M465:IndependentProject

The Effect of Environment on the Microbiota

For your independent project, you have chosen to fund a proposal focused on environmental variables that may alter or impact the Drosophila microbiota. Each team will focus on one of the three variables below:
1) Temperature (18C, 25C, 28C)
2) Glyphosate (0 ug/mL, 0.1 ug/mL, 0.5 ug/mL)
3) Antibiotics (0 ug/mL, 5 ug/mL, 50 ug/mL)

For each variable, you will have three conditions (outlined above in parentheses). You will begin with wild type flies lacking any Wolbachia infection.

Below is a rough outline of the next few weeks - things may be adjusted depending on how well the flies grow, etc.

Tuesday, October 25th: Set up flies

Today we'll be establishing our flies in each of our conditions. Below are protocols for each of the three variables above. Remember that each team will focus on one of these variables. Each student will have their own set of three experimental lines for this experiment.

Glyphosate

• Protocol to be performed as a group such that you standardize your food across the team.
  

1. Each student will collect three vials of food from your instructor. These vials have 5 mL of food in them each. In addition, you will need two small beakers.
2. Microwave the food vials for 10 seconds at a time until the food is liquified. Pour the food into the small beaker.
3. Using a plastic graduated pipette, take 5 mL of the food and place into one of the previously used glass vials. Label this vial CONTROL. Create four control vials
4. Calculate the amount of glyphosate needed for the remaining food. For example, if you have four people in your group, you will have liquified total of 60 mL of food, out of which you will have used 20 mL for the CONTROL vials. That would leave 40 mL. Remove 20 mL of food and place in a new beaker.
5. Create a food mixture with 0.1 ug/mL glyphosate and one with 0.5 ug/mL glyphosate. Stir well and dispense using the graduated pipettes.
6. Each student in the group should have one control, one 0.1 ug/mL vial and one 0.5 ug/mL vial.

Antibiotic treatment

• Protocol to be performed as a group such that you standardize your food across the team.
  

1. Each student will collect three vials of food from your instructor. These vials have 5 mL of food in them each. In addition, you will need two small beakers.
2. Microwave the food vials for 10 seconds at a time until the food is liquified. Pour the food into the small beaker.
3. Using a plastic graduated pipette, take 5 mL of the food and place into one of the previously used glass vials. Label this vial CONTROL. Create four control vials
4. Calculate the amount of antibiotic needed for the remaining food. For example, if you have four people in your group, you will have liquified total of 60 mL of food, out of which you will have used 20 mL for the CONTROL vials. That would leave 40 mL. Remove 20 mL of food and place in a new beaker.
5. You are provided with 10 mg/mL tetracycline stock. Create a food mixture with 5 ug/mL tetracycline and one with 50 ug/mL tetracycline. Stir well and dispense using the graduated pipettes.
6. Each student in the group should have one control, one 5 ug/mL vial and one 50 ug/mL vial.

Temperature 1. Each student in the group should collect three vials of food from your instructor.
2. Label each vial with the temperature: either 18C, 25C or 28C.

Collecting your flies

• This part of the protocol will be performed upstairs in the Newton Laboratory after your vials ready.
  

1. For each of your vials you will need 10 female flies and 5 male flies. Collect these on the CO2 pad and place them in your vials to wake.
2. After you have established your lines, place them in the Newton Lab incubator (for tetracycline and glyphosate treatments) or in the temperature incubators appropriate to the treatment (for the temperature experiment).
3. Make sure your vials are labeled properly with your initials, your team, and the condition being tested!

Thursday, October 27th: CFU analysis

Today you will perform your first analysis of colony forming units for your adult flies in each condition. Remember that we are going to be looking at the offspring of these flies for our experimental results. Looking at the adults now, especially in the control conditions, will help us establish a baseline for what type of microbiota we should expect from these flies.
Work with your flies will be performed using the fly laboratory in Jordan Hall.

Plating Fly Lysates

1. Obtain your fly vials from each of your three conditions. For each of these vials, you will plate one fly lysate. Ahead of time, label three 1.5 mL tubes with the condition and fill each tube with 500 ul of PBS
2. Collect three female flies from each of your vials using the CO2 pad and dissecting scope.
3. Place the three flies in the appropriate 1.5 mL tube with 500 ul of PBS.
4. Homogenize the flies in PBS using a sterile pestle.
5. For each condition, perform a dilution series. Label three tubes 1,2,3. Add 0.9 mL of PBS to each of these tubes.
6. Add 0.1 mL of your original fly lysate to tube 1. Cap the tube and mix by vortexing.
7. Add 0.1 mL of the liquid in tube 1 to the liquid in tube 2. Cap tube 2 and mix by vortexing. Tube 2 will be our 10^2 dilution.
8. Add 0.1 mL of the liquid in tube 2 to the liquid in tube 3. Cap tube 3 and mix by vortexing. Tube 3 will be our 10^3 dilution.
9. At this point you are ready to plate your dilutions. For each condition, label four solid agar plates (two of MRS and two of LB, you will plate on both) with your initials, team name, the experimental condition, and the dilution factor. "make sure you label the agar side!". You should have a total of 12 plates at this point.
10. Uncap your agar plate. Take 200 ul of your dilutions (tube 2) and spot it onto the center of the agar. Using sterile glass beads, spread the liquid as evenly as possible across the surface of the agar.
11. Replace the cap and allow your sample to sit, undisturbed, agar side down, for ~2 minutes.
12. Repeat the plating and spreading for dilution tube 3 and for each of the two media types.

You will incubate your fly lysates at 30C.
Remember to come back to the laboratory on Saturday to count colonies!