HISTO LEC: Module 1 - Part III

DO NOT EXPLOIT.

Removal of specimen

First step in tissue processing; cytological purpose and for the best histological preparation, the material should be removed from an anesthetized animal, preferably from the death of an animal.

Fixation

Second step in tissue processing; mainly preserves the protoplasm with the least alteration from the living state.

TRUE

TRUE OR FALSE: The first requirement for preservation of protoplasmic structures is to interrupt the dynamic process of the cells as promptly as possible and to stabilize the structure with minimal change since these tissues are perishable.

Fixatives

• Chemicals which render the structural components in soluble solution or precipitate proteins by replacing associate water and the hardened tissue

• It should also prevent evaporation, autolysis, swelling, shrinkage, and attack by bacteria or mold.

TRUE

TRUE OR FALSE: Good fixatives are: neutral formalin, osmic acid, zenker’s fixative, bouin’s and muller’s fixatives.

Formaldehyde or formalin

Widely used fixative for preserving tissues; concentration of the solution is around 5% to 10%.

Washing the specimen

Third step in tissue processing; uses a large volume of water so as to remove the excess fixative.

Dehydration

Fourth step in tissue processing; tissue will be immersed in ascending grades of alcohol solutions from 60% to absolute 100% to remove the water.

Clearing

Fifth step in tissue processing; the tissue is made translucent by such reagents as xylene, tuluol, chloroform and benzene and also removes any dehydrating agents.

Embedding

• Sixth step in tissue processing; the dehydrated tissue is encased in an embedding medium (e.g. paraffin wax, gelatin, or celloidin).

• Provides rigid support in the tissue block upon sectioning.

Sectioning

• Seventh step in tissue processing; Thee block of paraffin is then cut into very thin slices with a microtome.

• Ideal thickness of a tissue section ranges from 2-10 μm (micrometers).

Staining

• Eighth step in tissue processing; purpose of this step is to enhance the natural contrast and to make more evident various cell and tissue components and extrinsic materials.

• Staining method that is usually used is hematoxylin-eosin (H&E).

Hematoxylin

It is used as the basic dye.

Eosin

It is the acid dye in staining.

Mounting

• Ninth step in tissue processing; stained sections are placed on a slide that hardens.

• Examples are Canada balsam and colorless nail polish.