General information in impregnation, embedding and microtomy

• Impregnation (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 and give a firm consistency to the specimen
• Embedding (Casting or Blocking)


  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.
• PARAFFIN WAX IMPREGNATION


  the simplest, most common and best embedding medium used for routine tissue processing
• Paraffin wax i


  polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils.
• It is solid at room temperature but melts at temperatures up to about 65°C or 70°C.
• The traditional advice with paraffin wax is to use this about 2°C above its melting point.
• To decrease viscosity and improve infiltration of the tissue, technologists often increase the temperature to above 60°C or 65°C.
• High molecular weight mixtures melt at higher temperatures than waxes comprised of lower molecular weight fractions. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C.
• Longer time is required for thicker tissues.
• The 56°C wax is normally used for routine work.
• laboratory with temperature ranging from 20-24°C, paraffin wax with a melting point of 54-58°C is indicated
• Manual Processing

  At least four changes of wax are required at 15 minutes intervals in order to insure complete removal of the clearing agent from the tissue
• Automatic Processing


  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.
• Automatic Processing


  only 2- 3 changes of wax are required to remove the clearing agent and properly impregnate the specimen
• Vacuum Embedding


  involves wax impregnation under negative atmospheric pressure inside an embedding oven

  reduces the time when tissues are subjected to high temperatures thus minimizing heat-induced tissue hardening
• Vacuum Embedding


  It facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination
• With vacuum embedding, the time required for complete impregnation is reduced by 25% -75% of the normal time required for tissue processing.
• The vacuum embedding oven consists of a flat-bottomed heavy brass chamber covered with a heavy glass lid resting on a wide and thick rubber valve which produces an airtight seal when the chamber is being used.
• Infiltration in overheated paraffin (above 60°C) will also produce shrinkage and hardening of tissues and destroy lymphoid tissues completely.
• Paraffin wax must be pure, i.e. free from dust, water droplets and other foreign matter. Fresh wax should be filtered before use in a wax oven at a temperature 2°C higher than its melting point.
• Paraplast


  mixture of highly purified paraffin and synthetic plastic polymers, with a melting point of 56-57°C.

  more elastic and resilient than paraffin wax thereby permitting large dense tissue blocks such as bones and brain to be cut easily with the same result as in double embedding
• Paraplast with a melting point of 56 to 58 oC is recommended. During the winter, 54 to 56 oC Paraplast may be used if the tissue is cut in a cool room.
• Embeddol

  synthetic wax substitute similar to Paraplast with a melting point of 56-58°C. It is less brittle and less compressible than Paraplast. Bio/aid is a semisynthetic wax recommended for embedding eyes. Tissue Mat is a product of paraffin, containing rubber, with the same property as Paraplast.
• Ester Wax


  lower melting point (46-48°C), but it is harder than paraffin. It is not soluble in water, but is soluble in 95% Ethyl Alcohol and other clearing agents; hence, it can be used for impregnation without prior clearing of the tissue
• Water Soluble Waxes


  plastic polymers, mostly polyethylene glycols with melting points of 38-42°C or 45-56°C. Polymer waxes are incorporated in the majority of proprietary histological paraffin wax blends presently available to improve adhesion, hardness and plasticity.
• Carbowax


  polyethylene glycol containing 18 or more carbon atoms, which appears solid at room temperature
• Dimethyl sulphoxide (DMSO)


  added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning
• Celloidin (Collodion)


  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
• Wet Celloidin Method


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


  preferred for processing of whole eye sections. The principle and procedure of this method is similar to wet celloidin method, except that 70% alcohol is not used for storage before cutting
• Gilson's mixture

  made up of equal parts of chloroform and cedarwood oil
• 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. 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.
• EMBEDDING

  tissue is placed into a mold containing the embedding medium and this medium is allowed to solidify.
• Ideally the embedding medium should match the tissue type in strength and hardness
• If the embedding medium is too soft for the material, the tissue will not be supported and sections will be torn or shredded. If the medium is too hard for the tissue, sections will be brittle and will shatter. To infiltrate the tissues with supporting embedding medium, tissues must be free of all 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 5-10°C above its melting point and then cooled rapidly in a refrigerator at -5°C or immersed in cold water to solidify.
• 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
• Leuckhart’s Embedding Mold


  Blocks produced are even, with parallel sides, and with a fairly shaped initial setting of the wax
• Leuckhart’s Embedding Mold


  mold is adjustable to give a wide variety of sizes to fit the size of the tissue block for casting

  recommended for routine use, although, too slow and cumbersome for use in a busy laboratory