The behaviour of tissue slices in chromate solutions: a
study of the comparative importance of the tonicity and
osmotic concentration of fixative systems
By GEORGE R. BERNARD and GAIL G. WYNN
(From the Department of Anatomy, Medical College of Georgia, Augusta, Georgia, U.S.A.)
Summary
A study was made of the weight changes undergone by slices of rat liver, kidney, and
other organs immersed in certain fixatives containing chromium. In some experiments the data could be interpreted as indirect measurements of the fixative's 'effective' osmotic pressure, or 'tonicity', a property which usually differs from the fluid's
'total' osmotic pressure. In the case of kidney slices exposed to aqueous potassium
dichromate, the rapidly occurring and reproducible responses seem proportional to
the fixative concentration; no clear-cut differences were observed with liver slices
similarly exposed. Altering the concentration of chromic acid did not affect quantitatively or qualitatively the response patterns of either tissue. Responses of slices to
combinations of dichromate and formalin suggest cumulative action of the two
moieties. The response patterns to the latter fluids could be modified by the addition
of 'indifferent' substances such as sucrose.
Introduction
BAKER (1958, pp. 83-84), after reviewing the literature and his own experiences, stated that in making up fixatives '. . . there is no virtue in isotonicity' and that'. . . the total osmotic pressure exerted by the constituents of a
fixative is unrelated to the swelling or shrinking of cells'. The evidence for the
latter statement is conclusive. However, the term 'isotonicity' when applied to
fixative solutions made up in physiological saline solutions may be misleading.
As Potts and Parry (1964) have emphasized, one solution is isosmotic with
another if it exerts the same osmotic pressure, i.e. has the same solute particle
concentration (usually determined by measuring some resultant property
such as freezing-point depression). Tonicity, on the other hand, is defined in
terms of the response of cells immersed in a solution. A solution is isotonic
with a cell (or tissue) if the cell neither swells nor shrinks when immersed in
it. Sometimes an isotonic solution is also isosmotic. We have recently shown
(Bernard and Wynn, in press) that a 4% formaldehyde solution, although
grossly hyperosmotic to plasma as measured cryoscopically, caused slices of
rat liver, kidney cortex, and thigh muscle to swell, but not as much as they
would in a distilled water environment. The formalin solution, then, should
be considered hypotonic to these tissues. We attributed the impedance to
'total swelling' (such as occurs in distilled water) to the 'effective' osmotic
concentration of some constituent of the fixative other than the formaldehyde
molecules. Modification of the formalin solution by increasing the concentration or by addition of certain quantities of 'indifferent salts' can be employed to reduce the swelling to a minimum.
[Quart. J. micr. Sci., Vol. 106, pt. 1, pp. 23-30, 1965.]
24
Bernard and Wynn—Tonicity of chrornate fixatives
Regaud's fluid, a freshly-prepared aqueous 20% formalin/2-4% potassium
dichromate mixture, is generally recommended for the demonstration of
mitochondria by light microscopy. Since mitochondria are remarkably
sensitive to osmotic changes in their environment (Lewis and Lewis, 1914-15;
Hogeboom, Palade, and Schneider, 1948; Zollinger, 1948), it might be
supposed that Regaud's fluid is roughly isotonic with the contents of the
mitochondria. Hirsch and Jacobs (1926) reported that the freezing-point
depression (A) is greater than — 2-8° C (theoretically, the A value should be
greater than —4-65° C, the value given for 20% formalin by Kraut in 1890).
Since the A of plasma is about —o-6° C, Regaud's fluid is grossly hyperosmotic. The dichromate moiety, if used alone, is hypo-osmotic, since its A is
slightly less than —0-41° C. The A of closely related chromium trioxide at
1% concentration is —0-35° C (Hirsch and Jacobs, 1926), and it too is hypoosmotic to the plasma. We investigated the tonicity of this group of related
fluids by the use of tissue slices.
Materials and methods
Well-nourished, adult Sprague-Dawley rats were decapitated. The organ—
liver, kidney, rectus femoris muscle, muscular portion of the diaphragm,
spleen, or pancreas—was removed quickly, extraneous tissue (e.g. fascia,
renal medulla) dissected away, and slices less than 0-5 mm thick were cut by
hand with a fresh razor-blade. The muscle was sliced longitudinally; the
other organs, in random planes. In the case of the diaphragm, serosal surfaces were left intact and one leaf made up the 'slice'. Each slice was pierced
by a tared platinum hook (27-gauge wire fashioned like a barbless fish-hook
complete with eye), which served for suspension during all subsequent manipulations. After weighing on a torsion balance (Roller-Smith type, 500 mg
capacity), the slice was immersed in 35 ml of one of the test solutions.
The temperature of the solutions was 23° to 25° C. A wire resting on the rim
of the beaker held the slice and hook in such a way that the slice was completely immersed but not touching the beaker. After exposure for 5 min to
the fixative the hook and slice were removed from the beaker, the slice gently
blotted on cotton gauze, reweighed, and re-immersed. The same process was
repeated at 15, 30, 60, 240, and 1,200 min after immersion. In one set of
experiments the first interval was 2 min. Weight changes were tabulated as
percentages of the original weight.
Slices were immersed in the following fluids: (1) distilled water; (2) 1-2%
or 2-4% aqueous potassium dichromate (K 2 Cr 2 0 7 ), or 0-5%, i-o%, or 3-0%
aqueous chromium trioxide (CrO3) solutions (the aqueous solution is called
chromic acid); (3) Regaud's fluid (80 volumes of 3% K 2 Cr 2 0 7 and 20 volumes
of 100% formalin (80:20); (4) Regaud's fluid components at different proportions (e.g. 75:25); and (5) Regaud's fluid to which sucrose was added.
Chromic acid solutions are closely related ionically to dichromate solutions
(Casselman, 1955), but because of their lower pH, act as coagulant fixatives.
Bernard and Wynn—Tonicity
of chrornate
fixatives
25
Results
Comparison of slice response to distilled water, potassium dichromate, and chromic
acid solutions
Liver. Sections immersed in distilled water rapidly gained weight and,
although considerable variability was noted, the immediate response was a
FIG. I. Weight changes with time of rat tissue slices exposed to various fluids. In
the figures time is expressed as the square root of the number of min of exposure,
in order to emphasize early changes.
linear one. After exposure for 15 min the rate of weight-gain diminished
(perhaps indicating that the elasticity of the system was beginning to counteract inflow forces), and after several hours the swollen slice shrank somewhat,
but by this time autolytic processes must have changed the system drastically.
The data are tabulated in table 1 (see Appendix, p. 30), and the initial response is included in fig. 1, A. The responses of liver slices to 1-2% and 2-4%
aqueous potassium dichromate solutions were very similar, so that the two
curves almost overlap (fig. 1, A). This indicates similarity in their tonicities
(although the solute concentration is doubled). After a brief lag the weight
increased linearly for the first hour and then the rate diminished. During the
following 16 h the weight diminished slightly, but there was still a net gain of
44% after immersion for 20 h.
Ordinarily, chromic acid solutions are used as fixatives at concentrations of
26
Bernard and Wynn—Tonicity of chrornate fixatives
i% or less. The response of liver slices to 0-5 or 1% CrO3, although less than
the responses of sister slices to the dichromate solutions, was similar qualitatively in that the immediate weight gain was followed by a period of stability
in the case of 0-5% CrO3 and some weight loss in the case of the 1% CrO3.
Initially the two response patterns are remarkably similar and it would appear
that the effective osmotic concentration (or tonicity) of the two chromic acid
solutions are similar. 3% CrO3 is so powerful an oxidant that liver slices,
after increasing in weight for the first hour (as was expected), suddenly
shrivelled and usually became too friable for weighing.
Kidney cortex. Slices responded to a distilled water environment much like
liver slices (compare fig. 1, A and B). However, after exposure for 30 min the
kidney slices began to lose weight slowly. With 1-2% dichromate there is
also remarkable similarity between the two organs. However, with 2-4%
dichromate the response pattern indicates greater effective osmotic concentration and the magnitude of the response is more proportional to the solute
concentration. It should also be noted that the narrow span of the data obtained during the first hour's exposure to these 3 chromic acid solutions with
their sixfold range of concentration strongly suggests that the total solute
concentration is not involved in determining the tonicity of the solution for
either test system. The 3% CrO3 solution is apparently isotonic for kidney
cortex (whereas it is hypertonic for liver slices), but as with liver slices it ultimately distorts the tissue grossly.
Formalin-dichromate mixtures
Of the 4 test fluids—75:25 (75 volumes 3-0% K 2 Cr a 0 7 to 25 volumes
100% formalin), 80:20 (Regaud's fluid), 85:15, and 90:10—the 75:25,
because of its greater formaldehyde concentration, is theoretically the most
'hyperosmotic'. Liver tissue was used in all fluids, kidney cortex slices in the
75:25, 80:20, and 90:10, and slices from the rectus femoris muscle, the
muscular leaves of the diaphragm, pancreas and spleen in the 80:20 fluid
only. Table 2 summarizes the data (see Appendix, p. 30).
Liver. Slices immersed in either 80:20 or 75:25 responded similarly.
After a 15-min period without any appreciable weight-change, a slight
temporary increase in weight was observed (fig. 1, A). However, slices in both
the 85:15 and 90:10 solutions gained weight, the greater increase occurring
in the 'less hyperosmotic' 90:10 solution. After the first hour some of the
increment was lost. It is noteworthy that all four response patterns were
approximately parallel. Although in the 80:20 mixture the slice weight remained rather constant, dynamic changes must have been occurring.
Kidney cortex. Again the slices reacted similarly in both 75:25 and 80:20
solutions. The sudden drop in weight, 5% in 2 min, certainly was not expected (fig. 1, B). This weight loss is followed by a temporary recovery period
(1-4 h), thus yielding a biphasic curve. These slices may contain a considerable amount of connective tissue, in addition to the parenchyma, thus making
it difficult to assign reasons for the changes observed. Unlike the liver-slice
Bernard and Wynn—Tonicity
of chrornate
fixatives
27
response, where apparently we witnessed the additive effect of the osmoticallyactive particles in the formalin and dichromate moieties, kidney cell membranes apparently are affected differently by the dichromate, and a rapid
outward movement (presumably of water) was induced.
Pancreas. Regaud's fluid caused no change in slice weights.
Spleen. After a 15-min period of 'equilibrium', the slices lost a small
amount of weight in Regaud's fluid.
Muscular tissue. The weights of slices from the rectus femoris muscle
changed very little during the period of observation. Muscular leaves of the
diaphragm, on the contrary, lost more than 10% of their original weight
during the first 5 min of exposure, gained slightly during the next 25 min,
then lost weight. The biphasic response is quite similar to that of the kidney
cortex. Since the muscular diaphragm was partially invested with either
parietal pleura or peritoneum, or both, plus some connective tissue, the relative proportions of muscle fibres may be less in the diaphragm than in the
thigh muscle, and some or all of the change recorded may have been due to
serosal and connective tissues.
Regaud's fluid with sucrose added
In making Regaud's fluid, the concentrated formalin and 3% K 2 Cr 2 0,
solutions have to be mixed immediately before use because of the complex
chemical reactions which occur between the components of the two solutions.
The addition of sucrose to this reactive mixture not only alters the solute
concentration (or 'total' osmotic concentration) but also increases the complexity of the chemical reactions. To some freshly prepared Regaud's fluid
enough sugar was added to increase (theoretically) the total osmolar concentration by 200 mOsm/1; to another aliquot, enough to raise the total
osmolar concentration by 500 mOsm/1. (Mammalian plasma has an osmolar
concentration of approximately 300 mOsm/1.) Rat liver slices were exposed
to these solutions as well as to the unmodified fluid. The sugar-modified
Regaud data are summarized in fig. 2. They show that under the conditions
of the experiment rat liver slices react to these 3 solutions as though the unmodified Regaud's fluid is very slightly hypotonic, while both fluids that
contain sucrose are hypertonic, the fluid with more sucrose being more
hypertonic.
Discussion
There is no facile explanation for the above results, because both systems—
the fixative environment and the tissue slice—although superficially simple,
are really quite complex. Aqueous solutions of potassium dichromate,
chromium trioxide, or formaldehyde are sometimes called 'simple' fixatives.
But are they ? In the case of the chromium compounds the solutions may contain, in addition to the undissociated molecules, several species of solute
particles such as potassium ions, bichromate ions (HCrO 4 ~), dichromate ions
(Cr 2 O 7 = ), chromic acid (H 2 Cr0 4 ), chromate ions (CrO 4 = ), and hydronium
28
Bernard and Wynn—Tonicity of chromate fixatives
ions (Casselman, 1955). A similar situation exists in formalin, where, in
addition to the formaldehyde molecules, cells are exposed to the hydrated
form of the molecule (H2C(OH)2) plus various polymers of the molecule,
and the contaminants, methyl alcohol, formic acid, formate ions, and
hydronium ions.
E 100
v/t
in min
FIG. 2. Weight changes with time of rat liver slices exposed to certain fluids.
Northrop's (1927) analysis of published data on osmotically induced
swelling of Arbacia eggs showed that the eggs swell much like a collodion
sac system, whereas slices of carrot swell in distilled water like gelatin blocks.
In the latter type of system the entire mass offers resistance to the passage of
water and the resistance is not confined to surface layers. We assume that
animal organ slices behave much like carrot slices. In fact, our studies (in
press) of the behaviour of thin gelatin blocks in formaldehyde environments
show qualitative similarities to that of rat organ slices.
When a tissue slice is immersed in a fixative solution, the blood remaining
in the larger vessels may flow or be flushed out. Lymph, secretions, or excretory fluids as well as interstitial fluid and cytoplasm from ruptured cells also
may be lost. The mass of these natural fluids, however, would be comparatively small and the spaces would be reoccupied by fixative solution. With
fixative solutions composed of a combination of several fixative ingredients,
this passive perfusion process plus the use of thin slices minimizes the effects of
different diffusion rates of the fixatives' components.
When a cell is bathed with a fixative several changes may occur. Fixative
molecules, in order to be effective, must diffuse into the cell through the
cellular membrane, and it is possible that in this passage the fixative molecules
may alter the permeability of the membrane. Assuming, however, that the
Bernard and Wynn—Tonicity
of chr ornate
fixatives
29
permeability to water or to the fixative particles is not immediately affected,
we may suppose that water would move into or out of the cell, as a result of
the relative osmotic potentials of the fixative and the intracellular environments. If the fixative is an aqueous solution of a nearly pure compound such
as potassium dichromate, the ions from which might be capable of passing
through the plasma membrane, then the fixative environment (although it
does possess some osmotic pressure as measured cryoscopically) would be
grossly hypotonic and would act much as a distilled water environment.
Water would then pass readily into the cell along with the fixative molecules
or ions, thus increasing the mass of the slice. If, on the other hand, certain
particles were present in the fixative environment which were not capable of
penetrating the cell, they could exert osmotic forces which might overcome the
osmotic attractiveness of particles within the cell for water. Water would then
pass out from the cell and weight would be lost. The weight accruing through
the entrance of the fixative molecules might be insufficient to counterbalance
this loss of water.
The process of fixation not only alters protoplasmic constituents so that
they resist autolysis, but also prepares the cells for the sequential damaging
processes of dehydration, infiltration, embedding, and microtomy. The
'quality' of fixation has therefore generally been appraised by the appearance
of the end-product, the tissue section. An example of such a study is Zirkle's
(1928) report upon the fixation picture of Zea mays root tips after fixation in
different chromium salt fixatives. Yet each step in the preparative process
influences the final appearance, as Tellyesniczky (1898), Patten and Philpott
(1920-21), Tarkhan (1931), and others have shown. We measured one parameter, weight change, during the initial phase of the preparative sequence.
Our data only permit inferences as to what material movements caused the
weight changes observed. But it is only through such experimentation that
the mechanisms involved in the fixation process will be elucidated.
This work was supported in part by a grant from the USPHS (GM 9047)
and in part by grants from the Professional Research Fund of the Medical
College of Georgia.
References
BAKEK, J. R., 1958. Principles of biological microtechnique. London (Methuen).
BERNARD, G. R., and WYNN, G. G., 1964. Anat. Rec. (in press).
CASSELMAN, W. G. B., 1955. Quart. J. micr. Sci., 95, 321.
HIRSCH, G. C , and JACOBS, W., 1926. Z. Zellforsch. mikr. Anat., 3, 198.
HOCEBOOM, G. H., SCHNEIDER, W. C , and PALADE, G. E., 1948. J. biol. Chem., 172, 619.
KRAUT, 1890. Liebig's Ann. Chem., 258, 103. (Cited by Hirsch and Jacobs, 1926.)
LEWIS, M. R., and LEWIS, W. H., 1914-15. Amer. J. Anat., 17, 339.
PATTEN, B. M., and PHILPOTT, R., 1920-21. Anat. Rec, 20, 393.
POTTS, W. T. W., and PARRY, G., 1964. Osmotic and ionic regulation in animals. New York
(Macmillan).
TARKHAN, A. A., 1931. J. roy. micr. Soc, 51, 387.
TELLYESNICZKY, K., 1898. Arch. mikr. Anat., 52, 202.
ZIRKLE, C , 1928. Protoplasma, 4, 201.
ZOLLINCER, H. U., 1948. Amer. J. Path., 24, 569.
30
Bernard and Wynn—Tonicity
of chromate fixatives
Appendix
TABLE I
Weight changes (expressed as % of original weight, mean i standard error of
rat liver (L) and kidney cortex (K) slices during the first 20 h of immersion in
various fixatives containing chromium
Time (min)
1-2%
K a Cr a O,
2-4%
K a Cr a O,
0-5% CrO s
i-o% CrO 3
3-0% CrO 3
5
15
30
60
240
1,20c
8
6
6
6
i23±5-o
I26±2'5
iO9±i-3
i53±6-7
53±2-i
i68±7-8
59±2-8
i83±8'o
i83±9-o
io4±8-3
150 = 5-0
II6±I-6
29±2-O
IO
IO9±I-2
IO4±I'5
IO6±I-I
I8±I-I
37±226±I'
N
L
K
L
K
L
K
L
K
L
K
L
K
6
8
6
8
6
6
6
ZI±I'I
09 ± I • I
J3±i-7
11 ±i-3
11 = 1-7
io6±o6
io4±fo
io6±i'i
IOl4;O-6
IO2±O'8
O9±2-O
OI±I-O
14-
HaO
Organ
If-
Solution
i8±2I4±i-c
i6±2I4±iI2±3'3
00 = 0-5
147=4-6
I37±I-4
I4I±I-8
I34±i'3
Il8±2-O
I24±3'3
ii3±o-6
I2i4j3'6
ii4±o-7
II6±2-9
9 8±i-o
150 = 2-6
i52±i-8
I48±2'2
'37±5'4
J35±4'7
109 ± 1 0
I24±2'6
iO9±o-7
8
7±3'3
73±i'4
>44 = 3'i
i39 = 4-3
144=16
133 = 5-3
111 = 10
II4 = 4'2
IO7=I'2
•
t
• Only 3 weighings possible: 37=1-5t Only 4 weighings possible: 27±0-7.
TABLE 2
Weight changes (expressed as % of original weight; mean ± standard error)
of rat liver (L), kidney cortex (K), rectus femoris muscle (M), muscular portion
of diaphragm (D), pancreas (P), and spleen (S) slices during the first 20 h
of immersion in various combinations of potassium dichromate and formalin.
N equals number of observations
Time (min)
Solution
75:25
80:20
85:15
90:10
Organ N
L
K
L
K
M
D
P
S
L
L
K
4
6
24
12
3
5
3
3
4
4
4
2
5
103 i 1 0
94±o 5
ioo±O'4 IOI±O 4
95±o-6 93 ±0 6
ioo±o'9 IOO±O 4
93±o-6 89=0 8
99 + 0-5 IOO±O 5
ioo±o-8 99±o S
io4±o 9
iO4±i 0
94±i 5
15
3°
60
IOI±O 7 io 4 ±o- 9 io7±o 3
92=0 9
92±o'8 94±o 6
IOI±O 5 iO4±o-7 io6±o 8
92±O 6 93±o-8 96 = 0 8
99 = 0 8 98 = 07 99 i 1 0
9i±i 0
95± 1-2 93±o 8
98 ± I 2 99±o-6 IOO±O 7
97±o-6 96 = 0 8
9 8±o 7
IO6±I 5 IO8±I-7 110=1 0
IIO±I 0 112=07 116 = 0 7
96±o 8 99±o-8 IO 3 ±I
240
1,200
103=0 7 98 = 3.3
88 = o-8
92=0 5
104=0 7 100 = >7
95±i 0 91 = •3
93 + 0
9 8±o 9
9>±o 9
ioiii
92±
-1
1
97 = 0
96 ± 0 6 94 3: <J 8
108 ± 0 9 103 ± -0
H 3 ± i 0 107 = • i
96 = c •9
IOI±I 0