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BISC209/F13: Lab7: Difference between revisions
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→Antagonistic and Mutualistic Interactions Analysis
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<font color = "red"> *NOTE: You must remember to set up fresh nutrient broth cultures for your isolates 1-3 days before lab to do this test! </font color = "red"> <br> | <font color = "red"> *NOTE: You must remember to set up fresh nutrient broth cultures for your isolates 1-3 days before lab to do this test! </font color = "red"> <br> | ||
The microbial community living in soil is a complex one with many different microorganisms. As is true of any environment, these microbes interact with each other - both functionally and physically. Do selected bacteria from your community help each other or harm each other while trying to find a niche in your soil community? Today, you will try to answer that question by testing your cultured isolates for examples of mutualism or antagonism (co-operation or competition)by culturing them in controlled communities. Some of these bacteria may prevent the growth of others through the production of chemical inhibitors; others might promote the growth of their neighbors by producing metabolites that are needed. We are going to look for both positive and negative interactions.<br> | The microbial community living in soil is a complex one with many different microorganisms. As is true of any environment, these microbes interact with each other - both functionally and physically. Do selected bacteria from your community help each other or harm each other while trying to find a niche in your soil community? Today, you will try to answer that question by testing your cultured isolates for examples of mutualism or antagonism (co-operation or competition) by culturing them in controlled communities. Some of these bacteria may prevent the growth of others through the production of chemical inhibitors; others might promote the growth of their neighbors by producing metabolites that are needed. We are going to look for both positive and negative interactions.<br> | ||
'''PREPARING THE ISOLATES''':<BR> | '''PREPARING THE ISOLATES''':<BR> | ||
<font size="+1">Interaction Assay Set Up</font size="+1"> | <font size="+1">Interaction Assay Set Up</font size="+1"> | ||
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[[Image:Interactions_slide3.jpg]]<BR> | [[Image:Interactions_slide3.jpg]]<BR> | ||
1. You will inoculate 50 µl of each of the 8 unique isolates to be tested into the illustrated row of wells. Use the concentration adjusted culture tube you made to match the turbidity of the McFarland 0.5 standard since you are trying to control for similar numbers of organisms in your inoculum. (A1 is Isolate 1, A2 is Isolate #2 etc through A8) | |||
[[Image:Interactions_slide4.jpg]] <BR> | [[Image:Interactions_slide4.jpg]] <BR> | ||
Beginning with Isolate #2, inoculate a second 50 μl of each of your isolates into the column wells B1, C1, etc. (indicated by the green color). | 2. Beginning with Isolate #2, inoculate a second 50 μl of each of your isolates into the column wells B1, C1, etc. (indicated by the green color). | ||
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Add 100 μL of nutrient broth to each of the wells containing your isolates (row wells A1-A8 and column wells B1-H1) | 3. Add 100 μL of nutrient broth to each of the wells containing your isolates (row wells A1-A8 and column wells B1-H1) | ||
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Gently move the 96 well plate in a circular motion to mix.<BR> | 4. Gently move the 96 well plate in a circular motion to mix.<BR> | ||
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[[Image:Interactions_slide5.jpg]] <BR> | [[Image:Interactions_slide5.jpg]] <BR> | ||
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Transfer 10 μl of the contents of the 7 wells - A2 (containing isolate #2 etc) through A8 to the empty wells in each column as indicated by the yellow arrows. You will need to remove the first tip from a multichannel pipette. If you are using the multichannel pipette, be sure that you work slowly and check that each pipette tip is evenly filled. You may need to tighten the tips by hand, if so be sure to only touch the part of the tip that sits on the multichannel pipette, you wouldn't want to contaminate your wells with human organsisms!<BR> | 5. Transfer 10 μl of the contents of the 7 wells - A2 (containing isolate #2 etc) through A8 to the empty wells in each column as indicated by the yellow arrows. You will need to remove the first tip from a multichannel pipette. If you are using the multichannel pipette, be sure that you work slowly and check that each pipette tip is evenly filled. You may need to tighten the tips by hand, if so be sure to only touch the part of the tip that sits on the multichannel pipette, you wouldn't want to contaminate your wells with human organsisms!<BR> | ||
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Transfer 10 μl of the contents of wells A1 (containing isolate #1 etc) through H1 to each well in the row as indicated by the red arrows. <BR> | 6. Transfer 10 μl of the contents of wells A1 (containing isolate #1 etc) through H1 to each well in the row as indicated by the red arrows. <BR> | ||
Again gently mix the contents of the well by moving the plate in gentle circles.<BR> | 7. Again gently mix the contents of the well by moving the plate in gentle circles.<BR> | ||
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NEXT, we will inoculate a square (NUNC) tray containing nutrient agar medium with about 5 μl of the contents of the wells we just prepared. For this step we will use either a tool called a "frogger" or a multichannel micropipette. If using the frogger, dip the tips into 96 wells to attract a drop of inoculum onto the end of each steel tip and then touch the those tips to the surface of the sterile NA square NUNC plate. Do not break the surface of the agar but make sure your pressure is even so every steel tip has touched the agar surface and deposited the same inoculum. Be sure to disinfect the frogger by dipping it into a series of disinfectant and rinse solutions that you will find at the '''cleaning station''' prepared for you . <BR><BR> | 8. NEXT, we will inoculate a square (NUNC) tray containing nutrient agar medium with about 5 μl of the contents of the wells we just prepared. For this step we will use either a tool called a "frogger" or a multichannel micropipette. If using the frogger, dip the tips into 96 wells to attract a drop of inoculum onto the end of each steel tip and then touch the those tips to the surface of the sterile NA square NUNC plate. Do not break the surface of the agar but make sure your pressure is even so every steel tip has touched the agar surface and deposited the same inoculum. Be sure to disinfect the frogger by dipping it into a series of disinfectant and rinse solutions that you will find at the '''cleaning station''' prepared for you . <BR><BR> | ||
If the frogger is not available, use an 8 channel multichannel pipet set to 5µl and remove 5μL of culture from each well of your culture dish and deposit all of it onto an area of the NA square agar NUNC plate that is in the same location as in the 96 well culture dish. Again, be sure the tips are on tightly before loading the pipet. Repeat this procedure, with new tips, for each ROW of 8 wells until you have completed depositing the full array in the same orientation as the 48 wells.<br><BR> | 9. If the frogger is not available, use an 8 channel multichannel pipet set to 5µl and remove 5μL of culture from each well of your culture dish and deposit all of it onto an area of the NA square agar NUNC plate that is in the same location as in the 96 well culture dish. Again, be sure the tips are on tightly before loading the pipet. Repeat this procedure, with new tips, for each ROW of 8 wells until you have completed depositing the full array in the same orientation as the 48 wells.<br><BR> | ||
10. Wait for your inoculated spots to dry, seal or cover the NUNC square tray, and incubate at Room Temp. The 96 well plate was used only to mix the cultures so you can discard this in the appropriate manner. In 24 hours we will need to transfer a small sample from each colony for analysis by MaldiTof Spectroscopy. If so, your instructor will provide more information. You will check on your assay and note any differences in the appearance of the colony growth of each isolate, alone vs mixed next lab. <br> | |||
==Pictionary® Tournament== | ==Pictionary® Tournament== | ||
