TWO STEP COLD STAINING METHODS FOR AFB DETECTION Vol 40 No. 4 July 2009 765 Correspondence: Mr Soham Gupta, Department of Microbiology, St Johns Medical College, Ban- galore- 560034, Karnataka, India. Tel: 080-2206 5052 E-mail: soham.micro@gmail.com COMPARATIVE EVALUATION OF TWO COLD STAINING METHODS WITH THE ZIEHL-NEELSEN METHOD FOR THE DIAGNOSIS OF TUBERCULOSIS Soham Gupta 1 , Vishnu Prasad 2 , Indira Bairy 2 and S Muralidharan 1 1 Department of Microbiology, St Johns Medical College, Bangalore, Karnataka; 2 Department of Microbiology, Kasturba Medical College, Manipal, Karnataka, India Abstract. In developing countries pulmonary tuberculosis is usually diagnosed by detecting acid-fast bacilli (AFB) in sputum using a Ziehl-Neelsen (Z-N) staining method. However, in the field the traditional method of staining is difficult to carry out. This study evaluates the efficiency of two cold staining methods, namely Gabbet’s and a modified two reagent cold staining method compared with the Z-N taken as the gold standard. Triplicate smears were prepared from 267 sputum samples and stained by the Z-N, Gabbet’s, and a modified cold staining method, the smears were positive for AFB in 21 (7.87%), 18 (6.74%) and 19 (7.12%), respectively. The sensitivities for the Gabbet’s and modified cold stain were 85.7% and 90.5%, respectively. The positive agreement between the Z-N and Gabbet’s (92.3%) and Z-N and modified cold stain (95%) were good. The modified cold staining method was less time consuming and easier to perform in the field. INTRODUCTION Tuberculosis (TB) is a disease that has affected mankind since early times. It is prevalent in India and a leading cause of death. Demonstration of acid-fast bacilli (AFB) in the sputum is a method used to diagnose pulmonary tuberculosis through- out the world (Selvakumar et al, 2002). Sputum smear microscopy is a low cost procedure suited to developing countries but poses problems in the field. The Ziehl- Neelsen (Z-N) method is used in the Directly Observed Treatment, Short-course (DOTS) as per Revised National Tuberculosis Con- trol Program (RNTCP) guidelines initiated in 1993 (Rao, 2007). The Z-N staining method is cumbersome because it requires the ap- plication of heat during carbol-fuchsin stain- ing resulting in the necessity for a flame source (Gokhale et al, 1990). To achieve suc- cess in tuberculosis control in developing countries, modifications to eliminate techni- cal and administrative problems in an af- fordable and practical way should be con- sidered (Jagannath, 1990). Cold staining using Gabbet’s methylene blue as a decolorizer and counter stain has been advocated as an alternative staining technique (Gokhale et al, 1990). Even a novel two step cold staining method for detecting AFB in sputum smears has been developed (Reuben, 1989; Tripathi DG et al, 2001), elimi- nating heating and combining the decoloriz- ing and counter staining stages. However, its use in peripheral laboratories has not been adequately studied. The present study compares Gabbet’s cold staining method and a modified two