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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

Ampaa, Ronn Gerald S. Atienza, Mark Kevin A. Gote, Najmah M. Salamat, Rachelle Arah B. Ubaa, 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

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

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 researchers 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 womens 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

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 1. Staining capability of Morning Glory (Ipomoea tricolor) and Gumamela (Hibiscus rosa sinensis) dye, in terms of: Staining time Clarity Differentiation of cellular structures o Nucleus o Cytoplasm 2. Significant difference of the staining 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.

Process Collection of flowers Extraction of dyes Staining of epithelial tissues Histological examinations

Output Ipomea tricolor and Hibiscus rosa sinensis dye as an alternative to Hematoxylin and Eosin in staining Epithelial tissues

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.

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. 1.2. 1.3. Staining time Clarity 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. 2.2. 2.3. Preparation Affordability 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)?

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.

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

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.

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: Stain:

Altered or caused by disease. A dye used to color tissues and other specimens for microscopical examination.

Tissue:

A collection of cells specialized to perform a specific function.

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

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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 dont 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 womens 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

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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, its 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

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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

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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

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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

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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

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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

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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 Willsttter 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,

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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 Ampaa (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

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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

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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

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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 Bian, 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

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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 Bian, Laguna. Full blossomed Morning Glory and Gumamela flowers will carefully be handpicked and oven dried for 1.5 hours at 55C 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 55C

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 2 minutes Stains clearly Nucleus is well demonstrated ----5.5 Eosin 1 minute Stains clearly ----Cytoplasm is well demonstrated 4.75

Staining time Clarity Cellular structure Nucleus Cytoplasm pH

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 10 minutes Stains clearly Nucleus is well demonstrated ----3.53 Eosin 5 minutes Stains clearly ----Cytoplasm is well demonstrated 4.75

Staining time Clarity Cellular structure Nucleus Cytoplasm pH

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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 5 minutes Stains clearly Nucleus is well demonstrated ----5.5 Gumamela 10 minutes Stains clearly ----Cytoplasm is well demonstrated 6.25

Staining time Clarity Cellular structure Nucleus Cytoplasm pH

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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. 30

Table 4 Staining Time, Clarity, Differentiation of Cellular Structures Using Morning Glory and Gumamela as a Stain Morning Glory 10 minutes Stain is pale Nucleus is poorly demonstrated ----3.53 Gumamela 10 minutes Stains is pale ----Cytoplasm is poorly demonstrated 6.25

Staining time Clarity Cellular structure Nucleus Cytoplasm pH

Buccal mucosa cell stained with Morning Glory and Gumamela Using LPO (10x magnification)

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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 5 minutes Stains fairly Nucleus is demonstrated --------Gumamela ---------------------

Staining time Clarity Cellular structure Nucleus Cytoplasm pH

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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 cells 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.

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