Performed ClO2 exposure to P. larvae 9545 on pine wood chips.
A 100 uL volume of Spore Stock #14 was added to the top of a small pine chip purchased from Tractor Supply. The volume was allowed to evaporate and adhere to the chips (placed in petri plates) in the biological safety cabinet for one hour. The volume was rather large for the size of most of the chips. After the one hour time, some spots could be seen under the chips on the petri plate. It appears the 100 uL volume was enough to fully saturate the pine chip and some soaked through to the bottom of the petri plate. I hope I did not lose too much of the spores on the plates.
Three pine chips with adhered spores were transferred to modified anaerobic chambers. A volume of 50 mL of ddH2O was also added to the chambers to increase humidity. Dry weight concentrations of ClO2 reagents were mixed in PCR tubes and place within the chambers.
Total dry weights of reagents:
0 mg
25 mg
50 mg
100 mg
After the reagents were combined in the PCR tubes and mixed the tube was placed within the chamber (while open) and the chamber was sealed using the cover. The metal stir bar was used again inside the chambers in order to mix the gas more effectively. The chambers were protected from light and incubated at room temperature for six hours.
Unfortunately, the magnetic stir bar was not longer spinning in the 100 mg container after six hours. The bar apparently spun out of control and knocked over the petri plate containing the pine chips and the PCR tube containing the reagents. This prevented a good mix of the two reagents, and likely I will not be able to use the results from this treatment group. The ppm of chlorine gas was checked in each container after six hours.
ppm of Cl in each container after six hours:
| mg | ppm Cl |
| 25 |
15
|
| 50 |
20
|
The determined ppm inside the containers were about half of what they were when using the glass cover slips. The reduced ppm of Cl in the containers could be due to the presence of the pine chips. It could be that they are serving as a sink for the gas similar to how water does.
The pine chips were removed from the modified anaerobic chambers and re-suspended in 1 mL of sterile ddH2O in 50 mL conical tubes. The tubes were vortexed for 30 seconds and left to incubate at room temperature for 5 minutes. The volume was diluted and plated onto MYPGP agar. After about five days I will be able to count colonies and determine CFU/mL of the spores subjected to ClO2 treatment.
ND Isolate Spore Stocks
The P. larvae spores extracted from ND isolated (from honey) were determined today once the colonies were countable on the MYPGP agar. Below are the calculated CFU/mL concentration of these spores stocks. The stocks are stored in 4C and will be used in the near future for the ClO2 study.
| ND Stock # | CFU/mL |
| 2a -A | 1.56E+06 |
| 2a -B | 1.03E+06 |
| 4a -A | 7.30E+04 |
| 4a -B | 3.50E+04 |
| 5a -A | 3.25E+06 |
| 5a -B | 2.25E+06 |
| 22a -A | 5.00E+06 |
| 22a -B | 3.75E+06 |
| 55a-A | 7.50E+05 |
| 55a-B | 7.75E+05 |
//EWW
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