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Free Content Hydrochloric vs. sulphuric acid in water for Ziehl-Neelsen staining of acid-fast bacilli

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SETTING: Damien Foundation Bangladesh tuberculosis (TB) control projects.

OBJECTIVES: To compare 25% sulphuric acid in water (H2SO4) with hydrochloric acid in water (HCl) to differentiate acid-fast bacilli in sputum smears stained with 1% carbolfuchsin.

DESIGN: For 1 year, all 158 microscopy laboratories used either H2SO4 or 3%/6%/10% HCl for their routine work, alternating monthly between H2SO4 and HCl. Each month a sample of five smears per laboratory was rechecked blind. After recording qualitative staining aspects, all sample smears were restained before rechecking, using H2SO4 for destaining.

RESULTS: A total of 368 059 H2SO4 and 335 436 HCl smears were routinely read, yielding 7.2% positive or scanty results in both groups. Of these, 9492 were rechecked. There was no difference in false-negatives detected (0.66%, 95%CI 0.44–0.95 for H2SO4 vs. 0.68%, 95%CI 0.46–0.98 for HCl), but apparently there were more false-positives with H2SO4 (2.12%, 95%CI 0.92–4.14 vs. 0.28%, 95%CI 0.00–1.54, P = 0.05). Qualitatively, only 3% HCl yielded significantly inferior differentiation results.

CONCLUSIONS: HCl 6–10% in water can be recommended for Ziehl-Neelsen destaining above H2SO4. Diluting is easier and safer, and it may cause less confusion with false-positives during rechecking, including a restaining step.

Keywords: Ziehl-Neelsen method; acid-fastness; acids; destaining; tuberculosis

Document Type: Regular Paper


Affiliations: 1: Damien Foundation Bangladesh, Dhaka, Bangladesh 2: International Union Against Tuberculosis and Lung Disease, Paris, France; Mycobacteriology Unit, Institute of Tropical Medicine, Antwerp, Belgium

Publication date: July 1, 2011

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  • The International Journal of Tuberculosis and Lung Disease publishes articles on all aspects of lung health, including public health-related issues such as training programmes, cost-benefit analysis, legislation, epidemiology, intervention studies and health systems research. The IJTLD is dedicated to the continuing education of physicians and health personnel and the dissemination of information on tuberculosis and lung health world-wide.

    Certain IJTLD articles are selected for translation into French, Spanish, Chinese or Russian. They are available on the Union website

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