Abstract
BackgroundTwo 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.FindingsWe 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.ConclusionsThis 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.
Highlights
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
Several methods have been reported for livecell time-lapse imaging of bacteria which can be crudely divided into two general approaches: growing bacteria on thin agar pads [1,2,3] or growing bacteria in microfluidic chambers [4,5]
Micro-fluidic devices usually constrain cell growth to one or two dimensions to allow for single cell tracking, and the cells have to be attached to a substratum strong enough to resist the fluid flow [6]
Summary
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. Several methods have been reported for livecell time-lapse imaging of bacteria which can be crudely divided into two general approaches: growing bacteria on thin agar pads [1,2,3] or growing bacteria in microfluidic chambers [4,5] A cover slip containing a micro-channel is produced and the cells/culture fluid added to the channel, which is covered with a porous membrane [5] In this technique, the system is not sealed so gaseous exchange can occur freely and agents can be added to the culture fluid mid-experiment, so longer and more varied manipulations can be performed. The physical constraint in micro-channels may prevent significant aspects of cell division from occurring, such as snapping in rod-shaped actinomycetes [2]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
