CULTIVATION OF ANAEROBIC BACTERIA. 79 



If we make an inoculation of one of the species which is not 

 strictly anaerobic into a test tube containing nutrient gelatin or agar- 

 agar, we may have a development all along the line of puncture, 

 and this may be more abundant below, as in Fig. 47. But when we 

 make a long stick culture with a strict anaerobic the development 

 occurs only near the bottom of the line of puncture (Fig. 48). 



We may then, if we have a pure culture to start with, propagate 

 these anaerobic bacilli in long stick cultures. It is best to use tubes 

 which have been recently sterilized, as boiling expels the air from 

 the culture medium ; and a very slender needle should be used in 

 making the inoculation. To prevent the absorption of oxygen a 

 layer of sterilized olive oil may be poured into the tube after the in- 

 oculating puncture has been made, or it may be filled up with agar 

 jelly which has been cooled to about 40 C. Roux has proposed to 

 prevent the absorption of oxygen by the culture medium by plant- 

 ing an aerobic bacterium Bacillus subtilis upon the surface, after 

 making a long stick culture with the anaerobic species. The agar 

 jelly is first boiled and quickly cooled ; the inoculation is then made 

 with a slender glass needle ; some sterile agar cooled to 40 C. is 

 poured into the tube, and when this is- solid the aerobic species is 

 planted upon the surface. The top of the test tube is then closed 

 hermetically and it is placed in the incubating oven. The aerobic 

 species exhausts the oxygen in the upper part of the tube by its 

 growth on the surface of the culture medium, and the anaerobic 

 species grows at the bottom of the tube. To obtain material for a 

 new culture or for microscopical examination the test tube is broken 

 near its bottom. 



Cultures in liquid media may be made by exhausting the air in 

 a suitable receptacle or by displacing it with hydrogen gas. The 

 first-mentioned method has been largely used in Pasteur's laboratory, 

 but methods in which hydrogen gas takes the place of atmospheric 

 air in the culture tube are more easily applied and require simpler 

 apparatus. The flask shown in Fig. 49 may be used in connection 

 with an air pump. The sterile culture liquid is first introduced into 

 a long-necked flask and inoculated with the anaerobic bacillus to be 

 cultivated. The neck of the flask is then drawn out in a flame at c. 

 The open end is then connected .with a Sprengle's pump or some 

 other apparatus for exhausting the air. The flask is placed in a 

 water bath at 40 C. , which causes ebullition at the diminished pres- 

 sure, and the exhaustion is continued for about half an hour. The 

 narrow neck is then sealed at c by the use of a blowpipe flame. 



The flask shown in Fig. 49, which can be made from a test tube, 

 may also be used in connection with a hydrogen apparatus. In this 

 case a slender glass tuba is passed into the flask, as shown in Fig. 



