824 PRINCIPLES OF SOIL MICROBIOLOGY 



agar cultures. Each pot weighing about 680 grams can be used to 

 inoculate one bushel of seed or one acre of land (Wilson) . Peat cultures 

 of the nodule bacteria are also being used quite extensively. 24 



To test cultures of legume bacteria for the abundance and vitality 

 of the particular organism, two methods are used: 



1. The culture is diluted to 1: 10,000 or 1: 100,000, then 1 cc. of the 

 final dilution is added to 9 cc. of agar medium (Temple used a medium 

 consisting of 10 grams sucrose, 1 gram KH 2 P0 4 , 15 grams agar, 1000 cc. 

 tap water, pH = about 6.5 to 7.0) and plates prepared. These are 

 incubated for 6 to 7 days at 25°C. ; the number of viable bacteria, as well 

 as abundance of contaminations, can then be determined. It is fre- 

 quently difficult to differentiate on the plate between Radiobacter and 

 Bad. radicicola 25 and it is also impossible to learn which particular 

 groups of the organism are present in the soil. 



2. To identify the strain, direct inoculation tests must be employed. 

 Either bottles with sterilized sand containing 20 per cent moisture or 

 tall cylinders containing sterile 0.75 per cent agar media must be used. 

 The seeds are sterilized by treatment for fifteen minutes with 0.1 per 

 cent corrosive sublimate, 1 per cent formaldehyde or 5 per cent hypo- 

 chlorite, then rinsed in sterile water and germinated on moist filter 

 paper in a moist chamber. The sprouted seeds are then removed with 

 sterile forceps, dipped in the inoculating material and dropped upon 

 the substrate, in which they are expected to grow. Controls should 

 always be employed. The formation of nodules is an index of the activ- 

 ity of the culture. The purity of culture can also be tested on sterilized 

 potato, upon which nodule bacteria do not grow (some give some growth 

 in 4 weeks), while common contaminations and Bad. radiobacter produce 

 a growth in 5 to 7 days at 28°C. 26 



Bad. radicicola multiplies very rapidly in sterile soil and the develop- 

 ment of the organism is greatly diminished when the sterile soil is 

 mixed with non-sterile soil, indicating that normal soil is not a very 

 favorable medium for their development. 27 



Biological types of legume bacteria. It has been pointed above that, 



24 Earp-Thomas, G. H. Peat as a carrier for bacteria. Jour. Amer. Peat Soc, 

 16: 18-23. 1922. 



25 Joshi, N. V. Studies on the root nodule organism of the leguminous plants. 

 India Dept. Agr. Mem. Bact. Ser., 1: 219-276. 1920. 



2 «Lohnis and Hansen, 1921 (p. 126). 



27 Duggar, B. M., and Prucha, M. J. The behavior of Pseudomonas radicicola 

 in the soil. Centrbl. Bakt. II, 34: 67. 1912. 



