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Biology: Lab 5: Cell Respiration

Excerpts from AP Biology Teachers' Discussion Group

General Overview
Equipment and Supply Modifications
Pre-Lab Preparations
Procedure Modifications
Trouble Shooting
Conducting Lab Using CBL System
Alternate Lab Ideas

General Overview
"Regarding pea germination -- there are really two experiments going on here.

Experiment 1: How does the respiration rate of germinating peas compare with the respiration rate of nongerminating peas? Keep in mind that nongerminating peas are alive, but simply going through a dormant state as has already been explained by other listmates.

Experiment 2: How does temperature affect the rate of respiration? For this we are comparing both the germinating and the nongerminating peas at two different temperatures. However, the rate of the nongerminating peas at the two different temperatures is not too revealing since it is near zero in both cases.

Now, regarding the glass beads -- the purpose of this part of the experiment is to account for any pressure changes in our respirometers, which are due to factors other than pea respiration. We subtract these pressure changes from those observed in the other respirometers in order to get the CORRECTED PRESSURE DIFFERENCE. There are several factors that may cause a pressure change other than pea respiration. (1) A change in pressure due to a temperature change in the system during the course of the experiment. (2) When the experiment starts there is already some CO2 in the respirometer. Having that react with the KOH would cause a very, very slight drop in the pressure. (3) There may be other factors, in theory, such as an interaction of the cotton with KOH or whatever. This experiment is simple, but elegant and probably teaches our students much about how to correctly set up a controlled experiment. It is my personal favorite."
-- Bob Goodman, Hunter College High School, New York City. 2/11/99

Equipment and Supply Modifications
Tip: "For trays I use very large, white, plastic trays that are deep enough for the experiment. I will check on Monday for a supplier. Some students do seem to have a problem sighting the water level and so I took sheets of different colored paper and laminated them. These are placed under the respirometers as a background. It is interesting to see that some students prefer no background, and others different colors. There does not seem to be any pattern. One modification that I have made to this lab is to increase the equilibration time and this seems to give more consistent results. Before we did this, we had those lively glass beads respiring faster than the old 'dead' peas."
-- Peter Gardiner, St. Michaels University School, Victoria, British Columbia, Canada. 10/16/99

Tip: Two possibilities for trays that are cheap: First, the wallpapering trays from Wal Mart or Home Depot or such. I have gray ones and the pipettes are more visible than they are in black or aluminum trays. Second, the trays that hold the little plants at the garden store. They are usually very cheap around this time of year. Unfortunately, they are usually black or dark green. However, it's always nice when some young student with 17-year-old eyes has to ask me to find the meniscus for them!!"
-- Israel Solon, Greenhill School, Dallas, Texas. 10/16/99

Question: "I'm having trouble finding nonabsorbent cotton for the lab."

Answer 1: "Carolina Biological sells it. I have not tried it but others say if you use KOH pellets you do not need it. It can be called bacterial plugging cotton (AA-71-2600). It costs about $8.50 a roll, which will last you for years for that lab."
-- Charlotte Freeman, Girls Preparatory School, Chattanooga, Tennessee. 11/11/99

Answer 2: "Polyester batting or polyester fill works. Ask the home economics teacher for some if you don't have any around the house; a little bit goes a long way."
-- Nancy Hein, Hawley High School, Hawley, Texas. 11/11/99

Tip:"....I have used or known people to use pinto beans, kidney beans, and even lima beans from the store. Soak them overnight, then spread them out between layers of very soggy paper towels for a couple of days. After two days in the towels, it is easy to see which seeds are starting to grow hypocotyls -- I instruct the students to pick out some nice vigorous ones, and we usually have good results.
-- Israel Solon, Greenhill School, Dallas, Texas. 10/16/99

Tip: "I have been using germinating peas that are purchased at a health food store. Being in California we have all sorts of 'weird' things at organic markets. The peas are found in plastic containers in the produce section and work great. It saves me from having to begin the germination process ahead of time and from dealing with the potential of fungal infection. You can also get a mixed selection of germinating seed (lentils, beans, etc.) that can be used to compare respiration rates of different seeds. Overall, it works great."
-- David Knight, University High School, Irvine, California. 10/15/99

Tip: "The variety of peas matters; the sweeter ones have a lower water potential and swell up more when you soak them. You have to do a 'dry run' in advance, soaking them and seeing how many will fit in your respirometer."
-- Barbara Beitch, Hamden Hall Country Day School, Hamden, Connecticut. 10/24/99

Tip: "One of the best tips I ever had for the respiration lab was to forget the KOH solution and use KOH pellets instead -- roughly enough to cover the bottom of the vial. This is much easier for students to handle, and it works."
-- Leslie Haines, Walter Williams High School, Burlington, North Carolina. 10/25/99

Tip: "Problems about respirometer set ups keep coming across the AP Biology mailing list: Maybe this will help. Years ago, I discovered that by using a large test tube fitted with a #4 rubber stopper and a Kimble 2 mL disposable plastic pipette (which fits very snuggly), there were never leaks! I also use 40 peas (which gives more gas consumption) and soda lime pellets instead of KOH liquid that gets all over everything, and I put my large hex nut and bolt from the microtome part of transpiration inside as my weight. Using wallpaper troughs (put foil on the bottom if visualizing the bubble is a problem), we have pretty flawless results (although we almost always pick a day when the barometric pressure is changing)."
-- Carolyn Schofield, Robert E. Lee High School, Tyler, Texas. 11/1/99

Pre-Lab Preparations
Question: "Can KOH go bad? Should I mix it fresh from year to year?"

Answer: "Yes! KOH solution does indeed go bad, because it gradually absorbs CO2 from the air, forming K2CO3 which slowly precipitates. However, this does not happen rapidly, so KOH solution should be good for several weeks if it is kept tightly sealed. Similarly with KOH pellets -- they react with atmospheric CO2 and are gradually converted to potassium carbonate. Again, it doesn't happen overnight or even in a few weeks, but eventually it will. It happens more slowly if the container is kept tightly closed. But as Dave Hall said in an earlier post, you really need to make up a fresh solution rather than keeping it year to year. And if you have a bottle of KOH pellets more than 5 to 7 years old, it may be time to throw it out and get a new supply, unless you have been diligent in keeping it tightly sealed when not in use."
-- C.O. Patterson, Texas A & M University, College Station, Texas. 10/30/00

Tip: "I soak the peas overnight. The next day I wrap them up in small amounts in the normal brown school paper towels. The paper towels should be wet. I just sprinkle on some water as I am rolling them up. I put the rolled up peas and paper towels in a grocery store paper bag, sprinkle on some water with my hand until they look wet -- but no water is running out. Then I fold down the paper sack and put a big rubber band around it. You can put the entire thing into another grocery sack if you have gotten things too wet. Then I put them in my bacteria oven overnight -- about 90º F. Any warm place will do. They have never failed to sprout. If you leave them a second night, put some more water on them. I've never had fungus. Also, I buy English peas at the local farm feed and seed store. They always have several species. I just pick one.

Once I kept my peas over to the next year. I just left them in the paper bag from the store. The next year I had a disaster. Some sort of very small insect eggs were in the peas. They hatched. Every pipette was filled with those pesky little critters, and they had laid eggs all over everything. What a mess. Since then I have never kept peas from year to year.

If you have never done the lab before, this might be helpful. Warn the students not to shove the end of the pipette into a pea when they stopper the respirometer. The mashed pea will prevent gas exchange. This has happened several times in my classes, and they can't figure out why they aren't seeing anything happen."
-- Thomas Strayhorn, Snyder, Texas. 10/16/99

Tip: "I have discovered that it takes a lot of water to germinate those peas. I put them in a large beaker and cover them with water -- repeatedly for two days. The wet paper towel method used for most seeds is not sufficient."
-- Deborah A. Hill, Norman High School, Norman, Oklahoma. 4/21/00

Question: "Is it possible to set up the pea vials the day before?"

Answer: "Two years ago, I set the vials up on day one, sealed them with parafilm, and put them in the fridge overnight. It worked great. I would do it again without hesitation."
-- Ruth Tummey, Manahawkin, New Jersey. 10/23/00

Procedure Modifications
Tip: "If anyone has problems doing the respiration lab in the time allotted, here is a suggestion: Germinate the peas three days ahead of time and take readings every two minutes instead of every five minutes. I tried this and it worked very well. It was the best data we ever collected in the eight years I have been teaching AP Biology. It offers the following advantages: only 10 minutes of data collection instead of 30, less time for temperature and pressure changes, less time for the respirometers to develop leaks, and the students can actually see the water move up the pipette in the respirometers containing germinated peas."
-- Steve Ianniello, Enka High School, Enka, North Carolina. 9/29/00

Tip: "…. if you are time challenged, as I am, you may have to take some shortcuts. I count out the needed number of peas for the students. Once they set up the respirometers, I look at the clock and see how much time remains in the 65-minute lab, and modify the time between readings accordingly. Don't cut down the equilibration time any more than you have to, however; it affects the data. Of course, they do have the glass bead control to deal with the temperature equilibration problem. Anyway, that can be instructive -- interpreting the data. They sometimes conclude that GLASS BEADS RESPIRE!"
-- Barb Beitch, Hamden Hall Country Day School, Hamden, Connecticut. 10/16/99

Tip: "A hint for those hard-to-read pipettes. Just before submerging the pipettes under the waterline, place a dot of food color at the tip (the open end) of the pipette. After the pipette is submerged, the color will migrate with the water as it moves downward/inward. It's a good idea to place white paper under the apparatus to make reading easier, unless your pans are white.

I have found it is necessary to reapply the silicon cement on a yearly basis: off with the old, on with the new... just to be safe. I can usually get a student to volunteer to do it during a free period or lunch period."
-- Pam Tidswell, Rancocas Valley Regional High School, Mt. Holly, New Jersey. 10/18/99

Question: "In the pea lab, at first, the water moved down the pipette as the peas were respiring but then the water started backing out of the pipette."

Answer 1: "Did that happen in the controls also? It could be due to a change in atmospheric pressure (storm approaching, for instance), in which case it would affect all tubes. Another possibility that happened in my class last year is that a student caused localized warming in the water bath by playing with the ice cubes!"
-- Margaret Kaminsky, Penfield High School, Penfield, New York. 10/29/00

Answer 2: "Often times the students do not moderate the water bath temperatures properly. (In fact, I've caught a few with the thermometers in upside down!) This, plus slight changes in atmospheric pressure (weather) and loose stoppers on the respirometers, can cause the gas volume to alter. Check the control. By the way, I have the students keep the respirometers underwater by weighing them down with burette clamps. If the respirometers are floating and are not completely level under the bath, the water will flux in and out as the respirometers pivot." -- Stew Brittner, Millburn High School, Millburn, New Jersey. 10/30/00

Answer 3: "I had that same problem of the gas production pushing the water out of the pipette. I solved it by doubling the concentration of KOH and decreasing the amount of nonabsorbent cotton that I was using (in case it was preventing the CO2 from reaching the KOH). So much for only having one independent variable. It worked. I will determine which was more important next year. My gut feeling is that I had too much nonabsorbent cotton between the peas and KOH so that the gas could not diffuse to the KOH fast enough."
-- Barb Fuller, Hall High School, West Hartford, Connecticut. 10/30/00

Answer 4: "To prevent the water going out the pipette rather than in: (1) Instruct the students to make sure they have a tight seal between the stopper and the container that houses the seeds. Warn them to be careful about breaking the glass when inserting the pipette and the stopper if that is what you are using. (2) Don't use too much nonabsorbent cotton -- a thin layer should be fine. (3) Use a fresh solution of KOH and, unless the pellets are relatively new, increase the percentage of KOH in solution. (4) The students should not touch the apparatus, including the trays, once the experiment is underway -- slight movements can cause pressure changes."
-- Bruce Faitsch, Guilford High School. Guilford, Connecticut. 10/31/00

Conducting Lab Using CBL System
Question: "Which computer probes would you suggestion using for the cell respiration lab?"

Answer 1: "I recently completed the respiration lab using the CO2 probes -- the results were excellent, the set up was ridiculously minimal."
-- Israel Solon, Greenhill School, Dallas, Texas. 11/27/00

Answer 2: "The CO2 and O2 probes both work... and, yes, they require the least fussing. But I still opt to use the gas pressure sensor. The gas pressure sensor is least expensive. But that aside, when measuring pressure changes, this lab teaches the students more about experimental design than any other lab we do in AP Biology. The methodology is 'elegant.' In fact, I feel that the results are kind of obvious and it is the 'experimental' design that makes this experiment so cool to do. Using the glass beads to correct for environmental differences in pressure...and then subtracting those differences to get corrected pressure changes shows the students how one could cleverly rule out 'unwanted' variables in the experiment."
-- Bob Goodman, Hunter College High School, New York City. 11/27/00

Answer 3: "You can also do it with both the CO2 and O2 probes simultaneously using a PVC tee. We just measured the O2 levels of a mouse in a closed container (not the bottle) this morning and had excellent results."
-- Gretchen Stahmer DeMoss, Vernier Software & Technology, Beaverton, Oregon. 11/28/00

Tip: "I have found a way to save some time with this AP mass of material. A few tradeoffs but next year I plan to use my idea. This year I did the pea respiration lab three ways: (1) As prescribed in the AP manual and as I have done for years; I assigned one team to this. (2) Four groups used the Vernier pressure sensors and did the lab as written in the Vernier lab manual. (3) One group used the CO2 sensor from Vernier.

The major modification I made was to substitute soda lime pellets for the liquid KOH. Wow, did that simplify things and work better!

The old-fashioned way got good data and finished in the double period lab I have. Three of the four groups using the Vernier pressure sensors got good data, but because there are only two probes, they had to make three runs and, of course, did not finish during the lab period. I had to spend lunch hour doing the last run myself and then printing the results and cleaning up.

Hands down -- no question -- the CO2 sensor is the way to go. In fact it is so easy that it makes one wonder why we would even bother doing the lab. I will do it as a demonstration, projecting it for the whole class to watch next year. From start to finish you can accomplish the lab objectives in 30 minutes. For those of you not familiar with this, it is a probe from Vernier that measures CO2 directly in the air. It comes with a plastic bottle to fit the stopper and that is the 'respirometer.' I first took 25 germinating peas and put them in the bottle, put the sensor on, and clicked on 'collect' for five minutes. The data points climbed diagonally up the screen almost too perfectly. Then I put those peas in ice water, washed and dried the flask, put dry peas in there, and collected data for five minutes. I got a nearly flat line! While I was doing that the germinating peas were chilling. I put them in the flask and collected for another five minutes and got a nice line just slightly above the flat line and a small slope. Click on regression curve and a nice slope prints out!

It was classic perfect data -- no KOH, no controls, nothing -- and that is the trade off. It is so direct and simple that the students learn nothing about setting up controls, no math, etc. So I have to struggle with the slower, harder way that teaches more about getting up and designing an experiment and all that. But to be able to do the entire experiment in 30 minutes and practically no muss and fuss is really appealing. The probe is expensive (around $200 I think) and, of course, you need the interfacing equipment, but for schools that already have serial box interfaces or ULIs, purchasing one probe might be a good option. I am lucky enough to have a projector to attach to the computer, so I can have the entire class watch in real time. I played around with it, and found that if I put a handful of peas in, the results were more dramatic. I also discovered that one needs to wash and dry the flask completely -- apparently the residue left after removing the peas is still emitting CO2 and you get a reading. Drying the flask is hard because it has a small neck, therefore, I will probably try to buy three containers so I will not have to reuse the same one immediately. The probe needs 90 seconds to equilibrate and you need to be sure the probe is completely through the stopper so all holes are exposed. The rate is going to be calculated as ppm/minute, which is a little weird, but I can live with that.

It is certainly a way to do that lab and show the effect of germination on oxygen consumption and the effect of temperature. It makes one wonder if doing the lab is actually worth demonstrating the obvious. Data Logger Pro from Vernier has some really nice features, such as replaying the data in real time or 10 times faster or instantly. So, if saved on a disk, a good run could be replayed one year when the other things mess up and you do not get data."
-- Charlotte Freeman, Girls Preparatory School, Chattanooga, Tennessee. 1/18/00

Tip: ….This lab is notoriously problematic. I use CBLs, and we do dry peas with beads to control for volume, peas in three water baths -- room temperature, warm, and cool. The data is a little screwy -- dry peas and beads show respiration, etc. But, it is an excellent lab for getting students to look at the data and figure out what might be going on -- such as what sorts of things did we not control or not do a good job of controlling, leakage, inadequate time to come to constant experimental temperature, expansion of atmospheric air in respirometer, and so on. The students were at first very disappointed, but came to see how it could help them write a better lab design on a free-response question. Isn't AP great! These kids always seem to take lemons and make lemonade! If you don't already have them, try to get them. A CBL system allows you to collect 600 data points in 10 minutes versus the few that you can get otherwise. The students can then manipulate the data in several ways on the computer for different kinds of graphs and analyses."
-- Deborah A. Hill, Norman High School, Norman, Oklahoma. 4/21/00

Tip: "In the respiration lab, one of the objectives is listed as 'how a respirometer works in terms of the gas laws.' My feeling is that when we use probes, we should make sure that our students also have the background and knowledge to do the experiment if we did not have the probes available. The probes are very useful, but we should not lose sight of the concepts that the tools are 'doing for us.'"
-- Steve Rierson, Hopkins High School, Hopkins, Michigan. 6/22/01

Alternate Lab Ideas
Tip: "Alternate cell respiration lab: I use yeast and measure the production of carbon dioxide and it works well. In fact we test it at different temperatures and with different sugars (sucrose, glucose, and lactose). The yeast suspension goes into a syringe that is submerged in water. Gas is collected in a graduated cylinder. The students use a 10 mL syringe without needle. In the syringe goes 4 mL of a yeast suspension, 4 mL of a 10 to 20 percent sugar solution (a variable), and the rest, air. A short piece of rubber tubing (about 5 cm) goes on the end of the syringe and curls up into the 10 mL graduated cylinder.

The cylinder is full of water (and upside down) initially but emerges from the water bath. Fill it underwater. The syringe is angled slightly so that carbon dioxide -- and not the solution -- escapes. We collect for 10 min or 10 mL, whichever comes first. The water bath temperature can be a variable. With warm temperatures be sure to disregard the first few bubbles as they are just expanding air. Allow the first bubbles to come out before putting the rubber hose into the syringe. The rate of respiration is proportional to the CO2 production. I use the lab in three different ways:
  1. Three sugars: glucose, sucrose, and lactose. The lactose doesn't do much. The glucose and sucrose are statistically the same. We pool class data and do t-tests on this.
  2. Temperature: use sucrose at various temps from 5º C to 55º C and you get a respectable curve. Again data is pooled.
  3. Lactaid: Same set up using lactose at 35º C but with a pinch of ground-up Lactaid tablet. The yeast will eat it up. This can be compared to controls: yeast/lactose, yeast/Lactaid, lactose/Lactaid (no yeast)."
    -- Howard Lovejoy, International School Nido de Aguilas, Santiago, Chile. 10/17/99

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