618 W. S. PERRIN. 
living cell. The addition of small quantities of vital staining 
reagents, neutral-red, brillant-cresyl-blau and methylene blue 
failed to disclose any further details of structure. For 
making permanent preparations of the parasite, films fixed 
and stained in various ways were found to be alone admissible. 
Sections of 24 thickness cut in paraffin were found to be of 
but little value. 
Films were prepared in two ways: 
1, The Malpighian tubules were cut up into small pieces, 
which were spread over a coverslip. ‘lhe coverslip was then 
allowed to dry, immersed in absolute alcohol for ten minutes, 
dried, and stained with one of Giemsa’s modifications of the 
Romonowsky-Nocht stain. The best results were given by 
leaving the films over night in a mixture of an aqueous solu- 
tion of eosin and azur II freshly prepared according to 
Giemsa’s well-known recipe.! The coverslips were washed 
with tap-water after staining, dipped fora moment in absolute 
alcohol to prevent overstaining, washed again with tap-water, 
dried, and mounted in cedar wood oil. 
When a preparation was required immediately, Giemsa’s 
ready prepared eosin-azur solution was employed. The 
specially powerful mixture of azur I, azur II, and eosin used 
by Schaudinn for the Spirocheete of syphilis was found to stain 
too deeply and was discarded. 
The above method of fixation and staining, though appar- 
ently drastic, gives excellent results. Careful comparison 
with the living material and preparations fixed with osmic 
vapour shows that the trophozoites themselves are but. very 
little deformed in general outline, frequently not at all, while 
no other method of staining differentiates nucleus and proto- 
plasm so sharply (see figs. on left hand side of plate). 
The method has an additional advantage in that the 
nuclei stain differently. ‘his point will be referred to later. 
Nuclei of the cells of the Malpighian tubules upon the other 
hand are completely spoilt by this method (see fig. 15). The 
1 Ten parts of a solution of 1 gr. eosin B.A. in 1000 c.c. water is added 
to 1 part of a solution of ‘8 gr. Azur II (Griibler) in 1000 c.c, of water, 
