ASTM E2839-21

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of the standard as published by ASTM is to be considered the official document.
Designation: E2839 − 18 E2839 − 21
Standard Practice for
Production and Storage of Spores of C. difficile for Use in
Efficacy Evaluation of Antimicrobial Agents
This standard is issued under the fixed designation E2839; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope Scope*
1.1 This practice specifies the procedures for producing and storing standardized suspensions of Clostridioides diffıcile spores for
the evaluation of the sporicidal activity of antimicrobial formulations using the Quantitative Method for Testing Antimicrobial
Agents against Spores of C. diffıcile on Hard, Non-porous Surfaces or other procedures.
1.2 This practice may involve hazardous materials, chemicals, and microorganisms and should be performed only by persons with
formal training in microbiology.
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 and health practices and determine the applicability of regulatory
limitations prior to use.
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.
2. Referenced Documents
2.1 ASTM Standards:
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
E3218 Test Method for Quantitative Method for Testing Antimicrobial Agents against Spores of C. diffıcile on Hard,
Non-porousNonporous Surfaces
3. Terminology
3.1 For definition of terms used in this practice refer to Terminology E2756.
This practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Oct. 1, 2018June 1, 2021. Published September 2019July 2021. Originally approved in 2011. Last previous edition approved in 20112018 as
E2839–11.–18. DOI: 10.1520/E2839-18.10.1520/E2839-21.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2839 − 21
3.2 Definitions of Terms Specific to This Standard:
3.2.1 density gradient medium, adj/n—HistoDenz (trademarked) is a non-ionic gradient medium used here to separate spores
from vegetative cells and cell fragments on the basis of density.
3.2.2 pre-reduced medium, n—an agar or broth manufactured in an oxygen-free environment and packaged in air-tight sealed
pouches or bags.
3.2.3 purified spores, n—level of quality based on when spore concentration reaches ≥95 %, as vegetative cells and cell fragments
are separated by the density gradient medium.
3.3 Acronyms:
CDC = Centers for Disease Control and Prevention
CFU = colony-forming unit
VFS = vegetative frozen stock.
4. Summary of Practice
4.1 This practice provides detailed instructions for the culture, maintenance, sporulation, and storage of spores of C. diffıcile.
Spores are harvested from an agar medium following incubation in an anaerobic environment for 10 days. Upon harvesting, spores
are washed several times with phosphate-buffered saline with polysorbate 80, exposed to heat to inactivate any vegetative cells,
and purified using a density gradient medium. Purified spores are enumerated and assessed for quality using a carrier-based test
employing two concentrations of sodium hypochlorite (NaOCl). Spores of C. diffıcile are stored for up to 90 days at -80-80 °C 6
5 °C.
5. Significance and Use
5.1 This practice describes a procedure for preparing and storing a suspension of C. diffıcile spores that meets the following
acceptance criteria: (1) spore titer of approximately 5.0×10 spores/mL, (2) spore purity of ≥95 %, and (3) a mean log reduction
(LR) value >5.0 for 3 carriers exposed to 5000 ppm and a mean LR of <3.0 for 3 carriers exposed to 1500 ppm sodium
hypochlorite. These acceptance criteria are necessary in order to use the spore suspension to evaluate the performance of
antimicrobial formulations using Test Method E3218.
6. Apparatus
6.1 Calibrated micropipettes (for example, 200 μL and 1000 μL)—1,000 μL pipettes with corresponding tips for transferring
reagents and spores. 200 μL pipettes with corresponding tips for deposition of test substance on carrier.
6.2 Sterile centrifuge tubes—Polypropylene, 15 mL and 50 mL graduated plastic centrifuge tubes with conical bottoms.
6.3 Microcentrifuge tubes—Siliconized, sterile 1.5 mL low-retention microcentrifuge tubes. Use for dilutions and processing of
spores during purification.
6.4 Centrifuge with swinging-bucket rotor—To allow sedimentation of spores for washing and/or concentration.
6.5 Microcentrifuge—To allow sedimentation of spores for washing and/or concentration.
6.6 Inoculating loop—10 μL loop to streak plates.
6.7 Vortex mixer.
The sole source of supply of HistoDenz (trademark) (Cat. No. D2158) known to the committee at this time is Sigma-Aldrich, St. Louis, MO. If you are aware of
alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible
technical committee, which you may attend.
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6.8 Polyethersulfone membrane filter (PES)—For recovery of test spores, 47 mm diameter with 0.2 μm pore size. Use any filtration
apparatus including filtration units (reusable or disposable).
6.9 Anaerobic chamber—Supported by a gas mixture consisting of at least 5 % H with the balance comprising any inert gas such
as CO , N , or Ar; refer to chamber manufacturer’s recommendations. Alternatively, an activated anaerobic jar can be used
2 2
according to manufacturer’s instructions for ensuring an anaerobic environment.
6.10 Incubator—Use an incubator at 36 6 1 °C inside an anaerobic chamber to support the growth of the organism. Alternatively,
place anaerobic jars inside an aerobic incubator at 36 6 1 °C.
6.11 Microscope with 10× eyepiece and 100× (oil) objective with phase contrast option—To evaluate purity of spore suspension.
6.12 Timer—Any timer that can display time in seconds.
6.13 Cell scraper/spreader—To aid in the removal of spores from agar plates.
6.14 Laboratory film (or sealable bag)—To seal inoculated plates during extended incubation (>48 h).
6.15 Refrigerator (2-8 °C)—For short term storage of spore suspension during the purification process.
6.16 Freezer (-80 6 5 °C)—For long term storage of stock cultures and spore suspensions.
6.17 Carriers—Disks Flat disks (1 cm in diameter) made from approximately 0.8 mm thick sheets of brushed and magnetized
stainless steel (AISI 430) for use in spore qualification using NaOCl. of AISI Type 304 Stainless Steel with 150 grit unidirectional
finish on one side. See Test Method E3218 for carrier specifications.
6.17.1 The top of the disk is brushed and has rounded edges; only the top is visually screened and inoculated. Carriers are
single-use only. See Test Method E3218 for carrier specifications.
6.18 Calibrated 10 μL positive displacement pipette with corresponding 10 μL tips—For carrier inoculation.
6.19 Bottle-top dispensers, squirt bottles, pre-measured volumes in tubes, or pipettes—For rinsing vials and filters.
6.20 Sterile forceps—To handle membrane filters and to pick up the carriers for placement in vials.
6.21 Filter paper—150 mm diameter, to line Petri plates.
6.22 Magnet—Use to hold carrier in place in the vial while liquid is being dispensed into filter unit.
6.22 Sterile vials with lids (plastic or comparable)—for holding inoculated carriers to be exposed to the test chemical and for
accommodating the neutralizers. Flat-bottomed and wide-mouthed to accommodate addition and removal of the carriers. Use vials
at least 25 mm in neck diameter and capable of holding at least 20 mL of liquid.
6.23 Desiccator (with gauge to measure vacuum) with fresh desiccant (for example, CaCO )—For drying the inoculum on the
carriers.
6.24 Vacuum source—In-house line or suitable vacuum pump (0.068 to 0.085 MPa) for drying carriers and for filtering.
6.25 Digital titrator kit—To measure total chlorine. Alternate titration methods may be used.
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7. Media and Reagents
7.1 Culture Media:
7.1.1 Reinforced clostridial medium (RCM)—For use in rehydrating lyophilized/frozen vegetative culture of C. diffıcile. Prepare
RCM according to manufacturer’s instructions, and pre-reduce in an anaerobic environment for 2424 h 6 2 h prior to use.
7.1.2 RCM plus 15 % glycerol (Cryoprotectant)—For use as cryopreservation medium for vegetative frozen stock (VFS) cultures.
Prepare RCM and add 15 % (v/v) glycerol, autoclave for 20 min at 121 °C; pre-reduce in an anaerobic environment for at least
24 6 2 24 h 6 2 h prior to use.
7.1.3 CDC anaerobic 5 % sheep blood agar (CABA)—Sporulation medium commercially available pre-reduced.
7.1.4 Recovery media for enumeration of viable spores—Pre-reduced brain-heart infusion agar with yeast extract, horse blood and
sodium taurocholate (BHIY-HT).
7.2 Reagents :
7.2.1 Phosphate-buffered saline (PBS)—For use as a rinsing agent and to prepare PBS containing 0.1 % (v ⁄v) Tween-80 (PBS-T)
and PBS-T with 0.1 % (w/v) sodium thiosulfate; PBS pH is 7.0 6 0.5.
7.2.2 Phosphate-buffered saline containing 0.1 % Tween 80 (PBS-T)—Diluting and washing reagent; PBS-T pH is 7.2 6 0.2.
7.2.3 PBS-T with 0.1 % (w/v) sodium thiosulfate—Neutralizer for sodium hypochlorite-based test chemicals; PBS-T with sodium
thiosulfate pH is 7.2 6 0.2.
7.2.4 Purity of Water—all references to water as diluent or reagent shall mean de-ionized water or water of equal purity.
7.2.5 Reagent-grade sodium hypochlorite (NaOCl) with total chlorine ≥4%—Use to prepare 5000 6 250 ppm and 1500 6 150
ppm total chlorine solutions to qualify spores.
7.2.6 Tween-80 (polysorbate 80)—Use to prepare PBS-T.
TM
7.2.7 HistoDenz —Use as a density gradient medium for spore purification. Prepare a 50 % (w/v) solution in deionized water.
Pass the solution through a sterile 0.45 μm filter to sterilize. Store at 2-5 °C.
7.2.8 Soil load—Use in assay to qualify spore suspension. Soil load is a mixture of the following stock solutions:
7.2.8.1 Bovine serum albumin (BSA)—Add 0.5 g BSA (radio immunoassay (RIA) grade or equivalent) to 10 mL of PBS, mix, and
pass through a 0.2 μm pore diameter membrane filter to sterilize.
7.2.8.2 Yeast extract—Add 0.5 g yeast extract to 10 mL of PBS, mix, and pass through a 0.2 μm pore-diameter membrane filter
to sterilize.
7.2.8.3 Mucin—Add 0.04 g mucin (from bovine submaxillary gland or equivalent) to 10 mL of PBS, mix thoroughly until
dissolved, and autoclave (15 min at 121 °C). pass through a 0.2 μm pore diameter membrane filter.
7.2.8.4 Aseptically aliquot soil stock solutions and store for up to one year at -20 6 5°C.-20 °C 6 5 °C. The stock solutions of
the soil load are single use only; do not refreeze once thawed.
NOTE 1—Other volumes of the stock solutions may be prepared following the same ratio.
The sole source of supply of the CABA (Cat. No. AS-646) and BHIY-HT (Cat. No. AS-6463) known to the committee at this time is Anaerobe Systems, Morgan Hill,
CA. If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a
meeting of the responsible technical committee, which you may attend.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference Materials, American Chemical Society, Washington, DC. For
suggestions on the testing of reagents not listed by the American Chemical Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and
the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
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8. Test Organism
8.1 Clostridioides diffıcile (ATCC 43598), formerly known as Clostridium diffıcile, a toxigenic strain (tcdA-, tcdB+), obtained from
a reputable vendor. The strain produces Toxin B only (presence of tcdB gene by PCR). The organism is a Gram-positive, strictly
anaerobic, spore-forming bacterium that produces flat, gray, irregular colonies on the surface of CABA plates within 48 h at
3636 °C 6 1 °C.
9. Hazards
9.1 The test organism (C. diffıcile, ATCC 43598) must be incubated under strict anaerobic conditions and in accordance with local
biosafety practices or those recommended by the U.S. Centers for Disease Control and Prevention/National Institutes of Health
(CDC/NIH) for organisms at Biosafety Level II (1). Processing of spores can be conducted in an aerobic environment (for example,
inside a BSC); all incubation for growth, however, must be performed anaerobically.
10. Preparation of Frozen Stock Cultures of Test Organism (Vegetative Form)
10.1 Preparation of Inoculum:
10.1.1 C. diffıcile received in lyophilized vegetative form:
10.1.1.1 In an anaerobic environment, reconstitute contents of the lyophilized culture with 0.5 mL of sterile pre-reduced RCM
according to manufacturer’s instructions.
10.1.1.2 After rehydration, aseptically transfer the vial contents to a tube containing 44 mL 6 1 mL of pre-reduced RCM, and mix
by gentle vortexing.
10.1.2 C. diffıcile received as frozen vegetative culture:
10.1.2.1 Thaw frozen culture at room temperature.
10.1.2.2 In an anaerobic environment, transfer the contents to a tube containing 461 4 mL 6 1 mL of sterile pre-reduced RCM
and mix by gentle vortexing.
10.2 Inoculation of CABA Plates for Vegetative Stock Culture:
10.2.1 Spread plate 100 μL of the reconstituted culture in RCM on each of five CABA plates.
10.2.2 Streak one CABA plate for isolation to check for culture purity.
10.2.3 Invert plates and incubate anaerobically at 36 6 1 °C36 °C 6 1 °C for 48 6 4 48 h 6 4 h. Observe CABA plate for purity
and characteristics of C. diffıcile; discard the CABA plates if not pure and reinitiate a new stock culture.
10.3 Harvest of CABA Plates for Stock Culture:
10.3.1 Following incubation (10.2.3), add approximately 2 mL of sterile and pre-reduced cryoprotectant (7.1.2) to each CABA
plate.
10.3.2 Using a sterile cell scraper, gently scrape culture from the surface of one plate, aspirate with a pipette and transfer to a
15-mL15 mL conical tube. Repeat this process for the remaining plates.
10.3.3 Pool the suspensions, mix thoroughly, and pipette approximately 0.40.4 mL 6 0.1 mL aliquots into cryovials; cap tightly.
10.3.4 Store the cryovials at -80-80 °C 6 5 °C. These vials contain the Vegetative Frozen Stock (VFS) Cultur
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