1、Designation: D 6503 99 (Reapproved 2009)Standard Test Method forEnterococci in Water Using Enterolert1, 2This standard is issued under the fixed designation D 6503; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last r
2、evision. 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 covers a simple procedure for thedetection of enterococci in water and wastewater. It is based onIDEXXs pat
3、ented Defined Substrate Technology (DST).2Thisproduct, Enterolert, utilizes a nutrient indicator that fluoresceswhen metabolized. It can detect these bacteria at one colonyforming unit (CFU)/100 mL within 24 h. The presence of thismicroorganism in water is an indication of fecal contaminationand the
4、 possible presence of enteric pathogens.1.2 This test method can be used successfully with drinkingwater, source water, recreational (fresh and marine) water, andbottled water. It is the users responsibility to ensure thevalidity of this test method for waters of untested matrices.1.3 The values sta
5、ted 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 an
6、d health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D 1129 Terminology Relating to WaterD 1193 Specification for Reagent WaterD 2777 Practice for Determination of Precision and Bias ofApplicable Test Methods of Committ
7、ee D19 on WaterD 3370 Practices for Sampling Water from Closed Conduits3. 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 enterococci, na gram positive bacteria possessingthe enzyme b-D-g
8、lucosidase, which cleaves the nutrient indi-cator and produces fluorescence under a long wave length (366nm) ultraviolet (UV) light.3.2.2 most probable number (MPN), na statistical methodfor determining bacterial density based on the Poisson distri-bution.3.2.3 presence-absence, na term used to indi
9、cate if en-terococci is present in a water sample. It is a qualitative value,“yes” or “no” for reporting results.3.2.4 quanti-tray2, na system for the quantification ofenterococci. It consists of a sealer and trays which havemulti-wells and can enumerate up to 2000 CFU/100 mLwithout dilution.3.2.5 s
10、nap pack, na package containing Enterolert reagentfor testing 100-mL sample either in the P/A format or quanti-tatively, that is, Quanti-Tray2system).4. Summary of Test Method34.1 This test method is used for the detection of enterococci,such as E. faecium, E. faecalis in drinking water, source wate
11、r,recreational waters (marine water and fresh), and bottled water.When the reagent is added to the sample and incubated at 41 60.5C for 24 h, Enterolert can detect these bacteria at 1CFU/100 mL. Fluorescence is produced when enterococcimetabolizes the nutrient indicator. Enterolert can be used as ap
12、resence-absence test or for quantification (5-tube, 10-tubeMPN, 15-tube serial dilution or the Quanti-Tray system).5. Significance and Use5.1 This test provides an easy and reliable method for thedetection of enterococci in water within 24 h. For recreationalwater (fresh and marine) testing is perfo
13、rmed to insure areasare safe for swimming. Enterolert also can be used for testingbottled water and drinking water.6. Interferences6.1 The presence of Bacillus spp. can interfere with thetesting of marine water samples. To eliminate interference, a1:10 dilution is required with sterile water (deioni
14、zed ordistilled).7. Apparatus7.1 Ultraviolet Lamp, 6-watt long wavelength (366 nm).7.2 41C Incubator (60.5C), air or water bath.7.3 Vessels, sterile, nonfluorescent.7.4 Quanti-Tray Sealer.21This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of S
15、ubcommittee D19.24 on Water Microbiology.Current edition approved May 1, 2009. Published June 2009. Originallyapproved in 1999. Last previous edition approved in 2005 as D 6503 99 (2005).2Trademark of IDEXX Laboratories, One Idexx Dr., Westbrook, ME 04092.3For referenced ASTM standards, visit the AS
16、TM 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.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, Unit
17、ed States.7.5 Quanti-Tray or Quanti-Tray 2000.28. Reagents and Materials8.1 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water conformingto Specification D 1193, Type IV. Sterilize the water by eitherautoclaving or by sterile filtration (0.22 micr
18、on-filtered water).8.2 Enterolert Test Kit.29. Precautions9.1 The analyst must observe the normal good laboratorypractices and safety procedures required in a microbiologylaboratory while preparing, using, and disposing of cultures,reagents and materials and while operating sterilization equip-ment
19、and other equipment.10. Sampling10.1 Collect the sample as described in detail in the USEPAmicrobiological methods manual4and in accordance withPractices D 3370.10.2 Sample Storage Temperature and HandlingConditionsIce or refrigerate water samples at a temperatureof 2 to 8C during transit to the lab
20、oratory. Use insulatedcontainers to ensure proper maintenance of storage tempera-tures. Take care that sample bottles are not totally immersed inwater during transit or storage.10.3 Holding Time LimitationsExamine samples, as soonas possible, after collection. Do not hold samples longer than6 h betw
21、een collection and initiation of analyses.11. Quality Control Check11.1 Check and record temperatures in incubators daily toensure temperature is within stated limits.11.2 Quality control should be conducted on each new lot ofEnterolert. See package insert for the recommended qualitycontrol procedur
22、e, which consists of the following protocol:11.2.1 For each type of the American Type Culture Collec-tion (ATCC) bacterial strain listed below, streak the cultureonto labeled TSA or blood agar plates and incubate at 35C for18 to 24 h.11.2.2 For each bacterial strain, touch a 1-l loop to acolony and
23、use it to inoculate a labeled test tube containing5 mL of sterile deionized water. Close cap and shake thor-oughly.11.2.3 For each bacterial strain, take a 1-l loop from thetest tube and use it to inoculate a labeled vessel containing100 mL.11.2.4 Follow the Enterolert presence/absence steps listeda
24、bove to test these controls. Compare the test results to thefollowing expected results:Control ATTC No. Expected ResultEnterococcus faecium 335667 FluorescenceSerratia marcescens (g, ) 43862 No fluorescenceAerococcus viridians (g, +) 10400 No fluorescence12. Procedure12.1 Presence/AbsenceSee package
25、 insert.12.1.1 Samples should be brought to room temperature (18to 30C).12.1.2 Carefully separate one snap pack from the strip.12.1.3 Tap the snap pack to insure that all of the powder istowards the bottom of the pack.12.1.4 Open the pack by snapping back the top of the scoreline. Do not touch the o
26、pening of pack.12.1.5 Add the reagent to a 100-mL water sample, which isin a sterile, transparent, nonfluorescent vessel.12.1.6 Aseptically cap and seal the vessel.12.1.7 Shake until dissolved.12.1.8 Incubate Enterolert for 24 h at 41 6 0.5C,12.1.9 Read results at 24 h. If the sample is inadvertentl
27、yincubated over 28 h without observation, the following guide-lines apply: Lack of fluorescence after 28 h is a valid negativetest. Fluorescence after 28 h is an invalid result.12.1.10 Check for fluorescence by placing a 6-W 366-nmUV light within 5 in. of the sample in a dark environment. Besure the
28、 light is facing away from your eyes and towards thevessel. If fluorescence is observed, the presence of enterococciis confirmed.12.2 MPNQuanti-tray enumeration test procedure for100-mL sample (see package insert).12.2.1 Follow steps 12.1.1-12.1.7.12.2.2 Pour the reagent sample into the Quanti-Tray
29、avoid-ing contact with the foil tab and seal the tray according to theQuanti-Tray package insert.12.2.3 Incubate for 24 h at 41 6 0.5C.12.2.4 Follow the same interpretation instructions from12.1.9 through 12.1.10, and count the number of positive wells.Refer to the MPN table (see Table 1) provided w
30、ith theQuanti-Tray to determine the CFU/100 mL.12.3 MPN5-tube 3 20 mL, 10-tube 3 10 mL and 15-tubeserial dilution.12.3.1 Follow 12.1.1-12.1.7.12.3.2 sterile nonfluorescent tubes or transfer 20 mL of thereagent sample into five sterile nonfluorescent tubes.12.3.3 Incubate for 24 h at 41 6 0.5C.12.3.4
31、 Follow 12.1.9 and 12.1.10 for interpretation.12.3.5 Refer to the MPN tables (see Tables 2-4) to deter-mine the CFU/100 mL.13. Calculation13.1 For P/A, there are no calculations. For quantification,refer to Quanti-Tray MPN tables and for the 5, 10, and 15 tubetest results refer to the respective MPN
32、 tables.514. Report14.1 Report as positive or negative for presence/absencetesting.14.2 Reporting of results is based on calculation of entero-cocci density determined from the appropriate MPN tables.4Bordner, R.H., Winter, J.A., and Scarpino, P.V., Eds., Microbiological Methodsfor Monitoring the En
33、vironment, Water, and Wastes , EPA-600/8-78-017.5Standard Methods for the Examination of Water and Waste Water, 19th Edition.D 6503 99 (2009)215. Precision and Bias615.1 PrecisionA limited collaborative study was con-ducted. Nine technicians from three laboratories tested threedifferent matrixes at
34、three levels following Practice D 2777.Outliers were rejected in accordance with the statistical testsoutlined in Practice D 2777. All data from one technician wasrejected for recreational water-marine and single values wererejected for both recreational water-fresh at the low level andfor recreatio
35、nal water-marine at the low level. The mean count,the overall standard deviation (St), and the single operatorstandard deviation (so), are indicated in Table 5.15.2 BiasThe mean value obtained for the samples(drinking water, recreational water fresh and marine) from thenine technicians for the low-,
36、 mid- and high-spiked samples allfall within the 95 % confidence interval (poisson distribution)of the actual values obtained from plating on blood agar.15.3 Results of this collaborative study may not be typicalof results for matrices other than those studied.16. Keywords16.1 bottled water; drinkin
37、g water; enterococci; Enterolert;most probable number; presence-absence; Quanti-Tray; recre-ational water; source waterTABLE 1 51-Well Quanti-Tray MPN TableNo. of Wells GivingPositive ReactionMPN/100-mL Sample95 % Confidence LimitsLower Upper0 200.5 146.1 infinite6Supporting data have been filed at
38、ASTM International Headquarters and maybe obtained by requesting Research Report RR: D191167.D 6503 99 (2009)3TABLE 2 IDEXX Quanti-Tray/2000 MPN Table (cfu/100 mL)No. LargeWellsPositiveNo. Small Wells Positive0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 230 2419.2D650399(2009)5TABLE 4
39、MPN Index and 95 % Confidence Limits for Various Combinations of Positive Results When Five Tubes are Used/Dilution(10 mL, 1.0 mL, 0.1 mL)ACombination ofPositivesMPN Index/100 mL95 % Confidence LimitsCombination ofPositivesMPN Index/100 mL95 % Confidence LimitsLower Upper Lower Upper4-2-0 22 9.0 560
40、-0-0 2 4-2-1 26 12 650-0-1 2 1.0 10 4-3-0 27 12 670-1-0 2 1.0 10 4-3-1 33 15 770-2-0 4 1.0 13 4-4-0 34 16 805-0-0 23 9.0 861-0-0 2 1.0 11 5-0-1 30 10 1101-0-1 4 1.0 15 5-0-2 40 20 1401-1-0 4 1.0 15 5-1-0 30 10 1201-1-1 6 2.0 18 5-1-1 50 20 1501-2-0 6 2.0 18 5-1-2 60 30 1802-0-0 4 1.0 17 5-2-0 50 20
41、1702-0-1 7 2.0 20 5-2-1 70 30 2102-1-0 7 2.0 21 5-2-2 90 40 2502-1-1 9 3.0 24 5-3-0 80 30 2502-2-0 9 3.0 25 5-3-1 110 40 3002-3-0 12 5.0 29 5-3-2 140 60 3603-0-0 8 3.0 24 5-3-3 170 80 4103-0-1 11 4.0 29 5-4-0 130 50 3903-1-0 11 4.0 29 5-4-1 170 70 4803-1-1 14 6.0 35 5-4-2 220 100 5803-2-0 14 6.0 35
42、5-4-3 280 120 6903-2-1 17 7.0 40 5-4-4 350 160 8205-5-0 240 100 9404-0-0 13 5.0 38 5-5-1 300 100 13004-0-1 17 7.0 45 5-5-2 500 200 20004-1-0 17 7.0 46 5-5-3 900 300 29004-1-1 21 9.0 55 5-5-4 1600 600 53004-1-2 26 12 63 5-5-5 $1600 ABased on Standard Methods for the Examination of Water and Wastewate
43、r,19thed.TABLE 5 Mean Count, Overall Standard Deviation and Single Operator Standard DeviationNOTE 1All calculations were made from the statistical summary given as Table One in the study fileSummary Table Low Level Middle Level High Leveln X St So n X St So n X St SoBackground MPN/100 mL % % MPN/10
44、0 mL % % MPN/100 mL % %MatrixDrinking water 9 9.1 27.7 24.2 9 26.2 15.1 15 9 61.0 10.2 9.5Rec. water 9 9.4A30.2A23.4A9 29.1 14.1 6.9 9 64.3 10.6 11FreshRec. water 8 7.9A25.2A17.7A8 29.4 17.4 13 8 67.5 8.0 7.6MarineAOne value rejected to make this estimate.ASTM International takes no position respect
45、ing the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This stan
46、dard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM Internatio
47、nal Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.
48、This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).D 6503 99 (2009)6
