Assessment and Interpretation of Bacterial Viability by Using the LIVE/DEAD BacLight Kit in Combination with Flow Cytometry

Abstract

The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium E. coli as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive Enterococcus faecalis, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.

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".…”

A study of the control of oral plaque biofilms via antibacterial photodynamic therapy

“…The reliability of the determination of the total bacterial counts by plate culture has been questioned as it is a retrospective method of bacterial viability (Berney et al, 2007) and its reproducibility is affected by factors such as transport and culture media, temperature, atmosphere and incubation period (Lehtinen et al, 2004). These problems can potentially lead to an underestimation of the total bacterial counts or overestimation in very rare cases.…”

Implication of the NorB Efflux Pump in the Adaptation of Staphylococcus aureus to Growth at Acid pH and in Resistance to Moxifloxacin

“…Since norB expression was increased shortly after a shift to pH 4.5, we wished to determine whether susceptibility to moxifloxacin, a NorB substrate (24), was also affected by a shift to pH 4.5. Cell viability was measured in response to moxifloxacin at 30 min after a shift to pH 4.5 or pH 7.0, using rapid LIVE/DEAD cell staining with a Fishman-R cytometer (4). At 90 min after addition of moxifloxacin, there was an approximately 6-fold-greater reduction of live FIG.…”