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CHAPTER 1

INTRODUCTION

Background of the Study

In the recent years, the antimicrobial actions have received much attention. This is so because

of the increasing interest in human health and have been studied in vitro and in vivo by many

researchers. The natural antimicrobial agents protect living organisms from damages resulting in

the prevention of various diseases. There is a growing interest in substances exhibiting

antimicrobial properties that are supplied to human and animal organisms as specific

pharmaceutics. Although, much work has been done on the antimicrobial effects of different

plants species. It has been well known that essential oils and plant extracts have antimicrobial

effects.

This kind of Jasminum gathers more than 200 species originating from all the continents. The

species Jasminum sambac, probably originating in tropical India and Burma, is cultivated for its

perfume. Jasminum sambac is a persistent shrub, which often reaches 5 feet height in pots. The

sheets are persistent, green-beds, brilliant and opposite. The flowers are white. The flowers are

used in the manufacture of the perfumes and aromatizing. The plant is used in the form of liana

to decorate the hair and the neck in the form of collar. The traditional use of this plant suggests

analgesic, antidepressant, anti-inflammatory, antiseptic, aphrodisiac, sedative, expectorant and

tonic (uterine) effects. Essential oil of J. sambac is used as fragrance for skin care products.

Jasmine oil and absolute reduce skin inflammation, tones the skin and lifts up your mood.

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Objectives of the Study

General Objective:

The study aims to evaluate the effectiveness of maceration method to draw out

antibacterial property of Jasminum sambac.

Specific Objective:

• To determine the antibacterial activity in terms of average zones of inhibition of

Jasminum sambac extract using maceration method against the following:

Gram (-) bacteria:


1.1 Escherichia coli

Gram (+) bacteria:


1.2 Staphylococcus aureus

• Compare the results of zones of inhibition between the Jasminum sambac extract

in the test organisms.

Null Hypothesis

There is no significant correlation between the antibacterial activity of Jasmine Sambac

extract against selected gram-negative and gram-positive bacteria.

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Significance of the Study

The significance of this study is vital to the doctors, pharmaceutical companies,

researchers, medical students and patients. The result we gain from this study will provide

doctors new information regarding the emergence anti- microbial property that can be observe

from plant or plant materials Jasminum sambac extract thereby promoting the use and

prescription of organic herbal medications rather than of synthetic drugs. Researchers can

significantly benefit from this study since this can somehow provide idea and information

regarding the presence of antibacterial activity of Jasminum sambac extract. They can further

conduct, improve or modify a similar research. The result we gain from this study gives new

ideas to pharmaceutical companies to develop new drugs which is safer, more natural, and cost

efficient not only to consumers but also means financial gain to these companies. This study will

help the medical students provide knowledge with the use and properties of Jasminum sambac

extract. Students can now be able to apply what they learned and use them into practice in the

near future. This research would also promote the use of alternative medicine and become

actively involved in the field of research development. This would increase awareness regarding

the presence and effectiveness of the antibacterial activity of Jasminum sambac plant. Become

involve in promotion of the use of organic and alternative medicine rather than synthetically

prepared medicines which is more expensive, harder to prepare and may cause side effects.

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Scope of Delimitation of the Study

This study focuses in determining the antibacterial activity of In vitro evaluation of crude

extract of Jasminum sambac on E. coli and S. Aureus based on the minimum inhibitory

concentration or (MIC). The effectiveness will be based on the ability of the extract to inhibit the

colonial growth reflected by the zone of inhibition of E. coli and S. areus after the administration

of the extract.

The study will be conducted in the Research and Microbiology laboratory of Remedios

Trinidad Romualdez Medical Foundation upon the preparation of bacteria, its cultivation and

preservation. Jasminum sambac will be obtained within the vicinity of Tacloban City.

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Theoretical Framework

The continuous emergence of antibiotic- resistant microbes has struck researchers

worldwide to discover new antimicrobial agents that are effective against the resistant microbial

pathogens. Introduction of plants with potential therapeutic values have been utilize for

preventing and treating various elements and food borne diseases. Flower extract and essential

oils are also considered to be potential natural antimicrobial agents. Available reports indicate

their efficacy and possess a broad spectrum of antimicrobial activity against various spoilage and

pathogenic microorganisms, which is attributed to their bioactive constituents.

Infectious disease and Food borne


illness causes severe health effects
that can lead to death.

Introduction of antimicrobial
agents derived from plant
materials

Reducing total dependence on antibiotics reducing development of


antibiotic resistance by pathogens microorganisms, controlling cross-
contamination and strengthening immune systems in humans.
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Conceptual Framework

From the information that was gathered from theoretical framework, we came up with a

conceptual framework of our study following the organization of theories. This represents the

predictive relationship among the different variables and how they correlate with each other.

Upon using S. aureus and E. coli this experimentation will provide us with the extract to inhibit

microbial growth. Upon reacting the extract to our prepared microbes it is appropriate in

reducing and inhibiting microorganism’s growth will determine how accurate and effective the

extract would be.

Gram (+) S. aureus


Gram (-) E. coli

J. sambac extract; extracted & was made to inhibit the


growth of E. coli and S. aureus

Culture, experimentation and analysis

Extract’s effectiveness in inhibiting microorganism’s


growth (S. aureus and E. coli)
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CHAPTER II

REVIEW OF RELATED LITERATURE

Based on the article ofCentre for P. G. Studies. Department of Microbiology, Orissa

University of Agriculture and Technology, Bhubaneswar-751 003, India, Infectious diseases

an(Nascimento and others 2000; Thaller and others 2010) foodborne illnesses can cause severe

health effects and can even lead to death among the residing population, especially in the

developing regions of the world. The continual emergence of antibiotic-resistant microorganisms

has prompted researchers’ world over to search for new antimicrobial agents that are more

effective against the resistant microbial pathogens Structural modification of the antimicrobials

(against which microbial resistance has been developed) is reported to improve the effectiveness

of antimicrobial agents against bacteria, fungi, and viruses. However, of late, research efforts

have been put forth to improve the effectiveness of antimicrobial drugs by developing novel and

a new class of antimicrobial drugs that can effectively work on multitargeted sites or organisms.

Traditionally, plants with potential therapeutic or medicinal values have been

successfully utilized for preventing and treating various ailments and foodborne illnesses. Since

time immemorial, various plants and their products have been used in traditional medicine to

cure some of the common disorders and degenerative diseases in humans as well as in animals

(such as Ayurvedic and traditional Chinese medicinal practices). The effectiveness of these

procedures has been attributed mainly to the presence of active phytochemicals or bioactive

compounds in plants.

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Given the scope of searching new antimicrobial agents, antimicrobials derived from plant

materials are often regarded as natural and safe compared to industrial chemicals. Of late, plant-

based medicine has become more popular due to the increasing concern of consumers with

regard to the use of synthetic chemical preparations and use of artificial antimicrobial

preservatives, especially in modern food protection practices.

Some of the hoped-for advantages of using natural antimicrobials include: reducing total

dependence on antibiotics, reducing development of antibiotic resistance by pathogenic

microorganisms, controlling cross-contaminations by foodborne pathogens, improvizing food

preservation technology, and strengthening immune system in humans Today, growing market

trends indicate a rapid increase in the number of natural plant-derived products (such as green

tea, herbal decoctions, or herbal medicines) that may include aerial parts, seeds, fruits, roots,

rhizomes, and flowers. Among these, flowers have attained high priority and found various

applications. Floral extracts and their isolated essential oils are traditionally believed to be rich in

phytochemicals exhibiting rich bioactivity. These compounds are of interest to the local industry

as well as to the general population and are actively being explored for various commercial

applications (such as tea, bakery products, and more). Floral extracts and essential oils are also

considered to be potential natural antimicrobial agents. Available reports indicate their efficacy

and to possess a broad spectrum of antimicrobial activity against various spoilage and pathogenic

microorganisms, which is attributed to their bioactive constituents. Based on these facts, the

present review focuses mainly on providing baseline information on exploring some of the

common and wild (edible and nonedible) flowers possessing potential antimicrobial activities.

The details on these aspects are hopefully expected to be useful for the commercial exploitation

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of flowers to develop natural preservative preparations with applicability in the food and

pharmaceutical industries.

Although it has been reported that Sampaguita has possible anti-microbial activity against

Staphylococcus aureus and Escherichia coli, the active components have not been isolated and

identified. Isolation, purification and characterization of the major bioactive components of

several other plants have been reported. In the isolation, purification and characterization of the

major bioactive components of the rhizomes of Ethlingeria elatior, the sample was prepared by

air drying. The crude extracts of the air dried samples were extracted using DCM as solvent and

isolated using column chromatography (Budoy et al., 2003). The antioxidant compounds of

avocado, was extracted by solvent extraction by using organic and aqueous solvents. The crude

extract from avocado was partitioned between hexane and chloroform. Purification of the

chloroform fraction was done by isocratic and gradient elution column chromatography (De Asis

and Espeso, 2003). Adoption of the above mentioned extraction and isolation methods can be

done to sampaguita to successfully isolate, purify and partially characterize the components

responsible for its antibacterial activity against Staphylococcus aureus and Escherichia..

Maceration became a popular and inexpensive way to get essential oils and other plant

healing properties to the people. With the appearance of pharmaceutical drugs in the late 1880’s,

homemade preparations like macerations fell out of favor. Little was written down and much of

the process was lost. The 1960’s saw a questioning of “modern practices” and a turning to more

natural ways of living. The pharmaceutical industry was held in suspicion and making your own

herbal preparations were revisited. Many looked to the Native Americans and other ancient

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cultures to relearn or regain information about making these preparations. The old ways of

producing essential oils inexpensively were lost, but are now being rediscovered.

Maceration oils, also called infused oils, are carrier oils that have been used as a solvent

to exctract the therapeutic properties of a certain plant or plants. The base oils commonly used

are Olive or Sunflower and the process is quite simple because of microbiological infection

caused by wet herbs infusing in oil, it is recommended to use only properly dried herbs and

flowers for this process.

Pathogens

Escherichia coli is a Gram-negative, facultative anaerobic, rod-shaped bacterium. E.coli

is the most common cause of Urinary Tract Infection.

Staphylococcus aureus is a Gram-positive coccal bacterium that is a member of

Firmicutes, and is frequently found in the nose, respiratory tract and on the skin.

Escherichia coli and Staphylococcus aureus, are among the most prevalent species of

gram-positive and gram-negative bacteria, respectively that induce mastititis an inflammation of

woman’s breast, usually as a result of bacterial infection.

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CHAPTER III

METHODOLOGY

Research Design

This study utilized an experimental research to determine the anti-bacterial activity

present in the macerated extract of Jasminum sambac (Sampaguita) against selected species of

gram (-) and gram (+) bacteria.

Subject of the Study

The study focused on the antibacterial agent present in the macerated extract of

Jasminum sambac. After thorough extraction, antimicrobial susceptibility testing will follow;

utilizing the Kirby-Bauer method or Disc Diffusion method and then measurement of zones of

inhibition will proceed. The investigation used 1 specie of gram (-) and 1 gram (+) pathogens

namely: Escherichia coli and Staphylococcus aureus because both are common pathogens to

human.

Sample Collection and Identification of Plant Material

Jasminum sambac (Sampaguita) is acquired from the sampaguita vendors at Sto. Nino

church of Tacloban City. The plant sample was presented to DENR, Ecosystems Research and

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Development Service Region 8, Tacloban City for verification and certification by Ms. Emma

M. Germano as the Senior Science Research Specialist The collected plants were then washed

under running tap water to remove any soil contaminants.

Extract Preparation

Maceration Method

In the maceration method of extraction, quantities of Jasminum sambac flower materials

were washed with tap water and was dried as possible. The plant material was chopped finely to

break the plant cell walls and encourage more of the plant's oil-soluble compound to infused into

the base oil. It is placed into a sterilized airtight container until the plant material was covered

with olive oil. The dried plant matter and oil in an airtight container such as glass jar was placed

in a warm sunny location for up to three weeks, the sunshine gently heats the oil and extract

many of plant's properties. Every week or so, the macerated flower material were replaced so

that it will continue to extract more therapeutic properties into base oil. This mixture is allowed

to sit for three weeks and agitated and mixed periodically.

After some time has passed the macerated plant material is removed and the oil is filtered

and strained of any remaining plant particles. The resulting oil is now infused with and contains

the plant’s essential oil molecules.

Test Organism

The study utilized gram (-) and gram (+) pathogens as test organisms for the

antimicrobial susceptibility testing. The pure stock cultures of microorganisms were obtained in

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the Laboratory of Doña Remedios Trinidad Romualdez Medical Foundation with permission.

The test organisms include gram (-), Escherichia coli and gram (+), Staphylococcus aureus.

Laboratory Assay

Standardization of Inoculums

The cultured microorganisms were standardized using of densitometer following the 0.5

Mcfarland standard prior to antimicrobial susceptibility testing. The medium to be used for

antimicrobial susceptibility testing was the standard agar based medium, Mueller-Hinton Agar

(MHA).

Preparation of Positive and Negative Control

The antibiotic used as a positive control was Penicillin and Ampicillin. The chosen

controls was based on the recommended antibiotics for the selected microorganisms. For the

negative control, a 6mm-filter paper was soaked in distilled water.

Antibacterial Assay

Antimicrobial susceptibility testing was done utilizing the Kirby-Bauer method following

the extraction. 6mm-filter paper discs were soaked in the base oil of Jasminum sambac

(Sampaguita) prior to the testing. A total of 3 MHA plates per bacteria were used to occupy 3 of

the soaked discs per MHA plate leaving a total of 9 tests/discs for each plant extract. After

placing of the discs accordingly, the plates were incubated at 37C for 24 hours. Positive and

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negative control per test bacteria were incubated together with the test sample. Observation and

measurement of zones of inhibition followed.

Statistical Analyses

After the antibacterial assay, the mean the zone of inhibition were calculated. To check

the variance of the data gathered, the standard deviation from the means of each sample trial was

calculated. And from this calculation, a two-tailed independent group t-test was followed to

analyze and check the differences of data gathered from the antibacterial assay which includes

the test, negative and positive control. The choice of test also involves the disparity between the

postive control and negative control when compared to test. Formula and calculations of data are

found in the appendix D.

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Chapter IV

Results & Discussion

Result:

Table 1: Average zone of ohobition of Macerated oil extract from Jasminum sambac

(Sampaguita) Against selected Gram (-) and Gram (+) Pathogens

Extracts Average Zone of Inhibition Average Zone of Inhibition

against Echerichia coli against Staphylococcus

aureus

Macerated extract 6mm 6mm

Positive control 32mm 30mm

Negative control 6mm 6mm

(Value of 6mm is the diameter of disc used which is equal to zero of inhibition)

Table 1 presents the result of the Jasminum sambac macerated extract against gram

negative & gram positive test organisms based on their average zone of inhibition together with

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the positive control Ampicillin (gram +) & penicillin (gram -) and negative control (distilled

water). It was observed that both Escherichia coli & Staphylococcus aureus showed no

difference with mean zone of inhibition of 6mm on macerated oil extract. In the both positive

control (penicillin & ampicillin) showed an expected value of zone of inhibition.

Table 2: Summary of computed t-value of test organisms between the macerated extract of

Jasminum sambac & negative control

Average zone
of inhibition
Category Test (-)control SD DF Tabular Computed Interpretation
(distilled
water) (Test) Value t-value

A 6 6 0 58 2.0017 0 NS

B 6 6 0 58 2.0017 0 NS

Where:
A= Comparison between macerated extract and the negative control using S. aureus
B= Comparison between macerated extract and the negative control using E. coli
S = Significant
NS= Not Significant

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Table 3: Summary of computed t-value of test organisms between the macerated extract of

Jasminum sambac & positive control (Ampicillin & Penicillin)

Average zone of
inhibition
Category Test (+) control SD DF Tabular Computed Interpretation
(Ampicillin
& Penicillin) (Test) Value t-value

A 6 30 0 58 2.0017 0 NS

B 6 32 0 58 2.0017 0 NS

Where:
A= Comparison between macerated extract and the positive control (Ampicillin) using S. aureus
B= Comparison between macerated extract and the positive control (Penicillin) using E. coli
S = Significant
NS= Not Significant

Table 2 & 3 summarizes the comparison between the macerated extract of Jasminum

sambac against Negative & positive control using test organisms. Using two-tailed paired t-test,

it was determined that their was no significant difference between the computed t-values of

macerated extract against our positive controls (Ampicillin & Penicillin) as seen in the table.

Compared to the positive & negative control the mean zone of inhibition using macerated extract

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on both E.coli & S.aureus yielded a computed t-value of zero. Since these computed t-values are

zero, therefore the results are considered not significant. Further, it implies that our macerated

extract has no potential to inhibit the growth of E.coli & S.aureus.

Discussion:

The sampaguita flowers bloom either singly or as bundles of blossoms at the top of the

branches. Blooming all through the year, sampaguita are pure white, small, dainty, star-shaped

blossoms. The flowers open at night and wilt less than a day.

Sampaguita’s distinct sweet, heady fragnance is its unique feature. The essential oil from

the flowers is similar jasmine (Jasminum grandiflores). Sampaguita flowers do not bear seeds,

therefore the plant is cultivated by cuttings. Sampaguita was imported into the Philippines in 7th

century from Himalayan areas. The Sampaguita is a native part of the Philippine landscape for

centuries. The plant is originally from India and is grown throughout India today. About eight

cultivars are generally listed for Sampaguita. Some varieties Sampaguita can grow as large as

small roses in India.

Jasminum sambac (Sampaguita) has many medicinal properties like anti-depressant,

antiseptic, cicatrisant, aphrodisiac, expectoral, anti-spasmodic, galactogogue, sedative,

parturient, uterine etc. The Jasminum sambac is used for removing intestinal worms and is also

used for jaundice and venereal diseases. The flower buds are useful a treating ulcers, vesicle,

boils, skin disease and eye disorders. The leaves extracts against breast tumours. The leaves are

antiseptic and are useful for wounds and acne when used as a poultice. Drinking Jasmine tea

regularly helps in curing cancer.

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The dried flowers of Jasminum sambac are used by the Chinese to flavour Jasmine tea.

Jasmine tea is most commonly consumed with or after meals as a digestive aid. It aslo used for

making perfumes, creams, shampoos, soaps and incense. Its flowers are used to flavour Jasmine

tea and other herbal or black tea.

According to the study of Phytochemicals of Jasminum sambac it yielded alkaloids, glycoside,

flavonoid, terpines, tannin, resin and salicylic acid. Study showed all extracts with antimicrobial

activity against pathogen, scoring highest with S.typhi and lowest with S.aureus. the study

supports its traditional use for infections.

The test organisms utilized in this study, Escherichia coli and Staphylococcus aureus,are

among the most prevalent species of gram-positive and gram-negative bacteria, respectively that

induce mastititis.

Thus, this present study aims to evaluate if there is antimicrobial activity exhibited by

the macerated extract of Jasminum sambac. The extraction of said compounds can only be done

by using the right method as the components of phytochemicals can only be extracted upon the

solubility and polarity of the right materials used.

After that thorough extraction, testing antimicrobial property followed which utilized the

modified Kirby-Bauer method or disc diffusion method. A standard 6-mm whatmann filter paper

disc were soaked in the extract and the other for the negative control were soaked in distilled

water. Penicillin and Ampicillin disc was the choice of the antibiotic as the positive control. The

test were inoculated on to the surface of the Mueller-Hinton Agar followed by the addition of

negative control, positive control and the sterile paper disc containing the plant extract. It was

incubated for 24 hours at 37C. The inhibition of microbial growth was indicated by a clear area

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(zone of inhibition) around the disc. The zone of inhibition was measured to its diameter which

is expressed in millimeter.

The utilization of standard analytical quantization technique was employed to obtain their

statistical values and somehow to determine their significant difference between the mean of

macerated extract, positive and negative control based on the zone of inhibition. The one-sample

independent t-test was utilized, a summarized in Tables 2, 3, and 4, respectively. It showed that

all result from using macerated Jasminum sambac extract as antibacterial have statistical

similarity when compared to negative control. This means that macerated Jasminum sambac

extract do not possess potent antibacterial properties against the two test organisms used in the

study. However, upon comparing the test result against that of the positive control (Ampicillin

and Penicillin), a high statistical difference was conveyed, therefore the macerated Jasminum

sambac extract is not potent to be used as antibiotic to Escherichia coli and Staphylococcus

aureus.

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CHAPTER V

CONCLUSION AND RECOMMENDATION

Conclusions

Based on the zone of inhibition obtained using maceration method for the extraction of

Jasminum sambac (Sampaguita) against the gram negative and gram positive test organisms, it

was found out that Escherichia coli and Staphylococcus aureus exhibited no average zone of

inhibition of 6mm.

Among the test organisms that were used, it was found out that Jasminum sambac extract

showed no bactericidal effect on both Staphyloccocus aureus and Escherichia coli in terms on the

averages of zone of inhibition observed.

Recommendations

The researchers recommend that further test on antimicrobial property from Jasminum

sambac extract to be conducted using other pathogenic bactteria.

It is also highly recommended exploring other means of extraction that would enable the

extract to express its beneficial property at its best and to properly isolate the active components

of the plant with antibacterial properties.

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Bibliography

Betty A. Forbes, Daniel F. Sahm, Alice S. Weissfel. Bailey and Scott’s Diagnostic Microbiology,
Twelfth Edition, Morsby Publishing. P157
Hasina Yasmi1, Md. Abdul Kaisar2, Md. MoklesurRahman Sarker3, Mohammed Shakifur
Rahmman4 and Mohammad A. Rashid (2009). Preliminary anti-bacterial activity of some
indigenous plants of Bangladesh, dhak. Univ. J. Pharm. Sci. 8(1):61-65.
John Bernard Henr, M.D. Clinical Diagnosis and Management by Laboratory Methods,
Nineteenth Edition. W.B SAUNDERS COMPANY. Part 6, Medical Microbiology,
p. 1152-1153
Nimri, LF; Meqdam, MM and Alkofahi, A (1999). Antibacterial activity of Jordanian medicinal
plants. Pharmacologycal Biology. Vol. 37(3), 196-201

Websources:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792499/

https://en.m.wikipedia.org/wiki/steam_distillation - Steam Distillation

https://en.m.wikipedia.org/wiki/Minimum_inhibitory_concentration-Minimum-inhibitory
concentration

https://cvi.asm.org/content/11/3/463.full

https://www.stuartxchange.com/Sampaguita

https://everything-lavender.com/methods-of-producing-essential-oils.h

https://www.airase.com/extraction-methods-for-essential-oils/

https://www.ethnoleaflets.com/leaflets/naveen.html

https://www.naha.org/explore-aromatherapy/about-aromatherapy/how-are-essential-oils-

extracted

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APPENDICES A

MEASUREMENT OF PERFORMED TRIALS

Escherichia coli

TRIAL JASMINE POSITIVE NEGATIVE

MACERATED OIL CONTROL CONTROL

EXTRACT
(Penicillin)

1 6mm 32mm 6mm

2 6mm 32mm 6mm

3 6mm 32mm 6mm

AVERAGE 6mm 32mm 6mm

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Staphylococcus aureus

TRIAL JASMINE POSITIVE CONTROL NEGATIVE CONTROL


MACERATED OIL (Ampicillin)
EXTRACT

1 6mm 10mm 6mm

2 6mm 10mm 6mm

3 6mm 10mm 6mm

AVERAGE 6mm 10mm 6mm

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APPENDICES B

Model for hypothesis testing using one-sample, independent groups t-test

Step 1: Hypothesis
𝑯𝒐 : 𝑿𝟏 = 𝑿𝟐 (There is no significant difference in the average zones of inhibition
between the test organisms)
𝑯𝑨 : 𝑿𝟏 ≠ 𝑿𝟐 (There is a significant difference in the average zones of inhibition
between the test organisms)

Step 2: Test Statistics: two-tallied paired t-test

Step 3: Value of α:0.05

Step 4: Critical Value: These is derived from table of critical values. The critical value of t-
distribution 3+3-2 degrees of freedom is 2.000

Step 5: Formulas:

𝟐
∑(𝑿 − 𝑿𝟐 )𝟐
𝑺 =
𝒏−𝟏

𝑿𝟏 − 𝑿𝟐
𝒕=
(𝒏𝟏− 𝟏)𝒔𝟏 𝟐 𝒏𝟏+𝒏𝟐
√[ 𝒏𝟏+𝒏𝟐 −𝟐
][
𝒏𝟏𝒏𝟐
]

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Legend:
𝑿𝟏 = mean of sample 1
𝑿𝟐= mean of sample 2
𝒏𝟏= number of subjects in sample one
𝒏𝟐= number of subjects of sample two
𝒔𝟏= standard deviation of sample one
𝒔𝟐= standard deviation of sample two

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APPENDICES C

CALCULATION
I. Variance
Jasminum sambac Macerated extract (mm)

Escherichia coli Staphylococcus Negative Positive control


TRIALS aureus control

(𝑿𝟏 − 𝑿𝟐 ) (𝑿𝟏 − 𝑿𝟐 )
E.coli S.aureus
X X

1 6 0 6 0 6 32 30

2 6 0 6 0 6 32 30

3 6 0 6 0 6 32 30

𝑋1 6 0 6 0 6 32 30

∑ 0 0 0 0 0

𝑆2 0 0 0 0 0

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I. Variance/ Standard Deviation
A. Macerated extract using Escherichia coli
∑(𝑋1− 𝑋2 )2 0 0
𝑆2 = = = =0
𝑁−1 3−1 2
B.Maceration extract using Staphylococcus aureus
∑(𝑋1− 𝑋2 )2 0 0
𝑆2 = = = =0
𝑁−1 3−1 2
C.Positive Control (Penicillin & Ampicillin)
∑(𝑋1− 𝑋2 )2 0 0
𝑆2 = = = =0
𝑁−1 3−1 2

D. Negative Control (Distilled water)

2
∑(𝑋1− 𝑋2 )2 0 0
𝑆 = = = =0
𝑁−1 3−1 2
II. T-test Calculations
Escherichia coli
Sample x (Zone of inhibition) ∑(𝑋1− 𝑋2 )2 𝑆2

Macerated extract 6 0 0
Positive Control 32 0 0
Negative Control 6 0 0

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A. Macerated extract (𝑿𝟏 )vs Negative Control (𝑿𝟐 )

𝑋1 − 𝑋2
𝑡=
(𝑛1 −1)𝑠1 2 (𝑛1− 1)𝑠2 2 𝑛1+𝑛2
√[ ][ ]
𝑛1+𝑛2 −2 𝑛1𝑛2

6−6
𝑡=
(3−1)0(3−1)0 3+3
√[ ][ ]
3+3−2 (3)(3)

0
𝑡= 0 6
=0
√[ ][ ]
4 9

B. Positive Control (𝑿𝟏 ) vs Macerated extract (𝑿𝟐 )

𝑋1 − 𝑋2
𝑡=
(𝑛1 −1)𝑠1 2 (𝑛1− 1)𝑠2 2 𝑛1+𝑛2
√[ ][ ]
𝑛1+𝑛2 −2 𝑛1𝑛2

32 − 6
𝑡=
(3−1)0(3−1)0 3+3
√[ ][ ]
3+3−2 (3)(3)

26
𝑡= 0 6
=0
√[ ][ ]
4 9

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Staphylococcus aureus
Sample x (Zone of inhibition) ∑(𝑋1− 𝑋2 )2 𝑆2

Macerated extract 6 0 0
Positive Control 10 0 0
Negative Control 6 0 0

A. Macerated extract (𝑿𝟏 )vs Negative Control (𝑿𝟐 )

𝑋1 − 𝑋2
𝑡=
(𝑛1 −1)𝑠1 2 (𝑛1− 1)𝑠2 2 𝑛1+𝑛2
√[ ][ ]
𝑛1+𝑛2 −2 𝑛1𝑛2

6−6
𝑡=
(3−1)0(3−1)0 3+3
√[ ][ ]
3+3−2 (3)(3)

0
𝑡= 0 6
=0
√[ ][ ]
4 9

30
B. Positive Control (𝑿𝟏 ) vs Macerated extract (𝑿𝟐 )

𝑋1 − 𝑋2
𝑡=
(𝑛1 −1)𝑠1 2 (𝑛1− 1)𝑠2 2 𝑛1+𝑛2
√[ ][ ]
𝑛1+𝑛2 −2 𝑛1𝑛2

10 − 6
𝑡=
(3−1)0(3−1)0 3+3
√[ ][ ]
3+3−2 (3)(3)

4
𝑡= 0 6
=0
√[ ][ ]
4 9

APPENDIX D
COMMUNICATION LETTERS

31
APPENDIX E
DOCUMENTATION

32