EN ISO 20776-1:2020

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
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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
...