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PRACTICAL: HISTOLOGICAL TECHNIQUES
Introduction: Histological techniques refer to the procedures used to prepare tissues for
microscopic examination
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Harvesting of biological tissues-tissues can be obtained from human or animal sources
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g
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Wash the organs in normal saline for 20 minutes
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Gross examination-involves trimming, cassetting, labelling
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3
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g
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The tissues can then be transferred into 70% alcohol
indefinitely
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85g of sodium chloride and transfer to a
clean conical flask; add 90ml of distilled water and mix to dissolve the powder
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b) Fix the harvested tissues
4
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g
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Test endpoint of decalcification periodically
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Add 10mls of concentrated nitric acid gently
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Label and store
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Tissue processing-involves taking the tissues through 4 processes, namely dehydration,
clearing, infiltration and embedding to obtain a tissue block
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a) Dehydration- this is done by taking the tissue through various concentrations of diluted
alcohols in an ascending order i
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70%, 80%, 90%, 95%, 100%, 100%, 100% at an interval
of 1hour in each alcohol
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It also removes the fixative
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This is done to remove alcohol and replace it with the
medium that is wax soluble
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This is done by
passing the tissue through 2 or 3 bathes of molten paraffin wax (set at 20C above melting
point of wax e
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600C) for 1hr in each bath or overnight
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It is done using embedding
mold and embedding machine
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Microtomy-slice the tissue block into thin slices of 3-5 microns thick
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Practical exercise: perform microtomy
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Practical exercise: perform floatation
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Practical exercise: perform fishing
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This is done to ensure complete
drying and removal of excess wax
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Staining-routine Haematoxylin and Eosin (H&E) staining technique is applied on the
sections
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Mix to dissolve
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Label and store
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Dewax sections in 2 changes of xylene each 5 minutes
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Remove xylene in 2 changes of absolute alcohol each 3 minutes
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Hydrate sections in descending grades of alcohol ie 90%, 80% and 70% each 3 minutes;
followed by tap water for 5 minutes
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Stain nucleus in Haematoxylin stain for 15-30 minutes
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Rinse in tap water
vi
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vii
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viii
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Rinse in tap water
x
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xi
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xii
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Leave overnight on a flat bench for mountant to harden
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Microscopy-examine the structures seen on the stained section using x10 and x40 objectives
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Taking slide images from stained
mounted slides

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