PHYTOCHEMICAL SCREENING FOR SECONDARY PLANT METABOLITES Extraction of Fresh Plant Material 1.1 Weigh 100g dried ground plant in an Erlenmeyer flask 1.2 Treat sufficient 80% ethyl alcohol to completely submerge the material *Note the volume alcohol used 1.3 Stopper the flask and keep the material soaked for 24-48 hours. 1.4 Filter through buchner funnel with gentle suction. 1.5. Rinse the flask and plant material with fresh portions of alcohol. 1.6 Transfer washings and plant material to the funnel, combining the washings with the first filtrate 1,7 Apply gentle suction to complete collection of the plant extract. 1.8 Discard the plant residue. 1.9 Concentrate the filtrate under vacuo at temperature below 50°C to about 20mL, Measure the exact volume of the concentrated extract.Prior to use, shake the turbidity standard vigorously on a mechanical vortex mixer; This standard can be kept up to 6 months, if stored in tightly sealed screwcapped tube in the dark The broth culture Preparation of the nutrient broth Refer to label for the preparation of 1000 mi solution. Take a loopful of bacteria, gram positive or gram negative, from the culture slant and inoculate in 50m! nutrient broth; Incubate the culture broth for 18-24 hours at 35° Observe the culture broth for turbidity, indicative of microbial growth Adjusting the turbidity of the inoculum The adjusted turbidity serves as the inoculum for the microbial assay. This will serve as the inoculum which will be swabbed onto agar plates. Use within 15 minutes after adjusting the turbidity. Aseptically transfer 5 mi of the culture broth in sterile screw-capped tubes. Agitate on a vortex mixer the bacterial suspension and immediately compare against the 0.5 Mcfarland standard prepared. If the bacterial suspension does not appear to be of the same density as the Mcfarland standard, adjust the turbidity by adding sterile saline solution or culture broth and subsequently compare the resulting turbidity to the standard,sheet of white paper on which 2 sharp - | rei acusting inocu tarbsity against a MiFavand bavi sulfate standacd Preparation of plates Prepare plates depending on the number of test organism and replications fequired. Common practice requires 2 replicates for each extract and for each test organism. Pour approximately 15 ml of melted nutrient agar into dry and sterile petri dishes. Let the medium solidify Moisten a sterile cotton swab into the test organism (inoculum) suspension, Use cotton swab with wooden applicator handles. Dip a sterile cotton swab into a suspension of the test organism/inoculum. Press and rotate the moistened swab firmly against the inside walll of the tube just above the fluid level to remove the excess liquid Cotton swabbing Aseptically swab the test organism into a solidified nutrient agar by streaking the swab over the entire surface of the agar plate ‘3X, rotating the plate 60 degrees after each application to ensure an even distribution of the inoculumFigure M4, Swabbed agar plate on the surface of the medium. Let the swabbed plates stand for 5 minutes. per disc diffusion method Materials: Screw-capped tube of test organism Nutrient agar Forcep 6 mm or 13 mm paper disc, sterilized in petri dish (Pack disc in sets of 10) Using the forceps pick out one paper disc and immerse the paper disc into the plant extract. Lay the moistened filter disc gently on the seeded agar plate.Figure M7. Placing paper discs conte the agar plate Gently tap disc with forcep to ensure maximum full contact of the dic with the agar medium, Incubate the plates inverted. Technique for handling the paper disc and moistening with the plant extract: With the forcep on one hand and the petri dish containing the serilized filter discs on the other hand, open the dish and pick out one paper disc with the forcep. Immerse the disc into the assay solution. Remove excess liquid by letting the moist paper disc to touch the inside wall of the container of the assay solution. IV. Reading the say plates, Look for a “HALO” or “Clearing” around the discs. This is known as the zone of inhibition. Invert the plates and with a ruler measure the diameter of each inhibition zone in millimeters. Express results as mm diameter zone of inhibition. Record the diameter of the paper disc used in the assay in mm.IV. Analyzing the results <10 mm may be expressed as inactive 10-13 mm, partially active 14-19 mm, active >19 mm, very active