M465:Communityanalyses
Antagonistic and Mutualistic Interactions
*NOTE: You must remember to set up fresh nutrient broth cultures for your isolates 1-3 days before lab to do this test!
The microbial community living in your environments 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 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.
PREPARING THE ISOLATES:
You will inoculate 50 µl of log phase (young culture) isolate grown in fresh nutrient broth into the assigned well(s). Once again try to control for similar numbers of organisms in your inoculum by starting with normalized optical density measurements (start with a concentration of 0.5 OD600)
Interaction Assay Set Up
You will use 64 of the wells on a 96 well plate for this assay. Each pair will use 8 unique isolates to test for interactions. Use Excel to keep track of which organisms that will be inoculated into each well as described and illustrated below.
FOLLOW THE TEMPLATE CAREFULLY!!!!!! It is easy to get this inoculation messed up, but don't!
Transfer 50 μL of each of 8 unique isolates to be tested into the illustrated row of wells (A1 is Isolate 1, A2 is Isolate #2 etc through A8)
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).
Add 100 μL of nutrient broth to each of the wells containing your isolates (row wells A1-A8 and column wells B1-H1)
Gently move the 96 well plate in a circular motion to mix.
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!
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.
Again gently mix the contents of the well by moving the plate in gentle circles.
At the end of these transfers, measure the optical density in each well using the plate reader. Then put your 96-well plate in a plastic tupperware container at 37C to grow. Take measurements on the optical density daily to confirm growth inhibition or stimulation.
Antibiotic Resistance
Biofilm Assay
The crystal violet assay is great to quantify biofilm formation quickly in a high-throughput way. We will be growing our cultures in polystyrene dishes (12 well) on PVC coverslips.
"Biofilm disruption solution"
- 33% acetic acid
"Protocol"
- The day before, create an overnight culture of your isolates to be tested by inoculating a single colony into a tube of nutrient broth (or other appropriate medium).
- In the morning, measure tho OD of the culture and dilute to 0.05. Use this dilution to inoculate at least 3 wells in your polystyrene dish (add ~5 ml of media to each well). Include a medium only control.
- Incubate for 24-48 hours at 37°C, monitoring turbidity and growth daily.
- At the completion of incubation, aspirate the liquid from the wells using a pasteur pipette.
- Wash the wells once with deionized, distilled water.
- Stain the sample with crystal violet by adding 1-2 drops from your droppers into each well and waiting 2 minutes at room temperature.
- Wash the sample again with water twice.
- Fill each well with acetic acid
- Shake for 30 mins at room temp
- Measure OD600
