1、Designation: D 3863 87 (Reapproved 2003)Standard Test Method forRetention Characteristics of 0.40 to 0.45-m MembraneFilters Used in Routine Filtration Procedures for theEvaluation of Microbiological Water Quality1This standard is issued under the fixed designation D 3863; the number immediately foll
2、owing 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 metho
3、d covers a procedure to test membranefilters for their ability to retain bacteria whose diameter is equalto or slightly larger than membrane filters with pore size ratedat 0.40 to 0.45 m.1.2 The procedures described are for the use of userlaboratories as differentiated from manufacturers laboratorie
4、s.1.3 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
5、 Documents2.1 ASTM Standards:2D 1129 Terminology Relating to WaterD 1193 Specification for Reagent Water3. Terminology3.1 Definitions:3.1.1 For definitions of terms used in this method refer toTerminology D 1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 Grams staina routine bacterial
6、stain that dividesbacteria into two categories, depending on whether they can bedecolorized with acetone, alcohol, or aniline oil after stainingwith one of the rosaniline dyes such as crystal violet, methylviolet, or gentian violet and treating with iodine. Those thatresist decolorization remain blu
7、e or violet and are designatedGram-positive; those that are decolorized and take up the redcounterstain, such as neutral red, safranin, or dilute carbolfuchsin are termed Gram-negative.3.2.2 vacuumfor the procedure used, a source of suctionthat can produce a reading of 500 to 600 mm Hg on a vacuumga
8、ge.4. Summary of Test Method4.1 This test method is based on the cultivation of organ-isms whose diameters are equal to or slightly larger than poresof the membrane filter to be tested and then filtering a specificaliquot containing organisms through the membrane followedby an examination of the fil
9、trate after incubation for sterility. Asterile filtrate indicates complete retention of the organism andvalidates the ability of the membrane to retain bacteria equal toor slightly larger than the stated pore size.5. Significance and Use5.1 Microbiological water testing procedures using mem-brane fi
10、ltration are based on the premise that all bacteria withina specific size range will be retained by the membrane filterused. If the membrane filter does not retain these bacteria, falsenegative results or lowered density estimates may occur thatcould have serious repercussions due to the presence of
11、unrecognized potential health hazards in the water being tested,especially in drinking water.5.2 This procedure as devised will enable the user to testeach membrane filter lot number for its ability to retain allbacteria equal to, or larger than, the stated membrane pore size.6. Apparatus6.1 Membran
12、e Filtration Units, six.6.2 Vacuum Source with trap vessel.6.3 Filtering Flasks, 1-L, with vacuum tubing into which aglass tube and aY-tube have been incorporated as in Fig. 1. Thefree end of the Y-tube is connected by tubing to a sterilebacterial air vent. The tubing to air vent is clamped shut dur
13、ingfiltration and released after filtration.6.4 Forceps, blunt-nosed, and small beaker of 95 % ethanol.6.5 Incubator, 20 to 25C.6.6 Pinch-Cock Clamps.6.7 Autoclave or Other Sterilizing Equipment.6.8 Appropriate Equipment for producing reagent gradewater.1This test method is under the jurisdiction of
14、 ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.08 on Membranes and IonExchange Materials.Current edition approved March 27, 1987. Published July 1987.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm
15、.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, United States.6.9 Appropriate Laboratory Glassware.6.10 Sterile Rubber Stopp
16、ers, to fit 1-L filtering flask.6.11 Expendables:6.11.1 Double-Strength Broth, 140-mL aliquots.6.11.2 Sterile Pipets, 1 and 10-mL.6.11.3 Sterile 0.1 % Peptone in 99-mL quantities.6.11.4 Sterile 0.1 % Peptone as rinse water.6.11.5 Broth Cultures of Serratia marcescens,186 2h.6.11.6 Sterile Membrane F
17、iltersTest membranes.6.11.7 Petri Dishes, 50-mm, containing 6 to 8-mL of agar.7. Reagents and Materials7.1 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 on Analyti
18、cal Reagents of the American Chemical Society,where such specifications are available.3Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.7.2 Purity of Water Unless otherwise in
19、dicated, referenceto water shall be understood to mean reagent water conformingto Specification D 1193, Type II, with 0.2-m membranefiltration. In addition, suitability tests for determining thebactericidal properties of the reagent grade water should beperformed.7.3 Bacterial SuspensionAdd1mLofan18
20、6 2-h brothculture of Serratia marcescens to 99 mL of 0.1 % peptonewater, to obtain a working concentration of approximately 103to 104organisms per millilitre. Prepare five bottles. Incubatortemperature of suspension is 25C.7.4 Peptone Water (0.1 %)Prepare a 10 % stock solutionof peptone in water. D
21、ilute a measured volume of the 10 %stock solution to obtain final solution of 0.1 % peptone inrequired amount. Sterilize at 121C for 15 min.7.5 Test OrganismSerratia marcescens ATCC strain14756, also called FDA strain PC1 1107. Red pigmentedSerratia marcescens strains other than the above may also b
22、eused.7.6 Tryptic Soy Agar and Tryptone Soya Agarare inter-changeable and henceforth referred to as agar medium, formu-lated, prepared, and dispensed in accordance with the manu-facturers specifications.7.7 Tryptone Soya and Tryptic Soy Brothare interchange-able and henceforth referred to as broth m
23、edium, formulated,prepared, and dispensed in accordance with the manufacturersspecifications.8. Procedure8.1 Place 140 mL of double-strength broth into six 1-Lvacuum flasks with the attached vacuum tubing, Y-tube, andbacterial air vent. Wrap in kraft paper and sterilize by auto-claving at 121C for 1
24、5 min.8.2 Aseptically assemble membrane filtration apparatusonto each flask, connect to the vacuum source, and asepticallyplace the test membrane filter into the a filter holder and secure.Place the clamp on tubing between the Y-tube and sterilebacterial air vent.8.3 Pour the culture suspension (100
25、 mL) into a filter funneland turn on the vacuum.8.4 After the suspension has been filtered, wash down thesides of the funnel with two 20-mL peptone water rinses andimmediately turn off the vacuum when the last drop of peptonewater has been filtered. Release the clamp between Y-tube andbacterial air
26、vent to allow air in the flask to equilibrate. Afterequilibration has taken place, place the clamp on the vacuumtubing in front of the glass tube at point ( 1) and remove theglass tube and the rest of vacuum tubing.8.5 Repeat the process using sterile equipment and sterile0.1 % peptone as a negative
27、 control inoculum.8.6 Aseptically remove the membrane filtration apparatusand place the sterile stopper in the flask and then incubate thestoppered flask for 48 h at 25C.3Reagent Chemicals, American Chemical Society Specification, AmericanChemical Society, Washington, DC. For suggestions on the test
28、ing of reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, UK, and the United States Pharmacopeia andNational Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,MD.FIG. 1 Apparatus Required for Testing Retention C
29、haracteristics of Membrane FiltersD 3863 87 (2003)28.7 Using flamed forceps, aseptically remove the membranefrom the membrane-filter holder and place a filter on agar(50-mm petri dish) for 48 h at 25C and check the membranesfor growth outside of filtering area. Growth outside of thefiltering area wi
30、ll indicate a faulty filtering apparatus and mayresult in a false positive test.8.8 Note any signs of turbidity in the liquid medium as anindication of growth and thus failure of the membrane to retainbacteria larger than 0.45 m. Confirm turbid broths by streak-ing to an agar plate and check for str
31、ain purity. Apply Gramsstain test and perform biochemical tests if in doubt.8.9 Examine control broth culture after 48 h. If any sign ofturbidity occurs, this indicates technique failure and the testand control procedures should be repeated.8.10 Test a minimum of five randomly selected membranesfrom
32、 five randomly selected packages. Take the control mem-brane from the same package as one of the test membranes.9. Precision and Bias9.1 Since this is a positive or negative test, precision andbias statements are not applicable to this procedure.10. Keywords10.1 filter; membrane; microbiological; wa
33、ter qualityANNEX(Mandatory Information)A1. INTERPRETATION OF TEST RESULTSA1.1 Failures in Retention of Test OrganismThe appear-ance of test organisms downstream of the test membrane filterin a retention test may be due to one or more of three possiblefactors: ( 1) inherent failure of the membrane, (
34、2) failure of themembrane filtration unit to form a proper seal, or damage to themembrane from improper handling during the test. Beforeconcluding that a membrane filter is inherently faulty, items (2)and (3) must be considered as possible causes of an apparentfailure.A1.2 Membrane Filtration Appara
35、tusThe performance ofequipment used to evaluate membrane filters for bacterialretention should be examined prior to its use. Membranefiltration units that employ a positive seal such as those fittedwith a sealing O-ring are ideal for this application. Onceequipment is qualified to ensure proper seal
36、ing, it may then bereserved exclusively for membrane-retention testing.A1.3 Damage Due to Improper HandlingExercise care atall times when manipulating dry membranes. While handlingwith smooth-tip filter forceps, examine membranes visuallyprior to testing. Discard any filters showing visible flaws su
37、chas cracked edges or holes.ASTM International takes no position respecting 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 infr
38、ingement of such rights, are entirely their own responsibility.This standard 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 standa
39、rd or for additional standardsand should be addressed to ASTM International 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 vie
40、ws known to the ASTM Committee on Standards, at the address shown below.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 3863 87 (2003)3
