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2.7 BIO- AUTOGRAPHIC ASSAY METHOD
The extracts were prepared by dissolving 1 g of the plant powder in 10 ml of
dichloromethane. The mixture was covered with foil, shaken overnight with a
mechanical shaker and filtered. The filtrate was used for the bio-autographic assay.
Thin-layer bio-autographic assay was carried out by placing 5 µl of the extract on the
silica gel thin-layer chromatography Merck-manufactured TLC plates (Alugram R Sil
G/UV254, 0.2 mm). The plates were developed with acid mixture (72% toluene, 20%
dioxane, 8% acetic acid). Two TLC plates were prepared for each extract under
identical conditions: one to be incubated with the test microorganism Staphylococcus
aureus (ATCC 2600) and Mueller-Hinton agar and a reference TLC plate without a
test microorganism and agar. Both were kept at room temperature. The developed
TLC plate was dried and sterilized for one hour under ultraviolet (UV) light. It was
then placed on to the agar. The Mueller Hinton agar (15 ml) containing
Staphylococcus aureus, (approximately 1 x10 6 inoculums) was overlaid on top of the
TLC plates. The plates were allowed to pre-diffuse for one hour at 4 ºC; and,
thereafter, the plates were incubated for 24 hours at 37 ºC. The plates were sprayed
with iodonitro-tertrazolium (INT) (0.2 mg/ml) and compared to the reference TLC
plate (Van Vuuren, 2007).
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