122 LABORATORY MANUAL OF MICROBIOLOGY 



nitrate and asparagin salt, previously analyzed for total nitrogen 

 should be used. 



(a) Control — not inoculated. 



(6) Pure culture of unknown organism capable of bringing 

 about denitrification. 



(c) Pure culture of unknown organism capable of bringing 

 about denitrification. 



{d) B. pyocyajieus, B. fluorescens liquefaciens, or B. hartlehii. 



After 2 to 3 days incubation at 28°C. make qualitative tests of 

 each culture for ammonia, nitrites, and nitrates. After 7 to 10 

 days incubation make quantitative determinations of nitrates 

 and total nitrogen. 



Use the modified Kjeldahl method to include nitrates. For total 

 nitrogen analysis take portions of 50 cubic centimeters each of 

 the cultures. 



Nitrates. — Take 10-cubic centimeter portions of the control, 

 dilute with 100 cubic centimeters of distilled water, and of this 

 evaporate 10-cubic centimeter portions to dryness. 



In the case of the inoculated cultures with nitrates present, 

 take 2 samples and proceed as follows: (a) Evaporate 10 cubic 

 centimeters to dryness, and (b) dilute 10 cubic centimeters to 100 

 cubic centimeters and evaporate 10 cubic centimeters of this 

 to dryness. 



Exercise 36 

 Denitrification in Soil 



Prepare eight 100-gram samples of field soil in tumblers. 

 Add to each sample of soil 60 milligrams of nitrogen in the 

 form of potassium nitrate. 

 Treat the series as follows: 

 (a) 1 and 2, control untreated. 

 (6) 3 and 4, add 2.5 grams of glucose. 



(c) 5 and 6, control untreated. 



(d) 7 and 8, add 2.5 grams of glucose. 



Mix these materials thoroughly by means of a spatula. 



To soil portions 1 to 4, add sterile water to bring the moisture 

 content to about one-half saturation. 



To soil portions 5 to 8 add sterile water to bring moisture up to 

 total saturation. 



