to  be  inferred,  not  only  from  the 
birefringence,  but  also  from  the  anisotropy  of  the  absorption  of  light. 
Iodine-zinc  chloride  solution  and  Congo  red  stain  decalcified  and 


304  FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

cleaned  chitinous  tendons,  as  they  do  bast  fibres^  dichroically;  the 
direction  of  the  stronger  light  absorption  coincides,  as  in  cellulose, 
with  the  fibre  axis.  The  similarity  in  the  dichroic  coloration  of  chitin 
and  cellulose  is  interesting  in  that  it  tends  to  show  that  the  dichroism 
of  these  colour  reactions  characterizes  not  so  much  a  certain  chemical 
compound  as  its  micellar  structure  with  orientated  inner  surfaces. 

The  egg-shell  of  ^j-^r^m  provided  Schmidt  (1936)  with  an  object 
in  which  the  submicroscopic  chitin  rodlets  scatter,  thus  forming  a 
wall  of  foliate  texture.  The  plan  ot  the  eggs  shows  them  to  be  isotropic, 
but  the  optical  cross-section  through  the  wall  exhibits  a  negative 
spherite  cross.  This  optical  behaviour  is  produced  by  an  arrangement 
of  the  submicroscopic  ordered  lattice  regions  as  represented  in  Fig.  1 48c. 

The  sporangiophores  of  Aspergillus  niger  must,  from  their  optics, 
be  presumed  to  have  a  fibroid  texture  with  scattering  (Fig.  i48d; 
Frey,  1927a).  We  do  not  3^et  know,  however,  whether  this  membrane 
is  stratiform  like  Phycowjces;  for  in  that  fungus,  with  particularly  large 
sporangiophores  several  centimetres  in  length,  Oort  and  Roelofsen 
(1932)  found  an  outer  primary  skin  of  tubular  texture,  under  which 
there  is  a  thickened  secondary  wall  layer  of  fibrous  texture  exhibiting 
slight  scattering;  it  is  by  reason  of  its  predominant  bulk  that  only  this 
appears  on  the  X-ray  photograph.  It  is  assumed  that  at  the  core  there 
is  another,  very  thin  layer  of  steep  spiral  texture  (Fig.  i48e). 

These  results  of  the  indirect  methods  are  only  partly  corroborated 
by  the  electron  microscope.  The  cell  wall  of  the  sporangiophore  of 
Phycomyces  consists  of  chitinous  microfibrils  which  are  similar  to  those 
in  cellulose  walls  (Frey- Wyssling  and  Muhlethaler,  1950;  Roelof- 
sen, 195  la).  There  is  a  homogeneous  cuticle  devoid  of  any  structure, 
a  primary  wall  with  interwoven  microfibrils  and  a  thick  parallel 
textured  secondary  wall  (Fig.  86c,  p.  128).  Roelofsen  differentiates 
the  primary  wall  in  an  outer  layer  with  a  network  texture  and  an  inner 
layer  with  almost  transverse  oriented  microfibrils.  The  texture  of  the 
uniform  secondary  wall  runs  almost  parallel  to  the  cell  axis.  There  is 
no  pronounced  spiral  texture  and  no  special  internal  wall  layer  as  had 
previously  been  found  in  the  polarizing  microscope  (Fig.  1486). 

Spiral  growth.  The  end  of  the  sporangiophore  is  conspicuously  of 
spiral  growth  (Oort,  193 i;  Castle,  1937a,  1942).  This  fact  can  be 
verified  by  placing  a  mark  above  the  zone  of  growth  which  was  found 
^  Walchli  (1945). 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


305 


not  only  to  travel  upwards,  but  at  the  same  time  to  rotate  around 
the  axis  of  the  sporangiophore  (Fig.  149).  There  is  nothing  in  the 
submicroscopical  texture  of  the  primary  wall  which  might  account  for 
this  behaviour.  Oort  and  Roelofsen  (1932)  state  that  the  isolated 
wall  is  flabby  and  flexible  and,  as  it  tears  impartially  in  all  directions, 
is  not  ot  parallel  texture.  This  is  con- 
firmed by   the   electron   microscopic 
evidence.    However,    if  the  interior 
pressure  in   the   zone   of  growth  is 
artificially  enhanced,  the    membrane 
bursts    through    a    very  steep  spiral 
longitudinal  tear,  which  may  be  at- 
tributed to  the  anisotropic   states  of 
tension  in  all  tubular  walls  described 
on  page  289.   Artificial  extension  of 
the  zone  of  growth  is  accompanied 
bv  a  rotation  which,  after  relaxation, 
recovers.  Thus  the  optics  point  to  a 
woven    tubular    texture,    while    the 
mechanical  properties  require  a  spiral 
texture.  Castle  (1942)  discovered  ad- 
ditional complications;  he  was  able  to  show   that   at   first   there  is 
regularly  a  left  tendency  in  growth,  which  then  suddenly  changes 
for  an  hour  to  a  right-hand  spiral  and  then  reverts  again  to  a  left 
spiral.  He  tried  to  account  for  this  by  suggesting  the  preformation 
of  both  a  left-hand  and  right-hand  screw  in  the  primary  wall;  that 
is  to  say,  it  would  be  a  crossed  system  indistinguishable  from    the 
tubular    texture.    Preston    (1948)    has   even   developed  a  formula 
for  calculation  of  the  change  of  rotation  from  the  elastic  properties 
of  the  cell  wall  which  alter  during  its  diff'erentiation.  But  all  these 
considerations  are  based  on  a  spiral  texture  (Preston,  1934,  1936) 
which  obviously  is  not  realized  in  the  growth  zone  of  the  Phjcomyces 
sporangiophore  (Roelofsen,  1949/50,  1951a).  Therefore,  the  simplest 
assumption  is  that  intercalary  growth  in  the  zone  of  extension  travels 
in  a  circle;  this  must  be  so,  since  the  slender  conical  shape  of  the  zone 
of  growth  could  hardly  be  maintained  if  the  surface  grew  simul- 
taneously on  all  sides.  In  fine  with  this  is  the  fact  observed  by  Oort 
and  Roelofsen,  viz.,  that  in  Phjcomjces  Blakeskeanns  var.  piloboides 


Fig.  149.  spiral  growth  of  Phycomyces 

(from     Castle,     1937  a).     Zone     of 

growth   dotted;    •    marks    to    trace 

rotation. 


3o6         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  HIT 

with  a  characteristically  inflated   sporangiophore,   the   sporangium 
does  not  rotate. 

It  follows  from  this  discussion,  that  there  must  be  a  local  growth  by^ 
intussusception,  the  mechanism  of  which  is  not  yet  fully  understood. 

Crossed  lamellar  systems.  The  growth  in  area  of  membranes  which; 
are  obviously  cross-textured  is  equally  difficult  to  explain. 

An  instructive  example  of  a  microscopically  laminated  structure  is. 
provided  by  the  inner  layer  of  the  lobster  shell.  As  an  entity,  this, 
layer  behaves  like  a  uniaxial,  optically  negative  composite  body;  i.e.,. 
seen  trom  the  surface,  it  is  isotropic.  In  cross-section,  on  the  other 
hand,  strongly  birefringent  (positive  with  reference  to  the  lamellation) 
and  isotropic  layers  are  seen  to  alternate.  Older  investigators  (Bieder- 
MANN,  1903)  thought  these  lamellae  possessed  cross-wise  fibrillation 
at  right  angles.  Were  this  true,  it  should  be  possible  to  cut  cross-^ 
sections  at  45°  to  the  two  fibrillar  directions  through  the  composite 
body  in  which  all  the  lamellae  would  show  the  same  behaviour  in 
the  polarizing  microscope.  This,  however,  is  not  the  case,  for  cross- 
sections,  in  whatever  direction,  through  the  lobster  shell  all  invariably^ 
disclose  the  same  pattern  of  lamellation.  Schmidt  (1924,  p.  238) 
therefore  assumes  that  the  iibrillae  in  consecutive,  very  thin,  parallel- 
fibred  layers  very  gradually  change  direction,  so  that  two  layers  at 
a  certain  distance  from  each  other  will  contain  fibrillae  crossed  at  right 
angles,  but  those  in  between  will  contain  fibrillae  in  any  of  the 
transitions  from  0°  to  90°.  An  arrangement  such  as  this  is  indicated 
in  Fig.  i48bi  (p.  303).  This  should  be  verifiable  optically  for,  in  the 
transition  from  lamella  to  lamella,  the  light  retardation  should  drop- 
following  a  sine  curve  from  the  maximum  value  to  nil.  X-ray  analysis, 
would  likewise  show  whether  all  possible  fibrillar  directions  are  before 
us.  It  seems  to  me  an  important  point  that  the  hypothetical  layers  are 
submicroscopically  thin  for,  were  they  of  microscopical  dimensions, 
it  would  mean  that  this  is  a  comparable  case  to  the  spiral  texture  of 
cotton  fibre ;  that  is  to  say,  owing  to  the  obliquely  crossed  layers,  the 
top  view  of  the  interior  layer  of  the  shell  could  not  be  isotropic,  but 
would  have  to  transmit  some  light  under  all  azimuths. 

Instead  of  assuming  submicroscopic  lamellae  consisting  of  parallel 
microfibrils  superimposed  in  different  directions  of  orientation  (Fig^ 
148b,  p.  303),  it  would  be  equally  plausible  to  picture  the  micro- 
fibrils as  interweaving. 


I  CARBOHYDRATES,  CHITIN  AND  CUTIN  507 

Vegetable  cellulose  membranes  were  studied  (Frey-Wyssling, 
1 941)  with  the  object  of  discovering  whether  in  laminated  systems 
the  individual  layers  are  of  parallel  texture,  or  whether  it  is  a  matter 
of  interweaving.  We  have  examples,  such  as  the  algae  \^alonia  (Van 
Iterson,  1933;  Preston,  Nicolai,  Reed  and  Millard,  1948)  and 
Chaetotnorpha  (Nicolai  and  Frey-Wyssling,  1938),  the  laminated  cell 
walls  ot  which  can  be  split  up  into  single  lamellae  of  a  few  tenths  of 
a  //  in  thickness ;  these  lamellae  are  made  up  of  strictly  parallel  fibrillae, 
which  accounts  for  their  striking  cleavability  parallel  to  the  fibre 
direction.  In  consecutive  lamellae  the  fibre  directions  cross  at  approxi- 
mately right  angles  (in  'Valonia  at  78°);  consequently  the  optical 
anisotropy  of  the  individual  lamellae  is  to  a  large  extent  mutually 
neutralized  and,  in  transmitted  light,  the  appearance  is  roughly  that 
ofstatistically  isotropic  packets  of  layers.  (Cf.  Preston,  1947;  Picken, 
Pryor  and  Swann,  1947). 

As  opposed  to  these  systems  of  membranes  with  uniform  parallel 
texture  of  the  individual  lamellae,  we  have  the  fine-structure  of  the 
primary  wall  of  cotton  fibres.  This  thin  membrane  exhibits,  according 
to  Anderson  and  Kerr  (1938),  three  different  systems  of  striations, 
one  of  which  runs  perpendicular  to  the  fibre  axis,  the  two  others 
falhng  symmetrically  at  an  angle  of  about  30°  obliquely  from  the  left 
and  right.  As  the  membrane  cannot  in  this  case  be  split  up  into  three 
lamellae,  presumably  there  are  three  different  fibrillar  directions  in  one 
and  the  same  lamella.  It  may  be  supposed  that  submicroscopic  fibriUae 
are  interwoven  in  the  three  directions  after  the  manner  of  a  textile 
fabric. 

The  observations  made  by  Rosin  (1946)  on  the  tails  of  tadpoles 
would  support  the  latter  possibility.  Judging  by  the  arrangement  of 
the  pigment  cells,  which  rest  on  a  basal  membrane  of  connective 
tissue,  it  would  seem  that  the  intrinsic  texture  of  this  membrane  must 
consist  of  orthogonally  trellised  submicroscopic  fibrillae  of  collagen. 
As  it  cannot  be  split  up  into  two  lamellae,  the  two  systems  of  fibrillae 
apparently  lie  in  the  same  plane.  Rosin  was  able  to  show  how  the 
orthogonal  fibrillar  system  grows  by  "afline"  enlargement  of  the 
surface,  the  trellising  of  the  two  fibrillar  systems  always  remaining 
rectangular  (Fig.  150).  Intussusception  is  responsible  for  surface 
enlargement,  inasmuch  as  new  submicroscopic  fibrils  are  embedded 
in  parallel. 


30,8 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


In  nature,  therefore,  there  are  very  probably  crossed  micro- 
structural  systems,  the  fibrillar  structural  elements  of  which  interweave 
orthogonally  or  at  other  angles.  The  establishment  of  this  fact  may 
assist  very  materially  in  clarifying  the  submicroscopic  texture  of  ex- 
ceedingly thin  membranes. 


Fig.  150.  Affine  growth  (from  Rosin,  1946).  a  may  grow  larger  progressively  in  pro- 
portion to  h,  or  towards  one  side  {c,d,e) ;  the  crossed  system  thereby  remaining  orthogonal. 


It  is  satisfactory  that  these  speculations  can  be  substantiated  by 
electron  microscopy.  In  primary  cell  walls  (Frey-Wyssling,  Muhle- 
THALER  and  Wyckoff,  1948)  and  in  the  Tunicata  mantle  (Frey- 
Wyssling  and  Frey,  195  i)  interwoven  crossed  microfibrils  are  visible 
(Fig.  86d,  p.  128).  Therefore,  in  one  and  the  same  microscopic 
lamella,  fibrillar  elements  may  be  laid  down  in  different  directions  and, 
if  they  cross  at  90°,  their  optical  anisotropy  is  mutually  cancelled  when 
polarized  light  is  transmitted  perpendicularly. 

Rodlet  and  intrinsic  birefringence  of  the  chi  tin  frame.  Chi  tin  was  the  first 
biological  object  in  which  the  interaction  of  textural  and  intrinsic 
birefringence  —  discovered  by  Ambronn  in  artificial  gels — could  be 
demonstrated  (Mohring,  1922).  When  decalcified  specimens  of 
chitin  (lobster  shell  or  lobster  tendons)  are  immersed  in  solutions  of 
potassium  mercuric  iodide  of  increasing  refractive  power,  the  bire- 
fringence decreases,  falls  to  nil,  changes  its  sign,  reaches  a  minimum 
in  the  negative  region,  becomes  nil  a  second  time  and  then  returns 
to  positive  (Fig.  151).  The  inference  from  this  is  that  chitin  is  marked 
by  a  pronounced  positive  form  anisotropy,  i.e.  rodlet  birefringence, 
and  a  sHghtly  negative  intrinsic  birefringence. 

In  his  imbibition  experiments  Castle  (1936)  finds  reversal  of  the 
birefringence  with  mercuric  iodide  of  potassium  and  iodobenzene  in 
xylene,   but  not  with   other   organic  liquids    (methylene   iodide  in 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


309 


0.012 

0.010 

0.008 

0.006 

0.004 

0.002 

0 

-0.002 

-0.004 


\ 

\ 

\ 

\ 

•^ 

\ 

\ 

\ 

k 

\ 

\, 

V 

^- 

\ 

- — ■ 

^ 

137  141  145  149  153  1.57  1.61  IdSn^ 


xylene,  iodobenzene  in  alcohol).  From  this  he  concludes  that  the 
source  of  the  negative  birefringence  is  not  natural  chitin,  but  chitin 
chancred  chemically  by,  say,  potassium  mercuric  iodide.  This  con- 
clusion is,  however,  incorrect,  for  Diehl  and  Van  Iterson  (1955) 
found  with  mixtures  of  glycerol  and  quinoline,  and  Schmidt  (1936) 
with  a-monobromo-naphthalene  (mixed 
with  xylene)  negative  minima  of  the 
rodlet  birefringence  curve,  even  though 
these  curves  are  not  identical  for  various 
imbibition  mixtures  (Fig.  1 5 1).  What  was 
demonstrated  in  cellulose  (Frey-Wyss- 
LiNG,  1936b)  probably  applies  here,  viz., 
that  the  difference  in  the  adsorptive 
power  of  the  micellar  frame  with  respect 
to  the  components  of  the  imbibition  li- 
quid is  responsible  for  the  displacement  of 
the  curves.  From  the  data  now  available, 
therefore,  it  may  confidently  be  asserted 
that  the  submicroscopic  chitin  rodlets 
have  a  negative  intrinsic  birefringence. 

Cellulose    likewise  becomes  optically 
negative  by  nitration  and  complete  acetyl- 

ation  (triacetyl  cellulose),  i.e.,  by  the  esterification  of  the  polar  OH- 
groups.  It  may  therefore  reasonably  be  presumed  that  it  is  the  acetyl 
side  chains  of  the  chitin  which  cause  the  negative  birefringence.  It  is 
nevertheless  a  curious  fact  that  for  chitin  only  one  acetyl  group  per 
glucose  residue  is  required  for  this,  whereas  three  are  necessary  in 
cellulose;  presumably,  therefore,  the  amino  group  of  the  glucosamine 
also  tends  to  produce  negative  birefringence. 

The  negative  intrinsic  birefringence  of  chitin  does  not  hamper  the 
approach  to  the  micellar  texture  of  chitinous  composite  bodies  by 
polarizing  optics  if  the  imbibition  agents  used  are  Hquids  whose 
refractive  index  is  below  1.48,  i.e.,  water  or  glycerol.  It  should, 
however,  be  realized  that  the  determination  of  the  micellar  orientation 
does  not  then  take  place  on  the  basis  of  the  positive  intrinsic  aniso- 
tropy  of  the  submicroscopic  frame  of  the  membrane,  as  in  cellulose, 
but  rests  on  the  positive  rodlet  birefringence  of  the  chitin  skeleton. 
Another  interesting  fact  has  been  discovered  based  on  the  optical 


Fig.  15 1.  Rodlet  birefringence  curve 
of  chitin  sinews  (from  Diehl 
and  Van  Iterson,  1935);  A  with 
quinoline-glycerol,  B  with  mer- 
curic iodide  of  potassium.  Ab- 
scissa: Refractive  index  n^  of  the 
imbibition  liquid.  Ordinate:  Bi- 
refringence n^-no. 


3IO         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

properties  of  chitin.  Young  Lepidopferan  scales  yield  a  curve  of  rodlet 
birefringence  with  a  minimum  at  ng  =  1.57  instead  of  1.61  (Fig.  151). 
From  this  behaviour  Picken  (1949)  concluded  that  growing  scales  are 
not  yet  chitinous,  and  he  proved  that  they  consist  at  this  stage  of 
protein  similar  to  muscle  protein  (n  =  1.57)  or  keratin  (n  =  1.55)- 
No  intrinsic  double  refraction  is  then  visible ;  it  appears  only  in  fully 
grown  scales  after  the  formation  of  chitin  with  its  typical  negative 
character. 

d.  Starch  Grains  (Amylose  and  Amylopectin) 

Molecular  structure  of  starch.  The  reserve  carbohydrates  sucrose, 
maltose  and  starch  are  a-glucosides  (see  page  60),  in  contradis- 
tinction to  the  skeletal  carbohydrates  cellulose,  xylan,  etc.  which  are 
jS-glucosidic.  Compared  to  the  straight  cellulose  chains,  the  glucosan 
chains  with  a- 1-4  bonds  are  rather  kinked  (Meyer  and  Mark,  1930). 
The  result  is  that  a  spatial  lattice  of  such  chains  must  be  less  compact 
and,  therefore,  is  more  soluble,  as  indeed  its  physiological  function 
as  a  reserve  material  requires  it  to  be.  Evidently  the  voids  formed  by 
this  particular  molecular  configuration  are  partly  filled  with  water 
molecules.  Even  the  simplest  a-glucoside,  maltose,  crystallizes  with 
water  of  crystallization,  and  loosely  bound  water  molecules  also  play 
an  important  part  in  the  crystal  lattice  of  starch.  They  do  not,  ad- 
mittedly, escape  from  the  lattice  as  easily  as  from  protein  crystals,  but 
when  grains  of  starch  are  crushed,  their  lattice  structure  is  likewise 
wrecked  as  the  result  of  loss  of  water;  they  become  amorphous,  the 
birefringence  and  their  X-ray  diagram  (Sponsler,  1922)  vanishing. 
Hence  additive  water  molecules  apparently  stabilize  the  lattice  order 
of  starch,  as  is  the  case  in  the  reserve  proteins. 

The  chemistry  of  starch  is  complicated  by  the  presence  in  the  starch 
grains  of  two  chemically  distinct  substances,  viz.,  amylose  and  amylo- 
pectin. Amylose  is  soluble  in  hot  water  and  is  stained  blue  by  iodine, 
whereas  amylopectin  swells  in  boiling  water  and  gives  a  violet  iodine 
coloration.  Thus,  when  the  starch  grains  'form  into  a  paste,  amylose 
goes  into  solution,  while  the  amylopectin  becomes  a  swollen,  in- 
soluble jelly.  Neither  component  exhibits  any  reducing  power  upon 
Fehling's  solution,  which  signifies  that  neither  contains  free  aldehyde 
groups.  K.  H.  Meyer  (1940b)  has  discovered  the  difference  in  con- 
stitution between  amyloses  and  amylopectin.  He  states  that  amyloses 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


311 


consist  of  unbranched  chains,  whereas  amylopectin  is  made  up  of 
branched  chains  (Staudinger  and  Husemann,  1937;  Staudinger, 
1937  b)  which  together  form  a  gel  framework  (Fig.  152);  consequently 
amyloses  are  soluble,  which  amylopectin  is  not.  Their  other  properties 
are  given  in  Table  XXVIII.  In  different  starch  samples  the  content  of 


^P?P^$:R>R?Ri3^PR? 


b)  c) 

Fig.  152.  Diagram  of  the  molecular  shapes  of  starch  molecules.  Glucose  residues  repre- 
sented as  small  rings:  they  are  far  more  numerous  (degree  of  polymerization)  than  shown 
here,  a)  Expanded  amylose  chain  (cf.  cellulose),  b)  Amylopectin  (from  K.  H.  Meyer, 
1943):  branched  chain  molecule.  At  x  signs  of  the  activity  of  the  sugar-forming  amylase: 
splitting  off  of  the  disaccharide  maltose.  In  the  absence  of  the  dextrin-forming  amylase, 
■degradation  ceases  if  maltose  has  split  off  from  all  the  free  terminals  up  to  the  branching 
place,  c)  Glycogen:  highly  branched  starch  molecule. 


TABLE  XXVIII 
COMPARISON  BETWEEN  AMYLOSES  AND  AMYLOPECTIN 


Amyloses 


Amylopectin 


Molecular  configuration 

Molecular  weight  (osmot.) 

j3-Amylase 

Pasting 

Films 

Tetramethyl  glucose 
from  maize  starch 


Unbranched  chain 
1 000c  -  1 00000 
Complete  hydrolysis 
Forms  no  paste 
Solid  film 

0.51% 
almost  nil 

branches/molecule 


Branched  molecule 
5  0000  -  1 000000 
Malto  dextrin 
Forms  paste 
Friable  film 

3-7% 
about  100 

branches/molecule 


amylose  varies  from  34%  to  0%  (Table  XXIX).  The  blue  starch 
reaction  with  iodine  is  limited  to  amyloses  with  crystallized  chains, 
i.e.,  unbranched  chains  orientated  in  parallel  (Meyer  and  Bernfeld, 
1941a),  or  to  individual  amylose  chains  wound  up  into  a  helix. 


312  FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 


TABLE  XXIX 
AMYLOSE  CONTENT  OF  STARCH 
(from  bates,  FRENCH  AND  RUNDLE,   1 943) 


Starch 


%  Amylose 


Ketan  {Ory^a  sativa  f.  glutimsa)  . 
Waxy  Corn  {Zea  mays  f.  saccharatd) 
Tapioca  {Manihot  utilissimd)    .    . 

Rice  {Oryxa  sativa) 

Banana  {Musa  sap/enfum) .    .    .    . 

Corn  (Zea  mays) 

Potato  {Solanum  tuberosum)  .  .  . 
Wheat  {JTriticum  aestivum)  .  .  . 
Sago  {Mefroxylon  spec.)  .... 
Lily  bulb  {JLilium  spec.)  .... 


o 
o 

17 
17 
20.5 

21 
22 

24 
27 
34 


Freudenberg,  Schaaf,  Dumpert  and  Ploetz  (1939)  as  also 
RuNDLE  and  Edwards  (1943)  argue  that  the  chains  of  dissolved  and 
precipitated  amylose  molecules  are  spirals,  with  six  successive  glucose 
rings  to  one  revolution.  Just  as  there  are  H-bonds  between  the  neigh- 
bouring chain  molecules  of  cellulose,  so  might  there  also  be  H-bonds 
between  neighbouring  turns  of  the  same  chain  in  the  spiral  model  of 
the  starch  molecule.  The  six  glucose  rings  per  revolution  can  be 
compared  with  Schardinger's  dextrins^,  the  molecules  of  which 
contain  six  to  seven  glucose  residues  (Hanes,  1957).  Then,  the  inside 
of  the  hollow  cylinders  formed  by  the  spiral  chain  provides  the 
necessary  space  for  the  infiltration  of  iodine  causing  the  blue  starch 

reaction. 

Dextrins  obtained  as  de- 
gradation products  in  starch 
hydrolysis    give   no  iodine 
colour  reaction  when  they 
contain  only  six  or   fewer 
glucose  units.  Dextrins  con- 
taining  eight  to  twelve  glu- 
cose units  produce  red  ra- 
ther than  blue  complexes.  Only  the  longer  amylose  chains  give  the 
typical  blue  iodine  colour.  It  is  believed  that  the  \  molecules  are 
arranged  along  the  centre  of  the  amylose  helix  (Fig.  153). 
^  Kratky  and  Schneidmesser  (1938). 


Fig.  153.  Model   of  iodine-filled   amylose   helix, 
(from  RuNDLE,  Foster  and  Baldwin,  1944). 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


513 


Molecules  with  branched  chains  produce  red  (amylopectin)  or  even 
brown  colouring  (glycogen)  with  iodine. 

The  branching  is  due  to  glucosidic  bonds  from  the  aldehyde  group 
of  one  amylose  chain  to  another  chain  (Fig.  154).  Such  bifurcations 


CH.OH      r 

-0,    ' 


CHfiH 


non-reducing 
end  groups 


branching 
member 


additional       aldehyde 
branching      end  groups 


Fig.  154.  End  branching  of  amylopectin  (from  Frey-Wyssling,  1948  c). 

are  frequently  repeated  and  it  can  be  shown  that  most  of  them  corre- 
spond to  1-6  bonds  (Myrback,  1938;  Gibbons  and  Boissonnas, 
1950).  When  branched  chains  are  methylated  and  then  hydrolyzed 
(Irvine,  1932),  considerable  quantities  of  dimethyl  glucose  (from 
branching  junctions)  and  tetramethyl  glucose  (from  the  end  members 
of  the  side  chains)  are  formed  in  addition  to  trimethyl  glucose.  For 
amylopectin  the  amount  of  2, 3 -dimethyl  glucose  formed  is  similar  to 
that  of  2,3,4, 6-tetramethyl  glucose  (5-5%).  This  means  that  the 
number  of  end  members  ///  is  about  the  same  as  the  number  of  bifur- 
cations b.  A  dichotomous  branching  would  satisfy  this  relation, 
because  it  yields  h  ^  m  —  i  (Fig.  155). 


aldehydic    end  member 


1  bifurcation 

2  bifurcations 


16    end  members 


64   end  members 
(b) 


Fig.  155.  Dichotomous  branching  of  amylopectin,  a)  in  a  plane,  b)  in  space  (from  Frey- 
Wyssling,  1948  c). 


314         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

For  amylopectin  of  Ys  million  molecular  weight  (Table  XXVIII, 
p.  311),  which  corresponds  to  a  degree  of  polymerization  of  2000, 
64  bifurcations  and  as  many  end  members  would  be  found.  The 
average  length  of  the  branches  with  1 5  glucose  units  would  measure 
about  50  A,  so  that  the  brush-like  molecule  of  Fig.  155b  would  cover 
350  A  in  an  expanded  state. 

The  amylopectin  content  varies  in  the  different  kinds  of  starch, 
which  accounts  for  the  familiar  specific  differences  between  them. 
Potato  starch  has  a  higher  amylopectin  content  than  wheat  starch 
(Meyer  and  Bernfeld,  1941b).  Ketan,  the  starch  grains  of  which 
are  dyed  red  bv  iodine,  contains  only  amylopectin  of  high  molecular 
weight  (Meyer  and  Heinrich,  1942).  Amylopectin  possesses  weakly 
acid  properties  and  can  therefore  be  separated  by  electrophoresis  from 
amyloses,  which  are  absolutely  neutral  (Lamm,  1937).  Presumably  the 
acid  groups  in  amylopectin  are  responsible  for  the  fact  that  only  basic 
dyes  can  stain  starch  grains  with  a  colour  which  is  fast  to  washing, 
Samec  (1927)  says  they  consist  of  phosphoric  acid.  Meyer  and  Mark 
questioned  in  1950  the  existence  of  phosphoric  ester  bridges  between 
the  glucose  chains,  and  nowadays  amylopectin  is  regarded  as  free 
from  phosphoric  acid  (Meyer  and  Brentano,  1936;  Samec,  1942), 

The  discovery  by  Hanes  (1940)  that  the  enzymatic  degradation  of 
starch  is  a  phosphorolysis,  and  not  hydrolysis,  invests  the  phosphorus 
content  of  starch  grains  with  a  particular  significance.  This  knowledge 
led  to  the  synthesis  of  starch  in  vitro.  Starting  from  phosphorylized 
glucose,  Hanes  united  it  with  the  enzyme  phosphorylase ;  when  equi- 
librium sets  in  between  glucose- 1 -phosphate  and  starch,  this  com- 
pound, owing  to  its  insolubility,  is  synthesized.  Hanes'  synthesis  of 
starch  is  the  first  instance  of  an  artificial  manufacture  of  a  high 
polymeric  natural  product. 

The  decomposition  of  starch  is  a  highly  complicated  process  of 
fermentation  (Myrback,  1938;  Myrback  and  co-workers,  1942). 
Amylase,  the  enzyme  which  decomposes  starch,  consists  of  two 
different  constituents,  viz.,  the  dextrinogenous  a-amylase  and  the 
saccharogenous  /9-amylase,  both  of  which  have  been  isolated  and 
crystallized  (Meyer,  Fischer  and  Bernfeld,  i  947 ;  Meyer,  Fischer 
and  PiGUET,  195 1 ;  Meyer,  K,  H,,  195  i).  The  latter  splits  off  maltose 
(twin  groups  of  glucose)  from  the  aldehyde  end  of  the  starch  chains 
(Fig.  152b,  p.  311),  but  is  unable  to  break  up  the  branch  junctions 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


315 


of  the  amylopectin.  a-amylase  is  able  to  break  down  the  amylopectin 
into  soluble  fragments  (dextrins)  without  at  first  generating  maltose; 
subsequently  saccharification  sets  in  by  degrees  (Meyer  and  Bern- 
FELD,  1941c).  The  branching  junctions  of  the  amylopectin  are  in- 
accessible to  the  /5-amylase,  for,  besides  the  usual  1-4  bond  between 
the  glucose  residues,  there  is  an  additional  6-1  bond  passing  into  the 
side  chain,  the  splitting  of  which  needs  yet  another  enzyme. 

The  microscopic  structure  of  starch  grains.  The  microscopic  structure  of 
starch  grains  has  been  dealt  with  so  often  and  so  exhaustively  (see 
Badenhuizen's  comprehensive  review  1937)  that,  to  avoid  repetition, 
I  shall  here  touch  only  on  a  few  points  which  appear  to  me  of  par- 
ticular importance  (Samec,  1942/43). 

The  familiar  arrangement  in  layers  of  starch  grains  is  brought  about, 
in  the  unanimous  opinion  of  the  majority  of  investigators  from 
Nageli  (1858)  to  our  contemporaries,  by  alternate  layers  of  stronger 
and  weaker  refractive  power,  or  containing  a  smaller  or  larger 
percentage  of  water.  Now  if  a  weakly  refractive,  narrow  layer  were 
bordered  both  inside  and  outside  by  neighbours 
of  higher  refractivity,  it  would  shine  brightly  in 
the  microscope  at  low  adjustment  and,  when  the 
tube  is  raised,  the  bright  Becke  lines  on  both 
sides  should  pass  over  into  the  optically  denser 
layers.  This,  however,  does  not  take  place  (Frey- 
Wyssling,  1936a,  page  287).  With  pronounced 
stratification,  especially  of  eccentric  starch  grains 
immersed  in  water  (potato,  Pellionia,  etc.),  it  may 
be  seen  distinctly  how  the  Becke  line  at  the  edges 
of  all  layers  moves  outivards  only  when  the  tube  is 
raised.  True,  there  are  cases  when  a  pale  lustre 
can  be  seen  to  shift  inwards,  but  on  the  outside 
it  is  always  incomparably  stronger.  From  this  we 
may  confidently  conclude  that  every  layer  is  more  refractive  on  the 
inside,  the  refractive  power  outwards  as  a  rule  diminishing  quite 
gradually,  and  then  suddenly  coming  up  against  a  layer  of  higher 
refractive  index.  Thus,  in  a  section  through  the  grain  the  refractive 
power  in  the  various  layers  is  not  equally  high  or  low,  but  there  is 
a  continuous  decrease  towards  the  edges  and  discontinuous  increase 
at  the  outer  edges  of  the  layers,  as  represented  in  diagram  bv  the 


0  12    3      4       5 


Fig.  156.  Microscopic 
lamination  of  starch 
grains.  Diagram  of 
the  refraction.  Ab- 
scissa: 1-5  layer  edges. 
Ordinate:  Refractive 
index  «. 


3l6         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

serrated  line  in  Fig.  1 5  6.  The  arrangement,  therefore,  of  the  layers  is 
not  dense/light/dense,  but  rather  a  gradual  reduction  in  density  in 
each  layer  up  to  the  edge  of  the  next  layer,  at  which  point  the  density 
suddenly  rises  again  to  its  initial  level.  The  fact  noted  by  Young 
(1938)  that  the  layers  of  the  starch  grains  of  Canna  are  more  easily 
stained  outside  than  inside  is  probably  due  to  the  looser  structure  of 
the  outer  portions  of  the  layers. 

The  inference  from  the  foregoing  as  to  the  apposition  growth  of 
starch  grains  is  that,  in  the  formation  of  a  new  layer,  the  deposition 
is  at  first  dense,  becoming  looser  little  by  little  until,  at  a  given  degree 
of  impoverishment,  growth  ceases  altogether.  The  sugars  consumed 
have  then  probably  to  be  made  good  before  the  process  can  start 
afresh.  As  Van  de  Sande  Bakhuizen  (1925)  showed,  if  external  con- 
ditions are  constant,  lamination  does  not  occur,  because  nutritive 
material  is  then  always  available  in  the  same  concentration  and, 
therefore,  there  is  no  impoverishment  during  growth.  The  same 
applies  to  the  lamination  of  cotton  fibres,  which  likewise  depends 
largely  upon  external  conditions  of  growth,  viz.,  temperature  (Kerr, 
1937),  or  can,  indeed,  be  prevented  altogether  by  constant  exposure 
to  light  and  the  exclusion  of  fluctuations  in  temperature  (Anderson 
and  Moore,  1937).  During  their  entire  growth,  the  starch  grains  are 
enclosed  within  the  amyloplast,  which  produces  them;  this  stretches 
very  considerably  in  the  process  and  finally  becomes  an  exceedingly 
thin,  scarcely  perceptible  pellicle  enveloping  the  grain. 

Radial  structures  have  for  long  been  observed  in  addition  to  lami- 
nation ;  they  take  the  form  of  corroded  patterns  during  the  mobili- 
zation of  the  starch  in  the  germinating  seeds,  or  of  thin  radial  cracks. 
The  starch  grains  have  therefore  been  thought  to  be  of  spherite  tex- 
ture. This  would  seem  to  receive  support  from  the  optical  fact  that 
a  positive  spherite  cross  always  occurs  (see  Fig.  66,  p.  96),  because 
the  starch  chains,  like  cellulose  chains,  are  optically  positive  as  referred 
to  their  long  axis. 

The  starch  grains  can  be  split  up  tangentially  and  radially  by  chemi- 
cal means  into  minute  blocks  of  i  ^  edge  length  (Hanson  and  Katz, 
1934;  Badenhuizen,  1937)  and  these  particles  have  been  said  to  be 
pre-formed  elementary  units  of  the  starch  grain  (Fig.  157).  Structures 
of  the  kind  are  obtained  if  starch  granules  are  treated  for  days  with 
7^4%  hydrochloric  acid  and  are  then  swollen  in  2  molar  Ca(N03)2 


I  CARBOHYDRATES,  CHITIN  AND  CUTIN  517 

(known  as  "Lintnerization").  Hanson  and  Katz  suppose  that  the 
blocks  consist  of  packets  of  amyloses  and  that  the  swollen  inter- 
mediate substance  is  amylopectin.  This  view  is  not  borne  out  by  the 
staining  properties  of  the  substances,  since  the  basic  dyestufF,  fuchsine, 
stains  the  blocks  a  deep  red,  whereas  rvOOCZl^^ 

the  supposed  amylopectin  intermed-  r^P*^  nn^'^'^Q^ 

iate  substance  remains  colourless.  r^rr€^^^:^rf4=^lf]^^^ 

Seeing  that  this  block  structure  is      c96^<^K^^°^^^m>  ^S\ 
formed  only  after  the  application  of     8§Hmo^^o\P§^ 
strong  hydrolysis  with  hydrochloric     RR  QQfx'^^        °n!  )o^ '^'^ 
acid,   the   view   of  pre-formation  is 
hardly  tenable;  it  is  more  likely  to  be 
a  case  of  hydrolysis  patterns   (Frey- 
Wyssling,  1936a,  p.  290).  This  kind 
of  partitioning  of  objects  made  up  of 
high-polymeric    chain    molecules   has       Fig.  157.  Microscopic  block  structure 

likewise  been  observed  in  theproduc-       "^  "linjnerized"  wheat  starch  (from 

^  Hanson  and  Katz,  1934). 

tion  of  chemical  cross-sections  through 

cellulose  fibres  with  sulphuric  acid  (Kelaney  and  Searle,  1930),  the 
decomposition  of  cotton  into  "dermatosomes"  by  hydrochloric  acid 
(Farr  and  Eckerson,  1934)  and  m  the  decomposition  of  muscle  fibres 
in  acid  alcohol  (Schmidt,  1937a,  p.  180).  Thus  in  all  these  cases 
hydrolytic  agents  are  necessary  to  produce  the  reported  dissociations. 
Considering  how  sensitive  high-polymeric  main  valence  chains  with 
glucoside  or  peptide  bonds  are  to  hydrolysis,  it  is  out  of  the  question 
that  the  reagents  used  would  merely  have  a  dissolving  effect;  thev 
surely  cannot  fail  to  induce  break-down  and  decomposition.  Cellulose 
chains,  for  instance,  are  broken  down  by  i  n  HCl  (=  3.6%)  at  53°  C. 
in  six  hours  from  1660  to  445  degree  of  polymerization  (Staudinger 
and  Sorkin,  1937a)  and  amylose  chains  are  shortened  in  only  3^/, 
minutes  by  2  n  HCl  from  940  degree  of  polymerization  to  one-fifth 
their  length  (Staudinger  and  Husemann,  1937).  Nor  is  it  surprising 
that  this  hydrolytic  degradation  should  take  place  mainly  across  the 
particular  texture  (fibrous  or  spherite),  since  the  hydrolysis  occurs 
perpendicularly  to  the  alignment  of  the  thread  molecules.  The  par- 
titioning parallel  to  the  axis  of  orientation  need  not  necessarily  be  of 
a  hydrolytic  nature;  it  is  as  likely  to  take  place  in  a  less  drastic, 
physical  way  (radial  cleavage,  cracks  due  to  drying,  fibrillation  through 


3l8         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

swelling),  for  in  this  direction  there  are  chiefly  secondary  and  not 
primary  valencies  that  have  to  be  overcome. 

A  question  which  it  is  difficult  to  answer  is  why  the  hydrolysis 
should  occur  with  such  characteristic  rhythm  in  the  case  of  these 
microscopically  homogeneous  structures.  If  the  microscopic  segments 
which  are  formed  correspond  to  the  chain  lengths  of  the  macro- 
molecules,  as  Staudinger,  Staudinger,  and  Sauter  (1937)  assume 
that  they  do  in  the  laminate  break-down  of  synthetically  produced 
polyoxymethylene  crystals,  a  mechanical  cleavage  perpendicularly 
to  the  crystal  axis  should  occur;  but  starch  molecules  are  not  of 
microscopic  length.  Therefore,  any  such  interpretation  would  not 
apply  to  starch  grains.  Another  possible  explanation  is  that  maybe  the 
submicroscopic  capillary  system  of  the  object  in  question,  corre- 
sponding to  the  hydrolysis  pattern,  is  periodically  fine  and  coarse. 
Without  any  such  auxiHary  hypotheses,  however,  it  is  possible  to 
suppose  that  in  the  hydrolytic  break-down  of  fibrous  or  spherite 
structures,  fragments  of  uniform  size  are  produced,  just  as,  in  the 
mechanical  pulverization  of  crystals  or  glass,  only  segments  or 
splinters  of  approximately  the  same  size  split  off,  this  size  having 
nothing  to  do  with  the  structural  elementary  units,  but  depending 
solely  upon  the  method  of  comminution  applied.  Macroscopically  as 
well,  objects  of  entirely  uniform  structure  can  be  split  into  pieces  of 
similar  size  which  have  not  been  pre-formed;  thus,  when  ice  is  broken 
up,  a  perfectly  homogeneous  slab  of  ice  may  split  up  into  floes  of 
equal  proportions,  the  size  of  which  is  by  no  means  predetermined. 
Under  certain  circumstances  and,  of  course,  to  an  enormously  en- 
larged scale,  the  pattern  of  the  floes  may  be  strikingly  reminiscent  of 
the  block  structure  represented  in  Fig.  157.  In  the  opinion  of  Baden- 
HUiZEN  (1938)  the  "blocks"  certainly  are  not  pre-formed  in  the 
structure  of  the  starch  grains. 

The  submicroscopic  structure  of  starch  grains.  Katz  and  Derksen  (1933) 
have  established  that  different  kinds  of  starch  do  not  produce  the 
same  X-ray  spectrum.  For  example,  the  gramineous  starch  of  wheat, 
rice,  corn  and  oats  produces  what  is  known  as  an  A  spectrum, 
whereas  potato  starch  has  a  B  spectrum,  and  both,  when  formed  into 
a  paste,  produce  a  third,  called  the  V  spectrum.  Starches  with  a  B 
spectrum  have  been  converted  at  higher  temperatures  to  the  A  kind 
(Katz  and  Derksen,  1933);  it  has  also  been  shown  that  the  V  spec- 


I  CARBOHYDRATES,  CHITIN  AND  CUTIN  519 

trum  reverts  to  a  B  spectrum  in  the  so-called  retrogradation  of  paste, 
in  which  process  the  quantity  of  bound  water  plays  a  certain  part. 
Thus  the  following  conversions  may  be  observed  in  wheat  starch 
which  is  pasted  up  and  then  retrogresses:  A  ^  V  ^  B. 

Several  investigators  (Sponsler,  1923;  v.  Naray-Szabo,  1928)  have 
attempted  to  deduce  the  size  of  the  elementary  cell  of  crystallized 
starch.  Bear  and  French  (1941)  find  for  B  starch  an  orthorhombic 
cell  with  a  volume  of  930  A^  and  for  A  starch  a  triclinic  cell  with 
843  A^  volume.  This  is  much  more  than  the  cellulose  cell  which 
occupies  only  670  A^.  This  proves  that,  besides  glucose  residues, 
water  molecules  are  enclosed  in  the  cell.  But  these  results  are  doubtful, 
as  starch  produces  only  powder  diagrams,  i.e.,  Debye-Scherrer 
rings.  Recently  Kreger  (1946,  195 1)  has  succeeded  in  irradiating  only 
part  of  the  large  starch  grains  oi  Phajus  grandifolius  by  a  special  micro- 
method.  In  this  way  he  gets  a  fibre  pattern,  which  enables  him  to 
calculate  the  cell  of  B  starch  more  exactly.  Rundle,  Daasch  and 
French  (1944)  were  able  to  prepare  artificial  amylose  threads,  which 
yielded  a  fibre  period  of  10.6  A,  whereas  that  of  cellulose  is  only 
10.3  A.  They  think  that  the  two  glucose  residues  of  the  glucosan  chain 
is  somewhat  stretched  in  crystallized  B-starch,  whereas  Kreger 
(195 1)  places  three  helically  arranged  a-glucose  rings  into  the  distance 
of  10.6  A.  When  the  results  of  the  investigators  mentioned  are 
combined,  the  following  orthorhombic  unit  cell  is  found  for  crystal- 
lized starch  (B-diagram) : 

a  :  b  :  c  =  9.0  :  10.6  :  1 5.6  A. 

Of  these  periods  a :  c  show  the  ratio  i  :  -\/5,  indicating  a  hexagonal 
symmetry.  This  is  in  accordance  with  a  threefold  screw  axis  along  the 
chains  suggested  above.  The  hexagonal  unit  cell  has  the  periods 
a  :  b  =  18  :  10.6  A  and  contains  18  chains,  i.e.  54  glucose  residues  and 
54  water  molecules.  The  density  of  starch  under  water  of  1.60-1,63 
is  in  agreement  with  this  unit  cell  which  is  illustrated  by  Fig.  158 
showing  two  possible  arrangements  of  the  starch  chains  (Kreger, 
1951). 

Senti  and  Witnauer  (1946)  have  shown  that  in  the  A  spectrum 
of  starch  there  is  also  a  fibre  period  of  10.6  A.  From  this  it  follows 
that  in  the  starch  grain  of  either  A  or  B  type  the  amylose  chains  are 
expanding,   forming   a   spiral  pitch   of   10.6  A   with   three   glucose 


320         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

residues.  Consequently  it  is  only  in  solution  that  they  contract  to  a 
pitch  of  7.8  A  (RuNDLE  and  Edwards,  1943)  formed  by  six  glucose 
residues.  It  is  obviously  misleading  when,  in  analogy  to  the  proteins, 
the  extended  amylose  chain  is  called  "denatured"  amylose,  because 
its  natural  state  in  the  starch  grain  seems  to  be  the  expanded  modi- 
fication. 


«>         /   ._; \ 1       /      1      \ 

/  •      '      •  \!/  •      '      •  \ 

Fig.  158.  Cross-sections  of  the  two  possible  unit  cells  of  crystallized  starch  (from  Kreger, 

1951). 

The  optical  behaviour  of  the  starch  grains  rules  out  contracted 
chams  in  their  structure  (Frey-Wyssling,  1940  c).  Since  the  con- 
tracted amylose  chains  show  their  highest  polarizability  (corre- 
sponding to  n^)  perpendicular  to  the  helical  axis  which,  according  to 
X-ray  evidence,  runs  radially,  the  starch  grains  ought  to  be  optically 
negative.  But,  as  mentioned  above,  they  represent  optically  positive 
spherites.  Therefore,  they  must  contain  expanded  chains  which  have 
their  highest  polarizability  (n^,)  parallel  to  the  fibre  axis.  The  iodine 
dichroism  points  in  the  same  direction.  Contracted  amylose  chains 
(Fig.  1 5  3,  p.  312)  have  their  highest  absorption  coefficient  (k^)  parallel 
to  the  fibre  axis,  which  consequently  runs  perpendicular  to  n^.  In 
expanded  chains,  however,  k^  and  n,,  coincide.  Since  this  coincidence 
is  characteristic  for  starch  grains,  it  must  be  taken  for  granted  that 
they  consist  of  expanded  chains. 

Further  proof  of  radially  orientated  elements  is  provided  by  the 
existence  of  rodlet  birefringence  in  starch  grains  (Speich,  1941).  This 
raises  the  question  as  to  what  type  of  submicroscopic  spaces  permits 
the  penetration  of  imbibition  liquids.  They  cannot  have  the  same 
character  as  in  cellulose,  because  hitherto  no  submicroscopic  struc- 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


321 


0.074 


0.013 


tural  elements  of  starch  have  been  found  with  the  electron  microscope 
(unpublished  data)  so  that  its  texture  must  be  amicroscopic.  On  the 
other  hand,  those  spaces  must  be  fairly  wide,  since  they  are  accessible 
to  the  big  molecule  I.^  whose  diameter  measures  2.7  and  5.3  A,  and 
to  organic  dyestuffs.  It  is  doubtful  whether  these  molecules  can  be 
inserted  into  the  intermolecular  spaces  of  the  crystal  lattice  shown  in 
Fig.  158,  where  the  cross-section  of  the  starch  chains  is  not  drawn  in 
its  actual  dimensions,  but  is  merely  symbolized  by  a  black  dot. 
However,  there  is  the  possibility  of  gaps  in  the  chain  lattice,  or  of 
a  widening  of  the  crystal  lattice  by  water,  in  the  same  way  as  is  known 
to  occur  in  zeolites. 

The  analysis  of  the  rodlet 
birefringence  supports  this  view\ 
Series  of  aldehydes,  monovalent 
alcohols  and  polyvalent  alcohols 
(glycol,  glycerol)  including  wa- 
ter give  three  different  curves  of 
rodlet  birefringence  (Fig.  159), 
disclosing  different  intrinsic 
double  refractions  in  these  three 
groups  of  liquids.  This  is  due  to 
the  different  interaction  of  these 
compounds  with  the  starch 
chains.  Lipophilic  liquids  (amyl- 
bromide,  xylene,  toluene,  ben- 
zene, chlorobenzene,  bromoben- 
zene,  and  a-bromonaphthalene) 
do  not  penetrate  into  the  starch 
grain,  because  they  have  no  affin- 
itv  for  starch ;  the  double  refrac- 
tion  therefore  does  not  change  in 
a  series  of  lipophilic  liquids  with 
increasing  refractive  power. 

Since  crystallized  starch  attracts  water  molecules  which  penetrate 
between  the  molecular  chains,  the  starch  grains  ought  to  dissolve  in 
water. 

Such  a  dissolution  is  possible  with  another  reserve  carbohydrate,  viz., 
mannan,  which  is  obtained  from  the  tuber  of  Amorphophallus  konjak  and  is 


0012 


OOII 


1.60  ni" 


Fig.  159.  Rodlet  birefringence  curves  of 
potato  starch  (Speich,  1941).  A  in  lipophilic 
liquids;  B  in  aldehydes;  C  in  monovalent 
alcohols  (except  ethanol);  D  in  water, 
ethanol,  glycol,  glycerol  and  their  mixtures. 


322 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


marketed  in  Japan  under  the  name  of  "Konyaku".  The  technical  com- 
mercial product  consists  of  irregularly  bordered  granules  which  light  up 
in  a  quite  irregular  manner  between  crossed  nicols  and  reveal  no  ordered 
structure  (Fig.  i6oa).  But  when  these  granules  of  konyak  are  observed  in 
water,  they  are  seen  to  undergo  a  remarkable  change.  Under  the  very  eyes 
of  the  observer,  they  swell  and  assume  a  spherotexture,  exhibiting  a  positive 


a) 


b) 


Fig.  1 60.  Alannan  granules  of  konyaku  {Amorphophallns  konjak)  in  the  polarizing  micro- 
scope. Embedding  medium  a)  xylene;  h)  water;  there  is  the  transient  appearance  of  a 

spherite  cross. 


spherite  cross  (Fig.  i6ob).  After  a  time  the  appearance  becomes  fainter 
and  eventually  vanishes  altogether,  because  the  swelling  is  not  limited,  but 
continues  until  solution  takes  place. 

This  phenomenon  may  be  interpreted  as  follows:  The  mannan  chains, 
which  are  comparable  to  the  amylose  chains,  in  the  dry  konyak  granule 
(which  it  is  best  to  examine  in  a  hydrophobic  embedding  medium,  such 
ac  xylene  or  Canada  balsam)  are  arranged  in  bundles  that  accumulate  in 
the  granule  without  any  defined  orientation.  This  explains  why  the  polar- 
izing picture  is  irregular.  As  soon  as  water  is  added  to  these  chains, 
however,  they  are  hydrated,  become  mutually  mobile  and  align  themselves 
radially  and  this  results  in  a  spherotexture.  The  conditions  are  much  the 
same  as  those  in  myelin  figures  (see  Fig.  47,  p.  56),  except  that  hydration 
is  not  limited  to  one  hydrophilic  pole  of  the  molecule,  but  encompasses 
the  entire  mannan  chain  and  ultimately  spreads  to  such  an  extent  that  the 
individvial  thread  molecules  become  independent  of  each  other  and  go  into 
solution. 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


3^5 


Fig.  i6i.  Diagram  of  the  submicro- 
scopic  structure  of  a  layer  of  a  starch 
grain,  a  Outer  portion  looser,  less 
refractive,  with  little  interlinking;  / 
inner  portion  denser,  more  refractive, 
more  closely  interlinked. 


The  stage  of  voluntary  spherite  formation  is  comparable  to  the 
structure  of  starch  grains.  The  starch  molecules  are  obviously  also  fixed 
in  a  radial  direction  by  water  of  hydration.  In  this  case,  however, 
the  water  does  not  function  as  a  solvent,  but  participates,  as  a  loosely 
bound  constituent,  in  the  build-up  of  the  spatial  lattice.  The  starch 
chains  are  far  more  highly  polymeric 
than  the  molecules  of  konyak  mannans . 
Notwithstanding  this,  the  amy  loses 
are  soluble  and,  if  the  starch  grain 
seems  nevertheless  to  swell  only  to  a 
limited  degree,  there  must  be  some 
particular  hindrance  to  solution.  This 
is  probably  to  be  found  in  the  amylo- 
pectin,  the  glucosan  chains  of  which 
are  interlinked.  There  is  good  reason 
to  believe  that  these  amylopectinous 
linkages  occur  in  each  individual  layer 
of  the  starch  grains  in  the  inner,  denser 

and  more  refractive  portions  and  that  independent,  amylose  chains 
are  accumulated  in  the  outer,  looser  portions  of  the  layers.  Jaloveczky 
(1942)  states  that  the  lamellae  containing  amvlopectin  are  isotropic, 
whereas  those  containing  amylose  are  anisotropic  and  can  be  stained. 

It  has  been  suggested  that  all  the  amylopectin  is  localized  in  the 
outermost  marginal  layer  of  starch  grains,  which  is  resistant  when  they 
are  made  into  paste.  It  would  seem  more  probable,  however,  that  the 
starch  is  liable  to  every  conceivable  transition  from  the  easily  soluble 
amyloses  to  the  virtually  insoluble  constituent  of  the  amylopectin, 
which  resists  even  enzymatic  degradation.  Thus  it  might  be  supposed 
that  amylopectins  occur  in  the  denser  portions  of  all  the  layers,  though 
not  in  the  same  degree  as  in  the  insoluble  outside  layer  which  resists 
when  starch  is  made  into  paste.  On  this  assumption  the  submicro- 
scopic  structure  of  a  starch  grain  layer  was  represented  in  1938  as  in 
Fig.  161. 

This  diagram  takes  into  account  the  following  observed  facts :  The 
density  and  refractive  index  at  the  core  of  a  layer  diminish  gradually 
towards  the  outer  regions  and  then  increase  suddenly  at  the  boundary 
of  the  layer.  The  solubilitv  is  not  equal  everywhere  within  the  layer. 
The  water  of  constitution  between  the  chains  is  partially  bound  as 


324 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


hydration  water  of  the  lattice  and  is  partially  mobile  as  swelling  water. 
The  swelling  maximum  of  the  grains  is  governed  by  the  linkage  of 
the  chains.  Adjacent  layers  have  coalesced.  The  structure  is  wide- 
meshed  and  porous,  causing  colourability  and  rodlet  birefringence 
(Speich,   1 941).  In  the  process  of  pasting,  the  loosely  linked,  or 


Fig.  162.  Possibilities  of  fine-structure  in  a  layer  of  a  starch  grain  (n  =  degree  of  poly- 
meri2ation).  a)  Fine-structure  of  amy  lose  (n'-^2  5o);  />)  fine-structure  of  amylopectin 
(n'-^8ooo);  c)  mixture  of  amylose  {n^^z'^o)  and  amylopectin(  n -^  2,000,000);  d) 
amylopectin  with  inward  pointing  aldehyde  group ;  e)  amylopectin  with  outward  pointing 
aldehyde    group;  /)   amylopectin   molecules    with   opposite   orientation    (from   Frey- 

Wyssling,  1948  c). 

unlinked  glucosan  chains  go  into  solution  as  amylose,  whereas  the 
strongly  linked  amylopectin  chains  agglutinate  throughout  the  paste. 
Minor  specific  or  individual  variations  in  linkage  may  be  responsible 
for  the  peculiar  resistance  of  different  kinds  of  starch,  or  of  different 
grains  within  the  same  kind  of  starch.  For  instance,  there  are  grains 
of  potato  starch,  the  peripheral  layer  of  which  is  so  resistant  to 
■enzymes  that  some  of  them  may  pass  unaffected  through  the  intestines 
(Weichsel,  1936). 

To-day  a  more  detailed  discussion  of  the  fine-structure  in  the  starch 
grain  is  possible,  because  the  chemical  constitution  of  amyloses  and 
amylopectins  has  been  cleared  up  since  1938.  Fig.  162  shows  some 
possible  arrangements  of  these  molecules  with  different  degrees  of 
polymerization  n  in  a  layer  i  ^  thick  of  a  starch  grain  (Frey-Wyssling, 
1948c).  The  simplest  case  is  represented  by  a),  where  only  amylose 
molecules  with  n--^  250  (0.088  ix  length)  are  drawn.  However,  since 
the  amyloses  form  only  a  minor  portion  of  the  starch  grains  (Table 


CARBOHYDRATES,  CHITIN  AND  CUTIN 


325 


XXIX,  p,  5 1 2),  the  arrangement  of  the  amylopectins  is  more  important, 
b)  and  c)  show  such  molecules  of  n  '-^  8000  and  n  -^  2,000,000;  it  is 
remarkable  that  a  dichotomous  amylopectin  molecule  of  n  ~'  2,000,000 
should  have  only  the  same  length  {ca.  0.09  ;*)  as  an  amylose  molecule 
of  n/--'  250.  The  many  end  members  of  the  amylopectin  molecule  are 


Fig.  163.  Interpenetration  of  amylopectin  chains  of  opposite  orientation  (from  Frey- 
Wyssling,  1948  c).  Arrows  indicate  the  non-reducing  end  of  the  chains.  Dotted  areas 

=  crystalline  regions  (cf.  Fig.  iGzf). 

not  aldehyde  in  character;  only  the  glucose  residue  at  the  starting 
point  of  the  bifurcated  high  polymer  has  an  open  aldehyde  group. 
Therefore,  it  is  likely  that  such  a  molecule  grows  by  adding  new 
glucose  molecules  with  the  active  aldehyde  group  to  the  brush  end. 
This  is  the  reason  why  in  Fig.  162  the  molecules  have  been  oriented 
in  such  a  way  that  their  growth  direction  coincides  with  that  of  the 
apposition  growth  of  the  starch  grain.  Since  the  amylopectin  is 
attacked  by  the  /5-amylase  from  the  non-aldehydic  end,  this  would 
explain  why  the  enzymatic  dissolution  of  the  starch  grain  often  starts 
at  the  outer  boundary  of  its  layers.  However,  such  an  arrangement 
would  cause  a  higher  density  of  the  layer  in  its  outer  portion  as  seen 


326         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

from  d),  and  this  is  in  contradiction  to  the  optical  result  which  proves 
the  outer  portion  of  the  layers  to  be  less  dense  than  their  inner  portion 
(Fig.  1 5  6,  p.  515).  Therefore,  an  arrangement  as  indicated  in  e)  would 
better  correspond  to  the  optical  behaviour  of  the  grains.  But  then 
the  inner  portion  of  the  layers  ought  to  be  attacked  first  by  ^-amylase. 
This  contradiction  and  the  fact  that  no  chemical  polarity  of  the  layers 
has  ever  been  observed,  make  a  compromise  probable  as  shown  in  f). 
If  the  amylopectin  molecules  grow  in  both  directions,  the  layer  will 
be  chemically  uniform.  Further,  branches  running  in  opposite 
directions  may  crystallize  with  each  other  (Fig.  163).  Since  in  the 
crystal  lattice  of  cellulose  the  glucosan  chains  run  also  in  opposite 
directions,  such  a  structure  for  starch  is  quite  probable.  The  diagram 
of  Fig.  163  would  allow  of  a  mixed  crystallization  of  amylose  with 
amylopectin  and  it  shows  how  gaps  may  arise  in  the  crystal  lattice 
of  starch.  Since  the  X-ray  diagram  is  that  of  a  fibre  texture,  the  two 
directions  of  the  bifurcating  chains  cannot  be  crossed  as  in  Fig.  163, 
but  must  run  almost  parallel. 

Of  all  the  theories  so  far  developed  for  the  structure  of  starch 
grains,  that  propounded  by  A.  Meyer  (1895)  comes  nearest  to  the 
views  set  forth  here.  Instead  of  his  dendritic  branching,  however,  we 
assume  that  there  is  all-round  interlinking,  and  that  the  dimensions 
of  the  structure  are  reduced  by  some  orders  of  magnitude  to  the 
molecular. 

§  2.  Proteins 

a.  Reserve  Protein 

There  is  a  fundamental  difference  between  reserve  proteins  and 
fibrous  proteins.  First  and  foremost,  the  reserve  proteins  are  soluble 
in  water,  dilute  salt  solutions,  acids  and  alkalies,  whereas  the  distin- 
guishing feature  of  the  frame  substances  is  their  pronounced  in- 
solubility. Reserve  proteins  frequently  tend  to  crystallize  if  the  solvent 
is  withdrawn  in  the  proper  way,  as,  for  instance,  by  natural  means  in 
the  formation  of  aleurone  granules  owing  to  the  drying  up  of  vacuoles 
in  vegetable  storage  tissues.  Polyhedral,  crystallized  corpuscles  are 
then  formed,  different,  however,  from  real  crystals  in  that  they  are 
liable  to  swell  and  to  take  up  stain.  Nageli  (1862)  therefore  called 
them  crystalloids.  Notwithstanding  the  fact  that  the  term  "crystalloid" 
was  later  applied  by  Graham  in  quite  another,  and  etymologically 


2  PROTEINS  327 

incorrect,  sense  to  real  solutions  of  substances  of  low  molecular 
weight,  Nageli's  original  definition  was  retained  by  botanical 
cvtologists,  for  to  this  very  day  the  enclosures  of  the  aleurone  granules 
in  the  seeds  of  Ricinns  (Pfeffer,  1872),  Momordica  (Zimmermann, 
1922),  Telfairia  (Leuthold,  1935),  etc.  are  called  crystalloids. 

The  crystal  lattice  of  globular  proteins  are  often  cubic  or  hexagonal ; 
witness  the  occurrence  of  cubic  or  rhombohedral  crystal  shapes  in  the 
crystallized  reserve  proteins  of  vegetable  seeds.  The  globular  ele- 
mentary units  of  the  molecular  lattice  (see  p.  26)  are  so  big  as  to 
produce  a  large  spaced  lattice  (Fig.  90a,  p.  136),  into  the  meshes  of 
which  swelling  agents  and  dyestuffs  can  penetrate.  The  swelling  of 
the  rhombohedral  protein  crystalloids  is  anisotropic,  being,  as 
Nageli  (1862)  had  already  discovered,  different  parallel  to  the  crystal 
axis  from  what  it  is  perpendicular  to  it.  Up  to  1939  only  seven  of  all 
the  many  crystallizing  globular  proteins  had  been  examined  by  X-ray 
crystallography,  these  being  pepsin,  insulin,  excelsin,  lactoglobulin, 
haemoglobulin,  chymotrypsin  and  tobacco  seed  globulin  (Crowfoot, 
1939,  1941).  For,  in  spite  of  repeated  attempts,  it  was  long  before  any 
success  crowned  the  efforts  to  obtain  X-ray  photographs  of  mono- 
crystals  of  the  crystalloids.  Thus,  for  example,  the  crystalloids  of  the 
seed  globulin  excelsin  of  the  spruce  have  threefold  symmetry,  and 
those  of  pepsin  hexagonal,  but  this  fact  was  in  no  way  revealed  by 
the  X-ray  photograph  of  a  single  crystal.  On  the  contrary,  until  a 
short  while  ago  all  monocrystal  photographs  of  globular  proteins,  and 
particularly  in  the  case  of  the  well  "crystallized"  pepsin  (Astbury  and 
LoMAx,  1934),  only  produced  Debye-Scherrer  rings  with  lattice 
spacings  of  4.6  and  11.5  A,  which  unexpectedly  proved  to  have  the 
backbone  thickness  and  the  side  chain  spacing  of  polypeptide  chains. 
In  view  of  the  large  molecular  weight  of  the  crystallized  proteins,  it 
was  anticipated  that,  instead  of  such  spacings,  there  would  be  very 
large  periods  which  would  produce  interference  dots  quite  near  the 
centre  of  the  photograph.  Although  some  such  large  lattice  spacings 
had  been  found  in  insulin  (Clark  and  Corrigan,  1932)  and  in  pepsin 
(Fankuchen,  1934),  Bernal  and  Crowfoot  (1934)  were  the  first  to 
be  entirely  successful  in  obtaining  monocrystal  X-ray  diagrams.  The 
secret  of  their  success  lay  in  the  fact  that  they  irradiated  the  pepsin 
crystalloids  (hexagonal  bipyramids  2  mm  in  height)  in  their  mother 
liquor.  In  this  way  they  discovered  a  wide-meshed  crystal  lattice,  the 


3z8 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


nr 


elementary  regions  of  which  harbour  globular  macromolecules  of 
about  40,000  molecular  weight,  a  figure  that  tallies  with  the  values 
found  in  the  ultracentrifuge. 

Fig.  164  shows  the  result  of  such  an  investigation  of  crystallized 
insuUn  by  Crowfoot  (1938,  1941).  It  is  characterized  by  contour  lines 


Fig.  164.  Patterson-Fourier  diagram  of  crystallized  insulin  (from  Crowfoot,  1938). 


in  the  unit  cell  of  the  lattice  which  are  derived  from  intensity  measure- 
ments of  the  X-ray  diffraction  pattern.  The  resulting  so-called 
Patterson-Fourier  diagram  shows  the  trigonal  symmetry  of  the 
crystal  lattice  in  a  most  instructive  way. 

The  moment  the  crystalloids  are  removed  from  the  mother  liquor^ 
however,  and  are  exposed  to  the  air,  they  denature  and  produce  only 
powder  diagrams.  Although  they  retain  their  crystallographic  shape 
outwardly,  apparently  the  internal  regular  crystal  lattice  order  can 
only  exist  for  just  so  long  as  the  solvent  is  distributed  between  the 
macromolecules . 

It  would  seem  that  there  is  some  relationship  between  globular 
reserve  and  fibrillar  frame  proteins,  notwithstanding  the  great 
differences  between  them  in  point  of  solubility  and  the  morphology 
of  the  molecular  elementary  units,  for  Astbury,  Dickinson,  and 
Bailey  (1935)  succeeded  in  producing  filaments  and  films  from  the 
seed  globulin  edestin  and  from  egg  albumin  which,  when  elongated,, 
exhibit  the  ^-keratin  type  of  fibre  diagram.  Astbury  therefore  assumes 
the  presence  of  folded  polypeptide  chains  in  the  crystalloids  of  the 
reserve  proteins,  as  represented  in  Fig.  165.  In  this  way  certain  self- 
contained  isodiametric  areas  might  be  imagined,  corresponding  ap- 


2  PROTEINS  3^9 

proximately  to  the  globular  molecules  of  the  reserve  proteins,  but  only 
capable  of  existence  in  equilibrium  with  molecules  of  the  solvent. 
Where  there  is  denaturation,  these  loosely-knit  complexes  would  dis- 
sociate and  long  chains  would  begin  to  form  across  the  intervening 
spaces.  This  would  explain  why  denatured  reserve  proteins  become 

NH.  ,C0 

—  -NH-CHR-COy^     iNH-CHR-CO-NH CO-NH-CHR-CO  ^NH-CHR-CO-  — 

CO-CHR-NH'     *C0-CHR-NH-C0 NH-Cn-CHR-NH  ,CO-CHR-NH— 

—NH-CHR-CO^     ,NH-CHR-CO-NH CO-NH-CHR-CO  ^NH-CHR-CO— 

— NH-CHR-NH'     ^CO-CHR-NH-CO NH-CO-CHR-NH.  CO-CHR-NH— 

CO  *NH 

Fig.  165.  Molecular  structure  of  a  protein  crystalloid.  The  arrows  mark  the  bonds  which, 

in  "degeneration"  to  a  fibre  protein,  are  resolved  to  form  bridges  over  the  intermediate 

spaces  (which  contain  solvent)  to  the  neighbouring  molecules,  by  which  means  straight 

chains  come  into  existence  (from  Mark  and  Philipp,  1937). 

less  digestible,  since  in  this  process  the  polypeptide  compounds  pass 
from  a  loosened  soluble  form  to  the  insoluble  chain  lattice  form  ,of 
the  frame-protein  type  (see  Fig.  90,  p.  136). 

Miss  Wrinch  (1937)  suggests  that  ring  formation  of  polypeptide 
chains  may  be  responsible  for  the  globular  shape  of  reserve  protein 
molecules.  According  to  her  "cyclol  theory",  the  chains  would  form 
hexagons  by  ring  folding  and  forming  a  bridge  bond  at  the  open 
position  between  the  NH  and  CO  groups.  If,  by  tTiis  scheme,  six 
amino  acids  are  assigned  to  a  cyclol  six-ring,  the  result  is  three  regular 
hexagons  arranged  trigyrically  around  a  central  hexagon.  This  ternate 
arrangement  falls  into  line  with  the  trigonal  or  hexagonal  crystal 
system  of  the  crystallized  reserve  proteins.  For  each  bridge  formed, 
an  alcoholic  C(OH)  group  comes  into  existence  (see  page  158), 
all  the  hydroxyls  of  which  lie  on  the  same  side  of  the  ring  system; 
this  will  therefore  have  a  hydrophilic  and  a  hydrophobic  side  and 
there  will  thus  be  a  tendency  towards  double  layer  formation.  On  this 
view,  the  protein  crystalloids  are  to  be  conceived  as  packets  of  double 
layers  of  this  kind,  the  hydrophilic  planes  being  responsible  for  the 
ability  of  the  crystal  lattice  to  swell. 

In  recent  years,  it  has  become  doubtful  whether  in  globular 
proteins  the  peptide  linkages  characteristic  of  fibrillar  proteins  are 
already  preformed  (Jordan,  1947;  Scheibe,  1948).  Because  of  their 


330 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


pronounced  dipole  character,  there  is  a  strong  mutual  attraction 
between  amino  acids.  In  +H3N.CH3.C02~  this  leads  to  the  formation 
of  molecular  layers  when  glycine  crystallizes.  For  the  other  amino 
acids  there  is  a  steric  hindrance  due  to  the  side  chains  R,  which  prevent 
the  formation  of  closelv  packed  layers.  Those  amino  acids  therefore 


-T 


-  -I 


X'-- 


Fig.  1 66 


Fig.  167 


I 


Fig.  166.  Aggregation  of  amino  acid  dipoles  by  three  causes  threefold  symmetry.  Peptide 
bonds  are  not  yet  realized  (from  Scheibe,  1948).  Fig.  167.  Plate  of  24  amino  acid  groups 
of  three.  Groups  I  (double  rings)  lie  in  a  somewhat  higher  plane  than  groups  II  (simple 
rings) ;  opposite  borders  of  the  hexagon  differ  from  each  other,  causing  polarity  in  the 
direction  of  the  arrows.  X'  —  Z  cross-section  (from  Scheibe,  1948). 


associate  in  threes  with  the  side  chains  R,  pointing  in  three  different 
directions  in  the  plane  in  which  -NH3+  and  -COO"  lie  (Fig.  166). 
These  groups  of  three  attract  each  other,  forming  hexagonal  rings. 
In  such  a  ring  three  groups  lie  in  a  somewhat  higher  plane  and  three 
in  a  lower  one  (Fig.  167).  The  hexagonal  rings  represent  a  molecular 
layer  with  trigonal  symmetry.  These  layers  can  be  superimposed, 
yielding  a  hexagonal  crystal  lattice.  The  peculiarity  of  such  a  crystal 
is  that  it  consists  of  amino  acids  which  still  retain  their  individuality 
and  are  not  tied  together  to  form  polypeptide  chains.  It  represents 
a  "protein"  without  peptide  bonds. 

Denaturation  would  then  imply  the  formation  of  peptide  bonds 
between  adjacent  amino  acids.  Arguments  in  favour  of  such  a  view 


PROTEINS 


351 


are  these:  There  are  globular  proteins,  such  as  haemoglobin,  which 
are  not  attacked  by  polypeptidase  until  they  are  denatured  (Hauro- 
wiTZ,  1949),  but  since  many  proteolytic  enzymes  work  under  con- 
ditions which  cause  denaturation  (e.g.  pepsin  at  p^  i),  this  fact  is  often 
obscured.  Further,  it  seems  that  the  three  amino  acids  of  the  pre- 
formed groups  in  the  molecular  layer  (Fig.  166)  form  tripeptides 
when  denatured  (tripeptols  of  Jordan,  1947). 

On  the  other  hand,  there  is  no  indication  of  how  such  an  arrange- 
ment  leads  to  molecules  with  a  definite  weight.  When  4  layers  as  seen 
in  Fig.  167  with  24  x  3  amino  acids  are  superposed,  a  molecule  of 
288  amino  acids  is  obtained,  which  would  correspond,  for  instance, 
to  insulin.  It  is  not  clear,  however,  why  piles  of  only  four  layers  exist, 
and  why  aggregations  of  such  fourfold  layers  by  2,  4  etc.  occur 
according  to  the  Svedberg  series.  It  seems  likely,  therefore,  that  the 
binding  forces  inside  the  molecules  are  stronger  than  those  which 
cause  the  aggregation  of  globular  protein  molecules  to  multiples  and 
crystal  lattices  (Fig.  84,  p.  126). 

b.  Si/k  (Silk  Fibroin) 

Microscopic  and suhmicroscopic  structure.  A  cross-section  of  the  cocoon 
thread  of  the  silk-moth  {Bombyx  mori)  reveals  two  halves  in  mirror 
symmetry,  which  owe  their  existence  to  the  paired  silk-glands.  These 
■produce  two  discrete  fibroin  threads  which  are  covered  with  a  layer 


Fig.  168.  Fine-structure  of  silk,  a)  Microscopic  cross-section  through  the  cocoon  filament 
(after  Ohara,  1933a);  b)  suhmicroscopic  structure  of  the  fibroin  thread,  i  Skin  and  2 
cortex  (fibroid  texture  with  tangential  scattering)  of  the  sericin  layer.  3  Skin,  4  cortex 
(fibroid  texture  with  radial  scattering)  and  5  central  zone  (fibrous  texture)  of  the  fibroin 

filament. 


of  sericin  (Fig.  168).  The  regular  structure  as  seen  in  Fig.  168  a  is 
apparently  disturbed  where  the  threads  cross  in  the  cocoon,  which 
would  go  to  show  that  the  thread  is  still  plastic  when  it  leaves  the 


332  FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

silk-gland.  A  finer  structure  is  revealed  both  by  the  sericin  layer  and 
by  the  fibroin  threads  (Ohara,  1933  a).  On  the  outside  is  a  very 
weakly  birefringent,  almost  amorphous  membrane  of  sericin,  under 
which  comes  a  strongly  birefringent  layer  of  a  fibroid  texture.  The 
sericin  layer  is  separated  from  the  fibroin  threads  by  an  isotropic 
lamella.  It  is  here  that  the  sericin  becomes  detached  from  the  fibroin 
when  the  silk  is  degummed  in  a  dilute  soap  solution.  Two  degummed 
silk  threads  are  then  formed  from  every  cocoon  filament  or  raw  silk 
thread.  The  difference  between  raw  silk  and  ordinary  degummed  silk 
is  therefore  that  the  former  is  still  surrounded  by  the  sericin  cortex, 
though  admittedly  this  often  suffers  considerable  mechanical  damage. 

The  fibroin  filaments,  which  are  now  to  form  our  main  topic,  have, 
according  to  Ohara,  three  zones  which  are  optically  distinguishable, 
i.e.,  a  central  zone  of  fibrous  texture,  a  cortical  layer  around  this  of 
fibroid  texture  and,  finally,  at  the  outside  a  skin  layer.  This  is  only 
slightly  anisotropic,  yet  its  texture  is  apparently  slightly  fibroid.  It  is 
interesting  to  note  that  here  the  prevailing  direction  of  orientation 
— i.e.,  deviation  of  the  optically  positive  submicroscopic  fibroin 
rodlets — is  not  tangential,  as  in  the  sericin  layer  or  in  cellulose  fibres, 
but  radial  (see  Fig.  i68b).  It  seems  that  in  the  process  of  degumming, 
the  character  of  the  scattering  in  the  coating  layer  changes  from  radial 
to  tangential,  for,  after  the  hot  water  treatment,  the  large  axis  of  the 
index  ellipse  lies  tangentially.  The  scattering  of  the  fibroid  texture  of 
the  cortical  layer  is  likewise  radial.  Thus  in  a  cross-section  through 
the  cocoon  threads,  sericin  and  fibroin  are  easily  distinguishable  by 
their  different  optical  behaviour  in  a  polarizing  microscope  in  which 
a  selenite  test  plate  has  been  inserted,  in  that  the  sericin  wrapping 
produces  a  negative,  and  the  fibroin  cortex  a  positive  spherite  cross 
(cf.  Fig.  66a,  b,  p.  96).  In  the  cross-section  the  central  zone  appears 
to  be  isotropic. 

The  zoning  of  the  fibroin  filament  is  brought  out  clearly  by  dichroic 
gold  and  silver  staining.  In  conformity  with  its  fibrous  texture,  the 
central  zone  exhibits  pronounced  dichroism;  in  the  cortical  zone,  on 
the  other  hand,  with  its  far  inferior  orientation,  the  coloration  is  not 
dichroic,  and  in  the  coating  layer  there  is  none  at  all.  According  to 
Ohara  (1933  a),  this  is  how  the  fibroin  filament  laminates  as  a  result 
of  coagulation:  The  coating  layer  is  the  first  to  coagulate  on  leaving 
the  silk-gland,  before  there  is  any  opportunity  for  an  ordered  sub- 


2  PROTEINS  333 

microscopic  structure  to  be  formed.  A  little  later,  the  cortical  layer, 
the  jfibroin  thread  molecules  of  which  are  already  to  some  extent 
orientated,  coagulates.  The  fibroin  mass  in  the  central  zone  remains 
plastic  for  a  longer  period  and  the  chain  molecules  of  the  silk  fibroin 
are  all  able  to  orientate  with  parallel  axes  before  they  combine  to  form 


•fs!e*-.«=5S«.-., 


^^"^  ik 


7> 


-  -c? 


r 


v< 


a)  b) 

Fig.  169.  Fine-structure  of  silk  (from  Ohara,  1933b).  a)  Beading  with  Ca(N03)2  solution; 

b)  fibrillar  formation  with  hypobromite. 


a  micellar  frame.  As  rayon  filaments  often  display  a  similar  structure 
(Preston,  1933),  Ohara's  hypothesis  is  attractive,  but  it  should  be 
pointed  out  that,  unlike  natural  silk,  the  cortical  part  of  viscose  is 
submicroscopically  better  orientated  than  the  central  part  of  the 
filament  ("skin  effect"  according  to  Preston,  1933).  In  rayon,  the 
stretching  process  brings  about  an  orientation  of  the  peripheral  region, 
whereas  the  thread  molecules  of  the  still  uncongealed  mass  in  the 
centre  of  the  filament  are  not  effectively  held  by  the  orientating  forces, 
owing  to  their  mobility.  Hence  the  optical  conditions  prevailing  in 
natural  silk  which  conflict  with  this  interpretation  must  be  explained 
in  some  other  way. 

Since,  like  vegetable  bast  fibres,  the  silk  fibroin  filaments  possess 
a  central  portion  of  a  fibrous  texture  and  a  skin  with  a  pronounced 
deviation  of  the  microfibrils  from  the  direction  of  the  fibre  axis,  their 
swelling  and  hydrolysis  phenomena  are  similar  to  the  cellulose  walls 
■of  fibre  cells.  Thus  Ohara  (1933b)  finds  a  beaded  appearance  in  silk 


534         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

similar  to  that  found  in  cellulose  fibres,  when  the  central  portion, 
expanding  powerfully,  is  pressed  through  weakened  spots  of  the  skin 
layer  (Fig.  169).  Furthermore,  the  central  zone  can  be  split  up  bv 
bromine  lye  (hypobromite)  into  fibrillae,  which  then  disintegrate  into 
short  bundles  of  fibrillae,  as  in  cellulose  fibres.  These  facts  are  im- 
portant, in  that  they  imply,  contrary  to  Ludtke's  statement  (1936) 
about  cellulose  fibres,  that  beading  and  hydrolytic  disintegration 
perpendicular  to  the  fibre  axis  do  not  depend  upon  any  partition  across 
the  fibre ;  for  there  can  be  no  question  of  the  formation  of  any  such 
hypothetical  segmentation  during  the  generation  of  the  silk  thread. 

Just  as  with  other  fibrous  structures,  a  system  of  submicroscopic 
rodlets  is  to  be  expected;  and,  in  fact,  Ohara  (1933a)  found  rodlet 
double  refraction  in  silk.  However,  as  the  minimum  of  the  curves  he 
has  published  (n  ^  i-47)  does  not  agree  with  the  average  refractive 
index  of  silk  fibroin,  his  measurements  do  not  seem  altogether 
reliable.  Hegetschweiler  (1948)  finds  (n,,)^  =  1.5960  and  (nj^ 
=  1.5454  yielding  the  double  refraction  An  =  0.0506  for  silk.  These 
figures  give  an  average  of  (n^,  -f  2nJ/3  =  1-565,  which  is  quite  in- 
compatible with  n  =  1.47  mentioned  above.  For  this  reason,  Heget- 
schweiler (1950)  repeated  the  imbibition  experiments  of  Ohara  and 
found  that  liquids  which  do  not  swell  silk  fibroin  cannot  penetrate 
and,  therefore,  do  not  change  its  birefringence.  Since  the  cross- 
section  of  the  fibroin  thread  is  triangular  (Fig.  168  a,  p.  331),  so  that 
the  thickness  corresponding  to  a  retardation  of  light  F  observed  can 
only  be  measured  after  rotation  of  the  thread  through  90°,  and  as  the 
swelling  in  aqueous  solutions  is  considerable,  it  is  very  difficult  to 
obtain  reliable  figures  reflecting  small  changes  of  the  birefringence  of 
silk  by  the  formula  Zl  n  =  F/d.  If  all  the  necessary  precautions  are 
taken  and  numerous  measurements  made  in  the  same  liquid  in  order 
to  obtain  reliable  average  values,  it  can  be  proved  that  silk  fibroin  is 
not  a  mixed  Wiener  body. 

This  optical  finding  is  borne  out  by  electron  microscope  investi- 
gations (Hegetschweiler,  1950).  Unlike  native  cellulose,  silk  fibroin 
does  not  consist  of  individual  microfibrils.  There  is  a  distinct  fibrillar 
texture,  but  the  diameter  of  the  visible  strands  depends  on  the  method 
of  preparation.  The  same  fibroin  threads  show  rather  coarse  (o.i  ju) 
or  very  fine  (0.0 1  /u)  strands,  or  both  types  together  with  intermediate 
grades,  depending  on  the  way  in  which  they  have  been  hit  during 


2  PROTEINS  355 

disintegration  in  the  blender.  There  is  a  similarity  here  with  rayon  in 
which  different  types  of  fibrillar  strands  are  visible  in  the  electron 
microscope  (Frey- Wyssling  and  Muhlethaler,  i  949  c)  without  any 
evidance  of  individual  microfibrils.  It  seems  that  during  the  spinning 
process  a  less  regular,  more  compact  body  is  formed  than  during 
o-rowth,  when  innumerable  uniform  microfibrils  originate  from  a 
living  matrix.  There  is  therefore  a  pronounced  difference  between  the 
submicroscopic  texture  of  grown  and  spun  fibres. 

Molecular  structure.  Silk  fibroin  consists  of  expanded  polypeptide 
chains  which  crystallize  in  a  chain  lattice.  This  is  why  silk  has  a  high 
tensile  strength  and  a  large  intrinsic  double  refraction,  similar  to  those 
of  the  chain  lattice  of  cellulose.  It  is  noteworthy  that  this  similarity 
has  no  chemical  background  whatsoever,  since  silk  fibroin  and  cellu- 
lose belong  to  quite  different  classes  of  chemical  compounds.  It  is  only 
the  fundamental  morphological  principle  of  parallel  macromolecular 
chains  with  a  high  polarizability  parallel  to  the  fibre  axis  which  is 
responsible.  This  shows  how  important  morphological  considerations 
are  for  the  analysis  of  the  properties  of  high-polymer  substances. 

According  to  Bergmann  and  Niemann  (1957)  silk  fibroin  consists 
of  2^  X  3^  =  2592  amino  acids  (mol.wt.  --^-^  220,000).  Half  of  these 
are  considered  to  be  glycine,  1/4  alanine,  1/16  tyrosine,  1/2 16  arginine, 
1/648  lysine  and  1/2592  histidine.  In  addition  to  these  constituents 
Drucker  and  Smith  (1950)  have  found  by  paper  chromatography 
small  amounts  of  aspartic  acid,  glutamic  acid,  serine,  threonine,  valine, 
leucine,  phenylalanine  and  proline.  From  viscosity  measurements  they 
assign  a  molecular  weight  of  33,000  to  fibroin,  which  is  almost  one 
order  smaller  than  that  of  Bergmann  and  Niemann  (1937).  This 
discrepancy  is  no  doubt  due  to  the  fact  that  the  determination  of 
amino  acids  in  very  small  quantities  is  open  to  considerable  error. 

Since  three  quarters  of  silk  fibroin  consists  of  the  smallest  amino 
acids  glycine  and  alanine,  a  relatively  simple  chain  lattice  can  be 
derived  from  X-ray  analysis  (Meyer  and  Mark,  1928),  if  the  other 
amino  acids  (tyrosine  etc.)  are  considered  to  exist  as  amorphous 
substances  without  participating  in  the  crystal  lattice.  This  view  is 
supported  by  the  observation  of  Drucker  and  Smith  (1950)  that 
tryptic  hydrolysis  of  short  duration  leaves  glycine,  alanine  and  serine, 
i.e.  the  simplest  amino  acids,  undissolved,  whereas  all  the  other  amino 
acids  are  found  in  the  hydrolysis  liquor. 


356 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


In  profile  the  crystallized  chains  present  the  familiar  picture  of  the 
zig-zag  line  with  consecutive  CO,  NH  and  CH  groups  (Fig.  170a). 
AsTBURY  (1935a)  calls  the  distance  between  neighbouring  chains  the 
"backbone  spacing"  and  it  measures  4.5  A,  It  may  therefore  be  said 
that  the  depth  which  a  chain  requires  in  the  plane  of  the  zig-zag  line, 


\ 
/ 

RCH 


NH 


/ 

NH 

\ 


CO 


/ 

CO 

\ 


'CHR 


NH 


RCH 
\ 

/ 

NH 
\ 


CO 


Profile 


CO 

RCH 

/ 

NH 

\o- 

/ 

RCH 

\ 

NH 
/ 
CO 

RCH 

/ 

NH 

\ 

CO- 
/ 


I 

-co- 
I 

CH 
I        °^ 

NH      ^ 


:;r 


CH, 


■CO- 

I 
-CH 

I 
NH 


C>5 


,„J 


CO 

CH-CH,—(~^0H 

I 
NH 

I 

—-co 

I 


to 


>  P 


Front 

a)  b) 

Fig.  170.  Molecular  structure  of  silk  fibroin. 


or  the  "backbone  thickness", 
amounts  to  4.5  A.  The  side 
chains  of  the  CH  groups  are 
not  seen  in  the  profile  view  of 
Fig.  170  a,  as  they  stand  off,  like 
ribs,  perpendicular  to  the  back- 
bone plane,  suggestive  of  a 
vertebrate  skeleton.  The  thread 
molecule  has,  therefore,  to  be 
seen  from  the  front  to  get  the 
side  chains  in  their  proper  place 
(Fig.  170b).  Thus  the  kinked 
chain  appears  as  a  straight 
line  with  foreshortened  valence 
bonds,  while  the  side  chains  lie 
in  the  plane  of  the  drawing.  The  glycine  residue  can  scarcely  be  said  to 
have  a  side  chain,  which  in  this  case  is  represented  only  by  the  insigni- 
ficant H  atom;  but  with  the  alanine  residue  it  consists  ofa  methyl  group. 
The  side  chains  are  not  fitted  in  pairs  like  actual  ribs  but  point  alter- 
nately to  left  and  right  on  consecutive  CH  groups,  with  the  result  that 
every  two  neighbouring  amino  acid  residues  together  form  a  morpho- 
logical unit,  which  in  the  X-ray  diagram  becomes  the  fibre  period.  In 
silk  fibroinitamountsto6.95  A.  From  this  it  may  be  concluded  that  the 
length  of  each  backbone  segment,  or  in  other  words  the  extension  of 
each  individual  amino  acid  member,  is  3.5  A.  This  length  is  quite 
irrespective  of  the  nature  of  the  amino  acids  in  the  primary  valence 
chain.  Thus  all  the  entirely  different  components,  glycine,  alanine  and 
even  tyrosine,  represent,  as  members  of  the  chain,  sections  of  the 
thread  molecule  of  exactly  the  same  length  (Astbury,  1933b).  They 
can  therefore  be  interchanged  without  thereby  causing  any  alteration 
in  the  fibre  period  or  the  backbone  thickness. 

The  distance  apart  of  the  main  chains  depends  on  the  length  of  the 
side  chains  which,  as  may  be  seen  in  Fig.  170  b  where  tyrosine  is 


2  PROTEINS  337 

added,  is  very  unequal.  In  order  to  preserve  rigid  regularity  in  this 
respect  as  well,  Meyer  and  Mark  (1950)  assumed,  as  mentioned 
above,  that  only  glycine  and  alanine  residues  form  crystallized  silk 
iibroin.  It  does  not  seem  likely  that  this  view  can  be  maintained,  for 
up  to  the  present  it  has  not  been  possible  to  define  an  undoubted 
elementary  cell  of  the  crystal  lattice  as  in  cellulose  (Kratky  and 
KuRiYAMA,  1951;  Sakurada  and  Hutino,  1935;  Brill,  1943).  The 
reason  may  be  a  certain  irregularity  caused  by  the  side  chains  of  other 
amino  acids.  Friedrich-Freksa,  Kratky  and  Sekora  (1944)  treated 
silk  fibroin  with  iodine  and  found  by  X-ray  analysis  a  new  period  of 
70  A  perpendicular  to  the  fibre  axis.  As  it  is  likely  that  the  iodine  is  in- 
troduced into  the  tyrosine  residue,  every  20th  amino  acid  of  the  poly- 
peptidic  chain  should  be  tyrosine.  This  would  agree  with  the  statement 
of  Bergmann  and  Niemann  (1937)  that  out  of  16  amino  acid  residues 
one  is  tyrosine.  It  is  therefore  probable  that  tyrosine  belongs  to  the 
crystallizing  polypeptide  chains.  The  primary  valence  chains  are  held 
together  by  hydrogen  bonds  (see  p.  148)  forming  a  chain  lattice 
(Brill,  1941). 

In  the  glands  of  the  silkworm  the  fibroin  exists  probably  as  globular 
protein  called  fibroinogen.  Kratky,  Schauenstein  and  Sekora  (1950) 
find  that  air-dried  glands  yield  an  X-ray  diagram  similar  to  F-actin 
(see  p.  352).  It  is  called  silk  I,  whereas  the  usual  diagram  is  silk  II. 
By  stretching,  silk  I  can  be  transformed  into  silk  II  of  the  spun  thread. 
Only  the  transition  silk  I  ->  silk  II  has  been  observed,  the  reverse 
being  apparently  impossible.  This  favours  the  view  that  the  formation 
of  the  silk  thread  consists  in  the  denaturation  of  an  originally  soluble 
globular  protein.  If  these  protein  molecules  contain  a  certain  pro- 
portion of  all  the  amino  acids  found  in  silk  fibroin,  it  would  be  likely 
that  the  polypeptide  chains  formed  by  denaturation  comprise  not  only 
glycine  and  alanine,  but  also  the  other  amino  acids.  The  portions  of 
the  chains  with  unwieldy  side  branches  would  then  not  crystallize 
(Fig.  54b,  p.  70)  and  might  therefore  be  more  easily  susceptible  to 
hydrolysis  than  the  smooth  glycine  and  alanine  portions  of  the  chain 
which  can  crystallize. 

Mercer  (195  i)  finds  that  microfibrils  of  fibroin  (100  A  thick  and 

3  500  A  long)  are  formed  spontaneously  from  a  solution  of  fibroinogen 
in  water.  This  seems  to  be  a  favourable  object  for  studying  the  trans- 
formation of  a  globular  protein  into  fibrils  with  the  electron  microscope. 


558  FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  Ul 

c.  Ho}  ny  Substances  (Keratin) 

Microscopic  structure  and  birefringence  of  hair.  The  great  technicaf 
importance  and  the  remarkable  elastic  behaviour  of  wool  and  other 
hairs  were  the  incentive  to  research  on  keratin. 

Microscopically,  hairs  consist  of  three  layers,  viz.,  a  scaly  and  un- 
pigmented  epidermis  which  is  covered  by  a  very  thin  cuticle  or 
epidermicula  (Lindberg,  Philip  and  Gralen,  1948;  Schuringa  and 
Algera,  1950),  a  thick,  fibrous  cortical  layer  containing  pigment,  and 
a  parenchymatous  pith.  Sometimes  there  is  no  pith,  as  in  Merino  wool. 
The  surface  skin,  which  covers  the  cortex  with  scales  that  are  ring- 
shaped  or  like  roofing  tiles,  may  likewise  disappear  owing  to  mechanic- 
al chafing,  and  yet  the  elastic  and  optical  properties  of  the  hair  will 
not  radically  change.  Their  source  is,  therefore,  the  keratin  fibre  cells 
of  the  cortex,  which  consist  of  numerous  tonofibriUae  orientated  in 
parallel.  In  the  electron  microscope  the  fibrillae  can  be  seen  to  unravel 
into  still  finer  subfibrillae  (Reumuth,  1942).  The  tonofibriUae  vary  in 
length  between  50  f.i  and  a  few  millimetres,  being  about  80  pL  (Hohnel,. 
1887)  in  sheep.  They  are  usually  flattened.  Although  a  hair  appears 
to  be  optically  homogeneous,  it  is  not  comparable  cytologically  to- 
a  single  bast  fibre,  but  to  multicellular  strands  of  bast  fibres  consisting 
of  relatively  short  fibre  cells,  as  they  occur  in  Monocotyledons  (sisal^ 
Manila  hemp,  etc.). 

.  Unlike  cellulose  fibres,  horn  fibres  are  extremely  elastic.  In  cold 
water  a  hair  can  be  stretched  reversibly  by  50  to  70^0,  whereas  bastr 
fibres  of  good  fibrous  structure  break  when  stretched  only  a  feu^ 
per  cent.  The  elastic  elongation  of  the  hairs  is  especially  impressive 
under  the  polarizing  microscope  (Pochettino,  1913).  Although  the 
cross-sectional  area  of  the  hair  decreases  owing  to  the  elongation,  the 
retardation  increases  considerably,  and  this  is  apparent  from  the  sharp- 
rise  in  interference  colours.  It  is  a  fascinating  spectacle  to  watch  the 
polarization  colours  of  weakly  pigmented  (fair)  hair  changing  as  the 
hair  is  stretched  and  released.  Whereas  photo-elastic  effects  of  this 
kind,  however,  are  usually  brought  about  by  slight  changes  of 
distance  in  the  crystal  lattice  which  are  not  detectable  by  X-rays- 
(Wiener,  1926b),  the  molecular  frame  of  keratin  is  completely  re- 
formed during  elongation. 

In  curly  wool  the  stretched  outer  side  takes  basic  dj-es  (Janus  green^ 
neutral  violet,  pyronine)  more  easily,  and  has  a  lower  refractive  index. 


PROTEINS 


559 


in  a  radial  direction,  than  the  inner  side  of  the  curl  (Ohara,  1958^ 

1939)- 
Alol^cular  structure  of  keratin.  Astbury  (1935  c)  has  demonstrated 

that  stretched  hairs  produce  quite  a  different  X-ray  diagram  from 

unstretched    ones.    The    difference    is    especially   evident   when    the 


\ 


NH 


CO' 


rf 


CO-' 

\ 


CHR 


CHR 


NH 


<o 

Co 
>; 

<r> 

II 

I5 


NH 


CO 


C0-- 


CHR 


NH 
fi- Keratin 


y  CH.R)-C0-NH- 

NH 

I 
CO 


■R':CH 

'■'    I 
NH 
CO 

CH^ 


CH  (R\-NH'  CO-\R)CH  ^ 
NH 

CO 

.    I 
IR)CH 

NH 
I 


^CH 

NH 

I 
CO 

CH® 


'.     ..■■'  CO 

■,RyCO-NH-(§)CH'' 


a-Kerafin 


b) 


Fig.  171.  a)  ^-keratin  (after  Astbury,  1933c).  h)  Folding  of  the  polypeptide  chain;  R  side 

chains  (after  .'\stbury  and  Bell,  1941). 


elongation  takes  place  in  a  vapour-saturated  chamber  at  100°  C,  where 
about  100%  elongation  can  be  attained.  The  X-ray  picture  shows  the 
distance  between  the  members  of  the  chain  to  be  3.38  A.  This  tallies 
well  with  the  chain  period  of  silk  fibroin,  viz.  3.5  A  and  it  may 
therefore  be  assumed  that  elongated  primary  valence  chains  of  poly- 
peptide thread  molecules  are  also  present  in  stretched  wool.  As  the 
fibre  period  in  unstretched  wool  is  5 .06  A,  some  other  modification, 
which  Astbury  designates  as  a-keratin,  must  be  involved.  The  keratin 
in  stretched  wool  is  known  as  /5-keratin.  By  folding  the  polypeptide 
chain,  he  derives  a-keratin  from  jS-keratin,  arguing  that  by  the  mutual 
attraction  of  two  NH  and  CO  groups  separated  by  five  valence  bonds, 
pseudo-diketopiperazine  rings  are  liable  to  be  formed.  Taking  into 
account  the  rules  of  distance,  the  fibre  period  of  a-keratin  for  a  chain 


34©         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

thus  folded  comes  to  5.06  A.  As  the  diagram  shows  (see  Fig.  171a), 
the  chain  length  is  doubled  at  full  stretch  (100%  elongation). 

The  ingenious  theor)^  of  folding  to  form  piperazine  rings  is  con- 
fronted with  steric  difficulties ;  for  the  side  chains  R,  which  point  in 
the  same  direction,  come  so  close  together  that  they  hinder  each  other 
spatially.  Astbury  and  Bell  (1941)  have  therefore  drawn  up  a  new 
folding  diagram  for  the  /5-a  transformation,  which  satisfies  the 
following  conditions : 

I.  The  a-form  must  be  about  half  as  long  as  the  ^^-form.  2.  The 
density  must  remain  practically  constant.  3.  The  folds  must  repeat  at 
a  distance  of  about  5.1  A.  4.  The  side  chains  must  stand  out  alternately 
on  one  side  and  the  other  of  the  plane  of  the  fold.  5 .  The  folds  must 
be  nowhere  so  sharp  as  to  have  insufficient  room  for  the  side  chains. 

This  diagram  is  reproduced  in  Fig.  171b.  Side  chains  pointing 
upwards  are  marked  R  enclosed  in  a  full-line  circle  and  those  pointing 
downwards  by  R  within  a  dotted  circle.  The  side  chains  standing  on 
the  same  side  form  groups  of  three,  which  in  the  diagram  appear  as 
the  angles  of  the  triangles  indicated. 

The  R  side  chains  are  particularly  important.  If  hairs  stretched  in 
steam  at  100°  C  are  dried  in  the  extended  state,  the  elongation  loses 
its  reversibility  and  is  retained.  The  side  chains  of  neighbouring  poly- 
peptide chains  enter  into  spatial  relationship  and  connect  the  primary 
valence  chains  to  a  kind  of  grid  (Fig.  172).  The  distance  between  the 
bars  of  the  grid  is  9.8  A;  hence  the  side  chains,  which  at  intervals  of 
3.38  A  stand  off  more  or  less  perpendicularly  from  the  primary  chains 
to  the  right  and  left,  should  have  half  that  length.  The  thickness  of 
the  grid  corresponds  to  the  backbone  thickness  of  the  stretched, 
zig-zag  polypeptide  chains  and  is  therefore  4.5  A. 

Glutamic  acid,  arginine  and  cystine  are  among  the  most  important 
products  of  the  hydrolysis  of  wool  (see  Fig.  88,  p.  133).  Assuming 
amidic  linking  between  glutamic  acid  and  arginine,  there  will  be 
a  kind  of  rung  linking  two  primary  valence  chains,  as  represented  in 
Fig.  172  b.  Retaining  the  tetrahedral  angle,  this  side  connection  would 
be  about  12.5  A  long.  It  is,  therefore,  of  the  order  of  magnitude  of 
the  length  found  by  X-ray  measurement,  viz.,  9.8  A,  for  it  is  quite 
conceivable  that  the  chains  may  somehow  be  shortened. 

Cystine  is  the  most  interesting  of  the  three.  This  contains  two  amino 
acid   residues  united  by  a  sulphur  bridge.  It  is  assumed  (Astbury, 


PROTEINS 


341 


1933c;  Mark  and  Philipp,  1957)  that  such  sulphur  bonds  hold  the 
polypeptide  chains  together  in  keratin,  for  sulphur  plays  a  similar 
part  in  vulcanized  rubber.  It  connects  the  free  polyprene  chains 
of  the  raw  rubber  laterally,  in  this  way  producing  a  molecular  frame, 
and  thus  enhances  the  elastic  properties  of  the  raw  rubber,  while 


1       1       '                            1 

1    1    1     1    1     1    1     1    1 

— 

Sjde__ 
chains 

CO 

"1" 

6 
— tt" 

ft8/J 

:i 


o) 


NH 


NH 

CH' 

I 

CO- 

I 

NH 

RCH 

ho 

I 

NH 

^: 

CO 

I 


CH,       CO 


I 
(^0 

NH 

CHy  CH2         I 


^CHl'^.N'H'^f^^CtC      '^"^ 


Glutamic     I 
acid 


-^0 
Arginine         j^^ 

I 


CM. 


CH. 


S S' 


Cystine 


ho 

NH 

-ho 


CHR 

ho 
f 

RCH 


Fig.  172.  a)  Keratin  frame  as  lattice  grid;  b)  side  chains  of  keratin. 


its  plasticity  deteriorates.  If  too  many  sulphur  bridges  are  intro- 
duced, however,  the  material  will  lose  its  elasticity,  being  "vulcanized 
to  death",  and  hard  rubber  or  ebonite  results.  Now  there  is  some 
analogy  between  raw  rubber  and  vulcanized  rubber,  on  the  one  hand, 
and  actomyosin  (free  from  sulphur,  p.  3  5  2)  and  keratin  (containing  sul- 
phur), on  the  other.  By  way  of  comparison,  therefore,  the  tonofibrillae 
have  been  termed  "vulcanized'  muscle  fibres,  which  would  explain 
the  loss  of  contractility  and  their  great  strength. 

Despite  illuminating  comparisons  such  as  these,  which  are  very 
helpful  to  a  qualitative  interpretation,  there  remain  serious  quanti- 
tative obstacles  to  a  complete  understanding  of  the  submicroscopic 
structure  of  keratin.  Above  all,  the  length  of  the  cystine  molecule  does 
not  agree  with  the  X-ray  evidence  as  to  the  length  of  the  keratin  side 
chains.  As  is  apparent  from  Fig.  172  b,  the  sulphur  bridge  is  by  no 
means  long  enough  to  span  the  distance  of  9.8  A  from  primary  chain 
to  primary  chain.  Hence  the  molecular  frame  cannot  be  as  simple  and 


542         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

orderly  as  it  is  represented  in  Fig.  172  a;  possibly,  therefore,  some  other 
amino  acid  besides  cystine — say  glutamic  acid — assists  in  bridging 
this  great  distance. 

There  is,  however,  another  reason  why  the  molecular  frame  is 
unlikely  to  be  a  simple  structure.  Astbury  (1933  c)  advances  plausible 
arguments  to  show  that,  in  the  re-transformation  of  the  /5-keratin  of 
stretched  hairs  into  the  folded  a-keratin,  side  bridges  must  be  broken 
off.  This,  with  a  planar  molecular  frame,  would  be  avoidable  only  if 
all  the  parallel  2ig-2ag  polypeptide  chains  could  be  folded  simultane- 
ously perpendicular  to  the  projection  plane  of  Fig.  172a  without 
breaking  the  cross  links.  If,  however,  the  primary  valence  sheets  are 
linked  in  various  directions,  the  individual  polypeptide  chains  can  no 
longer  be  folded  without  breaking  up  the  side-chain  bonds. 

It  is  very  significant  that  ordinary  water  is  capable  of  disrupting 
the  bonds  in  question  in  the  case  of  /3-keratin;  for  a  hair  stretched 
to  double  its  length  and  then  dried  has  only  to  be  placed  in  water 
to  regain  its  reversible  elasticity.  This  means  that  drying  brings  about 
only  temporary,  and  not  permanent,  set.  Nevertheless,  if  a  hair 
elongated  100%,  is  left  for  half  an  hour  in  a  steam  bath,  it  loses 
the  capacity  to  contract  again  to  its  original  length,  being  now 
permanently  set  and  retaining  this  imposed  length  even  when  wetted 
in  what  is  known  as  "permanent  set".  This  fact  is  put  to  use  in  the 
hairdressing  profession,  for  it  is  only  when  the  hairdresser  succeeds 
in  imparting  permanent  set  to  the  /5-keratin  produced  at  the  curved 
places  in  the  hair  that  he  can  claim  to  have  provided  a  "permanent 
wave".  The  permanent  setting  of  the  /5-keratin  is  said  to  be  achieved 
by  the  prolonged  action  of  the  steam,  whereby  so  many  strong 
bridges  are  laid  between  the  keratin  chains  that  hot  water  is  subse- 
quently unable  to  disrupt  them. 

Elod,  Nowotny  and  Zahn  (1940)  oppose  Astbury 's  theory  that 
keratin  contains  grid  frames  connected  by  sulphur  bridges  in  the  side 
chains.  Treatment  of  the  wool  with  metallic  mercury  will  convert 
50%  of  the  keratin  sulphur  to  HgS.  Removing  half  of  the  -S-S- 
bridges  should  weaken  the  molecular  frame,  involving  modification 
of  the  properties  of  the  wool.  This,  however,  is  not  the  case  and  these 
investigators  therefore  assume  that  it  is  not  the  side  chains  which 
build  up  the  frame  but,  as  in  silk  fibroin,  hydrogen  bonds  (see  Fig.  98, 
p.  148)  between  the  primary  chains  in  the  backbone  planes  (Nowotny 


a  PROTEINS  345 

and  Zahn,  1942).  The  side  chains,  they  say,  stand  perpendicular  to 
the  planes  of  the  frame  and  it  is  therefore  of  no  consequence  if  they 
differ  in  length.  It  is  assumed  that  the  grids  form  a  laminar  structure 
parallel  to  the  surface  of  the  hair  or  nail. 

Other  arguments  against  the  salt  link  theory  are  advanced  by 
LiNDLEY  (1950).  The  basic  amino  acid  residues  arginine,  lysine  and 
histidine  have  a  constant  ratio  12:4:1,  whereas  the  other  members 
of  the  keratin  polypeptide  (cystine,  tyrosine,  glutamic  and  aspartic 
acid)  show  considerable  fluctuations  depending  on  the  wool  sample 
chosen  (Block,  1939).  Fractional  hydrolysis  yields  peptides  of  low 
molecular  weight  with  numerous  acid  residues  clustered  together, 
whilst  the  basic  amino  acids  are  regularly  distributed  along  the  poly- 
peptide chains. 

MiDDLEBROOK  (1951)  thinks  that  cystine  which  amounts  to  about 
1/8-1/4  of  the  total  number  of  amino  acid  residues  is  concentrated  in 
definite  regions  along  the  polypeptide  chains,  and  that  these  regions, 
w^ith  a  periodicity  of  about  200  A,  cannot  assume  the  a-folds  because 
of  steric  hindrance.  Therefore,  a-keratin  would  always  contain  peri- 
odical short  segments  with  straight  /^-constellation. 

If  a  hair,  which  has  been  stretched  100%  and  temporarily  set,  is 
placed,  free,  in  a  steam  bath  for  a  short  time,  it  will  contract,  not 
only  to  its  original  length,  but  considerably  further;  a  super  con- 
traction takes  place.  This  fact  implies  that  the  polypeptide  chains  in 
the  a-keratin  are  not  entirely  free  and  independent  of  each  other; 
rather,  it  would  seem  that  they  too  are  mutually  stabilized  by  certain 
bridges.  Apparently,  however,  the  treatment  breaks  up  these  linkages 
and  enables  the  polypeptide  chains  to  fold  far  more  than  before. 
Restrictive  lateral  bridges  of  this  kind  are  also  supposed  to  be  re- 
sponsible for  the  fact  that  a  hair  is  only  50-70%  extensible  in  cold 
water;  they  weaken  in  hot  water  and  the  polypeptide  chains  can  then 
be  fully  stretched  (about  100%). 

If  keratin  is  exposed  for  a  short  time  to  the  action  of  hot  water  or 
vapour,  connecting  bridges  between  protein  chains  are  evidently 
broken  down.  Since  dilute  caustic  soda  similarly  loosens  the  chains, 
this  might  be  a  case  of  hydrolytic  decomposition  of  acid  amide 
bridges.  Yet  the  self-same  treatment,  if  more  prolonged,  will  facilitate 
the  formation  of  new,  stronger  bonds.  In  view  of  the  theory  regarding 
the  structure  of  cytoplasm  developed  in  this  book,  this  behaviour  is 


544         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

very  significant,  as  it  shows  how  readily  the  frame  of  proteins  can  be 
destroyed  and  built  up  again.  Seeing  that  hot  water  suffices  to  initiate 
this  process  in  keratin,  it  is  not  difficult  to  imagine  how,  in  the  far 
more  labile  cytoplasm,  the  protein  thread  molecules  are  constantly 
forming  new  combinations  and  side  bonds,  while  others  are  con- 
tinually being  broken  down,  so  that  a  definite  molecular  framework 
is  always  in  existence,  despite  the  apparent  liquid  state  of  the  material. 

Fine-structure  of  finger  nails.  Finger  nails  are  built  up  of  submicro- 
scopic  fibrillae.  X-ray  analysis  shows  that  the  keratin  fibril lae  run,  not 
parallel,  but  perpendicular  to  the  longitudinal  axis  of  the  nail  (Derk- 
SEN,  Heringa,  and  Weidinger,  1957).  As  in  the  elongation  growth 
of  the  plant  cell  wall,  therefore,  the  micelles  are  orientated  perpen- 
dicular to  the  direction  of  growth.  The  alignment  of  the  micellar 
strands,  therefore,  is  not  a  passive  process  due  to  the  forces  of  growth 
pushing  the  nail  forward;  there  are  special  formative  forces  at  work, 
building  up  submicroscopic  textures  with  due  regard  to  their  future 
functions. 

By  maceration  with  NaoS  a  thin,  100  A  thick  membrane  can  be 
detached  from  the  surface  layers  of  human  skin  and  finger  nails.  It  is 
compared  with  the  epicuticle  of  wool  (Lagermalm,  Philip  and 
Lindberg,  195  i). 

Fsather  keratin.  Not  all  horny  substances  are  naturally  in  the  state 
of  a-keratin.  Instead  of  the  fibre  period  for  mammalian  hair,  viz., 
5.06  A  in  the  direction  of  the  primary  chain,  that  of  quills  in  the  un- 
extended  state  is  3.1  A  (Astbury  and  Marwick,  1932).  By  elongation 
it  can  be  increased  continuously  and  reversibly  to  3.3  A  but,  if 
subjected  to  further  elongation,  the  quill  breaks.  From  this  fact  it 
may  be  concluded  that  the  polypeptide  chains  in  quill  keratin  are 
stretched  approximately  in  the  same  way  as  in  elongated  hairs  or  in 
silk  fibroin.  The  fact  that  the  length  of  the  members  of  the  primary 
chains  is  neither  3.38  A  nor  3.5  A  is  said  to  be  due  to  slight  corrugation 
(so-called  "primary  folding")  of  the  polypeptide  chains  in  the  feather 
keratin,  owing  to  a  certain  interaction  of  the  side  chains.  This  slight 
primary  folding  is  also  supposed  to  be  responsible  for  the  shortness, 
as  compared  with  silk  fibroin,  of  the  amino  acid  residues  of  ^-keratin. 
The  far  sharper  kinks  in  the  a-keratin  chains  are  distinguished  from 
this  slight  corrugation  as  "secondary  folding".  Thus  the  sclero- 
protein  of  quills  is  a  modification  of  keratin  in  which  there  is  no 


PROTEINS 


545 


secondary  folding.  The  keratin  primary  valence  chains  are  therefore 
used  by  the  animal  body  for  the  building  of  the  horny  tissues,  either 
heavily  folded,  or  in  more  or  less  stretched  condition. 

d.  Connective  Tissue  {Collagen) 

Molecular  structure  of  collagen  and  elastoidin.  Tendons  and  decalcified 
bones  consist  of  the  gelatinous  protein  collagen.  Glue  and  gelatin  are 
relatively  little  changed  decomposition  products  of  this  insoluble 
frame  substance  which  have  become  soluble  in  hot  water  owing  to 
sHght  hydrolytic  degradation. 

TABLE  XXX 

CHEMICAL  COMPOSITION  OF  COLLAGEN 
(SCHAUENSTEIN  AND  STANKE,   I951) 


0/ 

/o 

Arginine  .    . 

4-9 

Histidine  .    . 

0.5 

Lysine  .    .    . 

2.8 

Oxylysine .    . 

0.8 

Glutamic  acid 

4.0 

Aspartic  acid 

2.6 

Glycine.    .    . 

34.6 

Alanine.    .    . 

10.5      ; 

Valine  .  .  . 
Leucine.  .  . 
Proline .  .  . 
Oxyproline  . 
Phenylalanine 
Serine  .  .  . 
Threonine    . 


/o 


2.8 

4-1 

12.7 

II. 2 

2.4 

5.0 

98.6 


Collagen  is  a  protein  the  chemical  composition  of  which  differs 
remarkably  from  the  amino  acid  content  of  reserve  proteins.  It 
contains  a  considerable  amount  of  proline  and  oxyproHne  (Table 
XXX)  but  no  tyrosine,  tryptophane,  cysteine  nor  methionine. 

Tendons  and  elongated  gelatin  both  produce  the  same  X-ray 
pattern  (Gerngross  and  Katz,  1926).  It  shows  8.4  A  as  the  fibre 
period  which,  divided  among  three  amino  acid  residues,  shows  the 
length  of  the  members  of  the  primary  chain  to  be  2.8  A.  Moreover, 
there  are  two  interferences  on  the  equator  of  the  diagram,  which 
correspond  to  4.65  A  (backbone  thickness  of  the  primary  chain)  and 
10.0  A  (length  of  the  side  chains).  The  resemblance  to  the  conditions 
in  /5-keratin  is  striking,  except  that,  as  compared  with  the  amino  acid 
residues  of  silk  fibroin  and  of  keratin,  the  primary  chain  period  of 
2.8  A  would  appear  to  be  rather  short.  This  may  be  due  to  the 


346 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


N 


/ 

CH 


COH- 


I 
N 

II 
-COH 


N 


I 

HC 

I 


■CH-. 


\ 


'\ 


/ 


CH, 


CH. 


/ 

CH, 


co- 


COH- 


CHR 
\ 


/ 

CH 


\ 


Profile 


COH- 


HOCH 
\ 


CO 

I 

CH^ 

N 

II 
- COH 

I 

CHR 

I 

N 

II 
COH 

/"^^Ih 


Proline 


Glycine 


■  Amino  acid  R 


presence  in  the  collagen  of  about  24%  of  proline  and  oxyproline  in 
addition  to  34%  of  glycine.  The  many  five-membered  rings  cannot, 
of  course,  all  act  as  chain  end  groups  (see  Fig.  88,  p.  133);  they 
must  surely  be  built  into  the  primary  chains  (Fig.  173),  causing  con- 
siderable primary  folding  (Astbury,  1940).  Collagen,  like  the  other 

frame  proteins,  appears  after  all  to 
be  built  up  according  to  the  diagram 
of  polypeptide  chains  of  indeter- 
minate length. 

Tautomeric  rearrangements  help 
to  explain  the  shortening  of  the 
members,  for  if  within  the  stretched 
chain  the  hydrogen  of  every  second 
NH  group  is  transferred  to  the 
neighbouring  CO  group,  double 
bonds  -N  =  C(OH)-  are  formed 
which  entail  the  stereoisomeric  poss- 
ibilities of  the  cis  and  trans  configur- 
ation. If  the  cis  position  is  assumed, 
the  members  of  the  chain  are  short- 
ened to  the  value  of  2.86  A  ascert- 
ained experimentally  (cf.  Halle, 
1937;  Ch ampetier  and  Faure  -  Fre- 
MiET,  1938).  The  enolic  peptenol 
form  [^  C  (OH)]  of  the  polypeptide 
chain  (Fig.  173)  has  been  shown  by  UV  absorption,  since  ^  C  =  C<^ 
bonds  yield  a  characteristic  UV  band  between  2400  and  2600  A.  Since 
the  aromatic  amino  acids  tyrosine  and  tryptophane,  which  have  over- 
lapping absorption  bands,  are  absent,  the  peptenol  group  can  be  studied 
in  collagen  by  this  method  better  than  in  any  other  protein 
(ScHAUENSTEiN  and  Stanke,  195  i). 

There  is  a  similar  small  fibre  period  of  2.9  A  in  elastoidin  (Cham- 
petier  and  Faure-Fremiet,  1937),  which  is  the  frame  substance  of 
the  fin  rays  oiSelachii  (Faure-Fremiet,  1936).  Its  thermal  and  sweUing 
properties  are  comparable  to  those  of  collagen,  from  which  elastoidin 
is  distinguished  by  slight  chemical  differences  in  resistivity  to  trypsin 
and  by  sulphur  content. 

Optical  and  swelling  behaviour  of  tendons.    Optically,    tendons   and 


CH:; 


I 

,N 

I 


Oxyproline 


Front  view 


Fig.  173.  Diagram  of  a  gelatin  chain. 


2  PROTEINS  347 

welatin  filaments  are  positively  uniaxial  as  referred  to  the  fibre 
direction,  i.e.,  the  same  as  silk  and  hairs.  Rodlet  birefringence  is  also 
evident  if  the  tendons  are  tanned  before  imbibition  (Kuntzel,  1929). 
The  tendons  are  very  liable  to  swell  in  the  presence  of  most  imbibition 
liquids,  or  to  shrink  (e.g.,  with  xylene).  Collagen  behaves  peculiarly 
on  tanning;  for  whereas  the  optical  character  of  the  tendons  is 
retained  with  mineral  tanning  materials  (chromic  salts)  and  formol, 
it  is  reversed  and  becomes  negative  with  pyrogallic  tanning  agents 
(tannin,  sumach)  and  other  phenols  (trinitrophenol)  and  aldehydes 
(eugenol,  cinnamic  aldehyde).  Schmidt  (1934)  imagines  that  the 
optical  negative  reaction  is  brought  about  by  orientated  adsorption, 
as  the  non-tanning  univalent  phenols  and  aldehydes  may  be  washed 
out  again,  whereupon  the  normal  optically  positive  reaction  returns. 
Personally,  I  am  inclined  to  believe  that  it  is  rather  a  matter  of  chemi- 
cal- changes  in  the  side  chains.  Tanning  depends  upon  the  permanent 
connecting  of  one  polypeptide  chain  molecule  to  another  by  strong 
side-group  linkages.  Moreover,  the  pyrogallic  tanning  agents  must 
thereby  change  the  polarity  of  the  side  groups  in  a  manner  similar 
to  what  takes  place  in  the  nitration  or  acetylation  of  cellulose.  In  view 
of  the  lability  of  many  side  chain  reactions  of  the  polypeptide  chains, 
it  is  not  surprising  that  washing  out  of  the  non-tanning  phenols  should 
easily  upset  the  chemical  changes  brought  about  by  trinitrophenol, 
eugenol,  etc.  Rodlet  birefringence  and  X-ray  analysis  thus  provide 
evidence  for  the  submicroscopic  fibrous  structure  of  tendons. 

It  is  not  only  the  strange  optical  behaviour  of  tendons  which  has 
for  long  attracted  attention  (v.  Ebxer,  1894),  but  also  their  re- 
markable swelling  power.  In  water  they  swell  by  50%  in  thickness, 
which,  as  X-ray  evidence  shows,  involves  expansion  up  to  35  %  of 
the  crystal  lattice  (Kuntzel  and  Prakke,  1933),  while  the  fibre  period 
remains  unchanged.  Hence  the  swelling  is  not  intermicellar  as  in 
cellulose,  but  intramicellar,  inasmusch  as  the  individual  primary 
valence  chains  are  pushed  apart.  This  explains  why  the  swelling  of 
tendons  may  assume  unprecedented  dimensions.  In  dilute  acids  and 
alkalies,  which  obviously  completely  hydrolyze  the  side-chain  bonds, 
they  are  liable  to  swell  550*^0  in  thickness,  though  admittedly  they 
shorten  at  the  same  time  by  30%.  Despite  this  shortening,  the  increase 
in  volume  due  to  the  infiltration  of  fluid  may  amount  to  as  much  as 
4500%  (Kuntzel  and  Prakke,  1933). 


348  FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

Reduction  in  length  becomes  more  striking  when  the  tendons  are 
placed  in  hot  water  (60  to  70°  C).  They  suddenly  contract,  while 
swelling,  and  at  the  same  moment  the  birefringence  and  X-ray  dia- 
gram vanish.  This  unusual  reduction  in  length  imparts  rubber-like 
elasticity  to  the  tendon.  After  careful  elongation  the  X-ray  diagram 
reappears  and  continued  stretching  will  finally  restore  and  establish 
the  inelastic  collagen  fibre.  All  this  resembles  the  behaviour  of  rubber 
which,  when  unstretched,  produces  no  X-ray  diagram,  but  gives  a 
pattern  after  it  has  been  considerably  stretched.  Meyer  and  Mark 
(1930)  point  out  another  interesting  property  common  to  both 
materials.  If  the  contracted  tendons  or  unstretched  rubber  be  frozen 
in  liquid  air  and  the  objects  be  then  smashed,  they  crumble  to  a  friable 
mass,  like  sand;  whereas  under  similar  treatment  native  tendons  or 
elongated  rubber  will  split  up  into  a  fibrillar  mass.  From  this  it  may 
be  inferred  that  the  polypeptide  chains  of  the  collagen  fibres  contract, 
as  in  /9-keratin,  and  fold  up.  But  whereas  folding  of  the  ^  ->  a-keratin 
type  is  limited,  with  collagen  it  is  so  violent  that  the  straight  protein 
chains  shrivel  up  completely.  Evidently,  the  impulse  of  polypeptide 
chains  to  shorten  in  the  free  state  is  very  widespread  and,  if  means 
are  found  to  make  this  process  reversible  and  to  regulate  it,  a  model 
will  be  provided  for  the  contractile  muscle  fibres. 

Suhfnicroscopic  striation  of  collagen  fibres.  While  collagen  fibrils  are 
perfectly  smooth  in  the  ordinary  microscope,  they  appear  to  be 
striated  in  the  electron  microscope.  This  striation  was  first  reported 
by  ScHMiTT,  Hall  and  Jakus  (1942)  and  by  Wolpers  (1944).  The 
period  of  the  cross-bands  in  collagen  fibrils  of  the  human  tendons 
and  human  skin  is  640  A  (Schmitt,  Hall  and  Jakus,  1943;  Gross, 
1950).  This  corresponds  to  the  macroperiod  found  in  kangaroo 
tendons  by  X-ray  small  angle  diffraction  (Bear,  1944).  X-ray  analysis 
has  also  revealed  the  remarkable  fact  that  the  extended  collagen  chains 
do  not  form  a  three-dimensional  lattice,  the  direction  of  their  side 
chains  changing  arbitrarily  in  the  chain  lattice  (Bolduan  and  Bear, 
1950). 

Pratt  and  Wyckoff  (1950)  have  shown  that  in  the  particularly 
clean  fibrils  of  collagen  from  dog  heart,  the  segments  are  bordered 
by  pairs  of  cross  striae  (Fig.  174).  Sometimes  a  third  cross-band  is 
seen  in  each  segment  between  the  pairs.  Then  the  fibril  appears 
continuously  cross-striated  with  a  period  of  ca.  640  A/3    =   210  A. 


PROTEINS 


349 


These  authors  think  that  the  third  cross-band  is  due  to  remains  of 
a  second  transverse  system  of  fibrils  which  bind  the  separate  collagen 
fibres  into  a  fabric-like  system.  It  is  likely  that  it  is  the  pairs  of  cross 
striae  which  adsorbs  more  easily  silver  than  the  rest  of  the  segment 
(Dettmer,  Neckel  and  Ruska,  195  i). 


0^ 


^'.  - 


Fig.  174.  Striation  of  collagen  fibrils,  34,000:  i  (from  Pratt  and  Wyckoff,  1950). 


The  reason  why  collagen  fibrils  display  a  submicroscopic  seg- 
mentation is  obscure.  Possibly  there  is  some  relation  to  the  globular 
state  of  proteins  in  solution.  Bahr  (1950)  and  Noda  and  Wyckoff 
(195 1)  succeeded  in  reconstituting  tendons  dissolved  in  dilute  acetic 
acid  into  collagen  fibrils  by  precipitation  of  a  dilute  collagen  solution 
of  0.75  to  0.05%  with  salts,  (0.7  to  1.5%  at  p^  3.8  to  7.0).  This 
reconstitution  furnishes  segmented  collagen  fibrils  with  a  period  of 
635  A  or  650  A  which  can  be  dissolved  again  by  dialysis  against  water 
and  acetic  acid.  Since  dissolved  collagen  represents  a  globular  protein, 
as  known  from  gelatin  (see  p.  93),  precipitation  may  join  these 
spheres  in  a  linear  way  causing  beaded  chains ;  if  such  chains  associate 
laterally  to  form  fibrils,  denser  and  less  dense  cross-bands  are  likely 
to  be  produced. 

In  the  work  of  Schmitt,  Hall  and  Jakus  (1942,  1943,  1945)  the 
dense  segments  are  marked  A,  the  more  transparent,  B.  Upon  arti- 
ficial elongation  the  B  segments  increase  in  length  at  the  expense  of 
the  A  segments ;  the  period  can  be  raised  to  as  much  as  6000  A.  It  is 
:Supposed  that  the  polypeptide  chains  are  more  tightly  folded  in  the 


550         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 


A  segments  than  in  the  B  segments  and  that  they  unfold  partially  when 
stretched.  When  collagen  fibrils  are  stained  with  phosphotungstic 
acid,  the  electron  microscope  reveals  not  merely  one  dark  and  one 
bright  segment  per  period,  but  a  series  of  bands  (e.g.,  5)  within  the 
dark  segment.  Apparently  these  bands  combine  preferentially  with  the 
phosphotungstic  acid. 

Suhmicroscopk  structure  of  hones  and  teeth.  Bone  represents  a  complex 

system  of  collagen  fibres,  its  inter- 
fibrillar  substance  being  calcified  by 
hydroxyl-apatite  Caio(P04)6(OH)2 
(Brandenberger  and  Schinz, 
1945).  It  is  a  Wiener  body,  which 
can  only  be  imbibed,  however, 
with  liquids  of  variable  refractive 
indices,  if  one  of  the  two  com- 
ponents is  destroyed.  This  can  be 
done  either  by  decalcification  or 
by  ignition  of  the  organic  com- 
ponent. AscENZi  (1950)  has  mea- 
sured the  form  birefringence  of 
the  organic  substance  (ossein)  and 
the  inorganic  substance.  The  re- 
sult is  represented  in  Fig.  175.  The 
ossein  has  almost  no  intrinsic  bire- 
fringence, whilst  that  of  the  incin- 
erated bone  is  strongly  negative. 
This  is  due  to  the  optical  properties 
of  the  hydroxyl-apatite  which  cry- 
stallizes in  optically  negative  hex- 
agonal prisms.  The  minimum  of 
the  rodlet  birefringence  curve  at 
n  =  1.600  is  caused  by  the  optical  properties  of  apatite  (1.634  — 1.638 
=  —  0.004). 

In  the  electron  microscope  the  collagen  fibrils  are  visible  with  their 
striation  (Rutishauser,  Huber,  Kellenberger,  Majno  and  Rouil- 
LER,  1950;  Huber  and  Rouiller,  195  i). 

Teeth  have  a  similar  submicroscopic  structure  to  that  of  bones.  Of 
special  interest  is  the  enamel  which  covers  the  dentine  as  a  specially 


0.009 


0.008 


0.007 


0.006 


0.005  - 


O.OOi, 


0.003 


0.002 


0.001 


0  001 


0002 


■  0.003 


-0.00^ 


0 

0 

0 

0 

0 

0 

0 

0 

0 

10 

0 

IT) 

0 

10 

0 

LO 

0 

U-) 

IT) 

U1 

0 

10 

x-~* 

r-^ 

Fig.  175.  Birefringence  of  human  femoral 
diaphysis  (from  Ascenzi,  1950).  A)  In- 
organic bone  fraction,  B)  ossein,  C)  total 
bone.  Abscissa:  refractive  index  of  the  im- 
bibition liquid.  Ordinate:  birefringence. 


Z  PROTEINS  351 

hard  and  resistant  layer.  It  consists  of  parallel  prisms  of  hydroxyl- 
apatite  orientated  perpendicular  to  the  enamel  surface.  Its  optics  have 
been  investigated  by  W.  J.  Schmidt  (1923,  1936/37).  In  a  very  young 
state  this  layer  is  optically  positive  with  respect  to  the  axis  of  the 
prisms,  whereas  in  full-grown  teeth  the  enamel  assumes  an  optically 
negative  character.  This  change  is  explained  by  the  following  facts: 
In  the  embryonic  state  the  enamel  prisms  represent  a  Wiener  body 
with  submicroscopic  spaces  between  submicroscopic  crystallites  of 
hydroxyl-apatite;  hence,  its  positive  double  refraction  is  caused  by 
'marked  rodlet  birefringence.  Later,  when  the  enamel  hardens,  the 
submicroscopic  spaces  are  filled  with  material  of  a  refractive  power 
similar  to  that  of  the  crystallites ;  consequently  the  form  birefringence 
disappears  and  the  optically  negative  intrinsic  birefringence  of  the 
hydroxyl-apatite  becomes  visible.  This  behaviour  proves  that  the 
optical  axis  of  the  submicroscopic  crystallites  must  run  parallel  to  the 
axis  of  the  microscopically  visible  prisms. 

It  had  been  assumed  that  the  filling  material  would  be  exclusively 
inorganic,  but  the  electron  microscope  has  shown  that  it  is  not.  Scott 
and  Wyckoff  (1946/47)  have  developed  a  method  for  preparing  thin 
replicas  of  pofished  and  slightly  etched  tooth  sections.  In  order  to 
obtain  undamaged  replicas  they  must  be  freed  by  dissolving  the  section 
in  18  %  HCl  and  2  %  pepsin,  whereupon  the  replica  is  shadowed. 

In  such  preparations  the  microscopic  enamel  prisms  appear  to  be 
surrounded  by  a  thin  organic  sheath  and  inside  the  prisms  there  is 
a  very  fine  organic  matrix  (Frank,  1950).  Enamel  is  not  therefore 
simply  an  inorganic  coat  of  the  tooth,  but  contains  an  organic  frame 
as  well.  This  explains  why  even  completely  intact  teeth  are  subject 
to  decay. 

Elastic  tissue.  Elastic  tissue  as  found  in  the  back  of  the  neck  (liga- 
mentum  nuchae)  of  the  vertebrates  differs  from  the  connective  tissue 
of  collagen  in  several  characteristic  properties.  Being  highly  elastic 
and  resistant  to  tryptic  digestion,  its  protein  has  been  given  a  special 
name,  viz.,  "elastin". 

Threads  of  elastin  have  little  birefringence,  but  their  double 
refraction  can  be  enhanced  by  stretching  (Schmidt,  1924).  It  was 
therefore  thought  that  elastin  w^ould  behave  like  rubber,  with  dis- 
ordered chain  molecules  in  the  relaxed  state  and  parallel  molecules  in 
the  stretched  state  (cf.  Gross,  1949). 


552         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

€.  Muscle  Fibres  (Actomyosin) 

Proteins  of  muscle  fibres.  Fresh  striated  muscle  contains  about  20  % 
of  protein.  On  extraction  of  minced  muscle  with  water,  the  soluble 
protein  myogen  is  dissolved;  but  this  protein  does  not  appear  to  be 
involved  in  contractility. 

The  contractile  substance  is  contained  in  the  insoluble  fraction.  If 
treated  with  a  slightly  alkaline  salt  solution  the  protein  myosin  can  be 
extracted.  Its  I.E. P.  is  5.3.  Szent-Gyorgyi  (1943)  succeeded  in 
obtaining  crystallized  myosin  and  established  in  this  way  that  ordinary 
myosin  solutions  are  contaminated  with  another  protein,  actin.  Under 
the  electron  microscope  crystallized  myosin  appears  to  be  a  fibrillar 
protein  (Astbury,  1947/49).  Its  molecular  weight  is  1.5  million 
(Snellman  and  Erdos,  1948). 

When  myosin  is  properly  extracted  from  muscle  tissue,  the  main 
part  of  the  acfin  remains  in  the  residue.  After  drying  this  solid  fraction 
with  acetone,  the  actin  can  be  dissolved  (I.E. P.  4.7).  The  solution  is 
perfectly  clear  and  has  a  low  viscosity.  When  left  in  the  presence  of 
KCl,  it  becomes  more  viscous  and  ultimately  turns  into  a  thick  thixo- 
tropic  gel.  This  gelation  is  due  to  a  transformation  of  globular  protein. 
Both  modifications  are  visible  in  the  electron  microscope.  The 
globular  actin  has  been  called  G-actin  and  the  fibrillar  modification 
F-actin.  The  particles  of  G-actin  are  ellipsoidal  with  the  dimensions 
300  A  X  100  A.  Rosza,  Szent-Gyorgyi  and  Wyckoff  (1949)  have 
shown  how  these  particles  form  F-actin  in  situ  by  linear  aggregation. 
The  filaments  of  F-actin  are  100  A  thick  and  appear  to  be  segmented 
with  a  period  of  300  A.  They  aggregate  laterally  forming  cross- 
striated  bands. 

The  G-actin  as  seen  in  the  electron  microscope  would  have  a 
molecular  weight  of  about  1.5  million,  whereas,  according  to  measure- 
ments in  the  ultracentrifuge,  it  consists  of  only  4  Svedberg  units 
(MW  =  70,000;  Straub,  1948).  The  particles  of  G-actin  visible  in 
the  electron  microscope  therefore  consist  of  about  24  actin  molecules. 

Neither  myosin  nor  F-actin  is  contractile.  But  if  these  two  proteins 
are  brought  together  they  react  with  each  other  forming  the  con- 
tractile substance  F-actomyosin.  There  is  an  optimal  reaction  with 
a  ratio  of  2.5  parts  myosin  to  i  part  F-actin.  Snellman  and  Erdos 
(1949)  conclude  from  this  fact  that  there  is  a  stoichiometrical  ratio 
of  these  two  components  in  the  contractile  muscle  protein. 


2  PROTEINS  355 

When  adenosine  triphosphate  (ATP)  is  added,  F-actomyosin  con- 
tracts violently.  Under  the  electron  microscope  F-actomyosin  consists 
of  fine  filaments  and,  after  treatment  with  ATP,  coarse  threads. 
However,  as  there  is  no  change  in  the  X-ray  pattern,  the  syneresis 
which  occurs  has  been  declared  to  be  intermolecular  and  not  intra- 
molecular (Perry,  Reed,  Astbury  and  Spark,  1948).  A  gel  of  2-3% 
actomyosin  throws  out  so  much  water  by  dehydration  as  to  become 
a  dense  gel  of  50%  protein.  The  mechanism  of  this  contraction  is  not 
vet  thoroughly  understood. 

Optics  of  striated  muscle  fibres.  The  safest  way  to  assess  the  micro- 
scopic structure  of  the  highly  differentiated  striated  muscle  fibre  is 
between  crossed  nicols  (Vles,  191  i;  v.  Muralt,  1933;  Schmidt, 
1937a).  This  circumvents  many  sources  of  error,  such  as  the  com- 
plicated diffraction  phenomena  of  striated  sj^stems  (Pfeiffer,  1942  b; 
L.\NGELAAN,  1 946),  and  the  changes  in  structure  which  are  greatly, 
though  sometimes  wrongly,  feared  in  the  fixation  of  tissues. 

The  muscle  fibre  is  lo  to  loo  ^  in  width  and  is  enclosed  in  a  thin 
skin,  the  sarcolemma.  It  disintegrates  into  optically  resolvable  fibrillae 
about  I  [x  thick  and  at  roughly  0.5  ji  distance  from  each  other.  The 
visible  fibrillae  consist  of  bundles  of  parallel  submicroscopic  ele- 
mentary fibrils  (HiJRTHLE,  1931).  The  sarcoplasm,  which  surrounds 
the  fibrils  on  all  sides,  lies  in  between  the  myofibrils.  Essentially  it 
consists  of  muscle  albumin,  or  myogen,  while  the  fibrils  are  identical, 
in  the  main,  with  muscle  globulin,  or  actomyosin.  The  sarcoplasm  is 
always  isotropic,  but  myofibrils  are  birefringent  and  exhibit  the 
familiar  segmentation  into  bright,  so-called  Q  and  A  bands  and  dark, 
very  weakly  birefringent  (usually  called  isotropic)  I  bands,  which  are 
subdivided  by  a  stronger  birefringent  Z  band.  There  are  accumu- 
lations of  nucleic  acids  of  the  adenyl  nucleotide  type  in  the  semi- 
isotropic  I  sections  (Caspersson  and  Thorell,  1941).  The  re- 
markable part  of  this  structure  is  that  all  the  fibrils  of  a  muscle  fibre, 
though  independent,  have  their  bright  and  dark  bands  at  exactly  the 
same  level,  with  the  result  that  the  entire  fibre  is  evenly  striated. 

The  coincidence  of  the  strongly  and  weakly  birefringent  bands  is 
due  to  the  division  of  the  individual  fibrils  after  the  striation  of  the 
original  mother  fibrils  has  occurred.  Despite  the  conspicuous  optical 
differentiation,  the  fibrils  are  not  transversally  subdivided,  but  run  in 
uninterrupted  succession  through  the  entire  length  of  the  fibre.  Their 


354         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  HI 

cleavability,  which  betrays  no  mechanical  inhomogeneity  at  the 
boundaries  of  the  segment,  is  an  argument  in  favour  of  their  uni- 
formity. Further,  very  young  fibrils  are  uniformly  birefringent  (later 
the  striation  appears  gradually  differentiated  from  the  middle  towards 
the  extremities),  while  the  cross  striation  may  disappear  in  explanted 
skeletal  muscle  cells  through  dedifferentiation  (Schmidt,  1937a,  pp. 
215,  223). 

Whereas  the  fibrils  are  probably  continuous,  the  sarcoplasm  appeaxs 
to  be  subdivided  by  transverse  septa;  for  in  the  centre  of  the  dark  I 
band  there  is  always  a  narrow  Z  band,  easily  identified  by  staining, 
which  shines  brightly  between  crossed  nicols  (Fig.  176).  It  is  supposed 
to  be  a  cross  membrane,  continuous  with  the  sarcolemma,  the  myo- 
fibrils thrusting  through  it  without  hindrance.  When  the  muscle 
contracts,  these  regions  do  not  thicken  appreciably,  so  that  the 
sarcolemma  is  thrown  into  festoons. 

The  segment  of  the  myofibril  from  one  Z  band  to  the  next  is  called 
the  sarcomere.  Its  length  is  about  2  //.  In  a  growing  muscle  fibre,  the 
sarcomeres  are  added  to  the  end  of  the  fibre  originating  from  one 
single  cell.  The  sarcomeres  at  the  two  ends  are  less  differentiated 
during  growth  than  in  the  middle  of  the  fibre  (Haas,  1950). 

On  both  sides  of  the  Z  band  slightly  birefringent  N  bands  occur, 
often  joining  the  Z  band.  Matoltsy  and  Gerendas  (1947)  suppose 
the  lack  of  optical  anisotropy  in  the  I  band  to  be  caused  by  the  inter- 
calation, between  the  myofibrils,  of  an  optically  negative  substance,, 
called  N-substance,  which  compensates  the  positive  double  refraction 
of  the  actomyosin  (Gerendas  and  Matoltsy,  1947).  The  UV  ab- 
sorption of  the  N-substance  is  the  same  as  that  of  nucleic  acid,  which 
is  an  optically  negative  substance  (see  p.  220).  Muscle  fibres  extracted 
with  0.3  Af  KCl,  which  dissolves  myosin,  lose  their  isotropic  bands 
(Snellman  and  Gelotte,  1950). 

The  retardation  of  the  Q  bands  in  the  fibre  decreases  considerably 
during  contraction,  notwithstanding  the  appreciable  increase  in 
thickness;  the  optical  term  for  this  is  negative  fluctuation.  The  fact 
established  by  v.  Muralt  (1932)  that  negative  fluctuation  also  occurs 
with  isomeric  contraction — i.e.,  when  the  muscle  is  forcibly  held 
to  its  original  length  during  contraction — is  of  great  importance. 

Besides  intrinsic  birefringence,  which  is  manifested  as  birefringence 
of  flow  in  myosin  solution  (v.  Muralt  and  Edsall,  1930),  the  myo- 


PROTEINS 


555 


fibrils  exhibit  distinct  rodlet  birefringence  (Stubel,  1925).  It  follows 
from  this  that  the  fibrils  are  not  uniform  in  structure,  but  are  of  the 
class  of  rodlet  composite  bodies.  Boehm  and  Weber  (1932)  produced 
composite  bodies  of  this  kind  artificially  by  injecting  myosin  solutions 
into  water.  The  resulting  filaments  displayed  the  same  optical  proper- 
ties, both  qualitatively  and  quantitatively,  as  the  Q  sections  of  the 
myofibrils  (Weber,  1934).  It  is  surprising  to  find  how  well  the 
measured  birefringence  agrees  with  that  calculated  from  Wiener's 
formula  (see  p.  84),  for  the  assumptions  of  Wiener's  composite 
bodies  are  hardly  applicable  to  hydrophilic  micellar  systems.  Above 
all,  the  theory  requires  that  there  should  be  a  well-defined  phase 
boundary  between  the  rods  and  the  imbibition  liquid,  which  there 

cannot  be  with  a  swellable  protein  which  adds  on 
water  molecules  to  its  macromolecular  chains.  A 
further  assumption,  which  is  more  to  the  point  in 
this  case,  is  that  the  submicroscopic  rodlets  have 
practically  unlimited  length.  Weber,  it  is  true, 
assumes  a  particle  length  of  only  500  A  and  Wor- 
SCHITZ  (1935)  has  X-ray  evidence  for  lengths  up 
to  2050  A,  but  no  reliance  can  be  placed  on  X-ray 
determinations  of  particle  length  with  dimensions 
beyond  500  A  (see  Frey-Wyssling  1937a,  p.  376). 
It  may  therefore  be  assumed  with  equal  justice  that 
the  optically  identified  rodlets  are  bundles  of 
primary  valence  chains  of  unknown  length  which 
run  parallel  through  the  myofibrillae. 

X-ray  analysis.  X-ray  analysis  gives  us  some  in- 
formation about  the  inner  structure  of  elementary 
fibrils.  Myosin  filaments  produce  the  same  X-ray 
diagram  as  relaxed  muscles  (Boehm  and  Weber, 
1932),  which  proves  the  identity  between  the 
fibrillar  substance  and  myosin.  Model  experiments 
can  therefore  be  carried  out  with  m^^osin  films  and 
it  is  in  this  way  that  Astbury  and  Dickinson 
(1935  a)  found  that  the  X-ray  picture  of  muscle 
protein  corresponds  to  that  of  keratin.  The  a  ?^ 
/3-keratin  conversion  can  be  attained  by  elongation, 
but  in  the  relaxed  muscle  it  is  not   the   stretched 


Fig.  176.  Striated 
muscle  fibres  between 
crossed  nicols  (by 
courtesy     of     Prof. 

W.    J.    Schmidt, 

Giessen).     Wide    Q 

sections  and  narrow, 

weakly  luminous    Z 

stripes. 


356 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


jS-form,  but  the  folded  a  condition  that  is  found.  True,  the  modifi- 
cation of  myosin  to  the  ^-form  can  also  be  forced  upon  the  muscle 
by  artificial  extension  (Astbury  and  Dickinson,  1935  b),  but  the 
a-form  always  occurs  in  the  natural  state.  Hence  it  must  be  assumed 
that  the  polypeptide  molecules  in  the  relaxed  muscle  run,  as  in  un- 
stretched  hairs,  in  folded  chains  parallel  to  the  fibre  through  the 
fibrillae.  This  is  where  the  X-ray  method  is  at  a  distinct  disadvantage 
as  compared  with  polarization  optics,  for  it  fails  to  distinguish  the 
more  strongly  birefringent  Q  sections  of  striped  muscles  from  the 
almost  isotropic  I  bands. 


Fig.  177.  Electron  micrographs  of  striated  muscle  fibres  (from  Hall,  Jakus  and  Schmitt 
1946).  Above:  relaxed;  below:  contracted. 


2  PROTEINS  557 

Electron  microscopy.  The  electron  microscope  provides  a  means  of 
checkine  the  conclusions  derived  from  the  results  of  indirect  methods. 
WoLPERS  (1944)  and  Hall,  Jakus  and  Schmitt  (1946)  find  the 
following  micrographs  of  striated  muscle  fibres  (Fig.  177):  the  Q 
segment  is  dark  and  interrupted  by  a  cross-band  M,  whilst  the  I 
segment  is  clear;  i.e.,  there  is  a  denser  packing  of  protein  in  Q  and 
a  much  looser  arrangement  in  I.  The  most  surprising  result  is  the 
complete  blackness  of  the  Z  zone  in  contrast  to  the  lack  of  electron 
scattering  in  the  adjacent  N  zones  (Fig.  177  above).  In  that  part  we 
must  assume  the  presence  of  heavy  atoms  and,  as  Caspersson  and 
Thorell  (1941)  have  found  more  nucleic  acids  in  the  semi-isotropic 
sections  of  the  fibres,  it  is  likely  that  phosphorus,  besides  metallic 
cations  like  potassium,  is  accumulated  in  the  Z  zone.  It  might  also 
be  possible  that  the  Z  zone  has  a  special  adsorbing  power  for  heavy 
metals,  since  osmium  fixation  (Wolpers,  1944)  or  phosphotungstic 
staining  of  the  fibres  has  been  used  in  the  previous  treatment.  In 
contracted  muscle  fibres  much  electron  scattering  material  is  found 
in  the  I  band  (Fig.  177  below).  F.  O.  Schmitt  (1950a)  assumes  that 
on  irritation  there  is  a  migration  of  Q-substance  into  the  I  band, 
causing  the  much  discussed  reversal  of  striation. 

The  microscopic  myofibrils  consist  of  parallel  submicroscopic 
microfibrils  of  100-150  A  diameter.  Like  the  myofibrils,  these  micro.-, 
fibrils  run  straight  through  the  segments  and  across  their  border  lines. 
For  this  reason  earlier  attempts  to  explain  the  weak  optical  anisotropy 
of  the  I  bands  by  a  disorientation  of  submicroscopic  elements  must 
be  discarded.  The  microfibrils  produce  the  X-ray  interferences  of  both 
actin  and  myosin ;  hence  they  are  considered  to  consist  of  actomyosin 
(AsTBURY,  1947/49).  X-ray  diffraction  discloses  a  long-range  axial 
period  of  400  A  and  a  short-range  spacing  of  27  A,  while  in  F-actin 
54  A  has  been  found  (Schmitt,  1950a). 

In  palladium  shadowed  electron  micrographs  Rozsa,  Szent- 
Gyorgyi  and  Wyckoff  (1950)  offer  evidence  of  the  incrusting 
materials  in  the  myofibrils.  They  find  a  heavy  incrustation  in  the  Z 
and  M  zones.  Further,  the  whole  Q  band  is  rich  in  interfibrillar 
substance  except  two  narrow  zones,  called  H  zones,  adjacent  to  the 
M  stripe  which  intersects  the  Q  segment.  Unexpectedly  the  I  bands, 
with  the  exception  of  the  Z  stripe,  are  free  from  such  substances.  The 
authors  consider  the  microfibrils  to  represent  pure  F-actin  and  discuss 


358         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

whether  myosin  could  be  a  part  of  the  incrusting  material.  This  does 
not  seem  likely,  since  the  ratio  of  myosin  to  actin  is  2.5  (or  even  3) 
to  I  (Snellman  and  Erdos,  1949)  so  that  myosin  cannot  be  an  ac- 
cessory substance  in  the  muscle  fibre,  but  must  be  incorporated  in 
the  fibrillar  material.  It  is  likely  that  potassium  ions  are  part  of  the 
dense  substance  of  the  Q  bands,  which  is  rich  in  ash,  as  disclosed  by 
microincineration.  All  incrusting  substances  can  be  removed  by 
washing  without  disturbing  the  course  of  the  microfibrils,  whereupon 
a  perfectly  smooth  myofibril  results. 

Present  information  on  the  fine-structure  of  myofibrils  is  detailed 
and  extensive,  but  still  confusing.  Matoltsy  and  Gerendas  (1947) 
claim  to  have  found  an  optically  negative  N-substance  incrusting  the 
I  segments,  whereas  this  segment  is  free  from  interfibrillar  material 
according  to  Rgzsa,  Szent-Gyorgyi  and  Wyckoff  (1950),  so  that 
its  semi-isotropy  is  difficult  to  understand.  Further,  on  the  ground  of 
the  negative  fluctuation  of  the  birefringence  during  contraction,  it  is 
generally  accepted  that  the  Q  segments  shorten  more  than  the  I 
segments.  Hall,  Jakus  and  Schmitt  (1946),  on  the  contrary,  have 
observed  in  the  electron  microscope  that  the  Q  band  of  contracted 
myofibrils  does  not  change,  whereas  the  I  band  is  shortened  con- 
siderably, accounting  for  almost  the  whole  contraction,  which 
amounts  to  40%  of  a  sarcomere  (relaxed  about  2  /z,  contracted  1.2  fj). 

By  staining  with  phosphotungstic  acid.  Hall,  Jakus  and  Schmitt 
(1945)  were  able  to  detect  a  submicroscopic  banding  in  smooth 
muscle,  the  fibre  period  being  725  A.  It  would  therefore  seem  that 
the  banding  of  protein  fibrils  is  a  common  property,  resulting,  as  the 
electron  microscope  discloses,  from  the  periodic  dense  and  loose 
packing  of  protein  or  phosphorous  substances. 

The  mechanism  of  muscular  contraction.  There  are  several  ways  of  at- 
tacking the  important  problem  of  muscular  contraction:  thermo- 
dynamic, chemical  and  morphological  views  may  help  to  find  a 
consistent  explanation.  The  thermodynamic  approach  has  tried  to 
make  the  disorientation  of  molecular  elements  responsible  for  the 
liberation  of  energy  when  the  fibre  contracts  (cf.  Bailey,  1942).  Bio- 
chemical investigations  show,  however,  that  the  energy  is  liberated 
by  the  reaction  of  myosin  and  adenosine  triphosphate,  this  nucleotide 
being  dephosphorylated  and  the  liberated  phosphoric  acid  used  for 
the  phosphorolysis  (see  p.  314)  of  glycogen.  The  enzyme  adenosine 


2  PROTEINS  359 

triphosphatase  is  intimately  tied  to  myosin  or  may  even  be  a  part  of 
this  protein  molecule  (Needham,  1942  a,  b;  Potter,  1944).  The 
intimate  interrelation  of  the  mechanics  of  the  contractile  muscle  with 
chemical  reactions  is  shown  by  Hill  (1950). 

We  have  first  to  discuss  the  morphological  side  of  the  problem. 
When  muscle  contracts,  the  polypeptide  chains  coil  up.  Both  extended 
and  relaxed  muscle  have  the  a-keratin  structure,  which  becomes  dis- 
orientated on  contraction  (Huxley  and  Perutz,  195  i).  Actually  the 
same  thing  occurs  as  in  the  supercontraction  of  the  keratin  chains, 
with  the  difference,  of  course,  that  in  the  case  of  muscle  the  phe- 
nomenon is  reversible  and  can  be  voluntarily  induced.  A  relaxed 
muscle  frozen  in  liquid  air  splits  up  into  fibres,  whereas  a  contracted 
muscle  disintegrates  into  small  lumps  (Meyer  and  Mark,  1950). 
Furthermore,  contraction  wipes  out  the  X-ray  diagram.  Roughly 
speaking,  a  contracted  muscle  is  amorphous  like  unextended 
rubber,  whereas  in  the  relaxed  state  it  is  crystalline  like  elongated 
rubber. 

Notwithstanding  the  enormous  mass  of  literature  on  the  physio- 
logical processes  involved  in  muscular  contraction  (Verzar,  1945; 

Faure-Fremiet,  1946),  we  do  not  yet 
(^-co^  know  what  special  process  it  is   that 

Hydration-  Induccs  the  folded  polypeptide  chains 

to  supercontract.  K.  H.  Meyer  (1929) 
^fooj  \<:Zi:>NH^       suggests  that  fundamentally    it  is  the 

_,^„  ><CZII>coo"      mutual  repulsion  of  groups  bearing  the 

— ^  ^^^^coo~      same  charge,  e.g.,-COO~groups,  which 

M~    -^  V.  prevents  the  chains  in  the  relaxed  muscle 

Ph^^  ^"^'^^   ^        from  crumpling.  This  occurs  when  the 

Fig.  178.  Contraction  of  the  poly-        chain  is  in  repose  at  a  Ph  of  7.4,  viz.,  in 

peptide  chains  in  the    isoelectric  ^^  ^[y^^Unc  medium  (see  Fig.  I78).  Now 

state  (I.E. P.)  (after  K.  H.  Meyer,  ^  , 

ic,2c,),  if  by  some  physiological  process  the 

pjj  of  the  muscle  serum  is  reduced  to 
4.7,  which  corresponds  to  the  isoelectric  point  of  the  protein  actin, 
the  amino  groups  become  positively  charged  and  the  groups  with  the 
opposite  electric  charge  are  attracted  to  the  point  of  contact  and  the 
chains  coil  up.  Kuhn  and  Hargitay  (195  i)  have  calculated  these 
attractive  and  repulsive  forces  foi  the  case  of  polyacrylic  acid,  which 
contracts  in  an  acid  and  expands  in  an  alkaline  medium.  They  find 


360         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

values  compatible  with  the  stress  measured  in  contracting  threads  of 
polyacrylic  acid  when  placed  in  o.oz  N  HCl. 

The  matter  is  probably  not  quite  as  simple  as  this,  for  the  charges 
of  the  polypeptide  thread  molecules  are  not  in  the  primary  chain,  but 
at  the  extremity  of  the  end  groups  of  the  side  chains.  In  a  later  work, 
Meyer  and  Picken  (1937)  prove  by  thermoelastic  investigations  on 
stretched  muscle  fibres  that,  in  a  state  of  rest,  the  polypeptide  chains 
are  mobile  as  in  a  liquid,  whereas  fixed  bonds  are  established  as  soon 
as  the  muscle  is  irritated ;  thus  the  molecular  framework  of  the  muscles 
passes  from  an  apparently  "fluid"  to  a  solid  state.  It  should  be  noted 
that  the  comparison  with  rubber  ceases  to  be  valid  under  these  circum- 
stances, for  in  that  material  the  polyene  chains  are,  conversely,  more 
mobile  in  the  contracted  state  and  are  interlocked  in  the  elongated  con- 
dition. The  interesting  reaction  involved  in  muscular  induration  must 
surely  take  place  between  the  end  groups  of  neighbouring  side  chains. 

This  is  a  good  example  demonstrating  the  consistency  of  our 
theory  of  junctions.  Contracted  muscle  fibres  exhibit  an  extreme  gel 
structure,  whereas  relaxed  fibres  show  a  less  tightened  structure.  We 
may  thus  compare  muscle  relaxation  with  the  transformation  of  the 
plasmagel  to  the  plasmasol  in  protoplasmic  flow,  when  junctions  must 
similarly  be  freed  to  allow  displacement  of  the  structural  elements. 

f.  Nerves  (Neurokeratin  and  Neuronin) 

The  wjelin  sheath.  Myelinated  nerves  in  Vertebrates  consist  of  a 
central  strand  enveloped  in  a  highly  birefringent  sheath.  The  bire- 
fringence of  this  sheath  is  produced  by  the  embedded  myelin,  which 
produces  the  myelin  forms  described  on  p.  54  upon  the  addition  of 
water.  Like  myelin  tubes,  the  mvelin  sheath  is  optically  negative  as 
referred  to  the  axial  direction.  Referred  to  the  radial  direction, 
however,  the  birefringence  is  positive.  Thus  in  a  cross-section  through 
the  nerves  the  sheath  shows  a  positive  cross,  while  the  axoplasm 
appears  as  isotropic.  Since  myelin  comprises  lecithin  (Fig.  47,  p.  56), 
cephalin,  cholesterol  (Fig.  92,  p.  138)  and  other  anisodiametric  optic- 
ally positive  molecules,  they  must,  judging  by  the  birefringence,  be 
orientated  in  the  sheath  with  the  longitudinal  axis  running  radially. 
Isolated  myelin  substances  produce  X-ray  periods  corresponding  to 
double  the  molecular  length.  There  must  therefore  be  bimolecular 
lipid  layers  in  the  nerves.  The  thicknesses  of  the  layers  are  given  in 


PROTEINS 


361 


Table  XXXL  The  averay-e  distance  between  the  moIecul?r  chains  is 

4.8  A  (BOEHM,   1933). 

Small-angle  X-ray  diffraction  furnishes  layer  periods  of  186  A  for 
fresh  and  1 5  8  A  for  dried  mammalian  nerves.  This  shrinkage  shows 
that  hydration  water  lies  between  the  lamellae.  Since  a  drv  double 
layer  of  neural  myelin  is  only  66  A  thick,  it  is  likely  that  the  macro- 
period  of  1 5  8  A  not  only  includes  two  myelin  double  layers  but  also 
structural  protein  (Schmitt,  1950b).  Cf.  Fig.  48,  p.  57. 


TABLE  XXXI 

THICKNESS  OF  BIMOLECULAR  LAYERS  OF  LIPIDS  IN  NEURAL 

MYELIN  (after  BEAR,  PALMER,  AND  SCHMITT,    I941) 


Spacing  in  A 


Substance 


Determined  by 


Calculated  from 
atomic  distances 


Lecithin  .  .  . 
Cephalin  .  .  . 
Sphyngomyelin 
Kerasin.  .  .  . 
Phrenosin.    .    . 


65 
64 
64 


The  myelin  sheath  does  not  entirely  lose  its  birefringence  when  the 
myelin  substances  are  extracted  with  fat  solvents,  but  there  then 
appears  a  negative  cross  on  the  cross-section  (Schmidt,  1937a,  b; 
Schmitt  and  Bear,  1939).  This  birefringence  decreases  appreciably 
when  the  extracted  cross-sections  of  the  nerves  are  transferred  from 
alcohol  to  Canada  balsam.  There  is  therefore  lamellar  form  bire- 
fringence, for  the  radial  direction  remains  the  optical  axis,  just  as 
before  extraction  of  the  myelin.  The  submicroscopic  layers  must 
consist  of  neurokeratin,  which  is  to  be  considered  as  the  frame 
substance  of  the  sheath.  The  polypeptide  chains  of  this  protein  cannot 
have  any  preferred  orientation,  for,  if  they  had,  there  would  be  no 
optical  axis  in  the  radial  direction.  The  submicroscopic  lamellae  of 
protein  must  therefore  be  fohate  in  texture.  Schmidt  (1937a,  p.  306, 
Fig.  80)  assumes  that  there  are  individual  submicroscopic  particles  of 


362 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


protein  which  are  to  some  extent  independent  of  each  other.  This 
conflicts  with  the  idea  of  these  insoluble  protein  lamellae  as  frame 
substance. 

Fig.  179  represents  the  submicroscopic  structure  of  the  nerve  sheath 
according  to  Schmidt  (1937b).  Neurokeratin  lamellae  running 
tangentially  alternate  with  bimolecular  lipid  layers.  It  is  difficult  to 
say  what  the  physiological  significance  of  this  foliate  fine-structure 
may  be.  It  should  be  noted  that  if  this  is  destroyed,  say  by  melting 
of  the   myelin   substances,   nerves   lose  their  electric  conductivity. 


a) 


^3 


b) 


Fig.  179.  Fine-structure  of  medullated  nerves,  a)  Optics.  N  neurofibrillar  string,  positively 
uniaxial  as  referred  to  the  axial  direction.  M  myelin  sheath  positively  uniaxial  as  referred 
to  radial  direction  (after  Ambronn  and  Frey,  1926).  h)  Submicroscopic  structure  of  the 
medullary  sheath  (after  Schmidt,  1937b).  A  lamellae  of  protein.  L  bimolecular  lipid  layers. 


(Further  details  in  F.  O.  Schmitt,  1936;  O.  Schmidt,  1942;  v. 
MuRALT,  1946.)  Another  interesting  fact  is  reported  by  Taylor  (1942), 
who  found  that  in  nerves  having  approximately  equal  conduction 
velocities,  the  product  of  fibre  diameter  and  sheath  birefringence  is 
roughly  constant. 

The  laminated  fine-structure  of  the  myelin  sheath,  found  by  in- 
direct methods,  has  been  made  visible  in  the  electron  microscope 
(Fernandez-Moran,  1950a,  b).  The  periodicity  of  the  lamination  is 
80  A,  which  is  half  the  long-range  X-ray  diffraction  period  of  1 5  8  A 
reported  above. 

Schmidt  (1937a)  detected  a  similar  arrangement  of  lipid  molecules 
orientated  perpendicular  to  the  parallel  layers  of  protein  in  the  outer 
members  of  the  retinal  cells  in  the  eyes  of  Vertebrates,  which  has  been 


PROTEINS 


363 


substantiated  by  electron  micrographs  (Sjostrand,  1949);  and  in  this 
monograph  (Fig.  151b,  p.  259)  such  an  arrangement  has  been  shown 
to  be  probable  in  the  microstructure  of  the  chloroplasts.  It  looks, 
therefore,  as  if  submicroscopic  lamellar  protein-lipid  systems  of  the 
kind  are  fairly  common  in  biological  material. 

The  axon.  The  protein  of  the  nerve  axon  has  been  termed  neuronin 
(Bear,  Schmitt  and  Young,  1937).  In  the  living  nerve  it  constitutes 
only  3-4%  of  the  fibre  weight,  the  rest  being  an  aqueous  solution. 


r 


Fig.  180.  Submicroscopic  structure  of  an  internodal  segment  of  a  myelinated  nerve  fibre 

(from  Fernandez-MorAm,  1950,  1952a).  N  neurilemma,  M  myelin  sheath,  Ax  axolemma, 

A  axon,  C  collagen  fibrils,  E  dark  smooth  fibrils. 


It  is  for  this  reason  that  X-ray  absorption  micrographs  record  5  to  8 
times  less  mass  in  the  nerve  axon  (0.05  X  io~'^'^  gj fji^)  than  in  the 
myelin  sheath  (0.3  to  0.4  X  lo'^^g^^s.  Engstrom  and  Luthy,  1949, 
Engstrom  and  Lindstrom,  1950).  This  high  dilution  makes  it  un- 
certain whether  the  axoplasm  exists  as  a  sol  or  as  a  gel  in  the  living 
state.  Flaig  (1947)  reports  that  its  viscosity  is  considerably  increased 
during  nerve  activity,  indicating  a  sol  —  gel  equilibrium  similar  to 
that  involved  in  protoplasmic  flow. 

In  fixed   axoplasm,   neurofibrils   become   visible.    Their   diameter 
ranges   down   to   the   resolving   limit   of  the   ordinary   microscope. 


364         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

However,    in    the    electron    microscope    much    finer  filaments  with 
100-200  A  diameter  are  visible  (Fernandez-Moran,  1952b). 

Like  other  fibrous  protein  substances,  the  neurofibrils  are  posi- 
tively uniaxial,  but  their  birefringence  is  very  weak  and  is  pushed  into 
the  background  by  the  very  strong  anisotropy  of  the  myelin  sheath 
(Fig.  179a).  The  axoplasm  shows  form  birefringence  (Bear,  Schmitt 
and  Young,  1935).  When  heated,  it  shrinks  lengthwise,  like  col- 
lagen fibres  (Schmitt  and  Wade,  1935). 

The  intrinsic  birefringence  of  neuronin  is  0.005,  which  is  near  to 
that  of  myosin  (0.008).  The  mean  refractive  index  as  indicated  by  the 
minimum  of  the  form  birefringence  curve  amounts  to  1.57-1.60^ 
a  value  which  coincides  with  that  of  neurokeratin  1.58  and  muscle 
myosin  1.576  (H,  H.  Weber,  1934). 

Fine-stnicture  of  nerves.  Thin  sections  have  yielded  very  instructive 
electron  micrographs  which  settle  several  controversial  points  of 
nerve  cytology.  A  distinct  neurolemma  which  envelopes  the  myelin 
sheath  is  visible.  At  the  nodes  of  Ranvier  the  axon  is  constricted  but 
not  intercepted  (RozsA,  Morgan,  Szent-Gyorgyi  and  Wyckoff, 
1950a,  b). 

Fernandez-Moran  (1950,  1952a)  has  compiled  the  results  of  his 
electron  microscopic  studies  in  a  diagrammatic  outline  which  is  re- 
produced in  Fig.  180. 

There  is  a  200  A  thick  granular  neurolemma  (N)  with  dark  smooth 
fibres  (E)  which  resemble  elastic  fibres,  and  adhering  cross-striated 
collagen  fibres  (C).  The  sheath  (M)  consists  of  about  50  thin  con- 
centric lamellae  with  an  average  periodicity  of  80  A.  The  interlamellar 
spaces  are  locally  inflated.  The  sheath  is  separated  from  the  axon  by 
a  reticulate  membrane,  the  axolemma  (Ax),  formed  by  beaded 
filaments  100-200  A  in  width.  In  the  axis  cylinder  a  verv  fine  reticulum 
is  visible. 

g.  Fibrillar  Proteins.  Recapitulation. 

The  important  frame  proteins  are  of  the  fibrillar  type.  Their  poly- 
peptide chains  have  a  strong  tendency  to  crystallize  by  forming  chain 
lattices.  X-ray  diffraction  studies  have  disclosed  two  types  of  axial 
spacings  in  these  lattices,  which  have  been  classified  as  the  keratin- 
myosin  and  the  collagen  group  (Astbury,  1947;  Marks,  Bear  and 
Blake,  1949). 


PROTEINS 


365 


3.5i 


Fig.  181.  \^olume  of  an  amino  acid  residue. 


Keratin-myosin  group.  In  extended  crystallizing  polypeptide  chains 
the  space  needed  by  an  amino  acid  residue  in  the  direction  of  the 
chain  axis  is  3.5  A.  This  spacing 
is  called  main  chain  spacing;  the 
fibre  period  found  by  X-rays  is 
usually  a  multiple  of  this  value. 
The  lateral  distance  of  the  main 
chains  in  the  direction  perpen- 
dicular to  the  plane  of  the  side 
chains,  termed  backbone  spacing 

by  AsTBURY,  is  4.5  to  4.6  A.  In  the  third  direction,  the  side  chain 
spacing  depends  on  the  length  of  the  radicals  R  of  the  amino  acids 
involved.  With  the  exception  of  silk  fibroin,  this  spacing  is  astonishingly 
constant,  indicating  an  average  length  of  the  amino  acid  residues  of 
about  10  A.  Therefore,  the  average  volume  of  an  amino  acid  residue 
is  roughly  3.5  A  X  4.6  A  X  10  A  =  161  (A)=^  (Fig.  181). 

In  Mammals  ectodermal  formations  such  as  hairs,  feathers,  epi- 
dermis, nails,  horns  and  the  mesodermal  proteins  muscle  myosin, 
blood  fibrinogen  and  fibrin  correspond  to  this  type.  Astbury  (1947) 
has  therefore  called  it  keratin-myosin-epidermis-fibrinogen  (k-m-e-f) 
group. 

The  special  interest  of  this  group  is  the  possibility  of  the  ^^^a 
transformation  of  its  polypeptide  chains  (Fig.  171,  p.  339),  whereby 
the  main  chain  spacing  of  three  amino  acid  residues  is  reduced  from 
10.5  A  to  5.1  A.  Many  natural  fibrous  proteins  exist  in  this  folded 
form  (Table  XXXII).  As  shown  by  Astbury,  the  a-chains  can  be 
reversibly  transformed  into  the  ^^-configuration ;  hence  they  display 
an  inherent  elasticity  and  potential  contractility.  The  muscle  protein 
of  Invertebrates  is  also  of  this  type  (clam  muscle  of  Molluscs)  and 
even  the  bacterial  flagella  of  Proteus  vulgaris  show  the  characteristic 
spacing  of  5.1  A  (Astbury  and  Weibull,  1949). 

Silk  fibroin  differs  from  the  fibrous  proteins  of  the  k-m-e-f  group 
not  only  by  its  short  side  chain  spacing  but  also  by  its  lack  of  any 
/S  ^  a  transformation. 

The  specificity  of  the  different  proteins  in  the  k-m-e-f  group  is  due 
to  the  special  share  of  the  different  amino  acids  and  their  arrangement 
along  the  polypeptide  chain.  It  is  thought  that  bonds  between  the 
side  chains  stabilize  the  chain  lattice  both  of  the  ^-  and  of  the  a-form. 


366 


FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES 


III 


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2  PROTEINS  567 

When  the  side  bridges  of  these  chains  are  detached,  the  primary- 
valence  chains  crumple  and  contract  with  great  force.  Cystine-sulphur 
bridges,  which  only  relatively  drastic  treatment  can  rupture,  are  sup- 
posed to  be  active  in  a-keratin.  The  side  chain  bonds  in  actomyosin, 
on  the  other  hand,  are  far  more  labile,  with  the  result  that  only  a 
slight  change  in  the  reaction  of  the  surrounding  medium  is  needed 
for  contraction.  On  account  of  its  lability,  actomyosin  has  been  com- 
pared with  raw  rubber  and  keratin  with  vulcanized  rubber  and,  as 
already  stated,  the  tonofibrils  have  been  described  as  "vulcanized" 
myofibrils  (Mark  and  Philipp,  1937).  Convenient  as  such  com- 
parisons undoubtedly  may  be,  they  should  be  applied  only  with  the 
utmost  discretion,  particularly  as  long  as  our  knowledge  of  acto- 
myosin is  no  fuller  than  it  is  at  the  present  time. 

Collagen  group.  The  proteins  of  the  collagen  type  are  wide-spread 
in  the  animal  kingdom.  They  occur  as  collagen  proper  in  the  meso- 
dermal tissues  of  Vertebrates  (connective  tissue,  tendons,  bones),  as 
elastoidin  in  scales  and  fins  of  fishes,  as  ovokeratin  in  the  egg  capsule 
of  rays,  as  ichthyocol  in  the  swimbladder  of  fishes,  as  bysso-keratin 
in  the  byssus  threads  of  Pinna  nohilis  (Mollusca),  as  ascaro-collagen 
in  the  cuticle  of  Ascaris  (Nematoda),  as  connective  tissue  in  the 
peristome  of  the  sea  urchin  Arbacia  (Echinodermata),  in  the  axial 
stalk  of  the  sea  pens  (Coelenterata),  as  spongin  in  the  Porifera  etc. 
(IVIarks,  Bear  and  Blake,  1949).  All  these  fibrous  proteins  have  a 
main  chain  spacing  of  2.8  A,  whilst  the  backbone  and  side  chain 
spacings  are  similar  to  those  of  the  k-m-e-f  group  with  the  main  chain 
spacing  of  3.5  A  (Table  XXXII).  The  shortening  of  the  collagen 
fibre  spacing  by  20%  is  due  to  the  cis-position  of  the  chain  member 
>CHR  related  to  the  peptide  bond  -NH  -  CO-  or  -N  =  COH- 
(Fig.  173,  p.  346).  Thus  the  fibre  proteins  of  the  collagen  group  are 
composed  of  polypeptide  chains  in  the  cis-form,  whilst  those  of  the 
k-m-e-f  group  assume  the  trans-form.  The  latter  are  capable  of  re- 
versible contractions,  whereas  those  of  the  collagen  group  have  a 
strong  tendency  towards  an  irreversible  supercontraction  when  the 
lateral  bonds  of  the  chain  lattice  are  destroyed;  as  an  extreme  result, 
globular  proteins  can  be  formed  (e.g.  gelatin). 

Long-range  spacings.  Besides  the  short  main  chain  spacings  which 
characterize  the  keratin  and  the  collagen  group,  there  are  long-range 
spacings  in  the  fibre  proteins  which  are  disclosed  by  low  angle  X-ray 


368         FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

diffraction.  The  results  of  such  studies  are  collected  in  Table  XXXII. 
In  silk  fibroin  Friedrich-Freksa,  Kratky  and  Sekora  (1944)  found 
a  period  of  70  A,  which  corresponds  to  20  amino  acid  residues.  In 
feather  keratin  a  somewhat  longer  spacing  of  95  A  is  reported.  In 
porcupine  quill  there  is  a  long-range  spacing  of  198  A  and,  as  its 
keratin  is  present  in  the  a-form,  where  3  amino  acid  residues  cover 
5.1  A,  116  residues  (which  is  near  to  2^  x  3^  =  108)  would  constitute 
such  a  period.  In  the  adductor  muscle  of  the  mollusc  Venus  mercenaria 
a  small-angle  spacing  of  even  725  A  has  been  reported,  which  seems  to 
be  divided  into  four  subspacings  of  145  A.  In  this  spacing  426  amino 
acids  can  be  lodged  (which  can  be  associated  with  2^  X  3^  =  432). 

Bear  (1944),  who  has  measured  these  long  fibre  periods,  discovered 
transverse  long-range  spacings  as  well;  they  amount  to  about  0.4  of 
the  fibre  spacings  reported  (Table  XXXII).  Consequently,  there  is  not 
only  a  repetition  of  definite  sequences  of  amino  acids  in  the  main 
chains,  but  at  the  same  time  distinct  numbers  of  polypeptide  chains 
are  collected  into  crystallographic  units. 

In  contrast  to  this  behaviour,  which  shows  that  the  proteins  of  the 
keratin-myosin  group  exist  in  a  three-dimensional  crystalline  state, 
BoLDUAN  and  Bear  (1950)  have  only  found  a  long-range  fibre  spacing 
in  the  collagen  group  but  no  transverse  spacings.  The  inference  is, 
therefore,  that  there  is  no  true  crystal  pattern  in  the  collagen  proteins, 
but  simply  an  arrangement  of  parallel  chains,  similar  to  that  of  liquid 
smectic  crystals  with  only  a  unidirectional  periodicity. 

The  long-range  fibre  period  seems  to  be  the  same  for  all  collagen 
proteins  investigated ;  it  measures  640  A  (Marks,  Bear  and  Blake, 
1949)  and  agrees  with  the  striation  seen  in  the  electron  microscope, 
corresponding  to  228  amino  acid  residues.  There  seems  to  be  more 
uniformity  in  the  proteins  of  the  collagen  group  than  in  those  of  the 
keratin-myosin-epidermis-fibrinogen  group. 

General  occurrence  of  striated  protein  fibrils.  Microscopic  histology 
considered  the  striated  muscle  fibrils  as  a  special  case  of  protein  fibres. 
The  electron  microscope  has,  however,  revealed  the  fact  that  banding 
is  a  general  feature  of  fibrillar  proteins,  the  period  of  this  striation 
being  submicroscopic.  It  has  been  found  in  smooth  muscle  fibrils 
(Hall,  Jakus  and  Schmitt,  1945),  collagen  fibrils  (Fig.  174,  p.  349), 
precipitated  blood  fibrin  (Wolpers  and  Ruska,  1939),  ejected  tricho- 
■cysts  oi  Paramecium  (Jakus,  1945;  Wohlfahrt-Bottermann,  1950; 


Z  PROTEINS  569 

Knoch  and  Konig,  195  i)  etc.  In  collagen  fibres  the  striation  period 
of  640  A  corresponds  to  the  long-range  spacing  discovered  by  small- 
angle  X-ray  diffraction.  This  method  discloses  even  in  keratin  a  long- 
range  periodicity  of  200  A  (McArthur,  1945). 

At  first  sight  this  widespread  occurrence  of  a  submicroscopic 
striation  in  fibrous  proteins  seemed  rather  enigmatic.  But  its  formation 
can  be  studied  nowadays,  since  there  are  soluble  proteins  which  yield 
striated  fibrous  proteins  on  precipitation.  Such  an  example  is  blood 
fibrin.  Even  more  interesting  is  the  fact  that  dissolved  collagen  can 
be  reconstituted  to  precipitated  collagen  fibrils  with  a  striation  period 
of  635  A  (Bahr,  1950). 

These  experiments  favour  the  view  that  the  striated  microfibrils  are 
formed  by  linear  aggregation  (Fig.  104a,  p.  160)  of  globular  particles. 
In  this  way  the  submicroscopic  striation  is  easily  understood,  but  it 
is  difficult  to  explain  how  a  chain  lattice  with  polypeptide  chains  very 
much  longer  than  the  diameter  of  the  dissolved  protein  particles  is 
formed.  In  this  dilemma  a  helpful  suggestion  may  be  that  in  globular 
proteins  the  amino  acids  are  only  loosely  bound  and  not  yet  tied 
together  by  firm  peptide  bonds  (see  p.  329).  Then,  on  denaturation 
by  precipitation,  not  only  should  peptide  bonds  be  formed  inside  the 
globular  protein  macromolecule,  but  should  also  bridge  the  amino 
acids  of  the  adjacent  molecule,  the  result  being  polypeptide  chains 
running  straight  through  numerous  protein  particles.  It  is  more  likely 
that  some  such  mechanism  is  involved  than  that  preformed  poly- 
peptide chains  curled  up  in  the  globular  particle  should  unfold  com- 
pletely to  form  straight  threads,  which  would  be  necessarily  entangled 
before  a  chain  lattice  can  be  formed. 

Chemical  changes  of  the  protein  molecule  due  to  the  transformation 
globular  ->  fibrillar  of  its  shape  have  been  recorded  in  fibrinogen 
(Bailey,  Bettelheim,  Lorand  and  Middlebrook,  195  i).  When 
blood  clots,  fibrinogen  (M.W.  500,000)  is  transformed  into  fibrillar 
fibrin  by  the  enzyme  thrombin.  This  change  is  associated  with  the 
appearance  of  amino-terminal  residues  of  glycine  by  specific  hy- 
drolysis. Whereas  fibrinogen  has  no  such  end  groups,  five  terminal 
glycine  residues  appear  per  mole  of  fibrinogen  when  converted  into 
fibrin.  It  should  be  emphasized  that  ordinary  denaturation  does  not 
cause  this  effect  and  that  only  thrombin  is  capable  of  inducing  it. 


370  FINE-STRUCTURE  OF  PROTOPLASMIC  DERIVATIVES  III 

Conclusion 

Whatever  the  final  explanation  of  these  important  molecular  trans- 
formations may  be,  the  typical  properties  of  the  polypeptide  chains 
may  be  said  to  be  the  general  tendency  to  agglomerate  into  fibrous 
strands  and  their  widespread  poiver  of  contracting  (actomyosin,  keratin, 
collagen).  Thus  the  very  structure  of  protoplasmic  polypeptides 
furnishes  the  fundamental  conditions  for  fibrillar  differentiation  and 
contractility. 


RETROSPECT 


A  revolutionary  fact  which  emerged  from  the  synthesis  of  organic 
compounds  was  that,  in  chemistry,  there  is  no  fundamental  difference 
between  living  and  inanimate  matter.  The  complicated  process  of 
metabolism  is  not  controlled  by  some  special  vital  principle,  but  has 
its  being  in  the  co-ordination  of  innumerable  reactions,  each  and  all, 
being  separately  accessible  to  causal  investigation.  Yet  no  simple 
mechanistic  interpretation  can  account  for  their  delicately  attuned 
harmony  and  their  purposiveness.  Morphological  formations  in  the 
submicroscopic  world  present  an  exactly  similar  case.  Whoever  had 
expected  to  find  special  formative  principles,  alien  to  the  inanimate 
world,  in  these  invisible  regions,  is  doomed  by  the  results  of  research 
into  natural  substances  of  high  molecular  weight  to  as  great  a  dis- 
appointment as  was  at  one  time  suffered  by  the  believers  in  mysterious 
life  forces  which  alone  were  deemed  capable  of  building  up  organic 
compounds.  The  formative  forces  in  protoplasm  and  its  derivatives 
are  no  different  from  those  operating  within  inanimate  Nature.  There 
is  no  evidence  of  the  existence  of  formative  principles  beyond  the 
atomic  valency  and  the  various  molecular  cohesive  forces  in  their  vari- 
ous patterns.  This  need  cause  no  surprise  if  it  be  remembered  that,  in 
the  molecular  world,  the  chemical  and  formative  properties  merge  into 
each  other.  In  that  realm,  chemistry  and  morphology  become  in- 
separably one,  since  every  morphological  change  which  a  molecule 
undergoes  inevitably  involves  chemical  changes.  All  metabolic 
processes  therefore  run  parallel  to  changes  in  molecular  form.  For 
this  reason  substance  and  form  are  closely  interrelated,  not  only  in 
the  inanimate  world,  where  every  compound  can  be  clearly  classified 
by  its  molecular  or  crystal  structure,  but  in  living  matter  as  well.  The 
idea  of  an  essential  difference  between  the  morphology  of  the  animate 
and  that  of  the  inanimate  world  has  no  place  in  the  theory  of  sub- 
microscopic  morphology. 

Just  as  organic  chemistry  grew  out  of  inorganic  chemistry  and  has 


37^  RETROSPECT 


its  roots  in  the  fundamental  principles  of  the  latter,  so  should  bio- 
morphology  be  considered  simply  as  a  highly  developed  system,  evolved 
from  molecular  and  micellar  morphology  to  the  shaping  of  cells  and 
organisms.  Only  the  first  step  in  this  development  at  present  lends 
itself  to  deductive  reasoning,  viz.,  the  transition  from  molecular  to 
micellar  morphology.  This  has  been  made  possible  by  the  modern 
evidence  on  the  structure  of  highly  polymeric  chain  molecules  and 
globular  macromolecules. 

There  are  two  guiding  principles,  of  the  utmost  importance  to 
biomorphology,  which  are  already  recognizable  in  the  configuration 
of  chain  molecules.  They  are:  i.  The  principle  of  repetition,  which 
is  the  foundation  of  all  lattice  structures  and  of  every  form  of  banding, 
and  2.  The  principle  of  specificity.  The  first  principle  is  represented, 
on  the  one  hand,  by  the  ever-recurring  members  of  the  chain  (intra- 
molecular spacing)  and,  on  the  other,  by  the  assemblage  into  a  lattice 
pattern  of  kindred  chains  (intermolecular  spacing),  as  for  example 
frame  substances,  reserve  substances,  and  lipid  layers.  Only  if  all  the 
members  of  a  certain  kind  of  chain  are  of  exactly  the  same  structure 
can  true  intermolecular  repetition  take  place.   This  law  does  not 
normally  apply  to  polypeptide  chains,  since  their  side  groups  are  often 
of  different  structure.  In  consequence,  we  find  the  second  principle 
holding  sway,  i.e.,  the  capacity  of  otherwise  similar  molecular  ele- 
mentary units  to  assume  a  specific  arrangement  which  may  be  repeated 
for  its  part  in  long-range  periods.  We  do  not  yet  know  how  the 
visible  specific  forms  of  cellular  organelles,  cells,  tissues  and  organ- 
isms grow  out  of  this  specificity,  but  doubtless  causal  relations  do 
■exist  between  molecular  morphology  and  morphogenesis,  as  fore- 
shadowed by  enzyme  chemistry  and  the  asymmetrical  synthesis  of 
organic  compounds. 

A  problem  no  less  difficult  than  causal  morphological  development 
is  that  of  the  molecular  morphology  of  heredity;  for,  assuming  that 
€very  kind  of  visible  form  owes  its  origin  to  particular  configurations 
of  concrete  hereditary  entities  which  cannot  arise  spontaneously,  then 
their  complicated  structure  must  be  constantly  reproducing  their  like. 
Although  the  multiplication  of  the  virus  molecules  presents  some 
analogy  to  this,  we  have  nothing  to  go  upon  to  build  up  a  clear  picture 
of  the  auto-reproduction  of  those  complicated  structures,  the  genes. 
Fox  the  present,  submicroscopic  morphology  has  been  successful 


RETROSPECT 


573 


Only  in  so  far  as  specificity  is  ignored,  but  within  this  modest  sphere 
the  knowledge  acquired  is  most  significant.  The  substratum  in  which 
life  is  inherent  is  not  a  disperse  phase  with  individual  particles  or 
ultramicrons ;  it  possesses  2l  structure.  Its  active  centres,  which  control 
development,  are  arranged  in  a  given  order.  They  are  not  intermingled 
by  mere  laws  of  chance  and  Brownian  molecular  movement ;  the  fact 
is  rather  that  they  arrange  themselves  into  a  delicate,  very  plastic  and 
flexible  pattern,  actuated,  as  it  were,  by  a  purposeful,  co-ordinative 
impulse.  No  more  than  leaves,  blown  by  autumnal  winds  from  the 
twig  and  fluttering  helplessly  in  the  air,  are  able  to  assimilate  for  the 
parent  tree,  can  independent,  ambulant,  reactive  molecules  take  part 
in  any  organized  work.  It  is  not  surprising,  therefore,  that  the  active 
groups  of  the  enzymes  should  only  be  capable  of  acting  in  association 
with  a  carrier  of  a  given  structure.  For,  orderly  biological  processes 
are  unthinkable  without  presupposing  structure,  and  it  is  therefore 
out  of  the  question  that  any  living  constituent  of  protoplasm  could 
consist  of  structureless,  fluid,  independently  displaceable  particles.  It 
is  for  this  reason  that  colloid  chemistry,  based,  as  it  is,  upon  the 
disperse  principle,  has  thrown  so  little  light  upon  the  submicroscopic 
structure  of  protoplasm.  For  the  cell  certainly  is  not  a  pouch  filled 
with  ultramicrons  suspended  in  a  fluid,  whirling  about  haphazardly 
and  in  confusion;  it  is,  on  the  contrary,  a  wonderful  system,  the 
intrinsic  structure  of  which,  could  it  but  be  seen,  would  assuredly  fill 
every  observer  with  an  enthusiasm  equal  to  that  which  microscopic 
cytomorphology  inspires. 

It  is  true  that  metabolic  centres  (lyoenzymes,  mitochondria,  ery- 
throcytes, chloroplasts)  are  independent  of  each  other;  but  their 
movement  does  not  obey  the  law  of  entropy;  they  are  actively 
directed  to  the  localities  where  their  biochemical  capacity  is  needed. 
On  the  other  hand,  the  special  cytological  and  histological  systems 
which  facilitate  an  appropriate  production  and  distribution  of  those 
metabolic  centres  (protoplasmic  flow,  blood  capillaries,  glands)  must 
have  some  coherent  structures  at  their  disposal.  The  organization  of 
these  semi-solid  structures  is  responsible  for  the  creation  of  biological 
objects  of  any  shape  or  form  and,  therefore,  is  the  very  foundation 
of  morphogemsis. 

In  the  inanimate  world,  crystallization  will  at  times  produce 
structures  from  an  amorphous   mass;   but  the  structures  of  living 


374  RETROSPECT 

protoplasm  cannot  be  spontaneous!}"  generated  from  unformed 
solutions  because,  complicated  and  delicately  inter-adjusted  as  they 
are,  they  can  only  actualize  in  contact  with  already  existing  structures. 
Hence  the  supreme  axiom  of  cytology,  namely,  that  all  cells  derive 
from  their  like,  applies  equally,  though  in  a  wider  sense,  to  invisible, 
submicroscopic  cytogenesis : 

STRUCTURA  OMNIS  E  STRUCTURA 


I 


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J.  c;ll.  comp.  Physiol.  17.  357. 
Bear,  R.  S.  and  Schmitt,  F.  O.,  1936.  (Nerve  myelin,  optics.)  J.  opt.  Soc.  Amer.  26.  206. 
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London  123.  505. 
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Becker,  W.  A.,  1935.  (Youngest  cell  wall.)  Z.  Zellforsch.  23.  253. 
Becker,  W.  A.,  1936.  (Vital  staining.)  Protoplasma  26.  439. 
Becker,  W.  A.  and  Kozbial,J.,  1937.  (Chromosomes,  double  refraction.)  Acta  Soc.  Bot. 

Polon.  14.  239. 
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Bensley,  R.  R.,  1938.  (Plasmosin.)  Anat.  Rec.  72.  351. 
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LITERATURE  377 

Bernal,  J.  D.  and  Fankuchen,  I.,  1937.  (Cr}'stalli2ed  virus,  X-ray  diffraction.)   Nature 

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Bernhard,  W.,  Gautier,  A.  and  Oberling,  Ch.,  195 1.  (Liver  cells,  beaded  chains.)  C.  r. 

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Acta  2.  339. 
Bessis,  M.  and  Bricka,  M.,  1949.  (Erythrocyte,  electron  microscopy.)  Arch.  Anat.  micr. 

38.  190. 
Biedermann,  W.,  1903.  (Solid  secretions.)  Z.  allg.  Physiol.  2.  395. 
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Block,  R.  J.,  1939.  (Wool,  ratio  of  amino  acids.)  J.  biol.  Chem.  128.  181. 
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Arbeitsmeth.  v.  Abderhalden,  Abt.  II,  Teil  3,  p.  3939. 
Boehm,  G.  and  Weber,  H.  H.,  1932.  (Myosin,  X-ray  diffraction.)  Kolloid-Z.  61.  269. 
Bogen,  H.  J.,  1938.  (Permeability  to  urea  and  glycerol.)  Planta  28.  535. 
BoLDUAN,  O.  E.A.  and  Bear,  R.  S.,  1950.  (Collagen,  long-range  spacing.)  J.  Polymer  Sci. 

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Bonner,  J.,  1935.  (Cell  elongation.)  Jahrb.  wiss.  Bot.  82.  377. 
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Bonner,  J.,  1936b.  (Respiration  of  Avena  coleoptile.)  J.  gen.  Physiol.  20.  i. 
Borries,  B.  von,  1941.  (Electron  microscopy.)  Stahl  u.  Eisen  61.  725. 
Borries,  B.  von  and  Ruska,  E.,  1939a.  (Electron  microscopic  image.)  Naturwiss.  27.  281. 
Borries,  B.  von  and  Ruska,  E.,  1939b.  (Electron  microscope.)  Z.  w'ss.  Mikr.  56.  517. 
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BoT,  G.  M.,  1939.  (Chloroplasts,  proportion  of  P.)  Diss.  Leiden. 
BoTTiNi,  O.,  1937.  (Intramicellar  base  exchange.)  Kolloid-Z.  80.  56. 
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Acta  A  12.  Suppl.  XVI. 
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Univ.  Istanbul  B  8.  30. 
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Bretschneider,  L.  H.,  1948.  (Sperm,  fine-structure.)  Vakbl.  voor  Biologen  28.  137. 
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Proc.  Conf.  Electron  Micr.  Delft. 
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378  LITERATURE 

Bretschneider,  L.  H.,  1950b.  (Sperms,  electron  microscopy.)  Proc.  Acad.  Sci.  Amster- 
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Bretschneider,  L.  H.,  1950c.  (Differently  fixed  cytoplasm,  electron  microscopy.)  Proc. 
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Brill,  1941.  (Natural  silk,  action  of  spinning.)  Naturwiss.  29.  221. 

Brill,  R.,  1943.  (Silk  fibroin.)  Z.  phys.  Chem.  B  53.  61. 

Brown,  D.  E.  S.,  1934a.  (Cytoplasm,  high  pressure  experiments.)  J.  cell.  comp.  Physiol. 

4-  257- 

Brown,  D.  E.  S.,  1934b. (Viscosity  of  plasm  depending  on  pressure.)  J.  cell.  comp.  Physiol. 

5-  335- 

Brown,  G.  L.,  Callan,  H.  G.  and  Leaf,  G.,  1950.    (Nuclear  sap,    chromatography.) 

Nature  165.  600. 
Brown,  H.  P.,  1945.  (Hairy  flagella,  electron  microscopy.)  Ohio  J.  Sci.  45.  247. 
Bull,  H.  B.,  1947.  (Protein,  monolayers.)  Advances  in  Protein  Chem.  3.  95. 
BuNGENBERG  DE  JoNG,  H.  G.,  1932.  (Coaccrvation.)  Protoplasma  15.  no. 
Bungenberg,  de  Jong,  H.  G.  and  Bonner,  J.,  1935.  (Films  of  phosphatides.)  Protoplas- 
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Burstrom,  H.,  1942.  (Cell  elongation,  osmotic  conditions.)  Lantbrukshogskolans  Anna- 

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Burstrom,  H.,  1951.  (Cell  elongation.)  Physiol.  Plantarum  4.  199. 
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BiJssEM,  W.  and  Weyl,  W.,  1936.  (Glass,  amicroscopic  structure.)  Naturwiss.  24.  324. 
Callan,  H.  G.,  Randall,  J.  T.  and  Tomlin,  S.  G.,  1949.   (Nuclear  membrane.)  Nature 

163.  280. 
Callan,  H.  G.  and  Tomlin,  S.  G.,  1950.   (Nuclear  membrane.)  Proc.  Roy.  Soc.  London 

B  137.  367. 
Caspersson,  T.,  1936.  (Chromosomes,  ultra  violet  absorption.)  Skand.  Arch.  Physiol. 

Suppl.  8  of  vol.  73. 
Caspersson,  T.,  1940a.  (Nucleus,  distribution  of  proteins).  Chromosoma  i.  562. 
Caspersson,  T.,  1940b.  (Chromomeres,  distribution  of  nucleic  acids.)  Chromosoma  i.  605. 
Caspersson,  T.,  1941.  (Protein  metabolism  in  the  cell.)  Naturwiss.  29.  33. 
Caspersson,  T.  and  Thorell,B.,  1941.  (Muscle,  adenyl  nucleotide.)  Naturwiss.  29.  363. 
Castle,  E.  S.,  1936.  (Chitin,  rodlet  double  refraction.)  J.  gen.  Physiol.  19.  797. 
Castle,  E.  S.,  1937a.  (Spiral  growth  in  Phycomyces.)  J.  cell.  comp.  Physiol.  9.  477. 
Castle,  E.  S.,  1937b.  (Cell  wall  extension.)  J.  cell.  comp.  Physiol.  10.  113. 
Castle,  E.  S.,  1942.  (Spiral  growth  in  Phycomyces.)  Amer.  J.  Bot.  29.  664. 
Chambers,  R.,  1925.  (Microdissection.)  Amer.  J.  Anat.  35.  385. 
Chambers,  R.,  1928.  (Microinjection.)  Biol.  Bull.  55.  369. 

Champetier,  G.  and  Faure-Fremiet,  E.,  1937.  (Elastoidin  fibres.)  J.  chim.  phys.  34.  197. 
Champetier,  G.,  and  Faur£-Fremiet,  E.,  1938.  (Collagen,  X-ray  analysis.)  J.  chim.  phys. 

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Chargaff,  E.,  1950.  (Nucleic  acids,  specificity.)  Experientia  6.  201. 
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Christiansen,  G.  S.  and  Thimann,  K.  V.,  1950.  (Cell  wall,  protein   content.)    Arch. 

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Clark,  G.  L.  and  Corrigan,  K.  E.,  1932.  (Insulin,  X-ray  diffraction.)  Phys.  Rev.  40.  639. 
Claude,  A.,  1944.  (Mitochondria,  constitution.)  J.  exp.  Med.  80.  19. 


LITERATURE  379 

Claude,  A.,  1946.  (Mitochondria,  isolation  by  centrifugation.)  J.  exp.  Med.  84.  51. 
Claude,  A., and  FuLLAM.E.F.,  1 945.  (Mitochondria,  electron  micrograph.)  J. exp.  Med.  81. 5 1. 
Claude,  A.  and  Fullam,  E.  F.,  1946.  (Liver  sections,  rotating  high   speed    microtome.) 

J.  exp.  Med.  83.  499. 
CoHN,  E.  J.  and  Edsall,  J.  T.,  1943.  Proteins,  amino  acids  and  peptides.  New  York. 
Collander,  R.,  1932.  (Lipid  filter  theon,'.)  Handb.  d.  N'aturwiss.  Jena.  2  Aufl.  7.  804. 
Collander,  R.,  1937a.  (Lipid  filter  theory-.)  Schr.  phys.-okon.  Ges.  Konigsb.  69.  251. 
Collander,  R.,  1937b.  (Permeabilit^^)  Ann.  Rev.  Biochem.  6.  i. 
Collander,  R.  and  holmstrom.  A.,  1937.  (Adenoid  action  of  cytoplasm.)  Acta  Soc. 

Fauna  Flora  fenn.  60.  129. 
Comar,  C.  L.,  1942.  (Chloroplast,  chemical  composition.)  Bot.  Gaz.  104.  122. 
Correns,  C,  1893.  (Cell  walls  of  algae.)  Zimmermanns  Beitr.  Morphol.  Physiol.  Pflanzen- 

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Crick,  F.  H.  C.  and  Hughes,  A.  F.  W.,    1950.  (Cytoplasm,  magnetic  particle  method.) 

Exp.  Cell  Research  i.  37. 
Crowfoot,  D.,  1958.  (Cn-stallized  insulin,  Patterson  -  Fourier  analysis.)  Proc.  Roy.  Soc. 

London  A  164.  580. 
Crowfoot,  D.,  1939.  (Protein  crystals,  X-ray  analysis.)  Proc.  Roy.  Soc.  London  A  170.  74. 
Crowfoot,  D.,  1941.   (Cr\'stals  of  insulin,  horse  methaemoglobin  and  lactoglobulin.) 

Sci.  Chem.  Rev.  28.  215. 
Curtis,  H.  J.,  1936.  (Plasmalemma.)  J.  gen.  Physiol.  19.  929. 
CzAjA,  A.  Th.,  1930.  (Ultrafiltration.)  Planta  11.  582. 
Danielli,  J.  F.,  1936.  (Plasmalemma.)  J.  cell.  comp.  Physiol.  7.  393. 
Danielli,  J.  F.,  and  Harvey,  E.  N.,  1935.  (Plasmalemma.)  J.  cell.  comp.  Physiol.  5.  483. 
Danon,   D.  and   Kellenberger,  E.,  1950.  (Microtome  for  electron  microscopy.)  Arch. 

Sci.  Geneve.  3.  169. 
Davson,  H.  and  D.anielli,  J.  F.,  1943.  The  permeability  of  natural  membranes.  Cam- 
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Denny,  F.  E.,  1935.  (Ethylene,  stimulating  substance.)  Contr.  Boyce  Thompson  Inst. 

7-  97- 
Derksen,  J.  C,  Heringa,  G.  C,  and  Weidinger,  A.,  1937.  (Keratin,  cornification.)  Acta 

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Dervichian,  D.  G.,  Fournet,  G.  and  Guinier,  A.,  1947.  (Erythrocyte.)  C.  r.  Acad.   Sci. 

Paris  224.  1848. 
Dettmer,  N.,  Neckel,  J.  and  Ruska,  H.,    195 i.  (Collagen  fibres,  electron  microscopy.) 

Z.  wiss.  Mikr.  60.  291. 
Deuel,  H.,  1943.  (Pectins.)  Diss.  E.  T.  H.  Zurich. 

Deuel,  H.,  1947a.  (Pectin,  artificial  cross-links.)   HabiUtiationsschrift  E.  T.  H.  Zurich. 
Deuel,  H.,  1947b.  (Esterification  of  polygalacturonic  acid.)  Experientia.  3.  151. 
Devaux,  H.,  1935a.  (Films  of  albumin.)  C.r.  Soc.  Biol.  Paris  119.  1124. 
Devaux,  H.,  1935b.  (Films  of  albumin.)  C.r.  Acad.  Sci.  Paris  200.  1560,  201.  109. 
DiEHL,  J.M.  andVAN-lTERSON,  G.,  1935.  (Chitin,  rodlet  double  refraction.)  Kolloid-Z.  73. 

142. 
DoBRY,  A.,  1938.  (Coacervation.)  J.  Chim.  phys.  35.  387. 
DoBRY,  A.,  1940.  (Coacervation.)  Bull.  Soc.  Chim.  biol.  22.  75. 
Doutreligne,  J.,  1955.  (Chloroplasts,  grana.)  Proc.  Acad.  Sci.,  Amsterdam  38.  886. 
Drawert,  H.,  1937.  (Fixed  tissues,  staining.)  Flora  32.  91. 
Drawert,  H.,  1948.  (Permeability  to  urea.)  Planta  35.  579. 

Drucker,  B.  and  Smith,  S.  G.,  1950.  (Silk,  paper  chromatography.)  Nature  165.  196. 
Ebner,  V.  VON,  1894/96.  (Collagen,  optics.)  S.B.  Akad.  Wiss.  Wien  103.  162,  105.  17. 
Elod,  E.,NowoTNY,H.andZAHN,H.,  1940a.  (Wool,  chemical  reactions.)  Kolloid-Z.  93. 50. 


380  LITERATURE 

Elod,  E.,  NowoTNY,  H.  and  Zahn,  H.,  1940b.  (Wool,  fine-structure.)  Melliand  Textilber. 

No.  8. 
Elvers,  I.,  1943.  (Chromosomes,  electron  micrograph.)  Acta  Horti  Berg.  13.  149. 
Emerson,  R.  and  Arnold,  W.,  1932.  (Unit  of  assimilation  )  J.  gen.  Physiol.  16.  191. 
Emerson,  R.  and  Lewis,  C.  M.,  1939.  (Photosynthesis.)  Amer.  J.  Bot.  26.  808. 
Engstrom,  A.  and  Lindstrom,  B.,  1950.   (X-ray  absorption  of  microscopic  objects.) 

Biochim.  Biophys.  Acta  4.  351. 
Engstrom,  A.  and  Luthy,  H.,  1949.  (Nerve,  X-ray  absorption.)  Experientia  5.  244. 
Engstrom,  A.  and  Ruch,  F.,  195 i.  (Giant  chromosomes,  mass  distribution.)  Proc.  nat. 

Acad.  Sci.  Wash.  37.  459. 
Euler,  H.  von,  Bergman,  B.  and  Hellstrom,  H.,  1934.  (Chloroplasts  of  Elodea,  chloro- 
phyll concentration.)  Ber.  dtsch.  bot.  Ges.  52.  458. 
Eymers,  J.  G.  and  Wassink  E.G.,  1938.  (Purple  sulphur  bacteria,  COj  assimilation.)  Enzy- 

mologia  2.  258. 
Fajans,  K.,  1923.  (Deformation  of  ions.)  Naturwiss.  11.  165. 
Fajans,  K.,  1925.  (Deformation  of  ions.)  Z.  Kristallogr.  61.  18. 
Fankuchen,  J.,  1934.  (Pepsin,  X-ray  diffraction.)  J.  Amer.  Chem.  Coc.  56.  2398. 
Fankuchen,  J.,  1941.  (Proteins,  X-ray  analysis.)  Cold  Spr.  Harb.  Symp.  quant.  Biol.  9. 198. 
Farr,  W.  K.  and  Eckerson,  S.H.,  1934.  (Cotton  hairs,  dermatosomes.)  Contr.  Boyce 

Thompson  Inst.  6.  189,  309. 
Faure-Fremiet,  E.,  1936.  (Elastoidin  fibres.)  Arch.  Anat.  micr.  32.  249. 
Faure-Fremiet,  E.,  1946.  (Cytology,  review  1940-1946.)  Anne  biol.  22.  57. 
Faure-Fremiet,  E.,  Bessis,  M.  and  Thaureaux,  J.,  1948.  (Hyaloplasm,  electron  micros- 
copy.) Microscopic  (Paris)  i.  41. 
Felix,  K.,  195 i.  (Frankfurt  a.  M.)  Oral  communication. 
Fernandez-Moran,  H.,  1950a.  (Myelinated  nerve,  fine- structure  of  sheath.)  Exp.  Cell 

Research  i.  143. 
Fernandez-Moran,  H.,  1950b.    (Myelinated   nerve,   electron  microscopy.)  Experientia 

6.  339. 
Fernandez-Moran,  H.,  1952a.  (Myelinated  nerve  fibre.)  Inaugural  Diss.  Uppsala. 
Fernandez-Moran,  H.,  1952b.  (Axon  of  nerve  fibre.)  Exp.  Cell  Research  3.  i. 
Feulgen,  R.  and  Rossenbeck,  H.,    1924.  (Nucleal  staining.)    Hoppe  Seyler  Z.  physiol. 

Chem.  135.  203. 
Fischer,  A.,  1894.  (Hairy  flagella.)  Jahrb.  wiss.  Bot.  26.  187. 
Fischer,  F.  G.,  1942.  (Nucleic  acids,  molecular  structure.)  Naturwiss.  30.  377. 
Fischer,  H.  et  al.,  1935.  (Chlorophyll,  molecular  structure.)  Liebigs  Ann.  519.  209, 520.  88. 
Fitting,  H.,  1927.  (Protoplasmic  flow  by  hi  .tidine.)  Jahrb.  wiss.  Bot.  67.  427. 
Fitting,  H.,  1936.  (Protoplasmic  flow  by  histidine.)  Jahrb.  wiss.  Bot.  82.  613. 
Flaig,  J.  v.,  1947.  (Nerve  axon,  colloidal  state  of  neurin.)  Neurophysiol.  10.  211. 
Foster,  E.,  Baylor,  M.  B,.  Meinkoth,  N.  A.  and  Clark,  G.  L.   1947.   (Hairy  flagella. 

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Foster,  F.  J.  and  Samsa,  E.  G.,  1950.  (Ovalbumin,  birefringence  of  flow.)  Science  112. 475. 
Franck,  J.,  1935.  (Theory  of  COg  assimilation.)  Naturu'iss.  23.  226. 
Frank,  R.,  1950.  (Teeth,  fine-structure  of  enamel.)  Rev.  mens.  Suisse  Odontol.  60.  H09. 
Freudenberg,  K.,  1933.  Stereochemie,  Wien. 
Freudenberg,  K.,  Schaaf,  E.,  Dumpert,  G.  and  Ploetz  T.,  1939.  (Starch  molecule, 

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Freundlich,  H.,  1922.  Kapillarchemie,  Leipzig. 
Freundlich,  H.,  1937.  (Properties  of  gels.)  J.  phys.  Chem.  41.  901. 
Freundlich,   H.,    1942.   (Thixotropy.)    In  W.   Seifriz,  The  structure  of  protoplasm. 
Ames-Iowa,  p.  85. 


LITERATURE  .  381 

Freundlich,  H.  and  Lindau,  C,  1932.  (Mechanochemistty.)  Handb.  d.  Naturwiss.  v. 
Abderhalden,  2.  Aufl.  6.  831. 

Freundlich,  H.,  Stapelfeldt,  F.  and  Zocher,  H.,  1924.  (Double  refraction  of  flow.) 
Z.  phys.  Chem.  114.  161,  190. 

Frey,  a.,  1924.  (Types  of  double  refraction.)  Kolloidchem.  Beih.  20.  209. 

Fret,  A.,  1925.  (Dichroism  of  fibres,  microtechnics.)  Z.  wiss.  iVIikr.  42.  421. 

Frey,  A.,  1926a.  (Micellar  theory.)  Ber.  dtsch.  bot.  Ges.  44.  564. 

Frey,  A.,  1926b.  (Cell  walls,  submicroscopic  structure.)  Jahrb.  wiss.  Bot.  65.  195. 

Frey,  A.,  1926c.  (Closterium,  viscosity  of  cell  sap.)  Rev.  gen.  Bot.  38.  273. 

Frey,  a.,  1927a.  (Aspergillus,  sporangiophore,  optics.)  Rev.  gen.  Bot.  39.  277. 

Frey,  A.,  1927b.  (Dichroism  of  fibres.)  Jahrb.  wiss.  Bot.  67.  597. 

Frey,  A.,  1928a.  (School  of  Ambronn.)  Kolloid-Z.  44.  6. 

Frey,  A.,  1928b.  (Micellar  science.)  Protoplasma  4.  139. 

Frey,  R.,  1950.  (Chitin  of  fungi.)  Diss.  E.  T.  H.  Zurich  1950;  Ber.  schweiz.  bot.  Ges. 
60.  199. 

Frey-Wyssling,  a.,  1930.  (Micellar  textures.)  Z.  wiss.  Mikr.  47.  i. 

Frey-Wyssling,  a.,  1932.  (Latex  tubes.)  Jahrb.  wiss.  Bot.  77.  560. 

Frey-Wyssling,  A.,  1935a.  Die  Stoffausscheidung  der  hoheren  Pflanzen.  Berlin. 

Frey-Wyssling,  A.,  1935b.  (Theory  of  junctions.)  Proc.  VI.  Int.  Bot.  Congr.  Amster- 
dam 2.  294.  Illustrations  to  this  topics  in  1936a. 

Frey-Wyssling,  a.,  1935c.  („Nutrition  line".)  Naturwiss.  23.  767. 

Frey-Wyssling,  A.,  1936a.  (Structure  of  cell  walls.)  Protoplasma  25.  261. 

Frey-Wyssling,  A.,  1936b.  (Cellulose,  optical  dispersion.)  Helv.  chim.  Acta  19.  900. 

Frey-Wyssling,  A.,  1936c.  (Filaments,  optics.)  Ber.  dtsch.  bot.  Ges.  54.  445. 

Frey-Wyssling,  A.,  i936d.  (Cellulose  fibres,  permutoid  reactions.)  Protoplasma  26.  45. 

Frey-Wyssling,  a.,  1937a.  (Intermicellar  system.  X-ray  analysis.)  Protoplasma  27.  372. 

Frey-Wyssling,  A.,  1937b.  (Intermicellar  system,  ultramicroscopic  analysis.)  Proto- 
plasma 27.  563. 

Frey-Wyssling,  A.,  1937c.  (Chloroplasts,  structure.)  Protoplasma  29.  279. 

Frey-Wyssling,  A.,  i937d.  (Submicroscopic  morphology.)  Ber.  dtsch.  bot.  Ges.  55.  (i  19). 

Frey-Wyssling,  A.,  1938.  (Micellar  science.)  Kolloid-Z.  85.  148. 

Frey-Wyssling,  A.,  1940a.  (Cytoplasm,  fine-structure.)  J.  R.  micr.  Soc.  60.  128. 

Frey-Wyssling,  a.,  1940b.  (Starch  grains,  optics.)  Ber.  schweiz.  bot.  Ges.  50.  321. 

Frey-Wyssling,  A.,  1940c.  (Starch  grains,  optics.)  Naturwiss.  28.  78. 

Frey-Wyssling,  a.,  1941.  (Crossed  micellar  systems.)  Protoplasma  35.  527. 

Frey-Wyssling,  a.,  1942.  (Plant  cell  walls  with  tubular  texture.)  Jahrb.  wiss.  Bot.90.  705. 

Frey-Wyssling,  a.,  1943a.  (Scattering  in  gels.)  Helv.  chim.  Acta  26.  833. 

Frey-Wyssling,  A.,  1943b.  (Chromosomes,  scattering  of  nucleic  acid  chains.)  Chromo- 
soma  2.  473. 

Frey-Wyssling,  A.,  1944a.  (Chromosomes,  distribution  of  nucleic  acids.)  Schweiz.  med. 
Wochenschr.  74.  330. 

Frey-Wyssling,  A.,  1944b.  (Genes,  structure  and  size.)  Arch.  Klaus-Stift.  19.  451. 

Frey-Wyssling,  a.,  1945a.  (Cell  extension.)  Arch.  Klaus-Stift.  20.  Erganzungsbd.  p.  381. 

Frey-Wyssling,  a.,  1945b.  Ernahrung  und  Stoffwechsel  der  Pflanzen.  Zurich. 

Frey-Wyssling,  A.,  1947/49.  (Plasma  gel,  protoplasmic  flow.)  Exp.  Cell  Research 
Suppl.  I,  Stockholm,  p.  33. 

Frey-Wyssling,  A.,  1948a.  (Extension  growth,  energetics.)  Viertelj.  schr.  Naturf.  Ges. 
Zurich  93.  24. 

Frey-Wyssling,  a.,  1948b.  (Cell  walls,  growth  in  area.)  Growth  Symp.  12.  151. 

Frey-Wyssling,  a.,  1948c.  (Starch  grains,  fine -structure.)  Schweiz.  Brauerei  Rundschau 
1948,  No.  1. 


382  LITERATURE 

Frey-Wyssling,  a.,  1949a.  (Cytoplasm,  physicochemical  behaviour.)  Research  2.  300. 
Frey-Wyssling,  a.,  1949b.  (Chloroplasts,  lipoproteins.)  Faraday  Soc.  Disc.  1949,  No.  6. 

p.  130. 
Frey-Wyssling,  A.,  195 1.    Elektronenmikroskopie.    Neujahrsblatt    der    Naturf.    Ges. 

Zurich  195 1. 
Frey-Wyssling,  A.  and  Frey,  R.,    1950.    (Tunicin,  electron  microscopy.)  Protoplasma 

39,  656. 
Frey-Wyssling,  a.  and  Muhlethaler,  K.,  1944.  (Gels,  electron  microscopic  studies.) 

Viertelj.  schr.  Naturf.  Ges.  Zurich  89.  214. 
Frey-Wyssling,  a.  and  Muhlethaler,  K.,  1946.   (Electron  microscopy  of  bacterial 

cellulose.)  J.  Polymer  Sci.  i.  172. 
Frey-Wyssling,  A.  and  Muhlethaler,  K.,  1949a.  (Chloroplasts,  electron  microscopy.) 

Viertelj.  schr.  Naturf.  Ges.  Zurich  94.  179. 
Frey-Wyssling,  A.,  and  Muhlethaler,  K.,  1949b.  (Root  hairs,  electron  microscopy.) 

Mikroskopie  (Wien)  4.  257. 
Frey-Wyssling,    a.    and   Muhlethaler,    K.,    1949c.    (Rayon,   electron    microscopy.) 

Schweiz.  Bauzeitg.  67.  51. 
Frey-Wyssling,  A.  and  Muhlethaler,  K.,  1950.  (Chitinous  cell  walls,  electron  micros- 
copy.) Viertelj  .schr.  Naturf.  Ges.  Zurich  95.  45. 
Frey-Wyssling,  A.,  Muhlethaler,  K.  and  Wyckoff,  R.  W.  G.  1948.  (Cell  walls,  micro- 
fibrils.) Experientia  4.  475. 
Frey-Wyssling,  a.,  and  Speich,  H.,  1942.  (Cellulose  fibres,  deficit  of  density.)  Helv. 

chim.  Acta  25.  1474. 
Frey-Wyssling,  A.and  Stecher,  H.,  1951.  (Cell  walls,  growth  in  area.)  Experientia  7.  420. 
Frey-Wyssling,  A.  and  Steinmann,  E.,  1948.  (Chloroplasts,  layer  birefringence.)  Bio- 

chem.  Biophys.  Acta  2.  254. 
Frey-Wyssling,  H.  and  Walchli,  O.,  1946.  (Silver  dichroism.)  J.  Polymer  Sci.  i.  266. 
Frey-Wyssling,  A.  and  Weber,  E.,  1941.  (Double  refraction  of  flow,    measurement.) 

Helv.  chim.  Acta.  24.  278. 
Fricke,  H.,  1925.  (Erythrocytes,  electrical  capacity.)  J.  gen.  Physiol.  9.  137. 
Friedel,  G.,  1922.  (Mesophases.)  Ann.  Phys.  Paris  18.  358. 
Friedrich-Freksa,  H.,  Kratky,  O.  and  Sekora,  A.,  1944.  (Silk  fibroin.)  Naturwiss. 

32.  78. 
Fritz,  F.,  1935.  (Cuticular  layers.)  Jahrb.  wiss.  Bot.  81.  718. 
Fritz,  F.,  1937.  (Rhythmical  cutinisation.)  Planta  26.  693. 

Gaffron,  H.  and  Wohl,  K.,  1936.  (Theory  of  COg  assimilation.)  Naturwiss.  24.  81,  103. 
Gaumann,  E.  and  Jaag,  O.,   1936.   (Cuticular  transpiration.)  Ber.  schweiz.  bot.  Ges. 

45.  411. 
Gause,  G.  F.,  1936.  (Chemical  asymmetry.)  Ergebn.  Biol.  13.  54. 
Geitler,  L.,  1934.  Grundriss  der  Zytologie.  Berlin. 
Geitler,  L.,  1937.  (Chloroplasts,  grana.)  Planta  26.  463. 
Geitler,  L.,  1938.  Chromosomenbau.  Berlin. 
Geitler,  L.,  1940.  (Endomitosis.)  Ber.  dtsch.  bot.  Ges.  58.  131. 
Gerendas,  M.  and  Matoltsy,  A.  G.,  1947.  (Striated  muscle,  optics.)  Hung.  Acta  Physiol. 

I.  No.  4. 
Gerngross,  O.,  Herrmann,  K.  and  Abitz,  W.,  1930.  (Gelatin.)  Biochem.  Z.  228.  409. 
Gerngross,  O.,  Herrmann,  K.  and  Lindemann,  R.,  1932.  (Gelatin.)  KoUoid-Z.  60.  276. 
Gerngross,  O.  and  Katz,  J.  R.,  1926.    (Gelatin,  X-ray  diffraction.)  Kolloid-Z.  39.  181. 
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Acta  33.  1477. 
Gicklhorn,  J.,  1932a.  (Myelin  forms.)  Protoplasma  15.  90. 


LITERATURE  585 

GiCKLHORN,  J.,  1932b.  (Nucleus,  protein  crystals.)  Protoplasma  15.  276. 

GiROUD,  A.,  1928.  (Mitochondria,  optics.)  C.r.  Acad.  Sci.  Paris  186.  794. 

GoLDACRE,  R.  J.  and  Lorch,  I.  J.,  1950.  (Plasm  flow.)  Nature  166.  497. 

GoLDSCHMiDT,  V.  M.,  1926.  (Diameter  of  atoms.)  Naturwiss.  14.  477. 

GoLDSCHMiDT,  V.  M.,  1927.  (Diameter  of  atoms.)  Ber.  dtsch.  chem.  Ges.  60.  1263. 

Gorter,  E.  and  Grendel,  F.,  1925.  (Erythrocytes,  lipid  coating.)  J.  exp.  Med.  41.  439. 

GoRTER,  E.  and  van  Ormondt,  J.,  1935.  (Myosin,  spreading.)  Biochem.  J.  29.  48. 

Gorter,  E.,  van  Ormondt,  J.  and  Meyer,  T.  M.,  1935.  (Complex  proteins,  spreading.) 
Biochem.  J.  29.  38. 

GouGH,  A.,  1924.  (Erythrocytes,  shape.)  Biochem.  J.  i8.  202. 

Graham,  Th.,  1861.  (Colloid  particles.)  Philos.  Trans.  1861,  p.  183. 

Graham,  Th.,  1862.  (Dialysis.)  Liebigs  Ann.  i2i.  i. 

Grakick,  S.,  1938.  (Chloroplasts,  method  of  isolation.)  Amer.  J.  Bot.  25.  561. 

Granick,  S.,  1948.  (Haem  and  chlorophyll.)  Harvey  Lectures  (Springfield,  111.)  44. 
220. 

Granick,  S.,  1949.  (Chloroplast,  number  of  grana.)  In  Franck  and  Loomis,  Photosyn- 
thesis in  plants.  Ames,  Iowa,  p.  113. 

Granick,  S.  and  Porter,  K.  R.,  1947.  (Chloroplasts,  electron  microscopy.)  Amer.  J.  Bot. 

34-  545- 
Gross,  J.,  1949.  (Elastic  tissue,  electron  microscopy.)  J.  exp.  Med.  89.  699. 
Gross,  J.,  1950.  (Collagen  fibres,  segmented.)  Ann.  N.Y.  Acad.  Sci.  52.  964. 
Guilliermond,  a.,  1930.  (Protoplasm,  ultramicroscopy.)  Rev.  gen.  Bot.  42.  327. 
GuiLLiERMOND,  A.,  Mangenot  ,G.  and  Plantefol,L.  1933.  Traite  de  Cytologic  vegetale, 

Paris. 
GuNDERMANN,    J.,   Wergin,    W.  and  Hess,  K.,    1937.  (Waxes  of  cell  walls.)  Ber.  dtsch. 

chem.  Ges.  70.  517. 
Haas,  J.  N.,  1950.  (Muscle  fibre,  tip  growth.)  Growth  14.  277. 
Haase-Bessel,  G.,  1936.  (Substratum  of  genes.)  Planta  25.  240. 
Hadorn,  E.,  1939.  (Pupation,  caused  by  hormones.)  Mitt.  Naturwiss.  Ges.  Thun  1939, 

Heft  4,  p.  I. 
Hakansson,  a.  and  Levan,  A.,   1942.  (Nucleoli,  formation.)  Hereditas    (Lund)  28.  436. 
Hall,  C.  E.,  Jakus,  M.  A.  and  Schmitt,  F.  O.,  1945.  (Muscle,  electron  stains.)  J.  appL 

Phys.  16.  459. 
Hall,  C.  E.,  Jakus,  M.  A.  and  Schmitt,  F.  O.,  1946.  (Striped  muscle  fibres,  electron 

micrograph.)  Biol.  Bull.  90.  32. 
Halle,  F.,  193 i.  (Length  of  rodshaped  molecules.)  Kolloid-Z.  56.  77. 
Halle,  F.,  1937.  (Protein  molecules,  structure.)  Kolloid-Z.  81.  334. 
Hanes,  C.  S.,  1937.  (Starch,  hydrolysis.)  New  Phytol.  36.  loi. 

Hanes,  C.  S.,  1940.  (Synthesis  of  starch.)  Proc.  Roy.  Soc.  London  128.  421,  129.  174. 
Hanson,  E.  A.  and  Katz,  J.  R.,  1934.  (Starch  grains.)  Z.  phys.  Chem.  A  168.  339. 
Hansteen-Cranner,  B.,  1926.  (Phosphatides  in  the  cell  wall.)  Planta  2.  438. 
Harder,  R.,  1937.  (Chitin  of  fungi.)  Nachr.  Ges.  Wiss.  Gottingen  (math. -phys.  Kl.) 

Nachr.  Biol.  3.  i. 
Harris,  J.  E.,  1939.  (Nuclear  thixotropy.)  J.  exp.  Biol.  16.  258. 
Harris,  J.  E.,  1952.  (Nucleus,  thixotropy.)  In  A.  Frey-Wyssling,  Deformation  and 

flow  in  biological  systems.  Amsterdam,  p.  486. 
Harvey,  E.  B.,  1933.  (Cell  fragments,  development.)  Biol.  Bull.  64.  125. 
Harvey,  E.  B.,  1936.  (Centrifuged  cells.)  Biol.  Bull.  71.  loi. 

Harvey,  E.  N.,  1936.  (Surface  tension,  sessile  drop.)  J.  cell.  comp.  Physiol.  8.  251. 
Harvey,  E.  N.,  1937.  (Cytoplasm,  surface  tension.)  Trans.  Faraday  Soc.  33.  943. 
Harvey,  E.  N.  and  Loomis,  A.  L.,  1930.  (Centrifuging  microscope.)  Science  72.  42. 


384  LITERATURE 

Harvey,  E.  N.  and  Danielli,  J.  F.,  1936.  (Films  of  proteins,  elasticity.)  J.  cell.  comp. 

Physiol.  8.  31. 
Haurowitz,  p.,  1949.  (Protein  molecules,  internal  structure.)  Experientia  5.  347. 
Hausermann,  E.,  1944.  (Intercellular  spaces,  wettability.)  Diss.  E.  T.  H.  Ziirich. 
Haworth,  W.  N.,  1925.  (Sugars,  pyranose  structure.)  Nature  116.  430. 
Haworth,  W.  N.,  1929.  The  constitution  of  sugars.  London. 
Hecht,  K.,  1912.  (Plasmolysis.)  Beitr.  Biol.  Pfl.  ii.  137. 
Hegetschweiler,  R.,  1948.  (Silk,  double  refraction.)  Viertelj.schr.  Naturf.  Ges.  Ziirich 

93-  57- 

Hegetschweiler,  R.,  1950.  (Silk  fibroin,  optics.)  Diss.  E.  T.   H.    Zurich;    Makromol. 

Chem.  4.  156. 

Heidenreich,  L.  D.  and  Matheson,  L.  A.,  1944.  (Surface  orientation,  electron  microsco- 
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Heierle,  E.,  1935.  (Pigments  of  leaves,  spectrometric  analysis.)  Diss.  E.T.H.  Zurich. 

Heilbronn,  a.,  1914.  (Cytoplasm,  viscosity.)  Jahrb.  wiss.  Bot.  54.  357. 

Heilbronn,  a.,  1922.  (Cytoplasm,  displacement  of  magnetic  particles.)  Jahrb.  wiss. 
Bot.  61.  284. 

Heilbrunn,  L.  v.,  1930.  (Cytoplasm,  viscosity.)  Protoplasma  8.  58. 

Heitz,  E.,  193 1.  (Nucleoli,  number.)  Planta  12.  775. 

Heitz,  E.  1932.  (Chloroplasts,  grana.)  Planta  18.  616. 

Heitz,  E.,  1935.  (Chromosomes,  structure.)  Z.  indukt.  Abstamm.- u.  Vererblehreyo.  402. 

Heitz,  E.,  1936a.  (Chloroplasts,  structure.)  Ber.  dtsch.  bot.  54.  362. 

Heitz,  E.,  1936b.  (Chloroplasts,  structure.)  Planta  26.  134. 

Hengstenberg,  J.,  1928.  (Intramolecular  periods.)  Z.  Kristallogr.  67.  583. 

Hengstenberg,  J.  and  Mark,  H.,  1928.  (X-ray  determination  of  particle  size.)  Z.  Kristal- 
logr. 69.  271. 

Hermans,  P.  H.,  1938.  (Deformation  of  gels.)  Kolloid-Z.  83.  71. 

Hermans,  P.  H.,  1941.  (Swelling  of  gels.)  Kolloid-Z.  97.  231. 

Hermans,  P.  H.,  1946.  Contribution  to  the  physics  of  cellulose  fibres.  Monograph  on  the 
progress  of  research  in  Holland  during  wartime.  Amsterdam. 

Hermans,  P.  H.,  1949.  Physics  and  Chemistry  of  Cellulose  Fibres.  New  York  and 
Amsterdam. 

Hermans,  P.  H.  and  De  Leeuw,  A.  J.,  1937,  1941.  (Isotropic  cellulose  fibres.)  Kolloid-Z. 
8i.  300,  97.  326. 

Hermans,  P.  H.  ,  Kratky,  O.  and  Treer,  R.,  1941.  (Gels,  orientation  during  extension.) 
KoUoid-Z.  96.  30. 

Hermans,  P.  H.  and  Vermaas,  D.,  1946.  (Cellulose  fibres,  density.)  J.  Polymer  Sci.  i.  149. 

Hermans,  P.  H.  and  Weidinger,  A.,  1949.  (Crystallinity  of  cellulose.)  J.  Polymer  Sci. 
4.  135. 

Herzog,  R.  O.,  1927.  (Cellulose  „crystallits".)  Handb.  d.  Textilfasern  7.  i.  Berlin. 

Hess,  K.,  Kiessig,  H.  and  Gundermann,  J.,  1941.  (Cellulose  crushed  in  ball  mill.) 
Z.  phys.  Chem.  B.  49.  64. 

Hess,  K.,  Kiessig,  H.,  Wergin,  W.  and  Engel,  W.,  1939.  (Cell  wall,  formation.)  Ber. 
dtsch.  chem.  Ges.  72.  642. 

Hess,  K.,  Trogus,  C.  and  Wergin,  W.,  1936.  (Meristems,  „Primarsubstanz".)  Planta 

25.  419. 

Heyn,  a.  N.  J.,  1931.  (Cell  elongation,  optics.)  Diss.  Utrecht. 

Heyn,  a.  N.  J.,  1933.  (Epidermis,  X-ray  analysis.)  Proc.  Acad.  Sci.  Amsterdam  36.  560. 

Heyn,  A.  N.  J.,  1934.  (Meristems,  X-ray  analysis.)  Protoplasma  21.  299. 

Hill,  A.  V.,  1950.  (Muscle,  mechanics  of  contraction.)  Nature  166.  415. 

HiLLiER,  J.,  1946.  (Electron  microscopy.)  Bull.  Amer.  Ceramic  Soc.  25.  438. 


LITERATURE  385 

HiLLiER,   |.  and  Gettner,  M.  E.,   1950.   (Electron  microscopy,  thin  sections.)  Science 

112.  520. 

HiRSCHLER,  J.,  1942.  (Chromosomes,  contents  of  lipids.)  Naturwiss.  30.  642. 

HoAGLAND,  D.  R.  and  Broyer,  T.  C,  1936.  (Resorption  and  respiration.)  Ann.  Bot. 
Lond.  50.  345. 

HoBER,  R.,  1922.  Physikalische  Chemie  der  Zelle  und  der  Gewebe,  Leipzig. 

Hoerr,  N.,  1943.  (Mitrochondria,  isolation.)  Biol.  Symp.  10.  185. 

HoFLER,  K.,  193 1.  (Plasmic  membranes.)  Ber.  dtsch.  bot.  Ges.  49.  (79). 

HoFLER,  K.,  1932.  (Tonoplast.)  Protoplasma  15.  462. 

HoFLER,  K.,  1934.  ("Intrability".)  Z.  wiss.  Mikr.  Kiister-Festschr.  51.  70. 

HoFLER,  K.,  1936a.  (Compared  permeability.)  Mikrochem.  Molisch-Festschr.  p.  224. 

HoFLER,  K.,  1936b.  (Rhodophyceae,  centrifuging  experiments.)  Protoplasma  26.  377. 

HoFLER,  K.,  1940.  (Cap  plasmolysis.)  Ber.  dtsch.  bot.  Ges.  58.  292. 

HoFLER,  K.,  1942.  (Specific  permeability  series.)  Ber.  dtsch.  bot.  Ges.  60.  179. 

HoFMEiSTER,  L.,  1935.  (Compared  premeability.)  Bibl.  bot.  Stuttgart  113.  i. 

HoFMEiSTER,  L.,  1940.  (Plastids  of  diatoms.)  Z.  wiss.  Mikr.  57.  259. 

HoGEBOOM,  G.  H.,  Claude,  A.  and  Hotchkiss,  R.  D.,  1946.  (Mitochondria,  fixed  oxida- 
ses.) J.  biol.  Chem.  165.  615. 

HoHNEL,  F.  VON,  1887.  Mikroskopie  der  Faserstoffe.  Leipzig. 

HoiwiNK,  A.  L.,  1951.  (Flagellum  of  Euglena  gracilis.)  Proc.  Acad.  Sci.  Amsterdam  C 
54.  132. 

HuBER,  L.  and  Rouiller,  Ch.,  195  i.  (Collagen  fibre  of  bone.)  Experientia  7.  338. 

Hubert,  B.,  1955.  (Chloroplasts,  molecular  structure.)  Diss.  Leiden. 

HiJRTHLE,  K.,  193 1.  (Muscle  fibre,  fine-structure.)  Pfiiig.  Arch.  ges.  Physiol.  227.  610. 

HusEMANN,  E.  and  Ruska,  H.,  1940.  (Molecules  of  glycogen.)  J.  prakt.  Chem.  156.  i. 
Naturu'iss.  28.  534. 

HusKiNS,  C.  L.,  1941.  (Chromonema,  spiral  structure.)  Cold  Spr.  Harb.  Symp.  quant. 

Biol.  9.  13. 
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Ames-Iowa,  p.  109. 
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Induni,  G.,  1945.  (Swiss  electron  microscope.)  Viertelj.  schr.  Naturf.  Ges.  Zurich  90.  181. 
Irvine,  J.  C,  1932.  (Starch  methylated.)  Nature  (Lond.)  129.  470. 
Itschner,  v.,  1935.  (Glucosamin,  constitution.)  Diss.  Univ.  Zurich. 
Jaccard,  p.  and  Frey,  A.  1928.  (Tracheids,  spiral  texture.)  Jahrb.  wiss.  Bot.  68.   844. 
Jakus,  M.  a.,  1945.  (Trichocysts  of  Paramecium,  electron  micrograph.)  J.  exp.  Zool. 

100.  457. 
Jaloveczky,  p.,  1942.  (Starch  grains,  lamellar  structure.)  Bot.  Kozlen  (Ungarn)  39.  610. 
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Kamiya,  N.,  1940  (Protoplasmic  flow,  propulsive  pressure.)  Science  92.  462. 
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386  LITERATURE 

Karrer,  p.,  1935.  (Carotenoids,  constitution.)  Schweiz.  med.  Wochenschr.  65.  898. 

Karrer,  p.,  1941.  Lehrbuch  der  organischen  Cliemie.  7.  Aufl.  Leipzig. 

Karrer,  P.,  1944.  (Vitamins.)  Schweiz.  Z.  Pathol.  Baicteriol.  7.  303. 

Karrer,  P.,  1946.  (Carotenoids,  epioxides.)  Bull.  Soc.  Chim.  bid.  28.  688. 

Karrer,  P.  Peyer,  I.  und  Zegar,  Z.,    1923.    (Cellulose   in  cork.)   Helv.   chim.   Acta 

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Katz,  J.  R.,  1924.  (Swelling.)  Ergebn.  exakt.  Naturwiss.  3.  332. 
Katz,  J.  R.  and  Derksen,  J.  C,  1933.  (Starch,  X-ray  diffraction.)  Z.  phys.  Chem.  A 

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Katz,  J.  R.  and  De  Rooy,  A.,  1933.  (Blood  fibrin.)  Naturwiss.  2X.  559. 
Kausche,  G.  A.  and  Ruska,  H.,  1940.  (Chloroplasts,  electron  micrograph.)  Naturwiss. 

28.  303. 
Kelaney,  M.  a.  El  and  Searle,  G.  O.,  1930.  (Cellulose  fibres,  chemical  cross-sections.) 

Proc.  Roy.  Soc.  London  106.  357. 
Keller,  R.,  1932.  Die  Elektrizitat  der  Zelle.  3.  Aufl.  Mahrisch  Ostrau. 
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KiESEL,  A.,  1930.  Chemie  des  Protoplasmas.  Berlin. 
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Knapp,  a.,  1946.  (Nucleic  acid,  double  refraction  of  flow.)  Diss.  Bern. 
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Chem.  122.  763. 
Kohler,  a.,  1904.  (UV  microscopy.)  Z.  wiss.  Alikr.  21.  129,  273. 
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Kratky,  O.,  1933.  (Orientation  in  gels.)  Kolloid-Z.  64.  213. 
Kratky,  O.,  1934.  (Short-range  order.)  Kolloid-Z.  68.  347. 
Kratky,  O.,  1938.  (Orientation  in  gels.)  Kolloid-Z.  84.  149. 
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Kratky,  O.  and  Platzek,  P.,  1938.  (Intrinsic  double  refraction,  depending  on  swelling.) 

Kolloid-Z.  84.  268. 
Kratky,  O.,  Schauenstein,  E.  and  Sekora,  A.,  1950.  (Silk  I  and  silk  IL)  Nature  165.  319. 
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Kreger,  D.,  1946.  (Starch  grain.  X-ray  diffraction.)  Nature  158.  199. 
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KuHN,  R.,  1937.  (Carotenes.)  Naturwiss.  25.  225. 

KuHN,  W.  and  Hargitay,  B.,  1951.  (Chain  molecules,  contraction.)  Experientia  7.  i. 
KiJNTZEL,  A.,  1929.  (Tendon,  rodlet  double  refraction.)  Collegium  4.  207. 
KuNTZEL,  A.,  1941.  (Collagen  fibre.)  Kolloid-Z.  96.  273. 

KuNTZEL,  A.  and  Prakke,  F.,  1933.  (Collagen,  swelling.)  Biochem.  Z.  267.  243. 
KiJSTER,  E.,  1933.  (Plastids,  anisotropy.)  Ber.  dtsch.  bot.  Ges.  51.  523. 
KiJSTER,  E.,  1934a.  (Protein  spindles.)  Ber.  dtsch.  bot.  Ges.  52.  564. 
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LITERATURE  587 

KiJSTER,  E.,  1935c.  (Plastids,  formation  of  strands.)  Ber.  dtsch.  bot.  Ges.  53.  834. 

KiJSTER,  E.,  1937.  (Plastids,  anisotropy.)  Z.  wiss.  Milcr.  54.  88. 

KuwAD.\,  Y.  and  Nak.\ml'r.\,  T.,  1934.  (Chromonema.)  Cytologia  6.  78. 

Lagerm.\lm,  G.,  Philip,  B.  and  Lindberg,  J.,  195 1.  (Finger  nail,  surface  film.)  Nature 
168.  1080. 

Lamm,  O.,  1957.  (Potato  starch.)  Nova  Acta  Soc.  Sci.  upsal.  10.  No.  6. 

Landolt-Bornstein,  1923.  Physikalisch-chemische  Tabellen.  Berlin. 

Langelaan,  J.  W.,  1946.  (Muscle  fibre,  interference  pattern.)  Verb.  Akad.  Wet.  Amster- 
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Langmuir,  J.,  1917.  (Surface  films.)  J.  Amer.  chem.  Soc.  39.  1848. 

Laue,  M.  von,  1926.  (Particle  size  by  X-ray  analysis.)  Z.  Kristallogr.  64.  115. 

Lea,  D.  A.,  1940.  (Size  of  genes  as  determined  by  ionizing  rays.)  Nature  146.  137. 

Lecher,  E.,  1919.  Lehrbuch  der  Physik  Rir  Mediziner.  Leipzig. 

Lehmai^tn,  F.  E.,  1937a.  (Disturbance  in  development  by  LiCl.)  Naturwiss.  25.  124. 

Lehma>jn,  F.  E.,  1937b.  (Disturbance  in  development  by  LiCl.)  Arch.  Entw.  Mech.  Org. 
136.  112. 

LEHM.A>rN,  F.  E.,  1940.  (Tubifex  eggs,  polarity.)  Rev.  Suisse  Zool.  47.  177. 

Lehmann,  F.  E.,  1945.  Einfuhrung  in  die  physiologische  Embryologie.  Basel. 

Lehm.ann,  F.  E.,  1947.  (Biosomes.)  Rev.  Suisse  Zool.  54.  246. 

Lehmann,  F.  E.,  1948.  (Egg  of  Tubifex,  polar  plasm.)  Rev.  Suisse  Zool.  55.  i. 

Lehmann,  F.  E.,   1950.  (Cytoplasm,  submicroscopic  globular  particles.)  Experientia  6. 
382 

Lehmann,  F.  E.,  195  i.  (Beaded  chains  in  Amoeba.)  Kolloqu.  dtsch.  Ges.  physiol.  Chem. 
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Lehmann,  F.  E.  and  Biss,  R.,  1949.    (Tubifex  egg,  electron   microscopy.)   Rev.   Suisse 
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Lehmann,  O.,  1917.  (Liquid  crystals.)  Ergebn.  Physiol.  i6.  255. 

Lepeschkin,  W.  W.,  1927.  (Er}'throcyte,  membrane.)  Protoplasma  2.  143. 

Leuthardt,  F.,  1949.   (Mitochondria,  metaboHsm.)  Viertelj.schr.  Naturf.  Ges.  Zurich 
94.  132. 

Leuthold,  p.,  1933.  (Aleurone  grains.)  Ber.  schweiz.  bot.  Ges.  42.  31. 

Lewis,  E.  B.,  1950.  (Genes,  screened  by  heterochromatin.)  Adv.  in  Genetics  3.  73. 

Lewis,  W.  H.,  1942.   (Plasm  viscosity.)  In  W.  Seifriz,  The  structure  of  protoplasm. 
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Liebich,  H.,  1941.  (Chloroplasts,  contents  of  iron.)  Z.  Bot.  37.  129. 

Lieser,  Th.,  1940.  (Cellulose,  supramolecular  constitution.)  Cellulose-Chem.  18.  121. 

LiESER,  Th.  et  al.,  1941.  (Cellulose,  supramolecular  constitution.)  Liebigs  Ann.  548,  195, 
204,  212. 

Lindberg,  J.,  Philip,  B.  and  Gralen,  N.,1948.  (Wool,  epidermicula.)  Nature  162.  458. 

LiNDLEY,  H.,  1950.  (Wool  structure,  review.)  Research  3.  509. 

LoEB,  J.  and  Beutner,  R.,  1912/13.  (Potential  in  membranes.)  Biochem.  Z.  41.  i,  44.  303, 
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LoEWY,  A.  G.,  1949.  (Protoplasmic  flow.)  Amer.  Philosoph.  Soc.  93.  326. 

LoTMAR,  W.  and  Picken,  L.  E.  R.,  1950.  (Chitin  of  Polychaeta  and  Mollusca.)  Exper- 
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LiJDTKE,  M.,  1936.  (Cellulose  fibres,  "Querelemente".)  Planta  25.  791. 

Lundegardh,  H.,  1922.  Zclle  und  Zytoplasma.  Berlin. 

LuNDEGARDH,  H.  and  Burstrom,  H.,  1933.  (Resorption  and  respiration.)  Planta  18.  683. 

LiiscHER,  E.,  1936.  (Cork  waxes.)  Diss.  Bern. 

LuYET,  B.  J.,  1937.  (\^itrification  of  gels.)  Biodynamica  1937,  No.  29. 

LuYET,  B.  J.,  1939.  (Cytoplasm,  freezing.)  Arch.  exp.  Zellforsch.  22.  487. 


388  LITERATURE 

LuYET,  B.  J.  and  Ernst,  R.,  1934a.  (Nuclear  frame  and  nuclear  sap.)  Biodynamica  1934. 

No.  2. 
LuYET,  B.  J.  and  Ernst,  R.,  1934b.  (Nuclear  membrane.)  Proc.  Soc.  exp.  Biol.  31.  1225. 
McArthur,  I.,  1943.  (Wool,  X-ray  analysis.)  Nature  152.  38. 
McClintoc,  B.,  1950.  (Genes,  influence  of  heterochromatin.)  Proc.  nat.  Acad.  Sci.  Wash. 

36.  344. 
Mackinney,  G.  and  Milner,  H.  W.,  1933.  (Carotenes.)  J.  Amer.  chem.  Soc.  55.  4728. 
Magnus,  A.,  1922.  (Numbers  of  coordination.)  Z.  anorg.  Chem.  124.  291. 
Manegold,  E.,  1941.  (Systematics  of  gels.)  KoUoid-Z.  96.  186. 
Manton,  I.  and  Clarke,  B.,  1950.  (Spermatozoid  of  Fucus,  electron  microscopy.)  Nature 

166.  973. 
Mark,  H.,  1940.  AUgemeine  Grundlagen  der  hochpolymeren  Chemie.  Leipzig. 
Mark,  H.  and  Philipp,  H.,  1937.  (Proteins,  crystal  lattice.)  Naturwiss.  25.  119. 
Mark,  H.  and  Schossberger,  F.,  1937.  (Organic  compounds,  crystal  structure.)  Ergebn. 

exakt.  Naturwiss.  16.  183. 
Marklund,  G.,  1936.  (Compared  permeabiHty.)  Acta  hot.  fenn.  18. 
Marks,  M.  H.,  Bear,  R.  S.  and  Blake,  C.  H.,  1949.  (Collagen  family.)  J.  exp.  Zool. 

III.  55. 
Marsland,  D.  a.,  1942.  (Cytoplasm,  high  pressure  experiments.)  In  W.  Seifriz,  The 

structure  of  protoplasm.  Ames-Iowa,  p.  127. 
Martens,  P.,  1927/29.  (Karyokinesis,  vital  observation.)  Cellule  38.  67,  39.  167. 
Martens,  P.,  1934.  (Cuticle,  origin.)  Cellule  43.  289. 
Martin,  L.  C,  1938.  (Electron  microscope.)  Nature  142.  1062. 

Matoltsy,  a.  G.  and  Gerendas,  M.,  1947.  (Striated  muscle,  optics.)  Nature  159.  502. 
Matoltsy,  A.  G.,  Gross,  J.  and  Grignolo,  A.,   1951.   (Vitreous  body  of  eye,  beaded 

chains.)  Proc.  Soc.  exp.  Biol.  76.  857. 
Matthey,  R.,  1941.  (Chromosomes,  spiral  structure.)  Arch.  Klaus-Stift.  16.  630. 
Mazia,  D.  and  Jaeger,  L.,  1939.  (Chromosomes,  digestion  by  nuclease.)  Proc.  nat.  Acad. 

Sci.  Wash.  25.  456. 
Meeuse,  a.  D.  J.,  1938.  (Sclerenchyma  fibres,  growth.)  Rec.  Trav.  bot.  neerl.  35.  288. 
Meeuse,  a.  D.  J.,  1941.  (Sliding  growth.)  Diss.  Delft. 
Menke,  W.,  1934a.  (Chloroplasts,  myelin  forms.)  Protoplasma  21.  279. 
Menke,  W.,  1934b.  (Chloroplasts,  double  refraction.)  Protoplasma  22.  56. 
Menke,  W.,  1938a.  (Cytoplasm,  chemistry.)  Z.  Bot.  32.  273. 
Menke,  W.,  1938b.  (Chloroplasts,  method  of  isolation.)  Hoppe  Seyler  Z.  physiol.  Chem. 

257-  43- 
Menke,  W.,  1938c.  (Chloroplasts,  fine-structure.)  Kolloid-Z.  85.  256. 
Menke,  W.,  1940a.  (Chloroplasts,  electron  micograph.)  Protoplasma  35.  115. 
Menke,  W.,  1940b.   (Chloroplasts,  contents  of  chlorophyll  and  ash.)   Hoppe  Seyler  Z. 

physiol.  Chem.  263.  100,  104. 
Menke,  W.,  1940c.  (Carotenoids  of  plastids  as  chromoproteins.)  Naturwiss.  28.  31. 
Menke,  W.,  i94od.  (Chloroplasts,  lamination.)  Naturwiss.  28.  158. 
Menke,  W.,  1943.  (Plastids,  dichroism.)  Biol.  Zbl.  63.  326. 
Menke,  W.,  and  Jacob,  E.,  1942.  (Chloroplasts,  contents  of  lipids.)  Hoppe  Seyler  Z. 

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Menke,  W.  and  Kuster,  H.  J.,  1938.  (Chloroplasts,  dichroism.)  Protoplasma  30.  283. 
Mercer,  E.  H.,  1951.  (Fibroinogen  of  silk.)  Nature  168.  792. 
Mestre,  H.,  1930.  (Phyllochlorin.)  Contr.  Marine  Biol.  Stanford  Univ.  Press. 
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quant.  Biol.  9.  23. 


LITERATURE  3^9 

Metzner,  p.,  1937.  (Chloroplasts,  fluorescence.)  Ber.  dtsch.  bot.  Ges.  55.  (16). 

Meyer,  A.,  1883.  Das  Chlorophyllkom.  Leipzig. 

Meyer,  A.,  1895.  Untersuchungen  iiber  die  Starkelcorner.  Jena. 

Meyer,  K.  H.,  1929.  (Muscle  fibre,  contractibility.)  Biochem.  Z.  214.  253. 

Meyer,  K.  H.,  1930.  (Model  of  micelles.)  Kolloid-Z.  53.  8. 

Meyer,  K.  H.,  1935.   (Theory  of  selective  permeability.)  Lecture  29th  Nov.  Ass.  des 

Chimistes  de  Geneve. 
Meyer,  K.  H.,  1940a.  Die  hochpolymeren  Verbindungen.  Leipzig. 
Meyer,  K.   H.,  1940b.  (Ramified  and  unramified  starch  components.)   Naturwiss.   28. 

397- 
Meyer,  K.  H.,  1943.  (Starch,  review.)  Melliand  Textilbcr.  1943,  No.  3. 

Meyer,  K.  H.,  1951.  (Amylases.)  Angew.  Chem.  63.  153. 

Meyer,  K.  H.  and  Bernfeld,  P.,  1941a.  (Starch,  blue  iodine  reaction.)  Helv.  chim.  Acta 

24.  389. 
Meyer,  K.  H.  and  Bernfeld,  P.,  1941b.  (Potato  starch).  Helv.  chim.  Acta  24.  378. 
Meyer,  K.  H.  and  Bernfeld,  P.,  1941c.  (Action  of  a-amylase.)  Helv.  chim.  Acta  Fest- 

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Meyer,  K.  H.  and  Bernfeld,  P.,  1946.  (Membrane  structure,  potentiometric  analysis.) 

J.  gen.  Physiol.  29.  353. 
Meyer,  K.  H.  and  Brentano,   W.,    1936.    (Corn  starch.)   C.   R.   Soc.   Phys.   Hist.  nat. 

Geneve  53.  iii. 
Meyer,  K.  H.,  Fischer,  E.  H.  and  Bernfeld,  P.,  1947.  (a-amylase,  crystallisation.)  Ex- 

perientia  3.  106,  455. 
Meyer,  K.  H.,  Fischer,  E.  H.  and  Piguet,  A.,  195 1.  (/S-amylase,  crystallisation.)  Helv. 

chim.  Acta  34.  316. 
Meyer,  K.  H.  and  Heinrich,  P.,   1942.   (Starch,  content  of  amylopectin.)  Helv.  chim. 

Acta  25.  1639. 
Meyer,  K.  H.  and  Mark,  H.,  1928.  (Silk  fibroin  as  main  valency  chain  lattice.)  Ber.  dtsch. 

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Meyer,  K.  H.  and  Mark,  H.,  1930.  Der  Aufbau  der  hochpolymeren  organischen  Natur- 

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Meyer,  K.  H.  and  Misch,  L.,  1937.  (Cellulose,  crystal  lattice.)  Helv.  chim.  Acta  20.  232. 
Meyer,  K.  H.  and  Pankow,  G.  W.,  1935.  (Chitin,  crystal  lattice.)  Helv.  chim.  Acta  18.  589. 
Meyer,  K.  H.  and  Picken,  L.  E.  R.,  1937.  (Muscle  fibre,  thermodynamics.)  Proc.  Roy. 

Soc.  London  B  124.  29. 
Meyer,  K.  H.  and  Sievers,  J.  F.,  1936.  (Theory  of  selective  permeability.)  Helv.  chim. 

Acta  19.  649,  665,  948,  987. 
Meyer,  K.  P.,  1939.  (Chlorophyll,  spectroscopy.)  Helv.  phys.  Acta  12.  349. 
Meyer,  M.,  1938.  (Cuticular  layers,  submicroscopic  structure.)  Diss.  E.T.H.  Zurich. 
MiCHAELis,  L.,  1933.  Oxydations-  und  Reduktionspotentiale.  Berlin. 
Michel,  K.,  1940.  Grundziigc  der  Mikrophotographie.  Jena. 
Michener,  H.  D.,  1935.  (Ethylene,  stimulating  substance.)  Science  82.  551. 
Middlebrook,  W.  R.,  195 1.  (Wool  keratin,  chemical  composition.)  Biochim.  Biophys. 

Acta  7.  547- 
Minder,  W.  and  Liechti,  A.,  1945.  (Irradiation,  effects  in  biology.)  Experientia  i.  298. 
MiRSKY,  A.  E.,  1936.  (Formation  of  protein  frame  after  fertihzation.)  Science  84.  333. 
MiRSKY,  A.  E.  and  Pollister,  A.  W.,  1943.  (Fibrous  nucleoproteins.)  Biol.  Symp.  10.  247. 
MiTCHisoN,  J.  M.,  1950a.  (Amoeba,  birefringence.)  Nature  166.  313. 
Mitchison,  J.  AL,  1950b.  (Erythrocyte,  membrane.)  Nature  166.  347. 


590 


LITERATURE 


MiTTASCH,  A.,  1936.  (Catalysis.)  Natunviss.  24.  770. 

MoHRiNG,  A.,  1922.  (Chitin,  rodlet  double  refraction.)  Diss.  Jena.  Summary  in  Ambronn- 

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392 


LITERATURE 


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Pallmann,  H.,  1937.  (Hydration.)  Schweiz.  med.  Wochenschr.  67.  528. 

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Pekarek,  J.,  1932.  (Absolute  viscosity  in  cells.)  Protoplasma  17.  i. 
Pekarek,  J.,  1933.  (Phase  separation  in  cell  sap.)  Protoplasma  20.  251. 
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Biochim.  Biophys.  Acta  2.  674. 
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Perutz,  M.  F.,  1951.  (Polypeptide  chains,  spiral  configuration.)  Nature  167.  1053. 
Perutz,  M.  F.  and  Mitchison,  J.  M.,  1950.  (Erythrocyte,  sickle  shape.)  Nature  166. 

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Pfeiffer,  H.,  1941a.  (Chromosomes,  micro  surgery.)  Chromosoma  2.  77. 
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Pfeiffer,  H.,  1942a.  (Leptones.)  KoUoid-Z.  100.  254. 
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Nature  159.  434. 
Pilnik,  W.,  1946.  (Pectin,  double  refraction  of  flow.)  Diss.  E.  T.  H.  Ziirich. 
Pirschle,  K.,  1930.  (Series  of  ions.)  Jahrb.  wiss.  Bot.  72.  335. 
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LITERATURE  393 

Pratt,  A.  W.  and  Wyckoff,  R.  W.  C,  1950.  (Collagen  fibres,    striation.)    Biochim. 

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Preston,  R.  D.,  1936.  (Phycomyces,  spiral  growth.)  Proc.  Leeds  Phil.  Soc.  3.  327. 
Preston,'  R.  D.,   1946.   (Conifer  tracheids,   fine-structure.)    Proc.    Roy.    Soc.    London 

B  133-  327- 
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Preston,'  R.  D.,  Nicolai,  E.,  Reed,  R.  and  Millard,  A.,  1948.  (Valonia,  microfibrils.) 

Nature  162.  665. 
Priestley,  J.  H.,  1943.  (Cuticle  in  Angiosperms.)  Bot.  Rev.  9.  595. 
Priestley,  J.  H.  and  Irving,  A.  A.,  1907.  (Chloroplasts,  microscopic  structure.)  Ann.  Bot. 

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Prokofyeva  Belgorskaja,  a.  a.,  1948.  (Genes,  screened  by  heterochromatin.)  J.  Genetics 

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Exp.  Cell  Research  I.  I. 
Ranzi,  S.,  1951.  (Viscosimetric  distinction  of  fibrillar  and  globular  proteins.)  Experientia 

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Raven,  Ch.,  and  Bretschneider,  L.  H.,   1942.   (Egg,  stratification  by  centrifiigal  force.) 

Arch.  Neerl.  Zool.  6.  255. 
Reinders,  D.  E.,  1940.  (Water  resorption  depending  on  respiration.)  Diss.   Groningen. 
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Renner,  O.,  1915.  (Cohesion  of  water.)  Jahrb.  wiss.  Bot.  56.  617. 
Reumuth,  H.,  1942.  (Wool,  fine-structure.)  Klepzigs  Textil-Z.  1942,  p.  288. 
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Roelofsen,  P.  A.,  1951b.  (Cotton  hair,  primary  wall.)  Biochim.  Biophys.  Acta  7.  43. 

Roelofsen,  P.  A.  and  Kreger,  D.  R.,  195 i.  (Crystallized  pectin  in  coUenchyma.)  J.  exp. 
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RoTHELi,  A.,  Roth,  H.  and  Medem,  F.,  1950.  (Fish  sperms.)  Exp.  Cell  Research  i.   115. 

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Rothmund,  V.,  1898.  (System  phenol/water.)  Z.  phys.  Chem.  26.  433. 

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594  LITERATURE 

Ro2sa,G.,Szent-Gyorgyi,  A.and  Wyckoff,  R.  W.  G.,  1950.  (Myofibrils,  fine-structure.) 

Exp.  Cell  Research  1.  194. 
RozsA,  G.  and  Wyckoff,  R.  W.  G.,  1950.  (Mitosis,  electron  microscopy.)  Biochim. 

Biophys.  Acta  6.  335. 
RozsA,  G.  and  Wyckoff,  R.  W.  G.,  195 i.  (Cytoplasm,  electron  microscopy.)  Exp.  Cell 

Research  2.  630. 
RucH,  F.,  1945.  (Chromosomes,  spiral  structure.)  Viertelj.  schr.  Natuf.  Ges.  Zurich  90.  214. 
RucH,  F.,  1949.  (Meiotic  chromosome,  spiral  structure.)  Diss.  E.  T.  H.  Zurich;  Chro- 

mosoma  3.  358. 
RuHLAND,  W.,  1912  (Ultrafilter  theory'.)  Jahrb.  wiss.  Bot.  51.  376. 
RuHLAND,  W.,  1950.  (Permeability,  ultrafilter  theory.)  Proc.  VII  th  Int.  Congr.  of  Botany, 

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RuNDLE,  R.  E.,  Daasch,  L.  and  French,  D.,  1944.  (Starch,  X-ray  analysis.)  J.  Amer, 

Chem.  Soc.  66.  130. 
RuNDLE,  R.  E.,  and  Edwards,  F.  C.  J.,   1943.   (Amylose,  helical  structure.)  J.  Amer. 

Chem.  Soc.  65.  2200. 
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J.  Amer.  Chem.  Soc.  66.  21 16. 
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Runnstrom,  J.,  1944.  (Asterias  egg,  fertilization  membrane.)  Acta  Zool.  25.  159. 
Runnstrom,  J.  and  Monne,  L.,   1945.   (Sea  urchin  egg,  surface  changes.)  Ark.  Zool. 

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Runnstrom,  J.,  Monne,  L.  and  Wicklund,  E.,  1946.  (Sea  urchin  egg,  surface  studies.) 

J.  Colloid  Sci.  I.  421. 
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Siemens  Werke  Werkstoft'-Sonderh.  Berlin  1940,  p.  372. 
Ruska,  H.,  1939.  (Electron  microscope,  preparation  technics.)  Naturwiss.  27.  287. 
Rutishauser,  E.,  Huber,  L.,  Kellenberger,  E.,  Majno,  G.  and  Rouiller,  Ch.,  1950. 

(Bone,  electron  microscopy.)  Arch.  Sci.  (Geneve)  3.  175. 
Sadron,  G.,  1937.  (Double  refraction  of  flow,  theory.)  J.  Phys.  Radium  8.  481. 
Sakurada,  I.  and  Hutino,  O.,   1933.   (Silk  fibroin,  crystal  lattice.)  Sc.  Pap.    I.P.C.R. 

21.  266. 
Samec,  M.,  1927.  Kolloidchemie  der  Starke.  Dresden. 

Samec,  M.,  1942.  Die  ncuere  Entwicklung  der  Kolloidchemie  der  Starke.  Dresden. 
Samec,  M.,  1942/43.  (Starch,  review.)  Kolloidchem.  Beih.  54.  147,  177,  435. 
Sauter,  E.,  1937.  (Cellulose,  X-ray  diffraction.)  Z.  phys.  Chem.  B  35.  83,  117. 
Scarth,  G.  W.,  1924.  (Chloroplasts,  double  refraction.)  Quart.  J.  exp.  Physiol.  14.  99. 
ScARTH,  G.  W.,  1927.  (Plasm,  microsurgery.)  Protoplasma  2.  189. 
Scarth,  G.  W.,    1942.   (Cytoplasm,  fine-structure.)    In  W.   Seifriz,    The   structure   of 

protoplasm.  Ames-Iowa,  p.  99. 
ScHAEDE,  R.,  1927.  (Protoplasm,  microscopic  structure.)  Protoplasma  3.  145. 
Schauenstein,  E.  and  Stanke,  D.,  1951.  (Collagen,  UV  absorption.)  Makromol.  Chemie 

5.  262. 
ScHEiBE,  G.,  1948.  (Protein  crystals.)  Naturwiss.  35.  168. 
Scherrer,  a.,  1914.  (Chloroplasts  and  chondriosomes.)  Diss.  Univ.  Zurich. 
Scherrer,  p.,  1920.  (Particle  size  measured  by  X-rays.)  Kolloidchemie  von  Zsigmondy, 

3.  Aufl.  p.  387. 
Schimper,  a.  F.  W.,  1885.  (Chloroplasts,  grana  structure.)  Jahrb.  wiss.  Bot.  16.  i. 
Schmidt,  O.,  1942.  (Nerves,  conduction  of  stimuli.)  Naturwiss.  30.  644. 
Schmidt,  W.  J.,  1923.  (Tooth  enamel,  double  refraction.)  Ber.  niederrhein.  Ges.  Natur- 

kunde  Jahrg.  1923,  Bonn  1925. 


LITERATURE  395 

Schmidt,  W.  J.,  1924.  Die  Bausteine  des  Tierkorpers  im  polarisierten  Licht.  Bonn. 

Schmidt,  W.  J.,  1928.  (Chromatin,  double  refraction.)  Zool.  Jalirb.  45.  177. 

Schmidt,  W.  J.,   1934.   (Submicroscopic  structure  of  cells  and  tissues.)   Handb.  biol. 

Arbeitsmeth.  v.  Abderhalden,  Abt.  V.  Teil  10,  p.  435- 
Schmidt,  W.  J.,  1936.  (Ascaris,  envelope  of  ovum.)  Z.  Zellforsch.  25.  181. 
Schmidt,  W.  J.,  1936/37.  (Tooth  enamel,  double  refraction.)  Naturwiss.  24.  361 ;  25.  283. 
Schmidt,  W.  J.,  1937a.  Die  Doppclbrechung  von  Karyoplasma,  Zytoplasma  und  Meta- 

plasma.  Berlin. 
Schmidt,  W.  J.,  1937b.  (Nerve  sheath,  fine-structure.)  Z.  wiss.  Mikr.  54.  159. 
Schmidt,  W.  J.,  1937c.  (Chromosomes,  double  refraction.)  Naturwiss.  25.  506. 
Schmidt,  W.  J.,  1938.  (Form  double  refraction  in  sols.)  Z.  wiss.  Mikr.  55.  476. 
Schmidt,  W.  J.,  1939a.  (Nuclear  spindle,  double  refraction.)  Chromosoma  i.  253. 
Schmidt,  W.  J.,  1939b.  (Anisotropic  chromosomes,  microtechnics.)  Z.  wiss.  Mikr.  56.  i. 
Schmidt,  W.  J.,  1939c.  (Nuclear  membrane,  double  refraction.)  Protoplasma  32.  193. 
Schmidt,  W.  J.,  1941a.  (Chromosomes,  double  refraction.)  Chromosoma  2.  86. 
Schmidt,  W.  J.,  1941b.  (Protplasm,  double  refraction.)  Ergebn.  Physiol,  biol.  Chem.  u. 

Pharmakol.  44.  27. 
ScHMiTT,  F.  O.,  1936.  (Nerve  fibre,  ultrastructure.)  Cold  Spr.  Harb.  Symp.  quant. Biol.4. 7. 
Schmitt,  F.  O.,  1938.  (Nuclear  membrane,  double  refraction.)  J.  appl.  Phys.  9.  109. 
ScHMiTT,  F.  O.,  1950a.  (Muscle,  fine-structure.)  Biochim.  Biophys.  Acta  4.  68. 
Schmitt,  F.  O.,  1950b.  (Nerve,  myelin  sheath.)  Mult.  Sclerosis  &  Desmyel.  Diseases 

28.  247. 
Schmitt,  F.  O.  and  Bear,  R.  S.,  1937.  (Nerve  axons,  optics.)  J.  cell.  comp.  Physiol  9.  261. 
Schmitt,  F.  O.  and  Bear,  R.  S.,  1939.  (Nerve  axon  sheath,  fine-structure.)  Biol.  Rev. 

14.  27. 
Schmitt,  F.  O.,  Bear,  R.  S.  and  Ponder,  E.,  1936.  (Erythrocytes,  optics.)  J.  cell.  comp. 

Physiol.  9.  89. 
Schmitt,  F.  O.,  Bear,  R.  S.  and  Ponder,  E.,  1938.  (Erythrocytes  as  composite  bodies.) 

J.  cell.  comp.  Physiol.  11.  309. 
Schmitt,  F.  O.,  Hall,  C.  E.,  and  Jakus,  M.  A.,  1942.   (Collagen,  electron  microscopy.) 

J.  cell.  comp.  Physiol.  20.  11. 
Schmitt,  F.  O.,  Hall,  C.  E.,  and  Jakus,  M.  A.,  1943.  (Protoplasmic fibrils,  fine-structure.) 

Biol.  Symp.  10.  261. 
Schmitt,  F.  O.,  Hall,  C.  E.,  and  Jakus,  M.  A.,  1945.  (Collagen  fibrils,  macroperiod.) 

J.  appl.  Phys.  16.  263. 
Schmitt,  F.  O.  and  PaImer,  K.  J.,  1940.  (Water  absorption  by  myelin  substances.)  Cold 

Spr.  Harb.  Symp.  quant.  Biol.  8.  94. 
Schmitt,  F.  O.    and    Wade,    L.    J.,    1935.    (Nerve,    thermal    shortening.)    Amer.    J. 

Physiol.  III.  159. 
Schmucker,  Th.,  1930.  (Assimilation,  quantum  efficiency.)  Jahrb.  wiss.  Bot.  73.   824. 
Schneider  et  al.,  1936.  (Pectin,  esterification.)  Ber.  dtsch.  chem.  Ges.  69.  2530,  2537- 
Schoch-Bodmer,  H.,  1936.  (Filaments,  elongation.)  Planta  25.  660. 
Schopfer,  W.  H.,  1936.  (Growth  factors.)  Arch.  Mikrobiol.  7.  571. 
ScHOPFER,  W.  H.,  1937.  (Growth  factors.)  Ber.  schweiz.  bot.  Ges.  47.  460. 
ScHULTZ,  J.,  1941.  (Nucleic  acids,  importance  in  chromosomes.)  Cold  Spr.  Harb.  Symp. 

quant.  Biol.  9.  55. 
ScHULZ,  G.  v.,  1936.  (Chain  length  of  macromolecules,  osmotic  determination.)  Z.  phys. 

Chem.  A  176.  317. 
ScHULZ,  G.  v.,  1939.  (Criticism  of  junction  theory.)  Z.  angew.  Chem.  52.  498. 
ScHiJRHOFF,  P.  N.,  1924.  Die  Plastiden.  Berlin. 
Schuringa,G.  J.andAi,GERA,L.,  1950.  (Wool, epidermis.)  Biochim.  Biophys.  .'\cta6.  325. 


596  LITERATURE 

Scott,  D.  B.  and  Wyckoff,  R.  W.  G.,  1946/47.  (Teeth,  electron  microscopy.)  U.S.A. 
Public  Health  Rep.  61.  697,  1397;  62.  1513. 

Seifriz,  W.,  1927.  (Erythrocytes,  physical  properties.)  Protoplasma  i.  345. 

Seifriz,  W.,  1929.  (Cytoplasm,  contractility.)  Amer.  Nat.  63.  410. 

Seifriz,  W.,  1936.  Protoplasm.  New  York  and  London. 

Seifriz,  W.,  1937.  (Protoplasmic  flow.)  Science  86.  397. 

Seifriz,  W.,  1938.  (Protoplasm,  fine-structure.)  Science  88.  21. 

Seifriz,  W.,  1942.  (Protoplasm,  physical  properties.)  The  structure  of  protoplasm.  Mono- 
graph Amer.  Soc.  Plant  Physiol.  Ames-Iowa,  p.  245. 

Seifriz,  W.,  1943.  (Protoplasm,  streaming.)  Bot.  Rev.  9.  49. 

Seifriz,  W.,  1945a.  (Protoplasm,  stnacture.)  Bot.  Rev.  11.  231. 

Seifriz,  W.,  1945b.  (Torsion  in  protoplasm.)  J.  Colloid  Sci.  i.  27. 

Seifriz,  W.,  1950.  (Anesthesis  of  protoplasm.)  Anesthesiol.  11.  24. 

Seifriz,  W.  and  Plowe,  J.Q.,  1931.  (Cytoplasm,  extensibility.)  J.  Rheology  2. 

Semmens  and  Bhaduri,  1939.  (Nucleoli,  differential  staining  with  methyl  green.)  Stain 
Tech.  14.  I. 

Senn,  G.,  1908.  Gestaltveranderungcn  der  Pflanzenchromatophoren.  Leipzig. 

Senti,  F.  R.  and  Witnauer,  L.  P.,  1946.  (A-starch,  X-ray  analysis.)  J.  Amer.  Chem.  Soc. 
68.  2407. 

Serra,  J.  A.  and  Queiroz-Lopes,  A.,  1944.  (Nucleolus,  basic  proteins.)  Chromosoma 
2.  576. 

Seybold,  a.,  1941.  (Leaf  pigments.)  Bot.  Arch.  42.  254. 

Seybold,  a.  and  Egle,  K.,  1940.  (Chlorophyll,  physical  state  in  chloroplast.)  Bot.  Arch, 

41.  578. 

Seybold,  A.,  Egle,  K.  and  Hulsbruch,  W.,  1941.  (Chlorophyll  in  algae.)  Bot.  Arch. 

42.  239. 

Sharp,  W.  L.,  1934.  An  introduction  to  cytology.  3rd  ed.  New  York. 

Siedentopf,  H.  and  Zsigmondy,  K.,  1903.  (Ultramicroscope.)  Ann.  Phys.  Lpz.  (4)  10.  i. 

Signer,  R.,  1930.  (Double  refraction  of  flow.)  Z.  phys.  Chem.  A.  150.  257. 

Signer,  R.,  1933.  (Double  refraction  of  flow.)  Z.  phys.  Chem.  A  165.  161. 

Signer,  R.,  Caspersson,  T.  and  Hammarsten,  E.,  193S.  (Nucleic  acids,  double  refraction 

of  flow.)  Nature  141.  122. 
Signer,  R.  and  Gross,  H.,  1933.  (Double  refraction  of  flow.)  Z.  phys.  Chem.  A  165.  161. 
Sjostrand,  F.  S.,  1949.  (Retinal  rods,  layering.)  J.  cell.  comp.  Physiol.  33.  383. 
Smith,  J.  H.  C,  1936.  (Carotins,  light  absorption.)  J.  Amer.  chem.  Soc.  58.  247. 
Smith,  J.  H.  C,  1937.  (Leaf  pigments.)  Ann.  Rev.  Biochem.  6.  489. 
Snellman,  O.  and  BjornstAhl,  Y.,  1941.  (Birefringence  of  flow.)  Kolloid.  Beih.  52.  403. 
Snellman,  O.  and  Erdos,  T.,   1948.   (Myosin,  molecular  weight.)  Biochim.  Biophys. 

Acta  2.  650. 
Snellman,  O.  and  Erdos,  T.,  1949.  (F-actomyosin,  actin-myosin  quotient.)   Biochim. 

Biophys.  Acta  3.  523. 
Snellman,    O.    and    Gelotte,    B.,   1950.   (Muscle  fibre,  myosin  extracted.)  Exp.  Cell 

Research  i.  234. 
Soding,  H.,  1 93 1.  (Cell  wall,  extensibility.)  Jahrb.  wiss.  Bot.  74.  127. 
SoDiNG,  H.,  1934.  (Cell  wall  structure  of  growing  cells.)  Jahrb.  wiss.  Bot.  79.  231. 
SoLLNER,  K.,  1950.  (Selective  permeability.)  J.  Electrochem.  Soc.  97.  139. 
Speich,  H.,  1941.  (Starch  grains,  optics.)  Diss.  E.  T.  H.  Zurich. 

Spemann,  H.,  1936.  Experimentelle  Beitrage  zu  einer  Theorie  der  Entwicklung.  Berlin. 
Spoehr,  H.  a.,  1935.  (Leaf  pigments.)  Year  Book  Carnegie  Inst.  Wash.  34.  192. 
Sponsler,  O.  L.,  1922.  (Starch,  X-ray  diff'raction.)  Amer.  J.  Bot.  9.  471. 
Sponsler,  O.  L.,  1923.  (Starch,  crystal  lattice.)  J.  gen.  Physiol.  5.  757. 


LITERATURE  597 

Sponsler,  O.  L.  and  Bath,  J.  D.,  1942.  (Protoplasm,  molecular  structure.)  In  W.  Seifriz, 
The  structure  of  protoplasm.  Ames-Iowa,  p.  41. 

Sprecher,  a.,  Heierle,  E.  and  Almasi,  F.,  1955-  (Leaf  pigments,  quantitative  spectrosco- 
py.) Biochem.  Z.  283.  45. 

Stanley,  W.  M.,  1935.  (Crj'stallized  virus  protein.)  Science  81.  644. 

Stanley,  W.  M.,  1936.  (Crystallized  virus  protein.)  J.  biol.  Chem.  115.  673. 

Stanley,  W.  M.,  1938a.  (Virus  protein.)  Handb.  d.  Virusforsch.  v.  Doerr  and  Hallauer 

Bd.  I,  p.  447,  Wien. 
Stanley,  W.  M.,  1938b.  (Progress  in  virus  investigation.)  The  Sigma  Xi  Lextures.  New 

Haven  (Yale  Univ.  Press). 
Staudinger,  H.,  1932.  Die  hochpolymeren  organischen  Verbindungen.  Berlin. 
Staudinger,  H.,  1936a.  (Macromolecular  chemistry.)  Z.  angew.  Chem.  49.  801. 
Staudinger,  H.,  1936b.  (Polymer  homologous  cellulose  series.)  ZellstofF-Faser  1956,  Heft 

Nov./Dez. 
Staudinger,  H.,  1937a.  (Cellulose,  constitution.)  Svensk  kem.  Tidskr.  49.  3. 
Staudinger,  H.,  1937b.  (Cellulose,  starch,  glycogen.)  Naturwiss.  25.  673. 
Staudinger,  H.  and  Husemann,  E.,  1937.  (Starch,  constitution.)  Lieb.  Ann.  527.  195. 
Staudinger,  H.  and  Sorkin,  M.,  1937a.  (Cellulose,  h^^drolysis.)  Ber.  dtsch.  chem.  Ges. 

70.  1565. 
Staudinger,  H.  and  Sorkin,  M.,  1937b.  (Nitrocellulose.)  Ber. dtsch.  chem.  Ges.  70.  1993. 
Staudinger,  H.,  Staudinger,  M.  and  Sauter,  E.,   1937.   (Synthetic  high  polymers, 

microscopic  structure.)  Z.  phys.  Chem.  B  37.  403. 
Steinbrinck,  C,  1906.  (Shrinking  and  cohesion.)  Biol.  Zbl.  26.  637. 
Steward,  F.  C,  1932.  (Salt  resorption  and  respiration.)  Protoplasma  15.  497. 
Steward,  F.  C,  1933.  (Salt  resorption  and  respiration.)  Year  Book  Carnegielnst.  Wash. 

32.  281. 
Stocker,  O.,  1928.  (Halophytes.)  Ergebn.  Biol.  3.  265. 
Stoll,  a.,  1936.  (Chlorophyll,  constitution.)  Naturwiss.  24.  53. 
Stoll,  a.  and  Wiedemann,  E.,  1941.  (Chloroplastin.)  Verb.  Schweiz.  Naturf.  Ges.  Basel 

1941,  p.  125. 
Straub,  F.  B.,  1948.  (Actin,  molecular  weight.)  Hung.  Acta  Physiol,  i.  50. 
Straub,  J.,  1938.  (Chromosomes,  spiral  structure.)  Z.  Bot.  33.  65. 
Straub,  J.,  1943.  (Chromosomes,  structure.)  Naturwiss.  31.  97. 
Strugcjer,  S.,  1935/36.  (Vital  staining.)  Protoplasma  24.  108,  26.  56. 
Strugger,  S.,  1936/37.  (Chloropjasts,  vital  staining.)  Flora  31.  113.  324. 
Strugger,  S.,  1949.  Fluoreszenzmikroskopie  und  Mikrobiologie.  Hannover. 
Strugger,  S.,  1950.  (Proplastids.)  Naturwiss.  37.  166. 

Strugger,  S.,  195 i.  (Chloroplast,  microscopic  structure.)  Ber.  dtsch.  bot.  Ges.  64.69. 
Stuart,  H.  A.,  1934.  Molekiilstruktur.  Berlin. 
Stuart,  H.  A.,  1941.  (Short-range  order.)  Kolloid-Z.  96.  149. 
Stubel,  H.,  1923.  (Muscle  fibre,  rodlet  double  refraction.)  Pfliig.  Arch.  201.  629. 
Svedberg,  Th.,  1930.  (Protein  particles.)  Kolloid-Z.  51.  10. 
Svedberg,  Th.,  1931.  (Reserve  protein,  molecular  weight.)  Nature  127.  438. 
Svedberg,  Th.,  1938a.  (Ultracentrifuge.)  Industr.  Engng  Chem.  10.  113  (analyt.  edition). 
Svedberg,  Th.,  1938b.  (Proteins,  multiple  law.)  Kolloid-Z.  85.  119. 
Szent-Gyorgyi,  a.,  1943.  (Crystallized  myosin).  Studies  Inst.  Med.  Chem.  Univ.  Szeged 

3.  76. 
Takahashi,  W.  N.  and  Rawlins,  T.  E.,  1933,  1935.  (Virus  protem,  double  refraction  of 

flow.)  Science  77.  26,  284;  81.  299. 
Takahashi,  W.  N.  and  Rawlins,  T.  E.,  1948.  (Tobacco  mosaic  virus,  length.)  Phytopath. 
38.  279. 


398  LITERATURE 

Tavel,  p.,  1939.  (Cellulose,  ester  with  dicarbonic  acids.)  Diss.  Bern. 

Taylor,  G.  W.,  1942.  (Nerve,  conduction.)  J.  cell.  comp.  Physiol.  20.  359. 

Teorell,  T.,  1935.  (Selective  permeability,  theory.)  Proc.  Soc.  exp.  Biol.  N.Y.  33.  282. 

Thimann,  K.  v.,  1936.  (Growth  hormones,  chemistry.)  Curr.  Sci.  4.  716. 

Thimann,  K.  V.  and  Bonner,  J.,  1933.  (Cell  walls  in  meristems,  chemistry.)  Proc.  Roy. 

Soc.  London  B  113.  126. 
Thimann,  K.  V.  and  Bonner,  J.,  1938.  (Growth  hormones,  chemistry.)  Physiol.  Rev. 

18.  524. 
Thorell,  B.,  1948.  (Erythrocytes,  nucleic  acids.)  Cold  Spring  Harbour  Symp.  quant. 

Biol.  12.  247. 
TiMM,  E.,  1942.  (Chloroplasts,  amino  acids.)  Z.  Bot.  38.  i. 

Timofeeff-Ressovsky,  N.  W.,  1940.  (Mutation,  biophysical  analysis.)  Nova  Acta  Leo- 
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Tischler,  G.,  1921/22.  Allgemeine  Pflanzenkaryologie.  Berlin. 
ToRNAVA,  S.  R.,  1939.  (Plasmalemma,  capacity  of  expansion.)  Protoplasma  32.  329. 
Trillat,  J.  J.,  1925/27.  (Bimolecular  films.)  C.r.  Acad.  Sci.  Paris  180.  1838,  184.  812. 
Trurnit,  H.  J.,  1950.  (Formvar  film,  penetrability  of  tr^'psin.)  Science  112.   329. 
Tupper-Carey,  R.  M.  and  Priestley,  J.  H.,  1923.  (Cell  walls  of  meristems.)  Proc.  Roy. 

Soc.  London  95.  109. 
Ullrich,  H.,  1934.  (Anion  permeability.)  Planta  23.  146. 
Ullrich,  H.,  1936a.  (Protoplasm,  double  refraction.)  Planta  26.  311. 
Ullrich,  H.,  1936b.  (Cytoplasm  boundary  structure.)  Protoplasma  26.  183. 
Ullrich,  H.,  1941.  (Freezing  of  gels.)  KoUoid-Z.  96.  348. 
Ursprung,  a.,  1915.  (Cohesion  of  water.)  Ber.  dtsch.  bot.  Ges.  33.  153. 
Van  de  Sande  Bakhuizen,  H.  L.,  1925.    (Starch  grains,   without  layers.)   Proc.  Soc. 

Exp.  Biol.  N.Y.  23.  302. 
Van  Iterson,  G.,  1927.  (Cell  wall,  formation.)  Chem.  Weekbl.  24.  166. 
Van  Iterson,  G.,  1933.  (Cellulose  symposium.)  Chem.  Weekbl.  30.  i. 
Van  Iterson,  G.,  1935.  (Cell  wall,  fine-structure.)  Proc.  IV.  Int.  Bot.  Congr.  Amsterdam 

2.  291. 
Van  Iterson,  G.,  1937.  (Protoplasmic  flow  and  cell  wall  texture.)  Protoplasma  27.  190. 
Van  Iterson,  G.,  Meyer,  K.  H.,  and  Lotmar,  W.,  1936.  (Plant  chitin.)  Rec.  Trav.  Chim. 

Pays-Bas  55.  61. 
Vermaas,  D.,  1941.  (Adsorption  double  refraction.)  Diss.  Utrecht. 
Vermaas,  D.,  1942.  (Adsorption  double  refraction.)  Z.  phys.  Chem.  B  52.  131. 
Verzar,  F.,  1943.  Theorie  der  Muskelkontraktion.  Rektoratsprogramm  der  Univ.  Basel. 
Virtanen,  a.  I.,  1948.  (Cytoplasm,  enzymes.)  Oral  communication. 
Vischer,  E.  and  Chargaff,  E.,  1948.  (Purines  and  pyrimidines,  chromatography.)  J.  Biol. 

Chem.  176.  703. 
Vles,  F.,  1911.  Proprietes  optiques  des  muscles.  Paris. 
Vogel,  a.,  1950.  (Pollen  tubes,  electron  microscopy.)  Diplomarbeit  am  Pflanzenphysiol. 

Institut  der  E.T.H.,  unpublished. 
Vorlander,  D.,  1936.  (Supracrystalline  compounds.)  Naturwiss.  24.  113. 
Wakkie,  J.  G.,  1935.  (Chlorophyll,  light  absorption.)  Diss.  Leiden. 
Walchli,  O.,  1945.  (Cellulose,  staining  with  congored.)  Diss.  E.T.H.  Zurich. 
Warburg,  O.  and  Negelein,  E.,  1923.  (Assimilation,  quantum  efficiency.)   Z.   phys. 

Chem.  106.  191. 
Wassink,  E.  C,  Vermeulen,  D.,  Reman,  G.  H.  and  Katz,  E.,   1938.  (Chlorophyll, 

fluorescence.)  Enzymologia  5.  100. 
Waugh,  D.  F.,  1950.  (Erythrocyte,  membrane.)  Ann.  New.  York  Acad.  Sci.  50.  835. 
Weber,  E.,  1942.  (Plant  waxes,  Qptics.)  Diss.  E.T.H.  Zurich. 


LITERATURE  399 

Weber,  F.,  1921.  (Cell  sap,  viscosity.)  Ber.  dtsch.  bot.  Ges.  39.  188. 

Weber,  F.,  1932.  (Plasmalemma,  tonoplast.)  Protoplasma  15.  453. 

Weber,  F.,  1933.  (Chloroplast,  myelin  forms.)  Protoplasma  19.  455. 

Weber,  F.,  1936.  (Chloroplasts,  grana.)  Molisch-Festschr.  (Wien),  p.  447. 

Weber,  F.,  1937.  (Anisotropic  plastids.)  Protoplasma  27.  280,  460. 

Weber,  H.  H.,  1934.  (Artificial  myosin  fibres,  optics.)  Pfliig.  Arch.  235.  205. 

Weichsel,  G.,  1936.  (Potato  starch,  resistance  to  enzymes.)  Planta  26.  28. 

Weier,  E.,  1938.  (Chloroplasts,  review.)  Bot.  Rev.  4.  497. 

Weiss,  P.,  1939.  Principles  of  development.  New  York. 

Went,  F.  W.  and  Thimann,  K.  V.,  1937.  Phytohormones.  New  York  and  London. 

Wergin,  W.,  1937.  (Primary  cell  wall,  optics.)  Naturwiss.  25.  830. 

Wergin,  W.,  1942.  (Cellulose  fibres,  electron  micrograph.)  KoUoid-Z.  98.  131. 

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400  LITERATURE 

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AUTHOR  INDEX 


Abbe,  I,  119 

Abe,  188 

Adam,  49,  143 

Algera,  256,  257,  338 

Almasi,  250 

Ambronn,  4,  76,  77,  85,  87, 
89,  90,  96,  loi,  293,  298, 
308,  362 

Anderson,  293,  307,  316 

Andresen,  156 

Ardenne,  v.,  116,  120,  123, 
124,  127 

Arisz,  199,  288 

Arnold,  261 

Arnon,  162 

Aronoff,  251 

Ascenzi,  350 

Astbun,-,  158,  215,  229,  327, 
328,  336,  339'  340,  342, 
344,  346,  352,  353.  355, 
356,  357,  364,  365,  366 

Atkin,  366 

Baas  Becking,  252 

Badenhuizen,  315,  316,  318 

Bahr,  349,  369 

Bailey,  328,  358,  369 

Baker,  222 

Baldwin,  312 

Baltzer,  179 

Bank,  24 

Barlund,  199 

Bartholome,  146 

Bates,  312 

Bath,  132 

Baud,  217 

Beams,  195,  222 

Bear,  56,  86,  87,  270,  271, 

319,  348,  361,  363,  364,  366, 

367,  368 
Becke,  315 
Becker,  192,  218,  219,  230, 

281 
Belar,  222 

Bell,  215,  229,  339,  340 
Bensley,  172,  173,  184 
Berger,  240 


Bergman,  B.,  247,  261 
Bergmann,    AI.,    134,    142, 

335,  337 
Bernal,  127,  138,  185,  241, 

527 
Bernfeld,  205,  311,  314,  315 

Bernhard,  176 

Bersin,  180,  208 

Berthold,  184 

Bessis,  172,  174,  176,  273 

Bettelheim,  569 

Beutner,  203 

Beyer,  256 

Bhaduri,  221 

Biedermann,  306 

Binz,  244 

Biss,  176,  186 

Bjomstahl,  92 

Blackman,  261 

Bladergroen,  156,  158 

Blake,  364,  566,  367,  368 

Blank,  287 

Block,  343 

Boehm,  90,  355,  361 

Bogen,  205 

Boissonnas,  313 

Bolduan,  348,  368 

Bonner,  22,  201,  209,  280, 

281,  282,  288,  292 
Borries,  v.,  116 
Bosshard,  286 
Bot,  246 
Bottini,  80 
Bougault,  296 
Bouin,  174,  222 
Bourdier,  296 
Bourne,  173 
Bragg,  97 

Brandenberger,  350 
Brauner,  149,  199,  297 
Brenner,  173 
Brcntano,  314 
Bretschneider,      122,      174, 

175,  274,  275,  277 
Bricka,  174,  176,  273 
Brill,  337 
Brown,  D.E.S.,  164,  172 


Brown,  G.  L.,  217 

Brown,  H.  P.,  276 

Broyer,  199 

Bull,  51 

Bungenberg   de    Jong,    18, 

19,  20,  22,   23,   24,   265, 

266,  267,  268,  269 
Burstrom,  199,  288 
Burton,  115 
Biissem,  81 
Biitschli,  182 

Callan,  217,  218 

Caspersson,  173,  212,  213, 
214,  219,  220,  221,  227, 
229,  238,  239,  353,  357 

Castle,  289,  290,  304,  305, 
308 

Chambers,  163,  199 

Champetier,  346,  366 

Champy,  174 

Chargaff,  214 

Chibnall,  246,  292,  295,  296 

Chodat,  170 

Cholodny,  1 5 1 

Christiansen,  286 

Clark,  327 

Clarke,  276 

Claude,  122,  172,  173,   174 

Cohn,  134 

Collander,  197,  198,  199 

Comar,  246 

Corey,  241 

Correns,  290 

Corrigan,  327 

Crick,  171 

Crowfoot,  327,  328 

Curtis,  199 

Czaja,  100 

Daasch,  319 
Danielli,  197,  199,  200 
Danon,  122 
Davson,  197 
Debye-Scherrer,     99,     100, 

loi,  104,  106,  319,  327 
De  Leeuw,  105 


402 


AUTHOR   INDEX 


Denny,  210 

Derkscn,  318,  344 

De  Rooy,  366 

Dervichian,  263,  269 

Dettmer,  349 

Deuel,  60,  64,  68,  73,  201 

Devaux,  5 1 

Dickinson,  328,  355,  356 

Diehl,  301,  309 

Dobry,  21 

Donnan,  75,  202,  203,  204 

Doutrelignc,  243,  244 

Drawert,  161,  193,  205 

Drucker,  335 

Dumpert,  312 

Ebner,  v.,  347 
Eckerson,  317 
Edsall,  134,  354 
Edwards,  312,  320 
Egle,  251,  252 
Einstein,  10 
Elod,  342 
Elvers,  231 
Emerson,  261 
Engel,  280 
Engstrom,  226,  363 
Erdos,  352,  358 
Erickson,  215 
Ernst,  A.,  256 
Ernst,  R.,  217 
Euler,  v.,  247,  261 
Evans,  222 
Eymers,  261 

Fajans,  30 

Fankuchen,  127,  185,  327 

Farr,  317 

Faure-Fremiet,    172,    173, 

174,  346,  359,  366 
Fehling,  310 
Felix,  211 
Fernandez-Moran,  362,  363, 

364 
Feulgen,  176,  214,  215,  221, 

224,  226,  227 
Fischer,  A.,  275 
Fischer,  E.  H.,  314 
Fischer,  F.  G.,  213,  214 
Fischer,  H.,  248 
Fitting,  210 
Flaig,  363 

Flcmming,  182,   192,  255 
Foster,  93,  276,  312 
Fournet,  263,  269 
Franck,  261 
Frank,  35 1 


French,  312,  319 
Freudenberg,  33,  312- 
Freundlich,   9,    18,   42,   66, 

.172 
Frey,  R.,  128,  301,  308 
Fricke,  268 
Friedel,  52 
Friedlfinder,  275 
Friedrich-Freksa,   337,    366, 

368 
Fritz,  293 
FuUam,  122,  173,  174 

Gaffron,  261 

Gaumann,  301 

Gause,  207 

Gautier,  176 

Geitler,  217,  225,  240,  243 

Gelotte,  354 

Gerendas,  354,  358 

Gerngross,  69,  345 

Gettner,  122 

Gibbons,  313 

Gibbs-Thomson,  44,  48 

Gicklhorn,  55,  221 

Giroud,  173 

Goebel,  i 

Goldacre,  186 

Goldschmidt,  29,  148,  149 

Gorter,  51,  143,  268,  270 

Gosling,  228 

Gough,  263,  264 

Graham,  8,  326 

Gralen,  338 

Granick,  245,  256,  257,  258, 

265 
Grendel,  268,  270 
Grignolo,  176 
Gross,  H.,  9 
Gross,  J.,  176,  348,  351 
Guilliermond,   8,    173,    191, 

243,  244,  245 
Guinier,  263,  269 
Gundermann,  280,  292 

Haas,  354 

Haase-Bessel,  236 

Hadorn,  180 

Hagen-Poiseuille,  63,  65,  66 

Hakansson,  222 

Hall,    1 18,    348,    349,    356, 

357,358,368 
Halle,  34,  346 

Hammarsten,  214,  220,  229 
Hanes,  312,  314 
Hanson,  316,  317 
Hansteen-Cranner,  292 


Harder,  301 

Hargitay,  359 

Harris,  216 

Hartzog,  61 

Harvey,  E.  B.,  195,  223,  243 

Harvey,    E.    N.,    165,    166, 

194,  200 
Haurowitz,  331 
Hausermann,  295 
Haworth,  38 
Hecht,  170 
Hegetschweiler,  334 
Heidenreich,  120 
Heierle,  250,  251 
Heilbronn,  167,  168,  169 
Heilbrunn,    146,    147,    163, 

Heinrich,  314 

Heitz,   217,   219,   222,   225, 

226,  239,  243,  244,  245, 

257,  258,  261 
Hellstrom,  247,  261 
Helly,  174 

Hengstenberg,  34,  98 
Heringa,  344 
Hermans,     105,     106,     107, 

108,   109,   no,   112,   113, 

114 
Herrmann,  69 
Hertwig,  176 
Herzog,  78 
Hess,  121,  280,  292 
Heuberger,  121 
Hevn,  280,  287 
Hill  3  59 

Hillier,  116,  119,  122 
Hirschler,  210,  240 
Hoagland,  199 
Hober,  41,  43,  148,  202 
Hoerr,  173 
Hofler,   184,   197,   198,  200, 

205 
Hofmeister,    148,    151,    153, 

199,  243 
Hogeboom,  173 

Hohnel,  v.,  338, 

Holden,  2 1 5 

Holmstrom,  198 

Hotchkiss,  173 

Houwink,  276 

Huber,  350 

Hubert,  243,  252 

Hughes,  171 

Hiilsbruch,  251 

lliirthle,  353 

Husemann,     11,     118,    311, 

317 


AUTHOR   INDEX 


40 ; 


Huskins,  226 
Husted,  124 
Hutino,  337 
Huxley,  359 

Induni,  116,  117 
Irvine,  313 
Irving,  245 
Itschner,  302 

Jaag,  301 

Jaccard,  95 

Jacob,  247 

Jaeger,  227 

Jakus,   118,   348,   349,   356, 

357,  358,  368 
Jaloveczky,  323 
Jeener,  173 
Jensen,  275 

Joly,  51,  93>  176 
Jordan,  329,  331 
Jorpes,  265 
Jung,  264,  269,  273 

Kamiya,  187,  188 

Karrer,  208,  249,  250,  265, 

300 
Karstens,  256 
Katz,  E.,  251 
Katz,  J.  R.,  Ill,  316,  317, 

318,  345,  366 
Kausche,  256,  257 
Kekule,  58 
Kelansy,  317 
Kellenberger,  122,  350 
Kellner,  219 
Kerr,  307,  316 
Kiesel,  140,  183,  210,  211, 

214.,  238 
Kiessig,  280 
Kinsinger,  119 
Knapp,  215 
Knoch,  369 
Koepfli,  209 
Kohl,  1 1 5 
Kohler,  i 
Konig,  369 
Koning,  252 
Kopp,  31 

Kopscli-Regaud,  174 
Kossel,  211 
Koydl,  256 
Kozbial,  230 
Kratky,    78,    89,    106,    107, 

108,   115,  312,  337,  366, 

368 
Kreger,  280,  295,  319,  320 


Kuhn,  R.,  250 
Kuhn,  W.,  359 
Kundt,  253,  298 
Kiintzel,  72,  347 
Kuriyama,  337,  366 
Kiister,  171,  194,  220,  221, 

243,  244,  253,  256 
Kuwada,  230 

Lagermalm,  344 

Lamm,  314 

Landolt-Bornstein,  169 

Langelaan,  353 

Langmuir,  49 

Laue,  V.  98 

Lea,  232 

Leaf,  217 

Lecher,  42 

Lehmann,  F.  E.,  172,  174, 
176,  179,  186,  190,  195, 
196,  200,  276,  277,  278 

Lehmann,  O.,  17 

Lepeschkin,  264 

Leuthardt,  173 

Leuthold,  327 

Levan,  222 

Lewis,  C.  M.,  261 

Lewis,  E.  B.,  239 

Lewis,  W.  H.,  186,  187 

Liebich,  247 

Liechti,  232 

Lieser,  63,  68 

Lindau,  42 

Lindberg,  338,  344 

Lindley,  343 

Lindstrom,  363 

Loeb,  203 

Loewy,  189 

Lomax,  327 

Loomis,  194 

Lorand,  369 

Lorch,  186 

Loschmidt,  27,  142,  167 

Lotmar,  301,  302 

Liidtke,  334 

LundegS,rdh,  182,  199 

Liischer,  295,  296,  297 

Liithy,  363 

Luyet,  196,  217 

McArthur,  369 
McClintoc,  239 
Mackinney,  250 
Magnus,  29 
Majno,  350 
Manegold,  76 


Mangenot,  8,  173,  243 

Manton,  276 

Mark,  30,  31,  32,  38,  50,  59, 

77,  78,  97,  98,  302,  310, 

314,  329,  335,  337,  341, 

348,  359,  367 
Marklund,  199 
Marks,    364,    366,    367,    368 
Alarsland,  164,  172,  :86 
Martens,  215,  300 
Martin,  116 
Marwick,  344,  366 
Matheson,  120 
Matoltsy,  176,  354,  358 
Matthey,  226 
Maxwell,  92 
Mazia,  227 
Medem,  275 
Meeuse,  281 
Menke,  140,  245,  246,  247. 

248,  249,  253,  255,  256, 

257,  258,  260 
Mercer,  337 
Mestre,  247 
Aletz,  233 
Metzner,  245 
Meyer,  A.,  243,  326 
Meyer,  K.H.,  30,  31,32,  38, 

50,  59,  68,  77,  78,  97,  98, 

142,   185,  201,  203,  204, 

205,  301,  302,  310,   311, 

314,  315,  335,  337,  348, 

359,  360 
Meyer,  K.  P.,  252 
Meyer,  M.,  293,  294,   299, 

300 
Michaelis,  156 
Michel,  124 
Michener,  210 
Middlebrook,  343,  369 
Millard,  307 
Mikier,  250 
Minder,  232 
Mirsky,  184,  191 
Misch,  98 
Mitchison,    200,    264,    270, 

271 
Mittasch,  40 
Mohring,  77,  308 
Mollendorf,  v.,  i8i 
Mommaerts,  245,  247 
Monne,  170,  173,  176,  i8i, 

182,  190,  217,  277 
Monroy,  200,  276 
Montalenti,  276 
Moore,  316 
Morgan,  C,  364 


404 


AUTHOR  INDEX 


Morgan,  T.  H.,  230 

Morton,  100 

Mothes,  1 5  5 

Moyer,  186 

Miihlethaler,  121,  127,  128, 

129,   173,  256,  259,  282, 

283,  284,  304,  308 
Muller,    II,   233,   234,   255, 

335 
Muller,  A.,  34 
Muller,  A.  F.,  173 
Muller,   H.   O.,    120,    122, 

124,  125 
Muralt,  v.,  353,  354,  362 
Myrback,  313,  314 

Nageli,  4,  76,  77,  78,  79,  80, 

108,  315,  326,  327 
Nageotte,  55,  57 
Nakamura,  230 
Naray-Szabo,  319 
Nathanson,  197 
Nebel,  225 
Neckel,  349 

Needham,  156,  157,  359 
Negelein,  261 
Nelson,  113 
N6mec,  216 
Newman,  122 
Newton,  164,  170 
Nicolai,  252,  307 
Niemann,  134,  142,  335,  337 
Niggli,  27 

Noack,  245,  247,  252 
Noda,  349 
Nowotny,  342 

Oberling,  176 

O'Brien,  121 

Ogur,  215 

Ohara,  331,  332,  333,  334, 

..  339 
Ohman,  276 

Oltmanns,  169 

Oort,  303,  304,  305 

Oster,  177,  215 

Ostergreen,  224 

Ostwald,  8,  16,  18,  66 

Overbeck,  281 

Overton,  197,  198 

Pacsu,  68 
Painter,  230 
PaUmann,  31,  149 
Palmer,  57,  61,  361 
Pankow,  302 


Pasewaldt,  124,  125 

Pasteur,  207 

Patau,  227 

Patterson-Fourier,  328 

Pauling,  147 

Pekarek,  167,  168,  169,  191 

Perr}%  352 

Perutz,  135,  265,  270,  359 

Peterlin,  91,  108 

Peters,  181 

Peyer,  300 

Pfeffer,  76,  327 

Pfeiffer,  79,   154,  167,   168, 

170,  195,  217,  229,  353 
Philip,  338,  344 
Philipp,  113,  329,  341,  367 
Philippoff,  66 
Picken,  302,  307,  310,  360 
Piguet,  314 
Pilnik,  61,  92 
Piper,  292,  295,  296 
Pirschle,  153 
Pischinger,    193,    212,    215, 

216,  218 
Plantefol,  8,  173,  243 
Platzek,  115 
Ploetz,  312 
Plowe,  170,  205 
Pochettino,  338 
Poiseuille,  170 
Pollister,  184 
Poison,  125,  126 
Ponder,  167,  169,  262,  263, 

264,  270,  271,  273 
Porter,  256,  257 
Posternak,  238 
Potter,  359 
Prakke,  347 
Pratt,  348,  349 
Preston,  J.  M.,  333 
Preston,  R.  D.,  95,  305,  307 
Priestley,  245,  280,  300 
Prokofyewa  Belgorskaja, 

239 
Prudhomme  van  Reine,  170 
Pryor,  307 

Queiroz-Lopes,  221 

Rabinowitch,  246,  261 
Randall,  218,  275 
Ranvier,  364 
Ranzi,  136,  137 
Raven,  277 
Rawlins,  127,  241 
Reed,  307,  353 
Reinders,  199 


Reinke,  183,  184 
Reman,  251 

Renner,  42 
Reumuth,  338 
Rhumbler,  163,  164,  170 
Ries,  156 
Riley,  215 
Ringer,  56,  166 
Roelofsen,    280,    285,    303, 

304,  305 
Rohdewald,  247 
Romeis,  221 
Rosen,  215 
Rosenberg,  273 
Rosin,  307,  308 
Rossenbeck,  214 
Roth,  275 
Rotheli,  275 
Rothen,  159 
Rothmund,  46 
Rottenburg,  205 
Rouiller,  305 
Rozsa,   174,  2i6,  218,  222, 

352,  357,  358,  364 
Ruch,  226 
Ruhland,  197 
Rundle,  312,  319,  320 
Runnstrom,  170,  190,  276 
Ruska,  E.,  72,  116,  120,  129, 

368 
Ruska,    H.,    10,    118,    121, 

256,  257,  349 
Rutishauser,  350 
Ruttle,  225 

Sadron,  92,  1 1 5 

Sakurada,  337 

Samec,  314,  315 

Samsa,  93 

Sankewitsch,  1 5 1 

Sauter,  98,  318 

Sax,  215 

Scarth,   168,   i8i,   186,   199, 

200,  253 
Schaaf,  312 
Schaede,  216 
Schardinger,  312 
Schauenstein,  337,  345,  346 
Scheibe,  135,  329,  330 
Scherrer,  A.,  256 
Scherrer,  P.,  17,  77,  98 
Schiff,  214 
Schimper,  243 
Schinz,  350 
Schmidt,  O.,  362 
Schmidt,  W.  J.,  76,  92,  96, 

178,  217,  220,  222,  224, 


AUTHOR    INDEX 


405 


228,   229,   230,   255,   274, 
303,   304,   306,   317,   347, 

351.  353.  354.  355.  361, 

362 
Schmiedeberg,  210 
Schmitt,  F.  O.,  56,  57,  86, 

87,    118,    217,    229,    270, 

271.   348,  349.  356,  357, 

358,  361,   362,  363,  364, 

368 
Schmucker,  261 
Schneider,  64 
Schneidmcsser,  312 
Schoch-Bodmer,  281 
Schopfer,  243 
Schossberger,  31 
Schultz,  240 
Schulz,  G.  v.,  61 
Schulz,  J.,  185 
SchiirhofF,  243 
Schuringa,  338 
Schweizer,  62,  65,  300 
Schwendener,  76,  77 
Scott,  351 
Searle,  317 
Seeds,  228 
Seifriz,  67,  77,  170,  171,  172, 

182,  186,  187,  263,  264 
Sekora,  337,  368 
Semmcns,  221 
Senarmont,  294 
Senn,  243,  257 
Senti,  319 
Serra,  221 
Seybold,  251,  252 
Sharp,  243 
Siedentopf,  4,  8 
Sievers,  203 
Signer,  9,  90,  214,  220,  229, 

298 
Sjostrand,  363 
Smith,  J.  H.  C,  250 
Smith,  S.  G.,  355 
Snellman,  92,  352,  354,  35 8 
Soding,  281,  287 
Sollner,  201 
Sorkin,  65,  317 
Spark,  353 
Speich,  loi,  no,  113,  320, 

321.  324 
Spemann,  178 
Spoehr,  250 
Sponsler,  132,  310,  319 
Sprecher,  250 
Stanke,  345,  346 
Stanley,  11,  127,  240 
Stapelfeldt,  9 


Staudinger,    5,    58,    59,   61, 

62,  63,  64,  65,  67,  68,  78, 

157,  311,  317.  318 
Stecher,  286 
Steinbrinck,  no 
Steinmann,  254,  257 
Steward,  199 
Stocker,  153 
Stokes,    10,    167,    168,    170, 

191 
Stoll,  247,  248,  260,  261 
Stout,  162 
Straub,  F.  B.,  352 
Straub,  J.    220,  226 
Strugger,  162,  193,  245,  247, 

256,  257,  258 
Stuart,  31,  91,  108 
Stubel,  77,  355 
Svedberg,   10,   11,  61,   125, 

141,   142,   144,   159,  231, 

260,  265,  331,  352 
Swann,  307 
Szent-Gyorgyi,     352,     357, 

358,  364 

Takahashi,  127,  241 
Tavel,  68,  73 
Teorell,  201,  266 
Thaureaux,  172 
Thimann,  209,  280,  286,  292 
Thorell,  353,  357 
Thung,  256 
Timm,  247 
Timofeeff-Ressovsky,     231, 

,232,  233,  234,  235 
Tischler,  219,  221 
Tomlin,  218 
Tornava,  199 
Treer,  107 
Trillat,  56 
Trogus,  280 
Trurnit,  159 
Tupper-Carey,  280 
Tyndall,  8 

Ullrich,  90,   178,   199,  202, 

253 
Ursprung,  42 

Vance,  1 1 6 

Van   de    Sande   Bakhuizen, 

316 
Van  der  Waals,  31,  35,  61, 

93,  159,  185,  266 
Van  Dijk,  199 
Van  Iterson,  G.,  281,  290, 

291,  301,  307,  309 


Van  Iterson,  W.,  256 
Van  't  Hoff,  61 
Vermaas,  112,  115 
Vermeulen,  251 
Verne  van  Bremen,  222 
Verzar,  559 
Virtanen,  179 
Vischer,  214 
Vl^s,  353 
Vorlander,  52,  53 

Wade,  364 
Wiilchli,  103,  304 
Wakkie,  252,  253 
Warburg,  261 
Wassink,  251,  261 
Waugh,  50,  264 
Weber,  E.,  90,  299,  300 
Weber,  F.,   191,    192,   205, 

245,  247,  253 
Weber,  H.  H.,  355,  ^(64 
Weibull,  365,  366 
Weichsel,  324 
Weidinger,  113,  344 
Weier,  243 
Weiss,  179 
Went,  209 

Wergin,  121,  280,  281,  292 
Werner,  28,  33 
Wettstein,  v.,  242 
Weurman,  252 
Weyl,  81 
White,  220 
Wicklund,  170 
Wiedemann,  248 
Wiegner,  80 
Wieler,  244,  245 
Wiener,  82,  83,  84,  88,  89, 

loi,   115,  220,  270,  334, 

338,  350.  351.  355 
Wilbrandt,  202 
Wilkins,  228 
Williams,  125,  126 
Willstiitter,  247,  261 
Windaus,  138 
Winkler,  265,  266,  267,  268, 

269 
Wirth,  287 
Wissler,  92,  220 
Witnauer,  319 
Wohl,  261 

Wohlfahrt-Bottermann,  368 
Wolpers,  72,  129,  264,  268, 

271,  272,  348,  357,  368 
Worschitz,  355 
Wrinch,  158,  227,  329 
Wuhrmann  ,61,  121,  287 


4o6 


AUTHOR   INDEX 


Wuhfmann-Meyer,  292 

WyckofF,  115,  121,  122,  123, 

125,   126,   127,   129,   159, 

174,    i75>   216,   218,   222, 

241,   256,   257,   282,   308, 

348,  349,  351,  352,  357, 
358,  364 

Young,  316,  363,  364 


Zacek,  273 
Zacharias,  184 
Zahn,  342,  343 
Zegar,  300 
Zenker,  255 
Zernike,  i,  108 
Zetzsche,  296,  297 
Ziegenspeck,  no 
Ziifle,  113 


Zimmer,  231 
Zimmermann,  221, 
Zirkle,  245 
Zocher,  9,  52,  53 
ZoUikofer,  287 
Zollinger,  173 
Zsigmondy,   4,    8, 

17,  76 
Zworykin,  115,  116 


327 


10,    II, 


I 


SUBJECT   INDEX 


absorption,  anisotropic,  85 

light,  252 

ultraviolet,  219,  226 
achromatin,  219 
actin,  352 

active  elimination,  198 
active  group,  208 
active  plasma,  181 
actomyosin,  352 
adenoid  activity,  198 
adenosine  triphosphate,  358 
aerogel,  iii 
aggregation,  159,  330 
agon,  236 

aleurone  grains,  193,  326 
aliphatic  compounds,  34 
amino  acids,  132,  330 
amylase,  314 
amylopectin,  310 
amy  lose,  310 
angle  of  scattering,  90 
anisotropic  absorption,  83 
anisotropic  diffraction,  83 
anisotropy,  optical,  85 
anucleal,  224 
apo-enzyme,  208,  235 
apposition  growth,  290,  316 
arginine,  340 
aromatic  compounds,  36 
assimilation,  unit  of,  261 
atomic  distances,  29,  30. 
autocatalytic  reproduction, 

241 
auto-reproduction,  240 
auxins,  209 
axolemma,  364 
axon,  363 


backbone  spacing,  336,  365 
bast  fibres,  105 
beaded  chains,  93,  176 
Bergmann-Niemann  rule, 

biochemistry,  7 
biomorphology,  7,  372 
biosomes,  174 


birefringence  (cf.  double  re- 
fraction), 83,  85,  114 
form,  85,  254 
intrinsic,  88,  298,  308 
layer,  255 
lamellar  =  layer 
of  flow,  90,  298 
platelet  =  layer 
rodlet,  84,  114,  309,  321 

block  structure,  316 

blood  corpuscles,  red  s.  ery- 
throcytes 

bones,  350 

boundary  layers,  197 

cap-plasmolysis,  184,  197 
carboxylase,  236 
carotenes,  249 
carotenoids,  246 
carrier  hypothesis,  233 
cell  elongation,  281 
cell  extension,  282 
cell  polarity,  190 
cell  wall,  cutinized,  293 

meristematic,  279 

micellar  textures,  95 

primary,  279 

secondary',  279,  290 
cellobiose,  39 
cellodextrines,  62 
cellulose,  59,  62,  97,  280 
cellulose  frame,  286 
centrifuge  microscope,  194 
centromeres,  222,  224 
chain  lattice,  34,  36,  69 
chain  length,  61 
chains,  beaded,  93,  176 
chitin,  130,  301,  308 
chlorophyll,  246,  248,  252 
chloroplastin,  248,  258 
chloroplastin  symplex,  247 
chloroplasts,  243 
cholesterol,  138,  266 
choline,  138 
chondrioconts,  173 
chondriome,  173 
chondriosomes,  173 


chromatid  threads,  225 
chromatin,  219 
chromidia,  176 
chromocentres,  220 
chromomeres,  225 
chromonema  theor)',  225 
chromonemata,  225,  241 
cbromophily,  219 
chromoprotein,  248,  249, 

258,  265 
chromosomes,  217,  224,  231, 

234 
coacervation,  18 
coagulation,  directed,  183 
co-enzyme,  208,  235 
cohesion,  31 

molar,  32 
cohesive  bonds,  146,  185 
cohesive  forces,  31,  32,  185 

Van  der  Waals,  31 
cohesive  pressure,  42 
cohesive  tension,  42 
coleoptile,  287 
collagen,  307,  345 
collagen  fibres,  348 
collagen  group,  367 
colloid  chemistry,  4 
colloid  particles,  8,  15 
colloid  solutions,  64 
colloids,  15 

corpuscular,  74,  81 

globular,  126 

reticular,  74,  81 
composite  bodies,  82 
condensation,  59 
connective  tissue,  345 
constellation,  37 
contractility,  134 
contraction,  muscular,  358 

of  protoplasm,  187 
co-ordination,  28,  29 
corpuscular  colloids,  73,  8 1 
cotton  fibres,  307 
crossed  lamellar  systems, 

506 
crystal  lattice,  26,  69,  97 
crystal  structure,  26 


4o8 


SUBJECT  INDEX 


crystalline  liquid,  52 
crystalline,  super-,  53 
crystalloids,  194,  221,  326 
crystals,  liquid,  51 
cuticle,  293 

cuticular  layers,  293,  297 
cuticular  transpiration,   301 
cutin,  293,  297 
cutin  waxes,  294 
cutinized  cell  walls,  293 
cyanophily,  219 
cyclic  compounds,  37 
cysteine,  133,  155 
cystine,  155,  340 
cytoplasm,  132 

behaviour  of  proteins,  141 

different  phases,  191 

fixation,  174 

flow,  186 

ground-,  174,  179 

heredity,  242 

molecular  constituents, 

132 

molecular  morphology, 

207 

morphogenesis,  179 

permeability,  197 

physical  properties,  163 

submicroscopic  structure, 

172 

surface  tension,  165 

viscosity,  146,  166 

water  content,  177 
cytoplasmic  layers,  200 
cytoplasmic  membrane,  199 

Debye-Scherrer  diagram,  99 
deflection  discs,  9 
dehydration,  20,  177 
dehydrogenase,  207,  236 
dehydrogenation,  154 
denaturation,  136,  143,  329, 

330 
deplasmolysis,  198 
dermatosomes,  317 
desoxyribose,  212 
desoxyribose  nucleic  acids, 

237 
dextrins,  312 
dialysis,  8 
diamino  acids,  133 
dichroism,  83,  loi 
difi^iaction,  anisotropic,  83 
dipeptide,  132 
dipole,  19,  147 
disaccharides,  39 
dispersed  phase,  15 


dispersing  medium,  1 5 
dispersion  series,  70 
dispersoids,  15,  16 
Donnan  equilibrium,  202 
double  refraction  (cf.  bire- 
fringence), 83,  84,  114 
incidental,  88 
intrinsic,  88,  298,  308 
orientation,  89 
tension,  89 

ectoplasm,  186,  199 
eggs,  190,  194,  276 
elastic  tissue,  351 
elasticity,  65,  171 

of  flow,  164 

structural,  164 
elastin,  351 
elastoidin,  346 
electron  diff^raction  dia- 
grams, 120 
electron  microscope,  3,  116 
electron  microscopy,  115, 

123 

cell  wall,  283 

chloroplasts,  256 

erythrocytes,  271 

gels,  127 

globular  colloids,  1 26 

muscle  fibres,  357 
electron  rays,  115 
electrophoresis,  8 
electrosmosis,  75 
elementary  cell  =  unit  c, 

26,  97,  319 
elimination,  active,  198 
elongation  growth,  288 
enamel,  350 

enchylema,  181,  215,  217 
endo-enzyme,  208 
endoplasm,  186,  199 
energy  equivalent,  30 
enzymes,  180,  207,  235 
epidermis,  293 
equilibrium  liquid,  21 
erythrocytes,  262 
erythrophily,  219 
estolids,  296 
euchromatin,  220 
extension  growth,  283 
extinction  angle,  91 

fat,  137,  139 
fatty  acids,  137 
feather  keratin,  344 
Feulgen's  nucleal  reaction, 
214 


fibre  diagram,  97,  99,  104 
fibre  texture,  =  fibrous  t., 

94,  290,  303,  332 
fibres,  1 01 

bast,  105 

cellulose,  105 

collagen,  348 

cotton,  307 

muscle,  352 

ramie,  97,  loi,  104 
fibrillar  hypothesis,  227,  237 
fibrillar  proteins,  135,  364 
fibrillar  theory,  182 
fibrils,  origin  of,  194 

protoplasmic,  194 
fibrinogen,  369 
fibroid  texture,  94,  304,  332 
fibroin,  331 
fibroinogen,  337 
fibrous  texture  =  fibre  t. 
fine-structure,  5 
finger  nails,  344 
fixation,  75,  161,  174 
flagella,  hairy,  275 
Flimmergeisseln,  275 
flow,  birefringence  of,  90, 

298 

protoplasmic,  164,  186 
flow-birefringence  appar- 
atus, 299 
fluorescence,  162,  252 
focal  depth,  120 
foliate  texture,  96,  304 
form  birefringence,  85,  254 
framework,  176 

gel,  66,  68,  69 

mi  cellar,  73,  81 

molecular,  73,  80,  81,  201, 

341 
free  rotation,  37 
freeze -drying,  178 
freezing,  196 

galacturonic  acid,  60 

gametes,  274 

gel  framework,  66,  68,  69 

gel-sol  transition,  171,  187 

gel  solutions,  65 

gels,_5  8,  71,  82 

cristallinity,  112 

electron  microscopy,  127 

polarization  microscopy, 

82 

structure,  58,  66 

swelling,  109 

X-ray  analysis,  96 
gelatin,  21,  23,  93,  345 


1 

i 


SUBJECT  INDEX 


409 


genes,  230,  233,  240 
ghost,  264 
Gibbs-Thomson  theorem, 

44 
glasses,  81,  112 
globoid,  194 
globular  molecules,  135 
globular  proteins,  135,  141 
globulins,  212 
glucosamine,  301 
glucose,  37,  60,  313 
glucosidases,  40 
glucosides,  40,  310 
glutamic  acid,  340 
glutathione,  155 
glycine,  336,  346 
glycogen,  3 i i 
grana,  245,  247,  257 
growth,  apposition,  290, 

316 

elongation,  288 

extension,  283 

forces  of,  287 

in  area,  282,  307 

mosaic,  286 

spiral,  304 

substances,  209 

surface,  282,  307 

tip,  282 
guanyl  nucleic  add,  214 

haemocyanin,  125 
haemoglobin,  265,  269 
haemolysis,  264 
Haftpunkt-Theorie,  145 
Haftpunkte,  67 
Hagen-Poiseuille's  law,  65 
hair,  338 

hairy  flagella,  275 
Hecht,  strands  of,  170 
hemicelluloses,  61 
heredity,  230,  242 
heterocapillarity,  105 
heterochromatic  parts,   220 

225,  239 
heterocyclic,  37 
heterogeneous,  12 
heteropolar  lattice,  28 
hexane,  36 
high  polymers,  58 
histidine,  210 
histones,  212 
homocapillarity,  105 
homogeneity,  optical,  12 

physico-chemical,  12 
homogeneous,  statistically, 

12 


homopolar  lattice,  28 
honeycomb  theory,  182 
hormones,  208 
horny  substances,  338 
hydration,  19,  148,  163 
hydrogen,  208 
hydrogen  bonds,  147 
hydrogen  bridges,  147 
hydrogen  pressure,  156 
hydrogenation,  155 
hydrolysis  patterns,  317 
hydrophilic  groups,  47 
hydrophily,  44 
hydrophobic  groups,  47 

identity  period,  26 

I.E. P.  =  isoelectric  point, 

153.  154  _ 
imbibition  liquid,  84 
imbibition  water,  196 
incidental  double  refraction, 

88 
index  ellipsoid,  87 
insulin,  328 
interferences,  97 
intermicellar,  79,  81 
intermicellar  phase,  79 
intermicellar  portion,  79 
intermicellar  processes,   80, 

81 
intermicellar  spaces,  99,  100 
intermicellar  substances,  81 
intermicellar  swelling,  109 
intermolecular,  32 
interstitial  substance,  80,  8 1 
intrability,  197 
intramicellar,  80 
intramicellar  processes,  81 
intramicellar  swelling,  no 
intramolecular,  32 
intrinsic  birefringence   =  i. 

double  refraction,  88, 

298,  308 
intussusception,  285,  306, 

307 
ion  lattice,  28 
ion  radii,  148 
ion  series,  148 
ionization,  232 
isoelectric  s.  I.E.?. 

junctions,  67,  145,  159 
theory  of,  145,  184 

Kappenplasmolyse,  197 
karyokinesis,  238,  242 
karyolymph,  215,  217 
keratin,  338 


keratin- myosin  group,  365 
kinetochore,  224 
kinoplasm,  181,  200 
konyaku,  322 

lamellar  birefringence   = 

layer  b.,  255 
lamellar  structure,  255,  256 
lamellar  systems,  crossed, 

306 
lattice,  26 

chain,  34,  36,  69 

crystal,  26,  69,  97 

heteropolar,  28 

homopolar,  28 

ion,  28 

layer,  36 

primary  valency,  28 

molecule,  30,  35 
lattice  arrangement,  12 
lattice  plane,  26 
lattice  regions,  78 
layer  birefringence,  255 
layer  composite  body,  82 
layer  lattice,  36 
layer  structure,  255 
lecithin,  55,  138,  252 
leptonema,  234,  237 
leptones,  79 
light  absorption,  252 
lipid  filter  theory,  197,  267 
lipid  theory,  197 
lipidic  drops,  193 
lipids,  137,  267 
lipophilic  groups,  47 
lipophily,  44 
liquid  crystals,  5 1 
lintnerization,  317 
long-range  forces,  158,  177 
long-range  spacings,  367 
lyo-enzyme,  208 

macrocoacervation,  20 
macromolecular  chemistry, 

5,  59 
macromolecules,  59,  81, 

125,  160 
main  chain  spacing,  365 
main  valency  forces,  31 
maltose,  39,  314 
mannan,  59,  321 
mannose,  60 
matrix,  181,  226 
medullary  sheath,  362 
meristematic  cell  walls,  279 
meristematic  cells,  281 
mesophases,  51 


4IO 


SUBJECT    INDEX 


methylene  bridge,  155 

micellar,  79 

micellar  framework,  75,  81 

micellar  phase,  79 

micellar  strands,  73,  96 

micellar  structure,  77,  81, 
105 

micellar  theory,  76 

micellar  texture,  81,  94 

micelle,  76,  81 

microcoacervation,  20 

microfibrils,  104 

microscope,  centrifuge,  194 
electron,  3,  116 

microscopy, polarization,  82 

microsomes,  172 

Mischkorper,  83 

miscibility,  diagram  of,  46 

mitochondria,  173 

molar  cohesion,  32 

molecular  framework,  73, 
80,  81,  201,  341 

molecule  lattice,  30,  35 

monolayer,  49 

monomolecular  films,  49 

monosaccharides,  37 

morphogenetic  configura- 
tions, 179 

morphology,  i 

mosaic  growth,  286 

mosaic  theory,  197 

muscle  fibres,  352 

muscular  contraction,  358 

mutation  rate,  231 

myelin  forms,  54,  247 

myelin  sheath,  360 

myofibrils,  353,  357 

myogen,  352,  353 

myosin,  352 

nematic  state,  52 

nerves,  360 

neurofibrils,  363 

neurokeratin,  361 

neurolemma,  364 

neuronin,  363 

nucleal  reaction,  Feulgen's, 

214 
nuclear  membrane,  217 
nuclear  sap,  215,  217 
nuclear  spindle,  222 
nuclear  staining,  218 
nucleicacids,  23,  212,228, 237 
nucleolus,  221 
nucleoproteins,  210,  227, 

240 
nucleotide,  208,  212 


nucleus,  210,  242 

active,  215 

chromosomes,  224 

fine-structure,  215 

fixed,  216 

hereditary  processes,  230 

molecular  constituents, 

210 
nutrition  line,  162 

optically  negative,  53,  87 
optically  positive,  53 
organ,  i 
organizer,  179 
orientation  angle,  90 
orientation  double  refrac- 
tion, 89 
ossein,  350 
ovalbumin,  93 

paraplasm,  181 
pasting,  324 
Patterson-Fourier  diagram, 

328 
pectins,  60,  293 
pectic  substances,  60 
pentosans,  61 
permeability',  197 

selective,  201 

formula,  204 

theories,  199,  201 
permutoid  reaction,  iii 

PH,  156 
phase,  6,  15,  68 

meso-,  51 

pseudo-,  69 
phase  boundaries,  18,  40 
phase  separation,   191,   19-1, 

196 
phenol,  46 
pheron,  236 
phosphatides,  138 
phospholipids,  265,  266 
phosphorolysis,  314 
photo-elastic  effect,  89 
phragmoplast,  224,  281 
phyllochlorine  complex, 

247 
physiology,  7 
pigments,  248 
plasma,  active,  181 
plasma  gel,  186 
plasma  sol,  1 86 
plasmalemma,  199 
plasmic  strands,  170 
plasmolysis,  170 

cap-,  197 
plasmoptysis,  17c 


plasmosin,  184 

plastid  membrane,  256 

plastin,  183 

platelet  composite  body  = 

layer  composite  b.,  82 
points  of  attachment,  67 
polarity  of  cytoplasm,  190 
polarizability,  optical,  92 
polarization  microscopy,  82 
polyarabinan,  61 
polygalacturonic  acid,  60 
polymer  uniform  sub- 
stances, 64 
polymeric  homologous,  63 
polymerization,  58 
polypeptide  chains,  132, 

134,  339 
polysaccharides,  59 
polystyrene,  67 
porphyn  ring,  248 
proline,  133,  346 
proplastids,  245 
prostethic  group,  208 
protamines,  211 
protein  cr^'stalloids,  194, 

221,  326 
protein  fibrils,  striated,   368 
proteins,  132,  141,  184,  211, 

326 

fibrillar,  135,  364 

globular,  135,  141,  327 

reserve,  326 
protopectin,  281 
protoplasmic  flow,  164,  186 
protoplasmic  fibrils,  194 
pseudophase,  69 
purine,  213,  214 
pyrimidine,  212,  214 

ramie  fibres,  97,  loi,  104 

rays,  ionizing,  231 

red  blood  corpuscles  s.  ery- 
throcytes, 262 

redox  potential,  156 

reserve  proteins,  326 

resolving  power,  1,119 

reticular  colloids,  74,  81 

reticular  structures,  72 

reticular  systems,  70,  73,  81 

reticulum,  216 

rn,  136 

ribonucleic  acid,  215 

rigidity,  modulus  of,  171 

ring  diagram,  99 

ring  texture,  94 

rodlet  birefringence,  84, 
114,  309,  321,  334 


SUBJECT  I,NDEX 


411 


iodlet  composite  body,   82 
rodlet  double  refraction  s. 

rod  let  birefringence 
rubber,  59,  341 

sarcolemma,  353 
sarcomere,  354 
sarcoplasm,  353 
scattering,  angle  of,  90 
selective  permeability,  201 
selectivity  constant,  203 
sensitizer,  21,  266 
sericin,  331 
shadowing,  122 
short-range  order,  107 
shrinkage,  iii,  149 
sickle  diagram,  99 
side  chain  spacing,  365 
side  chains,  polypeptide, 

133'  135 
silk,  loi,  531 
silk  fibroin,  331 
smectic  state,  52 
sol-gel  transition,  187 
sol  solution,  65 
sols,  8,  75 
solvation  layer,  19 
spacing,  26 

backbone,  336,  365 

main  chain,  365 

side  chain,  365 
specificity,  134,  372 
sperm  nuclei,  220,  228 
spermatozoa,  274 
spherite  texture,  86,95,  316, 

322 
spherites,  86,  95 
spindle  fibres,  222 
spinning  capacity,  170 
spiral  growth,  304 
spiral  structure,  226 
spiral  texture,   95,  291,  304, 

305 
spongioplasm,  181 

sporangiophores,  304 
sporopoUenin,  297 
stabilizer,  266 
starch,  59,  310,  318 
starch  grams,  310,  315,  318 
sterines,  138 

stimulant  substances,  209 
Stokes'  law,  167 
strain  theory,  223 
stretching  experiments,  105 
stroma,  244,  256,  264 
stromatin,  265,  266 
structural  chemistry,  4,  24, 

33 


structural  elasticity,  164 
structural  principles,  24 
structural  viscosity,  64,  66 
structure,  81 

crystal,  26 

framework,  182,  201 

gel,  66 

micellar,  77,  81,  105 

reticular,  72 

spiral,  226 

surface,  48 
sturine,  21 1 
suberin,  297 
sugar,  37 
sulphur  bridges,  155,  340, 

342 
super-crystalline,  53 

surface  elasticity,  200 

surface  energy,  43 

surface  films,  47,  143 

surface  growth,  282,  307 

surface  skin,  41 

surface  structure,  48 

surface  tension,  16,  40,  47, 

165 
Svedberg  loile,  125 
Svedberg  unit,  141 
swelling,  55,  109,  148,  346 

intermicellar,  109 

intramicellar,  no 

limited,  67 
syneresis,  75 

tanning,  75,  347 
target  area,  232 
target  theory,  231 
teeth,  350 
tendons,  345,  346 
tension  double  refraction, 

89 
texture,  81 

fibre,  94,  290,  303,  332 

fibroid,  94,  304,  332 

fibrous  =  fibre 

foliate,  96,  304 

micellar,  81,  94 

lamellar,  255,  256 

ring,  94 

spherite,  86,  95,  316,  322 

spiral,  95,  291,  304,  305 

tube,  94,  282,  288,  305 

tubular  =  tube 
thixotropy,  66 
thymonucleic  acid,  214,  220 
tip  growth,  282 
tonofibrillae,  338 
tonoplast,  198,  200,  205 

membrane,  205 


topochemical  reaction,  111 
transpiration,  cuticular,  301 
tube  texture    =   tubular  t., 

94,   282,   288,   305 
tubular  texture  =  tube  t. 
tunicin,  129 
turgor  extension,  288 
turgor  pressure,  288 
turgor  tension,  289 
Tyndall  scattering,  8 
tyrosine,  133,  336 

ultracentrifuge,  10,  61,  141 
ultrafilter,  127,  199 
ultrafilter  theor}',  197 
ultrafiltration,  8 
ultraviolet  absorption,  219, 

226 
ultraviolet  dichroism,  229 
unit  cell  =  elementary  c, 

26,  97,  319 
unit  of  assimilation,  261 

vacuoles,  23,  191 
vacuolization,  22 
valency,  primary  =  main  v. 

28,  31 

residual,  147 

secondary,  31,  147 
valency  angle,  37 
valency  bonds,  29,  146,  153 
valency  forces,  main,  31 
valency  lattice,  primary,  28 
valency  rule,  1 5 1 
Van  der  Waals  cohesive 

forces,  31,  185 
virus,  125,  240 
virus  protein,  240 
viscose,  333 
viscosity,  146,  166,  181 

structural,  64,  66 
vital  staining,  192,  218 
vitamins,  208,  243,  249 
vitrification,  196 

wall  'ension,  289 
waxes,  137,  292,  295,  297 
Wiener's  formula,  84 
wool,  338 

xanthophylls,  246,  249,  250 

xerogel,  1 1 1 

X-ray  analysis,  25,  26 

gels,  96,  1 1 3 

muscle,  355 
X-rays,  25,  113 
xylan,  61,  310 

Zugfasertheorie,  223 
zygonema,  234 


PRINTED  IN  THE  NETHERLANDS  BY 
DRUKKERIJ    MEIJER  N.V.,  WORMERVEER  AND  AMSTERDAM 


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