114 LABORATORY MANUAL OF MICROBIOLOGY 



After 6 to 8 weeks examine for nodules. 



When mature, remove and analyze the tissue for total nitrogen. 



Exercise 24 



Direct Method for Demonstrating the Occurrence of Azotobacter 



IN Soil 



A. Macroscopic Method (Winogradsky).^ — To 100 grams of 

 freshly sieved soil, add 5 grams of pulverized starch. Mix the 

 starch thoroughly with the soil. 



Transfer the starch-soil mixture to a shallow dish and add 

 water in sufficient amount to make a thick paste of the soil. 

 Avoid too much water. 



Divide the soil paste between two Petri dishes. Press the soil 

 well into the Petri dishes, and smooth off the surface with a 

 wet glass slide. Make the top as smooth and even as possible. 



Incubate the plates at 28°C. for 48 hours. If Azotobacter is 

 present in the soil, small limpid colonies will be observed on the 

 surface of the plate. Stain these colonies of organisms for 2 to 

 3 minutes with thionin or erythrosin. Pure cultures may be 

 obtained from these colonies (see Exercises 25 and 26). 



B. Microscopic Method. — Thoroughly mix 0.5 gram of manni- 

 tol with 50 grams of soil. Place the mixture in a Petri dish, and 

 moisten the soil with water. 



Incubate the plate at 28°C. for 48 hours. 



Make an erythrosin or thionin stain of the soil. Azotobacter 

 cells will frequently be observed. 



Anaerobic nitrogen fixing organisms (CI. pasteurianum) can 

 be cultivated by adding 0.5 gram of glucose to 50 grams of soil 

 and mixing thoroughly. Divide the soil-glucose mixture between 

 three large test tubes and saturate the soil with water. 



Incubate the tubes at 28°C. and examine microscopically after 

 48 hours. Examine portions of the soil taken from different 

 depths in the tube. 



Exercise 26 

 Isolation of Azotobacter from Various Soils 



Prepare four small Erlenmeyer flasks of mannitol liquid 

 medium (Medium 77), about 20 cubic centimeters in each. 

 Avoid deep layers. 



1 WiNOGRADSKY, S., Ann. Inst. Past., 40: 455 (1926). 



