V49-12 MANUAL OF METHODS FOR PURE CULTURE STUDY 



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), Kulp 

 (1925), Koser and Gait (1926) and Kovacs (1928). The two im- 

 portant 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 tryp- 

 tophane is apparently more satisfactory. 



The medium used should, therefore, contain 1.0% of casein digest. 

 No other ingredients need be added if the organism under investi- 

 gation will 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. K necessary, add agar and perform the test on 

 agar slants. 



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

 ordinarily sufficient. In fact, with rapid-growing organisms, the 

 reaction may be positive in 24 hours, but negative the following day. 

 Therefore both 24-hour and 48-hour tests are recommended. The 

 test for indole may be performed by the technic of Ehrlich-Bohme, by 

 either the Gore or the Kovacs modification of the same, or by the 

 Gnezda technic. The Kovdcs method is especially simple and con- 

 venient. These procedures are as follows: 



Bohme (1905) called for the following solutions: 



Solution 1 



Para-dimethyl-amino-benzaldehyde 1 g. 



Ethyl alcohol (95%) 95 ml. 



Hydrochloric acid, concentrated 20 ml. 



Solution 2 

 Saturated aqueous solution of potassium persulfate (KjSaOg). 



To about 10 ml. of the culture fluid add 5 ml. of solution No. 1, then 5 ml. of solution 

 No. 2, and shake; a red color appearing in five minutes indicates a, positive reaction. 

 This test may also be performed (and sometimes more satisfactorily) by first shaking 

 up the culture with ether and adding solution No. 1 (Ehrlich's reagent) dropping down 

 the side of the tube so that it spreads out as a layer between the ether and the culture 

 fluid. After this method of applying, solution No. 2 seems to be unnecessary. 



The Got6 (1921) test uses these same solutions, but the method of application is as 

 follows: Remove the plug of the culture tube (which must be of white absorbent 

 cotton), moisten it first with four to six drops of solution No. 2, then with the same 



