BACKGROUND: Flow cytometry has become a choice methodology for microbiological research. However, functional cytometric assays in live bacteria are still limited. This is due, in part, to the cell wall impairing penetration of vital dyes in bacteria, thus imposing permeabilization procedures. These manipulations may affect cell physiology, provoke cell aggregation or lysis, and they are time-consuming. Escherichia coli B strains have been used for mutagenic assays because of an altered lipopolysaccharide that provokes increased membrane permeability. We assessed the use of these strains as possible alternatives for flow cytometric assays to avoid the permeabilization steps. METHODS: Suspensions of E. coli K-12 (strain AB1157) and E. coli B (strain WP2 uvrA/pKM101, denoted as strain IC188) were stained with several fluorochromes, including fluorescein isothiocyanate, propidium iodide, Nile Red, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, hydroethidine, and dihydro-dichlorofluorescein diacetate, under basal conditions and following permeabilization, impairment of membrane potential, inhibition of dye efflux pump, and oxidative stress. Fluorescent staining of both strains was compared by epifluorescence microscopy and flow cytometry. RESULTS: The E. coli B strain IC188 exhibited more efficient staining with vital fluorochromes than the E. coli K-12 strain AB1157 and maintained a similar membrane potential. In addition, IC188 showed higher sensitivity than AB1157 to reveal oxidative stress when challenged with prooxidants. CONCLUSIONS: E. coli B strains may be useful for biochemical and toxicological studies based on flow cytometry and fluorescence microscopy. Copyright 2002 Wiley-Liss, Inc.
BACKGROUND: Flow cytometry has become a choice methodology for microbiological research. However, functional cytometric assays in live bacteria are still limited. This is due, in part, to the cell wall impairing penetration of vital dyes in bacteria, thus imposing permeabilization procedures. These manipulations may affect cell physiology, provoke cell aggregation or lysis, and they are time-consuming. Escherichia coli B strains have been used for mutagenic assays because of an altered lipopolysaccharide that provokes increased membrane permeability. We assessed the use of these strains as possible alternatives for flow cytometric assays to avoid the permeabilization steps. METHODS: Suspensions of E. coli K-12 (strain AB1157) and E. coli B (strain WP2 uvrA/pKM101, denoted as strain IC188) were stained with several fluorochromes, including fluorescein isothiocyanate, propidium iodide, Nile Red, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, hydroethidine, and dihydro-dichlorofluorescein diacetate, under basal conditions and following permeabilization, impairment of membrane potential, inhibition of dye efflux pump, and oxidative stress. Fluorescent staining of both strains was compared by epifluorescence microscopy and flow cytometry. RESULTS: The E. coli B strain IC188 exhibited more efficient staining with vital fluorochromes than the E. coli K-12 strain AB1157 and maintained a similar membrane potential. In addition, IC188 showed higher sensitivity than AB1157 to reveal oxidative stress when challenged with prooxidants. CONCLUSIONS:E. coli B strains may be useful for biochemical and toxicological studies based on flow cytometry and fluorescence microscopy. Copyright 2002 Wiley-Liss, Inc.