Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Biotechnol Bioeng. 1993 Jun 5;42(1):30-6. doi: 10.1002/bit.260420105.
A novel method is described for the on-line determination of viable cell number. It has been tested in fermentations of Escherichia coli. The cells are transfected with the gene for firefly luciferase and fed low levels of luciferin in the medium. The reaction requires ATP, so the nonviable cells cannot produce light. Thus, light production is linear with viable cell density from innoculation through most of exponential growth. The light emitted by these cells is then conducted from the reaction vessel to the light detection equipment by an optical fiber. With the equipment described below, as few as a 10(6) cells/mL, or an OD(600) of 0.004, are easily detectable and concentrations greater than 10(10) cells/mL are well within range. The data are collected by a computer, so adaptation to on-line control applications is straightforward. During lag phase, this method is much more accurate then optical density measurements. At the end of exponential growth, rapid changes in light production mark carbon source depletion and the onset of cell lysis. A simple model accounts for the luciferin used during the fermentation and corrects the light detected to the proper cell density.
一种新的方法用于在线测定活细胞数量。该方法已经在大肠杆菌发酵中进行了测试。将萤火虫荧光素酶基因转染到细胞中，并在培养基中低水平地添加荧光素。该反应需要 ATP，因此非存活细胞无法产生光。因此，从接种到大多数指数生长期，光的产生与活细胞密度呈线性关系。然后，通过光纤将这些细胞发出的光从反应容器引导到光检测设备。使用下面描述的设备，很容易检测到低至 10^6 个/mL，或 OD600 为 0.004 的细胞，并且浓度大于 10^10 个/mL 的细胞也在检测范围内。数据由计算机收集，因此适应在线控制应用非常简单。在迟滞期，该方法比光密度测量更准确。在指数生长期结束时，光产生的快速变化标志着碳源的耗尽和细胞裂解的开始。一个简单的模型解释了发酵过程中使用的荧光素，并将检测到的光校正为适当的细胞密度。
