Evaluating Qualitative Biological Activity
Bioautography can determine how many biologically active compounds are
present in an extract or can be used to ensure that a compound isolated earlier is
not isolated again.
The components of an extract are separated, usually by TLC. If microorganisms
can grow on the plate it is usually easy to determine the Rf value of the compound
that inhibits growth. Some authors have tried blotting the wet TLC plate with filter
paper and then spraying the filter paper with a culture of the test organism. Others
have applied an agar layer over the chromatogram by spraying with semi-liquid
agar containing the test organism.
Neither of these techniques gave good results in our hands. We tried spraying
the culture directly on the chromatogram, but it is difficult to visualize microbial
growth. If a fungus that has visible spores is used this does work. We then used a
technique developed by Begue and Kline [41]. In this technique the eluent is re-
moved from the chromatogram, which is sprayed with a concentrated microbial
suspension and after overnight incubation, the chromatogram is sprayed with tet-
razolium violet. Microbial growth is indicated by red-purple areas and inhibition by
clear areas. This method worked very well with aerobic bacteria (Fig. 5.1). By add-
ing carbon dioxide and by including anaerocult the method also worked well with
micro-aerophilic/anaerobic bacteria [42].
The bioautography technique is more difficult with fungi because they grow
slower and contamination can be a problem. We have succeeded in developing a
technique that works well with several fungi including C. albicans [43].
Bioautographic techniques work extremely well if antioxidant activity is deter-
mined using the free radical DPPH (1,1-diphenyl-2-picryl-hydrazyl) as a spray re-
agent (Fig. 5.2).
By applying these qualitative techniques, one can obtain a good idea of which
species would be the most interesting to investigate further. By comparing bioau-
106 5 Plant Extracts Used to Manage Bacterial, Fungal, and Parasitic Infections in Southern Africa
tograms measuring different activities one can identify compounds with antibacte-
rial, antifungal, and antioxidant activities.
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