536 THE BACTERIOPHAGE AND ITS BEHAVIOR 



lus. He found that, even after 24 hours, there was some increased resist- 

 ance, and that the immunity was fully acquired by the 4th day. 



In a third series of experiments he tested the subcutaneous route as a 

 mode of administration of the bacteriophage suspension. A single 

 injection of 2 cc. by this route gave him the same results as had 1 cc. 

 by the intravenous route, provided the suspension was not more than 

 3 months old. Two injections of a suspension 2| years old perfectly 

 immunized the animals. 



Under the direction of Flu, Sardjito^*^" made a study of this question 

 the subject of his thesis for the doctorate. He prepared his suspensions 

 of the bacteriophage by causing a very virulent race of the bacteriophage 

 to act upon a suspension containing 300 million bacilh per cubic centi- 

 meter, the Shiga strain being very toxic. When, after bacteriophagy, 

 the fluid was perfectly limpid, he added a new quantity of a concentrated 

 suspension of bacilli, in such a way as to restore the count to 300 million 

 per cubic centimeter. Again after bacteriophagy he restored the bac- 

 terial suspension a third time, in the same way. When bacteriophagy 

 was finished, the suspension then containing the dissolved bacterial 

 substance of 900 million bacteria per cc, he filtered through a candle 

 and used the fresh filtrate for his injections.* 



Six rabbits inoculated by the subcutaneous route with 0.25 cc. of this 

 bacteriophage suspension were tested after a variable length of time 

 by the injection of 4 surely fatal doses of a culture of Shiga bacilli. 

 The results in this series were: 



Two rabbits, tested after 45 days, resisted without loss of weight. 



Three rabbits tested after 37 days, 2 resisted without loss in weight, 

 1 died. 



One rabbit tested after 31 days died. 



Therefore, after the injection of 0.25 cc. of a suspension of the bac- 

 teriophage 4 out of 6 rabbits had an antitoxic immunity sufficient to 

 enable them to resist the injection of 4 lethal doses of Shiga bacillus 

 culture. 



In a second series, 7 rabbits received a subcutaneous injection of 0.12 

 cc. of the bacteriophage suspension. They were tested by the inocula- 

 tion of 4 fatal doses, as follows: 



* This was a mistake. Sardjito thus lost 9 animals because of the toxicity of 

 the fresh filtrate. He mentions, however, that this course was taken because of 

 the urgency of circumstances. As I have shown elsewhere, the toxicity dimin- 

 ishes considerably as the suspension ages. When the time comes for applying 

 this method of immunization against bacillary dysentery to man, it will be advis- 

 able to age the suspensions for a least a month before use. 



