1、Designation: E2871 12E2871 13Standard Test Method forEvaluating Disinfectant Efficacy againstAgainstPseudomonas aeruginosa Biofilm Grown in CDC BiofilmReactor usingUsing Single Tube Method1This standard is issued under the fixed designation E2871; the number immediately following the designation ind
2、icates the year oforiginal 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 operationa
3、l parameters required to perform a quantitative liquid disinfectant efficacy testagainst biofilm bacteria.1.2 The test method was developed using a Pseudomonas aeruginosa biofilm grown in the CDC Biofilm Reactor (Test MethodE2562), modified to include borosilicate glass coupons as a hard nonporous s
4、urface and P. aeruginosa ATCC 15442.1.3 Disinfectant preparation and contact time are used in the assessment according to the manufacturers instructions for use.1.4 The test method uses a closed system to treat biofilm. A coupon is placed in a single tube for the treatment, neutralization,and sampli
5、ng steps to prevent the loss of cells.1.5 Verification of disinfectant neutralization is determined prior to conducting the test method.1.6 This test method describes how to sample and analyze treated and untreated control biofilms for viable cells. Biofilmpopulation density is recorded as log10 col
6、ony-forming units per coupon. Efficacy is reported as a log10 reduction of viable cells.1.7 Basic microbiology training is required to perform this assay.1.8 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.9 This standard do
7、es 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 health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Stand
8、ards:2E1054 Test Methods for Evaluation of Inactivators of Antimicrobial AgentsE2562 Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow usingCDC Biofilm Reactor2.2 Other Standards:Method 9050 C.1.a Buffered Dilution Water Preparation according
9、to Eaton et al (1)33. 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 gro
10、wthrate and gene transcription.3.1.1.1 Discussion1 This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2012Oct.
11、1, 2013. Published June 2012November 2013. Originally approved in 2012. Last previous edition approved in 2012 asE287112. DOI: 10.1520/E287112.10.1520/E287113.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of A
12、STM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.3 The boldface numbers in parentheses refer to a list of references at the end of this standard.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indi
13、cation 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 versionof the standard as published by ASTM is to be c
14、onsidered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1Biofilm may be comprised of bacteria, fungi, algae, protozoa, viruses, or infinite combinations of these microorganisms. Thequalitative characteristics of
15、 a biofilm including, but not limited to, population density, taxonomic diversity, thickness, chemicalgradients, chemical composition, consistency, and other materials in the matrix that are not produced by the biofilmmicroorganisms, are controlled by the physicochemical environment in which it exis
16、ts.3.1.2 contact time, npredetermined time that the biofilm is exposed to the activity of a disinfectant.3.1.3 coupon, nbiofilm growth surface.3.1.4 disinfectant, na chemical that destroys vegetative forms of microorganisms, but does not ordinarily kill bacterial spores.3.2 Acronyms:3.2.1 ATCCAmeric
17、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 tube method to evaluate the efficacy of a liquid disinfectant against aPseudomonas aeruginosa biofilm on a hard no
18、nporous surface grown in the CDC Biofilm Reactor. The test method consists ofadding a disinfectant (treated) or a control buffer (untreated) to individual coupons held in 50-mL conical tubes. Three couponsare treated with disinfectant and three coupons receive buffered dilution water. Neutralizer is
19、 added to the tubes after theappropriate contact time. A combination of vortexing and sonication are used to remove the biofilm from the coupon anddisaggregate the clumps. The cell suspension is serially diluted and plated on agar medium. Viable plate counts from treated anduntreated control coupons
20、 are used to calculate the log10 reduction of viable cells.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 (2). Altering e
21、ither the engineered system oroperating conditions will modify those characteristics as well as the physicochemical environment. The goal in biofilm researchand efficacy testing is to choose the growth reactor and operating conditions that generate the most relevant biofilm for theparticular study.5
22、.2 The test method was developed using Pseudomonas aeruginosa ATCC 15442 biofilm grown on borosilicate glass couponsin the CDC Biofilm Reactor and liquid disinfectants. Efficacy data developed using other bacteria, different shear, differentcoupons, or other standardized biofilm reactor systems, and
23、/or other forms of disinfectants may result in different log10 reduction(LR) values and repeatability and reproducibility standard deviations.5.3 The efficacy test was designed to determine the log10 reduction in bacteria after exposure to a disinfectant in a closed system.5.4 The test method was de
24、veloped using 50-mLconical tubes.The conical geometry allows for disinfectant exposure to biofilmon all surfaces of the coupon.5.5 Each efficacy test includes a single contact time and temperature for three untreated control coupons (exposed to buffereddilution water) and three treated coupons (per
25、disinfectant/concentration combination).6. Apparatus6.1 Conical centrifuge tubes, sterile, any with 50-mL volume capacity and secure leakproof lids.6.2 Ultrasonic water bath, any capable of maintaining a homogeneous sound distribution at 45 kHz with a variable powersetting and a volume large enough
26、to accommodate 50-mL conical tubes in a wet environment.6.3 Test tube rack, any capable of holding 50-mL conical centrifuge tubes.6.4 Micropipettes, continuously adjustable pipettes with volume capacity of 100 L and 1000 L.6.5 Sterile pipette tips, 100-L and 1000-L volumes.6.6 Bunsen burner, used to
27、 flame-sterilize Allen wrench and plate spreader.6.7 95 % Ethanol, used to flame-sterilize Allen wrench and plate spreader.6.8 Small Allen wrench, for loosening set screws and pushing coupons out of reactor rods.6.9 Timer, any that can display time in seconds.6.10 Vortex mixer, any vortex that will
28、ensure proper agitation and mixing of centrifuge tubes.6.11 Serological pipettes, sterile single-use pipettes with volume capacity of 1, 5, 10, 25, and 50 mL.6.12 Plate spreader, for spreading serial dilutions on agar plates.E2871 1326.13 Water bath, any capable of maintaining a constant temperature
29、 of 20 6 1C.6.14 Sterilizer, any steam sterilizer capable of producing the conditions of sterilization.6.15 Colony counter, any one of several types may be used. A hand tally for recording of the bacterial count is recommendedif manual counting is done.6.16 Environmental incubator, any capable of ma
30、intaining a temperature of 36 6 2C.6.17 Appropriate glassware/plasticware, as required to make media and agar plates.6.18 Volumetric flasks, used for preparing disinfectants.6.19 Magnetic stir bars, sterile, for mixing prepared disinfectant.6.20 Magnetic stir plate, any capable of mixing.7. Reagents
31、 and Materials7.1 Purity of Waterall references to water as diluent or reagent 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
32、-sterilized (prepared according to Method 9050 C.1.a Buffered Dilution Water Preparation (1).7.4 Disinfectantproduct to be tested.7.5 NeutralizerDey/Engley Neutralization Broth or one specific to the disinfectant being evaluated as determined foreffectiveness and toxicity according to Test Method E1
33、054.8. Culture/Inoculum Preparation8.1 Borosilicate glass coupons with mature Pseudomonas aeruginosa ATCC 15442 biofilm grown according to Test MethodE2562 through step 10.2.4.9. Procedure9.1 The test is conducted with three treated and three untreated control coupons.9.2 An overview of the procedur
34、e is shown in Fig. 1.9.3 Prepare Disinfectant:9.3.1 Prepare disinfectant according to manufacturers specifications in sterile volumetric glassware. Ensure that the disinfectantis adequately mixed. Use within 3 h of preparation or as specified in the manufacturers instructions.9.3.2 Place prepared di
35、sinfectant in water bath equilibrated to 20 6 1C for 10 to 15 min.FIG. 1 Single Tube Method OverviewE2871 1339.4 Remove Coupons from the CDC Biofilm Reactor:9.4.1 Prepare sampling materials: treatment tubes, rinse tubes, small flame-sterilized Allen wrench, and serological pipettes.9.4.2 Aseptically
36、 remove a randomly selected rod containing coupons with biofilm from the CDC Biofilm Reactor by pullingit straight up firmly.9.4.3 Rinse the coupons to remove planktonic cells. Orient the rod in a vertical position directly over a 50-mLconical centrifugetube that contains 30-mL sterile buffered wate
37、r. Immerse the rod with a continuous motion into the buffered water with minimalto no splashing, then immediately remove.Anew 50-mLconical tube containing 30-mLsterile buffered water is used for each rod.9.4.4 Hold the rod with one of the randomly selected coupons centered over an empty, sterile 50-
38、mL conical tube. Loosen theset screw and allow the coupon to drop directly to the bottom of the tube. If the coupon does not freely drop, press directly in thecenter of the coupon with the Allen wrench used to loosen the set screw.NOTE 1The use of 50-mL conical tubes allows for uniform disinfectant
39、contact around the entire coupon surface.9.4.5 Repeat coupon removal twice more for a total of three tubes each containing a coupon.NOTE 2All biofilm on the coupon must be exposed to disinfectant. Do not lose any biofilm from the coupon by allowing it to touch the top or theinner sides of the 50-mL
40、conical tube as it falls to the bottom of the tube. To ensure that the maximum biofilm 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 tube and/orheld coupons that were not a
41、ngled and replace them with new tubes and coupons.9.5 Conduct Effcacy Evaluation:9.5.1 Slowly pipette 4 mL previously prepared and equilibrated disinfectant (treatment) or equilibrated buffered dilution water(untreated control) into the tubes containing the coupons, being careful to completely cover
42、 the coupon. Note and record the time.NOTE 3The order of application of disinfectant or buffered dilution water is randomly selected.NOTE 4For a 10-min contact time, a 1-min interval between coupons is recommended.9.5.2 Tap each tube to release any air bubbles trapped below the coupon. Do not shake
43、the tubes.9.5.3 Incubate the tubes at 20 6 1C for the specified contact time.9.5.4 At the end of the contact time, add appropriate volume of neutralizer to each tube. Replace the cap and mix thoroughlyby vigorously shaking the tube several times.Allow the coupon to remain in the neutralized disinfec
44、tant at room temperature untilstep 9.6.NOTE 5It is recommended that a neutralization study be conducted prior to running the efficacy test (Test Method E1054) to determine the appropriateneutralizer formulation, concentration, volume, and neutralization time. For each efficacy test, the concentratio
45、n, volume, and neutralization time shouldbe the same for the control and treated coupons. Thirty-six (36) mL of neutralizer was used in the collaborative study.9.5.5 Obtain a set of three coupons for the remaining treatment(s) or control(s) as described in steps 9.4.2 through 9.4.5.9.5.6 Repeat step
46、s 9.5.1 through 9.5.4 with randomly selected treatment(s) or control(s).9.6 Remove and Disaggregate Biofilm:9.6.1 Vortex each tube on the highest setting for 30 6 5 s.9.6.2 Place all tubes into a test tube rack and suspend the rack in the ultrasonic water bath so that the liquid level in the tubesis
47、 even with the water level in the tank of the bath.9.6.3 Sonicate the tubes at 45 kHz for 30 6 5 s.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 are the 100 dilution.NOTE 6The results fr
48、om an interlaboratory study of this test method demonstrated the importance of following the disaggregation and removalprotocol exactly as written including using the recommended ultrasonic water bath and vortex mixer.9.7 Dilute and Plate Disaggregated Biofilm Samples:9.7.1 Serially dilute the sampl
49、e in buffered water.9.7.2 Culture each dilution in duplicate for colony growth using 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 to the plating method used.10. Data Analysis10.1 Calculate biofilm density.10.1.1 Calculate X, the arithmetic mean CFU from the replicate samples plated (3).10.1.2 The log10 density for each coupon is calculated as follows:Log10 CFU/coupon!5Log10 X/B!V/D!# (1)where:X = average CFU of the repli
