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UBC Theses and Dissertations

Studies on B. Salmonicida Stewart, Beatrice J. 1932-12-31

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STODIES ON 3. S ALMOIN I CxDA By B e a t r i c e - ^ Stewart — 0 O 0 - - A Thesis submitted f o r the Degree of Master of Arts i n the Department of Bacteriology — 0 O 0 — The University of B r i t i s h Columbia A p r i l , 1938, . 1 i ' isxi to e x p r e s s &y nppT-eeiat ioi i to"' D r . D. C. B. B u f f f o r M o a s s i s t a n c e i n t h i s work.' STUDIES OH B. SiLLMQSICIDA Contents H i s t o r i c a l Study of Furimculosis ...... ....... I Introduction.... *. *:................... «.,.,,.., «•*.......... X Examination of cultures »....-...< & ^b) Staining M e t h o d s * * t . r « * * * S : G.J -LUG1 uiXOcL-S- QX1Q. .LOStS XI.SGCL • * • * -* »• • •• .* * •••*»** •*• * * • * * •* .•»•..••.• ^  [d> Morphology of the Bacillus.......•......*...,....*B>. ..'^  } Staining Q u a l i t i e s ............ * f E f f e c t of pH. on the Growth and the Pigment Production.... .14 V i a b i l i t y of B. salapiiicida....................... <............ .15 • !Di s txXXcsci u£?x*.» *• •••* * o«* • »* *»cv a *, .* * .*. * «.*.«a••• ? ••. #. * ».X5 • (c.) Tap water plus sodium chloride...................16 E f f e c t of Temperature on B. salmohielda...................17 Relation to. Oxygen. ...„,**.......-...... . .18 Pathogenicity of B salmon! clda ..............<. .19 |a) Methods used. Ia. pathogenicity experiments........19 b) Detailed d e s c r i p t i o n of Go l d f i s h experiments.-Bi^'*^t- c^.) Other organ!sms i s o l a t e d from Goldfish.. J&j Table 111x3 strs. ting G o l d f i s h experiments. ,54 e) Experimental i n o c u l a t i o n of rabbits with Contents ( c o n t i n u e d ) . I n f l u e n c e o f the o r g a n i c c o n t e n t o f water on the organism,, 3 . s a l m o n i c i d a . . . . . . .» .*• . .• . «*.. . . , 61 [a} P r e p a r a t i o n o f water o f v a r y i n g o r g a n i c c o n t e n t ..61 rb^ Chemica l a n a l y s i s . »-.6S (c) T e s t i n g the I n h i b i t i v e A c t i o n o f O r g a n i c M a t e r i a l Upon the 'Growth o f B . s a l m o n l o l d a . , 65 (d) T a b l e i l l u s t r a t i n g r e c o r d o f growth on p l a t e s o f s e t number 3 .«,,....,.. 68 (e) Graph i l l u s t r a t i n g r e c o r d o f growth on p l a t e s o f .set nunfoer 3......................... . — ........69 C o n c l u s i o n * . . . . . . 70 ^1) Ca rbohydra te r e a c t i o n s 70 y 2) Pigment s o l u b i l i t y 70 p3) R e a c t i o n o f medium... *71 ^4; V i a b i l i t y o f the organism.* *.•».... » .» . . . . . . . . . . . . 7 1 ^ 5) Temperature .72 [6) R e l a t i o n t o o x y g e n . . . . . 7S 7) P a t h o g e n i c i t y Exper iments on F i s h . . . » „ 7S (s> P a t h o g e n i c i t y Exper iments on R a b b i t s . 75 (9) E f f e c t o f o r g a n i c m a t e r i a l on the growth o f the o r g a n i s m . „ ....... 76 Summary - . . . . . . . . . . ....*.........» ............. .78 B i b l i o g r a p h y . 79 STUDIES OH B. SALMONICIDA. H i s t o r i c a l Study o f P u r u n c u l o s i s . As e a r l y as 1894 Emmerich and I ' e i b e l i n Germany, i s o l a t e d and named B a c i l l u s o r B a c t e r i u m s a l m o n i c i d a . the c a u s a t i v e organ ism o f f i i r u n c u l o s i s I n the Sa lmon idae . Some y e a r s l a t e r Marsh i n M i c h i g a n , D. S . A . , d e s c r i b e d an organ ism w h i c h he had c a l l e d B a c t e r i u m t r u t t a e , and which he s t a t e d to be p a t h o g e n i c o n l y t o d o m e s t i c a t e d f i s h , n o t t o those found I n n a t u r a l w a t e r s . This.6rg|ni§m was l a t e r found t o be i d e n t i c a l w i t h the B. s a l m o n i c i d a Of Emmerich and w e i b e l , and t h e i r nom e n c l a t u r e was g i v e n p r e f e r e n c e . In 1911 P l e h n s t a t e d t h a t u n t i l 1909 the ep idemic had been seen o n l y i n d o m e s t i c a t e d w a t e r s , but a f t e r t h a t t ime r e p e a t e d ep idemics were r e p o r t e d i n n a t u r a l waters.j f i n a l l y s p r e a d i n g throughout c e n t r a l E u r o p e . A c c o r d i n g , t o h e r , o l d salmon were more s u s c e p t i b l e , t o the d i s e a s e than t h e young , : b u t t h a t a l a r g e number o f d e a t h s a l s o o c c u r r e d ;among; t^ he y e a r l i n g s . As w e l l as salmon and t r o u t ; p i k e , c a r p , arid t ench ; were a l s o a f f e c t e d by t h e < d i s e a s e . A . T . Master man i n h i s r e p o r t i n 1911 sugges ted tha t d u r i n g t h a t summer the h i g h m o r t a l i t y among' f r e s h water f i s h i n the r i v e r s o f Eng land was due i n some p a r t t o - t h e drought and the h i g h temperatures o f t h a t y e a r , but t h a t i n c e r t a i n r i v e r s the m o r t a l i t y was o u t s t a n d i n g , bo th I n i n t e n s i t y II.' • :and In l e n g t h . The g r a y l i n g , p e a l , and bream were a f f e c t e d as w e l l as the salmon and the t r o u t . These appeared to have d i e d . f rom some s p e c i f i c cause wh ich D r . A r k w r i g h t r e p o r t e d as a f u r u n c u l o s l s e p i d e m i c , a d i s e a s e , the p r o g r e s s o f w h i c h , when a t i t s h e i g h t , was ve ry r a p i d . The m o r t a l i t y was so r a p i d that: i n many cases no s i g n s o f the d i s e a s e were o b s e r v a b l e on the e x t e r i o r o f the f i s h . From the dead f i 3 h A r k w r i g h t i s o l a t e d an. o rgan ism which he s t a t e d to be p r o b a b l y i d e n t i c a l w i t h the B. s a l m o n i c i d a o f Emmerich and W e i b e l , and the B. t r u t t a e o f Marsh . F o l l o w i n g t h i s ep idemic A r k w r i g h t made an e x h a u s t i v e s t u d y o f the b a c t e r i u m . Mettarn, In 1914, r e p o r t e d on an o u t b r e a k o f f u r u n - c u l o s i s i n the R i v e r L i f f e y i n I r e l a n d . lie s t a t e d t h a t the f i s h dead f rom the d i s e a s e "show numbers o f w e l l deve loped b o i l s beneath the s k i n and i n the subcutaneous and muscu la r t i s s u e s " but tha t these symptoms may be absent i n o t h e r f i s h k i l l e d by the same o r g a n i s m . "Care and D i s e a s e s o f T r o u t " , the work, o f H . S . D a v i s i n 1988, g i v e s us a d e s c r i p t i o n o f f u r u n c u l o s l s and d e s  c r i b e s i t as o c c u r r i n g " i n ep idemic form o n l y among t r o u t and sa lmon" , though i t may a l s o a f f e c t a l a r g e number o f f r e s h water and mar ine f i s h . D a v i s a l s o remarks on the development o f b o i l s , and on the p a r t i c u l a r l y heavy t o l l among the young t r o u t . H o m e , i n A u g u s t , 1928, r e p o r t e d on the impor tance o f c a r r i e r s in, the s p r e a d o f the d i s e a s e . He found these to be the main r e s e v o i r o f i n f e c t i o n , f o r the o r g a n i s m - c o u l d be i s o l  a t e d f rom them when no f u r u n c l e s were p r e s e n t , s e v e n t y - s e v e n days a f t e r t h e i n c i d e n c e o f the d i s e a s e . P l e h n , i n her work, had a l r e a d y remarked t h a t the b a c i l l u s , i s met w i t h 0 f a i r l y f r e  q u e n t l y i n the g u t o f outward ly h e a l t h y f i s h i n i n f e c t e d w a t e r s 0 . ....... j. Two years , l a t e r I s o b e l W i l l i a m s o n made f u r t h e r r e p o r t s on f u r u n c u l o s i s o f the Sa lmonidae showing t h a t the ep idemic had i n c r e a s e d both i n r i v e r f i s h and I n t r o u t farms d u r i n g 1928. I n 1930 she advocated the d i s i n f e c t i o n o f the f i s h ova i n the f a r m s , s t a t i n g t h a t they a re a p o s s i b l e v e h i c l e o f I n f e c t i o n . W i t h E l e a n o r Anderson i n the same y e a r she found .33. s a l m o n i c i d a to be a homogeneous s p e c i e s and stated, t h a t I t c o u l d be i d e n t i f i e d by the c o m p l e m e n t - f i x a t i o n r e a c t i o n w h i c h , i n t h i s c a s e , r e q u i r e s a r a t h e r d i f f i c u l t t e c h n i q u e d e s c r i b e d by these workers i n "The I d e n t i f i c a t i o n o f B. s a l m o n i c i d a by the C o m p l e m e n t - F i x a t i o n T e s t - A F u r t h e r C o n t r i b u t i o n t o t h e Study o f F u r u n c u l o s i s o f the S a l m o n i d a e 0 . STDDIES OH B« SALMOSICIDA. Introduction. An i n v e s t i g a t i o n of the epidemic reported f o r the t h i r d year among the game f i s h of the E l k River near Fernie, B r i t i s h Columbia, was begun by Dr. D. C. 3. Duff oh August 12th, 1931. The area of water covered was of about f o r t y miles, extending from below Elko to almost f i f t e e n miles above Fernie. The deadfish, a l l g r a y l i n g , were recovered from the r i v e r . A l l bore cert a i n c h a r a c t e r i s t i c marks, namely :a haemorrhagic, almost c i r c u l a r area between the p e l v i c f i n s , which often extended to the f i n bases. Some specimens were found to be marked i n a s i m i l a r manner on the ventral surface. The peritoneum and the omentum were heavily I n f i l t r a t e d with .blood, and In two of the f i s h the poste r i o r h a l f of the - i n t e s t i n e was swollen and haemorrhagic. In the majority of cases the kidney was a f l u i d mass. The diseased f i s h which were obtained were examined ; at Fernie immediately a f t e r their'recovery from the stream. Pure cultures of a brown pigmenting organism c l o s e l y resembling the B. salmonicida of Emmerich and Weibel and the B. truttae : of Marsh were obtained from the kidney, the dorsal blood . vessel and the i n s i d e p e l v i c sore. The cultures obtained by Dr. Duff were grown on nutrient agar and kept f o r further examination. E x a m i n a t i o n o f c u l t u r e s . 2. ;:l[edla.,- : • • ^ • • ffutrient Agar - B a c t o Agar 1.5^~pH. 7 . 0 . B l o o d Aaar - B a c t o Agar 1.5>> w i t h 5 to 10 d r o p s f r e s h l y drawn h e a r t b l o o d from r a b b i t per p l a t e , y N u t r i e n t B r o t h - B a c t o . B r o t h ^ pH. 6 .8 *• Carbohydrate B r o t h s - S t e r i l e n u t r i e n t - b r o t h to wh i ch 1$ o f the s t e r i l i s e d s u g a r , and brom thymol b l u e , a t the r a t e o f I c e . o f a 1.6^ a l c o h o l i c s o l u t i o n to the l i t r e , as i n d i c a t o r , a re added i n an a s e p t i c manner. pH. 7 . 0 . G e l a t i n - Bac to N u t r i e n t G e l a t i n . L i tmus M i l k - F r e s h s e p a r a t e d m i l k to which a z o - l i t r a i n i s added as i n d i c a t o r . M t r a t e B r o t h - N u t r i e n t b r o t h to which 0.1;» o f p o t a s s i u m n i t r a t e i s added . P o t a t o - F r e s h raw p o t a t o soaked .24 hours i n r u n n i n g w a t e r . Tryptophane B r o t h - 1% s o l u t i o n o f Bac to T ryptophane B r o t h . Serum - Loef f ler»s B l o o d Serum. pH. 7 . 4 - 7 . 6 . S t a r c h Media - (a) N u t r i e n t b r o t h to which 0 . 2 ^ s o l u b l e s t a r c h i s added, pf l . 6 . 8 . (b) N u t r i e n t agar to which 0 . 2 $ s o l u b l e s t a r c h i s added.. pH. 6 . 8 . A l l media were s t e r i l i s e d f o r 20 minutes w i t h 15 pounds p r e s s u r e , w i t h the e x c e p t i o n o f m i l k , which was steamed f o r 30 minutes on t h r e e s u c c e s s i v e d a y s . 3. S t a i n i n g Methods . Wi th a S imple A n i l i n e Dye . F l o o d e d w i th s t a i n f o r one m i n u t e , washed, b l o t t e d , and ••examined.. Gram S t a i n . .F looded w i t h a n i l i n g e n t i a n v i o l e t f o r one h a l f minute. : l a s h e d i n w a t e r . F l o o d e d w i t h G r a n t s i o d i n e s o l u t i o n f o r one and one h a l f m i n u t e s . •Washed i n w a t e r . D e c o l o r i z e d w i t h 95>p a l c o h o l u n t i l no more b l u e c o l o u r Game o u t . Washed i n w a t e r . C o u n t e r s t a i n e d w i t h acqueous f u c h s i a one h a l f m i n u t e . Washed i n w a t e r , c l o t t e d d r y , examined . Wr ight*s S t a i n . 'X, F l o o d e d c o m p l e t e l y w i t h W r i g h t * s " s t a i n , l e f t t h r e e m i n u t e s , then added an e q u a l volume o f w a t e r , drop by d r o p . B l o t t e d d r y , and examined. Methods and T e s t s Osed : S t o c k c u l t u r e s o f a l l o rgan isms were sorai on n u t  r i e n t agar s l a n t s . pH. 7.0. C u l t u r e s were -'examined d a l l y , and any changes r e c o r d e d . The P r e p a r a t i o n o f N u t r i e n t Agar o f a H i g h A l k a l i n e pH. I t was found t h a t on s t e r i l i s a t i o n o f n u t r i e n t agar o f a h i g h a l k a l i n e p H . , when the pf l . was r a i s e d f rom pH. 7.0 to a pH . above 8.0, due to some c h e m i c a l a c t i o n , perhaps h y d r o l y s i s , the medium a c q u i r e d a d a r k brorai c o l o r a t i o n . T h i s was not s u i t a b l e f o r the e x a m i n a t i o n o f the pigment p r o d u c i n g a b i l i t i e s o f the organ ism so the f o l l o w i n g method was n s e d : The agar and the a l k a l i were s t e r i l i s e d s e p a r a t e l y , thenmixed In axi a s e p t i c manner a f t e r c o o l i n g s l i g h t l y . F i n a l l y t h i s was tubed , a l s o i n an a s e p t i c manner, s l a n t e d , and used f o r c u l t i  v a t i o n o f the o r g a n i s m . N i t r a t e s . :f T e s t e d f o r r e d u c t i o n o f n i t r a t e s to n i t r i t e s u s i n g t h r e e drops T r o a m s d o r f ' s r e a g e n t (100 c c . b o i l i n g 20% zinc c h l o r i d e s o l u t i o n t o 4 grains o f s t a r c h , A f t e r d i s s o l v i n g d i l u t e w i t h w a t e r , add B grams zin-c i o d i d e . D i l u t e to 1 l i t e r and f i l t e r ) , then 1 d rop d i l u t e s u l p h u r i c a c i d ( d i l u t e d IJS)., A b lue c o l o u r i n d i c a t e s the p resence o f n i t r i t e s . 5. Tryptophane b r o t h . 1. Tested, c rude c u l t u r e s u s i n g Clough*s M o d i f i c a t i o n o f the E h r l i c h t e s t , w i t h the f o l l o w i n g . reagents* (a) Pa rad imethy laminobenaa ldehyde . (b) H y d r o c h l o r i c a c i d d i l u t e d 1 : 3 . (c) C h l o r o f o r m 0. S . P . •Method;; . . . . . . To 5 c c crude c u l t u r e added. Q > 5 . cc» r e a g e n t (a) a n d l c c . r e a g e n t . (b) * P l a c e d i n b o i l i n g water f o r 20 s econds , . shak ing v i g o r o u s l y , placed, i n i c e water "k m i n u t e . E x t r a c t e d w i t h 1 c c , r e a g e n t (e) . Coin- pared w i t h s t a n d a r d s made i n the same way. A p i n k ; c o l o u r i n d i c a t e d a, p o s i t i v e r e a c t i o n . 2. T e s t e d the. d i a l y a e d c u l t u r e , u s i n g P l o u g h ' s M o d i f i c a t i o n o f / t h e E h r l i c h t e s t . F o r d i a l y s i s f o l l o w e d the method d e s c r i b e d f i n ^ D e t e c t i o n o f I n d o l i n B a c t e r i a l Cu l tu res" - - B u f f , • u s i n g c e l l o p h a n e a s t h e d i a l y z i n g membrane. T e s t e d f o r i n d o l . a f t e r 7 days* 14 days and 21 d a y s . H y d r o l y s i s o f s t a r c h . (a) U s i n g agar p l u s 0.2;£ s o l u b l e s t a r c h . S t r e a k e d p l a t e s and i n c u b a t e d . F l o o d e d the s u r f a c e o f the p l a t e w i t h a s a t u r a t e d s o l u t i o n o f i o d i n e I n o0% a l c o h o l . The c l e a r zone o u t s i d e the a r e a o f growth i n d i c a t e d the e x t e n t o f the s t a r c h d e s t r u c t i o n . . 6 . ' ^ . (b) U s i n g b r o t h p l u s 0.2,2 s o l u b l e s t a r c h . F o l l o w e d E c k f o r d * s method. Examined on the 2 n d , 4 t h , 7 t h , and 1 0 t h d a y s . T e s t e d f o r a c i d p r o d u c t i o n u s i n g brom thymol b lue as i n d i c a t o r . T e s t e d f o r h y d r o l y s i s o f s t a r c h by adding one drop d i l u t e i o d i n e s o l u t i o n t o one drop c u l t u r e on the spot p l a t e , it b lue c o l o u r i n d i c a t e s no h y d r o l y s i s , a r e d d i s h brown c o l o u r p a r t i a l h y d r o l y s i s w i t h the p r o d u c t i o n o f e r y t h r o d e x t r i n , c l e a r n e s s i n d i c a t e s complete h y d r o l y s i s ' w i t h the p r o d u c t i o n o f d e x t r i n o r perhaps g l u c o s e . T e s t e d f o r the r e d u c t i o n o f sugar w i t h F e h l i n g ! s s o l u t i o n by b o i l i n g 1 c c . F e h l i n g r s s o l u t i o n and 1 c c . s o l u t i o n 2 toge ther f o r f i v e m i n u t e s . Then added 2 c c . o f the c u l t u r e f i l t r a t e drop by d r o p . A y e l l o w to orange p r e c i p i t a t e i n d i c  a t e s the p resence o f s u g a r . McFar l and T u r b i d i t y S t a n d a r d s . Used on ly s t a n d a r d s number 1 , 2, and 4 . These were prepared i n the f o l l o w i n g manner. The t u r b i d i t i e s s e r e made by adding bar ium c h l o r i d e i n a l;t s u l p h u r i c a c i d s o l u t i o n forming d i f f e r e n t c o n c e n t r a t i o n s o f bar ium s u l p h a t e . Fo r number 1, 1 c c . o f bar ium c h l o r i d e s o l u t i o n was added to 99 c c . o f water? f o r number 2, 2 c c . o f bar ium c h l o r i d e s o l u t i o n were added to 98 c c . o f w a t e r ; and f o r number 4 , 4 c c . o f bar ium c h l o r i d e s o l u t i o n were added to 96 c c . o f w a t e r . These were made up i n t u b e s , sea led , .and l a b e l l e d . . 7. Morphology o f the b a c t e r i u m . The organ ism was found to be p l e o m o r p h i c . In young • agar c u l t u r e s s m a l l Gram n e g a t i v e c o c c o i d forms., i n c l i n i n g to o v o i d i n some c a s e s , were d i s c o v e r e d b o t h s i n g l y and i n masses The average d i a m e t e r o f these -forms was found to be 0 .5 m ic rons to 1 m i c r o n . In o l d e r c u l t u r e s , e s p e c i a l l y i n b r o t h , g e l a t i n and serum, on. wh ich the organ ism was l a t e r sown, rods or fcacillary forms w i t h rounded ends were o b s e r v e d . These were o f an average l e n g t h o f 0 .75 m i c r o n s t o 2 m ic rons w i t h a w i d t h o f 0 . 5 m ic rons t o 0 . 8 microns. . S p h e r i c a l l o rms were o c c a s i o n a l l y n o t i c e d among t h e s e . No s p o r e s o r c a p s u l e s were observed and no t r u e m o t i l i t y . S t a i n i n g qua l i t i es« . The organ ism s t a i n s e a s i l y , w i t h the o r d i n a r y a n i l i n e d y e s . The Gram s t a i n i s n e g a t i v e and I s o f t e n f a i n t . • •..3, G a l t u r a l C h a r a c te r i s t i c s . F o r o r d i n a r y maintenance o f growth n u t r i e n t agar w i th a pH. o f 7.0 was u s e d . F o r e x a m i n a t i o n o f the c u l t u r a l : c h a r a c t e r i s t i c s the organ isms used were those I s o l a t e d f rom f r e s h g r a y l i n g and sa lmon . The organisms were sown on g e l a t i n , - l i t m u s m i l k , n u t r i e n t b r o t h , n u t r i e n t agar, I c k f o r d ' s b r o t h , potato, n i t r a t e b r o t h , t r y p t o p h a n e b r o t h , ana . L o e f f l e r l s b l o o d • serum, w h i l e n u t r i e n t a g a r and n u t r i e n t agar p l u s 0,2$ s t a r c h wore used f o r p l a t i n g . The c u l t u r e s were examined d a i l y f o r •any changes i n - appearance and a l l r e s u l t s r e c o r d e d . Agar s l a n t s . In 2 days - w h i t e , modera te , e f f u s e , g l i s t e n i n g convex growth which was translneestfc and .slisiy... A b r o s s i s h p i g - : meat i n the medium... In 7 days - an. abundant , e f f u s e growth which was . g r a y i s h i n c o l o u r w h i l e the medium, was brown, t h r o u g h o u t . In IS days - the medium was a d a r k brown t h r o u g h o u t . G e l a t i n s t a b . In S days - f i l i f o r m growth , l i q u e f a c t i o n a t the top i n the form o f a s m a l l s a c . • In some cases a sac l i k e l i q u e f i e d p o r t i o n appeared a t the bottom o f the stab*. In S days - abundant growth w i t h s a c c a t e l i q u e f a c t i o n . * In 7 days - i n f u n d i b u l l f o r m l i q u e f a c t i o n . . Sediment c l i n g s to the s i d e s o f the t u b e s . In 8 days . - complete l i q u e f a c t i o n . In 20 days - a brown r i n g about one c e n t i m e t e r i n h e i g h t a t the top o f the medium* T h i s l a t e r extended throughout the medium. L o e f f l e r ? s b l o o d serum. In .1 day - a moderate , y e l l o w i s h , smooth, f i l i f o r m , g l i s t e n i n g g r o w t h . . In 4 - 5 days - the growth became e f f u s e , the medium had a brownish t i n g e , gas bubb les were n o t i c e d i n the c u l t u r e s . In 6 days - the serum was p a r t i a l l y d i g e s t e d and brown i n c o l o u r . L a t e r t h i s became a mahogany brown i n c o l o u r and a l l d i g e s t e d . N u t r i e n t b r o t h . In 2 days •- an abundant growth w i t h f l o c c u l e s I n the tube and sediment c l i n g i n g to the s i d e s o f the t u b e . In some cases l a r g e f i l m s o f growth were observed I n the c e n t r e o f the t u b e . In 7 days - the growth became more abundant and v i s c i d on a g i t a t i o n . A s l i g h t r i n g o f growth was n o t i c e d a t the top o f the c u l t u r e . 1 0 . In 7 - 20 days - a brown r i n g appeared a t the top o f the tube wh ich s p r e a d g r a d u a l l y throughout the medium u n t i l the whole became a d a r k brown i n c o l o u r . P o t a t o . I n 2 days - the growth was v e r y s c a n t o r a b s e n t . In 5 days - a s c a n t , creamy. r a i s e d , f i l i f o r m g r o w t h . In 7 days - the growth remained about the same, l a t e r becom i n g a d i r t y g r e y i s h c o l o u r . Marsh , D a v i s and Mettam a l l f a i l e d to o b t a i n v i s i b l e growth on p o t a t o , but when smears were made by Mettam a f t e r one month the organism v-as found on the s l i d e . L i tmus M i l k . In 2 days - no c h a n g e s . In 7 days - the m i l k was c o a g u l a t e d but n o t ac id . . I n 13 days - the m i l k was c o a g u l a t e d and p e p t o n i z e d . In 20 days - t h e 1 ' m i l k became a d i r t y y e l l o w c o l o u r . Bck fo rd*s B r o t h . O r i g i n a l pH. o f b r o t h 6 .9 - 7 . 0 . In 2 days pfi . was 6 . 0 . In 3 d a y s pH. was 5 . 8 . In 5 days pH. was 5 . 6 . In 9 d a y s pH. was 5 . 4 . Y/hen i o d i n e was added to the c u l t u r e s a f t e r two days the s o l u t i o n became c l e a r showing complete h y d r o l y s i s o f s t a r c h . . > • 1 1 . ' When t e s t e d w i t h F e h l i n g 1 s s o l u t i o n f o r sugar r e d u c t i o n no r e d u c t i o n o f the sugar c o u l d be o b s e r v e d even a f t e r n i n e d a y s . H i t r a t e b r o t h . when t e s t e d . w i t h T r o m m s d o r f 1 s r e a g e n t n i t r i t e s were observed w i t h i n 5 to 7 d a y s . Tryptophane b r o t h . In 7 days - t e s t e d c rude c u l t u r e f o r i n d o l , Ho i n d o l . In 14 days - no i n d o l i n crude c u l t u r e . In 21 days - no i n d o l I n crude c u l t u r e . When t e s t e d by the d i a l y s i s method as d e s c r i b e d by D. C. B. D u f f i n h i s work on " D e t e c t i o n o f I n d o l i n B a c t e r i a l C u l t u r e s * n e g a t i v e r e s u l t s were o b t a i n e d a f t e r 7, 14 and 21 d a y s . I n 2 1 days pigment was produced i n t r y p t o p h a n e b r o t h - a r i n g o f -2 c e n t i m e t e r s i n h e i g h t a t the top o f each c u l t u r e . 1 Agar p l a t e s . : The c o l o n i e s were v e r y s m a l l , c i r c u l a r , and f i n e l y g r a n u l a r w i t h e n t i r e e d g e s . They became s l i g h t l y convex and t r a n s l u c e n t . As they became o l d e r they c o u l d be s p l i t and pushed over the s u r f a c e o f the p l a t e . A f t e r 3 to 5 days the medium became a d a r k brown i n c o l o u r . I I • 12 . Agar p l u s 0.2/t s t a r c h . • A f t e r 7 days i o d i n e was poured over the p l a t e s which" had been s t r e a k e d . C l e a r h a l o e s appeared around the s t r e a k s showing^'the o rgan ism to u t i l i z e d s t a r c h m o d e r a t e l y . A s lower i growth than t h a t observed on n u t r i e n t agar p l a t e s , was n o t i c e - \ a b l e on these p l a t e s . B lood a g a r p l a t e s . In 2 days - a r e a s o f growth appeared w i t h c l e a r , t r a n s l u c e n t a reas around them. In 3 days - the c o l o n i e s were r o u n d , smooth, r a i s e d , e n t i r e and f i n e l y g r a n u l a r . C l e a r a reas around a l l c o l o n i e s . I n 5 days - the p l a t e s became a d a r k brown, the growth a d i r t y , g r e y i s h c o l o u r . In 7 days - the. growth became g r e e n i s h . Odour. cw, No odour was n o t i c e a b l e on any o f these c u l t u r e s . Carbohydra te r e a c t i o n s . The organ isms were sown on the f o l l o w i n g c a r b o  h y d r a t e s and the changes r e c o r d e d as below: g l u c o s e ( d e x t r o s e ) + i n 2 d a y s , l a c t o s e - i n 2 - 5 d a y s . - s a c c h a r o s e (sucrose.) - i n 1 - 2 d a y s , l a e v u l o s e t i n £ d a y s , manni te * i n 2 - 3 d a y s , s a l i c i n + In 2 - 3 d a y s . \ g a l a c t o s e + i n 2 d a y s , r a f f l n o s e - i n -2 - 3 d a y s . X3 *. i n n l i n - i n 2 - 3 days, maltose + i n 2 - 5 days, arahinose f i n 1 day. x y l o s e 4- In S days, Gas was produced i n glucose and mannite tubes, but was often very s l i g h t . P i gment S o l u b i l i t y . The pigment produced by the organism was found to DO soluble i n water, and when e t h y l alcohol was added to an agar slope the pigment diffused out Into the alcohol. (V- IT '14.: ' E f f e c t o f pE . on the Growth and the Pigment P r o d u c t i o n . C u l t u r e s o f B. s a l r a o n i c l d a were sown on n u t r i e n t agar s l a n t s o f v a r y i n g pH, to t e s t f o r the range o f growth and o f pigment p r o d u c i n g a b i l i t y o f the organism.. A l k a l i n e M e d i a . Agar o f pEL 9,0- - moderate growth w i t h pigment p r o d u c t i o n c o n f i n e d m a i n l y to the growth a r e a . Pigment was produced a f t e r seven d a y s . Agar o f pfi. 9 . 2 - s c a n t growth w i t h no pigment p r o d u c t i o n . Agar o f p H . . 9 . 4 - n o g r o w t h . A c i d M e d i a . Agar o f pH. 5.4 - moderate growth w i t h moderate pigment p r o d u c t i o n i n f i v e d a y s . Agar o f pH. 5.2 - s can t growth w i t h no pigment p r o d u c t i o n . Agar o f pEL 5.0 - ve ry s c a n t growth w i t h no pigment 4 p r o d u c t i o n . Agar o f pH. 4 . 8 - no g r o w t h . There appeared to be a g r e a t e r amount o f pigment produced I n a s h o r t e r t ime on a s l i g h t l y a l k a l i n e medium than on a s l i g h t l y a c i d medium. The optimum pEL f o r pigment p r o d u c t i o n was between 7.4 and 7 .8 1 5 . V i a b i l i t y o f B.« s a l m o n i c i d a . (a) D i s t i l l e d w a t e r , I n d i s t i l l e d water the organ ism was n o t o f t e n r e c o v e r e d a f t e r 24 h o u r s , but i n some cases c o u l d be r e c o v e r e d a f t e r .3 d a y s . - The method used was t h e f o l l o w i n g : * - 250 c e . s t e r i l e d i s t i l l e d water were p l a c e d i n a f l a s k and i n o c u l a t e d w i t h 1 cc. o f a number one M e F a r l a n d s u s p e n s i o n o f the o r g a n i s m . B e f o r e p l a t i n g . e a c h f l a s k was t h o r o u g h l y s h a k e n . P l a t e s were made e v e r y t w e n t y - f o u r h o u r s , and I n d u p l i c a t e i n a l l c a s e s . R e s u l t s were r e c o r d e d as be low. Sample A - made £ 7 / 1 1 / 3 1 - none r e c o v e r e d 5 0 / i l / f e l . -Sample 3 - -Sample 0 - Sample D ** Sample B — Sample Sample G Sample fi * Sample I - Sample J - n ft i i IT W £7/11/51 - 4/12/31 - 4/13/31 - 25/2/32 - 23/2/32 - 1/3/32 - 3/5/3® - 15/3/32 - 15/3/32 - n t? « •a R- H -Ti ff 11 30/11/31. 5/12/31,. 7/12/31. 24/2/32. 24/2/32. 4/3/32, tJ'-SJ'* 19/3/32,. (b) Tan w a t e r . S t e r i l e tap water c u l t u r e s were made i n the same manner, and t e s t e d d a i l y f o r the appearance o f growth on n u t r i e n t agar p l a t e s . Sample A ~ made Sample B - tt Sample C - Sample D - J! Sample E - Sample F - Sample G - tt Sample fi - n Sample I - a Sample J - K Sample £ - It Sample L - n - 2/12/31 - none r e c o 2/12/31 7/12/31 7/12/31 23/2/ 25/2/32 1/3/32 1/3/32 15/3/32 15/3/32 15/3/32 15/3/32 _ © _ n _ » '« ered 5/12/31. 5/12/51. 10/12/31. 10/12/31. 25/2/32. 25/2/32. 13/3/32. IS/3/32. 22/3/32, 22/3/32. 26/3/38- £6/3/52. 18.. (c) Tap g a t e r p l u g , s o d i u g c n l o r i d e , Then the v i a b i l i t y o f the organ ism was t e s t e d on tap water p l u s v a r y i n g amounts o f sodium c h l o r i d e . Eaeh f l a s k c o n t a i n e d 250 c c - s t e r i l e tap water p l u s a c e r t a i n percentage of sodium c h l o r i d e . As i n the above cases one c u b i c c e n t i  meter o f a number one M c F a r l a n d s u s p e n s i o n was i n o c u l a t e d , i n t o each f l a s k . . C o n t r o l s o f s f c e r i l a tap water were made a t the same t i m e , and i n the same manner. F l a s k s were p r e p a r e d i n d u p l i c a t e i n a l l c a s e s . Sample Number % Had.. Date made A . .85 ,»o 0.0 2/12/31 2/12/31 p/l 0/7.1 2/12/31 none r< .# • *. 5 COVC >red 8/12/31. 9/12/31. 22/12/31. 5/12/31. B. 1.0 .85 0,0 ;: 23/2/32 23/2/3-2 23/8/32 23/2/32 •ir. •.fl ip , #• ® IT Tt 3/3/32. o/iS/oS •• 16/3/32. 25/2/32. C, 3.0 1.0 .35 •••5 ' 0,0 29/8/32 29/2/32 29/2/32 29/2/32 29/2/32 iff r ••ii * • -.IT' n '» n n n 9/3/32. 11/3/32. 14/3/32. 24/3/32. 11/3/32. • • •17. g f f e c t o f g e m u g ^ a j ^ r ^ ^ B . . , s a l s o n i c l d a . Sotved foiar n u t r i e n t b r o t h c u l t u r e s w i t h one l o o p f u l o f the -organism from a n u t r i e n t agar s l a n t and k e p t samples a t 37°C,. 28°C- 14°C ( a p p r o x i m a t e l y ) and a t room t e m p e r a t u r e . T e s t e d d a i l y by sub c u l t u r e s on n u t r i e n t b r o t h . Sample £. • Sowed 2/13/51 - Samale B. Sowed 6/1/32 - Sample C. Sowed 18/2/32 - 37°C - no growth on s u b c u l t u r e s o f 18/12/31. 28°C - growth o n s u b c u l t u r e s o f 30/3/32. 14°C ~ growth on s u b c u l t u r e s o f 30/3/32. room - growth on s u b c u l t u r e s o f 30/S/32. 37°C '28°C 14°C no growth on s u b c u l t u r e s o f 2S/3/32. growth on s u b c u l t u r e s o f 50/5/32. growth on s u b c u l t u r e s o f 30/3/32. 37°C - growth on s u b c u l t u r e s o f 30/3/32. 28°C - growth on s u b c u l t u r e s o f 30/3/32.. 14°G - growth on s u b c u l t u r e s o f 30/3/32. 18. R e l a t i o n t o oxygen . Ho work was done i n r e g a r d t o t h e growth o f the organ ism a n a e r o b i c a l l y . I t was o b s e r v e d t h a t when c u l t u r e s v*ere s e a l e d w i t h p a r a f f i n Immediate ly a f t e r i n o c u l a t i o n growth v/as moderate but no p igment was p roduced even a f t e r f o u r t e e n d a y s . When the p a r a f f i n was removed o r when a s u b - c u l t u r e was sown on f r e s h n u t r i e n t a g a r p igment was produced i n two d a y s . '19. -: Pat/hogealci ty -, o-.f B *, gal-;soni€t&& - - ; .1 H e a l t h y l i v i n g trout- d i d n o t s u r v i v e under l a b o r a - I fcory c o n d i t i o n s * As a r e s u l t o f t h i s t h e f o l l o w i n g exper iments 'aveve pe r fo rmed u s i n g g o l d f i s h as t/ie h o s t . The g o l d f i s h nsjnb- v ered 1 t o o l i n c l u s i v e . Methods u s e d I n P a t h o g e n i c i t y exper iments . . I n o c u l a t i o n o f f i s h . C u l t u r e s o n agar o b t a i n e d f rom o t h e r f i s h . , t h r e e to f o u r days o l d , were washed o f f w i t h a s t e r i l e , normal s a l i n e s o l u t i o n and p o o l e d . These were t h e n a d j u s t e d , w i t h the use . o f s a l i n e s o l u t i o n s , , t o the degree o f t u r b i d i t y r e q u i r e d . a c c o r d i n g t o the M c F a r l a a d s t a n d a r d s . The f i s h were h e l d i n :. the. palm o f the hand d u r i n g i n o c u l a t i o n , and a s t e r i l e s y r i n g e : was u s e d . V a r y i n g q u a n t i t i e s o f the. s u s p e n s i o n were u s e d , and the f i s h were i n o c u l a t e d i n t r a m u s c u l a r l y and i n t r a p e r i t o n e a l l y . , rAutopsy o f the f i s h . F o r the au topsy the i n s t r u m e n t s were s t e r i l i s e d by : b o i l i n g . . A s m a l l wooden board was washed t h o r o u g h l y w i t h 95$ . a l c o h o l o r ^%5^ p h e n o l , and the spec imen t o b e . e x a m i n e d , r i n s e d .; o f f w i t h tap w a t e r , t h e n wiped ?d.ih a b s o r b e n t c o t t o n soaked i n - fcu5# p h e n o l . The s c a l e s were removed w i t h s t e r i l e f o r c e p s i n : o r d e r to examine the e x t e r i o r o f the f i s h f o r the- p r e s e n c e o f • haemorrhagic a r e a s . The f i s h was then p l a c e d , abdomen u p , on • the b o a r d , f a s t e n e d i n p o s i t i o n by means o f p i n s p l a c e d i n the head- and tho t - i i l . end t h e abuoui inal - s a i l c u t - b e g i n n i n g a t the a n a l e n d . The s k i n was removed,, p i n n e d t o t h e beard and t h e v i s c e r a l c o n t e n t s were e:vaisined. C u l t u r e s were t a k e n f rom the organs w i t h a s t o r i l e l o o p , sown on n u t r i e n t a g a r , and i n c u b  a t e d , i l f t e r two to t h r e e days p l a t e s were s t r e a k e d from these • c u l t u r e s and i n c u b a t e d . C o l o n i e s r s s ^ e ; a b l i n g those o f B.  s a l a o n i c l d a wore p i c k e d , sown on n u t r i e n t agar end examined f o r p igment p r o d u c t i o n . Tnese were t h e n exaa ined f u r t h e r f o r c a r b o h y d r a t e a c t i o n and t h e i r growth on g e l a t i n , a n d on l i t m u s m i l k . •'21.. D e t a i l e d d e s c r i p t i o n o f G o l d f i s h experiments. G o l d f i s h number 1. 30/10/31 - i n o c u l a t e d i n t r a m u s c u l a r l y w i t h 2/10 c u b i c c e n t i  meters number 1 M c F a r l a n d s u s p e n s i o n o f 3 . s a l m o n i - • c i d a . L e f t a t room t e m p e r a t u r e . 31/10/31 - f i s h remained q u i e t i n w a t e r , w h i t e a t s i t e o f i n o c u l a t i o n , s w e l l i n g below i n o c u l a t i o n s i t e . 2/11/31 - s w e l l i n g , below i n o c u l a t i o n s i t e was l a r g e r . 3/11/31 - f i s h found dead a t 8 A . M. Found a t bottom o f b o w l , e r e c t w i t h t a i l t o u c h i n g the bot tom. ; A u t o p s y . E x t e r i o r e x a m i n a t i o n . The i n o c u l a t i o n s i t e was covered w i t h a s o f t , s l i m y , e a s i l y removed mass . Under low power t h i s appeared to ; be dead t i s s u e . F i s h appeared b l o a t e d and pu lpy around the musc les near the i n o c u l a t i o n s i t e . I n t e r i o r e x a m i n a t i o n . Cut a square open ing around the i n o c u l a t i o n s i t e . Found a l a r g e a b s c e s s f i l l e d w i t h w h i t e p u r u l e n t m a t t e r . The a b s c e s s , which was f o u r m i l l i m e t r e s deep a t the deepest p o i n t and one c e n t i m e t r e i n d i a m e t e r , was surrounded by a l a r g e , . haemorrhagic a r e a o f 1*| c e n t i m e t r e s i n d i a m e t e r . The musc les around t h i s a r e a appeared c o n g e s t e d . The lower p o r t i o n o f the • f i s h , and a l s o the g i l l s appeared n o r m a l . Cut abdomen o f f i s h . The g a l l b l a d d e r was ve ry l a r g e , and p i n p o i n t b l a c k d o t s covered the caeca and the . 2 2 , i n t e s t i n e . The i n t e s t i n e did n o t appear abnormally- congested. . The l i v e r was soft:, p u l p y , and a l a o s t colourless.; while the .kidney was covered with wh i te .spots. The swim bladder appeared- -nonnal,, • S l i c e d f i s h t r a n s v e r s e l y . Found a. d i s t i n c t red spot: below the abscess, on the dorsal region.* Occasional r e d spots were observed i n the muscle .area* .Sowed' cultures: from t h e l l v e r > the kidney.,, the-guts,, the d e p t h of the a b s c e s s and from the o u t s i d e o f the abscess. Examined s l i d e s taken from the w h i t e , pu lpy mass o u t s i d e the sore* the l i v e r , , and the depth o f the a b s c e s s . S t a i n e d these w i t h methy lene b l u e , Gram s t a i n , and «'right Ts s t a i n . R e s u l t s o f m i c r o s c o p i c e x a m i n a t i o n o f s l i d e s . .1 . O u t s i d e sore.. „ . . (a) Methy lene b l u e - the s l i d e c o n t a i n e d i n the m a j o r i t y s m a l l rods w i t h rounded e n d s , s i n g l e and i n p a i r s , 0 . 5 to 0 .7 mic rons i n l e n g t h and 0 . 3 mic rons i n w i d t h . Some o f these were a l m o s t coccus l i k e i n shape . 0°) Gram s t a i n . - The r o d s appeared f a i n t l y Gram n e g a t i v e . (c.) Wr igh t*s s t a i n - no change i n the appearance o f the r e d s . The s l i d e s a l s o c o n t a i n e d t i s s u e c e l l s . 2. L i v e r . (a) Methy lene b l u e - the smear c o n t a i n e d a ve ry few s h o r t rods o f from 0 .5 t o 1 m ic ron i n l e n g t h by 0 .2 - 0 . 5 m ic rons i n w i d t h . 2 3 . (b) Gram s t a i n - the rods appeared to be Gram n e g a t i v e . ( c ) Wr ight*s s t a i n - no change i n the appearance o f the r o d s . 3.. Depth o f a b s c e s s . (a) Methy lene b l u e - an a lmost pure smear o f s h o r t rods o f 0 . 5 — 1 m i c r o n s by .2 - 0 . 5 m ic rons i n l e n g t h . (b) Gram s t a i n - the r o d s appeared Gram n e g a t i v e . (e) Wr ight*s s t a i n - no change i n the appearance o f the r o d s . Wi th f / r i g h t ' s s t a i n i n a l l cases clumps o f c e l l appeared to be p r e s e n t which were thought to be due to the d e g e n e r a t i o n o f c e l l u l a r e l e m e n t s . There were few p e r f e c t c e l l s . Some c e l l l i k e b o d i e s were a l s o f a i n t l y v i s i b l e i n t h e methy lene b l u e s m e a r s . . These appeared egg l i k e i n shape w i t h a dark b l u e s t a i n e d n u c l e u s . The f a c t t h a t the r o d s I n s i d e the body o f the f i s h were l a r g e r than those f rom the o u t s i d e seems to i n d i c a t e tha t the a b s c e s s d i d n o t open to the o u t s i d e . Pure c u l t u r e s o f the o rgan i sm were i s o l a t e d from the a b s c e s s , the l i v e r and the g u t s , i G o l d f i s h number 2 . 30/10/31 - i n o c u l a t e d i n t r a m u s c u l a r l y w i t h 2/10 c c . number one McFar land s u s p e n s i o n o f B. sa lmon i c ida . . L e f t a t room t e m p e r a t u r e . . . . 31/10/31 - whi te m a t e r i a l covered i n o c u l a t i o n s i t e . 2/11/31 - s w e l l i n g a t i n o c u l a t i o n s i t e , whi te m a t e r i a l around g i l l o p e n i n g . 24. •.. 3/11/31 - f i s h remained i n a c t i v e near the bottom o f the b o w l . The i n o c u l a t i o n s i t e was s w o l l e n and d i s c o l o r e d . 4/11/31 - f i s h had d i f f i c u l t y i n r e t a i n i n g b a l a n c e . 4/11/31 - s w e l l i n g a t i n o c u l a t i o n s i t e l a r g e r , d i s c o l o r a t i o n a d a r k e r r e d . The wound was c o v e r e d w i t h a wh i te fungus l i k e g r o w t h . The f i s h had d i f f i c u l t y i n r e t a i n i n g i t s b a l a n c e and the e x t e r i o r was c o m p l e t e l y c o v e r e d w i t h w h i t i s h m a t e r i a l . 7/11/31' - the f i s h was more a c t i v e , , the s w e l l i n g l e s s r e d and l e s s w h i t e m a t e r i a l covered the f i s h . The whi te m a t e r i a l on m i c r o s c o p i c e x a m i n a t i o n appeared t o be clumps o f c e l l and t i s s u e , w i t h few p e r f e c t c e l l s p r e s e n t . 8/11/31 - a t t imes the f i s h appeared a lmost d e a d . 9/11/31 - the wound appeared l o n g e r and d a r k e r u n d e r n e a t h . it d a r k spo t appeared on the s i d e o p p o s i t e the wound, the f i s h appeared f a i r l y a c t i v e . 10/11/31- a d a r k e r r e d l i n e appeared around the wound, l e s s w h i t e appeared on the wound. 12/11/31- the s o r e was open , a l l w h i t e m a t t e r had d i s a p p e a r e d except around the edges o f the s o r e , b l a c k mat ter c o v e r e d the s o r e . B l a c k s p o t s appeared on the f i n s , the t a i l , the g i l l , and on the s i d e s . 13/11/31- the open s o r e was one c e n t i m e t e r i n d iameter w i t h b l a c k around the e d g e s , around the g i l l s l i t s , and on the s i d e s . 14/11/31 - the s o r e was l a r g e r . The f i s h had p e r i o d s o f extreme a c t i v i t y , then i n a c t i v i t y . 18/11/31 - f i s h appeared more a c t i v e . : 19/11/31 - a l l b l a c k marks "had d i s a p p e a r e d . '. 25/11/31 - k i l l e d f i s b f a f t e r 26 d a y s . Per formed a u t o p s y . ; A u t o p s y . E x t e r i o r e x a m i n a t i o n . F i s h appeared a c t i v e , a l l b l a c k s p o t s had d i s a p  p e a r e d . The s o r e was b e g i n n i n g t o c l o s e . I n t e r i o r e x a m i n a t i o n . Cut a s q u a r e open ing around the i n o c u l a t i o n s i t e . Found a s h a l l o w , s o f t wound c o n t a i n i n g r e d d i s h m a t t e r . The f l e s h around the wound appeared f i r m and w h i t e . The g i l l s appeared n o r m a l . Cut abdomen.. The i n t e r i o r o rgans appeared normal w i t h the e x c e p t i o n ox the l i v e r which was s o f t and p u l p y . Sowed c u l t u r e s f rom the open wound, the l i v e r , the h e a r t , t he k i d n e y , the g a l l b l a d d e r and the d o r s a l b l o o d v e s s e l . Made smears f rom the o u t e r s u r f a c e , the open wound the s p l e e n , . i n s i d e the wound, the h e a r t , , and the g a l l b l a d d e r S t a i n e d w i t h methy lene b l u e and examined.. R e s u l t s o f m i c r o s c o p i c examina t i on o f s l i d e s . .1. O u t s i d e wound - o n l y one or two coccus l i k e organisms and one o r two v e r y s m a l l r o d s w i t h rounded ends o f .45 - .65 microns by .25 - 0.3 m i c r o n s . 2 . L i v e r - a v e r y few s n o r t r o d s wi th/rounded ends., a l l about the same s i z e , o f 0*7 mic rons by 0 .2 m i c r o n s . Some c e l l u l a r s u b s t a n c e s were a l s o p r e s e n t . 5 . H e a r t ~ s m a l l r o d s w i t h rounded ends o f 0 . 5 - 1 microns by 0 . 2 - 0 .S m i c r o n s . A l s o some c o c c i i n masses and some c e l l u  l a r m a t e r i a l , 4 . I n s i d e wound - a few s m a l l r o d s o f 0 . 5 - 1 m i c r o n s by .25 - p . 3 m i c r o n s . A l s o o b s e r v e d the p r e s e n c e o f some c e l l u l a r : m a t e r i a l * . . . . 5 . S p l e e n - a v e r y few s m a l l r o d s o f 0 . 5 - 0 .8 m i c r o n s by 0 . 2 - 0 . 3 m i c r o n s . " 6 . G a l l b l a d d e r - c o n t a i n e d s m a l l r o d s o f 0 . 5 - 1 microns by .0.2 - 0 . 3 m i c r o n s . I s o l a t e d pure c u l t u r e s o f B. s a l m o n i c i d a from the i n s i d e o f the s o r e , the l i v e r , the h e a r t , and the k i d n e y . A p p a r e n t l y the f i s h had been a b l e t o r e s i s t the d i s e a s e . I f such were the case t h e n a f i s h which had been s c a r i f i e d would p r o b a b l y n o t s u f f e r any i l l e f f e c t s . G o l d f i s h number 3 . 30/10/31 - s c a r i f i e d f i s h and rubbed c u l t u r e o f organ ism i n t o s o r e . 31/10/31 - o b s e r v e d a s m a l l , wh i te s p o t on the s o r e . Ifo o ther changes were o b s e r v e d . The f i s h remained i n a p p a r  ent good h e a l t h . 2/12/31 - i n o c u l a t e d f i s h i n t r a m u s c u l a r l y w i t h 2/10 c c . 21. . number. 1 M c F a r l a n d s u s p e n s i o n o f B. s a l m o n i c i d a . 3/12/31 - f e d f i s h w i t h -suspension o f o rgan ism mixed, w i t h f i s h food-. The f a e c e s was l o n g and i n a t h i n ; s t r a i g h t l i n e . The i n o c u l a t i o n s i t e was covered l ;. . . . w i t h , w h i t e m a t e r i a l , . .4/12/31 -. o b s e r v e d a d a r k , r e d a r e a around t n s i n o c u l a t i o n s i t e , , and a ne rvous t w i t c h i n g o f the d o r s a l f i n . . 5/12/31 - f i s h f o u n d dead a t 8 JU M . Pe r fo rmed a u t o p s y , ,Autopsy o f f i s h . E x t e r i o r examinat ion . . . "i ... •. - •' . Ho w h i t e m a t t e r on the o u t s i d e o f s o r e but a dark r e d l i n e was o b s e r v e d below i t . . I n t e r ! o r e x a m i n a t i o n . The I n s i d e o f the so re was dark r e d and f i l l e d w i t h p u r u l e n t m a t t e r . The musc le a r e a around the s o r e s o f t , but i n o t h e r p a r t s o f the body was f i r m and w h i t e . Found a l a r g e b l o o d c l o t -underneath the a b s c e s s e d s p o t . The i n t e r n a l organs .of ••the f i s h appeared n o r m a l . Sowed c u l t u r e s on nu t r i en t - agar f rom .the o u t s i d e of the a b s c e s s , the I n s i d e o f the a b s c e s s , the. l i v e r , the s p l e e n , the k i d n e y , the d o r s a l b l o o d v e s s e l , and. the h e a r t . Hade smears from the d e p t h o f the abscess.., the l i v e r , and the spleen. . . S t a i n e d w i t h Gram s t a i n and examined. R e s u l t s o f m i c r o s c o p i c e x a m i n a t i o n o f s l i d e s . 1) The d e p t h o f the a b s c e s s - the s l i d e c o n t a i n e d ve ry small Gram n e g a t i v e r o d s o f 0 .45 - 0 .8 mic rons i n l e n g t h and 2 8 . 0*2 - -0-.3 mic rons i n w i d t h , a l s o Gram n e g a t i v e c o c c i o f a p p r o x i m a t e l y 0.6 m i c rons i n diameter> 2) The L i v e r - the s l i d e c o n t a i n e d some l a r g e Gram p o s i t i v e . . r o d s and some Gram n e g a t i v e r o d s o f 0 . 5 - 1 m ic rons i n !. l e n g t h and 0 .2 - 0 . 3 m i c r o n s i n w id th* 3) The s p l e e n - the s l i d e c o n t a i n e d Gram n e g a t i v e rods o f , 4 5 0 . 9 m i c r o n s i n l e n g t h and 0 .2 - 0*3 microns- i n . w id th* a l s o numerous Gram . n e g a t i v e c o c c i o f 0.6 m ic rons i n diameter» I s o l a t e d pure c u l t u r e s o f 3 . s a l m o n i c i d a f rom the h e a r t , the s p l e e n , and the l i v e r . G o l d f i s h i n u m b e r 4 . 30/10/31 - s c a r i f i e d f i s h and rubbed c u l t u r e o f the organism i n t o the .sore... 31/10/51 - s o f t wh i te fungus l i k e m a t e r i a l was observed on the s c a r i f i e d s p o t . Ho o t h e r changes were observed i n the f i s h . : The f i s h remained i n good h e a l t h and a c t e d i n a normal manner. 2/12/31 - i n o c u l a t e d 2/10 c c . number 1 McPar land s u s p e n s i o n o f B. s a l a o n i c i d a i n t r a m u s c u l a r l y . 3/12/31 - w h i t e fungus l i k e m a t e r i a l was observed a t the i n o c u l a t i o n s i t e . Added a b r o t h c u l t u r e o f the organ ism t o the f i s h f o o d . 4/12/31 - The i n o c u l a t i o n s i t e was d a r k red, . 5/12/31 - The i n o c u l a t i o n s i t e was s w o l l e n and d i s c o l o r e d 29:...,' but the f i s h - remained, a c t i v e , M i r e d b r o t h c u l t u r e o f the o rgan i sm w i t h the f i s h food* 7/12/51 - the f i s h remained a c t i v e but the wound was -swollen and- discoloured» The f a e c e s was two -and one h a l f i n c h e s l o n g , was t h i n and w h i t e . 8/12/31 - o b s e r v e d a l a r g e r e d haemorrhag ic a r e a around the s o r e * The f i s h remained near the top -of the bowl,. had c o n v u l s i v e movements w i t h l a t e r p e r i o d s o f i n a c t i v i t y * 9/12/31 - t h e red. .area, s u r r o u n d i n g t h e s o r e w a s - l a r g e r . The f i s h remained n e a r the s u r f a c e o f the bowl . 10/12/51 - -observed a l a r g e open s o r e . The f i s h remained l e s s a c t i v e , 11/12/31 - a t 9 A. M. found the f i s h f l o a t i n g o n i t s s i d e i n the b o w l . Per formed a u t o p s y . Autopsy., E x t e r i o r e x a m i n a t i o n . A l a r g e h o l e , 1 c e n t i m e t e r i n d iameter and 4 .5 m i l l i m e t r e s i n d e p t h , was observed at. the s i t e o f i n o c u l a t i o n . The s o r e was sur rounded by w h i t e , d e c o l o r i s e d s c a l e s . The t i s s u e i n the h o l e was h a r d , the s c a l e s s o f t and e a s i l y removed. I n t e r i o r e x a m i n a t i o n . Cut abdomen o f f i s h , removed s k i n . Connected w i t h the open wound was a l a r g e a b s c e s s e d a r e a o f 1 c e n t i m e t e r i n d i a m e t e r . T h i s was f i l l e d w i t h whi te p u r u l e n t m a t t e r , and t>0». the a r e a s u r r o u n d i n g the a b s c e s s was s o f t , A r e d haemorrhagic a r e a was found on the musc le below the s o r e . The l i v e r and the k idney were s o f t and p u l p y , and t h i t i s h i n c o l o u r . The swim bladder . , t h e h e a r t , and the s p l e e n were a i l a p p a r e n t l y n o r m a l . Sowed c u l t u r e s from the d e p t h o f the s o r e , the sore from the o u t s i d e , the l i q u i d i n the a b s c e s s , the depth o f the abscess, , the c l o t under the a b s c e s s > the h e a r t , the d o r s a l b lood v e s s e l ^ the sp leen. , the l i v e r , the k i d n e y , and the l i q u i d i n the d o r s a l a r e a . Made .smears, from- the o u t s i d e o f the so re and the depth o f the a b s c e s s . S t a i n e d w i t h Gram s t a i n and examined. R e s u l t s o f m i c r o s c o p i c e x a m i n a t i o n o f s l i d e s . 1) . O u t s i d e of, the s o r e - observed numerous s m a l l Gram n e g a t i v e rods- w i t h rounded ends and l e n g t h o f .45 - 0 .9 m ic rons w i t h a w i d t h o f 0 , 8 ~ 0.3 m i c r o n s . 2) D e p t h , o f the a b s c e s s - an a lmost pure smear o f s m a l l Gram n e g a t i v e rods o f 0 . 5 m ic rons i n l e n g t h and 0 .2 - 0 . 3 m ic rons i n w i d t h . I s o l a t e d pure c u l t u r e s o f B. s a l a o n i c l d a from the h e a r t , the s p l e e n , the l i v e r , the d o r s a l b l o o d v e s s e l , the d e p t h . o f the a b s c e s s , and the c l o t under the a b s c e s s . These c u l t u r e s d i d n o t produce any pigment on agar u n t i l s u b c u l t u r e d f o u r t i m e s , w i t h a p e r i o d o f one week between each two s u b c u l t u r e s . G o l d f i s h number 5 and number 6 . 30/10/31 - added 15 c c . t h r e e day o l d b r o t h c u l t u r e o f • 31». B. s a l a o n l c i d a to the w a t e r . Ho a p p a r e n t e f f e c t s were o b s e r v e d . The f i s h remained i n good h e a l t h * 2/12/31 - i n o c u l a t e d 2/10 c e . number 2 M c F a r l a n d s u s p e n s i o n i n t r a m u s c u l a r l y . 3/12/31 - o b s e r v e d w h i t e m a t t e r around t h e i n o c u l a t i o n s i t e . The f a e c e s was l o n g , t h i n and c o l o u r l e s s . 4/12/51 - a d a r k r e d d i s c o l o r a t i o n , was n o t i c e a b l e around the i n o c u l a t i o n , s i t e * 5/12/31 - the t i s s u e was s w o l l e n around the s o r e but the f i s h remained a c t i v e . 7/12/31 — a r e d haesorr .hag ic a r e a was o b s e r v e d around the sore , - w h i t e m a t t e r was . g i v e n o f f • 9/12/31 - t h e f i s h remained near the s u r f a c e , o f the w a t e r . 12/12/31 - the f i s h grew mors a c t i v e u n t i l 29/12/31 - when I t was p l a c e d i n the i n c u b a t e r a t S6°C. 16/1/32 - i n o c u l a t e d a g a i n w i t h 3/10- c c , number 1 McFar land s u s p e n s i o n o f B . s a l m o n i c i d a . 18/3/32 - n o t i c e d a . s l i g h t s w e l l i n g around the i n o c u l a t i o n s i t e . Ho o t h e r e f f e c t s were o b s e r v e d . 2/2/32 - d e a t h o f g o l d f i s h Ho . 6 due to acc ident . . Ho c u l t u r e s -were o b t a i n e d , 9/2/32 - Removed g o l d f i s h Ho. 5 to room t e m p e r a t u r e . G o l d f i s h number 7 . 2/12/31 i n o c u l a t e d i n t r a m u s c u l a r l y w i t h 2/10 c c . number 1 M c F a r l a n d s u s p e n s i o n o f B . s a l m o n i c i d a . 4/12/31 - r e d haemorrhag ic a r e a s u r r o u n d e d the i n o c u l a t i o n • s i t e . 5/12/31 - the t i s s u e around t h e i n o c u l a t i o n s i t e was s w o l l e n , and red. . The f i s h remained a c t i v e . 16/1/32 - the f i s h was a p p a r e n t l y r e c o v e r i n g from i t s p r e v i o u s I n o c u l a t i o n . * R e i n o c u l a t e d i n t r a m u s c u l a r l y w i t h 3/1Q c c . number 1 I c F a r l a n d s u s p e n s i o n o f B . s a l m o n i c i d a . P l a c e d i n i n c u b a t o r a t 26 °C. 17/1/32 - f ound f i s h dead a t 5 ? . I . Per fo rmed a u t o p s y . Autopsy o f f i s h number 7. E x t e r i o r e x a m i n a t i o n . (a) l e f t s i d e - The p e c t o r a l f i n was turned a n t e r i o r a l l y . A t the base o f the p e c t o r a l f i n was a haemorr - ; h a g i c a r e a of 2 by 4 m i l l i m e t r e s . A t the base o f the c a u d a l f i n was a haemorrhag ic a r e a 2 mm. square,. The anus appeared normal, . (b) r i g h t s i d e . - The p e c t o r a l f i n was normal* The s i t e o f i n j e c t i o n was not haemorrhag ic but there was a marked haemorrhag ic a r e a above the c a u d a l f i n . On the a n t e r - ; i o r b e l l y was a s u f f u s e d a r e a o f 1 cm. by 3 mm. between the p e c t o r a l f i a s > ; • I n t e r i o r e x a m i n a t i o n . There were numerous d a r k spots o f J m i l l i m e t e r i n d iameter on the w a l l s o f the i n t e s t i n e and the per i toneum. The swim b l a d d e r was i n t a c t , the l i v e r s o f t , and the upper i n t e s t i n e f i l l e d w i t h g a s . The deep musc le l a y e r s c o n t a i n e d 3 3 . no h e m o r r h a g i c a reas , but -when the s c a l e s were .scraped off- many s m a l l haemorrhag ic a r e a s were observed a l l a long the f l a n k . Sowed c u l t u r e s 021 n u t r i e n t agar fro© the l i v e r , the body c a v i t y , and the h e a r t * Incuba ted a t 2S°C. I s o l a t e d pure c u l t u r e s o f B. s a l m o n i c i d a from a l l o f t h e s e . . G o l d f i s h , H q , ft, 2/1-2/31 - i n o c u l a t e d i n t r a m u s c u l a r l y w i t h 2/10 c c . number 1 M c F a r l a n d s u s p e n s i o n o f &. s a l m o n i c i d a . From 3/12/31, j to- 29/12/31 - the symptoms were the same as those observed i n g o l d f i s h number 5* 16/1/3.8 - i n o c u l a t e d i n t r a m u s c u l a r l y w i t h 3/10 c e - number I M c F a r l a n d s u s p e n s i o n o f B. s a l m o n i c i d a - Incubated a t 26°0, 17/1/32 - f l s b found de-?d. a t 3 P. M- Autopsy per fo rmed , .Autopsy of, f i s h . E x t e r i o r e x a m i n a t i o n - a) r i g h t s i d e ~ appeared n o r m a l . h ) l e f t s i d e - The p e c t o r a l was tu rned a n t e r i o r - a l l y and had a haemorrhag ic a r e a a t the base . Found a haemor- r b a g i c a r e a a t the base o f the opercu lum. Removed s c a l e s - There was no v i s i b l e haemorrhag ic a r e a w i t h the e x c e p t i o n o f one s o f t s p o t o n the under s i d e ;just below the d o r s a l f i n . The c o n t e n t s o f the body c a v i t y were s o f t and c o n t a i n e d much f l u i d . The swim b l a d d e r was i n t a c t , the i n t e s t i n e c o n s t r i c t e d i n spots. , and f u l l o f g a s . The l i v e r was a lmost f l u i d . The deep musc le was haemorrhag ic i n s p o t s . Sowed c u l t u r e s on n u t r i e n t agar f rom the p e r i t o n e a l f l u i d , the h e a r t , and the k i d n e y * Incuba ted a t 26°C. I s o l a t e d pure c u l t u r e s o f By s a l m o n i c l d a from a l l o f these* g o l d f i s h number 9 ana number 10. 4/1/52 - I n o c u l a t e d 2/10 c c . number 1 M c F a r l a n d s u s p e n s i o n o f B. s a l i a o n i c i d a b o t h i a t r a p e r i t o n e a l l y and i n t r a  m u s c u l a r l y * Incubated a t 26°G.. 5/1/52 - found b o t h f i s h f l o a t i n g on s i d e i n bowl a t 9 A. M. Per formed a u t o p s y . Au tqpsy o f f i s h . E x t e r i o r e x a m i n a t i o n . under the p e c t o r a l f i n s were d e e p , r e d , s o f t a r e a s . The whole o f e a c h f i s h was c o v e r e d w i t h s o f t , wh i te m a t t e r . About |- i n c h f rom the t a i l t n e r e was a dax vk,. r e d , b loody a r e a which d i d n o t go to any depth*. . I n t e r i o r e x a m i n a t i o n . A l l the organs o f the f i s h appeared n o r m a l . Sowed c u l t u r e s o f G o l d f i s h number 9 f rom the h e a r t , the i n t e s t i n a l c o n t e n t s , the p e c t o r a l f i n base , and the haemor rhage n e a r the t a i l . . Sowed c u l t u r e s o f G o l d f i s h number 10 from the b l o o d , the d o r s a l b l o o d v e s s e l , the r e d i n the musc le a r e a , the l i v e r , the i n s i d e o f the s o r e under the p e c t o r a l f i n s . , .und the v e n t . I s o l a t e d pure c u l t u r e s o f B. s a l i a o n i e i & a frosa a l l o f these c u l t u r e s . G o l d f i s h B o . 1 1 . 4/1/52 - i n o c u l a t e d w i t h 2/10 c c . number 1 McFar land suspens  i o n o f B . s a l m o n i c i d a b o t h i n t r a p e r i t o n e a l l y and i n t r a m u s c u l a r l y * 6/1/32 - o b s e r v e d r e d d i s h s p o t a t the I n o c u l a t i o n s i t e . 7/1/32 - f ound f i s h f l o a t i n g on s i d e a t 9 A.. M. Per fo rmed autopsy* : Autopsy o f f i s h . i l s d^iog examinat ion. . Observed a l a r g e d a r k p a t c h under the s c a l e s w i t h an abscess f a r t h e r down o p p o s i t e the p e l v i c f i n , about I c e n t i m e t r e f rom the i n o c u l a t i o n s i t e - . T h i s was s o f t , , d a r k , r e d and f i l l e d w i t h s o f t , r e d matter. . The f i s h i t s e l f was v e r y s o f t , the. s k i n b roke . e a s i l y , and the i n t e r i o r appeared, to be i n a s o f t decayed s t a t e . I n t e r i o r e x a m i n a t i o n . The i n t e s t i n e appeared s l i g h t l y c o n g e s t e d , the s p l e e n was v e r y s o f t and f e l l to p i e c e s e a s i l y . The body was haemorrhagic a l l th rough the musc le a r e a , even on the s i d e o p p o s i t e the i n o c u l a t i o n s i t e . Sowed c u l t u r e s on n u t r i e n t agar f rom the abscess b e f o r e s e a r i n g , the s e a r e d a o s c e s s , tho i n t e s t i n a l c o n t e n t s , the musc le o p p o s i t e the i n o c u l a t i o n s i t e , • t h e s p l e e n - the f l u i d near the back,. I s o l a t e d pure c u l t u r e s o f If. s a l m o n l c l d a f rom a l l rof t h e s e . •Go ld f i sh number 1 2 . 4/1/32 - i n o c u l a t e d w i t h 3/10 c c . number 1 MeFar land s u s - • p e n s i o n o f B. s a l m o n i e i d a . L e f t a t 26°C. 6/.1/32 - a s m a l l r e d s p o t a t the i n o c u l a t i o n s i t e . 7/1/32 - found dead a t S i 3 0 A . U. e r e c t i n bottom, o f j a r . Per formed autopsy,. •'•Autopsy o f f i s h . I n t e r i o r e x a m i n a t i o n , So d e f i n i t e a b s c e s s a t i n o c u l a t i o n p o i n t on the s h o u l d e r but the f l e s h was s o f t and haemorrhag i c . S e v e r a l b l a c k a r e a s on b o t h s i d e s . The bases, o f the p e c t o r a l s appeared : haemorrhag ic* I n t e r i o r e x a m i n a t i o n . The g a l l b l a d d e r was a lmost as large- as the l i v e r , the h i n d gut conges ted and s w o l l e n . S m a l l haemorrhag ic a reas were o b s e r v e d about the anus . Sowed c u l t u r e s on n u t r i e n t agar from the muyc le , the I n o c u l a t i o n s i t e , the p e r i t o n e a l f l u i d , the mid g u t , the l i v e r , the b i l e f rom the g a l l b l a d d e r , the k i d n e y , the d o r s a l b l o o d v e s s e l , the s o f t musc le p a t c h , and from a d o r s a l deep muscle b l o c k , i n c u b a t e d a t 26°C. D i d n o t o b t a i n c u l t u r e s o l 5 . s a l m o n i c i d a fro.a the fcianey, the d o r s a l b l o o d v e s s e l , and the d o r s a l deep musc le ! b l o c k . I s o l a t e d , p u r e c u l t u r e s from a l l the" r e s t . . G o l d f i s h number 1 5 . 9/1/52_ r i n o c u l a t e d f i s h w i t h 2/10 c c number 1 McFar land s u s p e n s i o n o f B»salmonicida i n t r a m u s c u l a r l y . L e f t . a t 26°C. 18/1/32- the i n o c u l a t i o n o f 9/1/32 d i d n o t seem to have a f f e c t  ed the f i s h i n any way. R e l n o e u l a t e d i n t r a p e r l t o n - e a l l y w i t h 3/10 c c . number 1 M c F a r l a n d s u s p e n s i o n o f B. s a l m o n i c i d a . L e f t a t 2 6 ° C 17/1/52- F i s h found dead a t 3:30 P. M. Per formed a u t o p s y . -Autopsy o f : f i s h . E x t e r i o r e x a m i n a t i o n . (a) R i g h t s i d e - the p e c t o r a l f i n was turned o u t , and had a s m a l l haemorrhag lc a r e a a t the b a s e . A .haemorrhagic a r e a on the pedunc le showed through the s c a l e s as d i d a c o n  g e s t i o n a t the i n o c u l a t i o n s i t e . Vfhen the s c a l e s were removed : many minutes haemorrhag ic s p o t s ' were o b s e r v e d over the e n t i r e s ide* (b) L e f t s i d e - The l e f t p e c t o r a l f i n was n o r m a l . When the s c a l e s were removed many minutes haemorrhagic s p o t s were o b s e r v e d over the e n t i r e s i d e . I n t e r i o r e x a m i n a t i o n . The body w a l l s were s o f t and r o t t e n . Thsre was 38* much f l u i d i a the boey c a v i t y , Ths sr;i;i bladdar J/?.S i n t a c t . •Dark spots wore observed on the peritoneum. Several"definite ; ) soft baemarr'aagic areas ?fere observed below the s k i n . These ; iwere of B vim. i n diameter with a pin point of pus i a the centre- of e a c u Sowed only one culture and t h i s ti-cm the peritoneum, "on nutrient agar. Incubated at 26 °C. Obtained a pure culture of B, salmonicida. from the •peritoneum. G oIdfIsh number.14. 9/1/32 - inoculated Intramuscularly tvith 2/10 cc. number 1 McFarland suspension of 5> sal.-.ionieida.. "16/1/32 - the Inoculation of 3/1/32 did not seem to have affected the f i s h i n any way. Re-Inoculated i n t r a - p e r i t o n e a l l y with 3/10 c c . number 1 McFarland suspension o f . B . salmonicida. Left at 26°G. Found f i s h dead at 3 P, M. Death was due to .injury to the swim bladder. Uo cultures of B . salmonicida were obtained. •.Goldfish number 15. 9/1/32 - Inoculated f i s h with 2/10 cc. number 1 McFarland suspension of B . salmonicida. Incubated at 28°C. 16/1/32 - reinoculated witb 3/10 cc. number. 1 McFarland suspension of b, salmonicida i n t r a p e r i t o n e a l ^ . Incubated at 26°C. 5 9 . .17/1/52 - found dead a t 3:30 P. M. Per formed a u t o p s y . iAutopsy . E x t e r i o r e x a m i n a t i o n . (a) L e f t s i d e - p e c t o r a l f i n tu rned out w i t h haemor-j r h a g i c a r e a o f 5 mm. by 3 mm. a t the b a s e . Haemorrhagic spo t i a t I n o c u l a t i o n s i t e . (t>) R i g h t s i d e - p e c t o r a l f i n t u r n e d out w i t h •haemorrhagic a r e a o f 5 mm. by 2 .5 mm. a t the b a s e . A f t e r remova l o f s c a l e s , b o t h s i d e s showed s m a l l haemorrhag ic s p o t s . I n t e r i o r e x a m i n a t i o n . The f l e s h was too s o f t to o b s e r v e . The swim b l a d d e r was i n t a c t . Soiled one c u l t u r e o n l y , and t h i s f rom the p e r i t o n  aeum'. Incubated a t 26°C. F i n a l l y i s o l a t e d a p u r e . c u l t u r e o f 3 . s a l m o n i c i d a f rom t h i s . : G o l d f i s h number 1 6 . 16/1/32 - i n o c u l a t e d i n t r a p e r i t o n e a l l y w i t h 5/10 c c . number 1 M c F a r l a n d s u s p e n s i o n o f B. s a l m o n i c i d a . Incubated a t 26°C. 17/1/52 - f i s h found dead a t 3:30 P. M. Per formed au topsy . A u t o p s y . E x t e r i o r e x a m i n a t i o n . (a) R i g h t s i d e - the p e c t o r a l f i n was tu rned down- . ward, and had a s l i g h t haemorrhag ic a r e a a t b a s e . Removed ' '40. • s c a l e s , found o n l y one o r two haemorrhag ic s p o t s on the b e l l y . (b) L e f t s i d e - Observed a haemorrhagic a r e a the d o r s a l s i d e o f the o p e r c u l u m . O therw ise the l e f t s i d e appeared n o r m a l . Removed s c a l e s , no haemorrhagic s p o t s were } o b s e r v e d . I n t e r i o r e x a m i n a t i o n . There were no v i s i b l e s p o t s o r haemorrhages i n the m u s c l e . The v i s c e r a l organs appeared i n t a c t . The b e l l y was p u n c t u r e d a t the s i t e o f i n o c u l a t i o n . O b t a i n e d o n l y one pure c u l t u r e of B» - s a l m o n i c i d a . T h i s was from the M o n e y . G o l d f i s h number•17. .16/1/38 - i n o c u l a t e d f i s h i n t r a p e r i t o n e a l l y w i t h 3/10 c c . number 1 M c F a r l a n d s u s p e n s i o n o f B. s a l m o n i c i d a . L e f t a t 28°C. 18/1/32 - f i s h found dead a t 9 A . M. Per formed a u t o p s y . : Au t o p s y . E x t e r i o r e x a m i n a t i o n . (a) R i g h t s i d e - The p e c t o r a l f i n was turned a n t e r i o r a l l y . The whole body o f the f i s h was d a r k , the anus r e d d i s h , and the l e f t f i n tu rned o u t . A l a r g e haemorrhagic a rea o f 2 - 3 mm. i n d iameter was observed j u s t below the p e c t o r a l f i n . Below t h i s was a l a r g e d a r k a r e a a lmost § i n c h i n d i a m e t e r . P u r u l e n t mat te r was exuding from the haemorrhagic a r e a . 4 1 . (b) L e f t s i d e •-• s u f f u s e d haemorrhag ic a reas were observed on t h e l e f t s i d e . I n t e r i o r e x a m i n a t i o n . Dark s p o t s were found on the i n t e s t i n a l w a l l and on the p e r i t o n e u m . The swim b l a d d e r was i n t a c t , the i n t e s t i n e c o n g e s t e d . The l ower h a l f o f the f i s h c o n t a i n e d no haemor r h a g e s . Sowed c u l t u r e s f rom the l i v e r , the haemorrhage, the s e a r e d a b s c e s s , the d o r s a l b l o o d v e s s e l , and the p e r i t o n e a l - f l u i d . . I s o l a t e d p u r e - c u l t u r e s o f B. s a l m o n i c i d a from a l l o f these c u l t u r e s . . G o l d f i s h number 1 8 . 21/1/32 - . i n o c u l a t e d f i s h i n t r a p e r i t o n e a l l y w i t h 3/10 c c . number 1 M c F a r l a n d s u s p e n s i o n from c u l t u r e s from t h e k i d n e y and the p e r i t o n e a l f l u i d o f f i s h H o . 8 . Incubated a t 26°C. 22/1/52 - found f i s h f l o a t i n g i n water a t 9 A . H . Per formed an a u t o p s y . •Autopsy o f f i s h . E x t e r i o r e x a m i n a t i o n . The f i s h appeared n o r m a l . I n t e r i o r e x a m i n a t i o n . The l i v e r appeared v e r y s o f t and covered w i t h s m a l l ' dark s p o t s . O t h e r w i s e the organs appeared In a normal c o n d i  t i o n . Sowed c u l t u r e s on n u t r i e n t agar from the l i v e r spots , the l i v e r , the h e a r t , the s p l e e n , and the p e r i t o n e a l f l u i d . Incubated a t 26°C. I s o l a t e d pure c u l t u r e o f B. s a l m o n i c i d a f rom the h e a r t , the l i v e r , the l i v e r s p o t s and the p e r i t o n e a l f l u i d , G o l d f i s h number 19. 21/1/32 - I n o c u l a t e d t h e f i s h i n t r a p e r i t o n e a l ! } - w i t h 3/10 c c . number 1 M c F a r l a n d s u s p e n s i o n o f c u l t u r e o f B. s a l - . m o n i c i d a i s o l a t e d f rom f i s h number 8. L e f t a t 26°C. 22/1/32 - found f l o a t i n g a t 9 A . M. Per formed a u t o p s y . A u t o p s y o f f i s h . E x t e r i o r e x a m i n a t i o n . There was no e x t e r i o r e v i d e n c e o f the d i s e a s e . I n t e r i o r e x a m i n a t i o n . The l i v e r was v e r y s o f t and f l u i d , and covered w i t h s m a l l s p o t s . A l l o t h e r organs appeared to be i n a normal c o n d i t i o n . Sowed c u l t u r e s on n u t r i e n t agar from the l i v e r , the b i l e , the h e a r t , the p e r i t o n e a l f l u i d , the s p o t s on. the l i v e r . Incubated a t 26°C. I s o l a t e d pure c u l t u r e s o f B. s a l m o n i c i d a from the h e a r t , the l i v e r , the b i l e - , the p e r i t o n e a l f l u i d . , and the s p o t s on the l i v e r . G o l d f i s h number 20, 21/1/32 - i n o c u l a t e d the f i s h i n t r a p e r i t o n e a l l y w i t h 3/10 c c . number 1 M c F a r l a n d s u s p e n s i o n o f c u l t u r e o f B. s a l m o n i c i d a i s o l a t e d from f i s h number 8. L e f t a t room t e m p e r a t u r e . 22/1/32 - found f l o a t i n g a t t o p o f bowl a t 12 A . M. Performed;: a u t o p s y . Au topsy o f f i s h . . E x t e r i o r examinat ion• A l a r g e open s o r e sur rounded by a l a r g e haemorrhag ic a r e a was p r e s e n t a t t h e i n o c u l a t i o n s i t e . T h i s had b loody mat te r exuding f rom - i t f o r one hour b e f o r e d e a t h o f the f i s h . The musc le a r e a s u r r o u n d i n g the anus , and t h a t under the p e c t o r a l f i n s , were a l l h a e m o r r h a g i c . A l e s i o n had formed under the c a u d a l f i n> and the musc le between the p e l v i c f i n s was a l s o h a e m o r r h a g i c . I n t e r i o r e x a m i n a t i o n . The I n t e s t i n e was v e r y haemorrhag ic a l l the way from the anus to the s tomach . The o v a r i e s were l a r g e . A l l I o rgans were haemorrhag ic i n p a t c h e s . The swim b l a d d e r was . u n a f f e c t e d . On c r o s s s e c t i o n i n g a l i n e o f haemorrhage was found which extended a l l a long the back o f the f i s h . S m a l l haemorrhag ic s p o t s were observed deep i n the musc le and s u r r o u n d i n g the back bone. These e x i s t e d even i n the head : a r e a . Solved c u l t u r e s on n u t r i e n t agar from the peritoneum, the p o s t e r i o r i n t e s t i n a l c o n t e n t s , the b i l e , and the k i d n e y . I s o l a t e d pure c u l t u r e s o f B. s a l m o n i c i d a f rom a l l these organs.. •; G o l d f i sh number 21. 21/1/32 - i n o c u l a t e d f i s h i n t r a p e r i t o n e a l l y w i t h 3/10 c c , number 1 M c F a r l a n d suspens ion . B. s a l m o n i c i d a o b t a i n e d f rom G o l d f i s h number 8. L e f t at room temperature* 22/1/32 - found f l o a t i n g a t top o f bowl a t 1 P. M. Per formed autopsy. . •  Autopsy o f f i s h . E x t e r i o r e x a m i n a t i o n . The f i s h was very b l o a t e d i n appearance w i t h a haemorrhage o f 0.5 cm, i n w i d t h f rom the s i t e o f i n o c u l a t i o n up the s i d e f o r 3 cm. The p e c t o r a l f i n s and the anus were . a l s o haemorrhag ic and a s m a l l haemorrhagic spo t was found d i r e c t l y a c r o s s f rom the s i t e o f i n o c u l a t i o n . . I n t e r i o r .examination.:. The i n t e s t i n a l organs were haemorrhag ic and the p o s t e r i o r end o f the i n t e s t i n e was s w o l l e n to a d i s t a n c e o f •2 cm* f rom the anus* B l o o d c l o t s were numerous i n the -omentum.. On c r o s s s e c t i o n i n g s m a l l haemorrhag ic spots were round around t h e back bone , a l l a l o n g the l a t e r a l l i n e and . th roughout a l l the musc le a r e a . C u l t u r e s were sown on n u t r i e n t agar from the p e r i t o n e u m , the h e a r t , the k i d n e y , the i n t e s t i n a l c o n t e n t s , and from one o f the b l o o d c l o t s . • Pare c u l t u r e s o f 3 , s a l i s o n i c i d a were i s o l a t e d f rom the h e a r t , the p e r i t o n e u m , the k i d n e y , the i n t e s t i n a l contentsj j B o t h G o l d f i s h number 20 and G o l d f i s h number SI remained v e r y n e a r the s u r f a c e o f the water b e f o r e d e a t h . G o l d f i s h number 22. • •26/2/52 - i n o c u l a t e d f i s h i n t r a p e r l t o n e a l l y w i t h 2/10 c c . o f j a 1 :100,000 d i l u t i o n o f a number 4 McFar land s u s  p e n s i o n o f B. s a l m o n i c i d a . L e f t a t 26°C. 1/2/32 - no v i s i b l e changes were o b s e r v e d . On t h i s da te f i s h d i e d as the r e s u l t o f an a c c i d e n t , Bo c u l t u r e s were o b t a i n e d . G o l d f i s h number 2 3 . 26/1/32 - i n o c u l a t e d f i s h i n t r a p e r i t o n e a l l y . w i t h 2/10 c c . o f .; I s 1 0 0 , 0 0 0 d i l u t i o n o f a number 4 McFar land s u s  p e n s i o n o f B. s a l m o n i c i d a . L e f t a t 28°C, 9/2/32- - no changes were v i s i b l e so removed . to room temperature ' . 20/3/52 - F i s h was a c t i v e , and u n a f f e c t e d by the i n o c u l a t i o n . G o l d f i s h number 2.4. 26/1/52 - i n o c u l a t e d f i s h i n t r a p e r i t o n e a l l y w i t h 2/10 c c . o f a 1;100 d i l u t i o n o f a number 4 McFar l and suspens ion , o f B . s a l m o n i c i d a . L e f t a t 26°C. 9/2/32 - no changes had been observed so removed to room t e m p e r a t u r e . 46.. . 24/3/52 ~ b l a c k s p o t s were o b s e r v e d on the s i d e s o f the f i s h . and on the t a i l . 29/2/32 - the f i s h had a tendency to l o s e i t s b a l a n c e . The s p o t s were becoming larger.., and e x i s t e d around the p e l v i c f i n s and g i l l o p e n i n g s . The f i s h remained m o t i o n l e s s near t h e top o f the water . 2/3/32 - the f i s h remained i n the same i n a c t i v e s t a t e . K i l l e d a t 12 A.. M. Per formed autopsy o f f i s h . Autopsy o f f i s h . E x t e r i o r , e x a m i n a t i o n . S m a l l haemorrhag ic spo ts were observed near the p e l v i c f i n s and a l o n g the muscle a r e a a f t e r the removal o f the s c a l e s . I n t e r i o r e x a m i n a t i o n . . A l l the o rgans appeared n o r m a l . The i n t e s t i n e was haemorrhagic n e a r the anus,. Sowed c u l t u r e s on n u t r i e n t a g a r f rom the h e a r t , the d o r s a l b l o o d v e s s e l , the k idney. , the l i v e r , the a n t e r i o r p o r t i o n o f the i n t e s t i n e , the p o s t e r i o r p o r t i o n o f the i n t e s t i n e , and the s p l e e n . I s o l a t e d a pure c u l t u r e o f B. s a l m o n i c i d a from, the p o s t e r i o r p o r t i o n o f the i n t e s t i n e o n l y . G o l d f i s h number 2 5 . 26/1/32 - i n o c u l a t e d f i s h i n t r a p e r i t o n e a l l y w i t h 2/10 c c . o f a 1 :100 d i l u t i o n o f a number 4 McFar l and s u s p e n s i o n 47. o f B. s a l m o n i c i d a . l e f t afc-28°C. 1/2/52 «- the f i s h remained active*• A haemorrhage appeared n e a r the p e c t o r a l f i n o n the r i g h t s i d e . 5/2/32 - d e a t h due to a c c i d e n t . Ho c u l t u r e s o b t a i n e d . G o l d f i s h number 2 8 . 28/1/32 - i n o c u l a t e d f i s h i n t r a p e r i t o n e a l ! ; / w i t h 2/10 c c . o f a 1:100 d i l u t i o n o f a number 4 McFaTland s u s p e n s i o n o f B. sa lmonic ida. . - L e f t a t room tempera tu re . 27/1/32 - no e x t e r n a l changes were v i s i b l e but the f i s h was i n a c t i v e and remained near the s u r f a c e o f the . .waters . 2/2/52 ' - f i s h found dead a t 1 P. M. Per formed a u t o p s y . Autopsy o f f i s h . - ; BEtef f foy symptoms. The o n l y v i s i b l e s i g n was a s m a l l h o l e a t the s i t e o f i n o c u l a t i o n . I n t e r i o r e x a m i n a t i o n . . The anus was r e d a t t h e o p e n i n g , the i n t e s t i n e haemor rhag i c . and s m a l l haemorrhages were observed i n the musc le l a y e r s . O therw ise the organs appeared u n a f f e c t e d . Sowed c u l t u r e s on n u t r i e n t agar from the h e a r t , the i n t e s t i n a l c o n t e n t s , the b i l e , the f l u i d a t back and p e r i t o n e a l f l u i d - I s o l a t e d pure c u l t u r e s o f B. s a l m o n i c i d a from the h e a r t , the I n t e s t i n a l c o n t e n t s , the b i l e , the f l u i d a t the "back, and the p e r i t o n e a l f l u i d . G o l d f i s h number 2 7 . ;28/l/32 - i n o c u l a t e d f i s h i n t r a p e r i t o n e a l l y w i t h 2/10 c c . o f a 1:100 d i l u t i o n o f a number 4 racFarland s u s p e n s i o n o f 3 . s a l m o n i c i d a . L e f t a t room, tempera ture . 27/1/32 - the f i s h remained i n a c t i v e . ;2/2/32 - the f i s h remained near the s u r f a c e o f the w a t e r . 3/2/32 - the f i s h found dead a t S A. M. Performed a u t o p s y . Autopsy o f f i s h . E x t e r i o r examinat ion . . A t the i n o c u l a t i o n s i t e was a l a r g e h o l e w i t h a s o f t a r e a s u r r o u n d i n g i t . There were r e d haemorrhagic s p o t s under the s c a l e s near the t a i l , the base o f the a n a l f i n was haemorrhagic. . and o n . p r e s s u r e r e d m a t t e r exuded from the a n a l o p e n i n g . A l a r g e dark p a t c h was o b s e r v e d on the s i d e o p p o s i t e the i n o c u l a t i o n s i t e . I n t e r i o r e x a m i n a t i o n . The i n t e s t i n e was conges ted and haemorrhagic to one e i g h t h o f an i n c h f rom the a n a l opening... O therwise the v i s c e r a l o rgans appeared n o r m a l . The musc le a r e a was haemor r h a g i c t h r o u g h o u t . Bowed c u l t u r e s on n u t r i e n t agar s l a n t s from the per i toneum and the- h e a r t . Osed f i s h f o r s e c t i o n i n g . flaomhorragie areas were found from the t a i l to the head and s u r r o u n d i n g the backbone. 49, G o l d f i s h number 23» 26/1/52 - I n o c u l a t e d f i s h I n t r a p e r i t o n e a l l y w i t h 2/10 c c number 4 M c F a r l a n d s u s p e n s i o n o f c u l t u r e o f B. s a l  m o n i c i d a . Incubated a t 2S°C„ 28/1/32 -~ t h e ' f i s h was f l o a t i n g a t 9 A. M» Per formed a u t o p s y . Autopsy o f f i s h . E x t e r i o r e x a m i n a t i o n . The anus was dark r e d and surrounded by a haeraor*- r h a g i c a r e a , s m a l l r e d s p o t s appeared around the mouth, and dark pa tches were v i s i b l e th rough the s k i n . I n t e r i o r e x a m i n a t i o n . The haemorrhage a t the anus was exud ing a dark red l i q u i d , a haemorrhage was observed on the r i g h t s i d e near the p e c t o r a l f i n . T h i s was o f a d iameter o f 0 . 5 c e n t i m e t r e s . The i n t e r i o r o f the f i s h was i n f l a t e d and c o n g e s t e d , b l o o d c l o t s were found i n the p e r i t o n e u m . On s l i c i n g haemorrhages were found throughout the musc le l a y e r s , and down the l a t e r a l l i n e . Sowed c u l t u r e s on n u t r i e n t agar from the I n t e s t i n a l c o n t e n t s , the back f l u i d , haemorrhage a long the l a t e r a l l i n e , the b i l e f rom the g a l l b l a d d e r , the anus , the haemorrhage at the t a i l , the haemorrhage on the r i g h t s i d e , the p e r i t o n e a l f l u i d , c l o t i n the p e r i t o n e u m , and the h e a r t . Obta ined c u l t u r e s o f 3. s a l m o n i c i d a from a l l o f these c u l t u r e s . oO« G o l d f i s h number 2 9 . 26/1/32 - i n o c u l a t e d f i s h i n t r a p e r i t o n e a l l y v/itii 2/10 c c , number 4 M c F a r l a n d s u s p e n s i o n o f c u l t u r e o f B* sa l -* mon i c ida* i n c u b a t e d a t S6°G.. 28/1/32 - the f i s h was found dead a t the bottom of the bowl on i t s s i d e a t S A . M, Per formed a u t o p s y . Autopsy o f f i s h . E x t e r i o r e x a m i n a t i o n . A t the i n o c u l a t i o n s i t e was a h o l e w i t h a s m a l l haemorrhage, 4 cm. i n d i a m e t e r . Red mat te r exuded from the a n u s . I n t e r io jL^Samina^i^a * The I n t e s t i n e was haemorrhagic and d i s t e n d e d . C l o t s were found i n the p e r i t o n e u m . The o ther organs appeared n o r m a l . Sowed c u l t u r e s on n u t r i e n t agar f rom the i n t e s t i n a l c o n t e n t s , c l o t i n the p e r i t o n e u m , the p e r i t o n e a l f l u i d , the h e a r t , the anus , ana the haemorrhage at the i n o c u l a t i o n s i t e . C u l t u r e s o f B.. s a l m o n i c i d a were not o b t a i n e d from the c l o t In the per i toneum or from the anus . G o l d f i s h . n u m b e r 5 0 . 26/1/32 ~ mixed 10 c c - o f a number 4 McFar land s u s p e n s i o n o f B. s a l m o n i c i d a w i t h the f i s h f o o d . L e f t a t room, temperature « 1/2/52 - i n o c u l a t e d the f i s h i n t r a p e i - i t o n s - a l l y w i t h 2/10 c c . 5 1 . nnsiDsr 4 M c F a r l a n d s u s p e n s i o n o f B. salmonicida» L e f t a t room t e m p e r a t u r e . 2/2/32 - found f i s h f l o a t i n g a t 9 A . &. Per formed a u t o p s y . Autopsy o f f i s h , E x t e r i o r e x a m i n a t i o n . There were no e x t e r i o r symptoms, w i t h the e x c e p t i o n o f a s m a l l h o l e under the s c a l e s a t the I n o c u l a t i o n s i t e . This was sur rounded w i t h s o f t t i s s u e . I n t e r i o r e x a m i n a t i o n . The i n t e s t i n e was h a e m o r r h a g i c . Otherwise the organs appeared n o r m a l . A b l o o d c l o t was observed i n the per i toneum. . Sowed c u l t u r e s on n u t r i e n t agar from the h e a r t , the p e r i t o n e a l f l u i d , the f l u i d a t the back , the c l o t , the l i v e r , the o i l s from the g a l l b l a d d e r , and the i n t e s t i n a l c o n t e n t s . C u l t u r e s o f B. s a l m o n i c i d a were I s o l a t e d from a l l these c u l t u r e s . G o l d f i s h number 51 . 26/1/82 - mixed 10 c c . o f a number 4 M c F a r l a n d suspens ion o f 3 . s a l m o n i c i d a w i t h the f i s h f o o d . L e f t a t room temperature , . 1/2/52 - i n o c u l a t e d the f i s h i n t r a p e r i t o n e a l l y w i t h 2/10 c c , number 4 M c F a r l a n d s u s p e n s i o n o f 3. s a l m o n i c i d a * L e f t a t room temperature* 52. 2/2/32 - found f i s h f l o a t i n g a t I P . &. Per formed a u t o p s y . Xntow o f f i s h * Interior exa-njLnr.tion. The f i s h was v e r y b l o a t e d i n appearance- A s m a l l h o l e was observed a t the s i t e o f i n o c u l a t i o n . I n t e r i o r e x a m i n a t i o n . The i n t e s t i n e was s l i g h t l y haemorrhagic a t the a n a l e n d . O t h e r w i s e the o rgans appeared to be i n a normal c o n d i t i o n . B l a c k s p o t s were found on the l i v e r - , and a rvlood c l o t on the p e r i t o n e u m . Sowed c u l t u r e s on n u t r i e n t agar f rom the b l a c k s p o t s , the l i q u i d a t the back , the c l o t i n the p e r i t o n e u m , the h e a r t , the por i tone-a l f l u i d , the I n t e s t i n a l c o n t e n t s , and the b i l e f rom the g a l l b l a d d e r * C u l t u r e s o f B> s a l r a o n i c l d a were I s o l a t e d f rom a l l but the b i l e . 5 5 . .-Other orgaz i l sas i s o l a t e d from g o l d f i s h . . . In n e a r l y every case an o r g a n i s e s i m i l a r to the B a c i l l u s o f the Pr-otous group r e f e r r e d to by A rkwr igh t was found i n the c u l t u r e s I s o l a t e d from the f i s h . These organisms were Gram- n e g a t i v e , r o d shaped o r g a n i s m s , o f t e n c o c c o i d i n f o r m , which l i q u i f i e d g e l a t i n r a p i d l y , fermented d e x t r o s e , . s a c c h a r o s e , m a l t o s e , and mann i te , but n o t r-aff inose. , l a c t o s e , and i u u l l n . G a l a c t o s e fvnd lr-evulose were a l s o f e r m e n t e d . M i l k was d i g e s t e d and o f t e n became c o l o u r l e s s . The organism was a l s o m o t i l e , and grew w e l l a t room t e m p e r a t u r e , 2 8 ° C and S7°C 54, T a b l e i i l u s t r a - i l n . ' . G o l d f i s h . exi>erfJ3eht s: r i s h Doso Eo. 2 6 7 8 9 Temper- Sate of Date of atare Inocula- Death Pure cultures from: 2/10 cu Room #1 McF. 2/10 cc Room. #1 -McF. 2/10 cc Room #1 McF. 2/10 cc Room §1 McF. 2/10 cc #1 McF. 3/10 #1 McF Room 2/10 cc #1 McF. 3/10 cc #1 McF. 2/10 cc #1 McF, 3/10 cc #1 McF. 2/10 cc #1 McF. 3/10 cc #1 McF> 3/10 cc #1 McF. 2e°c, .23° C. 30/10/31 3/11/31 . .abscess., kidney*, guts. .30/10/31 S i l l e d - . 23/11/31 inside- of p e l v i c sore, liv e r , , hearty kidney- - .2/12/31 5/1S/31 heart, spleen, l i v e r , , 2/12/51 -11/12/31 heart., .spleen, l i v e r , d orsal blood vessel, depth of abscess., c l o t under the abscess-. 2/12/51 18/1/32 r e i n o c u l a t e d Room 2/12/31 16/1/32 2/2/32 reinoculated Room 2/12/31 16/1/32 17/1/32 reinoculated Room 2/13/31 16/1/32 17/1/32 reinoculated 26° C. 4/1/32 5/1/52 10 3/10 cc 2S°C. 4/1/32 5/1/52 not affected by. the org ani ss *. death due to accident no 'cultures obtained. liver,.body c a v i t y , heart. peritoneal fl u i d . , heart, kidney.. heart, i n t e s t i n a l conteats, haemorrhage near t a i l , pectoral f i n base. inside- sore-,, blood,, dorsal blood vessel, rea i n muscle area, l i v e r , vent. F i s h Dose H a . Temper- 11 S/10 cc S6°C. #1 K c F . 12 S/10 cc 23°C. #1 McF. 13 2/10 cc 26°C. • #1 McF* 3/10 cc 26 X . #1 licF.. 14 2/10 cc 2S°C. #1 L4cF. 3/10 cc 26°G. #1 McF. 15 2/10 ee 26°G. f i McF, 3/10 co 26°C. 16 5/10 cc 26 °C. #1 McF. I T 3/10 cc 2S°e. #1 McF . 13 S/10 cc 26 °C, 19 5/10 cc 26°C, #1 McF. 20 3/10 cc Room 55 . Date o f Date o f Pure c u l t u r e s f rom: Inoeula— Death t i o n .. , 4/1/32 7/1/S2 4/1/32 7/1/32 l a r g e a b s c e s s , musc le o p p o s i t e i n o c u l a t i o n s i t e , i n t e s t i n a l c o n  tents . , s p l e e n , f l u i d near back , seared a b s c e s s . musc le i n o c u l a t i o n p o i n t , mid g u t , p e r i  t o n e a l f l u i d , k i d n e y , l i v e r , b i l e , s o f t musc le p a t c h . 9/1/32 16/1/52 17/1/32 p e r i t o n e u m . 9/1/32 16/1/32 16/1/32 d u e to i n j u r y to swim r©inoculated b l a d d e r — no c u l t u r e s •obtained*. 9/1/32 16/1/32 17/1/32 p e r i t o n e u m . 16/1/32 17/1/52 k i d n e y . IS/1/32 21/1/52 m / i / 5 g 18/1/32 l i v e r , seared a b s c e s s , haenor rhage , peri tout - ' e a l f l u i d , d o r s a l b l o o d v e s s e l . 22/1/52 b l a c k spots on l i v e r , l i v e r . , pe r i toneum, hear t. 22/1/52. 21/1/52 28/1/32 h e a r t , l i v e r , b i l e , per i toneum, , b l a c k s p o t s on l i v e r * k idney , , h e a r t , b i l e , per i toneum, , i n t e s t i n a l c o n t e n t s . ' I sh Dose Ho . Tempei a t u r e 21 3/10 cc hoom #1 McF. 22 3/10 co 26°C. 1:100,000 a i l T n #4 McF- 23 3/10 cc ee°c. 1:100,000 d i l*» #4 McF, B4 3/3.0 cc 26°C . 1:100 l i l * n #4- McF* Room 25 3/10 cc 28°C. 1:100 d l l - ' n #4 McF. 26 3/10 cc Room 1:100 d i l»e #4 McF, t h e n 3/10 cc Room #4 fficF. 27 3/10 cc Room 1:100 a i l * n t h e n 5/10 cc Room #4 McF. 2S 3/10 cc 26°C #4 McF. Date o f Pat© o f Pure c u l t a r e s from.: I n o c u l a - D e a t h t l o n , ; . ;.'„ B l / l / 5 2 2-2/1/52 h e a r t * peritoneum... kidney-, i n t e s t i n a l c o n t e n t s . 26/1/32 -1/2/52: dea th due t o a c c i d e n t - no c u l t u r e s obta ined. . 2 6 / 1 / 5 2 : •26/1/52 S/2/52 2/3/32 p o s t e r i o r I n t e s t i o a a l . contents*. =26/1/52 3/2/52 d e a t h due to a c c i d e n t - no c u l t u r e s o b t a i n e d . 26/1/52 1/2/32 jge/i/ie 2/2/52 i n t e s t i n a l c o n t e n t s , h e a r t , b l u i d i n back , p e r i toneum, b i l e . .1/2/52 h e a r t , per i toneum. -26/1/32 -28/1/32 f l u i d i n back , haemor rhage i n l a t e r a l l i n e . , p e r i t o n e u m , b l o o d c l o t , haemorrhage- on . r i g h t s h o u l d e r , haemorrhage on t a i l , i n t e s t i n a l contents>. anus , h e a r t , b i l e . 57. Bo. 29 2/10 cc #4 McF. Temper- Date of Bats of Pure cultures from* ature Innocula- Death t i o n _ . 26/1/5 28/1/52 hearty peritoneum* ' I n t e s t i n a l contents,. ' haemorrhage, .o0 cu f uur.e fed. then 5/10 cc 51 c u l t u r e . f e d then 5/10 cc #4 McF. 2&/1/32 ftoom 1/2/52 Boom 26/1/SB 2/2/52. heart, peritoneum, blood c l o t , , l i v e r , b i l e , , i n t e s t i n a l con t e n t s , back f l u i d . Room 1/2/52 2/2/52 p e r i toaeuai* black spots on. l i v e r , i n t e s f i n a l contents, heart 53.. E x p e r i m e n t a l I n o c u l a t i o n o f R a b b i t s w i t h B . s a l m o n i c i d a . S i n c e t h e organ ism had remained v i a b l e i n n u t r i e n t . b r o t h a t 37 *C. f o r a p e r i o d o f f i f t y - f i v e days an- at tempt was made to s e t up I n f e c t i o n i n r a b b i t s . Sub c u l t u r e s were sown from the f i r s t b r o t h c u l t u r e s , a g a i n on n u t r i e n t b r o t h , and i n c u b a t e d a t 37*0. A f t e r t?/o days sub c u l t u r e s were sown f rom these on t o n u t r i e n t b r o t h . These were a l s o i n c u b a t e d a t 37 93. T h i s p r o c e d u r e was c o n t i n u e d u n t i l f i v e s u c c e s s i v e sub c u l t u r e s had been made and i n c u b a t e d a t t h a t t e m p e r a t u r e . Prom t h e f i n a l sub c u l t u r e s , c u l t u r e s were sown on n u t r i e n t b r o t h c o n - j t a i n i n g a p h y s i o l o g i c a l s a l i n e s o l u t i o n . These were i n c u b a t e d j a t 37 c O . , and the same p rocedure f o l l o w e d u n t i l f i v e s u c c e s - j s i v e sub c u l t u r e s had been made. H u t r i e n t agar s l a n t s were j sown from the f i n a l tubes and i n c u b a t e d a t 37°C. A f t e r t h r e e ! days pigment was p r o d u c e d . A number 4 M c F a r l a n d s u s p e n s i o n o f the p o o l e d c u l t u r e s was made i n a s t e r i l e p h y s i o l o g i c a l i s a l i n e s o l u t i o n . . . • ij The f o l l o w i n g amounts were i n j e c t e d i n t r a v e n o u s l y i n t o r a b b i t s . I n j e c t i o n s were made i n the e a r w i t h a s t e r i l e I s y r i n g e . R a b b i t 1 A - 0 .5 c c . R a b b i t I B - 0.5 c c . \ R a b b i t 2 A - 0.5 c c . R a b b i t 5 A - 1.0 c c . R a b b i t 5 B - 1.0 cc* A f t e r one day R a b b i t 2 A was found dead and an autopsy p e r f o r m e d . The musc le a long the p o s t e r i o r s u r f a c e o f the b e l l y was g r e e n i s h i n c o l o u r w h i l e the p e r i c a r d i a l f l u i d was b l o o d y . The l u n g s appeared h a e m o r r h a g i c , and the b l o o d v e s s e l s o f the a n t e r i o r p o r t i o n o f the i n t e s t i n e were engorged . The l i v e r was a d a r k p u r p l e c o l o u r . A l l o t h e r o rgans appeared to be I n a norma l c o n d i t i o n . C u l t u r e s were sown on n u t r i e n t agar f rom the p e r i  c a r d i a l f l u i d , the l u n g , the s p l e e n , the l i v e r , and the h e a r t . These were o b t a i n e d i n the f o l l o w i n g manner. The organ was s e a r e d , then c u t w i t h a s t e r i l e k n i f e . A s t e r i l e P a s t e u r p i p e t t e was f o r c e d i n t o the c u t , the f l u i d drawn i n t o the p i p e t t e , and sown on the agar s l a n t s . A t the same t ime smears were made from a l l these organs and s t a i n e d w i t h Gram's s t a i n . On e x a m i n a t i o n these were found to c o n t a i n t i s s u e c e l l s o n l y . The c u l t u r e s were i n c u b a t e d a t 37°C. A l l proved to be s t e r i l e w i t h the e x c e p t i o n o f t h a t f rom the p e r i c a r d i a l f l u i d . The o n l y organism i s o l a t e d from I t was a Gram n e g a t i v e b a c i l l u s which appeared to form s p o r e s , and which showed a white w r i n k l e d growth on n u t r i e n t a g a r . I t s a c t i o n on ca rbohydra tes .was no t a t a l l s i m i l a r to t h a t o f B. s a l m o n i c i d a . d e x t r o s e + i n 1 d a y . ma l tose + i n 1 day . s u c r o s e + i n 1 day . s a l i c i n w i t h p e l l i c l e f o r m a t i o n i n 2 days, x y l o s e s l i g h t a c i d i t y w i t h p e l l i c l e f o r m a t i o n i n 2 d a y s , mannite .+ w i t h p e l l i c l e f o r m a t i o n i n 2 d a y s . r a f f i n o s e r w i t h p e l l i c l e f o r m a t i o n i n 2 days.. The o t h e r r a b b i t s were no t a f f e c t e d i n any way by the i n t r a v e n o u s i n o c u l a t i o n w i t h B. s a l m o n i c i d a . As R a b b i t 6 0 . 2 A bad p r e v i o u s l y been i n i l l h e a l t h d e a t h a t t h i s time was p r o b a b l y the r e s u l t o f s h o c k . 6 1 . I n f l u e n c e o f the o r g a n i c c o n t e n t o f water on the o rgan i sm, •;B.. s a l m o n i c i d a . P r e p a r a t i o n o f water o f v a r y i n g o r g a n i c c o n t e n t . On 9 / 8 / 3 2 o b t a i n e d samples o f sewage from the C i t y Sewers Department . U n t i l used t h i s was kept i n corked f l a s k s i n the I c e box . A f t e r 1 2 hours s t a n d i n g the e f f l u e n t was decanted o f f and the s o l i d m a t e r i a l d i s p o s e d o f . T h r e e 5 0 0 cc.: samples o f e f f l u e n t were k e p t f o r c h e m i c a l a n a l y s i s and e i g h t  een 1 , 0 0 0 c c . f l a s k s were p r e p a r e d i n the f o l l o w i n g manner. S e t s , numbers 1 and 5 S e t number 2 F l a s k C - 5 0 0 c c , s t e r i l e tap w a t e r . F l a s k C - 5 0 0 c c . s t e r i l e tap w a t e r . F l a s k 0 - 5 0 0 c c . u n d i l u t e d e f f l u e n t . F l a s k 0 - 5 0 0 c c . u n d i l u t e d . F l a s k 1 - 5 0 0 c c . 1 : 1 0 d i l u t i o n e f f l u e n t i n s t e r i l e tap w a t e r . F l a s k 1 - 5 0 0 c c . 1 : 1 G L d i l u  t i o n e f f l u e n t i n s t e r i l e tap w a t e r . F l a s k 2 - 5 0 0 c c . 1 : 1 0 0 d i l u  t i o n e f f l u e n t i n s t e r i l e t ap w a t e r . F l a s k 2 - 5 0 0 c c . 1 : 1 0 0 d i l u  t i o n e f f l u e n t i n . s t e r i l e tap w a t e r . F l a s k 3 - 5 0 0 c c . 1 : 1 0 0 0 d i l u  t i o n e f f l u e n t i n • s t e r i l e tap w a t e r . F l a s k 3 - 5 0 0 c c . 1 : 1 0 0 0 d i l u  t i o n e f f l u e n t I n s t e r i l e tap w a t e r . F l a s k 4 - 5 0 0 c c . 1 : 1 0 , 0 0 0 : F l a s k . 4 - 5 0 0 c c . 1 : 1 0 , 0 0 0 d i l u t i o n e f f l u e n t i n s t e r i l e tap w a t e r . d i l u t i o n e f f l u e n t i n s t e r i l e tap w a t e r . F l a s k 5 - 5 0 0 c c . 1 : 1 0 0 , 0 0 0 d i l u t i o n e f f l u e n t I n s t e r i l e t ap water* F l a s k 5- - 5 0 0 c c . 1 : 1 0 0 , 0 0 0 d i l u t i o n e f f l u e n t i n s t e r i l e tap w a t e r . F l a s k 6 - 5 0 0 c c . 1 : 1 , 0 0 0 , 0 0 0 d i l u t i o n e f f l u e n t i n s t e r i l e tap w a t e r . F l a s k D - 5 0 0 c c . 1 : 1 , 0 0 0 , 0 0 0 d i l u t i o n e f f l u e n t in s t e r i l e tap w a t e r . 6 2 . S e t s , number 1 and 3 w e r e . t h e n s t e r i l i s e d , w h i l e s e t number 2 rema ined n o n - s t e r i l e . >Chemical a n a l y s i s . P l a c e d the t h r e e 300 c c . sewage e f f l u e n t samples . i n l a r g e beakers and evapora ted over a water b a t h , washing d o w n 1 a l l d e p o s i t s u s i n g tap water and a r u b b e r p o l i c e m a n , u n t i l a l l had become a t h i c k , s y r u p y mass. Poured these samples i n t o c a r e f u l l y weighed and d r i e d c r u c i b l e s , put on water b a t h , and e v a p o r a t e d u n t i l a l m o s t d r y . Then d r i e d i n an oven a t 103°C. f o r h a l f an h o u r , we igh ing and r e p e a t i n g u n t i l a c o n s t a n t we ight had been r e a c h e d . F i n a l l y heated c r u c i b l e s s l o w l y a t f i r s t over a c l a y t r i a n g l e w i t h a bunser b u r n e r , r a i s i n g the temperature g r a d u a l l y u n t i l a r e d heat had been r e a c h e d . Kept a t t h i s temperature f o r 50 m i n u t e s , c o o l e d and we ighed . Repeated the p r o c e d u r e , u n t i l a c o n s t a n t weight was r e a c h e d . The samples were kep t i n a d e s i c c a t o r , when n o t i n u s e . ; Sample A . Constant weight o f empty c r u c i b l e . . . . . 3 1 . 1 3 2 0 grams. • Weight o f c r u c i b l e + s o l i d s . . . . . . . . . . . . 31.7872 grams. Weight o f s o l i d s . 6552 grams. Cons tant weight o f c r u c i b l e + a s h . . . . . . . . . . . 3 1 . 3 5 5 5 grams. Weight o f ash .2235 grams. Weight o f s o l i d s - Yreight o f a s h .4317 grams. /Sample B. C o n s t a n t we igh t o f empty c r u c i b l e . . 3 1 . 8 4 2 0 grams. Weight o f c r u c i b l e + s o l i d s 3 2 . 4 9 5 grams. Weight o f s o l i d s . . . * . . * . . . . ^ ... .6530 grams* Cons tan t weight o f c r u c i b l e + a s h . . . . . . . . . 52 .0570 grams.. V/signt o i 0-Sii •«...»»*••..»••- .»...»••..»»**...«. . 2-loQ grams. Weight o f s o l i d s - we ight of- ASh--. -•.».-. .4380 grams. Sample C . C o n s t a n t , w e i g h t o f c r u c i b l e . . . . . . . - . . . « . . . • . * . 3 3 . 0 0 4 4 grams. Weight o f c r u c i b l e ^ s o l i d s . . . . . . . . . . . . . . . . . 3 3 . 6 5 7 1 grams. Weight o f s o l i d s . .8527 grams. Cons tan t weight o f c r u c i b l e + a s h . . 3 3 . 2 4 5 0 grams. Vveight o f ash. . . . . -» .2414 grams. Weight o f s o l i d s - we igh t o f a s h * . . . . 4113 grams. : Results. , "of a n a l y s i s . Sample A - The we igh t o f o r g a n i c s o l i d s i n 500 c c . u n d i l u t e d e f f l u e n t was .4317 grams o r 4 3 1 . 7 mgm. which would be 8 8 3 . 4 mgm,. p e r l i t r e , . . Sample B - The we igh t o f o r g a n i c s o l i d s i n 50O c c . u n d i l u t e d e f f l u e n t was .4580 grams o r 876 .mgm. pe r l i t r e . Sample C - The we ight o f o r g a n i c s o l i d s i n 500 c c . u n d i l u t e d e f f l u e n t was .4113' grams o r 822.6 mgm. per l i t r e . The average, weight o f o r g a n i c s o l i d s i n 1 l i t r e u n d i l u t e d e f f l u e n t was t h e n 8 5 4 . 0 mgm. 6 4 . Then, i n a 1 : 1 0 d i l u t i o n - o f e f f l u e n t there «ould be S o . 4 mgm. o r g a n i c s o l i d s pe r l i t r e o f s o l u t i o n ; . In a 1 : 1 0 0 d i l u t i o n o f e f f l u e n t t h e r e would be 8 . 5 4 ragm. o r g a n i c s o l i d s per l i t r e o f s o l u t i o n . In a 1 . 1 , 0 0 0 d i l u t i o n o f - e f f l u e n t t h e r e wou ld be . 8 5 4 agm. o r g a n i c s o l i d s p e r l i t r e o f s o l u t i o n . I n a 1 : 1 0 . , 0 0 0 d i l u t i o n o f e f f l u e n t t h e r e would be . 0 8 5 4 mgm. o r g a n i c s o l i d s per l i t r e o f s o l u t i o n . In a 1 : 1 0 0 , 0 0 0 d i l u t i o n o f e f f l u e n t t h e r e would be . 0 0 8 5 4 mgm. o r g a n i c s o l i d s per l i t r e o f s o l u t i o n . In a 1 : 1 , 0 0 0 , 0 0 0 d i l u t i o n o f e f f l u e n t t h e r e would be . 0 0 0 8 5 4 mgm. o r g a n i c s o l i d s per l i t r e o f s o l u t i o n . o o , T e s t i n g the I n h i b i t ! v e A c t i o n o f O r g a n i c M a t e r i a l Opon- the  Growth o f B. s a l m o n i c i d a . Method To each o f the 18 l i t r e f l a s k s were added 0 .2 c c . o f a number 1 M c F a r l a n d s u s p e n s i o n o f poo led t h r e e day o l d p igment ing c u l t u r e s o f B> s a l m o n i c i d a i s o l a t e d from g o l d f i s h . T h i s amount had p r e v i o u s l y been found to be the amount which would form from 300 to 400 c o l o n i e s on n u t r i e n t agar p l a t e s a f t e r one day when.-sown i n 500 c c . s t e r i l e t a p w a t e r . A l l f l a s k s Yiere l e f t a t room t e m p e r a t u r e . P l a t e s were sown d a l l y f rom each f l a s k , u s i n g a 1 c c . sample , a f t e r f i r s t t h o r o u g h l y shak ing each f l a s k . R e s u l t s . (a) Se t number 2 . T h i s work may be i n a c c u r a t e because o f the d i f f i  c u l t i e s e x p e r i e n c e d i n a s c e r t a i n i n g which were the t y p i c a l B. s a l m o n i c i d a c o l o n i e s . Many s m a l l , c i r c u l a r , t r a n s l u c e n t c o l o n i e s were observed which were n o t o f t h a t t y p e . The p l a t e s were made d a i l y f o r t h i r t y - f i v e d a y s , and each day were crowded w i t h g r o w t h . The growth o f the B. s a l m o n i c i d a c o l o n i e s seemed to i n c r e a s e d a i l y and f a r exceeded the o r i g i n a l l i m i t o f 300 t o 400 c o l o n i e s . A f t e r f i f t e e n days the numbers o f B. s a l m o n i c i d a c o l o n i e s showed a d e c l i n e on a l l p l a t e s , and c o n t i n u e d to d e c l i n e d a i l y . I t appeared as i f the growth o f B. s a l m o n i c i d a was more . p r o l i f i c on the higher d i l u t i o n s * w h i l e more .growth o f the . sewage organisms appeared on the l o w e r d i l u t i o n s . Growth on the c o n t r o l p l a t e s was absent a f t e r ': ••••eleven d a y s . •(b) S e t s numbers 1 and 5. The appearance and e x t e n t o f the growth on the p l a t e s made from these s e t s o f f l a s k s can be exp la ined , most r e a d i l y by the t a b l e and the g r a p h on the f o l l o w i n g p a g e s . Growth on the p l a t e s sown from the c o n t r o l f l a s k s j o f s t e r i l e water was absent a f t e r e l e v e n d a y s , a f t e r showing : a g r a d u a l d e c l i n e . Oh the p l a t e s sown f rom the f l a s k o f u n d i l u t e d e f f l u e n t growth was p r o l i f i c f rom the f i r s t day u n t i l the end o f t h i r t y - f i v e days* A d a i l y i n c r e a s e was n o t e d i n the :•• growth from these f l a s k s . In each o f the d i l u t e d s o l u t i o n s o f e f f l u e n t the number o f c o l o n i e s g r a d u a l l y i n c r e a s e d , sudden ly r e a c h i n g a • h i g h uncountab le l i m i t . T h i s l i m i t was r eached i n ten days i n the 1:10, the" 1:100* and the 1:1,000 d i l u t i o n s , w h i l e i t was n o t u n t i l a f t e r s e v e n t e e n days t h a t t h i s peak was reached i n the case o f the 1:10,000, the 1:100,000, and the 1:1,000,000 d i l u t i o n s . A f t e r t h i r t y - o n e days the number o f c o l o n i e s on •a l l p l a t e s s t i l l remained u n c o u n t a b l e , though a g r a d u a l d e c r e a s e i n the number o f c o l o n i e s was no ted on p l a t e s poured from d i l u t i o n s 1:10,000, 1:100,000, and 1:1,000,000 '•  a f t e r t w e n t y - t h r e e d a y s . A d e c r e a s e i n the number o f co lon ies : 67. - on p l a t e s poured 'from d i l a t i o n s . I f l O G and l s l , 0 O 0 was observed, a f t e r -tfsentjMQine. days • • B n f o r t u n a t e l y because -of t ime s h o r t a g e t h i s work was n o t completed. . . • 68. xn o <<5 O H O P*4 * lO W CD H O «3< ra in r-i o •I. r-i o ta •} •« H «H . too o o i o ta «: B d H bO £15 o a> 53 hO w o 1+3- H O f-i ..^ S tsQH to Ad «5 to r H C D t\a o S H ra • b O H ?-f (4 O a> o -ta»' S CQ ^ SB H «5 'O ® H & H • 14 O fi) feCOOfflPt 3 r-i bp O & *H .. . tn -sf «fj • H i O fe» CO £i i d »-3 u o © O M R o g o CI K$ *H Cj t 3 H ft H <0 U O CO Pt-i co o to Pi o © H ?-f 44 "H 0 wi U a! <D 6} rH ^ i i , CO rQ CM 1>J S O G5 13 Q g^i tO ^ to rH H Ol H 03 H rH O i O -P O S O 4J> O fli 0> H H f f l O H O i O r i P H . £ - Oit>- O H O © C 0 H U O H © c !> 3 O © tt> J4 o . . . U ttl . U Pr a) CI O ^ CD i-Q ^ £2 CQ CO LO O © f-i O ^ H H O ^ r i O S H O H O 3 05 T* CO !M CO « tO >» c5 t3 W O O uO tO 05 ^ rH r- to co o "e- O £- rH rH O H - • $ H iO «2 <> t- O £ in co £- co to co 3 vft O 5) CJ O +3 ES SS Cf- *(S- O 3 ^ O H O Ci3 CO to ^  o K) o H iO CI cp tO LO too 0> lO IO o 3 . -© H 3D «8 +> S3 3 12 O O C C: e LO 05 ^' tO tO to- CO to o to H H H CD r-i to m IS jr # US O o P G> 0 i ixi O -<5'H CO to c3 p O o 13 C' IS: K f t B e ts c t4 C O t> <?s • O H O £ > 0 0 O O O - O CXiH © 3 H C O o H CVS £<3 ^ lO t - Q O r i CQ CO I> r*"4 r*J rH rH rH CD tQ CO u 'U co o <y es * CH 5)0) CO ^4 . C5 OJ O -P t>, 9c< 4 4 T T ^TlCQ^h2 i l l r n 11 I T TT ITT 1 M i l l Q+gara.vc raafeV^I ore fee^oojlk ^ 0.5aJnzoriici<4a~ - Soralr+ol^QsK: ' — H E / / ItwtraV d i a l e d I • too . 69, 70. /•.Conclusion. 1» C a r b o h y d r a t e r e a c t i o n s . On g l u c o s e the B. t r u t t a e o f Marsh produced gas -without sugar f e r m e n t a t i o n . A rkwr igh t* work ing on 3. sa lmon- , i c i d a . found both a c i d and gas p r o d u c t i o n i n t h a t s u g a r . y U e l t h e r P l e n a n o r D a v i s d e s c r i b e d the c a r b o h y d r a t e r e a c t i o n s o f 3. s a l m o n i c i d a . W i l l i a m s o n r e p o r t e d the p r o d u c t i o n o f a c i d w i t h gas i n some i n s t a n c e s , i n g l u c o s e and manni.te. She a l s o ; s t a t e d t h a t s a c c h a r o s e and l a c t o s e were n o t fermented but t h a t the medium became a l k a l i n e i The s t r a i n s i s o l a t e d from the E l k r i v e r f i s h and : t h e g o l d f i s h produced a c i d , w i t h o c c a s i o n a l l y a s l i g h t .amount o f gas p r o d u c t i o n , i n both mannite and g l u c o s e t u b e s . I n s a c c h a r o s e , l a c t o s e , r a f f i n o s e and i n u l i n the medium. became a l k a l i n e and the growth was abundant . In l a e v u l o s e , s a l i c i n , g a l a c t o s e , maltose. , a r a b i n o s e and x y l o s e the sugar was fe rmented w i t h a c i d p r o d u c t i o n i n a l l c a s e s . S» Pigment s o l u b i l i t y . Marsh r e p o r t e d t h a t the pigment o f h i s B. t r u t t a e was s o l u b l e i n a l c o h o l , w h i l e A rkwr igh t found tha t - f t i f . 'absolute a l c o h o l ?/ere poured on to a m o i s t pigmented agar c u l t u r e the watery s o l u t i o n o f the pigment d i f f u s e d out i n t o '. the a l c o h o l , and t h a t the pigment remained i n s o l u t i o n i n • the Impure alcohol.® The s t r a i n s o f B. s a l m o n i c i d a u s e d i n these e x p e r i  ments produced a pigment w h i c h d i f f u s e d f rom the medium i n t o 7 1 . the a l c o h o l , and remained i n s o l u t i o n i n the a l c o h o l . 5. R e a c t i o n o f medium - i t s e f f e c t on the growth and pigment p r o d u c t i o n o f B. s a l m o n i c i d a . Marsh*s B. t r u t t a e was ve ry s e n s i t i v e w i t h r e g a r d to the r e a c t i o n o f the medium on which i t was c u l t i v a t e d w h i l e W i l l i a m s o n found the optimum pH. f o r p i g m e n t . p r o d u c t i o n t o he pH. 7.6. P o o l e d s t r a i n s o f B. s a l m o n i c i d a grew on agar s l a n t s a t room tempera ture w i t h i n a pH. range o f 5 .0 to 9.2 i n  c l u s i v e . P igment was produced w i t h i n a pH. r a n g e o f 5.2 t o 9.2 i n c l u s i v e . A f t e r seven days growth w i thout pigment p r o d u c t i o n o n an agar s l a n t o f pH. 5.0 when sub c u l t u r e s were sown on agar w i t h a pH. o f 7.0 pigment was a g a i n produced i n t h r e e o r f o u r d a y s , 4 . V i a b i l i t y o f the o r g a n i s m . Workers seem to agree that, c l e a r water i s d e l e t e r  i o u s t o the development and growth of the f u r u n c u l o s l s o r g a n i s m . P l e h n found t h a t p u r e water was d i sadvantageous to the b a c t e r i u m , t h a t i n i t B. s a l m o n i c i d a was dead a f t e r t h r e e d a y s , w h i l e A r k w r i g h t found i t a l i v e , though i n d i m i n  i s h e d numbers , a f t e r f i v e days i n s t e r i l e w a t e r . D r . A r k  w r i g h t a l s o o b s e r v e d t h a t sea water was i n j u r i o u s to the growth o f the o rgan i sm, t h a t the organ ism cannot be r e c o v  ered a f t e r n i n e t e e n h o u r s . Our s t r a i n s o f B. s a l m o n i c i d a d i d n o t l i v e l o n g i n s t e r i l e d i s t i l l e d w a t e r . In sub c u l t u r e s on n u t r i e n t agar 72. p l a t e s , -in some I n s t a n c e s no growth was r e p o r t e d a f t e r one day. In o t h e r c a s e s the organ ism remained a l i v e f o r th ree d a y s , and i n one i n s t a n c e f o r f o u r d a y s . I n s t e r i l e tap water the ox'ganism s u r v i v e d f o r a l o n g e r p e r i o d . Even a f t e r e l e v e n and twelve days c o l o n i e s were found on n u t r i e n t agar p l a t e s . These were i n ve ry s m a l l numbers.• I n o t h e r f l a s k s c o n t a i n i n g the same amount o f s t e r i l e tap water the organ ism c o u l d no t be r e c o v e r e d a f t e r two or t h r e e n a y s . A r k w r i g h t found t h a t i n d i s t i l l e d water c o n t a i n i n g 5^ sodium c h l o r i d e B. s a l m o n i c i d a soon d i e d . Us ing the s t r a i n s o f B. s a l m o n i c i d a i s o l a t e d from' g o l d f i s h the organism was r e c o v e r e d i n s m a l l numbers a f t e r e i g h t days i n "£$>. s a l t water.. No growth was r e p o r t e d on the n i n t h day . I n 1% s a l t water the organ ism remained a l i v e f o r n i n e and e l e v e n d a y s , w h i l e i n .85>£ s a l t water the average t ime r e p o r t e d was t e n d a y s . I t would seem t h e n , tha t n e i t h e r c l e a r water nor water c o n t a i n i n g sodium c h l o r i d e a re p a r t i c u l a r l y f a v o u r a b l e f o r the growth o f B. s a l m o n i c l d a . The s h o r t t ime t h a t i t can remain a l i v e I n such water however may be t ime enough f o r the organ ism to be c a r r i e d to many f i s h , thus caus ing i n f e c t i o n . 5. Temperature . A r k w r i g h t r e p o r t e d tha t the organ ism soon d i e d when i n c u b a t e d a t 36°C. D a v i s secured s i m i l a r r e s u l t s , s t a t i n g t h a t the organ ism cannot l i v e l o n g a t 5 7 D C . W i l l i a m s o n found no growth a t 37°C. P o o l e d s t r a i n s o f B. s a l m o n i c i d a i s o l a t e d from g o l d f i s h c o u l d s u r v i v e over a p e r i o d o f from s i x t e e n to e i g h t y days a t 37°C. When sub c u l t u r e s were made f rom t h e s e , r a p i d growth took p l a c e a t the same t e m p e r a t u r e . Pigment was a l s o produced by these c u l t u r e s a t 37°C. The organ ism remained v i a b l e a t 28°C. and a t 14°C. f o r a t l e a s t t h r e e months. Pigment p r o d u c t i o n a l s o took p l a c e a t these t e m p e r a t u r e s . I t may be t h a t the o r g a n i s m i n i t s passage through the more r e s i s t a n t g o l d f i s h , and I t s i n c u b a t i o n a t 26°C. . had become f o r t i f i e d to a g r e a t e r e x t e n t a g a i n s t changes i n t e m p e r a t u r e . 6 . R e l a t i o n to o x y g e n . The o r g a n i s m , a c c o r d i n g to W i l l i a m s o n , i s an aerobe and a f a c u l t a t i v e anaerobe . Ho c o n c l u s i o n s can be . drawn from our work. . 7. P a t h o g e n i c i t y Exper iments on F i s h . From the I n o c u l a t i o n exper iments i t would appear t h a t B. s a l m o n i c i d a when i n j e c t e d i n t r a p e r i t o n e a l l y o r i n t r a m u s c u l a r l y . i n t o g o l d f i s h i s a b l e t o produce s e p t i c a e m i a , and d e a t h , I n some c a s e s w i th the presence o f l o c a l abscesses and haemorrhages , I n o t h e r s w i t h no such symptoms. F e e d i n g and s c a r i f y i n g exper iments d i d n e t seem to a f f e c t g o l d f i s h i n any way, perhaps because o f the hardy n a t u r e o f the f i s h . C e r t a i n o f the f i s h were a b l e to r e c o v e r a f t e r what appeared to be a t y p i c a l a t t a c k o f f u r n c u l o s i s — take f o r example g o l d f i s h number S which a f t e r t w e n t y - f i v e days showed s i g n s o f recovery , , w h i l e g o l d f i s h numbers 5 and 6 , i n s p i t e , o f two i n o c u l a t i o n s w i t h the o rgan i sm, and a change from room temperature to 2-6°C, were both a b l e to r e s i s t the d i s e a s e . G o l d f i s h number 5 appeared a c t i v e and i n good h e a l t h th roughout the y e a r . Suspens ions o f the organ isms In v e r y h i g h d i l u t i o n s d i d n o t seem t o a f f e c t the f i s h , w i t n e s s g o l d f i s h number 23 wh ich remained a c t i v e d e s p i t e i t s i n o c u l a t i o n w i t h a 1 :100,000 d i l u t i o n o f a number 4 McFar land s u s p e n s i o n o f the o r g a n i s m . I n the c a s e s o f g o l d f i s h numbers 7 and 8 which were r e s i s t a n t to a f i r s t i n o c u l a t i o n and I n c u b a t i o n a t room t e m p e r a t u r e , r e i n o c u l a t i o n and i n c u b a t i o n a t 26°C. seemed t o d e c r e a s e t h i s power o f r e s i s t a n c e . G o l d f i s h numbers 13 and 15 were u n a f f e c t e d by a f i r s t i n o c u l a t i o n w i t h i n c u b a t i o n a t 28°C. but q u i c k l y succumbed a f t e r a second d o s e . I n b c u l a t i o n w i t h i n c u b a t i o n a t 26°C. seems to cause more r a p i d d e a t h w i t h e x t e r i o r symptoms o f the d i s e a s e a t the same t i m e , w h i l e a t room temperature dea th was o f t e n caused J u s t as r a p i d l y but w i thout the appearance o f these 75. e x t e r n a l s i g n s . As an e x c e p t i o n to the r u l e , however, we have g o l d f i s h number 20 ana 21 a t room temperature , and numbers 13 and 19 at 26°C. L a b o r a t o r y exper iments show, t h e n , tha t the pigment fo rming b a c t e r i u m B. s a l m o n i c i d a i s a b l e to produce many o f the most c h a r a c t e r i s t i c f e a t u r e s o f f u r u n c u l o s i s as d e s c r i b e d by Met tarn, A r k w r i g h t and P l e h n , namely e-.bsces.ses, haemorrhagic a r e a s i n the m u s c l e s , c o n g e s t i o n o f the I n t e s t i n e , p a r t i c u  l a r l y the l o w e r , and a d i s c h a r g e o f a ' p u r u l e n t l i q u i d from the anus» There a r e c a s e s when some or a l l these symptoms were n o t observed but where d e a t h o c c u r r e d and the i n j e c t e d organism was r e c o v e r e d f rom the f i s h . 8 . P a t h o g e n i c i t y E x p e r i m e n t s . o n Babb i t s . . Though B, s a l m o n i c i d a had been grown on b o t h n u t r i e n t b r o t h , n u t r i e n t agar and o n n u t r i e n t b r o t h c o n t a i n i n g a p h y s i o l o g i c a l s a l i n e , s o l u t i o n f o r some time a t 57°G. when. I n j e c t e d i n t o the m a r g i n a l ear v e i n o f r a b b i t s i t had no apparent e f f e c t o n . t h e rabb i t s - d u r i n g a p e r i o d o f twenty-two d a y s . The h a b i t a t o f the o rgan i sm, then., seemed to be r e s t r i c t e d to trie c o l d b looded an ima ls where I t i s an a c t i v e p a r a s i t e c i r c u l a t i n g w i th the b l o o d and a t t a c k i n g the t i s s u e s o f the h o s t . Even here i t s a c t i v i t y i s r e s t r i c t e d f o r , a c c o r d i n g to most w o r k e r s , B. s a l m o n i c i d a i s n o t pa thogen i c to f r o g s . Ho work was done on amphibians i n c o n n e c t i o n w i t i r t h i s r e s e a r c h . 9 . E f f e c t or o r g a n i c m a t e r i a l on the growth o f the o r g a n i s m . P l e h n s a i d t h a t though the f u r u n c u l o s l s b a c t e r i u m d i e d i n c l e a r water w i t h i n one to three days B. s a l m o n i c i d a -••would grow i n the p resence o f a g r e a t d e a l o f c o n t a m i n a t i o n i n the water . F u r u n c u l o s l s as i t o c c u r s i n sewage p o l l u t e d Y/aters and i n water c o n t a i n i n g d i s p o s a l s f rom b r e w e r i e s and d i s t i l l e r i e s I s commonly more I n f e c t i o n s and more d i s  g u s t i n g than t h a t found i n c l e a r water , accord ing, to P l e h n . The r e s u l t s o f h e r work show t h a t the numbers o f B. sa lmon- . i c i d a I n c r e a s e d by m i l l i o n s i n water r i c h i n o r g a n i c m a t t e r , i n the same t ime r e q u i r e d f o r i t to d i e out i n the same amount o f c l e a r w a t e r . W i l l i a m s o n , i n 1928, found no growth o f B. sa lmon- . i c i d a i n sewage a f t e r f o r t y - e i g h t h o u r s , but t h a t y?arm weather and low water f a v o u r the development o f B. s a l m o n i c i d a . Borne found no growth o f the organ ism i n sewage a f t e r f o r t y - e i g h t h o u r s , w h i l e D a v i s remarked t h a t the . o r g a n i s m ^may l i v e f o r weeks I n water c o n t a i n i n g an a p p r e c  i a b l e amount o f o r g a n i c m a t t e r 1 5 . From our exper iments i t would appear t h a t , when sown i n non s t e r i l e sewage p o l l u t e d w a t e r , the organism . f i r s t showed a r a p i d i n c r e a s e i n numbers, but tha t a f t e r t e n days t h e r e was a g r a d u a l d e c r e a s e i n the numbers o f B. s a l m o n i c i d a . On the o t h e r hand, i n s t e r i l e sewage p o l l u t e d water t h e r e was f i r s t a d e c l i n e i n the numbers o f the o rgan ism p r e s e n t , then i n t e n days t h e r e was a r a p i d 77. and u n a c c o u n t a b l e I n c r e a s e i n numbers i n the lower d i l u t i o n s . In the u n d i l u t e d e f f l u e n t t h e r e was no l a g phase but the numbers i n c r e a s e d d a i l y . I n the h i g h e r d i l u t i o n s the i n c r e a s e "In numbers d i d n o t take p l a c e u n t i l t he s i x t e e n t h o r the . s e v e n t e e n t h day i n the 500 c c . samples . The numbers o f the organisms s t i l l remained a t an u n c o u n t a b l e h e i g h t even a f t e r t h i r t y days-*- but a f t e r twenty-one days a. s l i g h t d e c r e a s e i n the numbers was n o t i c e d : , and the growth o f the organisms became s l o w e r . U n f o r t u n a t e l y t h e r e was n o t time enough l e f t to c a r r y these exper iments f u r t h e r , , though many new and i n t e r  e s t i n g problems p r e s e n t e d themselves f o r f u t u r e s t u d y . 73V Summary o f the c h a r a c t e r i s t i c s o f the Elk- -River s t r a i n o f B. s a l m o n i c i d a . 1 . B. s a l m o n i c i d a i s a p leomorph i c Gram n e g a t i v e organism s t a i n i n g e a s i l y , w i t h o r d i n a r y a n i l i n e d y e s . 2. B. s a l m o n i c i d a grows w e l l on o r d i n a r y n u t r i e n t a g a r , n u t r i e n t b r o t h , L o e f f l e r J s b l o o d serum,, g e l a t i n and l i t m u s mi lk . . I t p roduces h a e m o l y s i s -on b lood agar p l a t e s , does n o t produce i h d o l , and i s a moderate •starch l o v e r . 3 . The p igment produced by B« s a l m o n i c i d a i s s o l u b l e i n water and d i f f u s e s out i n t o a n a l c o h o l s o l u t i o n . 4 . B. s a l m o n i c i d a does n o t t h r i v e on a medium o f d i s t i l l e d w a t e r , s t e r i l e tap water , o r s t e r i l e tap water c o n t a i n i n g sodium c h l o r i d e , 5. B . s a l m o n i c i d a s t r a i n s i s o l a t e d from g o l d f i s h w i l l 'grow f o r some t ime when Incubated a t 37°C., 2 8 ° C , and 14°C. 8 . B. sa lmon ic ida . produces- no ..pigment i n the absence d f •oxygen. 7. B. s a l m o n ! c i d a I s p a t h o g e n i c f o r g o l d f i s h , p r o d u c i n g l e s i o n s and d e a t h I n most c a s e s , but no t f o r r a b b i t s . 8. B, s a l a o n l c i d a grows abundant l y i n sewage p o l l u t e d water and remains v i a b l e f o r a t l e a s t t h i r t y - o n e d a y s . 79 . B i b l i o g r a p h y . A r k w r i g h t . J . A . . Repor t to the Board o f A g r i c u l u r e and F i s h e r i e s , J o u r n a l of H y g i e n e , 12 , 1915, 3 9 1 . Blake» I s o b e l ana A n d e r s o n , E l e a n o r J . M. The I d e n t i f i c a t i o n o f B a c i l l u s s a l m o n i c i d a by the Com p l e m e n t - F i x a t i o n T e s t - A F u r t h e r C o n t r i b u t i o n to the Study o f F u r u n c u l o s i s o f the Sa lmon idae . F i s h e r i e s , S c o t l a n d , Salmon F i s h , 1930,. Ho. I . B l a k e . I s o b e l The E x t e r n a l D i s i n f e c t i o n o f F i s h Ova - With R e f e r e n c e to the P r o p h y l a x i s o f F u r u n c u l o s i s . F i s h e r i e s , S c o t l a n d , Salmon F i s h , 1930, Ho. I I . D a v i s . H . S . Care and D i s e a s e s o f T r o u t . D. S . Department o f Commerce - Bureau o f F i s h e r i e s - Document N o . 1061. D u f f . D. C. B. D e t e c t i o n o f I n d o l i n B a c t e r i a l C u l t u r e s . Amer ican J o u r n a l o f P u b l i c H e a l t h - V o l . XX. l o . 9 September, 1930. H o m e , J. B. F u r u n c u l o s i s i n T r o u t and the Importance o f C a r r i e r s i n the Spread o f the D i s e a s e . J o u r n a l o f Hyg iene , 28 , No. 1, A u g u s t , 1928. A More Complete D e s c r i p t i o n o f B a c t e r i u m T r u t t a e . B u l l e t i n . U. S . F i s h Commiss ion, V o l * X X I I , 1902 (1904) p p . 411 - 415 . Mas ter man, A . T, R e p o r t by A . T . Masterman, D . S c * , upon the E p i d e m i c Amongst Sal-nonidae. i n the Summer o f 1911. Board o f A g r i c u l t u r e and F i s h e r i e s . Mettam. A* E. Report on the Outbreak o f F u r u n c u l o s i s f i n the R i v e r L i f f e y i n 1913, F i s h e r i e s , I r e l a n d , S c i e n t i f i c I n v e s t i g a t i o n s , . 1914., I I . (1915) . P l e h n . M. P rak i tum der F i s c h k r a n k h e i t e n 179 pp* XXX.,, 1924. E. S c h w e i s e r b a r t f s che Ver lagsbuc l i i i and lung j S t u t t g a r t . W i l l i a m s o n , I s o b e l J . F . F u r u n c u l o s i s o f the Salmonidae 1928. F i s h e r i e s , S c o t l a n d , Salmon F i s h , 1928, H o . V . F u r t h e r O b s e r v a t i o n s on F u r u n c u l o s i s o f the Salmonidae 1928. F i s h e r i e s , S c o t l a n d , Salmon F i s h 1929, l o . I . 

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