Procter & Gamble

Microbiology Standard Operating Procedure (RESTRICTED)

Title Microbiological Media Control and Equivalency

Global Microbiology
010 GCAS.Revision 92319714.005
Organization (GMO)
Ute Krone 30 Days after
Originator Dawn Widener
Implementation Date Effective Date
Approver Approver
Ingrid Bosch (AD) Angelique Boyer (AD)
Industrial Microbiology R&D Corporate Bioscience
Applies to Industrial Microbiology and R&D Microbiology

SCOPE

This Standard Operating Procedure (SOP) refers to microbiological media prepared in-house, commercially available ready-
made microbiological media, and media-reagents used for quality control or research purposes within the microbiological
laboratories. The quality control testing applies to the initial generation of a batch. Contract manufacturing sites and/or
laboratories are expected to maintain similar programs, but are not accountable to the details of this SOP. At a minimum they
have to perform the same tests according to the Media Making Requirements (MMR), as well as comply with applicable
compendial or regulatory guidance concerning those tests.

PURPOSE

The purpose of this SOP is to ensure the quality and functional properties of microbiological media and media-reagents prior
to use in routine testing. The SOP also describes the process by which a manufacturer’s microbiological media are deemed
equivalent to the respective Media Making Requirement (MMR) standard(s).

RESPONSIBILITIES

Ready-made purchased media In-house prepared media

Verify Vendor Certificate of Analysis Perform Quality Control Testing per MMR;
Microbiology Analyst
(CoA) report Non-Conforming Results

Confirm data from CoA Review and approve media quality control testing;
investigate Non-Conformance
Media Laboratory Owner,
Site Microbiologist, Assess if media are equivalent (according to MMR) or if an assessment via Global R&D
or qualified designee Micro Media Owner is needed

Submit requests for reduced frequency testing (Attachment IV)

Review and approve requests for reduced frequency testing (Attachment IV)

Approve supplier qualification as needed (Attachment II)

Regional Microbiologist
Approve media equivalency assessment request as needed (Attachment III) or forward to
R&D Microbiologist

Update “Microbiological Media Manufacturer Approved Media List” on current Microbiology

(PS) Share Point

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 Procter & Gamble
Microbiology Standard Operating Procedure (RESTRICTED)

Title Microbiological Media Control and Equivalency

Global Microbiology
010 GCAS.Revision SOP-92319714.005
Organization (GMO)

Approve supplier qualification as needed (Attachment II)

Global R&D Micro Media Approve media equivalency assessment request as needed (Attachment III)
Owner or delegate
Validate media expiry date (Attachment V) and
Not applicable
update Attachment I

DEFINITIONS

Term Definition
A unique number assigned to a grouping of material having been uniformly processed and having
identical character and quality within specified limits.
Unique processing example: A prepared suspension of broth is processed in two different autoclaves.
Batch/Lot This requires two batch number assignments.
Identical character / quality example: A prepared suspension of broth is dispensed in 9 ml and 90 ml
quantities and sterilized in a single autoclave. Due to volume differences, two batch number
assignments must be given as the identity is different.
Differential Media that differentiates closely related organisms or groups of organisms. Presence of certain dyes or
Media chemicals in the media produce the differential characteristic changes or growth patterns.
Performed to verify test conditions, like the aseptic process, of all individual procedures. It is performed
using the chosen lot code of diluent in place of the test sample material in accordance to procedure
requirements for each individual examination test.
Negative
For example, for the specified test for E. coli, the test diluent will be exposed to the full test procedures
Control
including the same lot codes of media as the test sample. Minimally, this is to be done for each new
media lot used in the test. There must be no growth of microorganisms. A failed negative control
requires an investigation to determine validity of results.
The original source of the organism. If obtained directly from ATCC, the lyophilized source is considered
Master Seed the master seed (0). Subsequent passage of this source is to be considered passage 1. Companies
Lot which market ATCC strains may be several passages removed from the original ATCC source. Consult
the CoA to determine passage number.
A test article or an aliquot of that test article for quality control testing purposes.
Media
For example, large volume articles (>90 ml) may have 10 ml aliquots dispersed into sterile containers as
Sample
test samples for certain quality control testing purposes.
Media which inhibits one genre of microorganism while promoting growth of another.
Selective
For example, MacConkey Agar selects for gram negative organisms while inhibiting gram positive
Media
organisms.
Sterility as referred to in this procedure reflects a check in the verification of successful media
Sterility sterilization. Sterility is performed on unopened containers only. Sterility referred in this procedure is
defined within each MMR.
Reference standard plates upon which the standard average colony forming unit (cfu) value of the
Verification
challenge inoculum is determined. Reference standard plates are considered previously qualified
of Inoculum
batches / lots.

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 Procter & Gamble
Microbiology Standard Operating Procedure (RESTRICTED)

Title Microbiological Media Control and Equivalency

Global Microbiology
010 GCAS.Revision SOP-92319714.005
Organization (GMO)

Media Excipients in media are defined as those ingredients not related to neutralization. They may encompass
Excipient peptones, carbohydrates, buffers, selective agents, dyes/indicators, agar, etc.
Ingredients within a microbiological formula that are designed to inactivate preservative / hostile
Neutralizers
compounds present in a product formulation.

PROCEDURE

Each microbiological medium used in P&G test methods is assigned an individual GCAS number referred to as Media Making
Requirement (MMR). The MMR contains the medium identity or standard formula that was used in method suitability and
quality control parameters. All approved microbiological media and manufacturers may be found on the “Approved
Manufacturer and Media List” on the current microbiology (PS) share point. If a Manufacturer or medium is not in the list, the
Supplier Qualification Form (Attachment II) and the Media Equivalency Form (Attachment III) need to be completed and
approved.

1. General

1.1. Country regulations for the control of media have to be followed if they are more stringent than the process
described below.

1.2. All materials, reagents, and equipment required for this procedure should be of appropriate design, condition of
cleanliness / sterility, and within calibration / expiration dating as appropriate for their intended use. All operators
who will execute this procedure are properly trained.

1.3. After initial sterilization, all agar media may only be remelted once. The maximum storage time for molten agar
stored in a water bath is 8 hours at a recommended temperature of 44-48 ºC. Molten agar which is intended for
use more than 24 hours after sterilizing will be allowed to cool immediately until the agar gels, and then it must be
refrigerated at 2-8°C (room temperature requires additional validation of shelf life).

1.4. Media making capabilities established in-house will have properly documented and qualified / validated equipment
(autoclaves, dispensers, etc.), appropriate quality control equipment process checks, and validation prior to making
media or pursuing reduced frequency testing.

2. Manufacturer / Supplier and Media Qualification

Manufacturers for dehydrated media or ready-made media must be qualified (Attachment II).

2.1. Confirm that media composition meets composition specified in MMR.

2.2. Confirm that manufacturer tests each batch according to MMR and provides a CoA.

2.3. Perform manufacturer assessment or receive sufficient manufacturer information (e.g. certifications, accreditations).

2.4. Test 3 lots of each media and confirm results meet specified parameters in MMR.

2.5. Get approval for manufacturer qualification from Regional Microbiologist / R&D Microbiologist.

3. Media Equivalency

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 Procter & Gamble
Microbiology Standard Operating Procedure (RESTRICTED)

Title Microbiological Media Control and Equivalency

Global Microbiology
010 GCAS.Revision SOP-92319714.005
Organization (GMO)

3.1. When assessing microbiological media excipients, it is understood that the manufacturer’s formula may in some
circumstances differ slightly from the MMR reference standard as biological materials are varied in composition.
The manufacturer may adjust or supplement the media to meet performance expectations through additions,
deletions, or adjustments of ingredient excipients.

3.2. Media are considered equivalent if

 the media manufacturer states “Meets United States Pharmacopeia (USP), European Pharmacopoeia (EP)
and / or Japanese Pharmacopoeia (JP)” in the performance specifications or uses similar wording (media not
developed by Procter & Gamble).
 the media composition meets composition specified in the MMR and salts or agar amounts do not deviate by
more than 10%.

3.3. For media considered equivalent according to the principles described above the Site Microbiologist or R&D
Microbiology Analyst provides the information and tests requested in Attachment III to the Regional Microbiologists
or Global R&D Micro Media Owner for approval.

3.4. In case of deviations in the media composition beyond salt or agar amount, the equivalency needs to be assessed
by the Global R&D Micro Media Owner. The Site Microbiologist or R&D Microbiology Analyst has to provide the
information requested in Attachment III and send to the Regional Microbiologist or Global R&D Micro Media Owner.
The Regional Microbiologist will check the information in the form and forward it to the Global R&D Micro Media
Owner.

3.4.1.Provided that the additional neutralizers do not bridge across MMR GCAS standards the media are
considered equivalent, e.g. Modified Letheen Broth has four different configurations based on added
neutralizers : Letheen Broth, Modified Letheen Broth, Modified Letheen Broth + Tween, Modified Letheen
Broth + Tween + Lecithin.

3.4.2.If a manufacturer’s formula has additional neutralizers that conform more to a different MMR standard or
contains interfering substances, it is not equivalent.

Common Neutralizing Agents

Interfering Substances Neutralizing Agents
Glutaraldehydes, mercurials Sodium hydrogen sulfite (sodium bisulfite)
Aldehydes Glycine
Quaternary ammonium compounds, parahydroxy-
Lecithin
benzoates (parabens), bisbiguanides
Quaternary ammonium compounds, iodine, parabens Polysorbate
Mercurials Thioglycollate
Mercurials, halogens, aldehydes Thiosulfate
EDTA (edetate) Mg or Ca ions

3.5. As all media undergo quality control tests or qualification tests including appearance, pH, quantitative growth
promotion or qualitative growth check, sterility, and volume verification, the variances in the supplier formula will be
verified for media growth promoting qualities.

4. Individual Component Weight Variance

4.1. In-house prepared media, where no weight variance for the individual components is specified in the MMR, will
adhere to the following principles:

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 Procter & Gamble
Microbiology Standard Operating Procedure (RESTRICTED)

Title Microbiological Media Control and Equivalency

Global Microbiology
010 GCAS.Revision SOP-92319714.005
Organization (GMO)

MMR Specified Weight Decimal Example Weight Allowed Weight Variance

Whole number 1-4 g ± 5%
≥5g ± 0.5 g
Tenths place 7.2 g ± 0.1 g
Hundredths place 0.85 g ± 0.01 g

5. Medium / Media-Reagent Receipt

5.1. Upon receipt of media and / or media-reagents, the Microbiology Analyst shall ensure that all shipping containers
are not damaged or compromised.

5.2. The microbiologist shall ensure that the supplier testing listed on the CoA matches the testing parameters listed on
the MMR.

5.3. Purchased chemical media-reagents which are not assigned an expiration date from the supplier shall be assigned
an expiry date of 30 days until the validation of the expiry date is completed.

5.4. Media prepared in-house will be assigned an expiry date not to exceed the time frames in Attachment I. If the media
is not listed in Attachment I, an expiry date of 30 days shall be assigned until the validation is completed by R&D
Microbiology (see Attachment V for example of protocol).

6. Quarantine and Release Program

6.1. The Microbiology Laboratory shall employ a quarantine and release program to control microbiological media and
media-reagents. Upon acceptable review and approval of quality control testing, each batch may be released for
use.

6.2. If microbiological media and reagents need to be utilized concurrently with quality control testing, the laboratory is to
follow applicable site procedure(s) for processing of exceptions including the following principles:

6.2.1. Media exception occurrences should be infrequent.

6.2.2. The Reviewer or Site Microbiologist agrees that all products analyzed with media being concurrently
approved, are subject to hold until all media qualification results are acceptable and approved.

6.2.3. Should quality control testing of media or reagents result in failing disposition, the Reviewer or Site
Microbiologist accepts responsibility that all results generated for test samples utilizing those media or
reagents are invalid and the testing must be repeated with qualified media.

7. Test Parameters and Requirements (according to MMR)

7.1. In-house prepared media

Manufacturing facilities or laboratories which manufacture and / or test regulated drug products are to follow all
testing parameters. If a manufacturing facility or laboratory manufactures or tests products that are both drug
regulated and non-drug-regulated or change to drug related status, the media quality control program is to default
to the highest standard.

7.2. Ready-made purchased media

For sites that are purchasing ready-made media meeting the requirements listed in the MMRs from qualified

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 Procter & Gamble
Microbiology Standard Operating Procedure (RESTRICTED)

Title Microbiological Media Control and Equivalency

Global Microbiology
010 GCAS.Revision SOP-92319714.005
Organization (GMO)

manufacturers, testing may be reduced once approved by the Regional Microbiologist / Global R&D Micro Media
Owner via Attachment IV Reduced Testing Request. CoA’s are to be reviewed and retained from each lot received.
To qualify for reduced testing frequency the media storage temperatures have to be met and documented for the
transportation process.

7.3. Testing Frequency

Group 1
Media used for testing of products which are considered drugs, quasi drugs, over the counter (OTC) drugs, or
medical devices in country of sale

Media Sterility, Volume Inhibition Growth Promotion Growth

pH Check

Dehydrated CoA Not Each lot Each lot Not

applicable applicable

In-house Each lot 1 lot / Each lot Each lot Not

prepared quarter* applicable

Ready- CoA CoA CoA CoA Each lot if

made no growth
Annual Annual Quarterly verification Quarterly verification
purchased promotion /
verification verification with all with all microorganisms
inhibition
microorganisms according to MMR
performed
according to MMR
* see 7.7

Group 2
Media used for testing of products which are not considered drugs, quasi drugs, over the counter (OTC) drugs,
or medical devices in country of sale

Media Sterility, Volume Inhibition Growth Promotion Growth

pH Check

Dehydrated CoA Not Each lot Each lot Not

applicable applicable

In-house Each lot 1 lot / Quarterly verification Quarterly verification Each lot if
prepared quarter* with all with all microorganisms no growth
microorganisms according to MMR promotion /
according to MMR inhibition
performed

Ready- CoA CoA CoA CoA Not

made applicable
Annual Annual Annual verification with Annual verification with
purchased
verification verification all microorganisms all microorganisms
according to MMR according to MMR

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 Procter & Gamble
Microbiology Standard Operating Procedure (RESTRICTED)

Title Microbiological Media Control and Equivalency

Global Microbiology
010 GCAS.Revision SOP-92319714.005
Organization (GMO)

* see 7.7

Group 3
Experimental media without MMR

Media Sterility, pH Volume Inhibition Growth Promotion Growth Check

Data Owner / Reviewer and Micro Media Lab Owner are establishing test parameters, test
requirements, and test frequency.

7.4. Appearance
All plates / tubes / jars of each lot should be checked for appearance imperfections, e.g. clumping, cracking,
desiccation, or excessive moisture, before they are used.

7.5. Sterility
If no information is available in the MMR for turbid broths, streak out onto TSA (GCAS 92347889) and incubate at
30-35°C for at least 18-24 h. Medium is suitable if no microbial growth is observed.

7.6. pH
Allow sample to equilibrate to room temperature. Take pH before dispensing and sterilization and adjust to be
within specification. For validation of the media making process the final pH has to be taken after sterilization to
confirm the medium is within the specified range.

7.7. Volume Verfication

For media that require a method specified volume, a volume verification is performed either volumetrically or by
weighing by difference. Results are acceptable if the volume is within 5.0 % of the intended target.
Volumes can be validated for each specific load / container / volume autoclave configuration. In this manner, the
volume lost during sterilization will be accounted for initially and then verified after sterilization to ensure the volume
is within 5.0 % of the intended target. After each specific load / container / volume configuration is validated,
volume verifications can be reduced to one batch per quarter for each validated volume.

7.8. Inhibition
Inoculate containers with >100 cfu of the challenge organism. Dilutions needs to be prepared for the
microorganisms. TSA is used to confirm inoculum is >100 cfu.

7.9. Growth Promotion

7.9.1.Microorganism Challenges
Microorganisms used must be no more than than 5 passages removed from the master seed lot (national
repository) and must be individual. Mixed pools of microorganism challenges may not be used.
Quantitative delivery systems such as Microbiologic EZ CFU may be used to deliver the ≤100 cfu challenge
organism. For some selective and differential media, it may be necessary to prepare a fresh 18-24 hour
culture and dilute to ≤100 cfu to obtain suitable characteristic growth.

7.9.2.Inoculation of Test Media

Inoculate samples of the test media and verification media with ≤100 cfu for each of the specified growth
promotion microorganisms and incubate accordingly. Verification media are previously qualified batches of the
same type of media or TSA. For selective media only previously qualified batches of the same type of media
are applicable as verification media. Test media and verification media are to be incubated at the same
temperature and for the same time.

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 Procter & Gamble
Microbiology Standard Operating Procedure (RESTRICTED)

Title Microbiological Media Control and Equivalency

Global Microbiology
010 GCAS.Revision SOP-92319714.005
Organization (GMO)

7.9.3.Results Interpretation – Quantification Properties

Plate counts should be recorded to confirm that the medium was spiked with <100 cfu and compared to a
control plate to determine pass / fail. Media pass if there are 50-200 % recovery on the new batch of media
compared to the verification plates.

7.9.4.Results Interpretation – Agar for Presence / Absence Purposes (i.e. selective and differential agar)
Medium has passed growth promotion if growth is observed (if applicable with the appropriate characteristics
like color or halos) within the incubation parameters.

7.9.5.Results Interpretation – Liquids (i.e. general nutrient, or selective and differential)

Samples must show growth for the challenge microorganism(s). If the liquid is inherently turbid due to the
media ingredient formulation, the microbiology analyst may streak out the liquid onto TSA (GCAS 92347889)
after the specified incubation period and incubate at 30-35°C for 18-24 h. Medium has passed growth
promotion if growth is noted on the TSA plate(s).

7.10. Qualitative Growth / Inhibition Check

7.10.1. In case of reduced testing for in-house prepared media a qualitative check for growth or inhibition has to be
done according to the table above once the process is qualified and approved according to Attachment IV.

7.10.2. Microorganisms according to MMR are streaked onto plates or inoculated in broths. Incubation time and
incubation temperature according to MMR are used. No reference plate is needed.

7.10.3. Results Interpretation

 Medium has passed growth check if growth is observed and if applicable with the appropriate
characteristics like color or halos within the incubation parameters. Medium has passed if no
growth is observed for inhibition check within the incubation parameters.

 If a liquid is inherently turbid due to the media ingredient formulation, the microbiology analyst may
streak out the liquid onto TSA (GCAS 92347889) after the specified incubation period and incubate
at 30-35°C for 18-24 h. Medium has passed growth promotion if growth is noted on the TSA
plate(s).

8. Results not meeting Acceptance Criteria

8.1. If results are outside of the acceptance criteria specified, perform a laboratory investigation to determine root cause.
If laboratory error is found, the original test is void and the assay is to be re-executed.

8.2. If no immediate attributable root cause can be found, the lot may be re-tested in duplicate. All repeated tests must
meet acceptance criteria for the lot to be released. Should any of the repeated replicates fail, the lot as-made
cannot be used.

UPDATE

Version 005

 Updated naming convention (e.g. GMCO to Global R&D Micro Media Owner and Regional GM&LO to Regional
Microbiologist)

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 Procter & Gamble
Microbiology Standard Operating Procedure (RESTRICTED)

Title Microbiological Media Control and Equivalency

Global Microbiology
010 GCAS.Revision SOP-92319714.005
Organization (GMO)

 Added content of SOP 22 Equivalency of Microbiological Media Manufacturers (GCAS 96550084)

 Changed title to “Media Control and Equivalency”
 Updated and simplified wording
 Simplified process to define media equivalency
 Added individual component weight variance
 Changed reduced testing frequencies for growth promotion or inhibition
 Added qualitative checks in case of reduced testing for growth promotion or inhibition
 Changed method for volume check and allowed reduced testing
 Removed and / or updated attachments, added new Attachment IV for Reduced Testing Request and new
Attachment V with example of media expiration dating protocol

REFERENCES

 USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests

 USP <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
 USP <1117> Microbiological Best Laboratory Practices

ATTACHMENTS

All attachments can be found on the current Microbiology (PS) Share Point.

Attachment I – Recommended Expiration Dates for Media Prepared In-House

Attachment II – Manufacturer / Supplier Qualification Form
Attachment III – Media Equivalency Form
Attachment IV – Reduced Testing Request
Attachment V – Example of Media Expiration Dating Protocol

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