LESSON 2: TISSUE PROCESSING:
mitochondria
FIXATION:
 Process adopted to kill, harden and
preserve tissue materials (from destruction
and post-mortem changes)
 Basically process adopted to kill, harden
and preserve for microscopic study
EFFECTS OF FIXATION
 Denaturation of proteins rendering them
insoluble.
o It gives them a gel-like state into
semi-solid state
 Tissue become resistant to the effect of
subsequent technical processing
o That actually can cause damage or
distortion.
 Staining will be strongly influenced because
it will act as a mordant or accentrator
o Note: some fixatives have the
disadvantages of inhibiting or
interfering with dye reactions.
o Formalin – intensify
o Osmium tetroxide – would actually
inhibit hematoxylin stain.
 Inhibits bacterial decomposition
 Soft and friable tissues are hardened
making handling and cutting easier
 Optical differentiation of cells and tissue
components is increase, rendering them
more visible during examination.
 Safer handling & processing
o Thus reducing the risk of infection
EFFECTS OF AUTOLYSIS
 AUTOLYSIS – self-digestion, refers
destruction of self through their own
enzymes. Digestion of an enzyme by an
molecule of the same enzyme.
o When tissue is remove it will
undergo autolysis, that’s why it need
to be fixed to preserve the
morphology and to keep the
antigenicity or the RNA structure of
your tissue
o Under fixation can result to bad
morphology
o Over fixation – can result to
masking of the antigen or strong
nonspecific background staining
 Disappearance of the nuclei
 Cytoplasm becomes cloudy and losing its
staining affinity
 Surface of the cell are lost by desquamation
 Fine intracellular structures are lost like
 Disintegration – putrefaction
 Bacterial growth – decomposition
o Saprophytic bacterial growth
FIXATION REQUIREMENTS
 Tissue size:
o 2 cm2/10mm X 4 mm thick (light
microscopy)
o 1 – 2 mm2 (electron microscopy)
o For LUNG EDEMA
 1 -2 cm thick
 Volume of fixative: routine 10 – 25 times
the volume of the specimen.
o But for museum 50 -100 X the
volume of the specimen
 Osmium tetroxide – 5 – 10 X of the
volume
 Museum: Not <50 – 100 X
 TISSUE CONTAINER: adequate in size,
wide mouth and non-rusting.
TISSUE/ORGAN PREPARATION:
 Hollow organs should be packed with
cotton soaked in fixative before immersion
or dilate with cotton soaked in fixative
 AIR-FILLED LUNGS – must be covered
with several layers of gauze.
 HUMAN BRAIN – should be washed with
ringer’s lactate (intravascular perfusion),
fix first before sampling.
o This is usually suspended by a cord
tied in the circle of willis.
o Brain should be fixed: 2 – 3
WEEKS TO ENSURE FIXATION
prior to sectioning.
 WHOLE ORGANS (EYES) – should be
injected with fixative (formol alcohol) as
well as immersed in it.
 HARD TISSUES
o Lendrum’s method: washed out
with running water over night and
immersed in 4% aq. Phenol solution
for 1 – 3 days
 MUSCLES:
o Stretched with sutures on each end
o Laid flat in moist filter paper
OTHER TYPES OF TISSUES:
 LIPIDS: Mercuric Chloride or KCrO2
o Usually remove in large portions:
cryostat or frozen sections are
recommended followed by
general stains.
 o Phospholipids: baker’s formol-
calcium
o CHOLESTEROL: Digitonin
 CHO/CARBOHYDRATES: alcoholic
fixatives (ROSSMAN’S FLUID - glycogen)
 CHON/PROTEIN: Neutral buffered formol
saline or formaldehyde vapour
 ELECTRON MICROSCOPY: 2 FIXATIVES
OTHER FACTORS TO BE CONSIDERED:
 pH for fixation: 6 – 8 outside of this pH there
may be damage to tissue or ultrastructure of
the organ.
o Storage granules of adrenalin and
nor-adrenaline are most stable at the
pH of 6.5 using formalin fixative
o GASTRIC MUCOSA are best at the
pH of 5.5
 TEMPERATURE: Room temperature (20 –
22 oC)
o 45oC – RNA; 65oC – DNA
(uncoiling)
o For electron microscopy and
some histochemistries low
temperature are needed: 0 – 4O it is
to slow down the autolysis
o For fixation in bacteriology like in
cases of leprosy and tuberculosis
and blood film: heat may be use
o For urgent biopsy: formalin can be
heated up to 60OC.
 NOTE: fixation can be done within five
hours at 40OC but higher temp would
deteriorate some antigens
  BUFFERING – formalin is buffered to 7.0
 OSMOLALITY: slightly hypertonic
solutions in other references slightly
isotonic
o Isotonic and hypotonic can cause
cells to swell and can lead to poor
fixation
 FIXATIVE CONCENTRATION:
o 10% buffered formalin
o 3% glutaraldehyde
o Or saturated solution of picric
acid or mercuric chloride
o Change of concentration can be
change by with the change of pH or
with addition of buffer.
 DURATION OF FIXATION:
o Can be shortened by heat, agitation
and microwave.
o BUFFERED FORMALIN – 2- 8 hrs
or less than 24 hrs
 Prolong fixation can lead to
shrinking or hardening of the
tissues
FACTORS AFFECTING FIXATION
 SIZE AND THICKNESS OF THE TISSUE –
that why we have specific measurements
and thickness depending on the method
we’re going to use (LIGHT MICROSCOPY
OR ELECTRON MICROSCOPY)
 PRESENCE OF MUCUS – presence of
mucus prevents penetration, thus you have
to wash it in NSS
 PRESENCE OF FAT – you have to cut the
samples in thin sections
 PRESENCE OFBLOOD – flush with NSS
 COLD TEMPERATURE – inactivate
enzymes
ENHANCED BY:
 Size and thickness of the tissue
 AGITATION
 Heat (37 – 56OC)
 CONCENTRATION
o Adjusted to Lowest possible
concentration because you will
spend less.
o FORMALIN – is best at 10%
glutaraldehyde which is best which
is generally made up of .24% to 4%
o TOO HIGH CONCENTRATION –
may adversely affect the tissue and
produce ARTIFACTS that is similar
to excessive use of heat
with PHOSPHATE
 PENETRATION – this will depend upon the
diffuse ability of each individual fixative
which is actually constant
o Formalin and alcohol penetrates
the best.
o GLUTARADEHYDE PENETRATES
THE WORST
o MERCURIALS AND OTHERS – are
somewhat in between
o ONE WAY TO GET AROUND THIS
PROBLEM IS SECTIONING THE
TISSUES THINLY WHICH IS
AROUN 2 -3 MM
o PENETRATION INTO THIN
SECTION WILL OCCUR MORE
RAPIDLY THAT TO THICK
SECTIONS
 TIME INTERVALS – the faster you can
remove the tissue and fix it the better
 o ARTIFACTS –will be introduce by  Lead f
drying, so if tissue is left out make  Trichloroacetic acid f
sure tissue are MOISTENED with  Osmium tetroxide f
NORMAL SALINE  Gl. Acetic acid
o The longer you wait the more
cellular oranelles will be lost and
more nuclear shrinkage and artefact CHARACTERISTIC OF GOOD FIXATIVE:
actual clumping will occur  Cheap and economical
 Stable and safe to handle
CLASSIFICATION OF FIXATIVES:  Fast acting, permits rapid and even
penetration
 Inhibits bacterial decomposition and
AS TO CHEMICAL COMPOSITION autolysis
 SIMPLE – made up of only one component  Must harden tissues
 COMPOUND – made up of 2 or 3  At least isotonic
components
 Must render tissues insensitive to
AS TO COAGULABILITY
subsequent processing
 NON-COAGULABLE: does not precipitate
 Must be compatible with many staining
proteins
procedures
o Like formalin
 COAGULABLE: precipitates protein
o Alcohol
AS TO FUNCTION
 ROUTINE OR GENERAL: intended
demonstration of general relationship
among cells, tissues and organs.
 SPECIAL: intended for specific cellular
details
AS TO ACTION
 CYTOLOGIC: preserve specific cellular
elemnts/constituents
o NUCLEAR FIXATIVES: usually
contain gl.HAc with pH 4.6 or less
 EXAMPLES: Flemming’s,
Carnoy’s, Bouin’s,
Newcomer’s, Heidenhain’s
Susa
o CYTOPLASMIC FIXATIVES
WITHOUT ACETIC ACID with pH
4.6
 EXAMPLE: Flemming’s;
Helly’s; Formalin (with post-
chroming); regaud’s ; Orth’s
 HISTOCHEMICAL: they preserve the
chemical constituents of the cells and the
tissues
 MICROANATOMICAL: act on the smallest
part of the tissue the purpose of this is to
preserve the structural pattern and
intracellular relationship of the tissues
AS TO REAGENTS Group
 Aldehyde f
 Metallic f
 Alcohol f
 Picric acid f