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Studies on the Streptococcus Viridans Bligh, Una Maud 1936

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STUDIES  ON T H E S T R E P T O C O C C U S  Una  Bligh  B.A.  Thesis submitted degree of MASTER OF  ARTS  October,  1936  University  of  VIRIDANS  British  for  Columbia  STUDIES ON THE STREPTOCOCCUS VIRIDANS  I  INTRODUCTION.  II  METHODS AND TESTS USED.  III  (a)  Media  (b)  S t r a i n s used•  used.  (c)  T e s t f o r peroxide.  (d)  D e s c r i p t i o n of apparatus used f o r o b t a i n i n g d e f i n i t e mixtures of carbon d i o x i d e and oxygen*  EXPERIMENTS AMD' RESULTS. (a) (b) (o) (d)  Production o f Peroxide. Production o f A c i d . Production of Haeraolysin. Examination f o r e n t e r o - , necr-oov 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 c h a r a c t e r i s t i c a l l y found as a saprophyte upon the mucous membrane of the upper part of the alimentary canal and the respiratory passages of man.  I t can be  demonstrated i n p r a c t i c a l l y a l l mouths In the entire absence of any recognizable pathological process. I t i s associated frequently with tooth abscesses, and may be present i n middle ear diseases and i n f e c t i o n of the accessory sinuses of the nose.  I t i s not infrequently found i n  the t o n s i l l a r crypts and has been known to cause mild forms of t o n s i l l itis.  The viridans i s frequently associated with sub-acute vegetative  endocarditis.  In such cases the organism causes r e l a t i v e l y firm  vegetations, usually on the mitral but sometimes also on the a o r t i c valves, extending occasionally along the walls of the a u r i c l e s .  The  organisms can be readily cultivated from the blood stream where they may remain f o r weeks and months. H.D. Wright ( l ) writes, "In endocarditis the o r i g i n a l i n f e c t i o n 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 t r a c t and presumably come from this situation."  Because of these f s c t s ^ i t was  thought  that i t  would be of i n t e r e s t to make some s t u d i e s , i n t h i s l a b o r a t o r y , on t h i s organism.  S© attempt  to f i n d out i f under c e r t a i n  by  'conditions of atmosphere arrNusing d i f f e r e n t media the S t r e p t o Subs'ta.ticaS  coccus v i r i d a n s  could be made to produce some t o x i c o f f o o t s ,  which might account f o r i t s a c t i o n i n some of the d i s e a s e s f o r which i t i s r e s p o n s i b l e .  I t was  thought  t h a t i t would be of  i n t e r e s t a l s o t o determine whether or not Streptococcus v i r i d a n s produced  a ^"haemolytic-llke  There i s no d i r e c t  substance.  evidence i n the l i t e r a t u r e  that Streptococcus v i r i d a n s produces haemolysin,  although  much work has been ctone on the e n d o c e l l u l a r and  extracellular  p r o d u c t i o n of haemolysin  streptococci  by both the haemolytie  (2) and the pneumooocci ( 3 ) .  Much work has  also been done  on the p r o d u c t i o n of peroxide by both the pneumococoi (3) and haemolytie s t r e p t o c o c c i (30» and  there i s evidence i n the  l i t e r a t u r e t h a t the v i r i d a n s s t r e p t o c o c c i produce peroxide ( 4 ) . The o b s e r v a t i o n o f the f i r a L . phenomenon of haemolysis, was  first  made by Marmorek i n 1895.  Marmorek be-  l e i v e d t h a t there was  a d i r e c t r e l a t i o n s h i p between v i r u l e n c e  and haemolytie power•  Other i n v e s t i g a t o r s , however, notably  Schotmuller, b e l o v e d , from the beginning, that the power was changeable lence.  haemolytie  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-  by experimental enhancement or r e d u c t i o n of v i r u -  I t i s g e n e r a l l y b e l o v e d by the workers of today that  an exact p a r a l l e l i s m between v i r u l e n c e and haemolytlo i n Streptococcus s t r a i n s does not e x i s t , s i n c e many  activity  independent  observers have found that haemolysin p r o d u c t i o n may continue a c t i v e i n a s t r a i n . w h i c h has l o s t i t s v i r u l e n c e * I t was decided to t e s t f o r haemolysin t i o n by two methods:  produc-  ( 1 ) p l a t e method ( 2 ) tube method.  I t was S c h o t t m u l l e r s i n t r o d u c t i o n o f the blood f  agar p l a t e which l e d to the d i f f e v e n t r a t i o n of those atral ns produoing haemolysin from those producing methaemoglobin.  The  appearance of a S t r e p t o c c a l c u l t u r e on a blood agar p l a t e which i s accepted as evidence of haemolytic a c t i v i t y i s the development of c l e a r , c o l o u r l e s s t r a n s p a r e n t 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.  T h i s i s not simply haemolysis, i t Is haemolysis  p l u s d e s t r u c t i o n or removal o f blood pigment and S t a p p a r e n t l y it • may occur although the coccus concerned c r e t e a haemolytic t o x i n .  does not r e a d i l y s e -  These d i s c r e p a n c i e s between the  p l a t e method a n d the tube method of observing haemolysis have been noted by s e v e r a l workers ( 5 ) ( 6 ) ( 7 ) .  The u s u a l d i s c r e -  pancy i s t h a t s t r a i n s appearing to be haemolytic a r e not found to produce haemolysis when the f l u i d blood  c u l t u r e s are added t o  suspensions. Brown (£) who has devoted  a n extensive monograph  to t i l l s s u b j e c t of d i f f e r e n t types o f haemolysis  on blood agar  p l a t e s d e s c r i b e s an oC type which corresponds t o the Streptoccus  -4virldans,  a  ^  Streptococcus haemolytic  type  pyogenes  production Neil  haemolysin exposure  to  which  looses  nor  and  its  suitable  and  corresponds an  V  green  Mailory  activity reducing  type  to  the  actively  haemolytic  w h i c h gives~ n e i t h e r  discolouration. (0)  have  shown t h a t  on o x i d a t i o n agents®  but  streptococcal  regains  it  an  MATERIAL AM) TESTS  Veal entire  infusion  experiment.  1%  proteoae  m e d i u m made u p t o a p h o f 0 . 01$ o f y e a s t broth. filter  extract  The b r o t h paper  flasks^  1,  and  entire The  actions  isolated  and opaque,  organisms  saline.  and t h e broth,  to the  infusion  filtered  a Seitz  the  infusion  was added  b r o t h were  the c u l t u r a l  the colonies,  period  ox-bile.  and y e a s t  was a d d e d ,  F o r the yeast  (Orla-Jensen)  six strains  2 and 3 were  dome-shaped  7.4.  throughout  E.K.  through into  coarse  sterile  tubes.  done w i t h  obtained,  peptone  and t h e n r u n t h r o u g h  All was  b r o t h was u s e d  They  from normal  agar,  and they  remained  classified  ^tg - S~t • #3-  V«-KI  St. viT,  were  When  small,  i n a 1:10 mixture spontaneously  first smooth,  J an%  C<*ccorcf.tn<f  C  -  f5  »  of in  the  diffuse. Bacto 0.85$  Fermentation  as:  Jans  Strains  so throughout  be d e m o n s t r a t e d .  the strains  paper  viridans.  T h e g r o w t h i n b r o t h was  did not agglutinate could  i n this  throats.  on blood  not solubile  No c a p s u l e s  reported  of Streptococcus  o f the work. were  work  73ttTya.y)  "  )  re-  S t r a i n #4 was  i s o l a t e d from an abscessed t o o t h .  On sheep:-  blood agar the c o l o n i e s were opague, c o a r s e l y g r a n u l a r , f l a t tened w i t h a c e n t r a l e l e v a t i o n and i r r e g u l a r edges. first  When  I s o l a t e d and d u r i n g 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 b r o t h . s t r a i n was  not s o l u b l l e i n a 1:10 mixture of B a c t o - b i l e .  capsule could be demonstrated. nate spontaneously i n 0.85^ classified  The No  The organism d i d not a g g l u t e -  saline.  Fermentation r e a c t i o n s  the s t r a i n as Streptococcus  v/r/c/a»s  - »<>n  /«7s «/, st  aa  j,'c,n  **-rm  S t r a i n #5 - i s o l a t e d from a c a s e of mastoids. C u l t u r a l c h a r a c t e r i s t i c s were the. same as No. 4, but the f e r mentation r e a c t i o n s 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 - i s o l a t e d from a Mood c u l t u r e of 'bacterial endocarditis.  When f i r s t  obtained the c o l o n i e s on  sheep blood agar were s m a l l , smooth, dome-shaped and Upon subsequent  opaque.  t r a n s f e r they became l a r g e r , c o a r s e l y granular,  f l a t t e n e d ? w i t h a c e n t r a l e l e v a t i o n and i r r e g u l a r  edges.  On the f i r s t s u b c u l t u r e , the c o l o n i e s a s m a l l r i n g of green d i s c o l o u r a t i o n ; upon subsequent a c l e a r r i n g of haemolysis, was  produced transfer  produced, and t h i s remained  c h a r a c t e r i s t i c throughout the experiment.  a  The s t r a i n grew as a coarse f l o c c u l a r ment i n b r o t h . mixture  The organism was  of Bacto o x - b i l e .  t i o n i n 0.85$ s a l i n e .  not s o l u b i l e i n a  There was  sedi1:10  spontaneous a g g l u t i n a -  Uo capsule could be demonstrated.  Fermentation r e a c t i o n s 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 f o r Peroxide P r o d u c t i o n . To 0.5  c.c* of f i l t r a t e i n a s m a l l c u l t u r e  tube add a s m a l l piece of f r e s h l y cut potato (about 2 mm. 2 mm.  x 4 mm.)  x  and three drops of f r e s h l y prepared s a t u r a t e d  g l a c i a l a c e t i c a c i d s o l u t i o n of b e n z i d i n e .  I n the  of peroxide the potato takes on a b l u e c o l o u r .  The  presence intensity  of the c o l o u r depends on the amount of peroxide present.  -h + +• f + ++ + + +  Very s l i g h t blue c o l o u r .  A l i g h t blue c o l o u r , Dark b l u e c o l o u r . i n t e n s e blue c o l o u r .  Apparatus f o r and O x y g e n .  Obtaining ~  Definite ~~  Mixtures of  ^  Carbon dioxid e : ——  "6' Y  ft  &uction(f-  lo%K)/oumji  =9The water with  carbon dioxide  means  of  the  and  principle  filled  with  of  carbon dioxide  20%  the  carbon  dioxide  is  into  run  and  B -  a  the  flask  litre  the  saturated  attached  to  which  about  run of  is  into the  B at  an  B.  into  level B in  atmospheric  carbon dioxide  is  absorbed  dissolving water. the  three  a  and  and  times  results.  is  scale  is  of  and  oxygen  one  test  of  of  gas  can be  gas  to  on  one  is  tank of  B  about  Then " a " a  Is  used  oxygen  the mixture  of  gases  i s done  is  c"  then run in  B.  the  to  is  tt  on  The  in  dioxide  This  in  closed. mixture  carbon made  dioxide by  200 grams absorbed of  analysis  insure  are  level  the  percentage  the  dioxide  the  calculated. of  mixture  to  carbon  mixture,  each mixture  and  Potassium Hydroxide  of  a  carbon  litre  Potassium Hydroxide  amount  say  brought  When t h i s  of  and  litre,  A is  leave  completely  b e e n marked  five  By  attached  oxygen from  in  using.  B is  displaced. -  saturated  before  "a" is  has  about  water to  of  is  obtaining  opened  oxygen present  the  volume  for  .  n  volumes  -  In  oxygen,  rough  solution  By t a k i n g  dioxide  c  analysis  100 grams  original  M  pressure.  of  flask  siphon,flask  Q0%  order  A gas  mixing  water*  water  pure  99%  in  by  the  oxygen g e n e r a t o r  The  water  of  generator,  flask  the  oxygen each time  saturated  dividing of  in  of from  carbon is  accurate  done  The broth are means "d ,  of  rushes The  a  "b"  H  and  Is  the  c  tt  0 and  flask  slightly  below  G will  is  the  amount  of  the  slight  rise  volume  of  the  when 0  quite  in  in  of  G will  b  the is  n  tubes  air  is  means with  level the  n  this  in  the  when i n c u b a t e d  slight to  flask at  B in  a  C.  B closed.  pump> a n d the A is  order  two kept  that  the  difference  certain  G cue  37°  in  of in  by and  t t  are  suction  water  in  "g  "b"  mixture  the  compensate  to  gases  and  M  infusion  withdrawn  of  a  the  water -  d  of  of  of  attached  filled  filled  pressure  gas  is  filled the  n  or  The m i x t u r e  f r o m G; b y  level  be  gas  Then  opened.  time  the  plates  from which  again  last  not  agar  pump.  withdrawn  This  in  C,  are  fl  to  gases.  flask  in  suction  over  gas  then  placed  blood  to  the  degree  in for  increase  E X P E R I M E N T S AND  Peroxide  RESULTS  Production. A l o o p f u l of-'atwenty-four hour b r o t h c u l t u r e of  each of the s i x s t r a i n s of Streptocooous  v i r i d a n s was  inocu-  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) yeast i n f u s i o n b r o t h i n a 10 c.c. o u l t u r e tube and  incubated a t 37° C. i n atmos-  pheres o f : (a) a i r (b) 100$ (c) 80$ (d) 60$ (e) 40$ ( f ) 20$ (g) 100$  oxygen oxygen oxygen oxygen oxygen carbon  and 20$ and 40$ and 60$ and 80$ dioxide  carbon carbon carbon carbon  dioxide dioxide dioxide dioxide  Every twelve hours from each tube 0.5 f i l t r a t e was  removed and  t e s t e d f o r the presence  c.c. of  of peroxide.  A l o o p f u l ofvtwenty-four b r o t h o u l t u r e of each of the s t r a i n s of Streptococcus v i r i d a n s was  inoculated into  10 c.c. of: (1) i n f u s i o n b r o t h (2) yeast i n f u s i o n b r o t h (3) dextrose i n f u s i o n b r o t h (1$ contained i n a 125  370 ; c  i n  a i r  .  dextrose)  c.c. e r l y a ^ f a - f l a s k and incubated a t  —12«»  Every twelve hours from each tube 0.5 c . c . of f i l t r a t e was removed and t e s t e d f o r the presence of p e r o x i d e . The r e s u l t s are shown i n Table 1 and 2. Table 1. R e l a t i v e amount o f peroxide produced by the s i x s t r a i n s of Streptococcus v i r i d a n s growing i n i n f u s i o n b r o t h i n a c u l t u r e tube I n ; a i r . Hours  12  24  S t r a i n s #1  -  —  S t r a i n s #2  -  S t r a i n s #3  -  -  Strains #4 S t r a i n s #5 S t r a i n s #6  -  36  48  60  72 +  —  _  -  -  -  -  -  -  -  -  —  + +  — —  -  +• •*•  84 +  96  +•  -i-  +  +  -h-h  i-  +  4- -i-  •hi-*  i-  +•  +•  4-  108  •h i- +  i-  +  + -h  + +-  + +  + +••• -h  Table 2. R e l a t i v e amount o f peroxide produced by 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 growing i n i n f u s i o n b r o t h i n 125 c . c . iaa£xta±soi f l a s k s i n a i r .  12  Hours S t r a i n s #1 Strains #2 Strains #3  +  +  +  + +  +  +h+  + i  h-h  +-+'  h h - h  S t r a i n s #4  •hh-f-f-  Strains #5 S t r a i n s #6  24  36  48  +  +  +  72  h+h+  84  96  108  •hh^-t-  y- V-*  f—i-t- +  +++  +• -f-f—f- -h-f-+-f  + •& + +  + + -f- +  •h  60  .•t—t-++  hh+-t  -t-*-i-t  -t-+~l~t  -h-h-f-*  4-+i-  -h-f-  + •h +  •hi-  h-f-f  These r e s u l t s were the same f o r the three types of medium. Peroxide was produced  w i t h i n twelve hours by  the s i x s t r a i n s o f Streptococcus v i r i d a n s when grown i n f l a s k s which allowed a l a r g e s u r f a c e exposure t o a i r i n p r o p o r t i o n to the volume o f b r o t h p r e s e n t . reached until  The p r o d u c t i o n of peroxide  i t s h e i g h t i n twenty-four hours j i t remained  constant  the n i n e t y - s i x t h hour a f t e r which time i t began t o de-  crease. The b r o t h o u l t u r e tubes of the s i x s t r a i n s grown i n a i r d i d not show the presence about the seventy-second  hour.  of peroxide  until  The peroxide increased t o a  s m a l l extent u n t i l the n i n e t y - s i x t h hour, but d i d not become g r e a t e r a f t e r t h i s time.  -ItThe the  different  little  same w a s  atmospheres  difference  production produced  in  in  could  the  about  the  remained ance  of  the  about  peroxide was  However,  batches  were  of  amount This  test  acid was  the  carbon  the  a  different The  slight  different  in  oxygen. of  Very  peroxide  no  peroxide  was  dioxide.  peroxide  time.  to  amount  However,  was  evident  media  time  of  extent  batches  on t h e whole  of  same f i l t r a t e s peroxide  produced. done  every  1  of  4 c.c.  results  water are  were c.c.  twelve  was  The  of  three  in  standards  blue.  100$  same  Brora-thymol-blue added  in  and  grown  from  used  and  the  appear-  when  of  at  the  infusion  these  different  Acid.  presence of  of  varied  organisms  similiar.  - The the  the  mixtures.  the  results  very  found  the  repeated  broth.  for  in  for  carbon dioxide  presence  time  until  Production  be  an atmosphere  same  experiment  of  various  The  true  were and  that  were u s e d  also  used  of  used.  shown i n  for  Table  5.  was  ninety-six  To 1 c . c .  drops  testing  testing  each f i l t r a t e  hours  eight  in  in  of  of  0.04$  for  the  used. hours.  filtrate brom-thymol-  -15Table  Ph o f f i l t r a t e s Streptococcus different  viridans  atmospheres  twenty-four  growing of  Strains  #2  Strains  #3  Strains  #4  Strains  #5  Strains  in infusion  -  6-9  ..  ..  -  •  C-9  ••  C?  #6  C-9  Ph o f t h e f i l t r a t e the f i r s t filtrate  .»•• •  C?  ••  ct  remained  hours,  t h e Ph o f v a r i o u s  media  concentrations.  infusion  and y e a s t  When t h e d e x t r o s e  the f i l t r a t e so.  "  growing  which  of  time  6.8 -  sz  6.9  cultures  carbon dioxide  The Ph o f  t h e two m e d i a ,  increasing  b r o t h w a s t h e same a t  hours  were  and  with  i n twelve  •  t h e Ph o f  b r o t h was u s e d ^ h o w e v e r ,  was 6 . 0 o r l e s s  *-3  decreased  infusion  'S-Z.  i n a i r , the  7.4 to  When t h e b r o t h  atmospheres  carbon dioxide broth  after  ,  C-3  -  from an i n i t i a l  constant.  the d i f f e r e n t  6-3  .. <£•*-  changed  6-3  '•"  - c $~  were  6- 3  ,  .. c &  C-6  C?  twenty-four  ,  >• C&  C- 8  oxygen,  mained  for  ft  /*A  CC  6?  in  of  i n a i r and  CO?o  c^  £•?  growing  stages,  broth  and oxygen  coz  When t h e o r g a n i s m s  the  of  fo ?o Ox  9  /% C-S  "  in  the s i x s t r a i n s  hours. 3o7o  #1  of  carbon dioxide  a. iv  Strains  3  the a l l  the Ph  and i t  re-  -1BHAEMOLYSIN  PRODUCTION hoar  A loopful each  of  lated  the  into  six  of  strains  5 c.c.  of  a  Streptococcus  (2) 25 c . c .  pheres  yeast  culture  one  tube  was  removed  The  supernatant haemolysin.  of  red  0.45  was  inocu-  broth  infusion  tube  blood except  c . c  of  air 100$ 90$ 80$ 70$ 60$ 50$ 40$ 30> 20$ 10$ 100$  At  the  of  each  and  of  tube,  viridans  of  and  broth  incubated  at  37°  C.  in  atmos-  and and and and and and and and and  10$ 20$ 30$ 40$ 50$ 60$ 70$ 80$ 90$  oxygen oxygen oxygen oxygen oxygen oxygen oxygen oxygen oxygen  of I (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (1)  hours  culture  of:  ( 1 )_ i n f u s i o n  In a  twenty-fourvbroth  end of  of  seventeen  the  centrifuged  six  t h e n removed  For  each' s t r a i n  there first  0.85$  was of  saline.  hours  strains  in  a t J3000 r . p . m .  was  cell the  carbon dioxide carbon dioxide carbon dioxide carbon dioxide carbon dioxide carbon dioxide carbon-dioxide carbon dioxide carbon dioxide carbon dioxide oxygen  a  each  and in  and the  for  tested  for  thirty-six two  one h a l f the  e a c h medium and  series  of  series,  six there  media  tubes.  hour«  presence each To  had b e e n  type each  added  -19each o~f Tovthe was  added  was  shaken w e l l  and  0.45  the  second  and  added  was  repeated  0.45  c.c.  sixth tube red  0.45  was  until  of  c.c. to  the  of  the  the  fluid.  mixture  third  tube  was  fifth  of  tube  the The  was and  this The  second  discarded.  The  saline  in  it.  added  0.05  c.c.  10$ s u s p e n s i o n  of  thrice  cells the  from  a  citrated  racks  were  blood.  placed  in  Each an  tube  To  was  incubator  from  procedure  c.c.  of  of  was  mixture  removed  reached.  tube  series  with 0.45  each washed  well  at  37°  for  C.  hour. During  several the  times  the  first  but  were n o t first  of  the  and  placed  at  room t e m p e r a t u r e  the  degree  of  haemolysis  sheep  and  rabbit  exactly  5 mg.  of  the  blood  sodium  the  tubes  during  the  second  for  read. used. that  taken from  an hour, The This into  red  were half  the  which  cells  of  each  tube  hour.  incubator  after  procedure  shaken  time  human,  was  repeated  was  put  hydrosulphite.  different  results  was  hour  they were  same way e x c e p t  This three  hour  were  half  touched  end  in  tubes  supernatant  fifth  of  second  left  shaken and  At  the  the  mixture  and  was  blood  one  tube  of  tube  c.c.  first  obtained  experiment  batches the  of  first  was  performed  three  infusion  broth.  time  experiment  the  Table was  times 4.  with  shows  done.  the  -19There was some evidence  of haemolysin  t i o n as shown by the r e s u l t a n t a c t i o n of the yeast b r o t h f i l t r a t e s on the red blood c e l l s no evidence  o f haemolysis  of sheep.  produc-  infusion There was  of the human or r a b b i t red blood  c e l l s by the same f i l t r a t e s .  N e i t h e r was there any evidence of  Haemolysis by f i l t r a t e s of i n f u s i o n b r o t h o u l t u r e s when tested on the red blood c e l l s  o f humans , sheep or r a b b i t s , a f t e r  growing i n a i r and the d i f f e r e n t atmospheres of carbon d i o x i d e and  oxygen.  When the experiment was repeated  d i f f e r e n t batches  of i n f u s i o n b r o t h , no haemolysin  could be shown i n the f i l t r a t e s and  rabbit c e l l s ,  twice again w i t h production  when t e s t e d on human  9  sheep  At no time d i d the a d d i t i o n of 5 mg. of  sodium h y d r o s u l p h i t e i n c r e a s e the amount of haemolysis. Haemolysin p r o d u c t i o n was a l s o tested f o r on blood agar p l a t e s .  Two types of agar media were used •  i n f u s i o n agar and v e a l i n f u s i o n yeast agar, 1.5$ agar been added t o the b r o t h media.  Veal having  1 2 c.c. of agar were used f o r  each p l a t e and to t h i s was added 0.6 c.c. of c i t r a t e d  blood,  Human, sheep and r a b b i t c i t r a t e d blood was used. Prom a twenty-four hour broth c u l t u r e of t h e s i x s t r a i n s of Streptococcus v i r i d a n s , blood agar spread p l a t e s were made.  These p l a t e s were incubated a t 37° c . f o r t h i r t y -  s i x hours i n atmospheres o f :  -20-  (a) (b) (c) (d) (e) (f) (g)  and  after  examined  thirty-six  different  Table  carbon carbon carbon carbon carbon oxygen  hours  immediately,  p haemolys!n a  air 100$ 80$ 60$ 40$ 20$ 100$  with  production.  batch  5.  of  dioxide dioxide dioxide dioxide dioxide  these  plates  the aid  ^ W ^ — ^  broth.  ^  ^  ^  <w%' to it,  Strain* 1  J  X  ,  JL.  ,  tfjf  *4  4.  J.  J.  -Cd  J  /.  JL  JB  A.  J.  JL  X  jft  J.  J.  JL  4(3  -~  -i  -l  J. (3  p  JL  u  JL  JL  . »s  • so* ;  i~  JL  a  n  Streptococcus and  at  produce red  cells  The r e s u l t s  are  ^ ^ ^ ^ ^  5. /oaPo  •  A.  J.  d(3 •  A.  *L  el  A  J.6  A  JL.  A  JL.G  JL  A  A  JL. el  A&  JL  -J. ,. .r*> -w (3  a  of  on blood  of  causes  ei.eJ.ei c<  JL.  oL  Jl J.  J_fl U(i  J.  Uft  J.  j(i  <(3  A(2 oi/S eld  J.  *L *L o l  growing  which  and n o t  d&  JL.  -4 JL  -<  d@  A.  JL  ft  -k  O*.  sa*  *C  JL  in  -  J.(i  -L  using  shown  A  >L(3  and  was r e p e a t e d  Jl  *  atmospheres  a substance  blood  A  and  for d  >±  viridans  different  *t>Z to*  oxygen oxygen oxygen oxygen  microscope,  —  Table  . ~£  of  20$ 40$ 6 0$ 80$  were' removed  The e x p e r i m e n t  infusion  ^.  and and and and  J^.  A.  agar  oxygen and  plates  carbon  the haemolysis  t h e human o r  rabbits  of  in  air  dioxide the  sheep  erythrocytes,  -21The infusion agar  blood  plates.  different  agar The  batch  Production  of  of  enterotoxio-, strains  prepared  same  Infusion  the  the  into  in six  a  20  30  six  hours  also  the  infusion  blood  v/ith  a  Products.  and  toxic-like  Streptococcus  of  on  broth.  necrotoxic-,  following  same  obtained  the  strains  c.c,  were  the  yeast  for  the  c.c.  on the  results  A loopful of  as  v/ere  test  of  in  obtained  plates  Toxic-like In  six  results  possible  viridans  production  substance  the f i l t r a t e s  by  of  an  the  used  were  manner: of  a  twenty-four  Streptococcus  hour  viridans  culture  was  of  each  inoculated  of: (1)  infusion  (2)  yeast  infusion  broth  (3)  serum i n f u s i o n  broth  culture  tube  broth  and  incubated  (20$) at  37°  c.  for  inj (a) (b)  air 20$  oarbon  dioxide  and  80$  oxygen  thiity-  -22A f t e r 36 hours the c u l t u r e s were removed from the i n c u b a t o r , f i l t e r e d through a No. 1 f i l t e r paper and then •through a S e i t z E.K. i n t o s t e r i l e tubes.  The c u l t u r e " t u b e s  c o n t a i n i n g these s t e r i l e f i l t r a t e s were plugged w i t h  sterile  rubber stoppers and placed i n the i c e box where they were kept u n t i l used. and  0.5 c.c. of f i l t r a t e was added t o s t e r i l e  incubated  broth  f o r twenty-four hours t o t e s t f o r 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  filtrates  obtained  by the method described above, of each o f  the s i x s t r a i n s of t h e Streptococcus  v i r i d a n s grown i n :  (1) i n f u s i o n b r o t h (2) yeast i n f u s i o n b r o t h (3) serum i n f u s i o n b r o t h (20%) and  incubated i n (a) a i r (b) 20$ carbon d i o x i d e and 80$ oxygen  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 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 a l s o made.  T h i s experiment was repeated  on a white Angora r a b 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 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 the back of a white r a b b i t .  C o n t r o l s 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 a l s o made. Angora r a b b i t .  This experiment was repeated  on a white  -asIii'these the  backs  of  the  rabbits  0.2 filtrates  was  of  yeast  inoculated  trols  of  a  experiments  c.c.  of  broth  (a)  air  (b)  20$  b y means a 1:1  of  growing  the  of  a L:l  removed  of  the  from  clipper* same  sterile  in:  and  into  of  was  an e l e c t r i c  carbon dioxide  dilution  hair  dilution  intracutaneously  1:1  the  the  yeast  80$  oxygen  back  of  a  cat.  i n f u s i o n medium  Con-  were  made. Ql filtrates  were  of  c.c.  infusion  inoculated  broth  (a)  air  (b)  20$  dilution  grown  the  sterile  in:  carbon dioxide  intracutaneously  of  into  and the  80$  oxygen  arm of  a  human  volunteer. After 1:50  dilution  grown  was  the  sterile  of  o n e w e e k 0.1  filtrates  of  c.c.  infusion  of  broth  in:  inoculated  human  of  an. i n t e r v a l  (a)  air  (b)  20$  carbon dioxide  intracutaneously  volunteer.  into  and  the  80$  arm  of  oxygen the  same  a  All negative 'a  very  results.  light  pink  but  All  reactions  little  The  they  larger  reactions  than  those  ly  no  r e a c t i o n s of  had  in  on  In  all  area  than  reactions  on the  back  well  of  as  gave  dilutions  were  by  -  little  the  on  red.  were  control  a  the  a  filtrates  of  serum  than  almost  the white  done  the  larger  the  were  of  reactions  as  no  colour  those  rabbits  caused  different  rabbit  controls  the  1:1  were  Angora  skin  the  pink  the  on  those  the  The  only  reactions. more  Intense  rabbit.  the  cat  filtrates  were  gave  ne-  absolute-  reaction.  filtrates  and  filtrates  tion  except  a deeper  On t h e  The  dilutions  reactions  from  The gative .  The  These  others,  a  1:10  colour  broth f i l t r a t e s .  the  the  of  this  media  human v o l u n t e e r  controls and  the  controls  that  dilution  of  gave  were  no  neither  1:1  reactions  dilution  of  the  reaction  the  the  control  were  same  was nor  a  very  size.  1:50 the  of  the  violent.  The  dilu-  dilution.  filtrate  gave  any  reaction.  4. of  Streptococcus  c.c.  of  viridans  sterile grown  (1)  infusion  (2)  yeast  (3)  serum b r o t h  filtrate  in:  broth  Infusion  broth  (20$)  of  the  six  strains  each  int  were at  (a)  air  (b)  20$  inoculated  two d a y  interperitoneally  six  each  was  into  six  80$  oxygen  kittens  six  times  filtrates  showed  no  observable  effects  kittens.  1 organisms  and  intervals. The  on t h e  carbon dioxide  grown  c.c.  of  sterile  filtrate  of  the  six  different  in: (1)  serum b r o t h  (2)  yeast  (3)  infusion  (a)  air  (b)  20$  (20$)  infusion  broth  broth  in:  inoculated  times  at  two  vable  effects  carbon dioxide  intraperitoneally  day  intervals.  on the  mice.  The  into  and  80$  six  white  filtrates  oxygen mice,  showed  no  six obser-  -2BSIGNIFICANCE  The -grown i n  six  different  Carbon d i o x i d e  production  of  remained  tion,  media usually  culture ted  tubes  before  an  no  increased  not  of  tension  100$  results  In  oxide  products  duction .given,  of the  could  flask.  reached  hours.  the  time  of  of  their  The  Ph  observa-  production  strength  to  peroxide  could  be  in  six  be  in  demonstra-  in  peroxide have  a  small was  de-  organisms  Except  different  this  viridan small  s t r a i ns amount  twelve  that  a (3 £ [ a e m o l y s i n - l l k e  no  be  in  that  could  been  in be  more  atmospheres  quantity not  of  practical  with  the-laboratory.  within time  the  seen.  probably  However,  uoed  occurred one  air,  growing  by u s i n g  a relatively  The  of  would  for  When t h e flasks  of  production  tested  apparatus  atmospheres  peroxide  carbon dioxide  peroxide  large  The  viridans  hours.  effect  The  large  during  observable  been  limited  same  presence  of  in  twenty-four  When t h e  could  the  as  sufficient  if  a  well  different  was  conclusive  medium i n  under  hours.  demonstrated.  have  Streptococcus  ninety-six  oxygen  an atmosphere  of  before  the  seventy-two  monstrated  and  o x y g e n as  maximum a c i d the  RESULTS  strains  media  and  OP  of  grown  broth,  in  the  per-  hours.  an i n d i c a t i o n  substance  maximum h a e m o l y s i n was  were  in  produced  a  of  the  culture  protube  lay s e v e n t e e n  was  hours.  -23-  This  remained  time  no  of  constant  oxidized  oxidized of  tional  reduced  readily  haemotoxin  haemotoxin  amount  filtrate  by  treating  appeared  slowly^ as colony  and  the  then a  produced  the  type  ture  ?  by  than a  suggested  on b l o o d  clear  when p e r o x i d e  peroxide  production  H ion  in  an  atmosphere  of  20$  in  an atmosphere  of  100$  greatest  greatest  and  addito  the  not  be  called it  diffused  organism  discolour  or  blood  tempera-  an antagonistic  to  and  results  action protect only  ceases." due  of  carbon dioxide is  on  producing  refrigerator  carbon dioxide  H ion  surrounding  the  not  production  of  or  Haemolysis  diminishes  concentration  so  appearance  have  acid•  Is  this  the  at  tending  haemolysin  of  the  the  the  of  haemolysis.  growing  acid  the  could  methaeraoglobln  explai ning  and  as  b e e n added  peroxide,  of  "peroxide  This as  zone  c.  by  have  amount  lack  used.  of  37°  them f r o m h a e m o l y s i s  small  which  The  significant  plates,  at  corpusles,  not the  the  ring  at  demonstrated.  might  agar  alternately  haemolysin  and  methods  Hagan^nen agar  however,  blood  was  be  hour  sodium h y d r o s u l p h i t e ,  later  there  thirty-sixth  could  which  with  with  the  small  haemotoxin  observed  haemolysin  the  is,  h a e m o l y s i n was  On t h e  more  until  to  the  haemolysin and in  80$  increase takes  place  o x y g e n and  which  produced.  not  atmosphere  -28The  peroxide does not seem t o be a f f e c t e d ^  by a g r e a t e r c o n c e n t r a t i o n o f H i o n s i n c e i n the fermented .dextrose i n f u s i o n b r o t h the p r o d u c t i o n of peroxide was unaffected. That an i n d i c a t i o n a t l e a s t o f the p r o d u c t i o n of a  h a e m o l y s i n - l i k e substance  was obtained  i n one batch of  i n f u s i o n b r o t h and not i n two others i s i n keeping with the f i n d i n g s o f Todd^^wtien working on S t r e p t o l y s i n .  He w r i t e s  "there i s a great v a r i a t i o n i n the y i e l d  of S t r e p t o l y s i n  from d i f f e r e n t batches of b r o t h prepared  i n the same way and  as the e s s e n t i a l f a c t o r r e s p o n s i b l e f o r t h e i r v a r i a t i o n i s unknown, b r o t h which i s s a t i s f a c t o r y f o r S t r e p t o l y s i n prod u c t i o n has been secured  by t e s t i n g every batch of broth made  from the r o u t i n e work of the l a b o r a t o r y and r e s e r v i n g s u i t a b l e batches f o r S t r e p t o l y s i n p r o d u c t i o n .  B  A f t e r t r y i n g two batches of media other  than  that i n which the (I haemolysin was produced, i t was decided not t o t r y f u r t h e r t o produce the haemolysin, as i n t h i s l a b o r a t o r y , where only s m a l l q u a n t i t i e s of i n f u s i o n b r o t h are made up a t one time, i t was not p r a c t i o a l . I n t e s t i n g f o r the p r o d u c t i o n of a n enterot o x i c - l i k e substance and  Gockorofty^o  t o x i n , were used.  the t e s t s devised by Dolman, Wilson,  show the presence of Staphlococcus  entero-  -29-  Th© r e s u l t s were negative i n every case. . This can be I n t e r p r e t e d t o mean one o f three t h i n g s :  the entero-  t o x i n produced by the S t r e p t o c o c c i i s not shown by t h i s  test,  t h a t the exact c o n d i t i o n s f o r the p r o d u c t i o n of A n t e r o t o x i n by Streptococcus  v i r i d a n s were not used, t h a t the s i x s t r a i n s  of Streptocou3 v i r i d a n s used do n o t produce an i n t e r o t o x i n l i k e substance.  Prom the r e s u l t s of these experiments no  d e f i n i t e conclusions  can be reached.  In t e s t i n g f o r the p r o d u c t i o n of a n e c r o t o x i c l i k e substance the r e a c t i o n s were so s m a l l as to make the results inconclusive.  This means that the c o n d i t i o n s under  which the s i x s t r a i n s were grown were not s u i t a b l e f o r the maximum p r o d u c t i o n of n e c r o t o x i n . No t o x i c products  could be demonstrated i n the  Of  f i l t r a t e s o f b r o t h , asd serum b r o t h c u l t u r e s using s u i t a b l e tests.  T h i s can be taken t o i n d i c a t e that e i t h e r no t o x i c  substance i s produced by any of the s i x s t r a i n s under  investi-  g a t i o n , or t h a t the c o n d i t i o n s f o r the p r o d u c t i o n of such t o x i c substances were not used• Summary of R e s u l t . Under defined c o n d i t i o n s , u s i n g d i f f e r e n t media and  atmospheres of v a r y i n g mixtures of carbon d i o x i d e and  oxygen the f i l t r a t e s of the s i x s t r a i n s of Streptococcus v i r i d a n s caused the haemolysis of the red c e l l s of sheep.  -SO-  Under lite® c o n d i t i o n s the red c e l l s o f sheep i n agar p l a t e s were haemolysed by f i v e o f the s i x s t r a i n s o f S t r e p t o . coccus v i r i d a n s , Under these same c o n d i t i o n s and using a v a r i e t y o f l a b o r a t o r y animals, the p r o d u c t i o n of t o x i c - e n t e r o t o x i o - or n e c r o t o x i c - l i k e substances were not able t o be demonstrated i n the f i l t r a t e s of b r o t h c u l t u r e s of these same s i x s t r a i n s *  REFERENCE (1) (2)  H. D. Wright, 1925, A System of B a c t e r i o l o g y , V o l . 2, P. 105. Stevens, 1919, J . Exp. Med. 30, 553. P. K r u i f , 1920, J . Enf . D i s . 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. " 1930, B r i t . J . Exp. Path. 11, 391. 1930, B r i t . J . Exp. Path. 11, 479. " " 1932, j * Exp. Med, 55, 278. " " 1932, B r i t . J . Exp. Path. 13, 258. Hodge, 1933, J . Exp. Med, 58, 284. R. L a n c e f i e l d , 1934, J . Exp. Med. 59, 468, 11  H  M  w  11 w  (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, " " • J . E x p Med, 39, 366. " " " J , Exp. Med. 39, 744. - " * . J . Exp. Med. 39, 755. " " J . Exp. Med, 40, 422, 427. N e i l & Avery, 1925, J , Exp. Med. 41, 285. J . N e i l , 1926, J . Exp. Med. 44, 273, N e i l & Hemming, 1927, J . Exp. Med. 46, 735. S. E . Cawan, 1934, J . B a c t . & Path. 38, 61. n  w  s  11  M  w  n  K  (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, R o c k e r f e l l e r I n s t , f o r Med, Research.  (9)  N e i l & M a l l a r y , 1926, J . Exp. Med, 44, 213. S. E. Cawan, 1934, J . Bact, & Path. 38, 61.  (10) Hagan, 1925, J . I n f . D i s . 37, 1. (11) E . W, Todd, 1932, J . Exp. Med. 55, 278. (12) Dolman, W i l s o n , C o c k c r o f t , 1936, (Paper i n process of p u b l i c a t i o n ) .  

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