Final Post: Ethanol resistance in V. fischeri

For anyone reading my blogs it might seem like my first post, which contained my abstract, didn’t cover aspects of my research the way that my weekly blogs might have. It might also seem like my abstract contained many more procedures and topics than did my weekly blogs. Both of these statements are, in part, true because of the nature of my experiment, and of research in general. I started off the summer with a very clear goal in mind of the steps that I would take to complete my experiment, starting with determining the maximum concentration of ethanol that would allow V. fischeri strains to survive in LBS media, then evolving the strains by putting them in media containing high ethanol concentrations, and finally seeing how this affected the V. fischeri symbiosis with the Hawaiian bobtail squid. In doing this research, I realized that my initial assessment of the project was way too ambitious, but also neglected to assume that their would be setbacks (which I detailed in my weekly blogs throughout this process). I also didn’t realize that through working through those setbacks I would pick up new aspects of my project, adding to both the complexity and relevance of my research.

[Read more…]

Weeks 8 & 9

These weeks were spent doing a variety of evolution and growth curve experiments with the bacteria on a minimal media (M9) with ethanol. My first growth curve was successful, as I was able to show that the absorbance readings, a measure of the number of bacterial colonies, went up steadily each half hour throughout the 12 hour period during which I took readings. After seeing the growth curve work with small amounts of LBS present, I took on the next step, which was introducing the bacteria to M9 media without the presence of LBS. I used a new technique for this which involved washing the cells that removed any LBS media left in the culture, and tried to grow the bacteria on the M9 this way, but this led to the bacteria being unable to grow on M9 at all. After meeting with my professor about this, he discussed several reasons why this might occur, namely that the centrifugation required for the washing process was too severe for the bacteria, particularly since their membranes were being agitated and they were then being placed into a media containing ethanol, which contributes further to the destruction of their membrane. Another option might be that the bacteria need to be evolved to withstand a minimal media with ethanol in it first, so the extra LBS in the media might have been facilitating this evolution. To figure out which of the two options was more likely, I inoculated strains in M9 minimal media straight off the plate and tried to perform growth curve experiments with the strains using the minimal media starter cultures as well as the LBS cultures that were washed. Though nothing grew in either condition, my PI advised me to try evolving them over longer periods of time (re-culturing either every 12 hours or every day) to see whether the bacteria would grow when re-cultured. If they do, this will provide a more straightforward approach to use to evolve the bacteria to ethanol. If they do not, I will need to continue introducing them into the M9 starting with LBS, evolving them to survive and be culturable in M9 media.

Week 7

This week I took readings from my previous experiment with M9 minimal media containing 0.5% ethanol as the carbon source. The data from this experiment was very promising, as it showed growth well above what is considered “minimal growth” for all of the strains. Though most of the OD600 readings were between 0.30 and 0.80, a few strains had OD600 readings as high as 0.300. Since the standard for “minimal growth” is an OD600 reading at or above 0.010, these results are quite high for bacteria that have not yet been allowed to evolve to be resistant to ethanol. After meeting with my PI, I started a preliminary procedure to test the growth of the strains over 5 days and found that the bacteria grew incredibly well even on the first day in most strains, showing that most strains only need 24 hours to grow to their maximum numbers, which is important to know for the next stages of the experiment. This week I also tested the strains SAIG and VF7744 at ethanol concentrations of 4.0-4.9% again, as per recommendation by my PI, to see if the data I was getting could be replicated. Both strains grew in all ten percentage points in all five trials, suggesting that the ethanol resistance in these strains is likely much higher than expected. Given this data, I will re-test the given strains at higher percentages of ethanol (specifically at 5.0% and 6.0%) to see whether these strains can truly grow at higher concentrations of ethanol than the data from previous students has shown.

Week 6

I met with my advisor this week to discuss the strange results in the M9 media and he provided more insight into the possible reasons for the results I got. He seemed to think that it was possible that the 0.5% ethanol concentration was too high for the V. fischeri, since I wasn’t giving them extra nutrients and they had not previously been exposed to ethanol (I used new cultures rather than the ones that I had tested in ethanol in my other experiments). He suggested trying the M9 experiment at the 0.5% and 0.25% concentrations without ethanol to see if there might be at least one mutant, and then transferring any bacteria that does grow into new test tubes containing M9 with the same concentration of M9 in order to allow the bacteria to evolve in the presence of M9. He also suggested a separate procedure in case nothing grows in the current M9, wherein I’d expose bacteria to ethanol and allow it to go through several evolutions before transferring it to the M9 media. This week, the squid eggs arrived in the lab, which will need to be hatched and raised before any experiments on squid-Vibrio symbiosis can be done (which would be a future phase of my experiment). Since this week was short due to Independence day, I was not able to complete any experiments but I did set up an experiment testing all strains that we have in the lab in 0.5% ethanol and a selection of strains in 0.25% ethanol, both in M9 media. I’ll be taking readings for these experiments next Monday.

Week 5

This week I continued my experiments testing V. fischeri strains in 4.0-4.9% ethanol. I tested the strains SAIG and SL518 in 4.0-4.9% ethanol. This week, I also performed a five day experiment with M9 medium containing both glycerol and 0.5% ethanol, which gave some strange results. In theory, the M9 media with glycerol should contain enough glycerol to allow the V. fischeri to grow to a point considered “significant growth”, since the glycerol contains a carbon source that this bacteria is equipped to use. However, my data showed significant growth in some but not all tubes, and the results were inconsistent even within a specific strain. I only tested a few strains just to see what the overall results might be, so I can’t compare different strains’ growing ability, but the strains that I did use were the strains that could grow at higher percentages of ethanol in LBS. My hypothesis as to why they didn’t grow as well as expected was that the presence of extra nutrients in the LBS allows the bacteria to expend the extra energy reducing the ethanol toxicity in some form. This process that would not be as easy to perform in the M9, since the M9 does not contain any extra nutrients and requires the bacteria to metabolize a small set of ingredients into whatever they need to grow. If this is the case, my next thought was that reducing the concentration of ethanol in the M9 media might work better. Another possibility, of course, is that I made an error in the making of the M9 and not all nutrients were available to the bacteria. Either way, next week’s experiments will include M9 at the 0.5% ethanol concentration and the 0.25% ethanol concentration to see if either one performs better.