METHODOLOGY

A. Experimental Design

This experiment consists of three replications in which each replication was

incubated for two days. A tabular form of each recorded data was taken as daily

observation. After the experiment, the gathered data was evaluated to select the best

replication which reacted to S. aureus and P. aeruginosa bacteria among the three

(antibiotic, distilled water and Crescentia cujete crude extract).

B. Materials

The materials used in this study were: 600 grams of Miracle Fruit Meat, scissors,

1000ml Erlenmeyer flask, 500ml of Erlenmeyer flask, 6 petri dish, lab gown, face mask,

hair net, ethanol, 4 liters of distilled water, cotton swab, alcohol, labeling stickers,

permanent marker, aluminum foil, cotton, funnel, digital camera, filter paper, engineer's

field notebook, agar, surgical gloves and shoe cover.

C. Test Organisms

A total of two species of bacteria were utilized in the study; one of which belong

to Gram-positive group, namely; Staphylococcus aureus while the other one belong to

Gram-negative group, namely; Pseudomonas aeruginosa.

D. Collection of Miracle Fruit Meat

The researchers provided their own Miracle Fruit Meat. The Miracle Fruit Meat

were picked freshly and after the meat was gathered; they were washed.

E. Extraction of the Miracle Fruit Meat

The Miracle Fruit Meat were washed with distilled water and dried under the heat

of the sun. The miracle fruit meat were cut into small pieces and placed inside the

1000ml Erlenmeyer flask. Ethanol was poured gently inside the 1000ml Erlenmeyer

flask and the opening was covered with cotton and was wrapped with aluminum foil. It

was placed inside the refrigerator for 48 hours. After 48 hours it was filtered using a filter

paper and funnel, the extract was placed in the 500ml Erlenmeyer flask.

F. Rotary Evaporation (RotaVap) Process of Miracle Fruit Meat Extract The solvent

collection flask of the unit should always be emptied prior use to prevent accidentally

mixing of incompatible chemicals or contamination. The flask with the solution is placed

on the rotary evaporator. A bump trap prevents the solution from accidentally splashing

into the condenser. A metal or Keck clip is used to secure the flask and the bump trap.

The aspirator vacuum is turned on. The flask is lowered into the water bath. The water

bath temperature should not exceed the boiling point of the solvent. For small amounts

of common solvents the bath heater is not needed. The solvent should start collecting

on the condenser and drip into the receiving flask. Some solvents (such as diethyl ether

or dichloromethane) are so volatile that they will also evaporate from the receiving flask

and be discharged down the drain. To prevent this, a cooling bath on the receiver or (on
some models) use a dry-ice condenser can be used. In addition, an additional trap (with

dry-ice or liquid nitrogen) can be placed between the vacuum source and the condenser

unit. Once all the solvent evaporated (or whatever is desired at this point), the vacuum

is released, the flask is raised out of the water bath and the spinning is discontinued.

The bump trap has to be cleaned and the receiving flask is emptied upon completion of

the evaporation. (http://www.chem.ucla.edu/~bacher/specialtopics/rotavap.com

G. Preparation of Culture Media

Test organisms were cultured in Nutrient Agar. This was prepared following the

recommended formulation, 28g/1000mL. The medium Was autoclaved at 15 psi for 20

minutes. It was cooled to about 45 C prior to pouring in plates.

H. Preparation of bacterial Inoculum

Each culture medium was inoculated with the test organisms. The inoculated

media were incubated for 24 hours at 37 C. Following incubation, cells were harvested.

It was kept until used.

I Preparation of Antibiotic Standard

Amoxicillin 500mg capsule was used in carrying out the antimicrobial assay. The

standard antibiotic solution, 5mg/mL was prepared by dissolving the contents of the

capsule in 100mL sterile water.

J. Filter Disc Treatment

Sterile filter discs were treated by soaking in filter-sterilized Miracle Fruit Meat

extract and antibiotic solution. Excess solvents in discs were allowed to drip for a While

prior to antimicrobial assay.

K. Antimicrobial Assay

All petri dish were sterilized. After it was sterilized, nutrient agar was placed

inside and was preserved. On the other hand, filter papers were put in the antibiotic

solution (gram positive), in distilled water (gram negative) and in Miracle fruit meat

crude extract. At the same time, a little amount of P. aeruginosa bacteria was swabbed

above the agar which was placed inside the petri dish using the cotton swab. All cotton

swabs should be disposed after it has been used to avoid contamination. There were 3

petri dish allotted for the P. aeruginosa bacteria which represent the three replications

or the R1, R2 and R3. After this, one filter paper from Miracle fruit meat crude extracts,

antibiotic and distilled water was put on the petri dishes on which P. aeruginosa bacteria

was placed into it. There should be enough space from one filter paper to another to
see the results or the zone of inhibition. For the S. aureus bacteria, all petri dish were

sterilized and nutrient agar was put inside it. After, S. aureus bacteria were swabbed

above the nutrient agar. Next, filter papers which were put in gram positive (antibiotic),

gram negative (distilled water) and in Miracle fruit meat crude extract were placed

above the S. aureus bacteria which was swabbed above the nutrient agar. There should

also be enough space from one filter paper to another to clearly see the zone of

inhibition. (http://www.antimicrobialscreening.com/