scholarly journals Bacterial Flow Cytometry and Imaging as Potential Process Monitoring Tools for Industrial Biotechnology

Fermentation ◽  
2020 ◽  
Vol 6 (1) ◽  
pp. 10 ◽  
Author(s):  
Sumana Kadamalakunte Narayana ◽  
Sanjaya Mallick ◽  
Henrik Siegumfeldt ◽  
Frans van den Berg
Keyword(s):  
Flow Cytometry ◽  
Physiological State ◽  
Cluster Formation ◽  
Membrane Integrity ◽  
Process Variations ◽  
Industrial Biotechnology ◽  
Cell Size Distribution ◽  
Industrial Processing ◽  
Imaging Results ◽  
Visual Confirmation

Minimizing process variations by early identification of deviations is one approach to make industrial production processes robust. Cell morphology is a direct representation of the physiological state and an important factor for the cell’s survival in harsh environments as encountered during industrial processing. The adverse effects of fluctuating process parameters on cells were studied using flow cytometry and imaging. Results showed that altered pH caused a shift in cell size distribution from a heterogeneous mix of elongated and short cells to a homogenous population of short cells. Staining based on membrane integrity revealed a dynamics in the pattern of cluster formation during fermentation. Contradictory findings from forward scatter and imaging highlight the need for use of complementary techniques that provide visual confirmation to interpret changes. An atline flow cytometry or imaging capable of identifying subtle population deviations serves as a powerful monitoring tool for industrial biotechnology.

scholarly journals Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

2002 ◽  
Vol 68 (11) ◽  
pp. 5209-5216 ◽  
Author(s):  
Kaouther Ben Amor ◽  
Pieter Breeuwer ◽  
Patrick Verbaarschot ◽  
Frank M. Rombouts ◽  
Antoon D. L. Akkermans ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Salt Stress ◽  
Bile Salt ◽  
Cell Sorting ◽  
Physiological Activity ◽  
Membrane Integrity ◽  
Multiparameter Flow Cytometry ◽  
Bifidobacterium Adolescentis ◽  
Multiparametric Flow Cytometry ◽  
Plate Counts

ABSTRACT Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

scholarly journals Physiological State, Growth Mode, and Oxidative Stress Play a Role in Cd(II)-Mediated Inhibition of Nitrosomonas europaea 19718

2008 ◽  
Vol 74 (8) ◽  
pp. 2447-2453 ◽  
Author(s):  
Kartik Chandran ◽  
Nancy G. Love
Keyword(s):  
Oxidative Stress ◽  
16S Rrna ◽  
Physiological State ◽  
Membrane Integrity ◽  
Fold Increase ◽  
Nitrosomonas Europaea ◽  
Growth Mode ◽  
Batch Cultures ◽  
Growth Modes ◽  
The Impact

ABSTRACT The goal of this study was to determine the impact of physiological growth states (batch exponential and batch stationary growth) and growth modes (substrate-limited chemostat, substrate-sufficient exponential batch, and substrate-depleted stationary batch growth) on several measures of growth and responses to Cd(II)-mediated inhibition of Nitrosomonas europaea strain 19718. The specific oxygen uptake rate (sOUR) was the most sensitive indicator of inhibition among the different responses analyzed, including total cell abundance, membrane integrity, intracellular 16S rRNA/DNA ratio, and amoA expression. This observation remained true irrespective of the physiological state, the growth mode, or the mode of Cd(II) exposure. Based on the sOUR, a strong time-dependent exacerbation of inhibition (in terms of an inhibition coefficient [Ki ]) in exponential batch cultures was observed. Long-term inhibition levels (based on Ki estimates) in metabolically active chemostat and exponential batch cultures were also especially severe and comparable. In contrast, the inhibition level in stationary-phase cultures was 10-fold lower and invariable with exposure time. Different strategies for surviving substrate limitation (a 10-fold increase in amoA expression) and starvation (the retention of 16S rRNA levels) in N. europaea cultures were observed. amoA expression was most negatively impacted by Cd(II) exposure in the chemostat cultures, was less impacted in exponential batch cultures, and was least impacted in stationary batch cultures. Although the amoA response was consistent with that of the sOUR, the amoA response was not as strong. The intracellular 16S rRNA/DNA ratio, as determined by fluorescence in situ hybridization, also did not uniformly correlate with the sOUR under conditions of inhibition or no inhibition. Finally, Cd(II)-mediated inhibition of N. europaea was attributed partially to oxidative stress.

scholarly journals The use of fluorescent probes to assess viability of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis by flow cytometry

2006 ◽  
Vol 31 (4) ◽  
pp. 349-356 ◽  
Author(s):  
Luiz G. Chitarra ◽  
Peter Breeuwer ◽  
Tjakko Abee ◽  
Ruud W. Bulk
Keyword(s):  
Flow Cytometry ◽  
Fluorescent Probes ◽  
Membrane Integrity ◽  
Pathogenic Bacterium ◽  
Plant Pathogenic Bacterium ◽  
Clavibacter Michiganensis ◽  
Clavibacter Michiganensis Subsp ◽  
Alternatives Assessment ◽  
Heat Treated ◽  
Calcein Am

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with PI.

scholarly journals Exosomes derived from HEK293T cells interact in an efficient and noninvasive manner with mammalian sperm in vitro

Nanomedicine ◽  
2020 ◽  
Vol 15 (20) ◽  
pp. 1965-1980
Author(s):  
Teresa Vilanova-Perez ◽  
Celine Jones ◽  
Stefan Balint ◽  
Rebecca Dragovic ◽  
Michael L Dustin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Sperm Function ◽  
Sperm Analysis ◽  
Mammalian Sperm ◽  
Boar Sperm ◽  
Computer Assisted Sperm Analysis

Aim: To investigate exosomes as a noninvasive delivery tool for mammalian sperm. Materials & Methods: Exosomes were isolated from HEK293T cells and co-incubated with boar sperm in vitro. Results: Internalized exosomes were detected within 10 min of co-incubation. Computer-assisted sperm analysis and flow cytometry demonstrated that even after 5-h of exposure to exosomes, there were no significant deleterious effects with regard to sperm motility, viability, membrane integrity and mitochondrial membrane potential (p > 0.05), thus indicating that exosomes did not interfere with basic sperm function. Conclusion: HEK293T-derived exosomes interacted with boar sperm without affecting sperm function. Exosomes represent a versatile and promising research tool for studying sperm biology and provide new options for the diagnosis and treatment of male infertility.

scholarly journals Effects of extender and equilibration time on post-thaw motility and membrane integrity of cryopreserved Gyr bull semen evaluated by CASA and flow cytometry

2010 ◽  
Vol 120 (1-4) ◽  
pp. 31-38 ◽  
Author(s):  
Ticiano Guimarães Leite ◽  
Vicente Ribeiro do Vale Filho ◽  
Rubens Paes de Arruda ◽  
André Furugen Cesar de Andrade ◽  
Lucas Luz Emerick ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Membrane Integrity ◽  
Equilibration Time ◽  
Bull Semen

Biocalorimetry and laser flow cytometry for characterization of the physiological state of microorganisms

1996 ◽  
Vol 277 ◽  
pp. 7-16 ◽  
Author(s):  
Brigitte Zentgraf
Keyword(s):  
Flow Cytometry ◽  
Physiological State

scholarly journals A Possible Flow Cytometry-Based Viability and Vitality Assessment Protocol for Pathogenic Vibrio cholerae O1 and O139 Postexposure to Simulated Gastric Fluid

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Atheesha Singh ◽  
Tobias George Barnard
Keyword(s):  
Flow Cytometry ◽  
Gastric Acid ◽  
Vibrio Cholerae ◽  
Fluorescent Probes ◽  
Membrane Integrity ◽  
Gastric Fluid ◽  
Simulated Gastric Fluid ◽  
Assessment Protocol ◽  
Pathogenic Vibrio ◽  
Vibrio Cholerae O1

During the intake of contaminated water, for diarrheal disease to occur, Vibrio cholerae must survive through the bactericidal digestive secretion of gastric fluid during passage through the stomach. Determining the viability of these bacteria is challenging, with the standard cultivation methods for viability being time-consuming and unable to culture cells that may still function accordingly. This study assessed the use of enzyme action and membrane integrity as alternatives for determining vitality and viability, respectively, in gastric acid-stressed pathogenic Vibrio cholerae O1 and O139, using fluorescent probes thiazole orange (TO) for viability based on membrane integrity, carboxyfluorescein diacetate (CFDA) with acetoxymethyl ester (AM) for vitality based on metabolic activity, and propidium iodide (PI) for cell death/damage due to loss of membrane integrity, with flow cytometry. Simulated gastric fluid-treated bacterial cells were labelled with blends of TO+PI and CFDA-AM+PI, and these stained cells were separated into heterologous populations based on their fluorescence signal. The gastric acid exposed cells presented with high green fluorescence signals after staining with the metabolic probe CFDA-AM, which indicated intact (live) cells due to being metabolically active, whereas when the same cells were stained with the DNA probe (TO), these appeared to be in a “stressed state” due to loss of membrane integrity. Damaged cells (dead cells) showed high red fluorescence levels after staining with PI probe. The use of flow cytometry with fluorescent probes is a favorable method for evaluating the vitality and viability of bacteria when cells are labelled with a combination of CFDA-AM+PI.

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