1、Designation: D6503 99 (Reapproved 2009)D6503 14Standard Test Method forEnterococci in Water Using Enterolert1,2This standard is issued under the fixed designation D6503; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of l
2、ast 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 This test method covers a simple procedure for the detection of enterococci in water and wastewater. It is based onIDEX
3、XsIDEXXs patented Defined Substrate Technology (DST).2 This product, Enterolert, utilizes a nutrient indicator thatfluoresces when metabolized. It can detect these bacteria at one colony forming unit (CFU)/100 mL within 24 h. The presence ofthis microorganism in water is an indication of fecal conta
4、mination and the possible presence of enteric pathogens.1.2 This test method can be used successfully with drinking water, source water, recreational (fresh and marine) water,wastewater, and bottled water. It is the usersusers responsibility to ensure the validity of this test method for waters of u
5、ntestedmatrices.1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standar
6、d to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD2777 Practice for Determination of Precision and Bias of Ap
7、plicable Test Methods of Committee D19 on WaterD3370 Practices for Sampling Water from Closed Conduits3. Terminology3.1 DefinitionsFor definitions of terms used in this test method, refer to Terminology D1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 enterococci, na gram positive bact
8、eria possessing the enzyme -D-glucosidase, which cleaves the nutrient indicator andproduces fluorescence under a long wave length (366(365366 nm) ultraviolet (UV) light.3.2.2 most probable number (MPN), na statistical method for determining bacterial density based on the Poisson distribution.3.2.3 p
9、resence-absence, na term used to indicate if enterococci isare present or absent in a water sample. It is a qualitativevalue, “yes” or “no” for reporting results.3.2.3.1 DiscussionIt is a qualitative value, “yes” or “no” for reporting results.3.2.4 quanti-trayQuanti-Tray2, na system for the quantifi
10、cation of enterococci. It consists of a sealer and trays which havemulti-wells and can enumerate up to 2000 CFU/100 mL without dilution.3.2.4.1 Discussion1 This test method is under the jurisdiction of ASTM Committee D19 on Water and is the direct responsibility of Subcommittee D19.24 on Water Micro
11、biology.Current edition approved May 1, 2009Aug. 1, 2014. Published June 2009October 2014. Originally approved in 1999. Last previous edition approved in 20052009 asD6503 99 (2005).(2009). DOI: 10.1520/D6503-99R09. 10.1520/D6503-14.2 Trademark of IDEXX Laboratories, One Idexx Dr., Westbrook, ME 0409
12、2.3 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.This document is not an ASTM standard and is intended only
13、to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
14、of the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1It consists of a sealer and trays which have multi-wells and can enumerate up to 2419 MPN/100 mL without di
15、lution.3.2.5 snap pack, na package containing Enterolert reagent for testing 100-mL sample either in the P/A format orquantitatively, that is,with the Quanti-Tray2 system).system.4. Summary of Test Method4.1 This test method is used for the detection of enterococci, such as E. faecium, E. faecalis i
16、n drinking water, source water,recreational waters (marine water and fresh), wastewaters, and bottled water. When the reagent is added to the sample andincubated at 41 6 0.5C for 24 h and up to 28 h, Enterolert can detect these bacteria at 1 CFU/100MPN/100 mL. Fluorescenceis produced when enterococc
17、i metabolizes the nutrient indicator. Enterolert can be used as a presence-absence test or forquantification (5-tube, 10-tube MPN, 15-tube serial dilution or the Quanti-Tray system).5. Significance and Use5.1 This test provides an easy and reliable method for the detection of enterococci in water wi
18、thin 24 h. For recreational water(fresh and marine) testing is performed to insure areas are safe for swimming. Enterolert also can be used for testing bottled waterwater, wastewater, and drinking water.6. Interferences6.1 The presence of Bacillus spp. can interfere with the testing of marine water
19、samples. To eliminate interference, a 1:10dilution is required with sterile water (deionized or distilled).7. Apparatus7.1 Ultraviolet Lamp, 6-watt long wavelength (366(365366 nm).7.2 41C Incubator (60.5C), air or water bath.7.3 Vessels, sterile, nonfluorescent.7.4 Quanti-Tray Sealer. 27.5 Quanti-Tr
20、ay or Quanti-Tray 2000. 28. Reagents and Materials8.1 Purity of WaterUnless otherwise indicated, references to water shall be understood to mean reagent water conforming toSpecification D1193, Type IV. Sterilize the water by either autoclaving or by sterile filtration (0.22 micron-filtered water).8.
21、2 Enterolert Test Kit. 29. Precautions9.1 The analyst must observe the normal good laboratory practices and safety procedures required in a microbiology laboratorywhile preparing, using, and disposing of cultures, reagents and materials and while operating sterilization equipment and otherequipment.
22、10. Sampling10.1 Collect the sample as described in detail in the USEPAmicrobiological methods manual4 and in accordance with PracticesD3370.10.2 Sample Storage Temperature and Handling ConditionsIce or refrigerate water samples at a temperature of 2 to 8Cduring transit to the laboratory. Use insula
23、ted containers to ensure proper maintenance of storage temperatures. Take care thatsample bottles are not totally immersed in water during transit or storage.10.3 Holding Time LimitationsExamine samples, as soon as possible, after collection. Do not hold samples longer than 6 h8 h between collection
24、 and initiationincubation of analyses.samples.11. Quality Control Check11.1 Check and record temperatures in incubators daily to ensure temperature is within stated limits.11.2 Quality control should be conducted on each new lot of Enterolert. See package insert for the recommended quality controlpr
25、ocedure, which consists of the following protocol:11.2.1 For each type of the American Type Culture Collection (ATCC) bacterial strain listed below, streak the culture ontolabeled TSA or blood agar plates and incubate at 35C for 18 to 24 h.4 Bordner, R.H., Winter, J.A., and Scarpino, P.V., Eds., Mic
26、robiological Methods for Monitoring the Environment, Water, and Wastes, EPA-600/8-78-017.D6503 14211.2.2 For each bacterial strain, touch a 1-l loop to a colony and use it to inoculate a labeled test tube containing 5 mLof steriledeionized water. Close cap and shake thoroughly.11.2.3 For each bacter
27、ial strain, take a 1-l loop from the test tube (11.2.2) and use it to inoculate a labeled vessel containing100 mL.100 mL of sterile deionized water.11.2.4 Follow the Enterolert presence/absence steps listed above to test these controls. Compare the test results to the followingexpected results:Contr
28、ol ATTC No. Expected ResultEnterococcus faecium 335667 FluorescenceSerratia marcescens (g, ) 43862 No fluorescenceAerococcus viridians (g, +) 10400 No fluorescence12. Procedure12.1 Presence/AbsenceSee package insert.12.1.1 Samples should be brought to room temperature (18 to 30C).12.1.2 Carefully se
29、parate one snap pack from the strip.12.1.3 Tap the snap pack to insure that all of the powder is towards the bottom of the pack.12.1.4 Open the pack by snapping back the top of the score line. Do not touch the opening of pack.12.1.5 Add the reagent to a 100-mL water sample, which is in a sterile, tr
30、ansparent, 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 and up to 28 h at 41 6 0.5C,12.1.9 Read results at 24 h and up to 28 h. If the sample is inadvertently incubated over 28 h without observation, the followinggu
31、idelines apply: Lack of fluorescence after 28 h is a valid negative test. Fluorescence after 28 h is an invalid result.12.1.10 Check for fluorescence by placing a 6-W 366-nm365366-nm UV light within 5 in. of the sample in a darkenvironment. Be sure the light is facing away from your eyes and towards
32、 the vessel. If fluorescence is observed, the presence ofenterococci is confirmed.12.2 MPNQuanti-tray enumeration test procedure for 100-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 avoiding contact with the foil tab and seal th
33、e tray according to theQuanti-Tray package insert.12.2.3 Incubate for 24 h and up to 28 h at 41 6 0.5C.12.2.4 Follow the same interpretation instructions from 12.1.9 through 12.1.10, and count the number of positive wells. Referto the MPN table (see Table 1) Table 1) provided with the Quanti-Tray to
34、 determine the CFU/100MPN/100 mL.12.3 MPN5-tube 20 mL, 10-tube 10 mL and 15-tube serial dilution.12.3.1 Follow 12.1.1 12.1.7.12.3.2 sterile nonfluorescent tubes or transfer 20 mL of the reagent sample into five sterile nonfluorescent tubes.12.3.3 Incubate for 24 h and up to 28 h at 41 6 0.5C.12.3.4
35、Follow 12.1.9 and 12.1.10 for interpretation.12.3.5 Refer to the MPN tables (see Tables 2-4) Tables 24) to determine the CFU/100MPN/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 tube testresults refer to t
36、he respective MPN tables.514. Report14.1 Report as positive or negative for presence/absence testing.14.2 Reporting of results is based on calculation of enterococci density determined from the appropriate MPN tables.15. Precision and Bias615.1 PrecisionA limited collaborative study was conducted. N
37、ine technicians from three laboratories tested three differentmatrixes at three levels following Practice D2777. Outliers were rejected in accordance with the statistical tests outlined in PracticeD2777. All data from one technician was rejected for recreational water-marine and single values were r
38、ejected for bothrecreational water-fresh at the low level and for recreational water-marine at the low level. The mean count, the overall standarddeviation (St), and the single operator standard deviation (so), are indicated in Table 5.Table 5.5 Standard Methods for the Examination of Water and Wast
39、e Water, 19th Edition.6 Supporting data have been filed at ASTM International Headquarters and may be obtained by requesting Research Report RR:D19-1167. Contact ASTM CustomerService at serviceastm.org.D6503 14315.2 BiasThe mean value obtained for the samples (drinking water, recreational water fres
40、h and marine) from the ninetechnicians for the low-, mid- and high-spiked samples all fall within the 95 % confidence interval (poisson distribution) of theactual values obtained from plating on blood agar.15.3 Results of this collaborative study may not be typical of results for matrices other than
41、 those studied.16. Keywords16.1 bottled water; drinking water; enterococci; Enterolert; most probable number; presence-absence; Quanti-Tray; recreationalwater; source waterwater; wastewaterTABLE 1 51-Well Quanti-Tray MPN TableNo. of Wells GivingPositive Reaction MPN/100-mL Sample95 % Confidence Limi
42、tsLower Upper0 200.5 146.1 infiniteD6503 144TABLE 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 2419.2D6503146ASTM International takes no position respecting the validity of any patent right
43、s 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 standard is subject to revision at any t
44、ime 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 International Headquarters. Your comments will
45、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.This standard is copyrighted by ASTM
46、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 t
47、hrough the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the Copyright Clearance Center, 222Rosewood Drive, Danvers, MA 01923, Tel: (978) 646-2600; http:/ 4 MPN Index and 95 % Confidence Limits for Various Combinations of Positive Results When Five
48、Tubes are Used/Dilution(10 mL, 1.0 mL, 0.1 mL)ACombination ofPositives MPN Index/100 mL95 % Confidence Limits Combination ofPositives MPN Index/100 mL95 % Confidence LimitsLower Upper Lower Upper4-2-0 22 9.0 560-0-0 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.
49、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 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
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