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A gentle technique for preparing spheroplasts of Capnocytophaga ochracea strain 25 is described. Cells in the exponential phase were washed with 1.0 M-NaCl, agitated in 1.0 M-NaCl for 2 h at 30°C and exposed to lysozyme in a Tris/salts buffer, pH 7.0. This procedure resulted in 98% spheroplast formation with complete removal of the peptidoglycan layer as detected by both phase-contrast and electron microscopy in combination with chemical analysis.