TITLE: Animal Slide Preparation Technique

INTRODUCTION:

Histology is the study of how cells are organized into tissue. The objective of most
histologists is to accumulate enough knowledge of structure in order to understand the functions
of living and dead (fixed or preserved) tissue. Besides that, histology also is the study of the
microscopic anatomy of cells and tissues of plants and animals. It is performed by examining a
thin slice (section) of tissue under a light microscope or electron microscope. The ability to
visualize or differentially identify microscopic structures is frequently enhanced through the use
of histological stains. Histology is an essential tool of biology and medicine.

A microtome is a mechanical instrument used to cut biological specimens into transparent

thin sections for microscopic examination. Microtomes use steel, glass, or diamond blades
depending upon the specimen being sliced and the desired thickness of the sections being cut.
Steel blades are used to prepare sections of animal or plant tissues for light microscopy
histology. Glass knives are used to slice sections for light microscopy and to slice very thin
sections for electron microscopy. Industrial grade diamond knives are used to slice hard
materials such as bone, teeth and plant matter for both light microscopy and for electron
microscopy. Gem quality diamond knives are used for slicing thin sections for electron
microscopy. The most common applications of microtomes are traditional histological technique
that tissues are hardened by replacing water with paraffin. The tissue is then cut in the microtome
at thicknesses varying from 2 to 25 µm (micrometers) thick. From there the tissue can be
mounted on a microscope slide, stained with appropriate aqueous dye(s) after prior removal of
the paraffin, and examined using a light microscope.

Microtome blades are extremely sharp, and should be handled with great care. Safety
precautions should be taken in order to avoid any contact with the cutting edge of the blade. If
one should accidentally drop never try to catch it with the unprotected hand. A recent
development is the laser microtome, which cuts with a femtosecond laser instead of a mechanical
knife. This method is contact-free and does not require sample preparation techniques. The laser
microtome has the ability to slice almost every tissue in its native state. Depending on the
material being processed, slice thicknesses of 10 to 100 µm are feasible. Biological tissue has
little inherent contrast in either the light or electron microscope.

Staining is employed to give both contrasts to the tissue as well as highlighting particular
features of interest. Where the underlying mechanistic chemistry of staining is understood, the
term histochemistry is used. Hematoxylin and eosin (H&E) is the most commonly used light
microscopical stain in histology and histopathology. Hematoxylin stains nuclei blue; eosin stains
the cytoplasm pink. Uranyl acetate and lead citrate are commonly used to impart contrast to
tissue in the electron microscope. Special staining: There are hundreds of various other
techniques that have been used to selectively stain cells and cellular components. Other
compounds used to color tissue sections include safranin, oil red o, Congo red, fast green FCF,
silver salts, and numerous natural and artificial dyes that were usually originated from the
development dyes for the textile industry. Histochemistry refers to the science of using chemical
reactions between laboratory chemicals and components within tissue. A commonly performed
histochemical technique is the Perls Prussian blue reaction, used to demonstrate iron deposits in
diseases like hemochromatosis.

Histology samples have often been examined by radioactive techniques. In historadiography

a slide (sometimes stained histochemically) is X-rayed. More commonly, autoradiography is
used to visualize the locations to which a radioactive substance has been transported within the
body, such as cells in S phase (undergoing DNA replication) which incorporate tritiated
thymidine, or sites to which radiolabeled nucleic acid probes bind in in situ hybridization. For
autoradiography on a microscopic level, the slide is typically dipped into liquid nuclear tract
emulsion, which dries to form the exposure film. Individual silver grains in the film are
visualized with dark field microscopy.

Recently, antibodies are used to specifically visualize proteins, carbohydrates, and lipids: this
is called immunohistochemistry, or when the stain is a fluorescent molecule,
immunofluorescence. This technique has greatly increased the ability to identify categories of
cells under a microscope. Other advanced techniques, such as nonradioactive in situ
hybridization, can be combined with immunochemistry to identify specific DNA or RNA
molecules with fluorescent probes or tags that can be used for immunofluorescence and enzyme-
linked fluorescence amplification (especially alkaline phosphatase and tyramide signal
amplification). Fluorescence microscopy and confocal microscopy are used to detect fluorescent
signals with good intracellular detail. Digital cameras are increasingly used to capture
histological and histopathological image. Alternative techniques include cryosection. The tissue
is frozen and cut using a cryostat. Tissue staining methods are similar to those of wax sections.
Plastic embedding is commonly used in the preparation of material for electron microscopy.
Tissues are embedded in epoxy resin. Very thin sections (less than 0.1 micrometer) are cut using
diamond or glass knives. The sections are stained with electron dense stains (uranium and lead)
so that they can possibly be seen with the electron microscope.

By prepared animal or plant slide, indirectly we know the technique of making the slides.
Besides that, we can observe the tissues and cells of certain organs easily in order to get some
knowledge in this field. We are grateful of having sophisticated instruments that help us in
animal slide preparation technique. Furthermore, we are very proud and grateful of God creation
that some cells and tissues have their own functions that make up an organism.

MATERIALS:

Mould Absolute alcohol + xilen (1:1)

Forceps Absolute alcohol
Slides and cover slides Alcohol90%
Microtomes Alcohol 80%
Beakers Alcohol75 %
Normal saline solution Distilled water
70%, 80%, 95% and 100% Ethanol Hematoxilin solution
Bouin’s solution 0.5% Hydrochloric solution
Toluena solution 0.5% sodium bicarbonate solution
Paraffin solution Scott’s solution
Xilen 1 Eosin
Xilen 2 DPX medium/Balsam Canada
METHODS:

TISSUE PREPARATION:

1. An animal was given to each group.

2. The animal was operated and the organs was taken

a. Liver d. Esophagus g. Uterus

b. Lung e. Heart

c. Intestine f. Ovary / testis

FIXATION:

1. After the organs were separated from the animal, the organs were rinsed with the normal
saline solution that had been prepared.

2. The organs that were rinsed before this were placed into the Bouin’s solution for a night.

DEHYDRATION:

1. After a night, the organs were transferred into the 70% Ethanol solution for 24 hours.

2. Next, the organs were transferred into the 80%, 95% and 100% Ethanol solution for an
hour each of the solution.

CLEARANCE

1. The organs were transferred into the Toluena solution for a night.

2. Do the procedure carefully as the solution has an unpleasant smell and dangerous.
EMBEDDED TECHNIQUE:

1. The different organs were put into the different small beakers that contain the paraffin
solution (60°C) for 1 hour.
2. The paraffin solution was poured into the large beaker and was replaced by another
paraffin solution for another 1 hour.
3. Then, after the paraffin solution was removed from the beaker, the paraffin solution was
pour into a mould and put the organ it by the using of forceps carefully. The bubbles are
absent in the mould.
4. The solid paraffin was removed from its mould.

SLICING TECHNIQUE:

1. The paraffin block was sliced after 5 days.

2. Paraffin block was inserted into the microtome holder.
3. The organ sample was sliced in 5 μm thick.
4. The sliced paraffin tapes were transferred onto the slides that have a few drops of
distilled water by using drawing brush. The albumin not the distilled water was drop on
the slide for kidney tissue and liver tissue.
5. The slides were put into the 30°C oven.
SLIDES COLORING:

Harris Haematoxylin-Eosin coloration was used during slides coloring.

1. The slides that contain paraffin tape were taken from the oven.
2. There are 21 solutions in 21 different jars. The slides were put into the solutions
according to the duration that was decided.

SOLUTIONS DURATION(min)

1. Xilen 1 10

2. Xilen 2 5

3. Absolute alcohol + xilen (1:1) 3

4. Absolute alcohol 3

5. Alcohol90% 3

6. Alcohol 80% 3

7. Alcohol75 % 3

8. Distilled water 3

9. Hematoxilin solution 2

10. 0.5% Hydrochloric solution One dip

11. Water flow 10

12. 0.5% sodium bicarbonate solution 5

13. Water flow 10

14. Scott’s solution 5

15. Eosin 5

16. Alcohol 75% 3

17. Alcohol 95% 3

18. Absolute alcohol 3

19. Absolute alcohol + xilen (1:1) 3

 20. Xilen 10

21. Xilen 10

3. Then, DPX medium/Balsam Canada was drop on the slides and was covered by cover
slides.
4. Then, the slides were put inside the 37°C oven for one week before observed with light
microscope.
5. The slides were labeled.
RESULT:

Small intestine of hamster

Magnification power: 10×10

Small intestine of hamster

Magnification power: 10×10

DISCUSSION:

Along this experiment, many diligent and dedicated works have been applied to complete
this task. From the first procedure tissue preparation, we did not have any problems that avoid us
to fulfill the task. We just use our dissecting set and cut through the hamster skin using the right
apparatus and knives. After the skin of the hamster was peel, the organs removed carefully from
the body of hamster. We just took the organs as the procedure statement. During fixation step,
dehydration step, and clearance step we not facing any problems. We just follow the procedure
provided.

During embedded technique, we are instructed to place the small beakers that contain the
paraffin solutions and organs into the oven. But, after an hour, the paraffin solutions are not
melting, so that Pn. Ira instructed us to transfer all the small beakers into the embedder system,
so that the paraffin solutions can melt within an hour. Besides that, in the third step of embedded
technique, we are difficult to lose the bubbles present in the paraffin solutions because it is
concentrated and the solutions are easily to become solids.

In slicing technique, we are able to use the microtome properly, but we are difficult to get
the good paraffin tapes. For some organs like small intestine and ovary, we are easily got the
good paraffin tapes. For another organs, we need to make many paraffin tapes to get the good
ones. We are lucky because we prepared many paraffin block in each organ. During the slicing
technique, we took a long time to finish the slicing of all the paraffin blocks. We can’t finish all
the paraffin blocks in one day, but 3 days.

Then, in the slides coloring, we need to immerse our slides in the 21 jars that contain
solutions that were provided by laboratory assistant. During this step, several tissues are peel off
from the slides. This is because, we need to immersed in 21 jars of solutions and maybe some of
the tissues not stick well to the slides and it is easy to peel off from the slides. Then, during we
drop the DPX medium on the slides, bubbles are present in some of the slides. This is because,
when we put the cover slide on the slide, we need to press the cover slide smoothly because the
cover slide is very thin and to avoid it from broken, so the bubbles still present on the slides.
 After a week, we observed our slides by using the light microscope. Although there are
many bubbles on the slides, we are able to differentiate between the bubbles and tissues. Many of
our slides can be observed and we select the best slide in each organ to submit.