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-05-09

Date Revised: 08-29-12

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SOP No. MB-05-09
Date Revised 08-29-12
Page 1 of 17

SOP Number

MB-05-09

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.

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, dilutions,
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:





Data SOP issued:



Controlled copy number:



Date SOP withdrawn:

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SOP No. MB-05-09
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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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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 TestLD 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

n

geometric mean density of 1.0 x 10 ); a mean TestLD below 6.0 and
above 7.0 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 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 TestLD below 5.0 and above 6.0 invalidates the test,
except for two retesting scenarios (outlined in the study protocol).

3.	If contamination is present in the test system for more than one carrier for
a passing product; no contamination is acceptable for a product test in
which the product fails to meet the standard.

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 (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 AOAC Methods 955.15, 964.02, and
955.14 may be substituted.

a. Synthetic broth. Use for (10 mL) daily transfers and (10 mL) 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

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(S & L Aerospace Metals, Maspeth, NY or Fisher Scientific catalog
number 07-907-5Q as of July 2012). Use only carriers that passed
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 disinfectant. 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.

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).

The AOAC 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 to a tube containing 10
mL of growth medium (e.g., synthetic broth) and incubate at 36 ±
1°C for 24 ± 2 h. Only one daily transfer is required prior to the
initiation of the final test culture. Up to 5 daily transfers into 10 mL
of broth may be conducted prior to the inoculation of the final test
cultures. Do not vortex the 24 h culture prior to final subculture
transfer.

b.	For the final subculture transfer, inoculate a sufficient number of 20

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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

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. Pool the test culture from each 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. 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
transfer to a sterile flask; pool cultures in the flask and swirl to mix.
Aliquot 20 mL portions into sterile 25 x 150 mm test tubes. Prepare
a minimum of 4-20 mL tubes.

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 min. 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 20 mL portions into
sterile 25 x 150 mm test tubes. Prepare a minimum of 4-20 mL
tubes.

f.	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.

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g.	Allow carriers to remain in inoculum for 15 ± 2 min.

h.	Drain the inoculum from the carriers 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.

i.	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.	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. Select one carrier from each of 6 Petri dishes.

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 sonication on the AOAC 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. Refer to the AOAC Use-Dilution
Test Carrier Counts Form (see section 14). 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.

e.	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. Dilutions 10"2 through 10"4 should produce plates
with CFU in the appropriate range. Spread inoculum evenly over
the surface of the agar. Plates must be dry prior to incubation.

f.	Incubate plates (inverted) at 36 ± 1°C for up to 48 ± 2 h.

g.	Count colonies. Plates that have colony counts over 300 will be
reported as TNTC. Record counts on the AOAC Use-Dilution Test

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Carrier Counts Form (see section 14). See section 13 for data
analysis.

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 AOAC 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 AOAC 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. For a contact time of 10 min, the carrier
must be deposited in the tube within ± 5 s of the prescribed drop
time.

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
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

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secondary tube of sterile medium after a minimum of 30 ± 5 min
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. Record the
results for both tubes (a carrier set) after this time.

g. Record timed events on the AOAC 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 AOAC 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 AOAC 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 AOAC Use-Dilution Test Results Sheet (see section
14).

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 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. Use viability
controls for comparative determination of a positive tube. 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

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microbe using isolations streaks on TSA or BAP.

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.

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 or 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 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:

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Organism Culture Tracking Form MB-05-09 Fl.docx

Test Microbe Confirmation Sheet (Quality MB-05-09 F2.docx
Control)

AO AC Use-Dilution Test Time Recording Sheet MB-05-09 F3.docx
for Carrier Transfers

AOAC Use-Dilution Test Information Sheet MB-05-09 F4.docx

AOAC Use-Dilution Test Results Sheet (1°) MB-05-09_F5.docx

AOAC Use-Dilution Test Results Sheet (l°/2°) MB-05-09_F6.docx

Test Microbe Confirmation Sheet MB-05-09 F7.docx

AOAC Use-Dilution Test Carrier Counts Form MB-05-09 F8.docx

AOAC Use-Dilution Test Processing Sheet MB-05-09 F9.docx

Carrier Count Spreadsheet MS Excel spreadsheet: MB-05-09_F10.xlsx
Carrier Count Template_UDT_v2

15. References

1.	Official Methods of Analysis. July 2012. 18th Ed., AOAC
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. 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-Wellcol ex* Colour Salmonella. Remel. Part no.
R30858301. Revised 10/17/07.

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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

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
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.

Attachment 2

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Culture Initiation and Stock Culture Generation Flow Chart for S. aureus, P. aeruginosa, and S.
enterica

® Rehydrate ampule.

i

© 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 (iL
culture from TUBE A; incubate.

Harvest inoculum
from plates.

i

© Culture ID & Quality Control

Prepare frozen
stock cultures

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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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-05-09
Date Revised 08-29-12
Page 15 of 17

Attachment 3

Confirmation Flow Chart for S. aureus

S. aureus Identification

Identified as S. aureus

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SOP No. MB-05-09
Date Revised 08-29-12
Page 16 of 17

Attachment 3 (cont.)

Confirmation Flow Chart for P. aeruginosa

P. aeruginosa Identification

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SOP No. MB-05-09
Date Revised 08-29-12
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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