VI42-16 MANUAL OF METHODS FOR PURE CULTURE STUDY 



nitrite and after incubation apply the regular nitrite test. It is as- 

 sumed that an organism not able to destroy this small amount of 

 nitrite cannot destroy or consume it as rapidly as it may be produced 

 from nitrate. 



The matter of action on nitrates can well be summarized by listing 

 a series of questions that must be answered before this action can be 

 thoroly understood. These questions are: 



1) Does the organism utilize completely 1% of KNO3? In what 

 media? 



2) If not, does it utilize part of the KNO3 furnished? What per- 

 centage? 



3) If either of these qviestions are answered in the affirmative, does 

 the organism actually reduce nitrate? Before answering this question 

 the following subordinate questions must be answered: 



a) Does it produce nitrite or gas in a nitrate medium but not in 

 the same medium without nitrate? 



b) Does it produce ammonia in a nitrate medium containing no 

 source of nitrogen other than the nitrate? 



c) If both these questions are answered in the negative, is the 

 organism able to destroy 2 p. p.m. KNO2 in a medium in which it is 

 being studied? If so, it may still be a nitrate-reducer, the accumu- 

 lation of nitrite being prevented by its action on nitrite. It must not 

 however, be assumed to reduce nitrite as it may utilize it as a source 

 of nitrogen without reduction, or may conceivably convert it to 

 nitrate. 



Recommendations: It must be distinctly understood that the Com- 

 mittee does not recommend making such a study as the above to 

 determine the action on nitrates in securing data for routine descrip- 

 tions of organisms. For such routine purposes the tests given in Leaf- 

 let V are ordinarily sufficient; but it is emphasized that in recording or 

 publishing negative results of such tests one must not make the state- 

 ment "nitrates not reduced". A negative nitrite test should merely be 

 recorded "no nitrite produced from nitrate under the conditions of 

 the experiment". 



The further tests outlined in this Leaflet are to be regarded as re- 

 search methods to be employed when information is desired concern- 

 ing the true action on nitrate of any organism under investigation. 



Action on Erythrocytes 

 Certain organisms during their growth cause a number of changes 

 in the pigment of red blood corpuscles. Some organisms break down 

 the corpuscles, liberating the hemoglobin, due to the action of hemo- 

 lytic substances. Some of these substances are analogous to exotoxins 

 and can be found in the filtrate of broth cultures. Other organisms 

 change the hemoglobin in the cells to methemoglobin or sulfhemo- 

 globin, producing a greenish coloration. While these organisms are 



