Cascade Team
Monthly Report (June 7 - July 14)

There are several granitic fracture zone hot springs near our hometown of Cascade, Idaho that have healthy-looking bacterial mats. Because it would seem to be very difficult for bacteria that survives in such an extreme environment to migrate, we wondered if perhaps the bacteria found in our local hot springs was somehow genetically unique. By the end of the summer, we hope to be able to answer this question by sequencing and comparing the DNA of Thermus Aquaticus found in Vulcan Hot Springs (located near Cascade) to a standard piece of T. Aquaticus DNA. To achieve this, we realized that we needed to learn how to lyse cells, extract DNA, culture T. Aquaticus, and run PCR. Furthermore, we also needed to collect samples from Vulcan Hot Springs to see if we even had any T. Aquaticus living there.

We began practicing our lysing procedure on an Escherica Coli sample, and after lysing, we ran electrophoresis, which resulted in a few strands of visible DNA, indicating that we had lysed the samples correctly. However, because at this point we had not begun culturing any samples of T. Aquaticus on our own, we could not begin lysis on T. Aquaticus, so we moved to the next item on our list - practicing and learning PCR techniques.

We had ordered some cheek cell/hair PCR kits from the Carolina Company, so we began working with those. However, we soon ran into a few problems; we just couldn’t seem to get any bands to show up when we electrophoresed the samples we had PCRed. Finally, after we adjusted the concentration of our agarose gel to 1.5% , we were able to see the bands on the gels. Apparently the 0.8% concentration we had been using was not dense enough because our strands of DNA were relatively small. Also, the Proteinase K enzyme that digests the hair sheaths didn’t seem very efficient and none of the hair samples yielded much DNA. When we ran the cheek cells in conjunction with the hair cells, the cheek cells yielded bright bands of DNA compared to the very faint hair cells. This led us to believe that our PCR procedure was successful. While we were working with these kits, we realized that the kit had not come with a standard piece of human DNA that we could use as a positive control. However, we did run a sample without adding the target DNA to serve as a negative control.

While we were working with the PCR kits, we were also learning how to culture T. Aquaticus. Mark and Megan showed us the proper aseptic techniques and procedures for making Castenholz-TYE Media for T. Aquaticus and gave us a sample of T. Aquaticus from their lab to start a culture of our own. We have been subculturing the sample roughly every three days, keeping the samples at 70°C and a pH of 8.2 and have made some slides with these samples to get an idea of what T. Aquaticus looks like.

We made a trip up to Vulcan Hot Springs, where we took water samples using aseptic techniques as well as sticks, rocks, and matting from eight sites, including the source of the hot springs, and inoculated the samples directly into the media at five of those sites. While there, we also measured the temperature, pH, and distance from the source of each site and took several pictures with the digital camera. We kept all of our media samples and half of our water samples in an oven at approximately 70°C until we returned to the lab. We kept the other half of the water samples in a refrigerator.


Once we returned to our lab, we subcultured the five media inoculations and inoculated four other samples into media. We made slides of all of the samples and thought we saw T. Aquaticus in a few. In the meantime, we are continuing to subculture these samples every three days in preparation for lysis.

We have been storing both our samples from Vulcan and the samples from Mark in a water bath at about 70°C. However, the bath blew a fuse and no longer shakes. We also had problems with evaporation, but once we ran duct tape along the edges of the water bath, evaporation decreased. We were concerned that our samples weren’t aerating enough, but we thought that as long as we continued to subculture the samples, we were, in a sense, aerating them. While we are still concerned that the samples are not aerating properly, we haven’t been able to develop any feasible solutions yet.

We are currently in the beginning stages of attempting to lyse our samples of T. Aquaticus. So far, we have applied the lysis procedure we used on the E-coli samples to our Thermus samples. When we ran electrophoresis on these T. Aquaticus samples, we did end up with some very light DNA bands. Along with our T. Aquaticus samples, we should have had one E. Coli sample to lyse to ensure that our procedure was correctly executed, so we plan to repeat this experiment.

If this lysis procedure doesn’t work out, we have brainstormed a couple of other ideas to try. We asked ourselves, “When are cells most efficient at absorbing things through their cell walls and membranes?” We decided that when they are growing, it is necessary for them to absorb nutrients, and that is therefore when they would most readily take in things. When does T. Aquaticus grow? At 70°C. When we lyse E. Coli and other bacteria, we are running the majority of the experiment while the bacteria is at its most vulnerable to absorbing the chemicals we add. When we lyse T. Aquaticus, however, we run the majority of the experiment below its optimal temperature, thus causing it to “shut down”, and perhaps making it more resilient to our procedures. However, if we could design and run a lysis experiment in which the majority of the experiment was run at 70°C, perhaps the bacteria would be more vulnerable and easier to break down.

Another more difficult idea we have talked about involves pressure changes. We wondered if we could slowly put the T. Aquaticus samples under more and more pressure over a few days and suddenly release it, hopefully causing their cells to rupture. However, because this would be more complicated, we haven’t really developed it.

During our final weeks here, we hope to develop a procedure to correctly lyse T. Aquaticus. If we can do this, and if we have time, we then hope to compare the DNA (gene 16s rRNA) of our Vulcan sample using PCR to the DNA of a Yellowstone or standard sample.