Académique Documents
Professionnel Documents
Culture Documents
Microbiological Laboratories
Accreditation for
Microbiological
Laboratories
Second edition
This document has been produced by Eurachem. It provides
microbiological laboratories with appropriate information and
guidance on how to be prepared in order to fulfil the
requirements of ISO/IEC 17025. It supersedes EA-
4/10:2002.
Editors
Mary Eleftheriadou, European University Cyprus
Kyriacos C. Tsimillis, Cyprus Accreditation Body
English edition
ISBN: 978-91-87017-92-6.
Copyright © 2013
The copyright of this text is held by Eurachem. Enquiries regarding the translation, and production and distribution of new editions
of this document should be directed to the Eurachem secretariat. The text may not be copied for resale.
CONTENTS
Main Standards used for laboratory accreditation 3.3 The laboratory management shall ensure that
ISO/IEC 17025, General requirements for the all personnel have received adequate training for
competence of testing and calibration the competent performance of tests and the
laboratories [1] operation of equipment. This should include
ISO 15189, Medical laboratories – Requirements training in basic techniques, e.g. plate pouring,
for quality and competence [2] counting of colonies, aseptic technique, etc., with
acceptability determined using objective criteria.
Terminology Personnel may only perform tests on samples if
ISO 9000, Quality management systems – they are either recognised as competent to do so,
Fundamentals and vocabulary [6] or if they do so under adequate supervision. On-
ISO/IEC Guide 99, International vocabulary of going competence should be monitored objectively
metrology – Basic and general concepts and with provision for retraining where necessary.
associated terms (VIM 3) (Available as JCGM 200 Where a method or technique is not in regular use,
from www.bipm.org) [7] verification of personnel performance before testing
is undertaken may be necessary. The critical
Basic standards interval between performance of tests should be
ISO 7218, Microbiology of food and animal feeding established and documented. The interpretation of
stuffs – General requirements for microbiological test results for identification and verification of
examinations [8] micro-organisms is strongly connected to the
ISO/TS 19036, Microbiology of food and animal experience of the performing analyst and should be
feeding stuffs – Guidelines for the estimation of monitored for each analyst on a regular basis.
measurement uncertainty for quantitative
determinations (incl. Amd 1: Measurement 3.4 In some cases, it may be more appropriate to
uncertainty for low counts) [9] relate competence to a particular technique or
ISO 29201 Water Quality – The variability of test instrument rather than to methods.
results and the uncertainty of measurement of
microbiological enumeration methods [10] 3.5 The competence of personnel to perform tests
ISO 8199, Water quality – General guidance on the shall be documented in relation to the results of
enumeration of micro-organisms by culture [11] internal and external quality control. The
effectiveness of the training programme, as well as
A detailed list of documents to be considered the identification of further training needs, should
appears in the References. also be evaluated based on these results.
3 Personnel
4.3 Hygiene
4.3.1 There shall be a documented cleaning
programme for laboratory fixtures, equipment and
surfaces. It should take into account the results of
environmental monitoring and the possibility of
AML 2013 Page 4
Accreditation for Microbiological Laboratories Eurachem Guide
5 Validation and Verification of test 5.2.2 For quantitative microbiological test methods,
the specificity, sensitivity, relative trueness, positive
methods deviation, negative deviation, repeatability,
reproducibility and the limit of quantification within a
ISO/IEC 17025, paragraph 5.4.5 defined variability should be considered and, if
See also ISO/TS 12869 [12], ISO 7218 [8], ISO necessary, quantitatively determined. The
17994 [13], ISO/TR 13843 [14], ISO 16140 [15] differences due to the matrices must be taken into
account when testing different types of samples.
The results should be evaluated with appropriate
5.1 Selection of test methods statistical methods.
The laboratory shall use appropriate test methods
to meet the specific needs in each case. To this 5.2.3 Laboratories shall retain validation data on
end it is preferable to use standard methods, i.e. commercial test systems (kits) used in the
the ones published in international, regional or laboratory. This validation data may be obtained
national standards, or in scientific textbooks or by through collaborative testing and from validation
widely recognised and reputable organisations. data submitted by the manufacturers and subjected
These methods have normally been validated. to third party evaluation (e.g. ΑFNOR, NordVal,
However, other methods could be used provided Microval, AOAC). If the validation data are not
that they are validated by the laboratory as available, or not wholly applicable, the laboratory
described in 5.2.1 and 5.2.2. This is the case with shall be responsible for completing the validation of
laboratory-designed/developed methods and the method.
standard methods used outside their intended
scope or modified in a way that an influence on the 5.2.4 If a modified version of a method is required
test results must be assumed. Further to this, the to meet the same specification as the original
detailed procedure to be followed in each case method, then the modified method must be
varies with the nature of the method, i.e. validated for those parameters that are likely to be
qualitative, semi-quantitative and quantitative. The affected by the revision. Experimental design and
method/technique used will dictate the aspects of analysis of results must be statistically valid.
validation to be considered.
5.2.5 Even when validation is completed, the user
The following standards can assist laboratories in will still need to verify, as appropriate, (e.g. when
obtaining method validation data: ISO/TR 13843, there is a change in the critical factors) that the
ISO 16140 and ISO 17994. documented performance can be met. This can be
accomplished by the use of spiked samples or
reference materials incorporating relevant matrices.
5.2 Validation
5.3 Verification
The validation of microbiological test methods
should reflect actual test conditions. This may be In the preferable case of standard and validated
achieved by using naturally contaminated products methods being used, the laboratory is still required
or products spiked with a predetermined level of to prove that it can implement them in a reliable
contaminating organisms. The analyst should be way. This is called verification – see further
aware that the addition of contaminating organisms “Eurachem Terminology in Analytical
to a matrix only mimics in a superficial way the Measurement”. For verification of quantitative
presence of the naturally occurring contaminants. methods the laboratory has, in most cases, to
However, it is often the best and only solution determine repeatability, measurement uncertainty
available. The extent of validation necessary will (see section 6) and limit of quantitation, and for
depend on the method and the application. The qualitative methods the limit of detection.
laboratory shall validate standard methods applied
to matrices not specified in the standard procedure.
(a) Autoclaves should be capable of meeting pipettes and disposable pipettes may all be used in
specified time and temperature tolerances. the microbiology laboratory. Laboratories should
Pressure cookers fitted only with a pressure gauge carry out initial verification of volumetric equipment
are not acceptable. Sensors used for controlling or and then make regular checks to ensure that the
monitoring operating cycles require calibration and equipment is performing within the required
the performance of timers should be verified. specification. Verification should not be necessary
for glassware which has been certified to a specific
(b) Initial validation should include performance tolerance. Equipment should be checked for the
studies (spatial temperature distribution surveys) accuracy of the delivered volume against the set
for each operating cycle and each load volume (for several different settings in the case of
configuration used in practice. This process must variable volume instruments) and the precision of
be repeated after significant repair or modification the repeat deliveries should be measured.
(e.g. replacement of thermo-regulator probe or
programmer, modification of loading arrangements (b) For ‘single-use’ disposable volumetric
or operating cycle) or where indicated by the equipment, laboratories should obtain supplies
results of quality control checks on media. from companies with a recognised and relevant
Sufficient temperature sensors should be quality system. After initial validation of the
positioned within the load (e.g. in containers filled suitability of the equipment, it is recommended that
with liquid/medium) to enable location differences random checks on accuracy are carried out. If the
to be demonstrated. In the case of media supplier has no recognised quality system,
preparators, where uniform heating cannot be laboratories should check each batch of equipment
demonstrated by other means, the use of two for suitability.
sensors, one adjacent to the control probe and one
remote, would generally be considered appropriate. 7.2.7 Thermal cyclers
Validation and re-validation should consider the Laboratories should carry out verification of
suitability of come-up and come-down times as well temperature, ramp rate, overshoots/undershoots,
as time at sterilisation temperature. hold time.
(c) Clear operating instructions should be provided 7.2.8 Other equipment
based on the heating profiles determined for typical
uses during validation/re-validation. Conductivity meters, oxygen meters, pH meters
Acceptance/rejection criteria should be established and other similar instruments should be verified
and records of autoclave operations, including regularly or before each use. The buffers used for
temperature and time, maintained for every cycle. verification purposes should be stored in
appropriate conditions and should be marked with
(d) Monitoring may be achieved by one of the an expiry date. Where humidity is important to the
following: outcome of the test, hygrometers should be
(i) using a thermocouple and recorder to calibrated, the calibration being traceable to
produce a chart or printout; national or international standards. Timers,
(ii) direct observation and recording of including the autoclave timer, should be verified
maximum temperature achieved and time at using a calibrated timer or national time signal.
that temperature. Where centrifuges are used in test procedures, an
assessment should be made of the criticality of the
In addition to directly monitoring the temperature of centrifugal force. Where it is critical, the centrifuge
an autoclave, the effectiveness of its operation will require calibration.
during each cycle may be checked by the use of
chemical or biological indicators for
sterilisation/decontamination purposes.
Autoclave tape or indicator strips should be used
only to show that a load has been processed, not
to demonstrate completion of an acceptable cycle.
8.2 In-house prepared media 8.3.3 As part of this performance evaluation, the
8.2.1 The suitable performance of culture media, user laboratory needs to have adequate knowledge
diluents and other suspension fluids prepared in- of the manufacturer's quality system and the
house should be checked, where relevant, with product specifications, which include at least the
regard to: following:
• recovery or survival maintenance of target • name of the media and list of components,
organisms; including any supplements;
• inhibition or suppression of non-target • shelf-life and the acceptability criteria applied;
organisms; • storage conditions;
• biochemical (differential and diagnostic) • sterility check;
properties; • check of growth of target and non-target control
• physical properties (e.g. pH, volume and organisms used (with their culture collection
sterility). references) and acceptability criteria;
• physical checks and the acceptability criteria
Quantitative procedures for evaluation of recovery applied;
or survival should be performed according to ISO • date of issue of specification.
11133.
All the above, including the validation exercise,
8.2.2 Raw materials (both commercial dehydrated should be taken into consideration by the
formulations and individual constituents) should be laboratory to set acceptability criteria for incoming
stored under appropriate conditions, e.g. cool, dry batches of media appropriate for its testing needs.
and dark. All containers, especially those for
dehydrated media, should be sealed tightly. 8.3.4 Batches of media should be identifiable. Each
Dehydrated media that are caked or cracked, or one received should be accompanied by evidence
show a colour change, should not be used. Distilled that it meets the quality specification. The user
deionised, or reverse osmosis produced water, free laboratory should ensure that it will be notified by
from bactericidal, inhibitory or interfering the manufacturer of any changes to the quality
substances, should be used for preparation unless specification.
the test method specifies otherwise.
Note:
8.2.3 Shelf-life of prepared media under defined Batch: A homogeneous and fully traceable unit of a
storage conditions shall be determined and culture medium or reagent referring to a defined
verified. amount of bulk, semi-finished product or end-
product, which is consistent in type and quality and
which has passed the requirement of production
8.3 Ready-to-use-media (in-process control) and performance testing and
which has been produced within one defined
8.3.1 All media, including diluents and other production period, having been assigned the same
suspension fluids procured ready-to-use or partially number.
complete, also require performance evaluation as
in 8.2.1 before use. Evaluation of performance in
AML 2013 Page 9
Accreditation for Microbiological Laboratories Eurachem Guide
8.4 Labelling
Laboratories shall ensure that all reagents
(including stock solutions), media, diluents, and
other suspending fluids are adequately labelled to
indicate, as appropriate, identity, concentration,
storage conditions, date of opening, preparation
date, validated expiry date and/or recommended
storage periods. The person responsible for
preparation should be identifiable from records.
10 Sampling
ISO/IEC 17025, paragraphs 5.7 and 5.8 ISO/IEC 17025, paragraph 5.8
See also ISO 7218 [8], paragraph 8, ISO 6887 See also ISO 7218 [8]
[24] and ISO 19458 [23]
The correct disposal of contaminated materials
11.1 Microbial flora may be sensitive to factors may not directly affect the quality of sample
such as temperature or duration of storage and analysis, although procedures should be designed
transport, so it is important to check and record the to minimise the possibility of contaminating the test
condition of the sample on receipt by the environment or materials. However, it is a matter of
laboratory. good laboratory management and should conform
to national/international environmental or health
11.2 The laboratory shall have procedures that and safety regulations.
cover the delivery of samples and sample
identification. If there is insufficient sample or the
sample is in poor condition due to physical
deterioration, incorrect temperature, damaged
packaging or deficient labelling, the laboratory
should consult with the customer before deciding
whether to test or refuse the sample. In any case,
records should be maintained and the condition of
the sample should be indicated on the test report.
14 Test reports
Calibration Operation that, under specified conditions, in a first step, establishes a relation
between the quantity values with measurement uncertainties provided by
measurement standards and corresponding indications with associated measurement
uncertainties and, in a second step, uses this information to establish a relation for
obtaining a measurement result from an indication.
NOTE 3 Often, the first step alone in the above definition is perceived as being
calibration.
[VIM 3]
Limit of detection Applied to qualitative microbiological tests: The lowest number of micro-organisms
that can be detected, but in numbers that cannot be estimated accurately.
Negative deviation Occurs when the alternative method gives a negative result without confirmation
when the reference method gives a positive result. This deviation becomes a false
negative result when the true result can be proved as being positive.
Positive deviation Occurs when the alternative method gives a positive result without confirmation when
the reference method gives a negative result. This deviation becomes a false positive
result when the true result can be proved as being negative.
Reference cultures Collective term for reference strain, reference stocks and working cultures.
Reference material Material, sufficiently homogeneous and stable with reference to specified properties,
which has been established to be fit for its intended use in measurement or in
examination of nominal properties.
[VIM 3]
Reference method Thoroughly investigated method, clearly and exactly describing the necessary
conditions and procedures, for the measurement of one or more property values that
has been shown to have trueness and precision commensurate with its intended use
and that can therefore be used to assess other methods for the same measurement,
particularly in permitting the characterisation of a reference material. Normally a
national or international standard method.
Reference stocks A set of separate identical cultures obtained by a single sub-culture from the
reference strain. [ISO 11133]
Reference strains Micro-organisms defined at least to the genus and species level, catalogued and
described according to its characteristics and preferably stating its origin.
[ISO 11133]
Normally obtained from a recognised national or international collection.
Relative trueness The degree of correspondence of the results of the method under evaluation to those
obtained using a recognised reference method.
Sensitivity The fraction of the total number of positive cultures or colonies correctly assigned in
the presumptive inspection. [ISO/TR 13843]
Specificity The fraction of the total number of negative cultures or colonies correctly assigned in
the presumptive inspection. [ISO/TR 13843]
Validation The confirmation by examination and the provision of objective evidence that the
particular requirements for a specific intended use are fulfilled. [ISO/IEC 17025]
Note: Primary validation. An exploratory process with the aim of establishing the
operational limits and performance characteristics of a new, modified or otherwise
inadequately characterised method. It should result in numerical and descriptive
specifications for the performance and include a detailed and unambiguous
description on the target of interest (positive colony, tube or plaque). [ISO 13843]
Provision of objective evidence that a given item fulfils specified requirements.
Verification
[VIM 3]
All parts of the process shall be fully documented and detailed records of all steps must be maintained
Check weight(s) Check against calibrated weight or check on Every two years
balance immediately following traceable
calibration
Temperature controlled (a) Establish stability and uniformity of (a) Initially, periodically, at documented
equipment (incubators, temperature frequency, and after repair/
baths, fridges, freezers) (b) Monitor temperature modification
(b) Daily .each use
Sterilising ovens (a) Establish stability and uniformity of (a) Initially, periodically, at documented
temperature frequency, and after repair/
(b) Monitor temperature modification
(b) Daily/each use
Safety cabinets (a) Establish performance (a) Initially, every year and after repair/
(b) Microbiological monitoring modification
(c) Air flow monitoring (b) Weekly
(c) Daily/each use
Laminar air flow cabinets (a) Establish performance (a) Initially, and after repair/modification
(b) Check with sterility plates (b) Weekly
Gravimetric diluters (a) Check weight of volume dispensed (a) Daily/each use
(b) Check dilution ratio (b) Daily/each use
Pipettors/pipettes Check accuracy and precision of volume Regularly (to be defined by taking account of
dispensed by gravimetric method the frequency and nature of use)
Spiral platers (a) Establish performance against (a) Initially and annually
conventional method (b) Daily/each use
(b) Check stylus condition and the start and (c) Monthly
end points
(c) Check volume dispensed
Laboratory environment Monitor for airborne and surface microbial Weekly for total count and moulds:
contamination using, e.g. air samplers, settle
plates, contact plates or swabs Biannually for pathogens or as otherwise
decided by the laboratory based on activities
and historical trends and results
Water baths Empty, clean, disinfect and refill Monthly, or every 6 months if biocide used
Laboratory (a) Clean and disinfect working surfaces (a) Daily, and during use
(b) Clean floors, disinfect sinks and basins (b) Weekly or more frequently if required
(c) Clean and disinfect other surfaces (c) Every 3 – 12 months depending on
type of laboratory work
References
1. ISO/IEC 17025:2005, General requirements for the competence of testing and calibration laboratories.
2. ISO 15189:2012, Medical laboratories – Requirements for quality and competence.
3. WHO Handbook: good laboratory practice (GLP): quality practices for regulated non-clinical research
and development - 2nd ed. ISBN 978 92 4 154755 0.
4. WHO Good Manufacturing Practices for Pharmaceutical Products: Main Principles. WHO Technical
Report Series, No. 961, 2011, Annex 3.
5. WHO Guidelines for good clinical practice (GCP) for trials on pharmaceutical products, WHO Technical
Report Series, No. 850, 1995, Annex 3.
6. ISO 9000:2005, Quality management systems – Fundamentals and vocabulary.
7. ISO/IEC Guide 99:2007, International vocabulary of metrology – Basic and general concepts and
associated terms (VIM) (also published as BIPM, Report JCGM 200, www.bipm.org).
8. ISO 7218:2007 (Amd2:2013), Microbiology of food and animal feeding stuffs – General requirements
and guidance for microbiological examinations.
9. ISO/TS 19036:2006 (Amd1:2009), Microbiology of food and animal feeding stuffs – Guidelines for the
estimation of measurement uncertainty for quantitative determinations.
10. ISO 29201:2012, Water quality – The variability of test results and the uncertainty of measurement of
microbiological enumeration methods.
11. ISO 8199:2005, Water quality – General guidance on the enumeration of micro-organisms by culture.
12. ISO/TS 12869:2012, Water quality – Detection and quantification of Legionella spp. and/or Legionella
pneumophila by concentration and genic amplification by quantitative polymerase chain reaction
(qPCR).
13. ISO 17994:2004 Water Quality – Criteria for establishing equivalence between microbiological
methods.
14. ISO/TR 13843:2000, Water quality – Guidance on validation of microbiological methods.
15. ISO 16140:2003 (Amd1:2011), Microbiology of food and animal feeding stuffs – Protocol for the
validation of alternative methods.
16. EA-4/16 G:2003, EA guidelines on the expression of uncertainty in quantitative testing, European co-
operation for Accreditation.
17. QSOP 4:2005, Uncertainty of measurement in testing, Health Protection Agency, UK.
18. ISO/IEC Guide 98-3:2008, Uncertainty of measurement – Part 3: Guide to the expression of uncertainty
in measurement (GUM:1995) (also published as BIPM, Report JCGM 100, www.bipm.org).
19. Guidance document on measurement uncertainty for GMO testing laboratories, S. Trapmann, M.
Burns, H. Broll, R. Macarthur, R. Wood, J. Zel, JRC/IRMM Guidance EUR 22756 EN/2 2009.
20. ILAC P10:01/2013: ILAC Policy on the Traceability of Measurement Results.
21. ISO/TS 11133-1:2009, Microbiology of food and animal feeding stuffs – Guidelines on preparation and
production of culture media – Part 1: General guidelines on quality assurance for the preparation of
culture media in the laboratory.
22. ISO/TS 11133-2:2003 (Amd1:2011), Microbiology of food and animal feeding stuffs – Guidelines on
preparation and production of culture media – Part 2: Practical guidelines on performance testing of
culture media.
23. ISO 19458:2006 Water Quality – Sampling for microbiological analysis.
24. ISO 6887-Pt 1-5:1999-2010, Microbiology of food and animal feeding stuffs – Preparation of test
samples, initial suspension and decimal dilutions for microbiological examination – Parts 1-5.
25. EA-4/18 TA:2010, Guidance on the level and frequency of proficiency testing participation, European
co-operation for Accreditation.
26. Selection, Use and Interpretation of Proficiency Testing (PT) Schemes by Laboratories, I. Mann, B.
Brookman (Eds), 2nd Edition, 2011, Eurachem www.eurachem.org.
27. ISO/IEC 17043:2010 Conformity Assessment-General requirements for proficiency testing.
28. S.L.R. Ellison and A. Williams (Eds), Eurachem/CITAC guide: Quantifying Uncertainty in Analytical
Measurement, Third edition (2012), ISBN: 978-0-948926-30-3. Available from www.eurachem.org.