FURTHER BIOCHEMICAL METHODS vi,.-15 



sibly to all three. Apparently reduction often accompanies or is pre- 

 liminary to utilization of nitrate by bacteria, and confusion often 

 arises in not distinguishing between the two processes. Thus, if an 

 organism is furnished with either nitrate or nitrite and that com|)ound 

 disappears, one sometimes finds the statement made that it has been 

 reduced, when it may well have been utilized without reduction. 



Before beginning the study of any organism in regard to this point, 

 one must first decide the object of his study — whether he merely 

 wishes to make a test for diagnostic purpo.ses in identifying his cul- 

 ture with some published description, or desires to know just what 

 the organism actually does to nitrate. In the former case he must 

 remember that if an organism has been described in the literature as 

 reducing or not reducing nitrate, such a statement ordinarily means 

 that it does or does not produce nitrite in a nitrate medium. For 

 diagnostic purposes, therefore, one can ordinarily get along with a 

 nitrite determination alone, if the test is made under the proper con- 

 ditions and is properly interpreted. This use of the nitrate reduction 

 test for diagnostic purposes alone is discussed in Leaflet V (page V44-9). 



If, on the other hand, the investigator desires to know the real 

 action of his culture on nitrate, a series of tests is often needed, since a 

 negative result is meaningless unless supported by evidence from 

 other tests. In case of a negative nitrite test, several possibilities are 

 to be considered: 1) nitrite may be demonstrable if some other nitrate 

 medium is used^; 2) nitrate may be utilized by the bacteria without 

 reduction; 3) nitrates may be reduced to ammonia or free nitrogen 

 without accumulation of nitrite in detectable quantit}^; 4) no action 

 on the nitrate may have occurred. Methods for determining which of 

 these explanations applies have been recently discussed by Conn 

 (1936). To make this determination often means a small research 

 problem in the case of any organism under investigation. Tests called 

 for in such an investigation are as follows: 



Qualitative tests for nitrate: (good only in the absence of nitrite.) 

 Zinc dust test: See Leaflet V, p. V44-IO. 

 Diphenylamine test: See idem. 



Quantitative test for nitrate: This is necessary if it is desired to 

 know whether the nitrate has been partially consumed even tho no 

 end-products can be detected. For details of procedure see Methods 

 of Analysis of the A.O.A.C. (1934) Chapter XXXVH, Sec. 16 and 17. 



Qualitative test for ammonia: (Significant only if the organism has 

 been growing on a synthetic medium with no nitrogen source other 

 than the nitrate.) The Thomas test as employed by Hucker and Wall 

 (1922) may be employed; but the modification of this test described 

 by Hansen (1930), using hypobromite instead of hypochlorite, seems 

 to be more reliable. 



Test for nitrite consumption: In instances when it is suspected 

 that nitrite may be consumed as rapidly as it is formed from the 

 nitrate, Bronfenbrenner and Schlesinger (1920) inoculate the organ- 

 isms in question into a medium containing only 2 p. p.m. of potassium 



'Thus ZoBell (1932) finds semi-solid agar (0.3%) invaluable in studying nitrate re- 

 duction of the Brucella and Salmonella groups. 



