The difficulty in staining tubercle bacilli is believed to be related to the complex surface structure containing a large amount of unsaponifiable wax. Any staining technique which can counteract the influence of this wax could therefore be expected to give better results. The standard method in vogue is the application of heat which renders the bacilli permeable to aqueous dyes. Several attempts have been made to develop a cold staining method for tubercle bacilli as for other organisms. Since this wax is soluble in chloroform, a cold staining method using carbol fuchsin containing chloroform was developed and the results of staining by this new method have been compared with the conventional Ziehl-Neelsen (ZN) method in the present study. Triplicate smears were made from 186 specimens and these were stained by ZN, Cold Staining (CS) and Fluorescent Microscopy (FM) methods. In addition, single smears of 343 specimens previously examined by FM were randomly divided into two subgroups and restained by ZN and CS methods respectively.

The results of examination of duplicate smears by ZN and CS methods showed a high degree of correlation with 75%(140/186) showing identical grading and only 8 were positive by one and negative by the other method. Of the 8 smears positive by CS alone, 7 were confirmed by culture, whereas 3 were positive by culture out of the 8 positive by ZN method. This indicates that those positive by CS alone are likely to be real cases, whereas those positive by ZN alone may include some false positive cases. As far as false negatives are concerned, there was no difference between ZN and CS methods. The reliability of these methods was judged on the basis of culture results and agreement among themselves. The cold staining method was found to be as efficient as ZN method in detecting different gradings of culture positives. In addition, preparation of stain, training of personnel for CS was also found to be as simple as ZN method.

Under the National Tuberculosis Programme, Ziehl-Neelsen method (ZN) is the recommended procedure for AFB staining. However, at the Peripheral Health Institutions, the applicability of this procedure appears to be adversely affected due to non- availability of alcohol for heat fixing, heating and decolourisation steps. Hence, it has become necessary to look for procedures when the use of alcohol could be avoided completely. In this study, three different methods of cold staining, wherein the use of alcohol is avoided, have been tried in 489 sputum specimens. One set of smears was fixed with acetone (CSB method), the second set with candle flame (CSD) and third set was not fixed (CSC). All were stained by a modified cold staining procedure. The (a) sensitivity (b) specificity (c) degree of agreement with culture and the standard ZN method and also (d) applicability of these methods, were studied. All the specimens were subjected for culture and ZN smear examinations. ZN method was significantly superior to other methods. As far as the agreement with culture is concerned, ZN method was found to be superior to CSB method by 29.26%, to CSC method by 24.3% and to CSD method by 31.7%.

The sensitivity and specificity of various methods were: ZN-76.1%, 98.4%, CSB-72%, 96.6%, CSD-72.7%, 96% and CSC-76.6%, 96.9% respectively.