26 Annals Entomological Society oj America [Vol. XI, 



EXPERIMENTATION WITH CULTURES. 



In order to test the pathogenicity of the separate cultures 

 laboratory experiments were performed with Bacillus poncei 

 and with d'Herelle's cultures labeled "Souche Cham" and 

 "Souche Sidi." I attempted no experiments with the culture 

 obtained from Messrs. Du Porte and Vanderleck for the reason 

 that my systematic study showed this culture to be identical 

 with d'Herelle's "Souche Sidi" strain. 



In all of my experiments the most painstaking bacteriological 

 technicalities were observed, so I shall not undertake to describe 

 all of the tiresome and well known methods in vogue such as 

 using sterile instruments, etc., for injecting and operating upon 

 a grasshopper. Suffice it to say, that sterile containers in the 

 form of battery jars were found extremely useful in performing 

 my experiments. Glass plates covered the jars in order to keep 

 the hoppers from jumping out. These plates had the further 

 advantage of keeping the corn leaves, with. which we fed the 

 insects, fresh. Prior to the injection the hoppers were always 

 washed with 95% alcohol. This must not be used too freely, 

 otherwise, the grasshoppers may die and after its use one must 

 wait a minute or so for the alcohol to evaporate before injecting. 

 A small amount of alcohol entering the wound, however minute, 

 caused by the hypodermic needle usually ends fatally. The 

 inoculations were always performed between the metathorax 

 and the first abdominal segment on the ventral side. In order 

 to avoid rupturing the gut or otherwise injuring the insect, two 

 operators are absolutely necessary to perform successful inoc- 

 ulations. One person must carefully, but firmly hold the insect 

 while the other inoculates. I performed a number of tests in 

 order to determine whether my technical precautions were 

 sufficient and I found them satisfactory. For example: I 

 washed ofT a large series of grasshoppers with alcohol and then 

 injected with sterile water. Some of the insects I permitted 

 to live until they seemed to die of natural causes; others I 

 killed after periods of one and two weeks in order to inoculate 

 culture tubes with some blood obtained by bathing the tro- 

 chanter and femur with alcohol and then breaking the joint 

 by a swift movement. The culture tubes remained perfectly 

 sterile. 



