1、Designation: D5259 92 (Reapproved 2012)D5259 14Standard Test Method forIsolation and Enumeration of Enterococci from Water by theMembrane Filter Procedure1This standard is issued under the fixed designation D5259; the number immediately following the designation indicates the year oforiginal adoptio
2、n 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 covers a membrane filter (MF) procedure for the detection
3、and enumeration of the enterococci bacteria inwater. The enterococci, which include Entero-coccus faecalis (E. faecalis), E. faecium, and their varieties are commonly found inthe feces of humans and other warm-blooded animals. Although some strains are ubiquitous and not related to fecal pollution,e
4、nterococci in water are an indication of fecal pollution and the possible presence of enteric pathogens. These bacteria are foundin water and wastewater in a wide range of densities. The detection limit is one colony forming unit (CFU)/volume filtered.1.2 This test method has been used successfully
5、with temperate fresh and marine ambient waters, and wastewaters. It is theusers responsibility to ensure the validity of this test method for waters of untested types.1.3 The values stated in SI units are to be regarded as the standard.1.4 This standard does not purport to address all of the safety
6、concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use. For specific hazard statements, see Section 9.2. Referenced Documents2.1 ASTM St
7、andards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD3370 Practices for Sampling Water from Closed ConduitsD3870 Practice for Establishing Performance Characteristics for Colony Counting Methods in Microbiology (Withdrawn 2000)33. Terminology3.1 DefinitionsDefinitions:Fo
8、r3.1.1 For definitions of terms used in this test method, refer to Terminology D1129. definitions of terms used in this test method,refer to Terminology D1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 EnterococcusInin this test method, Enterococcus species are those bacteria that prod
9、uce red to maroon colonies withblack or reddish-brown precipitate on underside, after incubation on mE agar and subsequent transfer to EIAmedium. Enterococciinclude E. faecalis, E. faecium, E. avium, and their variants.3.2.1.1 DiscussionEnterococci include E. faecalis, E. faecium, E. avium, and thei
10、r variants.1 This test method 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 June 1, 2012Aug. 1, 2014. Published August 2012October 2014. Originally approved in 1992. Last previous editio
11、n approved in 20062012 asD5259 92 (2006).(2012). DOI: 10.1520/D5259-92R12.10.1520/D5259-14.2 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 Sum
12、mary page on the ASTM website.3 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 n
13、ot be technically possible to adequately 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
14、Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States14. Summary of Test Method4.1 The procedure given in this test method provides a direct count of bacteria in water based on the development of colonieson the surface of the membrane filter.4 A water sample is filtered through the mem
15、brane that retains the bacteria. Followingfiltration, the membrane containing the bacterial cells is placed on a selective, medium, mE agar, and incubated for 48 h at 41C,then transferred to EIA agar and held at 41C for 20 min. Enterococci develop as red to maroon colonies with black orreddish-brown
16、 precipitate on the underside of the filter.5. Significance and Use5.1 The enterococci are indicators of the bacteriological quality for potable water, shellfish growing waters, ambient, andrecreational waters. A direct relationship between swimming, associated gastroenteritis, and enterococci has b
17、een establishedthrough epidemiological studies and marine and fresh water bathing beaches. These studies have led to the development of criteriathat can be used to establish bathing water standards based on established health-water quality relationships.5.2 Since small or large volumes of water or d
18、ilutions thereof, can be analyzed by the membrane filter technique, a wide rangeof levels of enterococci in water can be enumerated and detected.6. Interferences6.1 Water with high levels of colloidal or suspended materials can clog the membrane filter pores and prevent filtration. Also,suspended ma
19、terials cause spreading colonies that could interfere with target colonies and thereby prevent accurate counting.6.2 Smaller sample size or sample dilution can be used to minimize the interference of turbidity or high-background (non-target)bacterial densities. Replicates of smaller sample volumes o
20、r dilutions of sample may be filtered and the results combined. If themembrane filter technique is not applicable, the most probable number (MPN) method for fecal streptococci is recommended, withverification.6.3 In some samples, chemicals may have toxic effects on the target organism.7. Apparatus7.
21、1 Stereoscopic Microscope, wide-field type with magnification of 10 to 15X.7.2 Microscope Lamp, producing diffuse light from a cool, white fluorescent lamp adjusted to give maximum visibility.7.3 Counting Device, hand tally or electronic.7.4 Pipet Container, stainless steel, aluminum, or borosilicat
22、e glass, for glass pipets.7.5 Pipets, sterile tip delivery bacteriological or Mohr, glass or plastic, of appropriate volume.7.6 Graduated Cylinders, 100 to 1000 mL, covered with aluminum foil or kraft paper and sterile.7.7 Membrane Filtration Units, (filter base and funnel), glass plastic or stainle
23、ss steel, wrapped in aluminum foil or kraft paperand sterilized.7.8 Ultraviolet Unit, for disinfecting the filtration unit (optional).7.9 Line Vacuum, Electric Vacuum Pump, or Aspirator, for use as a vacuum source. In an emergency or in the field, a hand pumpor a syringe equipped with a check valve
24、to prevent the return flow or 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.7.11 Forceps, straight or curved, with smooth tips to handle filters without damage.7.12 Thermometer, checked against a Nati
25、onal Institute of Standards and Technology (NIST) certified thermometer, or onetraceable to an 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 dilu
26、tions. Dilutionbottles marked at 90 mL or tubes marked at 9 mL may be used for 1:10 dilutions.7.15 Inoculation Loops, at least 3 mm diameter, and needles, nichrome or platinum wire, 26 B and S gage, in suitable holders.7.16 Incubator maintained at 41 6 0.5C.7.17 Waterbath maintained at 44 to 46C for
27、 tempering agar.7.18 Test Tubes, 150 by 20 mm, borosilicate glass or plastic.4 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 Health, 69, 1979, pp. 690696.C
28、abelli, V. J., Dufour, A. P., Levin, M. A., McCabe, L. J., and Haberman, P. W., “Relationship of MicrobialIndicators to Health Effects at Marine Bathing Beaches,” American Journal of Public Health , Vol 69, 1979, pp. 690696.D5259 1427.19 Caps, aluminum or autoclavable plastic, for 20 mm diameter tes
29、t tubes.7.20 Test Tubes, screw-cap, borosilicate glass, 125 by 16 mm or other appropriate size.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that allreagents conform to the specifications of the Committee
30、 on Analytical Reagents of the American Chemical Society where suchspecifications are available.5 Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purityto permit its use without lessening the accuracy of the determination.8.1.1 The agar used in pre
31、paration of culture media must be of microbiological grade. Whenever possible, use commercialculture media as a means of quality control.8.1.2 Purity of WaterUnless otherwise indicated, references to water shall be understood to mean reagent water as defined byType III of Specification D1193.8.1.3 E
32、thanol, Methanol or Isopropanol, in a small, wide-mouth container, for flame-sterilization of pipets.8.2 Membrane Filters, sterile, white, grid marked, 47 mm diameter, with 0.45 6 0.02 m pore size or other pore sizes for whichthe manufacturer provides data demonstrating equivalency.8.3 Buffered Dilu
33、tion Water/Buffered Rinse Water:8.3.1 Composition/Litre:Sodium dihydrogen phosphate (NaH2PO4) 0.58 gSodium monohydrogen phosphate (Na2HPO4) 2.50 gSodium chloride 8.50 g8.3.2 PreparationDissolve the ingredients in 1 L of water in a flask and dispense in appropriate amounts for dilutions inscrew-cap b
34、ottles or culture tubes or into containers for use as rinse water, or both. Autoclave after preparation at 121C (15 lbpressure at sea level) for 15 min. The final pH should be 7.4 6 0.2.8.4 mE Agar:8.4.1 Composition of Basal Medium/Litre:Peptone 10.0 gSodium chloride 15.0 gYeast extract 30.0 gEsculi
35、n 1.0Actidione 0.05 gSodium azide 0.15 gAgar 15.0 gWater 1000 mL8.4.2 Preparation of Basal MediumAdd 71.2 g of the above mE basal medium to 1 L of water in a flask and heat to boilinguntil ingredients dissolve. Autoclave at 121C and 15 lb pressure for 15 min and cool in a 44 to 46C water bath.8.4.3
36、Reagents Added After SterilizationMix 0.25 g nalidixic acid in 5 mL water, add 0.2 mL of NaOH solution (400 g/L) todissolve, and add to the litre of basal medium. Add 0.15 g triphenyl tetrazolium chloride separately to the basal medium and mix.8.4.4 Preparation of mE Agar PlatesPour the mE agar into
37、 50 mm petri plates to a 4 to 5 mm depth (approximately 4 to 6mL), and allow to solidify. The final pH of medium should be 7.1 6 0.2. Store in a refrigerator.8.5 EIA Agar:8.5.1 Composition of EIA Medium/Litre:Esculin 1.0 gFerric citrate 0.5 gAgar 15.0 gWater 1000 mL8.5.2 PreparationAdd 16.5 g of deh
38、ydrated EIA medium to 1 L of water in flask and heat to boiling until ingredients aredissolved. Autoclave the EIA medium solution at 121C (15 lb pressure at sea level) for 15 min and cool in a 44 to 46C waterbath. After cooling, pour the medium into 50-mm petri dishes to a depth of 4 to 5 mm (approx
39、imately 4 to 6 mL and allow tosolidify. The final pH should be 7.1 6 0.2 before autoclaving. Store in a refrigerator.8.6 Brain Heart Infusion (BHI) Broth:8.6.1 Composition:5 “ReagentReagent Chemicals, American Chemical Society Specifications,”Specifications, Am.American Chemical Soc.,Society, Washin
40、gton, DC. DC, www.chemistry-.org. For suggestions on the testing of reagents not listed by the American Chemical Society, see “Analar Standards for Laboratory Chemicals,” BDH Ltd. Poole, Dorset,U.K.U.K., and the “United States Pharmacopeia.”United States Pharmacopeia and National Formulary, U.S. Pha
41、rmacopeial Convention, Inc. (USPC), Rockville, MD,http:/www.usp.org.D5259 143Calf brain infusion 200.0 gBeef heart infusion 250.0 gPeptone 10.0 gSodium chloride 5.0 gDisodium phosphate 2.5 gDextrose 2.0 gWater 1000 mL8.6.2 PreparationDissolve 37 g of dehydrated BHI broth in 1 L of water. Dispense in
42、 8 to 10 mL volumes in screw-cap tubesand autoclave at 121C (15 lb pressure at sea level) for 15 min. If the medium is not used the same day as prepared and sterilized,heat in boiling water bath for several min to remove absorbed oxygen, and cool quickly without agitation, remove absorbedoxygen, and
43、 cool quickly without agitation, just prior to inoculation. The final pH should be 7.4 6 0.2.8.7 BHI Broth with 6.5 % NaCl:8.7.1 CompositionBHI broth with 6.5 % NaCl is the same as BHI broth in 8.6 with additional NaCl.8.7.2 PreparationDissolve 60.0 g NaCl per litre of prepared BHI broth. Since most
44、 commercially available dehydrated mediacontain sodium chloride, this amount is taken into consideration in determining the final NaCl percentage above.8.8 BHI Agar:8.8.1 CompositionBHI agar contains the same components as BHI (see 8.6) with the addition of 15.0 g of agar per litre ofBHI Broth.8.8.2
45、 PreparationAdd 15.0 g of agar and 37.0 g of BHI dehydrated broth to 1 L of water. Heat to boiling until ingredientsare dissolved. Dispense 10 to 12 mL of medium in screw-cap test tubes and sterilize for 15 min at 121C (15 lb pressure at sealevel). Slant after sterilization. The final pH should be 7
46、.4 6 0.2.8.9 Bile Esculin Agar (BEA):8.9.1 Composition/Litre:Bacto beef extract 3.0 gBacto peptone 5.0 gBacto oxgall 40.0 gBacto esculin 1.0 gFerric citrate 0.5 gBacto agar 15.0Water 1000 mL8.9.2 PreparationAdd 64.5 g of dehydrated BEAto 1 Lwater and heat to boiling to dissolve. Dispense in 8 to 10
47、mLvolumesin tubes for slants or into flasks for subsequent plating. Autoclave at 121C (15 lb pressure at sea level) for 15 min. Overheatingmay cause darkening of the medium. Cool to 44 to 46C and dispense into sterile petri plates. The final pH should be 6.6 6 0.2.Store in a refrigerator.8.10 Gram S
48、tainPrepare according to APHA document.69. Hazards9.1 The analyst/technician must know and observe the normal good laboratory practices and safety procedures required in amicrobiology laboratory while preparing, using, and disposing of cultures, reagents, and materials, and while operating steriliza
49、tionand other equipment and instrumentation.9.2 Mouth-pipetting is prohibited.10. Sample Collection, Preservation, and Holding Times10.1 Sampling procedures are described in detail in Section II, A of the USEPA manual7 and Practice D3370 and adherence tosample preservation procedure and holding time limits is critical to the production of valid data. Samples should not be analyzedif these conditions are not met.10.1.1 Storage Temperature and Handling ConditionsIce or refrigerate bacteriological samples at a temperature of 1 to 4Cduring transi
