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

Hi guys-

In order to prepare for the biofilm tests that are coming up this week, it is necessary to show you one of the essential aspects of biofilm tests, and quite frankly, working with microbes at all: serial dilutions.

Luckily, serial dilutions are pretty simple.

For the general procedure, I took 6 test tubes for each microbe, filled each with 3mL of autoclaved water, lined the test tubes up in a row, and filled the first with 300 uL of inoculated stock. I put the test tube in a vortexor( it basically spins the test tube to mix stuff and ensure there are no clumps of bacteria floating around that could affect the results). While it is natural for stuff to settle at the bottom of a test tube, if it is not mixed properly, it could alter the concentration, OD, etc. and change results.  Then I  took 300 uL from that first test tube, put it in the next one, and did the same thing. It looks like this:

After that, for all three types of bacteria, I took agar plates, split them up into 6 sections (one for each dilution) and put 15 uL of diluted stock into each section. Then into the incubator they went to grow overnight! If you did it right, it should look like this:

Based on the kind of procedure you're doing with these plates, you can either perform an enumeration(count the plates to ensure the bacteria are growing well) or you could be performing a biofilm test. The biofilm test is different, but I will go over it in the next couple of days.

Well I hope that little nugget of information was helpful. You can generally do this kind of thing with different types of microbes, but at least I hope you are now educated on one of the most important microbiological procedures and that you are ready to hear about the biofilm tests!

увидимся (see you!)

Mackenzie

Ladies and gentlemen, here it is, as promised, part of my first set of reliable data!
 Here's what we did:
  The e.coli, staph. epidermidis, and lactobacillus were originally diluted 20 ug/mL, 20 ug/mL and 10 ug/mL, respectively (because Lactobacillus originally had better growth, so we needed to balance out the concentrations). After being put in the detergent, this cut the dilutions in half (10, 10 and 5 ug/mL).Since the lactobacillus was diluted down from 5 ug/mL, the second column represents that concentration of lacto. Then the third column would show that diluted in half, so 2.5 ug/mL, and so on.
To find the MIC, we had to take the control column (the last column) and divide it by 10. In the lactobacillus  case, that is about 0.11. Since the MIC is what happens when there is 10% growth, around 0.11 would be the number we're looking for.
This set of data is from the Lactobacillus MIC with Cascade (top three rows) and Dawn (bottom three rows).

2 3 4 5 6 7 8 9 10 11 0.058 0.058 0.058 0.058 0.058 0.059 0.059 0.059 0.059 0.059 0.36 0.981 1.046 1.113 1.163 1.124 1.132 1.078 1.156 1.222 0.447 0.944 0.949 0.973 1.027 1.096 1.083 1.024 1.072 1.164 0.376 1 0.952 0.94 0.978 1.031 1.011 0.988 1.077 1.074 0.072 0.081 0.102 0.408 0.426 0.926 1.047 1.004 1.027 1.081 0.37 0.117 0.038 0.083 0.436 0.877 1.085 1.006 1.129 1.078 0.536 0.096 0.238 1.093 0.417 0.66 0.76 1.061 1.201 1.123 0.057 0.054 0.055 0.059 0.064 0.06 0.058 0.06 0.059 0.059
Dilutions by column: 5 ug/mL   2.5 ug/mL  1.25 ug/mL...........AND SO ON.

 Below is the data for e.coli and staph. epidermidis, respectively, but the calculations show that neither detergent had much of an effect on them.

0.062	0.06	0.078	0.049	0.046	0.05	0.063	0.059	0.042	0.07
0.551	0.552	0.505	0.639	0.606	0.6	0.827	0.66	0.829	0.457
0.436	0.728	0.746	0.584	0.584	0.563	0.673	0.577	0.735	0.714
0.299	0.716	0.732	0.739	0.677	0.608	0.612	0.623	0.762	0.597
0.672	0.52	0.484	0.487	0.604	0.621	0.782	0.749	0.774	0.658
0.573	0.495	0.554	0.473	0.534	0.624	0.747	0.934	0.726	0.769
0.695	0.527	0.469	0.459	0.554	0.568	0.783	0.914	0.802	0.731
0.063	0.054	0.055	0.055	0.046	0.045	0.06	0.06	0.075	0.062
2	3	4	5	6	7	8	9	10	11
0.043	0.039	0.039	0.04	0.039	0.04	0.04	0.039	0.042	0.039
0.336	0.63	0.069	0.844	0.847	0.805	0.796	0.842	0.725	1.03
0.463	0.321	0.072	0.949	0.95	0.914	0.912	0.86	0.776	0.951
0.119	0.122	0.074	0.676	0.747	0.962	0.938	0.859	0.826	0.94
0.289	0.582	0.069	0.258	0.367	0.728	0.834	0.824	0.884	0.903
0.657	0.613	0.074	0.309	0.884	0.699	0.657	0.872	1.053	0.945
0.101	0.069	0.069	0.324	0.411	0.528	0.67	0.875	1.018	1.03
0.055	0.054	0.054	0.058	0.048	0.046	0.041	0.056	0.041	0.039

 10 ug/mL   5ug/mL   2.5ug/mL.......AND SO ON

 Since e.coli did not have these "magic numbers" for a true MIC, that means that the MIC for it would be much greater than the original concentration, so:

MICecoli= >10 ug/mL because it could take a lot more detergent to kill off the e.coli.


Since s. epidermidis responded slightly to the Dawn detergent (bottom 3 rows), its MIC would be 10->10 ug/mL.
And since Lactobacillus responded greatly to Dawn, its MIC would be 2.5 ug/mL, where the highlighted part of the table shows.

											0.066
											0.061

The purpose of an MIC is to determine the toxicity of a detergent- how many cells it kills. This test showed that Cascade was generally non-toxic to these gut flora (say Hallelujah, dishwasher lovers) and while the Dawn was generally non-toxic to e.coli and epidermidis, it was toxic to the Lactobacillus. Of course, these liquids were toxic at relatively high levels, so we are unsure of how much detergent a person would actually consume. One study by Mercurius- Taylor argued that it was somewhere around 100 ug/g weight.

But the more important thing is the biofilm tests. Gut flora accumulate in biofilms, rather than pockets of broth. Since the detergents didn't seem to kill the bacteria that much, it is quite possible that they just sweep up biofilms and carry them away. While this may not seem like a bad thing, if a colony of gut flora lives in one part of the intestine and is swept away to another part, it may overpopulate its new area and cause damage. So next week, I will be doing biofilm tests to see if these dish detergents remove the biofims as opposed to kill the bacteria inside  them.

 Пока Пока!! (Bye bye!!)

Mackenzie

Alas, it's Thursday, nearing the end of week 3 of my bubbly adventure. Luckily, I'm not dying, but hopefully within the next 24 hours, some unfortunate bacteria will. I am sorry to say that I do not yet have the MICs completed quite yet, but they will be done tomorrow. Tomorrow, I will post a short summary of the MIC results as to not leave you hanging, looking at random pictures of agar and well plates. Hopefully, what I have below will suffice. And I'm sorry for the microbiology jargon you will encounter if you read ahead.

Studying bacteria is a slow and tedious process. The bacteria grow best overnight, and between procedures, there is quite a bit of waiting involved. So here's a run-down of what I did this week:
I re-made my bacterial stocks so they would grow better, but the new Lactobacillus stock did not grow. We think there is another type of broth (media) that Lactobacillus best grows in, so we might get our hands on some MRS broth in the near future. I diluted all the new samples and plated them. I streaked an agar plate for Lactobacillus for good measure. Here are what the plates looked like the next day...

The e.coli plate

The Lactobacillus plate- ain't it purdy?

S.epidermidis- better growth!

I then took the OD of each new stock. They were as follows for Lactobacillus, e.coli and s.epidermidis: 2.29, 1.52, 0.9. So why was this important? We used these OD's to calculate the CFU (colony-forming units) per optimum density (OD) at the bacteria's highest growth rate. This number happened to be 0.4. After using a spreadsheet to do calculations, the CFU/ OD 0.4 were calculated using the following equation:

0.4(Avg CFU per mL/ OD measured). This yielded the results:

E.coli= 2.4E7 CFU/ OD 0.4
S.epidermidis= 2.18E7 CFU/OD 0.4
Lactobacillus= 5.24E7 CFU/OD 0.4

This meant that there was a good concentration of bacteria at the optimum growth rate, so we decided to go along and do the MIC. The optimum CFU/OD 0.4 of most bacteria is around 2.5E5. We diluted the e.coli and s. epidermidis samples 20uL/ mL and the Lactobacillus 10 uL/mL in test tubes. Then, I took a 96- well plate and organized it like this:

I started out with 200 uL of dish soap in column 2, then took my pipettes and moved 100 uL down the line, leaving the end row untouched by the detergent. When it was time to add the bacteria, I put 100 uL of diluted bacterial stock in each well.  I did this for all three types of bacteria, and Voila! Three beautiful plates ready to go in the incubator- one for each strain.

Clockwise: E.coli, S.epidermidis, Lactobacillus.

So while I must wait until tomorrow to get usable data, it is nice to finally be getting used to the procedures. I am left unsupervised most of the time, but I think I've done everything basically right. With the greater concentration of dish detergent, hopefully tomorrow we will get some solid data.

Now I wait for my well plates to grow, slowly. While writing this post, I'm eating yogurt, and it's pretty awkward to basically eat what you're studying in the lab, but oh well. I guess gut flora taste delicious.

до завтра (until tomorrow),

Mackenzie

Whew! That was fun. The first week of being a lowly high school kid who doesn't know what they're doing is over. No bubbles yet, but we're getting there. We started out with the basic stuff, lab procedures, the baseline necessities of the project. Before anything else, however, I had to make broth to grow the bacteria. First was LB broth, or Lysogeny broth, which contains a rich cocktail of proteins, yeast extract, and salt, among other things. I made quite a bit of it (1L) and 1L of this MH broth, which is similar, but has slightly different components. But both of them still smell like chicken stock. Next, I made agar, this gelatin-like substance derived from seaweed, to culture the bacteria in petri dishes. After streaking the plates with bacteria, they were incubated overnight. Today, I came into the lab, and Voila! There were little white dots all over my petri dishes. My last visit for this week, I prepared glycerol stocks of my bacteria, E.coli and Staph. epidermitis* (the lactobacillus hasn't come in yet).

Now here comes the part where I talk about the obstacles I have faced. So far my greatest obstacle has been not being entirely sure what to do. Although I have great instruction from Dr.Koppisch and can ask one of the intimidating grad students how to do basic procedures, sometimes I just had trouble. Like following a grad student's incorrect advice to autoclave fresh broth or thinking that a pipette measured in microliters (uL) instead of mL. Oh well, one must make mistakes. Hopefully I will know my way around the lab better and know what to do next week. But enough with the talking! Here are some pictures.

Signing out,

Mackenzie

Where it all starts: broth mix.

*Change of plans: due to the virulence of Staph. aureus, I am instead working with a similar bacterium, Staph. epidermitis. Even though it is not found in the stomach or intestine, it is found in the mouth (still part of the GI tract) and is used as a substitute for aureus. So, we should be all good.

Finished agar plates

Finished broth!

 And, of course, here are the bacteria.

Staph. epidermitis

E.coli