64 ISOLATION AND CULTIVATION OF ANTAGONISTS 



then streaked with the test organism, each side being equally inocu- 

 lated by separate streaking. This can be done by using a loop bent at 

 nearly right angles; the charged loop is moved from the circumfer- 

 ence toward the glass rod. The loop is then sterilized, recharged with 

 the test culture, and the streak continued on the other side of the 

 plate. The inoculation with the test organism may be made soon 

 after the plate is poured, or the antagonist may be given an opportu- 

 nity to develop before the test organism is streaked thus making the 

 antagonistic effect more striking. This method has also been used 

 (261, 267, 270) 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 (844). This method is particu- 

 larly convenient for detecting antagonists that possess lytic prop- 

 erties. 



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 difiFuse through the agar and reduce 

 the growth of the test bacterium (571). 



Semisolid media are used for testing the action of antagonists against 

 motility of bacteria (173). 



METHODS OF GROWING ANTAGONISTIC 



ORGANISMS FOR THE PRODUCTION 



OF ANTIBIOTIC SUBSTANCES 



Once the antagonistic action of any organism has been established, 

 the next step is to determine the nature of the antibiotic substance pro- 

 duced by the antagonist and to measure quantitatively this antibiotic 

 action. Before this can be done, however, the organism must be grown 

 upon suitable media and suitable conditions must be established for the 

 favorable production of the antibiotic substance. 



