THE STUDY OF MICROORGANISMS IN THE SOIL 123 



Incubate for two weeks at 28°C. 



At the end of this time remove a sample for nitrate determina- 

 tion and dry the remainder for total nitrogen analysis. Use the 

 modified Kjeldahl method to include nitrates (see p. 67). 



From these results calculate the percentage of the nitrogen 

 denitrified, and note the effect of excessive moisture and excessive 

 organic matter on the loss of nitrogen. 

 Tabulate results. 



NITRIFICATION 



Exercise 37 



Nitrification in Impure Cultures 



A. Nitrite Formation (Qualitative) 



(a) Prepare five 150-cubic centimeter Erlenmeyer flasks with 

 20-cubic centimeter portions each of "ammonia" solution 

 (Medium 48). 



(5) Inoculate two of the flasks and incubate at 28°C. 



1. Add approximately 0.1 gram of field soil. 



2. Add approximately 0.1 gram of garden soil. 



(c) At regular intervals of 7 to 10 days remove, with a sterilized 

 platinum needle or pipette, 1 drop of the solution from each 

 flask and test as follows : 



1. Nitrites — Trommsdorf's reagent. 



Place 3 drops of Trommsdorf's reagent in a depression of a 

 spot plate. Add 1 drop of dilute sulphuric acid (1:3). Remove 

 a loopful of the solution to be tested and touch to surface of 

 reagent. A blue color indicates the presence of nitrites. 



2. Ammonia — Nessler's reagent. 



Place in a depression on a spot plate 1 drop of Nessler's solu- 

 tion. Remove a loopful of the solution to be tested and touch to 

 surface of reagent. Do not stir. A yellow-brown color indicates 

 ammonia. 



(d) As soon as the culture shows the presence of nitrites and 

 absence of ammonia, make subinoculations into a sterile flask of 

 the same medium. If it is desired to study the nitrite bacteria 

 in enrichment cultures, repeated subinoculations may be made. 



B. Nitrate Formation (Qualitative). — Prepare five 150-cubic 

 centimeter Erlenmeyer flasks with 20-cubic centimeter por- 

 tions each of nitrite solution (Medium 51). 



