42 ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 
SECTION 15: THE NITROGEN CYCLE IN NATURE 
EXERCISE 58 
AMMONIFICATION 
Treat five tubes of’a one per cent peptone solution as follows: 
(1) Leave one tube as a sterile control. 
(2) Inoculate with a loopful of soil. 
(3) Inoculate with Bacillus mycoides. 
(4) Inoculate with Pseudomonas fluorescens. 
(5) Inoculate with Proteus vulgaris. 
Incubate these tubes at 30° C. 
At the end of 48 hours test the tubes for the presence of ammonia. Place a 
drop of Nessler’s reagent on a spot plate; touch this with a loopful of the sub- 
stance under test. The presence of ammonia is indicated by a yellow color. 
Nessler’s Reagent . 
To 50 grams of potassium iodide dissolved in distilled water add a satur- 
ated solution of mercuric chloride until a slight precipitate remains. Add 400 
c.c of a 50 per cent solution of sodium hydroxide which has been clarified by 
sedimentation. Dilute to 1000 c.c. by adding distilled water, and allow to settle 
for one week before using. Decant the clear solution and keep in well stoppered 
bottles away from light. 
EXERCISE 59 
NITRIFICATION: NITRITE FORMATION 
Inoculate a flask of the medium furnished with about 0.1 gram of soil. 
Incubate at laboratory temperature. 
The medium used for this purpose has the following composition: 
Ammonium sulphate (NH,), SO, 2.0 grams 
Dibasic potassium phosphate (K,HPO,) nsec 1.0 gram 
Magnesium sulphate (Mg SO,) 0.5 gram 
Ferrous sulphate (Fe SO,) 0.4 gram 
Sodium chloride (NaCl) 0.4 gram 
Water 1000.0 grams 
Magnesium carbonate (Mg Co,) Excess 
Test the culture every week for ammonia and nitrites. A test for nitrites 
should appear after a week or two and after several weeks the test for ammonia 
should fail owing to the complete oxidation of the ammonia present to nitrites. 
The test for ammonia may be made with Nessler’s solution by placing a drop 
of the solution on a spot plate and then touching this with a loopful of the 
culture. 
