The Fixation of Adipose Fat by Potassium Dichromate
By J. T. Y. CHOU
{From the Cytological Laboratory, Department of Zoology, University Museum, Oxford)
SUMMARY
Ordinary adipose fat globules can be rendered resistant to paraffin embedding by
the immersion of suitably fixed tissues in a saturated solution of potassium dichromate
at 370 C for 7 weeks. Xylene has less tendency to remove the postchromed fat than
chloroform, benzene, or toluene.
Adipose fat is usually dissolved out of the tissues during dehydration or embedding.
Empty spaces are therefore usually seen in paraffin sections wherever fat globules
were present during life. Several authors (Kaufmann and Lehmann, 1926 a, b; 1928;
Smith, Mair, and Thorpe, 1909) have claimed that potassium dichromate can in
certain circumstances be used to fix adipose fat in such a way that it is retained in
paraffin sections, but a full study of this subject has not previously been made. My
object has been to find a reliable method of fixing adipose fat by potassium dichromate
in such a form that it is not dissolved away when paraffin sections are made.
MATERIAL AND METHODS
T
HE skin of the mouse was used for this investigation, as the subcutaneous
adipose cells are large and abundant. It was shaved with a razor-blade
and cut into pieces about 1 cm square.
The skin was always fixed for 2 days in Ciaccio's fluid (formalin 20 ml;
potassium dichromate, 5% aq., 80 ml, glacial acetic acid, 5 ml). This was
chosen because it was known not to be destructive of lipids. After washing
in running water for 24 h, the skin was soaked in 3 % potassium dichromate
for 24 h at room temperature, and then transferred to a saturated solution of
potassium dichromate in an incubator at 370 C. The period in the saturated
solution varied from 1 to 7 weeks. The intention was to discover whether
potassium dichromate would render adipose fat insoluble in the fluids used
for dehydration and embedding, and if so, what period of postchroming was
necessary.
A standardized process of dehydration was used throughout. After the tissue
had been washed for 24 h in running water it was left for \ h in each of the
following grades of ethanol: 50%, 70%, 80%, 90%, 95%, and absolute.
Four different antemedia were tried: namely, chloroform, benzene, toluene,
and xylene. The intention was to discover which of these had the least tendency to dissolve the fixed adipose fat. The piece of tissue was first left for
\ h in a mixture of equal volumes of absolute alcohol and antemedium, and
then for \ h in the antemedium alone. The piece was then transferred to
melted paraffin wax (m.p. 560 C) and left for 1 h (with one change of wax).
Sections were cut at 15 p and were coloured for 5 min in a saturated solution
of Sudan black in 70% ethanol. They were rinsed for 5 sec in 70% ethanol
[Quarterly Journal of Microscopical Science, Vol. 98, part 4, pp. 431-433, Dec. 1957.]
432
Chou—The Fixation of Adipose Fat by
and i min in 50%; then they were washed in distilled water and mounted in
Farrants's medium.
RESULTS
Different degrees of preservation of the fat were shown in different preparations. In many cases no sudanophil material survived dehydration and
embedding. When a little remained, it usually appeared in the form of a network extending across an otherwise empty space. When a considerable amount
remained, it was mainly aggregated at the periphery of the globule, while
the centre either contained a network of sudanophil material or else was
empty. (The partial solution of fat globules was studied long ago by Starke
(1895), who showed that the undissolved remnant formed a rim round an
empty space.) When the whole of the globule was preserved, it appeared
homogeneously black. In some cases the globules were well preserved near
the edge of the piece of tissue, but imperfectly towards the centre.
The various degrees of preservation of the fat are shown by symbols in
table 1. The symbols are explained in the legend to the table.
TABLE I
The effect of different periods of postchroming and of different
antemedia on the preservation of adipose fat
Period of0 postchroming
at 37 C (weeks)
Antemedia
Chloroform
Benzene
i.
O
o
2
o
o
o
+
++
+++
3
4
S
6
7
Toluene
Xylene
o
o
o
o
o
o
o
+
++
++
++
+
++
++
++
+
+ T
+++
++++
+ + + + Full coloration by Sudan black.
+ + + Fat globules near the edge of the section were solid black, while those near the
centre of the section showed a network.
+ + The margins of the globule were solid black, but the interior showed a network.
4- means that only a network of sudanophil material was left.
o No sudanophil material was present.
The table shows that no adipose fat is preserved unless the period of postchroming exceeds 3 weeks, whichever antemedium is used. With longer
periods of postchroming the preservation is progressively improved. Of the
various antemedia, xylene has the least tendency to extract the fat, and benzene
and toluene the most, chloroform is intermediate. If postchroming is continued for 7 weeks, and xylene is used as an antemedium, the fat droplets
appear homogeneously black in Sudan black preparations.
DISCUSSION
It was discovered by Smith, Mair, and Thorpe (1909) that triolein can be
rendered insoluble in ethanol, xylene, and ether by prolonged postchroming.
Potassium Dichrornate
433
Kaufmann and Lehmann (1926 a, b; 1928) claimed that all unsaturated lipids
could be rendered insoluble in lipid solvents by the action of potassium
dichromate, different lipids requiring different periods. The preservation of
various conjugated lipids by short postchroming is a familiar process of microtechnique. The facts presented in the present paper show that ordinary
adipose fat can be successfully postchromed, but the period has to be very
long. The postchromed fat is less soluble in xylene than in other antemedia
commonly used in paraffin embedding.
I wish to express my gratitude to Dr. J. R. Baker for suggesting and supervising this investigation, and also for helping in correcting this paper; to
Professor Sir Alister Hardy, F.R.S., for providing me with facilities for working in his Department.
The work was done during the author's tenure of an Inter-University Council
Fellowship through the Carnegie Corporation of New York, and study leave
from the Department of Zoology, University of Hong Kong.
REFERENCES
KAUFMANN, C , and LEHMANN, E., 1926a. Centralbl. allg. Path., 37, 145.
19266, Virchows Arch., 261, 623.
1928. Ibid., 270, 360.
SMITH, J. L., MAIR, W., and THORPE, J. F., 1909. J. Path. Bact., 13, 14.
STARKE, J., 1895. Arch. Anat. physiol., physiol. Abt. (no vol. number), 70.