Contagious Diseases of Insects. ' 271 



Artificial Cultuees of Bacteria. 



Methods of Culture. — The modes of culture used in all the 

 experiments reported in this paper were based, unless otherwise 

 specified, on those of Klein, as described in his paper " On the 

 Relation of Pathogenic to Septic Bacteria," in the Journal of 

 the Royal Microscopical Society for January, 1883, differing 

 only by modifications which will appear in the description. 

 The cultures were usually made in beef broth (rarely in in- 

 fusion of cabbage) in test tubes plugged with sterilized cotton, 

 or in ordinary flasks similarly closed. The broth was prepared 

 by boiling lean beef from a half hour to an hour in a 

 porcelain-lined vessel, and then filtering and carefully neu- 

 tralizing wdth caustic soda. The tubes, flasks, and cotton 

 were sterilized by heating in a tin oven, over a gasoline stove, 

 for several hours, at a temperature of not less than 275 degrees 

 or more than 300, as determined by a thermometer inserted in 

 the oven. The heat was sufficient to considerably scorch the 

 cotton without actually charring it. While still within the hot 

 oven the tubes and flasks were securely plugged by means of 

 steel forceps freshly heated in the flame of an alcohol lamp, the 

 cotton plugs, from two to three inches in depth, being pushed 

 firmly in. Most frequently the mouths of these plugged 

 vessels were covered wath a cap of sterilized cotton, held in 

 place by an inverted beaker also carefully sterilized by dry heat. 

 In charging the vessels with the fluids the plugs were rapidly 

 withdrawn and as promptly returned, the infusions being intro- 

 duced boiling hot and afterwards boiled for several minutes to 

 destroy any germs which might have entered during the instant 

 before the plug was replaced. It was not found necessary to 

 test the sterilization of the tubes by protracted incubation, as 

 all the check tubes and the stock flasks in which the store of 

 prepared infusion was preserved, remained unchanged through- 

 out the entire season. Neither was any incubator required, the 

 ordinary temperature of the air during the weeks when these 

 experiments were in progress being never below 60^ by day, and 

 ranging commonly above 70" both day and night. 



When a culture tube or flask was infected with the fluids 

 of a larva, the following process was invariably used. From 



