ZOOLOGY AND BOTANY, MICROSCOPY, ETC. 757 



B. Technique.* 

 CI) Collecting- Objects, including- Culture Processes. 



Cultivating' Trypanosomes.f — Thiroux cultivated Trypanosoma 

 duttoni in the following medium : beef or rabbit broth, 1000 grm. 

 (125 grm. of meat macerated in 1 litre of distilled water), Witte's 

 pepton 20 grm., salt 5 grm., agar 20 grm., carbonate of soda solution 

 (58 grm. to the litre) 10 c.cm. 



The materials are prepared and mixed in the customary way, except 

 that the medium is not clarified with white of egg. When made, it is 

 sterilised in the autoclave for 20 minutes at 110°, and preserved in tubes 

 covered with caoutchouc caps. 



When required for use the necessary quantity is melted in a water 

 bath and when cooled down to 45°, two volumes of defibrinated rabbit's 

 blood are added. It is then made into slopes, and next day the inocu- 

 lations are made in the condensation water, the blood being taken from 

 the heart of a mouse. The first cultures develop in from 10-15 days ; 

 from these sub-cultures are made, and so on until development occurs 

 on the 4th day. 



In order to stain the Trypanosomes, thin films are necessary. The 

 preparations are fixed in absolute alcohol and stained by Laveran's 

 method.J 



Cultivation of Amoebae. § — A. Lesage inoculated gelose with mucus 

 from dysenteric stools. The gelose, which had been washed in running 

 water for 8 days and afterwards sterilised, was placed in Petri's capsules 

 or in tubes. The temperature ranged from 18°-25°. In a few days 

 amoeba;, often motionless, were found buried among the bacteria. 

 Cultivations were also made on plates on which paracolon bacilli were 

 growing. By this method living amoebae were obtained from the human 

 intestine without passing through the encysted stage. 



Another method was to allow the amoebae to become encysted, and to 

 cultivate the cysts thus obtained. For this purpose some mucus and a 

 little sterilised water were placed in a covered capsule. The mucus 

 dried slowly at a temperature of 18°-25°. After a few days the dried 

 mucus was sown on gelose plates. In this way about one vessel out of 

 ten was found to contain amoebae. Each successful plate served to 

 obtain fresh cultures of the pure mixed cultures. Each time the plates 

 were inoculated the amcebaa were sown at the bottom of the plate while 

 held vertically, the upper end being inoculated with the food bacterium. 

 The plates were incubated at 20°. After a few days the amoebae reached 

 the upper end, and from this part fresh plates were inoculated, and so on. 



* This subdivision contains (1) Collecting Objects, including Culture Pro- 

 cesses; (2) Preparing Objects ; (3) Cutting, including Imbedding and Microtomes ; 

 (4) Staining and Injecting; (5) Mounting, including slides, preservative fluids, &c. ; 

 (6) Miscellaneous. t Ann. Inst. Pasteur, xix. (1905) pp. 566-9 (1 pi.). 



% See this Journal, 190:-?, p. 117, itud 1904, p. 120. 



§ Ann. Inst. Pasteur, xix. (1905) pp. 10-16 (2 pis.); Comptes Rendus, cxxxix. 

 (1904) pp. 1237-9. 



