Chapter II

Methodology
Research Design
This study shall utilize an experimental research design with emphasis to quantitative-
qualitative research measures. This research will determine the antibacterial properties of ten
citrus fruits, compare and contrast the efficacy of the antibacterial properties of the 10 citrus
fruits, and assess the efficacy of citrus extracts and uices against E. coli. !uantitative data
shall be obtained in measuring the zones of inhibition demonstrated by the antibacterial
properties of citrus fruits. The gathered data will be qualitatively analyzed.
Materials and Methods
Citrus Fruits and E. coli Collection
The "itrus fruits necessary in this research are lemon #Citrus limon), calamansi #Citrus
x microcarpa$, pomelo #Citrus maxima$, sweet orange #Citrus x sinensis$, lime #Citrus
aurantifolia), %umquat #Citrus japonica), "hinese orange #Citrus reticulata$, dalandan
#Citrus aurantium$, grapefruit #Citrus paradisi$, and &affir lime #Citrus hystrix$. Citrus
limon, Citrus x microcarp, Citrus maxima, Citrus reticulata, and Citrus hystrix shall be
obtained at the 'ta. "ruz (ar%et, )amboanga "ity. *n the other hand, Citrus aurantifolia
and Citrus paradisi shall be gathered from mar%ets in (anila while Citrus japonica and
"ittrus aurantium shall be purchased from mar%ets in +avao. The strain of ,. coli shall be
acquired at -teneo de )amboanga .niversity (icrobiology laboratory.
Culture Media and other Chemicals
The research shall utilize nutrient agar in order to carry out the experiment.
-dditionally, ethanol shall also be utilized in the extraction process. +istilled water and
chloroamphenicol shall also be used which serve as negative and positive control
respectively.
Preparation of Fruit Juices
The procured fruits shall be washed with tap water and shall be air dried at room
temperature. Twenty five milliliters of fruit uices shall be manually extracted from each
fruit. - filter paper shall also be utilized to avoid the inclusion of seeds. ,ach fruit uice
shall be %ept in separate air tight containers with proper labeling. /emaining fruit peels
shall be %ept in separate containers which shall be used in the extraction process.
Preparation of Fruit Extracts
*nly fruits peels of C. limon, C. x microcarpa, C. x sinensis, C. reticulata, and C.
maxima shall be utilized in the extraction process. 0t shall be air dried for 1 hours, and will
be cut into fine pieces. The pieces of fruit peels shall then be pounded by a mortar and
pestle. The ground substance shall be placed in a container, and shall be diluted to 100
m2 of 304 solution of ethyl alcohol. 0t shall be left for two days in order to complete the
extraction process. -fter which, it shall be strained using filter paper in order to separate
the extract from the ground lemon peels. The extract shall now then undergo simple
distillation in order to remove the ethyl alcohol content.
Well Diffusion ssay
0n order to determine the antibacterial property of the extract and uices, antibacterial
susceptibility assay shall be performed in the form of agar well diffusion assay. 'terile
nutrient agar shall be prepared and shall be poured into 15 sterile petri dishes and it shall
be allowed to solidify. 6ifty 7l of inoculums will be spread on each plate using sterile
swabs. The inoculum shall be left to dry for five minutes. -fterwards, four wells of 1 mm in
diameter shall be bored using a sterile cor% borer in each agar plate. 6ifty 7l of the ten
fresh fruit uices and of the five extracts shall be poured into the wells of the 18 agar
plates with the help of sterile syringes. 6ifty 7l of autoclaved distilled water and
chloroamphenicol #9.8 mg:ml$ shall also be poured into the wells of the remaining agar
plates which shall serve as the negative and positive control. The plates shall be allowed
to diffuse at a refrigerator for ;0 minutes and shall be incubated at ;5< "elsius for 9=
hours. -fter incubation, the plates shall be observed for zones of inhibition.
Disposal !reatment
The apparatuses, glasswares, culture plates, and other materials shall be
autoclave for ;0 minutes. +isposable materials shall be placed in tightly sealed plastic
bags and then shall be placed in appropriate trash bins. -ll apparatuses that are used
in the experiment shall be washed with liquid detergent, dried, and stored properly.
>roper disinfection after the experiment shall be observed in the wor%place of the
laboratory
Data Gathering
The clear zones around the wells in the agar plates are %nown to be the zone of
inhibition, and it shall be measured in millimeters using a vernier caliper or a ruler.
Data Analysis
The mean of the zones of inhibition for each agar plate shall be obtained and
tabulated. 'tandard deviation shall also be obtained for each sample in order to assess the
concentration around the mean. The presence of a zone of inhibition in a treatment shall
determine the antibacterial property of the sample. *ne-way -?*@- shall be also employed
to assess the significant difference between treatments. 6urther comparisons shall be made
with the gathered data. 2astly, the average zones of inhibition of five extract treatments and
the average zones of inhibition of the five fruit uice treatments of the same five species shall
be obtained in order to assess the efficacy between fruit uice and fruit extracts against E.
coli. - 9-sample T-test shall be employed in determining significant differences between fruit
uices and extracts.