THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 129 



sufficient agar in the original dish so that a perfect seal is formed when the special lid 

 is added. Recommended for routine use in hospital laboratories, and particularly 

 for mobile laboratories, where anaerobic cultures for pathogens may be encountered. 

 Disadvantages. Surface moisture may result in film formation in some instances; this 

 may be reduced by using a porcelain top (see footnote 1 on page 124) on the regular 

 dish prior to the Brewer anaerobic lid or by drying the plates in incubator before 

 streaking. Some organisms apparently are inhibited by the reducing agents. This is 

 not serious, since the reports indicate that all pathogenic types are easily cultured by 

 this method. The Brewer anaerobic lids are, at the present time, relatively expensive. 



TECHNICS FOR STUDY OF ANAEROBIC BACTERIA^ 



In the above section the various pieces of apparatus and methods for 

 their use with anaerobic bacteria have been considered. Formulas for 

 the particular media which are recommended may be found in Chap. III. 

 The remainder of this chapter will be devoted to a discussion of the details 

 of certain technics which should aid the worker who has not had previous 

 experience with anaerobes. 



It may not be amiss to insert here a precautionary note concerning the 

 necessity of very careful inspection of the purity of cultures. There are 

 instances on record, in the older literature, where two species grew sym- 

 biotically on plate culture with such constancy that recorded observations 

 were made of the colony type of mixture, the investigator being unaware 

 of the existence of more than one type. In all studies concerning obligate 

 anaerobes, a check on the purity of the culture should be made with 

 regard to aerobic contaminants. The following test is suggested: For 

 most cultures, streak a glucose nutrient agar slant and incubate it at 

 37°C; but for anaerobic species having a lower or higher optimum tem- 

 perature, incubate a second agar slant at the temperature which is 

 optimum for the anaerobe. If the culture appears free of aerobic types, 

 investigate the purity with respect to anaerobic contaminants. Make 

 repeated platings, and scrutinize intensely the colonies which develop. 

 For cultures which will grow on the egg-yolk agar (with 0.3 to 0.5 per 

 cent glucose added for the butyric group) of McClung and Toabe (1947), 

 contamination in cultures is more readily revealed on this medium than 

 on media not containing egg yolk. 



Preliminary Microscopic Examination 



If the sample is suitable, one should make preliminary examination 

 using the gram stain. The conventional method of staining a smear, heat 



^ In this chapter reference will be made to the "pathogenic group " and the "butyric- 

 butyl group." The former term is used to designate such organisms as Clostridium 

 tetani, C. septicum, C. histolyticum, C. chauvoei, C. perfringens, C. parahotulinum, C. 

 hotulinum, and C. sporogenes. In the butyric-butyl group are included C. butyricum, C, 

 beijerinckii, C. butylicum, C. pasteurianum, C. acetobutylicum, C. felsineum, C. roseum, 

 and C. thermosaccharolyticum. 



