US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Production of Clostridium difficile Spores for Use in Efficacy
Evaluation of Antimicrobial Agents
SOP Number: MB-28-01
Date Revised: 02-05-14

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SOP No. MB-28-01
Date Revised 02-05-14
Page 1 of 13
SOP Number
MB-28-01
Title
Production of Clostridium difficile Spores for Use in Efficacy
Evaluation of Antimicrobial Agents
Scope
Describes the test methodology, based on the ASTM Standard E2839-
11, for producing C. difficile spores.
Application
For use in the evaluation of antimicrobial products with C. difficile
claims.


Approval Date
SOP Developer:

Print Name:
SOP Reviewer

Print Name:
Quality Assurance Unit

Print Name:
Branch Chief

Print Name:


Date SOP issued:

Controlled copy number:

Date SOP withdrawn:

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SOP No. MB-28-01
Date Revised 02-05-14
Page 2 of 13
TABLE OF CONTENTS
Contents	Page Number
1.
DEFINITIONS
3
2.
HEALTH AND SAFETY
3
3.
PERSONNEL QUALIFICATIONS AND TRAINING
3
4.
INSTRUMENT CALIBRATION
3
5.
SAMPLE HANDLING AND STORAGE
3
6.
QUALITY CONTROL
3
7.
INTERFERENCES
3
8. NON-CONFORMING DATA
3
9.
DATA MANAGEMENT
3
10.
CAUTIONS
3
11.
SPECIAL APPARATUS AND MATERIALS
4
12.
PROCEDURE AND ANALYSIS
5
13.
DATA ANALYSIS/CALCULATIONS
9
14.
FORMS AND DATA SHEETS
10
15.
REFERENCES
10

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SOP No. MB-28-01
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1. Definitions
1.	Pre-reduced medium: Medium free of oxygen.
2.	Density gradient medium: HistoDenz™ is a non-ionic gradient medium
used to separate spores from vegetative cells and cell fragments on the
basis of density.
3.	Purified spores: Spore concentration reaches >95%.
4.	Toxigenic strain: Possesses either toxin A gene (tcdA+) or toxin B gene
(tcdB+) or both.
2. Health and
Safety
Follow procedures specified in SOP MB-01, Laboratory Biosafety. The Study
Director and/or lead analyst should consult the Material Safety Data Sheet for
specific hazards associated with chemicals.
3. Personnel
Qualifications
and Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
Refer to SOP EQ-02-06, EQ 05-05 for details on method and frequency of
calibration.
5. Sample
Handling and
Storage
Not applicable
6. Quality Control
1.	The organism is a Gram-positive, strictly anaerobic, spore-forming
bacterium that produces flat, gray, and irregular colonies on the surface of
CAB A medium within 48 h at 36±1°C.
2.	Other Quality Control information is documented in the method and on
the appropriate form(s) (see section 14).
7. Interferences
The test organism must be incubated under strict anaerobic conditions. If an
anaerobic environment is not maintained, the elevated oxygen will compromise the
viability of C. difficile.
8. Non-
conforming
Data
Management of non-conforming data will be specified in the standard test
method; procedures will be consistent with SOP ADM-07, Non-Conformance
Reports.
9. Data
Management
Data will be archived consistent with SOP ADM-03, Records and Archives.
10. Cautions
1.	Seal culture plates with Parafilm, or equivalent, to prevent dehydration during
the extended anaerobic incubation.
2.	Ensure media is pre-reduced prior to use.
3.	Inoculated plates and broth should be placed under anaerobic conditions within
2 hours.

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SOP No. MB-28-01
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11. Special
Apparatus and
Materials
1.	Biosafety cabinet—For maintaining an aseptic work environment.
2.	Sterile centrifuge tubes—Polypropylene, 15 niL and 50 niL graduated
plastic centrifuge tubes with conical bottoms.
3.	Centrifuge with swinging-bucket rotor—To allow sedimentation of spores
for washing and/or concentration.
4.	Micropipette—Calibrated.
5.	Positive displacement pipette—To inoculate steel carriers with spores.
6.	Timer—Any certified timer that can display time in seconds.
7.	Test tubes—Reusable or disposable 20 x 150 mm for cultures/subcultures.
8.	Inoculating loop—10 (.iL transfer loop.
9.	COY Anaerobic chamber—Supported by a gas mixture consisting of 10 %
Hydrogen, 5 % C02, and 85 % N2.
10.	Anaerobic incubator—Use the incubator at 36 ± 1°C inside the COY
anaerobic chamber to support the growth of the organism.
11 .Microscope with 1 OX eyepiece and 40X and 100X (oil) objectives with
phase contrast option.
12.	Vortex mixer.
13.	Serological pipettes—Sterile single-use pipettes of 10.0, 5.0, 1.0 niL
capacity.
14.	Cell Scraper—To gently scrape plates to remove spores for harvesting.
15.	Plate spreader—To spread inocula on agar to create a uniform lawn.
16.	Microcentrifuge tubes—Sterile 1,5-mL low-retention (siliconized)
microcentrifuge tubes.
17.	Cryovials—Sterile 2.0 niL cryovials.
18.	Parafilm™.
19.	Media and Reagents
a.	Reinforced clostridial medium (RCM)—For use in rehydrating
lyophilized/frozen vegetative culture of test organism. Prepare RCM
according to manufacturer's instructions, and pre-reduce in an
anaerobic environment for 24 ± 2 h prior to use.
b.	RCM plus 15 % glycerol (Cryoprotectant)—For use as maintenance
and cryopreservation medium for vegetative frozen stock (VFS)
cultures. Prepare RCM and add 15 % glycerol, autoclave for 20 min
at 121 °C, and pre-reduce in an anaerobic environment for 24 ± 2 h
prior to use.
c.	CDC anaerobic 5 % sheep blood agar (CABA) —Commercially

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SOP No. MB-28-01
Date Revised 02-05-14
Page 5 of 13

available pre-reduced, used for sporulation.
d.	Brain-heart infusion agar with yeast extract, horse blood and sodium
taurocholate (BHIY-HT) —Pre-reduced recovery media for
enumeration of viable spores.
e.	Phosphate-buffered saline (PBS)—Prepare 1 OX stock solution of
PBS by dissolving 492 g PBS powder in 5 L of deionized water.
Dilute 1:10 (1 part 1 OX solution plus 9 parts deionized water) to
obtain IX solution, distribute into bottles and autoclave for 20 min at
12 r°c.
f.	Phosphate-buffered saline (PBS) containing 0.1 % Tween 80
(ST80)—Washing reagent; add 2.0 mL of polysorbate 80 (Tween 80,
or equivalent) to 2.0 L of PBS (IX) solution in a 2 L volumetric flask
and bring solution to volume with PBS. Distribute into bottles and
autoclave for 20 min at 121°C.
g.	Water—Use sterile deionized water as diluent.
h.	Certified Hydrochloric acid—Use 2.5 M HQ for quantitative acid
resistance test.
i.	HistoDenz™—Prepare a 50 % (w/v) solution in deionized water.
This is a density gradient medium for spore purification. Pass the
solution through a sterile 0.45 \im filter to sterilize.
20. Test Organism:
a. Clostridium difficile (ATCC 43598), a toxigenic strain (tcdA-,
tcdB+), obtained from ATCC or another reputable vendor. The
strain produces Toxin B only (presence of tcdB gene by PGR).
12. Procedure and
Analysis

12.1 Preparation of
Frozen Stock
Cultures of
Test Organism
a.	Clostridium difficile received in lyophilized vegetative form.
To reinitiate a new stock culture, reconstitute contents of the
lyophilized culture with 0.5 mL of sterile pre-reduced RCM in the
CO Y anaerobic chamber as per the manufacturer's instructions.
After rehydration, aseptically transfer the vial contents to a tube
containing 4 ± 1 mL of pre-reduced RCM, and mix by gentle
vortexing.
b.	Clostridium difficile received as frozen vegetative culture.
To reinitiate a new stock culture, thaw frozen culture at room
temperature. Transfer the contents to a tube containing 4 ± 1 mL of
sterile pre-reduced RCM in the CO Y anaerobic chamber, and mix
by gentle vortexing.

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SOP No. MB-28-01
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c.	Spread plate 100 |iL of the reconstituted culture on five CAB A
plates. Also streak one CAB A plate for isolation to check for culture
purity. Invert plates and incubate anaerobically at 36=s=l°C for
48±4 h.
d.	Following incubation, add 2 niL of cryoprotectant to each CAB A
plate.
e.	Using a sterile cell scraper, gently scrape culture from the surface of
the plate, aspirate with a pipette and transfer to a 15-mL conical
tube.
f.	Repeat this process for the remaining plates. Pool the
cryoprotectant suspensions, mix thoroughly, and pipette 1 to 1.5 niL
aliquots into cryovials; cap tightly.
g.	Store the cryovials at -80±5°C. These tubes are the Frozen Stock
Culture (FSC).
h.	After a minimum of one week of freezing, thaw a FSC cryovial at
room temperature inside the COY chamber.
i.	Vortex suspension thoroughly, and dilute 1 niL in a 1:10 series out
tolO"6 in ST80. Spread-plate 100 |iL of diluted suspension on
BHIY-HT in duplicate. Invert plates and incubate anaerobically at
36± 1 °C for 48±4 h.
j. Record the number of CFlJ/plate to determine the CFlJ/mL. The
titer should be >8 log/mL to ensure that the FSC contains a
sufficiently high titer.
k. Prior to preparation of a spore preparation (See 12.2), streak three
CAB A plates with the FSC. Incubate two plates anaerobically, and
the third one aerobically at 36±1°C. Do not use the culture if
there is any growth on the plate incubated aerobically. Inspect
plates incubated anaerobically for purity and colony characteristics
typical of C. difficile.
12.2 Preparation of
a spore
suspension
from FSC
a.	Using one of the two plates from 12.1 k, inoculate 10 niL of p re-
reduced RCM with an isolated colony and mix well by vortexing.
Incubate anaerobically at 36± 1 °C for 24±2 h.
b.	After incubation, inoculate each of a minimum of ten CAB A plates
with 100 |iL of the RCM broth culture. Spread the inoculum
evenly using a disposable sterile spreader to create a lawn.
c.	Seal culture plates with Parafilm, or equivalent, to prevent
dehydration during incubation. Invert plates and incubate
anaerobically for 7 to 10 days at 36±1°C and >70% relative humidity.
d.	Open one or two plates after 24±2 h of incubation to inspect for
confluent growth. Reseal the plate and continue incubation. If growth

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SOP No. MB-28-01
Date Revised 02-05-14
Page 7 of 13
is not confluent, inspect the remaining plates and discard.
e.	Over the 7-10 day incubation time frame, periodically prepare wet-
mount samples of C. difficile from a sample plate and inspect under
phase-contrast microscopy. Spores appear bright and ovular, while
vegetative cells appear dark and rod-shaped.
f.	Note degree of conversion of vegetative cells to spores and estimate
the approximate ratio of spores to vegetative cells to determine the
optimal time for harvesting.
g.	When the percent of spores reaches > 90% (Attachment 1),
discontinue incubation and place the CAB A plates inside a BSC.
h.	Harvest growth from each plate by adding 5 niL of ST80 to each
plate, and gently scraping the surface of the plate with a cell scraper
or spreader to dislodge the spores. Do not break the surface of the
agar.
i.	Using a 10 niL sterile serological pipette, aspirate as much of the
microbial suspension as possible from each plate, and pool it in
sterile 50 niL plastic conical tubes.
j. Centrifuge tubes at 4500 g for 15 min.
k. Discard the supernatant and resuspend the pellet in 20 to 30 niL of
ST80. Cap the tubes tightly and disaggregate the pellet by
vortexing. This step is the first wash.
1. Repeat the washing step two more times. Note that resuspended
contents collected from two or more tubes can be combined in one
tube after pellets have been disaggregated. Mix by vortexing.
m. After the third wash, discard the supernatant and resuspend the pellet
in 4 niL of ST80. Mix well by vortexing to disaggregate the pellet.
Heat the spore suspension in a heat block for 10=1= 1 min at 65±2°C.
n. To ensure that the spore suspension has reached 65±2°C, place a
thermometer in an identical tube containing the same volume of
deionized water alongside the spore suspension and start the timer
once the temperature of the water has reached 65±2°C.
o. After heat treatment allow the suspension to cool to room
temperature.
p. Prepare a wet-mount of the well-vortexed, heat-treated spore
suspension and observe at least five fields using a phase-contrast
microscope. The percent of spores to vegetative cells should be
approximately 90%.
q. Perform serial 10-fold dilutions of the spore suspension out to 10"6
in ST80.

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SOP No. MB-28-01
Date Revised 02-05-14
Page 8 of 13

r. Spread-plate 0.1 niL of the appropriate dilutions on BH1Y-HT in
duplicate.
s. Once the inocula have dried, invert plates and incubate
anaerobically at 36=s=l°C for 48±4 h. Record the numbers of
CFU. The titer should be >108 viable spores/mL.
12.3 Spore
Purification
a.	Make a 50% (w/v) solution of HistoDenz in sterile deionized water
and pi pet 5 niL into each of four sterile 15 niL plastic conical tubes.
Layer 1 niL of spore suspension on top of the HistoDenz in each of
the four 15 niL tubes.
b.	Centrifuge tubes at 4,500 g for 10 min using a swinging bucket
rotor (see Note 1).
NOTE 1: Use of a swinging bucket rotor is essential for proper layer
removal and spore retention.
c.	Carefully remove, using a 1 niL pi pet, the top three layers - an upper
clear layer, a dense second layer, and a clear third layer -
(Attachment 3) and discard, leaving the pellet and the 3 to 4 mm
cloudy layer above the pellet undisturbed.
d.	Use a pipette to resuspend the pellet, vortex and transfer 1 niL
aliquots to siliconized micro- centrifuge tubes until the entire volume
has been transferred.
e.	Centrifuge the microcentrifuge tubes at 16,000xg for 5 min. Discard
the supernatant and resuspend the pellet in 1 to 1.5 niL of cold (2 to
5°C) ST-80.
f.	Cap the tubes and mix by vortexing to thoroughly disaggregate the
pellet.
g.	Centrifuge the microcentrifuge tubes at 16,000xg for 2 min. Discard
the supernatant and resuspend the pellet in 1 to 1.5 niL of cold (2 to
5°C) ST80. Cap the tubes and mix by vortexing to thoroughly
disaggregate the pellet. This step is the first wash.
h.	Repeat (h) procedure two additional times, for a total of three
washes.
i.	Discard the supernatant and resuspend the pellet in each
microcentrifuge tube with 0.5 mL of sterile ST80 and pool. This is
the final purified spore suspension (Attachment 2).
j. Determine spore purity using procedures stated in microscopic
evaluation of spore suspension. Calculate purity of the spore
suspension using the formula presented below in 13.1. The purity of
spores should be >95%.
k. Perform procedures specified in evaluation of titer and calculate the

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SOP No. MB-28-01
Date Revised 02-05-14
Page 9 of 13
titer of the purified spore suspension using the formula presented in
13.2. The titer of spores should be >8 logio/mL.
12.4 Quantitative
Hydrochloric
Acid (HC1)
Resistance Test
a.
b.
c.
d.
e.
f.
g-
Place 990 |iL of 2.5 M HQ into one 1.5 mL low-retention
(siliconized) microcentrifuge tube; for the control, place 990 |iL of
ST80 into one 1.5 mL low-retention (siliconized) microcentrifuge
tube.
Using a positive displacement pipette, transfer 10 |iL of purified
spore suspension (spore titer of >8 logm/mL) into each
microcentrifuge tube to result in a suspension containing >106
spores/mL. Vortex each tube.
Incubate the acid/spore suspensions and the control tubes for 10 min
±30 sec at room temperature.
At the end of each incubation period, transfer 0.1 mL from the
acid/spore tube and the control tube to tubes containing 900 |iL of
ST80 to dilute/neutralize the acid.
Serially dilute the neutralized suspensions out to 10"6 in ST80 and
spread-plate 0.1 mL aliquots from appropriate dilutions, in duplicate,
on BH1Y-HT. Invert plates and incubate for 48=1=4 h at 36± 1°C-
under anaerobic conditions.
The spores are considered acid-resistant if the log reduction is
between 0 to 2 following 10 min of exposure, as compared with the
control.
Determine the logio reduction following the HQ treatment using the
formula presented in 13.3.
12.5 Long Term
Spore Storage
a.
b.
Once the spore suspension fulfills all the required criteria (e.g., titer,
spore purity and acid resistance), assign a control number.
Make aliquots in cryovials, label appropriately and store at -20°C for
up to 1 year.
13. Data Analysis/
Calculations
1.	Determine spore suspension purity by the following calculation:
A
% Purity = 100 % x 			
17	A + B
Where A = mean spore count, and B = mean vegetative cell count.
2.	Determine the titer of the spores in suspension using the following
calculation:
A x B
Spores as CFU/mL = 100 % x —-—
Where A = mean colony count at dilution plated, B = reciprocal of

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SOP No. MB-28-01
Date Revised 02-05-14
Page 10 of 13

dilution used, and C = volume plated.
3. Determine the logio reduction following HC1 treatment:
Logio Reduction (LR) = LC-LH
Where
LC= Logio of viable spores after control treatment, and
LH= Logio of viable spores after HC1 treatment.
14. Forms and Data
Sheets
1.	Attachment 1: Monitoring percent sporulation of C. difficile.
2.	Attachment 2: Purified C. difficile spores, using a density gradient medium
(HistoDenz), depicting >99% purity.
3.	Attachment 3: Separation of vegetative cells (A) and cell fragments (B)
from C. difficile spores upon purification with HistoDenz.
4.	Test Sheets: Test sheets are stored separately from the SOP under the
following file names:
C. difficile Spore Titer Form MB-28-01 Fl.docx
HC1 Resistance Test Form MB-28-01 F2.docx
HC1 Resistance Test Dilution and Results Form MB-28-01_F3.docx
15. References
1.	ASTM E2839-1 1, Standard Test Method for Production of Clostridium
difficile Spores for Use in Efficacy Evaluation of Antimicrobial Agents.
ASTM International, West Conshohocken, PA, 201 1.
2.	EPA Guidance for the Efficacy Evaluation of Products with Sporicidal
Claims against Clostridium difficile, http://vvvvvv.epa.gov/oppadOO 1 / cdif-
guidance.html, 2009.
3.	Hasan, J. A., Japal, K. M., Christensen, E. R., & Samalot-Freire, L. C.,
"Development of methodology to generate Clostridium difficile spores for
use in the efficacy evaluation of disinfectants, a pre-collaborative
investigation," J. AO AC bit. Vol 94, 2011, pp. 259-272.
4.	Standard Methods for the Examination of Water and Wastewater,
American Public Health Association, Washington, D C., 2012.
5.	Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th
Ed., Centers for Disease Control and Prevention, and National Institute
of Health, Washington D C., 2009.

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Attachment 1
SOP No. MB-28-01
Date Revised 02-05-14
Page 11 of 13
Monitoring percent sporulation of C. difficile (ATCC 43598) during incubation at 36 ± 1°C
under phase contrast microscopy (magnification 1000X)

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SOP No. MB-28-01
Date Revised 02-05-14
Page 12 of 13
Attachment 2
Purified C. difficile spores (ATCC 43598), using HistoDenz, depicting >99% purity
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Attachment 3
SOP No. MB-28-01
Date Revised 02-05-14
Page 13 of 13
Separation of vegetative cells (A) and cell fragments (B) from C. difficile spores (ATCC 43598)
upon purification with HistoDenz.

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