Claußen, Lisa

Huckfeldt, Jonas

Soria-Dengg, Sally

Westphal, Bjarne

OceanRep

SFB 754 The	  Resazurin-­‐Agar	  Method:	  	  A	  Quick	  Test	  to	  Determine	  Water	  Quality	  Jonas Huckfeldt*, Lisa Claußen and  Bjarne Westphal Humboldt- Schule Kiel   GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel, Germany Structure-of-Resazurin-Structure of Resazurin Resazurin	  is	  a	  blue	  redox-­‐indicator	  which	  turns	  irreversibly	  to	  the	  pink	  coloured	  resoruﬁn	  when	  reduced	  by	  bacteria.	  Thus	  the	   degree	   and	   speed	   of	   colour	   change	   is	   an	   indica>on	   of	   microbial	   ac>vity	   and	   bacterial	   numbers.	   We	   tested	   the	  suitability	  of	  resazurin	  agar	  plates	  to	  test	  the	  microbial	  ac>vity	  in	  small	  volumes	  of	  diﬀerent	  water	  samples.	  	  Laboratory	  Tests	  •  The	  resazurin	  was	  reduced	  in	  the	  presence	  of	  yogurt	  bacteria	  in	  the	  ﬁlters.	  	  •  A	  discoloura>on	  gradient	  with	  regard	  to	  the	  concentra>on	  of	  bacteria	  was	  recognizable.	  •  A	  strong	  contamina>on	  by	  foreign	  bacteria	  caused	  reduction of resazurin and resorufin in the plates after 78 hrs. Preliminary	   experiment	   using	   a	   stock	   suspension	   of	   yogurt	  bacteria	   was	   prepared.	   Diﬀerent	   dilu>ons	   of	   the	   suspension	  were	  placed	  onto	  ﬁlters	  and	  the	  ﬁlters	  were	  placed	  face	  down	  on	  the	  resazurin	  agar	  plates.	  24 hrs 78 hrs •  The	  test	  is	  quick.	  Results	  are	  obtainable	  within	  24	  hours	  as	  compared	  to	  other	  screening	  tests	  like	  	  	  	  	  	  	  	  BOD	  (Biological	  Oxygen	  Demand),	  which	  need	  several	  days	  before	  results	  can	  be	  evaluated.	  •  It	  is	  simple	  and	  can	  be	  performed	  with	  a	  minimum	  of	  eﬀort	  and	  expense,	  with	  several	  screenings	  	  	  	  	  	  cos>ng	  less	  than	  $1.	  •  Using	  cell	  well	  plates,	  mul>ple	  assays	  can	  be	  done	  simultaneously	  lessening	  the	  >me	  for	  processing	  numerous	  samples.	  •  The	  test	  can	  be	  used	  for	  freshwater	  and	  marine	  	  environmental	  samples.	  	  •  The	  method	  is	  very	  simple	  and	  can	  thus	  be	  adopted	  for	  microbiology	  and	  ecology	  lessons	  in	  schools.	  The	  Resazurin-­‐	  Agar-­‐	  Method	  for	  es>ma>ng	  microbial	  ac>vity	  and	  thus	  water	  quality	  has	  several	  advantages	  :	  	  Lake	  Water	  Samples	  •  More	  reduced	  resazurin	  was	  observed	  in	  the	  sample	  from	  the	  site	  where	   the	   water	   was	   warmer.	   The	   higher	   temperatures	   there	  might	  have	  caused	   faster	  mul>plica>on	  of	   the	  bacteria	   leading	   to	  higher	  numbers	  of	  microbes.	  	  	  •  Cell	  wells	  were	  found	  to	  be	  prac>cal	  for	  this	  kind	  of	  test,	  enabling	  mul>ple	   assays	   with	   diﬀerent	   water	   samples.	   This	   also	   makes	  comparison	  of	  water	  samples	  easier.	  	  The	   method	   was	   veriﬁed	   using	   bacteria	   collected	   from	   2	   diﬀerent	  sources	   in	   a	   pond.	   One	   source	   was	   near	   a	   sub-­‐surface	   pipe	   which	  drains	   warm	   water	   into	   the	   pond.	   The	   second	   source	   was	   on	   the	  opposite	  side	  of	  the	  pond,	  where	  the	  water	  is	  colder.	  	  Start 61 hrs Seawater	  Samples	  Water	   samples	   from	   diﬀerent	   depths	   at	   two	   sta>ons	   in	   the	  Bal>c	   Sea	   were	   collected	   and	   ﬁlter	   frac>onated	   to	   separate	  diﬀerent	   sizes	   of	   bacteria.	   The	   oxygen	   concentra>ons	   at	   the	  sta>ons	  were	  measured	  separately	  using	  the	  Winkler	  method.	  	  •  In	   water	   samples	   collected	   from	   the	   sta>on	   with	   lower	  oxygen	   concentra>ons	   a	   higher	   resazurin	   reduc>on	   was	  observed.	  High	  microbial	  ac>vity	  may	  have	  contributed	  to	  the	  low	  oxygen	  levels	  at	  the	  sta>on.	  	  	  •  Water	   samples	   collected	   lower	   in	   the	  water	   column	  showed	  higher	  microbial	  ac>vity.	  •  Par>cles	   collected	   on	   the	   3.0	   μm	   ﬁlter	   showed	   higher	  resazurin	   reduc>on	   probably	   due	   to	   bacteria	   aYached	   to	  sinking	  organic	  maYer.	  	  O2-concentration         (ppm) 8,45 8,30 O2 -concentration         (ppm)    7,65       7,25 Surface   Mid depth Bottom   0,2    0,45  0,8    3,0  0,2     0,45   0,8   3,0 Sta>on	  1	   Sta>on	  2	  Filterpore	  size	  (μm)	  * Corresponding Author: Jonas Huckfeldt         jonas.huckfeldt@web.de Start 1	  2	  3	  4	   5	  6879	  10	  11	  Materials	  used	  for	  making	  the	  agar	  plates	  Informa>on:	  	  The	  numbers	  indicate	  decreasing	  concentra>ons	  of	  bacteria	  in	  the	  ﬁlters.	  Each	  step	  corresponds	  to	  ½	  dilu>on	  of	  the	  preceeding	  step.	  	  	  Discoloura>on	  was	  	  more	  intensive	  in	  the	  lower	  numbers	  aber	  24	  hours.	  	  Study	  site	  Water	  samples	  were	  collected	  on	  board	  a	  research	  vessel	  (A)	  from	  diﬀerent	  depths	  with	  a	  RoseYe	  water	  sampler	  (B	  &	  C).	  A	  C	  B	  Filter	  frac>ona>on	  of	  the	  water	  samples	  Sample	  collec>on	  sites:	  (1)	  warm	  water	  (red	  arrow)	  and	  (2)	  cold	  water	  (blue	  arrow)	  (1)	  	  (2)	  	  26 hrs Site	  2	  Samples	  were	  diluted	  with	  dis>lled	  water	  	  	  	  	  	  	  	  	  	  decreasing	  dilu>on	  of	  sample	  Site	  1	  Water	  samples	  

The Resazurin-Agar Method – a Quick Test to Determine Water Quality

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