Formvar support film

For negative staining transmission electron microscopy (TEM), ~20 μL of samples were transferred and left for 5 min on grids covered with Formvar support film (SPI supplies, West Chester, USA) and negatively stained with 1% (w/v) aqueous uranyl acetate for 5 s. To reduce polymeric background otherwise present in SYM media, B. subtilis were grown in SYM without yeast extract. The samples were fixed immediately to prevent further modifications. Excess staining solution was removed by a filter paper. Stained bacteria were air dried at room temperature and examined with a Philips CM 100 electron microscope at 80 keV.

For negative staining transmission electron microscopy (TEM), ~ 20 μl of samples were transferred and left for 5 min on grids covered with Formvar support film (SPI supplies, USA) and negatively stained with 1 % (w/v) aqueous uranyl acetate for 5 s. B. subtilis and E. coli were grown in SM (SYM^{44 (link)} without yeast extract) and M9 medium (6.4 % (w/v) Na₂HPO₄ × 7H₂O, 1.5 % (w/v) KH₂PO₄. 0.25 % (w/v) NaCl, 0.5 % (w/v) NH₄Cl, 2 mM MgSO₄, 0.4 % (w/v) glucose, 0.1 mM CaCl₂), respectively, to reduce the polymeric background otherwise present in SYM and LB media. The samples were fixed immediately to prevent further modifications. Excess staining solution was removed by a filter paper. Stained bacteria were air dried at room temperature and examined with a Philips CM 100 electron microscope at 80 keV.