Viability states of bacteria—Specific mechanisms of selected probes

Abstract

Single cell techniques like flow cytometry combined with viability staining can help to obtain information on viability states of bacteria. Many fluorescent dyes are available for this purpose and can be chosen according to the available excitation source, the species used, and the background of scientific questions and relevant specifications. Within this short overview, we focus on two diverse groups of bacteria: the gram2 Escherichia coli and representatives of the gram+ Mycobacterium to demonstrate differences and similarities in dye uptake principles, processing and binding. We call for attention to possible diverse responses of different species to various viability assays. The cell surface structure of bacteria and the chemical properties of fluorescent probes considerably determine the success of a certain staining practice. Particular focus was drawn on analysis of membrane integrity, uptake of substrates and transformation of fluorogenic substrates. '

A novel flow cytometric protocol for assessment of yeast cell adhesion

“…To optimize microsphere concentration for adhesion test preventing limitated amount of microsphere (which may lead to a reduced yeast adhesion profile), several concentrations ranging from 1 3 10 5 to 1 3 10 9 microspheres ml 21 were tested. Concentrations below 1 3 10 7 microspheres ml 21 were minimal compared with the concentration of yeast cells (1 3 10 6 cells ml 21 ); this unbalanced yeasts/microspheres relation caused a negative bias in the quantification of adhesion. We found a direct relation between yeast adhesion and microsphere concentration until reaching a saturation point with the concentration 1 3 10 7 microspheres ml 21 (Fig.…”

“…Concentrations below 1 3 10 7 microspheres ml 21 were minimal compared with the concentration of yeast cells (1 3 10 6 cells ml 21 ); this unbalanced yeasts/microspheres relation caused a negative bias in the quantification of adhesion. We found a direct relation between yeast adhesion and microsphere concentration until reaching a saturation point with the concentration 1 3 10 7 microspheres ml 21 (Fig. 2).…”

“…Briefly, the FN or GEL solutions were prepared in MES buffer at the required concentration (0.01 mg ml 21 ) and 5 ml of microsphere stock solution (2% aqueous suspension of microspheres) was added. On 15 min of room temperature incubation, EDAC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (4 mg ml 21 ) was mixed with the coating microspheres solution.…”

“…2). Concentrations above 1 3 10 7 microspheres ml 21 showed no differences in the percentage or in the distribution pattern of R3 population; therefore, this concentration was selected for further procedures. Incubation time was set to 30 min, allowing adhesion without microsphere or yeast aggregation (Fig.…”

“…Above this concentration, no differences were found in the percentage of cells with adherent microspheres or in the distribution pattern (data not shown). (B) To determine incubation time, 1 3 10 6 cell ml 21 were incubated with 1 3 10 7 microspheres ml 21 for periods of time ranging from 15 to 120 min. Thirty minutes of incubation was the selected time.…”