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HISTOTECHNIQUES - PART-2
Dr Swati Patil
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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
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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
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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
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DEHYDRATION
Definition:Alcohols of different
strengthsMechanism of
action:Time required for
dehydration:Volume of
dehydrating agent:
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STEPS OF PROCESSING
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DEHYDRATING FLUIDS
• Ethanol: • Industrial methylated
spirit: (denatured alcohol) ethanol +1% methanol
• Methanol: • Propanol: • Acetone:
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ANHYDROUS CuSO4:
Indicator of dehydration
Changes into blue colour
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CLEARINGDealcoholisation:Definition:Clearing agents:
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Criteria for suitable clearing agents: -Speedy removal of dehydrating agents. -Ease of removal of molten paraffin wax. -Minimum tissue damage. -Flammability -Toxicity -Cost
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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:
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-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
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IMPREGNATION
Definition: -placement of tissues with medium
- that fill natural cavities, spaces, replaces clearing agent
-supports tissue -firmness to tissue -without any
injurious effects
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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
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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
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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.
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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
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Other waxes:-Bees wax-Candle wax-Ester wax-Carbowax
Advantages of carbowax-soluble & miscible with water-lipids & natural fats-enzyme histochemistry- thinner sections
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PARAFFIN EMBEDDING
Advantages-Reasonable speed of processing
-Good consistency for serial sectioning
-A wide range of section thickness & durability of block
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STEPS IN PARAFFIN EMBEDDING
Selection of proper size of L shaped moulds smeared with glycerol
Filling with paraffin wax
Selection of tissueOrientation of tissueInsertion of labelCooling of block
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L MOULDS
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Automatic 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
AUTOMATED TISSUE PROCESSING
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Advantages:- Reduce time taken for
processing- Superior results are
obtained- Multiple blocks can be
processed together- Less laborious
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MICROTOMES
To cut smallCutting engines3 main srtuctures-block holder-knife carrier-rachet device
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TYPES OF MICROTOME
Cambridge rocking microtome
Rotary microtomeSliding & base
sledge microtomeFreezing
microtome
Rotary microtome
Sledge microtome
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TYPES OF MICROTOME
Freezing microtomeUltramicrotome
Cryomicrotome
Freezing microtome
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MICROTOME KNIVES
Made up of steel
Parts – blade -knife carrier -handle if
knife -bevel
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Bevel angle:Breadth of knife:
- Bevel angle less, breadth will be large
- Bevel angle is more, narrow bevel
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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
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MICROTOME KNIVES
Profile• Profile A: biconcave• Profile B:
planoconcave• Profile C: wedge
shaped - Wide
application• Profile D: chisel
shaped
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CARE OF MICROTOMES
Cover properlyAvoid rustApplication of light
oilRemoving of waxCleaning of metallic
parts
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CARE OF MICROTOMES KNIVESSharpening of
microtome: manual sharpening
-Removal of gross nicks
-Honing – removal of fine nicks
-motion always be from heel to toe
-Stropping – sharpen knife which is free of nicks
-on leather strop -reverse motion of honing
HONING
STROPPING
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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
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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
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Ready to stain
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THANK YOU