Microscopy! PCR!

On Monday, we spend the whole day in the microscopy lab! Our group used strains KR-65 (suspect of Geodermatophilus) and MCC-66 (a B. jujuensis suspect) to look at in the microscopy lab. In order to look at our samples under the microscope, we had to scrape colonies off of our plate and suspend them in .5 mL of water. We looked at a small amount of these suspensions (about 2 uL) on a slide under light microscopy (which allows us to veiw live cells). We stained these slides with DAPI, which allows one to see where the DNA is in the cell. The DAPI, which is a dye that intercalates between base pairs, fluoresces under UV light. This technique did not work (probably because the cells are resistant to the DAPI--remember, the are hydrophobic!! They might need to be treated with something else first, like toluene, which makes the cells permeable). We also viewed the slides using DIC microscopy. Some interesting things we saw using DIC included: strain #40 which showed motile, twitching cells, #66 which showed clumps of cells that were irregularly shpaed and looked stressed/dead, and #75 which also showed motile, twitching, fast moving cells. Lastly, we viewed our samples using a TEM microscope. The electrons that shine down on the slide when using this technique allow us to see the difference between closely spaced objects (in other words, it increases resolution). We also added liquid nitrogen to a tank on the side of the microscope. This is used to help keep a vacuum going and to condense vapor. To use this microscope, we put 50uL drop of cell suspension on parafilm and floated a copper-looking circle on top of the drop. The we dropped the copper circle on top of uranium acetate. However, when we viewed the circle (what is the actual name for this object? didn't she call it a grid, or something?) under the microscope, we couldnt find anything. Dr. Henk tried a different technique and we finally saw a dense, irregular blob of something from strain 66. All in all, this technique was not very sucessful.On Wednesday, we analyzed our strains that we placed in 10C, 45C and 50C. Most of our strains grew at 10C, some grew at 45C, but none grew at 50C. We also analyzed our strains that were plated on media containing xylan. To analyze these results, we covered the plate with iodine. A clearing in the iodine indicates a positive test (NONE of our strains were positive). Does this test for the presence of xylanase? Next, we analyzed our strains that had been plated on media containing avicell and granular cellulose. We first poured Congo Red on the plate, let it sit for 15 minutes, and then poured it off. Then, we poured a NaCl solution on the plate, let it sit for 15 minutes, and then poured it off into a beaker. A positive test shows up as a clearing in the red dye. A very small number of our strains were weakly positive and the rest were negative. Lastly, we ran our PCR products on an agarose gel. From our collection, strains 53, 54, 55, 60, 65, 73 showed up on the gel at band 565. GB20 also appeared on the gel. The rest of our strains did not show up on the gel, which most likely indicates improper extraction of the DNA.