This technique consisted in placing about 10 drops of broth 

 from a Pasteur pipette on the surface of a primary agar plate (one 

 spread direct from a "sterile" lithium-chloride tube, showing no groTrth 

 after incubation). Just before drawing up the "sterile" broth into the 

 pipette, the tip of the latter was bent in a flame to a slight angle. 

 This allowed the pipette, after delivery of the broth, to be used as a 

 spreader to distribute the fluid over the svirface of the agar. A portion 

 of the fluid was then collected in the pipette, transferred to the 

 surface of two fresh sterile agar plates and spread as before. After 72 

 hours' incubation at room temperature, providing no G-tjiDe colonies 

 appeared, one of two secondary plates was selected and the process was 

 repeated as required. 



IVhen growth appeared, agar slant cultures were made from the 

 colonies. The growth on these appeared as a flat transparent film on 

 moist slants but tending to form discrete colonies on dried agar. 

 Subcultures to broth after a few transfers on agar gave rise to uniform 

 clouding in 72 hours. 



Goldfish were inoculated intraperitoneally with this organism. 

 No micro-organisms were recovered from these fish and no lesions 

 developed. 



Reference may now be made to the small colonies which appeared 

 on agar plates of lithium-chloride broth series on one or two occasions 

 during the phase dissociation of the parent culture. As previously 

 recorded these had been transferred to agar for future examination. 

 Replating showed that they were identical in all respects with the small 

 colonies just described as having been recovered by special methods from 

 apparently sterile lithium- chloride tubes. These earlier cultures were 

 morphologically and biochemically identical with those obtained from 

 "sterile" tubes. 



Duff (1937) felt that the appearance of a minute colony forTi during 

 dissociation, and later from apparently sterile tubes in lithium-chloride 

 broth series of B. salmonicida corresponded most closely with the 

 appearance under similar circumstances of G-type variants of the Shiga 

 dysentery bacterium and of other species recorded by Hadley et al . 

 (1931). 



Recovery of Typical B. salmonicida from G-Variants (Duff 1937) 



Of a number of stock G-strains maintained on nutrient agar, three 

 reverted spontaneously to the E form after 12 months. Cultures of the 

 R form, having exactly the same biochemical characteristics as the 

 reverted G-strains, had previously been further changed to the pathogenic 

 S form thus making a similar procedure unnecessary with these strains. 

 There is, therefore, evidence of the existence of true G forms in B. 

 salmonicida. 



24 



