scholarly journals Flow cytometric analysis reveals culture condition dependent variations in phenotypic heterogeneity of Limosilactobacillus reuteri

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nikhil Seshagiri Rao ◽  
Ludwig Lundberg ◽  
Shuai Palmkron ◽  
Sebastian Håkansson ◽  
Björn Bergenståhl ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Size ◽  
Pulse Width ◽  
Flow Cytometric Analysis ◽  
High Growth ◽  
Optimal Growth ◽  
Cultivation Conditions ◽  
Oxygen Levels ◽  
Physiological Fitness ◽  
Initial Ph

AbstractOptimisation of cultivation conditions in the industrial production of probiotics is crucial to reach a high-quality product with retained probiotic functionality. Flow cytometry-based descriptors of bacterial morphology may be used as markers to estimate physiological fitness during cultivation, and can be applied for online monitoring to avoid suboptimal growth. In the current study, the effects of temperature, initial pH and oxygen levels on cell growth and cell size distributions of Limosilactobacillus reuteri DSM 17938 were measured using multivariate flow cytometry. A pleomorphic behaviour was evident from the measurements of light scatter and pulse width distributions. A pattern of high growth yielding smaller cells and less heterogeneous populations could be observed. Analysis of pulse width distributions revealed significant morphological heterogeneities within the bacterial cell population under non-optimal growth conditions, and pointed towards low temperature, high initial pH, and high oxygen levels all being triggers for changes in morphology towards cell chain formation. However, cell size did not correlate to survivability after freeze-thaw or freeze-drying stress, indicating that it is not a key determinant for physical stress tolerance. The fact that L. reuteri morphology varies depending on cultivation conditions suggests that it can be used as marker for estimating physiological fitness and responses to its environment.

scholarly journals Flow Cytometric Analysis Reveals Culture-condition Dependent Variations in Phenotypic Heterogeneity of Limosilactobacillus Reuteri

2021 ◽  
Author(s):  
Nikhil Seshagiri Rao ◽  
Ludwig Lundberg ◽  
Shuai Palmkron ◽  
Sebastian Håkansson ◽  
Björn Bergenståhl ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Size ◽  
Pulse Width ◽  
Flow Cytometric Analysis ◽  
High Growth ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Cultivation Conditions ◽  
Oxygen Levels ◽  
Physiological Fitness

Abstract Optimisation of cultivation conditions in the industrial production of probiotics is crucial to reach a high-quality product with retained probiotic functionality. Flow cytometry-based descriptors of bacterial morphology may be used as markers to estimate physiological fitness during cultivation, and can be applied for online monitoring to avoid suboptimal growth. In the current study, the effects of temperature, pH and oxygen levels on cell growth and cell size distributions of Limosilactobacillus reuteri DSM 17938 were measured using multivariate flow cytometry. A pleomorphic behaviour was evident from the measurement of light scatter and pulse width distributions. A pattern of high growth yielding smaller cells and less heterogeneous populations could be observed. Analysis of pulse width distributions revealed significant morphological heterogeneities within the bacterial cell population under non-optimal growth conditions, and pointed towards low temperature, high pH, and high oxygen levels all being triggers for changes in morphology towards cell chain formation. However, cell size did not correlate to survivability after freeze-thaw or freeze-drying stress, indicating that it is not a key determinant for physical stress tolerance. The fact that L. reuteri morphology varies depending on cultivation conditions suggests that it can be used as marker for estimating physiological fitness and responses to its environment.

scholarly journals Flow cytometric analysis of normal human megakaryocytes

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1244-1252 ◽  
Author(s):  
A Tomer ◽  
LA Harker ◽  
SA Burstein
Keyword(s):  
Flow Cytometry ◽  
Cell Membrane ◽  
Cell Size ◽  
Cell Structure ◽  
Surface Membrane ◽  
Flow Cytometric Analysis ◽  
Light Scatter ◽  
Membrane Expression ◽  
Color Flow ◽  
The Mean

Megakaryocytes from normal routine human bone marrow aspirates were analyzed by flow cytometry for size, fine cell structure and granularity, membrane expression of glycoprotein (GP) IIb/IIIa and ploidy. Marrow cells were initially enriched for megakaryocytes by a Percoll density gradient and megakaryocytes were labeled with a fluoresceinated monoclonal antibody directed to the GPIIb/IIIa complex. The cells were fixed with paraformaldehyde and stained with propidium iodide (PI) for DNA quantitation. Using two-color flow cytometry, megakaryocytes were identified by their high membrane immunofluorescence and their ploidy was determined according to the relative fluorescence intensity of the PI. Forward light scatter (FSC), correlating with cell size, 90 degrees side light scatter (SSC), reflecting primarily cell internal fine structure and granularity, and total cell membrane fluorescence were examined. To evaluate independently the relationship between size and cell membrane fluorescence obtained by flow cytometry, megakaryocytes were sorted directly on slides and analyzed by a laser-based anchored cell analyzer (ACAS). There was a strong correlation among size, SSC, and the level of membrane fluorescence. The mean diameter of megakaryocytes was 28.1 +/- 12.3 micron. The modal ploidy distribution was 16N with approximately one-fifth of the cells less than or equal to 4N. The mean FSC and SSC levels increased with increasing ploidy. However, the marked overlap observed between the ranges of these parameters in adjacent ploidy classes suggested that size and SSC increase continuously rather than by discrete steps as is characteristic for ploidy. The total surface membrane fluorescence was correlated with cell size (r = 0.98) as measured by FSC or directly by the ACAS (r = 0.85), and with cell ploidy (r = 0.99) indicating an augmentation in total membrane GPIIb/IIIa expression with an increase in cell size and ploidy. However, estimated GPIIb/IIIa fluorescence density was inversely correlated with FSC suggesting that the GPIIb/IIIa surface epitope density is decreased with increasing cell maturity. We conclude that flow cytometry is a useful technique for the rapid analysis of human megakaryocytes obtained by marrow aspiration, and should be applicable to studies of pathologic states.

scholarly journals Flow cytometric analysis of normal human megakaryocytes

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1244-1252 ◽  
Author(s):  
A Tomer ◽  
LA Harker ◽  
SA Burstein
Keyword(s):  
Flow Cytometry ◽  
Cell Membrane ◽  
Cell Size ◽  
Cell Structure ◽  
Surface Membrane ◽  
Flow Cytometric Analysis ◽  
Light Scatter ◽  
Membrane Expression ◽  
Color Flow ◽  
The Mean

Abstract Megakaryocytes from normal routine human bone marrow aspirates were analyzed by flow cytometry for size, fine cell structure and granularity, membrane expression of glycoprotein (GP) IIb/IIIa and ploidy. Marrow cells were initially enriched for megakaryocytes by a Percoll density gradient and megakaryocytes were labeled with a fluoresceinated monoclonal antibody directed to the GPIIb/IIIa complex. The cells were fixed with paraformaldehyde and stained with propidium iodide (PI) for DNA quantitation. Using two-color flow cytometry, megakaryocytes were identified by their high membrane immunofluorescence and their ploidy was determined according to the relative fluorescence intensity of the PI. Forward light scatter (FSC), correlating with cell size, 90 degrees side light scatter (SSC), reflecting primarily cell internal fine structure and granularity, and total cell membrane fluorescence were examined. To evaluate independently the relationship between size and cell membrane fluorescence obtained by flow cytometry, megakaryocytes were sorted directly on slides and analyzed by a laser-based anchored cell analyzer (ACAS). There was a strong correlation among size, SSC, and the level of membrane fluorescence. The mean diameter of megakaryocytes was 28.1 +/- 12.3 micron. The modal ploidy distribution was 16N with approximately one-fifth of the cells less than or equal to 4N. The mean FSC and SSC levels increased with increasing ploidy. However, the marked overlap observed between the ranges of these parameters in adjacent ploidy classes suggested that size and SSC increase continuously rather than by discrete steps as is characteristic for ploidy. The total surface membrane fluorescence was correlated with cell size (r = 0.98) as measured by FSC or directly by the ACAS (r = 0.85), and with cell ploidy (r = 0.99) indicating an augmentation in total membrane GPIIb/IIIa expression with an increase in cell size and ploidy. However, estimated GPIIb/IIIa fluorescence density was inversely correlated with FSC suggesting that the GPIIb/IIIa surface epitope density is decreased with increasing cell maturity. We conclude that flow cytometry is a useful technique for the rapid analysis of human megakaryocytes obtained by marrow aspiration, and should be applicable to studies of pathologic states.

Food waste bioconversion into new food: A mini-review on nutrients circularity in the production of mushrooms, microalgae and insects

2021 ◽  
pp. 0734242X2110381
Author(s):  
Francesca Girotto ◽  
Laura Piazza
Keyword(s):  
Food Waste ◽  
Optimal Growth ◽  
World Population ◽  
Cultivation Conditions ◽  
Health Security ◽  
Global Challenge ◽  
Factors Analysis ◽  
Growing Medium ◽  
The World ◽  
Food And Feed

The global challenge of feeding an ever-increasing world population is leading scientists’ attention towards nutritious and sustainable foods whose production should have low impacts on environment, economy and society. In case the input feedstock can be waste nutrients, the label of such productions becomes even greener. Nutrients circularity is nowadays an important circular economy practice. This mini-review focuses on the valorisation of food waste as precious biomass to grow new food and feed. In particular, three functional edibles are discussed in the present paper: mushrooms, microalgae and insects. These foods are part of people diets since ages in certain areas of the world and the original aspect of their cultivation and breeding found on waste nutrients recovery is here reviewed. Proofs of such food waste biorefinery viability are already given by several researches featuring the main traits of a suitable growing medium: optimal pool of nutrients and optimal pH. However, lot of work still needs to be done in order to assess the optimal growth and cultivation conditions and the health security of the harvested/bred edibles. A SWOT factors analysis was performed.

scholarly journals Flow cytometric analysis of megakaryocytes from patients with abnormal platelet counts

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 594-601 ◽  
Author(s):  
A Tomer ◽  
P Friese ◽  
R Conklin ◽  
W Bales ◽  
L Archer ◽  
...  
Keyword(s):  
Cell Size ◽  
Flow Cytometric Analysis ◽  
Distribution Analysis ◽  
Membrane Expression ◽  
Platelet Destruction ◽  
Platelet Production ◽  
Flow Cytometric ◽  
High Ploidy ◽  
The Right ◽  
Primary Thrombocytosis

Abstract Megakaryocytes (MKs) from 40 patients with quantitative platelet disorders and 19 normal volunteers were analyzed by flow cytometry for size, fine cell internal structure and granularity, membrane expression of the glycoprotein (GP) IIb/IIIa complex, and for ploidy distribution. Analysis was performed on unfractionated minimally manipulated marrows obtained from routine bone marrow aspirates. MKs were labeled with a fluorescent lineage-specific monoclonal antibody to the GPIIb/IIIa complex followed by DNA staining with propidium iodide. Eight hundred to 3,000 MKs were analyzed in each sample. The modal ploidy distribution in normals was 16N, comprising about half of the megakaryocytic population, with 22.6% of the cells less than or equal to 8N and 22.0% greater than or equal to 32N. Twelve thrombocytopenic patients with decreased marrow MKs on biopsy (mean platelet count [MPC] 44,600/microliters) showed an increase in low ploidy cells with 53.2% less than or equal to 8N (P less than .01); cell size was reduced in three patients when compared to normal cells of identical ploidy (P less than .05). Eight thrombocytopenic patients with enhanced platelet destruction (with normal or increased MKs on biopsy and shortened platelet survival; MPC 41,400/microliters) showed an increased proportion of high ploidy cells greater than or equal to 32N to 39.2% (P less than .01). Increased cell size and granularity were found in four of these patients (P less than .05). Six patients with thrombocytopenia secondary to multiple mechanisms affecting both platelet production and destruction (MPC 66,700/microliters) showed no shift in ploidy. Four patients with primary thrombocytosis (two with thrombocythemia and two with polycythemia vera; MPC 822,500/microliters) showed a marked shift toward high ploidy cells with 42.3% greater than or equal to 32N and 7.6% greater than or equal to 64N cells (P less than .01). The shift was accompanied by a marked increase in cell size and granularity in the patients with thrombocythemia. Ten patients with thrombocytosis secondary to chronic blood loss, malignant or inflammatory disorders (MPC 714,000/microliters), showed variable distributions with four patients exhibiting a shift in ploidy to the right similar to that found in the patients with increased platelet destruction. Based upon the present data, flow cytometric ploidy distribution may be diagnostically useful in thrombocytopenic patients by discriminating between disorders of platelet production and destruction.

scholarly journals Multiparameter Flow Cytometric Analysis of Antibiotic Effects on Membrane Potential, Membrane Permeability, and Bacterial Counts of Staphylococcus aureus andMicrococcus luteus

2000 ◽  
Vol 44 (4) ◽  
pp. 827-834 ◽  
Author(s):  
David J. Novo ◽  
Nancy G. Perlmutter ◽  
Richard H. Hunt ◽  
Howard M. Shapiro
Keyword(s):  
Staphylococcus Aureus ◽  
Flow Cytometry ◽  
Membrane Potential ◽  
Membrane Permeability ◽  
Micrococcus Luteus ◽  
Flow Cytometric Analysis ◽  
Bacterial Counts ◽  
Flow Cytometric ◽  
Permeability Parameter ◽  
Particle Counts

ABSTRACT Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus andMicrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC2(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC2(3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 μg/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 μg/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of <1 μg/ml did not change permeability, while a tetracycline concentration of 4 μg/ml permeabilized 50% of the bacteria; 4 μg of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.

scholarly journals Variations of bacterial-specific activity with cell size and nucleic acid content assessed by flow cytometry

2002 ◽  
Vol 28 ◽  
pp. 131-140 ◽  
Author(s):  
P Lebaron ◽  
P Servais ◽  
AC Baudoux ◽  
M Bourrain ◽  
C Courties ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Cell Size ◽  
Acid Content ◽  
Specific Activity ◽  
Nucleic Acid Content

scholarly journals Chromosome doubling in Cattleya tigrina A. Rich

2019 ◽  
Vol 15 (11) ◽  
Author(s):  
Thays Saynara Alves Menezes-Sá ◽  
Maria de Fátima Arrigoni-Blank ◽  
Andréa Santos da Costa ◽  
Janay De Almeida Santos-Serejo ◽  
Arie Fitzgerald Blank ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Chromosome Doubling ◽  
Flow Cytometric Analysis ◽  
Leaf Tissue ◽  
Stomatal Index ◽  
Chromosome Duplication ◽  
Flow Cytometric ◽  
Young Leaves ◽  
Commercial Value

Chromosome doubling induction in orchids may benefit their production for resulting in flowers of higher commercial value, larger size and higher content of substances that intensify the color and fragrance when compared with diploid orchids. This work aimed to induce and confirm artificial polyploidization, using flow cytometry and stomatal analysis. Explants were treated with colchicine at concentrations of 0, 2.5, 7.5, and 12.5 mM, for 24 and 48 hours and with oryzalin, at concentrations of 0, 10, 30, and 50 μM, for three and six days. For the flow cytometric analysis, a sample of leaf tissue was removed from each plant, crushed to release the nuclei and stained with propidium iodide. In addition to flow cytometry, the ploidy of the antimitotic treated plants was evaluated by stomata analysis. Young leaves were used where the density, functionality and stomatal index were evaluated. Colchicine provided induction of satisfactory polyploidy in C. tigrina at all concentrations and times of exposure, obtaining a greater number of polyploid individuals in the concentration of 12.5 mM for 48 hours. Oryzalin did not induce chromosome duplication at the tested concentrations.

scholarly journals Handheld Microflow Cytometer Based on a Motorized Smart Pipette, a Microfluidic Cell Concentrator, and a Miniaturized Fluorescence Microscope

Sensors ◽  
2019 ◽  
Vol 19 (12) ◽  
pp. 2761 ◽  
Author(s):  
Byeongyeon Kim ◽  
Dayoung Kang ◽  
Sungyoung Choi
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Flow Cytometric Analysis ◽  
Sample Volume ◽  
Fluorescence Microscope ◽  
Comprehensive Approach ◽  
Optical Systems ◽  
Simple Protocol ◽  
Microflow Cytometer ◽  
On Chip

Miniaturizing flow cytometry requires a comprehensive approach to redesigning the conventional fluidic and optical systems to have a small footprint and simple usage and to enable rapid cell analysis. Microfluidic methods have addressed some challenges in limiting the realization of microflow cytometry, but most microfluidics-based flow cytometry techniques still rely on bulky equipment (e.g., high-precision syringe pumps and bench-top microscopes). Here, we describe a comprehensive approach that achieves high-throughput white blood cell (WBC) counting in a portable and handheld manner, thereby allowing the complete miniaturization of flow cytometry. Our approach integrates three major components: a motorized smart pipette for accurate volume metering and controllable liquid pumping, a microfluidic cell concentrator for target cell enrichment, and a miniaturized fluorescence microscope for portable flow cytometric analysis. We first validated the capability of each component by precisely metering various fluid samples and controlling flow rates in a range from 219.5 to 840.5 μL/min, achieving high sample-volume reduction via on-chip WBC enrichment, and successfully counting single WBCs flowing through a region of interrogation. We synergistically combined the three major components to create a handheld, integrated microflow cytometer and operated it with a simple protocol of drawing up a blood sample via pipetting and injecting the sample into the microfluidic concentrator by powering the motorized smart pipette. We then demonstrated the utility of the microflow cytometer as a quality control means for leukoreduced blood products, quantitatively analyzing residual WBCs (rWBCs) in blood samples present at concentrations as low as 0.1 rWBCs/μL. These portable, controllable, high-throughput, and quantitative microflow cytometric technologies provide promising ways of miniaturizing flow cytometry.

Sign in / Sign up

Export Citation Format

Share Document