The Combinatorial Of M.oleifera And J. Curcas On Bacteria And Fungi

CHAPTER THREE
3.0 MATERIALS AND METHODS
 Collection and Identification of plant leaves
Healthy leaves of Jatropha curcas and Moringa oleifera were harvested from saw-mill area garden Moniya Ibadan,Nigeria in October 2017.the taxonomical identification of  the plant was confirmed by a plant taxonomist Mr Bolu Ajayi University Ilorin, Kwara  State .voucher number UILH/001/1008/UILH/002/1018
The fresh leaves of Jatropha curcas and Moringa oleifera were harvested, rinsed with tap water and air-dried under shade for 7 days.
3.1    RESEARCH DESIGN – Experimental design
3.2    SAMPLE AND SANPLING TECHNIQUES
Bacterial and fungal isolates (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus, vulgaris, Salmonella typhi, and Candida albican and Aspergillus niger) were tested against different concentrations of Jatropha curcas and Moringa oleifera leaf extracts (mg/ml) 1.0, 0.5, 0.25, 0.125, 0.0625.
The study was carried out using purposeful sampling techniques a type of non-probability sampling method which allows the researcher to make use of laboratory testing on bacteria and fungi isolate.
3.3    PREPARATION OF THE EXTRACT
      These were carried out using soxhlet extraction method. About 30g grounded powder plant materials of Jatropha curcas and Moringa oleifera were subjected  to sequential extraction using 150ml ethanol each for 6 hours. The ethanol extract was concentrated into dryness by evaporation of the solvent in water bath and the weight was 3.19g of Moringa oleifera and 3.04g of Jartropha curcas were extracted from 30g powder of sample. The extract was stored in a sterile container at 4oC in a refrigerator until require for use.
3.4    COLLECTION OF TEST ORGANISM
Pathogenic bacteria isolates of Escherichia coli, staphylococcus aureus, pseudomonas aeruginosa , proteus vulgaris, salmonella typhi,streptococcus pneumonia . Two fungal candida albican and Aspergilus niger were obtained from the Microbiology Department of UITH Ilorin, Kwara State, were used for the study. They were isolated and purified on blood agar, macCkey, Potatoes dextrose agar plates, characterized and identified.
ANTIMICROBIAL SUSCEPTIBILITY TEST/ZONE OF INHIBITION.
Antimicrobial activity of the extracts was determined by Agar- well diffusion method using the test organisms; Escherichia. coli and Staphylococcus. aureus.
An agar media was prepared and a sterile cork borer of about 6 mm in diameter was used to bore holes on the agar media.
The already characterized test organisms were swabbed on the solid agar media.
Aliquots of 0.2 ml of the extract was introduced in the holes made on the agar media containing each isolate of organisms, at different concentrations of (1.0 ,0.5, 0.25, 0.125, 0.0625mg/ml) of the two (2) extracts (Moringa. oleifera and Jartropha curcas).
A control hole where the solvent used for the extraction was added and the plates were incubated at 37 °C for 24 hrs in the incubator.
The ability of the various extracts to inhibit growth of the clinically significant bacteria was measured and recorded as diameter of the zone of inhibition in cm.