BISC209/S11: Assignment 209 BIOLOG
Assignment: Community Physiological Profiling of a Soil Community by Carbon Source Utilization
What to do with the DATA you have collected (A590nm) from your BIOLOG™ plate?
Your group should have about a week and a half of daily measurements recorded on the Excel workbook we provided as a template. to organize the recording and analyzing of the carbon source utilization data. This template is pre-formatted for the calculations you will do from these data. It includes the formulas to average replicate measurements each day and it will automatically subtract the background (readings in the water wells). There is a normalization for background that will be subtracted automatically (this threshold absorbance is provided by the manufacturer and was determined to be 0.25 absorbance units for each carbon source). These calculations are the first step in figuring out what you can learn from these data that provides evidence for one or more of our investigative goals. You have been asked to calculate Average Metabolic Response (AMR) and Community Metabolic Diversity (CMD). Does AMR or CMD provide evidence for abundance and diversity in your soil community? How so? Clearly these numbers will be more meaningful in relationship to other soil communities or when compared to some standard or reference point, but for the time being (for this assignment) you are only asked to calculate values for your soil community. When you write your final paper, you will need to make comparisons to make this evidence meaningful. We ask you to calculate AMR and CMD, but these parameters are certainly not the only way to use these data for the information you seek. Keep in mind that neither AMR or CMD sheds light on carbon source utilization patterns in your community. Is that something that would be useful to look at? How can you use the carbon utilization pattern you obtained to provide evidence that microorganisms in a community both compete and co-operate to fulfill metabolic needs of the community? Please think about additional or alternate ways to use your data to provide evidence for any of the questions we are exploring in this investigation. You should also keep in mind the limitations of this test in providing the answers you seek and make sure that when you use this evidence that you discuss (briefly) those limitations WITHOUT completely undermining all your reader's confidence that the test can shed light on the issues you address.
Calculating Average Metabolic Response (AMR) and Community Metabolic Diversity (CMD)
CALCULATING AMR: Average Metabolic Response over time and between soil sites and Average Carbon Source Utilization Ccore by the microbial community
Average Metabolic Response(AMR)= Σ(mean A590nm of triplicate carbon source tests - Mean A590nm of the three control wells) and less a threshold absorbance of 0.25nm / 31 (the number of carbon sources tested)
The calculation of AMR provides a method to compare total metabolic activity by a soil community among aerobic or facultative microbes. The AMR is calculated as the average difference between A590nm values of the three replicates of each C source and the 3 water control wells. The following calculations were built into the Excel template workbook: Take the average of each of the 3 replicate wells and subtract the average of the control wells from each of the carbon sources. A background absorbance threshold absorbance of 0.25 is substracted also. If you double click on the cells, you can check to see that the formulas and calculations are correct. The final calculation that is already built in is a division by 31 (the number of carbon source wells).
A high score indicates that there were many useable carbon sources for the community, a low score indicates that few of the carbon sources were able to be metabolized well. Calculate AMR for every date read. On the last page of the template workbook, please fill in the AMR table by day and overall average absorbance. Graph these data. Collect this AMR table data for the other 2 soil sample sites that you lab section collected and graph the 3 sites' average AMR over time as decribed in the next section. How do the three soil communities compare?
CALCULATING CMD: community metabolic diversity. CMD is a simple way to represent the total number of substrates able to be effectively metabolized by the microbial community. It's a measure of diversity in use of carbon sources. CMD is calculated by summing the number of positive responses (wells with a positive A595nm value after all the corrections) at each incubation time. Any negative values are considered 0 absorbance.
On your worksheet enter either a 0 or a 1. Zero indicates that there was a negative value or 0 for corrected A590nm. A 1 indicates a postive value of greater than zero. Once you have entered a 1 or a 0 as the "corrected" absorbance for all the cells, the template will calculate the CMD for that day. Complete the final CMD table on the last page of the template workbook and then collect these data for the other soil sampling sites for your lab section. Graph the average CMD over time as described below. How do the three soil communities compare?
GRAPHING THE DATA:
Both AMR and CMD should be calculated for each measurement date.
Once your team is finished collecting the data, make figures for AMR, CMD, and Carbon Utilization patterns.
To graph AMR: You will plot your soil sample's AMR values on the y axis against time on the x to provide a quick comparison of metabolic kinetics.
To graph CMD: Plot the calculated soil sample's CMD values on the y axis versus time on the x to get a sense of community functional metabolic richness.
Carbon source utilization pattern:
Neither AMR nor CMD analysis provides information about the pattern of carbon substrates used in each soil community.
To examine the pattern of carbon sources used across communities, you could plot the average A590nmabsorbance on the final day of data collection on the y axis and the 31 different carbon sources on the x axis in one figure that compares all 3 work-sites.
Can you think of another way to compare the patterns of substrates?
