340 C. E, SCHWERDT 



somewhat tedious and complex in execution. A glass coverslip coated with a 

 film of collodion is placed on the bottom of a specially designed analytical 

 ultracentrifuge cell which is fiUed with the virus suspension. The virus 

 particles are sedimented onto the film, which is then prepared for examination 

 in the electron microscope. The number of particles comited per unit area of 

 collodion membrane is related to their concentration in suspension and to the 

 volume from which they were sedimented. The counts obtained in this way 

 appear to be closely correlated with the dilution factor of the original suspen- 

 sion and with the particle concentration estimated indirectly from physical 

 data (Section III, B), 



This technique can be applied to the counting of virus particles from 

 suspensions of purified virus or even from crude suspension, provided the 

 virus particle has been previously identified and possesses a readily recogniz- 

 able size and shape distinguishing it from the nonviral particulate matter 

 released by disrupted cells. It offers the advantage of permitting counts of 

 particles suspended in relatively low concentrations (lO'^/ml.), but suffers the 

 disadvantage of requiring distilled water rinses after sedimentation to remove 

 salts which, when dried, may interfere with the examination of the specimen 

 in the electron microscope. Such rinsings may remove particles as weU as 

 salts if conditions are not carefully controlled. 



A modification of this basic method has been described by Sharp and 

 Beard (1952) which entails the sedimentation of the virus particles upon an 

 agar block instead of a collodion film. The sedimented particles are fixed with 

 osmic acid, removed quantitatively from the agar by means of a collodion 

 pseudoreplica, and subsequently counted in the electron microscope. This 

 improvement in design permits the sedimentation of virus particles from salt 

 solutions, since the salts diffuse into the agar and away from the virus on its 

 surface. Thus, high local concentrations of salts are avoided in the vicinity of 

 the virus particles during drying, minimizing possible distortion of particle 

 shape and obscuration of particle image in the electron microscope. Excellent 

 agreement was found by Sharp and Beard (1952) among the results obtained 

 by the two sedimentation methods and the spraying technique of particle 

 counting described below. 



2. Spraying 



The technique of electron microscopic counting of virus particles in 

 sprayed microdrops was developed in 1950 by Backus and Williams. It is 

 easily applied to virus suspensions of high concentration (10^ to 10^" particles/ 

 ml.) in water or volatile salt solutions, such as ammonium acetate and 

 ammonium bicarbonate. The same requirement holds here as for any electron 

 microscopic method, namely, ready identification of the virus particle on the 

 basis of size and morphology. 



