56 MANUAL OF MICROBIOLOGICAL METHODS 



yolk of one and the whites of two fresh eggs (preferably in a Waring 

 blender). Add 500 ml of distilled water, and adjust pH to 7.6. Stir 

 well, or mix in blender. Add 1 part of above to 5 parts of nutrient 

 broth, tube in deep columns, and sterilize for 20 min at 121°C. The final 

 medium should be an opaque, whitish liquid. During growth proteolysis 

 is indicated by progressive clearing of the medium. 



Nitrate broth. Add 0.1 per cent of KNO3 to a nutrient broth or agar. 

 For obligate anaerobes, also add 0.1 per cent of glucose and 0.1 per cent of 

 agar to basal medium and tube in deep columns. For organisms not 

 reducing nitrates in a peptone medium the following synthetic medium of 

 Dimmick (1947) is recommended: K2HPO4, 0.5 g; NaCl, 0.5 g; MgS04' 

 7H2O, 0.2 g; NaNOs, 2.0 g; glucose, 10.0 g; agar, 15 g; distilled water 

 1,000 ml. If the organism being studied requires more calcium, add 

 0.05 g of CaCl2 to the above; in this case it is important that the final pH 

 be 7.2; to assure this after sterilization, adjustment before sterilization 

 should be to about 7.8. 



Indole determination. Use 1 per cent concentration of a peptone high 

 in tryptophane, such as those prepared by enzymatic digestion of casein 

 or lactalbumin (see Chap. VII for methods of testing for indole). 



H2S production. If the lead acetate paper tests recommended in 

 Chap. VII are not desired, use dehydrated media or consult the original 

 papers of Vaughn and Levine (1936), Hunter and Crecelius (1938), and 

 Untermohlen and Georgi (1940) for directions for preparation of media 

 containing lead, bismuth, or iron salts which will precipitate as the 

 sulfides in the presence of II2S. 



Methyl red and Voges-Proskauer reaction. Prepare basal medium 

 for these tests as follows: peptone, 7.0 g; glucose, 5.0 g; K2HPO4, 5.0 g. 

 Since all peptones are not suitable, use for peptone an enzymatic digest of 

 casein. For details concerning these reactions consult Chap. VII. 



Medium for determination of utilization of citrate. Within the gram- 

 negative nonsporef orming bacilli, differentiation of some types is based on 

 utilization of citrate as the sole source of carbon. Koser's synthetic 

 medium is suitable for this and is prepared as follows: KH2PO4, 1.0 g; 

 MgS04, 0.2 g; NaNH4P04, 1.5 g; NaaCeHBOy, 3.0 g; water, distilled, 

 1 liter. Growth, as evidenced by turbidity, in the water-clear medium 

 indicates utilization of the citrate radical as a carbon source. One should 

 make certain that a small enough inoculum is used in this medium so that 

 it is not noticeably turbid before incubation. This can be accompHshed 

 by using a straight needle for inoculation or by using a loopful of a sus- 

 pension in sterile water. 



Litmus milk. Prepare saturated aqueous solution of litmus. Add a 

 sufficient quantity of the solution to give a light lavender color to fresh 

 skimmed milk (some grades of dried milk powder may be substituted, but 



