ASTM E3092-18

Standard Practice for Evaluating Efficacy of Vaporous Decontaminants on Materials Contaminated with <emph type="bdit">Bacillus</emph> Spores and Contained Within 0.2&#xb5;m Filter-Capped Tubes

SIGNIFICANCE AND USE
5.1 The practice can be used to evaluate coupon materials of any composition, insofar as the coupon can be prepared small enough to fit inside a 50-ml conical tube.  
5.2 This practice defines procedures that are quantitative, scalable, rapid, sensitive, safe, reduces consumables, minimizes labor and addresses statistical confidence (1, 2, 4).  
5.2.1 Quantitative—The total number of spores per coupon is determined by dilution-plating, and all spores remaining on the coupon are assayed for activity in the extraction tube to provide confidence that 100 % of spores were assayed.  
5.2.2 Statistical Confidence—The use of five independent preparations of spore inoculum for a statistical N of 5.  
5.2.3 Sensitivity—Allows for complete detection of all viable spores inoculated on a coupon, including the spores that remain attached to the coupon.  
5.2.4 Safety—Inoculated coupons are contained within 0.2 µm filter-capped 50-ml conical tubes. The 0.2 µm filter allows vaporous decontaminants to pass through while preventing escape of spores, thereby providing an important level of containment when working with pathogenic strains.  
5.2.5 Simplicity of Testing—Tests and extractions are performed in the same 50-ml conical tube to minimize handling steps.  
5.2.6 Scalable and Rapid—A maximum of 36 samples can be processed in 1 h by two technicians; a total of 300 samples have been processed by six technicians in 5 h (1-3).  
5.2.7 Wide application for numerous Bacillus species and strains.
Note 1: This practice differs from similar quantitative methods (E2111, E2197 and E2414) in the size and variety of coupon materials available for testing, in the practical confidence of the statistics, the application of the decontaminant, scalability and sensitivity.
SCOPE
1.1 This practice is used to quantify the efficacy of vaporous decontaminants on Bacillus spores dried on the surface of coupons made from porous and non-porous materials and contained within 0.2µm filter-capped tubes.  
1.2 This practice should be performed only by those trained in microbiological techniques, are familiar with antimicrobial (sporicidal) agents and with the end use of such products.  
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: 28-Feb-2018
• ICS: 11.080.20 - Disinfectants and antiseptics
• ICS: 71.100.35 - Chemicals for industrial and domestic disinfection purposes
• 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 E2111-12(2018) - Standard Quantitative Carrier Test Method to Evaluate the Bactericidal, Fungicidal, Mycobactericidal, and Sporicidal Potencies of Liquid Chemicals
• Effective Date: 15-Sep-2018
• Refers: ASTM E2756-18 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Apr-2018
• Refers: ASTM E2197-17e1 - Standard Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals
• Effective Date: 01-Dec-2017
• Refers: ASTM E2197-17 - Standard Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals
• Effective Date: 01-Dec-2017
• Refers: ASTM E2111-12 - Standard Quantitative Carrier Test Method to Evaluate the Bactericidal, Fungicidal, Mycobactericidal, and Sporicidal Potencies of Liquid Chemicals
• Effective Date: 15-May-2012
• Refers: ASTM E2197-11 - Standard Quantitative Disk Carrier Test Method for Determining the Bactericidal, Virucidal, Fungicidal, Mycobactericidal and Sporicidal Activities of Liquid Chemical Germicides
• Effective Date: 01-Jan-2011
• Refers: ASTM E2756-10 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-May-2010
• Refers: ASTM E1054-08 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 01-Apr-2008
• Refers: ASTM E2111-05 - Standard Quantitative Carrier Test Method to Evaluate the Bactericidal, Fungicidal, Mycobactericidal, and Sporicidal Potencies of Liquid Chemical Microbicides
• Effective Date: 01-Nov-2005
• Refers: ASTM E2414-05 - Standard Test Method for Quantitative Sporicidal Three-Step Method (TSM) to Determine Sporicidal Efficacy of Liquids, Liquid Sprays, and Vapor or Gases on Contaminated Carrier Surfaces (Withdrawn 2014)
• Effective Date: 15-Jun-2005
• Refers: ASTM E1054-02 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 10-May-2002
• Refers: ASTM E2197-02 - Standard Quantitative Disk Carrier Test Method for Determining the Bactericidal, Virucidal, Fungicidal, Mycobactericidal and Sporicidal Activities of Liquid Chemical Germicides
• Effective Date: 10-Apr-2002
• Refers: ASTM E2111-00 - Standard Quantitative Carrier Test Method To Evaluate the Bactericidal, Fungicidal, Mycobactericidal and Sporicidal Potencies of Liquid Chemical Germicides
• Effective Date: 10-Oct-2000

Overview

ASTM E3092-18 is a standard practice developed by ASTM International for evaluating the efficacy of vaporous decontaminants on materials contaminated with Bacillus spores, where coupons are contained within 0.2 µm filter-capped conical tubes. This standard provides a quantitative, sensitive, and scalable approach to assess the decontamination performance of vaporous agents across a wide variety of material types. The method ensures safety and minimizes handling by containing the spores and extractions within the same tube, which also permits rapid throughput and reliable statistical confidence.

Key Topics

• Quantitative Evaluation: Determines the total number of viable Bacillus spores remaining on test surfaces after exposure to vaporous decontaminants, using precise dilution and plating methods.
• Statistical Confidence: Employs five independent spore inoculum preparations (N=5) to provide robust data for statistical analysis.
• Sensitivity: Capable of detecting residual viable spores, ensuring accurate measurement of decontaminant efficacy, including spores firmly attached to the test coupons.
• Safety: All test coupons are contained within 0.2 µm filter-capped 50-ml tubes, permitting the passage of decontaminant vapors but preventing the escape of spores, which is essential when working with pathogenic strains.
• Simplicity and Efficiency: Consolidates testing and extraction into a single tube to reduce labor and material usage while supporting rapid processing (up to 36 samples/hour by two technicians).
• Material Versatility: Applicable to a range of coupon materials (porous, non-porous, hard, flexible, metallic, non-metallic) as long as they fit within the conical tube.

Applications

The ASTM E3092-18 standard is valuable in any context where it is crucial to verify the effectiveness of vaporous decontaminants against Bacillus spores, including:

• Biological Decontamination:

  • Validating disinfection procedures for environments and materials that may be exposed to Bacillus anthracis or surrogates.
  • Assessing materials used in public health, bioterrorism response, biosafety, and laboratory research.
  • Supporting emergency response efforts with statistically reliable and rapid assessments.

• Industry and Environment:

  • Testing efficacy of decontamination agents and technologies on healthcare equipment, building materials, and transportation vehicles.
  • Research and development of new antimicrobial and sporicidal products.
  • Ensuring safety in pharmaceutical manufacturing, food processing, and clinical settings where contamination risks must be managed.

• Regulatory Compliance and Product Claims:

  • Providing recognized, standardized data to support regulatory submissions.
  • Demonstrating product efficacy to clients, regulators, or stakeholders.
  • Supporting development and labeling of products with sporicidal claims.

Related Standards

When applying ASTM E3092-18, it is beneficial to be aware of other relevant ASTM standards on antimicrobial and sporicidal efficacy testing:

• ASTM E1054 – Evaluation of inactivators of antimicrobial agents
• ASTM E2111 – Quantitative carrier test for evaluating bactericidal, fungicidal, mycobactericidal, and sporicidal potencies of liquid chemicals
• ASTM E2197 – Quantitative disk carrier method for determining bactericidal, virucidal, fungicidal, mycobactericidal, and sporicidal activities of chemicals
• ASTM E2414 – Withdrawn method related to sporicidal efficacy of liquids and vapors on contaminated surfaces (for historical context)
• ASTM E2756 – Terminology relating to antimicrobial and antiviral agents

These standards complement ASTM E3092-18, offering a comprehensive framework for decontaminant efficacy testing and helping ensure laboratory quality, safety, and regulatory acceptance.

---

Relevant keywords: ASTM E3092-18, vaporous decontaminant efficacy, Bacillus spores, spore decontamination, antimicrobial testing, sporicidal efficacy, filter-capped tubes, biological decontamination 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: E3092 − 18
Standard Practice for
Evaluating Efficacy of Vaporous Decontaminants on
Materials Contaminated with Bacillus Spores and Contained
Within 0.2µm Filter-Capped Tubes
This standard is issued under the fixed designation E3092; 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 ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,
and Sporicidal Activities of Chemicals
1.1 Thispracticeisusedtoquantifytheefficacyofvaporous
E2414Test Method for Quantitative Sporicidal Three-Step
decontaminants on Bacillus spores dried on the surface of
Method (TSM) to Determine Sporicidal Efficacy of
coupons made from porous and non-porous materials and
Liquids, Liquid Sprays, and Vapor or Gases on Contami-
contained within 0.2µm filter-capped tubes.
nated Carrier Surface (Withdrawn 2014)
1.2 This practice should be performed only by those trained
E2756Terminology Relating toAntimicrobial andAntiviral
in microbiological techniques, are familiar with antimicrobial
Agents
(sporicidal) agents and with the end use of such products.
3. Terminology
1.3 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
3.1 For definitions of terms used in this guide, see Termi-
standard.
nology E2756.
1.4 This standard does not purport to address all of the
3.2 For inactivators and neutralizers of decontaminants see
safety concerns, if any, associated with its use. It is the
Test Methods E1054.
responsibility of the user of this standard to establish appro-
3.3 Definitions of Terms Specific to This Standard:
priate safety, health, and environmental practices and deter-
3.3.1 decontaminant, n—a physical or chemical agent or
mine the applicability of regulatory limitations prior to use.
process that destroys pathogenic or potentially pathogenic
1.5 This international standard was developed in accor-
microorganisms in/on surfaces or objects.
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the 3.3.2 endospore, n—a dormant, robust and non-
Development of International Standards, Guides and Recom- metabolically active structure produced by certain bacteria
mendations issued by the World Trade Organization Technical from the Firmicutes phylum.
Barriers to Trade (TBT) Committee.
3.3.3 exosporium, n—the outermost layer of spores of Ba-
cillus anthracis and its close relatives Bacillus thuringiensis
2. Referenced Documents
and Bacillus cereus.
2.1 ASTM Standards:
3.3.4 macrobacillus, n—a Bacillus endospore that possess
E1054Test Methods for Evaluation of Inactivators of Anti-
an exosporium.
microbial Agents
3.3.5 microbacillus, n—a Bacillus endospore that does not
E2111Quantitative Carrier Test Method to Evaluate the
possess an exosporium.
Bactericidal,Fungicidal,Mycobactericidal,andSporicidal
3.3.6 vapor,n—asubstanceinthegasphaseatatemperature
Potencies of Liquid Chemicals
lower than its critical temperature, such that it can be con-
E2197Quantitative Disk Carrier Test Method for Determin-
densed back into a liquid by increasing the pressure on it
without reducing the temperature.
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides,
3.3.7 vaporous decontaminant, n—for the purpose of this
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
practice, a vaporous decontaminant can be interpreted to
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved March 1, 2018. Published May 2018. DOI: 10.1520/ includegases,vapors,fogs,mistsandthermaldecontaminants.
E3092–18
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 last approved version of this historical standard is referenced on
the ASTM website. www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3092 − 18
4. Summary of Practice 5.2.4 Safety—Inoculated coupons are contained within
0.2µm filter-capped 50-ml conical tubes. The 0.2µm filter
4.1 This practice quantitatively evaluates the efficacy of
allows vaporous decontaminants to pass through while pre-
vaporous decontaminants on coupons contaminated with Ba-
venting escape of spores, thereby providing an important level
cillus spores (pathogenic and non-pathogenic strains). Spores
of containment when working with pathogenic strains.
are dried on coupon surfaces, and the coupons are then
5.2.5 Simplicity of Testing—Tests and extractions are per-
transferred to 0.2µm filter-capped 50-ml conical tubes (1-3).
formed in the same 50-ml conical tube to minimize handling
4.2 Coupon material is selected according to the claims or
steps.
intended use of the decontaminant. Coupons may be made of
5.2.6 Scalable and Rapid—A maximum of 36 samples can
any material – hard, flexible, porous, non-porous, metallic, or
be processed in1hbytwo technicians; a total of 300 samples
non-metallic. Flat (2 × 2 cm) coupons are preferred; however
have been processed by six technicians in 5 h (1-3).
non-flat coupons and smaller coupons have been tested using
5.2.7 Wide application for numerous Bacillus species and
this practice.
strains.
4.3 Fifty-ml conical tubes are used for extraction vessels.
NOTE 1—This practice differs from similar quantitative methods
This allows for greater flexibility in coupon material selection,
(E2111, E2197 and E2414) in the size and variety of coupon materials
accommodating materials that are difficult to manufacture in available for testing, in the practical confidence of the statistics, the
application of the decontaminant, scalability and sensitivity.
extremely small sizes, for example, concrete, asphalt, carpet
and wallboard.
6. Apparatus
4.4 Contaminated test coupons are subjected to decontami-
6.1 Autoclave.
nation procedures. Control coupons are subjected to identical
procedures without the decontaminant. 6.2 Shaking Incubator.
4.5 Solution controls (spores suspended in aqueous solu- 6.3 Incubator.
tion) will represent the 100% recovery reference for calculat-
6.4 Phase-Contrast Microscope.
ing spore survival after decontamination treatment and analy-
6.5 Centrifuge.
sis.
6.6 Water Bath.
4.6 Spore extraction percentage will be calculated by divid-
ingthenumberofsporesrecoveredfromeachspore-inoculated 6.7 Single-tube Vortex Mixer.
control coupon by the number of spores recovered from the
6.8 Multi-tube Vortex Mixer.
solution controls.
6.9 Analytical Balance.
4.7 The number of surviving spores from decontamination
6.10 -80 °C Freezer.
tests will be divided by the extraction percentage to determine
-1
the number of surviving spores in CFU ml . This spore 6.11 Stopwatch or Electronic Timer.
concentrationisthenmultipliedby10mltogiveatotalnumber
6.12 Manual or Electronic Pipettes.
of spores surviving (CFU) from each test sample. A log
6.13 Bio-Safety Cabinet (BSC).
transformation of the total surviving spores will then be
performed (log (total CFU + 1)).
6.14 Environmental Chamber, capable of maintaining tem-
perature 62°C and relative humidity 65% of target param-
5. Significance and Use
eters; must be capable of maintaining vapor concentration.
5.1 Thepracticecanbeusedtoevaluatecouponmaterialsof
6.15 Appropriate PPE, for example, gloves, safety glasses,
any composition, insofar as the coupon can be prepared small
lab coats, etc. (5).
enough to fit inside a 50-ml conical tube.
7. Reagents and Materials
5.2 This practice defines procedures that are quantitative,
scalable, rapid, sensitive, safe, reduces consumables, mini- 7.1 Reagents :
mizes labor and addresses statistical confidence (1, 2, 4).
7.1.1 Bacillus anthracis—Ames, Sterne, ∆Sterne.
-
5.2.1 Quantitative—The total number of spores per coupon 7.1.2 Bacillus thuringiensis—Al Hakam, cry HD-1.
is determined by dilution-plating, and all spores remaining on
7.1.3 Tryptic Soy Broth (TSB).
the coupon are assayed for activity in the extraction tube to 7.1.4 Tryptic Soy Agar (TSA).
provide confidence that 100% of spores were assayed.
7.1.5 Nutrient Broth (NB).
5.2.2 Statistical Confidence—The use of five independent 7.1.6 Tween 80.
preparations of spore inoculum for a statistical N of 5.
7.1.7 L-Alanine.
5.2.3 Sensitivity—Allows for complete detection of all vi-
able spores inoculated on a coupon, including the spores that
Reagent Chemicals, American Chemical Society Specifications, American
remain attached to the coupon.
Chemical Society, Washington, DC. For Suggestions on the testing of reagents not
listed by the American Chemical Society, see Annual Standards for Laboratory
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
this standard. MD.
E3092 − 18
A
TABLE 2 Extraction Buffer (pH 7)
7.1.8 Inosine.
Reagent Defined Amount
7.1.9 Sporulation Broth—0.8% (w⁄v) Nutrient broth or
1× Extraction Buffer
2.5% (w/v) Nutrient broth and salts, as defined in Table 1,pH
TSB 10 g
7 (see Annex A1 for preparation instructions) (1, 6, 7).
L-Alanine 100 mM
7.1.10 Extraction Buffer—pH7,asdefinedinTable2 (2, 3).
Inosine 1 mM
Tween 80 1 ml
7.1.11 pH-adjusted Bleach—0.6% (v/v) hypochlorite, 0.2%
Sterile, 18-megaohm water Add water to 1000 ml final volume
(v/v) acetic acid, pH 7.
2× Extraction Buffer
7.1.12 90 % (v/v) Ethanol.
TSB 20 g
L-Alanine 200 mM
7.2 Materials:
Inosine 2 mM
Sterile, 18-megaohm water Add water to 1000 ml final volume
7.2.1 Sterile 50-ml conical tube.
A
7.2.2 Sterile 50-ml conical tubes with 0.2µm filter cap, for
Can include a chemical neutralizer if necessary to neutralize any sporicidal
activity of a chemical vapor.
example, TPP TP87050; Techno Plastic Products .
7.2.3 Baffled flasks.
7.2.4 Sterile Petri dishes.
7.2.5 50-ml conical tube and microfuge tube racks. 9.3 Acrystalliferous strains of Bacillus thuringiensis–Al
-
7.2.6 Pipette tips. Hakam, cry HD-1.
7.2.7 Parafilm.
9.4 Other macrobacillus and microbacillus strains, vegeta-
7.2.8 L-shaped sterile spreaders.
tive bacteria, bacteriophage and viruses may also be tested
7.2.9 1.5-ml sterile microcentrifuge tubes.
using this practice.
7.2.10 Coupon materials—All coupon materials must be a
standardized surface area, preferably flat,2×2cm; however, 10. Preparation of Inoculum
it is understood that not all materials are easily adaptable to
10.1 Prepare five independent spore inocula from five inde-
these size constraints.
pendent spore preparations.
7.2.11 Sterile forceps.
10.2 Transfer concentrated spores from -80°C directly to a
50°C water bath for at least 30 min. This temperature is
8. Hazards
maintained during spore inoculation to mitigate the risk of
8.1 It is the responsibility of the individual user(s) of this
spore clumping prior to and during coupon inoculation.
practice to follow all safety guidelines and to be knowledge-
able about these procedures. Individual users should consult 10.3 Vortex concentrated spores for 15-30 s.
their safety authority and establish detailed safety plans and
10.4 Transfer concentrated spores into pre-labeled 50-ml
risk assessments prior to using this practice. Users are strongly
conical tubes containing preheated (50°C) sterile 0.1% (v/v)
urgedtoconsultthe Biosafety in Microbiological and Biomedi-
Tween80.Sporesfromeachindependentsporepreparationare
cal Laboratories (5).
used to prepare its corresponding independent spore inoculum.
The volume of 0.1% Tween 80 is set to achieve a target
9. Test Organisms
8 -1
concentration of 1-2 × 10 spores ml . Pipette tips should be
9.1 Specific organisms are recommended, but the choice of
rinsed by pipetting up and down twice in the 50-ml conical
organism(s)shouldberelevanttotheenvironmentinwhichthe
tube in order to rinse spores from the plastic tips.
decontaminant is expected to perform.
10.5 Hold the diluted spore inoculum at 50°C until coupon
9.2 Pathogenic and non-pathogenic stains of Bacillus an-
inoculation, which should occur within 24 h of preparing the
thracis – Ames, Sterne, ∆Sterne.
inocula.
10.6 In order to titer the spore inoculum, transfer 0.1 ml of
spore inoculum into 0.9 ml of 0.1% Tween 80, serially dilute
The sole source of manufacturing of the apparatus known to the committee at
andplateonTSAplates.Invertplatesandincubateat35 62°C
this time is Techno Plastic Products, Trasadingen, Switzerland. There are multiple
for 16 62 h. Count and record data. The time and temperature
sourcesforpurchasing.Ifyouareawareofalternativemanufactures,pleaseprovide
this information toASTM International Headquarters. Your comments will receive
of plate incubation can be adjusted for strains, for example, B.
careful consideration at a meeting of the responsible technical committee, which
thuringiensis HD-1 strains produce large colonies and this
you may attend.
7 strain is incubated at 30 62°C for 16 62 h in order to ensure
Trademarked by Bemis Corporate 2301 Industrial Drive Neenah, WI 54956.
countable plates.
TABLE 1 Sporulation Broth (pH 7)
10.7 Optional: Spores may be mixed with inorganic debris
Reagent Amount prior to coupon inoculation. Kaolin (Al Si O (OH) ) has been
2 2 5 4
-1 -1
Nutrient Broth 2.5 % (2.5 g l )or0.8%(0.5gl ) selected as a potential inorganic debris in published tests (2).
-1
KH PO 2.15 g
2 4
Kaolin was suspended in 0.1% Tween 80 at 100 g l kaolin
K HPO 4.35 g
2 4
andautoclave-sterilizedfor30minonawetcycle.Sporeswere
CaCl ·2HO0.15g
2 2
MnCl ·2H O 0.016 g suspended in kaolin at a final concentration of 1-2×10 spores
2 2
-1 -1
ZnCl 0.068 g
ml ,0.1%Tween80,50gl kaolin.Atatestconcentrationof
FeCl ·6H O 0.0003 g
3 2 8 -1
1-2×10 spores ml , kaolin was 250-500× excess over spores
Sterile, 18-megaohm water Add water to 1000 ml final volume
by weight.
E3092 − 18
10.8 Optional: Spores may be mixed with organic debris 12.1.2.6 Cap solution control tubes and store at ambient
prior to coupon inoculation. Humic acid suspended in spent (22 63°C) laboratory conditions until use.
sporulation medium (SSM) has been selected as a potential
12.2 Test and Controls—Coupons or controls, or both are
organic debris in published tests (2). The SSM collected after
incubatedattheappropriateenvironmentaltestconditionswith
spore harvest (see Refs (1, 6, 7) and Annex A1) was 0.2µm
or without chemical vapor. Test and control coupons and
filter-sterilized and then stored at -75 65°C. Humic acid was 7
solution controls contain ≥1×10 spores.
-1
suspended in SSM at 10 g l humic acid and autoclave-
12.2.1 Incubate test coupons at the test environmental
sterilized for 30 min on a wet cycle. Spores were then
conditions with or without chemical vapor (5 test coupons per
suspended in the humic acid + SSM at a final concentration of
coupon material).
8 -1 -1
1-2×10 spores ml , 0.05% (v/v) Tween 80,5gl humic
12.2.2 Incubate control coupons at ambient laboratory con-
acid, 0.5× SSM. At a test concentration of 1-2 × 10 spores
-1 ditions (22 63°C) (five (5) control coupons per coupon
ml ,thehumicacidwas25-50×excessoversporesbyweight.
material).
12.2.3 Incubate negative controls (uninoculated coupons) at
11. Preparation of Coupon
ambient laboratory conditions (22 63°C) (1 negative control
11.1 Rinse coupons with 18-megaohm water. Dry on absor-
per coupon material).
bent paper in an autoclave-safe container.
12.2.4 Incubate test solution controls (4.9 ml of 0.1%
Tween 80 plus 0.1 ml of spore inoculum) at the test environ-
11.2 Autoclave coupons at 121°C for 30 min on a wet
mental conditions with or without chemical vapor (five (5) test
cycle. Materials that are temperature sensitive should be
solution controls).
soaked in pH-adjusted bleach for 10 min, followed by a 90%
ethanol rinse. Store sterilized coupons in sterile containers at 12.2.5 Incubate control solution controls (4.9 ml of 0.1%
ambient (22 63°C) laboratory conditions until use. Tween80plus0.1mlofsporeinoculum)atambientlaboratory
conditions (22 63°C) (five (5) solution controls).
12. Test Procedure 12.2.6 There are a total of 21 samples per coupon material,
for example, five (5) coupon materials = 125 samples.
12.1 Carrier Inoculation—Confirm inoculum titer on the
day of coupon inoculation.
12.3 Extraction—Samples are processed in sets of 10-12
12.1.1 Coupons: samples at a time. See Fig. 1 (1).
12.1.1.1 Vortexpre-warmed(50 62°C)inoculumfor15-30
12.3.1 Coupons—Add 10 ml of 1× extraction buffer to each
s. sample.
12.1.1.2 Use a P-1000 pipette to transfer a single 100 µl
12.3.2 Solution Control—Add 5 ml of 2× extraction buffer
drop of clean spores or spores mixed with inorganic debris or
to each sample.
spores mixed with organic debris per inoculation. The pipette
12.3.3 Incubate samples at 26 62°Cfor1h.
tip should be immersed half way into the inoculum when
12.3.4 Vortexsamplesfor2minonamulti-tubevortexerset
removing aliquots.
at 70% full speed (for example, 70 out of 100 for a GlasCo
12.1.1.3 Inoculate 12-18 coupons at one time. Then return
vortexer) at ambient (22 63°C) laboratory conditions.
inoculum back into the 50°C water bath.
12.3.5 Within 20 min following vortexing, serially dilute
12.1.1.4 Select the next inoculum, working through all five
samples in 0.1% Tween 80 and plate on TSA plates. Directly
independent preparations, performing Steps 12.1.1.1 through
plate 1 ml across four plates (approximately 250 µl per plate),
12.1.1.3 until all coupons have been inoculated.
and 0.1 ml on one plate. Transfer 0.1 ml of sample into 0.9 ml
12.1.1.5 Allow coupons to dry, uncovered, overnight in
of 0.1%Tween 80, serially diluting and plating onTSAplates
BSC at ambient (22 63°C).
at ambient (22 63°C) laboratory conditions.
12.1.1.6 Transfer inoculated coupons into pre-labeled filter-
12.3.6 Incubateall50-mlconicaltubeswiththecouponand
capped tubes. Store at ambient (22 63°C) laboratory condi-
remaining 8.8 ml of extraction medium at 35 62°C for 16 62
tions until use.
h. Invert TSA plates and incubate at 35 62°C for 16 62h.
12.1.2 Solution Controls:
12.3.7 Score tubes for growth/no growth, count plates and
12.1.2.1 Aseptically add 4.9 ml of sterile 0.1% Tween 80
record data.
into 50-m
...