56 SOIL BACTERIOLOGY 



3. From these suspensions of soil bacteria prepare dupli- 

 cate plates with starch-nitrate agar. 



4. When the colonies are well developed, pour over the 

 surface of one of the duplicate plates a very dilute solution 

 of potassium iodid in dilute hydrochloric acid. Allow to 

 react for a moment or two, then pour off. The production 

 of a blue zone around colonies indicates a reduction of 

 nitrates to nitrites. 



KI + HCl = KCl + HI 



2HI + 2HNO2 = 2H2O 4- 2NO + I2 



5. Note the relative proportion of organisms capable of 

 reducing nitrates to nitrites. 



6. Note the general characteristics of such colonies, and 

 from similar colonies upon the untreated plates make 

 transfers to nitrate agar slopes (m. 33). 



Exercise 21 



Reduction of Stains by Denitrifying Organisms 



I Inoculate duplicate tubes of nitrate solution (m. 33) 

 with pure cultures of denitrifying organisms. 



2. Add to each tube 0.5 c.c. of a sterile i : 1000 (highest 

 purity) methylene-blue solution and mix thoroughly. 



3. In order to exclude partially the oxygen, pour paraffin 

 oil to a depth of about 2 cm. in one-half of the tubes. 



4. Incubate at 28^ C. 



5. Note each day the change in color. This change 

 furnishes a method for detecting nitrites. As long as 

 nitrites are present the solution remains blue. A colorless 

 solution indicates that all of the nitrite nitrogen is destroyed. 



