QUANTITATIVE CELL-LYSIS OF INDIGENOUS MICROORGANISMS AND RAPID EXTRACTION OF MICROBIAL DNA FROM SEDIMENT

This study reports improvements in two of the key steps, lysis of indi genous cells and DNA purification, required for achieving a rapid nons elective protocol for extracting nucleic acids directly from sodium do decyl sulfate (SDS)-treated sediment rich in organic matter. Incorpora tion of bead-mill homogenization into the DNA extraction procedure dou bled the densitometrically determined DNA yield (11.8 mu g of DNA.g [d ry weight] of sediment(-1)) relative to incorporation of three cycles of freezing and thawing (5.2 mu g of DNA.g [dry weight] of sediment(-1 )). The improved DNA extraction efficiency was attributed to increased cell lysis, measured by viable counts of sediment microorganisms whic h showed that 2 and 8%, respectively, survived the bead-mill homogeniz ation and freeze-thaw procedures. Corresponding measurements of suspen sions of viable Bacillus endospores demonstrated that 2 and 94% of the initial number survived. Conventional, laser scanning epifluorescence phase-contrast, and differential interference-contrast microscopy rev ealed that small coccoid bacterial cells (1.2 to 0.3 mu m long) were l eft intact after combined SDS and bead-mill homogenization of sediment samples. Estimates of the residual fraction of the fluorescently stai ned cell numbers indicated that 6% (2.2 X 10(8) cells.g [dry weight] o f sediment(-1)) of the original population (3.8 X 10(9) cells.g [dry w eight] of sediment(-1)) remained after treatment with SDS and bead-mil l homogenization. Thus, lysis of total cells was less efficient than t hat of cells which could be cultured. The extracted DNA was used to su ccessfully amplify nahR, the regulatory gene for naphthalene catabolis m in Pseudomonas putida G7, by PCR. By scaling down the mass of sedime nt extracted to 0.5 g and by using gel purification and SpinBind DNA p urification cartridges, the time required to extract DNA from whole se diment samples was reduced to 2 h.