Agar Dilution - An Overview - ScienceDirect Topics

13:55, 24/02/2022 Agar Dilution - an overview | ScienceDirect Topics
Agar Dilution
The agar dilution involves the incorporation of different
concentrations of the antimicrobial substance into a nutrient agar
medium followed by the application of a standardized number of
cells to the surface of the agar plate.
From: Handbook of Algal Science, Technology and Medicine, 2020
Related terms:
Agar, Inoculum, Minimum Inhibitory Concentration, Etest, Bacterium, Dilution,

Disk Diffusion, Broth Dilution, Antibiotic Sensitivity
Antibiotic Resistance Diagnostic Methods for

Pathogenic Bacteria
Ahmed Marroki, Leila Bousmaha-Marroki, in Reference Module in Biomedical
Sciences, 2021
Agar dilution method

Agar-dilution method is currently the reference standard susceptibility method for
anaerobic bacteria. The test is performed in Petri dish to determine the MICs to
multiple tested strains in single plate. Through this assay, a various antibiotics
agents are prepared usually at twofold dilution series and incorporated into molten
agar medium plates in different concentrations. Then inoculation of the medium
with a standardized suspension adjusted to 0.5 McFarland standard containing a
concentration of 5 × 108 CFU mL− 1 (CLSI, 2012a) was conducted. The bacteria are
diluted to around 107CFU mL− 1, and 2 μL are spotted with multi-point into agar
plates contain approximately 104 CFU/spot (Clinical and Laboratory Standards
Institute, 2015), and one agar plate used as control seeded without antibacterial
agent. After incubation in anaerobic condition at 35 °C for 16–18 h for non-
fastidious bacteria and for 18–24 h fastidious organisms (Barry, 1986), the agar
plates are examined by visually comparing the growth. The MICs is determined
based on this test as the lowest concentration of drug tested that stopped the
growth of bacteria (CLSI, 2012b; Brook et al., 2013). Currently, the agar-dilution
technique is considered as the reference susceptibility testing recommended by
CLSI for fastidious bacteria such as Helicobacter spp. and Campylobacter (CLSI,
https://www.sciencedirect.com/topics/immunology-and-microbiology/agar-dilution 1/10
2010; CLSI, 2017) and anaerobic bacteria (CLSI, 2012b), and for evaluate the
antifungal susceptibility (Benkova et al., 2020). In the study conducted by Liu et al.
(2016a,b), disk-diffusion and agar dilution methods used for Neisseria gonorrhoeae
AST were compared. The result obtained demonstrated a good correlation and
level categorical agreements has been obtained between the agar diffusion test and
standard agar dilution method. Similar conclusion obtained by Luangtongkum et
al. (2007) by comparison of agar dilution and the agar disk diffusion test applied for
Campylobacter. The advantage of agar dilution method is its suitability when few
antibacterial agents are tested against a large number of microorganisms (Barry,
1986; Liu et al., 2016a,b). One drawback of agar dilution method is time-
consuming and high degree of inherent error.
Read full chapter

URL: https://www.sciencedirect.com/science/article/pii/B9780128187319001336
Potential of anaerobic bacteria in bioremediation of

metal-contaminated marine and estuarine environment
M.B. Binish, ... Mahesh Mohan, in Microbial Biodegradation and Bioremediation
(Second Edition), 2022
15.9.2.4 Mercury tolerance testing

Agar dilution procedure was utilized for the identification of tolerance to mercury
(CLSI, 2012). Plates containing 20 mL of Wilkins–Chalgren anaerobic agar (casein
enzymichydrolysate 10.00 g L−1; peptic digest of animal tissue 10.00 g L−1; yeast
extract 5.00 g L−1; dextrose 1.00 g L−1; sodium chloride 5.00 g L−1; l-arginine
1.00 g L−1; sodium pyruvate 1.00 g L−1; hemin 0.005 g L−1; menadione
0.0005 g L−1; and agar 10.0 g L−1) with different concentrations of Hg2+ as mercury
chloride (0.1–5.0 µg mL−1) were streaked with the isolate. Uninoculated medium
and inoculated medium without mercury were used as negative and positive
control, respectively. The plates were then incubated in an anaerobic jar with
anaero gas pack (HiMedia, India) at 37°C for 14 days. The bacteria which showed
high tolerance toward mercury was selected and identified.
Read full chapter

Gonorrhea
C.A. Ison, D.A. Lewis, in Atlas of Sexually Transmitted Diseases and AIDS (Fourth
Edition), 2010
Agar-dilution susceptibility testing

Agar-dilution susceptibility testing is the reference method for measuring the
antimicrobial susceptibilities of strains of N. gonorrhoeae. Resistance to
antimicrobial agents is measured as the MIC of the agent that inhibits growth of
an isolate. Determination of susceptibilities to antimicrobial agents used for
therapy should be tested such as the third generation cephalosporins, cefixime and
ceftriaxone although other agents, such as ceftobutin, are used in different parts of
the world. The methodology for susceptibility testing of N. gonorrhoeae can vary
between countries, differing primarily in the base medium used; GC agar base
(CLSI, North America), Isosensitest (Australia) and Diagnostic Sensitivity Agar
(Europe). For therapeutic antimicrobial agents this has historically been most
important for the concentration of penicillin considered resistant; which can differ
from ≥2 mg/L (GC base agar) and ≥1 mg/L (Isosensitest and DST agar). Other
antimicrobials agents are affected less but this should be remembered when
comparing different methodologies.
Susceptibility testing is performed on the base medium of choice containing 1%
(vol/vol) IsoVitaleX or an equivalent supplement; antimicrobial agents are
incorporated into the base medium in serial twofold dilutions. Isolates to be tested
are grown overnight on chocolate agar and suspended in Mueller–Hinton broth (or
equivalent) to an optical density equivalent to a 0.5 McFarland standard, containing
approximately 108 colony-forming units (CFU)/mL. The suspensions are diluted 1 :
10 in Mueller–Hinton broth, and 104 CFU/mL are inoculated onto the surface of
the antibiotic-containing media and an antibiotic-free control medium with a
Steer's replicator, multipoint inoculator or a calibrated loop. Plates are incubated at
35–36°C in a CO2-enriched atmosphere for 24 hours and then examined for
growth. The MIC of the antimicrobial agent for an isolate is the lowest
concentration that inhibits its growth (Fig. 2.47). A modification of the full MIC is
the use of breakpoints which uses a similar method but with medium containing
one or two concentrations of antibiotic that can be used to categorize isolates as
being resistant or having decreased susceptibility. This is useful for screening
isolates, particularly for high-level resistance.
An alternative method for determining the MIC is the use of the Etest. This uses a
strip, impregnated with antibiotic at different concentrations which is placed on a
previously seeded agar plate which results in the antibiotic immediately being
released into the medium. After overnight incubation the MIC is determined where
the elipse crosses the strip (Fig 2.48). This is a very useful method for testing single
strains but is also useful for confirming MICs determined by the agar dilution
method.
Read full chapter

Antimicrobial Susceptibility Testing☆

F.C. Tenover, in Encyclopedia of Microbiology (Fourth Edition), 2019
Agar dilution
The agar dilution method involves preparing a series of agar plates containing the
antimicrobial agent to be tested in increasing concentrations, usually in doubling
dilutions (i.e., 1, 2, 4, 8, 16, 32 μg ml− 1, etc.). A suspension of the organism to be
tested is prepared to equal the turbidity of a 0.5 McFarland standard (∼ 1 × 108
colony forming units (CFU) ml− 1), and 1–5 μl of this suspension is placed on each
of the series of plates with increasing concentrations of the antimicrobial agent
using a replicator device (final inoculum is ∼ 5 × 104 CFU/spot). Thirty different
bacterial isolates (plus quality control organisms) can be tested simultaneously on
each agar plate. Nonfastidious organisms are incubated at 35 °C for 16–18 h
usually in ambient air, while fastidious organisms, such as Streptococcus
pneumoniae, are tested on blood-containing media, incubated from 18 to 24 h,
typically in a CO2-enriched atmosphere. The agar dilution method, while laborious
due to the time required to prepare each set of agar plates for each antimicrobial
agent to be tested, is often cost-effective for laboratories that test large numbers of
bacterial isolates against a limited set of antimicrobial agents. The testing medium
is usually Mueller–Hinton agar for nonfastidious organisms and Mueller–Hinton
agar containing 5% sheep blood for fastidious organisms. The exceptions are
Haemophilus influenzae isolates, which require HTM (CLSI) or MH-F (EUCAST), and
N. gonorrhoeae, which requires GC medium.
Read full chapter

Laboratory Diagnosis of Infection Due to Bacteria,

Fungi, Parasites, and Rickettsiae
John C. Christenson, ... Ryan F. Relich, in Principles and Practice of Pediatric
Infectious Diseases (Fifth Edition), 2018
Mycobacteria.
An agar dilution test has been the standard for testing mycobacteria. A
standardized concentration of test organism is inoculated onto agar (i.e.,
Middlebrook 7H10) containing the antimicrobial agent to be tested. After
incubation for up to 3 weeks (35°C in CO2), susceptibility is determined by
comparing growth on the antibiotic-containing medium with that on antibiotic-
free medium.
The BACTEC 460TB radiometric system has provided a major advance in the
susceptibility testing of slow-growing Mycobacterium spp. BACTEC 12B or
Middlebrook 7H12 broth containing an antimicrobial agent is inoculated with the
test organism and incubated for 5 days at 37°C. The growth index is compared
with that of a control bottle containing no antimicrobial agent.123 Automated
nonradiometric systems such as BACTEC MGIT 960 and VersaTREK appear to be
as reliable as older methods.124–127
Read full chapter
The scientometric analysis of the research on the algal

biomedicine
Ozcan Konur, in Handbook of Algal Science, Technology and Medicine, 2020
Other research fronts

Wiegand et al. (2008) study both the agar and broth dilution methods to determine
the ‘minimal inhibitory concentration’ (MIC) of antimicrobial substances, inhibiting
the growth the aerobic bacteria in a paper with 1328 citations. The agar dilution
involves the incorporation of different concentrations of the antimicrobial
substance into a nutrient agar medium followed by the application of a
standardized number of cells to the surface of the agar plate. This protocol can be
completed in 3 days.
Guerin et al. (2003) review the applications of astaxanthin from Haematococcus
pluvialis for human health and nutrition in a paper with 648 citations. As the
astaxanthin is a strong coloring agent and a potent antioxidant, they focus on the
antioxidant, UV-light protection, anti-inflammatory and other biomedical
properties of astaxanthin. They assert that protecting body tissues from oxidative
damage with daily ingestion of natural astaxanthin might be a practical and
beneficial strategy in health management.
Read full chapter

Isolation and Identification of Aerobic and Anaerobic

Bacteria
Jane E. Sykes, Shelley C. Rankin, in Canine and Feline Infectious Diseases, 2014
Dilution Methods
Dilution methods include broth macrodilution, broth microdilution, and agar dilution.
The most widely used method in North America is broth microdilution, whereby
twofold dilutions of antimicrobials are made in a broth medium in a microtiter
plate (Figure 3-9). Pre-prepared frozen or freeze-dried microtiter plates for
inoculation are available commercially (e.g., Sensititre plates, TREK Diagnostic
Systems, Cleveland). The results can be determined using visual examination of the
plates for the inhibition of bacterial growth, or by the use of semiautomated or
automated instrumentation. Dilutions can also be performed in agar, each dilution
being poured onto a plate in a standardized fashion and allowed to set before
inoculating it with the organism(s) of interest. Agar dilution can be used to perform
susceptibility testing of fastidious bacteria with special medium requirements, such
as Campylobacter spp.
The MIC for each antimicrobial drug tested against the organism is reported to the
clinician on the susceptibility panel. It is the lowest concentration of antibiotic
(usually in µg/mL) that inhibits growth of the organism in vitro. The lower the MIC,
the more potent is the antimicrobial at inhibiting bacterial growth.
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URL: https://www.sciencedirect.com/science/article/pii/B978143770795300003X
Challenges in Clinical Microbiology Testing

Laura Chandler, in Accurate Results in the Clinical Laboratory, 2013
Antimicrobial Susceptibility Testing Errors

Antimicrobial susceptibility testing (AST) is one of the most important functions
performed by the clinical microbiology laboratory. With improvements in testing
methods and quality control and quality assurance programs, AST errors are
infrequent and are usually detected and corrected before results are released to the
clinician. Although laboratory methods are designed to accurately detect most
resistance mechanisms, microorganisms are constantly evolving and in the setting
of antimicrobial usage, resistance mechanisms emerge. Resistance is complex, and
organisms have many methods by which they can acquire or express resistance to
antimicrobials. Organisms may acquire new resistance determinants by sharing
plasmids or other genetic elements with resistant bacteria, or they may express
resistance mechanisms inducible in the presence of an antimicrobial agent.
The laboratory must routinely update its policies and procedures to stay current
with susceptibility methods, known resistance patterns, and reporting strategies.
Errors in AST can have significant impact on patient care. Although there are many
potential sources of error in AST, most errors can be prevented by the use of well-
standardized, well-controlled policies and procedures by the laboratory. By using
well-established, standardized methods, and with good-quality control programs,
error rates in AST can be held to a minimum. The laboratory should also ensure
that the repertoire of agents for testing and reporting are matched to the hospital
formulary.
Potential sources of error in AST occur in several areas: method differences,
technical errors with performing and interpreting the assays, reporting errors, and
inherent problems with certain organism–drug combinations. Each of these can be
minimized by careful adherence to guidelines and recommendations, laboratory
procedures, and the quality control program. There are well-established standards
available through the Clinical and Laboratory Standards Institute (CLSI) that
laboratories should implement. Emerging resistance is always a consideration in
susceptibility testing. Finally, subtle resistance mechanisms or inducible resistance
mechanisms that are not manifested until therapy is implemented should always
be a consideration when the susceptibility testing results indicate an organism is
susceptible but the therapy fails.
Method Differences
The main methods for susceptibility testing are broth dilution, agar dilution, and
disk diffusion. In addition, several systems for automated AST are available. It is
important to note that each method may have limitations for certain organism–
drug combinations and that there may be minor differences between the results
generated by the automated systems. For example, certain organism–drug
combinations cannot be reliably tested on automated systems. Laboratories should
ensure that they have implemented the recommended changes to their software if
using automated instruments.
New Resistance Mechanisms or Emerging Resistance
AST systems may not accurately detect heteroresistance (subpopulations of bacteria
with resistance mechanisms present). When resistance determinants are present
but not being expressed by the organism at a high level, the isolate may appear
susceptible in in vitro testing, but use of antimicrobials may allow for expression of
the resistance gene. This phenomenon has been noted for several groups of
organisms. An example is the emergence of carbapenem resistance in Gram-
negative bacteria such as Klebsiella [23]. Automated systems may fail to detect
carbapenem resistance [24–26]. Laboratories should ensure that screening and
confirmatory testing are in place to detect potential carbapenem resistance. The
emergence of resistance mechanisms in bacteria can result in testing and reporting
errors. Unusual results should be reviewed and investigated; occasionally, aberrant
results can be traced back to technical errors. If no technical errors have occurred,
organisms with unusual resistance patterns should be tested with a different
method. Unusual susceptibility testing results should be verified prior to release;
laboratories should have documented protocols in place for verification of unusual
results. Often, investigation reveals that a technical issue led to the unusual or
unexpected result.
Inducible Resistance Mechanism Present, Not Detected
Inducible resistance mechanisms can be difficult to identify in the laboratory.
Methods are available for detection of inducible resistance to clindamycin in
staphylococci; sensitivity and specificity of these tests are adequate, and very few
errors occur. Inducible resistance to β-lactam agents can be more difficult to
detect, especially in Gram-negative organisms such as Acinetobacter.
Case Report: Fatal Acinetobacter Baumannii Infection with

Discordant Carbapenem Susceptibility
Initial susceptibility testing of an Acinetobacter baumannii strain isolated from blood
and respiratory cultures indicated the organism was susceptible to imipenem. The
patient was treated with meropenem but did not respond clinically; the patient
died. Subsequent testing of the bloodstream isolate using a disk diffusion test
revealed that the organism was susceptible to imipenem but resistant to
meropenem [27]. This case illustrates the potential for resistance mechanisms to be
missed when only one drug is tested. Testing of only one agent in a class is very
common when using panels on automated systems because of the limitations of
space on the panels. Laboratories must be aware of the potential for failure of the
automated susceptibility testing system to detect carbapenem resistance in Gram-
negative bacteria.
Read full chapter

Volatile oils: Potential agents for the treatment of

respiratory infections
A. Pasdaran, ... D. Sheikhi, in The Microbiology of Respiratory System Infections,
2016
3.1.3 The dilution method (agar or liquid broth)

Although the modification with liquid broth is mostly practical for fungi, the serial
dilution agar method is also common for bacteria and fungi. The difference is that
agar broth cultures are grown in Petri dishes or tubes, but liquid broth cultures are
cultivated in conical flasks filled with 100 mL medium or test tubes with 2.5–5 mL
medium (bacteria and moulds). The inhibitory growth index is determined for the
liquid broth in conical flasks (percent changes in mould’s biomass comparing to
the control culture). There is an inhibitory effect of essential oil which appears in
the test tube cultures and is measured turbidimetrically or with the plate count
method. The estimation of essential oil activity both in agar and liquid broth would
be simplified through counting the tested microorganisms that can remain in the
membrane. The lowest essential oil concentration in the broth that results in the
lack of visible microorganism growth changes is known as the MIC. The
microorganisms are then transferred from the lowest essential oil concentration
medium (with no visible microorganism growth) into a new broth medium and
incubation to determine the lethal activity of essential oil.78,79
Read full chapter

Laboratory Diagnosis and Therapy of Infectious

Diseases
Melissa B. Miller, Peter H. Gilligan, in Principles and Practice of Pediatric
Infectious Diseases (Fourth Edition), 2012
Methods of Susceptibility Testing

The reference method or “gold standard” for phenotypic susceptibility testing is
MIC determinations. Depending on the organism, testing can be done by either
broth microdilution or agar dilution.218 Automated commercial test systems, such
as Vitek (bioMérieux, Hazelwood, MO), Phoenix (BD Bioscience, Sparks, MD), and
MicroScan (Dade-Behring, Sacramento, CA), which employ modification of these
methods, are used widely. These systems are fast, mechanically reliable, highly
reproducible, offer significant cost efficiencies (including interfacing with clinical
information systems), and combine susceptibility testing and organism
identification in one system. Automated susceptibility testing, however, is not as
accurate as reference methods, particularly for detecting inducible resistance and

other emerging resistance.219 Software changes are introduced to address
problems but organisms evolve to develop resistance faster than changes can be
developed and validated. Perpetually, systems may be a step behind. For example,
in mid 2010, CLSI lowered carbapenem susceptibility breakpoints for meropenem
and imipenem 4-fold (from ≤4 µg/mL to ≤1 µg/mL) because some carbapenemase-
producing Enterobacteriaceae have carbapenem MICs as low as 0.125 µg/mL.220
But because the automated susceptibility systems are diagnostic devices regulated
by the FDA, susceptibility test system manufacturers must perform a complex
validation process before breakpoints can be changed on their systems, thus
causing conundrums for both laboratorians and clinicians.
In addition to microdilution methods, used by approximately 80% of laboratories
in the U.S., disk diffusion susceptibility is used by approximately 15% of
laboratories.219 Disk diffusion testing relies on the use of disks impregnated with
specific concentration for each drug. Antimicrobial containing disks are applied to
a lawn of bacteria inoculated on a standard medium and, after 16 to 24 hours’
incubation, zones of inhibition are measured (Figure 290-1A). Zones of inhibition
correlate reasonably well with specific MIC values. Breakpoints for susceptible,
intermediate, or resistant have been previously determined for each antibiotic disk
based on zone size. Disk diffusion consistently is more accurate than automated
commercial systems compared with reference methods,219 is more flexible for
choosing drugs for testing, and may better detect emerging resistance
problems.219 Disadvantages of disk diffusion testing are its labor-intensiveness,
less reproducibility because of manual measurement of inhibition zones, lack of
breakpoints for all clinically relevant bacteria, and proneness to clerical errors
because of manual entry of results into clinical information systems.
Etest (AB Biodisk, Solna, Sweden) is performed in a manner similar to disk
diffusion but instead of a disk being placed on a lawn of bacteria, a plastic strip
impregnated with a gradient of a specific antimicrobial agent is tested.221 After
appropriate incubation, the MIC is read where the elliptical zone of inhibition of
growth meets the strip. Although not as highly standardized as disk diffusion,
Etests are particularly useful and accurate compared with reference MIC methods
for determining antimicrobial susceptibility for organisms that are fastidious or
have a narrow range between drug concentrations considered as susceptible and
resistant. For example, determining MICs of penicillin and ceftriaxone for S.
pneumoniae is best performed by Etest222 (Figure 290-1B).
Read full chapter

Recommended publications
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13:55, 24/02/2022 Agar Dilution - an overview | ScienceDirect Topics

Agar, Inoculum, Minimum Inhibitory Concentration, Etest, Bacterium, Dilution,

Antibiotic Resistance Diagnostic Methods for

Agar dilution method

Read full chapter

Potential of anaerobic bacteria in bioremediation of

15.9.2.4 Mercury tolerance testing

Read full chapter

Agar-dilution susceptibility testing

Read full chapter

Antimicrobial Susceptibility Testing☆

Read full chapter

Laboratory Diagnosis of Infection Due to Bacteria,

Read full chapter

The scientometric analysis of the research on the algal

Other research fronts

Read full chapter

Isolation and Identification of Aerobic and Anaerobic

Read full chapter

Challenges in Clinical Microbiology Testing

Antimicrobial Susceptibility Testing Errors

Case Report: Fatal Acinetobacter Baumannii Infection with

Read full chapter

Volatile oils: Potential agents for the treatment of

3.1.3 The dilution method (agar or liquid broth)

Read full chapter

Laboratory Diagnosis and Therapy of Infectious

Methods of Susceptibility Testing

accurate as reference methods, particularly for detecting inducible resistance and

Read full chapter

Clinical Microbiology and Infection

Diagnostic Microbiology and Infectious Disease

Journal of Global Antimicrobial Resistance

Copyright © 2022 Elsevier B.V. or its licensors or contributors.

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