ELPHAS SHIKOKOTI SIAYI BSC MLS
(MMUST)
Antimicrobial susceptibility testing
Methods of assaying antibiotics may be broadly divided into three:
1. Conventional chemical assays, e.g. titrations, spectrophotometry and high-
performance liquid chromatography (HPLC)
2. Enzyme-based and immunoassays, where the antibiotic is, respectively, the
substrate for a specific enzyme or the antigen with which a specific antibody
combines.
3. Biological assays in which biological activity – in this case bacterial growth
inhibition - of the 'test' solution is compared with that of a reference standard.
Advantages and disadvantages of biological and non-biological methods
Biological antimicrobial assays
There are two main types of biological antibiotic assays, or bioassays
1. Agar diffusion assay
2. Turbidimetric assay
, Agar diffusion assay
In this technique the agar medium in a Petri dish or a larger assay plate is inoculated
with the test organism, wells are created in it by removing circular plugs of agar, and
these wells are filled with a solution of the chemical under test. As an altenative to wells
the antibiotic may be introduced on to the agar using absorbent paper discs, metal
cylinders or 'fish spine' beads (beads having a hole drilled in them which contains the
liquid).
Assay Principle
The chemical diffuses through the gel from A towards B (see diagram above) and the
concentration falls steadily in that direction. The concentration in the region A to X is
sufficiently high to prevent growth, and it is an inhibitory concentration.
Between X and B the concentration is sub-inhibitory and microbial growth occurs.
The concentration at X is known as the critical inhibitory concentration (CIC).
After incubation the gel between A and X is clear and that between X and B is opaque
as a result of microbial growth which, with the common test organisms, is usually
profuse. A zone of inhibition is therefore created, the diameter of which will increase as
the concentration of chemical or susceptibility of test organism to the chemical increases.
(MMUST)
Antimicrobial susceptibility testing
Methods of assaying antibiotics may be broadly divided into three:
1. Conventional chemical assays, e.g. titrations, spectrophotometry and high-
performance liquid chromatography (HPLC)
2. Enzyme-based and immunoassays, where the antibiotic is, respectively, the
substrate for a specific enzyme or the antigen with which a specific antibody
combines.
3. Biological assays in which biological activity – in this case bacterial growth
inhibition - of the 'test' solution is compared with that of a reference standard.
Advantages and disadvantages of biological and non-biological methods
Biological antimicrobial assays
There are two main types of biological antibiotic assays, or bioassays
1. Agar diffusion assay
2. Turbidimetric assay
, Agar diffusion assay
In this technique the agar medium in a Petri dish or a larger assay plate is inoculated
with the test organism, wells are created in it by removing circular plugs of agar, and
these wells are filled with a solution of the chemical under test. As an altenative to wells
the antibiotic may be introduced on to the agar using absorbent paper discs, metal
cylinders or 'fish spine' beads (beads having a hole drilled in them which contains the
liquid).
Assay Principle
The chemical diffuses through the gel from A towards B (see diagram above) and the
concentration falls steadily in that direction. The concentration in the region A to X is
sufficiently high to prevent growth, and it is an inhibitory concentration.
Between X and B the concentration is sub-inhibitory and microbial growth occurs.
The concentration at X is known as the critical inhibitory concentration (CIC).
After incubation the gel between A and X is clear and that between X and B is opaque
as a result of microbial growth which, with the common test organisms, is usually
profuse. A zone of inhibition is therefore created, the diameter of which will increase as
the concentration of chemical or susceptibility of test organism to the chemical increases.
