Chlorine Dioxide Pilot Study

From 10-18-14.

Want to determine if the chlorine dioxide gas is able to kill P. larvae at all. This pilot probably should have been performed right away at the start of this project, as the effect of ClO2 on Bacillus spp has been evaluation, but it has never been done on Paenibacillus. So, I will first adhere complete P. larvae 9545 cells (vegetative and spores) onto a sterile glass cover slip and expose it to an arbitrarily high concentration of ClO2 gas for six hours in order to gauge its effect. I should hopefully see a decline in CFU in the groups exposed to the gas.

Required Materials

P. larvae 9545 broth culture in BHI+thiamine (incubated for 2 days at 37C)

Glass cover slips (used for microscopy)

50 mL conical tubes

P200 + sterile tips

Petri plates

Sterile forceps

Chlorine Dioxide Gas components 

1. Wash several glass cover slips in ethanol and transfer them to a glass petri dish

2. Allow cover slips to dry (ethanol evaporates) and autoclave using dry cycle

3. Aseptically transfer cover slips to a petri dish using a forceps (flamed for sterility)

4. Carefully add 50 uL of P. larvae broth culture onto each cover slip

5. Allow broth culture to dry onto cover slip (takes about 1 hour in the fume hood)

6. Aseptically transfer cover slips containing bacteria to 50 mL conical tubes

7. At the bottom of the experimental tubes, mix each ClO2 components shortly before adding the cover slip

8. Transfer tubes to a shaking incubator protected from light - gently rotate shaker at room temperature for six hours

9. Aseptically transfer the cover slip to a new 50 mL conical tube
10. Add 10 mL of sterile ddH2O to the tube and vigorously vortex for 10 seconds
11. Ten fold serially dilute the now re-suspended bacteria

12. Plate onto MYPGP agar using the drop plate method
13. Incubate plates at 37C overnight not inverted, invert plates after 24 hours and allow to continue incubating for an additional 24 horus
14. Count CFU and determine concentration

//EWW