Microscopy

On Monday of this week, we went to the microscopy lab while Dr. Rainey was in Mexico. Each group took two of their strains to observe using two different techniques. We first looked at our strains using light microscopy. To prepare slides, we first scraped of cells into a tube containing water. Then we place 1.5µl of the liquid culture onto a cover slip. Then the labeled slide was placed onto the cover slip. DAPI (4',6-diamidino-2-phenylindole) was then added to the slides. DAPI is a fluorescent stain illuminated by U.V. light that binds to the DNA by passing through the intact cell membrane. Next the slides were viewed using Differential Interference Microscopy. It works by separating a polarized light source into two beams that pass through the sample. When the beams recombine, it is gives a 3D image. Pictures were taken of each sample, then we attempted to view the DAPi under U.V. light. None our samples showed any illumination from DAPI. This may have been because it was unable to pass through our highly hydrophobic samples. We probably needed to get rid of the polysaccharides so the DAPi could inter the cell.
Then we view was our samples using the transmission electron microscope (TEM). The sample were prepared by placing a drop of the liquid culture on a small copper grid. The grid contains a thin carbon film that holds the bacteria between the grid. Then grid was placed onto a drop of uranium acetate. The sample was placed into the TEM. TEM works by shining a beam of electron through the bacteria in a vacuum. The vacuum is maintained by adding liquid nitrogen to the machine which condenses the vapor within the machine. The image is formed by the interaction of electrons through the sample which is magnified and focused. We were able to observe the bacteria and find structures such as flagella on the bacteria. Pictures were taken of each sample on the TEM also.
On Wednesday, we recorded our results from the xylan, Avicel, and granular cellulose. We also recorded the result from the 45ºC, 50ºC, and second 10ºC temperature trials. We found that in some of our samples the strains appear a slightly different color at different temperatures. Iodine was added to the xylan plates to look for clearing. Congo red was added to the Avicel and granular cellulose plates to look for clearings also. Then our PCR results were run on a 1% agrose gel. We found that the bands that did appear were about the same size (approximately 565 base pairs) as reported in the paper.