EN ISO 14161:2009

SLOVENSKI STANDARD
01-januar-2010
1DGRPHãþD
SIST EN ISO 14161:2001
Sterilizacija izdelkov za zdravstveno nego - Biološki indikatorji - Navodilo za izbiro,
uporabo in predstavitev rezultatov (ISO 14161:2009)
Sterilization of health care products - Biological indicators - Guidance for the selection,
use and interpretation of results (ISO 14161:2009)
Sterilisation von Produkten für die Gesundheitsfürsorge - Biologische Indikatoren -
Leitfaden für die Auswahl, Verwendung und Interpretation von Ergebnissen (ISO
14161:2009)
Stérilisation des produits de santé - Indicateurs biologiques - Directives générales pour la
sélection, l'utilisation et l'interprétation des résultats (ISO 14161:2009)
Ta slovenski standard je istoveten z: EN ISO 14161:2009
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 14161
NORME EUROPÉENNE
EUROPÄISCHE NORM
September 2009
ICS 11.080.01 Supersedes EN ISO 14161:2000
English Version
Sterilization of health care products - Biological indicators -
Guidance for the selection, use and interpretation of results (ISO
14161:2009)
Stérilisation des produits de santé - Indicateurs biologiques Sterilisation von Produkten für die Gesundheitsfürsorge -
- Directives générales pour la sélection, l'utilisation et Biologische Indikatoren - Leitfaden für die Auswahl,
l'interprétation des résultats (ISO 14161:2009) Verwendung und Interpretation von Ergebnissen (ISO
14161:2009)
This European Standard was approved by CEN on 31 July 2009.

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 CEN Management Centre 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 CEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, 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: Avenue Marnix 17, B-1000 Brussels
© 2009 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14161:2009: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
This document (EN ISO 14161:2009) 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 March 2010, and conflicting national standards shall be withdrawn at
the latest by March 2010.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 14161:2000.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, 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 the United Kingdom.
Endorsement notice
The text of ISO 14161:2009 has been approved by CEN as a EN ISO 14161:2009 without any modification.

INTERNATIONAL ISO
STANDARD 14161
Second edition
2009-09-15
Sterilization of health care products —
Biological indicators — Guidance for the
selection, use and interpretation of
results
Stérilisation des produits de santé — Indicateurs biologiques —
Directives générales pour la sélection, l'utilisation et l'interprétation des
résultats
Reference number
ISO 14161:2009(E)
©
ISO 2009
ISO 14161:2009(E)
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©  ISO 2009
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ii © ISO 2009 – All rights reserved

ISO 14161:2009(E)
Contents Page
Foreword .v
Introduction.vi
1 Scope.1
2 Normative references.1
3 Terms and definitions .2
4 General .5
5 Characteristics of biological indicators .7
5.1 General .7
5.2 Test organism suspension for direct inoculation of products.7
5.3 Inoculated carriers .8
5.4 Self-contained biological indicators.8
5.5 Other biological indicators.9
6 Selection of supplier .9
6.1 General .9
6.2 Documentation .10
7 Biological indicators in process development.11
7.1 General .11
7.2 Overkill approach .12
7.3 Combined biological indicator and bioburden method.12
7.4 Bioburden method.13
8 Biological indicators in sterilization validation.14
8.1 General .14
8.2 Placement and handling of biological indicators .14
8.3 Sterilizer qualification .14
8.4 Performance qualification .14
8.5 Review and approval of validation .15
8.6 Requalification.15
9 Biological indicators in routine monitoring.15
9.1 General .15
9.2 Placement and handling of biological indicators .15
9.3 Process challenge device (PCD).16
10 Results.16
10.1 General .16
10.2 Interpretation of results .16
11 Application of biological indicator standards .17
11.1 General assessment of biological indicator performance by the user.17
11.2 Nominal population of test organism.17
11.3 Resistance determination.18
11.4 z value determination .20
11.5 F equivalent sterilization value determination .22
(T, z)
11.6 Establishing spore-log-reduction (SLR) .23
11.7 Sterility assurance level (SAL) calculation.23
11.8 Test equipment .24
12 Culture conditions.24
12.1 General .24
12.2 Incubation temperature.24
ISO 14161:2009(E)
12.3 Incubation period.25
12.4 Choice of growth medium.25
13 Third-party requirements .26
13.1 General.26
13.2 Minimum requirements for replicates and total number of biological indicators .26
13.3 Test equipment .26
14 Personnel training .27
15 Storage and handling .27
16 Disposal of biological indicators .27
Annex A (informative) Microbiological inactivation kinetics and enumeration techniques.28
Annex B (informative) Process challenge devices .34
Annex C (informative) Formulae for fraction negative methods for D value calculations .35
Annex D (informative) Examples of documentation for biological indicators prepared by the user .50
Annex E (informative) Calculation of z value.54
Annex F (informative) D value determination by survivor curve method.57
Annex G (informative) Survival-kill response characteristics .61
Bibliography .62

iv © ISO 2009 – All rights reserved

ISO 14161:2009(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 14161 was prepared by Technical Committee ISO/TC 198, Sterilization of health care products.
This second edition cancels and replaces the first edition (ISO 14161:2000), which has been technically
revised.
ISO 14161:2009(E)
Introduction
This International Standard provides guidance regarding the selection, use and interpretation of results of
biological indicators when used to develop, validate and monitor sterilization processes. The procedures
described in this International Standard are of a general nature and do not, of themselves, constitute a
comprehensive development, validation or monitoring programme with regard to the sterilization of health care
products. The intent of this International Standard is not to mandate the use of biological indicators in a
process but, if they are used, to provide guidance for their proper selection and use in order to obviate
misleading results.
In this International Standard, users will find guidance on selection of the correct biological indicator for their
particular sterilization process and critical parameters as well as guidance on its appropriate use.
The user should select a biological indicator that is appropriate for the particular process to be used. There is
a wide variety of sterilization processes in common use, and biological indicator manufacturers are not able to
foresee all possible uses of their product. Manufacturers, therefore, label biological indicators according to
their intended use. It is the responsibility of the users of biological indicators to select, use, recover and
interpret the results as appropriate for the particular sterilization process used.
The certified performance of a biological indicator can be adversely affected by the conditions of storage and
transport prior to its use, by the use of the biological indicator or by the sterilizer process parameters. In
addition, the incubation procedure used after exposure to the process, including outgrowth temperature and
culture medium type, supplier and specific lot, can affect measured resistance as a function of recovery and
growth. For these reasons, the recommendations of the biological indicator manufacturer for storage and use
should be followed. After exposure, biological indicators should be aseptically transferred (if applicable) and
incubated as specified by the biological indicator manufacturer.
It should be noted that biological indicators are not intended to indicate that the products in the load being
sterilized are sterile. Biological indicators are utilized to test the effectiveness of a given sterilization process
and the equipment used, by assessing microbial lethality according to the concept of sterility assurance level.
Suitably trained personnel should conduct these studies.

vi © ISO 2009 – All rights reserved

INTERNATIONAL STANDARD ISO 14161:2009(E)

Sterilization of health care products — Biological indicators —
Guidance for the selection, use and interpretation of results
1 Scope
This International Standard provides guidance for the selection, use and interpretation of results from
application of biological indicators when used in the development, validation and routine monitoring of
sterilization processes. This International Standard applies to biological indicators for which International
Standards exist.
NOTE 1 See, for example, the ISO 11138 series.
NOTE 2 The general information provided in this International Standard can have useful application for processes and
biological indicators not currently addressed by existing International Standards, e.g., new and developing sterilization
processes.
This International Standard does not consider those processes that rely solely on physical removal of
microorganisms, e.g., filtration.
This International Standard is not intended to apply to combination processes using, for example, washer
disinfectors or flushing and steaming of pipelines.
This International Standard is not intended to apply to liquid sterilization processes.
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 11135-1, Sterilization of health care products — Ethylene oxide — Part 1: Requirements for development,
validation and routine control of a sterilization process for medical devices
ISO 11138-1:2006, Sterilization of health care products — Biological indicators — Part 1: General
requirements
ISO 11138-2, Sterilization of health care products — Biological indicators — Part 2: Biological indicators for
ethylene oxide sterilization processes
ISO 11138-3, Sterilization of health care products — Biological indicators — Part 3: Biological indicators for
moist heat sterilization processes
ISO 11138-4, Sterilization of health care products — Biological indicators — Part 4: Biological indicators for
dry heat sterilization processes
ISO 11138-5, Sterilization of health care products — Biological indicators — Part 5: Biological indicators for
low-temperature steam and formaldehyde sterilization processes
ISO 11737-1, Sterilization of medical devices — Microbiological methods — Part 1: Determination of a
population of microorganisms on products
ISO 14161:2009(E)
ISO 14937, Sterilization of health care products — General requirements for characterization of a sterilizing
agent and the development, validation and routine control of a sterilization process for medical devices
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 18472:2006, 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
accreditation
procedure by which an authoritative body gives formal recognition that a body or person is competent to carry
out specific tasks
[3]
NOTE 1 See ISO/IEC 17011 .
NOTE 2 Accreditation does not itself qualify the laboratory to approve any particular product. However, accreditation
can be relevant to approval and certification authorities when they decide whether or not to accept data produced by a
given laboratory in connection with their own activities.
3.2
aseptic technique
conditions and procedures used to exclude the introduction of microbial contamination
3.3
bioburden
population of viable microorganisms on or in a product and/or sterile barrier system
[ISO/TS 11139, definition 2.2]
3.4
biological indicator
BI
test system containing viable microorganisms providing a defined resistance to a specified sterilization
process
[ISO/TS 11139, definition 2.3]
3.5
D value
D value
time or dose required to achieve inactivation of 90 % of a population of the test microorganism under stated
conditions
[ISO/TS 11139, definition 2.11]
3.6
holding time
period for which the sterilization variable within the sterilizer and at all points within the load are continuously
within the limits specified for the sterilization stage
3.7
inoculated carrier
supporting material on or in which a defined number of viable test organisms have been deposited
NOTE 1 See ISO 11138-1.
NOTE 2 The test organism is a microorganism used for the manufacture of inoculated carriers.
2 © ISO 2009 – All rights reserved

ISO 14161:2009(E)
3.8
installation qualification
IQ
process of obtaining and documenting evidence that equipment has been provided and installed in
accordance with its specification
[ISO/TS 11139, definition 2.22]
3.9
inoculation
addition of a defined amount of a characterized microbial entity into or on to an item
3.10
log reduction
LR
reduction in number of viable microorganisms, expressed in log units
3.11
operational qualification
OQ
process of obtaining and documenting evidence that installed equipment operates within predetermined limits
when used in accordance with its operational procedures
[ISO/TS 11139, definition 2.27]
3.12
performance qualification
PQ
process of obtaining and documenting evidence that the equipment, as installed and operated in accordance
with operational procedures, consistently performs in accordance with predetermined criteria and thereby
yields product meeting its specification
[ISO/TS 11139, definition 2.30]
3.13
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]
3.14
process challenge location
PCL
site that simulates “worst case” conditions as they are given for sterilizing agent(s) in the goods to be sterilized
3.15
process parameter
specified value for a process variable
NOTE The specification for a sterilization process includes the process parameters and their tolerances.
[ISO/TS 11139, definition 2.34]
3.16
process variable
condition within a sterilization process, changes in which alter microbicidal effectiveness
EXAMPLE Time, temperature, pressure, concentration, humidity, wavelength.
[ISO/TS 11139, definition 2.35]
ISO 14161:2009(E)
3.17
reference microorganism
microbial strain obtained from a recognized culture collection
[ISO/TS 11139, definition 2.39]
3.18
resistometer
test equipment designed to create defined reference combinations of the physical and/or chemical variables of
a sterilization process
NOTE 1 Adapted from ISO 11138-1, definition 3.15 and ISO 18472:2006, definition 3.11.
NOTE 2 Also referred to as Biological Indicator Evaluator Resistometer (BIER).
3.19
spore-log-reduction
SLR
log of initial spore population, N , minus the log of the final population, N
0 F
3.20
sterile
free from viable microorganisms
[ISO/TS 11139, definition 2.43]
3.21
sterility assurance level
SAL
probability of a single viable microorganism occurring on an item after sterilization
−6 −3
NOTE The term SAL takes a quantitative value, generally 10 or 10 . When applying this quantitative value to
−6 −3
assurance of sterility, an SAL of 10 has a lower value but provides a greater assurance of sterility than an SAL of 10 .
[ISO/TS 11139, definition 2.46]
3.22
sterilization
validated process used to render product free from viable microorganisms
NOTE In a sterilization process, the nature of microbial inactivation is exponential and thus the survival of a
microorganism on an individual item can be expressed in terms of probability. While this probability can be reduced to a
very low number, it can never be reduced to zero.
[ISO/TS 11139, definition 2.47]
3.23
sterilization cycle development
procedure for determination of the appropriate processing parameters that are consistent with attaining the
desired specifications and label claims for a given product or group of products
3.24
sterilization cycle validation
documented procedure for obtaining, recording and interpreting the results required to establish that a process
will consistently yield product complying with predetermined specifications
3.25
supplier
organization or person that provides a product
EXAMPLE Producer, distributer, retailer or vendor of a product, or provider of a service or information.
4 © ISO 2009 – All rights reserved

ISO 14161:2009(E)
NOTE 1 A supplier can be internal or external to the organization.
NOTE 2 In a contractual situation, a supplier is sometimes called “contractor.”
[ISO 9000, definition 3.3.6]
3.26
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)
[ISO 11138-1, definition 3.18]
3.27
third party
person or body that is recognised as being independent of the parties involved, as concerns the issue in
question
[1]
NOTE 1 See ISO/IEC Guide 2 .
NOTE 2 Parties involved are usually supplier (“first party”) and purchaser (“second party”) interests.
3.28
user
person or body employing biological indicators for a given purpose
[4]
NOTE 1 See ISO 9000 .
NOTE 2 The user is the customer who is the recipient of a product provided by the supplier. In a contractual situation,
the user is called “purchaser.” The user could be the customer, beneficiary or purchaser. The user can be either external
or internal to the organization and represents the “second party.”
3.29
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
4.1 This International Standard provides guidance on biological indicators that can apply generally for any
sterilization process, including new sterilization processes not yet covered by International Standards.
4.2 The use of biological indicators is normally documented in procedures and/or instructions.
[7]
NOTE Employing quality management systems such as ISO 13485 usually satisfies this provision.
4.3 Biological indicators should always be used in combination with physical and/or chemical
measurements in demonstrating the efficacy of a sterilizing process. When a physical and/or chemical
variable of a sterilization process is outside its specified limits, the reason for the sterilizer's inability to achieve
its process parameters should be evaluated and the problem corrected. Systems and/or procedures should be
established to evaluate any deviations from the cycle process limits, and reasons for accepting any deviation
should be fully documented.
4.4 A suitable biological indicator consists of carrier material and packaging and has a microbiological
component that is known to be suitable for handling without special containment facilities. The growth
conditions should be well documented, and the use of the indicator should be as simple and well described as
possible to avoid misinterpretation by the user.
ISO 14161:2009(E)
4.5 No formal approval system exists, internationally, for biological indicators that are marketed and used
for stated purposes or under stated conditions. Some national regulatory authorities, however, have particular
requirements for biological indicators and for the choice and use of biological indicators for the validation and
control of products marketed as sterile or sterilized.
4.6 A biological indicator represents a microbiological challenge to a sterilization process and is used to
verify that a sterilization process has the ability to inactivate microorganisms that have a known resistance to a
referenced sterilization process. Test organisms employed in biological indicators typically have resistance to
sterilization which exceeds that of common bioburden microorganisms, although some organisms can exhibit
a resistance to sterilization in excess of that of the test organisms. The appropriate biological indicator
provides a challenge to the sterilization process which exceeds that of the bioburden through a combination of
population and resistance. If there is reason to believe that the goods to be processed could be contaminated
with particularly resistant organisms, extended sterilization processing, based on the bioburden, could be
required.
4.7 Biological indicators are not intended for use in any process other than that specified by the
manufacturer on the product labelling. Microbial species and strains are selected as biological indicator
candidates based on their known resistance to the specific method of sterilization as certified by the
manufacturer. The use of an inappropriate biological indicator can give misleading results.
The user should ensure that the biological indicator has been qualified for use with the particular range of
sterilization conditions that are used. This could require information in addition to that given in the labelling.
When biological indicators are used outside reference conditions, the user can require information on the
reaction expected from the indicator, e.g., the effect of sub-optimal moisture conditions on the biological
indicators used in an ethylene oxide process. Users who employ biological indicators outside the
manufacturer's labelled recommendations should thoroughly characterize the resistance of the biological
indicators to the particular sterilization process. The relationship of the response of the biological indicator to
process parameters should be clearly demonstrated.
4.8 It is incumbent upon those responsible for the sterilization of product to ensure that the type of
biological indicator employed to validate and/or routinely monitor a given sterilization process is appropriate
for that use.
4.9 The manufacturer's recommendations for the use and storage of the biological indicators should always
be followed. Failure to do so can compromise the integrity of the biological indicator. If the user removes the
inoculated carrier from the biological indicator's primary packaging, changes in the resistance characteristics
can occur. Guidance should be sought from the manufacturer on the extent of this change, or the user can
evaluate changes in the resistance characteristics. The user should document that the performance
characteristics of the inoculated carrier are appropriate for their use.
4.10 Biological indicators should not be used beyond the expiration date stated by the manufacturer.
4.11 Those who employ biological indicators for validation and/or routine monitoring of sterilization should be
properly trained in their use. The time between completion of the sterilization process and the testing of the BI
should be justified as described in 8.2.4. Transferral of microorganisms exposed to the sterilization process to
the appropriate recovery medium should be done using aseptic technique.
4.12 The ISO 11138 series gives requirements for the information that the manufacturer should provide for
biological indicators. The information may be provided on the label, as a packet insert or as a general
specification accompanying the biological indicators. This series of International Standards also includes
minimum requirements for resistance characteristics. Testing conditions and methods are given as reference
methods.
4.13 Users of biological indicators come from a wide variety of industries, private enterprises and health care
facilities. Users are not generally required to perform resistance assays on biological indicators but can have
differing requirements for their quality assurance systems, which include audits of vendors and/or
manufacturers (see 6.2.2).
4.14 The verification of resistance characteristics by the user is an alternative to and/or complementary to an
audit, when necessary.
6 © ISO 2009 – All rights reserved

ISO 14161:2009(E)
5 Characteristics of biological indicators
5.1 General
5.1.1 Biological indicators provide means to assess directly the microbial lethality of a sterilization process
(see References [8] and [9]). When used in conjunction with physical and/or chemical process monitors,
biological indicators can provide an indication of the effectiveness of a given sterilization process.
5.1.2 A sterilization process should be considered as satisfactory only when the desired physical and/or
chemical parameters and microbiological results, as determined by an appropriate sterilization cycle
development, validation and monitoring programme have been realized. Failure to achieve the desired
physical and/or chemical parameters and/or microbiological challenge forms the basis for declaring the
[7] [22]
sterilization process as nonconforming (see ISO 13485 and ISO 9001 ).
5.1.3 Biological indicators consist of a defined population of test organisms presented in such a manner as
to allow their recovery following sterilization processing. For example, test organisms employed for ethylene
oxide sterilization processes can be spores of a suitable strain of Bacillus subtilis or Bacillus atrophaeus, as
noted in ISO 11138-2. For steam sterilization or moist heat sterilization, the test organisms employed can be
spores of a suitable strain of Geobacillus stearothermophilus, as noted in ISO 11138-3. Test organisms other
than bacterial spores can be used if they have been shown to provide appropriate resistance to the
sterilization process.
5.1.4 The basis of all formulae used to determine biological indicator resistance characteristics such as
D values is that the inactivation reaction follows first-order kinetics, with the requirement that the value for the
coefficient of determination, r , for the linearity of the survivor curve be not less than 0,8 (see Annex E and
Annex F). The strain, the production method, the suspension fluid, the carrier and packaging materials all
affect the resistance characteristics of the finished product (see ISO 11138-1).
5.1.5 The design and construction of a biological indicator can result in unique resistance characteristics
and can vary depending on whether the biological indicator is intended for use in the development and
validation of a sterilization process or for use in routine monitoring. If the design of the biological indicator for
use in routine monitoring differs from that employed to validate the sterilization processes, the challenge to the
process during validation should be correlated with the challenge to the process during routine monitoring.
5.1.6 The resistance characteristics of biological indicators vary according to the manufacturing methods
and the testing conditions. Depending upon placement within the load and the specific lethal conditions at
those discrete locations, biological indicators from the same lot may show different survival capabilities
(see 7.2.3). Users of biological indicators should note that ten indicators spread throughout the load are not
considered replicates due to the differences in lethality that exist throughout the chamber and load (see Note
to 11.3.1).
5.2 Test organism suspension for direct inoculation of products
5.2.1 Direct inoculation of test organisms on or in product can be necessary in cycle development and other
studies when the use of a biological indicator is not feasible. Direct inoculation can be appropriate for
assessing factors such as product sterilizability, identification of the more difficult to sterilize locations within
the device, and localized microbiological effects, e.g., moist heat versus dry heat environments.
The rationale for the selection of the “most difficult to sterilize” site(s) on a medical device or within a
sterilization load should be documented based on experimental data or derived from prior knowledge of the
particular sterilization methodology. In practice, the “most difficult to sterilize” site represents those locations
that are most likely to provide high resistance to the sterilization process. One should refer to specific
sterilization standards (e.g., ISO 17665-1 and ISO 11135-1) for guidance in determining and selecting difficult-
to-sterilize locations.
5.2.2 To assess the efficacy of sterilization at a particular site or location on the product, the desired
species and population of test organisms can be inoculated at those sites. The use of suspensions of test
organisms to prepare inoculated carriers or inoculated products requires caution. The materials on to which
test organisms are inoculated can alter the test organisms' resistance characteristics. The resistance can be
ISO 14161:2009(E)
higher or lower due to deposition as a monolayer or multilayer, coating effects, and/or bacteriostatic or
bactericidal effects of the material. The methods employed to recover the test organisms following processing
should be validated to ensure an adequate level of recovery from the product (see ISO 11737-1). Test
organism recovery should be expressed in terms of percent recovery of the population of the original inoculum.
5.2.3 Direct inoculation of products or materials with a test organism suspension can cause prolonged or
decreased survival of test organisms. This may affect the observed percent recovery of the original inoculum
relative to what is expected under specified sterilization conditions. Inoculated products may be assayed with
either survivor curve (enumeration/direct counting) or fraction-negative procedures (see Figure A.4). These
assays require aseptic techniques.
5.2.4 The D value and, when appropriate, the z value, are constant values only under determined and
defined conditions. The resistance characteristics of a spore suspension provided by a manufacturer or
supplier of biological indicators might not correspond to the resistance characteristics for direct product
inoculation studies. The resistance characteristics should be measured for the carrier employed (solid carrier
material or fluid) as well as for the specific sterilization cycle employed.
5.3 Inoculated carriers
5.3.1 Inoculated carriers consist of a defined population of test organisms inoculated on or in a suitable
carrier material (see ISO 11138-1:2006, Annex B). Caution should be exercised to ensure that the integrity of
the carrier material selected is sufficient to withstand sterilization processing without degradation and to
minimize the loss of the inoculated test organisms during transport and handling.
5.3.2 The resistance characteristics of a test organism in suspension can be considerably changed upon
deposition on or in carriers. Several factors can influence the resistance characteristics, such as the surface
on to which the suspension is inoculated (e.g., solid materials, viscous products or fluids), the way the spores
are dispersed and otherwise treated, the methods of drying, etc.
5.3.3 If an inoculated
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