Shapes of bacterial colonies and fluidity of media

     Does the roundness of colonies of Bacillus subtilis – a bacteria – change depending on the fluidity of the medium ? In the laboratory, we grow bacteria in different media ; for Bacillus subtilis it is the LB medium. To make this medium solid, we add agar : the more agar we add, the more the medium will be solid – until the point where the agar is in excess. The usual agar concentration is 1.5%, which means that 1.5% of the medium is composed of agar. In this medium, the colonies, which are groups of bacteria that emerged from one cell that divided again and again, are about round, even though they are not perfect circles. Studying how is the shape of colonies affected when the agar concentration is changed could help us model the growth of bacteria and understand better the way they spread. I chose to study Bacillus subtilis because it a bacterium with flagellas, appendages that allow a cell to move around in liquids ; its capacity to move makes this bacterium more likely to form weird-shaped colonies.

A cell of Bacillus subtilis.

 Picture by Allon Weiner, public domain

  
    To verify that, I took very few cells of B. subtilis that I spreaded on plates with different agar concentration, then I put them in an incubator at 37°C, an ideal temperature for a bacterium that live notably in human guts ! A few hours after that, the bacteria grew into colonies. I could measure the area and perimeter of single colonies and make a ratio of area/perimeter², which I call circularity ratio (CR). For a circle, this ratio is constant ; thus we can know how round is a figure by measuring how different is its CR from the one of circles.

     On the x-axis we have the concentration of agar and on the y-axis is the CR. Each dot is the CR of one colony. Dots are in 4 groups because I used 4 different agar concentrations – 1%, 1.5%, 2% and 4% - thus each set of dots corresponds to all the observed colonies for one conentration. The green line represents the CR of circles, which is equal to 1÷(4 x π). The red lines are means of CR for each set of dots. The further is the red line of one set from the green line, the less the colonies for this concentration are round. Those results suggest that there is a link between agar concentration and roundness below 2% since the average CR increases between 1 and 2%. However, we can observe no particular link between 2 and 4%, the concentration stays about constant.

     I observed only 421 colonies, which is not enough to be sure of anything, even though it is a good evidence that there could be a link between agar concentration and roundness of colonies. In order to know if there is indeed a link, we should make more similar observations, especially with different agar concentrations, like 0.7, 1.2 or 3%. This would allow to draw much more accurate graphs !

Samuel Churlaud, September 2016.

If you want to know more :

Why is the CR constant for circles ?

The area A of a circle is A=π x r² with r the radius

Its perimeter p is p=2 x π x r

Thus we have A=π x (p÷(2 x π))²

A=(π x p²)÷(4 x π²)

A=(p²)÷(4 x π)

A÷p²=1÷(4 x π)

The final result does not depend on any variable, which means that whatever the area and the perimeter, the CR of a circle will always be 1÷(4 x π).

My full report about this project, along with the data, script for data analysis and more is available here : https://tinyurl.com/jg9239h

If the cells of B. subtilis are many and dense enough on a plate, they can grow colonies in the shape of fractals ! This looks super cool, you can find some pictures on a search engine or in the studies I share below.

Some studies about the subject (technical) :

• Mitchell, A. J., & Wimpenny, J. W. T. (1997). The effects of agar concentration on the growth and morphology of submerged colonies of motile and non‐motile bacteria. Journal of applied microbiology, 83(1), 76-84
• Ben-Jacob, E., Schochet, O., Tenenbaum, A., Cohen, I., Czirok, A., & Vicsek, T. (1994). Generic modelling of cooperative growth patterns in bacterial colonies. Nature, 368(6466), 46-49
• Fujikawa, H., & Matsushita, M. (1989). Fractal growth of Bacillus subtilis on agar plates. Journal of the physical society of japan, 58(11), 3875-3878