30 THE BACTERIOPHAGE 



When the agar inoculation has shown that a bouillon suspension 

 contains an active bacteriophage this suspension is filtered through 

 infusorial earth and then through a bougie. A slightly turbid 

 suspension is prepared, using the bacterial strain against which 

 the bacteriophage has shown some activity, and into this suspen- 

 sion are introduced some four or five drops of the filtrate. After 

 an incubation period of twenty-four hours at 37C., if lysis has 

 not been produced, this second bacterial suspension is filtered 

 as before and a third suspension is inoculated with four or five 

 drops of the filtrate. Such transfers are continued until evident 

 lysis occurs. During the process it is easy to verify the presence 

 of the bacteriophage in each passage, and to detect any increase 

 in virulence, simply by spreading the successive cultures on agar 

 slants. Comparison of the cultures secured with each passage 

 reflects the degree of virulence. For example, the agar growth 

 obtained from the first passage shows a culture growth with ten 

 plaques, the second passage shows 100, with the third the layer 

 of bacillary growth is broken up with an abundance of the areas, 

 while with the fourth passage only a few isolated colonies of 

 bacteria are seen. It can be readily seen that the virulence of 

 the bacteriophage, that is, its ability to develop at the expense of 

 the bacteria, increases with each transfer until a point is reached 

 where lysis of the suspension is obtained. 



Successive transfers can be made upon agar slants, taking the 

 material from a tube showing the clear areas. With a platinum 

 wire material can be removed from the bacterial growth bor- 

 dering on a plaque and inoculated on a sterile slant. A sec- 

 ond, third, and fourth (or as many as may be desired) transfer 

 from agar to agar can be made. When a condition is reached 

 where the agar growth shows only fragments of bacterial culture 

 the surface of this tube is carefully washed off and filtered through 

 infusorial earth and a bougie. In the filtrate is found a bacterio- 

 phage sufficiently active to produce lysis of a bouillon suspension. 



As we shall see, the bacteriophage is not destroyed at 65C., 

 that is, at a temperature above the thermal death point of most 

 non-sporulating bacteria. Thus, instead of filtration the appli- 

 cation of heat may be employed. Heating at 58 to 60C. for 

 thirty minutes will kill the bacteria and not harm the bacterio- 



