UnitedStatesEnvironmentalProtectionAgencyEPA

1、united states environmental protection agency epa method 1602united states office of water epa 821-r-01-02<#990099'>9environmental protection washington, d.c. 20460 april 2001agencymethod 1602: male-specific (f+) andsomatic coliphage in water by singleagar layer (sal) procedureapril 2001ia

2、cknowledgmentsthis method was prepared under the direction of william a. telliard of the engineering and analysisdivision within the u.s. environmental protection agencys (epa) office of water. the epa technicallead was paul berger, of the standards and risk management division within the office of

3、water. thisdocument was prepared under epa contract no. 68-c-<#990099'>98-13<#990099'>9 by dyncorp information &amp; enterprisetechnology, inc.the contributions of the following persons and organizations to the development of this method aregratefully acknowledged:sobsey, mar

4、k, ming jing wu, and greg lovelace, university of north carolina, department ofenvironmental sciences and engineering, cb#7400, mcgavran-greenberg building, chapel hill,nc 275<#990099'>9<#990099'>9 hsu, fu-chih, and jim larkin, environmental health laboratories, 110 south hill st

5、reet, south bend, in46617chambers, yildiz, city of san diego marine microbiology laboratory, 5530 kiowa drive, la mesa, ca<#990099'>91<#990099'>942cliver, dean, tadesse mariam, and mulugeta tamene, university of california davis, department ofhealth and reproduction, school of ve

6、terinary medicine, davis, ca <#990099'>95616-7><8743danielson, richard, biovir laboratory, 685 stone road unit # 6, benicia, ca <#990099'>94510fujioka, roger and geeta rijal, university of hawaii, water resources center, holmes hall 283, 2540dole street, honolulu, hi <#9

7、90099'>96822karim, mohammad and dale young, american water works system research laboratory, 1115 southillinois street, belleville, il 62220-3731margolin, aaron and nicola ballester, university of new hampshire, department of microbiology,biological sciences building, rudman hall room 285, du

8、rham, nh 03824pillai, suresh and elisa camacho, texas a &amp; m university, department of poultry science, klebergcenter room 418d, college station, tx 77843pope, misty, kevin connell, jason kempton, ken miller, and jessica pulz, dyncorp information andenterprise technologies, 6101 stevenson ave

9、nue, alexandria, va 22304williams, fred and ron stetler u.s. environmental protection agency, 26 west martin luther kingdrive, cincinnati, oh, 45268yates, marylynn, omid bakhtar, and andre salazar, university of california riverside, department ofenvironmental sciences, 2217 geology, riverside, ca &

10、lt;#990099'>92521-0424iidisclaimermention of trade names or commercial products does not constitute endorsement or recommendation foruse.ivintroductioncoliphage presence in ground water is an indication of fecal contamination. method 1602 is a performance-based method for enumerating male-spe

11、cific (f+) and somatic coliphage in ground water and other waters.laboratories are permitted to modify or omit any steps or procedure, with the exception of the coliphagestock enumeration procedure (section 11.3), provided that all performance requirements set forth in thevalidated method are met. t

12、he laboratory may not omit any quality control analyses. this single agar layer procedure requires the addition of host bacteria, magnesium chloride, and double-strength molten agar medium to the sample, followed by pouring the total volume of the mixture into plates.all plates from a single sample

13、are examined for plaque formation (zones of bacterial host lawn clearing).the quantity of coliphage in a sample is expressed as plaque forming units (pfu) / 100 ml. this method is for use in the environmental protection agencys (epas) data gathering and monitoringprograms under the safe drinking wat

14、er act and the clean water act.questions concerning this method or its application should be addressed to: william a. telliardu.s. epa office of wateranalytical methods staff1200 pennsylvania nwmail code 4303washington, dc 20460email: requests for additional copies of this pub

15、lication should be directed to:water resource centermail code rc-4100401 m street, swwashington, d.c. 20460(202) 260-7786 or (202) 260-2814vitable of contents1.0 scope and application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12.0 s

16、ummary of method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13.0 definitions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14.0 interferences. . . . .

17、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25.0 safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26.0 equipment and supplies. . . . . .

18、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37.0 reagents and standards. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48.0 sample collection, preservation, and storage. . . . . . . . . .

19、. . . . . . . . . . . . . . . . . . . . . . . . . . . . .<#990099'>9<#990099'>9.0 quality control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1010.0 calibration and standardization. . . . . . . . . . . . .

20、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1511.0 enumeration of coliphage qc spiking suspensions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1512.0 single agar layer (sal) procedure for sample analysis. . . . . . . . . . . . .1813.0 data analysis and ca

21、lculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2014.0 method performance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2215.0 pollution prevention. . . . . . . . . . . . . . . .

22、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2316.0 waste management. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2317.0 references. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2418.0 flowcharts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .251<#990099'>9.0 glossary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

24、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2<#990099'>9april 20011method 1602: male-specific (f+) and somatic coliphage inwater by single agar layer (sal) procedureapril 20011.0 scope and application1.1 the single agar layer (sal) procedure detects and enumerates male

25、-specific (f+) and somaticcoliphages in ground water and other waters. this method is intended to help determine if groundwater is affected by fecal contamination. 1.2 although this method may be used for water matrices other than ground water, it has only beenvalidated for use in ground water.1.3 t

26、his method is based on procedures developed for the determination of coliphage in water in thesupplement to the 20th edition of standard methods for the examination of water andwastewater (reference 17.1).1.4 this method is not intended for use in biosolids samples or as a test for microorganisms ot

27、her thancoliphage. this method may be used in ground water and other water matrices where coliphage issuspected to be present.1.5 each laboratory and analyst that uses this method must first demonstrate the ability to generateacceptable results using the procedures in section <#990099'>9.0

28、.1.6 any modification of the method beyond those expressly permitted is subject to the application andapproval of alternate test procedures under 40 cfr parts 136.4 and 136.5, and/or summary of method2.1 method 1602 describes the single agar layer (sal) procedure. a 100-ml ground water sa

29、mple isassayed by adding mgcl2 (magnesium chloride), log-phase host bacteria (e. coli famp for f+coliphage and e. coli cn-13 for somatic coliphage), and 100 ml of double-strength molten trypticsoy agar to the sample. the sample is thoroughly mixed and the total volume is poured into 5 to 10plates (d

30、ependent on plate size). after an overnight incubation, circular lysis zones (plaques) arecounted and summed for all plates from a single sample. the quantity of coliphage in a sample isexpressed as plaque forming units (pfu) / 100 ml. for quality control purposes, both a coliphage-positive reagent

31、water sample (opr) and a negative reagent water sample (method blank) areanalyzed for each type of coliphage with each sample batch.3.0 definitions3.1 coliphages are viruses (bacteriophages) that infect e. coli and are indicators of fecalcontamination. this method is capable of detecting two types o

32、f coliphages: male-specific (f+) andsomatic. 3.2 f-factor is the fertility factor in certain strains of e . coli. it is a plasmid that, when present, codesfor pilus formation. the pilus allows for transfer of nucleic acid from one bacterium to another.method 1602: single agar layer (sal) procedureap

33、ril 2001 23.3 male-specific coliphages (f+) are rna or dna viruses that infect via the f-pilus of male strainsof e. coli.3.4 somatic coliphages are dna viruses that infect host cells via the outer cell membrane.3.5 definitions for other terms used in this method are given in the glossary in section

34、1<#990099'> interferences4.1 during the single agar layer procedure the sample and host bacteria should not remain in contactwith each other for more than 10 minutes prior to plating and after plating the agar must hardenwithin 10 minutes. increased contact time or agar hardening ti

35、me may result in replication ofphages such that the initial phage concentration is overestimated. the entire plating procedure fromcombining sample with host to hardening of single-agar layer plates should not exceed 20 minutes.5.0 safety5.1 the biohazards and the risk of infection by pathogens asso

36、ciated with handling raw sewage arehigh in this method. use good laboratory practices when working with potentially harmful samples.5.2 method 1602 does not purport to address all of the safety problems associated with its use. it is theresponsibility of the laboratory to establish appropriate safet

37、y and health practices prior to use ofthis method. the analyst/technician must know and observe the safety procedures required in alaboratory that handles biohazardous material while preparing, using, and disposing of cultures,reagents, and materials. the analyst/technician must use proper safety pr

38、ocedures while operatingsterilization equipment. equipment and supplies that have come into contact with biohazardousmaterial or are suspected of containing biohazardous material must be sterilized prior to disposalor re-use. field and laboratory staff collecting and analyzing environmental samples

39、are undersome risk of exposure to pathogenic microorganisms. staff should apply safety procedures used forhandling pathogens to all samples.5.3 the laboratory is responsible for maintaining a current awareness file of occupational safety andhealth administration (osha) regulations regarding the safe

40、 handling of the chemicals specifiedin this method. a reference file of material safety data sheets should be made available to allpersonnel involved in these analyses. additional information on laboratory safety can be found insection 16.0 waste management.5.4 samples may contain high concentration

41、s of biohazardous agents and must be handled with gloves.any positive reference materials also must be handled with gloves in an appropriate laboratoryhood. the analyst/technician must never place gloves near the face after exposure to media knownor suspected to contain pathogenic microorganisms. la

42、boratory personnel must change gloves afterhandling raw sewage or any other items which may carry pathogenic microorganisms.5.5 mouth pipetting is prohibited.method 1602: single agar layer (sal) procedureapril 200136.0 equipment and suppliesplease note: brand names, suppliers, and part numbers are f

43、or illustrative purposes only. noendorsement is implied. equivalent performance may be achieved using apparatus and materials otherthan those specified in this section, but demonstration of equivalent performance that meets therequirements of this method is the responsibility of the laboratory.6.1 e

44、quipment for collection and transport of samples 6.1.1 bottles for collection of watersterile, wide-mouth, polypropylene, 4-l (or smaller)bottles or carboys with screw caps6.1.2 ice chestigloo, coleman, styrofoam box or equivalent6.1.3 ice wet icepurchased locally, or ice packsblue ice

45、, utek cat. no. 42<#990099'>9, or equivalent, frozen for use6.1.4 bubble wrap6.2 equipment and supplies for growth of microorganisms6.2.1 sterile dilution tubes with screw capsreusable or disposable, 16 × 150 mm, or 16 × 100 mm6.2.2 test tube racksize to accommodate tubes specifi

46、ed in section .3 glass or plastic, plugged, sterile serological pipettesto deliver, of appropriatevolume(s) (falcon, kimble, or equivalent)6.2.4 pipet bulbs, automatic pipetterpipet-aid or equivalent6.2.5 inoculation loopsnichrome or platinum wire, disposable, sterile plastic loops, or woode

47、napplicator, at least 3 mm in diameter or 10 ?l volume (vwr, fisher, difco, orequivalent)6.2.6 micropipettors, adjustable10- to 200-?l, and 100- to 1000-?l, with appropriateaerosol resistant tips, gilson, eppendorf, or equivalent. please note: to avoid cross-contamination, micropipettors should be w

48、iped down with a 1 : 100 solution ofhousehold bleach followed by a 10% solution of sodium thiosulfate. alternatively,disposable pipets (serological, pasteur, or equivalent) may be used.6.2.7 burneralcohol, bunsen, fisher, or equivalent6.2.8 sterile disposable petri dishes100-mm -diameter dishes (fal

49、con # 102<#990099'>9) or 150-mm-dishes (falcon #1058) or equivalent6.2.<#990099'>9 incubator capable of maintaining 36°c ± 1.0 °c for growth of microorganisms 6.2.10beakers2- and 4-l, sterile, polypropylene, glass, or polycarbonate6.2.11polypropylene, glass, or pol

50、ycarbonate bottleswide-mouth, 100-ml or 1-l, square orround, autoclavable with screw cap6.2.12erlenmeyer flasks1-l and 2-l, sterile, corning, nalgene, kimble or equivalent6.2.13stir barfisher cat. no. 14-511-<#990099'>93, or equivalent6.2.14stir platefisher cat. no. 14-4<#990099'>

51、;93-120s, or equivalent6.2.15water bath capable of maintaining 36°c ± 1.0°c and 45°c to 48°c precision, vwrscientific, or equivalentmethod 1602: single agar layer (sal) procedureapril 2001 46.2.16sterilization filtration equipmentmillex type for syringe or larger millipore t

52、ype, sterile,0.22-?m pore size6.2.17sterile, cotton-tipped applicators6.2.18latex gloves for handling samples, supplies, and equipmentmicroflex, san francisco,ca, stock no. ul-315-l, or equivalent6.2.1<#990099'>9ph meterbeckman, corning, or equivalent6.2.20vortex mixervortex genie, or equi

53、valent6.2.21spectrophotometer or colorimeter (with wavelengths in visible range)spectronic 20,spectrum instruments, inc., or equivalent, with cell holder for ?; diameter cuvettes(model # 4015) or 13 mm × 100 mm cuvettes6.2.22cuvettes1-cm light path, beckman, bausch and lomb, or equivalent6.2.23

54、shaker flasksfluted erlenmeyer,125-ml with slip cap or sterile plug, fisher (0<#990099'>9-552-33, 10-140-6, 10-041-5a) or equivalent or equivalent6.2.24shaker incubatorcapable of 36°c ± 1.0 °c and 100 to 150 rpm, new brunswick,psychotherm, innova, or equivalent or an ordinar

55、y shaker in an incubator6.2.25flask weightsvwr 2<#990099'>9700-004 or equivalent6.3 supplies for collection and filtration of raw sewage (section 7.4.3) 6.3.1 disposable filter disks25-mm-diameter, 0.45-?m pore size, sterile, low protein binding(gelman acrodisc ht tuffryn, no. 4184, cellul

56、ose acetate corning no. 21053-25, orequivalent)6.3.2 syringessterile, disposable, 5-,10-, or 20-ml6.3.3 polypropylene dilution tubessterile, 10- to 20-ml, falcon or equivalent6.3.4 sterile glass or polypropylene 250-ml bottles for collection of raw sewage6.4 miscellaneous lab ware and supplies6.4.1

57、lint-free tissueskimwipes or equivalent6.4.2 weigh boats6.4.3 graduated cylinderssterile, polypropylene or glass, 100-ml, 250-ml, and 1-l6.4.4 autoclave6.4.5 thermometersrange of 0°c to 100°c6.4.6 balancecapable of weighing to 0.1 mg for samples having a mass up to 200 g6.4.7 freezer vials

58、sterile, 5-ml screw cap, nunc or equivalent6.4.8 light boxvwr 21475-460 or equivalent7.0 reagents and standards7.1 general reagents7.1.1 reagent watershould conform to specification d 11<#990099'>93, annual book of astmstandards (reference 17.5).7.1.2 10% (w/v) sodium thiosulfateadd 10 g sodium thiosulfate (na2s2o3) per <#990099'>90 ml reagentwater. mix until dissolved. b