Standard Operating Procedure for Disinfectant Towelette Test Against Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica SOP Number: MB-09-05 Date Revised: 01-30-13


US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Disinfectant Towelette Test Against Staphylococcus aureus,
Pseudomonas aeruginosa, and Salmonella enterica
SOP Number: MB-09-05
Date Revised: 01-30-13

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SOP No. MB-09-05
Date Revised 01-30-13
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SOP Number
MB-09-05
Title
Disinfectant Towelette Test: Testing of Staphylococcus aureus,
Pseudomonas aeruginosa, and Salmonella enterica
Scope
Describes the methodology used to determine the efficacy of
towelette-based disinfectants against Staphylococcus aureus,
Pseudomonas aeruginosa, and Salmonella enterica on hard surfaces.
The test is based on AO AC Method 961.02 (Germicidal Spray
Products as Disinfectants). See 15.1.
Application
For product evaluations under the Antimicrobial Testing Program
(ATP), a study protocol is developed which identifies the specific test
conditions for a product sample such as contact time, neutralizers, etc.
Although the default growth medium specified in this SOP is synthetic
broth, other growth media (e.g., nutrient broth) may be specified by
the study sponsor.


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Date SOP withdrawn: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-09-05
Date Revised 01-30-13
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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
10
14.
FORMS AND DATA SHEETS
10
15.
REFERENCES
11

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SOP No. MB-09-05
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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 and SOP COC-01.
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.
2.	The carriers inside the Petri dishes should be dry prior to inoculation.
Moisture can interfere with the concentration and drying of the
inoculum on the glass slide carrier.
3.	Any inoculated carrier that is wet at the conclusion of the carrier drying
period should not be used.
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 TestLD for carriers inoculated with S. aureus and P.
aeruginosa must be at least 5.0 (corresponding to a geometric
mean density of 1.0 x 105) and not above 6.5 (corresponding to a
geometric mean density of 3.2 x 106); a mean TestLD below 5.0
and above 6.5 invalidates the test, except for two retesting
scenarios (outlined in the study protocol).
b.	The mean TestLD for carriers inoculated with S. enterica must be
at least 4.0 (corresponding to a geometric mean density of 1.0 x
104) and not above 5.5 (corresponding to a geometric mean density
of 3.2 x 105); a mean TestLD below 4.0 and above 5.5 invalidates
the test, except for two retesting scenarios (outlined in the study
protocol).
3.	Management of non-conforming data will be specified in the study

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SOP No. MB-09-05
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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
There are time sensitive steps in this procedure including the use periods of
the inoculated carriers and the test chemical.
11. Special
Apparatus and
Materials
1.	Subculture media; use 20 mL aliquots (e.g., letheen broth, fluid
thioglycollate medium, and Dey/Engley broth). Note: Commercial
media made to conform to the recipes provided in AO AC Method
961.02 may be substituted.
2.	Test organisms. Pseudomonas aeruginosa (ATCC No. 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 Method 961.02 may be
substituted.
a.	Synthetic broth. Use for (10 mL) daily transfers and (10 mL) final
test cultures of S. aureus, P. aeruginosa and S. enterica.
b.	Nutrient broth. Alternatively, use for (10 mL) daily transfers and
(10 mL) final test cultures of P. aeruginosa.
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. Glass Slide Carriers, 25 mm x 75 mm (or comparable size)
borosilicate glass cover slips with number 4 thickness. 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 primary and secondary subculture media,
use autoclavable 38 x 100 mm glass tubes (Bellco Glass Inc., Vineland,
NJ). Cap tubes with closures before sterilizing.
8.	Sterile surgical gloves. For handling the towelette.
9.	Forceps. For manipulating glass slides.

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10.	Micropipettes. For performing culture transfers and serial dilutions.
11.	Positive displacement pipette. With corresponding sterile tips able to
deliver 0.01 mL.
12.	Timer. For managing timed activities, any certified timer that can
display time in seconds.
13.	Electronic Plate Scanning Device. 3M™ Petrifilm™ Plate Reader, 3M
Food Safety, St. Paul, MN, USA, Cat. No. 6499, or equivalent.
14.	3M™ Petrifilm™ Aerobic Count Plates. 3M Food Safety, St. Paul, MN,
USA, Cat. No. 6400.
12. Procedure and
Analysis
One towelette is used to wipe ten carriers/slides. The area of the towelette
used for wiping is folded and rotated so as to expose a new surface of the
towelette for each carrier.
The method may be altered to accommodate various towelette/carrier
combinations (e.g., more than one towelette per set of ten slides).
Prior to testing, perform the neutralization assay to determine if secondary
subculture tubes are necessary.
The Disinfectant Towelette Test Processing Sheet (see section 14) must be
used for tracking testing activities.
12.1 Test Culture
Preparation
a.	Defrost a single cryovial (see Attachment 2) 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 or nutrient 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 subcultured for up to
5 days; each daily culture may be used to generate a test culture.
For S. aureus and S. enterica only, briefly vortex the 24 h cultures
prior to transfer.
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 or nutrient broth) with 10 |iL per tube of the 24 h
culture then vortex to mix. Incubate 48-54 h at 36 ± 1°C. Do not
disturb the 48-54 h test culture. Record all culture transfers on the
Organism Culture Tracking Form (see section 14).
12.2 Carrier
Inoculation
a.	Inoculate approximately 80 carriers; 60 carriers are required for
testing, 6 for control carrier counts, and 1 for the viability control.
b.	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

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SOP No. MB-09-05
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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
12.2c.
c.	For S. aureus and S. en/erica, 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 transfer to a sterile flask; pool cultures in the flask and
swirl to mix.
d.	To achieve mean carrier counts within the appropriate range (see
section 8), the final test culture may be diluted or concentrated.
Dilution of the final test culture (e.g., one part culture plus one part
sterile broth) should be made prior to the addition of the OSL to
the inoculum. Concentration may be achieved using centrifugation
(e.g. 5000g for 20 min.) and re-suspending the pellet in the
appropriate volume of the sterile final test culture medium
necessary to meet the carrier count range. Note: The use of a
spectrophotometer to measure optical density (optical density at
650 nm) is recommended as a tool for assessing the need to dilute
the final test culture. Always use sterile broth medium to calibrate
the spectrophotometer.
e.	If organic burden is required for testing, the appropriate amount of
organic burden is added to the pooled test culture prior to the
inoculation of carriers. Swirl to mix. Aliquot a sufficient volume
of culture into sterile test tubes.
f.	Use a calibrated positive displacement pipette to transfer 0.01 mL
of the test culture onto the sterile test carrier in the Petri dish, at
one end of the slide. Do not place inoculum in the middle of the
slide. Vortex-mix the inoculum periodically during the inoculation
of carriers. Immediately spread the inoculum uniformly over one
third of the carrier surface using a sterile loop. Do not allow the
inoculum to contact the edge of the glass slide carriers during the
inoculation process. Cover dish immediately.
g.	Dry carriers in incubator at 36 ± 1°C for 30-40 min. Record the
timed carrier inoculation activities on the Disinfectant Towelette
Test Processing Sheet (see section 14). Perform efficacy testing
within two hours of drying.
h.	After completion of all slide inoculations, thoroughly wipe the
micropipette with 70% ethanol prior to removal from the BSC.
12.3 Enumeration
of viable
a. Assay dried carriers in 2 sets of three carriers, one set immediately
prior to conducting the efficacy test and one set immediately

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SOP No. MB-09-05
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bacteria from
carriers
(control
carrier
counts)
following the test. Randomly select 6 inoculated carriers for carrier
count analysis prior to efficacy testing.
b.	Place each of the inoculated, dried carriers in a 38 x 100 mm culture
tube or sterile 50 mL polypropylene conical tube containing 20 mL
of letheen broth. Vortex immediately - 60 ± 5 seconds fori5.
aeruginosa or 120 ± 5 seconds for S. aureus and S. enterica. Record
the time of vortexing on the Disinfectant Towelette Test Processing
Sheet (see section 14).
c.	After vortexing, briefly mix and make serial ten-fold dilutions in 9
mL dilution blanks of PBDW. Refer to the Disinfectant Towelette
Test Carrier Counts Form (see section 14). If the serial dilutions are
not made and plated immediately, keep the tubes at 2-5°C until this
step can be done. Complete the dilutions and plating within 2 h after
vortexing. Alternatively, pool the letheen broth from the tubes with
the carriers after vortexing. Briefly vortex after pooling. Serially
dilute and plate an aliquot of the pooled media (60 mL). The
average carrier count per set will be calculated.
d.	Briefly vortex each serial dilution tube prior to plating. 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.
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 Disinfectant Towelette
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.	Wipe the outside of the towelette packet or dispenser with 70%
ethanol and allow to air dry prior to opening.
12.5 Test
Procedure
a.	Record timed events on the Disinfectant Towelette Test Time
Recording Sheet for Carrier Transfers (see section 14).
b.	Wipe the outside of the towelette dispenser or packet with 70%
ethanol and allow to air dry.
c.	Aseptically remove several towelettes before aseptically removing

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SOP No. MB-09-05
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a towelette to initiate testing. Fold towelette in half lengthwise one
to two times depending on the size. Beginning at the bottom, fold
up towards the top five times. The following steps in the
"procedure" section are more conveniently done with two analysts
- one to manage the Petri dishes and slides, and the other to
perform the wiping procedure.
d.	Remove the lid from the Petri dish and aseptically remove the
inoculated slide and hold it firmly against the rim of the Petri dish.
e.	Wipe the slide back and forth three times lengthwise with the
towelette for a total of six passes across the inoculum or as
specified by the study sponsor. Wiping should be done within ±5
seconds of specified time. Place slide in Petri dish, close the lid,
and allow slide to sit undisturbed for the contact time. Maintain the
wiped carriers in a horizontal position.
f.	Repeat with four additional slides, folding the used section of the
towelette in such a way as to expose a new surface for wiping each
slide.
g.	After the fifth slide, unfold the vertical fold in the towelette and
reverse the towelette so that the used surface of the towelette faces
inward. Continue wiping an additional five slides, folding the
towelette between each slide to expose a new surface.
h.	After the last slide of a set (typically 10 slides) has been wiped and
the exposure time is complete, sequentially transfer each slide into
the primary subculture tube containing the appropriate neutralizer
within the ±5 second time limit. Drain the excess disinfectant from
each slide, without touching the Petri dish, and transfer into the
neutralizer tube. Perform transfers with sterile forceps. Place the
inoculated/wiped end of the slide into the primary subculture
medium.
i.	After the slide is deposited, recap the subculture tube and shake
thoroughly.
j. If a secondary subculture tube is deemed necessary to achieve
neutralization, then transfer the carrier from the primary tube to a
secondary tube. Within 25-60 min of the initial transfer, transfer
the carriers using sterile forceps 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.
k. Incubate all subculture tubes 48 ± 2 h at 36 ± 1°C.
12.6 Sterility and
viability
a. Viability controls. Place 1 (or 2) dried inoculated untreated
carrier(s) into separate tubes of the neutralizing subculture broth (if

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SOP No. MB-09-05
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Page 9 of 17
controls
primary and secondary media are different). Incubate tubes with the
efficacy test.
b. Sterility controls. Place 1 (or 2) sterile untreated carrier(s) into
separate tubes of the neutralizing subculture broth (if primary and
secondary media are different). Incubate tube with the efficacy test.
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 Disinfectant Towelette Test Results Sheet (see
section 14).
b.	Viability control. Growth should occur in all tubes.
c.	Sterility control. Growth should not occur in any tubes.
d.	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.
e.	Specialized neutralizer/subculture medium such as Dey/Engley
broth will not show turbidity; rather, the presence of pellicle at the
surface of the medium (fori5, 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.
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.

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SOP No. MB-09-05
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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.
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 from 0 through 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.	Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
Physical Screening of Carriers Record MB-03 Fl.docx
Organism Culture Tracking Form (Frozen Stock MB-06 F2.docx
Cultures)
Test Microbe Confirmation Sheet (Quality MB-06 F3.docx
Control)
Disinfectant Towelette Test Carrier Counts MB-09-05 F1 .docx
Form
Disinfectant Towelette Test Time Recording MB-09-05 F2.docx
Sheet for Carrier Transfers
Disinfectant Towelette Test Information Sheet MB-09-05 F3.docx
Disinfectant Towelette Test Results Sheet MB-09-05 F4.docx
(172°)
Disinfectant Towelette Test Results Sheet (1°) MB-09-05_F5.docx
Test Microbe Confirmation Sheet MB-09-05 F6.docx
Carrier Count Spreadsheet MS Excel MB-09-05 F7.xlsx
spreadsheet: Carrier Count Template_DTT_v3
Disinfectant Towelette Test Carrier Counts MB-09-05_F8.docx
Form (Pooled Carriers)

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Disinfectant Towelette Test MB-09-05 F9.docx
Processing Sheet
15. References
1.	Official Methods of Analysis. Revised 2013, publication pending.
AO AC INTERNATIONAL, Gaithersburg, MD, (Method 961.02).
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. L001237. 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

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SOP No. MB-09-05
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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
(-)
(+)
(-)
Typical Growth Characteristics on Solid Media
Mannitol Salt
No Growth
circular, small, yellow
colonies, agar turning
fluorescent yellow
N/A
Cctrimide
circular, small, initially
opaque, turning fluorescent
green over time; agar
fluorescent yellowish green
No Growth
N/A
Xylose lysine
dcoxycholate (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
co nvcx. no n-hc mo lytic
Typical Microscopic Characteristics
Cell dimensions
0.5-1.0 nm in diameter by
1.5-5.0 nm in length*
0.5-1.5 |im in diameter*
0.7-1.5 nm in diameter by
2.0-5.0 nm in 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
© Rehydrate ampule.
4
© Transfer entire
rehydrated pellet to
TUBE A.
Ampule
Incubate
TSB
TUBE A	TUBE A
(pre-incubation) (post-incubation)
©Stock Culture Generation
Inoculate TSA plates with 100 |liL
culture from TUBE A: incubate.
Harvest inoculum
from plates.
ill
Prepare frozen
stock cultures
© Culture ID & Quality Control
BAP
A
Selective
media
Gram
Stain
Additional
confirmation
steps (see
Attachment 3)
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, 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 "TUBE A". Mix well.
	c. Incubate broth culture (TUBE A) at 36 ± 1°C for 24 ± 2 hours. Record all

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SOP No. MB-09-05
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Page 14 of 17
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.
Store stock cultures up to 18 months. 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).

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SOP No. MB-09-05
Date Revised 01-30-13
Page 15 of 17
Attachment 3
Confirmation Flow Chart for S. aureus
S. aureus Identification
Identified as S1. aureus

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SOP No. MB-09-05
Date Revised 01-30-13
Page 16 of 17
Attachment 3 (cont.)
Confirmation Flow Chart for P. aeruginosa
P. aeruginosa Identification

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SOP No. MB-09-05
Date Revised 01-30-13
Page 17 of 17
Attachment 3 (cont.)
Confirmation Flow Chart for S. enterica
S. enterica Identification
Not Salmonella
Identified as S. enterica

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