38 THE BACTERIOPHAGE AND ITS BEHAVIOR 



isolated it from such cultures. Let us hasten to state, however, and 

 we will return to this important point, that such contaminated cultures 

 are rare. 



Methods of isolation 



The bacteriophagous principle is encountered but rarely in a sub- 

 stance which, m the usual sense of the word, is sterile. Usually it is 

 necessary to isolate it from a medium in which bacteria are also present. 

 Two methods may be followed to this end. Since the bacteriophage 

 will pass through porcelain filters and through ultrafilters, the material 

 under examination may be suspended in water or in bouillon and sub- 

 jected to filtration through porcelain or ultrafilters (d'Herelle^^°). 

 The bacteria are held back by the filter; the bacteriophage passes 

 through and is thus found in a pure state in the filtrate.* The bac- 

 teriophage resists temperatures of at least 60°C. Thus, if it is pres- 

 ent in a liquid medium containing non-spore-forming bacteria it is 

 only necessary to heat at 58°C. for a time sufficiently long to kill the 

 bacteria. The bacteriophage will then be found in a pure state (Bordet 

 and Ciuca^"). The bacteriophage will resist three successive heatings 

 made at this temperature (58°C.) and consequently it is possible to 

 isolate it by this means, even if the mixture contains spore-forming 

 organisms, since they will be ehminated by the method of fractional 

 steriHzation (Tomaselli^^^) . I have satisfied myseK that the bacterio- 

 phage may be isolated from feces by this method. 



In studying the influence of physical agents upon the bacteriophage 

 we will see that temperatures, even below 60°C., may cause some 

 attenuation. It is therefore advisable to utihze filtration wherever 

 possible and to resort to isolation through heating only in particular 

 cases and as a last resort. 



* This is true, naturally, only when the material does not contain, in addition 

 to the bacteriophage and the bacteria, some other filtrable organism, such as the 

 viruses of vaccinia, of rabies, of herpes, etc. In cases of this type a separation by 

 filtration or ultrafiltration can not be effected, since, as we shall see, all ultra- 

 viruses, the bacteriophage included, possess comparable, or possibly identical, 

 dimensions. This is the case, certainly, for the ultraviruses mentioned above, 

 as has been shown by Levaditi.*'^ Where there is an associated ultravirus separa- 

 tion by heating may be employed, at least in those cases where the associated 

 ultravirus is less resistant than the bacteriophage. In the opposite case (if, 

 for example, the mixture contains an ultravirus of the mosaic group) separation 

 can be accomplished by making several successive passages in the presence of a 

 susceptible bacterium. Here, only the bacteriophage will multiply, and at the 

 end of a few passages the associated ultravirus will have been eliminated by 

 dilution. 



