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

Status
Published
Public Enquiry End Date
14-Sep-2018
Publication Date
10-Aug-2020
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
14-Jul-2020
Due Date
18-Sep-2020
Completion Date
11-Aug-2020

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SLOVENSKI STANDARD
SIST EN ISO 20776-1:2020
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
SIST EN ISO 20776-1:2020 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 20776-1:2020
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SIST EN ISO 20776-1:2020
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.
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SIST EN ISO 20776-1:2020
EN ISO 20776-1:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 20776-1:2020
EN ISO 20776-1:2020 (E)
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

EN ISO 20776-1:2020 without any modification.
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SIST EN ISO 20776-1:2020
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SIST EN ISO 20776-1:2020
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
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SIST EN ISO 20776-1:2020
ISO 20776-1:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© 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

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
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SIST EN ISO 20776-1:2020
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 2019 – All rights reserved iii
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SIST EN ISO 20776-1:2020
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
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SIST EN ISO 20776-1:2020
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 .
© ISO 2019 – All rights reserved v
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SIST EN ISO 20776-1:2020
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SIST EN ISO 20776-1:2020
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 2019 – All rights reserved 1
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SIST EN ISO 20776-1:2020
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

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
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SIST EN ISO 20776-1:2020
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)
mP×
V = (2)
where
© ISO 2019 – All rights reserved 3
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SIST EN ISO 20776-1:2020
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 ev
...

SLOVENSKI STANDARD
oSIST prEN ISO 20776-1:2018
01-september-2018

3UHVNXVREþXWOMLYRVWLSRY]URþLWHOMHYLQIHNFLMQDGHORYDQMHDQWLPLNUREQRREþXWOMLYLK

QDSUDYGHO5HIHUHQþQDPHWRGD]DSUHVNXVDNWLYQRVWLLQYLWURDQWLPLNUREQLK
SRY]URþLWHOMHYQDYSOLYEDNWHULMSULQDOH]OMLYLKEROH]QLK ,62',6

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/DIS 20776-1:2018)
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/DIS 20776-1:2018)

Sensibilité in vitro des agents infectieux et évaluation des performances des dispositifs

pour antibiogrammes - Partie 1: Méthode de référence pour la détermination de la

sensibilité in vitro aux agents antimicrobiens des bactéries aérobies à croissance rapide

impliquées dans les maladies infectieuses (ISO/DIS 20776-1:2018)
Ta slovenski standard je istoveten z: prEN ISO 20776-1
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
oSIST prEN ISO 20776-1:2018 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 20776-1:2018
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oSIST prEN ISO 20776-1:2018
DRAFT INTERNATIONAL STANDARD
ISO/DIS 20776-1
ISO/TC 212 Secretariat: ANSI
Voting begins on: Voting terminates on:
2018-06-26 2018-09-18
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 pour la détermination de la sensibilité in vitro aux agents antimicrobiens

des bactéries aérobies à croissance rapide impliquées dans les maladies infectieuses

ICS: 11.100.20
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
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BEING ACCEPTABLE FOR INDUSTRIAL,
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Reference number
NATIONAL REGULATIONS.
ISO/DIS 20776-1:2018(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2018
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oSIST prEN ISO 20776-1:2018
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COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

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

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

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Published in Switzerland
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oSIST prEN ISO 20776-1:2018
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ISO CD 20776-1
13 Contents

14 Foreword ......................................................................................................................................................................... iv

15 Introduction..................................................................................................................................................................... v

16 1 Scope .......................................................................................................................................................................... 1

17 2 Normative references .......................................................................................................................................... 1

18 3 Terms and definitions .......................................................................................................................................... 1

19 4 Test procedures ..................................................................................................................................................... 3

20 4.1 General ...................................................................................................................................................................... 3

21 4.2 Medium ...................................................................................................................................................................... 3

22 4.3 Antimicrobial agents ............................................................................................................................................. 3

23 4.4 Preparation of inoculum ..................................................................................................................................... 5

24 4.5 Inoculation of micro-dilution trays ................................................................................................................. 5

25 4.6 Incubation of micro-dilution trays .................................................................................................................. 6

26 4.7 Reading results ....................................................................................................................................................... 6

27 4.8 Special test situations where the MIC result might give unreliable results ..................................... 7

28 5 Quality control ........................................................................................................................................................ 7

29 Annex A (normative) Requirements for Mueller-Hinton broth ................................................................... 8

30 Annex B (normative) Solvents and diluents for making stock solutions of selected

31 antimicrobial agents ................................................................................................................................... 11

32 Annex C (normative) Preparation of working dilutions of antimicrobial agents for use in

33 broth dilution susceptibility tests .......................................................................................................... 16

34 Annex D (Informative) Special test situations ................................................................................................. 17

35 Bibliography ................................................................................................................................................................. 18

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37 Foreword

38 ISO (the International Organization for Standardization) is a worldwide federation of national standards

39 bodies (ISO member bodies). The work of preparing International Standards is normally carried out

40 through ISO technical committees. Each member body interested in a subject for which a technical

41 committee has been established has the right to be represented on that committee. International

42 organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO

43 collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

44 electrotechnical standardization.

45 The procedures used to develop this document and those intended for its further maintenance are

46 described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

47 different types of ISO documents should be noted. This document was drafted in accordance with the

48 editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

49 Attention is drawn to the possibility that some of the elements of this document may be the subject of

50 patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any

51 patent rights identified during the development of the document will be in the Introduction and/or on

52 the ISO list of patent declarations received (see www.iso.org/patents).

53 Any trade name used in this document is information given for the convenience of users and does not

54 constitute an endorsement.

55 For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and

56 expressions related to conformity assessment, as well as information about ISO's adherence to the World

57 Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL:

58 www.iso.org/iso/foreword.html.

59 This document was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in vitro

60 diagnostic test systems.

61 This second edition cancels and replaces the first edition (ISO 20776-1:2006), which has been technically

62 revised.
63 The main changes compared to the previous edition are as follows:
64 — Revised to a broth micro-dilution only performance document
65 — Removal of S,I, R breakpoint definitions and information
66 — Moved embedded Tables to Annexes
67 — Removed quality control range Table.

68 — Updated Table (now Annex B) on solvents and diluents for antimicrobial agents used globally.

69 — Updated information on special culture media and method performance for specific currently used

70 antimicrobial agents.
71 A list of all parts in the ISO 20776 series can be found on the ISO website.
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73 Introduction

74 In vitro antimicrobial susceptibility tests are performed on micro-organisms suspected of causing

75 disease, particularly if the organism is thought to belong to a species that may exhibit resistance to

76 frequently used antimicrobial agents. The tests are also important in resistance surveillance,

77 epidemiological studies of susceptibility and in comparisons of new and existing agents.

78 Dilution procedures are used to determine the minimum inhibitory concentrations (MICs) of

79 antimicrobial agents for antimicrobial susceptibility testing. MIC methods are used in resistance

80 surveillance, defining identifying wild type phenotypes, comparative testing of new agents, to establish

81 the susceptibility of organisms that give equivocal results in routine tests, for tests on organisms where

82 routine tests may be unreliable and when a quantitative result is required for clinical management. In

83 dilution tests, micro-organisms are tested for their ability to produce visible growth in broth (broth

84 dilution) containing serial dilutions of the antimicrobial agent or on a series of agar plates (agar dilution).

85 The lowest concentration of an antimicrobial agent (in mg/l) that, under defined in vitro conditions,

86 prevents the appearance of visible growth of a micro-organism within a defined period of time is known

87 as the MIC. The MIC is a guide for the clinician to the susceptibility of the organism to the antimicrobial

88 agent and aids treatment decisions. Careful control and standardizationis required for intra- and inter-

89 laboratory reproducibility of broth MIC tests. The MICs generally span two to three doubling dilutions

90 with a dominant central value.

91 Broth dilution is a technique in which containers holding identical volumes of broth with antimicrobial

92 agent solutions in incrementally (usually geometrically) increasing concentrations are inoculated with a

93 known number of micro-organisms.

94 Broth micro-dilution denotes the performance of the broth dilution test in micro-dilution trays.

95 The method described in this part of ISO 20776 is intended for the testing of pure cultures of aerobic

96 bacteria that are easily grown by overnight incubation on agar and grow well in standardised micro-

97 dilution trays containing standardised Mueller-Hinton broth (volume of ≤ 200 µl), which may need to be

98 modified depending on the antimicrobial agent being tested.

99 The broth micro-dilution method described in this part of ISO 20766 is essentially the same as those used

100 in many countries, and as the methods published by the Clinical and Laboratory Standards Institute

101 (CLSI)[1] and the European Committee on Antimicrobial Susceptibility Testing (EUCAST)[2]. These

102 methods are based on those described by Ericsson and Sherris[3].
103
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104 Susceptibility testing of infectious agents and evaluation of
105 performance of antimicrobial susceptibility test devices — Part 1:
106 Broth micro-dilution reference method for testing the in vitro
107 activity of antimicrobial agents against rapidly growing aerobic
108 bacteria involved in infectious diseases

109 WARNING — The use of this part of ISO 20776 may involve hazardous materials, operations and

110 equipment. This part of ISO 20776 does not purport to address all of the safety problems associated

111 with its use. It is the responsibility of the user of this part of ISO 20776 to establish appropriate safety

112 and health practices and determine the applicability of regulatory limitations prior to use.

113 1 Scope

114 This part of ISO 20776 describes one reference method, broth micro-dilution, for determination of MICs.

115 The MIC may be a guide for the clinician, and reflects the activity of the drug under the described test

116 conditions, by taking into account other factors, such as drug pharmacology, pharmacokinetics, or

117 bacterial resistance mechanisms. This allows categorisation of bacteria as “susceptible” (S),

118 “intermediate” (I), or “resistant” (R). In addition, MIC distributions can be used to define wild type or non-

119 wild type bacterial populations. Although clinical interpretation of the MIC value is beyond the scope of

120 this part of ISO 20776, modifications of the basic method are required for certain antimicrobial agent -

121 bacteria combinations to facilitate clinical interpretation. These modifications are included in a separate

122 Annex of this document. It is necessary to compare other susceptibility testing methods (e.g. disc diffusion

123 or diagnostic test devices) with this reference method for validation, in order to ensure comparable and

124 reliable results.
125 2 Normative references
126 There are no normative references in this document.
127 3 Terms and definitions

128 For the purposes of this document, the following terms and definitions apply.

129 ISO and IEC maintain terminological databases for use in standardization at the following addresses:

130 — IEC Electropedia: available at http://www.electropedia.org/
131 — ISO Online browsing platform: available at https://www.iso.org/obp
132 3.1
133 antimicrobial agent

134 substance of biological, semi-synthetic or synthetic origin that inhibits the growth of or kills bacteria, and

135 is thus of potential use in the treatment of infections

136 Note to entry: Disinfectants, antiseptics and preservatives are not included in this definition.

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137 3.2 Antimicrobial agents — properties
138 3.2.1
139 potency

140 potency is a measure of drug activity expressed in terms of the amount required to produce an effect of

141 given intensity.

142 Note to entry: The potency is expressed as mass fraction in milligrams per gram (mg/g), or as activity

143 content in International Units (IU) per gram, or as a volume fraction or mass fraction in percent, or as an

144 amount-of-substance concentration (molar fraction) in mole per litre of ingredients in the test substance.

145 3.2.2
146 concentration
147 amount of an antimicrobial agent in a defined volume of liquid
148 Note 1 to entry: The concentration is expressed as mg/l.
149 Note 2 to entry: mg/l is the designated ISO unit.
150 3.3
151 stock solution
152 initial solution used for further dilutions
153 3.4
154 minimum inhibitory concentration
155 MIC

156 lowest concentration that, under defined in vitro conditions, prevents visible growth of bacteria within a

157 defined period of time
158 Note to entry: The MIC is expressed in mg/l.
159 3.5
160 breakpoint
161 BP

162 specific values of parameters, such as MICs, on the basis of which bacteria can be assigned to the clinical

163 categories “susceptible”, “intermediate” and “resistant”

164 Note to entry: For current interpretive breakpoints, reference can be made to the latest publications of

165 organisations employing this reference method (e.g. CLSI and EUCAST).
166 3.6
167 wild type

168 absence of known resistance mechanisms to the antimicrobial agent for a given strain

169 3.7
170 reference strain

171 catalogued, characterized bacteria with stable, defined antimicrobial susceptibility phenotypes and/or

172 genotypes

173 Note to entry: Reference strains are kept as stock cultures, from which working cultures are derived.

174 They are obtainable from culture collections and used for quality control.
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175 3.8 Susceptibility testing method
176 3.8.1
177 broth dilution

178 technique in which containers are filled with appropriate volumes of an antimicrobial solution,

179 employing incrementally (usually two-fold) increasing concentrations of the antimicrobial agent and

180 appropriate volumes of broth with a defined inoculum
181 Note to entry: The aim of this method is the determination of the MIC.
182 3.8.2
183 micro-dilution

184 performance of broth dilution in micro-dilution trays with a final test volume of ≤ 200 µl per well

185 3.9
186 broth
187 fluid medium used for the in vitro growth of bacteria

188 Note to entry: For the broth reference method the medium is standardised Mueller-Hinton broth (see

189 Annex A)
190 3.10
191 inoculum

192 number of bacteria in a suspension, calculated with respect to the final volume

193 Note to entry: The inoculum is expressed as colony-forming units per millilitre (CFU/ml).

194 3.11
195 inoculum effect
196 change in MIC related to change in inoculum concentration
197 4 Test procedures
198 4.1 General

199 The tests are performed in polystyrene micro-dilution trays. The method is based on the preparation of

200 antimicrobial agent working solutions, either in 50 µl volumes per well (with the addition of an inoculum

201 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

202 inoculum volume).
203 4.2 Medium

204 Mueller-Hinton broth shall be used (see Annex A for details and Annex D for special test situations).

205 4.3 Antimicrobial agents
206 4.3.1 General

207 Antimicrobial agents shall be obtained directly from the manufacturer or from reliable commercial

208 sources; pharmaceutical preparations for clinical use are not acceptable. The antimicrobial agents shall

209 be supplied as powders with a lot number, potency, an expiry date and details of recommended storage

210 conditions. Substances shall be stored in tightly closed containers in the dark, with a desiccant at the

211 recommended temperature of the supplier. Hygroscopic agents should be dispensed into aliquots, one of

212 which is used on each test occasion.
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213 To avoid condensation, allow containers to warm to room temperature before opening.

214 4.3.2 Preparation of stock solutions

215 The use of a calibrated analytical balance is required to weigh antimicrobial agents. Allowance for the

216 potency of the powder shall be made by use of the following formula to obtain the amount of antimicrobial

217 agent substance or the volume of diluent needed for a standard solution:
V
218 (1)
mP
V 
219 (2)
220 where
221  is the concentration of the stock solution, in mg/l;
222 m is mass of the antimicrobial agent (powder), in g;
223 P is the potency of the antimicrobial agent (powder), in mg/g;
224 V is the volume of diluent, in l.

225 Concentrations of stock solutions should be 1000 mg/l or greater, although the solubility of some agents

226 is a limiting factor. The actual concentrations of stock solutions depend on the method of preparing

227 working solutions (serial dilutions). Agents should be dissolved and diluted in sterile distilled water

228 unless the manufacturer states otherwise. Some agents require alternative solvents (see Annex B).

229 Note: For newer antimicrobial agents not identified in Annex B of this current document, consult

230 manufacturer information on the most appropriate solvent and diluent for the specific agent. Sterilisation

231 of solutions is not usually necessary. If required, sterilisation should be done by membrane filtration, and

232 samples before and after sterilisation should be compared by assay to ensure that adsorption has not

233 occurred.

234 Unless information is available on stability of stock solutions under specified storage conditions, they

235 should be prepared fresh for each test batch.
236 4.3.3 Preparation of working solutions

237 The range of concentrations selected for testing depends on the micro-organisms and antimicrobial

238 agent. The chosen range shall allow full endpoint MIC determination for appropriate reference strains. A

239 two-fold dilution series based on 1 mg/l is prepared in Mueller-Hinton broth. Dilutions should not be

240 prepared by serial dilution steps, but according to the procedure outlined in Annex C. Working solutions

241 shall be used the same day unless information is available on stability of the solutions under specified

242 storage conditions.
243 4.3.4 Preparation of micro-dilution trays

244 Working solutions are dispensed into micro-dilution trays at 50 µl per well with double the desired final

245 concentrations of antimicrobial agent, or at 100 µl per well in the desired final concentrations.

246 At least one well, containing 50 µl or 100 µl of antimicrobial agent-free medium, should be included as a

247 growth control for each strain tested. Likewise, a well containing 100 µl of antimicrobial agent-free

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248 medium should be included as an un-inoculated negative control well for each micro-organism type

249 tested.
250 4.3.5 Storage of micro-dilution trays

251 Filled trays may be used immediately or may be stored for up to three months or until documented

252 quality control or other evidence indicates degradation of the antimicrobial agent. For storage the filled

253 trays should be sealed in plastic bags and immediately placed in a freezer at ≤  60 °C unless the

254 antimicrobial agents are known to be stable at higher temperatures.

255 Trays shall not be stored in a self-defrosting freezer, and thawed antimicrobial solutions shall not be re-

256 frozen, as repeated freeze-thaw cycles accelerate the degradation of some antimicrobial agents,

257 particularly -lactams.

258 Allow frozen plates to thaw for up to 2 hours and inoculate by 4 hours of removal from the freezer.

259 4.4 Preparation of inoculum
260 4.4.1 General

261 Standardization of the inoculum is essential for accurate and reproducible broth micro-dilution

262 susceptibility tests. Therefore, purity checks and viable colony counts shall be performed on every isolate

263 tested with this reference procedure
264 4.4.2 Broth culture method

265 The inoculum may be prepared by diluting a broth culture or by suspending several morphologically

266 similar colonies (when possible) from an overnight culture on non-selective agar medium in broth with

267 a sterile loop or a cotton swab. When suspending colonies, morphologically similar colonies should be

268 picked to avoid contamination of other species or atypical variants of the same species.

269 The broth used shall not be antagonistic to the antimicrobial agent tested. The broth is incubated at 35

270 ±1°C until the growth reaches a turbidity equal to or greater than that of a 0.5 McFarland standard[4]. If

271 needed, the culture is adjusted with saline or broth to give a turbidity equivalent to the 0.5 McFarland

272 standard. This can be done by means of a photometric device (using 625 nm wavelength and a 1 cm path

273 cuvette, the absorbance will be 0.08 – 0.13), or by employing a suitably calibrated nephelometer.

274 Alternatively, this can be achieved visually by comparing the appearance of black lines through the

275 inoculum and 0.5 McFarland standard suspensions (the inoculum and McFarland standard shall be in

276 tubes of the same size) or any other method that gives reproducible CFU/ml. The final inoculum shall be

5 5 5
277 5 x 10 CFU/ml (target range, 2 x 10 CFU/ml to 8 x 10 CFU/ml).

278 NOTE A 0.5 McFarland standard can be produced by adding a 0.5 ml aliquot of 0.048 mol/l barium

279 chloride (BaCl ) (11.72 g/l BaCl 2H O) to 99.5 ml of 0.18 mol/l sulphuric acid (H SO ), with constant

2 2 2 2 4
280 stirring to maintain a suspension.
281 4.4.3 Direct colony suspension method

282 Several morphologically similar colonies from a non-selective nutritive agar medium (incubated at 35

283 ±1°C for 18 h to 24 h, unless longer incubation is required) are touched with a sterile loop and the growth

284 transferred to sterile broth or saline. The suspension is adjusted to give a turbidity equivalent to that of

285 a 0.5 McFarland standard, as described in 4.4.2 for the broth culture method.

286 For all micro-organisms, the concentration of viable cells in the final inoculum depends on the growth

287 phase of the culture. This effect is most pronounced for fastidious organisms such as Streptococcus

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288 pneumoniae, where use of older cultures can significantly reduce the number of viable cells in the

289 suspension.

290 A correctly adjusted suspension prepared by either method contains approximately 1 - 2  10 CFU/ml

291 for the common relevant bacteria.

292 The adjusted inoculum prepared as above is diluted in broth to give a final cell number concentration of

5 5 5

293 5  10 CFU/ml (target range 2  10 CFU/ml to 8  10 CFU/ml). The dilution required depends upon the

294 bacterial species being tested and the method used for inoculum delivery. Transfer of 0,1 ml of

295 standardized micro-organism suspension to a tube containing 9.9 ml (1:100 dilution) of broth results in

296 a suspension of 1  10 CFU/ml which, when 50 µl is added to an equal volume (50 µl) of antimicrobial

297 agent solution, results in a final inoculum of 5  10 CFU/ml with many Gram-negative bacteria (e.g.

298 Escherichia coli). If the wells already contain 100 µl of antimicrobial agents in broth, an appropriate

299 dilution to give the required final inoculum should be prepared prior to addition of up to10 µl of the

300 diluted suspension to each well. Different dilutions of the 0.5 McFarland suspension may be necessary,

301 as determined by colony counts in preliminary tests[5]
302 4.5 Inoculation of micro-dilution trays

303 The trays shall be inoculated within 30 minutes of standardizing the inoculum suspension, in order to

304 maintain viable cell number concentration. To each well containing 50 µl of diluted antimicrobial agent

305 in broth (see 4.3), a volume of 50 µl of bacterial suspension (see 4.4) is added. For tray wells that

...

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