48 THE BACTERIOPHAGE AND ITS BEHAVIOR 



of Shiga bacilli, after only 24 hours of incubation 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 utihzed, al- 

 most all of the bacteria are living after 24 hours at 37°C. A 24-hour 

 bouillon culture will, then, remain shghtly turbid when the bacterio- 

 phagic 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 dissolution is achieved. In this last case all 

 of the bacteria were living and susceptible to the attack of the bacterio- 

 phage. It is for this reason that it is preferable to effect bacteriophagy 

 in a suspension of bacteria rather than directly in a bouillon culture. 



Certain bacteria give a homogeneous growth in a young culture in 

 bouillon but when taken from agar they can be suspended only with 

 difficulty. B. pestis is such an organism. When working with such 

 bacteria it is preferable to have the bacteriophage act on a broth 

 culture in the following manner. A bouillon tube is hghtly seeded 

 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" will consist almost 

 entirely of young bacilli, readily subject to attack. 



The following experiment demonstrates clearly 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 

 "vaccinated" media) are without effect upon the phenomenon of 

 dissolution. 



Two cultures of B. dysenteriae Shiga, the one aged 15 days, the other, 

 18 hours are centrifugaHzed. The sediment from the first culture is 

 suspended in the supernatant fluid of the second, and the sediment 

 of the second culture is combined with the supernatant fluid of the 

 first. Both suspensions thus formed are inoculated with a drop of 

 a bacteriophage filtrate. The suspension consisting of "old" bacilli 

 and "young" medium remains turbid; that of "young" bacilli and 

 "old" medium becomes perfectly clear after 7 hours. 



But, although the products of bacterial metabolism are not inhibitory 

 for the process of dissolution, the products of dissolution, as we will 

 see, exert quite a different action. These products are the result of 

 the activity of the bacteriophagic process, and, as such, they impede 

 its activity. 



