Mmushi T MSc (Microbiology).pdf

each of the finely ground samples and each extracted with 10 ml of either hexane,
dichloromethane (DCM), acetone or methanol in 50 ml polypropylene tubes. The tubes
were vigorously shaken for 10 min at high speed. After centrifugation at 959 xg for 10
min, the supernatants were decanted into pre-weighed 50 ml Erlenmeyer flasks The
extraction process was repeated three times to thoroughly extract the plant material.
The solvents were evaporated in the fume cupboard at room temperature and the
remaining matter was quantified.
2.3. Test organisms
Antimycobacterial activity was tested against Mycobacterium smegmatis mc 2 155 and R.
erythropolis ATCC 4277. The bacterial strains were a gift from the School of Molecular
and Cell Biology, University of Witwatersrand. Mycobacterium smegmatis was
maintained on Middlebrook 7H9 broth containing 0.05% Tween 80 and 10% v/v ADC
supplement (Albumin Fraction V, Dextrose and Catalase) and R. erythropolis on Luria
Bertani broth (LB) at 37°C. The purity of the cultures were ensured by means of the
Ziehl-Neelsen staining and Gram staining, respectively, before being used in the
antimicrobial assays.
2.4. Screening of plants
2.4.1. Phytochemical screening
The main compounds of the plant extracts were analyzed by thin layer chromatography
(TLC) using aluminium-backed TLC plates (Merck, silica gel 60 F 254 ) according to the
method of Kotzé and Eloff (2002). The TLC plates were developed under saturated
conditions with each of the three mobile phases viz. (1) ethyl acetate: methanol: water
(40:5.4:4), [EMW] (polar/neutral); (2) chloroform: ethyl acetate: formic acid (30:24:6),
[CEF] (intermediate polarity/acidic); and (3) benzene: ethanol: ammonia hydroxide
(72:8:0.8): [BEA] (non-polar/basic). The separated compounds on the chromatograms
were visualized under UV light and sprayed with vanillin-sulphuric acid and heated at
110°C for colour development. The sprayed plates were scanned with a laser scanner
and analysed.
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