223
The Relative Oxidizing Properties of Certain Reagents and
Mixtures used for the Fixation of Tissues
By W. G. BRUCE CASSELMAN
(From the Cytological Laboratory, Department of Zoology and Comparative Anatomy, Oxford;
present address, Banting and Best Department of Medical Research, University of Toronto,
Toronto)
SUMMARY
The oxidation potentials of the fixing reagents studied range from I - I O V for
chromic acid to 0-23 V for formaldehyde. Potassium dichromate, mercuric chloride,
and osmium tetroxide have intermediate values. Commonly used mixtures prepared
from these compounds also exhibit a range of potentials from i-oo V for Champy's to
0-15 V for Regaud's. The oxidation potentials of Altmann's, Champy's, Flemming's,
Helly's, and Zenker's fluids remain relatively constant for at least 1 day, whereas those
of Regaud's and Sanfelice's fluids undergo appreciable change.
D
U R I N G an investigation of cytological fixation by reagents containing
anionic chromium, the oxidation potentials of various solutions of
chromic acid and potassium dichromate were determined (Casselman, 1955).
Because no previous reports regarding the redox potentials of fixing solutions
could be found in the literature, these studies were extended to include
certain other reagents and mixtures. The results are presented here.
METHODS AND MATERIALS
With the exception of osmium tetroxide, analytical grade reagents were
used. All solutions were prepared in distilled water. Measurements were made
on formalin which had been 'neutralized' with calcium carbonate as well as
on the 'unneutralized' solution. Only the former was used in the preparation
of mixtures.
Both hydrogen-ion concentrations and oxidation potentials were measured.
These were determined with a Marconi type T F 717A mains-operated pH
meter (Marconi Instruments Ltd., St. Albans, Hertfordshire). To ensure
stability, the instrument was left operating continually. For the pH measurements, it was equipped with a saturated-potassium chloride calomel reference
electrode, a glass electrode, and a temperature compensator. It was standardized frequently against the buffer provided for use with the pH meter
(Marconi, batch 016W, pH 6-46) and occasionally against 0-05 M potassium
hydrogen phthalate (pH 4-00). For the oxidation potential measurements,
the same calomel electrode and a gold electrode were used. All measurements
were made at least in duplicate at room temperature (i7 o -i0. 0 G.) and, usually,
on 50-ml. portions of the solutions or mixtures. In the case of the oxidationpotential measurements, sufficient time (45-75 minutes) was allowed for the
[Quarterly Journal of Microscopical Science, Vol. 96, part 2, pp. 223-226, 1955.]
224
Casselman—Relative Oxidizing Properties of
charge on the gold electrode to attain equilibrium with the solution (Kolthoff
and Furman, 1931; Elek and Boatman, 1953). The potential of the reference
electrode was taken as being 0-25 V at 200 C.
Measurements of hydrogen-ion concentrations and oxidation potentials
were made on solutions of the primary reagents as they are used in fixing
mixtures: 1 to 3-4 per cent, chromic acid, 10 per cent, formalin (approximately
4 per cent, formaldehyde), 1 to 5 per cent, mercuric chloride, 2 per cent,
osmium tetroxide, and 2-5 to 5 per cent, potassium dichromate.
Similar measurements were made after the addition of each solute during
the preparation of the fixing mixtures and, finally, after each mixture had
been left for 1 day at room temperature. The following mixtures were
studied: Altmann's, Champy's, Flemming's, Helly's, Regaud's, Sanfelice's,
and Zenker's.
OBSERVATIONS
The observed hydrogen-ion concentrations and oxidation potentials of
solutions of the primary fixing reagents are presented in table 1, which also
TABLE I
Primary fixing reagents
Reagent
pH
chromic acid, 3^4
1-7
ro
potassium
dichromate, 5-0
30
2-5
mercuric
chloride,
5-0
10
osmium
tetroxide,
E
Reaction
O'SO
1 05
I'2O
Cr 2 O 7 -+i 4 H+ + 6 e- ^ 2Cr++++7HIO
3-7°l
3-85
J
078
3-75]
3-25/
2-0
(4-75)
formaldehyde, 36
(io% formalin):
unneutralized
neutralized
34l
4-5SJ
2Hg++ + 2 e~ ^ Hgt+
0-64
OsO 4 +8H + +8 e~ ^ Os+4HSO
0-85
/HCOOH + 2H++2 e-^ HCHO + H2O
HCHO + 2H++2 e~ ^ CH3OH
0056
includes the reactions undergone by each reagent and its standard oxidation
potential (Latimer, 1952). The pH of the osmium tetroxide solution depends
upon that of the water in which it is dissolved.
The hydrogen-ion concentrations and potentials recorded at each step in
the preparation of Zenker's, Helly's, and Champy's mixtures are summarized
in table 2. Table 3 presents the observations on the various mixtures when
they were freshly prepared and, again, 1 day later. During the first 3-4 hours
Certain Reagents and Mixtures used for the Fixation of Tissues
225
TABLE 2
Stages in the preparation of Zenker's, Helly's, and Champy's fluids
E
PH
V
078
100 ml. 3'5% potassium dichromate
+ 1 gm. sodium sulphate
+ 10 gm. mercuric chloride
3-85
4-00
3-60
57 ml. above 'stock' mixture
+ 3 ml. glacial acetic acid (Zenker's)
3-60
2'5°
076
0-83
57 ml. 'stock' mixture
+3 ml. neutralized formalin (Helly's)
3'6o
370
076
074
35 ml. 3% potassium dichromate
+ 35 ml. 1% chromic acid
+20 ml. 2% osmium tetroxide (Champy's)
3-85
i-8o
1 80
078
1-03
100
(475)
3-90
1 80
064
076
100
20 ml. 2% osmium tetroxide
+ 35 ml- 3% potassium dichromate
+35 ml. 1% chromic acid (Champy's)
076
TABLE 3
Fixing mixtures
pH
E
When
prepared
After
1 day
When
prepared
After
1 day
Champy's (see table 2)
1 80
i-8o
V
i-oo
V
i-oo
Flemming's
15 vol. 1% chromic acid
4 vol. 2 % osmium tetroxide
1 vol. glacial acetic acid
i'4S
1-30
i-oo
i-oo
083
Mixture
Zenker's (see table 2)
250
2-50
0-83
Helly's (see table 2)
37O
4-20
075
0-70
Altmann's
1 vol. 5 % potassium dichromate
1 vol. 2 % osmium tetroxide
4-00
4-00
070
070
3 00
(063)
o-5S
4-85
(o-io)
0-15
Sanfelice's
16 vol. 1% chromic acid
1 vol. glacial acetic acid
8 vol. formalin
Regaud's
8 vol. 3 % potassium dichromate
2 vol. formalin
470
after preparation, there is a steady decline in the potentials of the mixtures
containing formaldehyde, especially Regaud's and Sanfelice's fluids. The
values within the parentheses are those at the end of the first hour. A moderate
226
Casselman—Oxidizing Properties of Fixatives
colour change from orange to brown was noted for Regaud's fluid and a
much more pronounced one, from orange to dark greenish-brown, for
Sanfelice's.
DISCUSSION
These observations emphasize the range of oxidative properties to be
found among some of the more commonly used fixing reagents and mixtures,
ranging from the quite strongly oxidizing chromic acid and Champy's and
Flemming's fluids to the more reductive formalin and Regaud's fluids. The
great changes in potential occurring in mixtures such as Sanfelice's and
Regaud's, like the changes in the colours of these solutions, indicate appreciable alterations in the composition of the mixtures. The changes will be
largely oxidation of formaldehyde, initially to formic acid, with reduction
of the chromic acid or dichromate to the trivalent chromium cation. In these
mixtures, therefore, fixation might not be entirely by anionic chromium.
The role of oxidative or reductive processes in general cytological fixation
is still unknown, especially with regard to any morphological changes which
are induced. Certain specific instances where these processes are important
can be cited; for example Ciaccio's method for rendering lipids less soluble in
organic solvents and other applications of 'postchroming' depend upon oxidation. Oxidative cleavage of certain tissue carbohydrates, which may be histochemically undesirable, can occur with certain mixtures containing chromic
acid, in the same way as it occurs under more controlled conditions in Bauer's
test.
It is a pleasure to acknowledge the many inspiring discussions and helpful
advice given by Dr. John R. Baker during this study. This investigation was
made during tenure of a Merck Postdoctoral Fellowship in the Natural Sciences
of the National Research Council of Canada.
REFERENCES
BAUER, H., 1953. Ztschr. f. mikr.-anat. Forsch., 33, 143.
CASSELMAN, W. G. B., 1955. Quart. J. micr. Sci., 96, 203.
ELEK, S. D., and BOATMAN, E. S., 1953. Nature, 172,
1056.
HEWITT, L. F., 1950. Oxidation-reduction potentials in bacteriology and biochemistry. 6th edn.
Edinburgh (Livingstone).
KOLTHOFF, I. M., and FURMAN, N. H., 1931. Potentiometric titrations. 2nd edn. New York
(Wiley).
LATIMER, W. M., 1952. Oxidation potentials. 2nd edn. New York (Prentice-Hall).