Production of spore spheroplasts of Clostridium botulinum and DNA extraction for density gradient centrifugation

A method is described whereby Clostridium botulinum 33A and 51B spore spheroplasts are produced in two sequential steps: (1) by reducing spore coats for 3 h with 50% mercaptoacetic acid – 8 M urea – 0.01 M EDTA, and (2) by lysis of the cortex for 2 h with 500 μg/ml lysozyme in 0.01 M Tris-HCl (pH 8.0) – 0.01 M EDTA – 1 mg/ml dithiothreitol and 25% lactose added as osmotic stabilizing agent.Optical density measurements (absorption spectra) of ultraviolet (UV)-absorbing material released after submitting spheroplasts to osmotic lysis in distilled water showed absorption maxima around 270 nm (dipicolinic acid) and 260 nm (nucleic acids). Radioactivity profiles of 3H-labeled TCA-insoluble material from centrifugation of alkaline sucrose gradients showed that the above schedule for obtaining spore spheroplasts resulted in optimal DNA extraction and did not cause extensive breakage of DNA.Phase-contrast microscopy indicated that spore appearance is not visibly affected by chemical coat reduction; however, spores become phase dark after treatment with lysozyme. Electron microscopy showed that after chemical reduction coat layers are less compact, of less electron density, and dissolved in some areas. Furthermore, lysozyme treatment disintegrates and to a large degree removes the cortex and structural details become visible in the core.