1、Designation: D 5392 93 (Reapproved 2006)Standard Test Method forIsolation and Enumeration ofEscherichia Coli in Water bythe Two-Step Membrane Filter Procedure1This standard is issued under the fixed designation D 5392; the number immediately following the designation indicates the year oforiginal ad
2、option or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method describes a membrane filter (MF)procedure for the de
3、tection and enumeration of Escherichiacoli, a bacterium found exclusively in the feces of humans andother warm-blooded animals. The presence of these microor-ganisms in water is an indication of fecal pollution and thepossible presence of enteric pathogens. These bacteria arefound in water and waste
4、water in a wide range of densities.Thedetection limit of this procedure is one colony forming unit(CFU) per volume filtered.1.2 This test method has been used successfully withtemperate fresh and marine ambient waters, and wastewaters. Itis the users responsibility to ensure the validity of this tes
5、tmethod for waters of other types.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limita
6、tions prior to use. For specific hazardstatements, see Section 9.2. Referenced Documents2.1 ASTM Standards:2D 1129 Terminology Relating to WaterD 1193 Specification for Reagent WaterD 3370 Practices for Sampling Water from Closed ConduitsD 3870 Practice for Establishing Performance Characteris-tics
7、for Colony Counting Methods in Microbiology3D 5465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating Methods3. Terminology3.1 DefinitionsFor definitions of terms used in this testmethod, refer to Terminology D 1129.3.2 Definitions of Terms Specific to This Standard:3.2.
8、1 Escherichia coli (E. coli)a species of bacteria that isa member of the total coliform group and known to originate inthe feces of warm-blooded animals.3.3 Performance Characteristics (Practice D 3870)3.3.1 precisionthe degree of agreement of repeated mea-surements of the same parameter expressed q
9、uantitatively asthe standard deviation or as the 95 % confidence limits of themean computed from the results of a series of controlleddeterminations.3.3.2 biasthe persistent positive or negative deviation ofthe average value of the test method from the assumed oraccepted true value.3.3.3 specificity
10、 the ability of a test method to select ordistinguish, or both, the target bacteria in the same watersample; the specificity characteristic of the method is usuallyreported as the percent of false positive and false negativeresults.3.3.4 upper counting limit (UCL)that colony count abovewhich there i
11、s an unacceptable counting error; the error may bedue to overcrowding or antibiosis.3.3.5 accuracythe proportion of the observed count to thetrue density of a sample.4. Summary of Test Method4.1 This two-step test method4provides a direct count ofbacterial colonies developing on the surface of the f
12、ilter whenplaced on a selective nutrient medium. The water sample ispassed through a membrane filter that retains the bacteria.Afterfiltration, the membrane filter containing the bacterial cells isplaced on a selective, differential medium, mTEC. The mem-brane on the medium is first incubated at 35C
13、 for2hsothatinjured or stressed bacteria can be resuscitated and then themedium is incubated at 44.5C for 22 h. Following incubationthe filter is transferred to a filter pad saturated with ureasubstrate.After 15 min all yellow or yellow-brown colonies arecounted with the aid of 10 to 153 magnifier a
14、nd a fluorescentlamp.1This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved July 1, 2006. Published July 2006. Originally approvedin 1993. Last previous edition approved in 2000
15、as D 5392 93 (2000).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn.4Dufour, A., Strickland, E., a
16、nd Cabelli, V., “Membrane Filter Method forEnumerating Escherichi coli,” Appl. and Environ. Microbiol. 41:11521158, 1981.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5. Significance and Use5.1 This test method is useful for measur
17、ing recreationalwater quality and chlorinated wastewaters, although it can beused for any water suspected of contamination by fecal wastesof warm-blooded animals.The significance of finding E. coli inrecreational water samples, especially samples obtained fromfresh recreational waters, is that there
18、 is a risk of gastrointes-tinal illness, directly related to the E. coli density, associatedwith swimming.55.2 Since small or large volumes of water or dilutionsthereof can be analyzed by the MF technique, a wider range oflevels of E. coli in water can be detected and enumerated thanwith other metho
19、ds.6. Interferences6.1 Water with high levels of colloidal or suspended mate-rials can clog the membrane filter pores and prevent filtration.Also, suspended materials cause spreading colonies that couldinterfere with target colonies and thereby prevent accuratecounting.6.2 Smaller sample size or sam
20、ple dilution can be used tominimize the interference of turbidity or high background(nontarget) bacterial densities. Replicates of sample volumesor dilutions of sample may be filtered and the results combined.However, the membrane filter techniques may not be appli-cable to high turbid waters with l
21、ow bacterial densities.6.3 In some samples, chemicals may have toxic effects onthe target organism.7. Apparatus7.1 Stereoscopic Microscope, wide-field type with magnifi-cation of 10 to 153.7.2 Microscope Lamp, producing diffuse light from a cool,white fluorescent lamp adjusted to give maximum visibi
22、lity.7.3 Counting Device, hand tally or electronic.7.4 Pipet Container, stainless steel, aluminum, or borosili-cate glass, for glass pipets.7.5 Pipets, sterile, T.D. bacteriological or Mohr, glass orplastic, of appropriate volume.7.6 Graduated Cylinders, 100 to 1000 mL, covered withaluminum foil or
23、kraft paper and sterile.7.7 Membrane Filtration Units (filter base and funnel),glass, plastic, or stainless steel, wrapped in aluminum foil orkraft paper and sterilized.7.8 Ultraviolet Unit, for sterilizing the filtration unit (op-tional).7.9 Line Vacuum, Electric Vacuum Pump, or Aspirator, foruse a
24、s a vacuum source. In an emergency or in the field, a handpump or a syringe equipped with a check valve to prevent thereturn flow of air, can be used.7.10 Flask, filter, vacuum, usually 1 L, with appropriatetubing. A filter manifold to hold a number of filter bases isoptional.7.11 Forceps, straight
25、or curved, with smooth tips to handlefilters without damage.7.12 Thermometer, checked against a National Institute ofStandards and Technology (NIST) certified thermometer, orone traceable to a NIST thermometer.7.13 Petri Dishes, sterile, plastic, 50 by 12 mm, withtight-fitting lids.7.14 Bottles, mil
26、k dilution, borosilicate glass, screw-capwith neoprene liners, marked at 99 mL for 1 to 100 dilutions.Dilution bottles marked at 90 mLor tubes marked at 9 mLmaybe used for 1 to 10 dilutions.7.15 Inoculation Loops, at least 3-mm diameter, andneedles, nichrome or platinum wire, 26B the MF is now held
27、between thefunnel and the base.12.3 Shake the sample bottle vigorously about 25 times todistribute the bacteria uniformly and measure the desiredvolume of sample or dilution into the funnel.12.4 For ambient surface waters and wastewaters, selectsample volumes based on previous knowledge to produceme
28、mbrane filters with 20 to 80 colonies. Sample volumes arenormally tested at half log intervals, for example 100, 30, 10,3 mL, etc.12.5 Use smaller sample size or sample dilution to minimizethe interference of turbidity or high bacterial densities. Mul-tiple volumes of the same sample or dilution of
29、sample may befiltered and the results combined.12.6 Filter the sample and rinse the sides of the funnel atleast twice with 20 to 30 mL of buffered rinse water. Turn offthe vacuum and remove the funnel from the filter base.12.7 Use sterile forceps to aseptically remove the membranefrom the filter bas
30、e and roll it onto the mTEC medium to avoidthe formation of bubbles between the membrane and the agarsurface. 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 resealablewater-proof plastic bags and t
31、ransfer them to a 44.5C incu-bator. Incubate the plates at 44.5 6 0.2C for 22 to 24 h.12.9 After incubation, remove the plates from the waterbathand aseptically transfer the membrane to a filter pad saturatedwith urea substrate medium. After 15 to 20 min incubation at7Bordner, R. H., Winter, J. A.,
32、and Scarpino, P. V., Eds., MicrobiologicalMethods 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,1978.D 5392 93 (2006)3room tempe
33、rature, examine the membrane for yellow toyellow-brown colonies. Count and record the number oftypical colonies.13. Calculation13.1 Use the following rule to calculate the E. coli count per100 mL of sample. If more than one sample volume containscolonies, select the membrane filter with 20 to 80 and
34、 calculatethe count per 100 mL according to the general formula:E. coli/100 mL 5No. E. coli colonies countedVolume in mL of sample filtered3 100 mL13.2 See Practice D 5465.14. Verification Procedure14.1 Yellow or yellow-brown colonies from the urease testcan be verified as E. coli. Verification of c
35、olonies may berequired in evidence gathering, and is also recommended as aQC procedure with initial use of the test and with changes insample sites, lots of commercial media or major ingredients inmedia compounded in the laboratory. The verification proce-dure follows:14.1.1 Using a sterile inoculat
36、ion loop, transfer growth fromthe centers of at least 10 well-isolated typical colonies tonutrient agar plates or slants and to tryptic soy broth. Incubatethe agar and broth cultures for 24 h at 35C.14.1.2 After incubation remove a generous portion of ma-terial from the nutrient agar with a platinum
37、 loop and depositon the surface of filter paper that has been saturated withcytochrome oxidase reagent prepared fresh that day or use thecommercial product. A positive test is indicated within 15 s bythe development of a deep purple color where the bacteria weredeposited.14.1.3 Transfer growth from
38、the tryptic soy broth to Sim-mons citrate agar, tryptone broth, and EC broth in fermenta-tion tubes. Incubate the Simmons citrate agar for 24 h andtryptone broth for 48 h at 35C. Incubate the EC broth at44.5C in a waterbath for 24 h. The water level must be abovethe level of the EC broth in the tube
39、. Add one-half mL ofKovacs indole solution to the 48-h tryptone broth culture andshake the tube gently. A positive test for indole is indicated bya deep red color that develops in the alcohol layer. E. coli is ECgas positive, indole positive, oxidase negative, and does notgrow on citrate medium.14.1
40、.4 Alternatively, use commercially available multi-testidentification systems to verify colonies. Inoculate the coloniesinto an identification for Enterobacteriaceae that includeslactose fermentation and/or o-nitrophenyl-b-Dgalactopyrano-side (ONPG) and cyto-chrome oxidase test tower case reac-tions
41、.15. Report15.1 Adjust counts based on verification and report theresults as E. coli per 100 mL of sample.16. Precision and Bias16.1 Single Laboratory Data:416.1.1 PrecisionThe mTEC method precision was foundto be fairly representative of what would be expected fromcounts with a Poisson distribution
42、.16.1.2 BiasThe bias of the E. coli MF with mTECmedium method has been reported to be2% of the truevalue.16.1.3 Other Statistics:16.1.3.1 Specificity The specificity of mTEC medium asreported for various environmental samples was 9 % falsepositive and less than 1 % false negative.16.1.3.2 Upper Coun
43、ting LimitThe upper counting limitfor E. coli on mTEC medium has been reported as 80 coloniesper filter.16.2 Collaborative Study Data:16.2.1 A collective study was conducted among elevenvolunteer laboratories, each with two analysts who indepen-dently tested reagent water, local fresh and marine rec
44、reationalwaters and sewage treatment plant effluent samples, in dupli-cate.16.2.2 The results of the study are shown in Fig. 1 where Soequals the pooled standard deviation among replicate countsfrom a single analyst for three groupings (counts less than 30,counts from 30 to 50, and counts greater th
45、an 50) and SBequalsthe pooled standard deviation between means of duplicatesfrom analysts in the same laboratory for the same groupings.The precision estimates from this study did not differ amongthe water types analyzed.16.2.3 By linear regression, the precision of the test methodcan be generalized
46、 as:So5 0.028 X! 1 6.11/plate, andSB5 0.223 X! 1 0.82 count/platewhere X = the count/plate or average count/plate.16.2.3.1 To convert the count/plate to the count/100 mL,multiply this count by the dilution factor as follows:dilution factor 5100volume of original sample filtered16.2.4 Because of the
47、instability of microbial populations inwater samples, each laboratory analyzed its own sample seriesand no full measure of recovery or bias was possible. However,all laboratories analyzed a single surrogate sample preparedfrom a freeze-dried culture of E. coli. The mean count (X) andthe overall stan
48、dard deviation of a single count (ST) includingthe variability among laboratories for this standardized E. colisample, were 31.6 colonies/membrane and ST= 7.61 colonies/membrane, respectively.17. Keywords17.1 ambient waters; Escherichia coli; fecal pollution;mTEC agar; recreational waters; two-step
49、membrane filtermethod; wastewatersD 5392 93 (2006)4ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdraw
