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TISSUE FIXATION micro anatomical, histological, cytological
- 1. TISSUE FIXATION (IN CYTOLOGICALAND HISTOCHEMICAL) NAME:- MANVI PAREKH ROLL NO.:- 16 SUBJECT:- ZOOLOGY(GIA) COURSE:- 404
- 2. INDEX • Introduction • Objectiveand Importance • Types of fixative • Mechanism and Function • Advantages and Disadvantages • Factors affecting fixation and fixative • Difference between simple & compound fixative • Conclusion
- 3. Introduction • Fixation isa complete and complex physiochemical process wherein cells and tissues are chemically fixed for further analyses. • Fixation is a complex series of chemical events which brings about changes in the various chemical constituents of cell like hardening, however the morphology of a cell and structural details are preserved. • A Fixative is a chemical solution used to preserve biological tissues and cells. • Fixatives are used for Solidification, Hardening, Optical differentiation, prevention of autolysis and tissue putrefaction.
- 4. Two Content Layoutwith Table OBJECTIVE In art, fixatives (like varnishes and natural resins) are used to preserve drawings and pastels, preventing smudging and degradation. In histology fixatives are crucial for preserving tissue samples for microscopic examination. The use of formaldehyde, developed in the late 19th century, became standard due to its effectiveness in cross-linking proteins, thus preserving cellular structure. Over time, various fixatives have been developed, including buffered formalin and alternatives like glutaraldehyde, alcohol and other substances were used. The evolution of fixatives reflects advancements in both art techniques and scientific methodologies, highlighting their importance in preserving and analyzing biological and artistic works.
- 5. Two Content Layoutwith SmartArt Importance • In order to study tissues with a microscope they must be preserved (fixed) and cut into sections thin enough to be translucent. • Fixation is a critical step in the preparation of histological sections. If it is not carried out under optimal conditions or if fixation is delayed, a tissue specimen can be irreversibly damaged. • The main goal of tissue fixation is to keep cells and tissues as close to their living state as possible, allowing for thin, stained sections for analysis • The choice of fixative and method used can vary based on what further processing and analysis are planned. While no fixative is perfect, formaldehyde is the closest option. IMPORTANCE
- 6. Add a SlideTitle - 2 TYPES OF FIXATIVE PHYSICAL FIXATIVE CHEMICAL FIXATIVE Physical method microwaving and cryo-preservation (freeze drying) Chemical fixation is usually achieved by immersion fixation, perfusion fixation,vapour-fix freeze-dried tissues. (eg: paraformaldehyde and osmium tetroxide)
- 7. CHEMICAL FIXATIVES SIMPLE FIXATIVES ->Formalin -> Mercuric chloride -> Picric acid -> Acetone -> Ethyl alcohol -> Osmium tetroxide Micro anatomical Cytological Histochemical -> Formal saline ->Neutral buffer formaline ->Zenker’s fluid ->Bouin’s fluid -> Acetone -> Acrolein/chromyl chloride -> Formaldehyde vapour Nuclear fixative -> Carnoy’s fluid -> Clarke’s fluid ->New Comer’s fluid Cytoplasmic fixatives -> Champy’s fluid- >Formal saline ->Formal Calcium COMPOUND FIXATIVES
- 8. COMPOUND FIXATIVES A compoundfixative is a solution used in histology and pathology to preserve tissue samples for microscopic examination. It usually contains multiple chemical agents that work together to stabilize and protect cellular structures while preventing decomposition and autolysis. Benefits: Compound fixatives provide a broader range of fixation, preserving different types of tissues more effectively than single-agent fixatives. Multiple agents can improve the quality of histological stains, making cellular structures more visible.
- 9. CYTOLOGICAL FIXATIVE Cytological fixativesare used to preserve the constituent elements of the cells. • Nuclear fixative:- These are the fixatives that primarily fix the nuclear components of the cells. Eg:- carnoy’s fluid. • Cytoplasmic fixatives: These are the fixatives that primarily fix the cytoplasmic components of the cell. For example, Champy’s fluid, Zenker’s fixative etc
- 10. NUCLEAR FIXATIVE 1. Carnoy'sfluid:- Rapid acting good nuclear preservation Nissl’s granules and glycogen are preserved. It lyses erythrocytes and dissolves lipids and can produce excessive hardening and shrinkage. Chemical composition is: Absolute alcohol = 60ml Chloroform = 30ml Glacial acetic acid = 10 ml
- 11. 2.Clarke’s fluid:- Usedon frozen sections and smears. In short time give fair result Preserves nucleic acids, but lipids are extracted. Fixation time is 3-4 hours Chemical composition is: Absolute alcohol = 75 ml Glacial acetic acid = 25 ml 3.New Comer's fluid:- Preservation of chromatin. Fixing polysaccharides. Small pieces of tissue should be used. Fixation is complete in12-18 hours. Chemical composition is:- Isopropanol = 60 ml Propionic acid = 40ml Petroleum ether = 10 ml. Acetone =10 ml. Dioxane =10 ml.
- 12. CYTOPLAMIC FIXATIVES 1.Champy's fluid:- Prepared fresh. Preserves the mitochondrial fat and lipids. Penetration slow and uneven. Section will be 1-2 mm thick not more than 3 mm. Pieces more than 2mm should be washed overnight after fixation After fixation specimen washed over in running tap water overnight to remove chromium and osmium compounds. Chemical Composition is: Chromium trioxide = 1g Potassium Dichromate = 3g Osmium tetroxide = 1g Distilled water = 250ml
- 13. 2. Formal saline Formaldehyde diluted in a saline solution Reduce autolysis and decay Maintain cellular structure It often produces formalin pigment. Fixation time is 12 to 24 hours. Chemical Composition is: 40% Formaldehyde = 100ml Sodium Chloride = 9g Distilled water = 900 ml 3. Formal Calcium Preserved lipids especially phospholipids. Fixation time is 12 to 24 hours. Also preserved mineralized tissue like bone. Chemical Composition is: 40% Formaldehyde = 100ml Calcium Chloride = 10g Distilled water = 900 ml
- 14. HISTOCHEMICAL FIXATION They areused for the Histochemical studies of the tissues where the minimum or no changes in the components to be demonstrated are required. for example, Buffered formalin or vapor fixatives include Formaldehyde vapor, Acetone, Acrolein etc.
- 15. HISTOCHEMICAL FIXATION 1.Acetone (CH3COCH3):- Similar action like alcohol Used as a fixative and dehydrant for tissue sample. Showing certain enzymes in tissues,usually when kept cold(4 °C). Because it is highly volatile and flammable it is generally not used on automatic tissue processors. Cold acetone is sometimes used as a fixative for the histochemical demonstration of some tissue enzymes like phosphatases and lipases. 2. Formaldehyde Vapour It is obtained by heating paraformaldehyde at temperature between 50° and 80 °C. Blocks of tissue require (3-5) hours whereas section require (½-1) hours.
- 16. 3. Acrolein /chromylchloride:- It used at 37 °C for 1-2 hours. Acrolein was introduced by Luft as a primary fixative agent, and it is a three carbon αβ Unsaturated monoaldehyde. Preservation of structural detail and conserves the virus antigenicity. It is also known as acrylic aldehyde. It reacts with macromolecules that result in formation of cross-links that are reversible. Acrolein is not commonly used because it is unstable at alkaline pH and forms insoluble polymers. Acrolein is highly reactive and is found to penetrate tissues rapidly. Acrolein fixatives are chiefly used in enzyme histochemistry.
- 17. MECHANISM OF FIXATION Twomain mechanisms of chemical fixations are:- • Cross linking • Coagulation Cross-linking:-involves covalent bond formation both within proteins and between them, which causes tissue to stiffen and therefore resist degradation. Coagulation:- is caused by the dehydration of proteins through the use of alcohols or acetone. These reagents remove and replace free water in cells and tissues and cause a change in the tertiary structure of proteins by destabilizing hydrophobic bonding. It is also called denaturation.
- 18. If a freshtissue is kept at room temperature it will become liquefied with a foul odour mainly due to action of pathogens i.e.; putrefaction and autolysis so the first and fore most aim of fixation is: 1. To preserve a tissue live and prevent from post mortem changes like autolysis and putrefaction . 2.Solidification: Converts the normal gel form into a sol form. The semifluid consistency of cells (gel) is changed into an irreversible semisolid consistency (solid) 3. Hardening: Easy manipulation of soft tissue like brain, intestines etc. is possible and maximally explored through Hardening via fixatives. 4. Optical differentiation: It alters to varying degrees the refractive indices of the various components of cells and tissues so that unstained components are more easily visualized than when unfixed. Function of fixatives
- 19. ADVANTAGES…. Molecular IntegrityPreservation: Fixation maintains the spatial arrangement and structural integrity of cellular components, which is crucial for accurate assessment of tissue morphology and for subsequent molecular analyse. Inhibition of Degradation Pathways: By rapidly cross-linking proteins and stabilizing nucleic acids, fixation prevents autolysis and necrosis. Optimization for Diagnostic Techniques: Fixed tissues are compatible with a variety of staining techniques, including histochemical, immunohistochemical, and molecular assays. This compatibility enhances the ability to visualize specific cellular markers and pathways relevant to disease diagnosis. Facilitation of Long-term Storage: Fixed tissues can be preserved for extended periods without significant degradation.
- 20. Disadvantages…. Tissue Shrinkage: Somefixatives can cause tissue shrinkage, which may distort the architecture and make it difficult to interpret results. Eg :Formalin and Mercuric chloride Toxicity: Many chemical fixatives are hazardous to health (e.g., formaldehyde is a known carcinogen), requiring careful handling and disposal. Delayed Processing: Chemical fixation often requires longer processing times compared to other preservation methods, which can delay results. Inconsistent Penetration: Fixatives may not penetrate tissues uniformly, leading to variable fixation quality, especially in larger specimens. Potential for Over fixation: Prolonged exposure to fixatives can lead to over fixation, which can obscure cellular details and complicate staining. Color Change: Some fixatives can cause discoloration of tissues, which may affect visual assessments and imaging. Environmental Concerns: Disposal of chemical fixatives can pose environmental risks if not managed properly, requiring adherence to specific regulations.
- 21. FACTORS AFFECTING FIXATION Length of Fixation:- Thinner tissues require less time to fix, while thicker tissues need more. Too long a fixation can make tissues brittle, while too short may lead to inadequate penetration. Overnight fixation is usually best. Concentration:- Each fixative has an optimal concentration. Low concentrations require longer fixation, while high concentrations can damage cells and enzymes. Temperature:- Fixation is most effective between 37°C and 45°C. Lower temperatures slow the process and may cause tissue breakdown, while excessive heat can also harm the tissue. Size:-Tissue thickness affects fixation; ideal specimens are 4 to 6 mm thick for good fixative penetration. Thicker samples may not be properly fixed and can break down. pH:- In the range of pH6-8. Otherwise the acidity and alkalinity can impact the fixation process and resulting tissue morphology.
- 22. Difference between Simpleand Compound fixatives
- 23. Fixation is akey step in histology and histopathology procedure. Each and every fixative has its own advantage and disadvantage. Various different fixatives perform various functions, and various factors such as size, concentration and temperature etc have direct effects on fixation procedure. The choice of fixative can significantly influence the quality of tissue preservation and the clarity of subsequent analyses CONCLUSION
- 24. REFERENCE:- https://research.unityhealth.to InternationalJournal of Clinical and Diagnostic Pathology 2021; 4(4): 113-11 Images:- google images