ASTM_D_5259_-_14.pdf

Designation:D525914Standard 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 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 covers a membrane filter(MF)proce-dure for the detection and enumeration of the enterococcibacteria in water.The enterococci,which include Entero-coccus faecalis(E.faecalis),E.faecium,and their varieties arecommonly found in the feces of humans and other warm-blooded animals.Although some strains are ubiquitous and notrelated to fecal pollution,enterococci in water are an indicationof fecal pollution and the possible presence of enteric patho-gens.These bacteria are found in water and wastewater in awide range of densities.The detection limit is one colonyforming unit(CFU)/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 testmethod for waters of untested types.1.3 The values stated in SI units are to be regarded as thestandard.1.4 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 limitations prior to use.For specific hazardstatements,see Section 9.2.Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD3370 Practices for Sampling Water from Closed ConduitsD3870 Practice for Establishing Performance Characteristicsfor Colony Counting Methods in Microbiology(With-drawn 2000)33.Terminology3.1 Definitions:3.1.1 For definitions of terms used in this test method,referto Terminology D1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 Enterococcusin this test method,Enterococcus spe-cies are those bacteria that produce red to maroon colonies withblack or reddish-brown precipitate on underside,after incuba-tion on mE agar and subsequent transfer to EIA medium.3.2.1.1 DiscussionEnterococci include E.faecalis,E.faecium,E.avium,and their variants.4.Summary of Test Method4.1 The procedure given in this test method provides adirect count of bacteria in water based on the development ofcolonies on the surface of the membrane filter.4Awater sampleis filtered through the membrane that retains the bacteria.Following filtration,the membrane containing the bacterialcells is placed on a selective,medium,mE agar,and incubatedfor 48 h at 41C,then transferred to EIA agar and held at 41Cfor 20 min.Enterococci develop as red to maroon colonies withblack or reddish-brown precipitate on the underside of thefilter.5.Significance and Use5.1 The enterococci are indicators of the bacteriologicalquality for potable water,shellfish growing waters,ambient,and recreational waters.A direct relationship betweenswimming,associated gastroenteritis,and enterococci has beenestablished through epidemiological studies and marine andfresh water bathing beaches.These studies have led to thedevelopment of criteria that can be used to establish bathingwater standards based on established health-water qualityrelationships.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 Aug.1,2014.Published October 2014.Originallyapproved in 1992.Last previous edition approved in 2012 as D5259 92(2012).DOI:10.1520/D5259-14.2For 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.3The last approved version of this historical standard is referenced onwww.astm.org.4Cabelli,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,Vol 69,1979,pp.690696.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 5.2 Since small or large volumes of water or dilutionsthereof,can be analyzed by the membrane filter technique,awide range of levels of enterococci in water can be enumeratedand detected.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 sample dilution can be used tomin