154 MANUAL OF MICROBIOLOGICAL METHODS 



Presence of nitrite shows the nitrate to have been reduced, and the 

 presence of gas is a strong indication that reduction has taken place. 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 question does make good growth by means of the 

 following modifications: increasing or decreasing the amount of peptone, changing the 

 amount of nitrate, altering the reaction, adding some readily available carbohydrate, 

 adding 0.1-0.5 per cent of agar to a liquid m.edium to furnish a semisolid substrate. 

 The appearance of nitrite in any nitrate medium whatever (while it is absent in a 

 sterile control) should be recorded as nitrate reduction. 



Absence of nitrite in the presence of good growth may indicate complete consump- 

 tion of nitrate or its decomposition beyond the nitrite stage as well as no reduction at 

 all. Test, therefore, for nitrate by adding a pinch of zinc dust to the tube to which the 

 nitrite reagents have been introduced and allowing it to stand a few minutes. If 

 nitrate is present, it will be reduced to nitrite and show the characteristic pink color. 

 Confirmation of the test may be obtained by placing a crystal of diphenylamine in a 

 drop of concentrated sulfuric acid in a depression in a porcelain spot plate and touching 

 with a drop of the culture (or of the liquid at the base of the slant if agar cultures are 

 used). The test will be more delicate if the culture is first mixed with concentrated 

 sulfuric acid and allowed to cool. A blue color indicates presence of nitrate, provided 

 nitrite is absent, but as nitrite gives the same color with diphenylamine, this test must 

 not be used when nitrite is present in the same or greater order of magnitude. 



If none of these tests indicate utilization of the nitrate, the organism 

 probably does not reduce nitrate, but to be certain of the fact further 

 investigation is necessary. It must be understood, however, that for 

 routine diagnostic work a determination of nitrite on standard nitrate 

 broth or agar is ordinarily sufficient; this is because most descriptions in 

 the literature containing the words "Nitrates not reduced" mean merely 

 that no nitrite is produced on this medium. But in recording such results 

 the student shotdd he careful to state only the observed fact, i.e., that nitrite is 

 or is not found in the nitrate medium employed. 



CHROMOGENESIS 



Color production should be recorded if observed in broth or on beef- 

 extract agar, gelatin or potato or if noticed to a striking extent on any 

 other medium (e.g., starch media). In the margin the space devoted to 

 chromogenesis refers to the color produced on beef-extract agar. This is 

 merely because chromogenesis on such a medium is referred to in the 

 literature more frequently than on the other media above mentioned. 

 As a matter of fact, agar containing peptone is a very poor medium for 

 the purpose. With some organisms, synthetic media are necessary to 

 bring out color. 



