Part II: Microscopy
smear preparation procedures

Sputum smear preparation
Smears should be prepared in manageable batches (maximum of 12 per batch). Labelling of smears should be done at the bench for incoming specimens using a permanent marker, eg. a diamond-point stylus or wax pencil. Avoid touching the surface of the slides.

The maximum chance of finding bacilli in unconcentrated specimens is in the solid or most dense particles of the sputum and the results of direct smear examination depend to a great extent on the choice of these particles.

The procedure for smear preparation is presented in Diagram 1 on page 21.

Value of smears on extra-pulmonary specimens
Because M. tuberculosis may infect almost any organ in the body, the laboratory could receive a variety of extra-pulmonary specimens, eg. body fluids, tissues, pus and urine. The benefit of microscopy on these specimens is limited and it is recommended that extra-pulmonary specimens be referred for culture.

Gastric washings
Direct smears should be avoided as the results can be misleading. Acid-fast bacilli are frequently present in food and water and hence in the stomach. There is no way of distinguishing such organisms from tubercle bacilli on microscopy and positive smears must be regarded with suspicion.

Laryngeal swabs
Direct smears are almost useless. A negative result is meaningless and it is best to conserve what little material there is for culture.

Pus and thick aspirates
Direct smears of these specimens should be very thin. Thick smears tend to float off the slide and even if they are retained, acid-fast bacilli may be difficult to see after staining. Problems may arise if a large amount of blood is present in the specimen since blood may sometimes produce acid-fast artefacts.

Pleural fluids
The fluid should be centrifuged and smears prepared from the sediment. Again, these should be thin or they may float off the slide.

Cerebrospinal fluids
Smears from cerebrospinal fluid are rarely positive and sediment from concentrated specimens should rather be cultured. If a smear is desired, two parallel marks, about 10mm long and 2mm apart, should be made on a clean glass slide. A loopful of the sediment is then spread between these marks and the smear allowed to dry. Another loopful is then spread over the first. When this is dry the process may be repeated, depending on how much sediment is available. This procedure clearly marks the area to be searched for acid-fast bacilli. It is desirable that the smears are examined by two independent microscopists. Clots should be saved for culture.

Smears of centrifuged urine deposits are very unreliable and should be avoided. Mycobacteria other than tubercle bacilli are sometimes present in urine, either when it is voided or as a result of poor collection techniques. The presence of acid-fast bacilli in urine should be viewed with suspicion.


Dr Martie van der Walt

Dr Roxanna Rustomjee
E-mail: roxanna.rustomjee@

Prof Valerie Mizrahi
E-mail: mizrahiv@

Prof. Paul van Helden


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