usually binding non-specifically to the bacteria as well as to the substrate surfaces, (2)
bacteria often being obscured and hidden by overlying particles and other cells and (3) the
fact that many bacteria tend to grow in microcolonies and hence are unevenly distributed
on attachment surfaces. Bacterial distribution is frequently non random in water (Daley,
1979 ; El-Shaarawi et al, 1981 ; Kirchman et al, 1982), sediments (Montagna, 1982) and
on surfaces of plants (Hossel and Baker, 1979), thus requiring special statistical treatments or homogenization (Daley, 1979).
The adherence of marine bacteria depends on the bacterial surfaces, the substrata, the
media separating the surfaces and environmental factors (Fletcher, 1980). Bacterial
adhesion is believed due, in part, to the extra-cellular polymers made up of acidic
polysaccharide (Marshall, 1973 ; Fletcher and Floodgate, 1973 ; Costerton et al, 1978)
and/or protein (Danielsson et al., 1977). The various factors effecting temporary and
permanent adhesion of bacteria to smooth surfaces in laboratory studies has been
discussed by Fletcher (1980). ,
Several investigators have attempted to disperse bacteria prior to counting by using
blender homogenization of water, sediment and plants (Daley, 1979 ; Montagna, 1982 ;
Scotten, 1971 ; Laycock, 1974 ; Mazure and Field, 1980) ; by sonication of sludge, soil,
sand grains and epiphytic contaminants (Pike et al., 1972 ; Banks and Walker, 1977 ;
Zvyagintsev and Galkina, 1967 ; Bingle, 1980 ; Weise and Rhineheimer, 1978, Polne et al,
1980) ; and by a combination of chemical treatments and ultrasound in sludge and soil
(Gayford and Richards, 1970 ; Pike et al., 1972 ; Banks and Walker, 1977 ; Bingle, 1980).
These attempts met with varying success. In addition, prolonged homogenization and
sonication lyse bacterial cells (Pike et al., 1972 ; Coakley et al., 1977 ; Paul and Myers,
1982) and therefore susceptibility of the cells is an important consideration.
The combination of chemical reagents (e.g. sodium tripolyphosphate and tetrasodium
pyrophosphate) with ultrasound has proven to be more successful than ultrasound alone
for dispersing bacteria present in flocs in activated sludge (Pike et al., 1972 ; Gayford and
Richards, 1970; Banks and Walker, 1977). The sequestering and deflocculating effects of
these reagents may aid in disaggregation and detachment of bacteria from various
surfaces.
We report upon the use of sodium pyrophosphate and ultrasound for dispersing marine
bacteria from surfaces to which they are attached into a suspension medium prior to
epifluorescence microscopic counting. This technique was used to examine marine subsurface and epibenthic waters, and sediment. Different aged blades of the lesser giant
kelp, Maerocystis integrifolia which had densities of bacteria, ranging from low on the
young blades to very high on the other blades, were also examined.
MATERIALS AND METHODS
Sample Collection
Water, sediment and Maerocystis integrifolia samples were collected by SCUBA in
January, 1983 from a kelp bed in Bamfield Inlet, Bamfield, Vancouver, Island, British
Columbia. The site was approximatively 10 m from shore and 4.5 m in depth.
Subsurface (0.1 m depth) and epibenthic (4m) water samples were collected with sterile 1
L polypropylene bottles. Water salinities were 22‰ and 26‰, respectively. A surface
sediment sample (4.5 m depth) was taken with a hand held sterile 50 ml syringe corer, the
end of which was immediately capped.
A frond of M. integrifolia with 29 blades and total length of 4.1 m was collected. The
250
bacteria often being obscured and hidden by overlying particles and other cells and (3) the
fact that many bacteria tend to grow in microcolonies and hence are unevenly distributed
on attachment surfaces. Bacterial distribution is frequently non random in water (Daley,
1979 ; El-Shaarawi et al, 1981 ; Kirchman et al, 1982), sediments (Montagna, 1982) and
on surfaces of plants (Hossel and Baker, 1979), thus requiring special statistical treatments or homogenization (Daley, 1979).
The adherence of marine bacteria depends on the bacterial surfaces, the substrata, the
media separating the surfaces and environmental factors (Fletcher, 1980). Bacterial
adhesion is believed due, in part, to the extra-cellular polymers made up of acidic
polysaccharide (Marshall, 1973 ; Fletcher and Floodgate, 1973 ; Costerton et al, 1978)
and/or protein (Danielsson et al., 1977). The various factors effecting temporary and
permanent adhesion of bacteria to smooth surfaces in laboratory studies has been
discussed by Fletcher (1980). ,
Several investigators have attempted to disperse bacteria prior to counting by using
blender homogenization of water, sediment and plants (Daley, 1979 ; Montagna, 1982 ;
Scotten, 1971 ; Laycock, 1974 ; Mazure and Field, 1980) ; by sonication of sludge, soil,
sand grains and epiphytic contaminants (Pike et al., 1972 ; Banks and Walker, 1977 ;
Zvyagintsev and Galkina, 1967 ; Bingle, 1980 ; Weise and Rhineheimer, 1978, Polne et al,
1980) ; and by a combination of chemical treatments and ultrasound in sludge and soil
(Gayford and Richards, 1970 ; Pike et al., 1972 ; Banks and Walker, 1977 ; Bingle, 1980).
These attempts met with varying success. In addition, prolonged homogenization and
sonication lyse bacterial cells (Pike et al., 1972 ; Coakley et al., 1977 ; Paul and Myers,
1982) and therefore susceptibility of the cells is an important consideration.
The combination of chemical reagents (e.g. sodium tripolyphosphate and tetrasodium
pyrophosphate) with ultrasound has proven to be more successful than ultrasound alone
for dispersing bacteria present in flocs in activated sludge (Pike et al., 1972 ; Gayford and
Richards, 1970; Banks and Walker, 1977). The sequestering and deflocculating effects of
these reagents may aid in disaggregation and detachment of bacteria from various
surfaces.
We report upon the use of sodium pyrophosphate and ultrasound for dispersing marine
bacteria from surfaces to which they are attached into a suspension medium prior to
epifluorescence microscopic counting. This technique was used to examine marine subsurface and epibenthic waters, and sediment. Different aged blades of the lesser giant
kelp, Maerocystis integrifolia which had densities of bacteria, ranging from low on the
young blades to very high on the other blades, were also examined.
MATERIALS AND METHODS
Sample Collection
Water, sediment and Maerocystis integrifolia samples were collected by SCUBA in
January, 1983 from a kelp bed in Bamfield Inlet, Bamfield, Vancouver, Island, British
Columbia. The site was approximatively 10 m from shore and 4.5 m in depth.
Subsurface (0.1 m depth) and epibenthic (4m) water samples were collected with sterile 1
L polypropylene bottles. Water salinities were 22‰ and 26‰, respectively. A surface
sediment sample (4.5 m depth) was taken with a hand held sterile 50 ml syringe corer, the
end of which was immediately capped.
A frond of M. integrifolia with 29 blades and total length of 4.1 m was collected. The
250