We stained inoculated biofilms and performed the Crystal Violet assay. Left in 37 C for 36 hours. We did 4 trials 100uL, 4 trials 200uL, and 4 trials 300uL. Biofilm formation was successful, with varying band thickness. We also replicated this with two 5mL LB petri dish inoculated with 20uL E.coli. We added 5mL of crystal violet each to the petri dishes to cover the whole plate.

We also performed an oxalic acid gradient with 0%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and 2.0% oxalic acid to give a final volume of 6mL. We added 20uL of E.coli to each 6mL tube. Left in 37 C.

We did a 1/100 dilution of E.coli into LB media, and left it in 37 C for (hopefully) 24 hours. We did 4 trials 100uL, 4 trials 200uL, and 4 trials 300uL.

Biofilm produced on 24h 96 well plate.

Testing to see if biofilm will produce on 12h 96 well plate (1 in 100 culture dilution).

And also doing 24h 96 well biofilm formation testing to see what detergents and chemical impacts have on the decrease in biofilm production.

*Biofilms placed at 8:00pm

Oxalic acid test run for 12 hours (0, 0.01, 0.05, 0.1, 0.15, 0.2, 0.4, 0.4, 0.5, 2.0%)

*Oxalic acid placed at 7:30pm

Tested the production of parafilm sacs for in vitro Varroa feeding. (using kimwipes and 2mL tubes as containment methods) .

Performed oxalic acid testing of E.coli growth in LB media at (0, 0.01, 0.05, 0.1, 0.15, 0.2, 0.4, 0.4, 0.5, 2.0%) concentrations. Placed at 6:50pm.

Produced more biofilms.

Had mites and created pouches including Water, LB, Supernatent, E.coli, E.coli RFP. Placed at room temperature and in chris’s drawer.

Took 48 and 24 hour biofilms and performed a bleach treatment on them (0.252%) for 30 minutes. .

Took oxalic acid readings after 23 hours of incubation

Readings are unrealiable as the cultures were left in the shaking incubator for over 23 hours. Still show the same general trend as past gradients.

Make oxalic acid samples again.

Make 24h biofilms.

Objective:Determine if we have successfully cultured mites in vitro using either paraffin pouches or bee pupae.

Table 2. Experimental design showing each of the treatments applied to the mites.

After 24 hours, all mites were found to be dead. Pupae showed some signs of black bacteria growth or browning on body. Will need to repeat or find another method of culturing mites in vitro.

Biofilm test performed on

Fantastic = Biofilm remained

Windex = Biofilm remained

Bleach = didn’t remain

Made overnight cultures for oxalic acid testing

13 hours left in shaker

Objective:Determine if the concentration of oxalic acid that kills bacteria will kill bees.

Procedure:Bees were obtained from the “Mighty Hive” near the penitentiary at 4:00 and placed into 5ml centrifuge tubes. The bees were transported to the University of Lethbridge and stored at room temperature until 8:00. Prior to experimentation, the tubes were supplemented with a sugar water solution to ensure that the bees would not starve. Following feeding, 2ml of each treatment were added to the 5ml tubes containing the bees and set horizontally along the bench. The abdomen of each bee was immersed in the treatment no matter what orientation the bee took. The bees were observed for 1 hour and subsequently euthanized in 70% EtOH.

Table 1. Observations of the bees following 60 minutes of exposure.

Discussion:The bees showed that they could survive well in concentrations of oxalic acid that were above the point where E.coli would die. Further tests must be done to determine the exact contact toxicity. Additionally, the contact toxicity to mites will need to be addressed.

Conclusion:The bees will be able to survive all of the oxalic acid that the E.coli can produce.

Bee: A More Specific Oxalic Acid Gradient

Objective: Determine a more specific concentration of oxalic acid that will prevent cell growth. Already we know that oxalic acid prevents cell growth between 0.05% and 0.2%. Now we want to determine a more specific concentration of oxalic acid that will prevent cell growth.

Cells were left in the shaker for 23 hours accidently. ODs were not taken because they had been left in the shaker too long.

Biofilms: Continued Growth of Biofilms.

Objective: Continue to determine what cleaners will destroy/degrade biofilms. Instead of using crystal violet dye, we are using E.coli DH5α cells with RFP (PSB4K5). 2 μL of KAN was added to 2 mL centrifuge tubes along with 1980 μL LB and 20 μL of culture.

300 μL of mix was pipetted into wells of 96 well plate. Biofilms left to grow 24 hours in 37˚C incubator.

Results: The biofilms did not grow on any of the plates as there was no red ring. Using fresh RFP cultures might fix this issue because the cultures we had used were prepared earlier in the month.

Objective: Due to the oxalic acid gradient from yesterday being left in the shaker too long, the gradient was repeated again.

Cells were left in the shaker for 15 hours and OD’ed the next morning.

Biofilms: Continued Growth of Biofilms

Objective: The biofilms made yesterday did not work, so fresh RFP cultures were prepared tonight for future biofilm testing. Colonies were picked from a streaked plate of RFP and placed in 5 mL of LB. 5 μL of KAN was added to each tube. Two picked colonies were prepared and left overnight in the 37˚C shaker.

Prepped bee pouches for in vitro mite/bee feeding.

Performed Bleach and Fantastic testing on RFP biofilms and did one with crystal violet to check for biofilm formation.

Made biofilms for July 3.

Can Bees Ingest Culture?

Objective: Determine if bees can ingest E.coli in liquid culture form with supplementation of sugar. Also, can mites survive on papers that have water and RFP liquid culture?

Procedure: Bees were obtained from the “Mighty Hive” near the penitentiary at 6:50 after a heinous struggle and lack of proper equipment. They were then fed RFP producing E.coli that had been supplemented with both glucose and sucrose sugars. The bees were then observed to determine if they were drinking the solution.

Mites were added to petri dishes following a sugar shake. The dishes had different paper derivatives soaked in treatment; H2O and RFP E. coli. They were then placed in the 27C iGEM incubator. They will be observed in the morning.

The bees were euthanized on EtOH, then surface sterilized in bleach, opened using a razor to expose the gut, and placed in LB to shake at 37C overnight. Problem: 50ul of kanamycin was added instead of 5ul. I suppose that’s what happens when you work a 13 hour day in the sun.

Results:

Table 1. Observations of the bees following 60 minutes of exposure.

Discussion: UPDATE TOMORROW

Conclusion: UPDATE TOMORROW

Objective: To continue growing biofilms and determine if certain cleaners will prevent further growth of the biofilms

Procedure: Same protocol used as June 30. Instead of using crystal violet dye, we are using E.coli DH5α cells with RFP (PSB4K5). 2 μL of KAN was added to 2 mL centrifuge tubes along with 1980 μL LB and 20 μL of culture.

300 μL of mix was pipetted into wells of 96 well plate. Biofilms left to grow 24 hours in 37˚C incubator.

Bees: Preparing Solutions for Oxalic Acid Gradient using Potassium Permanganate.

Objective: Solutions were prepared to be used for the oxalic acid assay that will see KMnO4 being oxidized by oxalic acid and therefore detect the presence of oxalic acid.

Procedure: To prepare the 1% and 20% Oxalic Acid concentrations, solid oxalic acid was weighed for a solution of 50 mL. 0.5 g of oxalic acid was weighed out and placed in a 50 mL falcon tube. 50 mL of d2H¬2O was added to the falcon tube to dissolve the oxalic acid and create the 1% Oxalic Acid concentration. Two falcon tubes of 1% Oxalic Acid was prepared.

10 g of oxalic acid was then weighed and dissolved in 50 mL of d2H¬2O in a 50 mL falcon tube for the 20% oxalic acid concentration. The 50 mL of 20% oxalic acid was then placed in the 60˚C oven to help dissolve the oxalic acid.

In addition 1 M H2SO4 was created using stock 5 M for the Wieden Lab. Both the 1 M H2SO4 and 1% oxalic acid were stored in the 4˚C fridge in E770.

Biofilms: Preparing More Biofilms and Reculturing the July 6 Biofilms

Objective: To continue growing biofilms for future testing and to determine if biofilms can be recultured from previous ones that had been degraded based on the July 6 biofilms.

Procedure: New Biofilms

New biofilms were created for July 7 testing. Same protocol used as June 30. Instead of using crystal violet dye, we are using E.coli DH5α cells with RFP (PSB4K5). 2 μL of KAN was added to 2 mL centrifuge tubes along with 1980 μL LB and 20 μL of culture.

300 μL of mix was pipetted into wells of 96 well plate. Biofilms left to grow 24 hours in 37˚C incubator.

Procedure: Degrading the July 6 Biofilms

The biofilms created yesterday successfully grew. To degrade the biofilms, bleach, Tilex and Truly cleaner solutions were used that had previously been created. The solutions of biofilms were disposed of in the biohazard waste and the microtiter plates placed in water. They were vigoursly shaken to dry and 300 μL of respective cleaning solution added to each well. Since three plates of biofilms were created, each cleaning solution received its on plate. 15 mins after the solutions was added, they were disposed of in the biohazard waste.

Procedure: Reculturing the July 6 Biofilms

To culture the biofilms from yesterday, 300 μL of LB media was added to one well of a microtiter plate. The media was then pipette up and down to mix with any remnants of the biofilms. The media was then pipetted back into the 5 mL tube of LB media. 5 μL of KAN was added to the 5 mL tube of LB media.

Process was repeated for the remaining two plates that used different cleaners.

Note: Before cultures were degraded using the different cleaners, Kieran had taken a sample of the biofilms and prepared a 5 mL tube of LB to culture the biofilm that would be ensured to grow.

Cultures were left to grow 24 hours in 37˚C incubator.