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
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.
Isolation of hydrocarbon-degrading bacteria:
1. Environmental samples
are diluted as above. Portions (0.1 mL) of each dilution are spread
onto BH agar and supplied with hydrocarbons as sole carbon and energy
source by placing it in a vapour tube (cut off micropipette tips,
sealed at one end with heat) in the lid of the plate. As a hydrocarbon
source it suggested you first try jet fuel or pure compounds like
naphthalene (just place a few crystals in the lid of the plate) or
dodecane. For jet fuel and dodecane place about 20 µL on filter
paper in the lid of the dish or use vapour tubes.
2. Control plates without
substrate are also inoculated.
3. Seal all plates with
parafilm and then incubate in a tupperware box (to reduce loss of
volatile hydrocarbons in to the lab). All plates are prepared in
triplicate for each dilution and incubated at 25oC for at least 1
Colonies larger and
different from those on the substrate-free control plates are selected
and purified for further investigation. Hydrocarbon-degrading bacteria
are removed from the isolation plates and purified on BH plates
supplied with hydrocarbons as sole source of carbon.
courtesy of Dr Jackie Aislabie)