Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry.
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.
Berney
Michael
M
Swiss Federal Institute of Aquatic Science and Technology, EAWAG, P.O. Box 611, CH-8600 Dübendorf, Switzerland.
Hammes
Frederik
F
Bosshard
Franziska
F
Weilenmann
Hans-Ulrich
HU
Egli
Thomas
T
D001419:Bacteria / Q000187:drug effects / Q000528:radiation effects; D018407:Bacterial Physiological Phenomena; D002462:Cell Membrane / Q000502:physiology; D002463:Cell Membrane Permeability; D004492:Edetic Acid / Q000494:pharmacology; D013293:Enterococcus faecalis / Q000502:physiology; D004926:Escherichia coli; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D005618:Fresh Water / Q000382:microbiology; D050296:Microbial Viability; D012486:Salmonella typhimurium / Q000502:physiology; D012763:Shigella flexneri / Q000502:physiology; D013194:Staining and Labeling; D014466:Ultraviolet Rays
D005456:Fluorescent Dyes; D004492:Edetic Acid