A simple, reliable M'Fadyean stain for visualizing the Bacillus anthracis capsule

The simple polychrome methylene blue (PMB) staining procedure for blood or tissue smears from dead animals (M'Fadyean reaction) established in 1903 remained accepted as a highly reliable, rapid diagnostic test for anthrax for six decades while that disease was still common in livestock throughout the world. Improvements in disease control led to anthrax becoming rare in industrialized countries and less frequent in developing countries with the result that quality controlled, commercially produced PMB became hard to obtain by the 1980s. Mixed results with alternative methylene blue-based stains then led to diagnosis failures, confusion among practitioners and mistrust of this procedure as a reliable test for anthrax. We now report that, for laboratories needing a reliable M'Fadyean stain at short notice, the best approach is to have available commercially pure azure B ready to constitute into a solution of 0.03 g azure B in 3 ml of 95% ethanol or methanol to which is then added 10 ml of 0.01% KOH (0.23% final azure B concentration) and which can then be used immediately and through to the end of the tests. Stored in the dark at room temperature, the shelf life is at least 12 months. Smears should be fixed with ethanol or methanol (95-100%), not by heat, and the stain left for 5 min before washing off for optimum effect.