and Laboratory Methods. 



1695 



3. By absorption of oxygen. 



4. By reduction of oxygen. 



5. By exclusion of atmospheric oxygen by means of various physical 

 principles and mechanical devices. 



6. By the combined application of any two or more of the above princi- 

 ples. 



METHODS FOR CULTIVATING ANAEROBIC BACTERIA IN A VACUUM. 



In the search for methods that would enable the investigator to obtain cul- 

 tures in the absence of oxygen, the principle of exhaustion was naturally one of 

 the first resorted to. It was much used in early research concerning anaerobic 

 bacteria. While absolute anaerobic conditions can be obtained in a vacuum 

 only, the difficulty of successful evacuation and the fact that a good vacuum 

 pump generally lies beyond the means of small laboratories, explain the reason 

 why the principle of exhaustion has been losing ground in recent years and is 

 gradually being replaced by more simple and less expensive means to produce 

 anaerobic conditions. 



Pasteur's Method. — Use a small flask with a long neck or test tube ; fill it 

 one-half (in case of flask) or one-third (in case of test tube) with the inoculated 

 medium. Constrict the neck at b (Fig. 1) and connect the end 

 of the neck c with the vacuum pump. While evacuating place 

 the bulb of the flask or the tube in a water bath at 37° C, caus- 

 ing the liquid to boil. Evacuate for thirty minutes; then, while 

 still exhausting, seal at b in the flame. 



According to Fitz this is the oldest and simplest method ; it 

 was introduced by Pasteur and tested by Cohn, Lister, Tyndall, 

 Aitken, and Fitz. Nenki introduced a method identical with the 

 above. 



Roux, in 1887, modified Pasteur's apparatus as shown in Fig. 2. 

 This type of apparatus has been in constant use in Pasteur's 

 laboratory for many years. The bulbs a and a' hold the medium, which 

 is introduced through the lateral capillary tubes b and b' by means of 

 suction at c. 



Method. — Push a loose cotton plug into tube 

 c. Seal the lateral tubes b and b' in the flame 

 and sterilize the apparatus in the hot eir sterilizer. 

 Break off the tips on the lateral tubes b and b' 

 and immerse the tubes in bouillon. Apply suction 

 at c. When the tubes a and a' are about one- 

 third full seal the ends of the lateral tubes again 

 and sterilize in steam sterilizer. When cool reopen 

 the lateral tubes and introduce the inoculating mate- 

 rial by suction at c. Melt off the lateral tubes in 

 the flame and connect c with a vacuum pump. 

 When completely evacuated seal tube c at its con- 

 striction and remove the apparatus to the incubator. y\g. 



Fig. I. 



