ASTM E3178-18

Standard Practice for Evaluating Static and Cidal Chemical Decontaminants against <emph type="bdit">Bacillus</emph> Spores using Centrifugal Filtration Tubes

SIGNIFICANCE AND USE
5.1 The practice can be used to evaluate coupon materials of any composition, insofar as the coupon can be small enough to fit inside filter units mentioned in 4.1.  
5.2 This practice defines procedures that are quantitative, scalable, rapid, sensitive, and safe, while minimizing labor and addressing statistical confidence (1, 2).  
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 all the spores were accounted for.  
5.2.2 Statistical Confidence—The use of five independent preparations of spore inocula for a statistical n of 5.  
5.2.3 Sensitivity—Allows for complete detection of all culturable spores inoculated on a coupon, including the spores that remain attached to the coupon.
5.2.3.1 The limit of detection is dependent on the culturability of fully matured spores to germinate, outgrow and divide in the presence of the extraction medium (1% tryptic soy broth, 100 mM L-Alanine, 1 mM inosine, 0.05% Tween 80) and/or on tryptic soy agar.
5.2.3.2 Results presented in Refs (1, 3) (and currently unpublished results) indicate that these media, combined with the test temperatures and conditions described herein will generate results with a high level of practical confidence for detecting culturable Bacillus spores.  
5.2.4 Safety—Inoculated coupons are contained within filter units.  
5.2.5 Simplicity of Testing—Tests and extractions are performed in the same filter unit to minimize coupon 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, 2).  
5.2.7 Wide application for numerous Bacillus species and strains. The method has also been modified and used for vegetative bacteria and viruses as well (1, 2).
SCOPE
1.1 This practice is used to quantify the efficacy of liquid or solid decontaminants on Bacillus spores dried on the surface of coupons made from porous and non-porous materials. This practice can distinguish between bactericidal and bacteriostatic chemicals within decontamination mixtures. This is important because many decontaminants contain both reactive compounds and high concentrations of bacteriostatic surfactants. All test samples are directly compared to pre-neutralized controls, un-inoculated negative growth controls, and solution controls on the same day as the test in order to increase practical confidence in the inactivation data.  
1.2 This procedure should be performed only by those trained in microbiological techniques, are familiar with antimicrobial (sporicidal) agents and the application instructions of the antimicrobial 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 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.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 30-Sep-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 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
• Refers: ASTM E1054-08 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 01-Apr-2008
• Refers: ASTM E1054-02 - Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents
• Effective Date: 10-May-2002

Overview

ASTM E3178-18 is a standard practice established by ASTM International for evaluating the effectiveness of both static and cidal (bactericidal) chemical decontaminants against Bacillus spores using centrifugal filtration tubes. This method is designed to quantify the decontamination efficacy of liquid or solid agents on Bacillus spores that have been dried on the surface of material coupons, which may be made from both porous and non-porous substances.

The methodology within ASTM E3178-18 emphasizes a quantitative, scalable, rapid, and sensitive approach that is safe and minimizes labor. It provides statistically robust data, allowing for a high level of confidence in the detection of culturable Bacillus spores. The practice is flexible, accommodating a wide range of coupon materials, and is applicable for multiple Bacillus species and even adaptable for use with other microorganisms, including vegetative bacteria and viruses.

Key Topics

• Quantitative Assessment: Determination of the total number of spores per coupon using dilution-plating. All spores, including those attached to the coupon surface, are assayed to ensure comprehensive data collection.
• Statistical Confidence: The use of five independent spore preparations enables a robust statistical analysis of results, improving the reliability of efficacy assessments.
• Sensitivity and Detection: The practice allows for the complete detection of all culturable spores on a coupon, relying on appropriate culturing media and conditions to achieve a high level of practical confidence in results.
• Safety and Simplicity: The entire test and extraction process is carried out within contained filter units, minimizing operator exposure and handling of hazardous biological materials.
• Scalability: The standard facilitates rapid processing, with the capacity to process up to 36 samples per hour for two technicians, and up to 300 samples in five hours with six technicians.
• Wide Applicability: The method is usable for numerous Bacillus species and strains, and is modifiable for assessment of vegetative bacteria and viruses.

Applications

• Validation of Decontaminants: This standard practice is vital for testing the effectiveness of new or existing decontamination products intended to inactivate Bacillus spores on various materials, helping manufacturers support regulatory submissions and marketing claims.
• Material and Surface Testing: The method supports the assessment of both porous and non-porous materials (e.g., fabrics, plastics, metals, painted surfaces) for decontamination efficacy, essential for healthcare, laboratory, and industrial settings.
• Regulatory Compliance: Using ASTM E3178-18 helps laboratories and product developers align with recognized international standards, supporting compliance with safety and performance requirements in regulated environments.
• Biodefense and Public Health: With Bacillus spores serving as models for biothreat agents, this practice is crucial for developing and verifying countermeasures intended for use in biodefense, emergency response, and public health.

Related Standards

• ASTM E1054: Test methods for evaluation of inactivators of antimicrobial agents, referenced for neutralization verification.
• ASTM E2756: Terminology relating to antimicrobial and antiviral agents, providing key definitions used in this practice.
• United States CFR Standards: Titles 7, 9, and 42 concerning possession, use, and transfer of select agents and toxins.
• World Trade Organization TBT Committee Principles: This international standard was developed in alignment with global principles, ensuring broad applicability and recognition.

Practical Value

ASTM E3178-18 offers a reliable, quantitative, and scalable procedure for laboratories and manufacturers needing to evaluate the effectiveness of decontamination technologies against resilient microbial spores. Its robust statistical approach, safety-oriented workflow, and adaptability to different test materials make it a valuable tool for R&D, quality assurance, and regulatory agencies involved in biosafety, biosecurity, and contamination control. By following this standardized method, users gain confidence in their decontamination results and ensure alignment with global best practices in microbial safety assessment.

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: E3178 − 18
Standard Practice for
Evaluating Static and Cidal Chemical Decontaminants
against Bacillus Spores using Centrifugal Filtration Tubes
This standard is issued under the fixed designation E3178; 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 2. Referenced Documents
1.1 This practice is used to quantify the efficacy of liquid or 2.1 ASTM Standards:
soliddecontaminantson Bacillussporesdriedonthesurfaceof E1054Test Methods for Evaluation of Inactivators of Anti-
coupons made from porous and non-porous materials. This microbial Agents
practicecandistinguishbetweenbactericidalandbacteriostatic E2756Terminology Relating toAntimicrobial andAntiviral
chemicals within decontamination mixtures. This is important Agents
3,4
because many decontaminants contain both reactive com- 2.2 CFR Standards:
pounds and high concentrations of bacteriostatic surfactants. Title 7 (Agriculture) CFR, Part 331Possession, Use, and
All test samples are directly compared to pre-neutralized Transfer of Select Agents and Toxins
controls, un-inoculated negative growth controls, and solution Title 9 (Animals and Animal Products) CFR, Part 121Pos-
controls on the same day as the test in order to increase session, Use, and Transfer of Select Agents and Toxins
practical confidence in the inactivation data. Title 42 (Public Health) CFR, Part 73Select Agents and
Toxins
1.2 This procedure should be performed only by those
trained in microbiological techniques, are familiar with anti-
3. Terminology
microbial(sporicidal)agentsandtheapplicationinstructionsof
3.1 For definitions of terms used in this Practice, see
the antimicrobial products.
Terminology E2756.
1.3 The values stated in SI units are to be regarded as
3.2 Definitions of Terms Specific to This Standard:
standard. No other units of measurement are included in this
3.2.1 decontaminant, n—a physical or chemical agent or
standard.
process that inactivates pathogenic or potentially pathogenic
1.4 This standard may involve hazardous materials,
microorganisms in or on surfaces or objects.
operations, and equipment. This standard does not purport to
3.2.2 endospore, n—a dormant, robust and non-
address all of the safety concerns, if any, associated with its
metabolically active structure produced by certain types of
use. It is the responsibility of the user of this standard to
bacteria from the Firmicutes phylum.
establish appropriate safety, health, and environmental prac-
tices and determine the applicability of regulatory limitations 3.2.3 exosporium, n—the outermost structural layer of mac-
robacillus spores including Bacillus anthracis, Bacillus th-
prior to use.
1.5 This international standard was developed in accor- uringiensis and Bacillus cereus.
dance with internationally recognized principles on standard-
3.2.4 macrobacillus, n—a Bacillus species that produces
ization established in the Decision on Principles for the
endospores that possess an exosporium including Bacillus
Development of International Standards, Guides and Recom-
anthracis, Bacillus thuringiensis and Bacillus cereus.
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
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
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides, the ASTM website.
Antimicrobials, and Alternative Control Agents and is the direct responsibility of United States Select Agent Program administered by the Center for Disease
Subcommittee E35.15 on Antimicrobial Agents. Control(CDC)and/ortheUnitedStatesDepartmentofAgriculture(USDA),Animal
Current edition approved Oct. 1, 2018. Published January 2019. DOI: 10.1520/ and Plant Health Inspection Service (APHIS)https://www.selectagents.gov/
F3178–18 https://www.access.gpo.gov
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3178 − 18
3.2.5 microbacillus, n—a Bacillus species that produces 4.8 The number of culturable surviving spores from decon-
endospores that do not possess an exosporium including tamination tests is divided by the extraction percentage to
-1
Bacillus subtilis and Bacillus atrophaeus. determine the number of surviving spores in CFU mL and
accountforanysurvivingsporesnotremovedfromthecoupon
4. Summary of Practice
during extraction. This spore concentration is then multiplied
4.1 For the purpose of this practice a decontaminant can be by 10 mL to give the total number of spores surviving (CFU)
interpreted to include liquids and solids that inactivate mi- from each test sample. A log transformation of the total
crobes. This practice quantitatively evaluates the efficacy of
surviving spores is then performed (log (total CFU + 1)).
decontaminants on coupons contaminated with Bacillus spores
(pathogenic and non-pathogenic strains). Spores are dried on
5. Significance and Use
coupon surfaces. The spore-inoculated coupons are then trans-
5.1 Thepracticecanbeusedtoevaluatecouponmaterialsof
ferred to Amicon Ultra-15 Centrifugal Filter Units with
anycomposition,insofarasthecouponcanbesmallenoughto
Ultracel-100 membranes (filter units) contained in sterile
fit inside filter units mentioned in 4.1.
50-mL conical tubes (1, 2).
5.2 This practice defines procedures that are quantitative,
4.2 Coupon material is selected according to the claims or
scalable,rapid,sensitive,andsafe,whileminimizinglaborand
intended use of the decontaminant. Coupons may be made of
addressing statistical confidence (1, 2).
any material - hard, flexible, porous, non-porous, metallic, or
5.2.1 Quantitative—The total number of spores per coupon
non-metallic. Flat (2 cm × 2 cm) coupons are preferred;
is determined by dilution-plating, and all spores remaining on
however, non-flat coupons and smaller coupons can be tested
the coupon are assayed for activity in the extraction tube to
using this practice. Coupons that have been tested are 0.1-0.3
provide confidence that all the spores were accounted for.
cm thick. Check that the coupons are thin enough to fit within
5.2.2 Statistical Confidence—The use of five independent
the filter units mentioned in 4.1.
preparations of spore inocula for a statistical n of 5.
4.3 Filter units contained in sterile 50-mL conical tubes (1,
5.2.3 Sensitivity—Allows for complete detection of all cul-
2) allows for greater flexibility in coupon material selection
turablesporesinoculatedonacoupon,includingthesporesthat
compared to microfuge tubes because these larger containers
remain attached to the coupon.
willaccommodatematerialsthataredifficulttomanufacturein
5.2.3.1 The limit of detection is dependent on the cultur-
extremely small sizes including fabrics and painted surfaces.
ability of fully matured spores to germinate, outgrow and
divideinthepresenceoftheextractionmedium(1%trypticsoy
4.4 Filter units contained in sterile 50-mL conical tubes (1,
broth, 100 mM L-Alanine, 1 mM inosine, 0.05% Tween 80)
2)allowthespore-inoculatedcoupons,whichmaycontainboth
and/or on tryptic soy agar.
cidal reactive compounds and static compounds including
5.2.3.2 Results presented in Refs (1, 3) (and currently
surfactants,tobesubmergedandthenbefilteredawayafterthe
unpublished results) indicate that these media, combined with
test. The membranes also allow for spores to be washed free
the test temperatures and conditions described herein will
fromanyresidualchemicalsthatmighthaveboundtothespore
generate results with a high level of practical confidence for
surfaces. Effectively, all the inoculated spores, including those
detecting culturable Bacillus spores.
bound to the coupons, can be recovered in the filter unit and
5.2.4 Safety—Inoculated coupons are contained within filter
assayed after filtering and washing the spore-inoculated cou-
units.
pons. Such data provide confidence that spores have been
5.2.5 Simplicity of Testing—Tests and extractions are per-
inactivated and the results are not merely the result of static
formed in the same filter unit to minimize coupon handling
activity.
steps.
4.5 Contaminated test coupons are subjected to decontami-
5.2.6 Scalable and Rapid—A maximum of 36 samples can
nation procedures. Control coupons are treated in an identical
be processed in1hbytwo technicians; a total of 300 samples
manner to test coupons but with a pre-neutralized decontami-
have been processed by six technicians in 5 h (1, 2).
nant. Recovery of viable spores from pre-neutralized decon-
5.2.7 Wide application for numerous Bacillus species and
taminant on the same day(s) as the test samples provides
strains. The method has also been modified and used for
greater confidence in the test data by eliminating time as a test
vegetative bacteria and viruses as well (1, 2).
variable.
4.6 Spores suspended in an aqueous medium represent the
6. Apparatus
spore recovery reference for calculating spore survival after
6.1 Autoclave.
decontamination treatment and analysis.
6.2 Shaking Incubator, capable of maintaining temperature
4.7 Sporeextractionpercentageiscalculatedbydividingthe
at 62°C within a minimum temperature range of 25-37°C.
numberofsporesrecoveredfromeachspore-inoculatedcontrol
coupon by the number of spores recovered from the aqueous
6.3 Gneral Purpose Microbiological Incubator (62 °C).
medium controls.
6.4 Phase-Contrast Microscope, oil immersion with magni-
fication ≥100×.
Trademarked by Millipore, Billerica, MA, USA, UFC9010096
6.5 Centrifuge, capable of ≥3,100xg that can hold a
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard. swinging-rotor bucket for 50-mL conical tubes.
E3178 − 18
TABLE 1 Sporulation Broth (pH 7)
6.6 Water Bath, capable of maintaining temperature at
62°C within a minimum temperature range of 50-65°C. Reagent Final Concentration
-1 -1
Nutrient Broth 25 g L (2.5%) or 8 g L (0.8%)
6.7 Single-tube Vortex Mixer.
-1 -1
KH PO (Molecular Wt 136.04) 2.15 g L (15.8 mmol L )
2 4
-1 -1
K HPO (Molecular Wt 174.18) 4.35 g L (25 mmol L )
2 4
6.8 Multi-tube Vortex Mixer.
-1 -1
CaCl ·2H O (Molecular Wt 147.01) 0.15 g L (1 mmol L )
2 2
-1 -1
6.9 Analytical Balance. MnCl ·2H O (Molecular Wt 197.9) 0.016 g L (0.1 mmol L )
2 2
-1 -1
MgCl (Molecular Wt 95.21) 0.095 g L (1 mmol L )
-1 -1
6.10 Ultra-Low Freezer, set at ≤-60°C.
ZnCl (Molecular Wt 136.3) 0.068 g L (0.05 mmol L )
-1 -1
FeCl ·6H O (Molecular Wt 270.3) 0.0003 g L (0.001 mmol L )
3 2
6.11 Stopwatch or Electronic Timer.
Sterile, 18 MΩ-cm water Add water to 1 L final volume
6.12 Manual or Electronic Pipettes.
6.13 Bio-Safety Cabinet (BSC).
A
TABLE 2 Extraction Buffer (pH 7)
Reagent Final Concentration
6.14 Appropriate PPE, for example, gloves, safety glasses,
lab coats, etc. (4). 1X Extraction Buffer
-1
Tryptic Soy Broth 10 g L (1%)
-1 -1
6.15 Pipettes, 200 µL and 1 mL.
L-Alanine (Molecular Wt 89.09) 8.91 g L (100 mmol L )
-1 -1
Inosine (Molecular Wt 268.23) 2.68 g L (1 mmol L )
-1
7. Reagents and Materials
Tween® 80 (Polysorbate 80) 0.5 mL L (.05%)
Sterile, 18-megaohm water Add water to 1 L final volume
7.1 Reagents:
2X Extraction Buffer
-1
7.1.1 Bacillus anthracis, cereus and thuringiensis— Tryptic Soy Broth 20 g L (2%)
-1 -1
L-Alanine (Molecular Wt 89.09) 17.82 g L (200 mmol L )
acquisition, holding, preparations and/or testing of virulent
-1 -1
Inosine (Molecular Wt 268.23) 5.36 g L (2 mmol L )
strainsrequireCDCorAPHISregistrationintheUnitedStates.
Sterile, 18 MΩ-cm water Add water to 1 L final volume
StrainscanbeobtainedfromATCC,theBacillusGeneticStock
A
Can include a chemical neutralizer if necessary to neutralize any sporicidal
Center, BEI resources repository at National Institute of
activity of a chemical vapor.
Health, or the Unified Culture Collection (UCC) at USA
8,9
Medical Research Institute of Infectious Disease.
7.1.1.1 B. anthracis strain examples include virulent Ames
(UCC BACI387), attenuated Sterne (UCC BACI397), and
7.1.11 90% (v/v) Ethanol.
attenuated ∆Sterne (UCC BACI056).
7.1.12 Sodium Thiosulfate (STS)—
7.1.1.2 B. cereus strain examples include a type strain
7.2 Materials:
(ATCC 10792), E33L (UCC BACI267), and 03BB102 (UCC
7.2.1 Amicon® Ultra-15 Centrifugal Filter Units with
BACI234).
Ultracel, 100K membranes (Millipore, Billerica, MA, USA,
7.1.1.3 B. thuringiensis strain examples include Al Hakam
UFC9010096) (filter units).
(UCC BACI229), cry- HD-1 (Bacillus Genetic Stock Center
7.2.2 Sterile 50-mL Conical Tube.
ID4A12).
7.2.3 Baffled Flasks.
7.1.2 Tryptic Soy Broth (TSB).
7.2.4 Sterile Petri Dishes.
7.1.3 Tryptic Soy Agar (TSA).
7.2.5 50-mL Conical Tube and Microfuge Tube Racks.
7.1.4 Nutrient Broth (NB).
7.2.6 Pipette Tips.
7.1.5 Tween-80. 0.1%, 3% and 20% stock solutions of
7.2.7 Parafilm .
Tween-80 suspended in deionized water.
7.2.8 L-shaped Sterile Spreaders.
7.1.6 L-Alanine.
7.2.9 1.5-mL Sterile Microfuge Tubes.
7.1.7 Inosine.
7.2.10 Coupon Materials—All coupon materials must be a
7.1.8 Sporulation Broth—0.8% (w/v) Nutrient broth or
standardized surface area, preferably flat, 2 cm×2cm;
2.5% (w/v) Nutrient broth and salts, as defined in Table 1,pH
however, it is understood that not all materials are easily
7 (see Appendix X1 for preparation instructions) (3).
adaptable to these size constraints.
7.1.9 Extraction Buffer—pH 7, as defined in Table 2 (4, 5).
7.2.11 Sterile Forceps.
7.1.10 pH-adjusted Bleach—0.6% (v/v) hypochlorite,
7.2.12 Sodium Thiosulfate (STS).
≥0.2% (v/v) glacial acetic acid, pH ≥6.5 and ≤7.0 within 1 h
that it is mixed. The pH will gradually drop to pH ≥4.0 and
8. Hazards
≤5.0 after storage for 7 d.
8.1 It is the responsibility of the individual user(s) of this
7 practice to follow all safety guidelines and to be knowledge-
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
Standard-Grade Reference Materials, American Chemical Society, Washington,
able about these procedures. Individual users should consult
DC. For suggestions on the testing of reagents not listed by theAmerican Chemical
their safety authority and establish detailed safety plans and
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
risk assessments prior to using this practice. Users are strongly
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
urgedtoconsulttheBiosafetyinMicrobiologicalandBiomedi-
copeial Convention, Inc. (USPC), Rockville, MD.
Virulent strains require CDC (cross reference citation in 2.2) orAPHIS (cross
cal Laboratories (4).
reference citation in 2.2) registration in the United States.
If you are aware of alternative suppliers, please provide this information to
ASTM International Headquarters. Your comments will receive careful consider-
1 10
ation at a meeting of the responsible technical committee, which you may attend. Trademarked by Bemis Company, Inc. Neenah, WI 54956
E3178 − 18
9. Test Organisms 10.8.2 Suspend spores in kaolin at a final concentration of
8 -1 -1
1-2×10 spores mL , 0.1% Tween 80 and 50 g L kaolin.
9.1 Specific organisms are recommended, but the choice of
8 -1
10.8.3 At a test concentration of 1-2×10 spores mL ,
organism(s)shouldberelevanttotheenvironmentinwhichthe
kaolin is 250-500 1-2×10 excess over spores by weight.
decontaminant is expected to be used.
10.9 Optional: Spores may be mixed with organic soil load
9.2 Pathogenic and non-pathogenic stains of macrobacillus
prior to coupon inoculation. Humic acid suspended in spent
including Bacillus anthracis – for example, Ames, Sterne,
sporulationmedium(SSM)wasselectedasanorganicdebrisin
∆Sterne.
published tests (3).
9.3 Acrystalliferous strains of Bacillus thuringiensis – for
10.9.1 Collect the SSM after spore harvest and centrifuga-
-
example, Al Hakam, cry HD-1.
tion (Ref (3) and Appendix X1).
10.9.2 Filter-sterilize the SSM through a 0.2µm filter. The
9.4 Other macrobacillus and microbacillus strains, vegeta-
SSMmaybestoredinanultra-freezerat≤-60°Cforlong-term
tive bacteria, bacteriophage and vertebrate viruses may also be
storage.
tested using this practice.
-1
10.9.3 Suspend humic acid in SSM at 10 g L humic acid
and autoclave-sterilize for 30 min on a wet cycle.
10. Preparation of Inoculum
10.9.4 Suspend spores in the humic acid + SSM at a final
10.1 Prepare five independent spore inocula from five inde-
8 -1
concentration of 1-2×10 spores mL , 0.05% (v/v) Tween 80,
pendent spore preparations. Spores from each independent
5 g L-1 humic acid, 0.5X SSM.
spore preparation are used to prepare its corresponding inde-
8 -1
10.9.5 At a test concentration of 1-2×10 spores mL , the
pendent spore inoculum.
humic acid is 25-50× excess over spores by weight.
10.2 Transfer 0.1% Tween 80 into a pre-labeled 50-mL
conical tube using a volume of 0.1% Tween 80 that will 11. Preparation of Coupon
8 -1
achieve a target concentration of 1-2×10 spores mL .
11.1 Rinse coupons with 18-megaohm water.
10.2.1 Preheatthe50-mLconicaltubeswith0.1%Tween80
11.1.1 Drycouponsonabsorbentpaperinanautoclave-safe
at 50 6 2°C.
container.
10.3 Transfer concentrated spores from an ultra-low freezer
11.2 Autoclave temperature-insensitive coupons at 121°C
(6.10) set at ≤-60°C directly to a 50 6 2°C water bath for at
for 30 min on a wet cycle.
least 30 min.
11.3 Soak materials that are temperature sensitive in pH-
10.3.1 Maintain spores at 50 6 2°C during spore inocula-
adjustedbleachfor10min,followedbysoakinginexcess90%
tion to mitigate the risk of spore clumping prior to and during
ethanol for at least 15 min. Air dry overnight in BSC.
coupon inoculation.
11.3.1 Store sterilized coupons in sterile containers under
10.4 Vortex concentrated spores for 15-30 s.
ambient (22 6 3°C) laboratory conditions until use.
11.3.2 Verify coupon sterility during testing when un-
10.5 Transfer concentrated spores into pre-labeled 50-mL
inoculated coupons are taken through the entire testing proce-
conical tubes containing preheated (50 6 2°C) sterile 0.1%
dure and checked for sterility.
(v/v) Tween 80 to achieve a target concentration of 1-2×10
-1
spores mL .
NOTE 1—The pre-neutralized controls in the test matrix will confirm
10.5.1 Rinsethepipettetipsbypipettingupanddowntwice there is no residual hypochlorite activity.
in the 50-mL conical tube in order to rinse spores from the
12. Test Procedure (see Fig. 1)
plastic tips.
12.1 Carrier Inoculation—Confirm inoculum titer on the
10.6 Hold the diluted spore inoculum at 50 6 2°C until
day of coupon inoculation.
coupon inoculation, which should occur within 24 h of
12.1.1 Coupons (Fig. 1 a-c):
preparing the inocula.
12.1.1.1 Vortex pre-warmed (50 6 2°C) inoculum for
10.7 In order to titer the spore inoculum, transfer 0.1 mLof
15-30 s.
spore inoculum into 0.9 mL of 0.1% Tween 80, serially dilute
12.1.1.2 Use a P-1000 pipette to transfer a single 100 µL
and plate 0.1 mL on TSA plates.
drop of clean spores or spores mixed with inorganic debris or
10.7.1 Invertplatesandincubateat35 62°Cfor16 62h.
spores mixed with organic debris per inoculation. The pipette
The time and temperature of plate incubation can be adjusted
tip should be immersed half way into the inoculum when
for strains, for example, B. thuringiensis HD-1 strains produce
removing aliquots.
large colonies and this strain is incubated at 30 6 2°C for 16
12.1.1.3 Since spores will settle in suspension, inoculate a
6 2 h in order to ensure countable plates.
maximum of 12-18 coupons at one time.Then return the spore
10.7.2 Count and record data.
suspension back into the 50 6 2°C water bath.
10.8 Optional: Spores may be mixed with an inorganic soil 12.1.1.4 Select the next inoculum, working through all five
load prior to coupon inoculation. Kaolin (Al Si O (OH) ) was
independent spore preparations, performing Steps 12.1.1.1
2 2 5 4
selected as an inorganic debris in published tests (3). through 12.1.1.3 until all coupons have been inoculated.
-1
10.8.1 Suspendkaolinin0.1%Tween80at100gL kaolin 12.1.1.5 Allow coupons to dry overnight in an operating
and autoclave-sterilize for 30 min on a wet cycle. BSC under ambient conditions.
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NOTE 1—Pre-neutralized decontaminant should be neutralized for at least 5 min prior to transferring to coupon. 5% sodium thiosulfate (STS) is the
neutralizer in the figure.
FIG. 1 Illustration of Decontamination Test Method to Remove Static and Cidal Chemicals from Coupons (Source: Ref 2)
E3178 − 18
(1)Record the temperature inside the BSC. 12.2.2.1 Prepare the pre-neutralized decontaminant by mix-
12.1.1.6 Transfer inoculated coupons into pre-labeled filter ing 2 mL of reactive decontaminant plus 2 mL of the
units (4.1). designated neutralizer.
(1)Store coupons under ambient (22 6 3°C) laboratory 12.2.2.2 Add the pre-neutralized decontaminant solution to
conditions until use.
spore-inoculated coupons.
12.2.2.3 Incubated for the designated contact time at ambi-
NOTE 2—Inoculated coupons can be stored indefinitely so long as there
ent temperature (22 6 3°C).
is no loss in spore titer. The recommended storage time is no longer than
12 months.
12.2.2.4 The pre-neutralized control provides confidence
that chemical neutralization was successful. A comparison of
12.1.2 Solution (Wet) Controls (not shown in Fig. 1):
this control and the solution controls with the test coupon
12.1.2.1 Aseptically add 27 mL of sterile 0.1% Tween 80
isolates reactive-ingredient contact time as the sole test vari-
into 50-mL conical tubes, and then place the filter insert into
able.
thetube.The27mLof0.1%Tween80preventsthesolutionin
12.2.3 Incubate negative controls (uninoculated coupons) at
the filter unit from dripping through the membrane over an
ambient laboratory conditions (22 6 3°C).
extended period of time, and the 100,000 molecular weight
cutoff membrane keeps spores in the filter unit. 12.2.4 Incubatesolutioncontrols(4.9mLof0.1%Tween80
plus 0.1 mL of spore inoculum) at ambient laboratory condi-
12.1.2.2 Aseptically add 4.9 mL of sterile 0.1% Tween 80
into the filter unit. tions (22 6 3°C).
12.1.2.3 Vortexthepre-warmed(50 62°C)sporeinoculum
12.3 Centrifugation and Washing (Fig. 1 h-m)—All spore-
for 15-30 s.
inoculated coupons are centrifuged and washed. The solution
12.1.2.4 Use a P-1000 pipette to transfer a single 100 µL
controls and negative coupon controls are not centrifuged and
drop of spores per solution control. The pipette tip should be
washed.
immersedhalfwayintotheinoculumwhenremovingaliquots.
12.3.1 Add 10 mLof sterile, autoclaved water to each filter
12.1.2.5 Inoculate solution controls in conjunction with
unit to dilute the neutralized decontaminant solutions.
coupons, up to 12-18 solution controls at one time.
12.3.1.1 Afte
...