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

• Published in: Journal of microbiological methods 2013-03, Vol.92 (3), p.264-269
• Main Authors: Owen, M.P., Schauwers, W., Hugh-Jones, M.E., Kiernan, J.A., Turnbull, P.C.B., Beyer, W.
• Format: Article
• Language: English
• Subjects: ambient temperature; Anthrax; Anthrax - diagnosis; Anthrax - microbiology; Azure B; Bacillus anthracis; Bacillus anthracis - cytology; Bacterial Capsules - metabolism; Bacteriological Techniques - methods; blood; Coloring Agents - metabolism; dead animals; developed countries; developing countries; disease control; ethanol; heat; livestock; M'Fadyean; McFadyean; methanol; methylene blue; Mfadyean; Polychrome methylene blue; potassium hydroxide; shelf life; Staining and Labeling - methods; washing
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2013.01.009

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 0.03g azure B in 3ml of 95% ethanol or methanol to which is then added 10ml 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 12months. Smears should be fixed with ethanol or methanol (95–100%), not by heat, and the stain left for 5min before washing off for optimum effect. ► The M'Fadyean stain remains important for the rapid diagnosis of anthrax. ► Reliable preparations of this stain have become unavailable commercially. ► An easy-to-prepare azure B-based formulation is supplied. ► The azure B formulation is reliable, stable and can be used immediately.