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|  | General Overview
Equipment and Supply Modifications
Question: "Are kits the way to go with this lab?"
Answer 1: "I used the Edvoteck enzyme lab this year and had a lot of trouble with it. The directions were pretty bad. I called the company with some questions and they could not answer them. It seems that the person in the company that designed the lab left several years ago and no one there could give me any sample data. I ended up spending a lot of time rewriting that lab so that students could follow directions."
-- Sande Ivey, Bangor Area Senior High School, Bangor, Pennsylvania. 8/20/99
Answer 2: "The Carolina lab kits are great, but I had trouble with the desiccated catalase enzyme. Probably my fault, but I couldn't get any activity out of it. I went back to my favorite source -- beef liver -- and it worked fine."
-- Jo Ann Burman, Andress High School, El Paso, Texas. 7/13/00
Answer 3: "I found the same trouble with the catalase from Carolina. It was very weak when I purchased some a few years ago. I find that liver produces excellent results -- follow the directions for preparation in the teachers' guide to the lab, make it fresh, and keep it on ice -- even at the kids work stations. In fact, you will probably have to dilute it from the recommendations in the lab book in order to get usable results."
-- Bruce Faitsch, Guilford High School, Guilford, Connecticut. 7/18/00
Question: "Is there any reason why frozen beef liver wouldn't work just as well as fresh liver for the enzyme lab? I have been to four different area grocery stores and everyone is out of fresh liver."
Answer 1: "I have always had better luck using fresh beef liver as a source of catalase, rather than the stuff from Sigma. For some reason, the dehydrated catalase that I had from Sigma was inactive. Raw potato also works."
-- Jo Ann Burman, Andress High School, El Paso, Texas. 8/20/99
Answer 2: "I often found stores have small containers of unfrozen chicken livers; they work well and there is little waste."
-- Fred Brown, William Hall High School, West Hartford, Connecticut. 8/31/00
Pre-Lab Preparation
Question: "Could you please post directions for preparing the catalase from the yeast?"
Answer: "I just dissolve some yeast in distilled water. I then mix 1 mL of the yeast extract with 20 mL of H2O2 in a 125 mL flask and measure the pressure change using Biogas Pressure Sensor interfaced to a TI-83 (or 83+) with a CBL. I measure the pressure change for three minutes, but I select out the first 15 seconds of data and determine the slope and use that as a rate change -- a rate change that could be compared under different conditions. As far as the amount of yeast used... that varies with the age of the yeast on the grocery shelf, so I try different concentrations each time I make it. I dilute it until I get a change of about 200 mmHg over the three minutes. This can be compared at different temperatures and substrate concentrations."
-- Bob Goodman, Hunter College High School, New York City. 8/24/99
Procedure Modifications
Trouble Shooting and Cleanup
Question: "We just did the catalase lab and got good results again, but we never seem to get good data for the spontaneous (uncatalyzed) rate of decomposition section. The amount of H2O2 consumed is usually equal to or less than the baseline. Is this because the amount that decomposes is so small that it is not accurately measurable by this method? Are we doing something wrong? Any ideas?"
Answer: "The uncatalyzed reaction is very, very slow; try setting out some H2O2 for a week or so, and then measuring how much is present."
-- Franklin M. Bell, Science Teacher, St. Mary's Hall, San Antonio, Texas. 10/12/99
Conducting Lab Using Probes and Computer/Calculator
Tip: "I have used the BSCS blue lab (on enzyme action) with great results. This lab procedure is also easily adaptable to use with the TI-83 calc, CBL, and gas pressure probe. I usually run the basic lab procedure looking at amount of enzyme vs. H2O2 produced. Then I have the kids design their own experiments. looking at the effect of pH, temp, etc. on catalase. One of my kids looked at salinity last year and found that salt water shuts down catalase almost completely."
-- Franklin M. Bell, St. Mary's Hall, San Antonio, Texas. 9/7/99
Question: "I want to try the enzyme lab using CBL-2 and TI-83 plus calculators. I noticed the manual gives two options for the enzyme lab -- one using the O2 sensor and the other using the gas pressure sensor. Does anyone have experience with these? Is one easier? Does one get better results than the other?"
Answer 1: "I have had much success doing the enzyme lab with the biogas (or gas pressure) sensor from Vernier -- using the CBL system. We monitored the rate of the reaction and also did the experiment using different substrate concentrations and at different temperatures. For the substrate concentration we showed an increase in the rate with increasing concentration. We did not get to a saturating concentration and I didn't feel comfortable (from a safety standpoint) working with higher concentrations of hydrogen peroxide. I attach a pipette and pump directly to the rubber stopper so that I can add the enzyme at a set time and then select the first 15 seconds of readings for measuring the velocity (rate) of the reaction."
-- Bob Goodman, Hunter College High School, New York City. 11/03/00
Answer 2: "The gas pressure sensor works great for this lab using the CBL 2 and TI-83 plus. The lab takes about 10 minutes and then could be repeated at different temperatures or substrate concentrations. It is far superior to the KMNO4 titration, which, frankly, calls for too many measurements (six) considering the small differences the kids are supposed to find in the end. Also, the same probe could be used for the transpiration, respiration, and water potential labs, so if you are on a limited budget, this is my probe of choice. There are some other great labs that can be done with this probe that are not part of the AP syllabus."
-- Bob Goodman, Hunter College High School, New York City. 9/26/00
Answer 3: "I have done the enzyme lab using the gas pressure sensor for the last two years and have found that it is a good idea to test the reaction before letting the students do it, because it sometimes occurs so rapidly that the pressure builds and the top pops off the vial before any time has passed. Therefore, I usually test various dilutions before letting the students do them."
-- Bob Heun, Brooks School, North Andover, MA. 9/27/00
Alternative Lab Ideas
Floating Disc Enzyme Assay
Question: "Re: the alternative enzyme lab using catalase soaked discs dropped into hydrogen peroxide: In doing a test run of the lab this morning, I found that the discs floated immediately. I first used undiluted 3 percent peroxide and found I had to dilute it many times to get the disc to stay on the bottom for any time at all. Is that extreme dilution necessary? In scrounging around our filter paper supply, I found lots of different grades and have been testing different ones. Should any filter paper work assuming the peroxide is dilute enough?"
Answer: "I am a first-year AP teacher and I did the enzyme lab with my kids using one large potato, diluted H2O2, and lab filter paper I found in my room. We peeled and cubed the potato, put it into a blender with chilled distilled H2O almost to the top, and pureed it. After straining this 'soup' through four layers of cheesecloth, the students made up the mixtures of enzyme and DH2O. We used discs of filter paper traced around a nickel; after being soaked in the enzyme for 5 seconds, and blotted on paper towels for 10 seconds, they were dropped into the H2O2 and sank to the bottom. It was most satisfying to watch them float at varying times, exactly as the lab said they would. Keep the enzyme cold by leaving it in an ice bath once it is made. We ran the lab twice more, changing the temperature of the enzyme just for fun, and because we had time (100-minute classes and seven students)."
-- Diana L. Latta, Palatka High School, Palatka, Florida. 10/8/99
Tip: "The catalase lab I do with my anatomy and physiology classes allows for quantitative comparison of catalase activity at different concentrations and temperatures might be of interest to you. Filter paper discs (any kind -- I've even used notebook paper with good results) are soaked in catalase and then carefully placed high on one side wall of a clean square dropper bottle (make sure the discs are not dripping by wiping them against the lip of the beaker before using). Five or ten mL of peroxide is then carefully funneled into the bottom of the bottle so it doesn't touch the discs yet (the amount you use will depend on the size of your bottle). As a stopper for the bottle, use a one-hole stopper with the glass part of an eye dropper inserted in the hole. Fill a pan with about two inches deep with water, into which a 50 ml graduated cylinder, stripped of its base and collar, is submerged and then turned upside down so the water stays in the cylinder as long as the mouth stays under water. Lay the bottle on its side with the disc side up (important). It is placed in the water with the open end of the dropper inserted under the mouth of the cylinder. Carefully turn the bottle 180 degrees to expose the discs to the peroxide, and start timing (I usually have them take measurements every 30 seconds for 10 minutes, but have shortened the total time to 6 minutes before also). The oxygen evolved can be measured as it displaces the water in the graduated cylinder.
I have each group do a different number of discs to test the effects of concentration, and then have them do it using ice cold water and very warm water to test the effects of temperature. It has always worked very well for me as long as I use fresh peroxide (I use 3 percent straight out of the bottle) and liver homogenate (I have used small pieces of the same slice now for five years -- I keep it in the freezer and blend a small piece of it for a few seconds in cold water, then filter through cheesecloth and keep on ice). Sometimes the students have trouble getting the discs in place in the bottle -- curved forceps work best for this."
-- Marcia Sloan, Cleburne High School, Cleburne, Texas. 10/9/99
Tip: "My class just performed this lab (catalase) today. I have used it for the last several years and find it to be excellent. 1.5 percent hydrogen peroxide works very well. The key is that it must be fresh. I always start with a new bottle. Also the catalase must be fresh. (And kept on ice.) I use potato or liver (usually potato). I have never tried yeast, but yeast and I have never gotten along very well! Filter paper has not been a concern -- I just use what I happen to have around."
-- Cher Callahan, The Savannah Country Day School, Savannah, Georgia. 10/8/99
Answer 2: "I use coffee filters and they work fine. I find a great deal of variation in actual H2O2 concentrations -- at times, I have to start with 'full' strength drug store 3 percent and at other times I have to dilute 3 percent down by 50 percent dilution series until it is only 6.125 percent of the original 'full' strength. This may sound confusing but I simply inform my students that the bottle of H2O2 is stock and our dilutions are measured from that. Over the years my students have investigated this protocol ad nausea -- partly this is because I make this a take-home lab. They actually have to investigate a variable of this protocol at home since everything is so cheap and safe. There is a big difference between the catalase sources. Be sure to caution students on the use of chicken liver and salmonella. When using yeast you'll find quite a bit of difference depending on the time of activation and whether it is an actively growing culture or not -- lots of cool variables to investigate."
-- Brad Williamson, Olathe East High School, Olathe, Kansas. 10/8/99
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