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
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.
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.
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
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
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.