Alright, here they are: the biofilm tests. In case you didn’t know or forgot, biofilms are basically little clusters of bacteria (with kind of a protective layer) that adhere to surfaces.  Examples of biofilms you may be familiar with include some types of pond scum, and especially plaque. They look like this:

Biofilms are pretty darn cool, even though they just kind of sit there in the well plates and act like they pay rent. But how did they get there in the first place?

We started out by making a culture using 5uL e.coli bacteria stock and 5mL LB media. After being in the incubator overnight, we did the same process again, this time just putting the bacteria in for a cozy 3 hours. When it was ready, we made 25 mL of a 1/500 dilution of our bacterial concoction with MH broth (that’s about 2uL bacteria/1 mL MH). After preparing a 96-well plate, we filled each remaining well with 100 uL of this diluted mixture. We parafilmed it and put it in the incubator. This was our 48-hour biofilm. We did the same thing the next day, just for a 24-hour biofilm. So why record the times? Even though it is not yet known the optimal time of growth for bacteria, we try to test at different times to see different types of growth and to get more variety out of the experiment.

Making biofilms

Incubating plates

When the biofilm plates were finally ready, we did something with them that might surprise you. We took them into the fume hood, took off the parafilm, got out an autoclave bucket, and shook them into the bucket. It doesn't see very scientific, but shaking the plate expels all these planktonic bacteria that would get in the way of the biofilm tests. We then added 100 uL of broth to each well, parafilmed the plates again and incubated them overnight.
The next morning, we did the whole shaking procedure over again for the 24hr. biofilm, rinsing with water in the same way. Then, I took two rows: Row C and Row E. I filled columns 2-10 of Row C with plain old LB broth for our control test. Then I filled columns 2-10 of Row E with 10 mg/mL Dawn detergent and let it sit on the counter for two hours:

When the plates were done, we put the biofilms on a sonicator to break them up. Then, I scooped up the liquid from 3 of the wells of my control row (about 300 uL), diluted them and plated them. I did the same thing with my other samples, so overall, I had used 6 plates and had 3 trials.

So, overall, it was pretty interesting. I have made a CRAP TON of agar plates, and chances are good I'm going to go through them fairly quickly. But overall, it's nice to be more comfortable in the lab setting, and next week I will have data on the effects of Dawn on e.coli biofilms, but for now, we wait.

До свидания! (Goodbye!)

Mackenzie