Aug. 19, 1922] THE BACTERIOPHAGE. [ M Jrcif Jor;.. 



supposition that the bacteriophagic principle is of corpuscular nature. The 

 following experiments give the proof thereof. 



Experiment B. 



Given a filtrate containing the bacteriophagic principle, let us determine its 

 force in the production of bare spaces. 



It is well known that to determine the number of living bacteria contained in 

 an emulsion agar plates are inoculated with a dilution of this emulsion; after incuba- 

 tion, the number of colonies which develop multiplied by the titre of the dilution 

 gives the number of bacteria per cubic centimetre of the primitive emulsion. The 

 enumeration of the bacteriophagic corpuscles contained in a filtrate is estimated in 

 exactly the same way. But as the bacteriophage only grows at the expense of living 

 bacteria, we must make the dilutions of the filtrate in a bacterial emulsion. For 

 this we pipette over agar the bacterial emulsion containing a given quantity of 

 filtrate: after incubation we obtain a bacterial layer strewn with circular bare spaces, 

 each of these spaces being a colony of bacteriophage issued from one corpuscle. 

 The number of bare spaces multiplied by the titre of the dilution gives the number 

 of ultramicroscopic bacteriophagic corpuscles contained in 1 c.cm. of the primitive 

 filtrate. 



This experiment shows that the behaviour of the bacteriophage is exactly 

 the same as that of any ordinary microbe. But this last develops at the expense 

 of the nutritive substances contained in the medium; the bacteriophage 

 develops at the expense of the bacterial bodies which constitute its nutritive 

 medium. The bare spaces represent places cleared up by the growth of the 

 ultramicroscopic bacteriophagic corpuscles. 



Experiment C. 



Now dilute a filtrate so as to obtain a dilution such that 1 c.cm. contains one 

 bacteriophagic corpuscle. Dilute this 1 c.cm. with 9 c.cm. of sterile water, and 

 inoculate ten tubes of bacterial emulsion each with 1 c.cm. It will be obvious that 

 only one of the ten tubes will contain the generator of a bare space; the nine others 

 will not contain any. Place the ten tubes in the incubator at 37° for twenty-four 

 to forty-eight hours; it will be seen that only one of the ten microbial emulsions 

 shows bacteriob'sis; the nine others will remain unchanged, the bacteria remain liv- 

 ing, normal, and subculturable. 



The lytic action, therefore, is complete when only one generator of a bare 

 space is introduced into a bacterial emulsion; the action is nil in the contrary 

 case. This experiment can only be explained on the supposition that the 

 bacteriophagic principle, the source of the lytic enzymes, is a corpuscle; and 

 that each corpuscle deposited on the agar in the midst of the bacteria gives 

 rise to a colony of these ultramicroscopic corpuscles, such a colony being repre- 

 sented by a bare space. 



Experiment D. 

 Inoculate a bacterial emulsion with a bacteriophagic filtrate of known corpuscular 

 strength (that is, of known bare spaces forming force); make agar slopes every hour 

 in the same way as in Experiment A. In this way, after six hours, we will have six 

 agar slopes inoculated, and, on incubation, it will be seen that the number of 

 bacteriophagic spaces (each bare space corresponding to a corpuscle) increases in 

 proportion as the lytic action progresses in the emulsion. The maximum number of 

 bare spaces is given when the lysis of the original emulsion is complete; at this 



