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Microbiology JoVE (Open Access) Citable · DOI

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

DOI: 10.3791/54752-v
What you'll learn
  • ✓ Prepare fluorescently labeled Bacillus subtilis strains for controlled co-inoculation experiments
  • ✓ Visualize spatial segregation patterns in microbial colonies using fluorescent microscopy
  • ✓ Examine how founder cell density influences assortment in evolving microbial populations
  • ✓ Interpret differences in colony growth patterns (swarming, sliding, biofilm) through imaging
Protocol

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

Difficulty
intermediate
Total time
~3–5 days (including culture preparation, inoculation, growth, and imaging)
Model organism
Bacillus subtilis (fluorescently labeled strains)
Biosafety
BSL-1

Steps

1
Prepare media and starter cultures

Prepare growth media and inoculate starter cultures of fluorescently labeled Bacillus subtilis strains. Allow cultures to grow to appropriate density for experimental co-inoculation.

▶ 00:47
2
Co-inoculate labeled strains for surface spreading

Mix fluorescently labeled bacterial strains at controlled founder densities and inoculate onto agar plates to observe surface spreading phenotypes (swarming or sliding).

▶ 01:54
3
Co-inoculate labeled strains for biofilm growth

Co-inoculate fluorescently labeled strains under conditions promoting biofilm formation on surfaces, maintaining controlled spatial distributions.

▶ 03:16
4
Image colonies using fluorescent microscopy

Acquire fluorescent microscopy images of growing colonies to visualize spatial segregation and assortment patterns of the two labeled strains.

▶ 04:32
5
Analyze spatial segregation patterns in colonies

Compare fluorescent imaging results across swarming, sliding, and biofilm phenotypes to demonstrate how growth mode influences spatial assortment of microbial populations.

▶ 05:36
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