FIXATIVES Flashcards
Procedure adopted to kill, harden and preserve materials for microscopic study, by means of a FIXATIVE
FIXATION
Fats, mucin, glycogen
Formaldehyde (Formalin)
CNS tissues and general post-mortem tissues
10% Formol-Saline
Preservation and storage of surgical, post-mortem and research specimens.
10% Neutral Buffered Formalin
Best fixative for tissues containing iron pigments
10% Neutral Buffered Formalin
Routine post-mortem tissues
Formol-Corrosive (Formol-Sublimate)
Routine light microscopy, and also electron microscopy
Glutaraldehyde
MOST COMMON FIXATIVE
Mercuric Chloride
Renal tissue, connective tissue, muscle, and fibrin
Mercuric Chloride
Small pieces of liver, spleen, connective tissue fibers and nuclei
Zenker’s fluid
Pituitary gland, bone marrow, and blood containing organs such as spleen and liver
Helly’s solution (Zenker-Formol)
Tumor biopsies especially of the skin; it is an excellent cytologic fixative
Heidenhain’s Susa solution
Fixative and decalcifier
Chromic acid
Fixative of choice for carbohydrates
Chromic acid
Preserves lipids and mitochondria
Potassium Dichromate
Chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues. Hemosiderin
Regaud’s (Moller’s) fluid
Study of early degenerative processes and tissue necrosis
Orth’s fluid
Acid MPS, fixes connective tissue mucin
Lead Fixatives
Glycogen
Picric acid
Embryos
Bouin’s solution
Soft and delicate structures such as endometrial curretings
Bouin’s solution
Glycogen
Brasil’s solution
Nuclear components of the cell, fixes and precipitates nucleoproteins, chromosomes and chromatins
Glacial Acetic Acid
Blood and tissue smears; preserves glycogen, nucleoproteins and nucleic acids; used for enzyme studies
70-100% Ethyl alcohol
MPS and nuclear proteins
Newcomer’s fluid
Fixation time and temperature using Newcomer’s fluid
12-18 hours; 18degCel
Fats, adrenal glands, myelin, peripheral glands, cytoplasmic structures
Osmium Tetroxide (Osmic Acid)
Fixative and weak decalcifying agent. Precipitates protein, softens dense fibrous tissues
TCA
Chromosomes, lymph glands and urgent biopsies
Carnoy’s fluid
Most common chrome-osmium acetic acid fixative used; nuclear preservation
Flemming’s solution
Fixative and stain
Osmium Tetroxide (Osmic Acid)
Cytoplasmic structures particularly the mitochondria
Flemming’s solution without acetic acid
Composed of chromic acid and osmic acid
Flemming’s solution without acetic acid
Study of water diffusible enzymes (phosphatases, lipases); fixes brain tissues for diagnosis of rabies
Acetone
Frozen tissue sections and bacteriologic smears
Heat Fixation
Most common type of fixatives
Aldehydes
Satisfactory for routine paraffin sections, electron microscopy, and when histochemical and enzyme studies are indicated
Aldehyde Fixatives
Simple fixatives
AMPOTAAAH
Aldehyde
Metallic fixatives
Picric acid
Osmiuim tetroxide
TCA
Acetic acid
Acetone
Alcohol
Heat fixatives
Microanatomical fixatives
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10% Formol saline
10% NBF
Formol sublimate
Heidenhain’s susa
Zenker’s fluid
Zenker’s formol
Bouin’s
Brasil
Nuclear fixatives
Flemming’s New Car Has Balls
Flemmings
Newcomers
Carnoy
Heidenhain’s susa
Bouin’s
Cytoplasmic fixatives
FOR Helly’s Fleng
Formalin with post chroming
Orth’s fluid
Regaud’s
Helly’s
Flemming’s fluid without acetic acid
Histochemical fixatives
10 FANAH
10% Formol saline
Acetone
Newcomer’s
Absolute ethanol
Process of placing an already fixed tissue in a second fixative in order
SECONDARY FIXATION
Purpose of Secondary Staining
To improve the demonstration of particular substances
To make special staining techniques possible
To ensure further and complete hardening and preservation of tissues
Secondary fixation where by a primarily fixed tissue is placed in aq. solution of 2.5-3% potassium dichromate for 24 hours
POST-CHROMATIZATION
Process of removing excess fixative from the tissue after fixation in order to improve staining and remove artifacts from the tissues; several solutions may be used
WASHING-OUT
Purpose of Post-Chromatization
To act as mordant for better staining effects
To aid in cytologic preservation of tissue
Solutions are used to remove excess fixative from the tissue after fixation in order to improve staining and remove artefacts from the tissues
Tap water
50-70% alcohol
Alcoholic iodine
Used to remove excess chromatin, formalin, and osmic acid
Tap water
Used to wash out excess picric acid
50-70% alcohol
Used to remove excess mercuric fixatives
Alcoholic iodine
Fixation is enhanced by:
Size and thickness of the tissue
Agitation
Moderate heat (37-56C)
Fixation is retarded by:
Size and thickness of the tissue
Presence of mucus, fat, blood
Cold temperature
Tissue:Fixative ratio
1:20
Special way of preserving tissues by rapid freezing (quenching) and removing water (dessication) by a physicaI process (vacuum drying apparatus)
Freeze-drying
Similar to freeze-drying but instead of being subjected to dehydration, is fixed in Rossman’s fluid (sat. picric acid in 95% alcohol, formaldehyde), or osmium tetroxide in 1% acetone, and dehydrated in absolute alcohol or acetone
Freeze substitution
Requires that the tissue be maintained in the frozen solid state during cutting of section, thereby supporting and protecting the tissue from damage and distortion by the knife during cutting
Fresh frozen section
Principle of Freeze-drying
Rapid freezing of tissue block to produce instant cessation of cellular activity, preventing chemical alteration of tissue constituents and displacement of tissue components;
Why does the the process of freezing must be rapid?
Freezing must be rapid, within 2-3 seconds to prevent the formation of ice crystal artifacts in tissue blocks, and produce optimum tissue preservation
Freezing agent commonly used
Liquid nitrogen (-160 to -180°C)
Aldehyde Fixatives
Formaldehyde
10% Formol Saline
10% NBF
Formol Sublimate
Glutaraldehyde
Mercurial Fixatives
BeHHZ
Zenker’s fluid
Helly’s solution
Heidenhain’s Susa solution
B-5 fixative
Chromate Fixatives
PORC
Potassium dichromate
Orth’s fluid
Regaud’s fluid
Chromic acid
Picric acid Fixatives
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Bouin’s solution
Brasil’s solution
Gendre’s
Alcohol Fixatives
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100% Methyl alcohol
70-100% Ethyl alcohol
Carnoy’s fluid
Newcomer’s fluid
Chrom-osmium fixatives
Flemming’s solution
Flemming’s solution without acetic acid