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Frozen section /orthodontic courses by Indian dental academy 

The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and
offering a wide range of dental certified courses in different formats.for more details please visit

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Frozen section /orthodontic courses by Indian dental academy 

  1. 1. 1 H. & E. STAINING IN FROZEN SECTION INDIAN DENTAL ACADEMY Leader in continuing Dental Education
  2. 2. 2 References :- • Bancroft J. D.,Stevens A., Turner R. D. ,Museum and other demonstration techniques, In Theory and Practice of Histological techniques, fourth edition, Churchill Livingstone. page no.699-712. • Culling C. F. A., Allison R. T. ,Museum Techniques, In Cellular Pathology Techniques, fourth edition, Butterworth. page no-523-540. • Drury R. A. B. ,Wallington E. A. Museum Techniques and injection methods, In Carleton's Histological Techiniques,fourth edition, Oxford University Press. page no-390-401.
  3. 3. 3 Contents 3. Microtomy a. CO2 freezing microtome b. Thermoelectric cooling devices c. The Cryostat d. Aerosol sprays 1. Preparation of the frozen sections 2. Preparation of the material
  4. 4. 4 4. Rapid Frozen Section Technique • Synthetic resin embedded tissue • Freeze-dried Tissue. • Freeze Substitution
  5. 5. 5 Introduction Frozen Section Uses :- 1. Rapid production of the sections for urgent diagnosis. 2. Use in diagnostic and research enzyme histochemistry where enzymes are labile. 3. Use in immunofluorescent method
  6. 6. 6 4. Use in immunocytochemical method. 5. Diagnostic & research non-enzyme histochemistry e. g.lipids and some carbohydrates. 6. Some silver methods, particularly in neuropathology
  7. 7. 7 ∙ When tissue is frozen the water within the tissue turns to be ice and in this state tissue is firm, the ice acting as an embedding medium. Frozen Section Theoretical Consideration :-
  8. 8. 8 ∙ The consistency of the frozen section block can be altered by varying the temperature of the tissue. ∙ The sectioning of the fixed tissue require a block temperature of the about minus 10 or warmer. Frozen Section
  9. 9. 9 • It is a refrigerated cabinet in which a modified microtome is housed. • All controls to the microtome are operated from outside the cabinet. • First microtome was introduced in the year 1954; at present rotary microtome is the type of choice. The Cryostat
  10. 10. 10 Various advancements in the design of the microtome are as : •Electronic temperature control. • Electronically controlled advancement and retreat of the block. • Specimen orientation facility. • Digital demonstration of the cryostat temperature and section thickness. • Mechanized cutting speed facility.
  11. 11. 11 The best quality cryostat sections are produced from fresh unfixed tissue, which has been rapidly frozen by one of the techniques outlined below. The conditions in the cryostat itself must be optimal - Block temperature correct for tissue being cut. Microtome operating correctly. Anti - roll plate adjustment correct. Cryostat Technique
  12. 12. 12 Methods of freezing are - Liquefied Nitrogen : - 190 0 C Isopentane cooled by liquid nitrogen : - 150 0 C Carbon dioxide ‘Cardice’ : - 70 0 C Aerosol sprays : - 50 0 C Freezing of the fresh unfixed tissue Tissue for freezing should be fresh, and freezing should be as rapid as possible.
  13. 13. 13 For almost all diagnostic purposes in routin laboratory cryostsat sections of unfixed tissues are suitable, although certain methods require post fixation in cold formal-calcium. Fresh Tissue & The Cryostat
  14. 14. 14 Tissue prepared in such a way is fixed under controlled conditions. The tissue must be absolutely fesh and placed in formal–calcium solution at 40 C. The tissue is fixed at this temperature for 18 hours
  15. 15. 15 When the tissue block is ready for sectioning ,the tempt of the microtome & cryostat chamber should be checked. Most unfixed material will section well between -15to -23 C. Cryostat Sectioning
  16. 16. 16 Unfixed tissues containing moderate to large amount of fat will require as cold a temperature as the cryostat is capable of producing.
  17. 17. 17 Most fixed tissues will section best within the range – 7 0 C to -120 C depending upon the hardness of the tissue. Cabinet temperature Microtome Blade or Knife Anti-roll plate Sectioning technique Soft tissue cut better at a slower speed and hard tissue cut at slightly higher speed. Fixed tissue is more difficult to cut
  18. 18. 18 Microtome Set-up
  19. 19. 19 Microtome
  20. 20. 20 Cryostat
  21. 21. 21 Freezing Tissue In The Histobath
  22. 22. 22 Processing
  23. 23. 23 Sectioning
  24. 24. 24 Tissue Sectioning
  25. 25. 25 Tissue Sectioning
  26. 26. 26 Tissue Sectioning
  27. 27. 27 Tissue Sectioning
  28. 28. 28 Frozen Section Stains
  29. 29. 29 Slides For Frozen Section
  30. 30. 30 Kidney Section Immediately Fixed
  31. 31. 31 Rapid Fixing In Broncho-alveolar Carcinoma
  32. 32. 32 It is a technique of rapid freezing of fresh tissue at -1600 C and subsequent removal of water molecules (in the form of ice) by sublimation in vacuum at a higher temperature e.g. – 400 C. Freeze Drying
  33. 33. 33 The freeze dried blocks are then raised to room temperature and either fixed by vapour fixative or embedded in a suitable medium. Freeze -drying can be considered in four stages:- a. Quenching b. Drying c. Fixation & embedding d. Subsequent treatment
  34. 34. 34 This instantly stops the chemical reaction and diffusion in the tissue. It brings the tissue into a solid state in which unbound tissue is changed into small ice crystals, which are subsequently removed in the drying phase. Quenching
  35. 35. 35 This part of the technique is the most time consuming as some tissue contains 70-80% water by weight and this has to be removed without damaging the tissue. Drying
  36. 36. 36 Drying can be divided into three distant stages - 1. Introduction of the heat to the tissue to cause sublimation of the tissue. 2. The transfer of the water vapor from the ice crystals through the dry part of the tissue. 3. The removal of the water vapor from the surface of the specimen.
  37. 37. 37 When the tissue is completely dry, it is allowed to come to room temperature. This delicate tissue is ready for embedding and processing. Vapor fixation - Fixatives used for vapor fixation are formaldehyde, glutaraldehyde and osmium tetra oxide. The most common and excellent fixative is formaldehyde because it gives excellent preservation of the tissue component and tissue can be used for all histochemistry with exception of the enzymes. Fixation & Embedding
  38. 38. 38 Application of Freeze drying • Demonstration of hydrolytic enzymes • Fluorescent antibody studies • Autoradiography • Microspectrofluorimetry of autofluroscent substances • Formaldehyde-induced fluorescence • Mucosubstances • Proteins • Scanning electron microscopy
  39. 39. 39 This technique originated by Simpson (1941) involves the rapid freezing of small pieces of tissues in a similar manner as for freeze drying. Substitution of the ice in such tissue by placing them in dehydrating agent at sub zero temperature. The preservation obtained with the freeze substitution is excellent. The technique produces considerable amount of shrinkage. Freeze Substitution
  40. 40. 40 THANK YOU.
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The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and offering a wide range of dental certified courses in different formats.for more details please visit


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