MLT GlobeHisto & Cyto Techniques: Fixation, processing & staining
94 Histo & Cyto Techniques MCQs on Fixation, processing & staining with answers and explanations.
Wet fixation of Pap smears is routinely done in:
95% ethanol dehydrates and precipitates proteins, preserving crisp nuclear detail. Formalin is a histology fixative, and xylene is a clearing agent.
Smears that must be sent by post to a distant laboratory are best fixed with:
Coating fixatives contain alcohol with a waxy polymer that forms a protective film, so slides travel dry. Air-drying distorts Pap morphology.
Which stain is standard for examining cell detail in exfoliated secretions?
The Pap stain gives transparent cytoplasm and crisp chromatin, showing differentiation and keratinization. The others are special or histology stains.
In a Pap-stained smear, Harris hematoxylin is responsible for:
Hematoxylin stains nuclear chromatin. OG-6 colors keratin orange and EA gives pink and green cytoplasm.
Which property would make a solution UNSUITABLE as a cytology fixative?
A fixative must halt autolysis by inactivating enzymes; a solution that promotes enzyme activity would let cells degrade.
Which statement about handling cytology specimens is correct?
Standard precautions apply to every body-fluid specimen; Pap smears are wet-fixed in alcohol, not formalin, and not all work is urgent.
The most widely used routine fixative in histopathology is:
10% NBF (about 4% formaldehyde) penetrates well and preserves morphology for H&E and most immunohistochemistry. Use at least 10 volumes of fixative per volume of tissue.
Formaldehyde fixes tissue mainly by:
Formaldehyde is an additive, non-coagulant fixative that forms methylene bridges between protein side chains. Alcohols fix by dehydration and coagulation.
What is the recommended minimum ratio of fixative volume to tissue volume for routine formalin fixation?
A fixative-to-tissue ratio of about 15–20:1 ensures enough formaldehyde is available for complete fixation. Smaller volumes become depleted, causing poor fixation.
A fixative containing picric acid, formaldehyde and acetic acid, often used for testis and GI biopsies, is:
Bouin's solution contains picric acid, formaldehyde and acetic acid and gives crisp nuclear detail. Zenker's is a mercuric chloride fixative.
Which fixative is preferred for tissue intended for transmission electron microscopy?
Glutaraldehyde, a dialdehyde, cross-links proteins rapidly and preserves fine ultrastructure. Alcohol-based fixatives cause shrinkage that ruins membrane detail.
A pathologist wants to show neutral lipid droplets in a liver biopsy with Oil Red O. How must the tissue be prepared?
Routine processing uses alcohols and xylene that dissolve lipids. Frozen sections avoid these solvents so fat remains for Oil Red O staining.
What is the correct order of steps in routine paraffin tissue processing after fixation?
Water is removed with graded alcohols, the alcohol is replaced by a clearing agent (e.g., xylene) that mixes with paraffin, then tissue is infiltrated and embedded.
What is the usual section thickness for routine paraffin sections stained with H&E?
Routine paraffin sections are cut at about 3–5 µm. Semithin plastic sections for EM are 0.5–1 µm, and thick sections are used for special purposes such as some nerve work.
In H&E staining, what is the purpose of placing slides in weak ammonia water or Scott's tap water after hematoxylin?
Bluing raises the pH, converting the soluble red hematein-aluminum lake to an insoluble blue form. Removal of excess hematoxylin is done by acid alcohol (differentiation).
Eosin stains cytoplasm pink because it is:
Eosin is an anionic acidic dye that binds to basic (positively charged) groups in cytoplasmic proteins and collagen, called acidophilic structures.
A kidney section stained with Congo red shows deposits that are salmon-pink under ordinary light. What finding under polarized light confirms amyloid?
Congo red-stained amyloid shows apple-green birefringence with polarized light because of its beta-pleated sheet structure. Color alone is not specific.
In Masson trichrome staining, collagen and muscle typically appear:
Masson trichrome stains collagen blue (aniline blue) or green (light green) and muscle and cytoplasm red, useful in liver fibrosis and muscle disease.
Alcohol-based fixatives such as ethanol and methanol act mainly by:
Alcohols remove water and denature (coagulate) proteins without forming cross-links, which can cause shrinkage and hardening. Methylene cross-linking is how formaldehyde works.
The main purpose of the clearing step in paraffin processing is to:
Paraffin does not mix with alcohol, so a clearing agent such as xylene, miscible with both, replaces the alcohol. Water is removed earlier by dehydration in graded alcohols.