trated on surfaces, but clearly there may be other controlling factors involved. A better
understanding of the conditions which promote attachment and the effect attachment has
on subsequent physiological activities should provide a more complete knowledge of the
importance of sessile bacteria in aquatic environments.
The present study describes the effect of surface attachment on nutrient uptake by a
brackish water sediment isolated of Enterobacter cloacae.
METHODS AND MATERIALS
Bacteria were isolated from sediments of Cerritos Channel in Long Beach, California in
an area where salinity varies seasonally from 0-12%. Isolates were selected on the basis of
their ability to : a) attach to surfaces, b) produce capsular exopolymers, and c) grow in the
presence and absence of O2. A bacterium exhibiting these characteristics that was
frequently isolated from the sediments was subsequently identified as Enterobacter
cloacae.
The isolate was cultured in a broth medium that encouraged capsule production (Salas
and Geesey, 1983). Cells were cultured anaerobically by placing inoculated culture flasks
in Gas Pak 100 jars (BBL) containing Gas Pak anaerobic generators. The jars were
incubated with agitation at 14°C. Subcultures were made, using as an inoculum, cells that
grew as a collar on the inner surface of the swirling culture flask. All transfers and
manipulations were conducted under a stream of sterile N2 gas. In this way, an adherent
cell population which produced capsular exopolymers was cultured and maintained
under anaerobic conditions.
Surface colonization of glass coverslips by cells of E. cloacae was performed according to
procedures outlined by Salas and Geesey (1983). Attached cell densities were determined
by epifluorescence microscopy.
Glucose uptake studies were conducted with cells attached to coverslips shortly after
maximum adherent cell density was obtained. Individual coverslips containing the attached cells were dipped in fresh sterile growth medium lacking organic constituents to
remove loosely attached cells and then introduced into a 20 ml volume of the same
medium supplemented with 3 H- glucose at a final concentration which ranged from
125-2,500 pg/ ml. After various periods of incubation with agitation , the coverslips were
removed and rinsed in fresh medium without labeled glucose and transferred to scintillation vials for counting. Uncolonized coverslips treated in a similar manner were used to
determine non-specific adsorption of radiolabeled glucose to the surface, and the resulting counts subtracted from those obtained from colonized coverslips to estimate cellassociate uptake.
Free cells were recovered from the culture flask at the time maximum adherent cell
density was achieved and washed free of culture medium by centrifugation. The cells were
resuspended in growth medium lacking organic constituents at a density which provided a
total cell number in a 5 ml volume approximately equal to that attached to a single
coverslip. Radioactive glucose was added at the concentrations described for the attached
cells and, after various incubation times, the reaction was stopped by filtering the cells
onto a 0.45 µm membrane. The membranes were dried and the associated radioactivity
determined by liquid scintillation. Corrections were made for differences in counting
efficiency caused by the different scintillation geometries of glass coverslip and membrane
samples. All uptake studies were conducted at 14°C under anaerobic conditions using an
N2 atmosphere.
244
understanding of the conditions which promote attachment and the effect attachment has
on subsequent physiological activities should provide a more complete knowledge of the
importance of sessile bacteria in aquatic environments.
The present study describes the effect of surface attachment on nutrient uptake by a
brackish water sediment isolated of Enterobacter cloacae.
METHODS AND MATERIALS
Bacteria were isolated from sediments of Cerritos Channel in Long Beach, California in
an area where salinity varies seasonally from 0-12%. Isolates were selected on the basis of
their ability to : a) attach to surfaces, b) produce capsular exopolymers, and c) grow in the
presence and absence of O2. A bacterium exhibiting these characteristics that was
frequently isolated from the sediments was subsequently identified as Enterobacter
cloacae.
The isolate was cultured in a broth medium that encouraged capsule production (Salas
and Geesey, 1983). Cells were cultured anaerobically by placing inoculated culture flasks
in Gas Pak 100 jars (BBL) containing Gas Pak anaerobic generators. The jars were
incubated with agitation at 14°C. Subcultures were made, using as an inoculum, cells that
grew as a collar on the inner surface of the swirling culture flask. All transfers and
manipulations were conducted under a stream of sterile N2 gas. In this way, an adherent
cell population which produced capsular exopolymers was cultured and maintained
under anaerobic conditions.
Surface colonization of glass coverslips by cells of E. cloacae was performed according to
procedures outlined by Salas and Geesey (1983). Attached cell densities were determined
by epifluorescence microscopy.
Glucose uptake studies were conducted with cells attached to coverslips shortly after
maximum adherent cell density was obtained. Individual coverslips containing the attached cells were dipped in fresh sterile growth medium lacking organic constituents to
remove loosely attached cells and then introduced into a 20 ml volume of the same
medium supplemented with 3 H- glucose at a final concentration which ranged from
125-2,500 pg/ ml. After various periods of incubation with agitation , the coverslips were
removed and rinsed in fresh medium without labeled glucose and transferred to scintillation vials for counting. Uncolonized coverslips treated in a similar manner were used to
determine non-specific adsorption of radiolabeled glucose to the surface, and the resulting counts subtracted from those obtained from colonized coverslips to estimate cellassociate uptake.
Free cells were recovered from the culture flask at the time maximum adherent cell
density was achieved and washed free of culture medium by centrifugation. The cells were
resuspended in growth medium lacking organic constituents at a density which provided a
total cell number in a 5 ml volume approximately equal to that attached to a single
coverslip. Radioactive glucose was added at the concentrations described for the attached
cells and, after various incubation times, the reaction was stopped by filtering the cells
onto a 0.45 µm membrane. The membranes were dried and the associated radioactivity
determined by liquid scintillation. Corrections were made for differences in counting
efficiency caused by the different scintillation geometries of glass coverslip and membrane
samples. All uptake studies were conducted at 14°C under anaerobic conditions using an
N2 atmosphere.
244