Methods For Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals Approved Guideline

Product Name: Infobase 2009 - Release Date: March 2009
M49-A
Vol. 26 No. 24
Replaces M49-P
Vol. 25 No. 22
Methods for Broth Dilution Susceptibility

Testing of Bacteria Isolated From Aquatic
Animals; Approved Guideline
This document provides the most up-to-date techniques for the determination of minimal
inhibitory concentrations (MICs) of aquatic bacteria by broth micro- and macrodilution,
and criteria for quality control testing.
A guideline for global application developed through the Clinical and Laboratory
Standards Institute consensus process.
(Formerly NCCLS)
This document is protected by copyright. Downloaded on 2/23/2009

Clinical and Laboratory Standards Institute

Advancing Quality in Healthcare Testing
The Clinical and Laboratory Standards Institute (CLSI, Most documents are subject to two levels of consensus—
formerly NCCLS) is an international, interdisciplinary, “proposed” and “approved.” Depending on the need for
nonprofit, standards-developing, and educational field evaluation or data collection, documents may also be
organization that promotes the development and use of made available for review at an intermediate consensus
voluntary consensus standards and guidelines within the level.
healthcare community. It is recognized worldwide for the
Proposed A consensus document undergoes the first stage
application of its unique consensus process in the
of review by the healthcare community as a proposed
development of standards and guidelines for patient
standard or guideline. The document should receive a wide
testing and related healthcare issues. Our process is
and thorough technical review, including an overall review
based on the principle that consensus is an effective and
of its scope, approach, and utility, and a line-by-line review
cost-effective way to improve patient testing and
of its technical and editorial content.
healthcare services.
Approved An approved standard or guideline has achieved
In addition to developing and promoting the use of
consensus within the healthcare community. It should be
voluntary consensus standards and guidelines, we
reviewed to assess the utility of the final document, to
provide an open and unbiased forum to address critical
ensure attainment of consensus (i.e., that comments on
issues affecting the quality of patient testing and health
earlier versions have been satisfactorily addressed), and to
care.
identify the need for additional consensus documents.
PUBLICATIONS
Our standards and guidelines represent a consensus opinion
A document is published as a standard, guideline, or on good practices and reflect the substantial agreement by
committee report. materially affected, competent, and interested parties
obtained by following CLSI’s established consensus
Standard A document developed through the consensus
procedures. Provisions in CLSI standards and guidelines
process that clearly identifies specific, essential
may be more or less stringent than applicable regulations.
requirements for materials, methods, or practices for use
Consequently, conformance to this voluntary consensus
in an unmodified form. A standard may, in addition,
document does not relieve the user of responsibility for
contain discretionary elements, which are clearly
compliance with applicable regulations.
identified.
COMMENTS
Guideline A document developed through the consensus
process describing criteria for a general operating The comments of users are essential to the consensus
practice, procedure, or material for voluntary use. A process. Anyone may submit a comment, and all comments
guideline may be used as written or modified by the user are addressed, according to the consensus process, by the
to fit specific needs. committee that wrote the document. All comments,
including those that result in a change to the document when
Report A document that has not been subjected to
published at the next consensus level and those that do not
consensus review and is released by the Board of
result in a change, are responded to by the committee in an
Directors.
appendix to the document. Readers are strongly encouraged
CONSENSUS PROCESS to comment in any form and at any time on any document.
Address comments to Clinical and Laboratory Standards
The CLSI voluntary consensus process is a protocol Institute, 940 West Valley Road, Suite 1400, Wayne, PA
establishing formal criteria for: 19087, USA.
• the authorization of a project VOLUNTEER PARTICIPATION
• the development and open review of documents Healthcare professionals in all specialties are urged to
• the revision of documents in response to comments volunteer for participation in CLSI projects. Please contact
by users us at customerservice@clsi.org or +610.688.0100 for
additional information on committee participation.
• the acceptance of a document as a consensus
standard or guideline.

M49-A
ISBN 1-56238-612-3
Volume 26 Number 24 ISSN 0273-3099
Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated
From Aquatic Animals; Approved Guideline
John P. Hawke, PhD Ron A. Miller, MS
Renate Reimschuessel, PhD, VMD Gilles Olivier, PhD
Takashi Aoki, PhD Thomas R. Shryock, PhD
Thomas A. Bell, PhD Peter R. Smith, PhD
Guillaume Blanc, PhD Clyde Thornsberry, PhD
Jeremy Carson, PhD Robert D. Walker, PhD
Beverly Dixon, PhD Jeffrey L. Watts, PhD, RM(AAM)
Ching Ching Wu, DVM, PhD
Abstract
Antimicrobial susceptibility testing is recommended to determine which antimicrobial agents should be considered for treating a
bacterial pathogen. Many bacteria that cause disease in aquatic animals require growth conditions that vary substantially from
routine terrestrial bacterial pathogens. It has thus become desirable to develop antimicrobial susceptibility testing standards for
organisms that prefer or require conditions, such as lower temperatures, semisolid media, or supplemented media (e.g., NaCl,
serum).
Clinical and Laboratory Standards Institute (CLSI) document M49-A—Methods for Broth Dilution Susceptibility Testing of
Bacteria Isolated From Aquatic Animals; Approved Guideline describes a standardized broth dilution method and quality control
criteria for testing Group 1 aquatic bacteria. These organisms grow readily in cation-adjusted Mueller-Hinton broth (CAMHB),
and are readily cultured at temperatures of 22 ± 2 °C and 28 ± 2 °C. Quality control ranges for Escherichia coli ATCC® 25922
and Aeromonas salmonicida subsp. salmonicida ATCC® 33658 when tested at 22 °C, 28 °C, and 35 ± 2 °C (E. coli only) are
listed for ten different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin,
ormetoprim-sulfadimethoxine, oxolinic acid, oxytetracycline, and trimethoprim-sulfamethoxazole).
Future editions of this document will incorporate additional data, as they become available. Still needed are methods for testing
other groups of aquatic bacterial pathogens, such as the gliding bacteria, obligate halophiles, and gram-positive cocci. In addition,
interpretive criteria will also need to be developed, which requires a correlation between pharmacokinetic/pharmacodynamic
properties of the drug, in vitro susceptibility data, and clinical outcomes.
Clinical and Laboratory Standards Institute (CLSI). Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From
Aquatic Animals; Approved Guideline. CLSI document M49-A (ISBN 1-56238-612-3). Clinical and Laboratory Standards
Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006.
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 healthcare 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, which is distributed to member
organizations, and to nonmembers on request. 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
(Formerly NCCLS)

Number 24 M49-A
This publication is protected by copyright. No part of it may be reproduced, stored in a retrieval system,
transmitted, or made available in any form or by any means (electronic, mechanical, photocopying,
recording, or otherwise) without prior written permission from Clinical and Laboratory Standards
Institute, except as stated below.
Clinical and Laboratory Standards Institute hereby grants permission to reproduce limited portions of this
publication for use in laboratory procedure manuals at a single site, for interlibrary loan, or for use in
educational programs provided that multiple copies of such reproduction shall include the following
notice, be distributed without charge, and, in no event, contain more than 20% of the document’s text.
Reproduced with permission, from CLSI publication M49-A—Methods for Broth

Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals; Approved
Guideline (ISBN 1-56238-612-3). Copies of the current edition may be obtained from
Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,
Pennsylvania 19087-1898, USA.
Permission to reproduce or otherwise use the text of this document to an extent that exceeds the
exemptions granted here or under the Copyright Law must be obtained from Clinical and Laboratory
Standards Institute by written request. To request such permission, address inquiries to the Executive Vice
President, Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,
Pennsylvania 19087-1898, USA.
Copyright ©2006. Clinical and Laboratory Standards Institute.
Suggested Citation
(Clinical and Laboratory Standards Institute. Methods for Broth Dilution Susceptibility Testing of
Bacteria Isolated From Aquatic Animals; Approved Guideline. CLSI document M49-A [ISBN 1-56238-
612-3]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,
Pennsylvania 19087-1898 USA, 2006.)
Proposed Guideline
August 2005
Approved Guideline
June 2006
ISBN 1-56238-612-3
ISSN 0273-3099
ii

Volume 26 M49-A
Committee Membership
Area Committee on Microbiology
Mary Jane Ferraro, PhD, MPH Jana M. Swenson, MMSc Robert P. Rennie, PhD
Chairholder Centers for Disease Control and University of Alberta Hospital
Massachusetts General Hospital Prevention Edmonton, Alberta, Canada
Boston, Massachusetts Atlanta, Georgia
John H. Rex, MD, FACP
James H. Jorgensen, PhD Michael L. Wilson, MD AstraZeneca
Vice-Chairholder Denver Health Medical Center Cheshire, United Kingdom
University of Texas Health Denver, Colorado
Science Center Melvin P. Weinstein, MD
San Antonio, Texas Advisors Robert Wood Johnson Medical
School
Donald R. Callihan, PhD Ellen Jo Baron, PhD New Brunswick, New Jersey
BD Diagnostic Systems Stanford Univ. Hospital & Medical
Sparks, Maryland School Matthew A. Wikler, MD, MBA,
Stanford, California FIDSA
Freddie Mae Poole Mpex Pharmaceuticals, Inc.
FDA Center for Devices and Lynne S. Garcia, MS San Diego, California
Radiological Health LSG & Associates
Rockville, Maryland Santa Monica, California Gail L. Woods, MD
University of Arkansas for Medical
David L. Sewell, PhD Richard L. Hodinka, PhD Sciences
Veterans Affairs Medical Center Children’s Hospital of Philadelphia Little Rock, Arkansas
Portland, Oregon Philadelphia, Pennsylvania
Thomas R. Shryock, PhD Michael A. Pfaller, MD

Elanco Animal Health University of Iowa College of
Greenfield, Indiana Medicine
Iowa City, Iowa
Subcommittee on Veterinary Antimicrobial Susceptibility Testing
Thomas R. Shryock, PhD Henry Heine, PhD Melanie R. Berson, DVM

Chairholder USAMRIID FDA Center for Veterinary
Elanco Animal Health Ft. Detrick, Maryland Medicine
Greenfield, Indiana Rockville, Maryland
Mark G. Papich, DVM, MS
Jeffrey L. Watts, PhD, North Carolina State University Carole A. Bolin, PhD
RM(AAM) Raleigh, North Carolina Michigan State University
Vice-Chairholder East Lansing, Michigan
Pfizer Animal Health Peter Silley, PhD
Kalamazoo, Michigan MB Consult Limited Diane M. Citron, M(ASCP)
Lymington, Hampshire, Santa Monica-UCLA Medical
Mike Apley, DVM, PhD United Kingdom Center
Kansas State University Santa Monica, California
Manhattan, Kansas Ching Ching Wu, DVM, PhD
Purdue University School of Rob P. Hunter, MS, PhD
Donald J. Bade Veterinary Medicine Elanco Animal Health
Microbial Research, Inc. West Lafayette, Indiana Greenfield, Indiana
Fort Collins, Colorado
Gary E. Zurenko Ronald N. Jones, MD
Steven D. Brown, PhD Micromyx, LLC JMI Laboratories
The Clinical Microbiology Institute Kalamazoo, Michigan North Liberty, Iowa
Wilsonville, Oregon
Advisors Carol L. Lemons
Jeffrey T. Gray, PhD North Carolina College of
University of Guelph Jo Abraham, DVM, MS Veterinary Medicine
Guelph, Ontario, Canada Bayer Health Care LLC Raleigh, North Carolina
Shawnee Mission, Kansas
iii

Number 24 M49-A
Advisors (Continued) Marilyn N. Martinez, PhD Clyde Thornsberry, PhD

FDA Center for Veterinary Focus Bio-Inova, Inc
Cindy Lindeman Medicine Franklin, Tennessee
Pfizer Animal Health Rockville, Maryland
Richland, Michigan John D. Turnidge, MD
Patrick McDermott, PhD Women’s and Children’s Hospital
Jennifer Lorbach FDA Center for Veterinary North Adelaide, Australia
Trek Diagnostic Systems Medicine
Cleveland, Ohio Laurel, Maryland Robert D. Walker, PhD
FDA Center for Veterinary
Carol W. Maddox, PhD Stefan Schwarz, PhD Medicine
University of Illinois Institut Für Tierzucht Laurel, Maryland
Urbana, Illinois Neustadt-Mariensee, Germany
S. Steve Yan, PhD
FDA Center for Veterinary
Medicine
Rockville, Maryland
Working Group on Aquaculture

Renate Reimschuessel, PhD, Jeremy Carson, PhD Robert D. Walker, PhD
VMD Department of Primary Industries, FDA Center for Veterinary
Co-Chairholder Water and Environment Medicine
FDA Center for Veterinary Tasmania, Australia Laurel, Maryland
Medicine
Laurel, Maryland Beverly A. Dixon, PhD Jeffrey L. Watts, PhD, RM(AAM)
California State University Pfizer Animal Health
John P. Hawke, PhD Hayward, California Kalamazoo, Michigan
Co-Chairholder
Louisiana State University Ron A. Miller, MS Ching Ching Wu, DVM, PhD
Baton Rouge, Louisiana FDA Center for Veterinary Purdue University
Medicine West Lafayette, Indiana
Takashi Aoki, PhD Laurel, Maryland
Tokyo University of Fisheries Staff
Tokyo, Japan Gilles Olivier, PhD
Gulf Fisheries Centre Clinical and Laboratory Standards
Thomas A. Bell, PhD Moncton, New Brunswick, Canada Institute
U.S. Fish & Wildlife Service Wayne, Pennsylvania
Arlington, Virginia Peter R. Smith, PhD
National University of Ireland
John J. Zlockie, MBA
Guillaume Blanc, PhD Galway, Ireland
Vice President, Standards
Ecole Nationale Vétérinaire de
Nantes Clyde Thornsberry, PhD
Tracy A. Dooley, BS, MLT(ASCP)
Nantes, France Focus Technologies, Inc.
Staff Liaison
Franklin, Tennessee
Donna M. Wilhelm
Editor
Melissa A. Lewis
Assistant Editor
iv

Volume 26 M49-A
Contents
Abstract ....................................................................................................................................................i
Committee Membership........................................................................................................................ iii
Foreword .............................................................................................................................................. vii
1 Scope..........................................................................................................................................1
2 Introduction................................................................................................................................1
3 Definitions .................................................................................................................................2
4 Antimicrobial Agents.................................................................................................................4
4.1 Source ...........................................................................................................................4
4.2 Weighing Antimicrobial Powders.................................................................................4
4.3 Preparing Stock Solutions.............................................................................................5
4.4 Number of Concentrations Tested ................................................................................6
5 Selection of Antimicrobial Agents for Routine Testing and Reporting.....................................6
5.1 Routine Reports ............................................................................................................6
5.2 Antimicrobial Classes ...................................................................................................6
5.3 Suggested Guidelines for Use and Selective Testing and Reporting............................8
6 Indications for Performing Susceptibility Testing .....................................................................8
7 Inoculum Preparation for Dilution Tests ...................................................................................9

7.1 Turbidity Standard for Inoculum Preparation...............................................................9
7.2 Direct Colony Suspension Method ...............................................................................9
8 Broth Dilution Procedures (Macrodilution and Microdilution) ...............................................10
8.1 Cation-Adjusted Mueller-Hinton Broth (CAMHB) Medium .....................................10
8.2 Preparing and Storing Diluted Antimicrobial Agents.................................................12
8.3 Broth Dilution Testing ................................................................................................13
9 Fastidious and Problem Organisms .........................................................................................14
9.1 Vibrionaceae and Photobacteriaceae (Obligate Halophilic Strains) (Group 2) ..........14
9.2 Gliding Bacteria (Group 3) .........................................................................................15
9.3 Streptococci (Group 4)................................................................................................16
9.4 Other Fastidious Organisms (Group 5).......................................................................16
10 Reporting of MIC Results........................................................................................................18
11 Quality Control Procedures......................................................................................................18

11.1 Purpose .......................................................................................................................18
11.2 Quality Control Responsibilities.................................................................................19
11.3 Reference Strains for Quality Control ........................................................................19
11.4 Storing and Testing Quality Control Strains...............................................................21
11.5 Control of Media and Materials..................................................................................21
11.6 Broth Dilution Quality Control Ranges ......................................................................21
11.7 Frequency of Quality Control Testing ........................................................................22
11.8 Corrective Action........................................................................................................22
v

Number 24 M49-A
Contents (Continued)
11.9 Other Control Procedures ...........................................................................................24
12 Limitations of Broth Dilution Test Methods............................................................................24
12.1 Application to Various Organism Groups ..................................................................24
References.............................................................................................................................................25
Appendix A. Antimicrobial Agents Used in Global Aquaculture and Status of Quality Control for
Broth Dilution Susceptibility Testing ...................................................................................................28
Appendix B. Aerobic Dilution Daily Quality Control Testing Protocol...............................................29
Appendix C. Aerobic Dilution Weekly Quality Control Testing Protocol ...........................................30
Table 1. Standard Methods for Broth Dilution Susceptibility Testing of Aquatic Bacterial
Pathogens ..............................................................................................................................................31
Table 2. Potential Modifications for Broth Dilution Susceptibility Testing of Aquatic Bacterial
Pathogens ..............................................................................................................................................32
Table 3. Frequently Isolated Bacterial Pathogens of Fish.....................................................................33
Table 4. Solvents and Diluents for Preparation of Stock Solutions of Antimicrobial Agents ..............34
Table 5. Scheme for Preparing Dilutions of Antimicrobial Agents to Be Used in Broth Dilution
Susceptibility Tests ...............................................................................................................................37
Table 6. Acceptable Quality Control Ranges of MICs (µg/mL) for Reference Strains When
Tested in Cation-Adjusted Mueller-Hinton Broth at 22 ± 2 °C After 24 to 28 Hours..........................38
Table 9. Acceptable Quality Control Ranges of MICs (µg/mL) for Reference Strains in
Cation-Adjusted Mueller-Hinton Broth (Except Where Noted) When Tested at 35 ± 2 °C After
16 to 20 Hours.......................................................................................................................................41
Summary of Comments and Subcommittee Responses........................................................................42
The Quality System Approach..............................................................................................................44
Related CLSI/NCCLS Publications ......................................................................................................45
vi

Volume 26 M49-A
Foreword
This CLSI guideline represents the collective efforts of the Subcommittee on Veterinary Antimicrobial
Susceptibility Testing - Aquaculture Working Group (VAST-AWG) to produce a guidance document
describing a standardized broth dilution susceptibility testing method for bacteria isolated from aquatic
species. The working group has relied heavily on the initial efforts of those who organized the Workshop
on MIC Methodologies in Aquaculture, Weymouth, 1998, and the subsequent publication of Alderman
and Smith’s draft protocols developed at the workshop.1 These documents outlined the problems
encountered when comparing data created by laboratories that were using different methods, since those
data usually varied greatly from laboratory to laboratory. The methods published by Alderman and Smith
were termed “tentative” by the authors to indicate there were a number of “unresolved issues.”
Members of the current VAST-AWG have expanded the work of the European group by targeting some
of these unresolved issues, such as the development of quality control ranges for quality control strains.
We have limited this guideline to the broth dilution susceptibility testing of Group 1 aquatic organisms.
This guideline contains the current best thinking of scientists in the field and their recommendations for
conducting a particular test. We have not addressed the issues of interpretive criteria. It is hoped that this
guideline and CLSI document M42—Methods for Antimicrobial Disk Susceptibility Testing of Bacteria
Isolated From Aquatic Animals for agar disk diffusion testing will evolve and include additional
standardized susceptibility testing methods and interpretive criteria for antimicrobial agents used to treat
bacterial infections in aquatic species.
Since we are attempting to harmonize standards as an international effort, we have chosen to include
agents that are used in some nations, but may not be used in other nations. In addition, concerns have
been raised about changes in susceptibility of bacteria exposed to antimicrobials in the environment, from
either human or veterinary use. It is therefore important to have standardized methods for testing drugs
used in other medical disciplines against bacteria isolated from the aquatic environment. Finally, if more
antimicrobials are approved for use in aquaculture, especially those already in use in other areas of
agriculture, standards will already be in place.
We have chosen to characterize two quality control strains based on their susceptibility profiles and
global availability. Aeromonas salmonicida subsp. salmonicida (ATCC®a 33658; NCIMBb 1102) and
Escherichia coli (ATCC® 25922; NCIMB 12210) are both susceptible to a wide range of antimicrobials,
grow well at low temperatures, and have proven to be stable after numerous passes in the testing medium.
It is proposed that both of these organisms be used as quality control organisms for broth dilution
susceptibility testing. With respect to Aeromonas salmonicida subsp. salmonicida, there is a ban on
importation of this organism in several nations and thus, E. coli may be used in its place.
We have optimized testing conditions primarily for the Group 1 (see Table 1) organisms and hope that
this guideline will engender future studies with other groups of bacteria. Such organisms include the
obligate halophiles, gliding bacteria, and gram-positive cocci.
The global aquaculture industry is comprised of many fish species, which have substantially different
bacterial flora and grow at different temperature optimums. Thus, quality control ranges have been
established at three different temperatures, 22 ± 2 °C, 28 ± 2 °C, and 35 ± 2 °C (E. coli only) (see Tables
6 through 9). These temperatures were chosen based on temperatures most frequently used for testing,
recommendations of the VAST-AWG, and also to coordinate our efforts with researchers from other
countries. In the case of zoonotic pathogens from aquatic sources or tropical fish species, clinicians may
request susceptibility data conducted at 35 ± 2 °C. In those cases, refer to Table 9 or CLSI/NCCLS
document M31—Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for
a
ATCC is a registered trademark of the American Type Culture Collection.
b
National Collection of Industrial and Marine Bacteria (NCIMB, www.ukncc.co.uk)
vii

Number 24 M49-A
Bacteria Isolated From Animals, for the appropriate QC organisms, ranges, and interpretive criteria.
Since this is a collective effort, recognition must go to the collaborating laboratories who conducted the
broth microdilution standardization study. In addition, we appreciate the assistance of the Subcommittee
on Veterinary Antimicrobial Susceptibility Testing, especially Dr. Thomas Shryock for his help with the
CLSI consensus process, and to Dr. Robert Walker for his guidance on developing control strains and
review of the document. We also thank the present and former members of the Aquaculture Working
Group and the organizers and participants of the Workshop on MIC Methodologies in Aquaculture,
Weymouth, 1998, who began this process. Special thanks must be given to Ron Miller from the Oak
Ridge Associated Universities, whose work has provided the data for the long-awaited criteria for quality
control testing. Finally, we must acknowledge the U.S. FDA and Oak Ridge Associated Universities for
providing support for this effort.
Renate Reimschuessel, PhD, VMD, Co-Chairholder

John P. Hawke, PhD, Co-Chairholder
Aquaculture Working Group
It is important for users of M49-A to recognize that commercial susceptibility testing devices are
not addressed in this guideline. The methods described herein are generic reference procedures
that can be used for routine susceptibility testing by clinical laboratories, or that can be used by
clinical laboratories to evaluate commercial devices for possible routine use. Some laboratories
could find that a commercial dilution, antibiotic gradient, colorimetric, turbidimetric,
fluorometric, or other method is suitable for selective or routine use.
Key Words
Antimicrobial susceptibility, aquaculture, aquatic, broth microdilution, macrodilution, minimal inhibitory

concentration (MIC)
viii

Volume 26 M49-A
Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From

Aquatic Animals; Approved Guideline
1 Scope
This guideline provides veterinary and aquatic animal disease diagnostic laboratories with currently
recommended antimicrobial broth dilution susceptibility testing methods for bacteria isolated from
aquatic animals, primarily Group 1 organisms (see Table 1), including criteria for quality control testing
with two quality control strains.
The document also provides tables outlining antimicrobial agents used in global aquaculture, methods for
preparing stock solutions and dilutions of antimicrobial agents, and a list of bacteria pathogenic to fish.
We have not addressed interpretive criteria in this guideline. Such criteria must be established using
pharmacokinetic and pharmacodynamic data, in vitro susceptibility testing data, and clinical efficacy data.
Developing interpretive criteria was beyond the scope of this document. As more aquatic animal-specific
information becomes available, this document, and CLSI document M42—Methods for Antimicrobial
Disk Susceptibility Testing of Bacteria Isolated From Aquatic Animals, will be revised to incorporate
those data.
2 Introduction
One of the great challenges of fish farming (aquaculture) is the control of disease outbreaks. Throughout
history, various compounds have been used to treat fish maladies, including salt, asphalt, and brandy.2
During the last century, major advances were made in isolation and identification of microorganisms
causing disease in aquatic animals.3-7 Concurrent with advances in diagnostic techniques, came advances
in production of antimicrobial substances.8 There has been quite an “evolution” of legislation and drug
approvals for aquaculture in many nations during the last 50 years. Using the United States as an
example, the first citations on the use of antimicrobials in fish in the U.S. described the use of sulfa drugs
to treat furunculosis in trout.9,10 The early 1950s, with the inclusion of a Veterinary Medical Branch in the
FDA, began the era of governmental regulation of veterinary drugs. To date, only four antimicrobial
agents are approved by the FDA, three of which are available for use in aquaculture fish species:
florfenicol to control enteric septicemia in catfish; ormetoprim-sulfadimethoxine to control furunculosis
in salmonids and enteric septicemia in catfish; and oxytetracycline monoalkyl trimethyl ammonium for
selected indications in salmonids, catfish, and lobsters (see U.S. FDA Center for Veterinary Medicine
website).11 Federal regulations, however, permit veterinarians to prescribe extra-label uses of certain
approved animal drugs and approved human drugs for minor species. A number of publications describe
the use of pharmaceuticals in aquaculture, both for food and ornamental species.12-14 Extra-label drug use
is a practice that occurs in many countries, and governmental agencies worldwide are currently grappling
with ways to provide proper veterinary care to minor species.15 Because of potential extra-label drug use
in aquaculture, any standardized methods for determining the susceptibility of microbes isolated from
aquatic species must include more drugs than those currently approved for use in aquaculture in any given
country. Antimicrobial agents mentioned in this document are not endorsed for use in fish-farmed species.
Clinicians must consult the regulations in their countries and also those of countries to which the fish may
be exported.
There are over 70 species of bacteria capable of causing disease in aquatic animals.16-21 Table 3 includes a
short list of the most commonly isolated aquatic pathogens. In addition to the organisms well-
characterized as pathogens of aquatic animals, there are numerous instances where isolated organisms are
either partially identified or unidentified. In such cases, as for identified pathogens, antimicrobial
susceptibility testing of the isolates is indicated prior to initiating therapy. Susceptibility tests are also
©
Clinical and Laboratory Standards Institute. All rights reserved. 1

Number 24 M49-A
indicated when there is concern that the organism belongs to a bacterial species capable of possessing
resistance mechanisms against commonly used antimicrobial agents.
Susceptibility testing of aquatic bacteria that are nonpathogenic is also indicated when examining the
epizoology of resistance in the aquatic environment. There is increasing concern about the potential for
public health impacts following antimicrobial use in aquaculture.22-26 These concerns are due, in part, to
detecting antimicrobial agent residues in fish farm sediment27-29 and to the occurrence of bacteria resistant
to antimicrobials following their use on fish farms.30,31 Much of this literature reports data derived using a
variety of antimicrobial susceptibility testing (AST) protocols. These procedures vary conditions of the
assay temperature, duration, media, and antibiotic concentration. In order to adequately assess the effect
antimicrobial use has on aquatic environmental bacteria, it is essential that standardized methods be
developed and used.
In 2003, Miller et al conducted an international multilaboratory trial to establish CLSI methods and
control criteria for antimicrobial disk susceptibility testing of bacteria isolated from aquatic animals.32
CLSI document M42—Methods for Antimicrobial Disk Susceptibility Testing of Bacteria Isolated From
Aquatic Animals details these methods. The current document describes the methods and control ranges
for dilution susceptibility testing.
3 Definitions
absorbance//optical density//A//OD – 1) in Optics, the capacity of a substance to absorb radiation;
NOTE: Absorbance is expressed as the logarithm of the reciprocal of the transmittance of the substance;
A = log (1/T) = -log (T)33; 2) decadic absorbance – the negative decadic logarithm of one minus the
absorptance.
Antimicrobial Susceptibility Test Interpretive Category – 1) classification based on a bacterium’s in

vitro response to an antimicrobial agent relative to that agent’s serum concentration (or other relevant
fluid concentration) that is attainable using standard of practice dose or the labeled target animal species
for that type of infection and infecting organism; 2) Susceptible Antimicrobial Susceptibility Test
Interpretive Category – a category that implies that an infection due to the isolate may be appropriately
treated with the dosage regimen of an antimicrobial agent recommended for that type of infection and
infecting species, unless otherwise indicated; 3) Intermediate Antimicrobial Susceptibility Test
Interpretive Category – a category that implies that an infection due to the isolate may be appropriately
treated in body sites where the drugs are physiologically concentrated or when a high dosage of drug can
be used; also indicates a “buffer zone” that should prevent small, uncontrolled, technical factors from
causing major discrepancies in interpretations; 4) Resistant Antimicrobial Susceptibility Test
Interpretive Category – resistant isolates are not inhibited by the usually achievable concentrations of
the agent with normal dosage schedules and/or fall in the range where specific microbial resistance
mechanisms are likely (e.g., beta-lactamases), and clinical efficacy has not been reliable in treatment
studies.
colorimeter – an instrument used for color measurement based on optical comparison with standard
colors.33(ASTM)
confidence limit – a number or pair of numbers that defines a confidence interval.
consensus – in CLSI documents, the substantial agreement by materially affected, competent, and
interested parties that is obtained by following the procedures specified for CLSI consensus approval;
NOTE: CLSI consensus does not always connote unanimous agreement, but it does mean that the
participants in the development of a standard or guideline have considered and resolved all relevant
comments and are willing to abide by the resulting agreement.
©
2 Clinical and Laboratory Standards Institute. All rights reserved.

Volume 26 M49-A
control material//control – a device, solution, or lyophilized preparation intended for use in the quality
control process; NOTE 1: The expected reaction or concentration of analytes of interest are known within
limits ascertained during preparation and confirmed in use; NOTE 2: Control materials are generally not
used for calibration in the same process in which they are used as controls; NOTE 3: See also standard.
minimal inhibitory concentration//MIC – the lowest concentration of an antimicrobial agent that

prevents visible growth (or a growth reduction of ≥ 80%) of a microorganism in an agar or broth dilution
susceptibility test.
optical density – deprecated term. See and use absorbance above.
precision (of measurement) – the closeness of agreement between independent test results obtained
under stipulated conditions (ISO Guide 30, ISO 3534-1)34,35; NOTE: Precision is not typically
represented as a numerical value but is expressed quantitatively in terms of imprecision—the standard
deviation (SD) or the coefficient of variation (CV) of the results in a set of replicate measurements.
quality assurance – part of quality management focused on providing confidence that quality
requirements will be fulfilled.36 (ISO 9000)
quality control – in Microbiology, the operational techniques that are used to ensure accuracy and
reproducibility.
range – 1) a measure of dispersion which is (ASQC)37 the difference between the largest and the smallest
observed value of a quantitative characteristic (ISO 3534-1)35 in a given sample; 2) measuring range
(working range) – a set of values of measurands for which the error of a measuring system instrument is
intended to lie within specified limits (VIM93)38; 3) dynamic range – the total span over which an
analysis can provide results; 4) reference range//reference interval//normal range – the range of test
values expected for a designated population of individuals (42 CFR §493)39; NOTE: For example, 95%
of individuals that are presumed to be healthy (or normal); 5) physiological range – the full range of
analyte levels to be expected in patient samples.
standard – 1) an authoritative “document” setting forth criteria for performance and characteristics;
NOTE 1: This definition is compatible with the use of “standard” in the U.S. Code of Federal
Regulations (CFR), as well as in the context of CLSI use; NOTE 2: As a consequence of the conflicting
use of the term as defined in definition 1, vs. the following definitions, any use of the term standard shall
be appropriately specified in context; 2) primary standard – a standard that is designated or widely
acknowledged as having the highest metrological qualities and whose value is accepted without reference
to other standards of the same quantity (VIM93)38; 3) secondary standard – a standard whose value is
assigned by comparison with a primary standard of the same quantity (VIM93)38; 4) reference
standard – a standard, generally having the highest metrological quality available at a given location or
in a given organization from which measurements made there are derived (VIM93)38; 5) working
standard – a standard that is used routinely to calibrate or check material measures, measuring
instruments, or reference materials (VIM93)38; NOTE 1: A working standard is usually compared against
a reference standard; NOTE 2: A working standard that is used routinely to ensure that measurements are
being carried out correctly is called a “check standard.” (VIM93)38
turbidity – the condition that exists when a liquid sample contains insoluble matter of sufficient particle
size to cause part of the incident light upon the sample to be scattered; NOTE: Turbidity causes
erroneously high absorption measurements; the magnitude of the error depends on the optical geometry of
the measuring instrument. Units of measurement include, but are not limited to McFarland units [0.5 = 80
to 88% transmittance (T), 1.0 = 67 to 77% T, 2.0 = 46 to 56% T, and 3.0 = 27 to 37% T]: nephelometric
turbidity units (NTUs) with a range of 0 to 10 000 NTU, or spectrophotometric units at a set wavelength.
©

Number 24 M49-A
4 Antimicrobial Agents
4.1 Source
Obtain antimicrobial standards or reference powders directly from the drug manufacturer, from the United
States Pharmacopoeia (www.usp.org, 12601 Twinbrook Parkway, Rockville, Maryland 20852, 800-227-
8772), or from other commercial sources. Do not use standard parenteral preparations for susceptibility
testing. Acceptable powders bear a label that states the drug’s generic name, lot number, potency (usually
expressed in micrograms [µg] or International Units [IU] per mg of powder), and expiration date. Store
the powders as recommended by the manufacturer or at ≤-20 °C in a desiccator (preferably in a vacuum).
When the desiccator is removed from the refrigerator or freezer, allow it to come to room temperature
before being opened to avoid condensation.
4.2 Weighing Antimicrobial Powders
All antimicrobial agents are assayed for standard units of activity. The assay units may differ widely from
the actual weight of the powder and often may differ between drug production lots. Thus, a laboratory
must standardize its antimicrobial solutions based on assays of the lots of antimicrobial powders used.
The value for potency supplied by the manufacturer should include consideration of measures of purity
(usually by HPLC assay), water content (e.g., by Karl Fischer analysis or by weight loss on drying), and
the salt/counter-ion fraction (if the compound is supplied as a salt instead of free acid or base). The
potency may be expressed as a percentage, or in units of µg/mg (w/w).
In some cases, a certificate of analysis with values for each of these components may be provided with
antibiotic powders; in this case, an overall value for potency may not be provided, but can be calculated
from HPLC purity, water content, and when applicable, the active fraction for drugs supplied as a salt
(e.g., hydrochloride, mesylate). However, if when applying these calculations, any value is unknown or is
not clearly determined from the certificate of analysis, it is advisable that the factors used in this
calculation be confirmed with the supplier or the manufacturer. The following demonstrates an example
calculation:
Example: meropenem trihydrate
Certificate of analysis data:
Assay purity (by HPLC): 99.8%
Measured water content (by Karl Fischer analysis): 12.1% (w/w)
Active fraction: 100% (supplied as the free acid, and not a salt)
Potency calculation from above data:
Potency = (Assay purity) x (Active fraction) x (1- Water Content)
Potency = (998) x (1.0) x (1- 0.121)
Potency = 877 µg/mg or 87.7%
Use either of the following formulas to determine the amount of powder and diluent needed for a standard
solution:
©

Volume 26 M49-A
Weight (mg) = Volume (mL) • Concentration (µg/mL) (1)

Potency (µg/mg)
or
Volume (mL) = Weight (mg) • Potency (µg/mg) (2)

Concentration (µg/mL)
Weigh the antimicrobial powder on an analytical balance that has been calibrated with National Institute
of Standards and Technology (NIST) weights (or other approved reference weights). If possible, weigh
more than 100 mg of powder. It is advisable to accurately weigh a portion of the antimicrobial agent in
excess of that required and to calculate the volume of diluent needed to obtain the final concentration
desired as in formula (2), above.
Example: To prepare 100 mL of a stock solution containing 1280 µg/mL of antimicrobial agent with
antimicrobial powder that has a potency of 750 µg/mg, weigh 170 to 200 mg of the antimicrobial powder.
If the actual weight is 182.6 mg, the volume of diluent needed is then as follows:
182.6 mg • 750 µg/mg

Volume (mL) = (Actual Weight) • (Potency) = 107.0 mL (3)
1280 µg/mL
(Desired Concentration)
Therefore, the 182.6 mg of antimicrobial powder is to be dissolved in 107 mL of diluent.
4.3 Preparing Stock Solutions
Prepare antimicrobial agent stock solutions at concentrations of at least 1000 µg/mL (e.g., 1280 µg/mL)
or ten times the highest concentration to be tested, whichever is greater. There are some antimicrobial
agents, however, of limited solubility that may require lower concentrations. In all cases, consider
directions provided by the drug’s manufacturer as part of determining solubility.
Some drugs must be dissolved in solvents other than water. In such cases:
• Solubilize the antimicrobial powder in a minimum amount of solvent.
• Make the final stock concentration with water or appropriate diluent as indicated in Table 4.
• For potentially toxic solvents, consult the material safety data sheets (MSDS) available from the
manufacturer (see Table 4).
Because microbial contamination is extremely rare, solutions that have not been sterilized are generally
acceptable. If desired, however, solutions may be sterilized by membrane filtration. Do not use paper,
asbestos, or sintered glass filters, which may adsorb appreciable amounts of certain antimicrobial agents.
Whenever filtration is used, it is important to document the absence of adsorption by appropriate assay
procedures.
Small volumes of the sterile stock solutions may be dispensed into sterile glass, polypropylene,
polystyrene, or polyethylene vials, carefully sealed, and stored (preferably at -60 ºC or below, but never at
a temperature warmer than -20 °C and never in a frost-free freezer). Adsorption may occur when sterile
glass is used with some antimicrobial classes (quinolones and tetracyclines), so specific cleaning
procedures may be required, such as acid washing, or the use of disposable glassware. Thaw and use vials
©

Number 24 M49-A
as needed the same day. Discard any unused drug at the end of the day. Store stock solutions of most
antimicrobial agents at -60 °C or below for six months or more without significant loss of activity. In all
cases, consider directions provided by the drug’s manufacturer, in addition to these general
recommendations. Any significant deterioration of an antimicrobial agent should be reflected in the
results of susceptibility testing using quality control strains.
4.4 Number of Concentrations Tested
The concentrations to be tested are at the discretion of the laboratory. However, it is advisable to choose a
range that allows at least one quality control organism to have on-scale values. Unusual concentrations
may be tested for special purposes.
5 Selection of Antimicrobial Agents for Routine Testing and Reporting

Selection of the most appropriate antimicrobial agents to test and to report is a decision best made by each
clinical laboratory in consultation with aquatic animal practitioners. Appendix A is a comprehensive list
of many of the antimicrobial agents used at varying frequencies in global aquaculture, for which the status
of quality control ranges (and methods for susceptibility testing) are listed.
5.1 Routine Reports
Routine reports to veterinarians and clinicians should include those antimicrobial agents appropriate for
therapeutic use. It may also be important for laboratories to monitor for changes in susceptibility to these
agents in bacteria isolated from the aquatic environment. Thus, the laboratory may need to conduct
susceptibility tests on nonapproved antimicrobial agents.
5.2 Antimicrobial Classes
To minimize confusion, report all antimicrobial agents using official nonproprietary (i.e., the active
ingredient) names; depending on the agent, use the ICD (International Chemical Denomination). To
emphasize the relatedness of the many currently available drugs in global aquaculture, they may be
grouped together by the following drug classes. Representatives of each group known to be used at
varying frequencies in global aquaculture are also listed.
5.2.1 Aminoglycosides
Members of this group inhibit bacterial protein synthesis at the ribosomal level. This group includes
members that are affected in various ways by aminoglycoside-inactivating enzymes, which results in
some differences in the spectrum of activity between agents. Aminoglycosides are used primarily to treat
aerobic, gram-negative infections or in synergistic combinations with cell-wall-active compounds against
some resistant, gram-positive bacteria, such as enterococci. Aminoglycosides (e.g., gentamicin,
kanamycin, streptomycin) have the potential to produce extended residue times in some animals (e.g., the
bovine); therefore, monitor their use carefully.
5.2.2 Aminopenicillins
Aminopenicillins have activity against penicillin-susceptible gram-positive bacteria, as well as some

gram-negative bacteria. Aminopenicillins are susceptible to destruction by β-lactamases and therefore are
not effective against bacteria that produce these enzymes. Ampicillin is acid-stable, but absorption is
slower and more irregular than that of some of the esters (bacampicillin and talampicillin) and analogs
(amoxicillin). In mammals, amoxicillin produces blood levels approximately twice those of ampicillin
after oral administration; however, its serum concentration-time curve resembles that for ampicillin.40 The
same findings have not yet been reported in aquatic species.
©

Volume 26 M49-A
5.2.3 Macrolides
Macrolides are structurally related antimicrobial agents that inhibit protein synthesis at the ribosomal
level. Erythromycin has been shown to exhibit in vitro bactericidal activity against some rapidly
replicating bacteria, but overall it is considered bacteriostatic.41 Drugs in this group are closely related and
with few exceptions, only erythromycin may need to be tested routinely.
5.2.4 Nitrofurans
The nitrofurans are synthetic antibacterial agents effective against many gram-negative and gram-positive
organisms.
Nitrofurans (e.g., furazolidone, nifurpirinol, nitrofurantoin, nitrofurazone) are commonly used in the
ornamental fish trade as an effective medication for the treatment of external bacterial infections, but in
the U.S. and Europe, this class has been banned for use in food-producing animals.
5.2.5 Phenicols
The phenicols are a group of compounds with broad-spectrum activity. These agents are generally
considered bacteriostatic, but they are bactericidal against some pathogens. The phenicol class can be
divided into the nonfluorinated compounds, such as chloramphenicol and thiamphenicol, and the
fluorinated compound, florfenicol. The fluorinated compounds retain activity against chloramphenicol
acetyltransferase-mediated resistance.
The p-nitro group of chloramphenicol has been associated with aplastic anemia, and in many countries
has been prohibited for use in food animals or aquaculture.
5.2.6 Quinolones/Fluoroquinolones
This group of antimicrobial agents functions primarily by inhibiting the DNA-gyrase activity of many
gram-positive and gram-negative bacteria. This group of compounds includes quinolones (e.g.,
flumequine, nalidixic acid, oxolinic acid, sarafloxacin) and fluoroquinolones (e.g., enrofloxacin,
piromidic acid). The U.S. FDA has prohibited extra-label use of fluoroquinolones in food animals.42
Some differences in spectrum may require separate testing of the individual agents.
5.2.7 Sulfonamides and Potentiated Sulfonamides
This group of compounds encompasses several chemotherapeutic agents with similar spectra of activity
resulting from inhibition of the bacterial folate pathway. Examples include: ormetoprim-
sulfadimethoxine, ormetoprim-sulfamonomethoxine, sulfamerazine, sulfamethazine, sulfisoxazole,
trimethoprim-sulfadiazine, and trimethoprim-sulfamethoxazole.
When sulfonamides are combined with trimethoprim or ormetoprim, two sequential steps in folate
metabolism are inhibited. Although ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole
combinations are most common in aquatic medicine, for susceptibility testing of other combinations, it is
recommended to test the combination in question to obtain the most reliable results.
5.2.8 Tetracyclines
These compounds inhibit bacterial protein synthesis at the ribosomal level. Drugs in this group are closely
related, and with few exceptions, only tetracycline may need to be tested routinely. Organisms that are
susceptible to tetracycline are also considered susceptible to doxycycline and minocycline. However,
©

Number 24 M49-A
some organisms that are intermediate or resistant to tetracycline may be susceptible to doxycycline or
minocycline or both.
5.3 Suggested Guidelines for Use and Selective Testing and Reporting
The drugs used for routine testing are at the discretion of the laboratory, in consultation with aquatic
disease practitioners. Some laboratories may choose to also test unapproved agents for a variety of
reasons. In these cases, it is important to note that the laboratory client or veterinarian assumes all
responsibility for efficacy, safety, and residue avoidance with the use of unapproved antimicrobial agents.
Listed in Appendix A are antimicrobial agents used in global aquaculture for which quality control ranges
have been established for dilution testing, and other agents reportedly used in aquaculture situations.
Each laboratory should include caveats appropriate for their respective country’s regulations when
reporting results. Since antimicrobial susceptibility testing may be done to monitor for changes in
susceptibility in the environment, it is not advisable to withhold data on prohibited antimicrobial agents,
but they should be identified as prohibited (e.g., chloramphenicol in the European Union and U.S.).
6 Indications for Performing Susceptibility Testing

Susceptibility testing is indicated for any bacterial pathogen that contributes to an infective process
requiring antimicrobial intervention, if its susceptibility cannot be reliably predicted from knowledge of
the organism’s identity. Unfortunately, not all pathogens have susceptibility test methods recommended,
quality control conditions developed, or interpretive criteria established. Susceptibility tests are most
often requested when the laboratory client believes the causative organism may belong to a bacterial
species capable of possessing resistance mechanisms to commonly used antimicrobial agents.
Mechanisms of resistance include production of drug-inactivating enzymes, alteration of drug targets, and
altered uptake or efflux. Some organisms still have predictable susceptibility to antimicrobial agents, and
empiric therapy is widely recognized. Susceptibility tests are important in studies of the epidemiology of
resistance and in studies of new antimicrobial agents.
Colonies of each type of bacterial pathogen isolated from an infectious disease process should be selected
from primary agar plates and used for susceptibility testing. A statistical model for assessing sample size
for bacterial colony selection has been proposed.43 Culture and identification procedures often precede in
vitro susceptibility tests. Several “short-cut” methods of susceptibility testing are to be discouraged, as
they will give misleading outcomes and could result in a poor treatment decision.
• Mixtures of different types of microorganisms should not be tested in the same susceptibility test
plate.
• The practice of conducting susceptibility tests directly with clinical material (e.g., normally sterile
body fluids and water samples) should be avoided.
• When the nature of the infection is not clear and the specimen contains mixed organisms or normal
flora, and the organisms probably bear little relationship to the infectious process, susceptibility tests
are often unnecessary, may be misleading, and result in inappropriate use of antimicrobial agents.
©

Volume 26 M49-A
7 Inoculum Preparation for Dilution Tests
7.1 Turbidity Standard for Inoculum Preparation
To standardize the inoculum density for a susceptibility test, use a BaSO4 turbidity standard equivalent to
a 0.5 McFarland standard44 or its optical equivalent (e.g., latex particle suspension). These standards are
readily available from various commercial sources. Alternatively, a BaSO4 0.5 McFarland standard may
be prepared as follows:
(1) Add a 0.5-mL aliquot of 0.048 mol/L BaCl2 (1.175% w/v BaCl2 • 2H2O) to 99.5 mL of 0.18 mol/L
(0.36 N) H2SO4 (1% v/v) with constant stirring to maintain a suspension.
(2) Verify the correct density of the turbidity standard using a spectrophotometer with a 1-cm light path
and matched cuvettes to determine the absorbance. The absorbance at 625 nm should be 0.08 to 0.13
for the 0.5 McFarland standard.
Alternatively, if a nephelometer or turbidimeter must be used for the preparation of a standard or

inoculum, standardize this procedure on a laboratory-to-laboratory basis due to optical geometry
differences in the measuring instrument. Use a turbidity equivalent in nephelometric turbidity units
(NTUs) to a 0.5 McFarland suspension.
(3) Transfer the barium sulfate suspension in 4- to 6-mL aliquots into screw-cap tubes of the same size as
those used for growing or diluting the bacterial inoculum.
(4) Tightly seal and store tubes in the dark at room temperature.
(5) Vigorously agitate the barium sulfate turbidity standard on a vortex mixer before each use and inspect
for a uniformly turbid appearance. If large particles appear, replace the standard. Mix latex particle
suspensions by inverting gently, not on a vortex mixer.
(6) Replace the barium sulfate standards monthly or verify their densities.
7.2 Direct Colony Suspension Method
(1) Prepare the inoculum by making a suspension directly in saline or broth. Deionized water has been
used to prepare inocula in other standardized dilutions methods (refer to the most current editions of
CLSI/NCCLS documents M31—Performance Standards for Antimicrobial Disk and Dilution
Susceptibility Tests for Bacteria Isolated From Animals and M7—Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria That Grow Aerobically), but since some aquatic pathogens may
experience osmotic stress in deionized water, it is generally advisable to avoid its use. Obligate
halophilic organisms should be diluted in broth or saline. Use three to five well-isolated colonies of
the same morphological type, selected from a 24- to 48-hour agar plate (a nonselective medium, such
as blood agar, should be used).
(2) Standardize inoculum to a concentration of 1 to 2 x 108 CFU/mL, which should correlate to an OD625
of 0.08 to 0.13 when using a spectrophotometer, a 0.5 McFarland standard44 when using a standard
colorimeter, or approximately 60 nephelometric turbidity units (NTUs) when using a turbidimeter.
The laboratory may use the 0.08 to 0.13 absorbance, 0.5 McFarland, or 60 NTU readings to estimate
the bacterial concentration, only if there are historical data showing a corresponding concentration of
approximately 1 to 2 x 108 CFU/mL for the given organism. Expect comparable cell densities with
many bacterial species; however, monitor densities closely using colony count determinations. To
adjust the turbidity of a suspension properly, use either a photometric device or colorimeter. These
data may need to be generated specifically for fish pathogenic bacteria. Until such data are available,
©

Number 24 M49-A
laboratories should check the validity of the application of this protocol for the strains they routinely
examine.
An alternative “line method” may be used in the absence of a colorimeter or spectrophotometer. A

heavy black line is drawn on a sheet of paper, and using a standardized 0.5 McFarland solution, the
bacterial suspension is brought to a turbidity equivalent to that of the 0.5 McFarland solution. The
turbidity of the two solutions is observed by looking at the line through both tubes, and when the line
on the sheet of paper appears the same through both tubes, the bacterial suspension should be
equivalent to a 0.5 McFarland suspension.
If the test strain autoagglutinates, then some difficulty may be experienced in preparing a
homogeneous suspension. In such cases, it is recommended that suspensions be mixed thoroughly on
a vortex mixer before their optical densities are examined, and that the bacterial concentration be
determined.
8 Broth Dilution Procedures (Macrodilution and Microdilution)

The methods described here represent the current recommendations of the Aquaculture Working Group of
the Subcommittee for Veterinary Antimicrobial Susceptibility Testing. Use these methods when
conducting broth dilution tests on organisms in Group 1 (see Table 1).
Group 1: Enterobacteriaceae
Aeromonas salmonicida (nonpsychrophilic strains)
Aeromonas hydrophila and other mesophilic Aeromonads
Pseudomonas spp.
Plesiomonas shigelloides
Shewanella spp.
Vibrionaceae and related bacteria (nonobligate halophilic strains)
8.1 Cation-Adjusted Mueller-Hinton Broth (CAMHB) Medium
(1) CAMHB is recommended as the medium of choice for broth dilution susceptibility testing of
commonly isolated, rapidly growing, aerobic or facultative organisms (see the most current edition of
CLSI document M7—Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That
Grow Aerobically). CAMHB demonstrates good batch-to-batch reproducibility for susceptibility
testing; is low in sulfonamide, trimethoprim, and tetracycline inhibitors; and yields satisfactory
growth of most pathogens. In addition, large bodies of data and experience have been gathered about
tests performed with this medium. This medium may be supplemented to support the growth of some
fastidious bacteria. Specific recommendations for supplementing CAMHB to support the growth of
some fastidious or problem organisms are provided in Section 9. However, no quality control ranges
have been established for broth dilution susceptibility testing of aquatic isolates under any conditions
in any media other than cation-adjusted Mueller-Hinton broth.
(2) Monitor routinely MIC performance and chemical characteristics of CAMHB. Check the pH of each
batch of broth with a pH meter after the medium is prepared; make sure the pH is between 7.2 and 7.4
at room temperature (25 ºC).
(3) Unless a batch of Mueller-Hinton broth has the correct concentrations of the divalent cations Ca++ and
Mg++ (20 to 25 mg of Ca++/L [50 mg/L when testing daptomycin] and 10 to 12.5 mg of Mg++/L),
MICs of aminoglycosides for P. aeruginosa and MICs of tetracycline for all bacteria and MICs of
daptomycin for gram-positive organisms will be different from those obtained on Mueller-Hinton
agar. Some manufacturers provide Mueller-Hinton broth that has already had cation content
adjustment. Therefore, follow the instructions for cation adjustments below only when the initial
©

Volume 26 M49-A
Mueller-Hinton broth has been certified by the manufacturer or measured by the user to contain no or
inadequate amounts of Ca++ and Mg++ when assayed for total divalent cation content by atomic
absorption spectrophotometry. Adding excess cations to Mueller-Hinton broth may lead to erroneous
results; however, for testing daptomycin, supplement the broth to 50 mg/L of Ca++.
Cation Adjustment of Mueller-Hinton Broth
(a) To prepare a magnesium stock solution, dissolve 8.36 g of MgCl2 • 6H2O in 100 mL of deionized
water. This solution contains 10 mg of Mg++/mL.
(b) To prepare a calcium stock solution, dissolve 3.68 g of CaCl2 • 2H2O in 100 mL of deionized
water. This solution will contain 10 mg of Ca++/mL.
Sterilize stock solutions by membrane filtration and store at 2 to 8 ºC.
(c) Prepare Mueller-Hinton broth as the manufacturer directs, autoclave, and chill overnight at 2 to
8 ºC or in an ice bath before cation addition if it is to be used the same day. Some Ca++ or Mg++ is
often present in the commercially prepared dehydrated medium. Account for this starting
concentration of cations when calculating the amount of Ca++ or Mg++ to add to the medium.
(d) With stirring, add 0.1 mL of chilled Ca++ or Mg++ stock solution per liter of broth for each desired
increment of 1 mg/L in the final concentration in the adjusted Mueller-Hinton broth. This
medium is called CAMHB. Adjustments of Ca++ or Mg++, or both, are not necessary when
Mueller-Hinton broth as received from the manufacturer already contains the correct
concentrations (20 to 25 mg of Ca++/L [50 mg/L for daptomycin] and 10 to 12.5 mg of Mg ++/L)
of divalent cations.
(4) CAMHB may be supplemented with 2.5 to 5% (v/v) lysed horse blood (LHB) for testing some of the
more fastidious organisms (e.g., streptococci) (see the most current edition of CLSI/NCCLS
document M31—Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for
Bacteria Isolated From Animals); however, quality control data have only been generated for testing
at 35 ± 2 ºC. Any data generated using LHB supplementation and testing at a temperature other than
35 ± 2 ºC cannot be considered to be in accordance with CLSI guidelines.
Preparation of Lysed Horse Blood (LHB)
(a) To prepare LHB, freeze and thaw defibrinated horse blood until the blood is thoroughly lysed
(about five to seven times). Aseptically mix equal volumes of the lysed blood and sterile, distilled
water (now 50% LHB).
(b) To be used in the broth test, the combination of broth and LHB must be clear, and this can be
accomplished by centrifuging the 50% LHB at 12 000 x g for 20 minutes. Decant the supernatant
and recentrifuge, if necessary. Add appropriate amounts of the 50% LHB to the broth medium to
yield a final concentration of 2.5 to 5% LHB.
(c) Check the pH after the aseptic addition of the blood to the autoclaved and cooled medium. Add
blood to the microdilution trays when first dispensed or after being thawed, and just prior to
inoculation. If added after plate preparation, the blood must be added along with the inoculum so
further dilution of the antimicrobial agent in the tray does not occur, and that the final
concentration of LHB in the well is 2.5 to 5%.
©

Number 24 M49-A
(5) Evaluate MIC performance characteristics of each batch of broth using a standard set of quality
control organisms (see Section 11.3). If a new lot of Mueller-Hinton broth does not yield the expected
MICs, investigate the cation content along with other variables and components of the test.
(6) To determine the suitability of the medium for sulfonamide and trimethoprim tests, perform tests at
35 ± 2 ºC with Enterococcus faecalis ATCC® 29212; NCIMBb 13280. Read end points as an 80% or
greater reduction in growth, or 20% growth intensity as compared to the positive control. If the
minimal inhibitory concentration (MIC) for trimethoprim-sulfamethoxazole is ≤ 0.5/9.5 µg/mL when
tested at 35 ± 2 ºC, the medium may be considered acceptable.
8.2 Preparing and Storing Diluted Antimicrobial Agents
8.2.1 Broth Macrodilution (Tube) Method
(1) Use sterile 13- x 100-mm test tubes to conduct the test.
(2) Use a control tube containing broth without antimicrobial agent for each organism tested.
(3) The tubes can be closed with loose screw caps, plastic or metal closure caps, or cotton plugs.
(4) Prepare the final twofold (or other) dilutions of antibiotic volumetrically in the broth. The schedule in
Table 5 indicates a convenient and reliable procedure for preparing dilutions. A minimum final
volume of 1 mL of each dilution is needed for the test. A single pipette can be used for measuring all
diluents and then for adding the stock antimicrobial solution to the first tube. Use a separate pipette
for each remaining dilution in that set. Because there will be a 1:2 dilution of the drugs when an equal
volume of inoculum is added, the antimicrobial dilutions are often prepared at double the desired final
concentration.
8.2.2 Broth Microdilution (Plate) Method
(1) This method is called “microdilution” because it involves the use of small volumes of broth dispensed
in sterile, plastic microdilution trays that have round or conical bottom wells. Each well should
contain 0.1 mL of broth. To prepare microdilution trays, antimicrobial agents may be diluted as
described in Table 4.
(2) The most convenient method to prepare microdilution trays is to use a dispensing device,
incorporating antimicrobial dilutions made in broth. These dilutions are used to dispense 0.1 (±0.02)
mL into each of the 96 wells of a standard tray. If the inoculum is to be added by pipette as described
in Section 8.3.2.2, the antimicrobial solutions are prepared at twice the desired final concentration,
and the wells are filled with 0.05 mL instead of 0.1 mL. Each tray should include a growth control
well and a negative (uninoculated) well.
(3) Seal the filled trays in plastic bags and immediately place in a freezer at ≤ -20 ºC (preferably at
≤ -60 ºC) until needed. Although the antimicrobial agents in frozen trays usually remain stable for
several months, certain agents (e.g., imipenem and ampicillin) are more labile. Do not store trays in a
self-defrosting freezer, and do not refreeze thawed antimicrobial solutions; repeated freeze-thaw
cycles accelerate the degradation of some antimicrobial agents, particularly β-lactams.
__________________________
b
National Collection of Industrial and Marine Bacteria (NCIMB, www.ukncc.co.uk)
©

Volume 26 M49-A
8.3 Broth Dilution Testing
8.3.1 lnoculum Preparation
A standardized inoculum for the macrodilution or microdilution broth methods may be prepared by
suspending colonies directly to achieve the same density as described in Section 7.
(1) Optimally, within 15 minutes of preparation, dilute the adjusted inoculum suspension in CAMHB
(macrodilution method), or sterile water, saline, or CAMHB (microdilution method) so that after
inoculation, each tube or well contains approximately 5 x 105 CFU/mL. Deionized water has been
used to prepare inocula in other standardized disk methods (refer to the most current editions of
CLSI/NCCLS documents M31—Performance Standards for Antimicrobial Disk and Dilution
Susceptibility Tests for Bacteria Isolated From Animals and M2—Performance Standards for
Antimicrobial Disk Susceptibility Tests), but since some aquatic pathogens may experience osmotic
stress in deionized water it is generally advisable to avoid its use. Obligate halophilic organisms and
control tests using A. salmonicida subsp. salmonicida ATCC® 33658 should be diluted in broth or
saline. The dilution procedure to obtain this final inoculum varies according to the method used to
deliver the inoculum to the individual wells or tubes and according to the organism being tested, and
it must be calculated for each situation. For microdilution tests, the exact inoculum volume delivered
to the wells must be known to make this calculation. For example, if the volume of medium in the
well is 0.1 mL and the inoculum volume is 0.005 mL, then the 0.5 McFarland suspension (1 x 108
CFU/mL) should be diluted 1:10 to yield 107 CFU/mL. When 0.005 mL of this suspension is
inoculated into the broth, the final test concentration of bacteria should be approximately 5 x 105
CFU/mL (or 5 x 104 CFU/well in the microdilution method).
(2) For testing streptococci, follow specific instructions in Section 9.3.
(3) Laboratories are encouraged to perform colony counts on inoculum suspensions periodically to
ensure that the final inoculum concentration routinely obtained closely approximates 5 x 105
CFU/mL. This can be easily accomplished by removing a 0.01-mL aliquot from the growth control
well or tube immediately after inoculation and diluting it in 10 mL of sterile saline (1:1000 dilution).
After mixing, a 0.1-mL aliquot is spread over the surface of a suitable agar medium. After incubation,
the presence of approximately 50 colonies would indicate an inoculum density of 5 x 105 CFU/mL.
8.3.2 Inoculating the Broth in Tubes or Microdilution Trays
8.3.2.1 Macrodilution (Tube) Broth Method
Within 15 minutes after the inoculum has been standardized as described above, add 1 mL of the adjusted
inoculum to each tube already containing 1 mL of antimicrobial agent in the dilution series (and a positive
control tube containing only broth). Mix each tube. This results in a 1:2 dilution of each antimicrobial
concentration and a 1:2 dilution of the inoculum.
It is advisable to check the purity of the inoculum suspension by subculturing an aliquot on a nonselective
agar medium for simultaneous incubation.
8.3.2.2 Microdilution Broth Method
Inoculate each well of a freshly prepared or thawed tray with a 0.05-mL pipette or pipette dropper or an
inoculum replicator. As in macrodilution, optimally dilute the inoculum and the broth within 15 minutes
after the inoculum is standardized. If the volume of the inoculum exceeds 10% of the volume of the well,
take into account the diluting effect of the inoculum on the antimicrobial agent. If a pipette is used to
inoculate the broth in a well, the resulting dilution is usually 1:2 (0.05 mL antimicrobial solution + 0.05
©

Number 24 M49-A
mL inoculum), but if a replicator is used, the resulting dilution is usually negligible (generally 5 to 10 µL
of inoculum in 0.1 mL antimicrobial solution).
It is advisable to perform a purity check of the inoculum suspension by subculturing an aliquot onto a
nonselective agar plate for simultaneous incubation. To prevent drying, seal each tray in a plastic bag,
with plastic tape, or with a tight-fitting plastic cover before incubation.
8.3.3 Incubation
Incubate the inoculated macrodilution tubes or microdilution trays at 22 ºC for 24 to 28 hours and/or 44 to
48 hours, or 28 ºC for 24 to 28 hours in an ambient air incubator. To maintain the same incubation
temperature for all cultures, it is preferable to stack microdilution trays no more than four high.
8.3.4 Determining MIC End Points
The MIC is the lowest concentration of antimicrobial agent that inhibits growth of the organism in the
tubes or microdilution wells as detected by the unaided eye. Viewing devices intended to facilitate
reading microdilution tests and recording of results may be used, as long as there is no compromise in the
ability to discern growth in the wells. Compare the amount of growth in the wells or tubes containing the
antimicrobial with the amount of growth in the growth-control wells or tubes (no antimicrobial) used in
each set of tests when determining the growth end points. For a test to be considered valid, acceptable
growth (≥ 2 mm button or definite turbidity) must occur in the positive control well. With trimethoprim
and the sulfonamides, antagonists in the medium may allow some slight growth; therefore, read the end
point as the concentration in which there is 80% or greater reduction in growth, or 20% of the growth
density, as compared to the control.
When a single skipped well or tube occurs in a dilution test, record the higher of the two MICs. Do not
report results for drugs for which there is more than one skipped well, and repeat this test. Generally,
MICs of gram-negative bacilli by microdilution are the same as or one twofold dilution lower than the
comparable macrodilution MICs.45
9 Fastidious and Problem Organisms

Aquatic animal bacterial pathogens for which standardized testing conditions exist (Group 1) and those
that prefer or require supplemented (e.g., NaCl, lysed horse blood) or diluted Mueller-Hinton media are
organized into “Groups” according to their growth preferences (see Tables 1 and 2).
If MIC tests are to be done with fastidious organisms, the medium, quality control procedures, and
eventually the interpretive criteria must be modified to fit each organism. The Aquaculture Working
Group hopes to approve a standardized susceptibility testing method for each of the Groups discussed
here. These methods will be invaluable tools to aquatic animal disease diagnosticians who routinely
observe mortalities attributed to these pathogens (see Table 3).
The testing conditions outlined here represent the most up-to-date opinions of members of the
Aquaculture Working Group. It is envisioned that the use of these provisional methods will provide
important data leading to useful, reproducible, and valuable standardized methods for testing the many
important groups of aquatic pathogens.
9.1 Vibrionaceae and Photobacteriaceae (Obligate Halophilic Strains) (Group 2)
The recommended methods of broth dilution susceptibility testing described below apply to obligate
halophilic members of Vibrionaceae and Photobacteriaceae. It is important to note that NaCl changes the
osmolarity of the medium and has a marked effect on the activity of the aminoglycosides gentamicin46
©

Volume 26 M49-A
and tobramycin.47 The effect NaCl supplementation may have on both the growth of the bacterium and
antimicrobial activity of other drugs has not been studied in detail and should be carefully analyzed prior
to developing a standardized method and establishing interpretive criteria.
9.1.1 Broth Medium
The Aquaculture Working Group recommends that tests of obligate halophilic strains be conducted in
CAMHB supplemented with 1% NaCl.48-50 Prior to performing susceptibility tests in media with
additional NaCl, the inability of a suspected obligate halophilic organism to grow in unsupplemented
CAMHB should be confirmed. Do not consider native NaCl concentrations in Mueller-Hinton broth
products in the 1% NaCl final concentration.
9.1.2 Test Procedure
The standard methods of inoculum preparation and inoculation apply as outlined in Sections 7 and 8.
Incubate tests on members of Vibrionaceae and related bacteria at 22 °C for 24 to 28 hours or 44 to 48
hours, or 28 °C for 24 to 28 hours, depending on the growth preferences of the test organism. Incubate
tests on slower growing members of Photobacteriaceae at 28 °C for 44 to 48 hours.
9.1.3 MIC Quality Control Data
No quality control data exist at this time for any organisms preferring or requiring NaCl supplementation
to CAMHB.
9.2 Gliding Bacteria (Group 3)
When conducting broth dilution tests on the gliding bacteria including Flavobacterium columnare, F.
branchiophilum, and F. psychrophilum, the Aquaculture Working Group recommends using diluted
forms of Mueller-Hinton broth described below.
9.2.1 Broth Medium
The recommended broth dilution testing medium for F. columnare is a diluted Mueller-Hinton broth
medium.51 Some members of the Aquaculture Working Group have found that a 1:7 dilution of the
standard CAMHB (see Section 8.1) yielded adequate growth for susceptibility tests (Aquaculture
Working Group, unpublished data, 2006).
Additional incubation time and supplementation of 5% horse or fetal calf serum or NaCl may be required
for tests with F. psychrophilum52 and F. branchiophilum.1
For tests on F. columnare, employ similar methods as outlined in Section 8. Prepare a suspension
equivalent to a 0.5 McFarland standard from an overnight culture of cells that were centrifuged and
resuspended in the test medium. The only other deviation from Section 8 is the use of a diluted Mueller-
Hinton broth medium (1:7). Conduct tests on F. columnare at 28 °C for 24 to 28 hours.
Susceptibility test conditions for F. psychrophilum and F. branchiophilum will likely require a lower
incubation temperature and extended incubation time of 15 to 18 °C for 44 to 48 hours and/or 68 to 72
hours.
©

Number 24 M49-A
9.2.3 MIC Quality Control Data
No quality control strains exist for the susceptibility testing of gliding bacteria under these recommended
growth conditions.
9.3 Streptococci (Group 4)
Standard CAMHB is inadequate for antimicrobial susceptibility testing of most streptococci.

Standardized methods of broth microdilution testing for most streptococci have been developed for tests
administered at 35 ± 2 ºC. However, many streptococcal fish pathogens prefer lower temperatures. The
Aquaculture Working Group recommends conducting broth dilution tests on the streptococcal fish
pathogens at either 22 or 28 °C (Group 4).
9.3.1 Broth Medium
Broth microdilution MIC testing using CAMHB supplemented with 2.5 to 5% lysed horse blood is
recommended. In Section 8.1(4), the method used for preparation of lysed horse blood and details about
its addition to the broth medium are provided.
Use the direct colony suspension procedure when preparing inocula for tests on streptococci (see Section
7.2).
The broth microdilution procedure steps, as described beginning in Section 8.3.2.2 for Group 1
organisms, should be followed. Incubate trays in ambient air at 22 °C for 44 to 48 hours or 28 °C for 24 to
28 hours or 44 to 48 hours.
9.3.3 MIC Quality Control Ranges
Although quality control ranges do exist for testing streptococci at 35 ± 2 °C (see Table 9), these ranges
are not applicable to tests at 22 or 28 °C. Thus, no quality control strains exist for the susceptibility testing
of streptococcal fish pathogens in CAMHB with 2.5 to 5% lysed horse blood at 22 or 28 °C.
Specific MIC interpretive criteria to be used when testing streptococci can be found in the most current
edition of CLSI document M7—Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That
Grow Aerobically. These interpretive criteria are for mammalian pathogens (based on a 35 ± 2 °C in vitro
incubation temperature) in mammalian models, however, and cannot be accurately correlated to the in
vivo situation in an aquatic animal model.
9.4 Other Fastidious Organisms (Group 5)
This group includes many organisms for which the Aquaculture Working Group felt they needed
additional data to make recommendations. Conditions for quality control are lacking for most of the
organisms in the sections below.
9.4.1 Psychrophilic Aeromonas salmonicida
No deviations from the standard methods of testing other than a lowered incubation temperature to 15 °C
are required for tests conducted on strictly psychrophilic strains of A. salmonicida. However, an attempt
to grow presumptive psychrophilic A. salmonicida strains at the standard 22 °C is recommended.
©

Volume 26 M49-A
9.4.2 Vibrio salmonicida and Moritella viscosa Strains
Recommended broth dilution susceptibility testing conditions for V. salmonicida and M. viscosa strains
cannot be made at this time. A study on V. salmonicida reported using standard Mueller-Hinton broth and
incubation temperatures of 4 °C and 15 °C for six days.53 The effect these extended incubation times may
have on accurate MIC determinations still needs to be elucidated.
9.4.3 Tenacibaculum maritimum (Formerly Flexibacter maritimus)
The Aquaculture Working Group recommends using dilute Mueller-Hinton broth (1:7) with inorganic ion
supplementation for broth dilution tests of T. maritimum isolates. This medium has been used with some
success in tests at 25 °C incubated for 48 hours.54,55 The effect inorganic ion supplementation may have
on both the growth of the bacterium and antimicrobial activity has not been studied in detail and should
be carefully analyzed prior to developing a standardized method and establishing interpretive criteria.
Inorganic ion supplementation per liter of distilled water:

- 25.0 g NaCl
- 5.0 g MgSO4 • 7H2O
- 1.0 g CaCl2 • 2H2O
- 1.0 g KCl
9.4.4 Renibacterium salmoninarum
Mueller-Hinton broth supplemented with 0.1% cysteine hydrochloride and kidney disease medium-2
(KDM-2) both have been used in broth microdilution studies56,57 on R. salmoninarum at 15 °C. Tests
conducted in the Mueller-Hinton broth were incubated for eight days, while those in KDM-2 were
conducted for 14, 21, and 30 days. The effect these extended incubation times may have on accurate MIC
determinations still needs to be elucidated. The Working Group cannot at this time recommend either of
these methods for determining MICs for R. salmoninarum.
9.4.5 Mycobacterium spp. and Nocardia seriolae
The Working Group refers researchers to the most current edition of CLSI/NCCLS document M24—
Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes for a more
comprehensive summary of standardized methods that may apply for testing acid-fast aquatic pathogens.
Also included in this document are proposed antimycobacterial agents and MIC values indicating
resistance of M. marinum, as well as broth microdilution breakpoints for testing Nocardiae and other
aerobic actinomycetes.
Most clinicians refrain from treating these nontuberculous acid-fast bacteria, due to the potential for
producing multidrug resistance. Mycobacterium marinum is a major pathogen of fish, and is also a
zoonotic pathogen that could prove life threatening for immunocompromised individuals; thus,
antimicrobial susceptibility testing may be warranted.
If treatment is warranted, veterinarians should take into account the poor penetrability of most
antimycobacterial drugs, the need for extended treatment times, and the fact that many diseased fish will
stop eating. All of these factors increase the likelihood for “underdosing” the fish, and consequently
“overdosing” the water, creating a potential health risk due to the propensity for bacterial resistance to
develop. Fish raised for human consumption should not be treated.
©

Number 24 M49-A
9.4.6 Erysipelothrix rhusiopathiae
Tests on this organism should follow methods described in M45 using CAMHB with 2.5 to 5% lysed
horse blood. Appropriate quality control organisms should also be used.
10 Reporting of MIC Results

Minimal inhibitory concentration values determined as described in this document may be reported
directly to the disease diagnosticians for potential treatment purposes. Currently, there are no
recommended interpretive criteria (resistant, intermediate, or susceptible breakpoints) for any
antimicrobial agents for use against any aquatic pathogens. Interpretive criteria need to be defined for
many antimicrobial agents used in aquaculture. Such criteria are extremely helpful to clinicians when
deciding what drug(s) to prescribe.
The interpretive criteria for some pathogens of terrestrial animals were established by the CLSI
Subcommittee on Veterinary Antimicrobial Susceptibility Testing and are contained in the most current
edition of CLSI/NCCLS document M31—Performance Standards for Antimicrobial Disk and Dilution
Susceptibility Tests for Bacteria Isolated From Animals. Interpretive categories were developed by
determining MICs and zone diameters at temperatures ≥ 35 °C for a large number of isolates. Second,
susceptibility test data were analyzed in relation to the pharmacokinetics of the drug from normal dosing
regimens. Third, whenever possible, the tentative in vitro interpretive criteria were analyzed in relation to
studies of clinical efficacy in the treatment of specific pathogens. These three parameters are the
foundation for establishing interpretive criteria for a given antimicrobial agent against a pathogen, and
should be included in efforts to establish criteria for aquatic pathogens.
Susceptible
The susceptible category implies that an infection due to the isolate may be appropriately treated with the
dosage regimen of an antimicrobial agent recommended for that type of infection and infecting species,
unless otherwise indicated.
Intermediate
The intermediate category implies that an infection due to the isolate may be appropriately treated in body
sites where the drugs are physiologically concentrated or when a high dosage of drug can be used; it also
indicates a “buffer zone” that should prevent small, uncontrolled, technical factors from causing major
discrepancies in interpretations.
Resistant
Resistant isolates are not inhibited by the usually achievable concentrations of the agent with normal
dosage schedules and/or fall in the range where specific microbial resistance mechanisms are likely (e.g.,
β-lactamases), and clinical efficacy has not been reliable in treatment studies.
11 Quality Control Procedures
11.1 Purpose
The goal of a quality control program is to assist in monitoring the following:
• the precision (repeatability) and accuracy (trueness) of the susceptibility test procedure;
©

Volume 26 M49-A
• the performance of reagents used in the test; and
• the performance of persons who carry out the tests and read the results.
The goals are best accomplished by, but not limited to the testing of quality control strains with known
susceptibility to the antimicrobial agents to be tested.
11.2 Quality Control Responsibilities
Modern laboratories rely heavily on pharmaceutical and diagnostic product manufacturers for provision
of reagents, media, or test systems for the performance of antimicrobial susceptibility tests. Although this
section is intended to apply to only the standard reference methods, it may be applicable to certain
commercially available test systems that are based primarily, or in part, on these methods.
A logical division of responsibility and accountability may be described as follows:
• manufacturers (in-house or commercial products):
— antimicrobial stability;
— antimicrobial labeling;
— potency of antimicrobial stock solutions;
— compliance with governmental quality system regulation;
— integrity of product; and
— accountability and traceability to consignee.
• laboratory (user):
— storage under the environmental conditions recommended by the manufacturer (to prevent drug
deterioration);
— proficiency of personnel performing tests; and
— adherence to the established procedure (e.g., inoculum preparation, incubation conditions,

interpretation of end points).
Manufacturers should design and recommend a quality control program that allows users to evaluate
those variables (e.g., inoculum density, storage/shipping conditions) that most likely could cause user
performance problems, and to determine that the test is performing correctly when used according to
established protocols.
11.3 Reference Strains for Quality Control
Ideal reference strains for quality control of broth dilution tests have MICs that fall near the middle of the
concentration range tested for all antimicrobial agents (e.g., an ideal control strain would be inhibited at
the fourth dilution of a seven-dilution series, but strains with MICs at either the third or fifth dilution
would also be acceptable). (See the most current edition of CLSI/NCCLS standard M31—Performance
Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals.)
©

Number 24 M49-A
In certain instances with newer, more potent antimicrobial agents, it may be necessary to test additional
quality control strains not normally tested in order to provide on-scale values.
When three or fewer adjacent doubling dilutions of an antimicrobial agent are tested by these methods,
quality control methods must be modified. One possible alternative is to use one control organism with a
modal MIC that is equal to or no less than one doubling dilution of the lower concentration and a second
control organism with a modal MIC that is equal to or no greater than one doubling dilution of the higher
concentration. The combination of results derived from testing these two strains should provide for at
least one on-scale end point. For users of commercial systems, this strategy might be used best by
selectively testing the most labile agents included in the panels.
It is currently recommended that either of the following strains be employed as quality control strains
when performing broth dilution tests at 22 ± 2 °C or 28 ± 2 °C with organisms in Group 1 (see Table 1).
The choice of a control strain for broth dilution susceptibility testing following the standard protocol
should be guided by the drug being investigated.
• Escherichia coli ATCC® 25922; NCIMB 12210; DSMc 1103
• Aeromonas salmonicida subsp. salmonicida ATCC® 33658; NCIMB 1102
E. coli (ATCC® 25922) was chosen as a quality control organism because of its wide temperature-
tolerance range, broad susceptibility pattern, and because it has already been extensively characterized in
susceptibility protocols for veterinary pathogens.
A. salmonicida subsp. salmonicida (ATCC® 33658) was also chosen as a quality control organism
because it has been shown to be susceptible to some drugs (e.g., erythromycin and chloramphenicol), for
which the E. coli strain has been shown to yield high MICs. Some Aeromonas salmonicida isolates
possess a surface layer (A-layer) shown to be associated with virulence and autoaggregation.58 After
repeated passes on culture media or incubation at supraoptimal temperatures, the A-layer positive
phenotype can be lost.59 The A-layer phenotype of various culture collection isolates of A. salmonicida
subsp. salmonicida ATCC® 33658; NCIMB 1102 has been shown to be A-layer negative. As the
presence/absence of the A-layer may affect susceptibility to some antimicrobials,60 laboratories should
check their isolates’ phenotype if problems arise in quality control tests using this strain.
Additional quality control strains have been established for testing veterinary pathogens at 35 ± 2 °C in
CAMHB with (for S. pneumoniae) and without 2.5 to 5% lysed horse blood (see Table 9). These quality
control strains may be used to test only those aquatic isolates that prefer this higher incubation
temperature over 28 °C or potential zoonotic pathogens.
• Escherichia coli ATCC® 25922; NCIMB 12210
• Staphylococcus aureus ATCC® 29213
• Pseudomonas aeruginosa ATCC® 27853; NCIMB 12469
• Enterococcus faecalis ATCC® 29212; NCIMB 13280
• Streptococcus pneumoniae ATCC® 49619
Quality control data generated when testing at 22 ± 2 °C, 28 ± 2 °C, or 35 ± 2 °C without the use of one
of the above (referenced) strains should not be reported as being in compliance with CLSI guidelines.
c
Deutsche Sammlumg von Mikroorganismen und Zellkulturen GmbH (www.dsmz.de)
©

Volume 26 M49-A
11.4 Storing and Testing Quality Control Strains
• For prolonged storage, these stock cultures may be maintained at -20 °C or below (preferably at
-60 °C or below or in liquid nitrogen) in a suitable stabilizer (e.g., 50% fetal calf serum in broth, 10 to
15% glycerol in tryptic soy broth, defibrinated sheep blood, or skim milk), or in a freeze-dried state
without altering their antimicrobial susceptibility significantly.
• Store working control cultures on tryptic soy agar slants at 2 to 8 °C and subculture each week for no
more than three successive weeks. Prepare new working cultures at least monthly from frozen, freeze-
dried, or commercial cultures.
• Test quality control strains using the standard broth dilution test procedure described herein, using the
same materials and methods that are used to test clinical isolates.
• Before testing, subculture the strains onto agar plates to obtain isolated colonies. Subculture frozen or
freeze-dried cultures twice prior to testing.
• Colonies are grown or suspended for testing according to the recommended inoculum preparation
procedures.
• A quality control culture can be used to monitor precision (repeatability) and accuracy (trueness) of
the broth dilution test as long as there is no significant change in the mean MIC that cannot be
attributed to faulty methodology. If an unexplained result suggests a change in the organism’s
inherent susceptibility, obtain a fresh culture of the control strain.
11.5 Control of Media and Materials
(1) Test each new batch or lot of macrodilution tubes or microdilution trays with the appropriate
reference strains to determine if MICs obtained with the batch fall within the expected range (see
Tables 6 through 9); if they do not, reject the batch.
(2) Incubate overnight at least one uninoculated tube or microdilution tray from each batch to verify
sterility of the medium.
(3) New lots of Mueller-Hinton broth used to prepare macrodilution tubes or microdilution trays may
need to be tested for acceptable cation content. For Mueller-Hinton broth, the MIC of gentamicin for
Pseudomonas aeruginosa ATCC® 27853 at 35 ± 2 ºC may be determined and compared with the
expected range (see Table 9). If the MIC is low, the broth may need to be supplemented with cations
as directed in Section 8.1(1).
(4) Keep records of the lot numbers of all materials and reagents used in performing susceptibility tests.
11.6 Broth Dilution Quality Control Ranges
Acceptable MIC quality control ranges for a single quality control test (single-drug/single-organism
combination) are listed in Tables 6 through 9. Monitor the overall performance of the test system using
these ranges by testing the appropriate control strains each day the test is performed or, if satisfactory
performance is documented (see Section 11.7.2.1), testing may be done weekly (see below).
©

Number 24 M49-A
11.7 Frequency of Quality Control Testing (see Appendix B)
The weekly quality control testing option outlined below is applicable to routine MIC tests only. Perform
quality control testing each test day for broth dilution tests performed infrequently.
11.7.1 Daily Testing
Performance is satisfactory for daily QC testing when no more than 1 out of 20 or 3 out of 30 consecutive
results for each antimicrobial agent/organism combination are outside the acceptable limit stated in
Tables 6 through 9. Corrective action by the laboratory is required when this frequency is exceeded.
11.7.2 Weekly Testing
11.7.2.1 Demonstrating Satisfactory Performance for Conversion From Daily to Weekly Quality Control
Testing
• Test all applicable control strains for 20 or 30 consecutive test days and document results.
• To convert from daily to weekly quality control testing, no more than 1 out of 20 or 3 out of 30 MICs
for each antimicrobial agent/organism combination may be outside the acceptable MIC ranges stated
in Tables 6 through 9.
11.7.2.2 Implementing Weekly Quality Control Testing
• Weekly quality control testing may be performed once satisfactory performance has been documented
(see Section 11.7.2.1 and Appendix C for a recommended weekly QC testing protocol).
— Perform quality control testing once per week and whenever any reagent component of the test
(e.g., a new lot of broth from the same manufacturer) is changed.
— If any of the weekly quality control results is out of the acceptable range, corrective action is
required (see Section 11.8).
• If a new antimicrobial agent is added or a different broth manufacturer is used, it must be tested for
20 or 30 consecutive days and satisfactory performance documented before it can be tested on a
weekly schedule. In addition, 20 or 30 days of testing are required if there is a major change in the
method of reading test results, such as conversion from a visual reading of MICs to an instrument
reading or conversion in the type of panel used (i.e., changing from breakpoint to MIC panels).
• These guidelines can also be used for testing systems in which an MIC is determined using three or
fewer adjacent doubling dilutions of an antimicrobial agent.
• For some drugs, quality control records may indicate the need for testing to be done more frequently
than once a week, because of the relatively rapid degradation of the drug (see Section 8.2.2).
11.8 Corrective Action
11.8.1 Out-of-Control Result Due to an Obvious Error
If there is an obvious reason for the out-of-control result, including:
• use of the wrong control strain;
©

Volume 26 M49-A
• obvious contamination of the strain or the medium; or
• inadvertent use of the wrong incubation temperature or conditions;
document the reason and retest the strain on the day the error is observed. If the repeated result is within
range, no further corrective action is required.
11.8.2 Out-of-Control Result Not Due to an Obvious Error
11.8.2.1 Immediate Corrective Action
If there is not an obvious reason for the out-of-control result, immediate corrective action is required.
• Test the implicated antimicrobial agent/organism combination on the day the error is observed and
monitor for a total of five consecutive test days. Document all results.
• If all five MICs for the antimicrobial agent/organism combination are within the acceptable ranges, as
defined in Tables 6 through 9, no additional corrective action is necessary.
• If any of the five MICs is still outside the acceptable range, additional corrective action is required
(see Section 11.8.2.2).
• Daily control tests must be continued until final resolution of the problem can be achieved.
11.8.2.2 Additional Corrective Action
When immediate corrective action does not resolve the problem, it is likely due to a system vs. a random
error. Investigate the following common sources of error to verify that:
• the turbidity standard has not expired, is stored properly, meets performance requirements (see
Section 7.1), and was adequately mixed prior to use;
• all materials used were within their expiration dates and stored at the proper temperature;
• the incubator is at the proper temperature and atmosphere;
• other equipment used (e.g., pipettors) are functioning properly;
• plates were stored at the proper temperature;
• the control strain has not changed and is not contaminated;
• inoculum suspensions were prepared and adjusted correctly; and
• inoculum for the test was prepared from a plate incubated for the correct length of time and in no
case, more than 48 hours old (72 hours in rare cases).
It may be necessary to obtain a new quality control strain (either from freezer storage or a reliable source)
and new lots of materials (including new turbidity standards), possibly from different manufacturers. If
the problem appears to be related to a manufacturer, contact the manufacturer. It may also be helpful to
exchange quality control strains and materials with another laboratory using the same method to
©

Number 24 M49-A
determine the root cause of unexplained system problems. Until the problem is resolved, it may be
necessary to use an alternate test method.
Once the problem is corrected, to return to weekly quality control testing, documentation of satisfactory
performance for another 20 to 30 consecutive days is required (see Section 11.7.2.1).
11.9 Other Control Procedures
11.9.1 Growth Control
Include in each microdilution broth tray or macrodilution broth series a growth control of basal medium
without antimicrobial agent to assess viability of the test organisms. With the broth tests, the growth
control also serves as a turbidity control for reading end points, which is particularly useful in testing
potentiated sulfonamides.
11.9.2 Purity Control
Following inoculation of broth dilution tests, streak a sample from a positive control well or tube on a
suitable agar plate and incubate overnight to detect mixed cultures and to provide freshly isolated colonies
in case retesting proves necessary. This step is particularly important for broth dilution tests where mixed
cultures are likely to go unrecognized.
11.9.3 Inoculum Control
Plate counts are performed periodically with representative inocula to ensure that the 0.5 McFarland
standard and the procedures for standardizing and diluting inocula remain under control. Samples for
plate counts are removed immediately after inoculation from the growth-control well of microdilution
trays or the growth-control tube of a macrodilution series.
11.9.4 End Point Interpretation Control
End point interpretation is monitored periodically to minimize variation in the interpretation of MIC end
points among observers. All laboratory personnel who perform these tests should independently read a
selected set of dilution tests. The results are recorded and compared to the results obtained by an
experienced reader. All readers should agree within ±1 twofold concentration increment of one another.61
12 Limitations of Broth Dilution Test Methods
12.1 Application to Various Organism Groups
The broth dilution methods described in this document are standardized for testing the nonfastidious,
rapidly growing pathogens in Group 1 (see Table 1), including most mesophilic (species with ideal
growth temperature of 20 °C to 45 °C) aquatic isolates, such as Enterobacteriaceae, Aeromonas
salmonicida, Aeromonas hydrophila and other aeromonads, Pseudomonas spp., Plesiomonas shigelloides,
Shewanella spp., and Vibrionaceae and related bacteria (nonobligate halophilic strains).
The quality control procedures described here cannot be used to control tests conducted at temperatures
other than 35 ± 2 °C using CAMHB with additional supplements (i.e., NaCl, lysed horse blood, serum).
©

Volume 26 M49-A
References
1
Alderman DJ, Smith P. Development of draft protocols of standard reference methods for antimicrobial agent susceptibility testing of
bacteria associated with fish diseases. Aquaculture. 2001;196:211-243.
2
Mitchell AJ. Finfish health in the United States (1609-1969): historical perspective, pioneering researchers and fish health workers, and
annotated bibliography. Aquaculture. 2001;196:347-438.
3
Marsh MC. Bacterium truttae, a new bacterium pathogenic to trout. Science. 1901a.;16:706.
4
Marsh MC. The brook trout disease. Trans Am Fish Soc. 1901b;30:66-81.
5
Davis HS. Culture and Diseases of Game Fishes. Berkeley, CA: University of California Press; 1953:332.
6
Sindermann CJ. Diseases of Marine Fishes. Neptune City, NJ: T.F.H. Publications; 1966:89.
7
Austin G, Austin DA. Bacterial Fish Pathogens; Disease in Farmed and Wild Fish. New York: Wiley; 1987.
8
Goldberg HS. Antibiotics: Their Chemistry and Non-Medical Uses. Princeton, NJ: D.Van Nostrand Company, Inc.; 1959:608.
9
Gutsell JS. Sulfa drugs and the treatment of furunculosis in trout. Science. 1946;104:85-86.
10
Gutsell JS, Snieszko SF. Response of brook, rainbow and brown trout to various dosages of sulfamerazine. Trans Am Fish Soc. 1947;77:93-
101.
11
U.S. Food and Drug Administration Center for Veterinary Medicine and Aquaculture. Available at:
http://www.fda.gov/cvm/aqualibtoc.htm. FDA Approved Drugs. Available at: http://www.fda.gov/cvm/4401.htm. FDA Chronological
History. Available at: http://www.fda.gov/cvm/chronological.htm. Animal Drugs for Minor Uses and Minor Species. Available at:
http://www.fda.gov/cvm/minortoc.htm.
12
Thorburn MA, Moccia RD. Use of chemotherapeutics on trout farms in Ontario. J Aquat Animal Health. 1993;5:85-91.
13
Stoffregen DA, Bowser PR, Babish JG. Antibacterial chemotherapeutants for finfish aquaculture: a synopsis of laboratory and field efficacy
and safety studies. J Aquatic Animal Health. 1996;8:181-207.
14
Shao ZJ. Aquaculture pharmaceuticals and biologicals: current perspectives and future possibilities. Adv Drug Deliv Rev. 2001;50:3:229-
243.
15
U.S. Food and Drug Administration NRSP-7 International Workshop on Minor Use and Minor Species (MUMS) October 2004. Available
at: http://www.fda.gov/cvm/mumsIntlMtg.htm.
16
Bullock GL, Wolf K. Infectious diseases of cultured fishes: current perspectives. U.S. Fish Wild Serv. 1986;Leafl 5.
17
Stoskopf MK, ed. Fish Medicine. Philadelphia, PA: W.B. Saunders Co.; 1993.
18
Noga EJ. Fish Disease: Diagnosis and Treatment. St. Louis: Mosby; 1996.
19
Woo PTK, Bruno DW, eds. Fish Diseases and Disorders, Volume 3: Viral, Bacterial and Fungal Infections. Oxon, U.K.: CABI Publishing;
1999.
20
Hawke JP. Bacterial disease agents. In: Stickney RR, ed. Encyclopedia of Aquaculture. New York: John Wiley & Sons, Inc.; 2000:69-97.
21
Plumb JA. Health Maintenance and Principal Microbial Diseases of Cultured Fish. Ames, Iowa: Iowa State University Press; 1999.
22
Garrett ES, dos Santos CL, Jahncke ML. Public, animal and environmental health implications of aquaculture. Emerg Infect Dis. 1997;3:4.
23
Alderman DJ, Hastings TS. Antibiotic use in aquaculture: development of antibiotic resistance potential for consumer health risk. Int J
Food Sci Technol. 1998;33:139-155.
24
Angulo F. Antimicrobial agents in aquaculture: potential impact on public health. APUA Newsletter. 2000;18(1).
25
Sorum H. Antibiotic resistance in aquaculture. Acta Vet Scand. 1999;99:29-36.
26
Benbrook CM. Antibiotic Drug Use in U.S. Aquaculture. Report for Institute for Agriculture and Trade Policy. March 2002.
27
Jacobsen P, Berglind L. Persistence of oxytetracycline in sediments from fish farms. Aquaculture. 1988;70:365-370.
28
Bjorklund H, Bondestam J, Bylund G. Residues of oxytetracycline in wild fish and sediments from fish farms. Aquaculture. 1990;86:359-
367.
29
Coyne R, Hiney M, O’Connor B, Kerry J, Cazabon D, Smith P. Concentration and persistence of oxytetracycline in sediments under a
marine salmon farm. Aquaculture. 1994;123:31-42.
©

Number 24 M49-A
30
Ervik A, Thorsen B, Eriksen V, Lunestad BT, Samuelsen OB. Impact of administering antibacterial agents on wild fish and blue mussels
Mytilus edulis in the vicinity of fish farms. Dis Aquat Org. 1994;18:45-51.
31
Herwig RP, Gray JP, Weston DP. Antibacterial resistant bacteria in surficial sediments near salmon net-cage farms in Puget Sound,
Washington. Aquaculture. 1997;149:263-283.
32
Miller RA, Walker RD, Baya A, et al. Antimicrobial susceptibility testing of aquatic bacteria: quality control disk diffusion ranges for
Esherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 at 22 and 28 degrees C. J Clin Microbiol.
2003;41:4318-4323.
33
American Society for Testing and Materials (ASTM). Compilation of ASTM Standard Definitions. 8th ed. Philadelphia: ASTM Committee
on Terminology; 1994.
34
ISO. Terms and definitions used in connection with reference materials. ISO Guide 30. Geneva: International Organization for
Standardization; 1992.
35
ISO. Statistics—Vocabulary and symbols. ISO 3534-1. Geneva: International Organization for Standardization; 1993.
36
ISO. Quality management systems – Fundamentals and vocabulary. ISO 9000. Geneva: International Organization for Standardization;
2000.
37
American Society for Quality Control (ASQC) Statistics Division. Glossary and Tables for Statistical Quality Control. 3rd ed. Milwaukee,
WI: ASQC Quality Press; 1996.
38
ISO. International Vocabulary of Basic and General Terms in Metrology. Geneva: International Organization for Standardization; 1993.
39
42 CFR §493; Laboratory Requirements. February 28, 1992.
40
Livermore DM, Williams JD. β-lactams: mode of action and mechanisms of bacterial resistance. In: Lorian V, ed. Antibiotics in Laboratory
Medicine. Baltimore, MD: Williams and Wilkins; 1996:502-578.
41
Stratton CW. Antimycobacterial agents. In: Lorian V, ed. Antibiotics in Laboratory Medicine. Baltimore, MD: Williams and Wilkins;
1996:579-603.
42
Payne MA, Baynes RE, Sundlof SE, et al. Drugs prohibited from extra-label use in food animals. JAVMA. 1999;215:28-32.
43
Singer RS, Johnson WO, Jeffrey JS, Chin RP, et al. A statistical model for assessing sample size for bacterial colony selection: a case study
of Escherichia coli and avian cellulitis. J Vet Diagn Invest. 2000;12:118-125.
44
McFarland J. The Nephelometer: an instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index
and for vaccines. JAMA. 1907;14:1176-1178.
45
Barry AL, Jones RN, Gavan TL. Evaluation of the micro-media system for quantitative antimicrobial drug susceptibility testing: a
collaborative study. Antimicrob Agents Chemother. 1978;13:61-69.
46
Medeiros AA, O’Brien TF, Wacker WE, Yulug NF. Effect of salt concentration on the apparent in-vitro susceptibility of Pseudomonas and
other gram-negative bacilli to gentamicin. J Infect Dis. 1978;124:Suppl-6.
47
Lamb JW, Mann JM, Simmons RJ. Factors influencing the microbiological assay of tobramycin. Antimicrob Agents Chemother.
1972;1:323-328.
48
Rigos G, Tyrpenou AE, Nengas I, et al. Pharmacokinetics of flumequine and in vitro activity against bacterial pathogens of gilthead sea
bream Sparus aurata. Dis Aquat Organ. 2003;54:35-41.
49
Thyssen A, Ollevier F. In vitro antimicrobial susceptibility of Photobacterium damselae subsp. piscicida to 15 different antimicrobial
agents. Aquaculture. 2001:259-269.
50
Zorrilla I, Chabrillón M, Arijo S, et al. Bacteria recovered from diseased cultured gilthead sea bream (Sparus aurata L.) in southwestern
Spain. Aquaculture. 2003;218:11-20.
51
Hawke JP, Thune RL. Systemic isolation and antimicrobial susceptibility of Cytophaga columnaris from commercially reared channel
catfish. J Aquat Anim Health. 1992;4:109-113.
52
Michel C, Kerouault B, Martin C. Chloramphenicol and florfenicol susceptibility of fish-pathogenic bacteria isolated in France: comparison
of minimum inhibitory concentration, using recommended provisory standards for fish bacteria. J Appl Microbiol. 2003;95:1008-1101.
53
Martinsen B, Oppegaard H, Wichstrom R, Myhr E. Temperature-dependent in vitro antimicrobial activity of four 4-quinolones and
oxytetracycline against bacteria pathogenic to fish. Antimicrob Agents Chemother. 1992;36:1738-1743.
54
Higgins MJ. An analysis of the diversity of marine flavobacteria on salmonid fish. Honours thesis. School of Aquaculture, University of
Tasmania, Australia, 2005.
©

Volume 26 M49-A
55
Lewin R, Lounsbery D. Isolation, cultivation and characterization of Flexibacteria. J Gen Microbiol. 1969;58:145-170.
56
Bandin I, Santos Y, Toranzo AE, Barja JL. MICs and MBCs of chemotherapeutic agents against Renibacterium salmoninarum. Antimicrob
Agents Chemother. 1991;35:1011-1013.
57
Hsu HM, Wooster GA, Bowser PR. Efficacy of enrofloxacin for the treatment of salmonids with bacterial kidney disease, caused by
Renibacterium salmoninarum. J Aquat Anim Health. 1994;6:220-223.
58
Sakai DK, Kimura T. Relationship between agglutinative properties of Aeromonas salmonicida strains isolated from fish in Japan and their
resistance to mechanisms of host defense. Fish Pathology (Tokyo). 1985;20:9-21.
59
Ishiguro EE, Kay WW, Ainsworth T, et al. Loss of virulence during culture of Aeromonas salmonicida at high temperature. J Bacteriol.
1981;148:333-340.
60
Henry MA, Secombes CJ. The A-layer influences the susceptibility of Aeromonas salmonicida to antibacterial peptides. Fish Shellfish
Immunol. 2000;10:637-642.
61
Barry AL, Braun LE. Reader error in determining minimal inhibitory concentrations with microdilution susceptibility test panels. J Clin
Microbiol. 1981;13:228-230.
©

Number 24 M49-A
Appendix A. Antimicrobial Agents Used in Global Aquaculture and Status of

Quality Control for Broth Dilution Susceptibility Testing
MIC QC Ranges for Testing at:
Antimicrobial Agents
22 °Ca 28 °Cb 35 ± 2 °Cc
Amoxicillind X
QC Ranges for Broth Dilution Testing of Aquatic Isolates
Drugs Used in Global Aquaculture With CLSI-Approved
Ampicillin X X X
Chloramphenicol X
Clindamycin X
Doxycyclinee X
Enrofloxacin X X X
Erythromycin X X X
Florfenicol X X X
Flumequine X
Fosfomycin X
Gentamicin X X X
Kanamycin X
Minocyclinee X
Nalidixic acid X
Nitrofurantoin X
Ormetoprim-sulfadimethoxinef X X X
Oxolinic acid X X X
Oxytetracyclinee X X X
Penicillin X
Rifampin X
Sulfisoxazole X
Tetracyclinee X
Tiamulin X
Trimethoprim-sulfamethoxazole X X X
Bicozamycin
Chlortetracyclinee
Approved QC Ranges for Broth Dilution Testing of
Drugs Used in Global Aquaculture With No CLSI-
Furanace
Furazolidone
Josamycin
Kitasamycin
Lincomycin
Miloxacin
Myroxacin
Aquatic Isolates
Novobiocin
Oleandomycin
Ormetoprim-sulfamonomethoxine
Penicillin dihydrostreptomycin
Piromidic acid
Sarafloxacin
Spiramycin
Streptomycin
Sulfamerazine
Sulfamethazine, sodium salt
Sulfamonomethoxine
Thiamphenicol
Tobicillin
Trimethoprim-sulfadiazine
Footnotes
a. See Tables 6 and 7.
b. See Table 8.
c. See Table 9.
d. Amoxicillin QC range exists only for testing in CAMHB with lysed horse blood (2.5 to 5% v/v).
e. Drugs in the tetracycline group are closely related and, with few exceptions, only oxytetracycline may need to be tested routinely.
f. Traditionally, trimethoprim-sulfamethoxazole may be used to predict susceptibility to ormetoprim-sulfadimethoxine; however, this has not been confirmed at
22 ± 2 °C or 28 ± 2 °C.
©

Volume 26 M49-A
Appendix B. Aerobic Dilution Daily Quality Control Testing Protocol
Test daily (Section 11.7.1)
≤1 out of 20 or 3 out of >3 out of 30 tests out

30 tests out of range of range
Corrective Action
Continue (Section 11.8)
daily
testing
Reason for error obvious Reason for error not obvious
Retest the same day Immediate Corrective Action

(Section 11.8.2.1)
Results in
range - Retest the same day and monitor
continue for five consecutive days
daily testing
All results in range Any results out of range
Additional Corrective Action

Continue (Section 11.8.2.2)
daily
testing
Investigate
possible
source of
errors
©

Number 24 M49-A
Appendix C. Aerobic Dilution Weekly Quality Control Testing Protocol
Demonstrate Satisfactory Performance

(Section 11.7.2.1)
≤1 out of 20 or ≤3 out of 30 results

out of range
Implement weekly testing

(Section 11.7.2.2)
Any result out of range

Corrective Action
(Section 11.8)
Reason for error obvious Reason for error not obvious
Retest the same day
Results in range Results out of range

Immediate Corrective Action
(Section 11.8.2.1)
Return to
weekly
testing Retest the same day and monitor for
five consecutive days
All results in range Any results out of range
Additional Corrective Action

(Section 11.8.2.2)
Return to
weekly
testing
Investigate
possible
source of
errors
©

Volume 26 M49-A
Table 1. Standard Methods for Broth Dilution Susceptibility Testing of Aquatic

Bacterial Pathogens
Organism Medium Incubation
a
Group 1: Nonfastidious bacteria
Enterobacteriaceae CAMHB 22 ºC (24-28 h and/or 44-48 h) or
Aeromonas salmonicida (nonpsychrophilic strains) 28 ºC (24-28 h)
Aeromonas hydrophila and other mesophilic Aeromonads
Pseudomonas spp.
Plesiomonas shigelloides
Shewanella spp.
Vibrionaceae and related bacteria (nonobligate halophilic
strains)
Footnote
a. Only Group 1 organisms have a standardized broth dilution susceptibility testing method.
©

Number 24 M49-A
Table 2. Potential Modifications for Broth Dilution Susceptibility Testing of Aquatic

Bacterial Pathogens
Organism Medium Incubation
Group 2: Vibrionaceae and Photobacteriaceae CAMHB + NaCl (1%) 22 ºC (24-28 h and/or 44-48 h) or
(obligate halophilic strains) 28 ºC (24-28 h and/or 44-48 h)
Group 3: Gliding bacteria
Flavobacterium columnare Diluted MHB (1:7) 28 °C (24-28 h and/or 44-48 h)
Flavobacterium psychrophilum and Diluted MHB (1:7)a 15 °C (44-48 h and/or 68-72 h)
Flavobacterium branchiophilum
Group 4: Streptococci
Lactococcus spp., Vagococcus salmoninarum CAMHB + LHB (2.5-5% v/v) 22 °C (44-48 h + CO2 if necessary
for growth)
Streptococcus spp., Carnobacterium maltaromaticum, CAMHB + LHB (2.5-5% v/v) 28 °C (24-28 h and/or 44-48 h +
and other streptococci CO2 if necessary for growth)
Group 5: Other fastidious bacteria
Psychrophilic Aeromonas salmonicida strains CAMHB 15 ºC (44-48 h)
Vibrio salmonicida and Moritella viscosa Unknown 4 °C or 15 ºC (6 days)
Tenacibaculum maritimum Diluted MHB (1:7) + inorganic 25 °C (44-48 h)
ion supplementation
Renibacterium salmoninarum Unknown 15 ºC (8 days)
Mycobacterium spp. and Nocardia seriolae See CLSI/NCCLS document See CLSI/NCCLS document
M24. M24.
Erysipelothrix rhusiopathiae CAMHB + LHB(2.5-5% v/v) See CLSI document M45.
Footnote
a. Recommended supplementation cannot be made at this time, but may include cations, horse or fetal calf serum, or NaCl.
©

Volume 26 M49-A
Table 3. Frequently Isolated Bacterial Pathogens of Fish

Bacterial pathogen Disease
Aeromonas caviae Motile aeromonad septicemia
Aeromonas hydrophila
Aeromonas sobria
Aeromonas salmonicida Furunculosis, Ulcer disease, Carp erythrodermatitis
Aerococcus viridans Gaffkemia
Carnobacterium maltaromaticum
Corynebacterium spp.
Edwardsiella ictaluri Enteric septicemia of catfish
Edwardsiella tarda Red pest disease, Edwardsiella septicemia
Flavobacterium branchiophilum Bacterial gill disease
Flavobacterium columnare Columnaris disease
Flavobacterium psychrophilum Cold-water disease, Rainbow trout fry syndrome
Lactococcus garvieae Lactococcosis, Lactococcus septicemia
Lactococcus piscium
Moritella viscosa Winter ulcer disease
Mycobacterium spp. Mycobacteriosis
Photobacterium damselae subsp. damselae Vibriosis
Photobacterium damselae subsp. piscicida Photobacteriosis, Fish pasteurellosis, Pseudotuberculosis
Piscirickettsia salmonis Piscirickettsiosis, Salmonid piscirickettsial septicemia
Plesiomonas shigelloides Winter disease
Pseudomonas spp. Pseudomoniasis
Pseudomonas anguilliseptica Red spot disease
Renibacterium salmoninarum Bacterial kidney disease
Shewanella putrefaciens
Streptococcus iniae Streptococcosis
Streptococcus difficilis Group B streptococcosis
Streptococcus dysgalactiae Group C streptococcosis
Tenacibaculum maritimum Salt-water columnaris, marine flexibacteriosis
Vagococcus salmoninarum Cold-water streptococcosis
Vibrio salmonicida Cold-water vibriosis, Hitra disease
Vibrio spp. Vibriosis
Yersinia ruckeri Enteric redmouth disease
©

Number 24 M49-A
Table 4. Solvents and Diluents for Preparation of Stock Solutions of Antimicrobial

Agents
Antimicrobial Agents Solvent Diluent
Amikacin Water
Amoxicillin Phosphate buffer, pH 6.0, 0.1 mol/L Phosphate buffer, pH 6.0, 0.1 mol/L
Ampicillin Phosphate buffer, pH 8.0, 0.1 mol/L Phosphate buffer, pH 6.0, 0.1 mol/L
Apramycin 95% ethanol Water
Azithromycin 95% ethanol or glacial acetic acidf Broth media
Azlocillin Water
Aztreonam Saturated solution sodium bicarbonate Water
Carbenicillin Water
Cefaclor Water
Cefadroxil Phosphate buffer, pH 6.0, 0.1 mol/L Water
Cefamandole Water
Cefazolin Phosphate buffer, pH 6.0, 0.1 mol/L Phosphate buffer, pH 6.0, 0.1 mol/L
Cefdinir Phosphate buffer, pH 6.0, 0.1 mol/L Water
Cefditoren Phosphate buffer, pH 6.0, 0.1 mol/L Water
Cefepime Phosphate buffer, pH 6.0, 0.1 mol/L Phosphate buffer, pH 6.0, 0.1 mol/L
Cefetamet Phosphate buffer, pH 6.0, 0.1 mol/L Water
Cefixime Phosphate buffer, pH 7.0, 0.1 mol/L Phosphate buffer, pH 7.0, 0.1 mol/L
Cefmetazole Water Water
Cefonicid Water
Cefoperazone Water
Cefotaxime Water
Cefotetan DMSOe,g Water
Cefoxitin Water
Cefpodoxime 0.10% (11.9 mmol/L) aqueous sodium bicarbonate Water
Cefprozil Water
Ceftazidime Sodium carbonated Water
Ceftibuten 1/10 vol DMSOg Water
Ceftiofur Water or broth Water or broth
Ceftizoxime Water
Ceftriaxone Water
Cefuroxime Phosphate buffer, pH 6.0, 0.1 mol/L Phosphate buffer, pH 6.0, 0.1 mol/L
Cephalexin Phosphate buffer, pH 6.0, 0.1 mol/L Water
Cephalothin Phosphate buffer, pH 6.0, 0.1 mol/L Water
Cephapirin Phosphate buffer, pH 6.0, 0.1 mol/L Water
Cephradine Phosphate buffer, pH 6.0, 0.1 mol/L Water
Chloramphenicol 95% ethanol Water
Cinoxacin 1/2 volume of water, then add 1 mol/L NaOH, dropwise to dissolve Water
Ciprofloxacin Water
Clarithromycin Methanole or glacial acetic acidf Phosphate buffer, pH 6.5, 0.1 mol/L
Clavulanic acid Phosphate buffer, pH 6.0, 0.1 mol/L Phosphate buffer, pH 6.0, 0.1 mol/L
Clinafloxacin Water
Clindamycin Water
Colistina Water Water
Dalbavancin DMSOg Water
Danofloxacin 1/2 volume of water, then add 1 mol/L NaOH, dropwise to dissolve Water
Daptomycin Water Water
©

Volume 26 M49-A
Table 4. (Continued)
Difloxacin 1/2 volume of water, then add 1 mol/L NaOH dropwise to dissolve Water
f
Dirithromycin Glacial acetic acid Water
Doripenem 0.85% physiological saline 0.85% physiological saline
Doxycycline Water
Enoxacin 1/2 volume of water, then 0.1 mol/L NaOH dropwise to dissolve Water
Enrofloxacin 1/2 volume of water, then add 1 mol/L NaOH dropwise to dissolve Water
Erythromycin 95% ethanol or glacial acetic acidf Water
Fleroxacin 1/2 volume of water, then 0.1 mol/L NaOH dropwise to dissolve Water
Florfenicol 95% ethanol Water
Flumequine 1/2 volume of water, then 0.1 mol/L NaOH dropwise to dissolve Water
Garenoxacin Water (with stirring)
Gatifloxacin Water (with stirring)
Gemifloxacin Water
Gentamicin Water
Imipenem Phosphate buffer, pH 7.2, 0.01 mol/L Phosphate buffer, pH 7.2, 0.01
mol/L
Kanamycin Water
Levofloxacin 1/2 volume of water, then 0.1 mol/L NaOH dropwise to dissolve Water
Linezolid Water
Loracarbef Water
Marbofloxacin 1/2 volume of water, then add 1 mol/L NaOH dropwise to dissolve Water
Mecillinam Water
Meropenem Water
Methicillin Water
Metronidazole DMSOe,g Water
Mezlocillin Water
Minocycline Water
Moxalactam
0.04 mol/L HCl (let sit for 1.5 to 2 h) Phosphate buffer, pH 6.0, 0.1
(diammonium salt)b
mol/L
Moxifloxacin Water
Nafcillin Water
Nalidixic acid 1/2 volume of water, then add 1 mol/L NaOH dropwise to dissolve
Netilmicin Water
Nitrofurantoinc Phosphate buffer, pH 8.0, 0.1 mol/L Phosphate buffer, pH 8.0, 0.1
mol/L
Norfloxacin 1/2 volume of water, then 0.1 mol/L NaOH dropwise to dissolve Water
Novobiocin Water
Ofloxacin 1/2 volume of water, then 0.1 mol/L NaOH dropwise to dissolve Water
Orbifloxacin 1/2 volume of water, then 1 mol/L NaOH dropwise to dissolve Water
Ormetoprim 0.05 mol/L lactice or hydrochlorice acid, 10% of final volume Water (may require heat)
Oxacillin Water
©

Number 24 M49-A
Table 4. (Continued)
Oxolinic acid 1/2 volume of water, then 1 mol/L NaOH dropwise to dissolve Water
Oxytetracycline 100% Methanol Water
Penicillin Water
Piperacillin Water
Pirlimycin Water
Polymyxin B Water Water
Quinupristin-dalfopristin Water
Rifampin Methanole [maximum concentration = 640 µg/mL] Water (with stirring)
Sparfloxacin Water
Spectinomycin Water
Streptomycin Water
Sulbactam Water
Sulfonamides 1/2 volume hot water and minimal amount of 2.5 mol/L NaOH to Water
dissolve
Tazobactam Water
Telavancin DSMOg Water
Telithromycin Glacial acetic acidf Water
Tetracycline Water
Tiamulin (if hydrogen Water
fumarate)
Ticarcillin Phosphate buffer, pH 6.0, 0.1 mol/L Phosphate buffer, pH 6.0, 0.1 mol/L
Tigecycline Water Water
Tilmicosin 95% ethanol Water
Tobramycin Water
Trimethoprim 0.05 mol/L lactice or hydrochlorice acid, 10% of final volume Water (may require heat)
Trimethoprim (if lactate) Water
Trospectomycin Water
Tylosin 95% ethanol Water
Vancomycin Water
Footnotes
a. The formulation of colistin used in antimicrobial susceptibility tests is colistin sulfate and not colistin methane sulfonate
(sulfomethate).
b. The diammonium salt of moxalactam is very stable, but it is almost pure R isomer. Moxalactam for clinical use is a 1:1
mixture of R and S isomers. Therefore, the salt is dissolved in 0.04 mol/L HCl and allowed to react for 1.5 to 2 hours to
convert it to equal parts of both isomers.
c. Alternatively, nitrofurantoin is dissolved in dimethyl sulfoxide.
d. Anhydrous sodium carbonate is used at a weight of exactly 10% of the ceftazidime to be used. The sodium carbonate is
dissolved in solution in most of the required water. The antibiotic is dissolved in this sodium carbonate solution, and water is
added to the desired volume. The solution is to be used as soon as possible, but it can be stored up to six hours at no more
than 25 °C.
e. These compounds are potentially toxic. Consult the material safety data sheets (MSDS) available from the product
manufacturer before using any of these materials.
f. For glacial acetic acid, use ½ volume of water, and then add glacial acetic acid dropwise until dissolved, not to exceed 2.5
µL/mL.
g. Dimethyl sulfoxide (DMSO)
©

Volume 26 M49-A
Table 5. Scheme for Preparing Dilutions of Antimicrobial Agents to Be Used in

Broth Dilution Susceptibility Tests
Antimicrobial Solution
CAMHBb Final
a
Step Concentration Source Volume + Volumea = Concentrationc Log2
1 5120 µg/mL Stock 1 mL 9 mL 512 µg/mL 9
2 512 Step 1 1 1 256 8
3 512 Step 1 1 3 128 7
4 512 Step 1 1 7 64 6
5 64 Step 4 1 1 32 5
6 64 Step 4 1 3 16 4
7 64 Step 4 1 7 8 3
8 8 Step 7 1 1 4 2
9 8 Step 7 1 3 2 1
10 8 Step 7 1 7 1 0
11 1 Step 10 1 1 0.5 -1
12 1 Step 10 1 3 0.25 -2
13 1 Step 10 1 7 0.125 -3
NOTE: This table is modified from Ericsson HM, Sherris JC. Antibiotic sensitivity testing. Report of an international
collaborative study. Acta Pathol Microbiol Scand. 1971;217 (suppl B):1-90.
Footnotes
a. The volumes selected can be any multiple of these figures, depending on the number of tests to be performed.
b. CAMHB, cation-adjusted Mueller-Hinton broth. Adjustment with cations, if necessary, occurs before this step.
c. Dilutions not centralized on 1 µg/mL (e.g., 0.75 µg/mL or 12 µg/mL) may be useful in some cases, but if tested, dilutions
including the established quality control ranges should be incorporated into any dilution test scheme.
©

Number 24 M49-A
Table 6. Acceptable Quality Control Ranges of MICs (µg/mL) for Reference Strains
When Tested in Cation-Adjusted Mueller-Hinton Broth at 22 ± 2 °C After 24 to 28
Hours
Aeromonas salmonicida subsp.
Escherichia coli
Antimicrobial Agent salmonicida
ATCC® 25922
ATCC® 33658a
Ampicillin 2 – 16 0.12 – 1
Enrofloxacin 0.004 – 0.015 0.008 – 0.03
Erythromycin — 4 – 16
Florfenicol 2 – 16 0.25 – 1
Flumequine 0.06 – 0.5 0.015 – 0.12
Gentamicin 0.12 – 0.5 0.25 – 1
Ormetoprim-sulfadimethoxine (1/19) 0.12/2.4 – 1/19 0.06/1.2 – 0.25/4.8
Oxolinic acid 0.03 – 0.25 0.008 – 0.03
Oxytetracycline 0.25 – 1 0.06 – 0.25
Trimethoprim-sulfamethoxazole (1/19) 0.03/0.6 – 0.12/2.4 0.03/0.6 – 0.12/2.4
NOTE 1: To determine whether the Mueller-Hinton medium contains excessive levels of thymidine or thymine, an E. faecalis
(ATCC® 29212 or 33186) should be tested with trimethoprim, sulfa compounds, or trimethoprim-sulfamethoxazole at
35 ± 2 °C. If excessive thymidine is present, an expected MIC within the susceptible category (trimethoprim-
sulfamethoxazole MIC ≤ 0.5/9.5 µg/mL) will shift to the resistant category (trimethoprim-sulfamethoxazole MIC >4/76
µg/mL).
NOTE 2: These MICs were obtained in several reference laboratories by broth microdilution. If four or fewer concentrations are
tested, quality control may be more difficult.
NOTE 3: For four-dilution ranges, results at the extremes of the acceptable range(s) should be suspect. Verify control validity
with data from other control strains.
Footnote
a. The A-layer phenotype of various culture collection isolates of Aeromonas salmonicida subsp. salmonicida ATCC®
33658; NCIMB 1102 has been shown to be A-layer negative. As the presence/absence of the A-layer may affect
susceptibility to some antimicrobials, laboratories should check their isolates’ phenotype if problems arise in quality
control tests using this strain.
©

Volume 26 M49-A
Hours
Escherichia coli
ATCC® 25922
ATCC® 33658 a
Ampicillin 4 – 16 0.25 – 1
Enrofloxacin 0.004 – 0.015 0.008 – 0.03
Flumequine 0.12 – 0.5 0.03 – 0.12
Gentamicin 0.25 – 1 0.25 – 2
Oxolinic acid 0.06 – 0.25 0.008 – 0.03
Oxytetracycline 0.5 – 2 0.12 – 1
µg/mL).
Footnote
©

Number 24 M49-A
Hours
Escherichia coli
ATCC® 25922
ATCC® 33658 a
Ampicillin 2 – 16 0.12 – 1
Enrofloxacin 0.008 – 0.03 0.004 – 0.03
Flumequine 0.12 – 0.5 0.015 – 0.12
Gentamicin 0.25 – 1 0.25 – 1
Oxolinic acid 0.06 – 0.25 0.008 – 0.03
Oxytetracycline 0.5 – 2 0.12 – 1
µg/mL).
Footnote
©

Volume 26 M49-A
in Cation-Adjusted Mueller-Hinton Broth (Except Where Noted) When Tested at
35 ± 2 °C After 16 to 20 Hours
Staphylococcus Enterococcus Escherichia Pseudomonas Streptococcus
Antimicrobial Agent aureus faecalis coli aeruginosa pneumoniae
ATCC® 29213a ATCC® 29212 ATCC® 25922 ATCC® 27853 ATCC® 49619b
Amikacin 1–4 64–256 0.5–4 1–4 –

Amoxicillin – – – – 0.03–0.12
0.03/0.015–
Amoxicillin-clavulanic acid 0.12/0.06–0.5/0.25 0.25/0.12–1.0/0.5 2/1–8/4 –
0.12/0.06
Ampicillin 0.5–2 0.5–2 2–8 – 0.06–0.25
Chloramphenicol 2–16 4–16 2–8 – 2–8
Clindamycin 0.06–0.25 4–16 – – 0.03–0.12
Doxycycline 0.12–0.5 2–8 0.5–2 – 0.015–0.12
Enrofloxacin 0.03–0.12 0.12–1 0.008–0.03 1–4 –
Erythromycin 0.25–1 1–4 – – 0.03–0.12
Florfenicol 2–8 2–8 2–8 – 1–4
Flumequine – – 0.25–1 – –
c
Fosfomycin 0.5–4 32–128 0.5–2 2–8 –
d
Gentamicin 0.12–1 4–16 0.25–1 0.5–2 –
Kanamycin 1–4 16–64 1–4 – –
Minocycline 0.06–0.5 1–4 0.25–1 – –
Nalidixic acid – – 1–4 – –
Nitrofurantoin 8–32 4–16 4–16 – 4–16
Ormetoprim-
– – 0.06/1.2–1/19 – –
sulfadimethoxine (1/19)
Oxolinic acid – – 0.06–0.25 – –
Oxytetracycline – – 0.5–4 – –
Penicillin 0.25–2 1–4 – – 0.25–1
Rifampin 0.004–0.015 0.5–4 4–16 16–64 0.015–0.06
e
Sulfisoxazole 32–128 32–128 8–32 – –
Tetracycline 0.12–1 8–32 0.5–2 8–32 0.12–0.5
Tiamulin 0.5–2 – – – 0.5–4
f
Trimethoprim 1–4 – 0.5–2 – –
Trimethoprim-
– – 0.03/0.6–0.12/2.4 8/152–32/608 0.12/2.4–1/19
sulfamethoxazole (1/19)
NOTE 1: Information in boldface type is considered tentative for one year.
NOTE 2: These MICs were obtained in several reference laboratories by broth microdilution. If four or fewer concentrations
are tested, quality control may be more difficult.
Footnotes
a. ATCC is a registered trademark of the American Type Culture Collection.

b. These quality control ranges for Streptococcus pneumoniae ATCC® 49619 are applicable only to tests performed by the broth
microdilution method using CAMHB with 2.5 to 5% lysed horse blood (v/v).
c. The approved MIC susceptibility testing method is agar dilution. Agar media should be supplemented with 25 µg/mL of glucose-6-
phosphate. Broth dilution should not be performed.
d. For control organisms for gentamicin and streptomycin high-level aminoglycoside screen tests for Enterococci, see Table 2D of the
M100 document.
e. This test should be performed by agar dilution only (see CLSI/NCCLS document M31—Performance Standards for Antimicrobial Disk
and Dilution Susceptibility Tests for Bacteria Isolated From Animals).
f. Very medium-dependent, especially with Enterococci.
©

Number 24 M49-A
Clinical and Laboratory Standards Institute consensus procedures include an appeals process that
is described in detail in Section 8 of the Administrative Procedures. For further information,
contact CLSI or visit our website at www.clsi.org.
Summary of Comments and Subcommittee Responses

M49-P: Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals;
Proposed Guideline
Section 4.1, Source
1. The last sentence does not indicate how long to leave out. M42-P quotes one to two hours at room temperature
to equilibrate before opening desiccators.
• Due to variations in room temperature, time is not specified. The one to two hours referenced in M42-P is
for removal of unopened disk containers from refrigeration stating “…and allow them to equilibrate to
room temperature before opening” without a timeframe specified. Users should refer to the
manufacturer’s recommendations.
Section 7.2, Direct Colony Suspension Method
2. This is the first time CAMHB is used, so it needs to be written out.
• This has been done as suggested.
Section 11.1, Purpose
3. Second bullet: Accuracy did not have (trueness) in brackets behind.
• This has been added.
Section 11.7.1, Daily Testing
4. Needs a reference to Appendix B like 11.7.2.2 refers to Appendix C.
• This has been added as suggested.
Appendix B. Aerobic Dilution Daily Quality Control Testing Protocol
5. The flowchart needs direction if 3 out of 30 occurs. Add a box and link it to corrective action. The start of the
chart is Section 11.7.1, but this does not refer to 1 out of 20. Remove this, add information to Section 11.7.1, or
change the reference to Section 11.7.2.2.
• Section 11.7.1 has been revised to reflect 1 out of 20 or 3 out of 30 with corresponding Appendix B
modified accordingly.
Appendix C. Aerobic Dilution Weekly Quality Control Testing Protocol
6. Add to the box “Implement Weekly Testing” the reference of Section 11.7.2.2.
• This has been added as suggested.
©

Volume 26 M49-A
NOTES
©

Number 24 M49-A
The Quality System Approach

Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system approach in the
development of standards and guidelines, which facilitates project management; defines a document structure via a
template; and provides a process to identify needed documents. The approach is based on the model presented in the
most current edition of CLSI/NCCLS document HS1—A Quality Management System Model for Health Care. The
quality management system approach applies a core set of “quality system essentials” (QSEs), basic to any
organization, to all operations in any healthcare service’s path of workflow (i.e., operational aspects that define how
a particular product or service is provided). The QSEs provide the framework for delivery of any type of product or
service, serving as a manager’s guide. The quality system essentials (QSEs) are:
Documents & Records Equipment Information Management Process Improvement

Organization Purchasing & Inventory Occurrence Management Service & Satisfaction
Personnel Process Control Assessment Facilities & Safety
M49-A addresses the quality system essentials (QSEs) indicated by an “X.” For a description of the other documents
listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the following page.
Purchasing &
Improvement
Organization
Management
Management
Information
Satisfaction
Assessment
Facilities &
Occurrence
Documents
Equipment
& Records
Service &
Personnel
Inventory
Control
Process
Process
Safety
X
M6
M23
M42
Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care.
Path of Workflow
A path of workflow is the description of the necessary steps to deliver the particular product or service that the
organization or entity provides. For example, CLSI/NCCLS document GP26⎯Application of a Quality
Management System Model for Laboratory Services defines a clinical laboratory path of workflow which consists of
three sequential processes: preexamination, examination, and postexamination. All clinical laboratories follow these
processes to deliver the laboratory’s services, namely quality laboratory information.
M49-A addresses the clinical laboratory path of workflow steps indicated by an “X.” For a description of the other
documents listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the following page.
Preexamination Examination Postexamination

receipt/processing
Sample collection
Results reporting
Sample transport
Results review
and follow-up
and archiving
Interpretation
Examination
Examination
management
ordering
Sample
Sample
X X X
M2 M2 M2
M7 M7 M7
M11 M11 M11
M42 M42 M42
Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care.
©

Volume 26 M49-A
Related CLSI/NCCLS Publications*

M2-A9 Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Ninth
Edition (2006). This document contains the current Clinical and Laboratory Standards Institute-recommended
methods for disk susceptibility testing, criteria for quality control testing, and updated tables for interpretive
zone diameters.
M6-A2 Protocols for Evaluating Dehydrated Mueller-Hinton Agar; Approved Standard—Second Edition
(2006). This document provides procedures for evaluating production lots of dehydrated Mueller-Hinton agar,
and for developing and applying reference media.
M7-A7 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved
Standard—Seventh Edition (2006). This document addresses reference methods for the determination of
minimal inhibitory concentrations (MICs) of aerobic bacteria by broth macrodilution, broth microdilution, and
agar dilution.
M11-A6 Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard—Sixth
Edition (2004). This standard provides reference methods for the determination of minimal inhibitory
concentrations (MICs) of anaerobic bacteria by agar dilution and broth microdilution.
M23-A2 Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters; Approved
Guideline—Second Edition (2001). This document addresses the required and recommended data needed for
the selection of appropriate interpretive standards and quality control guidelines for antimicrobial agents.
M42-A Methods for Antimicrobial Disk Susceptibility Testing of Bacteria Isolated From Aquatic Animals;
Approved Guideline (2006). This document provides the most up-to-date techniques for disk diffusion
susceptibility testing of aquatic species isolates, and criteria for quality control testing.
*
Proposed-level documents are being advanced through the Clinical and Laboratory Standards Institute consensus process;
therefore, readers should refer to the most current editions.
©

Number 24 M49-A
NOTES
©

Volume 26 M49-A
NOTES
©

Active Membership
(as of 1 April 2006)
Sustaining Members Centers for Medicare & Medicaid Eiken Chemical Company, Ltd. Associate Active Members
Services/CLIA Program Elanco Animal Health
Abbott Laboratories Chinese Committee for Clinical Electa Lab s.r.l. 35 MDSS/SGSAL (APO)
American Association for Clinical Laboratory Standards Enterprise Analysis Corporation 59th MDW/859 MDT/MTL (TX)
Chemistry Department of Veterans Affairs F. Hoffman-La Roche AG 78th Medical Group (GA)
AstraZeneca Pharmaceuticals Deutsches Institut für Normung Focus Bio-Inova, Inc. Academisch Ziekenhuis -VUB
Bayer Corporation (DIN) Future Diagnostics B.V. (Belgium)
BD FDA Center for Biologics Evaluation Gavron Group, Inc. ACL Laboratories (IL)
Beckman Coulter, Inc. and Research Gen-Probe ACL Laboratories (WI)
bioMérieux, Inc. FDA Center for Devices and Genaco Biomedical Products, Inc. Alexandria Hospital (IL)
CLMA Radiological Health Genomic Health, Inc. All Children’s Hospital (FL)
College of American Pathologists FDA Center for Veterinary Medicine Gentris Corporation Allina Health System (MN)
GlaxoSmithKline Iowa State Hygienic Laboratory Genzyme Diagnostics Allina Labs
Ortho-Clinical Diagnostics, Inc. Massachusetts Department of Public GlaxoSmithKline Alton Memorial Hospital (MN)
Pfizer Inc Health Laboratories Greiner Bio-One Inc. American University of Beirut
Roche Diagnostics, Inc. National Center of Infectious and Immunicon Corporation Medical Center (NY)
Parasitic Diseases (Bulgaria) Instrumentation Laboratory Anaheim Memorial Hospital (CA)
Professional Members National Health Laboratory Service International Technidyne Antwerp University Hospital
(South Africa) Corporation (Belgium)
AABB National Institute of Standards and I-STAT Corporation Arnett Clinic, LLC (IN)
American Academy of Family Technology Japan Assn. of Clinical Reagents Aspirus Wausau Hospital (WI)
Physicians National Pathology Accreditation Industries Associated Regional & University
American Association for Clinical Advisory Council (Australia) Johnson and Johnson Pharmaceutical Pathologists (UT)
Chemistry New York State Department of Research and Development, L.L.C. Atlantic Health System (NJ)
American Association for Health K.C.J. Enterprises Avista Adventist Hospital
Laboratory Accreditation Ontario Ministry of Health LabNow, Inc. Laboratory (CO)
American Association for Pennsylvania Dept. of Health LifeScan, Inc. (a Johnson & Johnson AZ Sint-Jan (Belgium)
Respiratory Care Saskatchewan Health-Provincial Company) Azienda Ospedale Di Lecco (Italy)
American Chemical Society Laboratory Medical Device Consultants, Inc. Barnes-Jewish Hospital (MO)
American Medical Technologists Scientific Institute of Public Health; Merck & Company, Inc. Barnes-Jewish St. Peters (MO)
American Society for Clinical Belgium Ministry of Social Micromyx, LLC Barnes-Jewish West County Hospital
Laboratory Science Affairs, Public Health and the MultiPhase Solutions, Inc. (MO)
American Society for Microbiology Environment Nanogen, Point-of-Care Diagnostics BayCare Health System (FL)
American Society of Hematology Div. Baystate Medical Center (MA)
American Type Culture Collection, Industry Members Nippon Becton Dickinson Co., Ltd. BC Biomedical Laboratories (Surrey,
Inc. Nissui Pharmaceutical Co., Ltd. BC, Canada)
ASCP AB Biodisk NovaBiotics (Aberdeen, UK) Bedford Memorial Hospital (VA)
Associazione Microbiologi Clinici Abbott Diabetes Care Novartis Institutes for Biomedical Boone Hospital Center (MO)
Italiani (AMCLI) Abbott Laboratories Research British Columbia Cancer Agency –
British Society for Antimicrobial Access Genetics Olympus America, Inc. Vancouver Cancer Center (BC,
Chemotherapy Acrometrix Corporation Optimer Pharmaceuticals, Inc. Canada)
Canadian Society for Medical AdvaMed Ortho-Clinical Diagnostics, Inc. Broward General Medical Center
Laboratory Science - Société Advancis Pharmaceutical (Rochester, NY) (FL)
Canadienne de Science de Corporation Ortho-McNeil Pharmaceutical Calgary Laboratory Services
Laboratoire Médical Affymetrix, Inc. (Raritan, NJ) (Calgary, AB, Canada)
Canadian Standards Association Agilent Technologies, Inc. Oxoid Inc. California Pacific Medical Center
CISMEL-SIMel Ammirati Regulatory Consulting Oxonica (UK) Canterbury Health Laboratories
Clinical Laboratory Management Anna Longwell, PC Paratek Pharmaceuticals (New Zealand)
Association Arpida Ltd Pathology Services Inc. Capital Health System Fuld Campus
COLA A/S ROSCO PathWork Informatics (NJ)
College of American Pathologists AstraZeneca Pharmaceuticals Pfizer Animal Health Capital Health System Mercer
College of Medical Laboratory Axis-Shield Diagnostics Pfizer Inc Campus (NJ)
Technologists of Ontario Axis-Shield POC AS Pfizer Italia Srl Carilion Consolidated Laboratory
College of Physicians and Surgeons Bayer Corporation – Tarrytown, NY Phadia AB (VA)
of Saskatchewan Bayer Corporation - West Haven, Powers Consulting Services Carolinas Medical Center (NC)
ESCMID CT PPD, Inc. Central Baptist Hospital (KY)
Hong Kong Accreditation Service Bayer HealthCare, LLC, Diagnostics Predicant Biosciences Central Ohio Primary Care
Innovation and Technology Div. - Elkhart, IN Procter & Gamble Pharmaceuticals, Physicians
Commission BD Inc. Central Texas Veterans Health Care
International Federation of Clinical BD Diabetes Care QSE Consulting System
Chemistry BD Diagnostic Systems Radiometer America, Inc. Centura Laboratory (CO)
Italian Society of Clinical BD VACUTAINER Systems Radiometer Medical A/S Chang Gung Memorial Hospital
Biochemistry and Clinical Beckman Coulter, Inc. Rapid Laboratories Microsystems (Taiwan)
Molecular Biology Beckman Coulter K.K. (Japan) Reliance Life Sciences Children’s Healthcare of Atlanta
Japanese Committee for Clinical Beth Goldstein Consultant (PA) Replidyne (GA)
Laboratory Standards Bio-Development S.r.l. Roche Diagnostics GmbH Children’s Hospital Medical Center
Joint Commission on Accreditation Bio-Inova Life Sciences Roche Diagnostics, Inc. (Akron, OH)
of Healthcare Organizations International Roche Diagnostics Shanghai Ltd. Children’s Hospital of Pittsburgh
Minneapolis Medical Research Biomedia Laboratories SDN BHD Roche Laboratories (Div. Hoffmann- (PA)
Foundation bioMérieux (NC) La Roche Inc.) Childrens Hospital of Wisconsin
National Academy of Clinical bioMérieux, Inc. (IL) Roche Molecular Systems Christian Hospital/Northeast/
Biochemistry bioMérieux, Inc. (MO) Sanofi Pasteur Northwest (MO)
National Society for Bio-Rad Laboratories, Inc. Sarstedt, Inc. Christus St. John Hospital (TX)
Histotechnology, Inc. Bio-Rad Laboratories, Inc. – France Schering Corporation City of Hope National Medical
Ontario Medical Association Quality Bio-Rad Laboratories, Inc. – Irvine, Schleicher & Schuell, Inc. Center (CA)
Management Program-Laboratory CA Seneca Medical Lab, Inc. Clarian Health - Methodist Hospital
Service Bio-Rad Laboratories, Inc. – Plano, SFBC Anapharm (IN)
RCPA Quality Assurance Programs TX Sphere Medical Holding Clendo Lab (PR)
PTY Limited Black Coast Corporation – Health Streck Laboratories, Inc. Clovis Community Hospital (CA)
SDS Pathology Care Systems Consulting Sysmex America, Inc. (Long Grove, CLSI Laboratories (PA)
Sociedad Espanola de Bioquimica Blaine Healthcare Associates, Inc. IL) Commonwealth of Kentucky
Clinica y Patologia Molecular Cepheid Sysmex Corporation (Japan) Community Care 5 (OH)
Sociedade Brasileira de Analises Chen & Chen, LLC TheraDoc Community College of Rhode Island
Clinicas Chi Solutions, Inc. Theravance Inc. Covance Central Laboratory
Sociedade Brasileira de Patologia Chiron Corporation Third Wave Technologies, Inc. Services (IN)
Clinica The Clinical Microbiology Institute Thrombodyne, Inc. Creighton University Medical Center
Taiwanese Committee for Clinical Comprehensive Cytometric THYMED GmbH (NE)
Laboratory Standards (TCCLS) Consulting Transasia Engineers Danish Institute for Food and
Turkish Society of Microbiology Control Lab Trek Diagnostic Systems, Inc. Veterinary Research (Denmark)
World Health Organization Copan Diagnostics Inc. TrimGen Corporation Dekalb Memorial Hospital (IN)
Cosmetic Ingredient Review Watin-Biolife Diagnostics and Detroit Health Department (MI)
Government Members Cubist Pharmaceuticals Medicals DFS/CLIA Certification (NC)
Cumbre Inc. Wyeth Research Diagnofirm Med Labs
Association of Public Health Dade Behring Inc. - Cupertino, CA XDX, Inc. Diagnósticos da América S/A (Sao
Laboratories Dade Behring Inc. - Deerfield, IL YD Consultant Paulo)
BC Centre for Disease Control Dade Behring Inc. - Glasgow, DE YD Diagnostics (Seoul, Korea) Dianon Systems (OK)
Caribbean Epidemiology Centre Dade Behring Inc. - Marburg, Dr. Everett Chalmers Hospital (New
Centers for Disease Control and Germany Trade Associations Brunswick, Canada)
Prevention Dade Behring Inc. - Sacramento, CA East Kootenay Regional Hospital
Centers for Medicare & Medicaid David G. Rhoads Associates, Inc. AdvaMed Laboratory (BC)
Services Diagnostic Products Corporation Japan Association of Clinical Evangelical Community Hospital
Digene Corporation Reagents Industries (Tokyo, Japan) (PA)
Faith Regional Health Services (NE)
FasTraQ Inc. (NV) Lewis-Gale Medical Center (VA) Pathology Associates of Boone (NC) Starke Memorial Hospital
Firelands Regional Medical Center L’Hotel-Dieu de Quebec (Quebec, Pediatrix Screening Inc. (PA) Laboratory (IN)
(OH) PQ) Penn State Hershey Medical Center State of Washington Department of
Fisher-Titus Memorial Hospital LifeCare Hospital Lab (PA) (PA) Health
(OH) Littleton Adventist Hospital Penticton Regional Hospital Stormont-Vail Regional Medical
Fleury S.A. (Brazil) Laboratory (CO) Laboratory (BC) Center (KS)
Florida Hospital East Orlando Long Beach Memorial Medical The Permanente Medical Group Sunnybrook & Women’s College
Fresno Community Hospital and Center (CA) (CA) Health Sciences Centre (Toronto,
Medical Center Long Island Jewish Medical Center Piedmont Hospital (GA) Ontario)
Gamma Dynacare Medical (NY) Pitt County Memorial Hospital (NC) Sunnybrook Health Science Center
Laboratories (Ontario, Canada) Magee Womens Hospital of Porter Adventist Hospital Laboratory (ON, Canada)
Gamma-Dynacare Laboratories UPMCHS (PA) (CO) Taiwan Society of Laboratory
(Brampton, Ontario) Magruder Memorial Hospital (OH) PPD (KY) Medicine
Geisinger Medical Center (Danville, Malmo University Hospital Presbyterian Hospital of Dallas (TX) Tan Tock Seng Hospital (Tan Tock
PA) (Sweden) Prince George Medical Lab (Prince Seng)
Geisinger Wyoming Valley Medical Manipal Acunova Pvt., Ltd. (India) George, BC) Temple Univ. Hospital - Parkinson
Center (Wilkes-Barre, PA) Martin Luther King/Drew Medical Provincial Health Services Authority Pav. (PA)
General Health System (LA) Center (CA) (Vancouver, BC, Canada) Texas Department of State Health
Hamad Medical Corporation (Qatar) Massachusetts General Hospital Provincial Laboratory for Public Services (TX)
Harris Methodist Fort Worth (TX) (Microbiology Laboratory) Health (Edmonton, AB, Canada) Timmins and District Hospital
Hartford Hospital (CT) MDS Metro Laboratory Services Quest Diagnostics, Inc (San Juan (Canada)
Health Network Lab (PA) (Burnaby, BC, Canada) Capistrano, CA) The Children’s University Hospital
Health Partners Laboratories (VA) Mease Countryside Hospital (FL) Quintiles Laboratories, Ltd. (GA) (Ireland)
High Desert Health System (CA) Mease Dunedin Hospital (FL) Regions Hospital Tri-Cities Laboratory (WA)
Hoag Memorial Hospital Medical Centre Ljubljana (Slovinia) Research Medical Center (MO) Tripler Army Medical Center (HI)
Presbyterian (CA) Medical College of Virginia Rhode Island Department of Health Tuen Mun Hospital (Hong Kong)
Holy Cross Hospital (MD) Hospital Laboratories Tuttle Army Health Clinic (GA)
Hôpital Maisonneuve - Rosemont Medical University of South Riverview Hospital (BC, Canada) UCSD Medical Center (CA)
(Montreal, Canada) Carolina (SC) Riyadh Armed Forces Hospital UCSF Medical Center China Basin
Hôpital Sainte - Justine (Quebec) Memorial Hospital (OH) (Riyadh) (CA)
Hospital Albert Einstein (Brazil) Memorial Medical Center Royal Inland Hospital Laboratory UNC Hospitals (NC)
Hospital Consolidated Laboratories (Napoleon Avenue, New Orleans, (BC) Union Clinical Laboratory (Taiwan)
(MI) LA) Rural Health Ventures (NE) Universita Campus Bio-Medico
Hospital for Sick Children (Toronto, Memorial Regional Hospital (FL) SAAD Specialist Hospital (Saudi (Italy)
ON, Canada) Methodist Hospital (TX) Arabia) University Medical Center (CA)
Hôtel Dieu Grace Hospital (Windsor, Missouri Baptist Medical Center SAE – Laboratorio Medico (Brazil) University of Chicago Hospitals
ON, Canada) (MO) St. Agnes Healthcare (MD) (IL)
Hunter Area Pathology Service (DE) Montreal General Hospital (Canada) St. Anthony Hospital Central University of Colorado Hospital
Hunterdon Medical Center (NJ) Mount Sinai Hospital (NY) Laboratory (CO) University of Debrecen Medical
Indiana University Mountainside Hospital (NJ) St. Anthony Hospital North Health and Science Center
Interior Health Authority MRL Europe (Zaventem) Laboratory (CO) (Hungary)
Island Hospital (WA) National Healthcare Group St. Anthony’s Hospital (FL) University of Illinois Medical
Jackson Health System (FL) (Singapore) St. Barnabas Medical Center (NJ) Center (IL)
Jackson South Community Hospital National University Hospital St. Christopher’s Hospital for University of Maryland Medical
(FL) (Singapore) Children (PA) System
Jacobi Medical Center (NY) NB Department of Health & St-Eustache Hospital (Quebec, University of MN Medical Center -
John C. Lincoln Hospital (AZ) Wellness (New Brunswick, Canada) Fairview
John H. Stroger, Jr. Hospital of Cook Canada) St. John Hospital and Medical University of the Ryukyus (Japan)
County (IL) NC State Lab of Public Health (NC) Center (MI) University of Virginia Medical
Johns Hopkins at Bayview (MD) The Nebraska Medical Center St. John Regional Hospital Center
Johns Hopkins Howard County New England Fertility Institute (CT) (St. John, NB, Canada) University of Washington
General Hospital (MD) New York University Medical St. Joseph’s Hospital (FL) UPMC Horizon Hospital (PA)
Johns Hopkins Medical Institutions Center St. Joseph’s Hospital and Medical U.S. Army Health Clinic – Vicenza
(MD) New Zealand Diagnostic Group Center (AZ) (APO)
Kadlec Medical Center (WA) NHG Diagnostics (Singapore) St. Joseph’s Hospital-Marshfield US LABS, Inc. (CA)
Kaiser Permanente (CA) Nichols Institute Diagnostics (CA) Clinic (WI) USA MEDDAC-AK
Kaiser Permanente (MD) NorDx (ME) St. Jude Children’s Research UZ-KUL Medical Center (Belgium)
Kantonsspital Aarau AG (Aarau, North Bay Hospital Hospital (TN) VA (Asheville) Medical Center (NC)
AG) North Coast Clinical Laboratory St. Louis Children’s Hospital (MO) Valley Health (VA)
Karolinska University Hospital (OH) St. Margaret Memorial Hospital Vejle Hospital (VA)
Kelowna General Hospital North Shore Hospital Laboratory (PA) Vernon Jubilee Hospital Laboratory
Laboratory (BC) (Auckland, New Zealand) St. Mary Corwin Regional Medical Virginia Beach General Hospital
King Abdulaziz Medical City – North Shore - Long Island Jewish Center Laboratory (CO) (VA)
Jeddah (Jeddah, WR, Saudi Health System Laboratories (NY) St. Michael’s Hospital (Toronto, Warren Hospital (NJ)
Arabia) Northern Plains Laboratory (ND) ON, Canada) Washington Hospital Center (DC)
King Fahad National Guard Hospital Northwestern Memorial Hospital San Antonio Community Hospital Waterford Regional Hospital
(Saudi Arabia) (IL) (TX) (Ireland)
King Faisal Specialist Hospital Ochsner Clinic Foundation (LA) San Francisco General Hospital Wellstar Health Systems (GA)
(Saudi Arabia) Orange Coast Memorial Medical (CA) West China Second University
Kootenay Boundary Regional Center (CA) Santa Clara Valley Medical Center Hospital, Sichuan University (P.R.
Hospital Laboratory (BC) Orlando Regional Healthcare System (CA) China)
Kosciusko Laboratory (IN) (FL) Shands at the University of Florida William Beaumont Army Medical
LabCorp (NC) Overlook Hospital (NJ) SJRMC Plymouth Laboratory (IN) Center (TX)
Laboratoire de Santé Publique du Parker Adventist Hospital Sonora Quest JV (AZ) William Beaumont Hospital (MI)
Quebec (Canada) Laboratory (CO) South Bend Medical Foundation (IN) Winn Army Community Hospital
Laboratory Alliance of Central New Parkland Health Center (MO) South Florida Baptist Hospital (FL) (GA)
York (NY) Pathology Associates Medical South Texas Laboratory (TX) Women’s Health Laboratory (TX)
Laboratory Corporation of America Laboratories (WA) South Western Area Pathology Woodlawn Hospital (IN)
(NJ) Service (Australia) York Hospital (PA)
Specialty Laboratories, Inc. (CA)
OFFICERS BOARD OF DIRECTORS
Robert L. Habig, PhD, Susan Blonshine, RRT, RPFT, FAARC Gary L. Myers, PhD
President TechEd Centers for Disease Control and Prevention
Abbott Laboratories
Maria Carballo Valerie Ng, PhD, MD
Gerald A. Hoeltge, MD, Health Canada Alameda County Medical Center/
President-Elect Highland General Hospital
The Cleveland Clinic Foundation Russel K. Enns, PhD
Cepheid Klaus E. Stinshoff, Dr.rer.nat.
Wayne Brinster, Digene (Switzerland) Sàrl
Secretary Mary Lou Gantzer, PhD
BD Dade Behring Inc. James A. Thomas
ASTM International
W. Gregory Miller, PhD, Lillian J. Gill, DPA
Treasurer FDA Center for Devices and Radiological Health Kiyoaki Watanabe, MD
Virginia Commonwealth University Keio University School of Medicine
Jeannie Miller, RN, MPH
Thomas L. Hearn, PhD, Centers for Medicare & Medicaid Services
Immediate Past President
Centers for Disease Control and Prevention
Glen Fine, MS, MBA,

Executive Vice President

940 West Valley Road T Suite 1400 T Wayne, PA 19087 T USA T PHONE 610.688.0100
FAX 610.688.0700 T E-MAIL: customerservice@clsi.org T WEBSITE: www.clsi.org T ISBN 1-56238-612-3
(Formerly NCCLS)

Methods For Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals Approved Guideline

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Methods For Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals Approved Guideline

Uploaded by

Copyright:

Available Formats

Product Name: Infobase 2009 - Release Date: March 2009

Methods for Broth Dilution Susceptibility

This document is protected by copyright. Downloaded on 2/23/2009

Clinical and Laboratory Standards Institute

This document is protected by copyright. Downloaded on 2/23/2009

This document is protected by copyright. Downloaded on 2/23/2009

Reproduced with permission, from CLSI publication M49-A—Methods for Broth

Copyright ©2006. Clinical and Laboratory Standards Institute.

This document is protected by copyright. Downloaded on 2/23/2009

Area Committee on Microbiology

Thomas R. Shryock, PhD Michael A. Pfaller, MD

Subcommittee on Veterinary Antimicrobial Susceptibility Testing

Thomas R. Shryock, PhD Henry Heine, PhD Melanie R. Berson, DVM

This document is protected by copyright. Downloaded on 2/23/2009

Advisors (Continued) Marilyn N. Martinez, PhD Clyde Thornsberry, PhD

Working Group on Aquaculture

This document is protected by copyright. Downloaded on 2/23/2009

Committee Membership........................................................................................................................ iii

Foreword .............................................................................................................................................. vii

7 Inoculum Preparation for Dilution Tests ...................................................................................9

11 Quality Control Procedures......................................................................................................18

This document is protected by copyright. Downloaded on 2/23/2009

Appendix B. Aerobic Dilution Daily Quality Control Testing Protocol...............................................29

Appendix C. Aerobic Dilution Weekly Quality Control Testing Protocol ...........................................30

Table 3. Frequently Isolated Bacterial Pathogens of Fish.....................................................................33

Summary of Comments and Subcommittee Responses........................................................................42

The Quality System Approach..............................................................................................................44

Related CLSI/NCCLS Publications ......................................................................................................45

This document is protected by copyright. Downloaded on 2/23/2009

This document is protected by copyright. Downloaded on 2/23/2009

Renate Reimschuessel, PhD, VMD, Co-Chairholder

Antimicrobial susceptibility, aquaculture, aquatic, broth microdilution, macrodilution, minimal inhibitory

This document is protected by copyright. Downloaded on 2/23/2009

Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From

This document is protected by copyright. Downloaded on 2/23/2009

Antimicrobial Susceptibility Test Interpretive Category – 1) classification based on a bacterium’s in

confidence limit – a number or pair of numbers that defines a confidence interval.

This document is protected by copyright. Downloaded on 2/23/2009

minimal inhibitory concentration//MIC – the lowest concentration of an antimicrobial agent that

optical density – deprecated term. See and use absorbance above.

This document is protected by copyright. Downloaded on 2/23/2009

4.2 Weighing Antimicrobial Powders

Example: meropenem trihydrate

Certificate of analysis data:

Assay purity (by HPLC): 99.8%

Measured water content (by Karl Fischer analysis): 12.1% (w/w)

Potency calculation from above data:

Potency = (Assay purity) x (Active fraction) x (1- Water Content)

Potency = (998) x (1.0) x (1- 0.121)

Potency = 877 µg/mg or 87.7%

This document is protected by copyright. Downloaded on 2/23/2009

Weight (mg) = Volume (mL) • Concentration (µg/mL) (1)

Volume (mL) = Weight (mg) • Potency (µg/mg) (2)

182.6 mg • 750 µg/mg

Therefore, the 182.6 mg of antimicrobial powder is to be dissolved in 107 mL of diluent.

4.3 Preparing Stock Solutions

• Solubilize the antimicrobial powder in a minimum amount of solvent.

This document is protected by copyright. Downloaded on 2/23/2009

4.4 Number of Concentrations Tested

5 Selection of Antimicrobial Agents for Routine Testing and Reporting

5.1 Routine Reports

5.2 Antimicrobial Classes

Aminopenicillins have activity against penicillin-susceptible gram-positive bacteria, as well as some