ASTM_D_5246_-_13.pdf

Designation:D524613Standard Test Method forIsolation and Enumeration of Pseudomonas aeruginosa fromWater1This standard is issued under the fixed designation D5246;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 The test method covers the isolation and enumeration ofPseudomonas aeruginosa.Testing was performed on spikedreagent grade water samples.1.2 It is the users responsibility to ensure the validity of thismethod for surface waters,ground waters,recreational watersfresh and marine),wastewaters.1.3 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard.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.Specific hazardstatements are given in Section 10.2.Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD2777 Practice for Determination of Precision and Bias ofApplicable Test Methods of Committee D19 on WaterD3370 Practices for Sampling Water from Closed Conduits3.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 Pseudomonas aeruginosaan aerobic,motile,gramnegative rod that produces fluorescent pigments and pyocya-nin.It is oxidase and caseinase positive,is able to grow at42C,is relatively resistant to many antibiotics,and may utilizeacetamide.3.2.2 refrigerationstorage at 2 to 8C.4.Summary of Test Method4.1 A water sample is passed through a 0.45 mm orequivalent membrane filter.The filter carrying the retainedorganisms is placed on a selective medium(M-PA-C)3and isincubated at 41.5 6 0.5C for 72 h.The resulting pink-brownto black colonies of Pseudomonas aeruginosa are counted andreported per 100 mL of the sample.Colonies may be verifiedon skim milk agar.5.Significance and Use5.1 Pseudomonas aeruginosa is an opportunistic pathogen,and has been linked as the causative agent of numerousinfections that may be transmitted through a contaminatedwater supply to a susceptible host.NOTE1Fecal waste is 95%E.coli which is found in humans andwarm bloodied animals.5.2 The membrane filtration procedure described is a rapidand reliable test method of detecting P.aeruginosa in water.6.Interferences6.1 For certain samples,bacterial cells may have beenexposed to adverse environmental factors that lower theirprobability for survival and growth on a membrane filtermedium.This effect may be pronounced in this test method dueto the presence of antibiotics and the elevated incubationtemperature.6.2 The selection of an appropriate dilution volume isessential.Too small a dilution volume may fail to detect any P.aeruginosa organisms,while too large a volume may cause anoverabundance of colonies that would interfere with an accu-rate count.6.3 Chemicals or a combination of chemicals in certainsamples can have a toxic effect upon P.aeruginosa whenconcentrated.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 May 1,2013.Published July 2013.Originally approvedin 1992.Last previous edition approved in 2004 as D5246 92(2004).DOI:10.1520/D5246-13.2For 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.3Available from BBL Microbiological Systems,Division of Becton Dickinsonand Co.,Cockeysville,MD 21030.Other suppliers may be utilized if equivalent.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 6.4 Turbidity in samples may clog filter or effect colordetection of organisms that develop on the filter.6.5 Water samples containing residual chlorine can bedetrimental to P.aeruginosa.Utilize the procedure defined inPractices D3370 to address chlorinated water samples.7.Apparatus7.1 Equipment for collection and transport of samples tolaboratory:7.1.1 Autoclavable sample container;Use sterile,non-toxic,glass or plastic containers with a leak-proof lid.Ensure that thesample container is capable of holding a 1-L sample withample headspace to facilitate mixing of sample by shakingprior to analysis.7.1.2 Ice chest.7.1.3 Ice packs.7.2 Rinse water bottles,sterile,polypropylene or glass.7.3 Pipettes,sterile,plastic or autoclavable glass pipetteswith a 2.5%tolerance with pipette b