450 BACTERIOPHAGES 



colorimeter or a nephelometer so that turbidity of a suspension 

 of bacteria can be used as a measure of bacterial concentration. 

 It must be remembered that the calibration curve so prepared 

 can be used only with bacteria grown under the same conditions 

 and in the same medium as those used in the calibration, since 

 a change in environmental conditions often conspicuously alters 

 size and morphology of the bacteria, reflected in changes in 

 turbidity. Also, changes in turbidity of a culture undergoing 

 lysis by phage cannot be interpreted in terms of numbers of 

 surviving bacteria, since the bacterial debris resulting from lysis 

 of a culture may be quite turbid and yet contain few or no 

 viable bacteria. Also, the phage particles themselves scatter 

 light appreciably. 



Assay of Phage by Agar Layer Method 



The agar layer method for plating bacterial viruses seems to 

 have been first described by Gratia (1936c), and is in general 

 use by practically all workers. 



Procedure. The host bacteria and virus particles are mixed in 

 a small volume of warm 0.7 per cent agar and the mixture is 

 poured over the surface of an ordinary agar plate and allowed 

 to harden to form a thin layer. The bacteria grow as tiny sub- 

 surface colonies in this layer and are nourished by the deep 

 layer of 1.5 per cent agar which is used as foundation. The 

 plaques appear as clear holes in the opaque layer of bacterial 

 growth. The soft 0.7 per cent agar is melted in a boiling water 

 bath and cooled in a 46 ° C. water bath. It is then transferred 

 with a warmed pipet in 2.5 ml, amounts to warmed test tubes 

 in the 46 ° C. water bath. The bacterial inoculum is prepared 

 by washing the bacteria from the surface of an agar slant with 

 5 ml. of broth. One drop of the resultant suspension is added 

 to each tube of soft agar. Then the diluted virus in any volume 

 up to 1 ml. is pipetted into the tubes of soft agar and the mixture 

 poured immediately over the surface of an agar plate. The 

 plate is rocked gently to mix the bacteria and virus particles and 

 set aside to harden. Both the 1.5 per cent agar and the soft 



