P. larvae germination in BAD medium assay

Repeated BAD germination experiment with several modifications.

1. Diluted Spore Stock A (3.05x10^6 CFU/mL) ten fold down to 10^-4.
           (100 uL spore stock + 900 uL ddH2O in eppendorf tubes for ten fold serial dilution.)
2. Added 100 uL of each spore stock dilution from 10^-1 to 10^-4 to 400 uL of BAD and also to 400 uL       of ddH2O in eppendorf tubes.
3. Incubated the tubes at 35C (same temperature that the honey bee larva is incubated at) shaking at                 150rpm for 2 hours.
4. Perform drop plate method to determine CFU/mL via colony counts.
          (plate five 10 uL drops onto MYPGP agar for each sample)
5. Further dilute each sample in a 10 fold dilution down 10^-2 further and repeat step 4.
          (100 uL sample + 900 uL ddH2O, repeat twice)
6. Transfer 200 uL of each original sample from both the BAD and H2O treatment groups that had been           incubating to new eppendorf tubes and heat them in a 65C water bath (Gibbs lab) for 15 minutes.
7. Repeat steps 4 and 5 using these samples.
8. Incubate all MYPGP agar plates at 37C not inverted for 24 hours.
9. Transfer plates to 37C 5%CO2 and invert them. Incubate an additional 48 hours and then perform colony     counts and compare.

If the P. larvae spores do not germinate in the BAD, I expect to see relatively the same CFU on both the heated and not heated samples in the BAD treatment compared to the H2O treatment group. If P. larvae spores are able to germinate in the BAD, then I expect to see lower CFU in the heated samples than the not heated samples in the BAD group compared to the H2O group.

Actual dilution on the plates includes the initial serial dilution from the stock (step 1) the 1:5 dilution from each treatment creation (step 2) and the 1:100 dilution from the drop plate (step 4). The samples were further diluted in step 6 as well and should also be factored in while determining CFU/mL in the future.

//EWW