at a low temperature. The viability of these suspensions frequently 

 varies depending upon the temperature at which they are stored, the 

 diluting media, the degree of desiocation before storage, and the period 

 of storage. 



An exploratory attempt was made in this investigation to obtain a 

 frozen stock suspension of this virus. Initially, whole moribund fish 

 were frozen and later thawed, blended, and filtered to obtain a suspen- 

 sion of the virus when needed for experimental purposes . This method 

 was unsatisfactory since it was not possible to obtain a consistent, homo- 

 geneous suspension. Moreover, the titer of material obtained in this 

 manner dropped from 10 ~° before freezing to 10" 2 after freezing, but 

 maintained this degree of viability when stored at 0° F. for a period 

 of 1 year. However, if the fish were thawed and refrozen more than once 

 they became innocuous when inoculated into experimental fish. 



In order that a more homogeneous and uniform suspension should be 

 available for experimental use, moribund fish were blended, diluted 1 to 

 10 with tap water, centrifuged, and stored in small glass vials which 

 were sealed with a gas-oxygen flame. These suspensions were then stored 

 at 0° F. The infectious level of these suspensions dropped from 10"° 

 before freezing to 10~2 after freezing; the virulence of this suspension 

 gradually decreased until h months after its preparation it was no longer 

 infectious. It was recognized that the maintenance of a suspension of 

 known virulence might be incumbent upon the temperature at which it was 

 stored and the degree of protein protection if diluted. Therefore, a 

 suspension from moribund fish was prepared by blending infected fish : 

 one-half of the emulsion was diluted 1 ;3 and the other half diluted 1 slO 

 before storage at -J4O F. This suspension had a titer of 10~" before 

 freezing. Three hours after freezing the material diluted lslO had a 

 titer of KT* while the material diluted ls3 had a titer of 10 -6 . The 

 virulence of the latter material decreased rapidly until at the end of 

 2 months it had a titer of 10 -2 . This infectious level decreased to 10 - -'- 

 after the suspension had been held at -hO" F. for a period of 1 year. 

 Virus material, when stored in a lslO glycerol suspension at 12° C, 

 remained viable for only 3 weeks. 



Effect of heat. --The infectious agent was consistently killed if 

 heated for 15 minutes at 60° C. However, in some experiments the in- 

 fectious nature of diseased tissue was destroyed if heated for 1$ minutes 

 at 50° C. or for 3 minutes at 60° C. 



Effect of pH. — Virus suspensions adjusted from pH h to pH 10 and 

 incubated for 1 hour before being adjusted again to neutrality and then 

 inoculated intraperitoneally into sockeye fingerlings were all virulent. 

 The only noticeable difference in mortalities was found in the group 

 inoculated with the suspension incubated at pH h; here, only one-half 

 the number of mortalities developed as compared with the other suspensions, 



23 



