CSI 102
Histological Technique
Tissue
Processing
Prepared by :
MUHAMMAD NURHAQEEM BIN ADAM
(2022845832)
MUHAMMAD ARAB HABIB BIN HAIRI
(2022610372)
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Topic Outline
INTRODUCTION PRINCIPLES
PROCEDURES PROBLEMS , CAUSES
& HOW TO SOLVE IT
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Introduction
This e-Book is explaining about the tissue processing
process which was learnt in a Histological Techniques.
Tissue processing is a crucial step in histological
techniques which to make the tissue more observable
because it embed the tissue in solid medium so that it
will be easier to be sectioned in the next step which is
tissue sectioning. This e-Book will be enlightened the
reader about principles of tissue processing,
procedures in the tissue processing process, and the
problems that will be encountered, causes and the
solutions. 3
Definition Importance
the process of To embed tissues in
impregnating fixed tissues solid medium
with the solid medium to To support the tissues
facilitate the production of To enable the knife to
microscopical sections and cut the section with
consists of three sequential little damage to knife
steps which are or tissues
dehydration, clearing, and
infiltration.
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PRINCIPLES OF TISSUE PROCESSING
Dehydration Clearing
Removal of water and Removal of dehydrating
fixative from tissue solutions, allow tissue
receptive to the
infiltrating medium, to
make tissue transparent
Infiltration & Embedding
impregnation
Orientating the tissue in a
Permeating the tissue with a support medium and allowing it
support medium
to solidify
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Dehydration Dehydrating agent
Method of dehydrating 1.ETHANOL 2.Methylated spirit 3.METHANOL
Tissues are passed through a Clear, colorless, Same Physical properties Consist of ethanol,
series of increasing flammable, hydrophilic, as ethanol methanol (1%),
concentrations of alcohol . miscible with water and isopropyl alcohol or
other organic solvents, fast Consist of ethanol, a combination of
acting and reliable methanol (1%), isopropyl alcohols
alcohol or a combination of
Suitable for electron alcohols
microscopy specimen and
routine procedure
4.acetone 5.butanol
Clear, colorless, Plant and animal histology
flammable, miscible with Slow dehydrant
water, ethanol and most Less shrinkage and
organic solvents hardening of tissue
Rapid in action
70 % alcohol 90 % alcohol 100 % alcohol 100 % alcohol Poor penetration
Cause tissue brittleness
(prolonged)
Remove lipid from tissue
during processing
With one change in each concentration
Duration of one hour in each
Keep covered to avoid evaporation
Tissue is immersed in alcohol for one hour
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CLEARING
Objectives 1.xylene 2. toulene 3.chloroform
The removal of dehydrating agents
from tissue Flammable, colorless, Similar properties to xylene Slower in action than
To make the tissue transparent miscible with most organic Less damaging (prolonged) xylene, less brittleness
solvents and paraffin More flammable and Block thickness greater
Method of clearing Routine histology, volatile than xylene than 1 mm
recyclable Tissue do not become
Tissue must less than 5 mm translucent
in thickness Non-flammable, highly
Over exposure – over- toxic,
hardening Heated form release
phosgene gas
Clearing agent For specimens of central
nervous system
4.Methyl benzoate
5.Citrus fruit oils
Slower in action
Double embedding techniques Extracts of orange and lemon
rinds
Xylene Xylene Xylene Non toxic, miscible with water
1 hour Strong pungent odor
1 hour 30 minutes 1 hour 30 minutes Dissolve minerals (copper,
calcium) in small tissue
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INFILTRATING AND Consequences
IMPEGRANATION
INADEQUATE IMPREGNATION -
Infiltration lead to drying and shrinking of
tissues
Clearing agent (xylene) is eliminated from tissues by
diffusion HIGH TEMPERATURE - harden
the tissues
Impregnation
INSUFFICIENT IMPREGNATION -
Wax diffuses into the tissue to replace the clearing develop crack and crumble
agent
Medium
Melted paraffin wax
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PRmaOuCatEnoDumaUalRteEd
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Manual automated
10% formalin 60°C 20min 1.4% formal saline 2 hrs porRvoeocruentsiisngihentg
Fresh acetone 20min 2.4 % formal saline 2 hrs
Fresh acetone 20min 3.70 % alcohol 1 hr 1. Formal saline
Fresh acetone 20min 4.90 % alcohol 1 hr 2.Formal saline
Xylene 10min 5.Absolute alcohol 1 hr 3.70% alcohol
Xylene 15min 6.Absolute alcohol 2 hrs 4.90% alcohol
Wax 30min 7.Absolute alcohol 2 hrs 5.100% alcohol
Wax 60min 8.Xylene 1 hr 6.100% alcohol
9.Xylene 11/2 hrs 7.100% alcohol
10.Xylene 11/2 hrs 8.Toluene 15min
11.Wax 2 hrs 9.Toluene 15min
12.Wax 3 hrs 10.Toluene 15min
11. Wax 15min
prosDcpaseeymscsitamiinlmlegenfor 12. Wax 15min
15min
15 min
15min
15min
15 min
30min
30min
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dehydrating clearing infiltration
Acetone 70% & impregnation
20 minutes
The tissue diffuses into melted
Acetone 80% paraffin wax
20 minutes 30 minutes
Acetone 100% Paraffin
20 minutes
Xylene Xylene
10 minutes 15 minutes
prewarming
chamber for
moulds
The tissue will The tissue kept in prewarming
become chamber and ready for embedding
transparent section
60 minutes
manual procedure 11
By using forceps , transfer all the casettes Insert and align the tissue processor organiser Place the tissue processor basket lid on top of
containing cutted tissue specimen into the tissue baskets into the basket hanger the organiser baskets
processor organising basket
Open the tissue processor operating head By using the hand-held controller,ensure each Press the "Auto Start" key to begin the tissue
door.Firmly attach the basket hanger into the steps for the processing time are correctly processing cycle.Close the operating head door
programmed
operating head slot
12 automated procedure
Notes:
Most automated tissues processor have 12 containers processing cycle
Some machine apply heat & vacuum to increase the rate of processing
PROBLEMS CAUSES SOLUTION
Shrinkage, Overheated Care must be taken
brittleness & not to overheat the
difficultiness in Inadequate tissues
sectioning Impregnation
Soak the tissue into
Tissue is cracked the right amount of
and crumble melted paraffin wax
Tissue is drying Insufficient Beakers and wax
and shrinking impregnation bath must be filled
to the correct fluid
level
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The tissue has Using the used Changing of
foreign particles and solution solution after
fragments using them for 2-3
The broken socket days
The machine is is used or
broken and not electrical Do a checkup at the
working disruption electric socket and
make sure it is not
Tissue is unfit to be Insufficient broken
observed due to amount of the
shrinkage and solutions Make sure to
brittle volume arrange the tissue
specimen in its rack
14 without any excess
Conclusion Srceafnerfeonrcoeusr
To sum up, tissue processing is an important process in a
histological techiniques that consist of four principles
which is dehydration , clearing, infiltration and
impregnation, and embedding. There is two method which
is automated tissue processing using the tissue processor
and manual method if the lab have no tissue processor.
Tissue processing objective is to make the tissue easier to
cut because the tissue is infiltrated with support medium
that is wax.
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