1、Designation: D 4198 82 (Reapproved 2003)Standard Test Methods forEvaluating Absorbent Pads Used with Membrane Filters forBacteriological Analysis and Growth1This standard is issued under the fixed designation D 4198; the number immediately following the designation indicates the year oforiginal adop
2、tion 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 These test methods cover the determination of thenutrient-holding capac
3、ity and the toxic or nutritive effect onbacterial growth of organisms retained on a membrane filter,when the absorbent pad being tested is used as a nutrientreservoir and medium supply source for the retained bacteria.1.2 The tests described are conducted on 47-mm diameterdisks, although other size
4、disks may be employed for bacterialculture techniques.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
5、of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 1129 Terminology Relating to WaterD 1193 Specification for Reagent WaterD 3508 Method for Evaluating Water Testing MembraneFilters for Fecal Coliform Recovery3. Terminology3.1 DefinitionsFor definitions of terms used
6、 in these testmethods, refer to Terminology D 1129.4. Summary of Test Methods4.1 Test Method A involves saturating a 47-mm absorbentpad with water and determining the volume of water held bythe pad by weighing the pad dry and when fully saturated.4.2 Test Method B involves culturing micro-organisms
7、fromsuspensions of pure cultures on a 0.45-m membrane filter,which is placed on the test absorbent pad saturated with theappropriate growth medium. The resultant cultures are com-pared to cultures grown on spread plates and to membranefilters placed directly on agar with no absorbent pad.5. Signific
8、ance and Use5.1 These test methods are appropriate for qualifying absor-bent pads used with membrane filters for bacteriologicalenumeration.5.1.1 The test methods described are applicable to qualitycontrol testing of absorbent pads by the suppliers and users ofthese pads and to specification testing
9、 of absorbent padsintended for use with membrane filters in bacteriologicalenumeration.5.2 Other pure culture organisms and their appropriateculture medium may be substituted for the E. coli and M-FCmedia for specification testing, as required.6. Apparatus6.1 Filtration Units for membrane filters wi
10、th side-armflask and tubing.6.2 Vacuum Source.6.3 Vortex Mixer or similar mixer.6.4 Forceps, flat-bladed.6.5 Incubator capable of maintaining temperatures of 44.56 0.2C.6.6 Stereoscopic Microscope and Illuminator.6.7 Illuminated Magnifying Stand for counting colonies onagar spread plates.6.8 Hand Ta
11、lly Counter.6.9 Autoclave.6.10 Analytical Balance readable to the nearest 1 mg.6.11 Petri Dish, 50-mm, nonsterile.6.12 Expendable Equipment:6.12.1 Filters (gridded, 0.45-m, 47-mm) sterile, for watertesting.6.12.2 Absorbent pads (47-mm), sterile for the growth test.6.12.3 Petri dishes, sterile 50-mm
12、and 100-mm.6.12.4 Pipets, sterile, 10-mL, 0.1 mL graduations, accuracyof 6 5%.6.12.5 Test tubes, sterile, 20-mL, with screw caps.6.12.6 Bent glass rod, sterile, for spreading bacterial cul-tures.1These methods are under the jurisdiction of ASTM Committee D19 on Waterand are the direct responsibiliti
13、es of Subcommittee D19.08 on Membranes and IonExchange Materials.Current edition approved Oct. 29, 1982. Published March 1983.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information,
14、 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.6.12.7 Burner, for flame sterilization.7. Reagents and Materials7.1 Purity of WaterUnless otherwise indicated, reference
15、to 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.7.2 M-FC Agar with Rosolic AcidFecal coliform mediumsp
16、ecific for the membrane filter technique.7.3 M-FC Broth with Rosolic AcidBroth nutrient forbacterial growth.7.4 Peptone Water, 0.1 %.7.5 E. coli (ATCC 11229).8. Preparation of Equipment and Materials8.1 Washing and CleaningClean all glassware and filtra-tion equipment thoroughly, using a suitable de
17、tergent in hotwater, rinse with hot water, and then rinse in reagent gradewater. Dry the equipment thoroughly prior to sterilization.8.2 SterilizationFollow standard microbiological labora-tory practices for preparing glassware and filtration equipmentprior to placing in the autoclave. Autoclave at
18、121C for 15min. Refer to Method D 3508 for details.8.3 IncubatorSet incubator at 44.5 6 0.2C.9. Media Preparation9.1 M-FC Broth with Rosolic AcidDissolve in reagentgrade water in accordance with the manufacturers instruc-tions.9.2 M-FC Agar with Rosolic AcidTo a solution of M-FC,add agar (15 g/1000
19、mL), mix while heating in accordancewith the manufacturers instructions, cool, and dispense into100-mm petri dishes.10. Culture Preparation10.1 Resuspend culture in accordance with the suppliersinstructions.10.2 Using a sterile loop, streak an agar plate with culture ofE. coli.10.3 Incubate 24 h at
20、44.5C.10.4 Using a sterile needle, inoculate M-FC agar withorganisms from a single colony on the streak plate.10.5 Let the culture incubate for 5 to6hat35C.10.6 Plate out a series of dilutions and store the remainderof the culture in the refrigerator. Incubate the plates for 18 62 h at 44.5C.10.7 Ba
21、sed on the 24-h plate count, dilute a portion of theculture to give a solution with 200 to 800 microorganisms permillilitre.11. Procedure11.1 Method AWater Retention:11.1.1 Weigh three dry 50-mm plastic petri dishes to thenearest 1 mg, for each lot of pads to be tested.11.1.2 Randomly select three a
22、bsorbent pads from each lot,place a dry pad in each of the dishes, cover, and weigh again.11.1.3 To each pad, add an excess of water.11.1.4 After the pads are fully saturated (20 to 30 s), pouroff the excess water and shake out any remaining excess.11.1.5 Cover the dishes and weigh again.11.2 Method
23、 BCulture Technique:11.2.1 Prepare a set of ten 100-mm sterile petri dishes with16 6 1 mL of M-FC agar. Make sure the agar plates are atroom temperature and that the surfaces are dry before using.11.2.2 Prepare a set of five 50-mm sterile petri dishes withthe sterile pads. To each pad add 1.8 mL of
24、sterile M-FC brothand pour off the excess.11.2.3 Test five replicate sets of three aliquots. Each repli-cate shall include (a) two membrane-filtered samples (one onagar, one on a pad), and (b) one spread plate.11.2.4 Add 0.1 mL of the diluted culture solution from 10.7to the agar plate from 11.2.1 a
25、nd using a sterile bent glass rod,spread over the surface of the agar. Cover the plate.11.2.5 Set up two sterile filter funnels with flasks so that thetwo membrane samples in the set can be run concurrently withthe spread plate.11.2.6 Place a sterile gridded 0.45-m membrane onto eachof the two filtr
26、ation bases and assemble the funnels.11.2.7 Add 0.1 mL of the diluted culture (20 to 80organisms/0.1 mL) from 10.7 to each of the two tubes, whichcontain 20 mL of sterile 0.1 % peptone.11.2.8 Cap the tubes and mix on a vortex mixer.11.2.9 Add the contents of one tube to each funnel and turnon the va
27、cuum.11.2.10 After the liquid has filtered through the membrane,carefully wash the sides of the funnel with about 20 mL ofsterile 0.1 % peptone solution.11.2.11 Turn off the vacuum and remove the funnel tops.11.2.12 Using sterile forceps, carefully remove one mem-brane and place on an agar plate. Re
28、move the other membraneand place on the pre-soaked test absorbent pad. Be careful notto trap air under the membranes as this will inhibit growth inthese areas.11.2.13 Repeat 11.2.4-11.2.12 four more times.11.2.14 Cover each plate after completing 11.2.12.11.2.15 Store 100-m plates inverted in sealed
29、 plastic bagswith a wet paper towel in each bag. Fifty-millimetre plasticpetri dishes do not require sealed plastic bags or wet towels,but should also be inverted.11.2.16 Place all of the plates into an incubator at 44.5 60.2C for 22 to 24 h.11.2.17 Remove the plates and count the number of colonies
30、on each plate.12. Calculation12.1 Method ACalculate the weight of water retained asfollows:C 5 B 2 T! 2 A 2 T!C 5 B 2 Awhere:C = weight of water retained,T = tare weight of dish,A = weight of dry pad plus dish, andB = weight of wet pad plus dish.12.2 Method B:D 4198 82 (2003)212.2.1 Average the five
31、 replicates for each of the three testsets and calculate the standard deviation.12.2.2 Compute percent recovery for the filter culture on theabsorbent pad (Rp) and the filter culture on the agar medium(Rm) as follows:% Rp5 Np/Na! 3 100% Rm5 Nm/Na! 3 100where:Np= the number of colonies recovered with
32、 the filter onthe pad,Na= the number of colonies recovered with the agarspread plate, andNm= the number of colonies recovered with the filter onagar directly.13. Results13.1 Compare % Rpwith % Rm.13.2 Report the volume of water retained by the absorbentpad, after converting weight of water to volume
33、.14. Precision and Bias14.1 No statement is made about either the precision or biasof these test methods for evaluating absorbent pads since theresults merely indicate whether there is conformance with therequirements for the intended use.15. Keywords15.1 absorbent pads; bacteriological; filter memb
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37、mmittee 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 4198 82 (2003)3
