1Department of Environmental Health Sciences, University of South Carolina (USC), 2Department of Biology, Syracuse University
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Taylor, R. G., Welch, R. D. Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography. J. Vis. Exp. (42), e2038, doi:10.3791/2038 (2010).
A swarm of the δ-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of signals to create complex, dynamic patterns as a response to environmental cues. These patterns are self-organizing and emergent; they cannot be predicted by observing the behavior of the individual cells. Using a time-lapse microcinematography tracking assay, we identified a distinct emergent pattern in M. xanthus called chemotaxis, defined as the directed movement of a swarm up a nutrient gradient toward its source 1.
In order to efficiently characterize chemotaxis via time-lapse microcinematography, we developed a highly modifiable plate complex (Figure 1) and constructed a cluster of 8 microscopes (Figure 2), each capable of capturing time-lapse videos. The assay is rigorous enough to allow consistent replication of quantifiable data, and the resulting videos allow us to observe and track subtle changes in swarm behavior. Once captured, the videos are transferred to an analysis/storage computer with enough memory to process and store thousands of videos. The flexibility of this setup has proven useful to several members of the M. xanthus community.
Part 1: Cell Preparation
Start by creating a sterile environment.
Part 2: Agar Preparation
Part 3: Nutritive Disk Construction
Part 4: Tracking Assay Preparation - Set Up Plate Complexes
Assemble the components, prepare the slide complexes, place the nutritive disk, pour the TPM media/agarose, separate and dry plate complexes, plate cells, and assemble tracking assay plate complexes.
Assemble plate complex components
Place nutritive disk
IMPORTANT: Steps 5 through 10 must be done to one slide complex at a time; otherwise the media/agarose could start to solidify resulting in poor movie quality.
Separate and dry plates
IMPORTANT: To prevent the media/agarose from drying out, steps 11 through 20 should only be performed on one slide complex at a time.
IMPORTANT: For best results, steps 11 through 13 should be performed at 4°C.
Part 5: Movie Preparation
Figure 1. Cartoon illustration of the TM plate complex. (A) shows the basic TM plate complex in exploded view and cross section. (B) shows the use of larger gaskets.
Figure 2. Microscope cluster. Each microscope node (inset) consists of a Nikon E400 microscope, objectives, a heated stage, an Insight camera, and a notebook computer. Each node is networked together and linked to a master controller computer. Two of the nodes are set up with fluorescence capabilities that consist of the EXFO light source and two Uniblitz shutters.
Figure 3. A 20X image of tracking assay apparatus at time = 0. Scale bar, 1 mm.
Figure 4. Adaptability of the TM plate complex. (A) a 100X image of M. xanthus gliding motility on CTTYE in 1.0% agar. (B) a 20X image of P. aeruginosa twitching motility. (C) a 20X image S. marcescens swarming motility. Both (B) and (C) were assayed on LB in 1.0% agar. (D) a 40X image of M. smegmatis sliding motility on LB in 0.5% agar. This image was captured using the alternative assay configuration seen in Fig 1B.
Video 1. A time-lapse video of an swarm subjected to the tracking assay.
Video 2. A time-lapse video of an M. xanthus swarm where 1% of the cells are fluorescently labeled. Alternating phase-contrast and fluorescent images were captured and overlaid to elucidate the position of fluorescent cells within the swarm. This video was captured on CTTYE in 1.0% agar.
Video 3. A time-lapse video of M. xanthus gliding motility. This video was captured on CTTYE in 1.0% agar.
Video 4. A time-lapse video of P. aeruginosa twitching motility. This video was captured on LB in 1.0% agar.
Video 5. A time-lapse video of S. marcescens swarming motility. This video was captured on LB in 1.0% agar.
Video 6. A time-lapse video of M. smegmatis sliding motility. This video was captured on LB in 0.5% agar using the alternative assay configuration seen in Fig 1B.
Time-lapse microcinematography (TM) has become a standard approach to studying prokaryotic motility 2-7. Traditionally, TM is performed by using filter paper wicks, thin agar pads, or agar slabs as substrates 8-11. These methods are adequate and cost effective when used to generate image sequences for general illustrations of bacterial movement. However, if image sequences must result in the generation of reproducible and quantitatively rigorous data, these methods are time consuming and somewhat unreliable. For example, variations in these techniques caused by human error could lead to a wide array of inaccuracies, from irregularities in the agar surface that could dramatically affect the behavior of the bacteria being studied to differences in the focal plane from one side of the assay substrate to the other. To solve these problems, we have designed a TM plate complex that is of sufficiently consistent quality to yield reproducible results by employing silicone gaskets that are both inexpensive and reusable (Fig. 1). In addition, the plate complex is highly modifiable and has proven to stay hydrated and sufficiently oxygenated for more than a week over a variety of media types and agar concentrations.
To facilitate the generation of time-lapse movies, 8 Nikon E400 microscopes were outfitted with 2X, 4X, and 10X objectives each. In addition, an Insight camera (Diagnostic Instruments, Inc.), and a heated stage (20/20 Technologies) was acquired for each microscope. Each camera was linked to a notebook computer on which the highly modifiable image acquisition software SPOT (Diagnostic Instruments, Inc.) had been installed. All the various components were assembled into nodes, each consisting of a microscope, three objectives, an Insight camera, a heated stage, and a controlling computer. Each of the eight nodes were networked together into a cluster and linked to a storage computer which is used to compile, analyze, and store the time-lapse videos generated by the cluster (Fig. 2). The storage computer was outfitted with a 1 terabyte RAID system, which is needed to store the vast amount of data generated by the cluster.
Once complete, the plate complex is placed on the heated stage of the microscope and tracking assay is initiated. Images are acquired at preset intervals that are determined based on the motility rate of the cells. For example, individual M. xanthus cells move at a rate of approximately one cell length per minute. If images are acquired of an M. xanthus swarm at that same rate (one image per minute), the resulting time-lapse video will appear smooth during playback. Once the image acquisition is complete, the images are compiled into a sequential matrix and played back at a sufficient rate that they appear to be moving (Video. 1). This time-lapse video can now be analyzed using a variety software packages.
Variations on Assay. The TM plate complex is highly modifiable and can be used to visualize many different microorganisms under a variety of conditions. Swarm visualization can be performed using phase-contrast microscopy (which records the behavior of the swarm as a single entity), fluorescence microscopy (which records the behavior of individual fluorescent cells that have been diluted in a non-fluorescent population), or a combination of both (which overlays alternating sequential phase-contrast and fluorescent images) (Video 2). The plate complex has been used to successfully generate time-lapse videos of several prokaryotic behaviors including M. xanthus gliding motility, Pseudomonas aeruginosa twitching motility, Seratia marcescens swarming motility, and the novel Mycobacterium smegmatis sliding motility (Fig. 4 and Videos 3-6).
No conflicts of interest declared.
This research was made possible by a National Science Foundation Career award (MCB-0746066, Characterization of Transcriptional Activators that Regulate Emergent Behavior) to R.D.W.
We are grateful to L. J. Shimkus, B. S. Goldman, G. Suen, M. Singer, L. G. Welch, K. A. Murphy, and H. G. Taylor for helpful discussions and comments on the manuscript.
|1.0% Casitone||Difco Laboratories|
|0.5% yeast extract||Difco Laboratories|
|Micro-sampling pipette||Fisher Scientific|
|100 Ál glass disposable tip||Fisher Scientific|
|2 x 2 cm, 0.5-mm-thick silicone rubber gasket||Grace Bio-Lab Inc.|