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Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester.

Krämer CE, Singh A, Helfrich S, Grünberger A, Wiechert W, Nöh K, Kohlheyer D - PLoS ONE (2015)

Bottom Line: Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity.Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase.Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.

View Article: PubMed Central - PubMed

Affiliation: IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany.

ABSTRACT
Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single cell cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity. Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.

No MeSH data available.


Related in: MedlinePlus

Mean CAM conversion rate constant and mean calcein efflux rate constant.(A) A mean maximal single cell reaction rate constant at 0.0025 min-1 was determined for CAM conversion under inhibition of calcein efflux due to carbon limitation. A maximal mean single cell calcein efflux rate constant was determined to be approximately twice as high at 0.005 min-1 (n = 5 colonies for each mean maximal single cell reaction rate constant). (B) Limitations in carbon supply inhibited cell division and energy driven transport of calcein out of the cell. Thus, mean single cell traces increase linearly over time until carbon supply was continued (every colored dotted line represent one individual cell, n = 15 cells).
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pone.0141768.g005: Mean CAM conversion rate constant and mean calcein efflux rate constant.(A) A mean maximal single cell reaction rate constant at 0.0025 min-1 was determined for CAM conversion under inhibition of calcein efflux due to carbon limitation. A maximal mean single cell calcein efflux rate constant was determined to be approximately twice as high at 0.005 min-1 (n = 5 colonies for each mean maximal single cell reaction rate constant). (B) Limitations in carbon supply inhibited cell division and energy driven transport of calcein out of the cell. Thus, mean single cell traces increase linearly over time until carbon supply was continued (every colored dotted line represent one individual cell, n = 15 cells).

Mentions: Media shift experiments were performed to discriminate and determine the influence of calcein efflux. Mean single cell reaction rate constants of CAM conversion to calcein in dependency of the extracellular substrate concentration were determined immediately after the initiation of carbon depletion (Fig 5A). After 10 h of famine condition the medium supply was switched back to CGXII + 4% GLC. After the re-supply of glucose, cells responded with an immediate and significant reduction of intracellular mean single cell fluorescence under feast conditions (Figs 4 and 5B). The efflux rate constants of calcein secretion were determined in the initial 50 min of the reestablished feast condition (Fig 5A). The maximal mean single cell reaction rate constant at 0.005 min-1 of the calcein efflux was found to be twice as high as the maximal mean single cell reaction rate constant at approximately 0.0025 min-1 of the CAM conversion. However, C. glutamicum cells always showed remaining mean calcein fluorescence depending on their relevant enzyme activity, cell size, and environmental cultivation conditions.


Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester.

Krämer CE, Singh A, Helfrich S, Grünberger A, Wiechert W, Nöh K, Kohlheyer D - PLoS ONE (2015)

Mean CAM conversion rate constant and mean calcein efflux rate constant.(A) A mean maximal single cell reaction rate constant at 0.0025 min-1 was determined for CAM conversion under inhibition of calcein efflux due to carbon limitation. A maximal mean single cell calcein efflux rate constant was determined to be approximately twice as high at 0.005 min-1 (n = 5 colonies for each mean maximal single cell reaction rate constant). (B) Limitations in carbon supply inhibited cell division and energy driven transport of calcein out of the cell. Thus, mean single cell traces increase linearly over time until carbon supply was continued (every colored dotted line represent one individual cell, n = 15 cells).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4625966&req=5

pone.0141768.g005: Mean CAM conversion rate constant and mean calcein efflux rate constant.(A) A mean maximal single cell reaction rate constant at 0.0025 min-1 was determined for CAM conversion under inhibition of calcein efflux due to carbon limitation. A maximal mean single cell calcein efflux rate constant was determined to be approximately twice as high at 0.005 min-1 (n = 5 colonies for each mean maximal single cell reaction rate constant). (B) Limitations in carbon supply inhibited cell division and energy driven transport of calcein out of the cell. Thus, mean single cell traces increase linearly over time until carbon supply was continued (every colored dotted line represent one individual cell, n = 15 cells).
Mentions: Media shift experiments were performed to discriminate and determine the influence of calcein efflux. Mean single cell reaction rate constants of CAM conversion to calcein in dependency of the extracellular substrate concentration were determined immediately after the initiation of carbon depletion (Fig 5A). After 10 h of famine condition the medium supply was switched back to CGXII + 4% GLC. After the re-supply of glucose, cells responded with an immediate and significant reduction of intracellular mean single cell fluorescence under feast conditions (Figs 4 and 5B). The efflux rate constants of calcein secretion were determined in the initial 50 min of the reestablished feast condition (Fig 5A). The maximal mean single cell reaction rate constant at 0.005 min-1 of the calcein efflux was found to be twice as high as the maximal mean single cell reaction rate constant at approximately 0.0025 min-1 of the CAM conversion. However, C. glutamicum cells always showed remaining mean calcein fluorescence depending on their relevant enzyme activity, cell size, and environmental cultivation conditions.

Bottom Line: Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity.Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase.Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.

View Article: PubMed Central - PubMed

Affiliation: IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany.

ABSTRACT
Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single cell cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity. Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.

No MeSH data available.


Related in: MedlinePlus