Appendix 2. Sputum smear process

2.1 Ziehl-Neelsen staining (hot method)

Equipment

– Gloves and respirator
– Water, distilled or filtered
– Carbol fuchsin
– 3% acid-alcohol (ethanol + hydrochloric acid)
– 0.3% methylene blue

Technique

– Flood the slide with carbol fuchsin (after filtering the carbol fuchsin).
– Gently heat the slide. Begin timing as soon as steam appears; let it steam for 5 minutes, without allowing the slide boil or dry out.
– Gently rinse the slide with distilled or filtered water until the water runs clear; drain.
– Cover the slide with 3% acid-alcohol solution, leave on for 3 minutes, then drain. Repeat this operation 2 or 3 times, until the slide is completely decolourized.
– Rinse the slide with distilled or filtered water, and drain.
– Cover the slide with methylene blue, and leave on for one minute.
– Gently rinse the slide with distilled or filtered water until the water runs clear, then allow to air dry.

Reading

A slide should be examined by an experienced laboratory technician on at least 300 fields (15 minutes on average) before giving a negative result. As a maximum, a technician can read 20-25 slides per day, otherwise quality is likely to suffer.
Tuberculosis bacilli stain red on a blue background, are straight or slightly curved, and often cluster in groups of 3 to 10.

Reporting

There are two types of grading scales: one by the Word Health Organization and the International Union against Tuberculosis and Lung Disease (WHO-IUATLD) and one by the US Centre of Disease Control and American Thoracic Society (CDC-ATS). Each field is a high powered field (HPF).

Grading AFB scale (WHO-IUATLD)

Number of acid-fast bacilli (AFB)

Report

No AFB

0

1−9 AFB per 100 fields

Scanty (report number of AFB)

10–99 AFB per 100 fields

1+

1–10 AFB per field

2+

More than 10 AFB per field

3+

Note that seeing 1-9 AFB per 100 HPF is reported as “scanty” and the exact number of bacilli should be recorded. For example, a report of “scanty 3” means there were three AFB seen per 100 HPFs and is not the same as a report of “AFB 3+”. Scanty results are considered as positive result.

Grading AFB scale (CDC-ATS)

Number of AFB

Report

0

Negative

1−2 AFB per 300 fields

+/-

1–9 AFB per 100 fields

+

1–9 AFB per 10 fields

++

1–9 AFB per 1 field

+++

More than 9 AFB per 1 field

++++

2.2 Auramin O staining

Equipment

– Gloves and respirator
– Water, distilled or filtered
– 0.1% auramin O solution
– 0.5 % acid alcohol
– 0.5% potassium permanganate solution
– Fluorescence microscope (or a LED device that can be attached to a standard light microscope)

Technique

– Flood the smear with 0.1% auramin solution and allow staining for 15 minutes ensuring that smears remain covered with stain.
– Rinse with distilled or filtered water until water runs clear and drain excess water from the slide. Do not use water containing chlorine (risk of interference with the fluorescence).
– Flood the smear with 0.5% acid-alcohol for 2 minutes to decolourize it.
– Rinse with distilled or filtered water and drain excess water from the slide.
– Flood the smear with 0.5% potassium permanganate solution and allow to counterstain for 2 minutes. Time is critical because counterstaining for longer may quench the fluorescence of AFB.
– Rinse with distilled or filtered water and drain excess water from the slide. Wipe the back of the slide with tissue paper.
– Allow smears to air-dry. Read as soon as possible after staining.

Note: to control the quality of the colouration, it is essential to include at least a known positive smear in the batch.

Reading

– 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.
– Look aspect of smear: black background without debris or artefacts.
– Read one length of the smear (about 40 fields).

Reporting

Number of AFB

Report

0 AFB per 1 length

Negative

1−19 AFB per 1 length

Scanty (report number of AFB)

20–199 AFB per 1 length

1+

5–50 AFB per 1 field

2+

More than 50 AFB per 1 field

3+

Notes:
– The technique need a skilled reader (artefacts are frequent).
– The fluorescence stain remains stable only for 3 days when sheltered from light. Quality control has to be organised accordingly.

2.3 Bleach sedimentation

Equipment

– Gloves and respirator
– 15 ml plastic conical screw capped tube
– 3.5% bleach solution (12° chl)1
– Vortex (optional)
– Transfer pipettes
– Slides

Technique

– Transfer the sputum to the 15-ml tube.
– Add an equal amount of 3.5% bleach.
– Screw the cap back tightly. Shake vigorously until the mixture is homogeneous.
– Let stand for sedimentation at room temperature for 15 to 18 hours.
– Using a pipette, carefully transfer the supernatant to a waste container containing a 1% chlorine solution.
– Mix the sediment with the remaining fluid.
– Transfer 2 drops of the sediment to a slide.
– Make a smear and let it air dry in a horizontal position.
– When the smear is completely dry, fix it by passing the smear through a flame 3 times.



Footnotes
Ref Notes
1

Check the actual available chlorine content of the bleach with a pool tester.