LAB 4: Community Level Testing for Richness as Functional Metabolic Diversity & for Community Co-operative Behavior
Community Level EXOENYMES PREVALENCE con't:
In Lab 3 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. Why would it be advantageous for a soil microbial community to include members that secrete enzymes that process starch, cellulose or phosphorus into other forms of nutrients? Would it be advantageous for all of the community members to possess all of these abilities? Why or why not? What might be the cost? Would the microbial community be different if NONE of its members secreted these exoenzymes? If microbial community membership is always in flux, trying to maintain a balance where its members can all survive and thrive, do you think the prevalence of these exoenzyme producers is constant or variable? If variable, what are the conditions that might have an effect on prevalence?
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. Your data should provide evidence of co-operative behavior among members of this soil community.
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 CFU. Use the soil extract dilution of the plates counted to calculate CFU/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 prevalence (in %) of exoezyme secreting microbes out of the total in the culturable community 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. Be sure to show all your data and calculations in your notebook.
There should be time in lab today for you to brain storm with your partners about how you will use these results in a figure/table and in the results narrative to help the hypothetic reader of your research report on this investigation to understand what your data mean in terms of our experimental questions.
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 NA 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.
Microscopic examination of isolates using a simple stain
Analysis of Carbon Source Utilization Data
You have been asked to calculate 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 measurement of diversity in use of carbon sources. Clearly a quantitative evaluation of CMD is more meaningful in relationship to other soil communities or when compared to some standard or reference point. If you are only analyzing your own soil community and have no other communities to compare, the reference comparison that you should use to make this quantitative evidence meaningful is to calculate a percentage of substrates used by the community (CMD) out of the total number possible (the number of substrates on the plate).
Calculating Community Metabolic diversity (CMD) & Percent of Carbon Sources Utilized
Your group should have a week of daily measurements recorded on the Excel workbook we provided as a template. 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.
CMD is calculated by summing the number of positive responses (wells with a positive A595nm value after all the corrections) at each reading day. On your worksheet enter for each carbon source either a 0 or a 1. One indicates a positive value for absorbance at A590nm after correction for background. (Remember that this correction is built into the formulas embedded in the template spreadsheet.) Zero should be entered for carbon sources that have either a negative value or a zero absorbance after built-in correction. Zero means that the community was unable to use that particular carbon source for its metabolic needs. Once you have entered a 1 or a 0 for each substrate, the template will sum the positive values. This number is your CMD for that day. It indicates the number of substrates usable by the community. Enter your daily CMD values into indicated column on the final page (CMD graph) of your worksheet template. A graph should be automatically generated showing average CMD on the y axis versus time (day) post-inoculation. Use the peak CMD value to calculate % of carbon sources utilized [number utilized divided by the total number available(31) x 100].
What to do with your data:
More information about how to analyze these data and on how to use your data as evidence for conclusions to our experimental questions can be found in the description of the Assignment for this week at BISC209/S13: Assignment_209_BIOLOG. You should have time in lab today to work with the other members of your group to analyze these data and think of ways to create effective tables/figures with appropriate legends.
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.
Analyze the prevalence of microbial starch & cellulose digesters and phosphate solubilizers in your soil community. Analyze your soil sample's community data for carbon source utilization; turn in spreadsheet with calculations and make graphs turned into figures/tables with legends.
Consult the full directions for this assignment found at: BISC209/S13: Assignment_209_BIOLOG.
Do before next lab:
Continue monitoring and following the appropriate protocols to isolate our targeted bacteria to pure culture. If you need to subculture several times this week to achieve a pure culture, do so! You must have pure cultures next week. Please make a fresh plate and a broth culture of each of your isolates (MUST BE PURE CULTURE!) before your lab by adding a single well-isolated colony from a plate with only one type of bacteria growing on it and incubate your broths at RT. Please be sure to subculture fast growing cultures no more than 24 hours prior to lab 5 and slower growing cultures, ~48 hours before lab so that we will have young cultures to work with. Use your judgement or ask your instructor for advice on how far in advance to subculture extremely slow growing isolates of groups such as Actinomycetes. Next week we will start our tests that characterize our isolates and we will also perform 16S rRNA gene amplification by PCR. If our amplifications are successful, we will send cleaned-up pcr products to a DNA sequencing facility for sequencing of the 16S rRNA gene of each of your isolates. When the sequences come back we will use a public database to compare these DNA sequences to others submitted to this bacterial database and, we hope, to identify a few members of the bacteria in your soil community.