Amniotic Fluid Bacterial Viability Analysis

The presence of bacteria in the amniotic fluid (n=66) was evaluated as previously described^{68 (link), 133 (link)}, using the LIVE/DEAD BacLight™ Bacterial Viability Kit (Cat# L7007, Life Technologies, Grand Island, New York) in a sterile biosafety cabinet. Briefly, 100μL of amniotic fluid were mixed with 900μL of sterile 1X phosphate buffered saline (PBS; Life Technologies). Three microliters of the dye mix (Component A and B were mixed at a 1:1 ratio) were added to the cell suspension and incubated for 15 min at room temperature in the dark. Next, the cells were centrifuged at 10,000 × g for 5 min and the supernatant was discarded. The cell pellet was then re-suspended in 5μL of 1X PBS, and a slide smear was prepared and air-dried. Lastly, the slide was gently rinsed with 1X PBS and mounted with ProLong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole or DAPI (Life Technologies). The presence of bacteria was evaluated using an Olympus BX 60 fluorescence microscope with an Olympus DP71 camera and DP Controller Software (Olympus Corporation, Tokyo, Japan).