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The Antibacterial Activity of

Wiregrass (Eleusine Indica) and


Pandanus specie leaves extract against
Escherichia Coli

S C I E N C E INVESTIGATORY PROJECT

EMALYN F. PUNZALAN
Researcher

Colegio de la Purisima Concepcion


The Antibacterial Activity of Wiregrass (Eleusine Indica) and

Pandanus specie leaves extract against Escherichia Coli

Emalyn F. Punzalan

Researcher

Abstract

This study aimed to determine the antibacterial activity of

Wiregrass (Eleusine Indica) and Pandanus specie leaves extract

against Escherichia Coli.

This study was conducted to create an affordable and easy

access antibacterial solution that would fight against

Escherichia Coli.

The researcher has four treatments in four replicates;

Treatment A: 100% Pandanus specie leaves extract; Treatment B:

100% Wiregrass leaves extract; Treatment C: 50% Pandanus specie

and 50% Wiregrass leaves extract; and Treatment D: positive

control (chloramphenicol).

Specifically, this study aims to find out if there is a

significant difference in the mean zones of inhibition using the

different concentrations of Wiregrass and Pandanus specie leaves

extract against Escherichia Coli.


At the end of the study, the researcher concludes that

Wiregrass and Pandanus specie leaves extract have an

antibacterial activity against Escherichia Coli.


RESEARCH PLAN

A. Tools and Materials

Tools:

Incubator, mortar and pestle, test tube, test tube brush,

cork, beaker, glass funnel, forceps, filter paper, tong, alcohol

lamp, forceps, refrigerator and agar plates.

Materials:

Wiregrass, Pandanus specie, pure cultured Escherichia Coli,

positive control (chloramphenicol), cotton, masking tape,

marking pen, laboratory gown, mask, and gloves.

B. General Procedure

1. Extraction Procedure

Wash and air-dry collected Wiregrass and Pandanus

specie leaves. The leaves are then pound using mortar and

pestle. Using chest cloth, the pounded leaves are squeeze

to produce extracts. These extracts are then filtered

using beaker, filter paper and glass funnel. The filtered

extracts are placed in a test tube covered with cork.

2. Preparation of Muller-Hinton agar plates

38 grams of dehydrated Muller-Hinton agar was placed

in a weighing scale with aluminum foil and transferred in an

Erlenmeyer flask. It was rehydrated with 1000 ml distilled water

and heated in a double boiler to melt. The melted medium was


sterilized in the autoclave at 121oC for 15 min at 15 psi (pounds

per square inch). The sterile medium was allowed to cool at

temperature between 40-45oC and transferred into sterile petri

plates at 20 ml each. The plates were allowed to cool and harden

and were set aside ready for use.

3. Standardization of bacterial culture of Escherichia Coli.

Pure cultured Escherichia Coli were aseptically inoculated from

Capiz Memorial Provincial Hospital (CMPH). Agar plates were

prepared where the bacteria will inhibit and grow. Using a

pasteur pipette, colony of Escherichia coli was place in a test

tube with distilled water ensuring that the water shall be

turbid. With a sterile cotton rod, the four replicates of the

Muller-Hinton agar plates as test plates were heavily streaked

with the bacterial suspension of Escherichia coli.

4. Bio-assay Proper

Muller-Hinton agar plates at test plates in four

replicates were divided into four parts each by means of a

marking pen and respectively labeled.

The four replicates of the streaked Muller-Hinton agar

plates as test plates were added with the different levels of

concentration of Wiregrass and ---- which were aseptically and

carefully placed at the center of its quadrant in the plate by

means of a sterile thump forcep. These plates were allowed to

stay at room temperature for 24 hours to give time for the


extracts to stick to the agar surface. These plates were then

secured properly with a masking tape and incubated at 400C.

After an overnight incubation, the plates were inspected

for the formation of the zones of inhibition. The diameter of

the zone was then measured by means of a ruler and the average

of the data was taken from the data in the y and x axis of the

zone. The data were plotted in the tabulated form and treated

statistically to determine the sensitivity of the plant extract

to Escherichia coli.
C. Procedural Design

Gathering of leaves, tools, and materials

Washing and air-drying of leaves, tools, and


materials

Preparation of

Treatments Muller-Hinton agar


plates

Treatment A: 100 %(PANDANUS) Bacterial culture of


Escherichia Coli
Treatment B: 100 %(WIREGRASS)

Treatment C: 50 %( PANDANUS)
and 50% (BAKONG) Incubation of the planted
bacteria
Treatment D: Positive Control

Application of different treatments on


cultured Escherichia Coli

Evaluation and gathering of data

Chapter I of collected data


Analysis and interpretation
INTRODUCTION

A. Background of the study

The use of chemicals and antibiotic drugs in animal

production both for exporting and local consumptions had created

the rug resistance bacteria. It was well known that the drug

resistance properties were transferable to other bacteria. Also,

drug resistance bacteria may enter the human via food from the

animal origins. (Noppins, et al., 2011)

For a long period of time, plants have been valuable source

of natural products for maintaining human health, especially in

the last decade, with more intensive studies for natural

therapies. The use of plant compounds for pharmaceutical

purposes has gradually increased in Brazil according to the

World Health Organization. (Santos, 1995)

Medical plants would be the best source to obtain a variety

of drugs. About 80% of individuals from developed countries use

traditional medicines, which compounds derived medicinal.

Therefore, such plants should be investigated to better

understand their properties, safety, and efficiency. (Elof,

1998)

The use of plant extracts and phytochemicals, both with known

antibacterial properties, can be great significance to


therapeutic treatments. In the last few years, a number of

studies have been conducted in different countries to prove such

efficiency. Many plants have been used because of their

antimicrobial traits, which are due to compound synthesized in

the secondary metabolism of plants. These products are known by

their active sustances, for example, the phenolic compounds

which are part of th essential oils, as well as in tannin.

(Saxena, 1994).

B. Statement of the problem

Generally, this study aims to determine if Wiregrass

and Pandanus specie leaves extract have an antibacterial

activity against Escherichia Coli.

Specifically, this study aims to answer this

question:

a.) Is there a significant difference in the mean zones of

inhibition using; Treatment A: 100% Pandanus specie

leaves extract; Treatment B: 100% Wiregrass leaves

extract; Treatment C: 50% Wiregrass leaves extract and

50% Pandanus specie leaves extract; and Treatment D:

Positive Control (chloramphenicol) against Escherichia

Coli.
C. Statement of the Hypothesis

a.) There is no antibacterial activity against Escherichia

Coli using the different treatments of Wiregrass and

Pandanus specie leaves extract.

b.) There is no significant difference in the mean zones

of inhibition using; Treatment A: 100% Wiregrass

leaves extract; Treatment B: 100% Pandanus specie

leaves extract; Treatment C: 50% Wiregrass leaves

extract and 50% Pandanus specie leaves extract; and

Treatment D: Positive Control (chloramphenicol)

against Escherichia Coli.

D. Objectives

General Objective:

Generally, this study aims to find out if Wiregrass

and Pandanus specie leaves extract have an antibacterial

activity against Escherichia Coli.

Specific Objective:

Specifically, this study aims to find out if there a

significant difference in the mean zones of inhibition


using; Treatment A: 100% Wiregrass leaves extract;

Treatment B: 100% Pandanus specie leaves extract; Treatment

C: 50% Wiregrass leaves extract and 50% Pandanus specie

leaves extract; and Treatment D: Positive Control

(chloramphenicol) against Escherichia Coli.

E. Significance of the Study

The study was conducted to create an antibacterial

solution that will fight against Escherichia Coli. Besides,

these abundant plants in the community will also serve as an

affordable and easy access natural antibiotic for the

different health illnesses caused by Escherichia Coli and

would be beneficial to those people living in the rural areas.

F. Scope and Limitation

This study is only limited in utilizing Wiregrass and

Pandanus specie leaves extract against Escherichia Coli.

Furthermore, the study will only utilize for treatments;

Treatment A: Treatment A: 100% Pandanus specie leaves extract;

Treatment B: 100% Wiregrass leaves extract; Treatment C: 50%


Wiregrass leaves extract and 50% Pandanus specie leaves extract;

and Treatment D: Positive Control (chloramphenicol).

To find out the antibacterial activity of Wiregrass and

Pandanus specie leaves extracts against Escherichia Coli, the

zones of inhibition were measured using a ruler and

Antimicrobial Indices (AI) were computed to get the average

diameter of the mean zones of inhibition. One-Way Analysis of

Variance (ANOVA) was used to identify the significant difference

among the different treatments.

G. Definition of terms

 Wiregrass- Wiregrass, also called pineland three awn, is

one of the most common grasses in the southern pine flat

woods and upland sand hills.

- In this study, it refers to one of the plant extract

applied on Escherichia Coli

 Pandanus specie

 Escherichia Coli- is a Gram-negative, facultative,

anaerobic, rod-shaped bacterium of the

genus Escherichia that is commonly found in the

lower intestine of warm-blooded organisms (endotherms).

- In this study, it refers to the experimental bacteria


 Chloramphenicol- is an antibiotic useful for the treatment

of a number of bacterial infections.

- In this study, it refers to the positive control

 Incubator- is a device used to grow and

maintain microbiological cultures or cell cultures.

- In this study, it refers to the machine where the

Escherichia Coli were cultured

 Agar plates- is a Petri dish that contains a growth

medium (typically agar plus nutrients) used to culture

microorganisms or small plants.

- In this study, it refers to the plates where the

bacteria were planted

 Muller-Hinton agar - is a microbiological growth

medium that is commonly used for antibiotic

susceptibility testing.

- In this study, it refers to the type of agar used in

the test plates

H. Review of related literature

Wiregrass

Wiregrass, also called pineland threeawn, is one of the most

common grasses in the southern pine flatwoods and upland

sandhills. Grass Family (Poaceae). Pineland threeawn is a


cool season, perennial bunch grass native to the U.S. The height

is between 1 1/2 and 2 1/2 feet. The leaf blade is mostly basal;

12 to 20 inches long; narrow; rolled inward; wiry; and hairy on

upper side at base. The ligule is hairy. The seed head is a

slender panicle 10 to 12 inches long; glumes have 3 awns about

1/2 to 3/4 inch long, one a little longer than other two.

Escherichia Coli

Escherichia coli is a Gram-negative, facultatively

anaerobic, rod-shaped bacterium of the genus Escherichia that is

commonly found in the lower intestine of warm-blooded organisms

(endotherms). Most E. coli strains are harmless, but

some serotypes can cause serious food poisoning in their hosts,

and are occasionally responsible for product recalls due to food

contamination. The harmless strains are part of the normal

flora of the gut, and can benefit their hosts by

producing vitamin K2, and preventing colonization of the

intestine with pathogenic bacteria.

E. coli and other facultative anaerobes constitute about

0.1% of gut flora, and fecal–oral transmission is the major

route through which pathogenic strains of the bacterium cause

disease. Cells are able to survive outside the body for a

limited amount of time, which makes them ideal indicator

organisms to test environmental samples for fecal contamination.


There is, however, a growing body of research that has examined

environmentally persistent E. coli which can survive for

extended periods outside of the host

The bacterium can be grown easily and inexpensively in a

laboratory setting, and has been intensively investigated for

over 60 years. E. coli is the most widely

studied prokaryotic model organism, and an important species in

the fields of biotechnology and microbiology, where it has

served as the host organism for the majority of work

with recombinant DNA. Under favorable conditions it takes only

20 minutes to reproduce.

(put diseases caused by E coli.)

Pandanus specie

-
Results and Discussions

Table 1. Inhibitory Effect of the different concentrations of

Wiregrass and Pandanus specie leaves extract against Escherichia

Coli.

Treatment Zone of Inhibition in millimeter (mm)

R1 R2 R3 R4 Mean

A- 12mm 20mm 17mm 10mm 14.75

B- 27mm 18mm 30mm 25mm 25

C- 25mm 27mm 32mm 25mm 27.25

D- 20mm 15mm 25mm 15mm 18.75

Results showed that the Treatment C got the highest mean of

27.25mm, followed by Treatment B with the mean of 25mm,

Treatment D with the mean of 18.75mm, and Treatment A with a

mean of 14.75mm.
Table 2. One way Analysis of Variance for the four different

treatments of Wiregrass and ------ based on their inhibitory

effect. (ANOVA Table)

Sum of

Squares df Mean Square F Sig.

Between
235.250 3 78.417 2.865 .081
Groups
Within
328.500 12 27.375
Groups

Total 563.750 15

ANOVA shows the summary of the different zones of

inhibition of Wiregrass and Pandanus specie against Escherichia

Coli. The null hypothesis is rejected and the difference lies

between the positive control and all the treatments.

Result shows that the Wiregrass and Pandanus specie leaves

extract have an antibacterial activity against Escherichia Coli.


CONCLUSION

Based on the experimentation performed, the researcher therefore

concludes that:

1. The different concentrations of Wiregrass and Pandanus

specie leaves extract have an antibacterial activity

against Escherichia Coli.

The researcher further concludes that:

1. There is no significant difference in the mean zones of

inhibition using; Treatment A: 100% Wiregrass leaves

extract; Treatment B: 100% Pandanus specie leaves extract;

Treatment C: 50% Wiregrass leaves extract and 50% Pandanus

specie leaves extract; and Treatment D: Positive Control

(chloramphenicol) against Escherichia Coli.

RECOMMENDATIONS

Based on the findings and conclusions, the following

recommendations are given:

1. Further studies about the importance and antibacterial

activity of the other parts of Wiregrass and Pandanus

specie.

2. Further studies about the antibacterial activity of

Wiregrass and Pandanus specie against other bacteria.


3. Further studies should be made on the antibacterial

properties of Wiregrass and Pandanus specie.

BIBLIOGRAPHY

A. Books

Santos, 1995

Elof, 1998

Saxena, 1994
APPENDICES

A. Plates

Gathering of Wiregrass and --------- leaves, tools, and

materials

Cutting of the leaves for pounding


Pounding of the leaves

Preparation of the Muller-Hinton agar plates


Bacteria culture of Escherichia Coli

Application of the different treatments on the cultured

Escherichia Coli
Incubation of the agar plates
B. Time Table

DATE TASK REMARKS


April 18, 2014 C o n ducted The task was don e
r e s earch about
t h e recent
p r o blems in the
e n v ironment
May 26, 2014 P r o ject proposal The task was don e
June 18, 2014 T h e project title
w a s finalized and
c o n structed
J u n e 2 1-2 2 , 2 0 1 4 C o n struction of The task was don e
the Review of the
R e l ated
L i t erature
J u l y 9- 1 1 C o n struction of The task was don e
the Background of
the Study,
O b j ectives,
S t a tement of the
P r o blem and
H y p othesis
August 8, 2014 C o n struction of The task was don e
t h e Research Plan
August 9, 2014 F i n alization of The task was don e
t h e Research Plan

C o n struction of
t h e Significance
o f the Study,
S c o pe and
L i m itations and
D e f inition of
T e r ms
A u g u s t 1 4, 2 0 1 4 C o n sultation of The task was done
t h e project at
C a p iz Memorial
P r o vincial
H o s pital
August 15, 2014 P r e paration of The task was don e
t h e materials for
the
e x p erimentation
August 16, 2014 P r o ject The task was don e
e x p erimentation
a t Capiz Memorial
P r o vincial
H o s pital
A u g u s t 1 7- 1 8 , The task was don e
M e a sured the
2014
z o n es of
i n h ibition and
g a t hering of data

D i s posal of the
b a c teria
A u g u s t 2 3- 2 4 , A n a lysis and The task was don e
2014 I n t erpretation of
c o l lected data
September 4 , C o n struction of The task was don e
2014 t h e Conclusion,
R e c ommendations,
a n d Bibliography
September 10 , C o n struction of The task was don e
2014 t h e Abstact and
F i n alization of
t h e Study
September 16, F i n al write - ups The task was don e
2014 o f the std was
c o m pletely
f i n alized
A CKNOWLEDGEMENT

The researcher would like extend her heartfelt

g r a t i t u d e a n d s i n c e r e appreciation to the following:

To Almighty God, for his guidance, bless ings,

protection during the r esearch expe rimentation and

d iv ine a s s i s t a n c e i n t he conduct of th i s study.

To Mrs. Elena Fr io , Scienc e and Technol ogy Class

teacher and adviser for her comments, suggest i on and

encouragement.

To Ms. Apr il An isco , for h er ass istance regard ing

t h e c o m p u t a tio n s n e c e s sary in the researcher s ' study.

To Mr. Normandy Balasa, for giving his extra effort and

time in reviewing and editing the manuscript.

To Mr. and Mrs. Eemmanuel F. Punzalan Sr., for their moral,

love and financial support. They are her inspiration in making

her study.

To their very supportive classmates and dearest

f r i e n d s f o r t h e i r e n c ouragement.

And to the r esearcher s ’ fam ily, for th e ir never

e n ding love and support: morally, sp ir itually and

f ina n ci ally and to those who extended the ir hel pin

m a king t h i s r e s e a r c h poss ible.