US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for Media and
Reagents: Preparation and Quality Evaluation
SOP Number: MB-10-06
Date Revised: 01-29-18

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SOP No. MB-10-06
Date Revised 01-29-18
Page 1 of 11
SOP Number
MB-10-06
Title
Media and Reagents: Preparation and Quality Evaluation
Scope
Describes the procedures used to log-in, prepare, and evaluate the
quality of media and reagents used in microbiological assays by the
Microbiology Laboratory Branch (MLB).
Application
For use in the quality evaluation of media and reagents used by
MLB.


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Date SOP withdrawn: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-10-06
Date Revised 01-29-18
Page 2 of 11
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
4
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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SOP No. MB-10-06
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1. Definitions
1.	General growth media = Media which support the growth of a broad
range of microorganisms and are used for their general cultivation and
maintenance. General growth media are non-selective. Examples
include nutrient agar (NA) and tryptic soy agar (TSA) which are used
to grow Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus
sub/ilis. Salmonella en/erica, etc.
2.	Selective media = Media that permit the growth of one type of
bacterium while inhibiting the growth of other types. Selective media
facilitate the isolation of a desired species. Examples include mannitol
salt agar and cetrimide agar.
3.	CFU = Colony forming unit.
4.	Additional 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 chemicals.
3. Personnel
Qualifications
and Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
Refer to SOP QC-13 (autoclaves), EQ-01 (pH meters), EQ-02
(thermometers), and EQ-03 (weigh balances) for details on method and
frequency of calibration.
5. Sample
Handling and
Storage
Store media and reagents as indicated on their Media/Reagent Preparation
Sheets.
6. Quality
Control
1.	Document the required information on the appropriate forms (see
section 14).
2.	Process re-usable glassware in Miele or Lancer dishwashers and check
glassware for detergent residues as noted in SOP QC-03, Glass
Washing and Detergent Residues Test.
3.	Use de-ionized water to prepare media and reagents. Water must meet
the quality indicators noted in SOP-QC-01, Quality Assurance of
Purified Water.
4.	Use appropriate autoclaving procedures (e.g., volume of media per
container, volume of media per autoclave run, etc.) as indicated in
QC-13.
7. Interferences
1. Discard media if a media preparation number or sterilization batch
number is illegible or missing and cannot be determined from the

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SOP No. MB-10-06
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Media/Reagent Preparation Sheet or the Daily Sterilization Record
Information Log Form (see section 14 and QC-13, respectively).
2.	Inspect all pre-sterilized laboratory supplies upon receipt for damage
or torn packaging; discard if supplies are damaged.
3.	Initiate routine sterility checks of pre-sterilized items if contamination
in a test is due to pre-sterilized items.
8. Non-
conforming
Data
Management of non-conforming data (e.g., inadequate media
performance/ sterility) will be specified in the test method; procedures
will be consistent with SOP ADM-07, Non-Conformance Reports.
If a non-conformance involving media/reagents is identified, conduct
an investigation into the cause of the non-conformance and the use of
the media/reagents.
9. Data
Management
Data will be archived consistent with SOP ADM-03, Records and
Archives. Examples include media/reagent preparation sheets,
sterility/performance forms.
Archive certificates of analysis for prepared media purchased by the
laboratory (e.g., TSA with 5% sheep's blood).
10. Cautions
1.	Do not use materials (e.g., media, reagents, pre-sterilized materials)
past the manufacturer's recommended expiration date.
2.	Use volumetric glassware as appropriate as indicated on Media
Preparation Sheets.
3.	Allow hot autoclaved media to equilibrate for a minimum of 30
minutes prior to plating or addition of heat-sensitive additives. If
using a water bath, ensure the temperature of the water bath is set to
45-50C or as appropriate for the media.
4.	Reassessment of the sterility of media and reagents not used in a
timely manner after preparation and/or after storage for more than one
month is strongly recommended prior to use.
11. Special
Apparatus and
Materials
1.	Water baths, for tempering of media.
2.	Inoculating loops, for isolation streaks and media inoculation.
3.	Volumetric glassware, for preparation of media/reagents as
appropriate.
4.	Microbial cultures used in the laboratory; refer to Attachment 2.
5.	Pre-sterilized filtration units with pore size of 0.2 |im such as Nalgene
Analytical Filter Units.

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SOP No. MB-10-06
Date Revised 01-29-18
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12. Procedure and
Analysis
1.	Utilize media/reagent preparation sheets approved by the Quality
Assurance Unit or designee; electronic versions of the preparation
sheets are archived on the shared network drive.
2.	On the electronic media/reagent preparation sheet, enter the total
volume of media/reagent to be prepared - the media/reagent
preparation sheet will automatically adjust the mass/volume of the
ingredients.
12.1 Preparation of
Media and
Reagents
a.	Assign a media preparation number for all media, reagents, and
carriers prepared in the laboratory. Record this number on the
Media/Reagent Preparation Log Form and the Media/Reagent
Preparation Sheet (see section 14). The media preparation
number consists of two parts:
i.	The first seven digits represent the date the medium or
reagent was prepared: P-MMDDYY where P=prepared,
MM=month, DD=day and YY=the last two digits of the
calendar year.
ii.	The suffix, where the digits after the dash act as a counter
for the number of preparations made on the same date.
For example, the first preparation made on January 8,
2018 would have the media preparation number P-
010818-01. The next item prepared on that same day
would have a suffix of -02; the third preparation made on
that same day would have a suffix of -03, etc.
b.	Label each preparation of media or reagent clearly with the name
of the preparation and the media preparation number.
c.	Strictly follow the specific directions for preparation of media
and reagents as listed on the Media/Reagent Preparation Sheet.
d.	Complete all fields on the media preparation sheet with the
appropriate information for that section. If a section is not
applicable to the item being prepared, place N/A (not applicable)
in that section.
e.	Sterilization of media and reagents (listed on the Media/Reagent
Preparation Sheet).
i.	Refer to QC-13 to generate sterilization batch number for
media and reagents that require autoclaving.
ii.	Record the sterilization batch number in the Sterilization
section on the Media/Reagent Preparation Sheet (see
section 14).

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iii.	For media and reagents that are filter sterilized, record the
sterilization mechanism in the Sterilization section on the
Media/Reagent Preparation Sheet (see section 14).
iv.	For media and reagents that do not require sterilization,
record N/A in the Sterilization section on the
Media/Reagent Preparation Sheet (see section 14).
v.	To ensure proper sterilization of media and reagents,
follow the Recommended Media/Reagent Sterilization
Procedures in QC-13.
f.	Verify the pH of the media as specified on the Media/Reagent
Preparation Sheet (e.g., before and/or after autoclaving, once per
lot, etc.).
g.	Record the pH of the media or reagent at room temperature (20C
to 25C) unless otherwise specified on the Media/Reagent
Preparation Sheet.
h.	Discard the batch of media if the pH falls outside of the desired
range as specified on the Media/Reagent Preparation Sheet.
i.	If using a water bath to equilibrate media, record the temperature
of the water bath at the time the media is used (e.g., just prior to
pouring in plates) on the Water Bath Temperature Record form
(see section 14). The temperature recorded must reflect
adjustments by the correction factor for the thermometer
specified.
12.2 Storage, Shelf
Life and
Inspection of
Media and
Reagents
a. The storage and shelf life requirements for the medium or reagent
are listed at the bottom of the Media/Reagent Preparation Sheet.
See Attachment 1 for detailed instructions.
12.3 Media
Performance
Assessment
Conduct the performance verification of media prior to or concurrently
with use, preferably with the organism that corresponds to the
anticipated use of the medium. Media controls used in neutralization
confirmation studies or titer assays may also be used in place of specific
media performance tests.
a. Culture Preparation: Use appropriate dailv culture, test culture,
stock culture or standardized spore suspension for the purpose of
inoculating media for performance assessment. Refer to the
relevant procedures for culture preparation (for example, SOPs
MB-05, MB-07, MB-25, etc.).

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SOP No. MB-10-06
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b.	Inoculation and Incubation of Solid Media: For solid media in
plates or tubes (e.g., TSA, NA, Middlebrook 7H11 (M7H11)),
use 6-8 plates or tubes to assess media performance. Spread
plate, in duplicate, 3-4 serial ten-fold dilutions of the test
microbe.
i.	Target counts of 30-300 CFU/plate are desirable and
should result from at least one of the dilutions plated -
this dilution will serve as the basis for determining media
performance. The dilutions should result in plate counts
which are too numerous to count (TNTC) through
extinction (no CFU) or near extinction levels.
ii.	For solid media in tubes (e.g., NA slants, M7H11 slants),
inoculate a minimum of 2 tubes per batch with an
undiluted culture of the test microbe.
iii.	For selective media in plates or tubes (e.g., mannitol salt
agar, cetrimide agar), streak- (for plates) or stab- (for
tubes) inoculate a minimum of 2 plates or tubes with an
undiluted culture of the appropriate target organism (i.e.,
the organism the media is designed to identify). Perform
an isolation streak on selective media in plates to aid in
the assessment of the medium's reaction and organism's
colony characteristics.
iv.	For selective liquid media (e.g., reinforced clostridial
media (RCM), Middlebrook 7H9 broth with 15% glycerol
(MADC)), inoculate a minimum of 2 tubes with an
undiluted culture of the appropriate organism.
v.	See Attachment 2 for detailed instructions.
c.	Inoculation and Incubation of Liquid Media: For liquid media
(e.g., letheen broth, nutrient broth, Modified Proskauer Beck
broth), evaluate 6-8 tubes for performance testing. Inoculate
tubes in duplicate with 0.1 mL aliquots of 3-4 serial ten-fold
dilutions of the appropriate culture.
i. Verify CFU/tube by spread plating in duplicate on the
appropriate medium. See Attachment 2 for detailed
instructions.
d.	Frequency of Media Performance
i. For commonly used media (see Attachment 3), conduct a
	media performance assessment for each lot of dehydrated

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SOP No. MB-10-06
Date Revised 01-29-18
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medium or every 6 months, whichever comes first.
ii. For all other media, conduct a media performance
assessment for each in-house preparation of media.
12.4 Performance
Results for
Solid Media
a.	Enumerate the colonies per plate, determine the average
CFU/plate and CFU/mL of diluted inoculum, and assess the
colony morphology.
i. Use whole numbers for the average CFU/plate and
CFU/mL, round up when necessary.
b.	Record findings on the Performance and Sterility Assessment of
Media in Plates form (see section 14).
c.	If growth occurs and exhibits typical morphology, record a "+."
If no growth is apparent, record a "0."
d.	If atypical growth is observed (not the test microbe), record as
contaminant.
e.	If the inoculum titer is significantly below the target of 30-300
CFU, then either the media performance is unsatisfactory or the
starting inoculum was substandard; repeat the media performance
assessment.
f.	For selective media, verify the performance oer the aoDrooriate
media reactions (e.g., agar turning fluorescent green for cetrimide
agar) or colony characteristics; refer to the appropriate method
SOP. Complete the appropriate form (see section 14) under the
"Performance Assessment" caption by checking either
Satisfactory or Unsatisfactory per the observations.
g.	For solid media in tubes, record the presence or absence of
growth on the Performance and Sterility Assessment of Media in
Tubes form (see section 14).
h.	For plates, enumerate the colonies per plate, determine the
average CFU/plate and CFU/mL of diluted inoculum, and assess
the colony morphology (refer to SOP MB-05, MB-07, MB-15,
MB-28, or MB-34 for colony characteristics). Record findings
on the Performance and Sterility Assessment of Media in Plates
form (see section 14).
12.5 Performance
Results Liquid
Media
a.	Record findings on the Performance and Sterility Assessment of
Liquid Media and Solid Media in Tubes form (see section 14).
b.	For each tube, record a "+" if growth is observed (indicated by
turbidity or growth) or a "0" if growth is not observed.

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c.	If atypical growth is observed (not the test microbe), record as
contaminant.
d.	Following incubation, assess the performance of each medium
and record observations on the Performance and Sterility
Assessment of Media in Tubes form (see section 14). Read plate
counts to determine the CFU/tube.
e.	Performance is judged to be satisfactory when at least one of the
two tubes in a dilution set that received a sufficiently low
challenge (1-50 CFU/tube) of the test microbe shows growth.
The number of CFU delivered to each tube in a set is based on
the corresponding averaged plate counts for that dilution. All
tubes in dilution sets receiving greater than the targeted challenge
should show growth as well. Based on this criterion under the
"Performance Assessment" caption on the form, check either
Satisfactory or Unsatisfactory.
12.6 Sterility
Verification of
Solid Media
a.	Verify sterility on a minimum of 2% of each preparation of
media.
b.	Place plates or tubes of solid media in an incubator for 3-10 days.
Use an incubation temperature consistent with the anticipated
organism of use. If necessary, place agar plates in sterile plastic
bags during incubation to prevent dehydration.
i. If desired, allow agar plates to remain at room
temperature prior to use to monitor their sterility.
c.	Following incubation, if no growth is observed, record a "0"
(satisfactory). If growth is observed, record a "+"
(unsatisfactory) on the appropriate Performance and Sterility
Assessment form (see section 14). On each form, fill in "Sterility
Assessment" by indicating either Satisfactory or Unsatisfactory
per the observations.
d.	A satisfactory result for sterility is typically based on no
microbial growth observed in the plates or tubes following
incubation. Although infrequent, an occasional bacterial or
fungal colony may appear on the surface of the agar; in these
instances, perform an additional sterility assessment. In addition,
examine the remaining plates of the preparation in question prior
to use to determine the extent of the contamination. If any
growth is observed in the second assessment, discard the
medium.
12.7 Sterility
a. If a medium or reagent is dispensed into multiple bottles,

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SOP No. MB-10-06
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Verification of
Liquid Media
and Reagents
evaluate at least one bottle per preparation for sterility.
b.	Use a pre-sterilized 0.2 |im filter unit and aseptically filter
approximately 2% of the total volume of medium or reagent.
c.	Do not place anything such as a pipette into the reagent bottle.
d.	Aseptically transfer the filter to a TSA, TSA with 5% sheep's
blood (BAP), or NA plate, incubate for 3-10 days, and assess
filter for presence of microbial growth. Use an incubation
temperature consistent with the anticipated organism of use. If
necessary, place agar plates in sterile plastic bags during
incubation to prevent dehydration.
e.	Following incubation of the filter, if no growth is observed,
record a "0" (satisfactory). If growth is observed, record a "+"
(unsatisfactory). Record the assay results and observations on
the appropriate form (see section 14).
f.	For media that cannot be filtered because of their viscosity (e.g.,
fluid thioglycollate medium (FTM), Kirchner's medium,
Middlebrook 7H9 broth), dispense at least 2% of the total volume
prepared into 10-20 mL aliquots in sterile tubes and incubate the
tubes for 3-10 days. Use an incubation temperature consistent
with the anticipated organism of use.
12.8 Prepared
Media and
Reagents
a. An in-house media performance and/or sterility assessment of
prepared media and reagents acquired from commercial vendors
that are accompanied by a certificate of quality/analysis is
optional.
12.9 Sterility of
Carriers
d.
For small carriers (e.g., 1 cm steel disks, steel or porcelain
penicylinders), place one carrier per autoclaved preparation into a
10 mL tube of FTM, letheen broth (LB), or tryptic soy broth
(TSB) and incubate for 3-10 days at 361C.
For glass slide carriers, place one carrier per autoclaved batch
into a 20 mL tube of FTM, LB, or TSB and incubate for 3-10
days at 361C.
Following incubation, if no growth is observed, record a "0" and
if growth is observed record a "+" on the Sterility Verification of
Carriers form (see section 14).
13. Data Analysis/
Calculations
Verify that the amounts of ingredients specified on the media/reagent
preparation sheet are accurate. For example, if a 2 L batch of FTM is
made, the prep sheet should accurately reflect the calculation of 29.8 g
for 1 L multiplied by 2: 29.8 x 2 = 59.6 g dehydrated medium required

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SOP No. MB-10-06
Date Revised 01-29-18
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to prepare 2 L of FTM.
2. Calculate the percent difference due to evaporation or loss during
autoclaving. The percent difference is determined using the formula:
Percent Difference = 100% - [(Measured Volume/Target Value) x
100]
14. Forms and
Data Sheets
1. Attachments/test sheets. Attachments and test sheets are stored
separately from the SOP under the following file names:
Storage and Inspection of Media and Reagents MB-10-06 A1 .docx
Inoculation and Incubation of Solid and Liquid MB-10-06_A2.docx
Media
Media with Reduced Frequency of MB-10-06_A3.docx
Performance Assessment
Media/Reagent Preparation Log Form MB-10-06 Fl.docx
Example Media/Reagent Preparation Sheet MB-10-06 F2.xlsx
Water Bath Temperature Record MB-10-06 F3.docx
Performance and Sterility Assessment of Solid MB-10-06 F4.xlsx
Media in Plates
Performance and Sterility Assessment of MB-10-06 F5.xlsx
Liquid Media and Solid Media in Tubes
Sterility Verification of Reagents MB-10-06 F6.xlsx
Sterility Verification of Carriers MB-10-06 F7.xlsx
15. References
1.	Official Methods of Analysis. Revised 2013. 18th Ed., AOAC
INTERNATIONAL, Gaithersburg, MD, (Methods 955.14, 955.15,
964.02, 961.02, and 966.04).
2.	Official Methods of Analysis. Revised 2012. 18th Ed., AOAC
INTERNATIONAL, Gaithersburg, MD, (Method 965.12).

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