A modified agar pad method for mycobacterial live-cell imaging.
A modified agar pad method for mycobacterial live-cell imaging.
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DOI：
10.1186/1756-0500-4-73
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发表时间：
2011-03-21
影响因子：
1.8
通讯作者：
Williams KJ
中科院分区：
文献类型：
--
作者：
Joyce G;Robertson BD;Williams KJ
到目前为止，原核生物活细胞成像的两种一般方法是在薄琼脂垫上培养细菌或在微通道中培养细菌。使用琼脂垫的方法在密封盖玻片之前将细胞“夹在”底部的琼脂垫和顶部的玻璃盖玻片之间。这种技术的优点是，它是简单和相对便宜的设置。然而，一旦盖玻片被密封，环境条件就不能被操纵。此外，琼脂垫的干燥和细胞在氧浓度逐渐下降的密封环境中的生长可能不允许进行更长期的研究，例如生长较慢的分枝杆菌所需的研究。我们在这里报告了一种改良的琼脂垫方法，其中细胞被夹在底部的盖玻片和盖玻片顶部的琼脂垫之间（而不是相反），并使用倒置显微镜从下面观察细胞。这种关键的修改克服了目前的一些限制与琼脂垫方法，并用于产生延时图像和电影的细胞生长的耻垢分枝杆菌和牛分枝杆菌BCG。该方法提供了对当前琼脂垫方法的改进，因为可以进行长期活细胞成像研究，并且允许在实验期间修改培养基。
Two general approaches to prokaryotic live-cell imaging have been employed to date, growing bacteria on thin agar pads or growing bacteria in micro-channels. The methods using agar pads 'sandwich' the cells between the agar pad on the bottom and a glass cover slip on top, before sealing the cover slip. The advantages of this technique are that it is simple and relatively inexpensive to set up. However, once the cover slip is sealed, the environmental conditions cannot be manipulated. Furthermore, desiccation of the agar pad, and the growth of cells in a sealed environment where the oxygen concentration will be in gradual decline, may not permit longer term studies such as those required for the slower growing mycobacteria. We report here a modified agar pad method where the cells are sandwiched between a cover slip on the bottom and an agar pad on top of the cover slip (rather than the reverse) and the cells viewed from below using an inverted microscope. This critical modification overcomes some of the current limitations with agar pad methods and was used to produce time-lapse images and movies of cell growth for Mycobacterium smegmatis and Mycobacterium bovis BCG. This method offers improvement on the current agar pad methods in that long term live cell imaging studies can be performed and modification of the media during the experiment is permitted.