NOTE: Make sure you make patches of your isolates for Tuesday!
The microbial community living within the cheese is a complex one with many different microorganisms. As is true of any environment, these microbes interact with each other - both metabolically and chemically. Today, you will be taking your isolates and testing them against each other for growth inhibition or growth benefit. Some of these organisms may prevent the growth of others through the production of chemicals we'll discuss in lecture; others might promote the growth of their neighbors by secreting metabolites that are needed.
1. Create a 96-well template for your isolates. Using the excel template found here Media:template.xls make up the map for this 96-well plate - you should know where each of your 8 chosen isolates will be.
2a. For each of your isolates, using sterile technique, take 1/2 of the patch and disperse it into 2 ml sterile media in 10 ml tubes. In the end you'll have liquid stocks of each of your individual isolates.
2b. Take 50 µl of your isolate from the liquid culture and place it into the appropriate well (according to your template). Add another 100 µl of sterile media to the well to dilute the culture
3. When you are finished loading the top and side wells, use the p 20 micropipette to move 10 µl from wells A2 - A8 into wells B2 - B8. Repeat this until H2 - H8 has 10 µl in it also.
4. Now we will add the organisms from wells B1 - H1 to the rest of the plate. Again, using the p 20, take 10 µl from wells B1 - H1 and mix into the wells B2 - H2. Repeat this process until you have mixed all of the possible combinations of your isolates on this plate.
5. Each of your wells now has isolates growing by themselves and isolates mixed together. We will inoculate from this plate onto a square agar tray in order to observe what these organisms look like when grown together. For this we will use the multichannel micropipette. Using the multichannel, take 5 µl of your inocula and deposit a drop on top of the square agar tray. Repeat this until you have completed the full array.
6. Wait for your spots to dry before placing your trays upside down.
You should come in to the lab a few times this week to check on your assay and note any interesting results