Accred Qual Assur (1996) 1: 1823

Q Springer-Verlag 1996 GENERAL PAPER

Dermot Hayes Quality assurance in the microbiology
laboratory
Abstract The pertinent issues nec-
essary for the establishment of
quality assurance in the microbio-
logy laboratory are discussed.
Quality assurance is a planned sys-
tem of control measures that ena-
bles management to ensure that
the analytical data produced in the
laboratory are valid. To introduce
quality assurance, all activities in
the laboratory that affect the pro-
duction of analytical data must be
documented and controlled. These
include sampling, method selection,
laboratory environment, equip-
ment, reagents and media, staff,
reference materials and internal
and external quality control. Labo-
ratory accrediation in accordance
with EN45001 and ISO Guide 25
enables laboratories demonstrate
to an external agency their ability
to perform analytical work and
produce valid analytical data. This
gives creditability to the laboratory
and allows management to have
confidence in the data produced.
Key words Microbiology
laboratory 7 Sampling 7
Micro-organisms 7 Reference
materials 7 Quality control
external 7 Quality control internal 7
Accreditation
Received: 6 June 1995
Accepted: 3 July 1995
Dermot Hayes (Y)
State Laboratory,
Abbotstown, Dublin 15, Ireland
Introduction
Quality assurance has been defined as the total inte-
grated management programme for assuring the relia-
bility of data [1]. In the microbiology laboratory its ad-
option has been slower than in other disciplines. Many
microbiologists consider it an unnecessary time-con-
suming bureaucratic process that is not applicable to
microbiological analysis. They consider that the com-
plexity inherently associated with living organisms
makes the process of quality assurance wasteful in time
and money. On the contrary, however, a laboratory
quality assurance scheme can save time and money by;
(I) reducing the need for costly repeat analysis,
(II) providing quality data with documented fail-safe
practices that ensure the acceptability of the results,
(III) training the staff to avoid costly mistakes by forc-
ing the adoption of written procedures, (IV) identify-
ing the staff requirements and training necessary to
provide an efficient quality service, (V) ensuring credi-
tability which is particularly important when there are
legal implications.
Quality assurance encompasses all aspects of the mi-
crobiology laboratorys analytical activities. In common
with other analytical testing laboratories, particularly
chemical testing laboratories [2], the most important top-
ics are considered to be (a) sampling (sample selection
and sample handling), (b) methodology, (c) environ-
ment, (d) equipment, (e) reagents and media, (f) staff,
(g) reference materials, and (h) internal and external
quality control.
The sample matrix, size, stability, condition, and
storage time before analysis all have significant effects
on the microbiological analysis. When it is considered
that the doubling time for many micro-organisms, such
as bacteria, may be as little as 30 min, a delay in testing
a sample or storage of the sample under inappropriate
conditions may make the analysis meaningless.
19
The environment of the microbiological laboratory
has a profound effect on the quality of the analyses car-
ried out. The laboratory design should facilitate the
testing requirements within the laboratory. Depending
on the type of microbiological analysis being carried
out, it may be necessary to have sterile work rooms,
various classes of laminar flow cabinets, or non-sterile
work bench areas.
The detection and quantification of micro-organism
depend on the method used (e.g. MPN, membrane fil-
tration), so proper methodology selection is essential.
A method suitable for one particular process or opera-
tion may be totally inadequate for another application.
Also the expense of carrying out the method, or the
time required for the analysis, may make the applica-
tion of a particular technique unsuitable. The equip-
ment used to carry out the tests must be shown to be
working properly and to give the correct results. All
media and reagents used in the tests must be of proven
quality and adequate for the test.
The use of reference materials and certified refer-
ence materials is the norm in other disciplines, but the
difficulty of obtaining stable reference materials with
viable cultures of micro-organisms is a further difficulty
for the microbiological analyst. Very few reference ma-
terials are available, and certified reference materials
do not exist [3]. The use of pure reference cultures of
the micro-organism is necessary in so far as it gives in-
formation on the cultural characteristics that are under
investigation, but it does not take into account the af-
fects of the matrix and the difficulties that are caused
by coextractants from the matrix.
The use of internal and external quality control pro-
cedures will check that the procedures in use in the la-
boratory are under statistical control and that the data
are precise and accurate. It is of vital importance that a
laboratory implements such quality control proce-
dures.
With less restrictions on trade throughout the world
the comparability of data among testing laboratories
becomes all the more important. In the past the differ-
ences in quality programmes operated in laboratories
made this difficult. Laboratory accreditation to quality
standards such as EN 45000 series and ISO Guide 25
[4] has given creditability to data produced by laborato-
ries and has ensured wider acceptability of the data.
Many European countries with national accreditation
bodies now have multilateral recognition agreements.
Sampling
Sample selection
The test result obtained from a sample should be indi-
cative of the sample lot. Sampling has been defined by
Garfield [5] as selecting a representative portion of
Fig. 1 Sampling in milk production may be at any of the critical
control points. Clinical samples may be taken to show the pres-
ence or absence of a pathogen
material, in some manner, to represent a larger body of
material, presumably for testing or analysis. The sam-
ple for analysis can only be representative when the
original bulk sample is homogeneous and stable. Also,
in discussing sampling Gy [6] explains homogeneous as
when all units are identical to one another.
Micro-organisms grow in discrete units and are not
spread throughout a bulk sample without thorough
mixing. With the exception of water, sample lots (such
as dense liquids, liquids with organic matter and foods)
do not readily mix, so the selection of a representative
sample is often very difficult.
The number of micro-organisms in a sample will
continuously change. If the sample is stored at P20 7C
before analysis, in order to prevent multiplication of
the micro-organisms, some will die or be injured. If the
sample is retained at higher temperatures, multiplica-
tion of the micro-organisms will inevitably occur. With
clinical samples, the need to maintain the samples at a
temperature as close as possible to 37 7C may be the
overriding consideration, particularly if one is trying to
isolate salmonellae or shigellae [7]. It may also be nec-
essary to maintain the samples in transport media while
awaiting laboratory analysis. The concepts more usual-
ly applied to analysis, such as trueness, bias, accuracy,
error, precision, uncertainty, proficiency testing, inter-
nal quality control, etc. can validly be applied to the
sampling process [8].
Sampling for microbiological analysis should be car-
ried out for a specific purpose (Fig. 1). If there is no
well-defined reason for the sampling, then the subse-
quent quality assurance of the analysis is to an extent
wasted.
On the production line, hazard analysis critical control
point (HACCP) analysis is used to identify sample
points which may show potential problems that require
continued monitoring.
20
Clinical samples are taken and tested to show the pres-
ence or absence of particular pathogens or their suscep-
tibility to chemotherapeutic agents. With certain clini-
cal samples such as those taken during surgery, it is
most unlikely that repeat samples can be obtained.
Thus it is essential that correct transport and storage of
these samples does not affect the viability of the micro-
organisms present.
Samples for regulatory purposes are drawn and tested
to show that the sample lot is safe or, if it is a foodstuff,
that it is fit for human consumption.
Samples that are taken which may involve litigation
should show an unbroken chain of custody from sample
point to laboratory and subsequently to the final report
from the laboratory. It is vital that this unbroken chain
is documented.
Sample handling
The microbiology laboratory should have a docu-
mented procedure for handling samples after delivery.
For example, (a) the sample should be given a unique
unambiguous identification code that traces the sample
from receipt to the end of the analysis, (b) the date and
time (if relevant) of receipt should be recorded togeth-
er with any identification number on the sample, and
(c) details of the sampling date and the condition of the
sample, including its temperature, and the name of the
sampler should be documented.
The sample used in the laboratory test may be pre-
pared by taking an aliquot, in the case of liquids, for
example, or may involve reconstitution and subcultur-
ing, for example in the case of dried products. Whatev-
er procedure is used to select the laboratory test sam-
ple, it must be shown to be representative of the sample
delivered to the laboratory.
Sample disposal
The safe retention and disposal of samples submitted
for laboratory analysis is essential, and in order to en-
sure this a procedure should be documented for de-
scribing their safe storage or disposal. It is important to
remember that certain samples may contain harmful
pathogens, which may require decontamination before
being discarded. It must also be remembered that even
non-pathogenic micro-organisms that have been cul-
tured on growth media may occur in such high numbers
that they are hazardous when released outside the labo-
ratory.
Methodology
Classical methods for the detection and enumeration of
micro-organisms depend on the ability of media to sup-
Fig. 2 As the selectivity of the medium (method) is increased, the
recovery of viable micro-organisms decreases
port the growth of the target organism. The choice of
medium is always a compromise between the ability of
the medium to inhibit the growth of non-target organ-
isms and the low recovery of target organisms in a high-
ly selective medium (Fig. 2).
Methods that differentiate organisms based on me-
tabolite production, or on the basis of cellular compo-
nents such as protein and fatty acid profiling, also de-
pend on the ability of the organisms to grow in a de-
fined culture medium and to show significant character-
istic differences. In general, immunological methods
(e.g. immunofluorescence, ELISA) depend on the
availability of antisera with high specificity. The more
recent molecular biology procedures such as polymer-
ase chain reaction (PCR) depend on the elucidation of
unique nucleic acid sequences that are specific for the
target organism.
The purpose for which the analysis is being perform-
ed will determine the choice of method. No method is
suitable for every purpose. It may be more appropriate
to try to detect a plant pathogen in a consignment of
potatoes by immunofluorescence microscopy, rather
than to attempt to culture the target pathogen on selec-
tive media. In a dairy, it may be more appropriate to
monitor contaminants by rapid conductance or impe-
dance procedures, while the official control laboratory
may use classical cultural methods.
The limit of detection, selectivity, time required to
carry out the tests and the number of tests that can be
carried out in a reasonable time all influence the choice
of procedure. Whatever the choice, the method should
be fully documented and be validated. Official methods
and methods from recognised national or international
organisations have already been validated and may not
require further validation. However, for all other meth-
ods, validation must be carried out and the data stored
for as long as is necessary.
Validation for quantitative and qualitative microbio-
logical test methods should estimate the specificity, re-
lative trueness, positive deviation, negative deviation,
limit of detection, limit of determination, matrix effect,
repeatability and reproducibility.
In microbiological tests the specificity refers to the
degree to which a method is affected by other compo-
nents (for example micro-organisms co-extracted) in
the sample. The relative trueness refers to the degree of
21
equivalence (for example the total count of micro-or-
ganisms) of the results of the method under investiga-
tion to those obtained using a reference method. The
positive and negative deviations refer to positive and ne-
gative results (e.g. the presence or absence of particular
micro-organisms) obtained with the method under in-
vestigation, while the reference method gives the oppo-
site result. The limit of detection refers to the lowest
number of micro-organisms that can be detected, but
they are too few in number to be estimated accurately.
The limit of determination refers to the lowest number
of micro-organisms with a defined variability that may
be determined under the experimental conditions of
the method under evaluation.
For validation of a microbiological method, natural-
ly contaminated products (if available) or products
spiked with various levels of micro-organisms should be
tested. It should be remembered that different sample
matrices can adversely effect the performance of a
method. Some international organisations, such as the
Association of Official Analytical Chemists (AOAC),
validate microbiological methods through collaborative
testing.
Environment
The microbiological analysis should be carried out in
facilities that do not adversely effect the result. Ideally,
the laboratory should be designed on a no way back
principle, but this is not always feasible, for example in
older buildings. In the design of the laboratory it is
preferable to have separate areas for (a) sample receipt
and storage, (b) sample preparation, (c) maintenance
of reference micro-organisms, and (d) sterilisation and
decontamination.
The laboratory floors, walls, ceiling and worktops
should be constructed of materials that are smooth and
easy to clean and disinfect.
Continuous documented monitoring of the laborato-
ry environment is essential, for example, by means of
contact plates and exposure plates [9]. Acceptable
background counts should be assigned and a docu-
mented procedure should be established to deal with
counts exceeding these limits. Analysts working in the
microbiology laboratory should wear protective cloth-
ing appropriate for the tests being carried out and
should remove them before leaving the area.
Equipment
All equipment used to carry out analyses must be kept
in good working order and the microbiology laboratory
must document and carry out a programme for mainte-
nance, calibration and performance verification of the
equipment. General service equipment, such as incuba-
tors, water baths, autoclaves, should be maintained by
cleaning and should be serviced regularly by trained
service engineers. Records of all cleaning and servicing
must be kept. A programme for calibration of the
equipment and verification of performance must also
be established; for example, thermometers in water
baths should be calibrated at least annually against a
reference thermometer at freezing point and at the
working temperature in the waterbath. The perform-
ance of equipment such as the autoclave should be de-
termined for a typical load, and it should be capable of
meeting the specified temperature at all points in the
typical load for sterilisation and/or decontamination.
The operation of the autoclave, including the tempera-
ture and time of each cycle, should be documented.
Thermocouples, maximum thermometers or visual ob-
servation of the maximum temperature achieved on the
autoclave temperature/pressure gauge should be used
to monitor the temperature. Chemical or biological in-
dicators may be used to check the effectiveness of the
autoclave. Autoclave tape should be used to indicate
that a batch has been processed; it should not be used
to indicate a successful sterilisation or decontamination
cycle.
Reagents
All reagents, media and solutions used to perform ana-
lytical tests in the microbiology laboratory should be
prepared according to documented procedures. Media
prepared in the laboratory should be checked to ensure
that they support the growth of specific microbial cul-
tures. Selective media should also be checked to ensure
that they inhibit the growth of non-target organisms.
All reagents must be used within their shelf-life. It
might be considered unnecessary to check media re-
ceived from reputable manufacturers, but Shanholtzer
et al. [10] concluded that the testing carried out by
manufacturers of media was not always adequate and
that failure of the media to recover significant patho-
gens would make the laboratorys skills in identification
irrelevant.
Staff
The microbiological tests must be carried out by or un-
der the direction of a person qualified in microbiology.
Staff performing the tests must have adequate training
for the techniques and instruments they use, and their
competence to carry out these tests should be contin-
ually monitored. They should be retrained if they fail to
achieve competence in the tests during subsequent
monitoring.
22
Fig. 3 The use of reference cultures of micro-organisms is similar
to the use of reference standards in chemical tests
Reference materials
The microbiological analyst is familiar with the use of
reference micro-organisms for control purposes in ex-
periments (Fig. 3). Reference strains give information
(a) on the cultural, biochemical and pathogenic charac-
teristics of the cultures under test, (b) on how the cul-
tures appear on different media and (c) on how they
behave at different incubation temperatures. They are
generally seen to be necessary for accurate comparative
work.
The use of reference micro-organisms in various ma-
trices is a relatively new concept. The use of micro-
organisms in this manner leads to consideration of tra-
ceability and uncertainty of measurement, which are
concepts not readily understood or used in microbio-
logical analyses. There is difficulty in obtaining such
reference materials and there are problems associated
with the stability of these materials, which need to be
overcome. Traceability of mass is readily understood
for physical measurements where mass is traceable
through an unbroken chain of comparisons to the pro-
totype kilogram (Bureau International des Poids et ei
Measures in Paris; BIPM). The mass measurement used
in the laboratory may be traceable to the prototype
kilogram, but the micro-organism isolated or quantified
is not traceable to some prototype micro-organism.
The solution proposed to solve this dilemma is simi-
lar to that established for chemical measurement [11],
i.e. the development of reference and certified refer-
ence materials. The materials used in chemical analysis
contain a guaranteed and predetermined level of ana-
lyte, with a given level of uncertainty (an estimate of
the range of values within which the true value lies).
To produce a similar type of reference material for
microbiological analyses it is necessary to inoculate var-
ious matrices with cultures of the micro-organisms that
are being tested and quantify the level through collabo-
rative studies. Such reference materials have been pro-
duced from spray dried milk and encapsulated with ge-
latin [12, 13]. Others have established similar materials
and stabilised them by freezing and freeze-drying [14].
The equivalent certified reference material have not
been produced, presumably because of the difficulty of
obtaining data with documented levels of uncertainty.
When using reference materials, the analyst assumes
that data obtained in from samples of a similar matrix are
correct if the data obtained using the reference material
are within the expected variation of the reference mate-
rial.
Since preparing reference materials is difficult in
many instances and impossible in others, it would be
preferable to add micro-organisms to the sample under
test and run this sample with the added micro-organ-
isms as a control sample. This could be routinely car-
ried out with bacteria, for example, by preparing stocks
of the pure culture and calculating the total viable
count in a dilution series. These diluted stocks could be
stored at P20 7C in 20% glycerol (or other cryo-protec-
tant) and used to spike samples. The viability of the di-
lution series of the pure cultures could be determined
regularly and the effect of the various matrices on their
recovery could be ascertained.
Internal / external quality control
The principle function of the microbiology laboratory is
to provide reliable data. Unreliable data can be danger-
ous, i.e. incorrect diagnosis of pathogens in clinical
samples or undetected pathogens in food and water
samples. An internal quality control programme in a la-
boratory covers the staff, methods, equipment, media
and reagents and is essential to the achievement of reli-
able data that are both precise and accurate.
Control charts are used in other disciplines to moni-
tor the day-to-day variation in the laboratory. In the
microbiology laboratory, a stable and homogeneous
quality control material may not always be available.
However, if spray-dried, freeze-dried or deep-frozen
reference material is available a portion of the material
can be analysed alongside each batch of samples. The
results of the analyses on the control material is plotted
on the ordinate of the control chart and the date plot-
ted on the abscissa. Lines representing acceptable re-
sults for the control material should be drawn. They are
usually set at the target value plus and minus 10%.
When the control material is outside these acceptable
limits the batch should be repeated. Control charts give
a rapid visual representation that the analyses are un-
der statistical control.
Participation in external quality control programmes
demonstrates the laboratorys ability to produce the
correct data, when testing for a specific analyte. Partici-
pation in proficiency testing schemes can be used by a
laboratory to ascertain its performance vis--vis other
laboratories. They have been used for the evaluation of
clinical laboratories for many years. Schemes for food
23
and water microbiology are also available, but unlike
clinical tests, these are often quantitative [15] and the
difficulty again arises for the distribution of stable ho-
mogeneous samples [16].
Accreditation
Accreditation bodies throughout the world have based
their assessment criteria on ISO/IEC Guide 25. In Eu-
rope, the European standard EN 45001 [17] is used and
is itself based on the ISO Guide 25. The microbiologi-
cal laboratories may be externally assessed by accredi-
tation bodies. A number of countries now have accredi-
tation bodies and there is mutual recognition between
most of these national accreditation organisations. Ac-
ceptance of analytical data between laboratories is fa-
cilitated by meeting accreditation requirements. The re-
quirements for accreditation cover all aspects of the
analytical work in the laboratory. Details concerning
every factor that influences the data are recorded and
documented and are available for inspection if neces-
sary. Among the topics covered in accreditation are;
(a) the laboratory management and organisation,
(b) the quality system in operation,
(c) audit and review procedures,
(d) the skills, experience and qualifications of the
staff,
(e) the calibration, performance checking and mainte-
nance of equipment,
(f) the methodology used,
(g) the laboratory environment,
(h) the handling and identification of samples,
(i) the recording of data and reporting of results.
These requirements for accreditation are written in
general terms and need interpretation for use in the mi-
crobiology laboratory. A guidance document that helps
this interpretation has been produced by EURA-
CHEM/EAL [18]. A laboratory may be accredited for a
specific test on a particular material, or it may be accre-
dited for techniques that are applicable in a number of
analytical areas. Before accreditation is granted, a labo-
ratory must demonstrate to the national accreditation
body that all the factors listed above that affect the
analysis are properly addressed.
Conclusion
In the majority of laboratories most of the elements of
a quality assurance programme are carried out, often
without a stated policy or without full documentation
of the process. Documenting the process may be time
consuming, but the pay-back is a more easily managed
laboratory where management can have confidence in
the data generated. When a laboratory gains accredita-
tion for its test procedures or techniques, it shows that
the practices used in the laboratory are managed to an
acceptable standard. The disadvantage of not gaining
accreditation is that results from a non-accredited labo-
ratory will be more liable to challenge. Since most of
the practices involved in a quality assurance pro-
gramme involve better management of laboratory re-
sources, it would be battling against the tide to resist
the introduction of such a programme.
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