ROUTINE TESTS FOR THE DESCRIPTIVE CHART V49-II 



to test a sterile control which has been kept under the same condition 

 to guard against errors due to absorption of nitrous acid from the air. 

 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 docs 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% agar to a liquid medium to furnish a semi-solid substrate. The ap- 

 pearance of nitrite in any nitrate medium whatever (while it is absent in a sterile con- 

 trol) 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 organ- 

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

 further investigation is necessary as outlined in Leaflet VI. It must 

 be understood, however, that for routine diagnostic work a determi- 

 nation of nitrite on standard nitrate broth or agar is ordinarily suffi- 

 cient; this is because most descriptions in the literature containing the 

 words "Nitrates not reduced" merely mean that no nitrite is pro- 

 duced on this medium. But in recording such results the student 

 should be careful to state only the observed fact, i.e. that nitrite is or is not 

 found in the nitrate medium employed. 



Chbomogenesis 



Color production should be recorded if observed in broth, 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 de- 

 voted to chromogenesis refers to the color j)roduced on beef extract 

 agar. Note difTerences, if any, in pigmentation of growth exposed to 

 air and shielded from air, or in presence or absence of light. Fre- 

 quently it is well to note the final H-ion concentration of the culture, 

 as some pigments act as H-ion indicators. 



