Ultrastructural Analysis of K. pneumoniae

The effect of LL-37, SP-A, rfhSP-A, and combinations thereof on the ultrastructure of K. pneumoniae was visualized by means of transmission electron microscopy, as described in (25 (link)). K. pneumoniae bacteria in the mid-logarithmic growth phase (2 x 10⁸ CFU/ml) were treated with 55.5 LL-37, of 0.3 µM SP-A, 1.75 µM rfhSP-A SP-A, mixed LL-37/SP-A and mixed LL-37/rfhSP-A at 37°C for 30 min in PBS buffer. Cells were spun down and PBS medium was removed. Cell pellets were then chemically fixed with 4% paraformaldehyde and 2.5% glutaraldehyde for 4 h at 4°C and washed three times with PBS. Next, bacteria were post-fixed with 1% osmium tetroxide for 1 h. Samples were then washed thrice with bi-distilled water and dehydrated using sequential exposure to acetone concentrations ranging from 30% to 100% for 15 min at room temperature. Next, infiltration and embedding were performed using Spurr’s resin. The samples were sectioned using an ultramicrotome with a diamond knife and were mounted on copper grids. Samples were examined on a JEOL JEM 1010 electron microscope (JEOL, Tokyo, Japan).