THK STRUCTURE OF TRYPANOSOMA LEWISI. 765 
gredient; (2) and (3) were either used directly on the wet 
film, or after previous exposure to osmic acid vapour. 
(1) Osmic acid.—I have employed osmic acid asa fixative 
of the films followed by various other fixatives, such as abso- 
lute alcohol, Flemming’s fluid, sublimate-acetic, etc. I will 
only deal in this section with its action when followed by 
alcohol, and discuss its action when combined with other fixa- 
tives in the sections dealing with those fixatives. Of all the 
methods I have tried, fixation with osmic vapour followed by 
fixation with alcohol (figs. 60-71) gives the best result as 
regards size, form, and general characters of the trypanosomes, 
so far as can be judged from a comparison with the standard 
(figs. 1-8). The osmic acid was used in the form of vapour 
given off from a 4 per cent. solution, combined with acetic 
acid in the following proportions: a 4 per cent. solution 
osmic acid, 20 drops, glacial acetic acid, 1 drop. 
The smears, after exposure to the osmic vapour, were in some 
cases allowed to dry off, either before further fixation or after 
staining; but as a rule they were transferred at once to 
absolute aleohol, and were stained and mounted in Canada- 
balsam without being allowed to dry at all. I will distinguish 
these two methods of treatment briefly as the dried-off osmic 
method and the never-dried osmic method respectively. ‘lhe 
dried-off method is most convenient for smears made on slides ; 
for the never-dried methods it is better to make the smears 
on coverslips. The dried-off osmic smears stain very well by 
the ordinary Romanowsky methods (Giemsa’s stain, azure- 
erythrosin, ete.), but I have never got good results with the 
iron-hematoxylin stain after drying off. The never-dried 
smears stain well with Romanowsky stain, and fairly well, but 
rather faintly, with iron-hematoxylin. The details of the 
flagellum and blepharoplast are difficult to make out aiter 
osmic-absolute fixation and iron-hematoxylin staining ; for 
these points fixation in sublimate mixtures is far superior. 
After the dried-off osmic method the trypanosomes do not 
show what I will term briefly a “ periplast-line,” that is to 
say, a clear border under the periplast, causing the periplast 
