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

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

Relations

• Refers: ASTM E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Nov-2019
• Refers: ASTM E2756-18 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Apr-2018
• Refers: ASTM E2756-10 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-May-2010

Overview

ASTM E3180-18 is a standardized test method developed by ASTM International for the quantification of a Bacillus subtilis biofilm that contains both vegetative cells and spores, using the colony biofilm model (CBM). This method specifies operational parameters and procedures necessary to grow and enumerate B. subtilis biofilms on a semipermeable membrane placed over an agar medium. The method is designed for use in static laboratory environments and supports a reproducible approach for sampling and analyzing biofilm populations. This standard is particularly significant for laboratories assessing the efficacy of antimicrobial agents against challenging biofilm structures involving both cell types.

Key Topics

• Biofilm Formation and Characterization: The test method enables the controlled development of B. subtilis biofilms with a mix of vegetative cells and endospores, embedded in extracellular polymeric substances (EPS).
• Colony Biofilm Model (CBM): Utilizes a modified CBM technique involving the growth of biofilms on semipermeable membranes atop tryptic soy agar plates to foster the formation and sporulation of biofilms over an 8-day incubation period.
• Quantification Procedures: Details the disaggregation of biofilm from membranes, followed by enumeration of total colony forming units (CFUs) and spores through dilution, plating, and heat-shock methods.
• Repeatability and Reproducibility: Provides guidelines to ensure repeatable outcomes, including a preliminary repeatability study evaluating test consistency.
• Safety and Training Requirements: Emphasizes that only personnel with basic microbiological training should perform this method and highlights the necessity of appropriate safety, health, and environmental practices.
• Use of SI Units: Specifies that all measurements and reported values adhere to SI units, ensuring global consistency.

Applications

ASTM E3180-18 is a valuable standard for a range of microbiological and antimicrobial efficacy studies. Key applications include:

• Antimicrobial Testing: The biofilm generated using this method presents a robust challenge for efficacy testing of disinfectants, biocides, and antibiotics as it contains both vegetative cells and spores, mimicking real-world resistance scenarios.
• Research & Development: Laboratories conducting R&D on new antimicrobial agents or surface treatments can employ this standard to evaluate biofilm resistance and product performance.
• Quality Assurance: Product manufacturers and quality control labs use this standardized method to ensure consistent biofilm quantification in routine testing.
• Regulatory Compliance: Meeting international standards and regulatory requirements for testing antimicrobial efficacy on biofilms, especially in the healthcare, pharmaceutical, and food safety industries.

Related Standards

For comprehensive studies and compliance, the following associated ASTM standards and references may be relevant:

• ASTM E2756 - Terminology Relating to Antimicrobial and Antiviral Agents: Offers definitions applicable to this method and broader antimicrobial testing.
• Current Protocols in Microbiology - For methodologies related to static biofilm growth and analysis.
• Referenced Methods for Antimicrobial Efficacy Testing: Other ASTM E35 standards pertaining to antimicrobial agents and surface testing.

Summary

The ASTM E3180-18 standard offers a reproducible methodology for the growth and quantification of Bacillus subtilis biofilms containing both vegetative cells and spores. Its use is highly impactful in antimicrobial efficacy testing, enabling research and quality assurance in fields where biofilm resistance is a significant concern. Adhering to this standard ensures robust, repeatable biofilm testing and supports regulatory and scientific rigor in microbiological analysis.

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
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
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-
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
tests. In this test method, membranes are inoculated and
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
Anderl, J. N., Franklin, M. J., & Stewart, P. S. (2000). Role of Antibiotic
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 [e-mail]. For Annual Book of ASTM
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 19428-2959. United States
E3180−18
the membrane. After the transfer, the colony biofilms are 7.1.2 Tryptic soy agar (TSA)—purchasedpre-madeplatesor
incubated for an additional seven (7) days on the same media prepared according to manufacturer’s instructions for a con-
plate without additional transfers to fresh medium as is centration of 40 g/Land poured into sterile 100 × 15 mm petri
typically performed in the published CBM. These growth plates.
conditions induce sporulation to occur within the biofilm. 7.1.3 Sterile water—purchased or sterilized distilled or re-
verse osmosis (RO) water.
4.2 After the growth phase, the biofilms grown on mem-
7.1.4 Tween 80—prepared to a concentration of 1% v/v in
branes are sampled by removing the biofilm from the mem-
distilled or RO water and filter sterilized.
brane surface and disaggregating the biofilm into a homoge-
neous cell suspension. The disaggregated cell suspension is
7.2 Materials:
split into two fractions. One fraction is directly diluted and 7.2.1 Inoculating loops—sterile,
plated for viable cell enumeration. The other fraction is heat
7.2.2 Test tubes—sterile, 50 mL volume capacity,
shockedpriortodilutingandplatingtokillthevegetativecells
7.2.3 Test tubes—sterile, any with a volume capacity of 10
and enumerate the spores in the sample.
mL and a minimum diameter of 16 mm and a cap that can
withstand 100°C. Recommended size is 25 × 150 mm boro-
5. Significance and Use
silicate glass with a threaded opening.
5.1 Biofilm characteristics such as thickness, matrix
7.2.4 Membrane filters—0.22 µm nitrocellulose, 13 mm
architecture, and population are dependent on factors such as
diameter,
shear and nutrient availability and composition. Additionally,
7.2.5 Beads—sterile, 6 mm borosilicate glass, and
sporulation can occur within biofilms, including those formed
7.2.6 Forceps—sterile.
by Bacillus subtilis. The purpose of this test method is to
7.3 Test organism:
define the parameters to grow and enumerate a B. subtilis
7.3.1 Bacillus subtilis, ATCC 35021.
biofilm comprised of vegetative cells and spores embedded in
extracellular polymeric substance (EPS). This type of biofilm
8. Culture Preparation
could provide a greater challenge to antimicrobials than
vegetativebiofilmorsporesalone.Thebiofilmgeneratedusing
8.1 Bacillus subtilis is the organism used in this test.
this method is suitable for efficacy testing of antimicrobials.
8.1.1 Thesourceculturemayoriginatefromafrozenculture
or spore suspension.
6. Apparatus
8.1.2 StreakaTSAplateforisolationwiththesourceculture
6.1 Biological safety cabinet,
and incubate at 32.5 6 2.5°C for 18-24 h.
6.2 Environmental chamber—capable of maintaining tem-
NOTE 1—Alternative strains can be substituted with the appropriate
perature at 32.5 6 2.5°C,
modifications to the method (that is, heat shocking temperature, etc).
Repeatability and reproducibility coefficients based on testing performed
6.3 Shaker, capable of maintaining 100 RPMs,
using B. subtilis ATCC 35021 will not apply to testing performed using
6.4 Variable volume pipetters, other strains.
6.5 UV lamp,
9. Procedure
6.6 Vortex mixer—any vortex mixer that will ensure proper
9.1 Inoculum Preparation:
agitation and mixing of test tubes,
9.1.1 Aseptically remove an isolated colony from a TSA
6.7 Ultrasonic bath—capable of maintaining 40 6 5 kHz.
plate and place into 20 mL of TSB in a 50 mL sterile tube.
9.1.2 Incubate the culture on a shaker in an environmental
6.8 Timer,
chamber at 32.5 6 2.5°C for 18 h -24 h at 100 RPMs.
6.9 Recirculating water bath,
9.1.2.1 Culturable cell density should equal ≥10 CFU/mL
6.10 Thermometer or thermocouple, and
and can be checked by serial dilution and plating.
6.11 Incinerator or Bunsen burner.
9.2 Inoculation of Membranes:
9.2.1 Prior to inoculation, decontaminate the membrane
7. Reagents and Materials
filters in a biological safety cabinet under an UV lamp for 30
7.1 Reagents:
min per side.
7.1.1 Tryptic soy broth (TSB)—purchased pre-made or pre-
9.2.2 Using sterilized forceps, aseptically place the mem-
pared according to manufacturer’s instructions for a concen-
branes on TSA plates.
tration of 30 g/L.
NOTE2—Multiplemembranescanbeplacedononeplateprovidedthey
do not touch and there is sufficient room between them for manipulations
Bra
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