Spring2013_Lab8

=Metabolism, Lab 8=

Quizzes:
We will have Quiz 5 on Tues and Quiz 6 on Thurs. Quiz 5 on Tuesday will cover Digestion (old material), Hormones (current material), and Metabolism (new material). Quiz 6 will cover Hormones (old material), Metabolism (new and current material). Current material will make up most of the quiz.

Experiments:
We'll be working on Tuesday with mice again, measuring respiration. We'll also use ourselves as test subjects on respiration. On Thursday, we will work with crayfish, and spend the entire lab measuring respiration in crayfish. We'll calculate the metablism of crayfish based on their respiration in water. So we will use a titration chemistry method to measure the chemicals in the water. We'll then combine our class data and you will analyze the entire class data in your worksheet.

Crawfish calculation hint:
-- 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).

Sample calculation:
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 3 mL by the standard solution number (1.43), and I get 2.2 O2 ml/L of H20. This is my O2 concentration for the jar I am working with. The units are "mL O2/L H20" for the water in that jar (control or experimental)

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 O2 concentration from 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 the O2 concentration for the cold control is .6 mL O2/L (I'm making these numbers up, by the way). And suppose the O2 concentration for the cold small crayfish is .5 mL O2/L.

Next, I can 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 mL O2 consumed" for the cold small crayfish.

SMR is O2/g/h. This 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 "mL 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. --> The link to the class data is right here:

Class data!
==> Please put in your group's data (O2 concentration, weight of animal, etc.) ==> Also, put in your group's SMR data.

8am Class data 10 am Class data


 * 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.

Worksheet hints:
For your worksheet, you'll need to make some charts. Each question (mouse, human, and crayfish) tells you to plot the data on the log-log chart. That's the chart with the weirdly-spaced lines. On a normal graph, the numbers on the axis go from 0, 10, 20, 30, etc. The steps between each interval is the same. So between 0 and 10 is goes 1, 2, 3, etc. all the way to 10. After 10 it goes to 11, 12, 13, 14, etc. all the way to 20.
 * Linear and log-log graphs:**

But on log-log paper, the axis interval changes. It starts with some number, and then goes up by multiples of 10. So it doesn't start with 0, because 0 times anything is still zero and that's a boring graph. It usually starts with .1, and then 1, 10, 100, 1000, etc.

The value of the lines between the axis intervals are weird, too. You take the first number, and then multiply by 2. So if first line .1, then after .1 the value of the next line is .2, then .3, .4, etc. Then the next interval begins with 1 (see the previous paragraph). After 1 the next line is 2, 3, 4, etc. Then 10. After 10 it is 20, 30, 40, etc. After 100 it is 200, 300, 400, etc. all the way to the next interval (1000). This continues until the end of the graph (something like 10 million).

You will plot all the data on Table 8.1 on the linear graph. You will also plot all of this data on the log-log graph. So you will plot it twice, but in to different graphs. Use different markers for ectotherms and for endotherms.
 * What to plot:**

Follow the instructions for the mouse and human questions as far as what markers to use.

In addition, you'll plot all your crayfish data on the log-log graph. Plot the crayfish so you know which is from the hot and cold temperatures.

You will also plot the crawfish data that includes SMR and temperature on linear paper. The room temperature was 25 C and the hot bath was about 35 C.

Lab procedure for Crawfish (Thurs):
Read through the introduction in your lab manual for metabolism and the crayfish experiment, and then read this to get an idea of the experimental procedure.

Pour the water slowly so no extra CO2 gets into the sample (smooth, bubble-less pouring).
 * 1) **PREPARE COLD JARS.**At the beginning of class, each group will get enough water to fill up all their jars (each jar is 240 mL). Pour the water slowly so no extra CO2 gets into the sample (smooth, bubble-less pouring).
 * 2) Each group will also get 2 crawfish, one big and one small. Put each crawfish in a jar each. 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. At this point you are about 0 minutes into the experiment (~:00)
 * 3) **MAKE CONTROL WATER**. Also at the beginning of class, each group will get in pairs of 2 groups. One person from each group will help make the cold water control.
 * 4) Here I will explain the titration process. Fill up the little jars (BOD bottles) with the liquid you want to analyze. Put on the cap, and it will flush out the extra water, leaving 60 mL of liquid. Go to the back room and put in the packets of chemicals, which will fix the oxygen in the water. First, put the first packet in and wait until the precipitate forms. Shake the bottle to do this. Next, put the next chemical in, and then shake it until the precipitate disappears. When you are done with the chemicals, put them in the chemical trash.
 * 5) Titration will be done with the long buret tubes. I'll explain this in class. When you are done with your liquid chemical, you can pour this into the hazardous waste jug in the back room.
 * 6) **TITRATE CONTROL WATER.**Get some water from your mason jars and titrate this water. This will be the cold-control for both groups. Two more people from each group will do the warm water control. Get 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.
 * 7) I will explain titration, and let the cold-control group titrate, while the hot-control watches. :20
 * 8) 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
 * 9) If we have time, here we'll take a quiz, or at least part of a quiz.
 * 10) **BEGIN COLD JAR EXPERIMENT; PREPARE WARM JARS**.
 * 11) After around 30 minutes for the cold-crayfish (:30), each group will get a sample of the water from each jar (from the small-cold jar and the big-cold jar). Be sure to pour the water slowly so no bubbles get into the small BOD bottle. :35
 * 12) **TITRATE COLD SAMPLES**. Split up the group to prepare the warm-crayfish jars and the other folks can titrate.
 * 13) Crayfish group members: Put the crayfish back into the jars and fill them with extra cold water, and put aeration stones in them for around :15
 * 14) Titration group members: Titrate the cold-small and cold-big samples. :50
 * 15) **BEGIN WARM JAR EXPERIMENT**. After 15 minutes, take out the stones from the warm crayfish-jars, re-fill them with hot-control water, and slide the lids on the jar. This is your small-warm and big-warm crayfish. Write down the time you put the lid on.
 * 16) Wait around 30 minutes for these warm-crayfish jars. All groups should be together at this point, with all the cold-crayfish that are now in warm-crayfish jars. :50
 * 17) If we have time here, we'll take a quiz, or finish a quiz. Or more presentations.
 * 18) **SAMPLE AND TITRATE WARM JARS.** After 30 minutes, get samples from the small-warm and big-warm crayfish water. 1:20
 * 19) Titrate the samples. 1:40
 * 20) If we have time, we'll do more presentations or take the quiz.

Pour the water slowly so no extra CO2 gets into the sample (smooth, bubble-less pouring).  Body weight Low Control O2 Conc. Low Temp O2 Conc. Low Temp SMR High Control O2 Conc. High Temp O2 Conc. High Temp SMR name!  1 1.6 0.979 1.119 0.979 0.629 Jessica  2 4.8 0.979 0.839 0.979 n/a Jessica  3 8.62 1.54 0.7 1.12 0 Caitlin  4 1.51 1.54 1.33 1.12 0.14 Caitlin  5 1.16 1.26 0.385 1.61 0.839 Mayumi  6 5.15 1.26 0.804 1.61 0.14 Mayumi  7 8.6 2.1 1.496 2 1.399 Ty  8 1.11 2.2 1.538 x x  9 1.2 1.96 1.89 1.4 1.33  10 7.9 1.96 1.54 1.4 0.42  11 15.24 2 1.39 1.7 1.18 justin  12 19.29 1.1 0.76 1.25 0.87 justin