プロタゴ−ル染色法

1. Cells were fixed for about 5 minutes in a 1:1 mixture of Bouin's fixative and saturated HgCl2.
   Bouin's fixative <-> A 1:1 mixture of saturated HgCl2 sol. and 2%(w/v) OsO4 in 0.1 M sodium phosphate buffer pH 7.2 でも可
2. Washed in tap water.
3. "enrobed" in a thin film of Mayer's albumin. to promote adhesion of cells on the slide, the slides were kept in an oven at 45ﾟC until the albumin film was nearly dry, and then the film was coagulated in 3 steps:
   1. a drop of methyl alcohol was placed over the albumin layer on the slide, and allowed to nearly evaporate.
   2. the slide was placed in a staining dish containing absolute methyl alcohol for 10 minutes.
   3. the slide was transferred to a 1:1 formalin-alcohol mix for 10-30 min.
4. The slides were then run down from 50% alcohol to distilled water, and exposed insuccession to 5% potassium permanganate (3-4 min.) and 5% oxalic acid (3-4 min.), with thoro washing in between.
5. After an additional distilled water wash, the slide were placed in fresh 1% Roque protargol (Roboz Surgical Instrument Co., 810 18th St. N.W., Washington, D.C.) at 45ﾟC. In general, 20 min impreqnation was adequate; longer periods often resulted in overstained cells. Copper was found not to be necessary.
6. Following protargol, the cells were washed in distilled water, and placed in the 1% hydroquinone 5% sodium sulfite solution.
7. The course of development was followed microscopically, and the time required was found to be highly variable. Some slides required only a few min, while slides on which cells appeared to darken very slowly were left in the hydroquinone overnight; some of the best slides were obtained in the latter fassion.
8. Following the hydroquinone step. the cells were washed, placed in 2% oxalic acid for about 2 min, washed very thoroly(?), then run up thru the alcohos to xylene and mounted.

1. Gortz, H-D.: The behavior and fine structure of the dorsal bristles of Euplotes minuta, E. aediculatus, and Stylonychia mytilus (Ciliata, Hypotrichida). J. Protozool. 29(3), 353-359 (1982).
2. Heckmann, K.: Paarungssystem und genabhangige Paarungstypdifferenzierung bei dem hypotrichen ciliaten Euplotes vannus O.F.Muller. ???