M465:Fieldtrip and inoculation

Field Trip and Inoculation

In this first lab you will learn:

1. How to sample wild Drosophila
   
2. Aseptic technique: don't contaminate yourself or your cultures!
   
3. The basic equipment and procedures used in microbiological investigation
   
4. Using a lab notebook to record the progress of your experiments
   
5. Inoculating your media for future use
   

Drosophila flies, otherwise known as "fruit flies" or "vinegar flies" or "wine flies", are attracted to decomposing fruit. It turns out that a lot of this has to do with the compounds being produced by the microbes colonizing the fruit as it ferments. Today we will drive to a local winery where we will sweep for fruit flies and also sample the environment in which they live (using sterile swabs). We will take these samples back to the lab and you will inoculate two different kinds of media with the fly lysates: Luria Bertani (LB) agar and de Man, Rogosa, Sharpe (MRS) agar. Each of these medias are selective for specific microbes, all heterotrophic. The LB agar will select enteric microbes, such as *Enterobacter*, commonly associated with the fruit fly. The MRS agar will select for lactic acid microbes, such as *Lactobacillus* and *Enterococcus* species also associated with the fruit fly.

Trip to the winery

Please arrive on time in JH 022. We will drive to the winery together. If you are able to drive, let your instructor know ahead of time.

Plating the microbes from Drosophila

Activity 1: Dilutions Worksheets:
Before we can get started on our study of these microbes, we need to grow them on our defined media. One measure that microbiologists use when determining how many microbes are in an environment is called "Colony Forming Units" or CFUs. In order to calculate the total CFUs in your sample, you need to know how you have diluted the sample before plating. Why do we dilute the sample before plating?
We will provide a dilutions worksheet to you. Please complete it to the best of your ability. We will go over the answers on Thursday.

Activity 2: Identifying Your Flies:
You have collected a large number of flies, some of which are Drosophila and some of which are other genera. We will use a dissecting scope and a simple key to identify different flies and group them by similar morphologies.
After you have sorted your insects into similar groupings, take one of each group and place it in a 1.5 mL tube using a sterile tweezer.
Place that 1.5 mL tube on ice. Take the rest each group of the same morphology and place in a labeled, 1.5 mL tube (use your initials and the date to label the tubes). Give this to your instructor to place in the -80C freezer.

Activity 3: Inoculating Your Media with Microbiome Isolates:
You each have an ice bucket in front of you with your sample of flies (1 fly per 1.5 mL tube). Your job is to first homogenize the flies using a sterile pestle and 200 ul of sterile water (this will be your original fly lysate) and then create a dilution series in the same. You will then plate two out of the 4 dilutions.

1. Remove four sterile 1.5 mL tubes at your bench (in the plastic containers with lids).
2. Label these tubes 1, 2, 3, and 4.
3. Add 0.9 mL of ddH20 to each of these tubes "what micropipette will you use?".
4. Add 0.1 mL of your original fly lysate to tube 1. Cap the tube and mix by vortexing.
5. Add 0.1 mL of the liquid in tube 1 to the liquid in tube 2. Cap tube 2 and mix by vortexing.
6. 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.
7. Add 0.1 mL of the liquid in tube 3 to the liquid in tube 4. Cap tube 4 and mix by vortexing. Tube 4 will be our 10^4 dilution.
8. At this point you are ready to plate your dilutions. Label two solid agar plates (MRS or LB, you will plate on both) with your initials and the dilution factor. "make sure you label the agar side!".
9. Uncap your agar plate. Take 200 ul of your dilutions (tube 3) 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.
10. Replace the cap and allow your sample to sit, undisturbed, agar side down, for ~2 minutes.
11. Repeat the plating and spreading for dilution tube 4 and for each of the two media types.

At the end of this exercise you will have four total plates - two dilutions on both MRS and LB - for each fly sample. For example, if you identified 3 different types of flies, you will have 3 x 4 = 12 total plates at the end of this exercise.

Activity 5: Preserving your Environmental Swabs:
You will have sampled four environments in the winery using a sterile swab. We will freeze the microbes associated with these swabs at -80C so that you can extract DNA from them at a later time point.

1. Label five 1.5 mL tubes with the same designations found on the 16 mm tubes containing your swabs. Make sure you write down in your lab notebook which sample came from which environment.
2. To each of these 16 mm tubes, add 1 mL of distilled, autoclaved water.
3. Vortex your sample containing the swab and 1 mL water for 1 minute. Remove and discard the swab.
4. Using a graduated pipette, remove the remaining liquid from the 16 mm tube and place it in the labeled 1.5 mL tube.
5. Place these tubes in your freezer box and give it to your instructor.

Activity 5: Clean Up:

- Bring all labeled, sorted, but unused fly samples to your instructor. These will be stored at -80C until Thursday.
- All pestles and lysates can be placed in the biohazardous waste bins by the hoods.
- Wrap your plates in colored tape. Mark with your initials and place all of your plates agar side up in the incubators at the back of the room (labeled M465).
- Empty your ice buckets in the sink.
- WASH YOUR HANDS