152 THE BACTERIOPHAGE AND ITS BEHAVIOR 



example, involving virulence determinations of three Shiga-bacterio- 

 phages derived from different sources; the first isolated from the feces 

 of a fowl, the second from the feces of a horse, both having undergone 

 but a single culture passage (+ in potency), the third isolated in 1916 

 from the stools of a convalescent from dysentery, and since that time 

 subjected to about 2000 culture passages. 



Take 3 tubes, each containing 10 cc. of a suspension of Shiga bacilli 

 with 250 million organisms per cubic centimeter. Inoculate 0.01 cc. 

 of the suspension of bacteriophage No. 1 into the first, 0.01 cc. of bac- 

 teriophage No. 2 into the second, and the same quantity of bacterio- 

 phage No. 3 into the third. After 24 hours at 32°C. examination shows 

 that suspension No. 1 is cloudy, suspension No. 2 is slightly cloudy, 

 and suspension No. 3 is clear. Filter the contents of the three tubes 

 through Chamberland L3 candles, yielding thus 3 bacteriophage sus- 

 pensions. Remove 0.55 cc. of suspension No. 1 and make a series of 10 

 dilutions, with 8 platings. This should be adequate to measure the 

 activity for this race is of but low potency. For races Nos. 2 and 3, 

 make the larger series of 12 dilutions and 10 spreadings. After incuba- 

 tion the results are as follows : 



/. Dilutions. After incubation at 32°C. for 24 hours the tubes con- 

 taining fluid media with successive dilutions of the filtrates show : 



Bacteriophage No. 1; the first 2 dilutions are cloudy, the others 



turbid. 

 Bacteriophage No. 2; the first 4 dilutions are clear, the others 



cloudy. 

 Bacteriophage No. 3; the first 5 dilutions are clear, the remainder 

 cloudy. 

 After incubation for 48 hours : 



Bacteriophage No. 1; all tubes are turbid. 



Bacteriophage No. 2; the first 5 are strongly clouded, the others 



turbid. 

 Bacteriophage No. 3; the first 9 are clear, the last 3 turbid. 

 It is obvious that these findings do not express the virulences of the 

 3 races in a precise manner. 



//. Platings. These are incubated for 3 days at 20°C., at the end 

 of which time the readings can readily be made, and are as follows : 

 Bacteriophage No. 1. 



Plating of dilution 10"^; bacterial layer abnormal, roughened. 

 Plating of dilution 10"^; bacterial layer abnormal, roughened. 

 Plating of dilution lO^^; bacterial layer abnormal, roughened. 



