AUDITING

OF
MICROBILOGICAL
LABORATORY
Presented by
Presented by
P.HEMA
P.HEMA
Q.A
Q.A
 CONTEXT
 Introduction
 Good house keeping
 Sampling and sample receipt
 Culture media
 Microbial cultures
 Microbial identification
 Control of reagents
 Microbiology laboratory equipment
 Method validation and qualification
 Bacterial endotoxin testing
 Water testing and sterility testing
 Antibiotic assays
 Environmental monitoring
 Biological indicators
 Out of specification procedures
 General documentation
 Data control and data integrity
 Training
 Biosafety
 Proficiency testing
 Waste management
 Space utilization and improving laboratory design
 Resourcing
 INTRODUCTION
Microbiology laboratories be subject to periodic audit , there will be specific areas that require examination.
This article addresses many of these microbiological-specific issues .

The audit should be seen as far more than a walk around a laboratory. The audit should be clear how the
level of compliance will be assessed.

Such issues will include, as USP <1117> points out: aseptic technique, control of media, control of test strains,
control of equipment, recording and evaluation of data, and appropriate training (1). In addition, the audit
may extend to issues of biosafety, waste management and the good optimization of space (where poor design
can lead to sample mix-ups or pose contamination risks to laboratory .

There are different types of audits that can be conducted: financial and efficiency. one starting point is to
review the strategy for microbiological control . The person auditing the microbiology laboratory has an
understanding of the nature of microbiology as a science and its variability, which are distinct from
analytical laboratories. .Since microbiology laboratories vary in their type of function and the range of tests
conducted, not every possible test or activity can be reviewed here. The article discusses general points about
laboratory controls and then focuses on some specific test examples.
Good housekeeping

The first thing that will catch the eye of any

auditor is the organization and cleanliness
of the laboratory.

The auditor may seek to verify cleanliness

of work stations and review cleaning and
disinfection procedures.

The auditor should enquire if these have

been qualified and how the expiry dates of
the agents has been established. The
laboratory should be practicing good
housekeeping. In addition, there must be
no expired
Sampling and sample receipt
For this review, appropriate questions to ask may include:
The auditor should assess how samples are How samples are taken in production?
received into the laboratory and stored Does the microbiology team provide sampling training to
operators to ensure samples are taken aseptically?
What protection is afforded to the sample once it has been
taken?
Traceability is important so assess Are samples stored in production until the end of the day or
how samples are handled and
controlled and how vital information submitted to the microbiology department immediately?
like batch numbers are recorded microbial growth or death during storage prior to submission
to the microbiology laboratory?
How are samples for microbiological testing logged in and
Control is also of importance, so good
practices such as clear labelling and stored?
adequate packaging to prevent ingress of How does the laboratory practice adequate segregation,
dust and thus microbial contamination is
essential storage and maintenance of samples?
How are samples referenced?
 As an example, when selecting a  Storage of the samples prior to and
numbering system samples it would during sample preparation, as well as
be useful to follow a system that is after its analysis should be
intuitive and meaningful performed under appropriate
conditions
Does the sample log book (or
electronic record)and who then took it
for testing (chain of custody)? Does the sample have an expiry time? How
is this controlled?

If samples are discarded, how What method validation supports the sample
is this controlled? storage conditions (e.g., water)?
 For instance, if the sample packaging
has been compromised or damaged
What site SOP governs what happens when
during shipping, for must be a sample time point is missed?
recorded.

What type of samples might be temporarily What happens if an electronic system is on-
stored while awaiting testing? line?

What are the required storage conditions in

terms of incubation temperature?
 Culture media
 culture media continues to play a major role in the microbiology laboratory . Hence, culture media remains the
bedrock of microbiology and a careful assessment of the use and controls relating to culture media is required.
It is important for the auditor to assess the laboratory is using the correct media. This includes assessing the
components in making media
 If media is prepared in-house, some appropriate audit points to consider and questions to ask are:

Review the SOPs for media preparation,

Ask ‘how do you control the quality of dehydrated media and water?

Review how reagents are prepared, assess lot numbers and expiration
dates, for both

Assess the calibration and daily verification of balances.

Check the suitability of glassware and washing procedures.

Assess the sterilization process

 For media batches that are provided by an external supplier, there should be
a certification of analysis providing details on the sterility and fertility.
 In terms of culture media storage, the auditor should assess:
 Segregation and labelling.
 That storage temperatures are appropriate.
 The system for putting media into quarantine.
 That media is stored away from direct sunlight (ideally in the dark).
 The system for releasing media.
 Media use, such as FIFO - first-in, first-out, meaning that the oldest inventory items are
used first.
 To assess that storage duration has been validated.
 Where solid media is used to prepare plates, that re-melting has been validated along
with the agar hold temperatures and periods.
 Assess temperature records for incubation devices.
Microbial cultures
 Some questions that an auditor could pose to the
microbiology laboratory are
 When purchasing microorganisms from a national culture
collection, what incoming QC tests
 are run for identity and purity?

To minimize this risk,

Best procedures
practices based should
on be in place and such
 Is the identification undertaken using genotypic analysis?
To Microbial
minimize cultures
this risk, purchasedand
procedures from
should external culture
procedures
so-called for
"seed-lot" preparation
technique for storagebeofinstock
place
resuscitation ofand such
cultures
cultures,
collections
proceduresneed
which involvesfor to be
should
using
identity
handled
preparation
be and
based
working
carefully
on and
resuscitation
the
cultures
to confirm
of
instructions
and never culturesthe
returning
 How are the # of passages of working cultures tracked,
should beand purity
based of the
on the culture
instructions
unused passages back to original stock and what is the maximum number
 permitted passages for cultures used?
 Ask to see the records of sub culturing a purchased
organism.
 How long can a working culture be used?
 How are cultures preserved for long-term storage
 Microbial identification
The identification of isolates is performed correctly and consistently.
TheInidentification of isolates is things
relation to identifications, performed correctly
can go wrong,and consistently.
ranging from
In Gram-stain
relation to identifications, things can go wrong, ranging from
over- decolourization to the selection of the incorrect
Gram-stain
test kit over- decolourization to the selection of the incorrect
test kit

During an audit, analysts are competent in the gram staining

techniques . Expiry dating of reagents should be checked.
Gram stain reagents should be replaced at appropriate
intervals

With automated identification systems, such technologies

tend to incorporate calibration routines such as standard
solutions, or ‘QC’ strains.
Control of reagents

As with other laboratories,

the microbiology laboratory
will have reagents that With endotoxin testing the With identification reagents
require control. Two Limulus Amebocyte Lysate and test kits, it is typically to
(LAL) method is the most carry out lot release testing
applicable areas are with widely used. using control strains
endotoxin testing and
microbial identifications.
Microbiology laboratory equipment
• New equipment needs to be qualified before use going through standard process of drawing . In terms of
regular use equipment use should be recorded in log books,to support the equipment to follow ongoing
checks by risk assessment.

• Assesment includes pippetors,and equipment includes temperature

monitoringdevics,incubators,refrigerators,freezers and there should be evidence of remapping.Schedule for
remapping due to power failures,drift and excessive opening

• Autoclave cycles are monitored and records, standard tests should be performed such as leak rate and Bowie and Dick
tests where vacuum stages are employed for certain cycles.
• Autoclaves should be regularly serviced and maintained transducers and control thermocouples. Where the autoclave is
used for culture media sterilization,
• The auditor may wish to assess that any load simulation probes should be checked and calibrated
• It is also important that equipment is frequently cleaned and the use of disinfectants is of particular importance where
growth conditions can occur, such as with equipment used to assess bioburden by filltration.
Method validation and qualification
 It is important that samples assayed against pharmacopeial methods have been appropriately qualified
and that any non-pharmacopeial methods have been appropriately validated.
 Sample qualification can include

Tests for bacteriostasis and fungistasis

Inhibition and enhancement

Methods of preparation and recovery and neutralisation

 The auditor should review the test procedures or protocols and the data and study the design and perform
requalification if needed.
Bacterial endotoxin testing

ELIZA methods
ELIZA methods

LAL test
LAL test
Some
laboratories tests Rabbit pyrogen test
Rabbit pyrogen test
will use

Automated or turbidometric
Automated or turbidometric

False results should be

False results should be
computerised
computerised
Water testing
The microbiology The sampling need sanitize an
laboratory have a procedure should outlet and/or to a
defined schedule for describe the way by hose prior to
sampling and testing which water is sampled sampling.

Some questions an auditor could ask include:

 How are water samples drawn?
 What is the flush time?
 Does the sample record contain time sample taken, name, site, time sample delivered to laboratory?
 Is the expiration date on those stored water sampling bottles ?
 What is the maximum number of hours that may elapse between sampling and bacterial enumeration
testing?
 Sterility testing
auditor should assess the auditor should documents should clearly
techniques for review the standard describe how the test is
transferring samples performed, the media to be
media and test equipment operating used, the incubation times
into the test environment procedures and and temperatures required
which may be an isolator specifications and the acceptance criteria

 If isolators are used, the auditor could ask:

 isolator decontamination process, whether a six-log sterilization kill has been confirmed in three
consecutive validation studies.
 decontamination agent assessment
 If the containers, media, filter sets, tubing, and other unaffected by penetration of the decontamination
agent.
 frequency of re-decontamination
 isolator gloves are subject to integrity testing.
 type of environmental monitoring
 review sterility failure rates
Antibiotic assays

samples for testing should be kept in the dark to

prevent potential degradation

control of antibiotic standards since errors in standard

preparation

auditor should check on the status of the analysis

software.

audit should consider the control and preparation

Environmental monitoring
for different monitoring locations and
frequencies as well as the specific tests to
be conducted

A documented procedure should also exist

to describes the limits applied and a
separate procedure is typically in place to
set out what action is required when these
limits are exceeded.
Questions and points an auditor may wish to pose could include:
 agar residues are removed post-sampling.
 Assess the appropriateness of exposure times for settle plates.
 Assess types of agars used and incubation conditions.
 Particle counting, active air sampling and airflow velocity monitoring of Unidirectional Air Flow
(UDAF) all require specialist equipment.
 The audit should ensure that these items of equipment are being regularly serviced, maintained and calibrated as
appropriate.
Biological indicators

Microbiology
laboratories
typically control the
supply of biological
Certificate
indicators used to of analysis Questions and points that the auditor could pose include:
assess sterilization
devices.
 Bacterial population and purity confirmed
for every supplier lot
 Is the D-value appropriate
procedures are
D value and the
in place to at
 D-value claimed by the supplier ever been
enumeration of verified by a qualified contract facility with
least verify the
spores present
on each
bioburden on  validation of production autoclaves a robust
the indicator as
indicator. logging procedure should be employed to
transport
record the number of biological indicators
issued and a check on those returned. A 100
percent reconciliation
Out-of-specification procedures
microbiology laboratory (OOS) needs to provide for an
should have a procedure in investigation that assess
place to address results that whether or not the observed
do not comply with a result is actually a true result or
specification. the result of laboratory error.
General documentation

review the correct and

a variety of
documentation up-to date
procedures and test A good microbiology SOP will contain
in the methods, work procedures the following areas:
microbiology instructions,
are being  Quality requirements
laboratory,calibration and
maintenanceused and  Media
from SOPs, to
records, validation
results sheets,protocols,that testing  Cultures
and histories are
guidelines,  Equipment
equipment
computerizedmanuals,accurate and  Training
systems complete.  Sample handling
 Lab operations
 Testing methodology
 Data handling/ responding /archiving
 Investigations.
 SOPs are the primary training tool
Data control and data integrity
areas of concern in relation to microbiology
Microbiology laboratories handle a lot of data, although unlike include:
analytical laboratories the data is more often an even mix of  colony forming units are miscounted;
quantitative and qualitative data  Missing samples, such as environmental
monitoring samples not being taken or
dropped on transit to an incubator;
 Failure to take exit suit plates
Paperwork and samples not being reconciled;
Examples of data The weight result  Failure to record a cleaning or disinfection
include the result from the weighing session;
of a settle plate of a raw material  A worksheet being ripped up and the results re-
exposed during the for use in the
environmental pharmacopeial written onto a second sheet;
monitoring of a Microbial Limits  Lack of reconciliation of printed forms;
cleanroom; Test and so on.
Concerns with computer systems e.g. lack of
passwords;
 Out-of-limits results not being investigated;
 Evidence of microbial identifications being re-
run
 Samples retested with no reason recorded.
 The reasons for these issues occurring can derive from:
 Lack of checking;
 Management competency;
 Not having audit trails (paper or electronic in place);
 Accuracy being too often assumed but not verified;
 Poor system security for electronic devices;
 No back-up or archiving of electronic devices;
 Cultural issues, such as seeking to avoid deviations.
 There are also specific concerns that can arise in relation to computerized systems
. five key data integrity questions are:
 Is electronic data available?
 Is electronic data reviewed?
 Is meta data (audit trails) reviewed regularly?
 Are there clear segregation of duties?
 Has the system been validated for its intended use?
 Data integrity in the laboratory is all about preserving and ensuring data over its life
cycle. When the laboratory can trust the information at hand, the organization
contributes to the overall success of the company.
Training Training
Record theis delivered
outcome
staff who are competent
by
Trainee observes test being conducted by a
of training and
to do so. (assuming a
Training
satisfactoryispass’
against
is a qualified staff member.
procedure.
obtained at each stage):
 number of practice sessions
 Trainee is assessed
 Trainee undergoes a final assessment by an
independent person
Assessments can take the form of:
 Observation against a series of set criteria
 By way of written assessment
 As an example, consider sterility testing. A
review of training in sterility testing
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 Risk assessments should address the predominant probable routes of transmission, which are via direct skin,
eye or mucosal membrane exposure to a microorganisms.
 In terms of protective measures, these will include the use of biosafety cabinets the use of personal
protective equipment (gloves, masks, laboratory coats),
Proficiency testing
 Some laboratories elect to self-audit
 test schemes is because all analytical procedures are subject to diverse sources of
systematic and random errors
 Test schemes for microbiology can include using random samples of laboratory
specimens

Waste management
waste disposed of will include direct
microbiological waste (such as culture microbiology waste along with
media, which will be considered as a containers/plates/tubes should
biohazard). either be autoclaved before
Other waste items will include
recycling/disposal or sent to-site
anatomical waste, waste sharps (such as
needles and glass), soiled waste, solid for incineration. Where recycling
waste, liquid waste, plus other types of is a requirement, the segregation
liquid waste generated from lab ware of non-contaminated waste at
washing and laboratory cleaning, and
chemical waste.
source can be undertaken
Space utilization and improving laboratory design

A failure to effectively size

the laboratory or to utilize
For example, areas where
available space to meet user
environmental monitoring
requirements can bench and
samples are read; or where
Not typically part of a formal equipment storage space,
water, or product samples are
audit into quality systems, inflexibility or limited
tested, handled and incubated,
laboratory space utilization is growth, and limited
need to be adequately
often important, especially for functionality in the laboratory.
separated from areas where
smaller organizations where This issue often embodies
there are tests that involve
space is a concern itself in laboratory spill-over
cultures or sub culturing like
into undesired spaces such as
microbial identification are
corridors and common spaces
conducted
result in a variety of safety
hazards.
To review the requirements for an existing laboratory
 power, data, and backup power requirements; and
 Planning for plumbing and HVAC services;
 Creating flexible engineering systems and casework that encourage research teams
 Establishing clearly defined circulation patterns;
 Provide interior glazing to allow people to see one another.
 Linked to the quality system is the topic of sustainability
 numbers of containment and exhaust devices;
 heat-generating equipment;
 microbiologists to have 24-hour access;
 Irreplaceable experiments require fail-safe redundant backup
 systems and uninterrupted power supply (UPS) or emergency power.
 Linked to this, with energy concerns being important,
 Reduction or elimination of harmful substances and waste,
 Improvements to the interior and exterior environments, leading to increased productivity.
 Efficient use of materials and resources,
 Recycling and increased use of products
Resourcing
laboratory must have
sufficient personnel and
funding in order to achieve
its remit.

While this is rarely a direct

part of the audit, it can
become an issue if non
compliances relate to
insufficient controls being
in place or lack of adequate
supervision of work.