incubated aerobically at 20° C. Colonies of B. salmonicida can be 

 recognized as growth develops hy the characters already described, and 

 the formation of a diffusible broT.vn pigment is specially significant. 

 Separate colonies are subcultured onto agar slopes, and their various 

 biological and biochemical characters ascertained. 



Pathogenicity can be determined? (1) by inoculating one-fifth of 

 an agar slope cultvire subcutaneously into a frog; (2) by applying 

 culture to a scarified area of the skin of a browi trout or ty injecting 

 it subcutaneously with a syringe. When the animal dies, cultures are 

 made from the heart blood to establish the nature of the infection. 



In examining fish which may possibly be carriers of the 

 bacterium, cultvires are made from the kidneys, heart blood, and 

 intestine. 



In both diseased fish and healthy carriers, the kidney is the 

 organ from which B. salmonicida is most readily isolated (Williamson 

 1929). 



The following diagnostic methods for obtaining specimens from 

 heart blood and kidney have been devised to examine salmon without 

 preliminary dissection or interference with the commercial value of the 

 fish. A 2 cc. syringe is employed, using a needle 3 to 4 inches in 

 length. The syringe and needle are sterilized by boiling for five 

 minutes before taking a sample. 



Heart blood 



The position from which the heart is to be reached is ascertained 

 ty the fingers. Actual measurements are useless, owing to variation in 

 size of fish. Pressure is applied on the midventral surface in front 

 of the bases of the pectoral fins, and the finger passed forvrard until 

 an arch of bone is felt. This is formed by the bones at the base of 

 the gill arches. The wedge-shaped area of soft tissue (isthmus), 

 ending anteriorly in a "V^ " is formed by the bones and overlies the 

 heart. The area is sterilized superficially by Tmshing T/ith methyl 

 alcohol, and the needle introduced in the midventral line, just in 

 front of the ejrtreme anterior attachment of the pectoral fins. It is 

 easily detected when the wall of the heart is penetrated. The desired 

 amount of blood (one drop is sufficient) is Tdthdrawn by aspiration and 

 placed on nutrient agar medium, on which it is spread with an inoculating 

 needle. 



Kidney 



A point is found on the side of the fish, vertically above the 

 insertion of the pelvic fins^ about one-quarter the distance from the 

 lateral line to the midventral surface. This position is chosen as being 

 most suitable, because other viscera are less likely to be injured in 



50 



