62 ISOLATION AND CULTIVATION OF ANTAGONISTS 



termined. Frost (302) 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 

 broth and placed in a flask containing the same kind of broth. The 

 test organism is inoculated into the medium inside the sac, and the 

 antagonist into the flask (302). 



Solid Media 



Solid media have also been used extensively for testing the action of 

 antagonists. These media offer certain advantages over liquid media. 

 The following methods are most commonly used : 



Simultaneous inoculation of antagonist and test organism. This method, 

 introduced by Garre (311) in 1887, consists in streaking the an- 

 tagonist and the test organism on the surface of a solidified agar or 

 gelatin medium. The streaks are alternate and may be parallel, radi- 

 ating from a common center, or intersecting at right angles (Fig- 

 ure 5). If the active substance produced by the antagonist does not 

 diffuse for any considerable distance into the medium, the method is 

 not satisfactory. Frost (302) modified this method by inoculating 

 the whole medium with the test organism and, when the medium 

 had hardened, streaking the antagonist across the surface. The first 

 of these came to be known as the anaxogramic method; the second 

 is often spoken of as the implantation method (705). The spotting 

 of the two organisms on the plate is illustrated in Figure 6. 



Successive inoculation of the test organism, after the antagonist has al- 

 ready made some growth, so as to enable the active substance to dif- 

 fuse. This method, suggested by Garre (311), consists in allowing 

 the antagonist to produce a good growth on the surface of the me- 

 dium; the mass of growth is then removed, and the test organism 

 inoculated into the same medium. 



Double plate methods (302). A Petri dish is divided into two parts by 

 means of a small glass tube or rod. After sterilization, one tube of 

 molten agar is heavily inoculated with the antagonist and poured 

 into one half of the plate. When the agar has hardened, another tube 

 of sterile agar is poured into the other half of the plate. Both sides are 



