Investigation 6: Cellular Respiration

Introduction
Today we are going to talk about cellular respiration, a vital process that cells perform every second. Constantly, cells are breaking and forming bonds, moving molecules around, expending energy and using up substances. Why are these cells working hard all the time? Well organisms need energy to perform vital functions such as moving, reproducing and growing, and the only way to get this energy from its surroundings is to perform cellular respiration. Cellular respiration works by breaking apart a sugar and then capturing energy released from breaking those bonds onto molecules called ATP, but for now we will focus on the process of cellular respiration, not the entire process of energy transfer.Today we are going to talk about cellular respiration, a vital process that cells perform every second. Constantly, cells are breaking and forming bonds, moving molecules around, expending energy and using up substances. Why are these cells working hard all the time? Well organisms need energy to perform vital functions such as moving, reproducing and growing, and the only way to get this energy from its surroundings is to perform cellular respiration. Cellular respiration works by breaking apart a sugar and then capturing energy released from breaking those bonds onto molecules called ATP, but for now we will focus on the process of cellular respiration, not the entire process of energy transfer.

This is the chemical equation for cellular respiration

On October 22nd, Vinay, Shreyan, Vikram, and I returned to the lab to perform an experiment to measure cellular respiration and determine the effects of sound on the respiration of a meal worm. The first problem that we encountered was how to measure the cellular respiration, a tiny chemical process, in a meal worm? The group could have measured any of the compounds formed by the chemical equation above, such as the decreasing amounts of glucose in the worm or increasing amounts of water in the worm, but these would be very difficult to measure, especially if we didn't want to kill the worms. The group instead chose to measure the increasing amounts of carbon dioxide in the air as this would show that cellular respiration was occurring. Also, the carbon dioxide sensors that Mr. Wong gave us only worked for that specific compound. 

A meal worm

After figuring out how to measure the rate of cellular respiration, the group decided how to use sound to affect a meal worm's rate of respiration. Our first thought was to use different types of music, such as a song with a slower beat versus one with a fast beat, soft sound versus loud, or a song with less bass or more bass. After some discussion, we decided to not use music because there are so many different types of sound that we wouldn't be able to track what specific difference between two songs cause a change in the worm's respiration. Instead, we decided to use extremely high and extremely low frequency sounds on the meal worms. We accomplished this by downloading a dog whistle application that had a frequency toggle on the sound it produced so that we could directly affect what our meal worms were listening to. Our hypothesis for this experiment was that the higher frequency sounds would increase the worms' rate of reaction because the energy of the sound would frenzy and eventually kill the worms. 

The purpose of this lab was to design and perform our own experiment using organisms and manipulating specific factors of their environment to increase or decrease the rate of cellular respiration in the worms. The purpose is also to apply knowledge learned in class to a practical situation and see if our knowledge of cellular respiration could expand through creating our own experimentation procedures.

Procedure
First, the lab group grabbed a beaker, a Vernier carbon dioxide sensor and rubber stopper for the top. Then we put two large meal worms into the beaker and brought all of our components to our lab station. I then connected the sensor to my iPad so it would record the data of the increasing levels of carbon dioxide and graph it on my tablet. After this, we stoppered the beaker, with the sensor attached, sealing off the worms from outside air and allowing the sensor to measure the respiration. We then set the timer for ten minutes and watched our worms move around. After the ten minutes were up we unstoppered the beaker, saved our data and put the worms back into the tub of worms. We did this so that our data would not be contaminated by worms full of cells that were oxygen deprived. We wanted fresh worms for every trial.

For our second trial, we followed the same procedure as before for gathering the worms and setting up the beaker, but then played a very high frequency dog whistle sound from the iPad into the side of the beaker at the worms. We continued this for ten minutes and then stopped the sound, recycled our worms, and saved the data. The third trial was the exact same as the second, but instead of a high frequency sound, we played a low frequency sound to the worms. 

Data/Results
Here is the graph of our data.
The Blue Line is our control experiment, the Yellow Line is our high frequency experiment, and the Red Line is our low frequency experiment

Here is a table containing all the values of carbon dioxide in the beaker every minute, and the total change in amount of carbon dioxide in the beaker. (The units are parts per million or ppm)

Trial/Time	Trial 1 (control) (ppm)	Trial 2 (High Frequency) (ppm)	Trial 3 (Low Frequency) (ppm)
0 min	660	377	495
1 min	643	441	574
2 min	666	501	610
3 min	696	549	655
4 min	720	592	697
5 min	758	636	735
6 min	795	674	775
7 min	833	716	814
8 min	867	751	840
9 min	902	792	866
10 min	938	831	895
Total Change (ppm)	+278	+454	+400

Here are pictures of the sensor-beaker system we set up.

Conclusion
The results of the experiment were much as the lab group had predicted. We hypothesized that the worms would respire more with a high frequency sound than with a low frequency sound or the control group, which did occur. With the high frequency sound, the worms added 454ppm carbon dioxide to the air, while with low frequency they only added 400ppm and a measly 278ppm by the control group. We did not observe the worms becoming frenzied as we had though they would in the high frequency trial, and we also discovered that the worms were alive after this trial. The lab group believed that the worms would be killed by the sound, or at least use up all of their oxygen faster because of it. We may have still been right and it just takes longer for the worms to die. One oddity that we came across in our data is the relatively high rate of cellular respiration by the worms at the beginning of trials 2 and 3. For trial 2, in the first 2 minutes alone, the amount of CO2 in the air increased by about 130ppm, almost a third of the overall increase of carbon dioxide. For the third trial, the rate was somewhat less drastic, increasing by 120ppm, but still was about one third of the total change in concentration of carbon dioxide.

I believe that the experiment was quite successful in hindsight. We saw the effects of respiration on the carbon dioxide content of the beaker, and we also observed that sound has an effect on the respiration of meal worms. In both of the sound trials, the respiration of the worms greatly increased in comparison to the silent first trial. We are not sure why this occurred, but it could be for a variety of reasons. First of all, the change could have been due to the experiment's change in location for the second and third trials. So that we would not bother or disturb other lab groups and their experiments with our sound, the lab group had to move outside. Perhaps the increase in respiration was due to the sunlight, temperature, or different colored surroundings that the worms were placed in. This could be fixed by confining the worms to a certain color background and temperature exposed to steady artificial lighting to control those variables. Second of all, the increase in carbon dioxide in the beaker could be because we used different worms for each trial, but the same beaker, so there was already an abnormal amount of carbon dioxide in the beaker. When the sensor was placed in the beaker, it would have taken it a little bit to totally adjust to the true amount of CO2 in the beaker, which explains the supposedly high rate of respiration in the opening minutes of both Trial 2 and 3. This could be easily fixed by having three concurrent trials in three different beakers that had been previously exposed to similar atmospheric environments. Thirdly, the worms might have actually had a higher rate of respiration during the trials with sound because meal worms seek dark and quiet habitats in nature, such as under rocks and in caves, so the sound could be instinctively programmed into their tiny brains as dangerous. More sound would mean less isolation and therefore more vulnerability for the worm. Also, the higher pitch sound's increased effect on respiration could have been because meal worms are primarily eaten by birds, many of whom have distinctively high pitched calls. As a result, when exposed to higher pitched sounds, the worms heart rate increases and they panic to try and escape the perceived danger. I think an interesting follow up experiment would be one using specific bird calls of birds that eat meal worms. In addition, perhaps exposing the worms to pheromones from these birds, thus supplementing the illusion of an impending attack, to examine the effects of more panic on cellular respiration on meal worms. 

Investigation 4.2 on Osmosis

Introduction
Membranes are one of the miracles of life. Present in all life forms, membranes regulate the flow of materials in and out of the cell and make sure that the every cell, from the smallest bacterium to the largest multicellular organism like humans, receive exactly what it needs and exactly how much. In most cells, there is a lipid bilayer that has both hydrophilic and hydrophobic regions to regulate the flow of water in and out of a cell. The transfer of water across a membrane is called osmosis, and the lab group and I carried out another experiment to model this.

A Cell's Phoso

On September 25th, Vinay, Shreyan, Vikram and I did an experiment to determine the effects of different solutions on osmosis out of a cell through measuring the rates of weight change in the tubes using dialysis tubes and solutions of different solutes. A dialysis tube is a selectively permeable membrane that will allow water and certain other solutes to flow through. The direction of the flow of the water is determined by the water potential of both solutions, but the flow will always be into the tube because we filled the tube with pure distilled water and the outside of the tube was comprised of water and a certain solute.

The solute potential of the solution outside of the dialysis tube is determined partially by temperature, but also by the ionization constant of each substance. The substance's ionization constant is the number of particles that it breaks up into when put into a solution, so for example, glucose, a sugar that doesn't ionize in a solution, has an ionization constant of 1 because there is one particle in solution for each particle of solute added. Sodium chloride, an ionic compound that ionizes into two particles while in solution, has an ionization constant of 2. This means that in solutions with higher ionization constants, the solute potential is higher.

The question is, though, how would the group observe osmosis if it occurred at such a small scale? We weren't able to actively see the water molecules go in and out of the tubing, and there was no other way to tell if water was entering the tubing. But that is the solution as well as the problem because we weighed the tubing and its contents every 2 minutes, noting the change in weight and attributing this to the flow of water into the dialysis tube. This was how we would quantitatively measure our data.

The purpose of this experiment was to model water potential and the effects of osmosis as it would occur in a cell. We wanted to see what effects different solutes had and also wanted more practice in the lab carrying out specific procedures so that our work could be replicated if it needs be. The group hypothesized that, because sodium chloride has a larger ionization constant, the difference in solute potentials in the solution and in the dialysis tubes would cause osmosis to occur the fastest when the tubing was put into a solution of NaCl.

Procedure
The lab group started our lab by first filling a beaker with deionized, distilled water, which would be the site of our osmosis experiment. Then we obtained a length of moist dialysis tubing. We didn't want dry tubing because the membrane could dry out and affect osmosis. Next we tied a knot on one end of the tubing that we decided was the "bottom" of the length of tubing. From that point on we kept this end towards the floor so as not to spill the contents of the tubing. Then we filled the tubing with 10mL of a 1.0M glucose solution and tied off the "top" of the tube. Because the membrane was selectively permeable, no fluid dripped out of the tubing even though we were moving the tube around.

We brought the tubing back to our experimental area and weighed the tubing and its contents as our base so that we could observe the changes in weight to the tube. Next we placed the tube in distilled water beaker from the first step of the experiment and waited. From that point on, we would remove the tubing, dry off any excess water and weigh the entire system every two minutes until 15 inutes had passed.

After we finished with the glucose tube, we repeated the same procedure but substituted 10mL of 1.0M solution of sucrose and 10mL of a 1.0M solution of NaCl for the glucose solution we had used. We did this to observe the changes in osmosis that occur due to different solutes and to create more experimental groups.

Data/Results

This is the data we directly received from weighing the dialysis tubing every 2 minutes.

	Mass of the Dialysis Tubing in grams
Time	Glucose	Sucrose	NaCl
0 min	11.7	12.0	11.8
2 min	12.2	12.4	12.3
4 min	12.7	12.6	12.6
6 min	13.1	12.8	13.0
8 min	13.3	13.2	13.2
10 min	13.6	13.5	13.6
12 min	13.6	13.8	13.8
15 min	13.7	14.0	13.9

The percent change was calculated using the measurements obtained from weighing the tubing. The initial weight was subtracted from the weight at each interval, then divided by that interval's weight and the whole thing multiplied by 100 to get a percentage.

	Percent Change in Dialysis Tubing Mass (%)
Time	Glucose	Sucrose	NaCl
0 min	0	0	0
2 min	4.27	3.33	4.24
4 min	8.55	5.00	6.78
6 min	11.96	6.67	10.17
8 min	13.68	10.00	11.86
10 min	16.23	12.50	15.25
12 min	16.23	15.00	16.95
15 min	17.09	16.67	17.80

This is a graph of the percent change in mass of the three different experimental tubes.

Conclusion
The results of the experiment were much as the lab group had predicted. We believed that water would flow from the solution into the dialysis tubing and would be seen in an increase of the tubing's weight. This definitely happened, with the glucose tube gaining 2 grams of water, sucrose gaining 2 grams as well, and sodium chloride gaining 2.1 grams. After 15 minutes, each of the tubes had similar percent changes, but as our group had predicted correctly, the greatest percent change occurred with the tube containing the NaCl solution. Whereas sucrose only increased its mass by 16.67% and glucose only saw an increase of 17.09%, the tube with NaCl saw a mass percentage gain of 17.80%!

The experiment was quite successful in hindsight. The concept of water potentials and osmosis that we had learned in class were confirmed by the fact that all three tubes gained mass. This was due to the fact that the solute potential inside the pseudo-"cells" was lower than that outside of the "cell" because the ratio of water to solute was lower inside than outside. Therefore, water rushed in, but since the solute could not diffuse out because of the selectively permeable dialysis tube, the particles were stuck and the tubing gained mass. Our hypothesis about NaCl gaining the most percent change was correct, so the effect of the ionization constant on water and solute potential has quite an effect on osmosis. Though the sodium chloride test did gain more mass by percentage, we had expected the mass to increase quite a bit more drastically. Because the ionization constant was twice that of glucose and sucrose, we believed that the sodium chloride would have somewhere around twice the percentage mass gained, not just a measly 0.71% more than the sucrose. This could be due to experimental error of not weighing our masses correctly, or perhaps as a lab group we overstated the effects of the ionization of sodium chloride on osmosis. For a follow-up experiment, we could use solutions of higher molarity to more clearly demonstrate the effects of osmosis over the relatively short period of time that is 15 minutes. Because the solute potentials would be so much different between higher molarity solutions and pure water, osmosis would occur at a faster rate. Another possible remedy for this problem would be the short amount of time that we tested each experiment. Perhaps the NaCl system had not reached equilibrium and therefore was not as telling of its effects on osmosis than it could be. A different follow up equation would be to test the effects of temperature on osmosis. To calculate solute potential of a solution, one must factor in temperature, so temperature change must have an effect on osmosis.