CHAPTER III

METHODOLOGY

Research Design
This study uses the experimental design in determining the inhibitory growth effect of
P.acidilactici and L. mesenteroides isolated from homemade Kimchi with varying concentrations
at 50%, 75% and 100% against S. pyogenes and the same goes for commercialized Kimchi. The
positive control will be clindamycin and sterile water will serve as the negative control. Each
treatment administered in this study will be replicated five times.
Variables of the Study
The main variables of the study are the zone of inhibition yielded by S. pyogenes and the
specific lactic acid bacteria isolated from homemade and commercialized kimchi with varying
concentrations.
The independent variable will be inclusive for P. acidilactici and L. mesenteroides isolated
from homemade and commercialized kimchi at different concentrations of 50%, 75%, and 100%.
This will be the cause of dependent variable.

The dependent variable will be the presence of zone of inhibition produce by S. pyogenes
and when it is planted with an antimicrobial agent which are P. acidilactici and L. mesenteroides.

This will be the effect of the independent variable, as to what extent the different concentrations
will inhibit the growth of the specified pathogens.

Research Procedure
A. Pre-data Collection Procedure
Collection and Preparation of Test Sample
The bacteria use in the study will be acquired from the National Institute of Molecular
Biology and Biotechnology (BIOTECH), University of the Philippines Los Banos, College,
Laguna 4031, Philippines. Upon its receipt, these will be subjected to its partial identification
through gram staining, H2O2 production, biochemical test reactions such as arginine hydrolysis
(ADH), citrate degradation, production of acetoine and carbohydrate fermentation profile.

Kimchi Preparation
1. Preparation of salting condition of cabbage
Prepare 3 kilograms of Chinese Cabbage (Brassica rapa subsp. Pekinensis) and cut each
whole cabbage from the head down the stalk carefully not cutting all the way through then
separate it with your hands. Wash the petchay in cold water then soak it in a solution of 15%
Kosher salt for 5 to 6 hours. The soaked Chinese cabbage are washed in tap water three
times and excess water is drained.
2.

Making of the Kimchi base

Mix 2 cups of distilled water and 2 tablespoons of sweet rice flour and bring them to
boil in a low heat while stirring it constantly until it reaches a thin soup-like consistency.
Then add 2 tablespoons of brown sugar and keep on stirring until sugar is completely
dissolved. Turn the heat off and let the porridge cool. Next, prepare 2 cups of thinly
sliced radish , cup of thinly sliced carrots one cup of chopped green onions or one cup of
chives.
Prepare 1 cup of garlic and 2 tablespoons of sliced ginger and 1 cup of chopped white
onion and puree it using a blender. Add cup of fish sauce , cup of fermented shrimp
and 2 cups of hot pepper flakes to your puree. Add the porridge mixture and cut vegetables
to the puree, mix well and now you have your kimchi paste.
3. Coating Chinese Cabbage with Kimchi paste
Properly rub or coat the drained salted Chinese cabbage with the kimchi paste into
each leaf and stalk and fold it to half then transfer it to a jar with cover. Press the coated
chinese cabbage making sure theres no space left in between.

4. Kimchi Fermentation
See to it that it is pressed well inside a jar and allow it to ferment for 36 hours in a room
temperature. To check if its fermented, it would give off a sour and sweet smell. After 36 hours
place it in the refrigerator with a temperature of 4 degrees Celsius. After 24 more hours the

kimchi is ready to be squeezed with an electric juicer for the collection of the filtrate that will be
subjected to centrifugation, dilution and plating.
Preparation of Culture Media for Lactic Acid Bacteria
A. Phosphate Buffered Saline
Phosphate Buffered Saline is a water based salt isotonic solution used for diluting
substances. The process of making dilution and controls in examining contaminated substances
and other specimens is through the use of phosphate buffered saline, pH 7.2. This solution
supplies ions such as potassium, sodium and phosphate ions which inhibits bacterial damage due
to alteration of pH in the medium. The said medium is independent from inhibitors and is well
buffered and provides conditions resuscitation of the cells.
Composition and instruction for usage
Phosphate buffered saline pH 7.2 comprises sodium chloride ( 8.500 g/l), Disodium
hydrogen phosphate (1.910g/l) and Potassium dihydrogen phosphate (0.380 g/l). This ingredients
are dehydrated powder and hygroscopic in nature which should be stored in a dry place in a wellsealed container below 25C and away from sunlight. The medium is prepared by dissolving
10.79gms in 1000ml distilled water. The medium is completely dissolved through heating to boil
with gentle swirling. It is then sterilize by autoclaving at 10 psi (115C) for 10 minutes. It is
finally cooled at room temperature prior to use.
B. de Man Rogosa and Sharpe Agar (MRS Agar)
MRS or Lactobacillus agar is a specialized agar used to isolate and cultivate Lactobacilli
specie. The medium contains carbon, nitrogen and vitamin sources namely: Proteose peptone,
beef extract and yeast extract these sources are used to satisfy the general growth requirements of
Lactobacilli in MRS agar. The fermentable carbohydrate and energy source is Dextrose. The

surfactant which supplies the fatty acids that are required for the facilitating metabolism uptake
of nutrients by Lactobacilli is Tween 80. Sodium acetate and Ammonium citrate acts as selective
inhibitory agent for e.g., Streptococci, moulds and many other microorganisms. Manganese and
Magnesium sulphate both provides cations used in metabolism while Disodium phosphate
provides good buffering action in the media. Lactobacilli appear as large white colonies
embedded in or on Lactobacilli MRS agar. The growth can or maybe subcultured into the
appropriate media for use in additional procedures. Lactobacilli require many layer of plates for
aerobic cultivation on solid media and they are microaerophilic. Its appearance is medium to
dark amber colour, clear to slightly opalescent gel.
For the preparation, dissolve 67.15 grams in 1000 ml of distilled water. Gently heat to
boiling with gentle swirling and dissolve the medium completely. Sterilize it by autoclaving at 15
psi (121C) for 15 minutes then cool the medium at 45-50C prior to dispense into sterile petri
plates.

Preparation and Isolation of Lactic Acid Bacteria from Kimchi Extract

After kimchi is fermented, a liquid juice is obtained from the 500 g of kimchi samples
being squeezed by the blender taken from the procedure of Jinhee Cho, et al. (2006), in their
study of Microbial population dynamics of kimchi, a fermented cabbage product. The sample is
subjected by 10-fold serial dilution with sterilized phosphate buffered saline and an aliquot of 0.1
ml was pipetted to the MRS agar plate using the multiple interrupted streak and was incubated
for 36 hours at 35C. The plates grown with single white colonies are subjected to pure plate

technique supported by the procedure of Keunho Ji, Na Young Jang, and Young Tae Kim (2015)
in their study of Isolation of Lactic Acid Bacteria Showing Antioxidative and Probiotic
Activities from Kimchi and Infant Feces with slight modifications. In selecting for colonies
grown from the pure plate, we isolate all colonies from a given plate to rule out possible bias and
to differentiate among the microbial population which is P. acidilactici and L. mesenteroides.
Lactic Acid Bacteria Differentiation
To examine for the physical and physiological properties of the single colony isolated
from the pure plate, each colony is morphologically differentiated with Gram staining. Results
from Gram staining that showed Gram positive, non-spore forming and catalase negative
diplococci will be subjected for further identification through its physiological properties using
biochemical reactions and carbohydrate fermentation.
A. Biochemical Reactions and Carbohydrate Fermentation
Gram positive, non-spore forming and catalase negative diplococci is tested for its
physiologic properties such as arginine hydrolysis, citrate degradation, and acetoin production
using Voges-Proskauer. Each samples are for their ability to ferment sugars such as arabinose,
maltose; rhamnose, mannitol, lactose, fructose, glucose, and sucrose. Their are other tests that are
used to further identify the specified lactic acid bacteria but it is excluded due to the limited
availability of materials in the stockroom.
I.

Antimicrobial Susceptibility Test: Agar Disk Diffusion Method

A. Preparation of Mueller-Hinton Agar Plate

Mueller-Hinton agar is commonly used for routine susceptibility testing of organisms for
nonfastidious using agar disc diffusion method. It has been chosen for bacterial isolation because
it contains animal infusion, casamino acids, and starch, and it can also support the growth of
most organism. We add human blood to the formulation to be able to perform the susceptibility
testing on streptococci. Starch is an ingredient on this agar as to protect the organisms against
toxic substances and it also serves as an energy source.
For the preparation, mix 17g of agar in distilled water and heat to boiling. Autoclave. Pour
into plates with a depth of 4mm. Cool the plates in a jar with lid so that excess moisture will be
inhibited from forming into the surface of the agar, or simply, to avoid contamination. For the
Mueller-Hinton agar with human blood as an addition, cool the basal medium first to 45 to 50 C.
Add 5% of defibrinated human blood into the cooled basal medium. Gently mix it then pour
quickly into the plates.
B. Preparation of the bacteria suspended in broth
Mueller Hinton broth (MHB) medium is known to be routinely used for antimicrobial
susceptibility test. Suspension of the bacterial isolate S. pyogenes is prepared by the use of the
Mueller Hinton plate with pure isolates of the organism, 6 to 8 colonies were inoculated to the 10
ml of the MHB. To match the 0.5 McFarland's Standard, the bacterial isolate suspended will be
incubated for 2-3hrs with prior monitoring at 37 C.

C. Preparation of Disk and Lawn of Bacteria

Whatman filter paper #1 is used for making paper disks. A two hole office puncher with
6mm diameter is used for punching on filter paper producing 75 pieces with the same size paper
disks. These disks are placed in a vial to be autoclaved to ensure sterility for future use.
Five Mueller Hinton agar (MHA) plates are prepared for inoculation of the S. pyogenes isolate
with labeled names. To check for sterility, 2 plates are used and are incubated for 24 hours at 35
degree Celsius. Multiple overlap technique is performed for the inoculation of S. pyogenes to the
five plates from broth suspension having the same content as McFarland's standard which is
approximately 1.5x108 CFU/mL saturated from the broth using sterile swab. Sterile swabs are
used in every plate inoculated were discarded.
The plates are divided equally to five regions for the impregnation of the paper disks with
the distance of 24mm lathered on the plate and incubated to check for zone of inhibition.
D. Preparation of Different Concentrations of Kimchi Extracts
Different concentrations of both P. acidilactici and L. mesenteroides are used against the
pathogenic bacteria, S. pyogenes. The two types of lactic acid bacteria is having 100%, 75%, and
50% concentration each. To be able to yield the concentrations, specified amount of kimchi is
mixed to a proportional amount of phosphate buffered saline water. Treatment preparations are
based on the formula: C1V1 = C2V2 .
Table I. Concentrations of specified LAB with their corresponding amounts
Concentration

P. acidilactici and L.
mesenteroides from
Kimchi

Phosphate Buffered
Saline Solution

Amount of Sample in
a Test Tube

50%

3.75 ml

1.25 ml

5 ml

75%

2.5 ml

2.5 ml

5 ml

100%

5 ml

0 ml

5 ml

E. Preparation of Controls
Cefotaxime discs serves as the positive control which is obtained from the Institute of
Clinical Laboratory Sciences and then, it is placed in a bacterial lawn of S. pyogenes. As for
negative control, the 6 mm disc is immersed in a sterile distilled water and is put in a plate with
S. pyogenes.

F. Testing of Extracts
The paper disc method procedure is based from Heejae Lee, et al. (2010) in their study of
Functional properties of Lactobacillus strains isolated from kimchi but with slight alterations.
Sterile 6 mm discs are immersed in the 5 ml extracts of P. acidilactici and L. mesenteroides at
different concentrations and were incubated for 24 hours. The discs are dried at room
temperature and placed on the agar seeded with bacterial lawn of S. pyogenes. The plate are
incubated for 24 hours at 37 C. After incubation, the presence of a clear zone is measured in
terms of millimeters.
1. Determination of Minimum Inhibitory Concentration
Antimicrobial susceptibility involving dilutions are one of the methods used to determine the
minimum inhibitory concentration which is defined as the lowest concentration of the

antimicrobial agent to prevent the growth of pathogenic bacteria. It is determined with the use of
a two-fold serial dilution and an addition of varying concentrations of antimicrobial agents.
1. Preparation of set-up
For the preparation of the set-up, broth/tube dilution method is performed for the
determination of MIC. To rule out any possible contaminants, aseptic techniques are applied
throughout the whole duration of laboratory process. A total of 54 sterile 5 ml tubes are used in
the experiments since the study involves homemade and commercialized kimchi extraction of the
specified lactic acid bacteria. The entire set-up is presented in tables 2-4 and is discussed in
detail.
a. MIC of 100% concentration. Nine sterile 5 ml tubes are used and are labelled
from 1-9. The first tube contains 300 ul of MHB, while the succeeding tubes contains 200
ul of MHB. 100 ul of the test extract specifically P. acidilactici is dispensed from the first
tube while the succeeding 2-9 tubes are serially diluted by 200 ul up to the seventh tube.
The last two tubes are for positive and negative controls respectively. Do the same with
the setup for L. mesenteroides.The presentation of set-up is in Table 2.

Table 2. MIC set-up using two-fold dilution for 100%

Tube Test Extract Volume Volume
Initial
Label
mixture of MHB
Dilution
(ul)
(ul)
1
P. acidilactici
100
300
1/5
With PBS

Volume of
Final
S. pyogenes Dilution
per tube
100
3/20

Total
volume
(ul)
400

Tube 1

300

200

1/10

100

3/40

400

Tube 2

300

200

1/20

100

3/80

400

Tube 3

300

200

1/40

100

3/160

400

Tube 4

300

200

1/80

100

3/320

400

Tube 5

300

200

1/160

100

3/640

400

Tube 6

300

200

1/320

100

3/1280

400

PC

200

NC

100

200

100

400
400

B. MIC of 75% concentration. Nine sterile 5 ml tubes are used and are labelled from 1-9. The
first tube contains 400 ul of MHB, while the succeeding tubes contains 200 ul of MHB. 100 ul of
the test extract is dispensed from the first tube while the succeeding 2-9 tubes are serially diluted

by 200 ul up to the seventh tube. The last two tubes are for positive and negative controls
respectively. Do the same with the setup for L. mesenteroides. The presentation of set-up is in
Table 3.

Table 3. MIC set-up using two-fold dilution for 75%

Tube Test Extract Volume Volume
Initial
Label
mixture of MHB
Dilution
(ul)
(ul)
1
P. acidilactici
75
400
1/5
With PBS

Volume of
Final
S. pyogenes Dilution
per tube
100
9/100

Tube 1

300

200

1/10

100

9/200

400

Tube 2

300

200

1/20

100

9/400

400

Tube 3

300

200

1/40

100

9/800

400

Tube 4

300

200

1/80

100

9/1600

400

Tube 5

300

200

1/160

100

9/3200

400

Tube 6

300

200

1/320

100

9/6400

400

PC

200

NC

100

200

100

Total
volume
(ul)
500

400
400

C. MIC of 50% concentration. Nine sterile 5 ml tubes are used and are labelled from 1-9. The
first tube contains 400 ul of MHB, while the succeeding tubes contains 200 ul of MHB. 100 ul of
the test extract is dispensed from the first tube while the succeeding 2-9 tubes are serially diluted
by 200 ul up to the seventh tube. The last two tubes are for positive and negative controls

respectively. Do the same with the setup for L. mesenteroides.The presentation of set-up is in
Table 4.
Table 4. MIC set-up using two-fold dilution for 50%
Tube Test Extract Volume Volume
Initial
Label
mixture of MHB
Dilution
(ul)
(ul)
1
P. acidilactici
50
400
1/5
With PBS

Volume of
Final
S. pyogenes Dilution
per tube
100
3/50

Tube 1

300

200

1/10

100

3/100

400

Tube 2

300

200

1/20

100

3/200

400

Tube 3

300

200

1/40

100

3/400

400

Tube 4

300

200

1/80

100

3/800

400

Tube 5

300

200

1/160

100

3/1600

400

Tube 6

300

200

1/320

100

3/3200

400

PC

200

NC

100

200

100

Total
volume
(ul)
500

400
400

SAMPLE VOLUME
The formula for the computation of the DILUTION= TOTAL VOLUME is used where the
sample volume represents the concentration of the volume of test extract and the total volume
was that of the sample volume of the mixture (300) plus the volume of the diluent (200). In
accordance with the following statement, DILUTION = 100/500= (previous dilution)
The formula for the computation of the dilution of the succeeding tubes,

FINAL DILUTION= INITIAL DILUTION x PREVIOUS DILUTION where the initial

dilution is the dilution that is made upon adding 300 ul of sample from the first tube to the next
tube giving a total of 600 ul (300 +300). Thus, the initial dilution is . The previous dilution is
the dilution made from the first tube. So,
FINAL DILUTION= () ()= This is the dilution of the second tube. This formula is the basis
for computing the successive dilutions.
There are two formula used in computing the final dilution of each tube after the addition
of the test bacterium.
SAMPLE VOLUME
DILUTION= TOTAL VOLUME
DILUTION= 300/400= 3/4
FINAL DILUTION= INITIAL DILUTION x PREVIOUS DILUTION
FINAL DILUTION= (3/4) ()= 3/20

2. Preparation of Bacterial Suspension of S. pyogenes

For the determination of Minimum Inhibitory Concentration, a bacterial suspension of 10 5
CFU/ml is use. To obtain the recommended CFU/ml, three 10 ml tubes containing each with
MHB labelled from 1-3 are used in the dilution. A freshly-prepared suspension is prepared from
an agar culture of S. pyogenes isolate to be used as an inoculum and these inoculum consisting of
5-8 colonies in MHB will be match with that of a turbidity standard (0.5% McFarland's standard
bearing approximately 108 CFU/ml). 1 ml is pipetted and dispensed to the first tube which is
equal to 107 CFU/ml from the standard that is made. From the first MHB tube, 1 ml is pipetted
again and dispense to the second tube which is equal to 106 CFU/ml. 1 ml is pipetted again from

the second MHB tube and is dispensed to the third MHB tube which is equal to 105 CFU/ml, the
ideal size and bacterial suspension for testing.
3. Inoculation of S. pyogenes suspension
From the tube 3 bacterial suspension that is equal to 105 CFU/ml, 100 ul (0.1 ml) is
pipetted and dispensed to a series of test tubes from 1-7 with growth medium and different
concentrations of the antimicrobial agent. 100 ul of the bacterial suspension is pipetted and
dispensed to tube 8, the positive control, which is expected to have growth and 100 ul is also
pipetted and dispensed to tube 9, the negative control, which is expected to no growth. Each
tubes are covered with cotton plugs and are incubated for 24 hours at 35 C. Visual examination
of the tubes is performed for the presence of turbidity to determine the minimum inhibitory
concentration with an exception to tube 9 since it is the negative control containing no bacteria.
4. Minimum Bactericidal Concentration Determination
The tubes from MIC determination with clear tubes is pour plated by pipetting 100 ul (0.1
ml) to the Mueller-Hinton Agar plate to determine the remaining number of viable bacteria and
to assess if the test extract is significant for bactericidal activity. Plates are cooled and are
incubated for 24 hours. Visible colony that is formed are counted and recorded.
Data Analysis
A. Antimicrobial Susceptibility Test
The obtained values of the zones of inhibition is tabulated as shown in Table IV.
Table IV. Zone of Inhibition of the different concentrations of Test extracts on Agar Disk
Diffusion Method
Treatment
Test Extracts

Zone of Inhibition (mm)

Disk 1

Disk 2

Disk 3

Disk 4

Disk 5

Mean

100% mixture (T1)

75% mixture (T2)
50% mixture (T3)
Controls
Positive Control
Cefotaxime
Negative Control
Sterile Water

The researchers use the Analysis of Variance technique in testing the hypothesis of the
study, determining whether there is a relationship of the variables and even checked if there is a
significant difference in the antimicrobial activity of homemade and commercialized kimchi as
measured by zone of inhibition brought about by the different concentrations of 50%, 75% and
100% of the test extracts.
The first, second, third, and fourth null hypothesis are subjected to Analysis of Variance
(ANOVA). The researchers use this parametric tool because it analyzes whether there is
significant differences among the group or whether there is an equal means for a given set of
populations. It is determined by computing first the means of each of the given populations and
by computing as well its variance which the latter is technically called as between groups
variance (MSSb). The population variances are combined together into one measure known as
within-the groups variance (MSSw). A computation is made for the ratio of (MSSb) and (MSSw)
and if the computed value of these ratio is large which is known as the computed F value, then
at least a pair of means differ significantly. Computed F value is considered large if it is greater

than its corresponding tabular F-value given a level of significance and degrees of freedom. A
comparison is made between the F value and tabular F-value to know if the null hypothesis will
be accepted or rejected.
Figure 1. F- Distribution Table (Retrieved from http://sites.stat.psu.edu/)

B. Minimum Inhibitory Concentration

The MIC test in this study is defined as the lowest concentration of lactic acid bacteria
isolated from kimchi that prevents the growth of S. pyogenes. The MIC tubes are examined for
turbidity after it will be incubated for 24 hours. Growth is expected in the positive control which
is cefotaxime while no growth is observed in the negative control which is the sterile water.
Table 5. Results for MIC Determination

Tube Number

Concentration (%)
Test Result
C. Viable Colony Count
Pour plate technique is performed for counting the viable bacteria that will grow in the plates.
The total number of colonies that will grow in the plate is equal to the number of viable
microorganisms in the sample. These colonies are obtained by diluting the original sample
several times and a small volume of diluted sample will be mixed with the molten agar. The total
number of colony forming unit on the plate is determined by multiplying the average number of
colonies per plate with the dilution factor. A bactericidal action of the test extract is an
implication that there is an absence of colonies in the actual number of CFU/ml.