58 ISOLATION AND CULTIVATION OF ANTAGONISTS 



and are retested for antagonistic properties, either by transfer to fresh 

 bacterial agar plates or by inoculating solidified agar plates and cross- 

 streaking with test organisms (934, 949). 



In the isolation of antagonistic fungi the same method is followed, 

 except that it is preferable to make the bacterial agar acid by using 

 KH2PO4 in place of K0HPO4. The resulting acidity (pH 4.5) inhibits 

 the growth of bacteria and actinomycetes. Since the soil contains fewer 

 fungi than bacteria, lower dilutions of soil are employed for this pur- 

 pose (i: loto i: 1,000). 



By the use of the soil enrichment and bacterial agar plate methods, 

 it is possible to demonstrate that ordinary soils contain a large popula- 

 tion of microorganisms that are antagonistic to bacteria, including both 

 gram-negative and gram-positive forms. The number of antagonists can 

 be greatly increased when the soil is enriched with living cells of 

 bacteria. 



Crowded Plate Method 



Ordinary field or garden soil is plated out on common nutrient (beef- 

 peptone) agar, very low dilutions (1:10 to 1:1,000) being used to 

 enable a large number of bacterial colonies to grow on the plate. The 

 resultant crowding of these colonies allows the development on the 

 plate of potential antagonists that are normally present in the soil. The 

 production of antibacterial substances by these antagonists inhibits the 

 growth of bacteria in close proximity to them and, in consequence, clear 

 zones are formed around the colonies (Figure 4). It is possible, by 

 means of this method, to demonstrate that many strains of spore-form- 

 ing bacteria possessing antagonistic properties are present in the soil and 

 can readily be isolated from it (857). 



Direct Soil Inoculation Method 



Nutrient agar plates are inoculated with the bacteria or fungi for 

 which antagonists are to be found, and the plates are incubated for 24 

 to 48 hours at 28° or 37° C. Particles of fresh or enriched soil placed 

 on the surface of the bacterial or fungus growth on the plate will give 

 rise to antagonistic organisms. These organisms will bring about the 

 killing or even the lysis of the original culture. By this method, or- 



