Week 1

Sporulation salts were made and used to make sporulation plates. Gridded microscopy plates were prepared using a wax pencil. Dead Varroa mites were gathered from the Unifarm location at Grebbedijk, Wageningen. These beehives were not treated against mites and contained a high number of them. The mites were not completely fresh.

Week 2

It was not known how many colonies could be isolated from one mite; therefore, microcentrifuge tubes were prepared with 1, 2, or 3 mites, 2 replicates each. The protocol for Bacillus isolation was followed, except the sterilization step was with 1% halamid solution and lasted 10 seconds.

Initial plates showed no colonies; the experiment was repeated with a ~2.5% bleach washing step.

The second set of plates also showed no colonies. After the heat treatment, the LB + mites was incubated for 24 hours and streaked with an inoculation loop.

Again, no colonies: a mite was dissected and its gut rubbed on a plate. Due to the small size of the mite, it was difficult to isolate the gut. Additionally, the same protocol as before was followed, except only 0.1 mL of the LB was heat treated and the rest was plated without a heat treatment.

This week, the protocol failed to yield any colonies. I also tried to plate mite samples without a sterilization step. During dissection of the dead mite, I was able to observe dessication; this indicated that the mites were too dry to avoid sucking up bleach during the sterilization step. Their guts were probably also sterile, which explains the lack of colonies.

To test this hypothesis, I repeated the isolation protocol without a sterilization step and only washed with sterile tap water. This yielded 3 colonies, but now I would be unable to exclude environmental contamination.

Week 3

More colonies grew on the plates from Week 2. They were streaked, even though plate 13's colony was yellow and plates 14 and 15 only had transparent colonies. Plate 17 had small and large white round colonies. Prepared more plates; also without sterilization step, but they were washed carefully. Up to plate 39 was prepared this week. I tried a different heat treatment: 3 minutes at 80 degrees Celsius. I observed the colonies with a stereo microscope, but images were unclear and largely useless.

Week 4

Prepared up to plate 49. A colony from plate 46 looked like Bacillus thuringiensis HD350! I prepared 16 samples for brightfield microscopy. B. thuringiensis HD350 (also referred to as Bt HD350) was used as a positive control; this strain was ordered from DSMZ (no. DSM-6030) and grown according to the supplied protocol. All samples were stained with Coomassie Brilliant Blue R according to the protocol. I used a Zeiss Axio Scope.A1 for imaging with the 100x oil immersion objective. Very little spore formation was observed.

Week 5

The streaks were now grown for 3 days to promote sporulation before they were imaged.

I made cell-free extracts of my isolates and Bt HD350. The protein content was quantified with the Bradford method, but in all cases, this amount came out very low or negative; the extraction failed. I also did 16s rRNA PCR on isolates 46 and 47; Bt HD350 and E. coli were used as positive controls, water as a negative control.

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 16

Week 17

Week 18

Week 19

Week 8

Week 20

Week 21

Week 22

Week 23