In addition, such methods must be capable of rejecting common artefacts such as imperfections in the agar, dust and edges of Petri dishes. Automating such counting procedures is not simple since colonies must first be isolated from the background and then, if they overlap, be separated. In different fields of microbiology, immunology and cellular biology, counting colonies of cells growing on agar plates is routine. Simultaneously, many optimised image processing algorithms and open-source libraries can be used on laptops and desktop computers. Nowadays, technologies such as digital cameras and webcams provide an increasingly high image quality and are increasingly inexpensive. Effectively, enumerating objects is a two-part process: image capture and image analysis. Given that such tasks are time-consuming and, to some extent, subjective, it is surprising that automation is still infrequent. It is therefore very common for biologists to enumerate objects such as pollen, eggs, seeds, nuclei, cells or organisms. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The author has declared that no competing interests exist.Ĭounting objects has always formed an important element of data collection in many fields of biology. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: This study is part of the project “Multidrug resistance and the evolutionary ecology of insect immunity” ( ) funded by the European Research Council. Received: Accepted: DecemPublished: February 15, 2013Ĭopyright: © 2013 Quentin Geissmann. PLoS ONE 8(2):Ĭentrum Wiskunde & Informatica (CWI) & Netherlands Institute for Systems Biology, The Netherlands Additonal cell counting resources and templates to help streamline your assays are also available.Citation: Geissmann Q (2013) OpenCFU, a New Free and Open-Source Software to Count Cell Colonies and Other Circular Objects. This protocol describes how to perform total nucleated cell counts with 3% Acetic Acid with Methylene Blue, and how to perform viable cell counts by Trypan Blue dye exclusion. In this case one would count the intact viable cells. The live cells remain intact and can be distinguished from dead cells by their ability to exclude the blue dye. Trypan Blue penetrates the cell membrane, thus it enters the cytoplasm of cells with compromised membranes (dead cells) to stain them blue. Alternatively, Trypan Blue is recommended for counting viable mammalian cells. Because mature red blood cells lack nuclei, they are excluded when counting. Acetic acid lyses the cellular membranes, and the methylene blue stains the exposed nuclei. ![]() When performing a total nucleated cell count, 3% Acetic Acid with Methylene Blue is recommended. Additionally, viable cell counts should be performed when a decrease in cell viability may be expected, for example, when working with cryopreserved cells or cells manipulated ex vivo.Ĭell counting can be performed using Trypan Blue or 3% Acetic Acid with Methylene Blue. It is recommended to perform an initial cell count prior to cell isolation this number can then be compared to the cell count after cell isolation to calculate cell recovery. Tissue and Cell Culture Dissociation ReagentsĬell counting is an integral part of determining cell concentrations for plating in culture, determining cell viability, and assessing the results of cell isolation procedures.Work at STEMCELL View Current Opportunities >
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