問題一覧
1
removal of calcium ions from a bone or calcified tissue through a histological process that makes them flexible and easier to cut
decalcification
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done after fixation and before impregnation, to ensure and facilitate the normal cutting of sections
decalcification
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allows sections to be obtained where the presence of calcium was not anticipated when the specimen was processed
surface decalcification
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face down on a pad of cotton or gauze saturated with ______ for approximately one hour.
10% hydrochloric acid
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most widely used agents for routine decalcification of large amounts of bony tissues because they are stable, readily available, and relatively inexpensive as compared to other decalcifying agents
acid decalcifying agents
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Due to the corrosive action of the acid, it is recommended that the thread be dipped in _____
melted paraffin wax
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The most common and the fastest decalcifying agent
nitric acid
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May be used as simple aqueous solutions with recommended concentrations of 5- 10%
nitric acid
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Decalcification time: NITRIC ACID
12-24hrs
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Reappearance of a white precipitate within ___
30mins
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*Reappearance of a white precipitate within __ is what?
presence of calcium ions
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When decalcification is complete, the acid must be removed by three changes of ____
70-90% ethanol
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inferior compared to nitric acid in its role as a decalcifying agent because of its slower action and greater distortion of tissue produced on the decalcified section
hydrochloric acid
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produces good nuclear staining and if used in 1% solution with 70% alcohol
hydrochloric acid
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moderate-acting decalcifying agent which produces better nuclear staining with less tissue distortion, and is safer to handle than nitric acid or hydrochloric acid
formic acid
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recommended for routine decalcification of postmortem research tissues
formic acid
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Best all around decalcifier
formic acid in 10% concentration
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A very weak decalcifying solution suitable only for minute pieces of bone
sulfurous acid
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It is highly corrosive to skin and mucous membranes and carcinogenic
chromic acid
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substances which combine with calcium ions and other salts
chelating agents
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substances which combine with calcium ions and other salts EXAMPLE
iron and magnesium
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preferred if preservation of nuclear DNA is important
chelating agents
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What is the example of chemating agents?
neutral edta
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most common chelating agent
edta and salt (VERSENE)
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optimal pH in edta
7-7.6
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In chelating agents: more appropriate for research applications:
immunohistochemistry flourescent in situ hybridization polymerase chain reaction
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Decalcification time: small bones or specimens
1-3 weeks
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Decalcification time: cortical bone
6-8 weeks
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In chelating agent, The solution should be changed every ___
3 days
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Hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions, thereby increasing solubility from the tissue
ion exchange resin
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Not recommended for fluids containing mineral acids such as nitric acid or hydrochloric acid
ion exchange resin
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The degree of decalcification may then be measured by
physical and x-ray method
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A process whereby positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution
electrophoresis
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In this method, hard tissues are placed in the decalcifying agent in a ____ for intermittent periods with regular changes of the solution till the end point is reached
microwave oven
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___ has been shown to speed up the process of decalcification significantly-from days to hours
microwave irradation
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FACTORS INFLUENCING THE RATE OF DECALCIFICATION
concentration fluid access size and consistency agitation temperature
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Optimal temperature
18-30
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spoils the staining of basophilic elements such as cell nuclei and in certain circumstances can cause maceration of the softer tissue elements
prolonged decalcification
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interfering with the normal cutting of sections and staining of specimens
incomplete decalcification
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Tissue volume for decalcification
20:1
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require manipulation, bending, probing or trimming of the specimen to "feel" for remaining calcified areas
physical test
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Simple, reliable and convenient method recommended for routine purposes, to detect the presence of calcium in the decalcifying solution.
chemical test
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Most expensive but is the most ideal, most sensitive and most reliable method of determining the extent of decalcification due to its ability to detect even the smallest focus of calcium which appears opaque
x-ray
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Water rinsing: small specimen
30mins
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Water rinsing: large specimens
1-4 hrs
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Other tissue softeners
molliflex 2% HCL 1% HCL in 70% alcohol
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37 degrees celsius
nuclear staining of Van gieson’s stain for collagen fibers
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55 degrees celsius
complete digestion within 24-48 hours
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Decalcifying fluid usually change every ___
24-48 hours
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A piece of __ is added to a test tube containing __
blue litmus paper 5ml
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If the litmus paper turn RED due to
acidity of the fluid
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Litmus paper turn to RED-BLUE
Alkalinity
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Presence of cloudiness
presence of calcium
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If solution remains clear after __, decalcification is __
30mins complete
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May act both decalcifyinga agent and tissue softener
perenyi’s fluid
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WHAT ARE THE TISSUE PROCESSING
fixation decalcification dehydration clearing infiltration embedding trimming section cutting staining mounting labelling
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Solutions of chemical test
ammonium hydroxide concentrated ammonium oxalate saturated