Things just got weird. Really weird. But it's ok, because science is full of surprises.

I found this week that the 1mg/mL detergent actually had a fairly small effect on the biofilms of s.epidermidis-- a 0.5 log reduction at maximum. This was less than I expected, as we were using a detergent at a much higher dosage than the 0.1 mg/mL tests.
 
 But then I noticed my error bars and a little issue with my agar plates. The error bars in my 0.1 mg/mL detergent tests had error bars that were at almost twice as big as the 1 mg/mL error bars. The bars were small enough for my data to still be considered relevant(when you compare them, even at their peak you still see a reduction) , but it shows that the 0.1 mg/mL detergents may have had less of an effect on your gut biofilms than you would think.

Yay! More plates!

Overall, this is good for your gut, and now hopefully you still feel comfortable with using dish soap. But here comes my second calamity--the agar plates. You might not think making agar plates is a particularly scary thing to do, but it actually takes a bit of attention and skill. You have to measure out the correct quantities of LB mix and agar mix( and make your own LB mix if you have run out), mix them with the correct amount of distilled water in a beaker and put the beaker on a hot plate to stir it. The ultimate goal is to dissolve all of the agar on the hot plate with a magnetic stirrer, but after watching the mixture for 15 minutes,  heating it, praying, and trying to stir it manually, sometimes you have to give up. And by give up, I mean scoop gobs of half-dissolved agar into the LB, autoclave it, and hope that there's enough agar in the mixture to help it set. It's really not difficult, but lately I have had problems with agar plates not solidifying and spilling all over the other plates.

But anyway, I'm sure y'all are super bored listening to me go on and on about agar plates. So here's something cool that happened this week. I got to watch two of my lab mates perform gel electrophoresis. Electrophoresis is a procedure that isolates RNA, DNA, and proteins by weight. It can be used to investigate gene sequences, identify organisms, and, in the case of my lab mates, look at a desired protein associated with a bacterium. They took agarose gel, which is kind of like a cross between Jell-O and a kitchen sponge with wells in it, filled each well with their desired mix of proteins, submerged it in a mixture of buffers (to help current flow) and applied an electric current to it. Since the short, light proteins move faster through the gel, they end up further away from where they started than the heavier proteins. The heavier proteins, therefore, which is what my lab mates were looking for, would end up closest to the wells. It's a labor intensive process, but when it is done, it basically looks like this:
(Image credit: http://www.bio-rad.com/webroot/web/images/lsr/solutions//technologies/protein_electrophoresis_blotting_and_imaging/protein_electrophoresis/technology_detail/pet11_img1.jpg)

So below are the detergent tests:

Artistic photo #2: Parafilmed epidermidis

 
Once again, I noticed that the bacteria were growing pretty well even after being suspended and plated in solutions of dish detergent.  And you might notice that the bacteria may not be growing as well as you might expect. After a certain amount of time, cultures that are kept in the fridge start to die, so old cultures may not grow as well compared to when they came out fresh from the freezer. However, this didn't really seem to affect my results in a negative way, and my error bars look good. Overall, things are looking pretty good. I'm excited to test my Lactobacillus next week with some fresh cultures.

Well, I'm kind of running out of ways to say "goodbye" in Russian, so I'll just end on this:

завтра выходные! Tomorrow's the weekend!


Mackenzie

привет! Hi!

For the remaining three weeks of my project, we decided to test all three types of bacteria against a solution of 1 mg/mL detergent. At the beginning of my project, we looked at the effects of  10 mg/mL solutions on e.coli and saw a HUGE change (like a 2-log reduction), and since we have results from the tiny, but more realistic concentration of 0.1 mg/mL, we thought it would be a good idea to try the happy medium. So far, the results of this test show almost what I expected: that it affected the bacteria a little more than the 0.1 concentration, but a little less than the 10 concentration. Although the graph is a bit small, it basically reads that there was about a 0.7-log reduction in the bacteria. Below are my plates-- I decided to add an artistic photo for your enjoyment.

1 mg/mL detergent plates

Ooh! An artistic picture of e.coli in Dawn

 I honestly expected a bigger reduction, for example, 1-log, but there may have been more error in this run due to hot autoclaved water. To dilute the bacteria for the biofilm tests, you have to fill your test tubes with autoclaved water to ensure nothing else is growing in them. However, yesterday we ran out of water, and I had to autoclave more. When things come out of the autoclave, they're usually steaming hot, so I waited about 30 minutes for the water to cool down. This may have not been enough and possibly killed some of the bacteria in the test tubes. Due to lack of time, however, I believe this data will suffice and I will talk about it in my presentation if you attend. As you can see below, it doesn't look like there's much of a difference between my controls and challenge tests.




The detergent tests once again showed significant growth of the bacteria floating around in solutions of Dawn and Cascade. So all in all, it's pretty nice to know that the detergents won't actually kill our gut biofilms, but rather move them around. Doing these biofilm tests has naturally brought on other questions one could research: what would happen if you tested toothpaste? What if you used different plates? I will no have time to answer these questions experimentally, but since toothpaste is probably ingested as much as, if not more than, common dish detergents, it leaves an open door for someone after me to test. In terms of new things I've learned, well, I learned the molecular structure of choline yesterday (thanks to my lab mate Kahla) and the importance of using cooler autoclaved water to do biofilm tests. It looks pretty cool.

Choline! woot woot!

Yay! Chemistry! But for now, I am off, so,

Всего наилучшего! All the best!

Mackenzie

I apologize for the fact that lab work is very repetitive. But at least it makes you appreciate the little things, like using different strains of bacteria for the same procedure.

Lactobacillus is an interesting little bacterium. People use it commercially for quite a few things- digestive supplements, yogurt, and even a type of beer called "sour beer" which uses my bacteria, Lactobacillus delbrueckii, during the fermentation process.

My plates..and they all pretty much look the same.

 Although my Lactobacillus seemed to be hurt badly by liquid dish soap concentrations, the biofilm tests showed that there was virtually no effect of the dish detergents on biofilms of Lactobacillus. There may have been some tiny reduction, like 10%, but really not significant enough to be concerning. There could have been experimental error involving possible soap in the test tubes or a detergent stock that had less detergent than desired, but this is certainly good news for your gut. Feel free to take your probiotics, drink your sour beer(if you are at least 21, of course) and wash your dishes. The interesting thing is that you can only tell that there has been next to no reduction when you change the graph axis units to a logarithmic scale. On a normal scale, even this puny reduction showed a HUGE difference between bar sizes. And this goes to show the nature of bacteria. There are so freaking many of them in one particular area at one particular time, even killing 100 colonies isn't a big deal, because you have 800000000 more.

My plates!.. which pretty much look the same.

Plating the detergents showed that the Lactobacillus grew quite well in the detergent solutions, once again showing that the detergents wash away the colonies instead of killing them.

Detergent plates

 
For the last couple weeks of my project, I will probably re-do my e.coli biofilm test just to make sure I have good data. And if I still have time after that, I may investigate the effects of other common cleaning products that contain SDS, such as toothpaste or possibly use different kinds of plates. We'll see as my adventures continue.

Прощай!(Goodbye!)

Mackenzie

Well it appears to have been another successful week of biofilm tests.

This week, I finally moved on from using e.coli to a relatively harmless strain of bacteria called staph.epidermidis. Epidermidis was used in place of a more virulent bacterium, staph.aureus, due to issues of safety and restrictions with having a minor work in a college lab, but luckily, it all worked out. It turns out that BOTH strains of bacteria can be considered gut flora, although epidermidis usually hangs out around the mouth area instead of the lower intestine. So it’s decently representative.

I did the same biofilm tests with the epidermidis as with e.coli over the last few weeks—same procedure, same test tubes, just no bleach. And it seems to have worked. The plate counts showed that there was a 0.5-log reduction of bacteria for BOTH 0.1 mg/mL detergents instead of just one. It’s interesting to see how after diluting the detergents 100 times, they still have a pretty big impact on the bacteria. It’s also interesting that both detergents can have the same effect on different strains of bacteria, especially because e.coli Is gram-negative and staph.epidermidis is gram-positive. (If you want to know the difference between gram-negative and gram-positive, click here: http://www.majordifferences.com/2013/10/difference-gram-positive-vs-gram_2.html#.Vv2CkjE73LJ

The epidermidis biofilm plates

After plating the detergents, I found that there was significant growth with both kinds of detergent, yet another piece of evidence to show that the dish detergents are sweeping biofilms away that would freely grow elsewhere. And here are the graphs!

detergent plates

Overall, these results support my and Dr.Koppisch’s hypothesis that the dish detergents sweep away biofilms instead of killing them. I can’t make any big statements about it yet, but I may be getting closer to understanding the relation of this experiment to the “hygiene hypothesis,” or, in short, the theory that our cleaning products make us more susceptible to illness. In terms of challenges, well, the only real challenge I can think of is remembering to complete all the steps. But overall, things are pretty smooth.

До скорого! (See ya!)

Mackenzie

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