REDUCTION OF NITRATES TO NITRITES 55 



Exercise 19 

 Isolation of Denitrifying Organisms 



1. Fill five test-tubes about two-thirds full of denitrifying 

 solution (m. 33). 



2. Inoculate as follows: 



(a) Uninoculated. 



(b) Inoculated with approximately o.i gram of garden soil. 



(c) Inoculated with approximately o.i gram of fresh manure. 



3. Incubate at 28° C. until aU nitrates have disappeared. 

 The destruction of nitrates is generally indicated by foam- 



4. At regular intervals, daily if possible, make qualitative 

 tests (spot plate) for the presence of nitrates, nitrites, and 

 ammonia. 



5. As soon as the nitrates are destroyed transfer a loopful 

 of the old culture to a new tube of denitrifying solution. 

 This may be repeated several times, although a pure 

 culture is readily isolated from the second transfer. 



6. Follow the same method of isolation as given in the 

 previous exercises. Pour plates of denitrifying agar, and 

 incubate them until the plates show a good growth. 



7. Now pick off several isolated colonies, making trans- 

 fers into tubes of sterile denitrifying solution. 



8. From the pure culture showing the most vigorous 

 denitrifi-cation make a transfer to denitrifying agar. Pre- 

 serve this pure culture for later study. 



Exercise 20 

 Reduction of Nitrates to Nitrites 



1. Prepare four tubes of starch nitrate agar (m. 35). 



2. Dilute two soil t3^es with sterile water until i c.c. 

 represents from 10 to 50 organisms. 



