458 BACTERIOPHAGES 



time much mucoid material was eliminated by spontaneous 

 precipitation. The lysate was clarified by filtration through a 

 10 in., 6 or 7 lb. Mandler candle. The filtered lysate was 

 passed through the 50 ml. concentration bowl of the Sharpies 

 centrifuge at a rate of 2 liters/hr. Bowl speed was 45,000 r.p.m. 

 (49,000 g). The lysate was followed by 1 liter of sterile 0.9 

 per cent saline, pH 6.5, at the same rate of flow. The bowl, 

 plugged and capped, was shaken vigorously and set in the 

 refrigerator overnight. The chilled bowl was again shaken 

 vigorously for 10 min., the 50 ml. of concentrate poured into a 

 sterile beaker, and the bowl washed with three 20 ml. portions 

 of sterile saline. The pooled concentrate and washings were 

 spun in 50 ml. Lusteroid tubes in the angle centrifuge at 2,000 

 g to remove particulate impurities. The supernatant fluid 

 showed a bright blue, opalescent Tyndall eflfect and contained 

 about 5 X 10^^ infectious particles/ml. with an over-all yield of 

 about 90 per cent. The phage in this concentrate was readily 

 sedimented in an ultracentrifuge at 20,000 g in 40 min., form- 

 ing a gel-like pellet. The pellets were easily resuspended in a 

 few milliliters of saline. This twice-concentrated material was 

 then spun in the angle centrifuge at 2,000 g to remove any 

 insoluble material. 



The final product represented a concentration of about 

 360-fold with an over-all yield of infectivity of better than 80 

 per cent. Actual yield of purified material was between 5 and 

 10 mg. /liter of broth lysate. The concentrate appeared to be 

 homogeneous and essentially free from extraneous material 

 when examined in the analytical ultracentrifuge and in the 

 electron microscope. If one were to apply this procedure to 

 other phages it might be well to add 10~^ M magnesium sulfate 

 to the saline to increase stability of the phage (see p. 454). [The 

 use of this method for phage T6 has been described by Putnam, 

 KozlofT, and Neil (1949) and with some improvements for 

 T4 by Jesaitis and Goebel (1955).] 



2. Another simple method for concentration of phage stocks 

 is by removal of water in vacuo. A phage stock clarified by 



