Method For Enumeration Of Cells Research Articles

Rapid cell counting and viability detection method of Escherichia coli Nissle using image cytometry

The improvement of cell enumeration methods for the counting of Escherichia coli (E. coli) is important as E. coli gains in popularity as a basis for biopharmaceutical applications. In the biopharmaceutical industry, enumerating, characterizing, and dosing the accurate number of cells is imperative. In this work, we demonstrate the utilization of a chip-based image cytometer using a thin-gap, low volume counting chamber consumable to directly enumerate E. coli in bright field and fluorescence, and measure their viability using SYTOX™ Green. The total E. coli particles can be counted accurately label-free by adjusting the focus and targeting the linear range of the instrument. The E. coli are stained with SYTOX™ Green to count the membrane-compromised dead bacterial cells in the green fluorescence channel, while the total cells are counted using the bright field channel. Optimization of the system settings, image focus, cell counting range, and staining conditions have yielded a precise, rapid, and accurate E. coli cell enumeration and viability measurement.

Cell Cytometry: Review and Perspective on Biotechnological Advances.

Cell identification and enumeration are essential procedures within clinical and research laboratories. For over 150 years, quantitative investigation of body fluids such as counts of various blood cells has been an important tool for diagnostic analysis. With the current evolution of point-of-care diagnostics and precision medicine, cheap and precise cell counting technologies are in demand. This article reviews the timeline and recent notable advancements in cell counting that have occurred as a result of improvements in sensing including optical and electrical technology, enhancements in image processing capabilities, and contributions of micro and nanotechnologies. Cell enumeration methods have evolved from the use of manual counting using a hemocytometer to automated cell counters capable of providing reliable counts with high precision and throughput. These developments have been enabled by the use of precision engineering, micro and nanotechnology approaches, automation and multivariate data analysis. Commercially available automated cell counters can be broadly classified into three categories based on the principle of detection namely, electrical impedance, optical analysis and image analysis. These technologies have many common scientific uses, such as hematological analysis, urine analysis and bacterial enumeration. In addition to commercially available technologies, future technological trends using lab-on-a-chip devices have been discussed in detail. Lab-on-a-chip platforms utilize the existing three detection technologies with innovative design changes utilizing advanced nano/microfabrication to produce customized devices suited to specific applications.

Tail or artefact? Illustration of the impact that uncertainty of the serial dilution and cell enumeration methods has on microbial inactivation

The estimation of the concentration of microorganisms in a sample is crucial for food microbiology. For instance, it is essential for prevalence studies, challenge tests (growth and/or inactivation studies) or microbial risk assessment. The application of serial dilutions followed by viable counts in Petri dishes is probably the most extended experimental methodology for this purpose. However, this enumeration technique is also a source of uncertainty.In this article, the uncertainty of the serial dilution and viable count methodology related to the sampling error is analyzed, as well as the approximation of the microbial concentration by the number of colonies in a Petri dish. We analyze from a theoretical point of view (statistical analysis) the application of the binomial and Poisson models, demonstrating that the Poisson distribution increases the variance when used to model individual serial dilutions. On the other hand, the binomial model produces unbiased results. Therefore, the Poisson distribution is only applicable when it is a good approximation of the binomial distribution, so the use of the latter is recommended.The relevance of this uncertainty is demonstrated by Monte Carlo simulations of a generic microbial inactivation experiment, where the only source of uncertainty/variability considered is the one generated by serial plating and viable cell enumeration. Due to both the uncertainty of the methodology and the omission of zero-count plates because of the log-transformation, the simulated survival curve can have a tail. Therefore, this phenomenon, which is usually attributed to biological variability, can be to some extent an artefact of the experimental design and/or methodology.

Cell-and-bound algorithm for chance constrained programs with discrete distributions

Chance constrained programing (CCP) is often encountered in real-world applications when there is uncertainty in the data and parameters. We consider in this paper a special case of CCP with finite discrete distributions. We propose a novel approach for solving CCP. The methodology is based on the connection between CCP and arrangement of hyperplanes. By involving cell enumeration methods for an arrangement of hyperplanes in discrete geometry, we develop a cell-and-bound algorithm to identify an exact solution to CCP, which is much more efficient than branch-and-bound algorithms especially in the worst case. Furthermore, based on the cell-and-bound algorithm, a new polynomial solvable subclass of CCP is discovered. We also find that the probabilistic version of the classical transportation problem is polynomially solvable when the number of customers is fixed. We report preliminary computational results to demonstrate the effectiveness of our algorithm.

Physiological comparison of cells with high and low alcohol dehydrogenase activities in bacterial populations consuming ethanol

Measuring the physiological heterogeneity of natural and industrial microbial populations is essential to studying, modelling and monitoring of microbial populations. It was discovered that populations of Escherichia coli and Bacillus megaterium growing in medium with ethanol as an external source of energy have two actively respiring but physiologically different subpopulations. Cells of one subpopulation have negligibly low alcohol dehydrogenase (ADH) activity (ADH-L cells) and cells of the other have high ADH activity (ADH-H cells). The subpopulation of ADH-H bacterial cells was measured using 10 min incubations of cells in a 1% solution of allyl alcohol for fast selective killing of cells with high activity of ADH and flow cytometry detection of dead cells after this incubation. The content of ADH-H cells during exponential phase of batch culture varied from 9 % to 90 % and lowered to zero for a few hours during starvation of the population. ADH-L cells are actively respiring cells and not depolarized cells. The simultaneous presence of ADH-L and ADH-H cells growing in the medium with ethanol can be explained by the fact that ADH-H cells oxidize actively external ethanol whereas ADH-L cells oxidize only intracellular storage carbohydrates. The method for enumeration of cells with high ADH activity can be used to monitor the heterogeneity of bacterial populations consuming ethanol as a sole source of carbon and energy.

An improved cell separation technique for marine subsurface sediments: applications for high-throughput analysis using flow cytometry and cell sorting.

SummaryDevelopment of an improved technique for separating microbial cells from marine sediments and standardization of a high-throughput and discriminative cell enumeration method were conducted. We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas ∼ 50% of cells were recovered from deep samples (100–365 m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 104–108 cells cm−3. We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high-throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single-cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere.

Quantitative PCR method for enumeration of cells of cryptic species of the toxic marine dinoflagellate Ostreopsis spp. in coastal waters of Japan.

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.

Optimization of a standard method for enumeration of total cell counts of colonial Microcystis aeruginosa in environmental samples collected from Boralasgamuwa Lake

Effectiveness of alkaline hydrolysis, heating and regression method for enumeration of cells of Microcystis aeruginosa was assessed using water samples collected from Boralasgamuwa lake during the bloom period from 15th February to 15th May 2013. Alkaline hydrolysis was performed using; 0.01, 0.1 and 1.0 M of NaOH with a control. Alkaline hydrolysis at 800C with NaOH for 15 minutes followed by 30s vortexing produced single cells of all the test samples. A total of 2.34 ± 0.11 x105 cells/ml of single cells were generated during hydrolysis with 0.01 M NaOH. The use of higher NaOH molarities resulted in cell losses. The control sample which contained only distilled water resulted highest numbers of single cells (2.89 ±0.01 X 105 cells/ml). The heating method was employed by heating M. aeroginosa colonies at 40 0C, 60 0C,70 0C,80 0C and 90 0C for 15 minutes. Heating at 800C for 15 min, followed by 30s vortex-mixing produced a suspension of single cells with 2.76±0.81x105 cells/ml. A standard plot was developed for the direct enumeration of cell number against the area of M.aeroginosa colonies using 30 selected colonies. The standard plot slightly overestimated the cell counts during enumeration (2.98 0±.51x105 cells/ml). Key words: Enumeration of cell counts, Microcystis aeruginosa, Alkaline hydrolysis, Heating method

Chemosensitivity Testing of Circulating Epithelial Tumor Cells (CETC) in <i>Vitro</i>: Correlation to in <i>Vivo</i> Sensitivity and Clinical Outcome

Background: Chemotherapy is a mainstay of tumor therapy, however, it is predominantly applied according to empirically developed recommendations derived from statistical relapse rates occurring years after the treatment in the adjuvant situation and from progression-free interval data in the metastatic situation, without any possibility of individually determining the efficacy in the adjuvant situation and with loss of time and quality of life in the metastatic situation if the drugs chosen are not effective. Here, we present a method to determine the efficiency of chemotherapeutic drugs using tumor cells circulating in blood as the part of the tumor actually available in the patient’s body for chemosensitivity testing. Methodology/Principal Findings: After only red blood cell lysis, omitting any enrichment (analogous to other blood cell enumeration methods, including rare CD34 cells), the white cells comprising the circulating epithelial tumor cells (CETC) are exposed to the drugs in question in different concentrations and for different periods of time. Staining with a fluorescence-labeled anti-epithelial antibody detects both vital and dying tumor cells, distinguishing vital from dying cells through membrane permeability and nuclear staining with propidium iodide. Increasing percentages of dying tumor cells are observed dependent on time and concentration. The sensitivity can vary during therapy and was correlated with decrease or increase in CETC and clinical outcome. Conclusions/Significance: Thus, we are able to show that chemosensitivity testing of circulating tumor cells provides real-time information about the sensitivity of the tumor present in the patient, even at different times during therapy, and correlates with treatment success.

On reduction of duality gap in quadratic knapsack problems

We investigate in this paper the duality gap between quadratic knapsack problem and its Lagrangian dual or semidefinite programming relaxation. We characterize the duality gap by a distance measure from set {0, 1} n to certain polyhedral set and demonstrate that the duality gap can be reduced by an amount proportional to the square of the distance. We further discuss how to compute the distance measure via cell enumeration method and to derive the corresponding improved upper bound of the problem.

Scheduling Flexible Manufacturing Systems with Petri Nets Based on the Cell Enumeration Method

In the field of modern manufacturing, flexible manufacturing systems (FMS) is very important because it can scheduleand optimize multipurpose machines to produce multiple types of products. When applying the FMS technology, Petri Net is used to model the machines, parts and the whole manufacturing progress. The core concern of FMS is to make sure that the manufacturing system can transfer from the original state to the final state, which is called reachabilty. Therefore, reachability analysis is one of the most important problems of FMS. When Petri Net is acyclic, the reachability analysis can be performed by finding a integer solution to a set of linear equation, named fundamental equation, which is known to be NP-complete. In this paper, a novel approach for finding the integer solution is applied by adopting a revised version of the cell enumeration method for an arrangement of hyperplanes in discrete geometry to identify firing count vector solution(s) to the fundamental equation on a bounded integer set with a complexity bound of O((nu)n¡m),where n is the number of nodes, m is the number of arcs and u is the upper bound of the number of firings for all individual arcs.

Reachability determination in acyclic Petri nets by cell enumeration approach

Reachability is one of the most important behavioral properties of Petri nets. We propose in this paper a novel approach for solving the fundamental equation in the reachability analysis of acyclic Petri nets, which has been known to be NP-complete. More specifically, by adopting a revised version of the cell enumeration method for an arrangement of hyperplanes in discrete geometry, we develop an efficient solution scheme to identify firing count vector solution(s) to the fundamental equation on a bounded integer set, with a complexity bound of O((nu)n−m), where n is the number of transitions, m is the number of places and u is the upper bound of the number of firings for all individual transitions.

A Novel Method for Reachability Determination in Petri Nets

Reachability is one of the most important behavioral properties of Petri nets and the past four decades have witnessed great efforts in developing various implementable methodologies in determining reachability of Petri nets. We propose in this paper a novel method for solving the fundamental equation in the reachability analysis, which has been known to be NP-complete. More specifically, by adopting a revised version of the cell enumeration method for an arrangement of hyperplanes in discrete geometry, we develop an efficient solution scheme to identify firing count vector solution(s) to the fundamental equation on a bounded integer set, with a complexity bound of O (( n ω) n–m ), where n is the number of transitions, m is the number of places and ω is the upper bound of the number of firings for every transition.

ISHAGE-based single-platform flowcytometric analysis for measurement of absolute viable T cells in fresh or cryopreserved products: CD34/CD133 selected or CD3/CD19 depleted stem cells, DLI and purified CD56+CD3− NK cells

Considering the growing use of immunotherapeutic strategies in paediatric stem cell transplantation associated with risk of graft-versus-host disease, an accurate method for the enumeration of residual T cells/kg recipient's body weight is of paramount importance. Therefore, we propose a multi colour-flow cytometric strategy for correct absolute vital T cell enumeration in manipulated cell preparations for clinical use. The gating strategy is based on the ISHAGE single-platform stem cell enumeration method in combination with experiences from lymphocyte subtyping, using low scatter, high expression of CD3 and CD45 antigens and 7-AAD staining in a no-wash-preparation with counting beads. In spiking experiments, the detection limit was determined to be at 0.7 +/- 0.5 CD3(+) cells/microl with a minimum of 50 T cell events acquired. The cell preparations analysed contained a median absolute CD3(+) T cell number of 221 x 10(3) (0.09%, CD34 selected grafts, n = 187), 900 x 10(3) (0.004%, CD3/CD19 depleted grafts, n = 15) and 283 x 10(3) (0.012%, CD3 depleted/CD56 enriched NK-cells, n = 14), respectively. The results differed of those from conventional T cell measurement in cell products after extensive manipulation. Our method provides reliable residual T cell enumeration even at extremely low concentrations.

Accurate apoptosis measurement requires quantification of loss of expression of surface antigens and cell fragmentation

The use of ratiometric cell enumeration methods emerges as a more accurate method of measurement of the occurrence of apoptosis in cell cultures. These new flow cytometry methods were used to quantify the impact of cell fragmentation and loss of lineage antigen (LAg) expression on measurement of apoptosis. Highly purified human lymphocyte populations were negatively sorted and cultured for 24 h. Apoptotic cells were identified using annexin V, 7-amino-actinomycin D and their LAgs were stained with antibodies. A new indicator, the apoptotic rate, was used to determine apoptosis occurrence and its validity compared with the widely accepted percentage of apoptotic cells (apoptotic index, AI). Loss of LAg expression and cell fragmentation were observed under all conditions assayed and for all cell populations studied. Current methods for quantifying of apoptosis involving AI systematically underestimate apoptosis occurrence in all populations and conditions, especially among cells undergoing spontaneous apoptosis.

Identification and enumeration of Alexandrium spp. from the Gulf of Maine using molecular probes

Abstract Three different molecular methods were used with traditional brightfield microscope techniques to enumerate the toxic dinoflagellate Alexandrium fundyense in samples collected in the Gulf of Maine in 1998, 2000, 2001, and 2003. Two molecular probes were used in fluorescent whole-cell (WC) microscopic assays: a large-subunit ribosomal RNA (LSU rRNA) oligonucleotide probe (NA1) and a monoclonal antibody probe thought to be specific for Alexandrium spp. within the tamarense / catenella / fundyense complex. Cell abundance estimates also were obtained using the NA1 oligonucleotide probe in a semi-quantitative sandwich hybridization assay (SHA) that quantified target rRNA in cell lysates. Here we compare and contrast the specificity and utility of these probe types and assay approaches. WC counts of the 1998 field samples demonstrated that A. fundyense cell densities estimated using the antibody approach were higher than those using either the NA1 oligonucleotide or brightfield microscopy due to the co-occurrence of A. ostenfeldii with A. fundyense , and the inability of the antibody to discriminate between these two species. An approach using cell size and the presence or absence of food vacuoles allowed more accurate immunofluorescent cell counts of both species, but small cells of A. ostenfeldii that did not contain food vacuoles were still mistakenly counted as A. fundyense . For 2001, a dual-labeling procedure using two oligonucleotide probes was used to separately enumerate A. ostenfeldii and A. fundyense in the WC format. In addition, the SHA was used in 2001 and 2003 to enumerate A. fundyense . Some agreement was observed between the two oligonucleotide methods, but there were differences as well. Not including samples with cell numbers below empirically determined detection limits of 25 cells l –1 , good correlation was observed for surface samples and vertical profiles in May 2001 and June 2003 when the SHA estimates were, on average, equivalent, and 1.5× the WC counts, respectively. The worst correlations were for virtually all samples from the June 2001 cruise where the SHA both over- and under-estimated the WC counts. Some differences were expected, since the SHA and the WC assays measure different, but related parameters. The former quantifies intact cells and particulate material that might contain non-viable cells or fragments, whereas the latter measures only intact cells that survive sample processing and are visible in a sample matrix. A variety of factors can thus affect results, particularly with the WC method, including variable uptake of the oligonucleotide probe due to cell permeability changes, cell lysis during sampling, preservation and processing; variable rRNA content or accessibility due to nutritional or environmental factors; and the variable detection of intact cells or cell fragments in fecal pellets and detritus. The SHA offers dramatic increases in sample throughput, but introduces uncertainties, such as those due to sample matrix effects (non-specific labeling and cross-reactions), variable rRNA levels in intact cells or to the possible presence of target rRNA in cell fragments, fecal pellets, or detritus. Molecular probes are powerful tools for monitoring and research applications, but more work is needed to compare and refine these different cell enumeration methods on field samples, as well as to assess the general validity of brightfield or fluorescent WC approaches.

Physical, Hematologic, and Plasma Biochemistry Values of Five Crocodile Monitors, Varanus salvadorii

ABSTRACT Body weight, body length, including snout/tail (ST) and snout/vent (SV) lengths, environmental temperature and humidity, core body temperature, complete blood count (CBC) and plasma chemistry values were determined monthly for one year from five (three male and two female) crocodile monitors, Varanus salvadorii, housed in similar environmental conditions. Summary statistics were calculated for hematologic and plasma chemistry values. Two white blood cell enumeration methods, phloxine B and Natt and Herrick, were compared. The effects of sex and season on the blood values of the sample population were evaluated. Values determined were found to be similar to other varanid species (ISIS Physiological Reference Values, 1999).

The viable-but-nonculturable state induced by abiotic stress in the biocontrol agent Pseudomonas fluorescens CHA0 does not promote strain persistence in soil.

The effects of oxygen limitation, low redox potential, and high NaCl stress for 7 days in vitro on the rifampin-resistant biocontrol inoculant Pseudomonas fluorescens CHA0-Rif and its subsequent persistence in natural soil for 54 days were investigated. Throughout the experiment, the strain was monitored using total cell counts (immunofluorescence microscopy), Kogure's direct viable counts, and colony counts (on rifampin-containing plates). Under in vitro conditions, viable-but-nonculturable (VBNC) cells of CHA0-Rif were obtained when the strain was exposed to a combination of low redox potential (230 mV) and oxygen limitation. This mimics a situation observed in the field, where VBNC cells of the strain were found in the water-logged soil layer above the plow pan. Here, VBNC cells were also observed in vitro when CHA0-Rif was subjected to high NaCl levels (i.e., NaCl at 1.5 M but not 0.7 M). In all treatments, cell numbers remained close to the inoculum level for the first 12 days after inoculation of soil, regardless of the cell enumeration method used, but decreased afterwards. At the last two samplings in soil, VBNC cells of CHA0-Rif were found in all treatments except the one in which log-phase cells had been used. In the two treatments that generated high numbers of VBNC cells in vitro, VBNC cells did not display enhanced persistence compared with culturable cells once introduced into soil, which suggests that this VBNC state did not represent a physiological strategy to improve survival under adverse conditions.