THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 121 



anaerobes usually are catalase-negative. For this reaction a plate culture 

 of the organism in question is flooded with a 10 per cent solution of 

 H2O2. The evolution of gas bubbles from the colonies denotes the 

 presence of catalase. 



If the proper material for the catalase reaction is not available or in 

 doubt, the following technic will usually suffice to characterize an anaer- 

 obic strain and to differentiate it from the aerobes: Inoculate, while the 

 agar is molten, several deep tubes (8- to 9-cm columns of medium) of a 

 suitable nutrient agar medium containing 1.0 per cent glucose; allow these 

 to solidify in an upright position and incubate the tubes at several tem- 

 peratures or at the optimum temperature for the organism in question; 

 adjust the seeding so that relatively few (e.g., 25-50) colonies per tube 

 will result. With an obligate anaerobe, all the colonies should be local- 

 ized in the bottom of the tube and none should appear on the surface or 

 in the upper 1-cm layer. Likewise, with pathogenic organisms cultured 

 in fluid thioglycollate medium, the growth should be confined to the 

 lower section of the medium and no growth should result in the upper 

 layer wherein the methylene blue is recolorized. If growth does occur in 

 the upper layer of either medium, the culture is not an obligate anaerobe 

 or is contaminated with an aerobic or a facultative species. In the case 

 of clinical cultures, in which speed is important, two blood agar or egg- 

 yolk plates (McClung and Toabe, 1947) may be inoculated and incubated 

 in parallel, one anaerobically, the other aerobically. The appearance of 

 growth in only the anaerobic environment is evidence of presence of an 

 obligate anaerobe. 



ANAEROBIC CULTURE METHODS AND EQUIPMENT 



All the procedures which have been devised for the cultivation of 

 anaerobic bacteria have the single purpose of excluding atmospheric 

 oxygen from the environment in which the growth is to take place. With 

 certain tubed media the oxygen potential may be reduced sufficiently by 

 constituents of the medium to permit anaerobic growth (Brewer, 1940; 

 Hewitt, 1950; Knight, 1931; Reed and Orr, 1943; and Holland, 1944). 

 Since this is rarely possible for -surface cultures on a solid medium, 

 usually plate and slant cultures are incubated within a closed container 

 from which the oxygen is removed by one or another means. A study of 

 the various methods shows that no single procedure may be proposed 

 as the best technic but the method of choice will depend upon the pre- 

 vailing circumstances. A procedure which is ideal for one situation may 

 be impractical or impossible to apply with other conditions. Each of the 

 technics outlined below is recommended within the limits proposed in the 

 discussions. 



