Chapter 3

Methodology and Research Design

Chapter Three consists of four parts: (1) Research
Design, (2) Materials, (3) Procedure, and (4) Data
Collection Procedure.
Part One, Research Design, presents and describes the
objectives and plans in carrying out the experiment.
Part Two, Materials, enumerates the instruments and
chemicals that will be used in the study.
Part Three, Procedure, presents a step-by-step process
in performing the study.
Part Four, Data Collection Procedure, gives the
details in deriving results from the study.

Research Design
This descriptive study is designed to assess the microbial
population of microorganisms in street-vended food from selected
mobile food stall in West Visayas State University La Paz, Iloilo
City. This research study was performed in West Visayas State
University, Research Laboratory.

Materials
The experimental materials are food samples from a selected
food stall in West Visayas State University La Paz, Iloilo City,
Mannitol Salt Agar, Nutrient Agar, E. Coli Broth and distilled
water.
The glassware include petri dishes, graduated cylinder,
Erlenmeyer flask, beakers, culture tubes, glass jar, and Pasteur
pipettes.
Autoclave, centrifuge, analytical balance, and electric
water bath and other equipments are classified under the nonglassware materials.

Methodology
Sterilization of Materials. The researcher washed the glass
wares dry and wrapped them with an old cloth or newspaper. Then,
they were placed inside the autoclave at 15 psi for 15-20 minutes
at 121C. After sterilization, the researchers dried them.
Afterwards, the materials were stored for later use.
Sample Collection. Only one vending site was considered.
Fish balls (cooked) prepared and/or sold by vendors were
collected for microbial analysis.
Five samples of about 10 g of each food on sale were placed
in sterile ziplock plastics adequately labelled and transported

to the laboratory for analysis within two-three hours of

collection.
Microbial Analysis of Street Food. Microbiological analysis
was performed in West Visayas State University Central Science
Laboratory. Identification of microorganisms will be limited to
its possible taxonomic classification. Standard microbiological
methods that were used are serial dilution, spread plate methods,
and multiple tube method.
Total Aerobic Plate Count and Identification of Bacteria.
The total aerobic plate count is intended to indicate the level
of microorganisms in a product. Portions of food weighing 10 g
was diluted at 1:10 with 90 ml normal saline solution and
homogenized with a blender for 2 minutes. Further tenfold serial
dilution was made and examined by means of 10-fold serial
dilution and spread plate method. All even dilutions (10-2, 10-4 ,
10-6 10-8 and 10-10 ) were plated, using prepared MSA plates and NA
plates. The plates were incubated at 35C for 24 hours, and two
replicate plates containing between 30 and 300 colonies was
counted.
Average counts in 2 replicates obtained were expressed as colony
forming units per gram of food (cfu/g) with replicates by
multiplying the number of bacteria by the dilution. The formula
is shown below.

Figure 2. Formula for CFU/g

Preparation of Different Culture Media

Mannitol Salt Agar. About 110g of MSA agar was weighed. It
was dissolved in 1L distilled water. This was thoroughly mixed
and sterilized. The prepared media was aseptically transferred to
several petri dishes. This was allowed to cool and store for
later use.
Nutrient Agar. About 42g of NA agar was weighed. It was
dissolved in 1L distilled water. This was thoroughly mixed and
sterilized. The prepared media was aseptically transferred to
several petri dishes. This was allowed to cool and store for
later use.
E. coli Broth. About 37g of EC broth was weighed. It was
dissolved in 1L distilled water. This was thoroughly mixed and
sterilized. The prepared media was aseptically transferred to

several culture tubes with durham tubes. This was allowed to cool
and store for later use.

Preparation of Diluents. The researcher dissolved 9g of NaCl

in 1L of distilled water. The researcher transfered the solution
in series of 25ml test tubes containing 9ml each. The researcher
maked up to 1010 dilution per sample. Autoclaving with appropriate
temperature and sample.
Isolation of Test Isolate Using Serial Dilution Method. For
each collected sample, 10g of sample were suspended in 90ml of
normal saline solution. Mixtures was allowed to settle, and
serial dilutions up to 10-10 were prepared using sterile normal
saline solution and agitated normally.
An aliquot of 0.1ml of each dilution from 10-2 , 10-4 , 10-6
and were taken and spread evenly over the surface of Mannitol
salt agar medium and Nutrient agar medium. The researcher
incubated the plates at 37 degrees Celsius and monitored after
24hours. The number of bacteria calculated from the original
sample using the formula
for plate counts with replicate as shown below:

= total number of colonies counted x Dilution Factor

Isolation of Test Isolate Using Multiple Tube Method. For

each collected sample, 10g of sample were suspended in 90ml of
normal saline solution. An aliquot of 10ml, 1ml, and 0.1ml of 100
dilution per sample was asceptically transferred in culture tubes
with EC broth and durham tubes. The researchers incubated the
plates at 37 degrees Celsius and observed for turbidity and
presence of gas inside the durham tubes after 24 hours. The
number of coliform bacteria in the food sample was estimated by
comparing the positive results to the MPN table by Garthright and
Blodgett (2003).

Data Collection Procedure. Data collection was based on the

observation of the five different set-ups. The microbial counts
of the fish balls were calculated based on CFU/gram. The number
of bacteria were calculated from the original sample using the
formula for plate counts with replicates. While Multiple Tube
Method was used to measure the MPN/g of coliforms present in the
sample.
Descriptive Data Analysis. Fishballs will be classified as
safe or unsafe depending on the corresponding values It will show
in the parameters.