METHODS OF ISOLATION 57 



the moisture of the soil is adjusted to optimum for the growth of aerobic 

 bacteria, which is about 6s per cent of the water-holding capacity of the 

 soil (20 to 50 per cent of the moist soil)j the containers are covered 

 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 (427). 



Bacterial Agar Plate Method 



This method was first used by Gratia and Dath (350) 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 K^HPO^. 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 

 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 3). The organisms are isolated from these colonies 



. V 

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lA-S^i- 



