Broth Dilution Methods
Difficulties with partitioning of hydrophobic compounds in agar and a desire to
more accurately monitor antibacterial activity over time has resulted in a move to
broth dilution methods for testing of plant extracts. In this method, bacteria are
grown in test-tubes in a liquid media in the presence of the test substance. At reg-
ular time intervals (e.g. every 10 min or every hour) a sample is removed and the
bacterial count determined by serial dilution of the sample, subsequent incubation
on agar and counting of colony-forming units. In contrast to the single data point
(e.g. 24 h incubation) utilized in disk diffusion and agar dilution assays, the broth
dilution method allows much finer evaluation of the antibacterial events over time
and features such as recovery from the effects of the test substance and proportion
of organisms killed at a given time point can be determined. However the method
is also time and resource intensive and can be impractical where very large num-
bers of test substances are to be screened.
As with other testing methods incorporation of hydrophobic compounds and es-
sential oils into the aqueous media is problematic, and as there is no solid phase to
trap these compounds they rapidly separate from the media and form a layer
across the surface of the media. For organisms sensitive to oxygen tension in the
media this can present an additional problem as the oil can inhibit gaseous ex-
change. Tween or ethanol may be used to enhance incorporation into the aqueous
media, however as previously discussed these compounds may interfere with the
assay results. Work in our laboratory has shown that essential oils can be stably in-
8.2 Antibacterial Assays 163
corporated into broth using 0.02% Tween-80 and that broth dilution assays are
more reliable and reproducible than either the disk/well diffusion or agar dilution
methods [20].
Micro-broth methods have also been developed, which utilize microtiter plates,
thus reducing the volume of extract needed, and have endpoints that can be deter-
mined spectrophotmetrically, either a measure of turbidity or use of a cell viability
indicator (e.g. resazurin, methylthiazoldiphenyltetrazolium (MTT)) [33]. Mann
and Markham [33] propose that the cell viability indicator is the best method of
endpoint determination for essential oils as the oil/water interface may interfere
with turbidity measures. While these micro-broth methods generally work well for
plant extracts, problems arise when the extract is heavily colored as this can inter-
fere with the measurement of the indicator chemical. Further, as these methods
use plastic microtiter plates, essential oils that have a solvent action on plastics (e.g.
Letospermum petersonii, Backhousia citriodora) cannot be used. We have also dem-
onstrated that the addition of essential oils to media changes its pH and this may
contribute to the observed antibacterial activity [19]; this might be expected to be
more significant in small volumes, for example in the micro-broth method.
Whether other plant extracts will also have the effect is unknown. Micro-broth
methods are also less time and resources intensive than other broth methods as
the need for multiple serial dilutions to determine bacterial count is eliminated.
Difficulties with partitioning of hydrophobic compounds in agar and a desire to
more accurately monitor antibacterial activity over time has resulted in a move to
broth dilution methods for testing of plant extracts. In this method, bacteria are
grown in test-tubes in a liquid media in the presence of the test substance. At reg-
ular time intervals (e.g. every 10 min or every hour) a sample is removed and the
bacterial count determined by serial dilution of the sample, subsequent incubation
on agar and counting of colony-forming units. In contrast to the single data point
(e.g. 24 h incubation) utilized in disk diffusion and agar dilution assays, the broth
dilution method allows much finer evaluation of the antibacterial events over time
and features such as recovery from the effects of the test substance and proportion
of organisms killed at a given time point can be determined. However the method
is also time and resource intensive and can be impractical where very large num-
bers of test substances are to be screened.
As with other testing methods incorporation of hydrophobic compounds and es-
sential oils into the aqueous media is problematic, and as there is no solid phase to
trap these compounds they rapidly separate from the media and form a layer
across the surface of the media. For organisms sensitive to oxygen tension in the
media this can present an additional problem as the oil can inhibit gaseous ex-
change. Tween or ethanol may be used to enhance incorporation into the aqueous
media, however as previously discussed these compounds may interfere with the
assay results. Work in our laboratory has shown that essential oils can be stably in-
8.2 Antibacterial Assays 163
corporated into broth using 0.02% Tween-80 and that broth dilution assays are
more reliable and reproducible than either the disk/well diffusion or agar dilution
methods [20].
Micro-broth methods have also been developed, which utilize microtiter plates,
thus reducing the volume of extract needed, and have endpoints that can be deter-
mined spectrophotmetrically, either a measure of turbidity or use of a cell viability
indicator (e.g. resazurin, methylthiazoldiphenyltetrazolium (MTT)) [33]. Mann
and Markham [33] propose that the cell viability indicator is the best method of
endpoint determination for essential oils as the oil/water interface may interfere
with turbidity measures. While these micro-broth methods generally work well for
plant extracts, problems arise when the extract is heavily colored as this can inter-
fere with the measurement of the indicator chemical. Further, as these methods
use plastic microtiter plates, essential oils that have a solvent action on plastics (e.g.
Letospermum petersonii, Backhousia citriodora) cannot be used. We have also dem-
onstrated that the addition of essential oils to media changes its pH and this may
contribute to the observed antibacterial activity [19]; this might be expected to be
more significant in small volumes, for example in the micro-broth method.
Whether other plant extracts will also have the effect is unknown. Micro-broth
methods are also less time and resources intensive than other broth methods as
the need for multiple serial dilutions to determine bacterial count is eliminated.
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