ROUTINE TESTS FOR THE IDENTIFICATION OF BACTERIA 153 



Some bacteriologists prefer to pour or to pipet the inoculated medium into 

 another sterile tube to ensure thorough mixing. 



Upon incubation, strict aerobes will be found to grow upon the surface 

 and in the upper layers only, microaerophiles will grow best just a few 

 millimeters below the surface, facultative anaerobes will grow throughout 

 the medium, and strict anaerobes will grow only in the depths, if at all. 



ACTION ON NITRATES 



Nitrate reduction should be indicated by complete or partial disappear- 

 ance of nitrate accompanied by appearance of nitrite, ammonia, or free 

 nitrogen. As quantitative nitrate tests are too time-consuming for 

 routine pure culture work, one must ordinarily be satisfied with tests for 

 the end products only. 



The following routine procedure is recommended: Inoculate into 

 nitrate both and onto 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 on the broth or by cracks in the agar. Then test for 

 nitrite with the following reagents. 



1. Dissolve 8 g of sulfanilic acid in 1 liter of 5iV acetic acid (1 part of glacial acetic 

 acid to 2.5 parts of water), or in 1 liter of dilute sulfuric acid (1 part of concentrated 

 acid to 20 parts of water). 



2. Dissolve 5 g of a-naphthylamine in 1 liter of 5N^ acetic acid or of very dilute 

 sulfuric acid (1 part of concentrated acid to 125 parts of water). Or dissolve 6 ml of 

 dimethyl-a-naphthylamine in 1 liter of 5N acetic acid. This latter reagent has 

 recently been recommended by Wallace and Neave (1927), and by Tittsler (1930), as 

 it gives a permanent red color in the presence of high concentrations of nitrite. 



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 

 control which has been kept under the same condition to guard against 

 errors due to absorption of nitrous acid from the air. 



A rapid method, calling for less than an hour's incubation, has been 

 devised by Bachmann and Weaver (1947), and a rapid microtechnic by 

 Brough (1950) and by Clarke and Cowan (1952). 



Among the micromethods, Brough's is detailed in a concise, easily followed form. 

 The culture is grown 18-24 hr at optimum temperature on a nutrient agar giving 

 good growth. The growth is washed off and sufficient to give high turbidity is added 

 to 1 ml (in a small test tube) of 0.1 per cent KNO3 in nutrient broth; Clarke and Cowan 

 show that the solution to which the KNO3 is added may be pH 6.8 buffer instead of 

 broth. The medium should be preheated in a water bath to 37°C before inoculation; 

 after inoculation it needs to be incubated only 15 min at that temperature before 

 adding the reagents of the nitrite test. 



