ASTM E2871-12
(Test Method)Standard Test Method for Evaluating Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor using Single Tube Method
Standard Test Method for Evaluating Disinfectant Efficacy against <emph type="bdit">Pseudomonas aeruginosa</emph> Biofilm Grown in CDC Biofilm Reactor using Single Tube Method
SIGNIFICANCE AND USE
Vegetative biofilm bacteria are phenotypically different from suspended planktonic cells of the same genotype. Biofilm growth reactors are engineered to produce biofilms with specific characteristics (2). Altering either the engineered system or operating conditions will modify those characteristics as well as the physicochemical environment. The goal in biofilm research and efficacy testing is to choose the growth reactor and operating conditions that generate the most relevant biofilm for the particular study.
The test method was developed using Pseudomonas aeruginosa ATCC 15442 biofilm grown on borosilicate glass coupons in the CDC Biofilm Reactor and liquid disinfectants. Efficacy data developed using other bacteria, different shear, different coupons, or other standardized biofilm reactor systems, and/or other forms of disinfectants may result in different log10 reduction (LR) values and repeatability and reproducibility standard deviations.
The efficacy test was designed to determine the log10 reduction in bacteria after exposure to a disinfectant in a closed system.
The test method was developed using 50-mL conical tubes. The conical geometry allows for disinfectant exposure to biofilm on all surfaces of the coupon.
Each efficacy test includes a single contact time and temperature for three untreated control coupons (exposed to buffered dilution water) and three treated coupons (per disinfectant/concentration combination).
SCOPE
1.1 This test method specifies the operational parameters required to perform a quantitative liquid disinfectant efficacy test against biofilm bacteria.
1.2 The test method was developed using a Pseudomonas aeruginosa biofilm grown in the CDC Biofilm Reactor (Test Method E2562), modified to include borosilicate glass coupons as a hard nonporous surface and P. aeruginosa ATCC 15442.
1.3 Disinfectant preparation and contact time are used in the assessment according to the manufacturer’s 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 sampling 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. Biofilm population density is recorded as log10 colony-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 does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation:E2871 −12
StandardTest Method for
Evaluating Disinfectant Efficacy against Pseudomonas
aeruginosa Biofilm Grown in CDC Biofilm Reactor using
Single Tube Method
This standard is issued under the fixed designation E2871; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
2.1 ASTM Standards:
1.1 This test method specifies the operational parameters
E1054 Test Methods for Evaluation of Inactivators of Anti-
required to perform a quantitative liquid disinfectant efficacy
microbial Agents
test against biofilm bacteria.
E2562 Test Method for Quantification of Pseudomonas
1.2 The test method was developed using a Pseudomonas
aeruginosa Biofilm Grown with High Shear and Continu-
aeruginosa biofilm grown in the CDC Biofilm Reactor (Test
ous Flow using CDC Biofilm Reactor
MethodE2562),modifiedtoincludeborosilicateglasscoupons
2.2 Other Standards:
as a hard nonporous surface and P. aeruginosa ATCC 15442.
Method 9050 C.1.a Buffered Dilution Water Preparation
1.3 Disinfectantpreparationandcontacttimeareusedinthe
according to Eaton et al (1)
assessment according to the manufacturer’s instructions for
use. 3. Terminology
3.1 Definitions:
1.4 The test method uses a closed system to treat biofilm.A
3.1.1 biofilm, n—microorganisms living in a self-organized
coupon is placed in a single tube for the treatment,
community attached to surfaces, interfaces, or each other,
neutralization, and sampling steps to prevent the loss of cells.
embedded in a matrix of extracellular polymeric substances of
1.5 Verification of disinfectant neutralization is determined
microbial origin, while exhibiting altered phenotypes with
prior to conducting the test method.
respect to growth rate and gene transcription.
1.6 This test method describes how to sample and analyze 3.1.1.1 Discussion—Biofilm may be comprised of bacteria,
treated and untreated control biofilms for viable cells. Biofilm fungi, algae, protozoa, viruses, or infinite combinations of
population density is recorded as log colony-forming units these microorganisms. The qualitative characteristics of a
per coupon. Efficacy is reported as a log reduction of viable biofilm including, but not limited to, population density,
cells. taxonomic diversity, thickness, chemical gradients, chemical
composition,consistency,andothermaterialsinthematrixthat
1.7 Basic microbiology training is required to perform this
are not produced by the biofilm microorganisms, are controlled
assay.
by the physicochemical environment in which it exists.
1.8 The values stated in SI units are to be regarded as
3.1.2 contact time, n—predeterminedtimethatthebiofilmis
standard. No other units of measurement are included in this
exposed to the activity of a disinfectant.
standard.
3.1.3 coupon, n—biofilm growth surface.
1.9 This standard does not purport to address all of the
3.1.4 disinfectant, n—a chemical that destroys vegetative
safety concerns, if any, associated with its use. It is the
forms of microorganisms, but does not ordinarily kill bacterial
responsibility of the user of this standard to establish appro-
spores.
priate safety and health practices and determine the applica-
3.2 Acronyms:
bility of regulatory limitations prior to use.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
This test method is under the jurisdiction of ASTM Committee E35 on contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct Standards volume information, refer to the standard’s Document Summary page on
responsibility of Subcommittee E35.15 on Antimicrobial Agents. the ASTM website.
Current edition approved April 1, 2012. Published June 2012. DOI: 10.1520/ The boldface numbers in parentheses refer to a list of references at the end of
E2871–12. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2871−12
3.2.1 ATCC—American Type Culture Collection. 6.3 Test tube rack, any capable of holding 50-mL conical
centrifuge tubes.
3.2.2 CDC—Centers for Disease Control and Prevention.
6.4 Micropipettes, continuously adjustable pipettes with
3.2.3 CFU—colony-forming unit.
volume capacity of 100 µL and 1000 µL.
4. Summary of Test Method
6.5 Sterile pipette tips, 100-µL and 1000-µL volumes.
4.1 This test method describes the use of the single tube
6.6 Bunsen burner, used to flame-sterilizeAllen wrench and
method to evaluate the efficacy of a liquid disinfectant against
plate spreader.
a Pseudomonas aeruginosa biofilm on a hard nonporous
6.7 95 % Ethanol, used to flame-sterilize Allen wrench and
surface grown in the CDC Biofilm Reactor. The test method
plate spreader.
consists of adding a disinfectant (treated) or a control buffer
(untreated) to individual coupons held in 50-mL conical tubes.
6.8 Small Allen wrench, for loosening set screws and
Three coupons are treated with disinfectant and three coupons pushing coupons out of reactor rods.
receive buffered dilution water. Neutralizer is added to the
6.9 Timer, any that can display time in seconds.
tubes after the appropriate contact time. A combination of
6.10 Vortex mixer, any vortex that will ensure proper agita-
vortexing and sonication are used to remove the biofilm from
tion and mixing of centrifuge tubes.
thecouponanddisaggregatetheclumps.Thecellsuspensionis
serially diluted and plated on agar medium.Viable plate counts
6.11 Serological pipettes, sterile single-use pipettes with
from treated and untreated control coupons are used to calcu- volume capacity of 1, 5, 10, 25, and 50 mL.
late the log reduction of viable cells.
6.12 Plate spreader, for spreading serial dilutions on agar
plates.
5. Significance and Use
6.13 Water bath, any capable of maintaining a constant
5.1 Vegetative biofilm bacteria are phenotypically different
temperature of 20 6 1°C.
from suspended planktonic cells of the same genotype. Biofilm
6.14 Sterilizer, any steam sterilizer capable of producing the
growth reactors are engineered to produce biofilms with
conditions of sterilization.
specific characteristics (2). Altering either the engineered
system or operating conditions will modify those characteris-
6.15 Colony counter, any one of several types may be used.
tics as well as the physicochemical environment. The goal in
A hand tally for recording of the bacterial count is recom-
biofilm research and efficacy testing is to choose the growth
mended if manual counting is done.
reactorandoperatingconditionsthatgeneratethemostrelevant
6.16 Environmental incubator, any capable of maintaining a
biofilm for the particular study.
temperature of 36 6 2°C.
5.2 The test method was developed using Pseudomonas
6.17 Appropriate glassware/plasticware, as required to
aeruginosa ATCC 15442 biofilm grown on borosilicate glass
make media and agar plates.
coupons in the CDC Biofilm Reactor and liquid disinfectants.
Efficacy data developed using other bacteria, different shear, 6.18 Volumetric flasks, used for preparing disinfectants.
different coupons, or other standardized biofilm reactor sys-
6.19 Magnetic stir bars, sterile, for mixing prepared disin-
tems,and/orotherformsofdisinfectantsmayresultindifferent
fectant.
log reduction (LR) values and repeatability and reproducibil-
6.20 Magnetic stir plate, any capable of mixing.
ity standard deviations.
5.3 The efficacy test was designed to determine the log
10 7. Reagents and Materials
reductioninbacteriaafterexposuretoadisinfectantinaclosed
7.1 Purity of Water—all references to water as diluent or
system.
reagent shall mean distilled water or water of equal purity.
5.4 The test method was developed using 50-mL conical
7.2 Bacterial Plating Medium—R2A agar is recommended.
tubes.Theconicalgeometryallowsfordisinfectantexposureto
7.3 Buffered Water—0.0425 g KH PO /L distilled water,
biofilm on all surfaces of the coupon.
2 4
filter-sterilized and 0.405 g MgCl·6H O/L distilled water;
5.5 Each efficacy test includes a single contact time and
filter-sterilized (prepared according to Method 9050 C.1.a
temperature for three untreated control coupons (exposed to
Buffered Dilution Water Preparation (1)).
buffered dilution water) and three treated coupons (per
7.4 Disinfectant—product to be tested.
disinfectant/concentration combination).
7.5 Neutralizer—Dey/Engley Neutralization Broth or one
6. Apparatus
specific to the disinfectant being evaluated as determined for
6.1 Conical centrifuge tubes, sterile, any with 50-mL vol-
effectiveness and toxicity according to Test Method E1054.
ume capacity and secure leakproof lids.
8. Culture/Inoculum Preparation
6.2 Ultrasonic water bath, any capable of maintaining a
homogeneous sound distribution at 45 kHz with a variable 8.1 Borosilicate glass coupons with mature Pseudomonas
power setting and a volume large enough to accommodate aeruginosa ATCC 15442 biofilm grown according to Test
50-mL conical tubes in a wet environment. Method E2562 through step 10.2.4.
E2871−12
9. Procedure 9.4.5 Repeat coupon removal twice more for a total of three
tubes each containing a coupon.
9.1 The test is conducted with three treated and three
untreated control coupons.
NOTE2—Allbiofilmonthecouponmustbeexposedtodisinfectant.Do
not lose any biofilm from the coupon by allowing it to touch the top or the
9.2 An overview of the procedure is shown in Fig. 1.
inner sides of the 50-mL conical tube as it falls to the bottom of the tube.
To ensure that the maximum biofilm surface area is in contact with the
9.3 Prepare Disinfectant:
disinfectant, the coupon should be at an angle in the bottom of the tube.
9.3.1 Prepare disinfectant according to manufacturer’s
Discard any tubes where the coupon touched the inner side of the tube
specifications in sterile volumetric glassware. Ensure that the
and/orheldcouponsthatwerenotangledandreplacethemwithnewtubes
disinfectant is adequately mixed. Use within3hof preparation
and coupons.
or as specified in the manufacturer’s instructions.
9.5 Conduct Effıcacy Evaluation:
9.3.2
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