Thin-Layer Chromatography–Bioautography
While the methods above are used to test whole extracts or extracts fractionated at
another time there is an increasing interest in bioassay-guided fractionation,
where the separation of extracts into fractions is completed simultaneously with
identification of bioactivity. In this method TLC is performed using crude extracts,
extract fractions, or whole essential oils. The developed TLC plate is then sprayed
with, or dipped into, a bacterial or fungal suspension (direct bioautography) or
overlain with agar and the agar seeded with the microorganism (overlay bioautog-
raphy) [34–37]. The latter method has been particularly used for determining the
activity of extract against yeasts such as Candida albicans, however Masoko and
Eloff [38] suggest that use of fresh cultures of yeasts and shorter incubation times
eliminated the previously reported difficulties of using the direct method with
yeasts [39]. Following incubation zones of inhibition are observed, either unaided
or following development with compounds such as MTT, around those com-
pounds with antifungal or antibacterial activity.
This method has been used to screen a range of crude and solvent-prepared ex-
tracts with the activity observed dependent on both the method of extraction and
solvents used in the TLC process [40–44]. While this method has the advantage of
combining both separation of extract constituents and simultaneous identification
of those fractions with bioactivity, it is not a suitable method for detecting activity
that is a product of synergy between two or more compounds. Further, the results
will be affected by the breakdown or alteration of compounds during the fraction-
ation phase.
164 8 Methods for Testing the Antimicrobial Activity of Extracts
While the methods above are used to test whole extracts or extracts fractionated at
another time there is an increasing interest in bioassay-guided fractionation,
where the separation of extracts into fractions is completed simultaneously with
identification of bioactivity. In this method TLC is performed using crude extracts,
extract fractions, or whole essential oils. The developed TLC plate is then sprayed
with, or dipped into, a bacterial or fungal suspension (direct bioautography) or
overlain with agar and the agar seeded with the microorganism (overlay bioautog-
raphy) [34–37]. The latter method has been particularly used for determining the
activity of extract against yeasts such as Candida albicans, however Masoko and
Eloff [38] suggest that use of fresh cultures of yeasts and shorter incubation times
eliminated the previously reported difficulties of using the direct method with
yeasts [39]. Following incubation zones of inhibition are observed, either unaided
or following development with compounds such as MTT, around those com-
pounds with antifungal or antibacterial activity.
This method has been used to screen a range of crude and solvent-prepared ex-
tracts with the activity observed dependent on both the method of extraction and
solvents used in the TLC process [40–44]. While this method has the advantage of
combining both separation of extract constituents and simultaneous identification
of those fractions with bioactivity, it is not a suitable method for detecting activity
that is a product of synergy between two or more compounds. Further, the results
will be affected by the breakdown or alteration of compounds during the fraction-
ation phase.
164 8 Methods for Testing the Antimicrobial Activity of Extracts
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