Antibiotics are molecules that kill bacteria or inhibit their growth. Common antibiotics target important cellular functions in bacteria (such as cell wall remodeling or protein synthesis). Believe it or not, many microbes are naturally resistant to antibiotics, either because they produce them or because that resistance is advantageous in the habitat they occupy. One place in which such selection is currently taking place, is in the human microbiome. Unfortunately, due to the overuse of antibiotics (both in medicine and agriculture), widespread antibiotic resistance has become the norm. The figure below captures the trend and shows the increasing prevalence of antibiotic resistance in human pathogens.
Today you will be testing whether or not your Drosophila isolates are resistant to antibiotics.
You will be using a soft agar overlay technique to test the degree of resistance in your bacterial isolates. First, you will take cellulose disks, impregnated with antibiotics, and put it on your agar plate. You will then take 2 mL of molten agar (45C water bath) and add 100 uL of an overnight culture of your isolate to that 2mL *very quickly* . Pour your soft agar over the surface of your agar plate, nutating the plate in order to spread the soft agar evenly. Don't take too long or the agar will solidify and you will not have time to pour it!
Lab Activity #1: Antibiotic Sensitivity
Set up an overnight culture the day before! For each isolate, perform the steps below. .
1. Grab a set of tubes with cellulose disks impregnated with antibiotic. You will need one for each antibiotic we are testing.
2. Label an agar plate of the appropriate medium for your isolate with your name, the date, and the isolate number. You will also want to label the plates into sections so that you can put one antibiotic disk in each section.
3. Using sterile tweezers, grab a single disk from the tube and place it on agar plate, in the appropriate quadrant. Gently tap disk into agar with sterile tweezers. Repeat procedure for other antibiotic disks making sure to spread the disks evenly around the agar surface.
The steps below must be performed quickly, and next to the water bath!!
4. Grab a tube of melted agar from the 45C bath.
5. Add 100 ul of your overnight culture of your isolate to the melted agar.
6. Vortex briefly to mix the bacteria into the soft agar and immediately pour onto the appropriate agar plate.
7. Tilt your agar plate back and forth to distribute the agar evenly over the surface.
8. After the agar hardens (about 10 minutes), invert the plate and incubate overnight at 30C.
9. Results should be visible in the next few days. Come in to check on your experiment and if you see growth in the agar lawn, take your plate out of the incubator and measure the zone of inhibition around the antibiotic disk.
Lab Activity #2: Antibiotic Production
Day 1 (Monday):
1. Using a sterile swab, dip the swab into your overnight culture and make an inoculation (as shown below) down the middle of a plate of appropriate agar media.
2. Repeat step 1. for each isolate to be tested. Label them carefully and incubate the plates at 30C until Wednesday.