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MOTHER MARGHERITA DE BRINCAT CATHOLIC SCHOOL, INC.
Kamias Road Brgy. Tucop Dinalupihan, Bataan

PLDT LANDLINE: (047) 633 – 2996 (ADMINISTRATION OFFICE)
(047) 633 – 2975 (SECONDARY DEPARTMENT)
“Peace and Good through Quality Education”
Chapter 1
Introduction
Background of the study
Cell is a basic membrane-bound unit that contains the fundamental molecules of life
in which all living things are composed. With the use of biological stain it can easily be
studied. This stain is a dye which is frequently used in biology and medicine to highlight
structures in biological tissues. It is used to create visual contrast between tissue elements as
tissues are essentially colourless.
In studying cell, cells are stained to highlight to know the differences of live and dead
cells. Utilizing stain is required to easily view the specimen clearer and to obtain an easier
observation. Slight differences in structure, dye content and its impurities are used for
staining which can result in failure of expected coloration of the specimen, leading to an
inability to properly identify microscopic structures. This problem was first noticed by the
earliest users of dyes and remains a concern until now.
In addition to that, nowadays, the commercial stain that is needed in studying cell is
somewhat expensive. It is because the raw materials being used to produce biological stain
were way too much expensive.
However, the researchers found an alternative material to produce an alternative

biological stain with the use of turmeric ginger (luyang dilaw) and 4o’clock flower. These
flowers usually have different colors such as yellow, white and pink. Many of the people
know flowers for its garden purposes only and others are also for medicinal purposes; in this
study the researchers used it in biology, for it is made as one of the main ingredients in
making alternative biological stain. The researchers chose the pink one for the production of
the stain for its high pigmentation of its petals. Its extract is needed to give highlights to a
particular cell that being studied by the researchers or even the students who will conduct
laboratory activities.
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We are a Catholic School providing holistic quality education to Foster a Catholic Education that will transform students into mature
equip students to be globally competitive and well-prepared in life. faithful Christians and promote peace and good to all.
On the other hand, luyang dilaw is known mostly in food purposes and even
medicinal purposes. In this study, the researchers also get the luyang dilaw’s extract that
provides yellow pigmentation.
The production of the alternative biological stain cannot harm the environment. The
researchers do not used processes that needed factory that can cause pollution to the air. This
alternative biological stain could also have a good advantage to those students who have
laboratory activities in identifying the parts of the cell. Also, this study can be a way to take
consideration to the materials used by the production of an alternative biological stain.
Statement of the Problem
This study seeks to answer the following questions:
1. How can Mirabilis jalapa and zingiber officinale be feasible to be used in production
of an alternative biological stain?
2. How may the alternative stain be describe in terms of:
a) Availability of the materials?
b) Efficiency?
c) Economy?
3. How does the alternative stain affect the visibility of the following cell parts?
a) Vascular bundles
b) Parenchyma cells
c) Epidermis
Significance of the Study
With the use of stain, cells can be easily seen and viewed a clearer image under the
microscope. The advantage of using stain is to reveal the details of the cells. The researchers
made a product that gives support to the use of biological stain that is readily available and
easy to reproduce.
The researchers believe that this study is beneficial to the community, teachers,
students and researchers:

To the community: this study will help the community, to understand the importance
of studying various types of plants. With this, the community will know that a simple part of
plants that can easily found around them can help other researchers to make something usable
in their studies. They will realize that plants have many advantages for them and with this;
healthy ecosystem can better withstand and recover from variety of disasters.
To the teachers, this study will help the teachers in studying the basic parts of the
cell. This can have a great advantage for them to quit using commercial stain for they can
have an alternative way of using biological stain.
To the students, this study will help the students to have an easier studying of parts
of the cells. This would serve as an avenue for student’s learning.
To the researchers, the findings of this study can be used as a reference and to give
ideas to create things that needed to solve problems in the community. This will help them to
have a better study when will undertake their own study.
Scope and Limitation
This study is conducted to test the feasibility of the Mirabilis jalapa and Zingiber
officinale as alternative materials to biological stain which is expensive. The materials used
in the production of the stain can be easily found around the community.
This research is only limited for the plants such as Mirabilis jalapa and Zingiber
officinale as the material used to stain cell. It can only test the effectiveness of indigenous
materials to plant cell specifically to dicot stem cell. The researchers did not use any other
part of the plant as it may affect the color due to its natural pigmentation. Lastly, the
limitation of this study is the lifespan of the alternative stain which can only last for a day
since there is no study yet done by the researchers as concerning with the expiration and
prolonging the lifespan of the product.

Chapter 2
Review of Related Literature
Historical Histological Staining Techniques in Medicine and Biological Studies
The history of staining indicates that the application of histological techniques is a

relatively new area of diseases diagnosis (Rodrigues et al., 2009). Historical staining
techniques by early pathologists and surgeons were borrowed from a seventeen scientist
Leeuwenhoek, who was instrumental in histology using substances such as Madder, indigo
and saffron to stain tissues and using rudimentary microscopes to study them (Titford, 2009).
Later, newer techniques were devised to enhance the study of cell structure in detail
using various laboratory chemicals to preserve tissues in their natural form before staining
(Titford & Bowman, 2012). Joseph Von Gerlach was viewed as the pioneer of microscopical
staining in 1858 when he used ammoniacal carmine successfully to stain cerebellum cells
(Costa, Brito, Gomes, & Caliari, 2010).
The early histologists used the readily available chemicals to prepare tissues for
microscopic studies; these laboratory chemicals were potassium dichromate, alcohol and the
mercuric chloride to hard cellular tissues (Iyiola & Avwioro, 2011). These fixatives and
staining agents were ingenious and after a period colored staining agents were developed
which are still applicable in current laboratory staining techniques (Black, 2012). Examples
of these ingenious colored stains still in use include the trichrome that is used in the liver and
renal biopsies as well as the silver nitrate that is used in other organisms (Musumeci, 2014).
Great development in histologic stains was shaped by the improved technologic

development of microscopes and the establishment of the histologic stains (aniline dye) in
1856 in Germany which manufacture a variety of new histological stains (Shostak, 2013). At
the same time, research and knowledge relating to anatomy and tissues of the human body
increased, and this knowledge was used to further research into new-histological techniques
for the study of diseased tissue (Titford, 2009).

In the wake of the nineteenth century, many medical centers hired physicians,
pathologists and surgeons to handle surgical issues (Titford & Bowman, 2012).The Gram
staining method was named after a Danish inventor Hans Christian Gram, who invented it as
an approach to differentiating bacteria species in 1875 (Anderson, 2011). It is while working
at the city morgue with his colleagues that Gram devised the technique of staining for the
purpose of distinguishing the type of bacterium infection and also as a way of making the
bacteria visible on selected and stained lung tissues during examination (Black, 2012).
Although this technique was found unsuitable for certain bacterium organisms, it is still used
today and competes fairly with modern molecular techniques of histology (Shostak, 2013).
Alternative Biological Stain
At the height of technological advancement and mass production of goods, people nowadays
would prefer to use natural and organic if given a chance to choose against the commercial,
chemically-synthesized products. Aside from reducing if not avoiding skin or body parts
irritation when they are used, the natural organic products have stood over the test of time,
giving people no side effects. Using natural organic products is environment-friendly aside
from the fact that they are more economical. The turmeric or the Curcuma longa Linn is an
example of the plant that a natural product can be derived. It is a tropical and sub-tropical
perennial herb belonging to the family Zingiberaceae which is related to ginger.
Aside from being recognized for its medicinal benefits, C. longa is used extensively as
traditional Indian spice (Krup et al., 2013), the source of coloring agent for foods, textiles,
and cosmetics (Dash et al., 2014). Its tuberous rhizomes have been used as a condiment, an
aromatic stimulant, and colorant. The bright yellow colored compound found in Turmeric is
called curcumin. Such main fluorescent component or the curcumin in Turmeric is so
dominant that it could be used as a stain. In biology laboratories where experiments using
microscope would be an exciting part of the student life on the school campus, Turmeric as
the alternative stain is interesting. A stain is a discoloration that can be clearly distinguished
from the surface, material, or medium it is found upon. They are generally used to add color
to plant and animal tissues, microbe, and spores to make them optically distinct (Korade et
al., 2014) [13].

Most stains in current use are synthesized from mixtures of various chemicals. Aside from
being expensive (Aguoru et al., 2016), they are also hazardous to human (Braide et al., 2011)
and animal health (Suryawanshi et al., 2017). Likewise, some staining procedures have been
abandoned because the chemicals needed have been scientifically proven to be lethal
(Alturkistani et al., 2016). Therefore, natural plant dyes have gained global interest and give
promising tissue staining results when tested. In Nigeria, many researchers have extracted
some colors from a variety of local plants (Abubakar et al., 2012).
Furthermore, natural dye from Curcuma longa provides a significant alternative as they are
safer to use without health hazards, biodegradable, have easy disposability, and can be used
as compost material for farming purposes after the necessary components have been extracted
(Suryawanshi et al., 2017). An excellent biological stain must be efficient, inexpensive, less
noxious, and the source must be accessible. Almost all stains have specific mechanisms
which could be used both in plant and animal tissues (Aguoru et al., 2015). Many researchers
have attempted to extract curcumin and used it as a substitute for synthetic biological dyes.
The study conducted by Kumar et al (2014) revealed that natural tint from turmeric could
stain tissues such as collagen and muscle fibers. In the investigations made by Bassey et al
(2012) and Inbnouf (2017), they concluded that Turmeric has good potential and a promising
histological dye that can excellently replace Eosin stain in the Hematoxylin and Eosin
routine. On the other hand, Chinyere et al (2017) found out that turmeric is not an ideal
alternative stain for tissues when compared to Hematoxylin and Eosin stains. This
experimental comparative study aimed to explore the capability of Turmeric (Curcuma longa
L.) rhizome extracts to replace chemically synthesized, noxious and expensive histological
stain. This research was conducted to discover the right modifications that would enable
turmeric to be suitable in replacing synthetic dyes in Histology practices.
Definition of Terms
Alcohol - Alcohols are among the most common organic compounds. They are used as
sweeteners and in making perfumes, are valuable intermediates in the synthesis of other
compounds, and are among the most abundantly produced organic chemicals in industry. The
two best-known alcohols are ethanol and methanol (or methyl alcohol). Ethanol is used in
toiletries, pharmaceuticals, and fuels, and it is used to sterilize hospital instruments while the

methanol is used as a solvent, as a raw material for the manufacture of formaldehyde and
special resins, in special fuels, in antifreeze, and for cleaning metals.
Biological Stains – are used for the medical and biological industries to aid in detection of
structures within tissues.
Cell – is the smallest structural and functional unit of an organism.
Cover slip – this is smaller sheet of glass that is usually between 18 and 25 mm on a side.
Cover slip serves two purpose the first one is to the microscope’s objective lens from
contacting the specimen and it creates an even thickness for viewing.
Dicot - any member of the flowering plants, angiosperm, which has a pair of leaves, or
cotyledons, in the embryo of the seed. There are about 175,000 known species of dicot most
are common garden plants, shrubs and trees, and broad-leafed flowering plants such as
magnolias and roses.
Epidermis – is a single layer of cells that covers the leaves, flowers, roots and stems of
plants. It forms a boundary between the plant and the external environment.
Histology – is the study of the microanatomy of cells, tissues, and organs as seen through a
microscope. It examines the correlation between structure and function. Histology guides
teachers the visual art of recognizing the structure of cells and tissues and understanding how
this is determined by their function.
Ipomoea batatas (sweet potato or kamote tops) - is a spreading, prostrate, herbaceous,

smooth or somewhat hairy vine. Stems or runners, sprawling several meters long, take root
when in contact with soil. Leaves are ovate to oblong-ovate, 6 to 14 centimeters long,
somewhat entire, with alternate heart-shaped or palmate lobed leaves, pointed at the tip, and
heart-shaped at the base.
Microscope – this is an instrument used to see objects that are too small to be seen by the
naked eye. An instrument that produces enlarged images of small objects that allows the
observer an exceedingly close view of minute structures at a scale convenient for
examination and analysis

Microscope Slide – is a small flat rectangular piece of glass on which specimens can be
mounted for microscopic study.
Mirabilis jalapa – the marvel of Peru or four o’clock flower, is the most commonly grown
ornamental species of Mirabilis plant, and is available in the range of colours. A single flower
can be plain yellow, pink or white, or have a combination of sectors, flakes and spots.
Furthermore, different combinations of flowers and patterns can occur on different flowers of
the same plant. Another interesting point is a color-changing phenomenon. For example, in
the yellow variety, as the plant matures, it can display flowers that gradually change to a dark
pink color. Similarly white flowers can change to light violet. The flowers usually open from
late afternoon (around 4'o clock) onwards, hence the first of its common names. Flowers then
produce a strong, sweet-smelling fragrance throughout the night, then close for good in the
morning. New flowers open the following day.
Parenchyma cells – a generalized plant cell type, parenchyma cells are alive at maturity.
This is typically composed of living cells that are thin-walled, unspecialized in structure, and
therefore adaptable, with differentiation, to various functions.
Staining – is a technique used to enhance contrast in samples, generally at the microscopic

level.
Vascular Bundles – is a part of the transport system in vascular plants. This is a strand of
conducting vessels in the stem or leaves of plant, typically with phloem on the outside and
xylem on the inside.
Zingiber officinale – or commonly known as ginger, is a spice consumed worldwide for

culinary and medical purposes.

Chapter 3
Methodology
Ingredients
In making this product, the researchers prepared all needed ingredients. The table
below shows the ingredients and the exact measurement used in the production of alternative
biological stain.
Ingredients Measurement
Four o’clock flower 20 grams
Luyang dilaw 20 grams
Alcohol 70% solution 75 ml.
Kamote tops (stem) 10 pcs.
Materials
The researchers also needed some equipment in the production of alternative stain.
The table below shows the equipment and its quantity used by the researchers.
Equipment Quantity
Microscope slides 20 pcs.
Cover Slips 20 pcs.
White Cloth 2 pcs. (6x6)
Blade 1o pcs.
Multi-purpose tool 1 pc.
Plastic cups 10 pcs.
Container 1 pc.
Grater 1pc.

Procedures
In making this alternative biological stain, the researchers prepare all the ingredients
such as Alcohol 70% solution (75 ml), 4 o’clock plant (20 grams), stem of kamote tops (10
pcs) and prepare all equipments that are needed in the project such as microscope slide (20
pcs), cover slip (20 pcs), white cloth 6x6 (2 pcs), multi-purpose tool and container (1 pc). The
researcher also used blade, scissors, plastic cups and grater.
Afterwards, the researchers sliced the dicot stem from the kamote tops into a very thin
cut. The sliced kamote tops were placed in the container with alcohol for the first set-up of
experimentation.
Then, the researcher produced an extract of luyang dilaw. By grating the luyang dilaw
the researchers produced the extract from the ingredient used and set aside it for a while. For
the mirabilis jalapa or the four o’clock flower, the researchers sliced the flower in to small
pieces and extract its juice using white cloth to get its desired consistency.
The researchers partly combined the two-alternative stain to form a mixture of

biological stain to be used for the coating of the cell with the aid of the juice of luyang dilaw
in about 5 minutes. Series of testing were observed to determine the possible application of
each biological stain.
Microscope was then used to determine whether the alternative biological stains
produced are working or not. Different set-ups were observed in identifying the parts of the
cell with the use of the biological stains. Identification of each part of the cell was also
observed to compare the original picture of the parts of the cell to the experimental biological
set-up.


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MOTHER MARGHERITA DE BRINCAT CATHOLIC SCHOOL, INC.

Kamias Road Brgy. Tucop Dinalupihan, Bataan

Background of the study

However, the researchers found an alternative material to produce an alternative

Our Vision Our Mission

Statement of the Problem

This study seeks to answer the following questions:

Significance of the Study

Our Vision Our Mission

Scope and Limitation

Our Vision Our Mission

Review of Related Literature

Historical Histological Staining Techniques in Medicine and Biological Studies

The history of staining indicates that the application of histological techniques is a

Great development in histologic stains was shaped by the improved technologic

Our Vision Our Mission

Alternative Biological Stain

Our Vision Our Mission

Our Vision Our Mission

Cell – is the smallest structural and functional unit of an organism.

Ipomoea batatas (sweet potato or kamote tops) - is a spreading, prostrate, herbaceous,

Our Vision Our Mission

mounted for microscopic study.

Staining – is a technique used to enhance contrast in samples, generally at the microscopic

Zingiber officinale – or commonly known as ginger, is a spice consumed worldwide for

Our Vision Our Mission

Our Vision Our Mission

The researchers partly combined the two-alternative stain to form a mixture of

Our Vision Our Mission

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