1. Examination of the contractile vacuole of Chilomonas paramecium during progressive impregnation by the Weigl osmic technique revealed that the so-called ‘osmiophil cortex’ appeared first as a number of perivacuolar osmiophilic granules. Prolonged impregnation caused these to fuse to form a closed ring which, after very prolonged incubation, became a solid osmiophilic mass.
2. Cultures fixed together and osmicated in separate tubes gave more consistent impregnations, in any one series, than those both fixed and osmicated separately.
3. With the two samples of osmium tetroxide used, after normal fixation, A consistently gave impregnations of 62-85 per cent., and C impregnations of 25-36 per cent.
4. Constant mixing during fixation and osmication was not advantageous, and gave more irregular results than normal methods.
5. Time of fixation, between the limits of 5 to 90 minutes, had no effect on the impregnation.
6. It was suggested that the irregularities of impregnation with a given sample of osmium tetroxide were due to the presence of a trace of some retarding agent, possibly introduced at the time of fixation.
7. It was shown that the osmiophily of the perivacuolar region in Chilomonas did not vary with the vacuolar phase.