THE EFFICACY OF Ipomoea tricolor (MORNING GLORY) AND

Hibiscus rosa sinensis (GUMAMELA) DYE AS AN

ALTERNATIVE TO HEMATOXYLIN AND EOSIN

IN STAINING EPITHELIAL TISSUES

An Undergraduate Research Presented

To the College of Medical Technology

Centro Escolar University

In Partial Fulfillment of the Requirements for the Degree

Bachelor of Science in Medical Technology

Ampaña, Ronn Gerald S.

Atienza, Mark Kevin A.

Gote, Najmah M.

Salamat, Rachelle Arah B.

Ubaña, Jose Joshua Mari G.

Villareal, Catherine Gem V.

October 2013
 CHAPTER 1

The Problem and Its Setting

Introduction

Over the years, researches have been engaged in order to improve and find

alternatives to certain processes. This processes when applied to health science may

be beneficial for the proper diagnosis and course of treatment to be given to patients

and therefore may help save precious lives. As the world constantly change and new

technologies are being discovered, synthetic materials are vogue and prevails over the

use of the natural ones. Depending on the rarity, materials of natural source can be

difficult to procure and hard to process, but the quality it provides is promising. “Staining

is the process of applying dyes on sections to see and study the architectural pattern of

tissue and physical characteristics of cells. This is made possible because different

tissue and cells display varying affinities for most dyes and stains” (Gregorios & Cohen

2012). These dyes and stains can be of natural source or be synthetically produced for

the use in the clinical laboratory. Hematoxylin and Eosin are the two most common stain

used in the microanatomical study of tissues.

The focus of this study involves the use of dyes extracted from Morning Glory

flowers (Ipomoea tricolor) as an alternative to Hematoxylin and dyes extracted from

Gumamela flowers (Hibiscus rosa sinensis) as an alternative to Eosin. The dyes

extracted from Morning Glory will stain the nuclear part of the cells while dyes from

Gumamela will stain the cytoplasm. The staining time, clarity and differentiation of

cellular structures using these alternative stains will be carried out in the process. The

study will also describe, enumerate and itemize the specific procedures that the

1
research intends to undergo as the materials are gathered, experiment are performed,

analyze the data and observe the results and proceed to qualify the hypotheses.

Furthermore, the study intends to provide cheaper and effective alternatives to

commercially prepared stain by using dyes extracted from natural sources.

Commercially prepared stain such as Hematoxylin and Eosin are both effective but are

more expensive. Morning Glory and Gumamela abounds not only in South East Asia

but also in different countries throughout the world and present variety of medicinal use.

The study aims to nurse and demonstrate the potential of these flowers as a stain which

may help in the diagnosis of disease. If the team can add more precious hours to make

life more desirable for all, and can make the Earth more habitable and livable, then this

study has reasons for its existence.

Background of the Study

Hematoxylin and Eosin staining is one of the standard procedure un preparing

specimens to be observed in Histology and Histopathology. Hematoxylin and Eosin

dyes are used for better examination of prepared tissue slides. The purpose of staining

the prepared specimens is to enhance natural contrast of the sample, and make the

various cell and tissue components and cell materials more evident.

The researchers aim to find cheaper and effective alternatives for Hematoxylin

and Eosin dye. The specimen chosen for the research are the pigments coming from

the flowers of Ipomoea tricolor “Morning Glory” as a substitute for Hematoxylin and

Hibiscus rosa sinensis “Gumamela” as a substitute for Eosin.

The research will be conducted at Centro Escolar University- Makati, Gil Puyat

Unit, a private, a non-sectarian, higher education institution that is highly recognized

2
with its programs in the health sciences including dentistry, pharmacy, medical

technology, optometry and nursing. The institution is located in the Makati Central

Business District along Sen. Gil Puyat Avenue.

Theoretical Framework

The researcher’s comprehension on the efficacy of Gumamela (Hibiscus rosa

sinensis) flower is based on the study conducted by Egbujo, E.C., Adison, O.J., and

Yahaya, A.B..

The study proves that the aqueous extract of Roselle (Hibiscus sabdariffa)

flower, a co-family of Gumamela (Hibiscus rosa sinensis) flower is effective for staining

lymph node and kidney biopsies. The study also shows that the Roselle dye molecules

are charged and that the ionic charges could be varied using acids or alkalis and

therefore its application as a nuclear or cytoplasmic stain made possible.

Moreover, the researchers also presumed that the Gumamela (Hibiscus rosa

sinensis) flower possess staining capacity whereas businessman of West Indies proved

that the flower can produce pigment that can make hair dye and shoe blackening.

In addition, Rummel (2005) stated that Gumamela (Hibiscus rosa sinensis) flower

are sometimes called “shoe flower” because it was used for polishing shoes and its

petals are also used to darken and enhance women’s eyebrows.

Biological studies of Morning Glory (Ipomoea tricolor) have shown that the most

ancestral type produced a pigment that made it a blue or purple color.

According to Rausher, the evolution of the color of the Morning Glory flower is an

excellent model to study such changes. Flower colors are produced by various

members of the group of plant compounds called anthocyanins. These pigment

3
molecules are synthesized by specialized biochemical pathways found in many plant

species.

The principle blue or purple pigment is called cyaniding; the red-pigment is called

pelargonidin. Zufall and Rausher compared the differences in anthocyanin pathways in

two types of Morning Glory to determine the subtle alterations in enzymes that created

the flower color changes.

Henceforth, researchers assume that it may be efficient as a main component in

making a Hematoxylin and Eosin stains.

Conceptual Framework

Input Process Output

1. Staining capability of Morning Glory  Collection of Ipomea tricolor and
(Ipomoea tricolor) and Gumamela (Hibiscus flowers Hibiscus rosa
rosa sinensis) dye, in terms of:  Extraction of sinensis dye as an
alternative to
 Staining time dyes
Hematoxylin and
 Clarity  Staining of Eosin in staining
 Differentiation of cellular structures epithelial Epithelial tissues
o Nucleus tissues
o Cytoplasm  Histological
2. Significant difference of the staining examinations
capability variable of Morning Glory
(Ipomoea tricolor) and Gumamela (Hibiscus
rosa sinensis) over Hematoxylin and Eosin.
3. Significant difference of the advantage
variable of Morning Glory (Ipomoea triclor)
and Gumamela (Hibiscus rosa sinensis) over
Hematoxylin and Eosin.

Statement of the Problem

This study determined the efficacy of Morning Glory (Ipomea tricolor) and

Gumamela (Hibiscus rosa sinensis) flower as an alternative to Hematoxylin and Eosin

stain.

4
 This study explicitly aims to answer the following questions:

1. What is the staining capability of Morning Glory (Ipomea tricolor) and Gumamela

(Hibiscus rosa sinensis) dye to Hematoxylin and Eosin stain terms of:

1.1. Staining time

1.2. Clarity

1.3. Differentiation of cellular structures

1.3.1. Nucleus

1.3.2. Cytoplasm

1.4. pH

2. What are the advantages of using Morning Glory (Ipomea tricolor) and

Gumamela (Hibiscus rosa sinensis) as an alternative to Hematoxylin and Eosin

stain, in terms of:

2.1. Preparation

2.2. Affordability

2.3. Availability

3. Is there a significant difference of the staining capability variables of Morning

Glory (Ipomea tricolor) and Gumamela (Hibiscus rosa sinensis)?

4. Is there a significant difference of the advantage variables of Morning Glory

(Ipomea tricolor) and Gumamela (Hibiscus rosa sinensis)?

5
Hypothesis

The researchers draw out the following hypotheses:

Ha1: There is a significant difference in the staining capability variables of Morning

Glory (Ipomea tricolor) and Gumamela (Hibiscus rosa sinensis) as an alternative

dye over Hematoxylin and Eosin.

Ho1: There is no significant difference in the staining capability variables of Morning

Glory (Ipomea tricolor) and Gumamela (Hibiscus rosa sinensis) as an alternative

dye over Hematoxylin and Eosin.

Ha2: There is a significant difference in the advantage variables of Morning Glory

(Ipomea tricolor) and Gumamela (Hibiscus rosa sinensis) as an alternative dye

over Hematoxylin and Eosin.

Ho2: There is no significant difference in the advantage variables of Morning Glory

(Ipomea tricolor) and Gumamela (Hibiscus rosa sinensis) as an alternative dye

over Hematoxylin and Eosin.

Scope and Limitations of the Study

The study primarily covers the identification of the efficacy of Morning Glory and

Gumamela dye over the commonly used Hematoxylin and Eosin dye. The researchers

would only use the flower of Gumamela and Morning Glory in the process. The

researchers would only test for the staining affinity of Gumamela dye and Morning Glory

dye to epithelial tissues that uses Hematoxylin and Eosin staining. The study focuses

only to the staining procedure in the entire tissue processing. The researchers will not

cover the preparation of their chosen tissue samples. Other types of tissues that also

use Hematoxylin and Eosin staining are not the concerned of this study.

6
Significance of the Study

The finding of this study aims to contribute to the following:

For students, the study may provide broader knowledge to different dyes that can

be used as an effective alternative in making stains.

For the College of Medical Technology particularly the Histopathology section, it

may give a new way of staining procedure for specific epithelial tissues.

For the economy, the study may aspire more economical way of making

Hematoxylin and Eosin stain by using common and popular flowers namely – Morning

Glory (Ipomea tricolor) and Gumamela (Hibiscus rosa sinensis) flower instead of the

logwood for Hematoxylin dye and the bromine and fluorescein for Eosin dye.

Moreover, the researchers would like to make best use of our natural and own

goods as a source of reliable and low cost end product that may give significant

advantage not only for Histopathology but also for the whole country.

Definition of Terms

Bromine: A non-metallic halogen element that is isolated as a deep red

corrosive toxic fuming liquid of disagreeable odor.

Cell: The basic unit of all living organisms, which can reproduce itself

exactly.

Cytoplasm: The jelly-like substance that surrounds the nucleus of a cell.

Eosin: Is a fluorescent red dye resulting from the action of bromine on

fluorescein. It can be used to stain cytoplasm, collagen and muscle

fibers for examination under the microscope. Structures that stain

readily with eosin are termed eosinophilic.

7
Fluorescein: A yellow or red crystalline dye with a bright yellow green

fluorescence in alkaline solution that is used as the sodium salt as

an aid in diagnosis (as of lesions and foreign bodies in the cornea

or of brain tumors).

Gumamela: Gumamela is a shrub that grows from one meter up to 4 meters

high. Gumamela is also known as: Hibiscus, China Rose and

Shoeflower. In the Philippines, Gumamela is cultivated as an

ornamental plant. The Gumamela flower comes in many colors:

red, yellow, orange, white, purple, pink and other color

combinations.

Hematein: Is an oxidized derivative of Hematoxylin, used in staining. Hematein

exhibits indicator-like properties, being blue and less soluble in

aqueous alkaline conditions, and red and more soluble in alcoholic

acidic conditions.

Hematoxylin: Is extracted from the wood of the logwood tree. When oxidized it

forms hematein, a compound with rich blue-purple color, and is

used, together with a suitable mordant (most commonly Fe(III) or

Al(III) salts, to stain cell nuclei prior to examination under a

microscope. Structures that stain with haematoxylin are called

basophilic.

Mordant: A substance such as alum or phenol, used to fix a stain in a tissue.

8
Morning Glory: A climbing plant of the bindweed family. Flowers: trumpet-shaped,

blue, purple, pink, or white, closing in the evening. Native to:

tropical regions.

Nucleus: The part of a cell that contains the genetic material, DNA. The

nucleus is separated by the cytoplasm by a double membrane, the

nuclear envelope.

Pathological: Altered or caused by disease.

Stain: A dye used to color tissues and other specimens for microscopical

examination.

Tissue: A collection of cells specialized to perform a specific function.

9
 CHAPTER 2

Review of Related Literatures

This chapter covers the review and related of both in local and foreign literature

and studies about Morning Glory (Ipomea tricolor) and Gumamela (Hibiscus rosa

sinensis).

The concept of this Chapter is to provide added information according to certain

criteria of relevance, interest and suitability that would be a good help for the

researchers to study and understand plant sample for further concern.

Foreign Literature

Morning glories are said to be a perennial growing vine that thrives in the full sun

and little water. It is a pest plant in Hawaii along with other members of Ipomoea genus.

The petals of blue morning glory, Ipomoea tricolor cv. Heavenly Blue is the first

example that proved the flower color change by pH change. The petals exhibit sky-blue

when full-opened, but in the bud stage the petal color is red although the responsible

pigment is the same heavenly blue anthocyanin, HBA during all the flowering stages.

The petal color change was in fact found to be due to an unusual increase of pH from

6.6 to 7.7 in colored cells.

Hibiscus rosa sinensis (Gumamela) is a native of the old world, but it is now

grown all over the tropics. It has long been cultivated in China and Japan and the

Pacific since the time of Europeans first reached Far East. At that time, the varieties

were introduced ranging from pink to white. It was in China where they encountered

yellow flowers. This plant scientifically called Hibiscus rosa sinensis linn,, is now

spontaneous throughout the Philippines. It belongs to the family Malvaceae. It is

10
cultivated most for ornamental purpose and is also planted as fence or hedges.

Bernard (1990) stated that, the flowers lasts only one day but if picked when first

opened and placed in a refrigerator until evening they will remain open until late at night

and does not have to be put in water, single bloom typically have five petals with a

staminal tube bearing 60 – 70 stamens that surround the style and five stigma pads,

Hibiscus rosa-sinensis is a flower in somewhat unique in, they don’t require water

usually last only one day during the warmer months ( a few cultivates bloom, however

two or even three days) bloom size commonly varies from 3 to 8 with even large size

appearing on some hybrids. Color combination and forms are extraordinary mixed in

almost endless variation of shade and petal arrangement (Black and a true

blue are the only hues not commonly available in the hibiscus world)

In the Botanical Beauty Book of Compendium of Cosmetics uses written by

Rummel (2005), he stated that Hibiscus rosa sinensis are sometimes called “shoe

flower” because it was used for polishing shoes, the petals where also used to darken

and enhance women’s eyebrows, and the fresh extract together with Virgin Coconut Oil

were used as a hair growth stimulant. The plant of Hibiscus rosa sinensis have a five

petal that can have a special use in terms of medicinal properties and serves as an

example of the bud of the flower that can used for cancerous swellings and mumps that

applied too.

According to the study of Staining Effect of Roselle (Hibiscus sabdariffa on the

Histologic Section of the Testis) conducted by Egbujo, E.C, Adisa, O.J. and Yohaya

A.B(2007) in their study base on a progressive nuclear stain substitute for Hematoxylin,

the Hibiscus sabdariffa was used as colorant in many tropical countries and its provides

11
a progressive blue stain for the cell nuclei of the tissue comparable to that seen with

hemalum, by simply adding a hot water extract, ferric chloride and acetic acid. By the

use of binatone blender the Hibiscus sabdariffa was ground until it reach the 10g of red

calyces, it’s been collected in conical flash with 200ml of distilled water and brought to

boil to create a brilliant red colored extract. After that Roselle immediately allowed to

cool and filtered it with a what man filter paper to give a clear extract of the Hibiscus

sabdariffa. And for compounding the solution 100ml of the extract, 5g of sodium

chloride, 1.2 ml of 10% ferric chloride solution and 3ml of glacial acetic acid. These

solutions were used to stain a paraffin section of formaldehyde fixed tissues with a

4micron in size along with parallel Hematoxylin and eosin used as controls. The results

showed that the staining of the nuclei with the extract could be a progressive nuclear

stain a substitute for hemalum in H&E procedures prior to its domestic availability.

Arnold (2008) stated on his book entitled “MUIRS Textbook of Pathology” that the

examination of sections stained by these simple tinctorial techniques that normal

Histology and the basic disease processes of inflammation, repair, degeneration and

neoplasia were defined. In the past century numerous chemical stains have been

developed to demonstrate, for example, carbohydrates, mucins, lipids and pigments

such as melanin and the iron-containing pigment hemosiderin.

According to Practical Manual of Histology for Medical Students written by Kote

(2009), Hematoxylin and Eosin staining is the most widely used staining technique in

Histology because it distinguishes the other parts of the cell. Hematoxylin stains blue on

the nucleus, on the other hand, Eosin stains pink in the cytoplasm, collagen fibers and

on muscle cells. The Hibiscus rosa sinensis flower petals of a large number of plant

12
species growing in the vicinity of our environment was screened for their antibacterial

activity. Gumamela is the National Flower of Malaysia.

E. Mazzio and K. Soliman (2009) stated that, the flower extracts from Hibiscus

rosa-sinensis were screened for antibacterial activity against human pathogenic

bacterial strain petals have some protective mechanism against microbial attack in most

of the plants. Furthermore, the flowers can be used as anti-asthmatic agents many

chemical constituents such as cyanidin, quercetin, hentriacontane, calcium oxalate,

thiamine, riboflavin, niacin and ascorbic acids have been isolated from this plant.

Resistance towards reveling antibiotics having become widespread among bacteria and

fungi, new class of antimicrobial substances are urgently required, and this studies

which reveal the presence of such compounds with antimicrobial properties in various

plant parts. For the procedure, Mazzio and Soliman initially separated the flowers from

the main body and rinsed with distilled water and shade dried and then homogenized

into fine powder and stored in air tight bottle, the only difference between in our

research is that the researchers will follow the procedure of maceration.

On the other hand Barnash, Paulina and Ross (2009) explained on their book

entitled Atlas of Descriptive Histology that Hematoxylin and Eosin can stained a simple

squamous epithelium (human mesovarium and kidney) and simple cuboidal epithelium

(human pancreas, lung and liver).

According to Practical Manual of Histology for Medical Students written by Kote

(2009), Hematoxylin and Eosin staining is the most widely used staining technique in

Histology because it distinguishes the other parts of the cell. Hematoxylin stains blue on

the nucleus, on the other hand, Eosin stains pink in the cytoplasm , collagen fibers and

13
on muscle cells. The Hibiscus rosa sinensis flower petals of a large number of plant

species growing in the vicinity of our environment was screened for their antibacterial

activity. Also conclude that a proper knowledge about the different cells and their

staining characteristic is upon on how the specimen done neatly and makes the

histological diagrams more attractive and meaningful.

Meshner (2010) supported the statement of Barnash, Paulina and Ross; on his

book Junqueiras Basic Histology he states that to study microscopic section, one must

typically be stained or dyed, due of most of the tissue are colorless. The methods of

staining tissue have therefore been devised that not only make the various tissue

components conspicuous but also permit distinctions to be made between them. The

dyes stain tissue components more or less selectively most of these days, behave like

acidic or basic compound and have a tendency to form electrostatic (salt) linkages with

ionizable radicals of the tissue.

Wiam, Sonfada, Oke, Et.al (2012) stated that based on their study named The

Lawsonia inermis and Hibiscus sabdariffa as possible stain, concluded that the extract

of two flowers named Lawsonia inermis and Hibiscus sabdariffa to be stain a

histological tissues that possess an ability of various concentrations as a aqueous

solution. By the use of the tongue and kidneys of the laboratory rat it was cut down at

6microns thickness that has been showed only the cellular cytoplasm was get stained,

Hence the combination of the 2 said extracts of Lawsonia inermis and Hibiscus

sabdariffa with eosin gave a good tissue staining with the high contrast and cellular

clarity. The results was used to compared with the usual staining with Hematoxylin and

eosin to be concluded that the extracts of Lawsonia inermis and Hibiscus sabdariffa can

14
be used as substitutes for eosin and Hematoxylin as routinely used for histological

preparations.

Bassey, Osinubi and Oremosu (2012) according to their feasibility study named

the staining effect of Hibiscus sabdariffa extract on the sperm cell morphology of

Sprague-dawleys in rats stated that, They evaluated there study in the use of Hibiscus

sabdariffa as stain to observe the sperm morphology, The sperm morphology was

analyzed by staining 10 slides of the smear with eosin that serves as a control and

ethanolic extract of Hibiscus sabdariffa dye was used to stain the sperm cells, they

viewed it in x400 magnification the sperms cells were stained in shades of reddish

brown. After the preliminary phytochemical screening of Hibiscus sabdariffa was

revealed that it contains alkaloid, saponin, flavonoids and tannins and has a potential for

use as a stain for studying the sperm morphology.

Local Literature

Morning Glory is a perennial climber that can grow to a height of 10feet and it

climbs the trellis, the porch railings and weaves themselves through the lawn ornaments

and grows quickly. “According to our ancestors this plants have a beautiful flowers that

are common in the Philippines because it is found anywhere and commonly mistaken

as a grass”. The flowers are hermaphrodite (possess both male and female organs), it is

about 5 cm long and pinkish purple to blue in color. When they bloom the flowers looks

like a funnel and when they are closed the buds looks like cine when they rolled up and

its leaves are slightly elongated heart.

According to Tolentino (1985), the vine was smooth, creeping or twinning and

wide spreading. The flowers are borne on pedicels in the axils of the leaves and are

15
usually as long as their stalks. The stalk is erect and bears up to six flowers, which often

open one at the time. The sepals are green, elliptic and around 8 mm long. The corolla

is purple, bell-shaped, around 5cm in diameter and slightly lobed. The capsules area is

smooth, ovoid, and about 1cm.

Gumamela species are found in the cultivation throughout the Philippines. This

plant belongs to the Malvaceae family and is native to warm temperatures, subtropical

regions like the Philippines. The flowers are large, conspicuous, trumpet shaped with

five or more petals with colors varying around red, pink, orange and yellow.

Foreign Studies

According to the study about Morning Glory colors reveals why evolution is stuck

in forward by Rausher and Zufall (2004), the evolution of the color of the morning glory

flower is an excellent model to study such changes. Flower colors are produced by

various members of the group of plant compounds called anthocyanins. These pigment

molecules are synthesized by specialized biochemical pathways found in many plant

species. Biological studies of Morning Glories have shown that the most ancestral type

produced a pigment that made it a blue or purple color. Those earliest flowers were

pollinated primarily by bees. But in some past period, another strain of red-flowered

morning glory evolved under pressure to become more attractive to another pollinator:

the hummingbird. The principal blue / purple pigment is called cyanidin; the red pigment

is called pelargonidin. Zufall and Rausher compared the differences in anthocyanin

pathways in the two types of morning glories to determine the subtle alterations in

enzymes that created the flower color changes.

According to Botanical Beauty Book of Compendium of Cosmetics uses written

16
Rummel (2005) stated that the plant of Hibiscus rosa sinensis have a five petal that can

have a special use in terms of medicinal properties and serves as an example of the

bud of the flower that can used for cancerous swellings and mumps that applied too.

The most commonly used stains in Histology are Hematoxylin and Eosin.

(Gartner and Hiatt 2002) to stain the tissue section, the vegetable dyes Hematoxylin

and eosin are traditionally used to distinguish between nucleus and cytoplasm, and to

identify some of the intracellular organelles. It is from examination of sections stained by

these simple tinctorial techniques that normal Histology and the basic disease

processes of inflammation, repair, degeneration and neoplasia were defined. In the past

century numerous chemical stains have been developed to demonstrate, for example,

carbohydrates, mucins, lipids and pigments such as melanin and the iron-containing

pigment hemosiderin. (Arnold 2008)

Bankroft and Gamble (2008) states that Hematoxylin and Eosin is the most

widely used histological stain. It is therefore popular based on its comparative simplicity

and has the ability to demonstrate clearly a huge number of different tissue structures.

Nowadays, commercially prepared Hematoxylin and Eosin solutions are commonly

used in laboratories for routine staining. Students must have a background of the dyes

and the preparation technique in order to troubleshoot and modify procedures for

specialized used. (Theory and Practice of Histological Techniques)

On the other hand Barnash, Paulina and Ross (2009) explained that Hematoxylin

and Eosin can stained a simple squamous epithelium (human mesovarium and kidney)

and simple cuboidal epithelium (human pancreas, lung and liver) The above statement

was supported by Meshner (2010), he states that to study microscopic section, one

17
must typically be stained or dyed, due of most of the tissue are colorless. The methods

of staining tissue have therefore been devised that not only make the various tissue

components conspicuous but also permit distinctions to be made between them. The

dyes stain tissue components more or less selectively most of these days, behave like

acidic or basic compound and have a tendency to form electrostatic (salt) linkages with

ionizable radicals of the tissue.(Junqueiras Basic Histology)

However Kote (2009) conclude that a proper knowledge about the different cells

and their staining characteristic is upon on how the specimen done neatly and makes

the histological diagrams more attractive and meaningful.

Katou, Okazaki, et.al (2009) also conducted a research and stated that beautiful

flower colors are mostly due to anthocyanins which change with the pH, like litmus,

becoming blue under alkaline conditions and red when acidic in vitro. Thus, the first

proposed idea for flower color difference was pH theory by Willstätter that blue petal

color was due to an alkaline cell sap and the red was caused by an acidic cell

condition.However, anthocyanins are dissolved in the central vacuoles of petal cells and

vacuolar pH (pHv) is generally kept as weakly acidic, in order to maintain various

secondary transport activities in the tonoplast and vacuolar functions. Therefore, with

respect to blue flower color development, there has long been a controversy between

pH theory and metal complex theories. Until now the metal complex theory was proven

by X-ray crystallographic analysis of blue pigments from Commelinacommunis and

Centaureacyanus.

Local Studies

According to Verona, Gemmalita Mrs. Acosta (1975) at Naguilian Central School,

18
Naguilian Isabela requires them to make a Gumamela jelly and Gumamela jam as a

part of their project by Detaching the petals from calyx and discard calyx of the

Gumamela flower then she chop petals finely and place in a very deep Pyrex bowl. She

Cover the petals with lemon juice and microwave on high for 4 minutes then she add a

boiling water and sugar and stir well. She Cook it for 2 minutes then stir. Then Cook

another 2 minutes and let it cooled for about 1 hour. Then it produced a reddish color

without adding any food coloring to make it red. According to Ampaña (2006), in his

investigatory project at Paco Catholic School when he was a 1st year high school, he

made a candy came from the petals of Gumamela flower. According to him he just

segregated the petals of Hibiscus rosa sinensis and weight it at least c of 10g and then

he add up a 1 cup of water then boiled it up to 15 - 20mins to secretes its juice . After

that he made a syrup came from refined sugar and add the juice with the final garnish of

tiny slices of the petals of Gumamela

The study of Gervin Tangdigan entitled “Alugbati (Basella rubra) Banana (Musa

sp) and Gumamela (Hibiscus rosa sinensis) as Biological stain” (2006) states that

Gumamela flowers are capable of being a biological stain. The researchers used the

extract of the Alugbati leaves, banana trunk and Gumamela flowers. They rate the

effectiveness of each dye on the two bacteria such as Escherichia coli and Vibrio. They

extracted the dye and then fixed it with heat and test it on the bacteria. It shows that the

rate of the dye depends on the number of the observed parts seen under the

microscope. The result of the images of the bacterial culture before and after staining

upon dropping of the plants extract, the bacterial culture of E. coli and Vibrio were

observed to be different from its previous image. It was more evident in all replicates of

19
Escherichia coli but not with Vibrio cholera. The replicates were only one part that was

observed is the cell wall.

Synthesis

The proponents believe that each and every literatures stated in this Chapter are

related to the present study and it will serve as a basis and guidelines . The proponent

relates and differentiates the research based on the flow of their network analysis from

the proposed study.

Natural dyes are derived from plants (flowers, barks, leaves and fruits) the

researchers desires to maximize the natural resources rather than synthetic materials

because it is less expensive and readily available. The purpose of the researchers is to

make an alternative tissue dye instead if Hematoxylin and Eosin dye the researchers

make use of Gumamela and Morning Glory flower extracts as their variable. The

difference of the present study to the other studies and literature stated in this Chapter

researchers use Gumamela as Eosin instead of Hematoxylin and the present study

make use of buccal mucosa cells instead of kidney, tongue and sperm cells. The

statement and theory of Rausher and Willstatter is related to the present study because

it proves that Morning Glory has a pigment that gives its blue color and has a capacity

to become a hematoxylin because it is an alkaline environment.

The statements of Wiam, Sonfada, Oke, et.al (2012) are related to the present

study because it confirms that Gumamela flowers have the capacity to give color and it

can be a stain for epithelial cells. Through series of exams, journals and literatures by

the researchers there is no single study that has ever been conducted in the Philippines

for morning glory and Gumamela flower. Moreover, this study is unique because the

20
researcher uses Morning Glory for Hematoxylin and Gumamela dye for Eosin stains

and there were no studies conducted involving the said variables in the present.

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 CHAPTER 3

Research Methodology and Procedures

This chapter presents and enumerates the specific procedure involved in order to

materialize the study. It comprises the discussion of research design, the subjects of the

study, the instruments used and the data gathering procedure.

Research Design

The researchers used experimental comparative method. This method includes

random assignment of two or more groups to a treatment. This was used to compare

the tissue staining affinity of Morning Glory (Ipomea tricolor) and Gumamela (Hibiscus

rosa sinesis) dye as an alternative to the traditional Hematoxylin and Eosin stains. The

experimental comparative method was used to determine the efficiency of Morning

Glory (Ipomea tricolor) and Gumamela (Hibiscus rosa sinensis) dye in staining epithelial

tissues.

According to Castillo (2002), experimental method is characterized as a research

design where in the cause and effect relationship of a treatment on a variable is

determined. Experimental comparative method on the other hand aims to seek for

cause-effect relationships between two sets of data (Leedy, 1997).

Moreover, a qualitative approach was applied in the experiment as subjects for

the study were purposively and carefully chosen.

Subjects of the Study

The researchers used purposive sampling method in choosing the subjects of the

study. Under the flowering plants of the genus Ipomoea, a medium sized, funnel shaped

and bluish purple flowers of the species tricolor were carefully chosen and were

22
consequently used as an alternative to Hematoxylin dye. On the other hand, under the

flowering and herbaceous plant of the genus Hibiscus, a large, bell-shaped and reddish

blossoms of the species rosa sinensis were carefully chosen and were consequently

used as an alternative to Eosin dye. All of the flowers acquired were from Biñan,

Laguna.

Research Instrument

The researchers used a self-made observation form. The observation form was

filled up by the researchers to assess trial and errors and to monitor under what

conditions does the Morning Glory (Ipomoea tricolor) and Gumamela (Hibiscus rosa

sinensis) dyes produced its best staining affinity to epithelial tissues.

The observation form consists of the following: Boiling, flooding and air drying

time, this was used to determine time it takes for the dyes extracted from the flowers to

stain best on epithelial tissues. Mordant used, this was used to determine which

mordant best facilitate fixing of stains in the tissues. Counter stain, this was used to

determine the stain used to color parts of the tissues not affected by another stain e.g. a

cytoplasmic stain (Gumamela dye) used to contrast with or enhance the nuclear stain

(Morning Glory). Pigment yield and strength of the pigment yield, this was used to

record the color produced of Morning Glory and Gumamela dyes on the stained tissues

after it was subject to various boiling, flooding and air drying time. Nuclear stain, this

was used to assess the quality or affinity of the stain from Morning Glory dye to the cell

nucleus after it was subject to various boiling, flooding and air drying time. Cytoplasmic

stain, this was used to assess the quality or affinity of the stain from Gumamela dye to

the cell cytoplasm after it was subject to various boiling, flooding and air drying time.

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Staining quality: this was used to assess the overall quality or affinity of the stain to the

tissues.

Figure 1
Observation form

Validation of Instrument

The instrument that was used in the course of the experiment was guided by

Medical Technology professors and laboratory technicians. The instrument was

validated by consulting the Research adviser, Genetics and Anatomy and Physiology

professor – Ms. Maricel Balatbat and was further approved by Mrs. Maria Carmen

Dizon, Head Coordinator for the Medical Technology program in CEU - Makati.

Data Gathering Procedure

The experimental procedures to acquire data will be performed in a laboratory

room at Centro Escolar University - Makati. The experiment will involve the extraction of

24
the dyes from the flowers and staining it to an epithelial tissue to assess the staining

quality of Morning Glory and Gumamela Dye.

Arrays of laboratory instruments will be used during the experimentation process

in order to aid the researchers and reinforce accuracy and precision. The instruments

will formally be requested from the laboratory instrumentation room at CEU - Makati.

The plants will be collected from Biñan, Laguna. Full blossomed Morning Glory

and Gumamela flowers will carefully be handpicked and oven dried for 1.5 hours at

55°C prior to extraction of dyes.

The dried petals of Morning Glory and Gumamela will be ground to powder form

using a mortar and pestle. A measured quantity of powdered flowers of Morning Glory

will be used to replace the Hematoxylin dye component of the Hematoxylin stain. The

same procedure will be done to the powdered flowers of Gumamela where it will replace

the Eosin dye of the Eosin stain.

Epithelial tissues will be collected by swabbing the buccal mucosa using a sterile

cotton swab. The specimen will be smeared into a clean glass slide. It will be air dried to

allow fixing of tissue into the slide and will be stained using the prepared alternative

Morning Glory and Gumamela stain.

The clarity, quality and differentiation of cellular structures will be observed under

the microscope and will be recorded using the observation form.

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Flowchart

Preparation of instruments and collection of flowers

Flowers will be oven dried for 1.5 hours at 55°C

Dried flowers will be ground to powder

Substitution of Dyes to Hematoxylin and Eosin

Morning Glory powder will replace Gumamela powder will replace
Hematoxylin dye component Eosin dye component

Collection of epithelial tissue

Collect through swabbing in the buccal mucosa

Staining of epithelial tissue

Morning Glory will use to stain the Gumamela will use to stain
nucleus cytoplasm

Check under the microscope

Clarity and quality of stain
Differentiation of cellular structures

Record result in observation form

Figure 2
Schematic Diagram of the Procedures

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 CHAPTER 4

Presentation, Analysis and Interpretation of Data

This chapter discusses the data gathered, their analysis and the interpretation of

results.

Table 1

Staining Time, Clarity, Differentiation of Cellular Structures of Commercially

Prepared Hematoxylin and Eosin

Hematoxylin Eosin
Staining time 2 minutes 1 minute
Clarity Stains clearly Stains clearly
Cellular structure
Nucleus is well -----
 Nucleus
demonstrated Cytoplasm is well
 Cytoplasm
----- demonstrated
pH 5.5 4.75

Buccal mucosa cell stained using Hematoxylin and Eosin

using HPO (40x magnification)

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 Buccal mucosa smear was prepared and was stained using the commercially

prepared Hematoxylin for 2 minutes and Eosin for 1 minute. The cell was clearly stained

and both nucleus and cytoplasm was well demonstrated. The pH for Hematoxylin is 5.5

on average and 4.75 for Eosin on average.

Table 2

Staining Time, Clarity, Differentiation of Cellular Structures Using Morning Glory

and Commercially Prepared Eosin as a Stain

Morning Glory Eosin

Staining time 10 minutes 5 minutes
Clarity Stains clearly Stains clearly
Cellular structure
Nucleus is well -----
 Nucleus
demonstrated Cytoplasm is well
 Cytoplasm
----- demonstrated
pH 3.53 4.75

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 Buccal mucosa cell stained with Morning Glory and Eosin
using LPO (10x magnification)

Buccal mucosa smear was prepared and stained using Morning Glory for 10

minutes and commercially prepared Eosin for 1 minute. The cell was clearly stained.

Both the nucleus and cytoplasm was well demonstrated, but the cytoplasm is a shade

lighter in contrary to the Hematoxylin and Eosin combination. The pH for the Morning

Glory stain is 3.53.

Table 3

Staining Time, Clarity, Differentiation of Cellular Structures Using Commercially

prepared Hematoxylin and Gumamela as a Stain

Hematoxylin Gumamela
Staining time 5 minutes 10 minutes
Clarity Stains clearly Stains clearly
Cellular structure
Nucleus is well -----
 Nucleus
demonstrated Cytoplasm is well
 Cytoplasm
----- demonstrated
pH 5.5 6.25

29
 Buccal mucosa cell stained with Hematoxylin and Gumamela
using HPO (40x magnification)

Buccal mucosa smear was prepared and stained using commercially prepared

Hematoxylin for 5 minutes and Gumamela for 1 minute. The cell was clearly stained.

Both the nucleus and cytoplasm was well demonstrated, but the nucleus is a shade

lighter in contrary to the Hematoxylin and Eosin combination. The pH for the Gumamela

stain is 6.25.

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 Table 4

Staining Time, Clarity, Differentiation of Cellular Structures Using Morning Glory

and Gumamela as a Stain

Morning Glory Gumamela

Staining time 10 minutes 10 minutes
Clarity Stain is pale Stains is pale
Cellular structure
Nucleus is poorly -----
 Nucleus
demonstrated Cytoplasm is poorly
 Cytoplasm
----- demonstrated
pH 3.53 6.25

Buccal mucosa cell stained with Morning Glory and Gumamela

Using LPO (10x magnification)

31
 Buccal mucosa cell stained with Morning Glory and Gumamela
using HPO (40x magnification)

Buccal mucosa smear was prepared and stained using Morning Glory for 10

minutes and Gumamela for another 10 minutes. The cell was able to absorb the stain

but offers a poor staining quality. Both nucleus and cytoplasm is distinguished but is

poorly demonstrated.

Table 5

Staining Time, Clarity, Differentiation of Cellular Structures Using Morning Glory

Dye Extract as a Stain

Morning Glory Gumamela

Staining time 5 minutes -----
Clarity Stains fairly -----
Cellular structure
 Nucleus Nucleus is demonstrated -----
 Cytoplasm ----- -----
pH ----- -----

32
Buccal mucosa cell stained with Morning Glory
using HPO (40x magnification)

33
 Buccal mucosa smear was prepared and stained using Morning Glory dye

extract for 5 minutes. The cell’s nucleus was stained with fair blue green color. The stain

was able to demonstrate the nucleus. This justifies the nuclear staining capability and

potential of Morning Glory.

34