Impregnation/Embedding

Cards (112)

  • Impregnation

    Clearing agent is completely removed from the tissues and replaced by a medium that will completely fill all the tissue cavities, thereby giving a firm consistency to the specimen, and allowing easier handling and cutting suitably thin sections without any damage or distortion to the tissue and its cellular components
  • Types of tissue impregnation
    • Paraffin wax
    • Celloidin (collodion)
    • Gelatin
    • Plastic
  • Paraffin wax impregnation

    1. Manual processing
    2. Automatic processing
    3. Vacuum embedding
  • Paraffin wax

    • Simplest, most common & best embedding medium
    • Thin individual serial sections may be cut with ease
    • Very rapid (within 24 hours)
    • May be stored for indefinite period of time
    • Many Staining procedures are permitted with good results
  • Overheated paraffin makes the specimen brittle
  • Prolonged impregnation will cause excessive tissue shrinkage & hardening
  • Inadequate impregnation will cause retention of the clearing agent
  • Tissues that are difficult to infiltrate need long immersion
  • Not recommended for fatty tissues
  • Paraffin wax impregnation by manual processing
    1. Fixation
    2. Dehydration
    3. Clearing
    4. Impregnation
    5. Embedding
  • Paraffin wax impregnation by automatic processing

    1. Makes use of an automatic tissue processing machine
    2. 2-3 changes of wax
    3. Example: Elliott Bench-Type processor
  • The frequency with the fluids are changed depends on the number, sizes, presence of odor of the clearing agent, and the dehydrating agents should be changed frequently
  • Fluid and wax containers must be filled to the appropriate level and correctly located in the machine
  • Wax bath thermostats should be set at least 3 degrees above the melting point of the wax, and timing should be checked when loading the machine
  • Paraffin wax impregnation by vacuum embedding
    1. Wax impregnation under negative atmospheric pressure inside an embedding oven
    2. Clear in 2 changes of xylene
    3. Place the tissue in molten wax, in vacuum chamber and make the oven airtight
    4. Exhaust the air slowly by means of a vacuum pump or Venture suction pump until there is a negative pressure of 400-500mmHg
    5. Leave for 15 mins, then slowly readmit air until normal atmospheric pressure is reached
    6. Place the tissue in fresh wax
    7. Repeat steps 3 and 4
    8. Place the tissue into fresh wax
    9. Repeat step 3 and leave for 30-45 mins
    10. Bring to normal atmospheric pressure and embed the tissue
  • Factors affecting paraffin wax impregnation
    • Nature and size of the tissues to be processed
    • Type of clearing agents to be used
  • Benzene and xylene are easily removed from the tissues, while chloroform & cedarwood oil are more difficult to remove and require more frequent wax changes
  • Addition of benzene may hasten displacement of cedarwood oil with less tissue shrinkage
  • Precautions observed in paraffin wax impregnation
    • Do not leave the tissues in the medium for longer periods of time
    • Infiltration (>60C) – shrinkage & hardening
    • Paraffin oven = 2-5C above the melting point of paraffin
    • Pure, free from dust, water droplets and other foreign matter
    • Fresh wax should be filtered
    • Wax trimmed may be reused & filtered using a coarse filter paper (Green's No. 904)
  • Paraplast

    Highly purified paraffin + synthetic plastic polymers, melting point: 56-57C, more elastic and resilient (bones and brain), more uniform, with better ribboning of sections
  • Ester wax

    Melting point: 46-48C, harder than paraffin, soluble in 95% Ethyl Alcohol, removal must be gradual - clearing agent + ester wax (3-6 hrs), sectioning is done on a heavy duty microtome (sliding or sledge type microtome)
  • Water soluble waxes

    Melting point: 38-42C or 45-56C, Carbowax - PEG (solid at RT, soluble and miscible with water), does not remove neutral fats and lipids, enzyme histochemical studies: tissues are not exposed to too much heat so that excessive hardening, shrinkage and brittleness of tissue is avoided, cytologic details are preserved, very easily dissolved in water: difficulty in floating out of sections and mounting, adding soap to water or using 10% PEG 900 in water will reduce tissue distortion and promote flattening & "floating-out" of sections
  • Celloidin (collodion)

    A purified form of nitrocellulose, soluble in many solvents, suitable for large hollow cavities, and large tissues, supplied in thin (2%), medium (4%), thick (8%) solutions of cellulose dissolved in equal parts of ether and alcohol
  • Advantages of celloidin impregnation
    • Processing of neurological tissues
    • Cut without undue distortion
    • Tissues are supported better
    • Does not require heat during processing, producing minimum shrinkage & tissue distortion
  • Disadvantages of celloidin impregnation
    • Slow penetration
    • Very thin sections
    • Serial sections are difficult to prepare
    • Inflammable, volatile
    • Photomicrographs are difficult to obtain
  • Wet celloidin method

    1. For bones, teeth, large brain sections and whole organs
    2. Placed in equal parts of ether & alcohol for 12-24 hrs
    3. Tissue is placed in thin celloidin (2-4%) for 5-7 days
    4. Transferred to medium celloidin (4-6%) for 5-7 days
    5. Thick celloidin (8-12%) 3-5 days
    6. Transfer to embedding medium
    7. Desiccator top is opened every now and then
    8. Block is stored in 70-80% alcohol until ready for cutting
  • Dry celloidin method

    1. For whole eye sections
    2. Principle same with wet method
    3. Gilson's mixture (equal parts of chloroform and cedarwood oil)
  • Nitrocellulose method

    LOW VISCOSITY NITROCELLULOSE (L.V.N), another form of celloidin, soluble in ether and alcohol, with a lower viscosity allowing it to be used in higher concentrations and still penetrate tissues rapidly, forms harder tissue block and makes cutting of thinner sections possible, more explosive
  • Gelatin impregnation is rarely used except when paraffin and celloidin are not suitable
  • CELLOIDIN IMPREGNATION - WET CELLOIDIN METHOD
    1. Placed in equal parts of ether & alcohol for 12-24 hrs
    2. Tissue is placed in thin celloidin (2-4%) for 5-7 days
    3. Transferred to medium celloidin (4-6%) for 5-7 days
    4. Thick celloidin (8-12%) 3-5 days
    5. Transfer to embedding medium
    6. Desiccator top is opened every now and then
    7. Block is stored in 70-80% alcohol until ready for cutting
  • CELLOIDIN IMPREGNATION - DRY CELLOIDIN METHOD
    1. Principle same with wet method
    2. Gilson's mixture (equal parts of chloroform and cedarwood oil)
  • NITROCELLULOSE METHOD

    • LOW VISCOSITY NITROCELLULOSE (L.V.N) - another form of celloidin, soluble in ether and alcohol, with a lower viscosity allowing it to be used in higher concentrations and still penetrate tissues rapidly
    • Forms harder tissue block and makes cutting of thinner sections possible
    • More explosive
  • GELATIN IMPREGNATION

    • Rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies
    • For delicate specimens and frozen tissue sections
    • Tissue should not be > 2-3mm
    • Volume of impregnating medium SHOULD be at least 25x the volume of the tissue
  • GELATIN IMPREGNATION Procedure

    1. Tissue is placed in 10% gelatin with 1% phenol for 24 hours
    2. 20% gelatin with 1% phenol for 12 hrs
    3. 20% gelatin with 1% phenol, cooled in ref until completed
    4. Transfer to 10% formalin for 12-24 hrs
  • EMBEDDING

    • CASTING / BLOCKING - OTHER NAMES
    • IMPREGNATED TISSUE IS PLACED INTO A PRECISELY ARRANGED POSITION IN A MOLD CONTAINING A MEDIUM WHICH IS THEN ALLOWED TO SOLIDIFY
    • PARAFFIN EMBEDDED TISSUES ARE ORIENTED IN THE MOLD WITH THEIR LABELS AND IMMERSED IN MELTED PARAFFIN AT A TEMP BET. 5-10c ABOVE ITS MELTING POINT AND COOLED RAPIDLY IN A REFRIGERATOR AT -5C OR IMMERSED IN COLD WATER TO SOLIDIFY
  • Embedding Procedure

    1. Open the tissue cassette, check requisition form entry to ensure the correct number of tissue pieces is present
    2. Select the mould; there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax
    3. Fill the mould with paraffin wax
    4. Using warm forceps select the tissue, taking care that it does not cool in the air at the same time
    5. Place the tissue in the mould according to the side to be sectioned. This side should be facing down against the mould
    6. Insert the identifying label
    7. Chill the mould on the cold plate, orienting the tissue & firming it into the wax with warmed forceps
    8. Add more paraffin into the mould to fill the cassette, cool the block on the cold plate
    9. Remove the block from the mould
  • Orientation

    • Correct orientation of tissue in a mould is the most important step in embedding
    • INCORRECT PLACEMENT : may result in diagnostically important tissue elements being missed or damaged during microtomy
    • TISSUE IS ARRANGED IN PRECISE POSITIONS IN THE MOLD DURING EMBEDDING, ON THE MICROTOME BEFORE CUTTING, AND ON THE SLIDE BEFORE STAINING
  • Blocking-out Molds

    • LEUCKHART'S EMBEDDING MOLD
    • COMPOUND EMBEDDING UNIT
    • PLASTIC EMBEDDING RINGS & BASE MOLDS
    • TISSUE TEK SYSTEM
  • Disposable Embedding Molds

    • PEEL-AWAY
    • PLASTIC ICE TRAYS
    • PAPER BOATS
  • Celloidin or Nitrocellulose Method
    • Recommended for hard tissues (bones, teeth) & large sections of whole organs (eyes)
    • Tissues are embedded in enamel pans covered by sheets of weighted glass
    • BELL JARS - control the rate of evaporation of the solvent
    • its use is now discouraged because of the special requirements needed for processing and the limited use of these types of sections in neuropathology