This document describes the process of tissue processing for microscopic examination. Tissue samples are first fixed in formalin to prevent decomposition. They are then dehydrated by passing through increasing concentrations of alcohol, cleared using xylene to remove the alcohol, and infiltrated with paraffin wax. The tissues are embedded in wax blocks and sectioned using a microtome. The sections are stained, most commonly with hematoxylin and eosin, which stain nuclei purple and cytoplasm pink. This allows examination under a microscope.

1. TISSUE PROCESSING

2. AT MICROSCOPIC LEVEL-
 HISTOLOGY
 Science of examination of normal tissues
 HISTOPATHOLOGY
 Examination of tissues for presence / absence of
changes in structure due to disease process

3. What happens to the SPECIMEN?
 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

7. TISSUE PROCESSING
1. Fixation
2. Dehydration
3. Clearing
4. Impregnation
5. Embedding and blocking
6. Section cutting
7. Routine staining

8. 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!

9.  Tissue get fixed in complete physical and partial
chemical state
 Principle : denaturation / precipitation of cell
proteins , soluble component is made insoluble

10. Fixatives produce the following effect…

11. IDEAL FIXATIVE

12. TYPES OF FIXATIVES
 [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

13. 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

19. 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

20. CLEARING
 Alcohol from tissues and cells is removed
(dealcoholisation) and replaced by a fluid in which
wax is soluble – makes tissue transparent
 Xylene (MC used) , toluene, benzene, chloroform,
cedar wood oil can be used

21. 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

22. TISSUE PROCESSOR
 Dehydration + clearing + impregnation
 Automated tissue processor
Open (hydraulic )
Closed (vaccum)

23. OPEN / HYDRAULIC PROCESSOR
 12 stations
 1 jar – formalin
 6 jars – grades of alcohol
 3 jars – xylene
 2 jars – molten paraffin wax

25. CLOSED / VACCUM PROCESSOR
 Different processing
fluids are moved in
and out of a single
station sequentially

26. EMBEDDING & BLOCKING
 Embedding – with molten wax
 Wax blocks –
 Metallic L (Leuckahart’s) blocks
 Plastic moulds

28. Embedding centre
 Wax reservoir
 Heated area for steel
moulds
 Wax dispenser
 Separate hot and cold
plates

32. SECTION CUTIING
 Microtome – equipment
 Microtomy – technique
 5 types of microtomes :
1. Rotatory – MC used
2. Sliding
3. Freezing
4. Rocking
5. Base - sledge

36. ROUTINE STAINING (H&E)
 Haematoxylin – nuclear stain
 Eosin – cytoplasmic stain
 Mounted in DPX/Canada balsm
 End result :-