Chapter XX: The Fixation and Hardening of Tissues

=GENERAL CONSIDERATIONS.= For the examination of material that may be injured by freezing, or when very thin sections are required for complicated staining procedures, it becomes necessary to prepare the tissue by _fixation_ and _hardening_, so that it can be _imbedded_ in some medium permitting the _cutting_ on the microtome of as thin sections as may be desired. Fixation is that process by which the appearances of the tissue are preserved as they were when it was taken for examination; hence in order to obtain pictures resembling as closely as possible those of the living tissue the material should be _fixed_ immediately upon its removal from the living body by operation, or as soon as possible after death when obtained by autopsy. _Fixing agents_ act by coagulating the cell albumins, in this way “setting” or “fixing” the constituents of the cell so that further change is stopped. Fixation, therefore, _hardens_ the cell, and all fixatives are also hardening agents. A practical distinction between fixing and hardening is made, however, resting upon the fact that not all fixatives harden the tissue so completely that the proper consistence for the cutting of thin sections is attained. To achieve this the tissue must be dehydrated. Alcohol and acetone are the only reagents fixing and hardening perfectly at the same time, as they remove the water from the tissue; for all other fixing agents an _after-hardening_ in alcohol is necessary. In the case of such reagents the division of the process into a _primary fixation_ and a _second hardening_ stage has been the cause of the divergence in meaning of the two terms.

The best fixing agents are those that kill the cells at once, but cause a slow coagulation with little or no shrinking. They must penetrate and diffuse through the tissues rapidly so that the deepest cells are quickly reached. Acid media, especially those containing small percentages of acetic acid, are therefore better than alkaline solutions. The tissue-elements, particularly the nuclei, must be preserved as perfectly as possible so that they will not be affected by further procedures of microscopic technique. The chemic properties of physiologic and pathologic substances must likewise be preserved. The preservation of karyokinetic figures is a criterion of good fixation. In pathologic work it is also desirable that the fixing agent should preserve the red blood cells, and permit of the staining of bacteria in sections. Since fixing media are more or less selective in their action, it follows that there is no one fixative that gives equally good results in all cases. Especial fixing reagents must be used for the demonstration of certain substances (fat, etc.), or for the use of certain staining methods. Some stains cannot be used at all after certain fixing agents have been employed. For general use that fixing agent having the widest range of usefulness should be employed; and for this reason fixing media composed of several fixing agents are often employed in preference to the use of a single one.

GENERAL RULES FOR FIXING AND HARDENING.

The tissue should be put into the fixing fluid as soon as possible after its removal from the body. It must not be allowed to dry. There should be an abundance of the fixing solution, 25-50 times the volume of the object to be fixed. The tissue should never be put into a dry vessel and the fixing fluid poured upon it; the vessel should first be filled with the fixing solution and the tissue then dropped into the latter. A slight agitation of the fluid will prevent the sticking of the tissue to the bottom or sides of the vessel. The size of the pieces must be adapted to the penetrating power of the fixing reagent, but as a rule the pieces should not be more than 2-3 cms. in thickness, and for some reagents 0.5 cm. is as thick as they can safely be. The reagents used should be changed when they become cloudy or discolored. The used solution may be filtered and used again, but some reagents can be used but once. Alcohol may be saved for redistillation. In the case of some reagents (mercuric chloride, chromic acid, osmic acid, etc.), the time limits of the fixation should not be exceeded, as over-fixation will ruin the staining-power of the tissues. Alcohol is practically the only solution in which tissues may be left indefinitely, but even with it there are certain limitations. As a general rule the time required for fixation may be shortened by keeping the reagents at incubator-temperature. As the different fixing reagents vary so greatly as to their especial advantages and disadvantages these will be considered separately. Only the best and most commonly used methods are here included.

1. =ACETONE.= A water-free acetone is employed by placing pure
dried white copper sulphate in the bottom of the bottle or vessel
in which the fixation-process is carried on. Several layers
of filter-paper are put over the copper sulphate to keep the
tissues from touching it, and the acetone is then poured into the
vessel. As soon as the copper-sulphate becomes blue it must be
again fused. It is only by this method of constant dehydration
that acetone can be employed to any advantage as a fixing agent;
if fused copper sulphate is not employed the amount of acetone
necessary to fix well is so great that the method becomes too
troublesome and expensive to be recommended. But with the simple
copper-sulphate method of constant dehydration acetone becomes the
cheapest, most rapid and one of the best fixing reagents. The use
of alcohol is avoided, and the period of infiltration in xylol
and in paraffin shortened. For very quick work the entire process
of fixation, hardening and dehydration may be achieved by the use
of acetone alone, small pieces of tissue being fixed ½-2 hours
in acetone and then transferred directly to soft paraffin. For
ordinary work pieces of tissue 0.5 cm. thick are put into acetone
over fused copper sulphate for 20-60 minutes. A judgment of the
degree of fixation can be obtained by pressing the tissue lightly
between the fingers; if it is of uniform consistence, and does
not give as if the inner portions were softer than the surface,
the fixation is complete. From the acetone the tissues may be
brought directly into xylol for 5-10 minutes, until they obtain a
cloudy transparency, thence into paraffin for 15-30 minutes and
then blocked. The whole process of fixation, imbedding, cutting
and staining can be carried out in 30 minutes. Acetone may be
combined with formol, alcohol or any of the other fixing agents,
but when it is desired to use any one of these for some especial
purpose it is better to fix first with the desired reagent and
then to use acetone for the dehydration-process alone, instead of
alcohol. We have found that formol-fixation followed by acetone
dehydration gives excellent results for general pathologic work.
=Formol-acetone= (acetone 100 cc., formol 10 cc.) may be found to
have advantages. The quick fixation in water-free acetone causes
less contraction than fixation with absolute alcohol; fixation with
graded acetone-solutions causes practically none.

=Advantages.= It is the cheapest and quickest method. It penetrates
well and causes little contraction of the tissues, shortens the
time in xylol and paraffin and makes more easy the cutting of dense
fibrous structures. Cell-division figures are as well preserved as
by alcohol fixation, and the staining of bacteria in the tissues
can be carried out as well after acetone-fixation as after alcohol.
It preserves lecithin, hence can be used for the fixation of
nerve-tissues. When combined with formol (formol-acetone) the red
blood-cells are well-preserved and take a brilliant eosin stain.

=Disadvantages.= The disadvantages are practically the same as with
alcohol fixation but not so marked. Fat is dissolved, cell-division
figures are not so well-preserved as with mercuric chloride and
Flemming’s solution, and the blood-cells not so well preserved as
with formol and mercuric chloride.

2. =ALCOHOL.= Absolute, 95-96 per cent alcohol, or graded alcohols
(70, 95 per cent. and absolute) may be used for fixation. For
this purpose the stronger alcohols are preferable, as the weaker
solutions do not fix quickly enough. On the whole the use of 95-96
per cent is to be advised. The pieces of tissue must not be thick.
Plenty of alcohol should be used and it should be changed several
times during the process of fixation, which for larger pieces
requires several days. For the last change absolute should be used.
Very small bits such as uterine curettings can be fixed in one
hour, by using three changes of absolute alcohol. Since alcohol
both fixes and hardens it has been generally used, but it is a
relatively poor fixative. For after-hardening it is indispensable.
Absolute alcohol may be made from 96 per cent by the use of fused
copper sulphate. To test the strength of alcohol mix a few drops
with pure water-free xylol; if no sediment appears when viewed
against a dark background the alcohol is absolute or practically
so. Many of the disadvantages of alcohol fixation can be obviated
by the use of =formol-alcohol= (95 per cent alcohol 100 cc.,
formalin 10 cc.).

=Advantages.= Cheapness, quickness, and ease of method. Can be used
for quick diagnostic work. Penetrates well, and can be used for
large pieces of tissue. Preserves glycogen, is especially good for
the staining of bacteria in sections, and the majority of stains
work well with alcohol-fixation.

=Disadvantages.= Causes much shrinking and loss of finer details;
preserves cell-division figures not at all or poorly; destroys
the red blood cells; dissolves fat and other chemic products:
does not permit of the use of certain specific staining methods
(nerve-tissues); causes excessive hardness of fibrous and elastic
tissues and makes cutting difficult.

3. =CHROMIC ACID AND SALTS.= Chromic acid is rarely used alone in
pathologic work, but is a constituent of Flemming’s solution (see
below). Its salts are employed in the form of:—

A. =Müller’s Fluid= (Potassium bichromate 25.0 grms., sodium
sulphate 10.0 grms., water 1,000.0 cc.) This was formerly the
favorite fixing solution, but is now used chiefly for the eye
and nervous tissues, either alone, or after formol fixation,
or mixed with formol. Large pieces may be used, even an entire
brain, but the process requires months or even a year for the
best results. Even small pieces take several weeks. The process
may be hastened in the incubator. The solution should be changed
whenever it becomes cloudy. When fixation is complete the fixed and
hardened tissue may be cut directly on the freezing-microtome or
after-hardened and dehydrated in alcohol or acetone when it is to
be imbedded. The dehydration in alcohol should be carried out in
the dark and without previous washing of the tissue in the case of
nervous tissue. Greenish or brownish chrome precipitates appear if
the dehydration takes place in the light; but for ordinary material
this precipitate can be avoided by washing the fixed material in
running water for 24 hours before transferring to alcohol. Moulds
grow luxuriantly in Müller’s fluid, but may be inhibited by the use
of pieces of camphor, thymol, etc.

=Advantages.= Cheap, penetrates well, causes little shrinking,
permits special nerve-stains, preserves red blood-cells, gives
beautiful results with ordinary stains, preserves fat.

=Disadvantages.= Slowness; does not preserve division-figures;
does not permit of staining for bacteria; does not give good
results with many special staining methods (fibrin, elastic tissue,
reticulum, etc.).

B. =Erlitzky’s Fluid= (Potassium bichromate 25.0 grms., copper
sulphate 5.0 grms., water 1,000.0 cc.). Used for fixation of
nervous tissue. The formation of pigment precipitates may lead to
misinterpretation: the artefacts may be removed by hot water or
dilute acetic acid.

C. =Orth’s Fluid= (Müller’s fluid 100 cc., formol 10 cc. Make
fresh before using, as the mixture precipitates on standing.).
Fix for 3-12 hours in the incubator, or for 24-48 hours at room
temperature. Wash in running water for 12-24 hours; cut on the
freezing microtome, or after-harden in acetone or alcohol and imbed.

=Advantages.= Combines the good features of Müller’s and formol
fixations, and obviates some of the disadvantages. It is a good
general fixing solution.

3. =FORMOL OR FORMALIN= (40 per cent solution of formaldehyde
gas). Used in a _ten per cent solution_ (water or physiologic salt
solution nine parts, formalin one part), often incorrectly called 4
per cent formol or formalin, the mistake arising from the confusion
with 4 per cent formaldehyde gas. For nerve-tissues it is better to
dilute the formol with physiologic salt-solution than with water.
Fix for 3-4-12 hours according to size of tissue. As it penetrates
well large pieces can be used. Wash in water before after-hardening
in alcohol, if tissue is to be employed for general work, otherwise
it can be transferred directly to the alcohol without washing.
Tissues can be kept in formol for some weeks, but after that time
the staining-power is slowly affected, and the finer structures
suffer.

=Advantages.= Probably the best fixing reagent for general
pathologic work. It is cheap; easily made and kept in solutions
of proper strength for fixing; hardens while it fixes; does not
require after-washing; penetrates well; causes little shrinking;
permits freezing directly from the fixing solution; preserves fat;
permits the use of after-hardening with bichromate solutions for
especial nerve-stains: preserves the red blood-cells: gives good
results with nearly all stains, and differentiates bile-pigment
from hæmatoidin. It is the best fixing reagent when tissues are to
be sent some distance, as over-fixation occurs only after several
weeks or even months.

=Disadvantages.= Affects many people unpleasantly, causing coryza,
eczema of the hands and arms, and affections of the finger-nails,
so that workers having acquired this idiosyncrasy cannot expose
themselves to formol vapor; it dissolves glycogen and uric acid;
does not fix cell-division figures as well as mercuric chloride
or Flemming’s solution; causes the precipitation of diffuse
hæmoglobin in the form of brown or black pigment-granules that
may be taken for melanin, malaria pigment or hæmosiderin; causes
a pseudo-ochronosis of cartilages; and, unless thoroughly washed
from the tissues before after-hardening in alcohol, it makes
carmine-staining difficult or unsatisfactory and affects also the
specific staining-reactions for amyloid and mucin; it is not as
good as alcohol or mercuric-chloride when the sections are to be
stained for bacteria. In spite of these disadvantages it can be
recommended as the best general fixing reagent.

For =Orth’s fluid=, =formol-acetone= and =formol-alcohol= see above.

4. =FREEZING AND DRYING.= The fresh tissue is frozen and dehydrated
in a vacuum over sulphuric acid, at a temperature of 20-30°C.; when
completely dried it is imbedded directly in paraffin. This method
has been especially recommended by Altmann on the ground that the
tissues are simply deprived of water without any change in volume.

5. =HEAT.= Physiologic salt-solution is heated to 80°C. Thin pieces
of tissue are placed in the hot water for two minutes, and then
after-hardened in alcohol. For larger pieces of œdematous tissues,
cysts, etc., that cannot be cut into thin pieces, the salt-solution
should be brought to 100°C and the tissues boiled for several
minutes. This method is advised particularly for the coagulation of
albumin in cysts, œdematous tissues, for the study of renal casts,
etc.

6. =MERCURIC CHLORIDE.= This is used most commonly in the form
of =a concentrated water solution= (mercuric chloride 7.5 grms.,
sodium chloride 0.5 grm., glacial acetic acid 5 cc., water 100.0
cc.), or as =Zenker’s solution= (mercuric chloride 5.0 grms.,
sodium sulphate 1.0 grm., potassium bichromate 2.5 grms., water 100
cc.; dissolve by heating, add 5 cc. glacial acetic acid just before
using. The use of a 5 per cent formol solution instead of acetic
acid is recommended). The pieces of tissue should not be thicker
than 5 mm. Fix 6-24 hours, then wash 24 hours in running water,
and after-harden in alcohol. Should the sections show mercuric
precipitates they should be treated with Lugol’s solution for 30-60
minutes, then washed in a dilute solution of lithium carbonate and
thoroughly washed out in water and alcohol. Much better stains can
be obtained by this treatment of the sections with Lugol’s. The
use of iodine in the alcohol during the process of after-hardening
is not advisable because of the action of the iodine upon the
albuminates of mercury. A 5 per cent solution of =sublamine= in
distilled water has recently been recommended by _Klingmüller_
and _Veiel_. Fix 1-3 hours, wash and after-harden in alcohol.
Precipitates are not formed, and good staining-results are obtained.

=Advantages.= The mercuric chloride solutions preserve well the red
blood-cells, mitotic figures and finer details of cell-structure,
and permit the staining of bacteria and animal parasites in
the sections. Certain especial staining methods (Mallory’s
reticulum-stain, etc.) can be used only after mercuric chloride
fixation, while many others (Heidenhain’s iron-hæmatoxylin,
Biondi-Heidenhain triple stain, etc.) give best results after
this fixation. For ordinary work the saturated mercuric chloride
solution is preferable to Zenker’s, as the latter does not give
good results with the commonly-used hæmatoxylin stains.

=Disadvantages.= More troublesome and expensive; require thorough
washing and subsequent removal of precipitates, and affect
(Zenker’s particularly) certain stains.

7. =OSMIC ACID.= Osmic acid is used alone in a 1 per cent solution,
or in such combinations as =Flemming’s solution= (chromic acid 1
per cent. sol. 15 cc., 1 per cent osmic acid 4 cc., glacial acetic
acid 1 cc.), =Hermann’s solution= (same as Flemming’s, with 15
cc. of a 1 per cent platinic chloride substituted for the chromic
acid), =Altmann’s solution= (5 per cent potassium bichromate
solution 50 cc., 2 per cent osmic acid solution 50 cc.), =Marchi’s
solution= (Müller’s fluid 2 parts, 1 per cent osmic acid solution
1 part), and that of =Pianese= (1 per cent sodium-chloroplatinate
15 cc., 2 per cent osmic acid 5 cc., ¼ per cent chromic acid
5 cc., formic acid 1 drop). The pieces of tissue must be very
thin, as osmic acid penetrates very slightly. Fix 6-24 hours in
the dark, and wash thoroughly in running water, and after-harden
in graded alcohols. These solutions have but limited use in
pathology, and are used chiefly for the study of fat (oleates) and
mitotic figures. Flemming’s and Hermann’s solutions are the best
for the study of mitotic figures, the latter bringing out plasma
details more clearly. Marchi’s fluid is used especially for the
study of nerve-degeneration, and Altmann’s for the demonstration
of Altmann’s granules. The method of Pianese is used for the
demonstration of cell-inclusions. The osmic-acid mixtures are all
expensive, penetrate poorly, cause precipitates, and affect greatly
the staining-power of the tissues, so that it becomes necessary
to use certain stains (safranin, carbol fuchsin, aniline gentian
violet, etc.) as counterstains.

8. =PICRIC ACID.= A saturated water solution of picric acid is
usually employed. Fix 12-24 hours, and wash in alcohol, not water,
and after-harden in graded alcohols. Preserves mitotic figures,
fine details of cell-structure, and is very good for bone and
calcified tissues, as it decalcifies and fixes at the same time.

Numerous modifications and combinations of the above methods
have been proposed such as _Flemming’s chrom-acetic solution_,
_Rawitz’s chromic-picric-nitric fluid_, _Rabl’s chrom-formic
mixture_, _Burckhardt’s chrom-osmic-nitric solution_, _Merkel’s
fluid (chromic acid-platinic chloride)_, _Carnoy’s mixture (glacial
acetic acid 1_, _absolute alcohol 6_, _chloroform 3_), and many
others. They have a limited use in pathologic work.

In the judgment of a section as to its fixation the following points may be of service: in alcohol fixation the red blood cells are haemolyzed, and there is much shrinking; with formol fixation the red cells stain copper-red with eosin; in mercuric chloride fixations the red cells stain rose-red with eosin, and pigment precipitates are present; in bichromate fixation the red cells preserve their natural color, and fat cells show a brownish color; osmic-acid fixation is shown by the black color of the oleates, and the failure of the tissue to stain by ordinary stains.