262 THE BACTERIOPHAGE AND ITS BEHAVIOR 



by the previous injection of sterile bouillon) and an anti-cholera serum 

 together with a culture of the cholera vibrio. 



Meissner states that the bacteriophage must be present in the intes- 

 tinal tract of the guinea-pig and pass through into the peritoneal 

 exudate because of the irritation caused by the injection, Flu^^o j^^g 

 suggested that we must not overlook the possibility that the strain of 

 cholera vibrio used in the experiment may have been naturally con- 

 taminated by a bacteriophage, that is, that it was a naturally mixed 

 culture. I am in accord with this view of Flu, particularly because of 

 my experience during 1918 and 1919, when, being very anxious to 

 obtain a Cholera-bacteriophage I subjected a great many guinea-pigs 

 to this test and always failed. The technique which I employed re- 

 sembled that of Meissner but the strains of vibrio which I used were 

 ultrapure. 



Flu^^o hag isolated a bacteriophage from a naturally mixed strain. 

 Here is the summary given by him in his paper. 



The author has isolated an anti-cholera bacteriophage from a pure culture of 

 Vibrio cholerae. The bacterial strain had been isolated in 1915, from the stools 

 of a patient in Java. 



In order to isolate the bacteriophage the strain was cultivated upon an agar 

 slant and the growth was suspended in a small amount of bouillon. The suspen- 

 sion was combined with an equal quantity of anhydrous sodium sulphate, thus 

 forming a dry mass. This mass was ground for a half-hour and suspended in 100 

 cc. of bouillon (pH 7.5). The suspension was then heated for an hour at 58°C. 

 and distributed in 10 cc. portions among 10 flasks, each of which contained 100 

 cc. of bouillon and one loopful of an 18-hour culture of a strain of cholera vibrio 

 differing from the ground up strain. After incubation for 1 or 2 weeks the ma- 

 terial was filtered through a candle and the filtrate was tested for a bacteriophage 

 against 10 strains of the cholera vibrio. 



Of the 10 strains examined one was found to be infected by the anti-cholera 

 bacteriophage. 



The anti-cholera bacteriophage is very sensitive and can not be cultivated 

 with lysogenic strains. 



In order to cultivate and preserve this bacteriophage it is necessary to inocu- 

 late bouillon with strains which are readily lysible and to use but a very small 

 amount of an 18-hour culture. This should be placed in the incubator and 

 should be watched continuously from hour to hour. When the tube containing 

 the bacteriophage is clear it should be filtered through a candle and a new in- 

 oculation of bouillon with the filtrate should be made. 



The bacteriophage became very active, still giving lysis in dilutions up to 10"'' 

 but it remained entirely inactive for the strain from which it had been isolated. 



For the cultivation of an anti-cholera bacteriophage from the feces of a cholera 

 patient, it is suggested that the feces be diluted with saline (0.9 per cent), 

 that the material be heated for an hour at 58°C., and that the cooled liquid be 



