468 BACTERIOPHAGES 



phage. An aliquot of the supernatant fluid is then accurately 

 assayed for residual free phage. The entire procedure from the 

 start of the adsorption process to the assay of free phage in the 

 centrifuge supernatant fluid must be completed before the end 

 of the latent period of phage growth to insure that lysis of in- 

 fected bacteria does not liberate additional free phage. The 

 dilution may be made into chilled medium and centrifugation 

 carried out in the cold to gain a little more time. This com- 

 plication can be avoided, of course, by using killed bacteria, 

 but it may be anticipated that the rate of adsorption will be 

 less than with growing bacteria. 



A plot of log per cent free phage remaining as a function of 

 time should give a straight line, the slope of which can be used to 

 calculate K. If the rate of adsorption is so slow that less than 20 

 per cent of the added phage is adsorbed during the available 

 time, this method cannot be used because the accuracy of phage 

 assay is not sufficient to give a reliable set of determinations of 

 free phage. If the proportion of phage adsorbed is much more 

 than 99 per cent, the method becomes unreliable because of 

 incomplete sedimentation of infected bacteria during the cen- 

 trifugation period, which leads to falsely high values of free 

 phage. This source of error can be reduced by centrifuging 

 for short periods in a high speed angle centrifuge instead of in 

 the relatively slow centrifuge usually used to sediment bacteria. 



[A recent method for measuring free (unadsorbed) phage is 

 to treat the infected culture with chloroform which inactiv^ates 

 the infected bacteria. In this procedure the adsorption mixture 

 is diluted into broth containing chloroform (4.5 ml. of broth 

 with 0.5 ml. chloroform), shaken vigorously and then assayed 

 for free phage. This method has been used successfully with 

 T2 and T4 but should be applicable to other phages which are 

 not inactivated by chloroform. Since infected bacteria con- 

 taining mature phage are lysed by chloroform to yield viable 

 phage (Sechaud and Kellenberger, 1956), it is essential to treat 

 the adsorption mixture with chloroform early in the latent period 

 of phage development. ] 



