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1
H. & E. STAINING
IN FROZEN SECTION
INDIAN DENTAL ACADEMY
Leader in continuing Dental Education
www.indiandentalacademy.c...
2
References :-
• Bancroft J. D.,Stevens A., Turner R. D. ,Museum and other
demonstration techniques, In Theory and Practi...
3
Contents
3. Microtomy
a. CO2 freezing microtome
b. Thermoelectric cooling devices
c. The Cryostat
d. Aerosol sprays
1. P...
4
4. Rapid Frozen Section Technique
• Synthetic resin embedded tissue
• Freeze-dried Tissue.
• Freeze Substitution
www.ind...
5
Introduction
Frozen Section Uses :-
1. Rapid production of the sections for urgent diagnosis.
2. Use in diagnostic and r...
6
4. Use in immunocytochemical method.
5. Diagnostic & research non-enzyme histochemistry
e. g.lipids and some carbohydrat...
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 ...
8
∙ The consistency of the frozen section block can be altered by
varying the temperature of the tissue.
∙ The sectioning ...
9
• It is a refrigerated cabinet in which a modified microtome is
housed.
• All controls to the microtome are operated fro...
10
Various advancements in the design of the microtome are as :
•Electronic temperature control.
• Electronically controll...
11
The best quality cryostat sections are produced from fresh
unfixed tissue, which has been rapidly frozen by one of the
...
12
Methods of freezing are -
Liquefied Nitrogen : - 190 0
C
Isopentane cooled by liquid nitrogen : - 150 0
C
Carbon dioxid...
13
For almost all diagnostic purposes in routin laboratory
cryostsat sections of unfixed tissues are suitable, although
ce...
14
Tissue prepared in such a way is fixed under controlled
conditions. The tissue must be absolutely fesh and placed in
fo...
15
When the tissue block is ready for sectioning ,the tempt of
the microtome & cryostat chamber should be checked.
Most un...
16
Unfixed tissues containing moderate to large amount of fat
will require as cold a temperature as the cryostat is capabl...
17
Most fixed tissues will section best within the range – 7 0
C to
-120
C depending upon the hardness of the tissue.
Cabi...
18
Microtome Set-up
www.indiandentalacademy.com
19
Microtome
www.indiandentalacademy.com
20
Cryostat
www.indiandentalacademy.com
21
Freezing Tissue In The Histobath
www.indiandentalacademy.com
22
Processing
www.indiandentalacademy.com
23
Sectioning
www.indiandentalacademy.com
24
Tissue Sectioning
www.indiandentalacademy.com
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Tissue Sectioning
www.indiandentalacademy.com
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Tissue Sectioning
www.indiandentalacademy.com
27
Tissue Sectioning
www.indiandentalacademy.com
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Frozen Section Stains
www.indiandentalacademy.com
29
Slides For Frozen Section
www.indiandentalacademy.com
30
Kidney Section Immediately Fixed
www.indiandentalacademy.com
31
Rapid Fixing In Broncho-alveolar Carcinoma
www.indiandentalacademy.com
32
It is a technique of rapid freezing of fresh tissue at -1600
C and
subsequent removal of water molecules (in the form o...
33
The freeze dried blocks are then raised to room temperature
and either fixed by vapour fixative or embedded in a suitab...
34
This instantly stops the chemical reaction and diffusion in
the tissue.
It brings the tissue into a solid state in whic...
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...
36
Drying can be divided into three distant stages -
1. Introduction of the heat to the tissue to cause sublimation of
the...
37
When the tissue is completely dry, it is allowed to come to
room temperature.
This delicate tissue is ready for embeddi...
38
Application of Freeze drying
• Demonstration of hydrolytic enzymes
• Fluorescent antibody studies
• Autoradiography
• M...
39
This technique originated by Simpson (1941) involves the
rapid freezing of small pieces of tissues in a similar manner
...
40
THANK YOU.
www.indiandentalacademy.com
41www.indiandentalacademy.com
42www.indiandentalacademy.com
43www.indiandentalacademy.com
44www.indiandentalacademy.com
45www.indiandentalacademy.com
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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 
www.indiandentalacademy.com

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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 www.indiandentalacademy.com
  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. www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  4. 4. 4 4. Rapid Frozen Section Technique • Synthetic resin embedded tissue • Freeze-dried Tissue. • Freeze Substitution www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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 :- www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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. www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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. www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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. www.indiandentalacademy.com
  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 well.www.indiandentalacademy.com
  18. 18. 18 Microtome Set-up www.indiandentalacademy.com
  19. 19. 19 Microtome www.indiandentalacademy.com
  20. 20. 20 Cryostat www.indiandentalacademy.com
  21. 21. 21 Freezing Tissue In The Histobath www.indiandentalacademy.com
  22. 22. 22 Processing www.indiandentalacademy.com
  23. 23. 23 Sectioning www.indiandentalacademy.com
  24. 24. 24 Tissue Sectioning www.indiandentalacademy.com
  25. 25. 25 Tissue Sectioning www.indiandentalacademy.com
  26. 26. 26 Tissue Sectioning www.indiandentalacademy.com
  27. 27. 27 Tissue Sectioning www.indiandentalacademy.com
  28. 28. 28 Frozen Section Stains www.indiandentalacademy.com
  29. 29. 29 Slides For Frozen Section www.indiandentalacademy.com
  30. 30. 30 Kidney Section Immediately Fixed www.indiandentalacademy.com
  31. 31. 31 Rapid Fixing In Broncho-alveolar Carcinoma www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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. www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  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 www.indiandentalacademy.com
  40. 40. 40 THANK YOU. www.indiandentalacademy.com
  41. 41. 41www.indiandentalacademy.com
  42. 42. 42www.indiandentalacademy.com
  43. 43. 43www.indiandentalacademy.com
  44. 44. 44www.indiandentalacademy.com
  45. 45. 45www.indiandentalacademy.com

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