Dear Readers (and Biofilm enthusiasts alike),


I am sorry that this must be my last SRP blog post.

My final presentation will take place May 10 at 7:00 p.m.

My MIC and Biofilm tests have generally shown that dilute concentrations of dishwashing detergents do not greatly affect bacteria of the human gut. Although the MIC tests showed significant reductions in Lactobacillus, 10mg/mL seems to be a rather large concentration that free-floating bacteria would come into contact with in the body.  While there were still reductions in bacterial counts for the biofilm tests for both 0.1 and 1 mg/mL detergent, they were not so large as to be particularly concerning.

 Additionally, the detergent tests showed that certain biofilms were washed away rather than killed and that these detergents could just move flora from one part of the gut to another. Therefore, based on my data and what we already know about human gut flora, consuming small quantities of dishwashing detergent shouldn't be too bad for you after all. However, until we find out more about the role of gut flora, it will be hard to determine whether the "swept away" biofilms would have any effect on our health.

In the 1980s, this scientist by the name of Stratchan hypothesized that the cleaning products we use were essentially making us sick by killing bacteria that would give us immunity to certain diseases. While it may be true upon further investigation using more toxic detergents and cleaning products (such as those containing the chemical triclosan), it does not appear to correlate with the data I have collected throughout this project.

However, to add to this experiment, it would be ideal first to complete my data set on 1 mg/mL detergent against Lactobacillus delbrueckii( using a live culture of course) and further investigate the topic as a whole. What would happen to the biofilms if you used toothpaste? We swallow toothpaste all the time, after all. One could investigate the effects of other products, such as window cleaners, or test the biofilms when grown on different plates. This kind of investigation could ultimately go in may directions, from just investigating gut flora to looking to remove biofilms from medical equipment ( a lot of research and effort has already been directed toward preventing biofilm growth on surgical implants and equipment). All in all, there are many new possibilities and continuations this investigation to follow, and science always leaves us with more questions.

But to close, I wanted to point out what a valuable experience this was for me. When I first was offered the opportunity to investigate this aspect of Microbiology and work in Dr.Koppisch's lab, I was interested, but still wondered how any of this bacterial stuff could affect me. I initially wanted to go down to Phoenix and shadow a doctor or witness a surgery, and I wasn't sure if this project would be fulfilling. But it truly, truly was.
 Despite the challenges of learning to work in a new kind of lab, with new techniques, procedures and precautions, I learned not only that microbiology was fascinating, but also the value of the "little things" in life (no pun intended). Sometimes you just have to stop riding your ego and see that even the things that many people would consider "boring' or "juvenile" may actually be some of the things that are the most important. Microbes may be small, but they are essential to our survival. I may have not been working with any super corrosive acids or toxic chemicals, but those dish soaps give us an insight into how the products we use in our daily lives affect our health.
I am honored to have worked in the Koppisch lab for the last couple months, and I have enjoyed the majority of my time in the lab, working with what I see as a little world at my fingertips, the little creatures that seem so small and insignificant, but also can have such a big impact on everything we do. From weighing down ships to contaminating surgical tubing, these biofilms truly do affect our lives.

While I still want to eventually be a physician, working in microbiology has definitely sparked my interest, and I look forward to taking some classes in college. And this experience, I believe, has truly helped prepare me for what lies ahead. Once again, I would like to thank Dr.Koppisch and his students for guiding me along the way and for not also being my colleagues, but also my friends.

Спасибо за прочитать. (Thank you for reading)

Спасибо , преподаватель Koppisch и ваших студентов. (Thank you, Dr.Koppisch and your students.)

я буду скучать по вам! (I will miss you guys!)

Mackenzie

(P.S. If you are interested in learning how to pronounce any of the Russian gibberish I've been throwing at you, I will be launching a website this summer called pronouncerus.weebly.com that, despite my nascent knowledge of the language, should help some of you curious folks learn some words.)

Although this is my last week of collecting data, I regret to inform you that I have no data to show. Let me explain.

Bacteria are pesky, capricious little buggers. They are organisms, and they do what they want, even if what they want includes dying. And I'm pretty sure that's exactly what my Lactobacillus cultures did to me this week. You see--bacteria can only live in a test tube in the fridge for a certain period of time until they die. That's why it's important to occasionally streak your plates to make sure that the bacteria are still growing. Luckily for me, I was able to rely on my other bacteria, e.coli and s.epidermidis to stay viable until I was done using them (although admittedly, my epidermidis  was on it's way out).

Lactobacillus being sassy.

However, this week, I knew something sketchy was going on with my Lacto. My overnight culture didn't grow, and I had to make my biofilms using a culture that had been growing for only three hours. When I made my biofilm plate, the OD of my bacteria was not ideal. It was 0.156 at 600 nm, and all my other cultures had been between 0.3 and 0.5 at the same wavelength. Then the morning of my dilutions, my biofilms had not appeared to grow, so I took their OD, which was around 0.23 and plate them anyway. An interesting thing to note, however, was that it took less time for my dilution droplets to soak into the agar than normal. My best plates usually had to sit for about half an hour before I could put them in the incubator, whereas this week's plates were ready in about 10 minutes. I'm not sure if the quantity of viable bacteria in the dilutions had anything to do with the absorption time, as it may have had to do with the quality of the agar, but it may be something interesting to investigate.

Middle tube: The culprit, Lactobacillus delbrueckii

But anyway, here's some good news. Two of my lab mates, Cody and Stephanie, made an undergraduate research poster with their biofilm work. Since our materials and procedures were similar, they used some of my data as part of their presentation. This means that I have my name on  poster. Woot woot! Although I can't add the poster on this blog due to formatting issues, the poster will be on display at the NAU Undergraduate Research event tomorrow, April 29. The title is "Effect of Decyl Rhamnoside on bacterial Biofilms." by Stephanie Arellano and William C. Crozier.
Decyl Rhamnoside is a surfactant, similar to my dishwashing detergents, and here is a comparison of the three:

Decyl Rhamnoside

NaCO3 (Sodium Carbonate in Cascade)

SDS (Sodium Dodecyl Sulfate) in Dawn detergent

 As for now, it's nice to know that I have some recognition in the scientific community. And as for the Lactobacillus plates that didn't grow, it leaves us with a prediction: a prediction that the detergents would not have really affected the bacteria much at all, similar to all my other trials. But if anyone really wants to make sure, it is open to investigation in the future. For now, however, these results are lost in the oblivion of uncertainty, and although we are not yet sure what would happen, we can make a reasonable, and hopefully accurate guess.


что случается, случается (What happens, happens)

Mackenzie

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