Microscopic study include: 1. Study of oral soft tissues Microscopic examination is the method used to study the histological structure of the oral tissues. The tissue should be appropriately prepared for microscopic examination. Microscopic study include: 1. Study of oral soft tissues Routine tissue processing 2. Study of dental hard tissues Ground sections Decalcified sections

For microscopic examination, tissue specimens must be thin enough (4-6µ) to permit the passage of transmitted light. The treatment of tissue specimens to allow paraffin wax embedding is called Tissue Processing The aim of tissue processing is to embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut, which can be viewed under microscope

Source of tissue specimen is biopsy / autopsy Tissue specimens should be immersed immediately in some solution to prevent autolysis and bacterial putrefaction Tissues are to be preserved at its most natural form that helps in visualizing the structure & structural defects of the tissue The solutions used for this purpose are called as Fixatives.

Soft tissue processing

Tissue processing can be Manual Automatic

Specimens got to lab should be examined for: Relevant details Adequate size of tissue specimen Labeling of the specimen bottle Fixative used Steps done to ensure complete fixation Sufficient volume of fixative The standard ratio of fixative to tissue volume is 10:1 Large tissues Cut into smaller size tissues Representative areas to be sent for processing

The procedure used for soft tissue processing can be grouped under As paraffin wax is immiscible with water, tissues fixed in aqueous fixatives must be treated to remove water. This is achieved by various stages of tissue processing The procedure used for soft tissue processing can be grouped under Fixation Dehydration Clearing Impregnation Embedding Sectioning Staining Mounting

Fixation First step & foundation in a sequence of events in tissue processing Process involving series of chemical events which results in the stabilization of proteins and makes the tissue resistant to damage during subsequent stages of processing & visualization. Objectives of fixation To preserve the living structure as closely as possible. To prevent autolysis of tissue To inhibit bacterial or fungal growth To make the tissue resistant to damage during subsequent stages of processing To prevent alteration of tissue volume and distort any part of the tissue structure.

Most commonly used fixative is 10% formalin To avoid interchanging specimen, a piece of paper with graphite pencil marking to be put in the tissue capsule Most commonly used fixative is 10% formalin Increases the cross linking and results in the stabilization of proteins. Other fixatives used: Glutaraldehyde Osmium tetroxide Chromic acid Methyl alcohol & ethyl alcohol Mercuric chloride Picric acid

Factors influencing the rate of fixation Specimen size (3-4 mm ideally) pH Agitation Heat Viscosity Vaccum Ultrasonics

Dehydration Done to remove the water content from tissues to allow the penetration of paraffin wax Done by passing through ascending grades of alcohol (to prevent sudden shrinkage of tissue) E.g.- 50%, …..70%, 90% & 100% Solutions used Methyl & Ethyl alcohol Isopropyl alcohol Acetone

Clearing Paraffin and alcohol are not miscible. So impregnation of tissue by paraffin is not possible unless alcohol is replaced by a fluid that is miscible with both alcohol & paraffin. This process is called clearing Xylene is one of the solutions that is miscible with both paraffin and alcohol

The term “clearing” comes from the fact that the clearing agents often have the same refractive index as proteins. As a result, when the tissue is completely infiltrated with the clearing agent, it becomes translucent/clear. The presence of opaque areas indicates incomplete dehydration.

Ideal requirements of a clearing solution Speedy removal of alcohol Minimum tissue damage & toxicity Cost factor Choice of a clearing agent depends on: The type of tissues to be processed The type of processing to be undertaken The processor system to be used Processing conditions ( temp, vaccum & pressure) Safety factors Cost and convenience

Reagents used Xylene (most commonly used) Chloroform Toluene Benzene CNP 30; Inhibisol Food oil derivatives Cedar wood oil

Impregnation Saturation of tissue cavities and cells by a supporting substance which is generally, but not always, the medium in which they are finally embedded Replaces xylene with paraffin by immersion in molten wax (600 C) Factors affecting impregnation Size & type of tissue Clearing agent employed Vaccum embedding

EMBEDDING/BLOCKING Process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidifies will provide sufficient external support during sectioning. Impregnated tissue transferred from wax bath to a mould filled with molten wax to get a block of wax with the tissue specimen at the center with the cutting surface facing the base of the block

Done using Leuckhart’s L shaped pieces Ice trays Paper boats Embedding cassette

Make sure that there are no air bubbles trapped between the tissue and the molten wax. The wax-filled mold containing the tissue is then allowed to cool. The wax blocks are labeled to make easier identification. Wax hardened block removed from the mould and trimmed. Wax block to be fixed on to a wooden/metal block to prevent wax block from crumbling during sectioning.

Sectioning Embedded tissues, to be cut into thin sections of 3- 5 µ with a microtome. Cut sections are, floated on a warm water bath Helps to spread the specimen and remove wrinkles. Floated sections are picked up on an adhesive coated glass slide. Glass slide kept on a slide warmer at 580 temp for 20 min to ensure adhesion Egg albumin with additives - commonly used adhesive

Manual Automatic

Staining Biochemical technique of adding a class-specific dye to a substrate (DNA, proteins, lipids, carbohydrates) to qualify or quantify the presence of a specific compound Commonly used Staining procedures Gram staining Haematoxylin and eosin (H & E) staining Papanicolaou staining (PAP) PAS staining Masson's trichrome staining

H & E stain/ Haematoxylin & Eosin stain Most popular staining method in histology. Most widely used stain in medical diagnosis. The staining method involves application of the basic dye haematoxylin, which colors basophilic structures with blue-purple hue, and alcohol-based acidic eosin Y, which colors eosinophilic structures bright pink.

Basophilic structures are usually the ones containing nucleic acids, such as the ribosomes and the chromatin-rich cell nucleus, and the cytoplasmatic regions rich in RNA. Eosinophilic structures are generally composed of intracellular or extracellular protein. Most of the cytoplasm is eosinophilic. Red blood cells are stained intensely red.

HEMATOXYLIN Hematoxylin is a natural dye which is extracted from the heartwood of the tree Hematoxylon campechianum. The dye is usually used in conjunction with a mordant which helps the stain to bind to the tissue Oxidation Mordant Hematoxylin Haematin STAIN

EOSIN Second component of the H & E is eosin, the counterstain Eosin is a red fluorescent dye resulting from the action of bromine on fluorescein. Eosin Y-Commonly used form of eosin Used to stain cytoplasm, collagen and muscle fibers Both water and ethanol soluble.

Procedure Steps involved Manual Automated Removal of wax (with xylene). Rehydrate the tissues (bring sections to water) Descending grades of alcohol Staining H & E Dehydrate: Ascending grades of alcohol Clearing (Xylene)

Mounting The stained section on the slide must be covered with a thin glass coverslip to protect the tissue from being scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section for years to come Mounting medium is used to adhere the coverslip to the slide

Two types of mounting media Water based mounting media Resinous mounting media DPX & Canada balsam commonly used

Allow it to dry and section viewed under microscope To mount a slide Apply drops of mounting medium upon tissue section. Hold coverslip at 45o allowing the drop to spread along the edge of the slip. Let go of slip and allow the medium to spread slowly. Allow it to dry and section viewed under microscope

Hard tissue processing

Study of dental hard tissues Ground sections Decalcified sections

Decalcified Sections Hard tissue specimens after decalcification are treated like routine soft tissue specimens All hard tissues except enamel can be studied after decalcification 96% mineralized; lost during decalcification

Decalcification Process by which calcium in the mineralized tissue is removed so that tissue becomes soft enough to make sections Decalcification is usually carried out between the fixation and processing steps Done by Acids (5% nitric acid, 15% formic acid) Chelating agents (EDTA) Electrolysis

Procedure Tissue to be fixed first Large tissue to be cut into smaller pieces Specimen to be put in decalcifying solution Check for completion of decalcification Consistency of tissue Piercing tissue with a needle Judicious bending of tissue Radiographs Chemical test Sodium hydroxide & ammonium oxalate Wash in running water

Ground sections Sections made of undecalcified specimens Helps in study of dental hard tissue, especially enamel Made using Manual Hard tissue microtome

Procedure Final reduction using a fine abrasive Tooth cut into 2-3 sections – diamond burs/discs Initial reduction using a rough abrasive (till 25-50µ) Arkansas stone Coarse wheel of a lathe Final reduction using a fine abrasive Domestic scouring powder followed by soap water Fine wheel of lathe Sections washed & dried (Dehydrated)\ Mounted & allowed to dry Viewed under microscope

Tissue specimen