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CHAPTER 1

THE PROBLEM AND ITS BACKGROUND

This chapter highlights information about the background and rationale of

conducting the study. It includes the introduction, statement of the problem, hypothesis,

significance of the study, scope and delimitation, and the definition of terms.

Introduction

In traditional herbal medicine, plants have been used for many years.

Therefore, plants have attained status of natural source of new and potent antimicrobial

agents. Medicinal plants are used as ethnomedicine in different countries around the

world and are source of natural products providing unlimited opportunity for new drugs

because of readily available medicinal diversity (Gull et al, 2013).

Traditional healers have long used plants to prevent or cure infectious conditions.

Plants are rich in a wide variety of secondary metabolites, such as chave antimicrobial

properties(Cowan, 2011).

Advanced methods of analytical, biotechnological, genomics, proteomics and

metabolomics are nowadays applied in medicinal plant research and contribute to the

advancement of alternative natural antimicrobial (Abdallah, 2011).

Magnifera indica, commonly called as mango, is a plant belonging to the family

Anacardiaeceae which consists of about sixty genera and six hundred species.

Conventionally, the mango plant has several biological properties as like to treat mouth

infections in children, diarrhea, dysentery, gastrointestinal tract disorders, typhoid fever,


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sore throat and scurvy. It is also a source of vitamin A when eaten in combinations with

salt and honey for the treatment of blood disorders. Ground seeds and leaves have been

used as remedy for diabetes, as colic and to treat irritations, scorpion and bee

stings(Hannan et al., 2013)

Kirby-Bauer disk diffusion susceptibility determine the sensitivity or resistance of

pathogenic aerobic and facultative anaerobic bacteria to various antimicrobial compounds

in order to assist a physician in selecting treatment options for his or her patients. The

pathogenic organism is grown on Mueller-Hinton agar in the presence of various

antimicrobial impregnated filter paper disks. The presence or absence of growth around

the disks is an indirect measure of the ability of that compound to inhibit that organism

(Hudzicki, 2010).

Staphylococcus aureus is a major human pathogen that causes a wide range of

clinical infections. It is a leading cause of bacteremia and infective endocarditis as well as

osteoarticular, skin and soft tissue, pleuropulmonary, and device-related infections. The

past 2 decades have witnessed two clear shifts in the epidemiology of S.

aureus infections: first, a growing number of health care-associated infections,

particularly seen in infective endocarditis and prosthetic device infections, and second, an

epidemic of community-associated skin and soft tissue infections driven by strains with

certain virulence factors and resistance to β-lactam antibiotics( Davis et al., 2015).

The researcher wants to conduct this study to produce an alternative antimicrobial

agent against pathogenic microorganisms such as Staphylococcus aureus using Mango

(Mangifera indica) leaf extract which is abundant in our country, cheaper, eco-friendly,
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and less hazardous. This study will give highlights on determining the capability of

Mango (Mangifera indica) leaf extract as an antimicrobial agent and its importance in

Microbiology section of the laboratory.

Statement of the Problem

The study was focused on determining the antimicrobial activity of Mango

(Mangifera indica) leaf extract against pathogenic microorganisms such as

Staphylococcus aureus.

The study seeks to answer the following questions:

1.) Is Mango (Mangifera indica) leaf extract capable on inhibiting

pathogenic microorganisms such as Staphylococcus aureus?

2.) Is Mango (Mangifera indica) can be alternative use for commercial

antimicrobial agents?

3.) Which is the most effective menstruum used for the extraction of Mango

(Mangifera indica)leaves to produce a zone of inhibition?

4.) What is the significance of the study among the researchers in the field of

Medical Technology?

Hypothesis

1. Mangifera indica extract is effective against Staphylococcus aureus.

2. There is a significant relationship between the capability of Mango (Mangifera

indica) leaf extract and Erythromycin to pathogenic Staphylococcus aureus.


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Significance of the Study

The study of the capability of Mango (Mangifera indica) leaf extract as an

antimicrobial agent will be beneficial to the following:

For Students.The study will enlighten the minds of the students aboutMango (Mangifera

indica) leaf extract as an antimicrobial agent. It will serve as their reference or guide in

creating their own study. It will also help the students who are taking Medical

Technology to continue or expand this study about Mango (Mangifera indica).

For Medical Technologists.The Medical Technologist will benefit on the said study in

the following terms: health – it is safer to use because of its organic components; Cost-

cutting – it is cheaper than the commercial antimicrobial products present in the market.

For Manufacturers. The manufacturers will benefit in the study because it will help

them to consume cheaper products in producing antimicrobial. Mango (Mangifera indica)

is abundant in our country or anywhere and it is less toxic than the chemical used in

making antimicrobial products. It will also give them the idea that .Mango (Mangifera

indica), is not only just a fruit we often buy at the market but also it can be use in the

laboratory as an alternative antimicrobial.

For the Community.The study will also be beneficial in the community because Mango

(Mangifera indica) can be purchased in any market that would give them the opportunity

to know its feasibility as an antimicrobial.

For the Future Researchers. The outcome of the study will be beneficial for the future

researchers. It would be a great help for the future researchers to be more creative and
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more productive in conducting a study. The research will give highlight on how

important Mango (Mangifera indica) is as an alternative antimicrobial.

Scope and Delimitation

The scope of the study was solely focused on the antimicrobial capability of

Mango (Mangifera indica) leaf extract against S. aureus. The researcher used Disk

diffusion method to determine the antimicrobial effects of aqueous, methanolic and

ethanolic extracts of M. indica leaves against clinical isolates of S. aureus. The

preparation of sample includes collecting, washing, grinding, and producing an extract

using maceration. It is as well focused on susceptibility testing and measurement of the

diameter of inhibitory zones of the Mango (Mangifera indica) leaf extract to determine its

activity against S. aureus.

Definition of Terms

The following terms are defined conceptually and operationally for better

understanding of this study.

Antimicrobial. An agent that stops the growth of S. aureus. In this study, the

Mango (Mangifera indica) leaf extract was tested for its antimicrobial action.

Capability. This refers to the ability Mango (Mangifera indica) leaf extract as an

antimicrobial agent.

Extract. A preparation containing the active ingredients of a substrate in

concentrated form. In this study, maceration method was used to produce the optimum

extract to be used as an antimicrobial product out of Mango (Mangifera indica) leaf.


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Mango (Mangifera indica). A specie of flowering plant in the sumac and poison

ivy family Anacardiaceae. It is native to the Indian subcontinent where it is indigenous.

In this study, Mango (Mangifera indica) leaf extract was tested if it is an effective

antimicrobial agent.

Staphylococcus aureus. A gram positive, non-moving small round shaped or non-

motile cocci and usually found in grape-like clusters. It is found in the environment and is

a normal human flora, located on the skin and mucous membranes of most healthy

individuals however, it is consider as a major bacterial human pathogen that causes a

wide variety of clinical manifestations. In this study, it is one of the bacteria that was

treated using the extracted Mango (Mangiferaindica) leaf.


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CHAPTER 2

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter includes information from the literatures that are related to this

research and studies done by other researchers that will correlate for the further

understanding of the study.

Related Literature

Mangiferaindica (MI), also known as mango, has been an important herb in the

Ayurvedic and indigenous medical systems for over 4000 years. Mangoes belong to

genus Mangifera which consists of about 30 species of tropical fruiting trees in the

flowering plant family Anacardiaceae. According to ayurveda, varied medicinal

properties are attributed to different parts of mango tree. Mango is one of the most

popular of all tropical fruits. Mangiferin, being a polyphenolic antioxidant and a glucosyl

xanthone, it has strong antioxidant, anti lipid peroxidation, immunomodulation,

cardiotonic, hypotensive, wound healing, antidegenerative and antidiabetic activities

(Shah et al., 2010).

In recent years, there has been a dramatic increase in number of reported poisoning

outbreaks caused by food-borne pathogenic bacteria. In 1996, one of the serious

outbreaks caused by Escherichia coli O157:H7 occurred in Sakai, Japan (National

Institute of Infections Diseases and Infectious Diseases Control Division, 1997).

Consequently, there has been considerable interest in preventing food contamination by

food-borne pathogens. Traditionally, various methods, such as heating, reducing water

activity, smoking, fermentation, and adding antimicrobial agents, have been used to
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prevent spoilage of foods. The addition of antimicrobial agents has been a particularly

effective method for controlling microbial contamination. Unlike chemically synthesized

antimicrobial agents, those from natural sources are acceptable to consumers. Phenolic

compounds, tannins and flavonoids in mango seed are found to be responsible for

antimicrobial property. They prevent microorganisms by inhibiting extracellular

microbial; growth and by avoiding oxidative phosphorylation. So mango seed kernel oil

is used to kill abdominal worm and also given as a cure for vomiting, diarrhea and

hyperacidity. (Kittiphoom,2011)

Infectious diseases are one of the main causes of morbidity and mortality worldwide.

Nowadays many infections are often caused by multi-resistant microorganisms resulting

in difficult to treat diseases and, consequently, substantial increases in healthcare costs.

Nature is a generous source of compounds, with the potential to treat diseases, including

infectious diseases. Studies exploiting the mechanism of action and the structure-activity

aspects of these natural compounds may provide both additional antimicrobial leads and

drugs, and also significant insight into potential possibilities to overcome the

antimicrobial resistance (Hayashi et al., 2013)

Plants are used for the treatment of diseases and novel drugs continue to be

developed through research from plants. There are more than 20,000 plant species

used in traditional medicines and they are prospective reservoirs for new

pharmaceuticals. Traditional medicinal plants have received significant attention

because their phytochemicals may lead to new drug discoveries.For several years,

there has been an increased interest in the study of different extracts obtained from

traditional medicinal plants as potential sources of new antimicrobial agents. The


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search for new antimicrobial agents is an important line of research because of the

resistance acquired by several pathogenic microorganisms (Compean, 2014).

The number of multidrug resistant (MDR) microorganisms is increasing.

Escherichia coli, methicillin-resistant S. aureus (MRSA), Pseudomonas

aeruginosa, and Candida species are but a few among the well-known microorganisms

and have shown an evolution in resistance to various pharmaceutical drugs. Because the

levels of resistant pathogenic microorganisms are on the rise, novel and effective

antimicrobial agents are needed to contain this epidemic. Natural products obtained from

various plant species have been used for maintaining health as evidenced by their

extensive use in traditional medicine (Vandal et al., 2015).

Plants have been used as food, spice, and medicine since ancient time. Much

before the discovery of the existence of microorganisms, plants were an integrated part of

the traditional medicine of many communities used for the treatment of various infectious

diseases (Bucar etal., 2014)

In the recent years, research on medicinal plants has attracted a lot of attentions

globally. Large body of evidence has accumulated to demonstrate the promising potential

of Medicinal Plants used in various traditional, complementary and alternate systems of

treatment of human diseases. Plants are rich in a wide variety of secondary metabolites

such as tannins terpenoids, alkaloids, flavonoids, etc, which have been found in vitro to

have antimicrobial properties (Sher, 2011).

Staphylococcus aureus is a bacterium that can cause a variety of illnesses through

suppurative or nonsuppurative (toxin-mediated) means. S aureus is a common cause of

skin and skin structure infections as well as osteoarticular infections in the pediatric
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population. S aureus is also identified in cases of septicemia, infective endocarditis,

pneumonia, ocular infections, and central nervous system infections. aureus has proved

adept at developing antibiotic resistance. This is accomplished through acquisition of

mobile genetic elements that transfer resistance and virulence from other bacterial species

and staphylococcal strains. External pressures (namely, overuse of antibiotics) can also

force a previously susceptible isolate to become resistant. Staphylococcus is considered a

part of normal human flora. A common body area of S aureus colonization is the anterior

nares. Colonization is a risk factor for infection, and infection often occurs with the

same S aureus strain that colonized the individual (Ondusko, 2018)

Related Studies

A. Foreign

Takudzwa et al., (2013) conducted a research entitled, “ Antibacterial Properties Of

Mangifera Indica On Staphylococcus Aureus” Antibacterial activity of Mango

(Mangiferaindica) stem bark extracts was determined using disk diffusion, agar and broth

dilution methods. In disk diffusion method, inhibition zone sizes were used to determine

the susceptibility of S. aureus to the extracts. The results showed that the stem-bark

extracts of M. indica have antimicrobial activity against S. aureus. Methanol extracts

showed the highest inhibition zone diameter of 25 mm, followed by ethyl acetate, water

and hexane extracts with inhibition zone diameter of 22 mm, 14 mm and 10 mm,

respectively. The antibacterial activities of different extracts were found to be

concentration dependent, in agar and broth dilution methods. The plant extracts were

shown to have a MIC range of 0.62 mg/ml to 4.17 mg/ml, in agar dilution method.

Results from the broth dilution method had a MIC range of 0.16 mg/ml to 1.25 mg/ml.
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The control (ampicillin) was however, more effective than plant extracts since only a

concentration of 0.03 mg/ml in agar dilution and 0.001 mg/ml in broth dilution method

were effective to inhibit the growth of S. aureus. The extracts were shown to be

bacteriostatic at low concentrations. Phytochemical screening of the extracts revealed the

presence of phyto-compounds such as alkaloids and tannins which are known to inhibit

bacterial growth by different mechanisms from those of synthetic drugs. These phyto-

constituents may be responsible for the M. indica antibacterial activity.

The study entitled, “Antimicrobial Activity Of Basella Rubra Leaves” conducted by

Rawal et al (2010), Basella rubra were evaluated by measuring the inhibition zones using

Disk Diffusion method. The test organisms are  Staphylococcus aureus, Escherichia coli,

Salmonella typhi, Pseudomonas aeruginosa, Aspergillus niger, and Vibrio cholerae.

Leaves were shade dried, powdered and then extracted with water, 95% ethanol and

petroleum ether for 48 hours using Soxhlet apparatus. The filterates were collected and

evaporated to dryness under reduced pressure using a Rota evaporator. The dried extracts

were stored in dry sterilized small containers at 4˚C. The ethanolic extract showed

maximum activity with zone of inhibition (14.3±1.82 mm) against E.coli, followed by

aqueous extract (13.4±1.2 mm) and petroleum ether (5.6±0.62 mm)  at a concentration of

50mg/ml. Ciprofloxacin was used as the standard drug having zones of

inhibition(17±0.34 mm) against E.coli and 19±0.18 mm against A. niger.


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Synthesis

According to the study of Takudzwa et al (2013), they concluded that Mango

(Mangifera indica) has an antimicrobial activity against bacterial cells. The study tested

Mango (Mangifera indica) plant for its antimicrobial property same with this study. The

study conducted by Takudzwa et al, used disk diffusion method on agar and broth, while

this study only used agar diffusion method. Takudzwa et al used methanol, water ethyl

acetate, and hexane as extracting solvents while this study used methanol, ethanol, and

water. The study used dried mango stem bark while this study used fresh mango leaves.

In the study conducted by Rawal et al (2010), they used diffusion method same with this

study. And also they used ethanolic, aqueous, and petroleum ether as an extracting

solvent. Soxhlet apparatus were used to obtain the extract while in this study maceration

technique was utilized.

Local

The study of Biswas et al., (2013) entitled “Antimicrobial Activities of Leaf

Extracts of Guava (Psidium guajava L.) on Two Gram-Negative and Gram-Positive

Bacteria”. The guava leaves were extracted in four different solvents of increasing

polarities (hexane, methanol, ethanol, and water). The efficacy of these extracts was

tested against those bacteria through a well-diffusion method employing 50 μL leaf-

extract solution per well. According to the findings of the antibacterial assay, the

methanol and ethanol extracts of the guava leaves showed inhibitory activity against

gram-positive bacteria, whereas the gram-negative bacteria were resistant to all the

solvent extracts. The methanol extract had an antibacterial activity with mean zones of
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inhibition of 8.27 and 12.3 mm, and the ethanol extract had a mean zone of inhibition of

6.11 and 11.0 mm against B. cereus and S. aureus, respectively.

Bandibas et al., (2017) conducted a studyentitled “Antimicrobial Test of Five

Ethnomedicinal Plants in an Ancestral Forest Area”. The assessment of plants was

utilized using the transect-plot method. Ethnomedicinal uses and the mode of

preparations were obtained using a semi-structured interview questionnaire. Five

ethnomedicinal plants: Crinum asiaticum L., Pavettaindica L., Bauhinia purpurea L.,

Mollugo pentaphylla L., and Cinnamomummercadoi S. Vidal were selected for the anti-

microbial test against cultured bacterial strains; the Escherichia coli and Staphylococcus

aureus using the disc diffusion method. The mode of preparation of the extracts follows

the traditional method of the indigenous people. Chloramphenicol, on the other hand, was

used as positive control. Thirty-three out of 61 identified species were locally claimed to

have medicinal value and are known to cure stomach disorders, poisoning, bleeding,

cough, fever and wounds, among others. Antimicrobial test showed greater inhibitory

effect of Pavettaindica and Bauhinia purpurea against Staphylococcus aureus and

Escherichia coli, correspondingly; however, the synthetic chloramphenicol exhibited

greater antibacterial action than any of the plant extracts.

Penecilla et al., (2011) conducted a study entitled “Antibacterial Activity of

Extracts of Twelve Common Medicinal Plants from the Philippines”. The antibacterial

activity of the n-hexane, acetone/dichloromethane, ethanol and aqueous extracts of

twelve common medicinal plants from the Philippines obtained through pounding and

solvent extraction was evaluated using disc Agar diffusion. The microorganisms tested

were: Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas


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aeruginosa. Susceptibility test results showed that different extracts (1000 mcg/disc) from

ten plants inhibited growth with a mean zone of inhibition range of 15 to 30 mm against

S. aureus, E. coli, and P. aeruginosa. The common medicinal plants which showed highly

positive activity were Psidium guajava (guava), Eucalyptus globulus, Mangiferaindica

(Indian mango), Nasturtium officinale (Watercress), Pterygospermumoleiferum

(Moringa), Carmona retusa (Wild tea), Citrus aurantifolia (Lemon), Citrus sinensis

(Orange), Allium sativum (garlic), and Allium cepa (onion). Preliminary phytochemical

screening revealed the presence of flavonoids, tannins, alkaloids, glucosides, saponins

and steroids/terpenes. The results suggest that the different plant extracts contain

bioactive constituent(s) particularly tannins, flavonoids, terpenoids and other glycosides

with very strong antibacterial activity and validates the ethno-medical use in the

treatment of bacterial skin diseases and other forms of bacterial infections. However,

many reputed plant antibacterials have very variable activities depending on certain

conditions.

Synthesis

According to the study conducted by Biswas et al., (2013) entitled “Antimicrobial

Activities of Leaf Extracts of Guava (Psidium guajava L.) on Two Gram-Negative and

Gram-Positive Bacteria”, guava leaf extracts was tested for its effectiveness as an

antimicrobial agent. The extraction method was also carried out through maceration

technique (less the drying up of leave). The similarity of their study from the researcher’s

study is, the researcher also used maceration technique to obtain the extract of the subject

which is Mango ( Mangifera indica) leaves. The study used hexane, methanol ,ethanol
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and, water were used as an extracting solvent while this study used ethanol, methanol,

and water.

The study of Bandibas et al., (2017) entitled “Antimicrobial Test of Five

Ethnomedicinal Plants in an Ancestral Forest Area” Ethnomedicinal plants were

extracted using traditional method, by boiling the leaves in tap water, while in this study

the researcher used maceration technique to extract Mango (Mangifera indica) leaves.

The similarity of their study to the researcher’s study is, they used the different subjects

but the same purposes, Bandibas et al., used the leaves of five ethnomedicinal plants:

Crinum asiaticum L., Pavettaindica L., Bauhinia purpurea L., Mollugo pentaphylla L.,

and Cinnamomummercadoi S., while the researchers used Mango (Mangiferaindica) leaf

extract as an antimicrobial and the study afore mentioned used positive control same with

this study.

According to the study entitled “Antibacterial Activity of Extracts of Twelve

Common Medicinal Plants from the Philippines”. conducted by Penecilla et al., (2011),

the twelve medicinal plant extract were obtained using maceration technique and it used

disk diffusion method to measure the susceptibility of plant extracts against the bacterial

cells being tested same methods used in this study And also both studies utilized

controls.

Conceptual Framework

The concept of this study is to determine the antimicrobial capability of Mango

(Mangiferaindica) leaf extract against Staphylococcus aureus. According to the


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information that the researcher gathered, foreign and local studies and literature, proves

that of Mango (Mangiferaindica) leaf extract has an antimicrobial activity against

bacterial cells.

In this study, the researcher used disk diffusion method, which is the most common

procedure in testing the susceptibility of antimicrobials. The researcher used maceration

technique in extracting Mango (Mangifera indica) leaves, 50% ethanol, 50% methanol,

and water were used as an extracting solvent. The researchers aim to determine the if

Mango (Mangiferaindica) leaf extractsreally is capable on inhibiting the growth of S.

aureus.

Paradigm

Mango (Mangiferaindica)
leaf extracts Resistant/Susceptible

Erythromycin Inhibit the growth of


(Control) Staphylococcus aureus

Fig. 1 Paradigm of the study

Figure 1 shows the scope of the the study to determine if Mango (Mangifera

indica) leaf extract can inhibit Staphylococcus aureus. The researchers used Mango
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(Mangifera indica) leaf extract as the experimental variable, while the erythromycin had

been used as a control variable. Maceration and kirby bauer disk diffusion are utilized.

The effectiveness of Mango leaf extract is determined by the zone of inhibition it

produced.

CHAPTER 3

RESEARCH METHODOLOGY

This chapter presents the method and procedure that will employed in this research.

It incorporates the research design, techniques, instruments and data gathering procedure.

Research Design

The study used an experimental type of research design where Mango

(Mangiferaindica)leaf extract was used as an experimental variable and subjected for its

antimicrobial action against S. aureus.

Research Subjects

The researcher used Mango (Mangiferaindica) leaf extract as the main subject and

it was used to test its sensitivity against S. aureus. Mueller Hinton Agar was used to test

the subject’s antimicrobial capability against S. aureus. The researchers used 3 MHA

plates, each plate were used for one trial.

Research Locale

The leaves of Mango (Mangiferaindica) is collected at La Paz, Tarlac and its

extract was subjected for identification tests to prove the presence of the active
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constituents. The ampoule which contains the S. aureus was gathered from the Medical

technology department laboratory, are propagate for culture and sensitivity testing. The

tests was be conducted at the laboratory of Central Luzon Doctors’ Hospital Educational

Institution located in Romulo Highway, San Pablo, Tarlac City.

Research Instruments
Materials Function
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Alcohol lamp for the sterilization of inoculating loop

during the planting of bacterial cells.

Tweesers Used to get the antibiotic in the cartridge

and place it on MHA. Data

Gathering
Analytical balance for weighing of Mango (Mangiferaindica)
Procedure
leaves and reagent for preparing agar plate.

Phase
Beaker Used to contain the pounded mango leaves
1: Collection
soaked in different menstruum.
and
Mueller hinton agar plate serves as a medium for antimicrobial
Preparation
susceptibility testing.
of Plant

Distilled water Used as one of the menstruum. Extract

Dropper for transferring small quantities of liquids. Fresh

Parafilm Used to cover the extract in the beaker to leaf samples

avoid contaminantion. were

collected
Filter paper impregnated with Mango (Mangifera
from the
indica) leaf extract and was placed on a
Mango
Mueller-Hinton (MH) agar plate,
(Mangifera
Funnel used when transferring liquid into a narrow
indica) trees.
mouthed container
Whole leaves

Incubator A device used for incubating the culture of were washed

S. aureus to achieve the bacterial optimum with tap water

growth.

Inoculating loop used to inoculate or plant bacterial cells in

the medium.
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then crush using mortar and pestle. Using beakers, crushed leaves was weighed 10 grams,

then soaked to 50% ethanol, 50% menthanol, and water respectively (1:1). Then covered

with parafilm to avoid evaporation and contamination for 24 hours at room temperature.

After 24 hours of soaking in different menstruum, the mixtures was filtered using filter

paper. The residues was discarded while the filtrates was collected.

Phase 2:Collection of Test Organisms

Clinical isolates of Staphylococcus aureus was collected from The Medical

Technolody Department laboratory. Collected isolates was sub-cultured for 24 hours and

were adjusted to 0.5 McFarland standard.

Phase 3: Authentication of Plant

The researchers went to Central Luzon State University to authenticate the plant

that was used in the study.

Phase 4: Preparation of the Sample Filter Paper Disk

Disks was prepared by the use of puncher to punch the filter paper into pieces.

Each measures 6 mm. The disks were soaked with Mango (Mangiferaindica) leaf extract

for 30 minutes.

Phase 5: Antimicrobial Susceptibility Testing using Disk Diffusion Antibiotic

Sensitivity Method

Inoculation of Plates
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Allow plates to come to room temperature before use.

1. Dip a sterile swab into the inoculum tube.

2. Rotate the swab against the side of the tube (above the fluid level) using firm pressure,

to remove excess fluid. The swab should not be dripping wet.

3. Inoculate the dried surface of a MH agar plate by streaking the swab three times over

the entire agar surface; rotate the plate approximately 60 degrees each time to ensure an

even distribution of the inoculum.

4. Rim the plate with the swab to pick up any excess liquid.

5. Discard the swab into an appropriate container.

6. Leaving the lid slightly ajar, allow the plate to sit at room temperature at least 3 to 5

minutes, but no more than 15 minutes, for the surface of the agar plate to dry before

proceeding to the next step.

Placement of the antibiotic disks

1. Place the appropriate antimicrobial-impregnated disks on the surface of the agar, using

either forceps to dispense each antimicrobial disk one at a time, or a multidisk dispenser

to dispense multiple disks at one time.

a. To use a multidisk dispenser, place the inoculated MH agar plate on a flat

surface and remove the lid.

b. Place the dispenser over the agar plate and firmly press the plunger once to

dispense the disks onto the surface of the plate.


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c. Lift the dispenser off the plate and using forceps sterilized by either cleaning

them with an alcohol pad or flaming them with isopropyl alcohol, touch each disk on the

plate to ensure complete contact with the agar surface. This should be done before

replacing the petri dish lid as static electricity may cause the disks to relocate themselves

on the agar surface or adhere to the lid.

d. Do not move a disk once it has contacted the agar surface even if the disk is not

in the proper location, because some of the drug begins to diffuse immediately upon

contact with the agar.

e. To add disks one at a time to the agar plate using forceps, place the MH plate

on the template. Sterilize the forceps by cleaning them with a sterile alcohol pad and

allowing them to air dry or immersing the forceps in alcohol then igniting.

f. Using the forceps carefully remove one disk from the cartridge.

g. Partially remove the lid of the petri dish. Place the disk on the plate over one of

the dark spots on the template and gently press the disk with the forceps to ensure

complete contact with the agar surface. Replace the lid to minimize exposure of the agar

surface to room air.

h. Continue to place one disk at a time onto the agar surface until all disks have

been placed.

2. Once all disks are in place, replace the lid, invert the plates, and place them in a 35°C

air incubator for 16 to 18 hours. Measure the zone of inhibition.

Interpretation of Results
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 Susceptible – implies that the isolates are inhibited by the usually

achievable concentrations of antimicrobial agent (Mangiferaindicatum leaf

extracts).

 Resistant – implies that isolates are not inhibited by the usually

achieveable concentrations of the antimicrobial agent

(Mangiferaindicatum leaf extracts).

 Intermediate – implies clinical efficacy of drugs are physiologically

concentrated or when bacterial cells respond to a higher than normal

dosage of a drug.

Statistical Treatment

The study in determining the antimicrobial capability of Mango

(Mangiferaindica) leaf extract against S. aureus, the researchers encoded the result in a

tabular form to compare the inhibitory activity of Mango (Mangiferaindica) leaf extract

against S. aureus.