Team:CLS CLSG UK/Results

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Results

Credit - National Cancer Institute@unsplash

• Overview

Results

Overview

While we were very happy with the results that we achieved, given we thought that any lab work was going to be impossible, we still had a few issues. The primary issue being that until we came to actually examine the plates under the light microscope we did not realise that we were unable to focus on the bottoms of the wells at a high power (400x). Given our initial plans to obtain quantitative data was to take images of the wells and then run them through an image processing software such as photoshop, we could not now do that. And while we could see the stark difference in the different wells we needed a new way of displaying and potentially quantifying the results. So we decided to construct a key with which each well’s coverage could be ranked on 100x magnification. By doing this it would create a system to present the results in a clear and easily understandable way, but also get quantitative data, though the use of this data given its subjective nature is contentious.

Looking at the plate layout (include link):

In order to make analysis of results easier we have divided the plate into quadrants:

One would expect that the highest level of adherence would be located in wells 1A to 5D, and that within this area there would be a decreasing gradient in the level of adherence due to the increased washing times. As well as this you would expect nothing to be present in columns 6 and 7 and that if any bacteria was present it would be in low quantities. As for the effect that washing with the synthetic sewer water rather than PBS would have we were not sure as there were arguments that it would increase and decrease the adherence of our bacteria.

First, looking at the plate washed only with PBS:

The first thing that is apparent, is that the increased washing has a significant effect on both strains of bacteria to adhere, as well as this column 6 and 7 remained empty. But outside of this we can also see that while there are areas where bacteria are able to adhere in very low amounts the level of adherence where both the SA E. coli and the GFP was present is clearly the highest. Comparing all four quadrants gives that the average density per well in quadrant 1 is 1.55, compared to 0.55 in quadrant 2 with 0.4 and 0.6 in quadrants 3 and 4 respectively.

Looking at the plate washed with our synthetic sewer water:

The same key was used for both plates so that we can compare them. The plate showed a similar trend to the first one regarding the effect of increased washing, as well as that, columns 6 and 7 remained empty. Similar to plate 1 the highest density of bacteria is in quadrant 1, where we would expect them, and other areas all have a very low adherence rate. By calculating the average well density for each quadrant, which is calculated by dividing the sum of all the values given to each quadrant by twenty, a bar chart can be made.

Displaying the results in this way clearly shows a number of things. Firstly, the synthetic sewer water had a significant effect on the ability of the E. coli to adhere, this can be seen through all the quadrants. In every quadrant the adherence in the synthetic sewer water is lower than that in the PBS by a sizable amount, this would indicate that the synthetic sewer water does have an effect on the ability of the bacteria to adhere to antigenic surfaces. We had a few theories as to why that is but we aren’t sure. Of course the presence of the ions caused this, but which ions specifically and how and why they caused this would require further experimentation. However, one conclusion that can be drawn that in sewer conditions the ability of the non-modified E. Coli to adhere was very close to zero, so the presence of the synthetic adhesin is very important to enable our bacteria to adhere to the surface.

The second thing that is apparent from this experiment is that no matter the conditions, the effect that the expression of the synthetic adhesin has on the adherence of the bacteria is large. This can be seen through the graphical representation of the results as well as the visual representation both of which are above. But we also wanted to see if we could show this mathematically and so ran an independent student's t-test. The two data sets used were the adherence of the S.A. E. Coli with GFP and the adherence of all of the other three conditions. This gives us:

This value can be rounded to 2.45 and when compared with the degrees of freedom table this value shows that these two sets of data are statistically significant to a 98% certainty. Therefore proving that the synthetic adhesin has a significant effect on the ability of the E. Coli to adhere.

Of course we are also aware that one could call this assumption flawed because of the manner with which the data was collected. While this is true the strong correlation between the quantitative and qualitative data as well as the fact that or findings are in line with our hypothesis and with the findings of the research paper published on this we are therefore able to assume that the expression of a synthetic adhesin when in the presence of a complimentary antigenic surface significantly increases the ability of that bacteria to adhere to the surface.