•ALBERTO •AUSTRIA •CHAN •MARCAYDA •STA ANA •RAMOS •UY

FIXATION:FORMALIN DEHYDRATION CLEARING

IMPREGNATION EMBEDDING TRIMMING
SECTIONING STAINING MOUNTING
1. Autopsy
2. Biopsy
•Loss of body supply and O2
•Accumulation of products of
metabolism
•Action of autolytic enzymes
•Putrefaction by bacteria
 Tissue specimen received in the
surgical laboratory have a request form
that list the patient information and
history along with a description of the
site of the origin
 The specimen will be identified by their
accesssion number
• It is the process in which a
specimen is treated by exposing
it to a fixative for a particular
period of time

• purpose: to preserve tissues

• principle:
denaturation/precipitation of cell
proteins, soluble component is
made insoluble
Fixative: Formalin
A fixative produce the following
effects:

1. Prevents petrification and autolysis

2. Hardens the tissue which helps in

section cutting

3. Makes cell insensitive to hypotonic

or hypertonic solutions

4. Acts as a mordant

5. Induces optical contrast for good

morphologic examination
Advantages:

• Rapid penetration

• Easy availability and cheap

• Does not over harden the tissue

• Fixes lipids for frozen sections

Disadvantages:

• Irritant to the nose, the eyes, and

mucous membranes

• Formation of precipitate of
paraformaldehyde which can be
prevented by adding 11-16%
methanol

• Formation of black formalin

pigment
• Process in which the water
content is completely reduced by
passing the tissue through
increasing concentrations of
dehydrating agents

 Dehydration is done so that the

wax i.e. Paraffin wax, which is
used for impregnation, can be
easily miscible as it is immiscible
by water
• The procedure wherein the
alcohol in the tissue is replaced
by a fluid which will dissolve the
wax for impregnating the tissues

 Dealcoholisation
 The various clearing agents used
are:

• Cedar wood oil: The best agent

but is expensive

• Benzene: It is carcinogenic

• Xylene: It is most commonly used

• Chloroform: Toxic and expensive

1. Cedar wood oil:
• Clearing time: indefinite until clear
-for central nervous system, skin, smooth
muscle and cytological work

2. Benzene:
• Clearing time: 15-60 mins
-best used for urgent biopsies

3. Xylene:
• Clearing time: 15-30 mins
-for urgent biopsies

4. Chloroform:
• Clearing time: 6-24 hours
-suitable for large tissue specimens, tough
tissues, nervous tissues, lymph nodes and
embryos
• The tissue is kept in a wax bath
containing molten paraffin for 6-
8 hours (empty spaces in tissues
and cells, after removal of
clearing agent, are taken by
molten wax)

• Melting point of wax: 54-65 C

 Hardens the tissue- helps in

section cutting
 The various waxes which can be
used are:

• Paraffin wax

• Paraplast

• Paraplast plus

• Gelatin

• Celloidin
• It is done by transferring the
tissue which has been cleared of
the alcohol to a mold filled with
molten wax & is allowed to cool
and solidify

• After solidification, a wax block

is obtained which is then
sectioned to obtain ribbons
Embedding
Embedding
• Purpose: to create an even, flat
surface in the area of interest in
the tissue so that histologist to
not have to face (cut with the
microtome) into the paraffin
block as deeply when trying to
get the first good sections for a
slide
• It is the procedure in which the
paraffin blocks which have been
prepared are cut or sectioned
and thin strips of varying
thickness are prepared

 Microtome:
Equipment/instrument

 Microtomy:
Technique/method
Sectioning
• Cells are virtually colorless and
so must be stained to be
visualised.
• It is done to highlight particular
features of the tissue as well as
to enhance the tissue contrast
• Most commonly used stain in
routine practice is Hematoxylin
and Eosin stain
• Hematoxylin and Eosin staining is used
routinely in histopathology as it provides
the pathologist/researchers a very
detailed view of the tissue.
• It achieves this by clearly staining cell
structures including the cytoplasm,
nucleus, and organelles and extra
cellular components.
• This information is often sufficient to
allow a disease diagnosis based on the
organization (or disorganization) of the
cells and also shows abnormalities or
particular indicators in the actual cells.
• Hematoxylin
-Reacts like a basic dye with a purplish
blue color. It stains acidic, or basophilic,
structure including the cell nucleolus
(which contains DNA and nucleoprotein),
and organelles that contain RNA such as
ribosomes and the rough endoplasmic
reticulum.
• Eosin
-an acidic dye that is typically reddish or
pink. It stains basic, or acidophilic,
structures which includes cytoplasm, cell
walls, and extra cellular fibers.
• The Embedding process must be reversed
in order to to get the paraffin wax out of
the tissue and allow Water soluble dye ss
to penetrate the sections. Therefore
before any staining can be done , the
slide are “deparaffinized” by running
them through xylenes(removes wax) to
alcohols(removes xylene) to water.
• Deparraffinization with xylene – Rinse in
xylene for 10 dips to remove wax

• Then rinse in graded alcohol (70%,80%,95%)

for 5 dips to remove xylene

• Stain rehydrated sections in Hematoxylin

solution for 20-40 minutes

• Wash in tap water for 1-5 minutes , until

sections turn blue

• Differentiate sections in 70% ethanol-

containing 1%HCl-for 5 seconds. This
removes excess dye, allowing nuclear details
to emerge.
• Wash 1-5 minutes in tap water until blue *Note the use of tap
• Stain in Eosin solution for 10 minutes water in washing steps—
• Wash 1-5 minutes in tap water tap water provides the
• Dehydrate, Clear, and Mount alkalinity necessry for
the bluing process.
Staining
 Adhesives used for fixing the
sections on the slides:
• Albumin solution (Meyer’s egg
albumin)
• Starch paste
• Gelatin

 Mountants:
• DPX
• Canada Balsam
• Colophonium Resin
• Terpene resin
Mounting
Mounting