RATIFICATION PAGE

The Genetics Practicum Report entitled “Chromosome Coloring by The

Giemsa Method” compiled by:
name : Siti Nurfatimah Mutmainnah R
ID : 210107510009
class : ICP of Biology Education
group : 02 (Two)
has checked and corrected by the assistant and assistant coordinator, then this
report is declared accepted.

Makassar, November 2022

Assistant Coordinator, Assistant,

Alfian Mubaraq Putri Nur Apriliani Basri

ID. 1814142033 ID. 1914140005

Knowed,
Lecturer in Charge of Practicum

Prof. Hartati, S.Si, M.Si, Ph.D

NIP. 19740405 200003 2002

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 TABLE OF CONTENTS

APPROVAL SHEET ............................................................................................. i

TABLE OF CONTENTS...................................................................................... ii

CHAPTER I INTRODUCTION ..........................................................................1

A. Background ................................................................................................1

B. Purpose .......................................................................................................2

C. Benefits ......................................................................................................2

CHAPTER II LITERATURE REVIEW .............................................................3

A. Chromosome ..............................................................................................3

B. Giemsa Method. .........................................................................................4

C. Chromosomal Behavior .............................................................................5

CHAPTER III OBSERVATION METHOD .......................................................6

A. Day and Date ............................................................................................7

B. Tools and material ......................................................................................7

C. Work Procedures ........................................................................................7

CHAPTER IV OBSERVATION RESULT .......................................................10

A. Observation Result .....................................................................................8

B. Discussion ..................................................................................................9

CHAPTER V CLOSING .....................................................................................12

A. Conclusion ...............................................................................................11

B. Suggestion ................................................................................................11

BIBLIOGRAPHY ................................................................................................12

ATTACHMENT...................................................................................................13

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 CHAPTER I
INTRODUCTION

A. Background
Living things are the subject of the science of biology. Plants, like humans
and other animals, are always linked to biology. Genetics is a branch of biology
that tries to explain the similarities and differences in traits that are passed down
from one generation to the next in living things. The Giemsa method of
chromosomal coloring is the topic of this practicum. However, before we get into
that, let's talk about what chromosomes are. Chromosomes are very important cell
structures that pass traits to offspring. Blueprint in the form of a biological trait
code for producing a phenotype is contained within genes, which occupy
particular positions or loci on the chromosomes. Genes are wrapped in one or
more chromosomes, which contain DNA. DNA is a polymer made up of
nucleotides, four different kinds of monomers.
Different things about living things are different. Division, class, family,
genus, species, and between species members are separated by the level of
difference. This is caused by all of a material's encoding characteristics. The
material that encodes these qualities is a quality which is a nucleic corrosive
macromolecule. Dioxyribo Nucleic Acid (DNA) is the nucleic acid macromolecule
in question. Chromosomes contain the DNA. All DNA and encoded traits are also
inherited from generation to generation because chromosomes are passed down
through a mechanism. Although DNA is not packaged into chromosomes, it can
be found in other organelles like mitochondria. DNA that isn't bundled into
chromosomes is called extrachromosomal DNA.
Weldeyer (1888) was the first person to use the Latin words "chroma"
(meaning "color") and "soma" (meaning "body") to describe a chromosome, so-
called because, when the preparation is colored, this body quickly absorbs the
dye. In point of fact, the cell nucleus is supported by the chromosomes.
Chromatin, which is derived from the Greek words "chroma" and "tin," which
means quiet, covers the chromosomes during the Interphase phase. Chromosomes

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are the shortening and thickening of chromatin that occur when division activity
begins. Chromids are the next stage after chromosomes double. In terms of
heredity, chromosomes are thread structures found in the cell nucleus.
In general, the chromosome structure is a functional unit that plays a
crucial role in the transfer of particular genes from the parent cell to the potential
individual daughter cells. Chromosomes are able to move certain genetic material
to the next generation of parental parents either through the process of cell
division by meiosis or through mitotic activity, both of which can be observed in
cells. As a first step toward determining the nature of the genetic material
contained in each individual chromosome of a new daughter cell, a number of
developmental studies began to examine the processes of mitosis and meiosis.
There are only chromosomes in living things.In higher organisms, each
somatic cell has the same basic number of chromosomes—one from each parent
and one from the father. Homologous chromosomes are pairs of chromosomes
that are identical to one another. The term "diploid" refers to these two sets of
chromosomes (2n). Except for the sex chromosomes, each chromosome in the
genome is named in order of length, with the longest chromosome being the first.
Animal chromosomes are smaller than those of plants. Karyotype is the
standard chromosome arrangement determined by an individual's somatic cell's
length, number, and shape. There are two main divisions in
chromosomes.specifically the chromosome arm and centromere. Protein bodies
known as kinetochores are found on the centromere's outer surface and are
responsible for chromosome replication and mitosis's chromosome separation.
The best way to find polynucleotides and the right chromosome number
and important information for research is through chromosome preparation. The
Giemsa method is typically used for chromosomal preparation. The most common
dye for staining chromosomes is the Giemsa method. It is necessary to carry out a
practical "Chromosomal Staining with the Giemsa Method" in light of the
preceding background.
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Based on the description above, the ICP Biology Education class

conducted a practicum on the Chromosome Coloring by The Giemsa Method, in
which we used giemsa solution as the main ingredient. In addition, the living
things used as experimental materials are frog muscles and fruit fly (Drosophila
melanogaster) with giemsa and alcohol solution and then observed under a stereo
microscope.
B. Purpose
The purpose of this practicum are:
1. Understand the theory of chromosomes as genetic material.
2. Understand the behavior of chromosomes in the cell cycle.
3. Understand the technique of chromosomal preparation either directly (dense
tissue technique).
4. Able to analyze the results of chromosomal preparation.
C. Benefits
The benefits of this practicum are:
1. Students can understand the theory of chromosomes as genetic material.
2. Students can understand the behavior of chromosomes in the cell cycle.
3. Students can understand the technique of chromosomal preparation either
directly (solid tissue technique).
4. Students are able to analyze the results of chromosomal preparation.
 CHAPTER II
LITERATURE REVIEW

A. Chromosome
Greek chromosomes:color and body are choma and soma, respectively.
Because they are able to quickly absorb color, chromosomes get their name.
Genes that are passed down through generations make up chromosomes. In every
cell, chromosomes are situated in the nucleus. During the process of mitotic and
meiotic division, the metaphase phase of chromosomes can be observed.
Chromosomes don't form bodies when they don't divide; rather, they form
chromatin threads, which are untangled threads. During cell division, it is possible
to see chromosomes with two chromatids (Yuliana & Fathurohman, 2019).
Although C. Von Nageli first wrote about chromosomes in 1842, it wasn't
until W. Waldeyer in 1888 that the term "chromosome" itself became widely used
because chromosomes can absorb color through histological methods.
Chromosomes are structures that are found in the cells of organisms and contain
genetic material known as genes. These genes influence the growth and
development of each organism and play a role in the process of inheritance of
traits (Aristya et al., 2018).
Proteins and DNA make up the solid structures of chromosomes. As a
method of packaging genes, chromosomes have a distinctive structure. To put it
another way, the position and location of a gene on a chromosome are known as
gene loci. A segment of DNA or DNA sequence that determines a protein is the
gene itself. During cell division, the metaphase stage, chromosomes take on a
variety of shapes. The centromere's position influences the shape of the
chromosomes; metacentric, submetacentric, acrocentric, and telocentric forms are
typical. The kinetochore, location where spindle fibers attach during cell division,
is located in the centromere, an indented region on the chromosome. At the stage
of cell division, the shape of the chromosomes is determined by the formation of
chromatids. In the process of growth or inheritance, the chromosomes replicate at
this stage, through both mitosis and meiosis (Arsal, 2018).

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As seen through a microscope, single chromosomes typically have a

folded form in the regional unit unit and complex internal organizations, but there
are distinct regions that interact with one another in a non-random and functional
manner. Global interaction networks are two categories in which their strengths
are weaker than those of intrachromosal tissue: exponencial spread and
chromosome-to-chromosome interaction (Sarnataro et al., 2018).
B. Giemsa Method
Staining is the process of sharpening an element to make a network's
components appear more contrasted and distinct. The procedure by which a dye
appears in the network and forms molecular bonds with it. Dyes are intricate
organic compounds with unique properties that help cells or tissues keep their
color. This is because the dye's content contains Chromophore and Auxohrome.
Peroxidase staining, Sudan Black, Rapid, BCB (Brilliant Cresyl Blue), Wright,
and Giemsa are among the various types of SADT staining. Among these stains,
the staining procedure ordinarily utilized for SADT is Giemsa, on the grounds that
the opposition of these colors is better with more clear staining results .However,
Giemsa also suffers from a weakness that prevents it from staining granulocyte
cells' granules. Additionally, the content of methylene blue, eosin, and azure B is
difficult to break down, resulting in waste that is toxic and flammable(Ayu, 2021).
The addition of a solution of methylene blue and eosin dissolved in
methanol results in the formation of black precipitation, which is the fundamental
component of Giemsa staining. Eosin, methylene azur, and methylene blue make
up the Giemsa stain, which can be used to stain blood cells by fixing them with
methyl alcohol. The best concentration of Giemsa must be determined in order to
get good results from a microscopic examination. However, this has some
drawbacks, such as the fact that microscopic observations still depend on the eye,
which can have different perceptions. When the value of the Giemsa color
intensity is high, the Giemsa solution's color is considered to be optimal, whereas
when the value of the Giemsa color intensity is low, the Giemsa solution's color is
less than optimal (Syaifudin et al., 2018).
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Giemsa stain is the dye of choice both domestically and internationally due
to its superior durability in tropical climates. Wright's stain is also used to stain
peripheral blood. When Giemsa staining is combined with Wright, it is hoped that
the advantages of both dyes will be realized, making the peripheral blood smear
more microscopically visible and lasting longer (Hormalia et al., 2018).
In the 19th century, the Romanowsky-Giemsa (RG) granules were created
to identify blood-borne plasmodia parasites. In later times, cytology and
hematology adopted GM staining as the standard method. With varying degrees of
success, GE staining has been applied to fixed-in-formalin paraffin-fixed sections
of the paraffin network. Sadly, the differentiation step frequently results in
inconsistent staining results, despite the fact that the majority of published work
on this subject describes a GE staining technique in which sections are folded
before being subjected to acid differentiation. There is no need for differentiation
if staining is carried out in the best possible conditions, with control over dye
concentration, pH, solution temperature, and staining time. The results of routine
hemalum and eosin staining were comparable (Teramoto et al., 2021).
Giemsa staining, which is commonly used in microbiology and
parasitology, provides a precise method for visualizing and counting reticulocytes
in individual blood samples, which is essential to its utility. The Giemsa stain
(Giemsa) is one of the stains that is encountered the most frequently in the field.
Deposits of hemosiderin, lipofuscin, and melanin are greenish, while calcium
deposits are blue (Wei et al., 2022).
C. Chromosomal Behavior
Each chromosome is made up of long, thin chromatin fibers, as stated by
Campbell in his book Biology, Eighth Edition (2013). This is true even when cells
are duplicating their DNA in preparation for cell division.However, when DNA is
duplicated, the chromosomes condense, and each chromatin fiber becomes tightly
coiled and folded. As a result, the chromosomes are much shorter and thicker than
what is visible under a light microscope.
The percentages of metaphase, anaphase, and telophase are low.In
accordance with the literature, which states that the interphase stage, which
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consists of three subphases, namely G1, S, and G2, takes a very long time, the
interphase is a phase that always appears every hour and is the phase with the
highest percentage compared to the mitotic phase.Stage G1 lasts six to twelve
hours, stage S six to eight hours, and stage G2 three (Kusumawati, 2018).
The cell grows by producing proteins and cytoplasmic organelles like
mitochondria and the endoplasmic reticulum in all three subphases.However, only
during the S phase do the chromosomes duplicate.As a result, the cell (G1)
divides (M), continues to grow while copying its chromosomes (S), and continues
to grow once more while completing the preparations for cell division (G2).The
cycle can then be repeated by the daughter cells (Campbell et al., 2013).
 CHAPTER III
PRACTICUM METHOD

A. Time and Place

Day / Date : Monday, October 31st 2022
Time : 14.40-16.50 WITA
The Place : Microbiology Laboratory 2nd floor, Department Makassar
State University Biology
B. Tools and Materials
1. Tools
a. Object glass (1 piece)
b. Drop pipette (1 piece)
c. Microscope (1 piece)
d. Surgical tools (1 piece)
e. Surgical board (1 piece)
2. Materials
a. Frog muscles (Enough)
b. Methanol (Enough)
c. Giemsa solution (Enough)
d. Aquadest (Enough)
e. Chloroform (Enough)
C. Work Procedures
1. Frog Muscle Observation

Frog muscle cut as

thin as possible.
Frog muscle is taken
from the frog's thigh.

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The object glass were

dropped with enough
methanol and set aside
for 5-7 minutes.

The Giemsa solution

The object glass were
was dissolved with
rinsed with distilled
distilled water. The
water, then dried.
object glass was
dripped with Giemsa
solution. Leave it for
15 minutes.

The object glass

observed under a
microscope.
2. Larvae Observation

Larvae were taken Larvae are placed in

from Erlenmeyer object glass.
using tweezers.
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The object glass was The object glass were

dripped with Giemsa dropped with enough
solution. Leave it for methanol and set aside
15 minutes. for 5-7 minutes.

The object glass

observed under a
microscope.
 CHAPTER IV
RESULT AND DISCUSSION

A. Observation Result
1. Table 4.1 Giant Chromosoms in frog (Rana Cancarivora) thigh muscles
No. Sample Observation Picture Note

1. Muscle of 1. Chromosome
Rana
cancarifora

2. Table 4.2 Giant Chromosoms in larvae

No. Sample Observation Picture Note

1. Larvae of 1. Chromosome
Drosophila
melanogaster

B. Discussion
During this practicum, we observe the Giemsa dye staining of
chromosomes.Chromosomes are the most important cell structures that transmit
traits to offspring. They are tiny bodies in the nucleus.A haploid cell's
chromosomes never have the same properties or size.DNA that is wrapped in one
or more chromosomes and contains a blueprint for the genetic code that produces
a phenotype is contained in chromosomes, which contain genes. In this instance,

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the size of the chromosome varies from species to species depending on the length
of the DNA sequence.The better hypotonic treatment accounts for the lower intra-
individual variation in the number of chromosomes obtained from good
dispersion, which ranges from 0.2 to 20 m.
We used frog (Rana cancarivora) material for this practicum.The muscle
is the part of the frog that is taken out.The thigh muscles are what we use.We used
the Biocular Microscope to observe the preparations that we had made.Giemsa
dye was used as the dye liquid so that the colored chromosomes of the prepared
preparations can be clearly seen through a microscope.In cells, structures called
chromosomes store genetic information.
Giemsa can be used to color chromosomes because it is made of a mixture
of eosin, methylene blue, and mehylene azure dyes. These dyes combine to form
eosinate, which makes the staining results more stable and makes it easier to see
the chromosomes clearly.Cytogenetic analysis is one of Giemsa dye's
capabilities.Giemsa staining is a method of microscopic staining first developed
by Gustac Giemsa in the field of microbiology.
Genes that are passed down through generations make up chromosomes.
In every cell, chromosomes are situated in the nucleus. During the process of
mitotic and meiotic division, the metaphase phase of chromosomes can be
observed. Chromosomes don't form bodies when they don't divide; rather, they
form chromatin threads, which are untangled threads.referred to as a chromosome
due to its ease of color absorption.
Chromosomal staining utilizing the Giemsa technique, here what is noticed
is the frog's muscle which is dribbled with Giemsa, then seen or saw under a
magnifying instrument. Whereas Giemsa staining combines eosin solution with
methylene blue solution, the blood preparation stained with this solution will
reveal pink erythrocytes, dark purple leukocyte nuclei, blue malaria parasite
cytoplasm, red parasite nuclei, and chromatin grains. Carmine-red becomes the
parasite.
Observations were made using a thin incision from the muscle of the rice
field frog (Rana cancrivora) which was placed on the slide and then immersed in
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methanol, the purpose of this immersion is so that in the Giemsa staining process
the color will stick to the incision used. This technique is also used in histology
because it is capable of Giemsa staining to color chromatin, of which chromatin is
part of a chromosome. The Giemsa staining solution was composed of a mixture
of eosin, methylene blue, and methylene azure dyes. A mixture of methylene
azure and methylene blue will form an eosinate which makes the staining result
more stable.
The addition of a solution of methylene blue and eosin dissolved in
methanol results in the formation of black precipitation, which is the fundamental
component of Giemsa staining. Eosin, methylene azur, and methylene blue make
up the Giemsa stain, which can be used to stain blood cells by fixing them with
methyl alcohol. The chromosomes in the frog's muscles can be seen when
examined under a microscope with a magnification of 10 x 40.
Observations that have been made are analyzing the chromosomes in frog
muscles to determine the characteristics of the observed species (karyotype).
Karyotype analysis is useful for determining the presence of genetic diseases,
chromosomal mutations, identifying species, identifying hybrids resulting from
crosses, monitoring sex and identifying the ploidy level of an organism.
Observation of the number of chromosomes during mitosis often creates
difficulties because the chromosomes overlap with one another and sometimes
still look blurry due to incomplete condensation.
Based on our observations, we found chromosomes in frog muscles that
looked like fine purplish threads due to the influence of Giemsa dye which had
been applied for 15-20 minutes. The condition of the preparations was not good
because the muscle cells in the frogs made from the preparations were not clearly
visible, perhaps because they were too thick when slicing, and the muscle areas
appeared purplish due to the influence of the Giemsa dye used.
 CHAPTER V
CLOSING

A. Conclusion
From the genetics lab of the Chromosome Coloring by The Giemsa
Method then it can be concluded that
1. Chromosomes are tiny cell bodies in the nucleus that carry substances with
biological properties. They are passed on to new daughter cells by inheriting
genetic traits from their parents to keep certain species of living things alive.
2. A smaller activity of a larger pattern of chromosomal behavior in a larger cell
cycle is the activity of cell reproduction through cell division.In order to
produce daughter cells, the process of cell division is divided into two major
processes.A chromosome's pattern of behavior during a cell cycle is divided
into two division processes: meiosis, which is responsible for the formation of
diploid stem cells, and mitosis, which is responsible for the division of the
parent cell into haploid gametes.
3. The direct and indirect manufacturing techniques for preparing chromium
preparations are well-known preparation methods.The direct method is used
to make chromosome preparations by taking the cells of the organ you want
to study directly from certain body parts.In contrast, the indirect method of
preparation involves cultivating tissue or cells in a culture to observe the
pattern of particular chromosome segments.
4. By directly observing muscle tissue, the results of the analysis of
chromosomal preparations using the preparation method produced zero
results.The findings of the observations did not result in the production of
chromosomes; rather, the chromosomes were only visible in the muscle tissue
that was left over from the treatment of too-thick incisions in frog
muscles.Frog chromosomes are difficult to observe, despite the fact that the
procedure was carried out in accordance with the working procedure.The cell
is entering the prophase, metaphase, anaphase, telophase, and interphase
phases, which depict the pattern of chromosome behavior in a cell cycle,

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 according to observations of chromosome preparation that ought to be
obtained.
5. Analysis of the results of chromosomal preparation can be seen from the
observation image that the black dots in the image represent chromosomes.
B. Suggestion
1. In this practicum, the practitioner should obey the rules and carry out the
practicum seriously, carefully and according to the procedure. So that it will
produce valid data as well as more effective and efficient time.
Communication between practitioners and assistants needs to be improved so
that the information conveyed can be received in its entirety. Practitioners
should also understand the material to be practiced.
2. For laboratory assistants, the supply of practicum materials is further
facilitated, in order to facilitate the practicum process.
3. Assistants are expected to always accompany the practitioner during the
observation or not in order to minimize the occurrence of errors during the
practicum.

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Aristya, G., R. Daryono, B., S. Handayani, N., S., N. Arisuryanti, T. (2018).

Karakterisasi Tumbuhan & Hewan. Yogyakarta: Gadjah Mada University
Press.

Arsal Faridah. 2018. Genetika Arif Memahami Kehidupan. Makassar : Badan

Penerbit Universitas Negeri Makassar.

Aristya, G., R. Daryono, B., S. Handayani, N., S., N. Arisuryanti, T. (2018).

Karakterisasi Tumbuhan & Hewan. Yogyakarta: Gadjah Mada University
Press.

Ayu Nirmala Sari, Alifa Tazkiya, Yudi Mafira. (2021). Ekstrak Air Bunga
Kencana Ungu (Ruellia simplex) Sebagai Pewarnaan Alternatif Preparat
Sediaan Apusan Darah Tepi (SADT). Jurnal Biologi Vol. 2. No. 1

Campbell, N.A., Reece, J.B., Urry, L.A., Cain, M.L., Wasserman, S.A., Minorsky,
P.V., Jackson, R.B. 2008. Biologi. Edisi Kedelapan Jilid 1. Jakarta:
Erlangga

Hormalia, Haitami, H., & Arsyad, M. (2018). Pengaruh Variasi Pengenceran

Giemsa Terhadap Pewarnaan Giemsa Plasmodium Sp Pada Pemeriksaan
Sediaan Darah Tipis. Jurnal ERGASTERIO VOL.5. No. 1

Kusumawati, S. A., Dwiranti, A.,  Salamah, A. 2018. Pengamatan Fase Mitosis

Hibiscus rosa-Sinensis L. Variasi Double Red pada Beberapa Waktu
Pengambilan Pucuk Daun. Proceeding of Biology Education. Vol 2 (1).

Mukh Syaifudin, Indah Irma , Dwi Ramadhani. (2018). Optimalisasi Pewarnaan

Giemsa Pada Apusan Darah Tipis Terinfeksi Plasmodium berghei Untuk
Mendukung Pengembangan Vaksin Malaria Iradiasi. Jurnal Biotek
Medisiana Indonesia Vol.7. No. 1.

Sarnataro Sergio, Andrea M. Chiariello Andrea Esposito Antonella Prisco Mario

Nicodemi. 2017. Structure of the human chromosome interaction network.
Journal Pone. 12 (11)

Teramoto, A., Yamada, A., Tsukamoto, T., Kiriyama, Y., Sakurai, E., Shiogama,
K., Michiba, A., Imaizumi, K., Saito, K., & Fujita, H. (2021). Mutual stain
conversion between Giemsa and Papanicolaou in cytological images using
cycle generative adversarial network. Heliyon, 7(2).

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Wei, H., Gao, W., Nie, H., Sun, J., & Zhu, M. (2022). Classification of Giemsa
staining chromosome using input-aware deep convolutional neural
network with integrated uncertainty estimates. Biomedical Signal
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