PROTOZOA 395 



Methods of study. The trypanosomes may be studied 

 in the fresh (living) state and in a fixed and stained condi- 

 tion. In the fresh condition a drop of blood from the infected 

 individual is placed on a slide and a cover-glass placed over it. 

 To study the structure of the organisms the blood must be in 

 a thin layer. The movement of the corpuscles caused by the 

 parasite enables one to locate them with a low magnification 

 (100 diameters). When they are rare it has been recom- 

 mended to centrifuge the blood after adding a small quantity 

 of sodium citrate solution to prevent coagulation. The try- 

 panosomes are found to be more numerous in the leucocytic 

 layer. The organisms can be studied for a considerable time 

 in a hanging drop preparation. 



In stained preparations, the following process was recom- 

 mended by Laveran. 1 



The blood is spread in a thin layer on a glass slide, dried very 

 rapidly, and fixed in absolute alcohol for five to ten minutes. The 

 following solutions should be prepared beforehand: 



1. Methylene-blue with silver oxide or Barrel 'blue. Take 50 or 

 60 cc. distilled water in a flask of about 150 cc. capacity, and add 

 some crystals of silver nitrate. When these are dissolved, fill the 

 flask with a concentrated solution of caustic soda and shake. A 

 precipitate of silver oxide is fopmed. This is washed several times 

 with distilled water, so as to get rid of any silver nitrate or excess 

 of soda. A saturated watery solution of methylene blue ("medicinal 

 methylene blue," Hoechst) is then poured on to the silver oxide and 

 allowed to remain for a fortnight, the flask being shaken from time 

 to time. 



2. Watery solution of eosin, 1 per 1,000 ('water-soluble' eosin, 

 Hoechst). 



3. Solution of tannin, 5 per cent, or better, a solution of tannin- 

 orange, which can be obtained ready made up. 



The staining mixture is prepared at the time of using, according 

 to the following formula: 



Solution of eosin (1 per 1,000) 4 cc. 



Borrel blue 1 cc. 



Distilled water 6 cc. 



The mixture is at once poured into a flat dish, such as a Petri 



1 Laveran. Compt. rendu de la Soc. de Biol., Vol. LII (1900) p. 

 549. 



