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September 1999

M26-A

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Methods for Determining Bactericidal Activity
of Antimicrobial Agents; Approved Guideline
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This document provides procedures for determining the lethal

activity of antimicrobial agents.

A guideline for global application developed through the Clinical and Laboratory Standards Institute consensus process.
Clinical and Laboratory Standards Institute
Setting the standard for quality in medical laboratory testing around the world.

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 Vol. 19 No. 18 M26-A
 
 
 
 
 
 
 
 
 
 
 
Methods for Determining Bactericidal Activity of Antimicrobial
Agents; Approved Guideline
 
 
 
Abstract
 

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Established laboratory methods that can assess the bactericidal activity of an antimicrobial agent are
needed, both because of the increase in the number of patients who do not have completely normal
host immune defenses and because of the new classes of antimicrobial agents that have been
introduced. Clinical cure depends largely upon host factors. Bactericidal tests can provide a rough

 
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prediction of bacterial eradication. It should be noted, however, that other factors (e.g., postantibiotic
effect and the growth-inhibitory effects of sub-MIC concentrations of antibiotics) may also impact
bacteriologic response of patients. The special susceptibility tests that assess lethal activity are not
routinely applied to all microorganisms, but are applied in unusual situations; e.g., endocarditis. Uniform
test procedures are thus needed to permit comparison of different datasets.

The methods for bactericidal testing are now evolving, but more work is needed with the methodological
aspects and clinical correlations. The techniques described in this document are intended primarily for
testing aerobic bacteria that grow after incubation in adjusted Mueller-Hinton broth or adjusted Mueller-
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Hinton broth supplemented with human serum or an ultrafiltrate thereof.
 
Clinical and Laboratory Standards Institute (CLSI). Methods for Determining Bactericidal Activity of
Antimicrobial Agents; Approved Guideline. CLSI document M26-A ( ISBN 1-56238-384-1). Clinical and
Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087.
 
 
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The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a
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i
 
 
 
M26-A
ISBN 1-56238-384-1
ISSN 0273-3099
September 1999
Methods for Determining Bactericidal Activity of Antimicrobial
Agents; Approved Guideline
 
 
 
 
 
 

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Volume 19 Number 18
 
Arthur L. Barry, Ph.D.
William A. Craig, M.D.

 
 
 
 
 
 
Harriette Nadler, Ph.D.
L. Barth Reller, M.D.
Christine C. Sanders, Ph.D.
Jana M. Swenson, M.M.Sc. PL
 
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 September 1999 NCCLS
 

Copyright ©1999 Clinical and Laboratory Standards Institute. Except as stated below, any reproduction of
content from a CLSI copyrighted standard, guideline, companion product, or other material requires
express written consent from CLSI. All rights reserved. Interested parties may send permission requests to
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CLSI hereby grants permission to each individual member or purchaser to make a single reproduction of
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Suggested Citation
CLSI. Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline.
CLSI document M26-A. Wayne, PA: Clinical and Laboratory Standards Institute; 1999.
 

 
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ISBN 1-56238-384-1
ISSN 0273-3099
 
 
iv
Vol. 19 No. 18 M26-A

Contents

Abstract.............................................................................................................................. i

Committee Membership ........................................................................................................ v

Active Membership ............................................................................................................. vii

Foreword...........................................................................................................................xv

1 Introduction ........................................................................................................... 1

1.1 Rationale .................................................................................................... 1

1.2 Methods ..................................................................................................... 1
1.3 Problems..................................................................................................... 1
1.4 Clinical Relevance ........................................................................................ 6

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2 Test Methods for Determining Bactericidal Activity ...................................................... 8

2.1 Preparation of Antimicrobial Solutions ............................................................. 8

2.2 Broth Medium.............................................................................................. 8

3
2.3
2.4
2.5

3.1
3.2
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Time-Kill Method .......................................................................................... 9
Dilution Methods........................................................................................ 10
Preparing Inoculum ..................................................................................... 12

Determining Endpoints........................................................................................... 13

Time-Kill Method ........................................................................................ 13

Agar Dilution Plate Count Method................................................................. 14
3.3 Minimal Bactericidal Concentrations .............................................................. 14
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4 Interpretation ....................................................................................................... 15

5 Quality Control ..................................................................................................... 16

5.1 Purpose .................................................................................................... 16

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5.2 Method Control.......................................................................................... 16

5.3 Selecting Reference Strains ......................................................................... 16
5.4 Suggested Quality Control Strains ................................................................ 16
5.5 Batch or Lot Control ................................................................................... 16
5.6 Other Control Procedures ............................................................................ 16

Table 1. Rejection Value and Calculated Sensitivity and

Specificity for Each Initial Concentration on the Basis
of Duplicate 0.01-mL Samples ................................................................................ 17

Table 2. Rejection Value and Calculated Sensitivity and

Specificity for Each Initial Inoculum Concentration on the
Basis of a Single 0.01-mL Sample ........................................................................... 18

Table 3. Suitable Quality Control Ranges for MICs and

MBCs With and Without Human Serum Using ATCC Strains ....................................... 19

xiii
September 1999 NCCLS

Contents (Continued)

References ....................................................................................................................... 20

Additional References......................................................................................................... 26

Summary of Comments and Subcommittee Responses ........................................................... 27

Related NCCLS Publications ................................................................................................ 29

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xiv
Vol. 19 No. 18 M26-A

Foreword

All of the susceptibility test methods commonly performed by clinical microbiology laboratories (e.g.,
disk diffusion, broth dilution, and agar dilution) measure the inhibitory activity (MIC) of an antimicrobial
agent.1,2,3 In most clinical situations, this is sufficient as the role of the antibiotic is to prevent the
spread of bacteria from the focus of infection by preventing microbial replication at new sites; the active
participation of the host's defense mechanisms finally achieves bacterial eradication and clinical cure.4
Antimicrobial assays can provide additional valuable information on the pharmacokinetics of the
agent(s) being used and, when combined with the MICs, can allow bacterial eradication to be predicted.

On occasion, it may be necessary to achieve bactericidal activity with an antimicrobial agent. This need
has been well documented for endocarditis5 and has been suggested by some for meningitis,6 for
osteomyelitis,7 as well as for infections in immunocompromised patients.8 The clinical occurrence of
tolerance9 may on rare occasion necessitate bactericidal testing.

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When assessment of bactericidal activity is deemed appropriate, an in vitro test method such as the
MBC determination or the use of time-kill kinetic methodology may be useful. Bactericidal activity
against the patient's isolate by the antibiotic tested allows eradication to be predicted based upon the
usual dosing of this antibiotic or based upon the results of an antimicrobial assay. When clinical
experience is lacking and assay methods are not readily available, the serum bactericidal test which

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integrates both pharmacodynamic and pharmacokinetic properties may be more useful. Depending on
certain modifications to the serum bactericidal test, the test can provide a quantitative assessment of
bactericidal activity relative to the MBC (the serum bactericidal titer), a dynamic assessment of rapidity
of killing over time (the serum bactericidal rate), or both the magnitude of serum bactericidal activity and
its duration (the area-under-the-bactericidal-titer-curve). In addition, methods using serum from persons
(e.g., volunteers) receiving antibiotics (ex vivo) can be used to assess antimicrobial bactericidal activity
across drug classes or between members of a class against a wide variety of microorganisms.

Because of the complexity involved with the serum bactericidal test (including the particular method
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used, the proper collection of timed serum specimens, and the interpretation of results), and the lack
of clinical data clearly documenting the usefulness of this test for most infections, it is recommended
that consultation with the microbiology laboratory be obtained as a prerequisite for this test. The
assistance of the laboratory's director is useful in (1) determining if such a test is needed; (2) selecting
NCCLS recommended methodology for testing; and (3) interpreting the results. Techniques for the
conduct of the serum bactericidal test may be found in the most current edition of NCCLS document
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M21—Methodology for the Serum Bactericidal Test.

This document describes the details of bactericidal testing and, in particular, the effects of variations
in methodology. This information has been obtained largely from published data. Use of these guidelines
should result in uniform methodology for bactericidal testing that is sufficiently practical for use in the
clinical microbiology laboratory. The methodology described in this document does not imply per se that
bactericidal testing is clinically relevant, but instead allows such testing to be used as a tool to assess
clinical relevance. The techniques described in this document are intended primarily for testing aerobic
bacteria that grow well after overnight incubation in either Mueller-Hinton broth or in Mueller-Hinton
broth supplemented with human serum as described in Section 2.2.1. Modifications for more fastidious
microorganisms such as anaerobes will be described in detail in the future.

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September 1999 NCCLS

Standard Precautions

Because it is often impossible to know what might be infectious, all human blood specimens are to be
treated as infectious and handled according to “standard precautions.” Standard precautions are new
guidelines that combine the major features of “universal precautions and body substance isolation”
practices. Standard precautions cover the transmission of any pathogen and thus are more
comprehensive than universal precautions which are intended to apply only to transmission of blood-
borne pathogens. Standard precaution and universal precaution guidelines are available from the U.S.
Centers for Disease Control and Prevention (Guideline for Isolation Precautions in Hospitals, Infection
Control and Hospital Epidemiology, CDC, Vol 17;1:53-80.), [MMWR 1987;36(suppl 2S):2S-18S] and
(MMWR 1988;37:377-382, 387-388). For specific precautions for preventing the laboratory
transmission of blood-borne infection from laboratory instruments and materials; and recommendations
for the management of blood-borne exposure, refer to NCCLS document M29—Protection of Laboratory
Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and
Tissue.

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Key Words

Bactericidal activity, minimal bactericidal concentration (MBC), minimal lethal concentration, serum
bactericidal concentration, time-kill determination

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xvi
Vol. 19 No. 18 M26-A

Methods for Determining Bactericidal Activity

of Antimicrobial Agents; Approved Guideline

1 Introduction MIC.10,11,12 This rate of killing is termed

time-dependent bactericidal activity.
1.1 Rationale
(2) The minimal concentration of drug needed
An increasing number of patients with to kill most ($99.9%) of the viable
infections do not have completely normal host organisms after incubation for a fixed length
immune defenses. In addition, classes of of time (generally 24 hours) under a given
antibiotics that have been considered to be set of conditions is the most common
bactericidal can no longer be assumed to kill estimation of bactericidal activity and is
every clinical isolate due to the possibility of known as either the minimal bactericidal
tolerance.9 Finally, as new classes of concentration (MBC) or the minimal lethal
concentration (MLC). It will be referred to in

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antimicrobial agents are introduced, there is a
need for established laboratory methods that this document as the MBC. Unfortunately,
can assess the bactericidal activity of these the definition of the MBC (99.9% killing of
agents. Such methods for assessing lethal the final inoculum) is somewhat arbitrary
activity should be considered special and separates the bacteria into two
populations – a segrega-tion which might

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susceptibility tests because they are not
routinely applied to all microorganisms; rather,
they are applied in unusual situations. Because
of their specialized nature, complexity, and
potential difficulty for interpretation, tests for
bactericidal activity should be done in the
context of consultation with appropriate
persons (such as the microbiology laboratory
not have biological relevance.13 The
determination of the MBC, moreover, is so
subject to methodologic variables that the
clinical relevance of MBCs is nearly
impossible to assess, particularly for certain
pathogen and drug combinations (e.g.,
staphylococci and beta-lactam agents).14
director) who are aware of the potential
(3) The serum of a patient receiving an
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problems involved in such testing.
antibiotic may be tested against the
1.2 Methods infecting microorganism. This can be done
using time-kill curve methodology (i.e.,
serum bactericidal rate) or using dilution
The killing effect of an antimicrobial agent on a
methodology (i.e., serum bactericidal titer).
microorganism can be assessed in several
The principles of these methods as well as
ways:
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the influence of biological and technical

factors are similar.
(1) Lethal activity may be expressed as the rate
of killing by a fixed concentration of drug
under controlled conditions. This rate is 1.3 Problems
determined by measuring the number of
viable bacteria at various time intervals. The Evaluating in vitro lethal effects of an
resulting graphic depiction is known as the antimicrobial agent is conceptually attractive
time-kill curve. Bacterial killing rates are, in and appears, at times, to be clinically
part, dependent on the class of antibiotic necessary. However, many biological and
and the concentration of this agent. With technical factors are known to interfere with
certain classes of antibiotics (e.g., such in vitro measurement of killing.
aminoglycosides and fluoroquino-lones), the
rate of killing increases with increased drug The biological factors include:
concentrations up to a point of maximum
effect.10 This is termed • Persisters
concentration-dependent bactericidal acti-
vity. In contrast, the killing rates of $-lactam • Paradoxical effect
agents and vancomycin are relatively slow
and continue only as long as the • Tolerance
concentrations are in excess of the
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September 1999 NCCLS

• Phenotypic resistance. been found to lyse RNA20 and this might be

related to the paradoxical effect.
The technical factors include:
There is also another mechanism of
• Growth phase of inoculum penicillin-induced cell death which is not related
to lysis21; the relationship of this mechanism to
• Inoculum size the paradoxical effect is unknown. A
paradoxical effect of aminoglycosides on the
• Insufficient contact growth of gram-negative bacilli also has been
described.22
• Volume transferred
The clinical relevance of the paradoxical effect
• Antibiotic carryover is unclear. However, Eagle and his coworkers23
infected mice with group B streptococci and
• Choice of media. demonstrated that the bacteria were killed in
vivo more slowly by high doses of penicillin.

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These variables create uncertainty in the There is at least one reported case24 where a
interpretation of bactericidal activity and are a reduction in dosage of penicillin (peak levels
major reason that professional consultation with decreasing from 36.7 to 11.3 :g/mL) resulted
the microbiology director is needed. in a marked increase in bactericidal activity in
the patient's serum (peak titers increasing from
1.3.1

1.3.1.1
Biological Factors

Persisters PL
For some well-studied antibiotics, e.g., $-lactam
agents, a small number (usually <0.1% of the
final inoculum) of bacteria were found to
survive the lethal effect of an antibiotic.15 If
1:8 to 1:256) with coincident improvement in
clinical status.

1.3.1.3 Tolerance

Tolerance means that the microorganism is able

to evade only the lethal action of the
antimicrobial agent; there is no change in the
these persisters are retested, they are just as MIC.9,18,25-27 At least four mechanisms have
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susceptible as the parent strain and no greater been described which enable clinical isolates to
proportion of cells persist. This phenomenon is survive during therapy with cell-wall-active
thought to be due to the fact that some cells agents. Two of these, persisters and the
are dormant or replicating slowly and paradoxic effect, have already been described.
consequently are not killed by the antimicrobial
agent.16 The rate of antimicrobial agent- Another mechanism is phenotypic tolerance.16
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induced killing is strictly proportional to the rate Phenotypic tolerance is a property of virtually all
of bacterial growth.17 Thus, the slower the rate strains of bacteria and is defined as decreased
of bacterial growth, the slower the bactericidal susceptibility to antimicrobial agents which is
effect of the antibiotic. As the growth of a manifested only under certain growth
microorganism reaches its maximum, the rate conditions.
slows and so does the killing effect.
The last mechanism of tolerance is that in
1.3.1.2 Paradoxical Effect which a microorganism possesses or acquires a
unique genetic property, such as a defective
Another factor known as the paradoxical effect autolytic system.27
occurs when the proportion of surviving cells
increases significantly as the concentration of All tolerant isolates, no matter which
the antimicrobial agent increases beyond the mechanism is responsible, exhibit unusually
MBC.18,19 This phenomenon is particularly high MBCs relative to their MICs, and tolerance
common for cell wall-active agents. It is has been defined as an MBC-MIC ratio of 1:32
thought that a high concentration of penicillin or greater after 24 hours of incubation.
inhibits protein synthesis to a degree which However, such a ratio cannot distinguish
prevents the growth necessary for expression of phenotypic tolerance from tolerance due to a
the lethal effect of the drug. Penicillin also has unique genetic property. Instead, a time-kill
kinetic study is needed to differentiate these

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Vol. 19 No. 18 M26-A

Related NCCLS Publications*

M2-A6 Performance Standards for Antimicrobial Disk Susceptibility Test—Sixth Edition;

Approved Standard (1997). This document provides current recommended techniques
for disk susceptibility testing, new frequency criteria for quality control testing, and
updated tables for interpretive zone diameters.

M7-A4 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically—Fourth Edition; Approved Standard (1997). This document provides
reference methods for the determination of minimal inhibitory concentrations (MIC) of
aerobic bacteria by broth macrodilution, broth microdilution, and agar dilution.

M11-A4 Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved

Standard—Fourth Edition (1997). This document provides methods for susceptibility
testing of anaerobic bacteria; description of reference agar dilution method, alternative

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agar methods (Wadsworth and limited dilutions), broth microdilution, and broth (macro)
dilution procedures, and quality control criteria for each procedure.

M21-A Methodology for the Serum Bactericidal Test; Approved Guideline (1999). This document
provides a direct method of antimicrobial susceptibility testing using a patient's serum

M29-A
patient.
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to measure the activity of the serum against the bacterial pathogen isolated from the

Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease

Transmitted by Blood, Body Fluids, and Tissue; Approved Guideline (1997). This
document provides guidance on the risk of transmission of hepatitis viruses and human
immunodeficiency viruses in any laboratory setting; specific precautions for preventing
the laboratory transmission of blood-borne infection from laboratory instruments and
materials; and recommendations for the management of blood-borne exposure.
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NOTES

* Proposed- and tentative-level documents are being advanced through the NCCLS consensus process; therefore, readers should
refer to the most recent editions.

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