METHODS OF PtJRE CULTURE STUDY 527 



a-methjl-indol. The Ehrlioh reagent, however (p-dimethyl- 

 arainobenzaldchyde), is rather too expensive to use in routine 

 laboratory work. For tliis reason tests with other reagents are 

 often preferred, as for example the Vanilin test. The Vanilin 

 test is a very convenient one to make and gives a more nearly 

 true indol reaction than does the Salkowski test. Frieber shows, 

 however, that it gives a positive reaction not only with the com- 

 pounds that give the Ehriich test but also with /3-methyl-indol. 

 In the light of this contribution to the subject the committee 

 suggests the following procedure, pending further investigation 

 of the method: Test all the cultures by the Salkowski reagent: 

 then test the positive culturco further with the Vanihi. test; and 

 lastly test those that are positive to Vanilin with the Ehriich 

 reagent. Record on the chart what reaction is obtained with 

 each test used, because it is very evident that the different 

 reagents indicate the presence of different compounds. 



The Salkowski test is made as follows: Mix 5 cc. of the culture with 

 about one-third its volume of 1:1 sulphuric acid. Then add on the 

 surface a small amount of a 0.02 per cent solution of sodium nitrite. 

 A positive reaction is indicated by a pink zone between the acidified 

 culture fluid and the nitrite solution. 



The Vanilin test is as follows: To 5 cc. of the culture add 5 drops of 

 a 5 per cent solution of vanilin in 95 per cent alcohol and 2 cc. of con- 

 centrated sulfuric acid. Indol gives a clear orange bj' this test which 

 reaches its greatest depth in two or three minutes. Tryptophane, on 

 the other hand, gives a reddish \Tiolet which develops more slowly and 

 deepens on standing or heating. 



The EhrUch test is made as follows: The reagent is a 2 per cent solu- 

 tion of para-dimethylaminobcnzaldehyde in 95 per cent alcohol. One 

 cubic centimeter of this reagent is added to the culture, then drop by 

 drop concentrated hydrochloric acid is added until a red zone appears 

 between the alcohol and peptone solution. Not more than 0.5 cc. of 

 the acid is required. On standing the zone deepens and widens. The 

 red color is soluble in chloroform and the test may be confirmed by 

 shaking the culture with chloroform to see if the pigment dissolves 

 If it proves soluble the test is considered positive. 



PRODUCTION OF HYDROGEN SULFIDE 



The method of determining hydrogen sulfide which seems to 

 be in most common use is the lead acetate agar method. This 



