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1、United States Environmental Protection Agency EPA Method 1602United States Office of Water EPA 821-R-01-029Environmental Protection Washington, D.C. 20460 April 2001AgencyMethod 1602: Male-specific (F+) andSomatic Coliphage in Water by SingleAgar Layer (SAL) ProcedureApril 2001iAcknowledgmentsThis m
2、ethod 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 Water. Thisdocument
3、was prepared under EPA Contract No. 68-C-98-139 by DynCorp Information & EnterpriseTechnology, Inc.The contributions of the following persons and organizations to the development of this method aregratefully acknowledged:Sobsey, Mark, Ming Jing Wu, and Greg Lovelace, University of North Carolina
4、, Department ofEnvironmental Sciences and Engineering, CB#7400, McGavran-Greenberg Building, Chapel Hill,NC 27599 Hsu, Fu-Chih, and Jim Larkin, Environmental Health Laboratories, 110 South Hill Street, South Bend, IN46617Chambers, Yildiz, City of San Diego Marine Microbiology Laboratory, 5530 Kiowa
5、Drive, La Mesa, CA91942Cliver, Dean, Tadesse Mariam, and Mulugeta Tamene, University of California Davis, Department ofHealth and Reproduction, School of Veterinary Medicine, Davis, CA 95616-78743Danielson, Richard, BioVir Laboratory, 685 Stone Road Unit # 6, Benicia, CA 94510Fujioka, Roger and Geet
6、a Rijal, University of Hawaii, Water Resources Center, Holmes Hall 283, 2540Dole Street, Honolulu, HI 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 Hamp
7、shire, Department of Microbiology,Biological Sciences Building, Rudman Hall Room 285, Durham, NH 03824Pillai, Suresh and Elisa Camacho, Texas A & M University, Department of Poultry Science, KlebergCenter Room 418D, College Station, TX 77843Pope, Misty, Kevin Connell, Jason Kempton, Ken Miller,
8、and Jessica Pulz, DynCorp Information andEnterprise Technologies, 6101 Stevenson Avenue, 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 Cali
9、fornia Riverside, Department ofEnvironmental Sciences, 2217 Geology, Riverside, CA 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. Me
10、thod 1602 is a performance-based method for enumerating male-specific (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 perf
11、ormance requirements set forth in thevalidated method are met. The 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 vo
12、lume of the mixture into plates.All plates from a single sample 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) d
13、ata gathering and monitoringprograms under the Safe Drinking Water 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: t
14、elliard.williamepa.govRequests for additional copies of this publication 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. . . . . . . . . . . . . . . . . . . . . . . . . . .
15、 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12.0 Summary of Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13.0 Definitions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16、 . . . . . . . . . . . . . . . . . .14.0 Interferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25.0 Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17、. . . . . . . . . . . . .26.0 Equipment and Supplies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37.0 Reagents and Standards. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48.0
18、Sample Collection, Preservation, and Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .99.0 Quality Control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1010.0 Calibration and Standardization.
19、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1511.0 Enumeration of Coliphage QC Spiking Suspensions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1512.0 Single Agar Layer (SAL) Procedure for Sample Analysis. . . . . . . . . . . . .181
20、3.0 Data Analysis and Calculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2014.0 Method Performance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2215.0 Pollution Prevention. . . .
21、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2316.0 Waste Management. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2317.0 References. . . . . . . . . . . . . . . . . . . . . .
22、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2418.0 Flowcharts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2519.0 Glossary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29April 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-specific (F+) a
24、nd 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 This method is ba
25、sed 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 other thancoliphag
26、e. 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 9.0.1.6 Any modification of the method
27、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 141.27.2.0 Summary of Method2.1 Method 1602 describes the single agar layer (SAL) procedure. A 100-mL ground water sample isassayed by adding MgCl2 (magn
28、esium 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 (dependent on plate size). After an ov
29、ernight 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 water sample (OPR) and a negative re
30、agent 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 of coliphages: male-specific (F+) and
31、somatic. 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) ProcedureApril 2001 23.3 Male-specific coliphag
32、es (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 19.3.4.0 Interferences4.1 During the
33、 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 time may result in replication ofphages such that the init
34、ial 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 associated with handling raw sewage arehigh in this method.
35、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 safety and health practices prior to use ofthis method. The a
36、nalyst/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 procedures while operatingsterilization equipment. Equipme
37、nt 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 are undersome risk of exposure to pathogenic microorgani
38、sms. 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 handling of the chemicals specifiedin this method. A re
39、ference 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 concentrations of biohazardous agents and must be handled with gloves
40、.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. Laboratory personnel must change gloves afterhandling raw
41、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 for illustrative purposes only. Noendorsement is implied.
42、 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 Equipment for collection and transport of samples 6.1.1 B
43、ottles 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 Ice6.1.3.1 Wet icepurchased locally, or6.1.3.2 Ice packsBlue Ice, UTek cat. no. 429, or equivalent, frozen for use6.1.4
44、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 specified in Section 6.2.16.2.3 Glass or plastic, plugged, sterile serological pipettesTo deliver
45、, 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 woodenapplicator, at least 3 mm in diameter or 10 ?L volume (VWR, Fisher, DIFCO, orequivalent)6
46、.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 wiped down with a 1 : 100 solution ofhousehold bleach followed by a 10% solution of sodium
47、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 (Falcon # 1029) or 150-mm-dishes (Falcon #1058) or equivalent6.2.9 Incubator capable of maintaining 36C 1.0