P. larvae Project Pilot

Performed P. larvae spore isolation and purification. I did not exactly follow the protocol from 6-24-14 (Link), I made several modifications discussed below. Used a P. larvae plate inoculated on 6-17-14 onto MYPGP (picture seen Link, top left picture). The plate had been incubating at 37C 5%CO2 for 7 days. This experiment was mostly to familiarize myself with the process.

Modified Paenibacillus larvae Spore Extraction from Agar

11. Wash the surface of the agar with 5 mL of sterile ddH₂O. Use a sterile spreader to gently rub off the colonies into suspension. Pipet off and collect the 5 mL of sterile ddH₂O.
12. Repeat the wash step two more time and combine all the wash volumes in a 50 mL conical tube (total of 15 mL)
13. Split the 15 mL volume into 15 1mL aliquots in 1.5 eppendorf tubes
14. Centrifuge the eppendorf tubes at 16,000 x g, 1 minute, at 4°C.
15. Carefully pipet off the supernatant. Very carefully attempt to remove the "white fluffy" precipitate that is covering the darker and more compact pellet.
16. Suspend the pellet in 1 mL of cold sterile ddH₂O.
17. Repeat steps 5-7 five additional times.Gradually consolidate the number of eppendorf tubes to just two (ie transfer the suspended pellets to another tube containing a pellet and suspend that one as well).
18. Suspend the pellet in 1 mL of cold sterile ddH₂O.
19. Heat eppendorf tubes at 65°C for 15 minutes in water bath. (Heating will kill vegetative cells).
20. Repeat wash steps 5-7 five times.
21. Suspend pellet in 500 uL of sterile ddH2O.
22. Store at 4°C.

I will eventually perform a serial dilution of the spores I've collected and plate them on MYPGP agar to perform colony counts. I need to get an accurate appraisal of how many spores are present in order to use them to calculate the lethal concentration (LC50) in honey bee larva.

Quick microscopic examination after step 1 revealed very low numbers of spores. In the future I will incubate plates for 10 days to allow for an increased spore production. I will also harvest from plates with nearly a lawn of bacteria. In the near future I will perform another microscopic examination of my purified spores to gauge their concentration.

//EWW