i

UTILIZATION OF Bixa orellana (ANNATTO) SEED EXTRACT AS

POTENTIAL PRIMARY STAIN ALTERNATIVE IN
ACID-FAST STAINING

A Thesis
Presented to the Faculty and Research Committee
Tagum Doctors College, Inc.
Tagum City

In Partial Fulfillment of the Requirements for the Degree of

Bachelor of Science in Medical Laboratory Science

CELINE B. BARCELON
NATHANAEL M. DALUGDOG
FRYNJHE AN A. HERNANDO

May 2024

JENRY KEN VINCENT MIBATO, RMT, MSMT

Research Adviser
 ii

Approval Sheet

This RESEARCH study entitled “UTILIZATION OF Bixa orellana (ANNATTO)

SEED EXTRACT AS POTENTIAL PRIMARY STAIN ALTERNATIVE IN ACID-
FAST STAINING” prepared by CELINE B. BARCELON, NATHANAEL M.
DALUGDOG and FRYNJHE AN A. HERNANDO in partial fulfillment of the
requirements for the degree of Bachelor of Science in Medical Laboratory
Science has been examined, approved and hereby endorsed.

JENRY KEN I. VINCENT MIBATO, RMT, MSMT

Research Adviser

PANEL OF EXAMINERS

APPROVED by the Research Committee on the Oral Examination with a

grade of PASSED.

Rey Anthony S. Perez, RMT, MSMT

Chairman

Noraine Princess G. Tabangcora, RMT, LPT, MSMT Ann Fatima G. Quindao, RMT, LPT, MPH
Member Member

ACCEPTED and APPROVED in partial fulfillment of the requirements for the

degree of Bachelor of Science in Medical Laboratory Science.

REY ANTHONY S. PEREZ, RMT, MSMT

Dean of Medical Laboratory Science
 iii

Acknowledgement

The researchers would like to express their heartfelt gratitude to Tagum

Doctors College, Inc., especially to Prof. Manuel Dennis E. Molina, MBA, the

President and CEO, Dr. Jesa S. Madelo, RPh, MPH, Ph.D., the Vice President

for Quality Assurance, Community Extension and Research and to all

administrative staff, for their invaluable approval and support to this research

endeavor.

To Prof. Rey Anthony S. Perez, RMT, MSMT, our Department Dean,

and all MLS Department Faculty for their guidance and support.

To Tagum Doctors Hospital, DRMC TB Culture and Laboratory,

WVN Testing and Research Laboratory, and all collaborating individuals and

groups for their invaluable contributions. Special thanks to Ms. Jisa Rizale J.

Yamas, RPh, and the entire laboratory staff at Tagum Doctors College, Inc. for

their assistance.

To Prof. Jenry Ken Vincent Mibato, RMT, MSMT, for his insightful

guidance as our research adviser, and to Prof. Altom Kent Tongol, RMT, for

his ideas, and Prof. Noraine Princess G. Tabangcora, RMT, MSMT, our

research instructor, for her expertise and mentorship.

To our dear panelist, Prof. Rey Anthony S. Perez, RMT, MSMT, Prof.

Noraine Princess G. Tabangcora, RMT, MSMT, and Prof. Ann Fatima G.

Quindao, RMT, LPT, MPH, thank you for your guidance and expert advises.

Our gratitude also extends to our validators and evaluators, Sir Ken

Ryan Dizon, MBA-HP, MN, RN, Prof. Raymundo R. Rosales, RMT, Prof.

Hiyasmin Gutierrez, RMT, MSMT, Prof. Ellaine H. Esteban, RMT, and Prof.

April Joy T. Abayon, RMT for their valuable inputs.

 iv

A word of gratitude to our statistician Sir Tom Paulie M. Tongol, LPT,

MAEd and grammarian Prof. Noraine Princess G. Tabangcora, RMT, MSMT,

for their huge contribution in writing this study.

We would like to thank our parents for their unwavering support and

financial assistance.

To our classmates, friends, and loved ones, thank you for your

understanding and encouragement.

Lastly, we acknowledge Almighty God as the source of all our blessings

and guidance throughout this endeavor.

C.B.B.

N.M.D.

F.A.A.H.
 v

Abstract

Carbol fuchsin, a stain crucial in diagnosing tuberculosis and leprosy, poses

great health and environmental risks. The aim of this study is to validate Annatto

seed pigment as a safer alternative primary stain for acid fast staining due to

its non-toxic, non-allergic, non-carcinogenic, and biodegradable nature. Tracing

plant extract dyes' history, especially in histopathology, reveals natural dyes'

efficacy over synthetic ones. Experimental results using Annatto seed extract

as an experimental sample and carbol fuchsin as a control showed promising

staining properties. However, the Annatto seed extract, while demonstrating

potential, did not fully stain the bacteria, likely due to extraction and

reconstitution methods that may have affected its binding to the mycolic cell

wall of the bacteria. Further refinement of extraction and reconstitution

processes is necessary to optimize staining efficacy. Nonetheless, the study

highlights the feasibility of using Annatto seed pigments as a safe and effective

alternative to carbol fuchsin in acid-fast staining, paving the way for safer

laboratory practices and environmental stewardship in medical diagnostics.

Keywords: Annatto seed extract, alternative primary stain, acid-fast staining,

Carbol fuchsin
 vi

Table of contents

TITLE PAGE i

APPROVAL SHEET ii

ACKNOWLEDGEMENT iii

ABSTRACT v

TABLE OF CONTENTS vi

LIST OF TABLES viii

LIST OF FIGURES ix

CHAPTER 1

INTRODUCTION

Background of the Study 1

Research Gap 3

Research Questions 3

Literature Review 5

Conceptual Framework 15

CHAPTER 2

METHODS

Study Design 17

Setting 17

Subjects 20

Instruments 21

Data Collection Procedure 23

Data Analysis 31

Ethical Consideration 32
 vii

CHAPTER 3

RESULTS

Results 35

CHAPTER 4

DISCUSSION

Discussion 40

CHAPTER 5

CONCLUSION AND RECOMMENDATIONS

Conclusion 46

Recommendations 48

References 51

Appendices 56

Curriculum Vitae 115

 viii

List of tables

TABLES TITLE PAGE

Table 1 The Phytochemical Analysis of the Bixa orellana 35

(Annatto Seeds)

Table 2.1 The Staining Characteristics of AFB with Annatto 36

Seed Extract (100g)

Table 2.2 The Staining Characteristics of AFB with Annatto 36

Seed Extract (300g)

Table 2.3 The Staining Characteristics of AFB with Annatto 37

Seed Extract (600g)

Table 2.4 The Staining Characteristics of AFB with Positive 38

Control

Table 3 The Significant Difference in Using Various 38

Concentrations of Annatto Seed Extract as

Primary Stain Alternative for Acid-Fast Organism

Table 4 The Significant Difference Between the Annatto 39

Seed Extract and Carbol Fuchsin (Positive

Control) on their Effectivity in the Primary Staining

of Acid-Fast Organism
 ix

List of figures

FIGURES TITLE PAGE

Figure 1 Annatto Tree with Seeds 5

Figure 2 Acid-Fast Staining 11

Figure 3 Conceptual Paradigm of the Study 15

Figure 4 Location of the Study 19

Figure 5 Diagram for Data Gathering Procedures 23

Figure 6 Concentration Formula 26

Figure 7 Flowchart of the Smearing Process 28

Figure 8 Staining Method 30

Figure 9 Pulverization and Extraction Process 72

Figure 10 Microscopic Characteristics of Annatto Seeds 85

 Chapter 1

Introduction

Background of the Study

Carbol fuchsin plays a crucial role in identifying acid-fast organisms, but

it is essential to recognize potential drawbacks and adverse effects in specific

contexts. Since the late 1800s, acid-fast staining has been used in medicine. It

was considered the gold standard for diagnosing tuberculosis and leprosy

(Prakoeswa et al., 2022). In this method, carbol fuchsin is used as the primary

staining agent, a mixture of phenol and basic fuchsin. Phenol, a component of

carbol fuchsin, is a toxic substance that can cause various complications of the

gastrointestinal, ocular, skin, neuropathy, and renal. A case report showed the

effects of carbol fuchsin poisoning in a patient wherein there is a rapid

progression of acute renal failure with metabolic acidosis (A Case of Carbol

Fuchsin Poisoning Successfully Treated With Hemoperfusion and Continuous

Venovenous Hemodiafiltration, n.d.). Basic fuchsin, which is also a component

of carbol fuchsin, is said to be carcinogenic (“Saudi Journal of Pathology and

Microbiology,” 2019). In the environmental aspect, carbol fuchsin is one of the

primary contaminants in industrial wastewater. When released, it enters the

environment and negatively impacts plants and wildlife (Koli et al., 2019). The

dyes from plants are safe because they are non-toxic, non-allergic, non-

carcinogenic, and biodegradable (Saha & Sinha, 2012). Dyes from the plants,

when oxidized, could be used as a suitable substitute for staining

microorganisms.

Tracing the history of using plant extract as stains or dyes had been

carried out effectively, especially mentioning the hematoxylin dye used in

 2

staining tissues in the histopathology section. Instead of synthetic dyes, which

are more expensive and negatively affect both people and the environment,

natural dyes derived from plants can be used to color different biological

tissues and pathogens (Hartika et al., 2021). The annatto seeds had been

widely used commercially as a strong food dye, and because of its strong

colors, many researchers studied their properties as a possible dye or stain.

According to Rd (2019), the extracted liquid can be used in cell staining; this

shows a higher possibility of this seed being used as an alternative primary

stain. Based on its coloring properties, the apocarotenoid bixin had higher

quantities of pigment and was the most abundant.

According to Ciscar (1965, as cited in Ulbricht et al., 2012), achiotin, an

extract of achiote seeds, can be used as a histologic stain for lipids. Ciscar

carried out the potentiality of the seed extract dissolved in a mixture of 70%

alcohol and anhydrous acetone to stain lipids, which was found effective. The

acid-fast organisms were known to have a lipid-rich cell envelope wherein the

carbol fuchsin attached. As the study shows the effectiveness of staining lipids,

it could also be possible in staining acid-fast organisms such as M.

tuberculosis.

To recognize the associated risks and environmental hazards of carbol

fuchsin, commonly used in acid-fast staining, this study aims to validate and

explore the potential of Annatto seed pigments as a safer alternative stain. The

objective is to assess its staining efficacy, compare it with conventional stains,

and recommend a more secure and ecologically friendly option. By studying

Annatto seed pigments, we seek a safer primary stain for acid-fast staining,

prioritizing laboratory safety without compromising accuracy in detecting AFO.

 3

Research Gap

The existing research has identified synthetic dyes, such as

carbolfuchsin, as posing significant health and environmental risks. Notably,

the adverse effects extend to various bodily systems, including the

gastrointestinal tract, ocular, skin, and nervous system A Case of Carbol

Fuchsin Poisoning Successfully Treated With Hemoperfusion and Continuous

Venovenous Hemodiafiltration, n.d.). Furthermore, evidence suggests that

carbolfuchsin is not only toxic but also carcinogenic, with reported cases of

poisoning. Additionally, the stain emerges as a prominent contaminant in

industrial water sources (Koli et al., 2019). Despite these findings, there

remains a critical research gap in fully understanding the comprehensive

scope and potential mitigation strategies for the health and environmental

impacts associated with carbolfuchsin and similar synthetic dyes.

This study tends to formulate a potential primary stain alternative in

acid-fast staining to stain acid-fast organisms. The researchers aim to create

an eco-friendly, cost-effective, and safe alternative primary stain. In

comparison to commercially available options, the annatto seed stands out as

a superior choice, offering distinct advantages.

Research Questions

The current study investigated the possibility to create a potential primary

stain alternative from Annatto seeds extract to identify or detect the presence

of acid-fast organism. This experimental intervention was designed to examine

the effectiveness of the alternative stain in conducting acid-fast staining. This

research sought to answer the following questions:

 4

1. What is the phytochemical profile of the identified Annatto seeds?

2. What are the staining characteristics of the AFB with Annatto seed

extract, and the positive control in terms of:

2.1 Intensity of the red color;

2.2 Clarity of shape (filamentous or rod-shaped);

2.3 Specificity in Staining (stains only AFB)?

3. Is there a significant difference in using various concentrations (100g,

300g, 600g) of Annatto seed extract as a primary stain alternative for

acid-fast organism?

4. Is there a significant difference between the Annatto seed extract and

carbol fuchsin (positive control) on their effectivity in the primary staining

of acid-fast organism?

Hypothesis

The following null hypotheses were formulated by the researchers based

on the foregoing study, and was tested at 0.05 level of significance:

1. There is no significant difference in using the Annatto seed extract as

a primary stain alternative for acid-fast organism in terms of the

concentrations of the extract.

2. There is no significant difference in the staining of acid-fast organism

using the Annatto seed extract and carbol fuchsin (positive control) on

determining its effectivity as an alternative primary stain.

 5

Literature Review

Annatto seed origin and components

Bixa orellana, popularly known as annatto, is a native of Central and

South America and was one of the first recognized natural dye-producing

plants. Annatto has long been used in traditional medicine to prevent and treat

a wide range of health problems. Because of the many traditional uses,

researchers have been encouraged to locate and isolate phytochemicals from

this plant's sections. tagys, apocarotenoids, terpenes, terpenoids, sterols, and

aliphatic chemicals are the major components found in all parts of this plant and

are reported to have various pharmacological effects. Annatto has undergone

a spike in scientific attention in recent years, owing partly to the discovery of a

yellow-orange natural pigment in its seeds that is extremely biodegradable,

non-toxic, and non-hazardous (Shahid-UI-Islam et al., 2016).

Figure 1. Annatto Tree with seeds. Retrieved from “Offset by Shutterstock”

annatto seeds on Offset. (n.d.).
https://www.offset.com/search/annatto+seeds. Copyright by Jittrapon
Kaicome
 6

Bixa orellana is a neotropical tree that is used for commerce. It may

reach heights of 2 to 8 meters and is perennial. It demonstrates phenotypic

variety in height, girth, flower color, pod color, and pod form. The tree

produces. "Annatto," a natural colorant that is the second-most sought-after

dye in the world (Bidyalaxmi et al., 2022).

Raddatz-Mota et al. (2016) studied the coloring components of the

annatto seeds. The bixin produces a red color, while the norbixin produces an

orange color.

Numerous phytochemical studies have focused on the isolation and

identification of carotenoids and apocarotenoids in Bixa orellana plants, with a

particular emphasis on extracts from seeds and seed coats. The primary

compound in B. orellana seed coats is bixin, identified as methylhydrogen-(9′Z)-

6,6′-diapocarotene-6,6′-dioate, constituting approximately 80% of the

carotenoid content, while trace amounts of other carotenoids are also present

(Shahid-UI-Islam et al., 2016).

Bixa orellana seeds are rich in carotenoids, especially apocarotenoids

like bixin, isobixin, and norbixin. Bixin is a diapocarotenoid soluble in oil,

containing two carboxylic acid groups, one of which is esterified. Norbixin is

derived from bixin through the hydrolysis of the ester group. These carotenoids

are the main pigments found in annatto seeds (Hirko & Getu, 2022).

Determining the total phenolic content of the seed is crucial, as this

phenol content facilitates the stain's penetration of the cell wall. According to a

study by Ahmed et al. (2020), the total phenolic content of annatto seed extract

using methanol is 186.02 mg/g of extract, expressed as milligrams of gallic acid

equivalent (mg GAE)/g of extract.

 7

Advantages

Natural colors, often known as biocolors due to their biological basis, are

typically taken from vegetables, fruits, roots, and microorganisms. Annatto dye

is a natural colorant of carotenoids derived from the pericarp of Bixa orellana

seeds. Annatto produces an orange-to-red color in dishes and can be used as

a natural pigment in various cuisines. The main dye component of annatto dye

is 9'-cis bixin, which is soluble in oil, while the main dye component of alkaline

extract is 9'- cis norbixin, which is soluble in water. In economics, annatto dye

is the world's second most often used natural additive color, and demand for

it is increasing (Yolmeh et al., 2015).

The thin, resinous arils of the tropical plant Bixa orellana seeds make

annatto dye, which is highly prized for its accessibility, affordability, and safety

in the food sector. Additionally, it is frequently used for dyeing, pharmaceutical,

and cosmetic reasons. The primary annatto colorants are bixin and norbixin,

which give food a red-to-yellow tint (Hirko & Getu, 2022).

Annatto extract is a popular natural dye that is in high demand around

the world. This is due to factors such as its abundance, diverse coloring

properties (ranging from yellow to reddish/orange), and stability, as well as the

ability to obtain hydrosoluble or liposoluble extracts from the same source,

depending on the extraction method (Cunha et al., 2008).

Annatto seeds, often known as achiote, lend a unique color to foods

while giving an interesting and healthful touch. This spice also has health

benefits because of its high concentration of carotenoid antioxidants, which

benefit the eyes and the immune system.

Due to its antioxidant characteristics, Bixin integration into nanofibers

 8

has already been used for therapeutic applications. Although it has not been

studied in the literature, it can be employed as an environmentally friendly

adsorbent agent on an industrial scale. The liposolubility of bixin allows for the

removal of adsorbent material after adsorption without loss of analyte or bixin

and provides a high-water remediation potential to the process (Domingues et

al., 2020).

One natural dye is dye from Bixa orellana (Annatto) seed, which can be

used as a dyeing agent for coloring textile fibers such as cotton, wool, and silk

and for manufacturing colorful "Gulal" in the food business. For this reason,

bixin, a pigment derived from red-colored seeds, can be utilized as a coloring

agent. It is non-carcinogenic and does not affect the human body or the

environment. The laboratory attempts to extract natural pigment from B.

orellana seeds for use in many industries, such as textiles, colorful powder,

and the food business (Saha & Sinha, 2012).

Potentiality as being dye or stain

Acetone and methanol extracts of Annatto efficiently stain molds but not

bacteria; however, when treated with glacial acetic acid and used as a

counterstain, they are proven to stain Escherichia coli. Ammonium Hydroxide

addition may also improve the staining capacity of Annatto only when it

decreases the ability of other plant extracts, such as Pterocarpus osun and

Lawsonia inamis, to stain (Badar et al., 2022).

Due to the ever-growing demand for environmentally friendly, non-toxic

colorants, natural dyes have become a viable alternative to relatively toxic

synthetic dyes. A study by Braide et al. (2010) shows the enhanced staining

potential of the annatto hot extract by Ammonium hydroxide treatment. Pure

 9

Cultures of Escherichia coli and Staphylococcus aureus were collected, as

isolated Aspergillus and Rhizopus species on Sabouraud Dextrose Agar

(SDA) were used for the study. Bacteria and molds were not stained from the

Annatto water extract, but the organic extracts were significantly stained after

acetic acid treatment.

Another study conducted by Siva et al. (2008) shows the effectiveness

of Annatto as an alternative tracking dye for gel electrophoresis. Bromophenol

Blue (BPB) is a regular dye used in electrophoretic techniques to determine

ion front. Still, due to several harmful effects, the study aims to find an

alternative tracking dye from natural sources. Annatto's production of orange-

to-red color dye was tested for its potential as a tracking dye. It also has the

same characteristics as BPB and does not interfere with the test proteins.

Electrophoretic migration of the sample using Annatto dye extract shows a

bright yellow-orange color on the front, and it was also observed that there

were no molecules present that could interfere with the electrophoretic

migration. A comparison of relative mobility was also done and showed

similarity between the two dyes.

A related literature used 11 plant extracts as test staining solutions,

including Bixa orellana extract (Annatto seed extract). It showed intensified

staining ability, but modified concentration, pH, and staining time are

considered for better natural histological stains (Sudharani, 2022).

As indicated in the study of Ayobami (2019), Bixa Orellana extract is

possible as a histologic dye. The study focuses on staining the liver, brain,

kidney, skin, and intestine as a counterstain to hematoxylin. The pigment was

extracted using the maceration extraction method using three reagents:

 10

acetone, ethanol, and water. Staining reactions depend on pH in relation to

the iso-electric points of the dye molecules.

Carbol fuchsin

Carbol fuchsin is a mixture of phenol and basic fuchsin (BF), a common

red dye commonly used for dyeing and organic dyeing of various materials such

as leather, paper, and cotton. Phenol-based compounds such as carbol fuchsin

can persist for long periods in the environment and cause soil and water

contamination. This is harmful to aquatic life and can have long-term ecological

effects. Disposal of solutions containing carbol fuchsin and materials used in

the staining process can be problematic. These materials are considered

hazardous waste and must be disposed of properly according to regulations,

which can be costly and require special handling (Abu-Zurayk et al., 2021).

Carbol Fuchsin is the key component of the Ziehl-Neelsen stain, which

consists of carbol and basic phenol. In the presence of acid, the Carbol Fuchsin

dye creates a yellow-brown compound that is well-retained by the waxy walls

of mycobacteria.

A case report described the consequences of carbol fuchsin poisoning

in a patient who developed metabolic acidosis and acute renal failure rapidly (A

Case of Carbol Fuchsin Poisoning Successfully Treated With Hemoperfusion

and Continuous Venovenous Hemodiafiltration, n.d.). When swallowed, the dye

may cause gastrointestinal discomfort, including nausea, vomiting, diarrhea,

and irritation of the respiratory system. Physical contact with the dye may cause

severe eye and skin irritation. Another potential outcome is organ damage from

ingestion or inhalation, including damage to the thyroid, liver, spleen, and blood.
 11

Repeated exposure can also impact the neurological system, along with

headache, lightheadedness, weariness, and tightening of the muscles. In

addition to being carcinogenic, its toxicity may also be mutagenic (Gupta et al.

2008).

One of the main pollutants in industrial effluent is carbol fuchsin. It enters

the environment through wastewater discharge and has a detrimental effect on

flora and fauna (Koli et al., 2019).

Acid-fast Staining

The most common way in determining our acid-fast bacilli (AFB) or

organisms is through acid-fast staining (AFS). The AFS was developed by Ziehl

and modified by Neelseen, that’s why the method used in identifying these

organisms is called Ziehl-Neelsen staining technique (Mokobi, 2022). On

principle basis the Ziehl-Neelsen method uses reagent that specifically

differentiates AFB from non-AFB.

B
A

Figure 2. A. Non-acid fast resulted to blue color B. Acid-fast resulted to blue

color Bright red to intensive purple, Red, straight or slightly curved rods,
 12

occurring singly or in small groups, may appear beaded, while. From

“Maricopa” by Anne Mason M.S., 2022,
https://open.maricopa.edu/myfirstbook/chapter/acid-fast-stain/

Carbol fuchsin is used to solubilize the lipoidal material in the

Mycobacterial cell wall. However, when heat is applied, carbol fuchsin not only

solubilizes but also penetrates the lipoidal wall, causing all cells to turn red.

Subsequently, a decolorizing agent (3% HCL in 95% alcohol) is applied to the

smear. The acid-fast cells resist decolorization due to their high lipoidal

material content, preventing the decolorizing solution from penetrating. In

contrast, non-acid-fast organisms lack this lipoidal material in their cell wall,

making them easily decolorized and leaving the cells colorless. The smear is

then stained with a counterstain, methylene blue. Only the decolorized cells

absorb the counterstain, resulting in a blue color, while acid-fast cells retain

their red color (Mokobi, 2022).

Acid-fast organisms

Acid-fast organisms are characterized by their staining characteristics in

retaining the color of the primary stain. These organisms belong to

Mycobacterium, Nocardia, Cryptosporidium, Legionella, and Rhodococcus.

Mycobacterium tuberculosis is the bacterium responsible for

tuberculosis (TB), an infectious disease primarily targeting the lungs and

transmitted through the air. When the bacteria are present in your body but

not active, it is referred to as a latent TB infection, detectable by tests but not

causing illness. In contrast, active TB disease occurs when the bacteria are

alive and proliferating, making you sick and contagious (Dallas, 2024).

The fifth most common cause of sickness and mortality in the general
 13

population in the Philippines is tuberculosis. It is one of the twenty-two nations

with a high burden of disease, which together account for 80% of all TB cases

worldwide. A third of all TB cases, the majority of which are smear-negative,

are new instances of pulmonary TB, with about 150,000 of those cases being

smear-positive each year. The issues with HIV and multi-drug resistance TB

in the country may further exacerbate this severe TB burden (Phelan et al.,

2019).

The first clinic is located at the Department of Health's Davao Regional

Hospital (DRH), one of the two tertiary hospitals southeast of the Philippines.

The clinic sees 420 tuberculosis patients annually on average. Patients who

are seen at the hospital and are thought to have tuberculosis are automatically

referred to the DRH TB DOTS. The second clinic, the Tagum City Health Office

(TCHO), is a government building. In addition, this clinic treats 420

tuberculosis cases annually on average (Vinson et. al, 2015).

Non-tuberculosis mycobacteria (NTM)

NTM are classified as the non-tuberculosis causing bacteria other than

the M. tuberculosis which is the causative agent of the aforementioned

disease. Nontuberculous mycobacteria are a group of bacteria naturally found

in soil, water and dust worldwide (American Lung Association, n.d.). These

group of bacteria still causes lung disease but is not known to be contagious

unlike the abovementioned organisms.

According to Nardell (2022) infections caused by these organisms have

been called atypical, environmental, and nontuberculous mycobacterial (NTM)

infections. Mycobacterium avium complex (MAC) and Mycobacterium

 14

abcessus are considered as common NTM that is prevalent around the world

producing NTM infections.

Preparation

The method for pulverizing and extracting the crude extract was

adapted from Venturina et al. (2020). First, ripened seeds were manually

separated and washed with distilled water. Sterilization was carried out at 60

degrees Celsius for 24 hours in a hot air oven. The seeds were then finely

crushed using a blender.

Next, 100 grams of pulverized seeds were mixed with 1 liter of methanol

and stirred for 12 hours using a magnetic stirrer. The mixture was then stored

away from sunlight at room temperature for an additional 12 hours. Filter paper

(Whatman Filter Paper No.1) was utilized, and the mixture was placed in an

IKA RV10 Rotary Evaporator at 40 degrees Celsius until the methanol

evaporated, leaving behind solid crude extract. The extract was stored in a

refrigerator before staining. The solution was then stored in amber bottle to

avoid any light exposure.

 15

Conceptual Framework

Independent Variables Dependent Variables

1. Annatto seed extract

Characteristics of the stained
1.1 100g (44.7%)
organisms with the extract from
1.2 300g (77.4%) Annatto seeds
1.3 600g (89.46%) 1. Intensity of red color
2. Carbol fuchsin stain 2. Clarity of shape
(positive control)
3. Specificity of staining

Figure 3. Conceptual Paradigm of the Study

In this experimental setup, the independent variable was the

concentration of extract derived from annatto seeds, specifically at three

different levels: 100g, 300g, and 600g. This concept was adapted from a study

conducted by Braide et al. (2010), where annatto seed extract was employed

for staining Escherichia coli and Staphylococcus aureus. Although the

mentioned study utilized only 100 grams of the seeds, experimental findings

revealed that the concentration was insufficient. Consequently, they

recommend augmenting the concentration by increasing the quantity of

pulverized annatto seeds. The annatto seeds extract was being explored as an

alternative primary stain for acid-fast bacilli in comparison to the conventional

carbol fuchsin stain. The choice of varying concentrations allowed researchers

to investigate the potential impact of different concentrations of the extract on

the staining process and subsequent visualization of acid-fast bacilli.

The dependent variable also evaluated the use of annatto seed extract

as a primary stain for acid-fast bacilli by assessing various characteristics of the

stained organisms. Firstly, the intensity of the red color produced by the annatto

seed extract served as a crucial parameter. This aspect reflected the

 16

effectiveness of the staining process, with a deeper and more vibrant red color

indicating a more successful and reliable staining outcome.

Secondly, the clarity of shape was another significant factor in the

evaluation. The stain should enable clear visualization of the bacterial

morphology, distinguishing between filamentous and rod-shaped structures.

This assessment contributed to the overall reliability and precision of the

staining method, ensuring that the characteristics of acid-fast bacilli were

accurately represented.

Lastly, the specificity of staining was a critical aspect to consider. The

ideal primary stain should selectively target acid-fast bacilli, ensuring that only

the intended microorganisms are colored. This specificity is essential for

accurate identification and diagnosis, as it minimizes the risk of false-positive

results. By evaluating these three characteristics — intensity of red color, clarity

of shape, and specificity of staining — the study can comprehensively assess

the efficacy of annatto seed extract as a primary stain for acid-fast bacilli

(Department of Health, 2021).

.
 Chapter 2

Methods

This chapter focused on the different methods and specific procedures

used during the study. Research methodology includes specific procedures or

techniques used to identify, select, process, and analyze information about a

topic. It helps evaluate a study's validity and reliability (LibGuides: Research

Support: Research Methodology, n.d.). The laboratory procedure of this study

was focused on testing the potential of the annatto seed extract in staining non-

tuberculosis mycobacteria (NTM).

Study Design

This study used a descriptive, experimental research design to assess

the potentiality of Annatto seed extract as a primary alternative stain in acid-

fast staining. The method used determine the efficacy of the hypothesis and

support its assumption in the study.

This research focused on describing the physical reaction of the stained

organism compared to the control group. Applying the prepared annatto seed

extract serve as the experimental scope of the study to test its effectivity as the

potential primary stain alternative.

Setting

This experimental study involved handling of bacteria hence proper

handling and personal protective equipment were practiced. NTM, are group of

bacteria that causes low-risk infections to human and high-risk only to those
 18

who are immunocompromised patients. Additionally, sputum samples that

tested positive were also used for experimentation. The bacterial specimen was

obtained from the TB Culture Laboratory of the Davao Regional Medical Center,

Apokon, Tagum City, a laboratory that mainly focuses on the growth of

tuberculosis causing bacteria. Smearing technique within a biosafety cabinet

as part of the experimental protocol was performed.

The annatto seeds were collected from the Municipality of Laak

specifically Tawinian, Poblacion Laak, Laak, Davao de Oro (see Figure 4.a).

The place was partially forested and the climate is often warm throughout the

year with high levels of humidity.

The experimentation was done at Tagum Doctors Hospital Laboratory

which was suitable for studying and processing the said specimen (see Figure

4.b). The laboratory was well-ventilated and well-lighted wherein air generally

flows from clean areas to potentially contaminated areas, and then it was

filtered and exhausted to the outside environment after inappropriate

decontamination.
 19

4.a

4.b

Figure 4. Location of the Study. 4a. Tawinian, Poblacion Laak, Laak, Davao de
Oro, Philippines and 4b. Tagum Doctors Hospital Laboratory. Image retrieved
from “Google maps”, “Wikipedia”, and “Tagum Doctors Hospital”
http://surl.li/ntqow and http://surl.li/ntqpn. Copyright 2023 by Wikipedia, Google
Maps, and Tagum Doctors Hospital.
 20

Subjects

Sufficient quantity of Annatto seeds were collected to obtain 1000g of

finely pulverized seeds. Prior to analysis, the authenticity of the seeds was

confirmed by a licensed agriculturist from the faculty of agriculture at the

University of Southeastern Philippines Tagum-Apokon Campus, ensuring the

accuracy and reliability of the research findings (Appendix A.7). Following this

verification, the annatto seeds underwent comprehensive phytochemical

analysis (Appendix E).

Sample of an isolate organism and sputum positive sample was sourced

from the TB Culture Laboratory of Davao Regional Medical Center in Apokon,

Tagum City, under the guidance of experts. These samples, obtained from the

mentioned laboratory was transported to the Tagum Doctors Hospital

Laboratory using triple packaging upon transport and serve as the sample for

the experiment, providing a baseline for comparison with the alternative method

utilizing annatto seeds extract. The control group undergone the smearing

technique within a biosafety cabinet. To ensure safe handling, the researcher

has diligently prepared and consulted the Pathogen Safety Data Sheet (PSDS)

(see appendix B.2).

The manufacturing process of stains used in the Ziehl-Neelsen method

for acid-fast staining involves three key components. First, carbolfuchsin, the

primary stain, is produced by dissolving fuchsin in phenol and adding a small

amount of detergent. This solution is then filtered and stored in a dark container.

Next, acid alcohol, the decolorizing agent, is prepared by mixing concentrated

hydrochloric acid with absolute ethanol. Lastly, methylene blue, the

 21

counterstain, is created by dissolving methylene blue powder in distilled water.

Each stain is carefully manufactured to specific concentrations and

compositions, ensuring their effectiveness in highlighting acid-fast bacilli during

the staining procedure while adhering to safety guidelines for laboratory use.

Instruments

This experimental study used a bright-field microscope that is good for

viewing organisms that use the Zhiel-Neelsen method (Khutlang et al., 2010).

The stained organisms are carefully viewed at 1000x total magnification using

oil immersion for enhancement using OLYMPUS CX23. The microscope

mentioned above is a bright-field microscope equipped with coplanar objectives

(CX23 | Educational Microscope for Students | Olympus LS, n.d.).

The study utilized the Ziehl-Neelsen method, a hot acid-fast staining

technique. This procedure involves three distinct stains: carbolfuchsin, acting

as the primary stain, is applied for 10 minutes with heat as the mordant.

Subsequently, a 3% acid alcohol solution serves as the decolorizer, followed

by methylene blue as the counterstain for 30 seconds to one minute. The

resulting color highlights acid-fast bacilli in red, reflecting the primary stain,

while non-AFB appears blue. Importantly, all reagents used in the process was

handled in strict accordance with the Material Safety Data Sheet (MSDS)

guidelines to ensure safety and accuracy in the experimental process (see

Appendix B.1).

After experimentation a comparison between the controlled sample and

the study sample was done through an evaluation tool (see appendix C). This

evaluation tool is based on the characteristics of the bacteria under the

 22

microscope. The tool involving the use of Likert rating scale was named after

by an American social scientist Rensis Likert. A Likert scale is a reliable method

for measuring opinions, perceptions, and behaviors. It is a 5 or 7-point scale,

often referred to as a satisfaction scale, that ranges from one extreme attitude

to another. Likert scales are popular because they provide quantifiable answer

options, making data analysis easier (Likert Scale: Examples and How to Use

It | SurveyMonkey, n.d.).

As the study involves microscopic examination, a camera was employed

for documentation purposes. The selected camera was stable and specifically

tailored for capturing images under the microscope. The Coolpix P530 is

Nikon's super-zoom bridge camera with a 16.1MP CMOS sensor and ISO

range of 100-6400. It features a 42x optical zoom lens (24-1000mm), Full HD

video recording, and a 3.0" 921k-dot monitor. Designed for enthusiasts, it

includes a DSLR-like mode dial for easy selection of aperture, shutter priority,

and full manual modes (Nikon CoolPix P530 Review | Photography Blog, 2014).
 23

Data Collection Procedure

Figure 5. Data Gathering Procedures

 24

A. Transmittal letter

The researchers obtained approval from the Office of the Research

Director and Dean of the Medical Laboratory Science Department at Tagum

Doctors College, Inc. The initial step involved formally informing the

institution about the planned study through a written communication.

Enclosed within this communication was a succinct overview of the entire

study, detailed in Appendix A.1 and Appendix A.2. This procedural approach

ensured adherence to established academic protocols and ethical

considerations, establishing a formalized framework for the research

endeavor.

B. Transmittal letter to TB Culture Laboratory & Tagum Doctors Hospital

In conducting the study, the researchers sent a transmittal letter to

the head of the laboratory of the TB Culture Laboratory of Davao Regional

Medical Center (Appendix A.3) located at Apokon, Tagum City to seek

approval and coordination for obtaining samples from their laboratory.

Another letter was sent to Tagum Doctors Hospital to conduct the said study

in their hospital laboratory (Appendix A.4). This methodological approach

ensured the acquisition of necessary permissions and cooperation from

relevant entities, adhering to established protocols within the scope of our

research.

C. Collection, Phytochemical Analysis and Extraction of the Dye

The researcher first gathered annatto seeds in Tawinian, Poblacion

Laak, Laak, Davao de Oro. The plant underwent confirmation by a licensed

 25

agriculturist (Appendix H.2). The Annatto seeds were handpicked from the

Annatto tree. The researchers obtained ample amount of the seeds to get

the desired total of 1000 grams of powdered seeds, since the study employs

a triplicate comparison with different concentrations.

Before the plant was subjected to pulverization and extraction

methods, a preliminary step involved conducting a thorough phytochemical

analysis to identify the precise chemical components present in the seeds.

This analytical process was carried out at WVN Research Laboratory,

Bangkerohan, Davao City, ensuring accuracy and reliability in capturing the

comprehensive phytochemical profile of the annatto seeds (Appendix E.1).

In the study by Venturina et al. (2020), annatto seeds were separated

by hand and then washed using distilled water. It was sterilized in a hot air

oven at 60 degrees Celsius for 24 hours. Using a blender, the seeds were

crushed to acquire 100g, 300g, and 600g of pulverized seeds. The

extraction of the different masses was done by adding 1 liter of methanol to

the pulverized seeds and stirring for 12 hours using a magnetic stirrer. The

mixture was stored without sunlight at room temperature for another 12

hours. Filter paper (Whatman Filter Paper No.1) was used, and the mixture

was placed in an IKA RV10 Rotary Evaporator at 40 degrees Celsius until

the methanol evaporates, leaving behind solid crude extract. The extract

remaining was stored in a refrigerator before staining. The solution was then

stored in amber bottles to avoid any light exposure (Appendix B).

After filtering the macerated solution through Whatmann Filter Paper

No.1, it was subjected to a Rotary Evaporator to remove the methanol in the

 26

solution and obtained pure solid crude extract. The yield was 63.2g of pure

solid crude extract from 600g, 67.8g from 300g, and 144.7g from 100g.

Following the method described by Sep (2016), the percentage yield of

crude dye was calculated using Equation 1, which relates to the original

weight of the dye material used for extraction. The equation is as follows:

(𝑊&' − 𝑊)' )
𝑊"# = 𝑥 100
𝑊&'

Figure 6. Concentration Formula

Where Wdy is the percentage yield of crude dye, Wbe is the dye

material weight before extraction in grams, and Wae is the crude solid dye

material weight after evaporation of extracts in grams. All weighing was

obtained using an electronic digital balance with an accuracy of 0.0001

grams.

The percentage yield of crude dye varied with the amount of dye

material used: for 100g, it was -44.7%, the yield for 100g of dye material

was recalculated to -1 multiplied by the initial percentage, resulting in a

positive value of 44.7%; for 300g, the yield was 77.4%; and for 600g, it

reached 89.47%, showing a higher efficiency in larger quantities.

D. Obtaining of Non-tuberculous mycobacteria (NTM)

The test organisms, NTM, were obtained from the TB Culture

Laboratory of Davao Regional Medical Center located at Apokon, Tagum

City. The specimen collected was transported to Tagum Doctors Hospital

Laboratory located in Rabe St., Visayan Village,Tagum City, Davao del

Norte. Triple packaging system was employed to ensure that the organism

would not be exposed to the external environment (Appendix G.6).

 27

E. Preparation of the Subjects

Personal protective equipment, including lab gowns, safety goggles,

gloves, and masks, were consistently worn in the laboratory (Appendix G.2).

Sealed containers with suitable labels were utilized for the storage of

solvents and bacteria to facilitate easy identification. Prior to handling NTM,

the materials were sterilized using an autoclave temperature of 121-131

Degrees Celsius for 15-30 minutes (Hossain et al., 2012). The handling of

bacteria was performed in a biosafety level 2 laboratory. The bacterial cell

cultures were appropriately labeled and placed in an incubator for storage

(Appendix G.6).

The reagents designated for staining purposes were meticulously

prepared in proximity to the laboratory sink. Furthermore, the primary stain

alternatives were meticulously stored in different amber glass containers,

each container reserved for specific concentrations 44.7%, 77.4%, and

89.4%.

For staining, the researchers prepared a total of 24 slides, dividing

them equally between isolates from a subculture of NTM and sputum

samples. Each group of 12 slides included three control slides, which were

subjected to staining using the traditional Ziehl-Nelseen method.

 28

7.a 7.b

Figure 7. Flowchart outlining the smearing process employed for the slides

using the bacterial isolates and slides using sputum samples.

F. Staining

Triplicates of the experimental setup and one for the control group

were made for the analysis(see Appendix B4.1 and B4.2). In scientific

experiments, the inclusion of triplicates holds significance as it serves to

authenticate empirical data or observed outcomes. Typically, this research

 29

incorporates three replicates to ensure the credibility and verification of the

results obtained from them (Biology Online, 2022).

F.1 Control group

The Ziehl-neelsen method of staining the NTM was used

(Appendix F). This procedure involves three distinct stains:

carbolfuchsin, acting as the primary stain, is applied for 10

minutes with heat as the mordant. Subsequently, a 3% acid

alcohol solution serves as the decolorizer, followed by methylene

blue as the counterstain for 5 seconds (Department of Health,

2021).

F.2 Test group

A thin film of sample was air dried and then heat fixed. The

slides were flooded with annatto seeds dye, then slides were

steamed using a lamp over the sink. Slides were set for 10

minutes followed by rinsing with water. Slides were then

decolorized using 3% acid alcohol for 3 minutes then rinsed with

water. Slides were then flooded with 0.1% methylene blue for 5

seconds as a counterstain and then rinsed with water. It was

allowed to dry and prepared for microscopic examination using oil

immersion objective (Instructions: Acid-Fast Staining, n.d.).

 30

8.a 8.b
Figure 8. Staining method for both 8.a control and 8.b experimental sample.

G. Examination and validation

To establish the efficacy of the primary stain alternative, three

validators were engaged for the study (Appendix A.8). These validators,
 31

registered medical technologists specializing in the microscopic

examination of Acid-Fast Bacilli (AFB), conducted evaluations on both the

control and test groups. Each validator meticulously examines three slides

for each concentration within the test group (Appendix G.9). The

assessment of potency was facilitated through the use of an evaluation tool,

details of which can be found in our provided Appendix C.2.

H. Interpretation of Data

The recorded results during the experimentation were used for data

analysis using both descriptive and inferential statistics to prove its

effectivity. Enhancing the credibility of our findings, three validators, each

registered medical technologists specializing in the relevant field and duly

trained for this specific study, utilized a rigorous evaluation tool (Appendix

C). This tool was specifically designed to assess and measure the

effectiveness of the results obtained. Their collective input and expertise

enhanced the credibility and validity of the data analysis process,

contributing to the overall robustness of our study.

Data Analysis

Proving the potential of the alternative stain required the use of both

descriptive and inferential statistics. This research study utilized the Statistical

Package for the Social Sciences version 29.0.10, a software that automatically

analyzed data (ICAR-Central Marine Fisheries Research Institute, n.d.). The

statistician interpreted the results of the study.

 32

This study employed ANOVA for robust statistical analysis to evaluate

the efficacy of the alternative primary stain. The evaluation tool was utilized to

derive values, which, in turn, was used to calculate the mean of all evaluators

to obtain an average value within the parameter limits (Appendix C.2).

Employing ANOVA, the study aimed to ascertain whether one of the stain

concentrations surpasses the others. T-test was used to compare control and

the possible one concentration that has the potential to stain AFB.

Ethical Consideration

Approval in accordance with the ethical standards for the use of the

Laboratories in Research was provided by the laboratory director of TDCI.

Approval was also obtained from the Laboratory head of TDH, Research

Adviser, Department of Environment and Natural Resources, and Research

Director of Tagum Doctors College Inc. for the accomplishment of the study.

Safety protocols (e.g., wearing proper personal protective equipment,

safety precautions, and avoiding hazards) were followed during the conduct of

the experiment. Reagents used for this experiment have product information,

precautionary labeling, first aid measures, disposal considerations, transport

information, handling and storage for it may also be hazardous when used.

Since the study includes handling of an organism specifically the NTM

which requires biosafety laboratory 2, Biosafety Level 2, was employed for

containment measures and practices designed to ensure the safe handling of

moderate-risk biological agents in laboratory settings. Laboratories designated

 33

as BSL2 are suitable for work involving agents that pose a moderate hazard to

personnel and the environment (CDC LC Quick Learn: Recognize the Four

Biosafety Levels, n.d.). The researchers selected an appropriate laboratory in

accordance with the required biosafety level for handling the organism.

Researchers underwent fit testing to ensure that respirators fit tightly to

their faces, protecting them from inhaling infectious aerosols containing

Mycobacterium tuberculosis and other airborne pathogens (Appendix G.1).

This measure was crucial for the safety of the researchers and the integrity of

the experimentation process (Respiratory Protection | Fact Sheets |

Publications & Products | TB | CDC, n.d.).

To manage the disposal of reagents, organisms, and used disposable

materials, the proponents adhered to the prescribed procedures outlined in both

the Pathogen Safety Data Sheet (PSDS) and Safety Data Sheet (SDS) as

detailed in Appendix B. Contaminated materials was securely sealed in bags

and promptly incinerated. Alternatively, infectious waste was collected in

autoclavable bags, subjected to autoclaving, and subsequently incinerated.

This rigorous disposal protocol ensured the safe and responsible handling of

materials, aligning with established safety guidelines for the protection of both

personnel and the environment.

The proponents, with their expertise and commitment to scientific rigor,

bring a wealth of knowledge and experience to the project. Their professional

backgrounds and adherence to ethical standards established a solid foundation

for the research's credibility. Additionally, the validators, registered medical

technologists specialized in the pertinent field, further bolster the study's

 34

trustworthiness. Their proficiency in microscopic examination of Acid-Fast

Bacilli (AFB) and dedication to precision contribute to the validity of the findings.

The combined commitment to transparency, ethical conduct, and expertise in

the subject matter positions both the proponents and validators as reliable

contributors to the scientific process, ensuring the study's outcomes can be

confidently accepted and applied within the relevant scientific community.

 Chapter 3

Results

This chapter focuses on the results of the data retrieved. The

researchers obtained the following results from a phytochemical analysis

conducted by WVN Testing and Research Laboratory (Appendix E). The ratings

in table 2 onwards were provided by evaluators trained in AFB microscopy.

Table 1
The Phytochemical Analysis of the Bixa Orellana (Annatto Seeds)
Test Parameter Results

pH (1:10) 3.5

Carotenoid, (as bixin) % 75.4

Note: The carotenoid (bixin) percentage yield was out of 100%.

Table 1 shows that annatto seeds possess a significantly higher

concentration of bixin, the principal coloring pigment found in the plant. The

analysis revealed that out of the total extract, an impressive 75.4% was

comprised of the bixin component found in Table 1. Additionally, pH was tested

resulting to an acidic pH of 3.5.

 36

Table 2

Table 2.1 The Staining Characteristics of AFB with Annatto Seed Extract (100g)

Parameters Result Descriptive

Mean SD Equivalent
a. Intensity of 2.0 0.9 Poor
the Red Color
b. Clarity of 2.3 1.3 Poor
Shape
c. Specificity in 3.0 1.7 Acceptable
Staining

Table 2.1 shows the staining characteristics of Acid-Fast Bacilli (AFB)

using a 100g or 44.7% concentration, evaluated across three parameters. The

intensity of the color yielded a mean descriptive equivalent of "poor" at 2,

indicating room for enhancement. Similarly, the clarity of the shape also

received a "poor" rating, with a mean of 2.3, suggesting a need for improvement

in this aspect. However, the specificity of the staining exhibited the highest

mean among all experimental samples, at 3, signifying an "acceptable"

descriptive equivalent and highlighting a strength in this area.

Table 2.2 The Staining Characteristics of AFB with Annatto Seed Extract (300g)

Parameters Result Descriptive

Mean SD Equivalent
a. Intensity of 1.3 0.5 Very Poor
the Red Color
b. Clarity of 2.3 1.3 Poor
Shape
c. Specificity in 2.3 2.0 Poor
Staining

Table 2.2 presents the staining characteristics of Acid-Fast Bacilli (AFB)

using a 300g or 77.4% concentration. In this analysis, the intensity of the red
 37

color was found to have a "very poor" descriptive equivalent, with a mean score

of 1.3, indicating a critical need for improvement in achieving a more robust

coloration. The clarity of the shape also showed a "poor" descriptive equivalent,

with a mean of 2.3, suggesting a need for refinement in this aspect. The final

parameter, which was not explicitly defined, also received a mean score of 2.3,

reflecting an overall "poor" descriptive equivalent and highlighting areas for

further optimization in the staining process.

Table 2.3 The Staining Characteristics of AFB with Annatto Seed Extract (600g)

Parameters Result Descriptive

Mean SD Equivalent
a. Intensity of 1.3 0.5 Very Poor
the Red Color
b. Clarity of 1.3 0.5 Very Poor
Shape
c. Specificity in 2.4 1.9 Poor
Staining

Table 2.3 details the staining characteristics of Acid-Fast Bacilli (AFB)

using a 600g or 89.46% concentration. The intensity of the red color and the

clarity of shape both received a mean of 1.3, indicating a "very poor" descriptive

equivalent for both parameters. This suggests a critical need for improvement

in achieving a more vivid coloration and clearer shape definition. Additionally,

the specificity in staining was rated as "poor," with a mean of 2.4, indicating an

area that could benefit from further refinement in the staining process.
 38

Table 2.4 The Staining Characteristics of AFB with Positive Control

Parameters Result Descriptive

Mean SD Equivalent
a. Intensity of 4.7 0.6 Very Good
the Red Color
b. Clarity of 4.7 0.6 Very Good
Shape
c. Specificity in 4.7 0.6 Very Good
Staining

Table 2.4 presents the staining characteristics of carbol fuchsin as a

primary stain. The data reveals a mean score of 4.7, indicating a high level of

consistency in the evaluations. Descriptively, this score corresponds to a rating

of "very good," suggesting that carbol fuchsin demonstrates strong staining

characteristics across the assessed parameters.

Table 3

The Significant Difference of Using Various Concentrations of Annatto Seed

Extract as Primary Stain Alternative for Acid-Fast Organism

Concentration Mean p η² Decision on Decision on

Difference 𝑯𝟎
α= 0.05
100g (44.7%) 2.444
300g (77.4%) 2.000 0.4 0.3 Not significant Not rejected
600g (89.46%) 1.703

Table 3 illustrates the comparison of staining characteristics among

three different concentrations (100g, 300g, 600g) of the staining solution. The

calculated p-value of 0.4 exceeds the predetermined level of significance of

0.05. This indicates that there is no significant difference in staining

characteristics among the three concentrations. Therefore, the null hypothesis,

 39

which posits that there is no difference in staining characteristics based on

concentration, is not rejected.

Table 4

The Significant Difference Between the Annatto Seed Extract and Carbol

Fuchsin (positive control) on their Effectivity in the Primary Staining of Acid-Fast

Organism

Independent Mean SD CV t p Decision Decision

Variables on on 𝑯𝟎
Difference
α= 0.05
Annatto 2.4 0.8 0.3
Seed Extract
(100g) 4.4 <.001 Significant Rejected
Carbol 4.7 0.6 0.1
fuchsin stain

Table 5 highlights a notable contrast between the annatto seed extract

concentration of 100g or 44.7% and carbol fuchsin, the positive control utilized

as the primary stain in acid-fast staining. The rejection of the null hypothesis is

supported by a p-value of less than 0.01, which falls below the predetermined

significance level of 0.05. This outcome strongly suggests a substantial

difference between the two independent variables in their ability to stain acid-

fast bacilli.
 Chapter 4

Discussion

The discussion of the study is based on the results of the phytochemical

analysis of B. orellana (annatto) seeds and the microscopic examination of both

cultured isolates and sputum within the control and experimental sample groups

(Appendix E). This analysis encompasses the evaluation of the annatto seed

extract's potential as a primary stain alternative in acid-fast staining.

The Phytochemical Analysis of Bixa orellana (annatto) seeds extract

determining its bixin component and the pH of the pure solid crude

extract.

The phytochemical analysis of annatto seed extract was done by

macerating the seeds in 1L of methanol and then evaporating the solution using

a rotary evaporator to obtain the pure extract.

Rd (2019) highlighted the significance of the apocarotenoid bixin, a

pigment, as the most abundant component in the seed. This observation is

supported by the trial conducted by the WVN Testing and Research Laboratory,

which reported a carotenoid content of 75.4% in the extract, indicating the

prevalence of bixin. Additionally, Shahid-UI-Islam et al. (2016) identified bixin

as the major component of annatto seeds in their study. According to Raju et

al. (2023), the seeds have a high carotenoid content, with bixin making up to

80% of the total pigment in some seeds. Bixin was the first cis-carotenoid to be

isolated from natural sources.

The high concentration of bixin in the extract suggests its potential as a

staining agent, given its coloring properties. Raddatz-Mota et al. (2016)

 41

conducted a comprehensive investigation into the coloring components of

annatto seeds, confirming that bixin produces a red color similar to carbol

fuchsin, a primary stain used in staining procedures.

Carbol fuchsin, a lipid-soluble stain, attaches or binds to the mycolic acid

cell wall of Acid-Fast Bacilli (AFB), as it is a major lipid component unique to

the AFB cell wall. Bixin, as the major component of annatto seed extract, is also

oil-soluble, similar to carbol fuchsin (Abud & Simon, 2022). This property of the

seed extract strongly supports its staining capacity, suggesting its potential as

a primary stain alternative in acid-fast staining techniques.

Furthermore, the pH compatibility of bixin (pH 3.5) with carbol fuchsin (3

to 4 pH) falls within the optimal range for effective staining solutions. The pH of

the staining solution directly impacts the dye's ability to dye a certain element

(Factors That Affect Dye Binding - LabCE.com, Laboratory Continuing

Education, n.d.). This alignment with established commercial stains

underscores the viability of our alternative stain, presenting it as a compelling

option for staining procedures.

The Staining Characteristics of the AFB with Annatto Seed Extract, and

the Positive Control

The staining characteristics of both annatto seed extract and the carbol

fuchsin as positive control to AFB were evaluated in terms of the intensity of the

red color, clarity of shape (filamentous or rod-shaped), specificity in staining

(stains only AFB). A parameter limit was used to determine the descriptive

equivalent seen in Appendix C.

The first concentration which was 100g or 44.7% result shows a more

staining capacity compared to other concentration. Both the intensity of the red
 42

color and the clarity of shape has a “poor” descriptive equivalent. This means

that low red color intensity with unclear shapes, and filamentous or rod-shaped

structure are somewhat visible based on its interpretation. The specificity of

staining parameter exhibits acceptable specificity, indicating a satisfactory

result.

The 300g or 77.4% concentration shows a “very poor” descriptive

equivalent on the first parameter which means that minimal red color intensity

was seen during the microscopic examination. Both clarity of shape and

specificity in staining has a “poor” descriptive equivalent meaning unclear

shapes, filamentous or rod-shaped structures are barely discernable and AFB

lacks specificity, suggesting an unreliable result.

Lastly, the highest concentration tested, 600g or 89.46%, exhibited a

lesser staining capacity. Both the intensity of the red color and the clarity of the

shape were rated as "very poor." This means that minimal red color intensity

with unclear shapes, and filamentous or rod-shaped structures are barely

discernible. The last parameter has a “poor” descriptive equivalent showing

poor specificity, indicating a less reliable outcome.

Among the experimental sample concentrations 100g (44.7%), 300g

(77.4%), and 600g (89.46%) of the annatto seed extract, the lowest

concentrations show more capacity to stain compared to the others. The

specificity in staining parameter shows an acceptable descriptive equivalent,

and the only one concentration who exhibit this descriptive equivalent.

Interestingly, the highest concentration tested, 600g or 89.4%, exhibited a

lesser staining capacity than the 100g concentration. This unexpected result
 43

could be attributed to the concentration being too high, leading to potential

saturation effects or interference with the staining process.

The specificity of the annatto seed extract stain was deemed acceptable,

as it did not interfere with the color reaction of the methylene blue counterstain,

which is crucial for distinguishing acid-fast bacteria from non-acid-fast bacteria.

This lack of interference indicates its potential as a staining agent with

acceptable specificity. The importance of specificity in acid-fast staining,

highlighted by Winn et al. (1994) and Forbes, Sahm, and Weissfeld (2007), is

crucial for accurately identifying Mycobacterium species. Specificity is achieved

through stains like carbol fuchsin, which target the unique cell wall components

of acid-fast bacteria, enabling their differentiation from other microorganisms in

clinical and laboratory settings.

This evaluation suggests that while annatto seed extract shows potential

as a staining agent, its current formulation and concentrations result in

suboptimal staining characteristics compared to carbol fuchsin. Further

refinement of the extraction process or exploration of different concentrations

may enhance its efficacy as a viable alternative stain for AFB.

The staining characteristics of Acid-Fast Bacilli (AFB) using carbol

fuchsin as the positive control, all parameters, including the intensity of the red

color, clarity of shape (filamentous or rod-shaped), and specificity in staining

(staining only AFB), have a mean of 4.667, corresponding to a descriptive

equivalent of "very good" (Appendix C.2).

This suggests that the commercially available primary stain, carbol

fuchsin, is highly effective in staining AFB organisms, providing a benchmark

 44

against which the staining characteristics of annatto seed extract can be

compared.

The Significant Difference of Various Concentrations (100g, 300g, 600g)

of Annatto Seed Extract as a Primary Stain Alternative for Acid-Fast

Organism

The lowest concentration, 100g or 44.7%, exhibits a mean of 2.444,

yielding a more acceptable result compared to other concentrations. The first

null hypothesis of the study, which posited no significant difference among the

concentrations in staining AFB, has not been rejected. This is supported by a

p-value of 0.406, which is greater than the set level of significance of 0.05

obtained through ANOVA. Additionally, the 26% variance or changes in the

outcome can be explained by the concentrations of the extract.

Therefore, the researchers concluded that the study recommendation by

Braide et al. (2010) to use a higher concentration is not feasible or might be

ineffective, same goes to the method used by the researchers according to

Venturina et al. (2020). This highlights the importance of optimizing the

concentration of annatto seed extract for effective staining of AFB, with the

lowest concentration showing promising results in this study.

The Significant Difference Between Annatto Seed Extract and Carbol

Fuchsin (Positive Control) on their Effectivity in the Primary Staining of

Acid-Fast Organism

The significant difference in the staining capacity between the 100g or

44.7% concentration of the annatto seed extract and carbol fuchsin as the

primary stain. Both samples underwent a T-test, resulting in a p-value of 0.01.

 45

This p-value is less than the set level of significance of 0.05, leading to the

rejection of null hypothesis number 2. This indicates a significant difference

between annatto seed extract and carbol fuchsin (positive control) in their

effectiveness in the primary staining of acid-fast organisms. Specifically, the

positive control, carbol fuchsin, is shown to be far more effective compared to

annatto seed extract.

These findings underscored the need for further refinement and

optimization of annatto seed extract as a staining agent. While it may not be as

effective as carbol fuchsin in its current form, additional studies could explore

ways to enhance its staining capacity, potentially through modifications to

concentration, extraction techniques, or combination with other substances to

improve its efficacy. Considering its natural origin and potential cost-

effectiveness, the exploration of annatto seed extract as a viable alternative

stain for acid-fast organisms remains a promising avenue for future research.
 Chapter 5

Conclusion and Recommendations

In this chapter, conclusions and their significance are presented.

Additionally, recommendations are provided which can be useful for future

research related to the findings of the study.

Conclusion

Based on the results of the study, the researchers conclude the following:

1. The comprehensive phytochemical analysis and experimental

evaluations conducted on Bixa orellana (annatto) seeds, particularly

focusing on its bixin component, affirm the potential of annatto seed

extract as an effective alternative primary stain in acid-fast staining

procedures. The findings, showing a high concentration of bixin (75.4%)

and favorable pH (3.50) compatibility with carbol fuchsin, support the

extract's suitability for use in staining applications. The consistent

evidence from various studies highlighting bixin as a significant

component enhances the credibility of this natural extract as a viable

staining agent. This alternative staining method could provide a cost-

effective and environmentally friendly option in microbiological and

pathological laboratories.

2. In this study, significant differences were observed in the staining

characteristics of Acid-Fast Bacilli (AFB) when comparing annatto seed

extract to the positive control, carbol fuchsin. While the annatto seed

extract showed potential as a staining agent, particularly at lower

concentrations, its staining capacity was generally inferior to that of carbol

 47

fuchsin. The lower concentrations of annatto seed extract demonstrated

better staining capacity compared to higher concentrations, suggesting

an optimal range for effective staining. However, even at the most

effective concentration, the staining capacity of annatto seed extract was

rated as "poor" to "very poor" in terms of red color intensity, clarity of

shape, and specificity in staining, indicating room for improvement.

Therefore, this indicates that there is a significant difference between

using Annatto seeds and the positive control for the staining

characteristics of AFB organisms.

3. There was no significant difference of various concentrations of the

Annatto seeds in staining AFB. Three concentrations were used: 44.7%

(100g), 77.4% (300g), and 89.47% (600g). The evaluation done by the

evaluators showed that the lower the concentration is, the higher the

intensity of the red color will be, but it is not effective in staining acid-fast

organisms. And based on our statistical results, since the p-value of 0.406

exceeds the significance threshold of 0.05, we do not reject the null

hypothesis. This indicates that there is no significant difference between

using different concentrations (100g, 300g, 600g) of Annatto seed extract

as an alternative primary stain for acid-fast organisms. Consequently, it

suggests that the different concentrations of Annatto seed extract

produce the same results.

4. The research findings demonstrate a significant difference in the staining

capacity between annatto seed extract and the positive control, carbol

fuchsin, for Acid-Fast Bacilli (AFB). The highest possible concentration of

annatto seed extract was found to be less effective than carbol fuchsin,
 48

as evidenced by the lower red color intensity and clarity of shape

observed in microscopic examination. This outcome, supported by a p-

value of 0.01, signifies a clear distinction in staining efficacy between the

two agents. While carbol fuchsin proved to be more effective in staining

AFB, the study highlights the potential of annatto seed extract as a

staining agent, especially at lower concentrations where it showed more

promising results. This indicates further that, as compared to the Annatto

seed extract, the positive control is considerably more effective.

Recommendations

Based on the data and conclusions of this study, the researcher

suggests the following:

1. In-depth studies are imperative to unveil the mechanisms of action

underlying the coloring properties and alcoholic content of B. orellana

(annatto). Understanding these mechanisms could reveal if the active

compounds have the ability to penetrate the mycolic cell wall of acid-fast

bacilli, potentially enhancing the efficacy of natural plant extracts.

Specifically, run an iodoform test to determine the carbonyl compounds

of the seed extract. Such studies would not only improve our

understanding of these extracts but also pave the way for developing

novel approaches in combating diseases caused by these pathogens.

2. Further trials should be undertaken to determine the optimal method for

utilizing the plant extract as a stain. Given its promising properties,

exploring various application methods could unlock the full potential of

 49

the extract as a primary stain alternative. This experimentation could

lead to the development of innovative staining techniques, offering more

efficient and cost-effective solutions in laboratory settings.

3. The formulation of the pure solid crude extract can be fine-tuned to

maximize its staining capacity. Future research endeavors could focus

on optimizing the extract's formulation by combining it with other

solutions that enhance its staining ability. This approach could result in

the development of a highly effective and versatile staining agent,

suitable for a wide range of laboratory applications.

4. Experimenting with the extract as a potential histological or

microorganism stain holds significant promise. This exploration could

provide insights into the extract's ability to stain various microscopic

samples, opening up new avenues for research and application. If

successful, this could revolutionize staining techniques in histology and

microbiology, offering new tools for researchers and practitioners alike.

5. Modifying the staining process, such as increasing the penetration time

of the primary stain, could significantly impact the staining capacity of

the extract. By extending the duration of contact between the extract and

the sample, there is a greater opportunity for the active compounds to

interact with the target structures, potentially enhancing the staining

efficiency. This modification could prove to be a simple yet effective way

to optimize the use of the extract as a staining agent, warranting further

investigation and experimentation.

6. Pure isolation of the bixin component of the annatto seed might also give
 50

more comparable results against the control, which is carbol fuchsin.

Bixin, a major carotenoid found in annatto, has shown promising staining

properties in previous studies. Isolating and purifying bixin from annatto

seeds could enhance its effectiveness as a staining agent by

concentrating the active component responsible for its dyeing properties.

This targeted approach could lead to a more consistent and reliable

staining performance, potentially matching or even surpassing that of

carbol fuchsin. A study by McCullagh and Ramos (2008) demonstrated

the potential of bixin in various applications, suggesting that its isolated

form could provide a more potent alternative for staining AFB. The

beforementioned study uses thin-layer and column chromatography for

separating the carotenoid bixin component from Annatto seeds.

7. Lyophilizing the seed extract could improve its stability and efficacy as a

staining agent. Lyophilization, or freeze-drying, helps in preserving the

bioactive compounds, ensuring consistent results in staining

applications. By removing water content through sublimation,

lyophilization stabilizes the extract, preventing degradation and

extending its shelf life. This method could enhance the practicality and

usability of annatto extract in laboratory settings, offering a more reliable

and robust staining solution. According to Quiroz et al. (2019),

lyophilization has been shown to preserve the functional properties of

bioactive compounds, making it an ideal method for preparing staining

agents from natural extracts. This process could ensure that the annatto

stain maintains its effectiveness over time, providing a consistent and

high-quality alternative to traditional staining methods.

 51

References

A Case of Carbol Fuchsin Poisoning Successfully Treated with Hemoperfusion

and Continuous Venovenous Hemodiafiltration. (2011).
https://www.krcp-ksn.org/journal/view.php?number=5927

Abud, E. M., & Simon, R. (2022). Provisional chapter title: food additives and
reactions: part 1. In Elsevier eBooks. https://doi.org/10.1016/b978-0-
323-96018-2.00009-2

Abu-Zurayk, R., Khalaf, A., Abbas, H. A., Nasr, R. A., Jamil, T. S., & Bawab, A.
A. (2021). Photodegradation of Carbol Fuchsin Dye Using an
Fe2−xCuxZr2−xWxO7 Photocatalyst under Visible-Light Irradiation.
Catalysts, 11(12), 1473. https://doi.org/10.3390/catal11121473

Ahmed, S., Moni, B. M., Ahmed, S., Gomes, D. J., & Shohael, A. M. (2020).
Comparative phytochemical, antioxidant, and antibacterial study of
different parts of Doigota plants (Bixa orellana L.). Bulletin of the National
Research Centre/Bulletin of the National Research Center, 44(1).
https://doi.org/10.1186/s42269-020-00349-1

American Lung Association. (n.d.). Learn about Nontuberculous Mycobacteria

(NTM). https://www.lung.org/lung-health-diseases/lung-disease-
lookup/nontuberculous-mycobacteria/learn-about-nontuberculosis-
mycobacteria

Ayobami, F. S. (2019). Staining Interactions of Bixa Orellana (Annatto) Seed

Extracts with Tissues of Wistar Rats. Annals of Advanced Biomedical
Sciences, 2(2). https://doi.org/10.23880/aabsc-16000144

Badar, R. a. D., Carmona, J. L. R., Collantes, J. G. C., Lojo, D. R., Ocampo, S.

M. B., Ursua, R. L. C., & Bercede, D. H. (2022). Staining capability of
plant extracts for the identification of gram-positive and gram-negative
bacteria: a systematic review. Asian Journal of Biological and Life
Sciences, 11(2), 276–284. https://doi.org/10.5530/ajbls.2022.11.37

Bindyalaxmi, K., Kumaran, K., Divya, M., Vennila, S., Raveendran, M., Radha,
P., & Priyanka, V. (2022). Pigment and oil content estimation in seeds of
Bixa orellana L. Pharma Innovation, 11(8), 419–423.
https://doi.org/10.22271/tpi.2022.v11.i8e.14679

Biology Online. (2022, February 24). Triplicate Definition and Examples -

Biology Online Dictionary. Biology Articles, Tutorials & Dictionary Online.
https://www.biologyonline.com/dictionary/triplicate

Braide, W., Akobundu, C., Nwaoguikpe, R. N., & Njiribaeko, L. C. (2010). The
use of extracts from four local Nigerian plants for the staining of selected
bacteria and moulds. African Journal of Microbiology Research, 5(1),
79–86. https://doi.org/10.5897/ajmr10.906
 52

CDC LC Quick Learn: Recognize the four Biosafety Levels. (n.d.).

https://www.cdc.gov/training/quicklearns/biosafety/

Cunha, F. G., Santos, K. G., Ataíde, C. H., Epstein, N., & Barrozo, M. a. S.
(2008). Annatto powder production in a Spouted Bed: an experimental
and CFD study. Industrial & Engineering Chemistry Research, 48(2),
976–982. https://doi.org/10.1021/ie801382d

CX23 | Educational Microscope for Students | Olympus LS. (n.d.).

https://www.olympus-lifescience.com/en/microscopes/upright/cx23/

Dallas, M. E. (2024, May 16). What is tuberculosis? Symptoms, causes,

diagnosis, treatment, and prevention. EverydayHealth.com.
https://www.everydayhealth.com/tuberculosis/guide/

Department of Health. (2021). National Tuberculosis Control Program Manual

of Procedures (6th ed.). https://doh.gov.ph/

Domingues, J. T., Orlando, R. M., Sinisterra, R. D., Pinzón-García, A. D., &

Rodrigues, G. D. (2020). Polymer-bixin nanofibers: A promising
environmentally friendly material for the removal of dyes from water.
Separation and Purification Technology, 248, 117118.
https://doi.org/10.1016/j.seppur.2020.117118

Factors that affect dye binding - LabCE.com, Laboratory Continuing Education.

(n.d.).
https://www.labce.com/spg363068_factors_that_affect_dye_binding.as
px

Forbes, D. F. S. B. A., & Trevino, P. B. E. A. (2007). Bailey & Scott’s diagnostic

microbiology (Issue 1). http://dlib.sbmu.ac.ir/site/catalogue/124481

Gupta, V., Mittal, A., Gajbe, V., & Mittal, J. (2008). Adsorption of basic fuchsin
using waste materials—bottom ash and deoiled soya—as adsorbents.
Journal of Colloid and Interface Science, 319(1), 30–39.
https://doi.org/10.1016/j.jcis.2007.09.091

Hartika, G., Zulharmita, Z., & Asra, R. (2021). Utilization of Natural dyes
Substances for histological staining: a review. Asian Journal of
Pharmaceutical Research and Development, 9(1), 149–158.
https://doi.org/10.22270/ajprd.v9i1.925

Hirko, B., & Getu, A. (2022). Bixa Orellana (Annatto Bixa): A review on use,
structure, extraction methods and analysis. Journal of Agronomy,
Technology and Engineering Management.
https://www.fimek.edu.rs/downloads/casopisi/jatem/issue/v5_1/2._Hirko
_and_Getu_2022_5(1)_687-696.pdf

Hossain, M. S., Balakrishnan, V., Rahman, N. N. N. A., Sarker, M. Z. I., & Kadir,
M. O. A. (2012). Treatment of clinical solid waste using a steam
autoclave as a possible alternative technology to incineration.
International Journal of Environmental Research and Public
 53

Health/International Journal of Environmental Research and Public

Health, 9(3), 855–867. https://doi.org/10.3390/ijerph9030855

ICAR- Central Marine Fisheries Research Institute. (n.d.). IBM SPSS Statistics:
An overview - CMFRI Repository. http://eprints.cmfri.org.in/17178/

Instructions: Acid-Fast staining. (n.d.).

https://www.atsu.edu/faculty/chamberlain/Website/lab/idlab/acidst.htm

Khutlang, R., Krishnan, S., Dendere, R., Whitelaw, A., Veropoulos, K.,
Learmonth, G. M., & Douglas, T. S. (2010). Classification of
Mycobacterium tuberculosis in Images of ZN-Stained Sputum Smears.
IEEE Transactions on Information Technology in Biomedicine, 14(4),
949–957. https://doi.org/10.1109/titb.2009.2028339

Koli, P. B., Kapadnis, K. H., & Deshpande, U. G. (2019). Transition metal

decorated Ferrosoferric oxide (Fe3O4): An expeditious catalyst for
photodegradation of Carbol Fuchsin in environmental remediation.
Journal of Environmental Chemical Engineering, 7(5), 103373.
https://doi.org/10.1016/j.jece.2019.103373

LibGuides: Research Support: Research Methodology. (n.d.).

https://libguides.wits.ac.za/c.php?g=693518&p=4914913

Likert scales: Examples, tips, and how to analyze the data. (n.d.).
SurveyMonkey. https://www.surveymonkey.com/mp/likert-scale/

McCullagh, J. V., & Ramos, N. (2008). Separation of the Carotenoid Bixin from
Annatto Seeds Using Thin-Layer and Column Chromatography. Journal
of Chemical Education, 85(7), 948. https://doi.org/10.1021/ed085p948

Mokobi, F. (2022, September 5). Ziehl-Neelsen Staining- Principle and

Procedure with Results. Microbe Notes. https://microbenotes.com/ziehl-
neelsen-staining/

MS, A. M., & Raymond, J., PhD. (2022, January 18). ACID-FAST STAIN.
Pressbooks. https://open.maricopa.edu/myfirstbook/chapter/acid-fast-
stain/

Nardell, E. A. (2022, July 15). Nontuberculous mycobacterial infections. MSD

Manual Professional Edition.
https://www.msdmanuals.com/professional/infectious-
diseases/mycobacteria/nontuberculous-mycobacterial-infections

Nikon CoolPix P530 Review | Photography Blog. (2014, May 27).

Photographyblog.
https://www.photographyblog.com/reviews/nikon_coolpix_p530_review

OFFSET by Shutterstock. (n.d.). https://www.offset.com/photos/close-up-on-

annatto-tree-can-be-use-for-dye-fabric-in-red-color-740974#
 54

Phelan, J. E., Lim, D. R., Mitarai, S., De Sessions, P. F., Tujan, M. a. A., Reyes,
L. T., Medado, I. a. P., Palparan, A. G., Naim, A. N. M., Jie, S., Segubre-
Mercado, E., Simoes, B., Campino, S., Hafalla, J. C., Murase, Y.,
Morishige, Y., Hibberd, M. L., Kato, S., Ama, M. C. G., & Clark, T. G.
(2019). Mycobacterium tuberculosis whole genome sequencing
provides insights into the Manila strain and drug-resistance mutations in
the Philippines. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-
019-45566-5

Prakoeswa, F. R. S., Rumondor, B. B., & Prakoeswa, C. R. S. (2022). Acid-

Fast staining revisited, a dated but versatile means of diagnosis. ˜the
œOpen Microbiology Journal, 16(1). https://doi.org/10.2174/18742858-
v16-e2206081

Quiroz, J. Q., Torres, A. C., Ramírez, L. M., García, M. S., Gómez, G. C., &
Camargo, J. J. R. (2019). Optimization of the Microwave-Assisted
Extraction Process of Bioactive Compounds from Annatto Seeds (Bixa
orellana L.). Antioxidants, 8(2), 37.
https://doi.org/10.3390/antiox8020037

Raddatz-Mota, D., Pérez-Flores, L. J., Carrari, F., Incani, M., Asis, R.,
Mendoza-Espinoza, J., León-Sánchez, F. D., & Rivera-Cabrera, F.
(2016). Chemical characterization and quantification of the pigment
extraction yield of seven mexican accessions of Bixa orellana. Revista
Mexicana De Ingeniería QuíMica, 15(3), 727–740.
https://doi.org/10.24275/rmiq/bio1021

Raju, S. K., Chandrasekar, S., Vengadhajalapathy, P., Sundaram, R.,

Periyasamyy, S., Chinnaraj, T., Sekar, P., & Kumar, S. (2023). Review
on phytochemical composition and pharmacological activities of Bixa
orellana. Journal of Pharmaceutical and Biological Sciences, 10(2), 57–
67. https://doi.org/10.18231/j.jpbs.2022.012

Rd, R. R. M. (2019, September 10). What is Annatto? Uses, benefits, and side
effects. Healthline. https://www.healthline.com/nutrition/annatto

Respiratory Protection | Fact Sheets | Publications & Products | TB | CDC.

(n.d.).
https://www.cdc.gov/tb/publications/factsheets/prevention/rphcs.html

Saha, P. D., & Sinha, K. (2012). Natural dye from bixa seeds as a potential
alternative to synthetic dyes for use in textile industry. Desalination and
Water Treatment, 40(1–3), 298–301.
https://doi.org/10.1080/19443994.2012.671169

Saudi Journal of Pathology and Microbiology. (2019). Saudi Journal of

Pathology and Microbiology. https://doi.org/10.21276/sjpm

Sep, C. (2016). Dye Yield, Color Strength and Dyeing Properties of Natural
Dyes Extracted from Ethiopian Dye Plants. www.academia.edu.
https://www.academia.edu/27485804/Dye_Yield_Color_Strength_and_
 55

Dyeing_Properties_of_Natural_Dyes_Extracted_from_Ethiopian_Dye_
Plants

Shahid-Ul-Islam, N., Rather, L. J., & Mohammad, F. (2016). Phytochemistry,

biological activities and potential of annatto in natural colorant production
for industrial applications – A review. Journal of Advanced Research,
7(3), 499–514. https://doi.org/10.1016/j.jare.2015.11.002

Sinha, K., Chowdhury, S., Saha, P. D., & Datta, S. (2013). Modeling of
microwave-assisted extraction of natural dye from seeds of Bixa orellana
(Annatto) using response surface methodology (RSM) and artificial
neural network (ANN). Industrial Crops and Products, 41, 165–171.
https://doi.org/10.1016/j.indcrop.2012.04.004

Siva, R., Mathew, G. J., Venkatratnam, A., & Dhawan, C. (2008). An alternative
tracking dye for gel electrophoresis. ResearchGate.
https://www.researchgate.net/publication/215465479_An_alternative_tr
acking_dye_for_gel_electrophoresis

Sudharani, S. J. . A. P. R., . M. C. S. ,. A. a. ,. B. M. K. S. ,. N. V. K. ,. V. K. S.
,. T. (2022). Screening of natural stains from Indian plants in rat skin
tissue for histological applications. www.pnrjournal.com.
https://doi.org/10.47750/pnr.2022.13.S08.97

Ulbricht, C., Windsor, R. C., Brigham, A., Bryan, J. K., Conquer, J., Costa, D.,
Giese, N., Guilford, J., Higdon, E. R., Holmes, K., Isaac, R., Jingst, S.,
Kats, J., Peery, L., Rusie, E., Savinainen, A., Schoen, T., Stock, T.,
Tanguay-Colucci, S., & Weissner, W. (2012). An Evidence-Based
Systematic Review of Annatto (Bixa orellanaL.) by the Natural Standard
Research Collaboration. Journal of Dietary Supplements, 9(1), 57–77.
https://doi.org/10.3109/19390211.2012.653530

Venturina, S. D., Comuelo, J. Z., Samaniego, W. R., & Jolito, F. C., III. (2020).
The utilization of methanolic bixa orellana seed extract as substitute for
safranin in gram staining. Department of Science and Technology,
Philippines. http://www.publiscience.org/wp-
content/uploads/2020/06/The-use-of-Bixa-orellana-extract-as-a-
substitute-for-safranin-in-Gram-staining.pdf

Vinson, R. C. K. T., Baruiz, C. P., & Pelegrino, M. (2015). Biopsychosocial

factors affecting the treatment outcomes of pulmonary tuberculosis
among patients enrolled in TB DOTS clinics. SPMC Journal of Health
Care Services, 1(1).

Winn, C. W. (1994). Koneman’s Color Atlas and Textbook of Diagnostic

Microbiology. http://ci.nii.ac.jp/ncid/BA74437508

Wikipedia contributors. (n.d.). File:Ph davao region.png - Wikipedia.

https://en.wikipedia.org/wiki/File:Ph_davao_region.png
 56

Yolmeh, M., Habibi-Najafi, M. B., Shakouri, S., & Hosseini, F. (2015).

Comparing antibacterial and antioxidant activity of Annatto dye extracted
by conventional and Ultrasound-Assisted methods. Majallah-i Taḥqīqāt-
i ̒Ulūm-i Pizishkī-i Zāhidān/Majallah-i Taḥqīqāt-i ̒Ulūm-i Pizishkī-i
Zāhidān./Zahedan Journal of Researches in Medical Sciences, 17(7).
https://doi.org/10.17795/zjrms1020
 57

Appendices

Appendix A

Transmittal letters

A.1 Transmittal Letter to the Research Director of TDCI

 58

A.2 Letter to the Office of the Dean of the Medical Laboratory Science
 59

A.3 TB Culture Laboratory

 60

A.4 Letter to Tagum Doctors Hospital Laboratory

 61

A.5 Letter for Plant verification

 62

A.6 Letter for Phytochemical Analysis

 63

A.7 Letter for Validators

64
65
 66

A.8 Letters for Evaluators

67
68
 69

Appendix B

Procedures

Pulverization & Extraction method

Appendix B 1: Annatto seed extract procedure by Venturina et al. (2020)
1. Annatto seeds are separated by hand and then washed using distilled

water.

2. Sterilize the seeds in a hot air oven at 60 degrees Celsius for 24 hours.

3. Using a blender, the seeds are crushed to acquire 100g, 300g, and 600g

of pulverized seeds.

4. 100g, 300g, and 600g of pulverized seeds are then added to 1L of

methanol and using magnetic stirrer, stirred for 12 hours and then stored

for another 12 hours without sunlight at room temperature.

5. Using Whatman Filter Paper No. 1, filter the extract and place it in IKA

RV10 Rotary Evaporator at 40 degrees Celsius the methanol had leaving

solid crude extract remained.

6. The solid extract is placed to amber glass bottle.

7. Store the extract in refrigerator prior to staining.

8. The crude extract is dissolved in 50 ml of methanol to form the solution.

Subculture

Appendix B 2: Subculture of the Cultured NTM Isolates

1. Put 5 drops of sterile distilled water into the screw-capped homogenizer

(tube with a 14ml screw-capped bottle, containing several 5-6mm sized

glass or plastic beads).

2. Harvest one loop full of growth with a 3mm diameter- loop from the

widespread surface of the growth.

3. Transfer to the homogenizer.

 70

4. Homogenize on vortex mixer for few minutes and let stand for 10

minutes.

5. Using disposable pipette, add 1 drop from the homogenize mixture to

your glass slide, make a smear and let it dry.

6. Proceed with smearing.

Smearing

This smearing procedure has been modified from the standard protocol

outlined by the Department of Health in 2021.

Appendix F 3.1: Smearing Procedure for Sputum

1. Prepare 12 clean glass slides.

2. Label each three glass slides respectively with 100g, 300g, 600g, and

Control.

3. Using wooden applicator transfer an appropriate amount of sample in a

clean glass slide.

4. Smear the specimen over an area with the length of 3cm and 2 cm width.

5. Allow it to dry.

6. Heat fix using alcohol lamp.

Appendix B 3.2: Smearing procedure for Isolates

1. Obtain a 12 clear, frosted-edged microscope slide.

2. Label each three frosted glasses with 100g, 300g, 600g, and control

3. Using a disposable pipette, add one drop of homogenize mixture to a

glass slide.

4. Make a thin smear.

5. Let the smear air dry.

6. Heat fix the smear using alcohol lamp.

 71

Staining

This staining procedure has been modified from the standard protocol

outlined by the Department of Health in 2021.

Appendix B 4.1: Staining Method (Controlled Sample)

1. Arrange the slides on the staining bridge consecutively. Keep an index

finger size distance between slides to prevent the transfer of

materials/solution running off the slides from one smear to another.

2. Begin at the edges, cover each slide completely with carbol fuchsin.

3. Heat each slide from below using an alcohol lamp or spirit cotton until

steam comes off from the stain. Do not boil or allow the slide to dry.

Leave the heated slide for 10 minutes.

4. Tilt the slide to drain excess stain. Wash the stains off with a gentle

stream of running water.

5. Cover the whole slide with 3% acid-ethanol solution for 3 minutes or until

no red color is seen. Rinse with water and drain

6. Apply 0.1% methylene blue counterstain on the smear for 5 seconds.

Flood the slide with the counterstain

7. Rinse with water and drain

8. Air dry the smear on a slide rack away from direct sunlight.
 72

Appendix B 4.2: Staining Method (Annatto Seed)

1. Arrange the slides on the staining bridge consecutively. Keep an index

finger size distance between slides to prevent the transfer of materials/

solution running off the slides from one smear to another.

2. Begin at the edges, cover each slide completely with differing

concentrations of annatto seed extract.

3. Heat each slide from below using an alcohol lamp or spirit cotton until

steam comes off from the stain. Do not boil or allow the slide to dry.

Leave the heated slide for 10 minutes.

4. Tilt the slide to drain excess stain. Wash the stains off with a gentle

stream of running water.

5. Cover the whole slide with 3% acid-ethanol solution for 3 minutes or

until no red color is seen. Rinse with water and drain

6. Apply 0.1% methylene blue counterstain on the smear for 5 seconds.

Flood the slide with the counterstain

7. Rinse with water and drain

8. Air dry the smear on a slide rack away from direct sunlight.
 73

Figure 9. A graphical representation of pulverization and extraction process

 74

Appendix C

Validation and Evaluation Tool

C.1 Validation Tool

75
76
 77

C.2 Parameter Limit

The parameter limit for the range of means among the three evaluators

ensures a stringent criterion for assessing the consistency in results when

employing Annatto seed as a primary stain alternative in Acid-fast staining.

This approach is amined to provide a reliable measure of the staining

effectiveness by establishing a narrow range within which the means of the

evaluators' observations must fall, thereby enhancing the precision and

reliability of the study's findings. This parameter range of means was adopted

from the study of Pahuriray and Algara (2021).

Range of means Descriptive equivalent Interpretation

(Pahuriray & Algara,
2023)
4.21 - 5.00 Very good Intense red color with
sharp and well-defined
shapes, making
filamentous or rod-
shaped structures easily
identifiable. Additionally,
staining of acid-fast
bacilli (AFB)
demonstrates very good
specificity, indicating a
highly reliable result.
3.41 - 4.20 Good High red color intensity
with clear shapes,
allowing for distinct
filamentous or rod-
shaped structures to be
discerned. Furthermore,
staining of AFB
demonstrates good
specificity, suggesting a
reliable outcome.
 78

2.61 - 3.40 Acceptable Moderate red color

intensity with reasonably
clear shapes, making
filamentous or rod-
shaped structures
discernible. Staining of
AFB exhibits acceptable
specificity, indicating a
satisfactory result.
1.81 -2.60 Poor Low red color intensity
with unclear shapes, and
filamentous or rod-
shaped structures are
somewhat visible.
Staining of AFB shows
poor specificity,
indicating a less reliable
outcome.
1.00 -1.80 Very poor No red color, unclear
shape, filamentous or
rod-shaped structures
are not observed, and
AFB is not stained.
79
80
81
82
83
84
 85

Appendix D

Classification of Annatto seeds (Bixa orellana)

Common/ Local Name Achiote, Achuete, Bija, Latkhan,

Roucou, Lipstick Tree, Urucu,
Natural Colour E1606.
Division Angiospermae
Class Dicotyledons
Order Malvales
Family Bixaceae
Genus Bixa
Species B. orellana

The table show the classification of Annatto seeds (Bixa orellana). The

botanist Mr. Edwin L. Solilap, RA, MSA, carried out the plant authentication at

the University of Southeastern Philippines, Tagum.

Physical characteristics of Annatto seeds (Bixa Orellana) extract.

Plant extract Appearance Color Odor

Annatto seeds Dark orange Orange-red slightly peppery

(Bixa orella) red liquid and with a hint of
free of lumps nutmeg.

The tables show the physical characteristics of the Annatto seeds (Bixa

Orellana) extract that was extracted using 100g, 300g, 600g of powdered

Annatto seeds and macerated in 1000 ml methanol for 24 hours. The physical

characteristics identified including the appearance, color, and odor.

Morphology characteristics of Annatto seeds (Bixa Orellana)

Leaves Spirally, simple, blade ovate

Margin Entire
Odor Characteristics
Taste Slightly bitter taste
Apex Acute
Flower Bisexual, regular, blade ovate
Fruit Globose or broadly to elongated ovoid
capsule
Seeds Bright orange- red fleshy seed coat
 86

The tables show the morphology characteristics of Annatto seeds (Bixa

Orellana) that was freshly picked from the tree. The morphology characteristics

identified including the leaves, margin, odor, taste, apex, flower, fruit and seeds.

Microscopic characteristics of Annatto seeds (Bixa orellana)

Figure 10. Microscopic Characteristics of B. orellana (Annatto seeds).

Retrieved from “PeerJ” https://shorturl.at/kDMT1. Copyright by Carballo-Uicab
2019.
 87

Appendix E

Raw data

E.1 Phytochemical Screening

 88

E.2 Evaluator’s Data

1. Intensity of the Red color

Evaluator Concentration Control Experimental Sample Descriptive

Slide Slide Slide Mea Equivalent

1 2 3 n

44.7% (100g) 5 1 1 1 1 Very poor

Evaluator 77.4% (300g) 5 1 1 1 1 Very poor

#1
89.46% (600g) 5 1 1 1 1 Very poor

Evaluator 44.7% (100g) 5 3 3 3 3 Acceptable

#2
77.4% (300g) 5 1 1 1 1 Very poor

89.46% (600g) 5 1 1 1 1 Very poor

Evaluator 44.7% (100g) 4 2 2 2 2 Poor

#3
77.4% (300g) 4 2 2 2 2 Poor

89.46% (600g) 4 2 2 2 2 Poor

 89

2. Clarity of Shape (rod-shaped or filamentous)

Evaluator Concentratio Control Experimental Sample Descriptive

n Slide Slide Slide Mean Equivalent

1 2 3

44.7% (100g) 5 1 1 1 1 Very poor

Evaluator 77.4% (300g) 5 1 1 1 1 Very poor

#1 89.46% 5 1 1 1 1 Very poor

(600g)

Evaluator 44.7% (100g) 5 4 4 4 4 Good

#2 77.4% (300g) 5 4 4 4 4 Good

89.46% 5 1 1 1 1 Very poor

(600g)

Evaluator 44.7% (100g) 4 2 2 2 2 Poor

#3 77.4% (300g) 4 2 2 2 2 Poor

89.46% 4 2 2 2 2 Poor

(600g)
 90

3. Specificity in Staining (stains only AFB)

Evaluator Concentration Control Experimental Sample Descriptive

Slide Slide Slide Mean Equivalent

1 2 3

44.7% (100g) 5 5 5 5 5 Very good

Evaluator 77.4% (300g) 5 5 5 5 5 Very good

#1 89.46% (600g) 5 5 5 5 5 Very good

Evaluator 44.7% (100g) 5 3 3 3 3 Acceptable

#2 77.4% (300g) 5 1 1 1 1 Very poor

89.46% (600g) 5 1 2 1 1.33 Very poor

Evaluator 44.7% (100g) 4 1 1 1 1 Very poor

#3 77.4% (300g) 4 1 1 1 1 Very poor

89.46% (600g) 4 1 1 1 1 Very poor

 91

Appendix F

Safety Data Sheet

F.1 Ziehl Neelsen Acid Fast Stains Kit Material Safety Data Sheet. From “LOBA
Chemie”, 2019, http://surl.li/nuahr. Copyright 2019 by Loba Chemie
92
93
94
95
96
97
98
99
 100

F.2 Pathogen Safety Data Sheet Mycobacterium spp. (non-tubercolous)

including M.ulcerans. From “Montana State University Office of Reseaerch

Compliance”, http://surl.li/nuajk, Copyright by Montana State University

 101

APPENDIX G

Documentation

G.1 Fit Testing

Researchers Fit testing for N95 Mask

 102

G.2 Materials

Materials for Washing, Pulverization, and Extraction

 103

G.3 Pulverization

Washing, Drying, Pulverization, Weighing, and Storing of the Annatto Seeds

powder
 104

G.4 Maceration

Maceration of the Pulverized seeds in 1L of Methanol

 105

G.5 Extraction

Filtration and Rotary Evaporator process of the Macerated seeds

 106

G.6 Transporting of specimen

Transporting of the bacterial specimen from DRMC TB Culture to TDH

Laboratory
 107

G.7 Smearing

Smearing of the cultured isolates and sputum in a Biosafety Cabinet

 108

G.8 Staining

Staining of the cultured isolates and sputum samples using the control and
the experimental stain
 109

G.9 Microscopic Examination of the Slides

Slides Cultured Isolates Sputum

Slide 1

Slide 2

Slide 3

Microscopic Examination of the Controlled slides

 110

Slides Cultured Isolates Sputum

Slide 1

Slide 2

Slide 3

Microscopic Examination of the 100g (44.7%) Annatto seed stain

 111

Slides Cultured Isolates Sputum

Slide 1

Slide 2

Slide 3

Microscopic Examination of the 300g (77.4%) Annatto seed stain

 112

Slides Cultured Isolates Sputum

Slide 1

Slide 2

Slide 3

Microscopic Examination of the 600g (89.46%) Annatto seed stain

 113

Appendix H

Certification
H.1 Bacterial Identification
 114

H.2 Plant Authentication

 115

H.3 Statistician
 116

H.3 Grammarian’s Certificate

 117

CURICULUM VITAE

CELINE B. BARCELON
Address: Purok Cacacho, Mankilam, Tagum City,
Davao del norte
Mobile No.: 09207852969
Email: celinebanudanbarcelon@gmail.com

PERSONAL INFORMATION

Civil Status : Single

Nationality : Filipino
Religion : Seventh-day Adventist
Date of Birth : December 10, 2002
Place of Birth : Davao City
Father’s Name : James Andrew L. Barcelon
Mother’s Name : Rebeilyn B. Barcelon

EDUCATIONAL ATTAINMENT

Tertiary: Tagum Doctors College, Inc. Present

Rabe Subd., Visayan Village, Tagum City, Davao del Norte
Secondary:
SHS Max Mirafuentes Academy 2020-2021
Manuel B. Suaybaguio Sr. St, Tagum, Davao del Norte
JHS Max Mirafuentes Academy 2018-2019
Manuel B. Suaybaguio Sr. St, Tagum, Davao del Norte

Elementary: Seventh-day Adventist Elementary School 2014-2015

Manuel B. Suaybaguio Sr. St, Tagum, Davao del Norte
 118

FRYNJHE AN A. HERNANDO

Address: Road 2, Purok Sampaguita, Seminary Drive,

Tagum City, Davao del Norte
Mobile No.: 09913342834
Email: frynjheanhernando@gmail.com

PERSONAL INFORMATION

Civil Status : Single

Nationality : Filipino
Religion : Roman Catholic
Date of Birth : February 9, 2002
Place of Birth : Tagum City, Davao del Norte
Father’s Name : Henry I. Hernando
Mother’s Name : Frelyn A. Hernando

EDUCATIONAL ATTAINMENT

Tertiary: Tagum Doctors College, Inc. Present

Rabe Subd., Visayan Village, Tagum City, Davao del Norte
Secondary:
SHS Tagum Doctors College, Inc. 2020-2021
Rabe Subd., Visayan Village, Tagum City, Davao del Norte
JHS Saint Mary’s College of Tagum, Inc. 2018-2019
National Highway, Tagum City, Davao del Norte

Elementary: Letran de Davao, Inc. 2014-2015

Seminary Drive, Tagum City, Davao del Norte
 119

NATHANAEL M. DALUGDOG
Address: Purok 5, Poblacion Laak, Laak, Davao de Oro
Mobile No.: 09669712233
Email: nathaanaeldalugdog@gmail.com

PERSONAL INFORMATION

Civil Status : Single

Nationality : Filipino
Religion : Christian
Date of Birth : August 22, 2003
Place of Birth : Sto. Tomas, Davao del Norte
Father’s Name : Arfel B. Dalugdog
Mother’s Name : Gemma M. Dalugdog

EDUCATIONAL ATTAINMENT

Tertiary: Tagum Doctors College, Inc. Present

Rabe Subd., Visayan Village, Tagum City, Davao del Norte

Secondary:
SHS Laak National High School 2020-2021
Poblacion Laak, Laak, Davao de Oro
JHS Laak National High School 2018-2019
Poblacion Laak, Laak, Davao de Oro

Elementary: Laak Central Elementary School 2014-2015

Poblacion Laak, Laak, Davao de Oro