TISSUE PREPERATION
Prelims
BSMT – 2B GIESSAN MAE LABRADO
TISSUE PROCESSING FIXATIVE PRODUCE THE FOLLOWING EFFECT
• terminate cell metabolism;
• prevent enzymatic degradation of cells and
tissues by autolysis (self-digestion);
• kill pathogenic microorganisms such as
bacteria, fungi, and viruses; and
• harden the tissue as a result of either
cross-linking or denaturing protein
molecules.
COMMONLY USED FIXATIVES
A crucial series of steps that prepares
biological tissues for microscopic examination. This FORMALIN
involves, dehydration, infiltration and embedding. • A 37% aqueous solution of formaldehyde will
Proper tissue processing is essential for maintaining preserve the general structure of the cell and
cellular details and ensuring accurate interpretation extracellular components by reacting with the
under a microscope for diagnosis and research. amino groups of proteins.
CHRONOLOGICAL STEPS • Does not react with lipids, it is a poor fixative
of cell membranes
1. Fixation
2. Dehydration OSMIUM TETROXIDE
3. Clearing
• Used as a secondary fixative
4. Infiltration
• It is valuable in electron microscopy because it
5. Embedding
enhances the contrast of lipid-containing
6. Sectioning
structures, such as cell membranes
7. Staining
8. Microscopy
DEHYDRATION
FIXATION
- a series of alcohol solutions of ascending
concentration as high as 100% alcohol to
remove water
It is a mixture of chemicals,
permanently preserves the
tissue structure for subsequent
treatments. Specimens should
be immersed in fixative
immediately after they are
removed from the body.
TISSUE PREPERATION
Prelims
BSMT – 2B GIESSAN MAE LABRADO
CLEARING • The infiltrated tissue is then embedded in a
block of the embedding medium.
• This provides the rigidity and support needed
The next step alcohol should be replaced by for sectioning.
paraffin wax. As paraffin wax is not alcohol soluble,
we replace alcohol with a substance in which wax is SECTIONING
soluble. This step is calls clearing.
- An organic solvent such as xylol or toluol, are
miscible in both alcohol and paraffin,
- This are used to remove the alcohol before
infiltration of the specimen with melted paraffin
CLEARING REAGENTS
1. Xylene (commonly use)
2. Chloroform
3. Benzene • The embedded tissue is then cut into very
4. Carbon tetrachloride thin slices, called sections, using a
5. Toluene microtome.
• These sections are typically a few
IMPREGNATION micrometers thick, allowing light to pass
through for microscopic examination
• The tissue is STAINING
permeated with the
embedding medium.
• Paraffin wax is
commonly used, but
other media like resins
are also employed.
• This provides support
to the tissue, allowing
it to be thinly sectioned
• Occur at melting point
temperature of
paraffin wax, which is • Staining involves applying dyes to the
54-60oC sections to enhance contrast and
distinguish between different cellular
components.
EMBEDDING • Hematoxylin and eosin (H&E) is a common
staining method
• The infiltrated tissue is o Hematoxylin stains cell nuclei
then embedded in a blue/purple
block of the embedding o Eosin stains the cytoplasm and
medium. extracellular matrix pink
• This provides the
rigidity and support
needed for sectioning.
TISSUE PREPERATION
Prelims
BSMT – 2B GIESSAN MAE LABRADO
MICROSCOPY
The prepared tissue sections are then
examined under a microscope. Light microscopes are
commonly used to visualize the stained tissue
structures.
