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á1117ñ MICROBIOLOGICAL BEST LABORATORY PRACTICES

Add the following:
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INTRODUCTION
MEDIA PREPARATION AND QUALITY CONTROL
Media Preparation
Media Storage
Quality Control Testing
MICROBIOLOGICAL MEDIA INCUBATION TIMES
MAINTENANCE OF MICROBIOLOGICAL CULTURES
LABORATORY EQUIPMENT
USING MODERN TECHNOLOGIES
LABORATORY LAYOUT AND OPERATIONS
SAMPLE HANDLING
INCUBATION TEMPERATURE EXCURSIONS
COMPETENCIES AND TRAINING OF PERSONNEL
QUALIFICATION OF ANALYSTS
CONSIDERATIONS FOR MICROBIOLOGICAL RISK ASSESSMENTS

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LABORATORY RESOURCES
Oversight of Contract Laboratories
Oversight of Suppliers
METHOD TRANSFER ci
DOCUMENTATION
MAINTENANCE OF LABORATORY RECORDS
QUANTITATIVE MICROBIOLOGY
DATA INTEGRITY OF MICROBIOLOGICAL DATA
INTERPRETATION OF ASSAY RESULTS
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Use of Negative Controls
Investigations
REFERENCES▲ (USP 1-Aug-2022)

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INTRODUCTION

Good ▲▲ (USP 1-Aug-2022) practices in a microbiology laboratory consist of activities that depend on several principles: aseptic
technique, control of media, control of test strains, operation and control of equipment, diligent recording and evaluation of
data, and training of the laboratory staff ▲in related competencies.▲ (USP 1-Aug-2022) Because of the inherent risk of variability in
microbiology data, reliability and reproducibility are dependent on the use of accepted methods and adherence to good
▲
practices in the laboratory. This chapter does not cover the unique and specific laboratory activities for virology and
mycoplasma recovery and detection.▲ (USP 1-Aug-2022)

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MEDIA PREPARATION AND QUALITY CONTROL

Media Preparation
Culture media are the basis for most microbiological tests. ▲Culture media may be either prepared in-house or obtained
commercially. The term “prepared media” used in this chapter covers both these types of media.▲ (USP 1-Aug-2022) Safeguarding
the quality of the media is therefore critical to the success of the microbiology laboratory. Media preparation, proper storage,
and quality control testing can ensure a consistent supply of high-quality media.
It is important to choose the correct media or components in making media based on the use of accepted sources or
references for formulas. The manufacturer’s formula and instructions for preparation routinely accompany dehydrated and
ready-made media. Because different media types may have different preparation requirements (e.g., heating, additives, and
pH adjustment), it is important to follow these instructions to ensure preparation of acceptable media quality. A certificate of
analysis describing expiration dating and recommended storage conditions accompanies ready-made media, as well as the
quality control organisms used in growth-promotion ▲testing, inhibitory and indicative property testing, as appropriate, and
the results of performance testing with acceptance criteria for that media. For in-house prepared media, similar quality control
criteria should be established.▲ (USP 1-Aug-2022)

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Water is the universal diluent for microbiological media. Purified Water is most often used for media preparation, but in certain
cases the use of deionized or distilled water may be appropriate. Water of lesser quality should not be used for microbiological
media preparation. The volume of the water used should be recorded.
Consistent preparation of media requires accurate weighing of dehydrated media or media constituents. A calibrated balance
with the appropriate weight range for the ingredients should be used (see Weighing on an Analytical Balance á1251ñ). Clean
weighing containers and tools (such as spatulas) should be used to prevent foreign substances from entering the formulation.
The weight of the components should be recorded.
Dehydrated media should be thoroughly dissolved in water before dispensing and sterilization. If heating is necessary to help
dissolve the media, care should be taken not to overheat the media because all culture media, to a greater or lesser extent, are
heat-sensitive. Equipment used in the preparation of media should be appropriate to allow for controlled heating, constant
agitation, and mixing of the media. Darkening of media (Maillard-type reaction or nonenzymatic browning) is a general
indication of overheating. When adding required supplements to media, adequate mixing of the medium after adding the
supplement should be performed.
Preparation of media in poorly cleaned glassware can allow inhibitory substances to enter the media. Inhibitory substances
can come from detergent residue after cleaning glassware or from prior materials used in the glassware. Be sure that the cleaning
process removes debris and foreign matter, and that the detergent is thoroughly rinsed out with Purified Water. See Cleaning
Glass Apparatus á1051ñ for additional guidance.
Sterilization of media should be performed within the parameters provided by the manufacturer or validated by the user.
Commercially prepared media should provide documentation of the sterilization method used. Autoclaving by moist heat is
the preferred sterilization technique, except in instances when boiling is required in order to avoid deterioration of heat-labile
components of the media. Sterilization by filtration may also be appropriate for some formulations.
The effects of the sterilization method and conditions on the media should be validated by sterility and growth-promotion
testing of the media. In addition, if sterilized by moist heat, the autoclave cycle should be validated to ensure proper heat

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distribution for selected loads and volumes. Typically, manufacturers recommend an autoclave cycle of 121° for 15 min using a
validated autoclave. These conditions apply to time at temperature of the media. As container size and the load configuration
of the autoclave will influence the rate of heating, longer cycles may be required for larger loads.▲▲ (USP 1-Aug-2022) Sterilization
time will be dependent on the media volume and autoclave load. Sterilization cycles in which the autoclave is slow to come up
▲
or come down▲ (USP 1-Aug-2022) to temperature may result in overheating of the media. Therefore, care must be taken to validate a
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sterilization cycle, balancing the need for sterile media against the tendency of the media to degrade under excessive heating.
Holding▲ (USP 1-Aug-2022) of the media in the autoclave after the liquid ▲or slow exhaust cycle▲ (USP 1-Aug-2022) is completed is not
recommended after cooling, as it may damage the media. Improper heating or sterilizing conditions—for commercially
prepared or internally prepared media—may result in a difference in color,▲▲ (USP 1-Aug-2022) loss of clarity, altered gel strength,
or pH drift from the manufacturer’s recommended range, as well as reduced growth-promotion activity and/or selectivity. ▲All
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sterilization cycle parameters should be documented.
Unless it is specified to confirm the pH of the medium prior to the sterilization process,▲ (USP 1-Aug-2022) the pH of each batch
of medium should be confirmed after it has cooled to room temperature (20°–25°) by aseptically withdrawing a sample for
testing. Refrigerated purchased media should be allowed to warm up to ambient room temperature if it is to be checked for
pH confirmation. A flat pH probe is recommended for agar surfaces, and an immersion probe is recommended for liquids. See
pH á791ñ for guidance with pH measurement and instrument calibration. The pH of media should be in a range of ±0.2 of the
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value indicated by the manufacturer unless a wider range is acceptable by the validated method.
Prepared media should be checked by appropriate inspection of plates and tubes for the following ▲quality and integrity
parameters:▲ (USP 1-Aug-2022)
• Cracked containers or lids
• Unequal filling of containers
• Dehydration resulting in cracks or dimpled surfaces on solid media
• Hemolysis
• Excessive darkening or color change
• Crystal formation from possible freezing
• Excessive number of bubbles
• ▲▲ (USP 1-Aug-2022)
• Status of redox indicators (if appropriate)
• Lot number and expiration date▲▲ (USP 1-Aug-2022)
• Sterility▲▲ (USP 1-Aug-2022)
• Cleanliness of plates (lid should not stick to dish)

Media Storage
It is prudent to ▲understand▲ (USP 1-Aug-2022) how the manufacturer or supplier transports and stores media before distribution
to the end user. Manufacturers of media should use transport and storage conditions that minimize the loss of moisture, control
the temperature, prevent microbial contamination, and provide mechanical protection to the prepared media.
Media should be labeled properly with batch or lot numbers, preparation and expiration dates, and media identification.
Media should be stored according to the manufacturer’s instructions. Media prepared in-house should be stored under validated
conditions. Do not store ▲media plates containing▲ (USP 1-Aug-2022) agar at or below 0°, as freezing could damage the gel structure.
Protect stored media from exposure to light, excessive temperature, ▲and temperature changes, as this may cause
condensation. Consideration should be given to storing agar plates in▲ (USP 1-Aug-2022) a sealed package or container to
▲
prevent▲ (USP 1-Aug-2022) moisture loss ▲from the plates.▲ (USP 1-Aug-2022)

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Remelting of an original container of solid media, ▲if not containing heat-labile components (1),▲ (USP 1-Aug-2022) should be
performed only once to avoid media whose quality is compromised by overheating or potential contamination. It is
recommended that remelting be performed in a heated water bath or ▲by other suitable methods that will not compromise
media quality.▲ (USP 1-Aug-2022) The use of microwave ovens and heating plates is common, but care should be taken to avoid
damaging media by overheating and to avoid the potential injury to laboratory personnel from glass breakage and burns. ▲It
is recommended that▲ (USP 1-Aug-2022) molten agar medium be held in a monitored water bath ▲until it has been
tempered to▲ (USP 1-Aug-2022) 45°–50°, ▲and not further held▲ (USP 1-Aug-2022) for ▲▲ (USP 1-Aug-2022) more than 8 h, ▲or as determined
appropriate for the medium composition.▲ (USP 1-Aug-2022) Caution should be taken when pouring the media from a container
immersed in a water bath to prevent water from the bath commingling with the poured sterile media. ▲Careful
wiping of▲ (USP 1-Aug-2022) the exterior of the container dry before pouring ▲is recommended.▲ (USP 1-Aug-2022)
Disposal of used cultured media (as well as expired media) should follow local biological hazard safety procedures.

Quality Control Testing

Although growth media can be prepared in a laboratory from individual components, many laboratories, for
▲
convenience,▲ (USP 1-Aug-2022) use dehydrated media or purchase commercially prepared media in plastic plates or glass
containers. Manufacturers of media attempt to standardize raw materials from biological sources but must constantly deal with
unavoidable differences in raw materials obtained from natural sources, and therefore lot-to-lot variability of media must be
considered. In addition, the performance of media prepared in a laboratory or by a manufacturer is highly dependent on
preparation and storage conditions. Improper media preparation can cause unsatisfactory conditions for microbial growth or
recovery and unreliable results.
Therefore, quality control tests should be performed on all prepared media, including media associated with swabs or media

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in strips ▲▲ (USP 1-Aug-2022). Tests routinely performed on in-house prepared media should include pH, growth promotion,
inhibition, and indicative properties (as appropriate). Periodic stability checks to confirm the expiration dating ▲are
recommended only until media expiry has been validated.
When media has not been terminally sterilized in its final container and secondary packaging (e.g., irradiated plates of media),
an inspection for absence of microbial contamination is recommended for plated or liquid media in tubes to prevent unintended
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use of contaminated media. The sterility inspection can be performed on a representative sample of the specific
media-and-container combination(s) on the basis of the risk of impact on its use in microbiological control testing. The intent
of the inspection should be to detect evidence of growth in the relevant medium, such as turbidity in liquid broth media or
colonies forming on plated media. Similar to the practice of pre-incubation of non-irradiated, plated media before use in an
aseptic manufacturing area, media incubation should be performed at the same temperature range and for at least the
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maximum time period for which the media is to be incubated in the test (e.g., if incubation time is 3–5 days in the test, the
minimum incubation time should be 5 days).
Every in-house sterilized batch of media should be tested for growth promotion.▲ (USP 1-Aug-2022) Test organisms may be selected
from the appropriate compendial test chapter. In addition, microorganisms used in growth-promotion testing may be based
on the manufacturer’s recommendation for a particular medium or may include representative environmental isolates (but
these latter ▲isolates▲ (USP 1-Aug-2022) are not to be construed as compendial requirements).
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▲
It is commonly stated that a satisfactory growth-promotion outcome includes growth that is visibly comparable to that
obtained with a previously tested and approved batch of medium. This is for a qualitative comparison; the comparison medium
should be of the same formula, consistency, and volume. If quantitative comparison is desired, follow guidance in Validation of
Microbial Recovery from Pharmacopeial Articles á1227ñ; the medium should be of the same formula, consistency, and volume.
For qualitative and quantitative comparison, direct physical comparison with a previously tested batch is not necessary.
Numerical results comparison in quantitative testing should be appropriate for the intended purpose, with adequate knowledge
of microbiological growth variability (i.e., factor of 2).▲ (USP 1-Aug-2022)
Expiration dates on media should have supporting growth-promotion testing to indicate that the performance of the media
still meets acceptance criteria up to and including the expiration date. The length of shelf life of a batch of media will depend
on the stability of the ingredients and formulation under specified conditions, as well as the type of container and closure.
When a batch of media does not meet the requirements of growth-promotion testing, an investigation should be initiated
to identify the cause. This investigation should include a corrective action plan to prevent the recurrence of the problem. Any
batch of media that fails growth-promotion testing is unsuitable for use. [NOTE—Failed growth-promotion test results may not
be used to negate positive test results.]
Some reagents are used for diagnostic purposes to help support identification of ▲microorganisms,▲ (USP 1-Aug-2022) e.g., Gram
stain, ▲▲ (USP 1-Aug-2022) oxidase, ▲and coagulase test▲ (USP 1-Aug-2022) reagents. These may have attributes that can be quality control
tested similar to microbiological media. Select the correct quality control standard microorganisms, following the
manufacturer’s instructions, and perform the testing before unknown sample diagnostic testing. All relevant diagnostic reagents
should be subjected to incoming quality confirmation before use.
Special care should be taken with media that are used in sterility tests (see Sterility Tests á71ñ for requirements) and in
environmental monitoring studies. Media used for environmental monitoring of critical areas should preferably be double
wrapped and terminally sterilized. If terminal sterilization is not performed, media should be subjected to 100% pre-incubation
and inspection before use within a critical area. [NOTE—Growth-promotion testing for this media ▲should▲ (USP 1-Aug-2022) be
performed ▲to qualify that▲ (USP 1-Aug-2022) pre-incubation ▲does not impact recovery.▲ (USP 1-Aug-2022)] This will prevent extraneous
contamination from being carried into controlled environments and will prevent false-positive results. A raised agar level for
surface contact plates should be verified.

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▲
MICROBIOLOGICAL MEDIA INCUBATION TIMES

Incubation times for microbiological tests with a duration of less than 3 days should be expressed in hours: e.g., “Incubate
at 30°–35° for 18–72 h”. Tests with a duration longer than 72 h should be expressed in days: e.g., “Incubate at 30°–35° for 3–
5 days”. For tests with incubation times expressed in hours, incubate for the minimum specified time and exercise good
microbiological judgment when exceeding that time. For tests with incubation times expressed in days, incubations started in
the morning or afternoon should generally be concluded at that same time of day in the morning or afternoon,
respectively.▲ (USP 1-Aug-2022)

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MAINTENANCE OF MICROBIOLOGICAL CULTURES

Biological specimens can be the most delicate standards to manage because their viability and characteristics are dependent
on adequate handling and storage. Standardizing the handling and storage of cultures by the user laboratory should be done
in a way that will minimize the opportunity for contamination or alteration of growth characteristics. The careful and consistent
treatment of stock cultures is critically important to the consistency of microbiological test results. Cultures for use in compendial
tests should be acquired from a national culture collection or a qualified secondary supplier ▲and have documented equivalency
to relevant ATCC strains (2).▲ (USP 1-Aug-2022) They can be acquired frozen, freeze-dried, on slants, or in ready-to-use forms.
Confirmation of the purity of the culture and the identity of the culture should be performed before its use in quality control

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testing. Ready-to-use cultures should be subjected to incoming testing for purity and identity before use. The confirmation of
identity for commonly used laboratory strains ideally should be done at the level of genus and species.
Preparation and resuscitation of cultures should follow the instructions of the supplier or a validated, established method.
The “seed-lot technique" is recommended for storage of stock cultures.
The original sample from the national culture collection or a qualified secondary supplier is resuscitated and grown in an
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appropriate medium. Aliquots of this stock culture (the first transfer or passage) are suspended in a cryoprotective medium,
transferred to vials, and frozen at –30° or below until use. If stored at –70° ▲or below,▲ (USP 1-Aug-2022) or in lyophilized form,
strains may be kept indefinitely. These frozen stocks can then be used to inoculate monthly or weekly working cultures. Once
opened, do not refreeze unused cell suspensions after culturing a working suspension. The unused portion should be discarded
to minimize the risk of loss of viability and contamination of the stock.
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The number of transfers of working control cultures should be tracked to prevent excessive subculturing that increases the
risk of phenotypic alteration or mutation. The number of transfers allowable for specific compendial tests may be specified in
that test. One passage is defined as the transfer of organisms from a viable culture to a fresh medium with growth of the
microorganisms. Any form of subculturing is considered to be a transfer/passage.

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LABORATORY EQUIPMENT

Most equipment (incubators, water baths, and autoclaves) are subject to standard validation practices of
▲
installation▲ (USP 1-Aug-2022) qualification, operational qualification, and performance qualification. ▲Some useful guidance can
be found in Analytical Instrument Qualification á1058ñ.▲ (USP 1-Aug-2022)Additionally, periodic calibration (generally annually ▲or
on a frequency based on a risk assessment▲ (USP 1-Aug-2022)) is commonly required. New equipment, critical to the operation of
the laboratory, should be qualified according to a protocol approved by the quality assurance unit (QAU). In addition, regular
cleaning and sanitization of equipment such as incubators, refrigerators, and water baths should be performed to minimize the
potential for contamination in the laboratory. Door seals of incubators and refrigerators should be cleaned and checked for
state of repair. ▲These activities should be incorporated into the equipment preventative maintenance program.▲ (USP 1-Aug-2022)
Instruments (pH meters and spectrophotometers) used in a microbiology laboratory should be calibrated on a regular
schedule and tested to verify performance on a routine basis. The frequency of calibration and performance verification will
vary based on the type of instrument and the importance of that equipment to the generation of data in the laboratory.
▲
Laboratory equipment with software should be evaluated for maintaining data integrity (Code of Federal Regulations Title 21
(21 CFR), Part 11).▲ (USP 1-Aug-2022)
Equipment that is difficult to sanitize (such as refrigerators and incubators) should be ▲segregated from▲ (USP 1-Aug-2022) aseptic
operations (such as storage of media for testing and incubation of sterility test samples) and live culture operations to minimize
the potential for inadvertent contamination of the tests.
Autoclaves are central to the operation of the laboratory and must have proper validation in place to demonstrate adequate
sterilization for a variety of operations. Autoclave resources must be available (and validated) to sterilize waste media (if
performed in that laboratory) as well as the media prepared in that laboratory. The choice of one or several autoclaves is not
driven by a need to separate aseptic and live operations (everything in the properly maintained autoclave is sterile after the
cycle), but rather driven by resource considerations (see below).

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USING MODERN TECHNOLOGIES

The use of new technologies in a microbiology laboratory requires new learning and training to ensure a proper
implementation in the laboratory (for examples, see Validation of Alternative Microbiological Methods á1223ñ, Microbial
Characterization, Identification, and Strain Typing á1113ñ), and Rapid Microbial Tests for Release of Sterile Short-Life Products: A
Risk-Based Approach á1071ñ. In addition to the laboratory specialists, the laboratory supervisors or quality assurance personnel
evaluating qualification of systems or deviations must also have the skills to analyze and interpret the complex data that may
be generated by new techniques. For each new technology, it is valuable to develop rules and principles that will build
consistency and accuracy when laboratory personnel are using that technology to perform the relevant procedure. The rules
and principles should be included in the written procedure.▲ (USP 1-Aug-2022)

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LABORATORY LAYOUT AND OPERATIONS

Laboratory layout and design should carefully consider the requirements of good ▲▲ (USP 1-Aug-2022) practices ▲in a
microbiology laboratory▲ (USP 1-Aug-2022) and laboratory safety. It is essential that cross-contamination of microbial cultures ▲or
DNA/RNA samples for PCR testing▲ (USP 1-Aug-2022) be minimized to the greatest extent possible, and it is also important that
microbiological samples be handled in an environment that makes contamination highly unlikely.
In general, a laboratory should be divided into clean or aseptic areas and live culture areas. Areas in which environmental

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▲
and other microbiological test▲ (USP 1-Aug-2022) samples are handled and incubated should be maintained completely free of live
cultures, if possible. If complete separation of live and clean culture zones cannot be accomplished, then other barriers and
aseptic practices should be employed to reduce the likelihood of accidental contamination. These barriers include protective
clothing, sanitization and disinfection procedures, and ▲containment by▲ (USP 1-Aug-2022) biological safety cabinets designated
for clean or aseptic operations only. Procedures for handling spills or mishaps with live cultures ▲or DNA/RNA▲ (USP 1-Aug-2022)
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should be in place, and all relevant technical personnel should be trained regarding these methods.
Some samples will demonstrate microbial growth and require further laboratory analysis to identify the contaminants. When
growth is detected, the sample should be taken from the clean section of the laboratory to the live culture section without
undue delay. Subculturing, staining, microbial identification, or other investigational operations should be undertaken in the
live culture section of the laboratory. If possible, any sample found to contain growing colonies should not be opened in the
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clean zone of the laboratory. Careful segregation of contaminated samples and materials will reduce false-positive results.
Staff engaged in sampling activities should not enter or work in the live culture handling section of a laboratory unless special
precautions are taken, including wearing ▲dedicated▲ (USP 1-Aug-2022) protective clothing ▲that should not be worn outside the
laboratory (e.g., coats)▲ (USP 1-Aug-2022) and gloves and carefully ▲washing and▲ (USP 1-Aug-2022) sanitizing hands upon exiting. Ideally,
staff assigned to sampling activities, particularly those in support of aseptic processing, should not work in the vicinity of live
culture laboratory operations.
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It is important to consider that microbial contamination of samples, which leads to false-positive results, is always possible
unless careful aseptic precautions are taken. Facilities should be designed so that raw material and excipient sampling can be
done under controlled conditions, including proper gowning and the use of sterilized sampling equipment. It may not always
be possible to sample utility systems, such as water systems, under full aseptic conditions; however, it should be noted that
when samples are not taken aseptically, their reliability is inevitably compromised.
Environmental sampling methods should require minimal aseptic handling in loading and unloading sampling instruments.
Whenever possible, sampling equipment should be loaded with its microbiological recovery media in the environment that is
to be sampled.
All testing in laboratories used for critical testing procedures—such as sterility testing of final dosage forms, bulk product,
seed cultures for biological production or ▲PCR testing of▲ (USP 1-Aug-2022) cell cultures used in biological production—should be
performed under controlled conditions. ▲Sterility tests should preferably be carried out in an isolator with ISO 5
classification.▲ (USP 1-Aug-2022) Isolators have been shown to have lower levels of environmental contamination than manned clean
rooms and, therefore, are generally less likely to produce false-positive results. Proper validation of isolators is critical both to
ensure environmental integrity and to prevent the possibility of false-negative results due to chemical disinfection of materials
brought into or used within isolators (see Sterility Testing—Validation of Isolator Systems á1208ñ). ▲Adequate disinfection of
materials prior to loading a sterility test isolator is another important step to reduce the possibility of false-positive results.
For less-critical microbiological testing, such as with nonsterile product or intermediate bioburden samples, testing under a
unidirectional airflow (UDAF) cabinet, or as applicable is preferable. During qualification, the UDAF air must meet the air quality
grade for which it is being qualified. In addition to the qualification, the cabinet’s environment (air and surfaces) may be tested
periodically. The testing frequency and levels defined would depend on the microbiological grade of the product tested, the
quality of air that can be delivered by the UDAF design and filters, the fact that nonsterile material may be present in the UDAF
and the cleaning/disinfection regime.▲ (USP 1-Aug-2022)

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SAMPLE HANDLING

Viable microorganisms in most microbiology samples—particularly water, environmental monitoring, and bioburden
samples—are sensitive to handling and storage conditions. Critical parameters in these conditions include product (or sample)

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composition, container composition, time of storage, and temperature of storage. Therefore, it is important to minimize the
amount of time between the sampling event and the initiation of testing and to control, as much as possible, the conditions
of storage. ▲Microbiological samples should never be stored in a frozen state before testing because this practice leads to loss
of cell viability.▲ (USP 1-Aug-2022) If the sample is to be transported to a distant location for testing, then the conditions of transport
(time, temperature, etc.) should be qualified as suitable for that test and sample. ▲For example, when monitoring water or
bioburden prior to bioburden-reducing steps or sterilizing steps, samples may be held at 2°–8° for up to 24 h from the time of
sample collection until the start of the analysis (3). When testing within 24 h is not possible (e.g., when contract laboratories
are used), the actual maximum hold time between collection and testing should be supported by experimental studies (e.g.,
challenge tests). For bacterial endotoxin testing, studies may be performed to demonstrate that endotoxins present in the
product and stored in the original container for a certain time may be adequately recovered. Storage conditions (e.g., 2°–8°)
in the laboratory should apply. Highly purified lipopolysaccharides (LPS) such as reference standard endotoxins (RSE) or control
standard endotoxins (CSE) might not be the most relevant endotoxin indicators for such studies and use of laboratory-derived
endotoxin indicators would provide a more realistic assessment as discussed in Endotoxin Indicators for Depyrogenation á1228.5ñ.
If environmental monitoring samples cannot be incubated within a reasonable time frame (e.g., due to the use of an off-site
testing laboratory), the time from sample collection to the start of incubation should be supported with experimental data.
All samples should be examined and the tests completed prior to release, whenever possible.▲ (USP 1-Aug-2022)
Product mixing before sampling may need to be evaluated and applied ▲▲ (USP 1-Aug-2022) to ensure ▲adequate
dispersal▲ (USP 1-Aug-2022) and representation in the sample aliquot.
All microbiological samples should be taken using aseptic techniques, including those taken in support of nonsterile products.
If possible, all microbiological samples should be taken under full aseptic conditions in specialized sampling areas. The areas
▲
and methods of transport of samples should be designed▲ (USP 1-Aug-2022) to minimize contamination▲▲ (USP 1-Aug-2022).
Samples submitted to the microbiology laboratory should be accompanied by documentation detailing source of the sample,

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date the sample was taken, date of sample submission, person or department responsible for the submission, ▲storage
conditions,▲ (USP 1-Aug-2022) and any potentially hazardous materials associated with the sample. The testing department should
acknowledge receipt of the sample and reconcile the identity and number of samples as part of this sample documentation.

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▲
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MICROBIOLOGICAL MEDIA INCUBATION TIMES

Incubation times for microbiological tests of less than 3 days’ duration should be expressed in hours: e.g., “Incubate at 30°
to 35° for 18 to 72 hours”. Tests longer than 72 hours’ duration should be expressed in days: e.g., “Incubate at 30° to 35° for
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3 to 5 days”. For incubation times expressed in hours, incubate for the minimum specified time, and exercise good
microbiological judgment when exceeding the incubation time. For incubation times expressed in days, incubations started in
the morning or afternoon should generally be concluded at that same time of day.▲ (USP 1-Aug-2022)

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▲
INCUBATION TEMPERATURE EXCURSIONS

Incubation equipment qualification and ongoing calibration provide the acceptable range for temperature conditions relative
to the expected procedural incubation temperatures or range. When an excursion occurs outside of the procedural incubation
temperature(s), an assessment should be performed to determine the potential impact on the test samples and what, if any,
action should be taken with the test samples. The effect of a higher or lower temperature on microbial recovery from the test
samples for the recorded period of time should be evaluated. An investigation of the excursion should be documented along
with the disposition of the test samples. For instance, a slightly lower temperature excursion (e.g., 1°–2°) for a brief time could
be compensated with an increase in incubation time; or, for a higher temperature excursion (e.g., greater than 40° for a total
aerobic microbial count or greater than 30° for a total yeasts and molds count) consider any negative impact on
recovery.▲ (USP 1-Aug-2022)

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▲
COMPETENCIES AND▲ (USP 1-AUG-2022) TRAINING OF PERSONNEL

Each person engaged in each phase of pharmaceutical manufacture should have the education, training, and experience to
do his or her job. The demands of microbiological testing require that the core educational background of the staff, supervisors,
and managers be in microbiology or a closely related biological science. They should be assigned responsibilities in keeping
with their level of skill and experience.
A coherent system of standard operating procedures (SOPs) is necessary to run the microbiology laboratory. These
procedures serve two purposes in a training program. Firstly, these SOPs describe the methodology that the microbiologist will
follow to obtain accurate and reproducible results, and so serve as the basis for training. Secondly, by tracking the procedures
in which a particular microbiologist has demonstrated proficiency, the procedure number or title also serves to identify what
training the microbiologist has received specific to his or her job function.
Training curricula should be established for each laboratory staff member specific to his or her job function. He or she should
not independently conduct a microbial test until qualified to run the test. Training records should be current, documenting the
microbiologist’s training in the current revision to the particular SOP.

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▲
Performing USP microbiology methods requires a quality control infrastructure that will support accuracy and consistency
of method outcomes. While this chapter provides information about effective microbiology practices, there are other USP
chapters that offer guidance in relevant areas where microbiology is integrated (see Guide to General Chapters and Chapter
Charts).▲ (USP 1-Aug-2022)
Periodic performance assessment is a wise investment in data quality. This performance testing should provide evidence of
competency in core activities of the microbiology laboratory such as hygiene, plating, aseptic technique, documentation, and
others as suggested by the microbiologist’s job function.
Microbiologists with supervisory or managerial responsibilities should have appropriate education and in-house training in
supervisory skills, laboratory safety, scheduling, budgeting, investigational skills, technical report writing, relevant SOPs, and
other critical aspects of the company’s processes as suggested in their role of directing a laboratory function.
Competency may be demonstrated by specific course work, relevant experience, and routinely engaging in relevant
continuing education. Achieving certification through an accredited body is also a desirable credential. Further, it is expected
that laboratory supervisors and managers have a demonstrated level of competence in microbiology at least as high as those
they supervise. Expertise in microbiology can be achieved in a variety of ▲ways▲ (USP 1-Aug-2022) in addition to academic course
work and accreditation. Each company is expected to evaluate the credentials of those responsible for designing, implementing,
and operating the microbiology program. Companies can thus ensure that those responsible for the program understand the
basic principles of microbiology, can interpret guidelines and regulations based on good science, and have access to individuals
with theoretical and practical knowledge in microbiology to provide assistance in areas in which the persons responsible for
the program may not have adequate knowledge and understanding. It should be noted that microbiology is a scientifically
based discipline that deals with biological principles substantially different from those of analytical chemistry and engineering
disciplines. Many times it is difficult for individuals without specific microbiological training to make the transition.

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QUALIFICATION OF ANALYSTS

The hiring of experienced personnel combined with periodic training and qualification of analysts, including regular vision
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checks, should limit to an acceptable level the variability of microbiological test results produced by each analyst’s handling
and reading of test results, as well as ensure that the defined good documentation practices are understood and followed.
These also apply to personnel who may be involved in visual inspection of media fill units.▲ (USP 1-Aug-2022)

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▲
CONSIDERATIONS FOR MICROBIOLOGICAL RISK ASSESSMENTS

A microbiological risk assessment can be beneficial and provide information to assist in decision-making about the impact
of microbiological quality concerns. (See Microbiological Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical
Preparations and Substances for Pharmaceutical Use á1111ñ for additional specific parameters for evaluating non-specified
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microorganisms that may be present in nonsterile products.)

Some examples of when a microbiological risk assessment might be used are: 1) elevated bioburden counts (but within
specification); 2) growth of atypical colonies after enrichment on selective agar plates for compendium-specified
microorganisms; 3) exceeded alert/action levels; and 4) identification of a recovered species of concern.
The nature and intent of a risk assessment can differ depending on the relevant situation. The following is a list of example
topics that could be included as part of the risk assessment.
• Scope of the Event—Providing information on the number of product lots that are directly or indirectly impacted on the
basis of shared raw materials, shared equipment, and similar manufacturing processes.
• Microorganism Identification—Identifying the microorganism(s) to the species level whenever possible.
• Characterization and Source of the Microorganism(s)—Researching the literature to determine where the natural
habitat for the microorganism can be found; the potential physiological states for the microorganism (e.g., spore vs.
vegetative, planktonic vs. biofilm); and the potential pathogenicity of the species, the disease potential of the
microorganism for the intended route of administration, and the number of microorganisms recovered.
• Manufacturing Process—Evaluating the product/material manufacturing process (or for an API or raw material) to
determine if it contains microbial ingress and/or growth-control points or growth-promoting, or microbial-reducing steps.
• Target Patient Base and Intended Use of the Product(s)—Identifying potential health effects to the probable user
group(s) targeted by the product based on the product insert and label information.
• Route of Administration of the Product(s)—Identifying the route of administration of the product (e.g., oral
non-aqueous/aqueous, topical, inhalant, etc.). If it is an API or raw material, identify the route of administration of the
product that it will be used to manufacture.
• Intrinsic Product(s) Characteristics—Evaluating the intrinsic characteristics of the product and the effect they could have
on the level of microorganism(s) over shelf life. This means identifying the nature of the product (such as its water activity
or pH) and whether the product can support growth or has antimicrobial activity.
• Assessment Conclusion—Providing a sound, scientifically-based determination. For example, determination that a
microorganism is objectionable or is not objectionable in the test article.
The risk assessment should include determination of the level of risk to the patient or product for an impacted material that
is used to manufacture finished product(s) or for a finished product that will be released to the market. An investigation to
determine the source of a recovered species of concern and its contamination risk may be required to be completed before a

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drug product is released to the market. Writing and approval of a microbiological risk assessment should be performed by
personnel with specialized education and experience in microbiology.▲ (USP 1-Aug-2022)

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LABORATORY RESOURCES

The laboratory management is responsible for ensuring that the laboratory has sufficient resources to meet the existing
testing requirements ▲(4).▲ (USP 1-Aug-2022) This requires some proficiency in budget management and in determining appropriate
measures of laboratory performance. A measure of laboratory performance is the number of investigations performed on tests
conducted by the laboratory, but this measure alone is not sufficient. In addition to tracking investigations, the period of time
between sample submission and initiation of testing should be tracked, as well as the period of time between end of test and
report release (or test closure). Significant delays in these measures are also indications of an under-resourced laboratory staff.
The laboratory management should have sufficient budget to meet testing requirements. Particular measures of budgetary
requirements will be specific to the given laboratory, but budgetary considerations related directly to the need of the laboratory
for sufficient resources must be addressed to ensure reliable testing results ▲(4).

Oversight of Contract Laboratories

Some companies do not have the laboratory space, specialized equipment, or capacity to conduct high-volume
microbiological analyses, so they may outsource this type of testing to contract laboratories. It is the company’s responsibility
to thoroughly investigate the reputation and quality control performance of any new or unfamiliar contract laboratory with

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which they engage in a quality agreement. Before signing an agreement, a manufacturer should confirm that the contract
laboratory complies with current good manufacturing practice (GMP) regulations regarding laboratory qualifications, can
perform the required USP compendial testing for microbial recovery and bacterial endotoxins (see Tests for Burkholderia Cepacia
Complex á60ñ, Microbial Enumeration Tests á61ñ, Tests for Specified Microorganisms á62ñ, á71ñ, and Bacterial Endotoxins Test á85ñ),
and an onsite audit can be conducted or, perhaps, a sample batch submitted for testing to ensure the quality of testing and
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data integrity. The qualifications of the laboratory management staff to interpret microbiological sample results need to be
documented, especially if these staff will serve as subject matter experts for data review or if a laboratory investigation of
questionable sample results ever needs to be conducted. Reports written by the contract laboratory should include complete
analytical worksheets from the analysis performed. The raw data should be available to and periodically reviewed by a qualified
microbiologist of the client company in order to verify the reliability of finished work. All the data used for calculations, including
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all the positive (growth-promotion results) and negative controls used during analysis, should also be included in the final report.
In contrast, a certificate of analysis with a summary of analytical results does not provide all the information needed to verify
sample test data or satisfy a regulatory audit. Relevant method-suitability test results should also be available to support the
subsequent testing.

Oversight of Suppliers
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The impact of the microbiological quality of materials from a supplier can be significant for the laboratory (e.g., media,
reagents, cultures) or for manufacturing (e.g., excipients, components); thus, microbiology knowledge and experience is critical
for adequate oversight of a supplier. Since the microbiology laboratory can perform specification tests for excipients and
products, alignment of knowledge and understanding with a supplier is valuable. Based on evaluation of the appropriate
microbiological quality for each material, adequate supplier oversight from a microbiological perspective can provide increased
knowledge, with consistent quality of the materials, and improved trust in the relationship with the supplier. For the laboratory
supplies, materials of good quality are an important input to systematically maintain laboratory capability for performing
consistent tests. For the manufacturing operations, an excipient supply of good quality provides the foundation for a consistently
produced product that is microbiologically safe for patients. The full understanding and evaluation of a supplier’s microbiological
testing strategy, along with sampling, test methodology, and training, provides a means for developing a relationship with
transparency and integrity. An experienced microbiologist should initiate ongoing communications in this type of customer–
supplier relationship and serve as a technical expert. The controls in place for testing and inspection of incoming lab supplies
are key to supply control.▲ (USP 1-Aug-2022)

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METHOD TRANSFER

Performance of validated methods can change from one laboratory to another. Differences in media, reagents, equipment,
procedures, and in levels of experience of personnel can all affect the outcome of a microbiological method. When transferring a
method from one laboratory to another, control of the change should be evaluated and directed so that there is minimal
negative impact. Each combination of method and test substance should be performed, according to the requisite procedure,
by both the sending and receiving laboratories in an appropriately defined and documented activity (with a defined number
of replicates) that will ensure that method testing capability and experience have been transferred effectively.
For method transfers, comparative testing of the same sample may not be relevant for microbiological methods due to the
non-homogeneity of microbial cells in the sample. Since no contamination is frequently observed, such testing would result in
comparing zero counts. Carrying out a method suitability verification of the analytical method of the product to be transferred

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would provide greater assurance that the transfer-receiving unit may adequately perform the tests under the conditions with
the material used (e.g. nutrient media).▲ (USP 1-Aug-2022)

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DOCUMENTATION

Documentation ▲(either manually or by an electronic laboratory management system)▲ (USP 1-Aug-2022) should be sufficient to
demonstrate that the testing was performed in a laboratory and by methods that were under control. This includes, but is not
limited to, documentation of the following:
• Microbiologist training and verification of proficiency
• Equipment validation, calibration, and maintenance
• Equipment ▲monitoring▲ (USP 1-Aug-2022) during test (e.g., 24-h/7-day chart recorders ▲for incubators▲ (USP 1-Aug-2022))
• Media preparation, sterility checks, growth-promotion,▲indicative, and inhibitory▲ (USP 1-Aug-2022) capabilities
• Media inventory ▲quality▲ (USP 1-Aug-2022) control ▲and shelf-life▲ (USP 1-Aug-2022) testing
• Critical aspects of test conducted as specified by a procedure
• Data and calculations verification
• Reports reviewed by QAU or a qualified responsible manager
• Investigation of data deviations (when required)
▲
Data integrity guidance will be addressed later in this chapter.▲ (USP 1-Aug-2022)

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MAINTENANCE OF LABORATORY RECORDS
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Proper recording of data and studies is critical to the success of the microbiology laboratory. The overriding principle is that
the test should be performed as written in the SOP, the SOP should be written to reflect how the test is actually performed,
and the laboratory notebook should provide a record of all critical ▲information▲ (USP 1-Aug-2022) needed to reconstruct the details
of the testing and confirm the integrity of the data. At a minimum, a laboratory write-up should include the following:
• Date
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• Material tested
• Microbiologist’s name
• Procedure number
• Documented test results
• Deviations (if any)
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• Documented ▲significant method steps and▲ (USP 1-Aug-2022) parameters (equipment used, microbial stock cultures used,
media lots used)
• Management/Second review signature
Every critical piece of equipment should be ▲documented▲ (USP 1-Aug-2022) in the write-up, and all should be on a calibration
schedule documented by SOP and maintenance records. Where appropriate, logbooks or forms should be available and
supportive of the laboratory notebook records. Equipment temperatures (water baths, incubators, autoclaves) should be
recorded and traceable.
The governing SOP and revision should be clearly noted in the write-up. Changes in the data should be crossed off with a
single line and initialed. Original data should not be erased or covered over.
Test results should include the original plate counts, allowing a reviewer to recreate the calculations used to derive the final
test results. Methods for data analysis should be detailed in cited SOPs. If charts or graphs are incorporated into
▲
paper▲ (USP 1-Aug-2022) laboratory notebooks, they should be secured with clear tape and should not be obstructing any data on
the page. The chart or graph should be signed by the person adding the document, with the signature overlapping the chart
and the notebook page. Lab notebooks should include page numbers, a table of contents for reference, and an intact timeline
of use. ▲Similar requirements should be established for electronic laboratory notebooks that capture this same
information.▲ (USP 1-Aug-2022)
All laboratory records should be archived and protected against catastrophic loss. A formal record-retention and -retrieval
program should be in place.

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QUANTITATIVE MICROBIOLOGY

Classical microbiology utilizes the colony forming unit (cfu) as the basis of quantitation for growth-based methods (5). The
heterogeneous nature of microbial contamination can lead to inaccuracies in quantitative methods. While representative
sampling is a high priority in all methods, calculating colony counts with averaging can lead to subjective and varied results
depending on the plate reader and analyst experience. It is important to understand that although averaging colony counts
from multiple plates can make the analysis simpler, a wide range of individual plate counts could lead to underestimation of
actual contamination. Rounding of cfu, another quantitative principle, should be carefully evaluated for fit with the method to

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avoid reporting inaccurate results. [NOTE—Microbial colonies represent biological entities; thus, fractions or decimals in colony
count results are meaningless.] There are methods, such as kinetic bacterial endotoxin testing, where decimal place calculations
are expected. Be careful of placing too much significance on the decimal portion of a result when evaluating a result compared
to an endotoxin limit specification.▲ (USP 1-Aug-2022)

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DATA INTEGRITY OF MICROBIOLOGICAL DATA

Microbiological methods are classically performed manually and on the basis of visual evaluation by an analyst performing
the test. Therefore, the interpretation of test results or the number of colonies tested may be prone to a certain subjectivity and
variability. To further improve data integrity and reduce subjectivity, alternative methods for the reading of plates, such as the
use of automated plate readers or high-resolution photographs of the plate, may be used. However, these systems can have
inherent challenges such as difficulty with the following: counting colonies embedded in the agar gel from pour-plated dishes,
counting satellite colonies, differentiating overlapping colonies, differentiating particles from colonies, and interpreting the
photo consistently from one individual to another. Automated enumeration methods that stack images to capture colonies
growing in time may overcome some of these challenges.
It should be noted that, in order to avoid jeopardizing the asepsis status required during testing, contemporaneous recording
of actions during execution of the microbiological testing cannot be followed in all cases. It is acceptable for recording of actions
to take place immediately after the working session upon exiting the UDAF hood instead of constantly having to go back and
forth from aseptic to non-aseptic areas to write down test actions executed.
Counts from Petri plates are considered original data on the day that the method requires the plates to be read and recorded.

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After reading, if these same plates are subsequently stored at room temperature or under refrigeration, it is not possible to
confirm the original results because the microbial counts may increase during storage. Many microbial colonies continue to
grow during refrigeration but at a slower rate. The colony counts derived from conducting a microbe test using a compendial
method depend on adherence to the incubation time and temperature stated in the prescribed method (i.e., á61ñ).
It is common practice for the raw data sheet that contains the original data, as well as the data entry from the raw data sheet
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to the laboratory information management systems (LIMS), to be reviewed for accuracy and completion by a qualified analyst,
an analyst who has not executed or evaluated the test, or the supervisor.
An important data integrity threat with microbiological testing resides with falsification of data and intentional omission of
testing results. To control this risk, the company culture and ethical standards are essential as well as the application of a rigorous
quality management system.
For the compendial sterility test that combines criticality of the test and higher risk of misinterpretation of results, it is now a
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standard practice to have a second analyst perform a contemporaneous evaluation of the sample (in test media) for microbial
growth. Nonetheless, applying uncritically a contemporaneous reading by a second analyst (four-eyes principle) for all samples
and microbiological tests is not recommended. Precision in counts may vary from one analyst to another (even if they are trained
and qualified) as colonies may overlap, swarm over media, etc., allowing for misinterpretation. Microbiology is a “logarithmic
science” ( á1223ñ); sample size is statistically weak and testing procedures have inherent variability. By tolerating no differences
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in counts, a high number of non-critical deviations will be generated, thus consuming resources unreasonably. As an alternative
to a contemporaneous enumeration, a contemporaneous verification by a second person that the testing activity is performed
correctly may be executed for higher risk tests. A second person could verify, for instance, if the reading of results is correctly
executed according to the procedure, if the result on the Petri plate is correctly transcribed onto the GMP recording sheet (i.e.,
if growth is observed this is captured in the GMP sheet), and if the description of the sample corresponds to the description on
the GMP recording sheet. An assessment of the risk due to a misinterpreted result and its impact on patient safety is performed
to determine the high risk test outlined above.▲ (USP 1-Aug-2022)

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INTERPRETATION OF ASSAY RESULTS

Analytical microbiological assay results can be difficult to interpret for several important reasons: 1) microorganisms are
ubiquitous in nature, and common environmental contaminants—particularly organisms associated with humans—
predominate in many types of microbiological analysis; 2) the analyst has the potential to introduce contaminating organisms
during sample handling or processing in the laboratory; 3) microorganisms may not be homogeneously distributed within a
sample or an environment; and 4) microbiological assays are subject to considerable variability of outcome. Therefore, apparent
differences from an expected outcome may not be significant.
Because of these characteristics of microbiological analysis, laboratory studies should be conducted with the utmost care
▲
(using aseptic techniques)▲ (USP 1-Aug-2022) to avoid exogenous contamination as previously discussed in this chapter. Equally
important, results must be interpreted from a broad microbiological perspective, considering not only the nature of the putative
contaminant but the likelihood of that organism(s) surviving in the pharmaceutical ingredient, excipient, or environment under
test. In addition, the growth characteristics of the microorganism should be considered (especially in questions of the growth
of filamentous fungi in liquid media).
▲
Microbiological data should be scheduled to be reviewed and trended to assess the capability of the measures to control
contamination and ensure the microbiological quality of products or raw materials remains within the acceptance criteria
defined and is not worsening. Trending of microbiological data can be performed in several ways, but the end goal is to
determine if an adverse trend is arising. An adverse microbiological trend is an early warning of a potential degradation or loss
of control within the environment, the utility, raw material, or product tested. Multiple designations may be used to define
adverse trends, and it is up to the user to define which is the most relevant definition in the context of the application. For

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instance, an adverse trend can be defined as repeating higher than usual counts or increasing amount and variety of
microorganisms or contamination occurrences over a certain time period. An adverse trend may be systematically related to
multiple events.
To reduce the role of subjectivity and to distinguish counts that are out of expectation or that differ from the norm, monitoring
levels could be calculated using statistical methods. There is no absolute standard that can be used to calculate levels using
historical data as data distribution may differ among the different microbiological tests. Microbiological data are generally not
normally distributed, may contain many zero counts (for highly controlled or aseptic areas), and are highly variable. Therefore,
classical process control tools based on normally distributed data may not apply. The percentile ranking method using the
negative binomial or gamma fit distribution has been shown to be reliable and covers a large diversity of microbiological data
(6). As an alternative, if enough values are available, a non-parametric fit may be used.

Use of Negative Controls

Some microbiological methods specify the use of a negative control, but their use in other situations should be relevant and
appropriate. For example, the testing of rinse fluid without product in a membrane filtration sterility test could provide useful
information in a laboratory investigation of an out-of-specification result. Also, for bioburden testing, a negative control can
provide ongoing trending of lab equipment sterilization and good aseptic techniques.

Investigations▲ (USP 1-Aug-2022)

When results are observed that do not conform to a compendial monograph or other established acceptance criteria, an
investigation ▲▲ (USP 1-Aug-2022) is required. There are generally two distinct reasons for the observation of microbial contamination

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that does not comply with a target or requirement: A laboratory error or laboratory environmental conditions may have
produced an invalid result, or the product contains a level of contamination or specific types of contaminants outside established
levels or limits. ▲In such cases, conduct an investigation using an appropriate investigative tool (e.g., Ishikawa "fishbone"
diagrams or the Kepner-Tregoe method).▲ (USP 1-Aug-2022) Laboratory management and, in most cases, the QAU should be notified
immediately. ▲Immediate actions may be required if the product’s quality is potentially impacted by the deviation. These may

campaign or already released on the market.▲ (USP 1-Aug-2022)

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include, for instance, putting the batch in quarantine and assessing the potential impact for batches produced in the same

A full and comprehensive evaluation of the laboratory situation surrounding the result should be undertaken. All
microbiological conditions or factors that could bring about the observed condition should be fully considered, including the
magnitude of the excursion compared to established limits or levels. In addition, an estimate of the variability of the assay may
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be required to determine whether the finding is significant.
The laboratory environment, the protective conditions in place for sampling, historical findings concerning the material under
test, and the nature of the material, particularly with regard to microbial survival or proliferation in contact with the material,
should be considered in the investigation. In addition, interviews with the laboratory analyst(s) may provide information
regarding the actual conduct of the assay that can be valuable in determining the reliability of the result and in determining
an appropriate course of action. If laboratory operations are identified as the cause of the nonconforming test outcome, then a
O

corrective action plan should be developed to address the problem(s). Following the approval and implementation of the
corrective action plan, the situation should be carefully monitored and the adequacy of the corrective action determined.
If assay results are invalidated on the basis of the discovery of an attributable error, this action must be documented.
Laboratories also should have approved procedures for ▲additional▲ (USP 1-Aug-2022) testing (retesting), and if necessary, resampling
where specific regulatory or compendial guidance does not govern the conduct of an assay investigation. ▲Since
microorganisms are not necessarily homogeneously distributed in a sample; microbial numbers evolve following storage; all
volume of the sample is used during testing in some cases; and sampling is a snapshot of a short time; the classical retesting
rules for chemical analysis should not be applied in microbiological testing. Additional testing of the original product, raw
materials, intermediates, environment, etc., however, may serve to provide supportive information for the root cause
investigation but not be used to invalidate the original results.▲ (USP 1-Aug-2022)

Add the following:

▲
REFERENCES

1. International Organization for Standardization. Microbiology of food, animal feed and water—Preparation, production,
storage and performance testing of culture media, ISO 11133; 2014.
2. Reference Strain Catalogue Pertaining to Organisms For Performance Testing of Culture Media. Version 29. World Data Centre
of Microorganisms. International Committee on Food Microbiology and Hygiene and Working Party on Culture Media;
Jan 2019.
3. American Public Health Association. Standard Methods for the Examination of Water and Wastewater. APHA; 2017.
4. International Council for Harmonisation. Pharmaceutical Quality System, Q10; 2008.
5. Sutton S. Accuracy of Plate Counts. J Val Technol. Summer, 2011; 42–46.
6. Gordon O, Goverde M, Pazda J, Staerk A, Roesti D. Comparison of different calculation approaches for defining
microbiological control levels based on historical data, PDA Journal of Pharmaceutical Science and Technology. 2015;
69:383–398.▲ (USP 1-Aug-2022)

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