1.Cell cultures of N. oceanica and N. gaditana were grown in the Cell-Hi CN medium at temperature 23°C with constant illumination at 100 μE m−2 s−1 and constant shaking on a Heidolph® Rotamax 120 Orbital Shaker (see Chapter II – Materials and methods – Growth conditions).
2.Agar plates of 9 cm diameter were used for the plating out of the cell culture. The agar medium contained seawater added Cell-Hi CN (1 mL to 1 L seawater) and antibiotic concentrations of following varies range from 5 to 300 µg/mL. The antibiotics used for the growth assay and their respective stock solution concentrations are listed in the table below.
Table 1: Antibiotics and their stock solution concentrations used in the cell growth assay.
|
Antibiotic |
Stock solution concentration (mg/mL) |
|
Ampicillin |
50 |
|
Apramycin |
30 |
|
Chloramphenicol |
30 |
|
Cycloheximide |
30 |
|
Gentamycin |
30 |
|
G418 Neomycin |
30 |
|
Hygromycin |
50 |
|
Kanamycin |
50 |
|
Spectinomycin |
50 |
|
Tetracaine |
30 |
3. For the growth tests the cell suspensions (3 µL) of both N. oceanica and N. gaditana species were spotted on antibiotic-containing with Cell-Hi CN medium plates at different cell concentrations (OD750nm of 1.0, 0.1, 0.055, 0.01 and 0.0055, corresponding to the cell numbers between 4.98×107 to 4.0×104 cells/mL, see table B.1, appendix B). Only cells of N. oceanica were used to test for lethal hygromycin concentrations (experiment # 2). The incubation of the cultures were performed under the same conditions as for the cell cultures` growth (see Materials and Methods). After two weeks of incubation, the results were recorded.
4. For determining the lethal antibiotic concentrations, the spotted cell concentration at 2.52×106 cells/mL (OD750nm of 0.055) was used, as higher concentrations might cause spontaneous mutations giving a feature of being able to grow on environment containing antibiotic. Intensity of the cell colonies of some cell growth experiments was analyzed by customer built Plate Imager analyzing the cell density on the agar-solidified medium in order to digitalize the visualized results.
Results
Figure 1: Comparison of effect of nine antibiotics on the growth of N. oceanica and N. gaditana cells (experiment # 1). N. oceanica and N. gaditana wild type cells were growing at 23°C and constant light intensity of 100 μE m−2 s−1 including continuous rotational shaking. The antibiotic concentrations are shown in µg/mL and range from 10 to 300 µg/mL. Growth characteristics were determined by spotting 3 µL of the cell suspension of different concentrations ranging from 4.98×107 to 4.0×104 cells/mL onto agar-solidified Cell-Hi CN medium added specific type of antibiotic at different concentrations. The positive cell growth is marked with black rectangle at certain cell and antibiotic concentration. In general, N. gaditana seemed to be less sensitive to all antibiotics compared to N. oceanica.
Table 2.: Comparison of the effect of nine antibiotics on N. oceanica and N. gaditana cell growth (summary of figure 1). The antibiotic concentrations ranged from 10 to 300 µg/mL. Growth characteristics were determined by spotting 3 µL of the N. oceanica and N. gaditana cell suspensions of different concentrations ranging from 4.98×107 to 4.0×104 cells/mL on agar-solidified Cell-Hi CN medium added certain type of antibiotic at certain concentration. The table shows the antibiotic concentration at which the cell growth is inhibited.
|
Antibiotic |
Lethal antibiotic concentration (µg/mL) |
|
|
N. oceanica |
N. gaditana |
|
|
Ampicillin |
≥ 300 |
≥ 300 |
|
Apramycin |
≥ 300 |
300 |
|
Gentamycin |
300 |
≥ 300 |
|
Cycloheximide |
10 |
10 |
|
Chloramphenicol |
10 |
50 |
|
Neomycin G418 |
10 |
100 |
|
Kanamycin |
≥ 300 |
≥ 300 |
|
Streptomycin |
50 |
300 |
|
Tetracaine |
50 |
50 |