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
-------
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
-------
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
-------
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
-------
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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