Biology > Bacteria > Enumeration Of Hydrocarbon

Site: http://www.nexusresearchgroup.com/microbiology/hcmicrobes.htm

Grade Category: High School

Subject Category: Biology

Sub Category: Bacteria

Enumeration of hydrocarbon-degrading bacteria in the environment


Oil pollution is relatively new problem for the environment. It is only recently since the discovery of crude oil that nations have refined it into different types of fuel and use oil as an engine lubricant. Supertankers have been built that travel the worlds oceans delivering this "liquid gold" as part of an industry that is worth many billions of dollars.

Cleaning up after oil spills at home, at sea or at an industrial complex is a messy business. What if we could use bacteria that can breakdown these hydrocarbons to do the cleaning up for us? So, if you are a school student, your first mission is to tackle questions 1, 2, 3, and 4 below:

  1. How common are these hydrocarbon-degrading microbes in the environment?
  2. Can we isolate them from water or soil?
  3. Can they degrade a wide variety of hydrocarbons or only a specific type?
  4. How long does it take to degrade hydrocarbons?
  5. Can we grow a batch of bacterial culture in large enough quantities to help clean up spills?
  6. As oil remediation agents, are bacteria as cost effective, efficient and environmentally friendly as chemical agents used at the moment?
  7. Do we need futher research in this area, and if so, who should pay for it?

Detecting hydrocarbon-degrading bacteria:

Equipment and chemicals

  • 90 mL 0.1% sterile sodium pyrophosphate solution containing 30 g of glass beads
  • 90 mL 0.1% sterile sodium pyrophosphate solution as diluent
  • 10 mL sterile BH medium (Bushnell Haas medium, available from Difco)
  • 10 mL of hydrocarbons (sterilised by filtration through 0.22 µm filter).
  • sterile 10 mL pipettes tips and micropipettors
  • BH agar (Bushnell Haas medium solidified with 1.5% purified agar(Oxoid))

Bushnell Haas Medium (made up in deionised water)

  • 0.2 g/L MgSO4.7H2O
  • 1.0 g/L K2HPO4
  • 1.0 g/L KH2PO4
  • 0.05 g/L FeCl3
  • 1.0 g/L NH4NO3
  • 0.02 g/L CaCl2
  • Adjust pH to 7-7.2 with HCl and sterilise (autoclave or pressure cooker; 15psi 15 min)


1. Dilute 10g (wet weight) of soil in 90 mL 0.1% sterile sodium pyrophosphate solution containing 30 g of glass beads (1:10 dilution). Shake for 1 hour.

2. Serially dilute soil solution (10 mL in 90mL sterile sodium pyrophosphate solution ) to 1:103 for pristine samples and 1:109 for the oil-contaminated samples. For the first dilution vortex for one minute to separate bacteria from soil particles. If using sea water or river water as your source of bacteria simply carry out the dilution series but make sure you dilute the sample in sterile sea or river water and use also use to make your media.

3. Transfer 1 mL of appropriate dilutions into 10 mL BH medium four times (1 tube will serve as a sterile control, the other three are triplicate counts).

4. Add 50 µL of hydrocarbons as sole carbon and energy source. Sources of hydrocarbons can be either volatile fuel oil, parrafin oil, or pure compounds like alkanes or aromatic hydrocarbons. It is suggested for your first experiment that you use something like jet fuel.

5. Controls are sterilised by autoclaving twice on consecutive days.

6. All tubes are incubated at 25oC for 3-6 weeks.


To determine if growth has occurred in the tubes they are compared with the controls and those that are both turbid and show disruption to the film of oil on the surface of the medium are scored as positive. Relate the dilution that still scored positive to the number of cells per gram of soil.