Introduction:
Chlamydomonas is a unicellular green alga that contains two flagellas that are used for swimming. It is found in fresh water, in oceans, and in soil. Chlamydomonas are easily grown and controlled. We study chlamydomonas because we are able to easily deflagellate them and then allow them to regenerate their flagella using different chemicals. Due to this characteristic, we are able to study how flagella growth is affected and the rate of flagella growth once deflagellated. Regeneration of flagella is easily observed under the microscope. The purpose of this lab series was to determine which cell activities are required for regeneration of the flagella and how each experimental variable we tested the chlamydomonas culture with affected …show more content…
We had 4, 1.5 mL microcentrifuge tubes with the chlamydomonas culture in it. Tubes #1, #2 and #3 received the acid shock and neutralization treatment (steps explained in paragraph 1). Tube #4 did not receive the acid shock and neutralization treatment. All four tubes received three washes (steps explained in paragraph 1) however their washes and last resuspensions were different. Tube #1 and tube #3 was washed and resuspended all three times in regular TAP medium. Tube #2 and tube #4 was washed and resuspended all three times in Ca-free TAP medium. In a separate microcentrifuge tube, we transferred 9 uL of culture from tube #1 and 6 uL of CaCl2 (5mM). We followed this step for each of the remaining tubes. We then transferred 12 uL of this mixture from 2 tubes at a time to the two different counting chambers of the hemocytometer. Tube #1 was our positive control so we expect to see regeneration of flagella. Tube #2 was our experimental group so the flagella will regrow or not regrow depending on the influence of EGTA. Tube #3 was our negative control so we will not expect regrowth of flagella. Tube #4 was conducted to test whether EGTA is lethal to the cells and will cause them to …show more content…
To ensure deflagellation, we treated the cells with the acid shock and neutralization treatment. We expected the cells to become immotile because they no longer had their two flagellas for swimming. Later they would be able to grow their flagella back and therefore become motile. (Taylor 1965) Our class data supported these expectations as well. At time=0 minutes, average number of cells were immotile and as we reached closer to time=60 minutes, the cells were becoming motile. Our data in tube #1 (positive control) and #3 shows that the number of immobile cells decreased, which means that the cells were mobile. In tube #1, the cells regenerated their flagella and in tube #3, the cells were never deflagellated so the addition of colchicine did not have an effect on the flagella. Colchicine affects polymerization of flagella, but in tube #3, polymerization already occurred beforehand. The class data shows similar results to our results. In Tube #2 (experimental group), the number of immobile cells remain constant with very little fluctuations, which is what was expected. This is because we deflagellated the cells and then added colchicine (does not allow regeneration of flagella), so throughout the whole process the cells did not have flagella to move around. I am confident about my conclusions because our controls behaved as expected and the class results and our results were similar to previous studies. Future directions for this research can be to have all three washes
Chlamydomonas is a unicellular green alga that contains two flagellas that are used for swimming. It is found in fresh water, in oceans, and in soil. Chlamydomonas are easily grown and controlled. We study chlamydomonas because we are able to easily deflagellate them and then allow them to regenerate their flagella using different chemicals. Due to this characteristic, we are able to study how flagella growth is affected and the rate of flagella growth once deflagellated. Regeneration of flagella is easily observed under the microscope. The purpose of this lab series was to determine which cell activities are required for regeneration of the flagella and how each experimental variable we tested the chlamydomonas culture with affected …show more content…
We had 4, 1.5 mL microcentrifuge tubes with the chlamydomonas culture in it. Tubes #1, #2 and #3 received the acid shock and neutralization treatment (steps explained in paragraph 1). Tube #4 did not receive the acid shock and neutralization treatment. All four tubes received three washes (steps explained in paragraph 1) however their washes and last resuspensions were different. Tube #1 and tube #3 was washed and resuspended all three times in regular TAP medium. Tube #2 and tube #4 was washed and resuspended all three times in Ca-free TAP medium. In a separate microcentrifuge tube, we transferred 9 uL of culture from tube #1 and 6 uL of CaCl2 (5mM). We followed this step for each of the remaining tubes. We then transferred 12 uL of this mixture from 2 tubes at a time to the two different counting chambers of the hemocytometer. Tube #1 was our positive control so we expect to see regeneration of flagella. Tube #2 was our experimental group so the flagella will regrow or not regrow depending on the influence of EGTA. Tube #3 was our negative control so we will not expect regrowth of flagella. Tube #4 was conducted to test whether EGTA is lethal to the cells and will cause them to …show more content…
To ensure deflagellation, we treated the cells with the acid shock and neutralization treatment. We expected the cells to become immotile because they no longer had their two flagellas for swimming. Later they would be able to grow their flagella back and therefore become motile. (Taylor 1965) Our class data supported these expectations as well. At time=0 minutes, average number of cells were immotile and as we reached closer to time=60 minutes, the cells were becoming motile. Our data in tube #1 (positive control) and #3 shows that the number of immobile cells decreased, which means that the cells were mobile. In tube #1, the cells regenerated their flagella and in tube #3, the cells were never deflagellated so the addition of colchicine did not have an effect on the flagella. Colchicine affects polymerization of flagella, but in tube #3, polymerization already occurred beforehand. The class data shows similar results to our results. In Tube #2 (experimental group), the number of immobile cells remain constant with very little fluctuations, which is what was expected. This is because we deflagellated the cells and then added colchicine (does not allow regeneration of flagella), so throughout the whole process the cells did not have flagella to move around. I am confident about my conclusions because our controls behaved as expected and the class results and our results were similar to previous studies. Future directions for this research can be to have all three washes