[2S] UNIT 6.2 Fixation Flashcards
First step in histotechnology
Fixation
Chemical process by which biological tissues are preserved from decay
Fixation
Process of preserving cells and tissue constituents in a
LIFE-LIKE manner
Fixation
Common specimens that are submitted in the laboratory
Surgery (Biopsy)
Dissection Room (Autopsy)
Tissue sample thickness should be?
5mm
Preserve the morphological and chemical integrity of
the cell
Harden and preserve tissue for further handling
Prevent or arrest degenerative processes
disruption of the cell due to lysosome
Autolysis
bacterial decomposition
Putrefaction
TYPES OF FIXATION
● Heat fixation
● Microwave fixation
● Cryo-preservation
Physical Method
TYPES OF FIXATION
● Immersion Fixation and Perfusion Fixation
● Coagulant fixatives
● Non-coagulant cross-linking fixative
Chemical Method
T/F: In immersion & perfusion fixation, the volume of the fixative is 10-20 or 25 times the volume of the specimen
T
BASIC MECHANISM IN FIXATION
Fixing agent is not incorporated into the tissue
NON-ADDITIVE FIXATION / COAGULANT FIXATIVE
BASIC MECHANISM IN FIXATION
● Non-coagulant cross-linking fixatives
○ Cross-linking of proteins (as in scaffolding of buildings)
■ Stabilizing physical characteristics of tissue
ADDITIVE FIXATION/ NON-COAGULANT FIXATIVES
BASIC MECHANISM IN FIXATION
Chemical constituent taken into the cell, forming molecular complexes and stabilizing proteins
ADDITIVE FIXATION/ NON-COAGULANT FIXATIVES
BASIC MECHANISM IN FIXATION
Formalin, Mercury and Osmium tetroxide
ADDITIVE FIXATION/ NON-COAGULANT FIXATIVES
BASIC MECHANISM IN FIXATION
● Dehydrant coagulant fixatives
○ Capable of dehydration/ removing of water molecules
for hardening or stabilizing tissue
NON-ADDITIVE FIXATION / COAGULANT FIXATIVE
BASIC MECHANISM IN FIXATION
● Alteration of tissue composition by removing bound water molecules at Hydrogen bonds within protein molecules
○ Stabilizes proteins by forming crosslinks after water molecule removal
NON-ADDITIVE FIXATION / COAGULANT FIXATIVE
BENEFITS OF FIXATION
T/F: Allows sectioning of the tissue by softening tissue
F; hardening
BENEFITS OF FIXATION
T/F: Prevents autolysis and inactivates infectious age
T
BENEFITS OF FIXATION
T/F: Improves cell avidity for special stains
T
MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC
T/F: High acidity decreases effectiveness
T
MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC
pH of a satisfactory fixation
ph 6 & 8 (neutral)
MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC
Best fixative
10% neutral buffered formalin
MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC
best blood buffer for systemic circulation
Bicarbonate
MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC
Common buffers
MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC
T/F: Outside the pH range, changes may occur that are
detrimental to ultrastructural preservation of the tissue.
T
MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC
T/F: High pH changes:
Precipitation of formalin pigments: Over time, formalin turns into formic acid, which has a lower pH
T
MAIN FACTORS AFFECTING FIXATION: TEMP
T/F: Increasing the temperature decreases the
rate of diffusion into the tissue and speeds up the rate of chemical reaction between the fixative and tissue elements
F; increases the rate of diffusion
MAIN FACTORS AFFECTING FIXATION: TEMP
Fixation of surgical specimens is done at what temp?
room temperature
MAIN FACTORS AFFECTING FIXATION: TEMP
T/F: The higher the temperature, the faster is the rate of fixation and the less time is needed for fixation
T
MAIN FACTORS AFFECTING FIXATION: TEMP
Fixation of surgical specimens can be followed by further fixation at temp up to [temp]?
40-45C
MAIN FACTORS AFFECTING FIXATION: TEMP
Fixative used for EM
Glutaraldehyde
MAIN FACTORS AFFECTING FIXATION: TEMP
Recommended temperature
body temperature
MAIN FACTORS AFFECTING FIXATION: TEMP
Temp for EM
0-4C
MAIN FACTORS AFFECTING FIXATION: TEMP
T/F:
Mast cells - Room temp
Nucleic acids - Not room temp
T
Light-colored areas = ___ areas
Dark colored areas = ____ areas
fixed
unfixed
THICKNESS OF SECTION
for EM
1 to 2 mm square
THICKNESS OF SECTION
for light microscopy
2 cm
T/F: Brain is usually suspended whole in 10% buffered formalin for 2-3 weeks to ensure fixation and some hardening prior to sectioning
T
OSMOLALITY
give rise to cell shrinkage
hypertonic
OSMOLALITY
cause swelling and poor fixation
Isotonic and Hypotonic fixatives
OSMOLALITY
The best result is usually obtained using ____ _______ solutions (400-450 mOsm)
slightly hypertonic
Concentration of formaldehyde and glutaraldehyde
- Formaldehyde – 10%
- Glutaraldehyde – 3%
Concentration of glutaraldehyde for immunoelectrochemistry
0.25%
CONCENTRATION
T/F: Presence of buffer causes polymerization of
aldehyde, with consequent increase in its effective concentration
F; decrease
DURATION OF FIXATION
________ in buffered formalin is usually carried out for 2-6
hours during the day the specimen is obtained
Primary Fixation
DURATION OF FIXATION
T/F: Prolonged fixation may cause shrinkage and hardening of the tissue
and may severely inhibit enzyme activity and immunological reaction
T
PRACTICAL CONSIDERATIONS OF FIXATION
The specimen should be placed in a fixative solution as soon as it is removed from the body to prevent autolysis and putrefaction
Speed
PRACTICAL CONSIDERATIONS OF FIXATION: PENETRATION
T/F: A commonly quoted rate of penetration for aldehyde
fixative is two to three millimeters per hour and slows down as it goes deeper into the tissue.
T
PRACTICAL CONSIDERATIONS OF FIXATION: PENETRATION
____ per hour fixative penetration
2-3 mm
PRACTICAL CONSIDERATIONS OF FIXATION: VOLUME
T/F: 10-25 times the volume of the tissue to be fixed
T
PRACTICAL CONSIDERATIONS OF FIXATION: VOLUME
T/F: The maximum effectiveness of fixation is
noted to be ____ the tissue volume.
20 times
PRACTICAL CONSIDERATIONS OF FIXATION: DURATION OF FIXATION
T/F: Small or loosely textured tissues such as biopsies / scrapings take longer than fibrous organs
F; Fibrous organs take longer than small or loosely textured tissues such as biopsies or scrapings
PRACTICAL CONSIDERATIONS OF FIXATION: DURATION OF FIXATION
T/F: Fixation time can be cut down by using heat, vacuum, agitation or microwave
T
PRACTICAL CONSIDERATIONS OF FIXATION: DURATION OF FIXATION
Negative pressure in a container that helps
fixative penetration to reach up to 5mm per hour
Vacuum
PRACTICAL CONSIDERATIONS OF FIXATION: DURATION OF FIXATION
The fixative container is slightly shaken so that the ions would not be relatively at rest but are excited and evenly distributed in the entire solution
Agitation
PRACTICAL CONSIDERATIONS OF FIXATION: MISCELLANEOUS CONSIDERATIONS
T/F: To maintain an adequate fixation time of 4-6 hours, the tissue size must be 2cm square
PRACTICAL CONSIDERATIONS OF FIXATION: MISCELLANEOUS CONSIDERATIONS
T/F: The tissue selected for sectioning should be thin enough to allow penetration by fixative within a reasonable amount of time
T
PRACTICAL CONSIDERATIONS OF FIXATION: MISCELLANEOUS CONSIDERATIONS
T/F: Refrigeration is used to fasten decomposition if the
tissue that needs to be photographed and cannot be fixed immediately
F; slow down decomposition
EFFECTS OF FIXATIVES
T/F: It hardens soft tissues and make the handling and cutting of section easier
T
EFFECTS OF FIXATIVES
T/F: It inhibits bacterial decomposition and reduces risks of infections during handling
T
EFFECTS OF FIXATIVES
T/F: Decreases optical differentiation of cells
F; Increases
EFFECTS OF FIXATIVES
T/F: Fixatives diluted and/or contaminated by bodily fluids (e.g. bile, blood, feces) will be increased in concentration and must be replaced regularly to ensure effectiveness
F; reduced in conc
EFFECTS OF FIXATIVES
T/F: Pinning specimens to a corkboard or inserting a paper or gauze “wick” into tubular structures can improve fixation and reduce tissue distortion
T
EFFECTS OF FIXATIVES
T/F: Prolonged fixation may be more difficult to reverse and may also result in loss of immunohistochemical
antigenicity
T
FIXATIVES ACCORDING TO COMPOSITION
made up only of one substance
Simple Fixatives
FIXATIVES ACCORDING TO COMPOSITION
○ Two or more fixatives to obtain optimal combined
effect
○ A combination of different simple fixatives
Compound Fixatives
FIXATIVES ACCORDING TO ACTION
General microscopic study of tissue structures
MICROANATOMICAL
FIXATIVES ACCORDING TO ACTION
Well preservation to the totality of physical characteristic or morphology of tissue
○ Not only used for the tissue but also the cytoplasm
○ Can preserve all tissue structures
MICROANATOMICAL
FIXATIVES ACCORDING TO ACTION
Preserve specific parts and particular microscopic
elements
CYTOLOGIC FIXATIVES
CYTOLOGIC: NUCLEAR OR CYTOPLASMIC
Preserves cytoplasmic structures in particular
Cytoplasmic
CYTOLOGIC: NUCLEAR OR CYTOPLASMIC
Preserve cytoplasmic
organelles better than
nucleus
Cytoplasmic
CYTOLOGIC: NUCLEAR OR CYTOPLASMIC
> 4.6 pH
Cytoplasmic
CYTOLOGIC: NUCLEAR OR CYTOPLASMIC
Flemming’s Fluid without
Acetic Acid
Helly’s Fluid
Formalin with
Post-Chroming
Regaud’s Fluid (Moller’s
Fluid)
Orth’s Fluid
Cytoplasmic
CYTOLOGIC: NUCLEAR OR CYTOPLASMIC
RNA preservation: Ethanol
(best) and Acetone
(alternative)
Cytoplasmic
CYTOLOGIC: NUCLEAR OR CYTOPLASMIC
Preserve nuclear structures
Nuclear
CYTOLOGIC: NUCLEAR OR CYTOPLASMIC
Preserve nucleus better
than cytoplasm
Nuclear
CYTOLOGIC: NUCLEAR OR CYTOPLASMIC
Glacial acetic acid as
primary component
Nuclear
CYTOLOGIC: NUCLEAR OR CYTOPLASMIC
<4.6 pH
Nuclear
CYTOLOGIC: NUCLEAR OR CYTOPLASMIC
Flemming’s Fluid
Carnoy’s Fluid
Bouin’s Fluid
Newcomer’s Fluid
Heidenhain’s Susa
Nuclear
FIXATIVES ACCORDING TO ACTION
Preserves chemical constituents of cells and tissues
HISTOCHEMICAL FIXATIVES
FIXATIVES ACCORDING TO ACTION
May preserve tissues, nucleus, and cytoplasm but aside from that, it can preserve the chemical component
HISTOCHEMICAL FIXATIVES
FIXATIVES ACCORDING TO ACTION
ODD ONE OUT: Histochemical Fixatives
○ 10% Formol Saline
○ Absolute Ethyl Alcohol
○ Acetone
○ Newcomer’s
○ Heidenhain’s Susa
Heidenhain’s Susa - Microanatomical
FIXATIVES ACCORDING TO ACTION
ODD ONE OUT: Cytoplasmic Fixatives
- Flemming’s Fluid without Acetic Acid
- Helly’s Fluid
- Bouin’s Fluid
- Formalin with Post-Chroming
- Regaud’s Fluid (Moller’s Fluid)
- Orth’s Fluid
Bouin’s Fluid - Nuclear
FIXATIVES ACCORDING TO ACTION
ODD ONE OUT: Nuclear Fixatives
- Flemming’s Fluid
- Formalin with Post-Chroming
- Carnoy’s Fluid
- Bouin’s Fluid
- Newcomer’s Fluid
- Heidenhain’s Susa
- RNA: ethanol and acetone
Formalin with Post-Chroming - Cytoplasmic
FIXATION FOR IMMUNOHISTOCHEMISTRY
Fixation methods generally fall into two classes
organic solvents and cross-linking reagents
FIXATION FOR IMMUNOHISTOCHEMISTRY
Use frozen sections and special stains (fixative: mercuric chloride and potassium dichromate)
Lipid Fixation
FIXATION FOR IMMUNOHISTOCHEMISTRY
such as alcohols and acetone remove lipids and dehydrate the cells while precipitating the proteins on the cellular architecture
Organic solvents
FIXATION FOR IMMUNOHISTOCHEMISTRY
(such as paraformaldehyde) form intermolecular bridges, normally through free amino
groups, thus creating a network of linked antigens
Cross-linking reagents
FIXATION FOR IMMUNOHISTOCHEMISTRY
T/F: Cross-linkers preserve cell structure better than organic solvents but may reduce the antigenicity of some cell components and require the addition of a permeabilization step to allow access of the antibody to the specimen
T
FIXATION FOR IMMUNOHISTOCHEMISTRY
Baker’s formal-calcium may be used to preserve phospholipids
Lipid Fixation
FIXATION FOR IMMUNOHISTOCHEMISTRY
T/F: 10% neutral buffer formalin would be effective
T
FIXATION FOR IMMUNOHISTOCHEMISTRY
T/F: Antigen retrieval phase is not necessary
F; is necessary
FIXATION FOR IMMUNOHISTOCHEMISTRY
Largely removed during processing
Lipid Fixation
FIXATION FOR IMMUNOHISTOCHEMISTRY
Alcoholic fixatives for better retention
Carbohydrate Fixation
FIXATION FOR IMMUNOHISTOCHEMISTRY
Aldehyde fixatives can be used in general
Lipid Fixation
FIXATION FOR IMMUNOHISTOCHEMISTRY
Glycogen can be demonstrated satisfactorily enough for diagnosis, although losses of
glycogen can be high (60-80%) in aqueous solution
Carbohydrate Fixation
FIXATION FOR IMMUNOHISTOCHEMISTRY
There is better retention of glycogen if the section is coated with celloidin
Carbohydrate Fixation
FIXATION FOR IMMUNOHISTOCHEMISTRY
Alcoholic formaldehyde is a better fixative in human skin compared with neutral
buffered formaldehyde.
Carbohydrate Fixation
FIXATION FOR IMMUNOHISTOCHEMISTRY
Neutral buffered formal saline
Protein fixation
FIXATION FOR IMMUNOHISTOCHEMISTRY
Routinely osmium tetroxide or glutaraldehyde is used with the whole procedure performed at 4°C.
Fixation for Electron Microscopy
FIXATION FOR IMMUNOHISTOCHEMISTRY
is the process of placing an already fixed tissue in a second fixative
Secondary Fixation
To make special staining techniques possible (with secondary fixative acting as
a mordant)
Secondary Fixation
To facilitate and improve the demonstration of particular substances
Secondary Fixation
To ensure further and complete hardening and preservation of tissues
Secondary Fixation
CHARACTERISTICS OF A GOOD FIXATIVES
T/F:
● It must be cheap.
● It must be stable.
● It must be safe to handle
T
CHARACTERISTICS OF A GOOD FIXATIVES
T/F: It must kill the cell slowly, thereby producing a minimum distortion of cell constituents
F; quickly
CHARACTERISTICS OF A GOOD FIXATIVES
T/F: It must be hypotonic, causing minimal physical and chemical alteration of the cells and their constituents (not strictly implemented).
F; isotonic
CHARACTERISTICS OF A GOOD FIXATIVES
T/F: It must inhibit bacterial decomposition and autolysis. It must produce minimum shrinkage of tissue
T
CHARACTERISTICS OF A GOOD FIXATIVES
T/F: It must permit rapid and even penetration of tissues
T
CHARACTERISTICS OF A GOOD FIXATIVES
T/F: It must harden tissues, thereby making the cutting of sections easier
T
CHARACTERISTICS OF A GOOD FIXATIVES
T/F: A good fixative can be slightly hypertonic to slightly
harden tissues
T
CHARACTERISTICS OF A GOOD FIXATIVES
T/F: It must make cellular components insoluble to hypotonic solutions and render them insensitive to subsequent processing.
T
CHARACTERISTICS OF A GOOD FIXATIVES
T/F: It must permit the subsequent application of many staining procedures to facilitate easier and more profitable examination
T
6 Fixatives in order of decreasing speed of
penetration
FAMOP
Formaldehyde > Acetic acid > Mercuric chloride > Ethyl/Methyl alcohol > Osmium tetroxide > Picric acid
The aldehydes form cross-links between proteins, creating a gel, thus retaining cellular constituents in their in vivo relationships to each other.
ALDEHYDE FIXATIVES
Gives some mechanical strength to the entire structure which enables it to withstand subsequent
processing.
ALDEHYDE FIXATIVES
T/F: Aldehyde fixatives are non-coagulant
T
ALDEHYDE FIXATIVES
Produced from oxidation of methyl alcohol/ methanol.
Formaldehyde
ALDEHYDE FIXATIVES
● Fixation time 24 hrs
● Irritating especially to mucosa (nose, throat) because of its fumes
Formaldehyde
ALDEHYDE FIXATIVES
If formaldehyde is not used immediately, it becomes acidic over time (formic acid), which forms a white precipitate called _________
paraformaldehyde
ALDEHYDE FIXATIVES: FORMALDEHYDE
● Cheap, Readily Available, Easy to Prepare
● Relatively Stable
● Compatible with many Stains
● Fumes Irritating to Nose and Eyes
● Solution is Irritating to Skin
10% FORMALIN
ALDEHYDE FIXATIVES: FORMALDEHYDE
For Routine Paraffin Sections, Electron Microscopy, Histochemistry & Enzyme Studies
10% FORMALIN
ALDEHYDE FIXATIVES: FORMALDEHYDE
Pigments removed by treatment with alcoholic picric acid
10% FORMALIN
ALDEHYDE FIXATIVES: FORMALDEHYDE
10% NaCl Solution as diluent
10% FORMOL-SALINE
ALDEHYDE FIXATIVES: FORMALDEHYDE
Fixation of central nervous tissues
10% FORMOL-SALINE
ALDEHYDE FIXATIVES: FORMALDEHYDE
Post-mortem tissues for histochemical examinations
10% FORMOL-SALINE
ALDEHYDE FIXATIVES: FORMALDEHYDE
● Fixation time: 24 hrs at 35 degrees/ 48 hrs at RT
● 10% formalin with NSS
10% FORMOL-SALINE
ALDEHYDE FIXATIVES: FORMALDEHYDE
ALDEHYDE FIXATIVES: FORMALDEHYDE
Components of 10% Formalin
900 mL Distilled H2O
100 mL Formaldehyde 40%
9 gm NaCl
ALDEHYDE FIXATIVES: FORMALDEHYDE
○ Fast penetration
○ Non-coagulant and additive
○ Prevents alterations during processing
10% Neutral Buffered Formalin
ALDEHYDE FIXATIVES: FORMALDEHYDE
Most recommended fixative
10% NEUTRAL BUFFERED FORMALIN
ALDEHYDE FIXATIVES: FORMALDEHYDE
● Buffered to a pH level near 7 so that there will be no
acidity and no precipitation
○ pH level makes the fixative usable for 1 month
10% NEUTRAL BUFFERED FORMALIN
ALDEHYDE FIXATIVES: FORMALDEHYDE
● Preservation and routine storage of surgical, post-mortem and research specimens
● Fixation time: 4-24 Hrs
10% NEUTRAL BUFFERED FORMALIN
ALDEHYDE FIXATIVES: FORMALDEHYDE
900 mL D H2O
100 mL Formaldehyde 40%
6.5 gm Disodium hydrogen
phosphate
3.5 gm Sodium dihydrogen
phosphate
10% Neutral Buffered Formalin
ALDEHYDE FIXATIVES: FORMALDEHYDE
○ Less shrinkage than other fixatives
○ Not osmotically active
○ Hardens tissue better (except alcohol and acetone)
10% Neutral Buffered Formalin
ALDEHYDE FIXATIVES: FORMALDEHYDE
○ Tissue can be stored in formalin indefinitely (except
for IHC)
○ Cheap and stable
10% Neutral Buffered Formalin
ALDEHYDE FIXATIVES: FORMALDEHYDE
Fixative of choice for immunohistochemistry and
molecular studies
10% Neutral Buffered Formalin
ALDEHYDE FIXATIVES: FORMALDEHYDE
10% Neutral Buffered Formalin Disadvantage
T/F: It is longer to prepare; hence, is time-consuming
T
ALDEHYDE FIXATIVES: FORMALDEHYDE
10% Neutral Buffered Formalin Disadvantage
T/F: Positivity of mucin to the periodic acid shift (PAS) is
increased
F; reduced
ALDEHYDE FIXATIVES: FORMALDEHYDE
10% Neutral Buffered Formalin Disadvantage
T/F: It may produce gradual loss in basophilic staining of
cells
T
ALDEHYDE FIXATIVES: FORMALDEHYDE
10% Neutral Buffered Formalin Disadvantage
T/F: Reactivity of myelin to Weigert’s iron hematoxylin
stain is reduced
T
ALDEHYDE FIXATIVES: FORMALDEHYDE
10% Neutral Buffered Formalin Disadvantage
T/F: It is inert towards lipids, especially neutral fats and
phospholipids
T
ALDEHYDE FIXATIVES: FORMALDEHYDE
● Brightens cytoplasmic and metachromatic stains
● Slow penetration
● After fixation, we can do the dehydration step immediately using absolute (100%) alcohol
FORMOL-CORROSIVE (FORMOL-SUBLIMATE)
ALDEHYDE FIXATIVES: FORMALDEHYDE
Sat. Aq. Mercuric chloride as diluent
ALDEHYDE FIXATIVES: FORMALDEHYDE
● Fixation Time: 3-24 Hrs
● Recommended for routine post-mortem tissues
FORMOL-CORROSIVE (FORMOL-SUBLIMATE)
ALDEHYDE FIXATIVES: FORMALDEHYDE
90 mL Sat aq Mercuric Chloride
10 mL Formaldehyde
FORMOL-CORROSIVE (FORMOL-SUBLIMATE)
ALDEHYDE FIXATIVES: FORMALDEHYDE
Polymerized form of formaldehyde usually obtained as a fine white powder (precipitate), which depolymerizes back to formalin when heated
PARAFORMALDEHYDE
ALDEHYDE FIXATIVES: FORMALDEHYDE
It is suitable for paraffin embedding and sectioning and also for immunocytochemical analysis
PARAFORMALDEHYDE
ALDEHYDE FIXATIVES: FORMALDEHYDE
also be stained for general histology, but the degree of fixation is less vigorous than Bouin’s, so the quality of the morphology obtained will be less
PARAFORMALDEHYDE
ALDEHYDE FIXATIVES: FORMALDEHYDE
a mixture of paraformaldehyde and glutaraldehyde.
Karnovsky’s Fixative
ALDEHYDE FIXATIVES: FORMALDEHYDE
It is suitable for use when preparing samples for light
microscopy in resin embedding and sectioning and for electron microscopy
Karnovsky’s Fixative
ALDEHYDE FIXATIVES
● 2.5% SOLUTION – small tissue fragments and needle
aspirates
● 4% SOLUTION – larger tissues
Glutaraldehyde
ALDEHYDE FIXATIVES: FORMALDEHYDE
T/F: Karnovsky should always be prepared fresh
T
ALDEHYDE FIXATIVES
● Less Irritating to nose and skin
● More Expensive
● Less stable
● Slow penetration
Glutaraldehyde
ALDEHYDE FIXATIVES
● For Routine Light Microscopy, also for EM
● Preserves cellular structures better
Glutaraldehyde
MECHANISM: Mercuric Chloride and Lead Fixatives’ Mercuric salts bind with sulfhydryl groups in acidic solutions
METALLIC FIXATIVE
Chromate Fixatives in water form Cr-O-Cr complexes that
have an affinity for –COOH and –OH groups of proteins,
so that complexes between adjacent molecules are
formed.
METALLIC FIXATIVE
METALLIC FIXATIVE: MERCURIC CHLORIDE
- Included in most compound Fixatives
- Nuclear components are
shown in fine detail
5-7% MERCURIC CHLORIDE
METALLIC FIXATIVE: MERCURIC CHLORIDE
- Excellent Trichome Staining
- Preservation of cell detail in
Tissue Photography
5-7% MERCURIC CHLORIDE
METALLIC FIXATIVE: MERCURIC CHLORIDE
Pigments removed by
treatment with Iodine
solution in 95% alcohol
5-7% MERCURIC CHLORIDE
METALLIC FIXATIVE: MERCURIC CHLORIDE
2.5 g Potassium Dichromate
1 gm Sodium Sulfate
100 mL DH2O
5-7% MERCURIC CHLORIDE
METALLIC FIXATIVE: MERCURIC CHLORIDE
With Glacial Acetic Acid for affinity to nuclear chromatin
ZENKER’S FLUID
METALLIC FIXATIVE: MERCURIC CHLORIDE
Permits Brilliant staining of nuclear and connective
tissue fibers
ZENKER’S FLUID
METALLIC FIXATIVE: MERCURIC CHLORIDE
- Fixation time: 12-24 hrs
- Lyses RBC and can make tissues brittle
ZENKER’S FLUID
METALLIC FIXATIVE: MERCURIC CHLORIDE
For Liver, Spleen, Connective Tissue Fibers, and Nuclei
ZENKER’S FLUID
METALLIC FIXATIVE: MERCURIC CHLORIDE
- Mercuric chloride, 5 gm
- Potassium dichromate, 2.5 gm
- Distilled water, 100 ml
- 40% formaldehyde 5 ml (to be added immediately before use)
ZENKER-FORMOL (Helly’s solution)
METALLIC FIXATIVE: MERCURIC CHLORIDE
- For Pituitary Gland, Bone Marrow and Blood Containing Organs
(e.g. Spleen and Liver) - Preserves Cytoplasmic Granules well
ZENKER-FORMOL (Helly’s solution)
METALLIC FIXATIVE: MERCURIC CHLORIDE
Recommended for Tumor biopsies of skin
HEIDENHAIN’S SUSA SOLUTION
METALLIC FIXATIVE: CHROMATE FIXATIVE
● Usually constituent of a compound fixative
○ NOT a simple fixative
● Preserves Carbohydrates and precipitates all proteins
● May produce sub-oxide precipitates
1-2% CHROMIC ACID
METALLIC FIXATIVE: CHROMATE FIXATIVE
● Preserves Lipids
● Preserves Mitochondria
● For post-chromatization
3% POTASSIUM DICHROMATE
METALLIC FIXATIVE: MERCURIC CHLORIDE
- Excellent Cytologic Fixative
- Fixation time 3-12 hrs
- Penetrates and fixes tissue rapidly and evenly
HEIDENHAIN’S SUSA SOLUTION
METALLIC FIXATIVE: MERCURIC CHLORIDE
● Commonly used for bone marrow biopsies
● Fixation time: 4 – 8 hours
● RAPID FIXATION: 1 1/2-2 hrs
● Unstable
● Can make tissues brittle
● Good and clear nuclear details
B-5 FIXATIVE
METALLIC FIXATIVE: CHROMATE FIXATIVE
● Fixation time 12-48 hrs
● Penetrates well
● Recommended for demonstration of Chromaffin tissues (phaeochromocytoma), Mitochondria, Mitotic Figures, Golgi Bodies, RBC, and Colloid-containing Tissues
● Unstable
● Poor nuclear staining
REGAUD’S FLUID (MULLER’S FLUID)
METALLIC FIXATIVE: CHROMATE FIXATIVE
● Recommended for study of Early Degenerative Processes and tissue Necrosis
● 2.5% Potassium Dichromate
● 40% Formaldehyde
● Fixation time: 36-72 hours
ORTH’S FLUID
METALLIC FIXATIVE: LEAD FIXATIVE
● Recommended for Acid Mucopolysaccharides
● Fixes Connective Tissue Mucin
● Takes up CO2 to form insoluble lead carbonates on standing
4% LEAD ACETATE
PICRIC ACID FIXATIVES
● Excellent for Glycogen Demonstration
● Penetrates and fixes small tissue rapidly
● Suitable for Aniline Stains
1% PICRIC ACID SOLUTION
● MECHANISM is still unknown
● Yellow color, which can be removed by lithium carbonate or washing with 50-70% ethanol
PICRIC ACID FIXATIVES
PICRIC ACID FIXATIVES
● With 37% Formaldehyde, Picric acid, Ethanol or Isopropyl Alcohol, and Trichloroacetic Acid
● Less messy than Bouin’s solution
BRASIL’S ALCOHOLIC PICROFORMOL FIXATIVE
PICRIC ACID FIXATIVES
● is recommended for fixation of embryos and pituitary biopsies
● Preferred fixative for connective tissue staining
BOUIN’S SOLUTION
PICRIC ACID FIXATIVES
● Yellow stain is useful for Fragmented Biopsies
● Minimal distortion of microanatomical structures
● Excellent for soft and delicate tissues
BOUIN’S SOLUTION
PICRIC ACID FIXATIVES
● Causes RBC hemolysis
● Not Suitable for Frozen Sections – causes it to crumble when cut
● Prolonged fixation makes tissue hard
1% PICRIC ACID SOLUTION
● Normally used in conjunction with other fixatives to form a compound solution
● It causes tissues (especially those containing collagen) to
swell.
GLACIAL ACETIC ACID
PICRIC ACID FIXATIVES
● Excellent for Glycogen
● Overnight tissue fixation by automatic processing
technique may utilize 3-4 changes of Brasil’s fixative at 1/2 to 2 hours each, succeeded directly by absolute alcohol.
BRASIL’S ALCOHOLIC PICROFORMOL FIXATIVE
ALCOHOL FIXATIVES
May be used as simple fixative
● Usually incorporated into compound fixatives for better results
● Fixes blood, tissue films, and smears
70-100% ETHANOL
● Fixes and precipitates nucleoproteins, Chromosomes and
Chromatin Materials
● For Nuclear Component studies
GLACIAL ACETIC ACID
MECHANISM: Rapidly denatures and precipitates proteins by destroying hydrogen bonds to stabilize the tertiary structure of proteins
Used both as Fixative and Dehydrating Agent
ALCOHOL FIXATIVES
ALCOHOL FIXATIVES
● The most rapid fixative (1-3 hrs only)
● Fixes and dehydrates at the same time
● Used to fix brain for diagnosis of rabies
CARNOY’S FLUID
ALCOHOL FIXATIVES
Recommended for fixing dry and wet smears, blood
smears and bone marrow tissues
100% METHANOL
ALCOHOL FIXATIVES
● Preserves Nucleoproteins and Nucleic Acids for Histochemistry and Enzyme studies
● Dissolves fat and lipids
● May shrink tissues
70-100% ETHANOL
ALCOHOL FIXATIVES
● May cause RBC lysis
● Shrinks and may harden tissues excessively
● Slow penetration
CARNOY’S FLUID
ALCOHOL FIXATIVES
FORMULA:
Ethanol (absolute) 75 ml
Acetic acid glacial 25 ml
Clarke’s solution
ALCOHOL FIXATIVES
- Fixation time: 1 - 6 hours
- Recommended Applications
- It is sometimes used to fix diagnostic cryostat sections. If used for
primary fixation, the specimens can be placed directly into 95% ethanol for processing.
Formol-acetic alcohol
ALCOHOL FIXATIVES
FORMULA:
* Ethanol absolute: 85 ml
* 40% formaldehyde: 10 ml
* Acetic acid glacial: 5 ml
Formol-acetic alcohol
ALCOHOL FIXATIVES
* Fixation time: 3 - 4 hours
* Has been used on frozen sections and smears
Clarke’s solution
ALCOHOL FIXATIVES
Gendre’s Fixative if with picric acid
○ 95% Ethanol
○ 40% Formaldehyde
○ Glacial Acetic Acid
Alcohol Formalin
ALCOHOL FIXATIVES
Post-fixation with phenol-formalin for 6 hours or more can enhance immunoperoxidase studies
Alcohol Formalin
ALCOHOL FIXATIVES
Useful for Sputum as it coagulates mucus
Gendre’s
ALCOHOL FIXATIVES
New alcohol
○ Isopropanol is used instead of the commonly used ethanol
Newcomer’s fluid
ALCOHOL FIXATIVES
Acts both as Nuclear and Histochemical Fixative
Newcomer’s fluid
permanganate fixatives
(potassium permanganate), potassium dichromate,
chromic acid, and osmium tetroxide
OXIDIZING AGENTS
ALCOHOL FIXATIVES
● Produces better reaction in Feulgen stain
○ Feulgen stain is used for staining DNA or nucleus
● Recommended for fixing mucopolysaccharides
● Fixation time: 12-18 hours at 3°C
Newcomer’s fluid
react with various side chains of proteins and other biomolecules, allowing the formation of crosslinks that stabilize tissue structure but cause extensive denaturation despite preserving fine cell structure and are used mainly as secondary fixatives
OXIDIZING AGENTS
OXIDIZING AGENTS
MECHANISM: Various hypotheses of lipid stabilization have been postulated
■ Oxidation of double bonds between adjacent carbon atoms to form monoesters and diesters
■ Binding of lipid to protein
■ Conversion of unsaturated fatty acids to stable glycol osmates.
OSMIUM TETROXIDE
OXIDIZING AGENTS: OSMIUM TETROXIDE
Fixes conjugated fats and lipids permanently by making them insoluble to alcohol and xylene during dehydration and clearing
6% OSMIUM TETROXIDE
OXIDIZING AGENTS: OSMIUM TETROXIDE
● Preserves cytoplasmic structures well
● Fixes materials for Ultrathin Sectioning in Electron Microscopy
6% OSMIUM TETROXIDE
OXIDIZING AGENTS: OSMIUM TETROXIDE
Drawback: forms black precipitates upon exposure to sunlight
6% OSMIUM TETROXIDE
OXIDIZING AGENTS: OSMIUM TETROXIDE
● With Glacial Acetic Acid for Nuclear affinity
● With 1% Chromic Acid as diluent
● Fixation time: 24-48 hrs (1-2 days)
FLEMMING’S SOLUTION
OXIDIZING AGENTS: OSMIUM TETROXIDE
● Recommended for nuclear preparation of such
sections
● Permanently fixes fat
● Excellent fixative for nuclear structures (e.g.
Chromosomes)
● Expensive
FLEMMING’S SOLUTION
OXIDIZING AGENTS: OSMIUM TETROXIDE
Recommended for cytoplasmic structures particularly the mitochondria
FLEMMING’S SOLUTION W/O GLACIAL ACETIC ACID
OTHERS
● Sometimes incorporated into compound fixatives
● It precipitates proteins
● Softening Effect on Dense Fibrous Tissues
● May be used as a weak decalcifying agent
● Poor penetration
TRICHLOROACETIC ACID
OTHERS
● This could be a backup alternative solution for
glutaraldehyde intended for electron microscopy; hence, it is used in cold temperatures.
● Dissolves fat
● Shrinks tissues excessively
● Used as a fixative and dehydrating agent
ACETONE
OTHERS
● Used at ice cold temperature (-5 to 4 degrees Celcius)
● Recommended for the study of water-diffusible enzymes (i.e., Phosphatases and Lipases)
● Used in Fixing Brain Tissues for diagnosis of Rabies
ACETONE
OTHERS
Stable medium for transport of fresh unfixed tissues, such
as renal, skin, and oral mucosa biopsies, which will
undergo subsequent frozen section and
immunofluorescence studies
MICHEL’S SOLUTION
OTHERS
Not suitable for transporting cells for flow cytometry or for tissues used for fluorescent in-situ hybridization (FISH)
MICHEL’S SOLUTION
OTHERS
● Specimens may be kept in it at room temperature for 5
days while in transport
● Not a fixative per se but a transport media
MICHEL’S SOLUTION
MECHANISM:
○ Involves Thermal Coagulation of tissue protein
○ For Rapid diagnosis
○ Employed for Frozen Tissue Sections and Bacteriologic Smears
○ Physical fixation
HEAT FIXATION
DIFFICULTIES CAUSED BY IMPROPER FIXATION
CAUSE: Failure to fix immediately (the tissue was
probably allowed to dry before fixing); Insufficient
fixative
a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure
b. Failure to arrest early autolysis of cells
DIFFICULTIES CAUSED BY IMPROPER FIXATION
CAUSE: Wrong choice of fixative
a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure
d. Removal of substances soluble in fixing agent
f. Loss or inactivation of enzymes needed for study
DIFFICULTIES CAUSED BY IMPROPER FIXATION
CAUSE: Overfixation
a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure
g. Shrinkage and swelling of cells and tissue structure
DIFFICULTIES CAUSED BY IMPROPER FIXATION
CAUSE: Incomplete washing of fixative
a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure
c. Presence of artifact pigments on tissue sections
DIFFICULTIES CAUSED BY IMPROPER FIXATION
CAUSE: Incomplete fixation
a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure
e. Tissues are soft and feather-like in consistency
DIFFICULTIES CAUSED BY IMPROPER FIXATION
CAUSE: Prolonged fixation
a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure
a. Tissue blocks are brittle and hard
Formalin pigment is a well-known artifact that may be
produced under acid conditions. The pigment may be eliminated or reduced by fixation in phenol-formalin.
FIXATION ARTIFACTS
FIXATION ARTIFACTS
is a well-known artifact that may be produced under acid conditions. The pigment may be eliminated or reduced by fixation in phenol-formalin
Formalin pigment
The use of neutral buffered formalin and phenol-formalin almost completely stops the formation of formalin pigment, which occurs with non-buffered formaldehyde solutions and also fixes tissues more rapidly.
FIXATION ARTIFACTS
FIXATION ARTIFACTS
may be found in surgical specimens, particularly in liver biopsies, associated with an intense eosinophilic staining at the center of the tissue in H&E stained sections. This may be due to partial coagulation of partially fixed protein by ethanol or by incomplete wax impregnation during subsequent histological processing.
“Crush artifact”
