Inefficiency of 0.3% Carbol Fuchsin in Ziehl-Neelsen Staining for Detecting Acid-Fast Bacilli

Abstract

We compared the sensitivity and specificity of a modified Ziehl-Neelsen (modified-ZN) staining method for acid-fast bacilli (AFB) with that of the standard Ziehl-Neelsen (standard-ZN) staining method, using culture results with Mycobacterium tuberculosis as the "gold standard." The sensitivity (72%; 101 of 140) of the modified-ZN staining method, which uses 0.3% carbol fuchsin, was significantly lower than that of the standard-ZN staining method (84%; 117 of 140); the modified-ZN method missed 21% of cases detected by the standard-ZN method and 11% more of culture-positive samples than the standard-ZN method. The World Health Organization recommendation of 0.3% carbol fuchsin in the ZN method for staining AFB needs to be reconsidered.In developing countries, sputum acid-fast bacilli (AFB) smear microscopy is the primary tool for detecting pulmonary tuberculosis (5). The Ziehl-Neelsen (ZN) method is commonly used for staining sputum smears because of its simplicity and low cost. Revised National Tuberculosis Control Programme (RNTCP) guidelines recommend the use of 1% carbol fuchsin in the ZN method (2). However, recent World Health Organization (WHO) guidelines recommend using carbol fuchsin at a concentration of 0.3% (5). However, the reasons for reducing the concentration of carbol fuchsin from 1 to 0.3% are not documented. The efficacy of 0.3% carbol fuchsin over 1% carbol fuchsin in the ZN staining method has not been studied previously.A total of 586 sputum samples were collected from the same number of patients with symptoms of pulmonary tuberculosis attending two tuberculosis detection centers during the second and third quarters of 2001 in Chennai, India: the Tuberculosis Research Centre, Chetput, and the Government Hospital for Thoracic Medicine, Tambaram Sanatorium. Many of these patients were receiving rifampin-containing short-course regimens for pulmonary tuberculosis and were followed up in controlled clinical trials.Preparation of reagents. Carbol fuchsin (1%) was prepared from 10 g of basic fuchsin (Hi-Media) dissolved in 100 ml of methanol (Qualigens) and 50 ml of melted phenol (Qualigens) in a flask maintained at 60°C in a water bath. This solution was made up to 1,000 ml with distilled water. Carbol fuchsin (0.3%) was prepared from 33 ml of the above solution diluted to 100 ml with distilled water before use. Sulfuric acid (25%) was prepared from 250 ml of concentrated sulfuric acid (Qualigens) slowly added to 750 ml of distilled water. Methylene blue (0.1%) was prepared from 1 g of methylene blue (Qualigens) dissolved in 1,000 ml of distilled water.Two direct smears were prepared from each of the 586 sputum samples and coded. One set was stained by the standard-ZN method, in which 1% carbol fuchsin was used, and the other set was stained using the modified-ZN method, in which 0.3% carbol fuchsin was used for staining. The RNTCP guidelines were followed to stain the smears. The air-dried smear slides were fixed over a flame three to five times for 3 to 4 s. The slides were then placed on a sta...

OptimisingMycobacterium tuberculosisdetection in resource limited settings

“…Four direct smears were made from each of the pooled sputum samples with applicator sticks on clean grease-free slides; measuring 2 cm×3 cm, and not too thick. The first slide was stained with Ziehl-Neelsen (ZN) stain according to the WHO recommended protocol described elsewhere 18. Briefly, the smear was air dried and fixed by gently passing it over a flame 2–3 times.…”

Same day sputum smear microscopy approach with modified ZN staining for the diagnosis of pulmonary tuberculosis in a microscopy centre at Rajahmundry

“…[7] Whereas in another study by Selvakumar et al, [8] it was concluded that use of 0.3% of BF may result in 20% smear-positive patients being missed. Later Pawan et al, [9] explained that reduced smear-positive results were due to lowered concentration of phenol to ~ 1.7%.…”

“…As per the studies, [8,9] it was very clear that in ZN staining, concentration of phenol should be 5% and BF should be 1%. Otherwise the smear positivity will be reduced automatically.…”

Investigating epidemic prone diseases: knowledge and practices of clinical health staff in Ghana

“…This can be attributed to the effort that is being placed on the polio eradication advocacy programs in the public health sector of the country 11 . A high level of awareness was seen in this study to the relevance of outbreak investigation by the clinical staff.…”