Standard Operating Procedure for Quantitative Three Step Method for Measuring the Efficacy of Liquid Sporicides against Spores of Bacillus subtilis on a Hard Nonporous Surface and Porous Surfaces SOP Number: MB-21-02 Date Revised: 01-06-14


US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Quantitative Three Step Method for Measuring the
Efficacy of Liquid Sporicides against Spores of
Bacillus subtilis on a Hard Nonporous Surface and Porous Surfaces
SOP Number: MB-21-02
Date Revised: 01-06-14

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SOP No. MB-21-02
Date Revised 01-06-14
Page 1 of 16
SOP Number
MB-21-02
Title
Quantitative Three Step Method for Measuring the Efficacy of Liquid
Sporicides against Spores of Bacillus subtilis on Hard Non-porous and
Porous Surfaces
Scope
This quantitative method is used to evaluate the sporicidal efficacy of
liquid disinfectants against spores of Bacillus subtilis (ATCC 19659)
on hard, non-porous and porous surfaces. This SOP is based on the
AO AC Method 2008.05 (see 15.1). Additional details for performing
the method with spores of Clostridium difficile are provided in
Attachment 1.
Application
Data from this method are used to generate the log reduction (LR)
values of viable spores of B. subtilis as the quantitative measure of
efficacy for liquid disinfectants on hard, non-porous and porous
surfaces.


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Date SOP withdrawn: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-21-02
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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-CONFORMING DATA
3
9.
DATA MANAGEMENT
3
10.
CAUTIONS
3
11.
SPECIAL APPARATUS AND MATERIALS
4
12.
PROCEDURE AND ANALYSIS
6
13.
DATA ANALYSIS/CALCULATIONS
12
14.
FORMS AND DATA SHEETS
13
15.
REFERENCES
14

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1. Definitions
Additional abbreviations/definitions are provided in the text.
1. ATCC = American Type Culture Collection
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 products.
3. Personnel
Qualifications
and Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
Refer to SOP QC-19 (Pipettes), EQ-02 (Thermometers and Hygrometers), and
EQ-05 (Timers) for details on method and frequency of calibration.
5. Sample
Handling and
Storage
Refer to SOP MB-22, Disinfectant Sample Preparation, and SOP COC-01,
Chain of Custody Procedures.
6. Quality Control
For quality control purposes, the required information is documented on the
appropriate form(s) (see section 14).
7. Interferences
1.	During exposure of the carrier to the disinfectant, do not allow the pipette
tip delivering the disinfectant to touch the inoculated carrier.
2.	To avoid cross-contamination, it is recommended that analyst analyze
chemical treatments from the most highly effective to the least effective,
followed by the untreated controls.
3.	Touching the interior sides of the microcentrifuge tube should be avoided
while the carriers are being lowered into the microcentrifuge tube and the
forceps are being removed.
4.	Prior to testing, use the Neutralization Confirmation procedure (see
section 12.8) to determine suitability of the neutralizer for the test
substance.
8. Non-
conforming
Data
1.	Management of non-conforming data will be specified in the study
protocol; procedures will be consistent with SOP ADM-07, Non-
Conformance Reports.
2.	Due to the occurrence of statistical variability in the log reduction (LR)
data, it is recommended that the analyst target carrier counts of 7-7.5 logs
to ensure confidence in a 6 LR.
9. Data
Management
Data will be archived consistent with SOP ADM-03, Records and Archives.
10. Cautions
To ensure the stability of a diluted sporicidal agent, prepare the dilutions
within three hours of the sporicidal treatment step unless specified otherwise.

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11. Special
1. Culture media.
Apparatus and
Materials
a.
Nutrient broth (NB) (dehydrated). For use in rehydrating test
organism and preparing nutrient agar.

b.
Nutrient agar (NA). For stock cultures slants. Add 1.5% (w/v)
Bacto-agar to unsterilized NB. Boil mixture until agar is dissolved.
If necessary, adjust pH to 7.2 ± 0.2. Dispense 5 mL portions into 16
x 100 mm screw-cap tubes. Larger tubes may be used as well.
Autoclave for 20 min at 121°C. Remove from autoclave and slant
tubes to form agar slopes. Dehydrated NA may be substituted;
suspend 23 g NA/L water and dissolve by boiling. If necessary,
adjust pH to 6.8 ± 0.2. Autoclave for 15 min at 121°C.

c.
Nutrient agar with 5 /ig/mL manganese sulfate monohydrate
(amendedNA). For spore production. Suspend 11.5 g NA in 495
mL water and add 5 mL 500 ppm MnS04'H20. Dissolve by boiling.
If necessary, adjust pH to 6.8 ± 0.2. Autoclave for 15 min at 121°C.
Pour agar into plates.

d.
Trypticase soy agar (TSA). Poured in plates for microbe isolation
and spread plating.

e.
Luria-Bertani (LB) broth. Dehydrated; suspend 25 g in 1 L water,
mix well, if necessary adjust pH to 7.0 ± 0.2, dispense in bottles, and
autoclave for 15 min at 121 °C; use as neutralizer.

f.
Modified Luria-Bertani broth. Neutralizer in HC1 resistance test.
Add 20 mL 1 MNaOH to 1 L LB broth, mix well, dispense in
bottles, and autoclave for 15 min at 121 °C.

g-
Luria-Bertani broth with 0.1% (w/v) sodium thiosulfate. Neutralizer
for sodium hypochlorite treatments. Add 1.0 g sodium thiosulfate to
1 L LB broth, mix well, dispense in bottles, and autoclave for 15 min
at 121°C.

2. Reagents.

a.
Manganese sulfate monohydrate (500ppm). Add 0.25 g manganese
sulfate to 500 mL water. Filter sterilize for use.

b.
Sodium thiosulfate. Neutralizing agent for sodium hypochlorite.

c.
Water. Either de-ionized distilled water or water with equivalent
quality for making reagent solutions and culture media.

d.
2.5 Mhydrochloric acid, e.g., certified HC1.

e.
Ethyl alcohol. 40 and 95%.

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SOP No. MB-21-02
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3. Test organism.

a. Bacillus subtilis (ATCC No. 19659) obtained directly from a

commercial supplier.

4. Apparatus.

a. Hard non-porous carriers.

i. Glass coupon. 5x5x1 mm (Erie Scientific Co.); custom

order Part No. EPA-1101 (minimum order of 1000 pieces),

single use.

ii. Stainless steel coupon. Stainless steel (EJ enterprise, Glen

Burnie, MD; type 316) cut into 5x5x1 single use.

b. Hard porous carriers.

i. Ceramic tile. Ceramic tile (United States Ceramic Tile Co.,

North Canton, OH; No. 078-66) cut into 5x5x1 use

unglazed side for inoculation, single use.

ii. Untreated pine wood. Non-treated select pine, No. 1 (Home

Depot) cut into 5x5x1 single use.

c. Microcentrifuge tubes. Sterile, 1.5 mL.

d. Centrifuge tubes. Sterile, polypropylene, 15 mL conical tubes with

conical bottoms.

e. Dissecting forceps.

f. Micropipette. Calibrated.

g. Positive displacement pipette.

h. Desiccator.

i. Water bath/chiller unit. Constant temperature, capable of

maintaining 20±1°C temperature or specified temperature.

j. Orbital shaker.

k. Microcentrifuge.

1. Microcentrifuge tube lid openers.

m. Sonicator (ultrasonic cleaner).

n. Floating microcentrifuge tube holder. To hold the Fraction A tubes

in a fixed upright position during sonication.

o. Hematology rotator, or a suitable mixer/shaker to provide gentle

agitation during incubation.

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SOP No. MB-21-02
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P-
Vortex mixer.


q-
Vortex adapters.


r.
Certified timer. Any certified timer that can display time in seconds.


s.
Test tubes. 25x 150 mm.


t.
Microscope. With 10X eyepiece and 40X and 100X (oil) objectives
with phase contrast option.
12. Procedure and
Analysis
1.
For non-porous and porous carriers, use the Three Step Method (TSM)
described in section 12.6.

2.
For non-porous carriers (glass and stainless steel), fractions B and C may
be consolidated into fraction BC using the modified Three Step Method
(mTSM) procedure outlined in section 12.7.

3.
Use Three Step Method Processing Sheet (see section 14) to track testing
activities.

4.
Conduct the testing (e.g., addition of soil load) as specified by the study
sponsor.
12.1 Culture
Initiation

a.
b.
c.
Initiate B. subtilis culture (e.g., use NB to rehydrate a lyophilized
culture, and incubate broth culture for 24±2 h at 36±1°C prior to
streak inoculation).
Streak inoculate a set (e.g., 6) of NA slants and incubate 24±2 h at
36±1°C. Perform purity and identification confirmation testing for
QC (e.g., colony morphology on TSA, Gram stain, or use of other
identification systems).
Following incubation, store at 2-5°C. Maintain stock culture on NA
slants by monthly (30±2 days) transfers.
12.2 Production of
B. subtilis
spore
suspension

a.
b.
c.
d.
Using growth from a stock culture tube, inoculate 10 mL tubes (e.g.,
2 tubes, depending on the amount of spore preparation desired) of
NB and incubate tubes 24±2 h on an orbital shaker at approximately
150 rpm at 36±1°C.
Use this culture to inoculate amended NA plates. Inoculate each
plate with 500 [xL broth culture and spread inoculum with sterile bent
glass rod or suitable spreading device.
Wrap each plate with parafilm or place in plastic bags. Incubate
plates inverted for 12-14 days at 36±1°C.
Following incubation, harvest the spores by adding 10 mL cold (2-
5°C) sterile water to each plate. Using a spreader (e.g., bent glass
rod), remove growth from plates and pipet suspensions into 15 mL

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sterile conical tubes (e.g., 10 plates = 14 tubes, -10 mL each).
e.	Centrifuge tubes at 5,000 rpm (4,500 x g) for approximately 10 min
at room temperature.
f.	Remove and discard supernatant. Resuspend pellet in each tube with
10 mL cold sterile water and centrifuge at 5,000 rpm (4,500 x g) for
10±1 min.
g.	Remove and discard supernatant. Repeat twice. Resuspend the
pellet in each tube with 10 mL sterile water. Store the spore
suspension at 2-5°C.
h.	Examine spore suspension with a phase contrast microscope or by
staining to assess quality of the spores. Examine a minimum of 5
fields and determine ratio of spores to vegetative cells (or sporangia).
Spores versus vegetative cells should be at least 95%. Record results
on the spore suspension preparation sheet.
i.	Spore suspension harvested from multiple plates can be combined
and re-aliquoted into tubes for uniformity.
j. Prior to inoculation of carriers, determine spore titer of the
concentrated spore suspension by plating serial dilutions (e.g., 10"6-
10"8) onto TSA. Incubate plates for 24±2 h at 36±1°C and determine
titer.
Note: When harvested and processed, 10 plates of amended NA
should provide 80-100 mL concentrated spore suspension. Diluting
the suspension prior to carrier inoculation will be necessary; a spore
titer of approximately 1.0 x 109 CFU/mL in the suspension should be
adequate to achieve the target carrier count.
12.3 Carrier
preparation
a.	Visually screen glass coupons (carriers) for scratches, chips, or
cracks. Discard those which are damaged or defective. Ensure pine
wood carriers are free of sawdust after cutting; no rinsing is required.
b.	Rinse glass, stainless steel, and ceramic wall tile carriers once with
water, 3 times with 95% ethyl alcohol, and finally 3 times with water.
Allow carriers to dry. Place in glass tubes (25 x 150 mm), 40
carriers per tube.
c.	Steam sterilize all carrier types 45 min at 121°C with a 30 min dry
cycle or sterilize for 2 h in hot air oven at 180°C. Cool. Transfer
carriers to sterile plastic Petri dishes for inoculation (approximately
40 carriers per dish).
12.4 Carrier
a. Transfer 10 [j,L spore suspension with a micropipette using aerosol
barrier tips or positive displacement pipette onto a 5 x 5 x l mm

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inoculation
sterile carrier coupon. Apply to one central spot on each carrier.
During carrier inoculation, mix inoculum frequently in Vortex mixer
to ensure uniform distribution of spores.
b.	Allow carriers to dry for minimum of 1 h in open Petri dish in a
biosafety cabinet, then for a minimum of 12±2 h in a desiccator.
Store inoculated carriers under desiccation for up to 30 days.
c.	Inoculated carriers must be discarded after use.
d.	Verify carrier counts (per the method for control carriers) prior to
testing; mean counts must be 5.0 x 106 to 5.0 x 107 spores/carrier.
12.5 Test chemical
(e.g., sporicide,
disinfectant)
sample
preparation
a.	Aseptically prepare test chemical samples as directed (refer to SOP
MB-22, Disinfectant Preparation).
b.	Place approximately 1.5 mL of each test chemical or control (sterile
water) in microcentrifuge tubes. Allow to equilibrate to appropriate
temperature, for approximately 15-30 min.
12.6 Test procedure
for non-porous
and porous
carriers
a.	A minimum of 3 carriers per test chemical and 3 carriers for the
water control (control carriers) are required per product test. Use one
pair of sterile forceps per fraction for each test chemical. Fractions
may be refrigerated briefly to allow for processing of other fractions.
It is recommended that 2 analysts perform this method so that
dilution and plating of the multiple fractions may be conducted as
soon as possible.
b.	Using sterile forceps, carefully transfer one inoculated carrier into
each microcentrifuge tube labeled Fraction A. Avoid touching
inoculated area of carrier and sides of microcentrifuge tube. Discard
carrier and tube if carrier touches sides of tube.
c.	Place Fraction A tubes containing carriers and tubes containing test
chemical(s) and sterile water (control) into chiller water bath at
20±1°C, or use a labtop cooler to maintain temperature of the tubes.
Equilibrate approximately 10 min.
d.	Add 400 |iL test chemical (test carriers) or 400 [xL sterile water
(control carriers) at 15 or 30±5 s intervals to appropriate
microcentrifuge tube (in triplicate). Allow contact of the carriers to
the test chemical or water in Fraction A tubes for the appropriate
exposure period.
e.	Following the exposure period, add 600 [xL of appropriate ice-cold
neutralizer (e.g., LB broth) to each test chemical Fraction A tube.
Add 600 |iL LB broth as neutralizer for water control Fraction A
tubes. Slightly agitate tubes to thoroughly mix liquid components.

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f.	Transfer each carrier using one pair of sterile forceps per carrier set
(i.e., 3 carriers) from Fraction A tube to corresponding Fraction B
tube. Fraction B tubes contain 400 [xL ice-cold (0-5°C) sterile water.
To prevent contamination, the use of a microcentrifuge tube cap
opener is recommended.
g.	Place Fraction A tubes in microcentrifuge, centrifuge for 6 min ±30
s at 13,000 rpm (15,500 x g).
h.	Remove 900 [xL from each tube without disturbing pellet. Discard
supernatant. Carefully add 900 [xL ice-cold LB broth to each tube.
Repeat 2 additional times.
i.	After third centrifugation, remove 900 [xL from each tube. Carefully
add 100 [xL ice-cold LB broth to each Fraction A tube and resuspend
pellet by mixing in a Vortex mixer 5 min ± 30 s (use the Vortex
adapter) at midrange speed.
j. Add 800 [xL ice-cold LB broth to each Fraction A tube. Proceed to
dilution and plating if another analyst is available, or store Fraction A
tubes in refrigerator.
Note: Fluid remaining in the Fraction A tubes contains spores
dislodged from carrier by exposure to the test chemical or water
control. Consistent orientation of the microcentrifuge tubes in the
microcentrifuge is important in locating the pellet. The pellet may
range in size and be difficult to visualize depending on the treatment.
Fractions B and C tubes can be evaluated while Fraction A tubes are
being centrifuged.
k. Sonicate Fraction B tubes 5 min ± 30 s using a floating
microcentrifuge tube holder placed inside an ultrasonic cleaner.
1. After sonication is complete, add 600 [xL ice-cold LB broth to
Fraction B tubes. Mix on a Vortex mixer approximately 1 min.
Transfer each carrier using one pair of sterile forceps per carrier set
from Fraction B tube to corresponding Fraction C tube (Fraction C
tubes contain 400 [xL ice-cold LB broth). Proceed to dilution and
plating if another analyst is available, or store Fraction B tubes at 2-
5°C; storage should be limited to 2 h.
Note: Fluid remaining in the Fraction B tubes contains spores
dislodged from the carrier by sonication.
m. Place Fraction C tubes in a hematology rotator inside incubator for
30±2 min at 36±1°C.
n. Remove Fraction C tubes after 30±2 min rotation/incubation from

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SOP No. MB-21-02
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incubator. Add 600 [xL ice-cold LB broth to each tube. The carriers
remain in the Fraction C tubes. Proceed to dilution and plating if
another analyst is available, or store Fraction C tubes at 2-5°C;
storage should be limited to 2 h.
Note: Fluid remaining in Fraction C tubes contains spores dislodged
from the carrier by gentle agitation for 30 min.
o. Mix on a Vortex mixer each microcentrifuge tube thoroughly prior to
making dilutions.
p. For each fraction and control tube, remove 100 [xL and serially dilute
10-fold in 900 [xL ice-cold LB broth. At a minimum for treated
carriers, prepare at least one 10-fold dilution for each fraction.
q. For each carrier, direct plate 100 [xL of the sufficient dilutions onto
TSA to ensure obtaining counts within the target range of 30-300
CFU/plate.
i.	For non-porous control carriers: the 10"3 and 10"4 dilutions for
Fractions A and B, and 10"2 and 10"3 for Fraction C should
result in countable plates.
ii.	For porous control carriers: the 10"2 and 10"3 dilutions for
Fraction A, 10"3 and 10"4 dilutions for Fraction B, and 10"2
and 10"3 for Fraction C should result in countable plates.
iii.	For non-porous control carriers using the mTSM: the 10"3 and
10"4 dilutions for both Fractions A and BC should result in
countable plates.
r. Incubate plates a minimum of 24±2 h at 36±1 °C. Record control
counts at 24±2 h. Record treated carrier counts at 24±2 and at 48±2
h.
s. Confirm the identity of a minimum of one representative colony
taken from at least one plate per treatment level (if available) using
Gram staining, general growth media (e.g., TSA), or other
confirmative procedure (e.g., VITEK 2 Compact). B. subtilis is a
large Gram-positive rod. On general growth media, B. subtilis
colonies are opaque, rough, round, low convex colonies with
irregular margins.
t. After plating, dilution tubes may be stored at 2-5°C until the results
are recorded; the tubes may be used for additional plating if initial
plate counts are beyond the recommended target range.
12.7 Alternative test
procedure
a. Process Fraction A as in 12.6, b-j.

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SOP No. MB-21-02
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(modified
Three Step
Method) for
non-porous
carriers
b.	Transfer each carrier using one pair of sterile forceps per carrier set
(i.e., three carriers) from Fraction A tube to corresponding Fraction
BC tube. Fraction BC tubes contain 400 [xL ice-cold (0-5°C) sterile
water. Fraction BC tubes can be processed while Fraction A tubes
are centrifuged.
c.	Sonicate Fraction BC tubes for 5 min ± 30 s using a floating
microcentrifuge tube holder placed inside an ultrasonic cleaner.
d.	After sonication is complete, mix on a Vortex mixer approximately 1
min.
e.	Place Fraction BC tubes in a hematology rotator inside an incubator
for 30 ± 2 min at 36 ± 1°C.
f.	Remove Fraction BC tubes from the incubator after 30 ± 2 min
rotation/ incubation. Add 600 [xL ice-cold LB broth to each tube.
The carriers remain in the Fraction BC tubes. Proceed to dilution and
plating if another analyst is available, or store Fraction BC tubes at
2-5°C; storage should be limited to 2 h.
Note: Fluid remaining in Fraction BC tubes contains spores
dislodged from the carrier by sonication, vortexing, and gentle
agitation for 30 min.
g.	Mix each microcentrifuge tube thoroughly on a Vortex mixer prior to
making dilutions.
h.	Prepare serial dilutions and plate as in 12.6, p-q.
12.8 Neutralization
confirmation.
a.	Use 12 microcentrifuge tubes. Add 400 [xL sterile water to tubes 1 -6
and 400 [xL test chemical to tubes 7-12. Allow tubes to equilibrate
approximately 10 min at 20±1°C (or other specified temperature).
b.	Add 600 [xL neutralizer in ice-cold LB broth (or only LB broth
depending on the product) to tubes 4-6 (neutralizer controls). Add
600 [xL neutralizer in ice-cold LB broth to tubes 7-9 (ability of
neutralizer to inactivate the test chemical). Gently mix.
c.	Add 10 |iL B. subtilis spore suspension (approximately 109
spores/mL) to each tube and mix in a Vortex mixer for
approximately 15 s.
d.	Incubate tubes for 30±2 min at 20±1 °C (or temperature specified by
test chemical manufacturer).
e.	After incubation, add 600 [xL ice-cold LB broth to tubes 1-3 (survival
controls). Add 600 [xL ice-cold LB broth to tubes 10-12 (test
chemical controls).

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f.	Serially dilute each tube (e.g., 10 [xL into 990 [xL ice-cold LB broth
or 100 |iL into 900 [xL ice-cold LB broth) to achieve plate counts of
30-300 CFU/plate.
g.	Plate 100 [xL of each dilution onto TS A. Incubate 24±2 h at 36±1°C.
Count colonies on each plate.
h.	LD (CFU/mL) in tubes 1-3 and 4-6 should reflect the original spore
suspension titer and should be within 1 log of each other. If the
difference in LD between tubes 1 -3 and 4-6 is greater than 1 log, then
the neutralizer has a sporicidal effect. If the test chemical is highly
effective, LD in tubes 10-12 should be approximately 5-6 logs lower
than LD in tubes 1 -6.
i.	To be an effective neutralizer, LD in tubes 7-9 should be within 1 log
of the LD in tubes 1 -6. For this assay, produce a spore preparation
according to the procedure for amended NA (refer to section 12.2).
Harvest growth from plates (e.g., 5 plates) per the method, except
resuspend pellet after final centrifugation step in approximately 100
mL aqueous (40%) ethanol.
12.9 HC1 resistance.
a.	Perform on each preparation of inoculated carriers. Conduct TSM
procedure with 2.5 M HC1.
b.	Follow procedure as specified in section 12.6 or 12.7 with 2 and 5
min exposure periods with 3 inoculated carriers per time period.
c.	Include 3 control (sterile water) carriers to determine control carrier
counts. Use LB broth modified with NaOH as the neutralizer
(instead of LB broth) for HC1 treatments. Perform test at 20±1 °C.
d.	Calculate LR. Spores should resist HC1 for > 2 min (i.e., based on
presence of viable spores after 2 min) to be qualified as resistant test
spores. Discard carriers if not resistant and repeat preparation of
carriers as previously described.
Note: Compared to the water control, anticipate LR of 0-3 at 2 min
exposure and LR of 2-6 following the 5 min exposure.
13. Data Analysis/
Calculations
1.	Use counts which fall within 0-300 CFU/plate for calculation of spore
titer.
2.	Obtain the total number of spores per fraction by dividing the number of
colonies counted in each fraction by its dilution, and account for volume
plated. To calculate the CFU/carrier when two (2) serial dilutions are
plated, use the following example formula:

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SOP No. MB-21-02
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f CFU for 10"' + CFU for 1(TZ 1
: 0.1
10 ^ + 10 z
V J
where 10"y and 10"z are the dilutions plated and 0.1 represents the volume
plated (0.1 mL).
a.	When TNTC values are observed for each dilution plated, substitute
300 for the TNTC at the highest (most dilute) dilution and scale up
accordingly for the calculations.
b.	When zeroes are observed for each dilution plated, substitute 0.5 for
the zero at the lowest (least dilute) dilution and scale up accordingly
for the calculations.
3.	Obtain the total number of spores per carrier by adding the total number of
viable spores per fraction for Fractions A-C. For the modified Three Step
Method, obtain the total number of spores per carrier by adding the total
number of viable spores per fraction for Fractions A and BC.
4.	Calculate the logio density (LD) of each carrier by taking the logio of the
spores/carrier.
5.	Calculate the mean logio density across treated carriers.
6.	Calculate the mean logio density across control carriers.
7.	Calculate the logio reduction (LR) for treated carriers:
logio reduction = mean logio control - mean logio treated
a. If no spores are recovered for any treated carrier, report the LR as
greater than or equal to the mean logio density for the control
carriers.
14. Forms and Data
Sheets
1.	Attachment 1: Quantitative Three Step Method for Measuring the Efficacy
of Liquid Sporicides against Spores of Clostridium difficile on Hard Non-
porous and Porous Surfaces
2.	Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
Three Step Method: Test Information Sheet MB-21-02 Fl.docx
Three Step Method: Time Recording Sheet MB-21-02 F2.docx
Three Step Method: Serial Dilution/Plating 21 02 F3 docx
Tracking Form -
Three Step Method: Results Sheet MB-21-02_F4.docx
Three Step Method Neutralization Test: Test MB-21-02_F5.docx

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SOP No. MB-21-02
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Information Sheet
Three Step Method Neutralization Test: Time
Recording Sheet
Three Step Method Neutralization Test: Serial
Dilution/Plating Tracking Form
Three Step Method Neutralization Test: Results
Sheet
Test Microbe Confirmation Sheet
Three Step Method Processing Sheet
Three Step Method Processing Sheet (mTSM)
Three Step Method Spreadsheet (3 Fractions)
Three Step Method Spreadsheet (2 Fractions)
MB -21 -02_F 6. docx
MB -21 -02_F7. docx
MB -21 -02_F8. docx
MB -21 -02_F9. docx
MB -21 -02_F 10 .docx
MB -21 -02_F 11 .docx
MB -21 -02_F 12 .xl sx
MB-21-02 F13.xlsx
15. References
1.	Official Methods of Analysis of AO AC INTERNATIONAL (Revised
2013) 18th Ed., AO AC International, Gaithersburg, MD. Method
2008.05.
2.	Tomasino, S. et. al., "Determining the Efficacy of Liquid Sporicides
Against Spores of Bacillus subtilis on a Hard Nonporous Surface Using
the Quantitative Three Step Method: Collaborative Study." Journal of
AOAC International Vol. 91, No. 4, 2008.
3.	Tomasino, S. et. al., "Use of Alternate Carrier Materials in AOAC Official
MethocfM 2008.05, Efficacy of Liquid Sporicides Against Spores of
Bacillus subtilis on a Hard, Nonporous Surface, Quantitative Three-Step
Method." Journal of AOAC International Vol.93, No.l, 2010.
4.	Rastogi, V. et. al., "Modified AOAC Three Step Method (OfficialMethod
2008.05): Consolidation of Fractions B and C " Journal of AOAC
International Vol.96, No.5, 2013.

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SOP No. MB-21-02
Date Revised 01-06-14
Page 15 of 16
Attachment 1 - Quantitative Three Step Method for Measuring the Efficacy of Liquid Sporicides
against Spores of Clostridium difficile on Hard Non-porous Surfaces. The following steps are
used when conducting efficacy evaluation of products with sporicidal claims against spores of C.
difficile (ATCC 43598):
Spores of Clostridium difficile are prepared according to MLB SOP MB-28.
Section 7: The test organism (C difficile ATCC 43598) must be incubated under strict anaerobic
conditions. The presence of oxygen will severely compromise the viability and growth of C. difficile.
Section 11.1: The recovery medium for C. difficile is brain-heart infusion agar with yeast
extract, horse blood and sodium taurocholate (BHIY-HT).
Section 11.2: Phosphate buffered saline (PBS) containing 0.1% Tween 80 (ST-80). Add 2.0 mL
of polysorbate 80 (Tween 80, or equivalent) to approximately 1.5 L of PBS (1X). Mix
thoroughly and (using a volumetric flask) bring solution to volume (2 L) with PBS. Distribute
into bottles and autoclave for 20 min at 12 PC. Used for control carriers. Fraction B tubes, and
serial dilutions.
Section 11.4: Use the COY anaerobic chamber and anaerobic incubator (supported by a
compressed gas mixture consisting of 10 % hydrogen, 5 % COj, and 85 % N2) to provide an
anaerobic environment.
Section 12.6, d: Add 400 |iL ST-80 to all control carriers.
Section 12.6, f: Fraction B tubes contain 400 |iL ice-cold (0-5°C) ST-80.
Section 12.6, r: For each treated and control tube, serially dilute in 900 |iL ice-cold ST-80.
Section 12.6, s: Use pre-reduced BHIY-HT recovery medium. Open each sealed package inside
the BSC just prior to placement of the filter. Do not expose BHIY -HT plates to oxygen for more
than 50=1=10 min.
Section 12.6, t: Incubate BHIY-HT from control carriers at 36=1= 1°C- for 48=1=4 h and from
treated carriers at 36±1°C for 5 days. Record results as CFU per carrier after 48=1=4 hrs of
incubation for control and after 5 days of incubation for treated carriers.
Section 12.6, u: See Table 2 for growth/diagnostic characteristics of C. difficile (ATCC 43598).
Growth from a typical colony from one or two plates will be processed either for spore staining
or to observe under phase contrast microscopy or using Vitek.
Section 12.7, b: Fraction BC tubes contain 400 [xL ice-cold (0-5°C) ST-80.

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SOP No. MB-21-02
Date Revised 01-06-14
Page 16 of 16
Table 2. Growth/Microscopic Characteristics of C. difficile (ATCC 43598)
Typical Diagnostic Characteristics
BHIY-HT plate
Growth circular, entire edge, convex, smooth and grey colonies.*
Phase-contrast
microscopy
Spores appear bright and ovular while vegetative cells appear dark and rod-shaped.
Spore staining
Spores appear green while vegetative cells appear red.
* After 48±4 h

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