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(TSM) to Determine Sporicidal Efficacy of Liquids, Liquid Sprays, and Vapor or Gases on Contaminated Carrier Surfaces (Withdrawn 2014)

SIGNIFICANCE AND USE
The quantitative micromethod described herein was designed to fulfill the following specifications:
4.1.1 To be quantitative (the number of viable spores loaded into carriers is determined by the spores quantitatively recovered in the controls),
4.1.2 Sensitive (sporicidal activity can be accurately measured up to 7 Log10 inactivation, see Section 12),
4.1.3 Reproducible (standard deviation of spore killing is smaller than 1 Log and usually closer to 1/10 of a Log, see Section 12),
4.1.4 Rapid (except for the overnight culture, it can be completed within 4 h),
4.1.5 Economical (being a micromethod, it uses carriers, dishes, and plastic wares that are small, inexpensive, and disposable), and
4.1.6 Environmentally friendly (using a microlitre volume of disinfectant agent, the test method can be considered for all practical purposes as nondestructive).
SCOPE
1.1 This test method determines the efficacy of sporicidal agents on microorganisms dried on the surface of solid carriers.
1.2 This test method can be used to evaluate sporicidal products (or decontaminant, disinfectant, and so forth), suspected, claimed, or assumed to have sporicidal activity. This test method allows:
1.2.1 Establishing the sporicidal efficacy of different disinfectants;
1.2.2 Identifying the effect, if any, of the surface materials on sporicidal efficacy; and
1.2.3 Comparing the relative resistance to disinfection of different microbial species or strains.
1.3 The values stated in SI units are to be regarded as the standard.
1.4 Strict adherence to the protocol is necessary for the validity of the test results.
1.5 Follow all the safety guidance of the institution in which the testing is being conducted. This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
WITHDRAWN RATIONALE
This test method determined the efficacy of sporicidal agents on microorganisms dried on the surface of solid carriers.
Formerly under the jurisdiction of Committee E54 on Homeland Security Applications, this test method was withdrawn in January 2014 in accordance with section 10.5.3.1 of the Regulations Governing ASTM Technical Committees, which requires that standards shall be updated by the end of the eighth year since the last approval date.

General Information

• Status: Withdrawn
• Publication Date: 14-Jun-2005
• Withdrawal Date: 06-Jan-2014
• ICS: 11.080.01 - Sterilization and disinfection in general
• Technical Committee: E54 - Homeland Security Applications

Relations

• Referred By: 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
• Effective Date: 15-Jun-2005
• Referred By: ASTM E2800-11(2017) - Standard Practice for Characterization of <emph type="ital">Bacillus</emph> Spore Suspensions for Reference Materials
• Effective Date: 15-Jun-2005

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E2414 − 05
StandardTest Method for
Quantitative Sporicidal Three-Step Method (TSM) to
Determine Sporicidal Efficacy of Liquids, Liquid Sprays, and
Vapor or Gases on Contaminated Carrier Surfaces
This standard is issued under the fixed designation E2414; 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 3. Summary of Test Method
3.1 Thistestmethoddeterminestheefficacyofdisinfectants
1.1 This test method determines the efficacy of sporicidal
on spores of Bacillus subtilis dried on carriers according to a
agentsonmicroorganismsdriedonthesurfaceofsolidcarriers.
general technique first described by J. L. Sagripanti and A.
1.2 This test method can be used to evaluate sporicidal
Bonifacino (1, 2) .
products (or decontaminant, disinfectant, and so forth),
3.1.1 Sporesof B. subtiliscanbeandhavebeenreplacedby
suspected,claimed,orassumedtohavesporicidalactivity.This
similar amounts of spores of Bacillus anthracis to substantiate
test method allows:
claims as may be needed in biodefense.
1.2.1 Establishing the sporicidal efficacy of different disin- 3.1.2 The material of the carrier is selected according to
fectants; claims or intended use, or both, of the disinfectant product.
General claims made to decontaminate metallic and polymeric
1.2.2 Identifying the effect, if any, of the surface materials
materials are tested on carriers made of stainless steel and
on sporicidal efficacy; and
silicone medical rubber, respectively. Flat coupons (0.5 by 0.5
1.2.3 Comparing the relative resistance to disinfection of
cm) are preferable. As may be required by claims or intended
different microbial species or strains.
use,thetestmethodcanbeaccuratelyusedalsoonavarietyof
1.3 The values stated in SI units are to be regarded as the
carriers with diverse geometrical characteristics (additional
standard.
examples of materials were reported by Sagripanti and Boni-
facino (1, 2).
1.4 Strict adherence to the protocol is necessary for the
3.1.3 Although the test method described herein refers
validity of the test results.
specifically to liquid disinfectants, the same procedure can be
1.5 Follow all the safety guidance of the institution in which
used to assess sporicidal activity of vapors and gaseous
the testing is being conducted. This standard does not purport
sporicidal agents, provided adequate containment is accom-
to address all of the safety concerns, if any, associated with its plished (see 10.4.5).
use. It is the responsibility of the user of this standard to
establish appropriate safety and health practices and deter- 4. Significance and Use
mine the applicability of regulatory limitations prior to use.
4.1 The quantitative micromethod described herein was
designed to fulfill the following specifications:
2. Referenced Documents
4.1.1 Tobequantitative(thenumberofviablesporesloaded
into carriers is determined by the spores quantitatively recov-
2.1 ASTM Standards:
ered in the controls),
E1054Test Methods for Evaluation of Inactivators of Anti-
4.1.2 Sensitive (sporicidal activity can be accurately mea-
microbial Agents
sured up to 7 Log inactivation, see Section 12),
4.1.3 Reproducible (standard deviation of spore killing is
smaller than 1 Log and usually closer to ⁄10 of a Log, see
This test method is under the jurisdiction of ASTM Committee E54 on
Section 12),
Homeland Security Applications and is the direct responsibility of Subcommittee
4.1.4 Rapid (except for the overnight culture, it can be
E54.03 on Decontamination.
Current edition approved June 15, 2005. Published July 2005. DOI: 10.1520/ completed within 4 h),
E2414-05.
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 Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
the ASTM website. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2414 − 05
4.1.5 Economical (being a micromethod, it uses carriers, 6.2 Sterile Luria—Bertani broth (LB) (if in powder form,
dishes, and plastic wares that are small, inexpensive, and prepared as recommended by the manufacturer).
disposable), and
6.3 HPLC quality sterile water and sterile phosphate-
4.1.6 Environmentally friendly (using a microlitre volume
buffered saline (PBS).
of disinfectant agent, the test method can be considered for all
6.4 Nutrient agar plates.
practical purposes as nondestructive).
6.5 Laboratory glassware, graduated cylinders, calibrated
volumetric flasks.
5. Apparatus
6.6 Cupric chloride (CuCl ·2H O); L-ascorbic acid, and
5.1 List of Equipment—Make and models are provided as
2 2
hydrogen peroxide if using cupric ascorbate as a positive
examples. Use the same or equivalents.
control.
5.1.1 Microcentrifuge, rated to 12000 g with rotor to hold
1.5-mL conical microcentrifuge tubes.
6.7 Purity of Reagents—Reagent grade chemicals shall be
5.1.2 Autoclave.
used in all tests. Unless otherwise indicated, it is intended that
5.1.3 Vortex mixer.
all reagents conform to the specifications of the Committee on
5.1.4 Sonicator, low-power water bath type, rated to 400 to Analytical Reagents of theAmerican Chemical Society where
500 W (generally used for cleaning small objects by immer- such specifications are available.
sion).
6.8 Carriers:
5.1.5 Class II biosafety cabinet.
6.8.1 Spotting/Flat—Preferable glass, but also rubber, stain-
5.1.6 Balance, accurate to 1 mg.
less steel, polymeric plastics, or other materials cut in squares
5.1.7 Refrigerator, able to maintain 0 to 5°C.
of 0.5 by 0.5 cm.
5.1.8 Freezer, able to keep −80°C.
6.8.2 Dipping—Carriers that do not hold liquid and need to
5.1.9 Incubator, able to maintain 37 6 1°C (usually with a
becontaminatedbyimmersion,forexample,screws,cylinders,
range from room temperature to 60°C).
and tubing. The size and shape of carriers should allow their
5.1.10 Colony counter.
introduction inside the 1.5-mL microcentrifuge tubes to be
5.1.11 Photomicroscope, providing 1000× maximal magni- immersed in the 400 µL of liquid and have a total volume
fication to control spore quality.
between 50 to 200 µL.
5.1.12 Micropipets, with corresponding sterile tips able to
6.9 Supplies:
delivervolumesintherangesof10,20,100,200,and1000µL.
6.9.1 Forceps/Tweezers, to handle carriers.
5.1.13 Timer, any certified timer/watch/clock that can dis-
6.9.2 Sterile Disposable Petri Dishes, 100 by 15 mm.
play time in seconds.
6.9.3 Sterile Disposable Petri Dishes, 47 mm in diameter
5.1.14 Rotator, able to provide 15 to 20 rpm (of the type
(preferable to test sprays).
used in hematology chemistry) with a rack to hold 1.5-mL
6.9.4 Sterile,disposable1.5-mLpolypropylenemicrocentri-
microcentrifuge tubes.
fuge tubes.
5.1.15 Shaker, able to control speed and inside temperature.
6.9.5 Sterile, disposable 15- and 50-mL conical-bottom
5.1.16 Anaerobic Jar, if testing gases.
centrifuge tubes.
5.1.17 Cooler, able to maintain temperature at 21 6 3°C 6.9.6 Sterile, disposable spreaders.
6.9.7 Nonsterile, latex examination gloves.
6. Reagents and Materials
7. Hazards
6.1 Spores of B. subtillis (Strains ATCC 1031 or ATCC
7.1 Allmanipulationsofthetestorganismarerequiredtobe
9372), Bacillus globigii (Renamed B. atrophaeus SB 512), or
performed in accordance with biosafety practices stipulated in
B. anthracis (Pathogenic Strains Albia BA 1029, Ames, NCTC
the institutional biosafety regulations. Use equipment and
1087, or Vollum or Nonpathogenic Strains Sterne, Delta-
facilities at the biosafety level indicated for the test microor-
Sterne, or Pasteur BC3132)—Stock suspensions are prepared
ganism.Forrecommendationsonsafehandlingofmicroorgan-
under the appropriate biosafety containment and produced at a
isms refer to Ref (5).
9 9
concentration between1×10 and5×10 colony-forming
7.2 Sporicidal products may contain a number of different
units/mL. A variety of media are available to grow spores of
active ingredients, such as heavy metals, aldehydes, peroxides,
Bacillus species. It is recommended the use of sporulation
phenol, halogen-containing substances, quaternary ammonium
media Media S whose formulation is described in Appendix
compounds, or any other reagent suspected, claimed, or as-
X1. Preparations (made as suggested in Appendix X1) are
sumed to have sporicidal activity. Gloves and personal protec-
accepted for use when consisting in more than 95% spores as
tive clothing or devices are worn during the handling of these
determined by both microscopic observation and acid resis-
tance. Microscopic observation of spores stained with trypan
blueshouldreveallessthan10vegetativecells(shapedasrods)
Reagent Chemicals, American Chemical Society Specifications, American
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
during the observation of 1000 spores (round shape). Testing
listed by the American Chemical Society, see Analar Standards for Laboratory
spores for acid resistance is described in Appendix X2 and
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
Appendix X3. Both tests for spore quality follow techniques
and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
published previously (1, 2, 4). MD.
E2414 − 05
materials. A chemical fume hood or other containment equip- specifications.After incubation with the disinfectant, ice-cold
ment is used when performing tasks with hazardous chemical LB medium with or without neutralizer is added.The carrier is
products. immediately transferred to a new 1.5-mLmicrocentrifuge tube
(labeled B) containing sterile distilled water at room tempera-
8. Sample Handling and Storage ture (21 6 3°C) and sonicated for 5 min. Ice-cold LB medium
is added and the carrier is transferred to a new 1.5-mL
8.1 Disinfectants are stored following manufacturer’s in-
microcentrifuge tube (labeled C) with LB medium. The Tubes
structions or at room temperature (21 6 3°C) if the product
C are incubated in a rotator inside of an incubator at 37°C for
labeldoesnotindicatespecialstorageconditions.Disinfectants
30 min. Ice-cold LB is added and the carrier discarded. The
requiring dilution or activation (or pH adjustment) before use
fluidcontainedinTubesA,B,andCcorrespondtoFractionsA,
are diluted or activated and tested within the time period
B,andC.Thesurvivingsporesineachfractionareenumerated
specifiedbythemanufacturerorwithintheshelf-lifetimeafter
byserialdilutionandspreadonpetridishescontainingnutrient
activation or dilution, if known for the particular disinfectant.
agar medium. Culture plates are incubated overnight (at least
If no information is available from the manufacturer or
12 h) at 37 6 1°C and colonies are counted. Total spores
shelf-life after dilution or activation is unknown, then test
surviving treatment with disinfectant are calculated by adding
within 4 h after dilution or activation.
the spores counted in Fraction A, plus spores in Fraction B,
8.2 Stocks of spores are stored refrigerated between 2 and
plussporesinFractionC.Thesporicidaleffectofadisinfectant
5°C.
is calculated by subtracting log of the total number of spores
surviving the treatment with disinfectant from the log of the
9. Calibration and Standardization
total number of spores in the controls incubated with sterile
9.1 Refer to the laboratory equipment calibration and main-
water. A summary of the method is shown in Figs. 1 and 2.
tenance standard operation procedures (SOPs) for details on 10.1.2 Use and change frequently nonsterile examination
test methods and frequency of calibration.
latex gloves for all handling during the procedure to avoid
spore carryover.
10. Procedure
10.2 Carrier Inoculation:
10.1 Brief Summary—This method recovers spores by dif-
10.2.1 Carrier Inoculation by Spotting:
ferential elution (in Fractions A, B, and C) by sequentially
10.2.1.1 Prepare a suspension of spores in sterile distilled
applying treatments of increasing dislodging strength. The
water at5×10 spores/mL with or without organic burden.
forces to dislodge spores in each fraction are different and not
10.2.1.2 The spores can be concentrated by centrifugation
interchangeable (loosely released by washing in A, sonication
or diluted in water if required. Resuspend spores thoroughly
in B, and incipient germination in C). These steps were
before spotting on carrier.
selected after trying many of possible combinations to maxi-
10.2.1.3 For general sporicidal claims, use glass carriers in
mize the recovery and accountability of spores.
0.5 by 0.5-cm squares (cut from microscope slides). For
specific claims, carriers can be made of, for example, silicone
NOTE 1—This procedure is to be performed by personnel trained in
medical rubber, light metal armor, building materials, or any
microbiology and with experience in the use of all laboratory equipment
listed. No standard or description can replace necessary training and
materialthatreflectsintendeduseofthetesteddisinfectantand
experience.Accordingly, the procedures in this test method are described
that can be cut into 0.5 by 0.5-cm squares, or a combination
at a level of detail that should allow their understanding and correct
thereof. Wash carriers three times with sterile distilled water
executionbyanybodyminimallyproficientinmicrobiologyandintheuse
andrinseoncewith95%ethanol.Thecarrierscanbesterilized
of general laboratory equipment.
by autoclaving or other procedures which will not affect the
10.1.1 Each clean and sterile carrier (either flat 0.5 by 0.5
properties of the carrier material.
cmordipping)receives10µLofasporesuspensioncontaining
10.2.1.4 Place the carriers flat inside the lower plate of a
9 9
between1×10 and5×10 organisms/mL (resulting in a
sterile plastic petri dish and load each carrier with 10 µLof the
microbialloadbetween1to5×10 sporespercarrier),withor
5×10 spore/mLsuspension as prepared in 10.2.1.1.The fluid
without organic load and dried for 10 to 20 h at 20 to 25°C.
must remain on the carrier or the carrier is discarded. Replace
The carrier loaded with spores is placed inside of a 1.5-mL
the cover of the petri dish and let the carriers dry 10 to 20 h
microcentrifuge tube (labeledA).The sporicidal product being
(overnight) inside a biosafety hood.
tested, is added to this tube, assuring that the inoculum in the
10.2.2 Carrier Preparation by Dipping—If odd-shaped ma-
carrier i
...