Procedure
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.
Results
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:
Procedure
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 month.
Results
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.
(Original protocol courtesy
of Dr Jackie Aislabie)
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