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Studies on the Streptococcus Viridans Bligh, Una Maud 1936

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STUDIES ON THE STREPTOCOCCUS VIRIDANS Una B l i g h B . A . T h e s i s s u b m i t t e d f o r d e g r e e o f MASTER OF ARTS O c t o b e r , 1 9 3 6 U n i v e r s i t y o f B r i t i s h C o l u m b i a STUDIES ON THE STREPTOCOCCUS VIRIDANS I INTRODUCTION. II METHODS AND TESTS USED. ( a ) M e d i a u s e d . (b) Strains used• (c) T e s t f o r peroxide. (d) Description of apparatus used f o r obtaining d e f i n i t e mixtures of carbon dioxide and oxygen* III EXPERIMENTS AMD' RESULTS. ( a ) Production o f Peroxide. (b) Production o f A c i d . (o) Production of Haeraolysin. (d) Examination for entero-, necr-o-ov t o x i c - l i k e s u b s t a n c e s * IV SIGNIFICANCE OF RESULTS. V SUMMARY OF RESULTS. INTRODUCTION Streptococcus viridans i s most characteristically found as a saprophyte upon the mucous membrane of the upper part of the alimentary canal and the respiratory passages of man. It can be demonstrated i n practically a l l mouths In the entire absence of any recognizable pathological process. It is associated frequently with tooth abscesses, and may be present i n middle ear diseases and infection of the accessory sinuses of the nose. It i s not infrequently found i n the tonsillar crypts and has been known to cause mild forms of t o n s i l l -i t i s . The viridans i s frequently associated with sub-acute vegetative endocarditis. In such cases the organism causes relatively firm vegetations, usually on the mitral but sometimes also on the aortic valves, extending occasionally along the walls of the auricles. The organisms can be readily cultivated from the blood stream where they may remain for weeks and months. H.D. Wright (l) writes, "In endocarditis the original infec-tion of the valves must result from an invasion of the blood stream by streptococci. These are i n most cases indistinguishable from those which are normally found i n the mouth and upper respiratory tract and presum-ably come from this situation." Because of these f s c t s ^ i t was thought that i t would be of interest to make some studies, i n this laboratory, on this organism. S© attempt to find out i f under certa i n by 'conditions of atmosphere arrNusing d i f f e r e n t media the Strepto-Subs'ta.ticaS coccus viridans could be made to produce some toxic offoots, which might account f o r i t s action i n some of the diseases for which i t i s responsible. I t was thought that i t would be of in t e r e s t also to determine whether or not Streptococcus viridans produced a ^ "haemolytic-llke substance. There i s no direct evidence i n the l i t e r a t u r e that Streptococcus viridans produces haemolysin, although much work has been ctone on the endocellular and e x t r a c e l l u l a r production of haemolysin by both the haemolytie streptococci (2) and the pneumooocci (3). Much work has also been done on the production of peroxide by both the pneumococoi (3) and haemolytie streptococci (30» and there i s evidence i n the l i t e r a t u r e that the viridans streptococci produce peroxide (4). The observation of the f i r a L . phenomenon of haemolysis, was f i r s t made by Marmorek i n 1895. Marmorek be-leived that there was a d i r e c t relationship between virulence and haemolytie power• Other investigators, however, notably Schotmuller, b e l o v e d , from the beginning, that the haemolytie power was a constant c h a r a c t e r i s t i c of certain s t r a i n s , un-changeable by experimental enhancement or reduction of v i r u -lence. I t i s generally b e l o v e d by the workers of today that an exact parallelism between virulence and haemolytlo a c t i v i t y i n Streptococcus strains does not ex i s t , since many independent observers have found that haemolysin production may continue active i n a strain.which has l o s t i t s virulence* -I t was decided to test for haemolysin produc-t i o n by two methods: ( 1 ) plate method ( 2 ) tube method. It was Schottmuller fs introduction of the blood agar plate which led to the d i f f eventration of those atral ns produoing haemolysin from those producing methaemoglobin. The appearance of a Streptoccal culture on a blood agar plate which i s accepted as evidence of haemolytic a c t i v i t y i s the development of clear, colourless transparent haloes i n the opaque medium i n the immediate v i c i n i t y o f the b a c t e r i a l colonies. This i s not simply haemolysis, i t Is haemolysis plus destruction or removal of blood pigment and St apparently it • may occur although the coccus concerned does not r e a d i l y se-crete a haemolytic toxin. These discrepancies between the plate method and the tube method of observing haemolysis have been noted by several workers ( 5 ) ( 6 ) ( 7 ) . The usual discre-pancy i s that strains appearing to be haemolytic are not found to produce haemolysis when the f l u i d cultures are added to blood suspensions. Brown (£) who has devoted an extensive monograph to t i l l s subject of different types of haemolysis on blood agar plates describes an oC type which corresponds to the Streptoccus -4-v i r l d a n s , a ^ t y p e w h i c h c o r r e s p o n d s t o t h e a c t i v e l y h a e m o l y t i c S t r e p t o c o c c u s p y o g e n e s and a n V t y p e w h i c h g i v e s ~ n e i t h e r h a e m o l y t i c p r o d u c t i o n n o r g r e e n d i s c o l o u r a t i o n . N e i l and M a i l o r y (0) h a v e s h o w n t h a t s t r e p t o c o c c a l h a e m o l y s i n l o o s e s i t s a c t i v i t y o n o x i d a t i o n b u t r e g a i n s i t a n e x p o s u r e t o s u i t a b l e r e d u c i n g a g e n t s ® MATERIAL AM) TESTS V e a l i n f u s i o n b r o t h was u s e d t h r o u g h o u t t h e e n t i r e e x p e r i m e n t . 1% p r o t e o a e p e p t o n e was a d d e d , and t h e medium made u p t o a p h o f 7 . 4 . F o r t h e y e a s t i n f u s i o n b r o t h , 0 . 0 1 $ o f y e a s t e x t r a c t ( O r l a - J e n s e n ) was a d d e d t o t h e i n f u s i o n b r o t h . T h e b r o t h and y e a s t b r o t h w e r e f i l t e r e d t h r o u g h c o a r s e f i l t e r p a p e r and t h e n r u n t h r o u g h a S e i t z E . K . i n t o s t e r i l e f l a s k s ^ and t u b e s . A l l t h e c u l t u r a l w o r k r e p o r t e d i n t h i s p a p e r was d o n e w i t h s i x s t r a i n s o f S t r e p t o c o c c u s v i r i d a n s . S t r a i n s 1 , 2 and 3 w e r e i s o l a t e d f r o m n o r m a l t h r o a t s . When f i r s t o b t a i n e d , t h e c o l o n i e s , o n b l o o d a g a r , w e r e s m a l l , s m o o t h , d o m e - s h a p e d and o p a q u e , and t h e y r e m a i n e d s o t h r o u g h o u t t h e e n t i r e p e r i o d o f t h e w o r k . T h e g r o w t h i n b r o t h was d i f f u s e . T h e o r g a n i s m s w e r e n o t s o l u b i l e i n a 1 : 1 0 m i x t u r e o f B a c t o o x - b i l e . T h e y d i d n o t a g g l u t i n a t e s p o n t a n e o u s l y i n 0 . 8 5 $ s a l i n e . No c a p s u l e s c o u l d be d e m o n s t r a t e d . F e r m e n t a t i o n r e -a c t i o n s c l a s s i f i e d t h e s t r a i n s a s : ^tg - S~t • V « - K I Jans C<*ccorcf.tn<f f5 73ttTya.y) # 3 - St. viT, J an% C - » " ) S t r a i n #4 was isolated from an abscessed tooth. On sheep:-blood agar the colonies were opague, coarsely granular, f l a t -tened with a central elevation and i r r e g u l a r edges. When f i r s t Isolated and during the time of the experiment the s t r a i n grew as a coarse f l o c c u l a r sediment i n broth. The s t r a i n was not solu b l l e i n a 1:10 mixture of Bacto-bile. No capsule could be demonstrated. The organism did not agglute-nate spontaneously i n 0.85^ s a l i n e . Fermentation reactions c l a s s i f i e d the s t r a i n as Streptococcus v/r/c/a»s - »<>n /«7sst«/, aaj,'c,n **-rm S t r a i n #5 - isolated from a case of mastoids. Cultural characteristics were the. same as No. 4, but the f e r -mentation reactions c l a s s i f i e d the s t r a i n as Streptococcus vir/<Jans S t r a i n #6 - isolated from a Mood culture of 'bacterial endocarditis. When f i r s t obtained the colonies on sheep blood agar were small, smooth, dome-shaped and opaque. Upon subsequent transfer they became larger, coarsely granular, flattened? with a central elevation and i r r e g u l a r edges. On the f i r s t subculture, the colonies produced a small ring of green discolouration; upon subsequent transfer a clear r i n g of haemolysis, was produced, and th i s remained a ch a r a c t e r i s t i c throughout the experiment. The s t r a i n grew as a coarse f l o c c u l a r s e d i -ment i n broth. The organism was not solubile i n a 1:10 mixture of Bacto ox-bile. There was spontaneous agglutina-t i o n i n 0.85$ s a l i n e . Uo capsule could be demonstrated. Fermentation reactions c l a s s i f i e d the s t r a i n as Streptococcus VIT/JanS CaecorJirtj to ~E>*rf• To Test for Peroxide Production. To 0.5 c.c* of f i l t r a t e i n a small culture tube add a small piece of freshly cut potato (about 2 mm. x 2 mm. x 4 mm.) and three drops of f r e s h l y prepared saturated g l a c i a l acetic acid solution of benzidine. In the presence of peroxide the potato takes on a blue colour. The i n t e n s i t y of the colour depends on the amount of peroxide present. -h Very s l i g h t blue colour. + +• A l i g h t blue colour, f + +- Dark blue colour. + + + + intense blue colour. A p p a r a t u s f o r O b t a i n i n g D e f i n i t e M i x t u r e s o f C a r b o n d i o x i d e and O x y g e n . ~ ~~ : — — "6' ^ Y ft &uction(f- lo%K)/oumji =9-T h e w a t e r i n t h e m i x i n g f l a s k i s s a t u r a t e d w i t h c a r b o n d i o x i d e and o x y g e n e a c h t i m e b e f o r e u s i n g . By means o f t h e p r i n c i p l e o f t h e s i p h o n , f l a s k B i s c o m p l e t e l y f i l l e d w i t h t h e s a t u r a t e d w a t e r * I n o b t a i n i n g s a y a m i x t u r e o f 20% c a r b o n d i o x i d e and Q0% o x y g e n , " a " i s a t t a c h e d t o t h e c a r b o n d i o x i d e g e n e r a t o r , M c n . i s o p e n e d and c a r b o n d i o x i d e i s r u n i n t o f l a s k B - a r o u g h s c a l e h a s b e e n marked o n B d i v i d i n g t h e f l a s k i n t o v o l u m e s o f one l i t r e , and a b o u t one l i t r e o f t h e s a t u r a t e d w a t e r i s d i s p l a c e d . T h e n " a " i s a t t a c h e d t o a n o x y g e n g e n e r a t o r - o x y g e n f r o m a t a n k I s u s e d w h i c h i s a b o u t 99% p u r e - and a b o u t f i v e l i t r e o f o x y g e n a r e r u n i n t o B . T h e l e v e l o f w a t e r i n A i s b r o u g h t t o t h e l e v e l o f t h e w a t e r i n B i n o r d e r t o l e a v e t h e m i x t u r e o f g a s e s i n B a t a t m o s p h e r i c p r e s s u r e . When t h i s i s done ttc" i s c l o s e d . A g a s a n a l y s i s t e s t i s t h e n r u n o n t h e m i x t u r e o f c a r b o n d i o x i d e and o x y g e n p r e s e n t i n B . The c a r b o n d i o x i d e i s a b s o r b e d b y a s o l u t i o n o f P o t a s s i u m H y d r o x i d e made b y d i s s o l v i n g 100 grams o f P o t a s s i u m H y d r o x i d e i n 200 grams o f w a t e r . B y t a k i n g t h e amount o f c a r b o n d i o x i d e a b s o r b e d f r o m t h e o r i g i n a l v o l u m e o f g a s m i x t u r e , t h e p e r c e n t a g e o f c a r b o n d i o x i d e and o x y g e n c a n b e c a l c u l a t e d . T h i s a n a l y s i s i s d o n e t h r e e t i m e s f o r e a c h m i x t u r e o f g a s t o i n s u r e a c c u r a t e r e s u l t s . The b l o o d a g a r p l a t e s o r t u b e s o f i n f u s i o n b r o t h a r e p l a c e d i n C , f r o m w h i c h t h e a i r i s w i t h d r a w n b y means o f a s u c t i o n pump. T h e n n b n i s a t t a c h e d t o " g t t and " d H , " b " and ttcfl a r e o p e n e d . The m i x t u r e o f g a s e s i n B r u s h e s o v e r t o 0 and when 0 i s f i l l e d n d M and " b " a r e c l o s e d . T h e g a s I s w i t h d r a w n f r o m G; b y means o f a s u c t i o n pump> and t h e n t h e f l a s k i s a g a i n f i l l e d w i t h t h e m i x t u r e o f t h e two g a s e s . T h i s l a s t t i m e t h e l e v e l o f t h e w a t e r i n A i s k e p t s l i g h t l y b e l o w t h e l e v e l o f t h e w a t e r i n B i n o r d e r t h a t t h e f l a s k G w i l l n o t be q u i t e f i l l e d - t h i s s l i g h t d i f f e r e n c e i n t h e amount o f g a s i n G w i l l c o m p e n s a t e t o a c e r t a i n d e g r e e f o r t h e s l i g h t r i s e i n p r e s s u r e i n t h e f l a s k G cue t o t h e i n c r e a s e i n v o l u m e o f t h e g a s when i n c u b a t e d a t 3 7 ° C . EXPERIMENTS AND RESULTS Peroxide Production. A loopful of-'atwenty-four hour broth culture of each of the s i x strains of Streptocooous viridans was inocu-lated into 5 c.c. of: (1) infusion broth (2) yeast infusion broth i n a 10 c.c. oulture tube and incubated at 37° C. i n atmos-pheres of: (a) a i r (b) 100$ oxygen (c) 80$ oxygen and 20$ carbon dioxide (d) 60$ oxygen and 40$ carbon dioxide (e) 40$ oxygen and 60$ carbon dioxide (f) 20$ oxygen and 80$ carbon dioxide (g) 100$ carbon dioxide Every twelve hours from each tube 0.5 c.c. of f i l t r a t e was removed and tested f o r the presence of peroxide. A loopful ofvtwenty-four broth oulture of each of the strains of Streptococcus viridans was inoculated into 10 c.c. of: (1) infusion broth (2) yeast i n f u s i o n broth (3) dextrose infusion broth (1$ dextrose) contained i n a 125 c.c. e r l y a ^ f a - f l a s k and incubated at 370 c ; i n a i r . —12«» Every twelve hours from each tube 0.5 c.c. of f i l t r a t e was removed and tested for the presence of peroxide. The results are shown i n Table 1 and 2. Table 1. Relative amount o f peroxide produced by the six strains of Streptococcus viridans growing i n infusion broth i n a culture tube I n ; a i r . Hours 12 24 36 48 60 72 84 96 108 Strains #1 - — + + -i- +• Strains #2 - - — _ + + + + -h-h Strains #3 - - - - i- + 4- -i- + -h Strains #4 - - - — +• •*• •hi-* •h i- + + +-Strains #5 - - - - — i- +• i- + + + Strains #6 - - — - +• 4- + +••• -h Table 2. Relative amount of peroxide produced by the si x strains of Streptococcus viridans growing i n in f u s i o n broth i n 125 c.c. iaa£xta±soi flasks i n a i r . Hours 12 24 36 48 60 72 84 96 108 Strains #1 + + + + +h+ + +++ f—i-t- + Strains #2 + + + i h-h •hh^-t- y- V-* Strains #3 h h - h +-+' + •& + + +• -f-f—f- -h-f-+-f -h-f-Strains #4 •hh-f-f- h+h+ .•t—t-++ hh+-t + Strains #5 + + -f- + -t-*-i-t -t-+~l~t •h + Strains #6 •h + + 4-+i--h-h-f-* h-f-f •hi-These results were the same fo r the three types of medium. Peroxide was produced within twelve hours by the s i x strains of Streptococcus viridans when grown i n flasks which allowed a large surface exposure to a i r i n proportion to the volume of broth present. The production of peroxide reached i t s height i n twenty-four hours j i t remained constant u n t i l the ninety-sixth hour after which time i t began to de-crease. The broth oulture tubes of the six strains grown i n a i r did not show the presence of peroxide u n t i l about the seventy-second hour. The peroxide increased to a small extent u n t i l the ninety-sixth hour, but did not become greater af t e r this time. - I t -T h e same was t r u e f o r t h e o r g a n i s m s g r o w n i n t h e d i f f e r e n t a t m o s p h e r e s o f c a r b o n d i o x i d e and o x y g e n . V e r y l i t t l e d i f f e r e n c e c o u l d be f o u n d i n t h e amount o f p e r o x i d e p r o d u c t i o n i n t h e v a r i o u s m i x t u r e s . H o w e v e r , n o p e r o x i d e was p r o d u c e d i n a n a t m o s p h e r e o f 100$ c a r b o n d i o x i d e . The p r e s e n c e o f t h e p e r o x i d e was e v i d e n t a t a b o u t t h e same t i m e i n t h e t h r e e d i f f e r e n t m e d i a u s e d and r e m a i n e d u n t i l a b o u t t h e same t i m e . T h e t i m e o f t h e a p p e a r -a n c e o f t h e p e r o x i d e v a r i e d t o a s l i g h t e x t e n t when t h e e x p e r i m e n t was r e p e a t e d i n d i f f e r e n t b a t c h e s o f i n f u s i o n b r o t h . H o w e v e r , r e s u l t s o n t h e w h o l e f r o m t h e s e d i f f e r e n t b a t c h e s w e r e v e r y s i m i l i a r . P r o d u c t i o n o f A c i d . - T h e same f i l t r a t e s t h a t were u s e d i n t e s t i n g f o r t h e p r e s e n c e o f p e r o x i d e w e r e a l s o u s e d i n t e s t i n g f o r t h e amount o f a c i d p r o d u c e d . 1 c . c . o f e a c h f i l t r a t e was u s e d . T h i s t e s t was d o n e e v e r y t w e l v e h o u r s f o r n i n e t y - s i x h o u r s . B r o r a - t h y m o l - b l u e s t a n d a r d s w e r e u s e d . To 1 c . c . o f f i l t r a t e was a d d e d 4 c . c . o f w a t e r a n d e i g h t d r o p s o f 0 . 0 4 $ b r o m - t h y m o l -b l u e . T h e r e s u l t s a r e shown i n T a b l e 5 . - 1 5 -T a b l e 3 Ph o f f i l t r a t e s o f t h e s i x s t r a i n s o f S t r e p t o c o c c u s v i r i d a n s g r o w i n g i n i n f u s i o n b r o t h i n a i r and d i f f e r e n t a t m o s p h e r e s o f c a r b o n d i o x i d e and o x y g e n f o r t w e n t y - f o u r h o u r s . a. iv 3o7o coz 9 fo ?o Ox CO?o S t r a i n s #1 /% C-S c^ /*A ft S t r a i n s #2 - 6-9 .. £•? .»•• CC , 6- 3 S t r a i n s #3 - .. C? • C-6 .. c & , 6-3 S t r a i n s #4 • C-9 6? >• C& '•" 6-3 , 'S-Z. S t r a i n s #5 •• C? C- 8 ct - c $~ - C-3 S t r a i n s #6 " C-9 •• C? .. <£•*- " *-3 • sz When t h e o r g a n i s m s w e r e g r o w i n g i n a i r , t h e Ph o f t h e f i l t r a t e c h a n g e d f r o m a n i n i t i a l 7 . 4 t o 6 . 8 - 6 . 9 i n t h e f i r s t t w e n t y - f o u r h o u r s , a f t e r w h i c h t i m e t h e Ph o f t h e f i l t r a t e r e m a i n e d c o n s t a n t . When t h e b r o t h c u l t u r e s w e r e g r o w i n g i n t h e d i f f e r e n t a t m o s p h e r e s o f c a r b o n d i o x i d e and o x y g e n , t h e Ph o f v a r i o u s m e d i a d e c r e a s e d w i t h i n c r e a s i n g c a r b o n d i o x i d e c o n c e n t r a t i o n s . The P h o f t h e two m e d i a , t h e i n f u s i o n b r o t h and y e a s t i n f u s i o n b r o t h was t h e same a t a l l s t a g e s , When t h e d e x t r o s e b r o t h was u s e d ^ h o w e v e r , t h e P h o f t h e f i l t r a t e was 6 . 0 o r l e s s i n t w e l v e h o u r s and i t r e -m a i n e d s o . -1B-HAEMOLYSIN PRODUCTION hoar A l o o p f u l o f a t w e n t y - f o u r v b r o t h c u l t u r e o f e a c h o f t h e s i x s t r a i n s o f S t r e p t o c o c c u s v i r i d a n s was i n o c u -l a t e d i n t o 5 c . c . o f : (1) _ i n f u s i o n b r o t h ( 2 ) y e a s t i n f u s i o n b r o t h I n a 25 c . c . c u l t u r e t u b e and i n c u b a t e d a t 3 7 ° C . i n a t m o s -p h e r e s o f I ( a ) a i r (b) 100$ c a r b o n d i o x i d e ( c ) 9 0 $ c a r b o n d i o x i d e and 1 0 $ o x y g e n ( d ) 8 0 $ c a r b o n d i o x i d e and 2 0 $ o x y g e n ( e ) 7 0 $ c a r b o n d i o x i d e and 3 0 $ o x y g e n ( f ) 60$ c a r b o n d i o x i d e a n d 4 0 $ o x y g e n ( g ) 5 0 $ c a r b o n d i o x i d e and 50$ o x y g e n (h) 4 0 $ c a r b o n - d i o x i d e and 60$ o x y g e n ( i ) 30> c a r b o n d i o x i d e a n d 70$ o x y g e n ( j ) 2 0 $ c a r b o n d i o x i d e and 8 0 $ o x y g e n ( k ) 10$ c a r b o n d i o x i d e and 9 0 $ o x y g e n ( 1 ) 1 0 0 $ o x y g e n A t t h e end o f s e v e n t e e n h o u r s and t h i r t y - s i x h o u r s one t u b e o f e a c h o f t h e s i x s t r a i n s i n t h e two m e d i a was r e m o v e d and c e n t r i f u g e d a t J3000 r . p . m . f o r one h a l f h o u r « T h e s u p e r n a t a n t was t h e n removed and t e s t e d f o r t h e p r e s e n c e o f h a e m o l y s i n . F o r e a c h ' s t r a i n i n e a c h medium and e a c h t y p e o f r e d b l o o d c e l l t h e r e was a s e r i e s o f s i x t u b e s . To e a c h t u b e , e x c e p t t h e f i r s t o f e a c h s e r i e s , t h e r e had b e e n a d d e d 0 . 4 5 c . c o f 0 . 8 5 $ s a l i n e . -19-each o~f T o v t h e f i r s t and s e c o n d t u b e s o f t h e s e r i e s was a d d e d 0 . 4 5 c . c . o f t h e s u p e r n a t a n t f l u i d . T h e m i x t u r e was s h a k e n w e l l and 0 . 4 5 c . c . o f t h e m i x t u r e was removed f r o m t h e s e c o n d t u b e and added t o t h e t h i r d t u b e and t h i s p r o c e d u r e was r e p e a t e d u n t i l t h e f i f t h t u b e was r e a c h e d . T h e s e c o n d 0 . 4 5 c . c . o f m i x t u r e o f t h e f i f t h t u b e was d i s c a r d e d . The s i x t h t u b e was l e f t w i t h 0 . 4 5 c . c . o f s a l i n e i n i t . To e a c h t u b e was added 0 . 0 5 c . c . o f a 10$ s u s p e n s i o n o f t h r i c e washed r e d b l o o d c e l l s f r o m c i t r a t e d b l o o d . E a c h t u b e was w e l l s h a k e n a n d t h e r a c k s w e r e p l a c e d i n a n i n c u b a t o r a t 3 7 ° C. f o r one h o u r . D u r i n g t h e f i r s t h a l f h o u r t h e t u b e s were s h a k e n s e v e r a l t i m e s b u t w e r e n o t t o u c h e d d u r i n g t h e s e c o n d h a l f h o u r . A t t h e end o f t h e f i r s t h o u r t h e y w e r e t a k e n f r o m t h e i n c u b a t o r and p l a c e d a t r o o m t e m p e r a t u r e f o r a n h o u r , a f t e r w h i c h t i m e t h e d e g r e e o f h a e m o l y s i s was r e a d . T h e r e d c e l l s o f h u m a n , s h e e p and r a b b i t b l o o d w e r e u s e d . T h i s p r o c e d u r e was r e p e a t e d i n e x a c t l y t h e same way e x c e p t t h a t i n t o e a c h t u b e was p u t 5 m g . o f s o d i u m h y d r o s u l p h i t e . T h i s e x p e r i m e n t was p e r f o r m e d t h r e e t i m e s w i t h t h r e e d i f f e r e n t b a t c h e s o f i n f u s i o n b r o t h . T a b l e 4 . shows t h e r e s u l t s o b t a i n e d t h e f i r s t t i m e t h e e x p e r i m e n t was d o n e . -19-There was some evidence of haemolysin produc-t i o n as shown by the resultant action of the yeast infusion broth f i l t r a t e s on the red blood c e l l s of sheep. There was no evidence of haemolysis of the human or rabbit red blood c e l l s by the same f i l t r a t e s . Neither was there any evidence of Haemolysis by f i l t r a t e s of infusion broth oultures when tested on the red blood c e l l s of humans , sheep or rabbits, after growing i n a i r and the di f f e r e n t atmospheres of carbon dioxide and oxygen. When the experiment was repeated twice again with d i f f e r e n t batches of infusion broth, no haemolysin production could be shown i n the f i l t r a t e s when tested on human9 sheep and rabbit c e l l s , At no time did the addition of 5 mg. of sodium hydrosulphite increase the amount of haemolysis. Haemolysin production was also tested for on blood agar plates. Two types of agar media were used • Veal infusion agar and veal infusion yeast agar, 1.5$ agar having been added to the broth media. 12 c.c. of agar were used for each plate and to this was added 0.6 c.c. of citrated blood, Human, sheep and rabbit citrated blood was used. Prom a twenty-four hour broth culture of the six strains of Streptococcus v i r i d a n s , blood agar spread plates were made. These plates were incubated at 37° c . f o r t h i r t y -s i x hours i n atmospheres of: - 2 0 -( a ) a i r ( b ) 100$ c a r b o n d i o x i d e ( c ) 8 0 $ c a r b o n d i o x i d e and 20$ o x y g e n ( d ) 60$ c a r b o n d i o x i d e and 40$ o x y g e n ( e ) 4 0 $ c a r b o n d i o x i d e a n d 6 0$ o x y g e n ( f ) 2 0 $ c a r b o n d i o x i d e and 80$ o x y g e n (g) 100$ o x y g e n and a f t e r t h i r t y - s i x h o u r s t h e s e p l a t e s w e r e ' removed and e x a m i n e d i m m e d i a t e l y , w i t h t h e a i d o f m i c r o s c o p e , f o r d and p h a e m o l y s ! n p r o d u c t i o n . The e x p e r i m e n t was r e p e a t e d u s i n g a d i f f e r e n t b a t c h o f i n f u s i o n broth . The r e s u l t s a r e shown i n T a b l e 5 . ^ W ^ — ^ ^ ^ ^ — ^ ^ ^ ^ ^ -T a b l e 5 . <w%' to it, *t>Z to* • /oaPo O*. ^. . ~£ • so* ; >± sa* Strain* 1 J JL u J. J. -Cd A * >L(3 -L JL Jl A J.(i *C ft -< Jl J_fl <(3 JL JL J /. JL JB A. -k d@ JL. A d& A. J. d(3 • A. *L el J. U(i A(2 . »s X J. JL X jft J. A. A& JL. A J.6 A JL. ei.eJ.ei J. Uft oi/S , tfjf JL. i~ JL J. JL 4(3 JL JL el A JL.G JL A c< oL JL. J. j(i eld -~ - i -l -4 -J. ,. .r*> -w J. , *4 4. a n J. (3 p JL (3 a *L *L o l J^. A. S t r e p t o c o c c u s v i r i d a n s g r o w i n g o n b l o o d a g a r p l a t e s i n a i r and a t d i f f e r e n t a t m o s p h e r e s o f o x y g e n and c a r b o n d i o x i d e p r o d u c e a s u b s t a n c e w h i c h c a u s e s t h e h a e m o l y s i s o f t h e s h e e p r e d b l o o d c e l l s and n o t o f t h e human o r r a b b i t s e r y t h r o c y t e s , -21-T h e r e s u l t s o b t a i n e d v/ere t h e same o n t h e i n f u s i o n b l o o d a g a r p l a t e s a s o n t h e y e a s t i n f u s i o n b l o o d a g a r p l a t e s . The same r e s u l t s w e r e a l s o o b t a i n e d v/ i th a d i f f e r e n t b a t c h o f I n f u s i o n b r o t h . P r o d u c t i o n o f T o x i c - l i k e P r o d u c t s . I n t h e t e s t f o r t h e p o s s i b l e p r o d u c t i o n o f a n e n t e r o t o x i o - , n e c r o t o x i c - , and t o x i c - l i k e s u b s t a n c e b y t h e s i x s t r a i n s o f S t r e p t o c o c c u s v i r i d a n s t h e f i l t r a t e s u s e d w e r e p r e p a r e d i n t h e f o l l o w i n g m a n n e r : A l o o p f u l o f a t w e n t y - f o u r h o u r c u l t u r e o f e a c h o f t h e s i x s t r a i n s o f S t r e p t o c o c c u s v i r i d a n s was i n o c u l a t e d i n t o 20 c . c . o f : (1) i n f u s i o n b r o t h (2) y e a s t i n f u s i o n b r o t h (3) s e r u m i n f u s i o n b r o t h (20$) i n a 30 c . c , c u l t u r e t u b e a n d i n c u b a t e d a t 37° c. f o r t h i i t y -s i x h o u r s i n j ( a ) a i r ( b ) 20$ o a r b o n d i o x i d e and 80$ o x y g e n -22-After 36 hours the cultures were removed from the incubator, f i l t e r e d through a No. 1 f i l t e r paper and then •through a Seitz E.K. into s t e r i l e tubes. The culture"tubes containing these s t e r i l e f i l t r a t e s were plugged with s t e r i l e rubber stoppers and placed i n the ice box where they were kept u n t i l used. 0.5 c.c. of f i l t r a t e was added to s t e r i l e broth and incubated for twenty-four hours to test for s t e r i l i t y . 0.2 c.c. of a 1:10 d i l u t i o n of the s t e r i l e f i l t r a t e s obtained by the method described above, of each of the s i x strains of the Streptococcus viridans grown i n : (1) infusion broth (2) yeast i n f u s i o n broth (3) serum in f u s i o n broth (20%) and incubated i n (a) a i r (b) 20$ carbon dioxide and 80$ oxygen was inoculated intracutaneously i n the back of a white r a b b i t . Controls of a 1:10 d i l u t i o n of the d i f f e r e n t media were also made. This experiment was repeated on a white Angora rab b i t . 0.2 c.c. of a 1:1 d i l u t i o n of the same s t e r i l e f i l t r a t e s was inoculated intracutaneously into the back of a white ra b b i t . Controls of a 1:1 d i l u t i o n of the d i f f e r e n t media were also made. This experiment was repeated on a white Angora rabbit. -as-I i i ' t h e s e e x p e r i m e n t s t h e h a i r was removed f r o m t h e b a c k s o f t h e r a b b i t s b y means o f a n e l e c t r i c c l i p p e r * 0 . 2 c . c . o f a 1:1 d i l u t i o n o f t h e same s t e r i l e f i l t r a t e s o f y e a s t b r o t h g r o w i n g i n : ( a ) a i r ( b ) 20$ c a r b o n d i o x i d e and 8 0 $ o x y g e n was i n o c u l a t e d i n t r a c u t a n e o u s l y i n t o t h e b a c k o f a c a t . C o n -t r o l s o f a 1:1 d i l u t i o n o f t h e y e a s t i n f u s i o n medium w e r e m a d e . Q l c . c . o f a L : l d i l u t i o n o f t h e s t e r i l e f i l t r a t e s o f i n f u s i o n b r o t h g r o w n i n : ( a ) a i r ( b ) 20$ c a r b o n d i o x i d e and 8 0 $ o x y g e n w e r e i n o c u l a t e d i n t r a c u t a n e o u s l y i n t o t h e a r m o f a human v o l u n t e e r . A f t e r an . i n t e r v a l o f one w e e k 0.1 c . c . o f a 1 : 5 0 d i l u t i o n o f t h e s t e r i l e f i l t r a t e s o f i n f u s i o n b r o t h g r o w n i n : ( a ) a i r ( b ) 2 0 $ c a r b o n d i o x i d e and 8 0 $ o x y g e n was i n o c u l a t e d i n t r a c u t a n e o u s l y i n t o t h e a r m o f t h e same human v o l u n t e e r . A l l t h e 1 : 1 0 d i l u t i o n s o n t h e r a b b i t s gave n e g a t i v e r e s u l t s . T h e r e a c t i o n s o f t h e 1 : 1 d i l u t i o n s were o f 'a v e r y l i g h t p i n k c o l o u r e x c e p t t h o s e c a u s e d b y t h e s e r u m b r o t h f i l t r a t e s . T h e s e r e a c t i o n s w e r e n o l a r g e r t h a n t h e o t h e r s , b u t t h e y had a d e e p e r p i n k c o l o u r - a l m o s t a r e d . A l l t h e r e a c t i o n s f r o m a l l t h e d i f f e r e n t f i l t r a t e s w e r e o n l y a l i t t l e l a r g e r i n a r e a t h a n t h o s e o f t h e c o n t r o l r e a c t i o n s . The r e a c t i o n s on t h e A n g o r a r a b b i t w e r e a l i t t l e more I n t e n s e t h a n t h o s e r e a c t i o n s o n t h e b a c k o f t h e w h i t e r a b b i t . The s k i n r e a c t i o n s d o n e o n t h e c a t were n e -g a t i v e . The c o n t r o l s a s w e l l as t h e f i l t r a t e s g a v e a b s o l u t e -l y n o r e a c t i o n . On t h e human v o l u n t e e r t h e r e a c t i o n s o f t h e f i l t r a t e s and c o n t r o l s o f t h e 1 : 1 d i l u t i o n w e r e v e r y v i o l e n t . T h e f i l t r a t e s and c o n t r o l s were o f t h e same s i z e . The d i l u -t i o n o f m e d i a t h a t g a v e no r e a c t i o n was a 1 : 5 0 d i l u t i o n . I n t h i s d i l u t i o n n e i t h e r t h e c o n t r o l n o r t h e f i l t r a t e gave any r e a c t i o n . 4 . c . c . o f s t e r i l e f i l t r a t e o f t h e s i x s t r a i n s o f S t r e p t o c o c c u s v i r i d a n s g r o w n i n : ( 1 ) i n f u s i o n b r o t h (2) y e a s t I n f u s i o n b r o t h (3) s e r u m b r o t h ( 2 0 $ ) e a c h i n t ( a ) a i r (b) 2 0 $ c a r b o n d i o x i d e and 8 0 $ o x y g e n w e r e i n o c u l a t e d i n t e r p e r i t o n e a l l y i n t o s i x k i t t e n s s i x t i m e s a t two d a y i n t e r v a l s . The f i l t r a t e s showed no o b s e r v a b l e e f f e c t s on t h e s i x k i t t e n s . 1 c . c . o f s t e r i l e f i l t r a t e o f t h e s i x d i f f e r e n t o r g a n i s m s g r o w n i n : ( 1 ) s e r u m b r o t h ( 2 0 $ ) ( 2 ) y e a s t i n f u s i o n b r o t h ( 3 ) i n f u s i o n b r o t h e a c h i n : ( a ) a i r ( b ) 20$ c a r b o n d i o x i d e and 8 0 $ o x y g e n was 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 i n t o s i x w h i t e m i c e , s i x t i m e s a t two d a y i n t e r v a l s . The f i l t r a t e s showed no o b s e r -v a b l e e f f e c t s on t h e m i c e . -2B-S I G N I F I C A N C E OP RESULTS The s i x s t r a i n s o f S t r e p t o c o c c u s v i r i d a n s -g rown i n d i f f e r e n t m e d i a and u n d e r d i f f e r e n t a t m o s p h e r e s o f C a r b o n d i o x i d e and o x y g e n as w e l l as i n a i r , r e a c h e d t h e i r maximum a c i d p r o d u c t i o n b e f o r e t w e n t y - f o u r h o u r s . The Ph o f t h e m e d i a r e m a i n e d t h e same d u r i n g t h e t i m e o f o b s e r v a -t i o n , u s u a l l y n i n e t y - s i x h o u r s . The p e r o x i d e p r o d u c t i o n i n c u l t u r e t u b e s was n o t o f s u f f i c i e n t s t r e n g t h t o b e d e m o n s t r a -t e d b e f o r e s e v e n t y - t w o h o u r s . When t h e p r e s e n c e o f p e r o x i d e c o u l d b e d e -m o n s t r a t e d no o b s e r v a b l e e f f e c t o f g r o w i n g t h e o r g a n i s m s i n a n i n c r e a s e d o x y g e n t e n s i o n c o u l d be s e e n . E x c e p t t h a t i n a n a t m o s p h e r e o f 1 0 0 $ c a r b o n d i o x i d e no p e r o x i d e c o u l d be d e m o n s t r a t e d . T h e r e s u l t s w o u l d p r o b a b l y h a v e b e e n more c o n c l u s i v e i f p e r o x i d e p r o d u c t i o n i n d i f f e r e n t a t m o s p h e r e s c o u l d h a v e b e e n t e s t e d f o r b y u s i n g a s m a l l q u a n t i t y o f medium i n a l a r g e f l a s k . H o w e v e r , t h i s was n o t p r a c t i c a l w i t h t h e l i m i t e d a p p a r a t u s uoed i n t h e - l a b o r a t o r y . When t h e s i x v i r i d a n s t r a i ns were g r o w n i n l a r g e f l a s k s I n a r e l a t i v e l y s m a l l amount o f b r o t h , t h e p e r -o x i d e p r o d u c t s o c c u r r e d w i t h i n t w e l v e h o u r s . The one t i m e t h a t a n i n d i c a t i o n o f t h e p r o -d u c t i o n o f a (3 £ [ a e m o l y s i n - l l k e s u b s t a n c e i n a c u l t u r e t u b e was . g i v e n , t h e maximum h a e m o l y s i n was p r o d u c e d lay s e v e n t e e n h o u r s . - 2 3 -T h i s r e m a i n e d c o n s t a n t u n t i l t h e t h i r t y - s i x t h h o u r a t w h i c h t i m e n o o x i d i z e d h a e m o t o x i n c o u l d be d e m o n s t r a t e d . The l a c k o f o x i d i z e d h a e m o t o x i n i s , h o w e v e r , n o t s i g n i f i c a n t as t h e amount o f h a e m o l y s i n was s m a l l and t h e s m a l l amount o f a d d i -t i o n a l r e d u c e d h a e m o t o x i n w h i c h m i g h t h a v e b e e n added t o t h e f i l t r a t e by t r e a t i n g w i t h s o d i u m h y d r o s u l p h i t e , c o u l d n o t be r e a d i l y o b s e r v e d w i t h the methods u s e d . On t h e b l o o d a g a r p l a t e s , t h i s s o c a l l e d h a e m o l y s i n a p p e a r e d l a t e r t h a n t h e p e r o x i d e , o r i t d i f f u s e d more s l o w l y ^ a s t h e r e was a r i n g o f m e t h a e r a o g l o b l n s u r r o u n d i n g t h e c o l o n y and t h e n a c l e a r zone o f h a e m o l y s i s . H a g a n ^ n e n e x p l a i n i n g t h e a p p e a r a n c e on b l o o d a g a r p r o d u c e d b y a l t e r n a t e l y g r o w i n g t h e o r g a n i s m p r o d u c i n g t h e t y p e h a e m o l y s i n a t 3 7 ° c. and a t r e f r i g e r a t o r t e m p e r a -t u r e ? s u g g e s t e d " p e r o x i d e and a c i d h a v e a n a n t a g o n i s t i c a c t i o n on b l o o d c o r p u s l e s , p e r o x i d e t e n d i n g t o d i s c o l o u r and p r o t e c t them f r o m h a e m o l y s i s b y t h e a c i d • H a e m o l y s i s r e s u l t s o n l y when p e r o x i d e p r o d u c t i o n d i m i n i s h e s o r c e a s e s . " T h i s h a e m o l y s i n I s n o t due t o the i n c r e a s e o f H i o n as t h e g r e a t e s t p r o d u c t i o n o f h a e m o l y s i n t a k e s p l a c e i n a n a t m o s p h e r e o f 20$ c a r b o n d i o x i d e and 80$ o x y g e n and n o t i n a n a t m o s p h e r e o f 100$ c a r b o n d i o x i d e i n w h i c h a t m o s p h e r e t h e g r e a t e s t c o n c e n t r a t i o n o f H i o n i s p r o d u c e d . -28-The peroxide does not seem to be affected^ by a greater concentration of H ion since i n the fermented .dextrose infusion broth the production of peroxide was un-affected. That an in d i c a t i o n at lea s t of the production of a haemolysin-like substance was obtained i n one batch of infusion broth and not i n two others i s i n keeping with the findings of Todd^^wtien working on Strep t o l y s i n . He writes "there i s a great v a r i a t i o n i n the y i e l d of Streptolysin from d i f f e r e n t batches of broth prepared i n the same way and as the essential factor responsible for their v a r i a t i o n i s unknown, broth which i s sat i s f a c t o r y for Streptolysin pro-duction has been secured by testing every batch of broth made from the routine work of the laboratory and reserving suitable batches f o r Streptolysin production. B After trying two batches of media other than that i n which the (I haemolysin was produced, i t was decided not to try further to produce the haemolysin, as i n this laboratory, where only small quantities of in f u s i o n broth are made up at one time, i t was not p r a c t i o a l . In testing for the production of an entero-t o x i c - l i k e substance the tests devised by Dolman, Wilson, and Gockorofty^o show the presence of Staphlococcus entero-toxin, were used. - 2 9 -Th© results were negative i n every case. . This can be Interpreted to mean one of three things: the entero-toxin produced by the Streptococci i s not shown by this t e s t , that the exact conditions for the production of Anterotoxin by Streptococcus viridans were not used, that the s i x strains of Streptocou3 viridans used do not produce an interotoxin-l i k e substance. Prom the results of these experiments no de f i n i t e conclusions can be reached. In testing for the production of a necrotoxic-l i k e substance the reactions were so small as to make the results inconclusive. This means that the conditions under which the s i x strains were grown were not suitable f o r the maximum production of necrotoxin. No toxic products could be demonstrated i n the Of f i l t r a t e s of broth, asd serum broth cultures using suitable t e s t s . This can be taken to indicate that either no toxic substance i s produced by any of the six strains under i n v e s t i -gation, or that the conditions for the production of such toxic substances were not used• Summary of Result. Under defined conditions, using d i f f e r e n t media and atmospheres of varying mixtures of carbon dioxide and oxygen the f i l t r a t e s of the s i x strains of Streptococcus viridans caused the haemolysis of the red c e l l s of sheep. - S O -Under lite® conditions the red c e l l s o f sheep i n agar plates were haemolysed by f i v e o f the s i x strains o f Strepto-. coccus viri d a n s , Under these same conditions and using a variety o f laboratory animals, the production of t o x i c - enterotoxio- or necrotoxic-like substances were not able to be demonstrated i n the f i l t r a t e s of broth cultures of these same s i x st r a i n s * REFERENCE (1) H. D. Wright, 1925, A System of Bacteriology, Vol. 2, P. 105. (2) Stevens, 1919, J . Exp. Med. 30, 553. P. Kruif, 1920, J . Enf . Dis. 26, 285. Avery & Morgan, 1924, J . Exp. Med. 39, 23 7. J . N e i l , 1926, J . Exp. Med. 44, 241. Gordon, 1929, B r i t . J . Exp. Path. 10, 192. E. W. Todd, 1928, J . Exp. Med. 48, 493. " 11 M 1930, B r i t . J . Exp. Path. 11, 391. H w 1930, B r i t . J . Exp. Path. 11, 479. " " 11 1932, j * Exp. Med, 55, 278. " " w 1932, B r i t . J . Exp. Path. 13, 258. Hodge, 1933, J . Exp. Med, 58, 284. R. Lancefield, 1934, J . Exp. Med. 59, 468, (3) Avery & Morgan, 1924, J . Exp. Med. 39, 287. Avery & Morgan, 1924, J . Exp. Med« 39, 345, Avery & N e i l , 1924, J . Exp. Med, 39, 355, n " " w• J . Exp s Med, 39, 366. " " " 1 1 J , Exp. Med. 39, 744. M - " n * . J . Exp. Med. 39, 755. w K " " J . Exp. Med, 40, 422, 427. Nei l & Avery, 1925, J , Exp. Med. 41, 285. J . N e i l , 1926, J . Exp. Med. 44, 273, Ne i l & Hemming, 1927, J . Exp. Med. 46, 735. S. E. Cawan, 1934, J . Bact. & Path. 38, 61. (4) Avery & Morgan, 1924, J . Exp. Med. 39, 287, (5) H. W. L y a l l , 192^, J . Med. Res, 30, 487. (6) L. Mishulow, 1921, J , Immunol, 6, 329, (7) W. M. Gumming, 1927, J , Path. & Bact. 30, 279. (8) Brown, 1919, Monograph No. 9, Rockerfeller Inst, f o r Med, Research. (9) N e i l & Mallary, 1926, J . Exp. Med, 44, 213. S. E. Cawan, 1934, J . Bact, & Path. 38, 61. (10) Hagan, 1925, J . Inf. Dis. 37, 1. (11) E. W, Todd, 1932, J . Exp. Med. 55, 278. (12) Dolman, Wilson, Cockcroft, 1936, (Paper i n process of publication). 

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