Holy Rosary College of Santa Rosa, Inc

Holy Rosary College of Santa Rosa, Inc.
CITY OF SANTA ROSA, LAGUNA
Senior High School Department
ANTIBACTERIAL ACTIVITY OF AVOCADO (Persea americana)
LEAF EXTRACT AGAINST Escherichia coli
A Research Study
Presented to the Faculty of
Holy Rosary College City of Santa Rosa Laguna
In Partial Fulfillment of the Requirements for Research
By:
Billen, John Clarence L.
Ibanez, Divina Marie B.
Javier, John Lloyd T.
Moral, Jada Eloise V.
Pelayo, Maria Allaine B.
Velasco, Paul Jordan C.
To:
Sir Digno C. Salamat
July 2019
Chapter 1
The Problem and Its Setting
Introduction
Diarrheal disease account for 1 in 9 child deaths globally; with children living in
remote communities being affected most often, making diarrhea the second leading cause
of mortality among children under the age of 5 (WHO, 2013). Enteric bacterial
pathogens, specifically enterohemmorhagic E. coli O157:H7 is the leading cause of
infectious diarrhea in approximately 1.7 billion cases among children per year (Slutsker
et. al, 1997). Infectious diarrhea alone in the country is a major public health concern
whereas, 10% of acute diarrhea cases in Philippines become persistent (Ocampo et.al,
1988). An estimated 600 million individual around the world fall ill due to the
consumption of contaminated food, wherein 420 000 die yearly, resulting in the loss of
33 million life years. Out of which 40% of the foodborne disease carriers are children
ages 5 and below, with 125 000 recorded deaths every year (WHO, 2015). According to
the Department of Health (DOH) over 32,000 cases of food and waterborne diseases in
the Philippines have been recorded as of September (2017).
Herbal medicine has become globally significant with medicinal and economic
implications, over the past few decades. In accord to the WHO, 80% of the society from
around the world utilizes traditional medicine coming from plants as personal healthcare
effort due to the increasing cases of antibiotic resistance (Ekor, 2014). Thus, producing
compounds: mainly antioxidant and analgesic properties that are effective against
antibiotic-resistant strain of bacteria (Cardoso, et.al, 2016). Moreover, with the aid of
phytochemical screening the results showed that positive avocado leaf extract contains
phytochemical substances such as flavonoids, saponins, tannins and steroids (Rahman et.
al, 2018).
The purpose of this study was to investigate on the antimicrobial activity of
extract of Persea Americana against the Gram-negative bacterium Escherichia coli.
Through further investigation on the zone of inhibition, researchers may identify the
opted measurement of concentration effective in reducing the growth of the said
bacterium. This may lead to the discovery of an alternative form of medication other than
antibiotics being used at present and the discovery of antimicrobial drugs by supporting
such studies, with respect to the usage of herbal medicines in the treatment of diarrheal
diseases (Nyila, 2014).
Theoretical/Conceptual Framework
The following theories/concepts are considered to serve as guide for this research.
In accord to The Evolutionary Ecology of Escherichia coli, Eguiarte (2002)
stated the different variations of E. coli. For instance, the harmless strain of E. coli called
2
K-12, widely used in genetic-engineering experiments, has been well studied and
sequenced by Frederick R. Blattner and his colleagues at the E. coli Genome Project at
the University of Wisconsin-Madison. Moreover, other strains of E. coli may differ in
terms of genomic structure. For instance, a dangerous enterohemorrhagic E. coli (EHEC)
known as O157:H7 appears to have acquired many of its traits through horizontal transfer
since it diverged from K-12 about 4 million years ago. Subsequently, horizontal or lateral
gene transfer takes place through bacterial conjugation, when two bacteria shares DNA;
through bacteriophages’ intervention, wherein they take up “loose” DNA from the given
environment.
Based on the Traditional Chinese Medicine, the usage of Chinese herbal
medicine (CHM) utilized over hundred of years ago, has been proven to be effective in
treating irritable bowel syndrome with diarrhea (IBS-D). In accordance to the tenets of
(TCM), cooling food work by clearing heat and toxins from the body, therefore
nourishing the yin, which is associated with earth, dark, and cold. Furthermore, certain
foods such as avocado may help, for it is high in monounsaturated fatty acids responsible
in nourishing the blood. Also, about (80%) of its caloric content is easily digested.
Rao (2013) stated in Wound healing activity of Persea Americana (avocado)
fruit: a preclinical study on rats that the extraction of the said fruit is rich in nutrient
waxes, proteins and minerals, as well as vitamins A, D, and E. Therefore, an excellent
3
source of enrichment for dry or damaged skin. Furthermore, the rate of wound
contraction supports the usage of Persea americana in terms of wound healing.
Operational Framework
The first frame contains the Avocado (Persea Americana) Leaf Extract and the
different concentrations that are considered to be the independent variable that will be
used in the study.
The second division contains the Escherichia coli is considered to be the
dependent variables that will be used in the study.
The figure below shows the paradigm of the study.

Antibacterial Activity of Avocado (Persea Americana) Leaf Extract against
Escherichia Coli
Independent Variable Dependent Variable
Avocado (Persea Americana) Leaf Escherichia coli

Extract
Different concentrations
Figure 1: Paradigm of the study
4
Statement of the Problem
The study strives to figure out the antibacterial activity of Avocado (Persea
Americana) leaves extract against Escherichia coli. Particularly, the study aspires to
answer the following questions:
1. How effective is avocado (Persea Americana) leaves extract in reducing the
growth of Escherichia coli in:
a. Number of E. coli
b. Zone of Inhibition
2. What is the effective concentration of avocado leaves extract between 20
milligrams, 50 milligrams, and 70 milligrams?
3. Is there a significant effect on the Escherichia coli when treated with 20
milligrams, 50 milligrams, and 70 milligrams of avocado leaves extract?
a. Number of E. coli
b. Zone of Inhibition
5
Statement of the Hypothesis
The following hypothesis have been formulated based on the sub-problems
needed for this research.
Ho: There is no significant effect on the Escherichia coli when treated
with 20 milligrams, 50 milligrams, and 70 milligrams of avocado leaves extract.
Ha: There is a significant effect on the Escherichia coli when treated with
20 milligrams, 50 milligrams, and 70 milligrams of avocado leaves extract.
Assumptions of the Study
The following assumptions have been formulated based on the sub-problems
needed for this research.
1. The E. coli bacteria can be destroyed by positive phytochemical substances.
2. The leaves of the avocado plant contain flavonoids, saponins, tannins and
steroids that could prevent the spread of bacteria such as Escherichia coli.
3. Products or consumables made with the components of the leaves of an avocado
plant such as Avocado tea can be an alternative in preventing the harmful effects
of the E. coli bacterium. According to the Columbia University Medical Center
avocado is one of the food remedies that may help against diarrhea.
6
Scope and Delimitation
The study intends to determine the antibacterial activity of avocado (Persea
americana) leaf extract against the Gram-negative bacterium Escherichia coli.
This study, intended for a non-expert audience will mainly concentrate on
discovering antibacterial activity of Avocado leaves extract on the bacteria Escherichia
coli. Moreover, its concern yearns to further investigate on the different zone of
inhibition of E. coli, therefore restricted to evaluating the extract on Escherichia coli.
Subsequently, the study will only make use of the Avocado’s leaves excluding the
other parts. However, the efficacy of the treatment at different amounts will not be
determined.
Due to the geographical state, the researchers will utilize a variety of Persea
Americana, known as the ‘Russell’ avocado, easily recognized by its gourd like shape.
Wherein, the fruit size ranges from 1.5 to 2.5 lbs (24 – 40 – oz). Its season varies from
July to August.
In concern with the study, limited amount of financial resources and time
framework will be utilized. The study will be conducted at Holy Rosary College, during
the first semester of S.Y. 2019 – 2020.
7
Significance of the Study
This study is expected to contribute additional information to serve the following
individuals and information.
For the community This study will help give new and valuable information about
avocado as a home remedy that would lessen the risk of diarrhea.
For the Department of Health The results of this study can be used for development of
future drugs or antibacterial substances that can be used against Escherichia coli.
For the students This study would help provide new knowledge and information about
avocado and encourages students to further research about avocado and its potential
effects to other bacterial entity.
For the future researchers This research may serve as guide or reference for academic
necessities to dissertations in the near future.
For the Department of Science and Technology Information gathered from this study
can pave the way for further research and advancements regarding the field of biology. It
can be used as a basis for finding other potentially antibacterial plants.
8
Definition of Terms
For better understanding of the study, the following terms are defined operationally:
Antibacterial Activity. Any activity out of which suppresses nor destroy the growth or
the reproduction ability of a certain bacterium.
Concentration. Amount of pure substances in comparison to another substance, which in
this case is water; expressed in percentage (%).
Diarrhea. A condition in which feces are discharged from the bowels frequently and in a
liquid form
Escherichia coli. A straight rod-shaped gram-negative bacterium used as an indicator in
public health of fecal pollution.
Extract. Liquid substance containing specific isolated phytonutrients or plant chemicals,
wherein will play a significant role in the study; it is the medium of which the variable
will be used as a treatment.
Phytochemicals. It refers to a variety of plant-derived compounds with therapeutic
activities such as anti-carcinogenic, anti-mutagenic, anti-inflammatory, and antioxidant
properties.
Phytochemical screening. It refers to the extraction, screening and identification of the
medicinally active substances found in plants.
9
Zone of inhibition. The area where there is no bacterial growth around an antimicrobial
agent. It is found with the help of disk-diffusion method. The diameter of the zone of
inhibition will determine the effectiveness of the extract.
10
Chapter 2
Review of Related Literature
This chapter presents the literature and studies reviewed which are significant to
the present research. This chapter will also present, a synthesis of the state of the art and
gaps in the review are presented.
State of the Art
The literatures and studies that were reviewed were found to have relevance on
the present study. The studies will serve as the underlying support for the
conceptualization of the study’s problems, methodology, and research design.
Antibacterial Effects of Avocado
The antibacterial activities of the methanolic leaf and bark extract of Persea
americana was tested in vitro on bacterial isolates namely: Streptococcus pyogenes,
Proteus mirabilis, Salmonella typhi, Klebsiella pneumoniae, Escherichia coli, Bacillus
subtilis (NCIB 3610), Staphylococcus aureus (NCIB 8588), Escherichia coli (NCIB 86),
Klebsiella pneumoniae (NCIB 418) and Pseudomonas aeruginosa (NCIB 950), using the
agar well diffusion method. The leaf extract was able to prevent the growth of the test
organisms at a concentration of 50.0mg/ml except Escherichia coli, Salmonella typhi,

Proteus mirabilis and Escherichia coli (NCIB 86), while the bark extract at the same
concentration inhibited the test organisms except Salmonella typhi, Escherichia coli,
Bacillussubtilis (NCIB 3610) and Escherichia coli (NCIB 86). (Ogundare & Oladejo,
2014)
Antimicrobial activity of seed extracts of Persia amerciana against Escherichia
coli, Klebsiella pneumoniae, Bacillus subtilis, Streptococcus pyogenes, Pseudomonas
aeruginosa, Staphylococcus aureus, Corynebacterium ulcerans, Salmonella typhi,
Neisseria gonorrhoea and Candida albicans was carried out using the disc diffusion
technique. The methanol, ethyl acetate and chloroform extracts showed promising
activity against the test organisms. The activity of methanol extract was more noticeable
against C. albicans (42mm) while that of petroleum ether extract was the least against E.
coli (6mm). However, S. typhi and E. coli were resistant to chloroform and methanol
extracts. The activity of the ethyl acetate, chloroform and methanol extracts compared
favorably with that of standard antibiotic streptomycin (30μgdics-1). Preliminary
phytochemical screening unveiled the presence of flavonoids, saponins, tannins, steroids
alkaloids and terpenoids. The spectra of antimicrobial activities showcased by the
extracts could be attributed to the presence of these phytochemicals and signifies the
potential of Persia amerciana as a source of therapeutic agents. This therefore, reinforces
the idea of traditional medicinal use of Persia amerciana and concludes that further
studies should be conducted to isolate and identify the active components of the extract
(Idris, Ndukwe and Gimba, 2009).
12
The seed and epicarp of Persea Americana Mill. var. Hass (Lauraceae), Persea
Americana Mill. var. Shepard, and Persea americana Mill. var Fuerte cultivars of mature
avocados were ground separately and extracted with both absolute ethanol and distilled
water. Extracts were observed for antimicrobial activity using the micro titer broth micro
dilution assay against four Gram-positive bacteria, six Gram-negative bacteria, and one
yeast. Antimicrobial activity against two molds was determined by the hole plate method.
The ethanol extracts showed antimicrobial activity (104.2–416.7 μg/mL) toward both
Gram-positive and Gram-negative bacteria (except Escherichia coli) (Dykes, 2010).
Remedies of Avocado Tea Leaves
Avocado leaves tea is very similar to traditional herbal tea, such as guava leaves
tea and are consumed in some parts of the world to treat diarrhea. This drink is part of
conventional medicines. Therefore, it provides a wide array of extraordinary healthy
properties. It is not surprising that a wide range of chemical elements that are contained
in avocado leaves tea provides a large number of health benefits. If the appropriate
medications are not available to you, you can drink avocado leaves tea to treat diarrhea.
It has the same effect on diarrhea as guava tea leaves. This herbal drink prevents diarrhea
and helps protect against bacteria and viruses that can later cause diarrhea (Okorie, 2019).
13
Why Use Kirby-Bauer Method
Among the list of antibiotic susceptibility tests only one is most commonly used
around the world. According to Sandle (2016) disc diffusion or the Kirby–Bauer test is
one of the classic microbiology techniques, and it is still very commonly used. Because
of convenience, efficiency, and cost. For most commonly isolated bacterial and fungal
pathogens, reliable antimicrobial susceptibility testing may be performed on hospitals,
community, public health, food, and veterinary laboratories. The most familiar methods
utilized are the disk diffusion susceptibility test method (also known as Kirby-Bauer) and
minimal inhibitory concentration (MIC) determination. (Who.int, 2019)
A recent study showed that there are two different types of antibiotic
susceptibility tests available; the Kirby-Bauer method and the liquid media based
microbroth dilution method. This method has become more widely used due to its ability
to become automated, however, the Kirby-Bauer method may be able to detect
subpopulations of resistant bacteria that would be visualized as inner colonies in the zone
of diffusion. Using Gram negative rod isolated collected from the UWMC clinical
microbiology laboratory, the presence of inner colonies was screened and both methods
were compared to determine if the microbroth dilution method was able to detect the
additional resistance of the subpopulations. The data went to show that the microbroth
dilution method was not detecting the additional resistance in about 68% of the isolates
14
tested. It was unable to be determined if the results were due a limitation of the
microbroth dilution method or whether it is an artifact of the testing methods (Luc, 2015).
Synthesis of the Reviewed Literature
The series of studies and literature written on this chapter discuss the concepts
and narrative detail of relevant and related information of the study. The information
gathered are vital in determining whether the different parts of the avocado can be used
as an antibacterial agent. The researchers and other writers and author like; Ogundare &
Oladejo, (2014), Idris, Ndukwe and Gimba, (2009), Dykes, (2010) have shown the
different results of different studies on different parts of the avocado whether it inhibited
or did not inhibited the organisms.
Likewise, Okorie (2019) revealed that avocado tea also helps the body in treating
diarrhea, if the appropriate medications are not available to you, you can drink avocado
tea to treat diarrhea. It has the same effect on diarrhea as guava tea leaves. This herbal
drink prevents diarrhea and helps protect against bacteria and viruses that can later cause
diarrhea.
Sandle (2016), Luc (2015) and the World Health Organization (WHO) also added
that the best method of antibiotic susceptibility test is the disk diffusion method or the
Kirby-Bauer method, for most commonly isolated bacterial and fungal pathogens,
15
reliable antimicrobial susceptibility testing can be performed by routine hospital,
community, public health, food, and veterinary laboratories. The most familiar methods
utilized are the disk diffusion susceptibility test method
Gaps Bridged by the Present Study
From the above review of the related literatures and studies, the following gaps
were determined:
1. There were little to no studies conducted on different acid-base extraction of
leaves of avocado.
2. There were little to no studies conducted on antibacterial activity of avocado
leaves extract using a different method of antibiotic susceptibility test.
3. There were little to no studies on the effectiveness of avocado leaves against
Escherichia coli
16
Chapter 3
RESEARCH METHODOLOGY
This chapter will discuss the procedure design needed to accomplish concerning
the study. Thus, containing the instrument utilized for data collection, including methods,
procedure and statistical treatment to maintain the validity and reliability of the
instrument.
Research Design
In order to satisfy the objectives of dissertation, a quantitative research was held.
It makes use of a Randomized Complete Block Design, specifically due to the
concentration of extract from the Avocado leaves that will be tested correspondingly to
inhibition zones of Escherichia coli. Therefore, determining the efficacy of Persea
americana against the Gram-negative bacterium Escherichia coli, with respect to its
different inhibition zones. In the words of Patidar (2013), he further stated that
Randomized Complete Block Design (RCBD) is used when differences between subjects
and experimental conditions are inherited. Subsequently, it concerns large experimental
groups, aided efficacy by bring homogeneity, with the means of variability reduction.
Materials
For the purposes of this research, certain materials will be used: ‘Russell’
Avocado (Persea americana), both the leaves and the fruit itself, and the Gram-negative
bacterium Escherichia coli grown on nine petri dishes.
Equipment
The inclusion of equipment for the dissertation is listed as follows: five
evaporating dishes, an electric stove, casserole, one amber bottle, four funnels, four
Erlenmeyer flasks, four filter papers, beaker, Bunsen burner, and refrigerator for
preservation. Subsequently, to decontaminate the culture media, an inoculating loop and
an autoclave will be used.
Maceration pertains to the process by which organized tissues is transformed into
a suspension of intact cells, as defined by (Katri et. al.). In order to get the crude extract
of the Avocado leaves, the researchers used ethyl alcohol, four Erlenmeyer flasks,
aluminum foils, masking tape, and the main ingredient, the Avocado leaves.
Procedure
As for the materials and equipment, the stated procedures are to be considered to
attain the required results. First, a bountiful amount of Avocado leaves was washed and
18
cut into smaller pieces. Thus, submerged in ethyl alcohol with the use of flasks. A small
foil was placed on the flasks’ mouth, serving as a lid to penetrate the quick evaporation of
alcohol despite its volatility.
Second, the solution is distributed into two beakers for further observations within
the span of seventy-two hours. The utilization of pulverized Avocado leaves from the
beaker was filtered using four Erlenmeyer flasks and four funnels. After filtration, the
filtered extraction was then transferred to the evaporating dishes, serving as a substance
known as the partial extraction of Persea americana leaves.
Third, the filtered Avocado leaves were evaporated to dryness with the aid of the
water-bath set-up to maintain cell lines. The process lasted for about 4-5 hours. Thus,
producing a homogenous, syrup-like solution due to the leaf extraction, that was the
placed in an amber bottle.
Fourth, the study also made use of Molten Basal Agar as the medium for the
Escherichia coli bacteria. to allow drying and solidification. The 20 ml of molten basal
agar medium (Mueller Hinton agar) is placed in each plate, allowing it to dry and
solidify.
Fifth, the microbial suspension (#0.5 MacFarland for bacteria and #2 MacFarland
for yeasts, fastidious bacteria and molds) were prepared and the seed layer medium/top
agar (Mueller Hinton agar) was melted and cool to 43-46°C.
19
Sixth, 0.1 of microbial suspension was seeded to the top seed layer medium/top
agar and pour or pipet 4 ml of seeded top agar (1% agar) into plate, allow solidifying and
drying. Each cylinder with 0.1 ml of sample, negative control, positive control and
solvent control was filled separately. Lastly, it was incubated at conditions and period
appropriate for the test organism and the diameter of each zone of growth inhibition to
the nearest 0.1 mm was measured.
20
Flow Chart
21
Sources of Data
The researchers will be using primary sources of data. The data will be taken from
the results and observation based on the conducted experiment in the laboratory.
Participants of the Study
The participants of the study are the researchers and with the help of trained
professional laboratory observers.
Data Blanks
Setup Trial 1 Trial 2 Trial 3
(20 mg) (50 mg) (70 mg)
Number of E. coli
Avocado leaves extract Diameter of zone of inhibition (mm)
Resistant Intermediate Susceptibility
20 milligrams
50 milligrams
70 milligrams
22
Statistical Treatment of Data
The Kirby-Bauer method, which was commonly known as the type of zone of
inhibition process is the data gathering technique that will be used in this study. In this
method, a sterile sensitivity disk will be used to test the effectiveness of the Avocado
leaves extract in inhibiting the growth of Escherichia coli bacteria. The expected results
of the method that will be used would be an area of media where the bacteria are unable
to grow due to the presence of Avocado leaves extract. The inhibition zone was measured
to determine the effectiveness the extract.
The data collected in this study will be organized and classified based on the
research design and the problems formulated. The data gathered will be tallied and
tabulated in order to facilitate the presentation and interpretation of results using the
following:
1. Analysis of Variance (ANOVA)
The Analysis of Variance gives the study different group means of each
concentration. From those results, the study determines whether the different
concentration affects the Escherichia coli bacteria differently. The formula used in this
technique will be:
Where:
F = Anova Coefficient
23
MST = Mean sum of squares due to treatment
MSE = Mean sum of squares due to error
Formula for MST is given below:
Where:
SST = Sum of squares due to treatment
p = Total number of populations
n = Total number of samples in a population.
Formula for MSE is given below:
Where:
SSE = Sum of squares due to error
S = Standard deviation of the samples
N = Total number of observations.
2.Pearson r Correlation Coefficient
This statistical tool will be used since it deals with measuring the degree of linear
relationship between two variables whether positive or negative. In this study, this tool
24
gives the answer by comparing the data gathered from the Kirby Bauer zone of inhibition
test and showing the effects of the different concentrations of the Avocado leaves extract
on the Escherichia coli bacteria.
The formula that will be used is:
Where:
N = number of pair of scores
∑xy = sum of the products of paired scores
∑x = sum of x scores
∑y = sum of y scores
∑x2 = sum of squared x scores
∑y2 = sum of squared y scores
25
In interpreting the correlation coefficient value (r) obtained, the scale below will
be used:
+1.00 perfect positive correlation

+0.99 - +0.75 very high positive correlation
+0.74 - +0.50 high positive correlation
+0.49 - +0.25 moderately small positive correlation
+0.24 - +0.01 very small positive correlation
0.00 no correlation
-0.01 - -0.24 very small negative correlation
-0.25 - -0.49 moderately small negative correlation
-0.50 - -0.74 high negative correlation
-0.75 - -0.99 very high negative correlation
-1.00 perfect negative correlation
26

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Holy Rosary College of Santa Rosa, Inc.

CITY OF SANTA ROSA, LAGUNA

Senior High School Department

ANTIBACTERIAL ACTIVITY OF AVOCADO (Persea americana)

LEAF EXTRACT AGAINST Escherichia coli

Presented to the Faculty of

Holy Rosary College City of Santa Rosa Laguna

In Partial Fulfillment of the Requirements for Research

Billen, John Clarence L.

Ibanez, Divina Marie B.

Javier, John Lloyd T.

Moral, Jada Eloise V.

Pelayo, Maria Allaine B.

Velasco, Paul Jordan C.

Sir Digno C. Salamat

Senior High School Department

The Problem and Its Setting

pathogens, specifically enterohemmorhagic E. coli O157:H7 is the leading cause of

the Philippines have been recorded as of September (2017).

Senior High School Department

The purpose of this study was to investigate on the antimicrobial activity of

extract of Persea Americana against the Gram-negative bacterium Escherichia coli.

opted measurement of concentration effective in reducing the growth of the said

diseases (Nyila, 2014).

In accord to The Evolutionary Ecology of Escherichia coli, Eguiarte (2002)

Senior High School Department

the University of Wisconsin-Madison. Moreover, other strains of E. coli may differ in

terms of genomic structure. For instance, a dangerous enterohemorrhagic E. coli (EHEC)

Based on the Traditional Chinese Medicine, the usage of Chinese herbal

Rao (2013) stated in Wound healing activity of Persea Americana (avocado)

waxes, proteins and minerals, as well as vitamins A, D, and E. Therefore, an excellent

Senior High School Department

contraction supports the usage of Persea americana in terms of wound healing.

used in the study.

The second division contains the Escherichia coli is considered to be the

dependent variables that will be used in the study.

The figure below shows the paradigm of the study.

Independent Variable Dependent Variable

Avocado (Persea Americana) Leaf Escherichia coli

Figure 1: Paradigm of the study

Senior High School Department

Statement of the Problem

answer the following questions:

1. How effective is avocado (Persea Americana) leaves extract in reducing the

growth of Escherichia coli in:

2. What is the effective concentration of avocado leaves extract between 20

milligrams, 50 milligrams, and 70 milligrams?

3. Is there a significant effect on the Escherichia coli when treated with 20

milligrams, 50 milligrams, and 70 milligrams of avocado leaves extract?

Senior High School Department

Statement of the Hypothesis

The following hypothesis have been formulated based on the sub-problems

needed for this research.

Ho: There is no significant effect on the Escherichia coli when treated

with 20 milligrams, 50 milligrams, and 70 milligrams of avocado leaves extract.

20 milligrams, 50 milligrams, and 70 milligrams of avocado leaves extract.

Assumptions of the Study

The following assumptions have been formulated based on the sub-problems