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  • 1. HISTOTECHNIQUES - PART-2 Dr Swati Patil
  • 2. INTRODUCTION • Histological technique deals with preparation of tissue for microscopic examination. Total or a selected part undergoes series of processes, namely, - Fixation - Dehydration - Clearing - Embedding - Cutting - Staining
  • 3. DECALCIFICATION• Tissue : bones & teeth• Criteria for decalcifying agent:- Complete removal of Ca salts- No damage to tissue- Lack of harmful effect on staining reactions- Speed for removal of Ca salts
  • 4. METHODS OF DECALCIFICATION• Chemical method -Acid reagents -Nitric acid -Trichloracetic acid -Formic acid -Chelating agents: - Ethylene Diamine Tetra acetic acid[EDTA]• Ion–exchange resin method( ammonium form of sulphonated polysterene resin)• Electrophoretic method
  • 5. DEHYDRATIONDefinition:Alcohols of different strengthsMechanism of action:Time required for dehydration:Volume of dehydrating agent:
  • 7. DEHYDRATING FLUIDS• Ethanol:• Industrial methylated spirit: (denatured alcohol) ethanol +1% methanol• Methanol:• Propanol:• Acetone:
  • 8. ANHYDROUS CuSO4:Indicator of dehydrationChanges into blue colour
  • 9. CLEARINGDealcoholisation:Definition:Clearing agents:
  • 10. Criteria for suitable clearing agents: -Speedy removal of dehydrating agents. -Ease of removal of molten paraffin wax. -Minimum tissue damage. -Flammability -Toxicity -Cost
  • 11. Clearing agents suitable for general use:-Xylene: -widely used -rapid in action -cheap, inflammable -shrinkage & overhardening - block 3-5mm : 15 – 30 mins- Benzene: -cheap, inflammable -vapors show toxicity -no shrinkage & overhardening-Toluene:
  • 12. -Chloroform: - expensive -high specific gravity, non inflammable – phosgene gas -slower in action, less transparency - little hardening- Cedar-wood oil: -thin fluid, expensive -used to clear both the paraffin & celloidin sections -penetration is well, improves section cutting -slow in action & less damaging to tissue -emersion in xylene necessary prior to paraffin impregnation
  • 13. IMPREGNATIONDefinition: -placement of tissues with medium - that fill natural cavities, spaces, replaces clearing agent -supports tissue -firmness to tissue -without any injurious effects
  • 14. EMBEDDINGDone by- Filling mould of suitable size with molten paraffin wax.- Orienting specimen in mould to ensure its being cut in right plane.- Cooling mass to promote solidification
  • 15. Types of embedding medium:-Ribboning media: eg. paraffin, soap-Non-ribboning media: eg. sugar, gum solution, gelatin Embedding media: -Water soluble media:- eg. gelatin, gum -Water insoluble media:- eg. Paraffin wax, nitrocellulose
  • 16. METHODS OF EMBEDDING• Fusion embedding method:- eg. Paraffin embedding: -simpler, better for routine use -allows thin section to be cut -about 10% shrinkage of section on cooling• Evaporation embedding method : eg. Celloidin embedding - suitable for specimens containing large cavities -slow, tedious, no serial sections -less shrinkage & distortion -no heat & heavy microtomeVolume of impregnating medium: at least 25 times of the volume of tissue.
  • 17. PARAFFIN WAX• Saturated hydrocarbons, very stable• Water insoluble, burns with smoky flame• Melting pt- 35-65 C• Strongly hydrophobic• Plastic point of paraffin wax• Low melting pt – thick sections• High melting pt – thin sectionsWax additives – hardness eg beeswax, rubber, ceresin etc
  • 18. Other waxes: -Bees wax -Candle wax -Ester wax -CarbowaxAdvantages of carbowax -soluble & miscible with water -lipids & natural fats -enzyme histochemistry - thinner sections
  • 19. PARAFFIN EMBEDDINGAdvantages-Reasonable speed of processing-Good consistency for serial sectioning-A wide range of section thickness & durability of block
  • 20. STEPS IN PARAFFIN EMBEDDINGSelection of proper size of L shaped moulds smeared with glycerolFilling with paraffin waxSelection of tissueOrientation of tissueInsertion of labelCooling of block
  • 21. L MOULDS
  • 22. Automatic Tissue Processing AUTOMATED TISSUE PROCESSINGPrinciple : Time required for tissue processing may be considerably reduced when tissue is suspended in fluid, continuously agitated, moved from one reagent to another whenever desired, not restricted by working hours.Processors are configured with preset interval for different schedules of suspension, agitation, & automatic changeover
  • 23. Advantages:- Reduce time taken for processing- Superior results are obtained- Multiple blocks can be processed together- Less laborious
  • 24. MICROTOMESTo cut smallCutting engines3 main srtuctures-block holder-knife carrier-rachet device
  • 25. TYPES OF MICROTOMECambridge rocking microtome Rotary microtomeRotary microtomeSliding & base sledge microtomeFreezing microtome Sledge microtome
  • 26. TYPES OF MICROTOMEFreezing microtomeUltramicrotome Freezing microtome Cryomicrotome
  • 27. MICROTOME KNIVESMade up of steelParts – blade -knife carrier -handle if knife -bevel
  • 28. Bevel angle:Breadth of knife:- Bevel angle less, breadth will be large- Bevel angle is more, narrow bevel
  • 29. MICROTOME KNIVESObliquity of knife: angle of knife edge & edge of tissue blockAngle of tilt: angle, plane passing through back edge of knife with plane of section
  • 30. MICROTOME KNIVESProfile• Profile A: biconcave• Profile B: planoconcave• Profile C: wedge shaped - Wide application• Profile D: chisel shaped
  • 31. CARE OF MICROTOMESCover properlyAvoid rustApplication of light oilRemoving of waxCleaning of metallic parts
  • 32. CARE OF MICROTOMES KNIVESSharpening of microtome: manual sharpening-Removal of gross nicks-Honing – removal of fine HONING nicks -motion always be from heel to toe-Stropping – sharpen knife which is free of nicks -on leather strop -reverse motion of honing STROPPING
  • 33. SECTIONINGInsert block into microtome chuckBlade should be angledDiscard paraffin ribbonWell ribboning block, pick with fine brush, float them on surface of water bathFloat sections onto glass slides
  • 34. SECTIONINGNo ribboning – place block on ice cubesPlace slides with parffin sections in 65 c oven for 20 minsSlides stored overnight at RT
  • 35. Ready to stain
  • 36. THANK YOU