Atypical forms of Borrelia burgdorferi (B31 strain) spirochetes induced by harmful culture conditions. A-D: Large agglomerates of atypical ring shaped and spherule forms of Borrelia burgdorferi after one week of BSKII culture followed by 5 minutes of osmotic shock generated by cold distilled water. A and C: Low and high power fields as revealed by dark field microscopy. B and D: Similar morphology in low and high power fields as revealed by immunohistochemistry using the anti-OspA monoclonal antibody. E and F: Low and high power atomic force microscopy (AFM) images showing similar morphology. In this case the inducing agent was 1 mg of Thioflavin S added to the medium at the commencement of one week of culture. See materials and methods for details. Bars: A, B = 30 μm, C-D = 20 μm; E = 5 μm, F = 1 μm.