Supplementary MaterialsSupplementary Information srep32104-s1

Supplementary MaterialsSupplementary Information srep32104-s1. in among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The mix of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally solved observations that deliver new insights into the dynamics of cellular behaviour. Alive or dead?, How dead is dead? or How red is dead? are pivotal questions posed during cellular live/dead determination, particularly when staining is performed with propidium iodide (PI). Although PI is a common cell death indicator, a gold standard protocol for its SR3335 use does not exist, and inconsistent staining results and pitfalls have been reported in the literature1,2,3,4,5,6. PI is a versatile indicator dye for dead cells that acts by intercalating with cellular DNA and emitting red fluorescence. Vital staining with PI is dependent on the impermeability of an intact cell membrane to this molecule. Live/dead staining with PI is commonly implemented to evaluate the viability of bacteria sampled from food products, clinical samples, and environmental or fermentation processes and to characterize vitality in eukaryotic cells1,7,8. This staining procedure has been employed for bacteria2,3, biofilms9, yeasts1, and a variety of mammalian cells10. However, the toxicities of fluorescence indicators or certain concentrations are rarely considered. Microscopic imaging approaches employing microfluidic devices containing cells prestained with PI and cell-wall permeant SYTO 9 have been reported for the live/dead quantification of bacterial cells11,12,13, sperm cells14, and yeast15 and are, in principle, comparable to SR3335 studies using fluorescence activated cell sorting (FACS). Conventional staining protocols using PI concentrations higher than 1?M intended for sorting4,14, confirmation of cell lysis16, or cellular analytics17,18,19,20 have been described for prokaryotes and eukaryotes. PI staining is generally performed as an endpoint measurement, frequently after cell fixation17,19,21. PI is SR3335 often, but not exclusively, used in combination with SYTO 9 as a counterstain2,4,5,22. PI is also combined with other cell-permeable DNA dyes, such as other SYTO dyes (and and was cultivated with minimal medium (CGXII?+?4% glucose (w/v) without PI) and used as the reference for three different PI concentrations (0.1?M, 1?M, and 10?M). growth was impaired by 10?M PI. PI permeated and slightly stained intact cells, but these bacteria continued to grow, although at a reduced rate. Bacterial growth was unimpaired by concentrations of 0.1 or 1?M PI (Fig. 1a). However, positively stained cells (PI+) were observed at frequencies of 0.01% for all three SR3335 PI concentrations due to spontaneous single cell death. Open in a separate window Figure 1 Determination of optimal propidium iodide concentration.(a) The model organism was stained continuously with 0.1?M PI, 1?M PI, and 10?M PI, and bacterial growth was normalized to the growth rate without PI addition. Total cell numbers are indicated with N. PI+ dead cells are marked by white arrows. (b) A PI concentration of 0.1?M was used with Gram-positive bacteria (ATCC 13032, DSMZ 14234 and 168), Gram-negative bacteria (MG1655 and ATCC 33867), and a small eukaryote (ATCC SR3335 13032 colony with a single PI+ cell. (d) DSMZ 14234 colony in the late exponential phase with distributed PI+ cocci. (e) DSMZ 14234 tetrad with the early appearance of a PI+ cell. (f) Densely grown 168 cell colony with the late appearance of a PI+ cell. (g) Early appearance of a PI+ MG1655 cell. (h) Segmented ATCC 33867 PI+ phenotype GIII-SPLA2 in a cell-packed region. (i) Dense colony with PI+ yeast cells. Based on these data, a PI concentration of 0.1?M was employed for our microfluidic analyses and validated by the addition of phenol during cultivation (see Supplementary Information Fig. S2). Furthermore, 0.1?M PI was found to be non-toxic and universally applicable, as revealed by testing a wide range of microorganisms.

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