Living in an Alkaline Environment

Teacher Preparation for Parts 1, 2, and 3

Part 1: How different is Mono Lake water from Distilled water?

A quart can supply 100-150 students working in pairs. If you want to have each pair mix its own solution, refer to the second row and make sure you have a good way to distribute the materials.

* NOTE: Mono Lake water has a pH of 10. If you want to use a 1-molar sodium hydroxide (NaOH) solution to raise the pH to 10 instead of solid crystals, use 50 ml of 1M NaOH for the class supply and 12.5 ml for the student team.

To make efficient use of materials, promote safe use of equipment, and avoid spilling the caustic Mono Lake water, Part 1's tests are best carried out at four activity stations. (Create as many sets of stations as necessary to accommodate the number of students in your class.) Provide the following materials:

• Station 1: Dropper bottle of simulated Mono Lake water (or have student teams use the water they prepared), dropper bottle of distilled water, way to measure a pH of 10 (e.g., meters, papers, or strips), towels or sponge
• Station 2: Dropper bottle of simulated Mono Lake water (or have student teams use the water they prepared and an eyedropper), dropper bottle of cooking oil, wax paper
• Station 3: Large beaker of distilled water, large beaker of simulated Mono Lake water (or have student teams use the water they prepared), way to measure density (e.g., specific gravity hydrometer or rods/spheres that indicate density by floating/sinking), dishpan of water for rinsing between tests, towels or sponge, egg white from two or three eggs, teaspoon, clear container (e.g., 100 ml beaker)
• Station 4: Beaker of simulated Mono Lake water (or have student teams use the water they prepared), heat source (e.g., Bunsen burner, candle, alcohol burner), distilled water, 2 spoons with insulated handles (or oven mitt) (label one spoon "Distilled Water" and the other "Mono Lake Water"), dropper bottle of vinegar, pan of water to rinse spoons
• Station 5: (Teacher Demonstration): The whites of 2 eggs, 2 Pyrex containers (e.g., beakers), boiling water
• Station 6: (Teacher Demonstration): Distilled water, simulated Mono Lake water, microscope whose image can be displayed on a computer or projection screen, microscope slide, cover slip, tissue or other absorptive material, cellular stain (if necessary for clarity)

Part 2: How does increasing alkalinity affect common soil bacteria?

Samples can be collected from a variety of sources. Soil and mud from ponds, roadside ditches, flowerpots, and gardens are rich sources of microbes. Sand is not as microbially rich and does not easily form a slurry, which makes colony formation take longer. You can also collect bacterially-rich water from standing water and birdbaths. Inoculate plates directly with this water. It might be interesting to collect samples from sites of different pH and observe growth patterns. For example, slag heaps are generally alkaline and swamps are generally acidic. To collect an environmental sample:

• Collect about 1 gram of soil/sediment from the environmental site in a tube (e.g., a capped test tube, disposable centrifuge tube, or culture tube).
• Add 10-15 ml of water from the same site, if possible. (If none is available, use sterile water).
• Shake to create a slurry.

• 2 Liter Flask
• 40-50 Petri dishes**
• Hot plate w/ stirrer
• Magnetic stir bar
• Aluminum Foil
• 1 Liter Water
• pH strips
• litmus paper, or pH meter
• 40 grams tryptic soy agar (TSA) (powder, pre-made, or Easy-Gel)
• Hydrochloric Acid (HCl) (0.1 M solution is easier than pellets)
• Sodium hydroxide (NaOH) (0.1 M solution is easier than pellets)

* Tryptic soy agar (TSA) is a nutrient-rich medium used for culturing many kinds of microorganisms. It is readily available from scientific vendors such as Ward's and Carolina. You may substitute with different types of agar, but results may not be the same.

** You will need to manipulate the agar's pH before pouring plates. Consequently, pre-poured plates will not work.

1. In a flask, add 40 g TSA to 1 L water and stir, using a magnetic stir bar and plate. Apply heat and bring just to a boil, stirring until agar is completely dissolved. CAUTION: agar will boil over quickly and can create a mess!
2. Once dissolved, remove the flask from the heat and cover it with foil.
3. Autoclave the flask, using the liquid setting. (For teachers with no autoclave, go to Step 5. If further dissolution is needed, heat in microwave. Fortunately, Part 2 of this activity does not require complete sterility.)
4. Remove from autoclave and let it cool to about 50-55 degrees C.
5. Measure the pH. It should be around 7. If not, adjust pH to 7 using NaOH to increase the pH or HCl to lower the pH. Pour about 15-20 ml agar into each plate. Pour slowly to avoid bubble formation. Place lids on each plate and set aside. (Consider practicing with water prior to this experiment in order to increase time efficiency when making plates.)
6. Place flask on the stirrer plate. Set it on low/medium heat so that the agar doesn't solidify. While stirring, add a few drops of NaOH. Measure the pH. Continue to add NaOH until pH is at 8. (If you go over 8, lower the pH with HCl.) Pour the second set of plates.
7. Repeat Step 6 for pH 9 and pH 10.
8. Let plates cool for 30-60 minutes. Once they have solidified, store them upside down to prevent condensation from falling on the agar. Note: A high pH can increase the length of time it takes for agar to solidify.
9. If your students will be inoculating the plates within the next day or two, you can store the plates at room temperature. Beyond two days, store them at 4 degrees C in a refrigerator.

To dispose of plates, autoclave in an autoclave-safe bag and then discard bag with normal trash. If you do not have access to an autoclave, add 2 cups of bleach to a dishpan of water. Submerge a plate and remove its lid. Keep the plate and top underwater. Repeat with all plates. Let them sit overnight. The next day, wash or discard.

Part 3: A WebQuest Exploring the Life and Ecology of Mono Lake

Set up a computer workstation with internet access for each student (or group). Provide field guides for reference. Tips for beginning Part 3 are located on the WebQuest Teacher Page.