1、Designation:E279911 Designation: E2799 12Standard 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
2、 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 specifies the operational parameters required to grow and t
3、reat a Pseudomonas aeruginosa biofilm in a highthroughput screening assay known as the MBEC (trademarked)2(Minimum Biofilm Eradication Concentration) Physiology andGenetics Assay. The assay device consists of a plastic lid with ninety-six (96) pegs and a corresponding receiver plate withninety-six (
4、96) individual wells that have a maximum 200-L working volume. Biofilm is established on the pegs under batchconditions (that is, no flow of nutrients into or out of an individual well) with gentle mixing. The established biofilm is transferredto a new receiver plate for disinfectant efficacy testin
5、g.3, 4The reactor design allows for the simultaneous testing of multipledisinfectants or one disinfectant with multiple concentrations, and replicate samples, making the assay an efficient screening tool.1.2 This test method defines the specific operational parameters necessary for growing a Pseudom
6、onas aeruginosa biofilm,although the device is versatile and has been used for growing, evaluating and/or studying biofilms of different species as seen inRefs (1-4).51.3 Validation of disinfectant neutralization is included as part of the assay.1.4 This test method describes how to sample the biofi
7、lm and quantify viable cells. Biofilm population density is recorded aslog10colony forming units per surface area. Efficacy is reported as the log10reduction of viable cells.1.5 Basic microbiology training is required to perform this assay.1.6 The values stated in SI units are to be regarded as stan
8、dard. No other units of measurement are included in this standard.1.7 ASTM International takes no position respecting the validity of any patent rights asserted in connection with any itemmentioned in this standard. Users of this standard are expressly advised that determination of the validity of a
9、ny such patent rights,and the risk of infringement of such rights, are entirely their own responsibility.1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and
10、 health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:6E1054 Test Methods for Evaluation of Inactivators of Antimicrobial Agents2.2 Other Standards:Method 9050 C.1.a Buffered Dilution Water Preparation according to Eaton et
11、al (5)3. Terminology3.1 Definitions:3.1.1 biofilm, nmicroorganisms living in a self-organized, cooperative self-organized community attached to surfaces,interfaces, or each other, embedded in a matrix of extracellular polymeric substances of microbial origin, while exhibiting an1This test method is
12、under the jurisdiction ofASTM Committee E35 on Pesticides,Antimicrobials, andAlternative Control MethodsAgents and is the direct responsibilityof Subcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2011. Published April 2011. DOI: 10.1520/E279911.Current edition approvedApr
13、il 1, 2012. Published June 2012. Originally approved in 2011. Last previous edition approved in 2011 as E2799 11. DOI: 10.1520/E279912.2The MBEC trademark is held by Innovotech, Inc., Edmonton, Alberta, Canada.3The sole source of supply of the apparatus known to the committee at this time is Innovot
14、ech Inc., Edmonton, Alberta, Canada. If you are aware of alternative suppliers,please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee,which you may attend.4The MBEC Assay is covered by a
15、 patent. Interested parties are invited to submit information regarding the identification of an alternative(s) to this patented item to theASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee, which you may attend.5The
16、 boldface numbers in parentheses refer to a list of references at the end of this standard.6For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document S
17、ummary page on the ASTM website.1This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recomm
18、ends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.altered phenoty
19、pes with respect to growth rate and gene transcription.3.1.1.1 DiscussionBiofilms may be comprised of bacteria, fungi, algae, protozoa, viruses, or infinite combinations of thesemicroorganisms. The qualitative characteristics of a biofilm including, but not limited to, population density, taxonomic
20、diversity,thickness, chemical gradients, chemical composition, consistency, and other materials in the matrix that are not produced by thebiofilm microorganisms, are controlled by the physicochemical environment in which it exists.3.1.2 disinfectant, nchemicals used on inanimate surfaces to rapidly
21、inactivate 99.9 % of the treated microorganisms at aspecific concentration 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 consisting of ninety-six (96) ide
22、ntical pegs.3.2.3 plate, nan 86- 3 128-mm standard plate consisting of ninety-six (96) identical wells.3.2.4 well, nsmall reservoir with a 50- to 200-L working volume capacity.3.3 Acronyms:3.3.1 ATCCAmerican Type Culture Collection3.3.2 BGCbiofilm growth check3.3.3 CFUcolony forming unit colony-form
23、ing unit3.3.4 MBECminimum biofilm eradication concentration3.3.5 rpmrevolutions per minute3.3.6 SCsterility 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 Assay in evaluating the efficacy
24、of a disinfectant against a Pseudomonasaeruginosa biofilm.Amature biofilm is established on pegs under batch conditions with very low shear produced by gentle rotationof the device on an orbital shaker. At the end of 24 h of growth, the pegs containing the biofilm are rinsed to remove planktoniccell
25、s and the peg lid is placed in a receiver plate. The wells in the receiver plate are filled according to an experimental design thatcontains the appropriate sterility, growth, and neutralizer controls as well as the disinfectants. After a specified contact time, thepeg lid is placed in a receiver pl
26、ate containing neutralizer, and the entire device is placed in a sonicator to remove the biofilm anddisaggregate the clumps. Samples from each well are then diluted, plated and the viable cells enumerated. The log10reduction inviable cells is calculated by subtracting the mean log10density for the t
27、reated biofilm from the mean log10density determined forthe untreated controls.5. Significance and Use5.1 Vegetative biofilm bacteria are phenotypically different from suspended planktonic cells of the same genotype. Biofilmgrowth reactors are engineered to produce biofilms with specific characteris
28、tics. Altering either the engineered system or operatingconditions will modify those characteristics. The goal in biofilm research and efficacy testing is to choose the growth reactor thatgenerates the most relevant biofilm for the particular study.5.2 The purpose of this test method is to direct a
29、user in how to grow, treat, sample and analyze a Pseudomonas aeruginosabiofilm using the MBEC Assay. Microscopically, the biofilm is sheet-like with few architectural details as seen in Harrison et al(5(6). The MBEC Assay was originally designed as a rapid and reproducible assay for evaluating biofi
30、lm susceptibility toantibiotics (2). The engineering design allows for the simultaneous evaluation of multiple test conditions, making it an efficientmethod for screening multiple disinfectants or multiple concentrations of the same disinfectant. Additional efficiency is added byincluding the neutra
31、lizer controls within the assay device. The small well volume is advantageous for testing expensivedisinfectants, or when only small volumes of the disinfectant are available.6. Apparatus6.1 Inoculating loopnichrome wire or disposable plastic.6.2 Petri dishsquare 100 3 100 3 15 mm, plastic, sterile.
32、 square 100- 3 100- 3 15-mm, plastic, sterile.6.3 Microcentrifuge tubessterile, any with a 1.5-mL volume capacity.6.4 96-well microtiter platesterile, 86- 3 128-mm standard plate consisting of ninety-six (96) identical flat bottom wells witha 200-L working volume.7NOTE 1Alignment corner must be in t
33、he H12 position of the plate for proper alignment with the MBEC lid (see Fig. 1).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, please prov
34、ide this information to ASTM International Headquarters. Your comments will receive carefulconsideration at a meeting of the responsible technical committee, which you may attend.E2799 1226.5 Vortexany vortex that will ensure proper agitation and mixing of microfuge tubes.6.6 Bath sonicatorany capab
35、le of an average sonic power of 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 nose pliersfor aseptic rem
36、oval and handling of pegs.6.11 Pipettecontinuously adjustable pipette with volume capabilitycapacity of 1 mL.6.12 Micropipettecontinuously adjustable pipette with working volume of 10 to 200 L.6.13 Sterile pipette tips200 L and 1000 L volumes. 200-L and 1000-L volumes.6.14 Sterile reagent reservoir5
37、0 mL polystyrene. 50-mL polystyrene.6.15 Analytical balancesensitive to 0.01 g.6.16 Sterilizerany steam sterilizer capable of producing the conditions of sterilization.6.17 Colony counterany one of several types may be used. A hand tally for the recording of the bacterial count isrecommended if manu
38、al counting is done.6.18 Environmental incubatorcapable of maintaining a temperature of 35 6 2C and relative humidity between 35 and 85 %.6.19 Orbital shakercapable of maintaining an orbit of 110 to 150 rpm.6.20 Reactor componentsthe MBEC Assay device is shown in Fig. 1. Fig.3Fig. 2 is a diagram of
39、the challenge plate.6.21 Sterile conical tubes50-mL, used to prepare initial inoculum.6.22 Appropriate glasswareas required to make media and agar plates.6.23 Erlenmeyer flaskused for growing broth inoculum.7. Reagents and Materials7.1 Purity of Waterall references to water as diluent or reagent sha
40、ll 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 manufacturers directions.7.3 Buffered Water0.0425 g KH2PO4/
41、L distilled water, filter-sterilized and 0.405 g MgCl6H2O/L distilled water; filter-sterilized (prepared according to Method 9050 C.1.a).7.4 Neutralizerappropriate to the disinfectant being evaluated (see Test Method E1054).96-well tissue culture plate (bottom) andcorresponding 96-peg lid (top).FIG.
42、 1 MBEC Assay DeviceE2799 1237.5 Disinfectantstock concentration.8. Culture/Inoculum Preparation8.1 Pseudomonas aeruginosa ATCC 15442 is the organism used in this test.8.2 Using a cryogenic stock (at 70C), streak out a subculture of P. aeruginosa on TSA.8.3 Incubate at 35 6 2C for 16 to 18 h.8.4 Ase
43、ptically remove isolated colony from streak plate and inoculate 200 mL of sterile bacterial growth broth (TSB).8.5 Incubate flask at 35 6 2C and 150 6 10 rpm for 16 to 18 h. Viable bacterial density should be $108CFU/mL and maybe checked by serial dilution and plating.8.6 Pipette 10 L from the incub
44、ation flask into 100 mL of TSB to adjust the inoculum to an approximate cell density of 105CFU/mL. Vortex the diluted sample for approximately 10 s to achieve a homogeneous distribution of cells.8.7 Perform 10-fold serial dilutions of the inoculum from 8.6 in triplicate.8.8 Spot plate 20 L of the se
45、rial dilutions from 100to 10-7on an appropriately labelled series of TSAplates. Incubate the platesat 35 6 2C for 16 to 18 h and enumerate (8).9. Procedure9.1 An overview of the procedure is shown in Fig. 2Fig. 3.9.2 Growth of Biofilm:9.2.1 Open the sterile package containing the MBEC device.9.2.2 T
46、ransfer 25 mL of the inoculum prepared in 8.6 into a sterile reagent reservoir.9.2.3 Using a micropipette, add 150 L of the inoculum to each well (exclude columns 9 to 11 and A12, B12, and C12) of the96-well tissue culture plate packaged with the MBEC device.NOTE 2Wells A12, B12, and C12 serve as st
47、erility controls and must NOT be filled with inoculum. Columns 9 to 11 are spare, empty wells.9.2.4 Place the peg lid onto the microtiter plate. Ensure that the orientation of the plate matches the orientation of the lid (thatis, peg A1 must be inserted into well A1 of the microtiter plate, otherwis
48、e the device will not fit together correctly, see Fig. 1).Label the device appropriately.NOTE 3Volume of inoculum used in this step has been calibrated such that the biofilm covers a surface area that is entirely immersed by the volumeof antimicrobial used in the challenge plate setup (Section 9.4).
49、 Using a larger volume of inoculum might lead to biofilm formation high on the peg thatphysically escapes exposure during the challenge step.9.2.5 Place the device on the orbital shaker in a humidified incubator (to prevent evaporation). Set shaker to 110 6 10 rpm toprevent spillover. 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 to minimize contact with attached biofilm, break off five (5) pegs D12,E12, F12, G12,
