21. fixatives.pptx in relation to oral cavity

It is the process by which the constituents of cells and tissue are fixed in a physical and chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of architecture. This is achieved by exposing the tissue to chemical compounds, called fixatives.

1. FIXATIVES Dr. Noori

2. CONTENTS INTRODUCTION AIMS AND OBJECTIVES PROPERTIES CLASSIFICATION PHYSICAL CHEMICAL FACTORS AFFECTING FIXATION ARTEFACTS CONCLUSION

3. FIXATION It is the process by which the constituents of cells and tissue are fixed in a physical and chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of architecture. This is achieved by exposing the tissue to chemical compounds, called fixatives.

4. FIXATIVE A fixative is defined as a substance which prevents post-mortem changes and stabilizes the microscopic cellular structure and composition in the specimen to allow subsequent processing and to preserve them for retrospective analysis.

5. AIMS & OBJECTIVES OF FIXATION To prevent autolysis and putrefaction. Rapid and even penetration of fixative. To preserve cells and tissues in a life like manner as possible. Elements that are to be demonstrated must remain in maximum concentration and precise localization. Stabilize labile elements.

6. Must be rigid to allow sectioning. Must allow staining Optical contrast must be induced for morphological examination. Allow long storage of tissues Must not react with the receptor sites & thus must not interfere with the staining procedure.

7. PROPERTIES OF AN IDEAL FIXATIVE : It should react rapidly and completely with the tissues, fixing all the constituents without removing any part of any of them. It should penetrate rapidly and deeply , without impeding its own progress by causing hardening or shrinkage of the outer layer. The full range of staining methods should be possible after its use.

8. Tissue should not be distorted by swelling or shrinkage during fixation and should be stabilized sufficiently to withstand the chemicals used at later stages of processing. Tissue should be made hard enough to be handled with ease at later stages but not over hardened. It should be cheap, non- toxic, non-inflammable and non-irritant.

9. CLASSIFICATION OF FIXATIVES

10. A) Baker classified fixatives according to their ability to coagulate a soluble protein as: 1) Coagulant 2) Non- coagulant B) Based on their effect on tissue protein : 1) Precipitants- Ethanol, Methanol 2) Non-precipitants-Osmium tetroxide, Potassium dichromate, formaldehyde.

11. C) Classification on the Basis of Type of Structures Fixed, Microanatomical Fixatives : 10% Formalin, Bouin’s fluid , Zenker’s Fluid Cytological Fixatives Cytoplasmic Fixatives : Formol Saline, Formol Calcium. Nuclear Fixatives : Alcohol, Chloroform, Glacial acetic acid 3. Histochemical Fixatives : Vapour Fixatives: Formaldehyde, Glutaraldehyde.

12. D) Based on the number of component present Simple Fixatives : Formaldehyde, Osmium Tetroxide, Picric acid Compound Fixatives : Formol Saline, Bouin’s Fluid , Zenker’s Fluid

13. E) Based on nature of fixation Immersion fixation Heat fixation Coating fixation Vapour fixation Perfusion fixation Freeze drying Microwave fixation

14. Physical Methods Of Fixation

15. HEAT FIXATION The simplest form of fixation is heat. Boiling or poaching an egg precipitates the proteins and on cutting, the yolk and egg white can be identified separately. Each component is less soluble in water after heat fixation than the same component of a fresh egg. In histopathology heat is primarily used to accelerate other forms of fixation as well as the steps of tissue processing.

16. MICROWAVE FIXATION The great advantage of microwave stabilization is that there are no chemicals involved which would extract important components from the tissue. Microwave heating speeds fixation and can reduce times for fixation of some gross specimens and histological sections from more than 12 hours to less than 20 minutes

17. Microwave-Stimulated Fixation with Fixatives : Formalin, is a solution containing methylene glycol and fomaldehyde . Normal formalin fixation takes place in three steps : First, the methylene glycol quickly penetrates the tissue (formalin will penetrate a 5 mm block in 4 hours); Second , some methylene glycol is slowly converted to formaldehyde by dehydration; Third , formaldehyde binds very slowly to the proteins in the tissue by cross-linking. All three of these steps can be accelerated by microwave exposure

18. Microwaving tissue in formalin results in the production of large amounts of dangerous vapors, so in the absence of a hood for fixation, or a microwave processing system designed to handle these vapors, this may cause safety problems. Recently, commercial glyoxal-based fixatives which do not form vapors when heated at 55°C have been introduced as an efficient method of microwave fixation.

19. FREEZE-DRYING AND FREEZE SUBSTITUTION Helpful in studying soluble materials and small molecules. Tissues are cut into thin sections, immersed in liquid nitrogen, and the water is removed in a vacuum chamber at -40°C. The tissue can be post-fixed with formaldehyde vapor. In substitution, specimens are immersed in cold (-40°C) fixatives, such as acetone or alcohol, which slowly remove water through dissolution of ice crystals, and the proteins are not denatured: bringing the temperature gradually to 4°C will complete the fixation process.

20. Ideal for Soluble material and very small molecules Advantages Excellent for enzymatic study No change of proteins No/ minimal shrinkage of tissue

21. CHEMICAL FIXATION

22. COAGULANT FIXATIVES Both organic and non-organic solutions may coagulate proteins making them insoluble. Cellular architecture is maintained primarily by lipoproteins and by fibrous proteins such as collagen; coagulating such proteins maintains tissue histomorphology at the light microscopic level.

23. Dehydrant Coagulant Fixative The most commonly used coagulating fixatives are alcohols (e.g. ethanol, methanol) and acetone. Removal and replacement of free water from tissue by any of these agents disrupt the tertiary structure of proteins ie , denaturation and change in physical properties causing insolubility and loss of function.

24. Other types of coagulant fixative Acidic coagulants such as picric acid and trichloroacetic acid change the charges of proteins and disrupt tertiary structures of proteins. Acetic acid coagulates nucleic acids but does not fix or precipitate proteins: it is therefore added to other fixatives to prevent the loss of nucleic acids

25. PICRIC ACID It is a protein precipitant forming picrates with amino acids, including those in nucleoproteins and with some carbohydrates. A coagulant fixative

26. Disadvantages : 1) Explosive. Crystals are not allowed to get dry. Thus, should be kept under water. 2) Penetrates very poorly. 3) Causes shrinkage but does not harden or distort

27. ACETIC ACID Coagulate nucleoproteins but otherwise has little or no fixing effect Used in several mixtures because it causes considerable swelling of tissues and so counteracts the shrinking effect of other constituents. Penetrates well and causes no hardening.

28. TRICHLOROACETIC ACID Never used alone Because of its swelling effect - may be employed usefully in a compound fixative A protein precipitant, and has some slight decalcifying properties May remove minerals ( e.g Ca, Fe, Cu) from tissues .

29. Non-coagulant cross-linking fixatives Several chemicals were selected as fixatives secondary to their potential actions of forming cross-links within as well as between nucleic acids and proteins. Examples include formaldehyde, gluteraldehyde and other aldehydes, metal salts such as mercuric and zinc chloride and other metallic compounds such as osmium tetroxide.

30. 1) Aldehydes: Aldehyde reacts by formation of cross-links with proteins

31. Formaldehyde (CH2O) A gas, commercially available as a 40% solution in water- termed formalin 10% formalin consists of: it actually contains 4% formaldehyde . Formalin- 10 ml Water - 90 ml

32. Advantages: 1) Cheap, easy to prepare, store and dispose. 2) Stable at room temperature. 3) Allows subsequent use of most staining procedures. 4) Penetrates tissue well. 5) Frozen sections can be made with formalin fixed tissues. 6) Allows the study of some tissues enzymes.

33. Disadvantages: 1) Allergy and dermatitis. 2) Fumes are irritant to nose and eyes. 3) Forms acid formalin hematin pigment which is dark brown pigment which can be seen in sites containing blood. 4) Becomes formic acid on storage and this decreases fixation abilities.

34. 5 ) Long term storage of concentrated formalin may be lead to the formation of a white precipitate of paraformaldehyde. 6) Causes shrinkage of collagen . 7) May be carcinogenic

35. Glutaraldehyde Commercially as a 25% solution Fixatives for electron microscopy because it fixes and preserve the ultrastructure. Most efficient cross-linking agent for collagen

36. Advantages Give a better preservation of ultrastructure More rapid fixing action than formaldehyde. Less cell shrinkage Preservation of protein is better

37. Disadvantages 1) Poor penetration and tissue should be thin. Brittleness of tissues that hinders microanatomy. Less stable compound

38. OSMIUM TETROXIDE Osmic acid- oxidizing agent It is now largely employed as a fixative in electron microscopy. A non-coagulant fixative.

39. Advantages: Excellent preservation of fine structures in electron microscopy. 2) It demonstrates lipids.

40. Disadvantages: Expensive With specimens greater than (2-3 mm3) in thickness, it penetrates poorly and unevenly. Section show over fixed blackened zone at the periphery and under fixed zone at the centre. 3) Unstable in air.

41. 4) Reduced by light, should be stored in dark or in brown bottles and stabilized by the addition of one drop of saturated aqueous mercuric chloride. 5) Irritant to the eyes can cause conjunctivitis. 6) Interfere with most staining procedures. 7) It doesn't fix carbohydrate and protein 8) Tissue swelling may occur.

42. Mercuric Chloride (HgCl 2 ) Protein precipitant - rapidly penetrates and hardens. Disadvantages : Formation of deposits of intensely black precipitates of mercuric pigment in the tissues. Excessive exposure produces excessive hardness and makes cutting of thin sections difficult. Mercuric salts are highly toxic and must not be disposed into sewage systems.

43. 4 ) Corrodes metal so must be stored in non-metallic containers. 5 ) Its presence in tissue inhibits freezing and makes frozen sections difficult to prepare .

44. Cross-linking fixatives for electron microscopy Cell organelles such as cytoplasmic and nuclear membranes, mitochondria, membrane-bound secretory granules, and smooth and rough endoplasmic reticulum need to be preserved carefully for electron microscopy. The lipids in these structures are extracted by many fixatives with dehydrants (e.g. alcohols). Therefore for ultrastructural examination it is important to use a fixative that does not solubilize lipids. The preferred fixatives are a strong crosslinking fixative such as glutaraldehyde , a combination of glutaraldehyde and formaldehyde,OsO4.

45. SPECIAL FIXATIVES DICHROMATE AND CHROMIC ACID FIXATION Dichromate-containing fixatives have primarily been used to prepare neuroendocrine tissues for staining, especially normal adrenal medulla and related tumors (e.g. phaeochromocytomas). Chromium trioxide dissolves in water to produce an acidic solution of chromic acid, with a pH of 0.85. Chromic acid is a powerful oxidizing agent which produces aldehyde from the 1, 2-diglycol residues of polysaccharides. Damage to skin. Thorough washing to remove oxides formed.

46. METALLIC IONS AS A FIXATIVE SUPPLEMENT Several metallic ions have been used as aids in fixation, including Hg2+, Pb2+, Co2+, Cu2+, Cd2+ and Zn2+. Mercury, lead, and zinc are used most commonly in current fixatives, e.g. zinc containing formaldehyde is suggested to be a better fixative for immunohistochemistry than formaldehyde alone.

47. Compound Fixatives Combination of fixatives are used to produce specific effects not possible with any of the reagent alone. The dehydrant ethanol, can be added to formaldehyde to produce alcoholic formalin. This combination preserves molecules such as glycogen and results in less shrinkage and hardening than pure dehydrants .

48. Types Choice - governed by the type of investigation required, both immediately and in the future Fixatives can be divided into three main groups : - Micro-anatomical - Cytological - Histochemical

49. Micro-anatomical Fixatives anatomy of the tissue, with the correct relationship of tissue layers and large aggregates of cells Fixatives for routine use should be drawn from this group

50. Cytological Fixatives Used when preservation of intracellular structures is of first importance These elements are preserved at the expense of even penetration, ease of cutting, and the loss of other cell structures

51. Histochemical Fixatives The fixative employed produces minimal changes in the element that is to be demonstrated The fixative used should be that recommended for the specific technique to be employed

52. MICROANATOMICAL FIXATIVES Routine formalin fixatives 10 % Formol-Saline : 10% formalin 0.9% NaCl Layer of marble chips/calcium carbonate added to neutralize formic acid production .

53. Buffered Gluteraldehyde Heidenhain’s Susa: It is a decalcifying solution that derives its name from the initial letters of sublimate (mercury bichloride saturated in 0.6% NaCl) and saure (trichloroacetic and glacial acetic acid), key ingredients of the fixative together with formalin, all in aqueous solution. It is considered a good general- purpose fixative, with rapid penetration. An excellent fixative for routine biopsy work and allows brilliant staining with good cytological detail. Gives rapid results, even penetration with minimum shrinkage. Tissue has to be left in it for over 24 hours. Tissue should be treated with iodine to remove mercury pigment impurity if any. Chemical composition is as: Mercuric chloride = 4.5gm Sodium chloride = 0.5 gm. Trichloroacetic acid = 2.0 gm. Acetic acid = 4.0 ml Distilled water to = 100 ml FORMALIN FIXATIVES FOR CARBOHYDRATES :

54. Advantages – Excellent fixative for routine biopsy work . Brilliant staining with good cytological detail . Rapid penetration, minimum shrinkage Disadvantages – intolerant fixative Tissue must be transferred to 96% absolute alcohol to avoid swelling of connective tissues

55. 3 . Zenker’s fluid: Mercuric chloride – 5g Potassium dichromate – 2.5g Sodium sulphate – 1g Distilled water to 100ml Glacial acetic acid immediately before use – 5ml Advantages – rapid & even penetration beneficial effect on cytologic, nuclear chromatin & fibre staining. Following its use, tissues must be washed in running water overnight to remove the excess dichromate and mercuric chloride pigment must be removed with iodine.

56. HELLY’S FLUID ( Zenker - Formol) Mercuric chloride 5g Potassium dichromate 2.5g Sodium sulphate 1g Distilled water 100 ml Add formalin immediately before use 5ml Excellent fixative for bone marrow, spleen It is irritational in that it contains pottassium dichromate which is an oxidising agent and formaldehyde which is a reducing agent, but it is an excellent microanatomical fixative.

57. Bouin's Fluid Picric acid, saturated aqueous solution- 75ml Formalin (40 per cent formaldehyde)- 25ml Glacial acetic acid- 5ml Penetrates rapidly and evenly and causes little shrinkage. Tissue fixed in it gives brilliant staining by the trichrome methods. The excess picric acid, to which the yellow colour of the tissue is due, should be removed from the section by treatment with alcohol or prolonged washing. Bouin's fluid can be used to demonstrate glycogen. Fixation is usually complete in 24 hours, but small pieces not exceeding 2—3 mm in thickness are fixed in 2—3 hours.

58. Cytological fixatives Subdivided into (A) nuclear (B) cytoplasmic.

59. Nuclear fixatives Carnoy’s fluid Absolute alcohol 60 ml Chloroform 30 ml Glacial acetic acid 10 ml Penetrates rapidly, gives excellent nuclear fixation, causes shrinkage, destroys most cytoplasmic elements. Recommended for the fixation of carbohydrates

60. Many lipids are also dissolved Used in the preparation of certain lipid rich tissues such as lipomas - help penetration of processing fluids

61. Clarke’s fluid- good nuclear detail, and also preserve cytoplasmic contents. Sanfelice’s fluid –good nuclear detail(mitotic figures and chromosome) Newcomer’s fluid –preserve chromosomes and chromatin. Flemming’s fluid –as secondary fixative of myelin following formalin.

62. CYTOPLASMIC FIXATIVES: Champy’s fluid : 3% pottassium dichromate 7 ml 1% chromic acid 7 ml 2% osmium tetroxide 4 ml Should be freshly prepared. Poor & uneven penetration Preserves mitochondria, fat, yolk, lipids

63. 2. Regaud’s fluid – mitochondria & chromaffin tissue 3. Muller’s fluid – bone specimens 4 . Zenker Formal - bone marrow, blood forming organs 5. Schaudinn’s fluid – For wet smears

64. Histochemical fixatives A good histochemical fixative should : 1.Preserve the constituent to be demonstrated, preferably preserving its morphological relationship . 2.Bind or otherwise preserve the specific tissue constituent, without affecting the reactive groups to be used in its visualization; 3. Not affect the reagent to be used in the process of visualization

65. Formal-saline -most common fixative Cold acetone - acetone at 0-4 C is widely used -study enzymes, particularly Phosphatases Absolute alcohol

66. FACTORS AFFECTING FIXATION

67. 1. Length of Fixation (Time) Different fixatives have different ideal fixation times. The fixative must diffuse into the specimen's center for fixation to take place, and then enough time must be allowed for the fixation reactions to take place. The optimal time will change depending on the fixative being used, as both diffusion time and reaction time depend on the specific reagent used. There is a lot of pressure in busy diagnostic laboratories to speed up turnaround times, and this can lead to the processing of incompletely-fixed tissues. Because poorly fixed tissue does not process effectively, this might result in low quality sections revealing tissue distortion and poor quality staining.

68. Ethanol will continue to fix the tissue if it is removed from formalin and placed in it during processing. As a result, the morphological image in the specimen's center will reflect ethanol fixation. Longer fixation times, however, lead to over-cross-linking, which makes samples fragile. If the fixation period is too brief, there won't be enough penetration into the tissues to cause cross-linking

69. 2. Temperature Increasing the temperature of fixation will increase the rate of diffusion of the fixative into the tissue and speed up the rate of chemical reaction between the fixative and tissue elements. It can also potentially increase the rate of tissue degeneration in unfixed areas of the specimen. Microwave fixation may involve the use of higher temperatures, up to 65°C, but for relatively short periods. For electron microscopic studies, 0°to 4°C is appraised as ideal temperature

70. 3. Concentration Fixative agents need prolonged time for fixation if concentration is low. If concentration of fixing agent is high, it results in damaging of cellular structures as well as obliterated enzyme activities. Too high a concentration may adversely affect the tissues and produce artifact similar to excessive heat. Different fixatives have different ideal concentration that is determined experimentally; for example, ideal fixative for oral soft tissue is formalin used in 10% concentrated solution

71. 4. Size Tissue thickness is one of the important factors for fixation. If the sample size is large, it is unfavorable for the fixative to penetrate and reach to the deeper part of the tissue, which would result in autolysis of epithelium. Ideally, 3mm to 5mm thick specimen is best suited for complete penetration by fixatives

72. 5. Osmolality If cells are fixed in a hypertonic solution, the cells may shrink. If the cells are fixed in a hypotonic solution, the cells may swell and burst. For that reason, it is recommended to use a normal phosphate buffered saline (PBS) based fixative. Hypertonic solutions give rise to cell shrinkage. Isotonic as well as hypotonic fixatives cause cell swelling and poor fixation. The best results are usually obtained using slightly hypertonic solution.

73. 6. Penetration rate The penetration rate of a fixing agent depends on its diffusion characteristics and varies from agent to agent. Formalin and alcohol penetrate the best and glutaraldehyde the worst. Mercurials and others are somewhere in between.

74. 7. Volume ratio It is important to have an excess volume of fixative in relation to the total volume of tissue because with additive fixatives, the effective concentration of reagent is depleted as fixation proceeds and in a small total volume this could have an effect on fixation quality. A fixative to tissue ratio of 20:1 is considered the lowest acceptable ratio but it would be better and faster if one can have a target ratio of 50:1. Agitation will also enhance fixation of the specimen. The use of small volumes of fixation fluids for larger specimens is the most frequent cause of poor tissue preservation

75. Fixation Artefacts

76. a . Inadequate fixation : CAUSES: Tissue sections not allowed enough time in fixative. • Inadequate amount of fixative relative to tissue volume. • Sections grossed too thick for good penetration. • Formalin solution is depleted.

77. Delay in fixation or inadequate fixation produces changes like : Altered staining quality of cells Cells appear shrunken and show cytoplasmic clumping Indistinct nuclear chromatin with nucleoli sometimes not seen Vascular structures, nerves and glands exhibit loss of detail

78. SOLUTIONS: Ensure that enough time is allowed for good fixation Ensure that the fixative volume is 15 to 20 times the tissue volume. • Ensure that the grossed sections are thin, preferably no more than3 mm thick. • Change formalin solutions frequently through out the process to prevent depletion. Hollow specimens like cystic cavities are either opened fresh or else fixed simultaneously from the outside and inside. Hollow specimen cavity is filled with formalin by syringe or catheter or packed with gauze or cotton impregnated with formalin. Cystic lesions are injected with formalin after the original fluid has been removed. Multilocular cysts require individual injection of larger cavities

79. b) Use of improper fixative: Fixation in alcohol results in poor staining of the epithelium and improper fixation of the connective tissue. Alcohol fixes tissues but causes severe shrinkage. Therefore, alcohol is not recommended as a substitute for formalin except in extreme emergencies. It also makes the tissue brittle, resulting in microtome sectioning artefacts with chattering and a Venetian blind appearance Currently, 10% neutral buffered formalin is highly recommended for routine fixation purposes. One excellent indicator of poor fixation is the loss of detail of extravasated RBC's.

80. c) Fixation artefact simulating acantholytic disease Tissue fixed in rehydrated formalin exhibits a prominent acantholysis of superficial epithelium with preservation and attachment of the basal cell layer to the underlying tissue. This acantholytic artefact simulates Pemphigus, Hailey-Hailey disease or Darier's disease. A similar artefact is produced by “fixing” the specimen in tap water.

81. Tissue immersed in water followed by routine processing

82. d . Dehydration artefact Tissues allowed to air-dry will dehydrate, particularly if placed on an adsorbent surface such as gauze sponge. Such tissue cannot be reconstituted and will show dehydration artefact.

83. e. Curling artefacts The tissue sometimes may twist by its own and get fixed in this unnatural position, after being excised. Particularly seen in the smaller or the thin specimen such as in case of delicate strip of the oral mucosa or the gingival specimen. Formalin fixation of the tissue causes shrinkage and curling which results in difficultly in the orientation of the tissue during the process of embedding.

84. The thin and small specimen place on the cardboard/ gauze to maintain its form before immersing it into the formalin. Non corrosive pins used to secure the tissue in the position over the cardboard.

85. g) Streaming artefacts This is due to diffusion of unfixed material to give false localization by coming to rest in a place other than its original location, for example, false localization with glycogen and enzymes in histochemistry. It is most often seen in formalin‑fixed tissue. It can be avoided by using glycogen fixatives (formol‑alcohol or Bouin ) or by freeze drying

86. h) Diffusion artefacts R efer to materials that may sometimes diffuse out of the tissue. Apart from large molecules, small molecules like inorganic ions and biogenic amines can be lost from tissues. Can be prevented by proper fixation for accurate localization and also by preventing the leaching of ions from the tissue

87. i) Artefacts due to microwave fixation Optimum temperature for microwave fixation in 45-55 Cº . Under heating results in poor sectioning quality whereas overheating above 65 Cº produces vacuolation, overstained cytoplasm and pyknotic nuclei. Remedy : Maintain optimal temperature during this procedure

88. j) Artefacts during freeze-drying-ice crystal artefacts During fixation using freeze drying method, the tissue must be plunged in liquid nitrogen immediately. Low temperature is important because unless the whole tissue is frozen, large ice crystals are formed causing disruption artifacts. This artifact cause total distortion of the tissue and diagnostic difficulty

89. k) Oxidation contamination: If the gasket of fixative container is loose, or the metal cap is not securely affixed to the container or the metal cap is rusty initially or the specimen is held in the fixative for long periods of time, fixatives such as formalin undergo oxidation and cause the metal cap to rust. This rust may contaminate the fixative within the container. This precipitate if included in tissue may resemble hemosiderin and even give a positive reaction with Prussian blue for iron.

90. l) Acid formalin hematin pigments: Acid formalin hematin pigment is produced by the reaction of formic acid of unbuffered formalin with the heme of the hemoglobin . It occurs in blood rich tissues which have been fixed in acid aqueous or alcoholic formalin which has a pH lower than 6 owing to the simultaneous action of acid and formalin on hemaglobin . The pigment is not formed in tissues fixed in neutral buffered formalin.

91. Formalin Pigment

92. n) Crush artifact which is associated with an intense eosinophillia at the centre of the tissue in H & E stained sections. Suggested mechanisms include protein coagulation by ethanol of partially fixed protein in subsequent histological processing. Incomplete wax impregnation has also been blamed.

93. Conclusion Fixation is a complex series of chemical events, and differs for the different groups of chemical substances found in the tissues It is clear that there is no universal fixative which will serve all requirements. Each fixative has specific properties and disadvantages and their many different effects emphasise the necessity for careful consideration and selection of the appropriate fixing reagent when studies of specific cellular substances are planned.

94. 10% buffered formalin and 2.5% glutaraldehyde is currently the most widely used fixatives for routine light microscopy and ultrastructural studies, but they too, have inherent disadvantages. The foundation of all good microscopic preparations is that the tissue should be appropriately and adequately fixed as soon as possible after removal.

95. References: Bancroft J.D., Stevens A., Turner D.R. Theory and Practice of Histological Techniques, 8 TH edition, Churchill Livingstone Edinburgh – 1996 Culling C.F.A., Allison R.T & Barr W.T. Cellular Pathology Technique, 3 rd edition, Butterworth & Co. (Publishers) Ltd., - 1985. Ajileye AB, Esan EO. Fixation and fixatives in histopathology: a review. Bayero Journal of Pure and Applied Sciences. 2022 Dec 9;15(1):231-43. Bhat AH, Hussein S. Fixation and different types of fixatives: Their role and functions: A review. International Journal of Clinical and Diagnostic Pathology. 2021 Oct 1;4(4):113-9. Chatterjee S. Artefacts in histopathology. Journal of Oral and Maxillofacial Pathology. 2014 Sep 1;18(Suppl 1):S111-6.