4 th lecture
 Five major groups of fixatives, classified according to mechanism
of action:

Aldehydes

Mercurials

Alcohols

Oxidizing agents

Picrates
 Six Factors affect fixation.
 Decalcification.
Aldehydes include formaldehyde (formalin) and glutaraldehyde:
The most widely utilized fixative in routine histopathology departments is 10%
formalin (4% formaldehyde) and its derivatives. Formalin has a pungent
odour, is a strong eye, skin and mucous membrane irritant and, in some
workers, may cause contact dermatitis. Personnel involved with formalin
usage should undergo annual respiratory sensitizer screening. Maximum
recommended exposure limits are one part per million and exposure levels
should be monitored on a regular basis. Formalin solutions should be
carefully handled in well-ventilated rooms/fume hoods and protective
clothing such as gloves, laboratory coats, goggles and respirators should be
worn.
The recommended time for fixation of tissues in formalin is 24–48 hours, the optimum
time being 7–10 days. Modern enclosed tissue processing machines allow for the
heating of reagents (40–45 oC) during processing and, thus, the process of fixation
can be expedited. No fixative is ideal but 10% formalin has the advantages of
preserving a wide range of tissues.
Gluteraldehyde: is often the fixative of choice for tissue requiring electron microscopy,
as it gives good preservation of the ultrastructure of the cell. It is a respiratory
sensitizer and is strongly linked to industrial asthma. Appropriate safety measures
should be observed when handling glutaraldehyde. It fixes very quickly so is good for
electron microscopy. It penetrates very poorly, but gives best overall cytoplasmic and
nuclear detail. The standard solution is a 2% buffered glutaraldehyde.
Fix tissue by an unknown mechanism. They contain
mercuric chloride and include such well-known fixatives
as B-5 and Zenker's. These fixatives penetrate relatively
poorly and cause some tissue hardness, but are fast and
give excellent nuclear detail. Their best application is for
fixation of hematopoietic and reticuloendothelial tissues.
Since they contain mercury, they must be disposed of
carefully.
Penetrates tissue rapidly and may be used in conjunction with other fixatives to
increase the speed of fixation. Absolute ethanol preserves glycogen but it
causes distortion of nuclear detail and shrinkage of cytoplasm. Carnoy’s
fixative is a good fixative for nucleic acids but causes shrinkage of the tissue
and lysis of red blood cells. It consists of absolute ethanol, chloroform and
glacial acetic acid. Methyl alcohol (methanol) and ethyl alcohol (ethanol), are
protein denaturants and are not used routinely for tissues because they
cause too much brittleness and hardness. However, they are very good for
cytologic smears because they act quickly and give good nuclear detail.
Spray cans of alcohol fixatives are marketed.
 They cross-link proteins, but cause extensive
denaturation. Some of them have specialized
applications, but are used very infrequently.
Oxidizing agents including:
 Osmium tetroxide is used as a secondary fixative in
electron microscopy, usually after primary fixation in
glutaraldehyde. It fixes lipids and also preserves the
fine structure of the cell. Osmium tetroxide is toxic
and appropriate safety precautions should be taken
when handling it.
 Potassium dichromate.
Include fixatives with picric acid. Foremost among
these is Bouin's solution. It has an unknown
mechanism of action. It does almost as well as
mercurials with nuclear detail but does not cause as
much hardness. Picric acid is an explosion hazard in
dry form. As a solution, it stains everything it touches
yellow, including skin.
 Various additives in fixatives may be used. For example,
tannic acid, phenol or heavy metal solutions may be added to
formalin to increase the rate of penetration, improve
preservation or enhance subsequent staining procedures.
 Vapour fixation.
 Microwave ovens can be used in fixation, either to preserve
the tissue with the action of the heat itself or to speed up the
process of fixation, as described earlier.
 Factors that will affect the fixation process:
1.
Buffering
2.
Penetration
3.
Volume
4.
Temperature
5.
Concentration
6.
Time interval
 Fixation is best carried out close to neutral pH, in the
range of 6-8. Hypoxia of tissues lowers the pH, so
there must be buffering capacity in the fixative to
prevent excessive acidity. Common buffers include
phosphate, bicarbonate HCO3- , cacodylate, and
veronal. Commercial formalin is buffered with
phosphate at a pH of 7.
 Penetration of tissues depends upon the diffusability
of each individual fixative, which is a constant.
Formalin and alcohol penetrate the best, and
glutaraldehyde the worst. Mercurials and others are
somewhere in between. One way to get around this
problem is sectioning the tissues thinly (2 to 3 μm).
Penetration into a thin section will occur more rapidly
than for a thick section.
 The volume of fixative is important. There should be
a 10:1 ratio of fixative to tissue. Obviously, we often
get away with less than this, but may not get ideal
fixation. One way to partially solve the problem is to
change the fixative at intervals to avoid exhaustion of
the fixative. Agitation of the specimen in the fixative
will also enhance fixation.
 Increasing the temperature, as with all chemical
reactions, will increase the speed of fixation, as long
as you don't cook the tissue. Hot formalin will fix
tissues faster, and this is often the first step on an
automated tissue processor.
 Concentration of fixative should be adjusted down to
the lowest level possible, because you will expend
less money for the fixative. Formalin is best at 10%;
glutaraldehyde is generally made up at 0.25% to 4%.
Too high a concentration may adversely affect the
tissues and produce artifact similar to excessive
heat.
 Also very important is time interval from of removal of
tissues to fixation.
 The faster you can get the tissue and fix it, the better.
Artefact will be introduced by drying, so if tissue is left
out, please keep it moist with saline.
 The longer you wait, the more cellular organelles will be
lost and the more nuclear shrinkage and artefactual
clumping will occur.
 Decalcification is the removal of calcium ions from
the bone or teeth or tissue (like lung) through
histological process thereby making the bone flexible
and easy for pathological investigation.
 End-Point of Decalcification:
1.
X-ray (the most accurate way)
2.
Chemical testing (accurate)
3.
Physical testing (less accurate and potentially
damage of specimen).
Other Method:
Ethylenediaminetetraacetic Acid (EDTA) is a calcium
absorbent that has gained popularity because of its
gentleness on tissue and antigens. It is a slow
decalcifies but the rate of decalcification can be
increased with use of microwave energy.