Histochemistry Of Oral Tissues Question And Answers

Histochemistry Of Oral Tissues

Question 1. Describe the stages of tissue processing and write about fixation.
Answer:

Tissue processing:

• It is a long procedure and requires 24 hours.
• Stages:

1. Fixation:

• It is an important step in the preparation of tissue for microscopic examination.
• Goals:
  • Preserves the structure.
  • It terminates any ongoing biochemical reaction.
  • It increases the strength and stability of treated tissue.

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• Purposes:
  • Coagulate proteins.
  • Prevents autolysis of cell
  • Fix cell constituents and tissue that withstand subsequent treatment with various reagents with minimum loss of anatomy.
• Steps:
  • Killing tissue.
  • Preservation of tissue.
  • It is done by fixatives, commonly used – 10% formalin.
• Temperature:
  • Done at room temperature.
• Time:
  • It takes several hours to several days.

2. Dehydration:

• After fixing the tissue, it is washed with running water.
• Water cannot mix with paraffin.
• Hence, the water content is to be removed.
• It is done by passing the specimen by increasing the percentage of alcohol.

3. Clearing:

• Alcohol is not miscible with paraffin.
• Thus, the tissue is first cleared of alcohol by a reagent like xylene.
• Xylene is miscible with both alcohol and paraffin.
  • Time:
    • A small piece of tissue is cleared in 0.5 1 hour.
    • Larger/thicker piece in 2-4 hours.

4. Infiltration:

• When xylene has completely replaced alcohol, the specimen is infiltrated with paraffin at 60°C.

5. Embedding:

• Tissue is embedded in a block of paraffin wax with the proper orientation of the specimen.
• Paraffin wax is used as it has hard consistency.

6. Cutting:

• The paraffin wax block is attached to a wooden cube which is clamped onto the microtome.
• It is adjusted to cut the desired piece of the specimen.

7. Mounting:

• The sections are mounted on microscope slides.

8. Staining:

• Mounted slides are stained to give color to a section.

Procedure:

• Put sections in xylene for 3 minutes
• Transfer to the increasing strength of alcohol for 3 minutes
• Wash in running water for 1 minute
• Put in hemotoxylin for 5 to 7 min.
• Wash in running water for 30 sec.
• Wash excess in 1% acid alcohol for 15 sec.
• Wash in running water for 30 sec.
• Give 2-3 dips of ammonia water solution until tissues attain blue color.
• Wash in running water for 30 sec.
• Counterstain it.
• Wash it
• Dehydrate it.

Question 2. Fixation procedures and fixatives.
Answer:

Fixation:

• It is a procedure to make cell constituents and tissue withstand subsequent treatment with minimum loss of anatomy.
• This is accomplished by using.
• Optimum osmotic conditions.
• Cold temperatures.
• Controlled pH of the fixing solution.
• Minimum possible exposure to the fixatives.

Procedures/types:

Physical:

• Heating
• Micro-waving
• Cryo-preservation or freeze drying.

Chemical:

• By immersing in fixatives.
• By perfusing the vascular system with fixatives.

Question 3. Preparation of the ground section of the tooth.
Answer:

• The labial surface of the tooth is ground down till the desired section is achieved using a coarse abrasive wheel.
• The coarse wheel is exchanged with the fine abrasive wheel.
• The tooth is further ground.
• An adhesive tape is wrapped around a wooden block with a sticky side outward.
• The grounded section of the teeth is stuck to it.
• A similar grinding procedure is repeated from the lingual surface.
• The finished ground section is soaked off with ether.
• It is then dried off for several minutes
• Longer drying leads to cracking.
• It is then mounted on microscopic slides.

Question 4. Microtome
Answer:

• It is a tool used to cut extremely thin slices of material, known as sections
• Used for preparation of samples

Blades used:

Applications:

• Traditional histological examination
• For frozen sections
• For electron microscopy
• For spectroscopy
• For fluorescence microscopy

Types:

Microtome knives:

1. Planar concave extremely sharp
2. Wedge-shaped More stable
3. Chisel shaped Has blunt edge

Question 5. Stages in soft tissue processing.
Answer:

Stages:

• Obtaining the specimen.
• Fixation Ideally with 10% formalin.
• Dehydration with increasing strength of alcohol.
• Clearing.
• By passing through xylene
• Infiltration by paraffin.
• Embedding the specimen in paraffin wax block.
• Cutting to obtain desired sections of tissues
• Mounting the sections on slides.
• Staining with the desired staining method.

Question 6. Declassification.
Answer:

It is a technique for removing minerals from bone and other calcified tissue so that good paraffin sections can be prepared.

Chemical used:

• 5% nitric acid.

Time required:

• 7-10 days for complete decalcification.

Tests:

Question 7. Xylene.
Answer:

Properties:

• Insoluble in water.
• Soluble in aromatic hydrocarbons.
• Flammable.

Uses:

• Result
• Easily pierced Absence of precipitate
• Used as a clearing agent to remove alcohol from the tissue. To remove paraffin from dried microscopic slides.
• After staining, slides are put in xylene prior to mounting. Used to dissolve gutta percha in endodontic treatment.
• As the solvent to remove synthetic immersion oil from the microscope.

Question 8. Alkaline phosphatase/Robinson’s Alkaline phosphatase.
Answer:

Alkaline phosphatase was first described by Robinson J.C.

• It is associated with the production of mineralized tissue.
• In hard connective tissue, it is found in the organic matrix.
• It hydrolyzes phosphate ions from organic radicals at alkaline pH.
• It cleaves pyrophosphate thereby permitting crystal growth to proceed.
• It is associated with osteogenesis and dentinogenesis.
• The osteoblasts and preosteoblasts exhibit alkaline phosphatase activity.
• It is seen in chondrocytes and cartilage matrix also.
• In human gingiva, the capillary endothelium of the lamina propria shows its activity.
• Alkaline phosphatase is involved in the mechanism of keratinization.
• Basement membrane associated with salivary gland acini and taste buds exhibits high alkaline phosphatase activity.

Question 9. Fixation of tissues:
Answer:

• It is an important step in tissue processing.
• It fixes the tissues to withstand subsequent treatment with various reagents with minimum loss of anatomy.
• The reagents used are called fixatives.

Purposes:

• Terminate ongoing biochemical reactions.
• Increases strength and stability of treated tissue.
• Preserves the structures
• Coagulate proteins.
• Prevents autolysis of cells.

Parameters:

• Minimal time between killing and fixation.
• The minimal size of the tissue
• Minimal tissue deformation.

Question 10. Frequently studied important enzymes.
Answer:

• Enzyme
• Alkaline phosphatize
• Acid phosphatize
• Oxidases and dehydrogenase
• Esterases
• Lysosomal sulfatase and adenyl cyclase

Question 11. Acid phosphatase.
Answer:

• Importance
• Related to the transfer of phosphate esters in the organic matrix of bone, enamel and dentin.
• Related to resorption of bone and dentin.
• Reflects metabolic activity of different oral tissues.
• Associated with the hydrolysis of carboxylic acid esters of alcohol Involved in the formation of cAMP.
• It is localized in membrane-bound vesicles called lysosomes.
• Types: Based on its interaction with substrates
• Para-nitro-phenyl phosphate
• Beta glycerophosphate.

Activity:

• In supranuclear and distal regions of secretory ameloblasts.
• During resorption, osteoclasts of bone and odontoblasts in resorbing dentin.
• Less activity occurs during the late secretory stage of amelogenesis.

Demonstration:

• Demonstrated cytochemical using naphthol ASBI phosphoric acid-fast red garnet GBC.
• In tissue sections using naphthol ASBI phosphoric acid pararosaniline.