The Antimicrobial Effect of Guava Leaf Extracts Psidium Guajava L. Against Pseudomonas Aeruginosa

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FEU MANILA- INSTITUTE OF ARTS AND SCIENCES -1-
The Antimicrobial Effect of Guava Leaf Extracts
(Psidium guajava L.) against
Pseudomonas aeruginosa
A Thesis Proposal
Presented to the
Department of Medical Technology
Far Eastern University Manila
In Partial Fulfillment
Of the Requirements of the Degree
Bachelor of Science in Medical Technology
By
Group 4
November 2020

Institute of Arts and Sciences
RESEARCH PROPOSAL ADVISER

ENDORSEMENT (FORM 3)
Date: 27/11/2020
Group Members:
Bosque, Darren Joseph P.
Lopez, Arjay Manuel S.
Masangkay, Edezon Joseph G.
Morales, Sheina T.
Ortega, Jeffrey Leynard F.
Sanchez, Neil Adrian D.
Velasquez, Tirso II H.
Title of Research

(Psidium guajava L.) against
Dear Francisco R. Gellecanao Jr., RMT, MPH

Program Head, Medical Technology Department
I would like to endorse this proposal manuscript for further review from the
selected members of the panel. I fully submit to the comments and suggestion that
the panel may find for the further improvement of the conduct of this study.
Thank you.
Lenzie P. Santillan, RMT
Research Adviser
Sign over
printed name
Date: 27/11/2020

Institute of Arts and Sciences
ADVISER ACCEPTANCE FORM

Name of Group Members
Date Submitted:
Last Name, Hagosojos Given Name, Bernardino M.I. M.
Name of Research 1 Professor:
Bosque, Darren
Lopez, Arjay
Masangkay, Edezon
Morales, Sheina
Ortega, Jeffrey
Sanchez, Neil Adrian
Velasquez, Tirso
Information on Proposed Research/Project
Working Title of (Psidium guajava L.) against

Proposed
Research
Brief description
of Proposal The objective of the study is to evaluate the efficacy of guava
(This should leaf extracts as potential and alternative antimicrobial
include solution to eliminate Pseudomonas aeruginosa. The method
objectives and that will be used to know the antimicrobial activity of guava
methods that the leaf extracts is disk diffusion method.
group will use to
achieve desired
results of the
study and should
be limited to 200
words or less)
I accept this research/project to be part of my advisee/s this Academic School

Year and agree to provide to the group the necessary intellectual and
technical guidance they will be needing for the completion of the project. I
have also agreed to provide the group ample time for inquiries they may
have regarding the conduct of the project/research.
LENZIE P. SANTILLAN, RMT

Date:OCTOBER 31, 2020
Signature over printed name of Adviser
Noted:
Francisco R. Gellecanao, Jr., RMT, MPH

Program Head, Medical Technology Dept
Research title: The Antimicrobial Effect of Guava Leaf Extracts (Psidium guajava
L.) against Pseudomonas aeruginosa
Research Adviser: Lenzie Santillan Research Instructor: Bernardino M.
Hagosojos
Semester: Second Section: 17
Group Members: Bosque, Darren Ortega, Jeffrey

Lopez, Arjay Manuel Sanchez, Neil Adrian
Masangkay, Edezon Velasquez, Tirso II
Morales, Sheina
Adviser’s Consultation form

ADVISER’S RESEARCH
SIGNATURE INSTRUCTOR’S
DATE REMARKS
AND DATE SIGNATURE
AND DATE
September Research proposal October 31,
20, 2020 and introduction. 2020
September Methodology October 31,

24, 2020 (concentration of 2020
extract, solvents to be
used and AST).
October Chapter 1 and 2 October 31,

31, 2020 checking. 2020
November Chapter 3 checking. November 24,

24, 2020 2020
ABSTRACT
This section is limited to 250-300 words, single-spaced and must include

at least four (4) keywords. Abstract should be confined in one page only.
ACKNOWLEDGEMENT
The researchers would like to thank the following for the help, guidance and
support extended to them:
To Prof. Bernardino Hagosojos and Prof. Nathaniel John Jumalon, Lecture and
Laboratory Professor, for their invaluable teachings and knowledge during
discussions in the course Introduction to Medical Technology Research.
To Prof. Lenzie Santillan, Research Adviser, for her guidance and important
suggestions during the writing of this research paper.
To their beloved parents, siblings, and friends for the moral support.
To God Almighty, for his endless blessings, wisdom, and strength to make this
research possible.
TABLE OF CONTENTS
Page
ABSTRACT ii
ACKNOWLEDGEMENT iii
LIST OF TABLES AND FIGURES vi
1.0. INTRODUCTION 1
1.1. Background of the study 2
1.2. Statement of the Problem 4
1.3. Hypotheses 4
1.4. Objectives of the Study 5
1.5. Significance of the Study 5
1.6. Scope and Limitations 6
1.7. Definition of Terms 7
LIST OF TABLES AND FIGURES
Tables
Table 2.3.2: Zone of inhibition (mm) of different bacterial

agents at varying concentrations of methanol extracts 19
obtained from P. guajava and standard antibiotic
norfloxacin
Figures
Figure 2.5.1: Conceptual Framework Diagram 23
Figure 3.1: Sample Collection and Processing 25

CHAPTER 1
INTRODUCTION
This chapter presents the background of the study, the statement of the
problem, the objectives of the research, its beneficiaries, hypothesis, scope and
limitation, and definition of terminologies.
1.1. Background of the study
A Guava leaf has a great potential as a functional ingredient for
development. They contain high levels of antioxidant, biological activities, and
phenolic compounds that serve as immunostimulatory agents.
Botanical Medicine, herbal medicine, medical herbalism, herbology,
phototrophy, or Herbalism, is a common practice in the folk period that is based
on the use of plants and plant extractions to cure ailments. According to Nafu et.
al (2017) , medical plants are the richest bioresource of drugs of traditional
systems not only in folk medicines but also in other fields such as modern
medicine, nutraceuticals, food supplements, folk medicines, pharmaceutical
intermediates, and chemical entities for synthetic drugs. As stated by Sofowora et.
al (2013), similarly, defines a “medical plant” in which some of its parts can be
used directly for the management of a disease. In addition, herbal medicines may
be made from cellular structures like leaf, petal, root, bark, or may be made out of
non-cellular structural agents like the latex and gum. In addition, it can also be a
decoction, which can be prepared in cold water or can be prepared by boiling and
allowing it to cool, similarly on how the herbal tea was made.
According to Abbiw (1990) as cited by Okafor, the ability of the herbs for
healing is acquired informally and improved upon with practice. The use of plant
extracts was the main method of treating various illnesses before the advent of
Western medicine. The plant extractions are continued to be used in rural
communities who do not have access to the hospitals or health clinics. Herbal
drugs are commonly promoted as natural and safe, claiming that they can cure a
disease in a natural way. The leaves and barks of a plant have a long history of
medical use. The decoction of the leaves and bark are used or believed to cure
diarrhea, dysentery, vomiting, and sore throats, and to regulate menstrual cycles.
As stated by Naseer, Hussain, and Naeem (2018), a guava is rich in
tannins, phenols, triterpenes, flavonoids, saponins, carotenoids, vitamins, essential
oils, lectins, fatty acids, and fibre. It is also higher in vitamin C than other citrus
fruits containing about 80 mg of vitamin C in every 100 grams. In addition, plants
have always played a role in combating diseases that affect humans. Various
plants exhibit antimicrobial activity due to their phytochemicals. Phytochemicals

are compounds found in plants which are used for their growth or defense against
competitors or predators. The guava (Psidium guajava) is a plant used in
traditional medicine for its supposed ability to treat and manage certain diseases.
Henceforward, the researchers would like to assess the effectiveness of
this plant against the bacteria- Pseudomonas aeruginosa. According to the Center
for Disease Control and Prevention (2019), P. aeruginosa is ubiquitously found
on soil and water and the leading cause of nosocomial infections to hospitalized
patients including bacteremia, pneumonia and other respiratory tract infections,
meningitis, septic shock, wound infections and etc. The pathogenicity of this
bacteria makes it interesting for the researchers to explore possible antimicrobial
solutions such as guava leaf extracts in an affordable way.
Further, the researchers would like to extend the knowledge of these
herbs, in particular to guava leaves, to determine its antimicrobial effect on
Pseudomonas aeruginosa and to evaluate relatively the foundation on how
phytochemical components fight this certain bacteria.With these, the researchers
may contribute for advancing and finding suitable antimicrobial agent against this
pathogenic entity.
1.2. Statement of the Problem
This research entitled “The Antimicrobial Effect of Guava Leaf Extracts
(Psidium guajava L.) against Pseudomonas aeruginosa” aims to test the
Effectiveness of Guava Leaf extract as a potential antimicrobial agent against
Pseudomonas aeruginosa.
Specifically, this aims to answer the following questions:
1. What are the phytochemical properties of guava leaves extract
against P. aeruginosa?
2. What are the mean zones of inhibition produced by the guava
leaves extracts (distilled, methanolic and ethanolic) at
concentration of 12.5 mg/mL against P. aeruginosa?
3. Is there a significant difference in the mean zone of inhibition
between the three extracts (distilled, methanolic and ethanolic) at
concentration of 12.5 mg/mL in inhibiting the growth of
Pseudomonas aeruginosa?
1.3. Hypotheses
The researchers hypothesize the following to be justified by scientific
evidence at the end of the study:
1.3.1 Null Hypothesis: Guava leaves extract has no phytochemical
properties against P. aeruginosa.
1.3.2 Alternative Hypothesis: Guava leaves extract has phytochemical
properties against P. aeruginosa.
1.3.3 Null Hypothesis: The three Guava leaves extracts (distilled,
methanolic and ethanolic) at concentration of 12.5 mg/mL will not produce a zone
of inhibition as tested against P. aeruginosa.
1.3.4 Alternative Hypothesis: The three Guava leaves extract (distilled,
methanolic and ethanolic) at concentration of 12.5 mg/mL will produce a zone of
inhibition as tested against P. aeruginosa.
1.3.5 Null Hypothesis: There is no significant difference in the mean
zone of inhibition between the three extracts (distilled, methanolic and ethanolic)
at concentration of 12.5 mg/mL in inhibiting the growth of Pseudomonas
aeruginosa.
1.3.6 Alternative Hypothesis: There is a significant difference in the
mean zone of inhibition between the three extracts (distilled, methanolic and
ethanolic) at concentration of 12.5 mg/mL in inhibiting the growth of
Pseudomonas aeruginosa.
1.4. Objectives of the Study
The main objective of the study is to determine the antimicrobial effect of
guava leaf extracts with varying combinations against Pseudomonas aeruginosa.
Specifically, the research aims the following:
1. Determine the specific phytochemical properties of guava leaf
extracts to combat Pseudomonas aeruginosa.
2. Determine the mean zones of inhibition produced by guava leaves
extract (distilled, methanolic, and ethanolic) at concentration of
12.5 mg/mL against Pseudomonas aeruginosa.
3. Determine if there is a significant difference in the mean zone of
inhibition between the three extracts (distilled, methanolic and
ethanolic) at concentration of 12.5 mg/mL in inhibiting the growth
of Pseudomonas aeruginosa.
1.5. Significance of the Study
The study of the Antimicrobial Effectiveness of Guava leaf (Psidium
guajava L.) extract on P. aeruginosa will benefit the following people as this
research will help to assess an alternative and natural antimicrobial solution to
eliminate P. aeruginosa that commonly found in soil and water.
This research will be great importance to the following beneficiaries:
For the Community. With this research, people will gain a wider knowledge
about the significance of guava leaf extracts against P. aeruginosa.
For the Medical Experts. This study will help the experts to look into the
antimicrobial effect of guava leaf extracts against the bacterium. Through this, the
experts would gain more knowledge about the guava leaf (Psidium guajava L.)
extracts and to consider the extracts as an antibacterial agent for P. aeruginosa.
Students. The students will benefit from this study by producing prerequisite
learnings on guava leaves extractions. With this, the students would therefore
have a great foundation of knowledge academically and experimentally.
Professors. The principal partners of students in the academe, the professors
would benefit from this study, would be given the opportunity to reflect on what
they know to add to their knowledge, and be the one to regulate the amount of
knowledge that will be introduced to the students. Eventually, the professors
would not only make good the students’ knowledge but also them to be more
meticulous in teaching.
Future Researchers. This would be of great help to the future researchers as this
study would be a basis for their work also. Not only would this study act as a
guideline for future frameworks, but also for review of related studies.
1.6. Scope and Limitations
The main goal of this research is to determine the inhibitory effect of leaf
extracts of guava (Psidium guajava L.) to the growth of Pseudomonas
aeruginosa. The extraction of guava leaves, experiment, and testing of the sample
for their antimicrobial properties are scheduled to take place at Far Eastern
University Manila Campus Laboratory during the second semester of the School
Year 2020 to 2021 under the supervision and guidance of their research adviser.
This experimental research study was limited to only one type of
microorganism Pseudomonas aeruginosa, which will be the subject to determine
the effectiveness of guava leaf extracts as an inhibitor to the growth of the said
bacterium. In addition, the researchers will only perform disk diffusion technique
to assess the antimicrobial property of the plant. Hence, minimum bactericidal
and minimum inhibitory concentrations will not be utilized in the study.

1.7. Definition of Terms
This section contains unfamiliar terms/ words that are defined to further
grasp the meaning of terminologies used in the study.
Antimicrobial.The ability of the material (natural or synthetic) to inhibit the
growth of certain bacteria or make them susceptible.
Bacteremia. A condition indicating presence of bacteria in blood.
Decoction. A method of plant extraction and herbal preparation through boiling
and heating to release the essence of the said plant.
Dysentery. A type of gastrointestinal infection by which intestinal lining is being
infiltrated by bacteria or parasites causing bloody diarrhea.
Lectin. A protein that acts as a protective barrier of plants as they grow;
commonly found in beans, tomatoes, potatoes,eggplant etc
Meningitis. A condition described as infection of the meninges surrounding the
brain.
Nosocomial. An infection or disease acquired or originated from the hospital.

FEU MANILA- INSTITUTE OF ARTS AND SCIENCES - 10 -
Phytochemicals. These are biochemical compounds produced by plants which
may play a role in defense against bacterial infections or other predators such as
animals.
Plant Extracts. A fluid came from plants through decoction or the use of
mechanical pressure or compression.
Saponins. A glycosylated triterpenes found in plants which provide protection
against pests and pathogens.
Septic shock. A severe condition caused by dysregulation of host's immune
response against an infectious pathogen.
Tannins. A class of polyphenolic compounds that binds and precipitate proteins
and amino acids.
Triterpenes. A thirty carbon compound that compose the surface waxes and
specialized membrane found in plants

CHAPTER 2
REVIEW OF RELATED LITERATURE
This chapter discusses and dissolves the relevant foreign and local
literature and studies; to explore connections and comparisons with varying
resources to support the foundation of this research.
2. 1 Psidium guajava L.
2.1.1 General Characteristics and Geographical Distribution
Psidium guajava L., commonly known as Guava or also called as “apple
of the tropics'' is habitually used as a treatment for several diseases in some
countries like China, India, and Indonesia. It is popular around the world due to
its affordable price, availability, and health benefits. In addition, the guava plant
arises from hot and subtropical regions like Hawaii, Florida, Egypt, and Asia. A
guava tree is a tiny tree wherein the leaves are 2 - 6 inches for length and 1 - 2
inches for the width.Whereas, Guava leaf is commonly used as a treatment for
diarrhea, gastroenteritis and other digestive complaints (Lally et al., 2015). Guava
(Psidium guajava L.) is a small tree or shrub up to 7–10m tall and belongs to the
genus Psidium of the Myrtaceae family - branches are in canopy forms and the
trunk can grow about 20 cm in diameter and leaves were 4–10 cm long and 2.5–6
cm wide (Medina & Herrero, 2016).

2.1.2 Phytochemical Components
Lately, there is lots of research that concentrates on making medications
and goods which are natural. Some plants that exhibit moderately massive
antimicrobial effect levels may be sources of mixtures that can repress foodborne
pathogens' maturity. Breaking of cell walls and membranes, intermittent
disturbance in the intracellular matrix can destroy bacteria cells when
administered with plant extracts. An example of those plants is a guava plant.
According to Biswas et al. (2013), guava is a phytotherapeutic plant that
they think possesses powerful elements that aid in handling and controlling
numerous conditions. In conventional medicine, people utilize several sections of
the plant in treating illnesses such as irritated gums, sore throat, wounds, cough,
toothache, diarrhea, vomiting, and a lot more. Also, it is vital in diabetes, obesity,
and hypertension regulation.There is a bioactive ingredient in those leaves that
can battle with pathogens, promote weight reduction, and manage glucose levels
in the blood. Guava leaves have inherent oils with many tannins, flavonoids,
resin, eugenol, malic acid, mineral salts, triterpenes, cellulose, and cineol,
chlorophyll, fats, and other essences. There are diverse approaches to extract the
guava plant such as infusion, digestion, percolation, Soxhlet extraction, aqueous-
alcoholic extraction by fermentation, maceration, decoction, supercritical fluid
extraction, and counter-current extraction, microwave-assisted extraction,

phytonic extraction, ultrasound extraction. Researchers decide that leaf extracts
from guava are effective against S. aureus; thus, identifying new possible sources
for antimicrobial products. The guava leaves have various modes of restraining
microorganisms. For example, they invade the cell membrane's lipid bilayer
through the cell membrane, causing it to be more porous, commencing to leakage
of essential cell components. The antibacterial movement of guava leaves is
useful in managing acne. Phytochemicals are non-nutritive substances created by
plants for their security and discovered to defend individuals against infection.
According to Metwally, Omar, Harraz, and Sohafy (2010), their study
reveals that antimicrobial testing displays that the extracts and components have
antibacterial and antifungal properties. In connection to this, it tells about the
folkloric application of these extracts against bacteria in cough and diarrhea
treatment. Also, it is potent in easing oral ulcers and reddened and irritated gums
and a wound. Guava leaves carry an enormous level of phenolic synthesis,
antioxidant, and physiological motions as immunostimulatory agents. Guava
leaves mouthwash has promising outcomes. It authorizes its acceptance as a
supplement to expert oral treatment as it possesses antimicrobial properties.
2.2 Selection of Bacteria for Testing
2.2.1 Representative Bacteria (Pseudomonas aeruginosa)

Pseudomonas aeruginosa is a ubiquitously distributed opportunistic
Gram-negative pathogen that inhabits soil and water, colonizing plant, animal,
and human hosts (Neves et al., 2014). Pseudomonas is a type of bacteria (germ)
that is found commonly in the environment, like in soil and in water. Of the many
different types of Pseudomonas, the one that most often causes infections in
humans is called Pseudomonas aeruginosa, which can cause infections in the
blood, lungs (pneumonia), or other parts of the body after surgery. (Centers for
Disease Control and Prevention, 2019).
Pseudomonas aeruginosa is a Gram-negative, rod-shaped, asporogenous,
and monoflagellated bacteria. It has a pearlescent appearance and grape-like or
tortilla-like odour. P. aeruginosa grows well at 25C to 37C, and its ability to grow
at 42C helps distinguish it from many other Pseudomonas species. P. aeruginosa
is a ubiquitous microorganism which has the ability to survive under a variety of
environmental conditions (Tang et al., 2015). All bacteria that inhabit the human
body fall into the heterotrophic group. However, nutritional needs differ greatly
within this group. Bacteria such as E. coli and Pseudomonas aeruginosa can use a
wide variety of organic compounds as carbon sources and grow on most simple
laboratory media (Mahon & Lehman, 2019).
Pseudomonas aeruginosa has become an important cause of gram-
negative infection, especially in patients with compromised host defense

mechanisms. It is the most common pathogen isolated from patients who have
been hospitalized longer than one week, and it is a frequent cause of nosocomial
infections. Pseudomonal infections are complicated and can be life-threatening
(Qureshi, 2020). P. aeruginosa exploits weaknesses in host defense to mount an
infection. Indeed, P. aeruginosa is the epitome of an opportunistic pathogen of
humans. The bacterium hardly infects uncompromised tissues, but it can invade
any tissue beleaguered by immunodeficiency. P. aeruginosa causes infection in
the urinary tract, respiratory system, dermis, soft tissue, bacteraemia, bone and
joint, gastrointestinal, and blood, particularly in patients with severe burns,
tuberculosis, cancer and AIDS (Tang et al., 2015)
Pseudomonas aeruginosa infections are generally treated with antibiotics.
Unfortunately, in people exposed to healthcare settings like hospitals or nursing
homes, Pseudomonas aeruginosa infections are becoming more difficult to treat
because of increasing antibiotic resistance. (Centers for Disease Control and
Prevention, 2019)
2.3 Selection of Method for Assaying Antibacterial Activity
2.3.1 Methods of Extraction
The separation/ extraction and purification of plant components and
constituents is usually carried out using one or other or a combination of different

techniques, mainly Paper chromatography (PC), Thin Layer chromatography
(TLC), gas liquid chromatography (GLC) and high performance chromatography
(HPLC), (J.B. Harborne, “Phytochemical Methods” 1984). Furthermore, there are
different types of solvents that are used in the extraction of different plants to
obtain its antibacterial activity, although for this study, ethanol and methanol will
be used since it is best used to show inhibitory activity of the guava leaves
against bacteria (Biswas et al., 2013). The guava leaves come into two different
forms of end product extraction, it can either be in powder or liquid form. For this
study, the group would use a liquid form of guava extract and to obtain this, it
would go under a specific method which will be further explained in the
procedure part of this research study.
2.3.2 Disk Diffusion (Kirby-Bauer Disk Diffusion Susceptibility
Testing)
Kirby-Bauer Disk Diffusion Susceptibility Testing is a standard
antimicrobial susceptibility method to detect whether a certain bacteria is
sensitive or resistant to the agent applied, this could be a synthetic antibiotic or
natural products such as plants (Hudzicki, 2009). The principle of testing implies
streaking the bacteria in the agar plate and the antimicrobial agent is placed
sequentially. After allowing the growth of bacteria, a clear zone of inhibition
surrounding the antimicrobial agent is seen indicating that bacteria is susceptible

to the agent applied. Results of said testing can be interpreted by either sensitive,
resistant, or intermediate depending on the diameter of the zone observed (Reller
et. al, 2009).
In a research conducted by Pathmanathan et. al (2008), the antibiotic
susceptibility testing through disk diffusion method was implied to determine the
characteristic of their isolate, Pseudomonas aeruginosa, against a series of
antibiotics such as gentamicin, ciprofloxacin, and amikacin. Results showed 99%
susceptibility to the said bacteria as opposed to enumerated medicines. Relatively,
the study suggests that the use of disk diffusion method is ideal and can be
adapted by the researchers to explore in vitro the antimicrobial effect against the
similar isolate P. aeruginosa which is the focal point of the study.
For selection of agar or media, several researchers employ the use of
nutrient media for antimicrobial susceptibility testing. The ingredient includes
gelatin peptone, sodium chloride, or magnesium chloride. Cetrimide- based agar
can also be used (Aryal,2018). In addition, other researchers also employed ready-
to-use media such as LB medium, MOPS (3-(N-Morpholino) Propane-Sulfonic
Acid), M9, PIA/ Difco™, and King A for antimicrobial testing of P. aeruginosa
(LaBauve and Wargo, 2012). Moreover, according to research conducted by
Javiya et. al (2008) on antibiotic susceptibility patterns of P. aeruginosa at a
tertiary care hospital in Gujarat, India. The media utilized was the Muller-Hinton
medium. Perhaps, the researchers will need to evaluate this different media used
by related literature and studies to pick the best method and media to be used for
antimicrobial susceptibility testing of P. aeruginosa (See Chapter 3).
Aside from antibiotic testing, several researchers also utilized the disk
diffusion method with the same principle applied pertaining to the use of natural
products such as plants or herbs. Dhiman et. al (2011) explore the antimicrobial in
vitro activity of methanolic leaf extract obtained from Psidium guajava L.
Bacterial isolates such as E. coli, S. aureus, B. subtilis have been subjected to
testing. In addition, a double strength nutrient agar is the media used for
antibacterial susceptibility testing. Preparation of said media includes 1 g of
peptone, 0.3 g of yeast, and 0.5 g of sodium chloride all diluted in water to make
50 ml. Sterilization of media is performed in an autoclave for 15 mins at a
pressure of 15 psi. Furthermore, in three separate 90 mm petri plates, 0.2 uL of
bacterial isolates were poured into 30 mL nutrient agar medium. Then, sterilized
filter paper immersed in methanolic leaf extract (with different concentrations of
methanol) are placed in the plates with varying organisms. Such isolates were
incubated at 37 C for 24 hours. The antimicrobial activity is also compared with
antibiotic Norfloxacin. Results of their study are shown in Table 2.3.2

Table 2.3.2: Zone of inhibition (mm) of different bacterial agents at varying concentrations of
methanol extracts obtained from P. guajava and standard antibiotic norfloxacin. (Adapted from Dhiman et.
al 2011)
In general, the results indicated in the table above shows that as methanol
concentration of leaf extracts increases, the zone of inhibition of bacterial isolates
is also increased. Thus, suggesting that leaf extracts obtained from P. guajava is
efficient in inhibiting the three indicated isolates. Several recommendations were
also proposed in the study which includes use of different extractions such as
ethanol, fractionation, and isolation of flavonoids compounds. In addition, other
bacterial isolates must also be considered which can also be utilized to explore
more the antimicrobial capacity of P. guajava.
Similarly, the research of Anas et. al (2008) also employed the use of
Psidium guajava L. against the clinical isolates of multidrug resistant (MDR) S.
aureus. Methods also implied the use of methanol leaf extraction. Methanolic and
aqueous extracts minimum bactericidal concentration (MBC) were set to 1.25 and
12.5 mg/ml respectively. Results revealed that the said methanolic extract killed
the multidrug resistant bacteria with a time-kill assay of 10 hours. In addition, no
hemolysis was found even in concentration higher than MBC as tested in human
RBC hemolytic assay. Thus, the research suggests that leaf extracts obtained from
Psidium guajava L. are (1) can kill a multidrug resistant S. aureus and (2) further
tested for therapeutic and medicinal ability where absence hemolytic side effects
are reported.
Other related literature includes the study of Rogers et. al (2013), where
they tested the efficacy of guava leaf extracts by utilizing agar well-diffusion
method and placing 50 uL of extracts in each plate. Results of antibacterial assay
showed that methanol and ethanol extracts from guava leaves inhibit the activity
of gram-positive bacteria, while gram negative bacteria showed resistivity.
Methanolic extracts displays activity with mean zone of inhibition of 8.27 and
12.3 mm; while ethanolic extracts displays activity with mean zone of inhibition
of 6.11 and 11.0 mm against gram-positive bacteria, specifically B. cereus and S.
aureus. The antimicrobial and pharmacological properties must be further
evaluated as suggested by their research.
Overall, the use of disk diffusion method based on relative literature and
studies presented in the preceding text suggest that (1) it is an ideal method to
determine the resistivity or susceptibility of bacteria to certain antimicrobial
agent; (2) the use of such method can also be applied to the researcher’s
representative bacteria P. aeruginosa ; (3) different media can applied for
antibacterial susceptibility testing suitable for the study; (4) disk diffusion is not
only limited to antibiotic testing but also for natural products such as guava leaf
extracts; lastly (5) there is a proven inhibitory role of Psidium guajava L. with
varying concentration and method of extraction against certain bacterial isolates
by which the researchers able to build bottom line for the method of the study.
2.4 Synthesis
The purpose of this review is to reconcile different studies and literature
with a minute relevance that provides deep and thorough information to
understand the background of the guava leaf, phytochemical analysis, the main
bacterial isolate, the methods implied for extraction, the principle of testing,
relevant results and conclusions adapted. The emergence of almost drug-resistant
P. aeruginosa is a challenge for health care workers and to the community in
which proper sanitation and disinfection must be considered to prevent serious
infection brought by these bacteria. Surprisingly, researchers of microbial interest
continue to maximize the use of natural products as an alternative aside from
synthetic ones. Perhaps, several researchers have moven into account for
discovering phytochemical mechanisms of plants in inhibiting bacterial
growth.Whereas, the focal point of the study is to determine whether Psidium
guajava L. upon extraction of varying chemical composition can inhibit the

growth of the main bacterial isolate. Such as methods for extraction, the media to
be used for antimicrobial susceptibility testing, and varying principles
incorporated must be consulted into different academic sources in order to
analyze and carefully examine the set examples before proceeding to testing. In
conclusion,the antimicrobial effectivity of guava leaf extracts depicted by the
results of different studies is a supportive basis in which this scientific inquiry
must be answered with thorough investigations.
2.5 Conceptual Framework
The figure below shows the research paradigm for the study “The
Antimicrobial Effect of Guava Leaves Extract against Pseudomonas aeruginosa.
Following the Input-Process-Output (IPO) method,the guava leaves are collected
as the main input for the study which aims to test its antimicrobial property.
Process and method used to attain answers from the problems stated in Chapter 1
includes phytochemical analysis of the said plant and the direct antimicrobial
susceptibility testing against the bacterial isolate. Plants extracts are also
subjected to varying chemical mixtures to determine which extracts are the most
satisfactory inhibitor. The output of this study is to discuss the phytochemical
properties of guava extracts and to evaluate the susceptibility of the plant to
Pseudomonas aeruginosa by measuring the zone of inhibition. With the basis of

statistical decisions, the rejection or acceptance of the hypothesis are settled and
the main problem has been concluded in this study.
Psidium guajava L.
Extraction and analysis phytochemicals of Guava

extract. (Ethanol and Methanol)
Antimicrobial susceptibility testing of Guava extract to

P. aeruginosa (Kirby-Bauer Diffusion Susceptibility
Testing)
P. aeruginosa is P. aeruginosa is not

susceptible to Guava susceptible to Guava
extract extract
Figure 2.5.1: Conceptual Framework Diagram

CHAPTER 3
Research Methodology
This chapter contains the procedures and techniques that the researchers
used for the completion of the study. In addition, this chapter shows how the
statement of the problem will be investigated by corresponding research design,
selection of testing specimens, laboratory methods implied, preparation and the
detailed procedure executed in order to attain results, research locale, statistical
treatment, and safety considerations.
3.1 RESEARCH DESIGN
This is a quantitative study utilizing true experimental research design that
specifically aims to test the scientific hypothesis whether guava leaves extract
(Psidium guajava L.) has or no inhibitory effect on the growth of Pseudomonas
aeruginosa. The approach of the study aims to conduct laboratory experiments
through methods discussed later in this chapter. In this study, the bacteria
Pseudomonas aeruginosa is the controlled group while the guava leaf extracts
with varying chemical compositions are the experimental group or the variables
being manipulated. Primarily, the purpose of this research is to determine the
antimicrobial effectivity of these extracts and to assess which extract is the most
suitable in inhibiting the main bacterial isolate.

3.2 SELECTION OF PLANT AND BACTERIAL STRAIN
3.2.1 Selection of Plant
In order to attain necessary data needed for the completion of the research,
selected plant materials, specifically Psidium guajava L. leaves, were collected in
Bocaue, Bulacan and placed in a zip lock bag with a label to be delivered at the
laboratory. The authentication of collected plants was done at the National
Museum of the Philippines by the Botany Division, Room 405, located at Padre
Burgos in the City of Manila.
3.2.2 Selection of Bacterial Strains
The microorganism used in the study, Pseudomonas aeruginosa, was
obtained from Fil-Anaserve, Incorporated at 20-B Maaralin Street Central District
Diliman, Quezon City.
3.3 METHODOLOGIES
I. Maceration
The researchers utilized the maceration technique for
herbal guava leaf extraction method. This involves the immersion
of the powdered plant in a solvent with varying chemical

compositions and concentration and allowing it to sit or contact at
room temperature (Maceration, n.d.)
II. Laboratory Examinations
Phytochemical Screening - The researchers will use
phytochemical screening to determine the presence or absence of
phytochemical components of the guava leaf extract. There are five
phytochemical analysis implied in the study which includes test
for saponins, test for phenols and tannins, test for terpenoids
(Salkowski’s Test), test for flavonoids (Shinoda Test), and test for
glycoside.
Antibiotic Susceptibility Testing - To identify the antibiotic
susceptibility of Pseudomonas aeruginosa, the study performed
disk diffusion using the testing conditions specified by EUCAST
(2020). The zones of inhibition observed are measured and
encoded for analysis to arrive with a conclusion.
3.4 RESEARCH PROCEDURE
3.4.1 Preparation of Psidium guajava L. extract
This section discusses the preparation of leaf extracts before proceeding to
experiment proper.
A. Equipment, Disposable, Reagents / Consumable
Psidium guajava L, Distilled water, methanol 80% v/v, ethanol 80% v/v,
beaker, stirring rod, funnel, Erlenmeyer flask, test tube, watch glass, blender,
refrigerator, centrifuge, analytical balance, Whatman No. 1 Filter paper,
aluminum foil, mortar and pestle.
B. Procedure
After collection from the site, the leaf samples are delivered at the
laboratory, wash with tap water and dried at room temperature. Guava leaves are
powdered using a blender to homogenize. In an Erlenmeyer flask, fluid extracts
are separated from the solid part through filtration using Whatman No. 1 Filter
paper and funnel.. The filtrides or solid powder extract was put on a watch glass
and was weighed on an analytical balance to acquire the desired weight of 12.5
mg respectively. In a three separate 100 mL beaker, the weighed extract was
mixed with corresponding solvent. Extract 1: 12.5 mg with 1 mL of Distilled
water; Extract 2: 12.5 mg + 1 mL of 80% v/v Methanol, and Extract 3: 12.5 mg +
1 mL of 80% v/v Ethanol to obtain the extract concentrations of 12.5 mg/mL.
The three beakers were wrapped in an aluminum foil to avoid evaporation and
exposure to light, and stored at room temperature for 3 days. After 3 days,
mixtures were transferred to 10mL test tubes and then it was centrifuged for 10
minutes at 4,000 rpm at 25 degrees celsius. After centrifugation,the supernatant
was collected and stored at 4 degrees celsius until further use and analysis.
3.4.2 Phytochemical Screening of Plant Extracts
This section discussed the testing methods implied for phytochemical
analysis of guava plant extracts.
A. Equipments, Disposable, Reagents/ Consumable
Test tube, Test tube rack, dropper, prepared guava extracts,
chloroform, magnesium ribbon fragments, hydrochloric acid,
glacial acetic acid, 2% solution of iron (III) chloride, concentrated
sulfuric acid
B. Procedure
B. 1. Test for Saponins
The extract was placed in a test tube and shaken vigorously. The
presence of saponins is indicated with a formation of stable foam.
B. 2. Test for Phenols and Tannins

The extract was mixed with a 2 mL of 2% solution of iron (III)
chloride. For the indication of presence of phenols and tannins, a
blue green or black coloration is present.
B. 3. Test for Terpenoids (Salkowski’s Test)
The extract was mixed with a 2 mL chloroform. Another 2 mL of
concentrated sulfuric acid was added carefully and shaken gently.
A reddish brown coloration of the interphase was shown for
positive results of the presence of terpenoids
B. 4. Test for Flavonoids (Shinoda Test)
The extract was mixed with magnesium ribbon fragments, and a
concentrated hydrochloric acid was added. The presence of
flavonoids will produce orange, red, pink, or purple coloration.
B. 5. Test for Glycoside
The extract was mixed with a 2 mL of glacial acetic acid
containing 2 drops of 2% of iron(III) chloride. The mixture is
poured to another tube that contains 2 mL of concentrated sulfuric
acid. The presence of glycoside will produce a brown ring at the
interphase.
3.4.3 Antimicrobial Susceptibility Testing
A.) Equipment, Disposables, Reagents/Consumable
Commercial blank disks, Psidium guajava L. Distilled water, Methanol
80% & Ethanol 80% Leaves Extract (12.5mg/mL), Bacterial Culture
(Pseudomonas aeruginosa), Mueller Hinton Medium (4 mm depth, 100mm
diameter circular plate), Incubator, Test Tube Rack, Test tube, Watch glass,
Normal Saline Solution, Inoculating Loop, Alcohol Lamp, Vortex, Sterile Swab,
Tweezer, Vernier caliper, 1000 µlMicropipette, Biobase® Biosafety cabinet, 0.5
McFarland Standard and Gentamicin Antibiotic (control).
B.) Preparation of Extracts Concentration
The Psidium guajava L. extract was put on a watch glass and was weighed
on an Analytical Balance. After acquiring the desired weight of 12.5mg, each
extract was diluted with 1 ml of Distilled water, 80% Methanol, and 80% Ethanol
to obtain the extract concentration of 12.5mg/mL.
C.) Procedure
The bacterium used (Pseudomonas aeruginosa) was based on the 0.5
McFarland Standard. The tests were run in triplicate. One Mueller Hinton Plate
was used per solution of guava leaves extract for a total of three plates. Each
Mueller Hinton Medium was swabbed by streaking in a forward and backward

motion, end to end.The medium was rotated at approximately 90° for the repeated
swabbing process. The plates with seeded bacteria were tested for susceptibility
by dispensing disks on the plates and incorporating a 20µL of each prepared
concentration of distilled water, methanol, and ethanolic extract on the dispensed
disks. The disks were applied firmly to the surface of the inoculated agar plate
within 15 minutes of inoculation. A Gentamicin antibiotic disk was also dispensed
in the plate for control. The agar plates were inverted and made sure disks do not
fall off the agar surface.
The Mueller Hinton plate containing the tested bacterium and disks of
different extract concentrations was incubated at 35±1°C in air for 18 ± 2 hours
(EUCAST, 2020).
The zone of inhibition was read using a Vernier caliper and the diameters
of the zones of inhibition were measured in millimeters (mm).
3.5 RESEARCH LOCALE
This experimental study was conducted in the Science Building laboratory
on Far Eastern University-Manila Campus, Nicanor Reyes St., Sampaloc, Manila.
A leading university that promotes principled and competent graduates. Since the
researchers are also students in this institution, it is more convenient and practical
to conduct preparation, extraction, and antimicrobial susceptibility testing within

the said locale to save time, money, and efforts for finding another vicinity to
perform such methods. The laboratory experiments have been implemented by
3rd year Bachelor of Science in Medical Technology students at 2nd Semester of
S.Y. 2020-2021 with guidance and supervision of their research adviser.
3.6 STATISTICAL TREATMENT OF THE STUDY
The researchers will use the following statistical methods to process the data
gathered and to answer the statement of the problem that the researchers presented
in Chapter 1.
3.6.1. Mean/ Average
The researchers will utilize mean as a measurement of central tendency.
The zone of inhibition, expressed in units of millimeters (mm), observed from the
disk diffusion technique is measured and averaged with total samples of 9 (n=9).
The mean data will be further deviated and used on One-way ANOVA. Data were
encoded, performed, and analyzed using a SPSS software tool.
3.6.2 Standard Deviation
The researchers will utilize standard deviation to measure precision or
variation of data gathered by researchers. This statistic showed how close the
zone of inhibition, expressed in units of millimeters (mm), varies from one
another. The standard deviation or variance data will be further deviated and used
on One-way ANOVA. Data were encoded, performed, and analyzed using a SPSS
software tool.
3.6.3 One-Way ANOVA (Analysis of Variance)
The researchers used One-Way ANOVA in order to know which of the
following three extracts used in disk diffusion method was the most satisfactory in
inhibiting the growth of Pseudomonas aeruginosa in accordance with their
respective mean zone of inhibition. Each extract is compared with one another:
Extract 1 vs Extract 2, Extract 1 vs Extract 3, and Extract 2 vs Extract 3. The
researchers assume that this is a parametric test, and the values are normally
distributed.
The researchers hypothesize the following: The null hypothesis states that
there is no significant difference of the mean zone of inhibition between the three
extracts in inhibiting the growth of Pseudomonas aeruginosa; the alternative
hypothesis states that there is a significant difference of the mean zone of
inhibition between the three extracts in inhibiting the growth of Pseudomonas
aeruginosa. This equates to one extract being more effective than the other two.
The researchers set the confidence interval at 95 % which corresponds to a level
of significance of 0.05 (α= 0.05). If p-value or significance is ≤ α 0.05, then the
researchers reject the null hypothesis. Reversely if p-value or significance is ≥ α

0.05, then the researchers do not reject the null hypothesis. Data were encoded,
performed, and analyzed using a SPSS software tool.
3.7 SAFETY CONSIDERATIONS
The following safety measures are settled by the researchers to ensure that
laboratory procedures done complied with standard protocols that ensured health
safety and prevention of common laboratory hazards.
Since the researchers used an opportunistic pathogen- Pseudomonas
aeruginosa for antimicrobial testing, it is necessary for the researchers to employ
safety precautions and proper disinfection techniques. Laboratory performance is
employed using personal protective equipment which consists of gloves,
laboratory gown, face mask, hair net, and eye protection. Proper donning and
doffing techniques are also executed. All microbial testing are done on biosafety
cabinet level 1. For disinfection of tabletops, a 1:10 dilution of sodium
hypochlorite was used as recommended by CDC (2008). Autoclave technique at
121 C, 15 psi, for 15 mins was used for sterilization of culture media and other
apparatus in contact with bacterial isolate, except glassware which utilized dry
heat at 160-180 C for 2 hours. All consumables that are in contact with bacterial
isolate are disposed of in a yellow infectious bin with a biohazard symbol.

Moreover, since researchers utilized hazardous chemicals for
phytochemical analysis, chemical safety are also taken into consideration. A
complete material safety data sheet is provided by the researchers before
execution of laboratory procedures involving chemicals. Appendix A shows the
MSDS of chemicals used in the study. Personal protective equipment is also
utilized. An eyewash and shower station is situated near the laboratory premises.
References:
Anas, K. et. al. (2008). In vitro antibacterial activity of Psidium guajava Linn. leaf
extract on clinical isolates of multidrug resistant Staphylococcus aureus.
Retrieved from https://pubmed.ncbi.nlm.nih.gov/18697570/
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gment%2C%20diffusing%20into%20the%20medium.
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International Journal of Microbiology 2013 (2013): 746165-7. Web.
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0657aa9185ad/Kirby-Bauer-Disk-Diffusion-Susceptibility-Test-Protocol-
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APPENDICES
A. Material Safety Data Sheet
Reagents Formula Health Flammability Reactivity Comments

Hazard
Distilled H2O 0 0 0 N/A

water
Methanol CH3OH 2 3 0 Poisonous
Ethanol C2H5OH 2 3 0 Eye

irritant
Chloroform CHCl3 2 0 0 Toxic
Magnesium Mg 0 1 1 Use No
ribbon water
Hydrochloric HCl 3 0 1 Acid

Acid
Glacial CH3COOH 3 2 0 Corrosive

acetic acid
Iron (III) FeCl3 2 0 0 Corrosive

chloride
Sulfuric acid H2SO4 3 0 2 Use No

Water
Hazard Rating: 0- Least; 1- Slight; 2-Moderate; 3-High; 4- Extreme
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Research Proposal · November 2020

Jeffrey Leynard Ortega

The user has requested enhancement of the downloaded file.

The Antimicrobial Effect of Guava Leaf Extracts

(Psidium guajava L.) against

Department of Medical Technology

Far Eastern University Manila

Of the Requirements of the Degree

Bachelor of Science in Medical Technology

Far Eastern University Manila

RESEARCH PROPOSAL ADVISER

The Antimicrobial Effect of Guava Leaf Extracts

Dear Francisco R. Gellecanao Jr., RMT, MPH

Lenzie P. Santillan, RMT

Far Eastern University Manila

ADVISER ACCEPTANCE FORM

The Antimicrobial Effect of Guava Leaf Extracts

Working Title of (Psidium guajava L.) against

I accept this research/project to be part of my advisee/s this Academic School

LENZIE P. SANTILLAN, RMT

Francisco R. Gellecanao, Jr., RMT, MPH

Semester: Second Section: 17

Group Members: Bosque, Darren Ortega, Jeffrey

Adviser’s Consultation form

September Methodology October 31,

October Chapter 1 and 2 October 31,

November Chapter 3 checking. November 24,

This section is limited to 250-300 words, single-spaced and must include

LIST OF TABLES AND FIGURES vi

1.1. Background of the study 2

1.2. Statement of the Problem 4

1.4. Objectives of the Study 5

1.5. Significance of the Study 5

1.6. Scope and Limitations 6

1.7. Definition of Terms 7

LIST OF TABLES AND FIGURES

Table 2.3.2: Zone of inhibition (mm) of different bacterial

Figure 3.1: Sample Collection and Processing 25

limitation, and definition of terminologies.

1.1. Background of the study

A Guava leaf has a great potential as a functional ingredient for

development. They contain high levels of antioxidant, biological activities, and

phenolic compounds that serve as immunostimulatory agents.

Botanical Medicine, herbal medicine, medical herbalism, herbology,

phototrophy, or Herbalism, is a common practice in the folk period that is based

al (2017) , medical plants are the richest bioresource of drugs of traditional

medicine, nutraceuticals, food supplements, folk medicines, pharmaceutical

allowing it to cool, similarly on how the herbal tea was made.

Western medicine. The plant extractions are continued to be used in rural

As stated by Naseer, Hussain, and Naeem (2018), a guava is rich in

tannins, phenols, triterpenes, flavonoids, saponins, carotenoids, vitamins, essential

fruits containing about 80 mg of vitamin C in every 100 grams. In addition, plants

plants exhibit antimicrobial activity due to their phytochemicals. Phytochemicals

competitors or predators. The guava (Psidium guajava) is a plant used in

Henceforward, the researchers would like to assess the effectiveness of

for Disease Control and Prevention (2019), P. aeruginosa is ubiquitously found

patients including bacteremia, pneumonia and other respiratory tract infections,

bacteria makes it interesting for the researchers to explore possible antimicrobial

solutions such as guava leaf extracts in an affordable way.

Further, the researchers would like to extend the knowledge of these

herbs, in particular to guava leaves, to determine its antimicrobial effect on