Biofilm formation was tested under conditions similar to that of seawater conditions using artificial seawater prepared in lab, which contains main salts and concentrations found in seawater. This test was done as a control to observe biofilm formation under standard conditions of artificial seawater.

Results indicated that biofilm was formed since absorbance is directly related to biofilm formation, and any increase in the absorbance is directly related to biofilm formation. We have found also that DH5 is capable of forming biofilm.

1. Minimal formation of Biofilm of SRB8 and DH5 at lower NaCl concentration range (0.05-1.6 M) in artificial sea water and 2X LB

DH5 cells were treated with artificial sea water with different NaCl concentrations and plated on a 96-well plate. The plate was incubated at 37C for ~48 hours. This was followed by staining with 0.1% crystal violet solution to measure the absorbance which corresponds to the biofilm formation.

DH5 cells were treated with 2X LB medium with different NaCl concentrations and plated on a 96-well plate. The plate was incubated at 37C for ~48 hours. This was followed by staining with 0.1% crystal violet solution to measure the absorbance which corresponds to the biofilm formation.

SRB8 cells were treated with artificial sea water with different NaCl concentrations and plated on a 96-well plate. The plate was incubated at 37C for ~48 hours. This was followed by staining with 0.1% crystal violet solution to measure the absorbance which corresponds to the biofilm formation.

SRB8 cells were treated with 2X LB medium with different NaCl concentrations and plated on a 96-well plate. The plate was incubated at 37C for ~48 hours. This was followed by staining with 0.1% crystal violet solution to measure the absorbance which corresponds to the biofilm formation.

2. Formation of Biofilm of SRB8 and DH5 at optimum NaCl concentration range (1.7-2.5 M) in 2X LB

DH5 cells were treated with 2X LB medium with different NaCl concentrations and plated on a tissue culture treated 96-well plate. The plate was incubated at 37C for ~48 hours. This was followed by staining with 0.1% crystal violet solution to measure the absorbance which corresponds to the biofilm formation.

SRB8 cells were treated with 2X LB medium with different NaCl concentrations and plated on a tissue culture treated 96-well plate. The plate was incubated at 37C for ~48 hours. This was followed by staining with 0.1% crystal violet solution to measure the absorbance which corresponds to the biofilm formation.

Analysis:

Figure 1:

An increase in the absorbance was observed from 0 M NaCl to 0.2 M NaCl. The maximum absorbance for DH5 in artificial sea water was at 0.2 M NaCl. Then the absorbance decreased from 0.2 M NaCl to 0.8 M NaCl. Followed by an increase through 0.8 M NaCl to 1.6 M NaCl.

Figure 2:

An increase in the absorbance was observed from 0 M NaCl to 0.1 M NaCl. The maximum absorbance for DH5 in 2X LB was at 0.1 M NaCl. After 0.1 M NaCl the absorbance started fluctuating through NaCl concentrations 0.1 M to 1.6 M.

Figure 3:

An increase in the absorbance was observed from 0.05 M NaCl to 0.2 M NaCl. The maximum absorbance for SRB in artificial sea water was at 0.2 M NaCl. After 0.2 M NaCl the absorbance started fluctuating through NaCl concentrations 0.2 M to 1.6 M.

Figure 4:

A decrease in the absorbance was observed from 0 M NaCl to 0.4 M NaCl. The maximum absorbance for SRB in 2X LB was at 0.8 M NaCl. Then the absorbance decreased from 0.8 M NaCl to 1.6 M NaCl.

Figure 5:

The absorbance was fluctuating through the various NaCl concentrations within the range of 1.7 to 2.5 M NaCl. The maximum absorbance for DH5 in 2X LB was at 0 M NaCl

Figure 6:

The absorbance was fluctuating through the various NaCl concentrations within the range of 1.7 to 2.5 M NaCl. The maximum absorbance for SRB in 2X LB was at 2.1 M NaCl