The antibiotic standards The antibiotic standard used in this bioassay is penicillin G sodium salt (benzylpenicillin) whose potency

will be treated against Bacillus subtilis or Staphylococcus aureus. Penicillin G is active against Gram-positive bacteria only. Plant extracts, that are active against Garm-positive bacteria, can be compared with the potency of penicillin G. For Gram-negative bacteria is general and Escherichia coli in particular ampicillin, gentamicin sulfate, tetracycline or choramphenicol can be used. Nystatin and amphoteracin B are active against the yeast Candida albicans or Candida tropicalis or better yet one can use the nonpathogenic yeast Sacharomkyces cerevisiae. The filamentous fungi biossay can be done against cyclohexamide. Penicillin G. The pure sodium salt of penicillin G, with a molecular weight of 365.38, has an assigned activity of 1667 units per mg of pure penicillin G. The pure potassium salt, with a molecular weight of 372.47, has an activity of 1595 units per mg. Preparation of the phosphate buffer solution 1.8 g KH2 PO4 8.2 g K2 HPO4 Dissolve in one liter of distilled water. With a pH meter, check the pH of the resulting solution and adjust to pH of 6.0. Use the following dilution equation if necessary: C1 V1= C2 V2 where: C1 is the initial concentration V1 the original volume C2 is the final diluted concentration V2 the final diluted volume

Use distilled water for dilution. Prepare stock solution of the antibiotic standards as given below: Weight 15.0 mg or 0.015 g penicillin G in a previously sterilized 150 mL beaker with the aid of a clean patula; Dissolve the antibiotic initially in 100 mL of sterile phosphate buffer pH 6.0; Transfer into a 250 mL volumetric flask, wash remaining solution with the pH buffer and dilute to mark with more phosphate buffer; The final solution should have a concentration of 0.06 mg/mL or 100 units/mL, based on the 1667 units/mg activity of penicillin G;

This serves as the stock solution. May be kept refrigerated for 1 week.

In five (5) previously sterilized regular sized test tubes, place in each test tube 10 mL of penicillin G with increasing potencies of 0.5,1.0,2.0, and 2.5 units/mL. as shown in the Table M2 below; Test Tube# Vol. of stock solution 0.05 0.10 0.15 0.20 0.25 Vol. in mL of buffer solution to be added 9.95 9.90 9.85 9.80 9.75 Final activity of the antibiotic in units/mL 0.5 1.0 1.5 2.0 2.5 Final concentration in ug/mL 0.3 0.6 0.9 1.2 1.3

1 2 3 4 5

3.3 The Assay Proper: Agar- well Diffusion Method Make 6 well/cup in each of the bioassay plates prepared in Section 3.2.2 with the use of a 6.0 mm sterilized cork borer; follow the procedure given in Section 1.3.1.1 Number the wells from 1 to 6 in all the assay plates. Refer to figure M10; Deliver the contents of each of the tubes #1 to 5 into their respective wells; In well #6 deliver the plant extracts; In well # 7 deliver the solvent used as negative control; Validity of assay By subjecting both plant extract and different concentrations of the antibiotic standard Under the same conditions, a comparison of the inhibition of the standard can be made with the plant extract. Some precautions The Pasteur pipettes are sterilized by lightly plugging the upper open end with cotton, wrapping sets of 5 pipettes in a paper or aluminum foil and autoclaving at 121C for 20 minutes Use one sterile Pasteur pipette for each antibiotic concentration and for each plant extract concentrations Allow the contents of the pipette to drip into each respective well by pinching the rubber bulb gently; The liquid should completely fill up the well to form a concave upper rim of liquid; Use the same number of droplets per well for all the wells; Care must be taken not to let the liquid overflow from the well. This will result in distorted inhibition zones.

Number of bioassay plates Three of the bioassay plates are used to assay the high concentration, 400 ug/mL plant extracts and the remaining 3 plates to assay the low concentration, 40 ug/mL plant extract. Incubate the plates at room temperature, preferably at 35C to 18 hours; After the incubation period, the solution in the well would have been completely absorbed in the agar medium, Invert the plate and measure the size of inhibition zone with use of a good ruler or a Vernier caliper; Inspect the two sets of plates, one set of 3 plates for the low plant concentration that gave defined measurable halo well within the set of mean inhibition zone produced by the standard antibiotics concentration.