Five grams of each soil sample was suspended in 5 ml
of sterile water by vigorous agitation on a vortex for
10 min. Following decantation of the coarse particulate
material, 200 μl of the suspension was seeded onto
plates containing YM medium (0.3% yeast extract,
0.3% malt extract, 0.5% peptone) supplemented with
2% glucose and 100 μg/ml chloramphenicol (YM-cm).
The plates were incubated at 4, 10, 15 and 22°C. Duplicate
of water sampling dishes were incubated at 4
and 10°C. The plates were incubated for 3 months and
periodically inspected for colony development. Once a
colony became visible, it was immediately transferred
to fresh YM-cm plates and incubated at the same
temperature as the source-plate. The procedure was
repeated for each soil sample to maximize the number
of isolates.