Once the images had been obtained, there came the process of extracting useful information from them. NaCl solution was chosen as the liquid because a)It was easy to obtain and create to our desired concentration, and b)It was hoped that, given the linear concentration range, we would also gain linear velocities that could be graphed nicely. The above yellow bead protocol was repeated using NaCl solutions of 1M, 3M and 5M. However, as it became apparent that the issues with the gas vesicles (see the relevant Project page here) were likely to extend beyond the 10 weeks allocated time, it was decided that we would more thoroughly test the capabilties of the system, by attempting to measure and compare the beads' sinking velocities in liquids of different densities.Įxperiment 3: Sinking Bead Velocities in Multiple NaCl Molarities Initially, the glass beads were only to be used to optimise the system for its final use in tracking bacteria. Also, the beads did sink very quickly – the majority of them had sunk in ~10 minutes, meaning the experiment could be run multiple times. Turned off, we were able to see the true brightness each time. This was due to the gain settings on the camera, which were still active. We had noticed in the previous experiment that the overall brightness had varied between images, making comparison tricky. Though the solution was less opaque than the previous, changes were still visible thanks to further changes in the camera setting. This set up was much better, in a number of ways. This was due to yellow beads letting out red and green light which slightly distorted the light intensity values. Only the blue light from the pictures were analysed.
#Spc900nc 00 software
The pictures were then sorted into a folder to be analysed.Īuto-click software () was used to allow the pictures to be taken automatically.This was so the quick changes of the first few minutes and the final steady state could be captured with minimal labour. From 10-60 mins one picture was taken every 10 minutes.For the first 10 minutes, after every minute another picture was taken.The solution with beads was then remixed with a pipette then quickly put in place and the first picture of the sequence was taken.A picture was taken of the background cuvette.Another cuvette was also filled with the same liquid, but with no beads, to act as a background.This cuvette was filled to 3ml with the appropriate solution (e.g.100µl was taken from the beads solution and added to a cuvette.The solution of 5µm glass beads was mixed using a vortex mixer.Preliminary calculations supported our hypothesis. Using glass beads 5um in diameter, and of density 200kg/m^3, the system could be tested multiple times per day. In order to be able to optimise the experiment, we would need a set-up which ran far quicker - otherwise, it could take days to get even the camera settings right! With this in mind, we procured some different beads for experiment 2.įor ease of repeatability, we needed to find a quicker experiment to run. One reason for this could be the shear number of beads we had used: our engineering adviser agreed that the concentration we'd used was far too high and we'd be unlikely to see anything. A quick calculation had put a preliminary estimate of 150 hours to travel the 3cm, but we had assumed changes would be visible in the time before this. In the end, this was also far too short, and even after 3 – 4 days there had been little change in the image brightness (again, nothing measurable). We changed the time between images to be 1 hour. Initially, the plan was to take an image every 10 minutes, but it very quickly became clear that there would be no changes in this time frame – at least no changes big enough to register. This was repeated at different times.(see below) Images were taken of the cuvette using the webcam.The LEDs were switched on, at a voltage of 3V.The cuvette was placed in the set-up as shown in the previous figure.This was again mixed to a uniform distribution This was added to a 3cm flat sided cuvette containing 3.5ml of water.200μl of double distilled water was added, and mixed to a uniform distribution.1ml of water was then removed, leaving the pellet of beads behind.This was centrifuged at 6G for 5 minutes.1ml of water was added to the 100μl of beads, and pipetted up and down to give a uniform distribution.100μl of this was pipetted into a second epindorf, in case the others were lost. 200μl of 1% red silicone bead suspension was remixed to return to a uniform distribution.With a very similar size (1.1um) and density (1100kg/m^3) to E.coli, we felt these would be an acceptable substitute for the cells. Phillips SPC900NC/00 Webcam, with laptop USB connectionįor the first experiment, we would be tracking the sedimentation of red silicone beads through water.Breadboard, with 2 parallel connected LEDs:.Connections to variable DC voltage supply.