Platings from the intermediate colony type showed a further 

 breaking up into: 



(1) An opaque, strongly convex, cream-colored type of colony. This type 

 v/as nonpathogenic, and was referred to as a "rough" type ("R"), 

 (see below). 



(2) A translucent, only slightly convex, bluish-green type of color^yo 

 This type was pathogenic, and v/as referred to as a "smooth" type 

 ("3"), (see below). 



Dissociation in Lithium Chlorid e Broth Series (Duff 1937) 



From lithium chloride the same stable variants arose, although 

 the time required for their production was much longer than in phenol 

 broth. The cream-colored opaque or "R" type of colony, as before, vras 

 nonpathogenic and the greenish-translucent or "S" was pathogenic. The 

 cream-opaque or nonpathogenic type was fuund to be more stable on 

 prolonged culture and produced flocculent growth in broth and friable 

 colonies on agar. The greenish- translucent or pathogenic type produced 

 on inoculation the typical lesions of the disease, was less stable on 

 prolonged culture, and gave butyrous colonies on agar. These cultural 

 types of B. sabnonicida thus shov/ respectix'ely the major characteristics 

 of the two classes of dissociant forms, rough (R) and smooth (S) first 

 described by Arkwright (1921) for members of the colon- typhoid-dysentery 

 group and since recorded hj others for many species of micro-organisms. 



Appearance of G-T:/'pe Cul tures (Duff 1937) 



During the period of dissociation of R and S type organisms in 

 lithium-chloride broth tubes, another colore form appeared on one or 

 two occasions. The colonies were very small (0,2 to 0.3 mm.), transparent, 

 and perfectljr circular, and only became visible after 72 hours' incubation. 

 Slide preparations from these colonies showed a very small. Gram-negative, 

 coccoid micro-organism (average 0,3 by 0,5 ;i), appearing in clumps in a 

 manner similar to the arrangement of the larger B. salmonicida . Colonies 

 of this third culture tj-pe were picked and inoculated on agar slants for 

 future investigation. 



After observing over twenty series in lithium chloride. Duff 

 (1937) noted a sudden auto-sterilization in the tubes. Drops from one 

 tube spread upon agar showed no visible growth even after 10 daj'-s' 

 incubation. Aware of the work of Hadley et al. (1931), he suspected 

 that a G-type organism might be present, and in order to demonstrate its 

 existence, he decided to apply the Hauduroy technique to the contents 

 of the tube. 



23 



