60 ISOLATION AND CULTIVATION OF ANTAGONISTS 



ture of the antibiotic substance or substances that they produce to use 

 more than one test bacterium, including one or more gram-positive and 

 one or more gram-negative bacteria. 



Most of the methods for testing antagonistic action are based upon 

 the growth of the test organisms in the presence of the living antago- 

 nists or of the antibiotic substances produced by them in liquid and on 

 solid nutrient media. Only a few of these methods are now utilized, 

 most of them being chiefly of historical interest. 



Liquid Media 



Several methods using liquid media have been proposed for testing 

 the antagonistic activities of microorganisms: 



Simultaneous inoculation of the medium with the antagonist and the test 

 organism. 



Inoculation of the medium with the antagonist first, followed after 6 to 

 48 hours by inoculation with the test organism. 



Inoculation of the medium with the test organism first, followed, after a 

 certain interval, by the antagonist. 



Effect of the metabolic products of the antagonist upon various micro- 

 organisms. In 1888, Freudenreich (298) first filtered the culture 

 through a Chamberland candle and inoculated the filtrate with the 

 test organisms. The culture filtrate is usually added to the fresh me- 

 dium, either previously inoculated with the test organism for the 

 purpose of establishing the lytic effect of the filtrate, or followed by 

 the test organism, whereby the bacteriostatic action is measured. 



Placing a porcelain filter or cellophane membrane between the cultures 

 of the antagonist and of the test organism. Frankland and Ward 

 (295) used a filter of the Pasteur-Chamberland type partly filled 

 with broth and placed in a beaker containing the same kind of broth; 

 the antagonist and test organism were inoculated into the two lots of 

 broth, and the effect of each upon the growth of the other was de- 

 termined. Frost (303) emphasized, however, that, although theo- 

 retically this is an ideal method, it is open to criticism since motile 

 bacteria are usually able to grow through the filter after a certain 

 lapse of time. 



Collodion sac method. Collodion sacs, prepared by means of test tubes 

 from which the bottoms have been cut out, are partly filled with 



