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To collect our samples, we go to Vulcan Hot Spring in central Idaho. It is about an 80-mile drive into the backcountry of Idaho, and much of the driving is on dirt roads. We then hike a mile to the origin of the spring. The water of the hot spring is boiling when it flows out of the vent and is extremely clear due to the high temperature. As the water flows further and further down the hill, you can see the temperature change by the different colors of bacteria on the rocks. The bacteria we use thrives between 70 and 80 degrees Celsius and has a PH between 8 and 9. We collect 5-7 samples of 75mL.While wearing gloves, we use a pipettor and 25mL pipettes to get the water from the hot spring and put it in a whirl pack. We then use the tweezers and tongs to collect small rocks and sticks, and place those in the whirl packs as well. We label the sample, then record the PH and temperature of the section of spring the sample was taken from, and use landmarks such as logs to describe the location. To get our samples, we collect water from the hot spring that contains bacteria. We also collect sticks and rocks from the water that the bacteria may be growing on. When we begin growing the samples, we add media to the water and bacteria. Media contains nutrients used to help the bacteria grow. 
Before leaving to collect our samples, we make media, set up the hot water bath, sterilize tweezers, tongs, and flasks in the autoclave, and gather together the necessary supplies to collect samples. These include: thermometers, sterile pipettes, tongs, PH probes, whirl packs, notebooks, markers to label samples, tweezers, gloves, and stripettors (used to fill pipettes). We then bring our samples to the lab and put 75mL of one of our samples into a flask, add 125mL of media, put a cap on the flask, and place it in the hot water bath. We then repeat this with all of the other samples we want to grow. Once they are all placed in the hot water bath, we turn on the shaker. The remaining sample can be left in the whirl packs and stored at room temperature. If our samples get contaminated, we can start over with the stored samples, add media, and begin it growing again. We grow these samples in a hot water bath at 75 degrees Celsius. During this step of the process, we have to be extremely careful to not contaminate our samples, as different types of bacteria could also be grown using our media. Every week we subculture our samples. This is done by removing 125mL of sample, which we freeze at 4 degrees Celsius to later use in isolation. Then we replace what we removed with 125mL of Castenholz TYE media, which we make in our lab. Here is a link to our recipe. At first, the samples do not contain enough bacteria to get a good sample, so we dispose of the 125mL we remove in bleach, but after a few weeks, the bacteria growing in our flasks turns into an orange-yellow-brown color. (below). At this point, we can use the samples we remove in isolation and further experiments. We found that evaporation of the samples was a big problem in our hot water bath; however, the samples needed a steady flow of oxygen, so we couldn't cork the flasks. To solve this problem we used plastic covers for our flasks (see photo below). These caused the evaporating water to condense on the plastic then drip back into the flask, while still allowing oxygen to enter from underneath the cover. We used plastic balls to keep the water in the hot water bath from evaporating. 
We also had a problem keeping the flasks upright in the hot water bath because we kept the shaker on. Our first attempt at solving this problem was unsuccessful. We tried using 2L soda bottles and cutting the tops off. We used screws to hold them to the bottom of the bath, and then placed the flasks inside them. This attempt failed because the bottles melted at the high temperatures. On our next attempt, we replaced these bottles with more durable plastic containers. We also found that we have to be sure to use stainless steel screws, because regular screws rust quickly in the hot water bath. Below is a picture of our hot water bath and samples. At the same time as we subculture, we record the color of each sample, and note if a significant amount of evaporation has taken place. If a great deal of sample has evaporated, we remove less and add more media when we subculture. We occasionally look at our samples under a microscope to ensure we are growing the right type of bacteria. The next step of our process is making the media for DNA isolation |
