EXAMINATION OF SOIL 175 



Section 4. The bacteria will finally appear in pure 

 culture. 



6. Determine the presence of nitrates. For this 

 determination proceed as follows: 



a) Preparation of phenol sulphonic acid: 30 grams 

 phenol are dissolved in 370 grams concentrated 

 sulphuric acid in a round-bottom flask. Immerse 

 completely in a water bath and heat for six hours. 



b) Dissolve 20 grams KOH in 20 c.c. distilled water. 



c) Evaporate a small amount of the culture to dry- 

 ness on a water bath. 



d) Add to the residue one c.c. phenol sulphonic 

 acid and rub with a glass rod. 



e) Add enough KOH solution to make the solution 

 alkaline. 



/) Dilute with distilled water. 

 If nitrates are present this will be indicated by the 

 appearance of a yellow color. 



EXERCISE 2. ISOLATION OF NITRATE BACTERIA 



These -bacteria may be isolated with agar or silica 

 jelly in the same manner as in Section 4, Exercise 2. 



SECTION 6 



THE ASSIMILATION OF FREE ATMOSPHERIC NITRO- 

 GEN AND ISOLATION OF THE BACTERIA 



EXERCISE I. BY LEGUME BACTERIA (TUBERCLE 



BACTERIA) 



Experiment i 



i. Secure the roots of a clover plant, or any other 

 legume. Note the appearance and distribution of 

 the tubercles. 



