1、Designation: D5392 93 (Reapproved 2006)D5392 14Standard Test Method forIsolation and Enumeration of Escherichia Coli in Water bythe Two-Step Membrane Filter Procedure1This standard is issued under the fixed designation D5392; the number immediately following the designation indicates the year oforig
2、inal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method describes a membrane filter (MF) procedure for
3、 the detection and enumeration of Escherichia coli, abacterium found exclusively in the feces of humans and other warm-blooded animals. The presence of these microorganisms inwater is an indication of fecal pollution and the possible presence of enteric pathogens. These bacteria are found in water a
4、ndwastewater 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 theusers responsibility to ensure the validity o
5、f this test method for waters of other types.1.3 The values stated in SI units are to be regarded as standard.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safet
6、y and health practices and determine the applicability of regulatorylimitations prior to use. For specific hazard statements, see Section 9.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD3370 Practices for Sampling Water from Close
7、d ConduitsD3870 Practice for Establishing Performance Characteristics for Colony Counting Methods in Microbiology (Withdrawn 2000)3D5465 Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods3. Terminology3.1 DefinitionsFor definitions of terms used in this test met
8、hod, refer to Terminology D1129.3.1 Definitions:3.1.1 For definitions of terms used in this test method, refer to Terminology D1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 Escherichia coli (E. coli)coli), na species of bacteria that is a member of the total coliform group and known
9、tooriginate in the feces of warm-blooded animals.3.3 Performance Characteristics (Practice D3870):3.3.1 accuracy, nthe proportion of the observed count to the true density of a sample.3.3.2 bias, nthe persistent positive or negative deviation of the average value of the test method from the assumed
10、or acceptedtrue value.3.3.3 precisionprecision, nthe degree of agreement of repeated measurements of the same parameter expressedquantitatively as the standard deviation or as the 95 % confidence limits of the mean computed from the results of a series ofcontrolled determinations.1 This test method
11、is under the jurisdiction of ASTM Committee D19 on Water and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved July 1, 2006Aug. 15, 2014. Published July 2006August 2014. Originally approved in 1993. Last previous edition approved in 20002006 asD5392 9
12、3 (2000).(2006). DOI: 10.1520/D5392-93R06. 10.1520/D5392-14.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.3
13、 The last approved version of this historical standard is referenced on www.astm.org.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to a
14、dequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Consho
15、hocken, PA 19428-2959. United States13.3.2 biasthe persistent positive or negative deviation of the average value of the test method from the assumed or acceptedtrue value.3.3.4 specificityspecificity, n the ability of a test method to select or distinguish, or both, the target bacteria in the same
16、watersample; the specificity characteristic of the method is usually reported as the percent of false positive and false negative results.3.3.5 upper counting limit (UCL)(UCL), nthat colony count above which there is an unacceptable counting error; the errormay be due to overcrowding or antibiosis.3
17、.3.5 accuracythe proportion of the observed count to the true density of a sample.4. Summary of Test Method4.1 This two-step test method4 provides a direct count of bacterial colonies developing on the surface of the filter when placedon a selective nutrient medium. The water sample is passed throug
18、h a membrane filter that retains the bacteria.After filtration, themembrane filter containing the bacterial cells is placed on a selective, differential medium, mTEC. The membrane on the mediumis first incubated at 35C for 2 h so that injured or stressed bacteria can be resuscitated and then the med
19、ium is incubated at 44.5Cfor 22 h. Following incubation the filter is transferred to a filter pad saturated with urea substrate. After 15 min all yellow oryellow-brown colonies are counted with the aid of 10 to 15 magnifier and a fluorescent lamp.5. Significance and Use5.1 This test method is useful
20、 for measuring recreational water quality and chlorinated wastewaters, although it can be used forany water suspected of contamination by fecal wastes of warm-blooded animals. The significance of finding E. coli in recreationalwater samples, especially samples obtained from fresh recreational waters
21、, is that there is a risk of gastrointestinal illness, directlyrelated to the E. coli density, associated with swimming.55.2 Since small or large volumes of water or dilutions thereof can be analyzed by the MF technique, a wider range of levelsof E. coli in water can be detected and enumerated than
22、with other methods.6. Interferences6.1 Water with high levels of colloidal or suspended materials can clog the membrane filter pores and prevent filtration. Also,suspended materials cause spreading colonies that could interfere with target colonies and thereby prevent accurate counting.6.2 Smaller s
23、ample size or sample dilution can be used to minimize the interference of turbidity or high background (nontarget)bacterial densities. Replicates of sample volumes or dilutions of sample may be filtered and the results combined. However, themembrane filter techniques may not be applicable to high tu
24、rbid waters with low bacterial densities.6.3 In some samples, chemicals may have toxic effects on the target organism.7. Apparatus7.1 Stereoscopic Microscope, wide-field type with magnification of 10 to 15.7.2 Microscope Lamp, producing diffuse light from a cool, white fluorescent lamp adjusted to g
25、ive maximum visibility.7.3 Counting Device, hand tally or electronic.7.4 Pipet Container, stainless steel, aluminum, or borosilicate glass, for glass pipets.7.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, of appropriate volume.7.6 Graduated Cylinders, 100 to 1000 mL, covered wit
26、h aluminum foil or kraft paper and sterile.7.7 Membrane Filtration Units (filter base and funnel), glass, plastic, or stainless steel, wrapped in aluminum foil or kraft paperand sterilized.7.8 Ultraviolet Unit, for sterilizing the filtration unit (optional).7.9 Line Vacuum, Electric Vacuum Pump, or
27、Aspirator, for use as a vacuum source. In an emergency or in the field, a hand pumpor a syringe equipped with a check valve to prevent the return flow of air, can be used.7.10 Flask, filter, vacuum, usually 1 L, with appropriate tubing. A filter manifold to hold a number of filter bases is optional.
28、7.11 Forceps, straight or curved, with smooth tips to handle filters without damage.4 Dufour, A., Strickland, E., and Cabelli, V., “Membrane Filter Method for Enumerating Escherichi coli,” Appl. and Environ. Microbiol. 41:11521158, 1981. Dufour, A.,Strickland, E., and Cabelli, V., “Membrane Filter M
29、ethod for Enumerating Escherichi coli,” Applied and Environmental Microbiology, Vol 41, 1981, pp. 11521158.5 Cabelli, V. J., Dufour, A. P., Levin, M. A., McCabe, L. J., and Haberman, P. W., “Relationship of Microbial Indicators to Health Effects at Marine Bathing Beaches,”American Journal of Public
30、Health, 69:690696, 1979.Cabelli, V. J., Dufour, A. P., Levin, M. A., McCabe, L. J., and Haberman, P. W., “Relationship of Microbial Indicatorsto Health Effects at Marine Bathing Beaches,” American Journal of Public Health, Vol 69, 1979, pp. 690696.D5392 1427.12 Thermometer, checked against a Nationa
31、l Institute of Standards and Technology (NIST) certified thermometer, or onetraceable to a NIST thermometer.7.13 Petri Dishes, sterile, plastic, 50 by 12 mm, with tight-fitting lids.7.14 Bottles, milk dilution, borosilicate glass, screw-cap with neoprene liners, marked at 99 mL for 1 to 100 dilution
32、s. Dilutionbottles marked at 90 mL or tubes marked at 9 mL may be used for 1 to 10 dilutions.7.15 Inoculation Loops, at least 3-mm diameter, and needles, nichrome or platinum wire, 26 B the MF is now held between the funneland the base.12.3 Shake the sample bottle vigorously about 25 times to distri
33、bute the bacteria uniformly and measure the desired volume ofsample or dilution into the funnel.12.4 For ambient surface waters and wastewaters, select sample volumes based on previous knowledge to produce membranefilters with 20 to 80 colonies. Sample volumes are normally tested at half log interva
34、ls, for example 100, 30, 10, 3 mL, etc.12.5 Use smaller sample size or sample dilution to minimize the interference of turbidity or high bacterial densities. Multiplevolumes of the same sample or dilution of sample may be filtered and the results combined.12.6 Filter the sample and rinse the sides o
35、f the funnel at least twice with 20 to 30 mL of buffered rinse water. Turn off thevacuum and remove the funnel from the filter base.12.7 Use sterile forceps to aseptically remove the membrane from the filter base and roll it onto the mTEC medium to avoidthe formation of bubbles between the membrane
36、and the agar surface. Reseat the membrane if bubbles occur. Close the dish, invert,and place it in a 35C incubator for 2 h.12.8 After two h incubation, place the plates in resealable water-proof plastic bags and transfer them to a 44.5C incubator.Incubate the plates at 44.5 6 0.2C for 22 to 24 h.7 B
37、ordner, R. H., Winter, J. A., and Scarpino, P. V., Eds., Microbiological Methods for Monitoring the Environment, Water, and Wastes, EPA-600/8-78-017, U.S.Environmental Protection Agency, Office of Research and Development, Environmental Monitoring and Support LaboratoryCincinnati, Cincinnati, Ohio,
38、1978.D5392 14512.9 After incubation, remove the plates from the waterbath and aseptically transfer the membrane to a filter pad saturated withurea substrate medium. After 15 to 20 min incubation at room temperature, examine the membrane for yellow to yellow-browncolonies. Count and record the number
39、 of typical colonies.13. Calculation13.1 Use the following rule to calculate the E. coli count per 100 mL of sample. If more than one sample volume containscolonies, select the membrane filter with 20 to 80 and calculate the count per 100 mL according to the general formula:E.coli/100 mL5 No. E.coli
40、 colonies countedVolume in mL of sample filtered3100 mL13.2 See Practice D5465.14. Verification Procedure14.1 Yellow or yellow-brown colonies from the urease test can be verified as E. coli. Verification of colonies may be requiredin evidence gathering, and is also recommended as a QC procedure with
41、 initial use of the test and with changes in sample sites,lots of commercial media or major ingredients in media compounded in the laboratory. The verification procedure follows:14.1.1 Using a sterile inoculation loop, transfer growth from the centers of at least 10 well-isolated typical colonies to
42、 nutrientagar plates or slants and to tryptic soy broth. Incubate the agar and broth cultures for 24 h at 35C.14.1.2 After incubation remove a generous portion of material from the nutrient agar with a platinum loop and deposit on thesurface of filter paper that has been saturated with cytochrome ox
43、idase reagent prepared fresh that day or use the commercialproduct. A positive test is indicated within 15 s by the development of a deep purple color where the bacteria were deposited.14.1.3 Transfer growth from the tryptic soy broth to Simmons citrate agar, tryptone broth, and EC broth in fermenta
44、tion tubes.Incubate the Simmons citrate agar for 24 h and tryptone broth for 48 h at 35C. Incubate the EC broth at 44.5C in a waterbathfor 24 h. The water level must be above the level of the EC broth in the tube. Add one-half mL of Kovacs indole solution to the48-h tryptone broth culture and shake
45、the tube gently. A positive test for indole is indicated by a deep red color that develops inthe alcohol layer. E. coli is EC gas positive, indole positive, oxidase negative, and does not grow on citrate medium.14.1.4 Alternatively, use commercially available multi-test identification systems to ver
46、ify colonies. Inoculate the colonies intoan identification for Enterobacteriaceae that includes lactose fermentation and/or o-nitrophenyl-Dgalactopyrano-oro-nitrophenyl-Dgalactopyrano-sideside (ONPG) (ONPG), or both, and cyto-chrome oxidase test tower case reactions.15. Report15.1 Adjust counts base
47、d on verification and report the results as E. coli per 100 mL of sample.16. Precision and Bias16.1 Single Laboratory Data:416.1.1 PrecisionThe mTEC method precision was found to be fairly representative of what would be expected from countswith a Poisson distribution.16.1.2 BiasThe bias of the E. c
48、oli MF with mTEC medium method has been reported to be 2 %be 2 % of the true value.16.1.3 Other Statistics:16.1.3.1 SpecificityThe specificity of mTEC medium as reported for various environmental samples was 9 % false positiveand less than 1 % false negative.16.1.3.2 Upper Counting LimitThe upper co
49、unting limit for E. coli on mTEC medium has been reported as 80 colonies perfilter.16.2 Collaborative Study Data:16.2.1 Acollective study was conducted among eleven volunteer laboratories, each with two analysts who independently testedreagent water, local fresh and marine recreational waters and sewage treatment plant effluent samples, in duplicate.16.2.2 The results of the study are shown in Fig. 1 where So equals the pooled standard deviation among replicate counts froma single analyst for three groupings (counts less than 30, count
