62 MANUAL OF MICROBIOLOGICAL METHODS 



gram-positive contaminants are suspected, add crystal violet in concen- 

 tration of 1 : 700,000. For differentiation of Brucella species on the basis 

 of bacteriostatic action of dyes, add 1 : 100,000 concentration of thionin 

 and 1 : 100,000 concentration of basic fuchsin. Brucella melitensis and 

 B. suis grow in the presence of thionin, but B. abortus in inhibited, 

 whereas on agar containing basic fuchsin, B. suis is inhibited by B. 

 melitensis and B. abortus is not inhibited. 



Media for cultivation of Corynebacterium diphtheriae. Perhaps the 

 most widely used medium for the cultivation of C. diphtheriae, which 

 yields cells of characteristic morphology and metachromatic granule 

 staining reactions, is coagulated blood serum. Although it may be pre- 

 pared from fresh serum (horse, cow, sheep, or pig) by adding 1 vol of 

 1 per cent dextrose broth to 3 vol of serum, users of small quantities of 

 this medium will find the dehydrated product more satisfactory. Special 

 precautions are necessary in the successful sterilization of this medium. 

 It may be sterilized by inspissation or by the following method: Add 

 desired quantity of dehydrated powder to warm (50°C) water, and main- 

 tain this temperature for 45 min during which time stir medium gently to 

 avoid bubbles. Tube in amounts such that a deep butt will not be pro- 

 duced when tube is slanted. Place tubes in slanted position in autoclave 

 equipped with manually operated air-escape valve. Cover tubes with 

 several layers of newspaper, or pack in metal box between layers of non- 

 absorbent cotton. Close door and air-escape valve of autoclave, and 

 admit steam, raising pressure of air and steam mixture quickly to 15 lb. 

 After 20 min open air-escape valve cautiously to permit escape of air 

 while maintaining steam pressure. When all air is removed, close escape 

 valve and continue heating at 121°C for 20 min. Reduce pressure 

 slowly, and open door when pressure is at zero. 



Other media, often containing potassium tellurite (0.03-0.05 per cent), 

 are valuable in the differentiation of this organism. The details of these 

 cannot be condensed here, but those interested should consult the original 

 papers, for example, Kellogg and Wende, 1946; Frobisher et al., 1948; 

 Petran, 1948; Whitley and Damon, 1949; Buck, 1949. 



Media for the cultivation of Neisseria gonorrhoeae. The problems 

 involved in the successful isolation of this organism from samples of 

 clinical material will not be discussed here. For suggestions concerning 

 the media to be used, consult Carpenter et al. (1949). 



Media for the cultivation of Mycobacterium tuberculosis. Many 

 media have been devised for the cultivation of this organism from sputum, 

 urine, pleural exudates, gastric contents, etc. The media of Dorset, 

 Loewenstein, Petragnani, Petroff, etc., have been used widely; descrip- 

 tions of such media are available in manuals for laboratory clinical 

 pathology, particularly Gradwohl (1948). Recently, Dubos and his 



