STLS: Staining Sputum Smear

After air drying and heat-fixing of the smear, it needs to be stained for the identification of MTB during microscopy.

The reagents required for the ZN staining procedure are shown in Figure 1. The steps involved in the staining procedure are shown in Figure 2.

Figure 1: Reagents Required for ZN Staining

Figure 2: Steps involved in ZN staining procedure

For a visual representation of the above-mentioned steps, please click the video below.

Resources

Laboratory Diagnosis by Sputum Smear Microscopy, GLI Initiative

Assessment

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 carol-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

After air drying and heat-fixing of the smear, it needs to be stained.

Fluorescence microscopy (FM) staining should always be carried out in a designated area. The reagents required for the FM staining procedure are shown in Figure 1. The steps involved in the staining procedure are shown in Figure 2.

Figure 1: Reagents Required for FM Staining

Figure 2: Steps involved in FM staining procedure

For a visual representation of the above-mentioned steps, please click the video below:

Resources

Laboratory Diagnosis by Sputum Smear Microscopy, GLI Initiative

Assessment

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