examining the change in absorbance and bacterial counts of the samples. The light
absorbance of two-2.5 ml samples was measured at 540 nm using a Carl Zeiss PMQ II
spectophotometer, and bacterial counts were carried out using two-100 µl subsamples
from each of the two samples for each of the sonication periods.
The tolerance of bacterial cells in water and sediment samples, with and without 3.7 %
formaldehyde was investigated. For each treament, two-5 ml epibenthic water samples
and two-100 µl sediment samples suspended in 5 ml of ASW were sonicated for time
periods ranging from 0 to 60 s and at power levels of 100 and 125 W (except where noted)
and enumerated.
Several chemical reagents (viz, hydrochloric acid, sodium carbonate, sodium hydroxide,
sodium periodate and tetrasodium pyrophosphate at concentrations ranging from 0.0001
M to 0.1 M dissolved in ASW) were tested for dispersal properties upon addition to
sediment and kelp discs from blade 10. Each sample was incubated for 15 min after
chemical addition and then treated with ultrasound. The samples were then examined
under epifluorescence microscopy and qualitatively assessed for gross changes in clumping or attachment of bacteria.
Since sodium pyrophosphate followed by sonication treatment had the greatest effect on
dispersion of bacteria in the sediment and kelp disc samples, the tolerance of bacterial
cells to the combination treatment was examined. Replicate 5 ml subsurface and epibenthic water samples were incubated with 0.001 M sodium pyrophosphate for 15 min and
sonicated at 100 W power level for time periods ranging from 0 to 60 s. Replicate 100 µl
sediment samples were each treated with 0.01 M pyrophosphate and sonicated at 75 to
125 W power level for up to 60 s. The samples were then examined for changes in bacterial
numbers.
RESULTS AND DISCUSSION
Effect of Ultrasound
Unfixed cells of the Gram negative rod shaped bacterial isolate were very sensitive to
sonication. Untreated cells were observed to be single or clumped in small aggregates.
Within 5 s of treatment at the 125 W power level, the bacterial count declined by 46 % and
after 90 s treatment, only approximatively 1 % of the original number remained (Fig. 1).
Light absorbance measurement of the unfixed samples after treatment with sonication
showed a similar trend (Fig. 1).
Many microorganisms treated with sonication have survival curves which show a negative exponential form (Coakley et al., 1977). A Gram negative and rod shaped bacterium
was chosen since it represented the most sensitive cell type to sonication (Coakley et al.,
1977). When the bacterial samples were fixed with 3.7 % formaldehyde (v/v) for 30 min,
there was in increase in tolerance of bacterial cells to sonication. After 5 s sonication at
125 W, there was a small increase in bacterial number (6 %) probably due to disaggregation of bacterial clumps. This was followed by a slow decline in numbers with time of
sonication until at 90 s, 85 % of the original bacterial numbers remained (Fig. 1). Absorbances of the sonicated samples were relatively constant for up to 30 s treatment and then
showed a small decline after 60 and 90 s treatment (Fig. 1) confirming the increased
tolerance of formaldehyde fixed cells to sonication.
Aldehyde fixatives form inter- and intra-molecular crosslinks with protein (Hayat, 1981)
and hence strengthen the cell components, including the cell walls, which results in an
increased tolerance of these cells to sonication. The fixation of proteins by formaldehyde
252
absorbance of two-2.5 ml samples was measured at 540 nm using a Carl Zeiss PMQ II
spectophotometer, and bacterial counts were carried out using two-100 µl subsamples
from each of the two samples for each of the sonication periods.
The tolerance of bacterial cells in water and sediment samples, with and without 3.7 %
formaldehyde was investigated. For each treament, two-5 ml epibenthic water samples
and two-100 µl sediment samples suspended in 5 ml of ASW were sonicated for time
periods ranging from 0 to 60 s and at power levels of 100 and 125 W (except where noted)
and enumerated.
Several chemical reagents (viz, hydrochloric acid, sodium carbonate, sodium hydroxide,
sodium periodate and tetrasodium pyrophosphate at concentrations ranging from 0.0001
M to 0.1 M dissolved in ASW) were tested for dispersal properties upon addition to
sediment and kelp discs from blade 10. Each sample was incubated for 15 min after
chemical addition and then treated with ultrasound. The samples were then examined
under epifluorescence microscopy and qualitatively assessed for gross changes in clumping or attachment of bacteria.
Since sodium pyrophosphate followed by sonication treatment had the greatest effect on
dispersion of bacteria in the sediment and kelp disc samples, the tolerance of bacterial
cells to the combination treatment was examined. Replicate 5 ml subsurface and epibenthic water samples were incubated with 0.001 M sodium pyrophosphate for 15 min and
sonicated at 100 W power level for time periods ranging from 0 to 60 s. Replicate 100 µl
sediment samples were each treated with 0.01 M pyrophosphate and sonicated at 75 to
125 W power level for up to 60 s. The samples were then examined for changes in bacterial
numbers.
RESULTS AND DISCUSSION
Effect of Ultrasound
Unfixed cells of the Gram negative rod shaped bacterial isolate were very sensitive to
sonication. Untreated cells were observed to be single or clumped in small aggregates.
Within 5 s of treatment at the 125 W power level, the bacterial count declined by 46 % and
after 90 s treatment, only approximatively 1 % of the original number remained (Fig. 1).
Light absorbance measurement of the unfixed samples after treatment with sonication
showed a similar trend (Fig. 1).
Many microorganisms treated with sonication have survival curves which show a negative exponential form (Coakley et al., 1977). A Gram negative and rod shaped bacterium
was chosen since it represented the most sensitive cell type to sonication (Coakley et al.,
1977). When the bacterial samples were fixed with 3.7 % formaldehyde (v/v) for 30 min,
there was in increase in tolerance of bacterial cells to sonication. After 5 s sonication at
125 W, there was a small increase in bacterial number (6 %) probably due to disaggregation of bacterial clumps. This was followed by a slow decline in numbers with time of
sonication until at 90 s, 85 % of the original bacterial numbers remained (Fig. 1). Absorbances of the sonicated samples were relatively constant for up to 30 s treatment and then
showed a small decline after 60 and 90 s treatment (Fig. 1) confirming the increased
tolerance of formaldehyde fixed cells to sonication.
Aldehyde fixatives form inter- and intra-molecular crosslinks with protein (Hayat, 1981)
and hence strengthen the cell components, including the cell walls, which results in an
increased tolerance of these cells to sonication. The fixation of proteins by formaldehyde
252