Experiment 1: Bacteria Isolation from Soil Samples

A typical soil sample has billions of microorganisms due to its moist, nutrient rich composition. Common soil-inhabiting bacteria include: Bacillus, Streptomyces, Pseudomonas, Micrococcus, coliforms (indicators of fecal contaminants), lactic acid bacteria, and Clostridium. Various fungi (molds) are also frequently found in soil samples. These organisms play a critical role in the different natural biogeochemical cycles. Bacteria meet their nutrient needs by metabolizing organic matter (decomposing plants and animals) in soil, thereby oxidizing and reducing the elements in the organic matter and returning those elements to the environment. In this experiment, you will examine the diversity of soil microorganisms by culture bacteria from a soil sample and performing some microbiological tests to attempt to identify the microbes.

Procedure

Prepare Nutrient Agar Plates:

1. Loosen or remove the cap on the nutrient agar bottle.
2. Place the bottle in a microwave (if you do not have a microwave, place the bottle in a heat-safe bowl and pour boiling water around the bottle) and heat until the entire bottle of agar is liquefied. You will need to remove the bottle and swirl every 10 seconds to distribute the heat.
   Note: If you notice the agar boiling over, STOP the microwave and let the bottle cool down before handling. Hot agar can violently explode out of the bottle if heated too quickly and/or shaken. After boiling has stopped, use a hot pad protecting your hands and remove the bottle from the microwave. Use caution when removing the bottle from the microwave as it will be HOT!
3. Gently swirl the bottle to mix the solution.
4. Slowly pour the liquefied agar solution into the bottom half of eight petri dishes so that it covers the entire bottom of the dish. It is important that the entire bottom is coated and that the agar is given time to spread out over the dish.
5. Place the lids onto the dishes and allow the agar to gel undisturbed. This should take approximately 30-60 minutes. If you will not be using the dishes immediately, store them upside down in the refrigerator after they have fully gelled. Remove from the refrigerator and allow them to sit at room temperature for at least on hour prior to use.
6. Measure and pour 10 mL of saline into one of the 1 - 15mL conical tubes.
7. Use a permanent marker to label this tube as “Stock Soil Solution”.
8. Use a permanent marker to label 4 of the remaining tubes as “Normal 10%”, “Normal 1%”, “Normal 0.1%”, and “Normal 0.01%”. Label the bottom of 4 of the petri dishes with the same titles.
9. Use a permanent marker to label the other 4 tubes as “Heated 10%”, “Heated 1%”, “Heated 0.1%”, and “Heated 0.01%”. Label the bottom of 4 of the petri dishes with the same titles.
10. Measure and pour 9 mL of saline into each of the 8 labeled tubes.
11. Locate a 1 tsp. soil sample. This can come from anywhere in your local environment.
12. Add the soil to the tube with 10 mL of saline (Stock Soil Solution) and invert 10 times to mix well.

Prepare a Serial Dilution of the Stock Soil Solution:

13. Using a sterile disposable transfer pipette, transfer 1 mL of the soil/saline mixture to the Normal 10% tube; invert 10 times to mix well.
14. Using the same transfer pipette, transfer 1 mL of the solution from the Normal 10% tube to the Normal 1% tube; invert 10 times to mix well.
15. Transfer 1 mL of the solution from the Normal 1% tube to the Normal 0.1% tube; invert 10 times to mix well.
16. Transfer 1 mL of the solution from the Normal 0.1% tube to the Normal 0.01% tube; invert 10 times to mix well.
17. Using a clean pipettor for each tube, transfer approximately 0.1 mL ( or about 4 drops) from each Normal tube to each corresponding petri dish. Spread the diluted soil solution evenly over the plate with a separate sterile spreader. Allow to air dry.
18. Tightly screw the cap on the Stock Soil Solution tube and immerse the tube in a boiling water bath for 10 minutes. Carefully remove the tube from the bath and immerse it in a glass of cold tap water for 5 minutes.
19. Prepare a serial dilution of this heated stock soil solution into the heated tubes as in steps 13 - 16.
20. Prepare the spread plates of the diluted, heated soil solution as in Step 17.
21. Incubate both sets of plates in a warm location (not to exceed 37.7 °C or 100 °F) for 1 - 2 days (until well defined bacterial colonies appear; this may take longer for the heated samples).
22. When you are finished with the experiment, seal the plates with Parafilm™ and reserve them for Lab 12 - Experiment 2.