5 Impregnation

• Impregnation or Infiltration is the process of permeating the tissue with a support medium

  • clearing medium is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities giving a firm consistency to the specimen, and allowing easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its cellular components
• Paraffin Wax Impregnation uses a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils.

  • simplest, most common
  • best embedding medium (routine process)
  • ease in cutting
  • permanent paraffin blocks
  • good staining results
  • not recommended for fatty tissue
• Overheated paraffin causes brittle specimen
• Inadequate paraffin wax impregnation causes clearing agent retention, where tissue will become soft and shrunken, tissue blocks crumble, break up when floated in water bath
• Prolonged paraffin wax impregnation causes tissue shrinkage and hardening
• Paraffin wax impregnation is very rapid, done within 24 hrs

  • May be stored in paraffin for an indefinite period of time after impregnation without considerable tissue destruction
• common paraffin wax melting points

  • 56 °C → Routinely used
  • Melting point of 54-58 °C (wax) at 20-24°C at room temp laboratories
  • Melting point of 50-54 °C (wax) at 15-18°C laboratory
• In paraffin wax impregnation, paraffin oven or an incubation is used which has been regulated at 55-60 °C
• Fresh wax should be filtered before use in a wax oven at temperature of 2°C higher than its melting point

  • solid at room temp but melts at 65 or 70 °C
  • most common for histological use is 56-58 °C
  • at melting point tends to be slightly viscous, decreases as temperature increases
  • to decrease viscosity and improve infiltration technologists increase temp to above 60 or 65 °C
• In manual or hand processing in paraffin wax impregnation, the specimen is immersed in another fresh solution of melted paraffin for approximately 3 hrs to ensure complete embedding or casting of tissues.

  • At least 4 changes of wax are required at 15 mins intervals in order to ensure complete removal of the clearing agent from the tissue
• A) 24

  B) 6

  C) 12

  D) 2

  E) 1

  F) 15

  G) 3

  
• Automatic processing in paraffin impregnation uses autotechnicon or Elliott Bench type processor; it has 12 processing steps

  • decreasing time and labor needed and MORE RAPID and less technicality
  • Wax bath temperature: 3 °C above the melting point
  • Dehydration is the most critical step
  • ADVANTAGE: “Constant agitation”
  • only 2-3 changes of wax are required to remove the clearing agent and properly impregnate the specimen
• Elliott Bench-Type Processor machine is mounted on rollers to permit the turning of platforms and easy access to beakers and wax baths
• Vacuum embedding in paraffin impregnation uses “under negative atmospheric pressure” inside an embedding oven

  • Facilitates complete removal of transition solvents, and prolongs the life of wax by reducing solvent contamination
  • Recommended for urgent biopsies, delicate tissues such as lung, connective tissues, decalcified bones, eyes, spleen, and CNS.
• Use of vacuum embedding gives the fastest results

  • Larger and denser tissue blocks e.g. bones, fibroids, brains require longer periods and more frequent changes of wax
  • Benzene and Xylene are easily removed from the tissues
  • Chloroform and 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
• Vacuum embedding

  • Temperature is maintained at 2-4 °C the melting point of the wax
  • Time required for complete impregnation is reduced by 25-75% of the normal time
  • Tissue is not over-exposed to heat; brittleness, shrinkage and hardening of tissues consequent to overheating is therefore prevented
  • Tissue can also be transferred after clearing to a heated bath of paraffin wax (air can be evacuated)
• Infiltration in overheated paraffin >60 °C will produce considerable shrinkage and hardening of tissues
• Paraffin oven must be maintained at a temperature of 2-5 °C above the melting point of the paraffin used
• Fresh wax should be filtered before use in a wax oven at temperature of 2 °C higher than its melting point
• Green’s No. 904 is a coarse filter paper used for the filtration of the “used” wax.
• Water must be removed by heating the wax to 100-105 °C (boiling point) thereby to raising its melting point.
• Paraffin wax may be used only twice and the paraffin wax must be changed.
• Paraplast is a mixture of highly purified paraffin and synthetic plastic polymers

  • MP: 56-57 °C.
  • More elastic and resilient than paraffin wax
  • For large tissue blocks
  • Blocks obtained more uniform → better ribboning of sections
  • Preventing formation of ice crystal artifacts
  • No deposit is left on slides after staining
  • Soluble in the majority of the clearing agent
• Paraplast with a melting point of 56-58 °C is recommended

  • During the winter = 54-56 °C
  • May be used if the tissue is cut in a cool room
  • Summer it may be necessary to use 60-63 °C, avoided if possible in order to not "cook" the tissue.
  • Cooked tissue does not section well or, if it does, not stain well and most details are destroyed
• Embeddol is a synthetic wax; substitute similar to paraplast.

  • Less brittle and less compressible
  • MP: 56-58 °C
• Bioloid is asemisynthetic wax recommended for embedding eyes
• Tissue Mat is a product of paraffin containing rubber, same property as paraplast
• Ester wax is harder than paraffin; it is insoluble = not soluble in water but they are soluble in 95% ethyl alcohol

  • MP: 46-48 °C
  • Uses Cellosolve or xylene
  • 3-4 changes of wax to ensure complete tissue impregnation
  • Should be done on a heavy duty microtome
• Water soluble wax is mostly polyethylene glycols (plastic polymers)

  • MP: 38-48 °C or 45-56 °C
• Carbowax is most commonly used water-soluble wax.

  • Soluble in and miscible with water → not require dehydration and clearing of the tissue
  • Used for enzyme histochemistry studies
  • Routine processing, 4 changes
  • Easily dissolved in water → add soap to water or using 10% Polyethylene Glycol 900 = promote flattening and floating out of sections
• Polyethylene glycol (18 or more C atoms) appears solid at room temperature
• Water soluble wax

  • Processing time is reduced thus harmful effects produced by ordinary dehydrating agents are avoided.
  • Does not remove neutral fats and lipids
  • Soluble in reagents used for routine processing with paraffin, allowing these substances to be demonstrated in thin sections.
• Celloidin or Collodion impregnation is recommended for “large hollow organs” which tend to collapse, for hard and dense tissues such as “bones and teeth” for large tissue sections of the whole embryo

  • Permits cutting of thicker tissue sections → recommended for neurological tissues
  • Crumbling of tissues avoided
  • Very slow (several weeks to months)
  • Thin sections difficult to cut
  • Serial sections difficult to prepare
• Celloidin is a purified form of nitrocellulose soluble in many solvents
• Wet celloidin is recommended for bones, teeth, large brain sections and whole organs

  • Tissue blocks is stored in 70-80% alcohol, to avoid, dehydration and shrinkage of tissues.
  • Ether and alcohol (equal parts) → 12-24 hours after usual fixation and dehydration
  • Thin celloidin → 2-4% for 5-7days
  • Medium celloidin → 4-6% for 5-7days
  • Thick celloidin (drain and poured)→ 8-12% for 3-5days (until impregnation)
• Dry celloidin method is preferred for the processing of whole eye sections

  • Gilson’s mixture (chloroform and cedarwood oil) is added (before hardening) to the block to make it transparent
  • Same with bioloid
  • Principle and procedure: Similar to wet celloidin method, except that 70% alcohol is not used for storage
  • This method does not make use of alcohol due to the presence of cedarwood oil in the block
• Nitrocellulose method is more explosive than celloidin

  • Tissue to crack → add plasticizers when embedding chrome-mordanted tissue
  • When dry, striking or dropping the container will cause explosion.
  • With lower viscosity allowing it to be used in higher concentration and still penetrate tissue rapidly
  • It is usually marketed while wet with alcohol → container must be kept tightly covered and protected from sunlight to avoid evaporation
• Low Viscosity Nitrocellulose has equal concentration of ether and alcohol
• Gelatin impregnation is rarely used except when dehydration is to be avoided → histochemical studies

  • Used for delicate specimen and frozen tissue section
  • Does not require dehydration and clearing
  • Low melting point
  • Tissue not more than 2-3mm thick
  • 1% phenol → prevent the growth of molds
  • 25:1 → ratio of impregnating medium to tissue
• Excess gelatin may be removed by floating the sections on to paper and trimming them with scissors