While the amplification of bacterial 16srRNA genes goes on in the thermal cycler, we have about an hour to work on the community profiling analyses that we started last week.
In Lab3 you started some quantitative assessments of your soil communities' ability to digest cellulose, starch, and to solubilize phosphates---all important functional roles. Today while the thermal cycler completes our polymerase chain reactions, you will have about an hour to complete the colony colony counts from the differential media that you inoculated with dilute soil extract last week. We will also talk about how your carbon source utilization profiling is progressing.
Community Soil Physiological Profiling: EXOENYMES PROTOCOL con't:
Examine the plates for processing of a particular nutrient (starch, cellulose or insoluble phosphates) in each 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 Culture-Dependent differential media, in that they allow you to visibly SEE the difference in particular groups of microbes---in our cases, 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.
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.
PART E: Isolation of Azotobacter, Hyphomicrobia, Spore Forming, or other interesting Bacteria
Continue to attempt to isolate to pure culture desired groups of bacteria. 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 3 pure cultures of DIFFERENT genera of bacteria from as many groups as possible.
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. Remember, if you successfully isolated hyphomicrobia your colony should not grow when streaked on nutrient agar. The other cultures may grow as well or better since the nutrient agar we use is rich in nutrients. 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.
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.
Write an Introduction section of final paper. Full directions and useful references can be found at Lab 4 Assignment: Assignment: Introduction
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.