P. larvae LC50 Pilot

Followed LC50 SOP from 7-2-14. Diluted Spore Stock A (3.05x10^6 CFU/mL) to create the 4 different experimental treatment groups (200, 400, 1000, and 10,000 CFU/mL) by following the dilution scheme shown in the image below. Each dilution was made in BAD as diluent. I will write up a more fluid and easy to follow procedure in the future.

Convoluted diluted scheme

It was somewhat difficult to accurately pipet the BAD at first, but once you allow enough time for the fluid to fill the pipet tip the volumes are what they should be. Tip: pipet slowly and leave the tip in the fluid longer than you would for less viscous liquids.

The honey bee queen from the "Woodchipper" hive at the USDA was placed in the Jenter box four days ago (Monday) and was freed the following day. Since then the Jenter box remained in the hive until it was brought back to the USDA today. 

Unfortunately, there weren't very many hatched larva after four days (despite literature review indicating that was when the first instar hatch). I was only able to retrieve 50 first instar larva from the USDA, less than a third of what I had hoped for. 

1        Larva were transferred from the Jenter box using camel hair paint brushed onto a petri plate                   containing a thin lawn of BAD.

2        Petri plates were wrapped in moist cloth and placed inside a Styrofoam container for transfer to               NDSU Van Es Fisher Lab.

3       Larva were then transferred from the petri plate to 24 well plates (one per well) using the same                type of paint brush (the thinner and shorter the brush bristles, the better for this experiment).

4       Because so few larva were obtained, only one treatment group was applied. 5 uL of the 200                  CFU/mL P. larvae spore-spiked BAD was added on top of each first instar larva

                    Note: 200 CFU/mL was chosen because literature review determined that was the                                         relative LC50 amount

5.     Three 24 well plates containing infected larva were incubated at 35C in the anaerobic container              (90% humidity) until tomorrow when the larva will be transferred to clean BAD on a new plate.

I am not very confident about the survival rate due to transfer this time. The petri plate containing the BAD was very dry in some spots containing the larva. In the future I will transfer the larva directly to the 24 well plates at the USDA and then transfer them to the NDSU lab. The small amount of Royal Jelly present on them that was placed there by the worker bees after hatching should be enough to sustain them long enough for transfer and prevent them from drying out.

It is disappointing there were so few hatched larva after four days. In the future I will retrieve the Jenter box early on the fifth day and select the smallest larva for my study. 

I will continue to monitor the survival of the larva.

//EWW