ROUTINE STEPS (No.

2)
(For Soft Specimens)
DEHYDRATION
 Dehydration

• The process of completely removing water from

the intercellular and extracellular parts of the
tissue following fixation and decalcification.
• Done in preparation for impregnation and
embedding. Many of the embedding media are
not miscible with water.
• The second routine step if decalcification is not
done.
• Done by using reagent fluids or dehydrating
agents or by freeze-drying.
 How it is done?
• Tissue is passed through a series or succession
of increasing strength or up-graded
concentration of dehydrating agent, ranging
from 70 – 95%.
• Purpose: To gradually remove water content
from cells /tissues.
• Direct immersion into 85 – 95% dehydrating
agent will produce considerable shrinkage and
hardening of tissues leading to distortion.
How….?
 Dehydration
• Concentrated dehydrating agents tend to
harden only the surface of the tissue while the
inner parts are not completely penetrated.
This will result in a relatively unequal
impregnation of tissue which will lead to poor
cutting of sections.
 Dehydration
• The strength of initial concentration depends
on:
• 1. size of tissue
• 2. nature of tissue
• 3. fixative used
 Dehydration
• For routine work:
*Initial concentration: usually 70%

• Prolonged storage in lower concentration

(below 70%) tends to macerate the tissue.
 Dehydration
• Dehydration time will depend upon the size
and nature of the tissue.
• An interval of 1 hour immersion in each
concentration
• Smaller and delicate tissues will require
shorter time interval.
 Dehydration
Characteristics of Dehydrating Agents:
1. Must dehydrate rapidly without producing
considerable shrinkage and distortion of tissues.
2. Must not evaporate quickly
3. Should not harden tissue excessively
4. Should not remove stains
5. Should be able to dehydrate even fatty tissues
6. Should not be toxic to the body
7. Should not be a fire hazard
 Dehydration
• Ethyl Alcohol / ethanol
• Best and most recommended dehydrating
agent.
 Preparation of Ethyl Alcohol
Class Activity:
• Prepare 200 ml. 70% alcohol from absolute
ethyl alcohol
• Prepare 200 ml. 70% alcohol from 95% ethyl
alcohol
• Prepare 500 ml. of 80% alcohol from 90%
ethyl alcohol
Formula:
CdVd = CsVs
 Routine Step No. 3
Dealcoholization/Clearing
 DEALCOHOLIZATION/CLEARING
• The process whereby alcohol is remove
(dealcoholization) from the tissue and replace
by a substance or a reagent that will dissolve
the wax with which the tissue is to be
impregnated (ex. paraffin) or the medium on
which the tissue is to be mounted (ex. canada
balsam).
• The medium used in this process is referred to
as clearing agent, hence, clearing process.
 Dealcoholization
• When used after alcohol dehydration, the
clearing agent serves to mix with alcohol and
remove it from the tissue.
• When used after the tissue had been stained,
the clearing agent will make microscopic
tissue preparations transparent due to their
high index of refraction.
 dealcoholization
• Due to this property of ‘high index of
refraction’ which makes tissue transparent,
solutions utilized for alcohol removal are also
referred to as ‘clearing agents’.
• Not all clearing agents exhibit this property.
 Characteristics of a good clearing
agent
1. Should be miscible with…..
a) alcohol to promote rapid removal of the
dehydrating agent from the tissue
b) paraffin to facilitate impregnation
c) mounting medium to facilitate mounting of
tissue sections
2. Should not produce excessive tissue shrinkage and
hardening
3. Should no dissolve out aniline dyes
4. Should not evaporate quickly in a waterbath
5. Should make tissue transparent
 Xylol / xylene
• Commonly used clearing agent for routine
work.
• A true clearing agent (removes alcohol and
clears tissue specimens)
• Clearing time: 1 hour
 Other true clearing agents
1. Toluene
2. Benzene
3. Cedar wood oil

• The process requires 2 – 3 changes of xylol

 Routine Step No. 4
Infiltration/Impregnation
 Infiltration/Impregnation
• The process whereby the clearing agent is
completely removed from the tissue and
replace by a medium that will completely fill
all the tissue cavities, thereby, giving a firm
consistency to the specimen, and allowing
easier handling and cutting of suitably thin
tissue sections without any damage or
distortion to the tissue and its cellular
components.
 Impregnation /Infiltration
• Commonly used medium: Paraffin
• When placed into tissues, it fills all tissue
spaces or cavities, hence, called ‘impregnating
or infiltrating medium’.
• Requires 3 changes of infiltrating medium
carried out in a paraffin oven / incubator using
a high temperature so that the medium is in
its melted state.
 methods
1. Paraffin wax impregnation
2. Celloidin / Collodion impregnation
3. Gelatin impregnation
 Paraffin Wax Impregnation
• The simplest, most common and best method
used for routine work.
• Medium: Paraffin wax with melting point of
56°C is recommended
• Lab temperature:
*20 – 24°C: MP 54- 58°C
*15 – 18°C: MP 50 – 54°C
• Hard tissues requires higher MP of paraffin
wax than soft tissues.
 Paraffin wax impregnation
• In the past, admixtures were used such as
paraffin wax plus 10 – 20% beeswax to give
extra hardness in very warm environments.
• Paraffin wax does not deteriorate with age.
• Older paraffin wax shows less tendency to
crystallize during setting.
• Recommended for urgent biopsies, dense and
hard fibrous tissues, lungs spleen and other
delicate tissues.
 Ways of Paraffin Wax Impregnation
1. Manual Processing

• After complete clearing, tissue is immersed in

2 – 4 changes of melted paraffin wax at 15
minutes interval, either in a paraffin oven or
an incubator which has been regulated at
55 – 60°C.
• Purpose of 2 – 4 changes:
To ensure complete removal of clearing
agent from the tissue.
 Paraffin wax impregnation
GENERAL RULE:
• The paraffin oven temp. for routine work
must be 2 – 5°C above the MP of the
infiltrating medium
 Manual Processing
About 3 mm. thick tissue
Fixation 10% BNF 24 hours
DEHYDRATION 70% Ethanol 1 hour
80% 1 hour
90% 1 hour
95% 1 hour
95% 1 hour
Dealcoholization / Xylol I 1 hour
Clearing Xylol II 1 hour
Xylol III 1 hour
Impregnation / Paraffin Wax I 15 mins
Infiltration Paraffin Wax II 15 mins
Paraffin Wax III 15 mins
Paraffin Wax IV 15 mins
Embedding Paraffin Wax 3 hours
 Factors affecting paraffin wax
impregnation
• Total impregnation time generally depends upon the
following:
1. Nature and size of tissues
• Larger and denser tissue blocks require longer time
and more frequent changes of wax.
2. Types of clearing agents used
• Benzene and xylene are easily remove from tissues
• Chloroform and cedarwood oil are more difficult to
remove and require more frequent wax changes.
 Paraffin wax impregnation
Precautions:
• Paraffin oven must be maintained at a temp. 2-5°C
above the MP of paraffin wax.
• Paraffin wax must be pure and free from dust, water
droplets and other foreign substances.
• Fresh wax should be filtered before use in a wax oven.
• Re-used wax: heat to 100 - 105°C to remove water and
raising its MP.
• Trimmed away wax may be re-used by melting and
filtering (Greene’s No. 904 coarse filter paper)
• Paraffin wax maybe used only TWICE.
 Substitutes for paraffin wax
1. Paraffin group
a) Paraplast
• A mixture of highly purified
paraffin and synthetic plastic
polymers.
• MP: 56 – 57°C
• More elastic and resilient than paraffin wax.
• permits large dense tissue blocks such as bones,
brain to be cut easily with the same result as in
double embedding
substitutes…..
b). Embeddol
• A synthetic wax substitute similar to paraplast
• MP: 56 – 58°C
• Less brittle and less compressible than paraplast
c). Bioloid
• A semi-synthetic wax recommended for
embedding eyes.
d). Tissue Mat
• A product of paraffin containing rubber
• Same property as paraplast
substitutes…..
2. Ester Wax
• Lower MP: 46 -48°C but harder than paraffin
• Not soluble in water but soluble in 95%
ethanol and other clearing agents, hence can
be used for impregnation without prior
clearing of the tissue.
• A heavy duty microtome such as sliding
microtome or sledge-type microtome is
required for sectioning due to relative
hardness of wax.
substitutes…..
3. Carbowax
• A water soluble wax made up of polyethylene
glycols
• MP: 38 – 42°C and 45 – 56°C
• Contains 18 or more carbon atoms, which appear
solid at RT.
• Soluble and miscible with water, hence, does not
require dehydration and clearing of the tissue.
• Tissue is fixed, washed-out and transferred
directly into the melted carbowax.
• Suitable for enzyme histochemical studies.
carbowax…..
• Due to its water soluble properties
(hygroscopic nature), tissue sections are very
difficult to float-out.
• Floating-out of tissue sections maybe done
using the following solutions:
• 1. Pearse solution
• 2. Blank and McCarthy Mixture
 carbowax processing
• For routine processing: at 56°C
• 4 changes of carbowax: with agitation
• 70% - 30 min
• 90%- 45 min
• 100% - 1 hour
• 100% - 1 hour
 carbowax processing
• Done at 56°C with agitation
Fixation
Impregnation 70% Carbowax 30 min
90% Carbowax 45 min
100% Carbowax 1 hour
100% Carbowax 1 hour
Embedding Fresh Carbowax

• Embedding is done at 56°C and rapidly cooled

in a refrigerator.
 2. Automatic Processing
• Uses automatic tissue processor / automatic
tissue processing machine.
• Automatically fixes, dehydrates, clears and
infiltrates tissues.
• With constant tissue agitation
Automatic Tissue Processor
 Automatic tissue processor
• Miles Tissue-Tek VIP 3000 Bench-To Model
• This automatic tissue processor provides ten
programs for fixing, dehydrating, clearing and
paraffin impregnation
 3. Vacuum Embedding
• Involves wax impregnation under negative
atmospheric pressure inside an embedding
oven.
 Vacuum Processing
• Time required for complete impregnation is
reduced from 25 – 75% of the normal time
required for tissue processing
• Particularly recommended for urgent biopsies,
for dense and hard fibrous tissues and delicate
specimens (lungs, spleen, eyes).
 Timetable Comparison
Paraffin Manual Tissue Automatic Tissue Vacuum Tissue
Processing Processing processing

32 hours 18 hours 5 hours

 2. Celloidin / Collodion Impregnation
• Celloidin / collodion is a purified form of
nitrocellulose soluble in many solvents.
• Suitable for specimen:
1. with large hollow cavities which tend to
collapse
2. hard and dense tissues
3. for large tissue sections ie. Whole embryo
celloidin….
• Used with 4 percentage preparations:
• Thin: 2 - 4%
• Medium: 4 - 6%
• Thick: 8 - 12 %
• Dissolved in equal parts of ether and alcohol
• Methods:
1. Wet celloidin method
2. Dry celloidin method
 Wet Celloidin Method
• Recommended for bones, teeth, large brain
sections n whole organs.
• Thick preparation (8%-12%) is used as embedding
medium.
• Dehydrating agent: equal parts alcohol-ether
mixture.
• 2-4% impregnation: 5 - 7 days
• 4-6%Impregnation: 5 - 7 days
• 8-12% impregnation: 3 - days
 Dry Celloidin Method
• Recommended for processing whole eye
sections
• Differs from wet method in storage of
specimen:
• Wet method: alcohol-ether mixture in a
dessicator
• Dry method: Gilson’s Mixture (equal parts of
chloroform and cedarwood oil)
 Nitrocellulose Method
• Uses low viscosity nitrocellulose ( LVN)
• Another form of cellolidin soluble in equal
parts of ether and alcohol.
• Forms harder tissue block and makes cutting
of thinner sections possible.
• the tendency of tissue to crack may be
prevented by adding plasticizers (oleum ricini
or castor oil when embedding chrome-
mordanted tissues.
 Nitrocellulose method
• more explosive than ordinary celloidin, so
must be handled with care.
• When dry, striking or dropping will cause
explosion.
• Marketed wet with alcohol.
 4. Gelatin Impregnation
• Rarely used except when dehydration is to be
avoided and for enzyme studies.
• Used as an embedding medium for delicate
specimens and frozen tissue section.
• Used in 2 percentage preparations:
• 10% with 1% phenol (prevent mold growth)
• 20% with 1% phenol (same % for embedding)
• Size to tissue block: not more than 2-3 mm thick
 Impregnation
• Ratio between impregnating medium and
tissue specimen:
• 25 times the volume of tissue specimen
Routine Step No. 5
Embedding
 Embedding
• Also referred to as “tissue blocking” or “tissue
casting”.
• The process by which the impregnated tissue
is placed in a precisely arranged position
(proper orientation) in a molder wherein
embedding medium is added until a tissue
block is formed (by allowing embedding
medium to solidify).
 Embedding: General Rule
• Embedding medium must be the same as the
one used in impregnation.
• Paraffin to paraffin, celloidin to celloidin,
gelatin to gelatin except in double embedding.
• Uses tissue molders or blocking-out molders.
 Tissue Molders
1. Leuckhart’s Embedding Molder
• Two L-shaped strips of heavy brass or metal.
 Tissue molders
2. Compound Embedding unit
• Ice trays either plastic or metal
 Tissue Molders
3.Plastic embedding rings and base molders
 Tissue molders
4. Disposable Embedding molders
4.a) Peel-away molders
 Tissue molders
4.b) Paper boats
• Cheap and easy to make
• For celloidin and paraffin blocks
 embedding process
• Before embedding, all the necessary materials
must be prepared: molder, pencil, strip of paper
for tissue code, alcohol lamp or burner.
• Two ways of embedding:
1. * After impregnation, the tissue is oriented at
the bottom of the mold and fresh embedding
medium is poured unto the molder.
*Place strip of paper with
tissue code on one side
of the molder.
 embedding process
2. *After impregnation, a fresh embedding
medium is poured into a molder, then the
infiltrated tissue is immediately oriented at
the bottom particularly at the center of the
molder.
* place the tissue code
tissue block formation
 Types of Embedding Method
1. Paraffin embedding method
• Most commonly requested
2. Celloidin / Nitrocellulose Method
• For hard tissues
3. Double-embedding Method
• Tissues are first infiltrated and embedded in
celloidin and subsequently embedded in
paraffin wax.
• For dense, firm tissues (brain)
4. Machine embedding Method
Embedding Machines
summary: embedding
 Routine Step No. 6
Trimming *(optional)
 Trimming (6)
• The process of cutting-off the excess wax on
all sides (sides, top and bottom) of the tissue
block and the sharp or pointed edges of the
block until all sides are perfectly level and
parallel, almost to the edge of the tissue
forming a ‘four-sided prism’ or ‘truncated
pyramid’.
• Distance from the edge of the trimmed block
to the tissue is about 2 mm.
 Precautions in trimming
1. do not trim near the tissue
2 . Only thin slices are taken out at a time to
prevent tissue block from cracking.

NOTE: trimming is not done in molders with

rounded ends.
 Materials for trimming
1. Ordinary kitchen knife
2. Sharp razor blade
3. Microtome knife
4. Scalpel

End…