HISTOTECHNIQUES - PART-2

Dr Swati Patil

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

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

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

DEHYDRATION

Definition:Alcohols of different

strengthsMechanism of

action:Time required for

dehydration:Volume of

dehydrating agent:

STEPS OF PROCESSING

DEHYDRATING FLUIDS

• Ethanol: • Industrial methylated

spirit: (denatured alcohol) ethanol +1% methanol

• Methanol: • Propanol: • Acetone:

ANHYDROUS CuSO4:

Indicator of dehydration

Changes into blue colour

CLEARINGDealcoholisation:Definition:Clearing agents:

Criteria for suitable clearing agents: -Speedy removal of dehydrating agents. -Ease of removal of molten paraffin wax. -Minimum tissue damage. -Flammability -Toxicity -Cost

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:

-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

IMPREGNATION

Definition: -placement of tissues with medium

- that fill natural cavities, spaces, replaces clearing agent

-supports tissue -firmness to tissue -without any

injurious effects

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

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

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.

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

Other waxes:-Bees wax-Candle wax-Ester wax-Carbowax

Advantages of carbowax-soluble & miscible with water-lipids & natural fats-enzyme histochemistry- thinner sections

PARAFFIN EMBEDDING

Advantages-Reasonable speed of processing

-Good consistency for serial sectioning

-A wide range of section thickness & durability of block

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

L MOULDS

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

Advantages:- Reduce time taken for

processing- Superior results are

obtained- Multiple blocks can be

processed together- Less laborious

MICROTOMES

To cut smallCutting engines3 main srtuctures-block holder-knife carrier-rachet device

TYPES OF MICROTOME

Cambridge rocking microtome

Rotary microtomeSliding & base

sledge microtomeFreezing

microtome

Rotary microtome

Sledge microtome

TYPES OF MICROTOME

Freezing microtomeUltramicrotome

Cryomicrotome

Freezing microtome

MICROTOME KNIVES

Made up of steel

Parts – blade -knife carrier -handle if

knife -bevel

Bevel angle:Breadth of knife:

- Bevel angle less, breadth will be large

- Bevel angle is more, narrow bevel

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

MICROTOME KNIVES

Profile• Profile A: biconcave• Profile B:

planoconcave• Profile C: wedge

shaped - Wide

application• Profile D: chisel

shaped

CARE OF MICROTOMES

Cover properlyAvoid rustApplication of light

oilRemoving of waxCleaning of metallic

parts

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

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

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

Ready to stain

THANK YOU