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Microscopy | Microscopy | ||
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Chemotaxis on Agar | Chemotaxis on Agar | ||
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+ | Conclusions | ||
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− | <p>From initial iterations of our community modelling, it became apparent that quantitative data on the growth rates of the bacteria were required in order to inform the model. For this, we observed changes in absorbance at 600 nm over 72 hours of the three nitrogen-fixing bacteria and | + | <p>From initial iterations of our community modelling, it became apparent that quantitative data on the growth rates of the bacteria were required in order to inform the model. For this, we observed changes in absorbance at 600 nm over 72 hours of the three nitrogen-fixing bacteria and E. coli in liquid culture at 30 °C using a ThermoFisher Scientific Varioskan LUX Microplate Reader.</p> |
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+ | <p>The data showed that A. brasilense grew at a slow, steady rate before sharply dying off after approximately 60 hours. The slow growth rate is likely to be because its optimal growth temperature is 37 °C rather than 30 °C. H. seropedicae and A. caulinodens showed very similar growth curves when grown at 30 °C: initial growth rate was very fast and then growth became very slow or static after 20 hours. E. coli grew at a medium pace to begin with and steadily slowed down with time. </p> | ||
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<section id='NaringeninMIC' class="s-services"> | <section id='NaringeninMIC' class="s-services"> | ||
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<p> An alternative method of observing chemotactic responses is through the use of microscopy. Brightfield microscopy allows direct observations of bacterial responses. This will allow comparisons of motility and morphology from our experimental data to that of the published literature that was used to underpin our first iteration of the community model [link to modelling]. Using microscopy enables the development of a cell density:optical density index (CD:OD index), a method of converting the two values. This index was also used in the community model to adapt the growth curve data collected during bacterial characterisation in standard laboratory conditions.</p> | <p> An alternative method of observing chemotactic responses is through the use of microscopy. Brightfield microscopy allows direct observations of bacterial responses. This will allow comparisons of motility and morphology from our experimental data to that of the published literature that was used to underpin our first iteration of the community model [link to modelling]. Using microscopy enables the development of a cell density:optical density index (CD:OD index), a method of converting the two values. This index was also used in the community model to adapt the growth curve data collected during bacterial characterisation in standard laboratory conditions.</p> | ||
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<p> </p> | <p> </p> | ||
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<p>The response index, developed by Pham and Parkinson [10], accounts for a ratio between the edge of the colony nearest the chemoattractant source and the edge furthest from the same source. This ratio is then used to determine if there has been positive chemotaxis (RI >0.52), no effect (RI = 0.48-0.52) or negative chemotaxis (RI <0.48).</p> | <p>The response index, developed by Pham and Parkinson [10], accounts for a ratio between the edge of the colony nearest the chemoattractant source and the edge furthest from the same source. This ratio is then used to determine if there has been positive chemotaxis (RI >0.52), no effect (RI = 0.48-0.52) or negative chemotaxis (RI <0.48).</p> | ||
<p>Results (Table 6) indicated that both <i>A. brasilense</i> (Figure 7a and 7b) and <i>H. seropedicae</i> (Figure 7c and 7d) experienced positive chemotaxis towards 50 μM between distances of 5-25 mm and 5-10 mm respectively. As such, further investigation utilised the distance that corresponded with the greatest RI value (15mm and 10mm respectively). For <i>H. seropedicae</i>, the colonies nearer the centre line again showed more constricted halos which may indicate that the naringenin concentration may still be too high. The response index of the control for all species at 5 mm was <0.48, suggesting chemorepulsion. This was anticipated as the control contains ethanol which possesses known antimicrobial properties and is commonly used to disinfect lab equipment.</p> | <p>Results (Table 6) indicated that both <i>A. brasilense</i> (Figure 7a and 7b) and <i>H. seropedicae</i> (Figure 7c and 7d) experienced positive chemotaxis towards 50 μM between distances of 5-25 mm and 5-10 mm respectively. As such, further investigation utilised the distance that corresponded with the greatest RI value (15mm and 10mm respectively). For <i>H. seropedicae</i>, the colonies nearer the centre line again showed more constricted halos which may indicate that the naringenin concentration may still be too high. The response index of the control for all species at 5 mm was <0.48, suggesting chemorepulsion. This was anticipated as the control contains ethanol which possesses known antimicrobial properties and is commonly used to disinfect lab equipment.</p> | ||
− | <font size="2">Table 6: Average Response Index and standard error of <i>A. caulinodans</i>, <i>A. brasilense</i>, <i>H. seropedicae</i> and <i>E. coli</i> colonies grown on 0.25% Minimal A Salt agar containing a gradient of either 100µM naringenin or 1.5% ethanol (control). RI = D1/(D1+D2) in which D1 represents distance between colony edge nearest chemical source to site of inoculation whilst D2 represents distance between colony edge furthest from chemical source to site of innoculation [10]. Bacteria were innoculated 15mm or 10mm from naringenin source and incubated at 30</font> | + | <font size="2">Table 6: Average Response Index and standard error of <i>A. caulinodans</i>, <i>A. brasilense</i>, <i>H. seropedicae</i> and <i>E. coli</i> colonies grown on 0.25% Minimal A Salt agar containing a gradient of either 100µM naringenin or 1.5% ethanol (control). RI = D1/(D1+D2) in which D1 represents distance between colony edge nearest chemical source to site of inoculation whilst D2 represents distance between colony edge furthest from chemical source to site of innoculation [10]. Bacteria were innoculated 15mm (<i>A. brasilense</i> and <i>E. coli</i>) or 10mm (<i>A. caulinodans</i> and (<i>H. seropedicae</i>) from naringenin source and incubated at 30</font> |
<table id="protocols"> | <table id="protocols"> | ||
<thead> | <thead> | ||
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<th>Naringenin Response Index</th> | <th>Naringenin Response Index</th> | ||
<th>Control Response Index</th> | <th>Control Response Index</th> | ||
+ | <th>Chemotactic Response</th> | ||
</tr> | </tr> | ||
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<tr> | <tr> | ||
<td><i>A. caulinodans</i> (ORS571)</td> | <td><i>A. caulinodans</i> (ORS571)</td> | ||
− | <td> | + | <td>0.478</td> |
− | <td> | + | <td>0.473</td> |
+ | <td>Negative</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><i>A. brasilense</i> (SP245)</td> | <td><i>A. brasilense</i> (SP245)</td> | ||
− | <td> | + | <td>0.552</td> |
− | <td> | + | <td>0.515</td> |
+ | <td>Positive</td> | ||
</tr> | </tr> | ||
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<tr> | <tr> | ||
<td><i>E. coli</i> (Z67)</td> | <td><i>E. coli</i> (Z67)</td> | ||
− | <td> | + | <td>0.472</td> |
− | <td> | + | <td>0.493</td> |
+ | <td>Negative</td> | ||
</tr> | </tr> | ||
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</table> | </table> | ||
<p> </p> | <p> </p> | ||
− | <p>The response index for <i>E. coli</i> and <i>A. caulinodans</i> indicated | + | <p>The response index for <i>E. coli</i> and <i>A. caulinodans</i> indicated negative chemotaxis in response to naringenin. This may be due to the fact that the naringenin wass dissolved in 1.5% ethanol which is commonly used to to sterilise due to ethanol's antimicrobial properties. As the other two species of nitrogen-fixers were demonstrated to show chemoattraction and both of which were motile, unlike <i>A. caulinodans</i>, it may be possible that the loss of motility combined with the antimicrobial properties of ethanol are triggering this result. This would mean the experimental set-up was not appropriate and thus requires further work. This will be work for the future.</p> |
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</section> | </section> |
Revision as of 15:01, 16 October 2018