138 CONN, HARDING, KLIGLER, FROST, PRUCHA AND ATKINS 



to those listed in table 1, differing only in that brom cresol purple 

 is used instead of brom thymol blue to show " neutrality" and 

 that the curdling point (pH=4.7) is used to separate between 

 "moderate" and " strong" acidity instead of the less definite 

 point of maximum red to methyl red. The same methods of 

 expression used in recording acidity in clear media should be 

 used in recording that of milk. 



Litmus milk often gives valuable information, showing not 

 only the production of acid, but also decolorization of the litmus 

 by organisms that are able to reduce it. More accurate results 

 as to acidity can be obtained by using brom cresol purple, as 

 shown by Clark and Lubs (1917 b). This indicator, however, 

 does not show the reduction phenomena which are sometimes 

 of diagnostic value in litmus milk cultures; its substitution for 

 litmus is not, therefore, always to be recommended. 



Reduction of nitrates. The following procedure is recom- 

 mended: Inoculate first into nitrate broth and onto slants of 

 nitrate agar, the media having the composition given on p. 128. 

 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 nitrite with the following 

 reagents : 



(1) Dissolve 8 grams sulphanilic acid in 1 litre of 5 N acetic 

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

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

 water) . 



(2) Dissolve 5 grams a-naphthylamine in 1 litre 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 conditions, 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 : 



