Laboratory technique and preparations

Transcription

1 Laboratory technique and preparations Bio 381 written by : Hind Alzaylaee Alshareef_ Maryam Alzayn Alshareef 9/17/2012

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3 graduated cylinder Funnel Flask beaker Dropping bottle Watch glass Petri dish Reagent bottle Microscope slides Staining boxes Coplin jars Vials Dissecting scissors Test tube Slide boxes Cover slips pipette Forceps Dissecting needle Scalpel

4 Necessary equipment in preparation microscopy Rotary Microtome Ultra-Microtome Freezing Microtome

5 Paraffin Oven Water bath Hot plate

6 Automatic Tissue Processor Automatic Tissue Processor Automatic Tissue Processor Automatic Tissue Processor Balances Automatic Tissue Processor

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8 Prepare some necessary solutions in laboratory preparations Some general rules for the preparation of solutions: 1- When preparing a 70% alcohol, for example, of the 95%: measured 70 ml of alcohol by graduated cylinder and then increase that amount to 95 ml by distilled water and in the same way can be prepared in any concentration is required. 1- When preparing 15% of the specific ways : we measure 15 ml of this liquid and add to it 85% of distilled water becomes final amount of 100 ml. 2- When preparing a 20% sodium chloride, for example: weigh 20 gm and dissolve in a certain amount of distilled water (50 ml, for example) and after completing the melting complete solution to 100 ml with distilled water. Solutions required preparation Physiological solutions Sodium chloride to wash samples focus 0.9% Sodium chloride gm. Distilled water ml. Fixatives Formal 10% Concentrated formalin. 10 ml. Distilled water ml. Dehydration water solutions (50%, 70%, 80%, 90%, ethyl alcohol) Clearing solutions Xylene Staining Hematoxylin, Eosin.

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11 Part Ocular (eyepiece) Nosepiece Functions use to look at the sample. usually it's enlarge power is (10x) or more than that (20x) holds objective lenses Use to magnify the image of sample Objective Lenses High power objective lens ( 40 x) (oily lens 100x ) Low power objective lens ( 4 x) (10 x ) Arm Stage supports upper parts of the microscope, used to carry the microscope where the slide is placed Stage Clips hold slide in place on the stage Condenser Collect light and direct it to the sample Diaphragm Control the amount of light which reaching to the condenser Coarse Adjustment Knob used to focus when using the low power objective Fine Adjustment Knob used to focus when using the high power objective Light Source provides light Base supports the microscope

12 1- When moving your microscope, always carry it with both hands, hold the arm with one hand and place the other hand under the base for support 2- Turn the nosepiece so that the lowest power objective lens is "clicked" into position 3-Use the coarse adjustmetnt knob to move the stage down. Your microscope slide should be prepared with a cover glass over the sample. This will help protect the objective lenses if they touch the slide. Place slide on the stage and fasten it with the stage clips 4- Look at the objective lens and the stage from the side and turn the coarse focus knob so that the stage moves upward. Move until the image comes into focus without touching the slide! 5- Now, look through the eyepiece and adjust the diaphragm for the greatest amount of light. 6- Slowly turn the fine adjustment knob. Continue until the image comes into focus. 7- Move the slide around until the image comes in center of the field of view and adjust the diaphragm for the clearest image. 8- Now, you should be able to change to the next objective lenses with only minimal use of the focusing adjustment. Use the fine adjustment, if available. If you cannot focus on your specimen, repeat steps 4 through 7 with the higher power objective lens in place. Do not allow the objective lens to touch the slide!

13 9The proper way to use a microscope is to look through the eyepiece with both eyes (this helps avoid eye strain). If you want close one eye when looking into the microscope, it's ok. Do not touch the glass part of the lenses with your fingers. Use only special lens paper to clean the lenses. 10- When finished, raise the tube (or lower the stage), click the low power lens into position and remove the slid. 11- Always keep your microscope covered when not in use. Dust is the number 1 enemy! Microscopes allow us to see micro organisms which are too small for the naked eye to see them such as bacteria,viruses,fungi and many important parts of the body like individual cells. Magnification power= multiply the objective lens (the one facing your specimen) with the ocular lens (the one through which you look). Most ocular lenses are 10x Example: If the eyepiece power x10,objective lens power x40 power magnification = 10 x 40 = 400 times

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15 Preparing temporary samples by loading Devices and tools required : Light microscope - anatomy Tools - toothpick - slides - Cover slip distilled water- Forceps - dropper - vegetable samples (onion tomato potato) A- Prepare plant cells in the skin of the onion: Method: Clean the slide and cover slip. Cut a piece of onion. Use Forceps to pull off a very thin piece of onion skin. Put drop of water on slide using dropper Place a small piece of onion skin on water drop. Carefully place cover slip on onion skin (figure1) Press the cover slip down carefully to remove any air bubbles. Place prepared slide onto stage of microscope. Look at onion cells under low power (4 x), then use medium power (10 x) and high power (40 x) and write the notes. x10

16 B - Prepare a Chromoplasts cells in tomato : Method: Clean the slide and cover slip. Cut a piece of tomato. Use Forceps to pull off a very thin piece of tomato pulp. put drop of water on slide using dropper Put a small piece of tomato pulp on water drop. Carefully place cover slip on sample. Carefully Press the cover slip down to remove any air bubbles.. Put prepared slide onto stage of microscope. Look at Chromoplasts in tomato cells under low power (4 x), then use medium power (10 x) and high power (40 x) and write the notes X40

17 C - Prepare starch granules in potato: Method Clean the slide and cover slip. Cut pieces of potato. Put potato pieces in a cup contained water for 10 minutes. Take a drop from solution and put it on slide using dropper Carefully place cover slip on drop. Carefully Press the cover slip down to remove any air bubbles. Put prepared slide onto stage of microscope. Look at starch granules under low power (4 x), then use medium power (10 x) and high power (40 x) and write the notes. X40

18 D- Prepare squamous cell epithelial lining of the mouth: Method: 1 - Put a small drop of distilled water in the center of clean microscopic slide. 2 - Rubbed several times lining of the oral cavity by the end transverse toothpick. (figure 1) 3 - Move the end of the toothpick in a small drop of water on a microscopic slide, even cells spread completely in the drop of water.. (figure 2) 4 - Placed a cover slide and examine the sample by using a microscope. (figure 3) X40

19 خاليا طالئية حرشفية مصبوغة بأزرق الميثلين كما تظهر تحت المجهر

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21 Tools required : Slides - medical swab - sterile needle - Light microscope Method: 1. Clean your slide by water then alcohol 95% 2. Cleans your finger by medical swab. 3. Make a puncture on a fingertip on sterile needle. 4. Wipe away the first drop of blood with clean gauze. 5.Touch the next drop of blood with a clean slide. 6. Bring a clean spreader slide, held at a 45 angle, toward the drop of blood on the specimen slide. 7. Wait until the blood spreads along the entire width of the spreader slide. 8. While holding the spreader slide at the same angle, push it forward rapidlyand smoothly. Then leave the blood smear to dry by air.

22 9- Stain the blood smear by using Leishman dye for 2-3 minute. 10- Remove the stain with distilled water then leave slid until dry. 11- examine the sample by using microscope.

23 :Tools used Bunsen flame or a candle - Slides - coverslip Bacterial culture, or yogurt Methylene blue dye Light microscope Inoculation loop Method: 1- cleans slide well and pass it on the flame before use several times 2- sterilize Loop before and after use by using the flame 3- takes Sample from yogurt, by puts point on the edge of the slide and then uniqueness and left to dry like blood smear 4- pass slide on a Bunsen flame several times to kill the bacteria and fixed it up to does not go away during the dye 5- put two drops of Methylene blue dye and leave for 3-5 minutes 6- clean the dye and rinsed by distilled water 7- dried in the air or on the flame put a drop of Cedar - wood oil on a slide and examined by the oily lens(100x) X 100

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25 Required tools and solutions: Distilled water - Himatoxlin stain Eosin stain - fatty tissue taken from any animal 70 % alcohol - formalin - Glycerin - slides Covers Sudan III stain. Method: 1. Prepare fat tissue on the slide. 2. Fixed on formalin 5-10 min. 3. Wash by distilled water. 4. Stain by Sudan III 15 min. 5. Wash on alcohol 70% until stain removed. 6. Wash by distilled water. 7. Stain by H.X 3-5 min.(optional) 8. Wash on tap water 5 min.(optional) 9. Dry the slide, add Glycerin, cover slip. Result : the fat cells will stain by orange color, nucleus by blue. x10

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27 Required tools and solutions: Distilled water - Hematoxylin stain Eosin stain - connective tissue Xylene - 70% alcohol - formalin - Glycerin - slides Cover slip Canada balsam - different concentration of alcohol(70%-80%- 90%- 100%) Method: 3- Preparation of the connective tissue (taken from a thin layer under the skin of the animal) on a glass slide 2-Fixed on formalin for 5-10 min. 3-Wash by distal water. 3-Stain by H.X (Hematoxylin stain) for 15 min. 4-Wash on tap water 10 min. 5- Wash by distal water. 6-Stain by Eosin 2 min. 7- Wash by distal water. 8-Pass the slide on different concentration of alcohol (ascending). 9-Clear in Xylene 5 min. 10- Drop of Canada balsam, mount the cover slip. Result: observe fibers and different cells. x10

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29 STEP 1 Fixation Obtaining a fresh specimen, wash by Saline to removed the blood Then Fixative by10% formalin or other fixatives for hours. Make sure you have enough fixative to cover tissues. Fixative volume should be 5-10 times of tissue volume. After that, rinsing the fixative from sample by running tap water for 12hour. STEP 2 Tissue Processing Specimen is placed in plastic cassettes, processed using Automatic Tissue Processor

30 Tissue Processing Including 1. Dehydration: (series of ethanol (alcohol) of increasing concentration until pure) 50% Ethanol, one changes, 1 hour. 70% Ethanol, two changes, 1 hour each. 80% Ethanol, one change, 1 hour. 90% Ethanol, one change, 1 hour. 95% Ethanol, one change, 1 hour. 100% Ethanol, three changes, 1hour each )Time depends on the size of the sample) 2. Clearing: By using clearing agent called Xylene. Causing we cannot infiltrate tissue with wax because wax and ethanol are largely immiscible. So we have to use intermediate solvent that is fully miscible with both ethanol and paraffin wax. Xylene, three changes, 1.5 hour each 3. Wax Infiltration : The paraffin wax based histological waxes are the most popular. Paraffin wax (58-60 ºC), two changes, 2 hours each Embedding tissues into paraffin blocks.

31 STEP 3 Embedding: By Embedding Machine STEP 4 Cutting, Trimming and Sectioning By Rotary Microtome

32 STEP 5 Floating Section is placed in floating bath.then loading on the slide. Staining : STEP 6 1. Put the slide in xylene, 2 changes for 2-5 min. to remove wax. 2. put the slide at descending series of alcohol (100%- 95%- 70%-50%-30%) for 2 min each concentration. 3. wash by tap water for 2 mint. 4. Staining by Hematoxylin for 5min. 5. Rinsing by running tap water for 2 min. 6. put the slide at ascending series of alcohol (30%-50%-70%- 95%) for 2 min each concentration. 7. Staining by Eosin for 2 min. 8.(95%-95% - 100%- 100%) for 2 min each concentration. 9. put slide at Xylene, 2 changes for 2 min.

33 STEP 7 Mounting drop canada balsam. Cover slip. Observation slide under microscope Final STEP RESULT: Hematoxylin= Nucleus (Blue color) Eosin= Cytoplasm (Pink color).

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35 Required tools and solutions: Clean slides - ascending series of alcohol - Canada balsam - Xylene insects (ants - bee - beetle)- cover slip. Method: Prepare parts of the insect ( wings- leg - mouth parts) or complete body if the insect is a small. Put the sample at ascending series of alcohol (70%-80%- 90%- 100%) for 5-10 minute each concentration. Put sample in Xylene even become transparent. Put sample on clean slides then covering by Canada balsam. Put cover slip and dried the sample in the oven at 37 degrees. examine the sample by using a microscope.