Agar Dilution Assay for Antimicrobial Susceptibility Testing

Agar Dilution Assay for Antimicrobial Susceptibility Testing

Agar dilution assay is a critical method for determining the antimicrobial susceptibility of bacteria that grow aerobically. This standard provides detailed procedures for broth macrodilution and microdilution techniques, ensuring accurate measurement of minimal inhibitory concentrations (MICs). Authored by experts in microbiology and infectious diseases, this eighth edition serves as a comprehensive guide for clinical laboratories and researchers. It includes quality control protocols, selection of antimicrobial agents, and specific methodologies for various bacterial species. Ideal for microbiologists and laboratory technicians, this document enhances the reliability of susceptibility testing in clinical settings.

Key Points

  • Describes standardized agar dilution methods for antimicrobial susceptibility testing.
  • Includes procedures for broth macrodilution and microdilution techniques.
  • Outlines quality control measures and responsibilities for accurate testing.
  • Provides guidelines for selecting appropriate antimicrobial agents for testing.
  • newtopiccyclegrowin
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January 2009
M07-A8
Vol. 29 No. 2
Replaces M07-A7
Vol. 26 No. 2
Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria That Grow
Aerobically; Approved Standard—Eighth
Edition
This document addresses reference methods for the determination of minimal inhibitory
concentrations (MICs) of aerobic bacteria by broth macrodilution, broth microdilution,
and agar dilution.
A standard for global application developed through the Clinical and Laboratory
Standards Institute consensus process.
M07-A8
ISBN 1-56238-689-1
Volume 29 Number 2 ISSN 0273-3099
Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
That Grow Aerobically; Approved Standard—Eighth Edition
Matthew A. Wikler, MD, MBA, FIDSA
Franklin R. Cockerill, III, MD
Karen Bush, PhD
Michael N. Dudley, PharmD, FIDSA
George M. Eliopoulos, MD
Dwight J. Hardy, PhD
David W. Hecht, MD
Mary Jane Ferraro, PhD, MPH
Jana M. Swenson, MMSc
Janet F. Hindler, MCLS, MT(ASCP)
Jean B. Patel, PhD, D(ABMM)
Mair Powell, MD, FRCP, FRCPath
John D. Turnidge, MD
Melvin P. Weinstein, MD
Barbara L. Zimmer, PhD
Abstract
Susceptibility testing is indicated for any organism that contributes to an infectious process warranting antimicrobial chemotherapy, if its
susceptibility cannot be reliably predicted from knowledge of the organism’s identity. Susceptibility tests are most often indicated when
the causative organism is thought to belong to a species capable of exhibiting resistance to commonly used antimicrobial agents.
A variety of laboratory methods can be used to measure the in vitro susceptibility of bacteria to antimicrobial agents. This document
describes standard broth dilution (macrodilution and microdilution [the microdilution method described in M07 is the same methodology
outlined in ISO 20776-1])
1
and agar dilution techniques, and it includes a series of procedures to standardize the way the tests are
performed. The performance, applications, and limitations of the current CLSI-recommended methods are also described.
The supplemental information (M100 tables) presented with this standard represents the most current information for drug selection,
interpretation, and quality control using the procedures standardized in M07. These tables, as in previous years, have been updated and
should replace tables published in earlier years. Changes in the tables since the previous edition (M100-S18) appear in boldface type and
are also summarized in the front of the document.
Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;
Approved Standard—Eighth Edition. CLSI document M07-A 8 (ISBN 1-56238-689-1). Clinical and Laboratory Standards Institute, 940
West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2009.
The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through
two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any
given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or
guideline, users should replace outdated editions with the current editions of CLSI/NCCLS documents. Current editions are
listed in the CLSI catalog and posted on our website at www.clsi.org. If your organization is not a member and would like to
become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax: 610.688.0700; E-Mail:
customerservice@clsi.org; Website: www.clsi.org
Volume 29
M07-A8
vii
Contents
Abstract .................................................................................................................................................... i
Committee Membership ........................................................................................................................ iii
Foreword ................................................................................................................................................. x
Summary of Major Changes in This Document ..................................................................................... x
Summary of CLSI Processes for Establishing Interpretive Criteria and QC Ranges ......................... xiii
CLSI Reference Methods vs Commercial Methods and CLSI vs FDA Breakpoints
(interpretive criteria) ............................................................................................................................ xiv
Subcommittee on Antimicrobial Susceptibility Testing Mission Statement ........................................ xv
1 Scope .......................................................................................................................................... 1
2 Introduction ................................................................................................................................ 1
3 Standard Precautions .................................................................................................................. 2
4 Terminology ............................................................................................................................... 2
4.1 Definitions .................................................................................................................... 2
4.2 Abbreviations/Acronyms .............................................................................................. 3
5 Indications for Performing Susceptibility Tests......................................................................... 4
6 Selection of Antimicrobial Agents for Routine Testing and Reporting ..................................... 5
6.1 Routine Reports ............................................................................................................ 5
6.2 Nonproprietary Names .................................................................................................. 5
6.3 Selection Guidelines ..................................................................................................... 8
6.4 Suggested Guidelines for Routine and Selective Testing and Reporting ..................... 8
7 Antimicrobial Agents ................................................................................................................. 9
7.1 Source ........................................................................................................................... 9
7.2 Weighing Antimicrobial Powders ................................................................................. 9
7.3 Preparing Stock Solutions ........................................................................................... 10
7.4 Number of Concentrations Tested .............................................................................. 11
8 Inoculum Preparation for Dilution Tests ................................................................................. 11
8.1 Turbidity Standard for Inoculum Preparation ............................................................. 11
8.2 Direct Colony Suspension Method ............................................................................. 11
8.3 Growth Method ........................................................................................................... 11
9 Agar Dilution Procedure .......................................................................................................... 12
9.1 Reagents and Materials ............................................................................................... 12
9.2 Preparing Agar Dilution Plates ................................................................................... 13
9.3 Preparing the Inoculum ............................................................................................... 14
9.4 Inoculating Agar Dilution Plates ................................................................................ 14
9.5 Incubating Agar Dilution Plates.................................................................................. 15
9.6 Determining Agar Dilution End Points ....................................................................... 15
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End of Document
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FAQs of Agar Dilution Assay for Antimicrobial Susceptibility Testing

What is the purpose of the agar dilution assay?
The agar dilution assay is designed to determine the minimal inhibitory concentration (MIC) of antimicrobial agents against bacteria that grow aerobically. By preparing a series of agar plates with varying concentrations of an antimicrobial agent, microbiologists can assess the effectiveness of the drug in inhibiting bacterial growth. This method is crucial for identifying resistant strains and guiding appropriate treatment options in clinical settings.
What are the key steps in performing an agar dilution assay?
Key steps in performing an agar dilution assay include preparing agar plates with different concentrations of the antimicrobial agent, inoculating the plates with a standardized bacterial suspension, and incubating them under controlled conditions. After incubation, the plates are examined for bacterial growth, and the MIC is determined by identifying the lowest concentration that prevents visible growth. Proper technique and adherence to quality control measures are essential for reliable results.
How does the agar dilution assay compare to other susceptibility testing methods?
The agar dilution assay is one of several methods used for antimicrobial susceptibility testing, alongside broth microdilution and disk diffusion techniques. Unlike disk diffusion, which provides qualitative results, the agar dilution assay offers quantitative data on MIC values. This method is particularly useful for fastidious organisms that may not grow well in broth culture. Each method has its advantages and limitations, making it important for laboratories to choose the appropriate technique based on the specific bacterial species and clinical context.
What quality control measures are recommended for the agar dilution assay?
Quality control measures for the agar dilution assay include using standardized bacterial strains with known susceptibility profiles to validate the testing process. Laboratories must regularly check the performance of their antimicrobial agents and agar media to ensure accurate results. Additionally, maintaining consistent inoculum preparation and incubation conditions is crucial for minimizing variability in test outcomes. These quality control protocols help ensure the reliability and reproducibility of susceptibility testing.
What types of bacteria can be tested using the agar dilution assay?
The agar dilution assay is suitable for testing a wide range of aerobic bacteria, including common pathogens such as Staphylococcus aureus, Escherichia coli, and Streptococcus pneumoniae. It can also be adapted for fastidious organisms that require specific growth conditions. The method is particularly valuable in clinical microbiology for identifying resistant strains and guiding appropriate antimicrobial therapy, making it an essential tool in infectious disease management.

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