PREPARATION OF MEDIA ii4,-15 



is not sufficient aid for many species. If other actively reducing sul)stances are neces- 

 sary, the following are the most suitable: glucose, sodium thioglycollate (and thiogly- 

 collic acid), sodium formaldehyde sulfoxylate, ascorbic acid, sodium formate, gluta- 

 thione, and cysteine. Glucose seems to be generally satisfactory, but some of the 

 other compounds are toxic for certain types. Methylene blue (1-500,000) may be 

 added to culture media to serve as an O/ll indicator. Obligate anaerobes will grow 

 only in the portion in which the dye remains decolorized following cooling after steriliza- 

 tion. 



The spore-forming anaerobes frequently have been divided into proteolytic and 

 saccharolytic groups. An organism of the former group possesses the ability to de- 

 compose complex proteins, usually with the production of offensive odors, sometimes 

 attacking a small variety of the simpler carbohydrates. The saccharolytic group, on 

 the other hand, usually show little action on complex proteins (except such com- 

 pounds as gelatin), but ferment a wide variety of the carbohydrates, usually with 

 copious production of gas. 



Reference in this Leaflet is also 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. welchii), C. sporogenes, 

 and C. parabotulinum, etc., which grow best in the richer animal tissue infusions and 

 require a high degree of anaerobiosis. Representatives of the butyric-butyl group 

 include C. butyricum, C. beijerinckii, C. butylicum, C. pasteurianum, C. acetobutylicum, 

 C. felsineum, C. roseum, and C thermosaccharolyticum; they are less exacting with re- 

 gard to oxygen exclusion and grow best when supplied a fermentable carbohydrate. 

 Due to the diversity of physiological types within the anaerobic group it will be neces- 

 sary frequently to recommend two or more media for the same purpose. 



All liquid media (except the thioglycollate medium and the semi-solid corn liver 

 medium) should be boiled 10 minutes, or heated in flowing steam for a similar period, 

 immediately prior to inoculation unless the medium is used on the same day it is initially 

 sterilized. The use of vaseline, mineral oil, or other seals at the surface of liquid media 

 is not recommended. If a liquid medium is used which will not remain reduced during 

 the desired incubation period, incubate the tubes in an anaerobic jar (see Leaflet III, 

 ^tk Ed.). 



1. ENRICHMENT AND GENERAL CULTIVATION MEDIA 



Dehydrated Thioglycollate Medium^. This medium (Brewer, 1940a, 

 b) is obtained in dehydrated form from the manufacturers. After 

 dissolving, it is essentially a liquid (the percentage of agar being too 

 small to affect the fluidity) in which sodium thioglycollate acts as a 

 reducing agent. It also contains meat infusion, peptone, NaCl and 

 a phosphate, with or without glucose and methylene blue; for most 

 purposes the presence of these last two ingredients is recommended. 

 The medium compares favorably with other infusion media in ability 

 to initiate growth from small inocula (McClung, 1940, 1943). 



The appropriate amount (indicated on bottle) of the dry powder is 

 dissolved in distilled water by brief heating, tubed or dispensed in 



^Dehydrated thioglycollate medium. Baltimore Biological Laboratory, Baltimore, 

 Maryland, or Difco Laboratories, Detroit, Michigan. If the commercially prepared 

 medium is not available, a satisfactory substitute can be prepared by adding 0.1% 

 agar and 0.1% sodium thioglycollate to a meat infusion base medium. 



