CULTURES OF ANAEROBIC BACTERIA 89 
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 1% glucose, and 2 fermentation tubes, 
one containing sugar-free bouillon, the other 1% glucose 
bouillon from a culture of 2. ée/ani (furnished). 
Read carefully in the text-books the methods for cultivating 
anaérobic bacteria. 
135. 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 culture 
of an anaérobic 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. 
136. The fermentation tubes for cultivating anaérobic bac- 
teria. If these tubes of bouillon have been properly sterilized, 
the closed branch is practically free from atmosphere. The 
obligatory anaérobe will grow in the closed branch-only, while 
the facultative anaérobe 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 aed and 
facultative anaérobic organisms. 
