CULTURES OF ANAEROBIC BACTERIA 97 



preparation. Stain with carbol fuchsin. Examine microscop- 

 ically in similar preparations the bacteria from one of the 

 fermentation tubes ; describe their appearance in each prepa- 

 ration and make a drawing of a few of them. 



Note the appearance of the cultures inoculated for the study 

 of the gas production. 



Inoculate (Liborius' method) 2 tubes of agar, one sugar 

 free, the other containing i% glucose, and 2 fermentation 

 tubes, one containing sugar-free bouillon, the other i / glucose 

 bouillon from a culture of B. tetani furnished. 



Read carefully in the text-books the methods for cultivating 

 anaerobic bacteria. 



141. Culture by Liborius' method. Liquefy 2 tubes of agar 

 and carefully pour them together. After this, boil the medium 

 for at least 5 minutes to expel the air, cool it down to a 

 temperature of 40 C., and then inoculate it from the cul- 

 ture of an anaerobic organism furnished, after which cool the 

 medium rapidly by standing it in cold water until it is 'set. In 

 inoculating the tube insert the loop nearly to the bottom and 

 stir very gently. In making the inoculations care must be 

 taken not to introduce air by shaking the liquid medium. 

 Place the culture in the incubator. 



142. The fermentation tubes for cultivating anaerobic bac- 

 teria. If these tubes of bouillon have been properly sterilized, 

 the closed branch is practically free from atmosphere. The 

 obligatory anaerobe will grow in the closed branch only, while 

 the facultative anaerobe will grow in both the open and closed 

 parts. If the organism is a gas producer, the gas will force the 

 cloudy liquid from the closed bulb into the open one, clouding 

 the otherwise clear liquid. To avoid the possibility of error 

 in interpreting these growths it is well to inoculate a tube 

 containing sugar-free bouillon, in which case the liquid in the 

 open bulb should remain clear, as gas will not be formed. 



These tubes are of equal value in testing obligatory and 

 facultative anaerobic organisms. 



