Paenibacillus larvae Spore Extraction from Agar

Paenibacillus larvae Spore Extraction from Agar

Fisher Lab. 2014.

Materials:

Broth culture of P. larvae	MYPGP Agar	37°C with 5% CO2
P200 pipet	P200 sterile pipet tips	65°C water bath
Pipettor	5 & 50 mL sterile pipets	Refrigerated centrifuge
Cold sterile ddH2O	50 mL conical tube	4°C refrigerator
Sterile spreaders

1. Create a 1:10 dilution from a two day broth culture of P. larvae in sterile ddH₂O. Plate 100 µL of diluted culture onto MYPGP agar using spreader.
2. Incubate plate at 37°C with 5% CO₂ for 6-7 days. (Plates with 50 to 5000 colonies is ideal)
3. Wash the surface of the agar with 5 mL of sterile ddH₂O. Use a sterile spreader to gently rub the surface of the agar to loosen the spores from the surface. Pipet off and collect the 5 mL of sterile ddH₂O.
4. Repeat the wash step two more time and combine all the wash volumes in a 50 mL conical tube (total of 15 mL)
5. Centrifuge the wash volume at 12,000 x g, 15 minutes, at 4°C.
6. Discard the supernatant.
7. Suspend the pellet in 30 mL of cold sterile ddH₂O.
8. Repeat steps 5-7 four additional times.
9. Suspend the pellet in 5 mL of cold sterile ddH₂O.
10. Store at 4°C.

Quantify Spore Concentration

11. Heat spore stock at 65°C for 15 minutes in water bath. (Heating will kill vegetative cells).
12. Serial dilute a portion of spore stock in sterile ddH₂O.
13. Plate 100 µL of each dilution onto MYPGP agar. Incubate at 37°C with 5% CO₂ for 6-7 days.

Optional: perform direct microscopic counting of spore stock. This will result in an overestimation – heat resistant spore counts are ~6% of direct microscopic counts.

Protocol adapted from de Graaf, et al. 2012. Standard methods for American foulbrood research. International Bee Research Association. Journal of Apicultural Research. DOI 10.3896/IBRA.1.52.1.11.

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