METHODOLOGY

A. Locale of the study

The study was conducted at Negros Prawn Producers Cooperative Analytical and

Diagnostic Laboratory, Bacolod City.

B. Materials and Equipment

1kg of Luhang Dalaga ( Pedilanthus tithymaloides ) leaves

12 Petri dish

3 Mask

6 Gloves

Vacuum rotary evaporator

1% sulphuric acid

Barium Chloride Dihydrate (BaCI2 2H2o)

Spectrophotometer

Forceps / Inoculation loop

Ruler

Vortex mixer

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 C. Delivery of the Luhang Dalaga Leaves

One (1) kg of Luhang Dalaga (Pedilanthus tithymaloides) fresh leaves were

delivered in the Negros Prawn Producers Cooperative Analytical and Diagnostic

Laboratory. It was freshly cut from the mother plant of Luhang Dalaga (Pedilanthus

tithymaloides) and was washed thoroughly to remove dirt and other impurities on the

leaves.

D. Drying and grinding of leaves

The leaves were then leave to dry in a controlled 400C room temperature. Then the dried leaves

were grinded by the use of a mechanical blender.

D. Soaking with 1:10 ethanol

Dried ground leaves was macerated in Ethanol (EtOH) overnight in a 1:10 ethanol.

F. Filtration

The grinded leaves of Luhang Dalaga that was macerated for 24 hours in an ethanol was

then filtrated by the use of a clean wool or cloth.

G. Rotary Evaporation Method

The solvent was evaporated 1:10 ethanol and controlled temperature (30 degrees celcius)

using a vacuum rotary evaporator.

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 H. Analysis using Kirby-Bauer Disc Diffusion Method:

Preparation of McFarland’s Standard: 100 mL of McFarland’s standard was prepared by

mixing 99.5 mL of 1% sulfuric acid and 0.5mL of 1.175% BaCI2 2H2o. The turbidity is

equivalent to 1.0 x 10 8 CFU/ml bacteria. Bacterial Isolates from pure culture of S. aureus and

fungal isolates from pure culture of C. albicans were then transferred to sterile buffered

phosphate or distilled water solution and the turbidity was adjusted using a spectrophotometer at

600 nm wavelength in comparison with the absorbance of the McFarland’s standard, 0.089.

I. Sensitivity Testing for S. aureus and C. albicans.

The sterile swab was dipped it into the broth culture of organism and was gently

squeezed against the inside of the tube in order to remove excess fluid in the swab. The swab

with the test organism was streaked evenly on each of the sterile and labeled Mueller-Hinton

agar (MHA) plates. After the streaking was completed, the plate was dried for 5 minutes, then

the discs (from Luhang Dalaga Crude Methanolic Extract) were placed on the surfaced of each

of the agar using sterilized forceps. The discs were gently press into the surface of the agar using

flame sterilized forceps or inoculation loop. The plates were then carefully inverted and

incubated for 24 hours at 37 degree C. After incubation , a ruler was used to measure the

diameter of the zone of inhibition

for each of the discs used. The measurement obtained from the individual discs were recorded

and compared with the standard table to determine the sensitivity zone to determine whether the

tested bacterial species is sensitive to the extract of Luhang Dalaga.

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J. Statistical Treatment

The statistical tool used was mean. It refers to the mean or average that is used to derived

the average of the Zone of Inhibition. It is determine by adding all the distance traveled by the

bacteria and then dividing to the total number of replicates.

K. Risk Assessment

A lab gown is use to provide protection of skin and personal clothing from incidental

contact and small splashes. Gloves help keep your hands clean and lessen your chance of getting

germs that can make you sick. A face mask also protects the wearer's nose and mouth from

splashes. No food or drink is allowed in lab unless food or drinks are provided as a part of the

lab. Even though lab tables and counters are wiped down before each lab set up, as a result of

some laboratory exercises, chemical residues may be present on the tables. Handle chemicals,

reagents, and stains carefully and follow all warnings. All bottles and containers are labeled as

to contents and potential hazards. If, for example, a label says avoid contact with substance and

fumes, do so. For potentially hazardous chemicals, information on the hazards, proper handling,

and clean-up is provided on Material Safety Data Sheets (MSDS). These are available in the lab.

It is highly recommended that you spend the first few minutes of the lab consulting the MSDS.

Label all test tubes and other containers with contents.

L. Waste Disposal

Used materials were properly disposed. Used equipment for the treatment were properly

disinfected and returned to its proper container after the treatment.

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