1、Designation: D3862 13Standard Test Method forRetention Characteristics of 0.2-m Membrane Filters Usedin Routine Filtration Procedures for the Evaluation ofMicrobiological Water Quality1This standard is issued under the fixed designation D3862; the number immediately following the designation indicat
2、es 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 covers a procedure to test m
3、embranefilters for their ability to retain bacteria whose diameter is equalto or slightly larger than the 0.2-m pore size of the membranefilter.1.2 The procedures described are for the use of userlaboratories as differentiated from manufacturers laboratories.1.3 The values stated in SI units are to
4、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 and health practices and
5、determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent Water3. Terminology3.1 Definitions:3.1.1 For definitions of terms used in this test method, referto Terminology D1129.3.2
6、Definitions of Terms Specific to This Standard:3.2.1 Grams stain, na routine bacterial 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,
7、 or gentian violet and treating with iodine. Those thatresist decolorization remain blue 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 vacuum, nfor the proce
8、dure used, source of suctionthat can produce a reading of 500 to 600 mm Hg on a vacuumgage.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 specif
9、icaliquot containing organisms through the membrane followedby an examination of the filtrate 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 siz
10、e.5. Significance and Use5.1 Microbiological water testing procedures using mem-brane filtration 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
11、 density estimates may occur thatcould have serious repercussions due to the presence ofunrecognized potential health hazards in the water being tested,especially in drinking water.5.1.1 This procedure as devised will enable the user to testeach membrane filter lot number for its ability to retain a
12、llbacterial equal to, or larger than, the stated membrane poresize.5.2 Since this membrane is often used to sterilize nonauto-clavable liquids, it is essential that the retention characteristicsof this membrane are stable.6. Apparatus6.1 Membrane Filtration Units, six.6.2 Vacuum Source, with trap ve
13、ssel.1This test method is under the jurisdiction ofASTM Committee 19 on Water andis the direct responsibility of Subcommittee D19.08 on Membranes and IonExchange Materials.Current edition approved Feb. 15, 2013. Published July 2013. Originallyapproved in 1990. Last previous edition approved in 2011
14、as D3862 80 (2001),which was withdrawn July 2010 and reinstated in February 2013. DOI: 10.1520/D3862-13.2For 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 standard
15、s Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States16.3 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
16、 is connected by tubing to a sterilebacterial air vent. The tubing to air vent is clamped shut duringfiltration and released after filtration.6.4 Forceps, blunt-nosed, and small beaker of 95 % ethanol.6.5 Incubator, 37C.6.6 Pinch-Cock Clamps.6.7 Autoclave or Other Sterilizing Equipment.6.8 Appropria
17、te Equipment for producing reagent gradewaters.6.9 Appropriate Laboratory Glassware.6.10 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 Ster
18、ile 0.1 % Peptone, as rinse water.6.11.5 Broth Culture of Pseudomonas diminuta,246 2h.6.11.6 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 otherw
19、ise indicated, it is intended thatall reagents shall conform to the specifications of the Commit-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 puri
20、ty to permit its use without lessening theaccuracy of the determination.7.2 Purity of WaterUnless otherwise indicated, referenceto water shall be understood to mean reagent water conformingto Specification D1193, Type II, for 0.2-m membrane filtra-tion. In addition, suitability tests for determining
21、 the bacteri-cidal properties of the reagent grade water should be per-formed.7.3 Bacterial Suspension4Prepare 100 mLof a suspensionof a Pseudomonas diminuta (ATCC 19146). Add 1.0 mL of a24 6 2 h saline lactose broth culture to 99 mLof 0.1 % peptonewater. This suspension will contain approximately 1
22、06 to 107organisms per millilitre.7.4 Peptone Water (0.1 %)Prepare a 10 % stock solutionof 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 OrganismPseudomonas diminuta ATCC strain1
23、9146, also called FDA strain PC1818.7.6 Tryptic Soy Agar and Tryptone Soya Agar are inter-changeable and henceforth referred to as agar medium,formulated, prepared, and dispensed in accordance with themanufacturers specifications.7.7 Tryptone Soya and Tryptic Soy Broth are interchange-able and hence
24、forth referred 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 attached vacuum tubing, Y-tube, andbacterial air vent. Wrap in kraft paper and sterilize by au
25、to-claving at 121C for 15 min.8.2 Aseptically assemble membrane filtration apparatusonto each flask, connect to the vacuum source, and aseptically3Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC, www.chemistry.org. For suggestions on thetesting o
26、f reagents not listed by the American Chemical Society, see AnalarStandards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and theUnited States Pharmacopeia and National Formulary, U.S. PharmacopeialConvention, Inc. (USPC), Rockville, MD, http:/www.usp.org.4For additional details on growin
27、g the challenge suspension, refer to thepublication: Leahy, T. and Sullivan, M., “Validation of Bacterial Retention Capa-bilities of Membrane Filters,” Pharmaceutical Technology, Vol 2, No. 11, 1978, pp.6575.FIG. 1 Apparatus Required for Testing Retention Characteristicsof Membrane FiltersD3862 132p
28、lace the test membrane filter into 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 mL) into a filter funneland turn on the vacuum.8.4 After the suspension has been filtered, wash down thesides of the funnel wi
29、th two 20-mL peptone water rinses andimmediately turn off the vacuum when the last drop of peptonewater has been filtered. Release the clamp between the Y-tubeand bacterial air vent to allow air in the flask to equilibrate.After equilibration has taken place, place the clamp on thevacuum tubing in f
30、ront of the glass tube at point (1) andremove the glass tube and the rest of vacuum tubing.8.5 Repeat the process using sterile equipment and sterile0.1 % peptone as a negative control inoculum.8.6 Aseptically remove the membrane filtration apparatusand place the sterile stopper in the flask and the
31、n incubate thestoppered flask for 48 h at 37C.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 37C and check membranes forgrowth outside of filtering area. Growth outside of the filteringarea will indica
32、te a faulty filtering apparatus and may result ina 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 retain0.2 m or larger bacteria. Confirm turbid broths by streaking toan agar plate and check for strain purity. Ap
33、ply Grams staintest and perform biochemical test 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 five randomly
34、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 filters; membrane; microbiological; retentionANNEX(M
35、andatory 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, (2) failure of theme
36、mbrane filtration unit to form a proper seal, or (3) damageto the membrane from improper handling during the test.Before concluding that a membrane filter is inherently faulty,items (2) and (3) must be considered as possible causes of anapparent failure.A1.2 Membrane Filtration ApparatusThe performa
37、nce 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 sealing, it may the
38、n 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 suchas cracked ed
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