Standard Operating Procedure for AOAC Sporicidal Activity of Disinfectants Test (Bacillus × porcelain component only) SOP Number: MB-15-02 Date Revised: 11-30-09


US Environmental Protection Agency
Office of Pesticide Programs

Office of Pesticide Programs

Microbiology Laboratory

Environmental Science Center, Ft. Meade, MD

Standard Operating Procedure for

AOAC Sporicidal Activity of Disinfectants Test

(Bacillus x porcelain component only)

SOP Number: MB-15-02
Date Revised: 11-30-09

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SOP No. MB-15-02
Date Revised 11-30-09
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EPA/OPP MICROBIOLOGY LABORATORY
ESC, Ft. Meade, MD

Standard Operating Procedure for the AOAC Sporicidal Activity of Disinfectants Test

(.Bacillus x porcelain component only)

SOP Number: MB-15-02

Date Revised: 11-30-09

Initiated By:		Date: / /

Print Name:	

Technical Review: 	Date: / /

Print Name:

Technical Staff

QA Review:		Date: / /

Print Name:	

QA Officer

Approved By:		Date: / /

Print Name:	

Branch Chief

Effective Date:	/ /

Controlled Copy No.:

Withdrawn By:	Date:	/	/

TABLE OF CONTENTS

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Contents	Page Number

I.0	SCOPE AM) APPLICATION	2

2.0	DEFINITIONS	3

3.0	HEALTH AND SAFETY	3

4.0	CAUTIONS	3

5.0	INTERFERENCES	4

6.0	PERSONNEL QUALIFICATIONS	4

7.0	SPECIAL APPARATUS AND MATERIALS	 4

8.0	INSTRUMENT OR METHOD CALIBRATION	7

9.0	SAMPLE HANDLING AM) STORAGE	7

10.0	PROCEDURE AM) ANALYSIS	7

II.0	DATA ANALYSIS/CALCULATIONS	19

12.0	DATA MANAGEMENT/RECORDS MANAGEMENT	19

13.0	QUALITY CONTROL	19

14.0	NONCONFORMANCE AND CORRECTIVE ACTION	20

15.0	REFERENCES	20

16.0	FORMS AND DATA SHEETS	20

1.0 SCOPE AND APPLICATION:

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1.1 This SOP describes the Sporicidal Activity of Disinfectants Test - Method II

methodology used to determine the sporicidal efficacy of liquid sporicidal agents
against Bacillus on hard surfaces (porcelain carriers). The method is based on
AO AC method 966.04 (see 15.1). In most cases, Bacillus subtilis (ATCC
#19659) is the test microbe selected for sporicidal testing; however, if requested,
other Bacillus species may also be used. Testing of suture loops and Clostridium
is not addressed in this SOP

2.0 DEFINITIONS:

2.1	AO AC = AO AC INTERNATIONAL

2.2	CFU = Colony Forming Unit

2.3	TNTC = Too Numerous to Count

2.4	References to water mean reagent-grade water, except where otherwise specified.

3.0 HEALTH AND SAFETY:

3.1	All manipulations of the test organism are required to be performed in accordance
with biosafety practices stipulated in SOP MB-01.

3.2	Disinfectants may contain a number of different active ingredients, such as
hydrogen peroxide/peracetic acid, sodium hypochlorite, glutaraldehyde, chlorine
dioxide, etc. Personal protective clothing or devices are recommended during the
handling of these items for purpose of activation, dilution, or efficacy testing. A
chemical fume hood or other containment equipment may be employed when
performing tasks with concentrated products. The study analyst may wish to
consult the Material Safety Data Sheet for the specific product/active ingredient
to determine the best course of action.

4.0 CAUTIONS:

4.1	To ensure the stability of a diluted sporicidal agent, use the diluted product within
three hours of preparation unless specified otherwise.

4.2	Use appropriate aseptic techniques for all test procedures involving the
manipulation of the test organisms and associated test components.

4.3	These microbiological methods are very technique sensitive and technique-
oriented, thus exact adherence to the method, good laboratory practices, and
quality control are required for proficiency and validity of the results.

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5.0 INTERFERENCES:

5.1 Touching the interior sides of the medication tube should be avoided while the
carrier is being lowered into the disinfectant and the hook is being removed as it
may lead to false positive results.

6.0 PERSONNEL QUALIFICATIONS:

6.1 Personnel are required to be knowledgeable of the procedures in this SOP.

Documentation of training and familiarization with this SOP can be found in the
training file for each employee.

7.0 APPARATUS AND MATERIALS:

7.1 Culture Media

7.1.1 Nutrient broth. For use in preparing nutrient agar. Add 5 g beef
extract (paste or powder), 5 g NaCl, and 10 g peptone (anatone) to
approximately 1 L water. Boil mixture for 20 minutes with constant
stirring. Readjust volume to 1 L with water and allow cooling to
around 50°C. Adjust pH to 6.8 ± 0.2 with IN HC1 or IN NaOH, if
necessary. Filter through paper (e.g., Whatman filter paper No. 4).
Dispense 10 mL portions into 20 x 150 mm culture tubes or 20 mL
portions into 25 x 150 mm culture tubes. Dehydrated nutrient broth
may be substituted - prepare according to the manufacturer's
instructions.

7.1.2	Nutrient agar. For stock cultures slants. Add 1.5% (w/v) Bacto-agar
to unsterilized nutrient broth. Boil mixture until agar is dissolved.
Adjust pH to 7.2 ± 0.2 if necessary. Dispense 5 mL portions into 16 x
100 mm screw cap tubes. Larger tubes may be used as well.

Autoclave for 20 minutes at 121°C. Remove from autoclave and slant
tubes to form agar slopes.

7.1.3	Nutrient agar with 5ug mL MnSO4-H20 (amendednutrient agar). For
spore production. Suspend 11.5 g nutrient agar in 495 mL water, add 5
mL 500 ppm MnSO^H^O. Dissolve by boiling. Adjust pH to 6.8 ±
0.2 if necessary. Autoclave for 15 minutes at 121°C. Pour agar into
plates.

7.1.4

Trypticase soy agar (TSA). Suspend 40 g dehydrated trypticase soy

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agar in 1 L water and heat gently while stirring. Boil one minute or
until completely dissolved. Adjust pHto 7.3 ± 0.2. Autoclave 15
minutes at 121°C. Pour agar into plates.

7.1.5	Fluid thioglycollate medium (FTM). Suspend 29.5 g of dehydrated
fluid thioglycollate medium in 1 L water. Heat to boiling to dissolve
completely. Adjust pH to 7.1 ± 0.2 if necessary. Dispense 10 mL
portions into 20 xl50 mm culture tubes and autoclave for 15 minutes
at 121°C. Store at room temperature. Protect from light. Note: If
after autoclaving the aerated portion of media consumes more than one
third of tube, media must be re-boiled by placing tubes in beaker of
boiling water. Media can only be re-boiled once.

7.1.6	Fluid thioglycollate medium with lMNaOH (modifiedFTM).- For
subculturing spores exposed to 2.5 M HC1. Suspend 29.5 g of fluid
thioglycollate medium in 1 L water. Heat boiling to dissolve
completely. Cool and adjust pH to 7.1 ± 0.2 if necessary. Add 20 mL
1M NaOH, mix well. Check final pH and record (pH between 8 and 9
is typical). Dispense 10 mL into 20 x 150 mm culture tubes and
autoclave for 15 minutes at 121°C. Store at room temperature. Protect
from light.

Note: Commercial dehydrated media made to conform to the specified
recipes may be substituted. Media can be stored for up to two months.

7.2	Manganese Sulfate Monohydrate. 500 ppm. Add 0.25 g of manganese sulfate
monohydrate to 500 mL water. Filter sterilize for use.

7.3	Dilute hydrochloric acid. 2.5M. Use to determine resistance of dried spores.
Standardize and adjust to 2.5M as in AO AC method 936.15 or purchase certified
2.5M HC1.

7.4	Sterile water. Use reagent-grade water. Reagent-grade water should be free of
substances that interfere with analytical methods. Any method of preparation of
reagent-grade water is acceptable provided that the requisite quality can be met.
Reverse osmosis, distillation, and deionization in various combinations all can
produce reagent-grade water when used in the proper arrangement. See Standard
Methods for the Examination of Water and Wastewater for details on reagent-
grade water.

7.5	Triton X-100

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7.6	Ethanol (40%)

7.7	Test organism-Bacillus subtilis (ATCC No. 19659) obtained directly from a
reputable supplier (e.g., ATCC).

7.8	Carriers.Penicylinders, porcelain, 8 ± 1 mm OD, 6 ± 1 mm ID, 10 ± 1 mm
length (Available from CeramTec Ceramic, Laurens, SC, www.ceramtec.com,
Cat. No. LP15819 0645.)

7.9	Glassware- For disinfectant, 25 x 100 mm culture tubes (Bellco Glass Inc.,
Vineland, NJ; reusable or disposable 20 x 150 mm (for cultures/subcultures); 16
x 100 mm screw cap tubes for stock cultures. Cap with closures before
sterilizing. Sterilize all glassware 2 hr in hot air oven at 180° C or steam sterilize
for a minimum of 20 min at 121°C with drying cycle.

7.10	Sterile centrifuge tubes -Polypropylene, 15 mL conical tubes with conical
bottoms (Corning), from Fisher, or equivalent.

7.11	Water bath/chiller unit. Constant temperature for test chemical, capable of
maintaining 20 ± 1°C temperature or specified temperature for conducting the
test.

7.12	Petri dishes -Plastic (sterile)

7.13	Filter paper -Whatman filter paper #2; placed in Petri dishes for storing carriers.

7.14	Test tube racks- Any convenient style.

7.15	Inoculating loop -Any convenient inoculation/transfer loop for culture transfer.

7.16	Wire hook-Vox carrier transfer. Make 3 mm right angle bend at end of 50 - 75
mm nichrome wire No. 18 B&S gage. Have other end in suitable holder.

7.17	Centrifuge.-Non-refrigerated (e.g., Eppendorf 5804 R).

7.18	Sonicator.-Ultrasonic cleaner (e.g., Branson Model 1510).

7.19	Orbital shaker- speed range from 25 to 500 rpm (e.g., VWR DS 500).

7.20	Vacuum desiccator-Vox carrier storage. With adequate gauge for measuring 27"
(69 cm) of Hg and fresh desiccant.

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7.21	Certified biosafety cabinet (Class I or ///-Recommended for use to maintain
aseptic work environment.

7.22	Certified Timer-Vox managing timed activities, any certified timer that can
display time in seconds.

8.0 INSTRUMENT OR METHOD CALIBRATION:

8.1 Refer to the laboratory equipment calibration and maintenance SOPs (SOP EQ
series) for details on method and frequency of calibration.

9.0 SAMPLE HANDLING AND STORAGE:

9.1 Sporicidal agents are stored according to manufacturers' recommendations or at
room temperature if the product label does not specify a storage temperature.
Those sporicidal agents requiring activation or dilution prior to use will only be
activated or diluted within three hours of testing unless label directions specify
otherwise.

10.0 PROCEDURE AND ANALYSIS:

10.1 Culture initiation, maintenance, culture identification and quality control.
10.1.1 Culture initiation.

10.1.1.1	Every 12 months (or sooner if necessary) initiate a new
stock culture from a lyophilized culture of Bacillus subtilis
(ATCC 19659) from the American Type Culture Collection
(ATCC) or other reputable supplier.

10.1.1.2	Open ampule of freeze dried organism as indicated by
ATCC.

10.1.1.3	Using a tube containing 5-6 mL of nutrient broth (NB),
aseptically withdraw 0.5 to 1.0 mL and rehydrate the pellet
for Bacillus subtilis.

10.1.1.4	Aseptically transfer the entire rehydrated pellet back into
the original tube of nutrient broth designated as "TUBE A,"
(see Attachment 1). Mix well.

10.1.1.5

Streak for isolation using a loopful of rehydrated
suspension on duplicate trypticase soy agar (TSA) or

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nutrient agar (NA) plates.

10.1.1.6	Incubate broth culture (TUBE A) and plate cultures at 30 ±
1°C for 24 ± 2 hours.

10.1.1.7	Record all manipulations on the Organism Culture
Tracking Form (see 16.17).

10.1.2 Culture identification.

10.1.2.1 Initial confirmation testing for quality control (QC) will be
performed using the 24 ± 2 hour NA or TSA plates from
step 10.1.1.5.

10.1.2.2	Following the incubation period (as stated in 10.1.1.6),
record the observed colony morphology on the NA or TSA
plates and selective media plates (including the absence of
growth) and stain reaction. See 10.8.9 for details on colony
morphology and Gram stain reaction.

10.1.2.3	Perform a Gram stain from growth taken from the TSA or
NA plates. Perform the Gram stain according to the
manufacturer's instructions. Observe the Gram reaction by
using brightfield microscopy at 1000x magnification (oil
immersion).

10.1.2.4	Perform VITEK™ analysis according to the manufactures'
instructions.

10.1.2.5	Record all confirmation results on the Test Microbe
Confirmation Sheet (Quality Control) (see 16.18).

10.1.3 Generation of stock cultures.

10.1.3.1	Use the 24 ± 2 hour TUBE A (see Attachment 1) broth
culture discussed in 10.1.1.4 to initiate stock cultures -
streak a minimum of six nutrient agar slants with B. subtilis
and incubate at 36 ±1°C for 24 ± 2 hours.

10.1.3.2	Following incubation, store the cultures at 2-5°C for 30 ± 2
days. These cultures are identified as the "stock cultures."
Begin stock culture transfers as outlined in section 10.1.5.

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Repeat the cycle for a maximum of one year.

10.1.3.3 From a set of six stock cultures, one is used every 30 ± 2
days for QC and to generate new stock cultures, four may
be used per month (one/week) for generation of test
cultures, and one is a back-up tube.

10.1.4	Monthly QC of stock cultures.

10.1.4.1	Monthly QC of stock cultures may occur just prior to or
concurrently with stock culture transfers. Use one
refrigerated stock culture tube and streak a loopful on a
plate of TSA.

10.1.4.2	Incubate the plates at 36 ± 1°C for 24 ± 2 hours (18-24
hours for use in the VITEK 2 Compact). Follow steps
outlined in section 10.1.2.2 to confirm the identity of the
organism.

10.1.5	Culture maintenance.

10.1.5.1	Every 30 ± 2 days inoculate a new set of stock culture
tubes from a current stock culture tube. Use the same
refrigerated stock culture tube used for Monthly QC
described in 10.1.4.1 to inoculate 6 new stock cultures
tubes as outlined in 10.1.3.1.

10.1.5.2	Incubate the new stock cultures as indicated in 10.1.3.1.

10.1.5.3	Following the incubation period, store the stock cultures at
2-5°C for 30 ± 2 days.

10.2 Production ofB. subtilis spore suspension.

10.2.1 Using growth from a stock culture tube, inoculate 10 mL tubes (e.g., 2
tubes, depending on the amount of spore preparation desired) of
nutrient broth and incubate tubes on an orbital shaker for 24 ± 2 hours
at approximately 150 rpm at 36 ± 1°C. Use this culture to inoculate
amended nutrient agar plates. Inoculate each plate with 500 |iL of
broth culture and spread the inoculum with a sterile bent glass rod or
suitable spreading device. In addition, verify the purity of this culture
by streak isolating on amended nutrient agar (incubate at 36 ± 1°C for

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24 ± 2 hours).Wrap each plate with parafilm or place in plastic bags.
Incubate plates inverted for 12-14 days at 36 ± 1°C.

10.2.2 Following incubation, harvest the spores by adding 10 mL cold sterile
water to each plate. Using a spreader (e.g. bent glass rod), remove
growth from plates and pipet suspensions into 15 mL sterile conical
tubes (10 plates = 14 tubes, -10 mL each). Centrifuge tubes at 5,000
rpm (4,500 x g) for approximately 10 minutes at room temperature.
Remove and discard supernatant. Re-suspend pellet in each tube with
10 mL cold sterile water and centrifuge at 5,000 rpm (4,500 x g) for
approximately 10 minutes. Remove and discard supernatant. Repeat
twice. Re-suspend the pellet in each tube with 10 mL sterile water.
Store the spore suspension at 2-5°C.

10.2.3 Examine spore suspension with a phase contrast microscope or by
staining to assess quality of the spores. Examine a minimum of five
fields and determine ratio of spores to vegetative cells (or sporangia).
Percentage of spores versus vegetative cells should be at least 95%.
Spore suspension from multiple plates can be combined and re-
aliquoted into tubes for uniformity.

10.2.4 Prior to inoculation of carriers, determine spore titer of the

concentrated spore suspension by plating 100 |iL aliquots of serial
dilutions (e.g., 1.0 x 10"5 through 1.0 x 10"7) using spread plating on
TSA plates or another comparable validated enumeration procedure.
Incubate plates for 24 ± 2 hours at 36 ± 1°C and determine titer. Note:
When harvested and processed, ten plates of amended nutrient agar
should provide 80-100 mL of concentrated spore suspension (approx.
109 CFU/mL). Diluting the suspension prior to carrier inoculation will
be necessary; a titer of 1.0 x 108 to 5.0 x 108 CFU/mL should be
adequate to achieve the target carrier count.

10.3 Preparation of porcelain carriers.

10.3.1	Prior to use, examine porcelain carriers individually and discard those
with scratches, nicks, spurs, or discolorations.

10.3.2	Rinse unused carriers gently in water three times to remove loose
material and drain.

10.3.3

Place rinsed carriers into Petri dishes matted with 2 layers of filter
paper in groups of 15 carriers per Petri dish or place carriers into 25 x
150 mm tubes (10 carriers per tube).

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10.3.4 Sterilize 20 minutes at 121°C. Cool and store at room temperature.

Note: Handle porcelain carriers with care when placing in Petri dishes.
Minimize carrier movement and avoid excessive contact between
carriers that might result in chips and cracks. Wash carriers with
Triton X-100 and rinse with water 4 times for reuse.

10.4	Inoculation of Porcelain Carriers.

10.4.1	Dilute the concentrated spore suspension as necessary with sterile
water to achieve carrier counts between 1.0 x 105 and approximately
1.0 x 106 spores/carrier. Dispense 10 mL diluted spore suspension into
an appropriate number of 25 x 150 mm tubes.

10.4.2	Add 10 sterile carriers to each tube containing 10 mL spore
suspension, slightly agitate, and let stand 10-15 minutes.

10.4.3	Remove each carrier with sterile hook and place upright in sterile Petri
dish lined with two sheets of filter paper, no more than 30 carriers per
Petri dish.

10.4.4	Air dry in biological safety cabinet for approximately 30 ± 2 minutes.
Place Petri dishes containing inoculated carriers in vacuum desiccator
containing CaCh and draw vacuum of 69 cm (27") Hg.

10.4.5	Dry carriers under vacuum for 24 ± 2 hours before use in HC1
resistance, efficacy testing or carrier counts. Maintain under vacuum
for up to three months.

10.4.6	Carriers may be used after three months if they meet the acceptable
HC1 resistance and carrier count criteria. Inoculated carriers should
not be used after one year of storage. Sterilize and reuse if necessary.

10.5	Spore Enumeration (Carrier Counts).

10.5.1	Prior to use, determine the carrier counts for each preparation of
carriers. Assay 3 to 5 randomly selected carriers per preparation.

10.5.2	Place each inoculated carrier into a 50 mL plastic, polypropylene
conical centrifuge tube containing 10 mL of sterile water.

10.5.3

Sonicate carriers for 5 minutes ± 30 seconds.

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Note: For sonication, place tubes into an appropriately sized glass
beaker with tap water to the level of sterile water in the tubes. Place
beaker in sonicator so that water level in the beaker is even with water
level fill line on sonicator tank. Fill tank with tap water to water level
fill line. Suspend beaker in sonicator tank so it does not touch bottom
of tank and so all three water levels (inside test tubes, inside beaker,
and sonicator tank) are the same.

Following sonication, vortex tubes for 2 minutes ± 5 seconds.

Dilute spore suspensions by transferring 1 mL aliquots to tubes
containing 9 mL sterile water. Dilute spore suspensions out to 1.0
xlO"4 and plate 100 |iL aliquots of dilutions 1.0 x 10"1 through 1.0 x
10"4. Plate each dilution in duplicate using spread plating with TSA or
another comparable validated enumeration procedure. Invert plates
and incubate for 24-48 hours at 36 ± 1°C.

Count colonies (by hand or with colony counter). Record all counts
less than 300 and use those counts for enumeration. Report plates with
colony counts over 300 as TNTC (Too Numerous to Count). Average
spore counts per carrier should be between 1.0 x io5 and
approximately 1.0 x 106 spores/carrier. Do not use carriers with
counts outside this range.

10.6 HCl resistance.

10.6.1	Equilibrate water bath to 20 ± 1°C. Pipet 10 mL of 2.5M HCl into two
25 x 100 mm tubes, place into water bath, and allow to equilibrate.
Start timer and rapidly transfer 4 inoculated penicylinders into a tube
with 2.5 M HCl using flamed hooks or forceps. Do not allow carriers
or transfer device to contact inside of wall of acid tube.

10.6.2	Transfer individual carriers after 2, 5, 10, and 20 minutes of HCl
exposure to a separate tube of modified FTM. Rotate each tube
vigorously by hand for approximately 20 seconds and then transfer
carrier to a second tube of modified FTM.

10.6.3	For viability control, place one unexposed inoculated carrier in a
separate tube of modified FTM. For media sterility, use one tube of
modified FTM.

10.6.4	Incubate all test and control tubes for 21 days at 36 ± 1°C. Record
results as growth (+) or no growth (0) at each time period. Spores

10.5.4

10.5.5

10.5.6

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should resist HC1 for > 2 minutes to be qualified as resistant test
spores. Discard carriers if not resistant and repeat preparation of
carriers as previously described.

10.7 Efficacy Test.

10.7.1	Prepare disinfectant samples according to MB-22. For a 30-carrier
test, place 10 mL product at dilution recommended for use or under
investigation into each of six 25 x 150 mm or 25 x 100 mm test tubes,
or use appropriate number of tubes assuming 5 test carriers per tube of
test chemical.

10.7.2	Place tubes in 20 ± 1°C water bath and let equilibrate to temperature.
Using a sterile hook (or forceps), transfer inoculated carriers
sequentially at 2 minute intervals in groups of 5 from Petri dish to test
tubes containing sporicidal agent. Use a certified timer to monitor
time.

10.7.2.1	Flame hook and allow cooling after each transfer. When
lowering carriers into test tube, neither carriers nor wire
hook may touch sides of tubes.

10.7.2.2	If interior sides are touched, note tube number - do not
count carrier set if any carrier from that group of 5 yields a
positive result. Testing another set of five carriers is
recommended.

10.7.2.3	Carriers must be deposited into test tubes within ± 5
seconds of the prescribed drop time. Return tubes to water
bath immediately after adding carriers.

10.7.3	After contact period has been achieved, transfer carriers in same
sequential timed fashion into primary subculture tubes containing
appropriate neutralizer (10 mL in 20 x 150 mm test tubes).

10.7.3.1	Remove the carriers one at a time from the test tube with
sterile hook, tap against interior side of tube to remove
excess sporicidal agent, and transfer into neutralizer tube
(primary tube).

10.7.3.2	All five carriers must be transferred during each 2 minute
interval. Flame hook between each carrier transfer. Move
remaining carriers into their corresponding neutralizer

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tubes at appropriate time.

10.7.3.3 Carriers may touch interior sides of neutralizer tube during
transfer, but contact should be minimized.

10.7.4 After each carrier is deposited, recap neutralizer tube and gently shake
to facilitate adequate mixing and efficient neutralization.

10.7.5 Within one hour from when last carrier was deposited into primaries,
transfer carriers using sterile wire hook to second subculture tube
(secondary tube) containing 10 mL of appropriate recovery medium,
one carrier per tube.

10.7.5.1 Move carriers in order, but movements do not have to be

timed. Gently shake entire rack of secondary tubes after all
carriers have been transferred.

10.7.6 Incubate primary (neutralizer) and secondary subculture tubes for 21
days at 36 ± 1°C. Report results as growth (+) or no growth (0).

10.7.6.1 A positive result is one in which medium appears turbid. A
negative result is one in which medium appears clear.
Shake each tube prior to recording results to determine
presence or absence of growth/turbidity.

10.7.6.2 Primary and secondary subculture tubes for each carrier

represent a "carrier set". A positive result in either primary
or secondary subculture tube is considered a positive result
for the carrier set.

10.7.7 Media sterility controls and system controls (check for aseptic
technique during carrier transfer process) are recommended.

10.7.7.1	For media controls, incubate 1-3 unopened subculture
medium tubes with the test sample tubes for 21 days at 36 ±
1°C.

10.7.7.2	For system controls, use sterile forceps or needle hooks to
transfer 3 sterile carriers into a tube of test chemical.

10.7.7.3

Transfer system control carriers to neutralizer medium as
follows: at start of sample test (prior to first tube), transfer
1 sterile carrier to tube of neutralizer medium. After one

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half of test carriers have been transferred to neutralizer
tubes, transfer a second sterile carrier to tube of neutralizer
medium. After all test carriers (last tube) have been
transferred to neutralizer tubes, transfer third sterile carrier
to tube of neutralizer medium.

10.7.7.4	Transfer system control carriers to secondary subculture
medium as follows: immediately prior to initiating transfer
of test carriers into secondary subculture medium tubes,
transfer first system control sterile carrier from neutralizer
medium to tube of subculture medium. After one half of
test carriers have been transferred to secondary subculture
medium tubes, transfer second system control sterile carrier
to tube of subculture medium. After all test carriers have
been transferred to secondary subculture medium tubes,
transfer third system control sterile carrier to tube of
subculture medium.

10.7.7.5	For each test, include a positive carrier control by placing
one inoculated carrier into tube of secondary subculture
medium. Incubate controls and test sample tubes together
for 21 days at 36 ± 1°C.

10.7.8 Perform identification confirmation on a minimum of three positive
carrier sets per test, if available, using Gram stain and/or plating on
TSA. Additional confirmation may be performed using VITEK, API
analysis or comparable method.

10.7.8.1 If fewer than three positive carrier sets, confirm growth

from each positive carrier set. If both tubes are positive in
carrier set, select only one tube for confirmatory testing.
For tests with 20 or more positive carrier sets, confirm at
least 20% by Gram stain. If Gram stains are performed
from growth taken directly from positive tubes, the staining
should be performed within 5-7 days of conducting the
efficacy test.

10.8 Neutralization Confirmation Procedure.

10.8.1 A neutralization confirmation test must be performed in advance or in
conjunction with efficacy testing. This assay is designed to simulate
the conditions (i.e., neutralizer, subculture medium, contact time,

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diluent, concentration of test substance) of the efficacy test and to
demonstrate the recovery of a low level of spores (e.g., 5-100).

Diluted inoculum (e.g., spores of B. subtilis) is added directly to the
various sets of subculture media tubes (see Table 1). This assay
provides for a quantitative approach to assessing the effectiveness of
the neutralizer and any bacteriostatic action resulting from the
neutralizer itself or neutralizer-disinfectant interactions.

10.8.2	Produce a spore preparation according to the procedure for amended
nutrient agar. Harvest growth from plates (e.g., five plates) per the
method, except re-suspend pellet after final centrifugation step in
approximately 100 mL aqueous (40%) ethanol.

10.8.2.1	Determine spore count by serial dilution and plating on
TSA. Desirable target of the initial working suspension is
1.0 x 108 to 1.0 x 109 CFU/mL. The suspension may
require adjustment to reach target titer.

10.8.2.2	Prepare serial ten-fold dilutions of the inoculum in sterile
water out to 10"7. Use 100 |iL aliquots of the 10"5, 10"6 and
10"7 dilutions to inoculate the neutralizer and subculture
media tubes - the target number of spores to be delivered
per tube in this assay is 5-100 per tube.

10.8.2.3	Determine spore titer by plating (spread plate or pour plate)
each of three dilutions in duplicate on TSA agar. Incubate
plates inverted for 24-48 hours at 36 ± 1°C. Count colonies
(by hand or with colony counter). Report plates with
colony counts over 300 as TNTC (Too Numerous to
Count).

Note: A standardized spore preparation adjusted to deliver
5-100 spores/mL may be substituted for the three dilutions
of spore inoculum. In addition, spores sheared from
inoculated carriers may be used as a working suspension.

10.8.3	Use 5 sterile porcelain carriers (only 3 to be used in the assay). Within
5 seconds, place a set of 5 carriers into a test tube (25 x 150 mm or 25
x 100 mm) containing test chemical; transfer carriers according to
section 10.7.2. Allow carriers to remain in test chemical per the
specified contact time and temperature.

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SOP No. MB-15-02
Date Revised 11-30-09
Page 17 of 41

10.8.3.1	After the contact time is complete, aseptically transfer three
of the five carriers individually into tubes containing the
neutralizer per section 10.7.3. This set of tubes is the
Neutralizer/Primary Subculture treatment.

10.8.3.2	Following the transfer of the last carrier into neutralizer
tube, transfer each carrier, in sequence, into tube containing
secondary subculture medium. This portion of assay is not
timed, but should be made as soon as possible. This set is
the Secondary Subculture treatment.

10.8.4	Following carrier transfer, inoculate each tube (Neutralizer/Primary
and Secondary Subculture treatment tubes) with 100 |iL of each of
three inoculum dilutions (10"5, 10"6and 10"7).

10.8.5	For controls, use three fresh unexposed tubes of neutralizer and three
tubes of the secondary subculture medium; also inoculate each control
tube with 100 |iL of each of three inoculum dilutions. Include one
uninoculated tube of neutralizer and secondary subculture media to
serve as sterility controls.

10.8.6	See Table 1 for tube inoculation scheme.

10.8.7	Incubate all tubes 5-7 days at 36 ± 1°C.

10.8.8 Record results as growth (+) or no growth (0). Note: The lack of

complete neutralization of the disinfectant or bacteriostatic activity of
the neutralizer itself may be masked when a high level of inoculum
(spores) is added to the subculture tubes.

Table 1. Neutralization confirmation procedure - inoculating treatment and control tubes
with diluted spore suspension*			

Ncutralizcr-Primary
Subculture Treatment

Secondary Subculture
Treatment (with Carrier)

Ncutralizcr-Primary
Inoculated Control

Secondary Subculture
Inoculated Control

100 (iL of 10"5 —» Tube 1
100 (iL of 10"6-> Tube 2
100 (iL of 10"7-> Tube 3

100 (iL of 105 —> Tube 1
100 (iL of 10"6-> Tube 2
100 (iL of 10"7-> Tube 3

100 (iL of 105 —> Tube 1
100 (iL of 10"6-> Tube 2
100 (iL of 10"7-> Tube 3

100 (iL of 105 —> Tube 1
100 (iL of 10"6-> Tube 2
100 (iL of 10"7-> Tube 3

*Use of 1.0 x 10"5 through 1.0 x 10"7 based on an approx. starting suspension of 108 spores/mL

10.8.9 Confirm a minimum of one positive per treatment and control (if

available) using Gram staining and colony morphology on TSA. For

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SOP No. MB-15-02
Date Revised 11-30-09
Page 18 of 41

each treatment and control group, conduct confirmation testing on
growth from tube with fewest spores delivered. B. subtilis is a Gram
positive rod and colonies on TSA are opaque, rough, dull, round, with
irregular margins, and low convex. Colonial variation may be
observed and is typical for this strain.

10.8.10	Growth in the inoculated controls verifies the presence of the spores,
performance of the media, and provides a basis for comparison of
growth in the neutralizer and subculture treatment tubes. Note: There
may be cases when the neutralizer is significantly different from the
secondary subculture media; in these cases, growth may not be
comparable. The uninoculated control tubes are used to determine
sterility, and must show no growth for the test to be valid.

10.8.11	The occurrence of growth in the Neutralizer/Primary Subculture and
Secondary Subculture treatment tubes is used to assess the
effectiveness of the neutralizer. No growth or growth only in tubes
which received a high level of inoculum (e.g., the dilution with plate
counts which are too numerous to count) indicates poor neutralization
and/or presence of bacteriostatic properties of the neutralizer or
neutralizer-disinfectant interactions.

10.8.12	For a neutralizer to be deemed effective, growth must occur in the
Secondary Subculture treatment tubes which received lower levels of
inoculum (e.g., 5-100 CFU/mL).

10.8.13	Growth in the Secondary Subculture inoculated Control verifies the
presence of the spores, performance of the media, and provides a basis
for comparison of growth in the neutralizer and subculture treatment
tubes. No growth or only growth in tubes which received high levels
of inoculum (e.g., a dilution with plate counts which are too numerous
to count) indicates poor media performance.

10.8.14	Growth in the Neutralizer-Primary inoculated Control should be
comparable to the Secondary Subculture inoculated Control if the
neutralizer is the same as the secondary subculture media. There may
be cases when the neutralizer is significantly different from the
secondary subculture media. In these cases, growth may not be
comparable to the Secondary Subculture inoculated Control.

10.8.15

The Neutralizer-Primary and Secondary Subculture uninoculated
Control tubes are used to determine sterility, and must show no growth
for the test to be valid.

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SOP No. MB-15-02
Date Revised 11-30-09
Page 19 of 41

11.0 DATA ANALYSIS/CALCULATIONS:

11.1 Data will be recorded on data sheets (see 16.2). Calculations will be computed
using a Microsoft Excel spreadsheet (see 16.3). Electronic copies of the
spreadsheet as well as hard copies will be retained.

11.2 To calculate CFU/mL per carrier:

(avg. CFU for\Qrw) + (avg. CFU forl(Tx) + (avg. CFU for\
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SOP No. MB-15-02
Date Revised 11-30-09
Page 20 of 41

13.2 For quality control purposes, the required information is documented on the
appropriate form(s) (see 16.0).

14.0 NONCONFORMANCE AND CORRECTIVE ACTION:

14.1 Any deviation from the standard protocol and the reason for the deviation will be
recorded on the appropriate record sheet (see 16.0); corrective action will be
expeditious.

15.0 REFERENCES:

15.1 Official Methods of Analysis (2006) 21st ED., AO AC INTERNATIONAL,
Method 966.04, Gaithersburg, MD, Chapter 6

15.2 Standard Methods for the Examination of Water and Wastewater. 21 st Ed.
American Public Health Association, 1015 15th Street, NW, Washington, DC

15.3 Tomasino, S.F. & Hamilton, M.A. (2006) JAOAC Int. 89, 1373-1397

16.0 FORMS AND DATA SHEETS:

16.1 Physical Screening of Carriers Record

16.2 Sporicidal Activity of Disinfectants Test: Organism Culture Tracking Form

16.3 Sporicidal Activity of Disinfectants Test: Test Microbe Confirmation Sheet
(Quality Control)

16.4 Sporicidal Activity of Disinfectants Test: Serial Dilution/Plating Tracking Form
for Carrier Counts

16.5 Sporicidal Activity of Disinfectants Test: Carrier Count Data Sheet

16.6 Carrier Count Spreadsheet

16.7 Sporicidal Activity of Disinfectants Test: Hydrochloric Acid Resistance Test Data
Sheet

16.8 Sporicidal Activity of Disinfectants Test: Information Sheet

16.9 Sporicidal Activity of Disinfectants Test: Time Recording Sheet for Carrier
Transfers

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Date Revised 11-30-09
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16.10 Sporicidal Activity of Disinfectants Test: Results Form (1-30)

16.11 Sporicidal Activity of Disinfectants Test: Results Form (31-60)

16.12 Sporicidal Activity of Disinfectants Test: Performance Controls Results Sheet

16.13 Sporicidal Activity of Disinfectants Test: Test Microbe Confirmation Sheet

16.14 Sporicidal Activity of Disinfectants Test: Neutralization Confirmation Assay
Information Sheet

16.15 Sporicidal Activity of Disinfectants Test: Neutralization Confirmation Assay
Results Form

16.16 Sporicidal Activity of Disinfectants Test: Neutralization Confirmation Assay
Time Recording Sheet for Carrier Transfers

16.17 Sporicidal Activity of Disinfectants Test: Neutralization Confirmation Assay
Serial Dilution/Plating Tracking Form

16.18 Sporicidal Activity of Disinfectants Test: Neutralization Confirmation Assay
Inoculum Enumeration Form

Attachment 1 Culture Initiation and Stock Culture Generation Flow Chart for B. subtilis

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Page 22 of 41

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SOP No. MB-15-02
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16.1

Physical Screening of Carriers Record
OPP Microbiology Laboratory

Carrier Information

Screening Results

Type

Vendor

Control Number

Date Screened/Initials

No. Pass

No. Fail

Prep # Assigned

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Date Revised 11-30-09
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16.2

ORGANISM CULTURE TRACKING FORM
OPP Microbiology Laboratory

Organism:

Bacillus sub tilis

Supply Control Number:

Source and Strain no.:

Lot Number:

MRME Number:

Date

Time

I nit.

Subculture Source

T ransfcr*

Media Inoculated
(and # inoc.)

Media Prep No.

Incubation
Conditions

Comments

Monthly

Daily

* "Monthly" indicates the monthly transfers for culture and "Daily" indicates a 24/48 hr serial transfer (added to control number)
NR = None Required, TC = Test Culture, applied after daily transfer number
NB = nutrient broth

16.3

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Page 25 of 41

TEST MICROBE CONFIRMATION SHEET (Quality Control)
OPP Microbiology Laboratory

Organism:

Bacillus subtilis

MRME Number:

Source and Strain no.:

Notes:

Source:
Tube/Plate
ID

Date/
Initials

Staining
Results*

Media Information

Results

Name

Prep. No.

Inc. Time/
Temp.

Date/
Initials

Colony Characteristics

Vitek #**

* Record Gram stain results (GPR = Gram positive rods)

* * Vitek tracking number
TSA = trypticase soy agar, NA = nutrient agar

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Date Revised 11-30-09
Page 26 of 41

16.4

Sporicidal Activity of Disinfectants Test: Serial Dilution/Plating Tracking
Form for Carrier Counts
OPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:

Test Dale

Carrier Type

Confirmed bv:

Dilution Tube

10-1

10~2

10-3

10-4

Starling volume of diluent

10 mL

9 mL

Volume added to serial dilution lube (1 mL)

1 mL

Volume plated (0.1 mL)

0.1 mL

Final dilution (used for calculations)*

10"2

10"3

10"4

10"5

Number of plates per dilution

Plating medium

Number of carriers evaluated

Starting volume of diluent

10 mL

Comments: The 10"1 dilution tube contains the carrier. *Adjusted for volume plated.

Dilution blank volume verified: ~ Yes ~ No Subculture medium volume verified: ~ Yes ~ No

REAGENT/MEDIA INFORMATION/Confirmed by:

Reagent/Media

Prep. No.

Reagent/Media

Prep. No.

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SOP No. MB-15-02
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16.5

Sporicidal Activity of Disinfectants Test: Carrier Count Data Sheet
OPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:

Test Dale

Carrier Type

RESULTS

Dale/Initials

Plating Method

Carrier No.

Dilution

CFU per Dilution Plate (2)

Comments:

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SOP No. MB-15-02
Date Revised 11-30-09
Page 28 of 41

16.6

Carrier Count Spreadsheet
OPP Microbiology Laboratory

Carrier Count Spreadsheet
OPP Microbiology Lab oratory

TEST INFORMATION/CoHfiiHied by:

EPA Reg. No.

Name

Sample No.(s)

Test Date

Onanism

SOP

MB-15

Test Type

Spoiicidal Activity of Disinfectants Test

^---^Camer No.
Dilution

CFU per Plate

CFU/caniisr

1 .E-02

l.E-03

1 .E-04

1 .E-05

Average CFU per carrier for all carriers tested:

Comments:

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Date Revised 11-30-09
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16.7

Sporicidal Activity of Disinfectants Test: Hydrochloric Acid Resistance Test

Results Sheet

OPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:

Test Dale

Results (+ = Growth. 0 = No Growth)

Dale/Initials:

2 Minutes

5 Minutes

10 Minutes

20 Minutes

Carrier Tracking. No.:

Primary

Secondary

Carrier Viability Control Results*: Growth No Growth

Medium Control**: Growth No Growth

Comments:

REAGENT/MEDIA INFORMATION/Confirmed by:

Reagent/Media

Prep. No.

Reagent/Media

Prep. No.

*Place one of each type of inoculated carrier in a separate tube of Modified FTM.
**One tube of uninoculated Modified FTM.

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Date Revised 11-30-09
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16.8

Sporicidal Activity of Disinfectants Test: Test Information Sheet
OPP Microbiology Laboratory

PRODUCT INFORMATION/Confirmed by:

EPA Reg. No.

Test Dale

Name

Test Organism

Sample No.

Comments:

Lot No.

TEST PARAMETERS/Confirmed by:

Diluent

Diluent Used

Hardncss/Datc/Inil.

/ /

Organic Soil

As Prcparcd/Dalc/Inil.

/ /

Neutralized 1°)

Recovery Medium (2°)

Temperature (°C)

Chiller Unit Display

Test Tube Waterbath

Before: After:

Contact Time (minutes)

Type of Carriers

Lot # or Preparation #

TEST MICROBE INFORMATION/Confirmed by:

Test Microbe

Avg. CFU/Carricr

REAGENT/MEDIA INFORMATION/Confirmed by:

Reagent/Media

Prep. No.

Reagent/Media

Prep. No.

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16.9

Sporicidal Activity of Disinfectants Test: Time Recording Sheet for Carrier Transfers
OPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:

Test Dale

Carrier Type

Product Reg. No.

Product Name

Lot No.

Contact Timc(s)

Organism

Initials/date

Disinfectant
Tube No.

Carrier No.

Carrier Drop Start Time
(into the disinfectant)

Carrier Drop End Time
(into the neutralizer media)'

Carrier Transfer
(into secondary)

Clock

Timer

Clock

Timer

Start Time"

Comments:

1 The time when the last carrier is dropped into neutralizer tube.

2 The time at which carriers are started to be transferred into secondary subculture; taken from clock.

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16.10

Sporicidal Activity of Disinfectants Test: Results Form (1-30)
OPP Microbiology Laboratory

PRODUCT INFORMATION/Confirmed by:

EPA Reg. No.

Test Dale

Name

Test Organism

Sample No.

Carrier Type

Lot No.

Comments:

CARRIER INFORMATION (to be completed by Analyst)

Carriers

Analyst Dropping Carriers

1-30

TEST RESULTS

Dale Recorded/Initials

Primary Subculture / Secondary Subculture (carrier)'

Results Summary

Number of Carrier Sets with Growth

Number of Carrier Sets without Growth

Modifications/Comments: Record positive (+) or negative (0) results as indicated by the presence or absence of typical

growth.

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16.11

Sporicidal Activity of Disinfectants Test: Results Form (31-60)
OPP Microbiology Laboratory

PRODUCT INFORMATION/Confirmed by:

EPA Reg. No.

Test Dale

Name

Test Organism

Sample No.

Carrier Type

Lot No.

Comments:

CARRIER INFORMATION (to be completed by Analyst)

Carriers

Analyst Dropping Carriers

31-60

TEST RESULTS

Dale Recorded/Initials

Primary Subculture / Secondary Subculture (carrier)'

Results Summary

Number of Carrier Sets with Growth

Number of Carrier Sets without Growth

Modifications/Comments: Record positive (+) or negative (0) results as indicated by the presence or absence of typical

growth.

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16.12

Sporicidal Activity of Disinfectants Test: Test Controls Results Sheet
OPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:

EPA Reg. No.

SOP

MB-15

Name

Test Dale

Sample No.

Comments:

Lot No.

RESULTS

Date Read/Initials:

Testing Controls

Type of Controls

Tube #1

Tube #2

Tube #3

Media Controls:
Nculralizer Tubes

Media Controls:
FTM Tubes

Beginning

Middle

End

System Controls:

Primary:

Secondary:

Positive carrier control: Growth

No Growth

Comments: Record results as growth (+) or no growth (0)

REAGENT/MEDIA INFORMATION/Confirmed by:

Reagent/Media

Prep. No.

Reagent/Media

Prep. No.

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16.13

Sporicidal Activity of Disinfectants Test: Microbe Confirmation Sheet
OPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:

EPA Reg. No.

Test Dale

Name

Test Organism

Sample No.

Comments

Lot No.

Source:
Tube/Plate ID

Dale/Initials

Slain
Results'

Media Information

Results

Type

Prep. No.

Inc. Time/
Temp.

Date/
Initials

Colony
Characteristics

Vitck ID
(if applicable)-

* GPR = gram positive rods, GPC = gram positive cocci, GNR = gram negative rods

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16.14

Sporicidal Activity of Disinfectants Test: Neutralization Confirmation Assay

Information Sheet

OPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:

EPA Reg. No.

SOP(s)

Product Name

Test Date

Product Sample No.

Neulralizer

Product Lot No.

Comments:

Expiration Date

TEST PARAMETERS/Confirmed by:

Diluent

Diluent Used

Hardncss/Datc/Inil.

/ /

Organic Soil

As Preparcd/Dalc/Inil.

/ /

Neulralizer (1°)

Recovery Medium (2°)

Temperature (°C)

Chiller Unit Display

Test Tube Watcrbath

Before: After:

Contact Time (minutes)

Type of Carriers (unseeded)

Lot # or Preparation #

TEST MICROBE INF ORMATION/Confirmed by:

Test Microbe

Org. Control No.

REGENT/MEDIA INF ORMATION/Confirmed by:

Reagent/Media

Prep No.

Reagent/Media

Prep No.

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16.15

Sporicidal Activity of Disinfectants Test: Neutralization Confirmation Assay

Results Form

OPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:

EPA Reg. No.

Test Dale

Product Name

Nculralizcr

Sample No.

Comments:

TEST RESULTS*: Date Recorded/Initials:

Treal me nls/Conlrol s

Inoculum Dilutions'

1 X 10"6

1 x 10"7

1 x 10"8

Ncutralizcr-Primary Subculture Treatment

Secondary Subculture Media Treatment (with Carrier)

Nculralizcr Inoculated Control

Subculture Media Inoculated Control

Nculralizcr Uninoculalcd Control Tube

Subculture Media Uninoculalcd Control Tube

*+ = growth, 0 = no growth

Reflects 100 |iL added to each tube from the 10~5, 10~6, and 10"7 dilution tubes, respectively.

SUMMARY OF RESULTS: Date/Initials:

Bacteriostatic Effect Observed?

Yes No

Comments:

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SOP No. MB-15-02
Date Revised 11-30-09
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16.16

Sporicidal Activity of Disinfectants Test: Neutralization Confirmation Assay Time Recording Sheet for

Carrier Transfers

OPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:

Test Dale

EPA Reg. No.

Product Name

Sample No(s).

Organism(s)

Neutralize r(s)

Carrier Type

Initials/dale

Disinfectant.
Tube No.

Carrier No.

Carrier Drop Start Time for carriers
(into the disinfectant)

Carrier Drop End Time for carriers
(into the nculralizcr)

Carrier Transfer
(into secondary media)

Clock

Timer*

Clock

Timer

Start Time1

Comments:

* = ±5 seconds
1= Taken from clock

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Date Revised 11-30-09
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16.17

Sporicidal Activity of Disinfectants Test: Neutralization Confirmation Assay
Serial Dilution/Plating Tracking Form
OPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:

EPA Reg. No.

Test Dale

Name

Neutrali/.cr(s)

Sample No.

Organism Control #

Confirmed bv:

Dilution Tube

10-1

10"3

10"4

10"5

10"6

10"7

Starling volume of diluent

9mL

9 mL

Volume added to serial dilution lube (1 mL)

1 mL

Volume plated (0.1 mL = 100 j.iL)

N/A

0.1 mL

Final dilution (used for calculations)*

N/A

10"6

10"7

10"8

Number of plates per dilution

N/A

Plating medium

Comments:

REGENT/MEDIA INFORMATION/Confirmed by:

Reagent/Media

Prep No.

Reagent/Media

Prep No.

16.18

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Sporicidal Activity of Disinfectants Test: Neutralization Confirmation Assay
Inoculum Enumeration Form
OPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:

EPA Reg. No.

Test Dale

Name

Organism

Sample No.

RESULTS: Date/Initials:

Plating Method

CFU per Dilution Plate

Average
CFU per niL

Dilution*

Plate 1

Plate 2

1 x 10"6

1 x 10"7

1 X 10"8

TNTC = Too Numerous To Count

Comments:*Reflects the volume plated (100 |iL).

REGENT/MEDIA INF ORMATION/Confirmed bv:

Reagent/Media

Prep No.

Reagent/Media

Prep No.

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Attachment 1

Culture Initiation and Stock Culture Generation Flow Chart for B. subtilis

® Rehydrate ampule.

Ampule

® Transfer entire rehydrated
pellet to TUBE A.

TUBE A

(vre-incubation)

TSA or NA

Gram VITEK
Stain

Incubate

TUBE A

(post-incubation)

CULTURE INITIATION

® Obtain lyophilized cultures annually from ATCC. Using a tube containing 5-6 mL of NB, aseptically withdraw
0.5 to 1.0 mL and rehydrate the pellet for B. subtilis.

© Aseptically transfer the entire rehydrated pellet back into the original tube of nutrient broth designated as "TUBE
A." Mix well. Use suspension in TUBE A for CULTURE ID & QUALITY CONTROL. Incubate TUBE A for B.
subtilis for 24 hours at 30 ± 1°C.

CULTURE ID & QUALITY CONTROL

CD Using a loopful of rehydrated suspension from TUBE A, streak for isolation on duplicate plates (NA or TSA).
Incubate plates at 30 ± 1°C for 24 hours. Record results on the Test Microbe Confirmation Sheet.

STOCK CULTURE GENERATION

© Using the 24 ± 2 hour TUBE A broth culture: initiate stock cultures by streak-inoculating six NA slants. Incubate
the slants at 36 ± 1°C for 24 ± 2 hours. Record all manipulations on the Organism Culture Tracking Form.

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