What Modern Microbes Could Survive on Early Earth?

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

Notes:

• Read all instructions before starting. Some steps will need to be timed, so it helps to be familiar with all the steps before you begin.
• Have an adult help you with handling the bleach. Read all warnings about handling the bleach before opening the container with an adult.

Collect Your Water Sample

1. Collect at least 600 mL of water from a natural water source.
   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.

Preparing Your Water Samples

1. Clean and sterilize the baby food jars by wiping the inside surface with a paper towel that 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. Pour approximately 100 mL (or approximately 0.4 cups) of the natural water sample into each of the 6 sterilized small glass jars.
   1. With the lid securely on the large jar, carefully turn the jar upside down a few times (i.e., invert it) so that the water is mixed before pouring out the samples.
   2. If you want to practice seeing how adding drops of vinegar or bleach changes the pH, you can prepare an additional (seventh) small jar for this.
3. Use the pH strips to check the pH of one of the small jars with the water sample. Record the pH in your lab notebook. This is the pH of the natural water sample.
   1. Read the instructions on how to use the pH strips to check the pH. If you have not used pH strips before, you can practice with any water. Depending on the pH strips, it can sometimes be hard to determine the exact pH based on the strip color. It may help to read the strips under bright, consistent lighting. Try to do your best!
   2. The natural water sample will likely have a pH of around 7, as shown in Figure 2.
   3. Label this jar with its pH.

Image Credit: Teisha Rowland / Science Buddies

Figure 2. This is an example of using pH strips with a sample of approximately pH 7.

4. To a second small jar with the water sample, use the eyedropper to add drops of distilled white vinegar. Carefully mix the sample and check the pH. Stop adding vinegar when the pH is approximately 6.
   1. Only add 2 drops of vinegar at a time so that the pH does not decrease too quickly.
      1. If the eyedropper touches any of the natural water sample, be sure to briefly sterilize the eyedropper afterwards by sucking up a small amount of isopropyl alcohol and squirting it out onto a clean paper towel. Rub the outside of the dropper with the paper towel. If the dropper does not look dry, wave it around a little for ten or more seconds until it looks dry before using it again. 
   2. Mix the sample using a clean, sterilized plastic or metal spoon.  
      1. Sterilize the spoon by wiping both sides with a paper towel made damp using isopropyl alcohol. If the spoon does not look dry, wave it around a little until it looks dry before using it again.
   3. Check the pH of the sample.
   4. Label the jar with its pH.
5. Repeat step 4 for two of the other small jars with the water sample, but this time keep adding vinegar until one jar is pH 5 and the other jar is pH 4.
   1. Tip: You will need to add increasingly more vinegar to lower the pH further. Because of this, for the lower pHs, you can try adding more drops at a time before measuring the pH.
6. Next, use bleach to increase the pH of the two remaining small jars with the water sample. Use the eyedropper to add drops of bleach to one of the remaining two small jars, carefully mix the sample, and check the pH. Stop adding the bleach when the pH is approximately 8.
   1. Caution: Have an adult help you when opening, handling, and pouring the bleach. Be sure to wear gloves, eye protection, and read all warnings and instructions on the bottle of bleach before opening it! Bleach is very hazardous.
   2. Only add 3 drops of bleach at a time so that the pH does not increase too quickly.
   3. You can again mix the sample using the eyedropper, but be sure not to accidentally add extra bleach while doing this.
   4. Label the jar with its pH.
7. Repeat step 7 for the remaining small jar with the water sample, but this time keep adding bleach until the jar's pH is 9.
8. You should now have prepared all 6 jars and have one jar with each of the following pHs (as shown in Figure 4): pH 4, pH 5, pH 6, pH 7, pH 8, pH 9. Let the jars sit undisturbed for 30 minutes.
   1. Use a timer or a clock to keep track of the time.
   2. This will help ensure that the change in pH affects the microbes in the water sample.

Image Credit: Teisha Rowland / Science Buddies

Figure 4. You should have 6 jars, one with each of the following pHs: pH 4, pH 5, pH 6, pH 7, pH 8, and pH 9. Let them all sit undisturbed for 30 minutes.

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, and plates.
2. Using a clean swab, dip the swab’s tip into one of the water samples in one of the jars. 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 pH on the back of each plate.
   3. When you are finished, you should have six prepared plates, as shown in Figure 5.

Image Credit: Teisha Rowland / Science Buddies

Figure 5. You should have 6 prepared plates, one for each pH you will test, labeled with the pH being tested on the back of each plate. (This image shows the backs of the plates.)

Growing Your Agar Plates and Making Observations

1. Stack the plates and flip them so that they are upside down (with the agar on the top of each plate).
2. Leave the plates inside where they will not be disturbed.
3. 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 pH 7 should have microbes that were not affected by a change in pH. Over time, colonies should appear, as shown in Figure 6.
   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 6. You should see colonies appear on the pH 7 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 pH conditions of the early Earth’s oceans?