Republic of the Philippines

Sultan Kudarat Polytechnic State College

Science Laboratory High School

ACCESS, EJC Montilla, Tacurong City

This study

Antimicrobial Activity of Bird’s Nest Fern

Extract Against Escherichia Coli

is presented to Division Science Fair 2009.

RESEARCHERS:

Breylle Bepitel

Earl Adam Prado

Rona Angelique Manuel

RESEARCHER ADVISER:

Prof. Merlyn Leysa

September 2009
 Dedication

This humble work is dedicated to all

Who have committed themselves

To the improvement

Of the life of our people.

 ACKNOWLEDGEMENT

The researchers recognize with the gratitude and appreciation their

indebtness to the following persons and institutions who have made valuable
contribution to the successful completion of this study:

To the Prof. Merlyn Leysa, our researcher adviser for her time, for
the guidance, patience in checking drafts, encouragement and for giving
enough consideration;

To the SKPSC faculty and staff and Dr. Herminia Dumadag for the
untiring support extended to us;

To the SKPSC Research Center , who provided us the materials and

equipment used in the conduct of this study;

To our classmates and friends who provided from the companionship

and inspiration for the completion of this study;

To our beloved parents who are supporting us for helping and

providing all we need and;

Most of all, God, who has endowed the researchers with intellectual
gifts and strong faith which sustained them through the different stages of
their research work.

The Researchers
 ABSTRACT

Breylle Bepitel, Earl Adam Prado, and Rona Angelique Manuel. A

project design “Antimicrobial Activity of Bird’s Nest Fern Extract
Against Escherichia Coli”. Sultan Kudarat Polytechnic State College-
Laboratory High School, Research Center , EJC Montilla, Tacurong City .
September 2009.

Advisers: Prof. Merlyn Leysa.

The study Antimicrobial Activity of Bird’s Nest Ferns Extract against

Escherichia Coli” generally aimed to determine the potential of bird’s nest
fern as an antibiotic for Escherichia Coli.

Specifically the study aimed to:

1. Determine the sensitivity of E. coli to bird’s nest fern extract

through Kirby-Bauer antibiotic testing.
2. Determine the treatment where the specimens of bacteria are
most sensitive to
3. Determine the significant differences among treatments and
replications conducted.
The major findings of this study were as follows:

1. Treatment 1(pure birds nest extract) got a general mean of its ZI

with 59.45 mm while Treatment 2 (5 ml bird’s nest extract + 5ml of
sterilized water) got a general mean of 0.67mm. The control
cefuroxime got a general mean of 51.11mm.
 2. Escherichia coli showed high sensitivity when exposed to pure bird’s
nest fern extract.

3. It is therefore found out that the treatment 1 is where Escherichia

coli is most sensitive to.

4. Based on the Analysis of Variance, treatments have significant

differences among each other in all trials conducted, while the
replicates and trials are not significant.

After the 24-hour incubation, the diameter of the zone of inhibition of

E. coli were determined as positive or negative to be sensitive to bird’s nest
fern.

Based on the findings of the test conducted, bacteria E. coli is

favorably sensitive to treatment 1, which is composed of pure bird’s nest
extract from its leaves as it were remarked sensitive. The treatments showed
highly significant differences while the replicates and trials are very precise
and showed not much significance. Likewise, the comparison between the
control group and the treatments used cannot be directly measured since
the control (cefuroxime) is manufactured considering the dosage for human
consumption while bird’s nest fern extract is made to combat E. coli in direct
contact with the bacteria itself.

Nevertheless, the study generally proved the great potential of

Bird’s Nest fern extract to be the radical antidote for the threatening public
health nightmares, the E. coli.
 TABLE OF CONTENTS

TITLE PAGE ………………………………………………………………………….i

Dedication ……………………………………………………………………………..ii

Acknowledgement……………………………………………………………………..iii

Abstract …………………………………………………………………………….....iv

THE PROBLEM ……………………………………………………………………..1

Background of the Study ……………………………………………………………..1

Objectives …………………………………………………………………………....2

Significance of the Study ……………………………………………………………...3

Hypotheses …………………………………………………………………………...3

Scope and Delimitations ……………………………………………………………....3

Definition of Key terms ..……………………………………………………………...4

REVIEW OF RELATED LITERATURE AND STUDIES …………………...........5

RESEARCH METHODOLOGY…………………………………………………. ….12

Research Design …………………………………………………………………....12

Material Gathering Procedure…..

………………………………………………............12

Preparation Of Experiment Set-Up….….………………………………………...……12

Materials and Equipment ….…………………………………………………………...13

Methodology .. ………………………………………………………………………..14

Data Collection Procedure …………………………………………………………..15

Statistical Analysis …………………………………………………………………......15

RESULTS AND DISCUSSIONS…….……………………………………………….16

SUMMARY OF FINDINGS, CONCLUSION, AND RECOMMENDATION………24

Findings ……………………………………………………………………………….24

Conclusions ……………………………………………………………………….......25

Recommendations……………………………………………………………………...25

APPENDICES

LITERATURE CITED
 CHAPTER 1

The Problem

Modernization like advancement in many fields grow faster, definitely

people are aware of this. It leads to open their minds to discover and expand
their knowledge through developing their concepts to solve different health
problems.

Today, antibiotic resistance is one of the major problems of medical

practioners. Antibiotic resistance is the ability of a microorganism to
withstand the effects of antibiotics. It is a specific type of drug resistance.
Antibiotic resistance evolves via natural selection acting upon random
mutation, but it can also be engineered by applying an evolutionary stress
on a population. Once such a gene is generated, bacteria can then transfer
the genetic information in a horizontal fashion (between individuals) by
plasmid exchange. If a bacterium carries several resistance genes, it is called
multiresistant or, informally, a superbug. The term antimicrobial resistance is
sometimes used to explicitly encompass organisms other than bacteria.

Antibiotic resistance can also be introduced artificially into a

microorganism through transformation protocols. This can aid in implanting
artificial genes into the microorganism. If the resistance gene is linked with
the gene to be implanted, the antibiotic can be used to kill off organisms that
lack the new gene.

The researchers have investigated the anti-bacterial properties of

various plants attemting to develop a new antibiotic out of natural
substances that could be used by communities.

Birds nest fern is effective against infecting Escherichia Coli and

Pseudomonas aeruginosa. They content the potent antibiotic components
like alkaloids, flavanoids, saponins and tannins. They have strong defensive
mechanism.

The study attempts to determine the potential of Bird’s nest fern

extract by evaluating the sensitivity of the Escherichia coli subjected to
Kirby-Bauer Antibiotic Testing.

Objectives of the study

General objectives

The study generally aimed to determine the potential of bird’s nest

fern extract as an antibiotic for Escherichia Coli.

Specific objectives

Specifically the study aimed to:

1 Determine the sensitivity of E. coli to bird’s nest fern extract through

Kirby-Bauer antibiotic testing

2. Determine the treatment where the specimens of bacteria are

most sensitive to
3. Determine the significant differences among treatments and
replications conducted.
Significance of the Study
 Specialists estimate that at least 90% of antibiotic prescriptions are
unnecessary or inappropriate. Mutation of bacteria happens and occurs
anytime by the transfer of resistance genes from the other bacteria.

Perhaps a new revolution of antibiotic is declared to be a necessity.

The study “Antimicrobial Activity of Bird’s Nest Ferns Extract against

Escherichia Coli has a vital importance in determining the effective and
cheap antibiotic out of commonly found in the locality like bird’s nest fern
which can also eradicate different kinds of bacteria, according to past
references. It is also significance in avoiding the use of harmful chemicals
which can pollute the air, kill other organism than the target pest and can
destroy the biodiversity.

Hypotheses

H0: Bird’s Nest Ferns Extract has no antibacterial effect on Escherichia

Coli,

H1: Bird’s Nest Ferns Extract has antibacterial effect on

Escherichia Coli,

Scope and limitations of the Study

This study was limited only on the detection of the sensitivity

of Escherichia Coli to Bird’s Nest Fern extract and to the stated objectives of
the research study.
 This study was conducted at Sultan Kudarat Polytechnic State
College- Laboratory High School Research Center , EJC Montilla, Tacurong
City from September 12-16 2009.

Definitions of Key Terms

Agar- is a gelatinous substance chiefly used as culture medium for

microbiological work.

Antibiotic- used to kill bacteria that give different diseases.

Antimicrobial agent- the bacteria causing agent. Its the transfer of

medium to medium.

Bird’s nest fern- is a tropical and sub-tropical plant which is native to the
rain forests of Asia, Africa, India , Polynesia and Australia .

Cefuroxime- is a second-generation cephalosporin antibiotic that has been

widely available.

Escherichia coli- can cause serious food poisoning in humans, and are
occasionally responsible for costly product recalls.

Extract- comes from the leaves or any parts of a plant that were subjected
to pounding and extracting.

Incubation - The development of an infection from the time the pathogen

enters the body until signs or symptoms first appear.

Kirby-Bauer antibiotic testing (KB testing or disk diffusion antibiotic

sensitivity testing)- is a test which uses antibiotic-impregnated wafers to
test whether particular bacteria are susceptible to specific antibiotics.
Sterilization – a process used to ensure that the materials are safe and
clean.

Zone of inhibition- This is an area around a paper disk or colony of

bacteria or mold where no other organisms are growing.

CHAPTER II

REVIEW OF RELATED LITERATURE AND STUDIES

 Related Literature
Escherichia coli is a Gram negative bacterium that is commonly

found in the lower intestine of warm-blooded organisms (endotherms). Most

E. coli strains are harmless, but some, such as serotype O157:H7, can cause

serious food poisoning in humans, and are occasionally responsible for costly
product recalls. The harmless strains are part of the normal flora of the gut,

and can benefit their hosts by producing vitamin K2, or by preventing the

establishment of pathogenic bacteria within the intestine.

E. coli are not always confined to the intestine, and their ability to

survive for brief periods outside the body makes them an ideal indicator

organism to test environmental samples for fecal contamination. The

bacteria can also be grown easily and its genetics are comparatively simple

and easily-manipulated or duplicated through a process of metagenics,

making it one of the best-studied prokaryotic model organisms, and an

important species in biotechnology and microbiology.

A strain of E. coli is a sub-group within the species that has unique

characteristics that distinguish it from other E. coli strains. These differences

are often detectable only on the molecular level; however, they may result in

changes to the physiology or lifecycle of the bacterium. For example, a strain

may gain pathogenic capacity, the ability to use a unique carbon source, the

ability to inhabit a particular ecological niche or the ability to resist

antimicrobial agents.

Different strains of E. coli are often host-specific, making it possible to

determine the source of fecal contamination in environmental samples.

For example, knowing which E. coli strains are present in a water

sample allows to make assumptions about whether the contamination

originated from a human, another mammal or a bird.

 New strains of E. coli evolve through the natural biological process of

mutation, and some strains develop traits that can be harmful to a host

animal. Although virulent strains typically cause no more than a bout of

diarrhea in healthy adult humans, particularly virulent strains, such as

O157:H7 or O111:B4, can cause serious illness or death in the elderly, the

very young or the immunocompromised.

Biology and biochemistry

Escherichia coli cells propel themselves with flagella (long, thin

structures) arranged as bundles that rotate counter-clockwise, generating

torque to rotate the bacterium clockwise.E. Coli is Gram-negative, facultative

anaerobic and non-sporulating. Cells are typically rod-shaped and are about

2 micrometres (μm) long and 0.5 μm in diameter, with a cell volume of 0.6 -

0.7 μm3. It can live on a wide variety of substrates. E. coli uses mixed-acid

fermentation in anaerobic conditions, producing lactate, succinate, ethanol,

acetate and carbon dioxide. Since many pathways in mixed-acid

fermentation produce hydrogen gas, these pathways require the levels of

hydrogen to be low, as is the case when E. coli lives together with hydrogen-

consuming organisms such as methanogens or sulfate-reducing bacteria.

Optimal growth of E. coli occurs at 37°C but some laboratory strains

can multiply at temperatures of up to 49°C.

 Growth can be driven by aerobic or anaerobic respiration, using a large

variety of redox pairs, including the oxidation of pyruvic acid, formic acid,

hydrogen and amino acids, and the reduction of substrates such as oxygen,

nitrate, dimethyl sulfoxide and trimethylamine N-oxide.

The Bird's Nest Fern, Asplenium nidus, is a tropical and sub-tropical

plant which is native to the rain forests of Asia, Africa, India , Polynesia and

Australia . In the wild, these largest of epiphytes live up in the crowns of

trees. Their means of nourishment is from nutrients found in dust particles

and rain which is collected in the plants deep, funnel-shaped rosette of

leaves. The genus name Asplenium (Spleenwort) is derived from the Greek

words 'a' meaning 'not', and 'splen', meaning 'spleen' and refers to the

medicinal properties the plant is supposed to have. The specific epithet or

species name 'nidus' means 'a nest', hence the common name Bird's Nest

Fern. The Bird's Nest Fern belongs to the 'Aspleniaceae' family under the fern

order 'Polypodiales'.

The leathery, tongue-like, glossy, pale green, wavy-edged fronds of

this fern are borne in a tight rosette at the rhizome, forming a bowl-shaped

clump with a fibrous center, which to many resembles a bird's nest, or at

least a seemingly sheltered spot for a bird's nest. Each frond is entire or

undivided and has a prominent, polished rib running down the center of the

frond. The new fronds uncurl from the hairy, dark scaled crown of the rosette
in typical fern manner. They are very soft and brittle when young and must

not be handled.

As the new fronds grow in, the older ones gradually die. Mature fronds

can grow to 3 feet in length and 8 inches in width, and under ideal

conditions, a plant can reach 4 to 6 feet in height, but, under normal home

conditions the plant rarely exceeds 15 inches in height. The stems of each

frond are stumpy and erect. Another commonly grown variety is Asplenium

crispafolium, which has deeply ruffled fronds.

Phytochemical Content of Leaf Extract

• Alkaloids are naturally occurring chemical compounds containing basic

nitrogen atoms. It protects plant from herbivores. They are anti-

spasmodic, anti-inflammatory,tranquilizer and vasodilator.

• Flavonoids (or bioflavonoids), also collectively known as Vitamin P and

citrin, are a class of plant secondary metabolites. They are anti-allergic

and antimicrobial and anti-inflammatory.

• Saponins, group of naturally occurring oily glycosides that foam freely

when shaken with water. They occur in a wide variety of plants,

including acacia, soapwort, soaproot, California pigweed, and many

others. Saponins have been, and sometimes still are, used as cleaning

agents and as foam producers, notably in fire-extinguishing fluids.

They have a bitter taste

 • Tannins , group of naturally occurring oily glycosides that foam freely

when shaken with water. They are antimicrobial, protect against

cancers and decrease blood lipids.

CHAPTER III

Research Methodology

This chapter discusses different mediums utilized in the research

design and studies as well the procedures and methods used in the conduct
of the study. Also included are the methods in gathering data as well as the
statistical tools in the research.

Research Design

The study is experimental and comparative in nature. The study

was conducted using the Analysis of Variance (ANOVA) of Completely
Randomized Design (CRD) with the three treatments replicated three times
in three trials with the same cultured bacteria per replication. The
treatments were as follows:

Treatment 1 -pure bird’s nest fern extract

Treatment 2 -5 ml bird’s nest fern extract + 5 ml water solution

Control - Cefuroxime
Material Gathering Procedure

The variables used in the study were bird’s nest fern and
Escherichia coli. The bacteria were collected from the urine oversaw by
SKPSC Research Center for culturing reasons. Bird’s nest fern were collected
at SKPSC garden and were gathered fresh for this contains more extract

Preparation of Experiment Set-Up

Escherichia Coli were cultured in the SKPSC Research Center for

the study. The three treatments, replicated thrice, were placed randomly on
every Petri dish with the cultured bacteria in it. Application of extract was
administered by impregnating Bird’s Nest fern extract unto sterile paper
disks. Three trials were conducted for the validity for the study.

Materials and Equipment

The following were used for the conduct of the study:

For Extracting:

Digital weighing machine

Mortar and pestle
Cheesecloth
Graduated cylinder

Sterilized water
Burner

Empty and clean bottle Bid’s Nest

fern

Crucible tongs

For Sensitivity Testing:

Bunsen burner
test tube

Inoculating loop
incubator

Sterile cotton swab

weighing scale

Petri dish
Erlenmeyer flask

Forceps
hot plate

Foil paper
Auto clave

Sterile paper discs

Mueller-Huinton agar (2%)

Sodium chloride

Tetracycline impregnated filter-paper disc

Bacteria (Escherichia Coli)

McFarland Standard

Agar used for incubating organisms:

Blood agar

MacConky Agar

Methodology:

The following methods were used in the study:

Extraction of Bird’s Nest fern:

1. Collect the materials needed and prepare the setup.

2. Pound the 20 grams of bird’s nest leaves fern using the mortar
and pestle
3. Squeeze the pounded leaves using the cheesecloth to obtain
the extract.
4. Treatment 1 requires concentrated amount of bird’s nest fern
extract and requires no boiling. For the treatment 2, add 5 ml
solution of pure bird’s nest fern extract to 5 ml sterilized water.
Dispense the mixture unto a test tube and set to boiling water.
 5. After obtaining the needed extract for both treatments, store
solutions securely unto sterilized empty bottles and seal.

Kirby-Bauer Antibiotic testing (direct method)

1. Growth for primary plate- inoculate into NSS

2. Streak to susceptible plate. E.coli MH with 2% NaCL
3. Let the inoculum dry for 3-5 minutes with the plates closed.
4. Placed the sterile paper disc impregnated with respective bird’s
nest fern extracts and cefuroxime(control) on the agar plate with
sterile forceps. Press down securely to ensure even contact.
-Disc must be equidistant with each other to allow zone of inhibition
(ZI) in millimeters

5. Incubate plates immediately or within 30 minutes for 24 hours

at degrees Celsius in an inverted position.
6. Read after 24-hour incubation.

Data Collection Procedure

Zone of Inhibition. After 24 hours, measure the diameter, in millimeters,

of the region where the Escherichia Coli were inhibited after 24 hours of
exposure to the treatments. Zone of inhibition will determine the remarks,
whether the bacteria were resistant or sensitive to the treatments. For the
control, it was based on the antidote’s SIR legend (on hospital standards).

Positive (+) for ZI = sensitive

Negative (-) for ZI = resistant

Statistical Analysis of Data

The zone of inhibition of each replicates and the mean of the three
replicates per treatments were computed. The data gathered were used in
answering the question: Which treatment will give the best result in terms of
antibacterial effect on Escherichia Coli after 24 hours?

Data gathered in this study was statistically analyzed using the

Analysis of Variance of Completely Randomized Design. The test for
significant differences among treatments means is subjected to Duncan‘s
Multiple Range Test (DMRT)
 CHAPTER IV

RESULTS AND DISCUSSION

This chapter presents the results of the tests conducted to

determine the potential of bird’s nest fern to be an antidote for Escherichia
coli and the statistical analysis performed on the data gathered. It is also
presents the interpretations of the results of the analysis.

Zone of inhibitions of Escherichia coli 24 hours after contact to

bird’s nest fern extract for the different trials are being shown in the
following table:

Table 1. Results of the sensitivity testing for the first trial.

Treatments R1 R2 R3 Total Mean Remarks

Zone of Inhibition

T1
(pure Bird’s Nest 55mm 54mm 56mm 165mm 55mm SENSITIVE
fern extract)
T2
(5 ml Bird’s Nest 0mm 2mm 0mm 2mm 0.67mm/ RESISTANT
fern w/ 5 ml undefined
H20)
Control SENSITIVE
(Cefuroxime) 46mm 50mm 48mm 144mm 48mm (SIR BASIS)

Positive (+) for ZI = sensitive

Negative (-) for ZI =resistant

Table1 shows that the result of the sensitivity test on Escherichia coli
after 24 hours of incubation and exposure to Bird’s Nest fern extract and the
control group on its first trial.

Treatment 1 which is a pure Bird’s Nest fern extract has a mean zone
of inhibition of 55 mm obtained from the zone of inhibition of its replicates:
R1=55mm, R2=54mm and R3= 56mm. treatment 2, a solution with bird’s
nest fern plus water extract has a zone of inhibition of 0.67mm with
replicates: R1=0mm, R2=2mm and R3=0mm. treatment 3 (Cefuroxime), the
control group, has a mean rate of 48 mm obtained from the mean rate of its
replicates: R1=46mm, R2=50mm and R3=48mm.

Based on the table 1, it was found out that Escherichia coli applied
with treatment 1 got the highest zone of inhibition and remarked “sensitive”,
which implies a good and effective antibacterial effect. Moreover, it is not
directly comparable to the control group since Cefuroxime is manufactured
for human dosage while Bird’s Nest fern extract intended for direct
inoculation to the bacteria. Conversely, treatment 2 (bird’s nest fern w/ H20)
showed the least ZI and therefore remarked as “resistant”.
Table 2. Results of the sensitivity for the second trial.

R1 R2 R3
Treatments Zone Of Inhibition Total Mean Remarks

T1
(pure bird’s nest 65mm 69mm 67mm 201mm 67mm SENSITIVE
fern extract)

T2
(5 ml bird’s nest 0mm 0mm 0mm 0mm 0mm/ RESISTANT
fern w/ 5 ml H20) undefined

Control
(Cefuroxime) 55mm 66mm 59mm 180mm 60mm SENSITIVE
(SIR BASIS)

Positive (+) for ZI = sensitive

Negative (-) for ZI =resistant

Table2 shows that the result of the sensitivity test on Escherichia

coli after 24 hours of incubation and exposure to Bird’s Nest fern extract and
the control group on its second trial. Treatment 1 which is a pure bird’s nest
fern extract has a mean zone of inhibition of 67mm obtained from the zone
of inhibition of its replicates: R1=65mm, R2=69mm and R3= 67mm.
Treatment 2, a solution with bird’s nest fern plus water extract has a zone of
inhibition of 0mm with replicates: R1=0mm, R2=0mm and R3=0mm.
Treatment 3 (Cefuroxime), the control group, has a mean rate of 60mm
obtained from the mean rate of its replicates: R1=55mm, R2=66mm and
R3=59mm.
 Based on the table 2, it was found out that Escherichia coli applied
with treatment 1 got the highest zone of inhibition and remarked “sensitive”,
which implies a good and effective antibacterial effect. Moreover, it is not
directly comparable to the control group since Cefuroxime is manufactured
for human dosage while bird’s nest fern extract intended for direct
inoculation to the bacteria. Conversely, treatment 2 (bird’s nest fern w/ H20)
showed the least ZI and therefore remarked as “resistant.”

Table 3. Results of the sensitivity testing for the third trial.

R1 R2 R3
Treatments Zone Of Inhibition Total Mean Remarks

T1
(pure bird’s nest 55mm 56mm 58mm 169mm 56.34mm SENSITIVE
fern extract)

T2
(5 ml bird’s nest 3mm 1mm 0mm 4mm 1.34mm RESISTANT
fern w/ 5 ml H20)

Control 45mm 43mm 48mm 136mm 45.34mm SENSITIVE

(Cefuroxime) (SIR BASIS)

Positive (+) for ZI = sensitive

Negative (-) for ZI =resistant

Table 3 shows that the result of the sensitivity test on Escherichia coli
after 24 hours of incubation and exposure to Bird’s Nest fern extract and the
control group on its third trial. Treatment 1 which is a pure bird’s nest fern
extract has a mean zone of inhibition of 56.34mm obtained from the zone of
inhibition of its replicates: R1=55mm, R2=56mm and R3= 58mm.
Treatment 2, a solution with bird’s nest fern plus water extract has a zone of
inhibition of 1.34mm with replicates: R1=3mm, R2=1mm and R3=0mm.
treatment 3 (Cefuroxime), the control group, has a mean rate of 45.34mm
obtained from the mean rate of its replicates: R1=45mm, R2=43mm and
R3=48mm.

Based on the table 3, it was found out that Escherichia coli applied
with treatment 1 got the highest zone of inhibition and remarked “sensitive”,
which implies a good and effective antibacterial effect. Moreover, it is not
directly comparable to the control group since Cefuroxime is manufactured
for human dosage while bird’s nest fern extract intended for direct
inoculation to the bacteria. Conversely, treatment 2 (bird’s nest fern w/ H20)
showed the least ZI and therefore remarked as “resistant.”

Table 4. Analysis of Variance of the first trial.

Source of df SS MS F -value F -tab

variance 5% 1%
Replication 6.94 18.00
Treatment 6.94 18.00
Error
Total
**Significant

*Not Significant

Based on the analysis of variance, the computed Frequency value

obtained for replication is __.The tabular frequency (2,4)=6.94 at 0.5 and 18
at 0.1. Hence, the replication is not significant. This means that replicates on
the second trial do not really differ with each other.
 For treatment, the computed f-value obtained is ______ and the
tabular F(2,4)=6.94 at 0.5level and 18 at 0.1 level of probability, the
treatment is significant which means that treatments really differ with each
other in terms of zone of inhibition.

Table 5. Analysis of Variance of the second trial.

Source of df SS MS F -value F -tab

variance 5% 1%
Replication 6.94 18.00
Treatment 6.94 18.00
Error
Total
**Significant

*Not Significant

Based on the analysis of variance, the computed Frequency value

obtained for replication is __.The tabular frequency (2,4)=6.94 at 0.5 and 18
at 0.1. Hence, the replication is not significant. This means that replicates on
the second trial do not really differ with each other.

For treatment, the computed f-value obtained is ______ and the

tabular F(2,4)=6.94 at 0.5level and 18 at 0.1 level of probability, the
treatment is significant which means that treatments really differ with each
other in terms of zone of inhibition.

Table 6. Analysis of Variance of the third trial.

Source of df SS MS F -value F -tab

variance 5% 1%
Replication 6.94 18.00
Treatment 6.94 18.00
Error
Total
**Significant
*Not Significant

Based on the analysis of variance, the computed Frequency value

obtained for replication is __.The tabular frequency (2,4)=6.94 at 0.5 and 18
at 0.1. Hence, the replication is not significant. This means that replicates on
the third trial do not really differ with each other.

For treatment, the computed f-value obtained is ______ and the

tabular F(2,4)=6.94 at 0.5level and 18 at 0.1 level of probability, the
treatment is significant which means that treatments really differ with each
other in terms of zone of inhibition.

Table 7. General tabulation of the trials conducted.

R1 R2 R3
Treatments Zone Of Inhibition Total Mean Remarks

T1
(pure bird’s nest 55mm 67mm 56.34mm 178.34mm 59.45mm SENSITIVE
fern extract)

T2
(5 ml bird’s nest 0.67mm 0mm 1.34mm 2.01mm 0.67mm RESISTANT
fern w/ 5 ml
H20)

Control SENSITIVE
(Cefuroxime) 48mm 60mm 45.34mm 153.34mm 51.11mm (SIR BASIS)

Positive (+) for ZI = sensitive

Negative (-) for ZI =resistant

 Table 7 shows the general tabulation of the trials conducted for the
sensitivity of Escherichia coli to different treatments used. Trials of treatment
1 which is a pure bird’s nest fern extract has a mean zone of inhibition of
59.45mm obtained from the zone of inhibition of its replicates:
Trial1=55mm, Trial 2=67mm and Trial 3= 56.34mm. Treatment 2, a
solution with bird’s nest fern plus water extract has a zone of inhibition of
0.67mm with replicates: Trial 1=0.67, Trial 2=0mm and Trial 3=1.34mm.

Treatment 3 (Cefuroxime), the control group, has a mean zone of

inhibition of 51.11mm obtained from the mean of its trials: Trial 1=48mm,
Trial 2=60mm and Trial 3=45.34mm.

Based on the table 7, it was found out that Escherichia coli applied
with treatment 1 got the highest zone of inhibition and remarked “sensitive”,
which implies a good and effective antibacterial effect. Moreover, it is not
directly comparable to the control group since Cefuroxime is manufactured
for human dosage while Bird’s Nest fern extract is intended for direct
inoculation to the bacteria. Conversely, treatment 2 (bird’s nest fern w/ H20)
showed the least ZI and therefore remarked as “resistant.”

Table 8.General ANOVA

Source of F -tab
variance df SS MS F -value 5% 1%
Replication 6.94 18.00
Treatment 6.94 18.00
Error
Total
**Significant

*Not Significant
 Based on the Analysis Of Variance, the computed Frequency value
obtained for trial is __.The tabular frequency (2,4)=6.94 at 0.5 and 18 at 0.1.
Hence, the trial are not significant. This means that result of the trial do not
really differ with each other.

For treatment, the computed f-value obtained is ______ and the

tabular F(2,4)=6.94 at 0.5level and 18 at 0.1 level of probability, the
treatment is significant which means that treatments really differ with each
other in trials conducted in terms of zone of inhibition.

Statistically, the analysis of variance of the first, second and third

trial showed almost the same results. It therefore implies that the study is
valid.

Results above showed high sensitivity of bacteria Escherichia coli to

pure bird’s nest fern extract as subjected to Kirby-Bauer Antibiotic Testing.

CHAPTER V

SUMMARY OF FINDINGS, CONCLUSION, AND RECOMMENDATION

This chapter discusses the findings, conclusions, and recommendations

of the research study on “Antimicrobial Activity of Bird’s Nest Ferns
Extract Against Escherichia Coli.” It was conducted at Sultan Kudarat
Polytechnic State College- Laboratory High School Research Center , EJC
Montilla, Tacurong City from September 12-16 2009 using the Analysis of
Variance of Completely Randomized Design (CRD) with three treatments
replicated three times.
*Major Finding

The major findings of this study were as follows:

1. Treatment 1(pure birds nest extract) got a general mean of its ZI

with 59.45 mm while Treatment 2 (5 ml bird’s nest extract + 5ml of
sterilized water) got a general mean of 0.67mm.The control cefuroxime
got a general mean of 51.11mm.

2. Escherichia coli showed high sensitivity when exposed to pure bird’s

nest fern extract.

3. It is therefore found out that the treatment 1 is where Escherichia

coli is most sensitive to.

4. Based on the Analysis of Variance, treatments have significant

differences among each other in all trials conducted, while the
replicates and trials are not significant

*Conclusions

The following conclusions were based from the major findings.

Based on the findings of the test conducted, bacteria Escherichia

coli is favorably sensitive to treatment 1, which is composed of pure birds
nest fern extract from its leaves as it remarked sensitive. The treatments
showed highly significant differences while the replicates and trials are very
precise and showed not so much significance.

Likewise, the comparison between the control group and the treatments
used cannot be directly measured since the control (i.e.Cefuroxime) is
manufactured considering the dosage for human consumption while bird’s
nest fern extract is made to combat E. coli in direct contact with the bacteria
itself.

Nevertheless, the study generally proved the great potential of

bird’s nest fern extract to be the radical antidote for the threatening public
health nightmares, the E. coli.

*Recommendations

Studies on the different mediums had shown significant findings.

In this connection, the researchers strongly recommend that:

1. The sensitivity testing using pure birds nest extract to another

sample of bacteria be conducted.
2. Determine the SIR legend for birds nest fern extract to determine
the accurate and precise comparison with commercial antibiotics.
3. Further study should be conducted to manufactured birds nest
fern in exact dosage intended for human consumption.