524 H. J. CONN, CHAIRMAN 



REDUCTION OF NITRATES 



The following procedure is recommended: Inoculate first into 

 nitrate broth and on to slants of nitrate agar (containing 0.1 

 per cent KNO3 plus beef extract and peptone as usual). 

 Test the cultures on various days as indicated on the chart. On 

 these days examine first for gas as shown by foam in the broth 

 or by cracks in the agar. Then test for nitrate with the follow- 

 ing reagents. 



1. Dissolve 8 grams sulphanilic acid in 1 hter of 5 N acetic 

 acid (1 part glacial acetic acid to 2.5 parts of water), or in 1 

 liter of dilute sulphuric acid (1 part concentrated acid to 20 

 parts water). 



2. Dissolve 5 grams a-naphthylamine in 1 liter of 5 N acetic 

 acid or of very dilute sulphuric acid (1 part concentrated acid 

 to 125 parts water). 



Put a few drops of each of these reagents in each broth culture 

 to be tested, and on the surface of each agar slant. A distinct 

 pink or red in the broth or agar indicates the presence of nitrite. 

 It is well to test a sterile check which has been kept under the 

 same condition to guard against errors due to absorption of 

 nitrite from the air. Presence of nitrite or of gas shows the 

 nitrate to have been reduced. A negative result does not prove 

 that the organism is unable to reduce nitrates; in such a case 

 further study is necessary as follows: 



In case the fault seems to lie in poor growth, search should 

 be made for a nitrate medium in which the organism in ques- 

 tion does make good growth by means of the following modifica- 

 tions: increasing or decreasing the amount of peptone; altering 

 the reaction; adding some readily available carbohydrate. 

 Presence of nitrite in any nitrate medium whatever should be 

 recorded as nitrate-reduction. Presence of gas should be simi- 

 larly interpreted, provided there is no other substance (e.g., 

 sugar) present in the medium, from which the organism under 

 investigation is able to produce gas. 



In the case of those organisms which are nitrite-negative and 

 produce no gas in nitrate-peptone agar or broth, but w^hich show 



