IMPREGNATION AND EMBEDDING Flashcards by Janus Palermo

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

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The impregnating medium should be at least _____ the volume of the tissue

25 TIMES

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Temperature for impregnation:

2 to 5C above the melting point of wax

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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

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temperature for emdedding

5 to 10C above the melting point of wax

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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

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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

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SIMPLEST, MOST COMMON and BEST EMBEDDING MEDIUM used for routine processing

PARAFFIN WAX

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melting point of paraffin wax normally used for routine work

56C

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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

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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)

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Water in the paraffin wax can be removed through heating wax to _____

100-105C

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How many times can paraffin wax be used?

Paraffin wax may be used only TWICE after which, FRESH WAX must be utilized

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METHODS OF PARAFFIN IMPREGNATION AND EMBEDDING

Manual processing (hand processing)
Automatic tissue processing (autotechnicon)
Vacuum embedding

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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)

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In Manual processing of embedding, approximately ____ hours to ensure complete embedding or casting of tissue

Approximately 3 hours

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How many processing steps does automatic tissue processing use?

12 processing steps

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Tissue processing steps that are included in automatic tissue processing:

Fixation
Dehydration
Clearing
Infiltration

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In automatic tissue processing, the wax bath temperature should be:

3C above the melting point of wax

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Advantage of automatic tissue processing:

CONSTANT AGITATION

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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:

1. WET celloidin method 2. DRY celloidin method 3. 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

1. Polyester plastics 2. Acrylic plastics 3. 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:

1. Leuckhart's embedding mold 2. Compound embedding unit 3. Plastic embedded rings and based mold 4. 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)