PREFINALS: IMPREGNATION AND EMBEDDING Flashcards
AKA Infiltration
IMPREGNATION
Removal of clearing agent from the tissue and replacing it with the infiltrating media
IMPREGNATION
This infiltrating media will completely fill all tissue cavities, thus giving firm consistency, as well as allow easy handling and cutting of thin tissue
IMPREGNATION
Incomplete Impregnation =
Airholes in tissue sections
Main factors: method of impregnation, nature and size of tissue, clearing agent used
IMPREGNATION
Types of Tissue Impregnation and Embedding Media:
- Paraffin impregnation
- Celloidin (Colloidin) impregnation
- Gelatin impregnation
- Plastic
Simplest, common and best media for routine processing
Paraffin
ADV: sections are cut easily without distortion; very rapid (24hrs); permits many staining procedures;
Paraffin
DADV: not for fatty tissues; must fully impregnate the tissue to avoid tissue crumbling
Paraffin
Paraffin
Melting point for routine work:
56C
Paraffin
Never overheat (>60OC):
causes brittleness, shrinkage, hardening; destruction of lymph tissue
maintain at 2 to 5C higher than MP of wax
Paraffin oven
Wax must be filtered first using coarse filter paper such as Green’s No. 904 in wax oven at 2OC higher than MP of wax
Paraffin
Used pure
Paraffin
Reusable only once, but remove water first by boiling to 100-105OC
Paraffin
Methods of Paraffin Impregnation:
- Manual
- Automatic
- Vacuum Embedding
Atleast 4 changes of paraffin every 15 minutes
Manual
Uses machines like Autotechnicon and Elliot Bench-Type Processor, which fixes, dehydrates, clears, and infiltrates tissues
Automatic
Infiltration is usually at stations 11 and 12
Autotechnicon
ADV: Has constant agitation speedy procedure
Autotechnicon
NOTE: Any odor in clearing agent indicates that the paraffin wax should be changed
Autotechnicon
Wax bath thermostat should be set atleast 3 degrees above the MP of paraffin
Autotechnicon
Fastest (25-75% reduction of usual impregnation time)
Vacuum Embedding
Uses embedding oven with negative atmospheric pressure rapid removal of air bubbles (e.g. lungs) and clearing agent rapid infiltration
Vacuum Embedding
For urgent biopsies, delicate tissues (e.g. CNS, eyes)
Vacuum Embedding
Paraffin Wax Substitutes:
- Paraplast
- Embeddol
- Bioloid
- Tissue Mat
- Ester Wax
- Water Soluble Waxes (Polyethylene glycol)
Mixture of pure paraffin and synthetic plastic polymer (Dimethyl sulfoxide); more elastic and resilient
Paraplast
MP: 56-57C
Paraplast
less brittle, and less compressible (MP: 56-57OC)
Embeddol
semisynthetic; for embedding of eyes
Bioloid
has rubber
Tissue Mat
Water insoluble but soluble in 95% ethanol, thus prior clearing is not needed
Ester Wax
MP: 46-48C
Ester Wax
Harder than paraffin thus used with sliding/sledge-type microtome
Ester Wax
But Cellosolve, and xylene may be used if indicated
Ester Wax
MP: 38-42OC or 45-56OC
Water Soluble Waxes (Polyethylene glycol)
Water Soluble Waxes (Polyethylene glycol) most common:
Carbowax
Carbowax
absorbs water; no need for dehydration and clearing
Hygroscopic