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      OpenCFU, a New Free and Open-Source Software to Count Cell Colonies and Other Circular Objects

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          Abstract

          Counting circular objects such as cell colonies is an important source of information for biologists. Although this task is often time-consuming and subjective, it is still predominantly performed manually. The aim of the present work is to provide a new tool to enumerate circular objects from digital pictures and video streams. Here, I demonstrate that the created program, OpenCFU, is very robust, accurate and fast. In addition, it provides control over the processing parameters and is implemented in an in- tuitive and modern interface. OpenCFU is a cross-platform and open-source software freely available at http://opencfu.sourceforge.net.

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          Most cited references 13

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          Optimized digital counting colonies of clonogenic assays using ImageJ software and customized macros: comparison with manual counting.

          To develop a digital method for counting colonies that highly replicates manual counting. Breast cancer cells were treated with trastuzumab-conjugated gold nanoparticles in combination with X-ray irradiation, (111)In labeled trastuzumab, or γ-radiation, followed by clonogenic assays. Colonies were counted manually or digitally using ImageJ software with customized macros. Key parameters, intensity threshold and minimum colony size, were optimized based on three preliminary manual counts or blindly chosen. The correlation of digital and manual counting and inter- and intra-experimenter variability were examined by linear regression. Survival curves derived from digital and manual counts were compared by F-test (P < 0.05). Using optimized parameters, digital counts corresponded linearly to manual counts with slope (S) and R(2) value close to 1 and a small y-intercept (y(0)): SK-BR-3 (S = 0.96 ± 0.02, R(2) = 0.969, y(0) = 5.9 ± 2.2), MCF-7/HER2-18 (S = 0.98 ± 0.03, R(2) = 0.952, y(0) = 0.74 ± 0.47), and MDA-MB-231 cells (S = 1.00 ± 0.02, R(2) = 0.995, y(0) = 3.3 ± 4.5). Both reproducibility and repeatability of digital counts were better than the manual method. Survival curves generated from digital and manual counts were not significantly different; P-values were 0.3646 for SK-BR-3 cells and 0.1818 for MCF-7/HER2-18 cells. Using blind parameters, survival curves generated by both methods showed some differences: P-values were 0.0897 for SK-BR-3 cells and 0.0024 for MCF-7/HER2-18 cells. The colony counting using ImageJ and customized macros with optimized parameters was a reliable method for quantifying the number of colonies.
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            A simple and fast method for determining colony forming units.

            To develop a flexible and fast colony forming unit quantification method that can be operated in a standard microbiology laboratory. A miniaturized plating method is reported where droplets of bacterial cultures are spotted on agar plates. Subsequently, minicolony spots are imaged with a digital camera and quantified using a dedicated plug-in developed for the freeware program IMAGEJ. A comparison between conventional and minicolony plating of industrial micro-organisms including lactic acid bacteria, Eschericha coli and Saccharomyces cerevisiae showed that there was no significant difference in the results obtained with the methods. The presented method allows downscaling of plating by 100-fold, is flexible, easy-to-use and is more labour-efficient and cost-efficient than conventional plating methods. The method can be used for rapid assessment of viable counts of micro-organisms similar to conventional plating using standard laboratory equipment. It is faster and cheaper than conventional plating methods.
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              Low-cost, high-throughput, automated counting of bacterial colonies.

              Research involving bacterial pathogens often requires enumeration of bacteria colonies. Here, we present a low-cost, high-throughput colony counting system consisting of colony counting software and a consumer-grade digital camera or document scanner. We demonstrate that this software, called "NICE" (NIST's Integrated Colony Enumerator), can count bacterial colonies as part of a high-throughput multiplexed opsonophagocytic killing assay used to characterize pneumococcal vaccine efficacy. The results obtained with NICE correlate well with the results obtained from manual counting, with a mean difference of less than 3%. NICE is also rapid; it can count colonies from multiple reaction wells within minutes and export the results to a spreadsheet for data processing. As this program is freely available from NIST, NICE should be helpful in bacteria colony enumeration required in many microbiological studies, and in standardizing colony counting methods.
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                Author and article information

                Journal
                1210.5502
                10.1371/journal.pone.0054072

                Computer vision & Pattern recognition, Quantitative & Systems biology

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