US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Mic ro bio lo gy La bo ra to ry
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Production of Spores of Clostridium difficile for Use
Efficacy Evaluation of Antimicrobial Agents
SOP Number: MB-28-03
Date Revised: 05-05-14
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SOP No. MB-28-03
Date Revised 05-05-14
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SOP Number
MB-28-03
Title
Production of Spores of Clostridium difficile for Use in the Efficacy
Evaluation of Antimicrobial Agents
Scope
Describes the test methodology, based on the ASTM Standard
E2839-11, for producing standardized spore suspensions of C.
difficile.
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Date SOP withdrawn:
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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- C ONF ORMIN G DATA
3
9.
DATA MANAGEMENT
3
10.
CAUTIONS
3
11.
SPECIAL APPARATUS AND MATERIALS
3
12.
PROCEDURE AND ANALYSIS
5
13.
DATA ANALYSIS/CALCULATIONS
10
14.
FORMS AND DATA SHEETS
10
15.
REFERENCES
10
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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 suspension exhibiting >95% spores following
processing with Histodenz.
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 SOPEQ-02-06 and EQ 05-05 for details on method and frequency of
calibration.
5. Sample
Handling and
Storage
Not applicable
6. Quality Control
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
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 when using an anaerobic chamber.
2. Ensure media (e.g. RCM) is pre-reduced for at least 24±2 h prior to use.
3. Inoculated plates should be placed under anaerobic conditions within 1 hour.
11. Special
Apparatus and
Materials
1. Biosafety cabinet—For maintaining an aseptic work environment.
2. Sterile centrifuge tubes—Polypropylene, 15 mL and 50 mL graduated
plastic centrifuge tubes with conical bottoms.
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3. Centrifuge with swinging-bucket rotor—To allow sedimentation of spores
for washing and/or concentration.
4. Micropipette—Calibrated.
5. Positive displacement pipette—To dispense 10|iL of 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% CO:, and 85% N:.
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, and 1.0 mL
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- retentio n (siliconized)
microcentri fuge tubes.
17. Cryovials—Sterile 2.0 mL cryovials.
18. Parafilm™—to seal plates.
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) plates—Commercially
available and pre-reduced, used for sponiation.
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d. Brain-heart infusion agar with yeast extract, horse blood and sodium
taurocholate (BHIY-HT)—Commercially available and pre-reduced,
used as recovery medium for enumeration of viable spores.
e. Phosphate-buffered saline (PBS)—Prepare 10X 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 niL of polysorbate 80 (Tween 80,
or equivalent) to 2.0 L of PBS (1X) 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 MHC1 for quantitati ve 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 PC R). The
organism is a Gram-positive, strictly anaerobic, spore-forming
bacterium that produces flat, gray, and irregular colonies when
grown anaerobically on the surface of CAB A medium within 48 h at
36±1°C.
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 niL 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 niL 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 niL of
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sterile pre-reduced RCM in the C0 Y anaerobic chamber, and mix
by gentle vortexing.
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±1°C for 48±4
h.
d. Following incubation, add 2 mL 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 suspensions,
mix thoroughly, and pipette 0.5 to 1 mL aliquots into cryovials; cap
tightly.
g. Store the cryovials at -80±5°C for 18 months. 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 mL in a 1:10 series out
to 1 0~(' in ST80. Spread-plate 100 |iL of diluted suspension on
BH1Y-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 C F U /mL. The
titer should be >1.0 x 108CFU/mL.
12.2 Preparation of
a spore
suspension
from FSC
a. Streak three CAB A plates with the FSC. Incubate two plates
anaerobically, and the third one aerobically at 36±1°C for 48±4 h.
Inspect plates incubated anaerobically for purity and colony
characteristics typical of C. difficile. Do not use the culture if there
is contamination on any plate.
b. Using one of the two plates (incubated anaerobically) from 12.2a,
inoculate 10 mL of p re-reduced RCM with a n isolated colony and
mix well by vortexing. Incubate anaerobically at 36±1°C for 24±2 h.
c. After incubation, inoculate ten CAB A plates each with 100 |.iL of the
RCM broth culture. Spread the inoculum evenly using a disposable
sterile spreader to create a lawn.
Note: See step 12.2m for inoculation of additional number of plates.
d. Seal inoculated plates with Parafilm, or equivalent, to prevent
dehydration during incubation in an anaerobic chamber. Invert plates
and incubate anaerobically for 10 days at36±l°C and approximately
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70% relative humidity.
e. Inspect one plate after 24±2 h of incubation to verify the presence
of a lawn (confluent growth on the plate). If growth is confluent
reseal the plate and continue incubation. If growth is not confluent,
discard the plates and inoculate a new set of ten plates.
Note: A plate may be inspected at day 7 to determine degree of
sponiation.
f On day 10, discontinue incubation and place the CAB A plates inside
a BSC. 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.
g. Degree of conversion of vegetative cells to spores should be
approximately 90%. See Attachment 1 for example of conversion
process over the incubation period.
h. Harvest growth from each plate by adding 5 niL of ST80 to each
plate, and gently scrape 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 lOmL sterile serological pipette, aspirate as much of the
microbial suspension as possible from each plate, and pool it in a
sterile 50 niL plastic conical tube. Divide evenly into two 50 niL
conical tubes. Mix well by through vortexing.
j. Centrifuge tubes at 4500 g for 15 min.
k. Discard the supernatant and resuspend the pellets with 30 niL of
ST-80. Cap the tubes tightly and disaggregate the pellet by
vortexing. This step is the first wash.
1. Repeat the washing step. After the second wash, disaggregate the
pellets from each tube, and combine into one tube for the third wash
with 30 niL ST-80. Mix well by vortexing. Centrifuge tubes at
4500 g for 15 min.
m. After the third wash, discard the supernatant and resuspend the pellet
in 4 niL of ST-80. Mix well by vortexing to disaggregate the pellet.
Note: For every 10 C AB A plates inoculated, resuspend the
resulting pellet in 4 niL of ST-80. Follow the ratio (4 niL per
10 plates) if additional plates are inoculated.
n. Heat the spore suspension in a heat block for 10± 1 min at 65±2°C.
To ensure that the spore suspension has reached 65 ± 2°C, place a
thermometer in an identical tube containing the same volume of ST-
80 alongside the spore suspension and start the timer once the
temperature of the water has reached 65±2°C.
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o. Following the 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% (or higher).
q. Perform serial 10-fold dilutions of the spore suspension (e.g., out to
10"7) in ST80.
r. Spread-plate 0.1 niL of the appropriate dilutions on BH1Y-HT in
duplicate.
s. Store remaining spore suspension at 4°C for up to 5 days prior to
purification.
t. Once the inocula have dried, invert plates and incubate
anaerobically at 36±1°C for 48±4 h. Record the numbers of CFU.
The titer must be > 10X viable spores/mL.
12.3 Spore
Purification
a. Make a 50% (w/v) solution of HistoDenz in sterile deionized water
and pipet 5 niL into each of four sterile 15 niL plastic conical tubes.
Note: If HistoDenz and spore preparation are refrigerated, bring to
room temperature before use.
b. Layer 1 niL of spore suspension on top of each of the four tubes of
HistoDenz.
c. Centrifuge tubes at 4,500 g for 10 min using a swinging bucket
rotor.
d. Following centrifugation, three distinct layers should be present in the
Histodenz. Using a 1 niL pipet, carefully remove the top three layers:
1) an upper clear layer, 2) a dense (opaque) second layer, and 3) a
clear third layer. Discard the top three layers, leaving the pellet and
the 3 to 4 mm cloudy layer above the pellet undisturbed.
e. Using a repetitive pipetting action, resuspend and mix the pellet
(without touching the pellet) with a 1 niL micro pipette. Bring the
volume up to approximately 1 niL with cold ST-80. Mix throughly by
vortex to break up the pellet (ensure absence of visual clumps or
fragments of the pellet) and transfer the contents of each tube to a
siliconized micro-centrifuge tube.
f Centrifuge the microcentrifuge tubes (four total tubes) at 16,000 g for
5 min. Discard the supernatant and resuspend the pellet in 1 mL of
cold (2-5°C) ST-80.
g. Cap the tubes and mix throughly by vortex to break up the pellet
(ensure absence of visual clumps or fragments of the pellet).
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h. Centrifuge tlie microcentrifuge tubes at 16,000 g for 2 min. Discard
the supernatant and resuspend the pellet in 1 mL of cold ST80. Cap
the tubes and mix throughly by vortex to break up the pellet (ensure
absence of visual clumps or fragments of the pellet). This step is the
first wash.
i. Repeat washing procedure two additional times, for a total of three
washes.
j. After the third wash, discard the supernatant and resuspend the pellet
in each microcentrifuge tube with 0.5 mL of sterile ST80 and pool
(e.g., pool into one 15 mL sterile conical tube). This is the final
purified spore suspension.
k. Determine spore purity using procedures stated in microscopic
evaluation of spore suspension (see 12.2 p). Calculate purity of the
spore suspension using the formula presented below in 13.1. The
purity of spores should be >95%. See Attachment 2 for example of a
purified spore suspension.
1. Determine titer as in 12.2 (q-s); use the formula presented in 13.2 for
calculations. Repeat titer assessment (i.e., two independent titer
evaluations). The average titer of spores from two determinations
should be approximately 109 viable spores/mL.
m. Adjust the spore concentration to 2.0 x 108to 8.0 x 108 viable
spores/mL using ST80 as the diluent.
Note: Store purified spore suspension at 4°C for up to 5 days during
titer adjustment and HCL resistance test (see 12.4).
12.4 Quantitative
Hydrochloric
Acid (HC1)
Resistance Test
a. Place 990 |iL of 2.5 M HQ into one siliconized microcentri fuge
tube (treatment tube) and 990 |iL of ST80 into one siliconized
microcentrifuge tube for the control.
b. In a timed sequence and using a positive displacement pipette,
transfer 10 |iL of the adjusted purified spore suspension into the
microcentrifuge treatment and control tubes and briefly vortex each
tube.
c. Allow the tubes to remain at room temperature for 10 min ±30 sec.
d. Following the 10 min exposure time, transfer 0.1 mL from each tube
(in sequential order) to tubes containing 900 |iL of ST80 (i.e., the
neutralizer).
e. Serially dilute the suspensions (e.g., out to 10~7)in ST80 and spread-
plate 0.1 mL aliquots from appropriate dilutions, in duplicate, on
BH1Y-HT. Invert plates and incubate for 48±4 h at 36±1°C under
anaerobic conditions.
f The spores are considered acid-resistant if the log reduction (control
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minus treated) is between 0 and 2.
g. Determine the log reduction following the HQ treatment using the
formula presented in 13.3.
12.5 Long Term
Spore Storage
a. The spore suspension is considered acceptable for use if all required
criteria have been met: 1) spore titer of 2.0 x 108to 8.0 x 10X viable
spores/mL), 2) spore purity of > 95% and 3) acid resistance (log
reduction between 0 to 2 following 10 min of exposure to 2.5 M
HC1.
b. Assign a preparation number to the suspension.
c. 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 formula:
A
% Purity = 100 % x
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
formul a:
Ax B
Spores as CFU/mL = —-—
Where A = mean colony count at dilution plated, B = reciprocal of
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. Test Sheets: Test sheets are stored separately from the SOP under the
following file names:
C. difficile Spore Titer Form MB-28-03 Fl.docx
HC1 Resistance Test Form MB-28-03 F2.docx
HC1 Resistance Test Dilution and Results Form MB-28-03_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.
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ASTM International, West Conshohocken, PA, 201 1.
2. EPA Guidance for the Efficacy Evaluation of Products with Sporicidal
Claims against Clostridium difficile, http://www.epa.gov/oppad001 / cdif-
guidance.html, 2009.
3. Hasan, J. A., Japal, K.M., Christensen, E. R. and Sanialot-Freire, L. C.,
"Development of methodology to generate Clostridium difficile spores for
use in the efficacy evaluation of disinfectants, a pre-collaborative
investigation," J. AOACInt, Vol 94, 201 1, 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
Percent sporulation of C. difficile (ATCC 43598) during incubation at 36±1°C under phase
contrast microscopy (magnification 1000X)
Spores at
day 8
Final harvested
spore prep
(approx. 90%
spores) at day 10
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Attachment 2
Purified C. difficile spores (ATCC 43598), using HistoDenz, depicting >99% purity
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