I have been screening plant extracts for bioactivity against the yeast Cryptococcus neoformans. I performed agar overlay bioautography on plant extracts separated on glass-backed TLC plates. I sprayed 2.5 mg/mL INT (p-iodonitrotetrazolium violet) dye onto the plates after incubation and incubated again as per protocol
Very faint inhibition zones were observed (slightly less pink than areas between separated compounds) after 2-3 days incubation at 37 °C.
I then placed the plates in the refrigerator at 4 °C. After leaving it there for 3 months, clear white zones were now present over a few of the separated compounds. The slightly pink, semi-inhibited zones from the initial incubation were now completely clear (white). How could the formazan dye move from the semi-inhibited zones to leave a clear zone? Are the compounds causing the pink formazan to change back to no colour (white) OR is it possible that the slow growing yeast at 4 °C was being inhibited after 3 months growth making the inhibition zones more apparent?
I don't understand how pink zones can turn into clear zones when using INT dye?
Hi Chris,
Regarding the principal of tetrazolium salt dye (detection of dehydrogenases enzymes present in all living cells), it's not logic to let your chromatogram for 3 months. The change in color during this period did not reflect the inhibition.
When you use bioautography you should examine the inhibition directly after incubation period to get significant results.
In your case, try direct bioautography. In direct bioautography, the use of tetrazolium salt is more convenient.
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