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Flow cytometry output (ECOBABe phase 1 experiment - microbial viability)

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posted on 15.06.2021, 05:12 by Brooke Wilson
Vaginal seeding is an unverified procedure where caesarean section newborns are exposed to their mother’s vaginal fluids in an attempt to seed them with beneficial microbes. Extracting microbes from a vaginal swab and orally administering them in solution is one method of delivering the microbes to the baby’s gut. However, the viability of vaginal microbes extracted in this manner is unclear. To address this, collection of vaginal microbes was conducted on three pregnant women using a gauze swab. The gauze swab was cut in half and mixed in 5 ml of either water or 0.9% saline to extract the microbes into solution. Microbial viability was assessed by flow cytometry using a combination of propidium iodide and SYTO BC cell staining.

Microbiota preparations were incubated for 5 minutes in the dark with 5 µM SYTO BC Green Fluorescent Nucleic Acid Stain (Thermo Fisher Scientific, Massachusetts, USA, #S34855) and 10 µg/ml propidium iodide (PI, Thermo Fisher Scientific, Massachusetts, USA, #P3566). Controls included an unstained fraction, separate incubations of SYTO BC and PI (for compensation), and a dead cell fraction (thermal shock at 70°C for 30 minutes, followed by PI staining). Samples were analysed on a LSR II flow cytometer (BD, New Jersey, USA). Microbial cells were filtered by size and gated into three fluorescently-distinct populations: viable cells (SYTO BC positive), dead cells (PI positive), and damaged cells (SYTO BC positive, PI positive) (Figure 1). The proportion of each subpopulation was calculated with reference to the total analysed cell count.

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