32 LABORATORY MANUAL OF MICROBIOLOGY 



Sterilize in small flasks in layers about 2 centimeters deep. 

 Allow the medium to stand for several days so that it becomes 

 saturated with oxygen and carbon dioxide. After standing, add 

 to each flask about 0.05 grams of sterilized iron dust and inoculate. 



Medium 75 



Manganese-ammonium Sulphate Solution 



Manganese bicarbonate, saturated solution 100.0 cc. 



Sodium bicarbonate (NaHCOa) 0.1 gm. 



Ammonium sulphate ((NH4)2S04) 0.1 gm 



Dipotassium phosphate (K2HPO4) trace 



Magnesium sulphate (MgS04-7H20) trace 



Tap water 900.0 cc. 



The manganese bicarbonate is prepared by saturating a sus- 

 pension of manganese carbonate with pure carbon dioxide. The 

 medium is sterilized in flowing steam for 25 minutes. 



Medium 76 



Ferrous Carbonate and Potassium Acetate Solution 



Potassium acetate (KC2H3O2) 0.5 gm. 



Ferrous carbonate (FeCOa) 0.5 gm. 



Tap water 1,000.0 cc. 



MEDIA FOR NITROGEN FIXING BACTERIA 



Medium 77 

 Nitrogen-free Mannitol Solution 



Mannitol 10.0 gm. 



Dipotassium phosphate (K2HPO4) 0.5 gm. 



Magnesium sulphate (MgS04-7H20) 0.2 gm. 



Sodium chloride (NaCl) 0.2 gm. 



Manganese sulphate (MnS04-4H20) trace 



Ferric chloride (FeCla-OHzO) trace 



Distilled water 1,000.0 cc. 



For a solid medium add 12.5 to 15 grams of washed agar to 

 each liter. For the isolation of Azotobacter prepare this liquid 

 medium in shallow layers and add sterilized CaCOs to each flask. 



