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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
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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
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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
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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


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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


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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


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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
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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)


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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.


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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.
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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.


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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


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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.


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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)


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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
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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
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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


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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


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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


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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


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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,


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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,
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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


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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.


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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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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)


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.
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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


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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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.
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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
Minimum Inhibitory Concentration
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


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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
Minimum Inhibitory Concentration
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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
Optical Density of the BB, PR, and CTE for the MIC Determination of
Cefoxitin against Staphylococcus aureus
Indicators Trial 1 Trial 2 Trial 3 Overall mean
Mean (SD) Mean (SD) Mean (SD) (SD)
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
Optical Density of the BB, PR, and CTE for the MIC Determination of
Chloramphenicol against Staphylococcus aureus
Indicators Trial 1 Trial 2 Trial 3 Overall mean
Mean (SD) Mean (SD) Mean (SD) (SD)
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


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because it has the highest overall mean while BB is the most sensitive

indicator.

Table 4-6
Optical Density of the BB, PR, and CTE for the MIC Determination of
Co-amoxiclav against Escherichia coli
Indicators Trial 1 Trial 2 Trial 3 Overall mean
Mean (SD) Mean (SD) Mean (SD) (SD)
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
Optical Density of the BB, PR, and CTE for the MIC Determination of
Meropenem against Escherichia coli
Indicators Trial 1 Trial 2 Trial 3 Overall mean
Mean (SD) Mean (SD) Mean (SD) (SD)
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.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Table 4-8
Optical Density of the BB, PR, and CTE for the MIC Determination of
Ampicillin-Sulbactam against Escherichia coli
Indicators Trial 1 Trial 2 Trial 3 Overall Mean
Mean (SD) Mean (SD) Mean (SD) (SD)
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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
Bromthymol 1.0748 0.9896 Brothymol blue vs 0.623
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.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Table 4-11
Significant Differences of the OD of BB, PR, and CTE for
Chloramphenicol MIC Determination
Indicators Mean S.D. F-value p-value Remarks/ Post Hoc p-value

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


*The mean difference is significant at 0.05 level

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
Indicators Mean S.D. F-value p-value Remarks/ Post Hoc p-value

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
CTE 2.4137 0.3574 CTE vs Phenol red *0.000
Total 4.5481
*The mean difference is significant at 0.05 level
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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
Indicators Mean S.D. F-value p-value Remarks/ Post Hoc p-value

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


*The mean difference is significant at 0.05 level

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
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
Bromthymol 1.8859 0.5792 Brothymol blue vs 0.633
Blue 20.2622 0.000 CTE
CTE 2.4267 0.2798 CTE vs Phenol red *0.000

Total 3.2268 3.2268


*The mean difference is significant at 0.05 level

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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).


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

References

Askim, A., Paulsen, J., Mehl, A., Solligard, E., Asvold, B., & Damas, J.

(2015) Epidemiology and outcome of Staphylococcus aureus

bloodstream infection and sepsis in a Norwegian county 1996-

2011: an observational study. Retrieved March 21, 2015, from

http://www.biomedcentral.com/ /1471-2334/15/116.

Carson, J., Miller, R., Walker, R., Coles, M, Coyne, R., Dalsgaard, I., Hsu,

H., Mathers, J., Papapetropoulou, M., Petty, B., Teitzel, C., &

Reimschuessel, R. (2005). Standardization of a broth microdilution

susceptibility testing method to determine minimum inhibitory

concentrations of aquatic bacteria. Retrieved from

http://www.ncbi.nlm.nih.gov/pubmed/15997819

Hughes, N. (2009). The Photoprotective Role of Anthocyanin Pigments in

Leaf

Tissues. Ann Arbor: UMI Dissertations Publishing. Retrieved March

21, 2015, from http://search.proquest.com/docview/305012023

Hung, L. (2011). Thermal Degradation of Blue Anthocyanin Extract of

Clitoria

TernateaFlower. Retrieved May 16, 2015, from

http://www.ipcbee.com/

vol7/12-ICBFS2011S035.pdf.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Kanchana, R., Fernandes, A., Bhat, B., Budkule, S., Dessai, Mohan, R.

(September

2013). Dyeing of Textiles With Natural Dyes An Eco Friendly

Approach.

International Journal of ChemTech Research.

Kraus, T. (November 2002). Tannins in nutrient dynamics in forest soils:

plant-litter-soil interactions. Netherlands: Kluwer Academic

Publications.

Li, Z. (2008). High Yield Anthocyanin Biosynthesis in Metabolic

Engineering of E.

Coli. Ann Arbor: UMI Dissertations Publishing. Retrieved May 18,

2015, from http://search.proquest.com/docview/89144922

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.

Turnidge, J., Rao, N., Chang, F., Fowler Jr., V., Kellie, S., Arnold, S., Lee.,

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

Microbiology.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Winn Jr, W. (2006).Konemans Color Atlas and Textbook of Diagnostic

Microbiology: 6th Edition.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

APPENDICES
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

ATCC 25923 :Staphylococcus aureus

Plate 1 Trial 1 (Ampicillin) Bromthymol blue

Plate 2 Trial 2 (Ampicillin) Bromthymol blue

Plate 3 Trial 3 (Ampicillin) Bromthymol blue

Plate 4 Trial 1 (Cefoxitin) Bromthymol blue

Plate 5 Trial 2 (Cefoxitin) Bromthymol blue

Plate 6 Trial 3 (Cefoxitin) Bromthymol blue

Plate 7 Trial 1 (Chloramphenicol) Bromthymol blue

Plate 8 Trial 2 (Chloramphenicol) Bromthymol blue

Plate 9 Trial 3 (Chloramphenicol) Bromthymol blue

Plate 10 Trial 1 (Ampicillin) Phenol red


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Plate 11 Trial 2 (Ampicillin) Phenol red

Plate 12 Trial 3 (Ampicillin) Phenol red

Plate 13 Trial 1 (Cefoxitin) Phenol red

Plate 14 Trial 2 (Cefoxitin) Phenol red

Plate 15 Trial 3 (Cefoxitin) Phenol red

Plate 16 Trial 1 (Chloramphenicol) Phenol red

Plate 17 Trial 2 (Chloramphenicol) Phenol red

Plate 18 Trial 3 (Chloramphenicol) Phenol red

Plate 19 Trial 1 (Ampicillin) - CTE

Plate 20 Trial 2 (Ampicillin) CTE

Plate 21 Trial 3 (Ampicillin) - CTE


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Plate 22 Trial 1 (Cefoxitin) - CTE

Plate 23 Trial 2 (Cefoxitin) - CTE

Plate 24 Trial 3 (Cefoxitin) - CTE

Plate 25 Trial 1 (Chloramphenicol) - CTE

Plate 26 Trial 2 (Chloramphenicol) - CTE

Plate 27 Trial 3 (Chloramphenicol) - CTE

ATCC 25922 :Escherichia coli

Plate 28 Trial 1 (Co-Amoxiclav) Bromthymol blue

Plate 29 Trial 2 (Co-Amoxiclav) Bromthymol blue

Plate 30 Trial 3 (Co-Amoxiclav) Bromthymol blue

Plate 31 Trial 1 (Meropenem) Bromthymol blue


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Plate 32 Trial 2 (Meropenem) Bromthymol blue

Plate 33 Trial 3 (Meropenem) Bromthymol blue

Plate 34 Trial 1 (Ampicillin-Sulbactam) Bromthymol blue

Plate 35 Trial 2 (Ampicillin-Sulbactam) Bromthymol blue

Plate 36 Trial 3 (Ampicillin-Sulbactam) Bromthymol blue

Plate 37 Trial 1 (Co-Amoxiclav) Phenol red

Plate 38 Trial 2 (Co-Amoxiclav) Phenol red

Plate 39 Trial 3 (Co-Amoxiclav) Phenol red

Plate 40 Trial 1 (Meropenem) Phenol red

Plate 41 Trial 2 (Meropenem) Phenol red

Plate 42 Trial 3 (Meropenem) Phenol red


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Plate 43 Trial 1 (Ampicillin-Sulbactam) Phenol red

Plate 44 Trial 2 (Ampicillin-Sulbactam) Phenol red

Plate 45 Trial 3 (Ampicillin-Sulbactam) Phenol red

Plate 46 Trial 1 (Co-Amoxiclav) - CTE

Plate 47 Trial 2 (Co-Amoxiclav) - CTE

Plate 48 Trial 3 (Co-Amoxiclav) - CTE

Plate 49 Trial 1 (Meropenem) - CTE

Plate 50 Trial 2 (Meropenem) - CTE

Plate 51 Trial 3 (Meropenem) - CTE

Plate 52 Trial 1 (Ampicillin-Sulbactam) - CTE

Plate 53 Trial 2 (Ampicillin-Sulbactam) - CTE


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Plate 54 Trial 3 (Ampicillin-Sulbactam) - CTE

Plate 55 Grinding of flowers using an osterizer

Plate 56 Grinded flowers soaked in 95% ethanol


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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


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Plate 60 Sterilized crude extract placed on an evaporating dish

Plate 61 Ampicillin & Cefoxitin (Trial 1)

Plate 62 Ampicillin & Cefoxitin (Trial 2)


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Plate 63 Ampicillin & Cefoxitin (Trial 3)

Plate 64 Augmentin & Meropenem (Trial 1)

Plate 65 Augmentin & Meropenem (Trial 2)


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Plate 67 Augmentin & Meropenem (Trial 3)

Plate 68 Chloramphenicol &Ampicillin+Sulbactam (Trial 1)

Plate 69 Chloramphenicol &Ampicillin+Sulbactam (Trial 2)


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Plate 70 Chloramphenicol &Ampicillin+Sulbactam (Trial 3)


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Figure 3 Computation for Stock Solution (Tube 1)

Figure 4 Computation for Stock Solution (Tube 2)


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Figure 5 Computation for Stock Solution (Tube 3)


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

APPENDIX E

Authentication of Plant
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

APPENDIX F

Phytochemical Analysis
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

APPENDIX G

Inserts

Plate 71- Cefoxitin


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Plate 72- Ampicillin- Sulbactam


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Plate 73- Ampicillin- Sulbactam


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Plate 74 Meropenem
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Table 75 Ampicillin
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

Table 46 Co-amoxiclav
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

She believes that one should learn to Put your heart, mind and

soul into even your smallest acts- this is the secret to success.
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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.


CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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
CENTRO ESCOLAR UNIVERSITY (COLLEGE OF MEDICAL TECHNOLOGY) 1

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