George H. Cook

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In this experiment we expressed our main focal point of our overall project and attempted a slow releasing distribution method of our VOC. This allowed us to test the effects of our VOC in small amounts of a chosen part per million as it is released over a period of time. Our objective was to keep P. destructans under constant treatment of the VOC. Our first attempt at this was a simple fragrance diffuser method. We executed this method by placing a 50 mL Erlenmeyer flask inside a large desiccator jar. We went forth and placed two 60 mm Petri dishes (uncovered), with one mycelial plug on each plate, in the jar. Adjacent to the plates we placed one 50mL flask containing a mixture of safflower oil (carrier oil) and our VOC (fragrance). Inside the the 50mL Erlenmeyer flask, with the oil/fragrance mixture, we put three diffuser sticks in order for the mixture to travel up the sticks and diffuse into the air.

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Since many of our past experiments focused on the effect our VOC had on mycelial plugs of P.destructans, in this experiment we wanted to shift our focus on the effect our VOC had on the spores of P.destructans. This experiment used our VOC at various parts per million against a dilution of spores going from 0, -1, -2, -3, and -4.  We placed duplicate plates, containing the appropriate dilution of spores, inside a mason jar and inoculated the inside wall of the mason jar with the appropriate ppm of our VOC.

This experiment was placed in the incubator for three weeks at 10˚C with ~30% humidity.

After the three weeks, we removed the plates and the results were as follows:

Spore	Control	0.5 ppm	1 ppm	2.5 ppm	5 ppm
0	Thick Lawn	Thick*	NG	NG	NG
-2	Thick Lawn	NG	Some growth*	NG	NG
-3	Lawn	NG	NG	NG	NG
-4	Spotty Lawn	NG	NG	NG	NG
-5	32 & 34 CFU	19 & 33 CFU*	NG	NG	NG

The results indicate that lower than the previously identified 5 ppm, was active against the P. destructans. The results labeled with an * was not expected. We suspect there was either difficulty with the VOC overcoming the spores, that the VOC dissipated more quickly in those jars, or that there was a human error when inoculating the jars with our VOC. The last suspected reason is most likely due to the very small quantity that needed to be placed in each jar.

Spore	Control	0.5 ppm	1 ppm	2.5 ppm	5 ppm
0	2 plates	2 plates	2 plates	2 plates	2 plates
-1	2 plates	2 plates	2 plates	2 plates	2 plates
-2	2 plates	2 plates	2 plates	2 plates	2 plates
-3	2 plates	2 plates	2 plates	2 plates	2 plates
-4	2 plates	2 plates	2 plates	2 plates	2 plates

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We simulated a cave-like environment in order to expose our VOC to the P. destructans in a more life-like environment. This was done by placing an inverted small mason jar inside a larger wide mouth mason jar. We then placed a 60mm Petri plate on top of the inverted mason jar. The Petri plate contained a P.destructans mycelial plug placed on PDA. The large mason jar was about 950mL in volume and was covered with a manipulated tin foil. We cut a small sized square shaped hole out of the middle of our tin foil and taped a membrane to the foil to cover the hole. This membrane allows the passage of our VOC without other particles entering as well. Our controls were contained in the same setup, but instead of free standing in the incubator, they were placed in a larger jar sealed with parafilm to prevent the VOC from entering.

The experiment was run for three weeks, keeping the large environmental chamber shut and dark for the entire time. This allowed us to maintain 10˚C and 5 ppm of our VOC over this time frame uninterrupted.

Our results, were as expected. The exposed jars did not have any growth from the mycelial plug, while the controls grew as expected. These plugs were removed and subcultured to grow without the presence of our VOC. After some time the VOC treated P. destrucansshowed as much growth as the control mycelial plugs. This indicated the VOC had a fungistatic effect on the P. destructans.

Row	1		2		3
A	Jar	VOC	Jar	VOC	Control
B	Control	VOC	Jar	VOC	Jar
C	Jar	VOC	Control	VOC	Jar