Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

Summary

To study Myxococcus xanthus swarm behavior, we have designed a time-lapse microcinematography protocol that can be modified for different assays. It employs standard growth conditions adapted for microscopy, and yields reproducible results by the use of inexpensive, reusable silicone gaskets. We have used this method to quantify multicellular chemotaxis.

Abstract

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.

Protocol

Supplies needed:

• Klett meter
• Pipette and tips
• 2.5 ml microcentrifuge tubes
• Microcentrifuge
• CTTYE media:
  • 1.0% Casitone (Difco Laboratories), 0.5% yeast extract (Difco Laboratories),
  • 10.0 mM Tris-HCl (pH 8.0), 1.0 mM KH₂PO₄, 8.0 mM MgSO₄
• TPM media:
  • 10.0 mM Tris-HCl (pH 8.0), 1.0 mM KH₂PO₄, 8.0 mM MgSO₄
• Agarose
• Water bath set to 55°C....

Discussion

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 un.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.0% Casitone	Difco Laboratories
0.5% yeast extract	Difco Laboratories
Micro-sampling pipette	Fisher Scientific
100 μl glass disposable tip	Fisher Scientific
2 x 2 cm, 0.5-mm-thick silicone rubber gasket	Grace Bio-Lab Inc.