blades were numbered from the frond apical meristem and 4 blades were sampled. Blade 1
was the first new blade free from the apical meristem while blade 22 was the oldest and
closest to the holdfast. Blade 4 was the longest complete blade while blade 10 was chosen
as the middle aged blade. All samples were preserved with 3.7 % formaldehyde (v/v). The
final pH of the sample after fixation was ca.7.8.
Bacterial Enumeration
The direct count method (Hobbie et al., 1977), as modified for 4’, 6 - diamino -2phenylindole (DAPI) by Porter and Feig(1980), was used for staining the bacteria, except
that the final DAPI concentration used was 1.0 µg/ml. After the specified treatment, the
samples were incubated with DAPI for 10 min and mixed vigorously for 15 s before being
filtered on to 0.2 µm pore size, 25 mm diameter, pre-dyed (Irgalan Black) Nuclepore
filters. An epifluorescence light microscope was used to examine and enumerate the
bacterial cells.
The volume of sample filtered was adjusted so that there were between 10 to 30 fluorescing
bacterial cells per grid and 20 to 30 randomly chosen microscopic fields were enumerated.
The volume of subsurface and epibenthic water filtered for bacterial enumeration was
5 ml. One hundred µl of the sediment sample was suspended in 5 ml of filter sterilized
26‰ sodium chloride solution (w/v) (ASW). After treatment, two subsamples of 1 ml
each were stained and made up to 5 ml before filtration.
The kelp blade was sampled with a sterilized 4 mm I.D. cork borer. The discs were stained
with DAPI, rinsed with ASW and placed on a glass slide. Warm glycerol gelatin was then
poured over each disc and a glass coverslip placed on top. After the glycerol had
solidified, the slide was examined using oil immersion. Discs treated with tetrasodium
pyrophosphate and ultrasound resulted in a dispersed mixture of microorganisms (including bacteria), kelp cells and mucillage. This mixture was stained and filtered in a similar
manner as the sediment suspensions. The maximum volume of the mixture that could be
filtered without clogging the filter was 3 ml from a total of 5 ml. For discs from blades 1,4
and 10, 3 ml were filtered, while for blade 22, 2 ml was filtered due to the much higher
density of bacteria encountered per grid.
Effect of Ultrasound and Chemical Reagents
A biosonik II generator with a high power level ultrasonic probe (Model BP-II) and a
standard 4 mm diameter titanium tip (Bronwill Scientific, N.Y.) was used for the ultrasound experiments. The volumes of all samples or samples plus suspending media were
kept constant at 5 ml in sterile disposable tubes, and the probe tip was kept 1 cm from the
bottom of the tube. The sample tubes were precooled to 0°C and packed in crushed ice
during sonication to dissipate heat. Treatment of samples which required sonication for
periods greater than 15 s were carried out in bursts of 15 s sonication followed by 45 s of
cooling in crushed ice before subsequent sonication.
The tolerance of bacterial cells to sonication was examined. A Gram negative rod shaped
bacterium was isolated from the sea water supply at Simon Fraser University. Two 500 ml
cultures of the isolate were grown in 21 Erylenmeyer flasks at 15°C using a reciproacting
incubator with a shake rate of 50 strokes/mn.
The exponential growth phase of the culture was between 2 to 25 h. Eight 5 ml aliquots
were sampled at 18 h from each flask. Four of the aliquots were fixed with 3.7 %
formaldehyde (v/v) for 30 mn. The tolerance of fixed and unfixed isolate cells to
sonication for durations ranging from 0 to 90 s at a power level of 125 W was assessed by
251
was the first new blade free from the apical meristem while blade 22 was the oldest and
closest to the holdfast. Blade 4 was the longest complete blade while blade 10 was chosen
as the middle aged blade. All samples were preserved with 3.7 % formaldehyde (v/v). The
final pH of the sample after fixation was ca.7.8.
Bacterial Enumeration
The direct count method (Hobbie et al., 1977), as modified for 4’, 6 - diamino -2phenylindole (DAPI) by Porter and Feig(1980), was used for staining the bacteria, except
that the final DAPI concentration used was 1.0 µg/ml. After the specified treatment, the
samples were incubated with DAPI for 10 min and mixed vigorously for 15 s before being
filtered on to 0.2 µm pore size, 25 mm diameter, pre-dyed (Irgalan Black) Nuclepore
filters. An epifluorescence light microscope was used to examine and enumerate the
bacterial cells.
The volume of sample filtered was adjusted so that there were between 10 to 30 fluorescing
bacterial cells per grid and 20 to 30 randomly chosen microscopic fields were enumerated.
The volume of subsurface and epibenthic water filtered for bacterial enumeration was
5 ml. One hundred µl of the sediment sample was suspended in 5 ml of filter sterilized
26‰ sodium chloride solution (w/v) (ASW). After treatment, two subsamples of 1 ml
each were stained and made up to 5 ml before filtration.
The kelp blade was sampled with a sterilized 4 mm I.D. cork borer. The discs were stained
with DAPI, rinsed with ASW and placed on a glass slide. Warm glycerol gelatin was then
poured over each disc and a glass coverslip placed on top. After the glycerol had
solidified, the slide was examined using oil immersion. Discs treated with tetrasodium
pyrophosphate and ultrasound resulted in a dispersed mixture of microorganisms (including bacteria), kelp cells and mucillage. This mixture was stained and filtered in a similar
manner as the sediment suspensions. The maximum volume of the mixture that could be
filtered without clogging the filter was 3 ml from a total of 5 ml. For discs from blades 1,4
and 10, 3 ml were filtered, while for blade 22, 2 ml was filtered due to the much higher
density of bacteria encountered per grid.
Effect of Ultrasound and Chemical Reagents
A biosonik II generator with a high power level ultrasonic probe (Model BP-II) and a
standard 4 mm diameter titanium tip (Bronwill Scientific, N.Y.) was used for the ultrasound experiments. The volumes of all samples or samples plus suspending media were
kept constant at 5 ml in sterile disposable tubes, and the probe tip was kept 1 cm from the
bottom of the tube. The sample tubes were precooled to 0°C and packed in crushed ice
during sonication to dissipate heat. Treatment of samples which required sonication for
periods greater than 15 s were carried out in bursts of 15 s sonication followed by 45 s of
cooling in crushed ice before subsequent sonication.
The tolerance of bacterial cells to sonication was examined. A Gram negative rod shaped
bacterium was isolated from the sea water supply at Simon Fraser University. Two 500 ml
cultures of the isolate were grown in 21 Erylenmeyer flasks at 15°C using a reciproacting
incubator with a shake rate of 50 strokes/mn.
The exponential growth phase of the culture was between 2 to 25 h. Eight 5 ml aliquots
were sampled at 18 h from each flask. Four of the aliquots were fixed with 3.7 %
formaldehyde (v/v) for 30 mn. The tolerance of fixed and unfixed isolate cells to
sonication for durations ranging from 0 to 90 s at a power level of 125 W was assessed by
251