A new method for the lysis and subsequent isolation of nucleic acids from Mycobacterium paratuberculosis is described using the technique of matrix solid-phage dispersion (MSPD). An E. coli strain containing a plasmid pUC19 was similarly treated for comparison and to show plasmid DNA could also be obtained. Bacteria were blended with octadecylsilyl (C18) derivatized silica to obtain complete cellular lysis. The blended material was used to prepare a column, which was eluted with various solvents and Tris-EDTA buffer (TE buffer). After subsequent purification by phenol-chloroform extraction and polyethylene glycol (PEG) precipitation, isolation of cellular nucleic acids was confirmed by spectrophotometric analysis and ethidium-bromide agarose gel electrophoresis. The highest yield of DNA was obtained when the nucleic acids were eluted with TE buffer alone, which was equally effective in recovering plasmid DNA from E. coli as the commonly used alkaline-lysis technique. Since the nucleic acids were effectively recovered with one solvent, the use of a column became unnecessary. Restriction endonuclease digestion of the DNA obtained was performed to show the applicability of the MSPD technique as a method for the lysis and subsequent isolation of nucleic acids from mycobacteria.