130 MANUAL OF BACTERIOLOGY. 



sulphuric acid and one c.c. of a o.oi per cent, solution of 

 sodium nitrite are added to the culture after it has been allowed 

 to grow for this length of time, and after this addition the 

 culture is observed at the end of one-half hour. The ap- 

 pearance of a pink color indicates the presence of indol. 



Another method, and quite a delicate one, after adding the 

 sodium nitrite, is to run in a layer of sulphuric acid beneath the 

 culture so as to form a layer of acid below and culture above. 

 The presence of indol is indicated by a pink ring at the point 

 of juncture of the acid and the culture. If the reaction is 

 obtained with sulphuric acid alone without the addition of the 

 nitrite, it indicates that both nitrites and indol are present in 

 the culture. Blank tests must be made with the same culture- 

 medium which is employed for the cultures, since, as was 

 shown by Wherry,* nitrates and probably also nitrites may 

 gain entrance to artificial culture, media from various sources, 

 such as the water, peptone and filter-paper used in preparing 

 the media. Wherry also showed that a sufficient quantity of i 

 nitrites may be absorbed from the air of the laboratory in a 

 few days to yield a distinct indol reaction. 



Rivasf found that a much more delicate test than the above 

 consists in the use of i c.c. of a 10 per cent, solution of 

 sodium hydroxide in place of the sodium nitrite, and the use 

 of i c.c: of 50 per cent, sulphuric acid. These are mixed 

 with the culture to be tested. This test gives a bright purple 

 or pinkish coloration with outlines of B. coli, but not with its 

 congeners. 



Check blank tests are especially demanded where the culture- 

 medium has been prepared by growing Bacillus coli in the 

 meat infusion to free it from sugar, since Bacillus coli itself 

 forms indol in appreciable amounts. 



The Committee on Methods of Identification of Bacterial; 



*Wherry. Journal of Infectious Diseases. Vol. 2, 1905, pp. 436-44^. 

 \Journ~ Infec. Dis. Vol. IV., No. 4, Nov. 15, 1907. pp. 642-646.' 



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