112 PRINCIPLES OF SOIL MICROBIOLOGY 



A fertile garden soil is dried, sieved and placed in a flask to a height of 5 cm. 

 The soil is moistened with water and sterilized in an autoclave for 45 minutes at 

 150°C. When the soil is found to be sterile, it is inoculated with an emulsion of a 

 fresh growth of the weakened culture grown on agar. The flasks are allowed to 

 incubate, one at room temperature under aerobic conditions and one at 28° under 

 anaerobic conditions, for one month, then at room temperature under aerobic 

 conditions. The soil has all dried out by this time. When two grams of it is 

 inoculated into sterile liquid medium, active growth and gas formation begins 

 in 12 hours, reaching a maximum in 36 hours. 



It was found that this invigorated culture would coagulate milk with 

 gas formation. Most strains, however, would not grow upon milk. Gel- 

 atin was not liquified and casein was not decomposed. Ammonia was 

 not formed from peptone, nitrates were not reduced. 



Nun-symbiotic nitrogen-fixing aerobic bacteria. When a simple medium 

 containing tap water, 0.02 per cent K 2 HP0 4 and glucose as a source of 

 carbon is inoculated with soil and incubated in the dark, CI. pastoria- 

 num, together with other bacteria, is obtained. When the glucose is 

 replaced by mannite (2 per cent) or by propionate of potassium or 

 sodium, another large organism predominates; Beijerinck called this or- 

 ganism Azotobacter chroococcum and found it in all soils and manures. 39 

 On repeated transfer to fresh lots of sterile media, the organism was 

 gradually purified from the majority of contaminating organisms and 

 finally isolated on mannite agar. In addition to the above simple 

 medium several other media are used successfully for the isolation of 

 Azotobacter: 



1. Beijerinck medium: 



Tap water 1000 cc. 



Mannite 20 grams 



K 2 HP0 4 0.2 gram 



The mannite can be replaced by dextrin, glycerol, calcium malate (0.5 per cent) 

 and other salts of organic acids. The water may be replaced by soil extract. 40 



2. Lipman's solution: 41 



Distilled water 1000 cc. MgS0 4 -7H 2 0.2 gram 



Mannite 15 grams CaCl 2 0.02 gram 



K2HPO4 0.2 gram FeCl 3 1 drop of 10 per cent solution 



39 Beijerinck, 1901 (p. 104). 



40 Lohnis, F. Beitriige zur Kenntnis der Stickstoffbakterien. Centrbl. Bakt. 

 II, 14: 582-604, 713-723. 1905; Landwirtschaftlich-bakteriologisches Praktikum. 

 1911. P. 131. 



41 Lipman, J. G. Further contributions to the physiology and morphology of 

 members of the Azotobacter group. N. J. Agr. Exp. Sta. 25th Ann. Rpt. 1904, 

 237-289. Lipman, J. G., and Brown, P. E. A laboratory guide in soil bacteri- 

 ology. 1911. 



