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Some curious effects of salts of metals and other chemicals on fixation.

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Osborn Zoological Laboratory, Y a l e University, N e w Haven, Connecticut
Ten years ago I published in Science several formulae of
fixing fluids containing phenol or its nitro derivatives. My
intention was to provide a fixing fluid for general use, which
would give satisfactory fixation and at the same time leave
the tissues as soft as possible. The cupric paranitrophenol
fluid no. 2 has been used extensively by myself and other
investigators since the publication of its formula. Prolonged
experience showed that fixation of mammalian and invertebrate material is fairly satisfactory, but that the fluid causes
peculiar changes particiilarly conspicuous in the nuclei of
amphibian tissues, Addition of formalin to the fixing fluid
at the time of use slightly improves fixation, but does not
give pictures comparable with such obtained after fixation
in fluids regularly used by cytologists. Determined to eliminate this distorting effect I continued my investigations with
a long series of salts of heavy metals and other ingredients.
In the course of these investigations the cause of the distorting effect was traced to its origin and finally successfully
eliminated. Once found, the solution proved to be so simple
that much time and labor could have been saved had it occurred
to me by some lucky chance at the beginning and not at the
end of the work. However, some additional observations were
made, showing other undesirable effects produced by ingredients which successfully counteract the distortion of nuclear
structures in amphibia. It seems therefore desirable to give
a more or less detailed account of the investigation and thus
put on guard other investigators who might be misled in the
interpretation of certain pictures due entirely to peculiarities
of fixing fluids.
My selection of salts and compounds of metals was governed
by a desire to get as complete precipitation of proteins as
possible. It is well known that precipitating agents of proteins
vary a great deal in this respect and that some proteins, even
when precipitated, may be again redissolved and thus washed
out of the cells. Experience has also shown that the presence
of other ingredients may make precipitation of proteins by
metallic salts more complete and permanent. Zirkle called
attention to differences in fixation produced by salts in which
the metal in solution forms a part of the cation, and such
in which it forms a part of the anion. As will be seen from
an examination of the following list, I experimented with
both types. I also tried such salts in which one metal forms
part of the cation and another metal part of the anion.
Naturally, the selection was also governed by the solubility
of the salts in water and alcohol, by the stability of the solution and to some extent by the cost and the possibility of
purchasing the chemicals in pure form. The sequence in which
they are listed is simply an alphabetic one and has nothing
to do either with their importance or with the progress of
the work.
Antimony : antimony trichloride, antimony trifluoride.
Chromium : chromic acid, chromic ammonium sulphate,
chromic potassium sulphate, chromic sulphate, potassium
bichromat e.
Cobalt : cobaltous nitrate.
Copper : cupric acetate, cupric bromide, cupric chloride,
cupric chromate, cupric bichromate, cupric nitrate, cupric
sulphate, cupric potassium cyanide, cuprous potassium
Mercury : mercuric acetate, mercuric bromide, mercuric
Thallium : thallous carbonate, thallous sulphate.
Uranium : uranyl nitrate.
Salts of iron, lead, silver, gold and platinum were tried
only in a few combinations with phenol derivatives.
The phenomenon of nuclear distortion after fixation in the
paranitrophenol-cupric fixing fluid consists in the following :
the chromatin condenses at the end of the nuclei opposite to
the exposed surface of the tissue. Chromatin threads can
be scarcely discerned. The mass of chromatin is so compact
that in preparations stained with haematoxylin-eosin it appears deep blue, almost black, while the rest of the nucleus
remains quite trasparent owing to the total lack of chromatin
in that region. Gradual elimination of the various ingredients of the paranitrophenol-cupric fixing fluid led to the
recognition of the totally unexpected fact that cupric nitrate
alone is responsible for the peculiar appearance of the nuclei.
Omission of the nitric acid increases the effect. Omission of
the alcohol, ether and paranitrophenol does not alter the
effect. Other salts of copper were then tried out and gave
the same result. The experiment was extended over the
entire list given above: some of the salts produced the effect,
others did not. The answer to the question as to the cause
of the difference is given by the dissociation of the solutes.
It may be formulated as follows:
1. Aqueous solutions of all heavy metals, in which the
cation alone contains the metal, produce the same distorting
effect on the nuclei (fig. 1). Additioii of ethyl alcohol, isopropyl alcohol, normal propyl alcohol, ether, formalin, phenol,
ortho-, meta-, para-nitrophenol or parabromophenol in no way
modifies or checks the effect.
2. Aqueous solutions in which the anion alone contains
the metal give normal fixation of nuclei and the same holds
triie in case of solutions of salts in which one metal forms
part of the anion, while another metal forms part of the
cation. Addition of formalin makes no difference (fig. 2).
Of the many fixing fluids containing potassium bichromate and
therefore belonging to this type, Regaud’s fluid gives in my
experience the most satisfactory fixation. Neither the formulae
proposed by Zirkle, nor such experimented with by myself,
equal it. Unfortunately Regaud’s fluid has serious faults :
it is unstable and begins t o decompose within a few hours, its
rate of penetration is so low that only very small pieces of
tissue can be fixed and it has the tendency to harden tissues
excessively. Notwithstanding such drawbacks it is used extensively for the study of mitochondria which it preserves
well. Rut mitochondria may be even better preserved by salts
in which the metal forms part of the cation if phenol is added
to the solution. I n such mixtures even cobalt gives good
results, while copper, mercury and uranium are superior to
potassium bichromate. Thallium, too, may be used to demonstrate mitochondria. Its salts give strongly alkaline solutions
of p1-I 10 and even 10.8 and must be adjusted to pH 7 by
the addition of hydrochloric acid. Even so the fixation of
structures other than mitochondria is not. satisfactory and
the cells look empty.
Antimony salts have a peculiar effect on the staining
properties of the cytoplasm of amphibian erythrocytes. The
cytoplasm becomes glass-clear and neither eosin nor acid
fuchsin have any power to stain it. This is unexpected in
view of the fact that antimony trifluoride was used in the
past as a mordant in the textile industry. Several other
undesirable effects of antimony on fixation of cytoplasm
and chromatin, as well as the instability of its salts make
its use troublesome and inferior to several other elements.
The most satisfactory salts in my experience are the salts
of copper. It is true that, alone, copper salts do not precipitate proteins as fully as do salts of mercury. But when used
in combination with acids and phenols complete precipitation
may be accomplished. The use of picric acid (trinitrophenol)
and trichloracetic acid for this purpose cannot be recommended although both are powerful precipitants of proteins.
I have tried both in many combinations. Picric acid affects
the cell structures in a manner which makes differential
staining impossible unless one is interested only in chromosomes. Trichloracetic acid forms a regular ingredient of the
so-called “ Susa” fixing fluid proposed by Heidenhain. It
leaves cells empty. I have tried it in numerous combinations
with other ingredients and had to discard it because of its
deleterious effect on cytoplasm. Ripart and Petit introduced
cupric chloride and cupric acetate as ingredients of their
fixing fluid. Cupric nitrate is an ingredient of my original
paranitrophenol fixing fluid. All soluble salts of copper serve
as powerful mordants f o r many dains and do not harden
tissues to the extent to which chromium, mercury and uranium
But let us return t o the problem of checking the condensation effect of chromatin by fluids in which the metal forms
part of the cation. Addition of nitric acid does it to a certain
degree, but not fully. Nitric acid, a strong oxidizer, forms
part of my paranitro fixing fluid. I n combination with paranitrophenol it helps complete precipitation of proteins, increases the speed of penetration and counteracts excessive
hardening. Acetic acid does not give as complete precipitation, but if added to an aqueous solution of a metallic salt
such as cupric nitrate or cobaltous nitrate, it prevents the
condensation of chromatin and induces normal fixation of
nuclei. But acetic acid destroys mitochondria or else makes
them invisible. It also has a bad effect on erythrocytes and
in some fixing fluids destroys them completely. However,
neither acetic acid, nor its haloid derivatives destroy mitochondria if used in combination with phenol or its derivatives.
The same is true of propionic acid. Instead, these acids introduce another deleterious effect, breaking up the cytoplasm of
amphibian erythrocytes into globules or granules (fig. 3).
After considerable experimentation I have found that this can
be easily prevented by the simple addition to or substitution
in place of acetic acid of formic acid. The latter is a strong
reducer, prevents the lumping of chromatin in the nuclei and
the breaking up of the cytoplasm (fig. 4).
The French edition of Lee’s Microtomists’s Vade-Mecum
published by him in collaboration with Henneguy in 1896,
contains on page 59 a statement not found in the American
editions. This statement refers to the use of bromine water
in connection with Ripart and Petit’s fixing fluid for fixation
of cells rich in protoplasm. As a student in Moscow University I tried this method the same year that the book was
published. At that time I worked on cockroaches and found
difficulties in getting good fixation of their salivary glands.
The use of Ripart and Petit’s fluid with the recommended
addition of bromine water proved to be a wonderful improvement. I still have in my possession a slide which I made at
that time and I have never since been able to get as good
fixation by any of the known fluids, my own sublimate and
paranitro included. With this experience in my mind I
decided to t r y out the introduction of a bromine ion in the
new fixing fluids with which I was experimenting. This time
I did not use bromine water as recommended by Ripart and
Petit, but selected the bromophenols, bromacetic acid and
cupric bromide because of the greater convenience in preparing solutions. Of the bromophenols only parabromophenol
can be used. The combination of the three in an alcoholic
solution gives excellent fixation, possesses great penetrating
power and leaves tissues soft. The granulation of erythrocytes is prevented by the addition of formic acid. Resting
and dividing nuclei are well fixed (fig. 5 ) . Mitochondria show
up clearly, especially if stained in iron-haematoxylin without
any counterstain, thus leaving the rest of the cell colorless
(fig. 6). I n fact, this fluid gives better fixation of mitochondria than does Regaud’s bichromate-formalin. Mitochondria in the mammalian liver are also preserved. What is
still more interesting is the fact that bile canaliculi become
easily visible, something very unusual in the case of fixation
without previous injection of the ducts (fig. 7). On the whole
I think I have finally solved the problem of making a fixing fluid
which would leave the tissues soft and at the same time give
good nuclear and cytoplasmic fixation.
The formulae of three new fixing fluids are given below.
Of these no. 1 is a modification of my old no. 1 phenol-cupric
fixing fluid with that difference that it is stable and may
be prepared in a single solution. It is free of the distorting
effect on the fixation of nuclei. Formula no. 2 is a modification
of my old no. 2 paranitrophenol-cupric. It is stable, can be
prepared in a single solution and gives normal fixation of
nuclei. Both fluids are rapid in their action and leave the
tissues about as soft as the old fluids. Either ethyl alcohol
or iso-propyl alcohol may be used. Normal propyl alcohol
may also be used, but has no advantage and is more expensive.
I have reduced the strength of the alcohol from 60% t o 40%
because experience has shown that the weaker solution is
sufficient to prevent even insect eggs from floating and is less
injurious to tissues rich in water. However, the strength may
be increased or decreased to meet special requirements.
Formula no. 3, the bromophenol-cupric fixing fluid, gives
better fixation and leaves tissues still softer, but is more
expensive and less stable when prepared in a single solution.
In about 10 days a colorless, oily, heavy fluid appears at the
bottom, apparently belonging to a bromine lacrimator type
of compound, high1y.irritating to the eyes. If the solvent is
iso-propyl alcohol the decomposition of the fluid is somewhat slower, taking not less than 3 weeks at room temperature. It is better, therefore, to prepare the fluid in two stock
solutions as given in the instructions and to mix them at the
time of use. Silver impregnation by the Bodian method gives
excellent results after fixation in the bromophenol-cupric fluid.
If no. 1or no. 2 is to be used for subsequent silver impregnation, a solution of pure formaldehyde, ca. 37% strong, should
be added in a ratio of no. I (or no. 2) 4 parts, formaldehyde
1 part. Formaldehyde cannot be added to the bromophenol
cupric fluid, as the mixture decomposes rapidly, but as just
stated, the fluid permits excellent impregnation when used
The relative proportion of the ingredients was worked out
by trial. I n some cases the amount of the 40% alcohol may be
increased from two to five times. However, the penetration
of the fluid is then less rapid and while that may be desirable
in some cases, it should be avoided whenever posible. I use
such weak solutions for the fixation of complete specimens of
invertebrates which are afterwards either to be cleared and
left in oil for purposes of demonstration of their internal
anatomy, or are to be used for dissection in weak alcohol.
The fixation is so gentle that the tissues remain semitransparent. If a large earthworm is first cleared of all soil in
its intestine by keeping it f o r days between wet sheets of
muslin, then anesthetized and allowed to stretch by putting
it into 5% alcohol with some ether at a temperature slowly
increasing to 50°C. and fixed in a weak solution of any of the
three fixing fluids, washed and transferred into 70% alcohol,
it may remain in the alcohol for many weeks retaining its
flexibility to such an extent that it may be wound up spirally
without damage and regains its shape when released. Dehydrated and cleared in cedar oil or xylene, almost the entire
anatomy of the worm becomes visible. I n the case of insects
fixed in one of the new fixing fluids, dissection in weak
alcohol or water is greatly facilitated because the malpighian
tubules, while remaining soft and semitransparent, acquire
sufficient tensile strength not to be broken while being
Tissues are best allowed to remain in the fixing fluid for
several hours or over night. They may be washed in running
water or in weak alcohol, increasing the strength gradually
to avoid shrinkage in view of their softness after fixation.
I n my paper published in Science in 1933 I pointed out that
tissues fixed in the paranitrophenol-cupric mixture increase
about 16% in volume after they are transferred into 70%
alcohol. No explanation of this phenomenon was offered. I
depended upon the displacement of water before and after
fixation. Recently Stowell applied a different method of
measurement consisting in careful measurement of the area
of one surface of a piece of kidney and calculating the change
in volume. He came to the conclusion that tissues fixed in the
paranitro mixture increase by about 30% in volume in 70%
alcohol and then shrink in the process of embedding in paraffin
to about 64% of their original volume. He further found
similar considerable swelling of tissues fixed in f ormalin.
The final shrinkage amounts in the case of formalin fixed
tissues to 60% of the original volume. Bouin’s, Susa and
Zenker produce no swelling, but cause a final shrinkage to
from 47% to 63% of the original volume. I cannot agree with
Stowell’s conclusion that the quality of fixation has anything to do with swelling and shrinkage of tissues. These are
strictly physical phenomena. Shrinkage after most fixing
fluids is considerably greater than after paranitro. The new
bromophenol-cupric fixing fluid causes slightly greater swelling than the old paranitro, yet fixation is much better. The
fact is that good fixation depends upon proper combination of
salts of heavy metals with acids and other ingredients, upon
their rate of penetration and upon their chemical action on
the cell constituents. Swelling and shrinkage are undesirable,
hut they are equally injurious in good as in bad fixation.
No. 1. Phenol-cupric fixing fluid, pH 1.5
40% ethyl (or iso-propyl) alcohol .........................
Ether ...................................................
Formic acid, spec. grav. 1.20, ca. 87% ......................
Phenol .................................................
Gupric bromide (or nitrate) ...............................
100 cc.
5 cc.
5 cc.
5 gm.
3 gm.
No. 2. Paranitrophenol-cupric fixing flnid, p H 1.5
40% ethyl (or iso-propyl) alcohol ..........................
Ether ...................................................
Formic acid, spec. grav. 1.20, ca. 87% .......................
Paranitrophenol .........................................
Cupric bromide (or nitrate) ...............................
100 cc.
5 cc.
5 cc.
5 gm.
3 gm.
No. 3. Bromophenol-cupric fixing fluid, pH 1.35
Solution A.
4001, iso-propyl (or ethyl) alcohol ........................
100 cc.
Ether .................................................
5 cc.
Formic acid, spec. grav. 1.20, ca. 87% ................... 1Occ.
Cupric bromide ........................................
Solution B.
40% iso-propyl (or ethyl) alcohol ........................
100 cc.
Ether .................................................
Bromacetic acid ........................................
10 gm.
Parabromophenol ......................................
For use mix : Solution A - 1part by volume, Solution B 1part. If desired, add 40% iso-propyl (or ethyl) alcohol from
1to 5 parts by volume.
COHEN, ISADORE 1934 Cytological fixation with salicylic and orthophosphorie
acids. Stain Technology, vol. 9, pp. 101-106.
GRAY,PETER1933 Notes on practice of fixation for animal tissues. J. R.
Microsc. Soc., vol. 53, pp. 13-19.
BOLLES,AND L. FBLIXHENNEGW1896 Trait6 des m6thodes
techuiques de 1'anatomie microscopique. Deuxihme 6dition. Paris.
Octave Doin, Bditeur.
LEE, ARTHURBOLLES 1937 The Microtoniist's Vade-Mecum. Tenth edition. P .
Blakiston 's.
ALEXANDER1933 New fixing fluids for general purposes.
Science, vol. 77, pp. 117-118.
ROBERT E. 1941 Effect on tissue volume of various methods of
fixation, dehydration and embedding. Stain Technology, vol. 16,
pp. 67-83.
ZIRKLE, CONWAY1928 The effect of hydrogemion concentration upon the
fixation image of various salts of chromium. Protoplasma, vol. 4,
pp. 201-227.
1929 Fixation images with ehromates and acetates. Protoplasma,
v01. 5 pp. 511-534.
1933 Cytological fixation with the lower f a t t y acids, their compounds and derivatives. Protoplasma, vol. 18, pp. 90-111.
1933 Some dicarboxylic acids a s components of fixing fluids.
Protoplasma, vol. 19, pp. 565-577.
1933 Aldehydes as cytological fixatives. Protoplasma, vol. 20,
pp. 169-179.
1934 Amines in cytological fixing fluids. Protoplasma, vol. 20,
pp. 473482.
Figures 1 t o 6 are microphotographs of sections through tissues of the common
newt, Triturus viridescens Raf., made at a linear magnification of 700. Figure 7
is a microphotograph of a section through the liver of cat at a linear magnification
of 1300.
1 Effect of metal cations on nuclei. Piece of skin fixed i n a solution of
cuprie nitrate in 40% alcohol with the addition of some formalin. Stained in
haematoxylin-eosin. Notice the condensation of chromatin at one end of the
nuclei. The same effect is produced by similar salts of cobalt, mercury, uranium
2 Effect of metal anions on nuclei. Piece of skin fixed in Regaud’s potassium
bichromate f ormalin fluid and stained i n haematoxylin-eosin. Notice t h a t the
nuclei appear normal.
3 Effect of acetic acid and its derivatives on the cytoplasm of erythrocytes
i n presence of phenols. Piece of liver fixed in a n alcoholic solution of cuprie
bronlide, bromophenol and bromacetic acid. Stained i n haematoxylin-eosin.
Notire the granulation of the cytoplasm.
4 Correcting effect of formic acid on fixatiou when added to the same
fixing fluid used i n figure 3. Skin, stained in haematoxylin-eosin. Notice normal
appearance of nuclei and cytoplasm.
5 Intestine fixed in no. 3 bromophenol cupric fixing fluid and stained in
6 Piece of liver fixed in no. 3 bromophenol-eupric fixing fluid and stained
in iron-haematoxylin without counterstain. Nothing but nuclei and mitochondria
appear stained.
7 Piece of eat’s liver fixed in no. 3 bromophenol-eupric fixing fluid and stained
in iron-hnematoxylin. Notice the bile canaliculi.
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