UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies on the viability of bacillus salmonicida in sewage water Thompson, Helen Gertrude 1936

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STUDIES  OH THE V I A B I L I T Y OF B A C I L L U S  SALMONIC  IH SEWAGE WATER  by  HELEN  A  Thesis  G. THOMPSON  submitted MASTER In  f o r t h e Degree of OF ARTS  the department of Bacteriology  The  University  of B r i t i s h  April,  1936  Columbia  ACKNOWLEDGEMENT.  I wish  to express  for  my  his interest  appreciation  t o D r . D. C . B . D u f f  and assistance  i n this  work.  HISTORICAL.  ;/  I n 1894,  a specific  bacillus  • s u f f e r i n g -from, s o - c a l l e d and  named  by  Michigan, which be  he  called  because In  1909, P l e h n  furunculosis, previous  reported The  first  was of  this  1909  Duff  among  i n Scotland.  throughout  t h e game  organism  diseased  organism  was  trout,  foun<4 t o  and W e i b e l ,  was  which  given she  Germany.  repeated  and  preference.  identified She  had been found  of the disease  reported  salmonicida.  Weibel,  stated  as that  only i n  epidemics  were  waters.  an outbreak  1932  B.  the disease  occurrence  have o c c u r r e d  epidemic  i n Southern  but from  from  i n Great  i n 1911 by M a s t e r m a n and A r k w r i g h t .  described  In  waters,  in natural  reported  emics  date  a disease  fish  I n 1.902, M a r s h i n  of Emmerich  nomenclature  reported  occurring  to t h i s  domesticated  their  This  from  by E m m e r i c h a n d  isolated  truttae.  salmonicida  of p r i o r i t y ,  isolated  salmonicida.  an organism  Bacillus  i d e n t i c a l wrthB.  first  furunculosis,  them, B a c i l l u s  described  was  He  that fish  Since  the c a u s a t i v e at E l k River,  has s u b s e q u e n t l y  i n other  I n 1914  then  the B r i t i s h  Britain  was  Mettam  numerous  epid-  Isles, organism British  reported  of an  Columbia, the  l o c a t i o n s i n the p r o v i n c e .  presence  INTRODUCTION.  The  means by  which  this  to  a n o t h e r has  never  if  an  parasite,  obligate  transmitted  considerable The  in  first  1909.  bottled ation ough  water In  the  and  show  to f i s h .  organic  n a t u r e were o f B.  Plehn's that  B.  w o r k was  agar  t w e n t y - f o u r hour She  hours  failed  from  the  sterile  laboratory  by  intervals  of  host.  d o n e by  by  she  inocul-  passage  (table can  Plehn,  in  thr-  1)  survive  conditions, pure  Great  and in  water.  lightly plated  in  polluted  a loopful  salmonicida  polluted  was  Britain,  salmonicida  and  to r e c o v e r B. lightly  the  for  W i l l i a m s o n , who  o f B. tap  be  survive  in relatively  Committee  distilled,  to  salmonicida  salmonicida  slant  organism,  waters. . Before  reported  the F u r u n c u l o s i s  of  experiments  content, than  a young  i t could  of  polluted  The  host  I f , however,  the body  this  one  require  leaving  time, under  c . c . of u n s t e r i l i z e d  forty-eight  fish  viability  filter.  further  emulsions.  of necessity  from  after  for a longer  At  demonstrated.  w e r e made p r e s u m a b l y  evidence to  under  spreads from  saprophytic ally,  of pure  "emulsified"  water. the  tested  samples  of high  working  15  She  1928,  would  investigations  a Berkfeld:  multiply  She  period  the waters  present  clearly  directly  organism cof/a e x i s t  the a  almost  been  infection  water.  of  after (table  2)  TABLE PLATE  1.  COUNTS E X P R E S S E D AS COLONIES P E R C . C .  TIME  P O L L U T E D WATER  Inoculum  5,800  24 h o u r s  '9,182  48 h o u r s  PURE  WATER  ' 6,336  273-  • 440,000  72 hours  0  8,803,000  0  T A B L E 2.  DAYS  DISTILLED  TAP  POLLUTED  WATER  WATER  WATER.  4-  + . -f •  9© - 9  *  scanty •  4* «  e  *  <» •  scanty • • «  very  scanty  •  0  4-  0  0  •  From  these  results  does not  exist  suggests  that  importation epizootic fish It  and  can  its  as the  of  B.  saprophyte  organism  fish  distributed  survive  Obviously, are  open  concluded  i s introduced  salmoncida  -  throughout  the  but  i n some way,  disease.  i s probably  for a period  that B.  i n p o l l u t e d waters,  c a r r y i n g the  salmonicida  wide-spread  ions  a  Williamson  In  the  liberated water,  sufficiently  case  from  where  long  as  by  of  an  diseased  apparently  to  allow  for  distribution.  the to  experimental  basis  of  Williamson's  conclus-  critism.  First. The  " e m u l s i f i c a t i o n " of  of  fluid  of  a l l relation  from ial  dead and  over  billions  to  the  sick  with  products,  ortion of  give  of  number  fish.  of  i t a  such substances  fluence,  at  bacterial  least  which  a  with  a  i n the to  test  a  fluid  some e x t e n t ,  enter large  amount; o f  the  c.c.  volume  definite,  d i s s o l v e d from  slant  per  could  small  considerable  together  nutrients  organisms  Furthermore,  g r o w t h when t r a n s f e r r e d to  carry ory  will  a whole" a g a r  and  mass of  be  small, The  c.c. out waters  bacter-  fluid,  bacterial  agar.  be  natural  of  though  will  i n 15  will  excretprop-  presence  likely  to  in-  t h e - s u r v i v a l time  of  the  population.  Second. 15 ume,  i n view  behaviour ly,  c.c.  to  of  of the  of the  fluid fact  organism  i t s probable  seems t o that  an  extremely  conclusions  in this  behaviour  be  volume  derived are  i n extremely  small from  applied,  large  volthe  direct-  volumes  under  natural  conditions.  Third. '  The  "was  quite  sample  taken  daily,  from  the  fluid  under  test,  many  plates  inadequate.  Fourth. W i l l i a m s o n makes no were as  made a t  to  the  suggest icida  each  use  the  of  without  sampling, any  the  among  use  of  as  to  how  w h e t h e r d i l u t i o n s were  differential  practical  colonies  statement  medium.  i m p o s s i b i l i t y of thousands  specific  of  Our  own  of  or  experiences  recognizing  colonies  used,  B.  sewage  salmonorigin  methods.  Fifth. Winslow Population"  points  unfavorable, phase  of  study  there  adjustment  a period This  of was  of  the  growth  Fuller  i n 1894, 3).  He  Although  a  should  bottled  live  out  that  on  by  the  "Rise  unless  i s always,  strikingly  stored  and  article  the  and  Fall  medium  in bottled  of  is  Bacterial  extremely  experiments,  b a c t e r i a l population,  a  followed  by  increase.  waters  table  i n an  illustrated  curve  in flasks at  the  at  have  work had come t o  experiment,  bacteria  30°C,  (see  Mi quel in a  been  her  she  i n p o l l u t e d waters,  bottled  graph)  done  notice,  states but  samples  dies  of  before yet,  that out  B.  i n 1891,  series  Lawrence Experimental  worked w i t h  this  of  by  and  also  Station  the  of  spring by (see  Williamson  basis  salmonicida  i n two  a  sewage.  that  on  of  in  or  three  of does  not  days.  5. 3.  TABLE  Bacteria  Originally  Time  per.p;e>  contained  1,190,000  2t hours Rose  108,000  s t e a d i l y t o maximum  35J- h o u r s  in  Fail  S~  In ion  steadily.  an e f f o r t  of Plehn  iments w i t h monicida agar  On  500  and Williamson,  could  Flask  sewage  of v a r y i n g  These  Finding  sewage  sal-  t h e raw only.  containing  d i l u t i o n s of e f f l u e n t . sewage .1  diluted.........  /  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  J  -  •••••••• ••••• •••••••3.  (f)  < •»t« * « « < « « « « « •  (^)  ««®»*<»'««<#4»»««e«9«»«eX  were  B.  nutrient  material  s e t up, e a c h  exper-  that  she abandoned  the s t e r i l i z e d  conclus-  began  (@)  salmonicida 400  sewage.  f l a s k s were  (a) u n d i l u t e d  \  i n 1931,  Stewart,  organisms,  with  of 2 l i t r e  lb)  on the opposing  n o t be d i s t i n g u i s h e d on o r d i n a r y  from  series  ....2,341,000  some l i g h t  raw a n d s t e r i l i z e d  plates  c.c.  8th day  to throw  sewage a n d c o n t i n u e d A  23,100,000  sterilized  w h i c h gave  b a c t e r i a p e r c.c.  and seeded with  a population Then 1  c.c.  i« *«»i>  * 10 J-UU  e  • -L 0 | 000 •  100  •  IJOOOJOOO  an inoculum  of approximately samples  y  0 00  of B. 300 t o  were p l a t e d  daily  6. on  nutrient In  the  with  B.  agar  undiluted  was  days.  a  In the  decrease  s u d d e n mceejgein t h e utions to  made w e r e  count,  decrease diluted the  and was  here  the  high  enough. day.  then a  multiplying,  bacteria the  were  (g)  and  a  the  would  (d)  on  day  the  ninth  As  were  the  too  subsequent  as  was  on  numbers  A  fall  slight  more  come on  until  the  the  sixteenth  day.  dilutions  made w e r e  not  noticed  seem  crowded  In the  t h e /nt*e<g*?did n o t  to  on  the  indicate  started  twenty-  that  took  to  when  longer  to  decrease  sooner. Since ized  Macarthur,  looked from  work  was  i n 1932,  sewage  organisms..  a g a r , Mc  Corkey's  Krumwiede's  devised  a maltose  would  After bile  brilliant blood  with  sewage  continued the  f o r a medium w h i c h  Endo's agar,  Stewart's  agar  that  had  research.  differentiate  trying salt  green  B.  many m e d i a  been  steril-  First  she  salmonicida such  as:-  agar,  eosin  methylene  medium,  and  blood agar,  medium.  a  dil-  experiment.  present the b a c t e r i a  their  i s , for  ( b ) , (c) and  c.c.  plates  increase  crowded,  decrease  was  per  crowded  that  t w e n t y - n i n t h day.  secondary  organic material  start  dilutions,  t h e r e was  results  experiment,  throughout  the  slight  These  the  remained  beginning,•. and  of  so  on  plates A  the  ( e ) , ( f ) and  and  plates  h i g h enough  noticed  day,  From  less  the  lower  number  remained  effluents  fourteenth  at  not  e l e v e n t h day,  third  effluent  salmonicida throughout  •thirty-five there  plates.  blue she  *  Baeto  Nutrient.Agar 1*5 jo J3H 7 »3 « » » * * • * •  « e « i o « # XOOc «c t  •  Endo's  .  Anhydrous  sodium  sulphite  25 g r a m s  9  Indicator  1$9.11oss  basic  ized The  added  agar.  separately open  side  blood  sulphite  fuchsin  To p r e v e n t  .,  5 c.c.  ..  i n 5 c . c . of water, the  above  to the melted  h a e m o l y s i s t h e s u g a r was  i n a thin walled  bulb  cotton,  blown  and c o o l e d  and t h e b u l b  t o 45°C  steril-  from glass  t h e s o l u t i o n , by the plug  A dehydrated nutrient  Ferments  1 ^rsiro  and t h e m i x t u r e t h e n added  When t h e a g a r was m e l t e d  *  of basic  • e c 4 o « « * « « « * » o « o  was d i s s o l v e d  e n d was p l u g g e d w i t h  the f l a s k ,  solution  • * ••«««*«••«  rabbit's  sodium  nutrient  10^ a l c o h o l i c  •«•••••»•  Gitrated The  .  was h e l d i n  of the f l a s k .  the bulb  medium p r o d u c e d b y  tubing.  was  Digestive  Co.  Composition. Baeto  beef  Sodium J33.G"to  extract  .,  3 grams  chloride &gs.r  8 grams  • • • « « • • • • • • • « » • • • • • • • • * • • « # » « • • * 1 *5 grs.ms 31  3.1 g r a m s water  of this  and the agar  nutrient  dissolved.  a g a r was a d d e d  grams  to 100 c . c .  of  broken rated not  and the sugar rabbit's blood  entirely  organisms maltose  dissolved.  At  was  T h i s medium,  satisfactory  which  blood,  show some  this  thee i t -  however, i s  a r e a few p o l l u t e d  resemblance  water  to B. s a l m o n i c i d a  medium, M a c a r t h u r  the  survival  ium  c o u l d n o t be depended  initely  as t h e r e  time  on  agar.  However, u s i n g  completely,  added..  t h e same  o f B. s a l m o n i c i d a  no  attempted  i n raw s e w a g e .  on t o d i f f e r e n t i a t e  q u a n t i t a t i v e count  shown t h a t m u l t i p l i c a t i o n  was  made,  of t h i s  to follow  B u t a s t h e med B.  salmonicida  b u t i t was  organism  took  defplace  EXPER I M E H T A L .  The .of  f o l l o w i n g experimentay/tsre  Stewart  ine  quant i t  inoculum  and a c a r t h u r .  The p r i m a r y  M  of . B  salmonicida i n bottled  attempt  differentiating  was made  medium.  starch blood  use  this  agar  agar,  by  water,  the retention  a viable  ether  was  of a reduced  the  salt  agar  were  as t h e mal-  some o f t h e s e w a g e o r -  number  nature  life,  o f sewage  as  o f the evidenced  organisms i n  Vapor.,.  effluent  with  were p l a c e d  a rubber  cork.  i n an o p e n v i a l ,  frequently, being  was s u b s t i t u t e d  careful  c o u l d no l o n g e r b e d e t e c t e d ,  flask.  I t was f o u n d  that  i n a 2 this  not to s p i l l  cork.  cork the  The f l a s k the ether.  a sterile  cotton  When t h e o d o r o f  samples were  t h e sewage  litre  the surface of the  to the v i a l .  evaporated,  f o r the rubber  From  above  T e n c . c . o f e t h e r were added  When t h e e t h e r h a d c o m p l e t e l y  ether  agar,  i t was d e c i d e d t o  to support  o f sewage w i t h E t h e r  was s u s p e n d e d  shaken  plug  bile  Therefore,  and i t s a b i l i t y  closed tightly  liquid.  satisfactory  starch blood  preserving the chemical  c . c . o f t h e sewage  flask,  a more  water.  condition.  Treatment 300  t o determ-  t o be a s s a t i s f a c t o r y  of Macarthur.  still  sewage  and a e s c u l i n  medium, b u t t o t r y a n d k i l l  ganisms, while polluted  to f i n d  S t a r c h agar,  t r i e d , , b u t none were f o u n d tose blood  objectwus  ati'velj t h e f l u c t u a t i o n s a n d t i m e o f s u r v i v a l o f a n  A further  maltose  a c o n t i n u a t i o n o f t h e work  plated  from  organisms which  were  10. left  c o u l d be  blood  agar  Before to  be  differentiated  from  B.  salmonicida  on  maltose  plates. inoculating  used  was  the  flask  with  B.  salmonicida  t e s t e d f o r morphology, pigment  the  production  strain and V'  sugar  fermentation.  Morphology. Small Nutrient  gram-rods Agar  whitish, effuse, glistening,  T h e r e was  Carbohydrate The the  coccoid)  Slant.  G r o w t h was lucent.  (almost  a brownish  pigment  convex and  i n the  trans-  medium.  Reactions.  o r g a n i s m was  changes  sown o n  the  f o l l o w i n g carbohydrates  and  recorded.  Maltose  4.  Pi X* Sib 1 HO S e L e i OijOSe  e«e » a « « e o o e $ « « « 9 « s » e @ —|—  » « 9 e « o e 9 « s « * « f t « * » 9 « « « « «  SlJ.GrOS e • *«««««««« «•««*« $*eaeo<» B. ular  salmonicida strain  The  SOOO  did  flask  monicida  u s u a l l y ferments  then  which would  b a c t e r i a per  c.c,  0.5  xylose,  but  this  partic-  not.  was  Determination 1  v  of  i n o c u l a t e d with give  an  inoculum  a p o p u l a t i o n of  o f B.  sal-  approximately  c.c. Amount  c.c.  and  of 0.3  Inoculum. c.c.  amounts  of  a  saline  suspen-  /  11. sion  of a f o r t y - e i g h t  turbidity were  of S o . 4  were  shaken  Then 1 c . c .  each.  P l a t e s of 200  inoculum  of  was  always  utions  the  flask  used.  thoroughly  t h e r e f o r e , the  face  the was  agar, and seen  technique  until  to B.  f o u r days  Table c.c  4  on  shaken before  found 1  no  way  of  to  stand f o r  1  c.c.  to  tap  water.  one  gave  approx-  o b t a i n an  initial  c . c - of hours  1  blood  a Ho. c.c.  agar.  samples  4  sus-  samples The  were  flask  taken.  water  the  cover  plate the  was  whole  Dil-  blanks.  there  high  plating.  was  enough.  to  count.  i n t h e number o f knowing  in  gently rolled  P l a t e s were  But  this  no  way  On  take  plates the  sudden d e c r e a s e  even be  the  using  counted at  beginning. that  place  the  made f r o m  t e n t h day  c o l o n i e s per  until  sur-  22°C.  i n organisms  of knowing  going to The  the  the  incubated  variation at  poured  p l a c e d on  s a l m o n i c i d a c o l o n i e s c o u l d not  after  crowded  slowly  surface.  a suddenificmaseright  as  very  s a m p l e s were  was  made w e r e n o t  fall  sterile  in sterile  t o grow  c.c.  e n d o u s m u l t i p l i c a t i o n was  den  maltose  the  i n d u p l i c a t e , from  10  twenty-four  quantitatively the  There  too  of  Therefore, c.c,  to  nephelometer,  inoculated with  shows  fortunately,  were  allowed  diluted  Technique.  plates,  this  Parland's  and  w e r e made w h e n n e c e s s a r y  of  culture  quantities  c.c.  Every  s a l m o n i c i d a was  liquid  c.c.  in triplicate  Plating B.  Mc  2000 c o l o n i e s p e r  plated  slant  amounts were p l a t e d  c o l o n i e s per  p e n s i o n were were  300  thoroughly  hour.  imately  agar  suspension,  inoculated into  These  hour  this  per  IJntrem-  dilutions a 1 dilution 1000  t h e r e was  a  sud-  c.c  Again,  there  would  occur,  and  was  this  12. time day  the d i l u t i o n s a gradual  when t h e r e • were  was  increase  from  sewage  eighth  o f B.  up  the twenty-fourth  B.  salmonicida  day  colonies  to and i n c l u d i n g  salmonicida  were  more  t h e p l a t e s were  the  sixty-  identity  onto  covered  n u t r i e n t agar  by pigment  o f such  regarding  checks  sewage  presumably  one  B.  t o make  and checked  as t o  When  t h e above  of B.  salmonicida  TABLE  4.  sixty-  salmonicida  and morphology.  i s able  low  bacteria.  a l s o ' made o n c a r b o h y d r a t e s .  the behaviour  complete  with  slants  production  l o w a c h e c k was  c o l o n i e s were v e r y  numerous, and on t h e  day, c o l o n i e s w h i c h were  were p i c k e d  use  decline.  t h e sewage  organisms  day  Each  became  until  On t h e f i f t e e n t h  day.  When t h e n u m b e r the  started,  a gradual  recovered  seventh  made w e r e n o t l o w e n o u g h .  their  counts By t h e  statements, "' 1  i n sewage, w i t h  confidence.  PLATE WITH ETHER  COUNTS OF B .  SALMONICIDA  "VAPOR E X P R E S S E D  DAYS 1  I N SEWAGE  AS COLONIES  COLONIES  per  C.C.  94,000 85,000 250,000  PER  TREATED  C.C.  OF  AVERAGE  89,000  • *««*«*  Dilutions high  not  400,000 +  enough. ri  4 0 0 , 0 0 0 -f-  SAMPLE.  </'  13c TABLE  4.  continued DAYS  COLONIES  4  p e r C.C.  AVERAGE.  Dilutions not  400,000^ high  enough  5  n  7  314,000 297,000 17,000  306,000  4,.280,000 1,390,000 1,990,000  1,690,000  8  10  400,000+-  Dilutions low  not  enough  11  12 . 14  15  17  18  170 240 210  210  120' 177 164  154  245 302 236  261  1,020 1,120 1,250  1,130  1,810 1,190 1,160  1,510  /  TABLE  4.  continued DAYS 19  21  COLONIES  per  1,490 1,630 1,740 3,200 4,300 5,100  C.C.  AVERAGE.  1,620  4,200  22 5,100 5,000 24  25  26  28  29  SO  31  33  1,810,000 1,430,000 1,570,000  1,600,000  550,000 530,000 560,000  550,000  480,000 500,000 470,000  480,000  420,000 400,000 450,000  420,000  200,000 190,000 220,000  200,000  140,000 160,000 150,000  150,000  60,300 61,-200 60,600  60,700.-  80,000 90,000 70,000  80,000  TABLE  4.  continued DAYS 35  .  36  37  38  39  40  42  43  COLONIES  p e r C.C.  AVERAGE.  91,000 94,000 93,000  92,000  98,000 93,000 94,000  95,000  111,000 88,000 79,000  93,000  65,000 79,000 75,000  73,000  60,000 55,000 61,000  59,000  52,000 36,000 43,000  44,000  31,000 30,000 35,000  32,000  28,000 29,000 32 000  30,000  10,000 11,000 9,000  10,000  5,300 5,700 6 ,100  5,700  1,800 1,500 1,900  1,720  8  45  47  49  TABLE  4.  continued -DAYS 50  51  52  53  54  57  58  59  60  63  64  COLONIES  p e r C.C.  AVERAGE.  451 473 487  470  39 45 52  45  2 4 5  4  48 62 72  61  12 17 15  15  56 55 44  52  66 71 66  68  59 65 52  •  59  20 25 23  23  11 11 13  12  8 22 10  12  TABLE  4.  continued DAYS  COLONIES p e r C X  •• AYE RAGE  9  65  7  7  5  66  13 4  8  6 7  6  6  67  0  68 0  0  o •  0  0  0  69  70  71 0  ;  0  72 0  o  0  0  0  0  0  0  73—87  93  99  18.  The  agar  slants picked  salmonicida, the  end  tinct  the  p l a t e s made f r o m  appeared to  types  1.  on  from  be  c o l o n i e s , presumably  the  contaminated.  f o r t y - n i n t h day T h e r e were  B.  until  two  dis-  of. g r o w t h :  A glistening,convex,  e f f u s e , and  translucent  growth.  Morphology. Small * Plated  gram-rods, on  haemolyzed  onies  skated  coccoid.  maltose blood  T y p i c a l B. was  almost  agar.  salmonicida and  the  colonies  sugar  appeared—the  fermented.  when p u s h e d w i t h  a  Also,  blood  the  col-  loop.  Morphology. Small  ,/  gram-rods,  colonies  to  al  salmonicida  o f B.  almost  n u t r i e n t agar was  coccoid.  slants. produced.  A  Picked  single  brown pigment, Sowed  into  typic-  carbohyd-  rates. Maltose  4-  Mannite  4.  Lose » • • • • • • • • « • 0 Arabinose XjQ.c"bose  . . . . . . . . 4.  ««««*««e*«  Sucros e  * a  T h i s was  loop,  tose  done b y  mixing  blood  agar  this and  touching  with  a drop  spreading.  0 0  a p o r t i o n of of  sterile  the broth  slant on  with  the  mal-  19. 2.  A few l a r g e  irregular  single  colonies,  shiny,  flat,  viscous,  with  edges,  Morphology. Large shape  gram-rods,  varying  on m a l t o s e  blood,  i n length,  and a few  clubbed  rods.  A.  Plated The  growth appeared  monicida—the  blood  agar.  t o b e t h e same a s t h a t  was h a e m o l y z e d  and t h e s u g a r  o f B.  sal-  fermented.  Morphology. Small gram-rods. pigment  gram-rods,  Picked  almost  coccoid,  single colonies  was p r o d u c e d ,  but there  and a few  to agar  were  slants.  two t y p e s  longer A  brown  of growth as  before ; (a)  a g l i s t e n i n g , convex  effuse  and  translucent  growth. (b)  a few l a r g e  single  colonies,  shiny,  flat,  viscous,  / with  irregular  • Subcultured agar  plate. (a)  from  Two  small  edges. the maltose  types round,  a g a r when p u s h e d  blood  of colonies shiny,  with  a  agar  plate  to nutrient  appeared.  convex,  regular  edge,  skated  loop.  Morphology. Small Picked  gram-rods,  to n u t r i e n t  Produced lb) a  heavy  larger creamy  almost agar  a brown  slant.  pigment,  colonies, centre.  coccoid.  flat,  viscous,  irregular  edge,  on  Morphology. large shape  gram-rods w i t h  rounded  ends.  A few c l u b b e d -  ^  rods.  Picked  to nutrient  agar  slant.  No b r o w n p i g m e n t was  produced.  So..ved i n t o  carbohyd-  ^  rates: Maltose  0  Mann i t e  ,  0  Arab i n o s e  1> 9.0 "t> 0 S 6 3 ULO B.  Plated Just  viscous,  JL 0 S 6 1  on n u t r i e n t one t y p e  irregular  0  •«•*•«••»«••  0 0  •«  agar.  of colony  appeared—large  edge, and a heavy  creamy  flat  colonies,  centre.  Morphology. Large shape For As  convenience  cultures same the  we  seemed  to bring were  technique results  (1) The  ends,  a n d a few  about  second  a reversion.  and t h e s e were  was  applied  identical,  Alternative "C" f o r m  this  type  clubbed-  that  of growth  this  of dissociation, experrlments  used  were  called  t o be a p o s s i b i l i t y  b e some f o r m  attempt  w i t h rounded  rods.  there  might  gram-rods  all,  be  The  i n a l l cases  recorded.  b r o t h to agar  grown f o r f o r t y - e i g h t  "C",  of the s i x t e e n  and s i n c e  one w i l l  sub c u l t u r e s f r o m  was  half  /  were begun t o  p i c k e d a t random.  t o them only  Only  form,  "C".  hours  plates. i n broth,  ^  a few  drops  incubated broth.  were  such  Serial  series  cultures  S u b c u l t u r e ! from change  after  (3) A  on  for forty-eight  Five  (2)  spread  seven  a nutrient hours.  Then  affected  no  a  plate  which  c o l o n y was  change  on n u t r i e n t plate  agar  was  sown  i n the  into;  morphology,  agar.  to p l a t e .  There  was  no  apparent  transfers,  Serial  subcultures  heavy,  turbid,  in  broth.  g r a n u l a r growth,  with a  surface  pell-  icle. Morphology. large plates).  The  Whether ociative iar to  gram-rods twelfth  the  form  "C"  b e h a v i o u r , , does the  survival  because  of  monicida colonies.  time  the f a c t  could  be ^  tube  form  o f B.  (longer  and  revealed  t u r n s out  thinner no  not  alter  of  the  that  by  the  proper  t o be  contaminant  accuracy of  original  organism.  treatment,  recovered i n every  from  morphological  eventually  s a l m o n i c i d a , or a  than  ease,from  the  changes.  a new  dis-  of p e c u l findings  This typical the  agar  is  L  as  true  B. s a l -  peculiar  DISCUSSION.  Plehn  stated  increased polluted it  and  by m i l l i o n s , under with  organic  i n sewage  more  ourable she  water least  line  noted  that  B.  found  that  which thirty  increase.  as h i g h  This  luted  1  d i d not l i v e  fav-  experiments and m u l t i p l y  salmonicida  grew  had been  sterilized,  and r e m a i n e d  days.  She f o u n d  of organisms  indicated  that  water  still  which  in  was  were  preserving  first by  a a  decrapid  too crowded t o made,  salmonicida  i s capable  content.  i n o c u l a t i n g w i t h B.  treated some  i n sew-  viable f o r  followed  organic  before  confused  and i t s a b i l i t y  B.  of high  t o t r y and k i l l those  while  there  present,  day the p l a t e s  t h e s e w a g e w a t e r was done  that  abundantly  enough d i l u t i o n s had n o t been  also  particularly medium,  infectious  and low w a t e r were  From her l a b o r a t o r y  the above e x p e r i m e n t s ,  was  as i t  i n c l e a r water.  warm w e a t h e r  m u l t i p l i c a t i o n i n water  monicida,  more  B.  On t h e t e n t h  Macarthur  In  required f o r  furunculosis,  i s commonly found  salmonicida  that  i n t h e number  count,  that  that  time  i n water  water.  Stewart age  Also,  colonies  conditions,  i n t h e same  p o l l u t e d waters  to furunculosis.  polluted  laboratory  water.  1  claimed  of B. s a l m o n i c i d a  material,  "disgusting ' than  Williamson  of  t h e number  to d i e out i n pure  occurs  at  that  with  ether  o f t h e sewage  vapor. organisms,  the use of the  the c h e m i c a l  to support  life.  differential  nature In  sal-  of the p o l -  this  menstruum,  i n o e u l a of B.  crease  i n numbers,  On  fifteenth  the  than  the  first,  organisms covered  per  from  Although uce  and  day and  the  then  a  on  the  can  first  tenth  increase  began to  decrease.  experiments  survive  can  day  B.  i n no  seem  to  a  m u l t i p l y i n water  the  "C"  in-  decrease  more  the  gradual  number  salmonicida  of  was  re-  days. way  be  indicate  and  a rapid  sudden  started,  sewage f o r s i x t y - s e v e n  these  showed  day  twenty-fourth  n a t u r a l c o n d i t i o n s , they  icida  the  second  on  c.c.  salmonicida  said  to  reprod-  t h a t B.  polluted  salmon-  with  organic  material. Whether  or  not  made f r o m  the  maltose  day  the  end,  until  cannot to  B.  agar  done  one  present  and  two  being  Many  dissociated  stated.  That  form  first  salmonicida.  the  tenth  of  polluted  i t w o u l d be  salmonicida  "C"  day.  water  have  water,  probably  by  form  and  bubbling  salmonicida.  on  and  the  the  Would any  sealed  also,  of  the  the  to  In  that  a i r through  "C"  form,  directly  sudden  oxygen  growth  is in  i n w a t e r w h i c h was  sterile  agar,  connection  i n v e s t i g a t e the  flasks,  this  on  appears.  biochemical  effect?  When  nutrient  other  colony  related  maltose  a p p l i e d to e x p l a i n t h i s  interesting  in tightly  polluted  o f B..  slants  forty-ninth  Whereas,' i f i t i s s t r e a k e d  t h e o r i e s c o u l d be the  the  n u t r i e n t agar.  B.  only  agar  s t r e a k i n g on  typical  agar  the  i t i s i n some way  s u b c u l t u r i n g to  described.  on  p l a t e s from  c o l o n i e s appear  on  this  growth  of  crease the  agar  i s s h o w n by  then  of  types  previously nutrient  blood  is a be  salmonicida  blood is  at  form  the  well  de-  demand with of  B.  non-aerated aerated,  polluted  water.  ^  Co-old t h i s which by  was  the  sudden  later  overcome?  other bacteria  salmonicida, organic  might  ognizable, chief  be  an  would  be  the  not  o f B.  be  sewage  to  some  may  be  some  cause  of  i n the  salmonicida,  to  organism, "G"  or  the  subsequent  form  reversion  of hours  have  might  something  objection  of  the  may  with  iant,  that  due  toxic  effect  be  produced  inhibitory  toxic  substance  to  B.  i n the  material.  dissociation  The  be  Some m e t a b o l i c p r o d u c t ,  i n the  or, there  Dissociation there  increase  such which  this  sewage w h i c h  at present  reversion  to t h e  typical  a theory is. that would  be  forms  days,  but  of months.  guick  powers  of r e v e r s i o n .  orded  in bacteriological  likely  the  to  the  type  of  Such  a form  literature.  has  is a  would  dissociant has  unrec-  dissoc-  to p a s s u n n o t i c e d ,  normal One  the  growth.  only  However, H a d l e y  "G"  "G"  cause  a form  of Hadley.  a new  would  That i s ,  into  of  to p o s t u l a t e  phenomenon.  not  stated,  matter,  therefore, with  extremely  y e t been  rec-  C O N C L U S I O N S .  1.  Bacillus  treated for  sewage w a t e r ,  sixty-seven ,2.  of  into  evaporation  of B a c i l l u s  i s at times  flasks  of  ether  of the ether,  survives  greatly  salmonicida  i n excess  colonies per c.c.  o f t h e number  inocul-  ( inoculum=2000 per c . c , maximum During  occurs 3. tially growth.  r e c o r d e d = l , 690,000 the p e r i o d  per c . c )  of o b s e r v a t i o n , wide  i n the c o n c e n t r a t i o n of t h i s The e t h e r alter  treatment  the c a p a c i t y  This (a)  B.  after  seeded  days.  The number  fluid  ated.  salmonicida,  i s evidenced  flucuation  o r g a a f i n the sewage.  o f t h e sewage  of the f l u i d  does n o t  to support  essen-  bacterial  by:  marked m u l t i p l i c a t i o n  of the i n i t i a l  inoculum  of  salmonicida. (b)  organisms.  eventual  multiplication  of the s u r v i v i n g  sewage  2,000, OOO  BIBLI0GR1PHY.  Arkwright,  J.  A,  Report  to the  Fisheries. Furunculosis  Interim  Committee.  Macarthur,  M.  I.  Report  H,  Stationary  Studies  of  on  the  Arts  at  1930.  1935.  Viability  the  391,  Office,  submitted  umbia,  C.  July,  and  I E , 1913,  o f j March,  Report,  M.  of A g r i c u l t u r e  Jour, Eyg.  Final  Thesis  M a r s h , M.  Board  f o r the  o f B.  salmonicida.  degree  of  University  Master  of B r i t i s h  (unpublished)  A More  Complete  Bulletin  o f U.  Description S.  Fish  o f B.  Commission  truttae, XXII,  190£, M a s t e r m a n , A.  T,  Report in  Stewart,  B.  J.  upon  of  Studies Thesis the  the E p i d e m i c  t h e Summer  Board  Col-  of  f o r the  University  Salmonidae  1911.  Agriculture  o n B.  amongst  411.  and  Fisheries.  salmonicida. degree  of Master  of B r i t i s h  of  Arts  Columbia. (unpublished)  at  Williamson,  Furunculosis  I. J . F.  of  F i s h e r y Board  the  for Scotland, 1928,  Winslow,  C.  E.  A.  and F a l l  No.  The  Rise  The  Newer K n o w l e d g e  Immunology, The  Salmonidae,  Chapt.  Salmon F i s h e r i e s  V.  of B a c t e r i a l  Populations,  of B a c t e r i o l o g y 6.  J o r d o n and  U n i v e r s i t y of Chicago  Press.  and  Falk. 1928.  


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