VIRUS PARTICLES AND THEIR FUNCTIONAL ACTIVITY 341 



Tlie virus concentrate is mixed with a suspension of polystyrene latex 

 reference particles of known concentration. Since the latter are spheres of 

 uniform diameter and density, their mass can be calculated and their con- 

 centration in suspension estimated with a fair degree of precision by 

 gravimetric means. The mixture is sprayed from a spray gun of simple 

 construction or from a commercially available nebulizer upon electron 

 microscope specimen grids covered with collodion. Individual droplets can be 

 examined in their entirety in the electron microscope and counts made of 

 both reference and virus particles per droplet pattern. The ratio of the 

 number of latex to the number of virus particles provides an absolute assay 

 of the latter per unit volume of the mixed suspension. Bovine albumin added 

 to such a mixture prior to spraying ensures the uniform dispersion of particles 

 and delineates the droplet pattern on the collodion film. Backus and Williams 

 (1950) found that the estimated standard deviation of the mean ratio of 

 virus to reference particle numbers coimted m 20 or more droplet patterns 

 generally lay close to the statistically anticipated value, suggesting that all 

 particles are randomly mixed in suspension and that each droplet is a 

 representative sample of the entire mixture. 



The method has been most effective in establishing the ratio of particle 

 numbers to infective units for several viruses of distinctive and character- 

 istic morphology, in particular the T series of coliphages (Luria et al., 1951), 

 meningopneumonitis virus (Crocker, 1954), the myxoviruses (Isaacs and 

 Donald, 1955), and several poxviruses (Dumbell et al., 1957). It has also been 

 used to identify the poliovirus particle as a sphere 28 m.fjL in diameter 

 (Bachrach and Schwerdt, 1954) and to establish the ratio of the number of 

 such spheres to plaque-forming units in tissue culture (Schwerdt and Fogh, 

 1957). 



3. Agar Filtration 



Kellenberger and Arber (1957) have devised a method for the quantitative 

 analysis of particle suspensions which embodies features of both the sedi- 

 mentation and spraying techniques. In this procedure a suspension of virus is 

 mixed with a known concentration of polystyrene latex spheres. An aliquot of 

 this mixture is spread over a collodion film formed on agar which contains 

 exactly the same medium as that used to suspend the virus and reference 

 particles. The liquid medium (with its dissolved salts) filters tlirough the 

 collodion membrane and diffuses into the agar gel. After such filtration, the 

 film is fixed over formol and sections are floated off the agar and collected on 

 specimen grids for electron microscopy. When a 0.1 -ml. aliquot of a suspension 

 containing 10^" particles per ml. is spread over a surface area equivalent to 

 that of an ordinary petri dish (50 cm.^), the number of particles observed per 

 field at 4600 X magnification averages 48. In this concentration range, the 



