The well plate was put inside an AnaeroPack container (Link and image below). The larger side container was filled with 15.5% glycerol in water to keep up humidity.
Anaerobic container was placed into an Innova 4400 shaker incubator set at 35C with no shaking (Link). I will monitor survival of the larva to see if they can survive in my environment. If they can survive until pupation, then I will introduce P. larvae to the next batch of honey bee larva I receive.
Isolated another stock of P. larvae spores that had been incubating at 37C 5%CO2 on MYPGP for 10 days (Colony concentration was similar to the image seen on this Link on the top left). Followed the same modified procedure as 6-24-14 (Link). There was significantly less of a viable pellet this time as opposed to 6-24-14. Maybe there are less spores present after 10 days compared to the previous seven days of incubation. Or maybe I did not successfully remove all the cell debris and vegetative cells the previous day. Regardless, I created a 10 fold serial dilution of the spore stock created today ("Stock C") and the two spore stocks created on 6-24-14 ("Stocks A and B"). Diluted down to 10-7 in sterile ddH2O. Plated 100 uL of each dilution onto MYPGP agar and incubated at 37C 5%CO2. Will perform colony counts to determine the concentration of my spore stocks.
Made 1 Liter of MYPGP for colony counts (Recipe). Note: did not add the 10% Glucose as per the recipe due to its temporary unavailability. Stored around 20 plates in the 4C walk in fridge.
//EWW
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