Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905, USA.
Biotechnol Bioeng. 1991 Aug 5;38(4):340-52. doi: 10.1002/bit.260380404.
Pronounced spatial nonuniformities in cell density, physiology, and activity frequently arise within densely packed immobilized cell supports. For a more fundamental understanding of immobilized cell phenomena, we have developed high-resolution microfluorimetric procedures to analyze local variations in both immobilized cell loading and growth rate. Fluorescent staining of total cellular DNA provides a measure of local biomass density. Actively growing (DNA synthesizing) cells are marked by pulse-labeling newly synthesized DNA with the thymine analog, bromouracil. An immunofluorescent technique allows subsequent detection of spatial variations in DNA synthesis rates. These procedures enable the influence of mass-transfer limitations and other immobilization effects on cell distribution and activity to be readily quantified. We demonstrate this approach through analysis of the patterns of growth of Escherichia coli entrapped within Sr-alginate gel beads. The experimental techniques are potentially applicable to a variety of other aggregate cell systems.
在密集固定化细胞载体中，细胞密度、生理学和活性常常呈现明显的空间非均一性。为了更深入地了解固定化细胞现象，我们开发了高分辨率微荧光分析程序，以分析固定化细胞负载和生长速率的局部变化。总细胞 DNA 的荧光染色可提供局部生物量密度的度量。用胸腺嘧啶类似物溴尿嘧啶对正在合成 DNA 的活跃生长（DNA 合成）细胞进行脉冲标记，可标记出正在合成 DNA 的细胞。免疫荧光技术可随后检测 DNA 合成速率的空间变化。这些程序可方便地量化传质限制和其他固定化效应对细胞分布和活性的影响。我们通过分析包埋在 Sr-藻酸盐凝胶珠中的大肠杆菌的生长模式来证明这种方法。该实验技术可能适用于各种其他聚集体细胞系统。
