Success tastes good. Very good.

I completed another biofilm test on e.coli this week, and this time, the bacteria didn’t get totally wiped out by test tube bleach. Instead, basically what we found out was that in extremely low concentrations, neither dish detergent had too huge of an impact on the biofilms. We noted a 0.5 reduction of bacteria (about 50% of the bacteria were gone) in the presence of Dawn and a very small reduction with Cascade. This means that the two dish detergents did not really kill any of the biofilms of e.coli that you would potentially find in your gut. 

e.coli plates

Detergent plates: Left-Dawn, Right-Cascade

When I diluted and plated the detergents, I found that there was significant bacterial growth, which means that both detergents swept away biofilms that were still able to grow. Most dishwashing detergents can be classified as surfactants, meaning that they lower the surface tension between two liquids or a liquid and a solid. Surfactants are part of the reason why it is easier to scrub away food residue from plates when using dish detergent as opposed to just water. So in this case, the dishwashing detergents acted as surfactants for the e.coli biofilms, sweeping them away from the colonies left on the well plate.

 

So what does this mean for you?

Well, basically, at least in terms of e.coli, this means that small concentrations of dishwashing liquid are not harmful to the bacteria. So if you’re in love with using that bottle of Dawn with the adorable little duckling on it to wash your dishes, you’re probably ok, because it’s not going to screw up your intestinal e.coli super badly. However, since both detergents seemed to sweep away bacterial biofilms, accidentally consuming small quantities of dish soap could move bacterial colonies from one part of your intestine to another. And since science hasn’t discovered enough about gut flora yet, we are unsure of whether this movement would be harmful. Just don’t chug a bottle of Dawn, please. That’s a really bad idea.

За здоровье! To your health!

Mackenzie

Just to let you guys know, I will probably be posting every Friday from now on, for the sake of having complete results.
 I decided to use a new concentration of dish soap for the biofilm tests that I did this week and that I'll carry out over the next couple of weeks. Instead of 10 mg/mL dish soap, the concentration will be 0.1 mg/mL. Here's why:
 Imagine you're washing a plate, by hand, with liquid detergent. You put one drop of soap on the plate and kind of swirl it around to get good coverage. Then you scrub, rinse, and dry. That drop of soap you put on the plate probably weighed about 0.1 g, or 100 mg. Now mix that with 10 mL of water, the water that you sipped at dinner or the water with which you washed the plate--that's a 10 mg/mL solution. But that's still an awfully large concentration of soap. If you ate off a plate coated in 10 mg/mL Dawn, you would probably taste more soap than food. So imagine that you rinse that plate REALLY well, how it should be rinsed. At this point, you can assume you've rinsed off the majority of the detergent, and thus 0.1 mg/mL of soap is probably a pretty good estimate of the maximum amount of dish soap you would eat off the plate, a hundred times less soap than you had before.

Now, this is the moment of truth.Science requires repetition.

When I walked into the lab Thursday morning and opened the incubator, I found that many of my plates had absolutely no bacteria growing on them at all. When I did my control enumerations, two of my three trial plates were completely empty. This didn't make sense, especially after following all the procedures and using a much smaller concentration of dish soap. But since there was no dish soap in my control row, the only logical explanation I could assign is that there was bleach in my test tubes that killed the bacteria, even though the tubes had been autoclaved. This being said, I will have to re-do my e.coli enumerations next week. But no biggie- I still have another six weeks or so, and three of those weeks have been set aside for this kind of thing.

My poor little plates...

I did the same biofilm challenge procedure with this new concentration, except I did one thing a little different. Instead of shaking off the detergent before sonicating the plate, I collected the detergents in test tubes and plated them using serial dilutions. This was done to determine whether the dish soaps penetrated sand killed bacteria inside the biofilm or simply swept them away. The detergent plates surprisingly grew more than my control plate, and still grew quiet a bit, which leads me to believe it's possible that the dish soaps are sweeping the biofilms away. However, this is unsure until I can get solid, unbleached data.

Detergent plates- top:Cascade, bottom: Dawn

But for now, I'm off to Oregon for spring break, so,
увидмся через две недели! (See you in two weeks!)

Mackenzie

And... we have results!

Last week's biofilm tests had invalid (and unreadable) data due to the agar plates evaporating, but I re-did the 24-hour biofilm tests for e.coli and a solution of 10 mg/mL Dawn and now we have numbers! Woot! The only big difference between last week's biofilm tests and those for this week was that rather than letting the plates sit in the hood for the weekend, we just put them in the  incubator overnight to keep the pesky agar form going awry. Below are some pictures of the plates after counting colonies:

My beautiful plates!

E.coli Dawn challenge plates

E.coli control plates

Since making you sift through six nasty, ugly-looking data tables would be rather cruel, here is a brief summary of the results:
The Dawn had a HUGE effect on the quantity of e.coli biofilms.

For example, a plate count we used for the 1st control group was 3.9E7 CFU/mL.
The corresponding Dawn challenge enumeration was 4.8E4 CFU/mL.
 The CFUs/mL for the 2nd group control and Dawn, respectively, were 1.8E7 and 4.62E5.
And for the 3rd group: 5.9E6 and 2.26E5.

This means that the Dawn detergent is greatly decreasing the quantity of plated colonies. However, we do not know whether this concentration of detergent is killing the bacteria in the biofilms or if it is just sweeping them away. I will probably not have time to perform that kind of test, but based on the MICs, we are fairly sure that the detergent is sweeping the bacteria away. It's interesting to see that the Dawn is actually having an effect on the e.coli biofilms, as this was fairly unexpected. But don't make any assumptions yet! I will probably decrease the concentration of detergent to a more realistic figure, like 5 or 3mg/mL in coming weeks.There is also a pretty good chance I will do this exact thing again with this different concentration.

I haven't really had many obstacles except for getting used to the procedures, and everything seems pretty smooth.This kind of procedure is probably what you guys will be seeing for the remainder of my project. But don't worry! I will test different types of bacteria with different types of detergent, so hopefully, you'll have an update on that every week.

до четверга (until Thursday),

Mackenzie

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