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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
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TABLE OF CONTENTS
LIST OF TABLES 2LIST OF FIGURES 2
ABSTRACT 3
1. INTRODUCTION 41.1 Research question 4
1.2 Research hypothesis 4 1.3 Background of the research question 4
1.4 Rationale for the study 51.5 Review of literature and conceptual framework 6
2. OBJECTIVES 12
3. METHODOLOGY 133.1 Research design 133.2 Method 15
4. RESULTS 294.1 Descriptive statistics 294.2 Inferential statistics 30
5. DISCUSSION 315.1 Limitations of the study 32
6. CONCLUSION AND RECOMMENDATIONS 346.1 Conclusion 346.2 Recommendations 34
REFERENCES 35
APPENDIX 37
2
LIST OF TABLES
Table 1. Zone of inhibition of peel extracts of different banana species 30using paper disk diffusion in different trials
LIST OF FIGURES
Figure 1. Conceptual framework 12Figure 2. Schematic diagram of the research design 14Figure 3. Allocation of treatment and control 18Figure 4. Demarcations for accepting or rejecting the null hypothesis 31
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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.
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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
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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
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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
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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)
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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
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peel extract was also shown to be more active against Staphylococcus (Gram-positive)
than Pseudomonas species (Gram-negative). Furthermore, the review of pharmacological
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
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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
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
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the experimental proper, wherein there may be discrepancies in measuring the outcome
observed.
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.
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CONTAMINATION
POSSIBLE CONFOUNDING FACTORS
Exposure to environmental factors
Preparation and storage techniques
Characteristics of the factor
Inherent defense mechanisms of the sample of interest
Characteristics of the factor
Type of solvent used Type of
phytochemicals present which contributes to antibacterial activity
Concentration of the extract
Inhibition of growth of Staphylococcus aureus
Antibacterial activity of
banana peel extract
Figure 1. Conceptual framework
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.
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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
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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 propagation.
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(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
microbial growth achieved with the least concentration of the peel extract. (18)(19) 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
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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)
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CONC.(A)
CONC. (B)
CONC. (C)
(+) CONTROL
(-) CONTROL
SOLVENT ONLY
CONC.(A)
CONC. (B)
CONC. (C)
(+) CONTROL
(-) CONTROL
SOLVENT ONLY
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.
Musa paradisiaca Musa balbisianana
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CONC.(A)
CONC. (B)
CONC. (C)
(+) CONTROL
(-) CONTROL
SOLVENT ONLY
Musa acuminata
Figure 3: Allocation of treatment and control
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)
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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
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)
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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
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
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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
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)
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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
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
24
(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
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
control, evaluating the antibacterial activity of the banana peel extracts (27). On the other
hand, distilled water will be used as the negative control for the study to serve as a
reference reflecting microbial growth (11). 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.
25
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.
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 = 0.975Z 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
26
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
antioxidant activities of the pulp and peel obtained from 8 different cultivars. (31)
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
27
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
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
proportional to the sensitivity of the organism to the antibiotic (18)(19), 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
28
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
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)
29
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.
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.
31
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 sapientum
0 0 0 0 0 0
Musa paradisiaca
0 0 0 0 0 0
Musa acuminata
0 0 0 0 0 0
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.
SST=∑ n(x−x )2 = 0
MST= SSTP−1
=0
SSE=∑ (ni−1 ) si2=0
MSE= SSEn−p
=0
F=MSTMSE
=0/Error
32
Figure 4. Demarcations for accepting or rejecting the null hypothesis (Retrieved from: https://statistics.laerd.com/statistical-guides/img/Hypotest_5.gif)
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
33
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
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
34
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
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.
35
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:
36
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.
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(2) Philippine Institute of Traditional and Alternative Health Care. http://www.doh.gov.ph/pitahc/Index.html
(3) Philippine Medicinal Plants: Banana (“Saging”). http://www.stuartxchange.org/Saging.html
(4) Provincial Government of Cavite. “Agri – P.Noy High Value Commercial Program” – Consolidated Monthly Report (August 2011).
(5) Levinson, W. (2006) Review of Medical Microbiology and Immunology. New York City: McGraw-Hill Professional.
(6) World Health Organization. “Initiative for Vaccine Research (VR): Bacterial infections”. Retrieved from http://www.who.int, 23 July 2011.
(7) World Health Organization. “Combating communicable disease: communicable disease surveillance and response”. Retrieved from http://www.wpro.who.int/nr/rdonlyres/572c5084-4adb-4634-8e85-ee8993da3e3d/0/05_csr.pdf, 23 July 2011.
(8) Carlos, C.C. (2006) The 2005 antimicrobial resistance surveillance data. PIDSP Journal, 10, (9 pages).
(9) Arakama, M., et. Al (2010) Emergence of methicillin-resistant Staphylococcus aureus among patients in a tertiary renal medical center. Philippine Journal of Microbiology and Infectious Diseases, 39, 28-33.
(10) World Health Organization. “Traditional medicine”. Retrieved from http://www.who.int, 23 July 2011.
(11) Mokbel, M.S. and Hashinaga, F. (2005). Antibacterial and antioxidant activities of banana (Musa AAA., cv. Cavendish) fruits peel. American Journal of Biochemistry and Biotechnology, 3, 125-131.
(12) Fagbemi, J.F.; Ugoji, E.; Tayo, A.; Omotoyin, A. (2009). Evaluation of the antimicrobial properties of unripe banana (Musa sapientum L.), lemon grass (Cymbopogon citratus S.), and turmeric (Curcuma longa L.) on pathogens. African Journal of Biotechnolog, 8 (7), 1176-1182.
37
(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.
(14) Banerjee, S.; Halder, B.; Barman, N.R.; Ghosh, A.K. (2010). An overview on different variety of Musa 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 Pharmacological Review. Journal of Applied Pharmaceutical Science, 1 (5), 14-20.
(16) Akter, S. and Imam, M.Z. (2011) Antimicrobial and cytotoxic properties of different extracts of Musa sapeintum, L. subsp. Sylvestris. Jourmal of Applied Pharmaceutical Science, 2(8).
(17) Hamid et al. Antimicrobial Activity of some Tropical Fruit Wastes (Guava, Starfruit, Banana, Papaya, Passionfruit, Langsat, Duku, Rambutan and Rambai)
(18) Mahon, C.R., Lehman, D.C., and Manuselis, G. (2007). Textbook of Diagnostic Microbiology. Missouri: Saunders.
(19) Forbes, B.A., Sahm, D.F., and Weissfeld, A.S. (2007) Bailey and Scott’s Diagnostic Microbiology. Missouri: Elsevier.
(20) Department of Microbiology and Parasitology, DLS-HSI College of Medicine (2011). “Laboratory Manual on Microbiology and Parasitology”.
(21) “Scientific control”. http://encyclopedia.thefreedictionary.com. Retrieved last 5 August 2011. (22) Edwards, B. G. (2003). Banana peel extract composition and method for extraction. Delft Pharma
International, Ogden Utah. Retrieved from: http://www.freepatentsonline.com/5989559.pdf
(23) Katzung, B. (2006). Basic and Clinical Pharmacology (10th edition) McGraw Hill. pp. 134-138.
(24) Lalitha, M. (2004) Manual on Antimicrobial Susceptibility Testing. Retrieved from: www.ijmm.org/documents/Antimicrobial.doc
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(26) Republic Act No. 9272 – “An Act Reapportioning the Province of Cavite into Seven (7) Legislative Districts”. Retrieved from http://www.senate.gov.ph last September 6, 2011.
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(28) UCLA Academic Technology Services. “What are the differences between one-tailed and two-tailed tests?”. http://www.ats.ucla.edu/stat/mult_pkg/faq/general/tail_tests.htm. Retrieved last 11 September 2011.
(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 extracts of Musa paradisiaca (var. Sapientum)”. African Journal of Biotechnology. Vol. 7 (12), pp. 1821-1825.
(31) Sulaiman, S.F. et. al. (2011) “Correlation between total phenolic and mineral contents with antioxidant activity of eight Malaysian bananas (Musa sp.)”. Journal of Food Composition and Analysis. Vol. 24 (1), pp 1-10. Retrieved from: http://www.sciencedirect.com/science/article/pii/S0889157510001985, 12 September 2011.
(32) McKubre, M.C.H. (2008) “The Importance of Replication”. ICCF-14 International Conference on Condensed Matter Nuclear Science.
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(33) The Three Main Types of Data Collection Tools. (n.d.). Retrieved October 1, 2011, from Scienceray: http://scienceray.com/technology/information/the-three-main-types-of-data-collection-tools/
(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 Copyright. http://books.google.com/
(36) Neelima Garg, K. L. Garg and K. G. Mukerji. (2010). Laboratory Manual of Food Microbiology. I. K. International Pvt Ltd. Copyright. pp. 75-78. http://books.google.com/
(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
(39) “What is the meaning of an F value less than 1 in one-way ANOVA?” Retrieved from http://stats.statexchange.com
APPENDIX
39
APPENDIX AData 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)
40
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)
MINIMAL INHIBITORY CONCENTRATION
SPECIES (1)
TUBE NO. 1 2 3 4 5 6 7 8 9 10
Antimicrobial agent concentration (μg/mL)
REPLICATE (1)
CONTROLREPLICATE (2)
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)
CONTROLREPLICATE (2)
REPLICATE (3)
Growth (+/- turbidity)
41
SPECIES (3)
TUBE NO. 1 2 3 4 5 6 7 8 9 10
Antimicrobial agent concentration (μg/mL)
REPLICATE (1)
CONTROLREPLICATE (2)
REPLICATE (3)
Growth (+/- turbidity)
APPENDIX BLaboratory Materials Used
42