Green fluorescent protein–propidium iodide (GFP‐PI) based assay for flow cytometric measurement of bacterial viability

Abstract

Background Several staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA‐binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual‐staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9‐PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability. Methods Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures. Results SYTO9‐PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP‐PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data. Conclusions In bacterial viability analyses, the GFP‐PI combination provided better distinction between current viability stages of E. coli cells than SYTO9‐PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed. © 2004 Wiley‐Liss, Inc.

Rose bengal uptake by E. faecalis and F. nucleatum and light-mediated antibacterial activity measured by flow cytometry

“…Previous studies that used FCM analysis of SYTO/PI labeled bacteria showed that not only intact (SYTO 9 positive) and damaged cells (PI positive) are observed, but that double-stained cells (SYTO 9/PI) are also detected [26][27][28][29]. For results, SYTO 9 positive cells were referred to as "live", PI positive cells as "dead", and cells displaying a double positive signal (SYTO 9/PI) were designated "injured".…”

Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria

“…However, since bacterial surface colonization starts with the adhesion of individual bacteria, the presented assay provides a versatile new tool for high spatial and temporal evaluation of bacterial viability on engineered surface coatings. The assay thus adds to the previously reported eGFP/PI flow cytometry assay that was limited to viability determination of suspended bacteria [21] and to the eGFP/PI endpoint viability study of groundwater E. coli [34].…”

“…We compared the performance of our eGFP/PI assay to the well-established SYTO ® 9/PI endpoint dual staining assay and found identical detection efficiencies of dead E. coli (Additional file 1: Figure S2). The SYTO ® 9/PI assay itself has been extensively compared to the above-mentioned viability tests [17,19,21,26,36] and showed comparative results to the solution based CFU assay as well as to other microscopy based endpoint viability protocols including the CTC assay. The added advantage of our assay is the ability to monitor the viability of adherent bacteria in real-time.…”

“…Furthermore, since both stains target DNA, the competitive displacement of the SYTO ® 9 (live stain) by the high affinity PI (dead stain) upon membrane breakdown can affect the staining reliability [20]. To eliminate the competitive displacement of the two DNA stains and the demand for prolonged incubation times caused by the passive diffusion of the SYTO ® 9 live stain through the bacterial membrane, it was suggested to replace SYTO ® 9 with green fluorescent protein (GFP) expressed by the bacteria as demonstrated previously for flow cytometry applications (Figure 1b) [21]. Although flow cytometry has been used to measure the viability of GFP expressing bacteria adsorbed to polystyrene beads functionalized with antimicrobial coatings [22], it cannot be applied for continuous in situ bacterial viability monitoring on planar surfaces.…”

Nonionic Surfactants Enhancing Bactericidal Activity at Their Critical Micelle Concentrations

“…PI stains dead bacteria in which cell membranes are destroyed. We were able to distinguish viable bacterial cells from the dead cells using these reagents 24,25 . Both reagents work within minutes, and we were able to determine penetration of these dyes in bacterial cells in a solution containing MEE.…”