64 A MANUAL OF BACTERIOLOGY 



Exercise 92. Nitrite Formation in Solution 



1. Prepare Winogradsky and Omelianski's solution for nitrite- 

 forming organisms (see Exercise 51). 



2. Divide the solution equally among five Erlenmeyer flasks 

 of 500 cc. capacity. Number the flasks consecutively. 



3. Inoculate flask No. 1 with 1-2 g. of fresh soil collected 

 about 10 cm. beneath the surface. Incubate the flasks at 25- 

 30 C. 



4. As soon as growth is evident (usually in four or five days), 

 inoculate flask No. 2 from flask No. 1, using the platinum loop. 

 Continue the serial inoculation from flask to flask. 



5. Examine the bacteria in a drop of Gram's iodine solution 

 and with simple stains. Test the solutions for nitrites with the 

 Griess-Ilosvay reagent, or with Trommsdorf reagent. 



Exercise 93. Nitrate Formation in Solution 



1. Prepare 1 liter of Winogradsky and Omelianski's solution 

 (see Exercise 52). Divide the solution equally among five Erlen- 

 meyer flasks of 500 cc. capacity. Sterilization is of no advan- 

 tage in this case, since none except nitrifying bacteria can develop 

 in this solution. 



2. Inoculate one flask with 1 g. of a soil sample collected 

 about 10 cm. from the surface. 



3. Watch the flask for evidence of bacterial growth. At the 

 first indications of growth inoculate the second flask with three 

 loopfuls of solution taken from the first. Carry through the 

 inoculation in this way from flask to flask, leaving one flask 

 uninoculated, to serve as a control. This method of serial trans- 

 fers gradually eliminates all bacteria except those fitted to de- 

 velop in this solution, that is, principally nitrifying bacteria. 

 At the end one obtains a nearly pure culture of these organisms. 



4. Make cover-slip stains from the flasks and examine the 

 organisms microscopically. Stain with warm carbol-fuchsin and 

 wash with acidulated alcohol, or stain with Loffler's alkaline 

 methylene blue. At the expiration of ten days test the solutions 

 for nitrates with phenol-sulphonic acid. 



