SIST EN ISO 20776-1:2020
(Main)Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices - Part 1: Broth micro-dilution reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases (ISO 20776-1:2019, including Corrected version 2019-12)
Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices - Part 1: Broth micro-dilution reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases (ISO 20776-1:2019, including Corrected version 2019-12)
This document describes one reference method, broth micro-dilution, for determination of MICs. The MIC can be a guide for the clinician, and reflects the activity of the drug under the described test conditions, by taking into account other factors, such as drug pharmacology, pharmacokinetics, or bacterial resistance mechanisms. This allows categorisation 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 document, modifications of the basic method are required for certain antimicrobial agent - bacteria combinations to facilitate clinical interpretation. These modifications are included in a separate annex of this document. It is necessary to compare other susceptibility testing methods (e.g. disc diffusion 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 20776-1:2019, einschließlich der korrigierten Fassung von 2019-12)
Dieses Dokument beschreibt eine Referenzmethode, Mikrodilution, zur MHK Bestimmung. Die MHK kann dem Kliniker als Orientierungshilfe dienen und spiegelt das Maß für die Aktivität einer Substanz unter den beschriebenen Testbedingungen wider. Hierbei können auch andere Faktoren wie die Pharmakologie und Pharmakokinetik des Wirkstoffs oder bakterielle Resistenzmechanismen berücksichtigt werden. Diese Vorgehensweise erlaubt die Einstufung von Bakterien 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. Obgleich die klinische Interpretation des MHK-Werts nicht im Anwendungsbereich dieses Dokuments liegt, sind für bestimmte Kombinationen von antimikrobiellen Substanzen/Erregern Modifikationen der Basismethode erforderlich, um die klinische Interpretation zu erleichtern. Diese Modifikationen sind in einem separaten Anhang dieses Dokuments aufgeführt. Es ist erforderlich, andere Methoden zur Empfindlichkeitsprüfung (z. B. mittels Plattendiffusion oder Diagnoseprüfgeräten) mit dieser Referenzmethode zur Validierung zu vergleichen, um vergleichbare und verlässliche Werte sicherzustellen.
Sensibilité in vitro des agents infectieux et évaluation des performances des dispositifs pour antibiogrammes - Partie 1: Méthode de référence de microdilution en bouillon pour la détermination de la sensibilité in vitro aux agents antimicrobiens des bactéries aérobies à croissance rapide impliquées dans les maladies infectieuses (ISO 20776-1:2019, y compris version corrigée 2019-12)
Le présent document décrit une méthode de référence, la microdilution en bouillon, pour déterminer les CMI. La CMI peut constituer un guide pour le clinicien et reflète l'activité du médicament dans les conditions d'essai décrites, en tenant compte d'autres facteurs tels que la pharmacologie du médicament, la pharmacocinétique 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 du présent document, des modifications de la méthode de base sont nécessaires pour certaines combinaisons agent antimicrobien-bactérie afin de faciliter l'interprétation clinique. Ces modifications sont incluses dans une annexe séparée du présent document. Il est nécessaire de comparer les autres méthodes d'essai de sensibilité (par exemple, les méthodes de diffusion en gélose ou les dispositifs d'essai de diagnostic) à cette méthode de référence à des fins de validation et pour garantir des résultats comparables et fiables.
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 20776-1:2019, vključno s popravkom verzije 2019-12)
General Information
Relations
Overview
EN ISO 20776-1:2020 (identical to ISO 20776-1:2019, corrected 2019-12) defines the broth micro-dilution reference method for determining minimum inhibitory concentrations (MICs) of antimicrobial agents against rapidly growing aerobic bacteria. The standard describes a standardized in vitro procedure used in antimicrobial susceptibility testing (AST) to quantify the activity of antimicrobial agents under controlled conditions. It is a reference method for validating other susceptibility methods and devices (e.g., disc diffusion, diagnostic test devices).
Key topics and technical requirements
- Test method: Detailed broth micro-dilution procedures including preparation of micro-dilution trays, stock and working solutions of antimicrobial agents, inoculum preparation (broth culture and colony suspension methods), inoculation, incubation and result reading.
- Medium: Requirements and guidance for standardized Mueller‑Hinton broth (including Annex A) and possible modifications for certain agents.
- Antimicrobial preparations: Solvents, diluents and storage for stock and working solutions (see Annex B and Annex C); corrected values are noted (e.g., ampicillin diluent pH correction in the corrected 2019-12 text).
- Quality control: Procedures to ensure reproducibility and reliability of MIC results across laboratories and over time.
- Special situations: Guidance on test situations that may produce unreliable MICs and recommended modifications (Annex D).
- Scope limits: The document focuses on MIC determination and method performance; clinical interpretation (defining clinical breakpoints S/I/R) is addressed separately and not defined within this standard.
Applications and who uses it
- Clinical microbiology laboratories use the method for precise MIC determination when quantitative results are required for patient management, epidemiology, or when routine tests are equivocal.
- Manufacturers of antimicrobial susceptibility test devices and in vitro diagnostic systems use EN ISO 20776-1:2020 as the reference method to validate and compare their devices against a recognized standard.
- Regulatory bodies and accreditation organizations reference this standard when assessing method conformity, device performance and lab quality systems.
- Public health and surveillance programs use MIC data produced to monitor resistance trends and to distinguish wild-type from non-wild-type bacterial populations.
- Research laboratories conducting comparative testing of new antimicrobial agents or pharmacodynamic studies.
Related standards
- Other parts of the ISO 20776 series cover evaluation and specific device performance and complementary methods; EN ISO 20776-1:2020 is intended as the laboratory reference for broth micro-dilution AST.
Keywords: EN ISO 20776-1:2020, ISO 20776-1:2019, broth micro-dilution, antimicrobial susceptibility testing, MIC, Mueller‑Hinton broth, AST, diagnostic devices, in vitro activity.
Standards Content (Sample)
SLOVENSKI STANDARD
01-september-2020
Nadomešča:
SIST EN ISO 20776-1:2007
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 20776-1:2019, vključno
s popravkom verzije 2019-12)
Susceptibility testing of infectious agents and evaluation of performance of antimicrobial
susceptibility test devices - Part 1: Broth micro-dilution reference method for testing the
in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved
in infectious diseases (ISO 20776-1:2019, including Corrected version 2019-12)
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 20776-1:2019, einschließlich der
korrigierten Fassung von 2019-12)
Sensibilité in vitro des agents infectieux et évaluation des performances des dispositifs
pour antibiogrammes - Partie 1: Méthode de référence de microdilution en bouillon pour
la détermination de la sensibilité in vitro aux agents antimicrobiens des bactéries
aérobies à croissance rapide impliquées dans les maladies infectieuses (ISO 20776-
1:2019, y compris version corrigée 2019-12)
Ta slovenski standard je istoveten z: EN ISO 20776-1:2020
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 20776-1
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2020
EUROPÄISCHE NORM
ICS 11.100.20 Supersedes EN ISO 20776-1:2006
English Version
Susceptibility testing of infectious agents and evaluation of
performance of antimicrobial susceptibility test devices -
Part 1: Broth micro-dilution reference method for testing
the in vitro activity of antimicrobial agents against rapidly
growing aerobic bacteria involved in infectious diseases
(ISO 20776-1:2019, including Corrected version 2019-12)
Sensibilité in vitro des agents infectieux et évaluation Labormedizinische Untersuchungen und In-vitro-
des performances des dispositifs pour antibiogrammes Diagnostika-Systeme - Empfindlichkeitsprüfung von
- Partie 1: Méthode de référence de microdilution en Infektionserregern und Evaluation von Geräten zur
bouillon pour la détermination de la sensibilité in vitro antimikrobiellen Empfindlichkeitsprüfung - Teil 1:
aux agents antimicrobiens des bactéries aérobies à Referenzmethode zur Testung der In-vitro-Aktivität
croissance rapide impliquées dans les maladies von antimikrobiellen Substanzen gegen schnell
infectieuses (ISO 20776-1:2019, y compris version wachsende aerobe Bakterien, die
corrigée 2019-12) Infektionskrankheiten verursachen (ISO 20776-
1:2019, einschließlich der korrigierten Fassung von
2019-12)
This European Standard was approved by CEN on 22 April 2020.
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-CENELEC 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-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20776-1:2020 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 20776-1:2020) has been prepared by Technical Committee ISO/TC 212 "Clinical
laboratory testing and in vitro diagnostic test systems" in collaboration with Technical Committee
CEN/TC 140 “In vitro diagnostic medical devices” 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 2021, and conflicting national standards shall
be withdrawn at the latest by July 2023.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 20776-1:2006.
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,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 20776-1:2019, including Corrected version 2019-12 has been approved by CEN as
INTERNATIONAL ISO
STANDARD 20776-1
Second edition
2019-06
Corrected version
2019-12
Susceptibility testing of infectious
agents and evaluation of performance
of antimicrobial susceptibility test
devices —
Part 1:
Broth micro-dilution reference
method for testing the in vitro activity
of antimicrobial agents against rapidly
growing aerobic bacteria involved in
infectious diseases
Sensibilité in vitro des agents infectieux et évaluation des
performances des dispositifs pour antibiogrammes —
Partie 1: Méthode de référence de microdilution en bouillon pour la
détermination de la sensibilité in vitro aux agents antimicrobiens des
bactéries aérobies à croissance rapide impliquées dans les maladies
infectieuses
Reference number
ISO 20776-1:2019(E)
©
ISO 2019
ISO 20776-1:2019(E)
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
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Published in Switzerland
ii © ISO 2019 – All rights reserved
ISO 20776-1:2019(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Test procedures . 3
4.1 General . 3
4.2 Medium . 3
4.3 Antimicrobial agents . 3
4.3.1 General. 3
4.3.2 Preparation of stock solutions . 3
4.3.3 Preparation of working solutions . 4
4.3.4 Preparation of micro-dilution trays . 4
4.3.5 Storage of micro-dilution trays . 4
4.4 Preparation of inoculum . 5
4.4.1 General. 5
4.4.2 Broth culture method . 5
4.4.3 Direct colony suspension method . 5
4.5 Inoculation of micro-dilution trays . 5
4.6 Incubation of micro-dilution trays . 6
4.7 Reading results . 6
4.8 Special test situations where the MIC result might give unreliable results. 6
5 Quality control . 6
Annex A (informative) Requirements for Mueller-Hinton broth . 8
Annex B (informative) Solvents and diluents for making stock solutions of selected
antimicrobial agents . .11
Annex C (informative) Preparation of working dilutions of antimicrobial agents for use in
broth dilution susceptibility tests .16
Annex D (informative) Special test situations .17
Bibliography .18
ISO 20776-1:2019(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in
vitro diagnostic test systems.
This second edition cancels and replaces the first edition (ISO 20776-1:2006), which has been
technically revised.
The main changes compared to the previous edition are as follows:
— revised to a broth micro-dilution only performance document;
— removal of S, I, R breakpoint definitions and information;
— moved embedded tables to annexes;
— removed quality control range table;
— updated table (now Annex B) on solvents and diluents for antimicrobial agents used globally;
— updated information on special culture media and method performance for specific currently used
antimicrobial agents.
A list of all parts in the ISO 20776 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
This corrected version of ISO 20776-1:2019 incorporates the following correction:
— Correction of the diluent pH value for ampicillin from 8,0 to 6,0 in Annex B.
iv © ISO 2019 – All rights reserved
ISO 20776-1:2019(E)
Introduction
In vitro antimicrobial susceptibility tests are performed on micro-organisms 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 for antimicrobial susceptibility testing. MIC methods are used in resistance
surveillance, defining identifying wild type phenotypes, 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, micro-organisms are tested for their ability to produce visible growth in broth (broth
dilution) containing serial dilutions of the antimicrobial agent or on a series of agar plates (agar
dilution).
The lowest concentration of an antimicrobial agent (in mg/l) that, under defined in vitro conditions,
prevents the appearance of visible growth of a micro-organism 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 standardization is required for intra- and inter-
laboratory reproducibility of broth MIC tests. The MICs generally span two to three doubling dilutions
with a dominant central value.
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 micro-organisms.
Broth micro-dilution denotes the performance of the broth dilution test in micro-dilution trays.
The method described in this document is intended for the testing of pure cultures of aerobic bacteria
that are easily grown by overnight incubation on agar and grow well in standardized micro-dilution
trays containing standardized Mueller-Hinton broth (volume of ≤200 µl), which may need to be
modified depending on the antimicrobial agent being tested.
The broth micro-dilution method described in this document is essentially the same as those used in
many countries, and as the methods published by the Clinical and Laboratory Standards Institute (CLSI)
[1] [2]
and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) . These methods
[3]
are based on those described by Ericsson and Sherris .
INTERNATIONAL STANDARD ISO 20776-1:2019(E)
Susceptibility testing of infectious agents and evaluation of
performance of antimicrobial susceptibility test devices —
Part 1:
Broth micro-dilution 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 document may involve hazardous materials, operations and
equipment. This document does not purport to address all of the safety problems associated
with its use. It is the responsibility of the user of this document to establish appropriate safety
and health practices and to determine the applicability of any other restrictions prior to use.
1 Scope
This document describes one reference method, broth micro-dilution, for determination of MICs.
The MIC can be a guide for the clinician, and reflects the activity of the drug under the described
test conditions, by taking into account other factors, such as drug pharmacology, pharmacokinetics,
or bacterial resistance mechanisms. This allows categorisation 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 document, modifications of the basic method are required for certain antimicrobial
agent - bacteria combinations to facilitate clinical interpretation. These modifications are included in a
separate annex of this document. It is necessary to compare other susceptibility testing methods (e.g.
disc diffusion or diagnostic test devices) with this reference method for validation, in order to ensure
comparable and reliable results.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
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 1 to entry: Disinfectants, antiseptics and preservatives are not included in this definition.
ISO 20776-1:2019(E)
3.2
potency
measure of drug activity expressed in terms of the amount required to produce an effect of given
intensity
Note 1 to entry: 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 (molar fraction) in mole per litre of ingredients in the test substance.
3.3
concentration
amount of an antimicrobial agent in a defined volume of liquid
Note 1 to entry: The concentration is expressed as mg/l.
Note 2 to entry: mg/l is the designated ISO unit.
3.4
stock solution
initial solution used for further dilutions
3.5
minimum inhibitory concentration
MIC
lowest concentration that, under defined in vitro conditions, prevents visible growth of bacteria within
a defined period of time
Note 1 to entry: The MIC is expressed in mg/l.
3.6
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 1 to entry: For current interpretive breakpoints, reference can be made to the latest publications of
organisations employing this reference method (e.g. CLSI and EUCAST).
3.7
wild type
absence of known resistance mechanisms to the antimicrobial agent for a given strain
3.8
reference strain
catalogued, characterized bacteria with stable, defined antimicrobial susceptibility phenotypes and/or
genotypes
Note 1 to entry: Reference strains are kept as stock cultures, from which working cultures are derived. They are
obtainable from culture collections and used for quality control.
3.9
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 1 to entry: The aim of this method is the determination of the MIC.
3.10
micro-dilution
performance of broth dilution in micro-dilution trays with a final test volume of ≤200 µl per well
2 © ISO 2019 – All rights reserved
ISO 20776-1:2019(E)
3.11
broth
fluid medium used for the in vitro growth of bacteria
Note 1 to entry: For the broth reference method the medium is standardised Mueller-Hinton broth (see Annex A).
3.12
inoculum
number of bacteria in a suspension, calculated with respect to the final volume
Note 1 to entry: The inoculum is expressed as colony-forming units per millilitre (CFU/ml).
3.13
inoculum effect
change in MIC related to change in inoculum in CFU/ml
4 Test procedures
4.1 General
The tests are performed in polystyrene micro-dilution 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
10 µl inoculum volume).
4.2 Medium
Mueller-Hinton broth shall be used (see Annex A for details and Annex D for special test situations).
4.3 Antimicrobial agents
4.3.1 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 as powders 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, with a desiccant at the
recommended temperature of the supplier. Hygroscopic agents should be dispensed into aliquots, one
of which is used on each test occasion.
To avoid condensation, allow containers to warm to room temperature before opening.
4.3.2 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
ISO 20776-1:2019(E)
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 Annex B).
NOTE For newer antimicrobial agents not identified in Annex B of this current document, consult
manufacturer information on the most appropriate solvent and diluent for the specific agent. 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.
4.3.3 Preparation of working solutions
The range of concentrations selected for testing depends on the micro-organisms and antimicrobial
agent. The chosen range shall allow full endpoint MIC determination for appropriate reference strains.
A two-fold dilution series based on 1 mg/l is prepared in Mueller-Hinton broth. Dilutions should not be
prepared by serial dilution steps, but according to the procedure outlined in Annex C. Working solutions
shall be used the same day unless information is available on stability of the solutions under specified
storage conditions.
4.3.4 Preparation of micro-dilution trays
Working solutions are dispensed into micro-dilution trays at 50 µl per well with double the desired
final concentrations of antimicrobial agent, or at 100 µl per well in the desired final concentrations.
At least one well, containing 50 µl or 100 µl of antimicrobial agent-free medium, should be included
as a growth control for each strain tested. Likewise, a well containing 100 µl of antimicrobial agent-
free medium should be included as an un-inoculated negative control well for each micro-organism
type tested.
4.3.5 Storage of micro-dilution trays
Filled trays may be used immediately or may be stored for up to three months or until documented
quality control or other evidence indicates degradation of the antimicrobial agent. For storage the
filled trays should be sealed in plastic bags and immediately placed in a freezer at ≤−60 °C unless the
antimicrobial agents are known to be stable at higher temperatures.
Trays shall not be stored in a self-defrosting freezer, and thawed antimicrobial solutions shall not be
re-frozen, as repeated freeze-thaw cycles accelerate the degradation of some antimicrobial agents,
particularly β-lactams.
Allow frozen plates to thaw for up to 2 h and inoculate by 4 h of removal from the freezer.
4 © ISO 2019 – All rights reserved
ISO 20776-1:2019(E)
4.4 Preparation of inoculum
4.4.1 General
Standardization of the inoculum is essential for accurate and reproducible broth micro-dilution
susceptibility tests. Therefore, purity checks and viable colony counts shall be performed on every
isolate tested with this reference procedure
4.4.2 Broth culture method
The inoculum may be prepared by diluting a broth culture or by suspending several morphologically
similar colonies (when possible) from an overnight culture on non-selective agar medium in broth with
a sterile loop or swab. When suspending colonies, morphologically similar colonies should be picked to
avoid contamination of other species or atypical variants of the same species.
The broth used shall not be antagonistic to the antimicrobial agent tested. The broth is incubated
at (35 ± 1) °C until the growth reaches a turbidity equal to or greater than that of a 0,5 McFarland
[4]
standard . If needed, the culture is adjusted with saline or broth to give a turbidity equivalent to the
0,5 McFarland standard. This can be done by means of a photometric device (using 625 nm wavelength
and a 1 cm path cuvette, the absorbance will be in the range of 0,08 to 0,13), or by employing a suitably
calibrated nephelometer. Alternatively, this can be achieved visually by comparing the appearance of
black lines through the inoculum and 0,5 McFarland standard suspensions (the inoculum and McFarland
standard shall be in tubes of the same size) or any other method that gives reproducible CFU/ml. The
5 5 5
final inoculum shall be 5 × 10 CFU/ml (target range, 2 × 10 CFU/ml to 8 × 10 CFU/ml).
NOTE A 0,5 McFarland standard can be produced by adding a 0,5 ml aliquot of 0,048 mol/l barium chloride
(BaCl ) (11,72 g/l BaCl ⋅2H O) to 99,5 ml of 0,18 mol/l sulphuric acid (H SO ), with constant stirring to maintain
2 2 2 2 4
a suspension.
4.4.3 Direct colony suspension method
Several morphologically similar colonies from a non-selective nutritive agar medium, incubat
...
Frequently Asked Questions
SIST EN ISO 20776-1:2020 is a standard published by the Slovenian Institute for Standardization (SIST). Its full title is "Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices - Part 1: Broth micro-dilution reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases (ISO 20776-1:2019, including Corrected version 2019-12)". This standard covers: This document describes one reference method, broth micro-dilution, for determination of MICs. The MIC can be a guide for the clinician, and reflects the activity of the drug under the described test conditions, by taking into account other factors, such as drug pharmacology, pharmacokinetics, or bacterial resistance mechanisms. This allows categorisation 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 document, modifications of the basic method are required for certain antimicrobial agent - bacteria combinations to facilitate clinical interpretation. These modifications are included in a separate annex of this document. It is necessary to compare other susceptibility testing methods (e.g. disc diffusion or diagnostic test devices) with this reference method for validation, in order to ensure comparable and reliable results.
This document describes one reference method, broth micro-dilution, for determination of MICs. The MIC can be a guide for the clinician, and reflects the activity of the drug under the described test conditions, by taking into account other factors, such as drug pharmacology, pharmacokinetics, or bacterial resistance mechanisms. This allows categorisation 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 document, modifications of the basic method are required for certain antimicrobial agent - bacteria combinations to facilitate clinical interpretation. These modifications are included in a separate annex of this document. It is necessary to compare other susceptibility testing methods (e.g. disc diffusion or diagnostic test devices) with this reference method for validation, in order to ensure comparable and reliable results.
SIST EN ISO 20776-1:2020 is classified under the following ICS (International Classification for Standards) categories: 11.100.10 - In vitro diagnostic test systems. The ICS classification helps identify the subject area and facilitates finding related standards.
SIST EN ISO 20776-1:2020 has the following relationships with other standards: It is inter standard links to SIST EN ISO 20776-1:2007. Understanding these relationships helps ensure you are using the most current and applicable version of the standard.
SIST EN ISO 20776-1:2020 is associated with the following European legislation: EU Directives/Regulations: 98/79/EC; Standardization Mandates: M/252, M/352. When a standard is cited in the Official Journal of the European Union, products manufactured in conformity with it benefit from a presumption of conformity with the essential requirements of the corresponding EU directive or regulation.
You can purchase SIST EN ISO 20776-1:2020 directly from iTeh Standards. The document is available in PDF format and is delivered instantly after payment. Add the standard to your cart and complete the secure checkout process. iTeh Standards is an authorized distributor of SIST standards.
SIST EN ISO 20776-1:2020 표준은 감염성 병원체에 대한 감수성 테스트 및 항균 감수성 테스트 장치의 성능 평가를 위한 중요한 기준을 제공합니다. 이 문서는 미생물 감수성 테스트의 기본 방법 중 하나인 broth micro-dilution 방식에 대한 상세한 지침을 제시하며, 이는 미생물의 적정 항균제 농도(MIC)를 정량하는 데 중점을 둡니다. 이 표준의 강점 중 하나는 광범위한 범위입니다. 항균제 활동을 평가할 때, MIC 값은 임상의에게 중요한 지침을 제공하며, 약물의 약리학, 약물동태학 및 세균의 내성 메커니즘과 같은 다양한 요소를 고려하여 해석됩니다. 이러한 측정을 통해 세균을 "감수성(S)", "중간(I)", "저항(R)"으로 분류할 수 있는 점은 임상적 실용성을 높여 줍니다. 더불어, MIC 분포는 야생형 또는 비야생형 세균 집단을 정의하는 데 유용하게 사용됩니다. 이 표준은 특정 항균제-세균 조합에 대한 방법 수정이 필요함을 인지하고 있으며, 이는 별도의 부록으로 포함되어 있습니다. 이로 인해 사용자는 임상 해석을 위한 보다 명확한 가이드를 받을 수 있습니다. 추가적으로, 이 기준서는 다른 감수성 테스트 방법(예: 디스크 확산법 또는 진단 테스트 장치)과 비교하여 유사하고 신뢰할 수 있는 결과를 보장할 수 있도록 요구하고 있는 점도 강조됩니다. SIST EN ISO 20776-1:2020 표준은 현대 의료의 필요에 부응하는 중요한 도구로, 항균제에 대한 감수성 테스트의 정확한 방법론을 제공함으로써 감염성 질환 치료에 있어 실질적인 기여를 할 수 있습니다. 이러한 점에서 이 표준은 감염병 관리 및 치료에 필수적인 문서로 자리잡고 있습니다.
The SIST EN ISO 20776-1:2020 standard provides a comprehensive reference method for antimicrobial susceptibility testing, specifically utilizing the broth micro-dilution technique. This method is crucial as it facilitates the determination of minimum inhibitory concentrations (MICs) for various rapidly growing aerobic bacteria implicated in infectious diseases. The standard thoroughly outlines the process for accurately identifying the MIC, which serves as a useful tool for clinicians to guide treatment decisions. One of the key strengths of this standard is its ability to reflect the activity of antimicrobial agents within specific test conditions, while also considering essential factors such as drug pharmacology, pharmacokinetics, and bacterial resistance mechanisms. This multifaceted approach enables the categorization of bacteria into "susceptible" (S), "intermediate" (I), or "resistant" (R) groups, aiding in the effective management of infections. Moreover, the standard offers insights into the interpretation of MIC distributions, which can be instrumental in delineating wild type versus non-wild type bacterial populations. This distinction is significant for understanding the dynamics of bacterial resistance and the effectiveness of different antimicrobial agents. While the document clearly establishes the broth micro-dilution method as the reference point for testing, it also recognizes that certain combinations of antimicrobial agents and bacteria necessitate modifications to facilitate clinical interpretation. These adaptations are detailed in an annex, ensuring that users can access vital information for specific scenarios. Lastly, the standard emphasizes the importance of validating other susceptibility testing methods, such as disc diffusion and diagnostic test devices, against the reference method to guarantee comparable and reliable results. This focus on validation underscores the standard’s relevance in laboratory settings and supports a unified approach to antimicrobial susceptibility testing across different methodologies. In summary, SIST EN ISO 20776-1:2020 stands out as a critical document that enhances the accuracy and reliability of antimicrobial susceptibility testing, providing a robust framework for clinicians and laboratories engaged in the fight against infectious diseases.
La norme SIST EN ISO 20776-1:2020 constitue une avancée significative dans le domaine du test de susceptibilité des agents infectieux et l'évaluation des dispositifs de test de susceptibilité antimicrobienne. Son champ d'application est clairement défini, se concentrant sur la méthode de micro-dilution en bouillon comme référence pour la détermination des Concentrations Minimales Inhibitrices (CMI). Cette approche est essentielle pour fournir des orientations cliniques précises, en tenant compte non seulement de l'activité des agents antimicrobiens, mais aussi de facteurs cruciaux tels que la pharmacologie et la pharmacocinétique des médicaments ainsi que des mécanismes de résistance bactérienne. Parmi les forces de cette norme, on note la capacité à classifier les bactéries en tant que « susceptible » (S), « intermédiaire » (I) ou « résistant » (R), une fonctionnalité qui est fondamentale pour le choix de la thérapie adéquate. De plus, le document traite des distributions des CMI, permettant de définir des populations bactériennes « type sauvage » ou « non type sauvage », ce qui est vital pour le suivi de l'évolution des résistances dans les souches bactériennes. La norme ne se limite pas à la simple description de la méthode de micro-dilution, mais elle souligne également l'importance de la validation des autres méthodes de test de susceptibilité, comme la diffusion sur disque ou les dispositifs de test diagnostiques, par rapport à cette méthode de référence. Cela garantit des résultats comparables et fiables, ce qui est indispensable dans le contexte clinique actuel où la précision des résultats de test est cruciale pour le traitement efficace des infections. En résumé, la norme SIST EN ISO 20776-1:2020 est non seulement pertinente mais aussi essentielle pour garantir la qualité et la fiabilité des tests de susceptibilité antimicrobienne, constituant ainsi un outil indispensable pour les professionnels de la santé dans la lutte contre les infections bactériennes.
SIST EN ISO 20776-1:2020は、感染症に関与する急速に成長する好気性細菌に対する抗菌剤のin vitro活性をテストするための、ブロス微希釈基準法を定義する重要な文書です。この標準の範囲は、MIC（最小発育阻止濃度）の決定に焦点を当てており、臨床医にとって有用なガイドとなります。MICの値は、テスト条件下での薬剤の活性を反映し、薬剤の薬理学、薬物動態、および細菌の耐性メカニズムなどの他の要因を考慮に入れています。 この標準の強みは、細菌を「感受性」（S）、「中間」（I）、「抵抗性」（R）のカテゴリに分類できる点にあります。さらに、MICの分布は野生型または非野生型細菌集団の定義にも利用可能であり、細菌学的研究の視点からも重要な役割を果たします。特に、この文書は、特定の抗菌剤と細菌の組み合わせにおいて、臨床解釈を容易にするために基本的な方法を修正する必要があることを明記しており、これらの修正は文書の別の付録に含まれています。 また、この標準は、他の感受性試験法（例：ディスク拡散法や診断テストデバイス）との比較検証が重要であることを強調しており、これにより比較可能で信頼性の高い結果を確保することができます。この点からも、SIST EN ISO 20776-1:2020は、感染症の診断と治療における抗菌剤感受性テストの実施に必須のガイドラインとして高い関連性を持っています。
Der Standard SIST EN ISO 20776-1:2020 bietet eine umfassende und präzise Anleitung für die Bestimmung der minimalen Hemmkonzentration (MIC) von antimikrobiellen Substanzen gegen rasch wachsende aerobe Bakterien, die in der Medizin von Bedeutung sind. Der Fokus auf die Methode der Broth-Mikrodilution als Referenzmethode stellt sicher, dass die Ergebnisse genau und reproduzierbar sind. Diese Methode ist besonders wichtig, da sie nicht nur die direkte Aktivität der Medikamente unter definierten Testbedingungen bewertet, sondern auch relevante Faktoren wie Pharmakologie, Pharmakokinetik und bakterielle Resistenzmechanismen berücksichtigt. Ein weiterer wesentlicher Aspekt des Standards ist die Möglichkeit, Bakterien in Kategorien wie "empfindlich" (S), "intermediär" (I) oder "resistent" (R) einzuteilen. Diese Kategorisierung ist entscheidend für die klinische Entscheidungsfindung und ermöglicht es den Fachleuten, fundierte Therapieentscheidungen zu treffen. Zusätzlich zu dieser Klassifizierung bietet die MIC-Verteilung wertvolle Informationen für die Definition von Wildtyp- und Nicht-Wildtyp-Bakterienpopulationen, was für das Verständnis von Resistenzmustern und Epidemien von großer Bedeutung ist. Der Standard umfasst auch wichtige Hinweise zur Vergleichbarkeit anderer Empfindlichkeitstestverfahren, wie z.B. Diskdiffusion oder diagnostische Testgeräte, mit der bereitgestellten Referenzmethode. Dies ist entscheidend für die Validierung alternativer Testmethoden, um vergleichbare und verlässliche Ergebnisse zu gewährleisten, was die Relevanz dieser Norm im klinischen Kontext weiter unterstreicht. Die separaten Anhänge, die spezifische Modifikationen der Grundmethode für bestimmte Kombinationen von antimikrobiellen Substanzen und Bakterien enthalten, zeigen den pragmatischen Ansatz des Standards, um sicherzustellen, dass klinische Interpretationen der Ergebnisse effizient durchgeführt werden können. Insgesamt stellt der SIST EN ISO 20776-1:2020 eine bedeutende Ressource dar, die sowohl für die Forschung als auch für die klinische Praxis unverzichtbar ist, indem sie eine evidenzbasierte Grundlage für die Beurteilung der Wirksamkeit antimikrobieller Behandlungen liefert.
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