Inoculation for Pure Culture Confirmation

2/17/09 – Tuesday Inoculation day
Using the yeast slurry from each of the four flasks used to propagate up the Avery 15 Brettanomyces sp., I inoculated three different mediums (Copper Sulfate, MYPG+cycloheximide and Lysine agar) prepared the day earlier. First technique was to loop cells from the slurry and streak inoculate each of the different mediums. Dipping the loop into the first flask I streaked onto a CuSO4 plate then flamed the loop and proceeded to inoculated from the same flask onto each of the other two media agars. I repeated these steps with the next flask until all four flasks had been streak inoculated onto each of the three mediums. The same was then done with serial dilutions I made from the four flasks. The serial dilutions were performed at 10^-2, 10^-4 and 10^-6 times the propagate yeast slurry attained from each flask. Again repeating as I’d done when streaking with each medium type, I pipetted .1 ml onto each plate and then dispersed with glass beads. When finished I placed the inoculated plates into the 28°C growth chamber and plan to check on their progress and the results throughout the week.

Copper Sulfate, MYPG+cycloheximide and Lysine agar plates

2/19/09 – Thursday Observation
Only the streaked plates had visible growth but no single colonies had formed yet. The pipetted plates had small cellular growth about the size of a pin head. Checking the colonies after 48 hours is too soon and four to five days seems to be a better wait time before first checking for colony growth. I will wait till next Monday (2/23/09) when growth should be sufficient enough to obtain single colonies to re-streak with and enumerate the colonies on the plates.

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~ by Chad on February 19, 2009.

 
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