P. larvae germination in BAD medium assay

Drop plates from 7-17-14 continue to incubate at 35C. No contamination has developed like seen before on the MYPGP agar. In the future all MYPGP agar will be poured and dried inside a fume hood for sterility.

Yesterday:

Read a paper about germination determination of P. larvae in an artificial media by using optical density changes. I performed a quick pilot study to determine if OD could even be quantified on a spectrophotometer using the thick BAD. The Beckman spectrophotometer in Fisher Lab was able to detect the OD and it wasn't beyond its scale.

Proceed with a time course study of germination. Diluted Spore Stock A in a 1:5 dilution in BAD in a 96 well plate. Using the Pruess Lab incubated spectrophotometer Laura N. created a program that would incubate the plate for 2 hours at 35C and take OD580 readings every 5 minutes.

I am unable to attach the raw data that is in an Excel file, but below is a quick graph of the data without standard deviations. Standard deviations remained rock solid between each of the two replicates in BAD. The variation between the two is likely due to minor pipetting error as it is difficult to accurately pipet the viscous BAD.

It appears that OD decreased over time when P. larvae is incubated in BAD. Below is the same assay performed in H2O and BHI+1ug/mL thiamine instead of BAD.

It appears OD increases (albeit slightly) over time when P. larvae spores incubate in H2O and remains relatively the constant in BHI. I plan to repeat this OD germination experiment again with more replicates and different spore stocks to see if these trends remain consistent.

According to Alvarado, 2013 a decrease in OD over two hours indicates that the P. larvae spores are germinating. This would mean that the P. larvae spores germinate in my BAD as evident by the decrease in OD over the two hours.

I would like to repeat this OD experiment using different Spore Stocks to confirm these results.

//EWW