Lab 1 Histology Micro-techniques PDF

BMS111 Lab 1 histology Micro-technique Dr Manal Shaaban Hafez Professor of histology and cell biology in Galala University Faculty of Medicine, Fall 2024-2025 Galala University...

BMS111 Lab 1 histology Micro-technique Dr Manal Shaaban Hafez Professor of histology and cell biology in Galala University Faculty of Medicine, Fall 2024-2025 Galala University gu.edu.eg MICRO TECHNIQUES AND MICROSCOPES  ILOs By the end of this section, you should be able to:  Identify the different micro techniques used in tissue preparation.  Demonstrate the steps of paraffin technique and the significance of each.  Recognize the components of the light microscope  Use the microscope to examine different histological sections. Histology is the study of cellular organization of tissues & organs. The light microscope is used in histological and pathological examinations.  Micro techniques Microscopic study of cells & tissues In tissue culture and In histological stem cells sections Paraffin Tech. Freezing Celloidin Tech. Tech PARAFFIN TECHNIQUE  1-Tissue Sampling Small pieces (1cm3) Cut with sharp instrument Immediately:  Biopsy.  After death.  After operation.  From experimental animals  2- Fixation 10% Formalin saline Aim:  Prevent putrefaction.  Prevent autolysis.  Preserve tissue structure.  Hardening of the tissue.  Coagulate proteins.  3-Dehydration Gradual removal of water from tissue  In ascending degree of alcohol (gradual dehydration).  To replace water by alcohol to prevent tissue shrinking. (50% , 70%, 90% then in 100%)  4-Clearing Replacement of alcohol in tissue by clearing fluid  In Paraffin solvent ex. benzene & xylol.  To replace alcohol.  Miscible with melted paraffin  Tissue becomes transparent (clear) Before After  5-Impregnation (Infiltration)  In melted soft paraffin in electric oven at 55-60OC.  To penetrate inter cellular spaces (The tissue is completely infiltrated with melted paraffin for easy cutting ).  6- Embedding In melted paraffin forming Paraffin block  7-Sectioning  Cutting the tissues in the block By Microtome  5-8 µm thin sections. Serial section/ ribbon  8-Mounting  On glass slides for easy handling  Stained for light Microscopy H&E  Hematoxylin & Eosin Hematoxylin (H) Eosin (E) Basophilic Acidophilic Basic stain Acidic stain Blue in color Red to pink in color Binds to acidic Binds to basic components components e.g. Nuclei (DNA,RNA) e.g. Collagen fibers  Ribosomes and Mitochondria ,  RER SER &  secretory granules.  FREEZING TECHNIQUE  Tissue samples removed during surgery & operations The biopsy is rapidly frozen in liquid nitrogen, making the tissue hard and ready for sectioning. Cutting sections by cryostat in a cabinet at subfreezing temperature. Microscopes  MICROSCOPES Based on electron interaction with Based on light interaction with tissue tissue Transmission Electron Light Microscopy microscopy (TEM) Scanning Electron microscopy (SEM) The magnification power = 1500 times The resolution power = 0.2 micrometer (µm)  LIGHT MICROSCOPE 3 lenses: 1. Condenser 2. Objective Beam used : Light beam from lamp or sun 3. Ocular /projector  MICROSCOPES Magnification= Resolution = magnification power of objective The smallest distance between 2 lens x particles at which they can be seen magnification power of ocular lens Magnifying power of LM.= 1000 -1500 as separate objects times The resolving power of L.M.= 0.2 µm  ELECTRON MICROSCOPES SEM TEM  Mention difference between transmission and scanning Electron MICROSCOPES TEM TEM Three Dimension images (3D images) Two dimension images (2D images) Beam of electrons scans only the surfaces of Beams of electron transmits through cells and cells and tissues tissues Examination of the surfaces of cells and organs Examination of internal structure of cells and tissues  PHASE CONTRAST MICROSCOPE It used to study growth and proliferation of living cells and tissue without stains Trials of OSPE Stations  Station 1: Answer the following questions 1- Fixation of the tissue is essential to: a. Remove water from tissue. b. Preserve tissue structure. c. Replace alcohol. d. Keep the cell alive. e. Give tissue a translucent appearance. 2- Resolution power of E.M. is: a. 0.2 mm. b. 0.2 nm. c. 0.02 nm. d. 0.02 µm e. 0.2 µm  Station 2: Answer the following: Answer: A. Microtome B. Cutting the tissues in the paraffin blocks C. Thin section about 5-8 µm A. Name the apparatus in the figure B. Mention its function C. What is the thickness of the sections for L/M? Answer Station 3 Light source /Electric lamb  Station 4: Answer the following B Q. Identify which of the letters A & B in H&E stained section refers to acidophilia and basophilia? A Answer: A refers to……………….. B refers to……………….. Answer: A refers to: basophilia B refers to: Acidophilia  Station 5 A. Identify the cells in the photo? B. Identify microscope used? A. Proliferating Stem cells without stains B. Phase contrast microscope.