Download - 2edited Research Output 7-Edited
-
8/3/2019 2edited Research Output 7-Edited
1/22
RESEARCH OUTPUT 7: Research Protocol
Adviser: Dr. I.A. Ilano
Submitted by: GROUP 9-A
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
Date:
November 8, 2011
Topic: "A comparative study on the antibacterial activity peel extracts obtained from Musa
acuminata, Musa balbisiana, and Musa paradisiacaagainst Staphylococcus aureus
-
8/3/2019 2edited Research Output 7-Edited
2/22
1
Table of Contents
I. Introduction 2
A. Research Question ........... 2
B. Research Hypotheses ... 2
C. Background of the Research Question .. 2
D. Significance of the Study .. 3
II. Research Objectives .. 3
III. Literature Review ........... 4
A. Epidemiology of Disease of Interest 4
B. Epidemiology of Exposure of Interest .. 4
C. Summary of Related Studies 5
D. Conceptual Framework . 6
IV. Methodology 9
A. Research Design 9
B. Experiment Procedure . 10
C. Sampling ............ 14
D. Data Collection ............. 17
V. Bibliography ............... 19
VI. Appendices ............... 20
-
8/3/2019 2edited Research Output 7-Edited
3/22
2
I. INTRODUCTION
A. Research question
Which species of banana peel extract has greater antibacterial activity against
Staphylococcus aureus?
B. Research hypotheses
i. Working hypothesis
The peel extracts obtained from the different species of banana have comparable
antibacterial activity against Staphylococcus aureus.
ii. Null hypothesis
The peel extracts obtained from the different species of banana do not have
comparable antibacterial activity against Staphylococcus aureus.
C. 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
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
-
8/3/2019 2edited Research Output 7-Edited
4/22
3
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 sapientumspecies 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.
D. Significance of 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 local
banana variants which may be used as active ingredients against drug-resistant
microorganisms such as Staphylococcus aureus.
II. RESEARCH OBJECTIVES
A. General objective
To determine which species of banana peel extract has greater antibacterial activity
against Staphylococcus aureus.
B. Specific objectives
1. To measure the zones of inhibition of peel extracts obtained from Musa acuminata,
Musa balbisiana, and Musa paradisiacaagainst Staphylococcus aureususing the
disk diffusion method.
2. To establish the respective minimum inhibitory concentrations of peel extracts
obtained from Musa acuminata, Musa balbisiana, and Musa paradisiaca against
Staphylococcus aureususing broth dilution test.
3. To compare the obtained zones of inhibition and minimum inhibitory concentrations
of the peel extracts from different banana species against Staphylococcus aureus.
-
8/3/2019 2edited Research Output 7-Edited
5/22
4
III. LITERATURE REVIEW
A. 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 agencys 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. aureusamong 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
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)
B. 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 bytheWorld 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
-
8/3/2019 2edited Research Output 7-Edited
6/22
5
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) Further discussions
regarding the designs and methods, as well as results of these studies are covered in the
next portion of the study, Summary of related/similar studies.
C. 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 extracts potency and enhanced its antimicrobial activity.
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 activity. Among the specific
compounds isolated from green banana peel, d-malic acid and 12-hydroxystearic
-
8/3/2019 2edited Research Output 7-Edited
7/22
6
exhibited the most active antimicrobial response, with an MIC varying between 140-750
ppm, respectively. (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 paradisiacaand Musa sapientumshowed 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
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 Musasapientum, L. subsp. Sylvestris fruits (MSSE). The methanolic extract of Musa
sapientumpeel 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 (Psidiumguajava); ripe, unripe and
leaves of starfruit (Averrhoacarambola); ripe and unripe banana (Musasapientumvariety
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 (Baccaureamotleyana) peel were evaluated for antimicrobial
activity against Gram-positive bacteria, Gram-negative bacteria, yeast and fungi(Staphylococcusaureus, Bacillussubtilis, 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
-
8/3/2019 2edited Research Output 7-Edited
8/22
7
yeast or fungi. Extracts from bananas, papayas, passionfruit peel, Lansiumdomesticum
peels and rambutan peels showed activity against Candida lypolyticawhile extracts from
guava showed strong activity against Saccharomycescerevisiae. Unripe banana showed
activity against all the bacteria except towards P. vulgaricus.(17) In the study conducted by
Mokbel and Hashinaga, fresh green and yellow banana peel of Musa, cv. Cavendishfruits
were treated with 70% acetone; and afterwards, partitioned with chloroform and ethyl
acetate. 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 whereas the antimicrobial activities of the extracts and isolated
components were evaluated using paper disc methods and minimum inhibition
concentration (MIC). Results of the study showed that the ethyl acetate and water soluble
fractions of green peel showed higher antimicrobial activity than yellow banana peel. The
antioxidant activity of the water extracts from green banana peel was comparable to those
of synthetic antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene.
(11)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.
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.
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.
-
8/3/2019 2edited Research Output 7-Edited
9/22
8
D. 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 banana peel
POSSIBLE CONFOUNDING
FACTORS
Exposure to environmentalfactors
Preparation and storagetechniques
CONTAMINATION
Characteristics of the
factor
Type of solvent used
Type of
phytochemicals
present which
contributes to
antibacterial activity
Concentration of the
extract
Characteristics of the
factor
Inherent defense
mechanisms of the
sample of interest
Inhibition of growth
ofStaphylococcus
aureus
-
8/3/2019 2edited Research Output 7-Edited
10/22
9
IV. METHODOLOGY
A. Researchdesign
The study will utilize an analytic experimental design, wherein the independent
variable under observation will be assigned particularly to a treatment group and will be
compared to a positive and negative control group. Figure 2 illustrates the design of the
study.
Operational definition of variables
1. Independent variable
The antibacterial activity of the different species of banana peel extracts on the
growth of Staphylococcus aureus serves 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 twomechanisms, 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 will be chosen as samples for the study.
2. Dependent variable
The expected result from the study will be the inhibition of the growth of
Staphylococcus aureuson nutrient agar medium. 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.(18) Therefore, strict compliance with the incubation of disk diffusion plates
within the allotted time of 24 hours for the Staphylococcispecies should be observed so
as to achieve reliable results and prevent possible growth of the organism. (19)
-
8/3/2019 2edited Research Output 7-Edited
11/22
10
Inhibition of growth will be determined qualitatively using the disk diffusion method,
as represented by zones of inhibition. Broth dilution test will also be employed to quantify
the degree of the antibacterial activity by determining the peel extracts minimum inhibitory
concentration. Minimum inhibitory concentration (MIC) of an antibiotic is the smallest
amount per unit volume that will inhibit the growth of a certain organism. In broth dilution
test, the end point tube will represent the peel extracts MIC. It is the tube wherein there is
complete inhibition of growth of bacteria with the least concentration of the peel extract,
which can be visually observed as absence of turbidity. (21)
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 may significantly influence the accuracy and analysis of the results. (18)(19)(21)
B. Experiment procedure
For the study, microbial growth inhibition will be 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 agentdiffuses 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
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 agents 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 ofthe test bacterium. This will be the parameter used in the study to reflect the presence of
microbial growth inhibition.(18)
Furthermore, control groups will be 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
-
8/3/2019 2edited Research Output 7-Edited
12/22
11
intended effect, thus minimizing the probability of false negatives. (20) For the study, the
drug of choice for Staphylococcus aureus, Vancomycin, will be utilized as the standard for
evaluating the antibacterial activity of the plant extracts. A negative control will also be set
up in order to confirm that the effect produced was not influenced by extraneous factors,
thereby minimizing the probability of false positives.(20) For the study, distilled water will be
used as a reference reflecting microbial growth, indicating that the treatment applied has
not been effective.
In addition, a solvent group will also be set up to evaluate the influence of the solvent
on the observed microbial growth inhibition. For the study, methanol will be used as
reference.
Quantitative measures, such as the minimal inhibitory concentration (MIC), will also
be applied to validate the degree of the antibacterial activity of the banana peel extracts.
Minimal inhibitory concentration refers to the smallest amount per unit volume that will
inhibit the growth of a certain organism.(18)(21) Broth dilution tests, wherein tubes containing
decreasing concentration of the plant extract tested are prepared by serial dilution, will be
conducted to determine the plant extracts respective MIC.(21)
Steps to be undertaken
a) Physical set-up
The study will use nutrient agar plates in observing the activity of peel extracts fromdifferent banana species (Musa paradisiaca, Musa balbisianana and Musa acuminata)
compared with the positive control, negative control and solvent control. Three replicates
will be used. Figure 3 illustrates the distribution of the treatments and control among the
bacterial culture.
Figure 3:Allocation of treatment and control
SPECIES
(A)
SPECIES
(B)
SPECIES
(C)
(+)
CONTROL
(-)
CONTROL
SOLVENT
ONLY
-
8/3/2019 2edited Research Output 7-Edited
13/22
12
b) Procurement of plant materials
Peels from different species of banana (Musa paradisiaca, Musa balbisianana
and Musa acuminata) will be collected from Dasmarias, Cavite and will be identified
taxonomically.
c) Preparation of extract
The banana peels (300 g for each variety) will be washed with water then coarsely
chopped. The peels will then be placed in a solvent, methanol, at a ratio of 1 gram banana
peel per 4.5 mL of methanol. The mixture will then be homogenized using a blender then
transferred into their respective reaction tanks which will be left for 3-48 hours at room
temperature. The mixture will be agitated or mixed periodically to prevent separation of
masticated peel from the solvent. Change of color from yellow to amber to an opaque
black liquid will indicate that the reaction is already complete. Upon completion of the
reaction step, the mixture will then be filtered through a screen mesh. The collected filtrate
will be further filtered through flat filter paper. The volume of the solution obtained after
filtration will be recorded. The extracts will also be stored in a desiccator at 5C. (22)
d) Preparation of peel extract stock solution
The crude extract with an initial concentration of 22 g/mL will be diluted with
distilled water to 0.001 g/mL (1000 ug/mL). To obtain the 1000 ug/mL final concentration
the concentration-volume ratio will be used.
(Initial Concentration) (Initial Volume) : (Final Concentraton) (Final Volume)
( 220 000 ug/mL) (Initial Volume) : (1000 ug/mL) (X)
e) Preparation of bacterial suspension
Staphylococcus aureus maintained on nutrient agar slants at 4C will be
aseptically transferred using a sterilized needle into fresh broth culture. The tubes will be
incubated at 37C for 24 hours. For standardization of bacterial density, the turbidity of the
fresh 24hour old cultures will be adjusted to 0.5 MacFarland standard (1 108 cells/ml)
by diluting with the sterile broth. (18)(21)
f) Paper disk diffusion assay
The plates with nutrient agar will be divided into 4 equal parts with a marking
pencil on the bottom of the plates. A sterile cotton swab will be aseptically dipped into the
-
8/3/2019 2edited Research Output 7-Edited
14/22
13
S. aureusbroth culture. The cotton swab will be rotated against the inside wall of the tube
to remove excess broth, then the swab will be streaked evenly over the surface of the
plate and in three directions, leaving no gaps in between strokes. The plate will be allowed
to dry for 3-5 minutes with the lid in place. Filter paper disks (6mm) will be impregnated
with the respective treatments and controls by dropping 1mL to each disk using a
micropipette then will be allowed to dry for 10 minutes. The individual disks will 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 will be incubated at 37C for 24 hours. The
zone of inhibition will be measured (in millimeters) after overnight incubation. (19)(23)
g) Determination of MIC
Two-fold broth dilution test method will be used in determining the minimum
inhibitory concentration of the plant extracts. In ten sterile tubes labeled 1 through 10,
0.5ml of sterile broth will be aseptically added. Then, 0.5 ml of the peel extract will be
added to tubes 1 and 2. Serial dilution of the peel extracts will be carried out from tube 2
until tube 9. All the tubes will then be inoculated with 0.5 mL of S. aureussuspension and
incubated at 37C for 24 hours. Tube 10, containing only the sterile broth and S. aureus
suspension will serve as the control tube. Bacterial growth in each tube will be examined
by observing turbidity. The highest dilution without growth is the minimal inhibitory
concentration.
Possible biases and limitations
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 (24), 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 the manner of
implementing the experiment procedure as well as in measuring the outcome observed.
The in vitrosetting of the study may be accounted to the limitation of the external
validity of the findings.(25) Extract activity observed in a controlled setting may be observed
differently when applied in the clinical setting.
The possible sources of errors in the study may come from faulty observation of
preparation and storage techniques of both the inoculum and plant extracts. Application of
the inoculum may contribute to the inaccuracy of results. Thickly applied inoculum onto the
-
8/3/2019 2edited Research Output 7-Edited
15/22
14
agar may result to a small zone of inhibition; whereas, a thinly applied inoculum may result
to a large zone of inhibition.(18)(21) Inadequate or haphazard application of plant extracts as
well as those of the controls on to the paper disks also contribute to the inaccuracy of
results.(18)
Control plan for biases and possible errors
The study will be conducted in an isolated controlled unit set at the temperature of
37C to minimize the possible effect of environmental factors. A facilitator with
microbiology expertise will be supervising the study in order to monitor errors in
preparation and storage techniques throughout the duration of the study. The principle of
blinding will be applied in the execution of the study and data collection. Three members
will be assigned solely to the execution of the experiment, observation, and data
collection. Another subgroup of three members will determine what type of treatment will
be assigned to a particular sample. The remaining three members will be responsible for
the result analysis, so as to minimize the occurrence of possible researcher bias.
Assigned members will also be oriented with the flow of the experiment proper (as
illustrated in the procedure schematic diagram) and proper data collection in order to
minimize the occurrence of observational bias.
C. Sampling
Selection of treatment and control
Selection for the type of banana species that will be utilized for the study will be
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 provinces
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
will be done at the most convenient district for the study, the 4 th district of Dasmarinas City
(26). Figure 4 depicts the selection process for the independent variable of the study.
-
8/3/2019 2edited Research Output 7-Edited
16/22
15
Figure 4: Selection process for banana species
One subgroup consisting of three members will be in-charge of treatment allotment.
Nutrient agar plate will be used for observing the activity of extracts from different banana
species (Musa paradisiaca, Musa balbisianana and Musa acuminata) in different
concentrations compared with a positive and negative control. Control groups as well as a
solvent group will be 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
aureuswill 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). The disk diffusion assay
will be replicated three times.
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 will be employed because the null hypothesis does not tell the
specific direction of difference.(28)
PROVINCE OF CAVITE
BANANA-GROWING
DISTRICTS
DISTRICT V DISTRICT VI DISTRICT VII
DISTRIBUTION/EXPORT OF
BANANAS TO OTHER DISTRICTS
DISTRICT IV
-
8/3/2019 2edited Research Output 7-Edited
17/22
16
In comparing the antibacterial activity of different species of banana, three controls
and three treatments will be used. Antibacterial activity of the peel extracts obtained from
Musa acuminata, Musa balbisiana, and Musa paradisiaca will be observed. For the
control, a positive control containing Vancomycin and a negative control containing
distilled water will be used. Methanol will be used for the solvent group. The zone of
inhibition observed for each concentration per treatment and control group 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 will be utilized in computing for the number of replicates: (29)
# of replicates = 2 (Z/2
+ Z)(/)2
Wherein Z/2 isassociated with Type I error; Z is associated with Type II error; is
associated with standard deviation; and 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 will be 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:
Z/2: = 0.05 (at 95% confidence level) Z: = 0.80/2= 0.025 Z value = 0.84
= 1 - 0.025= 0.975
Z value = 1.96
Value for standard deviation is set at = 0.1, based on previous experiments. Avalue of 0.14 is set for the anticipated true difference.
Calculation:
# of replicates = 2 (Z/2+ Z )(/)2
= 2 (1.96+0.84)(0.1/0.14)2
= 2 (2.8)(0.51)
= 2.86
~ 3 replicates will be used
-
8/3/2019 2edited Research Output 7-Edited
18/22
17
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
antioxidant activities of the pulp and peel obtained from 8 different cultivars. (31)
D. Data collection
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. Paper disk diffusion method and broth dilution tests will be
performed to collect the data needed in determining the antibacterial activity of different
banana species against Staphyloccous aureus. The zones of inhibition and minimal
inhibitory concentration can be obtained, respectively.
Method to be used
Results from the aforementioned tests will be determined through observation of the
presence or absence of microbial growth in the agar plate for the disk diffusion 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 is 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 will be 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 disk diffusion method
will be prepared to assure the precision of the observed antibacterial activity.
Prior to the experiment proper, the group will be 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
will be 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
-
8/3/2019 2edited Research Output 7-Edited
19/22
18
observational bias. The principle of blinding, as well as plans for controlling bias and other
possible errors have been discussed in the studys Research Design section under the
main point, Methodology.
In addition, available calipers or rulers will be calibrated to warrant accurate
measurement of zones of inhibition and extract concentration for the disk diffusion method
and broth dilution tests, respectively.
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 disk diffusion 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.(34) The measured zones of inhibition from the
three banana test species will be recorded in millimeters. (21)
Antibiotic sensitivity expressed in the context of the minimal inhibitory concentration
gives quantitative data not obtainable with the disk diffusion 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)Results of MIC will be recorded in g/mL.
The data obtained will then be organized in tables, as shown in the following section
(Tables I III). This functions as the data sheet but also aids in the analysis of the data.
The tables provide the analyst of the experiment the specific data required for the
evaluation of the hypotheses. Data collected from disk diffusion method will be 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 will be recorded and
their respective means standard deviation will be 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.
-
8/3/2019 2edited Research Output 7-Edited
20/22
19
Data collected from MIC, on the other hand, will be 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 will be recorded. Presence of
turbidity in the tube indicates growth. (36)
BIBLIOGRAPHY
(1) Bodeker G. and Kronenberg, F. (2002) A public health agenda for traditional, complementary, andalternative medicine. American Journal of Public Health, 92, 1582-1591.
(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 MonthlyReport (August 2011).
(5) Levinson, W. (2006) Review of Medical Microbiology and Immunology. New York City: McGraw-HillProfessional.
(6) World Health Organization. Initiative for Vaccine Research (VR): Bacterial infections. Retrieved fromhttp://www.who.int, 23 July 2011.
(7) World Health Organization. Combating communicable disease: communicable disease surveillance andresponse. Retrieved fromhttp://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 aureusamong patients in atertiary renal medical center. Philippine Journal of Microbiology and Infectious Diseases, 39, 28-33.
(10) World Health Organization. Traditional medicine. Retrieved fromhttp://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 unripebanana (Musa sapientum L.), lemon grass (Cymbopogon citratus S.), and turmeric (Curcuma longa L.) onpathogens. 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 AntibioticSubstances from the Banana (Musasapientum). 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 HerbalFormulations, 1 , 2-11.
(15) Akter, S. and Imam, M.Z. (2011) MusaparadisiacaL. and MusasapientumL. : A Phytochemical andPharmacological 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 Musasapeintum, 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)
http://www.doh.gov.ph/pitahc/Index.htmlhttp://www.doh.gov.ph/pitahc/Index.htmlhttp://www.doh.gov.ph/pitahc/Index.htmlhttp://www.stuartxchange.org/Saging.htmlhttp://www.stuartxchange.org/Saging.htmlhttp://www.stuartxchange.org/Saging.htmlhttp://www.who.int/http://www.who.int/http://www.wpro.who.int/nr/rdonlyres/572c5084-4adb-4634-8e85-ee8993da3e3d/0/05_csr.pdfhttp://www.wpro.who.int/nr/rdonlyres/572c5084-4adb-4634-8e85-ee8993da3e3d/0/05_csr.pdfhttp://www.wpro.who.int/nr/rdonlyres/572c5084-4adb-4634-8e85-ee8993da3e3d/0/05_csr.pdfhttp://www.wpro.who.int/nr/rdonlyres/572c5084-4adb-4634-8e85-ee8993da3e3d/0/05_csr.pdfhttp://www.who.int/http://www.who.int/http://www.who.int/http://www.who.int/http://www.wpro.who.int/nr/rdonlyres/572c5084-4adb-4634-8e85-ee8993da3e3d/0/05_csr.pdfhttp://www.wpro.who.int/nr/rdonlyres/572c5084-4adb-4634-8e85-ee8993da3e3d/0/05_csr.pdfhttp://www.who.int/http://www.stuartxchange.org/Saging.htmlhttp://www.doh.gov.ph/pitahc/Index.html -
8/3/2019 2edited Research Output 7-Edited
21/22
20
(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 Scotts Diagnostic Microbiology. Missouri:Elsevier.
(20) Scientific control.http://encyclopedia.thefreedictionary.com. Retrieved last 5 August 2011.
(21) Department of Microbiology and Parasitology, DLS-HIS College of Medicine (2011). Laboratory Manual onMicrobiology and Parasitology.
(22) Edwards, B. G. (2003). Banana peel extract composition and method for extraction. Delft PharmaInternational, Ogden Utah. Retrieved from:http://www.freepatentsonline.com/5989559.pdf
(23) World Health Organization. (2006). Blood Safety and Clinical Technology: Guidelines on Standard OperatingProcedures for Microbiology. Retrieved from: http://www.searo.who.int/en/section10/section17/section53/section482_1786.htm
(24) Researcher bias.http://www.pairbondpublications.com/for_students/online_glossary. Retrieved last 8August 2011.
(25) Food and Agriculture Organization of the United Nations (1997). Marketing Research and Information
Systems(Chapter 6: Experimentation). Retrieved from:http://www.fao.org/docrep/w3241e/w3241e07.htm#chapter 6: experimentation
(26) Republic Act No. 9272 An Act Reapportioning the Province of Cavite into Seven (7) Legislative Districts.Retrieved fromhttp://www.senate.gov.phlast September 6, 2011.
(27) World health Organization.http://www.who.int
(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. Siza 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 antioxidantactivity of eight Malaysian bananas (Musasp.). Journal of Food Composition and Analysis. Vol. 24 (1), pp1-10. Retrieved from:http://www.sciencedirect.com/science/article/pii/S0889157510001985, 12 September2011.
(32) McKubre, M.C.H. (2008) The Importance of Replication. ICCF-14 International Conference on CondensedMatter Nuclear Science.
(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/
http://encyclopedia.thefreedictionary.com/http://encyclopedia.thefreedictionary.com/http://encyclopedia.thefreedictionary.com/http://www.freepatentsonline.com/5989559.pdfhttp://www.freepatentsonline.com/5989559.pdfhttp://www.freepatentsonline.com/5989559.pdfhttp://www.pairbondpublications.com/for_students/online_glossaryhttp://www.pairbondpublications.com/for_students/online_glossaryhttp://www.pairbondpublications.com/for_students/online_glossaryhttp://www.senate.gov.ph/http://www.senate.gov.ph/http://www.senate.gov.ph/http://www.who.int/http://www.who.int/http://www.who.int/http://www.ats.ucla.edu/stat/mult_pkg/faq/general/tail_tests.htmhttp://www.ats.ucla.edu/stat/mult_pkg/faq/general/tail_tests.htmhttp://www.ndsu.edu/ndsu/horsley/ExptSize.pdfhttp://www.ndsu.edu/ndsu/horsley/ExptSize.pdfhttp://www.sciencedirect.com/science/article/pii/S0889157510001985http://www.sciencedirect.com/science/article/pii/S0889157510001985http://www.sciencedirect.com/science/article/pii/S0889157510001985http://scienceray.com/technology/information/the-three-main-types-of-data-collection-tools/http://scienceray.com/technology/information/the-three-main-types-of-data-collection-tools/http://books.google.com/url?id=F3tSac2PGicC&pg=PT57&q=http://www.bipgroup.com&clientid=ca-print-pub-7392917442895701&channel=BTB-ca-print-pub-7392917442895701+BTB-ISBN:8172253184&linkid=1&usg=AFQjCNFQuURFHID5U3RHCpaDOhRPBcbD3A&source=gbs_pub_info_s&cad=3http://books.google.com/books?idhttp://books.google.com/url?id=HcgGLfxDJSQC&pg=PA72&q=http://www.elsevierhealthindia.com&clientid=ca-print-reed_elsevier_india_pvt_ltd&channel=BTB-ca-print-reed_elsevier_india_pvt_ltd+BTB-ISBN:8131221636&linkid=1&usg=AFQjCNHSklgu2Bxdck2MwPtYxZ84C5sYPA&source=gbs_pub_info_s&cad=2http://books.google.com/books?id=HcgGLfxDJSQC&printsec=copyright&source=gbs_pub_info_s&cad=2http://books.google.com/http://books.google.com/http://books.google.com/http://books.google.com/url?id=8h4Ze5s6sFUC&pg=PA77&q=http://www.ikbooks.com&clientid=ca-print-pub-2792127120201681&channel=BTB-ca-print-pub-2792127120201681+BTB-ISBN:9380578016&linkid=1&usg=AFQjCNHqWlBEDx9hxlaHGnHzP0KasPTerA&source=gbs_pub_info_s&cad=3http://books.google.com/url?id=8h4Ze5s6sFUC&pg=PA77&q=http://www.ikbooks.com&clientid=ca-print-pub-2792127120201681&channel=BTB-ca-print-pub-2792127120201681+BTB-ISBN:9380578016&linkid=1&usg=AFQjCNHqWlBEDx9hxlaHGnHzP0KasPTerA&source=gbs_pub_info_s&cad=3http://books.google.com/books?id=8h4Ze5s6sFUC&printsec=copyright&source=gbs_pub_info_s&cad=3http://books.google.com/books?id=8h4Ze5s6sFUC&printsec=copyright&source=gbs_pub_info_s&cad=3http://books.google.com/url?id=8h4Ze5s6sFUC&pg=PA77&q=http://www.ikbooks.com&clientid=ca-print-pub-2792127120201681&channel=BTB-ca-print-pub-2792127120201681+BTB-ISBN:9380578016&linkid=1&usg=AFQjCNHqWlBEDx9hxlaHGnHzP0KasPTerA&source=gbs_pub_info_s&cad=3http://books.google.com/url?id=8h4Ze5s6sFUC&pg=PA77&q=http://www.ikbooks.com&clientid=ca-print-pub-2792127120201681&channel=BTB-ca-print-pub-2792127120201681+BTB-ISBN:9380578016&linkid=1&usg=AFQjCNHqWlBEDx9hxlaHGnHzP0KasPTerA&source=gbs_pub_info_s&cad=3http://books.google.com/http://books.google.com/books?id=HcgGLfxDJSQC&printsec=copyright&source=gbs_pub_info_s&cad=2http://books.google.com/url?id=HcgGLfxDJSQC&pg=PA72&q=http://www.elsevierhealthindia.com&clientid=ca-print-reed_elsevier_india_pvt_ltd&channel=BTB-ca-print-reed_elsevier_india_pvt_ltd+BTB-ISBN:8131221636&linkid=1&usg=AFQjCNHSklgu2Bxdck2MwPtYxZ84C5sYPA&source=gbs_pub_info_s&cad=2http://books.google.com/books?idhttp://books.google.com/url?id=F3tSac2PGicC&pg=PT57&q=http://www.bipgroup.com&clientid=ca-print-pub-7392917442895701&channel=BTB-ca-print-pub-7392917442895701+BTB-ISBN:8172253184&linkid=1&usg=AFQjCNFQuURFHID5U3RHCpaDOhRPBcbD3A&source=gbs_pub_info_s&cad=3http://scienceray.com/technology/information/the-three-main-types-of-data-collection-tools/http://www.sciencedirect.com/science/article/pii/S0889157510001985http://www.ndsu.edu/ndsu/horsley/ExptSize.pdfhttp://www.ats.ucla.edu/stat/mult_pkg/faq/general/tail_tests.htmhttp://www.who.int/http://www.senate.gov.ph/http://www.pairbondpublications.com/for_students/online_glossaryhttp://www.freepatentsonline.com/5989559.pdfhttp://encyclopedia.thefreedictionary.com/ -
8/3/2019 2edited Research Output 7-Edited
22/22