HIS H&E Staining & Coverslipping Flashcards
(37 cards)
Color of Nuclei, cartilage, microorganisms in H&E
Blue-purple to black
Color of collagen and elastin in H&E
Pale pink
Color of cytoplasm in H&E
Shades of pink
Color of muscle fibres in H&E
Dark pink to red
Color of RBCs and Eosinophils in H&E
Bright red
Source of hematoxylin dye
- from heartwood of logwood tree (Haematoxylum campechianum)
T or F: Hematoxylin is a dye
FALSE; hematoxylin is NOT a dye—it is oxidized to form hematin, the dye responsible for its purple color
Natural oxidation
- uses light and air to oxidize hematoxylin to hematin
- 2-4 month process
- stable for years
Ie. Ehrlich’s and Delafield’s hematoxylin
Chemical oxidation
- oxidizes hematoxylin to hematin
- formed instantly
- shorter shelf-life; continues to oxidize hematin to colorless compounds
Ie. Gill’s, Harris’, and Mayer’s hematoxylin
Hematin requires a __ to stain predictably
Hematin requires a MORDANT to stain predictably
What compounds are used as a mordant for Hematin?
Aluminum or iron compounds
- also chrome, tungsten, molybdenum compounds
Why is hematoxylin used at a low pH ?
To suppress non-nuclear staining
NOTE: pH 1.0 - 3.0
Which mordant for hematoxylin is most routinely used ? Why ?
Potassium ALUMINUM sulfate or ammonium ALUMINUM sulfate; produce good nuclear staining, quick and easy to use
Describe staining with Alum Hematoxylin
- potassium aluminum sulfate OR ammonium aluminum sulfate
- stains nucleus brick red
- needs bluing (alkaline) solution = blue-purple nucleus
- used progressively or regressively
List bluing agents
- Lithium carbonate
- dilute ammonia
-Scott’s tap water substitute (magnesium sulfate, sodium bicarbonate, and water)
Regressive staining vs Progressive
Regressive: Over-staining the tissue, followed by differentiation with acid alcohol (removes stain from less desirable tissue elements)
Progressive: No differentiation; uses pre-determined time to stain nucleus without staining other tissue elements
List some factors that determine staining time
- age of solution
- formulation
- progressive vs regressive staining
- fresh/ frozen or fixed tissue
Disadvantage of Alum hematoxylin
Decolorized by acidic stain solutions = CANNOT be used for special staining techniques using acidified stains (Masson trichrome)
Iron hematoxylin uses ferric iron as both the __ and the __.
Iron hematoxylin uses ferric iron as both the MORDANT and the OXIDIZER.
Iron hematoxylin
- uses ferric iron as the mordant + oxidizer
- less stable; over-oxidized within a day = hematoxylin and iron are stored separately and mixed immediately before use
- mostly used progressively
- prolonged staining times
Most frequently used iron hematoxylin formulation
Weigert’s hematoxylin:
A= hematoxylin dye + 95% alcohol
B= aqueous ferric chloride + HCl + dH2O
Form of Eosin mainly used in histology
Eosin Y (yellowish)
Optimal pH for Eosin stain. What can be added to adjust pH ?
Acetic acid = (pH 4.5 - 5.0)
When properly differentiated, eosin alone can stain tissues at least __ shades of __.
When properly differentiated, eosin alone can stain tissues at least THREE shades of PINK to RED).
