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CHAPTER II METHODOLOGY MATERIALS & EQUIPMENT

500 g Magkuno Plant flowers and leaves (250g flowers and 250g leaves) Water Ethanol Glass funnels Filter Paper Beaker Rotavap Machine Petri Dishes Ruler Clean Bench Micropipetor Test Tubes

Mueller Hinton Agar

Distilled Water Chloramphenicol Evaporating Dish L-rod Stirring Rod Medicine Dropper Filter Paper Dishes Vile Erlenmeyer Flasks Screw cup tubes Scissors

Micrococcus luteus (gram positive).Autoclave Note: All materials and equipment dealing with microorganisms will be sterilized and be disinfected before using. The researchers will use surgical gown. . All materials and equipment dealing with microorganisms will be sterilized before using. Escherichia coli (gram negative) and Bacillus subtilis (gram positive) from Microbiology Laboratory of Biology Department. gloves. The researchers will use pure stock culture of selected microorganisms such as Salmonella (gram negative). and mask in doing the experiment with the supervision of designated supervisor. MSU-IIT where the researchers will perform their antibacterial experiment for three days with the close supervision of the microbiologist.

7 10 11 14 11.3 10 12 17 13 13 13 12 12.7 21 21 21 21 10 10 10 10 13 15 15 14.7 12 13 12 12.3 15 14 12 13.7 10 9 10 9.7 12 17 12 13. 1st replicate 2nd replicate 3rd replicate Average Micrococcus luteus 1st replicate 2nd replicate 3rd replicate Average Escherichia coli 1st replicate 2nd replicate 3rd replicate Average Bacillus subtilis 1st replicate 2nd replicate 3rd replicate Average 10 11 11 10.RESEARCH DESIGN Table 2.7 24 24 24 24 10 10 10 10 20 17 20 19 15 16 17 16 13 15 16 14.3 22 22 22 22 10 10 10 10 11 11 11 11 .7 11 15 12 12.1: Reading of the Zone of Inhibition in mm upon application of the Magkuno 100% OF STOCK SOLUTION ZONE OF INHIBITION (in millimeters) 75% 50% 25% Positive (CHLORAMOF OF OF PHENICOL) STOCK STOCK STOCK SOLUTION SOLUTION SOLUTION Negative Salmonella sp.7 23 23 23 23 12 15 13 13.7 9 11 10 10 8 8 10 8.

Tested Microorganisms 100% flower extract Salmonella sp.Table 2.2 Average measurements of the Zone of Inhibition in millimeters (mm) on Four Test Organisms using Different Concentrations of Magkuno flowers and leaves Extract. Micrococcus luteus Escherichia coli Bacillus subtilis Experimental Set-up Control Set-up Negative Control Ethatnol 75% flower extract 50% flower extract 25% flower extract Positive Control Chloramphenicol TREATMENTS/GENERAL PROCEDURES Sample Collection and Preparation of the Magkono Plant Extract as .

250 g leaves).Antibacterial Agent Fresh flowers and leaves of Xanthostemon verdugonianus will be collected at Surigao City in August 2010. Preparation for Different Concentrations for Treatment (Dilution Process) . Out of 500 g of Magkuno sample a total of 50-ml pure extract will be collected. The extract was placed in a 200 ml of Erlenmeyer flask and cover with cork. The tiny pieces will be placed in a container containing ninety percent (90%) ethanol for soaking in 1 day. the collected flowers and leaves will completely cut into tiny pieces using sterilized scissors. the sample will then be separated from the extract using filter paper and will be ready for rotary evaporation process to separate the pure extract of the plant from the alcohol. The Magkuno flowers will be weighed using a triple beam balance with a mass of 500 g (250 g flowers. Flowers and leaves will be washed and air dried. Then will be ready for Phytochemical Analysis and Experimental testing. After 24 hours of soaking in ethanol. The air dried sample will then be prepared for extraction. On the extraction process.

The three parts were diluted by distilled water into levels of concentrations 10 ml each.The 30 ml pure “Magkuno Plant” extract was divided into three parts. each part contains 10 ml. 50%. The concentrations were prepared for the experimental set – up. . and 25% Magkuno Plant extract. The control set-up was prepared by dissolving 200 mg of Chloramphenicol in sixteen (16) controlled media. Twenty millimeter of pure extract was used all in all in the dilution of the treatments of concentrations 75%.