and Laboratory Methods. 1975 



Bacteria in the Soil. 



Toward the close of the course in Bacteriology at Cornell Medical College, 

 the subject of the soil bacteria is taken up, the students being given some garden 

 earth to plate out, count the colonies, and estimate the number per gram. They 

 are also told something about the Pathogenies found in the soil, but it seemed 

 that, in addition to this, it would be of interest to them to get an idea of the 

 various bacterial processes going on in the soil whereby the cycle of life is 

 maintained, and to this end the following plan was evolved. 



In the course of the previous exercises the students are shown how to test for 

 nitrites with sulfanilic acid and naphthylamine (Griess-Ilovsay solution), and for 

 ammonia with Nessler's solution, in order to determine if the bacteria they are 

 studying are denitrifiers or not. The cultures are grown in the following solution : 



Peptone, .-..-- 5 grams. 



KNO3, ------ 2 grams. 



NaCl, ------ 2.5 grams. 



Tap water, ----- 1000 c. c. 



After 6 or 7 days in the incubator the cultures are boiled for safety, 

 3 c. c. measured into an empty test-tube, and to this 2 c. c. of Griess-Ilovsay solu- 

 tion added. A red color shows the reduction of nitrates to nitrites. To the 

 remainder of the culture a drop of Nessler's solution is added ; a yellow color 

 showing the presence of ammonia to which the nitrites may have still further 

 been reduced. 



Control tubes of the original solutions are, of course, also tested. The 

 students then understand beforehand the tests for nitrites and ammonia. 



To demonstrate the process of nitrification in the soil, the following solution 

 is made up 6 or 8 weeks previously : 



Microcosmic salt, ----- 2 grams. 

 Potassium chloride, . . - . i gram. 



Sodium chloride, ----- 1 gram. 

 Magnesium sulfate, - - - - .5 grams. 



Water distilled, - - . - 1000 c. c 



This is distributed into several Erlenmeyer flasks, about 50 c. c. in each, and 

 sterilized. Into some of the flasks a few grams of garden earth are introduced, 

 and kept in the dark at room temperature. The inoculated flasks are tested at 

 intervals for nitrites, and when these are found to be present in considerable 

 quantities — the maximum is usually reached in 2 or 3 weeks — one-half the flasks 

 are boiled, in order to stop further action, and can then be kept indefinitely, for 

 demonstration of nitrosification. 



In the remaining flasks the nitrites gradually become oxidized to nitrates 

 by the nitrifiers, and in 6 or 8 weeks will give no greater reaction for nitrites 

 than the controls, and may be tested for nitrates. For this, evaporate a little of 

 the solution to dryness in a watch glass, add a few drops of phenol-sulfonic acid, 

 and then a little 10 per cent. NaOH solution. A yellow color indicates the 

 presence of nitrates. 



