HISTOPATH LEC-20-22

Infiltration and Embedding

Infiltration (Impregnation)

  • Definition: Process where the clearing agent is removed from tissue, replaced by a medium filling all cavities.

  • Purpose:

    • To provide firm consistency to the specimen.

    • Enables handling and cutting of thin sections without damage.

  • Importance:

    • Tissue cavities must be filled to ensure tissue firmness and facilitate easy cutting.

    • Essential step in tissue processing next to clearing.

  • Common Embedding Medium: Paraffin wax.

    • Most rapid infliltrating agent; used widely due to speed and compatibility with staining procedures.

  • Procedure:

    1. Place tissues in a container with paraffin wax.

    2. Use an oven to keep the wax melted and liquid.

    3. Requires four changes of wax at 15-minute intervals.

Paraffin Wax Infiltration

  • Advantages:

    • Rapid processing (can prepare sections within 24 hours).

    • Compatible with all staining methods.

  • Disadvantages:

    • Overheated paraffin can make specimens brittle.

    • Prolonged immersion can cause shrinkage and tissue hardening.

    • Not suitable for fatty tissues due to fat dissolution.

    • Requires a melting point of 56°C.

Methods of Paraffin Wax Infiltration

  1. Manual Process:

    • Requires a heating oven for melting wax.

  2. Automatic Process:

    • Uses an Autotechnicon for rapid infiltration with constant agitation.

  3. Vacuum Process:

    • Infiltration under negative atmospheric pressure.

Alternative Embedding Media

Celloidin

  • Overview:

    • A purified form of nitrocellulose, soluble in alcohol and ether.

    • Does not require heat, thus slower and difficult to obtain thin sections.

  • Procedure:

    • Requires large volumes of the impregnating medium (≥25 times the tissue volume).

  • Recommended Use:

    • Only for frozen sections or histochemical studies.

Other Embedding Materials

  • Paraplast:

    • Mixture of paraffin and synthetic polymers; melting point 56-57°C.

  • Carbowax:

    • Water-soluble wax; no need for dehydration/clearing but difficult to handle during cutting.

  • Gelatin:

    • Process includes a sequence of gelatin solutions to prevent mold growth and enhance tissue hardness.

Embedding Process

  • Definition: Placing infiltrated tissue in a mold with embedding medium to solidify.

  • Required Materials:

    • Embedding mold, embedding medium (e.g., paraffin wax), infiltrated tissue.

  • Temperature Requirement:

    • Melting point of paraffin should be 5-10°C above its melting point for effective embedding.

  • Result: Tissue block ready for further processing.

Trimming and Double Embedding

  • Trimming: Cutting excess paraffin to form a clean tissue block.

  • Double Embedding: Infiltrating with celloidin before embedding with paraffin; useful for dense tissues like the brain.

Plastic (Resin) Embedding

  • Types of Resins:

    • Epoxy (Araldite), Glycerol (Epon), Cyclohexene (Spurr).

    • Suitable for high-resolution microscopy, especially for thin tissue sections.

Embedding Molds

  • Disposable: Peel away molds, plastic ice trays, paper boats.

  • Non-disposable: Leuckhart’s and compound embedding units for processing multiple specimens efficiently.