Sporulation on Blood Agar

Goal: Improve my ability to extract P. larvae spores and increase their recovered concentration.

Made Columbian sheep's blood agar plates and slants. This type of agar was used by Genersch et al. (2005) for the propagation of P. larvae spores. Their method resulted in a ~10^7 spore concentration. To make the blood agar I autoclaved Columbian agar base and added 5% of defibrinated sheep's blood while stirring after it was cool enough to touch and pour. I made 40 blood agar slants by aseptically transferring 8 mL into sterile glass tubes (picture below). After the hot agar was added to the tubes, the tubes were tilted to achieve the slant. This was done using the racks used in the media kitchen as per Linda P.'s directions.

Columbian Sheep's Blood agar slant

Using a three day old broth culture of P. larvae in BHI, I added 300 uL to the Columbian blood agar slants. An image of what this looks like is seen below:

I will incubate the agar slants at 37C for 10 days, after which I will extract the spores by likely washing the agar slant and recovering the liquid suspension. I also inoculated 10 Columbian blood agar plates with 100 uL of the P. larvae broth culture. The plates are incubating at 37C 5%CO2 for seven days.

//EWW