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

TABLE OF CONTENTS

LIST OF TABLES ..................................................................................................................................... 2 LIST OF FIGURES ................................................................................................................................... 2 ABSTRACT................................................................................................................................................. 3 1. INTRODUCTION .................................................................................................................................. 4 1.1 Research question ...................................................................................................................... 4 1.2 Research hypothesis ................................................................................................................... 4 1.3 Background of the research question ........................................................................................ 4 1.4 Rationale for the study ................................................................................................................ 5 1.5 Review of literature and conceptual framework ........................................................................ 6 2. OBJECTIVES ...................................................................................................................................... 12 3. METHODOLOGY ............................................................................................................................... 13 3.1 Research design ....................................................................................................................... 13 3.2 Method ..................................................................................................................................... 15 4. RESULTS ............................................................................................................................................ 29 4.1 Descriptive statistics ................................................................................................................ 29 4.2 Inferential statistics .................................................................................................................. 30 5. DISCUSSION ...................................................................................................................................... 31 5.1 Limitations of the study ............................................................................................................ 32 6. CONCLUSION AND RECOMMENDATIONS ................................................................................ 34 6.1 Conclusion ................................................................................................................................ 34 6.2 Recommendations .................................................................................................................... 34 REFERENCES ........................................................................................................................................ 35 APPENDIX ............................................................................................................................................... 37

LIST OF TABLES

Table 1.

Zone of inhibition of peel extracts of different banana species ...................................... 30 using paper disk diffusion in different trials

LIST OF FIGURES

Figure 1. Figure 2. Figure 3. Figure 4.

Conceptual framework .................................................................................................... 12 Schematic diagram of the research design ...................................................................... 14 Allocation of treatment and control ................................................................................ 18 Demarcations for accepting or rejecting the null hypothesis .......................................... 31

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.

Chapter I INTRODUCTION 1.1 Research question

Which species of banana peel extract has greater antibacterial activity against Staphylococcus aureus?

1.2

Research hypothesis
1.2.1 Working hypothesis

The peel extracts obtained from the different species of banana have comparable antibacterial activity against Staphylococcus aureus.

1.2.2

Null hypothesis

The peel extracts obtained from the different species of banana do not have comparable antibacterial activity against Staphylococcus aureus.

1.3

Background of the research question

The contribution of various plants or their parts (roots, stems, leaves, fruit) in the treatment and management of certain health conditions has been growing in recognition. At present, the use of herbal medicine are becoming more common both in developing and developed countries. The World Health Organization (WHO) has estimated that about 70-95% of the citizens in majority of the developing countries utilize traditional medicine (including the use of herbal medicines) in managing their health and incorporate the practice in their primary health care to address emerging health-related needs.(1) Industrialized countries, such as Canada, France, Germany and Italy, share similar percentages in terms of the proportion of individuals who make use of traditional 5

medication.(1) In the Philippines, the use of plant extracts as medication has been passed on from one generation to another, and has established its importance in health delivery, considering the expensive Western treatment that most Filipinos cannot afford or cannot easily access.(2) The banana fruit (Musa sapientum), has been commonly known for its nutritional value; however, its medicinal properties have only been recently investigated, mostly in tropical and subtropical countries wherein the banana fruit is considered as one of their major agriculture products, such as India and other Southeast Asian countries. In these regions, other parts of the banana plant such as their young shoots and peels have been utilized as an alternative source of treatment for ulcers and wounds, especially in areas where access to conventional treatment is difficult.(3) In addition, majority of the studies conducted in these countries have been focusing on the most common variants of the Musa sapientum species available in their locality. The study aims to investigate the antibacterial activity of the most common local variants of banana grown in the province of Cavite, namely Musa acuminata (lakatan), Musa balbisiana (saba), and Musa paradisiaca (latundan)(4), and determine if the species variant plays a significant role in their respective antibacterial activity, specifically against Staphylococcus aureus, a common Gram-positive agent found in most health care-related infections.

1.4

Rationale for the study

This study may provide additional information regarding the antibacterial activity of the common variants of bananas in the region, and thus provide possible plant leads that can be used as alternative and less expensive sources of treatment for the benefit of the local residents. The additional information obtained may also contribute in evoking interest for further research on the phytochemical profile and antibacterial activity of the 6

local banana variants which may be used as active ingredients against drug-resistant microorganisms such as Staphylococcus aureus.

1.5

Review of literature and conceptual framework

1.5.1 Epidemiology of disease of interest Staphylococcus aureus is considered as one of the medically important pathogens, commonly causing abscess formation, various pyogenic infections (e.g. endocariditis), food poisoning, and toxic shock syndrome. It has also been among the prevalent causative agent for majority of hospital-acquired infections (e.g. pneumonia), septicemia, and surgical-wound infections.(5)(6) Infections caused by the bacteria have been more prevalent in the health care setting where these are treated with more frequent and intensive antimicrobial therapy as compared in the community setting.(6) Throughout the evolution of antimicrobial therapy against S. aureus, various strains of resistance have been developed by the agent, thus amplifying the disease burden. (6) In some countries in the Western Pacific Region, such as the Philippines, a growing trend in methicillin-resistant S. aureus (MRSA) had been recorded within a span of 7 years.(7) According to the 2005 Philippine Antimicrobial Resistance Surveillance report, wherein twelve out of the 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. aureus among patients. A study conducted in a tertiary medical institution revealed the presence of hospital-acquired MRSA (HA-MRSA) among patients suffering from chronic kidney disease and were 7

undergoing renal replacement therapy, such as hemodialysis. Strains of communityacquired 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)

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 extracts 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 12hydroxystearic exhibited the most active antimicrobial response.(11) Findings were supported in a phytochemical and pharmacologic review conducted by Akter et al. wherein it was demonstrated that the banana peel extract obtained from Musa paradisiaca and Musa sapientum showed better antibacterial activity against the test bacteria (Staphylococcus and Pseudomonas species) than the banana leaf extract. The peel extract was also shown to be more active against Staphylococcus (Gram-positive) than Pseudomonas species (Gram-negative). Furthermore, the review of pharmacological 9

activities suggests that the traditional uses of the banana plant in diarrhea, dysentery, ulcer, diabetes, hypertension and cardiac diseases are scientifically valid.(15) Other studies investigated if the difference in the subspecies of the Musa sapientum would yield varying degrees of antibacterial activity against a range of microorganisms. Akter et. al aimed to evaluate the antimicrobial and cytotoxic activities of different extracts of Musa sapientum, L. subsp. Sylvestris fruits (MSSE). The methanolic extract of Musa sapientum peel was investigated for antimicrobial activity by disk diffusion method and for cytotoxic activity by Brine shrimp lethality bioassay. The findings of the study demonstrated that the methanolic extract of Musa sapientum possessed good antimicrobial activity against Gram-positive and Gram-negative bacteria as well as against pathogenic fungi and affirmed the traditional use of the fruit to treat dysentery and diarrhea.(16) In addition, studies conducted by Hamid et al. as well as Mokbel et al. showed how a certain development of the banana fruit may contribute to its antibacterial activity by using both ripe and unripe banana peel extract. The type of solvent used was not specified. Extracts of ripe, unripe and leaves of guava (Psidium guajava); ripe, unripe and leaves of starfruit (Averrhoa carambola); ripe and unripe banana (Musa sapientum variety Montel); ripe and unripe papaya (Carica papaya); passionfruit (Passiflora edulis F. Flavicarpa) peel; two varieties of Lansium domesticum peels; rambutan (Nephelium lappaceum) peel and rambai (Baccaurea motleyana) peel were evaluated for antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, yeast and fungi (Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Lactobacillus bulgaricus; E. coli, Proteus vulgaricus, Pseudomonas aeruginosa, Salmonelli typhi; Saccharomyces

cerevisiae, Candida lypolytica; Rhizopus spp., Aspergillus niger, and Chlamydomucor spp). The antimicrobial activities were tested using both the disk diffusion and tube dilution assays. Most of the fruits showed some activity towards bacteria but poor activity against 10

yeast or fungi. Extracts from bananas, papayas, passionfruit peel, Lansium domesticum peels and rambutan peels showed activity against Candida lypolytica while extracts from guava showed strong activity against Saccharomyces cerevisiae. Unripe banana showed activity against all the bacteria except towards P. vulgaricus.(17) The study conducted by Mokbel and Hashinaga used both fresh green and yellow banana peel of Musa, cv. Cavendish fruits which were treated with 70% acetone; and afterwards, partitioned with chloroform (CH[Cl.sub.3]) and ethyl acetate (EtOAc). The antioxidant activities of the extracts were evaluated by using the thiocyanate method, beta-carotene bleaching method and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical elimination. Antimicrobial activities of the extracts and isolated components were evaluated using paper disk methods and minimum inhibition concentration (MIC). The EtOAc and water soluble fractions of green peel showed high antimicrobial and antioxidant activity. The antioxidant activity of water extracts was comparable to those of synthetic antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene. Among all isolated components, B-sitosterol, malic acid, succinic acid, palmatic acid, 12-hydroxystrearic acid, d-malic and 12-hydroxystrearic acid were most active against the Gram-negative and Gram-positive bacteria species tested. The MIC of d-malic and succinic acid varied between 140-750 ppm, respectively. (11) 1.5.3.2 Biases

Although there were no significant experimental biases recognized in the related literatures that were reviewed, a possible researcher bias, wherein the prior knowledge of the researchers might affect the analysis of the results, may be encountered in the study. Observational bias may also be encountered among the researchers along the course of the experimental proper, wherein there may be discrepancies in measuring the outcome observed. 11

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.

12

1.5.4

Conceptual framework

Figure 1 depicts the possible relationship of the exposure variable (application of plant extract) with that of the outcome variable (inhibition of microbial growth). The characteristics of the exposure variable must be taken into consideration as to how it would influence the outcome variable. Similarly, the characteristics of the outcome variable should also be taken into consideration as to how it may counteract the exposure variable and affect the result. Possible confounding factors such as exposure to environmental factors as well as experimental protocol (preparation and storage methods) were derived from literatures documenting various experimental processes in determining the antimicrobial activities of the plant extracts.

Antibacterial activity of banana peel extract

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

Characteristics of the factor Inherent defense mechanisms of the sample of interest

POSSIBLE CONFOUNDING FACTORS Exposure to environmental factors Preparation and storage techniques

CONTAMINATION

Figure 1. Conceptual framework

13

Chapter II OBJECTIVES
Given the previous data, the study conducted was generally aimed at determining which species of banana peel extract has greater antibacterial activity against Staphylococcus aureus. In order to achieve the general objective, the following specific objectives were met: 1. To measure the zones of inhibition of peel extracts obtained from Musa acuminata, Musa balbisiana, and Musa paradisiaca using the disk diffusion method. 2. To establish the respective minimal inhibitory concentrations of peel extracts from Musa acuminata, Musa balbisiana, and Musa paradisiaca using broth dilution test. 3. To establish the respective minimal bactericidal concentrations of peel extracts from Musa acuminata, Musa balbisiana, and Musa paradisiaca using the streak plate technique and incubation of Mueller Hinton agar plates. 4. To compare the obtained zones of inhibition, minimal inhibitory concentrations, and minimal bactericidal concentrations of the peel extracts against Staphylococcus aureus.

14

Chapter III METHODOLOGY 3.1 Research design

The study utilized an analytic experimental design, wherein the independent variable under observation was assigned particularly to a treatment group and was compared to a positive and negative control group. Figure 2 illustrates the design of the study.

Figure 2. Schematic diagram of the design

This type of study did not require obtaining subjects from a target population; therefore, an inclusion/exclusion criterion has not been defined. The application of

15

constructing a sampling design as well deriving a sample size calculation was also not appropriate for the study.

3.1.1

Operational definition of variables

3.1.1.1 Independent variable The antibacterial activity of the different species of banana peel extracts on the growth of Staphylococcus aureus served as the independent variable for the study. Antibacterial activity refers to the capacity of an agent to kill or suppress the growth of microorganisms, specifically bacteria.(18) This property may be further classified into two mechanisms, bacteriostatic and bacteriocidal. Bacteriostatic activity results into the

inhibition of microbial growth within a certain period of time. Microbial growth may be observed once environmental elements become suitable, or the microorganism has gained resistance to counteract the stimulus presented by the agent.(18) On the other hand, bacteriocidal activity results into the complete eradication of the species. In the study, significant bacteriostatic activity of the different species of bananas will be observed through disk diffusion method and broth dilution test. Musa acuminata (locally known as lakatan), Musa balbisiana (saba), and Musa paradisiaca (latundan) are considered as the most common group of species grown and commonly sold in the province of Cavite. Because of their wide availability and easy accessibility, these species were chosen as samples of plant extract for the study.

3.1.1.2 Dependent variable The inhibition of the growth of Staphylococcus aureus on nutrient agar medium served as the expected outcome of the study. Inhibition denotes a temporary cessation in microbial growth processes. This implies that there are still possible chances for growth, given that the environment becomes favorable once again for microorganism 16

propagation.(18) Therefore, strict compliance with the incubation of disk diffusion plates within the allotted time of 24 hours for the Staphylococci species were observed so as to achieve reliable results and prevent possible growth of the organism.(19) Inhibition of growth was determined qualitatively using the disk diffusion method, as represented by zones of inhibition. Broth dilution test was employed to quantify the degree of the antibacterial activity by determining the peel extracts 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 17

medium is unable to inhibit the growth of the test organism, thus forming well-demarcated borders, resulting in a distinct area known as the zone of inhibition.(18) This method only gives a qualitative value of an 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 of the test bacterium. This was the parameter used in the study to reflect the presence of microbial growth inhibition. (18) Furthermore, control groups were set up to validate that the microbial growth inhibition is indeed attributed to the antibacterial activity of the banana peel extract. A positive control will confirm that the treatment applied is competent to produce the intended effect, thus minimizing the probability of false negatives. (21) For the study, the drug of choice for Staphylococcus aureus, Vancomycin, was utilized as the standard for evaluating the antibacterial activity of the plant extracts. A negative control was also set up in order to confirm that the effect produced was not influenced by extraneous factors, thereby minimizing the probability of false positives.(21) For the study, distilled water was used as a reference reflecting microbial growth, indicating that the treatment applied has not been effective. In addition, a solvent group was set up to evaluate the influence of the solvent on the observed microbial growth inhibition. For the study, methanol was used as reference. Quantitative measures, such as the minimal inhibitory concentration (MIC), were also applied to validate the degree of the antibacterial activity of the banana peel extracts. Minimal inhibitory concentration refers to the smallest amount of the extract per unit volume that will inhibit the growth of a certain organism.(18)(20) Broth dilution tests, wherein tubes containing decreasing concentration of the plant extract tested are prepared by serial dilution, was conducted to determine the plant extracts respective MIC.(20) 18

3.2.1

Steps undertaken in the study 3.2.2.1 Physical set-up

The study used Mueller Hinton agar plates in observing the activity of peel extracts from different banana species (Musa paradisiaca, Musa balbisianana and Musa acuminata) compared with the positive control, negative control and solvent control. Three replicates were used. Figure 3 illustrates the distribution of the treatments and control among the bacterial culture.

CONC. (A) CONC. (B) CONC. (C) (+) CONTROL SOLVENT ONLY (-) CONTROL CONC. (B) CONC. (C)

CONC. (A) SOLVENT ONLY (-) CONTROL (+) CONTROL

Musa paradisiaca

Musa balbisianana

CONC. (A) CONC. (B) CONC. (C) (+) CONTROL SOLVENT ONLY (-) CONTROL

Musa acuminata
Figure 3: Allocation of treatment and control

19

3.2.2.2

Procurement of plant materials

Peels from different species of banana (Musa paradisiaca, Musa balbisianana and Musa acuminata) were obtained from Dasmarias, 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 45C for 48 hours. (22)

3.2.2.4

Preparation of peel extract stock solution

The stored extract was filtered using a flat filter paper on a funnel. The collected precipitate was then allowed to dry in the oven for 24 hours at 45C. 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 g/L). 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 (300g/L and 30g/L) and 1mL was set for use in MIC. The initial concentration obtained was set at 3,000 g. 10-fold dilution from this concentration was done until a concentration equivalent to that of the standard drug used

20

as control (Vancomycin = 30 g/L) 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 -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 g, 300 g, and 30 g.
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 g/L (or 0.3 mg/mL) (3000 g/L)(x) = (300 g/L)( 20 L) x = 2 L - to prepare 30 g/L (or 0.03 mg/mL) (3000 g/L)(x) = (30 g/L)(20 L) x = 0.2 L

Based from the above computations (B), to prepare 300 g/L concentration, 18uL of methanol was added to 2L of 3000 g/uL. 19.8 L of methanol was added to 0.2L of 3000 g/L to abtain the final concentration 30 g/L.

3.2.2.5

Preparation of the bacterial suspension

Staphylococcus aureus maintained on nutrient agar slants at 4C were aseptically transferred using a sterilized needle into fresh sterile broth culture. The tubes were incubated at 37C for 24 hours. For standardization of bacterial density, the turbidity of the

21

fresh 24 hour old cultures were adjusted to 0.5 MacFarland standard (1 108 cells/ml) by diluting with the sterile broth. (18)(20) In adjusting the turbidity of 24-hour old culture, both the standard and the inoculum tube were held side by side and no more than 1 inch from the face of the Wickerham card with adequate light present. The appearance of the lines through both suspensions was compared. If the bacterial suspension appears lighter than the 0.5 McFarland standard, more organisms were added to the tube from the culture plate. Whereas, if the suspension appears more dense than the 0.5 McFarland standard, additional sterile broth was be added to the inoculum tube in order to dilute the suspension to the appropriate density. In some cases it was easier to start over rather than to continue to dilute a bacterial suspension that is too dense for use. (18)

3.2.2.6

Paper disk diffusion assay

The plates with Mueller-Hinton agar was divided into 6 equal parts with a marking pencil on the bottom of the plates (Figure 3). A sterile cotton swab was aseptically dipped into the S. aureus broth culture. The cotton swab was rotated against the inside wall of the tube to remove excess broth, then the swab was streaked evenly over the surface of the plate and in three directions, leaving no gaps in between strokes. The plate was allowed to dry for 3-5 minutes with the lid in place. Filter paper disks of 6mm diameter has been shown to have the capacity to hold 0.005 mL(24). Thus each disk will be impregnated with 5 L (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 37C for 24 hours. The zone of inhibition was measured (in millimeters) after overnight incubation(19)(25). Zone diameters of the peel extracts with diameter of 7mm-14mm excluding the 6mm diameter of 22

the filter paper will be considered significant, this is based from the results of a previous study conducted.(11)

3.2.2.7

Determination of MIC and MBC

The concentration within the preceding smallest concentration of extract with significant zone of inhibition (7mm-14mm diameter) was used in MIC. Broth dilution test was performed in determining the minimum inhibitory concentration of the plant extracts. Two-fold serial dilution was used for this test, giving the following concentrations: 3840, 1920, 960, 480, 240, 120, 60, 30 and 15 g. 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 37C for 24 hours. Tube 10, containing only the sterile broth and S. aureus suspension served as the control tube. Bacterial growth in each tube was examined by observing turbidity. The lowest concentration without growth is the minimal inhibitory concentration. (20) Aliquots from all tubes that showed no growth and was inoculated in a Mueller-Hinton agar medium using the streak plating technique. The plates were incubated at 37 C for 24 hours. The plates were examined for bacterial growth by observing presence of S. aureus colonies. Minimum bactericidal concentration is the smallest amount of drug per unit volume that will kill the organism. Thus, plates with no growth indicated that the extract is bactericidal at that dilution.(20)

3.2.2.8

Control for possible biases and errors

The experiment proper was conducted in an isolated controlled unit set at the temperature of 37C to minimize the possible effect of environmental factors. A facilitator

23

with microbiology expertise supervised the editing of the experiment procedure in order to monitor errors in preparation and storage techniques throughout the duration of the study. The principle of blinding was also applied in the execution of the study and data collection. Members were assigned solely to the execution of the experiment, observation, and data collection. Unassigned members determined what type of treatment will be assigned to a particular sample and were responsible for the result analysis, so as to minimize the occurrence of possible researcher bias. Assigned members were oriented with the flow of the experiment proper and proper data collection in order to minimize the occurrence of observational bias.

3.2.3

Sampling 3.2.3.1 Selection of treatment and control

Selection for the type of banana species that were utilized for the study were based on the species availability and accessibility to the public. Different banana species that were common in the province of Cavite were determined. Based on the 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 was done at the most convenient district for the study, the 4th district of Dasmarinas City
(26)

One subgroup consisting of three members will be in-charge of treatment allotment. Six

plates of nutrient agar were planned to be prepared for the study. Two plates were allotted for observing the activity of extracts from different banana species (Musa paradisiaca, Musa balbisianana and Musa acuminata) in different concentrations compared with a positive, negative, and solvent control. Control groups were set up so as to validate that 24

the intended effect is indeed attributed to the independent variable of the study. Vancomycin, the drug of choice for Staphylococcus aureus will be used as a positive 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 control.
(11)

. Methanol was utilized to serve as the solvent

3.2.3.2

Sample size

The type of significance test to be done depends on the hypothesis. In this study, since the null hypothesis states that the peel extracts obtained from the different species of banana do not have comparable antibacterial activity against Staphylococcus aureus, therefore a two-tailed test was employed because the null hypothesis does not tell the specific direction of difference.(28) In comparing the antibacterial activity of different species of banana, two controls and three treatments were used. The zone of inhibition observed for each concentration per treatment will be measured using a 6-inch ruler. Determining the number of replicates to be used in an experiment is a matter of judgement and the available resources. The following formula incorporating the probabilities for type I and type II errors as well as the variance and true difference anticipated was utilized in computing for the number of replicates: (29) # of replicates = 2 (Z/2 + Z )(/)2 Wherein Z/2 is associated 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.

25

Values obtained for the formula were based from previous experiments on the antibacterial activity of a particular species.(11) The study was set a confidence level of 95%, with a power of 80%. Based on these percentages, the following Z values for Type I and Type II error are the following: Z/2: = 0.05 (at 95% confidence level) /2= 0.025 = 1 - 0.025 = 0.975 Z value = 1.96 Z: = 0.80 Z value = 0.84

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

3.2.3.3

Reference citations

In several studies investigating the antibacterial activity of unripe banana peels conducted by Alisi, et. al and Sulaiman, et. al, n=3 was used for the number of replicates with a significance of p = 0.05. Alisi, et. al made use of a two-way analysis of variance (ANOVA) to determine the inhibition of bacterial dehydrogenase activity reflected in dehydrogenase assays. (30) On the other hand, Sulaiman, et. al utilized one-way ANOVA testing to establish the correlations between total phenolic and mineral contents with the antioxidant activities of the pulp and peel obtained from 8 different cultivars.
(31)

26

3.2.4

Data collection 3.2.4.1 Sources of data (variables to be measured)

The independent variable in the study is the antibacterial activity of the banana peel extracts from different species whereas the dependent variable is the inhibition of growth of Staphylococcus aureus. Antibacterial susceptibility tests were performed to collect the data needed in determining the antibacterial activity of different banana species against Staphyloccous aureus. Thru antibacterial susceptibility tests such as Kirby-Bauer method and Minimum Inhibitory Concentration (MIC), the zone of inhibition and lowest inhibitory concentration can be obtained, respectively.

3.2.4.2

Method of data collection used

Results from the aforementioned tests were determined through observation of the presence or absence of microbial growth in the agar plate for the Kirby-Bauer method, as well as in the series of test tubes for MIC. The observation method is the preferred method in the context of a laboratory experimental study. Data collection was done by making use of standardized measurement methods and utilizing replications to achieve precision.(32) In the study, a schematic diagram of the step-by-step procedure was developed to ensure that uniformity in the observation process and data collection will be implemented among the assigned observers. Three replicates per banana species for the Kirby-Bauer method were prepared to assure the precision of the observed antibacterial activity. Prior to the experiment proper, the group was divided into three subgroups for the following tasks: 1) preparation of plant extract; 2) execution of antibacterial susceptibility tests and data collection; and 3) data analysis. Members assigned in a particular subgroup were oriented to the schematic process to ensure a smooth execution of the study and uniformity in the observation process, thus reducing a possible researcher and

27

observational bias. The principle of blinding, as well as plans for controlling bias and other possible errors have been discussed previously. In addition, available calipers or rulers were calibrated to warrant accurate measurement of zones of inhibition and extract concentration for the Kirby-Bauer method and MIC tests, respectively.

3.2.4.3

Data collection tool

Collection tools provide the necessary data for analysis; in this respect, tools should be strong enough in order for a research to yield a claim. Data collection tools are mainly categorized into three: secondary participation, in-person observations, case studies and analysis content.(33) In experimental designs however, these tools are rarely used. Instead, the researchers use an array of experimental procedures to obtain the data. The size of the zone of inhibition obtained from the Kirby-Bauer method is directly 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 the three banana test species were recorded in millimeters. Zone diameters of the peel extracts with diameter of 7mm-14mm excluding the 6mm diameter were labelled as S (sensitive). Diameters measuring below 7mm were labelled as R (resistant). Antibiotic sensitivity expressed in the context of the minimal inhibitory concentration gives quantitative data not obtainable with the Kirby-Bauer method. MIC is identified as the smallest concentration of antibiotic that inhibits the growth of the test bacterium; thus, these quantitative results are useful in predicting the amount of antibiotic that must be attained to assure inhibition.(19) In MIC, only antibiotics that show inhibitory activity towards 28

the bacterial isolate using the Kirby-Bauer method were tested further. The concentration of banana peel extract observed to have the most sensitive activity was serially diluted to make a range of antibiotic concentrations that encompass the concentration used in the Kirby-Bauer method.(35)(36) Results of MIC were recorded in g/mL. The data obtained were organized in tables, as shown in Appendix A. This functioned as the data sheet and also aided in the analysis of the data. The tables provided the analyst of the experiment the specific data required for the evaluation of the hypotheses. Data collected from Kirby-Bauer method were recorded in Tables I and II, for the treatment and control respectively. The measured zone of inhibition (mm) from the three replicates of different concentrations and species were recorded and their respective means standard deviation were computed. In this way, the analyst can assess the variation among the zones of inhibition observed due to possible errors and discrepancy of the measurements. Data collected from MIC, on the other hand, were recorded in Table III. The resulting concentration of peel extracts after serial dilution using a series of ten tubes as well as observation for microbial growth in each tube were recorded. Presence of turbidity in the tube indicated growth. (36)

3.2.5

Method of data analysis 3.2.5.1 Descriptive statistics

The mean diameter obtained among the three replicates was calculated in measuring the central tendency of the zone of inhibition produced by the peel extract. Measures of central tendency are inappropriate for the values obtained in conducting MIC and MBC since these tests are performed only once. Furthermore, the standard deviation was

computed from the value gathered from the zone of inhibition. The final data were then reported as mean + standard deviation. 29

3.2.5.2

Inferential statistics

One-way analysis of variance (ANOVA) was used as the test statistic since the study is dealing with more than two independent study groups. This is based on the premise that each peel extract groups 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 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 g, 300 g, and 3,000 g) 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 g. Several variables such as characteristics of the banana peel (e.g. color and storage duration), duration of blending the chopped peels, and the extract storage temperature were considered. Values for mean + standard deviation are then considered to be set at zero, since the values gathered per trial were null.(38) Table 1 shows the measurements of the zones of inhibition among the different banana species.

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

TRIAL 1 Musa sapientum Musa paradisiaca Musa acuminata 0

TRIAL 2 0

TRIAL 3 0

TRIAL 4 0

TRIAL 5 0

TRIAL 6 0

31

4.2

Inferential statistics
Since the values obtained were set at zero, replacing these on the F-ratio (i.e. statistic used in utilizing ANOVA) would reveal that the p-value would progress towards zero, implying that the value of statistical significance is within the non-rejection area of the normal distribution curve.(39) A conclusion can then be made that there are no sufficient evidences to reject that the peel extracts obtained from the different species of banana do not have comparable antibacterial activity against Staphylococcus aureus, in favor of the alternative hypothesis.

=0

Figure 4. Demarcations for accepting or rejecting the null hypothesis

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

32

Chapter V DISCUSSION
Akter et. al evaluated the antimicrobial and cytotoxic activities of different extracts of Musa sapientum, L. subsp. sylvestris fruits (MSSE). The findings of their study demonstrated that the methanolic extract of Musa sapientum pulp possessed good antimicrobial activity against all 13 Gram-positive and Gram-negative bacteria. The peel extract showed significant activity against all the organisms, while the seeds showed no activity against any organism.(16) Focusing on the peel extract, it showed activity against all the test organisms with a zone of inhibition of 7-9mm, which were in contrast with the result of the trials done on the 3 variants of bananas. Another study conducted by Mokbel and Hashinaga also explored on the antibacterial properties of banana peel extracts, this time utilizing the Cavendish variant. It also agreed with the study conducted by Akter et. al wherein these peel extracts displayed antibacterial properties against a range of Grampositive 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 g but showed no sign of antimicrobial activity against Staphylococcus aureus, as manifested by a 0mm zone of inhibition. It may have been due to the difference in the methods used. The procedure done in the study of Akter et. al with regards to extraction was more complex, utilizing a Soxhlet apparatus to obtain the extract, compared with the crude procedures done in this study.(16) On the other hand, the methodology incorporated an extraction procedure that was not done prior to this study: a compromise between the Soxhlet method of extraction and the soaking technique. This method, however, has not been used (or tested) insofar as the recent similar studies are concerned. The lack of materials such as a Soxhlet

31

apparatus may have had a bearing on the results, in terms of the quality of the plant extract produced. Another difference noted was on the degree of ripeness of the plants used. Both mature and unripe fruits were used whereas in the study, the researchers used only ripe bananas of the 3 variants. The ripeness of the bananas purchased were already at its peak, assuming that the active ingredients are at its potent state. However, in both

studies, the concerns raised up in the study such as the influence of plant characteristics (e.g. correlation of degree of ripeness with the potency of active ingredients) was not discussed. In addition, extract preparation and storage protocols which may have also influenced the optimal extraction of active ingredients were also not discussed in previous studies. Areas such as the establishment of the initial concentration (concentration prior to disc impregnation) and the impregnation of the paper disc are of great importance. In the matter of establishing the concentration, the initial concentration varied all throughout the trial. Utilization of an evaporator is commonly used to ensure that the initial concentration of the crude extract is almost pure or 100%. However, this experiment used passive evaporation technique that is time-consuming and erroneous. Thus, only an

approximation, not an accurate value, of the initial concentration was documented. Regarding the disc impregnation, inadequacy of the amount of extract in the paper disc could have resulted in the failure to elicit a zone of inhibition during the course of the trial. Further studies looking at the following concerns of the influence of confounding variables (i.e. plant characteristics, extract preparation and storage protocol) are needed.

5.1

Limitations of the study

Due to the initial results gathered, conducting trial sessions (paper disk diffusion assay) to confirm the antibacterial activity of the peel extracts were added to the 32

experiment procedure during the course of the study. This was deemed as necessary in order to avoid unwarranted expense of time and finances. Unfortunately, due to the time curb allotted for the experiment proper, a deeper investigation on the influence of confounding variables and modification of the experiment procedure in terms of extract preparation and storage protocol were not implemented; thus, other steps in the experiment procedure such as the determination of MIC and MBC were also not implemented.

33

Chapter VI CONCLUSION AND RECOMMENDATIONS

6.1

Conclusion
Based on the results, the proponents of the study conclude that the peel extracts obtained from the different species of banana do not have comparable antibacterial activity against Staphylococcus aureus. Furthermore, the zones of inhibition, minimal

inhibitory concentrations, and minimal bactericidal concentrations were not established since trials conducted via paper disk diffusion showed the absence of microbial growth inhibition.

6.2

Recommendations
In light of the limitations of the study and said conclusion, the following recommendations are proposed: 1) Careful re-evaluation of the method of extraction is highly suggested in order to establish its capability for optimal plant extraction. 2) Modification of the experiment procedure, specifically in the areas of establishing the initial concentration and impregnation of paper discs, ought to be re-evaluated to ensure proper documentation of the concentrations and to satisfy the adequacy of the extract. 3) A follow-up study should be conducted so as to provide further insight to the antibacterial properties of the Musa genus as suggested by other studies.

34

REFERENCES
(1) Bodeker G. and Kronenberg, F. (2002) A public health agenda for traditional, complementary, and alternative 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 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-4634-8e85ee8993da3e3d/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. (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 Scotts 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.

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(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 (25) World Health Organization. (2006). Blood Safety and Clinical Technology: Guidelines on Standard Operating Procedures for Microbiology. Retrieved from: http://www.searo.who.int/en/section10/section17/section53/ section482_1786.htm (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. (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. 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. 18211825. (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. (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 Copyright. http://books.google.com/ Microbiology & Immunology. Elsevier India

(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 pvalue. 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

36

APPENDIX

37

APPENDIX A Data Collection Tool

The zones of inhibition observed in the agar plates where different concentrations of the peel extracts obtained from three different banana species will be recorded in Table I in millimetres (mm); on the other hand, the zones of inhibition observed in the agar plates assigned as positive, negative, and solvent controls will be recorded in Table II and will also be recorded in millimetres (mm). Both measurements will be obtained using the paper disk diffusion assay. The peel extracts 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 (g/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 A SPECIES (1) SPECIES (2) SPECIES (3) B C A

R2 B C A

R3 B C A

M+SD B C A

INT B C

CONTROL
CONTROL

R1 A SPECIES (1) SPECIES (2) SPECIES (3) B C A

R2 B C A

R3 B C A

M+SD B C A

INT B C

38

MINIMAL INHIBITORY CONCENTRATION SPECIES (1)

TUBE NO. Antimicrobial agent concentration (g/mL) REPLICATE (1) REPLICATE (2) REPLICATE (3) Growth (+/- turbidity)
CONTROL

10

SPECIES (2)
TUBE NO. Antimicrobial agent concentration (g/mL) REPLICATE (1) REPLICATE (2) REPLICATE (3) Growth (+/- turbidity)
CONTROL

10

SPECIES (3)
TUBE NO. Antimicrobial agent concentration (g/mL) REPLICATE (1) REPLICATE (2) REPLICATE (3) Growth (+/- turbidity)
CONTROL

10

39

APPENDIX B Laboratory Materials Used

40