Preparation of B.

subtilis cell supernatant

B. subtilis was obtained from the Microbiology Laboratory, Department of Biology,

University of San Carlos. B. subtilis was maintained in nutrient agar. Cell supernatant was

prepared by inoculating B. subtilis in a 25mL of nutrient broth. The inoculated culture broth was

then incubated at 25ºC for seven days under shaking condition. After 7 days, the bacterial cells

and cell supernatant were then separated using Buchner funnel with whatman filter paper no. 1.

Preparation of Materials

Culture medium was prepared by dissolving 15grams of Potato Dextrose agar into 400ml

of distilled water. Normal Saline Solution (NSS) was also prepared for the inoculom. All

prepared materials were sterilized using autoclave at 121 degree Celsius for 15 minutes. Petri

dishes were sterilized using dry oven at 160 degree Celsius for 2hrs.

Preparation of Inoculum

48 hrs culture of Colletotrichum capsici was obtained. The microorganism was

inoculated to the sterile saline solution on tubes using inoculating loop until it became

comparable to the 0.5 McFarland standard. The fungal suspension was performed using aseptic

techniques inside the biological safety cabinet level 2.

Agar Diffusion Method

Sterile Potato Dextrose agar was cooled to 50-55C. The cooled medium was poured to

the individual sterile petri dishes. Agar wells were then made using a cork borer. Inoculum of

test fungi which is comparable to the standard were swabbed uniformly on solidified sterile

potato dextrose agar plates using sterile cotton swabs. 100µL of cell supernatant were then added
to the wells including with the positive control and the negative control.The inoculated plates

were incubated at 30°C for 48 h for fungi and the zones of inhibition that formed around the well

were measured using a microcaliper. Known antifungal such as Dithane M-45 (Neotec) was

introduced to the plates after the preceding procedure. Diameters of the inhibition zone by the

extract and the known drug was compared.

All procedures were done in three trials with three replicates at the Microbiology and

Microtechnique Laboratory, University of San Carlos, Talamban, Cebu City, Philippines.