488 TRANSACTIONS OF THE CANADIAN INSTITUTE. [VoL. VI. 
[II—METHOD OF STUDY, AND SPECIES STUDIED. 
The fixing reagents which I used were corrosive sublimate and 
picric acid, each in saturated aqueous solutions, the chrom-osmio-acetic 
mixture of Flemming, and a one per cent. solution of potassic iodide 
saturated with iodine. Undoubtedly for yeast cells the best of these, in 
my experience, are corrosive sublimate and Flemming’s fluid. They 
produce less alteration in structure than any other, and they do not, 
when properly used, alter the staining power of any part of the yeast 
cell. The most satisfactory method of employing them was to allow 
them to act in bulk on a large number of yeast cells, separated from the 
actively growing cultures by centrifuging the latter. After the reagents 
had acted sufficiently long the fluid was decanted, distilled water was 
peured upon the cells, which were immediately subjected to centrifuge 
action and thus separated, when they were treated with alcohols of 
gradually increasing strengths. With these reagents also I obtained 
reliable cover-glass preparations, in which drying of the cells was not a 
factor, by spreading a film of the yeast culture on the cover-glass, and 
then, with this face downward, placing it floating on a quantity of the 
solution, to remain there for from one to twenty-four hours. The fluid, 
at the moment of touching, removed very many of the elements, but 
enough were left adherent to make a good preparation. Afterwards 
the cover-glass so treated was placed in alcohol of 30, 50, 70, and 90 
per cent. strengths-successively. 
The cover-glass method of preparation could not be employed with 
the other reagents except to some extent in the case of iodine, as these 
remove from the cover all the cells. The only safe way of fixing with 
these solutions was to allow them to act on the yeast cells in the test 
tube. They were separated completely from the fluid, after the required 
time for complete fixation, by centrifugalizing the fluid. The hardening 
in these cases was completed by the use of alcohols of 30, 50, 70, and go 
per cent. strengths successively. The cells were completely separated 
in these cases by gravity. In the case of the iodine solution the method 
employed by some of allowing a film of yeast cells to dry on a cover- 
glass, and then placing it in the reagent, has no advantage over the one 
described, and I am not sure that it is free from objection. It is dif- 
ficult to believe that yeast cells can be unaffected when the fluid about 
them is completely removed by evaporation. I have, therefore, avoided 
this method of preparation, as well as that in which heat alone is used 
for the purpose of fixation. 
