EN ISO 11138-1:2006

SLOVENSKI STANDARD
01-september-2006
1DGRPHãþD
SIST EN 866-1:2000
Sterilizacija izdelkov za zdravstveno nego - Biološki indikatorji - 1. del: Splošne
zahteve (ISO 11138-1:2006)
Sterilization of health care products - Biological indicators - Part 1: General requirements
(ISO 11138-1:2006)
Sterilisation von Produkten für die Gesundheitsfürsorge - Biologische Indikatoren - Teil 1:
Allgemeine Anforderungen (ISO 11138-1:2006)
Stérilisation des produits de santé - Indicateurs biologiques - Partie 1: Exigences
générales (ISO 11138-1:2006)
Ta slovenski standard je istoveten z: EN ISO 11138-1:2006
ICS:
11.080.01 Sterilizacija in dezinfekcija na Sterilization and disinfection
splošno in general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 11138-1
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2006
ICS 11.080.01 Supersedes EN 866-1:1997
English Version
Sterilization of health care products - Biological indicators - Part
1: General requirements (ISO 11138-1:2006)
Stérilisation des produits de santé - Indicateurs biologiques Sterilisation von Produkten für die Gesundheitsfürsorge -
- Partie 1: Exigences générales (ISO 11138-1:2006) Biologische Indikatoren - Teil 1: Allgemeine Anforderungen
(ISO 11138-1:2006)
This European Standard was approved by CEN on 7 June 2006.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Central Secretariat or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11138-1:2006: E
worldwide for CEN national Members.

Foreword
This document (EN ISO 11138-1:2006) has been prepared by Technical Committee ISO/TC 198
"Sterilization of health care products" in collaboration with Technical Committee CEN/TC 102
"Sterilizers for medical purposes", the secretariat of which is held by DIN.

This European Standard shall be given the status of a national standard, either by publication of
an identical text or by endorsement, at the latest by January 2007, and conflicting national
standards shall be withdrawn at the latest by January 2007.

This document supersedes EN 866-1:1997.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

Endorsement notice
The text of ISO 11138-1:2006 has been approved by CEN as EN ISO 11138-1:2006 without any
modifications.
INTERNATIONAL ISO
STANDARD 11138-1
Second edition
2006-07-01
Sterilization of health care products —
Biological indicators —
Part 1:
General requirements
Stérilisation des produits de santé — Indicateurs biologiques —
Partie 1: Exigences générales
Reference number
ISO 11138-1:2006(E)
©
ISO 2006
ISO 11138-1:2006(E)
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ii © ISO 2006 – All rights reserved

ISO 11138-1:2006(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
1.1 General. 1
1.2 Exclusions . 1
2 Normative references . 1
3 Terms and definitions. 2
4 General manufacturing requirements. 4
4.1 Manufacturing controls. 4
4.2 Test organism . 5
4.3 Information supplied by manufacturer (labelling). 6
4.4 Storage and transport . 6
5 Specific manufacturing requirements . 7
5.1 Suspensions. 7
5.2 Carrier, primary and secondary packaging . 7
5.3 Inoculated carrier. 8
5.4 Biological indicators. 8
5.5 Self-contained biological indicators. 8
6 Determination of resistance . 8
6.1 General resistance requirements. 8
6.2 Test organism . 9
6.3 Population of test organisms . 9
6.4 Resistance characteristics . 9
6.5 Test conditions . 10
7 Culture conditions . 10
7.1 Incubator. 10
7.2 Growth medium. 10
7.3 Incubation. 10
Annex A (normative) Determination of viable count . 11
Annex B (normative) Determination of growth inhibition by carriers and primary packaging
materials exposed to sterilization processes . 13
Annex C (normative) D value determination by survivor curve method . 15
Annex D (normative) D value determination by fraction negative method. 19
Annex E (normative) Survival-kill response characteristics . 35
Annex F (informative) Relationship between components of biological indicators . 36
Bibliography . 37

ISO 11138-1:2006(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 11138-1 was prepared by Technical Committee ISO/TC 198, Sterilization of health care products.
This second edition cancels and replaces the first edition (ISO 11138-1:1994), which has been technically
revised.
ISO 11138 consists of the following parts, under the general title Sterilization of health care products —
Biological indicators:
⎯ Part 1: General requirements
⎯ Part 2: Biological indicators for ethylene oxide sterilization processes
⎯ Part 3: Biological indicators for moist heat sterilization processes
⎯ Part 4: Biological indicators for dry heat sterilization processes
⎯ Part 5: Biological indicators for low-temperature steam and formaldehyde sterilization processes
iv © ISO 2006 – All rights reserved

ISO 11138-1:2006(E)
Introduction
This part of ISO 11138 specifies general requirements for production, labelling, test methods and performance
requirements for the manufacture of biological indicators including inoculated carriers and suspensions
intended for use in validation and monitoring of sterilization processes. Subsequent parts of ISO 11138
provide additional specific requirements for biological indicators for defined sterilization processes.
A graphic description of a biological indicator and its components is presented in Annex F. The presentation
includes the two types of biological indicator which are covered by ISO 11138. This shows that inoculated
carriers can be presented directly to the sterilizing agent without prior packaging, or included in a primary
package that permits access by the sterilizing agent.
The resistance characteristics depend on the type of test organism, its numbers, the method of preparation
and the effects of the primary package. Advice on selection, use and interpretation of results of biological
[7]
indicators can be found in ISO 14161 .
For any individual sterilization process, including those covered in subsequent parts of ISO 11138, the
resistance of the biological indicator will also depend on its microenvironment during testing. In theory, this
could lead to an infinite variation in the preparation of biological indicators. Moreover, a sterilization process
could be manipulated in infinite variety to suit each possible set of conditions to which products could be
exposed. It has therefore been routine practice to manufacture biological indicators that, when exposed to a
set of conditions in a defined sterilization process, provide resistance characteristics expressed as D values
and, where relevant, z values. Such values are set out in the subsequent parts of ISO 11138.
ISO 11138, parts 1 to 5 represent the current “state-of-the-art” according to the experts representing
manufacturers, users and regulatory authorities involved in developing this International Standard.
Biological indicators for specific sterilization processes not covered by reference test conditions in subsequent
parts of ISO 11138 should comply with the general requirements in this part, including the resistance testing
procedures. Such biological indicators might not be well enough described, or might be used for novel
sterilization processes, or might be represented by isolated bioburden microorganisms. If microorganisms
[27]
other than risk group 1 (WHO,1993 ) are included in these biological indicators, the appropriate containment
and safety levels must be met.
Standards exist providing requirements for the validation and control of sterilization processes
(see Bibliography).
NOTE Some countries or regions might have published other standards covering requirements for sterilization or
biological indicators (see Bibliography).

INTERNATIONAL STANDARD ISO 11138-1:2006(E)

Sterilization of health care products — Biological indicators —
Part 1:
General requirements
1 Scope
1.1 General
1.1.1 This part of ISO 11138 provides general requirements for production, labelling, test methods and
performance characteristics of biological indicators, including inoculated carriers and suspensions, and their
components, to be used in the validation and routine monitoring of sterilization processes.
1.1.2 This part of ISO 11138 specifies basic and common requirements that are applicable to all
subsequent parts of ISO 11138. Requirements for biological indicators for particular specified processes are
provided in the subsequent parts of ISO 11138. If no specific subsequent part is provided, this part applies.
NOTE National or regional regulations may apply.
1.2 Exclusions
This part of ISO 11138 does not apply to microbiological test systems for processes that rely on physical
removal of microorganisms, e.g. filtration processes or processes that combine physical and/or mechanical
removal with microbiological inactivation, such as use of washer disinfectors or flushing and steaming of
pipelines. This part of ISO 11138, however, could contain elements relevant to such microbiological test
systems.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 8601, Data elements and interchange formats — Information interchange — Representation of dates and
times
ISO 11135:1994, Medical devices — Validation and routine control of ethylene oxide sterilization
ISO 11137-1, Sterilization of health care products — Radiation — Part 1: Requirements for development,
validation and routine control of a sterilization process for medical devices
ISO 11137-2, Sterilization of health care products — Radiation — Part 2: Establishing the sterilization dose
ISO 11137-3, Sterilization of health care products — Radiation — Part 3: Guidance on dosimetric aspects
ISO 11607-1, Packaging for terminally sterilized medical devices — Part 1: Requirements for materials, sterile
barrier systems and packaging systems
ISO 11138-1:2006(E)
ISO 11607-2, Packaging for terminally sterilized medical devices — Part 2: Validation requirements for
forming, sealing and assembly processes
ISO 11737-1, Sterilization of medical devices — Microbiological methods — Part 1: Determination of a
population of microorganisms on products
ISO 13485, Medical devices — Quality management systems — Requirements for regulatory purposes
ISO 15223, Symbols to be used with medical device labels, labelling and information to be supplied
ISO 17665-1, Sterilization of health care products — Moist heat — Part 1: Requirements for the development,
validation and routine control of a sterilization process for medical devices
ISO 18472, Sterilization of health care products — Biological and chemical indicators — Test equipment
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
biological indicator
test system containing viable microorganisms providing a defined resistance to a specified sterilization
process
[ISO/TS 11139, definition 2.3]
3.2
carrier
supporting material on or in which test microorganisms are deposited
3.3
colony forming unit
CFU
individual visible units of growth of microorganisms arising from a single cell or multiple cells
3.4
culture collection number
unique identification of the test organism allocated by a scientifically recognised service culture collection
3.5
culture conditions
combination of growth media and manner of incubation used to promote germination, growth and/or
multiplication of microorganisms
NOTE The manner of incubation may include the temperature, time and any other conditions specified for incubation.
[ISO/TS 11139, definition 2.10]
3.6
D value
D value
time or dose required to achieve inactivation of 90 % of a population of the test microorganism under stated
dose conditions
[ISO/TS 11139, definition 2.11]
2 © ISO 2006 – All rights reserved

ISO 11138-1:2006(E)
3.7
F value
BIO
product of the logarithm of the population and the D value where the F value is an expression of the
BIO
resistance of the biological indicator
3.8
inactivation
loss of ability of microorganisms to grow and/or multiply
[ISO/TS 11139, definition 2.21]
3.9
inactivation curve
graphical representation of inactivation of test organism against increasing exposure to the sterilizing agent at
stated conditions
3.10
inoculated carrier
supporting material on or in which a defined number of viable test organisms have been deposited
NOTE See Annex F.
3.11
nominal population
manufacturer’s stated number of viable microorganisms
NOTE This is generally expressed in log function (e.g., 10 ).
3.12
packaging system
combination of the sterile barrier system and protective packaging
[ISO/TS 11139, definition 2.28]
3.13
primary package
element of the packaging system which maintains the integrity of the product
NOTE The packaging system protects the inoculated carrier from damage and contamination without preventing
penetration of the sterilizing agent.
3.14
process challenge device
PCD
item designed to constitute a defined resistance to a sterilization process and used to assess performance of
the process
[ISO/TS 11139, definition 2.33]
3.15
resistometer
test equipment designed to create defined reference combinations of the physical and/or chemical variables of
a sterilization process
3.16
secondary package
container in which biological indicators are packed for transport and storage
ISO 11138-1:2006(E)
3.17
self-contained biological indicator
biological indicator presented in such a way that the primary package, intended for incubation, contains the
incubation medium required for recovery of the test organism
3.18
survival-kill window
extent of exposure to a sterilization process under defined conditions where there is a transition from all
biological indicators showing growth (survival time) to all biological indicators showing no growth (kill time)
3.19
suspension
viable test organisms suspended in a fluid
NOTE Suspension can be a biological indicator if ready to use in a sealed glass ampoule, or may be an intermediate
component used to produce an inoculated carrier or biological indicator.
3.20
viable count
actual number of recoverable colony-forming units or other appropriate units
NOTE See Annex A.
3.21
z value
change in exposure temperature of a thermal sterilization process, which corresponds to a tenfold change in D
value
NOTE See ISO 11138-3 and ISO 11138-4.
4 General manufacturing requirements
4.1 Manufacturing controls
4.1.1 Quality systems
The manufacturer shall establish, document and maintain a formal quality system (e.g. ISO 13485, GMPs or
other national or regional requirements) to cover all operations required by this part of ISO 11138. In particular,
the manufacturer shall take precautions at all stages of production to minimize contamination that would
adversely affect the performance of the biological indicator.
4.1.2 Traceability
4.1.2.1 Traceability of manufacturing components shall be maintained.
4.1.2.2 Manufacturing components shall include all materials incorporated in, or coming into direct
contact with, the test organism suspension, the inoculated carrier or its primary package.
4.1.3 End product requirements
The finished product shall comply with the requirements set out in this part of ISO 11138, see:
a) manufacturing (Clause 5);
b) labelling (4.3);
4 © ISO 2006 – All rights reserved

ISO 11138-1:2006(E)
c) resistance characteristics (6.4);
d) storage and transport (4.4).
NOTE 1 Advice on methods for the use of biological indicators is provided in ISO 14161.
NOTE 2 National and/or regional requirements might exist, for example in the various national or regional
pharmacopoeias.
4.1.4 Personnel
The procedures and methods in this part of ISO 11138 shall be carried out by suitably trained and
experienced laboratory personnel (see 4.1.1).
4.2 Test organism
4.2.1 Strain
4.2.1.1 Test organisms shall be of a defined strain, available through a recognised culture collection, and
shall be identified by appropriate test methods.
4.2.1.2 The test organism should be a strain that is:
a) suitable for handling without special containment facilities, does not need specific containment
procedures for handling and does not have specific transport or mailing requirements (e.g. Risk Group 1,
WHO 1993);
b) sufficiently stable to maintain its resistance characteristics for the duration of the stated shelf-life when
transported and stored in accordance with label directions.
NOTE Traditionally, the test organisms of biological indicators have been bacterial spores, usually derived from
Bacillus or Geobacillus species.
4.2.1.3 Test organisms other than bacterial spores may be used if they have been shown to provide
appropriate resistance to the sterilization process.
4.2.2 Originating inoculum for suspension
4.2.2.1 The initial inoculum for each batch of test organism suspension shall be:
a) traceable to the reference culture and available through a recognized culture collection;
b) verified as to its identity and purity.
4.2.2.2 The methods used for maintaining test organism cultures shall be designed to protect them from
contamination and to minimize any induced changes in the inherent properties of the test organisms.
4.2.2.3 Verification tests are specific for each strain of test organism and shall be documented and
validated by the manufacturer.
4.2.3 Test organism count
4.2.3.1 The viable test organism count of the suspension shall be determined in accordance
with Annex A.
4.2.3.2 If the user requires information on the growth index of the test organism, this shall be provided by
expressing the viable test organism count as a percentage of the total direct microscopic count.
ISO 11138-1:2006(E)
4.3 Information supplied by manufacturer (labelling)
4.3.1 The following information shall be provided on the label of each individual unit of suspension,
inoculated carrier packaging and biological indicator:
⎯ a unique code by which the manufacturing history can be traced;
⎯ the name of the test organism;
⎯ an indication of the sterilization process for which the suspension, inoculated carriers or biological
indicators are suitable;
⎯ the expiry date, expressed according to ISO 8601, e.g. YYYY-MM-DD;
⎯ the manufacturer’s name, trademark, address or other means of identification.
Internationally recognized symbols may be used where appropriate (see 4.1.3 and ISO 15223).
4.3.2 The information given in Table 1 shall be provided within the secondary packaging of each batch of
product.
4.3.3 Requirements for labelling may be achieved by the use of appropriate symbols (ISO 15223).
Table 1 — Information to be provided by the manufacturer
Inoculated Biological
Information requirement Suspension
carrier indicator
The name or abbreviation of the culture collection from
which the test organism has been obtained and the Required Required Required
reference number of the strain
The nominal volume of suspension, in ml Required — —
The process for which the product is suitable for use, the
resistance and the procedure and carrier used to determine Required Required Required
a
the resistance
The specified storage conditions Required Required Required
Disposal instructions Required Required Required
Directions for use, especially data about the medium,
incubation and other conditions to be used for recovery of test Required Required Required
organisms after exposure to the sterilization process
The number of test organisms per ml (suspension), or per
Required Required Required
a
unit (inoculated carrier or biological indicator)
The number of product units in the secondary pack — Required Required
A reference to this part of ISO 11138 Required Required Required
a
Test methodology used to determine resistance and population should be supplied by the manufacturer upon request.
4.4 Storage and transport
4.4.1 Storage and transport conditions for the test organism suspension shall be maintained such that the
test organism suspension complies with the requirements of this part of ISO 11138 and, where relevant, a
subsequent part of ISO 11138.
6 © ISO 2006 – All rights reserved

ISO 11138-1:2006(E)
4.4.2 If inoculated carriers are packaged, they shall be packaged in a way that does not affect the nominal
population or performance of individual inoculated carriers.
4.4.3 Storage and transport conditions for inoculated carriers shall be maintained such that the inoculated
carriers comply with the requirements of this part of ISO 11138 and where relevant, a subsequent part of
ISO 11138.
4.4.4 Individually-packaged biological indicators shall be placed in a secondary package for transport and
storage. Packaging for transport and storage shall ensure that biological indicators comply with this part of
ISO 11138 and, where relevant, a subsequent part of ISO 11138.
5 Specific manufacturing requirements
5.1 Suspensions
5.1.1 Culture medium and incubation conditions shall consistently produce test organism suspensions that
meet the performance requirements of this part of ISO 11138 and any relevant subsequent part of ISO 11138.
5.1.2 The suspending medium for the test organism suspension shall not adversely affect the stability of the
test organism and shall be compatible with the procedures and materials employed in the manufacture of
inoculated carriers and biological indicators.
5.1.3 The method of harvesting and subsequent treatment of suspensions to be used in the inoculation of
carriers should ensure that residues do not adversely influence the performance of the inoculated carrier or
biological indicator.
5.2 Carrier, primary and secondary packaging
5.2.1 The materials of the carrier and the primary and secondary packaging shall not contain any
contamination (physical, chemical or microbial) that would adversely affect the performance of the biological
indicator.
5.2.2 The carrier, the primary and secondary packaging, and the specified storage conditions shall be
designed so that the performance characteristics of the biological indicator meet the requirements of this part
of ISO 11138 throughout the stated shelf life of the product. The manufacturer shall provide the purchaser with
a statement of the maximum and minimum values of each dimension of the carrier on request.
5.2.3 During and after the sterilization process, the carrier and the primary packaging shall not retain or
release any substance to such an extent that, on transfer to the incubation medium, under the culture
conditions, the growth of low numbers of surviving test organisms will be inhibited. Testing shall comply with
Annex B.
5.2.4 The carrier, the primary packaging, and the secondary packaging shall withstand planned transport
and handling at the point of use, without breakage.
5.2.5 Raw materials used for the carrier and the primary packaging shall withstand exposure to the
sterilization process for which they are intended in such a way that the performance characteristics of the
inoculated carrier or biological indicator are maintained. Compliance shall be tested by observation of the
carrier and the primary packaging exposed to the extreme ranges and rates of change of the chemical and
physical variables of the sterilization process.
NOTE Reference sterilization conditions can be found in subsequent parts of ISO 11138.
5.2.6 Sterilization conditions likely to be used should be investigated by the manufacturer of biological
indicators and applied for testing of the applicability of the biological indicator.
ISO 11138-1:2006(E)
5.3 Inoculated carrier
5.3.1 The inoculated carrier shall be made of materials that will withstand exposure to the sterilization
process without distortion, melting, corrosion or other failure that would impair the use of the inoculated carrier.
5.3.2 Only one strain of test organism shall be used in a batch of inoculated carriers, unless the
manufacturer has demonstrated that the use of multiple strains does not significantly affect test organism
performance in the specified sterilization process.
5.3.3 Prior to inoculation, the carrier should be sterilized in accordance with ISO 17665-1, ISO 11135,
ISO 11137 parts 1 to 3 or other relevant sterilization methods. If sterilization is not practicable, acceptable
bioburden limits of the carrier prior to inoculation may be established in accordance with ISO 11737-1
(see Annex B).
5.3.4 Carriers shall be inoculated so as to maintain a consistent microbial population (see 6.3).
5.4 Biological indicators
5.4.1 Individually packaged biological indicators shall be prepared by placing individual inoculated carriers
in a primary pack.
5.4.2 The primary packaging shall be validated for its intended use (see Annex B).
5.4.3 Appropriate international or national standards for packaging should be used (see ISO 11607-1 and
ISO 11607-2).
5.5 Self-contained biological indicators
The performance of self-contained biological indicators shall be validated, including the ability of the culture
medium to promote growth of the test organisms after being subjected to the sterilization process.
6 Determination of resistance
6.1 General resistance requirements
6.1.1 The resistance of each lot/batch of biological indicators shall be tested to demonstrate conformance
with the performance requirements specified in this part of ISO 11138 and any relevant subsequent part of
ISO 11138.
6.1.2 Resistance characteristics of biological indicators intended for sterilization processes not specified in
any subsequent part of ISO 11138 shall be defined using the elements of this clause in which test conditions
for the subject processes are described.
6.1.3 It is recognised that the validation and monitoring of some sterilization processes may use biological
indicators that do not meet the minimum population and/or resistance criteria specified in ISO 11138. These
biological indicators are acceptable provided that:
a) all other requirements of ISO 11138 (including the method of test for population and resistance) are met;
b) the product information includes a clear statement of the population and resistance;
c) the product label carries a clear warning that the population and/or resistance (as appropriate) is below
the value specified in the relevant part of ISO 11138.
6.1.4 Resistance testing shall include determination of the viable count and determination of the resistance
characteristics (see 6.3 and 6.4).
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ISO 11138-1:2006(E)
6.1.5 The resistance of a biological indicator may be indicated by the term F value (see 3.7).
BIO
6.2 Test organism
The test organism shall be specified.
6.3 Population of test organisms
6.3.1 The viable count shall be determined (see Annex A).
6.3.2 The viable count shall be between 50 % and 300 % of the manufacturer's stated value when it is
determined by the manufacturer or a third party during the stated shelf life using the method specified by the
manufacturer.
6.4 Resistance characteristics
6.4.1 Resistance characteristics shall be determined by a combination of at least two of the following
methods:
d) determination of the D value through the construction of a survivor curve (see Annex C);
e) determination of the D value through a fraction negative method (see Annex D);
f) verification of the survival/kill response characteristics (see Annex E).
6.4.2 The values obtained by these methods shall be within the ranges specified in the relevant subsequent
parts of ISO 11138. At least two of these values shall be included in the labelling of the biological indicators
(see 4.3).
6.4.3 The D value shall be within ± 20 % of the manufacturer's stated value when determined by the
manufacturer during the stated shelf life using the method specified by the manufacturer.
Ideally, the survivor curve is linear over the full range of inactivation. In practice, deviations from this ideal
occur, but linearity must be maintained within acceptable limits. Construction of a survivor curve by
enumeration establishes the resistance for surviving populations greater than approximately 5 × 10 , whereas
the fraction negative method establishes a statistically based calculation of surviving test organisms below
that level. Good correlation of the D values obtained by the two methods can therefore be used to establish
that there are no serious deviations from a linear survivor curve.
Subsequent parts of ISO 11138 may require additional determinations (e.g. z value for biological indicators for
moist heat sterilization [ISO 11138-3] or dry heat sterilization [ISO 11138-4]).
The resistance characteristics specified in this part of ISO 11138 and any subsequent part of ISO 11138 apply
to the specific test conditions stated in those parts.
6.4.4 The survivor curve, when plotted as a semi-logarithmic curve of the log of the viable test organism
count against time, shall be linear with a correlation coefficient of at least 0,8 (see Annex C).
ISO 11138-1:2006(E)
6.5 Test conditions
Resistance characteristics shall be determined using specified testing conditions. See Table 2.
Table 2 — Minimum test samples according to method
Minimum number of test Minimum number of Minimum total number of
Test method per ISO 11138-1
samples exposure conditions test samples

a
4 — 4
Initial count of viable test organism
Annex C Survivor curve method 4 5 20
b b
b
Annex D Fraction negative method 20 5 100

Annex E survival-kill window 50 2 100
Minimum total number depending on choice of combination of methods: 124 or 204
NOTE Common test conditions for specific sterilisation methods have developed over time and are presented in subsequent parts
of ISO 11138.
a
The viable count of the unprocessed inoculated carrier or biological indicator.
b
The extra set of testing conditions at the exposure subsequent to t (see Table D.1) is not used in the calculations, but is a
condition for accepting the test results as valid.
7 Culture conditions
7.1 Incubator
7.1.1 The incubator shall be set to provide, and monitored to confirm, the specified culture conditions.
7.1.2 In addition to routine monitoring of temperature, the temperature distribution within the incubator
should be validated.
7.2 Growth medium
7.2.1 The growth medium shall be specified and demonstrated to support the growth of an inoculum of less
than 100 test organisms.
7.2.2 Labelling shall include information on culture conditions after exposure to the sterilization process
(see 4.3).
7.2.3 The growth medium shall be validated to ensure that it can neutralize any sterilizing agent residuals
that might influence the viability of the test organism (see 5.2.3).
7.3 Incubation
7.3.1 The incubation time and temperature shall be validated.
7.3.2 Manufacturers shall provide instructions for incubation (see Table 1). An incubation period is
commonly recognized to be 7 days for established sterilization processes, such as moist heat and ethylene
oxide, using well characterized test organisms, such as Geobacillus stearothermophilus and Bacillus
atrophaeus, respectively. Where sufficient data are not available to support a reference incubation period of
7 days for a novel sterilization method, at least 14 days shall be used as the reference incubation period on
which to base the validation.
NOTE National or regional requirements for incubation period validation may also exist.
10 © ISO 2006 – All rights reserved

ISO 11138-1:2006(E)
Annex A
(normative)
Determination of viable count
A.1 General
A.1.1 Enumeration techniques are used to determine the number of viable test organisms in suspension on
inoculated carriers, or from packaged biological indicators, by counting of distinct colony forming units (CFUs).
The method is used when the expected number of recoverable test organisms is above 5 × 10 CFUs.
A.1.2 The relevant products shall be examined for recoverable test organisms in accordance with A.2 to A.4.
This method applies to both processed and unprocessed test samples and can be used for the determination
of initial viable count (unprocessed samples) as well as for D value determination using the survivor curve
method (processed samples).
A.1.3 Alternative methods of enumeration with demonstrated equivalence to direct plating techniques may
be used.
A.2 Minimum number of test samples
A minimum of four test samples from each lot/batch or exposure shall be used.
A.3 Sample preparation and culture methods
A.3.1 The test samples shall be placed in an appropriate volume of suspending medium. The test
organisms shall be eluted from the test samples by a validated procedure (e.g. maceration with glass beads,
grinding and/or blending in a homogenizer and/or blender, vortexing, ultrasonication or other appropriate
procedure). (See ISO 11737-1.)
A.3.2 The concentration of microorganisms in the suspensions shall be adjusted by dilution, if necessary, in
appropriate sterile dilution fluid. Numbers of CFUs should be within a specified range for the method used
whenever possible.
For cultures poured into molten agar or spread on solidified agar in regular size Petri dishes, numbers of
CFUs between 30 and 300 are considered to be the most accurate.
A.3.3 An appropriate method for the enumeration of viable organisms shall be used.
Appropriate methods may include membrane filtration techniques, direct spreading on semi-solid agar growth
medium or mixing with molten tempered agar growth medium (see ISO 11737-1).
A.3.4 The biological indicator manufacturer shall identify or make available a suitable medium for recovery
of test organisms and/or complete data and instructions for the preparation of such a medium.
A.4 Incubation and enumeration
A.4.1 The plated samples or the membrane filters shall be incubated at temperatures and times specified by
the manufacturer.
ISO 11138-1:2006(E)
In general, the incubation periods and temperatures are 55 °C to 60 °C for not less than 48 h for thermophilic
microorganisms and 30 °C to 37 °C for not less than 48 h for mesophilic microorganisms.
NOTE Desiccation of the growth medium can adversely affect growth at elevated incubation temperatures.
A.4.2 After the appropriate incubation period, the numbers of colony forming units on the plates or
membrane filters shall be cou
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