MDSC 1401 Environment & Health - Introduction to Anat. PDF

MDSC 1401 Environment & Health Anatomy Unit, Department of Pre-clinical Sciences Faulty of Medical Sciences The University of the West Indies Introduction to Histology and Microscopy Anatomy Laboratory  WHAT IS HISTOLOGY? Histology - study of the microanato...

MDSC 1401 Environment & Health Anatomy Unit, Department of Pre-clinical Sciences Faulty of Medical Sciences The University of the West Indies Introduction to Histology and Microscopy Anatomy Laboratory  WHAT IS HISTOLOGY? Histology - study of the microanatomy of cells, tissues and organs and their structure. Histopathology - study of tissues affected by disease  How are histological slides produced Steps involved in producing histological slides 1. Obtain fresh specimen 6. Embedding 2. Fixation 7. Sectioning 3. Dehydration 8. Staining 4. Clearing 9. Slide mounting 5. Wax Infiltration 10. View slide  How are histological slides produced cont’d Obtain fresh specimen. Obtained from various sources, cut into 3 mm squares to facilitate fixation Fixation Fixation serves to retain the structure of the tissue and limit microbial growth that could otherwise make analysis more difficult or result in artefacts. Common fixatives used – formaldehyde, acetone, Bouin’s fluid  How are histological slides produced cont’d 3. Dehydration The tissue is progressively dehydrated by immersing it in successively higher concentrations of alcohol before transfer to an organic solvent (e.g. xylene) and finally embedding in wax 4. Clearing This involves immersing the tissue in an organic solvent like xylene to prepare it for wax infiltration  How are histological slides produced Wax Infiltration cont’d The most commonly used wax for infiltration is paraffin wax. Xylene is replaced with molten wax (60°C), which infiltrates the tissue. The tissues are immersed in a wax bath Embedding Immersing the tissue in a molten medium which is paraffin wax, which is then enclosed by a mould. This medium then solidifies to form a block  How are histological slides produced cont’d Sectioning Tissues are sectioned using a microtome of 3–5 μm thickness Cryostat: cuts sections from deep-frozen blocks, usually of unfixed tissue. Picture of a microtome  How are histological slides produced cont’d Staining techniques & Mounting of slides Most cells and cellular elements are virtually transparent - need for staining of histological slides. Most commonly used diagnostic stain for histology is hematoxylin and eosin, often referred to as ‘H&E’ staining. Typically nuclei are stained blue, whereas the cytoplasm and extracellular matrix are stained varying shades of pink. Slides are then dehydrated through an ascending series of alcohol, passed through an organic solvent (xylene) and covered with a coverslip to obtain a permanent preservation and retention of the specimen. H & E Staining https://www.youtube.com/watch? v=nUjK4n3_1C8&t=2s MICROSCOPY  MICROSCOPY cont’d Histology departments use light microscopes with transmitted light. This means that the light passes through the section to the objective lens, as shown in the following diagram. Here, light from the lamp is focused on the specimen by the substage condenser. The objective collects light from the specimen and focuses it to form an image within the barrel of the microscope. The eyepiece allows this image to be viewed as if it were a projection in the plane of the section. Figure 1: Elements of a light microscope.  MICROSCOPY cont’d USING A LIGHT MICROSCOPE 1. Switch on power supply to microscope. Clean slide to be viewed with lens paper and place in slide holder section of microscope. 2. Centre the slide so the tissue section is directly above the aperture. 3. Use the Coarse adjustment knob to bring the stage all the way up until it can go no further. Turn objective lens to the lowest magnification which is usually x4. 4. Use the fine adjustment knob to obtain a clear focus. 5. Once the slide is focused at low magnification, then switch to next magnification (x10) and adjust using the fine focus knob. https://www.youtube.com/watch?v=SUo2fHZaZCU  Acknowledgements Compiled and designed by the Technical and Support Staff, Anatomy Unit Nicole Bartholomew Alyshia Claverie Carol Duncan Denyse Friday Charmaine Lord-Pope Shankara Madivala Korie Phillip Sharon Vidale Additional support staff Dr Rajh from Vet School, Photography services Dexter Superville – Photography Services - CMSE Secretarial assistance from Lorraine Albelto