De La Salle University – Health Sciences Institute

COLLEGE OF MEDICINE
Department of Family and Community Medicine

A Comparative Study on the Antibacterial Activity of the Peel

Extracts Obtained from Musa acuminata, Musa balbisiana,
and Musa paradisiaca against Staphylococcus aureus

In partial fulfilment of the requirements in

Community Medicine II

Submitted by:
GROUP 9 – A

Adviser:
Dr. I.A. Ilano

Leader:
Holgado, Anna Victoria

Members:
Alcantara, Jan Christopher
Balandan, Patricia
Buenafe, Jonas Joaquin
Constantino, Erwin
Delos Santos, Kathrine Aira
Flores, Marie Felle
Hernandez, Kristeen Khae
Lopez, Edison

March 13, 2012

1
 TABLE OF CONTENTS

LIST OF TABLES ..................................................................................................................................... 2

LIST OF FIGURES ................................................................................................................................... 2

ABSTRACT................................................................................................................................................. 3

1. INTRODUCTION .................................................................................................................................. 4
1.1 Research question ...................................................................................................................... 4
1.2 Research hypothesis ................................................................................................................... 4
1.3 Background of the research question ........................................................................................ 4
1.4 Rationale for the study ................................................................................................................ 5
1.5 Review of literature and conceptual framework ........................................................................ 6

2. OBJECTIVES ...................................................................................................................................... 12

3. METHODOLOGY ............................................................................................................................... 13
3.1 Research design ....................................................................................................................... 13
3.2 Method ..................................................................................................................................... 15
4. RESULTS ............................................................................................................................................ 29
4.1 Descriptive statistics ................................................................................................................ 29
4.2 Inferential statistics .................................................................................................................. 30

5. DISCUSSION ...................................................................................................................................... 31
5.1 Limitations of the study ............................................................................................................ 32

6. CONCLUSION AND RECOMMENDATIONS ................................................................................ 34

6.1 Conclusion ................................................................................................................................ 34
6.2 Recommendations .................................................................................................................... 34

REFERENCES ........................................................................................................................................ 35

APPENDIX ............................................................................................................................................... 37

2
 LIST OF TABLES

Table 1. Zone of inhibition of peel extracts of different banana species ...................................... 30

using paper disk diffusion in different trials

LIST OF FIGURES

Figure 1. Conceptual framework .................................................................................................... 12

Figure 2. Schematic diagram of the research design ...................................................................... 14
Figure 3. Allocation of treatment and control ................................................................................ 18
Figure 4. Demarcations for accepting or rejecting the null hypothesis .......................................... 31

3
ABSTRACT

A comparative study on the antibacterial activity of peel extracts obtained from three

local banana variants (Musa acuminata, Musa balbisiana, and Musa paradisiacal) was done to

determine which among these variants had greater antibacterial activity against Staphylococcus

aureus, a common Gram-positive agent found in most health care-related infections. The zones

of inhibition evident in performing paper disk diffusion assay; respective minimal inhibitory

concentrations, and minimal bactericidal concentrations were used as parameters to evaluate

antibacterial activity. An experimental design was employed, keeping in mind the principle of

blinding in designating treatment and control groups. Positive, negative and solvent controls

were set to confirm that the observed antibacterial activity can be attributed to the peel extract.

Trials utilizing the paper disk diffusion assay were conducted to affirm antibacterial activity and

results showed the absence of microbial growth inhibition. A conclusion was made that the peel

extracts obtained from the different species of banana do not have comparable antibacterial

activity against Staphylococcus aureus. Careful re-evaluation of the extraction method

employed, modification of the experiment procedure, and a follow-up study on the antibacterial

properties of the Musa genus were some of the recommendations proposed.

4
 Chapter I
INTRODUCTION

1.1 Research question

“Which species of banana peel extract has greater antibacterial activity against

Staphylococcus aureus?”

1.2 Research hypothesis

1.2.1 Working hypothesis

“The peel extracts obtained from the different species of banana have comparable

antibacterial activity against Staphylococcus aureus.”

1.2.2 Null hypothesis

“The peel extracts obtained from the different species of banana do not have

comparable antibacterial activity against Staphylococcus aureus.”

1.3 Background of the research question

The contribution of various plants or their parts (roots, stems, leaves, fruit) in the

treatment and management of certain health conditions has been growing in recognition.

At present, the use of herbal medicine are becoming more common both in developing

and developed countries. The World Health Organization (WHO) has estimated that about

70-95% of the citizens in majority of the developing countries utilize traditional medicine

(including the use of herbal medicines) in managing their health and incorporate the

practice in their primary health care to address emerging health-related needs.(1)

Industrialized countries, such as Canada, France, Germany and Italy, share similar

percentages in terms of the proportion of individuals who make use of traditional

5
 medication.(1) In the Philippines, the use of plant extracts as medication has been passed

on from one generation to another, and has established its importance in health delivery,

considering the expensive Western treatment that most Filipinos cannot afford or cannot

easily access.(2)

The banana fruit (Musa sapientum), has been commonly known for its nutritional

value; however, its medicinal properties have only been recently investigated, mostly in

tropical and subtropical countries wherein the banana fruit is considered as one of their

major agriculture products, such as India and other Southeast Asian countries. In these

regions, other parts of the banana plant such as their young shoots and peels have been

utilized as an alternative source of treatment for ulcers and wounds, especially in areas

where access to conventional treatment is difficult.(3) In addition, majority of the studies

conducted in these countries have been focusing on the most common variants of the

Musa sapientum species available in their locality.

The study aims to investigate the antibacterial activity of the most common local

variants of banana grown in the province of Cavite, namely Musa acuminata (“lakatan”),

Musa balbisiana (“saba”), and Musa paradisiaca (“latundan”)(4), and determine if the

species’ variant plays a significant role in their respective antibacterial activity, specifically

against Staphylococcus aureus, a common Gram-positive agent found in most health

care-related infections.

1.4 Rationale for the study

This study may provide additional information regarding the antibacterial activity of

the common variants of bananas in the region, and thus provide possible plant leads that

can be used as alternative and less expensive sources of treatment for the benefit of the

local residents. The additional information obtained may also contribute in evoking

interest for further research on the phytochemical profile and antibacterial activity of the

6
 local banana variants which may be used as active ingredients against drug-resistant

microorganisms such as Staphylococcus aureus.

1.5 Review of literature and conceptual framework

1.5.1 Epidemiology of disease of interest

Staphylococcus aureus is considered as one of the medically important pathogens,

commonly causing abscess formation, various pyogenic infections (e.g. endocariditis),

food poisoning, and toxic shock syndrome. It has also been among the prevalent

causative agent for majority of hospital-acquired infections (e.g. pneumonia), septicemia,

and surgical-wound infections.(5)(6) Infections caused by the bacteria have been more

prevalent in the health care setting where these are treated with more frequent and

intensive antimicrobial therapy as compared in the community setting.(6)

Throughout the evolution of antimicrobial therapy against S. aureus, various strains

of resistance have been developed by the agent, thus amplifying the disease burden. (6) In

some countries in the Western Pacific Region, such as the Philippines, a growing trend in

methicillin-resistant S. aureus (MRSA) had been recorded within a span of 7 years.(7)

According to the 2005 Philippine Antimicrobial Resistance Surveillance report, wherein

twelve out of the agency’s 17 sentinel health care institutions contributed to the data

output, the overall MRSA rate among admitted patients significantly increased, from 17%

in 2004 to 31% in 2005.(8) An increase of 34% was also observed in urban areas such as

Metro Manila.(8)

Health care institutions in the Philippines constantly deal with the disease burden

brought about by various strains of methicillin-resistant S. aureus among patients. A study

conducted in a tertiary medical institution revealed the presence of hospital-acquired

MRSA (HA-MRSA) among patients suffering from chronic kidney disease and were

7
undergoing renal replacement therapy, such as hemodialysis. Strains of community-

acquired MRSA (CA-MRSA) were also isolated and were identified among patients with

no other underlying co-morbidities. CA-MRSA strains were commonly seen in skin and

soft tissue infections.(9)

1.5.2 Epidemiology of exposure (factor of interest)

Traditional medicine has been highly adopted throughout the world due to their

availability, affordability and cultural familiarity. It has been estimated by the World Health

Organization that in some Asian and African countries, 80% of the population depends on

traditional medicine.(10) They also identified the use of herbal treatments as the most

popular form of traditional medicine. Herbal medicines include herbs, herbal materials,

herbal preparations, and finished herbal products that contain parts of plants or other plant

materials as active ingredients. Among the components included in the current day

pharmaceuticals, it has been estimated that about seven thousand active ingredients are

of herbal origin. (10)

The antimicrobial properties of the banana plant (Musa sapientum) are not as widely

commercialized as compared to other local preparations. Nonetheless, its young leaves

have been used for a long time in local folk medicine as a cold dressing for inflamed and

blistered surfaces.(3) Scientific studies have presented evidences regarding the

antimicrobial activities of the banana plant. Commonly utilized parts among the studies

were its leaves, stem, peel, and fruit. Studies conducted by Mokbel and Hashinaga,

Fagbemi et. al, and Scott et. al showed high antimicrobial acitivity in peel and pulp extracts

of unripe bananas against certain bacteria, including S.aureus.(11)(12)(13)

8
1.5.3 Summary of related/similar studies

Majority of the experiments conducted made use of an analytical experimental

design wherein the exposure variable under observation was assigned particularly to a

treatment group and was compared to a control group. The method of extract preparation

and the different solvents used were some of the factors identified that may have

influenced the extract’s potency and enhanced its antimicrobial activity.

1.5.3.1 Method of preparation and solvents used for banana extracts

Banana (Musa sapientum), belonging to Musa species is considered as one of the

most useful plant species that carries a number of beneficial pharmacological effect such

as ulcer protective activity, antioxidant activity and mutagenic effect, antibacterial activity

and wound healing activity.(14)

Several studies have been conducted to show that banana extracts do have

antibacterial properties. In a study by Mokbel and Hashinaga, the antimicrobial and

antioxidant activity of fresh green and yellow banana peel extracts obtained from the

Cavendish variant were compared. Chloroform, ethyl acetate and water were used as

solvents for the peel extracts. Results showed that ethyl acetate and water soluble

fractions of green banana peel displayed high antimicrobial and antioxidant activity.

Among the specific compounds isolated from green banana peel, d-malic acid and 12-

hydroxystearic exhibited the most active antimicrobial response.(11)

Findings were supported in a phytochemical and pharmacologic review conducted

by Akter et al. wherein it was demonstrated that the banana peel extract obtained from

Musa paradisiaca and Musa sapientum showed better antibacterial activity against the test

bacteria (Staphylococcus and Pseudomonas species) than the banana leaf extract. The

peel extract was also shown to be more active against Staphylococcus (Gram-positive)

than Pseudomonas species (Gram-negative). Furthermore, the review of pharmacological

9
activities suggests that the traditional uses of the banana plant in diarrhea, dysentery,

ulcer, diabetes, hypertension and cardiac diseases are scientifically valid.(15)

Other studies investigated if the difference in the subspecies of the Musa sapientum

would yield varying degrees of antibacterial activity against a range of microorganisms.

Akter et. al aimed to evaluate the antimicrobial and cytotoxic activities of different extracts

of Musa sapientum, L. subsp. Sylvestris fruits (MSSE). The methanolic extract of Musa

sapientum peel was investigated for antimicrobial activity by disk diffusion method and for

cytotoxic activity by Brine shrimp lethality bioassay. The findings of the study

demonstrated that the methanolic extract of Musa sapientum possessed good

antimicrobial activity against Gram-positive and Gram-negative bacteria as well as against

pathogenic fungi and affirmed the traditional use of the fruit to treat dysentery and

diarrhea.(16)

In addition, studies conducted by Hamid et al. as well as Mokbel et al. showed how a

certain development of the banana fruit may contribute to its antibacterial activity by using

both ripe and unripe banana peel extract. The type of solvent used was not

specified. Extracts of ripe, unripe and leaves of guava (Psidium guajava); ripe, unripe and

leaves of starfruit (Averrhoa carambola); ripe and unripe banana (Musa sapientum variety

Montel); ripe and unripe papaya (Carica papaya); passionfruit (Passiflora edulis F.

Flavicarpa) peel; two varieties of Lansium domesticum peels; rambutan (Nephelium

lappaceum) peel and rambai (Baccaurea motleyana) peel were evaluated for antimicrobial

activity against Gram-positive bacteria, Gram-negative bacteria, yeast and fungi

(Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Lactobacillus bulgaricus; E. coli,

Proteus vulgaricus, Pseudomonas aeruginosa, Salmonelli typhi; Saccharomyces

cerevisiae, Candida lypolytica; Rhizopus spp., Aspergillus niger, and Chlamydomucor

spp). The antimicrobial activities were tested using both the disk diffusion and tube dilution

assays. Most of the fruits showed some activity towards bacteria but poor activity against

10
yeast or fungi. Extracts from bananas, papayas, passionfruit peel, Lansium domesticum

peels and rambutan peels showed activity against Candida lypolytica while extracts from

guava showed strong activity against Saccharomyces cerevisiae. Unripe banana showed

activity against all the bacteria except towards P. vulgaricus.(17) The study conducted by

Mokbel and Hashinaga used both fresh green and yellow banana peel of Musa, cv.

Cavendish fruits which were treated with 70% acetone; and afterwards, partitioned with

chloroform (CH[Cl.sub.3]) and ethyl acetate (EtOAc). The antioxidant activities of the

extracts were evaluated by using the thiocyanate method, beta-carotene bleaching

method and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical elimination. Antimicrobial

activities of the extracts and isolated components were evaluated using paper disk

methods and minimum inhibition concentration (MIC). The EtOAc and water soluble

fractions of green peel showed high antimicrobial and antioxidant activity. The antioxidant

activity of water extracts was comparable to those of synthetic antioxidants such as

butylated hydroxyanisole and butylated hydroxytoluene. Among all isolated components,

B-sitosterol, malic acid, succinic acid, palmatic acid, 12-hydroxystrearic acid, d-malic and

12-hydroxystrearic acid were most active against the Gram-negative and Gram-positive

bacteria species tested. The MIC of d-malic and succinic acid varied between 140-750

ppm, respectively. (11)

1.5.3.2 Biases

Although there were no significant experimental biases recognized in the related

literatures that were reviewed, a possible researcher bias, wherein the prior knowledge of

the researchers might affect the analysis of the results, may be encountered in the study.

Observational bias may also be encountered among the researchers along the course of

the experimental proper, wherein there may be discrepancies in measuring the outcome

observed.

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 1.5.3.3 Limitations

It can be noted that since almost all of the studies were conducted in vitro, the

occurrence of possible side effects or interactions of the extracts on actual clinical

infections cannot be identified.

1.5.3.4 Recommendations

Results from related literatures showed that the banana pulp and peel exhibits high

antimicrobial activity whereas in terms of solvent to be used, extracts using ethyl acetate,

ethanol and methanol exhibits high antimicrobial activity. These results should be taken

into consideration when choosing which part of the banana and what kind of solvent

should be used for extraction.

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1.5.4 Conceptual framework

Figure 1 depicts the possible relationship of the exposure variable (application of plant

extract) with that of the outcome variable (inhibition of microbial growth). The

characteristics of the exposure variable must be taken into consideration as to how it

would influence the outcome variable. Similarly, the characteristics of the outcome

variable should also be taken into consideration as to how it may counteract the exposure

variable and affect the result. Possible confounding factors such as exposure to

environmental factors as well as experimental protocol (preparation and storage methods)

were derived from literatures documenting various experimental processes in determining

the antimicrobial activities of the plant extracts.

Antibacterial activity of Inhibition of growth

banana peel extract of Staphylococcus
aureus
Characteristics of the
factor Characteristics of the
 Type of solvent used factor
 Type of  Inherent defense
phytochemicals
mechanisms of the
present which
contributes to sample of interest
antibacterial activity
 Concentration of the
extract

POSSIBLE CONFOUNDING
FACTORS

Exposure to environmental
factors
Preparation and storage
techniques

CONTAMINATION

Figure 1. Conceptual framework 13

 Chapter II
OBJECTIVES

Given the previous data, the study conducted was generally aimed at determining

which species of banana peel extract has greater antibacterial activity against

Staphylococcus aureus. In order to achieve the general objective, the following specific

objectives were met:

1. To measure the zones of inhibition of peel extracts obtained from Musa acuminata,

Musa balbisiana, and Musa paradisiaca using the disk diffusion method.

2. To establish the respective minimal inhibitory concentrations of peel extracts from

Musa acuminata, Musa balbisiana, and Musa paradisiaca using broth dilution test.

3. To establish the respective minimal bactericidal concentrations of peel extracts from

Musa acuminata, Musa balbisiana, and Musa paradisiaca using the streak plate

technique and incubation of Mueller Hinton agar plates.

4. To compare the obtained zones of inhibition, minimal inhibitory concentrations, and

minimal bactericidal concentrations of the peel extracts against Staphylococcus

aureus.

14
 Chapter III
METHODOLOGY

3.1 Research design

The study utilized an analytic experimental design, wherein the independent variable

under observation was assigned particularly to a treatment group and was compared to a

positive and negative control group. Figure 2 illustrates the design of the study.

Figure 2. Schematic diagram of the design

This type of study did not require obtaining subjects from a target population;

therefore, an inclusion/exclusion criterion has not been defined. The application of

15
constructing a sampling design as well deriving a sample size calculation was also not

appropriate for the study.

3.1.1 Operational definition of variables

3.1.1.1 Independent variable

The antibacterial activity of the different species of banana peel extracts on the

growth of Staphylococcus aureus served as the independent variable for the study.

“Antibacterial activity” refers to the capacity of an agent to kill or suppress the growth of

microorganisms, specifically bacteria.(18) This property may be further classified into two

mechanisms, bacteriostatic and bacteriocidal. Bacteriostatic activity results into the

inhibition of microbial growth within a certain period of time. Microbial growth may be

observed once environmental elements become suitable, or the microorganism has

gained resistance to counteract the stimulus presented by the agent.(18) On the other

hand, bacteriocidal activity results into the complete eradication of the species. In the

study, significant bacteriostatic activity of the different species of bananas will be observed

through disk diffusion method and broth dilution test.

Musa acuminata (locally known as lakatan), Musa balbisiana (saba), and Musa

paradisiaca (latundan) are considered as the most common group of species grown and

commonly sold in the province of Cavite. Because of their wide availability and easy

accessibility, these species were chosen as samples of plant extract for the study.

3.1.1.2 Dependent variable

The inhibition of the growth of Staphylococcus aureus on nutrient agar medium

served as the expected outcome of the study. Inhibition denotes a temporary cessation in

microbial growth processes. This implies that there are still possible chances for growth,

given that the environment becomes favorable once again for microorganism

16
 propagation.(18) Therefore, strict compliance with the incubation of disk diffusion plates

within the allotted time of 24 hours for the Staphylococci species were observed so as to

achieve reliable results and prevent possible growth of the organism.(19)

Inhibition of growth was determined qualitatively using the disk diffusion method, as

represented by zones of inhibition. Broth dilution test was employed to quantify the degree

of the antibacterial activity by determining the peel extract’s minimal inhibitory

concentration. The end point tube in the series of test tubes illustrates the absence of
(18)(19)
microbial growth achieved with the least concentration of the peel extract. Aliquots

from the tube with the least amount of drug that showed no growth and the two tubes that

immediately precede it were collected and inoculated in a nutrient agar medium using the

streak plating technique in determining the peel extracts’ respective minimal bacteriocidal

concentrations.

3.1.1.3 Confounding variables

Both the independent and dependent variables may face possible contamination

brought about by 1) their exposure to environmental factors, such as temperature and

foreign body contamination; 2) as well as their subjection to certain preparation and

storage techniques executed in the duration of the study. Contamination of the variables of

interest was considered to significantly influence the accuracy and analysis of the results.
(18)(19)(20)

3.2 Method

For the study, microbial growth inhibition was determined using the disk diffusion

method, also known as the Kirby-Bauer test. The principle behind the disk diffusion

method depends on the formation of a concentration gradient as the antimicrobial agent

diffuses instantaneously into the agar. The drug concentration decreases at increasing

distances from the disk. At a critical point, the amount of drug at a specific location in the

17
medium is unable to inhibit the growth of the test organism, thus forming well-demarcated

borders, resulting in a distinct area known as the zone of inhibition.(18) This method only

gives a qualitative value of an agent’s antimicrobial activity against a particular bacterial

species, as determined by the resulting zone of inhibition. The “zone of inhibition” refers to

the clear area surrounding an antimicrobial disk following overnight incubation that result

from the diffusion of the antimicrobial molecules into the agar and inhibition of growth of

the test bacterium. This was the parameter used in the study to reflect the presence of

microbial growth inhibition. (18)

Furthermore, control groups were set up to validate that the microbial growth

inhibition is indeed attributed to the antibacterial activity of the banana peel extract. A

positive control will confirm that the treatment applied is competent to produce the

intended effect, thus minimizing the probability of false negatives. (21) For the study, the

drug of choice for Staphylococcus aureus, Vancomycin, was utilized as the standard for

evaluating the antibacterial activity of the plant extracts. A negative control was also set up

in order to confirm that the effect produced was not influenced by extraneous factors,

thereby minimizing the probability of false positives.(21) For the study, distilled water was

used as a reference reflecting microbial growth, indicating that the treatment applied has

not been effective. In addition, a solvent group was set up to evaluate the influence of the

solvent on the observed microbial growth inhibition. For the study, methanol was used as

reference.

Quantitative measures, such as the minimal inhibitory concentration (MIC), were also

applied to validate the degree of the antibacterial activity of the banana peel extracts.

Minimal inhibitory concentration refers to the smallest amount of the extract per unit

volume that will inhibit the growth of a certain organism.(18)(20) Broth dilution tests, wherein

tubes containing decreasing concentration of the plant extract tested are prepared by

serial dilution, was conducted to determine the plant extracts’ respective MIC.(20)

18
3.2.1 Steps undertaken in the study

3.2.2.1 Physical set-up

The study used Mueller Hinton agar plates in observing the activity of peel extracts

from different banana species (Musa paradisiaca, Musa balbisianana and Musa

acuminata) compared with the positive control, negative control and solvent control. Three

replicates were used. Figure 3 illustrates the distribution of the treatments and control

among the bacterial culture.

CONC. CONC.
(A) (A)

CONC. CONC.
(B) SOLVENT (B) SOLVENT
ONLY ONLY

CONC. (-) CONC. (-)

(C) CONTROL (C) CONTROL

(+) (+)
CONTROL CONTROL

Musa paradisiaca Musa balbisianana

CONC.
(A)

CONC.
(B) SOLVENT
ONLY

CONC. (-)
(C) CONTROL

(+)
CONTROL

Musa acuminata
Figure 3: Allocation of treatment and control

19
 3.2.2.2 Procurement of plant materials

Peels from different species of banana (Musa paradisiaca, Musa balbisianana and

Musa acuminata) were obtained from Dasmariñas, Cavite public market.

3.2.2.3 Preparation of the extract

The banana peels (33.33 g for each variety) were washed with water then coarsely

chopped. The peels were then be placed in a solvent, methanol, at a ratio of 1 gram

banana peel per 4.5 mL of methanol. The mixture was then homogenized (for 5 minutes)

using a blender and filtered using a flat filter paper. Mechanical pressure was applied upon

the acquired mixture to ensure that every remnant of the extract from the mixture is

obtained. The collected residue was again placed in methanol (1g : 4.5mL) and the same

procedure during homogenization was followed. Residue collected from the second

homogenization was used in third homogenization, then discarded after all the filtrates has

been collected. The filtrates were properly labeled according to species and stored in a

beaker at 45°C for 48 hours. (22)

3.2.2.4 Preparation of peel extract stock solution

The stored extract was filtered using a flat filter paper on a funnel. The collected

precipitate was then allowed to dry in the oven for 24 hours at 45°C. From the collected

precipitate, 6 mg of the precipitate was redissolved in 2 mL of methanol to obtain an initial

stock solution of 3 mg/mL (or 3 000 µg/µL). The stock solution with a volume of 2 mL was

used for the duration of the experiment. From the 2mL, a total of 0.015 mL was used for

paper disk diffusion with three replicates, 0.003 mL was used for preparation of the other

test concentrations (300µg/µL and 30µg/µL) and 1mL was set for use in MIC.

The initial concentration obtained was set at 3,000 µg. 10-fold dilution from this

concentration was done until a concentration equivalent to that of the standard drug used

20
as control (Vancomycin = 30 µg/µL) was achieved. Vancomycin is an antibiotic that is

active only against gram-positive bacteria, particulary Staphylococcus. It is bactericidal for

most pathogenic staphylococci, including those producing β-lactamase, and those

resistant nafcillin and methicillin. (23)

Based on related studies, a 10-fold dilution from the starting concentration based on

the standard drug used was utilized to determine antibacterial activity. This will be the

value used as concentration factor, thus yielding the following concentrations for

observation: 3,000 µg, 300 µg, and 30 µg.

A) Dilution factor (DF) = stock solution (initial concentration)

final concentration
= 3 mg/mL
0.3 mg/mL
~ 1:10 dilution

(B) Sample to be obtained

C1V1 = C2V2
Wherein C1 – initial concentration available
C2 – final concentration desired
V2 – final volume of new concentration
V1 – volume of C1 required to make new concentration

- to prepare 300 µg/µL (or 0.3 mg/mL)

(3000 µg/µL)(x) = (300 µg/µL)( 20 µL)
x = 2 µL

- to prepare 30 µg/µL (or 0.03 mg/mL)

(3000 µg/µL)(x) = (30 µg/µL)(20 µL)
x = 0.2 µL

Based from the above computations (B), to prepare 300 µg/µL concentration, 18uL of

methanol was added to 2µL of 3000 µg/uL. 19.8 µL of methanol was added to 0.2µL of

3000 µg/µL to abtain the final concentration 30 µg/µL.

3.2.2.5 Preparation of the bacterial suspension

Staphylococcus aureus maintained on nutrient agar slants at 4°C were aseptically

transferred using a sterilized needle into fresh sterile broth culture. The tubes were

incubated at 37°C for 24 hours. For standardization of bacterial density, the turbidity of the

21
fresh 24 –hour old cultures were adjusted to 0.5 MacFarland standard (1× 108 cells/ml) by

diluting with the sterile broth. (18)(20)

In adjusting the turbidity of 24-hour old culture, both the standard and the inoculum

tube were held side by side and no more than 1 inch from the face of the Wickerham card

with adequate light present. The appearance of the lines through both suspensions was

compared. If the bacterial suspension appears lighter than the 0.5 McFarland standard,

more organisms were added to the tube from the culture plate. Whereas, if the suspension

appears more dense than the 0.5 McFarland standard, additional sterile broth was be

added to the inoculum tube in order to dilute the suspension to the appropriate density. In

some cases it was easier to start over rather than to continue to dilute a bacterial

suspension that is too dense for use. (18)

3.2.2.6 Paper disk diffusion assay

The plates with Mueller-Hinton agar was divided into 6 equal parts with a marking

pencil on the bottom of the plates (Figure 3). A sterile cotton swab was aseptically dipped

into the S. aureus broth culture. The cotton swab was rotated against the inside wall of the

tube to remove excess broth, then the swab was streaked evenly over the surface of the

plate and in three directions, leaving no gaps in between strokes. The plate was allowed to

dry for 3-5 minutes with the lid in place.

Filter paper disks of 6mm diameter has been shown to have the capacity to hold

0.005 mL(24). Thus each disk will be impregnated with 5 µL (0.005 mL) the respective

treatments and controls, then allowed to dry for 10 minutes. The individual disks were then

be placed and pressed lightly on the surface of the nutrient agar in the different sections

marked on the bottom of the plates. The plates were incubated at 37°C for 24 hours. The

zone of inhibition was measured (in millimeters) after overnight incubation(19)(25). Zone

diameters of the peel extracts with diameter of 7mm-14mm excluding the 6mm diameter of

22
the filter paper will be considered significant, this is based from the results of a previous

study conducted.(11)

3.2.2.7 Determination of MIC and MBC

The concentration within the preceding smallest concentration of extract with

significant zone of inhibition (7mm-14mm diameter) was used in MIC. Broth dilution test

was performed in determining the minimum inhibitory concentration of the plant extracts.

Two-fold serial dilution was used for this test, giving the following concentrations: 3840,

1920, 960, 480, 240, 120, 60, 30 and 15 µg.

In ten sterile tubes labelled “1” through “10”, 0.5ml of sterile broth was aseptically

added. 1 mL of peel extract was added to Tube 2 then serial dilution of the peel extracts

was carried out until Tube 9. All the tubes were then be inoculated with 0.5 mL of S.

aureus suspension and incubated at 37°C for 24 hours. Tube 10, containing only the

sterile broth and S. aureus suspension served as the control tube. Bacterial growth in

each tube was examined by observing turbidity. The lowest concentration without growth

is the minimal inhibitory concentration. (20)

Aliquots from all tubes that showed no growth and was inoculated in a Mueller-Hinton

agar medium using the streak plating technique. The plates were incubated at 37 °C for 24

hours. The plates were examined for bacterial growth by observing presence of S. aureus

colonies. Minimum bactericidal concentration is the smallest amount of drug per unit

volume that will kill the organism. Thus, plates with no growth indicated that the extract is

bactericidal at that dilution.(20)

3.2.2.8 Control for possible biases and errors

The experiment proper was conducted in an isolated controlled unit set at the

temperature of 37°C to minimize the possible effect of environmental factors. A facilitator

23
 with microbiology expertise supervised the editing of the experiment procedure in order to

monitor errors in preparation and storage techniques throughout the duration of the study.

The principle of blinding was also applied in the execution of the study and data collection.

Members were assigned solely to the execution of the experiment, observation, and data

collection. Unassigned members determined what type of treatment will be assigned to a

particular sample and were responsible for the result analysis, so as to minimize the

occurrence of possible researcher bias. Assigned members were oriented with the flow of

the experiment proper and proper data collection in order to minimize the occurrence of

observational bias.

3.2.3 Sampling

3.2.3.1 Selection of treatment and control

Selection for the type of banana species that were utilized for the study were based

on the species’ availability and accessibility to the public. Different banana species that

were common in the province of Cavite were determined. Based on the province’s

consolidated production report for the month of August 2011, Musa acuminata (lakatan)

garnered the top sales, followed by Musa balbisiana (saba) and Musa paradisiaca

(latundan).(4) Choices for banana species were further specified as to their distribution

among the districts of the province. Although majority of the local banana cultivars are

grown in the upland districts (i.e. Districts V – VII), the purchase of the banana samples

was done at the most convenient district for the study, the 4th district of Dasmarinas City
(26)
One subgroup consisting of three members will be in-charge of treatment allotment. Six

plates of nutrient agar were planned to be prepared for the study. Two plates were allotted

for observing the activity of extracts from different banana species (Musa paradisiaca,

Musa balbisianana and Musa acuminata) in different concentrations compared with a

positive, negative, and solvent control. Control groups were set up so as to validate that

24
the intended effect is indeed attributed to the independent variable of the study.

Vancomycin, the drug of choice for Staphylococcus aureus will be used as a positive
(27)
control, evaluating the antibacterial activity of the banana peel extracts . On the other

hand, distilled water will be used as the negative control for the study to serve as a
(11)
reference reflecting microbial growth . Methanol was utilized to serve as the solvent

control.

3.2.3.2 Sample size

The type of significance test to be done depends on the hypothesis. In this study,

since the null hypothesis states that the peel extracts obtained from the different species

of banana do not have comparable antibacterial activity against Staphylococcus aureus,

therefore a two-tailed test was employed because the null hypothesis does not tell the

specific direction of difference.(28)

In comparing the antibacterial activity of different species of banana, two controls and

three treatments were used. The zone of inhibition observed for each concentration per

treatment will be measured using a 6-inch ruler.

Determining the number of replicates to be used in an experiment is a matter of

judgement and the available resources. The following formula incorporating the

probabilities for type I and type II errors as well as the variance and true difference

anticipated was utilized in computing for the number of replicates: (29)

# of replicates = 2 (Zα/2 + Zβ )(σ/δ)2

Wherein Zα/2 is associated with Type I error; Zβ is associated with Type II error; σ is

associated with standard deviation; and δ is associated with the anticipated true difference

that might be detected among replicates.

25
 Values obtained for the formula were based from previous experiments on the

antibacterial activity of a particular species.(11) The study was set a confidence level of

95%, with a power of 80%. Based on these percentages, the following Z values for Type I

and Type II error are the following:

Zα/2: α = 0.05 (at 95% confidence level) Zβ: = 0.80

α/2= 0.025 Z value = 0.84
= 1 - 0.025
= 0.975
Z value = 1.96

Value for standard deviation is set at σ = 0.1, based on previous experiments. A value

of 0.14 is set for the anticipated true difference.

Calculation

# of replicates = 2 (Zα/2 + Zβ )(σ/δ)2

= 2 (1.96+0.84)(0.1/0.14)2

= 2 (2.8)(0.51)

= 2.86

~ 3 replicates will be used

3.2.3.3 Reference citations

In several studies investigating the antibacterial activity of unripe banana peels

conducted by Alisi, et. al and Sulaiman, et. al, n=3 was used for the number of replicates

with a significance of p = 0.05. Alisi, et. al made use of a two-way analysis of variance

(ANOVA) to determine the inhibition of bacterial dehydrogenase activity reflected in

dehydrogenase assays. (30) On the other hand, Sulaiman, et. al utilized one-way ANOVA

testing to establish the correlations between total phenolic and mineral contents with the
(31)
antioxidant activities of the pulp and peel obtained from 8 different cultivars.

26
3.2.4 Data collection

3.2.4.1 Sources of data (variables to be measured)

The independent variable in the study is the antibacterial activity of the banana peel

extracts from different species whereas the dependent variable is the inhibition of growth

of Staphylococcus aureus. Antibacterial susceptibility tests were performed to collect the

data needed in determining the antibacterial activity of different banana species against

Staphyloccous aureus. Thru antibacterial susceptibility tests such as Kirby-Bauer method

and Minimum Inhibitory Concentration (MIC), the zone of inhibition and lowest inhibitory

concentration can be obtained, respectively.

3.2.4.2 Method of data collection used

Results from the aforementioned tests were determined through observation of the

presence or absence of microbial growth in the agar plate for the Kirby-Bauer method, as

well as in the series of test tubes for MIC. The observation method is the preferred method

in the context of a laboratory experimental study. Data collection was done by making use

of standardized measurement methods and utilizing replications to achieve precision.(32) In

the study, a schematic diagram of the step-by-step procedure was developed to ensure

that uniformity in the observation process and data collection will be implemented among

the assigned observers. Three replicates per banana species for the Kirby-Bauer method

were prepared to assure the precision of the observed antibacterial activity.

Prior to the experiment proper, the group was divided into three subgroups for the

following tasks: 1) preparation of plant extract; 2) execution of antibacterial susceptibility

tests and data collection; and 3) data analysis. Members assigned in a particular subgroup

were oriented to the schematic process to ensure a smooth execution of the study and

uniformity in the observation process, thus reducing a possible researcher and

27
observational bias. The principle of blinding, as well as plans for controlling bias and other

possible errors have been discussed previously.

In addition, available calipers or rulers were calibrated to warrant accurate

measurement of zones of inhibition and extract concentration for the Kirby-Bauer method

and MIC tests, respectively.

3.2.4.3 Data collection tool

Collection tools provide the necessary data for analysis; in this respect, tools should

be strong enough in order for a research to yield a claim. Data collection tools are mainly

categorized into three: secondary participation, in-person observations, case studies and

analysis content.(33) In experimental designs however, these tools are rarely used. Instead,

the researchers use an array of experimental procedures to obtain the data.

The size of the zone of inhibition obtained from the Kirby-Bauer method is directly
(18)(19)
proportional to the sensitivity of the organism to the antibiotic , which in this particular

study is the sensitivity of Staphylococcus aureus to peel extracts of different banana

species. Infections due to organisms designated as sensitive to a given antibiotic are more

likely to respond clinically to that antibiotic than infections with strains designated as

intermediate or resistant.(34) Measured zones of inhibition from different concentrations of

the three banana test species were recorded in millimeters. Zone diameters of the peel

extracts with diameter of 7mm-14mm excluding the 6mm diameter were labelled as “S”

(sensitive). Diameters measuring below 7mm were labelled as “R” (resistant).

Antibiotic sensitivity expressed in the context of the minimal inhibitory concentration

gives quantitative data not obtainable with the Kirby-Bauer method. MIC is identified as

the smallest concentration of antibiotic that inhibits the growth of the test bacterium; thus,

these quantitative results are useful in predicting the amount of antibiotic that must be

attained to assure inhibition.(19) In MIC, only antibiotics that show inhibitory activity towards

28
 the bacterial isolate using the Kirby-Bauer method were tested further. The concentration

of banana peel extract observed to have the most sensitive activity was serially diluted to

make a range of antibiotic concentrations that encompass the concentration used in the

Kirby-Bauer method.(35)(36) Results of MIC were recorded in μg/mL.

The data obtained were organized in tables, as shown in Appendix A. This functioned

as the data sheet and also aided in the analysis of the data. The tables provided the

analyst of the experiment the specific data required for the evaluation of the hypotheses.

Data collected from Kirby-Bauer method were recorded in Tables I and II, for the treatment

and control respectively. The measured zone of inhibition (mm) from the three replicates

of different concentrations and species were recorded and their respective means ±

standard deviation were computed. In this way, the analyst can assess the variation

among the zones of inhibition observed due to possible errors and discrepancy of the

measurements.

Data collected from MIC, on the other hand, were recorded in Table III. The resulting

concentration of peel extracts after serial dilution using a series of ten tubes as well as

observation for microbial growth in each tube were recorded. Presence of turbidity in the

tube indicated growth. (36)

3.2.5 Method of data analysis

3.2.5.1 Descriptive statistics

The mean diameter obtained among the three replicates was calculated in measuring

the central tendency of the zone of inhibition produced by the peel extract. Measures of

central tendency are inappropriate for the values obtained in conducting MIC and MBC

since these tests are performed only once. Furthermore, the standard deviation was

computed from the value gathered from the zone of inhibition. The final data were then

reported as mean + standard deviation.

29
 3.2.5.2 Inferential statistics
One-way analysis of variance (ANOVA) was used as the test statistic since the study

is dealing with more than two independent study groups. This is based on the premise that

each peel extract group’s effect will be unique from the other as well as from the

established control groups. One-way ANOVA is helpful in determining the probability of

observing a difference existing among the means of several study groups at the same

time, minimizes the possibility of committing a type I error which may result from

conducting multiple t-tests.(37) The confidence interval for this study was set at 95%, with

the value for α at 0.05. These values have been derived from previous studies on the

antibacterial activity of plant extracts.(11)

3.2.6 Other considerations

The experiment conducted was not derived from an original study design.

Furthermore, ethical considerations in the form of safety, confidentiality, and informed

consent were found not to be applicable for the design employed, since the study included

the analysis of the antibacterial activity of chosen plant extracts in vitro.

30
 Chapter IV
RESULTS

4.1 Descriptive statistics

The antibacterial activity of the peel extracts obtained from Musa acuminata, Musa

balbisiana, and Musa paradisiaca were initially determined using the disk diffusion

method. Each species were prepared in three concentrations (30 µg, 300 µg, and 3,000

µg) and were observed for their respective antibacterial activity as evidenced by the zones

of inhibition produced. However, paper disk diffusion results revealed absence of inhibition

in all species. Difficulty in replicating the methods from previous studies wherein zones of

inhibition were observed brought about the need to conduct trials in determining the

antibacterial activity of the peel extract. In each trial, the extract concentration used was

set at 3,000 µg. Several variables such as characteristics of the banana peel (e.g. color

and storage duration), duration of blending the chopped peels, and the extract storage

temperature were considered. Values for mean + standard deviation are then considered

to be set at zero, since the values gathered per trial were null.(38) Table 1 shows the

measurements of the zones of inhibition among the different banana species.

Table 1. Zone of inhibition (mm) of peel extracts of different banana species using paper
disk diffusion in different trials

TRIAL 1 TRIAL 2 TRIAL 3 TRIAL 4 TRIAL 5 TRIAL 6

Musa
0 0 0 0 0 0
sapientum

Musa
0 0 0 0 0 0
paradisiaca

Musa
0 0 0 0 0 0
acuminata

31
4.2 Inferential statistics

Since the values obtained were set at zero, replacing these on the F-ratio (i.e. statistic

used in utilizing ANOVA) would reveal that the p-value would progress towards zero,

implying that the value of statistical significance is within the non-rejection area of the

normal distribution curve.(39) A conclusion can then be made that there are no sufficient

evidences to reject that the peel extracts obtained from the different species of banana do

not have comparable antibacterial activity against Staphylococcus aureus, in favor of the

alternative hypothesis.

∑ ̅ =0

Figure 4. Demarcations for accepting or rejecting the null hypothesis

(Retrieved from: https://statistics.laerd.com/statistical-guides/img/Hypotest_5.gif)

32
 Chapter V
DISCUSSION

Akter et. al evaluated the antimicrobial and cytotoxic activities of different extracts of

Musa sapientum, L. subsp. sylvestris fruits (MSSE). The findings of their study

demonstrated that the methanolic extract of Musa sapientum pulp possessed good

antimicrobial activity against all 13 Gram-positive and Gram-negative bacteria. The peel

extract showed significant activity against all the organisms, while the seeds showed no

activity against any organism.(16) Focusing on the peel extract, it showed activity against all

the test organisms with a zone of inhibition of 7-9mm, which were in contrast with the

result of the trials done on the 3 variants of bananas. Another study conducted by Mokbel

and Hashinaga also explored on the antibacterial properties of banana peel extracts, this

time utilizing the Cavendish variant. It also agreed with the study conducted by Akter et. al

wherein these peel extracts displayed antibacterial properties against a range of Gram-

positive and Gram-negative bacteria. Mokbel and Hashinaga also used methanol as

solvent for the extract.(11) In contrast, the peel extracts prepared with similar solvent in the

study were already set at the highest concentration of 3,000 µg but showed no sign of

antimicrobial activity against Staphylococcus aureus, as manifested by a 0mm zone of

inhibition. It may have been due to the difference in the methods used. The procedure

done in the study of Akter et. al with regards to extraction was more complex, utilizing a

Soxhlet apparatus to obtain the extract, compared with the crude procedures done in this

study.(16) On the other hand, the methodology incorporated an extraction procedure that

was not done prior to this study: a compromise between the Soxhlet method of extraction

and the soaking technique. This method, however, has not been used (or tested) insofar

as the recent similar studies are concerned. The lack of materials such as a Soxhlet

31
 apparatus may have had a bearing on the results, in terms of the quality of the plant

extract produced.

Another difference noted was on the degree of ripeness of the plants used. Both

mature and unripe fruits were used whereas in the study, the researchers used only ripe

bananas of the 3 variants. The ripeness of the bananas purchased were already at its

peak, assuming that the active ingredients are at its potent state. However, in both

studies, the concerns raised up in the study such as the influence of plant characteristics

(e.g. correlation of degree of ripeness with the potency of active ingredients) was not

discussed.

In addition, extract preparation and storage protocols which may have also influenced

the optimal extraction of active ingredients were also not discussed in previous studies.

Areas such as the establishment of the initial concentration (concentration prior to disc

impregnation) and the impregnation of the paper disc are of great importance. In the

matter of establishing the concentration, the initial concentration varied all throughout the

trial. Utilization of an evaporator is commonly used to ensure that the initial concentration

of the crude extract is almost pure or 100%. However, this experiment used passive

evaporation technique that is time-consuming and erroneous. Thus, only an

approximation, not an accurate value, of the initial concentration was documented.

Regarding the disc impregnation, inadequacy of the amount of extract in the paper disc

could have resulted in the failure to elicit a zone of inhibition during the course of the trial.

Further studies looking at the following concerns of the influence of confounding

variables (i.e. plant characteristics, extract preparation and storage protocol) are needed.

5.1 Limitations of the study

Due to the initial results gathered, conducting trial sessions (paper disk diffusion

assay) to confirm the antibacterial activity of the peel extracts were added to the

32
experiment procedure during the course of the study. This was deemed as necessary in

order to avoid unwarranted expense of time and finances. Unfortunately, due to the time

curb allotted for the experiment proper, a deeper investigation on the influence of

confounding variables and modification of the experiment procedure in terms of extract

preparation and storage protocol were not implemented; thus, other steps in the

experiment procedure such as the determination of MIC and MBC were also not

implemented.

33
 Chapter VI
CONCLUSION AND RECOMMENDATIONS

6.1 Conclusion

Based on the results, the proponents of the study conclude that the peel extracts

obtained from the different species of banana do not have comparable antibacterial

activity against Staphylococcus aureus. Furthermore, the zones of inhibition, minimal

inhibitory concentrations, and minimal bactericidal concentrations were not established

since trials conducted via paper disk diffusion showed the absence of microbial growth

inhibition.

6.2 Recommendations

In light of the limitations of the study and said conclusion, the following

recommendations are proposed:

1) Careful re-evaluation of the method of extraction is highly suggested in order

to establish its capability for optimal plant extraction.

2) Modification of the experiment procedure, specifically in the areas of

establishing the initial concentration and impregnation of paper discs, ought

to be re-evaluated to ensure proper documentation of the concentrations and

to satisfy the adequacy of the extract.

3) A follow-up study should be conducted so as to provide further insight to the

antibacterial properties of the Musa genus as suggested by other studies.

34
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(4) Provincial Government of Cavite. “Agri – P.Noy High Value Commercial Program” – Consolidated Monthly
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banana (Musa sapientum L.), lemon grass (Cymbopogon citratus S.), and turmeric (Curcuma longa L.) on
pathogens. African Journal of Biotechnolog, 8 (7), 1176-1182.

(13) Scott, W., H. McKay, P. S. Schaffer and T. Fontaine. The Partial Purification and Properties of Antibiotic
Substances from the Banana (Musa sapientum). Bureau of Agricultural and Industrial Chemistry,
Agricultural Research Center, Beltsrille, Maryland.

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species: importance and its enormous pharmacological action. Journal of Pharmacognosy and Herbal
Formulations, 1 , 2-11.

(15) Akter, S. and Imam, M.Z. (2011) Musa paradisiaca L. and Musa sapientum L. : A Phytochemical and
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(16) Akter, S. and Imam, M.Z. (2011) Antimicrobial and cytotoxic properties of different extracts of Musa
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Passionfruit, Langsat, Duku, Rambutan and Rambai)

(18) Mahon, C.R., Lehman, D.C., and Manuselis, G. (2007). Textbook of Diagnostic Microbiology. Missouri:
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(19) Forbes, B.A., Sahm, D.F., and Weissfeld, A.S. (2007) Bailey and Scott’s Diagnostic Microbiology. Missouri:
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(21) “Scientific control”. http://encyclopedia.thefreedictionary.com. Retrieved last 5 August 2011.

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(22) Edwards, B. G. (2003). Banana peel extract composition and method for extraction. Delft Pharma
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(29) North Dakota State University. “Size of an experiment – the number of replicates to use”. Retrieved from:
http://www.ndsu.edu/ndsu/horsley/ExptSize.pdf, 26 September 2011.

(30) Alisi, C.S., et. al. (2008) “Inhibition of dehydrogenase activity in pathogenic bacteria isolates by aqueous
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(32) McKubre, M.C.H. (2008) “The Importance of Replication”. ICCF-14 International Conference on Condensed
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(33) The Three Main Types of Data Collection Tools. (n.d.). Retrieved October 1, 2011, from Scienceray:
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(34) Vasanthakumari (2007). Practical Microbiology. BI Publications Pvt Ltd. Copyright. http://books.google.com/

(35) Parija,S.C.(2009).Textbook of Microbiology & Immunology. Elsevier India

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(37) “Analysis of Variance”. Retrieved from http://www.wikipedia.com

(38) MacMillan A., et al. (2007) “Basic statistics: mean, median, average, standard deviation, z-scores, and p-
value”. Retrieved from https://controls.engin.umich.edu

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http://stats.statexchange.com

36
APPENDIX

37
 APPENDIX A
Data Collection Tool
The zones of inhibition observed in the agar plates where different concentrations of the peel extracts obtained from
three different banana species will be recorded in Table I in millimetres (mm); on the other hand, the zones of inhibition
observed in the agar plates assigned as positive, negative, and solvent controls will be recorded in Table II and will also be
recorded in millimetres (mm). Both measurements will be obtained using the paper disk diffusion assay. The peel extract’s
antibacterial activity observed in the disk diffusion assay will be quantified by obtaining their respective minimal inhibition
concentration (MIC) using 10 test tubes in serial dilution. Observations will be recorded in Table III as micrograms per millilitre
(µg/mL). Specific names and concentrations of the species (treatment) as well as the controls will not be indicated in the form
to conform to the principle of blinding in the study, thus minimizing possible researcher bias.

OBSERVER:
DATE AND TIME OF OBSERVATION:

TREATMENT

CONCENTRATION OF BANANA PEEL EXTRACT

R1 R2 R3 M+SD INT

A B C A B C A B C A B C A B C

SPECIES (1)

SPECIES (2)

SPECIES (3)

CONTROL

CONTROL

R1 R2 R3 M+SD INT

A B C A B C A B C A B C A B C

SPECIES (1)

SPECIES (2)

SPECIES (3)

38
MINIMAL INHIBITORY CONCENTRATION

SPECIES (1)

TUBE NO. 1 2 3 4 5 6 7 8 9 10
Antimicrobial agent concentration (μg/mL)

REPLICATE (1)
REPLICATE (2) CONTROL

REPLICATE (3)

Growth (+/- turbidity)

SPECIES (2)

TUBE NO. 1 2 3 4 5 6 7 8 9 10

Antimicrobial agent concentration (μg/mL)

REPLICATE (1)

REPLICATE (2) CONTROL

REPLICATE (3)

Growth (+/- turbidity)

SPECIES (3)

TUBE NO. 1 2 3 4 5 6 7 8 9 10

Antimicrobial agent concentration (μg/mL)

REPLICATE (1)

REPLICATE (2) CONTROL

REPLICATE (3)

Growth (+/- turbidity)

39
 APPENDIX B
Laboratory Materials Used

40