VIRUS PARTICLES AND THEIR FUNCTIONAL ACTIVITY 345 



as to cause the dimerization of oiily a fraction of the red cells present; in other 

 words, the estimated number of hemagglutinating particles should be 

 considerably less than half the concentration of red cells. Since the paired red 

 cells will sediment more rapidly than the smgle cells, the optical density of 

 the suspension measured at some point midway between the meniscus and 

 the bottom of the tube will decrease more rapidly than that of the control 

 suspension without virus as the dimer boundary passes the light path. Once 

 the dimers have passed the beam of Hght, the slopes of the optical density 

 versus time curves from both the virus plus red cell and the red cell control 

 suspension should be equal. At this stage the difference in optical density 

 between the parallel slopes then represents the concentration of red cells 

 which have sedimented as dimers. One-half this number equals the number 

 of hemagglutinating particles present if the original premise is correct, namely, 

 that each particle present succeeds in agglutinating two red cells. 



This method was developed independently by Levine and associates (1953) 

 and by Horsfall (1954) and has been referred to as the "absolute assay" of 

 virus hemagglutinins. The former workers compared their hemagglutinin 

 comits with parallel particle counts by the electron microscopic spraying 

 technique of Backus and WiUiams (1950) and found them to be approximately 

 equal. 



The validity of this technique has been questioned by Isaacs (1957) and by 

 Tyrrell and Valentine (1957). Their principal objection is directed against the 

 original assumption that the hemagglutinating particles are 100 % efficient 

 in the formation of red cell dimers. They present a body of evidence from 

 which they conclude that the estimates of hemagglutinin particles by the 

 "absolute assay" method is erroneously low by a factor of approximately 10. 

 Levine et at. (1953) and Horsfall (1954), on the basis of their hemagglutinin 

 particle counts, estimated that, under optimmn conditions, 1 or 2 particles 

 represent 1 egg-infective unit. Donald and Isaacs (1954a), on the other hand, 

 fomid 10 particles visible by electron microscopy for each egg-infective unit 

 present in freshly harvested preparations produced mider equally optimum 

 circumstances. Tyrrell and Valentine (1957) made a comparative study of 

 particle counts by the hemagglutination technique and by the two electron 

 microscopic methods of spraying and adsorption on lysed red blood cells and 

 again found 10 times more physical particles by the latter direct methods 

 than by hemagglutination. They suggest that the correspondence Levine 

 et al. (1953) found between their "absolute assay" and spray droplet counts 

 was fortuitous and that the results by the electron microscopic method 

 might have been falsely low because of technical difficulties. Furthermore, 

 Donald and Isaacs (1954b) have shown filamentous influenza virus to be 7 

 to 8 timies more efficient than spherical virus in hemagglutination, thus 

 casting doubt on the presumed 100 % hemagglutinating efficiency of the 



