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ASTM E3286-21

(Practice)

Standard Practice for Preparation Of Cell Monolayers on Glass Surfaces for Evaluation of Microbicidal Properties of Non-Chemical Based Antimicrobial Treatment Technologies

Standard Practice for Preparation Of Cell Monolayers on Glass Surfaces for Evaluation of Microbicidal Properties of Non-Chemical Based Antimicrobial Treatment Technologies

SIGNIFICANCE AND USE
5.1 There are no reproducible standardized protocols for preparing specimens used to evaluate the microbicidal efficacy of non-chemical treatments such as ultraviolet (UV), highenergy electron beam, or other forms of non-chemical antimicrobial technologies.  
5.2 Conventional protocols for applying bioburdens to carriers (see Test Method E2197) cause cells to stack upon one another, thereby creating multiple cell layers in which cells in layers closer to the carrier are masked by cells in overlying layers, which makes relative comparison of different non-chemical antimicrobial treatments more difficult.  
5.3 Steel and other metal carriers have asperities that can shield a percentage of the applied cells from direct exposure to electromagnetic irradiation.  
5.4 The combined effects of 5.2 and 5.3 confound determination of the microbicidal effect of electromagnetic irradiation on test specimens.  
5.5 The practice addresses these two confounding factors by:  
5.5.1 Using glass microscope slides – the surfaces of which are asperity-free – as carriers.  
5.5.2 Reliably depositing bacterial cells onto the carrier as a monolayer.  
5.6 The resulting specimen ensures that all microbes deposited onto the carrier are exposed equally to the irradiation source thereby ensuring that the only variables are the controlled ones – starting inoculum concentration, wavelength (λ – in nm), exposure time(s), and resulting energy dose (J).
SCOPE
1.1 This practice provides a protocol for creating bacterial cell monolayers on a flat surface.  
1.2 The cultures used and culture preparation steps in this Practice are similar to AOAC Method 961.02 and US EPA MB-06. However, test bacteria are applied to the carrier using an automated deposition device (6.2) rather than as a suspension droplet.  
1.3 The carrier inspection protocol is similar to US EPA MB-03 except that carrier surfaces are inspected microscopically rather than visually, unaided.  
1.4 A monolayer of cells eliminates the confounding effect caused by the shadowing effect of outer layers of bacteria stacked upon other bacteria on test specimens – thereby attenuating directed energy beams (that is, ultraviolet light, high-energy electron beams) before they can reach underlying cells.  
1.5 An asperity-free surface eliminates the shadowing effect of specimen surface topology that can block direct exposure of target bacteria to non-chemical antimicrobial treatments.  
1.6 This practice provides a reproducible target microbe and surface specimen to minimize specimen variability within and between testing facilities. This facilitates direct data comparisons among various non-chemical antimicrobial technologies.  
1.6.1 Antimicrobial pesticides used in clinical and industrial applications are expected to overcome shadowing effects. However, this practice meets a need for a protocol that facilitates relative comparisons among non-chemical antimicrobial treatments.  
1.6.2 This practice is not intended to satisfy or replace existing test requirements for liquid chemical antimicrobial treatments (for example Test Methods E1153 and E2197) or established regulatory agency performance standards such as US EPA MB-06.  
1.7 This practice was validated using Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442) using a protocol based on AOAC Method 961.02. If other cultures are used, the suitability of this practice must be confirmed by inspecting prepared surfaces, by using scanning electron microscopy (SEM) or comparable high-resolution microscopy.  
1.8 The specimens prepared in accordance with this practice are not meant to simulate end-use conditions.  
1.8.1 Non-chemical technologies are only to be used on visibly clean, non-porous surfaces. Consequently, a soil load is not used.  
1.9 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.10 Th...

General Information

Status
Published
Publication Date
30-Sep-2021
ICS
07.100.01 - Microbiology in general
Technical Committee
E35 - Pesticides, Antimicrobials, and Alternative Control Agents
Drafting Committee
E35.15 - Antimicrobial Agents
Current Stage
Ref Project

Relations

Refers
ASTM D5465-16(2020) - Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
Effective Date
01-Jul-2020

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ASTM E3286-21 - Standard Practice for Preparation Of Cell Monolayers on Glass Surfaces for Evaluation of Microbicidal Properties of Non-Chemical Based Antimicrobial Treatment TechnologiesASTM E3286-21 - Standard Practice for Preparation Of Cell Monolayers on Glass Surfaces for Evaluation of Microbicidal Properties of Non-Chemical Based Antimicrobial Treatment Technologies
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ASTM E3286-21 - Standard Practice for Preparation Of Cell Monolayers on Glass Surfaces for Evaluation of Microbicidal Properties of Non-Chemical Based Antimicrobial Treatment Technologies
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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: E3286 − 21
Standard Practice for
Preparation Of Cell Monolayers on Glass Surfaces for
Evaluation of Microbicidal Properties of Non-Chemical
1
Based Antimicrobial Treatment Technologies
This standard is issued under the fixed designation E3286; 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 1.7 This practice was validated using Staphylococcus au-
reus (ATCC 6538) and Pseudomonas aeruginosa (ATCC
1.1 This practice provides a protocol for creating bacterial
15442) using a protocol based on AOAC Method 961.02. If
cell monolayers on a flat surface.
other cultures are used, the suitability of this practice must be
1.2 The cultures used and culture preparation steps in this
confirmed by inspecting prepared surfaces, by using scanning
Practice are similar to AOAC Method 961.02 and US EPA
electron microscopy (SEM) or comparable high-resolution
MB-06. However, test bacteria are applied to the carrier using
microscopy.
an automated deposition device (6.2) rather than as a suspen-
1.8 Thespecimenspreparedinaccordancewiththispractice
sion droplet.
are not meant to simulate end-use conditions.
1.3 The carrier inspection protocol is similar to US EPA
1.8.1 Non-chemical technologies are only to be used on
MB-03 except that carrier surfaces are inspected microscopi-
visiblyclean,non-poroussurfaces.Consequently,asoilloadis
cally rather than visually, unaided.
not used.
1.4 A monolayer of cells eliminates the confounding effect
1.9 Units—The values stated in SI units are to be regarded
caused by the shadowing effect of outer layers of bacteria
asstandard.Nootherunitsofmeasurementareincludedinthis
stacked upon other bacteria on test specimens – thereby
standard.
attenuating directed energy beams (that is, ultraviolet light,
1.10 This standard does not purport to address all of the
high-energy electron beams) before they can reach underlying
safety concerns, if any, associated with its use. It is the
cells.
responsibility of the user of this standard to establish appro-
1.5 Anasperity-freesurfaceeliminatestheshadowingeffect
priate safety, health, and environmental practices and deter-
ofspecimensurfacetopologythatcanblockdirectexposureof
mine the applicability of regulatory limitations prior to use.
target bacteria to non-chemical antimicrobial treatments.
1.11 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.6 Thispracticeprovidesareproducibletargetmicrobeand
ization established in the Decision on Principles for the
surface specimen to minimize specimen variability within and
Development of International Standards, Guides and Recom-
between testing facilities. This facilitates direct data compari-
mendations issued by the World Trade Organization Technical
sons among various non-chemical antimicrobial technologies.
Barriers to Trade (TBT) Committee.
1.6.1 Antimicrobialpesticidesusedinclinicalandindustrial
applications are expected to overcome shadowing effects.
2. Referenced Documents
However, this practice meets a need for a protocol that
2
2.1 ASTM Standards:
facilitates relative comparisons among non-chemical antimi-
D5465Practices for Determining Microbial Colony Counts
crobial treatments.
from Waters Analyzed by Plating Methods
1.6.2 This practice is not intended to satisfy or replace
E1153TestMethodforEfficacyofSanitizersRecommended
existing test requirements for liquid chemical antimicrobial
for Inanimate, Hard, Nonporous Non-Food Contact Sur-
treatments (for example Test Methods E1153 and E2197)or
faces
established regulatory agency performance standards such as
E2197Quantitative Disk Carrier Test Method for Determin-
US EPA MB-06.
ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,
1
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides,
2
Antimicrobials, and Alternative Control Agents and is the direct responsibility of For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Subcommittee E35.15 on Antimicrobial Agents. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Current edition approved Oct. 1, 2021. Published November 2021. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E3286–21 the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

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1.1 This test method specifies the operational parameters required to grow and treat a Pseudomonas aeruginosa biofilm in a high throughput screening assay known as the MBEC (trademarked)2 (Minimum Biofilm Eradication Concentration) Physiology and Genetics Assay. The assay device consists of a plastic lid with ninety-six (96) pegs and a corresponding receiver plate with ninety-six (96) individual wells that have a maximum 200 μL working volume. Biofilm is established on the pegs under batch conditions (that is, no flow of nutrients into or out of an individual well) with gentle mixing. The established biofilm is transferred to a new receiver plate for disinfectant efficacy testing.3, 4 The reactor design allows for the simultaneous testing of multiple disinfectants or one disinfectant with multiple concentrations, and replicate samples, making the assay an efficient screening tool.  
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Standard Practice for Preparing a Pseudomonas aeruginosa or Staphylococcus aureus Biofilm using the CDC Biofilm Reactor

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5.1 Bacteria that exist in biofilms are phenotypically different from suspended cells of the same genotype. Research has shown that biofilm bacteria are more difficult to kill than suspended bacteria (4, 5). Laboratory biofilms are engineered in growth reactors designed to produce a specific biofilm type. Altering system parameters will correspondingly result in a change in the biofilm. The purpose of this practice is to direct a user in the growth of a P. aeruginosa or S. aureus biofilm by clearly defining the operational parameters to grow a biofilm that can be assessed for efficacy using the Standard Test Method for Evaluating Disinfectant Efficacy Against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method (E2871).  
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5.1 This practice is to help in the development of protocols to assess the survival, removal and/or inactivation of human pathogens or their surrogates in indoor air. It accommodates the testing of technologies based on physical (for example, UV light) and chemical agents (for example, vaporized hydrogen peroxide) or simple microbial removal by air filtration or a combination thereof.  
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5.1 In the battle to reduce medical device and implant-related infections, prevention of bacterial colonization of surfaces is a logical strategy. Bacterial colonization of a surface is a precursor to biofilm formation. Biofilm is the etiological agent of many implant and device-related infections and once established, microorganisms in biofilm can be up to 1000 times more tolerant to antibiotic therapy. Often the best treatment strategy is removal of the implant or device at a high socioeconomic cost. Catheter associated urinary tract infections (CAUTI) are the most prevalent of the device-related healthcare associated infections. Catheter associated infections account for 37 % of all hospital acquired infections (HAI) and 70 % of all nosocomial urinary tract infections (UTI) in the U.S. (2, 3). The Intraluminal Catheter Model (ICM) was developed to evaluate the ability of antimicrobial catheters to inhibit biofilm growth on the catheter lumen.  
5.2 The purpose of this test method is to direct a user in how to grow, sample, and analyze an E. coli biofilm in a urinary catheter under a constant flow of artificial urine. The test method incorporates operational parameters utilized in similar published methods (4). The E. coli biofilm that grows has a patchy appearance that varies across the catheter. Microscopically, the biofilm is heterogenous, with large clusters in some areas, and flat sheets of cells or even single cells in others. By 24 h, the biofilm is developed in the control catheters. If the goal is to monitor early stage biofilm development, then tubing and effluent samples need to be collected prior to the 24 h sample collection. Monitoring biofilm development requires sampling. The biofilm generated in the Intraluminal Catheter Model is suitable for comparison testing between antimicrobial and control catheters.
SCOPE
1.1 This test method specifies the operational parameters required to assess the ability of antimicrobial urinary catheters to prevent or control biofilm growth. Efficacy is reported as the log reduction in viable bacteria when compared to a repeatable (1)2  Escherichia coli biofilm grown in the intra-lumen of a urinary catheter under a constant flow of artificial urine.  
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1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard  
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Standard Test Method for Determining the Antimicrobial Activity of Antimicrobial Agents Under Dynamic Contact Conditions

SIGNIFICANCE AND USE
5.1 Substrate–bonded, antimicrobial agents are not typically free to diffuse into their environment under normal conditions of use. This test method ensures good contact between the bacteria and the treated fiber, fabric, or other substrate, by constant agitation of the test specimen in a challenge suspension during the test period.  
5.2 The metabolic state of the challenge species can directly affect measurements of the effectiveness of particular antimicrobial agents or concentrations of agents. The susceptibility of the species to particular biocides could be altered depending on its life stage (cycle). One-hour contact time in a buffer solution allows for metabolic stasis in the population. This test method standardizes both the growth conditions of the challenge species and substrate contact times to reduce the variability associated with growth phase of the microorganism.  
5.3 Leaching of an antimicrobial is dependent upon the test conditions being utilized and the ultimate end use of the product. Additional testing may be required to determine if a compound is substrate-bound in all conditions or during the end use of the product.  
5.4 This test method cannot determine if a compound is leaching into solution or is immobilized on the substrate. This test method is only intended to determine efficacy as described in subsequent portions of the method.  
5.5 The test is suitable for evaluating stressed or modified specimens, when accompanied by adequate controls.
Note 1: Stresses may include laundry, wear and abrasion, radiation and steam sterilization, UV exposure, solvent manipulation, temperature susceptibility, or similar physical or chemical manipulation.
SCOPE
1.1 This test method is designed to evaluate the antimicrobial activity of antimicrobial-treated specimens under dynamic contact conditions. This dynamic shake flask test was developed for routine quality control and screening tests in order to overcome difficulties in using classical antimicrobial test methods to evaluate substrate-bound antimicrobials. These difficulties include ensuring contact of inoculum to treated surface (as in AATCC TM100), flexibility of retrieval at different contact times, use of inappropriately applied static conditions (as in AATCC TM147), sensitivity, and reproducibility.  
1.2 This test method allows for the ability to evaluate many different types of treated substrates and a wide range of microorganisms. Treated substrates used in this test method can be subjected to a wide variety of physical/chemical stresses or manipulations and allows for the versatility of testing the effect of contamination due to such things as hard water, proteins, blood, serum, various chemicals, and other contaminants.  
1.3 Surface antimicrobial activity is determined by comparing results from the test sample to controls run simultaneously.  
1.4 This test method may not be appropriate for all types of antimicrobial-treated articles or antimicrobial agents. The proper test methodology should be determined based on antimicrobial mode of action and end-use expectations (Guide E2922)  
1.5 Proper neutralization of all antimicrobials must be confirmed using Test Methods E1054.  
1.6 This test method should be performed only by those trained in microbiological techniques.  
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.8 This standard may involve hazardous materials, operations and equipment. 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.  
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SIGNIFICANCE AND USE
5.1 Bacteria that exist in a biofilm are phenotypically different from suspended cells of the same genotype. The study of biofilm in the laboratory requires protocols that account for this difference. Laboratory biofilms are engineered in growth reactors designed to produce a specific biofilm type. Altering system parameters will correspondingly result in a change in the biofilm. The purpose of this method is to direct a user in the laboratory study of biofilms by clearly defining each system parameter. This method will enable a person to grow, sample, and analyze a laboratory biofilm. The method was originally developed to study toilet bowl biofilms, but may also be utilized for research that requires a biofilm grown under moderate fluid shear.
SCOPE
1.1 This test method is used for growing a reproducible (1)2 Pseudomonas aeruginosa biofilm in a continuously stirred tank reactor (CSTR) under medium shear conditions. In addition, the test method describes how to sample and analyze biofilm for viable cells.  
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1.3 This test method describes how to sample and analyze biofilm for viable cells. Biofilm population density is recorded as log10 colony forming units per surface area (rotating disk reactor—efficacy test method (3)).  
1.4 Basic microbiology training is required to perform this test method.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 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.  
1.7 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.

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ASTM
ASTM D7847-22(Guide)
Standard Guide for Interlaboratory Studies for Microbiological Test Methods

SCOPE
1.1 Microbiological test methods present challenges that are unique relative to chemical or physical parameters, because microbes proliferate, die off and continue to be metabolically active in samples after those samples have been drawn from their source.  
1.1.1 Microbial activity depends on the presence of available water. Consequently, the detection and quantification of microbial contamination in fuels and lubricants is made more complicated by the general absence of available water from these fluids.  
1.1.2 Detectability depends on the physiological state and taxonomic profile of microbes in samples. These two parameters are affected by various factors that are discussed in this guide, and contribute to microbial data variability.  
1.2 This guide addresses the unique considerations that must be accounted for in the design and execution of interlaboratory studies intended to determine the precision of microbiological test methods designed to quantify microbial contamination in fuels, lubricants and similar low water-content (water activity  
1.3 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.  
1.4 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.

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ASTM
ASTM E2799-22(Test Method)
Standard Test Method for Testing Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm using the MBEC Assay

SIGNIFICANCE AND USE
5.1 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. Altering either the engineered system or operating conditions will modify those characteristics. The goal in biofilm research and efficacy testing is to choose the growth reactor that generates 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 and analyze a Pseudomonas aeruginosa biofilm using the MBEC Assay. Microscopically, the biofilm is sheet-like with few architectural details as seen in Harrison et al (6). The MBEC Assay was originally designed as a rapid and reproducible assay for evaluating biofilm susceptibility to antibiotics (2). The engineering design allows for the simultaneous evaluation of multiple test conditions, making it an efficient method for screening multiple disinfectants or multiple concentrations of the same disinfectant. Additional efficiency is added by including the neutralizer controls within the assay device. The small well volume is advantageous for testing expensive disinfectants, or when only small volumes of the disinfectant are available.
SCOPE
1.1 This test method specifies the operational parameters required to grow and treat a Pseudomonas aeruginosa biofilm in a high throughput screening assay known as the MBEC (trademarked)2 (Minimum Biofilm Eradication Concentration) Physiology and Genetics Assay. The assay device consists of a plastic lid with ninety-six (96) pegs and a corresponding receiver plate with ninety-six (96) individual wells that have a maximum 200 μL working volume. Biofilm is established on the pegs under batch conditions (that is, no flow of nutrients into or out of an individual well) with gentle mixing. The established biofilm is transferred to a new receiver plate for disinfectant efficacy testing.3, 4 The reactor design allows for the simultaneous testing of multiple disinfectants or one disinfectant with multiple concentrations, and replicate samples, making the assay an efficient screening tool.  
1.2 This test method defines the specific operational parameters necessary for growing a Pseudomonas aeruginosa  biofilm, although the device is versatile and has been used for growing, evaluating and/or studying biofilms of different species as seen in Refs (1-4).5  
1.3 Validation of disinfectant neutralization is included as part of the assay.  
1.4 This test method describes how to sample the biofilm and quantify viable cells. Biofilm population density is recorded as log10 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. 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 item mentioned in this standard. Users of this standard are expressly advised that determination of the validity of any such 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 responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.  
1.9 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.

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