Just
how hot are biofilms?
Have you ever wondered the extent of
biofilm protection? Well I have! For a week, I had the opportunity of working
on the influence of temperature on biofilms, and if the later were really
efficient when it came to protecting cells from a heat shock.
A biofilm is a clog of cells, linked together by a
matrix called Extracellular Polymeric Substance (EPS). When bacteria are close
enough, they send each other a signal, which tells them whether or not there is
enough of them to form bonds. When a bacterium receives enough signals, it will
modify its structure and create carbon and / or sugar bonds with each other. This
EPS is able to provide protection from the outside environment, such as heat
shock.
P.aeruginosa is
perfect to work with as they are obligate aerobes, meaning that they need
oxygen to live. This way, all the bacteria would come up to the surface to
breathe, thereby all being at the same place at the same time. This way the
bacteria will receive enough signals to form biofilms.
The
first step for answering my hypothesis was to prepare my tubes. In order to do
so, I put 1,5 mL of a mixture of a special growing media called LB, and P.aeruginosa, a bacteria that can easily
develop biofilm, in 20 different tubes. Half of these tubes went into an
incubator, and the other half in a thermo shaker (an incubator that moves non
stop). 24 hours later, I took 10 tubes out (5 of each) and exposed 4 to 50°C
for 5 minutes. I then vortexed the 10 tubes and plated them at 10^-4, 10^-5 and
10-6. Finally, I waited overnight for the colonies to develop, and counted
them.
This graph represents the results I got. As you can
maybe tell, they are not what I had expected… Once the plates had grown
colonies, I was able to count them and therby determine the living cells ratio.
First I had to count all the colonies. I obtained the following results: 63 and
49 for the plates that were in the incubator and that underwent heat shock (6,3
x 10^7 and 4,9 x 10^7 live cells); 123 and 98 for the samples that were in the
incubator but didn’t go through heat shock (1,23 x 10^8 and 9,8 x 10^7 live
cells). Regarding the samples that were in the thermo-shaker (and thereby had
developed biofilms), the plates had 51 and 49 colonies for heat shock (5,1 x
10^7 and 4,9 x 10^7 live cells) and 80 and 93 (8,0 x 10^7 and 9,3 x 10^7 live
cells) for no heat shock. What these results are showing is that in the samples
without biofilms, the cells survived better to heat shock. When we do the ratio, we obtain 0,51 for the cells that were protected by a biofilm, and 0,58 for the cells that weren't.
However, these results cannot approve nor disapprove
any hypothesis, as they are a single data. Moreover, when I looked at the
samples under the microscopes, all the bacteria were alive and moving. This
might suggest that the heat shock was not high enough. In order to answer my
hypothesis, there are many options that could be furthered. For example, I could try putting the
solution into test tubes instead of eppendorfs, or why not directly into petri
dishes, thereby creating more interface with oxygen and easing the process of
biofilm creation. Finally, as biofilms are supposed to protect cells from the
outside environment, we could try to determine the limit of the protection by
establishing the breaking point of the biofilm, meaning the point where we
wouldn’t need to vortex the solution as the heat shock would have broken the
biofilm itself.
If
you want to know more, you can check these links :
https://en.wikipedia.org/wiki/Biofilm#Properties
https://repositorium.sdum.uminho.pt/bitstream/1822/26324/1/PDF2.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568995/
Special thanks to Petunia center, the CRI and Paris Descartes
Daphné Guénée
@daphne_guenee
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