The document discusses the process of tissue processing which involves fixing, dehydrating, clearing, infiltrating, embedding, sectioning, staining, and mounting tissues for microscopic examination. Key steps include fixation to preserve tissue structure, dehydration to remove water, clearing to make tissue permeable to paraffin wax, infiltration and embedding in paraffin wax to harden tissue for sectioning, sectioning with a microtome to produce thin slices, staining for contrast, and mounting slices on slides for microscopic analysis. The goal is to histologically examine tissues at the microscopic level.

1. Tissue Processing
Dr. Muhammad Fakhar E Adil
D.V.M., M. Phil.

2. Introduction
Histology: Microscopic study of body tissues.
Histopathology: microscopic study of diseased tissue
Tissue source
Biopsy
Necropsy
The techniques for processing the tissues, whether
biopsies or tissues from necropsy so as to enable the
scientist to study them under the microscope

3. Source of Tissues
(human; animal)
Biopsy Removal of tissues Autopsy/Necropsy
Freezing Fixation
Cryomicrotomy Washing
Mounting Dehydration
Histochemical reactions Clearing
Fixation Infiltrating & Embedding
Staining or Counterstaining Microtomy (sectioning)
Cover slipping Mounting
Staining
Cover slipping
Microscopy
Photomicrography
Digital micrography

4. Protocols for paraffin sectioning
1. Receipt & Identification
2. Labeling of the specimen with numbering
3. Fixation
4. Dehydration
5. Clearing
6. infiltration
7. Embedding
8. Section cutting
9. Staining
10. Mounting

5. Specimen identification
 Patient information
 History
 Laboratory code (numbers that will identify each specimen for each
patient)

6. Fixation
 Preserve tissues permanently in as life-like a state as possible
 The fixative should be 15 – 20 times more in volume then the specimen
Aims
• Prevent autolysis
• Penetrate evenly and rapidly
• Harden specimen
• Antisepsis of the sample
• Increase optical density
Ideal Fixative
• Should not cause shrinkage or
swelling of cells
• Must not interfere with staining
• Cheap and easily available
• Should be good antiseptic
• Safe to use

7. Fixation
 The bits should of the size of approximately 1 cm3
 These bits are then placed in metal cassettes or capsules
which are then placed in the fixative

8. Fixatives
 10% Formalin
 10 ml formalin in 90 ml distilled water
 MOA – forms cross links between amino acids of proteins thereby
making them insoluble
 Advantages – rapid penetration, available & cheap, does not over
harden tissue, ideal for mailing
 Disadvantages – irritant, forms paraformaldehyde precipitates,
formation of black formalin pigment , acid formaldehyde hematin

9. Fixatives
 Other fixatives
1. Glutaraldehyde
2. Osmium tetraoxide
3. Potassium dichromate
4. Mercuric chloride
5. Picric acid
6. Zenker’s fluid
7. Zenker’s Formal (Helly’s fluid)
8. Bouin’s fluid

10. Fixatives
 Microanatomical fixatives:
 These are used to preserve the anatomy of the tissue.
 E.g. 10% formal saline (brain), buffered formalin
 Cytological fixatives:
 These are used to fix intracellular structures.
 E.g. nuclear fixatives (Carnoy’s fluid, Clarke’s fluid), cytoplasmic fixatives
(Champy’s fluid, Regaud’s fluid)
 Histochemical fixatives:
 These are used to demonstrate the chemical constituents of the cell.
 E.g. formal saline, cold acetone, absolute alcohol

11. Decalcification
 Removal of the calcium salts from the specimen
 Nitric acid, formic acid, picric acid, acetic acid, citric acid,
hydrochloric acid

12. Dehydration
 Process in which the water content in the tissue to be processed is
completely reduced by passing the tissue through increasing
concentrations of dehydrating agents
 E.g. Ethyl alcohol, Acetone, Isopropyl alcohol, Dioxane
 Dehydration is done so that the wax i.e. Paraffin wax can be easily
infiltrated in tissue as it is immiscible with water

13. Clearing
 The procedure where in the alcohol in the tissue is replaced
by a fluid which will dissolve the wax used for impregnating
the tissues
 Cedar wood oil : The best agent but is expensive.
 Benzene : It is carcinogenic.
 Xylene : It is most commonly used.
 Chloroform: Toxic and expensive

14. Infiltration
 In this the tissue is kept in a wax bath containing molten
paraffin wax for 6 – 8 hours.
 The wax is infiltrated in the interices of the tissue which
increases the optical differentiation & hardens the tissue &
helps in easy sectioning of the tissue.
 Paraffine wax
 Paraplast
 Paraplast plus
 Gelatin
 Celloidin

15. Embedding
 It is done by transferring the tissue which has been cleared of the alcohol to a
mould filled with molten wax & is allowed to cool & solidify.
 After solidification, a wax block is obtained which is then sectioned to obtain
ribbons.

16. Section cutting
 The procedure in which the blocks which have been prepared are cut or sectioned
and thin strips of varying thickness are prepared
 The instrument by which this is done is called as a Microtome
 Types of microtomes:
 Sliding
 Rotary
 Rocking
 Freezing
 Base sledge

17. Rotary microtome
 It is the most commonly used.
 Also known as Minnot’s Rotary microtome.
 In this the Block holder moves up and down while the knife remains fixed.
 It is suitable for cutting of small tissues & serial sections can be taken on it.

18. Parts of a Microtome (Rotary)
 Block holder
 Knife clamp screws
 Knife clamps
 Block adjustment
 Thickness gauge
 Angle of tilt adjustment
 Operating handle.

19. Tissue flotation bath
 It is a thermostatically controlled water bath with the inside colored black.
 It is maintained at a temperature maintained 5 – 6 degree below the melting point
of paraffin wax.

20. Staining
 Staining of the section is done to bring out the particular details in the tissue under
study
 The most commonly used stain in routine practice is Haematoxylin & eosin stain
 Nucleus – blue, cytoplasm – pink

21. Mounting
 Adhesives used for fixing the sections on the slides :
 Albumin solution (Mayor’s egg albumin)
 Starch paste
 Gelatin
 Mountants :
 DPX ( Distrene Dibutyl phthalate Xylene ).
 Canada Balsam
 Colophonium resin
 Terpene resin

23. "On the earth are Signs for those of
assured Faith; as also in your own selves:
And in yourselves. Then will you not see?"
(al-Dhariyat 51:20 - 21)