CHAPTER 1

The Problem and Its Background

Introduction
​ Oreochromis niloticus, the most common species amongst
Oreochromis. They thrive in brackish water and are suited for mass
production. It is widely cultivated in ponds and commercial facilities
which makes it accessible and affordable in the Philippines. It is sold in
different ways such as fresh, chilled, or live. In the Philippines, Nile
tilapia is produced for both domestic consumption and commercial sale,
utilizing methods such as intensive culture, improved aquafeed, and
effective pond fertilization techniques. The country’s continuous
production growth is driven by advancements in aquaculture practices,
which contribute to alleviating food scarcity in Asia (Serd, 2024).
​ By turning waste products from the fish industry into beneficial
biomedical resources, researchers are gaining a dual-purpose solution
reducing environmental impact while enhancing public health.
As research continues to explore the biochemical components of
fish scales, the potential applications in antifungal and antibacterial
treatments become increasingly possible. Promoting sustainable and
advancing approaches to healthcare and environmental conservation.
Background of the study

Candida albicans is a polymorphic fungus that naturally exists as

part of the human microbiome, primarily in the gastrointestinal,
respiratory, and urogenital tracts (Dadar et al., 2018). While typically
harmless in healthy individuals, it can transition to a virulent pathogen
in immunocompromised hosts, leading to systemic infections such as
invasive candidiasis (Dadar et al., 2018). The rising incidence of such
infections is compounded by the growing resistance of Candida albicans
to conventional antifungal therapies, particularly azoles like Fluconazole
(Moosa et al., 2004).

This issue is one aspect of a general problem of antimicrobial

resistance, which is not only present in fungal pathogens but also in
bacterial organisms. For instance, Staphylococcus aureus is a
gram-positive bacterium responsible for a wide range of clinical diseases,
from minor skin infections to life-threatening systemic conditions (Taylor
& Unakal, 2023). It is found most commonly on the skin and mucous
membranes and has humans as the primary reservoir. This bacterium is
common in community-acquired and hospital-acquired infections, and
with the increase in multi-drug resistant strains like
Methicillin-Resistant Staphylococcus aureus (MRSA), treating it is
becoming challenging. Research has estimated that half of all adults may
be colonized with S. aureus, and 15% may be persistently colonized in

2
the anterior nares. Certain populations—such as healthcare workers,
hospitalized patients, intravenous drug users, and immunocompromised
individuals—tend to have higher colonization rates, reaching up to 80%
(Taylor & Unakal, 2023). The growing burden of resistant pathogens like
MRSA shows the urgent need for new and effective antimicrobial agents
and natural compounds, including those derived from aquatic
organisms, which have gained attention as promising candidates.​

Among aquatic organisms, Oreochromis niloticus (Nile tilapia) is of

particular interest due to its wide availability. In fact, the Philippines is
one of the largest producers of Nile tilapia in Southeast Asia, which
highlights the potential for tilapia scales to be utilized as a sustainable
source of bioactive compounds. According to the literature by
Romana-Eguia et al. (2020), tilapia comprises 12% of the total
aquaculture production in the Philippines, with 277,006 metric tons
harvested in 2018. Nile tilapia, in particular, is favored for its rapid
growth, hardiness, and adaptability to tropical farming conditions. As
much as 70-80% of the world’s tilapia production is derived from Nile
tilapia (Romana-Eguia et al., 2020).

The use for Oreochromis niloticus (Nile tilapia) is also due to the
bioactive compounds present in its scales (Martins et al., 2018). Nile
tilapia scales, typically regarded as waste in fish processing, are rich in
collagen and hydroxyapatite, both of which have significant biomedical

3
and antimicrobial potential (Martins et al., 2018). Collagen from tilapia
scales has been shown to possess bioactive properties that can be
leveraged for various applications, including wound healing, tissue
engineering, and drug delivery systems (Martins et al., 2018; El-Rashidy
et al., 2015). Moreover, tilapia scales contain antimicrobial peptides
(AMPs), which are part of the fish’s innate immune system and play a
key role in defending against microbial pathogens, including fungi (Tai et
al., 2021). These peptides, such as tilapia piscidin 4 (TP4), have
demonstrated potent antimicrobial activity against a broad range of
microorganisms, including Candida albicans, by disrupting fungal cell
membranes and modulating immune responses (Tai et al., 2021).

Collagen extracted from tilapia scales has demonstrated potential

in biomedical applications, including wound healing, tissue engineering,
and drug delivery systems (Martins et al., 2018; El-Rashidy et al., 2015).
Biologically active components such as collagen and hydroxyapatite,
both of which exhibit significant antimicrobial and regenerative
properties (Martins et al., 2018). Furthermore, these scales contain
antimicrobial peptides (AMPs), such as tilapia piscidin 4 (TP4), which are
crucial components of the fish’s innate immune system. TP4 and other
fish-derived AMPs have exhibited potent antimicrobial activity against a
wide range of microorganisms, by disrupting fungal cell membranes and
enhancing immune modulation (Tai et al., 2021). The mechanism by
which piscidin exerts its antimicrobial effect involves disrupting

4
microbial membranes through toroidal pore formation, leading to cell
lysis and death (Campagna et al., 2007; Pan et al., 2007). This dual
antifungal and antibacterial activity, coupled with its low hemolytic
toxicity and effectiveness against resistant pathogens, highlights
piscidin’s potential as a promising natural compound for the
development of novel antimicrobial therapies (Hayden et al., 2015).

In light of these findings, the present study aims to investigate the

in vitro antimicrobial activity of Nile tilapia scales against Candida
albicans and Staphylococcus aureus. By evaluating the potential of tilapia
scales as an antimicrobial agent, this research contributes to the ongoing
search for alternative therapeutic options to combat the rise of
antimicrobial resistance and the growing burden of fungi like Candida
albicans and bacteria like Staphylococcus aureus globally. The results of
this study could open new avenues for the development of natural,
fish-derived antifungal agents that are both effective and sustainable.

5
Conceptual Framework

The figure below shows the research framework of the study which
was used by the researchers in experimenting the scales of Oreochromis
niloticus (Nile tilapia) in an In Vitro Antimicrobial activity against
Candida albicans and Staphylococcus aureus.

​ ​ ​ ​ Figure 1. Flow of the Study

​ The figure above shows the structure of the study which was used
by the researchers in experimenting the In Vitro Antimicrobial activity of
the scales of Oreochromis niloticus (Nile tilapia) against Candida albicans
and Staphylococcus aureus. The researchers obtained Nile tilapia fishes
from Navotas Fish Port Complex, Navotas, Philippines. Then the samples
obtained were transported to the National Fisheries Research and
Development Institute (NFRDI) in the Fisheries Building Complex, BPI

6
Compound, Visayas Ave, Quezon City, Philippines 1128 for
authentication and certification.
​ The process involved preparing the scales — cleaning the scales
with distilled water and drying the scales in an oven at 40-50°C. Once
the scales are dried, the scales will be pulverized. Following this, to
extract the bioactive compounds from the pulverized scales, different
approaches were used – Demineralization, Deproteinization, and
Deacetylation. The demineralization process involved soaking the 40g of
scales in an erlenmeyer flask containing 200 ml 1M HCl in 30°C for 24
hours, after soaking the scales were rinsed several times with distilled
water until the pH was neutral. The deproteinization process involved
soaking the demineralized scales in an erlenmeyer flask containing 0.5%
NaOH at a 1:1 (m/v) ratio overnight at room temperature, then
thoroughly rinsed several times with distilled water until the pH was
neutral. The deacetylation process involved adding 100 mL of 50% NaOH
then boiled at 120°C for 2 hours. After boiling and cooling, the scales
were rinsed continuously with distilled water until the wash became
clear. The residue was then filtered.
​ Two solvents were employed; Distilled water with Dimethyl
sulfoxide, and Ethanol with Dimethyl sulfoxide each with different
concentrations – 50%, and 100%. Then a Fourier Transform Infrared
Spectroscopy Analysis (FTIR) was performed to determine the
compounds present in the scales. After this, Antimicrobial testing used

7
are the agar well diffusion method for Candida albicans and agar disk
diffusion method for Staphylococcus aureus.
​ Lastly, the output assesses the zone of inhibition of both testing
methods to measure and analyze the efficacy of Oreochromis niloticus
extracts in inhibiting the growth of Candida albicans and Staphylococcus
aureus. Moreover, to evaluate the comparative effectiveness against
Fluconazole and Vancomycin as a standard antimicrobial agent.

Statement of the Problem

This study investigates the in vitro antifungal activity of
Oreochromis niloticus (Nile tilapia) scales against Candida albicans and
Staphylococcus aureus. The following questions are sought to be
answered:
1.​ How effective are Oreochromis niloticus scales in inhibiting the
growth of Candida albicans and Staphylococcus aureus?
2.​ How do differing treatments to pulverized Oreochromis niloticus
scales (ON-Aqua and Dimethyl sulfoxide; and ON-Eth Dimethyl
sulfoxide) affect the inhibition of Candida albicans and
Staphylococcus aureus growth?
3.​ How does the efficacy of Oreochromis niloticus scales differ from
commercially available microbial drugs (Fluconazole and
Vancomycin) against Candida albicans and Staphylococcus aureus?

8
Research Hypotheses
​ This study aims to analyze the in vitro antifungal activity of
Oreochromis niloticus scales against Candida albicans. The researchers
devised the following hypotheses:
1.​ Oreochromis niloticus scales are not effective for inhibiting the
growth of Candida albicans and Staphylococcus aureus.
2.​ There is no significant difference in the inhibition of Candida
albicans and Staphylococcus aureus growth between the different
treatments (ON-Aqua and Dimethyl sulfoxide; and ON-Eth
Dimethyl sulfoxide) to pulverized Oreochromis niloticus scales.
3.​ There is no difference in efficacy between Oreochromis niloticus
scales and commercially available microbial drugs (Fluconazole
and Vancomycin) against Candida albicans and Staphylococcus
aureus.

Significance of the Study

This study aims to investigate the potential antifungal activity of
Oreochromis niloticus (Nile Tilapia) scales against Candida albicans and
Staphylococcus aureus in vitro. The findings of this study will be valuable
to the following:
General Public. This study will raise awareness about the potential
use of fish scales in treating microbial infections, promoting other
alternative and sustainable treatment options. They will also learn about

9
the potential health benefits of fish scales as a remedy, encouraging
them to explore sustainable treatment options and promoting
sustainable healthcare practices.
Fishermen and Fish farmers. This study may provide an additional
source of income for fishermen and fish farmers by creating a demand
for fish scales, which are often discarded as waste. This could also help
them optimize their resource use, reduce waste, and contribute to a more
sustainable consumption and economically viable fishing industry.
Healthcare Professionals. The study may provide healthcare
professionals with an alternative treatment option for their patients in
treating microbial infections particularly those caused by Candida
albicans and Staphylococcus aureus, in addition to other existing
antimicrobial medications. This can help in recommending a more
diverse range of treatment options for their patients suffering from fungal
and bacterial infections, especially in cases where other treatment
options are limited or are not cost-effective.
Pharmaceutical industry. The study may offer valuable insights
that could serve as a foundation for developing new antimicrobial
medications from aquatic resources like fish scales. These developments
could help address the growing problem of antimicrobial resistance,
reduce the reliance on synthetic drugs, and potentially lead to more
affordable and accessible treatment options for people.

10
 Researchers. This study will aid in the advancement of scientific
knowledge on antimicrobial treatments and the use of aquatic resources
in the medical field, encouraging further investigation into the medicinal
properties of other often-overlooked natural resources, such as those
found in aquatic environments. This could lead to new discoveries in
biology, medicine, and healthcare, contributing to a better understanding
of biological mechanisms and the development of new treatments for
infectious diseases.

Scope and Delimitation

This study focuses on the In Vitro analysis of the antimicrobial
activity of Oreochromis niloticus (Nile tilapia) scales against Candida
albicans and Staphylococcus aureus. Specifically, it will measure the zone
of inhibition produced by the scales on Candida albicans and
Staphylococcus aureus.
The study will evaluate how different treatments to pulverized
Oreochromis niloticus scales (ON-Aqua and Dimethyl sulfoxide; and
ON-Eth Dimethyl sulfoxide) impact this inhibitory effect. Furthermore,
the study will compare the efficacy of Oreochromis niloticus scales with
Fluconazole and Vancomycin, which are standard treatments against
Candida albicans and Staphylococcus aureus, to provide an accurate
analysis of the scales’ effectiveness. Regardless of the sex of the fish, all
samples will be included, as the study focuses solely on the antimicrobial

11
properties of the scales, which are not influenced by the biological sex of
the fish.
This study will be conducted at Centro Escolar University - Makati
throughout the academic year 2024-2025. This research is limited to the
scales of Oreochromis niloticus as well as Candida albicans and
Staphylococcus aureus, thereby excluding other tilapia, bacteria, and
fungal species, respectively, that may react differently to the treatment.
The study will be conducted entirely in vitro, using standard
laboratory techniques such as agar well diffusion assay to ensure
controlled and measurable results.
The comparison with Fluconazole and Vancomycin is limited to
understanding the relative efficacy between the natural and synthetic
antimicrobial agents, as no other antimicrobial will be included. These
limitations ensure that the study remains focused on evaluating the
antimicrobial activity of Oreochromis niloticus scales against Candida
albicans and Staphylococcus aureus.

12
Definition of Terms
​ To facilitate better understanding of this study, the following terms
were defined:
​ Anti-microbial. Substances that can inhibit the growth or have an
ability to kill microbial pathogens.
​ Candida albicans. A common type of fungi used to test the
antifungal activity of extracts from Nile tilapia scales.
​ Dimethyl sulfoxide. A type of sulfur-containing organic chemical
solvent used to extract the potential bioactive compounds that can be
used as an antimicrobial from the scales of Nile Tilapia fish.
​ Distilled water. A solvent used to extract the potential bioactive
compounds that can be used as an antimicrobial from the scales of Nile
Tilapia fish.
​ Fluconazole. The standard antifungal drugs used to treat
infections caused by Candida albicans – a positive control used for the
antimicrobial activity of the scales of nile tilapia fish.
​ Ethanol. A type of alcohol used to extract the potential bioactive
compounds that can be used as an antimicrobial from the scales of Nile
Tilapia fish.
​ In Vitro. An experiment conducted to test the antimicrobial activity
of the Nile fish scales to Candida albicans and Staphylococcus aureus
and to determine its inhibitory effect.

13
​ Oreochromis niloticus. A common kind of tilapia that is the second
most important cultured species in the Philippines, also known as Nile
tilapia fish.
​ Scales. An external protective structure that covers and protects
the body of the Nile tilapia fish, composed of various bioactive
compounds that have been shown to possess antimicrobial properties.
​ Staphylococcus aureus. A common type of gram positive bacteria
used to test the antifungal activity of extracts from Nile tilapia scales.
​ Vancomycin. A type of antibiotic used to treat infections caused by
Staphylococcus aureus – a positive control used for the antimicrobial
activity of the scales of nile tilapia fish.

14
 CHAPTER 2

Review of Related Literature and Studies

This chapter contains a review of related literature that the
researchers considered to shed light on the subject at hand. The
literature presented in this chapter addresses relevant thoughts,
findings, and facts related to the antimicrobial properties of Oreochromis
niloticus scales against Candida albicans and Staphylococcus aureus,
helping to provide a better understanding of the significance of natural
antimicrobial agents. Moreover, this serves as a guide for the researchers
in developing the study and as a foundation for its validity.

Nile tilapia in the Philippines

​ Nile tilapia (Oreochromis niloticus) is a dominant species in the
Philippine aquaculture sector, accounting for a substantial portion of the
country's total aquaculture production. In 2018, tilapia production in the
Philippines reached 277,006 metric tons, with Nile tilapia representing
approximately 70-80% of the global tilapia production (Romana-Eguia et
al., 2020). This species is widely cultivated in regions such as Central
Luzon, Calabarzon, and Ilocos, where it thrives in freshwater ponds and
cages. Its significant presence underscores its economic importance and
accessibility. (Romana-Eguia et al., 2020)
Nile tilapia is valued in Philippine aquaculture for its rapid growth,
hardiness, and ability to adapt to the country’s tropical climate. It thrives
15
in various farming conditions, tolerating different water qualities and
temperatures, which enhances its suitability for diverse aquaculture
systems. The species' efficient feed conversion and relatively low
maintenance requirements make it economically viable, favoring its
widespread adoption among local fish farmers (Romana-Eguia et al.,
2020).
The species’ resilience and high productivity distinguish it from
other farmed fish in the region, reinforcing its role as a key species in the
Philippine aquaculture industry. Its superior adaptability and consistent
production also position it as an ideal candidate for biomedical research,
including antifungal studies.

Standard Maturity Size and Sexual Differentiation of Nile Tilapia

Standard maturity size is regarded as an important factor in
understanding the growth, reproduction, and ecological dynamics of
Oreochromis niloticus (Nile tilapia). In a study carried out in Lake
Coatetelco, Mexico by Gómez-Márquez et al., (2008) male and female O.
niloticus showed different growth patterns and sizes according to their
physiological and reproductive differences. The standard length range
was broader in females; 9.0–16.5 cm, compared to males, 8.9–14.8 cm.
This might suggest that females can attain larger sizes than males as
they are involved in reproduction and allocate energy differently. Males
and females grow at similar rates and have a maximum age of 2.5 years.

16
 An important aspect of the life cycle of Oreochromis niloticus is at
what age it reaches sexual maturity. According to Cuéllar Sáenz (2021),
in Nile tilapia generally, sexual maturity comes in about 3 to 4 months,
at a body weight of between 50 to 100 g, and a length of 10 to 12 cm;
these have been regarded as optimal for the initiation of the reproductive
cycle. Maturity is characterized by the differentiation of sexual organs:
males have two orifices; anus and urogenital orifice, while females have
three orifices; anus, genital pore, and urinary orifice. The ability to
distinguish between the sexes will make it easy to understand their
reproductive strategies​.

Antimicrobial properties of fish-derived compounds

The rise of fungal infections, particularly those caused by Candida
albicans, has become a pressing concern in clinical settings, largely due
to the increasing resistance to conventional antifungal therapies (Spettel
et al., 2019). This growing issue has prompted the search for alternative
therapeutic agents, including natural compounds derived from aquatic
organisms. Among these, fish-derived antimicrobial peptides (AMPs),
particularly from Oreochromis niloticus (Nile tilapia), have gained
attention for their broad-spectrum antimicrobial properties, including
significant antifungal activity.
Fish-derived AMPs are part of the innate immune system of fish,
playing a critical role in defending against pathogens (Tai et al., 2021).

17
The potential of these peptides to combat fungal pathogens, such as
Candida albicans, positions aquatic organisms such as Oreochromis
niloticus as promising candidates for alternative antifungal treatments.
In fact, these peptides, such as tilapia piscidin 4 (TP4), have
demonstrated potent antimicrobial activity against a variety of
microorganisms, including fungi. TP4, specifically, has shown significant
antifungal properties against Candida albicans in vitro, primarily by
disrupting fungal cell membranes and modulating immune responses
(Tai et al., 2021). This mechanism of action, involving both physical
disruption and immune system activation, highlights the dual
functionality of fish-derived AMPs as both antimicrobial and
immunomodulatory agents.
Furthermore, the potential of fish-derived peptides, particularly
TP4, has been further emphasized in studies investigating their use in
combating challenging fungal infections. Velumani et al. (2023) explored
the role of fish-derived peptides in the treatment of mucormycosis, a
severe fungal infection commonly found in diabetic patients. The study
demonstrated that peptides like TP4 could effectively target and
neutralize fungal pathogens, suggesting that these compounds could
serve as viable alternatives to conventional antifungal treatments. This
reinforces the idea that fish-derived AMPs, particularly those with
demonstrated antifungal properties, may play a crucial role in the

18
development of new therapeutic strategies for managing fungal
infections.
Fish-derived AMPs, such as TP4, not only show promising
antifungal activity but also possess antimicrobial properties that extend
beyond fungi to include bacteria and other pathogens. This
broad-spectrum activity enhances their potential as versatile therapeutic
agents. Furthermore, the development of recombinant forms of these
peptides, such as rTP4, facilitates large-scale production and makes
these peptides more accessible for use in clinical and agricultural
applications.

The composition and biomaterials of Nile tilapia scales

Tilapia fish scales, generally considered waste in fish processing,

are rich in bioactive compounds, notably collagen and hydroxyapatite,
which position them as valuable resources for biomedical,
environmental, and antifungal applications. Increasingly recognized for
their composition and bioactivity, tilapia scales exhibit potential in
multiple scientific domains, such as orthopedics, tissue engineering, and
drug delivery systems.

Structurally, tilapia scales are primarily composed of collagen

fibrils intertwined with hydroxyapatite, a calcium phosphate crystalline
form providing mechanical strength (Okuda et al., 2011). Biomedically,
tilapia scales have shown considerable promise. Research confirms that

19
tilapia fish scales exhibit antifungal properties, particularly against
Candida albicans, attributed to the collagen matrix that interacts with
fungal cell walls, potentially leading to degradation of pathogenic cell
walls (Chen et al., 2010). This antifungal activity opens avenues for
tilapia scales in developing alternative antifungal therapies.

Fish scales as antimicrobial and antifungal

Fish scale is the primary pollutant due to their high collagen
content which makes them highly resistant to natural degradation
leading to waste build-up and pollution, Hussain et al. (2017). This can
lead to foul smell and serve as a breeding ground for pathogenic bacteria
and fungi. In contrast, the recent study by Hussain et al. (2017) showed
that hydrolyzing fish scales can yield protein hydrolysates containing
bioactive compounds like peptides and amino acids, which exhibit
antimicrobial properties. Literature reports indicate that some of the
peptides may act as antifungal agents. There are peptides and amino
acids that act as antibacterial and even antiviral agents, Hussin et al.
(2017). Alkali hydrolysis, specifically, has been used to convert scales
from Cyprinus carpio into soluble protein hydrolysates that demonstrated
antibacterial efficacy against various human pathogens. This approach
applies the composition of scales in a natural way by breaking them
down to release active compounds that can inhibit bacterial growth as

20
mentioned in the study of Hussain et al. (2017) using this will effectively
target bacteria like Escherichia coli and Klebsiella pneumoniae.

In vitro antifungal susceptibility testing methods

Antifungal susceptibility testing (AFST) plays a crucial role in the
fight against fungal infections, particularly in the context of the rising
prevalence of antifungal resistance. Given the increasing challenge of
antifungal resistance, standardized and reliable testing methods are
essential to accurately determine the effectiveness of new compounds.
Various in vitro techniques have been developed to assess the antifungal
activity against pathogens such as Candida albicans.
In conclusion, in vitro antifungal susceptibility testing is a vital
component in the evaluation of new antifungal agents, including natural
products like tilapia fish scales. The findings from studies like Berkow et
al. (2020) and Turroni et al. (2022), highlight the importance of
methodological adaptation when testing natural products, ensuring that
results are both reproducible and comparable across different
experimental setups.

Health risks and resistance of Candida albicans

​ Candida albicans is a polymorphic fungus that normally exists as
a commensal organism in the human microbiome, particularly in the
gastrointestinal, respiratory, and urogenital tracts (Dadar et al., 2018).
While it typically poses no health threat, in immunocompromised or

21
immunologically stressed individuals, C. albicans can transition from a
harmless commensal to a virulent pathogen. The risk factors for invasive
candidiasis, including antibiotic therapy, neutropenia, surgery, central
venous access, and preexisting conditions like diabetes and HIV, further
complicate the clinical landscape (Dadar et al., 2018). In addition to
these factors, the emergence of antifungal resistance in C. albicans and
non-albicans species (e.g., Candida glabrata, Candida krusei) is a
growing concern. Resistance mechanisms, such as alterations in cell wall
synthesis, drug efflux pumps, and mutations in target enzymes,
complicate treatment and contribute to poor patient outcomes.
​ The clinical manifestations of Candida infection are extensive
which can range from infections of the mucous membrane to severe
spread of infection associated with sepsis. Candida spp. are considered
as a normal flora of the human skin, gastrointestinal, and genitourinary
microbiomes. Infection from this species occurs when the host's defenses
are hindered or an imbalance of microbiome, resulting in Candida
overgrowth. Clinical manifestations include oral candidiasis, esophageal
and vaginal yeast infections, and chronic mucocutaneous candidiasis.
Certainly, invasive candidiasis is serious and is often underdiagnosed
due to limited global surveillance (Bays et al., 2024).

22
Prevalence of Candida albicans infections
Approximately 20-25% of the world population suffers from
dermatophytosis, one of the most severe fungal infections that can
quickly damage tissue, organs, and nerves (Zhang et al. 2023). It has
become a global health concern. According to the study of (Zhang et al.
2023), an estimated 30-50% mortality rate in affected pathogens is more
prevalent.
According to Batac and Denning (2017), infectious diseases,
including fungal infections, remain a major cause of morbidity in the
Philippines, affecting approximately 1,852,137 people or 1.9% of the
population. Aspergillosis and Candidiasis are the leading causes of these
infections. Among them, recurrent vulvovaginal candidiasis is the most
prevalent, impacting 1,481,899 women. This is followed by allergic
bronchopulmonary aspergillosis and severe asthma with fungal
sensitization, as well as chronic pulmonary aspergillosis.

Antifungal activity of fluconazole against Candida albicans

​ According to Berkow et al. (2017), fluconazole is a key treatment
option despite its limitations due to resistance. The study points out that
understanding the importance of these resistance mechanisms to
preserve the effectiveness of fluconazole in treating fungal activity of
Candida spp. is important. Additionally, emergence of drug resistance

23
can be considered an inevitable consequence of the selective pressures
imposed by antifungal drugs.

Health risks and resistance of Staphylococcus aureus

​ Staphylococcus aureus is a known causative agent in a plethora of
infections. It is known to colonize various mucosal sites of the body and
considered as relevant and dangerous in modern age-medicine.
According to Gajdács (2019), between the 1960–1970s, MRSA infections
were predominantly associated with nosocomial outbreaks, until the end
of the 1970’s the termed hospital-acquired MRSA (HA-MRSA) was
predominantly used since hospitalized patients or outpatients that
frequently attend hospitals were associated with this infection. During
the 1990s the number of infections associated with acquiring HA-MRSA
increased significantly. Due to the increased risk of this pathogen, this
led to the emergence of community-associated MRSA (CA-MRSA)
infections that have spread and been reported from basically every region
on the planet in the past 10–20 years, owing to successful clones.
Because of the mutations of MRSA, several antibiotics appeared on the
market from 1980s and onward, allowing for the better management of
infections and observing the resistance of these drugs throughout the
years. However in the beginning of 2000s, Livestock-associated MRSA
(LA-MRSA) was reported which causes a significant cause of morbidity
and mortality in both the livestock/food industry since the outbreak.

24
Since the beginning of the 21st century, a concerning rise of
multidrug-resistant has been reported but still MRSA has proven to be
one of the most persistent drug resistant pathogens in both the
healthcare and community setting. Based on the assessments of the US
Centers for Disease Control and Prevention (CDC), MRSA has been
classified as a serious threat and S. aureus is included in the group of
"ESKAPE" bacteria and considered as the biggest concern for humanity.
Due to these reports, WHO is urging drug companies to invest in
antibiotics research. This proves that Staphylococcus aureus is a fine
example of a successful pathogen that can adapt even in modern day
and MRSA infections can be acquired all around the globe.

Prevalence of Staphylococcus aureus infections

​ Approximately 30% of the human population carries
Staphylococcus aureus in their normal flora. It is the major causative
agent of bacteremia and infective endocarditis, and is associated with
infections of bones, joints, skin and soft tissues, lungs, and may also
infect medical devices (Tong et al., 2015).

Antimicrobial activity of vancomycin against Staphylococcus

aureus
Based on the study of Zhang et al., (2023), Staphylococcus aureus
is a prevalent and virulent pathogen responsible for a range of serious
infections, notably S. aureus bacteraemia (SAB), which poses high

25
morbidity and mortality rates of up to 25%. The rise of
methicillin-resistant S. aureus (MRSA) has further complicated
treatment, making SAB a major contributor to severe conditions such as
sepsis, infective endocarditis, and meningitis. Vancomycin (VAN), a
glycopeptide antibiotic introduced over six decades ago, has been the
cornerstone therapy for MRSA infections due to its ability to inhibit
bacterial cell wall synthesis. It remains a first-line treatment for SAB in
international guidelines, including those from the United States, UK, and
China. However, VAN's relatively slow bactericidal activity, emerging
resistance, and the development of newer antimicrobial agents such as
ceftaroline, telavancin, and contezolid, have sparked debates about its
continued primacy in SAB management. While alternative treatments
like daptomycin and linezolid are also recommended, evidence remains
mixed, with most studies comparing VAN to only one or two agents at a
time. Thus, despite its widespread use, the need to reassess VAN's
efficacy and safety in light of evolving resistance patterns and new
therapeutic options remains critical.

Variables that might affect fish scales

​ According to the study of (Siddika et al., 2012), they concluded
that replacing fish meal with meat and bone meal did not negatively
affect the growth or survival of the fish. This is conducted in an
experiment where there are 4 experiment diets prepared with varying

26
levels of fish meal replaced by meat and bone meal, aimed at creating a
more cost-effective feed. Moreover, results showed that the fish fed with
the diet containing 100% meat and bone meal had the best outcomes in
terms of weight gain, specific growth rate, apparent net protein
utilization and survival rate. Generally, studies have shown that feed
quality can influence the overall health and development of fish,
including their scales.

Synthesis

The growing prevalence of microbial infections, particularly those

caused by Candida Albicans and Staphylococcus aureus and the
increasing resistance to conventional antimicrobial agents emphasize the
urgent need for alternative therapies. This scenario has focused scientific
interest on natural bioresources, particularly those originating from
aquatic species such as Ocreochromis niloticus (Nile tilapia), one of the
most widely farmed and economically important fish species in the
Philippines (Romana-Eguia et al., 2020). Due to its extensive availability
and biological properties, it is a favorable candidate for biomedical
purposes such as antimicrobial studies.

Several studies have identified fish-derived antimicrobial peptides

(AMPs), particularly Tilapia Piscidin 4 (TP4), as potent bioactive agents
capable of combating a broad spectrum of pathogens, including fungi
like C. albicans (Tai et al., 2021). These peptides work by disrupting

27
microbial cell membranes and modulating immune responses,
highlighting their dual antimicrobial and immunomodulatory roles.
Moreover, recombinant forms of these peptides have shown enhanced
accessibility and application in clinical settings (Velumani et al., 2023).

Tilapia scales, often regarded as waste products, have also

emerged as valuable bioresources due to their rich composition of
collagen and hydroxyapatite (Martins et al., 2018). These components
have demonstrated antifungal potential, as seen in studies where the
collagen matrix disrupts fungal cell walls (Tai et al., 2021). The process
of hydrolyzing fish scales further enhances their bioactivity by releasing
antimicrobial peptides and amino acids (Hussain et al., 2017), thereby
converting waste into therapeutically viable materials. This
transformation not only presents a sustainable waste management
strategy but also opens new avenues in antimicrobial drug development.
Supporting this, in vitro antifungal susceptibility testing methods have
proven critical in evaluating the efficacy of such natural products
(Turroni et al., 2022). Adaptations in these methodologies ensure the
reliability and comparability of test results, particularly when assessing
the antimicrobial activity of novel agents like fish-derived compounds.

The urgency of exploring alternative antimicrobial agents is further

emphasized by the increasing resistance of Candida albicans and
Staphylococcus aureus to conventional treatments such as fluconazole

28
and vancomycin. Both pathogens are associated with high morbidity and
mortality, particularly in immunocompromised individuals. As resistance
mechanisms continue to evolve, natural bioactive compounds offer a
promising solution to supplement or replace conventional antimicrobial
drugs.

The literature offers a solid basis for examining Nile tilapia scales'
antimicrobial activity. Their biochemical makeup, combined with the
increasing demand for sustainable and efficient antifungal agents, makes
them an attractive candidate for in vitro antimicrobial studies against
Candida albicans and Staphylococcus aureus.

29
 CHAPTER 3

Methods and Procedures

Introduction
​ This chapter discusses the experimental research design and
procedures used to investigate the antimicrobial activity of Oreochromis
niloticus (Nile tilapia) scales to inhibit the growth of Candida albicans and
Staphylococcus aureus. The study involves an in vitro analysis to assess
the effectiveness of fish scale extracts against the microbial growth of
Candida albicans and Staphylococcus aureus using ON-Aqua and
Dimethyl sulfoxide; and ON-Eth and Dimethyl sulfoxide. This aims to
examine the antimicrobial properties of the scales and provide valuable
insights into the potential of natural compounds as an alternative to
synthetic antimicrobial agents.
Research Methodology
The study employed an experimental quantitative research design,
specifically a two pair group design. This design includes two
experimental groups, each receiving distinct treatments, alongside two
control groups.
The researchers established two experimental groups: 1)
Oreochromis niloticus scale extract with distilled water (ON-Aqu) and
DMSO. Oreochromis niloticus scales extract with ethanol (ON-Eth) and
DMSO. Fluconazole and Vancomycin served as the control group. The

30
efficacy of each solution was evaluated by measuring the zones of
inhibition against Candida albicans and Staphylococcus aureus,
facilitating a comparative analysis of the antimicrobial activity of the
different scale solutions.

Research Procedure
The researchers used various procedures and techniques to gather
data on the in vitro antifungal activity of Oreochromis niloticus Scales
Against Candida albicans and Staphylococcus aureus.
1.​ CEU-IERB Application and Informed Consent Process
Prior to commencing the experiment, a research
proposal detailing the study on the antifungal activity of
Oreochromis niloticus scale extracts against Candida albicans
and Staphylococcus aureus was submitted to the
CEU-Institutional Ethics Review Board (CEU-IERB) for
evaluation and approval. The investigation proceeded only
upon receiving the necessary ethical clearance.
2.​ Preparation of Instruments to be Used
All of the following instruments and materials that were used
throughout the experiment were properly cleaned, sterilized,
and prepared to ensure reliable results. This includes:
●​ Triple Beam Balance
●​ Cork Borer

31
 ●​ Drying Oven
●​ Incubator
●​ Erlenmeyer Flasks
●​ Glass Funnel
●​ Graduated Cylinder
●​ Glass Rod
●​ Spatula
●​ Hot Plate
●​ Inoculating Loop
●​ Micropipettes and sterile tips
●​ Petri Dishes with Sabouraud Dextrose Agar (SDA)
Media
●​ Petri Dishes with Blood Agar Plate (BAP) Media
●​ Ruler
●​ Scalpel/Forceps
●​ Whatman Filter Paper
3.​ Collection and Authentication of Oreochromis niloticus (Nile
Tilapia)
Oreochromis niloticus (Nile tilapia) was collected from
the Navotas Fishing Port Complex, located on North Bay
Boulevard, Navotas City, Metro Manila. Prior to collection,
the researchers obtained permission from the management
of the fishing port to ensure compliance with local

32
 regulations and ethical standards regarding the use of tilapia
for research purposes. Following collection, the specimens
were transported to the National Fisheries Research and
Development Institute (NFRDI), situated in the Fisheries
Building Complex, BPI Compound, Visayas Avenue, Quezon
City, Philippines 1128, for certification and authentication.
4.​ Preparation of Oreochromis niloticus (Nile Tilapia) Scales
Upon successful authentication and collection, the fish
were transported to the laboratory. In a controlled
environment, scales were carefully excised from the
specimens, ensuring the removal of all adhering flesh. The
scales were then rinsed with distilled water to eliminate dirt
and potential contaminants. Subsequently, the scales were
dried in an oven at 40-50°C. Once adequately dried and
cooled, the samples are stored in a sterile container.
5.​ Fourier Transform Infrared Spectroscopy Analysis (FTIR)

Researchers aspirated a small amount of fluid from

the prepared sample solution, which was then analyzed
through FTIR to determine the bioactive components present
in the Oreochromis niloticus scale sample solution. The
findings were read at the Department of Science and
Technology (DOST), located on DOST Main Building, General

33
 Santos Avenue, Brgy. Central Bicutan, Taguig, Metro Manila,
Philippines.

6.​ Extraction of Oreochromis niloticus (Nile Tilapia) scales as

antimicrobial agent
To extract bioactive compounds from the scales of
Oreochromis niloticus (Nile Tilapia), different approaches are
used to obtain substances that can potentially inhibit
pathogenic microbes.
a.​ Demineralization
In the demineralization process, dried Oreochromis
niloticus scales were placed into an erlenmeyer flask
containing 200 ml 1M HCl and 40g of Nile tilapia scales,
soaked at 30°C for 24 hours. After soaking, the scales were
rinsed several times with distilled water until the pH was
neutral.
b.​ Deproteinization
The demineralized scales were then soaked in 40 ml
0.5% NaOH overnight at room temperature and thoroughly
rinsed several times with distilled water until the pH was
neutral.
c.​ Deacetylation
After the deproteinization process, 100 mL of 50%
NaOH were added to the sample scales, which were then

34
 heated at 120°C for 1 hour. After heating and cooling, the
scales were rinsed continuously with distilled water until the
wash became clear. The residue was then filtered.
d.​ Treatments
​ Two solvents were employed in the treatment of Nile
tilapia scales for antimicrobial extracts: distilled water
(ON-Aqu) and ethanol (ON-Eth), each with different
concentrations ( 50%, and 100%).
7.​ Antimicrobial Testing
The method that was used for antimicrobial testing
was the agar well diffusion assay and agar disc diffusion
assay.
a.​ Agar well diffusion assay for Candida albicans
A specified volume of Candida albicans culture
was inoculated onto an agar plate, and wells were
created using a sterile cork borer. The Oreochromis
niloticus scale extracts (ON-Aqu and Dimethyl
sulfoxide; and ON-Eth and Dimethyl sulfoxide) and 0.5
mg/mL Fluconazole, used as a positive control, and
were added to the wells, followed by incubation for 24
hours. After incubation, the zones of inhibition were
measured. Sabouraud Dextrose Agar was used for
subculturing Candida albicans in this experiment.

35
 b.​ Disc diffusion assay for Staphylococcus aureus
A specified volume of Staphylococcus aureus
culture was inoculated onto an agar plate. Pieces of
filter paper were infused with Oreochromis niloticus
scale extracts (ON-Aqua and Dimethyl sulfoxide; and
ON-Eth and Dimethyl sulfoxide) and 0.5 mg/mL
Vancomycin, the positive control, and were then added
to the plate, followed by incubation for 24 hours. After
incubation, the zones of inhibition were measured. A
Blood Agar Plate was used for subculturing
Staphylococcus aureus in this experiment.
8.​ Data Analysis
Following incubation, the zones of inhibition were
measured to evaluate the antifungal activity of Oreochromis
niloticus scale extracts (ON-Aqu and Dimethyl sulfoxide; and
ON-Eth and Dimethyl sulfoxide) against Candida albicans
and Staphylococcus aureus. The data obtained from the
zones of inhibition for these extracts were compared to the
zone of inhibition of Fluconazole and Vancomycin to assess
the efficacy of Oreochromis niloticus extracts in inhibiting the
growth of Candida albicans and Staphylococcus aureus and

36
 to determine their comparative effectiveness against
Fluconazole and Vancomycin, a standard antifungal agent.

Setting of the Study

​ The study was conducted at Centro Escolar University - Makati
laboratory room in a controlled and sterile environment for the
laboratory analysis. The marine sample, Oreochromis niloticus, was
obtained from Navotas Fishing Port Complex, North Bay Boulevard,
Navotas City, Metro Manila. Additionally, the culture of Candida albicans
and Staphylococcus aureus were bought from Bactefast Corporation
located at 9 Ocean Park, Novaliches, Quezon City, Metro Manila.
​ To ensure the accuracy of the sample, it was authenticated at the
National Fisheries Research and Development Institute (NFRDI) 101
Mother Ignacia Ave., Brgy, Diliman, Quezon City, 1103 Metro Manila.
This is critical for the study as it is the basis of the sample to be used in
the laboratory analysis.
Lastly, the researchers obtained Fluconazole and Vancomycin from
the pharmacy: Mercury Drug, to be diluted and be used as the control
for the in vitro analysis of the scales of Oreochromis niloticus against
Candida albicans and Staphylococcus aureus.

37
Subjects of the Study

The subject of this study involves three groups of organisms:

Candida albicans, a pathogenic yeast commonly associated with fungal
infections; Staphylococcus aureus, a bacteria known for causing various
infections in humans; and Oreochromis niloticus (Nile tilapia), believed to
have antimicrobial properties, particularly in its scales. The main
samples for this study were scales from Oreochromis niloticus and
cultured Candida albicans and Staphylococcus aureus, all selected based
on specific inclusion and exclusion criteria to ensure validity and
consistency in the results.

The inclusion criteria for Oreochromis niloticus (Nile tilapia) scales

are as follows: (1) scales must come from mature Nile tilapia; (2) the fish
must be healthy, showing no visible signs of disease, infection, or injury;
and (3) fish scales must be freshly obtained. The exclusion criteria for
Oreochromis niloticus scales are as follows: (1) scales that are not from
mature tilapia or scales from other fish species; (2) scales from fish
showing signs of disease, infection, or injury; and (3) scales from Nile
tilapia that have been stored for an extended period.

For Candida albicans and Staphylococcus aureus, the inclusion

criteria are: (1) strains must be obtained from a certified or accredited
laboratory, and (2) strains must be confirmed as Candida albicans and
Staphylococcus aureus. The exclusion criteria are: (1) strains not

38
obtained from a certified or accredited laboratory, and (2) strains not
identified as Candida albicans and Staphylococcus aureus.

Oreochromis niloticus samples were collected from Navotas Fishing

Port Complex, and immediately transported to the laboratory to ensure
freshness. Candida albicans and Staphylococcus aureus were obtained
from a recognized and accredited laboratory culture collection, Bactefast
Corporation, and stored under controlled conditions in the university
laboratory, with regular subculturing to maintain viability.

This study followed strict ethical protocols when working with

animals and a potentially pathogenic organism. The handling of Nile
tilapia adhered to the university’s animal care standards, while Candida
albicans and Staphylococcus aureus were managed according to biosafety
guidelines to prevent contamination and exposure. Necessary permits
were obtained, and proper handling procedures were strictly followed to
meet all required guidelines.

Sampling Techniques
First, purposive sampling was used to select Oreochromis niloticus
scales, as this sampling technique allows for the intentional selection of
samples directly related to the study's objectives. Since the focus is on
the antifungal activity of Oreochromis niloticus, purposive sampling

39
ensures that only scales from this specific species are chosen,
guaranteeing consistency and relevance to the study.

To increase reliability and accuracy, replicated sampling was

applied, wherein multiple trials were conducted for each treatment
group: ON-Aqu (aqueous solution) and ON-Eth (ethanol), and the
Fluconazole and Vancomycin control. By creating replicates for each
treatment, the study reduces variability and enhances the validity of the
results, providing a more accurate reflection of the antimicrobial efficacy
of each treatment on Candida albicans and Staphylococcus aureus.

Also, samples were randomly assigned to the treatment groups, a

process that minimizes bias by giving each sample an equal chance of
receiving any specific treatment. Random assignment supports a fair
comparison across the different treatments, which is essential for robust
statistical analysis.

40
Research Instruments
The researchers utilized an observation form in order to monitor
and evaluate the antimicrobial activities of the Oreochromis niloticus
scales against Candida albicans and Staphylococcus aureus in vitro. This
observation focused on the relationship between different concentrations
of the solutions and the zone of inhibition.
The observation form shown in Table 1 for the zone of inhibition
(mm) with different concentration of the Antimicrobial which was based
on the study of Nazer et al. (2023) entitled “Antibacterial and Synergistic
Potential of the Scale Extracts from Oreochromis mossambicus against
Bacterial Pathogens”.
The observation form is designed to record the data across various
solutions, specifically ON-Aqu and ON-Eth in different concentration
(50% and 100%), and the resulting zone of inhibition in millimeters and
any notable observations regarding the antimicrobial activity against
Candida albicans and Staphylococcus aureus. A Comparison with the
effectiveness of an antimicrobial, Fluconazole and Vancomycin were also
recorded for relative assessment.
Each of the entries on the observation form allowed the
researchers to record the zone of inhibition consistently for each
solution, analyzing the effectiveness of Oreochromis niloticus scale extract
inhibiting Candida albicans and Staphylococcus aureus growth at
different levels.

41
 Observation Form

Observed by:
Storage Condition:
Trial:

Test Solution Zone of Inhibition Interpretation

Positive Control
(Fluconazole)

ON-Aqua and DMSO

ON-Eth DMSO

Table 1. Observation Form for Antifungal Activity of Oreochromis niloticus

Scale Extract Against Candida albicans (Dummy table)

42
 Observation Form

Observed by:
Storage Condition:
Trial:

Test Solution Zone of Inhibition Interpretation

Positive Control
(Vancomycin)

ON-Aqua and DMSO

ON-Eth and DMSO

Table 2. Observation Form for Antifungal Activity of Oreochromis niloticus

Scale Extract Against Staphylococcus aureus (Dummy table)

Test Solution Trials Average Interpretation

1 2 3

ON-Aqua

ON-Eth

Positive

43
 Control
(Fluconazole)

Table 3. Summary of Trials (Candida albicans) (Dummy table)

Test Solution Trials Average Interpretation

1 2 3

ON-Aqua

ON-Eth

Positive
Control
(Vancomycin)

Table 3. Summary of Trials (Staphylococcus aureus) (Dummy table)

Statistical Treatment of Data

​ This section describes the statistical methods that were used to

analyze the data collected in the study. The following statistical tools will
be applied:

44
Descriptive Statistics: The mean and standard deviation of the zones of
inhibition, was calculated for each experimental group: ON-Aqu and
ON-Eth; and the Fluconazole as control. This will provide an overview of
the average inhibitory effect of each treatment on Candida albicans and
the variability within each group.

Inferential Statistics: To compare the data between the experimental and

control group are statistically significant:

●​ One-Way Analysis of Variance (ANOVA): A one-way ANOVA will be

used to compare the mean zones of inhibition across the different
treatments of Oreochromis niloticus scales (ON-Aqu and ON-Eth).
This analysis will determine if there is a statistically significant
difference in antifungal efficacy among the different treatments.
●​ Independent Samples t-Test: An independent sample t-test will be
used to compare the mean zone of inhibition of the most effective
Oreochromis niloticus scale treatment with that of Fluconazole.
This test will help determine if there is a statistically significant
difference in antifungal efficacy between the natural treatment
(Nile tilapia scales) and the synthetic antifungal (Fluconazole).
Moreover, the same test will be applied to compare the mean zone
of inhibition of the most effectiveOreochromis niloticus scale
treatment with that of Vancomycin.

45
●​ Kruskal- Wallis Test: This test will be used to determine if there is
a statistically significant difference in antimicrobial efficacy among
the different treatments.

46