Anthrax is an endemic zoonosis in many countries; it causes human disease following inoculation of the spores of Bacillus anthracis. B. anthracis was the first bacterial pathogen described by Koch and the model pathogen for 'Koch's postulates' (see Box 6.1, p. 100). It is a Gram-positive organism with a central spore. The spores can survive for years in soil. Infection is commonly acquired from contact with animals, paicularly herbivores. The ease of production of B. anthracis spores makes this infection a candidate for biological warfare or bioterrorism. B. anthracis produces a number of toxins that mediate the clinical features of disease. 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 repo that, for laboratories needing a reliable M'Fadyean stain at sho 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. Ref Harrison20th edition pg 1078