How Can Life Survive on a Planet with Extreme Tidal Locking?

This project follows the Scientific Method. Review the steps before you begin.

Note: Have an adult help you with using the oven.

Collect Your Water Sample

1. Collect at least 200 mL of water from a natural water source. If everything goes as planned, you will only need 120 mL, but it is easy to collect extra, and then you will have it available if needed.
   1. The water sample could be taken from natural water sources such as a pond, lake, river, ocean, fish tank, etc.
   2. Collect the water in a clean jar with a secure lid.
   3. Be sure to wash your hands with soap after handling the water.

Temperature Treat Your Water Samples

1. Clean and sterilize three baby food jars and one cup in the muffin tin tray. 
   1. Sterilize the jars and cup by wiping the inside surfaces with a paper towel you have made damp with isopropyl alcohol. Make sure any isopropyl alcohol has dried or evaporated before pouring in the water sample, or the alcohol may kill microbes in the sample.
   2. If you have a double oven (an oven that has two independent cooking compartments that be set to two different temperatures), you can use two muffin tin trays to test the two oven temperatures at the same time. If you will be doing this, sterilize one cup in two different muffin tin trays.
2. Pour approximately 20 mL (or approximately 4 teaspoons) of the natural water sample into each of the three sterilized small glass jars and the one (or two) muffin tin cup(s). 
3. Have an adult help you set the oven to the first oven temperature you will test, which should be 200°F. 
   1. If you have a double oven, have an adult help you set the second oven temperature to 300°F.
   2. Wait until the oven is completely preheated to the correct temperature before starting the next step.
4. Perform the temperature treatments as follows:
   1. Put one small glass jar with a water sample in the refrigerator. Leave the jar's lid off.
      1. Note: Leaving the jar's lid off will help it reach the correct temperature more quickly.
   2. Put one small glass jar with a water sample in the freezer. Leave the jar's lid off.
   3. Put one small glass jar with a water sample at room temperature, on a counter at room temperature.
   4. With an adult's help, put one (or two) muffin tray(s) with the water sample into the preheated oven.
   5. Record the time (or start a timer) when the samples were first placed in the refrigerator, freezer, or oven, and leave each sample at its designated temperature for 60 minutes. Be sure that the refrigerator, freezer, and oven remain closed during the testing time (so that the temperature does not significantly change). 
   6. Optional: You can use a thermometer, such as an infrared thermometer, to measure the temperatures inside of the refrigerator, freezer, oven, and room temperature. If you do this, check the temperature either at the very beginning of the testing period (immediately before placing your water samples at the correct temperature) or at the very end of the testing period (when you remove your samples). Be sure to still keep the refrigerator, freezer, and oven closed during the 60-minute testing period.
5. After the water samples have been treated at their designated temperatures for 60 minutes, remove the samples and place them at room temperature.
   1. Have an adult help you safely remove the muffin tray(s) from the oven using the oven mitts. Let the tray(s) sit at room temperature until it is cool enough to safely touch (about 10 minutes). Cover the cup with clean plastic wrap until you are ready to streak the agar plates so that more microbes do not get into the samples.
      1. Tip: For the sample treated at 300°F, some water likely evaporated. You only need a little water to use for the agar plates, so if there is any water in the cup, you should be able to use it. However, if there is no water left (in other words, it all evaporated), repeat the 300°F treatment sample but try slightly lowering the temperature (such as testing it at 275°F or 250°F) so that not all of the water evaporates and you still have some water left to use for the agar plates.
   2. Sterilize two baby food jar lids (as described in step 1, above) and put them back on the jars that were in the refrigerator or freezer, sealing the jars so more microbes do not get into the samples.
   3. When you remove the water sample from the freezer, it should be completely or nearly completely frozen. It may take about 45 to 60 minutes of being at room temperature for the sample to completely thaw so that you can use it in the next steps to streak the agar plates.
   4. If you do not have a double oven, you can now repeat the test for the second oven temperature by repeating steps 1 through 5, but this time set the oven to 300°F instead of 200°F.
   5. Label each jar or tray with the temperatures they were treated with.
6. You should now have prepared water samples treated with five different temperatures (as shown in Figure 3, below): freezer, refrigerator, room temperature, oven at 200°F, oven at 300°F.
   1. If you do not have a thermometer, here are some general temperature ranges for the conditions you tested:
      1. The general temperature of a freezer is around -10°F to 10°F (or -23°C to -12°C).
      2. The general temperature of a refrigerator is around 35°F to 38°F (or 2°C to 3°C).
      3. Note: Freezers and refrigerators may be a little warmer near their door (at the front).
      4. Room temperature is usually around 68°F to 74°F (or 20°C to 23°C).
      5. Ovens can cycle between hot and cold, and may be colder near the door (at the front). For example, an oven set to 300°F (149°C) may be around 270°F (132°C) near the door, or if it is set to 200°F (93°C), it may be around 170°F (77°C) near the door.
      6. In your lab notebook, record the temperatures you measured if you used a thermometer, or your best estimates based on the information here.

Image Credit: Teisha Rowland / Science Buddies

Figure 3. You should have treated water samples in jars and muffin trays for 60 minutes with the following temperature treatments: freezer, refrigerator, room temperature, oven at 200°F, oven at 300°F.

Preparing Your Agar Plates

1. Put on disposable gloves to avoid getting any microbes from your hands on the agar plates.
   
   1. While wearing the gloves, try to avoid touching anything other than the swabs, jars, muffin trays, and plates.
2. Using a clean swab, dip the swab’s tip into one of the water samples. Then carefully spread the swab’s tip across the agar plate in a zigzag motion, starting from the top and going all the way to the bottom, while also going all the way to the left and right each time. You can watch the video above for an example.
   1. Be sure to only touch the swab's tip to the water sample and then directly onto the agar.
   2. Apply even pressure with the swab, trying not to puncture the agar.
   3. Do not set the lid down on anything when you open the plates. Because of this, you may want a helper to open the plates for you. If you have a helper do this, be sure they wear disposable gloves too.
   4. After finishing, quickly put the lid back on the agar plate.
3. Repeat step 2 with each plate, preparing each plate one at a time.
   1. Try not to spend too much time on a plate (or with the lid off) because leaving its lid open could cause bacteria that are not from the water samples to grow on the plate.
   2. Using a permanent marker, write the temperature treatment on the back of each plate.
   3. When you are finished, you should have 5 prepared plates, as shown in Figure 4.

Image Credit: Teisha Rowland / Science Buddies

Figure 4. You should have 5 prepared plates, one for each temperature treatment, labeled with the treatment on the back of each plate. (This image shows the backs of the plates.)

Growing Your Agar Plates and Making Observations

1. Leave the plates on a counter or table somewhere inside where they will not be disturbed. Make sure each plate is set upside down, with the agar on top of each plate.
2. Check the plates each day for 3 days. Record observations each day, for each plate, when you check the plates. Wear gloves when handling the plates and do not open the lids.
   1. In your lab notebook, make a data table like Table 1. You can add more rows or make a new table for each day.
   2. Do you see any small spots on the plates that could be microbe colonies? If so, do the following:
      1. Make observations: Do some of the spots look different from others (in shape, texture, or color), possibly indicating that there are different types of microbe colonies?
      2. Try to count the total number of colonies on each plate and for each different type you observe.
      3. Compare the plates: Do some plates have more spots than others, or are any plates similar?
      4. Write your answers and any other observations in the data table in your lab notebook.
   3. The plate with the room temperature treatment should have microbes that were not affected by a temperature change. Over time, colonies should appear, as shown in Figure 5.
   4. Tip: If there is a lot of moisture on the inside of the lid, making it difficult to see any potential microbe colonies, you can hold the plate vertically (so it is not flat) and tap one end on a hard surface several times to make the moisture roll off the lid.
   5. If you have a camera, take some pictures of your plates for your lab notebook or your Science Fair Project Display Board.

Image Credit: Teisha Rowland / Science Buddies

Figure 5. You should see colonies appear on the room temperature plate over time. (This image was taken on black construction paper.)

4. If you are doing this experiment as a science fair project, repeat the procedure two more times. Scientists always repeat their experiments to make sure their findings are true and repeatable.
   1. If you want to finish the repeat procedures quickly, instead of waiting three days to set up your next set of plates, you could set up your second and third sets of plates on the first day as well, although this will require keeping track of more plates simultaneously. You can decide on your approach based on the time you have available.
5. What do your results tell you about whether your natural water sample had microbes that could survive the different temperature conditions that might be found on tidally locked planets? Do any of your results surprise you? Why?