IMPREGNATION

• IMPREGNATION (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.

  - promotes easier handling and for ease of cutting into thin sections
• EMBEDDING (Casting or Blocking)

  - is the process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify.
• TISSUE BLOCK


  end product of embedding
• 1. Paraffin wax

  2. Celloidin (Collodion)

  3. Gelatin

  4. Plastic


  TYPES OF INFILTRATING AND EMBEDDINGMEDIUM
• PARAFFIN WAX


  - simplest, most common and best embedding medium used for routine tissue processing.

  - most common for histological use: 56°C to 58°C
• 45°C, 52°C, 56°C, and 58°C

  Common waxes have melting points of
• 56°C wax


  ROUTINE WORK
• paraffin wax with a melting point of 54-58°C is indicated

  If the laboratory temperature is between 20-24°C
• the melting point of wax to be used should be between 50 and54°C

  If the laboratory temperature is between 15-18°C
• 1. By manual processing

  2. By automatic processing

  3. By vacuum embedding

  3 WAYS BY WHICH PARAFFIN WAX IMPREGNATIONAND EMBEDDING OF TISSUES MAY BE PERFORMED:
• MANUAL PROCESSING


  - 4 changes of wax are required at 15 minutes intervals in order to insure complete removal of the clearing agent from the tissue.

  - specimen is then immersed in another fresh solution of melted paraffin for approximately 3hours to insure complete embedding or casting of tissue.
• AUTOMATIC PROCESSING


  - 2- 3 changes of wax are required to remove the clearing agent and properly impregnate the specimen.
• VACUUM


  - involves wax impregnation under negative atmospheric pressure inside an embedding oven.

  - it facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination.

  - hastens the removal of air bubbles and clearing agent from the tissue block.

  - of the three methods of paraffin wax impregnation, vacuum impregnation gives the fastest result
• 1. Paraplast

  2. Embeddol

  3. Ester Wax

  4. Water Soluble Waxes

  5. Dimethyl Sulphoxide (DMSO)


  SUBSTITUTE FOR PARAFFIN WAX
• PARAPLAST


  - is a mixture of highly purified paraffin and synthetic plastic polymers; melting point of 56-57°C
• EMBEDDOL


  - synthetic wax substitute similar to Paraplast with a melting point of 56-58°C.

  - less brittle and less compressible than Paraplast.

  - bio/aid is a semisynthetic wax recommended for embedding eyes.
• ESTER WAX


  - has a lower melting point (46-48°C), but it is harder than paraffin.

  - not soluble in water, but is soluble in 95% Ethyl Alcohol and other clearing agents.

  - Cellosolve (ethylene glycol monoethyl ether) orxylene may be used as clearing agents.
• WATER SOLUBLE WAXES


  - most commonly used is Carbowax- are plastic polymers, mostly polyethylene glycols with melting points of 38-42°C or 45-56°C
• DIMETHYL SULPHOXIDE (DMSO)


  - added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.

  - possible health risks associated with the use of DMSO-paraffin wax are minimal if correct laboratory hygiene is observed.
• CELLOIDIN (COLLODION)


  - 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.
• Wet Celloidin Method

  Dry Celloidin Method


  TWO METHODS USED FOR CELLOIDINIMPREGNATION OF TISSUE:
• Wet Celloidin Method


  recommended for bones, teeth, large brain sections and whole organs.
• Dry Celloidin Method


  whole eye sections.
• NITROCELLULOSE


  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.
• GELATIN IMPREGNATION


  - rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies

  - 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
• 1. Leuckhart's Embedding Mold

  2. Compound Embedding Unit

  3. Plastic Embedding Rings and Base Mold

  4. Disposable Embedding Molds

  Celloidin or Nitrocellulose EmbeddingMethod

  SEVERAL TYPES OF BLOCKING-OUT MOLDS MAY BEUSED:
• LEUCKHART'S EMBEDDING MOLD


  - 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.
• COMPOUND EMBEDDING UNIT


  - 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.
• PLASTIC EMBEDDING RINGS AND BASE MOLD

  - consist of a special stainless steel base mold fitted with a plastic embedding ring, which later serves as the block holder during cutting.
• Peel-away


  - are simply peeled off one at a time, as soon as the wax has solidified giving perfect even block without trimming. It may be placed directly in the chuck or block holder of the microtome
• Plastic ice


  - used in ordinary refrigerators may be recommended for busy routine laboratories.

  - each compartment may be utilized for embedding one tissue block, which may then be removed by bending the plastic tray once the wax has solidified or by smearing the inner mold with glycerin or liquid paraffin before embedding.
• Paper boats


  - are normally utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks.

  - advantage of being cheap and easy to make.

  - provide easy and accurate identification of specimen, thereby avoiding confusion and interchange of tissue blocks.

  - rapid embedding of small or large volume of individual specimen is possible, since paper molds can be made to suit any size of tissue.
• CELLOIDIN OR NITROCELLULOSE EMBEDDINGMETHOD

  - 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.- Bell jars can be used to control the rate or evaporation of the solvent.
• DOUBLE-EMBEDDING


  - is 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.

  - is used to facilitate cutting of large blocks of dense firm tissues like the brain.
• PLASTIC (RESIN) EMBEDDING


  - are classified into epoxy, polyester, or acrylic, based on their chemical composition.
• Epoxy Embedding Plastics


  - made up of a carefully balanced mixture of epoxy plastic, catalysts and accelerators. Three types of epoxy plastics are used in microscopy, i.e., those based on either bisphenol A (Araldite), or glycerol(Epon), or cyclohexene dioxide (Spurr).
• Polyester Plastics


  - were originally introduced for electron microscopy in the mid- 1950s, but have been superseded by more superior epoxides, and are now seldom used
• Acrylic Plastics


  - made up of esters of acrylic or methacrylic acid, and are used extensively for light microscopy. Polyglycol methacrylate (GMA) has proved to be a popular embedding medium for light microscopy because it is extremely hydrophilic, allowing many staining methods to be applied, yet tough enough when dehydrated to section well on most microtomes.