PRACTICAL REPORT OF DEVELOPMENT LABORATORY PREPARATION

PRACTICAL REPORT OF DEVELOPMENT LABORATORY
PREPARATION
Lecture: Purwanti Widhy


Arranged by:
Tri Sulis Setyowati (09312241027)
C. Nulat Panggayuh (09312241035)
Sunjani (09312244033)
M. Ihzatul Faqih (09312244025)
Endah Dani (09312244034)

SCIENCE EDUCATION PROGRAM
MATHEMATICS AND SCIENCE FACULTY
YOGYAKARTA STATE UNIVERSITY
2010
PREPARATION

I. Goal
1. Knowing how to create a temporary preparations of plant
2. Knowing how to make the preparations while the animal

II. Basic Theory
There are many ways in making preparations of plant tissues, such as paraffin method. This method is now widely used, because almost all kinds of tissue can be cut properly when using this method. Merits of this method is that the resulting slice is much thinner than using the method or methods seloidin frozen. With the method of frozen, sliced average thickness above 10 microns, but with thick slices of paraffin method can achieve an average of 6 microns. Slices that are the series can be done easily when using this method. The procedure is much faster than the method seloidin. But this method also has the disadvantage of paraffin tissue becomes hard, shriveled and brittle. Large networks can not be doing, when using this method. Most of the enzymes will dissolve with this method (Suntoro, 1983).
Paraffin method is a way of making preparations to use paraffin as a medium planting (embedding). Steps in the manufacture of such stocks is (Tim MiTek Plant, 2002):
1. Shutdown and fixation: A lot of solutions that can be used for fixation, including the solution of FAA (Formaldehyde Acetic acid-Alcohol), with the following composition: 50% or 70% etilalkohol 90 cc, glacial acetic acid 5 cc 5 cc 40% Formalin. After the material is cut approximately 0.5 cm immediately incorporated into the FAA solution with a ratio of 1: 20 (material 1 / 20 volume FAA), should not exceed eight pieces in the vial. Old fixation in FAA for small or thin material a minimum of 12 hours while for large or heavy materials 24 hours.
2. Aspiration: Aspiration is done by use vacuum (aspirator) and is used with a short interval of time and many times, you can also use a syringe spet. .
3. Washing: Washing is done 2 times in 3 hours with 50% alcohol. Total solution used only appropriate cover material.
4. Dehydration with TBA (Tertiary Butyl Alcohol), and infiltration: Dehydration done with a mixture etilalkohol and TBA in a certain concentration of each solution is named I to V. Johansen
5. Investment (Embedding): Create a rather thick hard box with a size of about 5 X 2.5 X 2 cm (length X width X height), then fill with the liquid paraffin hard earlier in the vial, then enter the material before freezing paraffin . Arrange materials in paper boxes using a heated needle with alcohol or spritus lights and label. After paraffin and frozen material was not swayed, put the paper boxes in cold water. Let the frozen surface of the paraffin, then press all the boxes into the water until the paraffin freezes, or it can also put into the freezer until completely frozen throughout paraffin. Only after the paraffin may be removed from the box.
6. Cutting: Cut the paraffin block into smaller blocks, each of which contains an ingredient. Paraffin blocks were placed on wooden beams in the direction of the desired incision. Annealing is done by melt some paraffin blocks with a needle that has been heated, and then placed in paraffin blocks of wood. Do it several times so that the paraffin blocks firmly attached to the block of wood. The surface of the paraffin blocks that have been posted should be four square atu square. Note that the horizontal side must be absolutely parallel. Material in paraffin blocks cut with a rotary microtome (rotary microtome). Before the cut blocks that have been attached material and the blade is cooled first with cold water (refrigerator), so the temperature paraffin with a knife temperature. Wooden beams that have been affixed with paraffin blocks mounted on the holder contained in the microtome. Arrange the slice thickness (usually between 6-15 microns) by turning the screw on the right side of microtome. Put the knife on the microtome. At the time microtome player is run, the material in the paraffin that has been placed on the holder moves up and down and move forward. Hold paraffin cuts the ribbon-shaped with fine brushes. Paraffin ribbon incision results are stored in cardboard boxes or trays preparations. We recommend cutting is done in air-conditioned room.
7. Annealing the slice: How annealing is as follows:
• Put the glue on glass slide solution for small drops, rub it until the glue evenly on the slide with the fingertips to form a thin layer.
• Put a solution of formalin on glass objects that have been given the tape earlier. Place the slice on top, and place the glass slide above board heater for 30 minutes. Try to keep the paraffin slice evenly on the slide surface. Observe under the microscope dissection. Check if material has been flat incision is true.
• Absorb excess formalin solution contained in the suction side of the incision with the paper.
8. Staining: To color the material that has been affixed to it is to soak the glass slide into dye vessel (vessel coplin), coplin vessel is usually required approximately 12 pieces, depending on the dye that we use. Each vessel is labeled with the name of the substance therein, as well as with a lid.
Staining Safranin - Fast Green:
Xilol 100% 2 -5 minutes
Alcohol 100% 2 -5 minutes
Alcohol 95% 2 -5 minutes
Alcohol 70% 2 -5 minutes
Safranin 1% in Alcohol 70% 12 hours - 1 night
Alcohol 95% 2 -5 minutes
Fast Green 0.1% in Alcohol 95% 5-15 seconds
Alcohol 100% I 2 -5 minutes
Alcohol 100% II 2 -5 minutes
Alcohol 100%: Xilol 100% 1: 1 2 - 5 minutes
Xilol I 2 - 5 minutes
Xilol I 2 - 5 minutes
Examined under a microscope when they are visible colors contrase good then given the Canada balsam and covered with glass cover
http://www.bangkoyoy.com/2010/10/pembuatan-preparat-parafin-jaringan.html

How to make animal preparations
1. Narcotics
In making preparations for animal tissue using paraffin method typically used is either a mammal, for it is necessary for the process to kill the animal in question. Then the animal grugs with using cloroform.
2. Sectio
Clean feathers and dirt on the instrument or organ which will be made preparations. Organ slices approximately 3-5 mm and an area of approximately 1 cm2, so secured until a thorough penetration of the fixative the organ.
3. Labelling
Flakon fixed place of an organ that will be labeled according to type of organ.
4. Fixation
Desired object is cut and fixed in Bouin solution for about 12-24 hours.
5. Dehydration
After the fixative solution removed, replaced with alcohol 70%, 80%, 90% and 96% respectively as much as 3x and each of them for 10 minutes, shaking and overnight. After that, put into absolute alcohol for 10 minutes and shaken.
6. Dealkoholisasi
Dispose of alcohol and then replace it with a substance that is easy to expel alcohol, but then must be biased with paraffin. Which can use as a clearing agent is acetone, benzol, toluol and xilol. Clearing (dealkoholisasi) conducted at the latest 24 hours (overnight).
7. Infiltration
Infiltration is done in an incubator with a temperature of about 55 º C or 60 º C. Before entering parafin pure, then cut a better organ inserted into the mixture xilol: alcohol 1:1 first.
8. Cloaking
Make a cardboard box the size of 2x2x1.5 cm ± square. Object with a pipette dropper priest who has cut a narrow section quickly and put in cardboard boxes, add up to a box full of liquid paraffin. Then let the block at room temperature until hardened. For use of pipette futhermore miss the first flame to melt paraffin pipette attached to the wall. Parafin cloaking done at a temperature of 56 º C for 12 minutes.
9. Incision
Trim the block and the block forms a prism formation. Set microtome with a thickness of 5-10μm. Take a piece of the series with a brush, put on a slide that has given Mayer's albumin and drops slightly water. Heat the hot plate slides above the set temperature lukewarm (34-40) until firmly attached.
10. Coloration
Enter the preparations into xylol for 10 minutes (xilol on a slide using a tissue to inhaled were really dry). Then place it or enter into absolute alcohol, 96, 90, 80, 70, 60, 50, 40 and 30%. If you've finished cleaning with distilled water. Then enter into the preparations Hematoxylin solution for 30 seconds, dipped into distilled water and then wash under running water. When you have finished enter into alcohol 30, 40, 50, 60 and 70% respectively a few seconds. Enter into the preparations eosin solution for 1-2 minutes and then enter into the alcohol 70, 80, 90, 96% and absolute alcohol. Before inserted into xilol, alcohol, smoked until completely dry. Enter into xilol for 20 minutes.
11. Mounting
Is plaid stage. Slide drops with enthelan and then covered with glass cover. Place slide on hot plate to dry.
12. Labelling
Is a labeling phase of the preparations that we make. Label should contain the name of the network, thick slices, the direction of cutting, coloring and date of manufacture.

III. The Materials and Equipment
1. Leaves Sansivera
2. Catfish
3. Microscope
4. Glass preparation
5. Closing preparations6. Needle
7. Razor blade
8. Water


IV. Step Method
Steps to make preparations of plants (the leaf Sansivera)
1. Preparing fresh leaf Sansivera
2. Clean leaves Sansivera
3. Cut the leaves Sansivera into two sections transversely
4. Cutting the middle leaves (sliced crosswise), carefully, and as thin as possible with a razor blade.
5. Sliced formed, move slowly into the glass preparations using a needle.
6. Slices dripping leaves with little water.
7. Closing preparations with preparations closing, try not to have air bubbles.
8. Observing the preparations under a microscope.
9. Recording image making preparations.

Steps to make preparations Rhoe discolor leaves
1. Setting up the Rhoe discolor leaves
2. Clean the Rhoe discolor leaves
3. Cut the bottom surface of the leaf, the color purple, as thin as possible, using a razor blade.
4. Putting on the glass slice preparations by using a needle.
5. Adding a little water on the cut leaves
6. Closing preparations with preparations closing slowly so as not to form air bubbles.
7. Observing the preparations under a microscope.
8. Recording image making preparations.

Steps to create animal preparations (catfish)
1. Preparing fish catfish, which will be sampled.
2. Cut the flesh of fish Catfish
3. Cut the catfish meat, using a sharp razor blade so as to form a thin piece.
4. Put chops in glass preparations by using the needle slowly.
5. Give a little water, then slowly close it to cover preparations, try not to form air bubbles in the preparations.
6. Observing the preparations under a microscope.
7. Recording a picture on the preparations.
8. Repeat steps 2 through 7 with the use of fish Catfish grouse.

V. Data


The Rhoe discolor leave The Sansivera leave


Flesh of catfish Feeler of cutfish

VI. Discussion
In practice this time, that is about making preparations for the purpose of knowing how to manufacture fresh preparations plants and animals. Plants observed were Sansivera, or more commonly known as tongue-in-law plants, with an elongated shape and pointy toes and Rhoe discolor. While the animals used are catfish.


In making preparations plants, observed in this lab is part Sansivera plant leaves, transversely. Step-making leaf preparations are as follows, prepare fresh Sansivera leaves, then when the leaves look dirty, or clean a lot of land formerly attached to the water. Then slice the leaves crosswise into two large sections, then slice the middle leaves (sliced crosswise), carefully, and as thin as possible with a razor blade. After the incision is formed, move slowly into the glass preparations using a needle. Then slice the leaves dripping with water. Then close the lid preparations with preparations, try not to have air bubbles. The last step do is observe the preparations under a microscope. From this practice, implemented obtained the following results,
Leave of Sansivera
In making preparations for the second plant, used Rhoe discolor leaves, which are used are the bottom leaves that are purple. Step-making leaf preparations are as follows, setting up the Rhoe discolor leaves then clean it off the ground attached after it cut the lower surface of leaves, the color purple, as thin as possible, using a razor blade. If you've formed a thin slices and then put the slices on the glass preparations by using a needle. Then add a little water on the cut leaves the next step by closing preparations by closing preparations slowly so that no air bubbles formed. Preparations were then observed under a microscope. And from the lab that conducted the results obtained

Rhoe discolor leaves
Things to consider when making plant preparations are, first in preparing the leaves should prepare parts of the plants had just learned and in good condition. The ideal time for picking is a time through the morning, where the plants are in dry conditions (non-condensing), but not wilted from the heat. If after the process of picking, not directly given treatment, then the plants should be placed within the vase filled with water and placed in a cold room until ready to be dried. While the lab has done the leaves have been plucked a few hours before use, but it leaves no not stored in a vase filled with water or placed in a cold place, but only placed in plastic bags. Harvesting is done also in the morning, allowing the leaves are steamed or not in dry conditions. In preparations image visible presence of air bubbles, this happens because of the closure of preparations that are less precise, it can be tried with the following steps: hold the glass cover with the position of 45o to the glass object, touch the bottom edge of the glass cover on the surface of the medium and slowly lay so that the cover glass located above the glass slide. If there are any air bubbles repeat the work until there are no air bubbles. The placement of material on the glass slice preparations, should be done by dripping a little water on the glass and then put slices of material preparations in preparations that have been poured glass of water and then closed. But in practice that have been implemented there is a mistake, that slice is placed first and then drops with water. It cause air bubble formation in preparations. In the preparations observed Rhoe discolor leaf not seen the color purple as the color of the leaves are cut, it is because the incision that is too thin so that the leaf color is actually not visible, compared with preparations Rhoe discolor the following,

In the picture above, preparations Rhoe discolor leaves bottom looks purple, whereas in the preparations made in the lab this time the purple color does not look like the picture above.
While in the manufacture of animal preparations, in practice this will make the preparations of meat and tentacle (shaft) of catfish. Step-making animal preparations are as follows, which will prepare Catfish fish sampled, then cut the flesh of fish Catfish, then cut the flesh of fish Catfish, using a sharp razor blade that could form the thin pieces, then put a piece of meat on the glass preparations use the needle slowly. To clarify the observation Adding a little water, then slowly close it to cover preparations, try not to form air bubbles in the preparations. After the preparations were observed under a microscope. To make preparations for the grouse, by repeating the steps above. From this practice has been carried out following results are obtained,

Flesh of catfish Feeler of cutfish

In making preparations for these animals is also an error occurs, namely in the figure preparations visible presence of air bubbles, this happens because of the closure of preparations that are less precise, it can be tried with the following steps: hold the glass cover with the position of 45o to the glass object, touch the bottom edge glass cover on the surface of the medium and slowly lay thus located above the cover glass slide. If there are any air bubbles repeat the work until there are no air bubbles. The placement of material on the glass slice preparations, should be done by dripping a little water on the glass and then put slices of material preparations in preparations that have been poured glass of water and then closed. But in practice that have been implemented there is a mistake, that slice is placed first and then drops with water. It cause air bubble formation in preparations. The formation of air bubbles visible in both preparations, namely the catfish whiskers, black line on the edge of the object showed air bubbles, whereas in the first preparations, the catfish meat no visible air bubbles. But in the first preparations do not seem so obvious, it is because the magnification is not quite right or because of lack of sunlight, because when executed practicum overcast sky and the intensity of the sun just a little.

VII. Conclusion
REFERENS
http://www.bangkoyoy.com/2010/10/pembuatan-preparat-parafin-jaringan.html
http://id.wikipedia.org/wiki/Sansevieria
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