ASTM E3011-22

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Designation: E3011 − 15 E3011 − 22
Standard Test Method Practice for
In vitro production of Clostridium DifficileClostridioides difficile
Spores
This standard is issued under the fixed designation E3011; 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
1.1 This test method is designed to propagate spores of Clostridium diffıcile using liver broth.
1.2 It is the responsibility of the user of this test method to determine whether Good Laboratory Practices are required and follow
when appropriate.
1.3 This test method should only be performed by those trained in microbiological techniques.
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this
standard.
1.5 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.
2. Referenced Documents
2.1 ASTM Standards:
E2839 Practice for Production and Storage of Spores of C. difficile for Use in Efficacy Evaluation of Antimicrobial Agents
E2895 Practice for Producing High Titers of Viable and Semi-Purified Spores of Clostridium difficile using a Liquid Medium
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 Federal Standard:
40 CFR, Part 160 Good Laboratory Practice Standards
3. Terminology
3.1 Definitions: For definitions of general terms used in this test method, refer to Terminology E2756.
3.2 Definitions of Terms Specific to This Standard:
This test method 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 May 1, 2015Oct. 1, 2022. Published September 2015December 2022. Originally approved in 2015. Last previous edition approved in 2015 as
E3011–15. DOI: 10.1520/E3011–1510.1520/E3011–22.
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.
Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) 40 CFR Part 160, Good 185 Laboratory Practice Standards; Final Rule. 1989.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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3.2.1 frozen stock culture, n—a culture of vegetative bacteria propagated, and prepared for storage at ≤ –70°C in a liquid broth
medium containing a cryoprotectant such as glycerol.
3.2.2 spore suspension, n—harvested spores suspended in a liquid medium, sterile deionized water.
4. Summary of Test Method
4.1 This standard outlines a procedure for producing high-titer spore suspensions of C. diffıcile using a commercially available
liquid medium with 7 to 10 d of incubation under anaerobic conditions. Once adequate levels of spores are present in the liquid
medium, the spores are harvested and washed several times in cold sterile deionized water. The spore suspension is enumerated,
the spore purity is assessed and the acid resistance is verified.
5. Significance and Use
5.1 This test method describes a procedure for producing spore suspensions of C. diffıcile ATCC 700792, C. diffıcile ATCC 43598,
or C. diffıcile ATCC 43599. The spore suspensions may be used in antimicrobial efficacy testing, or other laboratory testing
requiring C. diffıcile spores. A spore crop is considered acceptable if the titer is >8 log spores/mL, purity of 95%, and is resistant
to 2.5M HCl after 10 min of exposure (see Test Method E2839).
6. Apparatus
6.1 Anaerobe jar—Any airtight jar or container that can be used in combination with gas packs to obtain an anaerobic
environment. An anaerobic chamber and anaerobic incubator may be substituted for an anaerobe jar.
6.2 Biological safety cabinet—To help maintain an aseptic work space.
6.3 Centrifuge—Any type or model capable of centrifuging up to 40 mL of liquid at 7500 × g.
6.4 Centrifuge tubes—Any type of sterile centrifuge tube with a 50 mL capacity.
6.5 Incubator—An incubator capable of maintaining 3661°C.
6.6 Laboratory glassware with closures—Glassware to hold up to 1L of media, and withstands autoclave temperatures. Closures
must be able to cover opening of glassware to protect media from environmental contamination.
6.7 Microcentrifuge tubes—Sterile 1.5 mL volume microcentrifuge tubes.
6.8 Micropipettors—Any suitable models capable of pipetting 10 μL or up to 1000 μL, or both. Calibrated micropipettors are
preferred.
6.9 Micropipette tips, sterile—Any sterile micropipette tips for use with Micropipettors in 6.8.
6.10 Microscope—Any microscope capable of 1000× magnification with phase contrast options.
6.11 Plate spreader—Any sterile spreader for spreading inocula on agar plates.
6.12 Serological pipettes—Any sterile, single use pipettes that are capable of pipetting 1.0 mL, 10 mL, or 50 mL volumes.
6.13 Sterile cheesecloth—Two layers of cheesecloth is placed inside an appropriately sized funnel and sterilized.
6.14 Vortex mixer.
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7. Reagents and Materials
7.1 Liver Broth —Used to sporulate C. difficile.
7.2 Butterfield’s Phosphate Buffer Stock Solution, 0.25M (PBSS)—Dissolve 34.0 g of monobasic potassium phosphate in 500 mL
of deionized water. Adjust pH to 7.2 with 10N NaOH, and dilute to 1 L.
7.3 Butterfield’s Phosphate Buffered Dilution Water (PBDW)—Add 1.25 mL of 0.25M PBSS to 1 L deionized water. Dispense into
9 mL or 99 mL portions. Autoclave for 20 min at 121°C.
7.4 pH adjusted Phosphate Buffered Dilution Water (pH adjusted PBDW)—PBDW with the pH adjusted with sterile 1 M Sodium
hydroxide (NaOH) to a pH that neutralizes 2.5 M Hydrochloric Acid (HCl) (pH 11-12 is suggested). The pH of PBDW may also
be adjusted with non-sterile 1 M NaOH prior to autoclave sterilization, provided the pH after sterilization is adequate to neutralize
2.5 M HCl.
7.5 Recovery Medium for Enumeration of Spore Suspension—Brain Heart Infusion Agar with yeast extract (5 g/L), horse blood
(70 mL/L) and sodium taurocholate (1 g/L) (BHIY-HT) , pre-reduced.
7.6 Hydrochloric acid (HCl)—2.5 M HCl is prepared from 5 M HCl.
7.7 Water—Sterile deionized water.
8. Hazards
8.1 C. diffıcile is a Biosafety Level 2 organism. Appropriate safety procedures, as recommended by the US Centers for Disease
Control and Prevention/National Institutes of Health or other local agency, should be used with this organism.
8.2 Consult Material Safety Data Sheets (MSDS) for the chemicals used in this method to determine the appropriate personal
protective equipment required for handling each chemical.
9. Test Organism
9.1 Frozen stock cultures of C. diffıcile ATCC 700792, C. diffıcile ATCC 43598, or C. diffıcile ATCC 43599. Frozen stock cultures
may be prepared from cultures obtained from a reputable vendor or culture collection agency.
9.2 Other strains of C. diffıcile may be sporulated using this method.
10. Procedure
10.1 Sporulation of Clostridium diffıcile in Liquid Medium:
10.1.1 Inoculate 1 L of Liver Broth with 0.25 mL to 0.5 mL of frozen stock culture of C. diffıcile. Other volumes may be used
as long as the ratio of Liver Broth to frozen stock culture is the same as previously stated.
10.1.2 Incubate Liver Broth under anaerobic conditions for 3661°C for 7-10 d, or until at least 95% spores are present. Some
strains of C. diffıcile may need more than 10 d to achieve the desired level of sporulation.
10.1.3 Use phase contrast microscopy, at 1000× magnification, to determine percent of spores present in the Liver Broth. Stir Liver
The sole source of supply for the Liver Broth (Cat. No. M928-500G) known to the committee at this time is HiMedia Laboratories, Marg, Mumbai, India. If you are
aware of alternative suppliers, please provide this information to ASTM International Headquarters. Composition of media available at www.himedialabs.com/TD/M928.pdf
accessed on February 23, 2015.
Centers for Disease Control and Prevention, and National Institutes of Health, Biosafety in Microbiological and Biomedical Laboratories, 5th ed., United States
Department of Health and Human Services, Washington, DC, December 2009.
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Broth prior to preparing slide for phase contrast microscopy. Count spores and vegetative cells in five fields of view. The broth
is ready to be harvested when the percent spores is ≥95% as determined using equation in 11.1. Checking the spore purity
beginning around d 7 is recommended.
10.1.4 Stir Liver Broth to resuspend any spores that have settled to the bottom. Filter the entire volume of Liver Broth through
sterile cheesecloth, and collect in sterile centrifuge tubes.
10.1.5 Centrifuge filtered broth at 7500 × g for 20 mins at 20 6 2°C. Dispose of the supernate and resuspend with filtered broth
until all broth has been centrifuged. Resuspend the final pellets in 20 to 30 mL of sterile deionized water.
10.1.6 Wash the final pellets four times by centrifuging at 7500 x g for 20 mins at 20 6 2°C and discard supernate. Resuspend
the pellet in sterile cold (2 - 8°C) deionized water.
10.1.7 Resuspend the final pellet in 10 to 30 mL of sterile cold deionized water to achieve the desired concentration of spores.
Store spore suspension at 2 - 8°C for up to 6 months.
10.1.8 Use phase contrast microscopy, at 1000× magnification, to determine percent of purity of the spore suspension. Mix the
spore suspension well. Count spores and vegetative cells in five fields of view. The spore suspension should have ≥95% spores
as determined using equation in 11.1.
10.1.9 Serially dilute the spore suspension in PBDW. Spread plate appropriate dilutions in duplicate on BHIY-HT. Incubate
inverted plates at 3661°C for 72 6 4 h under anaerobic conditions. Determine the log of the average CFU/mL of the spore
suspension.
10.1.10 The spore suspension may be diluted or concentrated as needed for efficacy testing.
10.2 Quantitative Acid Resistance Test—HCl Resistance (See Test Method E2839.)
10.2.1 Place 990 μL of 2.5 M HCl into three microcentrifuge tubes. Place 990 μL of sterile deionized water into one
microcentrifuge tube.
10.2.2 Using a micropipettor, preferably a positive displacement pipette, add 10 μL of the high titer (>8 log spores per mL) spore
suspension to each microcentrifuge tube prepared in 10.2.1. Vortex each tube. The microcentrifuge tubes should be held at ambient
temperature during the exposure time.
10.2.3 At the end of the 10 min exposure time for the HCl microcentrifuge tubes, transfer 0.1 mL from one tube to a
microcentrifuge tube with 900 μL of pH adjusted PBDW to neutralize. 0.1 mL from the control microcentrifuge tube is transferred
to a tube of 900 μL of pH adjusted PBDW after a 10 min exposure time.
10.2.4 Serially dilute each neutralized spore suspension in PBDW and spread plate 0.1 mL from appropriate dilutions in duplicate
onto BHIY-HT. Incubate inverted plates at 3661°C for 72 6 4 hours under anaerobic conditions.
10.2.5 A spore suspension is considered to be acid resistant if the Log reduction is between 0 and 2 at 10 mins of exposure as
compared to the control.
11. Calculation or Interpretation of Results
11.1 Percent Spores Present:
Average Spore Count
~100! (1)
Average Spore Count1Average Vegetative Cell Count
Precent Spores Present=
11.2 Log of the Average CFU/mL of the Spore Suspension:
~Mean CFUper plate!~Reciprocal of dilution!
~100! (2)
Volume Plated
Average CFU/mL=
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11.3 Log Reduction of HCl treatment:
Log Reduction = LC – LH
LC = Log of viable spores after control treatment
LH = Log of viable spores after HCl treatments
12. Precision and Bias
12.1 A precision and bias statement cannot be made for this test method at this time.
13. Keywords
13.1 C. diffıcile; Clostridium; liver broth; propagation; spores; spore crop; sporulation
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