MLT GlobeHisto & Cyto Techniques: Fixation, processing & staining – page 5
94 Histo & Cyto Techniques MCQs on Fixation, processing & staining with answers and explanations.
For fluid specimens that cannot be processed promptly, a commonly used pre-fixative is:
50% ethanol slows degeneration while limiting protein coagulation and cell hardening. Stronger alcohols clump proteins and make cells adhere poorly.
Carnoy's fixative is prepared from:
Carnoy's is a 6:3:1 mixture of ethanol, chloroform and glacial acetic acid. Ether-containing mixtures are different fixatives.
Denatured alcohol used as a cytology fixative is typically composed of:
The common formula is 90 parts 95% ethanol, 5 parts absolute methanol and 5 parts absolute isopropanol, which behaves like 95% ethanol.
During intraoperative consultation, a frozen section of breast tissue with much fat crumbles and will not cut at −18 °C. What adjustment is best?
Fat freezes at a lower temperature than other tissues, so fatty specimens need a colder cryostat, about −25 to −35 °C. Warmer settings make fat smear or crumble.
Frozen sections of a muscle biopsy show many small holes caused by ice crystals. How is this best prevented?
Very rapid freezing in isopentane chilled by liquid nitrogen prevents large ice crystals. Slow freezing, or freezing wet tissue, allows crystals to form and damage morphology.
A skin biopsy is suspected of leprosy. Which acid-fast method is preferred to show Mycobacterium leprae?
M. leprae is weakly acid-fast. The Fite method protects its lipid wall with peanut oil during deparaffinization and uses a gentler decolorizer. Strong acid alcohol may decolorize it.
In the Grocott methenamine silver (GMS) stain for fungi, what is the role of chromic acid?
Chromic acid oxidizes fungal wall polysaccharides to aldehydes, which then reduce methenamine silver to black metallic silver. Light green is the usual counterstain.
Alcian blue performed at pH 1.0 stains which mucins?
At pH 1.0 only strongly acidic sulfate groups are ionized, so only sulfated mucins stain. At pH 2.5 both carboxylated and sulfated mucins stain.
The von Kossa technique shows calcium deposits as black because silver ions:
Silver replaces calcium bound to phosphate or carbonate and is reduced by light to black silver. It therefore demonstrates the anions, not calcium itself.
B5 fixative, used for lymph nodes and bone marrow biopsies because it gives crisp nuclear detail, contains:
B5 is a mercury-based fixative with formaldehyde added just before use, favored for hematopoietic tissue. Picric acid–formaldehyde–acetic acid is Bouin's, and ethanol–chloroform–acetic acid is Carnoy's.
A paraffin ribbon curves to one side during sectioning on a rotary microtome. The most likely cause is:
When the upper and lower block edges are not parallel (or the block is wedge-shaped), one side of each section is longer and the ribbon curves. Trimming the edges parallel corrects it.
The Fontana–Masson method shows melanin as black because melanin is argentaffin, meaning it:
Argentaffin substances such as melanin reduce silver solutions by themselves. Argyrophilic structures, such as reticulin, bind silver but need an external reducing agent to show it.
A skin biopsy contains brown-black pigment. To confirm it is melanin, a serial section is treated before staining with:
Melanin is removed by strong oxidizing agents such as potassium permanganate followed by oxalic acid, while hemosiderin and formalin pigment are not bleached this way. Diastase removes glycogen.
Polymer-based immunohistochemistry detection is often preferred over the avidin–biotin complex (ABC) method on liver and kidney mainly because it:
Liver and kidney are rich in endogenous biotin, which can bind avidin and give false-positive staining with ABC. Polymer systems carry enzyme and secondary antibody without biotin.