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