APPROVAL SHEET
Complete report of genetics which entitle “Chromosomes Staining with
Giemsa Methods” arranged by :
name : Alifiah A.M. Pradina Fatshaf
ID : 1914441001
class : ICP of Biology Education
group : IV (four)
has been checked and corrected by Assistant and Assistant Coordinator, so this
report was accepted.
Makassar, October 14th 2021
Assistant Coordinator, Assistant,
Alfiqi Dwi Annisi Ainun Fitriski Utami
ID. 1614140010 ID. 1814040025
Known by
Responsibility Lecture
Hartati, S.Si, Ph.D
NIP. 19740405 200003 2 002
i
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 PREVIEW OF LITERATURE 3
A. Frog 3
B. Chromosomes 3
C. Giemsa Staining 4
CHAPTER III OBSERVATION METHOD 6
A. Day and Date 6
B. Tools and material 6
C. Work Procedures 6
CHAPTER IV OBSERVATION RESULT 8
A. Observation Result 8
B. Discussion 8
CHAPTER V CLOSING 11
A. Conclusion 11
B. Suggestion 11
BIBLIOGRAPHY 12
ATTACHMENT 13
ii
CHAPTER I
INTRODUCTION
A. Background
A cell is the smallest component of a living entity. Multicellular creatures'
cells are made up of a membrane, cytoplasm, and a cell nucleus. Chromosomes
are smooth rods that can be long or short, straight or curved, and are found in the
nucleus of the cell. Chromosomes take up residence in the cell's nucleus.
Chromosomes are a group of genes in the cell nucleus that play a part in
hereditary trait inheritance. Genes are located on the chromosome and contain a
blueprint for the biological trait code to produce a phenotype. The length of the
DNA sequence determines the size of the chromosome, which differs between
species. A haploid cell's chromosomes are never the same size. Homologous
chromosomes are those that have the same form and are typically seen in diploid
cells.
Chromosomal analysis, also known as karyotyping, can be performed to
assess the level of ploidy (diploid, triploid, and so on) and the features of a
species. Making chromosomal preparations allows you to observe the size and
number of chromosomes. As a result, unique talents are required in the
production of chromosomal preparations. There are two methods for making
chromosomal preparations: the solid tissue technique and the blood culture
technique. Because it is less expensive and takes less time, chromosomal
preparation with solid tissue technology is used in this activity.
Frogs are amphibians that live both on land and in water, have enormous
eyes, a loud voice, and a slippery body. Body tissue can survive for several
minutes to hours after the frog's leg is dissected. The network's culpability is
determined by the therapy provided. As a result, after the frog's legs are opened,
the muscle and nerve tissue must be continually moistened with Ringer's solution,
which has an ion concentration comparable to the frog's extracellular fluid. So
that the tissues's function is not hampered by dryness.
1
2
Because the chromosomes are at a condition of maximum condensation
during metaphase, the whole shape and number of chromosomes are easily
visible. Treatments used in the production of chromosomal preparations include
cell division inhibition (mitotic inhibitors), hypotonic therapy, fixation, staining,
and preparation closure. These procedures, known as dense tissue methods, are
also performed in this practicum. Each treatment serves a distinct purpose.
Because chromosomes are plainly visible during metaphase, cell division must be
stopped at this time.
B. Purpose
The aims of this experiment are :
1. To comprehend the concept of chromosomes as genetic material
2. To comprehend chromosomal behavior during the cell cycle
3. Understanding the technique of chromosomal preparation either directly
(solid tissue technique) or indirectly (liquid tissue technique)
4. In order to be able to interpret the outcomes of chromosomal preparation
C. Benefits
The benefits of this experiment are :
1. Students are known to comprehend the concept of chromosomes as genetic
material
2. Students are known to comprehend chromosomal behavior during the cell
cycle
3. Students are known to understanding the technique of chromosomal
preparation either directly (solid tissue technique) or indirectly (liquid
tissue technique)
4. Students can order to be able to interpret the outcomes of chromosomal
preparation
CHAPTER II
LITERATURE REVIEW
A. Frog
For people the world over and throughout history, frogs symbolize rebirth and
renewal. This association reflects three aspects of frogs’ biology, one of which is
their very essence: “Here today, gone tomorrow.” Frogs appear from nowhere
following a hard rain and then disappear. In temperate areas, they emerge with
warmer spring temperatures and vanish in the fall when temperatures plummet.
The Roman scholar Pliny the Elder claimed that frogs were reborn from mud
during the annual spring rains. Ancient Egyptians associated frogs with rebirth
because after the annual flooding of the Nile River, frogs that were assumed to be
dead mysteriously reappeared (Martha Crump, 2015).
Anura found in the Upper Opak part of both diurnal and nocturnal species, a
total of 8 species were found, namely Hydrophylax chalconotus (kongkang pond),
Occidozyga sumatrana (Sumatran swamp bancet), Occidozyga lima (green
bancet), Fejervarya limnocharis (moor frog), Polypedates leucomystax ( striped
tree frog), Microhyla palmipes (webbed peacock), Duttaphrynus melanostictus
(buduk toad), and Ingerophrynus biporcatus (jungle crow toad) (Table 2). Species
Hydrophylax chalconotus (kongkang pond) and Polypedates leucomystax (striped
tree frog) were found at the five sampling points upstream, both during the day
and at night (Donan Satria Yudha, et all., 2011).
B. Chromosomes
Living things have different characteristics. The level of difference provides a
grouping boundary between division, class, family, genus, species, and between
members of species. All traits encoded in a material are responsible for this. The
material that encodes these characteristics is a gene which is a nucleic acid
macromolecule. The nucleic acid macromolecule in question is Dioxyribo Nucleic
Acid (DNA). DNA is packaged into chromosomes. Chromosomes are passed
3
4
down from generation to generation through a mechanism, so that all DNA and
encoded traits are also inherited from generation to generation. Although there is
also DNA located in other organelles such as mitochondria, it is not packaged into
chromosomes. DNA that is not packaged into chromosomes is called
extrachromosomal DNA. The discussion in this chapter is not concerned with
extrachromosomal DNA (Andi Faridah Arsal, 2018).
The history of chromosomal research is divided into several eras, where each
era applies certain methods / technologies that continue to develop. In the modern
era, chromosomal research includes the development of four main technologies,
namely: (1) the discovery of a metaphase chromosomal spread method that allows
the observation of the number and shape of chromosomes (karyotype) (2)
Development of a chromosomal banding technique that allows identification of
homologous chromosomes/ mate (within karyotypes of the same type) and
homeolog (between karyotypes of different species) ( Joko Ridho Witono, 2008).
C. Giemsa Staining
The principle of Giemsa staining is the presence of black precipitation which
is formed from the addition of a solution of methylene blue and eosin dissolved in
methanol. To obtain effective microscopic examination results, it is necessary to
determine the optimal concentration of Giemsa although with some shortcomings,
including microscopic observations which still depend on the eye which can have
different perceptions (Mukh Syaifudin, et all., 2018).
Species and parasite asexual stage identification could be identified properly
from the thin smear stained according to Leishman, and this was subjectively
easier than in the Giemsa-stained slides. Themore mature trophozoite and schizont
stages sequester in the microcirculation of vital organs, compromising
microcirculatory flow and organ function. A preponderance of these more mature
parasite forms in the peripheral blood is thought to represent a larger sequestered
parasite burden and this is associated with more severe disease and poor disease
outcome (Sanghamitra Satpathy, et all., 2014).
5
While our results show that wet mount preparation from both staining
methods (NMB and Giemsa) provided almost identical reticulocyte counts, the
Giemsa method is marginally preferable to NMB due to its low cost and longer
shelf life. However, Giemsa requires longer staining time (15 minutes) than NMB.
Based on this study, a 5% Giemsa solution is recommended for the staining
procedure (Wenn Chayou Lee, et all., 2013).
Our use of the wet mounting method (NMB and Giemsa), kept viable
parasites alive during the examination period. The active movement of living
parasites were easily differentiated from the static reticulate matter of
reticulocytes. Dead parasites were differentiated from reticulocytes based on the
darker staining pattern of the latter. In most cases, parasitic and host reticular
matter possess a discernible difference in thickness, opacity, and refractive
indices; light that has travelled through these entities is subjected to different
degrees of optical interference. Such optical effects are further magnified by the
liquid medium of wet mount, which has refractive index higher than that of air.
However, such optical phenomena are not available on the dry smear. Hence, the
optical differentiation of living parasites, dead parasites, and reticulocytes is best
contrasted in wet mounting method. Certainly, our preference for wet mount
staining concurs with earlier work that used wet mounts of subvitally stained
blood from vivax malaria patients to confirm the preference of Plasmodium vivax
for reticulocyte invasion (Juan Jose Barcia, 2007).
CHAPTER III
RESEARCH METHODS
A. Date and Time
Day/ Date : Wednesday, October 6th 2021
Time : 13.00 – 14.40 pm
Place : 2nd floor Biology Laboratory, Department of Biology, Faculty of
Mathematics and Natural Sciences, University of Makassar State.
B. Tools and Materials
1. Tools
a. Surgical tools (1 Piece)
b. Surgical board (1 Piece)
c. Pin (Sufficiently)
d. Object glass (2 Pieces)
e. Drop pipette (1 Piece)
f. Microscope (1 Piece)
g. Glass jar (1 Piece)
2. Materials
a. Frog (Rana cancrivora) muscles
b. Methanol
c. Giemsa solution
d. Aquadest
e. Ether
f. Cotton
C. Work Procedures
6
7
Cut the frog (Rana
cancrivora) muscle
as thin as
possible.
Anesthetize the frog Place the chunks
(Rana cancrivora) using of frog (Rana
ether solution, let stand cancrivora)
a few minutes and make muscle meat on a
sure the frog is glass object.
unconscious.
Rinse with distilled Giemsa dye is
dissolved with Soak the glass slide
water, and allow to
distilled water, in a with methanol and
dry until observations
ratio of 1:20, then let it sit for 5-7
are made.
soak the glass minutes, then let it
preparations for 15 dry in the air.
minutes.
CHAPTER IV
RESULT AND DISCUSSION
A. Observation Result
Comparison Picture Observation Picture Notes
1. Arm (Telocentric)
2. Centromere
2 1
1 2
B. Discussion
The chromosomes were stained using the Giemsa method in the experiment.
where the chromosomes to be stained and viewed under a microscope are
obtained from frog muscles that have been thinly sliced with a razor blade, placed
on a table preparation, and stained with Giemsa's solution As a result, the
existence of telocentric chromosomes with apparent but relatively opaque shapes
is obtained. This is due to the fact that the frog's muscle slices are still thicker than
what is required. As a result, chromosomes are not readily apparent in several
studies. As a result, the data we use comes from a group that was able to obtain a
more clearly visible chromosomal observation than the others.
Chromosomes are macromolecular structures that contain DNA, which is
used to store genetic information in cells. On the nuclear membrane,
chromosomes suspended in a semifluid liquid. Chromosomes appear as elongated
entities that are difficult to see under a light microscope. They are known as
chromatids when they are in this state.
Meiosis, also known as reduction division, is a type of cell division that
results in daughter cells that have half the number of chromosomes as the parent
8
9
cell. Meiosis is critical for organisms that reproduce sexually, specifically in the
generation of gametes (gametogenesis). Meiosis occurs in two stages of division,
meiosis I and meiosis II. Meiosis I results in a reduction (reduction) in the number
of chromosomes, but meiosis II results in the same mechanism as mitotic division.
Mitosis is a type of cell division that takes place in somatic cells (the cells that
make up the body). During mitosis, each diploid (2n) parent cell creates two
diploid daughter cells (2n). The number of chromosomes in the daughter cell is
the same as the number of chromosomes in the parent cell. In mitotic division,
cells do not directly divide into two, but go through several phases (stages),
namely prophase, metaphase, anaphase, and telophase.
Chromosome preparation is the process of extracting chromosomes from
actively dividing cells. Chromosomes can be seen in tissue cells with a high rate
of cell division. The goal of chromosomal preparation is to achieve cells in the
metaphase stage by halting cell division. A chromosome is a lengthy DNA
molecule that includes some or all of an organism's genetic material. Most
eukaryotic chromosomes feature packing proteins called histones that, with the
help of chaperone proteins, attach to and condense the DNA molecule to maintain
its integrity.
Chromosomal analysis, also known as karyotyping, can be performed to
assess the level of ploidy (diploid, triploid, and so on) and the features of a
species. Making chromosomal preparations allows you to observe the size and
number of chromosomes. As a result, unique talents are required in the production
of chromosomal preparations. There are two methods for making chromosomal
preparations: the solid tissue technique and the blood culture technique. The solid
tissue technique of chromosomal preparation was used in this practicum since it is
less expensive and takes less time.
The chromosomes are stained so that they may be seen under a microscope.
Although the staining mechanism is not well understood, Giemsa is the most
commonly used dye to color chromosomes (Denton). Giemsa is employed in both
thin and thick review preparations. Giemsa's active ingredients include eosin Y
10
molecules and methylene blue. The quality of the staining results vary according
to the dye ratio utilized.
CHAPTER V
CLOSING
A. Conclusion
Based on the results of the experiments, it can be inferred that chromosomes
are macromolecular structures that contain DNA, which is used to store genetic
information in cells. On the nuclear membrane, chromosomes suspended in a
semifluid liquid. As a result, the existence of telocentric chromosomes with
apparent but relatively opaque shapes is obtained. This is due to the fact that the
frog's muscle slices are still thicker than what is required. As a result,
chromosomes are not readily apparent in several studies. As a result, the data we
use comes from a group that was able to obtain a more clearly visible
chromosomal observation than the others. Chromosome preparation is the process
of extracting chromosomes from actively dividing cells. Chromosomal analysis,
also known as karyotyping, can be performed to assess the level of ploidy
(diploid, triploid, and so on) and the features of a species.
B. Suggestion
According to the results of the trials, the practitioner should be more careful
in their activities using equipment. The assistant should pay attention to the
practitioner so that all of the practitioners are serious about the experiment. The
laboratory, on the other hand, is expected to be able to supplement the instruments
and materials that will be utilized in the experiment.
11
BIBLIOGRAPHY
Arsal, Andi Faridah. 2018. GENETIKA I Arif Memahami Kehidupan. Makassar:
Badan Penerbit Universitas Negeri Makassar.
Barcia, Juan Jose. 2007. The Giemsa stain: its history and applications. Int J Surg
Pathol. Vol. 15 No. 3
Crump, Martha L. 2015. Eye of newt and toe of frog, adder’s fork and lizard’s
leg: the lore and mythology of amphibians and reptiles. Chicago : The
University of Chicago Press
Lee, Wenn Chayou, Russell B, Lau YL, Fong MY, Chu C, et al. 2013. Giemsa-
Stained Wet Mount Based Method for Reticulocyte Quantification: A
Viable Alternative in Resource Limited or Malaria Endemic Settings.
PLOS ONE Journal. Vol. 8 No. 4
Satpathy, Sanghamitra, Mohanty, Akshaya, Parthasarathi, Mishra, Saroj, Behera,
Prativa, Patel, Goutam, Dondorp, Arjen. 2014. Comparing Leishman and
Giemsa staining for the assessment of peripheral blood smear preparations
in a malaria-endemic region in India. Malaria journal. Vol. 13
Syaifudin, Mukh., 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
Witono, Joko Ridho. 2008. Kilas Balik Penelitian Kromosom Palem Indonesia.
Berita Biologi JurnalIlmiah Nasional. Vol 9. No.2
Yudha, Donan Satria, Rury Eprilurahman, Trijoko, Muhammad Faisal Alawi,
Asmaa’anugerah Tarekat. 2011. Keanekaragaman Jenis Katak Dan Kodok
(Ordo Anura) Di Sepanjang Sungai Opak Propinsi Daerah Istimewa
Yogyakarta. Jurnal Biologi. Vol. 18 No.2
12
ATTACHMENT
