1909. | BLOOD-PARASITES OF FRESHWATER FISHES. 5 
dried. JI have tried various fixatives and get the best results with 
sublimate-acetic (HgCl, saturated in water 95 volumes, glacial 
acetic 5 volumes), Schaudinn’s fluid (HgCl, saturated in water 
2 volumes, absolute alcohol 1 volume, with addition of a few 
drops of glacial acetic), and Mann’s picrocorrosive-formol solution. 
The first two fixatives may be used directly on the wet smears, or 
with previous exposure to osmic vapour for a short time. I find 
it useless, however, to fix the smears on slides; the reason being 
that it is impossible, or at least very difficult, to put them into 
the fixatives without making them dive in with one end fore- 
most, a process which causes distortion in the wet and still unfixed 
elements of the smear. Satisfactory preparations can only be 
obtained by these methods of fixation, by making the smear on a 
coverslip and dropping it at once with the smear downwards into 
the fixative. I hold the coverslip with the fingers of my left 
hand, and a glass rod in my right hand ; a drop of blood is placed 
by an assistant on the coverslip; I then smear it out immediately 
with the glass rod and drop it instantly into the fixative. The 
whole process can be done most expeditiously, and with much less 
risk of partial drying than when dealing with slides. 
In staining trypanosomes with iron-hematoxyln, I find it best 
to let the mordant and stain act for a long time. The objects are 
first left in the iron-alum solution (34 °/,) over night; they are 
then, after brief and rapid washing with distilled water, transferred 
to the hematoxylin solution (4°/,) for at least 24 hours. The 
whole art of the process lies in the differentiation and extraction 
of the stain. My method is to put the coverslip into the iron- 
alum until colour is seen to be coming out; then the coverslip is 
dipped into tap-water to stop the extraction of the stain, and 
examined with moderate magnification (Zeiss Oc. 4, Obj. D). I 
the karyosome can be seen sharply and clearly in a trypanosome, 
the extraction is sufficient; if not, the coverslip is put into iron- 
alum again for a short time. Usually I do several smears of each 
sample of blood with different degrees of extraction of the stain. 
In most smears there are also thicker and thinner portions, and it 
is found that in thicker portions of the smear the stain is not 
so quickly extracted from the trypanosomes as in the thinner 
portions. Different degrees of extraction of the hematoxylin have 
their uses in showing up different points of structure. When the 
trypanosomes first come out of the stain they have an even, opaque 
black colour. I find that usually the stain is extracted first from 
the cytoplasm generally, then from the myonemes ; next from 
the coarse granulations of the cytoplasm; next from the flagella 
and the blepharoplasts; next from the karyosome, and last of 
all from the kinetonucleus which appears to give up the colour 
at its periphery first. There are exceptions, however, to this 
order, for in some trypanoplasms (e. g. 7’. gurneyorum) the 
cytoplasmic granules retain the stain as long as the karyosome. 
In one of my preparations which was under-extracted, I found 
that the trypanosomes and trypanoplasms showed their form and 
