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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 bacterial or fungal suspension (direct bioautography) or overlain with agar and the agar seeded with the microorganism (overlay bioautography) (Hamburger and Cordell, 1987; Rahalison et al, 1991, 1994; panda et al., 2010a, 2011c) (Plate-6).

The later method has been particularly used for determining the activity of extract against yeasts. This method has been used to screen a range of crude and solvent prepared extracts with the activity observed dependent on both the method of extraction and solvents used in the TLC process (Diallo et al., 2001; Nakamura et al, 1999; Sridhar et al, 2003). This method has the advantage of combining both separation of extract constituents and simultaneous identification of those fractions with bioactivity. However, 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 fractionation phase.
plate6Screening Methods in the Study-9
Plate 6 : Photograph of TLC and bioautography (Source-Panda et al., 2011)

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