ASTM_D_4201_-_96.pdf

Designation:D 4201 96An American National StandardStandard Test Method forColiphages in Water1This standard is issued under the fixed designation D 4201;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(e)indicates an editorial change since the last revision or reapproval.1.Scope1.1 This test method covers the determination of coliphagesinfective for E.coli C in water.The test method is simple,inexpensive,and yields rapid water quality data.Its sensitivityis limited to 5 coliphages per 100 mLof water sample.This testmethod is applicable to natural fresh water samples.1.2 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.2.Referenced Documents2.1 ASTM Standards:D 1129 Terminology Relating to Water2D 1193 Specification for Reagent Water2D 3370 Practices for Sampling Water from Closed Con-duits23.Terminology3.1 DefinitionsFor definitions of terms used in this testmethod,refer to Terminology D 1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 bacterial lawnconfluent growth of bacteria.3.2.2 coliphageas used in this procedure,bacterial viruscapable of replication using a specific strain of coliformbacteria(E.coli C)as a host.4.Summary of Test Method4.1 A measured water sample is added to a tube of meltedmodified nutrient agar.An E.coli C host culture is added to thetube,and the contents of the tube are mixed and poured into apetri dish.The dish is incubated at 35C.The coliphagespresent in the water sample lyse the bacteria and form plaques;the total number of plaques represents the number of coliph-ages in the volume of water sample tested.5.Significance and Use5.1 Coliphage organisms may serve as indicators of fecalpollution.The presence of coliphages in water in the absence ofa disinfectant indicates the probable presence of fecal contami-nation,but the absolute relationship between the number ofcoliforms and coliphages in natural waters has not beendemonstrated conclusively.3,4,55.2 The detection of coliphages in a water sample dependsupon the use of a sensitive-host strain in the coliphage assay.Coliphages may be detected in 4 to 6 h to provide importantsame-day information on the sanitary quality of a water.Thelower detection limit is 5 coliphages per 100 mL of fresh watersample.6.Interferences6.1 High salt concentrations,such as those found in saline orbrackish water,interfere in this test method by inhibitingplaque formation.6.2 Analysis for coliphages can be performed on disinfectedwaters.However,the quantitative relationship between coliph-ages and coliform bacteria is different from that observed innatural fresh waters.This difference is due to variations in thesurvival rates of coliphages and coliform bacteria exposed todisinfectants.For example,coliphages may have been shownto be more resistant to chlorine disinfection than fecal or totalcoliforms.67.Apparatus7.1 Water Bath,44.56 0.2C.7.2 Incubator,35 6 0.5C.7.3 Balance.7.4 Petri Dishes,sterile,100 by 15-mm.7.5 Pipets,plugged,sterile,1-mL and 5-mL.1This method is under the jurisdiction of ASTM Committee D-19 on Water andis the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved May 10,1996.Published July 1996.Originallypublished as D 4201 82.Last previous edition D 4201 82(1989).2Annual Book of ASTM Standards,Vol 11.01.3Kenard,R.P.,and Valentine,R.S.,“Rapid Determination of the Presence ofEnteric Bacteria in Water,”Applied Microbiology,Vol 27,1974,p.484.4Scarpino,P.V.,“Bacteriophage Indicators,”Berg,G.,editor,Indicators ofViruses in Water and Food,Ann Arbor Science,Ann Arbor,Mich.,1978,p.201.5Kott,Y.,Ari,B.,and Buras,N.,“The Fate of Viruses in a Marine Environment,”Proceedings 4th International Conference on Water Pollution Research,Jenkins,S.H.,editor,Pergamon Press,Oxford,1969,p.823.6Kott,Y.,Roze,N.,Sperber,S.,and Betzer,N.,“Bacteriophages as ViralPollution Indicators,”Water Research,Vol 8,p.165.1Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959,United States.7.6 Test Tubes with close fitting or airtight caps,16 by125-mm and 25 by 150-mm.7.7 Platinum Transfer Loop,sterilized by flaming.7.8 Erlenmeyer Flasks,125-mL.7.9 Sterile Vials,12 by 75-mm with caps.7.10 Spectrophotometer set at 520 nm.7.11 Refrigerator with non-frost-free freezer.8.Reagents8.1 Purity of ReagentsReagent grade chemicals shall beused in all tests.Unless otherwise indicated,it is intended thatall reagents shall conform to the specifications of the Commit-tee onAnalytical Reagents of theAmerican Chemical Society.7Other grades may be used,provided it is first ascertained thatthe reagent is of