Culture samples (prepared after rem

Culture samples (prepared after removal of
insoluble substrates) were centrifuged at 15000 g
for 15 min at 4°C and the cell-free supernatant was
used as the extracellular fraction. Cells were divided
into two portions; the 50 ml portion was suspended
in biological saline, washed twice in saline and resuspended
in saline for determining cell mass
gravimetrically while the second portion (150 ml)
was suspended in chilled McIlvaine or Na-acetate
buffer (pH 7) and called the insoluble fraction. This
fraction was disrupted by probe sonication in two
bursts of 1 min with a Braun sonicator 2000 on ice.
Supernatant from this disruption formed the third
fraction called the cytosolic fraction. The particulate
(or cell associated) fraction was obtained as a pellet
by centrifugation at 15000 g for 15 rain (4°C). The
pellet was suspended in buffer and formed the
fourth fraction.