PRACTICAL REPORT OF ANIMAL MICROTECHNIQUE

Group 2
LUSI KAROHMAH B1K014022
RELIGYA DEVYANTI B1K014023
LA GAYO BILLY MORA B1K014027
LUTFIA NIRWANA B1K014029
DEVI PRATIWI B1K014030

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2017
Title:
Blood Smear

INTRODUCTION
A. Background
Blood preparations are a permanent preparation, a preparation that is durable for
many years. This permanent preparation of the manufacturing process is quite
difficult, requires a variety of chemicals, need careful planning and thoroughness. The
purpose of making a permanent preparation is to provide the relevant object is always
available at any time is required in general, the procedure of making permanent
preparations through stages: fixation, washing, dehydration with staining insertion,
dealkoholization / clearing, mounting or closing and labeling (Rudyatmi, 2014).
Smear preparations are preparations that the process of making with the method of
applying or smear, ie by removing or making a thin film or film a substance in the form
of liquid or non-liquid over a glass of clean and fat-free objects. Next do the fixation,
coloring it, and cover it with a glass cover to be observed under a microscope. The
preparation of blood smear is to study the structure of erythrocytes, leukocytes, and
platelets.
Blood smear making using giemsa dye or also called Romanowski staining. The
principle of giemsa staining is the black precipitation formed by the addition of
methylene blue and eosin solution dissolved in methanol. Giemsa staining is used to
distinguish cell nuclei and cytoplasmic morphology from red blood cells
(erythrocytes), white blood cells (leukocytes), and platelets.
Blood is one connective tissue that has a function as a connector (transport device)
whose cells are retained and carried in a liquid (plasma) matrix. Blood is heavier than
water and more viscous. This fluid has a pH of 7.35 to 7.45. Blood color varies from red
to deep bluish red, depending on the oxygen levels brought by the red blood cells
themselves (Subowo, 1992).
More than half of the blood is a liquid (plasma), most of which contain dissolved salts
and proteins. The main protein in blood plasma is albumin. Other proteins are
antibodies (immunoglobin) and clotting proteins. Basically, blood has three main
functions: helping to transport food substances, protecting or protecting from foreign
matter and regulating the water content of the tissues, regulating body temperature
and pH setting (Subowo, 1992).
1. Red blood cells (Erythrocytes)
Erythrocytes are biconcave discs, which are rounded with a central curve and 7.65
micrometres in diameter. Erythrocytes are the most numerous cells compared to
other cells. Erythrocytes contain hemoglobin, which serves to bind red blood cells
and carry oxygen from the lungs and deliver it throughout the body's tissues.
2. White blood cells (leukocytes)
The amount is less, with a ratio of about 1 white blood cell to every 660 red blood
cells. There are 5 main types of white blood cells that work together to build the
main mechanisms of the body against infection, including producing antinodies.
Differentiated by the presence or absence of granules divided into granulocytes
(Neutrophils, Eusinophils, and basophils) and agranulocytes (lymphocytes and
monocytes).
A) Granulocytes
1. Neutrophils
Neutrophils work to help protect the body against infections of balteri, fungi
or harmful microorganisms that enter the body. It also plays a role in the
digesting or phagocytosis of foreign matter remnants of inflammation. There
are two types of neutrophils, ribbed and segregated neutrophils. Neutrophils
have 3-5 lobes connected by thin chromatin threads.
2. Eusinophils
Eusinophils have rough and large cytoplasmic granules. This cell has a dual
core and a diameter of 12-15 micrometers. It functions as a fat phagocytosic.
It will increase as allergies or parasitic diseases occur, but will decrease
during prolonged stress.
3. Basophils
Basophils have a large number of cytoplasmic and irregular shapes.
B) Agranulocytes
1. Lymphocytes
It has a smaller size than macrophages and neutrophils. Has a relatively large
core, rounded slightly concave on one side.
2. Monocytes
Leukocyte cells are relatively large 3-8% of normal leukocytes, diamternya 9-
10 micrometers. The core is usually eccentric, the indentation in the shape of a
horseshoe.
 3. Platelets
It is a relatively cell-like particle, smaller than the erythrocyte or leukocyte.
Play a role in blood clotting process after injury. Platelets collect in areas that
are bleeding and activated. Furthermore, platelets will attach to each other and
agglomerate to form filament or fibril closure cover.
B. Objectives
Student be able to prepare a blood smear preparation using the smear method and
staining, and student be able to analyze the blood smear by looking at the shape and
structure of the blood cells.

TOOLS, MATERIALS, AND METHODS

A. TOOLS
1. Glass object (glass object) and glass cover (cover glass/cover slip)
2. Franke sterile needle
3. Lancet Device + Auto Lancet
4. Spuit 1 ml
5. Drop pipette
6. Staining jarr
7. Sucking paper
8. Light microscope
B. Materials
1. Chicken blood.
2. Human blood.
3. Giemsa dyes
4. Tap water
5. Methanol (fixative).
C. Methods
1. Each group of practicum prepares 6 glasses of new objects, then cleaned with cotton
wetted with 70% alcohol solution and dried air.
2. Blood is taken from humans then before the blood is taken, the hands of probandus
are washed with soap and rinsed with running water. Wipe the fingertips to be
drawn with cotton wool that has been moistened with 70% alcohol. Blood is taken
from the tip of the finger no 2 or 3 or 4 by piercing one of these fingers using a
 sterile lancet needle. Stabbing is done in such a way that at least the tip of the needle
can enter as deep as 2-3 mm.
a. Chicken blood samples are taken using needle injection through the veins in the
wing, intracardi-cally or through the carotid artery.
b. Place a drop of blood on a clean glass. Hold the second glass as.
3. While maintaining contact on both glass objects, pull the object glass backwards so
that it touches the blood drops.
4. Maintain contact between the two glasses of the object and push the glass of the
object above to form a smear.
5. While maintaining contact on both glasses, pull the object glass backwards so that it
touches the drops of blood.
6. Maintain contact between the two glasses of the object and push the glass of the
object above to form a smear.
7. The smears are dried and then fixed with methanol solution for 3-5 minutes, dried
and then stained by dripping the Giemsa solution on top of the smear and sterilized
for 10 minutes or until the edges of the dye have appeared to dry out. Rinse the
stsining with running water at a small discharge.
8. A colored apple is dried and then observed under a low light magnification
microscope then with high magnification. Identify the type of visible blood cell,
observe the color absorption and calculate the proportion of each cell type.
Preparations are photographed as documentation and report material.
9. Record the result and submit the copy to the assistant as a temporary report.
10.Collect slides of lab results that have been labeled each group to the assistant

RESULTS AND DISCUSSION

A. Result

Figure 1. Microscopic of Blood Smear Method in Human Magnification 40 x 10

 Figure 1. Microscopic of Blood Smear Method in Chick Magnification 40 x 10

B. Discussion
Preparation of blood smear, this is done by smear method. The blood sample used is
human blood. Based on the results and photos obtained during observations under the
microscope, blood smear preparations with Giemsa staining is seen quite well and can
see the presence of erythrocytes and some kinds of leukocytes that appear protruding
with purple. The amount of erythrocytes appears most prominent when compared
with leukocytes.
Based on Murtiati, et al (2010), blood smear can also be used to identify the presence
of parasites such as malaria, microfilaria, and others. However, at this lab, only
observations were made to determine the description of the shape of the various blood
cells and to assess the percentage of observed blood cells.
Blood smear is done by using fresh blood substances derived from capillaries or OP
veins. OP on this lab is Lutfia Nirwana. First student draw blood from the tip of the
index finger of the left hand using blood lancet or injectable syringe then mix it with
EDTA so as not to freeze quickly. After that student put it to the glass object. Then
touched the cover to the blood drops until the blood widened. Next form an angle of
30-400 with a cover glass, then moved to the left form a blood smear that is not too
thin or too thick because if too thick then the observations under the microscope will
look unclear because of blood cells piled up.
After getting a good preparation (not thick and thin), then allow it to dry, after which
drip methanol onto the dye until the covered blood covered it all and leave it for 5
minutes. The function of methanol is to fix the blood so that blood is not lost when
observed. Subsequently the preparation is dripped with giemsa that has been diluted
with water and allowed it for 20 minutes and rinsed with water and dried. The
function of giemsa is to color the blood so that it is easily distinguished and can be seen
clearly when observed. This immersion time should not be too long because the blood
can not be seen due to the coloring that is too thick.
Furthermore, after the blood smear has been completed, then conducted by
observation by using a microscope to check blood smear. Before the observation of
blood smear droplets emersi oil first drip, the purpose of this emersi oil is to prevent
damage to the microscope.
At 10 x 10 magnification still looks a blood clot that closely attach and clearly visible
only erythrocytes with biconcave form while the structure and various forms of new
leucocytes can be observed clearly at magnification 40 x 10. The time required to
complete about 45-50 minutes. The discovery of large amounts of leucocytes in blood
smear shows that donors are experiencing pain related to leukocyte function as a form
of defense of the human body.
Erythrocytes appearing in transparent microscopes with rounded, disk-like shapes at
the inner (center) and non-core position, while leukocytes look like cells with a purple
core. The purple color that appears on the leucocytes is caused by a basic alkaline
leukocyte that easily absorbs the Giemsa dye. Leukocytes are seen only a few do not
see the whole range of erythrocytes such as eosinophil, lymphocytes and neutrophils.
The percentage of neutrophils is the most abundant in the blood, reaching 50-70% of
the number of leukocytes present, while the least amount is basophil with a percentage
of only 1% of the various leukocytes. But on observations of this blood smear
preparations look most rise the lymphocytes although the percentage is only 20% in
the blood. Other leukocyte counts such as eosinophil and neutrophil are only visible
1% alone as monocytes and basophils are not seen as possible because there are still
stacked blood films of blood because when blood film making is less thin and flat.
The formation of erythrocytes or erythropoiesis occurs in the red marrow located on
the spine, the sternum (the breastbone), the ribs, the skull, the shoulder blades, the
pelvis and the bones of the limbs (legs and hands). Erythrocytes are short-lived. The
absence of a nucleus in the erythrocytes causes the erythrocytes unable to synthesize
proteins to grow, or to multiply themselves (Dikaamelia, 2008). However, in the
absence of a nucleus in the erythrocytes and with a form of biconcave, the erythrocytes
have an optimal ability to bind oxygen so that the need for oxygen becomes fulfilled.
That's why when a person has sickle cell disease, a disease caused by an erythrocyte
structure shaped like a crescent moon, has less oxygen-binding ability that makes the
patient anemic and weak.
On observation in this lab is not found erythrocytes in the form of other than
biconcave, it means the OP does not suffer from abnormal erythrocyte structure.
Abnormalities in erythrocyte structures can be caused by genetic or environmental
factors.
Later obtained some types of leukocytes, but praktikan not able to identify whether
including basophils, eosinophils, stems, neutrophils, lymphocytes or monocytes. This is
due to the limitation of enlargement on the microscope that is used so that it can not be
seen clearly the shape of the leukocyte cell nucleus. Leukocyte classification into 5
kinds is a classification based on the size of the cell, the shape of the nucleus, and the
presence or absence of cytoplasmic granules so need more careful observation and
good microscope magnification and can also be assisted by using oil emersi.
Neutrophil cells have small pink granules in their cytoplasm. The nucleus has three to
five lobes associated with thin chromatin yarns. Its diameter reaches 9 m samapai 12
m. The eosinophil cell has a rough and large cytoplasmic granule, with a reddish
orange coloration. This cell has a double-dip nucleus, and a diameter of 12 m to 15
m. Serves as weak phagocytic. While basophils have a large number of large
cytoplasmic granules that are irregular in shape and will be purplish to black and show
an S.-shaped nucleus of about 12 m to 15 m (Sloane, 2003).
The leukocyte group of agranulocytes, lymphocytes and monocytes, data were
obtained based on the refers that the midline lymphocytes 6-8 m, 20-30% of the
blood leukocytes, had relatively large nuclei, rounded slightly concave on one side. The
cytoplasm is slight and its basophilic and azurophilic content is small (Efendi, 2003).
While monocytes are large leukocyte cells 3-8% of the normal leukocyte count,
diameter 9-10 um but on dry blood preparation diameter reaches 20 m or more. The
core is usually eccentric, the indentation in the shape of a horseshoe (Efendi, 2003).
The most common type of white blood cell is neutrophil with a percentage of 50-70%,
while the least is basophyl, that is 0.1-0.4%. Monocytes serve to kill bacteria, the
function of monocytes is the same as neutrophils, only the numbers are different. A
high number of monocytes show diarrhea as infection occurs. Based on observations,
the number of monsters is small, so neutrophils are less active in responding to tissue
destruction. In other words, the number of neutrophils in the blood that should have a
high amount / amount in the blood decreases in number. Lymphocytes serve as a key
element in the immune response. Lymphocyte levels are widely suspected because of
the small number of neutrophils in the blood. So as to maintain immunity, the
lymphocytes work actively.
Neutrophils are associated with the body's defenses against bacterial infections as well
as other small inflammatory processes, and usually also provide the first response to
bacterial infections; Activity and the death of neutrophils in large numbers leads to the
presence of pus. Eosinophils are particularly associated with parasitic infections, thus
increasing eosinophils signify the number of parasites. Basophils are primarily
responsible for giving an allergic antigenic reaction by removing the chemical
histamine that causes inflammation.
Lymphocytes are more common in the lymph system. Blood has three types of
lymphocytes: B cells make antibodies that bind pathogens and destroy them. (B cells
not only make antibodies that can bind pathogens, but after an attack, some B cells will
retain their ability to produce antibodies as a 'memory' system). T cells coordinate the
resistance response (which persists in the infection) as well as important to resist
intracellular bacteria. Natural killer cell is a natural killer cell (NK) that can kill body
cells that do not show a signal that he should not be killed because it has been infected
or has become cancer.
While the observed platelets are very small thrombocytes look like dots or spots that
are outside the cell and are purple. This is consistent with the theory that platelets are
un-nucleated, disc-shaped cells with a diameter of 1 - 4 micrometers and a volume of 7
- 8 fl. The normal value of platelets varies according to the method used. Normal
platelet count according to Deacie is 150 - 400 x 109 / L. When Rees Ecker method is
used the normal thrombocyte value is 140 - 340 x 109 / L, using the normal price
Coulter Counter 150 - 350 x 109 / L.
Based on the reference also mentioned that the percentage of red blood cells
(erythrocytes) in the body is the largest. Whereas leukocytes have fewer amounts than
erythrocyte cells. In Sloane (2003), it is mentioned that the number of erythrocytes in
healthy men reaches 4.2 to 5.5 million cells per mm3 and about 3.2 to 5.2 million per
mm3 in healthy women, whereas the normal number of leukocytes is 7000 to 9000 per
mm3 and platelets are 250,000 to 400,000 per mm3. This is in accordance with the
observations of the amount of erythrocytes> platelets> leukocytes. Although there are
at least three of the three blood cells present, the leukocyte function in the body is very
 important, where in a state of sickness or foreign body attacked the number of
leukocytes may increase.
The resulting preparations do not all show good results, it can caused by some error:
1. Error procedure performed by student at the time of making smears or blood film,
so that the cells there are damaged because of distress.
2. Lack of student skills in the use of microscopes, resulting in less lighting and
focusing.
3. The number of blood droplets during the filming of blood exceeds the capacity, so
there is no capillarity but blood layer buildup occurs.
4. The position of the glass of object B does not reach 45 at the end of the short edge
so that the friction of the blood film is not maximal.

CONCLUSSION
1. Blood preparations can be made by smear method.
2. Staining on blood smear preparations using Giemsa 3% dye that serves to distinguish
the non-colored chopsticks Giemsa clearly with leukocyte colored contrast so that can
be distinguished between the nucleus with the rest of the cell.
3. The form of red blood cells (erythrocytes) is biconcave round and has no nucleus, while
white blood cells (leukocytes) are larger in size with multiple forms, with both
granulocytes and agranulocytes. Leucocytes have a nucleus. This blood smear appears to
contrast with the purple color of the Giemsa dye.

REFERENCES
Lauralee, Sherwood. 2001. Fisiologi Manusia dari Sel ke Sistem. EGC: Jakarta.

Marianti, Aditya.2014. Petunjuk Praktikum fisiologi Hewan. Semarang: Biologi FMIPA

UNNES.

Murtiati, Tri dkk. 2010. Penuntun Praktikum Anatomi dan Fisiologi Manusia. Jurusan Biologi
FMIPA Universitas Negeri Jakarta.

Pearce, Evelyn C. 2002. Anatomi dan Fisiologi untuk Paramedis. Jakarta: Gramedia
Pustaka Utama.

Rudyatmi,Eli. 2014. Bahan Ajar Mikroteknik. Semarang: Jurusan Biologi FMIPA UNNES.

Sherwood, Lauralee. 1996. Fisiologi Manusia. Jakarta: ECG.

Sloane, Ethel. 2003. Anatomi dan Fisiologi Untuk Pemula. Jakarta: EGC.
Subowo. 1992. Histologi umum. Jakarta: PT.Bumi Aksara.

Ganong, William F. 1999. Buku Ajar Fisiologi kedokteran. Jakarta: ECG.

TITLE:
Span Preparations

INTRODUCTION
A. Background
These preparations are usually made at the time of surgery (sectio). Tissues that can be
used for span preparations include sub-cuticle tissue (loose connective tissue or tissue
containing fat cells), mesenterium, and pericardium (Mallory, 1936).
Span preparation is usually performed on cytochemical testing to see enzymes such as
phosphatase and hyaluronidase, or on cytological / histologic testing to see mast cell
morphology, fibroblasts, histiocytes, macrophage, collagen fibers, elastin fibers, reticular
fibers or connective tissue tissue matrices. This method is used to evaluate tissue
abnormalities and the presence of specific parasites (Mallory, 1936).
Span preparation requires skill and art because the tissues used are generally very thin
and tear easily (eg mesenterium and pericardium), although these two tissues are
relatively easy to absorb the dye. The sub-cutis tissue is relatively easier to take but the
presence of fat on the tissue is often disruptive to the coloring and mounting process (Ross,
2011).
Preparation of span are generated through several stages including: tissue taking, tissue
stretching over glasses, tissue fixation, staining, dehydration, clarification and closing with
a glass cover. Tissue retrieval, stretching and fixation should be done as soon as possible
after the animal is switched off, this is to maintain the freshness of the tissue so that the
tissue obtained. This condition is necessary for the structure and biochemistry of tissues to
be as close as possible to the conditions (Mobini, 2015).
As at the time the animal is alive. The delay in tissue retrieval and fixation will result in
a tissue that is in post-mortem condition. The commonly used fixative solution is methanol
whereas the commonly used dyes are Mallory Triplestains and Mason's Trichrome. With
Mallory dye, the collagen-containing fibrils will be strongly blue; Cartilage, bone, mucus
and amyloid blue-colored cryptic; Nucleoli, cell nuclei, myoglia and fibrin are red; The
blood corpuscles and myelin are yellow in color while the elastin fibrils are pale pink, pale
yellow or uncolored. With Mason's Trichrome dye, the nucleus of the cell is black;
Argentaffin granules are black or red; Cytoplasm of neuroglia fibers, keratin and
interselular fibers bright red; Strong blue collagen and blue mucus while the Golgi
apparatus is clear or uncolored (McFarlane, 1944)
B. Purpose
Able to made span connective tissue preparation

TOOLS, MATERIALS, AND METHOD

A. TOOLS
1. Dissecting kit
2. Object glass and cover glass
3. Staining jar
4. Tissue paper
5. Light Microscope
6. Timer
B. MATERIALS
1. Sub-cutis tissues from chicken
2. Malory Triple Stain
3. Dissolven Alcohol (70, 90, 100%), Xylol and entelan new
C. METHOD
1. Prepared a objects glass and a new cover glass and clean it with a 70% alcohol
solution.
2. Cutted the chicken neck with a knife.
3. Disected the chicken using surgical kit that has been provided so that the abdominal
cavity can be seen clearly.
4. Took the chicken sub-cutis tissue as wide as 3 cm2, then immediately stretched it
over the glass as flat and thin as possible.
5. Fixated the tissue in to methanol solution for a 3 minute.
6. Stained with 0.1% Acid Fuchsin sinus for 3 - 5 minutes and washed with aquades.
7. Inserted into PMA (Phospho Molybdic Acid) 1% during 5 minutes, washed with
aquades.
8. Moved into Malory dye (Mixture of: Oconnective tissue G 2.0 gr, Aniline blue 0.5 gr,
Aquadest 100.0 cc, Acidum Aceticum Glaciale 7.5 cc) for 2 minutes.
9. Transfered the preparation in to 95% alcohol solution by dipped it several times.
 10. Dehydration process was conducted in 100% alcohol solution for 1 minute, clear
in xylol solution, then covered with cover glass with entelan new or new balsam
adhesive.
11. Observed the connective tissue of preparations that have been colored under a light
microscope and observe the visible components and their color specifications.
12. Recorded the tissue components.

RESULT AND DISCUSSION

A. Result
Table 3.A.1. Span Connective Tissue Component Identified
No. Color Identified Chicken
1 Strong Blue -
2 Faded Blue +
3 Red +
4 Yellow +
5 Pink-pale or Yellow-pale or Unstained +

Interpretation:
+: Present
-: Absent
Color:
Strong blue : Fibrils containing collagen
Faded blue : Cartilage, bone, mucus and amyloid
Red : Nucleoli, cell nucleus, myoglia and fibrin
Yellow : Blood corpuscles and myelin.
Pink-pale/yellow-pale/unstained : Fibril elastin
 Figure 1. Light microscope view of chicken sub-cutis preparation
B. Discussion
Connective tissue consists of proteins, complex polysaccharides and water as different
mixtures depending on the type of tissue. In intramuscular connective tissues, the main
protein is collagen and another important protein is elastin. In addition, reticular fibers are
actually individual collagen fibrils (type III collagen) which form delicate networks around
muscle fiber. The intramuscular connective tissues in meat is in three hierarchical levels:
epimysium is the layer surrounding the whole muscle, perimysium contained the large
blood vessels and nerves of the muscle which surrounds bundles of muscle fibers , and
individual muscle fibers are surrounded by the endomysium (Puchtler,1958).
Mallory's trichrome stain is a stain utilized in histology to help with the imaging and
identified different macromolecules that make the cell. It uses three stains: aniline blue,
acid fuchsin, and orange G. As a result this staining technique can reveal collagen, ordinary
cytoplasm, and red blood cells. It is helpful, therefore, in examining the collagen of
connective tissue (Mallory, 1936).
For tissues that are not directly acidic or basic, it can be difficult to use only one stain
to reveal the necessary structures of interest. A combination of the three different stains in
precise amounts applied in the correct order reveals the details selectively. This is the
result of more than just electrostatic interactions of stain with the tissue and the stain not
being washed out after each step (Masson, 1929)
The fibres of dermal and loose connective tissue collagen are coloured green by the
Light Green counterstain, or fibre stain of the Mallory trichrome sequencethe collagen
fibres of tendon retain the initial red dye of the Acid Fuchsin. The collagen of tendons
which have been completely relaxed by previous surgical intervention no longer retains
the initial red stain, but is coloured by the counterstain. In other hand, the normally green-
staining dermal collagen fibres will retain the initial red stain throughout the staining
sequence if they are stretched before fixation These extraordinary differences in the
staining of collagen fibres (Mallory, 1936).
Mallory trichrome procedure which is to changes in the tensional state of the collagen,
are a kind like to the differences in the staining of stressed and unstressed bone. In
discussing the mechanism of the differential staining of collagen and other connective
tissue components by the Mallory trichrome procedure, the background dye is displaced by
phosphomolybdic acid so that collagen is coloured by the counterstain of Aniline Blue (or
in the case of the Mallory trichrome procedure, Light Green) (Mallory, 1936).

Conclusion
The method of spanning preparation was given a good quality of imaging and
interpetating the connective tissue from chicken of which stained by mallory stainer and
given a clear differences between component.

REFERENCE

Mallory FB. 1936. The Aniline Blue Collagen Stain. Stain technol. (11): 101-102

Masson P. 1929. Some Histological Methods. Trichome stainings and their preliminary
technique. J Techn Meth. (12): 75-90

McFarlane D. 1944. An Easily Controlled Regressive Trichomic Staining Method. Stain

techol. (19): 29-37

Mobini B. 2015. Histological Properties of Intramuscular Connective Tissue In Native

Chickens. GASJ. 2(1): 105-109

Puchtler H, Isler H. 1958. The Effect of Phosphomolybdic Acid on The Stainability Of

Connective Tissue By Various Dyes. J Histochem Cytochem. 262-270 p.

Ross, Michael H. 2011. Histology: A Text and Atlas. Philadelphia: Lippincott Williams &
Wilkins. 5-6 p.
Visual Histology [Internet]. c1994-2005. Long-Month (LM): American Histology
Association: [cited 2017 Jun 09]. Available from:
http://www.visualhistology.com/products/atlas/VHA_Chpt3_Connective_Tissue.html
Title:
Parafin Methods (Embedding Methods)

INTRODUCTION
A. Background
The organ is an arrangement of parts of the organism, whose purpose is to perform a
particular function or a closely related union. A network is a collection of cells that
have a particular function that is typical for its development. For example, the
epithelial tissue may consist of one or several layers of cells that have developed and
helped to cover the lining, other tissues, muscle tissue composed of muscle-forming
cells (Gunarso, 1989).
The structure of an organism consists of soft and hard parts. These structural
differences will determine the method used to prepare the preparations. The soft
structure generally uses the paraffin method (slice method). Paraffin method is a
method that will be made with the method of slices. There are 3 kinds of methods for
hardening the tissue or organ to be sliced, ie the frozen method, the seloidin method,
the paraffin method, and the double planting method. Currently the most commonly
used method is the paraffin method Karen almost any kind of tissue can be cut or sliced
properly when using paraffin method (Mannus & Robert, 1960).
Paraffin method is a way of making permanent preparations by using paraffin as an
embedding medium with a thickness of approximately 6 m-8 m slices. This method
has thinner slices than using a frozen method or a thick slice of seloidin method of
approximately 10 m. The process is also much faster than the method of seloidin. In
addition, the paraffin method also has an ugliness that the network becomes hard,
shrink and easily broken, large networks become unworkable (Gunarso 1986).
According to Sugiharto (1989), the paraffin method includes the slice method which is
a routine or standard method. Microscopic observation of a normal network of things
as well as a disease (pathological) will be better if done from well prepared
preparatory tissue, has been done thin enough, and given the appropriate coloring, so
that various elements of the network Researched more easily observed. Thus, not only
microanatomical research can be done, but also provides convenience in distinguishing
the various changes that occur in the tissue cells under study. Sometimes some types of
tissue require special treatment to be able to examine it, as in the case of the type of
staining that should be used for a certain type
 The paraffin method includes the widely used incision method, since almost all tissues
can be cut with this method. Microscopic observations of a tissue under various
conditions and various tissue elements can be observed or researched through a
permanent preparation prepared by paraffin method. Preparation of preparations by
paraffin method is the most commonly used method for making permanent
preparations, either in plants or in animals (Dasumiati 2008).
B. Objectives
The objectives of practical class this time are to know the process of making slices of
animal organs using embedding method with staining at the beginning.

TOOLS, MATERIALS, AND METHODS

A. TOOLS
1. Knives and surgical instruments
2. Bottle sample
3. Beaker glass volume 50 ml
4. Oven incubator with thermostat
5. Hot plate
6. Mold from cardboard
7. Wood block as holder
8. Pencils and labels
9. Microtom rotate
10. Brush and warm water bowl
11. Aluminum foil
12. Glass objects and glass cover
13. Staining the jar
14. Light microscope
B. Materials
1. Kidney organs, liver, gonads, heart and skeletal muscle tissue from chicken.
2. Neutral buffered formalin (NBF) fixative solution.
3. Alcohol solution 70%, 90% and 100%.
4. Xylol solution.
5. Regular paraplast (Sigma p3858).
6. Gelatin 1%
7. Mayer's haematoxylin dye and 1% Eosin akuosa
 8. The new Entelan
C. Methods
A. Organ sampling
1. Cut (horse) chicken and rabbit using a knife, turn off the fish with cervical
dislocation and turn off the fish damaging his brain.
2. Surgery of chicken, rabbit or mice and fish using a surgical tool that has been
provided.
3. Lift the heart, heart, kidneys, gonads and muscle tissue from each animal sample,
wipe off the blood and fixed with NBF in the sample bottle for at least 24 hours.
Note that the fixative volume is at least 10 times the volume of the network
sample. If the organ is too large, the organ can be cut into several parts. If the
sample will be fixed for more than 24 hours, then on the 2nd day. NBF is replaced
with a new one. In such conditions the sample may be left in the NBF for several
weeks as long as the fixed fixation solution is still clear
B. Network Processing For Embedding
1. Washing. If the sample is fixed for 1-3 days, wash the sample in alcohol 70% for 3
times then soak in 70% alcohol for 45 minutes and then proceed with
dehydration. If the sample was fixed for more than 3 days after washing with
70% alcohol, the sample was immersed in 70% alcohol for 6-12 hours and then
continued with dehydration. The purpose of leaching is to remove fixative from
within the network.
2. Dehydration. The dehydration stage is carried out by soaking the sample in an
alcoholic solution of stratum ranging from 70%, 80%, 90% and 2x100%
respectively for 45 minutes. The purpose of dehydration is to remove the water
contained in the tissue so that the clarification process can take place effectively.
3. Purification. When dehydration is performed in an alcohol solution, the cleansing
process is also called dealcoholization. The purpose of this stage is to remove
dehydran from within so that at the time of paraffin infiltration can enter
perfectly into the network. This stage is done by soaking the sample in mixture an
alcohol: xylol (1: 1), alcohol: xylol (1: 3), twice pure xylol each for 30 minutes.
Caution, do not allow too long samples in xylol solution because the sample will
become too hard.
4. Infiltration. The stage is carried out in the incubator oven at a temperature of 58-
60C (corresponding to the paraplast liquid point). The samples were immersed
 in a mixture of xylol: paraffin (1: 1), xylol: paraffin (1: 3) each for 45 minutes and
2 times in pure paraffin respectively for 60 minutes. Time can be changed
according to sample size. Caution, do not leave the sample too long in paraffin
because the sample will become too hard. Note also the incubator temperature,
try not more than 60C because the sample will be hard and easy to crack. The
error at this stage can not be fixed.
5. Embedding. For tissue planting in paraffin blocks, prepare prints of cardboard
according to the sample size (the size of the mold is approximately 3 times the
sample size). Pour the liquid paraffin into the mold about 2/3 of the height of the
mold. Place the mold over the ice block briefly so that the paraffin on the base of
the mold is slightly compacted. Implant the sample into a somewhat dense
paraffin, set its position according to the desired tissue slicing orientation. Add
liquid paraffins up to 4/5 prints then keep the holder from the labeled wooden
blocks. Allow paraffin to freeze in room temperature.
6. Evaluation of embedding results. Good tissue penetration results are
characterized by: paraffin blocks look clear without bubbles air, no visible
boundary between paraffin and sample, xylol odorless block and when sliced
paraffin or sample is cut well, not crushed, not felt hard and sample still bound by
paraffin tape. Some factors that can cause the embedding process to be imperfect
include: dehydration, clarity and infiltration that is less perfect.
7. Record your embedding result and submit the copy to the assistant as a
temporary report.

RESULTS AND DISCUSSION

A. Result

Figure 1. The Result of Liver Preparate with Embedding Method

B. Discussion
The first step in animal paraffin method is organ harvesting from the animal body, then
the animal organ is fixed with NBF solution. Fixation is a process done to maintain the
condition of the network. The purpose of fixation is to preserve all cell structures so
that they are as close to or equal to life as possible, to maintain cell morphology as
before, to prevent the occurrence of otolysis, and to prevent the growth of bacteria or
fungi. Several types of materials that can be used as fixation of a tissue, namely
formaldehyde, alcohol, carnoi solution, zenker solution, helly solution, bouin solution,
susceptibility, omium, and glutaraldehyde (Sudiana, 2005). According to Sulfiandini
(2015) the volume of Neutral Buffered Formalin (NBF) at least 10 times the volume of
the network.
After fixation, a multilevel dehydration is done by immersing successive pieces of
organs into alcohols of 70%, 80%, 90%, absolute alcohols I and II for some 45 minutes.
According to Fadilah et al. (2015) the purpose of the dehydration process is to draw
fluid present in the cytoplasm. Used alcohol is multilevel so that the water content
network can be out bit by bit until the expenditure of water to the maximum.
Dehydrated materials should be able to replace existing water in cells or tissues and be
able to continue the network's frigating fixative capability, as well as being able to
dissolve in the clearing agent.
The next step is dealkoholosasi using xylol: alcohols in stages with a ratio of 1: 3, 1: 1,
and 3: 1, this is done so that the solution of alcohol to be maximized so that the solution
can be bound to paraffin and for the organ can adapt, use xylol I and Xylol II, this step is
done so that the tissue is completely free of alcohol and there is only xylol. Xylol serves
to clarify (clearing) tissue from alcohol (dealkoholization), intermediates that can
dissolve in alcohol at once in paraffin.
The next stage is infiltration. This stage is carried out in the incubator at a temperature
of 58-60C. Samples were immersed in a mixture of xylol: paraffin (3: 1, 1: 1, 1: 3) each
for 45 minutes and 2 times in pure paraffin each for 60 minutes. The sample should not
be too long inside the paraffin because it will become too hard. Incubator temperature
should be noted, try not to exceed 60C because the sample will be hard and easy to
crack. The error at this stage can not be fixed. Paraffin solution is used in infiltration,
serves as a medium for embedding or embedding that is inserted into the tissue
gradually, a substance that occupies spaces within the tissues so that tissue sagging
does not alter its structure.
In the animal organs, embedding is the process of making an organ using a paper box.
This process makes it easy to make very thin slices using a microtome. Some
advantages of using a paper box in embedding that can make the incision direction and
mark a network. The tissue or sample will be planted on the box paper with the
paraffin first frozen at the bottom of the box and after tissue attachment followed by
the closure with paraffin to freeze (Khairul, 2001).
The process of slicing (sectioning) begins with the inclusion of paraffin blocks with
scalpel, so that the surface of paraffin blocks to be sliced with a rectangular microtome.
The location of the blade on the microtome produces the results obtained. The process
is continued with affixing the attachment of organ incision in glass object with albumin.
Albumin is a compound consisting of egg whites and glycerine which serves to glue the
tissue slices on the glass object. Albumin smeared on a glass object flat. When giving
albumin on the glass object should not be too much, because too much albumin will
result in the albumin participate colored which will cause the object glass becomes
dirty. The result of the incision is taken by using a brush carefully. The incision ribbon
is taped to the glass object by using meyer albumin. Glass the object is placed on the
table of the bath. Meyer albumin has an egg white content and glycerine and is an
excellent natural gluten (Hugo, 2008). The dyeing process was performed after the
preparation was depopulated by immersing the preparations on xylol (Khairul, 2001).
Deparafinizasion needs to be done before staining. This step is carried out by damping
the glass of the object containing the paraffin band into xylol I solution for 5 minutes,
then rehydrating it by transfer into xylol II solution for 5 minutes. The xylol solution
was washed with a 90% alcoholic alcohol solution, 80% alcohol, 70% alcohol
respectively 30-40 dye, then rinsed with distilled water.
Staining is done after paraffin is dissolved with xylol because it can not bind water,
whereas coloring binds water. Included in the hematoxylin dye aims to color the cell
nucleus (purplish blue), while the eosin to color the cytoplasm becomes pink. If the
staining is good enough, continue with dehydration by dipping the tissue into an
alcohol solution of 70%, 80% and 90%, absolute alcohols I and II, each 40 dyes. Clear
the tissue in xylol solution I and xylol II each for 40 dyes.
The last process is mounting. Closure (mounting) is maintained so that the network
should not dry. Mounting is done by using adhesive (entelan) then covered with glass
cover and dry. The preparations are labeled on the object glass, then examined
microscopically.
 Based on observations, some parts of the organs that are made are not clearly visible
due to the presence of water molecules. The presence of water molecules is due to
errors during the dehydration procedure. Dehydration which aims to attract water
molecules from cells does not run normally, so many water molecules are masil left in
the cell. Another error found when evaluating is that in some parts of the preparations
observed there appears to be piles or folds. This can be due to the fact that while
performing the procedure of afixing the paraffin ribbon does not stretch perfectly.
The results show that there is a well-stained preparation of the liver organ not
completely cut off so that the liver parts are incomplete, but there are too thick tissues
in the staining. According to Kurniawan (2010) there are some organs that fail to
become a preparation, this may be due to the lack of accuracy and skill when slicing
block paraffin when using mikrotom, so the network band sheets get too thick and
difficult to be observed under the microscope. In addition, some preparations can not
be clearly recognized which part is used from the experimental material because at the
time of the staining process, the washing and dyeing of the dosage into the alcohol
solution is faulty.

CONCLUSSION
Based on the discussion that has been submitted it can be concluded that how to make the
preparation of animal organ incisions by using embedding method is done through several
stages such as organ harvesting, fixation, dehydration, dealcoholization, infiltration, block
making, planting, pasting in holder, sectioning, afixing, deparafinizasion, rehydration,
coloring, dehydration, clearing, mounting, and labeling.

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