Bacillus subtilis as a model
On the colony level, it forms pellicles when grown on liquid media as a form of survival strategy (Prakash et al, 2003). These pellicles can dynamically form complex structures such as wrinkling (Pisithkul et al 2019) due to metabolic changes in their environment. B. subtilis also responds to mechanical stressors such as a difference in stiffness of the medium the bacteria is developing upon.
Advantages of using the MCAM™
Microbiology is a field that tends to use larger petri dishes or well plates to organize their samples. The larger media enable the maintenance and growth of the colonies to propagate the organism for subsequent experiment or for studies of larger organizations.
Well plates on the other hand are very convenient to control the environmental factors and introduce various treatments or perturbations (changes in the growth medium composition, in pH, addition of chemicals or drugs…).
The MCAM™ is a great tool to image a larger field of view while keeping resolution levels to observe subtle differences. It is also very well suited for well plate imaging due its multi camera architecture.
Bacillus subtilis observation over time under stressors
We acquired images of B. subtillis growing in 50 mm petri dishes as well as 24 well plates.
As can be seen in Figure 1, the bacterial colony grew over the 2% agar and started to form a 3D structure at the site of seeding in the center of the plate. The full plate image allowed us to distinguish detailed wrinkling in the central structure. Strikingly, when the bacteria was seeded in a full liquid medium, the pellicle formed wrinkles but without any particular spatial preferences inside the well (Fig. 1C).
Figure 1. (A,B) Full 50 mm diameter petri dish and Zoomed in Views of B. subtilis colony on 2% agar. (C )B. subtilis growing on liquid medium in a 24 well plate.
We then decided to take a further look into the pellicle and by extension wrinkle formation in B. subtilis colonies. We plated the bacteria in 24-well plates and treated each row with different chemicals and each column with different concentrations of the compounds. To assess the evolution of the colony, we acquired single snapshots of our well plate at 22, 48 and 72 hours post seeding. Figure 2 displays the experimental map and snapshots of this experiment. As expected, increasing concentrations of additives perturbed the growth of the colonies in most cases. Interestingly enough, this was not the case for the row of decreasing pH.
Figure 2. B. subtilis Pellicle Formation Assay with Additives at 22h, 48h, 72h in transmission (Top to bottom).
Finally we wanted to compare how different types of bacteria would compete for resources in the same environment. We seeded both B. subtilis and Saccharomyces cerevisiae in a 50 mm diameter petri dish and captured their development after 48 hours at 30C. The B. subtilis colony took over most of the plate engulfing the area around S. cerevisiae. A clear boundary was established between the two colonies and seemed to keep them separated.
Conclusion
Bacillus subtilis is a versatile tool to study changes in its environment and interactions with other bacteria. It is fairly easy to maintain and propagate, making it a perfect tool for classrooms and introductory courses in microbiology.
The MCAM™ Falcon used to image the samples above was designed for large areas while keeping a high level of resolution to be able to discern structures such as the wrinkle formation in the pellicle. Nevertheless, we were also able to image 24 well plates and perform dose response assays of common stressors on the B. subtilis pellicle. Other products in the MCAM™ line are specifically tailored to image well plates unlocking a faster pipeline from sample to data analysis.
References
Pisithkul T, Schroeder JW, Trujillo EA, Yeesin P, Stevenson DM, Chaiamarit T, Coon JJ, Wang JD, Amador-Noguez D. 2019. Metabolic remodeling during biofilm development of Bacillus subtilis. mBio 10:e00623–19.
Prakash, B., Veeregowda, B. M., & Krishnappa, G. (2003). Biofilms: a survival strategy of bacteria. Current science, 1299–1307.
Image Credit: Dr. Sunanda Sharma
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