730 PRINCIPLES OF SOIL MICROBIOLOGY 



bacteria may become limited in their development by the lack of suffi- 

 cient available phosphorus in the soil or in the medium. Since Azoto- 

 bacter cells may contain as much as 2 to 5 per cent P2O5, the rapid 

 development of this and other nitrogen-fixing bacteria, which produce 

 an extensive growth in the presence of an excess of available energy, 

 may be limited by the presence of this mineral. For every unit 

 of nitrogen fixed or assimilated by Azotobacter and synthesized into 

 microbial protein about half a unit of available phosphorus (P 2 5 ) is 

 required. The amount of phosphorus present in an available form 

 can be calculated from the amount of nitrogen fixed. The latter may 

 then become merely an index of the available phosphorus in the soil. 

 Four methods may be suggested 36 for measuring the nitrogen-fixing 

 and mannite decomposing power of a soil: 



1. The solution method consists of adding 1 or 5 grams of soil to 50 or 100 cc. of 

 a standard mannite solution (20 grams mannite, 0.2 gram MgS0 4 -7H 2 0, 0.2 gram 

 K2HPO4, 0.2 gram NaCl, 5.0 grams CaC0 3 , in 1000 cc. distilled water and made 

 neutral to phenolphthalein), incubating for 7 to 28 days, then determining the 

 increase in total nitrogen above the control (original solution + original soil 

 analyzed immediately for total nitrogen). This serves as an index of the activities 

 of the nitrogen-fixing flora of the soil and thus also, to some extent, of the 

 microbiological condition of the soil. 



2. The sml -method consists of adding 1 or 2 grams of mannite to 100 grams of 

 fresh sieved soil, bringing the latter to optimum moisture, incubating for 28 

 days, and determining the increase of nitrogen in the treated soil over the un- 

 treated soil, incubated under similar conditions. 



3. The -pure culture method 31 consists of adding 10 grams of the particular soil 

 to 100 cc. of a 2 per cent mannite solution, free from available phosphates, steril- 

 izing and inoculating with a vigorous culture of Azotobacter. After incubating 

 for 20 to 30 days, the increase in total nitrogen is determined. This can serve 

 as an index of the available phosphate in the soil. This method can also be 

 modified by adding to the above mineral solution, free from phosphorus, 10 per 

 cent of soil and estimating, from the amount of Azotobacter growth or from the 

 actual increase in total nitrogen, the amount of available phosphate in the soil. 

 In view of the fact that the majority of field soils contain only small quantities 

 of available phosphate, increasing quantities (0.0005 to 0.005 gram K2HPO4) 

 can be added to a series of flasks, which are then inoculated with the soil. 



4. The determination of residual mannite (or rather soluble organic matter 

 in the soil). 38 This consists of adding 2 per cent of mannite to air dry soil, 



36 Waksman, S. A., and Karunaker, N. Microbiological analysis of soil as 

 an index of soil fertility. IX. Nitrogen fixation and mannite decomposition. 

 Soil Sci , 17: 379-393. 1924. 



37 Niklewski, 1912 (p. 726); Stoklasa, 1925 (p. 621); Christensen, 1915 (p. 578). 



38 Christensen, H. R. Influence of soil conditions on bacterial life and changes 

 in the soil. II. Ability of soil to break down mannite. Soil Sci., 15: 329-360, 

 361-363. 1923. 



