Appendix 4. Sputum smear microscopy

Update: January 2022

4.1 Sputum smear preparation

Staff members present during sputum smear preparation should wear a respirator (FFP2 or N95) to prevent the inhalation of bacilli.

Sputum smears should be prepared promptly after sputum collection.

Equipment

• Gloves 
• New, clean glass slides (never re-use sputum smear slides)
• Wooden applicator sticks  

Technique

• Label one end of the slide with the date of sputum collection and laboratory serial number.
• Select a mucopurulent or blood-stained portion of the sputum specimen.
• Use an applicator stick to transfer to the slide.
• Smear the specimen over an area of 1.5 to 2 cm x 2 to 3 cm. Make it thin enough to be able to read through it. 
• Allow the smear to air dry for 15 minutes. Do not dry the smear in direct sunlight or over a flame.
• Fix the smear by passing the underside of the slide through a flame for 2 to 3 seconds. Repeat 3 or 4 times.  
• Allow to cool before staining.

4.2 Ziehl-Neelsen staining

Equipment

• Gloves 
• Distilled or filtered water
• 0.3% carbol fuchsin
• 3% acid-alcohol
• 0.3% methylene blue
• Binocular microscope with oil immersion objective (100x magnification)

Technique

• Flood the slide with 0.3% carbol fuchsin (after filtering the carbol fuchsin).
• Gently heat the underside of the slide. Begin timing as soon as steam appears. Let it steam for 5 minutes. Do not let the stain boil or dry. 
• Gently rinse the slide until the water runs clear, then drain off excess water.
• Flood the slide with 3% acid-alcohol for 2 to 3 minutes, then drain. Repeat this operation if the slide is not completely decolourised. 
• Gently rinse the slide, then drain off excess water.
• Flood the slide with 0.3% methylene blue for one minute, then drain.
• Gently rinse the slide until the water runs clear, then drain off excess water.
• Allow to air dry. Do not wipe or blot.

Reading

• The slides should be examined by an experienced technician. Technicians must be given sufficient time to accurately read slides.
• Before reading the slide, apply a drop of immersion oil to the left edge of the stained smear. Do not touch the slide with the immersion oil applicator (risk of AFB transfer into the oil bottle and onto another slide).
• Examine at least one length (100 high power fields, HPF) before giving a negative result (this should take at least 5 minutes). 
• AFB are red, straight or slightly curved rods. They may be found singly or in small groups. The background stains blue.

Reporting

Table 4.1 – Grading AFB scale (WHO-IUATLD) [1] Citation 1. European Centre for Disease Prevention and Control. Handbook on tuberculosis laboratory diagnostic methods in the European Union – Updated 2018. Stockholm: ECDC; 2018.
https://www.ecdc.europa.eu/sites/portal/files/documents/TB-handbook-updated-2018.pdf

4.3 Auramine O or auramine/rhodamine staining

Equipment

• Gloves 
• Distilled or filtered (not chlorinated) water
• 0.1% auramine O or auramine/rhodamine solution
• 0.5% acid alcohol
• 0.5% potassium permanganate or 0.3% methylene blue
• Fluorescence microscope (or a LED device that can be attached to a standard light microscope)

Technique

• Flood the slide with auramine O or auramine/rhodamine solution for 15 minutes. Ensure that the staining solution remains on the smear.
• Gently rinse, then drain off excess water. Do not use chlorinated water to avoid disturbing the fluorescence reading.
• Flood the slide with 0.5% acid-alcohol for one minute, then drain.
• Gently rinse, then drain off excess water.
• Flood the slide with 0.5% potassium permanganate solution or 0.3% methylene blue for one minute, then drain. 
• Gently rinse, then drain off excess water.
• Allow to air dry. Do not wipe or blot.

Note: to control the quality of the colouration include at least one known positive smear in the batch.

Reading

• The slides should be examined by an experienced technician (artefacts are frequent). Technicians must be given sufficient time to read slides.
• Use a 20x objective to screen the smear.
• Examine one length before giving a negative result. 
• Always read the positive control smear first. If the positive control is not positive do not continue with the patient smears, but re-stain the batch.
• AFB are bright yellow, straight or slightly curved rods. They may be found singly or in small groups. The background is dark. Non-specific debris stains pale yellow. 

Reporting

Table 4.2 – Grading AFB scale (WHO-IUATLD) [1] Citation 1. European Centre for Disease Prevention and Control. Handbook on tuberculosis laboratory diagnostic methods in the European Union – Updated 2018. Stockholm: ECDC; 2018.
https://www.ecdc.europa.eu/sites/portal/files/documents/TB-handbook-updated-2018.pdf

• 1-29 AFB per length is a positive result. Note that 1-29 AFB per length is reported as "scanty" followed by the exact number of AFB seen per length (e.g. "scanty 3" means there are 3 AFB per length).
  Do not confuse "scanty 3" (3 AFB per length) with AFB 3+ (more than 100 AFB per field).
• The fluorescence stain remains stable when sheltered from light for only 3 days. Quality control should be done within this time.