SAINT MARY’S ACADEMY OF CAPIZ

P. BURGOS ST., ROXAS CITY

Antibacterial Activity of Seaweed (Caulerpa lentilifera) and Neem Plant (Azachdiracta indica) Mixture

against Pseudomonas aeruginosa

A Quantitative Research

Presented to:

Saint Mary’s Academy of Capiz

By:

Kathleene Jade Fulgencio

Ma. Teresa Fave Molino

Janyn Anne Fanugon

Winfred Francis Rivera

March 2020

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APPROVAL SHEET

This Experimental Research entitled “Antibacterial Activity of Seaweed (Caulerpa lentilifera) and Neem plant

(Azachdiracta indica) mixture against Pseudomonas aeruginosa” at Saint Mary’s Academy of Capiz SY 2019-2020 ,

prepared and submitted by Kathleene Jade B. Fulgencio, Ma. Teresa Fave P. Molino, Janyn Anne B. Fanugon and

Winfred Francis Rivera in partial fulfilment of the requirements in Research Capstone (Grade 12- Science

Technology, Engineering and Mathematics) has been examined for approval and acceptance.

Mr. Jeres Andrin Garche, LPT

Research Adviser
_____________________________________________________________________________________________
Approved by the RESEARCH COMMITTEE ON ORAL EXAMINATION with a score of ________.

Mr. Aledel Christian Alejandro, LPT Ms.

Statistician Medical Technologist

Nida Abalayan, Ph.D Mercedita Abas, Ph.D

Vice Chairman Chairman

Accepted in Patial Fulfillment of the Requirements in Research Capstone (Grade 12- Science Technology,

Engineering and Mathematics)

Mr. Aledel Christian Alejandro, LPT S. Ma. Remy M. Villaraiz, RVM

Principal Directress

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Acknowledgement

The researchers would like to extend our sincere thanks to many individuals. This research becomes a reality

with the kind support and help of the following:

First and foremost, to our God Almighty for the wisdom he bestowed upon us and strength in order to finish

this research.

To Sr. Ma. Remy Villaraiz, RVM, School Directress, for entrusting us with this opportunity to have this kind of

school activity.

To Mr. Aledel Christian Alejandro, School Principal for the advises, corrections, comments and assistance to

improve this research.

To Mercedita D. Abas, Ph.D, Research Capstone Teacher, for the suggestions, corrections and motivating

messages to keep up with the research.

To Nida Abalayan, Ph.D, Senior High School Coordinator, for the unending support and kind assistance to do

better in this study.

To Mr. Jeres Andrin Garche, Research Adviser,

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Abstract

This study entitled “Antibacterial Activity of Seaweed (Caulerpa lentilifera) and Neem Plant

(Azachdiracta indica) Mixture against Pseudomonas aeruginosa” was conducted to evaluate “Antibacterial

Activity of Seaweed (Caulerpa lentilifera) and Neem Plant (Azachdiracta indica) Mixture against

Pseudomonas aeruginosa”. It also sought answers to the following questions: (1) What is the average

antibacterial activity of the different treatments made from seaweed and neem mixture against

Pseudomonas aeruginosa in terms of its zones of inhibition? (2) Is there a significant difference in the zones

of inhibition of Pseudomonas aeruginosa when treated with the treatments made from seaweed extract and

neem plant mixture? (3) 3. Is there a significant difference in the zones of inhibition of Pseudomonas

aeruginosa when treated with the different treatments made from seaweed and neem mixture as compared

to positive and negative control?

There were three (3) treatments and positive and negative control utilized in this study. Treatment A

contained 75% Seaweed and 25% Neem plant mixture; Treatment B contained 50% Seaweed and Neem

plant mixture; Treatment C contained 25% Seaweed and 75% Neem plant; Positive control used was

Cefalexin (500mg); Negative control used was Distilled water.

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Table of Contents
Page
Title Page i

Acknowledgement ii

Abstract

Table of Contents

List of Figures

List of Tables

List of Appendices

Chapter
1.INTRODUCTION TO THE STUDY

Background and Rationale of the Study

Statement of the Problem

Hypotheses

Conceptual Framework

Significance of the Study

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Scope and Delimitations of the Study

Definition of Terms

2.REVIEW OF RELATED LITERATURE

Seaweeds (Caulerpa lentilifera)

Neem (Azachdiracta indica)

Pseudomonas Aeruginosa

3.MATERIALS AND METHODS

Research Design

Materials

Procedures

Data Gathering Procedure

Statistical Tools

Procedural Design

4.RESULTS AND DISCUSSION

Descriptive Statistics of the Zone of Inhibition

Analysis of Variance in the Mean Rating of the Zone of Inhibition

Post Hoc Analysis between Treatments

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Discussions

5.SUMMARY, CONCLUSION AND RECOMMENDATION

Summary

Conclusions and Recommendations

References

Appendices

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List of Figures

Figure Page

1 Conceptual Framework of the Study

2 Laba-laba or Seaweed (Caulerpa lentilifera)

3 Bagalunga or Neem (Azachdiracta indica)

4 Extraction of Seaweed and Neem

5 Preparation of Five (5) Treatments

6 Impregnation of Paper disks

7 Preparation for Antibacterial Testing

8 Preparation of Culture Media

9 Pour Plating

10 Inoculating Microbe (Pseudomonas Aeruginosa)

11 Application of Treatments

12 Incubation of Petri dishes

13 Measuring Zones of Inhibition (mm)

14 Procedural Design

15 Zone of Inhibition

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List of Tables
Table Page
1 Table of Treatments

2 Interpretation Diameter of Zone of Inhibition

3 Descriptive Statistics of the Zone of Inhibition

4 Analysis of Variance in the Mean Rating of the Zone of Inhibition

5 Post Hoc Analysis between Treatment A,B,C,

Positive and Negative Control

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List of Appendices
Appendix Page

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Chapter 1

Introduction

Background and Rationale of the Study

As a human person, we are taught to take good care of ourselves so that we won’t get sick or

worse is be hospitalized. We often acquire bacteria which causes us sickness from places like hospitals or a

dirty environment. It is found in every habitat on Earth: soil, rock, oceans and even arctic snow. Some live in

or on other organisms including plants and animals including humans. There are approximately 10 times as

many bacterial cells as human cells in the human body.

Pseudomonas aeruginosa is member of the Gamma Proteobacteria class of Bacteria. It is a Gram-

negative, aerobic rod belonging to the bacterial family Pseudomonadaceae. It has a pearlescent

appearance and grape-like or tortilla-like odour. In fact, Pseudomonas aeruginosa is occasionally a

pathogen of plants. P. aeruginosa is a ubiquitous microorganism which has the ability to survive under a

variety of environmental conditions. It is a free-living bacterium, commonly found in soil and water. However,

it occurs regularly on the surfaces of plants and occasionally on the surfaces of animals.

(textbookofbacteriology.net)

It has become increasingly recognized as an emerging opportunistic pathogen of clinical relevance.

They usually do not cause infections in healthy people. If an infection does occur in a healthy person, it is

generally mild. More severe infections occur in people who are already hospitalized with another illness or

condition, or people who have a weak immune system. Infections can occur in any part of the body.

Symptoms depend on which part of the body is infected. The pathogen can often be acquired from a

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hospital or moist areas and are widely found in soil and water. The infection might occur after

exposure to hot tubs and swimming pools that are inadequately chlorinated. (healthline.com)

Pseudomonas aeruginosa is antibiotic resistant. Infections occur in blood, lungs, skin, eye, ear. The

organism only causes disease when a person’s immune system is already impaired. Conditions that may

increase the risk of infection include burn wounds, receiving chemotherapy for cancer, cystic fibrosis, HIV or

AIDS, presence of a foreign body, like a mechanical ventilator or catheter, undergoing an invasive

procedure, like a surgery. (healthline.com)

Natural ingredients are used in this study. The main ingredients to be used in the study, us, the

researchers conceptualized Caulerpa lentilifera (Seaweed) and Azachdiracta indica (Neem Plant). Neem

Plant (Azadirachta indica) is recognized as a medicinal plant well known for its antibacterial, antimalarial,

antiviral, and antifungal properties. Neem Plant is a very important traditional medicinal plant in India that

has been used extensively in Ayurveda, Unani, and homeopathic medicine and has become a focus of

modern medicine. The consensus is that the general antimicrobial activity of neem extracts is mainly due to

azadirachtin.

Neem seed extracts are bactericidal against gram-negative as well as gram-positive pathogens,

and thus have broad spectrum activity; they also have a synergistic interaction in combination with

antibiotics. Although crude extracts from various parts of neem have been used for medicinal applications

since time immemorial, modern drugs can be developed after extensive investigation of its

pharmacotherapeutics and toxicity, as well as proper standardization. (sciencedirect.com)

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Seaweeds are any large number of marine bethic algae. They are macroscopic, multicellular and

macrothallic in contrast with algae. It is often found in the seashore biome. Based on our research,

seaweeds species have been found to defend themselves from specific pathogens with naturally occurring

antibiotics, which could present opportunities for future nutraceuticals.

According to Julia Kubanek, an assistant professor of biology and chemistry at the institute,

“seaweeds live in constant contact with potentially dangerous microbes and they have apparently evolved a

chemical defense to help resist disease”. Also information on seaweed metabolites which are antimicrobial

in nature is also briefly reported. (nutraingreidients.com)

With these in mind, the researchers thought of creating a natural remedy to possibly address the

spread of Pseudomonas aeruginosa. As stated above, the plants have antibacterial properties that may also

be effective against the Pseudomonas aeruginosa that is made of natural ingredients.

Statement of the Problem

The main objective of the study is to evaluate the antibacterial effect of Caulerpa lentilifera (Seaweed)

and Azachdiracta indica (Neem Plant) mixture against Pseudomonas aeruginosa.

Specifically, this study seek to answer the following questions:

1. What is the average antibacterial activity of the different treatments made from seaweed and neem mixture

against Pseudomonas aeruginosa in terms of its zones of inhibition?

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2. Is there a significant difference in the zones of inhibition of Pseudomonas aeruginosa when treated with the

treatments made from seaweed extract and neem plant mixture?

3. Is there a significant difference in the zones of inhibition of Pseudomonas aeruginosa when treated with the

different treatments made from seaweed and neem mixture as compared to positive and negative control?

Hypotheses

1. There is no significant difference in the zones of inhibition of Pseudomonas aeruginosa when treated with the

treatments made from seaweed extract and neem plant mixture.

2. There is no significant difference in the zones of inhibition of Pseudomonas aeruginosa when treated with the

different treatments made from seaweed and neem mixture as compared to positive and negative control.

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Figure 1: Conceptual Framework of the Study

Independent Variable Dependent Variable

Different Concentrations
Of Seaweed and Neem Plant
Extract
Plant Extract Treatment A: 70% Method of
neem plant extract + 30%
seaweed extract Application:

Treatment B: 50% neem plant Disk-Diffusion

extract + 50% seaweed extract Zones of Inhibition (mm)
Method
Treatment C: 30% neem plant of P. aeruginosa
extract + 70 % seaweed extract
Positive Control:
Amoxicillin
Negative Control:
Distilled Water

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Significance of the Study

The results of this study will be of great benefit to the following:

Hospital community. The result of this study will benefit the hospital community in a way of giving

them ways of treating the pathogens that often occur in hospitals also it can prevent the sick patients to

acquire the pathogen and by any chance will enable them to have alternative medicine to diseases caused

by Pseudomonas aeruginosa.

Gardeners. The result of this study will be great help to the gardeners because they will be

protected from the pathogens which mostly came from soil and moist places and they will be treated with

natural ingredients.

Medical Doctors. The result of this study will help the medical doctors to have access to the best

level of healthcare cover in terms of treating the bacteria that causes infections to humans.

Researchers. The result of this study will provide information to the researchers about the

antibacterial property of seaweed extract and neem extract mixture.

Future Researchers. The result of this study can be used also in production of organic solution

against Pseudomonas aeruginosa. Furthermore, it can contribute to future studies since it may serve as

reference.

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Definition of Terms

Antibacterial. It is anything that destroys bacteria or suppresses their growth or their ability to reproduce.

(MedicineNet, 2018)

In this study, antibacterial refers to the content that the seaweed and neem plant contains that will be

used to kill the pathogens.

Seaweed. It is the common name for countless species of marine plants and algae that grow in the ocean

as well as in rivers, lakes, and other water bodies that contains anti-inflammatory and anti-microbial agents.

(OceanService, 2018)

In this study, seaweed refers to the plant that will be extracted and used as the main component in

making antibacterial mixture.

Neem. It is a natural herb that comes from Neem tree and has strong antioxidant, neutralizing free radicals

that may influence the development of some conditions and has antimicrobial effects which may be effective

against several types of bacteria, viruses, and fungi. (MedicalNewsToday, 2019 )

In this study, neem refers to the plant that will be extracted and used as the main component in

making antibacterial mixture.

Pseudomonas aeruginosa. It is member of the Gamma Proteobacteria class of Bacteria. It is a Gram-

negative, aerobic rod belonging to the bacterial family Pseudomonadaceae. (Textbookofbacteriology, 2012)

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In this study, Pseudomonas aeruginosa is the test pathogen used in the experimentation.

Extraction. It is a way to separate a desired substance when it is mixed with others. The mixture is brought

into contact with a solvent in which the substance of interest is soluble, but the other substances present

are insoluble. (Chemicool, 2007)

In this study, extraction refers to the process which will be used for liquefying the seaweed and neem

plant that will be used against P. aeruginosa.

Cefalexin. It is a cephalosporin (SEF a low spor in) antibiotic. It works by fighting bacteria in your body and

is used to treat infections caused by bacteria, including upper respiratory infections, ear infections, skin

infections, urinary tract infections and bone infections. (Drugs, 2018)

In this study, cefalexin is the positive control.

Distilled Water. It is a water that has been freed of dissolved or suspended solids and from organisms by

distillation (as for medical or chemical purposes) (Merriam Webster Dictionary, 2019)

In this study, distilled water is the negative control.

Disk-Diffusion. It is a method where a culturing surface inoculated with microbe is exposed to small disks

containing known amounts of a chemical agent resulting in a zone of inhibition (usually in millimeters) of

growth of the microbe corresponding to the susceptibility of the strain to the agent. (Reference.MD, 2012)

In this study, disk diffusion is the method that will be used to test the antibacterial effect of Seaweed

and Neem plant extract by impregnating the petri dishes.

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Zone of Inhibition. It is the clear region is an indication of the absence, or the effective inhibition, of microbial

growth by the antimicrobial agent. (Biology Online, 2019)

In this study, zone of inhibition refers to the clear region around the disk and at this point, no growth

will be observed.

Scope and Delimitation of the Study

This study is limited only in evaluating the antibacterial effect of Seaweed and Neem plant mixture

against Pseudomonas aeruginosa through disk-diffusion method.

Five(5) treatments will be used in the study. Treatment A: 70% neem plant extract + 30% seaweed

extract. Treatment B: 50% neem plant extract + 50% seaweed extract. Treatment C: 30% neem plant

extract + 70 % seaweed extract. For the positive control, Treatment D: Cefalexin (500 mg) and for the

negative control, Treatment E: Distilled Water.

The treatments will be extracted using only Extraction Method. The purpose of using this method is

to maintain the original composition of the plant intact to keep it organic and a chemical free antibacterial

treatment.

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Chapter 2

Review of Related Literature

Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a Gram-negative rod measuring 0.5 to 0.8 µm by 1.5 to 3.0 µm. Almost

all strains are motile by means of a single polar flagellum. The bacterium is ubiquitous in soil and water, and

on surfaces in contact with soil or water. Its metabolism is respiratory and never fermentative, but it will

grow in the absence of O2 if NO3 is available as a respiratory electron acceptor. Since the revisionist

taxonomy based on conserved macromolecules (e.g. 16S ribosomal RNA) the family includes only

members of the genus Pseudomonas which are cleaved into eight groups. Pseudomonas aeruginosa is the

type species of its group. which contains 12 other members. (Textbookofbacteriology, 2012)

Most strains of P. aeruginosa produce one or more pigments, including pyocyanin (blue-green),

pyoverdine (yellow-green and fluorescent), and pyorubin (red-brown). Previous investigations have

suggested that pyocyanin not only contributes to the persistence of P. aeruginosa in the lungs of CF

patients, but also interferes with many mammalian cell functions, including cell respiration, ciliary beating,

epidermal cell growth, calcium homeostasis and prostacyclin release from lung endothelial cells.

(Sciencedirect, 2015)

According to Flynn, R.J. 1963 the study entitled “Psuedomonas Aeruginosa Infection and

Radiobiological Research": the unresolved questions” was conducted at Argoone National Laboratory:

Effects, Diagnosis, Epizootiology, Control. Pseudomonas aeruginosa infection reduced the survival time of

lethally irradiated mice from 13.8 days to 6.0 days. Many radiobiological studies were affected, partioularly

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those concerned with protection against radiation injury. Various diagnostic procedures were studied

and evaluated. Tests based on P. aeruginosa's unique ability to produce pyocyanin were found to be the

most useful for the mass screening of mouse colonies. The moat efficient technique consisted of culturing

individual fecal or group water bottle samples in Wensinck's glycerol broth at 37 deg C for seven days and

then testing the culture for the chloroform-soluble pigment. The epizootiology of the infection was also

studied. One of 12 animal caretakers and about 26% of the cockroaches infesting the animal quarters were

found to be infected, but an extensive survey of various inanimate materials (building, equipment, feeds,

bedding) showed that when good sanitation is employed such materials are rarely infected. Control

procedures based on the removal of the probable source of infection (animate vectors) and the prevention

of the spread of existing infection through good sanitation are described. ( Flynn, R.J. 1963 )

According to Juangya, Lim and Bonifacio, 2017, the study entitled “Five-Year Antimicrobial

Susceptibility of Pseudomonas aeruginosa: the unresolved questions” was conducted at Local Tertiary

Hospital in Bacolod City, Philippines. Over five years, a total of 646 P. aeruginosa isolates was acquired

from different clinical specimens and their resistance to the commonly used anti-pseudomonal antibiotics

was determined. The majority of the isolates were from respiratory (60.99%) and urinary sources (23.22%)

while the least came from transudates and exudates (2.01%). Most of the samples were acquired from older

adults (77.55%), most of whom were admitted (67.03%). Amikacin was found to be the most effective drug

with a resistance rate of 7.5%, followed by piperacillin/tazobactam (8.5%) and gentamicin (13.5%). On the

other hand, 26.7% of the isolates were resistant to levofloxacin. Almost 100% of the isolates were screened

positive for AmpC production, which may suggest inducible resistance against expanded spectrum beta-

lactamase. Furthermore, for the last three years, P. aeruginosa isolates from this area have been noted to

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have decreasing resistance only to aztreonam and gentamicin. Also, for five years, a mean MAR

index of 0.17 was noted which indicates either proper antibiotic use or most isolates did not come from high-

risk areas. Moreover, there was no significant difference in the resistance of P. aeruginosa when compared

by specimen source (p = 0.662), but significant when compared by year band (p = 0.02). ( Juangya, Lim

and Bonifacio, 2017)

According to Laruan, Balangcod, Patacsil, Apostol, 2013 the study entitled “Phytochemical and

antibacterial study of Lagerstroemia speciosa (L.) Pers. and its ethnomedicinal importance to indigenous

communities of Benguet Province, Philippines" The methanolic extract of Lagerstroemia speciosa (L.) Pers.

leaves, locally known as Banaba, collected from Sablan, Benguet Province, Philippines was subjected to

phytochemical analysis for secondary metabolites and antibacterial screening against Escherichia coli,

Salmonella typhimurium, Staphylococcus aureus, and Pseudomonas aeruginosa using a modified Kirby-

Bauer method. Phytochemical analysis revealed the presence of anthraquinones, flavonoids, saponins, and

tannins. The extract exhibited high antibacterial activity against three of the bacteria in order of sensitivity as

Escherichia coli > Staphylococcus aureus > Pseudomonas aeruginosa, but had no activity against

Salmonella typhimurium. The medicinal value of this plant could be attributed to the presence of one or

more of the detected metabolites. Antibacterial activity possibly shows a basis for traditional use of the plant

as a local health remedy to the indigenous communities of Benguet Province, Philippines. ( Laruan,

Balangcod, Patacsil, Apostol, 2013 )

According to Lamberte, Cabrera, Rivera, 2011 the study entitled “Activity of the Ethanolic Extract of

Propolis (EEP) as a Potential Inhibitor of Quorum Sensing-Mediated Pigment Production in

Chromobacterium violaceum and Virulence Factor Production in Pseudomonas aeruginosa .": the

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unresolved questions” was conducted at Institute of Biology, College of Science, University of the

Philippines, Diliman, Quezon City, Philippines.This study investigated the QS inhibitory potential of the

ethanolic extract of propolis (EEP) in the test organism Chromobacterium violaceum ATCC 12472 and the

opportunistic organism Pseudomonas aeruginosa PAO1. Results of this study showed EEP as a potential

inhibitor of QSmediated violacein production in C. violaceum. EEP was thereby subjected to further testing

on its ability to interfere with virulence factor production and biofilm formation in P. aeruginosa. It was found

that EEP was able to significantly affect the LasA and LasB protease activities. In addition, changes in the

protease activity were observed with no significant effects on the growth of the organism. This implies that

changes in the enzyme activities are unrelated to bactericidal consequences. However, it was also found

that EEP inhibited the biofilm formation of P. aeruginosa PAO1 at lower concentrations but not at higher

concentrations. This suggests the need for further investigations to be made on the effect of EEP on the

maturation and differentiation of biofilms. ( Lamberte, Cabrera, Rivera, 2011)

According to Taganna, Quanico, Perono, 2010 the study entitled “Tannin-rich fraction from

Terminalia catappa inhibits quorum sensing (QS) in Chromobacterium violaceum and the QS-controlled

biofilm maturation and LasA staphylolytic activity in Pseudomonas aeruginosa.": the unresolved questions”

was conducted at Laboratory of Applied Microbiology, Institute of Biology, College of Science, University of

the Philippines, Diliman, Quezon City, 1101, Philippines. The study aimed to test the activity of Terminalia

catappa L. against bacterial quorum sensing (QS) in order to provide a potential scientific basis for the

traditional use of leaf extracts of this plant as an antiseptic. he anti-QS activity of the methanolic leaf extract

of Terminalia catappa was detected through the inhibition of the QS-controlled violacein pigment production

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in Chromobacterium violaceum. Fractions resulting from size-exclusion chromatography were

assayed. The most active fraction was characterized through qualitative phytochemical detection methods.

it was found that TCF12 was able to inhibit the maturation of biofilms of Pseudomonas aeruginosa, a

phenotype that has also been known to be QS-regulated. Therefore, tannin-rich components of Terminalia

catappa leaves are able to inhibit certain phenotypic expression of QS in the test strains used. ( Taganna,

Quanico, Perono, 2010)

Seaweed (Caulerpa lentilifera)

Seaweed is a water plant, especially saltwater algae. It  range from the size of a pinhead to having

large fronds (such as those of many kelps) that can be as much as 30.5 m (100 ft) in length. Certain species

are used for food (such as nori) and fertilizer, and others are harvested for carrageenan and other

substances used as thickening, stabilizing, emulsifying, or suspending agents in industrial, pharmaceutical,

and food products. Seaweed is also a natural source of the element iodine, which is otherwise found only in

very small amounts. (Yourdictionary, 2019)

Seaweeds are macro algae that are essential to marine life for food and habitat, and they have

been a key part of the human diet for thousands of years. Our Irish ancestors relied on seaweed as part of

their diet, handpicking it for their families from the edge of the shore. As far back as the 5th century,

monastic writings tell of its importance in Irish cooking. There are three main types of seaweed, based on

where we can find them on the shoreline. These are the green, red and brown seaweeds. (Thisisseaweed,

2019

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According to Santhanam, Manilal, Sugathan and Selvin, 2008, the study entitled “Antimicrobial

activity of seaweeds extracts against multiresistant pathogens” was conducted at Bharathidasan University

in India. The researchers used fourteen seaweeds collected from the intertidal zone of Southwest coast of

India and were tested against ten human pathogen bacteria and one human pathogen fungus using the well

diffusion test in the casitone agar medium. The researchers found that drying process has eliminated the

active principles in the seaweeds. In the study, methanol:toluene (3∶1) was found to be the best solvent for

extracting the antimicrobial principles from fresh algae and the findings revealed that the tested seaweeds

were highly active against Gram negative bacteria than Gram positive bacteria. The antimicrobial principle

from seaweed was found to be a lipophilic compound. The active principles of highly active seaweeds

Acrosiphonia orientalis and Stocheospermum marginatum were bactericidal. (Santhanam, Manilal,

Sugathan and Selvin, 2008)

According to Escobido and Orbita, 2016, the study entitled “Evaluation of the biochemical and

phytochemical components of green seaweed Caulerpa Lentilifera in Initao, Misamis oriental, Mindanao,

Philippines” was conducted at Mindanao State University. The green seaweed Caulerpa lentilifera that

occurred along the coast of Iligan Bay was analyzed for biochemical and phytochemical components.

Powdered samples of E. intestinalis were analyzed for carbohydrate, protein, fat, ash and moisture content.

Likewise, the presence of reducing sugar, tannins, phenols, saponins, anthraquinones, flavonoids, steroids,

terpenoids and alkaloids were determined using the standard methods. The amount of carbohydrate,

protein, fat, ash and moisture content was in considerable quantities and within the values specified for

seaweeds. The phytochemical analysis revealed the presence of saponins, steroids and terpenoids as well

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as alkaloids. This study further suggested that E. intestinalis possesses both nutritional and pharmaceutical

potential. (Escobido and Orbita, 2016)

According to Thanigaivel and Vijayakunar, 2014, the study entitled "Antioxidant and antibacterial

activity of Caulerpa Lentilifera against shrimp pathogen Vibrio parahaemolyticus" was conducted at Vella

Institute of Technology University. The aim of the present study was to analyse the bioactive compounds

extracted from ethanol extract of the seaweed Caulerpa Lentilifera by GC–MS study and to evaluate its

antioxidant and antibacterial activities for controlling bacterial infection in shrimp pathogens through

immersion and intramuscular infection study. The antioxidant activity of the selected seaweed was

estimated in order to evaluate the efficiency of its ethanol extract since this will help in finding novel

bioactive compounds with antioxidant & radical scavenging properties, which in turn can reveal the

unexploited properties of this seaweed which could be used in the food and pharmaceutical industry.

(Thanigaivel and Vijayakunar ,2014)

According to Baleta, Amar, Padilla and Quinitio, 2011, the study entitled "Biological Activity of

Extracts of Caulerpa Lentilifiera against Aquaculture Pathogenic Bacteria" was conducted at University of

the Philippines Visayas in Miagao Iloilo. Caulerpa Lentilifera was collected from the coastal area of Sta.

Ana, Cagayan, Philippines, and used in in vitro antibacterial assays against six pathogenic bacteria

commonly occurring in aquaculture. The extracts (methanol, n-hexane, dichloromethane, ethyl acetate,

aqueous) were screened against Vibrio harveyi, V. parahaemolyticus, V. alginolyticus, Flavobacterium

aurantiacum, Streptococcus faecalis, and Pseudomonas aeruginosa. The methanol extract showed strong

antibacterial activity against V. harveyi, S. faecalis, and P. aeruginosa and moderate activity against the rest

of the test pathogens. In general, V. harveyi was the most susceptible strain to all the extracts. This study

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suggests that extracts of Caulerpa Lentilifera may be promising sources of antibacterial agents for

use in aquaculture. (Baleta, Amar, Padilla, Quinitio, 2011)

According to Castillo, Cavalo and Laurente, 2018, the study entitled " Combined antibacterial Effects

of Kappaphycus alvarezii and Caulerpa lentilifera Ethanolic Extracts on Staphylocuccus aureus” was

conducted at University of Cebu, school of medicine. This research utilized a completely randomized

controlled in-vitro study design. After plant authentication and preparation of crude extracts, filtrates was the

subjected to Rotary Evaporation to obtain the pure extracts. Individual and combined preparations were

then compared with positive (vancomycin) and negative (ethanol) controls. Results of the study showed that

Ethanol extracts of Kappaphycus alvarezii  and Caulerpa lentillifera, individual and in combination, showed no activity

against Staphylococcus aureus, despite the positive control exhibiting a Zone of Inhibition. Individual and

combined biomedical properties of K. alvarezii and C. lentilifera have no significant antibacterial

effects against S. aureus. It is thus recommended to study the antibacterial activity of both

seaweed extracts on other organisms using methanol as solvent extract. ( Castillo, Cavalo and

Laurente, 2018)

Neem (Azachdiracta indica)

Neem is a compound that has a long history of use in both traditional Indian medicine and

Ayurveda. Many of the popular herbal treatments in these two systems are still derived from it. Neem is a

large evergreen tree, Azadirachta indica, in the mahogany family. It grows naturally in India and Sri Lanka,

and has been successfully transplanted to other regions including West Africa, Indonesia, and Australia.

The tree has small white flowers and produces a smooth, yellow-green fruit. All parts of the tree have

medical uses. In India, neem is sometimes called "the village pharmacy." Over 100 pharmacologically active

substances have been identified in this plant, and it has many traditional applications. (encyclopedia, 2005)

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Neem trees are attractive broad-leaved evergreens that can grow up to 30 m tall and 2.5 m in girth.

Their spreading branches form rounded crowns as much as 20 m across. They remain in leaf except during

extreme drought, when the leaves may fall off. The short, usually straight trunk has a moderately thick,

strongly furrowed bark. The roots penetrate the soil deeply, at least where the site permits, and, particularly

when injured, they produce suckers. This suckering tends to be especially prolific in dry localities. (NCBI,

1992)

According to Banerjee, Kim and Shariff, 2013, the study entitled “Antibacterial Activity of Neem

(Azadirachta indica) Leaves on Vibrio spp. Isolated from Cultured Shrimp”. Extracts of neem tree

(Azadirachta indica) leaves were tested against Vibrio parahaemolyticus and Vibrio alginolyticus isolated

from cultured shrimp. Aqueous extract of neem leaves did not produce any inhibitory zone while the neem

juice produced inhibitory zone that showed linear relationship to the concentration of neem juice on both

bacteria. The Minimum Inhibitory Concentration (MIC) for V. parahaemolyticus and V. alginolyticus was 3.13

and 6.25%, respectively. The Minimum Bactericidal Concentration (MBC) for V. parahaemolyticus and V.

alginolyticus was 12.50 and 25.00%, respectively. It is concluded that neem juice is an antibacterial agent

and is useful for inhibition of vibrios in shrimp. (Banerjee, Kim and Shariff, 2013)

According to Liberato, Nieves, and Toledo, 2011, the study entitled, “Antibacterial Potential of Carica

papaya, Persea americana and Azadirachta indica Extracts against Aquatic Pathogens” was conducted at

Caloocan City Science High School. The antibacterial activity of the aqueous and ethanolic extracts of plant

materials and were tested by disk diffusion method against Pseudomonas aeruginosa, Streptococcus

faecalis, Staphylococcus aureus, and Aeromonas hydrophila. All pure extracts displayed specific activity in

growth of inhibition of all bacteria tested at a concentration of 0.1-1 mg/ml. Antibiotic oxytetracycline (30 μg)

was used as positive control while the respective solvents were used as negative controls. Results showed

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that ethanolic extracts were more active compared to aqueous extracts (p < 0.05) and growth of

inhibition also decreased with decreasing concentration. In the 96 h acute toxicity test, plant extracts used in

this study manifested toxic effect in varying degree to both Nile tilapia and milkfish fry. Leaf extracts of A.

indica were the most toxic to both fish. However, treatment concentrations against bacteria were much

lower than the lethal concentrations. (Liberato, Nieves and Toledo, 2011)

According to Dr. Sarmiento, Gonzales and Maramba, 2011, the study “An in-vitro study on the

Antibacterial Effect of Neem (azadirachta indica) leaf extract on Methicillin sensitive and Methicillin-resistant

Staphylococcus Aureus” was conducted at Philippine General Hospital. An in vitro experimental study was

performed using Neem leaf, properly identified and verified, was subjected to ethanol extraction of its active

ingredients then diluted to produce 25%, 50%, 75%, and 100% concentrations. Standard strains of

Staphylococcus aureus and clinical isolates of MRSA where inoculated on blood agar plates and subjected

to the standardized disc susceptibility testing method. Zones of inhibition were measured for each test

extract and compared to currently used medications, namely oxacillin, vancomycin, mupirocin, andpovidone

iodine with the pure diluent as negative control. The data was analyzed using difference of means

hypothesis testing; it utilized the student’s t-test to determine significance. Results: A trend of increasing

antibacterial activity was noted with increasing concentration of the extract. Zones of inhibition started to

appear at 50% concentration for S. aureus and 75% for MRSA. The antibiotics were able to produce greater

zones of inhibition than the Neem extracts. Conclusion: Data from this study strongly suggest that the

ethanol extract from Neem leaves exhibits in vitro antibacterial activity against both Staphylococcus aureus

and MRSA with greatest zones of inhibition noted at 100% concentration. (Dr. Sarmiento, Gonzales and

Maramba, 2011)

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According to Allan and Coventry ,2001 , the study entitled “Microbiological and Chemical Analysis

of Neem (Azadirachta indica) Extracts: New Data on Antimicrobial Activity” .The antimicrobial effects of

extracts of neem seed (Azadirachta indica A. Juss.) were investigated using microbial growth inhibition

assays. A laboratory-prepared neem seed extract along with a commercially available formulated product,

were characterized using HPLC, and shown to be effective against a range of bacteria in an agar diffusion

assay. The active ingredient,i.e., the unformulated seed extract of the commercial product, also showed

activity and this was further investigated in a biochromatogram, using the sensitive bacterium Bacillus

mycoides. Results showed antibacterial activity as three discrete inhibition zones that did not correspond to

the Rf of the major neem metabolites, azadirachtin, nimbin and salannin. Experiments in liquid culture

suggested that the effect was fungistatic. Conidial germination of the commercially important obligate

pathogen Sphaerotheca fuliginea (powdery mildew) was reduced to 11%. The results show that neem seed

extracts possess antimicrobial activity with notable effects on some fungal phytopathogens. This Work

demonstrates that neem seed extracts have potential for controlling both microbial and insect pests. (Allan

and Coventry ,2001)

According to Juss, 2009, the study entitled “Isolation and Characterization of Pink Pigmented,

Facultative Methylotrophic (ppfm) bacteria from leaves of Neem, Azadirachta indica” was conducted at De

La Salle University-Manila. A total of twenty isolates of pink-pigmented, facultative methylotrophic bacteria

were obtained from the leaves of neem. All isolates exhibited pink to orange-pink pigmentation, entire

margin, round colonies with a smooth glistening surface, and convex elevation. Most of the colonies were

opaque with butyrous consistency. Staining revealed rod to coccobacilli shaped, Gram negative cells,

containing poly-β-hydroxybutyrate granules. Biochemical analyses showed that all were catalase positive;

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majority of them were positive for citrate utilization, urease and oxidase activities but were negative

for amylase activity. They can be cultivated on ammonium mineral salt (AMS) agar with methanol, glycerol

peptone agar (GPA) and tryptic soy agar (TSA) with variations in colonial morphology. Based on the

observed characteristics, the isolates obtained belong to the genus Methylobacterium. (Juss, 2009)

Chapter 3

Materials and Method

Materials

The following tools and materials will be used in the study: gloves, seaweed, neem leaves, distilled

water, stirring rod, agar plate, alcohol lamp, plastic bags, Pseudomonas aeruginosa bacteria, juicer, petri

dish, small containers, Erlenmeyer flask, Muslin cloth, filter paper, sterile cotton applicator, measuring cups

and weighing/measuring scale, beaker, Lysol spray.

Research design

The research design that will be used in the study is Experimental design. According to Blakstad, it

is a systematic and scientific approach to research in which the researcher manipulates one or more

variables, and controls and measures any change in other variables.

In this study, experimental design will be used to determine the sensitivity of the pathogen

(Pseudomonas aeruginosa) to the different treatments made from seaweed extract and neem plant extract.

This design will be used in this study in which the treatments will be made and will have designated

amount of extracts. The researchers’ controlled variables will be the three (5) treatments made from

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Seaweed extract and Neem plant extract, while the dependent variables will be the zones of inhibition of the

Pseudomonas aeruginosa.

Procedural Design

Gathering of Ingredients and Materials Gathering of Test Subjects

Extraction of Seaweed and Neem Plant Impregnating of Susceptibility Discs

Creation of treatments Preparation of Agar Plate

TREATMENT A TREATMENT B TREATMENT C TREATMENT E

TREATMENT D
70%. Neem plant 50 %. Neem Plant 30 % Neem Plant Negative Control –
Positive Control -
Extract + 30% Extract + 50% Extract + 70% Distilled Water
Amoxicillin
Seaweed extract Seaweed Extract Seaweed Extract

Gathering of Data

Disposal of Waste Materials

Analysis and Interpretation of Data

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Procedure

A. Gathering of Materials

The tools will be collected from the research laboratory. 100 grams Seaweeds will be bought in the market

and 100 grams Neem leaves will be collected from one of the researcher’s backyard in St. Francis.

B. Extraction

100 grams Seaweeds and 100 grams Neem leaves will be washed thoroughly first then weighed and will be

extracted separately using juicer. Afterwards, the ingredients will be transferred to a container.

C. Preparation of Treatments

Treatments Concentrations

Treatment A 70% Neem Plant Extract + 30% Seaweed Extract

Treatment B 50% Neem Plant Extract + 50% Seaweed Extract
Treatment C 30% Neem Plant Extract + 70% Seaweed Extract
Positive Control Amoxicillin (500 mg)
Negative Control Distilled Water
Table 1: Concentration of Treatments

D. Impregnation of Paper Disks

Varying concentrations of the Seaweed extract and Neem leaves treatments will be prepared and poured in

separate sterile petri dishes that will be labelled as Treatment A, B, and C. Four (4) paper disks will then be

impregnated in each petri dishes with varying concentrations of the Seaweed and Neem leaves extract. It

will stay overnight and will be set aside.

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E. Preparation of Culture Media

Ten (10) grams of Mueller Hinton Agar (MHA) will be weighed using Digital Weighing Scale. The culture

media will be transferred to a 500 ml. Erlenmeyer flask and 250 mL Distilled water will be added, stirring rod

will be used to dissolve the culture media and will be swirled until it is completely dissolved. It will be

sterilized for 20 minutes. The sterile culture media will be cooled in tap water for 10 to 15 minutes.

F. Pour Plating

20 mL of sterile culture media will be poured plated to four (4) Petri dishes, cooled until it is solidified. A

lighted alcohol lamp will be placed at the working table to ensure sterility of the working place against

airborne contaminants.

G. Inoculation

A sterile cotton applicator will be used to get 2 to 3 colonies of the P. aeruginosa isolates. The P.

aeruginosa isolates gathered will be inoculated to the solidified culture media using the clockwise

inoculating method. Inoculated plates will be placed in the working table in an inverted position.

H. Application of Treatments

The researcher will use disk-diffusion method. Impregnated paper disk with different treatment will be

planted into the incubated petri dishes divided into (4) quadrants each. Each inoculated petri dish will be

treated with different kind of treatments but the Treatment E control will be constantly placed at the center.

flamed, sealed, and covered.

I. Incubation of Petri Dishes

The petri dishes will be incubated for 16 hours with an optimal temperature of 37 degree Celsius in an

inverted position.

J. Measuring of Zone of Inhibition

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With the use of the Orion ruler, the zone of inhibition will be measured in millimeter. Disk diffusion test will

be utilized to determine the susceptibility of P. aeruginosa to the different treatments by measuring the zone

of inhibition (mm). Experiments will be replicated in the mean zone of inhibition will be calculated. The

results will be reported as susceptible, intermediate and resistant. Large zones of inhibition indicated that

the organisms are susceptible while small or no zone of inhibition indicates resistance. An interpretation of

intermediate was given for zones which fall between the accepted cut offs for the other interpretations.

K. Disposal of Cultured Media

Petri dishes will be sprayed with Lysol’s spray disinfectant and poured with an eucalyptus oil to further

disinfect the plates. Cover and let it stay for atleast 20 to 30 minutes. Using a spatula, cultured media will be

removed from the plates into a plastic bag; sprayed with Lysol and sealed. The researchers will dig a hole

for about 2 feet deep and bury the plastic bag containing the media and cover it with tap soil.

Statistical Tool
To interpret the collected data, the following statistical tools are used.

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Mean – to determine the antibacterial activity or zones of inhibition of the Pseudomonas aeruginosa when

exposed to different treatments of seaweed and neem plant extract mixture, including the positive and

negative control.

Standard Deviation – to determine the spread of the data which will reflect the consistency of the different

treatments of seaweed and neem plant extract mixture, including the positive and the negative control in

terms of zone of inhibition Pseudomonas aeruginosa.

One-Way ANOVA (Analysis of Variance) – to determine the significant difference exists between the means

of the different treatments of seaweed and neem plant extract mixture, including the positive and negative

control in terms of zone of inhibition of Pseudomonas aeruginosa at 5% level of significance.

Post Hoc Analysis – to determine which treatments exhibit the significant differences as compared to other

treatments.

Chapter 4

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Result and Discussions

Descriptive Statistics of the Zone of Inhibition

The descriptive statistics of the zone of inhibition in Pseudomona aeruginosa when using seaweeds

(Caulerpa lentillifera) and neem plant (Azachdiracta indica) crude extract is shown in the chart below.

Treatment A had inhibited bacterial growth with a mean of 9.00mm. and a standard deviation of .00;

Treatment B inhibited 9.50mm. growth zone with a standard deviation of 1.00; Treatment C had inhibited

the bacterial growth zone of 9.25mm. with a standard deviation of .50; and Treatment D which was the

Positive Control had inhibited 11.75mm. bacterial growth with a standard deviation of 2.22 while Treatment

E had 0mm. which was the Negative Control had not inhibited the growth of Pseudomona aeruginosa.

In general, Treatment A, B and C had caused resistant zone of inhibition when used against

Pseudomonas aeruginosa, however, Treatment D had caused intermediate zone of inhibition.

TREATMENTS REPLICATES MEAN STANDARD

1st 2nd 3rd 4th

DEVIATION

Treatment A 9 9 9 9 9.00 0.00

Treatment B 9 11 9 9 9.50 1.00

Treatment C 9 9 10 9 9.25 0.50

Treatment D 14 11 12 9 11.75 2.22

Treatment E 0 0 0 0 0 0.00

Table 3. Descriptive Statistics of the Zone of Inhibition

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Table 4 below present the Analysis of Variance in the zone of inhibition of Seaweed and Neem

extract against Pseudomonas aeruginosa to determine significant differences among treatments.

Result shows p-value of 0.00 which is lesser than 0.05 alpha level which rejects all the null

hypothesis and it only means that there is significant difference between Treatments A, B and C when

compared to the positive and negative control.

The results implied that varying concentrations of the extracts had did not vary in inhibiting the

bacterial growth zone of Pseudomonas aeruginosa bacteria.

Table 4: Analysis of Variance in the Mean Rating of the Zone of Inhibition

Treatments

Sum of Squares df Mean Square F Sig.

Between Groups 331.300 4 82.825 67.155 .000

Within Groups 18.500 15 1.233

Total 349.800 19

*p<0.05 = significant.

Post Hoc Analysis between Treatments

To determine which of the treatments exhibit the significant effect, a Post Hoc Analysis was utilized.

Table 5 shows the Post Hoc results of Treatment A, B, C, Positive Control and Negative Control. There

were ten comparison – Treatment A to Treatment B; Treatment A to Treatment C; Treatment A to Positive

Control; Treatment A to Negative Control; Treatment B to Treatment C; Treatment B to Positive Control;

Treatment B to Negative Control; Treatment C to Positive Control; Treatment C to Negative Control; and

Positive Control to Negative Control. Result shows that Treatment A to Treatment B had no significant

difference with a p-value of 0.967 indicating that Treatment A is just as comparable to Treatment B.

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Treatment A to C had no significant difference also with a p-value of 0.998 nor Treatment A and Positive

Control with a p-value of 0.023. Similarly, Treatment B to Treatment C with a p-value of 0.998; Treatment B

to Positive Control with a p-value of 0.075 have significant difference which has a mean of 9.5 for Treatment

B and 11.75 for Positive Control; Treatment C to Positive Control with a p-value of 0.042 had no significant

difference with a p-value greater than the set alpha value 0.05. Treatments A, B, C, and Positive Control to

Negative Control with a p-value of 0 have significant differences because p-value of each comparison is

lesser than the set alpha 0.05. Therefore, Treatment A, B, C and Positive Control are just as comparable

with each other and the four (4) treatments are only significant when compared to Negative Control. With

these, the researchers failed to reject the first null hypothesis, indicating a no significant difference among

the three treatments made by the researchers.

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Multiple Comparisons
Dependent Variable: Treatments
Tukey HSD

(I) Groups (J) Groups Mean Difference Std. Error Sig. 95% Confidence Interval
(I-J) Lower Bound Upper Bound

TreatmentB -.50000 .78528 .967 -2.9249 1.9249

TreatmentC -.25000 .78528 .998 -2.6749 2.1749

TreatmentA
TreatmentD -2.75000* .78528 .023 -5.1749 -.3251

TreatmentE 9.00000 *
.78528 .000 6.5751 11.4249
TreatmentA .50000 .78528 .967 -1.9249 2.9249
TreatmentC .25000 .78528 .998 -2.1749 2.6749
TreatmentB
TreatmentD -2.25000 .78528 .075 -4.6749 .1749
TreatmentE 9.50000 *
.78528 .000 7.0751 11.9249
TreatmentA .25000 .78528 .998 -2.1749 2.6749
TreatmentB -.25000 .78528 .998 -2.6749 2.1749
TreatmentC
TreatmentD -2.50000 *
.78528 .042 -4.9249 -.0751
TreatmentE 9.25000 *
.78528 .000 6.8251 11.6749
TreatmentA 2.75000 *
.78528 .023 .3251 5.1749
TreatmentB 2.25000 .78528 .075 -.1749 4.6749
TreatmentD
TreatmentC 2.50000 *
.78528 .042 .0751 4.9249
TreatmentE 11.75000 *
.78528 .000 9.3251 14.1749
TreatmentA -9.00000 *
.78528 .000 -11.4249 -6.5751

TreatmentB -9.50000* .78528 .000 -11.9249 -7.0751

TreatmentE
TreatmentC -9.25000 *
.78528 .000 -11.6749 -6.8251

TreatmentD -11.75000* .78528 .000 -14.1749 -9.3251

*. The mean difference is significant at the 0.05 level.

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Chapter 5

Summary, Conclusion and Recommendation

Summary

This study entitled “Antibacterial Activity of Seaweed (Caulerpa lentilifera) and Neem Plant

(Azachdiracta indica) Mixture against Pseudomonas aeruginosa” was conducted to evaluate the

antibacterial activity of Seaweed (Caulerpa lentillifera) and Neem (Azachdiracta indica) mixture

against Pseudomonas aeruginosa. It also sought answers to the following questions: (1) What is the

average antibacterial activity of the different treatments made from seaweed and neem mixture

against Pseudomonas aeruginosa in terms of its zones of inhibition? (2) Is there a significant

difference in the zones of inhibition of Pseudomonas aeruginosa when treated with the treatments

made from seaweed extract and neem plant mixture? (3) Is there a significant difference in the zones

of inhibition of Pseudomonas aeruginosa when treated with the different treatments made from

seaweed and neem mixture as compared to positive and negative control? There were (3)

treatments and positive and negative control utilized in this study. Treatment A contained 70%

Seaweed and 30% Neem extracts; Treatment B contained 50% Seaweed and 50% Neem extracts;

Treatment C contained 30% Seaweed and 70% Neem extracts; Positive Control used was

Amoxicillin (500mg); and Negative Control used was Distilled water. The antimicrobial activity of the

plant extracts were tested using the susceptibility testing through disk-diffusion method. The 6mm.

paper disks were impregnated with different treatments containing varied concentrations of seaweed

and neem plant extracts. Using the culture media, Mueller Hinton Agar (MHA), dissolved and

sterilized; and was cooled in tap water for 10 to 15 minutes. The culture media were pour plated to

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the petri dishes and were cooled until solidified; and the test pathogen was streaked plated

to each dish. Impregnated paper disks with different treatments were planted into the inoculated petri

dishes and were incubated for 16 hours with an optimum temperature of 37 degrees Celsius in an

upside down position. After 16 hours of incubation, petri dishes were observed with the presence of

clear zones around the disks; measured the zone of inhibitions using an Orion ruler calibrated in

millimetre (mm).

Zones of inhibitions showed that P. aeruginosa was resistant to the combination of Seaweed and

Neem plant extract and was comparable to the zones of inhibition of the Positive control (500 mg

Amoxicillin). The Analysis of the Variance in the mean rating of the zone of inhibition of P. aeruginosa with

varying concentration of seaweed and neem plant extracts showed was a significant difference with p-value

of 0.00 which is lesser than 0.05 alpha level which rejects all the null hypothesis. Multiple comparison

further showed that it was only the negative control that did not exhibit any inhibitory effect. However,

Treatments A, B, C all exhibited inhibitory effects and are not significant with each other failing to reject the

first null hypothesis. Interestingly, all three treatments did not exhibit significant difference with positive

control making all the three treatments as comparable to the commercial products.

Conclusions

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In the light of the results, the following conclusions were drawn.

1. Treatments A, B and C had caused resistant zone of inhibition against Pseudomonas aeruginosa,

however, Treatment D had caused intermediate zone of inhibition when used against Pseudomonas

aeruginosa.

2. There is no significant difference in the zones of inhibition of Pseudomonas aeruginosa using varied

concentrations of Seaweed (Caulerpa lentillifera) and Neem (Azachdiracta indica) .

3. There is only significant difference in the zones of inhibition of Pseudomonas aeruginosa after the

application of varied concentrations of Seaweed (Caulerpa lentilifera) and Neem (Azachdiracta indica)

extract when compared to the Negative Control and all three treatments did not exhibit significant

difference with positive control, only Treatment B is as comparable to the commercial product.

Recommendations

1. The use of ethanol in extract is highly recommended against Pseudomonas aeruginosa for the

efficacy and potency of the treatments.

2. Each plant must be further studied to know more its biological compounds that later on can be

formulated for new product.

3. Furthermore, most studies are ineffective without the use of ethanol which has only pure extracts.

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References

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