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.