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ED STATES ENVIRONMENTAL PROTECTION AGENCY
           WASWHQFTON, D.C. 20460
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    MICROSlOLOe-ICAI* CRITBEI2CWORKSHOP
        CHI ASSOCIATES, INC.
       *2045 N 15th Street
        Arlington, VA  22201

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                       I.   INTRODUCTION

      The  improved membrane  filter  technique described  below
 incorporates modifications  to  the  basic methodology  already in
 use  in many water testing  laboratories.  These modifications
 are  designed to enhance recovery of the specific indicator
 organisms, Escherichia coli and Enterococci, and are recommendations
 based on  extensive studies  that continued over a period of  ten
 years.
      Marine waters collected from  polluted and nonpolluted
 beaches were tested for six years.  The organisms recovered were
 evaluated as indicators for the occurrence of swimming-associated
 gastroenteritis in persons bathing in those waters.  Findings
 from the  first two years showed that neither the most  probable
 number (MPN) nor the membrane  filter method (MF) for total  coliforms
 gave  results that correlated well  with elevated swimming associated
 gastroenteritis rates  (1). This led to the exclusion of these  tests
 in the studies that followed.  Fecal coliforms and their component
 genera, E. coli, Klebsiella sp., Enterobacter sp. and  Citrobacter sp.
 were measured, as-well as Enterococci, Clostridium perfringens,
 (spores), Pseudomonas aeruginosa, Aeromonas hydrophila and  the
 pathogen  Vibrio parahaemolyticus. All of these organisms recovered
 from  the  membrane filters were sneciated during a period of  three
 years, and their presence evaluated as indicators.   Of the  organisms
 examined, Enterococci densities in the water correlated best  with
 swimming-associated gastroenteritis(1).  E.  coli was a weak  second.
 Total fecal coliforms, their component flora,  and other organisms
 tested showed no such relationship (2).
     The  series of studies on fresh waters  carried out during  1979
 through 1983 compared the indicator organisms  E. coli and Enterococci
 to the standard fecal coliforms.   They showed  a correlation  coefficient
with swimming related gastroenteritis for E.  coli to be slightly
 greater than Enterococci.   This came as  no  surprise as E.  coli are
 more numerous in feces and tolerate fresh water well, whereas
 Enterococci are salt tolerant and survive in marine waters better
than E.  coli.   However, on the  basis  of  survival quality of both

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                              - 2 -
marine and fresh water, Enterococci are the most dependable as a
single indicator organism.
     The two best indicator organisms were the subject of laboratory
studies designed to enhance their recovery rate.  The improved methods
presented in the procedure descriptions that follow, were developed
as a result of these studies.  The method for E. coli includes a
resuscitation period for weakened organisms that enhances recovery
rates to over 90% and quantifies them within 24 hours without requiring
further subculture and identification (3).  The method for Enterococci
results in a 10,000 fold reduction in background organisms and appears
to measure those species most closely associated with fecal wastes of
humans (4).  Verified recovery rates exceed those of the standard
KF method by one order of magnitude.

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             II.  METHOD FOR  RECOVERY  OF  ESCHERICHIA COLI

A.  Equipment needed
     (1)  Member  filter holders and  clamps
     (2)  Membrane  filters -  O^S^m pore size
     (3)  Plastic petri plates -  9 x 50 mm with  tight lid
     (4)  Polyethylene film water sample  bags
     (5)  Incubator  35°C
     (6)  Water bath 44.5°C
     (7)  Weights  (to ensure  immersion of petri  plates  in water bath)
     (8)  Container  to hold plastic  bag while immersed
     (9)  Forceps
     (10) Burner or  alcohol lamp
     (11) Vacuum source
     (12) Source of magnification 10 to 15 x
     (13) Autoclave or ultraviolet sterilizing apparatus.
     (14) Small beaker or other open mouth vessel to  hold alcohol
B.  Medium
                            mTEC Medium

    Proteose peptone No. 3 (Difco)                  5.0g
    Yeast extract  (Difco)                            3.0g
    Lactose  (Fisher Scientific)                    10.Og
    NaCl                                            7.5g
    K2HP04                                          3.3g
    KH2P04                                          l.Og
    Sodium Lauryl Sulphate (2003 Matheson,
     Coleman & Bell)                                0.2g
    Sodium Desoxycholate (Fisher Scientific)        O.lg
    Brom Cresol Purple (Nutritional Biochem. Co.)   O.OSg
    Brom Phenol Red (Matheson, Coleman & Bell)      O.OSg
    Agar (Difco)                                   IS.OOg
    Distilled water                             1000.00  ml
     Dissolve ingredients by stirring (and heating to dissolve
agar if medium is to be divided  into  smaller aliquots prior to
sterilization).   Sterilize by autoclaving at 121°C for 15 minutes.
Pour into 9 x 50 mm petri plates, at  4 ml.per plate. The pH of  the
medium should be 7.3 + 0.1.   When agar has solidified, ascertain
that the lids are tightly fixed.

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                              - 2 -

C.  Solutions and reagents

                        Urease Subtrate Solution


       Urea                                        2.0g
       Phenol red                                 lO.Oma
       Distilled water                           100.0ml.

          Adjust pH to 5.0 ± 0.2 (straw color). Store at 6 to 8°C
    for a period of no more than one week.


                        Phosphate Buffered Saline

       Can be purchased or prepared.

                             95% Alcohol

       Can be purchased.


D.  Performance of test

    (1)  Filtration

        (a)   Sterilize filter holders

             i.  Autoclave,15 Ibs pressure for 20 min., or

            ii.  Ultraviolet sterilizing apparatus for 2 min., or

           iii.  Place in  boiling water,one to two minutes

        (b)   Dip forceps  in alcohol and flame to sterilize

        (c)   Assemble filters using sterile forceps to place filter
               membrane in situ and adjust clamp to hold assembled
               filter in  place

        (d)   Shake sample vigorously to ascertain even distribution
               of organisms present

        (e)   If less than 20 ml of  sample is to be filtered,  moisten
               filter membrane by pouring approximately 20 ml of
               sterile phosphate buffer into filter.   If 20 or more
               ml of sample is to be filtered,  use 5.0 ml.

        (f)   Using a sterile pipette transfer a measured amount of
               sample into the filter.  Amounts  tested for unknown
               samples should range from 0.1 ml to 50.0 ml.
               Recommended amounts  for  testing  unknown sample are:

                 0 .1 ml,   0. 3 ml,   1. 0  ml,   3. 0 ml,

                10.0 ml,  30.0 ml, 100.0 ml

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                          - 3 -

           Filter holders can be re-used without re-sterilization
       if one works from smaller to larger amounts of test samples.

    (g)   Rinse filter holder with no less than 20 ml of sterile
          phosphate buffer. (Be certain to rinse down sides of
          the fulter so that organisms clinging to the sides will
          be wash down onto the filter membrane.

(2)   Culture technique

    (a)   Remove filter top exposing the filter pad placement area

    (b)   Dip forceps into alcohol and flame to sterilize

    (c)   Grasp filter membrane containing organisms with sterile
           forceps and place on surface of mTEC plate by first allow-
           ing  one  edge to make contact and then carefully rolling
           it onto the surface to avoid entrapment of air

(3)   Resuscitation and incubation

    (a)   Carefully close plate and press edges to secure lid.
           Place up to 4 plates in the sterile water tight
           plastic bags.   The open edge of the bag is then
           rolled tightly for several folds,  and held in place by
           bending the flaps at either edge.   Place the bag thus
           prepared in a 35° C incubator for  the 2 hour
           resuscitation period

    (b)   Remove the bags from the incubator and immerse them in
           a 44.5° C water bath.  Weight them  to keep plates
           immersed.   Incubate for approximately 22 hours.

(4)   Biochemical tests

    (a)   Remove bags from water bath,  dry and take plates from bags

    (b)   Remove top of petri dish   containing the culture,  and
           place a sterile filter membrane in it,  using a forceps
           sterilized by method previously described.  Saturate pad
           by pipetting approximately 1.5 ml  of urease agent onto it

    (c)   Dip forceps into alcohol and flame to sterilize

    (d)   Transfer filter membrane containing  colonies, and
           place  it  on top of the urease  saturated filter membrane,
           using the same roll on technique.

    (f)   Allow to stand 15 to  20  minutes

    (g)   Using lOx magnification,  count all yellow colonies  and
           record number.   This  count,  when adjusted on the  basis
           of amount of water  cultured,  is calculated to represent
           the  number  of  thermotolerant  E.  coli/100  ml of  sample.
           All  colonies  that are  dark  on  the mTEC  plates,  and  are
           urease  positive,  are not E.  cpli.

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                    III.  METHOD FOR RECOVERY OF ENTEROCOCCI

A.  Equipment needed

     The same as for E. coliy with exception that the only incubator

needed is one adjusted to 41°C.


B.  Media


                            mE Medium


     Peptone                                       10.Og
     NaCl                                          15.Og
     Esculin                                        l.Og
     Agar                                          15.Og
     Yeast extract                                 30.Og
     Actidione                                      O.OSg
     Sodium azide                                   0.15g
     Distilled water                             1000.0ml

       Autoclave at 121°C for 15 min. After autoclaving add:.

       Nalidixic acid                              0.24g
       Triphenyl tetrazolium chloride              0.15g

         Adjust pH to 7.1 + 0.1 and pour 4.0 ml amounts into sterile

     plastic petri dishes (9 x 50 mm) with tight lid.



                           EIA Medium (Esculin Iron Agar)

     Esculin                                        l.Og
     Ferric Citrate                                 0.5g
     Agar                                          15.Og
     Distilled water                             1000.0ml

         Adjust pH to 7.1 ± 0.1 before autoclaving at 121°C for 15 min,

     Pour 4.0 ml amounts into sterile 9 x 50 mm petri plates.


C.  Solutions

    Phosphate buffer - same as for E. coli


D.  Performance of test

    (1)   Filtration - same as for E.  coli

    (2)   Culture technique

         (a)   Proceed as for E. coli, except that filter membrane is
                placed on the mE plate

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                     -  2  -

(b)   Incubate  at 41°C for 48  hours

(c)   Remove  plates  from incubator

(d)   Dip  forceps in alcohol and flame to sterilize

(e)   Use  forceps to place membrane onto EIA plate,  using the
       same  roll on technique as previously described

(f)   Incubate  for 20 to 30 minutes at 41°C

(g)   Count dark  red to  purple colonies,  approximately 1-2 mm
      in  diameter,  with dark  halos, reading through  the bottom
      of  the plate.

(h)   Calculate the  number of  colonies grown from 100 ml sample
      as  for E.  coli

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                         LITERATURE CITED


1.  Water quality health effects criteria for marine and fresh
     recreational waters (in preparation, EPA)

2.  V.  J. Cabelli, A.  P. Dufour, L.J. McCafe, and M.A. Levin.,
   Swimming-associated gastroenteritis and water quality, Am. J.
   Epidemiol,  115; 606-616,  1982.

3.  A.P. Dufour,  E. R. Strickland and V. J. Cabelli., Membrane
   filter method for  enumerating Escherichia coli., Appl. and
   Environmental Microbiol.  41; 1152-8, 1981.

4.  M.A. Levin,  J. R.  Fischer, and V. J. Cabelli,  Membrane filter
   technique for enumeration of Enterococci in Marine Waters. Appl,
   Microbiol.  30: 66-71, 1975.

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