[M] Week 9 Decalcification, Dehydration, Clearing, Impregnation, and Embedding - Part 2 Flashcards
- or infiltration is the process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities and give a firm consistency to the specimen.
- This allows easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its cellular components.
Impregnation
IMPREGNATING MEDIUMS
The medium used to infiltrate the tissue is usually the same medium utilized for impregnation, and for general purposes is known as an
Embedding Medium
IMPREGNATING MEDIUMS
There are generally four types of impregnations and embedding medium, namely:
- Paraffin Wax (most common)
- Celloidin
- Gelatin
- Plastic: not commonly used
the simplest, most common and best
embedding medium used for routine tissue processing.
Paraffin
There are three ways by which paraffin wax impregnation and embedding of tissues may be performed, what are those?
- Manual Processing
- Automatic Processing
- Vacuum Processing
PARAFFIN WAX IMPREGNATION
At least four changes of wax are required at 15 minutes intervals in order to ensure complete removal of the clearing agent from the tissue.
Manual Processing
PARAFFIN WAX IMPREGNATION
- This method makes use of an automatic tissue processing machine (i.e.,Autotechnicon) which fixes, dehydrates, clears and infiltrates tissues, thereby decreasing the time and labor needed during the processing of tissues.
- This results in a more rapid diagnosis with less technicality. Usually, only 2- 3 changes of wax are required to remove the clearing agent and properly impregnate the specimen.
- This is made possible due to constant tissue agitation which accelerates and improves tissue penetration giving rise to more consistent results
Automatic Processing
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PARAFFIN WAX IMPREGNATION
Vacuum embedding involves wax impregnation under negative atmospheric pressure inside an embedding oven.
Vacuum Processing
SUBSTITUTE FOR PARAFFIN WAX
- Paraplast
- Ester wax
- Water soluble waxes
SUBSTITUTE FOR PARAFFIN WAX
- It is a mixture of highly purified paraffin and **synthetic
plastic polymers ** - Melting point: 56-57°C
- Comes in two forms— Embeddol and Tissue mat
Paraplast
Paraplast
- Is a synthetic wax substitute
similar to Paraplast - Melting point: 56-58°C
Embeddol
Paraplast
Is a product of paraffin, containing rubber, with the\ same property as Paraplast.
Tissue mat
SUBSTITUTE FOR PARAFFIN WAX
Has a lower melting point (46-48°C), but it is harder than paraffin
Ester wax
SUBSTITUTE FOR PARAFFIN WAX
- Are plastic polymers, mostly polyethylene glycols.
- Melting points of:
○ 38-42°C
○ 45-56°C - The most commonly used is Carbowax, a polyethylene glycol containing 18 or more carbon atoms, which appears solid at room temperature.
Water Soluble Waxes
IMPREGNATING MEDIUMS
- a purified form of nitrocellulose soluble in many solvents
- Suitable for specimens with large hollow cavities which tend to collapse, for hard and dense tissues such as bones and teeth and for large tissue sections of the whole embryo.
CELLOIDIN IMPREGNATION
Celloidin Impregnation are supplied in what percentage?
in thin (2%), medium (4%) or thick (8%) solutions of cellulose dissolved in equal parts of ether and alcohol.
Two methods are used for celloidin impregnation of tissue
Recommended for bones,
teeth, large brain sections
and whole organs
Wet Celloidin Method
Two methods are used for celloidin impregnation of tissue
Preferred for processing
of whole eye sections
Dry Celloidin Method
CELLOIDIN IMPREGNATION
Low Viscosity Nitrocellulose (L.V.N.) is another form of celloidin soluble in equal concentration of ether and alcohol, with a lower viscosity, allowing it to be used in higher concentrations and still penetrate tissues rapidly.
Nitrocellulose
impregnation
- Is rarely used except when dehydration is to be skipped or avoided and when tissues are to be subjected tohistochemical and enzyme studies.
- It is used as an embedding medium for delicate specimens and frozen tissue sections because it prevents fragmentation of tough and friable tissues when frozen sections are cut.
Gelatin Impregnation
- Also known as casting or blocking
- After impregnation, the tissue is placed into a mold containing the embedding medium and this medium is allowed to solidify. Basically, the impregnated tissue will be placed in a block of paraffin.
- Ideally, the embedding medium should match the tissue type in strength and hardness.
Embedding
Embedding
TOF
- If the embedding medium is too HARD for the material, the tissue will not be supported and sections will be torn or shredded.
If the medium is too SOFT for the tissue, sections will be brittle and will shatter.
- False (soft)
- False (Hard)
Embedding
- To infiltrate the tissues with supporting embedding medium,
tissues must be ____ (since usually embedding medium is not miscible with water). - Paraffin embedded tissues are arranged at the bottom of the mold together with their proper labels and immersed in melted paraffin at a temperature between____ above its melting point and then cooled rapidly in a refrigerator at ____ or immersed in ____ to solidify. This will allow hardening of tissues, giving them a firmer consistency and better support, thereby facilitating the cutting of sections.
- Free of all water
- 5-10°C
- -5°C or
immersed in cold water
TYPES OF BLOCKING-OUT MOLDS
- Leuckhart’s Embedding Mold
- Compound Embedding Unit
- Plastic Embedding Rings and Base Mold
- Disposable Embedding Molds
TYPES OF BLOCKING-OUT MOLDS
This consists of two L-shaped strips of heavy brass or metal arranged on a flat metal plate and which can be moved to adjust the size of the mold to the size of the specimen
Leuckhart’s Embedding Mold
TYPES OF BLOCKING-OUT MOLDS
- It is made up of a series of interlocking plates resting on a flat metal base, forming several compartments.
- It has the advantage of embedding more specimens at a time, thereby reducing the time needed for blocking.
Compound Embedding Unit
TYPES OF BLOCKING-OUT MOLDS
- It consists of a special stainless steel base mold fitted with a plastic embedding ring, which later serves as the block holder during cutting.
Plastic Embedding Rings and Base Mold
TYPES OF BLOCKING-OUT MOLDS
Includes peel-away, plastic ice trays, and paper boats.
Disposable Embedding Molds
- The process by which a tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining
- The surface of the section to be cut should be placed parallel to the bottom of the mold in which it is oriented.
ORIENTATION
OTHER EMBEDDING METHODS
- Celloidin or Nitrocellulose Embedding Method
- Double-Embedding Method
- PLASTIC (RESIN) EMBEDDING METHOD
OTHER EMBEDDING METHODS
Used to be recommended for embedding hard tissues such as bones and teeth, and for large sections of whole organs like the eye, since the delicate layers of the eyeball are difficult to keep intact when other media are used
Celloidin or Nitrocellulose Embedding Method
OTHER EMBEDDING METHODS
The process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with paraffin wax in which they are subsequently embedded.
Double-Embedding Method
OTHER EMBEDDING METHODS
Classified into epoxy, polyester, or acrylic, based on their chemical composition
PLASTIC (RESIN) EMBEDDING METHOD
