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Broth and Agar

The document discusses broth and agar dilution methods for determining the minimum inhibitory concentration (MIC) of antimicrobial agents against bacteria. Broth dilution involves testing varying concentrations of an antimicrobial agent against a bacterial suspension in liquid medium, either in tubes or microtitration plates. Agar dilution incorporates varying antimicrobial concentrations into agar plates, which bacteria are applied to. Both methods aim to find the lowest antimicrobial concentration that inhibits visible bacterial growth. Broth dilution is more reproducible and quantitative than disk diffusion, but agar dilution may provide more reliable MIC results and allows testing multiple bacteria simultaneously.

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89 views

Broth and Agar

The document discusses broth and agar dilution methods for determining the minimum inhibitory concentration (MIC) of antimicrobial agents against bacteria. Broth dilution involves testing varying concentrations of an antimicrobial agent against a bacterial suspension in liquid medium, either in tubes or microtitration plates. Agar dilution incorporates varying antimicrobial concentrations into agar plates, which bacteria are applied to. Both methods aim to find the lowest antimicrobial concentration that inhibits visible bacterial growth. Broth dilution is more reproducible and quantitative than disk diffusion, but agar dilution may provide more reliable MIC results and allows testing multiple bacteria simultaneously.

Uploaded by

Varghese
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Broth and agar dilution methods

The aim of the broth and agar dilution methods is to determine the lowest concentration of the assayed
antimicrobial that inhibits the visible growth of the bacterium being tested (MIC, usually expressed in μg/ml
or mg/litre). However, the MIC does not always represent an absolute value. The ‘true’ MIC is a point
between the lowest test concentration that inhibits the growth of the bacterium and the next lower test
concentration. Therefore, MIC determinations performed using a dilution series may be considered to have
an inherent variation of one dilution.
Antimicrobial ranges should encompass both the interpretive criteria (susceptible, intermediate and
resistant) for a specific bacterium/antibiotic combination and appropriate quality control reference organisms.
Antimicrobial susceptibility dilution methods appear to be more reproducible and quantitative than agar disk
diffusion. However, antibiotics are usually tested in doubling dilutions, which can produce inexact MIC data.
Any laboratory that intends to use a dilution method and set up its own reagents and antibiotic dilutions
should have the ability to obtain, prepare and maintain appropriate stock solutions of reagent-grade
antimicrobials and to generate working dilutions on a regular basis. It is then essential that such laboratories
use quality control organisms (see below) to assure accuracy and standardisation of their procedures.

Broth dilution
Broth dilution is a technique in which a suspension of bacterium of a predetermined optimal or appropriate
concentration is tested against varying concentrations of an antimicrobial agent (usually serial twofold dilutions) in
a liquid medium of predetermined, documented formulation. The broth dilution method can be
performed either in tubes containing a minimum volume of 2 ml (macrodilution) or in smaller volumes using
microtitration plates (microdilution). Numerous microtitre plates containing lyophilised prediluted antibiotics
within the wells are commercially available. The use of identical lots in microdilution plates may assist in the
minimisation of variation that may arise due to the preparation and dilution of the antimicrobials from
different laboratories. The use of these plates, with a documented test protocol, including specification of
appropriate reference organisms, will facilitate the comparability of results among laboratories.
Due to the fact that most broth microdilution antimicrobial test panels are prepared commercially, this
method is less flexible than agar dilution or disk diffusion in adjusting to the changing needs of the
surveillance/monitoring programme.
Because the purchase of antimicrobial plates and associated equipment may be costly, this methodology
may not be feasible for some laboratories.
• Agar dilution
Agar dilution involves the incorporation of varying concentrations of antimicrobial agent into an agar medium,
usually using serial twofold dilutions, followed by the application of a defined bacterial inoculum to the agar
surface of the plate. These results are often considered as the most reliable for the determination of an MIC
for the test bacterium/antimicrobial combination.
The advantages of agar dilution methods include:
i) the ability to test multiple bacteria, except bacteria that swarm, on the same set of agar plates at the
same time,
ii) the potential to improve the identification of MIC endpoints and extend the antibiotic concentration
range,
iii) the possibility to semi-automate the method using an inoculum-replicating apparatus. Commercially
produced inoculum replicators are available and these can transfer between 32 and 60 different
bacterial inocula to each agar plate,
Agar dilution methods also have certain disadvantages, for example:
i) if not automated, they are very laborious and require substantial economic and technical resources,
ii) once the plates have been prepared, they normally should be used within a week (or less, depending
on the antimicrobials tested),
iii) the endpoints are not always easy to read nor is the purity of the inoculum easy to verify.
Agar dilution is often recommended as a standardised AST method for fastidious organisms (CLSI, 2006c),
such as anaerobes and Helicobacter species.

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