Bioremediation of Gasoline
This is a screening test used to determine whether gasoline-degrading bacteria are present. Only one carbon source (gasoline) will be provided. The other macronutrients (nitrogen, phosphorous, and sulfur) are provided by the minimal salts. Gasoline is a relatively simple hydrocarbon so some bacteria should be able to use (degrade) it for their carbon and energy. In nature, it is likely that more than one species of bacteria is involved in degrading oil. One organism modifies an oil molecule so that it can be attacked by another until the molecule is finally degraded.
Degradation of the gasoline should result in growth on the plate containing minimal salts (nitrogen, phosphorous, sulfur, and magnesium) and gasoline as a carbon source.
Figure 1. Use of a spreading rod.
Minimal salts agar plates
Petri plate containing water and 1.5% agar
Gasoline (~10 mL)
Soil slurry (a pinch of soil mixed in a few milliliters of water). Use soils from different places, including soil likely to be contaminated with gasoline
Spreading rod (L-shaped glass rod or place a piece of No. 16 shrinkable Teflon tubing over a large, straightened (L-shaped) paper clip or metal spreaders can be purchased from Carolina Biological Supply), see Figure 1.
1. Teacher: Soak a piece of filter paper or paper towel in the gasoline and place the paper in the lid of one of the minimal-salts plates.
2. Place another gasoline-soaked paper in the lid of a water agar plate.
3. Place a water soaked paper towel in the lid of another minimal-salts plate. What is the purpose of each plate?
4. Pipette 0.51 mL soil slurry onto the agar in each plate.
5. Spread the soil over the surface of the agar using a spreading rod as shown in Figure 1.
6. Seal around the open edge of the plates with parafilm or a large rubber band to prevent escape of the gasoline.
7. Incubate the plates upside-down until colonies appear. Count the number of colonies on each plate.