66 A MANUAL OF BACTERIOLOGY 



aluminum sulphate) per liter to the soil suspension before filter- 

 ing, as directed above. 



4. Draw samples of 50 cc. or larger, as soon as possible, to 

 avoid the danger of denitrification. Determiae nitrate qualita- 

 tively, or quantitatively by the phenol-sulphonic colorimetric 

 method. Each 10 ec. of the extract represents 1 g. of the fresh 

 soil sample. By determining the water content of the soil 

 sample at the outset of the experiment, it will be possible to 

 reduce the nitrate determinations to a moisture-free basis. 



5. Compute the amount of nitrate formed iu the samples to 

 which ammonium sulphate was added. If soils from a variety of 

 sources under a variety of conditions were included in the plan 

 of the experiment, draw conclusions as to the effect of these 

 conditions upon nitrification. 



Note. The isolation of nitrite and nitrate organisms from soils involves the 

 preparation of silica-jelly media. The work is rather diflBcult for the begin- 

 ning student. Directions for procedure will be found in the following works 

 and others. 



Heinemann. Laboratory Guide in Bacteriology. 

 Lapae. Handbuch der teohnischen Mykologie 3 : 155. 

 Smith. Bacteria in Relation to Plant Diseases 1 . 36. 



Exercise 95. Reduction of Nitrates by Bacteria 



1. Prepare Giltay and Aberson's solution and fill several fer- 

 mentation tubes and test tubes. Sterilize. 



2. Inoculate some of the tubes with a small quantity of fresh 

 horse manure. Inoculate others with B. denitrificans, B. Hartlehii, 

 B. pyocyaneiis, or B. fluorescens liquefaciens. Keep the tubes in 

 the incubator for ten to fifteen days. 



3. Examine the fermentation tubes for free gases. Examine 

 the contents of the test tubes for nitrites and ammonia compounds. 



Exercise 96. The Reducing Action of Denitrifying Bacteria upon Nitrates 

 and Methylene Blue 



Fred. Centralbl. f . Bakt., 2te Abt., 32 : 421. 1912. 



Since denitrification is essentially a reducing process in which 

 oxygen is removed from its combination with nitrogen, the 

 action may well be demonstrated by adding a reducible dyestuff 



