Final Paper 4th Revision EGML

CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1
Clitoria ternatea Linn. Flower Extract as an Alternative Growth

Indicator for Minimum Inhibitory Concentration
Determination of Escherichia coli
And Staphylococcus aureus
A Research Study
Presented to the
College of Medical Technology
Centro Escolar University
In Partial Fulfillment
of the Requirements of the Degree
Bachelor of Science in Medical Technology
By
IMEE JOY R. BATUGAL CARL ALEXIS B. PARONE

ANGELA KRISNE H. BOCALBOS JOANNA L. PULPULAAN
DEANNA GEM D. CALARAMO JODILEN QUEZON
CATHERINE JOY T. CARVAJAL MELANIE AURA C. ROMERO
LOUIEGIE S. ESGUERRA JOHN IVAN RONALD V. SANTIAGO
SARAH C. EUGENIO CHARLENE D. SIMANGAN
ROSEAN A. FLORANZA CAMILLE M. URBANO
GINIA I. GONZALES PATRICIA ANN M. VICTORIO
TRICIA MARIE S. MACASPAC JOANNA MARIE JOYCE VELASCO
FRANCHETTE MERCADO MARIO N. VIDUYA
ANA FRANCESCA L. OCAMPO CHRISTINE JOY C. ZABAT
MAY 2015
ACKNOWLEDGMENT
The researchers would like to thank the Dean of the College of
Medical Technology, Dr. Charito M. Bermido, for her continuous support
throughout the conduction of the study.
To Secret Garden of Doris and the Garden of Silang, Cavite for
providing the researchers the flower samples.
To Mr. Romeo Ramos and Mr. Jerferson Permejo for lending the
researchers equipment needed to facilitate this experiment.
A very special thanks to Mr. Gil Soriano for teaching the
researchers the basics in Research Writing.
The researchers would like to express their deepest gratitude to
their adviser, Ms. Erika Gayle Lipana, for guiding them using her expertise
in this field, for her patience, and understanding.
A very special thanks to the families of the researchers who have
been supporting them morally and financially throughout the research
study.
Lastly, we would like to thank our Lord for giving them strength and
knowledge for them to be able to finish this research study.
The Researchers
DEDICATION
Four years of retrospect, the researchers have been through rough
waters toiling with their dreams of becoming professional medical
technologists. Needless to say, they are sprouting yet growing mushrooms
in the world of medicine. Their voyage was never smooth but they are now
travelling on the path leading to the actualization of our vision. The
researchers pay gratitude to the people who have generously, in a way, or
another, helped reach this momentum.
To the researchers parents, to whom their lives are indebted to,
they sincerely dedicate this to you. These pieces of paper resemble the
researchers hard work and perseverance. Thus, they offer this to you as a
partial fulfillment of their success.
To the future medical technology students, the researchers know
that you are a bit anxious about the world ahead of you. At first, you will be
virtual unknowns in this field we are engaging. But sooner or later, you will
solely be part of it. You might be dreading of what lies ahead. But
nonetheless, you must keep your cool because the challenges ahead can
be fate-testing.
This paper is dedicated to you, an epitome of the researchers
burning desire and passion to do whatever it takes to reach their goals.
The Researchers
ABSTRACT
Nowadays, many plants are being studied and explored to discover their
capabilities to prevent, cure, and identify pathogenic organisms in order to avoid
or stop spread of disease. Clitoria ternatea (Linn.) flower is a plant considered to
have potential and is valued for its forage and medicinal importance. Its
component, anthocyanin, is likely to produce an organic dye such as the blue dye
in its extract. The extract was tested against the antimicrobial activity of
Staphylococcus aureus and Escherichia coli. The capability of the extract was
compared to commercially made dyes such as phenol red and bromthymol blue.
Clitoria ternatea Linn. flower has been air dried and undergone ethanolic
extraction. The CTE has been sterilized, concentrated and undergone pH
adjustment. Staphylococcus aureus and Escherichia coli were used as a control
organisms in the study. This research aims to prove that Clitoria ternatea (Linn.)
flower extract can be a potential indicator for Minimum Inhibitory Concentration
determination in exchange to other locally and expensive indicators which is
commonly used in the laboratory. Accomplishment of the study is beneficial not
only in the medical field but also to the agricultural and research industry by
means of providing more job opportunity to farmers and new perspective to
researchers to extensively study this plant. The results shows that CTE is an
effective alternative indicator for MIC compared to commercially made dyes.
Keywords: Indicator, Pathogenic, Minimum Inhibitory Concentration

CHAPTER 1
INTRODUCTION
Background of the Study
Bacteria are the most common cause of disease which can lead to
mortality of humans, plants, and animals. It can easily spread through
direct contact or even thru aerosol transmission. Its ability to cause a
disease is dependent on its pathogenecity and virulence. Once an
individual gets infected, treatment is based on the action of antibiotics to
kill certain bacteria caused the disease.
There are different ways on how to identify the viability of
antimicrobials to eradicate infection. One of the methods used is the broth
dilution wherein the Minimum Inhibitory Concentration (MIC) is being
determined. MIC is the lowest concentration of an antimicrobial agent
required to inhibit the growth of a particular bacterial isolate (Mahon et al.,
2007). Broth and agar media are used to prepare serial dilution of test
organisms. MIC is then recorded as the lowest dilution in which there is no
microscopic growth after an overnight incubation. In order to conduct MIC
testing, certain dyes are used as indicators. An example of dye used is
Resazurin.
Clitoria ternatea Linn. belongs to family Fabaceae which is a
herbaceous perennial legume valued for its forage and medicinal
importance and has been originated in the traditional Indian system of

medicine for its phytochemical properties. (Pahune et al., 2013).Clitoria
ternatea Linn.iscomposed of anthocyanin, which is a type of delphidin,
flavonoids, and tannin. These pigments are of great potential in producing
an organic dye such as the blue dye from its flower extract. Our goal is to
prove that this blue dye would be capable of detecting bacterial growth for
MIC testing. If proven, many would benefit not only the medical field but
also the economic and agricultural industry by means of producing more
jobs to farmers.
SETTING OF THE STUDY
The plants cultivated were acquired from Silang Junction, Tagaytay
City while the flowers which were not cultivated by the researchers were
acquired from Secret Garden of Doris in Antipolo City. The study was
conducted in Centro Escolar University in Metro Manila, Philippines.
Centro Escolar University (CEU) is a private, non-sectarian higher
education institution with an enrollment of over 20,000 students in its three
campuses: Manila, Makati and Malolos. Established in 1907, Centro
Escolar de Seoritas, from a small school in Azcaraga has grown to
become one of the top universities in the country today. The
experimentation was done at the Centennial Research Laboratory of the
university. The said laboratory is fully-airconditioned and equipped with

highly precise and accurate machines, thus providing reliable results that
will be the basis in proving this study.
CONCEPTUAL FRAMEWORK
INPUT
Collection of Samples
Clitoriaternatea Linn. ethanolic
extraction
PROCESS
Culturing of the control (ATCC 25922 :
Escherichia coli, ATCC 25923 :
Staphylococcus aureus)
Phytochemical Analysis
OUTPUT
Evaluation of CTE as an indicator for
MIC determination
Analysis of the results using ELISA
Reader
Figure 1
Research Paradigm
The researchers used the IPO system shown in Figure 1. The
figure shows the actual step by step process of the research methodology.
Acquisition of the plant and subjecting this for ethanolic extraction and
series of phytochemical analysis will be the primary step of the study. The
process includes the microdilution MIC testing using 3 different sets of
antimicrobial agents for Escherichia coli and Staphylococcus aureus. For
easy interpretation of the microbroth MIC testing, several dyes were
added and were compared with the CTE. Lastly, the interpretation of the
study was done using sets of statistical tools. Reading of results was
based upon the reaction of the control organisms to the antibioticswhich
will be indicated by several dyes on the micro titer plate (MTP).
STATEMENT OF THE PROBLEM
The study determined whether Clitoria ternatea Extract (CTE)
would be capable of determining the MIC of antibiotics compared with
other MIC indicators. Specifically, the research answered the following
questions:
1. What is the MIC (in ug/mL) of the antibiotics (ampicillin, cefoxitin,
and chloramphenicol) against Staphylococcus aureus using the
following MIC indicators:
a. Phenol red (PR)
b. Bromthymol blue (BB)

c. CTE
2. What is the MIC (in ug/mL) of the antibiotics (ampicillin/ sulbactam,
co-amoxiclav, and meropenem) against Escherichia coli using the
following MIC indicators:
a. Phenol red (PR)
b. Bromthymol blue (BB)
c. CTE
3. Is there a significant difference among the optical densities of the
indicators used for MIC testing for Staphylococcus aureus?
4. Is there a significant difference among the optical densities of the
indicators used for MIC testing for Escherichia coli?
HYPOTHESES
Based on the information gathered, the hypotheses are as follows:
Ho: There is no significant difference among the optical densities
of the indicators used for MIC testing for Staphylococcus
aureus.
Ha: There is a significant difference among the optical densities of
the indicators used for MIC testing for Staphylococcus aureus.
Ho: There is no significant difference among the optical densities
of the indicators used for MIC testing for Escherichia coli.

Ha: There is a significant difference among the optical densities of
the indicators used for MIC testing for Escherichia coli.
SIGNIFICANCE OF THE STUDY
Developing of an alternative and organic growth indicator out of
the extract of Clitoria ternatea Linn.flower will be a great contribution not
only to the field of Medical Technology but to our economy as well.
Medical Technology Students. This study will serve as an
inspiration for the students. It willalso as a future reference to supplement
future studies related with this topic.
Economy. This study will be of great help especially in the
hospitals that cannot afford high cost of chemicals or reagents because
this CTE can be used as pH indicator for MIC for it is cheaper and easier
to make. This could also help those clinical laboratories in rural areas in
which plants are their only sources.
Agriculture: This study will be beneficial to the agricultural sectors
because development of CTE as an alternative growth indicator for MIC
needed mass production of the plant. In result, farmers will gain extra jobs
with it and gained profit from this.
Faculty. The results from this study will provide information about
the potentials of CTE as an alternative growth indicator for MIC during
bacterial isolation.
Future researchers. Findings for this study will serve as the basis
in the future studies regarding MIC alternative dye. It will also serve as a
future reference for researchers on the subject of Clitoria ternatea Linn.
flower an alternative growth indicator.
SCOPE AND LIMITATIONS OF THE STUDY
This study focused on the capability of CTE as an alternative
growth indicator for Minimum Inhibitory Concentration. The researchers
only focused on blue Clitoria ternatea Linn. flower and dye was extracted
using ethanolic extraction procedure. The researchers only focused on the
determination of the Minimum Inhibitory Concentration (MIC) of ampicillin,
chloramphenicol and cefoxitin for ATCC 25923Staphylococcus aureus and
for the MIC determination ATCC 25922 Escherichia coli, amoxicillin/
clavulanic acid (co-amoxiclav), meropenem and ampicillin/ sulbactam
(sulbactam) were used. The researchers referred to the CLSI Standards
2013 in choosing the antibiotics to be used in the study. Also, the
researchers used 3 commercially-made dyes, namely, bromthymol blue,
phenol red, and methyl red, which will be tested against CTE as the
indicators for the determination of MIC. During the ELISA reading, the
researchers set the wavelength to 550 nm which is based on the product
insert indicated on each and every indicator bottles.

The organisms that were subjected for the study were only limited
to ATCC 25923 Staphylococcus aureus and ATCC 25922 Escherichia coli
since they are the most commercially prepared strains of bacteria. No
further examinations for the other components of Clitoria ternatea Linn.
flower. The researchers limited the study to only its ability to produce a
blue dye. The researchers have no control on the process of the
cultivation of the plant; flowers were acquired from a botanical garden.
Also, the researchers limited the study on the potential of the flower to be
an organic pH indicator rather than on its medicinal and therapeutic uses.
OPERATIONAL DEFINITION OF TERMS
Anthocyanin - Pigments from flowers and plants that produce blue to red
coloring depending on the pH.
Antibiotics - An agent that either kills or inhibits the growth of the control
organisms in this study
Antimicrobial - agent that destroys microorganisms
ATCC - These were the controlled Escherichia coli and Staphylococcus
aureus that were used by the researchers
Cellulose Filtration -Sterilization technique used by the researchers in
order to prevent any contamination on the extract
Culture Media - Media used to support the growth of the organisms.
CTE - A dye extracted from Clitoria ternatea Linn. flower

Delphinidins - An anthocyanidin that occurs widely in plants in the form of
glycosides (such as delphinin)
Flavonoids- Are large family of compounds synthesized by plants that
have a common chemical structure.
Indicator - Dyes incorporated on the wells that change in color whenever
there is presence of growth of the organism
MIC - The lowest concentration of an antibacterial or antimicrobial that will
inhibit the growth of organisms.
Multi drug resistant - Bacteria that is resistant to a multiple number of
drugs.
Optical Density (OD) this is synonymous to absorbance
Reconstitution - The process of adding the sterile distilled water to a
powederized antibiotic (suspension) in order to turn it into liquid form
Serial Dilution - The series of sequential dilution used to reduce the
concentration of the antibiotic stock solution in order to determine the MIC
Tannins- An astringent, bitter plant polyphenolic compound that binds to
and precipitates proteins and various other organic compounds.

CHAPTER 2
REVIEW OF RELATED LITERATURE AND STUDIES
This chapter includes various literatures and studies relevant to
support the claims of the subject being investigated. Both foreign and local
literatures as well as foreign studies were reviewed giving the researchers
a more in depth insight of the problem.
Clitoria ternatea Linn. flower as pH Indicator
In the study by Pahune et al. (2013), entitled Antimicrobial Activity
of Clitoria ternatea Linn. flower extract and use as a natural indicator in
acid-base titration, it is stated that Clitoria ternatea Linn. is an ornamental
plant and as vegetation species, requires little care when cultivated. Its
roots fix nitrogen and therefore, this plant is also used to improve soil
quality. This plant is very useful for it has several therapeutic activities like,
anti-stress, anxiolytic, anti-depressant, anti-convulsant, tranquilizing, and
sedative agent. In Southeast Asia, the flowers are used to color food. In
animal tests, the methanolic extract of Clitoria ternatea Linn. roots are
found to process anti-depressant, anti-convulsant, and anti-stress activity.
The prepared flower extract was screened for its use as an acid-base
indicator in various acid-base titrations and the result of the screening,
compared with the result obtained by standard indicators, methyl red,
phenolphthalein, and mixed indicator (methyl orange & bromcresol green)

for strong acid versus strong base, strong acid versus weak base, and
lastly, weak acid versus strong base titrations respectively. It is concluded
from the data that the antimicrobial activity is directly proportional to
concentration. As the concentration of the solution increases, there is also
an increase in the zone of inhibition. The present research work about the
Clitoria ternatea Linn. flower extract alone can secure the purpose of the
indicator in weak acid and weak base titration, where generally, mixed
indicators are applied. The results obtained in all types of acid base
titrations lead the researchers to conclude that it was due to the presence
of flavonoid and anthocyanins and that is why color changes occurred at
the end point of the titrations.
According to the study that was conducted by Kanchana et al.
(2013), various experiments were conducted for the maximum extraction
of natural dye from Clitoria ternatea Linn. The sample were collected and
washed thoroughly with the help of grinder and the powdered samples
were used for the extraction of dyes. For them to find out the optimum
extraction conditions, experiment in aqueous extraction at various range of
pH (2-8) were conducted, in which in the pH solution the colour of butterfly
pea extract displayed a red colour; but in alkaline pH, the colour changed
to greenish blue. The change in colour and extraction rate of butterfly pea
solution depends on the change in equilibrium of four anthocyanin species
in its petals according to the prevailing pH. At lower pH, red colour
signified the presence of anthocyanins and in increasing pH, the colour
intensity transformed to blue colour due to the presence of quinonoidal
base and the yellow colour was for the chalcone.
Susceptibility of the bacterial strain to the natural dyes was
investigated using the disc diffusion method. Escherichia coli and
Klebsiella (gram negative) and Staphylococcus aureus and Bacillus sp.
(gram positive) culture were used. The antimicrobial activities of some of
these dyes are reported as potent owing to the existence of phenol, tannin
and quinine in there extracts. Clitoria ternatea Linn.shows a diameter of
zone of inhibition of the following bacteria: Escherichia coli (8mm),
Staphylococcus aureus (9mm), Klebsiella spp. (7mm), and Bacillus
spp.(10mm).
The result from these experiments indicated that these natural dyes
had antimicrobial activity both on solutions and substrate.
Clitoria ternatea Linn. commonly known as Butterfly pea is used as
folk medicines that contains anthocyanins. The Anthocyanins are
responsible for the red violet-blue pigment in the plant flowers. The
stability of anthocyanins depend on its structure, pH, temperature, oxygen,
light and water activity. The color of anthocyanins tends to red in very
acidic solution and blue in basic solution. The application of blue
anthocyanin contained in the flower has not been optimal yet. The extract
of the butterfly pea plant was used as an indicator in acid-base titration
based on its anthocyanin features. (Saptarini, 2015)
Staphylococcus aureus
Staphylococcus aureus is facultative anaerobic gram-positive cocci
which occur singly, in pairs, and irregular clusters. Staphylococcus aureus
is non motile and non-spore forming. Staphylococcus aureus is broadly
distributed in nature and is carried by 25-33% of normal individuals in the
anterior nares and skin. It can inhabit and infect both healthy,
immunologically competent people in the community and
immunocompromised patients. Staphylococcus aureus is one of the
common Gram-positive hospital-acquired organisms. (Turnidge, 2008)
Staphylococci can tolerate the osmotic pressure created by 7.5%
NaCl, while this concentration will inhibit the growth of most other gram-
positive and gram-negative bacteria. Mannitol Salt Agar contains mannitol
and uses phenol red as a pH indicator (pK = 7.8) in the medium. At pH
levels below 6.9, the medium is a yellow color. In the neutral pH ranges
(6.9 to 8.4) the color is red; while above pH 8.4, the color of phenol red is
pink. When mannitol is fermented by a bacterium it will produce an acid
which lowers the pH and results in the formation of a yellow area
surrounding an isolated colony on MSA. A non-fermenting bacterium that

tolerates the high salt concentration would result with a red to pink area
because of peptone breakdown.
All through staphylococcal growth in glucose-supplemented
medium, the pH of a culture starting close to neutrality usually decreases
by about 2 units as a result of the fermentation of glucose. Various
species can tolerate the resulting mildly acidic conditions which is pH 5.5
by increasing a cellular response, which acts to preserve the intracellular
pH and, in principle, to alter gene expression for optimal performance in a
slightly acidic infection site. Changes in staphylococcal gene expression
formerly thought to represent a glucose effect are largely the result of
declining pH. (Weinrick, 2004)
MSA is a selective medium for Staphylococcus aureus containing
7.5% sodium chloride that can tolerate high salt concentration of the agar.
It is also a differential medium that contains sugar mannitol, a food source
that will produce acidic byproducts of fermentation that lowers the pH of
the media. At pH levels below 6.9, the medium is a yellow color. In the
neutral pH (6.9 to 8.4) the color is red; while above pH 8.4, the color of
phenol red is pink. When mannitol is fermented by a bacterium, acid is
produced, which lowers the pH and results in the formation of a yellow
area surrounding an isolated colony on MSA. A non-fermenting bacterium
that withstands the high salt concentration would display a red to pink area
due to peptone breakdown. Staphylococcus aureus is capable of

fermenting mannitol that will cause the pH indicator, phenol red to become
yellow. (Shields, 2013)
Microorganisms may cause chemical changes depending on the
nutrients required for the growth of the microorganism. Release of colored
dyes and microbial enzymes is upon hydrolysis. This results the
production formation of colonies which will allow the identification of
pathogens easily (Perr & Freydierre 2007). Selective and differential
media are used for the identification of bacterial pathogens.
Staphylococcus can be found in the skin and in the mucous membrane of
healthy adults. It can grow on a media containing 10% NaCl. Once
Staphylococcus is isolated, a few tests are performed to determine the
species, it includes mannitol fermentation which is a medium that
examines Staphylococcus species.
On MSA, only pathogenic Staphylococcus aureus produces small
colonies surrounded by yellow zones. The reason for this color change is
that Staphylococcus aureus have the ability to ferment the mannitol,
producing an acid, which, in turn, the acidity of the media will cause the
pH indicator, phenol red changes the indicator color from red to yellow.
The growth of other types of bacteria is usually inhibited.
According to Askim et al. (2011) in their study of Epidemiology and
outcome of Staphylococcus aureus bloodstream infection and sepsis in a
Norwegian country, Staphylococcus aureus is one of the most common

and lethal causes of bloodstream infection and the incidence is increasing.
It carries a high case fatality rate, especially among those with severe
sepsis and septic shock and among those with a pulmonary or unknown
focus of infection there was no decrease in 30- or 90-day mortality risk
during the study period. This underscores the importance of continuing
surveillance and efforts to improve the outcome of this serious disease.
Escherichia coli
Escherichia coli is the head of the large bacterial
family, Enterobacteriaceae, the enteric bacteria, which are facultatively
anaerobic Gram-negative rods that live in the intestinal tracts of animals in
health and disease. The Enterobacteriaceae are among the most
important bacteria medically. A number of genera within the family are
human intestinal pathogens (e.g. Salmonella, Shigella, Yersinia). Several
others are normal colonists of the human gastrointestinal tract
(e.g.Escherichia, Enterobacter, Klebsiella), but these bacteria, as well,
may occasionally be associated with diseases of humans.
In response to change in temperature and osmolarity, it can vary
the pore diameter of its outer membrane porins to accommodate larger
molecules (nutrients) or to exclude inhibitory substances. With its complex
mechanisms for regulation of metabolism the bacterium can survey the
chemical contents in its environment in advance of synthesizing any

enzymes that metabolize these compounds. It does not wastefully
produce enzymes for degradation of carbon sources unless they are
available, and it does not produce enzymes for synthesis of metabolites if
they are available as nutrients in the environment (Todar, 2014).
The optimal growth pH for Escherichia coli is near
neutral. Escherichia coli cells can grow reasonably well over a range of
three pH units (from pH 5.5 to 8.5). Extreme pH beyond this range will
significantly decrease the cell growth rate and may sometimes even cause
cell death. The minimum and maximum growth pH for Escherichia coli are
pH 4.4 and 9.0 respectively. Escherichia coli cells appear to tolerate a low
pH better than a high pH. In fact, extended exposure of Escherichia
coli cells to a high pH causes cell lysis. At the saturation or stationary
phase, the pH of the Escherichia coli culture in commonly used media is
near its pH limits. pH is another limiting factor for cell growth in addition to
nutrition exhaustion and accumulation of toxic metabolites. The medium's
pH is determined by medium compositions, buffers, cellular metabolites,
and aeration conditions. After exhaustion these organic buffers, the
medium reaches the Escherichia coli cells' maximum pH limit. Escherichia
coli cells can also use sugars such as glycerol and glucose as carbon or
energy sources. When the Escherichia coli cells use these sugars as
carbon sources, they will produce acetic acid and therefore lower the
medium pH. Carefully balancing the phosphate buffer, organic buffers,

sugar contents, and aeration conditions can maintain the culture medium
pH near Escherichia coli optimum growth pH or within the range of the
three pH units. Low aeration conditions lead the cells to produce acids.
High aeration conditions allow the cells to use organic acids as carbon
source and increase medium pH. Selected aeration conditions can also
help cells maintain its medium pH.
Properties of Anthocyanins
According to Hughes (2009) in her study, The Photoprotective
Role of Anthocyanin Pigments in Leaf Tissues, anthocyanins are vacuolar
pigments, most commonly responsible for red to purple coloration in plant
tissues. Because they absorb strongly in the blue-green wave band,
anthocyanins effectively reduce internal light within the leaf, which may be
beneficial under high-light stress conditions. However, pigment patterns in
the natural system of ten appear inconsistent with a photoprotective
function and their absence in many species exposed to high-light stress
suggest that anthocyanins may not be necessary for photoprotection.
In the study of Li (2008), entitled High Yield Anthocyanin
Biosynthesis in Metabolic Engineering of E. coli, the largest subgroup of
flavonoids Sand a major group of plant pigments impart the eye-catching
hues to most flowers and fruits ranging from pink through red and purple
to dark blue. They are widely distributed in the human diet through crops,
beans, fruits, vegetables, and red wine and have been found to be present
in significant amounts in plants-based daily diets. Anthocyanins have been
given much attention because of their health-promoting properties,
including anti-oxidative, anti-inflammatory, anti-carcinogenic, anti-obesity,
anti-diabetic, and cardioprotective.
Minimum Inhibitory Concentration
A microdilution technique using commercially available media and
materials was developed and used to determine the minimal inhibitory
concentrations (MICs) of Clindamycin, Chloramphenicol, Tetracycline,
Minocycline, Ampicillin, Carbenicillin, Cephalothin, and Gentamicin. The
microdilution method allowed rapid performance of dilution susceptibility
tests with easily defined end points. During testing, multiple microtiter
plates are filled with a broth composed of Escherichia coli and
Staphylococcus aureus and supplements. Varying concentrations of the
antibiotics and the bacteria to be tested are then added to the plate. The
plate is then placed into a non-CO2 incubator and heated at thirty-five
degrees Celsius for sixteen to twenty hours. Following the allotted time,
the plate is removed and checked for bacterial growth. If the broth became
cloudy or a layer of cells formed at the bottom, then bacterial growth has
occurred. The results of the broth microdilution method are reported in

Minimum Inhibitory Concentration (MIC), or the lowest concentration of
antibiotics that stopped bacterial expansion. (Carson et al., 2005)
According to Kavre et al. (2013) in their study of Correlation of
Minimum Inhibitory Concentration of Ciprofloxacin to the therapeutic
response of patient with urinary tract infection caused by Escherichia coli,
the choice of an antibiotic depends solely on the identification of the
species by determination of the sensitivity characteristics of the
microorganism. Along with the determination of sensitivity pattern,
understanding the susceptibility pattern of particular strain isolated from
patient is equally important. Variation in patient and microorganism is
known to be key factor for predicting the outcome for individual patient and
establishing targets for clinical susceptibility. The clinician purpose in
prescribing an antimicrobial drug is to produce at the site of infection a
concentration high enough to kill or inhibit the growth of microorganism
Dosage adjustment in relation to minimum inhibitory concentration
(MIC) of drug, taking into account underlying pathogen might affect the
therapeutic response and hence improve clinical outcome of patient.
Therefore, E coli positive urine cultures of patients who were prescribed
Ciprofloxacin were collected and their MIC was determined by agar well
diffusion method. The response of patient was obtained by direct interview
with them after 3 days of Ciprofloxacin therapy. There is a direct
correlation between MIC and therapeutic outcome of antibiotic therapy.

Bromthymol blue as pH indicator
According to Lisboa et al. (2014) in their study Endophytic fungi
producing esterases: evaluation in vitro of the enzymatic activity using pH
indicator. Endophytic fungi release esterase enzyme which is a
hydrolase enzyme that is special in organic solvents. Esterase catalyzes
the breaking and formation of ester ligands, where its action is usually
restricted to short-chain fatty acids soluble in water. The colorimetric
method is one of the simplest methods to perform for the detection of
esterase enzyme. The fungi were obtained from plants and isolated as to
use in their experiment. They use bromthymol blue as a indicator in the
experiment, the reduction of the pH increases the concentration of the
indicator bromthymol blue which changes the color of the reaction
medium from blue to yellow that can be observed and measure
spectrophotometry at 616nm.
Another study conducted by Jackson et al. (1969) entitled
Bromothymol Blue and Bromocresol Purple as Indicators of pH Changes
in Chromatophores of Rhodospirillum rubrum and stated that BB can
easily indicate the external pH of the medium which is due the metabolic
end products of microorganism. This explains that BB can easily detect pH
change due to metabolic by-products, making this more sensitive in
determining the MIC for ampicillin antibiotic.

Phenol red as pH indicator
Acidimetric tests are less expensive to perform but are less
sensitive than the Nitrocefin assay. Opening of the B-lactam ring produces
penicilloic acid, which is more acid than penicillin. The change in pH is
detected by visual observation of an indicator dye, phenol red. A
suspension of phenol red and penicillin G is adjusted with NAOH to pH 8.5
(the point at which the color changes to purple) and stored at -20C for up
to 1 week. At the time of testing, a capillary tube was dipped into the
phenol red solution until 1 cm of the tube was filled. The filled end of the
tube is scraped across a bacterial colony to plug the tube, then incubated
at room temperature for 60 minutes. A change in the phenol red indicator
to yellow indicates the presence of B-lactamase. In every case the
Nitrocefin test has been the most sensitive and the most specific method
for measuring B-lactamases. It must be used when testing Moraxella
catarrhalis. There is little reason to use one of the other methods when the
Nitrocefin test serves all purpose. (Washington et al., 2006)
There have been attempts over the years to develop methods that
could enhance the apparent growth of bacteria and thereby reduce the
conventional overnight incubation required to determine MIC.
Investigations have used colorimetric indicators and leuco dyes to amplify
the macroscopic observation of turbidity. In macrodilution assays, phenol
red was added to assay tubes containing glucose and yeast extract to
enhance growth. If the test organism grew utilizing the glucose, tunes
would turn yellow as a result of the pH change. If the antimicrobial agent
inhibited the organism, the indicator would remain red. Nonfermenter
organisms could be detected by an orange-red color if they grew in the
antibiotic-free tubes. Redox indicators such as resazurin,
triphenyltetrazolium chloride, and methylene blue have been used as
indicators of growth. Because these dyes may be antibacterial, the
strategy was to add them to the control tube after a brief (3-5 hour)
incubation period. If sufficient growth was then detectable, the indicator
would be added to all assays. Barlett and Mazens enhanced the sensitivity
of triphenyltetrazolium chloride by the addition of phenazinemethosulfate,
which accelerated the formation of the red formazan precipitate resulting
from growth of the bacteria. (Lorian, 2005)

CHAPTER 3
METHODOLOGY
This chapter describes the procedure used by the researchers in
conducting the research study. It was started from the collection of the
sample up to the determination of the capability of CTE as an alternative
growth indicator for MIC.
3.1. Research Design
The study conducted was an experimental type of research which
involves the determination of the MIC of several antibiotics against the
tested organism thru the application of BB, PR and CTE as growth
indicators.
The collection of samples, authentication of the plant, extraction,
and sterilization of crude extract were the primary steps in conducting this
research. It was then followed by the phytochemical analysis, which was
done by DOST, rejuvenation of ATCC organisms, and biochemical testing
of ATCC organisms to ensure the purity and speciation. The last and most
crucial part of experimentation was the MIC determination. Figure 3.1
shows the research diagram on how the experimentation was performed.

Collection of Sample
Authentication of Plant
Extraction of Crude Extract
Sterilization of Extract Using Cellulose

Filter
Concentration and pH
Neutralization of the Flower Rejuvenation of ATCC
Extract organisms
Biochemical Testing of ATCC

Phytochemical Analysis Organisms Using API System
MIC Testing
Data Analysis
Figure 3.1 Schematic Diagram of Methodology

3.2 Methods and Materials
3.2.1 Collection of Sample
The researchers acquired the plant from Silang Junction, Tagaytay
City and Silang, Cavite City. The plant was then cultivated and the flowers
were picked on a daily basis while the flowers which were not cultivated by
the researchers were acquired from Secret Garden of Doris in Antipolo
City.
3.2.2 Authentication of the Plant
The plant used in this study was brought to the national herbaria at
the Botanical Taxonomic Identification and Classification at the National
Museum of the Philippines for authentication. It is important that the whole
plant will undergo a credible analysis for the confirmation of its botanical
taxonomic identification and classification. Confirmation ofits scientific
name, common name and local name were the subjects for authentication
as well.
3.2.3 Crude Extraction of Clitoria ternatea Linn. flowers
The total weight of the collected and dried flowers was
44.205grams. Dried Clitoria ternatea Linn. flowers were grinded using an
osterizer. Ground flowers were soaked into a sterile jar with 95% ethyl
alcohol and were agitated from time to time. After 24 hours, soaked
flowers were filtered using a 125mm whatman filter paper. The filtrated
CTE was evaporated under reduced pressure by the use of rotary
evaporation (ROTAVAP), to remove low boiling organic chemicals, usually
solvents from the mixture of compound. This procedure was adapted from
Degradation of Blue Anthocyanin Extract of Clitoria ternatea Flower by
Lee Kong Hung in 2011.
3.2.4 Sterilization of Crude Extract Using Cellulose Filter
Using a cellulose filter (0.45 um x 47 mm), the flower extract
undergone sterilization. Sterile amber bottles and a funnel were used to
avoid contamination of the extract. A maximum of 1ml of flower extract
were placed into the cellulose filter every 30 minutes and the cellulose
filter was replaced every 2 hours to ensure the sterility of the sample
extract.
3.2.5 Concentration and pH neutralization of the flower extract
The researchers weighed two sterile evaporating dishes covered
with foil on a double beam balance and prepared two beakers filled with
50mL tap water for the water bath process. This process was used for the
concentration of the CTE. Using a sterile syringe, the CTE was transferred
from the amber bottle to the evaporating dish to quantify the volume of the
extract, adding initial amount of 10mL to 20mL of extract. The two

evaporating dishes were heated on top of each beaker set up on a heating
pan. The CTE thickened and turned into paste. After the water bath
process, the evaporating dish with foil containing CTE was weighed on a
double beam balance extract to know the change in volume.
1 mg of CTE concentrate was diluted in 1 ml of sterile distilled
water. After dilution, a pH meter was used to check the initial pH of the
CTE concentration which is always acidic, ranging from pH 5 to pH 6. The
researchers titrated 10% NaOH and 10% HCl to adjust the CTE
concentrate to pH 7.
After neutralizing the CTE concentrate, it was transferred to a
sterile amber bottle using a funnel with cellulose filter paper while
practicing aseptic technique. The CTE was poured little by little to the
amber bottle set up. This process sterilizes the neutralized CTE. After
filtration, the amber bottle containing the CTE concentrate, was covered
with a sterile foil and screw cap to avoid contamination and was placed in
the refrigerator.
3.2.6 Phytochemical Analysis
The CTE was tested at the Department of Science and Technology
at the Industrial Technology Development Institute Standards and
testing Division (ITDI-STD). The ethanol extract was about 35mL of blue,
liquid extract in an amber bottle with a screw cap marked as CTE. It was
received on April 24, 2015 and tested last April 27,2015 and the results for
Phytochemical test for plant was: Sterol (-), Tripenens (++), Alkaloids (+),
Saponins (+), and Glycosides (++).
3.2.7 Preparation of Media
The researchers used specific culture media for the controlled
organisms. Mannitol Salt Agar (MSA), Nutrient Agar (NA), and Triple
Sugar Broth (TSB) were used for ATCC 25923 : Staphylococcus aureus
while MacConkey Agar (MAC), Nutrient Agar (NA), and Blood Agar Plate
(BAP) were used for ATCC 25922 : Escherichia coli.
3.2.8. Rejuvenation of Controlled Organisms
The researchers inoculated the controlled organisms on MSA, NA,
and TSB for ATCC 25923 : Staphylococcus aureus and MacConkey, BAP,
and NA for ATCC 25922 : Escherichia coli. This ensures the purity of the
isolate as well as rejuvenation of bacterial cells prior to MIC determination.
3.2.9. Confirmatory Testing Using API System
For ATCC 25922 Escherichia coli the speciation of the organism
was confirmedusing API-20-E system and for ATCC 25923 :
Staphylococcus aureus the speciation of the organism was confirmed

using Mannitol Salt Agar fermentation test, Coagulase test, Catalase test
and Oxidase test.
1.2.10. McFarland Standard
The researchers used the McFarland standard prior to MIC testing
to adjust the turbidity of bacterial suspensions so that the number of
bacteria was within a given range to standardize microbial testing.
In doing the McFarland standard, the researchers prepared
bacterial suspensions by using the control organisms in the study namely
ATCC 25923 : Staphylococcus aureus and ATCC 25922 : Escherichia
coli. These organisms were suspended on 3 ml Mueller Hinton Broth
(MHB) and incubated at 37 degrees Celsius for 20 to 30 minutes. After
incubation, the researchers made sure that the tubes were clean from any
interference. After which, the researchers set the wavelength of the
UV/VIS spectrophotometer at 640 nanometer, set the UV/VIS
spectrophotometer with the blank and McFarland standard, and lastly
,placed the bacterial suspension in the UV/Vis spectrophotometer for the
reading of the tubed-organisms. The tubed organisms were adjusted to
the McFarland standard which is 0.5 which is equivalent to 5 X 108
CFU/mL.
3.2.11 Preparation of Indicators
For the preparation of bromthymol blue, 0.1g of bromthymol blue
salt was dissolved in 100 ml of 20% ethanol. For methyl red, 0.1g of
methyl red salt was dissolved in 30 ml of ethanol and dilute to 100 ml of
distilled water. Lastly, for the preparation of phenol red, 0.1g of phenol red
salt was diluted in 100 ml of 20% ethanol.
3.2.12 Reconstitution of Antibiotics
The powdered-form Ampicillin, Cefoxitin, Chloramphenicol,
Ampicillin-Sulbactam, and Meropenem, which all weighed 1 g, were
reconstituted in 10 ml sterile distilled water to make a concentration of
100,000 ug/ml. While Co-amoxiclav, which weighed 1.2 g, was
reconstituted in 20 ml sterile distilled water to make a concentration of 100
ug/ml.
3.2.13 MIC Testing
A microtube dilution of MIC determination was used in this study.
The MIC determination had 3 trials of experimentation for each antibiotic.
The growth indicators used were bromthymol blue, phenol red, and the
CTE. The three antibiotics for susceptibility testing were Chloramphenicol,
Cefoxitin, and Ampicillin for ATCC 25923: Staphylococcus aureus while

Ampicillin-Sulbactam, Co-Amoxiclav (Augmentin) and Meropenem for
ATCC 25922 : Escherichia coli, as seen in Figure 1 of Appendix C.
All 96 wells contained 100 ul MHB. 500 ug/ml of the stock solutions
of Ampicillin, Cefoxitin, and Co-amoxiclav were dispensed to well 1 of their
respective columns while 256 ug/ml of stock solutions were dispensed to
well 2 of their respective columns, serially diluting them from well 2 to well
10, disposing 100 ul from the last well. On the other hand, 256 ug/ml stock
solutions of Meropenem and Ampicillin-Sulbactam were dispensed to well
1 of their respective columns, serially diluting them from well 1 to well 10,
disposing 100 ul from the last well. Lastly, 1000 ug/ml stock solution of
Chloramphenicol were dispensed to well 1 of its assigned columns, 500
ug/ml of stock solution were dispensed to well 2, and 256 ug/ml to well 3,
serially diluting them from well 3 to well 10, disposing 100 ul from the last
well.
Next, 100 ul of bacterial suspensions were dispensed to all wells
except for wells 12 because they served as negative control. After, all
MTPs were incubated at 37 degrees Celsius for 24 hours.
The indicators were then dispensed to the wells. 30 ul of
Bromthymol blue was dispensed to rows B and F, 30 ul of Phenol red to
rows C and G, and 10 ul of CTE to rows D and H.

3.3 Reading of results
Reading of the results was based upon the reaction of the control
organism to the antibiotics and reaction with dyes on each well which was
validated using an ELISA reader and was set at a wavelength of 550 nm.
3.4 Statistical Treatment
1. Mean - It was used to determine the overall index of the
absorption in all trials done for MIC testing.
2. Standard Deviation Used to determine the homogeneity and
heterogeneity of the absorption for MIC testing.
3. One-way ANOVA - It is used to know if there is a significant
difference among the indicators used for MIC testing of different
antibiotics.
CHAPTER 4
RESULTS AND DISCUSSION
This chapter includes the results and discussions followed by
analysis of data that have been gathered by the researchers from the
experimental work done. Data were presented in tabular form along with
their corresponding interpretations, thus, giving the comparison of each
indicator in terms of their sensitivity in determining MIC of the antibiotics
used for ATCC 25923 Staphylococcus aureus and ATCC 25922
Escherichia coli.
Table 4-1
MIC Determination of Ampicillin, Cefoxitin and Chloramphenicol
against Staphylococcus aureus using PR, BB, and CTE
Antibiotics Phenol red Bromthymol blue CTE
Ampicillin 64ug/ml 64ug/ml 64ug/ml
Cefoxitin 128ug/ml No MIC 64ug/ml
Chloramphenicol 256ug/ml 128ug/ml 128ug/ml
Table 4-1 shows that for the MIC determination of ampicillin, all the
three indicators were able to indicate the minimum inhibitory concentration
of the antimicrobial agent at 64 ug/mL concentration. For MIC
determination of cefoxitin, PR determined the MIC at 128ug/mL
concentration and followed by CTE at 64ug/mL concentration. However,
for BB, there was no MIC determined by the indicator. For

chloramphenicol, PR was able to determine the MIC at 256 ug/mL
concentration of the antibiotic while both BB and CTE determined the MIC
128 ug/mL.
Based from the gathered MIC results, PR showed the most
sensitivity in determining the MIC for cefoxitin at 128 ug/mL concentration
and chloramphenicol at 256 ug/mL concentration. While for the MIC
determination of ampicillin, all MIC indicators are at par with each other
indicating the MIC at 64 ug/mL
Table 4-2
MIC Determination of Ampicillin/Sulbactam, Co-amoxiclav and
Meropenem against Escherichia coli using PR, BB, and CTE
Antibiotics Phenol red Bromthymol blue CTE
Ampicillin-
32ug/ml 16ug/ml 32ug/ml
Sulbactam
Meropenem 32ug/ml 64ug/ml 64ug/ml
Co-amoxiclav 32ug/ml 128ug/ml 32ug/ml
Table 4-2 shows the comparison between the MIC of the three
antibiotics, where PR was able to determine the MIC of ampicillin/
sulbactam, meropemen and co-amoxiclav at 32ug/mL concentration. On
the other hand, BB was able to determine MIC at 16ug/ml of
ampicillin/sulbactam, 64 ug/ml of meropenem, and 128ug/mL of co-
amoxiclav. Lastly, CTE was able to determine MIC at 32ug/ml of

ampicillin/ sulbactam and co-amoxiclav and at 64ug/ml of meropenem
concentration.
Based from the following results, the table showed that for
ampicillin/sulbactam MIC determination, CTE and PR indicated the MIC at
a higher concentration of antimicrobial agent which is at 32 ug/mL. For
meropenem MIC determination, both CTE and BB indicated that the least
concentration of meropenem to inhibit bacterial growth is at 64 ug/mL. And
for co-amoxiclav, only BB showed to be the most sensitive in indicating
the MIC at 128 ug/mL.
Table 4-3
Optical Density of the BB, PR, and CTE for the MIC Determination of
Ampicillin against Staphylococcus aureus
Indicators Trial 1 Trial 2 Trial 3 Overall mean
Mean (SD) Mean (SD) Mean (SD) (SD)
Phenol red 3.752 (2.202) 7.276 (3.515 8.660 (2.823) 6.563 (2.531)
Bromthymol 1.193 (0.148) 1.287 (0.116) 1.254 (0.079) 1.245 (0.048)
Blue
CTE 1.667 (0.253) 1.545 (0.361) 1.815 (0.240) 1.676 (0.135)
Table4-3 shows that BB is the most sensitive indicator for the MIC
determination of ampicillin for Staphylococcus aureus with the lowest
overall mean of 1.254 followed by CTE and PR.
In relation to this findings, Jackson et al. (1969) conducted a study
entitled Bromothymol Blue and Bromocresol Purple as Indicators of pH
Changes in Chromatophores of Rhodospirillum rubrum and stated that
BB can easily indicate the external pH of the medium which is due the
metabolic end products of microorganism. This explains that BB can easily
detect pH change due to metabolic by-products, making this more
sensitive in determining the MIC for ampicillin antibiotic.
Table 4-4
Cefoxitin against Staphylococcus aureus
Phenol red 5.917 (3.514) 3.906 (2.143) 3.828 (2.174) 4.550 (1.184)
Bromthymol 1.070 (0.087) 1.086 (0.116) 1.071 (0.113) 1.076 (0.009)
Blue
CTE 1.375 (0.092) 1.316 (0.098) 1.721 (0.336) 1.471 (0.219)
Table4-4 shows that BB has the lowest overall mean of 1.076
which means that it can indicate bacterial growth of Staphylococcus
aureus at the lowest concentration among the three indicators tested.
Table 4-5
Chloramphenicol against Staphylococcus aureus
Phenol red 8.006 (3.209) 8.672 (2.799) 9.316 (2.159) 8.339 (0.655)
Bromthymol 1.239 (0.097) 2.123 (0.251) 1.606 (0.144) 1.681 (0.444)
Blue
CTE 1.669 (0.152) 2.951 (0.142) 2.716 (0.220) 2.31 (0.683)
Table 4-5 presents that PR is the least sensitive indicator for
the MIC determination of chloramphenicol for Staphylococcus aureus

because it has the highest overall mean while BB is the most sensitive
indicator.
Table 4-6
Co-amoxiclav against Escherichia coli
Phenol red 9.3063 (2.191) 9.3029 (2.201) 9.999 (0.000) 9.536 (0.401)
Bromthymol Blue 1.680 (0.439) 1.691 (0.401) 1.713 (0.404) 1.695 (0.017)
CTE 2.411 (0.400) 2.339 (0.361) 2.491 (0.380) 2.414 (0.076)
Table 4-6 represents the data which proves that for the MIC
determination of co-amoxiclav for Escherichia coli, BB is the most
sensitive indicator, having the lowest overall mean.
Table 4-7
Meropenem against Escherichia coli
Phenol red 5.731 (3.685) 7.295 (3.492) 5.943 (3.491) 6.323 (0.848)
Bromthymol 1.095 (0.110) 1.078 (0.090) 1.169 (0.105) 1.114 (0.048)
Blue
CTE 2.163 (0.413) 1.899 (0.110) 2.136 (0.207) 2.068 (0.147)
Shown in Table 4-7 is the MIC determination of meropenem for
Escherichia coli, it is best to use BB as indicator for it obtained the lowest
overall mean, meaning that it is the most sensitive among the three
indicators tested.
Table 4-8
Ampicillin-Sulbactam against Escherichia coli
Indicators Trial 1 Trial 2 Trial 3 Overall Mean
Phenol red 7.976 (3.257) 7.954 (3.294) 7.666 (3.842) 7.865 (0.173)
Bromthymol Blue 1.543 (0.544) 2.609 (0.635) 1.506 (0.569) 1.886 (0.626)
CTE 1.725 (0.365) 2.798 (0.187) 2.757 (0.187) 2.427 (0.608)
Table 4-8 shows that among the three indicators tested, BB is the
most sensitive with an overall mean of 1.886, followed by CTE, with an
overall mean of 2.427, and then PR, with an overall mean of 7.865,
because it has the lowest overall mean.
Table 4-9
Significant Differences of the OD of BB, PR, and CTE for Ampicillin
MIC determination
Indicator Mean S.D. F- p- Remarks/ Post p-
value value Hoc value
Phenol red 6.5671 1.6268 97.12 0.000 Phenol red vs *0.000
5 Brothymol blue
Bromthymol 1.2446 0.0604 Brothymol blue vs 0.575
Blue CTE
CTE 1.6743 0.2241 CTE vs Phenol *0.000
red
Total 3.1620 2.6205
*The mean difference is significant at 0.05 level of significance
A one-way ANOVA was used in order to establish whether there is
a significant difference between the indicators in terms of their sensitivity
in determining the MIC in specific antibiotic.
Table 4-9 shows the comparison of absorption of ampicillin with
each indicator. F value was 97.125 with p value of 0.000. Specifically,
multiple comparison tests were done to determine the difference between

groups. PR and BB show significant difference with p value of 0.000 at
0.05 level of significance. This means that BB is a much better indicator
for MIC testing compared with PR with a mean of 1.2446 than 6.5671.
Same findings were found in comparison with CTE and PR which shows
significant difference, which means that in the MIC determination of
ampicillin for Staphylococcus aureus. However, CTE and BB shows no
significant difference with p value of 0.575
Table 4-10
Significant Differences of the OD of BB, PR, and CTE for Cefoxitin
MIC Determination
Indicators Mean S.D. F-value p-value Remarks/ Post Hoc p-value
4.5504 1.6274 Phenol red vs *0.000

Phenol red
Brothymol blue
Blue 40.487 0.000 CTE
CTE 1.4707 0.1562 CTE vs Phenol red *0.000
Total 2.3653 1.8246
*The mean difference is significant at 0.05 level
Table 4-10 represents the data which shows that PR and BB, as
well as PR and CTE had significant difference having p-value of 0.000
with PR being less sensitive indicator with a mean of 4.5504. BB and CTE
on the other hand showed no significant difference with p-value of 0.623
which means that both indicators can be used interchangeably as
indicators for the MIC determination of cefoxitin for Staphylococcus
aureus.
Table 4-11
Significant Differences of the OD of BB, PR, and CTE for
Chloramphenicol MIC Determination
5.8967 1.1489 Phenol red vs *0.000

Phenol red
Brothymol blue
Bromthymol 1.6567 0.100 Brothymol blue vs *0.036
Blue 113.285 0.000 CTE
2.4446 0.131 CTE vs Phenol red *0.000
CTE
Total 3.3327 1.9813

Table 4-11 evinced that the three indicators used for the MIC
testing of chloramphenicol for Staphylococcus aureus had a significant
difference having an F-value of 113.285 and p-value of 0.000. Multiple
comparison test showed the same results with PR and BB, CTE and PR
having p-value of 0.000 and BB and CTE with p-value of 0.036
Table 4-12
Significant Differences of the OD of BB, PR, and CTE for Co-
Amoxiclav MIC Determination
9.5361 0.9760 Phenol red vs *0.000

Phenol red
Brothymol blue
Bromthymol 1.6947 0.3876 Brothymol blue vs *0.047
458.08
blue 0.000 CTE
4
Total 4.5481
Shown in table 4-12 is the comparison of absorption of co-
amoxiclav with each indicator. A significant difference between the three
indicators in the one way ANOVA was found with an F-value of 458.084
and p-value of 0.000. Specifically, significant difference was found among
all the indicators used with a p value of 0.000 for both PR and BB and CTE
and PR. In addition, significant difference was also found for BB and CTE
with a p-value of 0.047. The result means that when it comes to the MIC
determination of co-amoxiclav, BB, having a mean of 1.6947, is the most
suited indicator for the determination of co-amoxiclav followed by CTE and
PR.
Table 4-13
Significant Differences of the OD of BB, PR, and CTE for Meropenem
MIC Determination
6.3228 2.7135 Phenol red vs *0.000

Phenol red
Brothymol blue
Bromthymo 1.1139 0.029 Brothymol blue vs 0.381
l Blue 30.812 0.000 CTE
2.0669 0.044 CTE vs Phenol red *0.000
CTE
Total 3.1679 2.7622

Table 4-13 shows the comparison of absorption of meropenem with
each indicator. Its F value was 30.812 with p value of 0.000 which means
there was a significant difference between the indicators use in the
multiple comparison tests done to determine the difference between
groups. PR and BB showed significant difference with p value of 0.000 at
0.05 level of significance with BB being the better indicator. Same findings
were found in comparison with CTE and PR which showed significant
difference. However, CTE and BB showed no significant difference with p-
value of 0.381.
Table 4-14
Significant Differences of the OD of BB, PR, and CTE for Ampicillin/
Sulbactam MIC Determination
Brand Mean S.D. F-value p-value Remarks/ Post Hoc p-value
5.3679 2.1874 Phenol red vs *0.000

Phenol red
Brothymol blue
Blue 20.2622 0.000 CTE
Total 3.2268 3.2268

Table 4-14 represents the data which shows that PR, BB and CTE
have significant difference in MIC determination of ampicillin/sulbactam
with F-value of 20.2622and p-value of 0.000. Multiple comparison was
performed for each group of indicators. PR, BB and CTE showed a
significant difference with p-value of 0.000. On the other hand, BB and
CTE also showed a significant difference with a p-value of 0.633.

Despite the significant differences between CTE and BB in MIC
determination for ampicillin and cefoxitin for Staphylococcus aureus and
meropenem and ampicillin/ sulbactam for Escherichia coli, no literatures
were found to support the findings due a dearth of studies both locally
and internationally.
CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS
This chapter includes the summary, findings, and conclusion from
the experiment performed by the researchers. Also, this chapter includes
the recommendation/s for future researchers to improve their scientific
investigation regarding Clitoria ternatea Linn. flower extract as an
alternative growth indicator for MIC determination.
SUMMARY OF FINDINGS
Based on the gathered data and upon subjecting it for statistical
analysis, the concentration of antibiotics for Staphylococcus aureus and
Escherichia coli was determined. PR showed the highest capacity to
indicate MIC in all the trials conducted in all the antibiotics followed by
CTE and BB in all the antibiotics used.
Significant difference was found in all antibiotic concentration when
compared to PR and CTE as well as PR and BB. In addition, significant
difference was also found in MIC concentration in chloramphenicol and
co-amoxiclav between CTE and BB. However, no significant difference
was found in MIC determination for ampicillin, cefoxitin, sulbactam and
meropenem using BB and CTE as indicator.
The lowest concentration of antibiotic for Staphylococcus aureus in
ampicillin were 32 ug/mL in all indicators while cefoxitin has 32 ug/mL

using CTE. Chloramphenicol showed a MIC of 64 ug/mL using BB and
CTE.
The lowest concentration of antibiotic for Escherichia coli in
ampicillin-sulbactam was 8 ug/mL using BB while meropenem got 16
ug/mL using PR. Co-amoxiclav got 16 ug/mL for both PR and CTE.
CONCLUSION
Based on the gathered data, the researchers were able to conclude
the following:
The lowest concentration of antibiotic (MIC) for Staphylococcus
aureus that were indicated by PR are 64 ug/ml (ampicillin), 128 ug/ml
(cefoxitin), and 256 ug/ml (chloramphenicol). For BB, 64 ug/ml (ampicillin),
no MIC determined for cefoxitin, and 128 ug/ml (chloramphenicol). Lastly,
for CTE, 64 ug/ml (ampicillin), 64 ug/ml (cefoxitin), and 128 ug/ml
(chloramphenicol).
The lowest concentration of antibiotic (MIC) for Escherichia coli that
were indicated by PR are 32 ug/ml (ampicillin/sulbactam), 32 ug/ml
(meropenem), and 32 ug/ml (co-amoxiclav). For BB, 16 ug/ml
(ampicillin/sulbactam), 64 ug/ml (meropenem), and 128 ug/ml (co-
amoxiclav). Lastly, for CTE, 32 ug/ml (ampicillin/sulbactam), 64 ug/ml
(meropenem), and 32 ug/ml (co-amoxiclav).

PR and BB showed significant difference, same findings were
found in comparison with CTE and PR. However, CTE and BB showed no
significant difference in both Staphylococcus aureus and Escherichia coli.
RECOMMENDATIONS
For future researchers who plan on conducting a related study
regarding Clitoria ternatea Linn. flower as an alternative bacterial indicator,
the researchers recommend the following:
1. To test clinical samples instead of controlled organisms.
2. To try other means of CTE extraction like aqueous extraction.
3. Instead of the concentration procedure conducted by the
researchers, lyophilization can be performed to CTE.
4. Perform viability test on the crude CTE to check for its stability.
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Lisboa, H., Biasetto, C., de Medeiros, J., Araujo, A. Silva, D., Teles, H.,
Trevisan, H.,
(January 2014). Endophytic fungi producing of esterases: Evaluation
in
vitro of the enzymatic activity using pH indicator. Brazilian Journal of
Microbiology.
Lorian, V. (2005). Antibiotics in Laboratory Medicine: 5 th edition.
Mahon, C., Lehman, D., Manuselis, G. (2007). Textbook of Diagnostic
Microbiology: Third Edition. Missouri: SAUNDERS.

Pahune, B., Niranjane, K., Kishor, D., Bodhe, M., Rokade, V. (2013).
Antimicrobial
Activity of Clitoriaternatea L. flower extract and use as a natural
indicator
in acid base titration. J. Nat. Prod. Plant Resour., 2013, 3 (2):48-51
Saptarini, N., Suryasaputra, D., Nurmalia, H. (2015). Application of
Butterfly Pea (ClitoriaternateaLinn.) extract as an indicator of acid-
base titration. Journal of Chemical and Pharmaceutical Research.
7(2): pp. 275-280.
Shields, P., Tsang, A. (July 2013). Mannitol Salt Agar Plates
Protocol.American Society for Microbiology Microbe Library.
Todar, K. (2014, April 3). E. coli.Todars Online Textbook of Bacteriology.
Retrieved March 21, 2015, from www.textbookofbacteriology.net/e.coli.
html.
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B. (2008).
Retrieved May 18, 2015, from www.antimicrobe.org/sample_
Staphylococcus.asp
Weinrick, B., Dunman, P., McAleese, F., Projan, S., Fang, Y., Novick, R.
(2004). Journal of Bacteriology v. 186(24): American Society for
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Winn Jr, W. (2006).Konemans Color Atlas and Textbook of Diagnostic
Microbiology: 6th Edition.

APPENDICES
APPENDIX A
GANTT: Chart for Research
Research
Objectives
Nov Dec Jan Feb Mar April May
1. Writing of
Chapter 1
2. Writing of
Chapter 3
3. Writing of
Chapter 2
4. Pre-experiment
5. Research
Proposal Defense
6.Experimentation
7. Writing of
Chapters 4 and 5
8. Research
Defense
APPENDIX B
Table of Expenses
Quantity Amount
1. Gloves 7 boxes Php 970.00
2. Mask 2 boxes Php140.00
3. Pipette tips:
a. Yellow tips 2 boxes Php 800.00
b. Blue tips 1 boxes Php 400.00
4. Media:
a.Mueller Hinton Agar 1 bottle Php 2970.00
b. Mueller Hinton Broth 1 bottle Php 3564.00
c. Mannitol Salt Agar 1 bottle Php 2772.00
5. Reagents:
a. Methyl red 2 bottles Php 900.00
b. Bromothymol blue 2 bottles Php 3980.00
c. Phenol red 1 bottle Php 2500.00
6. Clitoriaternatea Linn. 1920 pcs. Php 9510.00
flowers
7. Red top 100 pcs. Php 450.00
8. Cellulose Filter Paper - Php 3033.00
9. Locker - Php 220.00
10. Printing & Computer - Php 669.50
services
11. Centennial Lab Php 4824.00
12. Syringe 12 pcs Php 80.00
13. Foil 9 tubes Php 796.75
14. Yellow bag 1 bag Php 58.00
15. Scissors 1 pair Php 15.00
16. Container - Php 40.00
17. Detergent & Lysol -- Php 288.75
18. Test Tube brush 2 pcs Php 10.00
19. Rugs - Php 97.00
20. Gauze - Php 20.00
21. Cotton balls 1 pack Php 70.00
22. Amber bottle 8 bottles Php 120.00
23. Folder - Php 40.00
24. Petri dish 1 pc Php 150.00
25. Masking tape 5 pcs Php 66.00
26. Sprayer - Php 52.50
27. Ziplock 1 box Php 140.00
28. Whatman Filter Paper 1 box Php 1000.00
29. Hand soap 2 sachets Php 16.25
30. Spatula 1 pc Php 55.00
31. Antibiotics
a. Meropenem (1g) 1 vial Php 550.00
b. Augmentin (1.2g) 1 vial Php 578.25
c. Cefoxitin (1g) 1 vial Php 921.00
d. Chloramphenicol (1g) 1 vial Php 45.00
e. Ampicillin (1g) 2 vials Php 70.00
f. Ampicillin+Sulbactam 2 vials Php 100.00
(750 mg)
32. Paper Towel 1 pack Php 71.75
33. Sterile Gloves 12 packs Php 180.00
34. Notebook 1 pc Php 41.75
35. Syringe 3 pcs Php 24.00
36. Safeguard (hand soap) 1 pack Php15.00
TOTAL Php 43239.50

APPENDIX C
Documentation of Tables, Plates, and Figues
Figure 1 Serial Dilution of Antibiotics
Well 11 (positive control) : 100ul MHB + 100ul bacterial suspension

Well 12 (negative control): 200ul MHB.
Co-
Ampicillin Cefoxitin Chloramphenicol Ampicillin- Meropenem
Amoxiclav
Sulbactam
(Augmentin)
Well 1
500 500 1000 256 256 500
(ug/ml)
Well 2
256 256 256 128 128 256
(ug/ml)
Well 3
128 128 128 64 64 128
(ug/ml)
Well 4
64 64 64 32 32 64
(ug/ml)
Well 5
32 32 32 16 16 32
(ug/ml)
Well 6
16 16 16 8 8 16
(ug/ml)
Well 7
8 8 8 4 4 8
(ug/ml)
Well 8
4 4 4 2 2 4
(ug/ml)
Well 9
2 2 2 1 1 2
(ug/ml)
Well
10 1 1 1 0.5 0.5 1
(ug/ml)
Table 2 Antibiotic concentration of each well

ATCC 25923 :Staphylococcus aureus
Plate 1 Trial 1 (Ampicillin) Bromthymol blue
Plate 4 Trial 1 (Cefoxitin) Bromthymol blue
Plate 7 Trial 1 (Chloramphenicol) Bromthymol blue
Plate 10 Trial 1 (Ampicillin) Phenol red

Plate 13 Trial 1 (Cefoxitin) Phenol red
Plate 16 Trial 1 (Chloramphenicol) Phenol red
Plate 19 Trial 1 (Ampicillin) - CTE
Plate 20 Trial 2 (Ampicillin) CTE
Plate 21 Trial 3 (Ampicillin) - CTE

Plate 22 Trial 1 (Cefoxitin) - CTE
Plate 25 Trial 1 (Chloramphenicol) - CTE
ATCC 25922 :Escherichia coli
Plate 28 Trial 1 (Co-Amoxiclav) Bromthymol blue
Plate 31 Trial 1 (Meropenem) Bromthymol blue

Plate 34 Trial 1 (Ampicillin-Sulbactam) Bromthymol blue
Plate 37 Trial 1 (Co-Amoxiclav) Phenol red
Plate 40 Trial 1 (Meropenem) Phenol red

Plate 43 Trial 1 (Ampicillin-Sulbactam) Phenol red
Plate 46 Trial 1 (Co-Amoxiclav) - CTE
Plate 49 Trial 1 (Meropenem) - CTE
Plate 52 Trial 1 (Ampicillin-Sulbactam) - CTE

Plate 55 Grinding of flowers using an osterizer
Plate 56 Grinded flowers soaked in 95% ethanol

Plate 57 Filtration of soaked flowers using Whatman filter paper
Plate 58 Extraction of crude extract of using ROTAVAP
Plate 59 Sterilization of crude extract using cellulose filter

Plate 60 Sterilized crude extract placed on an evaporating dish
Plate 61 Ampicillin & Cefoxitin (Trial 1)

Plate 64 Augmentin & Meropenem (Trial 1)

Plate 68 Chloramphenicol &Ampicillin+Sulbactam (Trial 1)


APPENDIX D
Antibiotic Preparation
Amount of
Antibiotics Amount of
Concentration
Antibiotic Diluent
Ampicillin 1g 10 ml 100,000 ug/ml
Cefoxitin 1g 10 ml 100,000 ug/ml
Chloramphenicol 1g 10 ml 100,000 ug/ml
Ampicillin-
1g 10 ml 100,000 ug/ml
Sulbactam
Meropenem 1g 10 ml 100,000 ug/ml
Co-Amoxiclav 1.2g 20 ml 100,000 ug/ml
(Augmentin)
Table 1 Reconstitution of Antibiotics
Figure 2 Preparation of Stock Solution

Figure 3 Computation for Stock Solution (Tube 1)


APPENDIX E
Authentication of Plant
APPENDIX F
Phytochemical Analysis
APPENDIX G
Inserts
Plate 71- Cefoxitin

Plate 72- Ampicillin- Sulbactam

Plate 73- Ampicillin- Sulbactam

Plate 74 Meropenem
Table 75 Ampicillin
Table 46 Co-amoxiclav
APPENDIX H
Curriculum Vitae
Imee Joy R. Batugal, born on November 6, 1995 in Delfin Albano,
Isabela. Both her parents graduated BS in Commerce. She went to San
Antonio Elementary School for primary school and St. Paul University
Philippines for her secondary years. She is now in her third year in Centro
Escolar University taking up BS in Medical Technology. She believes that
Education is the cure for poverty.

Angela Krisne H. Bocalbos, born on January 28, 1994 in Makati
City. She is residing at 383 Mangga St. Cembo, Makati City. She is the
daughter of Mr. Pedro F. Bocalbos and Mrs.Herlyn H. Bocalbos. She
finished her primary and secondary education at St. Paul College of
Makati with loyalty award and gold medalist of Glee Club. She also joined
the Red Cross Youth Council in high school. She is currently studying at
Centro Escolar University taking up Medical Technology. She truly
believes in the saying Nothing great ever came that easy.

Deanna Gem Calaramo was born on February 2 1995. Living at
Villa Corazon San Fernando Pampanga. She is the daughter of Mr Rowel
Calaramo and Mrs. Alma Calaramo. She finished her primary education at
Claveria West Central School and secondary education at New Era
University. She was 2nd place in the short story writing contest during the
English Week and 1st place for Ms. ASJ. She is currently pursuing
Bachelor of Science in Medical Technology at Centro Escolar University.
She believes that "Those who walk with God always reach their
destination.
Catherine Joy T. Carvajal, born on January 15, 1992. Living at
Katipunan Subdivision, Lucban, Quezon. She is the daughter of Mr. Arturo
P. Carvajal and Mrs. Belinda T. Carvajal. She finished her primary
education at Paaralang Elementrayang Lucban 1 and her secondary
education at Lucban Academy with a couple of award of being a achiever.
She is currently studying at Centro Escolar University taking up Medical
Technology. She believes in the saying Success isnt just about what you
achieve in life, its about what you inspire others to do.

Louiegie Salas Esguerra, born on January 03, 1996. He is
from 189 San Isidro, Laug, Mexico, Pampanga. He went from Laug
Elementary School from his primary and Saint Marys Angels College of
Pampanga from his secondary. And he is currently a Medical Technology
student at Centro Escolar University and a former basketball varsity of the
College of Medical Technology.

Sarah C. Eugenio, born on the 15th day of March 1994 at Taguig
City and was raised at Pasig City. She is the youngest daughter of Grace
Eugenio and Rodolfo Eugenio. She finished her primary education at
Colegio del buen Consejo, Pasig City where she became a consistent
honor student. She graduated high school at St. Marys Academy,
Guagua, Pampanga. She is currently studying at Centro Escolar
University taking up BS Medical technology. She was one of the deans
lister during her freshmen and sophomore years. Her motto in life is,
Believe in yourself in all that you are. Know that there is something inside
you that is greater than any obstacle.

Rosean A. Floranza, born on the 6th day of November, year 1995.
She came from a simple family that was well-raised and well-mannered by
her parents Fernando Floranza and Renalyn Floranza in the province of
Catanduanes. Reality check. Simplicity is beauty, and this woman is an
obvious proof of this. Behind her is a motivation and aspiration driven by
hardwork, perseverance, determination and a hope that someday she will
be able to be liberated from the bondage of ignorance and opens her
gateway to success and be able to impose her will, desire and passion for
others. She took her elementary days in Viga Rural Development High
School where she was a consistent honor student from grades 1 to 6 and
received several awards academically and in co-curricular activities.
Stakes went high as the levels of education and knowledge went deeper
and deeper, when she became a high school student at Viga Rural
Development High School. But with her willingness to make her parents
proud and well-equipped with enough intelligence and inspiration she
graduated as ahonorable mention, a recipient of different awards and was
recognized the best amongst the best of the schools batch 2011-2012.
Now, she is on her 3rd year of study in tertiary education in Centro Escolar
University taking up a Bachelors degree in Medical Technology and she
hopes to use this as her preparatory field of study when she takes up
Medicine. Success may still be a thousand miles away from being
achieved, yet she believes that with her courage, hardships, and God-
laden philosophy and ambition shell be able to embrace and fulfill it in
Gods time by Gods grace and for Gods purpose.

Ginia I. Gonzales, born on February 5, 1995 at San Juan, Metro
Manila. Currently resides at Cainta, Rizal. Her parents are Mr. Federico E.
Gonzales and Mrs. Maria I. Gonzales. She graduated primary and
secondary education at Siena College, Taytay where she was a CAT
officer, a varsity in Badminton and taking up Bachelor of Science in
Medical Technology in Centro Escolar University. Her motto is,
Everything in moderation, including moderation.

Tricia Marie S. Macaspac, born on September 21, 1995 in
Caloocan City. She resides at Happy Homes St. Brgy. San Vicente
Angono, Rizal. She is a daughter of Engr. Antonio R. Macaspac and Mrs.
Marissa S. Macaspac. She graduated elementary level at Angono Private
High School (2012), wherein she was a member of APHS Terpsichorean
Guild, she belonged to the pilot section, and one of the top students for
four consecutive years. She is currently taking Bachelor of Science in
Medical Technology in Centro Escolar University Manila. She believes that
she can do all things through Jesus Christ who gives her strength.
Franchette Mideth S. Mercado, born on September 5, 1995 at
Lucena City. She lives at Katipunan Avenue Phase III Calmar Homes
Subdivision Lucena City, Quezon Province. Her parents are Beth Mercado
and Ferdinand Mercado. She finished her primary and secondary
education at Maryhill College where she received a Loyalty and
Leadership award. She is also a consistent top ten in her class. She is
now taking up Bachelor of Science in Medical Technology at Centro
Escolar University where she became a Deans list. Her motto is Learn
from yesterday, live for today, hope for tomorrow. The important thing is
not to stop questioning.

Ana Francesca L. Ocampo, 21, was born on the 4th of October of
the year 1993. She is the third and youngest child in a family of five.
Having been born to parents both affiliated in the medical field, she has
always dreamt of becoming a Medical Technologist like her father,
Eduardo A. Ocampo and a doctor like her mother, Marilou B. Lupisan-
Ocampo. Having spent most of her educational life in the same school for
13 years, she finished her primary and secondary school at Colegio San
Augustin, Makati, wherein she received a loyalty award. With the dream of
becoming a Medical Technologist, she entered Centro Escolar University
and is currently taking up the degree of Bachelor of Science in Medical
Technology. Outside school, she enjoys a variety of activities, especially
sports football in particular. She also enjoys travelling, having been to
different places within the country and also outside.

He is Carl Alexis Parone, a believer of excellence over mediocrity.
He was born on February 6, 1995. He is a 20-year-old lad from the
province of Oriental Mindoro. His parents are Mr. Richard Parone and
Mrs. Jeneth Parone. He spent his Elementary years at Socorro Central
School, and graduated with flying colors. Way back in elementary, He was
a member of Rondallia Ensemble and represented his school in various
academic related contests such as quiz bee and essay writing contest.
finished his secondary education at Mina De Oro Catholic School. A
consistent honor student, he had received various awards in the field of
leadership wherein he was awarded outstanding achievement in
performing arts. Also, he was a finalist for Ayala Youth Leadership Awards
of the graduating batch. He excelled in both academic and extracurricular
activities inside and outside the campus. He p resented his Science
Investigatory Project (SIP) entitled "Mega-Lunggay Antibiotic against

diseases" on the search for the best SIP of the province. Also, he has
joined University of the Philippines- Tagisan ng talino at galing, on
Impromptu Public Speaking and landed fifth against other students across
the region. Certified scholar ng bayan during his high school years. At
present, he is studying at Centro Escolar University (Manila) taking up BS
Medical Technology. He was a president's lister during his second year
and CEU scholar for 1st year, 2nd year and 3rd year stay at CEU. He
believed in the saying, "It's not going to be easy, but it's going to be worth
it". He is an epitome of a dynamic working progress and an advocate of
excellence.
Joanna L. Pulpulaan, the eldest daughter of Mr. Alejo O. Pulpulaan
Jr. and Mrs. Roselyn Leal Pulpulaan. She is 19 years old who was born on
January 13, 1996 from the province of Cagayan Valley. She is currently
studying at Centro Escolar University with the course Bachelor of Science
in Medical Technology. She graduated Elementary in 2008 at Lasam West
Central School as second honourable mention with the special awards like
leadership award GSP and athlete of the year with a gold medal. She
graduated High school at San Lorenzo Ruiz Educational Institute in the
year 2012. She was given a leadership award with medal, Catholic
Christian service award with medal and a certificate of participation at the
Philippine Mathematical Olympiad, mathematical society of the
Philippines. She was awarded every end of the year during her high
school years being one of the top students. She believes that, If you have
discipline, drive and determination, nothing is impossible.

JodilenMaala Quezon, born on the 2nd day of June, year 1995. She
was born at Tiaong, Quezon and lives in San Pablo City, Laguna. She
was raised by Celedonio Quezon and Joyce Quezon, a loving and caring
parents. She graduated elementary from a catholic school in their city,
Canossa College, in the year 2008 and finished high school at the same
school in year 2012. Currently, she is enrolled at Centro Escolar University
in Mendiola, Manila and is taking up BS Medical Technology. She was a
consistent achiever during high school and a gold medalist in TagisTalino.
She believes that everything you did wrong today is a step closer to your
glory.
Melanie Aura C. Romero. She was born on June 2, 1996. She was
born in Brgy. Bagong Pook, Sta. Maria Laguna. She was the eldest
daughter of Mr. Cesar D. Romero and Marlyn C. Romero. She graduated
in Santa Maria Academy with consistent awards of being achiever, She
also got a Fourth honorable mention. She became an officer of the Filipino
club for two years and she is also a Student Volunteer Catechist. She is
now taking up Bachelor of Science in Medical Technology. Her motto in
life is To accomplish great things, we must not only act, but also dream;
not only plan, but also believe.

John Ivan Ronald V. Santiago, was born on the 9 th of September of
the year 1994 at St. James Hospital. He is the eldest son and have two
brothers and one sister. He lives at 553 purok 6 BO. Marinig, Cabuyao,
Laguna his parents are Ronaldo L. Santiago and Wilma V. Santiago. He is
currently studying at Centro Escolar University with the Degree of
Bachelor of Science in Medical Technology. He graduated his Grade
School in the year 2007 at Angels in Heaven School and he graduated his
high school in year 2011 at Lady of Rose Academy inc. with special
awards like athlete of the year, and he became an exchange student of
the Rotary Club of the Philippines, and became an eagle scout of the
Philippines. He believes in the saying that I can do impossible to possible.

Charlene D. Simangan, born on March 11, 1994 San Gabriel,
Tuguegarao City, currently resides at 1329-D Mignelin St. Sampaloc
Manila. Her parents are Mr. Carlos P. Simangan and Mrs. Shiela D.
Simangan. She finished elementary in Barasoain Memorial Elementary
School. She graduated High school in Sto. Angel dela Guardia Academy
as a Valedictorian with Special awards. She is taking Bachelor of Science
in Medical Technology in Centro Escolar University. She believes that
chance keep coming if you dont give up and that everything is possible if
you try enough. So if something doesnt work out, youre not trying
enough.
Camille M. Urbano, 19 years of age, residing in Brgy. San Isidro,
Zaragosa, Nueva Ecija. A loving daughter of Mr. Romualdo Urbano and
Mrs. Lyn Urbano, she finished her elementary education in Vincentian
Catholic Academy wherein she was one of the merit card holders. In her
secondary years, she graduated at College of the Immaculate Conception
where she was given an academic commendation award. Currently, she is
now taking up Bachelor of Science in Medical Technology at Centro
Escolar University wherein she was one of the deans lister for three
consecutive semesters. She believes that Success is not final, failure is
not fatal: it is the courage to continue that counts

Patricia Ann M. Victorio, 19, a student at Centro Escolar University
College of Medical Technology. She was born on March 23, 1996 and was
raised in Maybunga Pasig City. Her parents are Rex M. Victorio and Elena
Baby M. Victorio. She graduated as the Valedictorian of her batch in her
Elementary year and became the Vice President of the Student Council.
She then graduated as Valedictorian also in her High school year. She
also received Loyalty award from her former alma mater, Escuela de Sto.
Rosario. She became the President of the Student Council and won 1st
place in the Girl Scout Slogan Making Contest.
In her first year of college she became an entrance scholar for a
year. She was a Bobby C. Eusebio scholar in Pasig City until now. For a
year and a half, she became a University Student Council grantee and
worked at Student Affairs Office

She believes that one should learn to Put your heart, mind and
soul into even your smallest acts- this is the secret to success.
Joanna Marie Joyce C. Velasco, born on December 21, 1995 in
San Juan, Metro Manila. She was the eldest daughter of Mr. Dominador
P. Velasco and Mrs. Venice C. Velasco. She graduated her grade school
in K.I.D.S Montessori with a bronze medal. She graduated her high school
in Siena College of Taytay. She joined Dulaang Siena. She is now taking
up Bachelor of Science in Medical Technology. She got 50% scholarship
for one year and she was also a MTAA scholarship grantee. Her motto is
Aim high, so that you can attain high.

Mario N. Viduya, born in Manila on November 6, 1992.Son of Mr.
Mario and Mrs. Oflelia Viduya. Residing at Las Pinas City. He is currently
taking up Bachelor of Science in Medical Technology at Centro Escolar
University Manila. He finished primary school at So. Nino Elementary
School Paranaque City and his secondary school at PNHS-Don Galo
Annex where he was the First Honorable Mention and received Best in
Biology, Best in Social Science, and Best in MAPEH Awards. The future
belongs to those who believe in beauty of their dreams.

Christine Joy C. Zabat, born on April 7, 1995 at Baliuag, Bulacan.
She lives at Milflora Homes, Baliuag, Bulacan. A diligent daughter of
Fernando Zabat and Virginia Zabat. She went to Marian College of
Baliuag Inc. for her primary and secondary years where in she was given
an award of Most creative student in class for 5 succeeding years and
she also got the 3rd pace in BULPRISA Poster Making contest in provincial
level. And now shes taking up Bachelor of Science in Medical Technology
in Centro Escolar University, Manila. Her motto in life is Live boldly and
bloom to your fullest potential by taking small but fearless daily actions in
the direction of your dream.

Table of Contents
CHAPTER 1 ............................................................................................................... v
INTRODUCTION ....................................................................................................... v
Background of the Study ..................................................................................... v

SETTING OF THE STUDY ...................................................................................... vi
CONCEPTUAL FRAMEWORK ...............................................................................vii
CHAPTER 2 ............................................................................................................. xiv
REVIEW OF RELATED LITERATURE AND STUDIES.................................................. xiv
Clitoria ternatea Linn. flower as pH Indicator ................................................... xiv

Staphylococcus aureus ...................................................................................... xvii
Escherichia coli .................................................................................................... xx
Properties of Anthocyanins .............................................................................. xxii
Minimum Inhibitory Concentration ................................................................. xxiii
Bromthymol blue as pH indicator ..................................................................... xxv
Phenol red as pH indicator .............................................................................. xxvi
CHAPTER 3 ......................................................................................................... xxviii
METHODOLOGY ................................................................................................ xxviii
3.1. Research Design ...................................................................................... xxviii

3.2 Methods and Materials .............................................................................. xxx
3.2.1 Collection of Sample ............................................................................. xxx
3.2.2 Authentication of the Plant .................................................................. xxx
3.2.3 Crude Extraction of Clitoria ternatea Linn. flowers ............................. xxx
3.2.4 Sterilization of Crude Extract Using Cellulose Filter ............................ xxxi
3.2.5 Concentration and pH neutralization of the flower extract ............... xxxi

3.2.6 Phytochemical Analysis ...................................................................... xxxii
3.2.7 Preparation of Media ........................................................................ xxxiii
3.2.8. Rejuvenation of Controlled Organisms ............................................ xxxiii
3.2.9. Confirmatory Testing Using API System ........................................... xxxiii
3.2.10. McFarland Standard ....................................................................... xxxiv
3.2.11 Preparation of Indicators ................................................................. xxxv
3.2.12 Reconstitution of Antibiotics ............................................................ xxxv
3.2.13 MIC Testing ....................................................................................... xxxv
3.3 Reading of results ................................................................................... xxxvii
3.4 Statistical Treatment............................................................................... xxxvii
CHAPTER 4 ....................................................................................................... xxxviii
RESULTS AND DISCUSSION.............................................................................. xxxviii
CHAPTER 5 ............................................................................................................ xlix
CONCLUSIONS AND RECOMMENDATIONS .......................................................... xlix
SUMMARY OF FINDINGS................................................................................... xlix

CONCLUSION......................................................................................................... l
RECOMMENDATIONS .......................................................................................... li
APPENDIX A ...........................................................................................................lvii
GANTT: Chart for Research ...............................................................................lvii

APPENDIX B .......................................................................................................... lviii
Table of Expenses ............................................................................................. lviii

APPENDIX C .............................................................................................................lx
Documentation of Tables, Plates, and Figues......................................................lx

APPENDIX D ........................................................................................................ lxxiii
Antibiotic Preparation .................................................................................... lxxiii

APPENDIX E ........................................................................................................ lxxvi
Authentication of Plant ................................................................................... lxxvi

APPENDIX F ....................................................................................................... lxxvii
Phytochemical Analysis.................................................................................. lxxvii

APPENDIX G ...................................................................................................... lxxviii
Inserts ........................................................................................................... lxxviii

APPENDIX H ..................................................................................................... lxxxvii
Curriculum Vitae .......................................................................................... lxxxvii

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CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Clitoria ternatea Linn. Flower Extract as an Alternative Growth

IMEE JOY R. BATUGAL CARL ALEXIS B. PARONE

The researchers would like to thank the Dean of the College of

Medical Technology, Dr. Charito M. Bermido, for her continuous support

throughout the conduction of the study.

To Secret Garden of Doris and the Garden of Silang, Cavite for

providing the researchers the flower samples.

researchers equipment needed to facilitate this experiment.

A very special thanks to Mr. Gil Soriano for teaching the

researchers the basics in Research Writing.

The researchers would like to express their deepest gratitude to

in this field, for her patience, and understanding.

A very special thanks to the families of the researchers who have

been supporting them morally and financially throughout the research

knowledge for them to be able to finish this research study.

Four years of retrospect, the researchers have been through rough

waters toiling with their dreams of becoming professional medical

technologists. Needless to say, they are sprouting yet growing mushrooms

travelling on the path leading to the actualization of our vision. The

researchers pay gratitude to the people who have generously, in a way, or

another, helped reach this momentum.

To the researchers parents, to whom their lives are indebted to,

partial fulfillment of their success.

To the future medical technology students, the researchers know

This paper is dedicated to you, an epitome of the researchers

burning desire and passion to do whatever it takes to reach their goals.

capabilities to prevent, cure, and identify pathogenic organisms in order to avoid

or stop spread of disease. Clitoria ternatea (Linn.) flower is a plant considered to

extraction. The CTE has been sterilized, concentrated and undergone pH

adjustment. Staphylococcus aureus and Escherichia coli were used as a control

flower extract can be a potential indicator for Minimum Inhibitory Concentration

determination in exchange to other locally and expensive indicators which is

commonly used in the laboratory. Accomplishment of the study is beneficial not

means of providing more job opportunity to farmers and new perspective to

effective alternative indicator for MIC compared to commercially made dyes.

Keywords: Indicator, Pathogenic, Minimum Inhibitory Concentration

Background of the Study

mortality of humans, plants, and animals. It can easily spread through

direct contact or even thru aerosol transmission. Its ability to cause a

disease is dependent on its pathogenecity and virulence. Once an

individual gets infected, treatment is based on the action of antibiotics to

kill certain bacteria caused the disease.

There are different ways on how to identify the viability of

antimicrobials to eradicate infection. One of the methods used is the broth

dilution wherein the Minimum Inhibitory Concentration (MIC) is being

determined. MIC is the lowest concentration of an antimicrobial agent

required to inhibit the growth of a particular bacterial isolate (Mahon et al.,

organisms. MIC is then recorded as the lowest dilution in which there is no

microscopic growth after an overnight incubation. In order to conduct MIC

testing, certain dyes are used as indicators. An example of dye used is

Clitoria ternatea Linn. belongs to family Fabaceae which is a

herbaceous perennial legume valued for its forage and medicinal

importance and has been originated in the traditional Indian system of

medicine for its phytochemical properties. (Pahune et al., 2013).Clitoria

ternatea Linn.iscomposed of anthocyanin, which is a type of delphidin,

flavonoids, and tannin. These pigments are of great potential in producing

also the economic and agricultural industry by means of producing more

SETTING OF THE STUDY

The plants cultivated were acquired from Silang Junction, Tagaytay

conducted in Centro Escolar University in Metro Manila, Philippines.

Centro Escolar University (CEU) is a private, non-sectarian higher

education institution with an enrollment of over 20,000 students in its three

campuses: Manila, Makati and Malolos. Established in 1907, Centro