Determination of Mammalian Cell Counts,Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls^1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques⁶.Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.     

Rapid,Precise, and Accurate Counts of Symbiodinium Cells Using the Guava Flow Cytometer,and a Comparison to Other Methods

In studies of both the establishment and breakdown of cnidarian-dinoflagellate symbiosis, it is often necessary to determine the number of Symbiodinium cells relative to the quantity of host tissue. Ideally, the methods used should be rapid, precise, and accurate. In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer. We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument''s automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate. We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.     

The In Vitro Growth Response of Giardia Trophozoites from the Rabbit*

SYNOPSIS. A visual technic has been developed for determining concentration of Giardia trophozoites in culture tubes. Such a technic is desirable because the nature of Giardia growth makes routine enumeration of these organisms by hemocytometer or electronic cell counter expensive in both time and material. The visual method of counting Giardia trophozoites was correlated with counts of the same suspensions of organisms using an electronic particle counter. As a part of the correlation, the growth response, as measured by electronic cell counter, was established for 8 primary axenic cultures of Giardia trophozoites from the rabbit. The average starting number of organisms was 3.7 ± 0.6 × 10³ per ml, the average number of organisms at the peak of logarithmic growth was 1.78 ± 0.2 × 10⁵ per ml, and the generation time was 18.1 ± 1.6 hr. These data are compared with the available literature data quantitating Giardia growth.     

Evaluation of <Emphasis Type="Italic">Isochrysis galbana</Emphasis> (clone T-ISO) cell numbers by digital image analysis of color intensity

In microalgal cultivation, measuring cell numbers as a means to monitor growth rates is a long-standing problem. Many automated counting systems and schemes have been developed; among these are image analysis systems. However, such imaging systems have presented difficulties in dealing with the complexities of computer recognition of individual microscopic cells. It is known that the coloration of microalgae suspension is species specific and that color intensity increases are typically associated with increasing numbers. Using this qualitative insight, the present work describes the design, construction, and comparative performance of an inexpensive digital imaging system optimized for counting microalgal cells. The system circumvents the need to count individual cells and extracts cell numbers directly from the macroscopic color intensity of a microalgal suspension. The results suggest, using Isochrysis galbana (T-ISO) as an illustrative example, that this scheme is potentially useful for inexpensive and automated biomonitoring of microalgal cell numbers. Percentage difference comparisons with a standard Coulter Counter indicated that the three algorithms tested provided better than 10% accuracy over density thresholds of 1.52 × 10⁶ to 8.10 × 10⁶ cells mL⁻¹ with precision of 4% attainable at high density concentrations.     

Elutriation and Coulter Counts of Tetrahymena pyriformis Grown in Peanut and Cottonseed Meal Media

Growth of Tetrahymena pyriformis W has been used to evaluate nutritional quality of peanut and cottonseed meals. An efficient elutriation method is described for separating cells of this organism from particulate matter left in the substrate (enriched with basal medium) after 4 days of incubation. After elutriation the cells can be counted with a Coulter counter by using calibration procedures which are presented. Elutriation and Coulter counting provide a rapid and efficient method of measuring the growth response of T. pyriformis W. Utility of the method is demonstrated by agreement between Coulter counts and visual counts of the cells and by demonstration of a linear response of cell numbers to substrate nitrogen.     

Analysis of mammalian viable cell biomass based on cellular ATP

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.     

A 3D in situ cell counter reveals that breast tumor cell (MDA‐MB‐231) proliferation rate is reduced by the collagen matrix density

Many cell types require the biophysical and biochemical cues within the 3D extracellular matrix (ECM) to exhibit their true physiologically relevant behavior. As a result, cell culture platforms have been evolving from traditional 2D petridish plates into 3D biomatrices, and there is a need for developing analytic tools to characterize 3D cell culture. The existing cell counting method, using a hemocytometer or coulter counter, requires that cells are suspended in fluids prior to counting. This poses a challenge for 3D cell culture as cells are embedded in a 3D biomatrix. We use a facile 3D cell counting method that overcomes this limitation and allows for in situ cell counting in a 3D cell culture using equipment that is commonly available in a biology lab. Using a breast tumor cell line, MDA‐MB‐231, as a model system, we demonstrated that MDA‐MB‐231 cells (1) grow slower within a 3D collagen matrix than on a 2D substrate for an extended growth time (a week) with a comparable, initial cell‐to‐cell distance, (2) their cell growth rate decreases with the increase of collagen concentration, showing a linear growth rate rather than an exponential growth rate. Further work using flow cytometry showed that the observed growth rate reduction was consistent with the retardation of the transition to S (synthesis) phase in the cell cycle. This work demonstrates the validity of the 3D cell counting method and the importance of cell–ECM interactions in cell proliferation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:990–996, 2015     

Estimation of Immune Cell Densities in Immune Cell Conglomerates: An Approach for High-Throughput Quantification

Background

Determining the correct number of positive immune cells in immunohistological sections of colorectal cancer and other tumor entities is emerging as an important clinical predictor and therapy selector for an individual patient. This task is usually obstructed by cell conglomerates of various sizes. We here show that at least in colorectal cancer the inclusion of immune cell conglomerates is indispensable for estimating reliable patient cell counts. Integrating virtual microscopy and image processing principally allows the high-throughput evaluation of complete tissue slides.

Methodology/Principal findings

For such large-scale systems we demonstrate a robust quantitative image processing algorithm for the reproducible quantification of cell conglomerates on CD3 positive T cells in colorectal cancer. While isolated cells (28 to 80 µm²) are counted directly, the number of cells contained in a conglomerate is estimated by dividing the area of the conglomerate in thin tissues sections (≤6 µm) by the median area covered by an isolated T cell which we determined as 58 µm². We applied our algorithm to large numbers of CD3 positive T cell conglomerates and compared the results to cell counts obtained manually by two independent observers. While especially for high cell counts, the manual counting showed a deviation of up to 400 cells/mm² (41% variation), algorithm-determined T cell numbers generally lay in between the manually observed cell numbers but with perfect reproducibility.

Conclusion

In summary, we recommend our approach as an objective and robust strategy for quantifying immune cell densities in immunohistological sections which can be directly implemented into automated full slide image processing systems.     

An automated 3D-printed smartphone platform integrated with optoelectrowetting (OEW) microfluidic chip for on-site monitoring of viable algae in water

A sudden increase of algae and their associated toxins in aquatic ecosystems can detrimentally affect the quality of the water, causing serious socio-economic and public health problems. To prevent the spread of harmful algae in aquatic ecosystems, it is essential to track the water’s quality through rapid and in-situ monitoring systems. Conventional methods of algae quantification such as microscopy, hemocytometry, and UV–vis spectroscopy, however, are often unsuitable or inconvenient for in-situ assessment as they require skilled labor and expensive equipment. In this study, we developed a three-dimensional (3D)-printed smartphone platform integrated with a light-driven microfluidic chip operated by optoelectrowetting (OEW). This OEW-driven microfluidic chip not only allows multiplexed drop-wise functions such as droplet transportation, merging, mixing, immobilization on a detection zone, for on-chip water sample preparation but also fluorescent detection and counting of target algae cells using a commercially-available smartphone. Two freshwater algae (C. reinhardtii and M. aeruginosa) and two marine water algae (Amphiprora sp and C. closterium) were employed to validate the 3D-printed smartphone platform in this study. The fluorescence images of viable algae and the cell counting from the microfluidic chip were comparable to the results from a hemocytometer (P > 0.05). We have further conducted tests with spiked samples using freshwater and marine water that were directly collected from environmental samples, showing the same order of magnitude of cell numbers in the spiked and control cultures of algae cells (10⁶ cell/mL, P > 0.05). Unlike traditional quantification methods, the 3D-printed smartphone platform integrated with the OEW offers a highly portable, user-friendly, low-cost tool that enables simple on-chip sample preparation and detection of viable algae. Thus, this stand-alone technology has the potential for rapid and in-situ monitoring of water quality, while using the smartphone’s wireless communication capabilities to report the quality of the water in real-time.     

Quantitative cytocentrifugation in the evaluation of cerebrospinal fluid

Five hundred sixteen samples of cerebrospinal fluid (CSF) were subjected to cytocentrifugation to determine whether this technique is reliable in quantifying the cells present while simultaneously allowing precise cytologic identification of the types of malignant and atypical cells present. Cell counts obtained by the cytocentrifuge method were comparable to those obtained by the standard hemocytometer method. Because of the larger volume of fluid used in cytocentrifugation, cells (0.2/cu mm) were found in 264 specimens that would have been considered devoid of cells by hemocytometry. Six of these samples contained malignant cells. The Wright's-stained cytocentrifuged specimens also allowed precise identification of hematopoietic cell types. CSF cytocentrifugation offers the advantages of (1) a simple and rapid method of quantifying the number of cells present, (2) use of larger volumes than the hemocytometer method, thereby minimizing the possibility that the specimen will be classified as acellular, and (3) improved morphology of hematopoietic cell types by use of the Wright's stain. We conclude that the cytocentrifugation method is useful in the routine quantification and diagnosis of CSF specimens.     

An automated cell-counting algorithm for fluorescently-stained cells in migration assays

A cell-counting algorithm, developed in Matlab^®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells), images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r² = 0.99, P < 0.0001 for a total of 47 images), with no difference in the measurements between methods under all conditions. We conclude that our automated method is accurate, more efficient, and void of variability and potential observer bias normally associated with manual counting.     

Detection of reagent errors by internal quality control of the Coulter counter S]

By a simple program of quality control for the Coulter counter model S two deficient reagents could be detected. To high values of PCV associated with a decrease of MCHC were caused by a faulty Isoton batch. This error was revealed by native blood samples but not by the stabilized 4 C standard. Falsely elevated leukocyte counts due to insufficiently lysed giant platelets and platelet aggregates were produced by modified Lyse-S batches. Comparisons made with a Coulter counter model F using Zaponin and with a counting chamber yielded values at lower levels.     

Comparison of coulter volumes with radiometrically determined intracellular water volumes for cultured cells

Summary During methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells, proliferation is inhibited, morphologic and biochemical changes occur, and giant, often multinucleated, cells form. We have used the increase in cell volume as a marker of the mature syncytiotrophoblastlike phenotype. Uninduced and differentiated BeWo cells are not spherical, and theoretical considerations suggested that deviations in shape could result in significant errors in Coulter volume. To determine if the values obtained by electrical pulse sizing reflected the actual mass of BeWo cells, we have evaluated the relationship between Coulter volumes and intracellular water volumes obtained using a shape-independent estimate for eight cell types. A close correlation (r ²=0.97) was found, indicating that cell volume changes in populations of irregularly shaped cells can be accurately measured using a Coulter instrument. Supported by an operating grant from the National Cancer Institute of Canada. N.S.B. was a recipient of a studentship award from the Alberta Heritage Foundation for Medical Research. C.E.C. is a Senior Research Scientist of the National Cancer Institute of Canada. The McEachern Laboratory is a research facility of the Faculty of Medicine, University of Alberta, and the Cross Cancer Institute, Edmonton, Alberta.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.     

High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection

Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays.     

Quantitation of mast cell heparin by flow cytofluorometry.

Mast cells can be automatically identified in a mixed cell population by flow cytofluorometry after Berberine sulphate staining. Volume specific counts of the total number of cells and number of mast cells, as well as frequency distributions of fluorescence intensities of mast cells, based on a large number of cells, can be rapidly obtained. Results obtained by microscope fluorometry of cells identified by phase contrast microscopy showviously published results it may be inferred that the fluorescence intensity of individual mast cells is proportional to mast cell heparin content. The automated cell counts correlated very well with manual hemocytometer counts. Both cell counts and the determination of mean mast cell fluorescence showed excellent reproducibility.     

Kinetics of Cell Fusion Induced by a Syncytia-Producing Mutant of Herpes Simplex Virus Type I

We have isolated a number of plaque-morphology mutants from a strain of herpes simplex virus type I which, unlike the wild type, cause extensive cell fusion during a productive viral infection. After the onset of fusion, there is an exponential decrease in the number of single cells as a function of time after infection. At a multiplicity of infection (MOI) of 3.8 plaque-forming units per cell, fusion begins 5.3 h after infection with the number of single cells decreasing to 10% of the original number 10.2 h after infection. As the MOI is gradually increased from 0.4 to 8, the onset of fusion occurs earlier during infection. However, when the MOI is increased from 8 to 86, the onset of fusion does not occur any earlier. The rate of fusion is independent of the MOI for an MOI greater than 1. The rate of fusion varies linearly with initial cell density up to 3.5 × 10⁴ cells/cm² and is independent of initial cell density at higher cell concentrations. To assay cell fusion we have developed a simple quantitative assay using a Coulter counter to measure the number of single cells as a function of time after infection. Data obtained using a Coulter counter are similar to those obtained with a microscope assay.     

Quantitative extraction of microgram amounts of lipid from cultured human cells

A simple and efficient procedure based on the solvent system, ethyl acetate:acetone, was developed to extract the principal cellular lipids quantitatively from monolayer cultures of human fibroblasts with populations of even less than one-third million cells. The method was suitable for both radioisotope incorporation studies and studies involving quantitative analysis of fatty acid composition by gas-liquid chromatography. In a modified form the extraction procedure also allowed a 99% recovery of radioactive cell lipid from serum-containing growth medium. Included are results and discussion on the validity of cell number data obtained from monolayer cell cultures which are detached by trypsin and counted by a hemocytometer and a Coulter counting apparatus.