Mitosis 101

Summary of Mitosis

Cell Communication

Introduction:
Yeast cells have two "sexes," called a-type and alpha-type. They locate each other via specific secreted factors. The exchange of factors causes them to mate. When these two cultures mix, the haploid cells (meaning they only have one complete set of chromosomes) become gamete cells (a mature sexual reproductive cell). The yeast cells halt asexual reproduction and grow into pear-shaped gametes called schmoos. When a-type and alpha-type come into contact with each other, they fuse, and the haploid nuclei form a diploid nucleus.

Purpose: The purpose of the lab was to analyze the cell signaling that occurs between the alpha-type and a- type yeast cells. The mating interaction of the two yeast types mixed together was the focus of the experiment. Our results for the mixed culture should support the idea of yeast cells mating with the use of special factors that attach to the opposite mating type. The characteristics of each yeast cell type were also individually analyzed.

Methods: 

1.We labeled 3 agar plates and 3 culture tubes as alpha-type, a-type, or mixed.

2. We added about 2 mL of sterile water to each tube and transferred a small amount of each yeast type into its designated tube.

To do this we used a toothpick to gather yeast cells and mixed the yest cells with the sterile solution by swishing the toothpick in the solution. For the mixed culture, we gathered a-type cells with a toothpick and placed it into the solution. Then with a different toothpick we gathered alpha-type cells and placed it in the same solution. Drops of yeast suspension were placed on their designated plates. We used different cotton swabs like in the picture below to spread the suspension on the agar plates.

 

 We then looked at the yeast under the microscope at different intervals of time to see what occurred in the life cycle. We captured images at 0 minutes,30 minutes, and 24 hours later. Unfortunately, the 0 and 30 minute images are unable to upload, but below are images of the alpha-type, a-type, mixed-type, and experiment yeast suspensions on the agar plates after 24 hours.

Mixture of alpha and a type saw the most growth on the agar plate. Best cell communication

alpha type yeast saw some growth too, but still not as dense as the mixed plate. Mating factors of the different yeast type can signal the opposite sex type, which impedes cell communication.

a type yeast cells grew a bit

we placed alpha and a type yeast on opposite ends of the plate (separated). Growth occured in the sides of each individual type but failed to develop towards the middle of the plate since the mating factors never or barely came in contact.



Data/graph

 

Discussion 

A large part of analyzing this lab is noticing the differences in the yeast over a period of time. In order from least amount of change to most amount  of change overnight it goes; the mixed separate dish, a-type, mixed, and then alpha-type. The reasons that the separate mixed was the least had to do with the fact that we started the yeast on opposite sides of each other. This makes it difficult for the yeast cells to sense the actual amount of cells in the dish, and therefore they do not shmoo as much as they should. Even when they do, it is a far distance across the dish for these little cells to get to. This slows down the overall process even more. The reasoning for the mixed being one of the highest producing ones is because it was mixed. This gives more opportunity for diversity within the cells and makes them able to produce faster. The reasons, I believe, that alpha was the largest change, was just due to its genetic make-up. It was obviously more adapted to move and produce quickly. This experiment proves how cells communicate with one another. After they got to a certain density, they stopped producing. We were also able to see how they changed their cytoskeleton to reach one another. This experiment truly shows how cells signal one another. 

Conclusion:
This lab proves that yeast cells communicate through pheromones, also known as chemical signals transmitted between organisms. Because a-type and alpha-type cultures changed into their gamete form, they detected a signal from the opposite type. Also based on the fact that alpha-type yeast changed the most, we can conclude that this type releases the most mating factor.

Plant Pigments and Photosynthesis

Plant Pigment Chromatography
Purpose: In this experiment, we were trying to separate and identify pigments as well as other molecules found in plant extracts. We needed to calculate the Rf constant, which represents the relationship between the distance moved by a pigment to the distance moved by the solvent. This helped us determine the factors involved in the separation of pigments.

Introduction:  Paper chromatography separates the components of cell extract. Different molecules and pigments move up the paper at varied rates due to differences in solubility, molecular mass, and hydrogen bonds (1). Chlorophyll a, the primary pigment that absorbs light, absorbs mostly violet and blue light for photosynthesis. Chlorophyll b is an accessory pigment that broadens the absorption spectrum on the spinach leaf by absorbing different wavelengths than chlorophyll a. Chlorophylls contain oxygen and nitrogen and have a greater affinity for the paper (3). Carotenoids are also accessory pigments that absorb violet and blue-green light. Their function is to perform photoprotection to dissipate excessive light energy that could damage chlorophyll pigments. They are very soluble and form no hydrogen bonds with the paper. Xanthophyll is a division of the carotenoid group that has a similar structure to carotenes but contain oxygen atoms and create hydrogen bonds with the paper. (2).

Methods:
We got a 50mL graduated cylinder that had 1 cm of solvent in the bottom. Then we cut a piece of filter paper long enough to reach the solvent and made sure the end was cut into a point.
We used a coin to crush leaf cells about 1.5 cm above the point of the paper. By rubbing the coin against the leaf, we were able to extract pigments.

We put the filter paper in the cylinder so the pointed end was barely immersed in the solvent, and stopped the cylinder.

When the solvent moved about 1cm from the top of the paper, we removed the paper and marked the location of the solvent and the bottom of each pigment band. Next we measured the distance each pigment moved from the bottom of the pigment origin to the bottom of each band.

 

Data and Graphs

Discussion:

In this lab we found out that the solubility, size of particles, and their attractiveness to the paper are all involved in the separation of pigments.  The different solubility’s of the pigments would change the Rf values. The reaction center contains chlorophyll a. The other pigments collect different light waves and transfer the energy to chlorophyll a.  Xanthophylls went furthest up the paper. We examined that the closer the rf factor to each other the distance  of the pigment traveled is closer to the distance traveled by the solvent. The separation of pigment in chromatography allowed us to look at the different pigments there in the plant. We can tell if a plant will reflect the color that showed and doesn’t absorb as much from the light and wavelengths. The orange, yellow, and green light will be somehow reflected from the Spinach leaves. To find the RF you take the distance pigment migrated divided by the distance solvent. The RF for band 1 is .252 mm and RF band 2 is .326. Band 3 is .467 and RF band is .585. The larger the RF is, then the more distance that was traveled by the pigment. Pigments like the carotene have the highest RF factor since they are the least polar and travelled the most. The chlorophyll pigments are extremely polar and have a high affinity for the paper which slows them down.

 

  
Conclusion: The 5 different pigment bands seen on the paper demonstrate that a mixture of these pigments is needed for photosynthesis to occur. The light spectrum of each pigment provides the spinach leaves with a large source of light that can be used to power the light reactions of photosynthesis. The first line of pigment from the bottom appears to be chlorophyll b since it is the most polar and most soluble due to its carbonyl group. This high polarity inhibits this pigments ability to move up the paper. Chlorophyll a is the next most polar which makes it the 2^nd pigment line. The less polar pigments are able to travel up the paper since they are less likely to create interactions with the paper. Errors that could’ve have occurred might be calculation errors and measurements.

Sources:

1.     http://www.phschool.com/science/biology_place/labbench/lab4/concepts1.html

2.     http://en.wikipedia.org/wiki/Xanthophyll

3.     Lab Introduction

Photosynthesis:
Purpose: We were trying to test the hypothesis that light and chloroplasts are required for light reactions to occur. To do this we had to measure the transmittance and absorbance from four cuvettes, each of with contained a different mixture. By measuring transmittance/absorbance, we were able to determine photosynthetic rates. We also took data at different times to see how light intensity affects the rate of photosynthesis.

Introduction: Plants depend on light energy to fuel the light reactions of photosynthesis, which produces the reactants of the light independent reactions. The absorption of light occurs in a photosystem light-harvesting complexes which contain various pigments (discussed in lab 4A) that harvest light and send it to the reaction-center complex. Electrons are boosted to high energy levels and must be carried by the electron transport chain and another photosystem in order to return to a more stable condition (1). In this process, ATP and NADPH are produced. NADPH is produced by the reduction of NADP+.  DPIP replaces the electron acceptor NADP+ in dye-reduction. DPIP begins with a blue liquid and as it’s reduces it becomes colorless.

Methods:

We set up an incubation area that included a light and a water flask.

Then we prepared each cuvette. The first one included 1mL phosphate buffer, 4 mL H2O, and three drops of unboiled chloroplasts. This served as our blank, and we used it to calibrate the colorimeter (we measured the light transmittance through each of the other tubes as a percentage of light transmitted through this tube).

The other cuvettes all had the phosphate buffer, H2O an DPIP in them. Cuvette 2 was covered in foil and had 3 drops of unboiled chloroplasts in it. 3 had three drops of unboiled chloroplasts. 4 had 3 drops of boiled chloroplasts. 5 had no chloroplasts whatsoever.

After mixing cuvette 2, we removed the foil sleeve and put it into the colorimeter. Then we recorded % transmittance and absorbance at time 0. We replaced cuvette 2 in the sleeve and placed it in the incubation area. We took and recorded additional data at 5, 10, and 15 minutes. With each cuvette, we did the same steps, only no foil sleeves were involved.

Data and Graphs

 Discussion:

The graph shown above proves that there is an inverse relationship between transmittance and absorbable. As the amount of blue dye solution decreased, transmittance increased.

Each curvette had a specific purpose:

Cuvette 1 with no DPIP and chloroplasts was used to calibrate the colorimeter. The difference between unboiled and boiled chloroplasts is that boiling chloroplasts denatures them which changes the shape of their protein and might lead to changes in function. Boiling chloroplast reduces the efficiency of these chloroplasts which negatively impacts the rate of photosynthesis.

Cuvette 2 with unboiled chloroplast,DPIP, and no light (due to the aluminum foil wrapped around) supported the idea that light is needed for the rate of photosynthesis to increase. Cuvette 3 with unboiled chloroplasts, light, and DPIP demonstrates that perfect conditions can result in a quick rate of photosynthesis. The chloroplasts are functioning properly, light fuels the light reactions that produce products used in the dark reactions, and DPIP acts as a new electron carrier. Cuvette 4 had all the same elements of Cuvette 3 except that Cuvette 4 had denatured chloroplasts, which negatively impacted the rate of photsynthesis. Finally, Cuvette 5 was used to demonstrate that DPIP can't be reduced on its own. Chloroplasts are needed to reduce DPIP, even if light is present DPIP can't function without the light that excites electrons.

In this lab the DPIP is the electron acceptor in this experiment. The molecule that found in chloroplasts is DPIP and substitutes for the NADP molecules. The source of the electron that will reduce DPIP is the electrons that come from the photolysis water.  The amount of light transmittance is measured by a spectrophotometer. The effect of darkness will have no reaction take place. The effect of boiling the chloroplast on the subsequent reduction of DPIP will stop the reduction.   The difference in the percentage of transmittance between the live chloroplasts that were included in the light and those that were kept in the dark was the light energy. In the dark there isn’t a flow of electrons and photolysis water while the light does. In Cuvette one was our control and set to 100% transmittance. Cuvette two was light reaction work in dark.  In cuvette three was light reaction work in live chloroplast. Cuvette four boiled chloroplast.  Finally, in cuvette five shows us that chloroplast is needed in plants.

Conclusion: The spectrophotometer measured the light transmittance through the cuvettes and chloroplast solutions. The biggest change in transmittance (low to high) occurred in the cuvette with unboiled chloroplasts that was placed in the light. This proves that the rate of photosynthesis increased the most. Factors like light availability and denaturing of chloroplasts affected the rate of photosynthesis. Errors could have occurred from simple mistakes in calculations. The amount of time that the cuvettes spent in front of the light might not also be exact which could lead to skewed data.

Sources

1. Biology Book

Cell Respiration Lab

Purpose

The purpose of this lab was to see how as cell respiration happens, CO2 levels rise and O2 levels drop. Also, the purpose was to see the rate that these levels change and if the environment changes, how would these rates change. We also used it to analyze the difference between cell respiration in a germinated plant and a dormant one.

Introduction

  Every seed grows into plants. Plants don’t grow overnight they need soil, temperature and water.   Plants spouting of a seeds into growth are called germination. The cell respiration formula for ATP is C₆H₁₂O₆ + 6O₂ → 6CO₂ + 6H₂O + energy.  There are three steps in the cell respiration that are Glycolysis, Krebs cycle and Electron Transport Chain. The glycolysis is when the breakdown of a glucose molecule into three- carbon molecule.  It happens in cytosol and oxide glucose into  pyruvate. The Krebs cycle doesn’t always have to need oxygen.  It happens in the mitochondria.  The Krebs cycle begins with acetyl. It is broken down into carbon dioxide. The first two cycles make ATP, but not that much. The electron transport chain needs oxygen and produces the most energy. It happens in the inner membrane of the mitochondrion.

Methods

The first step of our experiment
was to take the temperature of
the room. It read 20 degrees
Celsius.

Secondly, we received 25 glass balls,
put them into the bio chamber,
and measured their CO2 and O2
levels for a control group.  

Thirdly, we got 25 non germinated
bean seeds.

We measured the level of CO2
and O2 just as we did the glass balls.

Next,  we got 25 of the
germinated bean seeds.

We measured the CO2 and O2
levels for these as well.

After that, we took the 25 germinated
seeds and put them into ice water, and
let them soak for about 2 minutes.

We measured the temperature of the
ice water; it was measured at 4 degrees
celsius. 

 

Finally, we took the CO2 and O2
levels of the cold germinated seeds.

Table 1
Condition	Temperature (°C)
Room	20°C
Cold Water	4°C

Table 2
Peas	Rate of Respiration (ppm/s)
Germinated, room temperature	.009 ppm/s
Non-germinated, room temperature	.019 ppm/s
Germinated, cool temperature	.140 ppm/s

Discussion:
Cell respiration is the chemical process that makes most of the energy in the cell. Respiring cells take in oxygen and give off carbon dioxide. Specifically, O2 is an input of oxidative phosphorylation and CO2 is an output of the Kreb's Cycle. If you took a glance at our glass bead graph, you'd notice that the CO2 slope is barely a slope at all. It was a whopping .009. In a perfect world, the slope would be 0, because beads don't grow, give off carbon dioxide, and/or undergo cellular respiration. But hey, you can't win 'em all. At least the class average was .03.
Non germinating beans the slope came out to .0191, which is slightly larger than the glass bead slope of CO2. This happens because even though these beans aren't growing (AKA they're dormant), all cells need energy to survive. Meaning basically they need to take in carbon dioxide and release oxygen in order to not die.
On the other hand, germinating beans gave us a CO2 slope of .139 and an O2 slope of -2.348x10^-4. In other words, CO2 concentration is increasing while O2 concentration in the chamber is decreasing. When a bean is germinating, it means it's still growing; it requires energy for growth and development. Therefore the beans are increasing O2 consumption.
Lastly, we have our cold germinated beans. As temperature decreases, the rate of cellular respiration should decrease as well. Enzymes essential to cellular respiration are known to work fastest at certain optimal temperatures. So when the temp is too low, the enzymes can't function as effectively. The class averages give a good example at this; the cold germinating beans had a CO2 slope of .16 while the room temperature beans that had a slope of .22. Oddly enough, our CO2 slope was .14, about the same as our germinated beans. There's a possibility that we didn't leave the beans in the water for long enough, or we took too much time transferring them to the chamber, or the water wasn't quite cold enough. So many possibilities for error. Next time, if there's a freezer available, it might be beneficial to store some beans and peas in there, as the temperature will be held constant for a longer period of time.
With the exception of the cold germinated beans, our results do support our hypothesis. We thought that the slope would be almost nonexistent for glass beads, that non germinating beans would give off a very slight amount of carbon dioxide, and that for germinating beans the oxygen slope would be negative while the carbon dioxide slope would be positive.

Conclusion

In the cell respiration lab, we found out the germinating beans produced  more co2. The Co2 concentration was rising while the O2 in the chamber was deceasing.  The predicted outcome was correct.  We predicted that if the environment changes the rates would change. Through our graphs we were able to prove this. In our experiment we showed how CO2 and O2 must have changed environments for rate would change.