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<p> Primary spectrophotometry analysis, by checking optical density at 600 nm of all triplicates with different concentrations of PEG 6000, as well as the control medium without PEG showed cell growth was inhibited with increasing PEG 6000 concentrations used for stress condition induction (Figure 1).</p> | <p> Primary spectrophotometry analysis, by checking optical density at 600 nm of all triplicates with different concentrations of PEG 6000, as well as the control medium without PEG showed cell growth was inhibited with increasing PEG 6000 concentrations used for stress condition induction (Figure 1).</p> | ||
− | <img class="ui centered image" src="https:// | + | <img class="ui centered image" src="https://static.igem.org/mediawiki/2015/1/1b/UFSCariGEM2015_Figure1_plasmolysis.jpg"> |
<h5 class="ui center aligned header"><b>Figure 1</b>: Experimental plot of absorbance versus time in different PEG 6000 concentration.</h5> | <h5 class="ui center aligned header"><b>Figure 1</b>: Experimental plot of absorbance versus time in different PEG 6000 concentration.</h5> | ||
+ | <p> Figure 1 shows no significant variation of weekly results with increasing PEG 6000 concentration. Optical density of bacterial suspension decreased with rising of PEG 6000 concentration. The growth could be verified since environment concerned an available carbon source for the microorganism, as glycerol. Cells number in the suspension influences the absorbance readings directly, however, possible cells arrangement and conformational changes could help formation of mobile cell complexes changes which affect considerably obtained data. It is due to light reflection direct linking with conformational and structural arrangement of these cells. To verify this, microscopy experiments were carried out through the Gram method (Figure 2).</p> | ||
+ | |||
+ | <img class="ui centered image" src="https://2015.igem.org/File:UFSCariGEM2015_Figure1_plasmolysis.jpg"> | ||
+ | <h5 class="ui center aligned header"><b>Figure 2</b>: Cells conformational changes in optical microscopy reveals cell arangement to reduce entropy in medium through hydrophobic interactions.</h5> | ||
+ | <p> The above images show that, with increasing PEG concentration in solution, occurs the formation of complex cell corresponding to Escherichia coli. This can demonstrate a thermodynamically favorable response to the presence of an extremely hydrophilic molecule in the medium, so it is expected that those cells that do not suffer osmotic stress and do not come into plasmolysis, lose viability even in solutions containing the osmolyte. | ||
+ | The viability of the cells after plasmolysis, as well as the viability of the control medium without polyethylene glycol was verified weekly by direct plating of 100ul of each triplicate on solid medium, also concurrently with the plating serial dilutions in solid medium. The counting forming units demonstrate that the viability showed no polyethylene glycol present in the control for about a month and one week; Recententes counts of colony forming units show that the control is gradually losing viability, whereas the viability of media with polyethylene glycol has been found to date, totaling seven weeks at room temperature.</p> | ||
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Revision as of 14:01, 17 September 2015
Results
What did we observe?
Plasmolysis
Main objective of this section is to verify the viability of Escherichia coli cells under treatments with polyethylene glycol (PEG 6000) for different time lapses, and the retaken of metabolism after plasmolysis. To this, we submited this organism to different concentrations of PEG 6000 and have assessed its growth hability. Assays of plasmolysis induction were performed using Escherichia coli K-12 strain Dh5α cells obtained from a culture in lysogeny broth until an optical density at 600 nm of ~ 1.5-2.0, finally cells were harvested by centrifugation and washed with sterile 0.9% saline. Cells were resuspended in plasmolysis media (2% (vo./vol.) glycerol, 50 mM sodium acetate, 10 mM zinc chloride) containing various different concentrations of PEG 6000 (5, 10, 15, 20, 25 and 30% wt/vol). Proportion of cells in the final solution was 0.1% wt/vol. All media and their corresponding sterile controls were kept in triplicate at room temperature.
Primary spectrophotometry analysis, by checking optical density at 600 nm of all triplicates with different concentrations of PEG 6000, as well as the control medium without PEG showed cell growth was inhibited with increasing PEG 6000 concentrations used for stress condition induction (Figure 1).
![](https://static.igem.org/mediawiki/2015/1/1b/UFSCariGEM2015_Figure1_plasmolysis.jpg)
Figure 1: Experimental plot of absorbance versus time in different PEG 6000 concentration.
Figure 1 shows no significant variation of weekly results with increasing PEG 6000 concentration. Optical density of bacterial suspension decreased with rising of PEG 6000 concentration. The growth could be verified since environment concerned an available carbon source for the microorganism, as glycerol. Cells number in the suspension influences the absorbance readings directly, however, possible cells arrangement and conformational changes could help formation of mobile cell complexes changes which affect considerably obtained data. It is due to light reflection direct linking with conformational and structural arrangement of these cells. To verify this, microscopy experiments were carried out through the Gram method (Figure 2).
![](https://2015.igem.org/File:UFSCariGEM2015_Figure1_plasmolysis.jpg)
Figure 2: Cells conformational changes in optical microscopy reveals cell arangement to reduce entropy in medium through hydrophobic interactions.
The above images show that, with increasing PEG concentration in solution, occurs the formation of complex cell corresponding to Escherichia coli. This can demonstrate a thermodynamically favorable response to the presence of an extremely hydrophilic molecule in the medium, so it is expected that those cells that do not suffer osmotic stress and do not come into plasmolysis, lose viability even in solutions containing the osmolyte. The viability of the cells after plasmolysis, as well as the viability of the control medium without polyethylene glycol was verified weekly by direct plating of 100ul of each triplicate on solid medium, also concurrently with the plating serial dilutions in solid medium. The counting forming units demonstrate that the viability showed no polyethylene glycol present in the control for about a month and one week; Recententes counts of colony forming units show that the control is gradually losing viability, whereas the viability of media with polyethylene glycol has been found to date, totaling seven weeks at room temperature.
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