3. Microscopic study include: 1. Study of oral soft tissues
Microscopic examination is the method used to study the histological structure of the oral tissues.
The tissue should be appropriately prepared for microscopic examination.
Microscopic study include:
1. Study of oral soft tissues
Routine tissue processing
2. Study of dental hard tissues
Ground sections
Decalcified sections

4. For microscopic examination, tissue specimens must be thin enough (4-6µ) to permit the passage of transmitted light.
The treatment of tissue specimens to allow paraffin wax embedding is called Tissue Processing
The aim of tissue processing is to embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut, which can be viewed under microscope

5. Source of tissue specimen is biopsy / autopsy
Tissue specimens should be immersed immediately in some solution to prevent autolysis and bacterial putrefaction
Tissues are to be preserved at its most natural form that helps in visualizing the structure & structural defects of the tissue
The solutions used for this purpose are called as Fixatives.

6. Soft tissue processing

7. Tissue processing can be
Manual
Automatic

8. Specimens got to lab should be examined for:
Relevant details
Adequate size of tissue specimen
Labeling of the specimen bottle
Fixative used
Steps done to ensure complete fixation
Sufficient volume of fixative
The standard ratio of fixative to tissue volume is 10:1
Large tissues
Cut into smaller size tissues
Representative areas to be sent for processing

10. The procedure used for soft tissue processing can be grouped under
As paraffin wax is immiscible with water, tissues fixed in aqueous fixatives must be treated to remove water. This is achieved by various stages of tissue processing
The procedure used for soft tissue processing can be grouped under
Fixation
Dehydration
Clearing
Impregnation
Embedding
Sectioning
Staining
Mounting

12. Fixation
First step & foundation in a sequence of events in tissue processing
Process involving series of chemical events which results in the stabilization of proteins and makes the tissue resistant to damage during subsequent stages of processing & visualization.
Objectives of fixation
To preserve the living structure as closely as possible.
To prevent autolysis of tissue
To inhibit bacterial or fungal growth
To make the tissue resistant to damage during subsequent stages of processing
To prevent alteration of tissue volume and distort any part of the tissue structure.

13. Most commonly used fixative is 10% formalin
To avoid interchanging specimen, a piece of paper with graphite pencil marking to be put in the tissue capsule
Most commonly used fixative is 10% formalin
Increases the cross linking and results in the stabilization of proteins.
Other fixatives used:
Glutaraldehyde
Osmium tetroxide
Chromic acid
Methyl alcohol & ethyl alcohol
Mercuric chloride
Picric acid

14. Factors influencing the rate of fixation
Specimen size (3-4 mm ideally) pH
Agitation
Heat
Viscosity
Vaccum
Ultrasonics

15. Dehydration
Done to remove the water content from tissues to allow the penetration of paraffin wax
Done by passing through ascending grades of alcohol (to prevent sudden shrinkage of tissue)
E.g.- 50%, …..70%, 90% & 100%
Solutions used
Methyl & Ethyl alcohol
Isopropyl alcohol
Acetone

16. Clearing Paraffin and alcohol are not miscible.
So impregnation of tissue by paraffin is not possible unless alcohol is replaced by a fluid that is miscible with both alcohol & paraffin.
This process is called clearing
Xylene is one of the solutions that is miscible with both paraffin and alcohol

17. The term “clearing” comes from the fact that the clearing agents often have the same refractive index as proteins.
As a result, when the tissue is completely infiltrated with the clearing agent, it becomes translucent/clear.
The presence of opaque areas indicates incomplete dehydration.

18. Ideal requirements of a clearing solution
Speedy removal of alcohol
Minimum tissue damage & toxicity
Cost factor
Choice of a clearing agent depends on:
The type of tissues to be processed
The type of processing to be undertaken
The processor system to be used
Processing conditions ( temp, vaccum & pressure)
Safety factors
Cost and convenience

19. Reagents used Xylene (most commonly used) Chloroform Toluene Benzene
CNP 30; Inhibisol
Food oil derivatives
Cedar wood oil

20. Impregnation
Saturation of tissue cavities and cells by a supporting substance which is generally, but not always, the medium in which they are finally embedded
Replaces xylene with paraffin by immersion in molten wax (600 C)
Factors affecting impregnation
Size & type of tissue
Clearing agent employed
Vaccum embedding

21. EMBEDDING/BLOCKING
Process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidifies will provide sufficient external support during sectioning.
Impregnated tissue transferred from wax bath to a mould filled with molten wax to get a block of wax with the tissue specimen at the center with the cutting surface facing the base of the block

22. Done using Leuckhart’s L shaped pieces Ice trays Paper boats
Embedding cassette

23. Make sure that there are no air bubbles trapped between the tissue and the molten wax.
The wax-filled mold containing the tissue is then allowed to cool.
The wax blocks are labeled to make easier identification.
Wax hardened block removed from the mould and trimmed.
Wax block to be fixed on to a wooden/metal block to prevent wax block from crumbling during sectioning.

27. Sectioning
Embedded tissues, to be cut into thin sections of 3- 5 µ with a microtome.
Cut sections are, floated on a warm water bath
Helps to spread the specimen and remove wrinkles.
Floated sections are picked up on an adhesive coated glass slide.
Glass slide kept on a slide warmer at 580 temp for 20 min to ensure adhesion
Egg albumin with additives - commonly used adhesive

28. Manual
Automatic

32. Staining
Biochemical technique of adding a class-specific dye to a substrate (DNA, proteins, lipids, carbohydrates) to qualify or quantify the presence of a specific compound
Commonly used Staining procedures
Gram staining
Haematoxylin and eosin (H & E) staining
Papanicolaou staining (PAP)
PAS staining
Masson's trichrome staining

33. H & E stain/ Haematoxylin & Eosin stain
Most popular staining method in histology.
Most widely used stain in medical diagnosis.
The staining method involves application of the basic dye haematoxylin, which colors basophilic structures with blue-purple hue, and alcohol-based acidic eosin Y, which colors eosinophilic structures bright pink.

34. Basophilic structures are usually the ones containing nucleic acids, such as the ribosomes and the chromatin-rich cell nucleus, and the cytoplasmatic regions rich in RNA.
Eosinophilic structures are generally composed of intracellular or extracellular protein. Most of the cytoplasm is eosinophilic.
Red blood cells are stained intensely red.

35. HEMATOXYLIN
Hematoxylin is a natural dye which is extracted from the heartwood of the tree Hematoxylon campechianum.
The dye is usually used in conjunction with a mordant which helps the stain to bind to the tissue
Oxidation
Mordant
Hematoxylin
Haematin
STAIN

36. EOSIN Second component of the H & E is eosin, the counterstain
Eosin is a red fluorescent dye resulting from the action of bromine on fluorescein.
Eosin Y-Commonly used form of eosin
Used to stain cytoplasm, collagen and muscle fibers
Both water and ethanol soluble.

37. Procedure Steps involved Manual Automated
Removal of wax (with xylene).
Rehydrate the tissues (bring sections to water)
Descending grades of alcohol
Staining H & E
Dehydrate:
Ascending grades of alcohol
Clearing (Xylene)

38. Mounting
The stained section on the slide must be covered with a thin glass coverslip to protect the tissue from being scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section for years to come
Mounting medium is used to adhere the coverslip to the slide

40. Two types of mounting media
Water based mounting media
Resinous mounting media
DPX & Canada balsam commonly used

41. Allow it to dry and section viewed under microscope
To mount a slide
Apply drops of mounting medium upon tissue section.
Hold coverslip at 45o allowing the drop to spread along the edge of the slip.
Let go of slip and allow the medium to spread slowly.
Allow it to dry and section viewed under microscope

44. Hard tissue processing

45. Study of dental hard tissues
Ground sections
Decalcified sections

46. Decalcified Sections
Hard tissue specimens after decalcification are treated like routine soft tissue specimens
All hard tissues except enamel can be studied after decalcification
96% mineralized; lost during decalcification

47. Decalcification
Process by which calcium in the mineralized tissue is removed so that tissue becomes soft enough to make sections
Decalcification is usually carried out between the fixation and processing steps
Done by
Acids (5% nitric acid, 15% formic acid)
Chelating agents (EDTA)
Electrolysis

48. Procedure Tissue to be fixed first
Large tissue to be cut into smaller pieces
Specimen to be put in decalcifying solution
Check for completion of decalcification
Consistency of tissue
Piercing tissue with a needle
Judicious bending of tissue
Radiographs
Chemical test
Sodium hydroxide & ammonium oxalate
Wash in running water

49. Ground sections Sections made of undecalcified specimens
Helps in study of dental hard tissue, especially enamel
Made using
Manual
Hard tissue microtome

50. Procedure Final reduction using a fine abrasive
Tooth cut into 2-3 sections – diamond burs/discs
Initial reduction using a rough abrasive (till 25-50µ)
Arkansas stone
Coarse wheel of a lathe
Final reduction using a fine abrasive
Domestic scouring powder followed by soap water
Fine wheel of lathe
Sections washed & dried (Dehydrated)\
Mounted & allowed to dry
Viewed under microscope

51. Tissue specimen