404 BACTERIOPHAGES 



ogous bacterial types, or host-range mutants able to multiply 

 in these types. The incidence of either of these two sorts of 

 particle may be sufficiently high (10~^ to 10"^) to cause confluent 

 lysis of a number of types when phages are applied undiluted, 

 and type distinction then becomes difficult if not impossible. 

 For these reasons, Craigie and Yen (1938) used each Vi-typing 

 phage in the highest dilution that would produce confluent 

 lysis on its homologous bacterial type. This dilution was origi- 

 nally designated the critical test dilution by Craigie and Yen but 

 is now more generally known as the routine test dilution (RTD) . 

 As the result of its use reactions on heterologous types are mini- 

 mized and type distinction becomes an easy matter. Although 

 no other phage-typing schemes exist that depend on multiple 

 adaptations of a single phage, it has nevertheless been found that 

 the application of a similar principle to schemes employing a 

 number of unrelated phages aids materially in establishing 

 characteristic reaction patterns for epidemiologically distinct 

 bacterial types. 



c. Technique 



The technique of phage typing is simple and is substantially 

 the same as that described by Craigie and Yen in 1938. The 

 culture to be examined is inoculated into nutrient broth and 

 incubated until a turbidity equivalent to about 5X10^ organisms 

 per ml. is attained. The initial inoculum is so adjusted that this 

 opacity will be reached in about 2 or 2V2 hours. The culture is 

 then spread on the surface of a nutrient agar plate, either as a 

 series of discrete areas or as a complete lawn, allowed to dry, and 

 the phages are spotted serially on to these areas in standard 

 amounts of the RTD delivered either by a standardized loop or 

 pipette. When the spots of phage are dry the plates are in- 

 cubated at a temperature suitable for the system concerned. 

 With the Enterobacteriaceae, first readings are usually possible 

 at 5 to 7 hours and second readings after i'urther overnight in- 

 cubation. The plates are, of course, suitably marked so that 

 the phages that have caused lysis are easily identified. Two 



