United States            Office of Water          EPA 821 -R-00-026
          Environmental Protection        Washington, D.C. 20460      September 2000
          Agency



 AEMI    Method 1605: Aeromonas in Finished


          Water by Membrane Filtration



          September 2000 - Draft
t-


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                                 Acknowledgments

This method was prepared under the direction of Mary Ann Feige of the Office of Ground Water and
Drinking Water's Technical Support Center within the U.S. Environmental Protection Agency's (EPA's)
Office of Water (OW). This document was prepared under EPA OW Engineering and Analysis Division
by DynCorp Information & Enterprise Technology, Inc.
                                      Disclaimer

This method is in draft form. It has not been released by the U.S. Environmental fto^ction
should not be construed as an Agency-approved method. It is being circulated for Comments
technical merit. Mention of trade names or commercial products does not constitute endors
recommendation for use.                                              '

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                                       Introduction

Aeromonas is a common genus of bacteria indigenous to surface waters, and may be found in non-
chlorinated or low-flow parts of chlorinated water distribution systems. Monitoring their presence in
distribution systems is desirable because some aeromonads may be pathogenic and pose a potential
human health risk. Method 1605 describes a performance-based membrane filtration technique for the
detection and enumeration of Aeromonas species. This method uses a selective medium that partially
inhibits the growth of non-target bacterial species while allowing Aeromonas to grow. Aeromonas is
presumptively identified by the production of acid from dextrin fermentation and the presence of yellow
colonies on ampicillin-dextrin  agar (ADA) medium. Yellow colonies are counted and confirmed by
testing for the presence of cytochrome c (oxidase test) and the ability to ferment trehalose.

This method is for use in the Environmental Protection Agency's (EPA's) data gaBesing
programs under the Safe Drinking Water Act.                             : :,!
                                                                    ,. ."^
Questions concerning this method or its application should be addressed to: j-.ji
                                                                   4B
Mary Ann Feige
U.S. EPA Office of Water
Office of Ground Water and Drinking Water
Technical Support Center
26 West Martin Luther King Drive
Cincinnati, OH 45268-1320
Requests for additional copies of this publication should be dir^cfed to: A;
Water Resource Center
Mail Code RC-4100
401 M Street, SW
Washington, D.C. 20460
(202) 260-7786 or (202) 260,2814        „ ,^
Note:  Prior to validation ofthiitjgajjkijid^ each sttjjtpfttijgynethod must be performed as written. Once
       the method is validated, tffijfcgsiMgyill beffeifjbrmance-based. At that point, the laboratory is
       permitted to modify or ff^^tny^^ps:i^proceyj^, provided that all performance requirements
       set forth in the vajjjfcfted method'S^/if^jll'T.he laboratory may not omit any quality control
       analyses. The teyns  "shall," "mu$ir>f'&jjjj$iay not" indicate steps and procedures required for
       producing reliable results. The terys -fjnould" and "may " indicate optional steps that may be
       modified o>f omitted if the laboratory can demonstrate that the modified, validated method
       produces results equivalent or superior to results produced by this method.
Note:  This method -has"'M9n revised based on reviewer comments on the April 2000 draft of this
       method.

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                                  Table of Contents
1.0    Scope and Application  .......




2.0    Summary of Method	




3.0    Definitions	




4.0    Interferences and Contamination




5.0    Safety	




6.0    Equipment and Supplies




7.0    Reagents and Standards




8.0    Sample Collection, Preservation, and Storage




9.0    Quality Control




10.0   Procedure




11.0   Data Analysis and Calculations
12.0    Method Performance	#*:-•  • • • • ;£^j$Slfc.-*'/	 15



13.0    Pollution Prevention	 .^m".	v^'	*,! .«..-	 15




14.0    Waste Management	      .... ^'f. ........ jf		 15




15.0    References	.. . . A, .^/.. . . ..         ^:&:^:.	 16



                                               Data . ^'Kigff:	 17
                        ,



16.0   Tables, Diagram^fFli ""
17.0   Glossary  ........TT
                                                                                        20
                                            IV

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  Method 1605: Aeromonas in Finished Water by Membrane Filtration

                              September 2000 - Draft
1.0    Scope and Application
1.1     This method describes a membrane filter (MF) procedure for the detection and enumeration of
       Aeromonas in finished water samples. Aeromonas is a common genus of bacteria indigenous to
       surface waters. Its numbers are more likely to be greater during periods of warmer weather and
       when increased concentrations of organic nutrients are present. It also is more likely
       in non-chlorinated, or low-flow parts of chlorinated water distribution systems. Som|4j
       species are opportunistic pathogens.
1 .2     This method is adapted from Havelaar et al. (1987) for the enumeratiofisof Aeromo.
       finished water by membrane filtration (Reference 15.1), and has bee||J|riefly desjjpL&ecl as
       proposed method in Standard Methods for the Examination ofWate^midWastater, 20"
       edition, Section 9260 (Reference 15.2). It is a quantitative assay
       which partially inhibits the growth of non-target bacterial species w
       grow. Aeromonas is presumptively identified by the production
       and bright yellow colonies that are greater than 0.5 mm in
       identified colonies are counted and confirmed by testing for the presence of c
       (oxidase test) and the ability to ferment trehalose.
1 .3     This method is designed to meet the monitoring
       Protection Agency. Aeromonas hydrophila was mclu
       (CCL) (Mar. 2, 1998, 63 FR 10274) and in the Revns to
       Monitoring Proposed Rule (UCMR) (September,^ 1999, 6Jjj)PR 50556rt?ottaminants in the
       UCMR are candidates for future regulation andlhay be inc|lfied in a monitoring program for
       unregulated contaminants. Unregulated contpnifiaht mooiioring wou|apE>e required for large
       systems and a representative sample of smalt and mediumisized \¥jlpr?distribution systems.
1 .4     EPA intends to conduct.a multi-lab validation of this methiJ'dliifjfinished waters.
                                                     •  •      *
                                                                            ive medium
                                                                            Aeromonas to
                                                                               fermentation
                                                                                  List
2.0   Summary of Metho
2.1
       The method provides a.d^^^fUttt of Aeromonas «i water based on the development of yellow
       colonies on the surface of tf Pfiem^aafeJilter using a selective media for Aeromonas species. A
       water sample is fpered through Ot^S'-^ia-pore-size membrane filter. The filter is placed on
       ampicillin-dexlpn agar and incuba'fedl^35°C ± 0.5°C for 22 to 26 hours. This medium uses 10
       mg/L ampicjJj|fL to inhibit non-Aeromonas species, while allowing most Aeromonas to grow. The
       medium uses dextrin as a fermentable carbohydrate, and bromothymol blue as an indicator of
       acidity produced by the fermen|^|fbn of dextrin. Yellow presumptively identified colonies are
       counted Sg^cojifirmed by testing for the presence of cytochrome c (oxidase test) and the ability
       to fermettt-trehalose.  ,.,,^- ••-'"
       The membraae'filtfaWbn procedure provides a direct count of culturable Aeromonas in water
       samples that is based on the growth of bacterial colonies on the surface of the membrane filter.
                                                                        September 2000 - Draft

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Method 1605 - Aeromonas
         If samples are to be archived for further analysis to determine species or hybridization group,
         from the nutrient agar plate, either inoculate a nutrient agar slant for short term use or shipment
         to another laboratory or inoculate a tube of nutrient agar broth for internal storage in the freezer.
  3.0     Definitions
  3.1     Aeromonas are bacteria that are facultative anaerobes, Gram-negative, oxidase-positive, polarly
         flagellated, and rod shaped. They are classified as members of the family Aeromonadaceae.
         Demarta et al. (1999) reported 15 Aeromonas species based on 16S rDNA sequences though no|
         all are officially recognized. Some, but not all, have been associated with human disa
         method, Aeromonas are those bacteria that grow on ampicillin-dextrin agajy'ADA)
         yellow colonies, are oxidase-positive, and ferment trehalose.
                                                                       $££•-$&&
  3.2    Definitions for other terms are provided in the glossary at the end of tf|*method (S<
4.0   Interferences and Contamination
4.1    This method is designed to be used with finished water. Water sampler
       suspended particulate material may clog the membrane filter and pr^ent
       spreading of bacterial colonies which could interfere with identification
4.2    Other ampicillin-resistant bacteria that are not aeromonads
       Some of these bacteria may also produce yellow
       byproducts from the fermentation of dextrin or some
       yellow pigment. Enterococcus bacteria are reportei
                                                                                   ;olloidal or
                                                                                      iuse
                                                                                        Medium.
                                                                                     id
                                                                                      produce a
                                                                                 iw colonies on
         ADA. Confirmation of presumptive Aeromonas colpnies is necessary taifiMpite false positives.
  5.0   Safety
  5.1

  5.2


  5.3
Since some strains
materials shoul
                     ^omonas ajjs Opportunistic
                         ed prior A>«!$a|riag or disposal."
                         *       *      ' ' '
The analyst/technici
microbiology laborato
materials.
                                                                     containers and waste
                                      and observe the normal safety procedures required in a
                                           , usittg and disposing of cultures, reagents, and other
       This method does jot address lal! safety' issues associated with its use. The laboratory is
       responsible forjplmtaining a safe W0tfc environment and a current awareness file of OSHA
       regulations regirding the safe handling .ephe chemicals specified in this method. A reference file
       of material safety data sheets (MSgSs) should be available to all personnel involved in these
       analyses. __;t
September 2000 - Draft

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                                                                        Method 1605 - Aeromonas
6.0   Equipment and Supplies
Note:  Brand names, suppliers, and part numbers are for illustrative purposes only. No endorsement is
       implied. Equivalent performance may be achieved using apparatus and materials other than
       those specified here, but demonstration of equivalent performance that meets the requirements of
       this method is the responsibility of the laboratory.
6.1
6.2

6.3
6.4

6.5
6.6
6.7
6.8
6.9
6.10
6.11
6.12
6.13
6.14
6.15
6.16
6.17
6.18
Equipment for collection and transport of samples to laboratory
6.1.1  Autoclavable sample bottles—1-L glass or plastic, sterile, with gufilcient
       mixing sample
6.1.2  Ice chest
6.1.3  Icepacks
Autoclavable dilution bottles—125-mL marked at 99 mL or 90 mL; B&^MfMally produced
dilution bottles may be used
Rinse water bottles
Sterile plastic or autoclavable glass pipettes with a 2.5% tolerance^-To delivieifflWBHbuid 10-
mL
Pipet bulbs or automatic pipetter
Autoclavable pipette container (if using glass pipettes)
Thermometer—with 0.5°C gradations checked aga^t a National Instira^of Sfandards and
Technology (NIST) certified thermometer, or one that meets file requirements of NIST
Monograph SP 250-23                     .^  !        ."
Inoculating loop—Sterile metal, plastic, or/wooden applfiftor sticks =
Burner—Flame or electric incinerato|ii&i,s^ilizing nietaIftGfa|pting loops and forceps
                                          or hani
                                   and lxt, sterile, polypropylene or glass
Colony counting
Hotplate stirrer
Magnetic stir bar
Graduated cylinders — IW
                  , •cS-v*--  ..
Balance — Capable w weighin
Weigh boats
            %*^
pH meter  ,f;;,;f'
Turbidimefe'(optional)
        Rvl'K'' '" j.
Equipment J5>f membrane filter procedure
6.1 8.1 McTibatoi^Hot air 'or water-jacketed microbiological type to maintain a temperature of
       6.1 8.2 PetrMi^hes — sterile, 50 x 9 mm or other appropriate size
                                                                           September 2000 - Draft

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Method 1605 -Aeromonas
  6.19
  6.20

  6.21


  7.0
  7.1
  7.2
  7.3
6.18.3 Membrane filtration units (filter base and funnel made of glass, plastic, or stainless steel)
       autoclavable and wrapped with aluminum foil or Kraft paper and sterilized
6.18.4 Vacuum source
6.18.5 Flasks—1-L vacuum filter with appropriate tubing; a filter manifold to hold a number of
       units is optional
6.18.6 Side-arm flask to place between vacuum source and filtration devices or filter manifold
6.18.7 Membrane filters—Sterile, cellulose ester, white, gridded, 47-mm-diameter with 0.45-/^m
       pore size
6.18.8 Forceps—Sterile, straight or curved, with smooth tips to handle filters withoiy
       damage
6.18.9 Ethanol or other alcohol in a container to sterilize forceps
Dissecting microscope—Low power (10X to 15X), binocular, illumiaajed
Autoclave—Capable of 121 °C at 15 psi. Must meet requirements seuftrth in 1
Certification of Laboratories Analyzing Drinking Water, 4* Edition^,
Membrane filters (for sterilization purposes)—Sterile, cellulose estefj*
                                                                         m pore size
                                                      Ications i]jjManual for the
                                                      Edition 0? Standard Methods for the
                                                                in 40 CFR Part 141),
Reagents and  Standards
Purity of reagents and culture media — Reagent-grade cher|ttei®Jia|l 6e used in all teffe Unless
otherwise indicated, reagents and culture media shall cj^pbnn to^thejp^ffi£ations-'to Standard
Methods for the Examination of Water and WastewateF (latest edjmoii a^l^V^ckbyEPA in 40
CFR Part 141), Section 9050 (Reference 15.2). Thg(jpgarused,p*prepar%lSSfiof1Culture media
must be of microbiological grade.
Purity of water — Reagent-grade water confgjujjng to spj
Certification of Laboratories Analyzing Drinjjing Watef,
Examination of Water and Wastewate$"(latest edition ;
Section 9020 (Referent&.l 5. 2).
Phosphate buffered:® fim«||t jvater
7.3. 1    Concentratevdstfe^|||bsphate buj^saWtion — Dissolve 34.0 g potassium dihydrogen
        phosphate  (Kfr^^f jn^OO mL reageat-geade water. Adjust the pH to 7.2 ± 0.5 with IN
        sodium hydrogd#|paiOH) and dilute to-ft with reagent-grade water. Autoclave or filter
        sterilize thjgf|pfa niter with 0.22 /^m pore size.
        Magnesium chloride solu^|^3^polve 81.1 g magnesium chloride hexahydrate
               H20) in reagent-gSfeiStfer and dilute to 1 L.
               phosphate buffered dilution water by adding 1.25 mL of concentrated stock
            hate buffer solution (Section 7.3.1) and 5.0 mL of magnesium chloride solution
               7.3.2)  to a 1-L graduated cylinder and adjust final volume to 1 L with reagent-
            s water. PreD|j|:a "portion of buffered dilution water in 1-L bottles for rinse water.
                ; or ftfflPiterilize through a filter with 0.22 //m pore size.
        Phosplate buffered dilution water may be stored indefinitely, unless turbidity is
        observed.
         7.3.2
         7.3.3
         7.3.4
September 2000 - Draft

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                                                                         Method 1605 - Aeromonas
7.4    Ampicillin-dextrin agar (ADA)— Tec Pac - Biolife Italiana cat. No. 401019 (M-Aeromonas
       Selective Agar Base [Havelaar]) Uile Monza, Milano, Italy., or equivalent. Follow procedure as
       specified on media for preparation. EPA highly recommends the use of commercial ADA.,
       however ADA may be prepared as follows, if necessary.
       7.4.1   5.0 g tryptose—Difco cat. no. 0124-17-2, or equivalent
       7.4.2   10.0 g dextrin—Difco cat. no. 0161-17-6
       7.4.3   2.0 g yeast extract—Difco cat. no. 0127-17-9, or equivalent
       7.4.4   3.0 g sodium chloride (NaCl)—Baker cat. no. 3624-01, or equivalent
       7.4.5   2.0 g potassium chloride  (KC1)—Fisher cat. no. P217-500, or equivalent
       7.4.6   0.2 g magnesium sulfate heptahydrate (MgSO4 7H2O)—Fisher cajbiip. M6
               equivalent                                               ^
               0.1 g iron (III) chloride hexahydrate (FeCl3 6H2O)—Sigma catlno. F-2877,<
7.4.7
7.4.8
7.4.9
                                                                                 icillin, sodium
                                                                                     .d filter
                                                                                       ons for
       0.08 g bromothymol blue—Baker cat. no. 1-D470, or equiv;
       15 g agar, bacteriological grade—Fisher cat. no. BP1423-51
7.4.10 Ampicillin, sodium salt—Sigma A0166, or equivalent. Add 1
       salt to 10 mL reagent water. Prepare on the same day that
       sterilize through a filter with 0.22 ^m pore size. Follow masufac
       appropriate storage temperature and length.
7.4.11 Sodium deoxycholate—Sigma cat. no. D-6750, or e
       deoxycholate to  10 mL of reagent water.  Filter
       pore size.
7.4.12 Add reagents in Sections 7.4.1 through 7.4.1% 1-L ojfeagent-
                                                                               iter, cool to room
               temperature, and adjust pH to 8.0 usiryjil'I'NaOH oif-lN HC1. ^jitoclave for 15 min,
               cool to 50 °C, and add the sterile ampiollin and steBle sodiumfifeoxycholate solutions.
Note:   The agar (Section 7.4$)^may be added after thepH is •adjusted, if it is more convenient for the
        laboratory. Agar tij^iQxf'dissolved before the media is aWoclaved.
                            * «"^>            x /' "   $y/
7.5
7.4.13 Add approximately |te|/0f ADA per 50 * 9" mm petri dish and allow to solidify. For
       larger pla-^8jiS!'vB^BnemjpiK)priateIy. ADA plates should be stored in a tight fitting
                 Pe. sealed plastl£§^^ at a temperature of 1 °C to 5°C  for no longer than 14
       days.jji
Pentahydrat^.CS Reagent grade sodium thiosulfate — Fisher cat. no. S446, or equivalent.
7.5.1  SfecM solution (3 % solution) — Add  3 g sodium thiosulfate to 100 mL reagent-grade
                              '
7.5.2
                      i&pf s°d|ti|*thiosulfate per L of sample to sample bottles prior to autoclave
                         . Alternatively, if using presterilized sample bottles, sodium thiosulfate
                          tbclaved for 15 minutes or filter sterilized through a filter with 0.22 ^m pore
               size before adding to the sample bottles.
                                                                            September 2000 - Draft

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Method 1605 - Aeromonas
  7.6
  7.7

  7.8


  7.9
  7.10

  7.1 1
  8.0
  8.1
  8.2
                                                                       ; Tests (Difco
Disodium salt of ethylenediaminetetraacetic acid (EDTA)—Sigma cat. no. E 4884, or equivalent.
EDTA should only be added to samples, if metals in water samples exceed 1.0 mg/L.
7.6.1   Stock solution—Add 372 mg EDTA to  1 L of reagent-grade water.
7.6.2  Working solution (15% solution)—Add 15 mL of the stock solution to 100 mL of
       reagent grade water and adjust pH to 6.5 using IN NaOH or IN HC1 before sterilization.
7.6.3  If EDTA is necessary to reduce metal toxicity, add 2.5 mL of the working solution per L
       of sample to sample bottles prior to autoclave sterilization. If using presterilized sample
       bottles, EDTA should be autoclaved for 15 minutes or filter sterilized through a filter   ^
       with 0.22 ^m pore size.
Positive control culture—Aeromonas hydrophila ATCC #7966; obtained torn the.
Type Culture Collection (ATCC, 10801 University Blvd, Manassas, VAvJ0MO-22
Negative control—One or more non-Aeromonas bacteria which grow fltrADA will ]
to be used as negative culture controls. The purpose of these is to hejpithe analysljecogma
other bacteria which may grow on ADA.                        '""
Nutrient agar —Difco cat. no. 0001-17-0 or equivalent
Oxidase reagents—Sigma cat. no. T3134, or Dry Slide - Oxidase DispJ
DF3530-75-3) or BBL Reagent Droppers (0.5mL) B-D 4361181  or
0.5% Trehalose confirmation reagent - add 5 g trehalose (Sigma  cat. no.
100 mL water and filter sterilize solution. Prepare purple
according to manufacturer directions as for one liter but
purple broth base.  Cool to room temperature.  Aseptic
purple broth base.  Store in refrigerator or preferablyj
accordingly. Dispense into 6mL or larger size tub|^Knd fill apfrfoxima
Sample Collection, Preservation, and Storage
Use 1-L glass or pla§%bottles (Section 6.1 J). Samplu^fflrp£C«!kfes are described in detail in
Standard Methodsj^ir^^Sxamin^S^^j^ter and fflatftewater, Section 9060 (Reference 15.2).
Adherence to saragtej^B®gtvation pre«Nwlllp%nd holding time limits is critical to the production
of valid data. Sample j^pb will be coMMtefcd invalid if those conditions are not met.
Sample collection—Samte^pastbe repres&rtaiiv^ of the drinking water distribution system.
Water taps used for sa^^to^KWMTie free of aerators, strainers, hose attachments, mixing type
faucets, and purifi^g^no^cBJd&l^Srter tapl should be used. The service line should be
cleared before s^ffipling by maintaining a Steady water flow for at least two minutes (until the
water changes-temperature).     ->W
8.2.1   Ad^f mL of sodium thiosulfate per L of sample (Section 7.5).
8.2.2  ll^ejials  in the sample ;eiceed l.Omg/L, add 2.5 mL of EDTA working solution per L of
              to reduce mel^poxicity (Section 7.6).
8.2.3  GglMptttniniuinlb? 1-L of sample in a sterile, non-toxic 1-L glass or plastic container
                      lid. Leave headspace to allow mixing.
                                                                         water.
                                                                           utionJcooled
September 2000 - Draft

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                                                                         Method 1605 - Aeromonas
8.3    Sample preservation and handling
       8.3.1   Immediately following sample collection, tighten the sample container lid(s) and place
               the sample container(s) in an insulated storage cooler with ice packs or in a refrigerator
               to chill prior to packing the cooler for shipment. Do not freeze the sample.
       8.3.2   Place the chilled sample(s) upright in the center of a plastic-lined, insulated container.
               Use insulated containers to ensure proper maintenance and storage temperature. Use
               enough freshly frozen ice packs to ensure that the samples will arrive at a temperature of
               1 °C to 10°C. Use a minimum of two ice packs per shipment and add extra ice packs for
               multiple samples. Place one or more ice packs on each side of the container tojitabilize  •
               samples.
       8.3.3   Samples must be maintained at a temperature of 1 °C to 10°C du
               must not be frozen.
                                                                       ,
Note:  Sample temperature during shipment is critical. Icepacks must be '^r
       immediately prior to shipment.
8.4    Refrigerate samples at 1 °C to 5 °C upon receipt at the laboratory and analyze
       after collection. Samples must be analyzed within 30 hours o
       study results are summarized in Section 16, Table 2.
9.0   Quality Control
Note:
9.1
9.2
The quality control requirements in Sectio,
Certification of Laboratories Analyr~
control requirem&i^^MJfed in
during review ofth^zfyfujft^ versl
after evaluation of the 'mi&$$Ojjatory w
                      rg U.S. EPA Manual for the
'king Watef^if^tfijXEdition, the analytical quality
  ted ConfS^mdnt Monitoring Rule, and comments
   ethod. These requirements are subject to change
 ten  results.
Each laboratory thftf uses this metfao4i|tji|wired to operate a formal quality assurance (QA)
program.  The.jpinimum requiren|«Hfl^^^^uality control program for this method consist of
initial and on|J|ing demonsrrations|^6tb7ratoiy capability through analysis of positive and
negative control samples and methjll blanks. Laboratory performance is compared to the
performanci||;riteria specified injjSection 9.4 to determine whether the results of the analyses
meet the performance characteristics of the method.
Specific ^a^^jtrpL^I^TOquirements for Method 1605 are provided below. QA and QC
criteria for1{%cil|Si^,.^aRiomel, and laboratory equipment, instrumentation, and supplies used in
microbioldfOTFanalyies must be followed according to Standard Methods for the Examination
of Water and Wastewater (latest edition approved by EPA in 40 CFR Part 141) and the U.S. EPA
                                                                            September 2000 - Draft

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Method 1605 - Aeromonas
    carefu
'at this
  , without
         Manual for the Certification of Laboratories Analyzing Drinking Water, Fourth Edition (March
         1997) (Reference 15.5).
  9.3    Dilution scheme for determining organism density. This procedure is adapted from Standard
         Methods for the Examination of Water and Wastewater, 19th Edition, Section 9020 B (Reference
         15.9). This entire process should be performed quickly to avoid loss of viable organisms. See
         Section 16, Flowchart 1, for an example of this dilution scheme.
         9.3.1   Inoculate organisms onto the entire surface of several nutrient agar slants with a slope
                 approximately 6.3 cm long in a 125 x 6 mm screw-cap tube. Incubate for 18 to 24 hours
                 at35°C.
         9.3.2   From the slant which has the best growth, prepare serial dilutions using four
                 bottles with 99 rnL of sterile buffered dilution water (A, B, C  and JD below
                                                                          *sP*&itaL
                 dilution bottle containing 90-mL of sterile buffered dilution wa6ejpB2 bel<
         9.3.3   Pipet 1 mL of buffered dilution water from bottle "A" to onerfkhe slants.
                 growth on the slant by gently rubbing the bacterial film with l|je
                 to tear the agar. Pipet the suspension back into dilution bottf^|
                 procedure a second time to remove any remaining growth o
                 disturbing the agar.
         9.3.4   Make  serial dilutions as follows:
                 9.3.4.1        Shake bottle "A" vigorously and pipette 1 niL to 1
                 9.3.4.2        Shake bottle "B" vigorously and               bottle'
                 9.3.4.3        Shake bottle "C" vigorously                        "D,l
                 9.3.4.4        Shake bottle "D" vigorously^dpipette|'6'i^a9^^^D2"; this
                               should result in a final dilujpn of apjM^ximate%JlTC¥0 / mL.
         9.3.5   Filter  1 - to 5-mL portions in triplicate|f)Wbottlesj;JT and "Disaccording to the
                 procedure in Section 10 to determinelifii.'number.djr'CFU in theHiilutions. The target
                                                il!ili         ~  •<*        N-lr1"^
                 dilution is one that produces 20 to gpeolonies per ADA pjatpt' Dilutions should be stored
                 at 1 °C to 5°C,and may be used:thri|ghout the day they.|reprepared.
                           A             • <*= '"
  Note:  Analysts may practice[^"iijj&tion scKeiAe bjjjjjiQfsing filters on nutrient agar plates instead of
         ADA plates. After a gri^K:pait&n is deter^&^^Kd..the analyst can accurately determine the
         target concentrations, ditml&8$jfram SectiofK^W^fay be filtered in duplicate. However,
         dilutions should be ant^^Atn-tmill&Ue wherinew cultures are used.
         9.3.6   Thera.?should be approximately \QW Aeromonas hydrophila CPU per slant. Therefore,
                 dil&lm bottles "A" through "D2" should contain approximately 1010, 108, 106, 104, and
                 l^^S'U per dilution botfle, respectively.
September 2000 - Draft

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                                                                         Method 1605 -Aeromonas
Note:  Depending on the growing conditions and the strain and species selected, these numbers may
       vary. As a result, until experience has been gained with the conditions and organisms used, more
       dilutions may need to be filtered to determine the appropriate dilution.
9.4    Analytical QC for the membrane filter (MF) procedure. The laboratory must successfully analyze
       a positive control sample (Section 9.4.1), negative control sample (Section 9.4.2), and method
       blank (Section 9.4.3) before performing any field sample analyses using this method.
       9.4.1   Positive control and positive control duplicate — this must be perfj
               that samples are analyzed or once per media batch, whichever
               9.4.1.1
               9.4.1.2
               9.4.1.3
Using a pure culture obtained from a
(Section 7.7), grow Aeromonas on;
Prepare a stock culture and dilute tc
10 CPU per mL by the procedure li
(positive control and positive control
volume of the appropriate dilution iato 5'
to obtain 20-80 CPU per filter. Filter i
Process positive control and pasi|tve control
to the procedure in Section<|0f;:,poi&m positive 'ccfitol and
positive control duplica^^^nt-^roc^ta® listed in" Section
10.11.             -/  '     ^     "* ',„  H, ^
Calculate the relatiye percenyfinerence (RPB)using the
following equafctojtf         -'        ^'"'
    .ed onq
      freque
 lifted outs
hour nurteiit agarl
        approximately^'
      Ltion 9.3. For each
      tyi spike enough
           agent water
                             RPD = 70Q:«4
               9.44,4
               9.4.3
   !D is the^fate*^&rcent difference
     the densjiy^f Aeromonas in the positive control sample
           mL)"
           .ensity of Aeromonas in the positive control
diiffl^»:tample (CPU /100 mL)
    percent difference between duplicate positive controls
snould not exceed [to be determined through method validation].
If target colonies do not appear on ADA agar, or do not
confirmed,  halt all sample analyses. Prepare new media and
culture dilutions and analyze a second positive control sample. If
results are still unacceptable, then culture viability or reagent
potency may have been compromised. To identify whether a
problem is due to the positive control rather than the ADA agar,
                                                                            September 2000 - Draft

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Method 1605 -Aeromonas
                                                                                     s according to
                                     it may be advisable to filter an extra positive control in 9.4.1.2
                                     and place the filter on a nutrient agar plate.
       9.4.2   Negative control—On an ongoing basis, the laboratory must perform, at a minimum, one
               negative control per media batch.
               9.4.2.1                Using pure cultures obtained from a qualified outside source
                                     (Section 7.8), grow negative control cultures on a 24 hour
                                     nutrient agar slant.
               9.4.2.2                Streak negative controls onto ADA agar to become familiar with
                                     the color and morphology of non-Aeromonas bacteria,;
       9.4.3   Method blank—On an ongoing basis, dilution/rinse water method blanks mu
               processed at the beginning and end of each filtration series to cheJiNfor pog
               contamination. A filtration series ends when 30 minutes or moj|pl|>se betwl
               filtrations. An additional method blank is also required for every 20 sample
               than 20 samples are processed during a filtration series. For^ample,
               plans to run 30 samples during a filtration series, a method ^^jk^shqultf be processedl
               the beginning, middle, and end of the filtration series.
               9.4.3.1                Process 100-mL dilution/rinse water i
                                     the procedures in  Section 10, as appropria
               9.4.3.2                No growth should appear in niethoJ blanks.
                                     halt all analyses.  Prepare new|^fitiorji/rinse
                                     the method blanks.
                                     new dilution/rinse watja^issess"
                                     reagents.
9.5    A tube of the 0.5% trehalose (Section  7.11) shouldtle incubatfd with every t>atch of samples to
       confirm sterility.                            , .          . :v
9.6    If the laboratory has two or more analysts, e&eh are requu^d to countjlpFget colonies on the same
       membrane from one positive sample per month. Comp$Wt%ch anaiitt's count of the target
       	                          .•;  • #        -^Nf:ilS*  • ' m?i%r
       colonies. Counts should fall within 1Q|4 between anal^^^wM^fs fail to fall within 10% of
       each other, analystef^hji|||d contintt^pa^^pn counts;Jtj§|pme number of target colonies
       counted fall                    anm^^S^jrrat least three consecutive samples. If there are no
       positive samples, thel^^K^ontrol cl^^^tsed for this determination.
9.7    Verify autoclave sterilizliiM'Aionthly by plibfaui Bacillus stearothermophilus spore suspensions
            J                   *'*<$£g$&ig&f ^,-^. J   •/ f  =, , , f*?-'f,',,'%;                  iff
       or strips inside glasswa^^p|^^p^i(.21 °C fo^||5'minutes. Place in trypticase soy broth tubes
       and incubate at            nOT^|fi^^;for growth to verify that sterilization was adequate. If
       sterilization wajjiladequate, deterjaiyftwfctepriate time for autoclave sterilization. Filter
       sterilization may be used as long aj|fi§^pne quality controls are instituted for the filtrate.
9.8    Participate,:5|i|:4nterlaboratoiy perfcfibance studies conducted by local, state, and federal agencies
       or commqrci0 organizations, if ajpiilable.
  10.0  Proce|i|
  10.1   The membflheTillteT (IvlF) procedure with ampicillin-dextrin agar (ADA) is used to enumerate
         Aeromonas in finished waters.
  10.2   Label each petri dish with sample identification, preparation date, and analysis start date/time.
September 2000 - Draft
                                               10

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                                                                         Method 1605 - Aeromonas
10.3   Use a sterile MF unit assembly (Section 6.18.3) at the beginning of each filtration series. A
       filtration series ends when 30 minutes or more elapse between sample filtrations.
10.4   Sterilize forceps with alcohol. Flame off excess alcohol. Alternatively, an electric incinerator
       may be used to sterilize forceps (Section 6.9). Using sterile forceps, place the MF (grid side up)
       over the sterilized funnel. Carefully place the top half of the filtration unit over the funnel and
       lock it in place.
10.5   Shake the sample bottle vigorously approximately 25 times to distribute the bacteria uniformly.
       Using aseptic technique, transfer one, 500-mL aliquot of sample to a single funnel. Use a
       graduated cylinder with a "to deliver" tolerance of approximately 2.5%.
Note:  Laboratories must filter the entire 500-mL sample volume unless thejilj&r clogs. If
       clogs, a minimum of 100 mL of sample must be filtered, which may require multMe'Ji
       If less than 500 mL are filtered and analyzed due to filter clogging, fflqjisure tjjjjresidual,
       unfiltered volume to determine the volume filtered, and adjust the re^^^^^jmit accordingly.
10.6   Filter each sample under partial vacuum (10-15 mmHg) through a sterile mer
       the funnel after each sample filtration by filtering three, 20-mL^o.40smL portioMsM|^^
       buffered dilution water.                                    ""ifc^ss^         ffc^ •-•: v
10.7   Upon completion of the final rinse, disengage the vacuuntarid refi
10.8   Using sterile forceps, immediately remove the MF anf-place it gpd-sfJS^§Q^ADA medium
       with a rolling motion to avoid trapping air under thSfilter. Respat the mpp^^ filter if bubbles
       occur. Place the inverted petri dishes in the 35jfl!jt).5°C ijjpbator irm&diately after
       preparation. Sterilize  forceps and filtering apfpptus between the prenjj!tion of each sample.
10.9   After 22 to 26 hours of incubation at 35°C^.5°C, cogl^nd recjoJ^ellow colonies with a
       diameter greater th;
10.10  Aeromonas confi:
                     5 mm under
                         Isolation:of
                            should )S<|
presumptive Aeromonas^^mss up to
cytochrome c (oxidase t
                                                ation
                                                                     microscope.
                                                w colonytli$%~*diameter greater than 0.5 mm on
                                                       a presumptive positive for Aeromonas. All
                                                      ile must be tested for the presence of
                                  „,„ __„	*.v,-v*,*	positive for the oxidase test must be
       tested for the ability tofea^^ate^jse (SecttbiriO.10.3). In this method, any presumptive
       colony that is posi^p^TOr oxiaa^-j^yfeaiientslSrehalose is considered to be Aeromonas.  Slight
       variations in colef 'Snd morphologji^y Ixsjjresent between different Aeromonas species grown
       on ADA meditM. The colonies         fbr confirmation should be representative of all the
       presumptivd^'positive colonies griffm'on the ADA plate. For example, if 30 bright yellow
       colonies affii20 dull yellow colonies are observed, then 6 bright yellow and 4 dull yellow
       colonies sKbuld be submitted to/tj&nfirmation.
                                              11
                                                                            September 2000 - Draft

-------
Method 1605 - Aeromonas
  Note:  It is important to record the number of colonies of each presumptively positive morphological
         type so that the final density of Aeromonas can be reported based on percent confirmation of
         each morphological type. Also, the laboratory may submit more than ten presumptively positive
         colonies to the confirmation step.
  Note:
         10.10.1    To confirm as Aeromonas, pick a colony and streak the colony onto a plate of
                    nutrient agar medium (Section 7.9) and incubate at 35°C ± 0.5°C for 22 to 26 hoursd
                    to obtain isolated colonies.
  Note:  If the streak plate has more than one morphological type of colony, s,
         the confirmation steps.
         10.10.2   The oxidase test can be performed with a freshly made s
                    available preparation of N,N,N,N-tetramethyl-p-phenyleni
                    (Section 7.10).  If using a freshly made solution, saturate
                    amount of a discreet colony from the nutrient agar to the'oxidai
                    wooden or plastic applicator. A blue/purple color reaction within
                    considered a positive oxidase test.
                                                                                commercially
                                                                                 °A>, aqueous)
                                                                                     a small
        Timing of the color reaction is critical, as some Gram-positivt
        after 10 seconds. Also, it is important to put jusf^mall amajnt of the
        Too much bacteria can also cause a false positive bxidas&jjfst.
     false positives
lyon the oxidase.
         10.10.3   If the oilfc,test is positive, then test fortrekjtose fermentation. Trehalose
                                            .bylnOCuJating a tube containing 3-10 mL (depending on
                                       sed - fill^b(^|yflffull) of 0.5% trehalose in purple broth base
                                          ilony                agar and incubating at 35°C for 24 ± 2
                                            "the medium from purple to yellow is considered a
                   If the
                   fermetttafibi1
                   the size
                   (Section 7.f'
                   hours. A
                   positiv^
        10.10.4    If ajpSny is both ox|ipa^i:iia«l'ft:ehalose positive, report as a confirmed Aeromonas
                   artdParchive the colonjk:for:fti&ier identification. As part of the UCMR monitoring,
                 ,
-------
                                                                      Method 1605 - Aeromonas
11.2
11.0  Data Analysis and Calculations
11.1   See Standard Methods for the Examination of Water and Wastewater (Reference 15.2) for
       general counting rules. The density of Aeromonas determined by the membrane filter (MF)
       procedure is calculated by direct identification and enumeration of yellow colonies by a
       dissecting microscope (Section 6.19) followed by oxidase and trehalose confirmation. Bacterial
       density is recorded as presumptive Aeromonas colony forming units (CPU) per 100 mL of
       sample and confirmed Aeromonas CPU per 100 mL.
       Counting colonies on ADA
       11.2.1    Record the number of presumptive Aeromonas CFU/lOOmL. If there is m
                 one morphological type that is considered to be presumptively positive,
                 number of presumptive positives for each morphological typ^as well
                 number of presumptive positives.
       11.2.2    If there are more than 200 colonies, including backgrourjjppolonies, n
                 too numerous to count (TNTC) and resample. If resampfbig is necejipry, an
                 undiluted 500-mL sample and a minimum of three diluttoiSPsfatQttW be analyzed.
                                                                 ^",; * •* s
                                                                       V^S^i.
       11.2.3    If the colonies are not discrete and appear to be growing tQ$e$fcraSLeport results as
                 confluent growth (CNFG) and resample.
       Confirmation and calculation of Aeromonas density
       11.3.1
11.3
                 All presumptive colonies that are oxidase positivejaft<|ftfnient tret
                 confirmed as Aeromonas. For the final density^^^^m^Aeromon^tj^^ist the
                 initial, presumptive count based on the posiliW€6iu%Si^iaft*^ercentageTOr each
                 presumptively positive morphological typirfind repo^^^lEiffled CPU per 100
                 mL.                               .-"         "
       11.3.2
                 Calculate the number of positive
                 morphological type from allfilh
    No. positively confirmed.,^
  No. submitted to confirmifiarl
                               sresumi
   ations_Jbr each presumptively positive
a given sample using-the following equation:
            = Confirmed Aeromonas / 100mL
                                                 v- «'
                                                 •emonas per 100 mL for each colony
       11.3.3    Record the fl«ffl^-g|conrlrme

                        «/> sP^  " ' ^' ^"<*^1f$^;l;''b '' ",
       11.3.4    SumJ^iiumber o^^^^^^Aeromonas per 100 mL for all presumptively positive
                 coloirf types (Section           report as the density of confirmed Aeromonas per
                 IQp'mL.
                                            13
                                                                         September 2000 - Draft

-------
Method 1605 - Aeromonas
1 1 .3.5
                   Example 1: In this example, 500 mL of sample was filtered and two different
                   morphological types of presumptively positive colonies were observed.
  Example 1
Morphological Description
Type A: Bright yellow, round,
opaque
Type B: Dull yellow, oval,
translucent
No,
presumptive
positive
colonies
30
20
No. submitted to
confirmation
steps
6
4
No.
positively
confirmed
6
3 ^
£-"jJ&>
'HaHj"^*
Total number of confirmed Aeromonas per sample:
No. of confirmed
Aeromonas per 1 00
ml
6
\ .;*
,;S-Sx ,^fl|l'
»

;vtta
V
                    fi     "\   4 nn
                    7"x 30 x —— = 6 Confirmed Type A Aeromonas /  100m
                    6     /   500
                    — x 20  x —— =  3 Confirmed Type B Aeromonas / 100$t
                   Example 1 results in 9 confirmed Aeromona,.
                                                           '-
         11.3.6



  Example 2
          Example 2: In this example, 200 mL of jBpnple wasj
          morphological types of presumptively positive cojifjnies w
   Morphological Description
   Type A: Dull yellow, round,
   Type B: Dull yellow, round,
   translucent
                              No.
                          presumptive
                            posit
                            cc
                              4cr
g^g.,,
8fcEi!i'x40
             ffe). submit!
             "t confirm
                  stepf!

                                                                    :ively
                                              nfirmed Aeromonas per sample:
  No. of confirmed
Aeromonas per 100
       mL
                                                 20
                                                                          12
                                                                     32 per 100 mL
                   t§'x 40) x ^52. = 20 IffnllfTed Type A Aeromonas / 100mL
                                        -
                            —— ^^2 Confirmed Type B Aeromonas / 100mL
                              csults in 32 confirmed Aeromonas 1100 mL.
September 2000 - Draft
                                     14

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                                                                     Method 1605 - Aeromonas
       11.3.7     If there were no presumptively positive colonies or if none of the presumptive
                 colonies are confirmed, then report the results as less than the detection limit (DL) in
                 CPU per 100 mL based on sample volume filtered. If less than 500 mL are filtered,
                 then adjust the reporting limit per 100 mL accordingly. The DL may be calculated as
                 follows:

                 DL  per 100 ml = 100 /volume filtered CPU per 100mL
12.0
12.1
12.2
12.3
12.4


13.0
13.1

13.2
14.0
14.1
              11.3.7.1       Example 3: If 500 mL of sample was filtered end there were
                            confirmed colonies, then report as <0.2 CFU/lOOmL.
              11.3.7.2       Example 4: If 100 mL of sample was filtered         we:
                            confirmed colonies, then report as <1.0 CFU/|w mL.
       Method Performance
       Specificity of media                                          Ji^
       12.1.1  Of the 30 Aeromonas strains tested, 21 grew will on ADA al'35°.
       12.1.2  ADA was able to support the growth of all the species msstrmften as
              disease.                                     x^"
       12.1.3  Efforts continue to identify colonies which giv<;
              media but do not confirm.
       Bias: To be determined through method validatioi
       Precision: To be determined through method
                                             :
       Method performance: To be determined thrgujpi"method.:¥aiidation .ylf
Pollution
The solutions and
recycled and managi
Solutions and reagents
the volume
                                                                                   human
                                                                        ive on the ADA
                                   in this            little threat to the environment when
                                           in^fff||iies consistent with laboratory use to minimize
14.2
       Waste Management
       It is the laboratory's responsibility to comply with all federal, state, and local regulations
       governing wMe management; particularly the biohazard waste identification rules and land
       disposal*re$||ftt;tdtts, ^dMjfrotect the air, water, and land by minimizing and controlling all
       releases ftijfa fume Ke^dsand bench operations. Compliance with all sewage discharge permits
       and regulation's is also required.
       Samples, reference materials, and equipment known or suspected of having bacterial
       contamination from this work must be sterilized prior to disposal.
                                            15
                                                                        September 2000 - Draft

-------
Method 1605 -Aeromonas
  14.3   For further information on waste management, consult "The Waste Management Manual for
         Laboratory Personnel" and "Less is Better: Laboratory Chemical Management for Waste
         Reduction", both of which are available from the American Chemical Society's Department of
         Government Relations and Science Policy, 1155  16th Street N.W., Washington,  D.C. 20036.
  15.0
  15.1


  15.2



  15.3

  15.4

  15.5

  15.6

  15.7

  15.8

  15.9



  15.10
References
Havelaar, A.H., M. During, and J.F.M. Versteegh. 1987. Ampicillin-dextrin agar medium for the
enumeration of Aeromonas species in water by membrane filtration. Journal of Applied
Microbiology. 62:279-287.
Standard Methods for the Examination of Water and Wastewater.
Eaton, L.S. Clesceri, and A. Greenberg. American Public Health AssodJjfflBri, Ameri
Works Association, and Water Environment Federation. American Pii4*c Health '
Washington, D.C., publisher.                                  fi
Demarta, A., M. Tonolla, A. Caminada, N. Ruggeri, and R. Peduzzi
within the  16S  rDNA sequences of Aeromonas popoffii. FEMS Mic:
Annual Book of ASTM Standards, Vol. 11.01. American Society for
Philadelphia, PA 19103.
Manual for the Certification of Laboratories Analyzing DrinkingJ&ter. 1997
815-B-97-001.  Office of Ground Water and Drinking
Moyer, N.  P. 1996. Isolation and enumeration of aeromojiia#T%'"^SIki&ls Aero,
Austin, M. Altwegg, P. Gosling, and S.  Joseph. John 1
                                             jil
Reagent Chemicals, American Chemical Society Sj^cificationC
Washington, D.C.                        J
Handfield, M.,  P. Simard, andR. Letarte. 19^pirffereni
waterborne Aeromonas hydrophila. Applied^vironm
Standard Methods forthe Examinationlof Water and
Eaton, L.S. Clescer^^l)^. Greenbfrt^tfiilrican Pul
Works AssociatidMte^ater Env'^
Washington,
Janda, J.M. and S.L. Ab'
expanding panorama
Clinical
                                          ature region
                                           172:239-246.
                                           .Materials.
                                                     PA-
                                              _jr
                                              ;%5. Eds. B.
                                             Chester, U.K.
                                               Society,
                         media fo| quantitative recovery of
                         Microbjpiogy 62:3544-3547.
                           ' If?995. 19th Edition. Eds. A.D.
                             Association, American Water
             federation. Alnerican Public Health Association,
  ", Evolving concepts regarding the genus Aeromonas: an
i|jd||?age presertfjajtions , and unanswered questions. Journal of
September 2000 - Draft
           16

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                                                        Method 1605 - Aeromonas
16.0  Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Growth of ATCC cultures on ADA at 30° and 35°C in 24 hrs
Collection #
ATCC 7966
ATCC 35654
AMC 12723-W
ATCC 51 108
AMC 14228-V
ATCC 3S6582
AMC 15228-V
ATCC 15468
MML 1685-E
ATCC 33907
AMC Leftwich
ATCC 233092
ATCC 35993

Muldoon SMHC
ATCC 9071
AMC1123-W
ATCC 437003
AMC1108-W
ATCC 49657"
NMRI 206
ATCC 5 1208
ATCC 49568
ATCC 49569
ATCC 35622
WR4659 j~
CECT 4342 **
LMG 175415
AMC (ATCC
35941)
Hybridization group
Group 1
Group 1
Group 1
Group 2
Group 2
Group 3
Group 3
Group 4
Group 4
Group 5
Group 5
Group 6
Group 7

Group 7
Group 8
Group 8
Group 12
, „» jft
!.GmuRi12 ^j?£
*^'
^iff*^" .
!"xi > -~
-•'?Group 9
v' ^fMr
!>4C"
-erf Group 9 :f
'•j| Group 10 ,,-:*
*•' <
-------
Method 1605 - Aeromonas
Collection #
AMC (ATCC
43946)
CDC 0434-84
Hybridization group
-
Group 3
Aeromonas species
Group 501
Motile Group 3
Growth at 30°C
+
+
Growth at 35°C
+1
+1
  (1) Organisms displayed brighter yellow color and/or were larger on ADA media at 35°C
  (2) Respective Aeromonas cultures grew on ADA medium at 35°C when streaked, but not when filtered.
  (3) Respective Aeromonas cultures grew when streaked on ADA medium, however filtration was not performed
  with these cultures.
  (4) Respective Aeromonas cultures did not grow on ADA medium when streaked.
  (5) Respective Aeromonas cultures grew poorly on ADA medium at both temperatures.
  Data for empty cells will be incorporated once analyses at different temperatures are com]
  ATCC = American Type Culture Collection, Rockville, MD. Other cultures were obtaiiM&lJlam Arrf
  University of Maryland. The wild type was obtained from Gene Rice, U.S.  EPA. All
  slant, and transferred to 100 mL of buffered dilution water. Serial dilutions represeni
  were filtered and the membrane placed on ADA medium as described in Section 10
  representing the same dilution for each of the respective cultures were placed on bn
  control. All  organisms tested oxidase and trehalose positive except the ATCC 35941
  Amy Carnahan.
  Table 2. Aeromonas recovery based on holding time and the addition of EDTA
        Species
   A. caviae
   A. hydrophila
   A. veronii/sobria
   A. bestiarum
                      Preservation
                          State
                        w/o EDTA
                        w/ EDTA
                        w/o EDTA
                        w/o EDTA
                        w/
                                                                            »,in-
                                                                             ures were
                                                                             approximate
                                                                            Iditional rae»
                                                                                   " K>n agar as a
                                                                                  fpositive culture from'
                                          HourO
                                         100
                                         100
                                         1 00
                                           100^4*

                                                        Hour 6
&*—
I.,'143
67
                                                           92
                                                         -| 20
                100
32
                                                                           86
                                                                          58
                                                                          52
                                                                         105
                302
                               Hour 30
                                 24
                 88
                                      28
                                                                                           92
                                 43
                                 58
                                 82
                 285
For the holding time studjpconducted for this
using ADA media stogM'with the preservative
Aeromonas were anaj|ised: A. caviae, A. hydr<
caviae and A. hydrtrnjiijia, indicating that wi
drop at 24 and 30 ||g^WhiIe those pre
                                                 afl samples were stored at 10°C. Results were compared
                                             TlPFwim those held with no preservative. Four species of
                                             ila, A. veronii/sobria and A. bestiarum. Results were similar for A.
                                           io preservative there was a slight drop at 6 hours and a significant
                                           with EDTA remained fairly stable throughout the 30 hour holding
  time. However fo^^/sobriaj^ple preserved with EDTA saw a 50% loss by Hour 6, and then remained
  stable (at the 50%1oss)i^S-HoU|S)pfn contrast, the non-preserved sample stayed fairly stable for the first 6 hours
  then exhibited a SO^fogg after one "day. The density of unpreserved A. bestiarum remained fairly stable, while A.
  bestiarum preserved with EDTA showed increased densities.
September 2000 - Draft
                                                 18

-------

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-------
Method 1605 -Aeromonas
  17.0  Glossary
  17.1   Symbols
°c
f^m
±
<
%
degrees Celsius
micrometer
plus or minus
less than
percent
  17.2   Alphabetical characters and acronyms
                              American Society for Testing and Materials
                              American Type Culture Collection
                              Code of Federal Regulations
                              ethylenediaminetetraacetic acid
                              gram
                              liter
                              milligram
                              milliliter
                              millimeter
                              sodium thiosulfate
                              National Institute of Standards an
                              Occupational Safety and Health i
                              pounds per square inch
                              quality control
                              too numerous to count
                              United States Environmental Prote
                              "times"
              ASTM
              ATCC
              CFR
              EDTA
              g
              L
              mg
              mL
              mm
              Na2S2O3
              NIST
              OSHA
              psi
              QC
              TNTC
              USEPA
              X

17.3   Definitions

Method blank—A lOO-i
through all portions of the
used to determine if the samp
through poor technique.
  Must—This action, activity?6r proced

  Negative control—J^.npn-Aeromonas bact|tia tfiSt is streaked onto ADA agar so that the analyst can
  become familiar wjjjijthe color and morphology" of non-Aeromonas bacteria.

  Positive control-J?Hl|00-mL reagent w|ter spiked with 20 - 80 CPU of Aeromonas. The positive control
  is analyzed exac|^p^^^ampleJM|purpose is to assure that the results produced by the laboratory
  remain within th^j^sfengpecifieStir?this method for precision and recovery.

  Selective medium—A culture medium designed to suppress the growth of unwanted microorganisms and
  encourage the growth of the target bacteria.

  Should—This action, activity, or procedural step is suggested but not required.
                                                 'ater that is'lrefated exactly as a sample and carried
                                                      negative or positive. The method blank is
                                                       the introduction of a foreign microorganism
September 2000 - Draft
                                             20

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                                                                        Method 1605 - Aeromonas
Presumptive positive colonies—Aeromonas spp. that grow as yellow colonies on ampicillin-dextrin agar.

Confirmed colonies—Presumptively positive colonies that test positive for oxidase and ferment
trehalose.
                                                       '•i*"
                                             21
September 2000 - Draft

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