Team:Glasgow/BiofilmResults

From 2011.igem.org

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<h1> Properties of <i>P. Aeruginosa Biofilms</i></h1>
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<p>Biofilm formation was also confirmed by SEM pictures that showed the extracellular matrix, such as Picture 1 below.</p>
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<b>Aims</b>
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1. Determine quantitatively the base rate of dissociation from a biofilm. This rate can be compared to the novel biofilm dispersing biobricks rate of dispersal to determine their effectiveness.  
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2. Examine the structure of biofilms after set periods of time. These images can be used to compare the 3D structure of Nissle 1917 biofilm.
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<img src="http://farm7.static.flickr.com/6177/6166139079_a35d6a5930_m.jpg" width="230" height="180" /><p><font size="1" color="grey">Picture 1: SEM image of Nissle biofilm </br>showing the extracellular matrix</font></p>
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<img src="http://farm7.static.flickr.com/6155/6169966778_9ee504c001_m.jpg"/>
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<p><font size="1" color="grey"> Picture 2: 400x A gram-stained 16-hour </br><i>E.coli</i> Nissle biofilm </font></p>
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<img src="http://farm7.static.flickr.com/6161/6169966780_264a3b3147_m.jpg"/>
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<p><font size="1" color="grey"> Picture 3: 100x A gram stained 16-hour </br><i>E.coli</i> Nissle biofilm</font></p>
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<p>The images below may show why <i>E.coli</i> Nissle has the biofilm forming that <i>E.coli</i> lab strains lack. In Picture 4 the fimbriae of Nissle are clearly visible. Since the <i>E.coli</i> lab strain in Picture 5 does not have them, they may be the reason <i>E.coli</i> Nissle 1917 are so adept at clinging to each other.</p>
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<b>Method</b>
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<img src="http://farm7.static.flickr.com/6156/6166741226_e4cfd217bd_m.jpg" /><p><font size="1" color="grey">Picture 4: 10,000x SEM image of Nissle </br>showing the fimbriae</font></p>
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<p>As we were trying to specifically disperse areas of biofilm it was necessary to establish a base rate of dispersal of the biofilm without using any of the dispersal mechanisms we designed. This would allow us to show quantitativly the increase in rate of dispersal that our different dispersal biobrick could generate when compared to a biofilm of non-transformed bacteria.</p>
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<p>To measure the base rate of dispersal glass slides were put into 50ml tubes containing 20ml of LB broth. The LB covered around a third of the glass slide, this is the area where the biofilm would form. The LB was then inoculated with 20μl of overnight culture of <i>Pseudomonas aeruginosa</i>. These tubes were then left on a bench top shaker at room temperature for a set amount of time (time points ranged from 1hr to 48hrs). After the biofilm had grown its alloted time the glass slide was carefully removed and placed into a fresh 50ml tube with 25ml of LB (which completely covered the biofilm) and left to allow the bacteria to disperse for 1hr. The slide was then transferred to a fresh 50ml tube with 25ml of LB. The biofilm is scraped off the slide using a thin flexible spatula. At this point both the dispersed cell and the biofilm scrapings were sonicated to stop clumping and plated in serial dilutions.</p>
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<img src="https://static.igem.org/mediawiki/2011/c/c7/Biofilmgraph.jpg" />
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<p><font size="1" color="grey"> Image 2: 15,000x EM of E.coli for comparison. </br>No fimbriae or EPS is visible. (courtesy of Rocky Mountain Laboratories)</font></p>
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<p><b>Figure 1: Number of viable cells in biofilm compared to number of cells dispersed from the biofilm in one hour.</b> The base rate of dispersal remains roughly proportional to the number of cells in the biofilm. The information for the time point hour 14 for the biofilm was not available.
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<p> To make the images of biofilm formation, biofilms were formed on glass slides inserted into 50ml tubes. The tube was filled with 20ml of LB broth and inoculated with 20μl of over night culture of <i>Psuedomonas Aeruginosa</i>. The biofilms were left to form for the time indicated on the images.</p>
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<b>Results</b>
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The number of cells that dispersed from the biofilm seemed to be proportional to the number of cells in the biofilm with a ratio of roughly 5 dispersed:1 in biofilm. Figure 1 shows the number of dispersed cells when compared to the number of cells in the biofilm in both a graph and a table.
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<img src="https://static.igem.org/mediawiki/2011/f/f0/Biofilmtimeseries.jpg" width="100%" /></br>
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<b>Figure 2: <i>Pseudomonas aeruginosa</i> biofilm growth over time.</b> These photographs were taken after 1hr, 14hrs and 48hrs of biofilm growth. They were stained using a Grams stain method that is designed specifically to avoid sheer forces being applied to the delicate biofilm structure. Details of this method are included in the <a href="https://2011.igem.org/Team:Glasgow/Lab_Book"><i>Pseudomonas aeruginosa</i> biofilms</a> lab book.
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Revision as of 00:38, 22 September 2011

Biofilm formation was also confirmed by SEM pictures that showed the extracellular matrix, such as Picture 1 below.

Picture 1: SEM image of Nissle biofilm
showing the extracellular matrix

Picture 2: 400x A gram-stained 16-hour
E.coli Nissle biofilm

Picture 3: 100x A gram stained 16-hour
E.coli Nissle biofilm

The images below may show why E.coli Nissle has the biofilm forming that E.coli lab strains lack. In Picture 4 the fimbriae of Nissle are clearly visible. Since the E.coli lab strain in Picture 5 does not have them, they may be the reason E.coli Nissle 1917 are so adept at clinging to each other.

Picture 4: 10,000x SEM image of Nissle
showing the fimbriae

Image 2: 15,000x EM of E.coli for comparison.
No fimbriae or EPS is visible. (courtesy of Rocky Mountain Laboratories)

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