Rapid assessment of the physiological status of Streptococcus macedonicus by flow cytometry and fluorescence probes

2006 ◽  
Vol 111 (3) ◽  
pp. 197-205 ◽  
Author(s):  
Konstantinos Papadimitriou ◽  
Harris Pratsinis ◽  
Gerhard Nebe-von-Caron ◽  
Dimitris Kletsas ◽  
Effie Tsakalidou
Keyword(s):  
Flow Cytometry ◽  
Rapid Assessment ◽  
Fluorescence Probes ◽  
Physiological Status

scholarly journals Rapid Assessment of the Physiological Status of the Polychlorinated Biphenyl Degrader Comamonas testosteroni TK102 by Flow Cytometry

2002 ◽  
Vol 68 (4) ◽  
pp. 2031-2035 ◽  
Author(s):  
Yoshinori Hiraoka ◽  
Kazuhide Kimbara
Keyword(s):  
Flow Cytometry ◽  
Polychlorinated Biphenyl ◽  
Rapid Assessment ◽  
Live Cells ◽  
Physiological Status ◽  
Comamonas Testosteroni ◽  
Double Staining ◽  
Permeabilized Cells ◽  
Degrading Bacterium ◽  
Double Staining Method

ABSTRACT The viability of the polychlorinated biphenyl-degrading bacterium Comamonas testosteroni TK102 was assessed by flow cytometry (FCM) with the fluorogenic ester Calcein-AM (CAM) and the nucleic acid dye propidium iodide (PI). CAM stained live cells, whereas PI stained dead cells. When double staining with CAM and PI was performed, three physiological states, i.e., live (calcein positive, PI negative), dead (calcein negative, PI positive), and permeabilized (calcein positive, PI positive), were detected. To evaluate the reliability of this double-staining method, suspensions of live and dead cells were mixed in various proportions and analyzed by FCM. The proportion of dead cells measured by FCM directly correlated with the proportion of dead cells in the sample (y = 0.9872 x + 0.18; R 2 = 0.9971). In addition, the proportion of live cells measured by FCM inversely correlated with the proportion of dead cells in the sample (y = −0.9776 x + 98.36; R 2 = 0.9962). The proportion of permeabilized cells was consistently less than 2%. These results indicate that FCM in combination with CAM and PI staining is rapid (≤1 h) and distinguishes correctly among live, dead, and permeabilized cells.

scholarly journals Flow cytometric measurements as a proxy for sporulation intensity in the cultured macroalga Ulva (Chlorophyta)

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Omri Nahor ◽  
Cristina F. Morales-Reyes ◽  
Gianmaria Califano ◽  
Thomas Wichard ◽  
Alexander Golberg ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Life Cycle ◽  
Abiotic Factors ◽  
Physiological Status ◽  
Seaweed Cultivation ◽  
Reproductive Growth ◽  
Biotic And Abiotic Factors ◽  
Flow Cytometric ◽  
Abiotic Stimuli ◽  
Fundamental Shift

Abstract Controlling the life cycle of the green macroalga Ulva (Chlorophyta) is essential to maintain its efficient aquaculture. A fundamental shift in cultivation occurs by transforming the thallus cells into gametangia and sporangia (sporulation), with the subsequent release of gametes and zoids. Sporulation occurrence depends on algal age and abiotic stimuli and is controlled by sporulation inhibitors. Thus, quantification of sporulation intensity is critical for identifying the biotic and abiotic factors that influence the transition to reproductive growth. Here, we propose to determine the sporulation index by measuring the number of released gametes using flow cytometry, in proportion to the total number of thallus cells present before the occurrence of the sporulation event. The flow cytometric measurements were validated by manually counting the number of released gametes. We observed a variation in the autofluorescence levels of the gametes which were released from the gametangia. High autofluorescence level correlated to phototactically active behaviour of the gametes. As autofluorescence levels varied between different groups of gametes related to their mobility, flow cytometry can also determine the physiological status of the gametes used as feedstock in seaweed cultivation.

scholarly journals Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.

1992 ◽  
Vol 40 (10) ◽  
pp. 1605-1612 ◽  
Author(s):  
G C Knowles ◽  
C A McCulloch
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Actin Polymerization ◽  
Direct Method ◽  
Dnase I ◽  
Fluorescence Probes ◽  
Fluorescence Labeling ◽  
Cell Type ◽  
Fluorescence Spectrophotometry ◽  
Actin Rearrangement

Previous studies of fluorescence probes for labeling the monomeric actin pool have demonstrated lack of specificity. We have used quantitative analytical methods to assess the sensitivity and specificity of rhodamine DNAse I as a probe for monomeric (G) actin. The G-actin pool of attached or suspended fibroblasts was stabilized by ice-cold glycerol and MgCl2. Formaldehyde fixation was used to clamp the filamentous (F) actin pool. G- and F-actins were stained by rhodamine DNAse I and FITC-phalloidin, respectively. Confocal microscopy indicated that the G- and F-actins were spatially separate in substrate-attached cells. Flow cytometry and fluorescence spectrophotometry demonstrated low co-labeling of the separate actin pools, although measureable background binding of rhodamine DNAse I was detectable. Estimates of the extent of actin polymerization after trypsinization demonstrated reciprocal changes of monomeric and filamentous actins, consistent with the formation of a perinuclear array of F-actin. The labeling and quantitation methods were also sufficiently sensitive to detect cell type-dependent variations in actin content. Dual labeling of cells with rhodamine DNAse I and FITC-phalloidin may provide a simple and direct method to image and quantify actin rearrangement in individual cells.

scholarly journals A new fluorescent probe for the equilibrative inhibitor-sensitive nucleoside transporter. 5′-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5′-thioadenosine (SAENTA)-x2-fluorescein

1991 ◽  
Vol 273 (3) ◽  
pp. 667-672 ◽  
Author(s):  
J S Wiley ◽  
A M Brocklebank ◽  
M B Snook ◽  
G P Jamieson ◽  
W H Sawyer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mammalian Cells ◽  
Total Concentration ◽  
Rapid Assessment ◽  
Tight Binding ◽  
Fluorescein Isothiocyanate ◽  
Molar Ratio ◽  
Nucleoside Transporter ◽  
Affinity Ligand ◽  
Leukaemic Cells

The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue. 5′-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5′-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi 2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi 2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi 2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [3H]NBMPR binding, only one of which was accessible to SAENTA-chi 2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi 2-fluorescein to leukaemic cells and a Kd of 6 nM was obtained. SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.

Feasibility of disinfection kinetics and minimum inhibitory concentration determination on bacterial cultures using flow cytometry

2008 ◽  
Vol 58 (4) ◽  
pp. 937-944 ◽  
Author(s):  
J. H. Cunningham ◽  
C. Cunningham ◽  
B. Van Aken ◽  
L.-S. Lin
Keyword(s):  
Escherichia Coli ◽  
Flow Cytometry ◽  
Minimum Inhibitory Concentration ◽  
Antimicrobial Agents ◽  
Inhibitory Concentration ◽  
Microbial Consortium ◽  
Silver Ion ◽  
Single Type ◽  
Physiological Status ◽  
Bacterial Cultures

Disinfection kinetics has been well established for selected antimicrobial agents on isolated bacterial strains. Due to the difficulties of culturing most bacteria, the majority of these studies have been limited to readily cultivable microorganisms of a single type or family. This study explores the feasibility of using flow cytometry for characterising the disinfection kinetics and minimum inhibitory concentration (MIC) of an Escherichia coli culture and a microbial consortium. The proposed method relies on fluorescent dye molecules to indicate the morphological and physiological status of numerous individual cells. Biocides of varying effectiveness and inactivation mechanisms (chlorine, iodine, and silver) were used to evaluate this novel application. Using pseudo-first-order kinetics, the coefficients of specific lethality of chlorine and iodine on Escherichia coli were 4.71 and 3.78×10−3 L mg−1 min−1 and MIC of silver ion was between 60 and 80 μg L−1. The coefficients of specific lethality of chlorine and iodine on the microbial consortium were 4.96 and 8.89×10−3 L mg−1 min−1 and MIC of silver ion was between 40 and 60 μg L−1. This method can be used to provide a rapid and consistent way of determining disinfection kinetics and MICs for pure and mixed bacterial cultures and can potentially be used to examine water and wastewater disinfection efficiency. However, caution should be used to ensure that the physiological and morphological status characterised by cytodyes is a result of the inactivation mechanisms of the disinfectants evaluated.

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