CULTIVATION OF ANAEROBIC BACTERIA. 81 
If we make an inoculation of one of the species which is not 
strictly anaérobic 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 stab culture with a strict anaérobic 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 anaérobic bacilli in long stab 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 aérobic bacterium—Bacillus subtilis—upon the surface, after 
making a long stab culture with the anaérobic 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 aérobic 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 aérobic 
species exhausts the oxygen in the upper part of the tube by its 
growth on the surface of the culture medium, and the anaérobic 
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 Pasteuar’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 anaérobic 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 tube is passed into the flask, as shown in Fig. 
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