ClO2 Study - P. larvae Spores on Glass

Goal: Expose P. larvae spores on a glass surface to chlorine dioxide gas in order to determine the gas's effect on the spores. The glass surface will serve as somewhat of a control, as metal, plastic, and possibly wood surfaces will be implemented in the future.

Procedure:

1. A 100 uL volume of P. larvae spore stock was added to the surface of a sterile glass cover slip. The volume was allowed to evaporate for about an hour inside the laminar flow hood. 
1. For this experiment. I used Spore Stock #16 from 4-7-15, so each cover slip was inoculated with 2.8x10^5 spores.
   

   

   100 uL volume of P. larvae spore stock on glass cover slip

   
   
2. The chlorine dioxide reagents (Part A & Part B) were weighed out using he analytical balance in the Pruess lab with reagents being temporarily stored in 1.5 mL eppendorf tubes. Note: Be careful not to mix the two reagents and to change out weigh paper and scoops between each reagent.
1. For this experiment, there was only two experimental groups using chlorine dioxide- 100 mg and 1000 mg concentrations. Once the reagents are mixed their final weight concentration will be 200 mg and 2000 mg.
3. The actual ClO2 gas exposure will take place inside of the modified anaerobic chambers as shown on 10-18-15 inside of a laminar flow hood. The exposure chambers were assembled and organized also similarly as to what had been previously done. A beaker containing 50 mL of ddH2O was added to the corner of the modified anaerobic chamber to increase humidity (higher humidity increases the rate at which ClO2 is generated). A metal stir bar in combination with a magnetic stir plate were utilized in order to evenly mix the gas once it is being generated. In order to get the metal stir bar to smoothly spin inside the chamber a number of glass microscopy slides were placed on the bottom of the chamber for the stir bar to spin on. Otherwise the stir bar would be spinning on the plastic.

1. 

   Set up anaerobic chamber similarly it what it was on 11-9-14. This image does not show the sachet containing the ClO2 reagents. Note: the metal stir bar in the center (blurry spot)- it is spinning on top of several glass microscope slides that have been placed on the bottom of the chamber.

   

   Chambers set up in laminar flow hood. The ClO2 exposure will be conducted within the hood. 

   
   
4. The modified anaerobic chambers were closed and allowed to spin for an hour to build up humidity (and while I prepped everything else)



How the modified anaerobic chamber was organized. Image created using Inkscape Software.

1. After three hours of the chamber being closed and spinning the humidity was 77% and was a temperature of 26.2C.
5. The glass cover slips containing the dried P. larvae spores were aseptically transferred to the modified anaerobic chambers using sterile forceps. The cover slips (three per container) were placed inside a sterile petri plate.
6. The chlorine dioxide gas reagents were quickly and thoroughly mixed inside small sachet bags and added to the modified anaerobic chambers. The tops of the sachet bags were pinched in the opening of the chamber lid and sealed shut. The light in the hood was turned off and the opening to the hood was sealing using foam boards
1. 
   

   

   Hood sealed with foam board to protect the anaerobic chambers from reaction - ClO2 will not be generated as efficiently in the presence of light. 

   
   
7. The exposure continued for six hours inside the sealed laminar flow hood with the lights off and metal stir bar spinning.
8. After six hours, the chlorine gas concentrations was measured in each modified anaerobic chamber using chlorine gas detection tubes (previously described 10-8-14) in conjugation with the GasTec (Link) pump. A total volume of 200 mL was removed from the container.
1. For this experiment, there was significantly more chlorine gas present in the anaerobic chamber with the larger weight of chlorine dioxide gas reagent, which makes logical sense. In the container with only 200 mg the Cl detection tube only detected 75 ppm Cl. In the container with 2000 mg, there was over 500 ppm of chlorine present. The detection tube was unable to determine exactly how much Cl was present in this container since it was beyond what the detection column could detect.



Chlorine gas detection tubes from each of the experimental groups, the left is from the container with 200 mg total of reagent (100 mg each), and the right is from the container with 2000 mg total weight.

In fact, the concentration of Cl was so high in the one container that the container itself turned yellow. Even the water inside that container turned yellow. The below image shows the difference between the two containers - there is no trick with the lighting, the container on the right now has a yellow tint to it compared to the one on the left.

Notice the color differences between the two chambers that were exposed to different levels of ClO2 gas



1. Additionally, the temperature inside the hood, but not the containers, after 6 hours was 30.4C.

9. The anaerobic chamber was opened inside the laminar flow hood and the cover slips containing the P. larvae spores were aseptically transferred to a 50 mL conical tube containing 1.0 mL of sterile ddH2O. The glass cover slip was broken (shattered) using a sterile forceps while inside the tube.

10. The tube and contents were vigorously vortexed for 30 seconds and allowed to incubate at room temperature for 5 minutes.

11. The re-suspended spores were diluted in sterile ddH2O and plated on MYPGP agar. Plates were incubated at 37C for three days until colonies can be counted and spore concentration can determined.

//EWW