1、Designation: E2799 12E2799 17Standard 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
2、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 treat a Pseud
3、omonas 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 (96) individ
4、ual wells that have a maximum 200-L 200 L working volume. Biofilm is established on the pegs underbatch conditions (that is, no flow of nutrients into or out of an individual well) with gentle mixing. The established biofilm istransferred to a new receiver plate for disinfectant efficacy testing.3,4
5、 The reactor design allows for the simultaneous testing ofmultiple disinfectants or one disinfectant with multiple concentrations, and replicate samples, making the assay an efficientscreening tool.1.2 This test method defines the specific operational parameters necessary for growing a Pseudomonas a
6、eruginosa 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 biofilm and
7、 quantify viable cells. Biofilm population density is recorded aslog10 colony forming units per surface area. Efficacy is reported as the log10 reduction 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 standard
8、. 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 any s
9、uch 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 hea
10、lth practices and determine the applicability of regulatorylimitations prior to use.1.9 This international standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guide
11、s and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:6E1054 Test Methods for Evaluation of Inactivators of Antimicrobial Agents1 This test method is under the jurisdiction of ASTM Committee E35 on Pesticides
12、, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2012April 1, 2017. Published June 2012May 2017. Originally approved in 2011. Last previous edition approved in 20112012 asE2799 11.E2799 1
13、2. DOI: 10.1520/E279912.10.1520/E279917.2 The MBEC trademark is held by Innovotech, Inc., Edmonton, Alberta, Canada.3 The sole source of supply of the apparatus known to the committee at this time is Innovotech Inc., Edmonton, Alberta, Canada. If you are aware of alternative suppliers,please provide
14、 this information to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee,1which you may attend.4 The MBEC Assay is covered by a patent. Interested parties are invited to submit information regarding the identification
15、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,1 which you may attend.5 The boldface numbers in parentheses refer to a list of references at the end of this standar
16、d.6 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.This document is not an ASTM standard and is intended only
17、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 recommends that users consult prior editions as appropriate. In all cases only the current version
18、of 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 States12.2 Other Standards:Method 9050 C.1.a Buffered Dilution Water Preparation according to EatonRice et al (5)3
19、. Terminology3.1 Definitions:3.1.1 biofilm, nmicroorganisms living in a self-organized community attached to surfaces, interfaces, or each other, embeddedin a matrix of extracellular polymeric substances of microbial origin, while exhibiting altered phenotypes with respect to growthrate and gene tra
20、nscription.3.1.1.1 DiscussionBiofilms may be comprised of bacteria, fungi, algae, protozoa, viruses, or infinite combinations of these microorganisms. Thequalitative characteristics of a biofilm including, but not limited to, population density, taxonomic diversity, thickness, chemicalgradients, che
21、mical composition, consistency, and other materials in the matrix that are not produced by the biofilmmicroorganisms, are controlled by the physicochemical environment in which it exists.3.1.2 disinfectant, nchemicals used on inanimate surfaces to rapidly inactivate 99.9 % of the treated microorgani
22、sms at aspecific concentration and desired exposure time.3.2 Definitions of Terms Specific to This Standard:3.2.1 peg, nbiofilm samplegrowth surface (base: 5.0 mm, height: 13.1 mm).3.2.2 peg lid, nan 86-86 128-mm 128 mm plastic surface consisting of ninety-six (96) identical pegs.3.2.3 plate, nan 86
23、-86 128-mm 128 mm standard plate consisting of ninety-six (96) identical wells.3.2.4 well, nsmall reservoir with a 50-50 to 200-L 200 L working volume 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 er
24、adication 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 of a disinfectant against a Pseudomo
25、nasaeruginosa 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 planktoniccells and the peg lid is placed in a rec
26、eiver 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 plate containing neutralizer, and the
27、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 log10 reduction inviable cells is calculated by subtracting the mean log10 density for the treated biofilm from the mean log10
28、 density 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 characteristics.Altering either the engineer
29、ed 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 user in how to grow, treat, sample
30、 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(6). The MBECAssay was originally designed as a rapid and reproducible assay for evaluating biofilm susceptibility to antibiotics(2).
31、The engineering design allows for the simultaneous evaluation of multiple test conditions, making it an efficient method forscreening multiple disinfectants or multiple concentrations of the same disinfectant.Additional efficiency is added by including theE2799 172neutralizer controls within the ass
32、ay device. The small well volume is advantageous for testing expensive disinfectants, or whenonly small volumes of the disinfectant are available.6. Apparatus6.1 Inoculating loopnichrome wire or disposable plastic.6.2 Petri dishsquare 100-100 100-100 15-mm, 15 mm, plastic, sterile.6.3 Microcentrifug
33、e tubessterile, any with a 1.5-mL 1.5 mL volume capacity.6.4 96-well microtiter platesterile, 86-86 128-mm 128 mm standard plate consisting of ninety-six (96) identical flat bottomwells with a 200-L 200 L working volume.7NOTE 1Alignment corner must be in the H12 position of the plate for proper alig
34、nment with the MBEC lid (see Fig. 1).6.5 Vortexany vortex that will ensure proper agitation and mixing of microfuge tubes.6.6 Bath sonicatorany capable 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-sterili
35、ze inoculating loop (if metal) and other instruments.6.9 95 % Ethanolused to flame-sterilize pliers.6.10 4-in. 4 in. bent needle nose pliersfor aseptic removal and handling of pegs.6.11 PipettePipette(s)continuously adjustable pipettepipette(s) with volume capacity of 1 mL.6.12 MicropipetteMicropipe
36、tte(s)continuously adjustable pipettepipette(s) with working volume of 10 to 200 L.6.13 Sterile pipette tips200-L and 1000-L 200 L and 1000 L volumes.6.14 Sterile reagent reservoir50-mL 50 mL polystyrene.7 The sole source of microtiter plates (Nunclon (trademarked) Catalogue No. 167008) that provide
37、 reproducible results is Thermo Fisher Scientific, Waltham, MA, USA,. If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive carefulconsideration at a meeting of the responsible technical committee,1 which you may att
38、end.96-well tissue culture plate (bottom) andcorresponding 96-peg lid (top).FIG. 1 MBEC Assay DeviceE2799 1736.15 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
39、hand tally for the recording of the bacterial count isrecommended if manual counting is done.6.18 Environmental incubatorcapable of maintaining a temperature of 35 6 2C and relative humidity between 35 and85 %.85 %.6.19 Orbital shakercapable of maintaining an orbit of 110 to 150 rpm.6.20 Reactor com
40、ponentsthe MBEC Assay device is shown in Fig. 1. Fig. 2 is a diagram of the challenge plate.6.21 Sterile conical tubes50-mL, used to prepare initial inoculum.6.21 Appropriate glasswareas required to make media and agar plates.6.22 Erlenmeyer flaskused for growing broth inoculum.7. Reagents and Mater
41、ials7.1 Purity of Waterall references to water as diluent or reagent 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
42、according to manufacturers directions.7.3 Buffered Water0.0425 g KH2PO4/L distilled water, filter-sterilized and 0.4050.4055 g MgCl6H2O/L distilled water;filter-sterilized (prepared according to Method (Method 9050 C.1.a).7.4 Neutralizerappropriate to the disinfectant being evaluated (see Test Metho
43、d E1054).7.5 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),70C), streak out a subculture of P. aeruginosa on TSA.8.3 Incubate at 35 6 2C for 16 to 18 h.8.4 Aseptically remove
44、 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 108 CFU/mL and maybe checked by serial dilution and plating.8.6 Pipette 10 L from the incubation flask int
45、o 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.Columns 1 through 5 are test disinfectant (n
46、=5). Column 6 serves as the neutralizer effectiveness control. Column 7 serves as the neutralizer toxicity control (N).Column 8 is the untreated control for each row (UC). Column 12, rows A to C are sterility controls for each experiment (SC), rows D to H are the biofilm growthcheck controls (BGC).
47、Lined out cells are spare (columns 9, 10 and 11). The numbers in columns 1 to 5 refer to the percentage of undiluted sample with 100representing 100 % concentration of the stock solution, 50 representing a 50 % concentration of the stock solution and so on.FIG. 2 Challenge Plate PreparationE2799 174
48、8.8 Spot plate 20 Lof the serial dilutions from 100 to 10-7 on 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. 3.9.2 Growth of Biofilm:9.2.1 Open the sterile package containing
49、 the MBEC device.9.2.2 Transfer 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 sterility 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
