SIST EN ISO 20776-1:2007
(Main)Clinical laboratory testing and in vitro diagnostic test systems - Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices - Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases (ISO 20776-1:2006)
Clinical laboratory testing and in vitro diagnostic test systems - Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices - Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases (ISO 20776-1:2006)
This part of ISO 20776 describes one reference method, broth microdilution, for determination of MICs. The MIC reflects the activity of the drug under the described test conditions, and can be interpreted for clinical management purposes by taking into account other factors, such as drug pharmacology or bacterial resistance mechanisms. This allows categorization of bacteria as "susceptible" (S), "intermediate" (I), or "resistant" (R). In addition, MIC distributions can be used to define wild type or non-wild type bacterial populations. Although clinical interpretation of the MIC value is beyond the scope of this part of ISO 20776, modifications of the basic method are required for certain antimicrobial agent;bacteria combinations to facilitate clinical interpretation. These modifications are included in a separate table. It is advisable to compare other susceptibility testing methods (e.g. routine methods or diagnostic test devices) with this reference method for validation, in order to ensure comparable and reliable results.
Labormedizinische Untersuchungen und In-vitro-Diagnostika-Systeme - Empfindlichkeitsprüfung von Infektionserregern und Evaluation von Geräten zur antimikrobiellen Empfindlichkeitsprüfung - Teil 1: Referenzmethode zur Testung der In-vitro-Aktivität von antimikrobiellen Substanzen gegen schnell wachsende aerobe Bakterien, die Infektionskrankheiten verursachen (ISO 20776-1:2006)
Dieser Teil der ISO 20776 beschreibt eine Referenzmethode (Mikrodilution) zur MHK-Bestimmung. Die MHK ist ein Maß für die Aktivität einer Substanz unter den beschriebenen Testbedingungen. Bei der Interpretation für klinische Zwecke können auch andere Faktoren wie die Pharmakologie des Wirkstoffs oder bakterielle Resistenzmechanismen berücksichtigt werden. Diese Vorgehensweise erlaubt die Einstufung bakterieller Isolate in die Kategorien sensibel (S), intermediär (I) und resistent (R). Die Verteilung der MHK-Werte kann zudem dazu dienen, bakterielle Wildtyp-Populationen von Nicht-Wildtyp-Populationen zu unterscheiden. Für bestimmte Antibiotika/Erreger-Kombinationen sind Modifikationen der Basismethode erforderlich, um die klinische Interpretation der MHK-Ergebnisse zu erleichtern. Obwohl die klinische Interpretation nicht zur Zielsetzung dieses Teils der EN ISO 20776 gehört, sind entsprechende Modifikationen in einer separaten Tabelle aufgelistet. Es ist ratsam, andere Methoden zur Empfindlichkeitsprüfung (z. B. mittels Routinetests oder Diagnosegeräten) anhand der Ergebnisse dieser Referenzmethode zu kalibrieren, um vergleichbare und reproduzierbare Werte sicherzustellen.
Systemes d'essais en laboratoire et de diagnostic in vitro - Essais de réceptivité d'agents infectieux et évaluation des performances des dispositifs de réceptivité antimicrobienne - Partie 1: Méthode de référence pour la détermination de la sensibilité in vitro aux agents microbiens des bactéries aérobies a croissance rapide impliquées dans les maladies infectieuses (ISO 20776-1:2006)
L'ISO 20776-1:2006 décrit une méthode de référence, la microdilution en bouillon, pour déterminer les CMI. La CMI reflète l'activité du médicament dans les conditions d'essai décrites, et peut être interprétée pour des besoins de gestion clinique en tenant compte d'autres facteurs tels que la pharmacologie du médicament ou les mécanismes de résistance bactérienne. Cela permet de classer les bactéries comme étant «sensibles» (S), «intermédiaires» (I), ou «résistantes» (R). En outre, les distributions de CMI peuvent être utilisées pour définir les populations bactériennes de type sauvage ou non sauvage. Bien que l'interprétation clinique de la valeur de la CMI se trouve au-delà du domaine d'application de la présente partie de l'ISO 20776, des modifications de la méthode de base sont nécessaires pour certaines combinaisons agent-bactérie afin de faciliter l'interprétation clinique. Ces modifications sont incluses dans un tableau séparé. Il est préférable de comparer les autres méthodes d'essai de réceptivité (par exemple les méthodes de routine ou les dispositifs d'essais de diagnostic) à cette méthode de référence à des fins de validation et pour garantir des résultats comparables et fiables.
Klinični laboratorijski preskusi ter dignostični preskusni sistemi in vitro - Preskus občutljivosti povzročiteljev infekcij na delovanje antimikrobno občutljivih naprav - 1. del: Referenčna metoda za preskus aktivnosti in vitro antimikrobnih povzročiteljev na vpliv bakterij pri nalezljivih boleznih (ISO 20776-1:2006)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 20776-1:2007
01-marec-2007
.OLQLþQLODERUDWRULMVNLSUHVNXVLWHUGLJQRVWLþQLSUHVNXVQLVLVWHPLLQYLWUR3UHVNXV
REþXWOMLYRVWLSRY]URþLWHOMHYLQIHNFLMQDGHORYDQMHDQWLPLNUREQRREþXWOMLYLKQDSUDY
GHO 5HIHUHQþQDPHWRGD]DSUHVNXVDNWLYQRVWLLQYLWURDQWLPLNUREQLK
SRY]URþLWHOMHYQDYSOLYEDNWHULMSULQDOH]OMLYLKEROH]QLK ,62
Clinical laboratory testing and in vitro diagnostic test systems - Susceptibility testing of
infectious agents and evaluation of performance of antimicrobial susceptibility test
devices - Part 1: Reference method for testing the in vitro activity of antimicrobial agents
against rapidly growing aerobic bacteria involved in infectious diseases (ISO 20776-
1:2006)
Labormedizinische Untersuchungen und In-vitro-Diagnostika-Systeme -
Empfindlichkeitsprüfung von Infektionserregern und Evaluation von Geräten zur
antimikrobiellen Empfindlichkeitsprüfung - Teil 1: Referenzmethode zur Testung der In-
vitro-Aktivität von antimikrobiellen Substanzen gegen schnell wachsende aerobe
Bakterien, die Infektionskrankheiten verursachen (ISO 20776-1:2006)
Systemes d'essais en laboratoire et de diagnostic in vitro - Essais de réceptivité d'agents
infectieux et évaluation des performances des dispositifs de réceptivité antimicrobienne -
Partie 1: Méthode de référence pour la détermination de la sensibilité in vitro aux agents
microbiens des bactéries aérobies a croissance rapide impliquées dans les maladies
infectieuses (ISO 20776-1:2006)
Ta slovenski standard je istoveten z: EN ISO 20776-1:2006
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
SIST EN ISO 20776-1:2007 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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EUROPEAN STANDARD
EN ISO 20776-1
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2006
ICS 11.100
English Version
Clinical laboratory testing and in vitro diagnostic test systems -
Susceptibility testing of infectious agents and evaluation of
performance of antimicrobial susceptibility test devices - Part 1:
Reference method for testing the in vitro activity of antimicrobial
agents against rapidly growing aerobic bacteria involved in
infectious diseases (ISO 20776-1:2006)
Systèmes d'essais en laboratoire et de diagnostic in vitro - Labormedizinische Untersuchungen und In-vitro-
Essais de réceptivité d'agents infectieux et évaluation des Diagnostika-Systeme - Empfindlichkeitsprüfung von
performances des dispositifs de réceptivité antimicrobienne Infektionserregern und Evaluation von Geräten zur
- Partie 1: Méthode de référence pour la détermination de antimikrobiellen Empfindlichkeitsprüfung - Teil 1:
la sensibilité in vitro aux agents microbiens des bactéries Referenzmethoden zur Testing der In-vitro-Aktivität von
aérobies à croissance rapide impliquées dans les maladies antimikrobiellen Substanzen gengen Bakterien, die
infectieuses (ISO 20776-1:2006) Infektionskrankheiten verursachen (ISO 20776-1:2006)
This European Standard was approved by CEN on 14 November 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 20776-1:2006: E
worldwide for CEN national Members.
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EN ISO 20776-1:2006 (E)
Foreword
This document (EN ISO 20776-1:2006) has been prepared by Technical Committee CEN/TC 140
"In vitro diagnostic medical devices", the secretariat of which is held by DIN, in collaboration with
Technical Committee ISO/TC 212 "Clinical laboratory testing and in vitro diagnostic test systems".
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 May 2007, and conflicting national standards shall
be withdrawn at the latest by May 2007.
This document has been prepared under a mandate given to CEN by the European Commission
and the European Free Trade Association, and supports essential requirements of EU Directive(s).
For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this
document.
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.
2
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EN ISO 20776-1:2006 (E)
ANNEX ZA
(informative)
Relationship between this European Standard and the Essential
Requirements of EU Directive 98/79
This European Standard has been prepared under a mandate given to CEN by the European
Commission and the European Free Trade Association to provide one means of conforming to
Essential Requirements of the New Approach Directive 98/79.
Once this standard is cited in the Official Journal of the European Communities under that Directive
and has been implemented as a national standard in at least one Member State, compliance with
the normative clauses of this standard confers, within the limits of the scope of this standard, a
presumption of conformity with the relevant Essential Requirements of that Directive and
associated EFTA regulations.
WARNING: Other requirements and other EU Directives may be applicable to the products
falling within the scope of this standard.
3
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INTERNATIONAL ISO
STANDARD 20776-1
First edition
2006-11-15
Clinical laboratory testing and in vitro
diagnostic test systems — Susceptibility
testing of infectious agents and
evaluation of performance of
antimicrobial susceptibility test
devices —
Part 1:
Reference method for testing the in vitro
activity of antimicrobial agents against
rapidly growing aerobic bacteria involved
in infectious diseases
Systèmes d'essais en laboratoire et de diagnostic in vitro — Essais de
réceptivité d'agents infectieux et évaluation des performances des
dispositifs de réceptivité antimicrobienne —
Partie 1: Méthode de référence pour la détermination de la sensibilité in
vitro aux agents microbiens des bactéries aérobies à croissance rapide
impliquées dans les maladies infectieuses
Reference number
ISO 20776-1:2006(E)
©
ISO 2006
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ISO 20776-1:2006(E)
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ii © ISO 2006 – All rights reserved
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ISO 20776-1:2006(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Terms and definitions. 1
3 Test procedures . 4
3.1 General. 4
3.2 Medium . 4
3.3 Antimicrobial agents . 4
3.4 Preparation of inoculum . 9
3.5 Inoculation of microdilution trays. 10
3.6 Incubation of microdilution trays. 10
3.7 Reading results . 10
3.8 Special test situations where the MIC result might give unreliable results . 10
4 Quality control. 11
Annex A (normative) Requirements for Mueller-Hinton broth. 16
Bibliography . 18
© ISO 2006 – All rights reserved iii
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ISO 20776-1:2006(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 20776-1 was prepared by the European Committee for Standardization (CEN) Technical Committee
CEN/TC 140, In vitro diagnostic medical devices, in collaboration with Technical Committee ISO/TC 212,
Clinical laboratory testing and in vitro diagnostic test systems, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
ISO 20776 consists of the following parts, under the general title Clinical laboratory testing and in vitro
diagnostic test systems — Susceptibility testing of infectious agents and evaluation of performance of
antimicrobial susceptibility test devices:
⎯ Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing
aerobic bacteria involved in infectious diseases
⎯ Part 2: Evaluation of performance of antimicrobial susceptibility test devices
iv © ISO 2006 – All rights reserved
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ISO 20776-1:2006(E)
Introduction
In vitro susceptibility tests are performed on microorganisms suspected of causing disease, particularly if the
organism is thought to belong to a species that may exhibit resistance to frequently used antimicrobial agents.
The tests are also important in resistance surveillance, epidemiological studies of susceptibility and in
comparisons of new and existing agents.
Dilution procedures are used to determine the minimum inhibitory concentrations (MICs) of antimicrobial
agents and are the reference method for antimicrobial susceptibility testing. MIC methods are used in
resistance surveillance, comparative testing of new agents, to establish the susceptibility of organisms that
give equivocal results in routine tests, for tests on organisms where routine tests may be unreliable and when
a quantitative result is required for clinical management. In dilution tests, microorganisms are tested for their
ability to produce visible growth on a series of agar plates (agar dilution) or in broth (broth dilution) containing
serial dilutions of the antimicrobial agent.
The lowest concentration of an antimicrobial agent (in mg/l) that, under defined in vitro conditions, prevents
the appearance of visible growth of a microorganism within a defined period of time is known as the MIC. The
MIC is a guide for the clinician to the susceptibility of the organism to the antimicrobial agent and aids
treatment decisions. Careful control and standardisation is required for intra- and inter-laboratory
reproducibility, as results may be significantly influenced by the method used. It is generally accepted that
broth MIC tests are reproducible to within one doubling dilution of the real end point (i.e. ± one well or tube in
a doubling dilution series).
Broth dilution is a technique in which containers holding identical volumes of broth with antimicrobial agent
solutions in incrementally (usually geometrically) increasing concentrations are inoculated with a known
number of microorganisms.
Broth microdilution denotes the performance of the broth dilution test in microdilution trays.
The method described in this part of ISO 20776 is intended for the testing of pure cultures of aerobic bacteria
that are easily grown by overnight incubation on agar and grow well in Mueller-Hinton broth, which may be
supplemented. The broth microdilution method described in this part of ISO 20776 is essentially the same as
[1] [2] [3] [4]
those used in many countries, including France , Germany , Sweden , the United Kingdom , and the
[5]
United States . The method is also essentially the same as the broth microdilution method published by the
[6]
European Committee on Antimicrobial Susceptibility Testing (EUCAST) . All these methods are based on
[7]
those described by Ericsson and Sherris .
© ISO 2006 – All rights reserved v
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INTERNATIONAL STANDARD ISO 20776-1:2006(E)
Clinical laboratory testing and in vitro diagnostic test
systems — Susceptibility testing of infectious agents and
evaluation of performance of antimicrobial susceptibility test
devices —
Part 1:
Reference method for testing the in vitro activity of
antimicrobial agents against rapidly growing aerobic bacteria
involved in infectious diseases
WARNING — The use of this part of ISO 20776 may involve hazardous materials, operations and
equipment. This part of ISO 20776 does not purport to address all of the safety problems associated
with its use. It is the responsibility of the user of this part of ISO 20776 to establish appropriate safety
and health practices and determine the applicability of regulatory limitations prior to use.
1 Scope
This part of ISO 20776 describes one reference method, broth microdilution, for determination of MICs. The
MIC reflects the activity of the drug under the described test conditions, and can be interpreted for clinical
management purposes by taking into account other factors, such as drug pharmacology or bacterial
resistance mechanisms. This allows categorization of bacteria as “susceptible” (S), “intermediate” (I), or
“resistant” (R). In addition, MIC distributions can be used to define wild type or non-wild type bacterial
populations. Although clinical interpretation of the MIC value is beyond the scope of this part of ISO 20776,
modifications of the basic method are required for certain antimicrobial agent - bacteria combinations to
facilitate clinical interpretation. These modifications are included in a separate table. It is advisable to compare
other susceptibility testing methods (e.g. routine methods or diagnostic test devices) with this reference
method for validation, in order to ensure comparable and reliable results.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
antimicrobial agent
substance of biological, semi-synthetic or synthetic origin that inhibits the growth of or kills bacteria, and is
thus of potential use in the treatment of infections
NOTE Disinfectants, antiseptics and preservatives are not included in this definition.
2.2 Antimicrobial agents — properties
2.2.1
potency
antimicrobially active fraction of a test substance, determined in a bioassay against a reference powder of the
same substance
© ISO 2006 – All rights reserved 1
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ISO 20776-1:2006(E)
NOTE The potency is expressed as mass fraction in milligrams per gram (mg/g), or as activity content in International
Units (IU) per gram, or as a volume fraction or mass fraction in percent, or as an amount-of-substance concentration
(mass fraction) in mole per litre of ingredients in the test substance.
2.2.2
concentration
amount of an antimicrobial agent in a defined volume of liquid
NOTE 1 The concentration is expressed as mg/l.
NOTE 2 mg/l ≡ µg/ml but it is not recommended to use the unit µg/ml.
2.3
stock solution
initial solution used for further dilutions
2.4
minimum inhibitory concentration
MIC
lowest concentration that, under defined in vitro conditions, prevents visible growth of bacteria within a defined
period of time
NOTE The MIC is expressed in mg/l.
2.5
breakpoint
BP
specific values of parameters, such as MICs, on the basis of which bacteria can be assigned to the clinical
categories “susceptible”, “intermediate” and “resistant”
NOTE For current interpretive breakpoints, reference can be made to the latest publications of organizations
employing this reference method (e.g. CLSI and EUCAST).
2.5.1
susceptible
S
bacterial strain inhibited in vitro by a concentration of an antimicrobial agent that is associated with a high
likelihood of therapeutic success
NOTE 1 Bacterial strains are categorized as susceptible by applying the appropriate breakpoints in a defined
phenotypic test system.
NOTE 2 This breakpoint can be altered due to changes in circumstances (e.g. changes in commonly used drug
dosages, emergence of new resistance mechanisms).
2.5.2
intermediate
I
bacterial strain inhibited in vitro by a concentration of an antimicrobial agent that is associated with uncertain
therapeutic effect
NOTE 1 Bacterial strains are categorized as intermediate by applying the appropriate breakpoints in a defined
phenotypic test system.
NOTE 2 This class of susceptibility implies that an infection due to the isolate can be appropriately treated in body sites
where the drugs are physiologically concentrated or when a high dosage of drug can be used.
NOTE 3 This class also indicates a “buffer zone”, to prevent small, uncontrolled, technical factors from causing major
discrepancies in interpretations.
NOTE 4 These breakpoints can be altered due to changes in circumstances (e.g. changes in commonly used drug
dosages, emergence of new resistance mechanisms).
2 © ISO 2006 – All rights reserved
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ISO 20776-1:2006(E)
2.5.3
resistant
R
bacterial strain inhibited in vitro by a concentration of an antimicrobial agent that is associated with a high
likelihood of therapeutic failure
NOTE 1 Bacterial strains are categorized as resistant by applying the appropriate breakpoints in a defined phenotypic
test system.
NOTE 2 This breakpoint can be altered due to changes in circumstances (e.g. changes in commonly used drug
dosages, emergence of new resistance mechanisms).
2.6
wild type
absence of acquired resistance mechanisms to the antimicrobial agent for a given strain
2.7
reference strain
catalogued, characterized bacteria with stable, defined antimicrobial susceptibility phenotypes and/or
genotypes
NOTE Reference strains are kept as stock cultures, from which working cultures are derived. They are obtainable
from culture collections and used for quality control.
2.8 Susceptibility testing method
2.8.1
broth dilution
technique in which containers are filled with appropriate volumes of an antimicrobial solution, employing
incrementally (usually two-fold) increasing concentrations of the antimicrobial agent and appropriate volumes
of broth with a defined inoculum
NOTE The aim of this method is the determination of the MIC.
2.8.2
microdilution
performance of broth dilution in microdilution trays with a capacity of u 200 µl per well
2.9
broth
fluid medium used for the in vitro growth of bacteria
2.10
inoculum
number of bacteria in a suspension, calculated with respect to the final volume
NOTE The inoculum is expressed as colony-forming units per millilitre (CFU/ml).
2.11
inoculum effect
change in MIC related to change in inoculum
© ISO 2006 – All rights reserved 3
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ISO 20776-1:2006(E)
3 Test procedures
3.1 General
The tests are performed in microdilution trays. The method is based on the preparation of antimicrobial agent
working solutions, either in 50 µl volumes per well (with the addition of an inoculum also in a volume of 50 µl),
or in a volume of 100 µl per well (with the addition of a maximum of 5 µl inoculum volume).
3.2 Medium
Mueller-Hinton broth shall be used (see Annex A for details).
3.3 Antimicrobial agents
3.3.1 General
Antimicrobial agents shall be obtained directly from the manufacturer or from reliable commercial sources;
pharmaceutical preparations for clinical use are not acceptable. The antimicrobial agents shall be supplied
with a lot number, potency, an expiry date and details of recommended storage conditions. Substances shall
be stored in tightly closed containers in the dark, at 4 °C to 8 °C, with a desiccant unless otherwise
recommended by the manufacturer. Hygroscopic agents should be dispensed into aliquots, one of which is
used on each test occasion.
Allow containers to warm to room temperature before opening them to avoid condensation.
3.3.2 Preparation of stock solutions
The use of a calibrated analytical balance is required to weigh antimicrobial agents. Allowance for the potency
of the powder shall be made by use of the following formula to obtain the amount of antimicrobial agent
substance or the volume of diluent needed for a standard solution:
V×ρ
m= (1)
P
mP×
V= (2)
ρ
where
ρ is the concentration of the stock solution, in mg/l;
m is mass of the antimicrobial agent (powder), in g;
P is the potency of the antimicrobial agent (powder), in mg/g;
V is the volume of diluent, in l.
Concentrations of stock solutions should be 1 000 mg/l or greater, although the solubility of some agents is a
limiting factor. The actual concentrations of stock solutions depend on the method of preparing working
solutions (serial dilutions). Agents should be dissolved and diluted in sterile distilled water unless the
manufacturer states otherwise. Some agents require alternative solvents (see Table 1). Sterilisation of
solutions is not usually necessary. If required, sterilisation should be done by membrane filtration, and
samples before and after sterilisation should be compared by assay to ensure that adsorption has not
occurred.
Unless information is available on stability of stock solutions under specified storage conditions, they should
be prepared fresh for each test batch.
4 © ISO 2006 – All rights reserved
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ISO 20776-1:2006(E)
Table 1 — Examples of solvents and diluents for making stock solutions of selected
antimicrobial agents
Antimicrobial agent Solvent Diluent
Amikacin Water
Amoxicillin Phosphate buffer 0,1 mol/l, pH 6,0 Phosphate buffer 0,1 mol/l, pH 6,0
Ampicillin Phosphate buffer 0,1 mol/l, pH 8,0 Phosphate buffer 0,1 mol/l, pH 6,0
a
Azithromycin Ethanol volume fraction 95 % or glacial acetic acid Water
Azlocillin Water
Aztreonam Saturated sodium bicarbonate solution Water
Carbenicillin Water
Cefaclor Water
Cefamandole Water
Cefazolin Phosphate buffer 0,1 mol/l, pH 6,0 Phosphate buffer 0,1 mol/l, pH 6,0
Cefdinir Phosphate buffer 0,1 mol/l, pH 6,0 Water
Cefditoren Phosphate buffer 0,1 mol/l, pH 6,0 Water
Cefepime Phosphate buffer 0,1 mol/l, pH 6,0 Phosphate buffer 0,1 mol/l, pH 6,0
Cefetamet Phosphate buffer 0,1 mol/l, pH 6,0 Water
Cefixime Phosphate buffer 0,1 mol/l, pH 7,0 Phosphate buffer 0,1 mol/l, pH 7,0
Cefmetazole Water
Cefonicid Water
Cefoperazone Water
Cefotaxime Water
Cefotetan Dimethyl sulfoxide Water
Cefoxitin Water
Cefpodoxime Mass concentration 0,1 % sodium bicarbonate solution Water
Cefprozil Water
Ceftazidime Saturated sodium bicarbonate solution Water
Ceftibuten 1/10 volume of dimethyl sulfoxide Water
Ceftizoxime Water
b
Ceftobiprole Dimethyl sulfoxide plus glacial acetic acid Water, vortex vigorously
Ceftriaxone Water
Cefuroxime Phosphate buffer 0,1 mol/l, pH 6,0 Phosphate buffer 0,1 mol/l, pH 6,0
Cephalothin Phosphate buffer 0,1 mol/l, pH 6,0 Water
Chloramphenicol Ethanol volume fraction 95 % Water
Cinoxacin Half volume of water, a minimum volume 1 mol/l NaOH Water
to dissolve, then make up to total volume with water
Ciprofloxacin Water
a
Clarithromycin Methanol or glacial acetic acid 0,1 mol/l phosphate buffer, pH 6,5
Clavulanic acid Phosphate buffer 0,1 mol/l, pH 6,0 Phosphate buffer 0,1 mol/l, pH 6,0
Clinafloxacin Water
Clindamycin Water
c
Colistin Water Water
d
Dalbavancin Dimethyl sulfoxide Water and dimethyl sulfoxide
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ISO 20776-1:2006(E)
Table 1 (continued)
Antimicrobial agent Solvent Diluent
Daptomycin Water Water
a
Dirithromycin Glacial acetic acid Water
Doripenem NaCl volume fraction 0,85 % NaCl volume fraction 0,85 %
Doxycycline Water
Enoxacin Half volume water, a minimum volume 0,1 mol/l NaOH Water
to dissolve, then make up to total volume with water
Ertapenem Phosphate buffer 0,01 mol/l, pH 7,2 Phosphate buffer 0,01 mol/l, pH 7,2
a
Erythromycin Ethanol volume fraction 95 % or glacial acetic acid Water
Faropenem Water Water
Fleroxacin Half volume water, a minimum volume 0,1 mol/l NaOH Water
to dissolve, then make up to total volume with water
Fusidic acid Ethanol volume fraction 95 % Water
Garenoxacin Water (with stirring)
Gatifloxacin Water (with stirring)
Gemifloxacin Water
Gentamicin Water
Imipenem Phosphate buffer 0,01 mol/l, pH 7,2 Phosphate buffer 0,01 mol/l, pH 7,2
Kanamycin Water
Levofloxacin Half volume water, a minimum volume 1 mol/l NaOH to Water
dissolve, then make up to total volume with water
Linezolid Water
Loracarbef Water
Mecillinam Water
Meropenem Phosphate buffer 0,01 mol/l, pH 7,2 Phosphate buffer 0,01 mol/l, pH 7,2
Methicillin Water
Mezlocillin Water
Minocyc
...
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