while some were in mexico...we worked hard......

So, this week Rainey decided to abandon us kids and go on a vacation! Luckily for us Cindy invited us to the microscopy lab. In the microscopy lab we worked with the light microscope and the electron microscope. In order to view the organisms under the light microscope we had to follow a few procedures. First we put the colonies into the tube with water. This step could have caused the cell to explode through osmotic pressure, which could have ended really badly. Next, we put a small amount (2 micro liters) of the solution on the cover slip of a slide. If we had put too much of the solution the cells would have moved around even if they were not motile. We then turned the slide upside down and let the cover slip adhere to the slide. We then put water on the slide so it wouldn’t dry out. After, we added DAPI on one side of the slide. DAPI staining intercalates between DNA bases; this form of fluorescent staining works with UV light. The DAPI cannot absorb all of the light without giving off visible light. After this was complete we moved to the room with the light microscope. Here Cindy gave us some tips on using the microscope and focused in on the organisms for us. Some things that we learned about the microscope was that focusing on the word on the slide would help us find the cells better, Also, we were told that a decrease in magnification cause the lens to appear further away from the slide. We used differential contrast and 80X magnification. Under the light microscope the strains appeared as follows:
14- nothing specific showed
17- resembled Deinococcus, suspect Modestobacter, moved a lot because of the liquied, aggregated at the edge of the water, Image 1
37- uniformly shaped cells; a lot present, lumpy in appearance, in pairs and clumps, motile (moved opposite directions, twitching), bigger than strain 17, image 2
40- twitching, possible motile, various shapes from round to long to short, looks like spirochetes, image 3
66- big clumps, irregularly shaped, lots of cells, big piles of cells, looks like Deinococcus, various shapes and sizes, blackish brown colored, looks like its outside the cells, looks stressed or dead, no fluorescent used, Image 4
75- motile, twitching, no fluorescence, vibrio shaped, moves really fast, not Deinococcus, Image 5
This completed part one of our day, next we worked with a biofilm found from the bottom of an aquarium. For this experiment collodion was on the slide to stick the bacteria to it so that it cannot move. We also added Dapi to the slide. This was examined under the light microscope under low magnification. On the slide you can see that the DNA fills up cells. This was apparent from the blue dots present which represented the DNA. The cells moved very quickly so taking a picture of it was quite a task. This is Image 6.
Part three of our experiment consisted of using the TEM (transmission electron microscopy). For this experiment our liquid solution was put on parafilm. The copper was placed dark side down on the liquid sample strain. A drop of uranium acetate was put on the slide. The copper was then picked up with a tweezer and put on filter paper. The uranium acetate formed a coat around the bacteria capsule so that the cells would be visible under the microscope. The TEM shined an electron beam down through the sample; however, we cannot see the electrons. The advantage of using this microscope was that we could tell the difference between two different spots close together because this type of microscope offered better resolution by making wavelength shorter, so the sample appeared more detailed. The screen gives off visible light when excited by electrons. The TEM utilized liquid nitrogen so that the vapors that are given off are condensed. The strains appeared as follows:
37- no image; nothing found
17- no image; nothing found
14- small, balloon like , round shaped
66- big crystal arrangements; dense; Image 7
After these strains appeared to be giving off very little Cindy used a different method. She soaked the strains in uranium acetate.
40- flagella present (1), oval shaped, lots of different cell shapes and sizes
14- occasional coccoids, various sizes some long, tiny, and thin, and some rectangles
66- clumps of cells together
75- nothing present
17- cells together; looks like dumbbells; four cells in a ring formation
37- group of cells stuck together, oval shaped, lumpy
That pretty much sums up Monday! Wednesday Rainey came back, we immediately got back to work. Our assignments consist of: analyzing temperature plates, analyzing and testing the xylan, avacel crystalline, and granular cellulose plates, and running our gel. Majority of the strains did not grow on 45 or 50 degree plates. We ran test on our xylan plates by putting iodine on it to test for the hydrolysis of amylase. We put congo red for 15 minutes than salt for 15 minutes on the CG and AC plates. Our plates test negative for both tests. By running our gel we found out that our isolates were approximately 564 base pairs. Those which showed up were 34, 44, 45, LRH 4, 9, 11, 12, and 14