ASTM_D_5392_-_14.pdf

Designation:D539214Standard 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 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 describes a membrane filter(MF)procedure for the detection 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 wastewater 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 testmethod for waters of other types.1.3 The values stated in SI units are to be regarded asstandard.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)3D5465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating Methods3.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 Escherichia coli(E.coli),na species of bacteria thatis a member of the total coliform group and known to originatein the feces of warm-blooded animals.3.3 Performance Characteristics(Practice D3870):3.3.1 accuracy,nthe proportion of the observed count tothe true density of a sample.3.3.2 bias,nthe persistent positive or negative deviationof the average value of the test method from the assumed oraccepted true value.3.3.3 precision,nthe degree of agreement of repeatedmeasurements of the same parameter expressed quantitativelyas the standard deviation or as the 95%confidence limits ofthe mean computed from the results of a series of controlleddeterminations.3.3.4 specificity,n 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.3.3.5 upper counting limit(UCL),nthat colony countabove which there is an unacceptable counting error;the errormay be due to overcrowding or antibiosis.4.Summary of Test Method4.1 This two-step test method4provides a direct count ofbacterial colonies developing on the surface of the filter 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 is1This 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.15,2014.Published August 2014.Originallyapproved in 1993.Last previous edition approved in 2006 as D5392 93(2006).DOI:10.1520/D5392-14.2For 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.4Dufour,A.,Strickland,E.,and Cabelli,V.,“Membrane Filter Method forEnumerating Escherichi coli,”Applied and Environmental Microbiology,Vol 41,1981,pp.11521158.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 placed on a selective,differential medium,mTEC.The mem-brane on the medium is first incubated at 35C for 2 h so thatinjured 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 15 magnifier and a fluorescentlamp.5.Significance and Use5.1 This test method is useful for measuring recreationalwater quality and chlorinated wastewaters,although it can beused f