MLT GlobeHistopathology: H&E and routine staining – page 2
45 Histopathology MCQs on H&E and routine staining with answers and explanations.
Why does nuclear chromatin take up basic dyes such as hematoxylin lake or methylene blue?
Phosphate groups of DNA are anionic, so chromatin binds cationic (basic) dyes and is called basophilic. Positively charged proteins in cytoplasm bind acidic dyes such as eosin.
Raising the temperature of a staining solution within reasonable limits usually:
Warmth increases diffusion and reaction rates, so many methods (e.g. Ziehl-Neelsen, some silver stains) use heat. Dye concentration, pH, fixation and time also influence staining.
In staining terminology, a 'lake' refers to:
When a dye combines with its mordant metal it forms a coloured complex called a lake, e.g. aluminium–hematein in alum hematoxylin. The lake then binds the tissue.
Which of these reagents serves as a mounting medium rather than a processing reagent?
Canada balsam is a natural resin used to attach the coverslip. Ethanol dehydrates, xylene clears and paraffin wax is used for embedding.
In regressive hematoxylin staining, sections are deliberately overstained. Which step then determines the quality of the nuclear stain?
Controlled removal of excess dye with acid alcohol leaves crisp nuclei and clear cytoplasm. Over- or under-differentiation spoils the stain; bluing then follows.
A crystal of thymol is added to an eosin solution. Its purpose is to:
Thymol is a preservative that inhibits mould growth in aqueous dye solutions. Acetic acid, not thymol, is added to sharpen eosin staining.
Which preparation is examined without any embedding medium?
Smears are spread directly on slides, fixed and stained. Paraffin, resin and celloidin sections all require embedding to support the tissue.
Which reagent acts as an oxidizing agent in histological staining?
Potassium permanganate oxidizes (bleaching, Gomori reticulin). Sodium sulfite is a reducer and aluminium sulfate a mordant.
A molecule that carries positive or negative net charge depending on the pH of the solution is described as:
Proteins are amphoteric: below their isoelectric point they carry a net positive charge and above it a net negative charge.
Why should a permanent mounting medium have a refractive index of about 1.5?
A mountant with a refractive index close to glass (about 1.52) makes the preparation optically uniform and transparent.
A mounted paraffin section looks cloudy and opaque in patches. The likely cause is:
Trapped air or water under the section scatters light, giving opaque patches; drying the slide properly avoids it.
In regressive haematoxylin staining, which step comes immediately after staining with haematoxylin?
Regressive staining deliberately overstains, then removes excess dye by differentiation before bluing and counterstaining.
Which salt is most often used as the mordant in Harris and Mayer's haematoxylin?
Aluminium from alum forms the dye lake with haematein; mercuric oxide or sodium iodate are oxidants that ripen haematoxylin.
Why are unstained paraffin sections hard to see in a brightfield microscope?
Stains selectively absorb light, colouring components differently and creating the contrast that unstained tissue lacks.
Hematoxylin on its own binds nuclei poorly. Why is a metal salt such as potassium alum or ferric chloride used with it?
Oxidized hematoxylin (hematein) forms a positively charged lake with the mordant metal, which then binds phosphate groups of nuclear DNA. Differentiation is done later with acid alcohol, not by the mordant.
Resinous mounting medium shrinks as its solvent evaporates. What problem results if too little medium is placed under the coverslip?
Because the medium contracts on drying, a slight excess is used so it fills the whole coverslip area. Insufficient medium lets air enter at the margins, which obscures and may fade the section.
Synthetic resinous mountants such as DPX are preferred over natural balsam. Which property would be a DISADVANTAGE, not an advantage?
Good synthetic mountants are neutral, colourless, chemically defined and dry fairly quickly. Acidity, as seen in aging Canada balsam, causes fading of hematoxylin and other basic dyes.
A mountant made from gelatin or gum acacia, sugar, glycerol and a phenol-type preservative belongs to which group?
These water-based media (e.g. glycerin jelly, Farrants' or Apathy's) are used for sections that cannot go through alcohol and xylene, such as fat stains and some fluorescent or metachromatic preparations.
In dye chemistry, the unsaturated groups such as C=C, C=O and N=N that absorb visible light and give a compound its colour are called:
Chromophores create colour; a benzene compound carrying them is a chromogen. Auxochromes are ionizing groups that let the chromogen bind tissue, making it a true dye.
A coloured chromogen cannot stain tissue until it carries an ionizable group that gives affinity for tissue components. This group is the:
Auxochromes (e.g. -NH2, -OH, -COOH, -SO3H) ionize and form salt links with tissue. The chromophore provides colour only; a mordant is a separate metal intermediary.