Oral Histology Lecture 1.pdf

 Introduction The microscopic examination of tissue sections and their proper preparation is essential in the study of oral tissue morphology. Therefore, a basic knowledge of the various types of microscopes and histological techniques is important to learn. T...

 Introduction The microscopic examination of tissue sections and their proper preparation is essential in the study of oral tissue morphology. Therefore, a basic knowledge of the various types of microscopes and histological techniques is important to learn. This helps to study the structure and function of oral tissues. 2  Microscopy There are many different forms of microscopy, but the most common types of microscopes for studying tissues are light microscope and electron microscope. Under the light microscope, tissues are examined via a light beam that is transmitted through the tissue. Because tissues and organs are usually too thick for light to pass through them, they must be sectioned to obtain thin, translucent sections and then attached to glass slides before they can be examined. 3  The general requirements for a specimen to be successfully examined using Light microscopy 1. That the cells and other elements in the specimen are preserved in a “life-like” state (this process is called “ xation”). 2. That the specimen is transparent rather than opaque, so that light can pass through it. 3. That the specimen is thin and at so that only a single layer of cells is present. 4. That some components have been differentially coloured (stained) so that they can be clearly distinguished. 4 fl fi  Preparation of specimen for microscopy Various techniques have been developed to prepare tissues for study so that they closely resemble their natural living state. Four techniques for oral tissue preparation are usually used for light microscopic examination. These are as follows: A. Paraf n embedded section of soft tissues. B. Decalci ed section for hard tissue. C. Ground sections for calci ed tissue. D. Frozen section for soft tissues. 5 fi fi fi A. Paraffin embedded section of soft tissues This is the most common technique used for soft tissues such as gingiva, cheek, tongue , lip, salivary gland , etc. That is, the tissue, which are not calci ed. The steps of tissue preparation in this type are: 1. Fixation of the specimen 2. Processing of the tissue 3. Embedding 4. Sectioning of the specimen 5. Mounting the cut sections 6. Staining the section 6 fi  1. Fixation of the specimen Fixation is a complex series of chemical events that differ for the different groups of substance found in tissues. The aim of xation: 1)To prevent autolysis and bacterial attack. 2)To x the tissues so they will not change their volume and shape during processing. 3)To prepare tissue and leave it in a condition which allow clear staining of sections. 4)To leave tissue as close as their living state as possible, and no small molecules should be lost. Golden note/ We used formalin because it react with hydrogen in amino acid to form bridges to make it stable. 7 fi fi  Fixation is coming by reaction between the xative and protein which form a gel, so keeping every thing as their in vivo relation to each other. The most commonly used xative agents for light microscopical examination are 10% neutral formalin and Bouin’s uid. Both of these substances are cross-linked proteins, so maintaining a life like image of tissue after removal from the body. After xation the tissue is washed overnight in running water. Factors affect xation : PH , Temperature, Penetration of xative, Volume of tissue. According to previous factors we can determine the concentration of xative and xation time. Golden note / 1- We use 10% of formalin because if it was : a-lower than 10% it will not fixed. b-higher than 10% outer layer will be fixed quick and become too dense, so the formalin can't penetrate into tissue, so the inner tissue will autolysis. 2- Fixation occurs in two steps : a-when the organ is transported to laboratory ,it stored in formalin. b-after the tissue is cut into slices 8 fi fi fi fi fl fi fi fi 2. Processing of the tissue (Dehydration, clearing and infiltration) Dehydration :mean remove xative and water from the tissue and replace them with dehydrating uid since water is not miscible with paraf n wax in which the tissue is embedded. Two widely used dehydrating agents are alcohol and acetone. The specimen is gradually dehydrated by being passed through a series of increasing percentages of alcohol ( 60% , 70% , 80% and 95% and absolute alcohol). 9 fl fi fi 2. Processing of the tissue (Dehydration,clearing and in ltration) Clearing:Then since paraf n and alcohol are not miscible ,the specimen is passed from alcohol through changes of xylene ,which is miscible with both alcohol and paraf n. This process is called clearing, since the tissue becomes transparent in xylene. Note / Xylene is a volatile hydrocarbon liquid. 10 fi fi fi 2. Processing of the tissue (Dehydration, clearing and in ltration) In ltration:Then the specimen is placed in a suitable container of melted paraf n wax, which has been in an oven at 65°C until it is completely in ltrated. The in ltrated process is done in order to distinguish the overlapping cells in a tissue and the extracellular matrix from one another. 11 fi fi fi fi fi  3. Embedding Is the process by which tissues are surrounded by a medium such as paraf n wax which when solidi ed will provide suf cient external support during sectioning. The specimen is embedded in melted paraf n wax after it has been completely in ltrated with paraf n. Once the tissue is impregnated with paraf n, it is placed into a small container, covered with melted paraf n, and then allowed to harden, forming a paraf n containing the tissue. The specimens is now ready to be sectioned on a microtome. 12 fi fi fi fi fi fi fi fi fi  4. Sectioning of the specimen The paraf n blocks are sectioned with a microtome, which is a device supplied with a stainless steel blade and an arm that can provide us with equal increments of the tissue thickness (usually from 4 to 10 microns). Then sections are placed on pre-cooled glass slides, permitted to come to room temperature, and stained with speci c dyes. 13 fi fi  5. Mounting the cut sections The section are placed (mounted) on a glass slides coated with suitable adhesive. The slide is then allowed to dry before staining with water–soluble stains for light microscopical study. 14  6. Staining the section Paraf n is rst removed from the section, then tissue is rehydrated and stain. The most commonly used stains in histology are hematoxylin and eosin, commonly referred to as H and E stain. Hematoxylin is a base, it colors the acidic components of the cells by bluish color. Because the most acidic components of the cells are DNA and RNA, the nucleus and some regions in the cytoplasm stain dark blue. These components are called basophilic. 15 fi fi  Eosin is an acid that dyes the basic components of the cells a pinkish color. Because many of the cytoplasmic constitutes have a basic PH, so they are stain pink in color. These elements are said to be acidophilic. 16 Properties Hematoxylin Eosin 1. Its nature Base Acid 2. The nature of components of the Acidic components Basic components cell DNA,RNA and some regions in 3. The components of cell Most of cytoplasm cytoplasm 4. Philicity of components Basophilic Acidophilic 5. The color of stain Bluish color Pinkish color 6. Example 17  B. Decalcified section for hard tissue The specimens in this section must be decalci ed ( the mineral substance removed by acid). This type is used for the tissue containing bone or teeth. Enamel of the tooth contains 96% minerals so it is completely destroyed if decalci ed unless it still not fully formed it can be seen. 18 fi fi  C. Ground sections for calcified tissue Specimens of calci ed tissue may be ground into thin section such as bone and undecalci ed tooth. This is done by slicing the undecalci ed specimen into a section of about 30-50 microns on a revolving stone or disc and then by grinding on lathe wheel or at stones. Golden note/In decalci ied section inorganic substance is dissolved and the organic substance remains while in ground section organic substance is burnt and inorganic substance remains 19 f fl fi fi fi  D. Frozen section for soft tissues This type is used to examined: 1. The pathological tissue specimens immediately. 2. When the reagent used for embedding would destroy the tissue characteristics that are to be studied. So specimen of soft tissue may be frozen and sectioned with freezing microtome (cryostat) without being embedded. 20 Reference 1. G S Kumar:Orban’s Oral Histology & Embryology, 13th edition. Elsevier Health Sciences,2011. 2. https://youtu.be/nUjK4n3_1C8 21 22