DENITRIFICATION BY PURE CULTURES OF BACTERIA 57 



This should be confirmed by qualitative tests with Tromms- 

 dorf's and diphenylamin reagents. 



Methylene-blue Leuco base 



CeHs— N— (CH3)2 CrH,™ N~(CH3)2 



CeHs = N~(CH3)2 C6H3~-N-(CH3)2 



CI 



Exercise 22 

 Denitrification by Pure Cultures of Bacteria 



1. Prepare ten 200-c.c. portions of denitrifying solution 

 (m. 33) in 300-c.c. Erlenmeyer flasks. 



2. Inoculate as follows: 



(a) I and 2, immoculated. 



(5) 3 and 4, pure cultuie of unknown denitrifier from Exercise 19. 



(c) 5 and 6, BaciUus pyocyaneus. 



(d) 7 and 8, Bacillus Hartlebii. 



(e) g and 10, Bacillus coli 



3. Incubate all cultures for two weeks at 28^ C. 



4. At the end of the incubation period make qualitative 

 tests of each culture for ammonia, nitrites, and nitrates. 

 If present, determine the amount according to quantitative 

 methods. 



5. In all of the cultures, inoculated and uninoculated, 

 determine the total nitrogen. Use the modified Kjeldahl 

 method (see page 147). For total nitrogen analysis take 

 duplicate portions of 50 c.c. each of the cultures. 



6. For determining the nitrate nitrogen take lo-c.c. 

 portions of the control, dilute with 500 c.c. of distilled 

 water, and of this evaporate lo-c.c, portions to dryness. 



