1、Designation: E2799 11Standard Test Method forTesting Disinfectant Efficacy against Pseudomonasaeruginosa Biofilm using the MBEC Assay1This standard is issued under the fixed designation E2799; the number immediately following the designation indicates the year oforiginal adoption or, in the case of
2、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 specifies the operational parametersrequired to grow and treat a Pseudomonas ae
3、ruginosa biofilmin a high throughput screening assay known as the MBEC(trademarked)2(Minimum Biofilm Eradication Concentration)Physiology and Genetics Assay. The assay device consists of aplastic lid with ninety-six (96) pegs and a correspondingreceiver plate with ninety-six (96) individual wells th
4、at have amaximum 200 L working volume. Biofilm is established onthe pegs under batch conditions (that is, no flow of nutrientsinto or out of an individual well) with gentle mixing. Theestablished biofilm is transferred to a new receiver plate fordisinfectant efficacy testing.3, 4The reactor design a
5、llows forthe simultaneous testing of multiple disinfectants or onedisinfectant with multiple concentrations, and replicatesamples, making the assay an efficient screening tool.1.2 This test method defines the specific operational param-eters necessary for growing a Pseudomonas aeruginosa bio-film, a
6、lthough the device is versatile and has been used forgrowing, evaluating and/or studying biofilms of differentspecies as seen in Refs (1-4).51.3 Validation of disinfectant neutralization is included aspart of the assay.1.4 This test method describes how to sample the biofilmand quantify viable cells
7、. Biofilm population density is re-corded as log colony forming units per surface area. Efficacy isreported as the log reduction of viable cells.1.5 Basic microbiology training is required to perform thisassay.1.6 The values stated in SI units are to be regarded asstandard. No other units of measure
8、ment are included in thisstandard.1.7 ASTM International takes no position respecting thevalidity of any patent rights asserted in connection with anyitem mentioned in this standard. Users of this standard areexpressly advised that determination of the validity of any suchpatent rights, and the risk
9、 of infringement of such rights, areentirely their own responsibility.1.8 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
10、 applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:6E1054 Test Methods for Evaluation of Inactivators of An-timicrobial Agents2.2 Other Standards:Method 9050 Buffered Dilution Water Preparation accord-ing to Eaton et al (5)3. Terminology3.1 Definitions:3
11、.1.1 biofilm, nmicroorganisms living in a self-organized,cooperative community attached to surfaces, interfaces, or eachother, embedded in a matrix of extracellular polymeric sub-stances of microbial origin, while exhibiting an altered pheno-type with respect to growth rate and gene transcription.3.
12、1.1.1 DiscussionBiofilms may be comprised of bacte-ria, fungi, algae, protozoa, viruses, or infinite combinations ofthese microorganisms. The qualitative characteristics of abiofilm including, but not limited to, population density,taxonomic diversity, thickness, chemical gradients, chemical1This te
13、st method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Methods and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2011. Published April 2011. DOI: 10.1520/E279911.2The MBEC trademar
14、k is held by Innovotech, Inc., Edmonton, Alberta, Canada.3The sole source of supply of the apparatus known to the committee at this timeis Innovotech Inc., Edmonton, Alberta, Canada. If you are aware of alternativesuppliers, please provide this information to ASTM International Headquarters.Your com
15、ments will receive careful consideration at a meeting of the responsibletechnical committee,1which you may attend.4The MBECAssay is covered by a patent. Interested parties are invited to submitinformation regarding the identification of an alternative(s) to this patented item tothe ASTM Internationa
16、l Headquarters. Your comments will receive careful consid-eration at a meeting of the responsible technical committee,1which you may attend.5The boldface numbers in parentheses refer to a list of references at the end ofthis standard.6For referenced ASTM standards, visit the ASTM website, www.astm.o
17、rg, orcontact ASTM Customer Service at serviceastm.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 Sposition, consiste
18、ncy, and other materials in the matrix thatare not produced by the biofilm microorganisms, are controlledby the physicochemical environment in which it exists.3.1.2 disinfectant, nchemicals used on inanimate surfacesto rapidly inactivate 99.9 % of the treated microorganisms at aspecific concentratio
19、n and desired exposure time.3.2 Definitions of Terms Specific to This Standard:3.2.1 peg, nbiofilm sample surface (base: 5.0 mm, height:13.1 mm).3.2.2 peg lid, nan 86 3 128 mm plastic surface consistingof ninety-six (96) identical pegs.3.2.3 plate, nan 86 3 128 mm standard plate consistingof ninety-
20、six (96) identical wells.3.2.4 well, nsmall reservoir with a 50 to 200 L workingvolume capacity.3.3 Acronyms:3.3.1 ATCCAmerican Type Culture Collection3.3.2 BGCbiofilm growth check3.3.3 CFUcolony forming unit3.3.4 MBECminimum biofilm eradication concentration3.3.5 rpmrevolutions per minute3.3.6 SCst
21、erility control3.3.7 TSAtryptic soy agar3.3.8 TSBtryptic soy broth3.3.9 UCuntreated control4. Summary of Test Method4.1 This test method describes the use of the MBEC Assayin evaluating the efficacy of a disinfectant against a Pseudomo-nas aeruginosa biofilm. A mature biofilm is established onpegs u
22、nder batch conditions with very low shear produced bygentle rotation of the device on an orbital shaker. At the end of24 h of growth, the pegs containing the biofilm are rinsed toremove planktonic cells and the peg lid is placed in a receiverplate. The wells in the receiver plate are filled accordin
23、g to anexperimental design that contains the appropriate sterility,growth, and neutralizer controls as well as the disinfectants.After a specified contact time, the peg lid is placed in a receiverplate containing neutralizer, and the entire device is placed ina sonicator to remove the biofilm and di
24、saggregate the clumps.Samples from each well are then diluted, plated and the viablecells enumerated. The log reduction in viable cells is calculatedby subtracting the mean log density for the treated biofilm fromthe mean log density determined for the untreated controls.5. Significance and Use5.1 V
25、egetative biofilm bacteria are phenotypically differentfrom suspended planktonic cells of the same genotype. Biofilmgrowth reactors are engineered to produce biofilms withspecific characteristics. Altering either the engineered systemor operating conditions will modify those characteristics. Thegoal
26、 in biofilm research and efficacy testing is to choose thegrowth reactor that generates the most relevant biofilm for theparticular study.5.2 The purpose of this test method is to direct a user in howto grow, treat, sample and analyze a Pseudomonas aeruginosabiofilm using the MBEC Assay. Microscopic
27、ally, the biofilm issheet-like with few architectural details as seen in Harrison etal (5). The MBECAssay was originally designed as a rapid andreproducible assay for evaluating biofilm susceptibility toantibiotics (2). The engineering design allows for the simulta-neous evaluation of multiple test
28、conditions, making it anefficient method for screening multiple disinfectants or mul-tiple concentrations of the same disinfectant. Additional effi-ciency is added by including the neutralizer controls within theassay device. The small well volume is advantageous fortesting expensive disinfectants,
29、or when only small volumes ofthe disinfectant are available.6. Apparatus6.1 Inoculating loopnichrome wire or disposable plastic.6.2 Petri dishsquare 100 3 100 3 15 mm, plastic, sterile.6.3 Microcentrifuge tubessterile, any with a 1.5 mLvolume capacity.6.4 96-well microtiter platesterile, 86 3 128 mm
30、 standardplate consisting of ninety-six (96) identical flat bottom wellswith a 200 L working volume.7NOTE 1Alignment corner must be in the H12 position of the plate forproper alignment with the MBEC lid (see Fig. 1).6.5 Vortexany vortex that will ensure proper agitation andmixing of microfuge tubes.
31、6.6 Bath sonicatorany capable of an average sonic powerof 180 W in a dry environment (7).6.7 Stainless steel insert trayfor bath sonicator.6.8 Bunsen burnerused to flame-sterilize inoculating loop(if metal) and other instruments.6.9 95 % Ethanolused to flame-sterilize pliers.6.10 4-in. bent needle n
32、ose pliersfor aseptic removal andhandling of pegs.6.11 Pipettecontinuously adjustable pipette with volumecapability of 1 mL.6.12 Micropipettecontinuously adjustable pipette withworking volume of 10 to 200 L.6.13 Sterile pipette tips200 L and 1000 L volumes.6.14 Sterile reagent reservoir50 mL polysty
33、rene.6.15 Analytical balancesensitive to 0.01 g.6.16 Sterilizerany steam sterilizer capable of producingthe conditions of sterilization.6.17 Colony counterany one of several types may beused. A hand tally for the recording of the bacterial count isrecommended if manual counting is done.6.18 Environm
34、ental incubatorcapable of maintaining atemperature of 35 6 2C and relative humidity between 35 and85 %.6.19 Orbital shakercapable of maintaining an orbit of 110to 150 rpm.6.20 Reactor componentsthe MBEC Assay device isshown in Fig. 1. Fig. 3 is a diagram of the challenge plate.6.21 Sterile conical t
35、ubes50 mL, used to prepare initialinoculum.7The sole source of microtiter plates (Nunclon (trademarked) Catalogue No.167008) that provide reproducible results is Thermo Fisher Scientific, Waltham,MA, USA, . If you are aware of alternative suppliers, pleaseprovide this information to ASTM Internation
36、al Headquarters. Your comments willreceive careful consideration at a meeting of the responsible technical committee,1which you may attend.E2799 1126.22 Appropriate glasswareas required to make mediaand agar plates.6.23 Erlenmeyer flaskused for growing broth inoculum.7. Reagents and Materials7.1 Pur
37、ity of Waterall references to water as diluent orreagent shall mean distilled water or water of equal purity.7.2 Culture Media:7.2.1 Bacterial Growth BrothTryptic soy broth (TSB)prepared according to manufacturers directions.7.2.2 Bacterial Plating MediumTryptic soy agar (TSA)prepared according to m
38、anufacturers directions.7.3 Buffered Water0.0425 g KH2PO4/L distilled water,filter-sterilized and 0.405 g MgCl6H2O/L distilled water;filter-sterilized (prepared according to Method 9050).7.4 Neutralizerappropriate to the disinfectant beingevaluated (see Test Method E1054).7.5 Disinfectantstock conce
39、ntration.8. Culture/Inoculum Preparation8.1 Pseudomonas aeruginosa ATCC 15442 is the organismused in this test.8.2 Using a cryogenic stock (at 70C), streak out asubculture of P. aeruginosa on TSA.8.3 Incubate at 35 6 2C for 16 to 18 h.8.4 Aseptically remove isolated colony from streak plateand inocu
40、late 200 mL of sterile bacterial growth broth (TSB).8.5 Incubate flask at 35 6 2C and 150 6 10 rpm for 16 to18 h. Viable bacterial density should be $108CFU/mL andmay be checked by serial dilution and plating.8.6 Pipette 10 L from the incubation flask into 100 mL ofTSB to adjust the inoculum to an a
41、pproximate cell density of105CFU/mL. Vortex the diluted sample for approximately 10s to achieve a homogeneous distribution of cells.8.7 Perform 10-fold serial dilutions of the inoculum intriplicate.8.8 Spot plate 20 L of the serial dilutions from 100to 10-7on an appropriately labelled series of TSA
42、plates. Incubate theplates at 35 6 2C for 16 to 18 h and enumerate (8).9. Procedure9.1 An overview of the procedure is shown in Fig. 2.9.2 Growth of Biofilm:9.2.1 Open the sterile package containing the MBEC de-vice.9.2.2 Transfer 25 mL of the inoculum prepared in 8.6 into asterile reagent reservoir
43、.9.2.3 Using a micropipette, add 150 L of the inoculum toeach well (exclude columns 9 to 11 and A12, B12, and C12) ofthe 96-well tissue culture plate packaged with the MBECdevice.NOTE 2Wells A12, B12, and C12 serve as sterility controls and mustNOT be filled with inoculum. Columns 9 to 11 are spare,
44、 empty wells.96-well tissue culture plate (bottom) andcorresponding 96-peg lid (top).FIG. 1 MBEC Assay DeviceE2799 1139.2.4 Place the peg lid onto the microtiter plate. Ensure thatthe orientation of the plate matches the orientation of the lid(that is, peg A1 must be inserted into well A1 of the mic
45、rotiterplate, otherwise the device will not fit together correctly, seeFig. 1). Label the device appropriately.NOTE 3Volume of inoculum used in this step has been calibrated suchthat the biofilm covers a surface area that is entirely immersed by thevolume of antimicrobial used in the challenge plate
46、 setup (Section 9.4).Using a larger volume of inoculum might lead to biofilm formation highon the peg that physically escapes exposure during the challenge step.9.2.5 Place the device on the orbital shaker in a humidifiedincubator (to prevent evaporation). Set shaker to 110 6 10 rpmto prevent spillo
47、ver. Incubate at 35 6 2C for 16 to 18 h.9.3 Biofilm Growth Check:9.3.1 Using flame-sterilized pliers held flush against lid tominimize contact with attached biofilm, break off five (5) pegsD12, E12, F12, G12, and H12.9.3.2 Place each peg into a separate sterile microfuge tubethat contains 1.0 mL of
48、buffered water.9.3.3 Float a stainless steel insert tray in the center of asonicator. Place the peg-containing tubes in the tray andsonicate on high for 30 6 5 min (7).9.3.4 Serially dilute by transferring 0.1 mL to sterile mi-crofuge tubes containing 0.9 mL buffered water and spot plateon TSA (7).
49、This serves as a biofilm growth check.9.4 Preparation of Challenge Plate:9.4.1 Using a sterile 96-well microtiter plate, the next stepswill describe how to aseptically prepare the challenge plate(Fig. 3).9.4.2 Prepare 100 mL stock solution of disinfectant.NOTE 4(Optional) Measure disinfectant concentration to ensure ac-curacy (9).9.4.3 Add 200 L of sterile TSB to well A12 of thechallenge plate. This will serve as the device sterility control(SC).9.4.4 Add 200 L of sterile neutralizer
