THE STRUCTURE OF TRYPANOSOMA LEWISI. 781 
kindly furnished me with the following directions for making 
up and using the stain: 
“To prepare the stain make up half-saturated watery solu- 
tions of neutral red and of Gribler’s light green, using 
distilled water. Place the neutral red solution in a large 
open vessel, and add to it sufficient light green solution to 
combine with the neutral red; the compound will form a 
precipitate. It is better not to have an excess of either stain, 
as the precipitate is difficult to wash. When the neutralisa- 
tion-point is reached, the water will contain a small quantity 
of both dyes in solution, giving it a dark appearance; the 
presence of both stains in solution can be easily tested by 
dropping a drop of the filtrate on to blotting paper ; this will 
spread out, leaving a light red central zone and a faint green 
outer zone. 
“No collect the precipitate it is better not to filter, for the 
stain soon blocks the pores of the filter-paper. If the 
solutions are warmed to about 30° or 40° C. before mixing, 
the precipitate will form in large sticky masses, some of 
which containing air-bubbles float to the surface and can be 
removed with a spatula. The rest settle and stick to the 
bottom and sides of the vessel, and can be collected after 
pouring off the fluid. The precipitate so collected is rinsed 
in distilled water and dried at 37°C. In this state it forms 
dark greenish masses, insoluble in water, somewhat soluble in 
ethyl alcohol, but soluble to a greater extent in methyl 
alcohol, and especially so if 5 per cent. of glycerine is added. 
“'lo make up the stain for use, it 1s best to pound up about 
0°25 grm. of the stain with some clean, sharp sand; this 
prevents the stain going into a sticky mass when the alcohol 
is added. ‘To the powder so obtained is now added some 
purest methyl alcohol, acetone-free, containing 5 per cent. by 
volume of glycerine. Pound up well to obtain a saturated 
solution ; then pour off and add a further quantity of alcohol 
elycerine solution and repeat the pounding; about 100 c.c. 
stain can be made from the quantity given. 
“The alcohol-glycerine mixture dissolves about ‘1 per cent. 
