Hydrangea macrophylla flower extracts as a natural dye for reticulocytes

Jesusa Marie Josephine S. Basallo, Concepcion J. Ancog, Rhea Marife Z. Costillas, Christian
Gabriel E. Cruz, Kris Nicole N. De Jesus, Bill Henry U. Ocampo, Mike Anthony Maui L. San
Miguel, Charlene D. Wong and Agnes A. Guzman

August 2019
 1

1.0 Introduction

For the past years it has become more and more certain that the increase in the circulation
of young red blood cells or reticulocytes, which contain reticular substance when supravitally
stained, is indicative of increased erythropoiesis. Clinical proof of this has been obtained by
innumerable examples of the production of reticulocyte crises in response to various therapeutic
procedures, and notably in cases of pernicious anemia following the institution of liver therapy,
first described by Minot and Murphy in 1926. The importance of these young cells becomes more
evident, the nature and behavior of the reticular or basophilic substance contained within them are
arousing an increasing amount of interest. This basophilic substance in erythrocytes, closely
related to the maturation of these cells, may well be a type of substance present in many other
cells, and indeed may prove to be important in the elucidation of many clinical problems, notably
those concerned with neoplastic diseases. (Clark W. Heath, M.D and Geneva A. Daland, S.B.)

The most common supravital stain is performed on reticulocytes using new methylene
blue or brilliant cresyl blue, which makes it possible to see the reticulo-filamentous pattern of
ribosomes characteristically precipitated in these live immature red blood cells by the supravital
stains. By counting the number of such cell, the rate of red blood cell formation can be
determined, providing an insight into bone marrow activity and anemia.

All chemical substances may be harmful depending on the dose, duration and conditions
of exposure. Nearly all the chemicals can be irritants, given sufficient exposure to tissue and can
cause reversible inflammation, specially, in eyes, skin and respiratory passages.

Many of the stains and dyes used for staining in a histopathology laboratory are
extremely harmful to humans and other animals. Toxicity, carcinogenicity, genotoxicity,
immunotoxicity are only some of the harmful effects of the standard stains and dyes available to
us.
Special stains are dyes that are used for special purpose and are not used routinely in a
histopathology laboratory. They are of special interest in research and diagnostic. Most of the
special stains are synthetic stains and are composed of harmful chemicals. Continuous and
regular exposure to them possesses serious risks to health; especially of pathologists and
laboratory scientists. Many of the synthetic special stains are found to be genotoxic, mutagenic,
 2

immunotoxic and carcinogenic. Their safe handling and disposal is very important from the point
of view of health and environmental safety. (Bharadwaj Bordoloi, et.al, 2017)

1.1 Statement of the Problem

This study determines the effectiveness of Hydrangea macrophylla flower

extracts as a natural dye for reticulocytes. Specifically, this study seeks to find answer to the
following questions.

1.1 Is there a significant difference in the staining property of the flower extract
Hydrangea macrophylla and Methylene blue in terms of:
1.1.1 Clarity;
1.1.2 Intensity;

1.2 Is the extract of Hydrangea macrophylla has the capability to stain reticulocytes?

1.2 Hypothesis

There is no significant difference in the staining property in regards to color, clarity,

intensity, and incubation period of Hydrangea macrophylla flower extract to Methylene Blue to
stain reticulocytes.

1.3 Significance of the Study

Synthetic stains are now widespread and mostly in use. It contains hazardous
chemical component that are harmful not only to its users but to the environment as well. Its
disposal is more complex. Thus, an effective natural stain has more reason to be produced in
order to lessen waste management and grants safety not only to the health professionals using it
but also to other people that comes in contact with. This study will be beneficial not only to the
greatest impact but to the least impact as well:

1. Laboratory Healthcare Workers. This study will assist healthcare

professionals, especially the medical technologist and pathologist, through the integration of
 3

Hydrangea macrophylla flower extracts as a natural dye for reticulocytes which is safe for use,
economical, non-toxic and non-irritating.

2. Community. The study will give great value to the Hydrangea macrophylla.
Aside from being an ornamental plant, it can be used as a natural dye. This study will serve as a
motivating factor for the community to engage themselves in the planting of more Hydrangea
macrophylla plants, thereby increasing its harvest.

3. Future Researchers. This research may serve as a basis for future researchers
employing the same approach. This will inspire researchers to provide relevant and broader data
for further investigation in relation to the experimental study which can be used as a reliable
source of information that would lead to the discovery and improvement of new natural dye.

1.4 Scope and Limitations

This study has only focused on the Hydrangea macrophylla flower extracts as a
natural dye for reticulocytes. Twenty-eight (28) Registered Medical Technologists; 19 from Our
Lady of Fatima University-Antipolo Campus College of Medical Laboratory Science and 9 from
Salve Regina Hospital were chosen to evaluate the Hydrangea macrophylla as a natural dye for
reticulocytes. This study helps us determine if Hydrangea macrophylla flower extracts could be
used as a natural dye for reticulocytes.
 4

2.0 Related Literature

2.1 Hydrangea macrophylla

Yoshida (2015), color of the sepals of hydrangea varies with the condition of
environment. Even though chemical and biological studies have a long history, small correct
knowledge was brought out about the development of color. All colored sepals contain the same
anthocyanin, delphinidin 3-glucoside. So, there must be some other way to develop a wide variety
of colors.

In hydrangea sepals, the cells of the epidermis appear colorless and only the second
layer of the cells is pigmented. Protoplast was prepared without any color change during enzyme
treatment of sepals, where we measured the vacuolar pH of each of the colored cells. We could
compare and relate the color of a single hydrangea cell with its vacuolar pH using a combination
of micro-spectrophotometry and proton selective microelectrode. Value for the vacuolar pH of
blue and red cells were 4.1 and 3.3, respectively, the vacuolar pH of blue cells being significantly
higher.

Jung (2016), Hydrangeaceae plant is natively found in the Korean mountains, known
as “soogook”, and is traditionally used as a folk medicine to treat a lot of diseases such as
diabetes and liver disorders. The major components of the Hydrangea macrophylla extract such
as phyllodulcin, hydrangenol, and hydrogenic acid was determined by the high-performance
liquid chromatography method.

Azima, (2014), the plant extract serves not only as a good source of bioactive compounds
but also as a natural pigment that can be applied as colorants in food and pharmaceutical
products.

Greer (2008), the most common supravital stain is used on reticulocytes using new
methylene blue has a certainty to see a reticulo-filamentous pattern of ribosomes
characteristically precipitated in these live immature red blood cells by the supravital stains. By
counting the number of such cells, the rate of red blood cell formation can be determined,
providing an insight into bone marrow activity and anemia. This is in contrast with vital staining,
when the dye employed is one that is excluded from the living cells so that only dead cells are
 5

stained positively. Vital stains include dyes like trypan blue and propidium iodide, which are
either too bulky or too charged to cross the cell membrane, or which are actively rapidly pumped
out by live cells.

2.2 Other Natural Dyes

Ademayo (2018), dyes are also called stains and can be used to add color to body
tissues, blood cells, organelles within individual cells as well as single cell and multi- cellular
microorganism such as bacteria, fungi and yeast to make them optically distinctive.

Anton van Leeuwenhoek was credited for the first use of stain or dye in histology. He
is the father of microbiology who worked with saffron, a natural dye extracted from saffron
crocus. Dyes are generally used to add some color stain to animal tissues, plant tissues, microbes
and spores to make them optically distinctive. Even though microorganisms can be seen in aid of
light microscope, they need to be properly fixed and stained to increase the visibility, preserve
morphological features for further study (Ademayo, 2018).

Most synthetic dyes contain toxic heavy metals such as chrome, copper and zinc which
are known to be carcinogenic. It causes allergic-like symptoms. Synthetic dyes have been
reported for being non-biodegradable and producing harmful waste to the environment, which is
harmful to man. Sources of synthetic dyes are also non-renewable, it also requires a professional
training due to the specificity of accuracy in measurement which has led to its unavailability
when needed, unlike natural dyes which are easy to prepare. Most synthetics dyes have been
found to be inflammable, which have resulted to some fire outbreak in some laboratories and
industries. Therefore, there is a need for environment friendly, easy to prepare, biodegradable and
non-toxic to man, source of dye (Ademayo, 2018).

United States forest service said plants as source for natural dyes have been used long
ago before the recorded history. Staining properties of plants were noted by humans and have
been used to produce and retain colors from plants throughout the history. Native plants and their
dyes have been used to enhance people’s lives decoration, fabrics, crafts, and even their bodies.
 6

Evidence of natural dyeing in ancient cultures has been discovered. A textile fragment
that was dyed red was from roots of an old-world species of madder that have been found in
Pakistan, dating around 2500 BC similar fabrics that were found in the tombs of Egypt.

2.3 Other Blue dyes

Leverette (2019) the Cornflower, Centaurea cyanus, is an annual plant that is native to
Europe with intense blue flowers, also called as Bachelor’s Button or Blue Bottle. As a native
wildflower species, it is now endangered due to growth of agriculture. However, it has been
cultivated to become a rather as garden flower. To create a dye bath, the petals are boiled with
alum and added to the water. The dye is then strained and is ready to color a cotton or wool
fabric.

Hyacinth, also called as Hyacinthus, Asparagaceae, is a group of plants grown from bulbs
in a range of colors including white, yellow, pink, purple, and blue. A Middle East native, the
bulbs became a garden standard in most of the world when the bulbs were cultivated
commercially in the Netherlands. To create a blue dye, use the blue and purple blooms in large
quantities boiled with water. (Leverette, 2019)

Indigofera tinctoria, also called as indigo, is a small tree found in many countries in the
Southeast Asia. The dye is derived from the leaves of the indigo plant that are red or purple in
color. The leaves are often divided into smaller leaflets. The fruit is a pod, usually with a thin
partition between the seeds. Indigo plants were brought to the American colonies and were grown
in the deep Southern states for a long time but never became the cash crop that many settlers
expected because of the complicated techniques needed to extract and create dye powder for
export. The crop was soon replaced by rice and cotton. The leaves are boiled to extract a deep,
true blue dye.

2.4 Supravital Staining

Supravital staining is a method used in microscopy to examine living cells that have been
removed from an organism. It has a greater toxicity, as only few cells need to survive in a short
while. As the cells are alive and unfixed, outside the body, supravital stains are temporary in
nature (Greer, 2008).
 7

According to Mallory, staining can be in-vivo or in-vitro. The difference between these
is that whereas In-vivo staining refers to the staining of a biological matter while it is still alive,
in-vitro staining refers to a staining technique where the biological matter is non-living.

Much better and much reliable results are obtained with the new methylene blue than
with brilliant cresyl blue. The new methylene blue is chemically much different from methylene
blue, which is a low-quality reticulocyte stain. New methylene blue stains the reticulo-
filamentous material in reticulocytes more deeply and more uniformly than brilliant cresyl blue,
which varies from sample to sample in its staining ability (Briggs, 2017).

2.5Methylene blue

According to American chemistry (2006), methylene blue is used commonly as stain to

help us see microscopic organism in brilliant color. Biologists usually add a drop or two of
methylene blue to bacteria on a glass slide before placing the slide under the microscope. The
blue color of bacteria helps microbiologist to observe their physiological characteristics. Bacteria
come in many varieties of shapes, ranging from tiny rod to spheres, spirals.

Shanks (2016) said that the earliest synthesized antimalarial drug was Methylene blue,
which is used as dye nowadays, having its first testing at the end of nineteenth century. Often
used in combination with other medication such as amodiaquine, it has shown to be effective for
falciparum malaria in African adults and children.

Methylene blue is a photoactive phenothiazine dye, when added to plasma it can inactivate
enveloped viruses in single units of plasma when exposed to light and has been used in Europe
for the past two decades (Marschner, 2013).

2.6 Reticulocytes

The enumeration of peripheral blood reticulocytes is often performed to obtain

information about the functional integrity of the bone marrow. Reticulocytosis is an increased
number of peripheral blood reticulocytes which occurs in anemic patients with functional bone
marrow while anemic patients with dysfunctional bone marrow produce decreased numbers of
reticulocytes and have decreased numbers of peripheral blood reticulocytes (i.e.,
 8

reticulocytopenia) in the absence of extra medullary hematopoiesis and other factors. In addition
to the evaluation of anemic patients, reticulocyte enumeration is also of value in monitoring bone
marrow regenerative activity after chemotherapy or bone marrow transplantation. In the
laboratory, the differentiation of the reticulocyte from the mature red blood cell is based on the
presence of RNA and other substances in the reticulocyte, which are lost during differentiation
into the mature red blood cell. Manual counting of reticulocytes by light microscopy with
supravital dyes for RNA was developed in the 1940s and remains the standard method of
reticulocyte enumeration. However, automated methods of reticulocyte enumeration developed
during the past decade are much more accurate, precise, and cost effective than manual counting
and are increasingly being performed in the clinical laboratory. In addition, the newer techniques
provide a variety of reticulocyte-related parameters, such as the reticulocyte maturation index and
immature reticulocyte fraction, which are not available with light microscopy and appear valuable
in the clinical diagnosis and monitoring of anemia and other diseases (Roger S. Riley, MD, PhD,
et al.).

Reticulocytes are non-nucleated immature red blood cells in peripheral blood, containing
residual RNA. The manual method for reticulocyte counts is the most commonly used in clinical
laboratories, and is based on microscopic observation of residual ribosomal RNA, easily
recognized by supravital staining. (Rev Bras Hematol Hemoter. 2014 Jan-Feb)

Reticulocytes are a type of blood cell made in the bone marrow. They are released into
the blood, where they mature into red blood cells according to Thompson (2018).

Health said in the past few years it has become more and more certain that the increase
of young red blood cells in the circulation, which contains reticular substance when supravitally
stained is indicative of increase erythropoiesis.

Importance of these young cells becomes more evident, the nature and behavior of the
reticular or basophilic substance contained within them are increasing in amount of interest. This
basophilic substance in erythrocytes is related to the maturation of these cells, may well be a type
of substance present in many other cells, and indeed may prove to be important in the elucidation
of many clinical problems, notably those concerned with neoplastic diseases.
 9

Reticulocytes are the immature non-nucleated RBCs that contain residual RNA and
mitochondria, which correspond to the polychromatophilic RBCs observed in Wright’s stained
blood smears according to Haschek (2010).

2.7 Related Studies

The Brassica oleracea var. capitata f. rubra is a variety of vegetable plant. It is

popularly known as the red cabbage due to its association with cabbage. The pH balance of the
soil where it is planted is a factor for its red color. The different parts of the plant can be extracted
to obtain acid-base indicators. Anthocyanin are the chemical compounds in the red cabbage
which are responsible for its indicator properties while the red color appearance is due to the
pigment cyaniding aglycone which is a kind of anthocyanins (Eco, 2014).

The purpose of this research is to find out if the color compound extract of Brassica
oleracea var. capitata f. rubra or red cabbage would be effective in staining the peripheral blood
smear. The examination of the morphology of the cells and assessment of the specific disease of
the patient could be done using the peripheral blood smear. Initial preparation of the crude extract
was done through osterizing or blending and utilizing 2.0 L 95% Ethanol to soak the leaves for 48
hours. A semi-solid crude extract was obtained by further evaporating the mixture with the use of
a water bath at 60 °C. Evaluation of the results were done based on the conducted surveys which
define how effective is Brassica oleracea var. capitata f. rubra in staining the peripheral smear.
The survey revealed that Wright’s stain remains to be more effective. With this finding, therefore,
Brassica oleracea var. capitata f. rubra may not be considered for routine blood smear stain.

Alturkistani (2015) said that the history of histology indicates that there have been a lot
of important changes in the techniques used for histological staining such as chemical, molecular
biology assays and immunological techniques, commonly referred to as histochemistry. Early
histologists used the readily available chemicals to prepare for microscopic studies of tissues;
these laboratory chemicals were potassium dichromate, alcohol and the mercuric chloride to
harden cellular tissues. Staining techniques used were carmine, silver nitrate, Giemsa, Trichrome
Stains, Gram Stain and Hematoxylin among others.

Alturkistani (2015) also said that the purpose of this research was to assess past and
current literature reviews, as well as case studies, with the aim of informing ways in which
 10

histological stains have been improved in the modern age. Results from the literature review have
indicated that there has been an improvement in histopathology and histotechnology in stains
used. There has been a rising need for efficient, accurate and less complex staining procedures.
Many stain procedures are still in use today, and many others have been replaced with new
immunostaining, molecular, non-culture and other advanced staining techniques. Some staining
methods have been abandoned because the chemicals are proven to be toxic. The study indicates
that in modern histology, a combination of different stain techniques is used to enhance the
credibility of the stained sample. Nowadays, improvement on histological stains, have been
recorded by modification and combination with other stains to improve their effectiveness.

Kusculuo (2015), pomegranate flower plant as natural dye, popularly known for the
treating various diseases, was not studied as a source of dye for cytological examination using
human blood cells. The importance of this research is to investigate dyeing result of pomegranate
flower extract on human blood cells. The natural dye source was pomegranate flower also called
as roselle and potassium aluminum sulfate was used as mordant or metal salt. Distilled water was
used as solvent. Fresh, clean and air-dried flowers were extracted with distilled water at 100 °C
for 30 minutes and then filtered.

2.8 Justification of the Study

The articles, journals related to the topic that has been studied, provided additional
information regarding to the staining properties of the flower that tested.

According to Ademayo (2018), dyes are also called stains and can be used to add color
to body tissues, blood cells, organelles within individual cells as well as single cell and multi-
cellular microorganism such as bacteria, fungi and yeast to make them optically distinctive.

Besides natural dye use in textiles, it is also used in the coloration of food, medicines,
and in leather processing. A lot of the dye yielding plants is used as medicines in various
traditional medicinal therapies. This research attempts to review the classification of natural dyes
and various sustainability issues involved in their production and application (Mansour, 2018).
 11

Most synthetic dyes contain toxic heavy metals such as chrome, copper and zinc which
are known to be carcinogenic. It also causes allergic-like symptoms. Synthetic dyes have been
reported for being non-biodegradable and producing harmful waste to the environment, which is
harmful to man. Sources of synthetic dyes are also non-renewable, it also requires a professional
training due to the specificity of accuracy in measurement which has led to its unavailability
when needed, unlike natural dyes which are easy to prepare. Most synthetics dyes have been
found to be inflammable, which have resulted to some fire outbreak in some laboratories and
industries. Therefore, there is a need for environment friendly, easy to prepare, biodegradable and
non-toxic to man, source of dye (Ademayo, 2018).
 12

3.0 Research Methodology

3.1 Research Design

This research entitled “Hydrangea macrophylla flower extracts as a natural dye for
reticulocytes” used qualitative type of experiment. The protocol of Nilgun Guler Kusculuo was
adopted for the experiment with some modifications.

The study used Methylene Blue as the control variable and the extract of Hydrangea
macrophylla as test subject. `

The researchers used Hydrangea macrophylla flower extracts as test subject to

determine its effectiveness as a natural dye for reticulocytes. The Hydrangea macrophylla was
purchased at Dangwa Flower Market in Dimasalang Road, Sampaloc, Manila, 1008 Metro
Manila.

3.2 Ethical Review

This research had used Hydrangea macrophylla flowers and human blood specifically,
reticulocytes as a test subject. This study has been submitted to the Ethical Board Committee of
Our Lady of Fatima University, Antipolo for its review. Proper handling of blood sample has
been observed in the course of the study.

3.3 Materials, Equipment and target specimen

Whole blood in Ethylenediaminetetraacetic acid (EDTA) tube, phlebotomy kit (multi

sample needle, tourniquet, 2x2 inches gauze pad, micro pore, and band aid), Pasteur pipette,
automatic pipette, test tube rack, test tube, dry bath, funnel, hot air oven, methanol, distilled
water, copper sulfate, blender, water bath, and glass slides. Methylene Blue as positive control
and Hydrangea macrophylla stain as test subject.
 13

3.4 Procedure

3.4.1 Extraction preparation

A thirty-gram amount was taken from dried Hydrangea petals and ground to a powder
using a blender then sifted. The Hydrangea powder was mixed in 150 ml distilled water, boiled
and filtered through filter paper in a funnel. The liquid extract was stored in a cold place/
refrigerator temperature for dyeing.

3.4.2 Peripheral blood film

A drop of blood was placed on a clean slide. Blood was spread on the glass slide by
another slide. This was repeated with several slides. Slides were dried and fixed with methanol.

3.4.3 Blood cells staining

A 150 ml extract of the plant and Copper sulfate solution as mordant of same quantity
was used and they were stirred with the stirring rod. Containers were placed in the oven at 50
degrees Celsius for one hour. After one hour, slides were taken from the containers, and left to
dry. In this way, the dyeing process was completed. This process was repeated at 100 degrees
Celsius for one hour. Photographs of blood cells on the slides were taken at 1000 magnification
by light microscopy.

3.5 Protocol for staining of human blood smear

Blood smears were prepared using ethylenediaminetetraacetic acid (EDTA) blood and
were air dried and dip in methanol for fixation for 30 seconds. The slides were stained with the
Hydrangea dye for 2 hours while the control groups were stained with Methylene blue.

3.6 Survey for the staining capacity and research instrument to be used

A survey form was distributed to the respondents to know the guidelines in evaluating the
stain. The respondents were twenty-eight (28) Registered Medical Technologists.
 14

3.7 Collection of data

The Bright-field microscope was used for viewing the blood smears and in determining
the staining capacity of the flower extract of Hydrangea macrophylla. The survey form had a
grading system which determines the effectiveness of the Hydrangea macrophylla to stain the
reticulocytes. The rating is 1 for poor, 2 corresponds to fair, 3 indicates satisfactory, 4 classifies
very satisfactory and 5 for excellent.

3.8 Data Analysis

Pearson-Chi square was used to evaluate the Hydrangea macrophylla as a natural dye for
reticulocytes. All statistical data was generated using the SPSS version 2.2.
 15

Research Flow

The Hydrangea macrophylla was bought from Dangwa Flower Market

Dried, Stored, Grind, Filtered, Refrigerate

Addition of Copper sulfate to Hydrangea dye, Preparation of smears stained

with control and Hydrangea dye.

Twenty-eight (28) evaluators viewed the slide in each table. A survey form
was provided.

Analyze the data and record the result.

Statistical analysis
 16

4.0 Data and Results

Table no. 1 Intensity Rating of Methylene blue compared to Hydrangea macrophylla

Intensity Methylene Hydrangea Total

Rating Blue macrophylla

Poor 0 9 9
Fair 2 8 10
Satisfactory 7 8 15
Very 9 3 12
Satisfactory
Excellent 10 0 10
Mean 3.9643 2.1796
Verbal Very Fair
Interpretation Satisfactory

Table no. 1 shows the rating of Methylene Blue compared to Hydrangea Dye as stain for
reticulocytes in terms of intensity. Out of 28 respondents for the effectiveness of methylene
blue, 10 have rated it with excellent, 9 gave a rating of very satisfactory, 7 gave a rating of
satisfactory, 2 gave a rating of fair while none gave a rating of poor, and of the same 28
respondents for the effectiveness of Hydrangea macrophylla dye, 9 have rated it with poor,
8 gave a rating of fair, 8 gave a rating of satisfactory, 3 gave a rating of very satisfactory
while none gave a rating of Excellent. The mean of all 28 respondents for the effectiveness
of Methylene Blue is 3.9643 with a verbal interpretation of Very Satisfactory while the
mean of all 28 respondents for the effectiveness of Hydrangea macrophylla is 2.1796 with
a verbal interpretation of Fair.
 17

Table no. 2 Pearson-Chi Square Intensity Rating of Methylene blue compared to

Hydrangea macrophylla

Pearson-Chi square 25.567

Degree of freedom 4
Critical Value 9.49
P-value .000

Table no. 2 shows that the Pearson-Chi square has a value of 25.567, with a value of 4 for degree
of freedom which is greater than the Critical Value of 9.49 and the P-value of .000 which is less
than the 5% level of significance.
 18

Table no. 3 Clarity rating of Methylene blue compared to Hydrangea macrophylla

Clarity Rating Methylene Hydrangea Total

Blue macrophylla

Poor 0 10 10
Fair 0 10 10
Satisfactory 10 6 16
Very 7 2 9
Satisfactory
Excellent 11 0 11
Mean 4.0357 2.000
Verbal Very Fair
Interpretation Satisfactory

Table no. 3 shows the rating of Methylene Blue compared to Hydrangea Dye as stain for
reticulocytes in terms of clarity. Out of 28 respondents for the effectiveness of methylene
blue, 11 have rated it with excellent, 7 gave a rating of very satisfactory, 10 gave a rating of
satisfactory, none gave a rating of fair and poor, and out of the same 28 respondents for the
effectiveness of Hydrangea macrophylla dye, 10 have rated it with poor, 10 gave a rating
of fair, 6 gave a rating of satisfactory, 2 gave a rating of very satisfactory while none gave a
rating of Excellent. The mean of all 28 respondents for the effectiveness of Methylene Blue
is 4.0357 with a verbal interpretation of Very Satisfactory while the mean of all 28
respondents for the effectiveness of Hydrangea macrophylla is 2.000 with a verbal
interpretation of Fair.
 19

Table 4 Pearson Chi-square and P-value Clarity rating of Methylene blue compared to
Hydrangea macrophylla

Pearson-Chi square 34.778

Degree of freedom 4
Critical Value
P-value .000

Table no. 4 shows that the Pearson-Chi square has a value of 34.778 with a value of 4 for degree
of freedom which is greater than the Critical Value of 9.49 and the P-value of .000 which is less
than the 5% level of significance.
 20

5.0 Discussion

Nowadays, anemic cells are counted in hematology laboratories by dyeing with

synthetic dyes such as New Methylene blue, Brilliant Cresyl blue, and methylene blue. This is
used to observe the reticulocytes. Although we have conducted work with mordant such as
copper sulfate and Hydrangea macrophylla extract, no studies were found on the use of natural
dyes in the dyeing of blood cells.

Based on the result on the survey form, using Hydrangea macrophylla as a natural dye
can help staining the reticulocytes but it is not distinguishable to other cell, because of the heat
stability of the components of the natural stain. Therefore, the researchers suggest removing the
chlorophyll of the plant, because it might be one of the reasons that altered the color of the stain
than what is expected. Also lower the temperature when incubating the plant because of its
sensitivity to heat, try to increase the amount of the petal’s weight in grams to milliliter of water
ratio and watch for the increase of gram, because it absorbs the water so fast and it turned the
stain in to mushy paste rather than a more dissolve solution.

This observations to the plant while conducting the experiment explicated that the plant
must be study further for more discoveries with its components and contents because it can be
useful and helpful for future researcher. This was also supported by the study conducted by
Yoshida (2015), color of the sepals of hydrangea varies with the condition of environment. Even
though chemical and biological studies have a long history, small correct knowledge was brought
out about the development of color and other component and Azima, (2014), said that the plant
extract serves not only as a good source of bioactive compounds but also as a natural pigment that
can be applied as colorants in food and pharmaceutical products.

White blood cells, red blood cells, reticulocytes were dyed in Hydrangea macrophylla
extract by copper sulfate mordant. Effective staining was not observed in cell elements at 50
degrees Celsius and without mordant.

In particular, the researchers found that effective dyeing of reticulocytes in healthy

individual was obtained in staining studies done at 100 degrees with mordant.
 21

As a result of staining studies, the researchers have conducted; the researchers believe
that Hydrangea macrophylla flower extracts in Copper sulfate mordant may be used to dye in
reticulocytes.

5.1 Conclusion

Hydrangea macrophylla is a good source of dye. Based on the data gathered, the
researchers concluded that Hydrangea macrophylla contains anthocyanin, delphinidin 3-
glucoside which contributes to the staining capacity of the flower.

Based on the results of the experimentation conducted by the researchers, it has been
concluded that the Hydrangea macrophylla flower extract had a positive effect on the blood cells,
especially on the reticulocytes. But the survey scores of the respondents for viewing the samples
slides with the natural dye and the control, showed low outcomes. Evidently, the results also
indicated that there were significant differences between the control and the natural dye.

Furthermore, it was seen in the slides that it is incomparable with each other. Because it
even stains the artifacts and the incubation period distort the colorization of red blood cells and
other parts of the smear is undistinguishable compare to the control but the reticulocytes is
stained fairly. Therefore, the result of the experimentation and the survey only attests that the
natural dye is effective in staining reticulocytes but not comparable to the control since they differ
in color.

5.2 Recommendations

Hydrangea macrophylla contains anthocyanin, delphinidin 3-glucoside which makes it a

good source of natural dye. It is recommended for future researchers to have a further study about
the major component of the flower in order to enhance its staining ability.

It is also recommended for future researchers to search for another method to remove
the chlorophyll so that it will not interfere with the color of the extract also conduct a
phytochemical test on the plant to know the components of the flower.
 22

It is also recommended to further study the Hydrangea macrophylla flower extracts if it

has the capability to stain microorganisms such as parasites, bacteria and viruses.

The researchers also recommend having at least fifty (50) Registered Medical
Technologists viewing the slides for better statistical data and results.

The researchers also recommend letting the dye aged to achieve the best quality of
staining cells; since aged dye is said to be the best choice to use when staining.

Further study is recommended for future researchers to use other natural dye to stain the
cells and make it completely organic, safe and non-toxic for laboratory scientists.