Background: Bleach-sedimentation may improve microscopy for diagnosing tuberculosis by sterilising sputum and
concentrating Mycobacterium tuberculosis. We studied gravity bleach-sedimentation effects on safety, sensitivity,
speed and reliability of smear-microscopy.
Methods: This blinded, controlled study used sputum specimens (n = 72) from tuberculosis patients. Bleach
concentrations and exposure times required to sterilise sputum (n = 31) were determined. In the light of these
results, the performance of 5 gravity bleach-sedimentation techniques that sterilise sputum specimens (n = 16) were
compared. The best-performing of these bleach-sedimentation techniques involved adding 1 volume of 5% bleach
to 1 volume of sputum, shaking for 10-minutes, diluting in 8 volumes distilled water and sedimenting overnight
before microscopy. This technique was further evaluated by comparing numbers of visible acid-fast bacilli, slidereading
speed and reliability for triplicate smears before versus after bleach-sedimentation of sputum specimens (n =
25). Triplicate smears were made to increase precision and were stained using the Ziehl-Neelsen method.
Results: M. tuberculosis in sputum was successfully sterilised by adding equal volumes of 15% bleach for 1-minute,
6% for 5-minutes or 3% for 20-minutes. Bleach-sedimentation significantly decreased the number of acid-fast bacilli
visualised compared with conventional smears (geometric mean of acid-fast bacilli per 100 microscopy fields 166,
95%CI 68-406, versus 346, 95%CI 139-862, respectively; p = 0.02). Bleach-sedimentation diluted paucibacillary
specimens less than specimens with higher concentrations of visible acid-fast bacilli (p = 0.02). Smears made from
bleach-sedimented sputum were read more rapidly than conventional smears (9.6 versus 11.2 minutes, respectively,
p = 0.03). Counting conventional acid-fast bacilli had high reliability (inter-observer agreement, r = 0.991) that was
significantly reduced (p = 0.03) by bleach-sedimentation (to r = 0.707) because occasional strongly positive bleachsedimented
smears were misread as negative.
Conclusions: Gravity bleach-sedimentation improved laboratory safety by sterilising sputum but decreased the
concentration of acid-fast bacilli visible on microscopy, especially for sputum specimens containing high
concentrations of M. tuberculosis. Bleach-sedimentation allowed examination of more of each specimen in the time
available but decreased the inter-observer reliability with which slides were read. Thus bleach-sedimentation effects
vary depending upon specimen characteristics and whether microscopy was done for a specified time, or until a
specified number of microscopy fields had been read. These findings provide an explanation for the contradictory
results of previous studies.