ASTM D5392-93(2006)

Standard Test Method for Isolation and Enumeration ofEscherichia Coli in Water by the Two-Step Membrane Filter Procedure

SIGNIFICANCE AND USE
This test method is useful for measuring recreational water quality and chlorinated wastewaters, although it can be used for any water suspected of contamination by fecal wastes of warm-blooded animals. The significance of finding E. coli  in recreational water samples, especially samples obtained from fresh recreational waters, is that there is a risk of gastrointestinal illness, directly related to the E. coli  density, associated with swimming.4  
Since small or large volumes of water or dilutions thereof can be analyzed by the MF technique, a wider range of levels of E. coli in water can be detected and enumerated than with other methods.
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.
This standard does not purport to address all of the safety concerns, 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.

General Information

• Status: Historical
• Publication Date: 30-Jun-2006
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: D19 - Water
• Drafting Committee: D19.24 - Water Microbiology

Relations

• Replaces: ASTM D5392-93(2000) - Standard Test Method for Isolation and Enumeration of <i>Escherichia Coli</i> in Water by the Two-Step Membrane Filter Procedure
• Effective Date: 01-Jul-2006
• Replaced By: ASTM D5392-14 - Standard Test Method for Isolation and Enumeration of <emph type="bdit">Escherichia Coli</emph> in Water by the Two-Step Membrane Filter Procedure
• Effective Date: 15-Aug-2014
• Referred By: ASTM D8506-23 - Standard Guide for Microbial Contamination and Biodeterioration in Turbine Oils and Turbine Oil Systems
• Effective Date: 01-Jul-2006
• Referred By: ASTM D5465-16(2020) - Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Jul-2006

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D5392 − 93(Reapproved 2006)
Standard Test Method for
Isolation and Enumeration of Escherichia 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Terminology
1.1 This test method describes a membrane filter (MF) 3.1 Definitions—For definitions of terms used in this test
procedure for the detection and enumeration of Escherichia method, refer to Terminology D1129.
coli, a bacterium found exclusively in the feces of humans and
3.2 Definitions of Terms Specific to This Standard:
other warm-blooded animals. The presence of these microor-
3.2.1 Escherichia coli (E. coli)—aspeciesofbacteriathatis
ganisms in water is an indication of fecal pollution and the
amemberofthetotalcoliformgroupandknowntooriginatein
possible presence of enteric pathogens. These bacteria are
the feces of warm-blooded animals.
foundinwaterandwastewaterinawiderangeofdensities.The
3.3 Performance Characteristics (Practice D3870)
detection limit of this procedure is one colony forming unit
3.3.1 precision—the degree of agreement of repeated mea-
(CFU) per volume filtered.
surements of the same parameter expressed quantitatively as
1.2 This test method has been used successfully with
the standard deviation or as the 95% confidence limits of the
temperatefreshandmarineambientwaters,andwastewaters.It
mean computed from the results of a series of controlled
is the user’s responsibility to ensure the validity of this test
determinations.
method for waters of other types.
3.3.2 bias—the persistent positive or negative deviation of
1.3 This standard does not purport to address all of the
the average value of the test method from the assumed or
safety concerns, if any, associated with its use. It is the
accepted true value.
responsibility of the user of this standard to establish appro-
3.3.3 specificity— the ability of a test method to select or
priate safety and health practices and determine the applica-
distinguish, or both, the target bacteria in the same water
bility of regulatory limitations prior to use. For specific hazard
sample; the specificity characteristic of the method is usually
statements, see Section 9.
reported as the percent of false positive and false negative
results.
2. Referenced Documents
2 3.3.4 upper counting limit (UCL)—that colony count above
2.1 ASTM Standards:
whichthereisanunacceptablecountingerror;theerrormaybe
D1129Terminology Relating to Water
due to overcrowding or antibiosis.
D1193Specification for Reagent Water
3.3.5 accuracy—the proportion of the observed count to the
D3370Practices for Sampling Water from Closed Conduits
true density of a sample.
D3870PracticeforEstablishingPerformanceCharacteristics
for Colony Counting Methods in Microbiology (With-
4. Summary of Test Method
drawn 2000)
D5465Practice for Determining Microbial Colony Counts
4.1 This two-step test method provides a direct count of
from Waters Analyzed by Plating Methods
bacterial colonies developing on the surface of the filter when
placed on a selective nutrient medium. The water sample is
passedthroughamembranefilterthatretainsthebacteria.After
This test method is under the jurisdiction ofASTM Committee D19 on Water
filtration, the membrane filter containing the bacterial cells is
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
placed on a selective, differential medium, mTEC. The mem-
CurrenteditionapprovedJuly1,2006.PublishedJuly2006.Originallyapproved
brane on the medium is first incubated at 35°C for2hso that
in 1993. Last previous edition approved in 2000 as D5392–93 (2000). DOI:
10.1520/D5392-93R06.
injured or stressed bacteria can be resuscitated and then the
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. Dufour, A., Strickland, E., and Cabelli, V., “Membrane Filter Method for
The last approved version of this historical standard is referenced on Enumerating Escherichi coli,” Appl. and Environ. Microbiol. 41:1152–1158, 1981.
www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5392 − 93 (2006)
medium is incubated at 44.5°C for 22 h. Following incubation 7.8 Ultraviolet Unit, for sterilizing the filtration unit (op-
the filter is transferred to a filter pad saturated with urea tional).
substrate.After15minallyelloworyellow-browncoloniesare
7.9 Line Vacuum, Electric Vacuum Pump, or Aspirator, for
counted with the aid of 10 to 15× magnifier and a fluorescent
useasavacuumsource.Inanemergencyorinthefield,ahand
lamp.
pump or a syringe equipped with a check valve to prevent the
return flow of air, can be used.
5. Significance and Use
7.10 Flask, filter, vacuum, usually 1 L, with appropriate
5.1 This test method is useful for measuring recreational
tubing. A filter manifold to hold a number of filter bases is
water quality and chlorinated wastewaters, although it can be
optional.
used for any water suspected of contamination by fecal wastes
7.11 Forceps, straight or curved, with smooth tips to handle
ofwarm-bloodedanimals.ThesignificanceoffindingE.coliin
filters without damage.
recreational water samples, especially samples obtained from
fresh recreational waters, is that there is a risk of gastrointes-
7.12 Thermometer, checked against a National Institute of
tinal illness, directly related to the E. coli density, associated
Standards and Technology (NIST) certified thermometer, or
with swimming. one traceable to a NIST thermometer.
5.2 Since small or large volumes of water or dilutions
7.13 Petri Dishes, sterile, plastic, 50 by 12 mm, with
thereof can be analyzed by the MF technique, a wider range of tight-fitting lids.
levels of E. coli in water can be detected and enumerated than
7.14 Bottles, milk dilution, borosilicate glass, screw-cap
with other methods.
with neoprene liners, marked at 99 mL for 1 to 100 dilutions.
Dilutionbottlesmarkedat90mLortubesmarkedat9mLmay
6. Interferences
be used for 1 to 10 dilutions.
6.1 Water with high levels of colloidal or suspended mate-
7.15 Inoculation Loops, at least 3-mm diameter, and
rials can clog the membrane filter pores and prevent filtration.
needles,nichromeorplatinumwire,26B&Sgage,insuitable
Also, suspended materials cause spreading colonies that could
holders.
interfere with target colonies and thereby prevent accurate
7.16 Incubator, air, maintained at 35 6 0.5°C.
counting.
7.17 Incubator, Waterbath, maintained at 44 to 46°C.
6.2 Smaller sample size or sample dilution can be used to
minimize the interference of turbidity or high background
7.18 Test Tubes,150by20mm,borosilicateglassorplastic.
(nontarget) bacterial densities. Replicates of sample volumes
7.19 Test Tubes, 75 by 10 mm, borosilicate glass.
ordilutionsofsamplemaybefilteredandtheresultscombined.
7.20 Caps, aluminum or autoclavable plastic, for 20 mm
However, the membrane filter techniques may not be appli-
diameter test tubes.
cable to high turbid waters with low bacterial densities.
7.21 Test Tubes, screw-cap, borosilicate glass, 125 by 16
6.3 In some samples, chemicals may have toxic effects on
mm or other appropriate size.
the target organism.
8. Reagents and Materials
7. Apparatus
8.1 Purity of Reagents—Reagent grade chemicals shall be
7.1 Stereoscopic Microscope, wide-field type with magnifi-
used in all tests. Unless otherwise indicated, it is intended that
cation of 10 to 15×.
all reagents conform to the specifications of the Committee on
7.2 Microscope Lamp, producing diffuse light from a cool,
Analytical Reagents of theAmerican Chemical Society where
white fluorescent lamp adjusted to give maximum visibility. 6
such specifications are available. Other grades may be used,
7.3 Counting Device, hand tally or electronic.
provided it is first ascertained that the reagent is of sufficiently
high purity to permit its use without lessening the accuracy of
7.4 Pipet Container, stainless steel, aluminum, or borosili-
the determination. The agar used in preparation of culture
cate glass, for glass pipets.
media must be of microbiological grade. Whenever possible,
7.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or
usecommercialculturemediaandreagentsasmeansofquality
plastic, of appropriate volume.
control.
7.6 Graduated Cylinders, 100 to 1000 mL, covered with
8.2 Purity of Water—Unless otherwise indicated, references
aluminum foil or kraft paper and sterile.
to water shall be understood to mean reagent water as defined
by Type III of Specification D1193.
7.7 Membrane Filtration Units (filter base and funnel),
glass, plastic, or stainless steel, wrapped in aluminum foil or
kraft paper and sterilized.
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 (2006)
8.3 Ethanol, Methanol, or Isopropanol, denatured, in a 9.2 Mouth-pipetting is prohibited.
small, wide-mouth container, for flame-sterilization or pipets.
10. Sample Collection, Preservation, and Holding Times
8.4 Membrane Filters, sterile, white, grid marked, 47-mm
10.1 Sampling procedures are described in detail in the
diameter,with0.45 60.02µmporesizeorotherporesizesfor
USEPA microbiological methods manual and in Practices
which the manufacturer provides data demonstrating equiva-
D3370. Adherence to sample preservation procedures and
lency.
holding time limits is critical to the production of valid data.
8.5 Buffered Dilution Water/Buffered Rinse Water:
Samples not collected according to these rules should not be
8.5.1 Composition Per Litre: analyzed.
Sodium Dihydrogen Phosphate (NaH PO)0.58g
2 4
10.2 Sample Storage Temperature and Handling Condi-
Sodium Monohydrogen Phosphate (Na HPO)2.50g
2 4
tions:
Sodium Chloride 8.50 g
10.2.1 Iceorrefrigeratewatersamplesatatemperatureof1
8.5.2 Preparation—Dissolve the ingredients in 1 Lof water
to4°Cduringtransittothelaboratory.Useinsulatedcontainers
in a flask and dispense in appropriate amounts for dilutions in
toe
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