Preparation of single cell suspension and survival rate improvement of microorganisms in fluorescence-activated cell sorting

Abstract:

[Background] High-throughput screening technologies represented by flow cytometry can efficiently screen the microbial strains with target characteristics. In fluorescence-activated cell sorting (FACS), the adhesion of microorganisms will lead to inaccuracy results and low purity. Therefore, a rapid and simple method for preparing single cell suspension is crucial for FACS to obtain reliable results. Desired mutants are usually screened out in fluorescence-based manners from the libraries generated by random mutagenesis with low positive mutation rates. However, the impurities and dead cells with strong spontaneous fluorescence tend to be included into the sorting gate, which will lead to false-positive results and reduce the survival rates of samples. It is urgent to improve the survival rate of microorganisms in FACS. [Objective] To establish a simple method for preparing single cell suspensions for FACS and improve the survival rate of sorted microorganisms. [Methods] Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and Saccharomycetes were used as the samples. The morphological characteristics and cell adhesion degrees of the live microorganisms were observed by microscopy. Four treatments, including ultrasound, digestive enzyme, surfactant, and ultrasound-surfactant, were then employed to alleviate cell adhesion. To improve the survival rate of microorganisms in FACS, we treated Saccharomyces cerevisiae HZ848 expressing green fluorescent protein by atmospheric and room temperature plasma (ARTP) to generate a mutant library. The top 0.5% of the whole cells and the negative cells after propidium iodide (PI) staining were separately sorted based on their GFP intensities, and the single-cell survival rates under both conditions were calculated. [Results] Yeast cells were effectively monodispersed by the treatment of 0.01% Tween-80 combined with ultrasound for 1 min, where the single cell rate was over 88%, and the breakage rate of PI-stained cells was lower than 1.4%. The single cell dispersion of C. glutamicum was achieved by the treatment with 0.01% Tween-80 combined with ultrasound for 5 min, where the single cell rate was over 97% and the breakage rate of PI-stained cells was lower than 1%. The survival rate of S. cerevisiae without PI staining was 4.3%, while this rate was raised to 18.3% for the PI-stained sample after removal of dead cells. [Conclusion] This study established a simple and rapid method to prepare single cell suspensions and provided a PI-staining strategy to significantly improve the survival rates of sorted samples in microbial FACS.