BACTERIOLYSIS 41 



expense of living bacteria, these being the ones which are lysed. 

 In the old suspension which we have mentioned in which there 

 is only about one organism in three hundred which is capable of 

 being dissolved, it can readily be comprehended that if lysis of a 

 suspension be taken as a criterion, the bacteriophage appears to 

 be without action in such a culture. 



It is useless to work with old cultures. In a broth culture of 

 Shiga, after only twenty-four hours of incubation, as has been 

 shown, about one-third of the organisms present are incapable of 

 producing colonies when planted on agar. If, on the other hand, 

 an agar slant culture is utilized, almost all of the bacteria are liv- 

 ing after twenty-four hours at 37C. A twenty-four hour bouillon 

 culture will, then, remain slightly turbid when the lytic process 

 is accomplished, while a suspension made in broth from a young 

 agar culture containing the same number of bacteria will be 

 perfectly limpid when the lysis is achieved. In this last case 

 all of the bacteria were living and susceptible to the attack of the 

 bacteriophage. It is for this reason that it is preferable to effect 

 cultures of the bacteriophage in a suspension rather than directly 

 into a bouillon culture. 



Certain bacteria give a homogeneous growth in a young cul- 

 ture in bouillon but when taken from agar they can be suspended 

 only with difficulty. B. pestis is such an organism. When work- 

 ing with such bacteria it is preferable to have the bacteriophage 

 act on a broth culture in the following manner. A bouillon tube 

 is lightly inoculated with the bacterium. When the culture has 

 clouded, the bacteriophage active for this bacterial strain is 

 introduced and at the same time the culture is diluted with an 

 equal volume of sterile medium. This dilution should be made 

 before the bacteriophage has had time to multiply sufficiently 

 to parasitize an appreciable number of bacteria. Thus, the 

 bacterial culture at the time of " departure," that is, when the 

 bacteriophagous organisms are sufficiently abundant, will consist 

 almost entirely of young bacilli, readily subject to attack. 



It has been demonstrated that the products of bacterial growth 

 as found in an old culture, products which, as is well-known, 

 inhibit the development of bacteria (as in the so-called "vac- 

 cinated" media) are without effect upon the lytic phenomenon. 



