HISTOLOGICAL SPECIMENS MICROTOMY by Sarita Solanki & Dr S Nayak

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PREPARATION OF HISTOLOGICAL SPECIMENS : MICROTOMY Mrs. Sarita Solanki Assistant Professor saritasinghsolanki@gmail.com Dr. S. Nayak Principal & Professor principal.bcp@gmail.com BANSAL COLLEGE OF PHARMACY, BHOPAL Visit : www. bansalpharmacy.com

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# A microtome (Greek mikros ,"small",and temnein ,"to cut") is a sectioning instrument that allows for the cutting of extremely thin slices of material, known as sections. Microtome sections can be made thin enough to section a human hair across its breadth, with section thickness between 0.05 μ m (50 nm) to 100 μ m. HISTORICAL BACKGROUND George Adams (1770) Alexander Cummings (1795) Andrew Prichard (1835) Wilhelm His. Sr (1865) Jan Evangelista Purkvne, the purkvne model is first in use. At the end of the 1800s, the development of microtome along with staining techniques allowed for the visualization of microscopic details Today, the majority of microtomes are available having changeable knife holder, a specimen holder and an advancement mechanism for controlling the thickness of sections..

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Steps in preparation of histological Specimens 1. Tissue Fixation 2.Tissue processing : (A) Dehydration. (B) Clearing. (C) Infiltration (D) Embedding. 3. Sectioning through microtome 4.Fixation of Sections 5. Staining and Mounting

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1.TISSUE FIXATION Fixation is the process of killing and hardening tissue. The first phase of fixation is the rapid killing of tissues; the second phase, the hardening of tissue. Tissue should be placed in the fixative immediately upon removal from the body to preserve tissue element as they were in life. The aim of fixation - To prevent tissue from bacterial attack. - To preserve tissue from degradation, and to maintain the structure of the cell and of sub-cellular components such as cell organelles. - To fix the tissues so they will not change their volume and shape during processing. - To prepare tissue and leave it in a condition which allow clear staining of sections. - To leave tissue as close as their living state as possible, and no small molecules should be lost.

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Factors affect fixation : PH. Temperature. Penetration of fixative. Volume of tissue. Types of fixative: 10% Formalin solution (most widely used) G lutaraldehyde (2.5% solution in phosphate buffer saline) Zenker’s fluid Absolute alcohol Bones and calcified tissues requires decalcification prior to fixation. Decalcification can be done by 5% Aqueous solution of nitric acid Formic acid –sodium citrate solution Sulfosalicylic acid method

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Mechanism : These fixatives preserve tissues or cells mainly by irreversibly cross-linking proteins. Aldehyde fixatives cross-link amino groups in proteins through the formation of CH 2 (methylene) linkage, in the case of formaldehyde, or by a C 5 H 10 cross-links in the case of glutaraldehyde. Drawback : Fixation can damage the biological functionality of proteins, particularly enzymes and can also denature them to a certain extent. Formalin fixation leads to degradation of mRNA and DNA in tissues. However, extraction, amplification and analysis of these nucleic acids from formalin-fixed, paraffin-embedded tissues is possible using appropriate protocols.

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TISSUE PROCESSING : (A) Dehydration For light microscopy, paraffin wax is most frequently used. Since it is immiscible with water, the main constituent of biological tissue, water must first be removed in the process of dehydration. Samples are transferred through baths of progressively more concentrated ethanol to remove the water. Dehydration from aqueous fixatives is usually initiated in 60%-70% ethanol, progressing through 90%-95% ethanol, then two or three changes of absolute ethanol before proceeding to the clearing stage. Duration of dehydration should be kept to the minimum consistent with the tissues being processed. Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol, blocks 5 mm thick require up to 90 minutes or longer in each change. Tissues may be held and stored indefinitely in 70% ethanol without harm. Some commonly used dehydrating agents : Ethanol, Methanol, Acetone.

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(B) Clearing Replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium. Choice of a clearing agent depends upon the following: - The type of tissues to be processed. - Speedy removal of dehydrating agent . - Ease of removal by molten paraffin wax . - Minimal tissue damage . Some clearing agents: Xylene, Toluene, Chloroform, Benzene (C) Infiltration Infiltration (Internal Embedding) is the process of replacing clearing agent by the embedding media. After dehydration and clearing, tissues are transferred to molten embedding media for specified period of time, according to the protocol.

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Processing methods and routine schedules Tissue processing takes at least 16 hrs, due to overburden of excess work, histology technician uses Histokinetic machine (Tissue processor), in which tissues forwarded automatically to next step after the specified period of time. Machine processing (B) Manual processing This can be achieved by changing fluid manually. It needs the persons physical appearance.

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(C) Embedding After the infiltration, biological tissue must be supported in a solid medium or hard matrix, firm enough to support the tissue and give it sufficient rigidity to allow thin sections to be cut, and yet soft enough not to damage the knife or tissue. Various Embedding materials are Paraffin wax, Agar, Gelatin , Epoxy resin, celloidin, nitrocellulose, carbowax. During this process the tissue samples are placed into moulds along with liquid embedding material which is then hardened. This is achieved by cooling in the case of paraffin wax and heating (curing) in the case of the epoxy resins. The acrylic resins are polymerised by heat, ultraviolet light, or chemical catalysts. The hardened blocks containing the tissue samples are then ready to be sectioned. Paraffin wax is most commonly used. The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax: improve ribboning, increase hardness, decrease melting point, improve adhesion between specimen and wax

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Precaution while embedding in wax The wax is clear of clearing agent. No dust particles must be present. Immediately after tissue embedding, the wax must be rapidly cooled to reduce the wax crystal size.

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There are four main mould systems presently in use : 1 - Traditional methods using paper boats 2 - Leuckart or Dimmock embedding irons or metal containers 3 - The Peel-a-way system using disposable plastic moulds and 4 - Systems using embedding rings or cassette-bases which become an integral part of the block and serve as the block holder in the microtome. Tissue processing embedding moulds: (A) paper boat; (B) metal boat mould; (C) Dimmock embedding mould; (D) Peel-a-way disposable mould; (E) base mould used with embedding ring (F) or cassette bases (G)

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General Embedding Procedure 1- Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present. 2- Select the mould, there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax. Fill the mould with paraffin wax. 3- Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat. 4- Insert the identifying label or place the labeled embedding ring or cassette base onto the mould.. Cool the block on the cold plate. 5- Remove the block from the mould. 6- Cross check block, label and worksheet. Tissue embedded paraffin blocks

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ORIENTATION OF TISSUE IN THE BLOCK Correct orientation of tissue in a mould is the most important step in embedding. Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy. . Elongate tissues are placed diagonally across the block. Tubular and walled specimens such as gastrointestinal tissues are embedded so as to provide transverse sections showing all tissue layers. Tissues with an epithelial surface such as skin, are embedded to provide sections in a plane at right angles to the surface (hairy or keratinised epithelia are oriented to face the knife diagonally). Multiple tissue pieces are aligned across the long axis of the mould, and not placed at random.

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(3) SECTIONING The results produced by microtomy depends the knives used to cut the sections, it is imperative that each operator know how to care for knife as well as how to use it. Microtome knives : The design of a microtome knife is dependant upon the material and preparation of the samples, as well as the final sample requirements (e.g. cut thickness and quality). STEEL KNIVES, NON-CORROSIVE KNIVES FOR CRYOSTATS, DISPOSABLE BLADES, GLASS KNIVES, DIAMOND KNIVES Knife Profile : Knives are characterized by the profile of the knife blade, categorised as Planar concave,Wedge shaped, Chisel shaped

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SECTIONING PROCEDURE The Paraffin block trimmed to correct shape. About 3 mm paraffin is left at the sides of object, while at the bottom is 7mm. The trimmed block attached to microtome block holder either directly or on the metal disc having rough surface. Previously sharpened and cleaned knife is fixed on knife holder. It should be inclined at an angle of about 8 o from the vertical. Wheel rotated carefully in order to bring the block close to the knife, the horizontal edge of block exactly parallel to the knife edge. The scale of the measuring thickness is placed at the required reading. Wheel rotated with a uniform motion. Each stroke cutting a section of block, continuous ribbon of serially cut section is formed. As the ribbon grows out, it is supported by a moist brush and after a ribbon of suitable length is cut it is detached and laid out on the slide, which is flooded with distilled water.

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(4) FIXATION OF SECTION The "ribbon" of these sections on the slide is allowed to float on the water bath. The temperature of the water is around 40°C (lower than the m.p. of paraffin) so that the paraffin spreads a little, straightening folds in the tissue. If the water is any hotter, the paraffin melts and the tissue is lost. After 2-3 minutes these ribbon sections are taken on a adhesive coated slides and allowed to cool on standing position on slide holder, excess of water can be dropped out on this position. Then slides kept on hot plate for fixation at temperature 45-50 0 C for 25 minutes, slides are removed and allowed to cool. Now the slides are ready for staining. Following adhesives can be used : Mayer’s Egg Albumin Eggs adhesive from dried albumin Gelatin adhesive

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(5) STAINING Hematoxylin and eosin (H & E stain) Staining : H & E stain is a charge-based, general purpose stain, it is used universally for routine histological examination of tissue sections. Hematoxylin, a basic dye, stains nuclei blue due to an affinity to nucleic acids in the cell nucleus; eosin, an acidic dye, stains the cytoplasm pink.

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H & E STAINING PROCEDURE Xylene, absolute alcohol, 95% alcohol, 70% alcohol then washed with water.(Deparaffinize and hydrate to water) If sections are Zenker-fixed, remove the mercuric chloride crystals with either lugol’s solution or 1% alcoholic iodine solution (10-15 min), wash with tap water. Treat with 5% sodium thiosulphate . Wash well with tap water. Harris hematoxylin for 15 minutes Wash in running tap water. Differentiate in acid alcohol(3-10 quick dips). Check the differentiation with the microscope- nuclei should be distinct and the background very light or colorless. Wash in tap water very briefly. Dip in Ammonia water or saturated lithium carbonate until sections are bright blue. Wash in running tap water for 10-20 minutes. Counter stain with eosin from 15 seconds to 2 minutes depending on the age of the eosin, and the depth of the counter stain desired. For even staining results dip slides several times before allowing them to set in the eosin for the desired time Dehydrate in 95% alcohols. Absolute alcohol –at least 2 changes Clear in xylene, two changes of 2 minutes each . Mount in Permount or balsam RESULTS : Nuclei - blue - with some metachromasia Cytoplasm - various shades of pink-identifying different tissue components

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APPLICATIONS OF MICROTOMY 1- Traditional histological technique: The tissue is cut in the microtome at thicknesses varying from 2 to 25 micrometers thick. From there the tissue can be mounted on a microscope slide, stained and examined using a light microscope. Can be achieved by rotary microtome. 2 - Botanical Microtomy Technique: Hard materials like wood, bone and leather require a sledge microtome. These microtomes have heavier blades and cannot cut as thin as a regular microtome.

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SLEDGE MICROTOME A sledge microtome is a device where the sample is placed into a fixed holder (shuttle), which then moves backwards and forwards across a knife. It is applicable for the preparation of large samples. Typical cut thickness achievable on a sledge microtome is between 1 and 60 µm. ROTARY MICROTOME The staged rotary action of wheel, slowly moves a block into the path of an extremely sharp steel knife. Modern blades are disposable and last for around 20 to 30 blocks. In a rotary microtome, the knife is typically fixed in a horizontal position. The typical cut thickness for a rotary microtome is between 1 to 60 µm.

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3- Cryosectioning technique : Water-rich tissues are hardened by freezing and cut in frozen state. Sections are stained and examined with a light microscope. This technique is much faster than traditional histology (5 minutes vs. 16 hours) and are used in operations to achieve a quick diagnosis. Cryosections can also be used in immunochemistry as freezing tissue does not alter or mask its chemical composition . For the cutting of frozen samples, many rotary microtomes can be adapted to cut in a liquid nitrogen chamber, in a so-called cryomicrotome setup.

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4- Electron microscopy : Extremely thin sections are required for transmission electron microscope (TEM) and serial block face scanning electron microscope (SBFSEM). The typical thickness of sections is between 40 and 100 nm for transmission electron microscopy . Sectioning can be achieved by the Ultramicrotone , in which a glass or gem grade diamond knife is used to cut very thin sections (typically 60 to 100 nm). Complementing traditional TEM techniques ultramicrotomes are mounted inside an SEM chamber so the surface of the a block face can be imaged at and then removed with the microtome to uncover the next surface which is ready for imaging. This technique is called SBFSEM. The thickness of these cuts is between 30 and 50 nm.

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VIBRATING MICROTOME : The vibrating microtome operates by cutting using a vibrating blade, allowing the resultant section to be made with less pressure than which is required for a stationary blade. The vibrating microtome is usually used for difficult biological samples. The cut thickness is usually around 10-500 µm. SAW MICROTOME : The saw microtome is especially for hard materials such as teeth or bones. The microtome of this type has a recessed rotating saw, which slices through the sample. The minimal cut thickness is approximately 30 µm, and can be made for comparatively large samples. LASER MICROTOME : Laser microtome cuts the target specimen with a infra-red laser instead of a mechanical knife. This method is contact-free and does not require sample preparation techniques. The laser microtome has the ability to slice almost every tissue in its native state. Depending on the material being processed, slice thicknesses of 10 to 100 µm are feasible.

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THANKS Mrs. Sarita Solanki Assistant Professor saritasinghsolanki@gmail.com Dr. S. Nayak Principal & Professor principal.bcp@gmail.com BANSAL COLLEGE OF PHARMACY, BHOPAL Visit : www. bansalpharmacy.com

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