The isolation of RNA from Streptococcus and other gram-positive bacteria has proven difficult because these organisms possess thick, rigid cell walls that prevent lysis. Generally, two methods are used to disrupt the cell wall:(i) degradative enzymes that digest and weaken the cell wall (4, 7) or (ii) mechanical agitation by mixing bacterial cells with zirconia/silica beads in a high-speed reciprocating shaker (1). Although enzymatic digestion is effective, it usually takes several minutes to complete. During this time, RNA processing/degradation can occur, resulting in poor quality and yield. Reciprocating shakers are a fast and effective means of lysing bacteria, but these instruments are often expensive. We describe the isolation of RNA from S. agalactiae using an inexpensive dental amalgamator as an oscillating shaker to lyse these bacteria.

Previously, we isolated RNA from S. agalactiae by an enzymatic method adapted from Ladin et al.(5) and Chomczynski and Sacchi (2). Briefly, 20 mL of cells were grown to the desired optical density (OD) 600 and chilled in a dry ice/ethanol bath. After pelleting, the cells were resuspended in 200 µL protoplasting buffer (20% sucrose, 20 mM Tris-HCl, pH 7.0, 10 mM MgCl2, 0.05% Triton® X-100, 10 mM vanadyl ribonucleoside complex). Mutanolysin (100 U; Sigma Chemical, St. Louis, MO, USA) was then added to digest the cell wall. The cells were then incubated at 37 C for 10–15 min or until clearing could be seen. To lyse the digested cells, the following were added: 400 µL lysis solution (4 M guanidinium isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M β-mercaptoethanol), 4.0 µL 2 M sodium acetate, pH 4.0, 400 µL watersaturated phenol and 80 µL chloroform: isoamyl alcohol (24: 1). The mixture was vortex-mixed and placed on