IMPREGNATION AND EMBEDDING Flashcards
Also known as INFILTRATION, process whereby the CLEARING AGENT IS COMPLETELY REMOVED from the tissue and replaced by a medium that will completely fill all the tissue cavities
IMPPREGNATION
The impregnating medium should be at least _____ the volume of the tissue
25 TIMES
Temperature for impregnation:
2 to 5C above the melting point of wax
Also known as CASTING or BLOCKING or MOLDING, process by which the impregnated tissue is placed in a precisely ARRANGED POSITION IN A MOLD containing a medium which is then allowed to solidify
EMBEDDING
temperature for emdedding
5 to 10C above the melting point of wax
Process by which a tissue is arranged in PRECISE POSITION IN THE MOLD DURING EMBEDDING, on the microtome before cutting, and on the slide before staining; MOST IMPORTANT STEP IN EMBEDDING
ORIENTATION
FOUR (4) TYPES OF IMPREGNATING MEDIUM AND ITS INDICATION
- PARAFFIN WAX: Routine
- CELLOIDIN WAX: For tough tissues
- GELATIN: Avoidance of dehydration (histochemistry and enzymes)
- PLASTCI: For electron microscopy
SIMPLEST, MOST COMMON and BEST EMBEDDING MEDIUM used for routine processing
PARAFFIN WAX
melting point of paraffin wax normally used for routine work
56C
The appropriate melting point of the paraffin if the current laboratory temperature is at:
MP IF LAB IS AT 20-24C:
MP IF LAB IS AT 15-18C:
The appropriate melting point of the paraffin if the current laboratory temperature is at:
MP IF LAB IS AT 20-24C: 54-58C
MP IF LAB IS AT 15-18C: 50-54C
Wax that has been trimmed away from the impregnated tissue may be melted and filtered for future use, with:
COARSE FILTER PAPER 9e.g. GREEN’s NO. 904)
Water in the paraffin wax can be removed through heating wax to _____
100-105C
How many times can paraffin wax be used?
Paraffin wax may be used only TWICE after which, FRESH WAX must be utilized
METHODS OF PARAFFIN IMPREGNATION AND EMBEDDING
- Manual processing (hand processing)
- Automatic tissue processing (autotechnicon)
- Vacuum embedding
In Manual processing of impregnation, at least ____ changes of paraffin at ____ minutes interval to ensure removal of the clearing agent
At least 4 changes of paraffin at 15 minutes interval
(15>15>15>15 = 60 minutes)
In Manual processing of embedding, approximately ____ hours to ensure complete embedding or casting of tissue
Approximately 3 hours
How many processing steps does automatic tissue processing use?
12 processing steps
Tissue processing steps that are included in automatic tissue processing:
Fixation
Dehydration
Clearing
Infiltration
In automatic tissue processing, the wax bath temperature should be:
3C above the melting point of wax
Advantage of automatic tissue processing:
CONSTANT AGITATION
Wax impregnation under NEGATIVE ATMOSPHERIC PRESSURE (400-500 mmHg) inside an embedding oven that hasten impregnation process by removing air bubbles within the tissue
Vacuum embedding
Temperature in vacuum embedding:
2-4C above the melting point of the wax
In vacuum embedding, the time required for complete impregnation is thereby reduced to ______% of the normal time required for tissue processing
25-75%
SUBSTITUTES FOR PARAFFIN WAX
- Paraplast
- Embeddol
- Ester wax
- Water soluble wax
- Dimethyl sulphoxide
A substitute for paraffin wax that is HIGHLY PURIFIED PARAFFIN and synthetic plastic polymers; recommended for large tissue blocks such as the bones and the brain
PARAPLAST
Melting point of paraplast:
56 to 57C
Synthetic wax substitute similar to paraplast with a melting point of 56-58C; less brittle and less compressible than paraplast
EMBEDDOL
A semisynthetic wax recommended for embedding EYES
BIOLOID
A product of paraffin containing RUBBER, with some property as paraplast
TISSUE MAT
Lower melting point (46-48C) but is harder than paraffin. Only soluble to 96% ethanol; It can be used to the tissue even without prior clearing of the tissue specimen
ESTER WAX
Plastic polymers , mostly polyethylene glycols with a melting point of 38 to 42C or 45 to 56C; Dehydration and clearing is skipped when used as impregnation medium
Water soluble wax
The most commonly used water-soluble wax; has a major disadvantage of DISSOLVING DURING FLOTAION. Because of the water-soluble properties of the carbowax
CARBOWAX
Purified form of nitrocellulose soluble in many solvents; Recommended for LARGE HOLLOW ORGANS, which tend to collapse, for HARD AND DENSE TISSUES as bones and teeth, for LARGE TISSUE SECTIONS of the whole embryo
CELLOIDIN IMPREGNATION
Celloidin impregnation types:
- WET celloidin method
- DRY celloidin method
- Nitrocellulose method
Recommended for BONES, TEETH, LARGE BRAIN SECTIONS and WHOLE ORGANS; tissue blocks stored in 70-80% ALCOHOL to avoid dehydration and shrinkage of the tissues until ready for cutting
WET CELLOIDIN METHOD
mnemonic:
“BWL and WET na Teeth-i”
Bones, Whole organs, Large brain sections, teeth in WET celloidin
Preferred for processing of WHOLE EYE SECTIONS;
DRY CELLOIDIN METHOD
mnemonic:
“DRY EYES”
GILSON’S MIXTURE is made up of
equal parts of CHLOROFORM and CEDARWOOD OIL
Note:
GILSON’S MIXTURE is added to celloidin blocks before hardening to make the tissue transparent. 70% alcohol is not used for storage before cutting instead Gilson’s mixture
Another form of celloidin which is soluble in equal concentration of ether and alcohol with a lower viscosity to allow it to be used in higher concentrations and still penetrate tissues rapidly
NITROCELLULOSE METHOD
RARELY USED except when dehydration is to be avoided and when tissues are subjected to histochemical and enzyme studies; used for DELICATE specimen and frozen sections
GELATIN IMPREGNATION
In gelatin impregnation, volume should be at least _____ the volume of the tissue
25 TIMES
In gelatin impregnation, tissue should NOT be more than _____ mm thick
2-3 mm thick
This is essential so that ULTRATHIN SECTIONS for ELECTRON MICROSCOPY (EM) may be cut with a minimum distortion
PLASTIC EMBEDDING
PLASTIC CLASSIFICATION BASED ON THEIR CHEMICAL COMPOSITION
- Polyester plastics
- Acrylic plastics
- Epoxy plastics
Plastic embedding used extensively for high resolution light microscopy and un-decalcified bone
ACRYLIC PLASTIC
mixture of epoxy plastics, catalysts, and accelerators:
BISPHENOL A (ARALDITE)
GLYCEROL (EPON)
CYCLOHEXENE DIOXIDE (SPURR)
MOLDS FOR EMBEDDING:
- Leuckhart’s embedding mold
- Compound embedding unit
- Plastic embedded rings and based mold
- Disposable embedding molds
2 ‘L’ shaped strips with a heavy brass which the size can be adjusted
Leuckhart’s embedding mold
Interlocking plates forming several compartments
Compound embedding unit
Examples of disposable embedding molds:
Peel-away
Plastic ice trays
Paper boats
recall: EXCEPT PAPER CUPS
In Double Embedding Method, tissue is first ________ and ________.
Tissue is first INFILTRATED WITH CELLOIDIN and EMBEDDED WITH PARAFFIN
mnemonic: ICEP (Infiltrated-Celloidin, Embedded-Paraffin)
