Determined CFU from ClO2 killing effect on P. larvae pilot that was performed recently. MYPGP plates have been incubating at 37C inverted since then (11 days).
There were no quantifiable CFU on any of the MYPGP plates that were treated with ClO2 gas. However, there were a number of colonies from the control treatment group.
Determining CFU/mL from colony count formula/equation:
[# colonies counted / volume pipetted onto plate in uL] * [ 1 / serial dilution made (ie 10^-2)] * [50 uL / 1 mL] = CFU/mL
| Control Treatment, No ClO2 | ||||||
| Replicate 1 | Replicate 2 | Replicate 3 | ||||
| 10^-2 | 10^-3 | 10^-2 | 10^-3 | 10^-2 | 10^-3 | |
| 25 | 3 | 24 | 1 | 22 | 3 | |
| 33 | 2 | 25 | 0 | 26 | 2 | |
| 31 | 0 | 25 | 2 | 25 | 3 | |
| 22 | 3 | 33 | 3 | 25 | 5 | |
| 21 | 2 | 19 | 4 | 29 | 2 | |
| Avg | 26.4 | 2 | 25.2 | 2 | 25.4 | 3 |
50 uL of P. larvae spore stock was added to a cover slip. This volume was resuspended in 5 mL of ddH2O after treatment (1:100), and then subsequently diluted 1:10.
P. larvae spores remaining after no ClO2 treatment:
1.28x10^4 CFU/50 uL
Meaning,
P. larvae spores remaining after ClO2 treatment:
< 500 CFU/50 uL
There was far less CFU (beyond what I could determine) in the experimental treatment group.
From 10-18-14.
Repeated pilot to determine if water and high humidity has an effect on the generation of chlorine dioxide gas. The experiment was repeated, with one difference - the electric fans were replaced by metal stir bars and a stir plate (picture below). This experiment took place in Van Es Hall Room 114, Fisher Lab, as opposed to the previous experiments which were performed at the USDA ARS building.
Metal stir bar plates were acquired from the T. Bergholz lab. Each of the plates were identical Barnstead Thermolyne Cimarec brand hot plates. The hot plate function was not used during this experiment. The metal stir function was set to "4" on the dial for each during the experiment.
Above is the anaerobic plate set-up. In order to prevent the metal stir bar from losing its magnetism to the plate and flying off damaging either the beaker of water or the sachet containing the ClO2 reagents a petri plate lid was affixed to the bottom of the chamber using double sided tape. A metal stir bar was placed within the petri plate cover. Unfortunately, there was a very fine line between efficient and consistent stirring and it being too fast or not enough to move the bar. This resulted in the dial being set to "4". There wasn't much air movement in my opinion due to this problem.
The sachets that were used were larger than the ones previously used as I was unable to find the more narrow ones. The larger sachet could have resulted in less mixing of the ClO2 reagents.
Only two modified anaerobic chambers were used in this pilot experiment. One containing a 50 mL beaker with 50 mL of ddH2O and the other just containing the beaker with no water added. This will determine if there is indeed a difference in ClO2 generation based on increased humidity as previously discussed. Chamber were set up next to each other on the Fisher lab bench. Humidity and temperature gauges were also added to each of the two containers. Reagents A and B were combined in the sachet and mixed briefly for 10 seconds by shaking the sachet. The sachet was placed in the side of the chamber and cover was added to the top, pinching the top of the sachet.
Chambers were allowed to incubate at room temperature, while stirring, for six hours. Chambers were protected from direct light by placing a large box over the tops of the two chambers during the entire time frame. Chlorine gas concentration was measured using the GasTech tool as before.
Results:
H2O experimental treatment after 6 hours:
79% humidity
25.6C
45 Cl conc
No H2O control treatment after 6 hours:
27% humidity
26.4C
45 Cl conc
Humidity was higher in the treatment group containing the 50 mL of ddH2O, which is to be expected with the additional water. Temperatures remained relatively similar after six hours in both treatment groups. Additionally, so did the quantified concentration of chlorine in each container. This amount was also similar to previous experiments that have been performed including the one that used the electric fan as a means to mix the gas more efficiently. This is unfortunate, as it appears that the addition of the metal stir bar did not have an effect on the dissemination of ClO2 gas. This value of 45 is far below the calculated concentration of gas that was supposed to be present relative to the amount of reagents added. I am beginning to wonder if there isn't a problem with circulating gas, but rather an issue with our calculations.
In the future I would like to determine the quantifiable concentration of ClO2 with increasing amounts of reagents that have been mixed in order to determine if there is indeed a gradient as their should be. I'll likely mix increasing amounts of Reagents A and B in anaerobic containers without any stirring mechanism and let the reaction occur for six hours. After which I'll measure the amount of gas in each chamber.
Interestingly, the electric fans used on 10-18-14 to mix the gas now seems to have a dramatically reduced functioning capacity. The reviews for the fan claimed that it would function for over nine hours with fresh batteries, however after four hours while being exposed to ClO2 gas the fans died. The fans were removed from the gas and fresh batteries were added, and now the fans only function for two hours before they stop. It would appear that the ClO2 gas effected the functionality of the fans, likely a corrosion issue due to the salt formation. For this reason, we likely wont be able to use any sort of electronic, battery powered, mixing tools in the future. I wonder what effect the gas is having on the temperature and humidity gauges that are also in the anaerobic chambers....
//EWW
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