Journal of Applied Microscopy. 267 



LABORATORY METHODS IN BACTERIOLOGY. 



Dk. F. G. Novy. 

 University of Michigan, Ann Arljor, Michigan. 



VI. — The Cultivation of Anaerobic Bacteria. 



While the majority of bacteria live in the. presence of air, there are some 

 which are not favored by the free access of oxygen. It is customary therefore 

 to designate as obligative anaerobic those bacteria which are obliged to live 

 under anaerobic conditions — that is to say, where there is no air. 



The exclusion of air from the culture medium can be accomplished, more or 

 less satisfactorily, by a variety of methods. In so far as the principles of the 

 methods employed are concerned, these may be spoken of under the following 

 heads : 



1. Exclusion of oxygen. 4. Displacement of air. 



2. Exhaustion of air. 5. Cultures apparently in presence of air. 



3. Absorption of oxygen. 6. Microbic association. 



EXCLUSION OF OXYGEN. 



Several methods can be grouped under this head. Thus, the earliest method 

 employed was to cover the liquid with a layer of oil. Although not perfect, yet 

 this method of Pasteur can at times be employed to advantage. Later on, Koch 

 endeavored to obtain colonies under anaerobic conditions by placing a thin 

 mica sheet on the surface of a gelatin plate. Better results are obtained by 

 covering the gelatin or agar plate with another sterile glass plate (Sanfelice). 



The well-known method of Liborius, culture in deep layers of gelatin or agar, 

 depends upon the exclusion of air. The method is simple and very convenient. 

 The tube should contain glucose agar or gelatin, and the medium should be 

 about two inches deep. Stab cultures are made in the usual manner. Growth 

 develops along the line of inoculation in the lower two-thirds of the medium, 

 while the upper third serves to exclude the air. In order to insure complete 

 exclusion of oxygen, the contents of an ordinary agar or gelatin tube can be 

 liquefied and then, with proper precautions against contamination, poured on 

 top of the inoculated medium and quickly cooled. This extra layer, however, 

 is not necessary if the medium has the depth given above. 



Isolated colonies can be obtained by the method just given. The liquefied 

 glucose agar or gelatin is inoculated in the usual manner as when making plates. , 

 The contents of the tube are then solidified and an extra layer of the medium 

 can be poured on top. 



The method of Roux deserves mention under this head. The inoculated 

 medium is drawn up into a sterile glass tube pipette, which is then sealed at both 

 ends by means of a flame. 



EXHAUSTION OF AIR. 

 Pasteur, in his studies on the bacillus of malignant oedema, employed 

 special tubes which were connected with an air-pump, and when a vacuum was 



