Microbial cell viability staining for infectious agents in AUP samples.

<p>Images are from oil immersion microscopy with the green (fluorescein) channel for sample #122, and green and red (rhodamine) channels for samples #146, #151 and #157. Image #122: AUP sample aliquot was incubated with SYTOX-green (5 μM in TBS) in the dark for 15 min, fixed on a glass slide at low heat (40°C), washed with water, and air-dried. C. albicans yeast forms are clearly visible. Red arrows point to dead cells (SYTOX-green stained), white arrows point to intact cells (unstained). Images #146 to #157: Sample aliquots were incubated with the live/dead differential staining kit (5 μM SYTO9 and 55 μM propidium iodide in TBS) in the dark for 15 min followed by centrifugation at 800 x g for 3 min, re-suspension in TBS, a 2^nd centrifugation step, and fixation with 4% paraformaldehyde for 15 min. #146: rod-shaped E. coli cells propidium iodide-stained (red arrow) are dead. #151: filamentous K. pneumoniae rods stained with SYTO9 are living cells (white arrow). Neutrophils (bright yellow stain) are surrounded by bacterial cells suggesting a failure of phagocytosis. #157: S. aureus cocci are trapped in NET-like structures with red and green dots indicating death and survival (red and white arrows, respectively). The #157 insert shows a cluster of dead bacterial cells.