of the Fishery Board for Scotland. 41 



By the above method I followed Houston's "Flaginac " basis of classification 

 for Bacillus Coli. When these tests are positive, the Bacillus Coli is 

 typical or indistinguishable from the typical Bacillus Coli of the human 

 intestine. If some of the tests are negative, or not well marked, then the 

 bacillus is considered atypical. Consequently the bacilli found were of the 

 type Bacillus Coli Communis. 



Blood Cultures. 



In three of the fish examined, growths of colonies appeared in the Agar 

 tubes. When tiiese were stained and examined they were found to be pure 

 growths of cocci, and gave the essential tests for the stapblococcus pyogenes 

 aureus. 



Peritoneal Fluid Cultures. 



These were all negative. 



Skctions 



Besides pursuing the ordinary methods, I also adopted the method of 

 preparation and staining of sections which was first introduced by Levaditi, 

 and described by him in the " Annales de Institut Pasteur," January 1906. 



Levaditi and many others who were working with him found great 

 difficulty in differentiating the Spirochaeta Pallida in sections by means of 

 the ordinary stains, and devised the following method which was adopted 

 here with the hope that the vibrio-like micro-organisms, which were seen in 

 all the smear preparations made from the ulcers while the fish were still 

 alive, might be exhibited, if present, in sections of the tissues. 



Method. 



(1) Small blocks of tissue were cut out through the ulcers about 1 mm. 

 thick, and fixed in 10 per cent. Formol for 24 hours. 



(2) Then washed and hardened in 96 per cent. Alcohol for 24 hours. 



(3) Washed in Aq. dest. for some minutes until the fragments fell to the 

 bottom. 



(4) The blocks were then impregnated at 38°C. for 5 days in 2 per cent, 

 silver nitrate. 



(5) Washed for a few minutes in Aq. dest. and reduced in the following 

 mixture at room temperature from 24 to 48 hours : — 



Ac. pyrogallic, . . . . 4 per cent. 



Formol, . . . . . . 5 cc. 



Aq. dest. . . . . . . 100 cc. 



(6) Washed in water, dehydrated in alcohol xylol, paraffin sections. 



(7) Some sections were mounted in xylol and Canada balsam, and some 

 were further treated by — 



{a) Giemsa's Solution. 



{h) Toluidin Blue (method of Manourlian). 



On examination the ulcers presented the appearance as already described. 

 The floor of the deeper ulcers extended down to the muscular tissue. Small 

 irregular masses of cocci were present in all, and were very numerous in most 

 of the preparations — especially in the subcutaneous tissues and at the 

 spreading margins. The cocci stain very readily with Ag. when precipitated 

 in the metallic state. 



Neither by any of these methods of preparation and examination of 

 sections, nor in any of the preparations from the culture media, were the 

 vibrio -like micro-organisms found, although they were undoubtedly present 



