336 C. E. SCHWERDT 



particular virus dilution is greater than would be expected if the pock count 

 truly reflected the random distribution of infective virus particles suspended 

 in the inoculum (Burnet and Faris, 1942; Fenner and Mclntyre, 1956; 

 Kaplan and Belyavin, 1957; Armitage, 1957). One possible reason for a 

 variability greater than expected on a Poissonian distribution of lesions is the 

 variability of susceptibility of individual chorioallantoic membranes (Kaplan 

 and Belyavin, 1957). Other considerations which may contribute to increased 

 variability of pock counts are: (1) nonspecific lesions resulting from injury to 

 the membrane, with a concurrent decrease in its susceptibility to pock 

 formation in the region of damage (Overman and Tamm, 1956a) and (2) the 

 appearance of secondary or satellite lesions, particularly on membranes that 

 remain moist after the initiation of a lesion. Improvements in the technique 

 of inoculation have succeeded in reducing the count scatter, although it still 

 remains in excess of that expected on a Poisson distribution (Westwood et al., 

 1957). 



The frequent observation that animal viruses propagating in monolayer 

 cultures of mammalian cells produced a grossly visible cytopathic effect 

 was quickly exploited by Dulbecco (1952) for the development of a plaque- 

 count assay. This opened the door to quantitative studies with animal 

 viruses equivalent in precision to those made with bacterial viruses. 

 Bacterial and animal virus plaque-count assays are analogous in principle. 

 The procedure in general is less susceptible to the errors and uncertainties 

 inherent in the pock count assay on chorioallantoic membranes of chick 

 embryos. 



Monolayer cultures for plaque assay are generally prepared by seeding 

 trypsinized cells from freshly excised tissues (Dulbecco and Vogt, 1954a; 

 Youngner, 1954) or from cultures of established cell lines (Scherer et al., 

 1953) in petri dishes or small flat bottles under an appropriate medium. The 

 cells attach to the glass surface and produce a confluent growth after incuba- 

 tion at 37°C. for a varying number of days. Virus inocula are then introduced 

 and the infected cultures layered with agar containing a maintenance medium. 

 The necrotic plaques which ultimately appear in the sheet of healthy cells 

 represent primary foci of infection. Secondary plaques fail to arise since virus 

 dissemination is prevented by the agar overlay. 



The plaque assay has been applied to an increasing number of animal 

 viruses. These include western equine encephalomyelitis and Newcastle 

 disease viruses (Dulbecco, 1952; Dulbecco and Vogt, 1954b); vaccinia (Noyes, 

 1953); the human polioviruses (Dulbecco and Vogt, 1954a); fowl plague 

 (Hotchin, 1955); certain strains of influenza virus (Ledinko, 1955; Granoff, 

 1955); foot-and-mouth disease (Sellers, 1955; Bachrach et al., 1957); Rift 

 Valley fever (Takemori et al., 1955); as well as Coxsackie and ECHO viruses 

 (Hsiung and Melnick, 1955). 



