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The Antimicrobial Effect of 100% Mature Psidium Guajava (Guava) Leaf Extract on the Growth of Pseudomonas Aeruginosa (Atcc # 27853) Essay

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THE ANTIMICROBIAL EFFECT OF 100% MATURE Psidium guajava (GUAVA) LEAF EXTRACT ON THE GROWTH OF Pseudomonas aeruginosa (ATCC # 27853) A Research Proposal Presented to the Faculty of the College of Medicine Cebu Doctors’ University Mandaue City, Philippines In Partial Fulfillment of the Requirement Research in Medicine II Block I, Module 2 by: Avancena, Glory O. Booc, Mark Henry C. Burgos, Dan Samuel S. Cabang, Eloise Maxine B. Conopio, Arnel Y. Cordova, Jose Reginald K. Gozo, Leoniza Gloria S. Pavillar, Lounard N. Tan, Marie Andone L.

Tancinco Aldo Ian A. Leah P. Acibar, M. D.

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Research Adviser August 2011 TABLE OF CONTENTS INTRODUCTION Background Review of Related Literature Significance of the Study Objectives Statement of Hypotheses Scope and Limitation MATERIALS AND METHODS Research Design Research Locale Research Respondents Research Instrument Data Collection Procedures Preliminary Procedure Experimental Procedure Actual Data Gathering Data Processing and Analysis DEFINITION OF TERMS BIBLIOGRAPHY APPENDICES A. Transmittal Letters B. Certification of Authenticity C. Data Collection Sheet D. Time Table E. Budget Summary F.

Sample Size Determination G.

Computation of Concentrations RESEARCHERS’ BIODATA 1 1 1 6 6 7 7 8 8 8 8 8 9 9 11 13 13 14 15 17 17 22 24 25 26 27 28 29 INTRODUCTION Background of the Study The increasing microbial resistance to drugs is a great threat that hampers the treatment of many illnesses. The indiscriminate abuse of antimicrobials together with the lack of proper identification of the causative organism and patient demands, have contributed to the rise of resistant strains of microbes to various drugs. Currently more than 70% of bacteria associated with hospital-acquired infections are now resistant to drugs that were once effective against it (Brunton et. l. 2006). Although new drugs are being created to overcome this problem, most of these new drugs are merely modifications of pre-existing drugs. It is feared that the rate of new drug production against currently resistant microbes might not be fast enough to counter the effects of increasing antimicrobial resistance (Brunton et. al. 2006). Rather than modifying pre-existing drugs, this research hopes to see if mature guava leaves will exhibit antimicrobial activity against Pseudomonas aeruginosa, a gram-negative bacillus, well-known for its increasing resistance to many drugs.

If this research proves successful, it is hoped that future researches will dwell deeper into the chemical properties of the leaves and identify its active antimicrobial component(s). This could potentially lead to a brand new drug which could be used against the increasing resistance of Pseudomonas, and possibly of other organisms. Review of Related Literature Psidium guajava, from the family Myrtaceae, is considered native to Mexico and extends throughout South America, Europe, Africa and Asia It grows in all the tropical and subtropical areas of the world and adapts to different climatic conditions but prefers dry climates.

P. guajava is a small tree with a height of 10m having thin, smooth, patchy, peeling bark. Guava leaves are opposite, short-petiolate, the blade oval with prominent pinnate veins, 5–15 cm long. Flowers are somewhat showy, petals whitish up to 2 cm long and with numerous stamens. Its fruit is yellow, fleshy, globose to ovoid berry about 5 cm in diameter with an edible pink mesocarp containing numerous small hard white seeds. There has been a tremendous interest in this plant as evidenced by the voluminous work (Gutierrez et al. 2008).

Different parts of the plant are used in the indigenous system of medicine for the treatment of various human ailments such as wounds, ulcers, and cholera. Pharmacological investigations indicated that its bark, fruit, and leaves possess antibacterial, hypoglycemic, anti-inflammatory, analgesic, antipyretic, spasmolytic, and CNS depressant activities (Macatol et. al 1998). In the Philippines, the unripe fruit, the leaves, the cortex of the bark and the roots are used for washing ulcers, wounds, astringent, and as treatment for diarrhea (Gutierrez et al 2008).

This study will make use of guava leaf, as previous studies showed that the leaf extracts demonstrated antimicrobial activity. alcoholic extracts of The inhibitory effects of aqueous and P. guajava leaf on the growth of Staphylococcus aureus, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella enteritidis, Bacillus cereus, Proteus spp. , Shigella spp. and Escherichia coli were examined using the in vitro agar well diffusion method. The results revealed that alcoholic extract of guava leaf prevented the growth of 81. % of the test organisms (Chah, et al, 2006). Flavonoids are hydroxylated phenolic substances that are known to be synthesized by plants in response to microbial infection, which is not surprising that they have been found in vitro to be effective antimicrobial substances against a wide array of microorganisms. Their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls. More lipophilic flavonoids may also disrupt microbial membranes (Cowan 1999).

In mature leaves, the greatest concentrations of flavonoids were found, namely, Myricetin (208. 44 mg kg? 1), quercetin (2883. 08 mg kg? 1), luteolin (51. 22 mg kg? 1) and kaempferol (97. 25 mg kg? 1) (Gutierrez et al. , 2008). P. aeruginosa is a gram-negative, motile, non-fermentative, pigment- producing, aerobic bacilli. This organism grows at 37-42°C in a wide variety of culture media, forming smooth round colonies with a fluorescent greenish color. non-fluorescent, bluish pigment called pyocyanin. It also produces a Aside from these pigments, it ometimes produces a sweet or grape-like or corn taco- like odor which is helpful in the identification of the organism. (Brooks 2010). It also has special structures, enzymes, and toxins which makes it highly virulent when introduced into areas devoid of normal defenses. The structures help the organism attach to host cells, avoid the action of phagocytes, and form biofilms, while the enzymes breakdown tissues causing necrosis. On the other hand, toxins produced by P. aeruginosa inhibit protein synthesis by interfering with adenosine diphosphateribosylation of elongation factor-2 (Braunwald 2008).

It is a bacterium responsible for severe nosocomial infections, life-threatening infections in immunocompromised persons, and chronic infections in cystic fibrosis patients. The bacterium’s virulence depends on a large number of cell-associated and extracellular factors. Cell-to-cell signalling systems control the expression and allow a coordinated, cell-density–dependent production of many extracellular virulence factors (Van Delden, 1998). This study will utilize P. aeruginosa as the test organism because of reported cases where the organism is highly resistant to commonly used antibiotics. Todar, 2011) The reason why it has a low antibiotic susceptibility is because of its efflux pumps with chromosomally encoded antibiotic resistance genes whose net effect is to effectively reduce the amount of drug that enter the bacterium. Since the drug does not reach high enough concentrations within the bacteria it is unable to kill it. Another tactic employed by the bacterium is the low permeability of the cell wall which further reduces the amount of drug that enters the cell. Most of the commonly-used antibiotics, particularly the penicillins and first generation cephalosporins, are not effective against Pseudomonas (Zinsser, 1992).

This study will use Amikacin as the positive control. Amikacin is a semisynthetic aminoglycoside antibiotic produced by acylation of Kanamycin A. It works by binding to the bacterial 30S ribosomal subunit interfering with the correct transcription of the mRNA resulting in the inhibition of bacterial protein biosynthesis. It is very effective against Pseudomonas species. It is recommended as a single dose or divided doses into two or three equal portions of 15 mg/kg/day but the dosage and interval must be altered for patients with renal failure.

Amikacin may be given intramuscularly or intravenously (Brunton, 2006). Amikacin resists degradation by most aminoglycoside inactivating enzymes known to affect gentamicin, tobramycin, and kanamycin (Bauer et al, 2004). Disc Susceptibility Test method, which requires the measurement of the diameters of the zone of inhibition, give the most precise estimates of antibiotic susceptibility. One such procedure has been recommended for use with discs to test susceptibility to Amikacin. Interpretation involves correlation of the diameters obtained in the disc test with MIC values for Amikacin.

When the causative organism is tested by the KirbyBauer method of disc susceptibility, a 30-mcg Amikacin disc should give a zone of 17 mm or greater to indicate susceptibility. Zone sizes of 14 mm or less indicate resistance. “Susceptible” indicates that the infecting organism is likely to respond to therapy while a report of “resistant” indicates that the infecting organism is not likely to respond to therapy. Since guava has been used against many types of ailments including those brought about by bacteria, it would be beneficial to test if its extracts are effective against P. aeruginosa.

Plants have been a source of many drugs since some of its compounds are known to be antimicrobial such as flavonoids. It is hoped that guava may have such compounds or possibly other unidentified compound which can aid against Pseudomonas. P. aeruginosa which utilizes many tactics such as efflux pump and a membrane that greatly limits entry of drugs is making it highly resistant to many drugs. If left untreated, it can be lethal. Significance of the Study This research will be conducted hoping that it will benefit the following: ? The general public, that they may be able to expand their knowledge of the eneficial effects of natural products. ? The Department of Health, Department of Science and Technology and other concerned agencies, that they do further studies on guava and determine the exact active component(s) to help in developing a new drug. ? Future researchers that they may use this study as basis for similar studies to be conducted that would involve determining the medicinal benefits of guava leaves. Objectives General Objective This study aims to determine the antimicrobial effect of 100% mature P. guajava (guava) leaf extract on P. aeruginosa (ATCC # 27853). Specific Objectives 1.

To determine the diameter of the zone of inhibition, in millimeters (mm) using Kirby-Bauer Method exhibited by the 100% mature guava leaf extract against P. aeruginosa. 2. To determine the diameter of the zone of inhibition, in millimeters (mm) using Kirby-Bauer Method exhibited by the positive control (Amikacin). 3. To calculate if there is a significant difference among the groups against P. aeruginosa. Statement of Hypothesis Null Hypothesis (Ho): There is no significant difference in the diameter of the zone of inhibition of P. aeruginosa with 100% P. guajava leaf extract compared to P. aeruginosa with Amikacin.

Alternative Hypothesis (Ha): The zone of inhibition of P. aeruginosa with 100% P. guajava leaf extract is less than or greater than the zone of inhibition of P. aeruginosa with Amikacin. Scope and Limitations The study aims to determine the effect of the guava leaf extract on the growth of the P. aeruginosa species only. This study will not determine the exact component that is responsible for the antimicrobial activity of P. guajava. concentrations of P. guajava leaf extract. It will not compare the effects of different MATERIALS AND METHODS Research Design This is a nonrandomized, open-label controlled trial.

Research Locale Extraction of the guava leaf extract will be conducted at the Research Laboratory, while the inoculation, incubation and reading of plates will be done at the Microbiology Laboratory, which are both located at 5th floor, #1 PV Larrazabal Jr. Avenue, Mandaue City, Cebu. Research Subjects The subjects of the study are P. aeruginosa (ATCC# 27853), which will be inoculated into 10 plated mediums. The organism will be obtained from the Department of Microbiology, Cebu Institute of Medicine. Research Instruments The researchers will utilize a data collection sheet for the tabulation of data.

Data Collection Procedures I. Preliminary Procedure Submission of Transmittal Letters A transmittal letter will be sent to Dr. Enrico B. Gruet, Dean of the College of Medicine of Cebu Doctors’ University for the approval of the implementation of the study. Upon approval by the office of the Dean, a letter would be submitted to the university research office again for the approval of the study. Transmittal letters will also be given to Dr. Roserie T. Quisumbing and Dr. Teresita E. Chiu asking their permission to conduct the study at the Microbiology Laboratory and the Research Laboratory, respectively.

Another transmittal paper will be addressed to Dr. Virginia Pato-Mesola, microbiologist of Cebu Institute of Medicine, to ask for P. aeruginosa ATCC #27853. Lastly, a transmittal letter will be given to a botanist, Dr. Franz Seidenschwartz, for the identification of the guava leaves. Collection and Preparation of Guava leaves for Extraction The Guava leaves will be taken from Lapu-Lapu City. The sample will be authenticated by a botanist from the University of San Carlos. The fresh mature leaves of Guava will be washed with tap water to remove the dirt and other unwanted materials. After washing, they will be chopped into pieces.

Extraction Procedure Maceration method will be used to produce the guava extract. 250 grams of chopped fresh guava leaves will be weighed and placed in a 500-ml Erlenmeyer flask. 450 ml of 95% Ethanol will be added, enough to submerge the leaves. The mixture will then be allowed to stand for 24 hours, after which it will be filtered. The filtrate will then be reduced to one-third of its original volume (150 ml) using a rotary evaporator producing the guava extract. The guava extract will be weighed to get 100 g. Sterile distilled water will be added to the extract to make a 100 mL solution, which is equivalent to 100% concentration.

Preparation of Filter Paper Disk Whatman Filter Paper No. 2 disks will be made with an approximate diameter of 6. 6 mm by using an ordinary office two-hole puncher. Precaution must be taken to avoid overlapping of holes, since the paper disks will have a tendency to curl after punching. They will be flattened by spreading them in a single layer on a clean smooth surface then pressed by rolling a bottle over it repeatedly. The disks will be placed in vials then autoclaved at 15 psi, 121? C for 15 minutes and then cooled. Impregnation of filter disk Using a sterile dropper, 1 mL of 100% guava leaf extract will be applied to filter paper disks.

To maintain sterility, the disks will be dried inside a laminar flow cabinet for two hours. For the positive control, commercially-made Amikacin antimicrobial disks will be purchased from a pharmaceutical company. Collection of P. aeruginosa organism The organism will be obtained from the Microbiology Department of Cebu Institute of Medicine. II. Experimental Procedure Preparation of Bacterial Suspension The tip of the wire loop will be sterilized through heating using an alcohol lamp. A loopful of colony from the stock culture will be isolated and suspended into a sterilized tube containing 5 mL of normal saline solution (NSS).

The suspension will be compared with the 0. 5 McFarland Standard to measure the appropriate turbidity of the bacterial suspension. When the turbidity of the bacterial suspension matches that of the standard, it will be incubated for 15 minutes at 37? C. Preparation of the Mueller-Hinton Infusion Agar Plates Nineteen grams of MH powder is dissolved in 500 ml of distilled water which should be mixed well and autoclaved to sterilize. After autoclaving, it will be allowed to cool down before pouring into the petri dishes, measuring approximately 150 x 15 mm. The depth will be controlled to approximately 4 mm to minimize variability in zone sizes.

Inoculation of the organism in the culture medium Mueller-Hinton Infusion Agar (MHIA) Within 15 minutes after adjusting the turbidity of the inoculum suspension, sterile cotton swab will be dipped into the suspension. Pressing firmly against the inside wall of the tube just above the fluid level, the swab will be rotated to remove excess liquid. It will be streaked over the entire surface of the medium three times, rotating the plate approximately 60 degrees after each application to ensure an even distribution of the inoculums. Finally, the edges of the agar surface will be swabbed with the bacterial suspension.

One person will perform the streaking method to avoid variation in technique. Disk application to the media Ten petri dishes with MHIA will be prepared. Each culture medium with the inoculated P. aeruginosa will be divided into two. Using sterile forceps, the Amikacin disks will be placed on one side while the impregnated disks of 100% Guava leaf extract will be placed, at least 24mm apart on the opposite side. Then, each disk will be pressed gently to ensure complete contact with agar. The culture medium will then be placed in the incubator in an upside down manner and incubated at 37? C for 24 hours.

III. Actual Data Gathering The agar plates inoculated with P. aeruginosa with the susceptibility disks will be placed on a table against a black background with reflected lighting. The readings on the zone of inhibition of the positive and experimental groups will be done by a Registered Medical Technologist using a vernier calliper and will be recorded on the data sheets. IV. Data Processing and Analysis The mean, standard deviation and variance will be computed and used for statistical analysis. One-way Analysis of Variants (ANOVA), Repeated Measures will be used to analyze the results of the study.

This is to accurately determine if there was a significant difference in the average zones of inhibition in the groups. Null hypothesis will be tested at 0. 05 ? and 0. 20 ?. DEFINITION OF TERMS Antimicrobial Effect – refers to the ability of the 100% guava leaf extract to inhibit the growth of P. aeruginosa as shown by a zone of inhibition of at least 17mm. Zone of Inhibition – clear zone surrounding the experimental test disk (100% guava leaf extract) and the positive control (Amikacin) with a diameter of 17mm which is devoid of P. aeruginosa growth. 00% Guava Leaf Extract – the liquid obtained from guava leaves after being subjected to maceration. BIBLIOGRAPHY Books Brooks, George F. , et al. Jawetz, Melnick & Adelberg’s Medical Microbiology. 25th ed. USA: McGraw-Hill Companies, 2010. Brunton, Lawrence C. , et al. Goodman & Gilman’s The Pharmacological Basis of Therapeutics. 11th ed. USA: McGraw-Hill Companies, 2006 Fauci, Anthony S. , et al. Harrison’s Principles of Internal Medicine. 17th ed. USA: McGraw-Hill Companies, 2008 Stary, F.. The Natural Guide to Medicinal Herbs and Plants. n. p. Sterling Publishing, 2005 Zinsser, Hans.

Zinsser Microbiology. 20thed. Norwalk CT: Appleton & Lange, 1992 Unpublished Papers Macatol, et al. “Effectiveness of guava leaf extract for wound healing among albino mice. ” Cebu Doctors’ University. 1998 Neviton, Rogerio, Sanches I, et al. “An evaluation of antibacterial activities of Psidium guajava (L. ). ”Universidad Estadual de Maringa, 2005 Online Journals Abdelrahim, S. I. , et al. “Antimicrobial Activity of Psidium guajava L. ” Fitoterapia (2002). Web. 10 August 2011 Abrue, P. R. C. , et al. “Guava Extract (Psidium guajava L. ) Alters the Labeling of Blood Constituents with Technetium. Journal of Zhejian University of Science 10. 1631 (2006). Web. 10 August 2011 Chah, K. F. , et al. “Antibacterial and wound healing properties of methanolic extracts of some Nigerian medicinal plants. ” Journal of Ethnopharmacology (2006). Web. 27 August 2011 Gutierrez, R. et al. “Psidium guajava: A Review of its Traditional Uses, Phytochemistry, and Pharmacology” (2008). Web. 3 July 2011 K. G. Kerr, A. M. Snelling. “Pseudomonas aeruginosa: A Formidable and ever-present adversary. ” (2009). Web. 3 July 2011 Metwall, A. M. , et al. “Phytochemical Investigation and Antimicrobial Activity of Psidium guajava L.

Leaves. Phcog Mag 6. 212 (2010). Web. 12 August 2011 Internet Sources Todar, Kenneth. “Online Textbook of Bacteriology. ” Web. 2011. 16 Aug 2011. APPENDIX A-1 TRANSMITTAL LETTER TO THE DEAN August 2011 Enrico B. Gruet, M. D. Dean, College of Medicine Cebu Doctors’ University #1 P. V. Larrazabal Ave. , Mandaue City Dear Dr. Gruet, Greetings! We, the second year students of Cebu Doctors’ University-College of Medicine, will be conducting a research study entitled “The Antimicrobial Effect of 100% Mature Psidium guajava (Guava) Leaf Extract on the Growth of Pseudomonas aeruginosa (ATCC # 27853)”.

With this regard, we are asking for your permission to conduct this study. It would be a great appreciation to have your approval on this study. Thank you and God Bless! Sincerely yours, Lounard N. Pavillar Group 9 Leader Noted by: Approved by: Leah P. Acibar, M. D. Research Adviser Enrico B. Gruet, M. D Dean, CDU-College of Medicine APPENDIX A-2 TRANSMITTAL LETTER TO THE HEAD OF THE MICROBIOLOGY DEPARTMENT August 2011 Roserie T. Quisumbing, M. D. Head, Microbiology Department Cebu Doctors’ University #1 P. V. Larrazabal Ave. , Mandaue City Dear Dr. Quisumbing, Greetings!

We, the second year students of Cebu Doctors’ University-College of Medicine, will be conducting a research study entitled “The Antimicrobial Effect of 100% Mature Psidium guajava (Guava) Leaf Extract on the Growth of Pseudomonas aeruginosa”. With this regard, we would like to ask permission from you to grant the group the privilege of using some of the equipments in the Microbiology Laboratory which are needed for our study. We are hoping for you kind approval. Thank you very much! Sincerely yours, Lounard N. Pavillar Group 9 Leader Noted by: Leah P. Acibar, M. D. Research Adviser

APPENDIX A-3 TRANSMITTAL LETTER TO THE CHAIRMAN OF THE MEDICAL EDUCATIONAL UNIT August 2011 Teresita E. Chiu, M. D. Chairman, Medical Educational Unit Cebu Doctors’ University PV Larrazabal Ave. , Mandaue City Dear Dr. Chiu, Greetings! We, Group 9, Level II students of Cebu Doctors’ University-College of Medicine, will be conducting a study on “The Antimicrobial Effect of 100% Mature Psidium guajava (Guava) Leaf Extract on the Growth of Pseudomonas aeruginosa (ATCC # 27853)”. We would like to ask your kind office if we can conduct our study in the Research Laboratory and if we can also use some of the equipments needed for our study.

We are hoping for your kind consideration regarding this matter. Thank you and God bless. Sincerely yours, Lounard N. Pavillar Group 9 Leader Noted by: Approved by: Leah P. Acibar, M. D. Research Adviser Teresita E. Chiu, M. D. Chairman – Medical Educational Unit APPENDIX A-4 TRANSMITTAL LETTER TO THE HEAD OF THE MICROBIOLOGY DEPARTMENT, CEBU INSTITUTE of MEDICINE August 2011 Virginia Pato-Mesola, M. D. Head, Microbiology Department, Cebu Institute of Medicine F. Ramos St. , Cebu City Dear Dr. Mesola, Greetings!

We, Group 9, Level II students of Cebu Doctors’ University-College of Medicine, will conduct a study on “The Antimicrobial Effect of 100% Mature Psidium guajava (Guava) Leaf Extract on the Growth of Pseudomonas aeruginosa (ATCC # 27853)”. In line with this study, we would like to purchase a pure culture of the said organism from your laboratory. Rest assured it will be used for scientific and educational purposes only. Your institution will not be held liable for any illnesses and accidents that may arise from the handling of the above culture. We are hoping for your kind consideration. Thank you and God bless.

Respectfully yours, Lounard N. Pavillar Group 9 Leader Noted by: Approved by: Leah P. Acibar, M. D. Research Adviser Virginia P. Mesola, M. D. Head, Microbiology Dept. CIM APPENDIX A-4 TRANSMITTAL LETTER TO THE BOTANIST FROM THE UNIVERSITY OF SAN CARLOS August 2011 Franz Seidenschwarz, PhD Botanist – University of San Carlos P. del Rosario St. , Cebu City Dear Dr. Seidenschwarz, Greetings! We, Group 9, Level II students of Cebu Doctors’ University-College of Medicine, will be conducting a study on “The Antimicrobial Effect of 100% Mature Psidium guajava (Guava) Leaf Extract on the Growth of Pseudomonas aeruginosa (ATCC # 27853)”.

We would like to ask for your assistance in the verification of the herbal plant the group will be utilizing in the research whether the plant is Psidium guajava. Thank you and God bless. Sincerely yours, Lounard N. Pavillar Group 9 Leader Noted by: Leah P. Acibar, M. D. Research Adviser APPENDIX B-1 CERTIFICATION FOR PSEUDOMONAS AERUGINOSA August 2011 Group 9, Level II Cebu Doctors’ University – College of Medicine This letter is to certify that the culture given has been verified as Pseudomonas aeruginosa (ATCC # 27853). Virginia Pato-Mesola, M. D. Microbiologist-Cebu Institute of Medicine

APPENDIX B-2 CERTIFICATION FOR PSIDIUM GUAJAVA August 2011 Group 9, Level II Cebu Doctors’ University – College of Medicine This letter is to certify that the leaves submitted for verification are indeed Psidium guajava. Franz Seidenschwarz, PhD Botanist – University of San Carlos APPENDIX C DATA COLLECTION SHEET PLATE POSITIVE CONTROL GROUP (Amikacin) (in mm) EXPERIMENTAL GROUP (100% mature guava leaf extract) (in mm) 1 2 3 4 5 6 7 8 9 10 APPENDIX D TIME TABLE SCHEDULE OF ACTIVITIES ACTIVITIES CONCEPTUAL PHASE 1. Formulation of Problem 2. Review of Related Literature 3. Identifying Variables and Formulating Hypothesis 4.

Developing research Framework DESIGN / PLANNING PHASE 5. Research Design 6. Identifying Population 7. Sample Plan 8. Statistical Measure 9. Finalized Proposal EMPERICAL PHASE 10. Collection of Data 11. Preparing Data for Analysis ANALYTIC PHASE 12. Analyze Data 13. Interpret Results DISSEMINATION PHASE 14. Communicate Findings 15. Utilize Findings MONTH JUNE JULY AUG SEPT OCT NOV DEC JAN FEB MAR APR MAY APPENDIX E BUDGET SUMMARY ITEM Bond Papers Printing (Ink) Photocopies Stock Culture of Pseudomonas aeruginosa Antibiotic Discs (Amikacin) Guava Leaves Ethanol Mueller Hinton Agar Plates Total QUANTITY 1 ream 500 1 vial 1 250 grams 500ml 1 15

PRICE (Php) 250 700 500 250 250 100 200 500 250 Php 3,000 APPENDIX F SAMPLE SIZE DETERMINATION Two-Sample T-Test Power Analysis Page/Date/Time 1 8/11/2011 5:01:17PM Numeric Results for Two-Sample T-Test Null Hypothesis: Mean1=Mean2. Alternative Hypothesis: MeanMean2 The standard deviations were assumed to be known and unequal. Power 0. 99966 0. 99997 N1 10 10 N2 10 10 Allocation Ratio 1. 000 1. 000 Alpha 0. 01000 0. 05000 Beta 0. 00034 0. 00003 Mean1 Mean2 17. 4 17. 4 21. 5 21. 5 S1 0. 8 0. 8 S2 2. 0 2. 0 Report Definitions Power is the probability of rejecting a false null hypothesis. Power should be close to one.

N1 and N2 are the number of items sampled from each population. To conserve resources, they should be small. Alpha is the probability of rejecting a true null hypothesis. It should be small. Beta is the probability of accepting a false null hypothesis. It should be small. Mean1 is the mean of populations 1 and 2 under the null hypothesis of equality. Mean 2 is the mean of population 2 under the alternative hypothesis. The mean of population 1 is unchanged. S1 and S2 are the population standard deviations. They represent the variability in the populations. Summary Statements Group sample sizes of 10 and 10 achieve 100% ower to detect a difference of -4. 1 between the null hypothesis that both group means are 17. 4 and the alternative hypothesis that the mean of group 2 is 21. 5 with known group standard deviations of 0. 8 and 2. 0 and with a significance level (alpha) of 0. 01000 using a two-sided two-sample t-test. APPENDIX G COMPUTATION FOR PLANT CONCENTRATION __ 100g solute_____ X 100% = 100% concentration 100 mL solution RESEARCHER’S BIODATA PERSONAL DATA Name: Sex: Place of Birth: Date of Birth: Age: City Address: Provincial Address: Contact Number: Civil Status: Citizenship: Religion: Glory O.

Avancena, RPh Female Cebu City April 28, 1989 22 Agus, Ibabao, Lapu-Lapu City Agus, Ibabao, Lapu-Lapu City 238-4879/0922-8293758 Single Filipino Independent Catholic EDUCATIONAL BACKGROUND Primary Education: Asian Learning Center Secondary Education: Asian Learning Center Tertiary Education: University Of San Carlos – TC Course: Post- Graduate Studies: Cebu Doctors’ University RESEARCH EXPERIENCE “Comparison on the Inhibitory Activity of the Fresh Leaves of Andropogon citrates Linn. (Lemon Grass) and Mentha arvensis Linn. Yerba Buena) on Human Platelet Aggregation” (2009). “An Assessment on the Mentoring Program of Cebu Doctors’ University – College of Medicine” (2011). Date Graduated: 2001 Date Graduated: 2005 Date Graduated: 2009 BS Pharmacy RESEARCHER’S BIODATA PERSONAL DATA Name: Sex: Place of Birth: Date of Birth: Age: City Address: Provincial Address: Contact Number: Civil Status: Citizenship: Religion: Mark Henry C. Booc Male Tagum City, Davao del Norte January 26, 1988 23 Agus, Ibabao Lapu-Lapu City Agus, Ibabao Lapu-Lapu City 09103741394 Single Filipino/American None

EDUCATIONAL BACKGROUND Primary Education: Manzanita Elementary School Secondary Education: Apollo High School Tertiary Education: Cebu Doctors’ University Course: Post- Graduate Studies: Cebu Doctors’ University RESEARCH EXPERIENCE “A Comparative Study of the Effect of Inorganic Chemical Fertilizer, and Vermicompost on the Growth of Zea mays (Corn)” (2010) “A Descriptive Study of the Food Handling Practices among Food Establishments within Canteens of Cebu Doctors’ University” (2011) Date Graduated: 2000 Date Graduated: 2006 Date Graduated: 2010 BS Biology

RESEARCHER’S BIODATA PERSONAL DATA Name: Sex: Place of Birth: Date of Birth: Age: City Address: Provincial Address: Contact Number: Civil Status: Citizenship: Religion: Dan Samuel S. Burgos Male Ormoc City, Leyte June 24, 1988 24 530 A. C. Cortes Avenue, Ibabao, Mandaue City 530 A. C. Cortes Avenue, Ibabao, Mandaue City 344-2823/09337201180 Single Filipino Christian

EDUCATIONAL BACKGROUND Primary Education: Saint Joseph College Secondary Education: Little Angels Montessori School Tertiary Education: University of the Visayas Course: Post- Graduate Studies: Cebu Doctors’ University EXPERIENCE IN RESEARCH: The Effects of the Orange Colored Highlighter on the short-term memory of Level I Nursing students in the University of the Visayas, (2009) The Level of Knowledge on Human Papillomavirus (HPV) and HPV Vaccination among Female Levels I, II, and III Medical Students of Cebu Doctors’ University, Mandaue City, Cebu. 2011) Date Graduated: 2001 Date Graduated: 2005 Date Graduated: 2009 BS Nursing RESEARCHER’S BIODATA PERSONAL DATA Name: Sex: Place of Birth: Date of Birth: Age: City Address: Provincial Address: Contact Number: Civil Status: Citizenship: Religion: Eloise Maxine B. Cabang, RN Female Cebu City May 1, 1985 26 3109 Maria Theresa Village 2 Guadalupe Cebu City 3109 Maria Theresa Village 2 Guadalupe Cebu City 255-2802/09173233936 Single Filipino Born Again Christian

EDUCATIONAL BACKGROUND Primary Education: Sacred Heart School for Girls Secondary Education: Sacred Heart School for Girls Tertiary Education: Cebu Doctors’ University Course: Post- Graduate Studies: Cebu Doctors’ University RESEARCH EXPERIENCE “The Effect of Quercetin in Allium cepa on Age Spots” 2005-2006 “An Assessment on the Mentoring Program of Cebu Doctors’ University – College of Medicine” 2010-2011 Date Graduated: 1998 Date Graduated: 2003 Date Graduated: 2006 BS Nursing

RESEARCHER’S BIODATA PERSONAL DATA Name: Sex: Place of Birth: Date of Birth: Age: City Address: Provincial Address: Contact Number: Civil Status: Citizenship: Religion: Arnel Y. Conopio Male Daan-Bantayan, Cebu August 8, 1989 22 A-13, Victoria Village, Talisay City, Cebu Tinubdan, Daan-Bantayan, Cebu 09065557939 Single Filipino Roman Catholic EDUCATIONAL BACKGROUND Primary Education: St.

Paul College Secondary Education: University of San Jose-Recoletos Tertiary Education: Cebu Doctors’ University Course: Post- Graduate Studies: Cebu Doctors’ University RESEARCH EXPERIENCE “A Comparative Study of the Effect of Inorganic Chemical Fertilizer, and Vermicompost on the Growth of Zea mays (Corn)” (2010) “Knowledge and Skills to Self Directed Learning Readiness of the Level Four Students of Cebu Doctors’ University-College of Medicine Problem Based Learning Curriculum Academic Year 2010 – 2011” (2011) Date Graduated: 2002 Date Graduated: 2006 Date Graduated: 2010 BS Biology

RESEARCHER’S BIODATA PERSONAL DATA Name: Sex: Place of Birth: Date of Birth: Age: City Address: Provincial Address: Contact Number: Civil Status: Citizenship: Religion: Jose Reginald K. Cordova, RN Male Bacolod City, Negros Occidental April 22, 1989 22 Mandaue City, Cebu Eroreco Village, Bacolod City 09162936147 Single Filipino Roman Catholic EDUCATIONAL BACKGROUND Primary Education: Don Bosco Technical Institute La Consolacion College – Bacolod Secondary Education: La Consolacion College – Bacolod Tertiary Education: University of St.

La Salle – Bacolod Course: Post- Graduate Studies: Cebu Doctors’ University RESEARCH EXPERIENCE “Awareness among the Residents of Zone 5 Purok Masinadyahon, Brgy. Bata, Bacolod City of the Influenza A (H1n1); A Basis for A Health Teaching Plan” (2009) “A Comparative Study Of Compliance Barriers To The Expanded Program Of Immunization (EPI) Between Mothers Whose Children Were Fully And Not Fully Immunized In Barangay S. N.

Pahina In The Year 2009” (2011) Date Graduated: 1995-1999 1999-2002 Date Graduated 2006 Date Graduated: 2010 BS Nursing RESEARCHER’S BIODATA PERSONAL DATA Name: Sex: Place of Birth: Date of Birth: Age: City Address: Provincial Address: Contact Number: Civil Status: Citizenship: Religion: Leoniza Gloria S. Gozo, RMT Female Cebu City December 9, 1986 22 Corner Gen. Maxilom and Echavez St. , Cebu City Corner Gen. Maxilom and Echavez St. , Cebu City 09166973161 Single Filipino Born Again Christian

EDUCATIONAL BACKGROUND Primary Education: Christ the King College Secondary Education: Christ the King College Tertiary Education: Cebu Doctors’ University Course: Post- Graduate Studies: Cebu Doctors’ University RESEARCH EXPERIENCE “The Anti-diuretic Effect of Euphorbia hirta on Selected Male Albino Mice” (2009) “The Correlation of the Level of Knowledge and Perspective Regarding the Main Points of the Reproductive Health and Population Development Acts of 2008 Among Selected Undergraduate Students of Cebu Doctors’ University for Academic Year 20102011” (2011) Date Graduated: 2001 Date Graduated: 2005 Date Graduated: 2009 BS Medical Technology RESEARCHER’S BIODATA

PERSONAL DATA Name: Sex: Place of Birth: Date of Birth: Age: City Address: Provincial Address: Contact Number: Civil Status: Citizenship: Religion: Lounard N. Pavillar, RMT Male Lapu-Lapu City November 11, 1986 24 Mactan Air Base, Lapu-Lapu City Mactan Air Base, Lapu-Lapu City 236-0931/09062295962/09227781553 Single Filipino Baptist EDUCATIONAL BACKGROUND Primary Education: Bob Hughes Christian Academy Secondary Education: Bob Hughes Christian Academy Tertiary Education: Velez College Course: Post- Graduate Studies: Cebu Doctors’ University RESEARCH EXPERIENCE “A Study on the Most Commonly Isolated Pathogens Found in Positive Blood Cultures of Newborn Babies Born in Cebu (Velez) General Hospital from 1995-2005. (2008) “The Level of Awareness and Actual Practice on Household Waste Segregation by Selected Residents of Brgy. Subangdaku, Mandaue City, Cebu. ” (2011) Date Graduated: 1999 Date Graduated: 2003 Date Graduated: 2008 BS Medical Technology RESEARCHER’S BIODATA PERSONAL DATA Name: Sex: Place of Birth: Date of Birth: Age: City Address: Provincial Address: Contact Number: Civil Status: Citizenship: Religion: Marie Andone L. Tan, RN Female Cebu City December 04, 1987 23 Dr. 4 Gallardo Apt. , Orchid St. , Capitol Site, CC Sitio Can-apog, Brgy. SubaVillaba, Leyte 412 0832; 09176273459; 09225386035 Single Filipino Roman Catholic EDUCATIONAL BACKGROUND Primary Education: St. Paul’s School of Ormoc Secondary Education: St.

Paul’s School of Ormoc Tertiary Education: Cebu Doctors’ University Course: Post- Graduate Studies: Cebu Doctors’ University RESEARCH EXPERIENCE “The Effects of 2gm raw Allium sativum (Garlic) on the Blood Cholesterol Levels of middle-aged individuals in Brgy. Guadalupe, Cebu City” “The Profile of CDU personnel manifesting with high total cholesterol and high low-density lipoprotein cholesterol levels from June 2007 to July 2010” Date Graduated: 2000 Date Graduated: 2004 Date Graduated: 2008 BS Nursing RESEARCHER’S BIODATA PERSONAL DATA Name: Sex: Place of Birth: Date of Birth: Age: City Address: Provincial Address: Contact Number: Civil Status: Citizenship: Religion: Aldo Ian A. Tancinco, RN Male Cebu City June 17, 1988 23 44-45 Anthurium St. Phase 2, Camella Homes, Lapu-lapu City 44-45 Anthurium St.

Phase 2, Camella Homes, Lapu-lapu City 2684644/ 09229511607 Single Filipino Born Again Baptist EDUCATIONAL BACKGROUND Primary Education: Sacred Heart School for Boys Secondary Education: Sacred Heart School for Boys Tertiary Education: Cebu Doctors’ University Course: Post- Graduate Studies: Cebu Doctors’ University RESEARCH EXPERIENCE “The Effect of kakawate leaves decoction on scabies infection among school age children” (2009) “The Knowledge, Attitude, and Practice with Regards to Contraception among Third Year Undergraduate Students of Cebu Doctors’ University” (2011) Date Graduated: 2001 Date Graduated: 2005 Date Graduated: 2009 BS Nursing

Cite this The Antimicrobial Effect of 100% Mature Psidium Guajava (Guava) Leaf Extract on the Growth of Pseudomonas Aeruginosa (Atcc # 27853) Essay

The Antimicrobial Effect of 100% Mature Psidium Guajava (Guava) Leaf Extract on the Growth of Pseudomonas Aeruginosa (Atcc # 27853) Essay. (2019, May 01). Retrieved from https://graduateway.com/the-antimicrobial-effect-of-100-mature-psidium-guajava-guava-leaf-extract-on-the-growth-of-pseudomonas-aeruginosa-atcc-27853/

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