44 THE BACTERIOPHAGE 



Experiment V. One hundred cubic centimeters of a suspension of young 

 Shiga bacilli are prepared in 0.85 per cent saline, neutral to litmus. This 

 suspension is inoculated with five drops of an earlier saline culture of the 

 bacteriophage, and the material is divided into five portions, 20 cc. in each 

 tube. The first of these remains as already prepared. The second is ren- 

 dered alkaline by the addition of NaOH in a quantity sufficient to give an 

 alkalinity equivalent to 10 mgm. of NaOH per liter. The remaining tubes 

 are also rendered alkaline, the third having a reaction equal to 25 mgm. of 

 NaOH per liter, the fourth equal to 50 mgm., and the fifth equal to 100 mgm. 

 After incubation for eighteen hours the first tube shows its original tur- 

 bidity, tube 2 is cloudy, and tubes 3, 4, and 5 are almost perfectly clear. 

 In these the turbidity is such that it can just be detected and is due to the 

 fact that a certain number of the bacilli had died in the saline before they 

 were attacked. When the lysis is completed in the last three tubes, to tube 

 1, which maintained its original turbidity, NaOH is added in an amount 

 equal to 100 mgm. per liter. Twelve hours later lysis has taken place; 

 the suspension has been transformed so that it possesses a transparency 

 comparable to tube 5. It has been demonstrated that this degree of 

 alkalinity by itself is without effect on the Shiga bacilli, since they develop 

 normally in bouillon containing as much as 500 mgm. of soda per liter. 

 From the cleared tubes all subcultures remain sterile. 



The bacteriophage can be indefinitely cultured in series in a 

 slightly alkaline saline solution. Indeed, the salt CNaCl) itself 

 can be dispensed with. Serial cultures have been maintained 

 in chemically pure water containing only 25 mgm. of soda per liter. 



Certain experiments with a synthetic medium, capable of main- 

 taining a culture of the Shiga bacillus, were performed and are 

 not without interest. Growth of the dysentery bacilli and com- 

 plete lysis by the bacteriophage are secured in a medium of the 

 following composition: water, 80 cc; sodium chloride, 1 cc; 

 potassium phosphate, 1 cc; sodium phosphate, 1 cc; and aspara- 

 gine, 3 cc, all in 10 per cent solutions. The medium is rendered 

 alkaline in accordance with the nature of the experiment to be 

 performed. 



In bouillon a relatively high alkalinity does not interfere with 

 lysis. 



Experiment VI. Five tubes, each containing 10 cc. of bouillon ren- 

 dered alkaline to — 8 are seeded with a suspension of the Shiga bacillus and 

 then inoculated with 0.02 cc. of a culture of the bacteriophage. A N/10 

 solution of NaOH is added; to the second tube 0.5 cc, to the third 1 cc, 

 to the fourth 1 . 5 cc, and to the last 2 cc. The first four tubes are perfectly 

 lysed after eighteen hours, only the fifth remains clouded. 



