Histology PDF

Introduction Fixation is the first step of any histological and cytological laboratory technique. It is the process by which the cells in the tissue are fixed in a chemical and physical state. All the biochemical and proteolytic activities within the cells are prevented so that the cells or tissues...

Introduction Fixation is the first step of any histological and cytological laboratory technique. It is the process by which the cells in the tissue are fixed in a chemical and physical state. All the biochemical and proteolytic activities within the cells are prevented so that the cells or tissues can resist any morphological change, distortion, or decomposition after subsequent treatment with various reagents. The fixation helps to maintain the tissue nearest to its original state in the living system. Essential Precautions for Fixation in General Certain essential precautions are necessary for proper fixation: The tissue should be free from excessive blood before putting it into fixative. Tissue should be thinly cut in 3--5 mm thickness. The fixative fluid should be 20 times more than the volume of the tissue. The tissue with fixative should be in a tightly screw-capped bottle. Aims of Fixation The primary aims of fixation are the following: To preserve the tissue nearest to its living state To prevent any change in shape and size of the tissue at the time of processing To prevent any autolysis To make the tissue firm/hard To avoid any bacterial growth in the tissue To make it possible to have a clear stain To have a better optical quality of the cells Ideal Fixative An ideal fixative should have the following qualities: Prevention of autolysis of the cells or tissue Prevention of decomposition of the tissue by bacteria Maintaining the volume and shape of the cell as far as possible. Consistently high-quality staining, mainly routine stains such as haematoxylin and eosin stains. Rapid action Cheap Non-toxic A large number of fixatives are available in the market. Each fixative has its advantages and disadvantages. Tissue Changes in Fixation The following changes may occur in tissue due to fixation: 1. Volume changes: Fixatives may change the volume of the cells. Some fixatives, such as osmium tetroxide, cause cell swelling. The exact mechanism of the volume change is not understood correctly. However, the volume change may be due to (a) altered membrane permeability, (b) inhibition of the enzymes responsible for respiration and (c) change of transport of Na+ ions. Formaldehyde may cause shrinkage of the volume by 33%. In an experiment, Bahr et al. noted that tissue shrinkage is inversely proportional to the formaldehyde concentration. Similarly, glutaraldehyde also causes significant tissue shrinkage. However, when glutaraldehyde and osmium tetroxide is used as fixations in epoxy resin, a 70% increase in cell size is noted. 2. Hardening of tissue: The fixation changes the tissue\'s consistency, and some hardening occurs due to fixation. 3. Interference of staining: Fixation may cause hindrance in the staining of enzymes. Formaldehyde inactivates 80% of the ribonuclease enzyme. It has been noted that osmium tetroxide inhibits haematoxylin and eosin staining. 4. Changes in optical density by fixation: The fixation may cause a change in the optical density of the nuclei, and the nuclei may look condensed and hyperchromatic. 1. Nature of fixation 2. Chemical properties 3. Component present 4. Action on tissue protein Description of Nature of Fixation Immersion fixation: This is the most typical way of fixation in laboratories. In this technique, the whole specimen is immersed in the liquid fixative, such as tissue samples immersed in 10% neutral buffered formalin or cytology smear in 95% ethyl alcohol. Coating fixation: This is commonly used in cytology samples. The spray fixative is used for easy transportation of the slide. The main advantages of spray fixatives are (a) Fixation of the cells, (b) impart a protective covering over the smear (c)  No need to carry liquid fixative in a bottle or jar. Vapour fixation: In this type of fixation, the vapour of chemicals is used to fix either a smear or tissue section. The commonly used chemicals for vapour fixation are formaldehyde, osmium tetroxide, glutaraldehyde and ethyl alcohol. The vapour converts the soluble material to insoluble material, and these materials are retained when the smear comes in contact with the liquid solution. Perfusion fixation: This is mainly used for research purposes. In this technique, the fixative solution is infused into the arterial system of the animal, and the whole animal is fixed. Perfusion fixation can also fix an organ such as the brain or spinal cord. Freeze-drying: In this technique, the tissue is cut into thin sections and then frozen at a very low temperature. Subsequently, the ice within the tissue is removed with the help of a vacuum chamber at a higher temperature (−30 °C). 6. Microwave fixation: Microwave is a type of electromagnetic wave with frequencies between 300 MHz and 300 GHz, and wavelength varies from centimetre to nanometre. Scientific and medical microwave ovens operate at 2.45 GHz and 0.915 GHz, respectively. The microwave creates an electromagnetic field, and the dipolar molecules, such as water, rapidly oscillate in this electromagnetic field. This rapid kinetic motion of these molecules generates uniform heat. The generated heat accelerates the fixation and also other steps of tissue processing. The essential characteristic of microwave heat generation is a homogeneous temperature increase within the tissue, and every part of the tissue is heatedCommonly Used Fixatives in the Laboratory. Formaldehyde Pure formaldehyde vapour dissolved in the water is available as formaldehyde in 37--40% concentration. This is also known as formalin and is considered 100% formaldehyde. In the laboratory, 10% of this formalin is used to make neutral buffered formalin for routine laboratory fixative. Rate of penetration: Formalin penetrates approximately 1 mm/h, and usually 24 h is needed for fixation of a 1 cm3 tissue. The volume of formalin: For proper fixation, the specimen should be sliced 5 mm apart, and the amount of formalin should be 20 times the volume of tissue. The specimen should be immersed entirely in formalin, not in direct contact with the container. There should be formalin-soaked clothes in between the container and the tissue. Removal of formalin from the tissue: As the cross-linking of the amino acids and proteins is a slow process, if the tissue is washed for 24 h in water, then 50% of formalin from the tissue is removed. Precaution: Formaldehyde is irritant to the eye and skin and toxic for inhalation. It is a carcinogenic element. Advantages: The penetration rate of formalin is high. Cell morphology is well preserved in formalin. Cheap. Stable. Easy to make the solution. Disadvantages; 1\. Slow fixation.\ 2. Formalin reaction with the tissue is reversible and can be removed by washing. 3\. Formalin fails to preserve acid mucopolysaccharides.\ 4. Highly vascular tissue may have dark-brown granules (artefacts)\ 5. Exposure to the skin may cause dermatitis. 6. Chronic inhalation may cause bronchitis. Glutaraldehyde Glutaraldehyde is a fixative for electron microscopy because it fixes and preserves the ultrastructure. The fixation occurs due to the extensive cross-linking of the proteins. The penetration power of glutaraldehyde is poor; therefore, only a tiny piece of tissue should be used for fixation. Glutaraldehyde does not react with lipids or carbohydrates; therefore, it should be used in combination with the other fixative. Advantages: Better fixation of ultrastructure. Less cell shrinkage. The preservation of protein is better. Good cross-linking with collagen. Less irritating. Disadvantages; Poor penetration and tissue should be less than 0.5 mm thick. Less stable compound. No lipid fixation. Costly. Osmium Tetroxide Osmium tetroxide is used for fixation in electron microscopy. It reacts with unsaturated bonds in the lipid molecules and fixes them. The penetration of the osmium tetroxide in the tissue is poor, and if it is used alone, then a good amount of protein and carbohydrates may be lost during fixation. Advantages: This is an excellent fixative for lipids. It preserves cytoplasmic organelles such as Golgi bodies and mitochondria, It does not make the tissue hard, Disadvantages: It does not fix proteins and carbohydrates; therefore, it should be used in combination with another fixative. Osmium tetroxide may react with the ribose group and cause DNA clumping. This can be prevented by pretreatment with potassium permanganate or post-fixation with uranyl acetate. Poor penetration in the tissue. Tissue swelling may occur. Toxic and volatilises at room temperature produce harmful vapour. This vapour is toxic to the eye and respiratory tract. Expensive. Methyl and Ethyl Alcohol Methyl alcohol (methanol) and ethyl alcohol (ethanol) are used as dehydrating agents, and these two alcohols are primarily used as fixatives of cytology smears. The tissue or smudge containing water should not be put directly in the higher concentration of alcohol as it may distort the cells due to the rapid rush of fluid from the cell. Therefore graded alcohol should be used for dehydration. Acetone It is mainly used for enzyme study and immuno-cytochemistry. It is a poor fixative for morphological preparation as it causes significant cell shrinkage. Acetone works by the dehydration of cells. Cold acetone is used at four °C for fixation. Bouin's Fixative Bouin's solution contains picric acid. This is an excellent fixative for glycogen. It reacts with protein and forms protein picrate. The tissue penetration rate of picric acid is high, and it fixes small tissue biopsies within 3--4 h. Bouin's fixative is unsuitable for quantitative DNA study as it damages the cell membrane and causes hydrolysis of nucleic acid. Advantages: 1\. It is a good fixative for connective tissue and glycogen. 2\. Rapid penetration rate. Disadvantages:\ 1. It produces a yellow stain on the tissue. Removal of yellow colour: The tissue should be washed thoroughly in 70% ethanol. This yellow colour can be removed by dipping the tissue in lithium carbonate in 70%alcohol. Processing of Tissue in Histopathology Laboratory *Principle of processing*: In tissue processing, the water within the tissue is removed, and another medium (usually paraffin wax) is impregnated in the tissue that provides adequate support. Therefore the essential steps in tissue processing. *Dehydration*: In this step, water is removed from the tissue. Water is immiscible with wax, and therefore to infiltrate the tissue with wax, it is necessary to remove water. *Clearing*: This is needed to clear the dehydrating agent and to facilitate the transition of the dehydration and impregnation stage. The clearing substance is usually miscible to both dehydrating agent and impregnating medium. *Infiltration and impregnation*: In this stage, the tissue is infiltrated with a supporting medium suitable to provide adequate tissue rigidity to make a thin section. Factors that Influence Tissue Processing The following factors influence tissue processing. 1\. *Size of the tissue sample*: The optimum tissue size is significant for effective processing---the smaller the tissue, the better the infiltration of the embedding medium. The optimum thickness of the tissue should be kept 3--4 mm only. 2\. *Agitation*: The tissue gets better contact with the surrounding medium if it is completely immersed and gently agitated. The agitation causes continuous fluid removal from the surface by a fresh medium. This has a better effect on the action of the fluid on the tissue. Most commercial tissue processors have the facility of agitation. It is important to note that too rapid agitation may damage the soft and delicate tissue. 3\. *Heat*: Heat increases the fluid penetration rate within the tissue, whereas low temperature impedes the process. The present commercial tissue processors have the facility to heat the tissue in all stages of processing. Overheating may produce hard and brittle tissue. 4\. *Viscosity*: The viscosity of the embedding media also affects the processing. The higher viscosity of the medium lowers the penetration rate in the tissue. Heat reduces the thickness of the medium and helps in better penetration. 5\. *Vacuum*: The application of negative pressure facilitates tissue processing. The vacuum helps remove the entrapped air from the tissue, thereby enhancing fluid penetration within the tissue. The negative pressure also increases the clearing agent\'s volatility and helps remove the fluid from the tissue. Dehydration Every tissue contains some amount of free or unbound water molecules. As the commonly used supporting medium (paraffin) is not miscible with water, removing the free water molecule from the tissue is necessary to impregnate the supporting medium successfully. The sharp difference in the concentration gradient between the tissues and the dehydrating fluid may cause a sudden fluid rush, damaging the delicate tissue. Therefore the dehydration should be done gradually from low. to the high concentration of dehydration fluid. The tissue should be kept in the dehydration fluid for optimal time because too much time in the dehydrating fluid may cause the tissue to become hard and brittle. Too little time in dehydration fluid may be insufficient to remove a free water molecule. Thin 2--3 mm tissue needs less time in dehydration fluid than thick 5 mm tissue. Dehydrating Agent Alcohol (Ethanol) Ethanol or ethyl alcohol is the most popular and most commonly used dehydrating agent. This is a clear and colourless fluid. Ethyl alcohol is a flammable liquid. This is a relatively rapid and efficient dehydrating agent. However, it needs a licence from the government to purchase ethyl alcohol for laboratory use. As a dehydrating agent, ethyl alcohol is used in 50, 70, 90 and 100% concentrations. The dehydration may be started for delicate tissue with a 30% concentration of ethyl alcohol. In the routine laboratory, 70, 90 and 100% alcohol for two h each is sufficient for dehydration of the tissue. If tissue is immersed in the ethyl alcohol for a long time, the removal of attached water from the carbohydrate and protein molecules causes hard and brittle tissue. Methanol Methanol is a clear, colourless, volatile and inflammable liquid. It can be used as a substitute for ethanol, but it is rarely used in laboratories because of its volatility and high cost. Butyl Alcohol *n*-Butanol, iso-butanol and tertiary butanol are dehydrating agents in animal tissue and plant histology processing. Butyl alcohol is a slowly-acting dehydrating agent that takes longer than ethyl alcohol for dehydration. However, the tissue shrinkage is less by butyl alcohol. Isopropyl Alcohol Isopropyl alcohol is available as isopropanol (99.8%). This is miscible with water and liquid paraffin. It is a relatively rapid-acting, non-toxic dehydrating agent causing minimal tissue shrinkage. It is a suitable lipid-dissolving solvent. Dehydrating Agents Other than Alcohol This is 1,4-diethylene dioxide. Dioxane is miscible with both water and molten paraffin wax. This is a rapidly acting dehydrating agent and produces minimal shrinkage. Tissue can be kept in dioxane without any harm. Dioxane liberates a highly toxic gas, and proper ventilation is mandatory for its use. Dioxane Ethylene Glycol It is also known as ethylene glycol mono-ethyl ether. It is a colourless and odourless fluid. Mono-ethyl ether is a rapidly acting dehydrating, and tissue can be kept in it prolong. Acetone Acetone is a colourless and inflammable liquid with a pungent ketonic smell. It is miscible with both water and alcohol. Acetone produces tissue shrinkage, and prolonged use may cause brittleness of tissue. It is best used in fatty tissue processing. Comparison of different dehydrating agents Clearing After removing free water molecules from the tissue, the next step of processing is to remove the dehydrating agent itself from the tissue because many dehydrating agents are not miscible with the impregnating material (paraffin wax). The clearing agent should be miscible with the dehydrating agent and the embedding medium. The refractive index of the clearing agent is similar to the tissue, giving a clear appearance of the anhydrous tissue. So the completely transparent tissue indicates the terminal point of the clearing process. Any opacity of tissue signifies incomplete dehydration. *Selection of appropriate clearing agent*: This depends on the following: *Type of tissue*: large tissue takes more time than smaller tissue *Type of processor*: manual versus automatic *Processing condition*: temperature and vacuum *Speed of removal* of a dehydrating agent Ease of replacement by molten wax Safety factors: flammability and toxicity Cost The molten wax easily and quickly removes the clearing agent with a low melting point. At the same time, a clearing agent with a high melting point takes time to be removed by the embedding medium. A clearing agent with high viscosity has a low penetration rate. Prolonged tissue exposure to a clearing agent may make the tissue brittle and more friable. Therefore the optimal time for clearing is necessary. The amount of clearing agent should be 40 times the tissue volume for clearing. Clearing Agents Xylene This is the most commonly used clearing agent in the laboratory. This is a clear and inflammable liquid. The small pieces of tissue are cleared rapidly by xylene within 30--60 min. Prolonged exposure to xylene may make the tissue hard and brittle. Toluene It has almost similar properties as that of xylene. However, it does not make the tissue hard even after prolonged exposure, and its action is slightly slower than xylene. Toluene is also flammable and toxic. Chloroform It is a highly volatile, non-inflammable, expensive and toxic agent. The penetrating power of chloroform is slower than xylene. However, it does not cause any tissue shrinkage and is mainly used in the uterus, muscle and other dense tissue. Presently chloroform is rarely used in laboratories. Esters The different esters are amyl nitrate, methyl salicylate and methyl benzoate. These are less toxic and may be used in manual processing. They do not cause tissue hardening even under prolonged exposure. Cedarwood Oil This expensive rapid-clearing agent is mainly used in clearing dense tissue. Infiltration and Embedding This is the next step after clearing. The process of diffusion removes the clearing agent within the tissue. The tissue space is now infiltrated with the embedding media. Usually, molten wax is used as the embedding medium. After cooling to room temperature, the molten wax is solidified to provide support for cutting into the thin section. *Ideal impregnating medium*: An ideal impregnating medium should have the following qualities: Miscible with clearing agent The liquid in higher temperatures (30--60 °C) and the solid at room temperature Homogenous and stable Non-toxic and cheap Transparent Fit for sectioning the tissue The time duration and the number of changes required for the impregnation in tissue depend on the following: 1. *Size of tissue*: Thicker large tissue takes more time to impregnate with the embedding medium. It also contains a more clearing agent to remove. 2. *Type of tissue*: Hard tissue, such as bone and cartilage, takes more time to embed than soft tissue. 3. *The type of clearing agent*: Certain clearing agents are easy to remove than others. Xylene and toluene are easy to remove than cedarwood oil. 4. *Type of processing*: Vacuum embedding enhances impregnation. Different Impregnating Medium Paraffin Wax Paraffin wax is a type of hydrocarbon and is produced as a by-product during the refining of crude petroleum. This is the most popular, universally accepted embedding medium for tissue processing. This is a non-toxic and inexpensive medium. The melting point of paraffin wax varies from 39 °C to 70 °C. The wax is sold according to its melting point. Paraffin wax with a low melting point is soft at room temperature, whereas paraffin wax with a higher melting point is much harder in consistency. Therefore, it is necessary to have paraffin wax that has a suitable melting point to get a reasonable ribbon of tissue. In this Indian subcontinent, the paraffin wax with a melting point of around 60 °C is the most suitable for laboratory use. A total of 3--4 hr of time in paraffin wax is sufficient to impregnate tissue with wax. *Advantages*: Tissue blocks can be stored for a long duration. Non-toxic. Cheap. Safe. It may cause tissue shrinkage and hardening in case of prolonged impregnation. Paraffin wax takes a long time to impregnate the bone and eye. Tissue Processing Methods Tissue processing can be done manually or with an automated processor. Manual tissue processing is done in a small laboratory with a few tissues. Automatic tissue processor is widely used in laboratories. *Automated tissue processor*: The basic principle of a tissue processor is to transfer the tissue into different fluids for a specified time in the desired environment. There are two types of tissue processors: Tissue and Fluid transfer. 1\. *Tissue transfer processor*: In this system, the bucket of tissue is transferred from one carousel to other after a specified time. There are several containers with reagents. Tissue remains in a basket with 30--100 cassettes. The tissue basket is submerged in the specific container for a particular time and then automatically transferred to the next container. Gentle agitation is created by vertical oscillation or by the rotatory movement of the tissue basket. A microprocessor determines the schedule and transfer of tissue in each container. 2\. *Fluid transfer processor*: This is a completely closed processor. Here the tissue is kept in the container, and the container is periodically filled with a particular fluid. After a certain period, the fluid is pumped out of the tissue container. It is again filled with the fluid required for the next step. In this processor, each step can be customised for vacuum, temperature, and time duration. Embedding The embedding medium has three critical functions: To give support to the tissue To prevent distortion of the tissue during cutting To preserve the tissue for archival use: *The choice of the embedding medium*: Various media are used for embedding, such as paraffin wax, epoxy resin, methacrylate, carbowax, etc. Paraffin wax is the most commonly used embedding medium. The choice of the embedding medium depends on the following factors: *Type of tissue*: The density of the tissue and the embedding medium should be close. Otherwise, tissue may not be adequately sectioned, and tissue will be deformed. *Type of microtome* *Type of microscope* Embedding Mediums *Paraffin wax*: Paraffin wax is a solid polycrystalline hydrocarbon. Paraffin wax is sold in the market with a different melting point. Paraffin wax with melting points ranging from 56 to 62 °C is used in our laboratory. Paraffin wax is cheaper and easy to use. Little supervision is needed to make a block by it. \(b) *Epoxy resin*: Epoxy resin is mainly used in electron microscopy as it provides better resolution and more incredible tissue details. \(c) *Acrylic medium*: Methacrylate monomer is miscible with ethanol. In the presence of a catalyst (benzoyl peroxide 2%), methacrylate monomer is polymerised and provides a hard and clear block. Methacrylate monomer is available in the market along with hydroquinone which should be removed with a weak alkali solution and thoroughly washed with water. The presence of water may lead to tiny bubbles within the block. \(d) *Agar gel*: Agar gel helps in the cohesion of friable and fragmented tissue, particularly in cytology samples, endometrial curetting, and small endoscopic biopsies. It does not provide good support for the tissue for section cutting. Agar-paraffin wax double embedding is a more suitable technique than agar alone. \(e) *Gelatin*: Gelatin is used in small friable tissues and frozen sections containing friable and necrotic tissue. The melting point of gelatin is 35--40 °C, and this low melting point makes it unsuitable for embedding. Different Types of Mould Used for Block Leuckhard Embedding Moulds: Leuckhard embedding moulds have two arms. One arm of the L is longer than the other arm. The two L-shaped arms are adjusted to make a convenient size for the block. An adequate lubricant, such as glycerine, is applied to the L arms and metal plate for easy tissue removal. The molten wax is poured into the space between two L arms, and the tissue is placed within the bottom of the liquid wax. The wax is cooled, and the block with tissue on one surface is removed for microtomy. Stainless Still Mould Here, the mould is made of stainless steel. The base of the mould Tissue Embedding Method The tissue embedding method is essentially the same for all types of embedding media. The following things are needed for tissue embedding: Tissue Embedding Method 1\. Molten paraffin wax\ 2. Mould with cover\ 3. Metal plate (cold plate) to work Tissue Orientation and Embedding The correct orientation of the tissue is significant for proper cutting and microscopic examination. Tissue is usually placed flat on the central part of the mould. It should be oriented in such a way that cutting is easy by the knife of the microtome. Some of the tissue needs special care as described below: 1\. The tubular tissue (fallopian tube, vas difference, artery, etc.) should be placed in such a manner so that we get a transverse section with all the layers. 2\. Tissue with epithelial surface should be placed vertically and at the right angle to the surface to get all the layers. 3\. Multiple tissue sections, such as endometrial curetting, should be placed in a central position. 4\. Linear long tissue should be placed diagonally. 5\. Muscle biopsy should be placed in longitudinal and transverse planes.\ 6. Long, membranous tissue, such as the amniotic membrane, should make a Swiss roll. Decalcification of Bony and Hard Tissue for Histopathology Processing The presence of calcium salt in the tissues makes them firm to hard, which may damage the knife. Therefore, it is often necessary to remove calcium salt from the tissue and make it soft to cut in a microtome. The process of removing calcium salt from the tissue is known as decalcification. *Aim*: The primary aims of decalcification are: Removal of calcium salt from tissue No damage to tissue morphology No significant effect in staining *Calcium-containing tissue*: The tissue containing a heavy amount of calcium salts is (1) the bone, (2) the tooth, (3) pathological tissue such as tuberculous lymph node, dystrophic calcification, and certain tumours such as teratomas, etc. *Requisites for successful decalcification*: The following measures are helpful for successful decalcification: *Consistency*: Exact assessment of the consistency of the tissue is required for successful decalcification. *Small pieces*: The tissue should be cut into 2--6 mm thick sections because thicker tissue may take longer to decalcify. *Fixation*: Adequate fixation of the tissue is necessary for proper decalcification. *Washing*: The fixed tissue should be washed thoroughly before decalcification. *Choice of decalcifying agent*: Suitable selection of the decalcifying agent is required. *Volume*: Optimum volume of the decalcifying agent is a prerequisite for proper decalcification. *Endpoint detection*: The endpoint of the decalcification should be determined correctly. Acid Decalcification This is the most standard method of decalcification in the routine laboratory process. Acid makes the soluble calcium salt, and calcium is removed from the tissue. Strong acids: Hydrochloric acid Nitric acid StrongAcid The strong acids are used in 5--10% concentration. They are rapid in action. However, careful attention is needed to prevent tissue damage. A neutraliser is also used to avoid any tissue distortion. Aqueous Nitric Acid This is rapid in action. It does not impair staining if the endpoint is not crossed. Weak acids: Formic acid Trichloroacetic acid Other Procedures of Decalcification The other uncommon procedures for decalcification include: *Ion-exchange resin method*: An ion-exchange resin (sulfonated polystyrene resin) is used with an organic acid as a decalcifying fluid. It also produces faster decalcification with the preservation of morphological details of the tissue. *Electrolysis method*: In this process, electrolysis of the tissue is done in a solution of hydrochloric acid and formic acid. Calcium from the tissue moves to the cathode plate. This is a very rapid decalcification method, taking only a few hours to decalcify the bone. However, there is a risk of tissue damage with this technique.  Tissue Microtomy: Principle and Procedure After embedding the tissue and preparing the block, the next step is microtomy. The word "microtomy" originated from the Greek language *ikros* means small, and *temnein* means to cut. So the word "microtomy" means to cut the tissue into thin sections. For successful micro- scopic examination, it is necessary to have thin tissue sections by microtomy. Microtomes It is the main instrument by which we cut the embedded tissue in the paraffin block as a thin section. The different types of microtomes in the traditional histology laboratory are: Rotary microtome Rocking microtome Base sledge microtome Sliding microtome Cryomicrotome Ultramicrotome Laser microtome *Rotary microtome*: This is the most commonly used microtome in the routine laboratory. The cutting blade is kept in a horizontal position, and the block containing tissue moves up and down with the help of a rotatory handle attached to the microtome. In each 360° rotation of the wheel handle, the block moves down followed by up, and the tissue is cut as a thin ribbon. This microtome can be semiautomated or automated with the adjustment and control of the movement of the block and the angle of the knife. *Advantages*: Good-quality 2--3 μm-thin section is possible. Heavy and stable automated rotary microtome reduces health hazards and gives the best-quality section. Good tissue ribbon production. Easy-to-cut various types of tissue: firm, fragile, small biopsy, etc. *Disadvantages*: Expensive. Unsuitable to cut large blocks. The knife faces up and so may be dangerous to the technical staff. \(b) *Rocking microtome*: The rocking microtome is also known as the Cambridge rocking microtome. The word "rocking" is used as a rocking action of the microtome, like an arm movement. In this type of microtome, the knife is static, and the block of tissue moves in a rocking motion (arc-like movement of the block). This is one of the oldest designs of the microtome. The microtome can cut thin sections with ribbons and is ideal for serial sections. The sections are slightly curved in this microtome. *Advantages*: Thin section Easy to operate Low-cost instrument and reliable *Disadvantages*: The tissue is curved, and the microtome does not provide a flat section. As the microtome is lightweight so vibration may occur. \(c) *Base sledge microtome*: In the sledge microtome, the block is fixed in a static position within a steel carriage. The knife slides to and fro over the top of the block. This microtome is the best for large tissue samples or hard tissue. The tissue sections are usually thick (more than 10μm) in the base sledge microtome. *Advantages*: Hard tissue can be cut. The large tissue sample can be cut. The best microtome for ophthalmology *Disadvantages*: Difficult to get thin sections. Large slides are required. \(d) *Sliding microtome*: In this microtome, the knife is static, and the block moves horizontally over the knife. *Advantages*: Large sections can be cut. Mainly used for celloidin-embedded tissue. Simpler design and easy maintenance. Brain sections can be cut better by this type of microtome. *Disadvantages*: The knife may glide in case of hard tissue and may jump. Long knives are difficult to sharpen. \(e) *Cryomicrotome*: This microtome is used to cut tissue for frozen samples. The sample is made hard in liquid nitrogen and then cut by the microtome in the chamber that contains liquid nitrogen. *Advantages*: To get a rapid section for rapid diagnosis To study nerve biopsy To study enzyme histochemistry *Disadvantages*: It needs continuous supervision to maintain the temperature. Freezing artefact is often seen. Costly instrument. Fixed tissue is very difficult to cut. \(f) *Ultramicrotome*: Ultramicrotome is used to cut ultrathin sections for transmission electron microscopy. Sections are cut between 40 and 100 nano micron thicknesses with the help of a glass knife or diamond knife. The tissue is at first trimmed to make a small block of 1 × 1 mm size, and then the section is cut by this ultramicrotome with the help of an optical microscope. The cut section is allowed to float on the water held by a boat and then finally picked up on a metal grid. \(g) *Laser microtome*: In this ultramodern microtome, the laser beam is used to cut the biological section without processing or embedding the material. An infrared laser beam with ultrashort pulse duration is applied, and therefore almost no heat is generated, and the tissue is cut without any thermal effect. Microtome Knife The microtome knife is vital to cut uniform and thin sections of tissue. These are made of steel. Various types of knife profiles are available for different types of microtomes. The most commonly used knife profile is Profile C or wedge profile. The various types of microtome knives include: 1\. *Plano concave (Profile A)*: One side of the knife is plain, and the other is concave. Originally these knives were used for cutting celloidin-embedded tissue. This is a very sharp knife and is used for cutting soft tissues. However, presently these knives are less frequently used. 2\. *Biconcave (Profile B)*: The knife is concave on both sides. The knife was used for rocking the microtome. The concavity of the knife is often difficult to identify. This is a less rigid type of knife and often vibrates during cutting. 3\. *Wedge (Profile C)*: This knife is plain on both sides. This is the most widely used knife for routine microtomy and is compatible with different types of microtomes. This type of knife is also easy to sharpen. 4\. *Tool edge (Profile D)*: The knife resembles a chisel used in woodworking. Both sides of the knife are plain; however, the cutting edge is steep. The tool edge knife is mainly used to cut hard tissue such as decalcified bone. The knife is difficult to sharpen and is not recommended presently. Disposable Knife Nowadays, the disposable blade is used in many laboratories to save time in sharpening. Two types of disposable blades are available: *Low-profile blade*: These blades cut small biopsies or large soft tissue. *High-profile blades*: These are used to cut firm to relatively hard tissue such as the uterus, heart, etc. *Advantages*: Easy to replace within seconds. No need to sharpen. The overall cost of the disposable blade system is low as there is no need for any knife sharpener or abrasive powder to sharpen the knife. *Disadvantages*: They are not rigid like an ordinary knife so that that vibration may be seen. Factors Involved in Cutting *Temperature*: Lowering the temperature facilitates section cutting. *The angle of the rake*: Higher rake angle helps in the smooth flow of ribbons. A lower rake angle is used for hard tissue. *Consistency of tissue*: Soft tissue is cut slower than hard tissue. Sectioning the Paraffin Block The following instruments are essential for section cutting: 1\. Microtome with blade\ 2. Water bath\ 3. Paraffin block with embedded tissue to cut 4. Ice tray\ 5. A blunt forceps or camel brush\ 6. Slide rack with slides The water bath is used to float the tissue after cutting. The temperature of the water bath is usually controlled automatically by a thermostat. The water temperature in the water bath should be 10 °C below the melting point of the embedded paraffin wax and usually kept at 40--50 °C. It is necessary to prevent the formation of any air bubbles within the water bath. One can add a few drops of alcohol or a little detergent for adequate tissue floating. This reduces the surface tension of the water, and tissue floats smoothly. Blunt Forceps and Camel Hair Brush Blunt forceps help to manipulate the floating tissue section. A Camel hair brush is used to clean the blade. Slide Rack with Clean Glass Slides The clean slides are kept in the slide rack. The slides can be labelled with a diamond pencil or on the frosted side with a lead pencil. Alternatively, this can be marked after lifting the tissue section. Adhesive In the case of routine section and staining, no adhesive is required. However, in certain situations, we use cell adhesive materials such as: Steps of Tissue Sectioning 1\. *Trimming the tissue*: Trimming the tissue is needed to expose the tissue piece within the paraffin wax for cutting. The block is fixed in the chuck of the microtome, and the paraffin is trimmed till the tissue is fully exposed. Adequate caution should be taken not to overcut tissue as this may produce various artefactual changes. 2\. *Cooling the block*: After the initial trimming, the blocks are kept for cooling for 15--20 min. This will help to maintain the same consistency of the paraffin and tissue. This helps in easy cutting. 3\. *Cutting proper*: The block is fixed in the chuck of the microtome. The cutting surface of the block should be parallel to the knife. The clearance angle should be only 2--5° to have a good section. Gentle, smooth and slow strokes cut the tissue in the block. The ribbon-like tissue sections are produced. The tip of the ribbon is held by forceps, and the end part of the ribbon is removed from the knife edge by a brush. In case of any difficulty in getting the flat section, the cutting surface should be gently warmed with warm water. 4\. *Floating the ribbon*: The ribbon of the tissue is floated in the water bath, which makes the tissue flat and removes any wrinkling. With the help of the forceps, the individual sections are separated. As mentioned before, the temperature of the water bath should be constantly maintained below the melting point of the paraffin wax. In case of temperature variation in the bath, air bubbles may be formed that may rupture the tissue. 5\. *Picking up the tissue*: The slide is placed vertically within the water bath in front of the tissue, and when the tissue is touched, the slide is withdrawn vertically from the water. The tissue pickup process must be gentle and smooth. To prevent any mix-up, the water bath should be cleaned immediately after cutting each block. 6\. *Drying the section*: The slide containing the picked-up section is kept in the rack. The slides are now kept in a hot oven to get dry. The oven\'s temperature should be slightly more than the melting point of the paraffin. Troubleshooting in tissue sectioning