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ASTM E3180-18

(Test Method)

Standard Test Method for Quantification of a Bacillus subtilis Biofilm Comprised of Vegetative Cells and Spores Grown Using the Colony Biofilm Model

Standard Test Method for Quantification of a <emph type="ital">Bacillus subtilis</emph > Biofilm Comprised of Vegetative Cells and Spores Grown Using the Colony Biofilm Model

SIGNIFICANCE AND USE
5.1 Biofilm characteristics such as thickness, matrix architecture, and population are dependent on factors such as shear and nutrient availability and composition. Additionally, sporulation can occur within biofilms, including those formed by Bacillus subtilis.5 The purpose of this test method is to define the parameters to grow and enumerate a B. subtilis biofilm comprised of vegetative cells and spores embedded in extracellular polymeric substance (EPS). This type of biofilm could provide a greater challenge to antimicrobials than vegetative biofilm or spores alone. The biofilm generated using this method is suitable for efficacy testing of antimicrobials.
SCOPE
1.1 This test method specifies the operational parameters required to grow and quantify a Bacillus subtilis biofilm comprised of vegetative cells and endospores (spores) using the colony biofilm method (CBM).2,3 The resulting biofilm is representative of static environments that can develop a sporulating biofilm rather than being representative of one particular environment.  
1.2 This test method utilizes a modified CBM to grow the biofilm. The CBM uses a semipermeable membrane on an agar plate as the biofilm growth surface and nutrient source.2,3 In this test method, membranes are inoculated and incubated for a total of 8 days to promote sporulation within the biofilm.  
1.3 This test method describes how to sample and analyze the biofilm for vegetative cells and spores. Biofilm population is expressed as total colony forming units (CFU) and spores per membrane.  
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.

General Information

Status
Published
Publication Date
30-Sep-2018
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 E2756-18 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
Effective Date
01-Apr-2018
Refers
ASTM E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
Effective Date
01-Nov-2019

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ASTM E3180-18 - Standard Test Method for Quantification of a <emph type="ital">Bacillus subtilis</emph > Biofilm Comprised of Vegetative Cells and Spores Grown Using the Colony Biofilm ModelASTM E3180-18 - Standard Test Method for Quantification of a <emph type="ital">Bacillus subtilis</emph > Biofilm Comprised of Vegetative Cells and Spores Grown Using the Colony Biofilm Model
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ASTM E3180-18 - Standard Test Method for Quantification of a <emph type="ital">Bacillus subtilis</emph > Biofilm Comprised of Vegetative Cells and Spores Grown Using the Colony Biofilm Model
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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:E3180 −18
Standard Test Method for
Quantification of a Bacillus subtilis Biofilm Comprised of
Vegetative Cells and Spores Grown Using the Colony
1
Biofilm Model
This standard is issued under the fixed designation E3180; 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 Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
1.1 This test method specifies the operational parameters
Barriers to Trade (TBT) Committee.
required to grow and quantify a Bacillus subtilis biofilm
comprised of vegetative cells and endospores (spores) using
2. Referenced Documents
2,3
the colony biofilm method (CBM). The resulting biofilm is
4
2.1 ASTM Standards:
representative of static environments that can develop a spo-
E2756Terminology Relating toAntimicrobial andAntiviral
rulatingbiofilmratherthanbeingrepresentativeofoneparticu-
Agents
lar environment.
1.2 This test method utilizes a modified CBM to grow the
3. Terminology
biofilm.TheCBMusesasemipermeablemembraneonanagar
3.1 Definitions—For definitions of terms used in this Test
2,3
plate as the biofilm growth surface and nutrient source. In
Method see Terminology E2756.
this test method, membranes are inoculated and incubated for
3.2 Definitions of Terms Specific to This Standard:
a total of 8 days to promote sporulation within the biofilm.
3.2.1 biofilm, n—microorganisms living in a self-organized
1.3 This test method describes how to sample and analyze
community attached to surfaces, interfaces, or each other,
the biofilm for vegetative cells and spores. Biofilm population
embedded in a matrix of extracellular polymeric substances of
isexpressedastotalcolonyformingunits(CFU)andsporesper
microbial origin, while exhibiting altered phenotypes with
membrane.
respect to growth rate and gene transcription.
1.4 Basic microbiology training is required to perform this
3.2.2 spore, n—a dormant, robust, and non-metabolically
test method.
active structure produced by certain bacteria enabling pro-
1.5 The values stated in SI units are to be regarded as longed survival and greater resistance to deleterious environ-
standard. No other units of measurement are included in this mental factors.
standard.
4. Summary of Test Method
1.6 This standard does not purport to address all of the
4.1 This test method utilizes a modified CBM to grow the
safety concerns, if any, associated with its use. It is the
biofilm. The CBM uses an inoculated semipermeable mem-
responsibility of the user of this standard to establish appro-
brane on an agar plate as the biofilm growth surface and
priate safety, health, and environmental practices and deter-
2
nutrient source. In the published method, the inoculated
mine the applicability of regulatory limitations prior to use.
membranes are transferred to fresh agar plates regularly
1.7 This international standard was developed in accor-
(usually every 8-24 h) to provide new nutrients to the
dance with internationally recognized principles on standard-
membrane-growncellsandareincubatedforatotalofapproxi-
ization established in the Decision on Principles for the
mately48hbeforesamplingorsubjectingthebiofilmtofurther
3
tests. In this test method, membranes are inoculated and
1
This test method is under the jurisdiction of ASTM Committee E35 on
incubated for 24 h before transferring the membrane to a new
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
media plate.This initial growth step is similar to the published
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Oct. 1, 2018. Published January 2019. DOI: 10.1520/ CBM and allows the colony biofilm to grow and spread across
E3180–18
2
Anderl, J. N., Franklin, M. J., & Stewart, P. S. (2000). Role of Antibiotic
4
Penetration Limitation in Klebsiella pneumoniae Biofilm Resistance to Ampicillin For referenced ASTM standards, visit the ASTM website, www.astm.org, or
and Ciprofloxacin. Antimicrobial Agents and Chemotherapy, 44(7) 1818–1824. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
3
Merritt,J.H.,Kadouri,D.E.,andO’Toole,G.A.(2011).Growingandanalyzing Standards volume information, refer to the standard’s Document Summary page on
static biofilms. Current Protocols in Microbiology. 22:B:1B.1:1B.1.1–1B.1.18 the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19
...

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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.  
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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.  
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  
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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.  
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Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow using CDC Biofilm Reactor

SIGNIFICANCE AND USE
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 (5, 7). 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. For example, research has shown that biofilm grown under high shear is more difficult to kill than biofilm grown under low shear (5, 8). The purpose of this test method is to direct a user in the laboratory study of a Pseudomonas aeruginosa biofilm by clearly defining each system parameter. This test method will enable an investigator to grow, sample, and analyze a Pseudomonas aeruginosa biofilm grown under high shear. The biofilm generated in the CDC Biofilm Reactor is also suitable for efficacy testing. After the 48 h growth phase is complete, the user may add the treatment in situ or remove the coupons and treat them individually.
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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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5.5.1 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.  
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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.  
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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...

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ASTM E3321-21(Test Method)
Standard Test Method for Intraluminal Catheter Model used to Evaluate Antimicrobial Urinary Catheters for Prevention of Escherichia coli Biofilm Growth

SIGNIFICANCE AND USE
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.  
1.2 The test method is versatile and may also be used for growing and/or characterizing biofilms and suspended bacteria of different species, although this will require changing the operational parameters to optimize the method based upon the growth requirements of the new organism.  
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1.5 Basic microbiology training is required to perform this test method.  
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.  
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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.  
1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in ...

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ASTM
ASTM E3226-19(Test Method)
Standard Test Method for Processing Cellulose Sponge-wipes to Detect Bacillus anthracis Spores Sampled from Environmental Surfaces

SIGNIFICANCE AND USE
5.1 This procedure describes a standardized method of processing cellulose wipes in a biosafety level 3 laboratory in order to detect and provide a semi-quantitative estimate of B. anthracis contamination after sampling of non-porous surfaces. Sampling may be conducted to characterize the extent of contamination or for clearance of an area after decontamination.
SCOPE
1.1 This test method covers a standardized method of processing cellulose wipes in a biosafety level 3 (BSL3) laboratory in order to detect and provide a semi-quantitative estimate of Bacillus anthracis contamination after sampling of non-porous surfaces. Sampling may be conducted to characterize the extent of the contamination, or for area clearance after decontamination.  
1.2 The laboratory procedures should be performed in a BSL3 laboratory by those trained for BSL3 microbiological techniques.  
1.3 This test method is specific to B. anthracis, but could be adapted for use with other organisms.  
1.4 The interlaboratory study was conducted with cellulose sponge wipes pre-moistened with neutralizing buffer. All reproducibility, sensitivity, and specificity data are based on the performance of these wipes. A review was conducted by subcommittee in 2019, and re-confirmed these ILS data are valid.  
1.5 Units—The values stated in SI units are to be regarded as standard. The values given in parentheses after SI units are provided for information only and are not considered 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 E3179-18(Test Method)
Standard Test Method for Determining Antimicrobial Efficacy of Ultraviolet Germicidal Irradiation against Influenza Virus on Fabric Carriers with Simulated Soil

SIGNIFICANCE AND USE
5.1 This test method determines the effectiveness of UVGI devices for reducing viable microorganisms deposited on carriers.  
5.2 This test method evaluates the effect soiling agents have on UVGI antimicrobial effectiveness.  
5.3 This test method determines the delivered UVGI dose.
SCOPE
1.1 This test method defines test conditions to evaluate ultraviolet germicidal irradiation (UVGI) light devices (mercury vapor bulbs, light-emitting diodes, or xenon arc lamps) that are designed to kill/inactivate influenza virus deposited on inanimate carriers.  
1.2 This test method defines the terminology and methodology associated with the ultraviolet (UV) spectrum and evaluating UVGI dose.  
1.3 This test method defines the testing considerations that can reduce UVGI surface kill effectiveness (that is, soiling).  
1.4 Protocols for adjusting the UVGI dose to impact the reductions in levels of viable influenza virus are provided (Annex A1).  
1.5 This test method does not address shadowing.  
1.6 The test method should only be used by those trained in microbiology and in accordance with the guidance provided by Biosafety in Microbiological and Biomedical Laboratories.2  
1.7 This test method is specific to influenza viruses  
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 Warning—Mercury has been designated by many regulatory agencies as a hazardous substance that can cause serious medical issues. Mercury, or its vapor, has been demonstrated to be hazardous to health and corrosive to materials. Use caution when handling mercury and mercury-containing products. See the applicable product Safety Data Sheet (SDS) for additional information. The potential exists that selling mercury or mercury-containing products, or both, is prohibited by local or national law. Users must determine legality of sales in their location.  
1.10 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.  
1.11 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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