Standard Operating Procedure for AOAC Use Dilution Method for Testing Disinfectants SOP Number: MB-05-11 Date Revised: 08-06-13

US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
AO AC Use Dilution Method for Testing Disinfectants
SOP Number: MB-05-11
Date Revised: 08-06-13

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SOP No. MB-05-11
Date Revised 08-06-13
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SOP Number
MB-05-11
Title
AO AC Use Dilution Method for Testing Disinfectants
Scope
Describes the Use-dilution methodology (see 15.1) used to determine
the efficacy of disinfectants against Staphylococcus aureus,
Pseudomonas aeruginosa, and Salmonella enterica on hard surfaces.
Additional testing guidance is provided for Carbapenem Resistant
Klebsiella pneumonia - refer to Attachment 4 for details.
Application
The methodology described in this SOP is used to evaluate the
performance of liquid disinfectants against the prescribed test
microbes.


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Date SOP withdrawn: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-05-11
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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
5
13.
DATA ANALYSIS/CALCULATIONS
11
14.
FORMS AND DATA SHEETS
11
15.
REFERENCES
12

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1. Definitions
Abbreviations/definitions are provided in the text.
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 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.	Any disruption of the Pseudomonas aeruginosa pellicle resulting in the
dropping or breaking of the pellicle in culture before or during its removal
renders that culture unusable in the use-dilution test.
2.	Transferring the inoculated carriers into the disinfectant is a critical,
technique-sensitive step. False positives can result from transfer of live
organisms to sides of tubes due to contact or aerosol formation.
3.	Viscous test chemicals may result in a substantial amount of product
remaining on treated carriers following the contact time, which upon
transfer to the primary subculture medium (neutralizer) produces
cloudiness in the medium. This cloudiness may impact the recording of
results.
8. Non-
conforming
Data
1.	Sterility and/or viability controls do not yield expected results.
2.	The mean log density for control carriers falls outside the specified range.
Note: The prescribed minimum and maximum carrier counts also account
for the addition of 5% organic soil to the inoculum.
a.	The mean Test LD for carriers inoculated with S. aureus and P.
aeruginosa must be at least 6.0 (corresponding to a geometric mean
density of 1.0 x 106) and not above 7.0 (corresponding to a geometric
mean density of 1.0 x 107); a mean Test LD below 6.0 and above 7.0
invalidates the test, except for two retesting scenarios (outlined in the
study protocol).
b.	The mean Test LD for carriers inoculated with S. enterica must be at
least 5.0 (corresponding to a geometric mean density of 1.0 x 105)

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and not above 6.0 (corresponding to a geometric mean density of 1.0
x 106); a mean Test LD below 5.0 and above 6.0 invalidates the test,
except for two retesting scenarios (outlined in the study protocol).
3.	No contamination is acceptable in the test system.
4.	Management of non-conforming data will be specified in the study
protocol; procedures will be consistent with SOP ADM-07, Non-
Conformance Reports.
9. Data
Management
Data will be archived consistent with SOP ADM-03, Records and Archives.
10. Cautions
1.	There are time sensitive steps in this procedure including the use periods
of the inoculated carriers and the test chemical.
2.	Verify the volume of dilution blanks, neutralizer tubes, and subculture
tubes in advance and adjust accordingly.
11. Special
Apparatus and
Materials
1.	Subculture media (e.g., letheen broth, fluid thioglycollate medium). Note:
Commercial media made to conform to the recipes provided in AO AC
Methods 955.15, 964.02, and 955.14 may be substituted.
2.	Test organisms. Pseudomonas aeruginosa (ATCCNo. 15442),
Staphylococcus aureus (ATCC No. 6538) and Salmonella enterica (ATCC
No. 10708) obtained directly from ATCC.
3.	Culture media. Note: Commercial media (e.g., synthetic broth) made to
conform to the recipes provided in AO AC Methods 955.15, 964.02, and
955.14 may be substituted.
a. Synthetic broth (10 mL tubes). Use for daily transfers and final test
cultures.
4.	Trypticase soy agar (TSA). For use in propagation of the test organism to
generate frozen cultures and as a plating medium for carrier enumeration.
Alternately, TSA with 5% sheep blood (BAP) may be used.
5.	Sterile water. Use reagent-grade water 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. See
Standard Methods for the Examination of Water and Wastewater and SOP
QC-01, Quality Assurance of Purified Water for details on reagent-grade
water.
6.	Carriers. Polished stainless steel cylinders, 8 ± 1 mm outer diameter, 6 ±
1 mm inner diameter, 10 ± 1 mm length; type 304 stainless steel, SS 18-8
(S & L Aerospace Metals, Maspeth, NY or Fisher Scientific catalog
number 07-907-5Q as of July 2012). Use only carriers that passed

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bioscreening; refer to SOP MB-03, Screening of Stainless Steel Cylinders,
Porcelain Cylinders and Glass Slide Carriers Used in Disinfectant Efficacy
Testing.
7.	Specialized glassware. For disinfectant, use autoclavable 25 x 100 mm
tubes (Bellco Glass Inc., Vineland, NJ). For glassware used to prepare test
chemical, refer to SOP MB-22.
8.	Recirculating chiller unit. For maintaining specified temperature of the
test chemical.
9.	Transfer loops. Make 4 mm inner diameter single loop at end of 50-75
mm (2-3 in.) Pt or Pt alloy wire No. 23 B&S gage or 4 mm loop fused on
75 mm (3 in.) shaft (available from Johnson Matthey, West Chester, PA
19380, USA). Fit other end in suitable holder. Bend loop at 30° angle
with stem.
10.	Micropipettes. For performing culture transfers and serial dilutions.
11.	Wire Hook. For carrier transfer. Make 3 mm right angle bend at end of
50-75 mm nichrome wire No. 18 B&S gage. Place other end in suitable
holder.
12.	Timer. For managing timed activities, any certified timer that can display
time in seconds.
13.	Sonicator (ultrasonic cleaner). For conducting control carrier counts.
14.	Electronic Plate Scanning Device. 3M™ Petrifilm™ Plate Reader, 3M
Food Safety, St. Paul, MN, USA, Cat. No. 6499, or equivalent.
15.	Vitek 2 Compact. Alternative for microbe confirmation.
12. Procedure and
Analysis
Prior to testing, perform the neutralization assay to determine if secondary
subculture tubes are necessary (refer to SOP MB-17, Neutralization
Confirmation).
Refer to Attachment 4 for details on testing with Carbapenem Resistant
Klebsiella pneumonia.
The AO AC Use-Dilution Test Processing Sheet (see section 14) must be used
for tracking testing activities.
12.1 Test Culture
Preparation
Refer to SOP MB-02 for the test microbe culture transfer notation.
a. Defrost a single cryovial at room temperature and briefly vortex to
mix. Add 10 |iL of the thawed frozen stock (single use) to a tube
containing 10 mL of growth medium (e.g., synthetic broth), vortex,
and incubate at 36 ± 1°C for 24 ± 2 h. One daily transfer is required
prior to the inoculation of a final test culture. Daily cultures may be

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subcultured for up to 5 days; each daily culture may be used to
generate a test culture.1 For S. aureus and S. enterica only, briefly
vortex the 24 h cultures prior to transfer.2
b. For the final subculture transfer, inoculate a sufficient number of 20
x 150 mm tubes containing 10 mL growth medium (e.g., synthetic
broth) with 10 |iL per tube of the 24 h culture; incubate 48-54 h at 36
±1°C. Do not shake the 48-54 h test culture. Record all culture
transfers on the Organism Culture Tracking Form (see section 14).
12.2 Carrier
Inoculation for
S. aureus, P.
aeruginosa,
and S. enterica
Inoculate approximately 80 carriers; 60 carriers are required for testing, 6 for
control carrier counts, and 1 for the viability control.
a.	For P. aeruginosa, remove the pellicle from the broth either by
decanting the liquid aseptically into a sterile tube, by gently
aspirating the broth away from the pellicle using a pipette, or by
vacuum removal. Avoid harvesting pellicle from the bottom of the
tube. Transfer test culture after pellicle removal into sterile 25 x 150
mm test tubes (up to approximately 20 mL per tube) and visually
inspect for pellicle fragments. Presence of pellicle in the final culture
makes it unusable for testing. Proceed as below in 12.2b.
b.	For S. aureus and S. enterica, using a vortex-style mixer, mix 48-54
h test cultures 3 -4 s and let stand 10 min at room temperature before
continuing. Remove the upper portion of each culture (e.g., upper 3/4
or approximately 7.5 mL), leaving behind any debris or clumps, and
pool culture into a sterile flask; swirl to mix. Measure and record the
OD at 650 nm. Use sterile broth medium to calibrate the
spectrophotometer.
Note: To achieve mean carrier counts within the appropriate range
(see section 8), the final test culture may be diluted (e.g., one part
culture plus one part sterile broth) prior to the addition of the OSL to
the inoculum using the sterile culture medium used to generate the
final test culture (e.g., synthetic broth). Use the diluted test culture
for carrier inoculation within 30 min.
c.	Add appropriate amount of organic burden if required. Swirl to mix.
d.	Drain the water from the carriers. Aseptically transfer 20 carriers
into each of the tubes containing the test culture. The test culture
must completely cover the carriers; reposition carriers as necessary to
ensure coverage. Alternatively, siphon off the water from the
carriers and add 20 mL test culture directly to the carriers without
1	Step not contained in the AO AC standard methods 955.14, 955.15, and 964.02.
2	Step not contained in the AO AC standard methods 955.14 and 955.15.

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transferring.
e.	Allow carriers to remain in inoculum for 15 ± 2 min.
f.	Drain the inoculum from the carriers with a pipette; avoid direct
contact of the carrier with the tip of the pipette.3 Briefly tap each
carrier against the side of the tube to remove excess culture and place
on end in vertical position in sterile Petri dish matted with 2 layers of
Whatman No. 2 (or equivalent) sterile filter paper, making sure that
carriers do not touch or fall over. Place no more than 12 carriers in a
Petri dish.
g.	Dry carriers in incubator at 36 ± 1 °C for 40 ± 2 min. Record the
timed carrier inoculation activities on the AO AC Use-Dilution Test
Processing Sheet (see section 14). Perform efficacy testing within
two hours of drying.
12.3 Enumeration
of viable
bacteria from
carriers
(control carrier
counts)
a.	Select one carrier from each of 6 Petri dishes, assay dried carriers in
2 sets of three carriers, one set immediately prior to conducting the
efficacy test and one set immediately following the test.
b.	Place each inoculated dried carrier into a tube containing 10 mL of
letheen broth and sonicate in an ultrasonic cleaner for 1 min ±5 s.
Record the time of soni cation on the AO AC Use-Dilution Test
Processing Sheet (see section 14).
c.	For sonication, place tubes into an appropriately sized glass beaker
with tap water to the level of the letheen broth in the tubes. Place the
beaker in an ultrasonic cleaner so that the water level in the beaker is
even with the water level fill-line on the tank. Fill the tank with tap
water to the water level fill-line. Hold the beaker so that it does not
touch the bottom of the tank and all 3 liquid levels (inside the test
tubes, inside the beaker, and inside the tank) are approximately the
same.
d.	After sonication, briefly mix and make serial ten-fold dilutions in 9
mL dilution blanks of PBDW. Briefly vortex and plate 0.1 mL
aliquots of appropriate dilutions in duplicate on TSA or BAP using
spread plating. Plate appropriate dilutions to achieve colony counts in
the range of 30-300 colony forming units (CFU) per plate. Spread
inoculum evenly over the surface of the agar. Plates must be dry
prior to incubation. If the serial dilutions are not made and plated
immediately, keep the sonicated tubes at 2-5°C until this step can be
done. Complete the dilutions and plating within 2 h after sonication.
3 Step not contained in the AO AC standard methods 955.14, 955.15, and 964.02.

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Alternatively, pool the letheen broth from the tubes with the carriers
and briefly vortex for each set of three carriers. Serially dilute and
plate 0.1 mL aliquots of the pooled media (30 mL).
The average carrier count per set will be calculated. Refer to the
AO AC Use-Dilution Test Carrier Counts Form (see section 14).
e.	Incubate plates (inverted) at 36 ± 1 °C for up to 48 ± 2 h.
f.	Count colonies. Plates that have colony counts over 300 will be
reported as TNTC. Record counts on the AO AC Use-Dilution Test
Carrier Counts Form (see section 14). See section 13 for data
analysis.
g.	Alternatively, Petrifilm may be used for enumeration of bacterial
organisms. Follow manufacturer's instructions for preparation and
incubation of Petrifilm cards. Note: A culture purity check should be
conducted on one dilution of one carrier.
12.4 Disinfectant
Sample
Preparation
a.	Prepare disinfectant sample per SOP MB-22.
b.	Equilibrate the water bath and allow it to come to 20 ± 1 °C or the
temperature specified (±1°C). Prepare the disinfectant dilutions
within 3 hours of performing the assay unless test parameters specify
otherwise. Record the time of disinfectant preparation on the AO AC
Use-Dilution Test Processing Sheet (see section 14).
c.	Dispense 10 mL aliquots of the disinfectant into 25 x 100 mm test
tubes, one tube per carrier. Place tubes in the equilibrated water bath
for approximately 10 min to allow disinfectant to come to specified
temperature. Record the temperature of the water bath and
recirculating chiller before and after testing on the AO AC Use-
Dilution Test Information Sheet (see section 14).
12.5 Test Procedure
a.	Sequentially transfer the carriers from the Petri dish to the test tubes
containing the disinfectant at appropriate intervals (e.g., 30 second
intervals).
b.	Add one carrier per tube and swirl the tube using 2-3 gentle rotations
before placing it back in the water bath. Avoid intense swirling and
agitation of the carrier. Add carrier within ±5 seconds of the
specified time for a contact time of 1 -10 minutes or within ±3
seconds for contact times <1 minute.
c.	Using alternating hooks, flame-sterilize the hook and allow it to cool
after each carrier transfer. When lowering the carriers into the
disinfectant tubes, neither the carrier itself nor the tip of the wire
hook can touch the interior sides of the tube. If the interior sides of

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the tube are touched, repeat the carrier.
d.	Following the exposure time, sequentially transfer the carriers into
subculture/neutralizer media. Remove the carrier from the
disinfectant with a sterile hook, tap it against the interior sides of the
tube to remove the excess disinfectant, and transfer it into the
subculture tube within ±5 s. Avoid tapping the carrier against the
upper third of the tube. Avoid contact of the carrier to the interior
sides of the subculture tube during transfer.
e.	Recap the subculture tube and shake thoroughly. Incubate at 36 ±
1°C for 48 ± 2 h.
f.	If a secondary subculture tube is deemed necessary to achieve
neutralization, then transfer the carrier from the primary tube to a
secondary tube of sterile medium after a minimum of 30 ± 5 min at
room temperature from the end of the initial transfer. Within 25-60
min of the initial transfer, transfer the carriers using a sterile wire
hook to a second subculture tube. Move the carriers in order but the
movements do not have to be timed. Thoroughly shake the
subculture tubes after all of the carriers have been transferred.
Incubate both the primary and secondary subculture tubes 48 ± 2 h at
36 ± 1°C.
g.	Record timed events on the AO AC Use-Dilution Test Time
Recording Sheet for Carrier Transfers (see section 14).
12.6 Sterility and
viability
controls
a.	Viability controls. Place 1 (or 2) dried inoculated untreated carrier(s)
into separate tubes of the neutralizing subculture broth (if primary
and secondary media are different). Incubate tubes with the efficacy
test. Report results as + (growth) or 0 (no growth) as determined by
presence or absence of turbidity. Growth should occur in both tubes.
Record results on AO AC Use-Dilution Test Results Sheet (see
section 14).
b.	Sterility controls. Place one sterile, uninoculated carrier into a tube
of neutralizing subculture broth. Incubate tube with the efficacy test.
Report results as + (growth), or 0 (no growth) as determined by
presence or absence of turbidity. Growth should not occur in the
tube. Record results on AO AC Use-Dilution Test Results Sheet (see
section 14).
12.7 Results
a. Gently shake each tube prior to recording results. Record results as +
(growth) or 0 (no growth) as determined by presence or absence of
turbidity, on the AO AC Use-Dilution Test Results Sheet (see section
14).

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b.	If secondary subculture tubes are used, the primary and secondary
subculture tubes for each carrier represent a "carrier set." A positive
result in either the primary or secondary subculture tube is
considered a positive result for a carrier set.
c.	Specialized neutral izer/sub culture medium such as Dey/Engley broth
will not show turbidity; rather the presence of pellicle at the surface
of the medium (for P. aeruginosa) or a color change to the medium
(yellow for growth of S. aureus or S. enterica) must be used to assess
the results as a positive or negative outcome.
i.	Use viability controls for comparative determination of a
positive tube.
ii.	If the product passes the performance standard, a minimum of
20% of the remaining negative tubes will be assayed for the
presence of the test microbe using isolations streaks on TSA
or BAP. Record preliminary results and conduct isolation
streaks at 48 ± 2 h, however, continue to incubate negative
tubes for up to an additional 24 hours to confirm the results.4
12.8 Confirmatory
Steps for Test
Microbes
a.	Confirm a minimum of three positive carrier sets per test. If there are
less than three positive carriers, then confirm each carrier. If
secondary subculture tubes are used and both tubes are positive in a
carrier set, select only the tube with the carrier for confirmatory
testing.
b.	For a test with greater than 20 positive carrier sets, confirm at least
20% by Gram staining, and a minimum of 4 positive carrier sets by
Gram staining, solid media, and appropriate biochemical and
antigenic analyses to ensure the identity of the organism.
c.	See Attachment 1 for Gram stain reactions, cell morphology, and
colony characteristics on solid media.
d.	For additional confirmation steps refer to the appropriate
Confirmation Flow Chart for S. aureus, P. aeruginosa, and S.
enterica (see Attachment 3).
e.	If confirmatory testing determines that the identity of the unknown
was not the test organism, annotate the positive entry (+) on the
results sheet to indicate a contaminant was present.
f.	Alternatively, the Vitek 2 Compact may be used for confirmation of
bacterial organisms. Follow manufacturer's instructions for use of
4 Step not contained in the AO AC standard methods 955.14, 955.15, and 964.02.

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SOP No. MB-05-11
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the Vitek 2 Compact.
12.9 Re-use of
Stainless Steel
Carriers
a. After use, autoclave all carriers. Carriers for which test results were
negative may be reused after cleaning. Carriers that are positive are
re-cleaned and screened biologically (see SOP MB-03, Screening
Carriers) before re-use. These carriers may be reused if the
biological screening test results in no growth. The extra inoculated
carriers, positive control,5 and those used for carrier counts may be
autoclaved, re-cleaned, and used again.
13. Data Analysis/
Calculations
Calculations will be computed using a Microsoft Excel spreadsheet (see
section 14). Both electronic and hard copies of the spreadsheet will be
retained. Counts up to 300 and their associated dilutions will be included in
the calculations.
14. Forms and Data
Sheets
1.	Attachment 1: Typical Growth Characteristics of strains of P. aeruginosa,
S. aureus, and S. enterica
2.	Attachment 2: Culture Initiation Flow Chart for S. aureus, P. aeruginosa,
and S. enterica
3.	Attachment 3: Confirmation Flow Charts for S. aureus, P. aeruginosa and
S. enterica
4.	Attachment 4: Confirmatory Testing Against Carbapenem Resistant
Klebsiella pneumoniae (ATCC# BAA-1705)
5.	Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
Organi sm Culture Tracki ng F orm MB -05-11 F1. docx
Test Microbe Confirmation Sheet (Quality MB-05-1 l_F2.docx
Control)
AO AC Use-Dilution Test Time Recording Sheet MB-05-11_F3 .docx
for Carrier Transfers
AO AC Use-Dilution Test Information Sheet MB-05-11 F4.docx
AOAC Use-Dilution TestResults Sheet (1°) MB-05-11_F5.docx
AO AC Use-Dilution TestResults Sheet (172°) MB-05-11_F6.docx
Test Microbe Confirmation Sheet MB-05-11 F7.docx
AOAC Use-Dilution Test Carrier Counts Form MB-05-11 F8.docx
AOAC Use-Dilution Test Processing Sheet MB-05-1 l_F9.docx
5 Step not contained in the AO AC standard methods 955.14, 955.15, and 964.02.

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Carrier Count Spreadsheet MS Excel spreadsheet: MB-05-1 lFlO.xlsx
Carrier Count Template_UDT_v3
AO AC Use-Dilution Test Carrier Counts Form MB-05-11_F1 l.docx
(Pooled Carriers)
15. References
1.	Official Methods of Analysis. July 2012. 18th Ed., AO AC
INTERNATIONAL, Gaithersburg, MD, (Methods 955.14, 955.15, and
964.02).
2.	Krieg, Noel R. and Holt, John G. 1984. Bergey's Manual of Systematic
Bacteriology Volume 1. Williams & Wilkins, Baltimore, MD.
P. aeruginosa p. 164, S. enterica p. 447.
3.	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.
4.	Package Insert - Gram Stain Kit and Reagents. Becton, Dickinson and
Company. Part no. 882020191JAA. Revision 07/2011.
5.	Package Insert - Catalase Reagent Droppers. Becton, Dickinson and
Company. Part no. LOO 1237. Revision 06/2010.
6.	Package Insert - Staphaurex Plus. Remel. Part no. R30950102. Revised
11/23/07.
7.	Package Insert - Oxidase Reagent Droppers. Becton, Dickinson and
Company. Part no. LOO 1133. Revision 06/2010.
8.	Package Insert - Wellcolex Colour Salmonella. Remel. Part no.
R30858301. Revised 10/17/07.
9.	CDC Protocol: Modified Hodge Test for Carbapenemase Detection in
Enterobacteriaceae. Download:
http://www.ndhealth. sov/microlab/Uploads/HodgeT est.pdf

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Attachment 1
Typical Growth Characteristics of strains of P. aeruginosa, S. aureus, and S. enterica (see ref.
15.2 and 15.3).	

P. aeruginosa*
S. aureus*
S. enterica*
Gram slain reaction
(-)
(+)
(-)
Mannilol Salt
No Growth
circular, small, yellow
colonies, agar turning
fluorescent yellow
N/A
Cclrimide
circular, small,
initially opaque,
turning fluorescent
green over time; agar
fluorescent yellowish
green
No Growth
N/A
Xylose lysine
dcoxycholalc (XLD)
agar
N/A
N/A
Round, clear red colonies
with black centers
Blood agar (BAP)
flat, opaque to off-
white, round
spreading (1),
metallic sheen,
slightly beta
hemolytic
small, circular, yellow
or white, glistening,
beta hemolytic
entire, glistening, circular,
smooth, translucent, low
convex, non-hemolytic
MacConkcy Agar
N/A
N/A
N/A
Typical Microscopic Characteristics
Cell dimensions
0.5-1.0 umin
diameter by 1.5-5.0
jim in length*
0.5-1.5 umin
diameter*
0.7-1.5 nm in diameter by
2.0-5.0 umin length*
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
straight rods, peritrichous
flagella
*After 24±2 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 edge, convex, rough, spreading, and translucent.
Pyocyanin is not produced.

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Attachment 2
Culture Initiation and Stock Culture Generation Flow Chart for S. aureus, P. aeruginosa, and S.
enterica
(D Rehydrate ampule
Ampule
TSB	TUBE A	TUBE A
(pre-incubation) fpost-incubation)
©Stock Culture Generation
Inoculate TSA plates with 100 jliL
culture from TUBE A; incubate.
Harvest inoculum
from plates.
1
Prepare frozen
stock cultures
© Culture ID & Quality Control
BAP	Selective
media
A
Gram Additional
Stain confirmation
steps (see
Attachment3)
Al. 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
(ATCC 15442), Staphylococcus aureus (ATCC 6538), and Salmonella enterica (ATCC
10708) from ATCC within 18 months.
b.	Open ampule of freeze dried organism as indicated by ATCC. Using a tube containing
5-6 mL of TSB for P. aeruginosa and S. aureus and 5-6 mL of NB for S. enterica,
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 designated as

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"TUBE A". Mix well.
c.	Incubate broth culture (TUBE A) at 36 ± 1°C for 24 ± 2 hours. Record all
manipulations on the Organism Culture Tracking Form (see section 14).
d.	Using a sterile spreader, inoculate a sufficient number of TSA plates (e.g., 5 to 10
plates per organism) with 100 |iL each of the culture. Incubate plates at 36 ± 1°C for
24 ± 2 h.
e.	Following incubation, add 5 mL cryoprotectant solution (TSB with 15% v/v glycerol)
to the surface of each agar plate. Re-suspend the cells in this solution using a sterile
spreader or a sterile swab and aspirate the cell suspension from the surface of the agar.
Transfer the suspension into a sterile vessel. Repeat by adding another 5 mL of
cryoprotectant to the agar plates, re-suspend the cells, aspirate the suspension and pool
with the initial cell suspension.
f.	Mix the pooled contents of the vessel thoroughly. Immediately after mixing, dispense
approximately 1.0 mL aliquots into cryovials (e.g., 1.5 mL cyrovials). Perform QC of
stock cultures concurrently with freezing (see section A2: QC of Stock Cultures).
g.	Place and store the cryovials at -70°C or below; these are the frozen stock cultures.
Stock cultures may be used up to 18 months; reinitiate using a new lyophilized culture.6
These cultures are single-use only.
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, for S. aureus and P. aeruginosa, streak a loopful onto both
selective media (MSA and Cetrimide); for S. enterica, streak a loopful onto XLD.
Incubate all plates at 36 ± 1°C 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 Attachment 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 15.5) 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 confirmation steps refer to the appropriate Confirmation Flow Chart for
S. aureus, P. aeruginosa, and S. enterica (see Attachment 3). Refer to 15.6-15.9 for
instructions.
e.	Record all confirmation results on the Test Microbe Confirmation Sheet (Quality
Control) (see section 14).
6 Step not contained in the AO AC standard methods 955.14, 955.15, and 964.02.

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Attachment 3
Confirmation Flow Chart for S. aureus
S. aureus Identification
S. aureus

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Attachment 3 (cont.)
Confirmation Flow Chart for P. aeruginosa
P. aeruginosa Identification
Not P. aeruginosa
P. aeruginosa

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Attachment 3 (cont.)
Confirmation Flow Chart for S. enterica
Salmonella Identification
Not Salmonella
Salmonella

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Attachment 4 - Testing Against Carbapenem Resistant Klebsiella pneumoniae (ATCC# BAA-
1705). The following modifications to this SOP are used when testing Carbapenem Resistant K
pneumoniae:
Stock cultures are prepared according to Attachment #2 using tryptic soy broth (TSB) to
rehydrate the lyophilized culture.
Test cultures are prepared according to Section 12.1; no pellicle is formed with K pneumoniae.
Section 8, #2: The mean TestLD for carriers inoculated with S. aureus and P. aeruginosa must
be at least 4.0 (corresponding to a geometric mean density of 1.0 x io4) and not above 5.0
(corresponding to a geometric mean density of 1.0 x 105); a mean TestLD below 4.0 and above
5.0 invalidates the test, except for two retesting scenarios (outlined in the study protocol).
Section 12.2: Inoculate approximately 15-20 carriers; 10 carriers are required for testing, 3 for
control carrier counts, and 1 for the viability control.
Section 12.2, c: Dilution of the final test culture, prior to the addition of the soil load, will be
necessary to achieve carrier counts within the required range. Typically, a 1:5 dilution will be
needed.
Section 12.3, a: Three control carriers will be evaluated at the end of the test period.
Section 12.8: Growth from all positive tubes (efficacy test) will be confirmed by Gram stain
(Gram negative rods), growth on blood agar (large, mucoid, non-hemolytic colonies), and
MacConkey agar (large, mucoid, lactose-fermenting pink colonies). See Table 1 below.
The carbapenem resistance of the organism shall be confirmed on cultures from each positive
tube using the modified Hodge test (see Reference 9). The following additional items will be
required to perform the modified Hodge Test:
1. 10 |ig meropenem susceptibility disks
2. Mueller-Hint on agar
3. 0.85% saline
4. Klebsiella pneumoniae (ATCC# BAA-1706) as a negative control
5. Escherichia coli (ATCC# 25922)

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Table 1. Growth Characteristics of K. pneumoniae on Growth Media
K. pneumoniae
Gram stain reaction
(-)
Typical Growth Characteristics on Solid Media
Blood agar (BAP)
Large, round, mucoid colonies.
Non-hemo lytic.
MacConkey Agar
Large, round, mucoid colonies.
Lactose fermenter (pink colonies)
Cell dimensions
0.5-1.0 nm in diameter by 1.5-5.0
jim in length*
Cell appearance
Straight rods, capsule may be
visible
*After 24±2 hours

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