FIXATION Flashcards
OVERVIEW:
1. primary purpose
2. secondary purpose
3. capable of inactivating ______
4. becomes part of the cross-link itself
5. removal of water in order for cross-links to form
- Preservation
- Protection
- lysosomes
- Additive
- Non-Additive
Factors to be Considered:
pH:
temp
1. Routine Manual
2. Routine Automated
3. Electron Microscopy
4. Formalin at 60C
5. Formalin at 100C
6. DNA
7. RNA
pH: 6-8
temp
1. room temp 20-22
2. 40C
3. 0-4C
4. very urgent biopsies
5. diagnosis of tuberculosis
6. 65
7. 45
Factors to be Considered:
size & thickness
1. Light Microscopy
2. Electron Microscop
3. Lung Edema
osmolality
1. light microscopy
2. electron microscopy
concentration
1. Formaldehyde
2. Glutaraldehyde
volume
1. Routine
2. Museum
3. Osmium Tetroxide
size & thickness
1. 2cm by 0.4cm
2. 1-2mm^2
3. 1-2cm
osmolality
1. hypertonic (400-450mosm)
2. isotonic (340mosm)
concentration
1. 10%
2. 3%
volume
1. 10-25x
2. 50-100x
3. 5-10x
Tissue/ Organ Preparations
1. Air-filled lungs
2. Hollow Organs
3. brain is suspended by cord tied under _____
- intravascular perfusion using ______
4. eyes should be injected with _____
5. hard tissues: ______ washed out / running
water overnight and immersed in ______
solution for 1-3 days
6. these are stretched / sutures on each end
- cover w/ several layers of gauze
- dilate w/ cotton soaked in fixative
- Circle of Willis
- Ringer’s Lactate - formol-alcohol
- Lendrum’s Method; 4% aq. phenol
- Muscles
Types of Fixatives
_____: preserve specific cellular components
- it is acid with pH
- contains _____ - it is basic with pH
Cytological
- Nuclear; 4.6 or less than
- glacial acetic acid - Cytoplasmic; more than 4.6
Types of Fixatives
_____: preserve chemical constituents
- Lipids
- Phospholipids
- Cholesterol
- Carbohydrates
- Glycogen
- Proteins
- Electron M.
- primary
- secondary
Histochemical
- mercuric chloride
- baker’s formol-calcium
- digitonin
- alcoholic fixatives
- rossman’s fluid/ cold absolute alcohol
- neutral buffered formol saline/ formaldehyde vapor
- Electron M.
- aldehydes
- osmium tetroxide
ACCORDING TO COAGULABILITY
1. ethanol, methanol, acetone
2. cross-linking: aldehyde, metal salts
ACCORDING TO FUNCTION
1. demonstration of general relationship among
cells, tissues, & organs
2. specific cellular details
- dichromate fixatives (2)
Mixture of Fixatives:
1. Karnovsky’s paraformaldehyde-
glutaraldehyde solution
2. mixture of glutaraldehyde or formaldehyde
ACCORDING TO COAGULABILITY
1. Coagulant
2. Non-Coagulant
ACCORDING TO FUNCTION
1. General/ Routine
2. Specific
- Periodic Acid Schiff &
argentaffin
Mixture of Fixatives:
1. Electron Cytochemistry
2. Acrolein
Aldehyde Fixatives
____: routinely used fixative
- gas produced by the oxidation of _______
- buffered at ph =
- hypoxia leads to acidity which favors the formation of ______ heme pigments (black, polarizable deposits) - pure stock: ______; commercial: _______
- dilution: ___ (10% sol’n); ____ (5% sol’n)
- conc. for fixation/working sol’n: ______
- white ppt. due to prolonged storage
- may be removed by filtration or addition of _____
Formaldehyde
- methyl alcohol
- 7-8
- Formalin heme pigments - pure stock: 40% formalin; commercial: 37-40% formaldehdye
- 1:10 (10% sol’n); 1:20 (5% sol’n)
- 10% formalin
- Paraformaldehyde
- 10% methanol
Aldehyde Fixatives:
- for enzymes, nucleoproteins, fats, & mucins
- composed of - best for frozen sections, iron pigments, & elastic fibers
- composed of - for lipids, especially neutral fats and phospholipids
- composed of - the demonstration of immunoperoxidase activity, glycogen,
microincineration; fix sputum
- composed of - electron microscopy
- 2 ____ linked by 3 carbon chains
- used in conjunction with
- 10% Formol-Saline
- Formaldehyde + Sodium Chloride - 10% Neutral Buffered Formalin
- F + Sodium - Formol-Corrosive/Sublimate
- F + Sat. Mercuric Chloride - Alcoholic Formalin/ Gendre’s
- F + Ethyl Alcohol + Gl. Acetic Acid - Glutaraldehyde
- formaldehyde residues
- osmium tetroxide
Metallic Fixatives: Mercuric Chloride
- for small pieces of liver, spleen, CT fibers & nuclei
- removal of mercuric deposits - for pituitary, bone marrow and blood-containing organs
- components - for tumor biopsies of the skin
- for bone marrow biopsies
- components
- Zenker’s Fluid
- removal of mercuric deposits - Zenker-Formol/
Helley’s
- Formaldehyde + Zenker - Heidenhain’s Susa Sol’n
- B-5 Fixative
- Mercury Chloride + F + Sodium Acetate
Metallic Fixatives: Chromate
- for carbohydrates
- for lipids & mitochondria
- for chromatin, mitochondria, mitotic figures, Golgi
bodies, RBC and colloid-containing tissues
- components - for early degenerative processes, tissue necrosis,
Rickettsiae and other bacteria
- components
- Chromic Acid
- Potassium
Dichromate - Regaud’s Fluid/
Moller’s
- Potassium Chromate + F - Orth’s Fluid
- Potassium Chromate + F +/- Sodium Sulfate
demonstration of acid mucopolysaccharides
- used in aq form:
—
Picric Acid
1. for embryos and pituitary biopsies
- components
2. for glycogen
- components
Lead Fixatives
1. 4%
—
Picric Acid
1. Bouin’s Solution
- Picric Acid + F + Gl. Acetic Acid
2. Brasil’s Alcoholic Picroformol Fixative
- Picric Acid + F + EA + TCA
Alcohol Fixatives
1. for glycogen: preserves but does not fix glycogen granules
2. for dry & wet smears, blood and bone marrow samples
3. for touch preparations
4. for chromosomes, lymph glands, urgent biopsies and brain fixation
for rabies diagnosis
- components
5. for mucopolysaccharides & nuclear proteins
6. for glycogen
may be though
- 95% Ethanol
- 100% Methanol
- 95% Isopropyl Alcohol
- Carnoy’s Fluid
- Abs. Alcohol + Chloroform + Gl. Acetic Acid - Newcomer’s
- Gendre’s Fixative
Other Fixatives
- for nuclear structures/ nucleoproteins
- for dense fibrous tissues
- for brain fixation for rabies diagnosis and water-diffusible enzymes
- use at cold temp (___ to ___)
- acts like ___ & ____
- Glacial Acetic Acid
- Trichloroacetic Acid
- Acetone
- (-5 to 40C)
- alcohol and dehydrant coagulate
Washing-Out: Removal of Excess Fixative
- tap water
- 50-70% alcohol
- alcoholic iodide
- chromates (Helly’s, Zenker’s
& Flemming’s), formalin, osmic acid - picric acid (bouin’s solution)
- mercuric fixatives
