Wk1 - P - Introduction to Microscopic Techniques.docx

Dubai Medical College for Girls Phase 1: Introduction to Human Biology (IHB102) Practical Session Y1S1 - B38 Prepared by Dr. Heba Ismail Supervised by Prof. Nadia Mahmoud - The materials were first developed for the Dubai Medical College for Girls (DMCG) 2023.\" - ![](media/image3.png...

Dubai Medical College for Girls Phase 1: Introduction to Human Biology (IHB102) Practical Session Y1S1 - B38 Prepared by Dr. Heba Ismail Supervised by Prof. Nadia Mahmoud - The materials were first developed for the Dubai Medical College for Girls (DMCG) 2023.\" - Please ensure the creative commons logo and text is included on all the slides/pages.**\ ** **Definition:** Microtechniques are the methods used in the preparation, processing, and examination of histological tissue samples at a microscopic level. These techniques are essential for preserving tissue structures, highlighting specific cellular components, and enabling their visualization under a microscope. **Types:** 1. **Paraffin Technique: most common** 2. Celloidine Technique: most perfect 3. Freezing Technique: fastest **[Paraffin Technique:]** The method involves obtaining fresh tissue, from a human being or experimental animal, preserving it (i.e., fixing it) to allow it to remain in as life-like a state as possible, cutting it into very thin sections (3--8 microns), mounting it on glass microscopic slides, and then staining the sections so that they can be observed under a microscope to identify different histological components within the tissue. **Section 1: Tissue Collection and Processing** 1. [Tissue acquisition:] Using the traditional tissue dissection, sterile sharp dissecting tools are used to minimize crushing the tissue while cutting for removal. Once dissected, place the tissue in fixatives for preservation. 2. [Fixation:] It the process in a which a chemical substance is used to preserve biological material prior to [microscopy](https://www.google.com/search?sca_esv=561629598&sxsrf=AB5stBgzBB50TZSQlvtdaKu7Hl1B1C6bFg:1693490930467&q=microscopy&si=ACFMAn9-5A9OMKPWcg180I9o9MndUZMrrOzQOPCu-J2ifX139GXd6IRJL0vEDQNkKAf6wjpZ-zQ7mIACqp1R18CNhIhYt-fDzQ%3D%3D&expnd=1). The fixative acts to: - Denature proteins, to prevent autolysis - Promote the attachment of dyes to particular cell components - Remove bound water to increase tissue refractive index to improve optical differentiation and contrast for viewing without staining. It is important to keep the sample size small, if possible (i.e., 2--3 mm3), as increased thickness will retard fixative penetration. The volume of the fixative should be 20--25 times the volume of the tissue. Formalin (10% buffered) is the most used fixative. 3. [Dehydration:] It is the process of gradual removal of water using alcohol. Tissues are placed in progressively increasing concentrations of a dehydrating agent (e.g., 70, 85, 95, and 100%) which is typically ethanol. 4. [Clearing:] It is the step in which the alcohol is replaced with a clearing agent, e.g. xylene. 5. [Infiltration/Impregnation (melted soft paraffin; melting point 50]°C[):] It is the step in which the clearing agent is removed from the tissue to allow a complete permeation of the tissue with paraffin wax. This will allow the paraffin to pass in the intracellular space and thus support its structure. Paraffin wax is commonly used and heated to a temperature that is 2--3°C above its melting point. 6. [Embedding (melted hard paraffin; melting point 55]°C)[:] It is the step in which the infiltrated tissue is placed in a mold to obtain a solid wax block containing the tissue. A high melting point of the wax (e.g., 55--60°C) increases the hardness and decreases the thickness to which the tissue may be sectioned. An embedding cassette is be placed on top of the mold and labeled with the name of the tissue, fixative, and date. The mold is filled and the tissue is placed at the base of the mold. After that, the mold is set out on a cooling tray and not disturbed until the wax has cooled and solidified completely. Once solidified completely, the paraffin block is now ready for sectioning. **Section 2: Tissue Sectioning and Staining** 1. [Microtomy:] A microtome is an instrument used for cutting the tissue into extremely thin sections (3-10 µm thick) for examination under a [microscope](https://www.google.com/search?sca_esv=561629598&sxsrf=AB5stBg8-8hi9XK1eFcePh5prQr9mQEUZw:1693492862793&q=microscope&si=ACFMAn9-5A9OMKPWcg180I9o9MndUZMrrOzQOPCu-J2ifX139GEBBVHDOzv508mq7x1B9s9R6sfQmPSQG2L5QkkYSqH1f3F1bw%3D%3D&expnd=1). The tissue block is passed across the knife of the microtome to produce a serial section called a ribbon. The ribbon is then is gradually lowered onto a water bath (floatation bath) to eliminate wrinkles. The individual sections are picked up by submerging a clean glass slide into the water bath at a \~45° angle, directly beneath the location of the section or ribbon. The slide should be lifted out of the water slowly to ensure that the sections lay on the slide. The slides should be drained vertically on a paper towel for several minutes before leaving them to dry overnight. The tissue is now ready for staining. 2. [Staining:] In order to allow water-soluble dyes to penetrate the sections, the process must be reversed to remove the paraffin wax from the tissue: clearing agent (e.g. xylene) à rehydration using descending grades of alcohol (100%, 95%, 85%, 70%, water)à staining using Hematoxylin & Eosin (H&E) that are the routine stains used commonly à dehydration and covering. Hematoxylin stains nuclei blue, eosin stains cytoplasm pink.