METHODS OF PURE CULTURE STUDY 125 



to 125 parts water) . Add a few drops of each of these reagents 

 to 3 to 5 cc. of the culture to be tested. A distinct pink or red 

 indicates the presence of nitrite. Always test a sterile check 

 that has been kept under the same conditions, to guard against 

 errors due to the absorption of nitrite from the air. 



Gas is rarely produced from nitrates, and when produced is 

 generally to be observed as bubbles in an open test tube. If 

 its presence is suspected, however, the culture should be tested 

 in a fermentation tube. 



In interpreting results, it must be remembered that the 

 absence of nitrite in a culture showing poor growth does not 

 indicate that the organism cannot reduce nitrates. If nitrate 

 reduction is observed in any medium whatever, the organism is 

 to be recorded as a nitrate-reducer; but unless the routine for- 

 mula is used, the exact composition of the medium must always 

 be given. 



Chromogenesis. Color production should be recorded if ob- 

 served in broth, on beef-extract agar, gelatin or potato, or 

 if noticed to a striking extent on any other medium. In the 

 group number, the point devoted to chromogenesis refers to the 

 color produced on beef-extract agar. 



Diastatic action on starch. Provisional method. Use beef- 

 extract agar containing 0.2 per cent of soluble starch. Pour 

 into a petri dish, and after hardening make a streak inoculation 

 on its surface. Incubate at 37° for organisms of series I and 

 III, at 25° for organisms of series II and IV. Determinations 

 for the group number shall be based upon results obtained on 

 the seventh day. To make the test, flood the surface of the 

 petri dishes with a saturated solution of iodine in 50 per cent 

 alcohol. The breadth of the clear zone outside of the area of 

 growth indicates the extent of diastatic action. If over 2 mm. 

 in width on the seventh day it shall be recorded as ''strong;" 

 if under 2 mm. as ''feeble;" if no clear zone is present, as 

 "absent." 



This method is advised by P. W. Allen (1918) and, although it 

 is the best method yet called to the attention of the committee, 

 it is recommended only for provisional use. It requires some 



