Question Answer

destruction/digestion of tissue and cells by enzymes autolysis

stabilize proteins fixation
substance/structure not normally present, produced by some
external action
artifact
heterogenous group of substances, contain enough color that they
are visible w/o staining
pigment
are primarily acetone and ethyl/methyl alcohol (flammables) Nonaqeous fixative
cloudiness, flocculation, and clot formation stages in coagulation coagulation
chemically link/add themselves to tissue additive fixative
solution that causes cells to swell hypotonic
fluids into which normal animal cells can be placed w/o causing
swelling or shrinkage of cells
isotonic
allows solution to penetrate or gain entry into the interior of the
tissue
coagulating fixatives
formaldehyde, osmium tetroxide, potassium dichromate and acetic
acid.
noncoagulant fixatives
do not allow good penetration by reagents noncoagulant fixatives
Alcohol, zinc salts, mercuric chloride, chromium trioxide and picric
acid
coagulating fixatives
stabilize and denature proteins heat
mercuric chloride, chromium trioxide, picric acid, formaldehyde,
gluteraldehyde, osmium tetroxide, and zinc sulfate or chloride
common additive reagents
act on tissue w/o combining with it acetone and alchol
alters tissue stabilize proteins, so that its resistant to further
change
fixative
kill the tissue so that postmortem activities of decay, putrefaction
and autolysis is prevented
Function of fixative
maintain proper relationship between cells and extracellular
substances
Function of fixative
will fail to stain severely autolysed tissue
must change solube contents of a cell into insoluble so that those
substances are not lost during processing
Fixative
causes proteins to be less capable of maintaining an intimate
relationship w/water and become more reactive, but fixative
molecule doesnot combine w/t protein
Denaturation
Render enzymes inactive, (brain, pancreas, liver) kill bacteria and
molds
Action of fixatives

Make tissue receptive to dyes, Modify tissue for the maximum
retention of form through subsequent processing
Action of fixative
Stabilize tissue elements so subsequent procedures will have
minimal effect
Action of fixative
Physical and Chemical Methods of Fixation
Heat and dessication Physical
Microwave - heat tissue in saline Heat method
Easily overheat, loss of enzyme activity and atingenicity, false
locatlization of nuceleic acid, lysis of RBC's
Disadvantage of heat method
Increase spee, less destructive to connective tissue Advantages of heat method
Another physical method of fixing proteins Dessication
Air Drying, Basic use: peripheral blood smears, Wright staining
most frequent use for this method
Dessication
Method that is rarely used Dessication
Primary method of protein stabilization Chemical fixation
additive vx nonadditive, Coagulant vx noncoagulant, Aqueous vx
nonaqueous
Reagents can be classified as
elements that affect the quality of fixation Fixation factors
Temperature, Size of tissue, Volume ration Factors influencing fixation
increased temp causes increased autolysis and increased diffusion
of cellular elements
Factors influencing fixation
Size- Sections should not be more than 3mm thick Factors influencing fixation
15 to 20 times greater than the tissue volume Fixative volume
Tissue should be placed in fixative immediately after surgical
removal
Time
tissue underlying epithelium lamina propria
Penetration, tissue storage, pH, osmolality
What to consider when choosing
a fixative
Number of particles in a solution Osmolality
Begins at Periphery of tissue and proceeds inward Fixation
achieve their full effect on tissue at any particular depth as soon as
they have penetrated to that depth at a concentration sufficient to
cause coagulation
Coagulant fixatives
Affected by heat, but not the concentration of the fixative Rate of penetration
Penetrates faster than any of the common fixatives ingridients Formaldehyde
Stored wet tissue storage
tissue may remain in this solution indefinitely Neutral buffered formalin
Solutions with a pH value above 7.0 are Alkali (base)
Solutions with a pH value below 7.0 are Acidic
Solutions with a pH value of 7.0 are Neutrals
is not important in light microscopy but is for electon microscopy pH of solution
Fixatives used for EM need to be at a physiological pH 7.2-7.4
most fixatives pH range between 4-9
pigment may be produced if pH drops below 4.0
have an osmolality of 340 mOsm Body fluids
is a semipermealbe membrane that allows water molecules to
pass through it very readily
Cell membrane
passes through the cell membrane toward the most concentrated
solution to equalize the concentration on both sides of the
membrane
Water
Water leaves the cell and the cell shrinks
When the cell is surrounded by a
hypertonic solution
The cell swells possibly rupturing its membrane (cell cytosol)
When the cell is placed in a
hypotonic solution
physiological or normal isotonic
Additive vx nonadditive, coagulant vx noncoagulant, aqueous vx
nonaqeuous
Fixative Classification
chemicaly combine with the protein molecules to changed them.
the protein is made insoluble by the addition of the fixative and
then becomes immobilitzed, remaining at is original location in the
cell.
additive fixatives
change tertirary structure of the protein additive fixatives
do not combine with protein nonadditive fixatives
they act on the proteins to coagulate them and precipitate them,
but do not change their chemical structures
nonadditive fixatives
The primary mechanism by whic these fixatives act is to dissociate
bound water molecules from tissue protein groups
nonadditve fixatives
Mercuric chloride additive
chromium tetroxide additive
picric acid additive
formaldehyde additive
gluteraldehyde additive
osmium tetroxide additive
zinc sulfate additive
zinc chloride additive
methyl alcohol nonadditive
ethyl alcohol nonadditive
acetone nonadditive
transformation of a liquid or a solid into a semi-solid or solid mass coagulant fixative
forms a semisolid network of molecules that allows other
preservative solutions to penetrate the tissue deeply
coagulant fixative
establishes a network in the tissue that allows solutions to readily
penetrate or enter most freely
Coagulation
act by creating a gel that makes penetration by a subsequent
solution difficult
noncoagulant fixative
forms a solid gel which does not allow for easy penetration of
solutions and are not used for easy penetration of solutions and
are not used for paraffin embedding
noncoagulant fixative
Alcohol coagulant
zinc salts coagulant
mercuric chloride coagulant
chromium trioxide coagulant
picric acid coagulant
Formaldehyde noncoagulant
gluteraldehyde noncoagulant
osmium tetroxide noncoagulant
potassium dichromate noncoagulant
acetic acid noncoagulant
Water based Aqueous fixatives
most commonly used Aqueous fixatives
formaldehyde Aqueous fixative
acetic acid Aqueous fixative
Picric acid Aqueous fixative
organic compound known as simple aldehyde Formaldehyde
occurs naturally as a colorless gas Formaldehyde
used interchangeably Formaldehyde and formalin
37-40% and are considered 100%formalin Commercial grade formaldehyde
100% formalin is diluted to 10% formalin, which means the
concentration of formaldehyde is 3.7-4%
Formalin fixation
Formalin additive fixative
it reacts primarily with the amino groups on the amino acids of
proteins to form bonds which cross link protein chains together
Formalin

nonacuagulating fixative forming a gel which makes the proteins
insoluble and keeps them in place and are able to with stand
further processing
formalin
causes less shrinkage to tissue Formaldehyde
It is hypertonic it is not osmotaically active Formaldehyde
preserves morphological detail so that tissue is life-like Formaldehyde
penetrates tissue rapidly Formaldehyde
Fixes tissue slowly up to 7days Formaldehyde
cross linking proteins because it is an additve fixative Formaldehyde
Chemically stable and kept at room temperature Formaldehyde
tissue can be stored long term without distorting the tissue Formaldehyde
hardens tissue more than other fixatives except ethanol and
acetone
Formaldehyde
can be used as a simple aqueous solution or w/ addition of sodium
chloride to achieve the correct osmolality
Formaldehyde
Buffers are added to maintain neutrality (pH of 6.8)and to prevent
pigments/percipitates from forming
Formaldehyde
organic acid Acetic acid
undiluted is called glacial acetic acid Acetic acid
Diluted is known as vinegar and is used in pickling Acetic acid
leaves tissue very soft Acetic acid
it doesnt fix lipids or carbohydrates Acetic acid
lyses red blood cells Acetic acid
Hypertonic effect on cells Acetic acid
coagulant fixative Picric acid
penetrates tissue well Picric acid
gives tissue a soft consistency Picric acid
Causes extreme shrinkage Picric acid
Not a good fixative for nucleic acids as it leaves DNA solube Picric acid
not the fixative of choice for nucelic acid stains Picric acid
Tissue must have picric acid washed out in 50% ethanol before
processing so distortion or obliteration of almost all cellular
structures does not occur
Picric acid
Toxic and explosive Picric acid
Other than formaldehyde or gluteraldehyde most fixative solutions
are combined so that the disadvantage of one component will be
counterbalanced by an advantage or disadvantage of another
Compound or combined fixatives
hypertonic/hypotonic balance Compound or combined fixatives
mercuric chloride, sodium acetate and formaldehyde B-5 fixative
used to fix hematopoietic and lymphoreticular tissues B-5 fixative
gives beautiful nuclear detail B-5 fixative
wash in iodine the sodium thiosulfate to remove mercury pigments
before processing
B-5 fixative
store wet tissue in 70% alcohol B-5 fixative
great for immunofluorescence and immunohistochemistry B-5 fixative
contains picric acid, acetic acid and formaldehyde Bouin solution
Good for nuclear preservation Bouin solution
Used for gastrointestinal and endocrine bx Bouin solution
Not used for EM studies or nucleic acid demonstration Bouin solution
Wash in 50-70% alcohol to remove yellow color of fixative Bouin solution
Not for long term storage, place tissue in 70-80% alcohol Bouin solution
NO water nonaqueous fixatives
coagulating, nonadditve fixative nonaqueous fixatives
are used only when the desired tissue components are destroyed
or dissolved by aqueous fixatives
nonaqueous fixatives
overharden tissue nonaqueous fixatives
shrink tissue nonaqueous fixatives
acetone nonaqeuous fixatives
ethyl/methyl alcohol nonaqueous fixatives
used for enzyme studies especially acid and alkaline phosphatase Acetone
used to fix brain tissue for diagnosis of rabies Acetone
frequently used on frozen sections used for cell surface antigens
by IHC
Acetone
Touch preps and blood smears Methyl alcohol
Preserves water soluble tissue components like glycogen and
urate crystals that are deposited in gout
Ethyl alcohol
preserves pigments, dissolves fat, overhardens and shrinks tissue Ethyl alcohol
color deposits which may be found in or on tissue Pigments
Result from chemical processing Artifact pigments
black acid hematain pigment Formalin pigment
brown crystalline pigments forms in tissue rich in blood when the
pH is below 6.0
Formalin pigment

it is removed by treating slides with a solution of alcohol saturated
w/picric acid or 70% alcohol w/ammonium hydroxide
Formalin pigment can be
removed
these pigments can be confused w/relative pigments or
microorganisms
Formalin pigment
found w/mercury containing fixatives (B-5) were not removed prior
to processing
Mercury pigment
red in color Mercury pigment
Treat w/Gram's or Lugol's iodine followed by sodium thiosulfate
Mercury pigment can be
removed
nucleus, proteins, lipids, carbohydrates cells that react w/fixatives
Dna, Rna and attached proteins are found in the Nucleus
acetic and carnoy solution fixatives for nucleic acids
produces nuclear bubbling where nuclei show coalescence of the
chromatin into strands with clear spaces
Formalin fixation
have a primary, secondary and tertiary structure which determine
the size and shape of protein molecule
Proteins
is determined by the sequence of the amino acids which are linked
by covalent bonds
Primary protein structures
is determined by hydrogen bonding between the amino acids Secondary protein structures
is determined by other types of chemical bonds between the amino
acids such as ionic bonds, hydrogen bonds and linkages with
sulfur atoms
tertiary protein structures
Many fixatives preserves lipids but only two fix them, so that they
are not lost in subsequent processing
Lipids
two chemicals that fix lipids
Osmium tetroxide and chromic
acid
some are lost in fixation Carbohydrates
the storage form of glucose glycogen
glycogen the storage form of glucose is thought to be retained by
entrapment of fixed proteins
carbohydrates
for immunofluorescence study or enzyme profile
specimen must be frozen w/o
fixation
tissue should be frozen, sectioned and briefly fixed in acetone
Immunohistochemistry T and B
cell markers for dx of lymphomas
Special stain choice of fixatives
for a trichrome fix in Bouins
for chromaffin granules found in cells of the adrenal gland fix in Orth's
for urate crystrals fix in Alcohols
when muscle cross-striations are to be stained with PTAH use Zenkers
cells shrink by releasing fluid hypertonic
resist extracgtion by most strong acids, water, alcohol or acetone Formalin pigments
highly polymeric form of formaldehyde Paraformaldehyde
strong tendency to polymerize to dimers and trimers formaldehyde
glutaraldehyde (most used in em) and osmium tetroxide Electron microscopy fixation
Zenker
skeletal muscle cross striation
(fixation)
use Orth solution when you need to demonstrate Chromaffin
granules
pheochromocytomas
require nonaqeous fixated absolute alcohol Urate crystals
Frozen w/o fixation immunofluorescence
Bouin tissue for trichrome (muscle)
frozen w/o fixation enzyme histochemistry
frozen sectioned and briefly fixed in acetone immunohistochemistry
a cell is a solution that is more concentrated than the cell cytosol hypertonic
NH2 amino group
-COOH Carboxyl group
Chromium, mercury and osmiums Heavy metals
are cations that combine with anion group of proteins Heavy metals
positvely charged cations
negatively charged anions
sulfhydryl, carboxyl and phosphoric combine w/anions
-Sh sulfhydryl
-PO4 phosphoric acid
most rapidly penetrating component of aqueous fixative water
has freezing point of 16.6C glacial acetic acid
prepared in water aqueous
B-5, Bouin, Gendre, Hollande, Zenker/helly, Orth, Zamboni, Zinc
formalin
compound fixative