Method Strengths Limitations
Broth dilution Allows monitoring of activity Essential oils may not remain in
over the duration solution for the duration of the assay;
emulsifiers and solvent may interfere
with the accuracy of results
More accurate representation Labor and time-intensive if serial
of antibacterial activity dilutions are used to determine cell
counts
Micro-broth methods can be Highly colored extracts can interfere
used to screen large numbers with colorimetric endpoints in
of samples in a cost-effective microbroth methods
manner
TLC-bioautography Simultaneous fractionation Unsuitable where activity is due to
and determination of component synergy
bioactivity
Dependent on extraction method and
TLC solvent used
Antiviral assays Allows simultaneous assess- Labor, time, and cost intensive
ment of cell toxicity with
antiviral assay
Few methods available there- Requires access to cell culture and viral
fore comparability across containment facilities
studies is high
Essential oils may not remain in
solution for the duration of the assay
Antiparasitic assays Methods are well documented Labor, time, and cost intensive
Some assays allow simul- May require access to cell culture
taneous assessment of cell facilities
toxicity
Essential oils may not remain in
solution for the duration of the assay
While the aforementioned methods are those most widely used for in vitro test-
ing of plant extracts for antibacterial activity, other methods have also been used.
For example, Garedew et al. [22] report on the use of a flow calorimetric method to
assess antibacterial activity of honey and demonstrated better sensitivity than oth-
er methods and Pitner et al. [23] propose the use of high throughput systems that
measure bacterial respiration via a fluorescent signal. However, the practicality of
these methods for screening of plant extracts is yet to be determined. An addition-
al method – thin-layer chromatography (TLC)–bioautography – allows for identifi-
cation of bioactive fractions of extracts within a single assay.
Plant extracts are obtained via aqueous or solvent extraction of flowers, roots, or
foliage or can be distilled, as an essential oil, from plant material; hence, there will
be a range of solubility and other characteristics that affect assay outcome. These
factors will be explored in the following sections. An additional group, the hydro-
sols (aqueous distillates), of a variety of plants have also gained a reputation as hav-
ing antimicrobial, among other, activities [24]. However, several studies conducted
in our laboratory have failed to show any antimicrobial activity in these plant ex-
tracts and hence they have not been discussed further [25, 26].
Semi-Solid Substrate Methods
Both the disk diffusion and agar dilution methods use bacteria grown on a solid
agar base to test antibacterial activity. These methods are relatively quick, inexpen-
sive, and do not require sophisticated laboratory equipment; however, they are not
without drawbacks.
Broth dilution Allows monitoring of activity Essential oils may not remain in
over the duration solution for the duration of the assay;
emulsifiers and solvent may interfere
with the accuracy of results
More accurate representation Labor and time-intensive if serial
of antibacterial activity dilutions are used to determine cell
counts
Micro-broth methods can be Highly colored extracts can interfere
used to screen large numbers with colorimetric endpoints in
of samples in a cost-effective microbroth methods
manner
TLC-bioautography Simultaneous fractionation Unsuitable where activity is due to
and determination of component synergy
bioactivity
Dependent on extraction method and
TLC solvent used
Antiviral assays Allows simultaneous assess- Labor, time, and cost intensive
ment of cell toxicity with
antiviral assay
Few methods available there- Requires access to cell culture and viral
fore comparability across containment facilities
studies is high
Essential oils may not remain in
solution for the duration of the assay
Antiparasitic assays Methods are well documented Labor, time, and cost intensive
Some assays allow simul- May require access to cell culture
taneous assessment of cell facilities
toxicity
Essential oils may not remain in
solution for the duration of the assay
While the aforementioned methods are those most widely used for in vitro test-
ing of plant extracts for antibacterial activity, other methods have also been used.
For example, Garedew et al. [22] report on the use of a flow calorimetric method to
assess antibacterial activity of honey and demonstrated better sensitivity than oth-
er methods and Pitner et al. [23] propose the use of high throughput systems that
measure bacterial respiration via a fluorescent signal. However, the practicality of
these methods for screening of plant extracts is yet to be determined. An addition-
al method – thin-layer chromatography (TLC)–bioautography – allows for identifi-
cation of bioactive fractions of extracts within a single assay.
Plant extracts are obtained via aqueous or solvent extraction of flowers, roots, or
foliage or can be distilled, as an essential oil, from plant material; hence, there will
be a range of solubility and other characteristics that affect assay outcome. These
factors will be explored in the following sections. An additional group, the hydro-
sols (aqueous distillates), of a variety of plants have also gained a reputation as hav-
ing antimicrobial, among other, activities [24]. However, several studies conducted
in our laboratory have failed to show any antimicrobial activity in these plant ex-
tracts and hence they have not been discussed further [25, 26].
Semi-Solid Substrate Methods
Both the disk diffusion and agar dilution methods use bacteria grown on a solid
agar base to test antibacterial activity. These methods are relatively quick, inexpen-
sive, and do not require sophisticated laboratory equipment; however, they are not
without drawbacks.
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