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 Cytometry Analysis of Changes in the DNA Content of the Polychlorinated Biphenyl Degrader Comamonas testosteroni TK102: Effect of Metabolites on Cell-Cell Separation

2002 ◽  
Vol 68 (10) ◽  
pp. 5104-5112 ◽  
Author(s):  
Yoshinori Hiraoka ◽  
Tohru Yamada ◽  
Keiko Tone ◽  
Yutaka Futaesaku ◽  
Kazuhide Kimbara
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Cell Separation ◽  
Polychlorinated Biphenyl ◽  
Gene Replacement ◽  
Comamonas Testosteroni ◽  
Single Chromosome ◽  
Degrading Bacterium ◽  
Intermediate Metabolites ◽  
Cell Cell

ABSTRACT Flow cytometry was used to monitor changes in the DNA content of the polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 during growth in the presence or absence of PCBs. In culture medium without PCBs, the majority of stationary-phase cells contained a single chromosome. In the presence of PCBs, the percentage of cells containing two chromosomes increased from 12% to approximately 50%. In contrast, addition of PCBs did not change the DNA contents of three species that are unable to degrade PCBs. In addition, highly chlorinated PCBs that are not degraded by TK102 did not result in a change in the DNA content. These results suggest that PCBs did not affect the DNA content of the cells directly; rather, the intermediate metabolites resulting from the degradation of PCBs caused the increase in DNA content. To study the effect of intermediate metabolites on the DNA content of the cells, four bph genes, bphA1, bphB, bphC, and bphD, were disrupted by gene replacement. The resulting mutant strains accumulated intermediate metabolites when they were grown in the presence of PCBs or biphenyl (BP). When the bphB gene was disrupted, the percentage of cells containing two chromosomes increased in cultures grown with PCBs or BP. When grown with BP, cultures of this mutant accumulated two intermediate metabolites, 2-hydroxybiphenyl (2-OHBP) and 3-OHBP. Addition of 2- or 3-OHBP to a wild-type TK102 and non-PCB-degrading species culture also resulted in an increase in the percentage of cells containing two chromosomes. Electron microscopy revealed that cell-cell separation was inhibited in this culture. This is the first report that hydroxy-BPs can inhibit bacterial cell separation while allowing continued DNA replication.

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 Method for Assessment of Viability and Morphological Changes of Bacteria in the Early Stage of Colony Formation on a Simulated Natural Environment

2006 ◽  
Vol 72 (7) ◽  
pp. 5037-5042 ◽  
Author(s):  
Yumi Shimomura ◽  
Ryuzo Ohno ◽  
Fusako Kawai ◽  
Kazuhide Kimbara
Keyword(s):  
Natural Environment ◽  
Colony Formation ◽  
Laser Scanning ◽  
Fluorescent Dyes ◽  
Polychlorinated Biphenyl ◽  
Early Stage ◽  
Morphological Changes ◽  
Membrane Filter ◽  
Physiological Status ◽  
Comamonas Testosteroni

ABSTRACT A quantitative analysis of changes in the physiological status of bacterial cells is a fundamental type of study in microbiological research. We devised a method for measuring the viability of bacteria in the early stage of colony formation on a simulated natural environment. In this method, a solid medium containing soil extract was used, and the formation of bacterial microcolonies on a membrane filter was determined by use of a laser scanning cytometer combined with live-dead fluorescent dyes. A polychlorinated biphenyl degrader, Comamonas testosteroni TK102, was used in this study. Surprisingly, approximately 20% of the microcolonies had their growth stopped and eventually died. In the presence of biphenyl, the growth arrest was increased to 50%, and filamentous cells were observed in the colonies. Predicted intermediate metabolites of biphenyl were added to the medium to determine the relationship between the change of viability and the production of metabolites, and the addition of 2,3-dihydroxybiphenyl showed low viability. The arrest was not observed to occur on nutrient-rich medium, suggesting that the change in viability might occur in a nutrient-poor natural condition. The results of this study demonstrated that toxic metabolites of xenobiotics might change cell viability in the natural environment.

scholarly journals Draft Genome Sequence of the Polychlorinated Biphenyl-Degrading Bacterium Comamonas testosteroni KF712 (NBRC 110673)

2015 ◽  
Vol 3 (5) ◽  
Author(s):  
Jun Hirose ◽  
Atsushi Yamazoe ◽  
Akira Hosoyama ◽  
Nobutada Kimura ◽  
Hikaru Suenaga ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Aromatic Compounds ◽  
Polychlorinated Biphenyl ◽  
Draft Genome ◽  
Gene Clusters ◽  
Draft Genome Sequence ◽  
Comamonas Testosteroni ◽  
Degrading Bacterium

We present a 5.89-Mb draft genome sequence of Comamonas testosteroni KF712 (NBRC 110673), a polychlorinated biphenyl degrader. The genome sequence clarified that KF712 harbors the gene clusters coding for the catabolism of biphenyl and at least seven other aromatic compounds.

Enzyme histochemical application and backscattered electron imaging to visualize the distribution of lymphatic capillaries

1990 ◽  
Vol 48 (3) ◽  
pp. 880-881
Author(s):  
Seiji Kato
Keyword(s):  
Staining Method ◽  
Double Staining ◽  
Reaction Products ◽  
Blood Capillaries ◽  
Backscattered Electron Imaging ◽  
Histochemical Observation ◽  
Backscattered Electron ◽  
Double Staining Method ◽  
Electron Imaging ◽  
Cryostat Sections

Previously, the author repeatedly confirmed the higher 5’-nucleotidase (5’-Nase) and lower alkaline phoaphatase (ALPase) activities in the wall of lymphatic capillaries reacted with the lead-based method relative to those of blood capillaries. The ALPase, on the other hand, is markedly higher in blood capillaries than in lymphatics. On the basis of these enzyme characteristics, the author has developed a 5’-Nase— ALPase double staining method to differentiate small lymphatics from blood capillaries at the level of the light microcsopy. Furthermore, we applied it to histochemical observation of the lead-containing reaction products of 5’-Nase in lymphatics on the same or adjacent cryostat sections using backscattered electron imaging (BEI) in scanning electron microscope (SEM). This paper presents a new applicability of 5’-Nase histochemistry by BEI-SEM to demonstrate the distribution of lymphatic capillaries in tissue blocks.

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

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 detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

2007 ◽  
Vol 2 (9) ◽  
pp. 2295-2301 ◽  
Author(s):  
Partha Mukhopadhyay ◽  
Mohanraj Rajesh ◽  
György Haskó ◽  
Brian J Hawkins ◽  
Muniswamy Madesh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Simultaneous Detection ◽  
Superoxide Production ◽  
Live Cells ◽  
Mitochondrial Superoxide

scholarly journals Immunohistochemical/histochemical double staining method in the study of the columnar metaplasia of the oesophagus

2014 ◽  
Vol 58 (1) ◽  
Author(s):  
D. Cabibi ◽  
A.G. Giannone ◽  
C. Mascarella ◽  
C. Guarnotta ◽  
M. Castiglia ◽  
...  
Keyword(s):  
Staining Method ◽  
Double Staining ◽  
Columnar Metaplasia ◽  
Double Staining Method

scholarly journals Characterization and Use of an Antibody Detecting the CBFβ-SMMHC Fusion Protein in inv(16)/t(16;16)-Associated Acute Myeloid Leukemias

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1882-1890
Author(s):  
David S. Viswanatha ◽  
I.-Ming Chen ◽  
Pu Paul Liu ◽  
Marilyn L. Slovak ◽  
Cathy Rankin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Type A ◽  
Detection Methods ◽  
Rt Pcr ◽  
Cytogenetic Abnormalities ◽  
Permeabilized Cells ◽  
Flow Cytometric ◽  
Acute Myeloid

The inv(16)(p13q22) and t(16;16)(p13;q22) cytogenetic abnormalities occur commonly in acute myeloid leukemia (AML), typically associated with French-American-British (FAB) AML-M4Eo subtype. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been recently developed to detect the presence of several variants of the resultant CBFB-MYH11 fusion gene that encodes a CBFβ-smooth muscle myosin heavy chain (SMMHC) fusion protein. We have now determined the clinical use of a polyclonal antibody [anti-inv(16) Ab] directed against a junctional epitope of the most common type of CBFβ-SMMHC fusion protein (type A), which is present in 90% of inv(16)/t(16;16) AML cases. Using flow cytometry, reproducible methods were developed for detection of CBFβ-SMMHC proteins in permeabilized cells; flow cytometric results were then correlated with cytogenetics and RT-PCR detection methods. In an analysis of 42 leukemia cases with various cytogenetic abnormalities and several normal controls, the anti-inv(16) Ab specifically detected all 23 cases that were cytogenetically positive for inv(16) or t(16;16), including a single AML case that was RT-PCR–negative. In addition to detecting all type A fusions, the anti-inv(16) Ab also unexpectedly identified the type C and type D CBFβ-SMMHC fusion proteins. Molecular characterization of one RT-PCR–positive and Ab-positive t(16;16) case with a non-type A product showed a novel previously unreported CBFB-MYH11 fusion (CBFB nt 455-MYH11 nt 1893). Flow cytometric results were analyzed using the Kolmogorov-Smirnov statistic D-value and the median value for positive samples was 0.65 (range, 0.35 to 0.77) versus 0.07 (range, −0.21 to 0.18) in the negative group (P < .0001). The overall concordance between cytogenetics and RT-PCR was 97%, whereas the concordance between flow cytometry and cytogenetics was 100%. Thus, using the anti-inv(16) Ab, all cytogenetically positive and RT-PCR–positive AML cases with inv(16) or t(16;16) could be rapidly identified. This study demonstrates the use of this antibody as an investigational tool in inv(16)/t(16;16) AML and suggests that the development of such reagents may have potential clinical diagnostic use.

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