26.6.13
Date: 26.6.13
Time: 8:14
Location: Dunn School of Pathology, Oxford, England
This morning began with a deflagellation experiment with Francois. Deflagellation is the process of removing the flagellum from the cells (in this case, leishmania) for further testing of the isolated component. This is done by taking the cells and first centrifuging them from their media then placing them in a PBS solution. Once in solution, the cells are counted. The deflagellation process has proved to only provide a 7% rate of recovery of useful material for Francois so he needs to count the cells with frequency to be sure he isn't losing too much of his sample.
After the cells were counted, the solution was spun down again in the super-centrifuge again at 100,000g. The supernatant was discarded and the cells were placed in fresh solution. The cells were then placed on special microscope slides that are divided into tiny little grids that can then be used to isolate cells for counting. The cells are then counted, which is very difficult since they are still alive and wriggling around! The cells are then treated and put through a syringe 100 times to remove the flagella. When substances have different densities, the more dense substances sink to the bottom while the less dense substances rise to the top and the two do not mix. Beds of sucrose are prepared and the samples are placed in them. The test tubes are then put in the ultracentrifuge, where the centripetal forces create an increase in gravity, forcing the more massive cell bodies to the bottom, through the sucrose, while the lighter flagellum remain at the interface between the sucrose and water. The flagellum can then be pipetted out. Then the tests can continue.
Unfortunately, the samples were not purified completely. When the cells should have dropped to the bottom of the sucrose during centrifugation, they did not. This could be due to too much sucrose, which would create too thick a bed that the cells would not be able to permeate. A contaminated sample, even slightly contaminated, is completely useless since the sample is being used to test flagellum and only flagellum.
When transferring the cells, the type of solution that is used is important. An isotonic solution keeps the osmotic pressure on the cell at normal, meaning there will be no change in the form of the cell. A hypotonic solution will decrease the osmotic pressure on the cell, so it will expand and sort of spread out. Finally, a hypertonic solution will increase the osmotic pressure on the cell, which will compress the cell into a smaller volume, increasing its density. For the purpose of this experiment an isotonic solution will be used because we do not want to change the shape or size of the cells.
Time: 8:14
Location: Dunn School of Pathology, Oxford, England
This morning began with a deflagellation experiment with Francois. Deflagellation is the process of removing the flagellum from the cells (in this case, leishmania) for further testing of the isolated component. This is done by taking the cells and first centrifuging them from their media then placing them in a PBS solution. Once in solution, the cells are counted. The deflagellation process has proved to only provide a 7% rate of recovery of useful material for Francois so he needs to count the cells with frequency to be sure he isn't losing too much of his sample.
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| Leishmania before deflagellation |
Unfortunately, the samples were not purified completely. When the cells should have dropped to the bottom of the sucrose during centrifugation, they did not. This could be due to too much sucrose, which would create too thick a bed that the cells would not be able to permeate. A contaminated sample, even slightly contaminated, is completely useless since the sample is being used to test flagellum and only flagellum.
When transferring the cells, the type of solution that is used is important. An isotonic solution keeps the osmotic pressure on the cell at normal, meaning there will be no change in the form of the cell. A hypotonic solution will decrease the osmotic pressure on the cell, so it will expand and sort of spread out. Finally, a hypertonic solution will increase the osmotic pressure on the cell, which will compress the cell into a smaller volume, increasing its density. For the purpose of this experiment an isotonic solution will be used because we do not want to change the shape or size of the cells.
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