Modern microscopy instrumentation and techniques permit the study of microorganisms at the single cell level and have been used extensively to study essential bacterial processes such as motility, cell division and the cell cycle. Several methods have been reported for live-cell time-lapse imaging of bacteria which can be crudely divided into two general approaches: growing bacteria on thin agar pads [1–3] or growing bacteria in micro-fluidic chambers [4, 5]. Both have their advantages and limitations.
The use of micro-channels is a sophisticated technique, often combined with micro-fluidic devices, and is consequently a more expensive method that requires specialised equipment and greater operator expertise. A cover slip containing a micro-channel is produced and the cells/culture fluid added to the channel, which is then covered with a porous membrane . 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. However, 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 . Furthermore, due to the spatial constraints, daughter cells either have to form as a chain, which is not necessarily the natural pattern for all bacteria, or they detach from the substrata and are lost in the flow . Finally, the physical constraint in micro-channels may prevent significant aspects of cell division from occurring, such as snapping in rod-shaped actinomycetes .
Agar pad methods consist of bacterial cells sandwiched between a thin agar pad and a glass cover slip on top, which is then sealed. The advantages of this technique are that it is simple, relatively inexpensive to set up and there are no physical barriers allowing expansive growth. However, the environmental conditions cannot be manipulated which limits the variety of experiments that can be performed and the growth of aerobic cells may be affected in a sealed environment. The agar pad itself is also prone to desiccation over time hampering longer term experiments . This technique is therefore best suited to short time studies where no manipulation of the culture conditions is required.
Time-lapse imaging of bacteria using existing agar pad methods have been used successfully to study a number of bacteria including Escherichia coli , Corynebacterium glutamicum , Vibrio cholerae , Caulobacter crescentus  and Pseudomonas aeruginosa . However, time-lapse imaging movies of the slower growing mycobacteria using existing agar pad methods are yet to be reported. Here we report the development of a modified agar pad method which overcomes some of the limitations of current protocols and present bacterial imaging and movie data obtained using this method.