56 ISOLATION AND CULTIVATION OF ANTAGONISTS 



times with sterile water. The contents of the plates are thoroughly 

 mixed in order to distribute the diluted soil suspension in the bacterial 

 agar. The plates are inverted and incubated at 28° or 37° C. 



After I to 10 days' incubation, depending on the nature of the or- 

 ganism used for the preparation of the plates, the presence of antago- 

 nists is manifested by the formation of clear zones surrounding their 

 colonies (Figure 2). The organisms are isolated from these colonies 

 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 (956, 978). 



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 K2HPO4. 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 (1:10 to 1:1,000). 



This method, like the soil enrichment method, does not always yield 

 desirable results. As shown in Table 6, some of the most important 

 antagonists, such as Ps. aeruginosa, S. antibioticus, A. jiavus, and P. 

 notatum-y do not develop on such a plate since they cause only limited 

 lysis of bacteria. On the other hand, B. brevis, S. griseus, A. fumigatus, 

 and A. clavatus cause extensive lysis of gram-positive bacteria and so 

 can readily be isolated. 



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 3). 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. 



