VIRUS PARTICLES AND THEIR FUNCTIONAL ACTIVITY 339 



cells to infective virus particles, thereby increasing the titration efficiency. 

 The greater plating efficiency of smaller- volume inocula of foot-and-mouth 

 disease can probably be attributed to the increased probability of effective 

 collisions between virus particles and cells. 



III. Methods of Determining Total Virus Particle Concentrations 



Estimates of virus concentrations by infectivity measurements are based 

 upon readily observable manifestations of local or systemic infection. While 

 such estimates may vary in accuracy, depending upon the virus-host system 

 employed, and will yield little if any information regarding the actual 

 number of virus particles present in suspension, they can, nevertheless, be 

 made objectively. Methods of estimating total virus particle concentration, 

 on the other hand, may be subject to greater degrees of micertainty. 



Perhaps the most satisfactory method of enumerating physical particles 

 depends upon their direct visualization in the electron microscope. The 

 obvious problem encountered in this approach is the identification of the 

 virus particle. When jDarticles of unique morphology are isolable only from 

 infected but not from analogous normal tissues there may be little question of 

 their viral identity. If tlie virus is small and sj^herical, however, distinguishing 

 it from the other, nonviral particulate matter of the ceU protoplasm can be 

 difficult. It may help to purify and concentrate such viruses partially and 

 then test the suspected particles for possible association with infective as well 

 as noninfective activities of the disease agent, e.g., agglutination by con- 

 valescent serum or adsorption and elution from red blood cells if it is a 

 hemagglutinating virus. The several criteria applicable to the identification of 

 a virus with particles seen by electron microscopy are reviewed by Bang ( 1 955 ) . 



Indirect methods have also been used for estimating virus particle concen- 

 tration. Here again the results are uncertain because assumptions must 

 necessarily be made which are difficult to verify. One procedure depends 

 upon estimates made of the virus particle's volume, density, and mass and 

 requires preparations of pure virus. Virus purity is a state for which there are 

 no completely satisfactory criteria. Another procedure is based upon an 

 estimate of the number of hemagglutinating particles present in suspension. 

 It is applicable to the myxoviruses and does not require purified preparations. 

 The assumptions which must be made, however, about the mechanics of 

 agglutination may not be justifiable. 



A. Direct Methods: Electron Microscopy 



1 . Sedimentation 



A method for enumeratmg virus particles by sedimentation on a collodion 

 membrane and subsequent examination in the electron microscope was 

 developed by Sharp in 1949. The procedure is simple in principle although 



