The document outlines the steps of tissue processing in pathology, detailing the aims and principles behind achieving sufficient rigidity for microscopic study. Key processes include dehydration, clearing, and embedding, with emphasis on the selection of appropriate agents and conditions that influence tissue processing quality. Factors affecting processing efficiency include tissue size, agitation, heat, viscosity, and vacuum application.

1. STEPS OF TISSUE
PROCESSING
DR.R.VENKATESH
PATHOLOGY PG 1ST
YEAR
MMCHRI

2. AIMS AND PRINCIPLE
•Aim: The basic aim is to provide sufficient
rigidity so it can cut into thin section for
microscopic study.
•Principle: Is to remove the water within the
tissue and impregnated with another
medium.

3. ESSENTIAL STEPS
Dehydration
Water molecule
is removed from
tissue
Clearing
Dehydration
agent is replaced
by clearing agent
Impregnation
Tissue is
infiltrated with
supporting
medium

4. FACTORS THAT INFLUENCE
TISSUE PROCESSING
•Size of the tissue sample: The smaller size, the
better processing
•Agitation: Facilitate the contact of tissue with
fresh solution

5. •Heat: Increase the better penetration
•Viscosity: The higher viscosity, lower the penetration
•Vacuum: Negative pressure to remove trapped air in
the tissue

6. DEHYDRATION
•Every tissue contains some amount of free or unbound
water molecules
•It is necessary to remove the water from tissue
•The sharp concentration gradient between the tissue
and the dehydration agent may cause rush of fluid, and
this may damage

7. •So the dehydration should be done gradually from low
concentration to the high concentration
•The tissue should kept in dehydration solution for
optimum time
•Too little time may insufficient to remove the water
molecules.

8. DEHYDRARTING AGENTS
•Ethanol alcohol
•Methylated spirit
•Methanol
•Butyl alcohol
•Isopropyl alcohol
•Other than alcohol: dioxane, ethylene glycol and
acetone.

9. CLEARING
•It’s the next step, after removal of water molecules
from the tissue
•The clearing agent should miscible with both
dehydrating agent and the embedding medium
•The refraction index of clearing agent is similar to the
tissue

10. SELECTION OF THE APPROPRIATE
CLEARING AGENT :
•Type of tissue
•Type of processor
•Processing condition
•Speed of removal of dehydrating agent
•Ease of replacement by molten wax
•Safety factors.

11. CLEARING AGENTS
•Xylene
•Toluene
•Chloroform
•Esters
•Cedarwood oil

12. PROPERTIES XYLENE TOLUENE CHLOROFORM ESTERS
TISSUE
SHRINKAGE
Yes Yes Minimum No
TISSUE
HARDENING
Yes No No No
INFLAMMABLE Yes Yes No Yes
HARMFUL
EFFECT
Irritant but less
harmful
Irritant Dangerous toxic gas
is liberate
Safe
COST Cheap Cheap Very expensive High cost

13. INFILTRATION AND EMBEDDING
•This is the next step after clearing
•The tissue space is infiltrate embedding medium
•Usually molten wax is used
•After cooling in room temperature, the molten wax is
solidified to provide support for thin section cutting

14. IMPREGNATING MEDIUM
An ideal impregnating medium should have following
qualities
• Miscible with clearing agent
• Liquid in higher temperature and solid in room temperature
• Homogenous and stable
• Non toxic and cheap
• Transparent
• Fit for section cutting

15. THE TIME AND NUMBER OF CHANGES
DEPENDS ON
• Size of the tissue
• Type of the tissue
• Type of the clearing agent
• Type of the processing

16. TISSUE PROCESSING METHODS
MANUAL
•Tissue transfer processor
•Fluid transfer processor
AUTOMATIC

17. TIME SCHEDULE FOE OVERNIGHT

18. THANK YOU