ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 43 
Test for nitrites by mixing on a spot plate three drops of Trommsdorf’s 
solution and two drops of sulphuric acid solution (1 part concentrated sulphuric 
acid to 3 parts water). Touch with a loopful of the culture. 
EXERCISE 60 
NITRIFICATION: NITRATE FORMATION 
Inoculate a flask of the medium furnished with about 0.1 gram of soil and 
incubate at laboratory temperature. 
The composition of this medium is as follows. 
Sodium nitrite (Na NO,) 1.0 gram 
Dibasic potassium phosphate (K,HPO,) --.-----.----------------- 1.0 gram 
Magnesium sulphate (Mg SO,) 0.3 gram 
Sodium carbonate (Na,CO,) 1.0 gram 
Sodium chloride (NaCl) 0.5 gram 
Ferrous sulphate (Fe SO,) 0.4 gram 
Water 1000.0 grams 
Test the flask each week for nitrites until the test fails due to the complete 
oxidation to nitrates. When a test is no longer obtained for nitrites test the cul- 
ture for nitrates. Since the test for nitrates also reacts for nitrites it can only be 
used in the absence of nitrites. 
The test for nitrates is made as follows: On a spot plate put one drop of 
diphenylamine reagent and two drops of concentrated sulphuric acid. Touch 
with a loopful of the culture. A positive test for nitrates will be shown by a 
dark blue color. 
Diphenylamine reagent 
To 28.8 c.c. of distilled water add 60 c.c. of concentrated sulphuric acid and 
0.7 gram of diphenylamine. Allow the solution to cool. Add slowly 11.3 c.c. of 
concentrated hydrochloric acid. 
EXERCISE 61 
DENITRIFICATION 
Inoculate a fermentation tube of nitrate broth with about 0.1 gram of soil. In- 
oculate another tube with a pure culture of Pseudomonas pyocyaneous. Incubate 
at 30° C. The nitrate broth is the same as ordinary nutrient broth with the addi- 
tion of one per cent of Na NO,. 
Determine the composition of the gas produced. If the gas is not absorbed 
by NaOH and does not form an explosive mixture with air, it is probably 
nitrogen. 
