Exercise 17. Nitrification 
MATERIALS: 
2 100 cc. Erlenmeyer flasks 
4 one-fourth pint milk bottles 
Ammonia and nitrite media 
25 cc. graduate 
Soil samples 
Milk bottle caps 
Cotton 
Nitrification includes the oxidization of ammonia (NH„) to nitrites (N o 0 3 ) 
and their subsequent oxidization to nitrates (N 3 0 5 ). The organisms which 
carry out the oxidization of ammonia to nitrites and nitrites to nitrates are 
present in most cultivated soils. Their activity in a given soil may vary with 
the cultivation and cropping of the soil. 
The solutions used for the cultivation of these organisms contain no organic 
matter—only in organic salts. 
A. Nitrite Formation 
In the culture medium for the nitrite-forming organisms, the source of 
nitrogen is ammonium sulphate (NH 4 ) 2 S0 4 and in the beginning thesolution 
contains no nitrites or nitrates. 
Ammonium sulphate (NH 4 ) 2 S0 4 )_ 2.00 grams 
Di-basic potassium phosphate (K,HP0 4 ) __ 1.00 “ 
Magnesium sulphate (MgS0 4 )_ 0.50 “ 
Ferrous sulphate (FeS0 4 )_ trace 
Sodium chloride (NaCl)_ 0.40 “ 
Water_1000.00 “ 
Magnesium carbonate (MgC0 3 )_ excess 
1. See that 3 milk bottles are clean 
2. The culture solution is on the desk in the white enamel graduate and is 
labeled “ammonia medium.” 
Stir thoroughly until the undissolved substances are in uniform suspension. 
Transfer by means of the 25 cc. graduated cylinder enough (about lOcc.) of the 
culture solution to each milk bottle and to the flask to form a layer of about 
one-fourth inch deep. 
3. Inoculate one bottle with 2 or 3 measurefuls of soil A and another 
with 2 or 3 measurefuls of soil B. Inoculate the solution in the flask with 
both soils. Label the flask in pencil on the etched portion. Be sure the label 
states the desk number as well as the notations about the experiment. 
4. Leave the flask properly plugged and labeled on the desk to be sterilized. 
5. Test each culture and the control once a week for: 
(a) Ammonia 
( b ) Nitrites 
When any culture no longer gives a positive test for nitrites, test it for 
nitrates. 
B. Nitrate Formation 
In the culture medium for the nitrate-forming organisms, the source of 
nitrogen is nitrite and the solution contains no nitrates at the beginning. 
