Methodology for Antimicrobial Studies

Plant material:

The plant materials of different plant species were collected in and around the Visakhapatnam district and shade dried. After complete dryness they are chopped into small pieces and are coarsely powdered in a wiley mill.

Preparation of plant extracts:

The extraction method employed here is a known amount of coarsely powdered plant materials were successively extracted with organic solvents like hexane, chloroform, methanol and water basing on order of polarity using soxhlet apparatus. The different extracts obtained were subsequently concentrated under reduced pressure to get their corresponding residues. The extracts were screened for anti-microbial activity using the method described under the section.

Organisms and Media:

The organisms used were purchased from Microbial Type Culture Collection & Gene Bank (MTCC), Chandigarh. The strains are maintained and tested on Nutrient Agar (NA) for bacteria and Potato Dextrose Agar (PDA) for fungi.

Antimicrobial assays:

The crude extracts of the different plant parts of different species were subjected to antimicrobial assay using the cup plate method of Murray et al (1995) modified by Olurinola (1996). 20 ml of nutrient agar was dispensed into sterile universal bottles these were then inoculated with 0.2ml of cultures mixed gently and pouredinto sterile petri dishes. After setting a number 3-cup borer (6mm) diameter was properly sterilized by flaming and used to make five uniform cups/ wells in each petri dish. A drop of molten nutrientagar was used to seal the base of each cup.The cups/wells were filled with 50µl of the different extracts of 100mg/ml, 200mg/ml, 300mg/ml, 400mg/ml and 500mg/ml, and allowed diffusing for 45 minutes. The solvents used for reconstituting the extracts were similarly analyzed. The plates were incubated at 37°c for bacteria and 25°c for fungal organisms. The standard antibiotic (Streptomycin)and anti-fungal (Bavistin) drugs were used at different concentrations to get MIC (Minimum inhibitory concentrations). The zones of inhibition were measured with Antibiotic zone scale in mm and the experiment was carried out in triplicates.