THE STUDY OF MICROORGANISMS IN THE SOIL 121 



Make moisture determinations on the soil at the time samples 

 are taken for nitrogen analysis. Tabulate results. 



DENITRIFYING BACTERIA 



Exercise 34 



Isolation of Denitrifying Bacteria 



Fill three test tubes about two-thirds full of asparagin nitrate 

 solution (Medium 55). 



Inoculate as follows: 



(a) Control — no inoculation. 



(h) Inoculate with approximately 0.1 gram of garden soil. 



(c) Inoculate with approximately 0.1 gram of fresh manure. 



Incubate at 28°C. until all nitrates have disappeared. The 

 destruction of nitrates is generally indicated by foaming. 



At regular intervals, daily if possible, make qualitative tests 

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



As soon as the nitrates are destroyed, transfer a loopful of the 

 old culture to a new tube of asparagin nitrate solution. This 

 may be repeated several times, although a pure culture is readily 

 isolated from the second transfer. 



Follow the same method of isolation as given in the previous 

 exercises. It is well to make a series of not less than four dilu- 

 tions. Pour plates of asparagin nitrate agar and incubate 

 until there is a good growth. 



Now pick off several isolated colonies, making transfers into 

 tubes of asparagin nitrate solution. 



From the pure culture showing the most vigorous destruction 

 of nitrates make a transfer to the agar medium. Preserve this 

 pure culture for later study. 



Exercise 35 



Denitrification by Pure Cultures of Bacteria 



Prepare four bottles, 125 cubic centimeters each, of asparagin 

 nitrate solution (Medium 55). Because of the total nitrogen 

 analyses to be made at the end of this experiment it is well to 

 prepare the medium with great care. Exact amounts of the 



