Fluorescent Microscopy: Properties of a well stained slide

Light-emitting Diode Fluorescence Microscopy (LED-FM) utilises the fluorescent dye Auramine-O to stain and detect Mycobacterium species in clinical samples.

Characteristics of a Well-stained Slide

• Auramine-O stained Mycobacterium species are visible as bright yellow/ green long slender rods, slightly curved, with variable lengths, single or in clumps, with uniform staining or granular appearance against a dark background (Figure A).

• Slides stained with Auramine-O should not have too much background fluorescence (Figure B)

      Possible reasons:

  • Smear thickness is not appropriate
  • Insufficient decolourisation
  • Counterstain too weak
  • Auramine-O dries on the smear

  • Auramine-O not filtered

    Possible solutions:

    • Internal quality control of prepared smears and stains
    • Use a stopwatch to time-staining steps
    • Filter Auramine-O before use
    • Add a sufficient quantity of stains to cover the smear

• Slides stained with Auramine-O should not have pale fluorescence (Figure C)

      Possible reasons:

  • Smear thickness is not appropriate
  • Low Auramine-O concentration
  • Excessive decolourisation time

  • Stained smears exposed to daylight

    Possible solutions:

    • Internal quality control of prepared smears and stains
    • Use a stopwatch to time-staining steps
    • Store Auramine-O and stained slides in the dark
    • Read stained slides as early as possible
• Non-fluorescent yellow/ green coloured bacillary shapes should not be considered as Mycobacterium species.

• Slides stained with Auramine-O may contain stained artefacts/ background debris which are not Mycobacterium species.

   

Figure: Slides stained with Auramine-O showing bright yellow/ green slender rods (A); slide with too much background fluorescence (B); slide with pale fluorescence (C); Source: Laboratory Diagnosis by Sputum Smear Microscopy

Resources

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

Assessment