Decalcification Flashcards
General Considerations for
Routine Acid Techniques
Thickness of the Specimen
1. dense/hard bone:
2. softer tissue:
Duration
1. ideal:
2. dense cortical bone (dense tissues):
Temperature & Heat
1. required temp
2. impairs nuclear staining w/ Van Gieson’s
3. tissues will undergo complete digestion within 24-48hrs
Thickness of the Specimen
1. 2-5mm thick
2. 4-6mm thick
Duration
1. 24-48hrs
2. 14 days
Temperature & Heat
1. 18-30C
2. 37C
3. 55C
General Considerations for
Routine Acid Techniques
Solution Used
1. conc. of solutions: directly proportional to rate of ________
2. _____: affects antigenicity of cells & tissue components
Agitation & Partial/Complete Vacuum
1. vigorous agitation:
2. low speed rotation, rocking, mechanical
stirrer, bubbling air into the solution
- protect tissues but slows down decalcification
- changing of the solution
- ____ volume of the tissue
Solution Used
1. decalcification
2. strong acids
Agitation & Partial/Complete Vacuum
1. sonication
2. gentle fluid agitation
- Additives
- once or twice a day
- 20x the volume of the tissue
General Decalcifying Methods
______: injurious to the organic ground substance of tissue
- principle
______: most common & fastest; rapid decalcifying agent: may inhibit nuclear stains & damage tissues
-1.recommended conc. when used as a simple solution
2. washing of tissue: acid removed by 3 changes of
3. washing slide: brought to water & placed in
4. causes causes _______ discoloration
5. if present in tissues: neutralize w/ ______
6. if present in solution: add ____ to pure conc nitric acid
Nitric Acid Formulations:
1. for silver impregnation of nerve fibers
2. slow-acting decalcifying agent
3. most rapid decalcifying agent (for urgent work)
Use of Acid Solutions/ Acid Decalcification
- Chemical Substitution
5-10% Nitric Acid
1. 5 to 10%
2. 70-90% ethanol
3. 1% aq. lithium
4. spontaneous yellow
5. 5% NaSO4
6. 0.1% urea
- De Castro’s Fluid
- Perenyi’s Fluid
- Phloroglucin-
Nitric Acid
Use of Acid Solutions/ Acid Decalcification other agents
- for small & large pieces of bones & teeth
- concentrated reagent:
- washing: neutralize w/ - slower action, w/ greater distortion
other acids
1. two for minute samples
2. for excellent nuclear and cytoplasmic staining
3. for very minute samples since it is a very weak decalcifying agent
Use of Acid Solutions/ Acid Decalcification other agents
- Formic Acid
- 90%
- 5% sodium sulfate - Hydrochloric Acid
other acids
1. TCA & Fleming
2. Citric Acid-Citrate Buffer
3. Sulfurous Acid
Use of Chelating Agents
- acid but does not work as w/ organic acids
- available in 2 formulations:
- salt at 7.0-7.4 (buffered since pH affects
the removal of calcium)
- pH adjusted to 7.4 using conc hac - duration
- small specimens
- dense cortical bone - pH
- inhibits calcium binding
- optimum binding (alkaline environment)
- allows binding & does not destroy tissue components
- EDTA
2.
- 5-10% EDTA Disodium
- EDTA Tetrasodium - duration
- 1-3 weeks
- 6-8 week - pH
- pH 3
- pH 8
- pH 7.0-7.4
Ion Exchange Resin
- uses ____ + _____
- volume of acid solution:
- 10 % and 20 % RAF:
- 2-3hrs:
- 4-8hrs: - reactivation of used resin
- 2washings w/
- final washing w/ - end-point of decalcification is measured using
- ammonium-sulfonated polysterene + formic acid
- 20-30
3.
- 2-3mm thick
- 5-6mmthick
4.
- N/10 HCl
- distilled water - Physical/ X-
Ray Methods
Electrophoresis
1. (+) charged _____ ions are attracted to a (–) electrode
2. temp
3. uses _____ and _____ as acid solution
- calcium
- 30 to 45 degrees Celsius
- 90%/88% formic acid and conc. HCl
Tests for Complete Decalcification
1. vague and not a reliable method
2. very expensive, most ideal, most sensitive & most reliable method
- DO NOT use for _____ fixed tissues
3. simple, reliable, & convenient method for routine purposes
- + ___ = Incomplete Decalcification
- Physical Test
- Radiologic Test (X-Ray)
- mercuric chloride fixed tissues - Chemical Test
- CALCIUM
