Bees experiments and protocols

Oxalic Acid Gradient:

1. In 15 mL falcon tubes, 5 mL of sulfuric acid, 2 mL of Potassium Permanganate, and x amount of oxalic acid.
2. The solutions would then sit for 10 minutes, being inverted every few minutes.
3. The optical density with a wavelength of 528 nm was taken for each falcon tube. The results were recorded.

Mite Trials:

1. Pouches were made using parafilm. This was done by stretching out the parafilm very thinly with the experimenter’s hands.
2. Using a pen cap, the parafilm was arranged so that there was a small pouch at the bottom for the mite and solution to fit in.
3. The pen was removed and the top of the parafilm was left open.
4. A Varroa mite was then placed into the pouch along with a solution such as water.
5. They were left overnight in the 27 ℃ incubator.

Bee Trials:

1. Bees were obtained from a bee hive.
2. The bees were then fed RFP producing E.coli with glucose and sucrose sugars. The bees were then observed to determine if they were drinking.
3. Mites were added to Petri dishes following a sugar shake. Paper derivatives were soaked in different treatments , water and RFP E.coli and were in the bottom of the Petri dishes.
4. The Petri dishes were then placed into the incubator for 24 hours at 27 ℃.
5. The bees were euthanized using EtOH, surface sterilized in bleach, opened using a razor to expose the gut, and placed into the shaker overnight at 37℃.

Biofilms experiments and protocols

Growing biofilms

In the lab for biofilms, our first focus was on trying to see if our team could grow biofilms. We successfully did this using the following protocol:

1. In a 50 mL falcon tube, 21 mL of LB media, 210 µL of RFP, and 21 μL of KAN were added. This solution was then mixed more thoroughly by putting the cap on the falcon tube and inverting it a few times.
2. 3 mL of this solution was pipetted into seven 35x10 mm Petri dishes.
3. The Petri dishes were then labeled and put into an incubator for 24 hours at 37℃.
4. After 24 hours, the solution was poured out of each dish into a bleach container. The dishes were then rinsed with distilled water twice.
5. ___ drops of crystal violet were placed around the rim of the Petri dishes. If a biofilm had formed, a ring of crystal violet would be seen.

Testing with home cleaners:

Once we knew that we could grow biofilms, we decided to test out the effectiveness of home cleaning products. The products we tested are Mr. Clean, Fantastik, Truly, Windex, Tilex, Bleach, Lysol, and GreenWorks. The protocol that we used for this is as follows:

1. Using biofilms made the previous day with protocol 1, the experimenter removed the contents in the dish in the bleach container. Then the experimenter rinsed the Petri dish with distilled water twice. The Petri dishes were laid out on paper towel to let the excess water drip out.
2. After the Petri dishes were thoroughly dry, 3.2 mL of one of the listed cleaning products was pipetted into a Petri dish. The cleaning product was left in the Petri dish for 30 minutes (If bleach was being used, only for 15 minutes).
3. The cleaning product was then disposed of in the bleach container. The Petri dish was then rinsed with water once.
4. To see if a biofilm was still remaining, 100 μL of LB media was pipetted onto the inside edge of the Petri dish.
5. The LB media was then pipetted back into the LB media tube along with 5 μL of KAN.
6. This was then put into the shaker for 24 hours at 37 ℃.

Testing Antibiotics:

We also tested antibiotics to see if they could get rid of biofilms as well. We used this protocol:

1. Biofilms made using protocol 1 were rinsed twice using distilled water.
2. In twelve 2 mL tubes, 1.65 mL of double distilled water was pipetted into each of them. Into two 2 mL tubes, 3.3 μL of AMP was pipetted into them.
3. Step 2 was repeated using CAM, TET, and water as a control.
4. Two of the 2 mL tubes for each of the antibiotic and control 2 mL tubes were each placed into a 35x10 mm Petri dish. This was repeated with five other Petri dishes.
5. Three of them were rinsed after 15 minutes while the other three and the control were rinsed after 30 minutes.
6. These were then recultured by pipetting 100 μL of LB media into the rim of the 35x10 mm Petri dish. This was then pipetted back into the tube along with 5 μL of KAN.
7. The Petri dishes were then put into an incubator for 24 hours at 37 ℃.

Typhoon:

To get quantitative data, we used the Typhoon.

1. An area on the grid of the typhoon was chosen depending on how many 35x10 mm Petri dishes we used.
2. Fluorescence scanning was chosen; the setting for that was 580 BP, AlexaFlour546, PMT: 600, and with a green laser.
3. The pixel size used was 100 microns.
4. The focal plane used was +3 mm so that it would scan through the plastic bottom of the Petri dish.