ENUMERATION OF BACTERIOPHAGE PARTICLES 29 



gave the phage concentration within the sampHng errors of the 

 two counts. The electron microscopic count was then compared 

 with the plaque count obtained per unit volume of mixture. 

 Such comparisons were made with a number of preparations of 

 phages T2, T4, and T6. The ratio of plaque-forming units to 

 visible particles varied from 0.4 to 1 .4, falling below unity for 8 

 preparations and rising above for 3. These results indicate that 

 the assay methods for T2, T4, and T6 have an absolute EOP 

 greater than 0.5. The low EOP for most of the preparations 

 probably indicates that they contained noninfective particles. 

 A mechanism of origin of noninfective particles of phage T2 

 has been identified by Sagik (1954), and it is likely that similar 

 principles apply to other phages. 



A considerably less accurate way of measuring the absolute 

 EOP involves determinations of the weight of the infective 

 particle by two independent methods. For phage T2 the dry 

 weight per infectious unit in purified preparations (Herriott 

 and Barlow, 1952) is about twice the weight of a single particle 

 as measured by hydrodynamic methods (Taylor, Epstein, and 

 Lauffer, 1955). The correspondence indicates an absolute EOP 

 of about 0.5 and also shows that the phage preparations are not 

 grossly impure. 



A modification of d'Herelle's plaque counting method, called 

 the agar layer method, is almost universally used by phage workers. 

 This method was invented by Gratia (1936c) and independently 

 by Hershey, Kalmanson, and Bronfenbrenner (1943a). About 

 2 ml. of melted 0.6 per cent agar is cooled to 45 ° C. and inocu- 

 lated with a drop of a concentrated suspension of the host 

 bacterium. A measured volume of phage suspension is then 

 added and the entire mixture poured over the surface of a 

 hardened layer of nutrient agar. After the upper agar layer has 

 solidified the plate is incubated. The bacteria grow as a multi- 

 tude of tiny colonies within the soft agar layer, fed by the layer 

 underneath, forming an opaque background against which 

 plaques are easily seen. Advantages of the agar layer method 

 are: (7) phage samples up to 1 ml. in volume can be plated per 



