460 BACTERIOPHAGES 



quantities (8-10 liters). The essential steps of the purification 

 of the lysate involve (/) separation of cell debris by filtration 

 through Hyflow Celite, or by settling and decantation at 5 °C. ; 

 (2) precipitation of the virus at 5 °C. by acidification to pH 

 4.0; (3) enzymatic digestion of extraneous DNA with DNAase, 

 which decreases the viscosity of the virus suspension, making 

 sedimentation easier, and increases the stability of the phage; 

 and (4) diflferential centrifugation, with selection of the fraction 

 remaining in the supernate at 5,000 g but sedimenting in 30 

 min. at 11,000 g. The recovery of active virus in the prin- 

 cipal fraction varied from 27 to 85 per cent. 



5. In many cases a high recovery of smaller amounts of 

 active phage has been achieved essentially through diflferential 

 centrifugation similar to the one described above, but omitting 

 the acid precipitation. Such a procedure should be worked 

 out to suit the properties of any individual phage and host to 

 which it is applied. 



As an example, for T2 or T4, a culture of E. coli B, grown in 

 M-9 synthetic medium and freshly lysed with phage, is either 

 centrifuged at low speed or filtered through Hyflow Celite as 

 described (Herriott and Barlow, 1952). The clear lysate is 

 passed through a Selas filter of porosity #02. It is then cen- 

 trifuged at high speed (12,000 g) for 90 min., the supernate is 

 poured oflf and the pellet resuspended in ^/so to Vs the original 

 volume of 0.15 M phosphate buffer containing 20 /xg. of gela- 

 tin per ml. The simplest method of resuspending the pellet is to 

 leave it undisturbed in the suspension medium a few houi^s with 

 occasional gentle agitation. (Resuspension of the pellet by 

 pipetting can result in loss of active titer.) The phage con- 

 centrate is then treated with DNAase (approximately 1 jug of 

 purified enzyme per ml. for 30-60 min at 37 °C.) and sometimes, 

 if desired, a similar treatment with RNAase. The preparation is 

 then subjected to one or more cycles of high and low speed 

 centrifugation, the final step being a removal of debris at low 

 speed.] 



