CHANGE OF INSOLUBLE PHOSPHATES 263 



SMITH, E. F.: Bacteria in Relation to Plant Diseases. Vol. II, 



pp. 96-138. 

 Inoculation with Nodule-forming Bacteria, Cir. 5, Mich. Exp. 



Sta., 1915. 

 FRED, E. B.: A Physiological Study of the Legume Bacteria, Ann. 



Kept. Va. Polytechnic Institute, 1911, 1912, pp. 174-201. 



EXERCISE 10. TO DEMONSTRATE THE CHANGE OF 

 INSOLUBLE PHOSPHATES TO A SOLUBLE FORM 

 THROUGH THE AGENCY OF MICROORGANISMS 



Apparatus. Dextrose; di- or tri-calcium phosphate; 

 tubes of soil-extract agar containing 2% dextrose; four 

 100 c.c. Erlenmeyer flasks. 



Culture. Soil. 



Method. 1. Place 0.1 gm. of di- or tri-calcium phos- 

 phate, and 60 c.c. of a 2% solution of dextrose in tap water 

 in each flask. Sterilize. 



2. To two flasks add 0.1 gm. soil each, leaving two for 

 controls. 



3. Incubate at 37 C., and after the fermentation has 

 continued for some days, make plates from the inoculated 

 flasks as follows: 



4. Sterilize about 0.1 gram of di- or tri-calcium phos- 

 phate in each of three test tubes. 



5. Place the contents of each tube in a sterile Petri dish; 

 make loop-dilution plates from flasks in soil extract agar 

 containing 2% dextrose, being careful to mix the phos- 

 phate well with the agar in the dish by carefully tilting. 



6. Incubate at 37 C. 



7. Note frequently the appearance of the plates. The 

 colonies of acid-producing bacteria developing at this 

 temperature dissolve the phosphate and thus become sur- 

 rounded by a clear area similar to that produced by lactic 

 acid-producing bacteria on dextrose calcium carbonate agar. 



8. Examine the colonies in a hanging drop for mor- 

 phology, motility, etc. 



9. How is the action noted in 7 made use of practically? 



