Standard Operating Procedure for Disinfectant Towelette Test: Testing of Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica


US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Mic ro bio lo gy La bo ra to ry
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Disinfectant Towelette Test: Testing of Staphylococcus
aureus, Pseudomonas aeruginosa, and Salmonella enterica
SOP Number: MB-09-07
Date Revised: 03-04-19

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SOP No. MB-09-07
Date Revised 03-04-19
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SOP Number
MB-09-07
Title
Disinfectant Towelette Test: Testing of Staphylococcus aureus,
Pseudomonas aeruginosa, and Salmonella enterica
Scope
Describes the methodology 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 official product testing, a study protocol is developed which
identifies the specific test conditions for a product sample such as
contact time, neutralizers, etc.


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Date SOP withdrawn: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-09-07
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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
11
15.
REFERENCES
11

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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 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 SOPs EQ-01 (pH meters), EQ-02 (thermometers), EQ-03 (weigh
balances), EQ-04 (spectrophotometers), EQ-05 (timers), and QC-19
(pipettes) 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.
2.	Prior to inoculation, ensure that the carriers are dry (inside Petri dishes).
Moisture can interfere with the concentration and drying of the
inoculum on the glass slide carrier.
3.	Do not use any inoculated carrier that is wet at the conclusion of the
carrier drying period.
4.	For neutralizes/subculture media that do not result in turbidity as the
outcome of growth, such as Dey/Engley broth, assess the interpretation
of a positive tube in advance of the test (see section 12.7.e).
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).

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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.	No contamination is acceptable in the test system.
4.	Management of non-conforming data will be consistent with SOP
ADM-07, Non-Conformance Reports.
9. Data
Management
Data will be archived per 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; 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

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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 or Fisherfinest®
Premium Frosted Microscope Slides (Fisher Scientific, Catalog number
12-544-2). 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.
10.	Micropipettes. For performing culture transfers and serial dilutions.
11.	Positive displacement pipette. With corresponding sterile tips able to
deliver 10 |iL.
12.	Timer. For managing timed activities, any certified timer that can
display time in seconds.
13.	3M™ Petrifilm™ Aerobic Count Plates. 3M Food Safety, St. Paul, MN,
USA, Cat. No. 6400.
14.	Vitek 2 Compact. Alternative for biochemical and antigenic analysis
component of microbe confirmation.
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
Refer to SOP MB-02 for the test microbe culture transfer notation. Refer to
Attachment 2 for culture initiation and generation of frozen stock cultures.
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.

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(Synthetic broth is used for S. aureus, P. aeruginosa, and S.
enterica. Nutrient broth may be used for P. aeruginosa). 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. To generate test cultures, 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 shake the 48-
54 h P. aeruginosa 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 1-2 for the viability
control(s). Set aside 1-2 uninoculated carrier(s) for sterility 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
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, S. enterica, and P. aeruginosa from 12.2.b, 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 3A), leaving behind any debris
or clumps, and transfer to a sterile flask; pool cultures in the flask
and swirl to mix. Measure and record the OD at 650 nm. Use
sterile broth medium to calibrate the spectrophotometer. Use the
test culture for carrier inoculation within 30 minutes.
d.	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 minutes.

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Note: Concentration of the final test culture may be used in the
event the bacterial titer in the final test cultures is too low.
Concentration may be achieved using centrifugation (e.g., 5000g
for 20 min) and resuspending the pellet in the appropriate volume
of the sterile final test culture medium necessary to meet the
carrier count range. Use the concentrated test culture for carrier
inoculation within 30 min.
e.	Add appropriate amount of organic burden if required. Swirl to
mix.
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
bacteria from
carriers
(control
carrier
counts)
a.	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. 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. 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

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prior to incubation. 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
and briefly vortex. Serially dilute and plate 0.1 mL aliquots of the
pooled media (60 mL).
d.	Incubate plates (inverted) at 36 ± 1°C for up to 48 ± 2 h.
e.	Count colonies. Plates that have colony counts over 300 will be
reported as TNTC. Record counts on the Disinfectant Towelette
Test Carrier Counts Form and calculate the mean counts (see
sections 13 and 14).
f.	Alternatively, Petrifilm may be used for enumeration of bacterial
organisms. Follow manufacturer's instructions for preparation and
incubation of Petrifilm cards. Note: At a minimum, conduct a
culture purity check (isolation streak) using suspension from one
dilution tube or letheen broth tube of one carrier or pooled set.
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.	Aseptically remove several towelettes before aseptically removing
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.
c.	Remove the lid from the Petri dish and aseptically remove the
inoculated slide and hold it firmly against the rim of the Petri dish.
d.	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.

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e.	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.
f.	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.
g.	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.
h.	After the slide is deposited, recap the subculture tube and shake
thoroughly.
i.	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.
j. Incubate all subculture tubes 48 ± 2 h at 36 ± 1°C.
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.
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(s) 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.

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c.	Sterility control. Growth should not occur in any of the 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 neutralizedsubculture 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.
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.	If confirmatory testing determines that the identity of the positive
tube was not the test organism, annotate the positive entry (+) on
the results sheet to indicate a contaminant was present.
e.	Alternatively, the Vitek 2 Compact may be used for confirmation
in place of biochemical and antigenic analyses. Follow
manufacturer's instructions for use of the Vitek 2 Compact.
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

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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.	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-09-07 F1 .docx
Form
Disinfectant Towelette Test Time Recording MB-09-07 F2.docx
Sheet for Carrier Transfers
Disinfectant Towelette Test Information Sheet MB-09-07 F3.docx
Disinfectant Towelette Test Results Sheet MB-09-07 F4.docx
(172°)
Disinfectant Towelette Test Results Sheet (1°) MB-09-07_F5.docx
Test Microbe Confirmation Sheet MB-09-07 F6.docx
Carrier Count Spreadsheet MS Excel MB-09-07 F7.xlsx
spreadsheet: Carrier Count Template DTT
Disinfectant Towelette Test Carrier Counts MB-09-07_F8.docx
Form (Pooled Carriers)
Disinfectant Towelette Test Processing Sheet MB-09-07_F9.docx
15. References
1.	Official Methods of Analysis. Revised 2013. AOAC
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,

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B altimore, MD. S. aureus p. 1015.

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

P. aeruginosa*
S. aureus*
5". enterica*
Gram stain reaction
(-)
(+)
(-)
Typical Growth Characteristics on Solid Media
Mannitol Salt
No Growth
circular, small, yellow
colonies, agar turning
fluorescent yellow
N/A
Cctrimidc
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
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.
© Transfer entire
rehydrated pellet to TUBI
A.
Ampule
Incubate
TSB
J
TUBE A	TUBE A
(pre-incubation) (post-incubation)
©Stock Culture Generation
Inoculate TSA plates with 100 |iL
culture from TUBE A; incubate.
Harvest inoculum from
plates.
Prepare frozen
stock cultures
© Culture ID & Quality Control
BAP
A
Selective
media
Gram Additional confirmation
Stain steps (e.g., Vitek)
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 fori5, 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 "TUBE A". Mix well.

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i.	Incubate broth culture (TUBE A) at 36±1°C for 24±2 h. Record all
manipulations on the Organism Culture Tracking Form (see section 14).
ii.	For QC purposes, perform a streak isolation of the TUBE A culture on a 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 h.
c.	Following incubation, use a sterile spreader to inoculate a sufficient number of TSA
plates (e.g., 5 to 10 plates per organism) with 100 |iL each of the 24±2 h culture.
Incubate plates at 36 ± 1°C for 24 ± 2 h.
d.	Following incubation, add 5 mL cryoprotectant solution (TSB with 15% v/v glycerol
for S. aureus and P. aeruginosa and NB with 15% v/v glycerol for S. enterica) to the
surface of each agar plate. Resuspend 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, resuspend the cells, aspirate the suspension and pool
with the initial cell suspension.
i. For QC purposes, use the pooled suspension to perform a streak isolation on a
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 h. Continue QC steps as per
sections g through i.
e.	Mix the pooled contents of the vessel thoroughly. Immediately after mixing,
dispense approximately 0.5 to 1.0 mL aliquots into cryovials (e.g., 1.5 mL cyrovials).
f.	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. These cultures are single-use only.
g.	Following the incubation period (see d.i.), record the colony morphology as observed
on the BAPs and selective media plates (including the absence of growth). See
Attachment 1 for details on cell and colony morphology, colony characteristics on
selective media, and stain reactions.
h.	For each organism, perform a Gram stain and Vitek 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).
i.	Record all confirmation results on the Test Microbe Confirmation Sheet (Quality
Control) (see section 14).

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