ASTM E3031-20

Standard Practice for Determination of Antibacterial Activity on Ceramic Surfaces

SIGNIFICANCE AND USE
5.1 Current solid surface test methodologies, such as the Test Method E2180 and ISO 22196, do not take into account the complexities associated with a ceramic surface. This includes, but is not limited to, differing chemistries incorporated into the glaze and desiccation due to water absorption through the bisque body. Each point will be elaborated below:  
5.1.1 The glaze composition of ceramic tiles can vary between manufacturers, lots, and product lines. Some glaze chemistries such as tin, silver and copper can negatively impact the testing conditions. Therefore, an untreated tile from the same lot is not always suitable for comparison. The control tile proposed herein is capable of supporting growth over the indicated time frame and nutrient level (see Section 9).  
5.1.2 Desiccation is a common problem when testing tile surfaces. This can be overcome by pre-hydrating the tile by placing the specimen on a moistened wipe and allowing incubation for 18 to 24 h before beginning the test. This reduces the number of false positive results and more accurately measures the ability of the antimicrobial to inhibit growth.  
5.2 This practice utilizes a low inoculum load and requires growth on the control substrate to demonstrate a valid testing environment. In addition, while some antimicrobials demonstrate activity against static cultures, others require growth of the bacteria to maintain activity. A low inoculum level will allow both types of antimicrobials to be examined with the same testing conditions.
SCOPE
1.1 This practice is designed to quantitatively evaluate the antibacterial activity of glazed ceramic surfaces that have been specifically designed to contain an antibacterial treatment as part of the glaze. This practice is meant to compare the efficacy of one ceramic surface to another ceramic surface using the stated conditions and is not meant to be extrapolated to other conditions.  
1.2 Knowledge of microbiological techniques is required for this practice.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 30-Sep-2020
• ICS: 81.060.20 - Ceramic products
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Refers: ASTM E2180-24 - Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) In Polymeric or Hydrophobic Materials
• Effective Date: 01-Apr-2024
• Refers: ASTM E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Nov-2019
• Refers: ASTM E2180-18 - Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) In Polymeric or Hydrophobic Materials
• Effective Date: 01-May-2018
• Refers: ASTM E2756-18 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Apr-2018
• Refers: ASTM E2180-07(2017) - Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) In Polymeric or Hydrophobic Materials
• Effective Date: 01-Apr-2017
• Refers: ASTM E177-14 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-May-2014
• Refers: ASTM E177-13 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-May-2013
• Refers: ASTM E691-13 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-May-2013
• Refers: ASTM E2180-07(2012) - Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) In Polymeric or Hydrophobic Materials
• Effective Date: 01-Apr-2012
• Refers: ASTM E691-11 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2011
• Refers: ASTM E177-10 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-Oct-2010
• Refers: ASTM E2756-10 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-May-2010
• Refers: ASTM E177-08 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-Oct-2008
• Refers: ASTM E691-08 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Oct-2008
• Refers: ASTM E1054-08 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 01-Apr-2008

Overview

ASTM E3031-20: Standard Practice for Determination of Antibacterial Activity on Ceramic Surfaces establishes a quantitative methodology for evaluating the antibacterial performance of glazed ceramic surfaces that incorporate an antibacterial treatment. Issued by ASTM International, this standard is specifically designed to address the complexities unique to ceramic tiles, such as variations in glaze chemistry and moisture absorption. It provides a scientific basis for comparing the efficacy of different treated ceramic surfaces under controlled laboratory conditions, supporting quality assurance and product claims for manufacturers and suppliers in the ceramic tile industry.

Key Topics

• Focus on Ceramic-Specific Conditions: Unlike prior standards for solid surfaces (e.g., ASTM E2180, ISO 22196), this practice considers the hierarchy of glaze chemistries (e.g., tin, silver, copper) and the risk of desiccation that can influence antibacterial test results on ceramics.
• Control Specimen Methodology: Control tiles must be selected to ensure robust bacterial growth over the test period, accommodating potential limitations of untreated tiles.
• Prevention of Desiccation: A unique pre-hydration step reduces false positives by counteracting water loss from the ceramic body, improving result accuracy.
• Low Inoculum Load: The method uses a low bacterial inoculum, ensuring that both bacteriostatic (growth-inhibiting) and bactericidal (killing) treatments can be effectively differentiated.
• Result Validity Criteria: Proper control growth is required for a valid test, based on SI-unit calculations and repeatability guidelines.

Applications

ASTM E3031-20 is particularly valuable in the following scenarios:

• Quality Assurance for Antibacterial Surfaces: Ceramic tile manufacturers can use this standard to validate and compare the effectiveness of antibacterial treatments integrated into tile glazes, supporting compliance and marketing claims.
• Research and Development: R&D laboratories can use this method to benchmark new antimicrobial glaze formulations and optimize performance.
• Third-Party Testing: Testing laboratories reference ASTM E3031-20 to deliver standardized, reputable measurement of antibacterial efficacy for clients in the construction and ceramics industries.
• Procurement and Regulatory Compliance: Facility managers and specifiers can request certification to ASTM E3031-20 to ensure that installed ceramic surfaces meet defined antibacterial performance benchmarks.

Practical Value

• Reliable Comparison: Enables fair comparison between different products or batches, accounting for lot-to-lot glaze variability.
• Routine Testing: Suitable for routine quality control and interlaboratory study use, with precision and repeatability defined.
• Health & Safety: Supports efforts to provide safer, more hygienic environments where ceramic tiles are used, such as healthcare, educational, and hospitality facilities.
• International Compliance: Developed in alignment with international standards principles (WTO TBT Committee), enhancing global applicability and acceptance.

Related Standards

• ASTM E2180 - Test Method for Activity of Incorporated Antimicrobial Agents in Polymeric or Hydrophobic Materials
• ASTM E1054 - Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• ASTM E691 - Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• ISO 22196 - Measurement of Antibacterial Activity on Plastics and Other Non-porous Surfaces
• ASTM E2756 - Terminology Relating to Antimicrobial and Antiviral Agents

Summary

ASTM E3031-20 provides a comprehensive, ceramic-specific protocol for quantifying antibacterial activity on treated glazed surfaces. Through careful attention to the unique characteristics of ceramic materials, this standard assures consistent, reliable, and reproducible assessment of antibacterial performance, supporting innovation, quality, and safety in the ceramics industry.

Keywords: ASTM E3031, antibacterial ceramic surfaces, standard practice, antimicrobial glaze, tile antibacterial testing, ceramic quality control, antibacterial efficacy, ceramic surface standard.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3031 − 20
Standard Practice for
Determination of Antibacterial Activity on Ceramic Surfaces
This standard is issued under the fixed designation E3031; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope E2180Test Method for Determining the Activity of Incor-
porated Antimicrobial Agent(s) In Polymeric or Hydro-
1.1 This practice is designed to quantitatively evaluate the
phobic Materials
antibacterialactivityofglazedceramicsurfacesthathavebeen
E2756Terminology Relating toAntimicrobial andAntiviral
specifically designed to contain an antibacterial treatment as
Agents
partoftheglaze.Thispracticeismeanttocomparetheefficacy
2.2 ISO Standard:
of one ceramic surface to another ceramic surface using the
ISO 22196Measurement of Antibacterial Activity on Plas-
stated conditions and is not meant to be extrapolated to other
tics and Other Non-porous Surfaces
conditions.
1.2 Knowledge of microbiological techniques is required
3. Terminology
for this practice.
3.1 For definitions of terms used in this practice refer to
1.3 The values stated in SI units are to be regarded as
Terminology E2756.
standard. No other units of measurement are included in this
4. Summary of Practice
standard.
4.1 This practice is used for evaluating the antibacterial
1.4 This standard does not purport to address all of the
effectofantimicrobialsincorporatedintoaceramicglaze.This
safety concerns, if any, associated with its use. It is the
standard does not seek to imitate all possible real-world
responsibility of the user of this standard to establish appro-
scenarios but to provide a standardized practice to compare
priate safety, health, and environmental practices and deter-
multipleantimicrobialtechnologiesthatcanbeincorporatedor
mine the applicability of regulatory limitations prior to use.
coatedonaceramicsurface.Theinherentnatureoftheceramic
1.5 This international standard was developed in accor-
tileallowsfordesiccation;therefore,eachceramicspecimenis
dance with internationally recognized principles on standard-
equilibrated to the testing environment for 18- 24 h. Once the
ization established in the Decision on Principles for the
tiles are equilibrated, bacteria are inoculated onto the surface
Development of International Standards, Guides and Recom-
followed by a 24-h exposure time. Bacteria are recovered in a
mendations issued by the World Trade Organization Technical
neutralizer broth and enumerated according to a validated
Barriers to Trade (TBT) Committee.
method. Log reductions are calculated for a treated versus an
2. Referenced Documents
untreated sample.
2.1 ASTM Standards:
5. Significance and Use
E177Practice for Use of the Terms Precision and Bias in
5.1 Current solid surface test methodologies, such as the
ASTM Test Methods
Test Method E2180 and ISO 22196, do not take into account
E691Practice for Conducting an Interlaboratory Study to
the complexities associated with a ceramic surface. This
Determine the Precision of a Test Method
includes, but is not limited to, differing chemistries incorpo-
E1054Test Methods for Evaluation of Inactivators of Anti-
rated into the glaze and desiccation due to water absorption
microbial Agents
through the bisque body. Each point will be elaborated below:
5.1.1 The glaze composition of ceramic tiles can vary
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides, between manufacturers, lots, and product lines. Some glaze
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
chemistriessuchastin,silverandcoppercannegativelyimpact
Subcommittee E35.15 on Antimicrobial Agents.
the testing conditions. Therefore, an untreated tile from the
Current edition approved Oct. 1, 2020. Published October 2020. Originally
samelotisnotalwayssuitableforcomparison.Thecontroltile
approved 2015. Last previous edition approved in 2015 as E3031–15. DOI:
10.1520/E3031–20
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Available from International Organization for Standardization (ISO), ISO
Standards volume information, refer to the standard’s Document Summary page on Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,
the ASTM website. Geneva, Switzerland, http://www.iso.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3031 − 20
proposed herein is capable of supporting growth over the 7.2 Growth medium.
indicated time frame and nutrient level (see Section 9). 7.2.1 Overnight culture—brain heart infusion broth pre-
5.1.2 Desiccation is a common problem when testing tile pared according to the manufacturer’s instruction.
surfaces. This can be overcome by pre-hydrating the tile by 7.2.1.1 Alternativemediamaybeusedforovernightculture
placing the specimen on a moistened wipe and allowing of the organism, such as tryptic soy broth, but details shall be
incubation for 18 to 24 h before beginning the test. This included in the final report.
reduces the number of false positive results and more accu- 7.2.2 Inoculation broth—1:500 dilution of nutrient broth as
rately measures the ability of the antimicrobial to inhibit defined below:
growth. 7.2.2.1 Prepare nutrient broth by dissolving 3.0 g of meat
(beef) extract, 10.0 g peptone, and 5.0 g of sodium chloride in
5.2 This practice utilizes a low inoculum load and requires
1000 mL of distilled or deionized water.
growth on the control substrate to demonstrate a valid testing
7.2.2.2 Dilute the nutrient broth with distilled or deionized
environment. In addition, while some antimicrobials demon-
water to a 500-fold volume and adjust the pH to a value
strate activity against static cultures, others require growth of
between 6.8 and 7.2 with sodium hydroxide or hydrochloric
the bacteria to maintain activity. A low inoculum level will
acid.
allow both types of antimicrobials to be examined with the
7.2.2.3 Sterilize by autoclaving at 120°C for 30 min.
same testing conditions.
7.3 Solid growth media—tryptic soy agar plates.
6. Apparatus
7.4 Sterile deionized water—or equivalent.
6.1 Incubator—capable of maintaining a temperature of 35
7.5 Neutralizer—A neutralizer should be selected that has
6 2ºC and >75% RH.
been shown to effectively neutralize the active according to
6.2 Pipetter—continuously adjustable between 100 µL and
Test Methods E1054.
1000 µL.
8. Culture Preparation
6.3 Sterilizer—any suitable steam sterilizer with conditions
that produce sterility of samples.
8.1 Bacteria: Escherichia coli American Type Culture
Collection,ATCC No. 8739. Grow a fresh 18 6 1 h culture in
6.4 Petri dish—sterile 150 mm by 15 mm for holding the
sterile brain heart infusion broth at 35 6 2ºC and shaking at
samples
110 r/min prior to beginning the test. Dilute this suspension
6.5 Culture tubes and closures—anywithavolumecapacity
appropriately in the inoculation broth described in 7.2.2 to
of 10 mL and a minimum diameter of 16 mm. Recommended
obtain 1-5 × 10 CFU/mL. This will be the working bacterial
size is 16 mm by 125 mm borosilicate glass with a threaded
stock solution.
opening.
8.2 Alternative microorganisms can be substituted relevant
6.6 Cover film—25 mm by 25 mm clean polyethylene or
to the environment and depending on the end use of the
other suitable material that does not impact bacterial growth.
product. Use of alternate organisms should be included in the
6.7 Large Water Absorbent Laboratory wipe—to facilitate
report, in addition to any other modification of media, buffer,
pre-hydration of samples similar to a Kimwipe Kimtech
bacterial concentration.
delicate task wiper 30 cm by 30 cm.
9. Untreated Control Specimen
6.8 Vortex mixer—to provide a homogenous bacterial sus-
9.1 Control tiles suitable for testing purposes may be
pension prior to inoculation of samples and prior to the
prepared from glaze ingredients that are free of elements that
enumeration technique that will be used.
contribute to antimicrobial activity. One example of a product
6.9 Plastic screw top jar—150 ml capacity that has an
that meets this criterion is F-524. However, glazed tiles are
opening large enough to insert the sample as a vessel for
generally acceptable as controls if they can be shown to meet
recovery.
the following criterion:
6.10 Wrist action shaker—torecoverbacteriafromsamples.
9.1.1 Cansupport>1.5loggrowthunderthetestconditions
given herein as calculated in 12.4.
6.11 Petri dish—100 mm by 15 mm for enumeration.
9.1.1.1 If a control tile, as described above, is not available
6.12 Shaking incubator—capable of maintaining 35 6 2ºC.
then the use of borosilicate glass squares, cut to the same
dimensions as described in 10.1, can be substituted as control
7. Reagents and Materials
specimens. Glass squares shall meet performance specifica-
7.1 Dilution fluid or diluent—sterile Butterfield’s buffered
tions indicated in 9.1.1.
phosphate.
Kimwipe is a registered trademark of Kimberly-Clark Dallas TX, USA The sole source of supply of the of the control tiles (F-524) suitable for testing
ACS Reagent Chemicals, Specifications and Procedures for Reagents and purposespreparedfromglazeingredientsthatarefreeofelementsthatcontributeto
Standard-Grade Reference Materials, American Chemical Society, Washington, antimicrobial activity and known to the committee at this time is Fusion Ceramics,
DC. For suggestions on the testing of reagents not listed by theAmerican Chemical Inc. (Carrollton, Ohio USA). If you are aware of alternative suppliers, please
Societ
...

This 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. Because
it 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
of the standard as published by ASTM is to be considered the official document.
Designation: E3031 − 15 E3031 − 20
Standard Test Method Practice for
Determination of Antibacterial Activity on Ceramic Surfaces
This standard is issued under the fixed designation E3031; 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
1.1 This method is designed to quantitatively evaluate the antibacterial activity of glazed ceramic surfaces that have been
specifically designed to contain an antibacterial treatment as part of the glaze. This test method is meant to compare the efficacy
of one ceramic surface to another ceramic surface using the stated conditions and is not meant to be extrapolated to other
conditions.
1.2 Knowledge of microbiological techniques is required for this test.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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.
2. Referenced Documents
2.1 ASTM Standards:
E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods
E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
E1054 Test Methods for Evaluation of Inactivators of Antimicrobial Agents
E2180 Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) In Polymeric or Hydrophobic Materials
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 ISO Standard:
ISO 22196 Measurement of Antibacterial Activity on Plastics and Other Non-porous Surfaces
3. Terminology
3.1 For definitions of terms used in this test method refer to Terminology E2756.
4. Summary of Test Method
4.1 This test method is used for evaluating the antibacterial effect of antimicrobials incorporated into a ceramic glaze. This
This test method practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility
of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Oct. 15, 2015Oct. 1, 2020. Published December 2015October 2020. Originally approved 2015. Last previous edition approved in 2015 as
E3031–15. DOI: 10.1520/E3031–1510.1520/E3031–20
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Available from International Organization for Standardization (ISO), ISO Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier, Geneva,
Switzerland, http://www.iso.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3031 − 20
standard does not seek to imitate all possible real world scenarios but to provide a standardized method to compare multiple
antimicrobial technologies that can be incorporated or coated on a ceramic surface. The inherent nature of the ceramic tile allows
for desiccation, therefore each ceramic specimen is equilibrated to the testing environment for 18- 24 h. Once the tiles are
equilibrated, bacteria are inoculated onto the surface followed by a 24-h exposure time. Bacteria are recovered in a neutralizer
broth and enumerated according to a validated method. Log reductions are calculated for a treated versus an untreated sample.
5. Significance and Use
5.1 Current solid surface test methodologies, such as the Test Method E2180 and ISO 22196, do not take into account the
complexities associated with a ceramic surface. This includes, but is not limited to, differing chemistries incorporated into the glaze
and desiccation due to water absorption through the bisque body. Each point will be elaborated below:
5.1.1 The glaze composition of ceramic tiles can vary between manufacturers, lots, and product lines. Some glaze chemistries such
as tin, silver and copper can negatively impact the testing conditions. Therefore, an untreated tile from the same lot is not always
suitable for comparison. The control tile proposed herein is capable of supporting growth over the indicated time frame and nutrient
level (see Section 9).
5.1.2 Desiccation is a common problem when testing tile surfaces. This can be overcome by pre-hydrating the tile by placing the
specimen on a moistened wipe and allowing incubation for 18 to 24 h before beginning the test. This reduces the number of false
positive results and more accurately measures the ability of the antimicrobial to inhibit growth.
5.2 This test method utilizes a low inoculum load and requires growth on the control substrate to demonstrate a valid testing
environment. In addition, while some antimicrobials demonstrate activity against static cultures, others require growth of the
bacteria to maintain activity. A low inoculum level will allow for both types of antimicrobials to be examined with the same testing
conditions.
6. Apparatus
6.1 Incubator—capable of maintaining a temperature of 35 6 2ºC and >75% RH.
6.2 Pipetter—continuously adjustable between 100 μL and 1000 μL.
6.3 Sterilizer—any suitable steam sterilizer with conditions that produce sterility of samples.
6.4 Petri dish—sterile 150 mm by 15 mm for holding the samples
6.5 Culture tubes and closures—any with a volume capacity of 10 mL and a minimum diameter of 16 mm. Recommended size
is 16 mm by 125 mm borosilicate glass with a threaded opening.
6.6 Cover film—25 mm by 25 mm sterile polyethylene or other suitable material that does not impact bacterial growth.
6.7 Large Water Absorbent Laboratory wipe—to facilitate pre-hydration of samples similar to a Kimwipes Kimtech delicate task
wiper 30 cm by 30 cm.
6.8 Vortex mixer—to provide a homogenous bacterial suspension prior to inoculation of samples and prior to the enumeration
technique that will be used.
6.9 Plastic screw top jar—150 ml capacity that has an opening large enough to insert the sample as a vessel for recovery.
6.10 Wrist action shaker—to recover bacteria from samples.
6.11 Petri dish—100 mm by 15 mm for enumeration.
Kimwipe is a registered trademark of Kimberly-Clark Dallas TX, USA
E3031 − 20
6.12 Shaking incubator—capable of maintaining 35 6 2ºC.
7. Reagents and Materials
7.1 Dilution fluid or diluent—sterile Butterfield’s buffered phosphate.
7.2 Growth medium.
7.2.1 Overnight culture—brain heart infusion broth prepared according to the manufacturer’s instruction.
7.2.1.1 Alternative media may be used for overnight culture of the organism, such as tryptic soy broth, but details shall be included
in the final report.
7.2.2 Inoculation broth—1:500 dilution of nutrient broth as defined below:
7.2.2.1 Prepare nutrient broth by dissolving 3.0 g of meat (beef) extract, 10.0 g peptone, and 5.0 g of sodium chloride in 1000
mL of distilled or deionized water.
7.2.2.2 Dilute the nutrient broth with distilled or deionized water to a 500-fold volume and adjust the pH to a value between 6.8
and 7.2 with sodium hydroxide or hydrochloric acid.
7.2.2.3 Sterilize by autoclaving at 120°C for 30 min.
7.3 Solid growth media—tryptic soy agar plates.
7.4 Sterile deionized water—or equivalent.
7.5 Neutralizer—A neutralizer should be selected that has been shown to effectively neutralize the active according to Test
Methods E1054.
8. Culture Preparation
8.1 Escherichia coli American Type Culture Collection, ATCC No. 8739 is the organism to be utilized for this test. Grow a fresh
18 6 1 h culture in sterile brain heart infusion broth at 35 6 2ºC and shaking at 110 r/min prior to beginning the test. Dilute this
suspension appropriately in the inoculation broth described in 7.2.2 to obtain 1-5 × 10 CFU/mL. This will be the working bacterial
stock solution.
9. Untreated Control Specimen
9.1 Control tiles suitable for testing purposes may be prepared from glaze ingredients that are free of elements that contribute to
antimicrobial activity. One example of a product that meets this criterion is F-524. However, glazed tiles are generally acceptable
as controls if they can be shown to meet the following criterion:
9.1.1 Can support > 1.5 log growth under the test conditions given herein as calculated in 12.4.
9.1.1.1 If a control tile, as described above, is not available then the use of borosilicate glass squares, cut to the same dimensions
as described in 10.1, can be substituted as control specimens. Glass squares shall meet performance specifications indicated in
9.1.1.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference Materials, American Chemical Society, Washington, DC. For
suggestions on the testing of reagents not listed by the American Chemical Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and
the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
The sole source of supply of the of the control tiles (F-524) suitable for testing purposes prepared from glaze ingredients that are free of elements that contribute to
antimicrobial activity and known to the committee at this time is Fusion Ceramics, Inc. (Carrollton, Ohio USA). 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.
E3031 − 20
10. Sample Preparation
10.1 Prepare five (5) replicates of each specimen, measuring 50 mm by 50 mm 6 1 mm (see Section 9). Wipe test specimens
to remove any debris from processing, place in a sterilization pouch/container and autoclave for at 120°C for 1 h.
NOTE 1—If the active ingredient is affected by autoclaving, then other types of sterilization can be used.
10.2 While test specimens are being sterilized, fold and place two large 1-ply laboratory wipes (30 cm by 30 cm) into a 150 by
15 mm sterile petri dish. Fold in such a way to get 18 layers in a 10 cm by 10 cm square. Moisten with sterile deionized water
until the wipe is saturated.
10.3 Remove a sterile test specimen (10.1) from sterilization pouch aseptically, place onto the wipe in the petri dish (10.2).
Visually monitor the dish during preparation to prevent excess water from accumulating in the dish.
10.4 Incubate the dish containing the sample at 35 6 2ºC with >75% RH for 18 to 24 h.
11. Procedure
11.1 Inoculation and Incubation:
11.1.1 Remove test specimens from the incubator and proceed to 11.1.2
11.1.2 Pipette 100 μL of the prepared bacterial stock solution (8.1) onto each pre-hydrated test specimen (see Section 10). The test
specimen will remain on the moistened wipe for the duration of the test. Addition of water may be necessary if the saturated wipe
has become dry.
11.1.3 Enumerate the inoculum by spread or pour plate.
11.1.4 Place a 25 mm by 25 mm polyethylene film on top of the inoculum to ensure even contact with the surface. Make sure that
no inoculum leaves the surface of the ceramic tile.
11.1.4.1 In accordance with 6.6, the cover film to be utilized should not affect bacterial growth or absorb water. If the test specimen
size is increased, the volume of inoculum and cover film shall be increased proportional to the surface area of the sample (a ratio
of 1μL:6.25 mm ).
11.1.5 Place each petri dish containing inoculated samples in the incubator at 35 6 2ºC with >75% RH for 24 6 1 h.
11.2 Recovery:
11.2.1 After the specified incubation time, remove the test specimen or control from the petri dish and loosen the cover film. Note
any desiccation that is observed for each sample.
11.2.2 Place the film and ceramic test specimen into a sterile 150-mL plastic screw top jar containing 100 mL of neutralizer. Shake
for 1 min on a wrist-action shaker set to the maximum speed.
NOTE 2—Alternative vessels and volumes may be utilized but their description will be included in the report. In addition, alternative recovery techniques,
such as vortex and sonication, may also be utilized. Use of other recovery methods should be noted in the test report.
11.2.3 Recover culturable organisms from appropriate dilutions by use of spread- or pour plate, spiral plate, or by other valid
microbial enumeration methods.
11.2.4 Incubate plates at 35 6 2°C for 24 h.
11.2.5 Count and record colony numbers for each dilution.
E3031 − 20
FIG. 1 Flowchart of Testing Operation
12. Calculation or Interpretation of Results
12.1 For each test specimen, determine the number of viable bacteria per specimen:
N 5 ~C 3 D 3 V! (1)
where:
N = is the number of viable bacteria recovered from test specimen;
C = is the average plate count;
D = is the dilution factor for the plates counted;
V = is the volume, in ml, of neutralizer added to the specimen;
If no colonies were recovered in any of the agar plates for a dilution series, then record the number of colonies counted as “<
V” (where V is the volume, in ml, of neutralizer added to the specimen). For calculating the average when there are no viable
bacteria recovered in a dilution series, consider the number of viable bacteria to be “V”.
EXAMPLE In the case of V = 100 ml, the number used for calculating the average will be 100.
12.2 Convert CFU to Log CFU for each specimen.
12.3 Calculate the geometric mean of the log value of the replicate samples.
12.4 Calculate the log difference between the Inoculum and T control specimens
24h
ΔLog Control5 A 2 B (2)
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where:
A = geometric mean of the Log T control specimens
10 24h
B = geometric mean of the Log Inoculum,
12.4.1 If ΔLog Control <1.5, the test is invalid and must be repeated.
12.4.2 If ΔLog Control ≥ 1.5, continue to 12.5.
12.5 Calculate the log reduction for all samples if the conditions for a valid test are met.
LogReduction5 C 2 T (3)
where:
C = geometric mean of the Log T control samples
10 24h
T = geometric mean of the Log T treated samples
10 24h
13. Interpretion
13.1 If Log reduction = ΔLog Control the treatment is bacteriostatic
13.2 If Log reduction > ΔLog Control the treatment is considered bactericidal
13.3 If Log reduction < ΔLog Control but >0.5 the sample is considered to display partial inhibition.
14. Precision and Bias
14.1 The precision of this test method is based on an intra-laboratory study of E3031 Test Method for Determination of
Antibacterial Activity on Ceramic Surface, conducted in 2014. A single laboratory participated in this study, testing the Log
reduction of Escherichia coli on ceramics treated with two different inhibitors. Every “test result” represents the average of five
determinations. The laboratory reported ten replicate test results for each ceramic material, as well as two growth control samples.
Except for the use of only one laboratory, Practice E691 was followed for the design and analysis of the data; the details are given
in ASTM Research Report No. RR:E35-1010.
14.1.1 Repeatability (r)—The difference between repetitive results obtained by the same operator in a given laboratory applying
the same test method with the same apparatus under constant operating conditions on identical test material within short intervals
of time would in the long run, in the normal and correct operation of the test method, exceed the following values only in one case
in 20.
14.1.1.1 Repeatability can be interpreted as maximum difference between two results, obtained under repeatability conditions that
is accepted as plausible due to random causes under normal and correct operation of the test method.
14.1.1.2 Repeatability limits are listed in Table 1 and Table 2 below.
TABLE 1 Repeatability Limits for Testing the Log Reduction of
Escherichia coli on Ceramics Treated with Two Different
Inhibitors
A
Treated Average Repeatability Repeatability
Ceramics Standard Limit
Deviation
s r
x¯
r
R2BZ treated 3.519 0.757 2.119
ceramic piece
A2B2Z2 treated 4.620 0.429 1.200
ceramic piece
A
The average of the laboratories’ calculated averages.
Supporting data have been filed at ASTM International Headquarters and may be obtained by requesting Research Report RR:E35-1010. Contact ASTM Customer
Service at [e-mail].
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TABLE 2 Repeatability Limits for Testing the Log10 (CFU) Growth
of Escherichia coli on Control Pieces
A
Control Average Repeatability Repeatability
Standard Limit
Deviation
s r
x¯ r
Control un- 1.624 0.964 2.700
treated ceramic
piece
Glass control 3.432 0.301 0.842
piece
A
The average of the laboratories’ calculated averages.
14.1.2 Reproducibility (R)—The difference between two single and independent results obtained by different operators applying
the same test method in different laboratories using different apparatus on identical test material would, in the long run, in the
normal and correct operation of the test method, exceed the following values only in one case in 20.
14.1.2.1 Reproducibility can be interpreted as maximum difference between two results, obtained under reproducibility conditions
that is accepted as plausible due to random causes under normal and correct operation of the test method.
14.1.2.2 Reproducibility limits cannot be calculated from a single laboratory’s results.
14.1.3 The above terms (repeatability limit and reproducibility limit) are used as specified in Practice E177.
14.1.4 Any judgment in accordance with statement 14.1.1 would normally have an approximate 95% probability of being correct,
however the precision statistics obtained in this ILS must not be treated as exact mathematical quantities which are applicable to
all circumstances and uses. The limited number of laboratories reporting replicate results essentially guarantees that there will be
times when differences greater than predicted by the ILS results will arise, sometimes with considerably greater or smaller
frequency than the 95% probability limit would imply. Consider the repeatability limit as a general guide, and the associated
probability of 95% as only a rough i
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