1、Designation: D3863 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 D3863; the number immediately follow
2、ing 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 () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method c
3、overs 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 laboratories.1
4、.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 Do
5、cuments2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent Water3. Terminology3.1 Definitions:3.1.1 For definitions of terms used in this method refer toTerminology D1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 Grams staina routine bacterial stain
6、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 blue or v
7、iolet 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 vacuumgage.4.
8、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 filtrate
9、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 filtrati
10、on 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 ofunreco
11、gnized 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 Membrane Filt
12、ration 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 duringfil
13、tration 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.1This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.08 on Membranes and IonExchan
14、ge Materials.Current edition approved March 27, 1987. Published July 1987. DOI: 10.1520/D3863-87R09.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Do
15、cument Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6.7 Autoclave or Other Sterilizing Equipment.6.8 Appropriate Equipment for producing reagent gradewater.6.9 Appropriate Laboratory Glassware.6.10
16、Sterile Rubber Stoppers, 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.
17、6 Sterile Membrane FiltersTest 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 C
18、ommit-tee on Analytical 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
19、 Unless otherwise indicated, referenceto water shall be understood to mean reagent water conformingto Specification D1193, 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 Sus
20、pensionAdd1mLofan186 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
21、peptone in water. Dilute 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 t
22、he above may also beused.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 ref
23、erred to as broth medium, 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-cl
24、aving at 121C for 15 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 cult
25、ure suspension (100 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-tub
26、e andbacterial air 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 % pe
27、ptone as a negative control inoculum.3Reagent Chemicals, American Chemical Society Specification, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, UK
28、, and the United States Pharmacopeia andNational Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,MD.FIG. 1 Apparatus Required for Testing Retention Characteristics of Membrane FiltersD3863 87 (2003)28.6 Aseptically remove the membrane filtration apparatusand place the sterile stopp
29、er in the flask and then incubate thestoppered flask for 48 h at 25C.8.7 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 th
30、efiltering area will 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
31、 and check for strain 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 sele
32、cted membranesfrom 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; m
33、icrobiological; water 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
34、of the membrane, (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
35、 Filtration ApparatusThe 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
36、ensure proper sealing, 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 showin
37、g visible flaws suchas 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, a
38、nd the riskof infringement 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 revis
39、ion of this standard 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 s
40、houldmake your views 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). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).D3863 87 (2003)3
