LAB 4: Community Level Testing for Richness as Functional Metabolic Diversity & for Community Co-operative Behavior
Community Level EXOENYMES PREVALENCE con't:
In Lab3 you started a quantitative assessment of the prevalence of microbial digesters of cellulose, starch, and for solubilizers of phosphates. Think about why we chose to test for your soil community's ability to process these particular nutrients. Consider the difference in soil microbial communities in different habitats. Should your microbial community have the same prevalence of starch, cellulose, or phosphate processors as other habitats, such as a real tropical habitat (rather than an artificial one) or compared to the New England cold temperate soil outside the greenhouse? What are the factors that might make the preval
Today you will complete the colony counts from the differential media that you inoculated with dilute soil extract last week. You and your group members will perform calculations that assess the prevalence of community members able to perform these valuable functions. We will also talk about how your carbon source utilization profiling is progressing.
Examine the plates for evidence of digestion or processing of a particular nutrient (starch, cellulose or insoluble phosphates) in each of the differential culture medium. Remember that these differential media are not selective (they aren't designed to inhibit the growth of any groups of soil microorganisms), but they are differential media, in that they allow you to visibly SEE the difference in particular groups of microbes---in our case, between those that produce and secrete a functional exoenzyme and those that don't. You will count the number of individual colonies showing a clear zone (halo) around the colony (using the plate with 30-300 total colonies) and compare those numbers with the number at the same soil dilution that grew on NA- a general purpose, non-differential medium. Why does a halo indicate digestion of starch, cellulose, or processing of insoluble phosphate into a soluble form?
1. Count the total number of colonies on the Nutrient Agar plate and assess total culturable CFUs. Use the soil extract dilution of the plates counted to calculate CFUs/gram of soil (wet weight) for each assessment medium. If you divide the number of colonies counted by the amount of inoculum plated times the dilution factor of that plate, you will obtain the number of cultivatable bacteria per gram of soil.
number CFU/dilution plated*dilution factor = number of CFU/gram
For example, if you counted 150 colonies on the 10-3 plate the calculation is:
150/(0.1ml plated*1X10-3dilution)= 150X104 which in scientific notation is written as 1.5X106 CFU/gram
2. Flood the starch plate with a thin layer of Grams iodine and count the number of colonies that show starch digestion activity as a clear zone or non-blue halo around the colony).
3. Count the number of colonies that show cellulose digestion activity as a clear zone or halo around the colony.
4. Count the number of colonies that show phosphate solubilizing activity as a clear zone or halo.
5. Calculate the % positive for the enzymatic activity for each assay (# positive colonies x dilution factor/total colony count x dilution factor [on nutrient agar] ) X 100. This correction for dilution factor allows you to compare the CFUs counted from different dilutions on plates. If you are able to use control (NA) and test plates from the same dilution (each has between 30-300 colonies), you can omit the dilution factor. This is the total number of CFUs/gram of wet soil of microorganisms able to perform the role of interest.
6. Add your data to the course spreadsheet on the instructor's computer. Be sure to click File Save after you enter your data.
Isolation of Interesting and Diverse Bacteria
Continue to isolate to pure culture interesting bacterial members of your soil community. Directions found in the Protocols section of the wiki at Cuture Media: General Purpose, Selective, Enrichment, Differential, & Assessment of Digestive Exo-Enzymes
Directions for Streaking for Isolation onto new solid media is found at Streaking for Isolation
Your goal is for each student to end up with ~4 pure cultures of genera of bacteria that are different from each other and different from those of your teamates.
Once you believe you have pure isolates, continue to subculture to fresh plates each week (isolation streak a colony onto a fresh plate), in subsequent labs you will make a bacterial smear and do a Gram stain and start other tests to explore the physical and metabolic characteristics of this isolate. Generally the medium used is the isolation medium, however, at some point you may want to test the ability of your isolates to grow on nutrient agar. If your organism grows well on nutrient agar, you can streak on this medium each week and stop using the original isolation medium. Ask you instructor if you are not sure what to do.
Analysis of Carbon Source Utilization Data
Calculating Community Metabolic diversity (CMD)
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.
GRAPHING THE DATA:
To graph community metabolic diversity(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 in carbon source utilization. What is important about this graph in providing evidence for our hypothesis that a soil microbial community must be able to utilize a wide variety of carbon sources to support and maintain its abundance?
Carbon source utilization pattern:
CMD analysis does not provide specific information about the pattern of carbon substrates used in your soil community.
To examine the pattern of use of carbon sources, you could plot on a column graph the average A590nm absorbance of the three replicates (with error bars) on the final day of data collection on the y axis and the 31 different carbon sources on the x axis in one figure. (Remember that there is no such thing as a negative value for Absorbance so count anything that is less than zero as zero. Why might you seem to have a negative value?). Should you arrange those carbon sources in the order they are on the BIOLOG plate or is there a better way to organize them on your graph to show your main message(s)? What kind of information do you need in the figure legend?
Brainstorm with your partners to see if you can you think of other ways to illustrate functional metabolic diversity in your soil community?
Work as a group to turn your graphs into effective figure(s) with appropriate legend information. Should the CD and carbon utilization pattern graphs be one composite figure? Why or why not? What information needs to be in the figure legend(s)? Remember that figures are independent of the results narrative; therefore, the legend must contain all the information that a reader (who is unfamiliar with the investigation and these tools) needs to be able to understand your main point AND what you measured and how it is a measurement of what you say it means in terms of answering your experimental question. It is not intuitive to such a reader that measuring color and turbidity in a spectrophotometer has anything to do with community metabolic diversity or carbon utilization unless you explain your how you indicator works and what is the same and different about each of the measurements. It is also not intuitive how you came up with one number for average CMD per day when there were 31 different carbon sources unless you explain from where that number came. You did three replicates of each measurement; should it be clear that you have plotted mean and, if so, is the variability around these means important enough to include? How might you do so?
1. All culture plates that you are finished with should be discarded in the big orange autoclave bag near the sink next to the instructor table. Ask your instructor whether or not to save stock cultures and plates with organisms that are provided.
2. Culture plates, stocks, etc. that you are not finished with should be labeled on a piece of your your team color tape. Place the labeled cultures in your lab section's designated area in the incubator, the walk-in cold room, or at room temp. in a labeled rack. If you have a stack of plates, wrap a piece of your team color tape around the whole stack.
3. Remove tape from all liquid cultures in glass tubes. Then place the glass tubes with caps in racks by the sink near the instructor's table. Do not discard the contents of the tubes.
4. Glass slides or disposable glass tubes can be discarded in the glass disposal box.
5. Make sure all contaminated, plastic, disposable, serologic pipets and used contaminated micropipet tips are in the small orange autoclave bag sitting in the plastic container on your bench.
6. If you used the microscope, clean the lenses of the microscope with lens paper, being very careful NOT to get oil residue on any of the objectives other than the oil immersion 100x objective. Move the lowest power objective into the locked viewing position, turn off the light source, wind the power cord, and cover the microscope with its dust cover before replacing the microscope in the cabinet.
7. If you used it, rinse your staining tray and leave it upside down on paper towels next to your sink.
8. Turn off the gas and remove the tube from the nozzle. Place your bunsen burner and tube in your large drawer.
9. Place all your equipment (loop, striker, sharpie, etc) including your microfuge rack, your micropipets and your micropipet tips in your small or large drawer.
10. Move your notebook and lab manual so that you can disinfect your bench thoroughly.
11. Take off your lab coat and store it in the blue cabinet with your microscope.
12. Wash your hands.
Full directions and useful references for this assignment can be found at Lab 4 Assignment: Assignment:
This assignment is due at the BEGINNING of Lab 5. Do not come late to lab because you are printing or otherwise completing this assignment and you may NOT work on it during lab. There is a 5% per day late penalty for work for this course and since you have a week or more to complete assignments, illness (unless it is lengthy and serious) does not excuse you from the late penalty.
Continue monitoring and following the appropriate protocols to isolate to pure culture our targeted bacteria.