Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris.
Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris.
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DOI：
10.1371/journal.pone.0097269
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Ross IL
中科院分区：
文献类型：
--
作者：
Chioccioli M;Hankamer B;Ross IL
干重生物量是藻类养殖中的一个重要参数。直接测量需要称量毫克量的干燥生物质，这对于包含少量细胞的小体积系统是有问题的，例如实验室研究和微孔板中的高通量分析。在这些情况下，必须使用间接方法，导致测量伪影的严重性随细胞类型和所用条件的不同而不同。在这里，我们利用流式细胞仪的脉冲宽度数据来估计细胞密度和生物量，以普通小球藻和莱茵衣藻为模型藻类，并将其与光密度法进行比较。用流式细胞仪测量细胞浓度比检测750 nm处的光密度(OD750)更敏感。然而，当生长条件不同时，细胞密度和光密度都不能很好地与生物量相关。与普通微囊藻在TAP(三醋酸酯-磷酸盐)中的生长相比，在TAP+葡萄糖中生长的细胞分裂速率减慢，干生物量积累比不含葡萄糖时增加2倍。伴随而来的是细胞体积的增加。利用流式细胞术中的激光散射特性来估计细胞直径，结果表明，流式细胞术的脉冲宽度与细胞干重、生物量之间呈现出经验的、非线性的关系。这种关系可以通过使用高渗条件(1M氯化钠)脱水细胞来线性化，如密度梯度离心法所示。与光密度测量相比，流式细胞术用于生物量估计具有简单、灵敏、提供更全面的信息等优点。此外，为了方便和准确，可以使用周期性的流式细胞仪测量来校准OD750测量。这种方法对于小样本特别有用，其中细胞特征，特别是细胞大小，预计在生长过程中会发生变化。
Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD750) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD750 measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth.
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