Sporulation on Blood Agar

From 3-24-15.

P. larvae ATCC #9545 was grown on Colombian sheep's blood agar for seven days at 37C 5% CO2.

1. The vegetative and endospores were extracted from the surface of the agar by adding 5 mL of sterile ddH2O and gently rubbing the surface with a sterile cell spreader.
2. The 5 mLs was collected in a 50 mL conical tube. This wash step was repeated an additional two times.
3. The volumes from three plates were combined into a single 50 mL conical tube. The tube was centrifuged at 18,000 x g for 2 minutes.

P. larvae 9545 on Colombian sheep's blood agar.

4. The supernatant was carefully decanted from the 50 mL tube. The pellet was re-suspended in 3 mL of sterile ddH2O.
5. The 3 mL volume was transferred to 1.5 mL eppendorf tubes in 1 mL volumes. The tubes were centrifuged at at 18,000 x g for 1 minute.
6. The supernatant and upper most layer of the pellet was carefully removed. This uppermost layer is composed of vegetative cells and debris. The endospores are more dense and thus are heavier so are at the bottom of the pellet.
7. The pellet was re-suspended in 1 mL of sterile ddH2O. This wash step was repeated five times.
8. After the last wash and upper pellet removal, the pellet was re-suspended in 1 mL sterile ddH2O again. The suspension was incubated at 60C for 15 minutes in a water bath.
9. The centrifugation and wash steps were repeated an additional five times.
10. QUANTIFYING SPORE STOCK: the suspension was diluted 1:10 down to 10^-8 and 10 uL volumes were plated onto MYPGP agar. Plates are incubated at 37C for 3 days until colonies are counted and CFU/mL is determined.

//EWW