50 MANUAL OF MICROBIOLOGICAL METHODS 



10 mg; FeS04-7H20, 5 mg; MnS04-4H20, 2.5 mg; NaMo04-2H20, 



2.5 mg; biotin, 3 jug; p-aminobenzoic acid, 50 /xg; sodium thiogly collate, 

 0.5 g; distilled water, 1 liter. The thiogly collate may be replaced by 

 0.2 g of Na2S-9H20 which is best added after steriUzation of the medium; 

 the growth factors may be replaced by 0.5-1.0 g of yeast extract. The 

 procedure is : Add heavy inoculum (5 per cent of black mud from fresh- 

 water or marine sources) and incubate culture at 35° C in completely 

 filled glass-stoppered bottles in order to exclude oxygen. To isolate pure 

 cultures, use a solid medium of the same composition in an anaerobic jar. 

 Use of bacteriostasis. A principle sometimes invoked in the enrich- 

 ment culture of a particular type of organism from a mixed population 

 is to utilize the inhibitory (bacteriostatic) property of a specific chemical 

 without which the medium would be suitable for many species in the 

 sample. For example, addition of crystal violet in a final concentration 

 of 1 : 100,000 will inhibit most gram-positive types without affecting the 

 gram-negative group. Similarly, the antibiotics penicillin and strepto- 

 mycin may be used for selective inhibition. Sodium azide (0.03 per cent) 

 has bacteriostatic action for gram-negative bacteria, the aerobic gram- 

 positive sporeforming bacilli, and certain other aerobes (Lichstein and 

 Soule, 1944). 



DIFFERENTIAL MEDIA 



The formulas presented in this section include media employed, com- 

 monly for original isolation, to determine differential reactions which may 

 permit presumptive identification of bacterial species. In some instances 

 the media constitute selective growth environments, often as a result of 

 the inclusion of specific compounds which inhibit the growth of those 

 organisms not under investigation. 



Blood agar. This medium is used frequently in the study of strep- 

 tococci and other groups exhibiting hemolytic properties. The medium 

 is valuable also in the routine cultivation of fastidious pathogenic species 

 which may or may not show hemolytic reactions. To prepare medium: 

 Add aseptically 5 per cent of sterile rabbit, sheep, horse, or human blood 

 to a satisfactory carbohydrate-free agar base medium containing 0.5- 

 0.85 per cent of NaCl (to maintain isotonicity). Blood agar seeded with 

 a light inoculum yields more typical hemolytic reactions than do heavily 

 streaked plates. Chocolate agar is prepared as above except that the 

 blood is added to the base medium at 80°C; this causes a distinct darken- 

 ing of the added blood (protein coagulation). 



Egg -yolk agar. This medium is used as a presumptive identification 

 medium for certain pathogenic Clostridia and for sporeforming aerobes. 

 Plates of an agar-base medium to which sterile egg yolk has been added 



