Introduction to Histology lecture.pdf

"Micro-techniques & Microscopy"  Dr. Amany Abd El-Fattah Mohamed Contact: Medical Histology & Cell Biology Department. E-mail: [email protected]. Mobile: 01006189202.  Learning outcomes At the end of the lecture, the students should be able to: 1- Identi...

"Micro-techniques & Microscopy"  Dr. Amany Abd El-Fattah Mohamed Contact: Medical Histology & Cell Biology Department. E-mail: [email protected]. Mobile: 01006189202.  Learning outcomes At the end of the lecture, the students should be able to: 1- Identify Histology and aim to study this science. 2- Classify the different techniques for preparation of tissue sections. 3- Enumerate types of Stains and their uses. 4- Identify different types of microscopes.  Case Scenario 45 years old female patient came with a swelling in her neck. Her clinical examination revealed firm non-tender but fully mobile mass. The physician asked for ultrasound which suggested that this mass may be malignant. What will be the next step?  Learning outcome 1 Identify Histology and aim to study this science.  What is Histology? The science that study the structure of the cells & tissues using Microscope  Why do we study it? Study microscopic structure of cells & tissues Correlate this structure to function Understand the pathology of diseases  Learning outcome 2 Classifying the different techniques for preparation of tissue sections. Micro-techniques. Microtechniques  Micro-techniques I-Preparing II- Staining of thin Sections this section.  I- Preparing thin Sections. 1- Paraffin 2- Freezing technique method  1- Paraffin technique. The routine method for histological preparation. Paraffin wax is used. Steps: 1- Obtain sample  2- Fixation. Put Small sample of tissue in fixative e.g. Formalin 10%. To prevent putrefaction and autolysis.  3-Dehydration. In ascending grades of alcohol (70%-90%-100% alcohol). To avoid shrinkage of the tissue.  4-Clearing. With Xylene to: Replace alcohol Paraffin solvent Renders the tissue transparent.  5-Impregnation. Infiltration with Paraffin wax in the oven.  6-Embedding in Paraffin In hard paraffin to obtain a paraffin block. 7- Sectioning Rotary Microtome Thickness of section is 5 um  Paraffin technique. 1-Obtain 6-Embedding 7-Cut sample in paraffin section. 8- Pick 2- Fixation 5-Impregnation section on slide. 3-Dehydration 4-Clearing  2- Freezing technique. Freezing of specimen by Cryocut. ❑Advantages: 1- Very rapid → rapid diagnosis in operating theatre. 2- Lipids and enzymes in the tissues are preserved→ histochemistry.  Learning outcome 3 Enumerate types of Stains and their uses.  II- Staining. Ordinary histological Special stains stain→ H&E  Stains → dyes used for staining sections. Acidic Basic Neutral Eosin (E) Leishman stain Haematoxylin (H) (Methylene blue +Eosin) Stain basic Stain acidic Two different structures structures colours. (cytoplasm) (nucleus) Acidic → basic structures. Acidophilic Basophilic Basic → acidic (pink) (blue) structures. Sections stained with H&E  Section stained with H&E Acidophilic cytoplasm Basophilic nucleus  Special Stains. 1- Carbohydrates: Periodic acid Schiff’s technique (PAS)→ Magenta color. Best's carmine (for glycogen)→ Red. 2- Lipid: - Frozen sections are used. ✓ Sudan III → Orange. ✓ Sudan black → Black. ✓ Osmic acid → Black.  Staining of living cells in vivo e.g. phagocytic cells Vital stain by injecting Trypan blue. Supravital Staining of living cells in vitro i.e. outside the stain body e.g. mitochondria by Janus green B. Metachro- Staining with a color different from the color of the dye (e.g. granules of mast cells are colored red matic stain when stained with toluidine blue).  Vital stain Macrophage with trypan blue Tattoo  Supravital stain Metachromatic S. Mitochondria with Janus green B Mast cell  Learning outcome 4 Identify different types of microscopes. Microscopy The microscope is an instrument which magnifies the image & reveals fine details of the object.  Microscopes Magnification up to 100.000 times. Light M. Electron M. (Student M.) Magnification Transmission 40, 100, 400, Other 1000. types Scanning Student Microscope Transmission E.M  Transmission E.M Needs extremely thin sections 0.08 um. Electron beam is used instead of light for illumination. The image appears black & white Dark components → electron dense White components → electron lucent Scanning E.M.  Blood Give 3D images cells Pollens TEM SEM 1 2  Other types of microscopes Phase Contrast Microscope:- used to study the living or fixed unstained cells. Fluorescence Microscope: used to detect fluorescent substances.  Case Scenario The case represents with a mass that may be benign or malignant tumor. A biopsy is needed for proper diagnosis.  Summary and wrap up ❑Finally, we discussed today: ✓Histology is the science that study the microscopic structure of cells and tissues of living body. ✓Micro-techniques for tissue processing include paraffin technique and freezing technique. ✓Ordinary histological stain used is H&E. ✓Types of microscopes include light microscope, electron microscope and other types  References or further readings Junqueira's Basic Histology Text & Atlas (15th ed.). https://www.youtube.com/watch?v=P0cZKCfyUwE https://www.youtube.com/watch?v=__D-DE0X0zk