Synthetic biology is a promising field in which unicellular and multicellular fungi have been harnessed to establish cell factories and produce fine and high value-added products. Therefore, haemocytometer counting remains the most commonly used technique to determine the concentration of eucaryotic microorganisms because of its ease of use and low cost. As a consequence, it is impossible to count microbial cells accurately without the insertion of a fluorescent protein. However, the sizes of most microbial cells vary widely, and a droplet may contain multiple cells. In addition, flow cytometry changes the breakpoint of droplets so that the size of droplets formed by sheath fluid can wrap a cell, but it demands a more uniform cell size and can be better detected when cells exhibit certain fluorescence signals. Professional yeast counters used in fermentation engineering are always expensive and may be assisted with stains that are unfavourable to the operator. Most cell counters can only count specific types of cells. ![]() Of course, this method relies on advanced expensive equipment and requires the pre-establishment of control genes, high-efficiency primers or the fluorescent dye propidium monoazide. Real-time quantitative PCR involves the application of related instruments and the drawing of accurate standard curves. Additionally, plate counting is also time consuming. The advantage of this method is that non-viable microbial cells cannot duplicate and form colonies on plates, but some shortcomings of this method are that the cell number only depends on different dilution concentrations and clumped cells will be registered as one count. A corresponding method for automatic colony counting with ImageJ software has been developed. ![]() The plate counting method is performed by spreading living cells on solid media to form colony-forming units (CFUs), which can be counted with the naked eye. At present, commonly used cell counting methods include plate counting, real-time quantitative PCR, haemocytometers, automatic cell counting instruments and flow cytometry counting in biological operation. These assays can be used to measure the results of yeast proliferation, to test the growth rate of yeast cells under different kinds of chemical, physical or environmental factors, and as an internal control to achieve consistent fermentations in industry. ![]() In summary, a convenient, rapid, reproducible and extremely low-cost method to count yeast cells and fungal spores is described here, which can be applied to multiple kinds of eucaryotic microorganisms in genetics, cell biology and industrial fermentation.Īssessing cell viability is a convenient and fundamental method to analyse the effects of various stressors on microbial cells in scientific research and in any fermentation process, in which cell counting-associated technologies, such as concentration calculations and spotting tests, are widely adopted to provide an estimation of viable cells. Taking the yeast Cryptococccus deneoformans and the filamentous fungus Pestalotiopsis microspora as examples, we observed that the customizable software algorithm reduced inter-operator errors significantly and achieved accurate and objective results, while manual counting with a haemocytometer exhibited some errors between repeats and required more time. The files processed through ilastik can be recognized by ImageJ, which can compute the numeric results with the macro ‘Fungal Cell Counter’. Briefly, learning from labels provided by the user, ilastik performs segmentation and classification automatically in batch processing mode and thus discriminates fungal cells from complex backgrounds. To answer this challenge, we explored and developed an easy-to-use fungal cell counting pipeline that combined the machine learning-based ilastik tool with the freeware ImageJ, as well as a conventional photomicroscope. In consideration of the drawbacks of manual cell counting, large quantities of fungal cells require methods that provide easy, objective and reproducible high-throughput calculations, especially for samples in complicated backgrounds. Measuring the concentration and viability of fungal cells is an important and fundamental procedure in scientific research and industrial fermentation.
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