Sporulation on Blood Agar

From 3-31-15.

It has been 10 days since the Colombian sheep's blood agar slants had been inoculated with 300 uL of P. larvae 9545 broth culture and incubated at 37C. The agar slants were washed and endospores were recovered.

A 3 mL volume of chilled sterile ddH2O was added to the surface of the agar slant. The surface of the agar was gently rubbed using a sterile inoculating loop to dislodge any of the attached cells. NOTE: be careful when rubbing as to not loosen or break any agar as it will become difficult to pipet and wash later!The volume was carefully transferred to a sterile 50 mL conical tube. The wash was repeated once more with 3 mL. The entire volume of five slants was combined into a single 50 mL tube. The tube and its contents was centrifuged at 15,000 x g for 5 minutes. The remainder of the wash and endospore extraction procedure was the same as previously performed .

P. larvae growth on the blood agar slants after 10 days

The biggest obstacle I encountered from extracting the spores from agar slants was that sometimes a small portion of the agar breaks off during the abrasion step during removal. This bit of agar can clog the pipet or can be easily transferred and become an issue during the wash steps. The agar is nearly invisible and I often times the only reason I noticed them is when they clogged my pipet. Fortunately, the agar is not as heavy as the endospores and will be removed with proper washing techniques. In the future, I will have to take better care as to limit the amount of agar that is dislodged during the initial steps.

Below is an image for future reference of what the endospore pellet looked like in size. Keep in mind, this is from five slants. I have never seen a pellet this size with P. larvae, and it was very easy to see the distinction between endospores and vegetative cells - the endospores were a much darker color whereas the vegetative cells and other debris were white/yellow.

Endospore pellet of P. larvae. This was the size of the darker bottom pellet (the spores).

A total of four P. larvae 9545 endospore stocks were made (each combined from five different slants). The stock was diluted and plated to determine CFU/mL. Microscopic examination revealed a large amount spores, a bit less than what has typically been seen after extracting B. thur spores. However, the spores in P. larvae were significantly more clumped together compared to Bacillus spores that underwent the exact same treatment and method of re-suspension. This clumping of P. larvae spores may be responsible for some of the erratic CFU counts in previous quantification attempts. After a few days of incubating at 37C I will quantify the concentration of spores.

//EWW