impreg

• Prolonged impregnation causes shrinkage, hardening – difficult to cut
• Prolonged immersion in paraffin is NOT advisable!
• Paraffin Wax Impregnation

  1. Paraffin is the SIMPLEST, MOST COMMON & BEST EMBEDDING MEDIUM used for Routine tissue processing

  2. NOT Recommended for Fatty tissue (Use Carbowax instead)
• Working Temperatures for Paraffin: Paraffin Oven/ Incubator, Lab Temp 20 – 24°C, Lab Temp 15 – 18°C, Flotation Bath Temp: 55 – 60°C, MP: 54 – 58°C, 50 & 54°C, 45 – 50°C (6 – 10° Below MP)
• Machine mounted on rollers to permit turning of platform & easy access to beakers
• Paraffin Wax Impregnation


  • Thin individual serial section may be cut without distortion
  • Process is very rapid (section prep in 24 hr)
  • Tissue blocks & Unstained sections may be stored for indefinite period of time
  • Good with most staining methods
• Precaution: Avoid spillage of reagent & Must be filled to the appropriate level, loaded well in machine
• Incomplete impregnation causes retention of the clearing agent – tissue crumble during sectioning, breaks up on floatation bath
• Automatic tissue processing (Autotechnicon – from Fixation to infiltration) decreases time & labor needed during processing resulting in Rapid diagnosis
• Enclosed thermostatically by WATER JACKET (usually maintained at 2 – 4°C above MP)
• Tissue is not exposed to heat and will not make it Brittle, Shrink, hardens
• The Exhaustion & Readmission of Air must be gradual or the specimen may be ruined
• Precaution: Wax Bath thermostat should be at least 3° above MP of the Wax
• Factors Affecting Paraffin Wax Impregnation: Larger & Denser tissue
• Exhaustion of 400 - 500 mmHg (Must never go beyond 500)
• Overheated Paraffin makes tissue Brittle
• Precaution: Presence of any odor of clearing agent in paraffin wax – Change the Wax
• Impregnation


  1. Process where clearing agent is removed & replaced by a medium that will completely fill all the tissue cavities

  2. Gives Firm consistency, Easier handling & cutting of thin section without any damage/ distortion to tissue
• Has 2-SCREW VALVES, 1 to allow readmission of Air, 1 with suction pump for exhaust (manometer gradual)
• Vacuum Embedding

  FASTEST METHOD (25 – 75% Time Reduction), Impregnation under Negative Atmospheric Pressure, Recommended for Urgent Biopsies of Dense & Fibrous Tissue such as: Brain & CNS tissues, Connective tissue, Bones, Lungs, Spleen, Delicate tissues such as the Eye
• Procedure for Vacuum Embedding

  Clear in 2 changes of Xylene for 1 hour each, Place tissue in molten wax in vacuum chamber & make the oven airtight, exhaust the air slowly until 400 – 500mmHg then leave for 15 min then slowly readmit air until N° pressure is reached, Place the tissue in fresh wax then repeat the process (15 minutes), Place the tissue in fresh wax then repeat the process (30 – 40 minutes)
• Tissue such as Bones, teeth, brain, eyes are difficult to impregnate (req. long immersion)
• Three Ways of Paraffin Impregnation & Embedding

  Manual Processing: Requires at least 4 changes of wax at 15 minutes interval (ensure removal of clearing agent), Specimen is then immersed in another fresh melted paraffin for 3 Hours (ensure infiltration), Automatic Processing: "Elliot Bench Type" with 12 Processes, 10 1L Beakers, 2 Wax Bath, Controlled arms, Electric Clock
• Precautions observed in Paraffin Impregnation

  • Prolonged immersion to melted paraffin causes excessive tissue shrinkage, hardening – difficult to cut
  • Overheated paraffin (Above 60°C) causes excessive tissue shrinkage, hardening & also destroys Lymphoid tissue
  • Maintain temperature of oven at 2 – 5°C above melting point of paraffin
  • Paraffin wax must be pure (free from dust, water droplets, foreign matter)
  • Paraffin wax may only be used twice
  • When using automatic tissue processing, the wax may be mixed with clearing agent
  • Fixed knife/heavy microtomes require harder paraffin with a higher melting point
• Polyethylene Glycol


  • Most commonly used
  • Solid at room temperature
  • Suitable for many enzyme histochemical studies
  • Tissue does not require dehydration & clearing process
• Embeddol


  • Synthetic wax substitute similar to Paraplast
  • Less brittle, less compressible
  • Recommended for eyes
  • Paraffin + Rubber with same properties as Paraplast
• Carbowax


  • Does not remove neutral fats & lipids
  • Suitable for many enzyme histochemical studies
  • Easily dissolved in water
  • Tissue sections are difficult to float out & mount due to extreme solubility in water
• Tissue processing: Wax impregnation/Embedding/Blocking

  1. Place the tissue in fresh wax then repeat the process (15 minutes)

  2. Place the tissue in fresh wax then repeat the process (30 – 40 minutes)
• Ester Wax

  • Harder than Paraffin wax
  • Not soluble in water but soluble in 95% alcohol & clearing agent
  • Tissue can be processed without clearing
  • Sectioning should be done on a heavy microtome
• Substitutes for Paraffin Wax

  • Paraplast
  • Paraffin + Plastic
  • Embeddol
  • Biolid
  • Tissue Mat
• Paraplast

  • More elastic & resilient than paraffin
  • Good for large dense tissue blocks such as bones, brain
  • Serial sections may be cut without cooling the tissue block
  • No deposit left on the slide after staining
  • Soluble in common clearing agents
• Factors affecting Paraffin Wax Impregnation

  • Larger & denser tissue blocks require longer periods & more frequent changes of wax
  • Benzene & Xylene are easily removed from tissue
  • Chloroform & Cedarwood oil are difficult to remove from the tissue & require more wax changes
  • Addition of Benzene may hasten displacement of Cedarwood oil
• Procedure for Carbowax processing

  1. 30 mins in 70% Carbowax

  2. 45 mins in 90% Carbowax

  3. 2 changes in 100% Carbowax 1 hour each

  4. Tissue to be embedded in fresh Carbowax at 50°C & cooled in refrigerator
• Water Soluble Waxes

  • Polyethylene Glycol
  • Carbowax
• Celloidin Impregnation

  Celloidin: 2% Thin, 4% Medium, 8% Thick
• Methods for Celloidin Impregnation

  1. Wet Celloidin

  2. Dry Celloidin
• Celloidin

  • Rubbery Consistency allows very hard tissue blocks to be cut
  • Does not Require heat during processing
  • Prevents crumbling of tissue such as hollow organs, eyes: Celloidin supports them better
• Very Volatile
• Very thin sections are difficult to cut
• Wet Celloidin

  1. Recommended for Bones, teeth, Brain, Large organs

  2. Tissue block is stored in 70 – 80% Alcohol to prevent dehydration & Shrinkage