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To validate our heuristic method, we ran two experiments where we tried to stabilize the concentration of living cells in a bacterial culture during 5 consecutive hours. Both were successful, and we will report in more detail one of them.
In the first part of the experiment, we first determined the parameters of the model that describe the behavior of our cell culture, according to the procedure described in the modeling part.
The first part of the experiment followed a protocol close to the other:
1.0-90 min : this first incubation was done in the dark to let the bacteria grow and produce KillerRed.
2. 90-330 min : a longer 4 hours period with illumination at maximal intensity. After that time, the effects of light were clear, and the parameters of the model were calculated to fit the observable kinetics.
3. 330-450 min : Then our model was used to calculate the light intensity that would stabilize the population of living cells : light was applied at 70% of its maximal intensity.
4. 450-720 min : After 2 hours, we noticed that the fluorescence and the absorbance were slowly drifting away from the predictions. Since the fluorescence was higher than expected and the OD was lower than expected, we reduced the illumination to 50% of its maximal intensity for 2 hours and then at 40% of its maximal intensity.
| Time (min) | 0 | 90 | 330 | 450 | 570 | 720 |
|---|---|---|---|---|---|---|
| illumination (%) | 0 | 100 | 70 | 50 | 40 | end |
On the curves below:
$\bullet$ The red surface is the prediction of absorbance caused by dead bacteria ($D$, in $OD_{600}$) on the absorbance curve, and the prediction of fluorescence caused by KillerRed stored in dead bacteria ($K_D$, in $UF$) on the fluorescence curve.
$\bullet$ The yellow surface is the prediction of absorbance caused by living bacteria ($C$, in $OD_{600}$) on the absorbance curve, and the prediction of fluorescence caused by KillerRed stored in living bacteria ($K$, in $UF$) on the fluorescence curve.
$\bullet$ The blue line follows the collected datas.
Step 1: The experiment is run for 5 hours without control. There is first a period of 1 hour and a half in the dark, then 3 hours and a half at maximal intensity. This process has 2 objectives : to fasten the emergence of the level of the amount of living bacteria and to improve the precision of model. At this point, parameters are found to fit best the curves observed, that is the reason why a long period is needed : it will improve the precision of parameters. Then, we serch for the light the will stabilize the population cell. It could have been 30% of maximal intensity all along, but again to fasten the apparition of the level, we decide to enlight bacteria first at 70% for 2 hours, and then decrease the intensity at 30%. Below are the fit of the 5 first hours and the predictions for the chosen intensities. The level should appear in 2 hours.
Step 2 ($t=330min$):
$\diamond$ modification of parameters to improve the fit.
$\diamond$ creation of prediction with different light intensity.
$\diamond$ selfction of the intensity that will stabilise the bacterial population, here $I=70%$ of maximal intensity.
Step 3 ($t=330min$):
We observed that the fluorescence and the absorbance were too low compared to the prevision, so light intensity was decreased at $I=50%$ of maximal intensity.
Step 4 ($t=570min$):
The fluorescence and the absorbance were still too low copmpared to the prevision, light intensity was decreased at $I=40%$ of maximal intensity.
Step 5($t=660min$):
The absorbance has grown up at a steady for more than 5 hours. We have to count the cells to prove that the living bacteria concentration has been constant for this period.
Step 6:
The absorbance has grown up at a steady for more than 5 hours. We have to count the cells to prove that the living bacteria concentration has been constant for this period.
The experiment has been continued to create a new set of parameters. Here are the new predictions, with the parameters value of the fifth experiment.
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