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.

The cleaned PCR products were sent to GATC for LightRun sequencing.

Week 6

I inoculated 100 mL LB in 500 mL erlenmeyers with Bt HD350, V46, V47 and E. coli strains expressing Cry1 and Cry2 (courtesy of Ruud de Maagd). These were grown for 3 days at 30 degrees Celsius. A French cell press was used for the protein extraction. The first SDS-PAGE failed, so it was repeated in Week 7.

Week 7

Supernatant, protein extract and the cellular debris were run on SDS-PAGE; however, the gel with cellular debris came out completely empty. Figure 5 and 6 show the two gels and the putative Cry toxin bands.

Week 8

I repeated the SDS-PAGE from Week 7, because the bands from 46 and 47 were unclear and I could not observe Cry1Aa from Bt HD350. The SDS-PAGE failed as the machine was interrupted by leaking buffer. Additionally, I started working on cloning Cry1 and Cry2 from the E. coli strains. This did not work out in the end.

Week 9

I performed another SDS-PAGE to observe Cry1Aa production. Additionally, we received Bacillus thuringiensis tenebrionis (also referred to as Bt tenebrionis) from DSMZ, a strain which produces Cry3Aa.

The SDS-PAGE gel shows that the Cry toxins are mostly present in the cellular debris and are not very soluble.

Week 10

I went to the Duurzame Bij in Veenendaal. These beekeepers do not treat their honeybees with pesticides, so they have a lot of Varroa mites. Dead mites were taken from the mite drawers below the hives with the help of Henk Kok. I filled 7 eppendorfs with approximately 100 mites each. With these fresh mites, I was able to perform the protocol as described and still get suitable colonies. I prepared up to plate 62. Plates 50, 55, 57, 58, 59, 60, 61 and 62 had colonies with the right colony morphology. Brightfield microscopy with the Coomassie stain showed that only isolate 60 had rod-shaped cells, but when I checked the streaked plate again, the plate turned out to be contaminated.

Week 11-14

Unable to do lab work due to the laboratory moving to a new building.

Week 15

Streaked out plates again, made some new glycerol stocks.

Week 16

I performed the isolation protocol and prepared up to plate 107. Plates 64, 68, 69, 71, 75, 81, 82, 87, 88, 90, 92, 93, 94, 95, 96, 97, 98, 99, 101, 103, 104, 105, 106 and 107 showed colonies; each of the colonies were streaked (2 to 3 per plate). Figure 9 shows the streaked plates.

Week 17

Week 18

Week 19

Week 8

Week 20

Week 21

Week 22

Week 23