GHH Lab (handout prelim)


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General Histology and Histotechnique 2012-2013; 1st sem; Prelim Handout

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GHH Lab (handout prelim)

  1. 1. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)Activity #1 B. Focusing parts: 1. Fine adjustment Microtechnique – it involves the instrument 2. Coarse adjustment microtome. C. Mechanical parts: Microtomy – sectioning instrument 1. Draw tube 2. Body tube Microtome 3. Adjustments screws- Indispensable in microtecnique 4. Revolving nosepiece 5. Dust shield3 essential parts: 6. Stage and stage clips1. Block holder 7. Inclination joint2. Knife carrier/knife 8. Pillar3. Pawl, ratchet feed wheel and adjustment 9. arm screws 10. basePawl- to line up the tissue block Other materials used in microtechnique- Adjust the thickness for successive  Staining bottle sectioning.  Glass slides  Cover slips Sliding Rotary  Pipettes-Slicing motion -Chopping motion  Vacuum oven-The object to be cut is -Knife is fixed in a  Tissue stretcherfixed and the knife is horizontal positioncarried obliquely intended to precisely  Staining rockacross it. cut equally thin  Slide box sections of different  Forceps materials.  Dissecting pan  Reagent boxMicroscope- Used to view micro-object- Used to check if the specimen is stained well or over-stain or not- Used to view cytoplasmic part of the cell and tissue.A. Illuminating & magnifying parts:1. Ocular/eyepiece2. Iris diaphragm3. Condenser4. Bulb5. Mirror
  2. 2. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)Activity #2: Physical HazardsCommon Reagents used in Microtechnique 1. Combustible - Substances that can ignite at certainReagents: temperature. Fixatives 2. ExplosivesEx: Ethanol, Xylene - Substances that can explode Dehydrants 3. Oxidizers Clearers - May initiate combustion Stains Embedding matrix Basophilic Adhesives Washing reagents Acidophilic Mountant MetachromaticReminders / precautions to be considered toproperly utilize reagents: For nature specifically such as flammable materials Toxicity For the safety precautions when handling compounds Method of disposal of hazardous waste Read labels before using. Use only one dropper per reagent to avoid mixing/contamination of reagentHealth hazards1. Biohazards- Can cause diseases in human- Infectious agents, contaminated solutions.2. Irritants- It causes reversible inflammatory effects to skin, eyes, & nasal passages.3. Corrosive chemicals- It destroys inanimate surface of living tissues.4. Carcinogens- Substances that causes cancer and tumors.5. Toxic Materials- Harmful; it can cause death upon ingestion.
  3. 3. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)Activity # 3: Disadvantages:Preparation of whole mounts 1. Processing kills and disintegrates many tissue components preventing theirWhole mount purposes: observations.- Specimen is mounted whole; thin specimen 2. Improper processing may also leave behind- To observe the gross anatomy certain by-products called artifacts that may- To preserve chemical and morphological interfere either the examination of slides. features of the cell. Qualities of a good whole mount:1. Temporary preparation - Transparent- Prepared in order to make possible the - Parts complete observations in the live of normal state. - Non-overlapping- Allows physiological observations to be - Not distorted made directly such as mitosis, phagocytosis etc. Selection of whole mount specimen:- Allows true and natural colors to be - Whole mount slide preparations is one observed. where the specimen is taken wholly without- Mountant used is water. part/s missing and carefully mounted as is is on a slide.Advantages: - specimen on character must be:- Allows one to observe living characteristics a. Adequate size of specimen. b. Adequate bulk- Can be prepared quickly. c. All parts present and free of distortion- Retain characteristics of the specimen d. Fresh specimen.- Requires no reagent, if not minimal hence cheap.Disadvantages:- Tissues prepared are thick and transparent- Cannot be used repeatedly since it is prone to dehydration.2. Permanent preparation- Have to undergo an elaborate processing of specimen.Advantages:1. Specimens may be sectioned thinly.2. Specimens are stained hence it enhances the resolution of tissue components3. Specimens may be stored for a long time.
  4. 4. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)Activity # 4: Separating Cells for Microscopic 4. Too much or too little stain is usedstudy: Squashing, Smearing, and Teasing 5. Staining time is not followed.Squashing TeasingQualities of a good squash preparation: Qualities of good slide:- Evenly stained cells  Cells are distinct and clear- Cells normal shape and contours are  Cells are isolated retained.  Cells exhibit correct form and shape.- Tissue components are well isolatedNote: Cells have the greater tendency to beunder-stained that to be over-stained.Applying too much pressure when crushing thestone cells may result to the: Distortion of the typical structure of the stone cells Uneven staining since the distorted parts of the cell may allow some stain to penetrate. Overlapping of stone cells Possible breakage of the glass slides.SmearingQualities of good smear preparations: Cells must be isolated from each other Different cells must be observable Staining makes the cells distinct Shape of the cell and nucleus should be normal and distinctBad qualities of Smearing:a. Clumping of cellsb. Distortionc. Homogeneityd. Under or over staining resulting to: 1. Too large drop of blood 2. Poor or uneven spreading 3. Pusher used has nicks
  5. 5. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)Activity # 5: Preparation of specimen for Activity #6: Tissue Processing part I; Fixation,microtome sectioning Washing, Dehydration, Clearing.Features to consider in selecting specimen: Fixation A specimen must be fresh, healthy, and in - To preserve cellular and structural live condition. elements with least alteration possible Specimen must be of sufficient numbers. - Prevents post-mortem conditions Specimen must be readily available when - Protects tissue by hardening naturally soft needed tissues - Increase visual differentiation of structuresReasons for cutting the specimen into 1x1 cm upon application of stains.dimensions To facilitate process of microtomy Washing To obtain full penetration and satisfactory - only necessary if the fixatives used contains fixation to avoid post mortem conditions to some reagents which may have undesirable occur. effects such as: For specimen to fit in congruence with the - Discoloration paraffin block during embedding for easy - Precipitation cutting. - Corrosion Note: washing must be done thoroughly and gradually Dehydration - Where does water to be removed in dehydration process come from? - Extracellular – comes from humidity and from water present around specimen - Intracellular – comes from vacuoles and cytoplasm of the cell Clearing - Renders the tissues translucent thus increasing the tissue’s refractive index. - Remove the opacity or darkening of the specimen. - Free specimen from opacity
  6. 6. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)Activity #7: Tissue processing part II; Activity #9: Staining microtome-sectionedInfiltration and embedding specimenInfiltration Hematoxylin - Natural stain used for nuclear stainingEmbedding - Stain the nuclei blue- To encase the tissue in block of solid paraffin. Eosin- Block must be contiguous, clear and - Synthetic acid stain for cytoplasmic staining homogenous, and free from crystallization. - Counterstained for hematoxylin- If bubbles appear only at the sides can be remedied by trimming off the block.- If bubbles are found up to the center of the block, re-embed the tissue.Measures on blade and microtome setting Sharpen the blade Clean the knife edge Tighten the screw Adjust knife Re-adjust angulations’ of knife.Activity #8: Microtome sectioningMicrotome sectioning- Also referred to as Microtomy- Involves 2 processes:1. Microtome setting which involves proper adjustment and alignment of microtome parts in preparation of the cutting proper.2. Sectioning which is the actual cutting of the tissue block.