Ou Fang, McGoverin Cushla, White Joni, Swift Simon, Vanholsbeeck Frédérique
Department of Physics, The University of Auckland, Auckland, New Zealand.
The Dodd-Walls Centre for Photonic and Quantum Technologies, Auckland, New Zealand.
Methods Mol Biol. 2019;1968:123-134. doi: 10.1007/978-1-4939-9199-0_11.
Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with characteristics of interest. Many flow cytometers cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. Here we describe the optimization and evaluation of a bead-based method for absolute cell counting applicable to basic flow cytometers without specialized counting features. Prior to the application of this method to an unknown concentration of a species of bacteria, a calibration experiment should be completed to characterize limits of detection and range of linearity with respect to the plate count method. To demonstrate the calibration process, mixtures of Escherichia coli or Staphylococcus aureus with proportions of live and dead cells ranging from 0% to 100% were prepared. These samples were stained using nucleic acid-binding dyes, and 6 μm reference beads were added (LIVE/DEAD BacLight kit). The calibration samples were analyzed using bead-based FCM as well as the agar plate count method, and the results from both methods were compared.
流式细胞术（FCM）基于对散射光和荧光的检测来识别具有感兴趣特征的细胞。许多流式细胞仪无法精确控制通过其检测点的流速，因此无法立即获得样品的体积和浓度。在此，我们描述了一种基于微珠的绝对细胞计数方法的优化和评估，该方法适用于没有专门计数功能的基本流式细胞仪。在将该方法应用于未知浓度的某一细菌种类之前，应完成校准实验，以确定相对于平板计数法的检测限和线性范围。为了演示校准过程，制备了活细胞和死细胞比例从0%到100%的大肠杆菌或金黄色葡萄球菌混合物。这些样品用核酸结合染料染色，并加入6μm的参考微珠（LIVE/DEAD BacLight试剂盒）。使用基于微珠的流式细胞术以及琼脂平板计数法对校准样品进行分析，并比较两种方法的结果。
