ZN Microscopy: Properties of a well stained slide

Characteristics of a Well-stained Slide

• Staining and appearance of Mycobacterium species in ZN staining.
  • The primary dye - carbol-fuschin stains all cells pink in sputum samples.
  • The bacilli retain the primary pink carbol-fuschin on decolourisation with acid-alcohol.
  • Epithelial, pus and mucous cells in the sputum decolourise on the addition of the acid-alcohol and take up the counterstain dye of methylene blue.
  • Thus, Mycobacterium species appears pink against a background of epithelial, pus and mucous cells that appear blue.
• ZN-stained Mycobacterium species are visible as pink, long, slender rods with granules or V-shaped or in clumps.
• Mycobacterium species should not be stained too dark or pale pink.
  • Possible reasons:
    • Smear thickness is not appropriate
    • Insufficient decolourisation
    • Low acid concentration
    • Carbol-fuchsin dries on smear
    • Primary staining/ decolourisation time not appropriate
    • Expired reagents used
  • Possible solutions:
    • Internal quality control of prepared smears and stains
    • Use a stopwatch to time staining steps.
    • Do not overheat carbol-fuchsin.
• The counter-stained cells should not be dark blue.
  • Possible reasons:
    • Smear thickness is not appropriate
    • Excessive counterstaining time
    • Inadequate washing after counterstaining
    • Methylene blue concentration is not appropriate
  • Possible solutions:
    • Internal quality control of prepared smears and stains
    • Use a stopwatch to time staining steps
• There should not be any deposits on stained smears.
  • Possible reasons:
    • Stains not filtered
    • Slides not clean
  • Possible solutions:
    • Internal quality control of prepared stains
    • Filter stains
    • Use clean, fresh, unscratched slides for smear preparation.

Resources

1. Laboratory Diagnosis of Tuberculosis by Sputum Smear Microscopy - The Handbook, GLI, 2013.
2. Module for Laboratory Technicians, RNTCP, CTD, 2005.

Assessment