Knock, Knock…who’s there...Lionel…Lionel who…Lionel get you nowhere, better tell the truth!! On Monday, surprisingly, we left almost an hour early! That never happens. But before we left we set up a carbon utilization test. This test uses 16 carbon sources plus a control to see what types of carbon each organism uses. In the Luedemann paper he said that he used basal media for his carbon test. The media we used contains 2.5 g/L of yeast extract (this is the buffer that sucks up the acid that the organism makes), 1g/L of calcium carbonate, 1% NaCl at 10g/L, then the carbon sources were added at 1g/L. Note that we added NaCl because most of the organisms that we are working with require salt to grow. We are hoping for more growth on the plates to which the carbon source was added, opposed to the control. To set up this experiment, we spotted 6, 10uL of each organism on the control and also on each carbon source plate. Eugene used stock plates to prepare the organisms for this experiment. Since the organisms were already growing on some type of media in which carbon was already added he had to wash them with saline, centrifuge them, wash them again with saline, and pipette off the saline to remove the carbon source from the plates that they were growing on.
On Wednesday we had ALOT to do. We started off by rating our pH plates that we had done a few weeks before. In looking at our plates we noticed some contamination, however, it wasn’t enough that we could not tell the amount of growth. In looking at the plates there wasn’t a common growth trend and this really seemed weird since the organisms we are working with are from the same family, Geodermatophilaceae. Although not all, but some organisms did show some sort of a growth trend. You can see that at pH’s 4-5 the organism 31 did not grow.

This is probably because the media was too acidic and prohibited the organism from growing. At pH’s 6,7, and 9 it grew well and did not grow at pH 8. Because the organism grew at pH 9 and not 8, it is a chance that those two plates were mixed up. So the pH 9 plate is probably the pH 8 plate, and vice versa. In looking at our plates we figured the optimum pH for these organisms is around 7-7.5. In reading the papers, this was confirmed.

After that, we started our DNA extraction which took FOR-EV-ER, FOR-EV-ER (as they said on the Sandlot)!! We used the MOBIO UltraClean Mega Soil DNA Kit. The items in this kit are much larger than those in the other DNA kit we used before. This is probably because this kit is used to extract DNA from soil, not directly from plated cultures. We put 10 g of our soil sample in the bead tube and vigorously vortexed it to mix the contents. We also vortexed our tube for an additional 30 minutes. While the tubes were being vortexed for 30 minutes, we enjoyed our lovely treat, Krispy Kreme and Milk from the Promiseland, courtesy of Dr. Rainey. OH, and I can’t forget the delicious potatoes that Manish’s wife made. After all of that we were near completion of the DNA extraction. We spinned (is this even a word) the Spin Filter 3 times, not 2, this was to get as much DNA on the filter as possible. We also added 4 mL of the MD5 solution instead of 8 to make the DNA “stronger.” After completion of our kit, we froze our DNA for later use on “next day,” as Dr. Rainey would say.