From June 6th to July 6th
We made different experiments to get the optimal conditions for our patch, such as agar concentration, trials with the nitrocellulose membrane and consistency of the polymer PVA.
180 mL of Nutrient broth was prepared with 0.9 g of peptone and 0.54g of meat extract. For solid medium, 15 mL of nutrient broth and 0.75 g of agar was necessary. To prove semi-solid medium, we trial different concentration, 4,6,8,10 and 12 (g/L) with 0.1,0.15,0.2, 0.25 and 0.3 g of agar respectively and all of them with 25 mL of nutrient broth.
70 mL of Nutrient broth was prepared with 0.35 g of peptone and 0.21g meat extract. Three new different concentrations of semi-solid medium was prepared, 2.5, 3 and 3.5 g/L each with 10 mL of broth and 0.07, 0.06 and 0.05 g of agar respectively.
In order to know which concentration of semi-solid medium was the one with better motility, we inoculated by puncture E.coli at the center of the tube, where the medium was placed. (All the concentrations were inoculated).
We checked the results of motility and the chosen one was between 3 and 3.5 g/L.
We choose two different agar concentration: 3 and 3.5 g/L. 50 mL of nutrient broth with 0.25 g of peptone and 0.15 g of meat extract; 25 mL of semi-solid medium was prepared.
The prepared media were placed at the center of petri dishes, it was tested with 500, 700 and 800 microliters.
The petri dishes were incubated by adding a filter paper and a membrane.
We decided to repeat the experiment.
All petri dishes were incubated at room temperature, starting at 5:00 p.m.
Membranes were in contact with the medium for 24 hours. Then, these were placed in liquid medium at 10:45 a.m.
About the second experiment, samples: M2, M3 and C3 showed the filter paper out of place. About the first experiment, the filter paper adhered to the tube wall was observed.
At 10:50 a.m., the tubes were vortexed and incubated at 37ºC. We observe the boxes with large filter papers.plated.
At 11:00 a.m., the plating of the second experiment began and transfer to liquid medium.
At 12:12 p.m., the positive control was plated and transferred to liquid medium.
Results from the previous experiment were observed. Nutrient broth where filter paper pieces from experiment 1 were put revealed no turbidity except for sample 3 (its initial Petri Dish even showed more colonies than inoculated at the beginning). From experiment 2 no bacterial growth was observed. Positive controls showed turbidity and therefore, bacterial growth. All membranes from both experiments were transferred to tubes containing nutrient broth and were incubated at 37ºC for 48 h.
Tubes with nutrient broth were checked once more. Same results than those reported on July 2nd. Membranes from all experiments incubated in nutrient broth showed turbidity. Membranes from controls showed no bacterial growth except for Control 1 from Experiment 2.
PVA was dissolved in distilled water (1g per 10 mL), heated at 70ºC (constant stirring) for 25 and 40 min.
Both protocols for polymer (PVA) preparation were carried out. For more information related to these experiments, see Methodology and Results.