METHUEN'S MONOGRAPHS
ON BIOLOGICAL SUBJECTS

General Editor

KENNETH MELLANBY

CYTOLOGICAL TECHNIQUE

The Principles underlying Routine Methods

Cytological Technique

THE PRINCIPLES UNDERLYING
ROUTINE METHODS

JOHN R. BAKER

D.Sc.(Oxon.), F.R.S.

Reader in Cytology in the University of Oxford

Joint Editor of the

Quarterly Journal of Microscopical Science

I

i

\K.

^^ m

LONDON: METHUEN ife CO LTD
NEW YORK: JOHN WILEY & SONS INC

First published 1933
Second edition, rewritten 1946

Third edition 1950

Fourth edition, rewritten 1960

Printed and bound in Great Britain

by Butler & Tanner Ltd, Frome and London

CATALOGUE NO (METHUEN) 2/4101/11

4.1

Dedicated
to the Memory of

GILBERT C. BOURNE, f.r.s.

Preface

A quarter of a century has passed since the first edition of this
book was pubHshed. During that period cytology has been
transformed. Seldom has any branch of science been so revolu-
tionized in a similar period by the invention of new techniques
and the revival of old ones. The subject-matter of the Cytological
Technique of 1933 is only a little fraction of the cytological
technique we know today. Nevertheless, the old processes of
fixation, embedding, dyeing, and moimting survive and flourish.
Indeed, it seems probable that even hundreds of years hence they
will still be necessary. Perhaps some cytologist of the distant
futiire, looking through the books on a forgotten shelf in the
basement of an old library, will read these words and agree that
I was not mistaken.

The old title of the earlier editions is retained at the special
request of the Publishers. The newer branches of the subject are
being covered in Biological Monographs written by other authors.
Dr W. G, B. Casselman's book on Histochemical Technique^^ has
already been published, and another by Dr J. Chayen on bio-
physical methods is in course of preparation.

The earlier editions have been subjected to the sincerest form
of flattery, for considerable parts have been taken from them
and reprinted in text-books with scarcely any change, and usually
without acknowledgement; and the Chinese have done me the
honour of pirating the whole book and producing it in their own
characters. All this is very encouraging and suggests that the
underlying plan was good. So, although this new edition has been
rewritten from start to finish, without the copying of a single
sentence, yet I have followed the original plan. That is to say,
I have chosen as few and as simple techniques as possible, and
have used them as illustrations of general principles.

vii

VIU PREFACE

Although this new edition is concerned primarily with the
principles rather than the practice of microtechnique, I have
thought it right to come down to earth occasionally by giving
detailed practical instructions. The intention is to show the
student some of the applications of the principles. No attempt is
made, however, to provide a consecutive course of instruction
in practical microtechnique, for there are many excellent text-
books devoted to this subject.^^' ^^' i"' "^' "^' ^^^

When the first edition was written, I had already given nine
annual courses in microtechnique to students of zoology at
Oxford. Since then I have given twenty-four more. If one keeps
up with progress in a particular subject over a long period, it
becomes increasingly difficult to write a short book about it. One
can scarcely make a single statement without thinking of excep-
tions and reservations, or being conscious that an accurate
exposition would require longer treatment. The temptation to
equivocate is strong, but must be resisted. It will be realized that
my object is to transmit a general grasp of the subject — to put
the reader in a position to understand the complications when he
or she comes up against them.

I hope that some readers may decide to pursue the subject
further in my larger book, Principles of Biological Microtechnique
(1958).^^ Those who have already seen the Principles may wonder
whether there is anything in the present book that might interest
them. The chief subject that is better treated here, I believe, is
the action of mordants. I have been carrying out research on this
since I wrote the Principles. I think it may be claimed that Chapter
9 of this book includes the first attempt to present a consistent
theory of mordanting in microtechnique, though certain parts of
the process have been admirably treated by Wigglesworth^^^ and
others. Much is known about the use of basic chromium salts as
mordants for azo-dyes in the textile industry, but this is a radically
different process. The discussion on fixative mixtures contains a
number of new ideas. There is also a clearer account of fixation
by mercuric chloride. In addition, the present book deals with
three subjects — embedding, mounting, and the structure of the
cell — that are only mentioned incidentally in the other.

PREFACE IX

In this new edition I must mention again the benefit I have
derived from the practical assistance of Mrs B. M. Luke (for-
merly Miss Jordan) over a period of many years. Mrs J. A. Spokes
has once more typewritten the whole book and helped me by
careful attention to detail throughout. Several colleagues have
given me valuable advice on particular problems. I must especially
thank Dr E. L. Bowen, Mr F. M. Brewer, Mr W. Llowarch, and
above all Dr L. M. Venanzi, who has generously put his know-
ledge of the chemistry of aluminium at my disposal. Any errors
must be attributed to myself alone, for my advisers have not seen
what I have written. Sir Alister Hardy has continued to support
cytological studies in the Department of Zoology at Oxford, and it
is a pleasure to acknowledge his help.

JOHN R. BAKER

Cytological Laboratory
Department of Zoology
University Museum
Oxford

Illustrations

Fig. 1 . A typical spermatogonium, seen in optical section.
The figure is intended to show the structure of the
living cell page 4

Fig. 2 (plate). Electron-micrographs representing the cyto-
plasm of a large phagocytic cell from the blood
of the domestic fowl, and of an acinar cell from
the pancreas of the rat facing page 10

Fig. 3. Graph showing the rate of penetration of mercuric

chloride into gelatine-albumin gel page 23

Fig. 4. A typical spermatogonium, as seen in a routine

microscopical preparation page 26

Fig. 5. Diagram showing the changes in volume imdergone
by gelatine-albumin gel when acted upon by
fixatives page 11

Fig. 6. Structural formulae for hydrated aluminium ions

page 113

Fig. 7. Diagram of a finished microscopical slide in
sectional view, showing adhesives and varnish

page 1 34

XHl

Abbreviations

aq aqueous (solution)
cm centimetre(s)
DNA deoxypentose nucleic acid
g gram(s)
ml millilitre(s)
mm millimetre(s)

H micron(s)
m// millimicron(s)
o.p. oxidation potential
RNA ribonucleic acid
v/v expresses the concentration of a solute in terms of the
number of ml of the solute in 100 ml of the solution.
w/v expresses the concentration of a solute in terms of the

number of g of the solute in 100 ml of the solution,
w/w expresses the concentration of a solute in terms of the

number of g of the solute in 100 g of the solution.
w/W expresses the concentration of a solute in terms of the
number of g of the solute in 100 g of the solvent.

In this book the concentrations of liquid solutes are expressed
as v/v. Thus 5% acetic acid means a solution containing 5 ml
of the acid in each 100 ml of solution.

The concentrations of solid and gaseous solutes are expressed
as w/v, except where the contrary is stated. Thus 4% formalde-
hyde means a solution containing 4 g of the gas in each 100 ml
of solution.

XV

CHAPTER 1

The Structure of the Cell

This book deals with the scientific principles underlying some of
the most ordinary processes of biological microtechnique : that is
to say, with the fixation, embedding, dyeing, and mounting of
small parts of organisms for subsequent study under the micro-
scope. It deals chiefly with the preparation of material for light-
microscopy; indeed, dyeing is not applicable if cells are to be

examined with the electron-microscope (p. 85).

The purpose of ordinary microscopical preparations is to
reveal minute structure. The study of this structure shows that
most organisms consist of cells and intercellular (or extra-
cellular) material; objects formed by the transformation of cells
(the red blood-corpuscles of mammals, for instance) often con-
stitute a third category of components. ^^ The primary com-
ponents are the cells, because the other constituents of the tissues
do not appear in their absence. The cells are, in fact, the proto-
plasm^^^' ^^^ or first-formed material, as indeed we can tell by
examining an early embryo; but since the expression first-formed
may suggest that something else formed them, it is more logical
to say that they are the plasson or formative material.^ ^ The
plasson constitutes the subject-matter of cytology, and this book
is concerned primarily (though not exclusively) with its fixation
and after-treatment in the routine processes of microtechnique.

It would be a convenience if one could describe a 'typical cell'
and then show how this object was aff"ected by the ordinary
processes of microtechnique. Unfortunately there is in reality no
such object, though pictures of it appear in various textbooks.
Cells show great diversity of structure in relation to their particu-
lar functions. Some are so obviously untypical that they could

B 1

2 CYTOLOGICAL TECHNIQUE

not possibly serve as representatives of other kinds. Extremes of
this sort are the flagellate spermatozoon, with strongly marked
polarity and scarcely any ground cytoplasm, and the ovum, with
a most unusual spherical or ovoid form and nearly always with
masses of reserve food-material preventing a clear view of the
other cytoplasmic inclusions. Embryonic cells are more un-
specialized. Those of animals are usually epithelial arid mesen-
chymatous; and since the latter are derived from the former, a case
can be made for the selection of an embryonic epithelial cell as
typical of cells in general. In fact, however, such cells usually
contain yolk granules, and these are much more evident than the
cytoplasmic inclusions that occur regularly in cells of all kinds.
The primary meristematic cells of plants are more unspecialized
than the embryonic cells of animals, and might indeed have been
selected for our purpose.

Animals do not maintain many reserve stores of unspecialized
cells, but rather produce new cells, when required, by the multi-
plication of those that have already become specialized for par-
ticular purposes. Spermatogonia and oogonia, however, do con-
stitute reserve stores of unspecialized cells, and an examination
of them will reveal what parts are necessary for the retention of
life itself, as opposed to the performance of special functions on
behalf of the organism as a whole. An ordinary spermatogonium
may be regarded as more or less typical of the unspecialized cells
of animals, and it will serve here as an approximation to the ideal
'typical cell'. We shall see later how its structure is altered arti-
ficially by many of the usual processes of microtechnique.

To determine the structure of a cell, it is essential to examine
it first while it is still alive. It can be examined in a body-fluid or
in a saUne solution of appropriate osmotic pressure. The cell
must be examined in its unaltered state by phase-contrast micro-
scopy, and also by direct microscopy after the use of vital dyes.
Subsequently it is proper to try every method of microtechnique
that can reasonably be supposed to throw light on the living
structure.

The first thing to notice is the shape of the cell. Spermatogonia
do not maintain a particular form by the secretion of a solid cell-

THE STRUCTURE OF THE CELL 3

wall. Their boundaries are usually determined by the pressure of
neighbouring cells and other tissue-constituents. They often occur
in cysts filled with similar cells. The external cells lie against the
wall of the cyst, but the others may be bounded by cells similar
to themselves. Mutual pressure gives a characteristic shape to
each cell. It has many sub-equal sides. These are nearly flat,
except where a cell abuts on the curved wall of the cyst or tubule
that contains the cells, or projects freely into the central cavity.
In section, most of the spermatogonia therefore appear polygonal
(fig. 1). The cell-membrane is exceedingly thin, far below the
limit of measurement by the light-microscope; but whether we
can actually see a cell-membrane or not in any particular cell, we
are compelled by convincing evidence to postulate the existence
of a thin surface layer having different characters from the
material within.

Beneath the cell-membrane lies the ground cytoplasm with
its inclusions. Spermatogonia commonly contain five kinds of
cytoplasmic inclusions that are large enough to be seen with the
light-microscope. Three of these appear to be invariably present
in ordinary cells. They do not occur in mature red blood-
corpuscles of mammals, and all of them do not occur in all
spermatozoa ; but they are present in young red blood-corpuscles
and also in spermatids, which are young spermatozoa. The three
kinds of cytoplasmic inclusions that are present in spermatogonia
and in all ordinary cells are the nucleus, mitochondria, and lipid
globules.

It may be thought degrading to the dignity of the nucleus to
call it a cytoplasmic inclusion, but logic seems to demand that it
should be included under this head. Some authorities would list
the 'Golgi apparatus' as a regular constituent of cells, but this
name appears to have been given to a variety of different objects,
not falling into a single category.^^^- i^- loo, 15, 170, 171. 19. 55, 56,

133, 22, 113

Spermatogonia commonly contain two other kinds of cyto-
plasmic inclusions, which are of frequent but not universal occur-
rence in cells. These are the centriole and idiozome.

The five kinds of cytoplasmic inclusions will now be briefly

CYTOLOGICAL TECHNIQUE

intercellular space

centriole

lipid ground cytoplasm

globules (in wide sense)

idiozome
\

nucleolus

centromere

heterochroma tic

segment of
chromosome

euchromatic segment of

chromosome
uJ

Wjj

Fig. 1. A typical spermatogonium, seen in optical section. The figure
is intended to show the structure of the living cell. Compare with
fig. 4, p. 26. {The contents of the nucleus are represented diagram-
matically; the rest of the figure is based on the spermatogonium
of the common newt, Triturus vulgaris.)^*

considered. The nucleus will be taken first, but it will be con-
venient to describe the rest of the inclusions in a different order
from that given above, mainly because the idiozome is such a
striking object in typical spermatogonia.

The nucleus is the largest single cytoplasmic inclusion in most
cells. In spermatogonia it is commonly eccentric (fig. 1), and this

THE STRUCTURE OF THE CELL 5

confers polarity on the cell, which is radially symmetrical about a
linear axis (though there is no precise symmetry of the flat sides).
The eccentricity of the nucleus leaves the bulk of the cytoplasm
at one end of the cell, and the idiozome lies near the middle
of this cytoplasmic mass. The nucleus of spermatogonia is usually
ovoid or spherical, but sometimes concave on the side towards
the idiozome. This concavity towards the idiozome is not unusual
in various cells, especially certain leucocytes. The nucleus is
bounded by a distinct membrane, but this is too thin to show any
structure with the light-microscope.

The nucleus contains spherical or subspherical bodies called
nucleoli. These commonly appear after mitosis, close up against
certain particular parts of particular chromosomes. Since the
latter are present in the form of two equal sets, the nucleoli are
usually present at first in equal numbers; but they have a ten-
dency to fuse, and many cells contain only one. The nucleoli are
particularly dense, heavy objects.^ ^ When a cell disintegrates
after death, the nucleoli and lipid droplets are distinguishable
after everything else has disappeared. Nucleoli contain basic
proteins and ribonucleic acid (RNA), and also, apparently, some
phospholipid. They are usually largest in cells that are actively
synthesizing protein in the cytoplasm, but no satisfactory explan-
ation of this fact has been produced.

Our knowledge of the structure of the part of the nucleus not
occupied by the nucleoli is unfortunately meagre. The electron-
microscope has given little help towards the solution of this
problem. Some authorities consider that during the late stages of
telophase the chromosomes swell until they touch one another;
others think that they spin out as thin threads that are separated
from one another by a continuous fluid or weak gel, the nuclear
sap. It is probable that both kinds of nuclei exist, but the weight
of evidence seems to suggest that in most cells the chromosomes
are largely in the form of very long, thin threads, not distinctly
visible with the light-microscope and not colourable by the dyes
that attach themselves so intensely to the mitotic chromosomes.
The whole of each chromosome, however, is not present in this
form, for certain particular regions, called the heterochromatic

6 CYTOLOGICAL TECHNIQUE

segments or karyosomes, do not undergo the same telophase
transformations as the rest, but remain as thick, somewhat
irregular bodies, easily visible in the living cell. The hetero-
chromatic segments are colourable without difficulty in fixed
preparations, since they contain deoxypentosenucleic acid (DNA),
which is the main substance responsible for the fact that the
mitotic chromosomes are so easily dyed. DNA has a strong
affinity for all members of the large group of colouring agents
known as basic dyes (p. 90).

DNA is not confined to the heterochromatic segments, but
appears to be distributed throughout the whole of the rest of the
nucleus (apart from the nucleolus), at a lower concentration than
in the heterochromatic segments. ^^^ The form in which it is
distributed is not known. Possibly it is strung out along the fine
threads that constitute the normal or euchromatic segments of
the chromosomes; possibly these threads are at this stage com-
posed of protein only, and the DNA is in colloidal solution in the
nuclear sap.

Before the chromosomes reappear distinctly in early prophase,
they have split longitudinally throughout their length. It is not
known exactly when this splitting occurred, and an arbitrary
decision must therefore be reached as to whether the chromosomes
should be represented in a diagram as split or unsplit. They are
represented in fig. 1 as single threads, swollen in particular places
into heterochromatic segments. Only two pairs of chromosomes
are represented in the diagram, but in the great majority of
animals the diploid number exceeds ten.

In dyed preparations of cells undergoing mitosis, each chromo-
some appears to be interrupted at a particular spot, where there
is a short segment lacking affinity for basic dyes. This segment is
the spindle-attachment or centromere. Long chromosomes are
attached to the spindle by this body, but short ones are wholly
embedded in the spindle. The centromere is the part of the
chromosome that leads the rest in the movement towards the
centriole in early anaphase. Since each centromere occupies an
unvarying position in each chromosome, it presumably persists
from telophase to the succeeding prophase.

THE STRUCTURE OF THE CELL 7

During interphase, the centriole may often be distinguished
near the middle of the mass of cytoplasm situated at the opposite
pole of the cell from the nucleus. This body is too small for the
light-microscope to reveal any structure in it. Astral rays and one
end of the spindle were centred on it at the preceding division.
We have no proof that it always persists from one cell-division to
the next. Shortly before the next division two centrioles are
present, close to each other.

The centriole is surrounded by a subspherical but rather
irregularly-shaped body. This was named Idiozom,^'^^ because it
forms a special envelope round the centriole (Greek idios^
special ; zdma, envelope). The word may be written 'idiozome' in
English, but it is wrong to spell it with an s in place of a 2, though
this is often done.^^^ It is difficult to see in the untreated, living
cell, but in some cases it can be made evident by the use of vital
dyes (neutral red, dahlia). It appears structureless when examined
by the light-microscope, and no definite limiting membrane can
be seen. It is present continuously from one cell-division to the
next, whether the centriole persists or not. The chemical com-
position of the idiozome has not been worked out fully. It is not
so markedly different from the rest of the cytoplasm in chemical
composition as to be readily dyed in a contrasting colour. It has,
however, a somewhat greater affinity for the colouring agents
called 'acid' dyes (p. 90). If a suitably fixed testis is soaked in a
solution of osmium tetroxide, the latter is reduced at its surface to
produce a deposit of the black hydrated dioxide. The idiozome
corresponds with what some authors have called the centrosome,
but it is probably best to avoid this word, since it has been applied
also to the centriole. The functional significance of the idiozome
remains obscure.

In many kinds of cells, especially blastomeres, there is a body
showing general resemblance to the idiozome, but the astral
rays of the preceding mitosis persist in continuity with it, so that
it appears spiky; it is commonly granular. This body in blasto-
meres was at first regarded as constituting the essential part of
the protoplasm: indeed, the name protoplasm was sometimes
restricted to the material of this particular object.*" The word

8 CYTOLOGICAL TECHNIQUE

Archoplasma was coined later to indicate the distinction between
ttiis material and the rest of the protoplasm.*^ It seems probable
that the archoplasm of blastomeres corresponds to the idiozome
of spermatogonia and other cells, though this has been
denied. ■^^

Sinuous rods or threads are seen in all parts of the cytoplasm ;
they are particularly abundant in the neighbourhood of the
idiozome, but they are never seen within the latter. These are the
thread-granules or mitochondria.^^ They often appear as granules
in fixed preparations, and not rarely as rows of granules; but
in life they are nearly always somewhat lengthened and usually
rod-shaped or filamentous. The diameter of all the mitochondria
in any one cell is commonly about the same, but the length varies
considerably: it is quite usual for the length and breadth to be
roughly proportional to those of a cigarette. The surface of
mitochondria is everywhere smooth, the ends rounded. Mito-
chondria very seldom branch. It is often said that they have a
capacity for independent movement, but it seems more likely that
motion is sometimes imparted to them by the ground cytoplasm.
We do not ordinarily see any objects in a cell that could be stages
in their growth from small rudiments, and there is strong reason
to believe that they usually multiply by transverse division.

A quarter of a century ago much was known about the
arrangement of mitochondria in different kinds of cells, but very
little about their significance for vital processes. The discovery
that they could be separated in bulk by differential centrifuging of
mashed tissue^ ^ made it possible to subject them to detailed
chemical and enzymological analysis, and today we probably
know more about their mode of action than about that of any
other part of the cell, the chromosomes not excepted. We find
in them the enzymes concerned with the degradation of pyruvic
acid to carbon dioxide and water: that is to say, with the parts
of the respiratory process known as the tricarboxylic acid cycle
and the cytochrome system. It remains to relate their function to
the complex internal structure revealed by the electron-micro-
scope.

Small lipid globules (lipochondria) are distributed in the

THE STRUCTURE OF THE CELL 9

cytoplasm, mostly in the vicinity of the idiozome (but never
within it). Nearly all cells contain lipid globules in the cytoplasm,
but they have been greatly overlooked, probably because they
are not seen in routine paraffin sections. These globules may con-
tain triglyceride, but usually consist mainly of more complex
lipids, especially phospholipids and cerebrosides. Their refractive
index is often low, and this suggests that they must contain water
associated with their phospholipid content. ^^^ Sometimes a lipid
cortex surrounds a non-lipid, probably aqueous core, but this
structure is perhaps rarer than superficial appearances would
suggest. ^8

The significance of lipid globules is obscure. Adipose fat
accumulates mainly in particular cells set aside for the purpose,
but nearly every cell contains globules of more complex lipids
that are not used up when the animal is starved. The lipids of
these globules are often highly unsaturated and therefore reduce
osmium tetroxide and silver nitrate easily.

Certain cytoplasmic inclusions are built on such a small
scale that the light-microscope cannot reveal their shape or
structure. We are forced to rely on the electron-microscope.
Since this instrument can only be applied to tissues that have been
completely dried, it is not possible to examine these inclusions
during the life of the cell. Fixed preparations must be used. A re-
action occurs between what was present in life on one hand, and
the fixative and other substances used in making the preparation
on the other. What is studied under the electron-microscope is the
reaction-product. 2" We lack the means of verification that are
available with all objects that are large enough to be studied
with the light-microscope during the life of the cell.

An electron-micrograph is generally regarded as 'good' if it
shows much minute detail, or if the constituent parts appear in
strong contrast with one another; but we have no means of
knowing whether the details represent the relative positions of
the parts in the living cell, nor do we know whether these parts
were sharply separated from one another.

The only way in which we can learn to interpret electron-
micrographs is by the study of known substances (especially

10 CYTOLOGICAL TECHNIQUE

proteins, lipoproteins, and lipids) that have been prepared for
electron-microscopy in the same way as pieces of tissue. This kind
of work is still in its infancy, but a most interesting start has
been made with phospholipids.^^® These appear in electron-
micrographs in the form of parallel membranes, often arranged
concentrically. Similar membranes are often seen in electron-
micrographs of cells.®" » ** They are called 'Golgi apparatus' by
many electron-microscopists. It seems better to regard them
provisionally as phospholipids.^"' ^^ These substances sometimes
occur free in the cytoplasm of the living cell, but sometimes they
are bound up in lipoprotein complexes. Certain fixatives and
other reagents used in microtechnique 'unmask' the phospho-
lipids of these complexes: that is to say, they set them free from
combination with protein.®^ In an electron-micrograph there may
be nothing to indicate in which form the phospholipid was
present in the living cell.^°' ^^

Reaction-products of two kinds occur repeatedly in the cyto-
plasm of very diverse cells. They appear to represent two distinct
objects, which may perhaps be of universal or almost universal
occurrence. These are the endoplasmic reticulum and the 'small
particles' of Palade (fig. 2).

The endoplasmic reticulum is a three-dimensional network

Fig. 2 {plate). A, electron-micrograph representing part of the
cytoplasm of a large phagocytic cell {macrophage) derived from
the blood of the domestic fowl. The micrograph is very exceptional
in representing not a section, but a whole mount of an extremely
thin cell.

f, fold in cell membrane; gc, ground cytoplasm {in the strict
sense); 1, lipid droplet; m, mitochondrion {unusual in being
branched); r, rounded component of the endoplasmic reticulum;
t, trabecular component of the endoplasmic reticulum.

B, electron-micrograph representing a section of a small part of
the basal region of an acinar cell from the pancreas of the rat.

cm, cell membrane ; qv, flattened components of the endoplasmic
reticulum, seen in transverse section; sp, small particles of Palade.

Reproduced from the Journal of Experimental Medicine^*' and the Journal of
Biophysical and Biochemical Cytology, '*" by kind permission of the Editors of
the journals and of Dr G. E. Palade and Dr K. R. Porter (authors of the papers
in which the micrographs first appeared). The lettering on the micrographs has been

altered.

r- TP" ffir-7

B

'■ €

• j^

cm -r-*i

:^^p

id

0-5

^

THE STRUCTURE OF THE CELL 11

exitending through most parts of the cytoplasm. It commonly
consists of rounded components (fig. 2, A, r), joined by narrower
elements that may be called trabeculae (t). The material forming
the network is seen in sectional view to be dark externally and
pale internally, and this has given rise to the opinion that the
reticulum is hollow. If so, the rounded components are vesicles
and the trabeculae are tubes, and all or nearly all the cavities are
continuous. There is, however, no rigorous proof that this is so.
It may be simply that the external layer has a greater affinity for
osmium than the internal part: we do not know which is the
denser.

The swollen components of the endoplasmic reticulum are not
always rounded. Sometimes they are flattened and seem to
resemble sacks piled one on another. In section the flattened
*sacks' appear as elongated spaces (fig. 2, B, er), though here
again we have no proof that the objects are in fact hollow. They
are connected with one another, like the rounded elements, by
trabeculae, which again may or may not be tubular.

Although the flattened components of the endoplasmic reticu-
lum are not individually recognizable under the light-micro-
scope, yet large piles of them do sometimes reach microscopical
dimensions. A well-known example is the striated material seen
at the base of the exocrine cells of the pancreas. Striated cyto-
plasm of this kind was long ago named ergastoplasme,^^^ ®^ and
the word is still used to denote this particular modification of the
endoplasmic reticulum. The Nissl bodies of the neurones of
vertebrates are similar in submicroscopic structure to the basal
cytoplasm of the exocrine cell.^*^

There is not yet any sure knowledge of the significance for the
life of the cell of the object that is represented by endoplasmic
reticulum in electron-micrographs.

The name 'endoplasmic reticulum' is an unfortunate one. The
object was first recognized in certain sarcoma cells, in which a
very narrow, hyaline, marginal zone could be distinguished from
the internal endoplasm. The reticulum was called endoplasmic
because it was retricted to the inner zone.^^^' ^^^ The difi"erentia-
tion of a special external zone or ectoplasm from a less hyaline

12 CYTOLOGICAL TECHNIQUE

endoplasm is found especially in free-living Protozoa and in
certain other cells that are directly exposed to a non-cellular
environment. It is far from being characteristic of cells in general.
It is unusual in those that are bounded by other cells, and does
not occur in spermatogonia. Yet it is thought likely that all cells
contain a cytoplasmic inclusion that would appear in electron-
micrographs as the 'endoplasmic' reticulum.

The 'small particles' associated particularly with the name of
the American cytologist Palade^*" are far too minute to be seen
separately by the light-microscope. There is strong reason to
suppose that they contain a high proportion of RNA, in the form
of ribonucleoprotein, and this acidic substance has an affinity
for basic dyes (p. 90). Cytoplasm that is seen by light-microscopy
to have a special affinity for these dyes can often be shown
by electron-microscopy to possess small particles in particularly
great abundance. In spermatogonia the small particles are few
and the cytoplasm has little affinity for such dyes.

It is not possible to tell exactly where the small particles were
in life, but in electron-micrographs many of them are usually
situated on the outer side of the endoplasmic reticulum, which
looks as though it has been peppered with them (fig. 2, B, sp).
In some kinds of cells, however, the small particles are very
numerous but separate from the reticulum. It is for this reason
that these two cytoplasmic inclusions, so often associated, must
be listed separately.

It is wise to regard with a certain amount of scepticism the
information about cellular constituents provided by the electron-
microscope. Nevertheless, one cannot fail to be struck by the
regularity with which the endoplasmic reticulum and small
particles appear in micrographs.

It remains to consider the ground cytoplasm. This term is used
in two distinct senses. In light-microscopy it means the whole of
the substance of the cell other than the visible inclusions. In this
wide sense it thus includes the endoplasmic reticulum and small
particles. When it is stated, for instance, that the refractive index
of the ground cytoplasm is commonly about 1-353^^^' ^^^ or that
its pH is generally between 6-4 and 7-2, the figures given must be

THE STRUCTURE OF THE CELL 13

taken to represent a summation of the refractive index and pH
of the substance, taken as a whole, that lies between the micro-
scopically-visible inclusions. In the strict sense, however, the
term 'ground cytoplasm' must apply only to the material in which
the submicroscopical inclusions are embedded (fig. 2, A). In this
sense, then, it excludes the endoplasmic reticulum and small
particles. Even when the reticulum is bulky and the particles
numerous, a large proportion of the cell is occupied by the
ground cytoplasm in the strict sense. It is regrettable that we
should know so little of this cellular component. In electron-
micrographs that are considered 'good', the space in question is
often criss-crossed by very fine fibres. These are thought to be
formed of protein. We can scarcely be far wrong in supposing
that the ground cytoplasm in the strict sense consists mainly of
water and protein, associated with one another through the
hydrophil groups of the protein (p. 20). The latter may be
supposed to exist partly as long, extended polypeptide chains
(p. 19), linked to one another here and there to form a weak gel,
partly as globules consisting of such chains wound up into
skeins. ^° The water must hold some of the cytoplasmic ions in
solution (principally potassium and phosphate, with some mag-
nesium and bicarbonate, and other ions in lesser amount).

Since this book is concerned mainly with light-microscopy, the
expression 'ground cytoplasm' is to be understood in the wide
sense, except where the contrary is distinctly indicated.

CHAPTER 2

Introduction to Fixation

There is only one really reliable criterion by which we can
determine whether the image that we see with the microscope is a
good representation of what existed in life, and that criterion is
comparison with the living cell. We have nowadays several
excellent ways of overcoming the natural transparency of this
object. Phase-contrast and interference microscopy are the chief
methods, and vital dyeing, though at the moment partially
eclipsed, remains potentially as illuminating as ever. Since we
possess these reliable means of obtaining knowledge, it may
reasonably be asked why we need also the elaborate procedures
that form the subject-matter of this book.

Many kinds of cells cannot be isolated for separate study while
still alive. Such cells can only be examined in permanent prepara-
tions. It is a great advantage to be able to cut tissues and cells
into thin slices. The relations of the cells to one another and to
the intercellular matter are much better shown in this way than
when cells are teased apart for direct study while still alive. The
living cell of many-celled organisms cannot be sectioned effec-
tively. Even if a slice could be cut, it would not remain alive nor
retain its form. Tissues and cells need to be stabilized in structure
and held firmly while being sliced. In fact, they require to be
'fixed' more or less in their living form and then embedded in
some solid material before they can be sectioned. When the pro-
cess of fixation has been carried out, nearly all their parts can be
dyed in contrasting colours, and this makes observation much
easier. Vital dyeing, invaluable though it is, is applicable only to
particular components of the cell. Again, few histochemical tests
are applicable to unfixed tissues. Beyond all this, it is a conveni-

14

INTRODUCTION TO FIXATION 15

epce to be able to store away permanent preparations, ready
for instant examination at any time.

For these reasons it is usual to fix a piece of tissue in a fluid
called a fixative; to embed it after fixation in some solid medium,
such as parafiSn wax, that will hold its constituent parts in the
right relation to one another during sectioning; then to section
it; next to dye the sections, often in two or more contrasting
colours ; and finally to mount the dyed sections in a medium that
renders them transparent.

In this book we are concerned with the principles underlying
fixation, embedding, dyeing, and mounting. We are not imme-
diately concerned with the application of histochemical tests, a
subject to which another volume in this series of Biological
Monographs is devoted. ^^ Nevertheless, the present work is
largely chemical in outlook, and the preparation of tissues for
study follows the same general routine whether a histochemical
test is to be applied or not. It is therefore hoped that the book may
be useful to those whose main interests are in histochemical or
cytochemical analysis. We shall study what happens when we fix,
embed, dye, and mount tissues (and especially the cells that they
contain).

If a piece of tissue is cut out of a living or recently dead organism
and no special care is taken to keep it alive or maintain its
structure, it will soon undergo marked changes. If left in the air,
it vwll lose water by evaporation and shrink; if left in a fluid, it is
likely to undergo osmotic swelling or shrinkage. If these distor-
tions are prevented, it will still be subject to attack by bacteria
and moulds. Even if these are excluded by asepsis, the tissue will
gradually fall to pieces by self-digestion or 'autolysis'."^* Cells
contain enzymes, collectively known as 'cathepsin', capable of
dissolving their own protein constituents when they die. These
enzymes (two proteinases, a carboxypeptidase, and an amino-
peptidase) show a remarkable resemblance to those of the diges-
tive tract. In life their function is presumably synthetic.

To preserve a piece of tissue one requires a fluid that will not
shrink or swell or dissolve or distort; will kill bacteria and

16 CYTOLOGICAL TECHNIQUE

moulds; and will render the autolytic enzymes inactive. Such a
fluid is a preservative. A fixative must do everything that a pre-
servative does, but in addition it must modify the tissues in such
a way that they become capable of resisting subsequent treat-
ments of various kinds. Of these treatments the ones that are
most likely to cause damage are embedding, sectioning, and
mounting (though the latter is not damaging if the tissue has
already been severely shrunken while being embedded). Apart
from the partial protection it gives against damage from these
processes, fixation usually makes many tissue-constituents
(especially chromatin) readily colourable by suitable dyes.

Not every tissue-constituent requires fixation, and some are
not capable of being fixed. Chitin, cellulose, starch-grains,
scleroproteins, amorphous silica, and certain inorganic crystals
are examples of hard, stable substances, scarcely or not at all
subject to distortion or decay. Other substances, such as the
soluble sugars, cannot be retained in their natural sites in the
tissues by any fixative. Glycogen is the only soluble carbohydrate
that is at all frequently retained in finished microscopical
preparations.

Though usually soft or indeed liquid, triglycerides do not re-
quire fixation if lipid-solvents are avoided in subsequent treat-
ment. Some fixatives have no effect on lipids, but this does not
by any means make them useless, for neither the cell nor inter-
cellular material is held together by lipids. If, however, the com-
mon proteins are not stabilized, tissues and cells fall to pieces.
The essential function of fixation is the stabilization of the
protein framework of the cell.

Fixation can be achieved by heat, but this method is seldom
used except for blood-films. It tends to cause distortion, but
presents the advantage that nothing is dissolved out of the cell.

Most fixatives are solids used in aqueous solution, but some
are organic liquids that can be used without dilution. The number
of chemical compounds that are useful as fixatives is very small.
The seven unmixed or 'primary' fixatives chosen for mention in
this book are listed below. All fixatives fall easily into two groups,
according to their obvious reactions with soluble proteins. Some

INTRODUCTION TO FIXATION 17

of them, when mixed with a solution of albumin, produce a
coagulum: others do not. The ones that produce a coagulum in
a test-tube transform homogeneous protoplasm into a micro-
scopical spongework.

Against each fixative in the list below is marked its 'standard
concentration'. This is the concentration at or near which it is
commonly used in fixation. Throughout the book, except where
the contrary is distinctly stated, it is to be understood that when
reference is made to one of the seven selected primary fixatives, it
was used at the concentration shown in the list (or at a concentra-
tion so close to this that no diff"erence in result could be
anticipated).

COAGULANTS

Ethanol (ethyl alcohol). C0H5OH. A light, colourless fluid,
miscible with water in all proportions. Standard concentration,
undiluted (absolute).

Mercuric chloride. HgCL. Colourless crystals, soluble in water
at about 7%. Standard concentration, saturated aqueous
solution.

Chromium trioxide. CrOg. Brownish-red crystals, giving a
strongly acid solution in water, in which it is extremely soluble.
Standard concentration, 0-5% aqueous.

NON-COAGULANTS

Formaldehyde. H2CO. Colourless gas, very soluble in water.
Standard concentration, 4% aqueous.

Osmium tetroxide. OSO4. Pale yellow crystals, soluble in water
at about 7%. Standard concentration, 1 % aqueous.

Potassium dichromate. KoCraOy. Orange-red crystals, giving a
weakly acid solution in water, in which it is soluble at about
10%. Standard concentration, 1-5% aqueous.

Acetic acid. H3C.COOH. Colourless liquid, miscible with
water in all proportions. Standard concentration, 5%.

Since each of the primary fixatives has its virtues and defects,
c

18 CYTOLOGICAL TECHNIQUE

it is usual in practice to mix two or more of them together.
Fixative mixtures will be considered in chapter 5 (p. 59). Their
effects on tissues can only be understood when those of their
components are known. Further, the proportions of the primary
fixatives in the mixtures have been very arbitrarily chosen. For
these reasons, any scientific account of fixation must start with
the primary fixatives and be concerned mainly with these.

Small pieces of tissue are best fixed by direct immersion, since
this brings the fixative most rapidly into contact with the cells
throughout the piece. When it is necessary to fix a piece of tissue
a centimetre or more thick, it is best to inject the fixative through
a blood-vessel in order to send it quickly to all depths. This
method of 'perfusion' has the disadvantage that the total amount
of fixative that can be contained in the blood-vessels is usually
small, and nothing outside the vessels is fixed until the fixative
substance has passed through their walls into the intercellular
spaces and thus reached the cells.

The purpose of fixation is usually to stabilize the tissues so that
they retain as nearly as possible the form they had in life, but
clearly it is not the purpose to leave their chemical composition
unchanged. This would indeed be the negation of fixation, for
the cells would be still alive or like recently dead ones. The
purpose is to change the chemical composition in such a way as
to confer structural stability. This fact by no means makes
histochemical studies impossible, for certain tissue-constituents
may be left unaltered, and others only altered in part.

The structural formula for amino-acids in general is con-
veniently written in the way shown here, because such formulae
may easily be joined together to make a protein chain. The letter
R in the formula represents any of the radicles that distinguish
the various amino-acids from one another. In forming a protein,
the amino-acids condense together with elimination of one

( II H
molecule of water at each peptide link \-C — N->

In the formula representing part of a protein chain, certain

conventions have been adopted. The radicles characteristic of

INTRODUCTION TO FIXATION 19

NH2
HCR HC.CHa^ )OH tyrosine

c=o

I
OH

General formula for HC(CH2)2C^ glutamic acid

amino-acids \ \OH

I

Three amino-acids as part
of a protein chain

each amino-acid are written on the right of the formula in each
case, although in fact they project in different directions; and the
repeated part or backbone of the formula is shown as straight,
though in nature it is folded, either to form a simple zigzag or in
more complex ways. These conventions will be found to simplify
the explanation of the reactions of proteins with fixatives and
dves.

The first requirement of a fixative is that it should not be
proteolytic. Any substance that breaks the peptide links and sets
free soluble amino-acids is the opposite of a fixative. The changes
produced by a fixative must tend in the other direction, towards
stability.

One may broadly distinguish two kinds of fixation of protein,
additive and non-additive. In the form_er, certain atoms of the
fiixative combine chemically with some part of the protein and
remain in combination. In the latter, no such obvious addition of
atoms occurs. Ethanol is a non-additive fixative in the sense that
none of its constituent atoms joins itself to protein, so far as is
known. The 'nature' of the protein is, however, profoundly
changed by its action, and the substance is therefore said to be

20 CYTOLOGICAL TECHNIQUE

'denatured'.^' ^' ^^^' ^^^ The most obvious change is loss of solu-
bihty with resultant coagulation.

When a denaturing fixative is added to a solution of albumin,
coagulation usually follov^s so quickly that it appears to be
instantaneous. In fact, however, denaturation can be shown to
proceed by stages. The first eff'ect is an increase in reactivity;
there then follows a flocculation or aggregation into minute
particles, which are soluble in weak acids and alkalis; finally the
flocculi join into a clot, soluble only by reagents that cause
proteolysis. Flocculation and clot-formation are both included
in the term coagulation. Throughout all these changes the back-
bone of the protein remains unaltered. Some authors mean by
denaturation almost any change in a protein that increases
reactivity but does not destroy the backbone. ^^^ In this book the
term will be used in a more limited sense to indicate a marked
change involving the relation of the protein with the surrounding
water. This change results in coagulation.

The chief hydrophil groups of proteins are the -NHg and

O

-C\ groups of basic and acidic amino-acids, the -OH group

I
of tyrosine, and the C=0 groups of the backbone. Whether a

particular protein be dispersed as a sol or held together by cross-
links to form a gel, it will ordinarily be related to water molecules
by such groups as these. When a protein is denatured, the rela-
tions with water are somehow disturbed. It may be supposed that
ethanol, a dehydrating agent, competes with the protein for the
water, and that the active groups, now no longer associated with
water, are more free to make fresh bonds with one another. The
formation of fresh bonds, so close as to exclude much of the
water that formerly lay between protein chains, would cause
coagulation. Interfaces between 'dry' protein and the surround-
ing fluid would thus be formed; and since these would scatter
light, the transparent sol or gel would change into a white
coagulum.

Various reactive groups that were present in the unaltered

INTRODUCTION TO FIXATION 21

protein in a latent condition are unmasked by denaturing and
now respond to tests for their presence. This applies to various
ionizing side-chains of the constituent amino-acids, especially
the -SH of cysteine, the -S— S- of cystine, the phenyl of tyrosine,
and the indolyl of tryptophane. The cause of this increase in
reactivity is not known, but it may be a straightening of much-
folded backbones and consequent exposure of previously-hidden
side-groups. The protein is now more accessible both to dyes and
to digestive enzymes. The specificity of the original protein is at
the same time generally lost. This is most evident if the protein is
or forms part of an enzyme.

Proteins can be denatured by very diverse means. Heat alone
is effective, if water be present; in the complete absence of
moisture, however, even a temperature of 100^ C does not suffice
to denature. Exposure to ultraviolet light or ultrasonic waves,
subjection to very high pressure, and extension in extremely thin
films can cause the denaturing of proteins, but these methods are
not ordinarily used in microtechnique.

In additive fixation a part or the whole of the fixative molecule
adds itself to the substance that it fixes, by making ionic or
covalent links. In the fixation of proteins, the link is usually with
the side-groups of one or more particular amino-acids. Additive
fixation is sometimes coagulant, sometimes not.

An additive fixative need not necessarily alter the reactions
of a protein very profoundly. For instance, it might act only on
tyrosine side-groups, and if these were sparsely represented in a
particular protein, most of the amino-acids might be able to
retain their character and show their usual responses to reagents.
The rest of the protein might undergo changes similar to those
that occur in non-additive fixation. Thus there need not be a very
sharp distinction between the two kinds of fixation, so far as
the protein as a whole is concerned. Nevertheless, it is convenient
to restrict the term 'denaturing' to cases in which the protein
does not undergo chemical combination.

Diff'erent primary fixatives penetrate into tissues at diff"erent
rates. Other things being equal, it is desirable that a fixative
should penetrate quickly, so that the tissue may be stabilized in

22 CYTOLOGICAL TECHNIQUE

Structure before autolysis has damaged it. In designing fixative
mixtures it is helpful to know the relative rates of penetration of
the components, for ideally one component should not outstrip
another.

To study the rate of penetration of fixatives, it is best to begin
by using a homogeneous protein gel in place of a piece of
tissue. ^^"' ^^ A suitable gel may be made by dissolving gelatine
at 15% in warm white-of-egg.^^ This material may be used as a
crude model of cytoplasm, for its refractive index (and hence its
protein-content) is about the same. While still warm it can be
drawn into glass tubes, and the gel then allowed to set. If one end
of the tube is inserted into a solution of a coagulant fixative, the
rate of penetration can easily be noted, since the material becomes
white and opaque on coagulation. To find how far a non-
coagulant fixative has penetrated, it is only necessary to turn the
tube upside down in a bowl of warm water, for the unfixed gel
then runs out, while the fixed material remains in the tube.^^ It
will be understood that what is measured in these experiments is
the distance through which the substance under test has moved at
the concentration that is just sufficient to fix the gel.

Fixatives penetrate into the gel rapidly at first and progres-
sively more slowly as time goes on. It has been shown that nearly
all fixatives move forward in accordance with the laws of
diffusion. ^^° If d is the distance penetrated in time /, and ^ is a
constant depending on the fixative used, then

d =K\/t.

Distance may be measured in mm and time in hours. K then
represents the distance in mm through which the fixative has
moved forward in one hour, at a concentration sufficient to fix
the protein.

It is convenient to transform the equation just given into its
logarithmic form,

log d ^\ogK + ^log /.

This, when graphed, will necessarily give a straight line. If a
set of figures for <i and /, obtained in an experiment, are converted
into logarithms and marked on a graph, it will at once be seen

INTRODUCTION TO FIXATION

23

whether they he approximately on a straight line. This is so with
nearly all fixatives. (See, for example, fig. 3.)
Where the straight line crosses the ordinate line, log / has

0-2

0-2 0-4 0-6 0-3

1-0 1-2
Log t

1-4 1-6 1-a 2-0 22

Fig. 3. Graph showing the rate of penetration of mercuric chloride
(at standard concentration) into gelatine-albumin gel. t is time in
hours; d is the distance penetrated in mm.'^^' '*

become zero and log d = log K. One can therefore read off the
value of log K on the ordinate line, and the antilogarithm of the
figure obtained is therefore K. In the case of mercuric chloride

24 CYTOLOGICAL TECHNIQUE

penetrating into gelatine-albumin gel, the K-va\ue is 2-27. Form-
aldehyde shows the highest ^- value (3-6) among the seven selected
primary fixatives. Chromium trioxide is slow {K = 1-12).

It is convenient to make the units of the abscissa-scale (log t)
one-half of those of the ordinate-scale (log d). Then, if the frac-
tion before log t in the logarithmic equation is ^ (that is, if d is
proportional to the square root of t), the slope of the line will be
45°. This is so with most fixatives (see fig. 3).

Few tissues are sufficiently homogeneous to give clear-cut
results in tests of rate of penetration. Liver, however, is suitable,
and certain coagulant fixatives leave a clear indication of how far
they have moved into this organ, since the fixed tissue is whitish
and opaque. Experiments with mercuric chloride show that here
also the laws of diffusion are obeyed. The X^-value of mercuric
chloride diff'using into liver is 0-84. So far as is known, all fixatives
penetrate much more slowly mto liver than into gelatine-albumin
gel, perhaps because they are held back by the lipids of cell-
membranes and ground cytoplasm. There is, however, a general
agreement in the results obtained with gels and liver. Thus
chromium trioxide penetrates much more slowly than mercuric
chloride into both.

It is important to realize how rapidly the rate of penetration
of fixatives falls off" with time. With a ^-value of 0-84, mercuric
chloride penetrates into liver a distance of 20fx (the diameter of a
large cell) in just over two seconds. That is to say, it penetrates
this small distance at the rate of 36 mm an hour; but actually it
only penetrates 0-84 mm in one hour, and it would take 77 days to
penetrate 36 mm. These facts emphasize the importance of using
small pieces of tissue for fixation. Pieces several cm thick are only
adequately fixed in their external parts, unless they are perfused
with the fixative.

Instructions are often given as to the time during which a
particular fixative should act, but it is usually safe to disregard
these. For light-microscopy it is convenient as a general rule to
fix overnight (say 18 hours). Little difference will in many cases
be noticed if the period is extended to several days, or even weeks
or years. A shorter time than 18 hours suffices for the pieces used

INTRODUCTION TO FIXATION 25

in cytology, which are seldom more than 3 mm thick, but over-
night fixation is not harmful. In electron-microscopy osmium
tetroxide is the most usual fixative. There is some evidence that
proteins may eventually be dissolved by this fixative, after having
been well fixed at first. Pieces about 1 mm thick are used, and it is
quite usual to fix only for an hour or two, or even for less than
an hour.

The structure exhibited in a fixed microscopical preparation is
alm.ost always to some extent artificial, for no fixative is perfect.
Artifacts are of two kinds, extrinsic and intrinsic.

Extrinsic artifacts are those formed of material brought in by
the fixative and deposited in the tissues. One may take as an
example the black granules often seen in material fixed in mer-
curic chloride solution (p. 36). Artifacts of this kind can usually
be avoided. They are commonly caused by the reaction of the
fixative left in the tissues with fluids in which the tissues are
subsequently immersed. An extrinsic artifact, once formed, may
often be removed by the use of special solvents.

The intrinsic artifacts are the distorted structures of the
tissue-constituents themselves. Very great changes are undergone
by a cell when it is fixed by a coagulant fixative, embedded in
paraffin, sectioned, dyed, and mounted; and fixation itself is the
chief cause of these changes. What happens may be studied by
comparing fig. 4 with fig. 1 (p. 4). The cell is irregularly
shrunken, and the ground cytoplasm is transformed into a
coagulum consisting of a spongework of protein strands. No
part of the original substance of the cell is left in the meshes of
the network, which are filled only with the mounting medium.
The size of the meshes depends on the particular fixative used.
They are often even larger than those shown in the diagram
(fig. 4). The coarseness of the coagulum is not always realized,
because the strands cross one another at different depths in the
thickness of an ordinary section, so as not to leave any very
obvious gaps, and rather feeble dyes are generally used for this
part of the cell ; but if a crudely coagulant fixative is used, and
very thin sections are strongly dyed with iron haematein (p. 121),
the gaps become obvious.

26

CYTOLOGICAL TECHNIQUE

spaces filled
with mounting
medium

coagulated

ground cytoplasm

precipitate
DMA

Fig. 4. A typical spermatogonium, as seen in a routine micro-
scopical preparation {diagrammatic). The cell has been fixed in
a coagulant fixative, embedded in paraffin, dyed, and mounted.

Compare with fig. 1 (p. 4).

No traces of the mitochondria or lipid droplets remain. The
centriole, if present, is intact; the idiozome sometimes but not
always persists. The nuclear sap has been coagulated, like the
ground cytoplasm. The heterochromatic segments of the chromo-
somes are still visible and strongly dyed, but the rest of the
DNA has been thrown down in the form of an irregular pre-
cipitate scattered here and there on the strands of the coagulum
and on the inside of the nuclear membrane. The nucleoli retain
their form but are somewhat shrunken.

The non-coagulant fixatives, especially osmium tetroxide and
formaldehyde, cause much less initial change in structure than
the coagulants. Indeed, while the cells still lie in these fixatives

INTRODUCTION TO FIXATION 27

they often look remarkably lifelike. Embedding in paraffin, how-
ever, usually causes serious distortion.
Ideally a fixative would leave the volume'of a piece of tissue

volume as percentage of original volume

100 200 300 400

chromium tri oxide

osmium tetroxide

mercuric chloride

formaldehyde

potassium dichromate

acetic acid

Fig. 5. Diagram showing the changes in vohime undergone by
gelatine-albumin gel when acted upon for 18 hours by the selected
primary fixatives. The latter were used at their standard

concentrations.-^

and of its constituent cells unaltered, and resistant to alteration
by fluids in which it was subsequently placed. Many fixatives
shrink tissues; some leave them scarcely changed in volume,
while others swell them, but in any case the tissue is liable to
subsequent shrinkage.

The results of shrinkage and swelling lend themselves more

28 CYTOLOGICAL TECHNIQUE

readily to quantitative analysis than do the other intrinsic arti-
facts caused by fixatives. Interesting experiments can be made
with the same gelatine-albumin gel as is used in studies of rate of
penetration. The results are shown in fig. 5. Acetic acid swells
strongly, and indeed this is one of the main reasons why it is
included in so many fixative mixtures, for the swelling counter-
acts the shrinkage caused subsequently by embedding, espec-
ially if the swollen cells are fixed in their swollen state by a
coagulant fixative (see p. 62). Fig. 5 shows that most of the
primary fixatives do not alter the volume of the gel greatly, but
ethanol shrinks it strongly.

Whole animals or whole organs may be measured before and
after fixation, and at various stages in the subsequent processes of
microtechnique; or cubes may be cut out of large organs and
treated in the same way. The most interesting results, however,
are those obtained by the study of single cells. It is best to choose
loose, spherical cells for this purpose, since they are simplest
from the geometrical point of view. The eggs of echinoids are
suitable in this respect. Some results are shown in Table 1.

TABLE 1

The volumes of unfertilized eggs of the echinoid, Arbacia pustulosa, at
various stages of preparation for microscopical study, expressed as
percentages of the volumes while alive in sea-water. Each figure is
calculated from the mean diameter of 10 to 15 eggs. {Data of Hertwig^^)

Fixative

Volume in

fixative,
24 hours

ethanol,

70%

ethanol,
abs.

xylene

formaldehyde (in

sea-water)
ethanol

105
48

70

50

48
41

Comparable results are shown by studies of the spermatocytes
of the common snail. Helix aspersa.^^^ When they have been
fixed in formaldehyde solution, embedded in paraffin, sectioned,

INTRODUCTION TO FIXATION 29

and mounted in Canada balsam, their volume is only 34% of
what it was when they were alive; yet formaldehyde is superior
to the six other primary fixatives in this respect. Ethanol is the
worst of all: the final volume is only 19% of the original.

The addition of 'indiff'erent' or non-fixative salts to fixatives
often improves the results obtained. A saline solution may be
prepared, having about the same osmotic pressure as the body-
fluids of the organism from which tissue is to be taken, and the
fixative substance dissolved in this instead of distilled water. It
follows that the complete solution is hypertonic. This would be
thought likely to cause great shrinkage of the cells, but in fact
shrinkage is reduced by the presence of the indifferent salt, and
indeed that is precisely why it is used. The way in which it acts is
obscure. ^^ The use of an indifferent salt is particularly helpful
when the fixative is chromium trioxide or formaldehyde.

To make a general study of the artifacts caused by fixation,
separate cells are often examined in a suitable saline solution
while still alive, and a fixative is then allowed to run under the
coverslip. The immediate effects are thus easily observed. The
classical work of this kind was done by dark-ground micro-
scopy.^" The introduction of phase-contrast microscopy resulted
in a renewal of interest in work of this kind.'*'' ^^' ^^' The method
is valuable but open to two objections. First, it is clearly a test
of preservation, not of fixation. Secondly, it shows only how
fixatives affect cells that come into immediate contact with them.
Many valuable fixatives damage the most superficial cells of a
piece of tissue but give good results at a little depth below the
surface. When separate cells are examined, they all react like
superficial cells, and the fixative may be wrongly condemned.

To overcome these objections, small pieces of suitable tissue
may be fixed, embedded, sectioned, dyed, and mounted, and the
resulting preparation carefully compared with what can be seen
in living cells from the same material. It is best to choose tissues
that are readily available but difficult to fix well. The testes of
various animals (mammals, urodeles, insects) are suitable. ^°' ^^
Embedding in paraffin is particularly likely to cause distortion,
and this medium should therefore be chosen for a rigorous test.

30 CYTOLOGICAL TECHNIQUE

The finished slides of the same material fixed in different ways
should be carefully compared by an observer who does not know
which is which. There are strong prejudices in favour of parti-
cular fixatives, and few cytologists would be able to judge the
slides impartially if they knew which fixative had been used in the
preparation of each.

The difficulty of judging electron-micrographs objectively has
already been mentioned (p. 9).

I

CHAPTER 3

Coagulant Primary Fixatives

In this chapter and the next each of the selected primary fixatives
(p. 17) will be considered separately. Its properties will be sum-
marized in a systematic way. The intention is to give as complete
a summary of the fixative action of each substance as is possible
in the present state of knowledge. The information will include
a good deal that has been scattered through chapter 2.

Since the present chapter and the next are likely to be used for
reference by some who have not read the earlier part of the book,
it is necessary to explain once more what is meant by 'standard
concentration'. This is a fixed concentration, approximately that
at which the substance in question is commonly used in fixation.
Throughout this book, except where the contrary is stated, every
remark about the action of a primary fixative refers to its action
at (or very near) its standard concentration.

The expressions w/v, w/w, &c. are defined on p. xv.

The oxidation-potentials (o.p.) quoted in chapters 3 and 4 are
taken from Casselman.^^- ^^ The Z^-values given in these chapters
refer to the rate of penetration into gelatine-albumin gel (p. 22),
or, in the case of acetic acid, into gelatine-nucleoprotein gel.

Fixatives that stabilize the proteins of the cell quite well may
leave much to be desired where cell-aggregates are concerned, for
these may be distorted during fixation or embedding, in such a
way as to leave artificial spaces. Groups of cells may become
separated from other groups or from basement membranes or
connective tissue. Although this book is primarily concerned with
cvtology, yet the needs of the histologist and microscopical
anatomist must be kept in mind, and the eff'ects of the selected
primary fixatives on cell-aggregates will therefore be briefly noted.

31

32 CYTOLOGICAL TECHNIQUE

The properties of the selected fixatives are summarized on the
pages Hsted here :

coagulant

ethanol . . . . . . . p. 32

mercuric chloride . . . . . p. 34

chromium trioxide . . . . . p. 37

non-coagulant

formaldehyde . . . . . . p. 40

osmium tetroxide . . . . . p. 45

potassium dichromate . . . . p. 49

acetic acid . . . . . . p. 52

When added at the proper concentration to a solution of
albumin, coagulant fixatives separate the protein from the water
as a curd or coagulum (p. 20). This reaction serves to define them.
They transform the proteins of the cytoplasm (and often the
nuclear sap as well) into a sponge-work. This does not necessarily
destroy structure at the microscopical level, since the meshes of
the spongework may be very fine ; but non-coagulant fixatives are
nearly always used in electron-microscopy. The production of a
spongework is helpful to the penetration of embedding media,
especially paraffin. The coagulant fixatives are much less diverse
in their action on cells than the non-coagulants are.

ETHANOL (ethyl alcohol)

Standard concentration. Undiluted (absolute).

Formula. C2H5OH.

Description. A light, colourless fluid, miscible with water in all
proportions.

Ionization. Not ionized.

Oxidation-potential. Low (95% ethanol, 0-45 volt).

Reactions with proteins. A non-additive or denaturing coagu-
lant of many proteins. It does not coagulate zein, gliadin, or
nucleoprotein. Ethanol is a powerful 'unmasking' agent for lipids ;
that is to say, it sets them free from combination with protein. It
acts in this way when used as a fixative^ ^ and also when used after

COAGULANT PRIMARY FIXATIVES 33

the tissue has been fixed by a substance that does not itself
unmask. ^^

Reactions with nucleic acids. Precipitates but does not fix them.

Reactions with lipids. These are not chemically changed by
ethanol, which tends rather to dissolve them than to fix them.
This tendency, however, is weaker than is commonly supposed.
Tripalmitin and tristearin are insoluble in cold ethanol, and so are
several other common lipids of the tissues. Triolein, lecithin, and
oleic acid are soluble; other fatty acids and cholesterol slightly so.

Reactions with carbohydrates. Precipitates glycogen without
fixing it.

Rate of penetration. Moderate.

Shrinkage or swelling. Shrinks excessively.

Hardening. Hardens excessively.

Method of washing out. Since ethanol is miscible in all pro-
portions with the antemedia (p. 73) used in embedding (and also
with water), and since it has no tendency to form an extrinsic
artifact, no special washing out is necessary.

Effect on the appearance of cells in microscopical preparations.
It produces a coarse coagulum in cytoplasm and nucleus, and
destroys mitochondria. Lipid droplets tend to fuse and may
dissolve.

In paraffin sections, cell aggregates are shrunken apart from
one another; cytoplasm is often piled up against the cell-
membrane on the side opposite to that from which the fixative
penetrated. Lipid droplets are not present; chromosomes are not
distinctly seen.

Most fixatives render tissues more basic or more acidic than
they were in the unfixed state, because they react with and block
the acidic or basic side-groups of certain amino-acids. As a result,
the affinity of the protein for basic and acid dyes is changed
(p. 90). Ethanol is exceptional in this respect. Since it does not
attack any of the side-groups, it leaves proteins neither more basic
nor more acidic than they were before fixation, and dyes may
therefore be used to find whether basic or acidic side-groups pre-
dominated in the living tissues. Nucleoprotein remains strongly
colourable by basic dyes, but it is not stabilized in position in the

D

34 CYTOLOGICAL TECHNIQUE

cell, and may even eventually be dissolved out. Ethanol is there-
fore a very poor fixative for nuclei as well as for chromosomes.
Compatibility with other fixatives. Compatible with mercuric
chloride, formaldehyde, and acetic acid. Since it tends to be oxid-
ized through aldehyde to acetic acid, it should not ordinarily be
mixed with chromium trioxide, potassium dichromate, or osmium
tetroxide (though its reaction with the two latter is slow).

MERCURIC CHLORIDE

Standard concentration. Saturated aqueous solution.

Formula. HgCL-

Description. Colourless, needle-shaped crystals. A covalent
compound, with much lower melting-point than most salts:
sublimes easily. Soluble in water at about 7 % ; readily soluble in
ethanol and in benzene. Very poisonous.

Ionization. Ionizes only partially, with hydrolysis, to give
[HgClJ^ (particularly abundant in the presence of other
chlorides), Hg++, hydronium ions (the pH of the standard solu-
tion is about 3-2), and other ions.

Oxidation-potential. Rather a strong oxidizer (o.p. about
0-75 volt).

Reactions with proteins. An additive, coagulant fixative. The
reactions depend on the pH at which the fixative is used.^^"

If the pH is well below (more acid than) the iso-electric point
of the protein, the -NHg groups of the basic amino-acids will

mostly be ionized as -NH3. There will therefore be electrostatic

!
NH

I +

HC(CH2)4NH3

c=o

I

Lysine forming part of a protein chain, in a solution
on the acid side of the iso-electric point

attraction between this positively charged group and the nega-
tively charged ion, [HgClJ^^, which could associate itself with

COAGULANT PRIMARY FIXATIVES 35

si::^h groups in two previously separate protein chains and thus
hold them together. The mercury thus adds itself to the protein in
the form of an ion. The reaction is promoted by the presence of
sodium chloride, since added chlorides increase the amount of the
reactive ion.

In the neighbourhood of the iso-electric point, the -NHo groups

I
NH

I
HC(CH,)4NH.,

I

c=o

I

Lysine forming part of a protein chain, in a solution
at or near the iso-electric point

of the basic amino-acids are not ionized. It is now the undissoci-
ated salt, HgCls, that reacts. Mercury can accept extra electrons
from electron-donor atoms, such as nitrogen. The compound
formed resembles the 'ammine' formed by the combination of
ammonia with mercuric chloride.^' ^ The lysine/mercuric chloride

H3N CI

\ /
Hg

/ \
H3N CI

77?^ compound of ammonia
with mercuric chloride

bond formed in this way is a very loose one. The reaction is
opposed by sodium chloride, and the coagulum produced may
indeed be dissolved (and fixation thus undone) by addition of this
salt.

In alkaline solutions the mercuric ion, Hg++, reacts with the
-COOH groups of acidic proteins; but fixatives are rarely
alkaline.

Mercuric chloride can also react with the cysteine groups of
proteins, forming a bridge that connects these groups in previ-
ously separate protein chains.

36 CYTOLOGICAL TECHNIQUE

NH NH NH

I I I

HC.CH2.SH HC.CH2.S— Hg— S— H2C.CH

C=0 C=0 C=:iO

1 I I

Cysteine as part of Mercury forming a link between cysteine
a protein chain side-groups in two protein chains

Mercuric chloride does not coagulate nucleoprotein solutions
very powerfully: there is flocculation, not formation of a coherent
clot. It acts on lipoproteins as an unmasking agent. ^^

Reactions vAth nucleic acids. Precipitates weakly.'^' ^^®

Reactions with lipids. Mercuric chloride leaves triglycerides
untouched. It forms compounds with phospholipids, but their
solubilities in lipid-solvents do not appear to have been studied.

Reactions with carbohydrates. None is described.

Rate of penetration. Moderate {K = 2-2). (See fig. 3, p. 23.)

Shrinkage or swelling. Shrinks gelatine-albumin gel and whole
livers slightly.

Hardening. Hardens moderately.

Method of washing out. Mercuric chloride leaves black par-
ticles, generally a few // in diameter, in the tissues. These are said
to consist of metallic mercury. ^^^ They can be removed by iodine
in alcoholic solution, presumably by formation of mercuric
iodide. Iodine colours the tissues, but it may be removed by
soaking in 70% ethanol or (much more quickly) by the action of
sodium thiosulphate.

2[So03l= + I2 -> [S40e]= + 21-

Thiosulphate Tetra- Iodide

thionate

Effect on the appearance of cells in microscopical preparations.
While the cell still lies in the fixative, it is better preserved than by
any other coagulant fixative. Its shape is well maintained, the
ground cytoplasm and nuclear sap are finely coagulated, and
mitochondria are not destroyed.

In paraffin sections the cytoplasm is rather badly shrunken and

COAGULANT PRIMARY FIXATIVES 37

thr: cells tend to separate from one another; chromosomes are not
well fixed ; mitochondria and lipid droplets are usually not present.

Proteins are more readily coloured by basic dyes than they are
after the action of any other fixative. The -COOH side-groups of
their acidic amino-acids are presumably intact in the usual cir-
cumstances of fixation. The special affinity for basic dyes must be
attributed in part to the coagulation of protein chains in a form
that permits the dye ions to penetrate easily between them to
reach their sites of action. There is some affinity for acid dyes,
and it is therefore clear that the amino-groups of the proteins are
not completely blocked.

Compatibility with other fixatives. Compatible with any of the
selected primary fixatives.

CHROMIUM TRIOXIDE

Standard concentration. 0-5% w/v aqueous solution.

Formula. CrOg.

Description. Brownish-red deliquescent crystals, extremely
soluble in water (a saturated solution is about 62% w/W).

Ionization. In water it forms chromic acid, HoCr04, which
cannot be isolated. A small amount of this remains undissociated
in water, while the greater part of it ionizes as dichromate,
[CroOv]^, and the rest mostly as hydrogen chromate, [HCrOJ",
with sufficient hydronium ions to give a 1 ^o solution a pH of

1.2.51,52

Oxidation-potential. This is much the strongest oxidizer of all
fixatives (o.p. about IT volt), the dichromate ion being readily
reduced to the chromic ion, Cr+++, or to chromic oxide, CrgOg.

Reactions with proteins. A powerful coagulant of albumin and
many other proteins, including nucleoprotein. Chromium trioxide
does not fix gelatine gels. It is probably an additive fixative, but
the reactions involved are not known. The affinity of proteins for
basic dyes is rather low after fixation by chromium trioxide, and
this suggests blocking of the -COOH groups of the acidic amino-
acids. It has been suggested, however, that acidic proteins are not
fixed but simply dissolve away.^^^

38 CYTOLOGICAL TECHNIQUE

Reactions with nucleic acids. DNA is precipitated from solution
in an insoluble form/^

Reactions with lipids. The fat of adipose tissue can be made
insoluble in lipid-solvents by prolonged treatment with chromium
trioxide/^*' ^^ but the ordinary period of fixation does not suffice.
The reactions with lipids have not been studied in detail, but they
probably resemble those of potassium dichromate (p. 50).

Reactions with carbohydrates. Polysaccharides are converted to
aldehydes, by oxidation, and will then respond to colour-tests for
aldehydes. ^^ It is not certain that chromium trioxide actually fixes
glycogen, but the aldehyde formed seems less liable to be dissolved
out of the tissues by water than glycogen itself is.

Rate of penetration. Slow {K = 1 0).

Shrinkage or swelling. Shrinks gelatine-albumin gel slightly,
tissues considerably.

Hardening. Moderate.

Method of washing out. It is usual to wash out in running
water. If any free chromium trioxide is left in the tissues, it may
subsequently be reduced to green chromic oxide, Cr^Oa, by
ethanol or some other reducer. The oxide is difficult to remove
from the tissues, as it is insoluble in ordinary solvents and
resistant to acids and other reagents.

Effect on the appearance of cells in microscopical preparations.
Ground cytoplasm is rather coarsely coagulated and there may be
some distortion of the shape of the cell ; the nucleus is on the
whole well preserved, the chromosomes excellently.

In paraffin sections, cell-aggregates are quite well fixed; so is
the nucleus, though the nuclear sap is changed to a coarse
coagulum; the chromosomes and nucleolus are particularly well
shown; mitochondria and lipid droplets are not seen.

Cytoplasm has a strong affinity for acid dyes and is therefore
easily dyed in a diff"erent colour from the nucleoprotein, though
the latter has not such a strong affinity for basic dyes as it has
after fixation by mercuric chloride or formaldehyde.

Compatibility with other fixatives. It can be mixed with mer-
curic chloride, osmium tetroxide, or acetic acid. If it is mixed with
potassium dichromate, the special properties of the latter sub-

COAGULANT PRIMARY FIXATIVES

39

Stance do not appear (see p. 50). As a general rule it is best not to
mix chromium trioxide with reducers, such as ethanol or form-
aldehyde, with which it reacts. A few useful fixatives, however,
contain chromium trioxide and formaldehyde.^ '^

>166

>. r :

K-

r^

^■P

CHAPTER 4

Non-Coagulant Primary Fixatives

The non-coagulants form rather a heterogeneous group. Form-
aldehyde and osmium tetroxide resemble one another in being
additive non-coagulants that harden protein gels without separat-
ing the water from the protein in them, and fix protoplasm with-
out producing microscopical spongeworks. Potassium dichromate
and acetic acid are anomalous. They are not fixatives for simple
proteins. The former is used chiefly for its action on certain lipids,
the latter for its swelling effect, which counteracts the shrinkage
caused by other reagents used in fixation and embedding.

Formaldehyde and osmium tetroxide are of outstanding impor-
tance in modern microtechnique. The former is much used in
histochemical studies, the latter in electron-microscopy. They will
therefore be treated at greater length than the other primary
fixatives selected for description in this book.

FORMALDEHYDE

Standard concentration. 4 % w/v aqueous solution.

Formula. HoCO.

Description. Colourless gas; it is very soluble in water as
HO(H2CO)nH. The monomer, HOCHoCO)!!, predominates at
the standard concentration. The commercial fluid, 'formalin', is
approximately at 40% w/v solution, containing some methanol.
Polymers predominate in this, and the high polymer, para-
formaldehyde (n = 100 or more), tends to be deposited as a white
precipitate.

Ionization. Formaldehyde itself ionizes to a minute degree, with
production of a negligible amount of hydronium ions. It is easily

40

NON-COAGULANT PRIMARY FIXATIVES 41

Oxidized, however, by atmospheric oxygen to formic acid, and the
standard solution, prepared by the dilution of commercial
formalin with distilled water, has a pH of about 4. If calcium
carbonate is present in excess, the pH is 6-4.^^"

Oxidation-potential. Formaldehyde can be reduced to methanol,
and thus may act as an oxidizing agent. Its oxidation potential,
however, is lower than that of any other fixative, so far as is
known (0-23 volt).

Reactions with proteins. This subject is particularly well under-
stood, because it has been necessary for industrial chemists to
study it carefully in connexion with the so-called 'tanning' of
leather. It will therefore be treated here at some length.

One of the most effective ways of studying additive fixation is
to prepare polypeptides consisting of a single amino-acid many
times repeated, and to find how much of the fixative they take up
from solution. It is only necessary to know how much polypep-
tide is present and the initial and final concentrations of the fixa-
tive substance. This method has been very successfully used in the
study of fixation by formaldehyde. Although no protein consists
of a chain composed entirely of similar links, yet some are made
up of very few amino-acids, and these also are useful in the study
of fixation.

Polyglycine is an artificial polypeptide consisting of nothing
but a group of glycine molecules, joined by peptide bonds. Simi-
larly, polyglutamic acid consists of a group of glutamic acid
molecules, joined in the same way. The fibroin of silk consists
mostly of alanine and tyrosine. All these substances bind very
little formaldehyde."^

NH NH NH o

I i I /•

HCH HC.CHg HCCCHOaC

I I I " \

C=0 C=0 C=0 NH,

J. I I

glycine alanine gliitamine

Structural formulae of certain amino-acids, as components of
polypeptides and proteins

42 CYTOLOGICAL TECHNIQUE

Polyglutamine is an artificial polypeptide consisting of nothing
but a group of glutamine molecules joined by peptide links. This
particular polypeptide, in striking contrast to polyglycine, poly-
glutamic acid, and silk fibroin, binds more formaldehyde than
any other macromolecule, so far as is known. "^ A glance at the
structural formulae shown here and on p. 19 suggests strongly
that the group that binds formaldehyde is the -NHo of poly-
glutamine. This conclusion is borne out by experiments made
with deaminized proteins; that is to say, with proteins in which
the -NH., groups of polyglutamine and other basic amino-acids
have been replaced by -OH. These deaminized proteins bind little
formaldehyde.

Lysine is the most abundant amino-acid possessing the amino-
group, and is thought to be particularly important in fixation by
formaldehyde. The reaction might be as follows:

-(CH2)4NH., 4- H,CO = -(CH.)iNH.H2COH

lysine side- formaldehyde

group

The -OH group shown in this equation is reactive, and thus
a methylene bridge (-CHo-) can be formed between two protein
chains, each possessing an amino-group.

-NH— CH2— NH-

A methylene bridge

One can readily imagine two lysine groups on neighbouring
protein chains being linked in this way, but analysis shows that
there is not a 1 :2 relation between the number of molecules of
formaldehyde taken up and the number of lysine groups linked to
them. It is not probable that two adjacent chains would have
many lysine radicles neatly arranged opposite one another, ready
to be linked by methylene bridges. Lysine may often in fact be
linked to glutamine, or may bind formaldehyde here and there
without the formation of a bridge.

Bridge-formation will harden a protein gel, but formaldehyde,
as we have seen, is not a coagulant fixative, and it is evident that
many of the hydrophil groups retain their relation with water
molecules. It is a remarkable experience to transfer a living or

NON-COAGULANT PRIMARY FIXATIVES 43

fresh jelly-fish from sea-water to formaldehyde solution (prefer-
ably dissolved in sea-water) and to see how marvellously its
transparency is preserved. This could not happen if the proteins
were coagulated. The specimen becomes opaque, however, if after
treatment with formaldehyde the water is removed by a dehydrat-
ing agent. Similarly, the ground cytoplasm of a cell fixed with
formaldehyde loses part of its initial homogeneity when passed
through dehydrating agents in the course of paraflfin embedding.
Formaldehyde does not stabilize the cytoplasm fully against the
destructive eff'ects of subsequent dehydration.

Formaldehyde does not coagulate nucleoproteins.

Reactions with nucleic acids. Does not precipitate DNA from
solution.

Reactions with lipids. Most lipids are well preserved by form-
aldehyde, though not necessarily fixed. Triglycerides and chol-
esterol, for example, are not dissolved. If lipid-solvents are
avoided in the subsequent treatment of tissues fixed by formalde-
hyde, the lipids of adipose tissue remain in their original sites.
Certain phospholipids, however, are very slowly dissolved by
aqueous solutions of formaldehyde.''"' ^^^' ^^^' ^^ (Lecithin is not
dissolved.) In dissolving, there is a partial separation of glycero-
phosphoric acid from the nitrogenous base.

Formaldehyde tends to render phospholipids insoluble in lipid-
sohents.^^^' ^°^' ^" It thus acts as a fixative for lecithin. The
chemistry of this process has not been fully worked out, but
reaction with the nitrogenous bases has been suggested. ^°^

There is some evidence that formaldehyde can also react with

_/-_/-_ j on their fatty

acid chains. ^^^ It would appear that in this reaction formaldehyde
acts as an oxidizing agent, with production of aldehyde groups in
the lipid. It does not appear to be proved that this change confers
insolubility in lipid-solvents.

Reactions with carbohydrates. Glycogen often exists in the cell
in intimate relation with protein, and formaldehyde fixes the
protein in such a way that this carbohydrate is not easily dis-
solved out by water. ^^^ Formaldehyde is therefore a valuable

44 CYTOLOGICAL TECHNIQUE

component of fixative mixtures intended for use in studies of
glycogen.

Rate of penetration. Fast {K = 3-6).

Shrinkage or swelling. Swells gelatine-albumin gel considerably,
but leaves the volume of liver almost unchanged.

Hardening. Hardens strongly.

Method of washing out. No special washing out is usually
necessary, since formaldehyde is very soluble in water and in
ethanol.

Effect on the appearance of cells in microscopical preparations.
The form of cells is rather well preserved, though there is a ten-
dency for little blebs of cytoplasm to be nipped off from those
directly exposed to the fixative (that is, not protected by overlying
cells). Cytoplasm is rendered very finely granular. Mitochondria
and lipid droplets are generally well preserved. The nucleus
remains remarkably lifelike.

In paraffin sections there is considerable distortion. Cell-
aggregates are separated from one another by wide artificial
spaces; cytoplasm is shrunk towards nuclei. Mitochondria are
sometimes retained; most lipid globules have been dissolved
away. The interphase nucleus retains a nearly lifelike appear-
ance, but mitotic and meiotic chromosomes are not well fixed.

Formaldehyde leaves proteins in a state in which they readily
take up basic dyes (see p. 90). This follows from the fact that their
acidic groups are unaffected by this fixative. Their basic groups,
on the contrary, are to a large extent blocked, and the affinity for
acid dyes is therefore greatly reduced. If the protein were allowed
to take up all the formaldehyde with which it was capable of
combining, all affinity for acid dyes would presumably be lost. In
practice, however, this stage is not reached,- because the reaction
between protein and formaldehyde proceeds slowly. If soluble
proteins or polypeptides be allowed to react with excess of form-
aldehyde for 8 hours at 70"" C, they take up about one-half of the
total amount that they are capable of binding, and the reaction is
still incomplete after 24 hours. '^^ The binding of formaldehyde is
of course even slower at room temperature.

Compatibility with other fixatives. Compatible with ethanol,

NON-COAGULANT PRIMARY FIXATIVES 45

mercuric chloride, and acetic acid. Reduces osmium tetroxide and
potassium dichromate slowly, chromium trioxide quickly.

OSMIUM TETROXIDE

Standard concentration. 1 % w/v aqueous solution.

Formula. OSO4.

Description. Pale yellow molecular (non-ionic) crystals, soluble
in water at about 7% w/W (in carbon tetrachloride at about
375 % w/W). The crystals melt at 4P C. They begin to sublime at
a much lower temperature than this; the gas given off is damaging
to the epithelium of the eyes, nose, and mouth. This is by far the
most expensive of all primary fixatives (1 g costs £3. 10^.).

Ionization. On solution, it takes up a molecule of water to
become hydrogen per-perosmate, HoOs05.^^^" This ionizes to
a minute extent to produce hydronium ions and HOSO5. The
substance can, however, scarcely be called an acid. Acetic is a very
weak acid, yet its ionization constant is well over 20 million times
as great as that of hydrogen per-perosmate.

Osmic acid, H0OSO4, is not used in fixation.

Oxidation-potential. A moderately strong oxidizer (the 2%
solution has o.p. 0-64 volt). On reduction, the brown or blackish
hydrated dioxide, OSO2.2H0O, is left.

Reactions with proteins. Osmium tetroxide gives no coagulum
with albumin solution, but on the contrary renders the protein no
longer coagulable by ethanol or heat.^^ It sets strong protein
solutions into gels, and stabilizes gelatine gels against solution by
warm water. It does not coagulate nucleoproteins.

Osmium tetroxide is an additive fixative, but the exact site of
its attachment to proteins is not known. It is capable, in certain

1

HC

0

I
HC 0

+

OsO, -

->

\
OsO.,

Hi

0

H(

: 0

Action of osmium tetroxide at the ends of a double bond

46 CYTOLOGICAL TECHNIQUE

circumstances, of simultaneous reaction at both ends of the
double bond present in many organic substances. ^^' ^' The
process is additive.

There are not very many suitable double bonds in proteins, and
indeed we have no actual proof that osmium tetroxide reacts with
proteins in this way. The evidence does point, however, in this
direction.

Most compounds of osmium, other than the tetroxide, are dark
or even black, and one can use this fact to find which substances
react with the tetroxide and which do not. Of the amino-acids,
tryptophane and histidine react strongly, forming dark precipi-
tates.^ These substances contain double bonds in their five-

NH, NH,

HC.CHa— C-

HCn

HC.CH>— C CH

C=0 N NH

C=0 N I \ /

I H OH C

OH H

Tnyptophane Histidine

membered rings. Among the various proteins, the ones that
contain a high proportion of tryptophane and/or histidine are
particularly reactive with osmium tetroxide. There is a positive
correlation between the tryptophane-content of a protein and the
capacity of that protein, in the form of an aqueous sol, to be gelled
by osmium tetroxide. ^^"^

The capacity of osmium tetroxide to gel protein sols and to
harden and stabilize protein gels (p. 45) would be more compre-
hensible if it could be shown that separate protein chains were

\ O /

HC— O ! O— CH

I Ml/

I Os

I / \
HC— O O— CH

/ \

Osmium acting as a bridge between two ring-compounds
{The latter are shown only in part.)

joined together by this substance. Of this we have no positiva

NON-COAGULANT PRIMARY FIXATIVES 47

ev idence, but it has been shown that osmium tetroxide can in fact
join certain double-bonded ring-compounds together.^"- ®^ The
double bond is lost in the process. An extra supply of oxygen is
required if this reaction is to occur.

Although reactions of this kind are probably concerned in the
fixation of proteins by osmium tetroxide, yet there are indications
that others also play a part. Tryptophane and histidine are not the
only free amino-acids that form a dark precipitate with osmium
tetroxide: cysteine (p. 36) behaves in the same way,^ yet its side-
group possesses no double bond.

There is no reason to believe that tryptophane and histidine
play any important part in the colouring of proteins by dyes, yet
it is characteristic of the proteins of tissues fixed by osmium
tetroxide that they show a remarkable lack of affinity for acid
dyes.^^ Since these dyes attach themselves to the -NHo groups of
the side-chains of basic amino-acids (especially lysine, p. 19),
there is strong reason to believe that osmium tetroxide somehow
blocks these groups. The mechanism of such blocking is, how-
ever, unknown. Some proteins that contain a lot of lysine and
arginine darken readily with osmium tetroxide, but the prota-
mines, which contain a particularly high proportion of arginine,
seem scarcely to darken at room-temperature.^ It would be
unjustifiable to assume that darkening is an invariable con-
comitant of reaction.

Reactions with nucleic acids. Does not precipitate DNA from
solution."^

Reactions with lipids. Osmium tetroxide blackens unsaturated
lipids of all kinds, but does not attack saturated ones.^ It is there-
fore clear that it reacts at the double bonds. Nearly all lipids in
organisms are mixtures containing some unsaturated components,
and blackening is therefore almost invariable, though it may be
very slow.

Osmium tetroxide is soluble in lipids. It may therefore enter
a saturated lipid and subsequently be reduced to black osmium
dioxide (p. 45) when the tissue is placed in ethanol. It follows that
the blackening of a lipid droplet does not necessarily prove that
the lipid was unsaturated.

48 CYTOLOGICAL TECHNIQUE

It is an unexplained fact that the conjugated Hpids of the tissues,
though usually highly unsaturated, are darkened by osmium
tetroxide much more slowly than the mixed triglycerides.

If suitable antemedia (p. 73) are used, tissues fixed by osmium
tetroxide may be embedded in paraffin without loss of unsatur-
ated lipids. Benzene and chloroform are particularly suitable
antemedia for this purpose.^^

If unsaturated lipid that has been blackened by osmium tetrox-
ide is bleached by hydrogen peroxide, the lipid is set free and is
now soluble once more in lipid-solvents, including benzene and
chloroform. ^^

After the fixation of tissues by osmium tetroxide, the unfixed
(saturated) component of a lipid globule may be dissolved out
during dehydration-' ^^^ or embedding. The fixed (unsaturated)
lipid is then left in a spherical cavity, which it does not fill. It
applies itself to the wall of the cavity, and may be seen in optical
section under the microscope as a ring, crescent, or 'cap'. These
appearances have often been misunderstood.^^

Reaction with carbohydrates. There appears to be no reaction
in the ordinary circumstances of fixation.

Rate of penetration. This is the only fixative, so far as is known,
that does not maintain a constant iC-value. It penetrates slowly.
The TiC-value during the first 16 hours is 1-0, but during the period
16 to 144 hours it is only 0-31.

Shrinkage or swelling. Gelatine-albumin gel shrinks very
slightly. There are no satisfactory figures for tissues and cells,
but there seems to be little change of volume.

Hardening. Leaves tissues rather soft.

Method of washing out. Osmium tetroxide is usually washed out
with running water, to prevent subsequent reduction in the tissues
by ethanol. In preparations for electron-microscopy, however,
tissues are sometimes transferred directly from the fixative to
50% or 70% ethanol.

Effect on the appearance of cells in microscopical preparations.
Preserves the structure of the living cell better than any other
primary or mixed fixative. It might almost be supposed that the
cell was still alive.

NON-COAGULANT PRIMARY FIXATIVES 49

The fixation is good for methacrylate embedding, but tliere is
very poor resistance to the distortion caused by embedding in
paraffin. Cell-aggregates are shrunken, so that wide artificial
spaces appear; the tissue tends to crack; the cytoplasm contracts
round the nuclei; chromosomes (especially in the first meiotic
prophase) are poorly fixed.

Acid dyes, as we have seen (p. 47), scarcely act on tissues fixed
bv osmium tetroxide. Basic dyes are taken up by both nuclear sap
and cytoplasm, and differential dyeing of chromatin is therefore
not obtained.

Compatibility with other fixatives. Osmium tetroxide reacts with
ethanol and formaldehyde, but is compatible with mercuric
chloride, chromium trioxide, and potassium dichromate. These
three substances prevent its reduction by daylight.

POTASSIUM DICHROMATE

Standard concentration. \-S% w/v aqueous solution.

Formula. K2Cr20;.

Description. Orange-red crystals, soluble at about 10 °o w/v in
water.

Ionization. The ions produced by the solution of potassium
dichromate are the same as those produced by the solution of
chromium trioxide, but the proportions are somewhat different.
In particular, the amount of hydronium ion is much less, the pH
of the standard solution being about 4-1. The chief ion containing
chromium is the dichromate, [CraO;]^, with some hydrogen
chromate, [HCrOJ".

Oxidation-potential. This is a strong oxidizer (o.p. 0-79 volt),
but not nearly so strong as chromium trioxide.

Reactions with proteins. Potassium dichromate is a non-coagu-
lant of proteins, including nucleoproteins. It very gradually
renders egg-white more viscous and eventually transforms it into
a weak gel. It gels hist ones more powerfully. ^^® With these excep-
tions, it seems doubtful whether potassium dichromate can be
regarded as a fixative for proteins, in the circumstances of its
ordinary use in microtechnique; but it must be remarked that

E

50 CYTOLOGICAL TECHNIQUE

gelatine gel can be rendered insoluble in warm water by the action
of potassium dichromate in bright light.

Since chromium trioxide is a vigorous coagulant, it is evident
that proteins react quite differently to the chrome anions accord-
ing to whether the pH is low or not.^®^ The 'critical range' of pH
is from 3-4 to 3-8.^^ At more acid pH than 3-4, the chrome anions
coagulate protein; above 3-8, they do not. Acidified potassium
dichromate acts like chromium trioxide, provided that the pH is
below 3-4.

It seems probable that unacidified potassium dichromate fixes
ground cytoplasm by reaction with the lipid component of lipo-
proteins, but this subject awaits adequate investigation.

Reactions with nucleic acids. Unacidified potassium dichromate
not only does not fix, but actually dissolves DNA.

Reaction with lipids. Potassium dichromate is important in
microtechnique chiefly for its fixative effect on certain lipids.

Adipose fat can be rendered insoluble in lipid-solvents by very
prolonged treatment with a solution of potassium dichromate. ^^
The evidence suggests that the unsaturated lipids are oxidized at

Q __ ^_) ; the uptake of chromium is not

concerned in the process. ^^ The reaction is too slow for practical
use.

Lipids that have a double bond near the end of the fatty acid
chain furthest from the carboxyl group have a special tendency
towards polymerization on oxidation; this is accompanied by
lessened solubility in lipid-solvents.

Certain lipids are able to take up chromium from solutions of
potassium dichromate, and in so doing to lose their solubility in
lipid-solvents. This is additive fixation. With the short post-
chroming that is usual in microtechnique (for instance, soaking
for 24 hours in a 5% solution of potassium dichromate at 37° C),
only phospholipids take up the metal. Colour-tests for chromium
will therefore reveal the sites of phospholipids in cells. ^^' ^^ The
way in which chromium links itself to the lipid is uncertain. The
phospholipids that occur in nature are highly unsaturated, but it
is stated that synthetic saturated ones (such as dipalmitoyl

NON-COAGULANT PRIMARY FIXATIVES 51

lecithin) are able to take up the metal. ^° It seems probable that
chromium attaches itself to the phosphate group, though other
suggestions have been made.^^^

The possibility has been mentioned above (p. 50) that potas-
sium dichromate may fix ground cytoplasm by acting on the lipid
component.

Reactions with carbohydrates. The unacidified salt is not known
to fix any carbohydrate. If acidified, it presumably acts like
chromium trioxide (p. 38).

Rate of penetration. Since proteins are neither coagulated nor
gelled in the ordinary period of fixation, the rate of penetration
cannot be measured by the method adopted with other fixatives.
There is no satisfactory information about the rate of penetration
of this substance.

Shrinkage or swelling. Gelatine-albumin gel is swollen by potas-
sium dichromate. Whole livers remain unchanged in volume in a
3 % solution, but they are subject to severe subsequent shrinkage
by ethanol.

Hardening. Tissues are left very soft.

Method of washing out. To avoid the possibility that insoluble
chromic oxide (CroOg) will be precipitated in the tissues through
subsequent reduction by ethanol, it is usual to wash out in run-
ning water. This reduction does not occur, however, if light be
excluded. ^^^

Effect on the appearance of cells in microscopical preparations.
The cell maintains its form rather well. Mitochondria are pre-
served, but filamentous ones may be changed into ovoids. The
nuclear sap becomes finely granular. The nucleolus shrinks (pre-
sumably through loss of RNA).

In paraffin sections there is a shrinkage apart of cell aggregates,
with production of artificial spaces ; the cytoplasm, though rather
homogeneous, is shrunk round the nucleus; mitochondria are
retained, but swollen ; mitotic and meiotic chromosomes and the
heterochromatic segments of the interphase nucleus are unfixed.
If the fixative solution is acidified below pH 3-4, the appearance
is the same as that given by chromium trioxide.

Both basic and acid dyes act quite strongly. The chromatin,

52 CYTOLOGICAL TECHNIQUE

though unfixed, cannot escape through the nuclear membrane; it
distributes itself at random within the nucleus. The colouring of it
by basic dyes is therefore not informative. This is an unsuitable
fixative for studies of the nucleus and chromosomes (unless
acidified).

ACETIC ACID

Standard concentration. 5% v/v aqueous solution.

Formula. H3C.COOH.

Description. A colourless liquid with a pungent smell, miscible
with water and ethanol in all proportions. The crystals produced
by cooling melt at 16-6'' C. The undiluted acid is often called
'glacial', because it is so easily frozen.

Ionization. A very weak acid. The pH at the standard con-
centration is about 2-3.^°®

Oxidation-potential. Can oxidize by being reduced to acetalde-
hyde (o.p. 0-77 volt).

Reactions with proteins. It neither coagulates nor gels most
proteins; non-additive. It extracts hist one from the tissues. A
thick precipitate is formed when acetic acid is added to a solution
of nucleoprotein. This is attributed to the action of the acetate
ion in splitting off" DNA from protein.

Reactions with nucleic acids. Precipitates DNA from solution.

Reactions with lipids. In writings on fixation it is often said
loosely that lipids are dissolved by acetic acid. It is true that some
of them (cholesterol and sphingomyelin, for instance) are soluble
in glacial acetic acid; but at the concentrations at which it is used
in fixation, acetic acid is not a lipid-solvent. It has, however, no
fixative effect on lipids.

Reactions with carbohydrates. Neither fixes nor destroys them.

Rate of penetration. In experiments to determine this, it is
necessary to substitute gelatine-nucleoprotein gel for gelatine-
albumin, for acetic acid leaves no visible mark to indicate its
progress into the latter. It penetrates rapidly {K = 2-75).

Shrinkage or swelling. Its capacity to swell protein gels and
tissues is the most striking character of acetic acid, and the main

NON-COAGULANT PRIMARY FIXATIVES 53

reason for its use as a component of fixative mixtures. Its action
offsets the shrinkage caused by other substances used in micro-
technique. Gelatine-albumin gel expands to between 4 and 5 times
its original volume in 18 hours (fig. 5, p. 27). Acetic acid has much
more swelling effect than the mineral acids have. This is partly
because the pH is not low enough to cause coagulation; partly, it
seems, because the undissociated acid is responsible for some of
the swelling. Thus, acetic acid has a swelling (or anti-shrinking)
effect even when it is used as a constituent of a non-aqueous
mixture, such as Clarke (see p. 59). In aqueous media acids are
thought to break the salt-links that connect protein chains; the
hydrophil groups thus exposed would draw water into the gel or
tissue.

Hardening. Leaves tissues much softer than any other fixative
does.

Method of washing out. Since acetic acid is miscible with
ethanol in all proportions and has no tendency to produce
extrinsic artifacts, no special washing out is necessary.

Effect on the appearance of ceils in microscopical preparations.
The external form of the cell is fairly well preserved. So, in general,
are the cytoplasmic inclusions, but the mitochondria become less
visible and may disappear. The nuclear contents are transformed
into a coarse network.

In parafhn sections, cell-aggregates tend to be widely separated,
with artificial spaces in between. Cytoplasm is sometimes coarsely
reticular, sometimes contracted round the nuclei. Mitochondria
are absent. The nuclear sap is coarsely reticular; the nucleolus is
swollen; metaphase and anaphase chromosomes are rather well
fixed; the mitotic spindle appears fibrous.

The acetate ion only produces its characteristic fixation-image
on the more acid side of pH 4 or thereabouts. ^^° On the less acid
side there is no fixation and the tissues macerate.

Cytoplasm takes acid dyes strongly, basic ones rather feebly.
The chromatin of interphase nuclei colours feebly with basic dyes,
scarcely at all with acid ones. Metaphase and anaphase chromo-
somes colour strongly with basic dyes.

Compatibility with other fixatives. Compatible with all other

54 CYTOLOGICAL TECHNIQUE

fixatives, but potassium dichromate behaves like chromium tri-
oxide if mixed with acetic acid, unless the amount of acid is so
small that the pH is on the less acid side of the critical range
(p. 50).

CHAPTER 5

Practical Fixative Solutions

The only primary fixatives that are commonly used without the
admixture of other primaries are formaldehyde and osmium
tetroxide. Solutions of primaries used in practice in this way may
be called simple fixatives to distinguish them from fixative mix-
tures, which contain two or more primaries. Simple fixatives
usually contain 'indifferent' or non-fixative salts (p. 29) in addi-
tion to the primary. Some of the indiff"erent salts that are used
with formaldehyde and osmium tetroxide will be mentioned in
the present chapter.

The great majority of practical fixative solutions are mixtures.
The formulae for a very large number of these have been pub-
lished. It is evident, from a study of the papers in which the
formulae first appeared, that most of them were not the product
of scientific experiment based on knowledge of the properties of
their components. On the contrary, they were put together in a
hit-or-miss fashion. In several cases the formula was relegated to
a footnote, with no indication of any reasons governing the
choice or concentration of the ingredients. Some of these empiri-
cal fluids gave good results and found favour, others did not. A
process of natural selection of almost random variations resulted
in the survival, on the whole, of the fittest; though many that are
used are superfluous.

To understand the action of fixative mixtures, it is best to make
a careful study of a few valuable ones. The principles involved will
emerge, and will be found widely applicable. The mixtures chosen
for study in this book are those of Clarke, Zenker, Flemming,
Helly, and Altmann.

The reader may care to refer to the general remarks on the
period of fixation given on p. 24.

55

56 CYTOLOGICAL TECHNIQUE

FORMALDEHYDE IN SIMPLE FIXATIVES

When used in this way, formaldehyde is generally dissolved in
a solution of an indifferent salt. The latter is used at a concentra-
tion that gives the same osmotic pressure as the body-fluids of the
organism from which a part is to be taken for fixation, or at a
concentration slightly less than this. The following is suitable for
the tissues of most vertebrates other than elasmobranchs, and for
many terrestrial and fresh- water invertebrates :

Distilled water 83 ml

Sodium chloride, 10% ^4- • • . 7 ml
Formalin . . . . . . 10 ml

Keep marble in the fluid. pH (in the presence of marble), 6-0. Wash
out with water or 50% ethanol.

In this and other formulae, the word 'formalin' means the com-
mercial fluid containing formaldehyde at approximately 40%
w/v. In the fixing solutions given here, formaldehyde is therefore
used at its standard concentration.

For the tissues of marine invertebrates, one may dilute formalin
with 9 times its volume of sea-water. The pH of the fluid is 7-6.

There can be no doubt that non-fixative salts improve fixation
by formaldehyde and certain other primary fixatives. Since they
are used at concentrations that give about the same osmotic
pressure as the body-fluids of the organism, the fixative solution
as a whole is hypertonic, for the osmotic pressure of the formalde-
hyde or other fixative is added to that of the non-fixative salt.
One would therefore expect the latter to cause shrinkage: in fact,
however, the virtue of non-fixative salts is that they reduce shrink-
age. It has already been mentioned (p. 29) that this has never been
satisfactorily explained. It has been suggested that when fixatives
are used without indiff"erent salts, the cells in the interior of a
piece of tissue behave at first— before the fixative substance itself
has arrived— as though the piece had been placed in distilled
water: that is to say, they swell up and burst. After bursting, they
are thought to shrink, and to be fixed subsequently in this
shrunken condition. If an indifl'erent salt is used, it diffuses into

PRACTICAL FIXATIVE SOLUTIONS 57

the tissue ahead of the fixative, and prevents the initial swelling
and bursting.^®' Certain difficulties in accepting this hypothesis as
a complete explanation have been discussed rather fully else-
where. ^^

Salts used for the purpose just mentioned may play another
role as well. Phospholipids have a tendency to alter their form by
sending out microscopical, tentacle-like 'myelin forms' into sur-
rounding water. Calcium salts prevent this change. ^°® In studies
of the lipid constituents of cells it is therefore reasonable to sub-
stitute calcium chloride for sodium chloride in fixative solutions. ^*
The concentration must be made slightly higher than that of
sodium chloride, if the same osmotic pressure is to be obtained.
The following formula is suitable :

Distilled water . . . . . 80 ml
Calcium chloride (anh.), 10% aq. . . 10 ml
Formalin . . . . . . 10 ml

Keep marble in the fluid. pH (in the presence of marble), 5-7. Wash
out with water or 50° o ethanol.

Formaldehyde is much used in these simple solutions in histo-
chemical studies, especially of lipids. To make sure that phospho-
lipids will be fixed, the tissue may be 'postchromed'. After quite
short fixation (6 hours) in the formaldehyde solution, it is trans-
ferred (without washing) to a solution of potassium dichromate.
A solution of this salt maintained at saturation in an incubator at
37^ C is suitable. Treatment for a day or two suffices. The tissue
must then be washed in running water overnight. More elaborate
methods of postchroming are also available. ^^' ^®

OSMIUM TETROXIDE IN SIMPLE FIXATIVES

Osmium tetroxide gives excellent results when unmixed with
other primary fixatives, provided that a suitable embedding
medium, such as methacrylate (p. 77), is used. Gross shrinkage
and distortion occur if tissues fixed in this way are embedded in
paraffin.-^ There is evidence^^^ that the submicroscopical struc-
ture of cells is best stabilized if the fixative solution is buff'e red just

58 CYTOLOGICAL TECHNIQUE

on the alkaline side of neutrality, though this has been disputed.
The buffer may also act as an indifferent salt. A modification of
Michaelis's buffer^^a jg usually used, probably because it is not
harmful to the cells of most vertebrates. The buffer at pH 7-4 is
this:

Sodium acetate and sodium veronal, both

M

at — m one solution, aq. . . . 20 ml
7 ^

Hydrochloric acid, 0-1 N. . . .20 ml

Sodium chloride, 8-5 aq. . . . . 8 ml

Distilled water . . . . . 52 ml

The acetate- veronal solution for use in this buffer and in Palade's
fixative (see below) is made by dissolving 9-714 g of sodium acetate,
SHoO, and 14-714 g of sodium veronal in distilled water, and making
up the volume to 500 ml by the addition of distilled water.

Michaelis's buffer has approximately the same osmotic pressure
as the body-fluids of mammals. Sodium veronal is a useful con-
stituent of buffers intended for biological work in the neighbour-
hood of neutrality. Boric-borate buffers are troublesome, because
boric acid forms complexes with carbohydrates and all other
organic di- and polyhydroxy-compounds. Sodium acetate acts in
buffers at a much lower pH than 7-4, and an equally good solu-
tion lacking this constituent could probably be devised.

Palade's fixative^ ^^ is Michaelis's buffer at pH 7-4, with the
addition of 1 g of osmium tetroxide and the substitution of dis-
tilled water for the sodium chloride solution. Thus it contains
osmium tetroxide at its standard concentration, and the saline
constituents are hypotonic to the body-fluids of mammals. It is
convenient to make up Palade's fixative thus:

Sodium acetate and sodium veronal, both

M

at — - in one solution (see above) . . 20 ml

7

Hydrochloric acid, 0-1 N . . .20 ml

Distilled water . . . . . 10 ml

To 1 ml of the fluid, add 1 ml of 2^'o osmium tetroxide solution.
pH of the fixative solution, 7-4.

PRACTICAL FIXATIVE SOLUTIONS 59

Use at about 5° C. Fix pieces not exceeding 1 mm in thickness for
1 to 4 hours. Wash out in several changes of distilled water; or pass
directly to 50% or 70% ethanol.

Palade's fixative and variants of it have been enormously used
in electron-microscopy, with great success. Since Michaelis's
buffer does not precipitate calcium from its salts, it is legitimate
to substitute 10 ml of calcium chloride solution for the 10 ml of
distilled water used in making up the buffer. ^^ The concentration
of this solution may be anywhere from 1% to 10% of the
anhydrous salt; the final concentration will be from 0-1 % to 1 %.
Calcium chloride is particularly valuable in the study of lipid
droplets.

60, 23

We turn now to fixative mixtures.

CLARKE (1851)«2

Absolute ethanol . . . . .3 vols
Acetic acid (glacial) . . . .1 vol

(The constituents are not ionized and pH has therefore no meaning.)
Wash out in absolute ethanol.

The formula for this fixative was given by the English neurolo-
gist no less than 35 years before Carnoy, the celebrated Belgian
cytologist, first published it.^^ In the intervening period it was
familiar in microtechnique as die Clarke' sche Vorschrift.'^^ This is
the most ancient of all fixative mixtures commonly used in micro-
technique today.

The advantage that may be gained by mixing certain primary
fixatives cannot be more vividly illustrated than by a considera-
tion of Clarke's hardy centenarian. By themselves, ethanol and
acetic acid are both very bad fixatives (see pp. 33 and 53), but
each compensates neatly for the defects of the other. The shrink-
age that ethanol alone would cause is off"set by the swelling action
of acetic acid; the latter stabilizes nucleoproteins, which are left
unfixed by the former; acetic acid fixes neither cytoplasm nor
nuclear sap, but both are fixed by ethanol (the former in a rather
coarse coagulum). The mixture is admirable in routine micro-

60 CYTOLOGICAL TECHNIQUE

anatomy and histology, of some value in studies of chromosomes,
and appHcable also in histochemistry whenever it is desirable to
avoid additive fixation and the resultant shift in the iso-electric
points of proteins; glycogen is preserved, but not fixed. The fluid
is a solvent of many lipids; for this reason, and because it
coagulates rather coarsely, it is unsuitable for the study of most
cytoplasmic inclusions.

Zenker's fluid (1894)^^^

Distilled water 100 ml

Mercuric chloride . . . . • ^ g

Potassium dichromate . . . . 2-5 g

Sodium sulphate . . . . . 1 g

To 20 ml, add 1 ml of glacial acetic acid immediately before use.

pH 2-5.^2

Pieces of tissue need not be very small.

If the tissue is to be dehydrated in grades of ethanol, pass it directly
from the fixative to 50'^ o ethanol acidified by the addition of 2% v/v of
concentrated sulphuric acid;^^ transfer from this to a 0-5% w/v
solution of iodine in 70% or 80% ethanol.

If the tissue is to be dehydrated by means of cellosolve (p. 74), pass
it directly from the fixative to 50° o cellosolve and from this to a 0-5 w/v
solution of iodine in undiluted cellosolve."^®

Zenker's fluid is chosen as our next example after Clarke be-
cause it is one of the best fixatives for use in routine histology and
in preliminary work with unknown tissues in biological micro-
technique of all kinds. It contains two protein coagulants (mer-
curic chloride and acidified potassium dichromate) and a sub-
stance that opposes shrinkage (acetic acid). No non-coagulant
fixative of protein is included in the mixture. Sodium sulphate is
an 'indiff'erent' salt; its eff'ect in this mixture is uncertain.

The ground cytoplasm and certain cytoplasmic inclusions are
far better preserved by Zenker than by most routine fixatives.
The fine texture of the protein coagulate is probably due to the
mercuric chloride, which also ensures easy dyeing. Acidified
potassium dichromate favours the action of acid dyes, which give
much more brilliant effects than after Clarke.

PRACTICAL FIXATIVE SOLUTIONS 61

The only notable defect of Zenker is a tendency to crumple
collagen fibres.

flemming's strong fluid (1884)"

Distilled water . . . . . 0-8 ml

Chromium trioxide, 5 ^o aq. . . 0-3 ml

Osmium tetroxide, 2% aq. . . 0-4 ml

Acetic acid, 20% (or less) aq. . . 0-5 ml

As a general rule, use the full concentration of acetic acid.

pH 1-4;^^ the amount of acetic acid used does not affect the pH.^*^

Use pieces 2 mm or less in thickness.

Wash out for several hours in running water or repeated changes.

Since 2 ml of the mixture are sufficient for the fixation of the
small pieces of tissue that should be used, it is desirable to follow
the formula given above. There is then no wastage of the ex-
tremely expensive osmium tetroxide. The fluid made up in this
way has the same composition as that which results from the use
of Flemming's own formula.

Flemming contains a trio of ingredients that are found over and
over again in successful mixtures ; namely,

(1) one or more coagulants of protein (in this case chromium
trioxide) ;

(2) a non-coagulant fixative of protein (in this case osmium
tetroxide) ;

(3) acetic acid.

One might suppose that the best results would be given by non-
coagulant fixatives in the absence of coagulants; but tissue that
has been treated with no other fixative of protein than a non-
coagulant does not give ready access to paraffin, and the prepara-
tion of good paraffin sections is often difficult. This particularly
applies to osmium tetroxide. The spongework produced by coagu-
lants provides spaces into which melted paraffin can enter (p. 75).
Beyond this, coagulants give the best fixation of chromosomes,
and chromium trioxide is pre-eminent in this respect. If, how-
ever, no non-coagulant fixative of proteins is included, the sponge-
work tends to be unduly coarse, and c)1;oplasmic inclusions are

62 CYTOLOGICAL TECHNIQUE

distorted mechanically or even destroyed. The coagulants and
non-coagulants thus compensate for one another's defects.

The chief effect of acetic acid in these mixtures is the prevention
of excessive shrinkage. Tissues fixed in acetic acid alone swell, but
are greatly shrunken on subsequent dehydration. If, however,
they are stabilized in the swollen condition by the simultaneous
action of a fixative for proteins, the shrinkage seen in the final
preparation is far less. When acetic acid is used in mixtures at 5 %
or thereabouts, there is no necessity to add an indiff"erent salt.
Acetic acid also plays a part in the fixation of nucleoprotein, but
this is not important when the coagulant is chromium trioxide.

A defect of Flemming is that the constituents penetrate at
different speeds. The most external part of the piece of tissue is
not capable of being dyed successfully, because osmium tetroxide
has exerted too powerful an eff'ect upon it (p. 49) ; the most in-
ternal part shows the effects of fixation by chromium trioxide and
acetic acid alone (apart from the blackening of lipid globules). In
the intermediate region, however, fixation is usually excellent.
Chromosomes are well fixed, as indeed one would expect; for the
famous German cytologist who designed the mixture did more
than anyone else to clarify the process of mitosis. The ground
cytoplasm preserves much of its homogeneity; various cyto-
plasmic inclusions, especially the idiozome, are very well shown.
Mitochondria are not necessarily destroyed, but as a rule they are
not easily shown after fixation by Flemming with full acetic. In
the study of these particular cytoplasmic inclusions the acetic acid
is commonly much reduced in amount^- or omitted.^"^- "

HELLY (1903)9 7

Distilled water 100 ml

Mercuric chloride . . . . 5 g

Potassium dichromate . . . . 2-5 g
Sodium sulphate . . . . . 1 g

To 10 ml add 0-5 ml of formalin (neutralized by marble) immediately

before use.
pH 3-7.^2

The fixative may be washed out in acidified 50° o ethanol and iodized

PRACTICAL FIXATIVE SOLUTIONS 63

70° 0 or 80% ethanol, in the same way as Zenker's fluid. Alternatively,
transfer the tissue directly from the fixative to a saturated solution of
potassium dichromate maintained at 37' C, leave for 24 to 48 hours,
wash overnight in running water, and dehydrate with through grades
of ethanol, with iodine treatment.

The Viennese anatomist's fixative is one of the most valuable
in the routine study of cytoplasmic inclusions in paraffin sections.
It contains a coagulant and a non-coagulant fixative of protein
(mercuric chloride and formaldehyde respectively), and also an
important fixative of many lipids (unacidified potassium di-
chromate). It is uncertain whether the indiff'erent salt (sodium
sulphate) plays a useful role in the mixture.

Helly is not nearly so similar to Zenker as the list of its con-
stituents might suggest. Formaldehyde and unacidified potassium
dichromate are profoundly diff"erent in their effects on tissues
from acetic acid and acidified potassium dichromate.

Mercuric chloride and formaldehyde, acting in conjunction, fix
ground cytoplasm smoothly, giving just sufficient sponginess to
allow easy penetration by paraffin. The constituents penetrate
well and it is not necessary to use very small pieces.

Postchroming is useful in studies of mitochondria. The latter
are usually well fixed, though those of mammalian liver tend to
shorten and round up.

ALTMANN (1894)=^

Osmium tetroxide, 2% aq. . . .1 vol.

Potassium dichromate, 5°o aq. . .1 vol.

pH 4-0.52

Use pieces of tissue 2 mm or less in thickness. In studies of mito-
chondria fix for 24 hours (postchroming is unnecessary).
Wash out overnight in running water.

Altmann was the first serious student of mitochondria, though
he did not know them under this name. The distinguished Leipzig
cytologist was mistaken about the nature of these cytoplasmic
inclusions, which he regarded as 'elementary organisms', living
within the cell ; but he designed a fixative that is still one of the
best for showing them in preparations for light-microscopy.

64 CYTOLOGICAL TECHNIQUE

The fluid differs from the mixtures already described and from
the great majority of fixative mixtures used in microtechnique, in
containing no coagulant. Ground cytoplasm is therefore fixed
very homogeneously. The cytoplasmic inclusions maintain their
form, partly because they are not distorted by coagulation of the
surrounding ground cytoplasm, partly because both the constitu-
ents of the fluid are lipid-fixatives. The dichromate, a stronger
oxidizer than osmium tetroxide, prevents excessive blackening of
the tissues and gives easy colouring by acid dyes ; unfortunately it
dissolves nucleoprotein, and the fixative is not adapted to studies
of nucleus or chromosomes.

The disadvantage of this fixative is that it does not give ready
access to melted paraffin. Embedding often results in shrinkage
and distortion, the tissue cracks, and sections are difficult to
flatten. When skill or luck overcomes these troubles, excellent
preparations result.

Ammonium dichromate may be substituted for potassium di-
chromate in this solution."^ The pH of 'NH4-Altmann' is slightly
more acid. The ammonium salt differs curiously from the other. ^^^
Mitochondria are not well fixed at the surface of the piece of
tissue, but in the interior they are better fixed than by potassium
dichromate: filamentous ones retain their form, instead of
shortening and thickening. For this reason it is best to cut out
rather large pieces of tissue when the ammonium salt is to be
used.

The great majority of fixative mixtures fall into one or other of
four groups. Fifteen of the most valuable are listed here.

Group A. Coagulant + acetic acid. These are primarily fixa-
tives for micro-anatomy and histology. Examples: Clarke,^^
Zenker. ^^^

Group B. Coagulant -f- non-coagulant + acetic acid. These are
mostly fixatives for detailed histology and general cytology : many
are used in the study of chromosomes. Examples: Flemming's
strong fluid,'^ Allen's 'B.15',i Bouin,=^9 Heidenhain's 'Susa',^«
Hermann (mammalian formula),^^ Sanfelice.^^^

Group C. Coagulant -f non-coagulant. These are routine fixa-

PRACTICAL FIXATIVE SOLUTIONS 65

tives for cytoplasmic inclusions. Examples: Helly,^' Champy,^^^
Lewitsky-saline,^°^' ^® Mann/^^ Zenker- without-acetic.

Group D. Non-coagulants only. This is a small group, adapted
to the study of cytoplasmic inclusions. Examples: Altmann,^
Regaud.i^^

It is curious that such a high proportion of fixative mixtures
contain one or more coagulants. This is probably connected with
the predominance of paraffin as an embedding medium. Group D
is likely to come to the fore in the future, as new plastics replace
paraffin ; but it remains to be seen whether any non-coagulant is
as useful as chromium trioxide in the fixation of chromosomes
for light-microscopy.

CHAPTER 6

Embedding

In the early days of histology and cytology, sections were cut by
hand. Skilled workers could cut ordinary plant tissue admirably,
for each cell was held in place by a firm cell-wall. Zoologists were
at a disadvantage, which they sought to overcome by using fixa-
tives that hardened the tissues. Indeed, their attention was focused
on this, rather than on the primary object of fixation ; and accord-
ingly they called their fixatives hardening agents. When micro-
tomes came into general use, it was found convenient to embed
tissues in suitable media which would hold the cells and intercellu-
lar matter in place while the razor slid past them. Animal tissues
are seldom sectioned today without having been embedded, and
it is therefore unnecessary that fixatives should have a hardening
eff"ect.

Embedding media must necessarily be substances that are
capable of easy conversion from liquid to solid form. The liquid
penetrates the tissues to a greater or lesser extent and is then con-
verted into a solid. We shall see that this conversion may involve
hydrogen bonding, covalent linkage, crystallization, or polymer-
ization.

It is sometimes suggested that embedding media may be divided
into two groups: those that penetrate the cells, and those that
merely surround them. Actually, it is doubtful whether any sharp
distinction can be drawn. Some embedding media certainly enter
cells, and indeed may penetrate their nuclei (p. 75) ; but whether
any particular medium penetrates any particular kind of cell or
merely surrounds it may depend on the fixative used. Those
fixatives that coagulate proteins in the form of a coarse sponge-
work leave the tissues in a state that favours the entry of embed-

66

EMBEDDING 67

ding media. Those that fix homogeneously, without coagulation,
tend to render the internal parts of cells inaccessible to most
embedding media. Failure to enter cells by no means necessarily
renders an embedding medium useless, for it may enter inter-
cellular spaces and give considerable support during sectioning.
Three substances have been chosen to represent diff"erent sorts
of embedding media. These three are gelatine, paraffin wax, and
butyl methacrylate. They have been chosen partly because they
illustrate three very different processes of solidification, partly
because they are among the most valuable of all embedding
media.

GELATINE

The outstanding advantage of gelatine as an embedding medium
is that the tissues need not at any stage be dehydrated. It follows
that most lipids, even if unfixed, remain in the tissue and can be
coloured by lysochromes (p. 86). Distortion by dehydration is
also avoided. There are, however, several disadvantages in gela-
tine embedding, as ordinarily practised. The gelatine cannot be
removed from the tissue; and since it is capable of taking up both
basic and acid dyes, it often tends to obscure the view. The
ordinary process cannot be relied on to provide sections thinner
than about 5 //, and they do not adhere to form ribbons. It has
recently been claimed, however, that by modifying the procedure
one may obtain ribbons of sections only 20 m/n thick, suitable for
examination with the electron-microscope.^*

It is commonly supposed that there is some special virtue in
formaldehyde that makes this the fixative of choice when tissues
are to be embedded in gelatine, but in fact almost any fixative may
be used. It is reasonable as a general rule to avoid those that
dissolve lipids.

To prepare tissues for embedding in gelatine, it is only neces-
sary to wash out the fixative, generally with running water. If the
fixative was one that fixes gelatine as well as the proteins of the
tissues, it is important to get rid of it by thorough washing; for
otherwise it would fix the gelatine as it started to diffuse into the

68 CYTOLOGICAL TECHNIQUE

tissue, and thus stop its progress. Among the common primary
fixatives, however, only formaldehyde and osmium tetroxide fix
gelatine.

Gelatine is a chemically modified form of collagen, an insoluble
protein distinguished by its high content of glycine, proline, and
hydroxyproline.

I
N-CH3

\
CHo

/
HC— CH2

1

1
Proline as part of a protein chain

{The middle ^CH, group is replaced by

^CH.OH in hydroxyproline.)

The collagen used in the preparation of gelatine is derived from
the organic matter in the bones of cattle and from the white con-
nective tissue fibres in the skin of cattle and pigs. Bones are first
decalcified with hydrochloric acid. The skin or bone is 'limed' for
several weeks in an aqueous solution of calcium hydroxide; the
soluble proteins are thus dissolved away. After having been
washed with water, the material that has resisted solution is kept
in clean water at about 60^ C for several hours. In this it dissolves.
It is filtered, cooled to produce a gel, and dried. ^°- The dry
material is sold in the form of sheets or powder. The latter is the
more convenient form for use in the laboratory.

The transformation that is undergone by collagen during the
long treatment with limewater is reflected in the change of the
iso-electric point from about pH 7-8 to pH 4-7.^^ This results from
the conversion of amide-groups in the asparagine and glutamic
acid of the protein chain to the carboxyl groups of aspartic acid
and glutamic acid respectively. It is this change that makes gela-
tine so much more readily colourable by basic dyes than collagen
is. Various bonds that tie the protein chains together, such

EMBEDDING 69

1

NH NH

O O

HC.CH2.C HC.CHo.C

//

NH, I OH

C=0 C=:0

I I

Asparagine as part of Aspartic acid as part of

a protein chain a protein chain

as those between carboxyl in one chain and basic groups in
another,^^ are also broken by the alkaline bath. When the
material is warmed, the loosened chains move apart from one
another, and are shortened by hydrolytic transverse breakage of
the peptide links. ^^^ Chains varying in molecular weight from
about 15,000 to 45,000 are thus set free in association with water
as a sol. The molecules are neither fully extended into rods nor
compacted into globular form, but each is about 50 times as long
as broad.

When the filtered sol is cooled, these molecules attach them-
selves to one another, probably by hydrogen bonding between
peptide groups in previously separate molecules.^® The bond is

c— o

HCR

NH . .

. o— C

1

CR

NH

C=0 HCR

I I

A hydrogen bond {. . .) between two peptide groups

in protein chains

very susceptible to heat. Over a wide range of concentration,
gelatine associates with water to form a solid (gel) below 20' C,
a fluid (sol) above 35' C, and a substance showing anomalous
viscosity (neither a solid nor a true fluid) between certain tem-
peratures intermediate between 20° C and 35° C. There are said
to be some free molecules even in the gel.'^-

70 CYTOLOGICAL TECHNIQUE

If gelatine be maintained for a long time at a temperature ex-
ceeding 60° C, and especially if it be boiled, the shortening of the
protein chain goes so far that the capacity to form a gel on cooling
is destroyed. The resulting substance is called metagelatine.
Gelatine gels that are intended for embedding should not be
melted frequently, since this also has a softening effect.

A piece of fixed tissue that has been washed in water may be
transferred directly to a gelatine gel melted at 37° C. There is no
reason, either theoretical or practical, why tissue should first be
placed in a melted gel of low gelatine content and then transferred
to a more concentrated one. A suitable gel may be made by plac-
ing 25 g of powdered gelatine in 100 ml of distilled water, and
leaving this in an incubator at 37^ C until the gelatine has
dissolved.

If the gel is to be kept in stock, it is necessary to take pre-
cautions against the growth of bacteria and moulds. A good
disinfectant for the purpose is sodium /7-hydroxybenzoate, since
this is particularly effective in preventing the growth of the bac-
terium that liquefies gelatine gels. It should be dissolved at 0-2 °o
in distilled water, and the gel made with this in place of distilled
water. 1^

A gelatine gel that has been formed by simple cooling is
scarcely hard enough to give the necessary support to embedded
tissue, and sections cut from it are inconveniently sticky. It is
possible to harden the gel by slow evaporation,^^ but the usual
method is to link the molecules more securely together by the
action of fixatives. Formaldehyde is suitable. It will be remem-
bered that it acts by forming methylene bridges between protein
chains (p. 42). When the covalent bonds of these bridges have
added their effect to that of the hydrogen bonds, the gel is harder;
it no longer melts on being warmed, and the tendency to swell in
acid solutions is greatly reduced. Sections are still slightly sticky,
however. The salts of aluminium are able to fix gelatine. Potas-
sium alum (p. 119) is suitable. It abolishes stickiness and gives
hard blocks; but it cannot be used alone, instead of formalde-
hyde, because sections of gelatine hardened in this way roll up
instead of remaining flat. 'Formalum' ^^ is a suitable solution for

EMBEDDING 71

hardening gelatine gels. It is made by diluting commercial forma-
lin with 4 times its volume of a 5 °o aqueous solution of potassium
alum crystals. Gelatine blocks may be preserved indefinitely in
formalum.

Formaldehyde solution should be used without the addition of
alum if for any reason it is necessary to avoid the mordanting
eifect of aluminium salts (for instance, in the acid haematein
test^^ for phospholipids ; on the subject of mordanting, see p. 1 10).

Beyond hydrogen bonding and the formation of covalent links,
a third hardening process is generally used before sections are cut.
The water contained in the fixed gelatine is frozen, usually by
allowing carbon dioxide at a low temperature to flow past the
block. If, however, the gelatine gel has been hardened by evapora-
tion, little water remains in it and the block can be cut with a glass
knife without cooling below room-temperature.®^

It is possible to attach gelatine sections to glass slides,^" but it
is simpler to dye them (or to treat them with lysochromes) while
they are still loose. If loose sections are mounted in an aqueous
mounting medium, such as dilute glycerine (p. 128) or Farrants's
medium (p. 129), the tissue will have been kept continuously wet
with water from the moment when the cells were still alive.

PARAFFIN

Solid paraffin (so-called paraffin 'wax') is more commonly used
than any other embedding medium. It certainly has great
advantages. The process of embedding is quick and simple; em-
bedded material may be stored indefinitely in the dry condition;
sections may be obtained regularly at all necessary thicknesses
from about 2 fx upwards, if suitable waxes are chosen; and each
section adheres automatically to the next as it comes off" the
microtome knife, so that ribbons are formed. These qualities
make paraffin the best embedding medium for most purposes in
micro-anatomy and routine histology, and it plays an important
part also in studies of chromosomes and other cellular con-
stituents that are not easily distorted or dissolved. The main

72 CYTOLOGICAL TECHNIQUE

disadvantages of this medium are the shrinkage that occurs
during embedding, and the solution of most lipids. Indeed, the
incompleteness of our knowledge of the lipid constituents of
cells must be attributed largely to the popularity of paraffin
among microtomists.

To prepare tissues for infiltration by melted paraffin wax, it is
necessary to dehydrate them thoroughly and soak them in a fluid
that is readily miscible with the embedding medium. Ethanol and
cellosolve are particularly suitable dehydrating agents.

If all the constituents of the fixative are freely soluble in or
miscible with 50% ethanol, the tissue may be transferred directly
to that fluid. If one or more of the constituents of the fixative
react with ethanol to produce insoluble material, it is usual to
wash the tissue thoroughly in running water after fixation.
Chromium trioxide, for instance, tends to be reduced by ethanol
to insoluble green chromic oxide, CroOg; osmium tetroxide to
black or brown dioxide (OsOa or Os02.2HoO). It is worth re-
marking, however, that tissues fixed in chromium trioxide solu-
tion may be transferred directly to 50% ethanol, if 2% v/v of
strong sulphuric acid has previously been added to the alcoholic
solution.^*

It is customary to transfer tissues through a series of aqueous
alcoholic solutions of higher and higher ethanol content, until
absolute ethanol is reached. A suitable series is 50%, 80%, 96%,
absolute. Many more grades than these are often used, and
special apparatus has been invented to achieve very gradual
dehydration. It is doubtful whether any benefit accrues from this.
However closely graded the series of ethanol solutions may be,
there is always considerable shrinkage at one concentration or
another, somewhere between 60% and 90 °o ethanol. ^^ It is a
useful experience to judge the appearances of dyed and mounted
sections of tissues that have been dehydrated in diff"erent ways. A
friend may be asked to fix two pieces of the same organ in the
same fixative, and to dehydrate them in two diff"erent ways : one
piece through a closely graded series of ethanols, the other
through a coarsely graded series, or even from the washing water
directly to absolute ethanol. If he makes a number of slides from

EMBEDDING 73

each piece of tissue and does not divulge which is which, it will
be found difficult or impossible to guess.

If the fixative contained mercuric chloride, iodine should be
dissolved at 0-5% w/v in one of the grades of ethanol (for
instance, 80%), to prevent the formation of a black deposit
(p. 36).

If the fixative contained no water (Clarke, p. 59, for instance),
the tissue should simply be washed in absolute ethanol.

Although the tissue has now been dehydrated, it cannot be
invaded by melted paraffin wax, because ethanol and paraffin
are immiscible. It is necessary that the tissue should pass through
a fluid that is freely miscible with both. Antemedia^ are fluids in
which tissues are soaked immediately before they are transferred
to embedding media. Some of them have about the same re-
fractive index as dehydrated protein. Since they fill up the spaces
in the tissue not occupied by dehydrated protein, they render it
more or less optically homogeneous and transparent. They are
therefore often called 'clearing' agents, but the capacity to 'clear'
is a useless attribute of certain antemedia, and some of the best
do not possess it. A large number of more or less harmless fluids
are miscible with both ethanol and melted paraffin, and one may
choose one's antemedium from among them. Toluene is as good
as any for routine use. This light, colourless liquid is so called
because it was first obtained by distillation of an oleo-resin

CH.

Toluene

exported from Tolu in Colombia; but nowadays it is got from
coal tar. Its boiling-point (1 10° C) is well above the melting-point
of paraffin wax. Tissues tend to be distorted (unevenly shrunk)
when passed directly from ethanol to toluene, and it is therefore
better to pass them instead to a mixture of ethanol and toluene in
equal volumes, and from this to pure toluene.

Ethylene glycol mono-ethyl ether ('cellosolve') may be used
instead of ethanol as a dehydrating agent. The relationship of

74 CYTOLOGICAL TECHNIQUE

this substance to ethylene glycol (the familiar 'anti-freeze') is
shown by the structural formulae. It is made by the action of

HO— CH2

HO CH2
HO CH2

^CHg
CH,

Ethylene

glycol

Ethylene

oxide

C0H50— CH2

Cellosolve

ethanol on ethylene oxide under pressure at high temperature.
It is a colourless, odourless liquid boiling at 134°C.^°^ It has
remarkable powers of dissolving diverse substances, including
cellulose nitrate and acetate (whence, presumably, its trade name).
It is miscible with water and with toluene in all proportions.

Since cellosolve is a less violent dehydrating agent than ethanol,
there is no advantage in using a graded series of mixtures of
cellosolve with water. Fixed tissues may be transferred from water
to a mixture of cellosolve with an equal volume of water, and
then to absolute cellosolve; indeed, it is possible to omit the
intermediate stage. If the fixative contained mercuric chloride,
iodine should be dissolved at 0-5% in the cellosolve; it must
subsequently be washed out by the pure solvent. Tissues pass
from absolute cellosolve to a mixture of this with an equal volume
of toluene, and thence to toluene itself.

Cellosolve causes less shrinkage and hardening than ethanol.
Indeed, there are some organs, such as mucous glands, that can
scarcely be sectioned in paraffin after dehydration by ethanol,
but present no difficulty when cellosolve is used instead. The
great solvent power of this substance is, however, a drawback for
certain kinds of work. It appears to dissolve some of the con-
stituents of mitochondria, even when appropriate fixatives have
been used.^^ In studies of cytoplasmic inclusions it is safer to pass
tissues through ethanol, unless it has been shown that the use
of cellosolve is not harmful.

Tissues may be transferred directly from toluene to melted
paraffin. It would not appear that there is any advantage in
soaking them first in a solution of paraffin in the antemedium.'^

Paraffin waxes are constituents of crude petroleum. They are
saturated, long-chain hydrocarbons of the methane series. The

EMBEDDING 75

commercial products are mixtures of molecules of different
molecular weights. The waxes commonly used in microtechnique
melt at various temperatures between 52° and 60° C. This sug-
gests that the number of carbon atoms in most of the molecules
is less than 30, for H3C(CHo)28CH3 melts at 66° C.

Paraffin wax should be maintained at a temperature only a
few degrees C above its melting-point. A wax of high melting-
point (that is, with long molecules) should be chosen if thin
sections are required, or if the microtome is to be used in a warm
room. There is no advantage in transferring tissues first to a wax
of low melting-point and then to one of high. Indeed, the wax of
low melting-point would be ousted from the tissue by the other
wax very slowly and probably incompletely, for these large
molecules replace one another slowly by diffusion.

When the tissue is placed in melted paraffin, the antemedium
passes out to mix with it, while the separate paraffin molecules
diffuse in to replace them. Great shrinkage of the tissue will occur
if the antemedium escapes much more quickly than it can be
replaced. If, on the contrary, it escapes too slowly (because it is
insufficiently soluble in or miscible with paraffin), replacement
is likely to be incomplete. Toluene possesses neither of these
defects.

It is best to change the melted paraffin once or twice, in order
to get rid of the antemedium that has escaped from the tissue and
thus to facilitate the escape of the remainder and its replacement
by paraffin.

Tissues are much more fully permeated by paraffin than by
gelatine. The embedding medium enters the cells, and indeed
there is proof that it sometimes penetrates their nuclei: for
paraffin crystals, being birefringent, advertise their presence when
sections are examined in polarized light. ^^^ Penetration takes
place readily if the proteins of the cells have been coagulated, but
with difficulty if they have been fixed homogeneously. It is partly
for this reason, in all probability, that coagulant fixatives have
retained their popularity, despite the fact that they necessarily
distort the finer structure of the cell. Protoplasm that has been
fixed by osmium tetroxide is scarcely porous, and paraffin

76 CYTOLOGICAL TECHNIQUE

molecules cannot enter freely. As a result the embedded tissue,
being dry, friable, and not properly supported, often cracks or
crumbles during sectioning.

When replacement of the antemedium is complete, the paraffin
is hardened by crystallization on cooling. Each long molecule
places itself parallel with its neighbours. The molecules are not
fully extended, for each forms a zigzag in one plane. ^^^ Crystal-
lization occurs readily because the molecules, unlike those of
certain other waxy constituents of petroleum, have no major
branches that would interfere with close packing. Not every
molecule has a branch ; such branches as occur arise near one end
of the molecule, and are usually only one carbon atom long.^"^^

The crystals take the form of plates or needles. Each molecule
is held in position only by van der Waals forces, and this accounts
for the softness of the material and its low melting-point. There
is a tendency to a lamellar structure in the crystal, for all the
molecular zigzags lie in parallel planes, and each molecule in each
monomolecular plane is somewhat more strongly attracted to its
neighbours in that plane than to the molecules of other planes. ^^^
It is presumably the slipping of the planes on one another that
gives paraffin waxes their greasy feel.

It is generally believed that paraffin blocks should be cooled
quickly, by plunging them into cold water. The wax then hardens
in the form of small crystals, and these are supposed to give
better cutting qualities than large crystals. An experiment was
performed to test this belief. The kidney of a mouse was cut in
two. Both the pieces were fixed in Zenker, dehydrated in ethanol,
and passed through toluene into melted wax. One piece of
wax was cooled suddenly, in the usual way. The cooling of the
other was done in stages: first an hour at 45° C, then an hour at
37° C, then further cooling in a warm room (22° C). Both pieces
were cut on the same microtome and the sections dyed and
mounted. The two blocks cut equally well and the final prepara-
tions were indistinguishable.^^

Sections of tissue that have been thoroughly permeated by
paraffin may be freely exposed to the air without risk of damage.
This makes it easy to stick them to glass slides, and as a result

EMBEDDING 77

paraffin sections are very seldom dyed in the loose state. Egg-
white is an excellent adhesive for this purpose. It is best to make a
stock solution by diluting it with an equal volume of l°o aqueous
sodium chloride solution; any insoluble material is thrown
down by centrifuging. In the presence of sodium /7-hydroxy-
benzoate at 0-2% w/v, this solution resists the attack of bacteria
and moulds and may be kept for an indefinite period." For
use, the stock solution is diluted with about 50 times its volume
of distilled water. The fluid is then spread on a glass slide and
the section floated on it. The slide is warmed on a hot plate
sufficiently to soften but not melt the paraffin, and any folds in
the section flatten out. The water is then drained off as far as
possible and the slide left on the hot plate to dry. When it has
dried, the minute quantity of albumen between the section and
the glass suffices to stick them firmly together.

Glycerine is often mixed with egg-white in the preparation of
the adhesive. ^^^ It serves no purpose."

To remove the paraffin from the dried slide, it is only necessary
to soak it for a few minutes in xylene. The slide is then passed
through absolute ethanol to 90 °o and then 70 °o. If the dye that
is to be used is dissolved in a weak ethanol solution, the slide
may be transferred to it from 70 ''o ethanol; if the dye is dissolved
in water, it is best to rinse in distilled water first.

BUTYL METHACRYLATE

Butyl methacrylate was introduced into microtechnique as an
embedding medium in 1949. i^' AUhough modern plastics of
other kinds have been tried since then and one or two of them are
promising, yet the methacrylates are still among the best, at any
rate for routine use.

The great advantages of this embedding medium are that it
causes little distortion and permits sections to be cut at any
thickness from 8 /< or more down to about 10 m/i (though for
very thin sections a mixture of butyl and methyl methacrylates is
used). If tissues are suitably fixed, sectioned in butyl methacrylate
at about 3 //, and mounted in an appropriate medium (p. 128),

78 CYTOLOGICAL TECHNIQUE

the appearance of the cells is astonishingly lifehke. Methacrylate
is seldom used when sections are to be dyed. It finds its chief
application in the preparation of very thin sections for electron-
microscopy. The main disadvantage of methacrylate is that the
process of embedding is rather complicated.

Very small pieces of tissue (often about 1 cubic mm) are
generally used. A solution of osmium tetroxide, buffered at about
pH 7-4, is the most usual fixative (p. 58). After fixation the tissue
may be washed with distilled water (though many workers use
weak ethanol solutions), and must then be dehydrated. Graded
ethanols are used for this purpose. It has not been found neces-
sary, even for the most delicate work, to use a closely graded
series of ethanols. The series recommended for use in paraffin
embedding (50%, 80%, 96%, absolute) is suitable. No special
antemedium is required. The tissue may be transferred to a
mixture of equal volumes of absolute ethanol and methacrylate,
or directly to methacrylate itself.

The embedding medium to which the tissue is transferred is
/7-butyl methacrylate, in monomeric form. This is a colourless,
mobile liquid, boiHng at about 163° C. It has a distinctive, rather
unpleasant smell. The vapour is somewhat toxic and it is desir-
able to increase the ventilation if the smell is noticeable. Butyl
methacrylate is miscible with absolute ethanol and with lipid-
solvents such as carbon tetrachloride, acetone, and toluene. It is
insoluble in water, on which it floats. It is highly inflammable.

The acrylic acids form a series similar to the fatty acids, but
two of the carbon atoms in the chain are joined by a double
bond. In the strict sense the series includes only those acids in
which the double bond is in the same position, in relation to the
carboxyl group, as in acrylic acid itself: that is to say, just beyond

H H
-C— C—

H

H

C=

=€

H

cf°

\OH

A a

*-ylic acid

Acrylic acid as a segmer
in a polymer

EMBEDDING 79

the next carbon atom from the carboxyl carbon. The aldehyde
corresponding to acryhc acid is acrolein, and from this exces-
sively pungent substance the acid derives its name.

From our point of view, the important character of the acrylic
acids is their strong tendency to polymerize, and in doing so to
become solids possessing special characters. ^°^' ^^^ Polymeriza-
tion occurs by the addition of one molecule to another, the double
bond becoming single in the process. There is no condensation:
each repeated unit or 'segmer' consists of the same atoms as the
monomer. The substance formed is thus an addition-polymer.

The hydrogen of the carboxyl group of the acrylic acids can be
replaced by methyl, ethyl, or other alkyl groups, without loss of
the tendency of the molecule to polymerize. The esters thus
formed are of greater practical value than the simple monomers.
Reaction (in a roundabout way) with normal butyl alcohol,
HO(CH2)3CH3, gives «-butyl acrylate. This, when polymerized,
is a very soft, rubbery substance, not adapted to the purposes of
microtomy. If, however, a methyl group be substituted for the
hydrogen attached to the next carbon to that of the carboxyl
group, a marked change occurs in the physical consistency of the

CH3
H H H I

— C— C— — C— C—

« i^O H \^^0

\0(CH2)3CH3 \0(CH,)3CH3

A segmer of n-butyl A segmer of n-butyl

acrylate polymer methacrylate polymer

polymer. We now have, in «-butyl methacrylate polymer, a
hard, colourless, transparent solid, admirably adapted to our
needs. All the methacrylates are harder than the corresponding
acrylates, but the length of the esterifying alcohol also affects the
hardness. The shorter the alcohol, on the whole, the harder the
polymer. Methyl methacrylate — better known under its commer-
cial name of perspex — is the hardest of all. It is often used as an
embedding-medium for electron-microscopy, usually with the
admixture of butyl methacrylate. The latter by itself, however,

80 CYTOLOGICAL TECHNIQUE

gives good sections for electron-microscopy as well as for light-
microscopy.

If the segmers of butyl methacrylate were only capable of
linking together in the way previously mentioned, the substance
produced would resemble a fibre rather than a resin. In fact, the
long chains branch and link in such a way as to form a three-
dimensional spongework devoid of any particular orientation.
The reactions involved in this process are mentioned below
(p. 82). It is this submicroscopic structure of the methacrylates
that fits them so well for use as embedding media, capable of
being sectioned equally well in any direction.

At room-temperature the monomer very slowly undergoes
spontaneous polymerization. An inhibitor must be added to
enable the methacrylate to be stored in monomeric form.
Quinone is suitable. It is stated to react with chain-forming
radicles to produce stable compounds. ^^ Manufacturers add a
small proportion of hydroquinone to methacrylate, and this

O
OH

OH

Hydroquinone

becomes converted to quinone by spontaneous oxidation. When
the methacrylate is about to be used, the inhibitor is removed by
shaking the fluid with an aqueous solution of sodium hydroxide.
The water, coloured by the inhibitor, separates from the metha-
crylate under the influence of gravity. The monomer is washed
free from sodium hydroxide by being shaken several times with
distilled water; the latter is then removed by the addition of
anhydrous sodium sulphate. Although uninhibited, the material
will remain unpolymerized for many weeks if kept in a refrigerator
maintained near 0° C.

The methacrylate is now ready to receive the tissue. The
molecules being fairly small, thorough penetration takes place
quite easily. The fluid is changed a few times to get rid of the

EMBEDDING 81

ethanol. It would then gradually harden by polymerization, if left
at room-temperature, but the process is too slow for practical
use. It is necessary to add an activator or so-called catalyst. The
latter name is inaccurate, for, as we shall see, the substance in
question actually forms part (though a very small part) of the
hardened material.

The extremely active substance that promotes polymerization
is the free phenyl radicle, H5C6. It will be remembered that free
radicles differ from the vast majority of chemical compounds in
possessing an uneven number of electrons in the sum-total of
their constituent atoms. In the formulae shown here, it will be

H

• • •

•. C . . C

H : C '• • C : H H : C *• '* C : H

H :C .• C:H H :C / ..C :H

•• C** •• C

• • ••

H H

Diagram of the structure Comparable diagram of

of benzene, showing the the structure of the free

valency-electrons phenyl radicle

noticed that 8 electrons of the outer shell are associated with each
carbon atom in benzene, but only 7 with one of the carbon atoms
in the free phenyl radicle. The formula for the latter is conveni-
ently written HjCe* to remind the reader of the existence of the
unpaired electron.

Benzoyl peroxide, (C6H5CO)202, is used to produce the free
phenyl radicle. This colourless, crystalline solid may be regarded
as hydrogen peroxide in which each of the hydrogen atoms has
been replaced by benzoyl, C6H5CO~. It is an explosive, and care
should be taken not to grind it with metal instruments; for
storage it is damped with water. The tissue is transferred from
the unmixed monomer to a weak, freshly prepared solution of
benzoyl peroxide in the monomer. It is left in this at room-
temperature until the peroxide has had time to penetrate the
tissue before active polymerization begins. Since the peroxide
was damped, it is necessary once more to dry the methacrylate

G

82 CYTOLOGICAL TECHNIQUE

with anhydrous sodium sulphate. The temperature is then in-
creased to about 50° C and the hardening starts.

Each molecule of benzoyl peroxide loses two of carbon
dioxide, and two phenyl radicles are set free. Each phenyl radicle
reacts with a monomer molecule, forming a covalent bond with

H
the carbon of the C= group. The compound necessarily has an

H
uneven number of electrons, but the odd electron now appears

CH3

H I

HaCe-C-C-

\0(CH,)3CH3

A phenyl radicle combined with butyl methacrylate
Note the unpaired electron.

in a new place, at the other end of the methacrylate. Meanwhile
the double bond has become single. The end of the molecule
provided with the odd electron has become as active as the
phenyl radicle was, and in exactly the same way. It combines
with another monomer, and an unpaired electron is thus pro-
duced at the extremity of the latter. So the process goes on, and a
high polymer is built up. The whole of it consists of methacrylate
monomers, except one extremity of the chain.

This process of polymerization leads to the production of very
long molecules, but it does not continue indefinitely. The growing
end of the chain may come up against another growing end, or
against a free radicle; in either case combination occurs, and the
chain becomes 'dead'.

The process that has been described would lead only to the
formation of molecules having the form of long, thin threads.
In fact, however, branching and net-formation occur. Branching
takes place in various ways in addition-polymers,"" but the
following is probably what happens in methacrylates.^^^ A
carbon atom, other than a terminal one, becomes active. The
atom is thought to be one of those in the butyl group. This
carbon atom lets go of one of its hydrogens, which departs with

EMBEDDING 83

its single electron and attaches itself to the growing point of
another molecule (which is thus terminated and becomes 'dead').
The carbon atom now has an uneven number of electrons, and
therefore acts as a new growing point, to which other monomers
add themselves one after another. The branch thus formed may

CH3
H I
— C— C—

H I .0
Cf H

\OC(CH,)2CH3

A segmer of butyl methacrylate, with an active carbon
(C) in the butyl radicle

itself be terminated and 'die' as a loose end; but if it meets another
growing branch, the two may fuse and thus accomplish a union
between two branched molecules that were previously separate.
Since the branches are not all formed in the same plane, a three-
dimensional meshwork results.

Tissues shrink slightly in butyl methacrylate while the latter is
still in monomeric form.^ Polymerization involves further shrink-
age, since the methacrylate itself becomes reduced in volume.
The various methacrylate esters differ from one another in this
respect. The longer the molecule of the esterifying alcohol, the
less the reduction in volume on polymerizing. Butyl methacrylate
contracts to about 85% of its former volume, methyl methacry-
late to about 79 %.^^^ The butyl ester is therefore preferable unless
a very hard block is required. Measurements of the volume of the
embedded tissue'' suggest that this shrinks more than the metha-
crylate. Shrinkage is less than with paraffin, and since it is nearly
equal in all directions, there is scarcely any distortion.

Blocks containing tissue can be stored indefinitely in the dry
condition. To obtain sections from 8 /< to 1 /^ thick, it is best to
soak the block overnight in 70 % ethanol and to cut on a sliding
microtome with an oblique knife flooded with 70% ethanol. ^^
The back of the knife should be raised rather higher above the
level of the cutting edge than is usual when cutting paraffin.
Sections may be stored in 70% ethanol.

84 CYTOLOGICAL TECHNIQUE

Thin sections for electron microscopy are usually cut with
knives made by breaking a piece of plate glass along a line at an
angle of 45° to one of its edges. "^ The cutting edge is the one at
the intersection of the two surfaces that subtend an angle of
45° to each other.

Methacrylate sections may be stuck to glass slides for study
by phase-contrast microscopy (p. 128) in the same way as paraffin
sections. 2^ They are brought from 70% ethanol to water, and
floated on very dilute albumen solution on a glass slide. To re-
move the embedding medium after the section has been attached,
it is only necessary to put the slide in ethyl acetate or toluene,
which dissolves the polymer. The slide is then passed through the
usual series of ethanols to distilled water, and mounted in diluted
glycerol (p. 128).

CHAPTER 7

Introduction to the Chemical
Composition of Dyes

It is best to give the general name of 'colouring agents' or
'colorants' to substances used in microtechnique to colour or
blacken the parts of organisms.

Colouring agents are used in very diverse ways. It is a strange
fact that the housewife is more careful in her terminology of
colouring agents than many microscopists are. She distinguishes
clearly between staining and painting the floor, while they often
use the word 'staining' without regard for the diversity of the
processes grouped by them under this single name. The 'staining'
of specimens by electron-microscopists has no connexion with
dyeing. The word was formerly used as a synonym for 'dyeing',
but has come to be treated so loosely in microtechnique that it is
avoided in this book.

• There are five principal ways of using colouring agents to
distinguish the microscopical parts of organisms. In this book we
are primarily concerned with only one of these, namely, dyeing \
but this one is best understood by contrast with the others. The
five will be briefly described here.

Injection of suspended coloured particles into closed spaces. We
may suspend minute, insoluble, coloured particles in a fluid and
inject this into the blood-vessels or other internal spaces of an
organism. The final distribution of the coloured particles will be
determined by their incapacity to penetrate the walls of the vessels
into which the fluid has been forced. The chemical nature of the
colouring agent is irrelevant, provided that it is insoluble.

Uptake of suspended coloured particles by phagocytic cells. We

85

86 CYTOLOGICAL TECHNIQUE

may provide a phagocytic cell with minute coloured particles
suspended in the fluid in which it lives, and allow it to eat them.
The distribution of the coloured particles at any time will be
determined by the vital activity of the cell (movements of the
cytoplasm, &c.)- The particles will not colour any pre-existent
object in the cell.

Solution of a lysochrome. We may dissolve a lipid-soluble
colouring agent (not a dye) in 70 '^o ethanol or some other suitable
medium, and soak a section of fixed tissue in it. The lipids of the
tissue will take up the colouring agent simply because it is more
soluble in them than in 70% ethanol, and the distribution of the
colour will be determined by this. Such colouring agents are
called lysochromes, because they colour by solution (Greek
/w^-zi-, solution). ^^

Local formation of a coloured substance. We may soak the fixed
tissues in a solution of a substance that will react with one or
more of the tissue-constituents to produce an indilTusible coloured
product; the solution used will be colourless or differently
coloured from the product. Thus a yellow solution of potassium
ferrocyanide will give a blue precipitate of insoluble Prussian
blue wherever there is ferric iron in the tissue. The distribution of
the colour will depend on the chemical properties of the reactive
substance or substances in the tissue. Many histochemical tests
fall into this category of colouring reactions.

Dyeing. We may soak tissues (living or dead) in a solution of a
dye. Certain tissue-constituents will combine with the coloured
ions of the dye and thus become coloured. The colour will
usually not change. The final distribution of the dye will depend
on the ability of the dye to penetrate the tissues and on the
affinity of the tissue-constituents for its coloured ions.

The nature of dyeing is only briefly indicated by what has just
been said, but it will be clear that the process is entirely diff"erent
from the first three described above. It shows some resemblance
to the fourth, but there are important differences. Dyes do not
usually change colour when their ions are taken up by the tissues;
and the uptake is generally far less specific, for most parts of the

THE CHEMICAL COMPOSITION OF DYES 87

cell are capable of being dyed to some extent by most dyes, if
sufficient time is allowed.

For the purposes of microtechnique, dyes may be defined thus.
They are aromatic, salt-like, crystalline solids, that dissolve in
water or aqueous solutions in the form of ions ; either the cations
or the anions (occasionally both) are coloured; the coloured ions
can link themselves chemically with proteins (and generally with
other tissue-constituents as well); when the linkage takes place,
the ions do not lose colour, and usually they do not change it.

Two questions present themselves. What causes dyes to be
coloured, and what causes their coloured ions to attach them-
selves to tissue-constituents ? The first question will be considered
here, the second in chapter 8.

Many of the familiar colours of nature — the brilliant wings of
many butterflies, for instance, and the iridescent feathers of
birds — owe their colour to parallel plates or parallel striations,
separated by distances varying from about a quarter of a wave-
length to a few wave-lengths of visible light. These 'structural'
colours disappear when the substance is dissolved, because they
are not due to any colour intrinsic in the molecules or lesser
particles of the substance. It is only when such particles them-
selves show a differential transmission of light of diff'erent wave-
lengths, that a substance remains coloured on solution.

If all substances that remain coloured on solution are examined,
it is found that most of them fall into a few major groups. It is a
familiar fact that the salts of chromium, iron, and cobalt are
coloured, and that colour is retained when they are dissolved.
These are only examples of the wide generalization that the ions
and ionic complexes formed by the transition elements are
coloured; or, to dig a little more deeply into causes, we may say
that the elements that give coloured ions are those metals that
possess an incomplete shell of electrons inside their outermost
shell. That is to say, the cause lies in intra-atomic structure. No
dye, however, owes its colour to the possession of a transition
element, nor indeed to the possession of any particular atom as
such: its colour is due to its inter-atomic, not its intra-atomic
structure.

88 CYTOLOGICAL TECHNIQUE

Many aromatic, but few aliphatic compounds are coloured;
and this suggests that there must be something in ring-structure
that favours the production of colour : that is to say, that favours
the absorption of light of particular wave-lengths. This is true;
and all dyes are aromatic compounds. Benzene itself, the simplest
of this group of substances, would appear coloured if we could
see a short way into the ultra-violet, for it has an absorption-
band at the wave-length of 256 mjn. The molecule may be thought

^^

..^^^

Alternative structural formulae for benzene

of as 'resonating' between different molecular configurations;
that is, undergoing rapid change from one to another.

The alternation of single and double bonds between carbon
atoms is favourable to resonance and the associated absorption
of electro-magnetic waves; but there are certain molecular
arrangements that excite the molecule to a particular degree, so

O

II I!

11

The paraquinonoid
ring

O

Parabenzoquinone

that light of greater wave-length is affected, and colour results. A
very frequent arrangement of this sort in dyes is the quinonoid.
All substances possessing a paraquinonoid ring are coloured.

Parabenzoquinone is the simplest of such substances. It is a
pale yellow solid, dissolving in water to give a pale yellow solu-
tion, but it is not a dye. It dissolves to form a solution of mole-
cules, whereas dyes dissolve as ions. Parabenzoquinone has no
special tendency to attach itself to proteins or other tissue-
constituents in such a way as to impart colour to them: dyes
have such a tendency, powerfully developed.

THE CHEMICAL COMPOSITION OF DYES

89

The structure of dyes may be illustrated by the one known
commercially 3.spararosaniIine. (The para in this word is not used
in its chemical sense.) This is a magenta solid, easily soluble in
water. To make it, one needs aniline and paratoluidine. Aniline

NHo

Aniline

NH,

Aniline

NH

NH,

CH,

NH, CI-
Pararosaniline

is a colourless fluid (if pure), paratoluidine a colourless, crys-
talline solid. Heat them in the presence of a mild oxidizing
agent and a source of chloride ions, and a brilliant colour
develops. Two molecules of aniline combine with one of para-
toluidine to give a substance in which one of the three rings is
quinonoid.

To which of the three rings is colour due: to those derived
from aniline, or to that derived from paratoluidine ? The formula

ci-

NH2

Pararosaniline : another
resonance position

90 CYTOLOGICAL TECHNIQUE

given first suggests the latter, but other formulae show the

structure equally well. In fact, there is resonance between

different structures : one may think of the positive electric charge

as being now in one position, now in another.

The dye possesses a quinonoid ring, which gives it colour, and

an electrically charged group, which enables it to attach itself to

proteins and other tissue-constituents. The quinonoid ring is the

chromophore or colour-bearer; the -NHo groups, all capable of

+
conversion to =NHo, are the auxochromes or colour-helpers,

which help the chromophore to attach itself, and often greatly

increase the intensity of the colours. The substance as a whole has

the general character of a salt.

There are not very many different auxochromes. Many dyes

+
have the same auxochrome as pararosaniline, namely =NH2.

Since this group of atoms is positively charged, the coloured ion

is the cation and the dye is called cationic or basic. The anion may

be chloride or sulphate or acetate; any inorganic anion will do,

provided that it gives sufficient solubility. Such dyes may be

represented by the formula R+C1~, if the anion is chloride. The

+
hydrogens of the ^NHo group are often substituted by methyl or

ethyl, or by aryl (the latter being a benzene ring with one or

more substitutions of hydrogen atoms).

In many dyes it is the negatively charged ion that is coloured ;
these dyes with coloured anions are called anionic or acid. Their
cation is usually sodium or potassium. If it is sodium we may
write the formula Na+R~; R~ means the coloured anion.
Various auxochromes give the necessary negative charge. The
most usual are the sulphonic, hydroxyl, and carboxyl groups,
which ionize to give -SOg", -0~, and -COO" respectively.

It is to be noticed that the so-called 'acid' dyes are seldom
acids. The possession of the potentially acidic groups (sulphonic,
hydroxyl, and carboxyl) gave rise to the name. Such groups are
called acid radicles, despite the fact that they can only form part
of an acid if the cation is hydrogen. In a few dyes this is so; the
simplest formula for such dyes is H+R~.

THE CHEMICAL COMPOSITION OF DYES

91

Beyond auxochromes and chromophores, dyes often possess
atoms or groups of atoms that are called modifiers. Thus rosani-

NH

NH,

+
Rosaniline

line differs from pararosaniline only in the possession of a single
methyl group; this modifies the colour, making it very slightly
bluer. (Basic fuchsine is a mixture of these two closely related
dyes.) The methyl group is here a modifier. It has been mentioned
that the hydrogens of basic auxochromes may also be replaced
by methyl or ethyl or aryl groups ; this again modifies the colour.
Each replacement of one of these hydrogens in pararosaniline by
methyl makes a bluer dye, and cr)^stal violet, in which all six are
replaced, is nearly blue. Ethyl and particularly aryl groups have
even more blueing effect, and purely blue dyes such as methyl
blue (p. 94) may be obtained in this way.

Dyes are classified into groups by their chromophores. Most
of these groups contain both cationic and anionic dyes, possessing
the auxochromes mentioned above ; many of them in all groups
have modifiers (frequently methyl or ethyl). The great majority
of the dyes used in microtechnique owe their colour either to the
quinonoid ring or to the azo-group, -N=N- (p. 96). The
quinonoid dyes, however, are so diverse that it is necessary to
divide them into sub-groups, each characterized by a particular
chromophore that can be represented in a skeleton formula. The
principles of dyeing in microtechnique can be explained by the
use of only a few dyes, belonging to a small number of groups.

92 CYTOLOGICAL TECHNIQUE

We shall concern ourselves with only live sub-groups of quin-
onoid dyes. All the dyes mentioned in this book are listed here
for convenient reference, under the names of their groups and
sub-groups.

Dyes are not known by their full chemical names, because
these would be inconveniently long. Shorter names are used,
many of which are to some extent descriptive. Initial letters,
written in capitals, often form part of a name; these serve to
distinguish a dye from others that are closely related. Thus SS
means spirit-soluble (soluble in ethanol, not in water); G means
gelb (yellow). The B of azure B is an arbitrary mark of distinction
from two closely related dyes called azures A and C.

QUINONOID DYES

Triarylmethane

Cationic. PararosaniHne, rosaniline, methyl violet, crystal

violet, aniline blue SS
Anionic. Methyl blue

Haematein

Anionic. Haematein

Anthraquinonoid

Anionic. Alizarine, purpurine, carminic acid

Xanthene

Anionic. Eosin

Thiazine

Cationic. Thionine, azure B

AZO DYES

Mono-azo

Anionic. Orange G

QUINONOID DYES

Triarylmethane dyes. These may be regarded as derived from
leuco-pararosaniline, which is a colourless substance, since it
possesses no chromophore. Leuco-pararosaniline itself is methane
in which three of the four hydrogens have been replaced by aniline.

THE CHEMICAL COMPOSITION OF DYES 93

The aniline rings are merely particular examples of aryl rings, and

NHc

NH.

NHo

ci-

Leitco-para-
rosanil'me

Skeleton-formula for
triarylmethane dyes

Triphenylpara-
rosaniline

all the many dyes that have three such rings held together in the

+

same way, whether any of the rings has an = NH2 group on it or
not, are therefore classified together as triarylmethane dyes. In
the skeleton-formula for this and other groups of dyes, the auxo-
chromes and modifiers are omitted, since these differ from dye to
dye.

Methyl violet is pararosaniline in which four or five of the
hydrogens of the amino-groups have been replaced by methyl.
This replacement has a strong blueing effect. When all six are
replaced, crystal violet is produced; this dye is almost blue. It
has already been mentioned (p. 91) that ethyl and aryl groups
have still greater blueing effect than methyl. This is well exempli-
fied by triphenyl pararosaniline, which, with another closely
related dye, constitutes the mixture known as aniline blue SS.
Triphenyl pararosaniline is a pure blue cationic dye. It is para-
rosaniline in which one of the hydrogens of each of the three

94 CYTOLOGICAL TECHNIQUE

amino-groups has been replaced by a phenyl group (that is, by a
simple, unsubstituted aryl group).

Methyl blue is a pure blue anionic triarylmethane dye that owes
its colour to three extra aryl groups acting as modifiers. Despite
its misleading name, it possesses no methyl group.

Haematein. This is a very small group of dyes that are derived
from natural products and are not made synthetically. As the

HO HO

HO^^f^^^Hs HO

CH,

HO^x^

Skeleton-formula for
haematein dyes

O

Haematein

Catechol

formula shows, this is not only an acid dye, but actually an acid,
related to catechol. It is of a brownish-red colour, but becomes
blue in alkaline solution. It is therefore used as an indicator of
pH, but it is not valuable by itself as a dye. When used with an
intermediary or 'mordant' (p. 110) between itself and the tissues,
it becomes one of the most important dyes used in micro-
technique.

Haematein is derived from the heart -wood of a small legum-
inous tree, Haematoxylon campechianum, native to Central
America. This contains a non-quinonoid substance, haematoxylin,
which is not a dye and is indeed colourless, since it lacks a
chromophore; but it is readily oxidized to haematein by weak
oxidizing agents, including atmospheric oxygen. Haematoxylin is
often sold in a partially oxidized form, as a brown powder that is
really a mixture of haematoxylin and haematein.

Anthraquinonoid dyes. These are related to anthraquinone, a
yellow, crystalline substance. The simplest is alizarine, the chief
coloured constituent of madder, a vegetable dye. Alizarine is
easily synthesized from anthraquinone. It is not used in micro-
technique. Its more complex relative, carminic acid, is a parti-

THE CHEMICAL COMPOSITION OF DYES

o o

95

O

Anthraqiiinone

OH

Alizarine

CO(CHOH)4CH.<.
OH

Carminic acid

cularly valuable dye in biological work. Carminic acid is soluble
(unlike alizarine) in distilled water, and also in ethanol. It occurs
naturally in the fat-body of the wingless females of the coccid
Dactylopiiis cacti. This is a scale-insect that sucks the juices of the
succulent plant Nopalea coccinellifera. The latter is cultivated in
various subtropical parts of the world to provide the insect with
food. The dried females constitute cochineal. Carmine is a crude
form of the dye, produced by precipitating an aqueous extract of
cochineal with alum (potassium aluminium sulphate). It is com-
posed mainly of carminic acid bound to aluminium and to pro-
tein derived from the insect. Carmine is insoluble in distilled
water; but solutions can be obtained (e.g. by the addition of acid
or alkali), and the crude dye is usable in microtechnique. It is
best to use the pure acid whenever one wants to know exactly
what reactions are occurring.

One of the main uses of carmine in microtechnique is explained
in the chapter on the use of mordants (p. 122).

Xanthene dyes. In these dyes, carbon and oxygen form links
between two rings. Not many members of this group are com-
monly used in microtechnique. Eosin, however, is particularly
familiar. It is a valuable 'background' dye: that is to say, it is
anionic, pale, diffuse in action, and used to give contrast with
objects picked out vividly in another colour by another dye.

96

CYTOLOGICAL TECHNIQUE

^ Br Br

C^\^

Na+ -O
Br

Br

COO- Na+

Skeleton-formula for
xanthene dyes

Eosin Y

The name refers to the pinkish-orange colour of the sky at dawn
(Greek eds, dawn). It will be noticed that eosin has two different
auxochromes, and that bromine is here used as a modifier. In
related dyes chlorine and iodine partially or wholly replace the
bromine, and a bluish tinge is thus imparted.

Thiazine dyes. Here sulphur and nitrogen link two rings (Greek
theion, sulphur; French azote, nitrogen; the a in thiazine is short,
as in azote). Thionine, a blue dye with a reddish tinge, is the

^^N

HoN

S. ^^ .NH.

N^\^

Skeleton-formula for
thiazine dyes

ci-

Thionine

simplest member of the group. The thiazines used in micro-
technique are all cationic. They are valuable dyes for nucleo-
proteins. Several of them, including thionine, are 'metachro-
matic'; that is to say, they impart quite different colours to
different substances (p. 104). This is a most valuable property,
since it gives important indications of chemical composition.
Azure B is another metachromatic thiazine dye. It is trimethyl
thionine ; that is to say, thionine modified by the substitution of
three methyls for three of the four hydrogens of the amino-
groups.

AZO DYES

These contain no quinonoid ring, apart from a few that have
both azo and quinonoid chromophores. The name refers to the

THE CHEMICAL COMPOSITION OF DYES 97

nitrogen atoms that link two rings together. (The name is derived
from French azote ; short a.)

SO3-

Na+

Na+ -O3S/ \

o— o

Q-N^N-Q

\ / \ /

HO

Skeleton-formula for
azo dyes

Orange G

The -N=N- group occurs once, twice, or thrice in the ion,
which may be a cation or an anion; the corresponding group-
names are mono-azo, disazo, and trisazo. The formula shows that
orange G is an anionic, mono-azo dye; there are two -SOg"
groups. The name refers to the colour, which is a very yellowish
orange. This is a typical background dye.

Not all azo substances are dyes. Sudan IV, for example,
possesses the azo chromophore and is coloured (red). The
formula shows, however, that it lacks an auxochrome. It is in-

CH3

Sudan IV

soluble in water, but very soluble in lipids. It is an important
lysochrome, used in histochemistry for the recognition of lipids.
It is presented to the tissues at saturation in 70% ethanol. Being
much more soluble in lipids than in this solvent, it accumulates in
lipid globules and colours them. As we shall see in the next
chapter, dyeing is a very different process from mere colouring
by solution.

H

CHAPTER 8

The Causes of Differential Dyeing

When a section is soaked in a solution of a single dye, the
various tissue-constituents react differently. There is differential
uptake, and the varying intensity of coloration serves to dis-
tinguish the constituents. The latter may also react differently to
different dyes, for a particular object may take up much of one,
little of another. These facts make it possible to produce striking
colour contrasts in what had been a transparent, colourless
object. That, indeed, is the purpose of dyeing. We are now con-
cerned with the causes that produce these effects.

Whether a particular object in a microscopical section is
strongly or weakly coloured by a particular dye depends on three
factors : the chemical ajfinity between the object and the dye, the
density of the object, and the permeability of the object by the
dye.

Chemical affinity

Some of the constituents of the tissues, in the form in which
they appear on the slide in sections, are acidic. Examples are
DNA and chromatin, RNA and ribonucleoprotein, the matrix of
cartilage, the mucous secretions of certain gland-cells, and certain
conjugated lipids. Other constituents, such as the ground-
cytoplasm of most cells and the contractile substance of muscle,
are neither particularly acidic nor particularly basic. They are
markedly amphoteric ; that is to say, their electric charge varies
easily with the pH of the fluid in which they lie. Others again,
such as collagen, the cytoplasm of red blood-corpuscles, and the
granules of eosinophil leucocytes, are basic.

DNA, RNA, and phospholipids owe their acidity to their

98

THE CAUSES OF DIFFERENTIAL DYEING 99

phosphoric groups. The acidity of the mucopolysaccharides of
cartilage and of certain mucous secretions is due to sulphuric
and carboxyl groups. The amphoteric proteins owe their char-
acter to a balance between acidic amino-acids (aspartic, glutamic,
hydroxyglutamic, &c.) and basic ones (lysine, arginine, and

I
NH

I
HC(CH2).,COOH glutamic acid

i

c=o

I

NH
I
HC(CH2)4NH., lysine

I

c=o

I
Part of a protein chain at the iso-electric point

histidine). The latter predominate in the basic proteins of
collagen and red blood-corpuscles.

When a protein is put in a fluid at a pH below its iso-electric
point, the amino-groups of its basic amino-acids tend to become

positively charged as -NH3. When the pH is above the iso-
electric point, the carboxyl groups of the acidic amino-acids
become ionized as -COO~. If the proportions of basic and acidic
amino-acids in a particular protein are about equal, the electric
charge of the protein as a whole is easily shifted by variation in
pH within the range of the fluids ordinarily used in microtech-
nique; but in fact all proteins can be shown to be amphoteric if
there is a sufficient change of pH.

We have seen in chapter 7 that all dyes dissolve as ions, and
that the coloured ions of some are positively charged, of others
negatively. This can easily be proved by experiment. An agar gel
is allowed to set in a vertical U-tube, and the dye-solution to be
tested is added to both limbs of the tube, above the agar. The
agar serves the mechanical function of holding the water in the
U-tube nearly still. Salt must be dissolved with the agar, to permit
the easy passage of an electric current. An electrode is placed in

100 CYTOLOGICAL TECHNIQUE

the dye-solution on each side. It is necessary to take the usual
steps to prevent polarization of the electrodes; full practical in-
structions for setting up the apparatus have been given else-
where.^^ When the current is switched on, the coloured ions begin
to move down one or the other limb of the U-tube, towards either
the cathode or the anode. The distance travelled in a given time
depends on the concentration of the dye and of the agar, on the
diffusibility of the dye, and on the strength of the electric current.
In the apparatus referred to,^^ a movement of 2 or 3 cm in 24
hours is usual. There is often a much smaller movement of the
coloured ions down the other limb of the U-tube. This is due to
simple diffusion.

Most dyes can be described unequivocally as cationic (= basic)
or anionic (= acid). Some, however, are amphoteric. Haematein
is an example. This dye is cationic below pH 6-6 (the iso-electric
point) but anionic above.

In general, the acidic (negatively charged) objects in tissues
attract the cationic dyes, while the basic (positively charged)
objects attract the anionic dyes. Since the former objects are
easily dyed by basic dyes, they are called basiphil; the latter, since
they are easily dyed by acid dyes, are called acidophil. It is to be
remembered that basiphil objects are acidic, acidophil objects
basic. Thus the nucleic acids, most nucleoproteins (ordinary
'chromatin', for instance), the matrix of cartilage, many mucous
secretions, and certain conjugated lipids are basiphil; while
collagen, the cytoplasm of red blood-corpuscles, and the granules
of eosinophil leucocytes are acidophil.

It is useful to form a concrete idea of the effects of cationic and
anionic dyes by studying sections of specially selected tissues. The
testis, intestine, and pancreas of the mouse are suitable. Most
fixatives leave the tissues in a state in which cationic dyes act well,
but some are unfavourable to anionic ones. It is well to use
Zenker's fluid for the present purpose, because it is especially
favourable to the action of anionic dyes. Sections should be
lightly dyed and then run quickly through the alcohols and xylene
into a resinous mounting medium.

In sections coloured by cationic dyes, the most prominent

THE CAUSES OF DIFFERENTIAL DYEING 101

feature is the strong colouring of chromatin. Metaphase and
anaphase chromosomes, both mitotic and meiotic, are more
deeply coloured than anything else; nuclei are conspicuous on
account of their chromatin content. The mucous secretion of the
goblet-cells in the intestine may be rather strongly dyed, fre-
quently in a different colour from anything else (p. 105). The
cytoplasm of most kinds of cells (such as spermatogonia and
spermatocytes, and the interstitial cells of the testis) is scarcely
tinged. Certain kinds of cells, however, have a large amount of
ribonucleic acid in the cytoplasm, and this takes the dye. The
epithelial cells of the crypts of Lieberkiihn are examples; the
bases of the exocrine cells of the pancreas are even more strongly
dyed, because of the particularly great amount of ribonucleic
acid associated with the massive endoplasmic reticulum of this
region. There is scarcely any colour, however, in the contractile
substance of smooth muscle, in collagen fibres, in the free border
of the intestinal epithelium, or in the large granules of the Paneth
cells in the crypts of Lieberkiihn. There is nothing to distinguish
the granules of the eosinophil leucocytes in the cores of the
intestinal villi, and these cells can scarcely be recognized. Red
blood-corpuscles are not dyed.

The whole aspect of sections of the same tissues coloured with
a typical anionic dye is entirely different. There is a general
colouring of the cytoplasm of most kinds of cells. The cytoplasm
of the interstitial cells of the testis takes up more of the dye than
that of the spermatogenetic cells, and groups of the former kind
of cells therefore stand out rather prominently under the low
power of the microscope. The basal part of the exocrine cells of
the pancreas is moderately deeply coloured. The cytoplasm of
the epithelium of the crypts is pale. The contractile substance of
smooth muscle takes the dye; so do collagen fibres. None of
these tissue-constituents, however, is nearly so strongly coloured
as certain cytoplasmic granules, namely those of the Paneth cells
and particularly the minute ones of the eosinophil leucocytes in
the cores of the villi. Chromatin shows no special affinity for the
dye and is only lightly coloured. The nuclear sap is colourless.
The nuclei therefore often appear almost as though they were

102 CYTOLOGICAL TECHNIQUE

cavities in the dyed cytoplasm, and they are not nearly so con-
spicuous as in sections coloured with a cationic dye. Red blood-
corpuscles will take any anionic dye that is able to enter them
(p. 108).

It must not be supposed that basiphil objects are necessarily
resistant to coloration by anionic dyes, or acidophil ones to
coloration by cationic dyes. Proteins that are predominantly
acidic contain some basic amino-acids, and conversely. Thus all
the proteins in a cell can eventually be dyed to some extent by any
dye, whether basic or acid. However, by controlling the times
during which the dyes act, it is easy to ensure that the basiphil
objects will be dyed by cationic (basic) dyes and the acidophil
ones by anionic (acid) dyes, each almost to the exclusion of the
other.

The amphoteric objects in cells can be dyed with either cationic
or anionic dyes, by controlling the pH. In practice one usually
wants to leave the cytoplasm uncoloured, or else to dye it differ-
ently from the chromatin. If a cationic dye is used in slightly
acid solution (methyl green, for instance, in a weak solution of
acetic acid), the chromatin will be coloured: but the amphoteric
cytoplasm, having been rendered basic by the acidity of the
solution, will have very little affinity for the cationic dye, and the
cytoplasm will therefore appear colourless or nearly so. Alterna-
tively one may use a cationic dye in acid solution first, and then
an anionic one of contrasting colour. The amphoteric cytoplasm
will have some affinity for the anionic dye, and it can thus be
coloured differentially.

Since most tissue-constituents will eventually be coloured to
some extent by any dye, it may be desirable to limit the period of
dyeing, so that some of them will appear colourless when others
have already been quite strongly dyed. In other words, dyeing
may be arrested at a particular stage instead of being allowed to
go to completion. This i?, progressive dyeing, so called because the
depth of colour becomes progressively greater until the desired
end is reached. In regressive dyeing, on the contrary, the tissues
are allowed to take up an excess of the dye, and the latter is then
partly removed, with careful control under the microscope, until

THE CAUSES OF DIFFERENTIAL DYEING

103

certain tissue-constituents have lost the colour while others
retain it. During this regressive process the parts become more
distinct from one another than they were when all parts held too
much of the dye. The regressive stage of the process is therefore
called differentiation.

Cationic dyes are frequently used regressively. It is customary
to use acids as differentiating agents. They act, in principle, in
exactly the same way as when they are mixed with the dye:
reliance is placed on the ability of the acid to render the am-
photeric tissue-constituents basic, and thus make them let go of
the cationic (basic) dye. Alkalis can be used in the same way to
differentiate acid dyes.

An alternative way of differentiating is to soak the tissue in a
fluid in which the tendency of the dye to remain ionized is
reduced or eliminated, but in which the dye is soluble. Ethanol,
either absolute or in strong aqueous solution, is often used for this
purpose. The dye is rapidly lost from those tissue-constituents
that hold little of it. If differentiation be stopped as soon as these
constituents appear colourless, the objects that still retain a
considerable amount of the dye will contrast strongly with them.
The tissue must now be brought quickly into a fluid, such as
xylene, in which the dye is insoluble.

Acidic tissue-constituents, lacking any basic groups, can in
certain circumstances be dyed by anionic (acid) dyes. Thus
methyl blue, an anionic triarylmethane dye, will colour cellulose
cell-walls. Indeed, it is used in the textile industry for dyeing
cotton, and is often called cotton blue. The structural formula
for cellulose shows the absence of any group that would be

H OH H.COH

O-i

r-O

H^COH H

Part of a cellulose molecule

OH

104 CYTOLOGICAL TECHNIQUE

thought Hkely to attract an acid dye. It is supposed that the
linkage is through a hydrogen bond," for it will be remembered
that hydrogen can in certain circumstances act as though it were
bivalent. Such a bond may tie the hydrogen of a hydroxy 1 group
of cellulose to the nitrogen of an amino-group (or substituted
amino-group) in a dye. Now methyl blue, and certain other
anionic triarylmethane dyes that will colour cellulose, do contain
nitrogen in a substituted amino-group.

The hydrogens of the peptide groups of nylon are thought to
form hydrogen bonds with the amino-groups of certain dyes in
much the same way. If so, it is likely that the peptide groups of

c=o

i
NH

I

Peptide group in nylon or protein

protein could behave similarly. This may account for the ten-
dency of certain acid dyes to colour tissue-constituents without
appearing to discriminate between those that are positively and
negatively charged.

It is thought by some that dyes are first brought close to
particular tissue-constituents by the interaction of electrical
charges, and are then tied closely to them by 'short-range' forces;
that is to say, by covalent or hydrogen bonds. ^^^ Whether a close
tying-up of this sort does or does not occur finally as a general
rule in microtechnical dyeing, the distribution of dyes is in the
main determined by the electrical charges, density, and per-
meability of the tissue-constituents.

It has already been remarked (p. 86) that dye-ions are usually
taken up without change of colour. An important exception to
this rule must now be briefly mentioned.

Certain pure (unmixed) dyes have the property of dyeing par-
ticular tissue-constituents in a colour that differs from that of the
solution. This is called metachromasy and the dye is called

THE CAUSES OF DIFFERENTIAL DYEING 105

metachromatic; the tissue-constituents that are dyed in a differing
colour are called chromotropes (colour-turners).

All the metachromatic dyes that are commonly used in micro-
technique are basic, and they all act on the same chromotropes.
The metachromasy of acid dyes will not be considered in this
book.

The metachromatic dyes do not form a chemical group by
themsehes. On the contrary, they are distributed among all the
major groups except the azo. Methyl violet is a metachromatic
triarylmethane dye, for instance; thionine and azure B are meta-
chromatic thiazines. (Commercial thionine is often adulterated
with a red dye, and may then give false metachromatic colours. ^°^)
It is a curious fact that when all the hydrogens of the amino-
groups of a dye are replaced by methyl, the resulting substance is
not metachromatic.^^" Thus crystal violet (p. 93) is not meta-
chromatic (though in commerce it is often adulterated with the
strongly metachromatic methyl violet).

The colour-shift caused by chromotropes is always in the
same direction. Green dyes become bluish; blue dyes are reddened
to purple or red; red ones become orange or yellow. This means
that the absorption-maximum of the dye moves in all cases
towards the shorter wave-lengths.

Chromotropes are necessarily acidic, since they have affinity
for cationic dyes. Their negative charges are due to the possession
of sulphuric, phosphoric, or carboxyl groups. Many of the most
familiar chromotropes are sulphuric esters of polysaccharides of
high molecular weight. ^^"^ These are often mucosubstances. Thus
the matrix of cartilage, the secretions of certain mucous glands,
and the granules of the basiphil cells (Mastzellen) of connective
tissue give metachromatic colours because they contain chon-
droitic acid, mucoitic acid, and heparin respectively. Not all
chromotropes, however, are mucosubstances. Agar, strongly
chromotropic, lacks an amino-group.

The cause of metachromasy has not been established with
certainty. The metachromatic dyes have a tendency to form
dimers and polym.ers, of the metachromatic colour, in aqueous
solution. Chromotropes appear to be substances that favour

106 CYTOLOGICAL TECHNIQUE

Strongly the dimeric and polymeric forms of the dye, and hold
the dye to themselves in its dimeric or particularly its polymeric
form.i^°' ^2^' ^^^' ^'^ A small molecule would not be able to hold
a high polymer of a dye, but a polymeric substance, with numerous
negative charges arranged on its surface at regular and convenient
distances apart, might be able to do so.^^^ It thus appears that the
mode of attachment of the dye causes the shift of colour.

Metachromasy is important in cytological technique because
it acts as a pointer towards chemical composition.

Density

Make two gelatine gels, one containing 5% and the other 20%
of gelatine. Fix these in formaldehyde solution (or in a mixture
containing formaldehyde). Cut a section of each on the freezing
microtome, at the same thickness (say 15 //). Allow an aqueous
solution of any dye to act on both sections for the same period,
which must be sufficient to allow complete permeation. Rinse
with distilled water. It is not surprising that one section is more
strongly coloured than the other. The result is due solely to the
fact that one section is denser than the other: that is to say, it
contains more colourable matter than the other, and therefore
takes up more dye.

One often reads that the cytoplasm of a particular cell is 'dense'.
How did the writer know ? The depth of colouring may be due to
quite different causes. If one had not made the gelatine gels one-
self, one would not have known the cause of the difference in
colouring. The density of the two sections might have been
exactly the same, but one of them might have been made of a
basic and the other of an acidic protein, and they would neces-
sarily have taken up different amounts of any particular dye.

Anyone who performs the experiment just described is likely
to be surprised by the darkness of the section that contains more
gelatine. There is only four times as much gelatine, but it appears
to have taken up much more than four times as much dye. This
is, in fact, an illusion. Let us suppose that we have used a black
dye, and that the section made from the v/eaker gelatine gel stops

THE CAUSES OF DIFFERENTIAL DYEING 107

one-half of the hght that is shone on to its surface. Make a pile
of four such sections, one on top of the other, and illuminate
them from below. The second from the bottom receives half the
original light, and sends up half of what it receives to the next;
this halves the light again, and so does the uppermost section.
Thus only one-sixteenth of the light gets through. But in these
four sections together there is the same amount of gelatine — and
therefore the same amount of dye — as in a single section cut from
the stronger gel. The latter, for the same reason, lets through
only one-sixteenth of the incident light, though the amount of
dye is only four times as great as in the section that let through
one-half. For this reason we are apt to form an exaggerated
opinion of the difference in the amount of dye taken up by
objects in microscopical preparations.

When dividing cells are coloured with a cationic (basic) dye,
each metaphase chromosome takes up far more of it than an
equal volume of cytoplasm does. This results partly from the
fact that nucleoprotein is much more acidic (basiphil) than the
cytoplasm, but partly also from the fact that the chromosomes
are denser. Their density is indicated by the fact that anionic
{acid) dyes also colour chromosomes more deeply than the cyto-
plasm; indeed, they are used to colour chromatin in several
familiar techniques. For instance, acid fuchsine is used for this
purpose in Mallory's method for the differential colouring of
collagen. ^^^ Whenever a particular object can be more deeply
dyed than the cytoplasm or nuclear sap by both anionic and
cationic dyes, it is likely to be denser (though one requires an
interference microscope for the actual measurement of density).
The converse, however, is not necessarily true: we cannot con-
clude that a particular object contains little matter, from the fact
that it takes up little or no dye. It may possess few or no acidic or
basic groups, capable of attachment to cationic or anionic dyes.
Thus triglyceride droplets, though very dense, cannot be dyed.

108 CYTOLOGICAL TECHNIQUE

Permeability

The amount of dye taken up by an object in a particular time
does not depend solely on the amount of matter it contains, or on
the abundance of charged groups. It depends also on whether the
dye can permeate the object easily. Certain dyes have a great
capacity for penetration, others very little. ^^' ^^' ^^°' ^^^ It is easy
to test this capacity. ^^ It is only necessary to allow some gelatine
or agar to set into gels in test-tubes, and then to add solutions of
different dyes, all at the same concentrations, to different tubes.
Some dyes diffuse quickly into the gel, some slowly. Eosin
(xanthene) and orange G (azo) are examples of rapidly permeat-
ing dyes, methyl blue (triarylmethane) of slowly permeating. In
general, the rapidly permeating dissolve as single ions (and not
very large ones); the slowly permeating tend to form colloidal
solutions, in which each particle is an aggregate of several ions.

In microtechnique we make use of the varying rates of penetra-
tion to colour differentially objects that carry the same electric
charge. Thus collagen and the red blood-corpuscles of vertebrates
both contain a high proportion of basic protein and are therefore
acidophil. It is quite easy, however, to colour them differently
with two different anionic (acid) dyes. The mixture of methyl
blue and eosin devised by the Oxford histologist, Mann, serves
the purpose well.^^^' ^^^ Methyl blue is much the more powerful
dye (partly, perhaps, because a single positive charge in the
object may be able to attract a whole ion-aggregate). Wherever
the two dyes can compete, methyl blue predominates and the
colour is blue or bluish. This applies to collagen fibres, which are
evidently loose-textured, for they are easily coloured by aggre-
gated dye-ions. Red blood-corpuscles, on the contrary, are very
close-textured, and they are much more easily entered by separate
dye-ions than by aggregates. The result is that Mann's methyl
blue eosin dyes collagen blue and red blood-corpuscles orange-
red.

The various tissue-constituents can be arranged in the order of
their permeability. Collagen is very permeable; cytoplasm less so;
the contractile substance of muscle less so again; red blood-

THE CAUSES OF DIFFERENTIAL DYEING 109

corpuscles are particularly impermeable. It is possible to dye
collagen in one colour, cytoplasm and contractile substance in
another, and red blood-corpuscles in a third, by the use of three
different anionic dyes. The result is achieved without any reliance
on different chemical affinities.

The three qualities in the tissue-constituents that make possible
the differential action of dyes — chemical affinity, density, and
permeability — may act together or may antagonize one another.
Chromatin, for instance, is chemically reactive (basiphil), dense,
but permeable : all these characters act together to render it easily
coloured by cationic dyes. Red blood-corpuscles, on the con-
trary, are chemically reactive (acidophil), dense, but very im-
permeable. A microscopical preparation coloured with two or
three dyes owes its variegated appearance to complex inter-
actions, and it is not always easy to disentangle the effects of the
three factors.

Those anionic dyes that penetrate readily tend to colour all
acidophil and amphoteric tissue-constituents about equally
deeply, and they are therefore diffuse in action. They are useful
'background' dyes, giving colour-contrast to a cationic dye used
for chromatin. If methyl blue or a similar anionic dye were used
instead of a diffuse one, it might spoil the effect by giving some of
its colour to chromatin.

It is best to choose background dyes of colours near the middle
of the visible spectrum (yellowish or green), since these appear to
the human eye as 'unsaturated' ; that is to say, they stimulate all
the colour-receptors in the retina to some extent and thus appear
pale, as though mixed with white. Orange G, being yellow, is suit-
able. For contrast the chromatin should be dyed in a 'saturated'
colour near one end of the spectrum (red, blue, or violet). Violet
gives the best contrast with yellow, because the colours are
complementary.

If chromatin be dyed black or grey, any background dye will
necessarily reduce the contrast.

CHAPTER 9

The Action of Mordants

up till now we have been concerned with the direct attachment of
dyes to tissue-constituents. In some of the most important pro-
cesses of dyeing, however, an intermediary or mordant stands
between the dye and the tissue. The dye attaches itself to the
mordant: the latter (as its name suggests) 'eats into' or grips the
tissue.

The great advantage of mordanting is that the colour is not
removable by neutral fluids, whether aqueous or alcoholic. One
may therefore colour progressively or regressively until the tissue
is properly displayed, and then dehydrate at leisure, or counter-
stain as desired. The fact that dehydration may be done as slowly
as one pleases is particularly helpful in making whole mounts. As
we shall see, it is chiefly chromatin that is coloured by most
mordant dyes. Nuclei are crowded together in the epithelial parts
of many organs, but are sparse in the connective tissues ; and there
may be cavities from which they are absent. Thus the dyeing of
chromatin gives clear micro-anatomical pictures in whole mounts,
even with quite low powers of the microscope.

The fastness of mordant dyes makes them suitable for use in
aqueous mounts, provided that acidity is avoided.

All dyes that act with mordants can also be used without them,
but if so their efl"ects are quite diff"erent. By no means all dyes can
be used with mordants. The chief ones so used in microtechnique
are carminic acid and haematein. The chief mordants used with
them are salts of aluminium and of ferric iron.

In the textile industry the chief dyes used with mordants are azo
dyes, and the chief mordants are complex basic salts of chromium.
The chemistry of the mordanting of azo dyes by chromium has

110

THE ACTION OF MORDANTS 111

been carefully worked out by the textile chemists. ^^' ^^^' ^^' ^^
Unfortunately it is radically different from the mordanting of
carminic acid or haematein by aluminium or ferric salts.

One of the simplest dyes that can be mordanted is purpurine,
which, like carminic acid, is an anthraquinone dye. Alizarine is
even simpler, but its low solubility in suitable solvents makes it
less convenient in practical use. The third -OH group, which

occurs in purpurine but not in alizarine, is rather unreactive, and
the two dyes tend to behave similarly with mordants. ^^^ Both dyes
occur naturally in the form of glucosides in the root of the madder
plant, Rubia tinctonim (Rubiaceae), but the synthetic products
are almost invariably used. Purpurine is unfortunately named,
for it is a red dye.

If purpurine is dissolved at saturation in 60 °o ethanol, it acts as
a typical though very weak acid dye. This can be shown by carry-
ing out the tests described on p. 101. If, however, the dye is dis-
solved in a solution of aluminium sulphate, the result is entirely
different. The following is a convenient solution.

Take 0-8 g of purpurine and 7-88 g of aluminium sulphate crystals
(I6H2O); add 450 ml of 60°o ethanol; boil with reflux condenser until
the solids have dissolved; cool; make up to 500 ml with 60° 0 ethanol.-^

The purpurine in this solution, if pure, is at M/160, the
aluminium sulphate at M/40. The solution may be called stand-
ard aluminium purpurine, or 'purpural' for short.

The ethanol in this solution increases the solubility of the dye
and thus makes the solution last longer when used repeatedly; it
slows down or prevents the growth of bacteria and moulds ; and
it decreases ionization and thus 'equalizes' the action on tissues
(that is to say, prevents the solution from over-dyeing locally).

112 CYTOLOGICAL TECHNIQUE

Restrainers such as ethanol and glycerol are particularly useful in
the dyeing of whole mounts, but they sometimes serve a useful
purpose with sections also, especially when dyes are used
progressively.

The various tissue-constituents mentioned on p. 101 are
coloured by purpural exactly as though it were a basic dye (p. 90).
There is, however, this difference, that whereas basic dyes can be
washed out of the tissues by neutral alcoholic solutions (50% or
70% ethanol, for instance), mordanted purpurine cannot.

Other soluble salts of aluminium (the chloride, for instance)
may be substituted for the sulphate: the action is again that of a
basic dye. If, however, sodium sulphate is used instead of the
aluminium salt, there is no mordanting and purpurine acts as a
very weak acid dye. Thus it is the cation that mordants.

It will be remembered that metallic aluminium has three elec-
trons in its outer 'shell' (the third or 'M' shell). In its salts these
are lost to the anion, and the cation is left with the same orbital
electrons as the inert gas neon. The cation has, however, the
ability to accept electrons from suitable donor atoms, and in the
presence of water it accepts no fewer than 12, two from each of
6 oxygens in water molecules. In solution the cation consists of
aluminium in the centre, with 6 molecules of water surrounding
j^- 45, 173 jj^g 5 dative covalency bonds are arranged in opposite
pairs, each pair being at right-angles to the other two pairs. Each
valency bond holds a molecule of water (fig. 6, left).

It may be wondered why exactly 6 bonds are formed, involving
the donation by water of 12 electrons. This is controlled partly by
the geometrical fact that 6 water molecules fit round a central
atom or ion more easily than (say) 5 or 7, but there is also another
cause. The reader may recall that the M shell will hold a total of
18 electrons, but these cannot all be accepted when the atomic
nucleus is that of aluminium. Twelve have the faculty of 'hybrid-
izing' with one another, without distinction as to whether they
belong to the s,p, or c^orbitals of the shell; and these 12 give the
dative covalency of six.

The complex water/aluminium ion, having 3 electrons less than
the total number required to neutralize the protons of its atomic

THE ACTION OF MORDANTS

113

4" ^'

♦^

O Q

HO-@CoH.

%

♦

0 'Q

'^

//^. 5. Structural formulae for hydrated aluminium ions, drawn in
perspective. The ion on the left carries three positive charges. That
on the right has lost a hydrogen ion and carries two positive charges.

nuclei (aluminium, oxygen, and hydrogen, together), may be re-
garded as holding 3 positive charges at the moment of its forma-
tion. The 6 water molecules, however, do not all remain intact.
One of them, at least, tends to lose a hydrogen ion,^^ which joins
a molecule of the solvent water to form a hydronium ion, HgO^,
and thus departs. Since it carries off a positive charge, the com-
plex ion (fig. 6, right) is only doubly charged; but the solution,
having gained a hydronium ion, has become acid. Thus the pH of
an M/40 solution of aluminium sulphate in 60% ethanol is 3-08.
When purpurine is dissolved in water or in solutions of ethanol
in water, it behaves like a phenol ; that is to say, like a weak acid.
A saturated solution in 60 % ethanol has a pH of 5-20. Some of the
hydrogens of the -OH groups are ionized and lost, and the dye is
now negatively charged. In the structural formula shown here,

O 1^-

OH

OH

O O

Ionized purpurine

OH

OHo

H,0 OH, J
77?^ aluminium purpurine ion

114 CYTOLOGICAL TECHNIQUE

one of the -OH groups is ionized. There are now two oxygen
atoms near one another, both capable of donating electrons to
suitable acceptors. If purpurine is added to a solution of an
aluminium salt, these two oxygen atoms will tend to replace two
of the water molecules that are combined with aluminium. Thus
the purpurine will form a complex or 'co-ordination compound'
with the aluminium. 1' 2

The purpurine ion might attach itself either to an aluminium
ion that was associated with 6 water molecules, or else to one that
was associated with 5 water molecules and one -OH group
(fig. 6, right); but the resulting compound would probably be
the same in both cases, for the acceptance of purpurine by the
aluminium atom would be likely to be accompanied by the accep-
tance of a hydrogen ion from a hydronium ion in the solution,
which would attach itself to the -OH group of the hydrated
aluminium ion.^^^

Since the fully hydrated aluminium ion has a triple positive
charge, while the purpurine ion brings with it a single negative
charge, the complex ion as a whole has a positive charge of two.

In the practical dyeing solution, standard aluminium purpurine
(p. Ill), there is only one molecule of purpurine to 8 atoms of
aluminium. The composition of the solution may be expressed in
terms of the mordant quotient ^"^^ that is to say, the number of
atoms of mordant metal in the solution, divided by the number of
dye molecules. In the standard aluminium purpurine solution,
then, the quotient is 8. It must therefore be supposed that the
majority of the hydrated aluminium ions in the solution that are
combined with purpurine at all are combined with only one ion
of it.

When ionized purpurine has associated itself with aluminium
in the way indicated on p. 1 13, a new ring has been formed, con-
sisting of 6 atoms arranged in the following order: aluminium,
oxygen, carbon, carbon, carbon, oxygen. This ring is particularly
stable. Since the purpurine sends out two bonds that grip the
aluminium like the chela of a lobster, the compound is said to be
chelate. ^^2

Since the aluminium-purpurine ion in the dye-solution is posi-

THE ACTION OF MORDANTS 115

tively charged, it will tend to combine with basiphil (negatively
charged) tissue-constituents, such as the nucleic acids. It will not
tend to combine with acidophil (positively charged) constituents,
such as the haemoglobin of red blood-corpuscles.

It might be thought that the hydrated aluminium ion, carrying
a triple positive charge, would have a greater tendency to be
attracted to sites of negative charge in the tissues than would the
doubly charged aluminium-purpurine ion. If so, little dyeing
would be likely to occur; yet the solution dyes strongly. The
explanation appears to be as follows. When some of the water
molecules of a coordination complex, such as the hydrated alu-
minium ion, are replaced by other molecules, the solubility of the
complex in water is reduced. This reduction in solubility will
favour the deposition of the aluminium-purpurine ion on the sites
of negative charge, while the hydrated aluminium ion has a strong
tendency to remain dissolved. ^^^

A few of the hydrated aluminium ions are likely to take up two
purpurine ions ; some may take up three. The result will be a reduc-
tion of the positive charge respectively to one or nought; in the
latter case there would be precipitation of an insoluble pigment.
On the analogy of the compound of purpurine with cobalt,^^^ it
must be regarded as probable that one aluminium atom can
accept chelate bonds from three molecules of purpurine. This,
however, is not certain. A great deal of study has been devoted to
the attempt to discover the exact composition of the flaming red
dye, Turkey red, which has been known in the East for many
hundreds of years. It appears certain that although this substance
contains calcium, yet the chelate bonds are all with aluminium,
and that only two alizarine molecules are thus joined to each
aluminium atom: of the two remaining covalencies of this metal,
one still retains a water molecule (and the other is linked through
oxygen to calcium)."^

Compounds between mordants and dyes are known as lakes.
The artists' water colour, madder lake, is of this nature. This
insoluble substance is similar in composition to Turkey red. It is
distributed by the artist's brush in the form of a fine suspension.
When a mordant and a dye are dissolved together, a lake may

116 CYTOLOGICAL TECHNIQUE

remain in solution, but there is often a tendency towards general
precipitation of an insoluble lake, in which the mordant metal has
combined with the maximum number of dye ions.

Although electrostatic attraction presumably draws the alu-
minium-purpurine ions towards sites of negative charge in the
tissues, yet a bond other than the ionic must eventually be estab-
lished, for otherwise the mordant/dye complex would be no more
tightly held to the tissues than basic dyes are, and like the latter it
would be washed away readily enough by neutral ethanol. It is
probable that covalent linkages are established between the
aluminium atom and acid groups in the tissues, such as the phos-
phoric group of the nucleic acids. If so, the tissue /mordant/dye
complex would have the structure shown here. It will be noticed

OH

OH

o o

HoO Al OHo

L H^O

o-

OH2

o-

+ +

0=F

0=F

sugar

O

O O

\/

sugar

O

sugar

sugar

0 0

0

\/

\/

sugar

sugar

base base base base base base

The mordant 'dye complex attracted to The tissue /mordant /dye
a site of negative charges (a nucleic acid) complex

in the tissue

Structural formulae showing hypothetically how purpurine
may be linked to nucleic acid through aluminium

that two water molecules are freed from association with alu-
minium, and that the double positive charge on the mordant/dye
complex is cancelled by two negative charges on the acid.

THE ACTION OF MORDANTS 117

Similar linkages are likely to be formed with carboxyl and other
acid groups in proteins and other tissue-constituents.

It is not always necessary to prepare a mordant/dye complex
first and subsequently to allow it to come into contact with the
tissues. On the contrary, one may first soak the tissues in a solu-
tion of the mordant and then in a solution of the dye. The basiphil
tissue-constituents will take up the positively charged hydrated
aluminium ion and the dye will displace water from the attached
aluminium complex when given the chance to do so. It might,
indeed, be said that the dye is being used as a reagent for the
detection of aluminium (though one might be misled if the tissues
happened to contain another metal capable of mordanting the
dye). Both the single-bath method (in which the dye is mixed with
the mordant) and the two-bath method (in which the tissue is put
first in the mordant and then in the dye) are of common use in
microtechnique.

When the single-bath method is used, one may dye either
regressively or progressively, but regressive dyeing is almost in-
variable with the two-bath process. There are two chief ways of
differentiating mordant dyes after deliberate over-dyeing. One
may extract the excess of dye by placing the tissue either in an
acid (often a weak solution of hydrochloric acid) or else in a solu-
tion of the substance that was used to mordant.

Acids attack both the links in the tissue /mordant /dye complex.
It is easy to prove that they attack the tissue /mordant link.^*
Haematein (p. 94) is a convenient dye for experiments of this
kind. It is only necessary to take two slides, treat both of them
with the mordant, then one only with an acid, and finally, after
washing away the acid, to put both slides in the dye for the same
length of time. The section that has been in the acid will be
more feebly dyed than the other, because the acid has removed
part of the aluminium from the tissues (and would remove it all,
if given sufficient time). The hydrogen ions of the acid compete
with the positively charged water /aluminium complex for attach-
ment to the negatively charged sites in the tissue.

That acids also attack the mordant/dye link is very easily shown
with those dyes that change their colour when they associate

118 CYTOLOGICAL TECHNIQUE

with a mordant. Haematein is an example. This has a dirty red-
dish colour when dissolved in water, but becomes clear blue in
the presence of the aluminium ion. The addition of acid restores
the original colour. Purpurine does not change colour markedly
when it associates with aluminium (though alizarine changes from
yellow to scarlet).

It always surprises students to learn that the mordant — the very
substance that attaches the dye to the tissue — can also be used to
remove the dye from it. The fact is that everything depends on the
relative abundance of mordant and dye. When a section has been
dyed, the total amount of dye in it is very small. If the section is
now placed in a solution of the mordant, the latter is present in
enormous excess. The dissolved aluminium competes with the
attached aluminium for the minute amount of purpurine that is
colouring the tissue. The dye redistributes itself. If sufficient time
be allowed, no visible trace of dye will remain in the tissue. In
practice one looks at the section under the microscope from time
to time. When the objects that it is desired to show clearly are still
strongly coloured, but the surrounding parts of the tissue have
become colourless or scarcely tinged, one washes away the
differentiating fluid with water.

While the dye is being extracted by the mordant, coloured
clouds may be seen in the solution surrounding the section. Thus
the solution extracts the dye, though both mordant and dye are
present in it. It follows that whether a mordant/dye solution dyes
or extracts depends on the relative abundance of dye and mor-
dant; that is to say, on the mordanting quotient. For any particu-
lar mordant and dye one can find a concentration of each that
neither increases nor decreases the intensity of colouring of
tissue-constituents that have already been dyed by the same mor-
dant and dye.^^ In experiments of this sort it is best to work with
sections that have been exposed to the standard aluminium
purpurine solution (p. Ill) until chromatin has been strongly
coloured but cytoplasm only feebly. The mordanting quotient of
an aluminium purpurine solution that neither increases nor de-
creases the intensity of colouring of such a section is called the
critical mordant quotient .^^ If the quotient of a solution is lower

THE ACTION OF MORDANTS 119

than the critical figure, the solution acts as a dye; if higher, as
a differentiating agent. In the case of aluminium purpurine, with
the aluminium salt dissolved at M/40 in 60*?o ethanol, the critical
quotient is 16.

If an iindyed section be placed in an aluminium purpurine solu-
tion in which the mordanting quotient is at the critical figure, dye-
ing will take place very slowly until chromatin is strongly and
cytoplasm feebly coloured; no further increase of colour will
occur, however long the section may remain in the dye.^^

Solutions in which the mordanting quotient is not very many
times less than the critical figure are particularly easy to use,
because there is not much tendency to over-dye. Standard alu-
minium purpurine ('purpural'), with a mordanting quotient of
8, is an example. It can be recommended to beginners as a
routine dye for chromatin and other negatively charged tissue
constituents. It is to be used progressively. The period of dyeing
varies according to the fixative used, but half-an-hour or an hour
usually suffices. The section is then simply washed in 50% or
70% ethanol.

Haematein reacts with aluminium salts in much the same way as
purpurine. As the formula on p. 94 shows, it possesses an =0
and a phenolic -OH, so situated in relation to one another as to
provide means of chelation with mordants.

The mordants commonly used with haematein in micro-
technique are potassium alum, Al2(S04)3.K2S02.24H20, and
ammonium alum, Al2(S04)3.(NH4)2S04.24H20. The colour of
aluminium haematein is always blue, whatever salt of aluminium
is used. The potassium and ammonium sulphates contained in
these alums play no part in lake-formation, but they may aff"ect
the pH of dye solutions and the solubility of dyes. The double
salts are easily crystallized and therefore easily purified. It was
probably for this reason that potassium alum was used when
mordant dyeing was invented in Turkey many centuries ago. The
traditional use of alums persists in microtechnique to the present
day, but there is no rigorous proof that alums are preferable to
simple salts of aluminium.

120 CYTOLOGICAL TECHNIQUE

Dye solutions are usually made with haematoxylin instead of
haematein. It will be remembered that the former, if pure, is
colourless (p. 94). It has the advantage of being more soluble
than haematein in water and in aqueous alcohoHc solutions.
Gradual oxidation by atmospheric oxygen keeps up the strength
of solutions for a long time. It is convenient to oxidize about
a quarter or a half of the haematoxylin by sodium iodate when
making up a solution, and to allow the rest to remain as a re-
serve. ^^ Solutions made in this way are ready for immediate use
and last a long time. The reserve is gradually oxidized.

Two of the most useful solutions containing aluminium haema-
tein are Delafield's and Ehrlich's. They are called haematoxylin
solutions, because it was intended by their inventors that they
should be made up with the unoxidized substance, reliance being
placed on atmospheric oxygen for gradual production of the dye.

In Delafield's haematoxyhn^^- the mordant is ammonium alum.
Glycerol and methanol act as solvents, disinfectants, and re-
strainers. The stock solution may be diluted as desired with dis-
tilled water. It is necessary to put the tissue in alkaline tap-water
or weak ammonia solution after soaking it in the mordant/dye
solution, for the alum is rather strongly acid and this prevents
lake-formation. The tissue changes in colour from dirty red to
clear blue, and the basiphil objects are then clearly revealed. This
is an excellent dye when used progressively by experienced
workers, but it is perhaps rather too quick in action for the be-
ginner. It is suitable for whole mounts as well as sections.

In Ehrlich's haematoxylin^^ the mordant is potassium alum.
Glycerol is contained in this solution, as in Delafield's, but
ethanol takes the place of methanol. The chief diff"erence from
Delafield's is that acetic acid is added to prevent the formation of
an insoluble lake. As a result the solution keeps almost indefin-
itely, and this is its great virtue. Dyeing takes much longer than
with Delafield's. Ehrlich himself worked progressively, but it is
usual to over-dye and extract the excess with weak aqueous
hydrochloric acid. 'Blueing' by an alkaline solution is necessary.

Ferric iron is a valuable mordant for haematein. Simple ferric
salts work well, but iron alum, Feo(S04)3.(NH4)2S04.24H20, is

THE ACTION OF MORDANTS 121

generally used. The ferric lake is black or blue-black. It attaches
itself more securely to the tissues than the aluminium lake does,
and is less easily removed by acids. It does not act simply as a
basic dye, but attaches itself also to certain objects that are not
basiphil: in particular, to mitochondria, centrioles, and certain
kinds of lipid droplets. It is probable that a covalent link is estab-
lished between the iron-haematein complex and the tissue. ^^^ The
difference between this link and that formed by the aluminium
haematein complex has not been satisfactorily explained.

When ferric salts and haematein are dissolved together, an in-
soluble lake tends to be deposited. For this reason the two-bath
method is generally used. The section is first soaked in iron alum
or some other ferric salt and then transferred to a simple solution
of 'ripened' (partly oxidized) haematoxylin. The latter is usually
dissolved in ethanol, in which it is very soluble, and the solution
subsequently diluted with water. The regressive method is used.
The excess of the dye is removed by soaking the section a second
time in the mordant.

Heidenhain's method^'^- ^^- ^^ is the best-known example of this
way of using iron haematein. Experienced workers can get pre-
cisely the result they require, but beginners find the differentiation
rather difficult, because the extraction by the mordant seems to go
faster and faster towards the end, just when one would like it to
go slowly. Heidenhain's is one of the most important of all
methods of dyeing in microtechnique. Almost everything in the
cell can be revealed by careful differentiation, if the tissue was
appropriately fixed. The black or blue-black colour gives excellent
images in the microscope and is very convenient for photomicro-
graphy. Preparations are permanent in hydrophobe mounting
media (p. 125), even in those (such as Canada balsam) that tend
to bleach certain dyes. It is generally best not to use an acid dye
in addition, because the contrast between black objects and their
surroundings is necessarily reduced by any background colour.

It will be remembered that carminic acid, like purpurine, is an
anthraquinonoid dye. It possesses an =0 and a phenolic -OH
placed in the same relation to each other as in purpurine. It

122 CYTOLOGICAL TECHNIQUE

behaves towards mordants very much like the latter dye. Its
colour (crimson) does not undergo much change when it links
itself to aluminium, the mordant most commonly used.

One of the best solutions is Mayer's carmalum,^^^ which con-
tains carminic acid, potassium alum, and water, with thymol,
salicylic acid, or sodium salicylate as disinfectant. It is suitable
for use with both sections and whole mounts. It may be used
either progressively or regressively. Potassium alum (5 % aqueous)
readily extracts any excess of colour. This is a very easy dye to use,
since it lends itself to leisurely progressive colouring. The colour
is well maintained in both hydrophil and hydrophobe mounting
media.

CHAPTER 10

Mounting

A mounting medium is a substance in which tissues are immersed
for examination under the microscope. The same name should
not be appHed to adhesives that merely stick sections to slides and
do not enclose the tissues at the moment of examination. Mount-
ing is necessarily the last process to which tissues are subjected in
the making of a microscopical preparation.

Solid, unshrinkable objects may be mounted dry. If so, air is
to be regarded as the mounting medium. In electron-microscopy
sections are examined dry, in a vacuum; if the embedding medium
has been removed, the tissue constituents are exposed directly to
the electron beam. With these exceptions, tissues are always
mounted in media that are fluid at the outset, though many
sohdify later. The liquid may be a single pure substance, a mix-
ture of liquids, or a solution of one or more solids in a liquid; it
must be optically homogeneous and stable. Volatility is allowable,
because the edge of the mount can be sealed (p. 133). The mount-
ing medium must fill even the minutest spaces formed within cells
by coagulant fixatives, for otherwise different parts of the speci-
men would be permeated by fluids of diff'erent refractive indices,
and the image would therefore be confused (see fig. 4, p. 26). The
mounting medium must be transparent and nearly or quite colour-
less. It must be capable of wetting the various tissue-constituents,
especially proteins (that is to say, it must be able to lie against
them without leaving any intervening spaces). It must not dissolve
or corrode the tissue-constituents that are to be studied, though
solvents of particular constituents (such as lipids) are permissible
if the intention is to study the insoluble remainder of the tissue. It
must not support the growth of moulds or bacteria.

123

124 CYTOLOGICAL TECHNIQUE

The refractive index of tlie medium must be related to the
means adopted to obtain contrast in the image produced by the
microscope. An example will make this clear. Let us imagine that
a piece of tissue has been fixed by formaldehyde, and that all
lipids, carbohydrates, and nucleic acids have been dissolved out
of it. The specimen will consist mainly of protein, and this, in its
fixed condition, will generally have a refractive index of about
1-536.^^ If the fixed tissue is now thoroughly permeated by a
colourless, transparent fluid of the same or very nearly the same
refractive index, such as methyl salicylate,^^ the whole object dis-
appears and no image of it can be obtained by the microscope.

A fluid that has this eff'ect is often called a 'clearing agent', but
the term is misleading. The fixed proteins of the tissue are trans-
parent and colourless, and require no clearing. To render the
tissue as a whole transparent, it is only necessary to fill all the
spaces intervening between the proteins with a transparent,
colourless fluid of the same refractive index. There is then no
diff'raction of light at the surfaces of the protein, and therefore no
image of it can be formed. The protein is unaff"ected. Methyl
salicylate no more 'clears' the proteins than a transparent, colour-
less fluid of the same refractive index as glass 'clears' a glass
object immersed in it.

To produce contrast and thus prevent invisibility caused in the
way just described, one may dye the tissue and then mount in the
same medium as before. The object becomes visible because light
of certain wave-lengths is reduced in intensity by the dye attached
to the proteins, but passes freely through the mounting medium.
The fact that the latter has the same refractive index as the pro-
teins is now helpful, because the complete homogeneity of protein
and medium in this respect is favourable to the production of an
optically perfect image.

Alternatively one may leave the object undyed, but render it
visible by mounting it in a fluid that diff'ers slightly from the pro-
teins in refractive index. The specimen should then be examined
by phase-contrast microscopy. To get the best efl'ect, the diff"er-
ence in refractive indices should be small. The retardation or
advancement of the light that passes through the object in relation

MOUNTING 125

to that which passes only through the mounting medium should
not greatly exceed a quarter of a wave-length of the light used for
examination, if phase-contrast is to be applied effectively.^'

A transparent object is rendered visible by direct^'' ('ordinary')
microscopy if it is mounted in a medium of much lower or higher
refractive index than its own. This is convenient if nothing more
is needed than a low-power view of a whole mount ; but wherever
the object comes up against the medium there will necessarily be
a Becke line^^^- ^- in the microscopical image, and this is a mis-
leading appearance, since it does not represent anything that was
present in the object. The resolution of minute detail is not
possible in such circumstances.

Mounting media may be classified as hydrophil and hydrophobe.
The hydrophil media are those that contain water or are miscible
with water. Sections or whole mounts may be transferred from
water to hydrophil media without the necessity to use an intermedi-
ary liquid not contained in the medium. It is reasonable, however,
to introduce the mounting medium gradually to the tissue. Thus,
in mounting in glycerol (p. 128) or Farrants's medium one may
pass the section or other object through a mixture of water and
glycerol before mounting in the medium itself. The two sub-
stances may conveniently be mixed in the proportion in which
they occur in Farrants's medium (50 g = 41*7 ml of glycerol with
100 ml of distilled water).

Hydrophobe media require the dehydration of the object, since
they are not miscible with water. It is usual to pass tissues through
a graded series of ethanols (often 70%, 90%, absolute). Tissues
that have been dehydrated by ethanol in the course of embedding
have undergone all the shrinkage of which they are capable, and
no harm could come from direct passage of sections from water
to absolute ethanol. However, the absolute ethanol would soon
be diluted if this were done repeatedly, and similarly the 90%
ethanol would soon lose its strength if direct passage were made
from water to ethanol of this concentration. The closer the
grading of the ethanols, the more accurately they will maintain
their concentration, but the more trouble will be involved in

126 CYTOLOGICAL TECHNIQUE

dehydration. The usual series provides a good compromise. The
dehydrated section is usually transferred to the solvent contained in
the mounting medium, and from this to the mounting medium
itself. Some hydrophobe media, however, contain no solvent.
Methyl salicylate is an example. The tissue is transferred directly
from absolute ethanol to such media.

Both hydrophil and hydrophobe mounting media are further
subdivisible into those that are adhesive (and thus bind the cover-
slip to the slide) and those that are not. If a preparation is to be
kept permanently, it is almost essential that the coverslip should
be bound to the slide. The easiest way of achieving this is to use
an adhesive mounting medium.

If perfect apposition can be obtained between two solid objects,
they will ordinarily adhere, because their molecules will be drawn
towards one another by the same forces that hold the molecules
together in the objects themselves.^^ There is no question, how-
ever, of causing coverslip and slide to adhere in this way, partly
because their surfaces cannot be made flat enough for perfect
apposition on the molecular scale, partly because a section or
other microscopical object must necessarily intervene. Some de-
gree of adhesion will be achieved if a fluid intervenes, provided
that the fluid is able to wet both the surfaces concerned.^^ It is
a fortunate circumstance that glass can be wetted both by water
and by the solvents used in hydrophobe mounting media. The
adhesion will be very poor, however, unless the liquid not only
adheres to the glass but also coheres within itself. In other words,
it must be viscous, or else be capable of actual conversion into
a solid. The presence of long molecules in the liquid will thus
favour adhesion. So that the surfaces may be easily wetted, it is
desirable that the adhesive should be at first of fairly low vis-
cosity. The necessary increase in viscosity or conversion to solid
may be achieved either by cooling or by the evaporation of a
solvent.

The adhesive hydrophobe mounting media, such as DPX
(p. 131), are hardened by evaporation of the solvent, which is
usually xylene. The commercial product that is sold under this
name is a mixture of three isomeric dimethyl benzenes with ethyl

MOUNTING 127

benzene. It has the percentage composition shown here, whether
CHs CH3 CH3

k^ k^CH3

o-xylene, 23% m-xylene, 43% p-xylene, 19% ethyl benzene,

15%

The components of xylene

it is derived from petroleum or coal.^"^ Xylene is chosen as a
solvent for hydrophobe mounting media because it evaporates
sufficiently slowly to allow the coverslip to be adjusted at leisure.
Hardening is usually hastened by putting the slide on a warm
plate after the coverslip has been arranged in position.

Hydrophil media are used when low refractive indices are re-
quired, or when it is desired to avoid dehydration at all stages.
Most hydrophobe media have high or rather high indices, approxi-
mating to those of proteins that have been fixed by formaldehyde
or by coagulant fixatives. These hydrophobe media generally give
more transparent preparations than hydrophil ones.

Non-adhesive media present the advantage that the final re-
fractive index is known, because there is (or should be) no loss of
a particular component (or components) by evaporation.

One cannot state the effective refractive indices of those adhe-
sive media that rely for adhesion on the evaporation of a volatile
component, because the exact amount of this component (if any)
that remains in the preparation at any particular time cannot be
controlled. One can only state the extreme limits of refractive
index : that is to say, the index of the complete medium, ready for
use, and the index of the medium from which the volatile com-
ponent has been completely driven off. It is unlikely that this
component is quite eliminated from ordinary microscopical
preparations.

Since high refractive index and firm adhesion of the coverslip
are desirable in most cases, adhesive hydrophobe media are more
often used than the others.

When non-adhesive media are used, it is generally desirable to

128 CYTOLOGICAL TECHNIQUE

Stick the coverslip to the slide by the use of a seal, which prevents
movement and also the evaporation of any volatile constituent.
Seahng is discussed on p. 133.

Four mounting media will be described here. They are repre-
sentative of the very large number that exist. They have been
chosen because they are simple in composition and therefore
demonstrate well the principles involved in the process of
mounting. The selected examples are these :

hydrophil
non-adhesive (fluid mount) . glycerol (see below)
adhesive .... Farrants's medium (p. 129)

hydrophobe
non-adhesive (fluid mount) . methyl salicylate (p. 130)
adhesive .... DPX (p. 131)

Glycerol (glycerine) is a familiar non-adhesive hydrophil
medium. It is useful in studies of lipids, which are preserved
whether fixed or not. Lysochromes (p. 86) maintain their colour
well in this medium, but certain dyes, including mordanted
haematein, are not perfectly stable. ^*^ Glycerol is very valuable as
a mounting medium for sections of tissues that have been em-
bedded in butyl methacrylate and are intended for study, without
dyeing, by phase-contrast microscopy. ^^ When preparing tissues
for electron-microscopy it is desirable to cut a few sections at
about 3 /^ and to examine these by phase-contrast. This shows at
once whether the tissue is well enough fixed to make it worth while
to cut thin sections and mount them on grids for electron-
microscopy. It also helps in the interpretation of electron-
micrographs, because the thickness of the section enables one to
focus up and down and thus obtain a three-dimensional view.
Material that has been fixed in Palade (p. 58), embedded in
methacrylate (p. 77), sectioned at about 3 ^, and mounted in
diluted glycerol, gives an extraordinarily lifelike appearance
under the microscope. A few authors^^^- ^^^ have studied meth-
acrylate sections by phase-contrast, but not nearly enough has
been done in this promising field.

It is generally supposed that the fixed proteins of the cell have

MOUNTING 129

a refractive index of about 1-535. This supposition rests on ob-
servations made with tissues fixed in formaldehyde solution and
embedded in paraffin. ^^ There is reason to believe that the pro-
teins of cells that have been fixed by osmium tetroxide and
embedded in butyl methacrylate have a considerably lower index
than this. It is desirable that the index of the mounting medium
should be somewhat lower again, if undyed sections are to be
examined by phase-contrast. The ground cytoplasm will then
appear very pale grey by positive contrast, while the mitochondria
and certain other cytoplasmic inclusions will be dark grey or
black. A mounting medium of refractive index 1-46 has been
recommended. ^^^ Media with even lower indices, such as 1-44 or
1 -42, seem to give still better results.-^ The refractive index of pure
glycerol is 1-474. By mixture with water, any lower refractive
index exceeding that of water itself (1-333) can be obtained. The
proportions of glycerol and water that give various refractive
indices are shown in published tables. ^^" To make a fluid of index
1-44, 78 g of glycerol are mixed with 22 ml of distilled water; of
index 1-42, 65 g of glycerol with 35 ml of water.

By the addition of cadmium chloride, the refractive index of
glycerol may be increased to 1-54 at saturation. ^^^ The salt im-
parts a pale yellow tint to this otherwise colourless medium.

Farrants's medium is a good example of an adhesive, hydrophil
medium, much used in studies of lipids. It is not nearly so good as
diluted glycerine for the study of undyed methacrylate sections
by phase-contrast.

Several diff'erent formulae have been published, all containing
the same three principal ingredients. The following-^ works well.

To 40 g of gum arabic Cpreferably powdered), add 40 ml of distilled
water. When the gum has dissolved, add 20 g of glycerol. A piece of
camphor may be put in]^the medium to act as a disinfectant.

The pH of this fluid is 4-1, whether camphor be added or not.^*

True gums are sticky, water-soluble substances that exude

through cracks in the bark of certain trees. Gum arabic (acacia

gum) is derived from species of Acacia (Leguminosae), especially

A. Senegal, a native of the Sudan and other parts of tropical

K

130 CYTOLOGICAL TECHNIQUE

Africa. The gum is taken from both wild and cultivated trees.*
Its name refers to the fact that it was first imported into Europe
from Arabian ports. The gum consists of the calcium salt of
arable acid,^°^ which owes its acidity to the carboxyl group of
glucuronic acid. Hydrolysis yields arabinose, rhamnose, and

HoCOH COOH

O

■ CHi

arabinose rhamnose galactose glucuronic acid

Skeleton-formulae of the hydrolytic products of gum arable

galactose, in addition to glucuronic acid. The exact way in which
these are bound together in the natural product is not known, but
the evidence suggests a branched chain, in which the components
are not arranged in a regularly repeated order. ^^^ The molecular
weight is about 240,000. ^'^^ The shape and size of the molecule
determine its high viscosity in aqueous solution.

The refractive index of the medium made up according to
the formula given above is 1-423 (or 1-424 if saturated with
camphor).^* It is best to drive off most of the water (and thus
increase the refractive index) by leaving the slide overnight in the
paraffin oven, after applying the coverslip. The latter adheres
firmly.

It has been stated in print that 'The ordinary media of the
Farrants' type have an index of refraction just over 1-3.' ^^ This
statement cannot be correct, whichever formula be followed; for
the refractive index of water itself is 1-333, while those of the other
constituents are very much higher.

The glycerine in Farrants's medium prevents the gum from
cracking when the water is driven off. It also helps to make the
tissue transparent, because it readily penetrates the minute spaces
in them, while the large molecules of the gum diffuse slowly. The
uneven distribution through the tissue of the two substances of
high refractive index in this medium is probably the cause of the
imperfection of the phase-contrast image.

Methyl salicylate.^'' Non-adhesive hydrophobe media are very

MOUNTING 131

seldom used, partly because most of the suitable substances are
inconveniently volatile, partly because it is so easy to prepare
hydrophobe media that will stick firmly to glass. Methyl sali-
cylate, however, has certain virtues. It is colourless and miscible

OH OCH3

Methyl salicylate

in all proportions with absolute ethanol. It gives excellent trans-
parency to most preparations, because its refractive index (1-537)
is very close to that of proteins fixed in the usual ways (see p. 124).
Since it is a single substance, there is no question of change of
refractive index by diff"erential evaporation of components. Basic,
acid, and mordanted dyes seem to maintain their colours well in it.

DPX.^^^ It has already been remarked that adhesive hydro-
phobe mounting media are more commonly used than any others.
They are valuable for making permanent preparations of dyed
objects. Dyes maintain their colours well in DPX, which possesses
the further advantage of being itself perfectly colourless. The
letters DPX are the initials of Distrene-Plasticizer-Xylene.

The most familiar of all media in this group is Canada balsam,
which was described in earlier editions of this book. Although it
has great virtues, it is omitted from the present edition, partly
because it has a tendency to bleach basic dyes, partly because it is
very complex in composition (there are at least 7 constituents in
addition to the solvent). ^'^

In adhesive hydrophobe media the essential constituent is a
'resin', in the wide sense of that term: that is to say, a solid,
amorphous organic compound, insoluble in water, having no
definite melting-point (because composed of molecules of varying
lengths). The natural resins that exude from the bark of certain
trees are oxidation-products of terpenes, but plastics having
similar physical characters nowadays receive the same name. The
particular plastic that gives high refractive index and adhesiveness
to DPX is polystyrene, a polymer having the same general char-
acter as acrylic acid (p. 78), but diff"ering in the substitution of a

132 CYTOLOGICAL TECHNIQUE

phenyl group for the carboxyl of the acid. The polymer that is
used in DPX has rather less than 800 segmers in most of its

H H

H H

C C

C C

«c/°

H

\OH

A segmer of acrylic acid A segmer of polystyrene

molecules. It is known by the trade name of 'distrene 80'. It is
a colourless solid, freely soluble in xylene.

If a section or other object be mounted in a solution of distrene
80 in xylene, the polymer retracts under the edges of the coverslip
as the solvent evaporates. This defect is mitigated or overcome by
the addition of a plasticizer to the medium. Typical plasticizers
are non-volatile solvents of the plastic on which they act. They
are supposed to associate themselves with reactive groups in the
polymer, which would otherwise attach themselves to other re-
active groups, and thus link the long molecules into a rigid mesh-
work ; but they do not combine with the polymer so as to form
a new substance. Thus they make the polymer softer than it would
otherwise be, and oppose its contraction when any volatile solvent
evaporates. The plasticizer in DPX is tri-/7-tolyl phosphate (often
called tricresyl phosphate) ; that is to say, an ester of phosphoric
acid, in which three/?-tolyl radicles have replaced three hydrogens.

CH,

The p-tolyl radicle

This plasticizer is a colourless, non- volatile liquid of high refrac-
tive index (about 1-56).

To prepare DPX, add to 100 ml of xylene 18-75 ml of tri-/?-tolyl
phosphate, and then 25 g of distrene 80. The latter is obtainable from
Messrs Honeywill and Stein, 21 St James's Square, London, S.W.I.
The plasticizer is irritating to the skin and contact should be avoided.

MOUNTING 133

The refractive index of DPX, while it still contains the original
amount of xylene, is 1 -532. Since the index of xylene is only about
1-496, that of the medium must increase as drying proceeds. The
final index is rather higher than that of the fixed proteins of
ordinary microscopical preparations. If all the xylene is deliber-
ately driven off" from DPX, the resultant substance (distrene
+ plasticizer) is a solid, melting above 100° C.^°^ Although the
plasticizer is a solvent for distrene, not nearly enough of it is
present to make a solution.

In whole mounts there is some tendency to retraction under the
coverslip, despite the presence of the plasticizer.

Permanent preparations can be made of objects mounted in non-
adhesive media, by sticking the periphery of the upper surface of
the coverslip to the slide (fig. 7). The gelatine gel used for embed-
ding (p. 70) is a good adhesive for this purpose.-^ It melts readily
in an incubator at 37° C. The slide is dried with a cloth up to the
edge of the coverslip, and the melted gel applied with a paint
brush. It hardens at first by becoming a gel again, and subse-
quently by evaporation of the water. In a day or two it becomes so
hard that it can scarcely be marked with the finger-nail. Both
hydrophil and hydrophobe mounting media can be sealed with
gelatine.

As an extra precaution against evaporation of the mounting
medium, it is wise to varnish the hardened gelatine and to extend
the varnish beyond the gelatine on to the upper surfaces of the
coverslip and slide (fig. 7). The varnish chosen for this purpose
should be one that makes perfect contact with glass surfaces and
is not readily softened or dissolved by the various fluids used as
immersion-oils. Gold size is convenient for the purpose. This is
a substance used by gilders in the preparation of surfaces to
receive gold foil. It consists essentially of an oleo-resin dissolved
with turpentine and linseed oil.^^° The oleo-resin (or resin dissolved
in an essential oil) is often wrongly called 'gum' animi. It exudes
from the bark of a leguminous tree, Hymenaea coiirbaril, a native
of the West Indies. ^^^ Two separate processes are involved in the
drying of this varnish: first, the evaporation of the essential oils;

134

CYTOLOGICAL TECHNIQUE

section

mounting medium

covers lip

slide

adhesive
attactiing section
to slide

Fig. 7. Diagram of a finished microscopical slide in sectional view,
showing adhesive s and varnish.

secondly, the oxidation by atmospheric oxygen of the Hnoleic and
other unsaturated fatty acids contained in the linseed oil. This
oxidation is helped by heating the oil with lead monoxide in the
preparation of the varnish. The hardening occupies some days
after the varnish has been applied.

All finished microscopical preparations of organisms and their
parts, other than those of living cells still lying in the fluid that
bathed them in their natural environment, are to be regarded as
products of the reaction between the living tissues and the various
media in which they have successively been soaked. The reaction-
products, which are what we study under the microscope, are only
informative about organisms and their life-processes if some
knowledge is available about the reactions involved; and this
presupposes knowledge of the reagents. It has been the purpose
of this book to supply information about the reagents that are
used in some of the simplest processes of routine microtechnique,
and about their reactions with tissue-constituents.

If an unknown tissue or cell be acted upon by an unknown re-
agent, no useful information can be obtained. Cytologists and
histologists should be as loath to use secret reagents as doctors
are to use secret medicines. Quite a lot of fancifully named embed-

MOUNTING 135

ding and mounting media of unstated chemical composition are
in use today, despite the fact that their reactions with tissue-
constituents cannot be understood while their formulae remain
secret. Even the field of electron-microscopy, in which a scientific
outlook might have been expected, has been invaded by makers of
secret mixtures. For instance, we are asked to use 'hardeners' and
'accelerators' to solidify an embedding medium, wdthout being
told what these reagents may be. We cannot interpret the resultant
electron-micrographs satisfactorily without this information, be-
cause it is necessary to know whether any tissue-constituents are
likely to have been dissolved or affected in some other way.
Reagents of all kinds used in microtechnique should be prepared
in the biological laboratory, or obtained from reliable manu-
facturers who announce the composition of their products. The
only possible exceptions are such substances as varnishes, which
do not come into contact with the tissues.

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LIST OF REFERENCES 143

176. Stoeckenius, W., 1957. Zeit. wiss. Mikr., 63, 210.

177. Strangeways, T. S. P., & Canti, R. G., 1927. Quart J. micr.

Sci.^lh 1-

178. Sylven, B., 1951. Ibid., 95, 327.

179. Tschirch, A., & Briining, E., 1900. Arch, der Pharm., 238,

487.

180. Tweney, C. F., & Shirshov, I. P., n.d. Hutchinson's technical

and scientific encyclopaedia. 4 vols. London (Hutchinson).

181. Venanzi, L. M. Personal communication.

182. Virchow, H., 1885. Arch. mikr. Anat., 24, 117.

183. Wehmer, C, 1929. 'Die P/lanzenstoffe.' 2nd ed. 2 vols. Jena

(Fischer).

184. Weil, A., 1929. /. biol. Chem., 83, 601.

185. Wigglesworth, V. B., 1952. Quart J. micr. Sci., 93, 105.

186. Wolman, M., & Greco, J., 1952. Stain Tech., 27, 317.
186a. Yost, D. M., & White, R. J., 1928. /. Amer. chem. Soc.

50,81.

187. Young, J. Z., 1935. Nature, 135, 823.

188. Zenker, K., 1894. Munch, med. Woch., 41, 532.

189. Zirkle, C., 1928^. Protoplasma, 4, 201.

190. 19286. Ibid., 5, 511.

Index

Where two or more consecutive pages are entirely devoted to the same
subject, only the first is mentioned in the Index.

Each reference to a particular subject that is distinctly more import-
ant than other references to the same subject, is distinguished by the
printing of the page-number in heavy type.

Acacia, 129

acetic acid, 17, 27, 28, 40, 52, 59

acid dyes, 90

acid fuchsine, 107

acid haematein test, 71

acidophil, 100

acinar cell of pancreas, fig. 2,

opp. p. 10; 11
acrolein, 78
acrylic acid, 78

activator of polymerization, 81
adhesives, 77, 126, 133
adipose fat, 50
agar, 99, 105
alanine, 41
alizarine, 111
Allen s 'B.15', 64
Altmann, 62, 65
aluminium ion, 112
aluminium purpurine ion, 113
aluminium sulphate. 111
alums as mordants, 119
amino-acids, 18, 19 (see also

under their separate names)
ammine, 35
ammonium alum, 119
amphoteric tissue-constituents,

98, 102
aniline, 89
anilme blue SS, 93
anionic dyes, 90
antemedia, 73
anthraquinone, 94
arabinose, 130
Archoplasma, 8
arginine, 47, 99

artifacts, 25
aryl groups, 91
asparagine, 68
aspartic acid, 68, 99
autolysis, 15
auxochrome, 90
azo-group, 91
azure B, 96

bacteria, 111

basic dyes, 90

basic fuchsine, 91

basiphil, 100

Becke line, 125

benzene, 48, 81, 88

benzoyl peroxide, 81

'blueing' of haematoxylin, 120

boric-borate buffers, 58

Bovven, ix

branching of butyl methacrylate,

83
Brewer, ix
7z-butyl acrylate, 79
«-butyl alcohol, 79
/i-butyl methacrylate, 77

cadmium chloride, 129

calcium chloride, 57

camphor, 129

Canada balsam, 121, 131

carbohydrates, fixation of, 38

carbon tetrachloride, 45

carmalum, 122

carmine, 95

carminic acid, 94, 110, 121

Carnoy, 59

145

146

INDEX

cartilage, 98, 100

Casselman, vii, 31

catechol, 94

cathepsin, 15

cationic dyes, 90

cell, structure of, 1

cell-membrane, 3

cellosolve, 73

cellulose, 16

centriole, 7, 121

centromere, 6

Champy, 65

Chayen, vii

chemical affinity for dyes, 98

chitin, 16

chloroform, 48

cholesterol, 43

chondroitic acid, 105

chromatin, 98, 100, 109

chromic oxide, 51, 72

chromium trioxide, 17, 24, 27, 37,

72
chromophore, 90
chromosomes, 5, 61, 107
chromotrope, 105
Clarke, 53, 59, 64, 73
'clearing agents', 73, 124
clot-formation, 20
coagulation, 20, 25, 32
collagen, 68, 98, 100, 101
colorants, 85
colour, causes of, 87
colouring agents, classification of,

85
cooling of paraffin blocks, 76
co-ordination compound, 114
coverslip, sealing of, 128, 133
critical mordant quotient, 118
'critical range' of potassium di-

chromate, 50
crypts of Lieberkiihn, 101
crystal violet, 91, 105
cysteine, 36, 47
cystine, 21
cytoplasmic inclusions, list of, 3

Dactylopiiis, 95

dahlia, 7

dark-ground microscopy, 29

dehydration, 72, 125

Delafield's haematoxylin, 120
denaturation, 20

density of tissue-constituents, 106
deoxypentose nucleic acid, see

DNA
dichromate ion, 37, 49
differential dyeing, 98
differentiation, 103

of mordant dyes, 117
distrene, 131, 132
DNA, 6, 26, 98
DPX, 131
dyeing

causes of differential, 98

definition of, 86

of chromosomes, 107

progressive, 102

regressive, 102, 121
dyes

ability to permeate, 108

amphoteric, 100

anionic, 100, 101

anthraquinonoid, 92, 94

azo, 92, 96

cationic, 100

chemical affinity for, 98

chemical composition, 85

comparison of cationic and
anionic, 101

disazo, 97

haematein, 92

mono-azo, 92

permeability to, 108

quinonoid, 92

thiazine, 92, 96

triarylmethane, 92

trisazo, 97

xanthene, 92, 95

ectoplasm, 12

eggs of echinoids, 28

egg-white, 77

Ehrlich's haematoxylin, 120

electric charge on dye-ions, 99

electron-donor atoms, 112, 114

electron-microscopy, 1, 5, 9, 25,

30, 67, 78, 79, 84, 123, 128,

135
embedding, 27, 29, 32, 65, 66
endoplasm, 12

INDEX

147

endoplasmic reticulum, 10

eosin, 95, 108

eosinophil leucocytes, 100, 101

ergastoplasme, 11

ethanol

as dehydrating agent, 38, 72, 78,
125

as differentiator, 103

as fixative, 17, 19, 27, 28, 29, 32,
59

as restrainer in dyeing, 112

as solvent for purpurine, 111
ethyl acetate, 84
ethylene glycol, 74
exocrine cells of pancreas, 101

Farrants's medium, 125, 129
fastness, 110
fibroin, 41
fixatives

additive, 19, 21

coagulant, 17, 25, 31

effect on dyeing, 33, 37, 38, 51,
53, 55, 59

effect on volume, 27

general remarks, 14

ii:-value, 22, 31

mixtures, 59
classification of, 64

non-additive, 19

non-coagulant, 17, 26, 40

oxidation-potentials of, 31

period of action, 24

practical, 55

primary, 16

rate of penetration, 21

shrinkage by, 56, 62

simple, 55

standard concentrations, 17, 31

swelling by, 53

tests of, 29

'trio' of ingredients, 61
Flemming, 61, 64, 76
flocculation, 20
formaldehyde

as fixative, 17, 26, 27, 28, 40, 56,
67

use in gelatine embedding, 70
formalin, 40
formal um, 70

formic acid, 41
frozen sections, 71

galactose, 130
gelatine

as embedding medium, 67

fixation of, 45, 50

for sealing coverslip, 133
gelatine-albumin gel, 22
gelatine-nucleoprotein gel, 52
glass knife, 84
glucuronic acid, 130
glutamic acid, 41, 68, 99
glycerol

as mounting medium, 125, 128,
129

as restrainer in dyeing, 112

in adhesive mixture, 77

refractive index, 129
glycine, 41, 68
glycogen, 16, 43
gold size, 133
'Golgi apparatus', 3, 10
ground cytoplasm, 12, 98
gum animi, 133
gum arable, 129

haematein, 25, 94, 100, 110, 117,

119
haematoxylin, 94

Delafield's, 120

Ehrlich's, 120

Heidenhain's, 121

oxidation of, 120
Haematoxylon, 94
Hardy, ix

heat as fixative, 16, 21
Heidenhain, 64, 121
Helix, 28
Helly. 62, 65
heparin, 105
Hermann, 64

heterochromatic segments, 5, 26
histidine, 46, 99
histochemical tests, 14, 15, 86
histone, 49

'hybridization' of electrons, 112
hydrochloric acid, as differentia-
tor, 120
hydrogen bond, 69, 104

148

INDEX

hydrogen chromate ion, 37, 49
hydrogen peroxide, 48
hydrogen per-perosmate, 45
hydrophil groups, 20
hydroquinone, 80
/7-hydroxybenzoate, 70
hydroxyglutamic acid, 99
Hymenaea, 133

Idiozom, 1
idiozome, 7

'indifferent' salts, 29, 56
interference microscopy, 14, 107
interstitial cells of testis, 101
intestinal epithelium, 101
iodine, 36
iron alum, 120
iron haematein, 121
iso-electric point, 99

karyosome, 6

lakes, 115

lecithin, 43

leuco-pararosaniline, 93

Lewitsky-saline, 65

linoleic acid, 133

lipid globules, 8, 26, 48, 121

lipids

conjugated, 98, 100

fixation of, 33, 36, 38, 43, 47, 50

mounting of, 128, 129
lipochrondria, 8
lipoprotein, 10
Llowarch, ix
Luke, ix

lysine, 19, 34, 42, 47, 99
lysochrome, 86, 97, 128

macrophage, fig. 2, opp. p. 10
madder lake, 115
Mallory, 107
Mann, 65, 108
Mastzellen, 105
Mayer's carmalum, 122
metachromasy, 96, 104
metagelatine, 70
methyl blue, 103, 108
methylene bridge, 42
methyl green, 102

methyl methacrylate, 77
methyl salicylate, 124, 126, 130
methyl violet, 93, 105
Michaelis's buffer, 58
mitochondria, 8, 26, 33, 36, 38,
44,51,53,62,63,64,121,129
modifiers, 91

mordant/dye complex, 116
mordanting

by aluminium sulphate. 111

by chromium salts, 110
mordant quotient, 114, 118
mordants, 94, 110
mounting media

adhesive, 126, 129, 131, 133

classification of, 128

general remarks, 123

hydrophil, 125

hydrophobe, 125

non-adhesive, 127, 128, 130

refractive index, 124
'M' shell of aluminium, 112
mucoitic acid, 105
mucopolysaccharides, 99
mucous secretions, 98, 100, 101,
105

neutral red, 7

'NH4-Altmann', 64

Nissl bodies, 1 1

Nopalea, 95

nuclear membrane, 5

nuclear sap, 5, 26

nucleic acids, 100 (see also DNA,
RNA)
dyeing of, by mordant dyes, 1 1 6
fixation of, 32, 36, 38, 52

nucleolus, 5, 26, 51

r.ucleoprotein, 52

nucleus, 3, 4

nylon, 104

orange G, 97, 108, 109

osmic acid, 45

osmium dioxide, 72

osmium tetroxide, 17, 25, 26, 27,

40, 45, 57, 72, 75, 78, 129
osmotic pressure, 19

Palade, 10, 12, 58, 128

INDEX

149

pancreas, acinar cell, fig. 2, opp.
p. 10; 11

Paneth cell, 101

parabenzoquinone, 88

paraffin, 71

paraformaldehyde, 40

paraquinonoid ring, 88

pararosaniline, 89, 93

paratoluidine, 89

penetration of fixatives, 21

peptide group, 18, 69

perfusion, 18

permeability of tissue-constitu-
ents, 108

perspex, 79

phagocytes, 85

phase-contrast microscopy, 14,
84, 124, 128

phenyl, free radicle, 81

phospholipids, 36, 43, 50, 57, 98

plasson, 1

plasticizer, 131, 132

polyglutamic acid, 41

polyglutamine, 41

polyglycine, 41

polymerization, 79, 82, 105

polystyrene, 131

Porter, fig. 2, opp. p. 10

postchroming, 57

potassium alum, see potassium
aluminium sulphate

potassium aluminium sulphate
as mordant, 119, 120, 122
in preparation of carmine, 95
use in gelatine embedding, 70

potassium dichromate, 17, 27, 40,
49
'critical range', 50

potassium ferrocyanide, 86

practical fixative solutions, 55

preservatives, 15

proline, 68

proteins
fixation of, 32, 34, 37, 41, 45, 49
refractive index of, 124, 127,

129
structure of, 19

protoplasm, 1

Protozoa, 12

Prussian blue, 86

purpural, 111

purpurine. 111, 121

compound with calcium, 115
compound with cobalt, 115
ionized, 113

quinonoid dyes, 92

reaction-products, 134

red blood-corpuscles, 1, 98, 100,
101, 102, 115

refractive index

of Farrants's medium, 130
of fixed protein, 124
of glycerol solutions, 129
of methyl salicylate, 131

Regaud, 65

regressive dyeing, 121

restrainers, 112

rhamnose, 130

ribonucleic acid, see RNA

ribonucleoprotein, 98

RNA, 5, 12, 98

Riibia, 111

salicylic acid, 122

Sanfelice, 64

saturated colours, 109

scleroproteins, 16

sealing of coverslip, 128, 133

secret reagents, 134

segmer, 79

shrinkage

by dehydrating agents, 72, 74

by fixatives, 27, 56, 62
silica, 16

single-bath mordant dyeing, 117
'small particles' of Palade, 10, 12
smooth muscle, 101
sodium acetate, 58
sodium iodate, 120
sodium 77-hydroxybenzoate, 77
sodium salicylate, 122
sodium veronal, 58
spermatogonium, 2
spindle attachment, 6
Spokes, ix
starch, 16

structural colours, 87
structure of cell, 1

150

Sudan IV, 97

sulphuric acid, 72

Susa, 64

swelling by fixatives, 27, 53

terpenes, 131
thionine, 96, 105
thiosulphate, 36
thymol, 122

tissue/mordant/dye complex,
toluene, 84
trabeculae, 10
triglycerides, 16, 43, 107
'trio' fixatives, 61
triphenylpararosaniline, 93
tri-/?-tolyl phosphate, 1 32
tryptophane, 21
Turkey red, 115
turpentine, 133
'typical cell', 1

INDEX

tryosine, 19, 21, 41

unmasking
of lipids, 10

of reactive groups in proteins,
21

116

varnishing of seal, 133
Venanzi, ix
vital dyeing, 14

volume changes caused by fixa-
tives, 27

Wigglesworth, viii

xylene, 77, 103, 127

Zenker, 60, 64
Zenker-without-acetic, 65

m

m