Agar Dilution Method
The agar dilution method is another relatively quick method that does not involve
the use of sophisticated equipment. Any laboratory with facilities for basic micro-
biological work can use this method. In this method the test substance is incorpo-
rated at known concentrations into the agar and, once set, bacteria are applied to its
surface. Replicate dishes can be set up with a range of concentrations of the test
substance and by dividing the surface of the agar into wedges or squares, a num-
ber of bacterial species may be applied to a single dish. In this way, a large number
of bacteria may be screened within a single assay run. The dishes are incubated for
24 h or more and the growth of the bacteria on the extract/agar mix is scored either
as present/absent or a proportion of the control (e.g. 0, 25%, 50%, 75%, 100%).
A criticism of this method is that when a scoring system is used it is difficult to
guarantee objectivity and to therefore compare one set of results with another.
This method suffers from several other limitations, including many that have
been discussed previously: use of larger volumes of test substance than in other
162 8 Methods for Testing the Antimicrobial Activity of Extracts
methods, confounding antibacterial actions from volatiles, difficulty of achieving
stable emulsions of essential oils in agar and restriction on the maximum concen-
tration that can be used before the agar becomes too dilute to solidify properly. Per-
haps the most frustrating of these is the difficulty of stably incorporating essential
oils and other hydrophobic extracts into aqueous environments. This problem oc-
curs not just in agar dilution assays but also in broth dilution and other antimicro-
bial assays. Many a researcher has thought they had incorporated their essential oil
into nutrient broth or other media only to find that, on return to the experiment af-
ter an hour or so, the oil had separated out and was floating on top of the media.
Griffin et al. [18] in their work on tea tree oil found that at concentrations above 2%
v/v the oil separated from the agar substrate and was seen as droplets on the agar
surface. The most commonly utilized method to overcome this problem is the use
of surfactants such as Tween-20, Tween-80, and alkyl dimethyl betaine (ADB). Sev-
eral authors have described the use of these products and the effect on antibacteri-
al activity. The results of their studies show that surfactants can interfere with cal-
culation of MIC values and the growth of some test organisms [31, 32], however it
has also been demonstrated that it is possible to use very small quantities of Tween
(<0.5% v/v) to emulsify the essential oil in media and thus avoid the effects on or-
ganism growth [18, 20]. Hammer et al. [31] also showed that inclusion of organic
matter such as bovine serum albumin in the agar also affected the antibacterial ac-
tivity of tea tree oil.
The agar dilution method is another relatively quick method that does not involve
the use of sophisticated equipment. Any laboratory with facilities for basic micro-
biological work can use this method. In this method the test substance is incorpo-
rated at known concentrations into the agar and, once set, bacteria are applied to its
surface. Replicate dishes can be set up with a range of concentrations of the test
substance and by dividing the surface of the agar into wedges or squares, a num-
ber of bacterial species may be applied to a single dish. In this way, a large number
of bacteria may be screened within a single assay run. The dishes are incubated for
24 h or more and the growth of the bacteria on the extract/agar mix is scored either
as present/absent or a proportion of the control (e.g. 0, 25%, 50%, 75%, 100%).
A criticism of this method is that when a scoring system is used it is difficult to
guarantee objectivity and to therefore compare one set of results with another.
This method suffers from several other limitations, including many that have
been discussed previously: use of larger volumes of test substance than in other
162 8 Methods for Testing the Antimicrobial Activity of Extracts
methods, confounding antibacterial actions from volatiles, difficulty of achieving
stable emulsions of essential oils in agar and restriction on the maximum concen-
tration that can be used before the agar becomes too dilute to solidify properly. Per-
haps the most frustrating of these is the difficulty of stably incorporating essential
oils and other hydrophobic extracts into aqueous environments. This problem oc-
curs not just in agar dilution assays but also in broth dilution and other antimicro-
bial assays. Many a researcher has thought they had incorporated their essential oil
into nutrient broth or other media only to find that, on return to the experiment af-
ter an hour or so, the oil had separated out and was floating on top of the media.
Griffin et al. [18] in their work on tea tree oil found that at concentrations above 2%
v/v the oil separated from the agar substrate and was seen as droplets on the agar
surface. The most commonly utilized method to overcome this problem is the use
of surfactants such as Tween-20, Tween-80, and alkyl dimethyl betaine (ADB). Sev-
eral authors have described the use of these products and the effect on antibacteri-
al activity. The results of their studies show that surfactants can interfere with cal-
culation of MIC values and the growth of some test organisms [31, 32], however it
has also been demonstrated that it is possible to use very small quantities of Tween
(<0.5% v/v) to emulsify the essential oil in media and thus avoid the effects on or-
ganism growth [18, 20]. Hammer et al. [31] also showed that inclusion of organic
matter such as bovine serum albumin in the agar also affected the antibacterial ac-
tivity of tea tree oil.
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