(1) At least 10 cultures should be used. If possible these should be subcultured from plates made direct from the water since all of the cultures obtained by plating from fermentation tubes may be descendants of a single cell in the water. If cultures from water plates are not available those obtained from plates made as prescribed under B (p. [101]) may be used.
(2) Inoculate each culture into dextrose potassium phosphate broth,[[H]] adonite broth, and gelatin. For additional confirmatory evidence inoculation may be made into tryptophane broth,[[I]] and saccharose broth. The dextrose broth must be incubated at 30°. Other sugar broths may be incubated at 30° or 37° as convenient. Gelatin should be incubated at 20°.
[H]. (a) Peptone Medium for the Methyl Red Test. To Make One Liter.
1. To 800 cc. of distilled water add 5 grams of Proteose-Peptone, Difco., or Witte’s Peptone (other peptones should not be substituted), 5 grams c. p. dextrose, and 5 grams dipotassium hydrogen phosphate (K2HPO4). A dilute solution of the K2HPO4 should give a distinct pink with phenolphthalein.
2. Heat with occasional stirring over steam for twenty minutes.
3. Filter through folded filter paper, cool to 20° C. and dilute to 1,000 cc. with distilled water.
4. Distribute 10 cc. portions in sterilized test-tubes.
5. Sterilize by the intermittent method for 20 minutes on three successive days.
[I]. Tryptophane Broth for Indol Test.
To 1,000 cc. of distilled water add 0.3 gram tryptophane, 5 grams dipotassium hydrogen phosphate (K2HPO4), and 1 gram peptone. Heat until ingredients are thoroughly dissolved, tube (6 to 8 cc.), and sterilize in autoclave for 15 minutes after the pressure reaches 15 pounds. Some American peptones are standardized to contain a uniform amount of tryptophane. If such peptone is used the tryptophane in the above formula may be omitted and the peptone increased to 5 grams.