Survival of tubercle bacilli in heat-fixed and stained sputum smears

Heat Inactivation Renders Sputum Safe and Preserves Mycobacterium tuberculosis RNA for Downstream Molecular Tests

“…Heat treatment is an established technique that has been used to decontaminate medical devices, ensure aseptic inoculation, and aid in therapeutic preparations (17,18). However, for M. tuberculosis, reports have shown that short slide flaming or drying on a hot block is insufficient to completely inactivate all bacilli (19,20). Heating M. tuberculosis cultures at 80°C for 20 min was shown to be effective at inactivating M. tuberculosis without compromising the integrity of DNA for downstream manipulation (21).…”

Improved Laboratory Safety by Decontamination of Unstained Sputum Smears for Acid-Fast Microscopy

“…Slides were then aseptically transferred to 16 by 150 mm tubes containing 10 ml of sterile distilled water, rinsed briefly, and cultured in selective lysed blood (SLB) medium. Our previous studies showed better results using this medium (6).…”

“…Depending on the distance and available means of transport, analysis of the sputum smears may be delayed for days. Some studies have shown that tubercle bacilli may survive in unstained heat-fixed sputum smears for at least 7 days (1,2,6,11,14) and may therefore present a potential risk of transmission of laboratory-acquired infections (9,17,19).The use of decontamination agents such as phenol (7), formaldehyde (2, 9), sodium hypochlorite (17), and glutaraldehyde (10, 11) to kill the tubercle bacilli present in smears has been documented. Decontamination of slides with these agents has improved the biosafety in transport, storage, or manipulation of slides containing sputa from patients suspected of having pulmonary tuberculosis.…”

“…Depending on the distance and available means of transport, analysis of the sputum smears may be delayed for days. Some studies have shown that tubercle bacilli may survive in unstained heat-fixed sputum smears for at least 7 days (1,2,6,11,14) and may therefore present a potential risk of transmission of laboratory-acquired infections (9,17,19).…”

“…To determine bacillary viability, duplicates of decontaminated slides (tests) and controls (slides with no decontamination) were cultured by placing each slide in 16 by 150 mm tubes containing 8 ml of SLB medium (1,6,18) and incubated at 37°C for 7 days. The slides were then removed from the SLB medium, rinsed briefly in 10 ml distilled water, decontaminated with 5% sodium hypochlorite for 10 min, stained by the Ziehl-Neelsen method (3,16), and examined with a compound microscope.…”