US Environmental Protection
Agency Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Germicidal and Detergent Sanitizing Action of
Disinfectants Test
SOP Number: MB-27-02
Date Revised: 10-28-16
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SOP No. MB-27-02
Date Revised 10-28-16
Page 1 of 16
SOP Number
MB-27-02
Title
Germicidal and Detergent Sanitizing Action of Disinfectants Test
Scope
This SOP describes the methodology used to determine the efficacy
of food contact sanitizers against Staphylococcus aureus and
Escherichia coli. The methodology is based on AO AC method
960.09 - Germicidal and Detergent Sanitizing Action of
Disinfectants - revision date 2013.
Application
For product evaluations under the Antimicrobial Testing Program
(ATP), a study protocol is developed which identifies the specific
test conditions for a test chemical sample including contact time,
dilution, and neutralizer.
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Date SOP withdrawn: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-27-02
Date Revised 10-28-16
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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
3
10.
CAUTIONS
3
11.
SPECIAL APPARATUS AND MATERIALS
3
12.
PROCEDURE AND ANALYSIS
7
13.
DATA ANALYSIS/CALCULATIONS
12
14.
FORMS AND DATA SHEETS
12
15.
REFERENCES
13
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SOP No. MB-27-02
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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 test chemicals.
3. Personnel
Qualifications
and Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
Refer to SOPs EQ-01 (pH meter), EQ-02 (Thermometer), EQ-03 (Weigh
Balance), and EQ-05 (Timers) 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). It is critical to maintain the highest
standards of good laboratory practices and aseptic technique during all
manipulations and handling of stock cultures.
7. Interferences
No contamination is acceptable in the treated or numbers control plates.
8. Non-
conforming
Data
1. Sterility and/or viability controls do not yield expected results.
2. The mean log density for numbers control plates falls outside the specified
range of 7.0 - 8.0 logsio/mL.
3. Manage non-conforming data as specified in the study protocol;
procedures are 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. Strict adherence to the protocol is necessary for the validity of the test
results.
2. Plating should be completed within 1 hour after the initiation of serial
dilutions.
3. For spread plating: ensure that the entire surface of the agar plate is dry
before adding inoculum. If necessary, leave the agar plates uncovered in
the biological safety cabinet (BSC) until the moisture has been completely
absorbed into the medium.
4. Use diluted test chemical within three hours of preparation unless
specified otherwise.
11. Special
Apparatus and
Materials
1. Test organisms. Escherichia coli (ATCC No. 11229) and Staphylococcus
aureus (ATCC No. 6538) obtained directly from a reputable supplier
(e.g., ATCC).
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SOP No. MB-27-02
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2. Culture media. Note: Commercial dehydrated media made to conform to
the recipes provided in AOAC Method 960.09 may be substituted, unless
indicated otherwise.
a. Trypticase Soy Agar (TSA). Prepare according to manufacturer's
instructions. Used for the generation of frozen stock cultures for S.
aureus.
b. Tryptic Soy Broth (TSB). Prepare according to manufacturer's
instructions. Used for the generation of frozen stock cultures for S.
aureus.
c. Nutrient broth. Boil 5 g beef extract (powder), 5 g NaCl, andlO g
peptone (anatone) in 1 L H2O for 20 minutes and dilute to volume
with de-ionized water; adjust to pH 6.8 ± 0.1. Filter through paper
(Whatman No. 4, or equivalent), place 10 mL portions in 20 x 150
mm test tubes, and steam sterilize 20 min at 121°C. Used for the
preparation of Nutrient agar plates.
d. Nutrient agar (AOAC): Dissolve Bacto agar to 1.5% (w/v) in nutrient
broth and adjust to pH 1.2-1 A. Steam sterilize for 20 min at 121°C.
Dispense into plates. Used for the generation of frozen stock cultures
for coli.
e. Nutrient agar -A (NA-A): Boil 3 g beef extract, 5 g peptone and 15 g
salt free agar in 1 L de-ionized water. Do not use premixed
dehydrated medium. Dispense 10 mL portions in 20 x 150 mm tubes
or 20 mL portions in 25 x 150 mm tubes and steam sterilize for 20
min at 121°C. Slant tubes after sterilization and let cool. Used for
daily transfers of test cultures.
f. Nutrient agar - B (NA-B): Boil 3 g beef extract, 5 g peptone and 30 g
salt free agar in 1 L de-ionized water. Do not use premixed
dehydrated medium. Steam sterilize for 20 min at 121°C. Temper
medium prior to dispensing 20-30 mL portions into sterile Petri
dishes. Used for development of the final test culture.
3. Subculture media: choose the appropriate recovery agar and neutralizer to
inactivate the test chemical, for example:
a. Tryptone glucose extract agar plus Neutralizer (TGEA-N): Combine
24 g of dehydrated medium with 975 mL de-ionized water and 25 mL
stock neutralizer if necessary. Steam sterilize for 15 min at 121°C.
Used for the recovery of test organisms from treated samples.
b. Tryptone glucose extract agar (TGEA): Prepare according to the
manufacturer's instructions. Used for the enumeration of numbers
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SOP No. MB-27-02
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control samples.
4. General Media and Reagents:
a. TSB with 15% [v/v] glycerol (Cryoprotectant solution). Suspend 7.5
g tryptic soy broth in 212.5 mL de-ionized water. Add 37.5 g glycerol
and stir until dissolved; boil to dissolve completely. Dispense into
bottles and steam sterilize for 15 minutes at 121°C. Used for the
preservation of frozen stock cultures.
b. Neutralizer Stock Solution (NSS): Mix 40 g Lecithin, 280 mL
polysorbate 80 and 1.25 mL 0.25 M phosphate buffer stock solution.
Dilute with de-ionized water to 1 L and adjust pH to 7.2. Dispense in
100 mL portions and steam sterilize for 20 min at 121°C.
c. Neutralizer blanks (NB): For use with < 200ppm quaternary
ammonium compounds. Mix 100 mL neutralizer stock solution, 25
mL 0.25 M phosphate buffer stock solution, and 1675 mL of de-
ionized water. Dispense into appropriate size vessel and steam
sterilize for 20 min at 121°C. Alternate neutralizers may be used as
necessary.
d. Phosphate buffer stock solution (PBSS): 0.25M. Dissolve 34 g
KH2PO4 in 500 mL de-ionized water in 1 L volumetric flask. Adjust
pH to 7.2 with 1 N NaOH, and dilute to 1 L volume mark. Sterilize
by filtration.
e. Phosphate buffer solution (PBS). Add 1.25 mL of phosphate buffer
stock solution and 8.75 g of NaCl to a volumetric flask; fill with de-
ionized water to the 1000 mL mark and mix. A pH of approximately
7.0 is desirable. Sterilize by either filtration or steam sterilization at
121°C for 15-20 min. Alternative PBS formulations with the same
pH may be used (e.g., dilute commercially prepared 10x PBS solution
to 1 x using de-ionized water).
f. Phosphate buffer dilution water stock solution (PBDW-SS). Dissolve
34.0 g of potassium dihydrogen phosphate (KH2PO4) in 500 mL de-
ionized water. Adjust pH to 7.2 ± 0.2 with 0.1 N NaOH or 0.1 N HC1
and bring to 1000 mL with de-ionized water. Alternative phosphate
buffers with the same pH may be used (e.g., commercially prepared
1 OX PBS solution).
g. Phosphate buffer dilution water (PBDW): Add 1.25 mL of 0.25 M
phosphate buffer stock solution to 1 L de-ionized water. Dispense
into appropriate size vessel and steam sterilize for 20 min at 121°C.
Used for numbers control assay and dilution blanks.
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h. Tween-80 (polysorbate 80).
i. PBS (lx) with 0.1% Tween 80 (PBS + T80): Add 100 mL PBS 10x
solution and 1 mL Tween 80 to a volumetric flask; fill with de-ionized
water to the 1000 mL mark and mix thoroughly. Sterilize by
filtration.
j. 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. Reverse osmosis, distillation, and deionization in various
combinations all can produce reagent-grade water when used in the
proper arrangement. 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.
k. Blood Agar (BAP). Commercially purchased plates. Used for the
presumptive identification of the test microbes.
1. Mannitol Salt Agar (MSA). Combine 111 g of dehydrated medium
with 1 L de-ionized water and mix thoroughly. Steam sterilize for 15
min at 121°C. Used for the presumptive identification of the test
microbes .Xylose lysine deoxycholate agar (XLD). Commercially
purchased plates. Used for the presumptive identification of the test
microbes.
5. Equipment and Glassware. For sanitizer efficacy and numbers control,
use 250 mL wide mouth Erlenmeyer flasks. For measuring sanitizers and
dilution blanks use 100 mL graduated cylinders. For glassware used to
prepare test chemical, refer to SOP MB-22.
a. Petri dishes. Sterile plates, 20 mm x 100 mm in size.
b. Recirculating chiller unit. For maintaining specified temperature of
the test chemical (capable of maintaining 25 ± 1°C).
c. Test tube racks. Any convenient style.
d. Transfer loops. Make 4 mm ID 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. Commercially available 4 mm ID transfer loops may
also be used. Volumetric transfer devices may be used instead of
transfer loops (e.g., micro volume pipette).
e. Timer. For managing timed activities, any certified timer that can
display time in seconds.
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f. Micropipettes. For performing culture transfers and serial dilutions.
g. Whatman No. 2 filter paper. Sterile.
h. Gram stain kit.
i. Vitek 2 Compact. For microbe identification and confirmation.
12. Procedure and
Analysis
Prior to testing, perform the neutralization assay to determine if the prescribed
neutralizer is appropriate for the test chemical.
12.1 Culture
Preparation
Refer to Attachment 2-A1 for preparation of the frozen stock cultures. Refer
to MB-02: Tracking of Test Microorganisms, Section 12 for the tracking and
transfer notations for the test microbes. Reinitiate new frozen stock cultures
every 18 months with a new lyophilized culture.
a. Defrost a single cryovial of frozen stock culture at room temperature
and briefly vortex to mix. Streak one loopful of the thawed frozen
stock onto a NA - A slant 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.
b. For the final test culture, add 5 mL of PBDW to a NA - A slant
(daily culture). Using a sterile loop, dislodge growth from agar
surface. Collect mixture and transfer to a flask containing 99 mL of
PBDW. Mix throughly. Add 200 |iL of the mixture to inoculate a
minimum of 5 NA - B plates to create a bacterial lawn. Incubate at
36 ± 1°C for 24 ± 2 h.
c. Record all culture transfers on the Organism Culture Tracking Form
(see section 14).
12.2 Test Culture
Harvesting
a. After incubation, add a minimum of 5 mL of PBS + Tween 80 to
each plate. Using a sterile rod, gently dislodge culture from agar
surface, avoid disrupting agar. Combine culture from all plates and
mix thoroughly.
b. Filter culture through sterile Whatman No. 2 filter paper using a
vacuum source; collect filtered test culture into a sterile vessel.
c. Standardize the filtered test culture, as necessary using PBDW to
achieve a final test culture microbe titer between 1.0 x io9 CFU/mL
and 1.0 x 1010 CFU/mL (9-10 logio/mL).
12.3 Test chemical
Sample
Preparation
a. Prepare test chemical sample per SOP MB-22 (Disinfectant
Product).
1) Ready-to-use test chemicals are tested as received; no dilution
is required.
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SOP No. MB-27-02
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2) If hard water is required as the diluent, prepare synthetic hard
water as described in SOP MB-22 (Disinfectant Product).
b. Equilibrate water bath and allow it to come to 25 ± 1°C or the
temperature specified (±1°C). Prepare the test chemical dilutions
within 3 h of performing the assay.
c. Dispense 99 mL aliquots of the diluted test chemical or ready-to-use
test chemical into sterile wide mouth Erlenmeyer flasks. Prepare
one flask per test microbe for each germicide to be tested.
d. Place flasks in the equilibrated water bath for approximately 10 min
to allow test solution to come to specified temperature.
e. Record the temperature of the water bath and recirculating chiller
before and after testing on the Germicidal and Detergent Sanitizing
Action of Disinfectants Method Test Information and Culture
Preparation Sheet (see section 14).
f. In addition, prepare a similar flask containing 99 mL of PBDW to
use for numbers control for each organism (see section 12.5)
12.4 Treated
Sample Test
Procedure
Flasks containing test culture x test chemical are referred to as the treated
samples.
a. Add 1 mL of test culture to the test flask as follows:
b. Whirl flask, stopping just before suspension is added, creating
enough residual motion of liquid to prevent pooling of suspension at
the point of contact with test sample.
c. Add suspension midway between center and the inner edge of the
flask with tip of pipette slightly immersed in test solution. Avoid
touching the neck or side of flask during addition. Swirl flask to
thoroughly mix contents.
d. At 30 ± 3 seconds after addition of the test culture, transfer a 1 mL
aliquot from the test flask (test culture x test chemical) to a tube
containing 9 mL neutralizer blank and mix well. This corresponds to
10"1 dilution tube. Record timed events on the Germicidal and
Detergent Sanitizing Action of Disinfectants Method Time
Recording Sheet for Transfers (see section 14).
e. Treated samples plating. — From 10"1 tube (i.e., 9 mL neutralizer
tube inoculated with 1 mL of exposed culture), plate four 1 mL
aliquots and four 0.1 mL aliquots onto TGEA - N plates, for a total
of 8 plates per treated sample. This will result in 10"1 and 10"2
dilutions respectively. Incubate plates at 36±1°C for 24-30 hours.
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f. Following incubation, count colonies on TGEA-N plates. Counts
over 300 are recorded as TNTC. Record plate counts on the
Germicidal and Detergent Sanitizing Action of Disinfectants
Method Results Sheet (see section 14).
12.5 Numbers
Control
Procedure
Flask containing 99 mL of PBDW is used for the numbers control procedure
and referred to as the numbers control sample.
a. Numbers control assay should be conducted within 5 minutes of
completion of treated samples.
b. In a sterile 250 mL wide mouth Erlenmeyer flask containing 99 mL
sterile PBDW - numbers control sample, add 1 mL of the test
culture (same test culture used for the treated samples) as follows:
c. Whirl flask, stopping just before suspension is added, creating
enough residual motion of liquid to prevent pooling of suspension at
the point of contact with test sample.
d. Add suspension midway between center and the inner edge of the
flask with tip of pipette slightly immersed in test solution. Avoid
touching to the neck or side of flask during addition. Swirl flask to
thoroughly mix contents.
e. Numbers control plating. — Within 30 seconds of addition of test
culture, transfer 1 mL aliquot from the test flask (test culture x
PBDW) into a tube containing 9 mL of neutralizer and mix well.
This corresponds to 10"1 dilution tube. Make serial 10-fold dilutions
in 9 mL PBDW, out to 10"6.
f. Plate four 1 mL aliquots and four 0.1 mL aliquots from the 10"6
dilution tube onto TGEA plates, for a total of 8 plates per numbers
control sample. This will result in 10"6 and 10"7 dilutions,
respectively.
g. Incubate plates at 36±1°C for 24-30 hours.
h. Following incubation, count colonies on TGEA plates. Counts over
300 are recorded as TNTC. Record plate counts on the Germicidal
and Detergent Sanitizing Action of Disinfectants Method Results
Sheet (see section 14).
i. For a valid test, the numbers control must fall between 7.0 - 8.0
logio/mL.
12.6 Sterility
controls
a. Neutralizer blank - plate 1 mL from a previously unopened tube
onto a TGEA plate.
b. Test chemical - Plate 1 mL of the test chemical used in the assay
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SOP No. MB-27-02
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onto a TGEA plate.
c. Diluent - Plate 1 mL of the diluent, if necessary onto a TGEA plate.
d. Incubate all plates at 36±1°C for 24 -30 hours, record results.
e. To be considered valid, no growth should be observed on any of the
sterility controls.
12.7 Neutralization
Confirmation
Test
a. A neutralization confirmation test should be performed prior to or
concurrently with the sanitizer evaluation assay. The neutralization
assay must demonstrate the recovery of a low level test organism
population (e.g., 10-100 CFU/mL) in the recovery media (i.e.
TGEA-N and TGEA).
b. Test Culture Titer (TCT) .— Add 0.1 mL of the test organism, which
has been serially diluted to target between 10-100 CFU/mL to 10
mL of PBDW and mix thoroughly. Dilutions 10"4 and 10"5 should
provide the range of 10-100 CFU/mL. Hold the mixture for a
minimum of two minutes. Plate 0.1 mL aliquots in duplicate onto
TGEA. Incubate plates at 36±1°C for 24-30 hours and record
number of colonies.
c. Neutralization Confirmation Treatment (NCT).— Add 1 mL of the
test chemical to 9 mL of the prescribed neutralizer and mix
thoroughly. Within 30 seconds, inoculate the sample with 0.1 mL of
the test organism used for the TCT. Mix thoroughly. Hold the
mixture for a minimum of two minutes. Plate 0.1 mL aliquots in
duplicate onto TGEA-N. Incubate plates at 36±1°C for 24-30 hours
and record number of colonies.
d. Neutralization Toxicity Treatment (NTT).—Add 0.1 mL of the test
organism used for the TCT to 10 mL of the prescribed neutralizer
and mix thoroughly. Hold the mixture for a minimum of two
minutes. Plate 0.1 mL aliquots in duplicate onto TGEA-N. Incubate
plates at 36±1°C for 24-30 hours and record number of colonies.
e. Plates that have colony counts over 300 are reported as TNTC.
Record the counts on the Neutralization Confirmation Assay Results
Sheet (see section 14).
f. Neutralization Results and Calculations.— In order to demonstrate
effective neutralization of the sanitizer, differences between
treatments should not exceed 1.0 log (e.g., TCT minus NCT).
To calculate CFU/mL for Neutralization Confirmation Test use the following
equation.
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SOP No. MB-27-02
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{(Mean CFU for 10 x) + (Mean CFU for 10 y)}
CFV/mL = w_x + w_y
where 10 x and 10 v are the dilutions plated. Two plates per dilution are
plated for all treatments. Alternatively if only one dilution is plated use the
same equation for only one dilution. Use counts of 0 to 300 for calculation
purposes. Score counts >300 as TNTC (too numerous to count).
NOTE: A spreadsheet will be used for data analysis and calculations
associated with the neutralization confirmation assay.
12.8 Results
a. For a valid test, numbers control counts must fall between 7.0 - 8.0
logs.
b. For the test chemical to be considered efficacious, a mean > 5 log
reduction is required with a contact time of 30 seconds.
c. Retesting guidance : For tests where the test chemical meets the
performance standard and the numbers control mean logio density
value is above 8.0, no retesting is necessary. For tests where the test
chemical fails to meet the performance standard and the numbers
control mean logio density is below 7.0, no retesting is necessary.
12.9 Confirmatory
Steps for Test
Microbes
a. Presumptive identification of the test microbes will be conducted
when results are indicative of a failing efficacy evaluation or when
results are inconclusive.
b. Representative growth from one plate per treatment sample should
be confirmed by Gram stain and growth characteristics on general
and selective media.
c. Gram stains are performed on smears taken from the treatment
sample plates. For the additional confirmatory tests, a smear of
culture from each selected treatment sample plate is streaked onto
BAP and selective media appropriate for the test organism and
incubated for 18-24 hours at 36 ± 1°C. See Attachment 1 for Gram
stain reactions, cell morphology, and colony characteristics on solid
media.
d. If characteristics on general and/or selective media are unusual, then
further confirmation will be conducted by VITEK, see SOP-QC-22:
VITEK 2 Compact: Use, Maintenance and Quality Control
Procedures for details.
e. If confirmatory testing determines that the identity of the organism
was not the test organism, the entry on the results sheet must be
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annotated 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.
To calculate CFU/mL, use the following equation.
, {(CFU for 10"x)+ (CFU for 10"^)}
CFU/mL = 10-*+10-„
where 10 x and 10 v are the dilutions plated. Four plates per dilution are plated
for treated samples and numbers control samples. Use counts of 0 to 300 for
calculation purposes. Score counts >300 as TNTC (too numerous to count).
a. Calculate the mean logio density (LD) for number control plates
b. Calculate the mean logio density (LD) for treated samples plates.
c. Calculate the logio reduction (LR) for treated samples:
Logio reduction = mean logio numbers control - mean logio treated sample
NOTE: If zeros are observed for treated sample, substitute 0.5 for the zero at
the lowest (least dilute) dilution and account for the dilution factor in the
calculations.
14. Forms and Data
Sheets
1. Attachment 1: Typical Growth Characteristics of strains of S. aureus and
E. coli
2. Attachment 2: Culture Initiation Flow Chart for S. aureus and E. coli
3. Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
Organism Culture Tracking Form MB-27-02 Fl.docx
Test Microbe Confirmation Sheet (Quality MB-27-02 F2.docx
Control)
Germicidal and Detergent Sanitizing Action of MB-27-02 F3.docx
Disinfectants Method: Test Information and
Culture Preparation Sheet
Germicidal and Detergent Sanitizing Action of MB-27-02 F4.docx
Disinfectants Method: Serial Dilution/Plating
Tracking Form
Germicidal and Detergent Sanitizing Action of MB-27-02 F5.docx
Disinfectants Method: Titer of Final Test culture
Form
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Germicidal and Detergent Sanitizing Action of MB-27-02 F6.docx
Disinfectants Method: Results Sheet
Germicidal and Detergent Sanitizing Action of MB-27-02 F7.docx
Disinfectants Method: Time Recording Sheet for
Transfers
Germicidal and Detergent Sanitizing Action of MB-27-02 F8.docx
Disinfectants Method: Test Microbe
Confirmation Sheet
Germicidal and Detergent Sanitizing Action of MB-27-02 F9.docx
Disinfectants Method: Neutralization
Confirmation Assay - Neutralization
Confirmation Control
Germicidal and Detergent Sanitizing Action of MB-27-02 FlO.docx
Disinfectants Method: Neutralization Toxicity
Control
Germicidal and Detergent Sanitizing Action of MB-27-02 F1 l.docx
Disinfectants Method: Neutralization
Confirmation Assay - Test culture Control
Germicidal and Detergent Sanitizing Action of MB-27-02 F12.docx
Disinfectants Method: Neutralization
Confirmation Assay Results Sheet
15. References
1. Official Methods of Analysis. 2013. 18th Ed., AOAC
INTERNATIONAL, Gaithersburg, MD. Method 960.09: Germicidal and
Detergent Sanitizing Action of Disinfectants. Revised First Action 2013.
2. Package Insert - Gram Stain Kit and Reagents. Becton, Dickinson and
Company. Part no. 882020191JAA. Revision 07/2011.
3. Package Insert - Catalase Reagent Droppers. Becton, Dickinson and
Company. Part no. L001237. Revision 06/2010.
4. Package Insert-Staphaurex Plus*. Remel. Part no. R30950102. Revised
11/23/07.
5. Package Insert - Rapid Indole Reagent Droppers. Becton, Dickinson and
Company. Part no. L001237. Revision 06/2010.
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SOP No. MB-27-02
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Attachment 1
Typical Growth Characteristics of strains of S. aureus and E. coli.
S. aureus*
E. coli*
Gram slain reaction
(+)
(-)
Typical Growth Characteristics on Solid Media
BAP
small, circular, yellow or while,
glistening, beta hemolytic
large, round, while colonies, non-
hemolytic.
Mannitol Salt
circular, small, yellow colonies, agar
turning fluorescent yellow
No Growth
XLD agar
No growth
large, flat, yellow colonies with no
black center.
Typical Microscopic Characteristics
Cell dimensions
0.5-1.5 |im in diameter
0.5 x 1-3 |im in diameter
Cell appearance
spherical, occurring singly, in pairs
and tetrads, sometimes forming
irregular clusters
varying from almost coccoid forms to
long rods, occurring singly, in pairs
and in short chairs. Motile or non-
motile, if motile with flagella.
*After 24±2 hours
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Attachment 2
Culture Initiation and Stock Culture Generation Flow Chart for S. aureus and E. coli.
© Rehydrate ampule.
© Transfer entire
rehydrated pellet to
TUBE A.
Ampule
Incubate
TSB or
NB
TUBE A TUBE A
(pre-incubation) (post-incubation)
©Stock Culture Generation
Inoculate TSA or NA plates with
100 |iL culture from TUBE A;
incubate.
Harvest inoculum
from plates.
© Culture ID & Quality Control
Prepare frozen
stock cultures
BAP
A
Selective
media
Gram Additional
Stain confirmation steps
(VITEK or
biochemical/antigenic
analyses)
A1. Preparation of Frozen Stock Cultures. Refer to SOP MB-02 for establishment of the
organism control number.
a. Initiate new stock cultures from lyophilized cultures of Staphylococcus aureus (ATCC
6538), and Escherichia coli (ATCC 11229) 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 S. aureus orNB for is. coli, aseptically withdraw 0.5 to 1.0 mL and
rehydrate the lyophilized culture. Aseptically transfer the entire rehydrated pellet back
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SOP No. MB-27-02
Date Revised 10-28-16
Page 16 of 16
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
manipulations on the Organism Culture Tracking Form (see section 14).
d. Using a sterile spreader, inoculate a sufficient number of TSA plates for S. aureus or
NA for E. coh, (e.g., 5 to 10 plates per organism) with 100 |iL each of the culture to
create a bacterial lawn. 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.
Repeat by adding another 5 mL cryoprotectant to the agar plates, resuspend the cells,
aspirate suspension, and pool with the initial cell suspension. Transfer the suspension
into a sterile vessel.
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. Store the cryovials at -70°C or below; these are the frozen stock cultures. Store stock
cultures up to 18 months. These vials 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 streak a loopful onto selective media (i.e.,
MSA); for E. coli, 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 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 conduct VITEK confirmation, see SOP-QC-22:
VITEK 2 Compact: Use, Maintenance and Quality Control Procedures for details.
e. Record all confirmation results on the Test Microbe Confirmation Sheet (Quality
Control) (see section 14).
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