Fall2011_Lab8

=Lab 8 - Metabolism=

Lab idea:
Metabolism is how the organism uses energy to maintain itself. It has to repair itself, build organs and muscle, and sometimes tear-down things that are broken. We can measure how much metabolism is going on in an animal by measuring the waste they produce from the metabolism (eg, carbon dioxide). In this lab we will measure the CO2 produced by mice, and by crawfish. We'll look at how body size and environmental temperature can affect how much metabolism is happening.

Monday:
We'll work with mice and the students, to measure different amounts of oxygen consumption.

Wed:
We'll have a quiz over Digestion and Hormone lab. Some of the questions will be over the lab procedure itself, such as how the experiments were set up, and why they were set up the way they were. What was the hypothesis, and what do you predict will happen, based on the set up of the different experiments, and what you know about digestion and hormones?

Mini-Lecture:
This lab is really all about maintaining temperature. If an organism is doing what it needs to live, it must use the energy in food to maintain its system, but this use of energy produces waste in the form of matter and heat. Too much heat, and an organism's biomolecules will melt, too little heat, and an organism will freeze. Enzymes also need an optimum temperature to work, for example. So how to organisms maintain the happy medium that they need for just the right amount of heat (remember the Goldilocks story)?

Endotherms use an internal heating-cooling mechanism to keep things in the right range of temperatures. Ectotherms use external sources of heat/cool to keep them cold. Endotherms include birds and mammals, and ectotherms include snakes, lizards, frogs, etc. The ectotherms are the focus of this lab. How is the metabolic rate affected by changes in the external environment? Metabolism is measured in O2 consumption per gram of the animal per hour that the animal is being observed. If it's hot outside, an endotherm will cool itself off. If it's cold outside, an endotherm will heat itself up. (Ecotherms can't do any of this, which is why they usually live in warm climates.) What do you think will happen to the organisms metabolism when it is cooling itself off or heating itself up? (In a way, the different possible environments (hot or cold) are the independent variables in the experiments of the crayfish.) Metabolism is directly affected by all the things an animal must do to keep itself alive, including heating and cooling behavior. The more an animal has to cool itself or heat itself, the more energy it needs to use to keep blood flowing, dilate blood vessels, sweat, etc, and this means the animal will consume more O2 in order to use the energy in the muscles and organs that will keep itself alive.

Another interesting thing to observe it how different sizes (and shapes) of endotherms can be factors in their metabolic rate. Would a big animal have a different rate of metabolism than a small animal? Which do you think would be a higher rate of metabolism, the small mouse or the large human? How would body size affect the ability of the organism to maintain its temperature (in the Goldilocks range)?

Think of the cast iron skillet vs. the thin aluminum skillet. After cooking with both of these kinds of skillet, the cast-iron skillet is hot for like 10 minutes after you are done cooking with it. The same concept can apply to large and small organisms. But the organism, after it loses its heat, must kick-start its heating/cooling system to maintain a steady temperature. So this loss of heat (using the skillet-principle) in small vs. large animals is an important factor in explaining the metabolic rate of the small animal compared to the large animal. In terms of physics, we can talk about heat conductivity. Heat can travel through stuff (like body mass), but the more body mass a thing has, the longer it takes for the heat to escape. Also, the more surface area there is, the faster heat will escape (think of a rabbit with huge floppy ears, compared to a rabbit with short stubby ears).

You will also plot your data on a special type of graph. Most graphs we've used had numbers on the axis that went like this: 1, 2, 3, 4, ... But the numbers for SMR are in such a huge range (on the order of 1 to 10 million grams), we will use a special graph that uses a different order for the numbers. The new order will just take the old numbers, but use them as exponents, and put a 10 underneath. So the new numbers go 10^1, 10^2, 10^3, 10^4, ..., where the "^" symbol is the sign for exponent. So 10^1 = 10 1.

Here is an example of a graph that has two axis with these logarithmic scales:

http://upload.wikimedia.org/wikipedia/commons/9/92/LogLog_exponentials.svg http://intmstat.com/exponential-logarithmic-functions/Image125.png

In these mouse and crayfish experiments, think about what the independent variables are, and what the dependent variables are. What are we measuring in the final calculation? This is usually the dependent variable. What kind of factors do we think will change the outcome of the dependent variable? What does the dependent variable depend on, so to speak? These are the independent variables.

Lab procedure for Crawfish (Wed):

 * 1) **GET 2 CRAWFISH PER GROUP**. At the beginning of class, each group will get 2 crawfish, one big and one small. Put them into a chamber (a mason jar, 240 mL) filled with water. Pour the water slowly so no extra CO2 gets into the sample (smooth, bubble-less pouring). Put lids on the crayfish-filled-jars and let them sit for 30 minutes. Write down the time the crayfishes began to sit in the jar. All groups should be together at this point, with cold-crayfish in jars. ~:00 into class at this point.
 * 2) **GET CONTROL WATER**. Also at the beginning of class, each group will get in pairs of 2 groups. One group will get some water and put it in a little bottle (BOD bottle, 60 mL). This will be the cold-control for both groups. The other group will get some more water, put it in a 3rd jar (a mason jar), put the jar in the hot water bath, and put aeration stones in the jar. This will be your hot-control for both groups. Let this control sit 10-15 minutes to warm up.
 * 3) **TITRATE CONTROLS**
 * 4) I will explain titration, and let the cold-control group titrate, while the hot-control watches. :20
 * 5) Then the hot-control will titrate. After titration, write down how much solution was used to titrate for the cold-control and hot-control. :30
 * 6) If we have time, here we'll take a quiz, or at least part of a quiz.
 * 7) **SAMPLE COLD JARS, PREPARE WARM JARS**.
 * 8) After around 30 minutes for the cold-crayfish (:30), each group will get a sample of the water from each jar (small-cold and big-cold). Be sure to pour the water slowly so no bubbles get into the small BOD bottle. :35
 * 9) **TITRATE COLD SAMPLES**. Split up the group to prepare the warm-crayfish jars and the other folks can titrate.
 * 10) Crayfish group members: Put the crayfish back into the jars and fill them with extra water, and put aeration stones in them.
 * 11) Titration group members: Titrate the cold-small and cold-big samples. :50
 * 12) **BEGIN WARM JARS**. After 10 minutes, take out the stones from the warm crayfish-jars and put the lids on the jar. This is your small-warm and big-warm crayfish. Write down the time you put the lid on.
 * 13) Wait around 30 minutes for them to use up some O2. All groups should be together at this point, with all the cold-crayfish that are now in warm-crayfish jars. :50
 * 14) If we have time here, we'll take a quiz, or finish a quiz. Or more presentations.
 * 15) **SAMPLE AND TITRATE WARM JARS.** After 30 minutes, get samples from the small-warm and big-warm crayfish water. 1:20
 * 16) Titrate the samples. 1:40
 * 17) If we have time, we'll do more presentations or take the quiz.

Titration:
You will use the little glass bottles with numbers on them. Write down the number of your jar for the different sample-types in your worksheet. So the 'cold control' should be #147, for example. This way you can find your bottle that you need. To titrate, we measure how much of some sort of chemical is in our sample. We can do this by combining chemicals, and using a chemical color-indicator that changes color when there a certain chemical reaction takes place. In this case, we want to measure how much O2 is in the water, and we can use a reaction that changes color when there is no more O2 in the water. But the only way we can do this is if we alter the O2 gas into something that will react with our color-indicator chemical. So we use two types of other chemicals to change the original water sample into a titration sample.

1st take the sample, and use one packet of a chemical from the plastic bag. Tear open the packet and put the chemical in the jar. Shake it up with the lid on the bottle. When this reaction is done, use the 2nd packet from the white plastic container, and shake it up until the color goes away. Then you can pour this into a beaker, and use the titration apparatus.

At the titration apparatus, write down how much chemical is in the long tube (the buret). You will slowly drip the buret (glass tube) chemical into the sample, slowly shaking the sample so everything is well-mixed. Put a white piece of paper under the sample so you can see when the sample changes color. Mark how much chemical was used, based on the volume marking on the buret tube. Now you know how much titration chemical it took to turn the sample into the new color. This is directly related to how much O2 is in the sample. Do the calculations based on the lab manual procedure, or read the hints below.

CRAWFISH LAB HINT:
Calculating the crayfish SMR.pdf -- This is a file helping you calculate the SMR data. I've also explained it below:

You will use this lab to plot the SMR data for the crayfish for the lab. Email me your SMR data, and I will post it on this website. The calculations might be tricky, but just email me if you have any question.


 * 1) Convert the **Standard Solution** you used (from the buret) into **O2 concentration** for hot and cold control temp, and also for the cold-big and warm-big & cold-small and hot-small crawfish (so 6 total).
 * 2) Calculate the **amount of O2** in the different jars (controls and experimental ones with the crawfish). For this calculation, you have to multiply the concentration of the O2 in the control jar with the **amount of liquid in the control** **jar** (in Liters). The jars held 240mL (minus any crayfish), but you'll have to convert the final amount into liters* (so 240 mL = .24 L). When calculating the **amount of liquid** in the jars with crayfish, just subtract 1mL for each 1 gram of crayfish (so if the crayfish weighed 1g, then you would have 240-1=239, which is 239 mL = .239 L). Then multiply the **amount of liquid** with the **concentration of O2** for that particular crayfish (or, if you are calculating the **amount of O2 for the control**, use the **control O2 concentration**). So the O2 concentration you are using in this multiplication will __not be the same__for the control and the sample.
 * 3) Remember, this amount gives you an **amount of O2**, for any jar. If the jar had a crayfish in it, it's the amount of O2 that is left in the jar after the crayfish had used some of it. So the **__control__ amount of O2** should be **larger** than the **amount of O2 for the __crayfish__**.
 * 4) Once you have the **amount of O2** in the different jars (you should have 6 values of different O2 amounts), you can see how much **O2 was consumed by the crayfish** by comparing the **amount of O2 in the control** with the **amount of O2 left in the crayfish jar**. When you are done, you should have 4 values.
 * 5) So, for example, the amount **O2 consumed** in the warm crawfish will be this: the amount of O2 in the warm crawfish minus the **amount of O2** in the **warm control**. This difference will give you the amount of **O2 consumed**.
 * 6) Use this amount of **O2 consumed** for the big and small warm/cold crawfish (4 values total), to calculate the **SMR**. SMR is in O2/g/h, so take your O2 amount, and divide by the **weight of the animal** (in grams) and then divide that result by the **amount of hours** (30 minutes = .5 hours).

Here is an example. Let say I used 3 mL of the standard solution after I titrated my cold small crayfish after 35 minutes (or 35/60 hours = .58 hours). I divide this by the standard solution number (something like 1.43?), and I get 2.2 O2 ml/L. This is my O2 concentration for the jar I am working with. I then have to figure out the volume of water in the jar with the small cold crayfish. The jar holds 240 mL This crayfish weighs 2 g, so this displaces 1 mL of water in the jar. The volume of water in the cold crayfish is 240 minus 2, or 238 mL. I convert this to L, using 100 mL = .1 L. Then, I take this volume in Liters and multiply it by the O2 concentration I calculated from the standard solution that I used when I titrated the sample from the cold crayfish jar. This number is 2.2, so 2.2 mL/L times .238 L gives me something like .5 mL of O2 in the jar with the cold crayfish. This is the O2 in the jar, after the crayfish sat for 30 minutes metabolizing the O2. I can do this same round of calculations, starting with the standard solution I used for the control jar sample (not the same as the amount of standard solution used for the cold crayfish sample), and figure out the O2 was in the cold control jar. Lets say it's .6 mL O2 (I'm making these numbers up, by the way). If I find the difference between the control jar and the experimental jar, then I can see how much O2 is "missing" from the jar due to the crayfish "breathing" it in. So .6 minus .5 is .1. This is gives me .1 O2 mL consumed for the cold small crayfish. SMR is the amount of O2 used by an organism, taking into account how long the organism was metabolizing and how big the organism actually is. So I can take this amount of O2 consumed and divide by how many grams the small crayfish weighs (2 g), and then divide that answer by how many hours the small crayfish experiment lasted (.58 hours). I do this and I get .086 O2/g/h as my SMR. I can do this entire process again for the small crayfish in the warm water, and for the big crayfish in the cold and warm water. Then I can put this info in the table on p. 100, and plot the SMR data on the log-log graph.

Email me this data that your group calculated (that is, email me all the info you have on p. 100 in your lab manual). This table is tricky, because in one row, it will ask for the control O2 concentration, and (in the first, smaller data table that you won't turn in) your control O2 concentration were not listed in the same row as your crayfish info. So for the table on p. 100 that you will turn in, for 2 animals you should have just 2 lines filled out, one row for each crawfish. --> Then I will post these numbers on this website, so you can fill out your worksheet.


 * When you are calculating your SMR, be sure to convert the mL volume of your jar into Liters. So 1000 mL = 1 L. You use this number, when you multiply the total volume of water (in Liters) by the O2 concentration. Then you can calculate your SMR using the same formula as with the mouse or human experiments on Monday.

media type="custom" key="10838049" 10-noon Lab:
 * Subject || Body Weight(g) || Low Control O2 Conc. || Low Temp O2 Conc. || Low Temp SMR || High Control O2 Conc. || High Temp O2 Conc. || High Temp SMR ||
 * 1-small (B) || 3.12 || 2.38 || 1.92 || .075 || 2.27 || 1.61 || .105 ||
 * 2-big (B) || 7.48 || 2.38 || 1.85 || .038 || 2.27 || .91 || .089 ||
 * 1-small (M) || 6.44 || 2.38 || .98 || .081 || 2.27 || .7 || .09 ||
 * 2-big (M) || 8.47 || 2.38 || 1.4 || .105 || 2.27 || 1.12 || .088 ||
 * 1-small (A) ||  ||   ||   ||   ||   ||   ||   ||
 * 2-big (A) ||  ||   ||   ||   ||   ||   ||   ||

8-10 lab:


 * Subject || Body Weight(g) || Low Control O2 Conc. || Low Temp O2 Conc. || Low Temp SMR || High Control O2 Conc. || High Temp O2 Conc. || High Temp SMR ||
 * 1-small (J) || 3.45 || 3 || 1.89 || 0.16 || 2.7 || 1.47 || 0.17 ||
 * 2-big (J) || 7.2 || 3 || 1.4 || 0.11 || 2.7 || 0.98 || 0.12 ||
 * 1 (M) || 5.27 || 2.67 || 1.69 || .093 || 1.96 || 1.12 || .079 ||
 * 2 (M) || 8.48 || 2.67 || 1.73 || .057 || 1.96 || .42 || .088 ||
 * 1-small (M) || 6.8 || 2.8 || 1.5 || .094 || 2.0 || 1.3 || .11 ||
 * 2-big (M) || 12.12 || 2.8 || 1.1 || .070 || 2.0 || .90 || .076 ||
 * 1-small (A) || 6.44 || 3.85 || 3.08 || .7 || 4.05 || .898 || 2 ||
 * 2-big (A) || 7.48 || 3.85 || 2.077 || .156 || 4.05 || .898 || .843 ||