METHODS OF ISOLATION 5 5 



with glass plates and placed in an incubator at 28° or 37° C. Washed 

 suspensions of living bacteria are added to the soil at frequent intervals, 

 care being taken to avoid puddling it with an excess of the fluid, so con- 

 ditions will not be made anaerobic. Samples of the enriched soil are 

 removed at intervals and tested for the presence of organisms antag- 

 onistic to the bacteria added. Fresh washed suspensions of the living 

 bacteria are inoculated with the enriched soil as soon as the presence of 

 antagonistic organisms is demonstrated j this results in the development 

 of the antagonistic organisms and the destruction of the bacteria in sus- 

 pension. Transfers are then made to fresh suspensions of the bacteria, 

 resulting in an enrichment of the antagonist, which can finally be iso- 

 lated in pure culture (201, 207, 442). 



The significance of the soil enrichment method and its application to 

 the isolation of specific antagonistic organisms has been questioned 

 (969). It was suggested that whereas there is no question concerning 

 the multiplication of microorganisms capable of decomposing a given 

 substance or of secreting enzymes active upon such a substance in re- 

 sponse to its introduction into the soil, there is still doubt whether 

 specific antagonistic organisms develop as a result of the introduction 

 of living cells into the soil. The reason for this was based upon the fact 

 that antibiotic reactions produced by antagonistic organisms do not 

 affect bacteria by simple digestive or oxidative mechanisms. 



Bacterial A gar Plate Method 



This method was first used by Gratia and Dath (357) for the isola- 

 tion of antagonistic agents, actinomycetes having been found readily 

 by it. 



To isolate antagonistic bacteria, agar (1.5 per cent) is washed in dis- 

 tilled water, then dissolved in water supplemented by i per cent glucose 

 and 0.2 per cent K0HPO4. Ten-milliliter portions of the sugar- 

 phosphate agar are placed in glass tubes and sterilized. The sterile agar 

 is melted, and the tubes are placed in a water bath kept at 42° C. A 

 washed, centrifuged suspension of living bacteria, grown on solid or in 

 liquid media, is then added and thoroughly mixed with the agar. This 

 "bacterial agar" is poured into a series of Petri plates containing one- 

 milliliter portions of fresh or enriched soil, diluted i : lOO to i : 10,000 



