The mixtures in the flasks were incubated at 20° C. for 24. hours. 

 Then equal quantities of culture from each flask were thoroughly mixed 

 together in a sterile flask and filtered through a Chamberland L5 filter. 

 The filtrate was kept at room temperature for 48 hours to test its 

 sterility and stored in an ice chest in glass ampoules or stoppered test 

 tubes until required. 



Technique in isolation and demonstration 



Christison and associates (1938) then made stroke inoculations 

 of the test organisms on agar plates with dense broth emulsions of 24. 

 hours' agar slope cultures as the inoculum. After diying in the incubator 

 at 37° Co 3 drops of the filtrates were placed on the strokes and the 

 plates were incubated at 25° C. for 48 hours. The results were then 

 readj the presence of phage being indicated by the partial or complete 

 absence of growth on the areas on which the drops had been placed. Four 

 filtrates and four strains were tested on one agar plate. 



A number of strains of B. salm.onicida were used for the isolation 

 of bacteriophage owing to the possibility of there being a number of 

 types of phage. In such a case one or more strains of the organism 

 might be resistant to one particular type of phage and thus prevent 

 its development and recognition. The use of twelve strains^ chosen at 

 random^ reduced this possibility to a minimum and rendered the isolation 

 of phage from any particular specimen of water more likely. 



The cultures of the strains to be used were also tested for the 

 presence of phage^ by growing them in broth in the same combinations 

 and under exactly the same conditions as in examining river waters^ and 

 testing each against the combined filtrates. Tliniile the results indicated 

 that weak phages were possibly present in some of the strains first 

 used^ it was very evident that the powerful phages obtained when river 

 waters were added could not be accounted for by the enrichment of phage 

 types alreacfy present in the cultures of the test strains. 



Occurrence 



In 1932 a bacteriophage for B. salmonicida was found in two 

 separate cultures of the organism isolated from sea trout which had 

 died of furunculosis in two Scottish rivers (Furonculosis Committee 

 1933). Todd (1933) also demonstrated a phage active towards B. 

 salmonicida in the waters of certain English rivers. The phage has 

 since proved to be widespread in natural waters throughout Great Britain 

 (Christison et ^ 1938). 



Various questions have arisen from the discovery of this lytic 

 principle fcr B. salmonicida in natural waters. Todd (1933) thought 

 that the presence of this phage in a river might be an indication of the 

 presence of furunculosis of fish in that river. However, the results 



27 



