Unit 2.2. Types of Fixatives

1. Simple

2. Compound

2 TYPES OF FIXATIVES ACCRDNG. TO COMPOSITION

simple

2 TYPES OF FIXATIVES ACCRDNG. TO COMPOSITION

• only one component substance

compound

2 TYPES OF FIXATIVES ACCRDNG. TO COMPOSITION

• made up of 2 or more fixatives which have been added together

• 10% Formol Saline = Formaldehyde + NaCL + Distilled Water

• Bouin’s Solution = Formaldehyde + Picric Acid + Glacial Acetic Acid

examples of compound fixative

1. Aldehyde Fixatives

2. Metallic Fixatives

3. Picric Acid Fixatives

4. Alcohol Fixatives

5. Osmium Tetroxide/Osmic Acid

6. Trichloroacetic acid

7. Acetone

7 TYPES OF FIXATIVE BASE ON THEIR ACTIVE COMPONENTS (UNDER SIMPLE FIXATIVES)

1. Formaldehyde

• 10% Formol Saline

• 10% NBF/Phosphate-buffered formalin

• Formol Corrosive

• Alcoholic-Formaline

2. Glutaraldehyde

3. Glyoxal

Enumerate Aldehyde Fixatives

1. Mercuric chloride

   • B-5 fixative

   • Heidenhain’s Susa solution

   • Zenker’s fluid

   • Zenker-Formol (Helly’s Fluid)

2. Chromic Fixatives

   • Chromic acid

   • Regaud’s fluid

   • Orth’s fluid

   • Potassium dichromate

3. Lead Fixative

Enumerate Metallic Fixatives

1. Bouin’s solution

2. Brasil’s alcoholic picroformol

Enumerate Picric Acid Fixatives

1. Isopropyl Alcohol (95%)

2. Carnoy’s fluid

3. Ethyl Alcohol (70-100%)

4. Methyl Alcohol (100%)

5. Newcomer’s fluid

Enumerate Alcohol Fixatives

1. Flemming’s solution

2. Flemming’s solution w/out acetic acid

Enumerate Osmium tetroxides (Osmic acid)

Formaldehyde

ALDEHYDES:

• most widely used fixative

• a gas produced by the oxidation of methyl alcohol

• soluble in water to the extent of 37-40% weight in volume

room temp

ALDEHYDES:

• formaldehyde fixation is performed at

• allergic rhinitis

• sinusitis

• excessive lacrimation

ALDEHYDES:

• formaldehyde fumes are irritating to the nose and eyes and can caue

True

T or F

Concentrated solutions of formaldehyde must never be neutralized since this might precipitate violent explosions

10% Formol-Saline

DILUTED FORMS OF FORMALDEHYDE:

• recommended for fixation of general and post-mortem tissues for central nervous system

• 1:10 dilution of the concentrated formalin

10% NBF

DILUTED FORMS OF FORMALDEHYDE:

• BEST general purpose tissue fixative

ph 7

phosphate buffer

in 10% NBF, formaldehyde is usually buffered at _____ with _____________

• 900 ml tap/distilled water

• 100 ml 37-40% concentrated formaldehyde

• 4 g sodium phosphate (monobasic, monohydrate)

• 6.5 g sodium phosphate (dibasic, anhydrous)

  • 1:10 dilution (ten-fold dilution)

How to prepare a 10% NBF

10% NBF

DILUTED FORMS OF FORMALDEHYDE:

• best for fats, surgical speimens, enzyme histochemistry, immunofluorescence & immunochemistry, nervous system tissues containing iron

Formol-Corrosive (Formol sublimate)

DILUTED FORMS OF FORMALDEHYDE:

• best for lipids, neutral fats, phospholipids tissues

• for routine post-mortem analysis

Formaldehyde + mercuric chloride

Formol-Corrosive (Formol sublimate) composition

Ethanol + Picric acid + GAA + Formaldehyde

Alcoholic-Formalin (Gendre’s Fixative) composition

Alcoholic-Formalin (Gendre’s Fixative)

DILUTED FORMS OF FORMALDEHYDE:

• used to fix sputum

• coagulates mucus

• for microincineration techniques (burn tissues into ashes)

add 10% methanol or remove by filtration

FORMALIN PIGMENTS:

• paraformaldehyde removal

Methanol

FORMALIN PIGMENTS:

• _________ acts as preservative, retarding formaldehyde breakdown and prevent the formation of formic acid or precipitation of paraformaldehyde

storage at room temperature

Paraformaldehyde prevention

Acid formaldehyde hematin

FORMALIN PIGMENTS:

• brownish black pigment granules produced by unstable formaldehyde fixatives

Acid formaldehyde hematin

FORMALIN PIGMENTS:

• formed from the reaction of formic acid and hemoglobin

Paraformaldehyde

FORMALIN PIGMENTS:

• white crystalline precipitate due to prolonged storage

buffer formalin at pH 7 with phosphate

FORMALIN PIGMENTS:

• Acid formaldehyde hematin prevention

1. Kardasewitch’s Method

2. Picric acid method

3. Alcoholic potassium hydroxide

4. Lillie’s method

FORMALIN PIGMENTS:

• Enumerate acid formaldehyde hematin removal methods

Kardasewitch's Method

FORMALIN PIGMENTS:

• Acid formaldehyde hematin removal:

  • 70% ethyl alcohol + 30% ammonia water → wash with water

Picric acid method

FORMALIN PIGMENTS:

• Acid formaldehyde hematin removal:

  • Saturated picrric acid → wash with running water

Lilie’s method

FORMALIN PIGMENTS:

• Acid formaldehyde hematin removal:

  • Hydrogen peroxide + acetone + ammonia water → wash with 70% alcohol

Glutaraldehyde

ALDEHYDES:

• made up of 2 formaldehyde residues linked by 3 carbon chains

osmium tetroxide

Glutaraldehyde fixation is followed by secondary fixation in ______________

Karnovsky’s Paraformaldehyde–Glutaraldehyde Fixative

ALDEHYDES:

• best for EM

• glutaraldehyde + acrolein

Glyoxal

ALDEHYDES:

• supplied as 40% aqueous solution

• fast-acting fixative

• smallest aldehyde fixative

36 hrs

45 mins

ALDEHYDES:

• In glyoxal fixation, surgical specimens are fixed within _____ & small biopsy speimens within _____

Mercuric chloride

METALLIC FIXATIVES:

• most common metallic fixative

• permits B-chromatic staining of cells

Mercuric chloride

METALLIC FIXATIVES:

• recommended for renal biopsies

• routine fixative of choice for preservation of cell detail in tissue photography

Dezenkerization

METALLIC FIXATIVES:

• mercurial fixatives used in ____________ must not go through drain disposal

Dezenkerization

• sodium thiosulfate treatment

• wash tissues w alcoholic iodine prior to staining

METALLIC FIXATIVES:

• how to remove black mercuric deposits?

Mercuric chloride

Chemical fixative most suitable for trichrome stains

B5 fixative

METALLIC: MERCURIC CHLORIDE FIXATIVES:

• for cytology of bone marrow biopsy

Heidenhain's Susa Fixative

METALLIC: MERCURIC CHLORIDE FIXATIVES:

• mainly for tumor skin biopsies

• excellent cytologic fixative

Heidenhain's Susa Fixative

METALLIC: MERCURIC CHLORIDE FIXATIVES:

• the only metallic fixative that does not produce mercuric chloride deposits

Zenker’s fluid

METALLIC: MERCURIC CHLORIDE FIXATIVES:

• for small spx of spleen

• contains

  • mercuric chloride

  • potassium dichromate

  • sodium sulfate

  • distilled water

  • Glacial acetic acid

Zenker’s Formol (Helly’s Solution)

METALLIC: MERCURIC CHLORIDE FIXATIVES:

• excellent microanatomic fixative for pituitary gland, bone marrow, blood, spleen, liver

Zenker’s Formol (Helly’s Solution)

METALLIC: MERCURIC CHLORIDE FIXATIVES:

• contains mercuric chloride, potassium dichromate, sodium sulfate, distilled water, and strong (40%) formaldehyde

1. Schaudinn's Ohlmacher

2. Carnoy-Lebrun Solution

Other 2 Metallic Fixatives

chromic acid (2%)

CHROMATE FIXATIVES:

• precipitates all proteins and adequately preserves carbohydrates

Regaud’s Fluid (Meuller’s Fluid)

CHROMATE FIXATIVES:

• for demonstration of chromatic mitochondria, mitotic figures, RBCs, chromatin, chromatin and colloid-containing tissues

Orth’s fluid

CHROMATE FIXATIVES:

• for the study of rickettsiae and other bacteria

• detection of early degenerative processes & tissue necrosis

3% potassium dichromate

CHROMATE FIXATIVES:

• best for lipids and mitochondria

Lead Fixative

METALLIC FIXATIVES:

• aqueous solution of basic lead acetate

• fixes connective tissue mucin and is recommended for acid mucopolysaccharides

Picric Acid Fixatives

PICRIC ACID 7 TYPES OF FIXATIVE BASE ON THEIR ACTIVE COMPONENTS (UNDER SIMPLE FIXATIVES)

• excellent fixative for glycogen demonstration

• highly explosive when dry, so must be kept moist with distilled water or saturated alcohol during storage

Picric Acid Fixatives

7 TYPES OF FIXATIVE BASE ON THEIR ACTIVE COMPONENTS (UNDER SIMPLE FIXATIVES)

• yellow stain taken in by tissues prevents small fragments from being overlooked

Picric Acid Fixatives

7 TYPES OF FIXATIVE BASE ON THEIR ACTIVE COMPONENTS (UNDER SIMPLE FIXATIVES)

• the only substance than can fix, differentiate, and stain all by itself

• a fixative, stain, & decalcifying agents

Bouin’s Solution

PICRIC ACID FIXATIVES:

• for fixation of embryos and pituitary biopsies

• excellent in preserving soft and delicate structures such as endometrial curettings

kidney structures

lipid

mucus

+++ fuelgen reaction

PICRIC ACID FIXATIVES:

• Bouins solution is not suitable for fixing

Formaldehyde

Picric Acid

GAA

PICRIC ACID FIXATIVES:

• Bouins solution composition

Brasil's Alcoholic Picroformol

PICRIC ACID FIXATIVES:

• better and less messy Bouin’s solution

• Excellent for glycogen

• Wash w/ saturated solution of lithium carbonate and 70% alcohol

• wash w/ water so on

PICRIC ACID FIXATIVES:

• Brasil's Alcoholic Picroformol stains tissues yellow. What is the remedy?

Hollande's Solution

PICRIC ACID FIXATIVES:

• used in GIT biopsies and so on

• modification of Bouin's fluid containing copper acetate

95% Isopropyl Alcohol

ALCOHOLIC FIXATIVES:

• for fixing touch preparations for certain special staining procedures such as Wright-Giemsa Stain

• both a fixative and dehydrating agent

Carnoy’s Fluid

ALCOHOLIC FIXATIVES:

• most rapid; 1-3 hrs fixation

• fixes & dehydrates at the same time

Carnoy’s fluid

ALCOHOLIC FIXATIVES:

• recommended for fixing chromosomes, lymph nodes, urgent biopsies, abd brain for diagnosis of rabies

GAA

Absolute Alcohol

Chloroform

ALCOHOLIC FIXATIVES:

• Carnoy’s fluid composition

100% Methyl Alcohol

ALCOHOLIC FIXATIVES:

• excellent for fixing dry and wet smear, blood smears, and bone marrow smears

Newcomer’s fixative

ALCOHOLIC FIXATIVES:

• reco for fixing mucopolysaccharides and nuclear proteins

• both a nuclear and histohemical fixative

70-100%

• lower concentrations leads to lysis of cells

ALCOHOLIC FIXATIVES:

• concentration range of alcoholic fixatives

Glycogen Polarization

ALCOHOLIC FIXATIVES:

• Disadvantage of Alcoholic fixatives

OSMIUM TETROXIDE (OSMIC ACID)

7 TYPES OF FIXATIVES BASE ON THEIR ACTIVE COMPONENTS (UNDER SIMPLE FIXATIVES)

• slow-acting fixative

• quite expensive

• for EM

• kept in a dark-colored, chemically clean bottle to prevent evaporation and reduction by sunlight

• not compatible with H&E staining because it inhibits Hematoxylin

• causes conjunctivitis

7 TYPES OF FIXATIVES BASE ON THEIR ACTIVE COMPONENTS (UNDER SIMPLE FIXATIVES)

• Major disadvantage of Osmium Tetroxide

Fleming’s Solution with GAA

OSMIUM TETROXIDE (OSMIC ACID):

• most common osmium tetroxide fixative

• recommended for nuclear section preparation

• excellent fixative for nuclear structures such as chromosomes

Fleming’s Solution with GAA

OSMIUM TETROXIDE (OSMIC ACID):

• 5-10:1 ratio

• fixes myelin, peripheral nerves, neurological tissues

Fleming’s Solution without GAA

OSMIUM TETROXIDE (OSMIC ACID):

• reco for cytoplasmic structures particularly the mitochondria

Glacial Acetic Acid

7 TYPES OF FIXATIVES BASE ON THEIR ACTIVE COMPONENTS (UNDER SIMPLE FIXATIVES)

• fixes and precipitates the nuclear proteins

• It precipitates chromosomes and chromatin materials; hence useful in the study of nuclear components of cells

• causes cells to swell

17°C

7 TYPES OF FIXATIVES BASE ON THEIR ACTIVE COMPONENTS (UNDER SIMPLE FIXATIVES)

• Glacial Acetic Acid solidies at ____

Trichloroacetic Acid (TCA)

7 TYPES OF FIXATIVES BASE ON THEIR ACTIVE COMPONENTS (UNDER SIMPLE FIXATIVES)

• for precipitation of proteins and nucleic acids

• A fixative & a weak decalcifying agent

• not a routine fixative

Acetone

7 TYPES OF FIXATIVES BASE ON THEIR ACTIVE COMPONENTS (UNDER SIMPLE FIXATIVES)

• Fixative and dehydrating agent

• for preservation of enzymes (lipase, phosphatase)

• fixes brain for rabies diagnosis

ice cold temperature (4-5°C)

Acetone temp requirement

• dissolves fats

• evaporates easily

• shabu component xD

7 TYPES OF FIXATIVES BASE ON THEIR ACTIVE COMPONENTS (UNDER SIMPLE FIXATIVES)

• Acetone disadvanatages

1. Microanatomical Fixatives

2. Histochemical Fixatives

3. Cytological Fixatives

3 FIXATIVES ACCRDNG. TO ACTION/PURPOSE

Microanatomical Fixatives

3 FIXATIVES ACCRDNG. TO ACTION/PURPOSE

• Permit general microscopic study of tissue structures without changing patterns and normal structures

osmium tetroxide

hematoxylin

3 FIXATIVES ACCRDNG. TO ACTION/PURPOSE

• Microanatomical Fixatives should never contain _________________ because it inhibits ______________

• 10% formol saline

• 10% NBS

• Heidenhain’s Susa

• Formol Sublimate (Formol Corrosive)

• Zenker’s Solution

• Zenker-Formol (Helly’s Solution)

• Bouin’s Solution

• Brasil’s Solution

3 FIXATIVES ACCRDNG. TO ACTION/PURPOSE

• Microanatomical Fixatives examples

Histochemical Fixatives

3 FIXATIVES ACCRDNG. TO ACTION/PURPOSE

• preserve the chemical constituents of cells and tissues (enzymes like phosphatase, lipase)

• 10% Formol-Saline

• Absolute Ethyl Alcohol

• Acetone

• Newcomer’s solution

3 FIXATIVES ACCRDNG. TO ACTION/PURPOSE

• Histochemical Fixatives Examples

Cytological Fixatives

3 FIXATIVES ACCRDNG. TO ACTION/PURPOSE

• preserves nucleus or cytoplasm parts and particular microscopic elements of the cell

3.1. Nuclear fixatives

3.2 Cytoplasmic Fixatives

2 SUBTYPES OF CYTOLOGICAL FIXATIVES

nuclear fixatives

2 SUBTYPES OF CYTOLOGICAL FIXATIVES

• preserve the nuclear structures (chromatin, chromosomes)

• usually contains GAA due to its affinity for nuclear chromatin

pH less than 4.6

2 SUBTYPES OF CYTOLOGICAL FIXATIVES

• pH requirement of nuclear fixatives

• Bouin’s,

• Fleming’s with GAA,

• Carnoy’s,

• Newcomer’s,

• Heidenhain’s Susa solution

2 SUBTYPES OF CYTOLOGICAL FIXATIVES

• Nuclear fixatives examples

Cytoplasmic Fixatives

2 SUBTYPES OF CYTOLOGICAL FIXATIVES

• preserves the cytoplasmic structures

GAA

mitochondria

golgi bodies

2 SUBTYPES OF CYTOLOGICAL FIXATIVES

• Cytoplasmic Fixatives should never contain _____ which destroys ___________ and ____________

pH of more than 4.6

2 SUBTYPES OF CYTOLOGICAL FIXATIVES

• pH requirement of Cytoplasmic Fixatives

• Fleming’s fluid without GAA

• Formalin with post-chroming

• Orth’s fluid

• Helly’s fluid

• Regaud’s fluid

2 SUBTYPES OF CYTOLOGICAL FIXATIVES

• Cytoplasmic Fixatives examples

• 4% formaldehyde

• Formol saline

Best fixative for Enzyme histochemistry