Friday, December 20, 2013

Cellular Communication Lab

Purpose: The purpose of this lab was to determine the effects of varied amounts of time on each type of yeast cell. The different types of yeast cells were A type, alpha type, and mixed. The independent variable was the amount of time the cells were left, and the dependent variable was the number of cells left at the end.

Introduction: Yeast cells use cellular communication to mate and form shmoos. Cellular communication is a method that cells use to talk to each other and mate. Specifically in yeast cells, the A type cells send out a signaling molecule specific to the receptor of the alpha type, and this forms a diploid zygote, which then turns into a diploid budding zygote and forms an ascus. An ascus is a mixture of alpha and A type cells. These types of cells  are only seen when a and alpha type yeast cells are mixed together. When on their own, we only see single cells and budding cells.
Mixed a and alpha cells are shown here. This we can see because there are many more cells viewable through the microscopes.

This shows the same mixture, yet fewer yeast cells can be seen. 

This is alpha type at 48 hours.

To begin the experiment, we got culture tubes and labeled them alpha-type, a-type, and mixed. We had 4 mL of sterile water in our culture tube, where then we put a small amount of yeast into each tube and mixed it around thoroughly to make sure all then yeast got into the water.  We were not allowed to use the same pick to get the yeast out for the alpha and a type, but for the mixed we could use either one. Then we went back to our lab table and used a piper to put 5 drops of yeast onto the designated slides. Once again, we couldn't use the same piper for alpha and a type. We put the coverslip over the drops of yeast that was in the slide next. We had to record the yeast at 0 time, 30 minutes, 24 hours and then 48 hours. Once we took out our microscopes and had them all set up, we put one of the slides and focused the yeast using the 10x lens, then once we saw the yeast cells, we changed the lens to objective 40x. Next, we took pictures through the microscope lens so then its would be easier to count all of the cells. We repeated these steps 3 other times per each type of yeast cell. 

First, we have the results for the mixture of a and alpha yeast cells. 
Then, we have the data for each type individually.


Below is the graph for alpha type yeast over time. The two lines depict single haploid cells and budding haploid cells, as per the key above. 
Then, we have the graph for a-type yeast. 
Below is the key for the mixed yeast culture. 

 We had two types of yeast cells, a and alpha, that were pre-labelled for our lab. If we did not know which was which, but we had one known sample, we could determine each type. For example, say we had a sample of a type yeast cells. When we combine it with other a type cells, we will only see single and budding cells. But if it is combined with alpha cells, shmoos would be visible under the microscope. This is because yeast cells have g-coupled protein receptors. In yeast, these receptors can receive signaling molecules from the opposite type of cell. Once this happens, kinases are activated that stimulate the growth of the cytoskeleton in the direction of the signal. This is where shmoos is created. For our results, there were some obvious patterns. First of all, the graphs of single and budding haploid cells for the a and alpha type cells were always inversely related. As one type's concentration went up, the other decreased. For a-type, the last reading could be a mistake. The microscope itself maybe have been too dirty or malfunctioning, but no other cells were visible as the screen was very dark. Looking at the mixed culture, we can see that the concentration of single haploid cells decreases sharply, which was expected. The number of budding zygotes and asci cells increased over time, which makes sense because a and alpha cells will be communicating with each other in the mixture. If they are mixed, then their proximity allows them to receive signals and mate. The only surprising result was that of the shmoos, which we thought would increase over time rather than stay relatively flat. 

The conclusion that we had was that obviously the mixed had a lot more yeast cells than the alpha and a type since it was a mixture of both of them. The other observation that we made was that the more time that elapsed, the less percentage of each cell in the mixed, except for the schmoos. Which could be what was supposed to happen or it could be a calculation error in some way. Also I believe the single haploid was eventually supposed to be less than the budding haploid, because they would all turn into those toward the end. That ended up happening in the alpha type and the a type, but the mixed culture seemed to have some differing results.

Monday, December 9, 2013

Plant Pigments and Photosynthesis Lab

Purpose: The purpose of this lab was to measure the different rates of photosynthesis in isolated chloroplasts using DPIP. We used a dye-reduction technique, which shows that light and chloroplasts are necessary for the light reactions to occur. The independent variable is boiled versus unboiled chloroplasts and the amount of time spent in the light. The dependent variable is  the percent transmittance. The control group is the group with no DPIP and the group with no chloroplasts. We were trying to see if the amount of time that each group of chloroplasts was left in the light would effect the percent transmittance.
For the chromatography portion, we wanted to see the distance that pigments in spinach leaves would travel with a solvent. 

Introduction: DPIP was the compound we used in place of the electron acceptor NADPH in photosynthesis. The dye-reduction technique was what showed us how many reactions took place in each group. Each time the DPIP accepted electrons, or was reduced, DPIP changed from blue to colorless.  This means that the more color was lost, the more reactions were taking place. The percent transmittance shows how much light was absorbed and how much was allowed to pass through. 

Methods: To set up the lab we had a flood light, in front of a heat sink, then eventually our five cuvettes would be lined up behind the heat sink. We first got our Labquest 2 set up and attached our colorimeter to it. We had five cuvettes that were all filled differently with our solutions. The 1st cuvette was the blank control and we were using that to collaborate our colorimeter. That had 1 mL of phosphate buffer, 4mL of distilled water, and 3 drops of unboiled chloroplast. The next was one that had unboiled chloroplasts dark which had 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP, and 3 drops of unboiled chloroplasts. Cuvette 2 was then covered in foil so that no light could get through to the solution. The 3rd cuvette was unboiled chloroplasts light, which had 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP, and 3 drops of unboiled chloroplasts. Cuvette 4 boiled chloroplasts light which had 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP, and 3 drops of boiled chloroplasts. The 5th cuvette was the no chlorplasts light, which was our negative control group. This had 1 mL of phosphate buffer, 3mL + 3 drops of distilled water and 1 mL of DPIP. We set the colorimeter to 1 by using the 1st. We weren't able to put the chlorplasts in until we were about to start the procedures. So we put all the chlorplasts in th colorimeter one at a time and at 0 min we measured the transmittance for all 5 cuvettes. Then we put all five in front of the heat sink where the light was shining through. Then at 5, 10, and 15 minutes, we took the cuvettes away from the light to measure the transmittance again and then put it back after each test. 
Test tubes receiving DPIP

Colorimiter being calibrated before the cuvettes were tested for time 0

The cuvettes and flood lamp

Below are pictures shown from the Chromatography Lab. Here, we used cells from spinach leaves on paper, with the end dipped in a solvent, to assess the pigment distribution.

For the chromatography lab, we have measurements of each band seen in the picture above. 

 This means that the Rf factor for the first band is 0.14, the second band is 0.27, the third is .35, the fourth is .51, and the final is the solvent front.
Then, we have our measurements from the Photosynthesis lab. In the first chart, we have our measurements from the first round of trials. Then, we have measurements from the second round of trials. The significant differences In results will be explained. 

Graphs and Charts:

Using this key, two graphs are shown. The first graph is from the first round of results, and we know these are inaccurate since transmittance can not be greater than 100%. The second graph depicts results from the second round of trials. 

In our first run through of the photosynthesis lab, we immediately knew something was wrong. In our first measurements at time 0, we had transmittance readings over 100%, which is not possible. For this reason, we increased the concentration of chloroplasts from our first round of trials. This way, the reactions would not be nearly finished taking place before we even took measurements in the colorimeter. After making this change, we saw an improvement in our results. Test tube 2, with unboiled dark chloroplasts, remained fairly constant. This makes sense due to the necessity of light for photosynthesis to take place and DPIP to be used. The transmittance of test tube 3, unboiled light chloroplasts, increased over time. This also makes sense due to the fact that the chloroplasts are functioning, and provided light to fuel their reactions. Test tube 4, boiled light chloroplasts, increased initially and then decreased. This is interesting because boiled chloroplasts become denatured, and therefore do not react. The graph should be relatively flat, and more trials could be taken to determine if a mistake was made. Finally, test tube 5 had no chloroplasts, and therefore no reactions taking place, which resulted in little or no change in the amount of transmittance. Excluding test tube 4, these results turned out the way we had hypothesized. 

In the chromatography lab, we used the below formula to determine the Rf factor for each pigment. The results come out different for each pigment because they are not equally soluble in the solvent. Beta carotene traveled the furthest because it was the most soluble, forming no hydrogen bonds. Xanthophyll, on the other hand, is less soluble and forms hydrogen bonds with cellulose, causing it to be found further from the solvent front. We did not have extensive prior knowledge about pigments such as these, but the information provided supported our results.

Conclusion: Cuvette 1 stayed similar in it's transmittance rates because it didn't have DPIP, and acted as the NADP+, which is the electron carrier in photosynthesis. Cuvette 2 didn't have any light going through to it so therefore there were no electrons being produced,so the color didn't change. Then Cuvette 4, which had unboiled chlorplasts which didn't cause much electron production or color change since some proteins had probably been denatured through the process of boiling them. Then, Cuvette 5 was our negative control that didn't have either type of chlorplast, so therefore it didn't have anything to react with and it basically had very similar transmittance each time like Cuvette 2 had.