ROUTINE TESTS FOR THE IDENTIFICATION OF BACTERIA 155 



Minor variations in pigmentations when a culture is grown under 

 different conditions should not be given too much emphasis, as they often 

 merely reflect ability to grow on the medium in question. Some species, 

 however (cf. Harrison, 1929), do show qualitative differences when the 

 environment is changed, and such should be noted. Similarly, differ- 

 ences in the presence or absence of air should be recorded. Frequently it 

 is well to note the final H-ion concentration of the culture, as some pig- 

 ments vary in their hue with reaction of the substrate. Cultures should 

 be allowed to develop completely before recording chromogenesis, not 

 making a final reading till after several days at room temperature. Fre- 

 quently pigmentation deepens when a culture is held in diffuse light. 



INDOLE PRODUCTION 



During the last 40 years, results of investigations on the indole test 

 have been published by Zipfel (1912), Frieber (1921), Fellers and Clough 

 (1925), Gore (1921), Holman and Gonzales (1923), Koser and Gait (1926), 

 and Kovacs (1928). The two important points brought out in these 

 papers are that the medium be of correct composition and that the test 

 used be specific for indole. 



The important consideration in regard to the medium is that a peptone 

 be employed containing tryptophane, which is not always present in 

 bacteriologic peptones. Peptones are ordinarily digests of lean meat, but 

 for the indole test a casein digest which contains tryptophane is appar- 

 ently more satisfactory. 



The medium used should, therefore, contain 1.0 per cent of casein 

 digest. No other ingredients need be added if the organism under 

 investigation w^ll grow in a solution of it alone. If the organism is not 

 able to grow in such a medium, add such ingredients as are needed to 

 assure its growth. If necessary, add agar and perform the test on agar 

 slants. 



If the organism produces good growth, 1-2 days' incubation is ordi- 

 narily sufficient. In fact, ^\dth rapid-growing organisms, the reaction 

 may be positive in 24 hr but negative the following day. Therefore both 

 24- and 48-hr tests are recommended. Arnold and Weaver (1948), how- 

 ever, with confirmation by Clarke and Cowan, (1952) show that 6-120 

 min incubation is enough by the following ''quick" method: 



The culture is grown on tryptone agar at optimum temperature till 

 good growth is obtained ; then heavy inoculation (to give high turbidity) 

 is made into preheated (to 37°C) tryptophane broth, 1 ml to the tube. 

 It is incubated at 37° for not over 2 hr, the test made with the Kovdcs 

 reagent, as given below. 



