Animal Slide Preparation Technique INTRODUCTION : Histology is the study of the microscopic anatomy of cells and tissues of plants

and animals. It is all about the study of structure and function of each tissue types, how those tissues are combined to form the organs and systems of the body, and how those combinations function together. It is performed by examining a thin slice 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. Fixation is a chemical process by which biological tissues are preserved from decay, either through autolysis or putrefaction. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues. Fixation is usually the first stage in a multistep process to prepare a sample of biological material for microscopy or other analysis. Therefore, the choice of fixative and fixation protocol may depend on the additional processing steps and final analyses that are planned. A microtome is a mechanical instrument used to cut biological specimens into transparent thin sections for microscopic examination. Microtome 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. There are some precaution when we are using the microtome while doing the slicing. Microtome use extremely sharp blades. All operations, with the blade, must be done with the

greatest of care. There are some operations where the hands must be brought close to the blade. For these procedures hand protection must be worn for example is cut resistant gloves. Then, the knives need to be cleaned. Disposable knives have an anticorrosion coating that must be removed. Diamond paste must be removed from knives when they are sharpened. The smallest silicon particle can ruin the sections. All dust must be removed. Such careful cleaning must bring the hands close to the blade with the attendant risk. Biological tissue has little inherent contrast in either the light or electron microscope. Staining is employed to give both contrast 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.

MATERIALS: Mould Forceps Slides and cover slides Microtomes Beakers Normal saline solution 70%, 80%, 95% and 100% Ethanol Bouin’s solution Toluena solution Paraffin solution Xilen 1 Xilen 2 METHODS: TISSUE PREPARATION: 1. An animal was given to each group. 2. The animal was operated and the organs was taken a. b. c. d. e. f. g. Liver Lung Intestine Esophagus Heart Ovary / testis Uterus Absolute alcohol + xilen (1:1) Absolute alcohol Alcohol90% Alcohol 80% Alcohol75 % Distilled water Hematoxilin solution 0.5% Hydrochloric solution 0.5% sodium bicarbonate solution Scott’s solution Eosin DPX medium/Balsam Canada

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 1. Xilen 1 2. Xilen 2 3. Absolute alcohol + xilen (1:1) 4. Absolute alcohol 5. Alcohol90% 6. Alcohol 80% 7. Alcohol75 % 8. Distilled water 9. Hematoxilin solution 10. 0.5% Hydrochloric solution

DURATION(min) 10 5 3 3 3 3 3 3 2 One dip

11. Water flow 12. 0.5% sodium bicarbonate solution 13. Water flow 14. Scott’s solution 15. Eosin 16. Alcohol 75% 17. Alcohol 95% 18. Absolute alcohol 19. Absolute alcohol + xilen (1:1) 20. Xilen 21. Xilen

10 5 10 5 5 3 3 3 3 10 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 :

Intestine of frog Magnification power : 10 x 10

Intestine of frog Magnification power : 10 x 10

DISCUSSION : In order to fulfill this task, a lot work and problem we had facing. At the first step, which is the process of dissecting the frog, we are not facing a big problem. The problem while doing the dissecting is because of the size of the frog. The frog is too smaller, so it is a little bit difficult for us to dissect the frog. Beside that, we also get a small organ from that frog. So, we only can make one block for each organ. As a result, we only have four organs from that frog, while the heart of the frog is damaged. During fixation step, dehydration step, and clearance step we not facing any problems. We just follow the procedure provided. A problem had happened when we are doing the embedded process. Firstly, we have to filled the block with paraffin until 2/3 full. Then, the organ will placed into the block which is 2/3 filled with the paraffin. But, when the organ is put into the block, paraffin at the lowest part of the block is already harden. So, when the block is fulfilled with the paraffin, the upper part of the paraffin is not as harden as the lower part. As a result, the tissue sample will easily to damage or breaking when the slicing technique. The present a lot of bubble also one of the main problem in this process. When the paraffin is filled into the block, a lot of bubble are present there. Then, when the organ is put into the block, the numbers of bubble is increased. so, we have to destroy the bubble, but the quantity of the bubble give a problem to us. we also worried if our organ will damaged if we break the bubble. The other problem we have to face during the embedded process is while placed the organ into the block filled with paraffin. The organs are not placed into the block carefully and in also not placed in the rigt position or orientation. So, when we slice the tissue sample, most of the paraffin tape are damaged. During slicing process, we also faced the problems. We did not get the best paraffin tape because the certain organ should be cut before placing into the block during the embedded process, for example stomach.

The next step is staining. During this process, several tissue are peels away from the slides. This is because the tissue were not really stick to the slides. Then, after staining process are done, we dropped the DPX medium on the slides but we have problem because the bubble are presented. 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. The last step was observing the slide under the light microscope. There are a lot of bubble on the prepared slide, but we still can differentiate between the cell and the bubble. The best slide are chosen to be submitted.

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