US Environmental Protection Agency
Office of Pesticide Programs

Office of Pesticide Programs

Microbiology Laboratory

Environmental Science Center, Ft. Meade, MD

Standard Operating Procedure for

OECD Quantitative Method for Evaluating Bactericidal Activity
of Microbicides Used on Hard, Non-Porous Surfaces

SOP Number: MB-25-02

Date Revised: 10-17-13


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SOP No. MB-25-02
Date Revised 10-17-13
Page 1 of 20

SOP Number

MB-25-02

Title

OECD Quantitative Method for Evaluating Bactericidal Activity of
Microbicides Used on Hard, Non-Porous Surfaces

Scope

To provide a quantitative procedure for testing the bactericidal activity
of liquid antimicrobial substances against Staphylococcus aureus and
Pseudomonas aeruginosa designed for use on hard, non-porous
surfaces. This SOP is based on OECD Guidance Document dated
June 21, 2013 (see ref. 15.1). Details for performing the method with
Mycobacterium terrae are provided in Attachment 2.

Application

This method measures log reduction (LR) as the quantitative measure
of efficacy for liquid disinfectants on a hard nonporous surface.





Approval Date

SOP Developer:



Print Name:

SOP Reviewer



Print Name:

Quality Assurance Unit



Print Name:

Branch Chief



Print Name:

Data SOP issued:



Controlled copy number:



Date SOP withdrawn:




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SOP No. MB-25-02
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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

4

10.

CAUTIONS

4

11.

SPECIAL APPARATUS AND MATERIALS

4

12.

PROCEDURE AND ANALYSIS

7

13.

DATA ANALYSIS/CALCULATIONS

12

14.

FORMS AND DATA SHEETS

12

15.

REFERENCES

13


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1. Definitions

Additional abbreviations/definitions are provided in the text.

1.	Eluate = recovered eluent that contains the test organism

2.	Eluent = any liquid that is harmless to the test organism(s) and that is
added to a vial containing the carrier to recover the test organism.

3.	Stock culture = frozen culture used to prepare the test culture

4.	Final test suspension = the test suspension with the addition of the soil
load

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 the test substance.

3. Personnel
Qualifications
and Training

1.	A reference standard (e.g., predetermined concentrations of sodium
hypochlorite) may be used to check method performance and analyst
proficiency.

2.	Refer to SOP ADM-04, OPP Microbiology Laboratory Training.

4. Instrument
Calibration

Refer to SOP EQ-01, EQ-02, EQ-03, EQ-04 and EQ-05 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.	Prior to efficacy testing, verify neutralizer effectiveness using the
procedure outlined in SOP MB-26 (Neutralization of Microbicidal
Activity using the OECD Quantitative Method).

2.	During testing, do not process carriers where the test substance runs off of
the carrier; replace with new carrier(s) and vial(s).

8. Non-
conforming
Data

1. The mean logio density for control carriers (referred to as the Test Logio
Density (TestLD)) falls outside the specified range. The TestLD for control
carriers must be between 0.5 and 1.5 logs higher than the performance
standard.

a. The TestLD must be at least 4.5 (corresponding to a geometric mean
density of 3.2 x 104) and not above 5.5 (corresponding to a geometric
mean density of 3.2><105); a TestLD below 4.5 or above 5.5
invalidates the test, except for two retesting scenarios (outlined in the
study protocol).


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9. Data

Management

Data will be archived consistent with SOP ADM-03, Records and Archives.

10. Cautions

Avoid extended soaking of the carriers in water or detergent and prolonged
rinsing to reduce risk of corrosion or rusting.

11. Special

Apparatus and
Materials

1.	Test microbes: Pseudomonas aeruginosa (ATCC #15442),

Staphylococcus aureus (ATCC #6538)

2.	Culture media. Refer to SOP MB-10, Media and Reagents Used in

Microbiological Assays, for QC of media and reagents.

a.	Cryoprotectant solution (TSB with 15% glycerol). Suspend 7.5 g
tryptic soy broth in 212.5 mL de-ionized water. Add 37.5 g glycerol
and stir, boil to homogenize. Dispense into bottles and autoclave for
15 minutes at 121C.

b.	Tryptic soy agar (TSA). Prepare according to manufacturer's
instructions.

c.	TSB. Prepare according to manufacturer's instructions.

d.	Synthetic broth (SB). Prepare according to manufacturer's
instructions.

3.	Reagents

a.	Neutralizer in eluent. The neutralizer is sterilized with or aseptically
added to PBS with Tween-80 prior to use. The final concentration of
Tween-80 in the eluent is typically 0.1% v/v. When the neutralizer is
heat-sensitive, prepare sterile double-strength solutions of both the
neutralizer and the Tween-80 in PBS and mix them in equal volumes.
Non-PBS based neutralizers may be used as deemed necessary.

b.	Phosphate buffer (PB) stock solution. Dissolve 34.0 g of potassium
dihydrogen phosphate (KH2PO4) in 500 mL de-ionized water.

Adjust pH to 7.2  0.2 with 0.1 N NaOH or 0.1 N HC1 and bring to
1000 mL with de-ionized water. Alternative phosphate buffers with
the same pH may be used (e.g., commercially prepared 10X PBS
solution).

c.	Phosphate buffered saline (PBS). Add 1.25 mL of phosphate buffer
stock solution and 8.75 g of NaCl to a volumetric flask; fill with de-
ionized water to the 1000 mL mark and mix. A pH of approximately
7.0  0.5 is desirable. Sterilize by filtration or autoclaving.
Alternative PBS formulations with the same pH may be used (e.g.,
dilute commercially prepared 1 OX PBS solution to IX using de-
ionized water).


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d.	Soil load. The recommended default standard soil load to be
incorporated in the test suspension is a mixture of the following stock
solutions in PBS:

i.	BSA: Add 0.5 g bovine serum albumin (BSA) to 10 mL of
PBS, mix and pass through a 0.2 |im pore diameter membrane
filter, aliquot and store at -20  5C.

ii.	Yeast Extract: Add 0.5 g yeast extract to 10 mL of PBS, mix,
and pass through a 0.2 |im pore diameter membrane filter,
aliquot and store at -20  5C.

iii.	Mucin: Add 0.04 g mucin (bovine or porcine) to 10 mL of
PBS, mix thoroughly until dissolved, and autoclave (15
minutes at 121C), aliquot and store at -20  5C.

The stock solutions of the soil load are single use only and
should not be refrozen once thawed; store up to one year at
-20  5C.

e.	Test substance. Refer to SOP MB-22, Disinfectant Sample
Preparation.

f.	Test substance diluent. The test substance diluent is 375 ppm hard
water (unless otherwise specified). Adjust the recipe for volumes
other than 1L.

i.	Prepare Solution A by dissolving 19.84 g anhydrous
magnesium chloride (or 42.36 g MgCh 6H20) and 46.24 g
anhydrous calcium chloride (CaCh) in de-ionized water and
dilute to 1000 mL. Sterilize by membrane filtration. Store
the solution in the refrigerator for no longer than one month.

ii.	Prepare Solution B by dissolving 35.02 g sodium bicarbonate
(NaHCOs) in water and dilute to 1000 mL. Sterilize by
membrane filtration. Store the solution in the refrigerator for
no longer than one week.

iii.	To prepare 1L of 375 ppm hard water, place 600-700 mL of
de-ionized water in a 1000 mL volumetric flask and add 6.0
mL of Solution A and then 8.0 mL of Solution B. Mix and
add water to the flask to reach 1000 mL. The pH of the hard
water should be 7.0  0.2 at room temperature. If necessary,
adjust the pH by using 1 N NaOH or 1 N HC1. Ensure
sterility of hard water prior to use in efficacy testing.

iv.	Prepare the hard water under aseptic conditions and use
	within 24 h of preparation. Measure the hardness of the water


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using a water hardness test kit or other suitable titration
method on the day of the test.

NOTE: The target hardness expressed as mg/L calcium
carbonate (CaCOs) is 375 mg/L +5%/-10% (338-394 ppm).
Other levels of water hardness may be used as appropriate.

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

h.	Tween-80 (polysorbate 80)

4. Apparatus

a.	Calibrated positive displacement pipettes (e.g., 10 |iL) for carrier
inoculation.

b.	Micropipettes (e.g., 200 |iL) for deposition of test substance on
carrier.

c.	Carriers: Disks (1 cm in diameter) made from 0.7 mm thick sheets of
brushed and magnetized stainless steel (AISI #430). The top of the
disk is brushed and has rounded edges; only the top is visually
screened and inoculated. Carriers are single-use only. See
Attachment 3 for complete specifications.

d.	Bottle-top dispensers, squirt bottles, pre-measured volumes in tubes,
or pipettes to assist in the rinsing of vials and filters.

e.	Forceps, straight or curved, non-magnetic, disposable with smooth
flat tips to handle membrane filters, appropriate to pick up the
carriers for placement in vials.

f.	Magnet strong enough to hold the carrier in place in the vial while
the liquid is being poured out of it for membrane filtration.

g.	Membranes (polyethersulfone) for filter sterilization and recovery, 47
mm diameter and 0.2 |im pore size. Filtration units (reusable or
disposable) may be used.

h.	Spectrophotometer; calibrated.

i.	Sterile vials (plastic or comparable) to hold test carriers: flat bottom
and wide-mouth to accommodate addition and removal of the
carriers, for holding inoculated carriers to be exposed to the test
substance and for accommodating neutralizer/eluent. Suitable vials
should be at least 25 mm in neck diameter and hold at least 20 mL of
liquid. Transparent vials are more desirable to facilitate application

	of 50 [iL test substance or PBS and to allow for the viewing of the


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carriers for removal of inoculum,
j. Certified timer

k. Desiccator with desiccant (e.g., CaCCb)

1. Vacuum source: in-house line or suitable vacuum pump

5. Hach kit. Total hardness, 10 to 4,000 mg/L as CaCC>3 (Hach Digital
Titrator Method 8213)

12. Procedure and
Analysis

12.1 Preparation
and

sterilization of

carriers

Visually check the brushed top surface of the carriers (with the
rounded edge) for abnormalities (e.g., rust, chipping, deep striations)
and discard if observed. Record physical screening of carriers on
form provided in section 14.

Soak visually screened carriers in a suitable detergent solution (e.g.,
Liquinox) free from any antimicrobial activity for 2-4 hours to
degrease and then rinse thoroughly in distilled water.

Prior to sterilization, place up to 20 clean dry carriers on a piece of
filter paper inside the bottom surface of a glass Petri dish (150 mm in
diameter). Cover the Petri dish with its lid and sterilize. After
sterilization, carriers may be transferred to sterile Petri dishes without
filter paper for inoculation.

12.2 Preparation of
test organisms

d.

e.

f.

Refer to Attachment 1 for preparation of the frozen stock cultures for
Pseudomonas aeruginosa (ATCC #15442) and Staphylococcus
aureus (ATCC #6538).

Defrost a cryovial; defrost rapidly to avoid loss in the viability of the
preserved cells (e.g., expose to running water to thaw). Each
cryovial is for single use only.

Pseudomonas aeruginosa: Add 100 [j,L of defrosted stock culture to
10 mL of synthetic broth, briefly vortex mix and incubate for 18-24 h
at 36  1C.

Staphylococcus aureus: Add 100 [j,L of defrosted stock culture to 10
mL TSB, briefly vortex mix and incubate for 18-24 h at 36  1C.

In addition, inoculate an agar plate (TSA with 5% sheep blood, BAP)
with a loopful of the test culture and streak for isolation. Incubate
plate with the test culture and examine for purity. Record results of
purity check on microbe tracking sheet (see section 14).

Following incubation, use the broth cultures to prepare a test	


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suspension for each organism.

g.	For Pseudomonas aeruginosa, inspect culture prior to harvest;
discard if pellicle has been disrupted (fragments in culture). Remove
visible pellicle on surface of medium and around associated interior
edges of the tube by pipetting or with vacuum suction. Using a
serological pipette, withdraw the remaining broth culture (approx. 7-
8 mL) avoiding any sediment on the bottom of the tube.

h.	Centrifuge the 18-24 h broth cultures as described below to achieve
the desired level of viable cells on the dried carrier.

i.	Centrifuge at -5,000 gN for 20  5 minutes and re-suspend the pellet
in 10 mL PBS. Dilute or concentrate the culture appropriately to
achieve the target carrier counts.

Note: Remove the supernatant without disrupting the pellet. For S.
aureus, disrupt the pellet using vortexing or repetitive
tapping/striking against a hard surface to disaggregate the pellet
completely prior to re-suspending it in 10 mL. If necessary, add 1
mL of PBS to the pellet to aid in the disaggregation.

j. To achieve carrier counts in the range of 4.5 to 5.5 logs for S. aureus,
resuspend the pellet in 10 mL PBS. If necessary, further dilute the
culture (e.g., 1:25, 40 |iL S. aureus + 960 |iL PBS) prior to preparing
the inoculum with the soil load.

k. To achieve carrier counts in the range of 4.5 to 5.5 logs fori5.

aeruginosa, resuspend the pellet in 10 mL PBS. Use this culture to
prepare the inoculum with the soil load.

1. Optical density/absorbance (at 650nm) may be used as a tool to
monitor/adjust the re-suspended test suspension.

12.3 Preparation of
the final test
suspension
with soil load

a.	Vortex the test suspension for 10-30 seconds or until re-suspended
(no more than 60 seconds) to evenly distribute the cells.

b.	To obtain 500 [xL of the final test suspension vortex each component
and combine the following:

i.	25 |iL BSA stock

ii.	35 |iL yeast extract stock

iii.	100 |iL mucin stock

iv.	340 |iL test suspension

12.4 Inoculation
and drying of

a. Following the addition of the soil load, vortex the final test
suspension for 10 seconds.


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carriers

b.	Inoculate the number of carriers required for the test plus extras.
Withdraw 10 [xL of the final test suspension with a calibrated
positive-displacement pipette and deposit it at the center of a clean
sterile screened carrier (a maximum of 20 carriers per Petri dish);
avoid contact with carrier and do not spread the test suspension with
the pipette tip. For consistency, use the same pipette tip to inoculate
each batch of carriers. Discard any inoculated carrier where the final
test suspension has run over the edge.

c.	Inside a biological safety cabinet, transfer the Petri dish with the
inoculated carriers into a desiccator and remove the lid of the Petri
dish. Close the desiccator and check that it is properly sealed.
Evacuate the desiccator using a vacuum source to achieve 20-25
inches mercury (508-635 torr; 677-847 mbar; 68000-85000 Pascal).

d.	Hold the inoculated carriers in the evacuated desiccator at 20-25C
for 60  10 minutes. If carriers are not dry within the specified time,
check the desiccator system (e.g., refresh desiccant if necessary). Do
not use carriers that are visibly wet.

12.5 Exposure of
the dried
inoculum to
the test
substance or
PBS (control
counts)

a.	Evaluate four control carriers and three treated carriers for each test
substance tested (one test organism and contact time/temperature
combination) unless specified otherwise. One set of control carriers
may be used for evaluating multiple test substances against one
organism on one test day.

b.	Use a certified timer to ensure that each carrier receives the required
exposure time (e.g., 5 min  3 sec).

c.	Using sterile forceps transfer each dried carrier with the inoculated
side up to a flat-bottom vial and cap the vial. Repeat until all carriers
are transferred.

Note: Prior to testing, inoculated carriers can be stored at 20-25C
for up to approximately one hour after drying.

d.	In a timed fashion, deposit 50 [xL of the test substance, equilibrated
to 20-25C, over the dried inoculum on each test carrier, ensuring
complete coverage, at predetermined staggered intervals. Use a new
tip for each carrier; do not touch the pipette tip to the carrier surface.
Do not cap the vials.

e.	Hold the test carriers at 20-25C for the selected contact period.

f.	Treat control carriers last - each control carrier receives 50 [j,L
phosphate buffered saline (PBS), equilibrated to 20-25C, instead of
the test substance. Hold the control carriers at 20-25C for the


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contact period.

12.6 Neutralization
of test

substance and
elution of test
organisms

The neutralizer for the control carriers is the same as that for the treated
carriers.

a.	Within  3 seconds of the end of the contact period, add 10 mL of
neutralizer at room temperature to each vial in the specified order,
including controls, according to the predetermined schedule (the
neutralized vial is documented as the 10 dilution). Briefly (2-3 sec)
vortex each vial following the addition of the neutralizer.

b.	Following the neutralization of the entire set of carriers, vortex each
vial for 30  5 seconds at high speed to recover the inoculum; ensure
that the carrier is vortexing along with the liquid in the vial. If
possible, visually examine each carrier and, in case of incomplete
elution, perform further vortexing to remove inoculum. Do not
remove the carrier from the vial.

12.7 Dilution and
recovery

a.	Initiate dilutions within 30 min at room temperature after
neutralization. Initiate filtration within 30 min of preparing the
dilutions.

b.	If necessary, serially dilute the eluate from the 10 dilution (vial with
the carrier) prior to filtration.

c.	Filter all samples, control and treated, through 0.2 |im PES
membrane filters. Direct plating is not allowed.

d.	Pre-wet each membrane filter with approximately 10 mL of sterile
PBS.

Note: Use separate membrane filters for each eluate; however, the
same filtration unit may be used for processing eluates from a given
carrier starting with the most dilute sample first.

e.	For the eluate in the 10 dilution (10 mL, or 9 mL if dilutions were
made), vortex the vial for 5 sec and holding a magnet at the bottom
of the vial to keep the carrier in place, pour the eluate into the filter
unit.

f.	Rinse the vial with -20 mL of PBS, vortex for 5 sec and keeping
magnet in place, pour the wash into the same filter unit. Repeat this
step one more time. For dilution tubes, rinse each tube once with
-10 mL of PBS and briefly vortex.

g.	Swirl the contents of the filter unit and apply the vacuum.

Note: If desired, the vacuum may be left on for the duration of the
filtration process beginning with 12.7d.


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h.	With the vacuum on, rinse the inside surface of the funnel unit with
an additional -40 mL PBS.

i.	Aseptically remove the membrane filter and place on the recovery
medium (TSA). Avoid trapping any air bubbles between the filter
and the agar surface.

j. Incubate the plates at 36  1C for 24-48 h.

k. The elution steps for control carriers are the same as for the test
carriers; however, eluates from control carriers will always require
10-fold dilutions to provide countable filters (up to 200 CFU/filter).

12.8 Recording
results

a.	Record results as CFU per carrier after 24-48 hrs of incubation.
Colony counts in excess of 200 should be recorded as Too Numerous
to Count (TNTC). If no colonies are present, record as zero.

b.	Inspect the growth on the filters for purity and typical characteristics
of the test microbe (see Table 1). Gram stain one representative
colony per carrier set with growth for treated and controls. Record
results on the Test Microbe Confirmation Sheet. Isolation streaks
may be performed for additional verification of the test organism.

Table 1. General diagnostic characteristics fori5, aeruginosa and S. aureus
(seeref. 15.7 and 15.8)

Aspect

P. aeruginosa*

S. aureus*

Gram slain
reaction

Negative

Positive

Mannitol Salt

Agar

No Growth

Circular, small, yellow colonies,
agar turning fluorescent yellow

Cclrimidc Agar

Circular, small, initially opaque,
turning fluorescent green over
time; agar fluorescent yellowish
green

No Growth

Blood agar (BAP)

Flat, opaque to off-white, round
spreading (1), metallic sheen,
slightly beta hemolytic

Small, circular, yellow or white,
glistening, beta hemolytic

Typical Microscopic Characteristics

Cell appearance

Straight or slightly curved rods,
single polar flagella, rods formed in
chains

Spherical, occurring singly, in pairs
and tetrads, sometimes forming
irregular clusters

*After 242 hours

(1) Test organism may display three colony types: a) circular, undulate edge, convex, rough
and opaque; b) circular, entire edge, convex, smooth and translucent; c) irregular, undulate


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edge, convex, rough, spreading, and translucent. Pyocyanin is not produced.

13. Data Analysis/
Calculations

1.	All colony counts are recorded and used in calculations to determine log
reductions.

2.	To calculate the CFU/carrier use the following equation:

f CFU Jr 10 ' +OT' f lirL, where 10 and 10* are the dilutions

[ (axlO-y) + (bxlQ-z) J

filtered, "a" and "b" are the volumes filtered at each dilution (typically 9
or 10 mL), and "c" is the volume of medium originally in the vial with the
carrier (10 mL). The calculations must account for the volume filtered.

a.	When TNTC values are observed for each dilution filtered, substitute
200 for the TNTC at the highest (most dilute) dilution and scale up
accordingly for the calculations.

b.	When zeros are observed for each dilution filtered, substitute 0.5 for
the zero at the lowest (least dilute) dilution and scale up accordingly
for the calculations.

3.	Calculate the log density of each carrier by taking the logio of the density
(per carrier).

4.	Calculate the mean logio density across treated carriers.

5.	Calculate the mean logio density across control carriers.

6.	Calculate the logio reduction (LR) for treated carriers:
logio reduction = mean logio control - mean logio treated

7.	For a set of 3 treated carriers: when the 10 dilution (the contents of the
vial with the carrier) is filtered either by itself or in addition to other
dilutions and the data for each carrier result in zeros for each dilution
filtered, 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: Procedures for Maintenance of Bacterial Cultures -
Preparation of Frozen Stock Cultures

2.	Attachment 2: Procedures for Maintenance of Mycobacterium terrae
Culture - Preparation of Frozen Stock Culture and Test Culture

3.	Attachment 3: Carrier Specifications

4.	Attachment 4: Confirmation Flow Charts for S. aureus and P. aeruginosa

5.	Test Sheets. Test sheets are stored separately from the SOP under the
following file names:

Physical Screening of Carriers Record Form MB-25-02 Fl.docx


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OECD Method for Bactericidal Activity: MB-25-02 F2.docx
Organism Culture Tracking Form

OECD Method for Bactericidal Activity: Test MB-25-02 F3.docx
Microbe Confirmation Sheet (Quality Control)

OECD Method for Bactericidal Activity: Test MB-25-02 F4.docx
Information Sheet

OECD Method for Bactericidal Activity: Time MB-25-02 F5.docx
Recording Sheet

OECD Method for Bactericidal Activity: Serial MB-25-02 F6.docx
Dilution Plating/Tracking Form

OECD Method for Bactericidal Activity: Results MB-25-02 F7.docx
Sheet

OECD Method for Bactericidal Activity: Test MB-25-02 F8.docx
Microbe Confirmation Sheet

15. References

1.	Guidance Document on Quantitative Methods for Evaluating the Activity
of Microbicides Used on Hard Non-Porous Surfaces (June 21, 2013)

2.	ASTM Standard E2197-11, 2011, "Standard Quantitative Disk Carrier
Test Method for Determining the Bactericidal, Virucidal, Fungicidal,
Mycobactericidal and Sporicidal Activities of Liquid Chemical
Germicides," ASTM International, West Conshohocken, PA.

3.	Package Insert - Gram Stain Kit and Reagents. Becton, Dickinson and
Company. Part no. 882020191JAA. Revision 07/2011.

4.	Package Insert - Catalase Reagent Droppers. Becton, Dickinson and
Company. Part no. L001237. Revision 06/2010.

5.	Package Insert - Staphaurex Plus*. Remel. Part no. R30950102. Revised
11/23/07.

6.	Package Insert - Oxidase Reagent Droppers. Becton, Dickinson and
Company. Part no. LOO 1133. Revision 06/2010.

7.	Krieg, Noel R. and Holt, John G. 1984. Bergey's Manual of Systematic
Bacteriology Volume 1. Williams & Wilkins, Baltimore, MD.

P. aeruginosa p. 164.

8.	Sneath, P., Mair, N., Sharpe, M.E., and Holt, J. eds. 1986. Bergey's
Manual of Systematic Bacteriology Volume 2. Williams & Wilkins,
Baltimore, MD. S. aureus p. 1015.


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Attachment 1

Procedures for Maintenance of Vegetative Bacterial Cultures - Preparation of Frozen Stock
Cultures

A1. Preparation of Frozen Stock Cultures. Refer to SOP MB-02 for establishment of the

organism control number.

a.	Initiate new stock cultures from lyophilized cultures of Pseudomonas aeruginosa
and Staphylococcus aureus from ATCC at least every 18 months.

b.	Open ampule of freeze dried organism per manufacturer's instructions. Using a
tube containing 5-6 mL of TSB, aseptically withdraw 0.5 to 1.0 mL and rehydrate
the lyophilized culture. Aseptically transfer the entire rehydrated pellet back into
the original tube of broth. Mix thoroughly. Incubate broth culture at 36  1C for
24  2 hours.

c.	After incubation, streak a loopful of the suspension on TSA to obtain isolated
colonies. Incubate the plates for 18-24 h at 36  1C. Refer to section A2 for QC
of stock cultures.

d.	Select 3-5 isolated colonies of the test organism and re-suspend in 1 mL of TSB.
For S. aureus, select only golden yellow colonies. Multiple phenotypes are present
fori5, aeruginosa - the stock culture should be representative of all phenotypes
present on the streak isolation plate. Spread plate 0.1 mL of the suspension on each
of 6-10 TSA plates. Incubate the plates for 18-24 h at 36  1C.

e.	Following the incubation of the agar plates from Aid, place approximately 5 mL
sterile cryoprotectant solution on the surface of each plate. Re-suspend the growth
in the cryoprotectant solution using a sterile spreader without damaging the agar
surface. Aspirate the suspension from the plate with a pipette and place it in a
sterile vessel large enough to hold about 30 mL. Repeat the growth harvesting
procedure with the remaining plates and continue adding the suspension to the
vessel (more than 1 tube may be used if necessary). Mix the contents of the
vessel(s) thoroughly; if more than 1 vessel is used, pool the vessels prior to
aliquoting culture.

f.	Immediately after mixing, dispense 0.5-1 mL aliquots of the harvested suspension
into cryovials; these represent the frozen stock cultures.

g.	Store the cryovials at -70C or lower for a maximum 18 months then reinitiate with
a new lyophilized culture.


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SOP No. MB-25-02
Date Revised 10-17-13
Page 15 of 20

A2. QC of Stock Cultures.

a.	Conduct QC of the pooled culture concurrently with freezing. Streak a loopful on a
plate of BAP. In addition, streak a loopful onto both MSA and Cetrimide. Incubate
all plates at 36  1C for 24  2 hours.

b.	Following the incubation period, record the colony morphology as observed on the
BAPs and selective media plates (including the absence of growth) and Gram stain.
See Table 1 for details on cell and colony morphology, colony characteristics on
selective media, and stain reactions.

c.	For each organism, perform a Gram stain (refer to 15.3) from growth taken from the
BAPs according to the manufacturer's instructions. Observe the Gram reaction by
using brightfield microscopy at 1000X magnification (oil immersion).

d.	For additional biochemical and antigenic analyses, refer to 15.4-15.5 for S. aureus
and 15.6 fori5, aeruginosa.

e.	Record all confirmation results on the Test Microbe Confirmation Sheet (Quality
Control) (see section 14).


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SOP No. MB-25-02
Date Revised 10-17-13
Page 16 of 20

Attachment 2

Procedures for Maintenance of Mycobacterium terrae Culture - Preparation of Frozen Stock
Culture and Test Culture

A1. Materi al s and reagents

a.	Growth medium. Middlebrook 7H9 broth containing glycerol and 10% ADC
Enrichment (MADC, 4.7 g Middlebrook 7H9 broth powder, 150 mL glycerol, 750
mL water, sterilize in autoclave. Add under aseptic conditions, 100 mL
Middlebrook ADC enrichment and then add sterilized water up to 1,000 mL. The
pH of the medium should be 6.60.2).

b.	Recovery medium. Middlebrook 7H11 Agar.

c.	Sterile Bijou bottles. Glass with aluminum caps; capacity 5-7 mL, with 10 glass
beads (3-5 mm in diameter).

A2. Preparation of Frozen Stock Cultures. Refer to SOP MB-02 for establishment of the

organism control number.

a.	Initiate new stock cultures from lyophilized cultures of Mycobacterium terrae
(ATCC 15755) from ATCC at least every 18 months. Open ampule of freeze dried
organism per manufacturer's instructions. Using a tube containing 6 mL of
Middlebrook 7H9 Broth with 10% ADC enrichment (MADC), aseptically withdraw
1.0 mL and rehydrate the lyophilized culture. Aseptically transfer the entire
rehydrated pellet back into the original tube of broth. Mix thoroughly.

b.	Spread 0.1 mL of the test organism suspension onto approximately 6-10 M7H9 or
M7H1 lagar plates. Incubate for 20-22 days at 361C. Refer to section A3 for QC
of frozen stock cultures.

NOTE: Each plate will yield -10 mL of harvested suspension and consequently
nine to ten cryovials, each containing >1 mL of frozen stock culture.

c.	At the end of the incubation period, add 5 mL MADC to the surface of each agar
plate. Re-suspend the cells in MADC using a sterile spreader without damaging the
agar surface. Aspirate the suspension from the plate with a pipette and place it in a
sterile vessel large enough to hold about 30 mL. Repeat by adding another 5 mL of
MADC to the agar plates, re-suspend the cells, aspirate the suspension and pool
with the initial cell suspension. Repeat the growth harvesting procedure with the
remaining plates and continue adding the suspension to the vessel (more than 1
vessel may be used if necessary). Mix the contents of the vessel(s) thoroughly; if
more than 1 vessel is used, pool the vessels prior to aliquoting the culture.

d.	Immediately after mixing, dispense >1 mL aliquots of the harvested suspension into
separate cryovials; these represent the frozen stock cultures.

e.	Store the cryovials at -70C or lower for a maximum of 18 months (from the date of
harvesting/freezing).


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SOP No. MB-25-02
Date Revised 10-17-13
Page 17 of 20

A3. QC of Stock Cultures.

a.	Conduct QC of the pooled culture concurrently with freezing. Streak a loopful on
M7H9 or M7H11 agar and incubate at 36  1C for 20-22 days.

b.	Following the incubation period, record the colony morphology as observed on the
plates. M. terrae typical colony morphology includes irregular margins, and
appears rough, erose, buff, opaque, and umbonate.

c.	Record all confirmation results on the Test Microbe Confirmation Sheet (Quality
Control) (see section 14).

A4. Preparation of test organisms from frozen stock cultures

a.	Defrost a cryovial; defrost rapidly to avoid loss in the viability of the preserved cells
(e.g., expose to running water to thaw). Each cryovial is for single use only.

b.	Add 1 mL thawed culture to a flask of 100 mL MADC. Inoculate 1-2 flasks using a
separate cryovial for each flask and incubate at 361C for 20-22 days.

c.	Aliquot 25 mL portions of the 20-22 day-old MADC broth culture into each of 2-50
mL conical screw cap tubes and centrifuge at 10,000g y for 205 minutes.

d.	Carefully remove the supernatant and re-suspend each pellet in 25 mL sterile
distilled/de-ionized (DI) water.

e.	Centrifuge the tubes a second time at 10,000g v for 205 minutes. After
centrifuging, re-suspend the pellets in a total of 5 mL sterile DI water (1/10 of the
starting volume), pool, and place in a bijou bottle (or equivalent) with 10 glass
beads; vortex for 5 min.

f.	The approximate titer of each freshly prepared and homogenized microbial test
suspension may be estimated spectrophotometrically at 650 nm, based on a standard
curve specific to the test organism.

g.	For mycobacterial claims, based on a potential performance standard of a log
reduction of 4, control counts should be within 4.5-5.5 logs per carrier.

h.	Prior to inoculation of carriers, aseptically add the soil load.

A5. Refer to sections 12.3 through 12.7h for preparation of the inoculum with soil, carrier
inoculation, and exposure of the carriers to the test substance or control fluid.

A6. For recovery of M. terrae test culture from exposed and control carriers, use M7H11 agar
plates. Incubate plates at 361C for up to 21-28 days; however, monitor filters for growth
and assess the number of visible colonies beginning at 12-14 days. If no colonies are
visible at the end of 14 days of incubation, re-incubate the plates for an additional 7-14
days before recording the final colony counts.


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SOP No. MB-25-02
Date Revised 10-17-13
Page 18 of 20

Attachment 3
Carrier Specifications

	Ferritic stainless steel: Consist of chromium (17%) and iron and essentially nickel-free.

	AISI Type 430 (European equivalent name X6Crl7 and number 1.4016) belongs to Group 2,
which is the most widely used family of ferritic alloys.

	Dimensions: 1 cm (0.39370 inch) in diameter; 0.7 mm (0.27559 inch) thick.

	AISI 430 - ASTM A240; Japanese Industrial Standard (JIS) G4305; EN 10088-2

	No. 4 Finish (EN 10088-2 1J/2J): A ground unidirectional finish obtained with 150 grit
abrasive (AISI).

	Passivation: A soak in a mild acid bath for a few minutes to remove any impurities and
accumulated debris from the disk surface.

	Tumbling: To remove the punching burrs from the edges of the discs they are tumbled in a
barrel together with ceramic chips and a cleanser.	


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SOP No. MB-25-02
Date Revised 10-17-13
Page 19 of 20

Attachment 4

Confirmation Flow Charts for S. aureus and P. aeruginosa

S. aureus Identification


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SOP No. MB-25-02
Date Revised 10-17-13
Page 20 of 20

Attachment 4 (continued)

P. aeruginosa Identification

Not P. aeruginosa

P. aeruginosa


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