Bacterial Viability Assessment Research Articles

Effectiveness of SYTOX Green stain for bacterial viability assessment.

The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations of Escherichia coli and Salmonella typhimurium. This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes. Its application to natural samples should be considered with caution.

Enumeration, viability and heterogeneity in Staphylococcus aureus cultures by flow cytometry

Several fluorochromes (rhodamine 123, bis-oxonol, propidium iodide, SYTO-13 and calcein) were tested by flow cytometry for their ability to determine cell density, viability and heterogeneity in Staphylococcus aureus cultures exposed to heating (60–70–80°C for 2 min), formaldehyde 2% for 20 min and gramicidin-S at 2–5–10 μg/ml for 20 min. Results were validated by viable plate count and counts performed with a particle analyser. Flow cytometry gave quicker results and more accurate information about intermediate states and heterogeneity of S. aureus cultures than viable plate counts. Rhodamine 123 and oxonol were found to be efficient dyes for the assessment of bacterial viability. SYTO-13 was an excellent marker for total counts and calcein can be used to assess metabolic activity.

Membrane hyperpolarisation by valinomycin and its limitations for bacterial viability assessment using rhodamine 123 and flow cytometry

The ionophore, valinomycin, was investigated as a possible means of bacterial viability assessment using the fluorescent membrane potential dye rhodamine 123. Membrane hyperpolarisation in Escherichia coli, Pseudomonas fluorescent, Enterobacter aerogenes and Arthrobacter globiformis was examined during exponential growth and during stress by brief starvation in a high sodium, low potassium buffer using flow cytometric analysis of rhodamine 123 uptake. Dye uptake was variable both between species and amongst cells from the same culture. Exponential phase cells showed no increase in dye uptake due to valinomycin treatment. Stressed P. fluorescens cells responded to valinomycin treatment by increased dye uptake, while stressed E. coli and A. globiformis cells showed no response. Approximately 50% of stressed Eb. aerogenes cells responded to valinomycin. The results demonstrate the limitations of rhodamine dye for viability analysing the viability of diverse bacterial communities and underline the degree of cell heterogeneity in batch cultures.

Membrane hyperpolarisation by valinomycin and its limitations for bacterial viability assessment using rhodamine 123 and flow cytometry

The ionophore, valinomycin, was investigated as a possible means of bacterial viability assessment using the fluorescent membrane potential dye rhodamine 123. Membrane hyperpolarisation in Escherichia coli, Pseudomonas fluorescent, Enterobacter aerogenes and Arthrobacter globiformis was examined during exponential growth and during stress by brief starvation in a high sodium, low potassium buffer using flow cytometric analysis of rhodamine 123 uptake. Dye uptake was variable both between species and amongst cells from the same culture. Exponential phase cells showed no increase in dye uptake due to valinomycin treatment. Stressed P. fluorescens cells responded to valinomycin treatment by increased dye uptake, while stressed E. coli and A. globiformis cells showed no response. Approximately 50% of stressed Eb. aerogenes cells responded to valinomycin. The results demonstrate the limitations of rhodamine dye for viability analysing the viability of diverse bacterial communities and underline the degree of cell heterogeneity in batch cultures.

Flow cytometry for rapid assessment of bacterial viability

Flow cytometry for rapid assessment of bacterial viability

Evaluation of the suitability of bis-(1,3-dibutylbarbituric acid) trimethine oxonol, (diBA-C 4(3) −), for the flow cytometric assessment of bacterial viability

The usefulness of oxonol ( bis-(1,3-dibutylbarbituric acid)trimethine oxonol) as a generally applicable indicator of bacterial viability was investigated using untreated and killed cultures of a variety of bacterial genera. Killing methods involved either heat or bactericidal antibiotics. For all strains tested, the fluorescent dye showed significantly more intense staining of killed than untreated cells. The sensitivity of Aeromonas salmonicida to gentamicin was assessed using oxonol. Although the bacterium was shown to be sensitive to the antibiotic, there was a delay between the time cells lost culturability, as judged by numbers of colony forming units, and that for which a dead cell population could be detected by flow cytometry.

Rapid assessment of bacterial viability by flow cytometry.

The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains: rhodamine 123 (Rh123); 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] and fluorescein diacetate (FDA). Rh123 was found to clearly differentiate viable from non-viable bacteria. The methodology for staining bacteria with this dye was optimised. Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDA than with Rh123. The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.

Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry

A.S. KAPRELYANTS AND D.B. KELL. 1992. The fluorescent dye rhodamine 123 (Rh 123) is concentrated by microbial cells in an uncoupler‐sensitive fashion. Steady‐state fluorescence measurements with Micrococcus luteus indicated that provided the added dye concentration is below approximately 1 mmol/1, uptake is fully uncoupler‐sensitive and is not accompanied by significant self‐quenching of the fluorescence of accumulated dye molecules. ‘Viable’ and ‘non‐viable’ cells are easily and quantitatively distinguished in a flow cytometer by the extent to which they accumulate the dye. The viability of a very slowly growing chemostat culture of Mic. luteus is apparently only about40–50%, as judged by plate counts, but most of the ‘non‐viable’ cells can be resuscitated by incubation of the culture in nutrient medium before plating. The extent to which individual cells accumulate rhodamine 123 can be rapidly assessed by flow cytometry, and reflects the three distinguishable physiological states exhibited by the culture (‘non‐viable’, ‘viable’ and ‘non‐viable‐but‐resuscitable’). Gram‐negative bacteria do not accumulate rhodamine 123 significantly because their outer membrane is not permeable to it; a simple treatment overcomes this. Flow cytometry using rhodamine 123 should prove of general utility for the rapid assessment of microbial viability and vitality.