124 LABORATORY MANAUL OF MICROBIOLOGY 



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. 



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

 platinum needle, 1 drop of the solution from each flask and test 

 as follows: 



1. Absence of nitrites — Trommsdorf's reagent. 



2. Presence of nitrates — Diphenylamine reagent. 



Place in a depression on a spot plate a drop of concentrated 

 sulphuric acid plus diphenylamine. Touch with a drop of the 

 solution to be tested. A deep-blue color indicates nitrates. 

 This test cannot be made in the presence of nitrites, chloric, and 

 selenic acids, ferric chloride, and many other oxidizing agents. 

 If the test for nitrites (Trommsdorf) is positive do not make this 

 test. 



As soon as the culture shows the presence of nitrates and 

 absence of nitrites, make loop subinoculations into a sterile flask 

 of the same medium. If it is desirable to study the nitrate bac- 

 teria in enrichment cultures, repeated subinoculations may be 

 made. 



Exercise 38 



Nitrification in Liquid Cultures (Quantitative) 



Prepare two flasks (750-cubic centimeter capacity) with 

 100 cubic centimeters each of Medium 52. 



Inoculate each flask with 1 cubic centimeter of enrichment 

 culture — ammonia oxidizing — and 1 cubic centimeter of nitrite 

 oxidizing culture. 



Incubate the flasks at 28°C. 



Once a week test the solution for the oxidation of ammonia and 

 nitrite. If this process is complete, add 1 cubic centimeter of a 

 10 per cent solution of ammonium sulphate. Repeat until the 

 oxidation of the ammonium sulphate ceases. If necessary, add a 

 small amount of magnesium carbonate to the culture. 



After the cultures cease to oxidize, make qualitative tests for 

 ammonia, nitrite, and nitrate. If nitrates are present, make 

 quantitative analyses by the phenoldisulphonic method as given 

 on page 63. 



