204 THE BACTERIOPHAGE AND ITS BEHAVIOR 



remains completely sterile) is spread over an agar surface the latter 

 remains sterile except for a very limited growth of fine colonies. This 

 restricted growth takes place at the extreme upper margin of the agar 

 at the point where the substrate forms only a very thin layer.* With the 

 tip of a very fine glass needle they removed the center of one of these 

 minute colonies and seeded it upon an agar slant which had been pre- 

 pared for a few days and was, therefore, dry. After 2 or 3 comparable 

 transfers, that is to say, after removing daily for seeding the center of a 

 fine colony located at the margin of the agar they obtained an ultra- 

 pure bacterial culture, one which no longer contained bacteriophage 

 corpuscles. t The ultrapure culture thus obtained is composed of 

 refractory bacilli insusceptible to the bacteriophage even if the latter is 

 of maximum virulence. A suspension of such bacilli inoculated with 

 any quantity whatever of bacteriophage suspension develops as in a pure 

 bouillon. 



They transplanted the ultrapure culture every day. At the end of 8 

 transfers they showed that the bacteria were still resistant but no longer 

 completely refractory for the inoculation of a bacteriophage into sus- 

 pensions caused bacteriophagy although the dissolution was only partial. 

 During the course of subsequent cultures upon agar resistance gradually 

 diminished but it was still demonstrable after 15 transplants. 



In brief, then, the conclusion of these authors confirms that of Bordet 

 and Ciuca insofar as it deals with the gradual loss of resistance. 



I have repeated this experiment, effecting the purification in another 

 manner, simply by the classical procedure of isolated colonies on agar, 

 operating on the basis of the fact which I had previously noted^^^ that 

 by implanting secondary cultures on agar one might obtain ultrapure 

 colonies. I have, therefore, very readily effected repeated purifications 

 by isolated colonies on agar following the old method described by E. 

 Roux.t Each of the tubes during the isolation should remain for 3 days 



* The formation of these colonies in spite of the presence of bacteriophage 

 corpuscles is due to the thinness of the agar layer. The products resulting from 

 l^acteriophagy can not diffuse into the substrate and the action of the bacterio- 

 phage is inhibited. 



t In what follows I will designate colonies, or a culture or a bacterial strain 

 which is free of bacteriophage corpuscles by the qualifying word "ultrapure." 



X This is a procedure which I can not recommend too highly for the isolation of 

 pure strains, not only in the case of a contamination by bacteriophage corpuscles 

 but also as a general procedure for purifying bacterial strains. The materials 

 needed are a tube of sterile bouillon (6 or 7 cc.) and an agar slant. The only con- 

 dition is that the agar slant contain some water of condensation, — if the amount 



