ASTM D5392-93(2000)
(Test Method)Standard Test Method for Isolation and Enumeration of Escherichia Coli in Water by the Two-Step Membrane Filter Procedure
Standard Test Method for Isolation and Enumeration of <i>Escherichia Coli</i> in Water by the Two-Step Membrane Filter Procedure
SCOPE
1.1 This test method describes a membrane filter (MF) procedure for the detection and enumeration of Escherichia coli , a bacterium found exclusively in the feces of humans and other warm-blooded animals. The presence of these microorganisms in water is an indication of fecal pollution and the possible presence of enteric pathogens. These bacteria are found in water and wastewater in a wide range of densities. The detection limit of this procedure is one colony forming unit (CFU) per volume filtered.
1.2 This test method has been used successfully with temperate fresh and marine ambient waters, and wastewaters. It is the user's responsibility to ensure the validity of this test method for waters of other types.
1.3 This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. For specific hazard statements, see Section 9.
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Designation:D5392–93(Reapproved2000)
Standard Test Method for
Isolation and Enumeration ofEscherichia Coli in Water by
the Two-Step Membrane Filter Procedure
This standard is issued under the fixed designation D5392; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 3.2 Definitions of Terms Specific to This Standard:
3.2.1 Escherichia coli (E. coli)—aspeciesofbacteriathatis
1.1 This test method describes a membrane filter (MF)
amemberofthetotalcoliformgroupandknowntooriginatein
procedure for the detection and enumeration of Escherichia
the feces of warm-blooded animals.
coli, a bacterium found exclusively in the feces of humans and
3.3 Performance Characteristics (Practice D 3870)
other warm-blooded animals. The presence of these microor-
3.3.1 precision—the degree of agreement of repeated mea-
ganisms in water is an indication of fecal pollution and the
surements of the same parameter expressed quantitatively as
possible presence of enteric pathogens. These bacteria are
the standard deviation or as the 95% confidence limits of the
foundinwaterandwastewaterinawiderangeofdensities.The
mean computed from the results of a series of controlled
detection limit of this procedure is one colony forming unit
determinations.
(CFU) per volume filtered.
3.3.2 bias—the persistent positive or negative deviation of
1.2 This test method has been used successfully with
the average value of the test method from the assumed or
temperatefreshandmarineambientwaters,andwastewaters.It
accepted true value.
is the user’s responsibility to ensure the validity of this test
3.3.3 specificity— the ability of a test method to select or
method for waters of other types.
distinguish, or both, the target bacteria in the same water
1.3 This standard does not purport to address all of the
sample; the specificity characteristic of the method is usually
safety concerns, if any, associated with its use. It is the
reported as the percent of false positive and false negative
responsibility of the user of this standard to establish appro-
results.
priate safety and health practices and determine the applica-
3.3.4 upper counting limit (UCL)—that colony count above
bility of regulatory limitations prior to use. For specific hazard
whichthereisanunacceptablecountingerror;theerrormaybe
statements, see Section 9.
due to overcrowding or antibiosis.
2. Referenced Documents
3.3.5 accuracy—theproportionoftheobservedcounttothe
true density of a sample.
2.1 ASTM Standards:
D1129 Terminology Relating to Water
4. Summary of Test Method
D1193 Specification for Reagent Water
4.1 This two-step test method provides a direct count of
D3370 Practices for Sampling Water from Closed Con-
bacterial colonies developing on the surface of the filter when
duits
placed on a selective nutrient medium. The water sample is
D3870 Practice for Establishing Performance Characteris-
passedthroughamembranefilterthatretainsthebacteria.After
tics for Colony Counting Methods in Microbiology
filtration, the membrane filter containing the bacterial cells is
D5465 Practice for Determining Microbial Counts from
placed on a selective, differential medium, mTEC. The mem-
Waters Analyzed by Plating Methods
brane on the medium is first incubated at 35°C for2hso that
3. Terminology
injured or stressed bacteria can be resuscitated and then the
medium is incubated at 44.5°C for 22 h. Following incubation
3.1 Definitions—For definitions of terms used in this test
the filter is transferred to a filter pad saturated with urea
method, refer to Terminology D1129.
substrate.After15minallyelloworyellow-browncoloniesare
counted with the aid of 10 to 153 magnifier and a fluorescent
lamp.
This test method is under the jurisdiction ofASTM Committee D19 on Water
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved April 15, 1993. Published September 1993.
Annual Book of ASTM Standards, Vol 11.01. Dufour, A., Strickland, E., and Cabelli, V., “Membrane Filter Method for
Annual Book of ASTM Standards, Vol 11.02. Enumerating Escherichi coli,” Appl. and Environ. Microbiol. 41:1152–1158, 1981.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D5392–93 (2000)
5. Significance and Use 7.11 Forceps, straight or curved, with smooth tips to handle
filters without damage.
5.1 This test method is useful for measuring recreational
7.12 Thermometer, checked against a National Institute of
water quality and chlorinated wastewaters, although it can be
Standards and Technology (NIST) certified thermometer, or
used for any water suspected of contamination by fecal wastes
one traceable to a NIST thermometer.
ofwarm-bloodedanimals.Thesignificanceoffinding E. coliin
7.13 Petri Dishes, sterile, plastic, 50 by 12 mm, with
recreational water samples, especially samples obtained from
tight-fitting lids.
fresh recreational waters, is that there is a risk of gastrointes-
7.14 Bottles, milk dilution, borosilicate glass, screw-cap
tinal illness, directly related to the E. coli density, associated
with neoprene liners, marked at 99 mL for 1 to 100 dilutions.
with swimming.
Dilutionbottlesmarkedat90mLortubesmarkedat9mLmay
5.2 Since small or large volumes of water or dilutions
be used for 1 to 10 dilutions.
thereof can be analyzed by the MF technique, a wider range of
7.15 Inoculation Loops, at least 3-mm diameter, and
levels of E. coli in water can be detected and enumerated than
needles,nichromeorplatinumwire,26B&Sgage,insuitable
with other methods.
holders.
7.16 Incubator, air, maintained at 35 6 0.5°C.
6. Interferences
7.17 Incubator, Waterbath, maintained at 44 to 46°C.
6.1 Water with high levels of colloidal or suspended mate-
7.18 Test Tubes,150by20mm,borosilicateglassorplastic.
rials can clog the membrane filter pores and prevent filtration.
7.19 Test Tubes, 75 by 10 mm, borosilicate glass.
Also, suspended materials cause spreading colonies that could
7.20 Caps, aluminum or autoclavable plastic, for 20 mm
interfere with target colonies and thereby prevent accurate
diameter test tubes.
counting.
7.21 Test Tubes, screw-cap, borosilicate glass, 125 by 16
6.2 Smaller sample size or sample dilution can be used to
mm or other appropriate size.
minimize the interference of turbidity or high background
(nontarget) bacterial densities. Replicates of sample volumes
8. Reagents and Materials
ordilutionsofsamplemaybefilteredandtheresultscombined.
8.1 Purity of Reagents—Reagent grade chemicals shall be
However, the membrane filter techniques may not be appli-
used in all tests. Unless otherwise indicated, it is intended that
cable to high turbid waters with low bacterial densities.
all reagents conform to the specifications of the Committee on
6.3 In some samples, chemicals may have toxic effects on
Analytical Reagents of theAmerican Chemical Society where
the target organism.
such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently
7. Apparatus
high purity to permit its use without lessening the accuracy of
7.1 Stereoscopic Microscope, wide-field type with magnifi-
the determination. The agar used in preparation of culture
cation of 10 to 153.
media must be of microbiological grade. Whenever possible,
7.2 Microscope Lamp, producing diffuse light from a cool,
usecommercialculturemediaandreagentsasmeansofquality
white fluorescent lamp adjusted to give maximum visibility.
control.
7.3 Counting Device, hand tally or electronic.
8.2 Purity of Water— Unless otherwise indicated, refer-
7.4 Pipet Container, stainless steel, aluminum, or borosili-
ences to water shall be understood to mean reagent water as
cate glass, for glass pipets.
defined by Type III of Specification D1193.
7.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or
8.3 Ethanol, Methanol, or Isopropanol, denatured, in a
plastic, of appropriate volume.
small, wide-mouth container, for flame-sterilization or pipets.
7.6 Graduated Cylinders, 100 to 1000 mL, covered with
8.4 Membrane Filters, sterile, white, grid marked, 47-mm
aluminum foil or kraft paper and sterile.
diameter,with0.45 60.02µmporesizeorotherporesizesfor
7.7 Membrane Filtration Units (filter base and funnel),
which the manufacturer provides data demonstrating equiva-
glass, plastic, or stainless steel, wrapped in aluminum foil or
lency.
kraft paper and sterilized.
8.5 Buffered Dilution Water/Buffered Rinse Water:
7.8 Ultraviolet Unit, for sterilizing the filtration unit (op-
8.5.1 Composition Per Litre:
tional).
7.9 Line Vacuum, Electric Vacuum Pump, or Aspirator, for
Sodium Dihydrogen Phosphate (NaH PO)0.58g
2 4
useasavacuumsource.Inanemergencyorinthefield,ahand
Sodium Monohydrogen Phosphate (Na HPO)2.50g
2 4
Sodium Chloride 8.50 g
pump or a syringe equipped with a check valve to prevent the
return flow of air, can be used.
8.5.2 Preparation—Dissolvetheingredientsin1Lofwater
7.10 Flask, filter, vacuum, usually 1 L, with appropriate
in a flask and dispense in appropriate amounts for dilutions in
tubing. A filter manifold to hold a number of filter bases is
optional.
Reagent Chemicals, American Chemical Society Specifications, American
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
listed by the American Chemical Society, see Analar Standards for Laboratory
Cabelli,V.J.,Dufour,A.P.,Levin,M.A.,McCabe,L.J.,andHaberman,P.W., Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
“RelationshipofMicrobialIndicatorstoHealthEffectsatMarineBathingBeaches,” and National Formulary,U.S.PharmaceuticalConvention,Inc.(USPC),Rockville,
American Journal of Public Health, 69:690–696, 1979. MD.
D5392–93 (2000)
screw-cap bottles or culture tubes, and/or into containers for holding time limits is critical to the production of valid data.
use as rinse water.Autoclave after preparation at 121°C for 15 Samples not collected according to these rules should not be
min. The final pH is 7.4. analyzed.
8.6 mTEC Agar: 10.2 Sample Storage Temperature and Handling Condi-
8.6.1 Composition Per Litre: tions:
10.2.1 Iceorrefrigeratewatersamplesatatemperatureof1
to4°Cduringtransittothelaboratory.Useinsulatedcontainers
Proteose Peptone 5.0 g
Yeast Extract 3.0 g
toensurepropermaintenanceofstoragetemperature.Takecare
Lactose 10.0 g
that sample bottles are not totally immersed in water during
Sodium Chloride 7.5 g
Dipotas
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