ISO/TS 16782:2016

TECHNICAL ISO/TS
SPECIFICATION 16782
First edition
2016-10-15
Clinical laboratory testing — Criteria
for acceptable lots of dehydrated
Mueller-Hinton agar and broth for
antimicrobial susceptibility testing
Détermination de la sensibilité aux antibiotiques — Critères
d’acceptabilité pour les lots d’agar déshydraté et de bouillon Mueller-
Hinton pour déterminer la sensibilité aux antibiotiques
Reference number
©
ISO 2016
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative reference . 1
3 Terms and definitions . 1
4 Requirements for Mueller-Hinton broth . 3
4.1 Components of Mueller-Hinton broth . 3
4.2 Physical and chemical characteristics . 3
4.2.1 Dehydrated powder or granules . 3
4.2.2 Prepared broth medium . 4
4.2.3 Cation supplementation and content for MHB . 4
4.2.4 Other medium components . . 4
4.2.5 Specific adjustments required by the manufacturer . 4
4.3 Manufacturers protocol for testing production lots of dehydrated Mueller-Hinton broth . 5
4.4 Interpreting the results . 5
4.5 Evaluating the results . 6
5 Requirements for Muller-Hinton agar . 6
5.1 Components of Mueller-Hinton agar . 6
5.2 Physical and chemical characteristics . 6
5.2.1 Dehydrated powder or granules . 6
5.2.2 Prepared agar medium . 7
5.2.3 Cation supplementation and content for MHA . 7
5.2.4 Other medium components . . 7
5.2.5 Specific adjustments required by the manufacturer . 7
5.3 Manufacturer’s protocol for testing production lots of dehydrated Mueller-Hinton agar . 8
5.4 Interpreting the results . 8
5.5 Evaluating the results .10
6 Testing new antimicrobial agents with production lots of dehydrated Mueller-
Hinton broth or agar .11
Annex A (informative) Mueller-Hinton medium .12
Annex B (informative) Preparing control cultures .14
Annex C (informative) Suggested data sheet for testing of production lots .16
Annex D (informative) Label statement .19
Bibliography .20
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing Technical Specifications 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 on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 212, Clinical laboratory testing and in vitro
diagnostic test systems.
iv © ISO 2016 – All rights reserved

Introduction
Historically, although various media have been recommended for susceptibility testing, Mueller-
Hinton broth (MHB) has been selected as the medium for the reference broth microdilution minimum
inhibitory concentration (MIC) method (ISO 20776-1) and Mueller-Hinton agar (MHA) is most widely
used for disc diffusion testing of rapidly growing bacteria.
Mueller-Hinton medium provides satisfactory growth of most non-fastidious pathogens, acceptable
batch-to-batch reproducibility, low sulfonamide, trimethoprim, and tetracycline inhibitors and a large
amount of data has been collected from antimicrobial susceptibility tests with this medium over several
decades.
This International Standard is the result of an effort to establish a standard description and protocol by
which manufacturers of dehydrated Mueller-Hinton agar (dMHA) and broth (dMHB) may determine its
acceptable performance characteristics.
The results of testing conform to defined quality control limit ranges for each combination of
antimicrobial agent and quality control strains. Each production lot is tested at least against these
combinations of antimicrobial agents and quality control strains.
This Technical Specification has been developed in part based upon two Clinical and Laboratory
[1]
Standards Institute (CLSI) documents, CLSI M6-A2 (protocols for evaluating dehydrated Mueller-
[2]
Hinton agar) and CLSI M32-P (evaluation of lots of dehydrated Mueller-Hinton broth for antimicrobial
[1]
susceptibility testing) with permission. Upon publication of ISO 16782, CLSI documents M6-A2 and
[2]
M32-P will no longer be available. Manufacturers can follow ISO 16782 to assess the performance
characteristics of their production lots of dMHA and dMHB.
TECHNICAL SPECIFICATION ISO/TS 16782:2016(E)
Clinical laboratory testing — Criteria for acceptable
lots of dehydrated Mueller-Hinton agar and broth for
antimicrobial susceptibility testing
1 Scope
This Technical Specification provides a standard description of the physical properties of dehydrated
Mueller-Hinton broth (dMHB) and Mueller-Hinton agar (dMHA) and performance criteria by which
manufacturers can assess the performance characteristics of their production lots of dMHA and dMHB.
Production lots of broth or agar can then be utilized by all users, including in vitro susceptibility testing
device manufacturers, as the test medium for performance of antimicrobial susceptibility testing.
This Technical Specification does not address supplements (e.g. blood or blood products) that are
[3][4][5][6]
added to the medium to support growth of fastidious bacteria . Those additives are provided
after the dehydrated medium is prepared in its liquid state as a final product and fall outside of the
scope of this Technical Specification. Although dMHA can be used for determination of MICs using the
[4][6]
agar dilution method or the gradient diffusion method, this Technical Specification only includes
performance testing of dMHA using disc diffusion methodology as described by the Clinical and
[5]
Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility
[3]
Testing (EUCAST) .
2 Normative reference
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 20776-1:2006, Clinical laboratory testing and in vitro diagnostic test systems — Susceptibility testing
of infectious agents and evaluation of performance of antimicrobial susceptibility test devices — Part 1:
Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic
bacteria involved in infectious diseases
CLSI M100, Performance Standards for Antimicrobial Susceptibility Testing; Informational Supplement
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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.
[SOURCE: ISO 20776-1:2006, 2.1]
3.2
antimicrobial disc
small paper disc containing known amounts of antimicrobial agents used for in vitro susceptibility testing
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 = µg/ml but it is not recommended to use the unit µg/ml.
[SOURCE: ISO 20776-1:2006, 2.2.2]
3.4
stock solution
initial solution used for further dilutions
[SOURCE: ISO 20776-1:2006, 2.3]
3.5
minimum inhibitory concentration
MIC
lowest concentration of antimicrobial agent 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.
[SOURCE: ISO 20776-1:2006, 2.4, modified — “lowest concentration that” has been modified to “lowest
concentration of antimicrobial agent that”]
3.6
reference strain
catalogued, characterized microorganism with stable, defined antimicrobial susceptibility phenotype
and/or genotype
Note 1 to
...