I. VIRUS PARTICLES 5 



and those isolated by our method, there is a good reason to believe 

 that the former particles are apparently a portion of the latter having 

 peculiar properties as will be fully discussed in a later chapter. Our 

 application of such a method in virus investigation might enable us to 

 succeed in finding facts other researchers might have failed to observe. 



Although the optimal agglutination pH varied to more or less 

 extent with varying kind of viruses, it was found to be about 5.5 with 

 vaccinia and also with many plant viruses, and 4.6 with phage. This 

 pH was markedly influenced by both the kind and the concentration of 

 inorganic salts present in the media. If due regard was not paid to 

 the salt, occasionally virus particles were isolated in an entirely in- 

 activated state, whereas application of distilled water containing no salt 

 yielded usually good results. It should be noted in this connection that 

 sometimes Ringer's solution proved to be better than distilled water 

 while physiological saline solution proved unfavourable (2) (3). Ringer's 

 solution contains, besides NaCI, minute quantities of other salts, espe- 

 cially that of Ca, which may play a role in this case as will fully be 

 discussed later. 



When a desirable precipitate cannot be obtained by a mere adjust- 

 ment of a virus solution to its optimal pH, the addition of alcohol at 

 about thirty per cent is recommended, thereby viruses may become 

 readily sedimentable, although alcohol tends to inactivate the virus. 



The substances isolated by our method are the aggregate of minute 

 particles composed of a protein and lipids. In order to prove these 

 particles to be the virus itself, we carried out a series of experiments 

 from various points of view. A number of reports, however, have been 

 made afterwards by some workers informing that considerable results 

 were obtained by a similar chemical method (4) (5) (6) (7). They used 

 mainly methanol instead of ethanol, and manipulations v/ere always 

 carried out at extremely low temperatures which must have been very 

 effective to prevent the virus from inactivation. 



It is most important, however, that such particles, composed of a 

 protein and lipids, are never peculiar to viruses, but that they can be 

 obtained from normal cells having no concern with viruses. For example, 

 bacterial culture lysed by a phage may give rise to a precipitate by a 

 proper addition of acetic acid, and the precipitate thus produced is 

 composed of the particles with phage activity, whereas similar particles 

 with no virus activity can be isolated from normal bacterial cells ground 

 up mechanically or lysed by proper chemicals. The particles never have 

 virus activity when the cells are decomposed by agents other than 

 viruses (8). 



Whether viruses are involved or not, the yield of the particles by 

 our method is so high that almost the total protoplasm appears to have 



