Procedure*
I. Prepare the acetocarmine stain.
1. Pour 45 ml of glacial acetic acid and 55 ml of distilled water
into a 1 liter flask. (caution. Glacial acetic acid can burn
skin or eyes. Use eye protection and a hood. Do not breathe
the fumes.)
2. Heat to boiling (in a hood)
3. Turn off the heat, add 0.5 grams of carmine, shake well, cool
and filter.
II. Prepare the Dippell stain.
1. Combine 10.5 parts of the acetocarmine, 4.5 parts of 45% acetic
acid, 2 parts of 1N HCl.
2. Then add 0.5 parts of 1% fast green which was dissolved in 95%
ethanol.
III. Test the stain for the right amount of green cytoplasm.
Test this solution on a few cells. You want a good green in the
cytoplasm that is not too dark. The purpose of the green is to
make the red nuclei clearer. If the solution is too light in
green, add a little more and keep testing until you like the results.
*Recipe was originally published in Ruth V. Dippell, A Temporary Stain
for Paramecium and Other Ciliate Protozoa, Stain Technology 30
(2): 69 - 7
|
|
Background and Use
Paramecium is a ciliate and shares with all members of that
group a large macronucleus with many copies of each gene and a small
micronucleus which is ordinarily diploid. The macronucleus is
where most of the mRNA is produced as a result of gene action. The
micronucleus serves as a gamete nucleus that may undergo meiosis when
sexual reproduction occurs.
You can see the macronucleus very easily with this stain. Ruth
Dippell was a zoologist at Indiana University who studied the anatomy of
Paramecium. She developed this simple stain to be able to
see whether the macronucleus was in one large piece, which is usually
is, or was fragmented into pieces. Fragments are characteristic of
the self-fertilization process called autogamy, found in the species of
P. aurelia species complex.
If you obtain some "P. aurelia" and grow them through about 20
fissions, many of them will then undergo autogamy if starved. This
will be seen with the Dippell stain as a dozen or so small fragments of
macronucleus rather than one large one.
Even if you have another species, just put a bunch of cells on a slide,
add a drop of Dippell's stain, and look for red nuclei on a green
background. Sometimes the micronucleus of P. caudatum" can
be seen, but most of the other common species will probably have
micronuclei too small to see with this stain.
|
