Reading a Fluorescent Stained slide

Smears stained with the fluorescent dye, Auramine, are read with a Light Emitting Diode Fluorescence Microscope (LED-FM).

• The fluorescent-stained smears are to be read within 24 hours of staining in a dark room as the fluorescent stain fades on exposure to light.
• The slides are to be stored in the slide box to avoid exposure to light. Alternatively, they may be stored wrapped in brown or black paper and kept away from light.
• The slides should not be stored in a fridge as 4°C does not prevent or delay fading of fluorescent dye.

After fluorescent staining, smears are examined at much lower magnifications (typically 200x) than used for Ziehl Neelsen (ZN)-stained smears (1000x). Each field examined under fluorescence microscopy, therefore, has a larger area than that seen with bright field microscopy.

Examination Procedure

The steps include:

1. Switch on the LED-FM in a dark room.

2. Focus the stained slide using a 20x or 40x objective lens and view through the 10x eyepiece (magnification of 200x or 400x).

- Does not require the use of oil immersion.

3. Read smear in a linear pattern.

4. Count bacilli that appear as slender bright yellow fluorescent rods against a dark background in 30-50 fields. Rule out any artefact.

- For a trained and experienced Lab Technician (LT), each smear would take approximately 2 minutes for 30-50 fields or three horizontal sweeps.

Reporting Procedure

The smears are reported as negative/ scanty/ 1+/ 2+/ 3+ per the grading scale (Table).

Table: A Comparison between ZN (1000x) and FM (400x and 200x) for Grading of Smears at Different Magnifications

Abbr: AFB: Acid Fast Bacilli; HPF: High Power Field

Resources

• Training Module (1-4) for Programme Managers and Medical Officers, NTEP, MoHFW, 2020.
• Manual for Sputum Smear Fluorescence Microscopy, RNTCP, MoHFW, 2007.
• Fading of Auramine-stained Mycobacterial Smears and Implications for External Quality Assurance.

Assessment