NITRIFICATION IN SOLUTION 49 



3. Incubate at 28*^ C. 



4. At regular intervals of four and six weeks remove, 

 with a sterilized platinum needle, i drop of the solution 

 from each flask and test as follows: 



(a) Absence of nitrites — ^Trommsdorf's reagent (see page 136). 



(b) Presence of nitrates — Diphenylamin reagent (see page 137) 



Use the spot plate for this test. Record the date and 

 results of tests in the table below. 



5. 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. 



6. If it is desirable to study the nitrate bacteria in en- 

 richment cultures, repeated subinoculations may be made. 



C. Nitrification Quantitatm: 



1. Place loo-c c. portions of medium 29 into eight i-liter 

 Erlenmeyer flasks. 



2. Inoculate each flask with i-c.c. portions of water 

 extract of different soils. 



Note. — Shake 50 grams of soil with 100 c.c. of sterile water and allow 

 to settle. 



(a) I, 2, 3, and 4, field soil. 



(b) 5, 6, 7, and 8, garden soil. 



3. Immediately after inoculation add 5 c.c. of con- 

 centrated sulphuric acid to flasks numbered i, 2, 5, and 6. 



4. Incubate all of the flasks at 28° C. for three to four 

 weeks. 



5. At the end of this time determine the amount of 

 nitrate nitrogen (see page X43). 



6. Tabulate results as shown on p. 50, 



