A Modified Procedure to Isolate Synchronous Cells from Yyeasts with Continuous Percoll Density Gradient and Their Raman Discri Mination
HUANG Shu-shi1, 2, LAI Jun-zhuo3, LU Ming-qian2, CHENG Qin4, LIAO Wei2, CHEN Li-mei1*
1. Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Chenggong 650500, China 2. Lab of Biophysics, Guangxi Academy of Sciences, Nanning 530007, China 3. Guangdong Institute of Arts Sciences, Zhanjiang 524400, China 4. Guangxi Subtropical Crops Research Institute, Nanning 530001, China
Abstract：A modified procedure of Percoll density gradient centrifugation was developed to isolate and fractionate synchronous cells from stationary phase (sp) cultures of different yeast strains，as well as Raman spectra discrimination of single yeast cells was reported. About 1.75 mL Percoll solution in 2 mL polypropylene centrifugal tube was centrifuged at 19 320 g, 20 ℃ with an angle rotor for 15 min to form continuous densities gradient (1.00～1.31 g·mL-1), approximately 100 μＬ sample was overlaid onto the preformed continuous density gradient carefully, subsequently, centrifuged at 400 g for 60 min in a tabletop centrifuge equipped with a angle rotor at 25 ℃. Yeast samples could be observed that the suspensions were separated into two cell fractions obviously. Both fractions of different yeast strains were respectively determined by differential interference contrast (DIC), phase contrast microscope and synchronous culture to distinguish their morphological and growth trait. The results showed that the lower fraction cells were unbudded, mostly unicellular, highly refractive, homogeneous and uniform in size, and represented growth characteristic synchronously; Their protoplasm had relatively high density, and contained significant concentrations of glycogen; all of which were accordant with description of quiescent yeast cells and G0 cells in previously published paper. It was shown that lower fraction was quiescent cells, synchronous G0 cells as well. A Raman tweezers setup was used to investigate the differences between two fractions, G0 cells and non G0 cells, at a single cell level. The result showed that both G0 cells and the non G0 cells had the same characteristic peaks corresponding biological macromolecules including proteins, carbohydrates and nucleic acids, but all characteristic peak intensities of G0 cells were higher than that of non G0 cells, implied that the macromolecular substance content of G0 cells was more higher. Principal component analysis (PCA) was performed between G0 cells and non G0 cells, the results showed that the chemical composition content among the synchronization G0 cells has less difference, and G0 cells were homogeneous but non G0 cells were heterogeneous, indicating single cell optical tweezers Raman spectroscopy could identify the synchronous and asynchronous cells. The modified method is feasible, economical and efficient highly. G0 synchronous cells of most yeast strains could be isolated by a modification of Percoll density gradient centrifugation.
Key words：Yeast;Percoll density gradient centrifugation;Synchronous cells;Raman spectrum
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