ClO2 Study - P. larvae Spores on Glass

From 5-15-15.

I was able to count CFU from the MYPGP plates that had the spores that were exposed to ClO2 gas for three hours on them. Spore stock #10 (1.1x10^7 CFU/mL) was initially inoculated onto the glass cover slips. Since a volume of 100 uL was added to the cover slips that means there is a concentration of 1.1x10^6 CFU on them before they were exposed to ClO2.

ClO2 Reagent	CFU/mL
0 mg	9.33E+05
50 mg	6.67E+05
100 mg	5.67E+05
200 mg	3.67E+05

There is a decreasing trend seen in spore survival as you increase the concentration of ClO2 reagents. This is a similar trend to what has been seen before after exposing for 6 hours, however the log killing was higher (due to the increase amount of exposure time). The standard deviations aren't very good, likely due to subtle decreases in spores between concentrations. For example, when 200 mg were used to treat spores on glass for 6 hours there was over a 3 log killing rate. It would appear that the length of exposure time has an effect on killing of spores, as well as generation of gas.

//EWW

ClO2 generation in chambers

Goal: Determine the rate at which ClO2 gas is being generated inside the modified anaerobic chambers. This information will be useful information for my ClO2 exposure study as an effective was of killing P. larva spores on apiary-related surfaces. Based on preliminary results, there appears to be a parabolic trend associated with gas generation inside the chambers, with Cl ppm peaking around 3 hours.

For this study, the modified anaerobic chambers were set up like they would be for the spore-exposure study (50 mL H2O, stir bar, etc.). A total weight of ClO2 reagents of 100 mg (50 mg from each Part A and Part B) was used for these experiments. The reagents were mixed inside a PCR tube and after mixing the tube was placed, open, inside the chambers. The ppm of Cl was checked every 30 minutes and were recorded.

Time	ppm Cl
0.5 hr	65
1.0 hr	75
1.5 hr	90
2.0 hr	100
2.5 hr	105
3.0 hr	105

I will continue the experiment with time points after 3.0 hrs up to 6.0 hours. 

//EWW

ClO2 Study - P. larvae Spores on Metal

From metal on 5-13-15.

I am able to count the CFU on the MYPGP from the spores on the metal coupons that were exposed to ClO2 gas for three hours. Unfortunately, I wasn't able to count any colonies from the treatment groups (even from the 10^-0 dilution!) as no colonies grew on the plates.

Spore stock #11 was added to the coupons, which means there was about 1.8x10^5 CFU on the coupon when it was exposed to the ClO2 gas. I was able to count CFU from the spores on metal that were exposed to ClO2 gas, unfortunately it was 2.2x10^4 CFU/mL, which means there was only about a 12% rate of recovery of the spore from the coupons! This was a similar problem that I had with the spores on wood, the spore recovery is very poor. I will need to adjust my re-suspension protocol for these two surfaces (pine chips and metal coupons). Perhaps increasing the time the surfaces are soaked in the 1 mL H2O would help with re-suspension.

Below is an image of one of the MYPGP dilution plates. There were only two colonies on the plate from the 10^-0 dilution. However, duplicate plates did not have any colony growth on them.

Spores on metal exposed to 50 mg weight of ClO2 reagent for 3 hours 

I will repeat this experiment in the future and adjust the re-suspension procedure.

//EWW

ClO2 Study - P. larvae Spores on Glass

From 5-10-15.

Spore stock #10 (1.1x10^6 CFU/mL) was used in a ClO2 exposure experiment for three hours on glass cover slips. A volume of 100 uL was added to sterile glass cover slips as before and they were exposed to ClO2 also as previously done, however, the exposure time was not six hours as before, but only three hours this time.

Three different ClO2 concentrations were used in this three hour exposure experiment: 50 mg, 100 mg, and 200 mg. A control of no ClO2 exposure was also used.

The ppm of chlorine gas in each container was checked using the columns as before by taking a 200 mL volume of air sample out after three hours. Interestingly, the ppm of Cl gas seem to agree with what was observed previously the last time this experiment was ran for only three hours. The ppm were about twice what was seen after six hours. This further indicates that there is likely a parabolic trend with generation of ClO2 gas inside the modified anaerobic chambers.


//EWW

ClO2 Study - P. larvae Spores on Wood

From wood on 5-10-15.
From metal on 5-13-15.

I was able to count colonies of P. larvae that grew on MYPGP agar. These spores were exposed to ClO2 gas on pine chips for six hours.

How to calculate CFU/mL
(CFU * Dilution Factor * Volume Factor)
or
(# CFU) * ( 1 / 10^ dilution factor) * ( 1 / volume plated in mL) = CFU / mL
ex:
3 * ( 1 / 10^ (-4)) *  ( 1 / 0.01mL) = 3x10^6 CFU /mL

ClO2 conc	CFU/mL	CFU/mL
0 mg	14000	1.40E+04
25 mg	9000	9.00E+03
50 mg	950	9.50E+02
100 mg	200	2.00E+02

Spore stock #14 was added to each pine chip. 1.6x10^6 CFU/mL was the concentration of this spore stock, which means there was 1.6x10^5 CFU added to each pine chip. That also means there was only about a 8.75% spore recovery from the pine chip that wasn't exposed to any ClO2 gas, which is terrible. Ideally, >90% recovery would be preferable. 

I attribute the poor spore recovery in part to the pine chips becoming saturated and portions of the spore stocks soaking through and onto the petri plate where it was drying. Another possible contributor could be due to the properties of the pine chip with retaining the spores once re-suspended. I will have to alter my procedure to account for these possibilities. Either by added smaller volumes of spores at a time and allow them to dry before adding the entire volume. I may also try to locate some thicker pine chips to use that wont become saturated as quickly with only 100 uL volume.

Also, there appeared to be a lot of different colony morphologies present on the MYPGP agar plates, more than usual for P. larvae. It could be related to other endospores already present in the pine chips that survived the autoclave sterilization process.

//EWW

Wax Worm/P. larvae LD50

From 5-8-15.

I added wax to three 96 well plates. Bee's wax was cut to small shavings using a sterile scalpel in a petri dish. Every pre-caution was taken to not contaminate the wax beyond its natural state. The wax itself is not sterile, but I didn't wish to add to it! The wax shavings were transferred to the 96 well plates using a sterile forceps.

Only the first and last four columns of each plate had wax added to them. This is because when the wax worms will be added (and an adhesive cover added) it will be easier to access them if they are not in the middle of the plate. The plates were heated in a ~65C water bath for ~5 minutes or however long it took to melt the wax to the bottom of the plate.

Hot plate water bath used to melt the wax in the 96 well plates. Notice, only the first and last 4 columns have was in them.

Bottom of 96 well plate after wax had melted. Only enough wax was added to cover the bottom of the well. In this case, less wax was preferable to more.

The plates were stored at room temperature. The spores have yet to be added to the wax, along with the early instar wax worms.

//EWW

ClO2 Study - P. larvae Spores on Metal

Spores on glass from 5-10-15.

Beekeepers use a number of metal tools when maintaining their hives (crowbars, scrapers, shovels, spades, smoke canisters, etc). The efficacy of ClO2 gas as a disinfectant of P. larvae spores on metal will be determined. I acquired 500 cut stainless steel coupons from NDSU shop foreman Kyle R. The coupons are made of 301 grade steel cut to 0.5 inch squares. P. larvae 9545 spores will be used in this experiment initially, followed by the North Dakota isolates.

100 of the metal coupons were autoclaved using the dry cycle in order to sterilize them. Spore stock 11 (1.8x10^6 CFU/mL)was used for this experiment. A 100 uL volume of spore stock was added to the tops of the sterile metal coupons. The spores were allowed to adhere to the surface of coupons by incubating at room temperature for 1 hour in a biological safety cabinet. 

100 uL of spores on metal coupons

The experiment was set up as before, with three coupons being placed inside a single modified anaerobic chamber. Total dry weights of chlorine dioxide reagents were mixed in PCR tubes and the reaction was allowed to take place for three hours this time. This is a change from the previous experiments that ran six hours.

After three hours, the concentration of chlorine gas was determined using the columns once again. 

Chlorine concentration in each container after 3 hours. The numbers indicate what the total weight of reagents were (in mg)

Dry wt	Conc Cl
0 mg	0
50 mg	50 ppm
100 mg	100 ppm

The detected ppm of chlorine was very surprising when compared to what has previously been seen using the glass cover slips at 6 hours. The 50 mg concentration of chlorine was pretty close to what had been previously seen, but the 100 mg concentration resulted in twice the detected ppm of chlorine gas after only three hours compared to the previously performed six hours. It could be that the gas peaks earlier than six hours and then lows due to exposure to the 50 mL of H2O being present. I could conduct an experiment of the gas generation inside the chamber in the future, but for now I will just continue with these experiments and see if this new trend continues.

The metal coupons treated with ClO2 gas were re-suspended in 1 mL of sterile ddH2O. The coupons were allowed to incubate at room temperature in the water for 5 minutes before diluted in H2O and plated on MYPGP agar. The plates were incubated at 37C for four days before colonies can be counted.

//EWW