METHODS OF TESTING ANTAGONISTIC ACTION 63 



antagonistic effect more striking. This method has also been used 

 (258) for testing the antibiotic properties of fungus cultures. 



Mixed culture inoculation. The cultures of the antagonist and the test or- 

 ganism are mixed and inoculated upon the surface of the solidified 

 agar or before the molten agar has been added to the plate. The colo- 

 nies of the antagonist will be surrounded by clear sterile zones, free 

 from any growth of the test organism. 



Spot inoculation of the antagonist upon an actively growing culture of a 

 bacterium or fungus on an agar plate. This method is particularly 

 convenient for detecting antagonists that possess lytic properties. 



A layer of molten sterile agar is used to cover the surface of an antagonist 

 that has made some growth in a plate, and the surface of the agar 

 layer is then inoculated with the test organism. The active substance 

 produced by the antagonist will diffuse through the agar and reduce 

 the growth of the test bacterium (609). 



Semisolid media are used for testing the action of antagonists upon 

 the motility of bacteria (182). 



A number of other methods, usually modifications of those outlined 

 above, have been used for testing the ability of fungi to produce anti- 

 biotic substances (724, 1016). Some of these methods, notably the agar 

 diffusion (cup, paper disc, cylinder) test, are used for the quantitative 

 estimation of the concentration of the antibiotic in the medium and for 

 isolation purposes. These methods can indicate the formation not only 

 of growth-inhibiting but also of growth-promoting substances (99). 



Raper et al. (765) removed plugs of agar of constant dimensions 

 from the fungus cultures being tested and placed them on the surface 

 of plates seeded with S. aureus. The plates were incubated at 37° C, 

 and the amount of penicillin present was estimated by the size of the 

 zones of inhibition. For the purpose of screening many cultures, a 

 modified Czapek's solution agar, i per cent by volume of corn steep 

 liquor {^$^ per cent solids) was used} the solution was adjusted to /jH 

 7.0, and 2 per cent agar was added. Twenty-milliliter portions were 

 placed in tubes, sterilized, and poured into sterile Petri dishes. The 

 plates were selected to insure that the agar layers were of uniform 

 depth. Single colonies were established by suspending spores of the cul- 

 ture to be tested in melted agar at 45 ° C. The agar was allowed to so- 



