laminar flow clean bench into dialysis chambers, prepared without supporting disks,
filters and screw-on ring at the upper side. After filling the chamber, the upper side was
covered by the supporting disks and filters, and tightened by the screw-on ring. Finally,
the chamber was securely closed by using a pair of pincers.
Determination of cell densities
At the beginning of the seawater experiments, a sample was withdrawn from the chamber
with a syringe, and the chamber was exposed again in the aquarium. We observed that the
air space left by the sample volume taken out remained in the chamber. It was therefore
decided to get the whole cell suspension from the chamber. From this suspension a 0.5 ml
subsample was diluted in sterile seawater, and 0.1 ml of appropriate dilutions were plated
on seawater agar. The plates were incubated at 24°C for 4 - 5 d. Three plates per dilution
were prepared and the mean values presented. The deviations from the mean values
amounted to 9 - 18%.
Three chambers were buried in the sediment of the same mud flat with the marked upper
sides upright. The upper sides of the chambers were covered with 1 cm of sediment layer.
The chamber was secured by two stainless steel wire clamps and flags were attached to the
wire for detecting the position of the chamber. After recovery the chambers were transported in a cool box to the laboratory. The outside wall of the chamber was first rinsed
with tap water and dried with a cellulose tissue. Subsequently the upper side was opened
under a laminar flow clean bench and 1 cm
3 of surface sediment was suspended in 4.5 ml
of sterile seawater. The suspension was homogenized with an Ultra-Turrax 18 KG
blender (IKA, Staufen) at 20000 rpm for 1 s. The Ultra-Turrax shaft was then cleaned in
4.5 ml of sterile seawater at 20000 rpm to get the remaining cells and sediment particles
off. Both suspensions were combined and further dilutions were made in sterile seawater.
Cell densities were also determined on seawater agar.
Experimental arrangement for culturing in seawater aquariums
The Teflon dialysis chambers were cleaned in concentrated nitric acid and rinsed several
times with bidistilled water before use. The chambers on magnetic stirrer holders were
kept in an aquarium and rotated by means of magnetic stirrers. The aquarium was built
from Lexan, a polycarbonate from General Electric Inc., New York. A 1 m
3 glass fibre
tank was used as reservoir. The tank and aquarium were cleaned with a brush, and rinsed
with 2 M HCl and seawater before starting the experiment. The two aquariums were
covered with black plastic sheets to retard algal growth in the seawater. A schematic
representation of the experimental arrangement is shown in Figure 4. This arrangement
was used for dialysis cultures in seawater without zinc addition. In toxicity experiments
with ZnCl 2 , a closed system with the seawater tank as reservoir was chosen. A ZnCl 2 stock
solution, 1000 fold in concentration, was made in 11 of bidistilled water, acidified with
0.5 ml of concentrated nitric acid. This stock solution was mixed to 1000 1 of seawater in
the reservoir, giving the desired final concentration of ZnCl 2 .
Zinc analyses
About 30 ml of the cell suspension was filtered on 0,4 pm Nuclepore filter. The cells were
washed twice with 5 ml of NaCl-MgCl 2 solution. Wet cells were dried at 80°C for 3 d, and
the dry weights determined. Dried cells on the filter were digested with a mixture of 3 ml
HCl and 1 ml HNO3 (both concentrated suprapure acids from Merck, Darmstadt) in a
Teflon autoclave at 150°C for 45 min. Zinc determinations were done with an atomic
absorption spectrophotometer 300 (Perkin-Elmer, Überlingen) equipped with a D2592
filters and screw-on ring at the upper side. After filling the chamber, the upper side was
covered by the supporting disks and filters, and tightened by the screw-on ring. Finally,
the chamber was securely closed by using a pair of pincers.
Determination of cell densities
At the beginning of the seawater experiments, a sample was withdrawn from the chamber
with a syringe, and the chamber was exposed again in the aquarium. We observed that the
air space left by the sample volume taken out remained in the chamber. It was therefore
decided to get the whole cell suspension from the chamber. From this suspension a 0.5 ml
subsample was diluted in sterile seawater, and 0.1 ml of appropriate dilutions were plated
on seawater agar. The plates were incubated at 24°C for 4 - 5 d. Three plates per dilution
were prepared and the mean values presented. The deviations from the mean values
amounted to 9 - 18%.
Three chambers were buried in the sediment of the same mud flat with the marked upper
sides upright. The upper sides of the chambers were covered with 1 cm of sediment layer.
The chamber was secured by two stainless steel wire clamps and flags were attached to the
wire for detecting the position of the chamber. After recovery the chambers were transported in a cool box to the laboratory. The outside wall of the chamber was first rinsed
with tap water and dried with a cellulose tissue. Subsequently the upper side was opened
under a laminar flow clean bench and 1 cm
3 of surface sediment was suspended in 4.5 ml
of sterile seawater. The suspension was homogenized with an Ultra-Turrax 18 KG
blender (IKA, Staufen) at 20000 rpm for 1 s. The Ultra-Turrax shaft was then cleaned in
4.5 ml of sterile seawater at 20000 rpm to get the remaining cells and sediment particles
off. Both suspensions were combined and further dilutions were made in sterile seawater.
Cell densities were also determined on seawater agar.
Experimental arrangement for culturing in seawater aquariums
The Teflon dialysis chambers were cleaned in concentrated nitric acid and rinsed several
times with bidistilled water before use. The chambers on magnetic stirrer holders were
kept in an aquarium and rotated by means of magnetic stirrers. The aquarium was built
from Lexan, a polycarbonate from General Electric Inc., New York. A 1 m
3 glass fibre
tank was used as reservoir. The tank and aquarium were cleaned with a brush, and rinsed
with 2 M HCl and seawater before starting the experiment. The two aquariums were
covered with black plastic sheets to retard algal growth in the seawater. A schematic
representation of the experimental arrangement is shown in Figure 4. This arrangement
was used for dialysis cultures in seawater without zinc addition. In toxicity experiments
with ZnCl 2 , a closed system with the seawater tank as reservoir was chosen. A ZnCl 2 stock
solution, 1000 fold in concentration, was made in 11 of bidistilled water, acidified with
0.5 ml of concentrated nitric acid. This stock solution was mixed to 1000 1 of seawater in
the reservoir, giving the desired final concentration of ZnCl 2 .
Zinc analyses
About 30 ml of the cell suspension was filtered on 0,4 pm Nuclepore filter. The cells were
washed twice with 5 ml of NaCl-MgCl 2 solution. Wet cells were dried at 80°C for 3 d, and
the dry weights determined. Dried cells on the filter were digested with a mixture of 3 ml
HCl and 1 ml HNO3 (both concentrated suprapure acids from Merck, Darmstadt) in a
Teflon autoclave at 150°C for 45 min. Zinc determinations were done with an atomic
absorption spectrophotometer 300 (Perkin-Elmer, Überlingen) equipped with a D2592