Instrumentation

• HISTOLOGY
• Science of examination of normal tissues
• HISTOPATHOLOGY
• Examination of tissues for presence / absence of changes in structure due to
disease process
• Specimen received in the lab (10% formalin)
• Grossed (appearance, measurements, noticeable pathological changes etc) and
kept for formalin fixation
• Bits given from representative areas ( not >4mm thick)
• Tissue processed…
• Final outcome : stained slide for microscopic examination
• Fixation
• Dehydration
• Clearing
• Impregnation
• Embedding and blocking
• Section cutting
• Routine staining
Fixation
• Any tissue once taken out of the body will decompose
• due to:-
• Loss of bloody supply and oxygen
• Accumulation of products of metabolism
• Action of autolytic enzymes
• Putrefaction by bacteria
• All the above changes PREVENTED BY FIXATION!
• Principle : denaturation / precipitation of cell proteins , soluble component is
made insoluble
Effects of Fixative
The Ideal Fixative
• [A]
• Simple (one substance) Eg. Formalin
• Compound (two or more) Eg. Bouin’s solution, Zencker’s solution

• [B]
• Microanantomical – preserves anatomy
• Cytological – cytoplasmic and nuclear features
• Histochemical – constituents and enzymes
Commonly used fixatives
• Formalin – MC – routine
• Glutaraldehyde – electron microscopy
• Picric acid(Bouin’s solution) – renal & testicular tissue
• Alcohol(Carnoy’s fixative) – cytologic smears, endometrial sampling
• Osmium tetraoxide – CNS tissues & electron microscopy
Dehydration
• Water removed from tissue s and cells – this space is occupied by wax
• Tissue sent through grades of alcohol : 70%, 80%, 95% and absolute alcohol
• Ethyl (MC used), methyl, isopropyl alcohol or acetone can be used
Clearing
• Alcohol from tissues and cells is removed (dealcoholisation) and replaced by a
fluid in which wax is soluble – makes tissue transparent
• Examples of chemical used include
• Xylene (MC used)
• toluene
• benzene
• chloroform
• cedar wood oil
Impregnation
• Empty spaces in tissues and cells , after removal of
• clearing agent, are taken by molten wax
• Hardens the tissue – helps in section cutting
• Melting point of wax – 54- 62 degree C
Tissue Processor
• Dehydration + clearing + impregnation
• Automated tissue processor
• Open (hydraulic )
• Closed (vaccum)
OPEN / HYDRAULIC PROCESSOR
• 12 stations
• 1 jar – formalin
• 6 jars – grades of alcohol
• 3 jars – xylene
• 2 jars – molten paraffin wax
• Different processing fluids are moved in and out of a single station sequentially
• Embedding is done with molten wax
• Wax blocks
• Metallic L (Leuckahart’s) blocks
• Plastic moulds
Embedding machine
• Wax reservoir
• Heated area for steel moulds
• Wax dispenser
• Separate hot and cold plates
Sectioning
• Microtome – equipment
• Microtomy – technique
• 5 types of microtomes :
• Rotatory – MC used
• Sliding
• Freezing
• Rocking
• Base - sledge