Advanced Technology Institute, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, Surrey GU2 7HX, United Kingdom.
Tuberculosis (Edinb). 2012 Nov;92(6):489-96. doi: 10.1016/j.tube.2012.06.006. Epub 2012 Sep 4.
Phenotypic heterogeneity in bacterial populations is thought to contribute to a number of important phenomena including sporulation and persistence. The latter has clinical implications in many diseases such as tuberculosis, where persistence of Mycobacterium tuberculosis within the human host is believed to be the root cause of latent tuberculosis and the ability of a minority population of cells to survive antibiotic exposure, despite being genetically identical to the bulk population that are killed. However, phenotypic variations caused by non-genetic mechanisms are difficult to study because of the transient nature of the persistent state and thereby the requirement to observe individual cells in real-time. Recently, microfluidics, combined with time-lapse microscopy, has become a powerful tool for studying population heterogeneity in bacteria. However, growth and replication of mycobacterial cells provide particular problems for the development of microfluidic systems due to their tendency to grow in three dimensions. We here describe a novel microfluidic device for the observation of growth and antibiotic killing in individual mycobacterial cells. We constructed a microfluidic device suitable for studying single cell behavior in mycobacteria. The growth of single cells of Mycobacterium smegmatis expressing green fluorescent protein was monitored using a confocal laser scanning microscope. Within the device M. smegmatis cells were tightly confined within a hydrogel matrix thus promoting planar growth. Cell growth and killing was observed in the device with dead cells highlighted by uptake of propidium iodide. Conclusions/Significance. We demonstrate that our device allows real-time analysis and long-term culture of single cells of mycobacteria, and is able to support the study of cell death during the application of antibiotics. The device will allow observation of individual cells' cell genealogy to be determined and direct observation of rare states, such as persistence.
细菌群体中的表型异质性被认为是许多重要现象的原因，包括孢子形成和持久性。后者在许多疾病中具有临床意义，例如结核病，据信结核分枝杆菌在人体宿主内的持久性是潜伏性结核病的根源，以及少数细胞能够在抗生素暴露下存活的能力，尽管它们与大量被杀死的细胞在遗传上是相同的。然而，由于持久性状态的瞬态性质，因此需要实时观察单个细胞，因此很难研究由非遗传机制引起的表型变异。最近，微流控技术与延时显微镜相结合，已成为研究细菌群体异质性的有力工具。然而，分枝杆菌细胞的生长和复制由于其倾向于三维生长，给微流控系统的开发带来了特殊问题。我们在这里描述了一种用于观察单个分枝杆菌细胞生长和抗生素杀伤的新型微流控设备。我们构建了一种适用于研究分枝杆菌单细胞行为的微流控设备。使用共聚焦激光扫描显微镜监测表达绿色荧光蛋白的耻垢分枝杆菌单细胞的生长。在该设备中，分枝杆菌细胞被紧密限制在水凝胶基质中，从而促进平面生长。在设备中观察到细胞生长和杀伤，死亡细胞通过摄取碘化丙啶被突出显示。结论/意义。我们证明，我们的设备允许实时分析和长期培养分枝杆菌的单个细胞，并能够支持抗生素应用过程中细胞死亡的研究。该设备将允许观察单个细胞的细胞谱系，并直接观察稀有状态，如持久性。
