In addition to the preparation of a direct, acid-fast stained smear, it is recommended that material (usually, but not always, sputum) should be cultured for M tuberculosis whenever this disease is clinically suspected. Some patients, in whom pulmonary tuberculosis is strongly suspected, may not cough up any sputum. A little sputum may, in fact, be produced but is immediately swallowed. In this case, the physician may send a specimen of fasting gastric juice (generally obtained early in the morning) to the laboratory. This should be treated in the same way as sputum. It is expensive to culture all sputum samples routinely for tubercle bacilli (although some unsuspected patients would be discovered), and this is not recommended.
Concentration (decontamination) procedure
Sputum from patients with tuberculosis often contains some solid particles of material derived from the lungs, and this material should be selected for culture whenever it is found. However, even tuberculous sputum is coughed up through the throat and mouth and contamination with the normal flora is inevitable. These contaminating bacteria must be killed, if the Löwenstein-Jensen cultures are not to become overgrown. A concentration (decontamination) procedure is therefore necessary for all specimens collected from a site where a normal flora is present. One of these procedures combines liquifying the sometimes mucoid sputum with destruction of the contaminating organisms using sodium hydroxide solution. However, this is partially toxic even to mycobacteria, and great care needs to be taken to ensure that:
• the final concentration of NaOH does not exceed 2%,
• the tubercle bacilli are not exposed to the sodium hydroxide for longer than 30 minutes, including the centrifugation time.
In a sealable small glass bottle or jar, equal volumes of sputum and 4% (40 g/litre) sodium hydroxide should be mixed together, shaken, and incubated at room temperature (25 - 30 °C) for 15 - 20 minutes. The mixture should be shaken regularly, with care, every 5 minutes (a mechanical shaker may be employed). In very hot countries, some degree of cooling may be needed, or the reaction time may be reduced (for example, to 10 - 15 minutes).
At the end of the 15 - 20 minutes, the specimen should be centrifuged in a sterile tube at high speed (> 13 000 g), but for not more than 10 minutes, or the mycobacteria will be killed by excessive exposure to the alkali. The supernatant should then be carefully discarded and the sediment immediately neutralized by adding, drop by drop, 2 mol/litre HCl containing 20 ml of phenol red solution per litre until the mixture remains pink. The neutralized deposit should then be inoculated on to at least three tubes of Löwenstein-Jensen medium. Excessive contamination (over 5%) of the Löwenstein cultures usually means that the decontamination procedure has not been effective.
Alternative concentration procedure
Some workers1 prefer to mix sputum (4 ml) with an equal volume of 10% (100 g/litre) trisodium phosphate (Na3PO4) solution2 and to hold the mixture overnight at room temperature or in an incubator at 35 °C, preferably with occasional shaking. This method is less aggressive against tubercle bacilli and neither the temperature nor the time require to be so accurately controlled. After the mixture has subsequently been centrifuged (> 13 000 g for 30 minutes) to recover the mycobacteria, it is again advisable to neutralize the deposit (using HCl 2 mol/litre, drop by drop, with phenol red as indicator) or to wash the sediment in 15 ml of sterile distilled water.
1 CORPER, H. J. & STONER, R. E. Improved procedure for diagnostic culture of mammalian tubercle bacilli. Journal of laboratory and clinical medicine, 31: 1364-71 (1946).
2 230 g of Na3PO4 . 12H2O per litre.
Interpretation of cultures for M. tuberculosis
The Löwenstein tubes should be incubated for 2 - 3 days at 35 - 37 °C in a horizontal position, with the tops loosened half a turn. The culture tubes should then be stored at 37 °C for 6 weeks and inspected for growth at weekly intervals. During these weekly inspections, the growth of any colonies of bacteria on the surface should be noted. A smear should be carefully made and stained by the Ziehl-Neelsen procedure. If the organisms are not acid-fast bacilli, then the culture may be recorded as contaminated.
Typical human strains of Mycobacterium tuberculosis are “rough, tough and buff”, and can sometimes be seen after 2-3 weeks of incubation (but seldom earlier).
Bovine strains (M. bovis) are generally smooth and whitish-cream in colour. Other, generally nonpathogenic, mycobacterial species may grow more quickly (sometimes after only several days) and may or may not produce pigmented growth (red, yellow, or orange).
If an isolate has the typical colonial appearance and the Ziehl-Neelsen stained smear from a colony is also typical, it should be reported as “Mycobacterium sp, probably M. tuberculosis”; the isolate should also be sent to the national or local reference laboratory for identification and susceptibility testing, as these are specialized procedures.