1、Designation: E2871 12Standard Test Method forEvaluating Disinfectant Efficacy against Pseudomonasaeruginosa Biofilm Grown in CDC Biofilm Reactor usingSingle Tube Method1This standard is issued under the fixed designation E2871; the number immediately following the designation indicates the year ofor
2、iginal 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 specifies the operational parametersrequired
3、 to perform a quantitative liquid disinfectant efficacytest against biofilm bacteria.1.2 The test method was developed using a Pseudomonasaeruginosa biofilm grown in the CDC Biofilm Reactor (TestMethod E2562), modified to include borosilicate glass couponsas a hard nonporous surface and P. aeruginos
4、a ATCC 15442.1.3 Disinfectant preparation and contact time are used in theassessment according to the manufacturers instructions foruse.1.4 The test method uses a closed system to treat biofilm. Acoupon is placed in a single tube for the treatment, neutraliza-tion, and sampling steps to prevent the
5、loss of cells.1.5 Verification of disinfectant neutralization is determinedprior to conducting the test method.1.6 This test method describes how to sample and analyzetreated and untreated control biofilms for viable cells. Biofilmpopulation density is recorded as log10colony-forming unitsper coupon
6、. Efficacy is reported as a log10reduction of viablecells.1.7 Basic microbiology training is required to perform thisassay.1.8 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.9 This standard does not purport to address all of
7、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 of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1054 Test Methods for Ev
8、aluation of Inactivators of An-timicrobial AgentsE2562 Test Method for Quantification of Pseudomonasaeruginosa Biofilm Grown with High Shear and Continu-ous Flow using CDC Biofilm Reactor2.2 Other Standards:Method 9050 C.1.a Buffered Dilution Water Preparationaccording to Eaton et al (1)33. Terminol
9、ogy3.1 Definitions:3.1.1 biofilm, nmicroorganisms living in a self-organizedcommunity attached to surfaces, interfaces, or each other,embedded in a matrix of extracellular polymeric substances ofmicrobial origin, while exhibiting altered phenotypes withrespect to growth rate and gene transcription.3
10、.1.1.1 DiscussionBiofilm may be comprised of bacteria,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, chemicalcompositio
11、n, consistency, 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 contact time, npredetermined time that the biofilmis exposed to the activity of a disinfectant.3.1.3 coupon, nbiofilm growth
12、 surface.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2012. Published June 2012. DOI: 10.1520/E287112.2Fo
13、r 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 standards Document Summary page onthe ASTM website.3The boldface numbers in parentheses refer to a list of referenc
14、es at the end ofthis standard.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.4 disinfectant, na chemical that destroys vegetativeforms of microorganisms, but does not ordinarily kill bacterialspores.3.2 Acronyms:3.2.1 ATCCAmeric
15、an Type Culture Collection.3.2.2 CDCCenters for Disease Control and Prevention.3.2.3 CFUcolony-forming unit.4. Summary of Test Method4.1 This test method describes the use of the single tubemethod to evaluate the efficacy of a liquid disinfectant againsta Pseudomonas aeruginosa biofilm on a hard non
16、poroussurface grown in the CDC Biofilm Reactor. The test methodconsists of adding a disinfectant (treated) or a control buffer(untreated) to individual coupons held in 50-mL conical tubes.Three coupons are treated with disinfectant and three couponsreceive buffered dilution water. Neutralizer is add
17、ed to thetubes after the appropriate contact time. A combination ofvortexing and sonication are used to remove the biofilm fromthe coupon and disaggregate the clumps. The cell suspension isserially diluted and plated on agar medium. Viable plate countsfrom treated and untreated control coupons are u
18、sed to calcu-late the log10reduction of viable cells.5. Significance and Use5.1 Vegetative biofilm bacteria are phenotypically differentfrom suspended planktonic cells of the same genotype. Biofilmgrowth reactors are engineered to produce biofilms withspecific characteristics (2). Altering either th
19、e engineeredsystem or operating conditions will modify those characteris-tics as well as the physicochemical environment. The goal inbiofilm research and efficacy testing is to choose the growthreactor and operating conditions that generate the most relevantbiofilm for the particular study.5.2 The t
20、est method was developed using Pseudomonasaeruginosa ATCC 15442 biofilm grown on borosilicate glasscoupons in the CDC Biofilm Reactor and liquid disinfectants.Efficacy data developed using other bacteria, different shear,different coupons, or other standardized biofilm reactor sys-tems, and/or other
21、 forms of disinfectants may result in differentlog10reduction (LR) values and repeatability and reproducibil-ity standard deviations.5.3 The efficacy test was designed to determine the log10reduction in bacteria after exposure to a disinfectant in a closedsystem.5.4 The test method was developed usi
22、ng 50-mL conicaltubes. The conical geometry allows for disinfectant exposure tobiofilm on all surfaces of the coupon.5.5 Each efficacy test includes a single contact time andtemperature for three untreated control coupons (exposed tobuffered dilution water) and three treated coupons (perdisinfectant
23、/concentration combination).6. Apparatus6.1 Conical centrifuge tubes, sterile, any with 50-mL vol-ume capacity and secure leakproof lids.6.2 Ultrasonic water bath, any capable of maintaining ahomogeneous sound distribution at 45 kHz with a variablepower setting and a volume large enough to accommoda
24、te50-mL conical tubes in a wet environment.6.3 Test tube rack, any capable of holding 50-mL conicalcentrifuge tubes.6.4 Micropipettes, continuously adjustable pipettes withvolume capacity of 100 L and 1000 L.6.5 Sterile pipette tips, 100-L and 1000-L volumes.6.6 Bunsen burner, used to flame-steriliz
25、eAllen wrench andplate spreader.6.7 95 % Ethanol, used to flame-sterilize Allen wrench andplate spreader.6.8 Small Allen wrench, for loosening set screws andpushing coupons out of reactor rods.6.9 Timer, any that can display time in seconds.6.10 Vortex mixer, any vortex that will ensure properagitat
26、ion and mixing of centrifuge tubes.6.11 Serological pipettes, sterile single-use pipettes withvolume capacity of 1, 5, 10, 25, and 50 mL.6.12 Plate spreader, for spreading serial dilutions on agarplates.6.13 Water bath, any capable of maintaining a constanttemperature of 20 6 1C.6.14 Sterilizer, any
27、 steam sterilizer capable of producing theconditions of sterilization.6.15 Colony counter, any one of several types may be used.A hand tally for recording of the bacterial count is recom-mended if manual counting is done.6.16 Environmental incubator, any capable of maintaining atemperature of 36 6 2
28、C.6.17 Appropriate glassware/plasticware, as required tomake media and agar plates.6.18 Volumetric flasks, used for preparing disinfectants.6.19 Magnetic stir bars, sterile, for mixing prepared disin-fectant.6.20 Magnetic stir plate, any capable of mixing.7. Reagents and Materials7.1 Purity of Water
29、all references to water as diluent orreagent shall mean distilled water or water of equal purity.7.2 Bacterial Plating MediumR2A agar is recommended.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 M
30、ethod 9050 C.1.aBuffered Dilution Water Preparation (1).7.4 Disinfectantproduct to be tested.7.5 NeutralizerDey/Engley Neutralization Broth or onespecific to the disinfectant being evaluated as determined foreffectiveness and toxicity according to Test Method E1054.8. Culture/Inoculum Preparation8.1
31、 Borosilicate glass coupons with mature Pseudomonasaeruginosa ATCC 15442 biofilm grown according to TestMethod E2562 through step 10.2.4.9. Procedure9.1 The test is conducted with three treated and threeuntreated control coupons.9.2 An overview of the procedure is shown in Fig. 1.E2871 1229.3 Prepar
32、e Disinfectant:9.3.1 Prepare disinfectant according to manufacturersspecifications in sterile volumetric glassware. Ensure that thedisinfectant is adequately mixed. Use within3hofpreparationor as specified in the manufacturers instructions.9.3.2 Place prepared disinfectant in water bath equilibrated
33、to 20 6 1C for 10 to 15 min.9.4 Remove Coupons from the CDC Biofilm Reactor:9.4.1 Prepare sampling materials: treatment tubes, rinsetubes, small flame-sterilized Allen wrench, and serologicalpipettes.9.4.2 Aseptically remove a randomly selected rod contain-ing coupons with biofilm from the CDC Biofi
34、lm Reactor bypulling it straight up firmly.9.4.3 Rinse the coupons to remove planktonic cells. Orientthe rod in a vertical position directly over a 50-mL conicalcentrifuge tube that contains 30-mL sterile buffered water.Immerse the rod with a continuous motion into the bufferedwater with minimal to
35、no splashing, then immediately remove.A new 50-mL conical tube containing 30-mL sterile bufferedwater is used for each rod.9.4.4 Hold the rod with one of the randomly selectedcoupons centered over an empty, sterile 50-mL conical tube.Loosen the set screw and allow the coupon to drop directly tothe b
36、ottom of the tube. If the coupon does not freely drop, pressdirectly in the center of the coupon with the Allen wrench usedto loosen the set screw.NOTE 1The use of 50-mL conical tubes allows for uniform disinfec-tant contact around the entire coupon surface.9.4.5 Repeat coupon removal twice more for
37、 a total of threetubes each containing a coupon.NOTE 2All biofilm on the coupon must be exposed to disinfectant. Donot lose any biofilm from the coupon by allowing it to touch the top or theinner sides of the 50-mL conical tube as it falls to the bottom of the tube.To ensure that the maximum biofilm
38、 surface area is in contact with thedisinfectant, the coupon should be at an angle in the bottom of the tube.Discard any tubes where the coupon touched the inner side of the tubeand/or held coupons that were not angled and replace them with new tubesand coupons.9.5 Conduct Effcacy Evaluation:9.5.1 S
39、lowly pipette 4 mL previously prepared and equili-brated disinfectant (treatment) or equilibrated buffered dilutionwater (untreated control) into the tubes containing the coupons,being careful to completely cover the coupon. Note and recordthe time.NOTE 3The order of application of disinfectant or b
40、uffered dilutionwater is randomly selected.NOTE 4For a 10-min contact time, a 1-min interval between couponsis recommended.9.5.2 Tap each tube to release any air bubbles trapped belowthe coupon. Do not shake the tubes.9.5.3 Incubate the tubes at 20 6 1C for the specifiedcontact time.9.5.4 At the end
41、 of the contact time, add appropriate volumeof neutralizer to each tube. Replace the cap and mix thoroughlyby vigorously shaking the tube several times.Allow the couponto remain in the neutralized disinfectant at room temperatureuntil step 9.6.NOTE 5It is recommended that a neutralization study be c
42、onductedprior to running the efficacy test (Test Method E1054) to determine theappropriate neutralizer formulation, concentration, volume, and neutral-ization time. For each efficacy test, the concentration, volume, andneutralization time should be the same for the control and treated coupons.Thirty
43、-six (36) mL of neutralizer was used in the collaborative study.9.5.5 Obtain a set of three coupons for the remainingtreatment(s) or control(s) as described in steps 9.4.2 through9.4.5.9.5.6 Repeat steps 9.5.1 through 9.5.4 with randomly se-lected treatment(s) or control(s).FIG. 1 Single Tube Method
44、 OverviewE2871 1239.6 Remove and Disaggregate Biofilm:9.6.1 Vortex each tube on the highest setting for 30 6 5s.9.6.2 Place all tubes into a test tube rack and suspend therack in the ultrasonic water bath so that the liquid level in thetubes is even with the water level in the tank of the bath.9.6.3
45、 Sonicate the tubes at 45 kHz for 30 6 5s.9.6.4 Vortex the tubes as described in 9.6.1.9.6.5 Sonicate the tubes as described in 9.6.2 and 9.6.3.9.6.6 Vortex the tubes as described in 9.6.1. These tubes arethe 100dilution.9.7 Dilute and Plate Disaggregated Biofilm Samples:9.7.1 Serially dilute the sa
46、mple in buffered water.9.7.2 Culture each dilution in duplicate for colony growthusing an accepted plating technique such as spread- or pour-plating.9.7.3 Incubate plates at 36 6 2C for 24 to 28 h.9.7.4 Count the appropriate number of colonies according tothe plating method used.10. Data Analysis10.
47、1 Calculate biofilm density.10.1.1 Calculate X, the arithmetic mean CFU from thereplicate samples plated (3).10.1.2 The log10density for each coupon is calculated asfollows:Log10CFU/coupon!5Log10X/B!V/D!# (1)where:X = average CFU of the replicate sample plates,B = volume plated,V = volume of disinfe
48、ctant or buffered water plus neutral-izer,D=10-k, andk = dilution.10.2 Calculate the mean log10density for each set of treatedand control coupons as follows:Mean LD 5 Log10Coupon A!1Log10Coupon B!1 Log10Coupon C!#/3 (2)10.3 Calculate the log10reduction for each disinfectant asfollows (4):LR 5 Mean L
49、og10Untreated Coupons Mean Log10Treated Coupons(3)10.4 Calculate the standard error of the mean LR as follows:SE of mean LR 5SD=m(4)where:SD = standard deviation of the LR, andm = number of experiments performed in a single labora-tory.10.5 Calculate the percent kill as follows:% Kill 5110LR! 3 100 (5)11. Precision and Bias11.1 Randomization is used whenever possible to reduce thepotential for systematic bias.11.2 Several potential biases exist for this method.11.2.1 A bias may occur between treated and control cou-pons due to increased
