THE IRON-ACETOCARMINE METHOD OF FIXING

AND STAINING CHROMOSOMES.

JOHN BELLING,
CARNEGIE INSTITUTION OF WASHINGTON.

A short description of this procedure was given in Vol. 55 of

the American Naturalist, pp. 573—574. The method has now
been used almost daily for five years, and certain improvements
have suggested themselves.
The solution can be prepared by mixing about equal volumes of
glacial acetic acid and water. To this is added powdered carmine
in excess. The liquid is then ‘¿justbrought to boil, cooled, and
decanted. To the red solution there are then added a few drops
of a solution of ferric hydrate in 50 per cent. acetic acid, to act
as a mordant. The amount of iron varies with different objects.
Too much produces a precipitate in a short time. The more
iron, the darker and bluer is the stain. A bluish red is usually
the best. Large cover-glasses (20 by 50 mm.) should be used,
and they should not be thicker than 0.17 mm. (Small or thick
covers prevent pressure being properly applied to individual
cells.) There should be a minimum thickness of liquid under
the cover-glass. The edges can be sealed with a honey-thick
solution of dammar in xylol applied with a brush; or better
perhaps, with melted soft paraffin. -
The following varied modes of preparation are useful in
different cases.
i. Pollen-mother-cells, etc., are obtained free by pressing or
tapping cut segments of anthers in a drop of iron-acetocarmine.
2. The cut-up anthers, or minute fragments of animal ovaries
or testes, are put in a tube with a large excess of iron-acetocarmine.
After 2 to 7 days, preparations are made in the usual way.
This gives good results with cancer tumors.
3. Smears of large anthers or testes are made on a cover-glass,
which is then laid on a drop of iron-acetocarmine on a slide.
4. For staining the vegetative and generative nuclei in pollen
grains or pollen-tubes, etc., crystals of chloral hydrate are dis
solved in a few drops of iron-acetocarmine until the liquid just
i6o
 THE IRON-ACETOCARMINE METHOD. 161

clears the specimens sufficiently. (Too much chloral hydrate

will cause shrinkage.) The pollen-grains are squeezed or partly
crushed in this liquid, and may be left to stain for a day or two.

FIG. I. Hyacinth—2n.

In the case of pollen-mother-cells, local pressure, with a small

roll of paper, on the cover-glass, often spreads out the cytoplasm
on the cover-glass or slide, with the chromosomes uninjured.
If required, these can be preserved in balsam, by replacing the
liquid by graduated mixtures of 45 per cent. acetic and alcohol.
Fig. I is a balsam preparation of the reduction metaphase in a
hyacinth, prepared in this way.

FIG. 2. Datura—2n—I.

The cytoplasm becomes quite clear in the acetic acid, and with
an appropriate green screen on the microscope, the bluish red
chromosomes stand out jet black. (Wratten green films Nos.
66 and 56, are most useful on a binocular, and No. 58 on a
monocular, with a good tungsten electric light.) Fig. 2, a pollen
mother-cell of Datura in the second metaphase, with 23 chromo
somes, in iron-acetocarmine, shows the sharpness of the images.
Pressure is usually best applied from 2 to 7 days after mounting,
but the time varies for different pollen-mother-cells. Well pre
pared slides will often keep for some months.

a.
162 JOHN BELLING.

The following notes may be of use.

Canna.—The pollen-mother cells are large and tender. The
anthers should be cut into segments about I mm. long, and
gently pressed out in a large drop of ironacetocarmine, being
left ten minutes or so to toughen before putting on the cover
glass. (Here, of course, as usual, the anther remains are previ
ously removed.)
Zea.—In maize the pollen-mother-cells are delicate, and the
same process is required as for Canna.
Nicotiana.—In tobacco the pollen-mother-cells are easily
pressed out and stained.
Datura.—Resembles a small tobacco. Pollen hard to stain.
Hyacinthus.—Yields excellently stained pollen-mother-cells,
and also pollen-grains. The latter require several days to stain
well. Pressing out is difficult.
Triticum and Secale.—In wheat and rye all the pollen-mother.
cells are connected and come out of the anther loculus in a string.
The young anthers are cut across once, and pressed out under the
cover-glass. The pollen-grains stain well.
Tradescantia.—Stains deeply and shows chromomeres well in
pollen-mother-cells, and in pollen-grains.
Uvularia.—Shows chromosomes like those of the Orthoptera in
the pollen-mother-cells. Pollen stains well. Some metaphase
chromosomes of the reduction division show compound rings.
Hosta.—Shows large and small chromosomes, the former with
marked chromomeres. Stains well.
Cypripedium acaule, and C. pubescens.—Both pollen-mother
cells and pollen-grains stain well. Ten pairs of chromosomes.
Scilla sibirica.—Pollen-mother-cells stain well. Shows six
pairs of chromosomes.
Narcissus.—Pollen-mother-cells and pollen-grains stain well.
Galanthus.—Pollen-mother-cells stain remarkably easily and
well. Several different sizes of chromosomes.
The above plants have chromosomes which are especially
worth studying, because of their size and for other reasons. In
Brassica, Gladiolus, Iris versicolor, Asparagus, Dahlia, Capsella,
Hemerocallis, Antirrhinum, Stizolobium, Phaseolus and Crocus,
the chromosomes are either small, or clumped and not easily
unravelled.