BIOLOGY

Experiment 1: Kidney Filtration

image5.jpgThe kidneys function to filter the blood in the body by waste removal. In this experiment, the dialysis bag represents a part of the kidney. The solution containing the Congo Red, yellow food coloring, and water symbolizes blood as it enters the kidney through the renal artery. As the experiment progresses, notice the filtration occurring with the kidney (dialysis tubing) and the resulting substances.

Materials

30 cm Dialysis Tubing 2 Small Rubber Bands Pipette 3 mL Congo Red 3 mL Yellow Food Coloring

(2) 250 mL Beakers 10 mL Graduated Cylinder *Water *You must provide

Procedure

1. Begin by placing the dialysis tubing in a beaker filled with 200 mL water. Submerge the tubing for 10 minutes.

2. Remove the tubing from the water after 10 minutes. Use your forefinger and thumb to rub the sides of the dialysis tubing apart. This will create a tube-like shape. Refill the beaker to 200 mL if the volume has decreased.

3. Secure a small rubber band around the bottom of the dialysis tubing to seal it. Wrap the rubber band around the dialysis tubing as many times as possible. Test that the dialysis tubing will not leak out of the bottom by placing a few drops of water into the tubing. If water leaks out the bottom, the rubber band has not been fastened tight enough. If water does not leak, pour the water out of the tubing into the sink. Set the tubing aside.

4. Grab one 250 mL beaker and fill it with 200 mL of water. Set this aside for now.

5. Use the 10 mL graduated cylinder to measure 3 mL of Congo Red. Pour this into the empty 250 mL beaker. Rinse the graduated cylinder.

6. Use the 10 mL graduated cylinder to measure 3 mL of yellow food coloring. Pour this into the 250 mL beaker with the Congo Red. Rinse the graduated cylinder.

7. Use the 10 mL graduated cylinder to measure 5 mL of water. Pour this into the 250 mL beaker with the Congo Red and yellow food coloring.

8. Take a pipette and mix the solutions in the 250 mL beaker. To do this, place the pipette in the solution and squeeze and release the bulb of the pipette while moving the pipette throughout the solution.

9. Pipette 10 mL of the mixed solution into the dialysis tubing, and complete Table 1 by indicating which solutions are present in each container.

10. When all 10 mL have been placed into the dialysis tubing, seal the top of the dialysis tubing by wrapping place a second rubber band around the top of the dialysis tubing.

11. Place the sealed dialysis tubing into the 250 mL beaker with 200 mL of water.

12. Wait 60 minutes. Look for any diffusion that may have occurred through the dialysis tubing (inbound or outbound). Indicate in Table 2 which solutions are present in each container.

Table 1: Solutions Present in Each Container Before 60 Minute Submersion

Solution

Dialysis Tubing

Beaker

Congo Red

Yellow Food Coloring

Table 2: Solutions Present in Each Container After 60 Minute Submersion

Solution

Dialysis Tubing

Beaker

Congo Red

Yellow Food Coloring

Post-Lab Questions

1. What specific part of the kidney does the dialysis tubing represent? What is this part’s function?

2. What does the yellow food coloring represent at the end of the experiment? What does the Congo Red represent?

3. Why is it important that the kidney filters the blood?

Experiment 2: Urinalysis

As was seen in Experiment 1, urine is the waste product filtered within the kidney. The urine is made up of many waste products as well as excess water. Urine is also a very helpful tool for doctors when diagnosing different conditions in patients. In this experiment, you will perform a urinalysis on four different samples of urine, testing a variety of different components. When all components have been tested, you will determine which of the urine samples are “abnormal” (use Table 3 for reference).

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Materials

4 Glass Test Tubes Test Tube Rack 25 mL Simulated Urine Sample A 25 mL Simulated Urine Sample B 25 mL Simulated Urine Sample C 25 mL Simulated Urine Sample D 100 mL Graduated Cylinder 16 Pipettes Permanent Marker

4 pH Test Strips 15 mL Benedict’s Solution 10 mL 3% Hydrogen Peroxide, H2O2 10 mL Biuret Solution Stopwatch *Hot Water Bath (boiling water in a deep bowl) *Hot Pad or Towel *You must provide

Procedure

Testing pH

1. Use the permanent marker to label each test tube as A, B, C and D.

2. Place the test tubes in the test tube rack.

3. Use a pipette to add five mL of Simulated Urine Sample A to the corresponding test tube.

4. Repeat Step 3 with samples B, C, and D. Use a new pipette each time.

5. Dip the reaction pad on one pH test strip into Sample A for 5 – 10 seconds and remove. Wait approximately 30 seconds and compare the resulting color on the pad to the color key (color key provided with the pH strips).

6. Record the pH of each of each sample in Table 4.

Glucose Test

1. Wash test tubes A – D. Re-label the tubes if the letters wash off.

2. Replace the test tubes in the test tube rack.

3. Use a pipette to add five mL of Simulated Urine Sample A to the corresponding test tube.

4. Repeat Step 3 with samples B, C, and D. Use a new pipette each time.

5. Add three mL of Benedict’s Solution to each test tube. Gently swirl each tube to mix the solutions.

6. Create a boiling water bath by retrieving a deep, heat-safe bowl.

7. Pour enough water into a pot or microwaveable bowl to cover the solutions in the test tubes. For example, if the solutions in the tubes are approximately 6.0 cm deep, fill the bowl with at least 6.1 cm of water.

8. Heat the water on a stove or in a microwave until boiling.

9. Use a hot pad or towel to carefully remove the water from the heat source, and place all four tubes into a boiling water bath for three minutes. If you do not want to hold the test tubes vertical for this time, you may place the test tubes in a container but monitor the apparatus to ensure that the tubes do not tip over.

10. Use a hot pad or towel to carefully remove the test tubes from the hot water. Place them in the test tube rack. Record their color change in Table 5. Note: A reducing sugar is present in the sample if a red, yellow or green precipitant forms.

Protein Test

1. Wash test tubes A – D. Re-label the tubes if the letters wash off.

2. Replace the test tubes in the test tube rack.

3. Use a pipette to add five mL of Simulated Urine Sample A to the corresponding test tube.

4. Repeat Step 3 with samples B, C, and D. Use a new pipette each time.

5. Add 25 drops of Biuret solution to each test tube.

6. Take Test Tube A out of the test tube rack and gently swirl the solutions. Watch for a color change as you swirl. Record any color changes in Table 6.

Yeast Test

1. Wash test tubes A – D. Re-label the tubes if the letters wash off.

2. Replace the test tubes in the test tube rack.

3. Use a pipette to add five mL of Simulated Urine Sample A to the corresponding test tube.

4. Repeat Step 3 with samples B, C, and D. Use a new pipette each time.

5. Add two mL of hydrogen peroxide to each test tube.

6. Observe the test tubes and record the presence or absence of bubbles in Table 7.

Ketone Test

1. Wash test tubes A – D. Re-label the tubes if the letters wash off.

2. Replace the test tubes in the test tube rack.

3. Use a pipette to add five mL of Simulated Urine Sample A to the corresponding test tube.

4. Repeat Step 3 with samples B, C, and D. Use a new pipette each time.

5. Using a wafting motion (pull your hand over the tube without bringing the tube directly to your nose; see Appendix for more guidance), notice the odor of each of the samples. Record your observations in Table 8.

Table 3: Urine Test

Test

Normal

Abnormal

pH

4.5 – 7.5

Acidic Urine (below 4.5) – Diabetes, starvation, dehydration, respiratory acidosis.

Alkaline Urine (above 7.5) – Kidney disease, kidney failure, urinary tract infection, respiratory alkalosis.

Glucose

None

Glucose present (red or green color after test); diabetes mellitus.

Protein

None

Protein present (violet color after test); kidney disease.

Yeast

None

Yeast present (bubbles form after test); yeast infection in urinary tract.

Ketones

Little or None

Large amount of ketones present (sweet smell of urine); starvation, prolonged vomiting, diabetes, hyperthyroidism, an other metabolic disorders

Table 4: Simulated Urine pH Test

Simulated Urine Sample

pH

A

B

C

D

Table 5: Simulated Urine Glucose Test

Simulated Urine Sample

Color Before Hot Water Bath

Color After Hot Water Bath

A

B

C

D

Table 6: Simulated Urine Protein Test

Simulated Urine Sample

Color Before Biuret Solution

Color After Biuret Solution

A

B

C

D

Table 7: Simulated Urine Yeast Test

Simulated Urine Sample

Bubbles Before Hydrogen Peroxide?

Bubbles After Hydrogen Peroxide?

A

B

C

D

Table 8: Simulated Urine Ketone Test

Simulated Urine Sample

Odor Observation

A

B

C

D

Post-Lab Questions

1. Fill in Tables 9 through 12. Refer to Table 3 to determine if each result was normal or abnormal. If abnormal, include the data which indicates this (e.g., a pH of 3.2 means that glucose is present).

Table 9: Sample A

Test

Test Results

pH

Glucose

Protein

Yeast

Ketones

Table 10: Sample B

Test

Test Results

pH

Glucose

Protein

Yeast

Ketones

Table 11: Sample C

Test

Test Results

pH

Glucose

Protein

Yeast

Ketones

Table 12: Sample D

Test

Test Results

pH

Glucose

Protein

Yeast

Ketones

2. Using the test results from each of the urine samples, along with the Table 3, diagnosis the condition(s), if any, that each of the sample patients is experiencing.

3. If you were a doctor and a patient’s urinalysis came back with high level of glucose, ketones and an acidic pH, what diagnosis would you immediately look into?

4. If you were a doctor and a patient’s urinalysis came back with an alkaline pH and high levels of protein, what diagnosis would you immediately look into?

5. What other conditions can urine be used to test for?

Experiment 4: Fetal Pig Dissection of the Urinary System

Like many other systems, the urinary system of the fetal pig provides a good representation of the anatomy of the human urinary system. The following experiment will guide you through an exploration of some of the structures of this system.

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Materials

Fetal Pig Dissection Tray

Dissection Tools Kit String (should still be tied onto pig’s hooves)

Procedure

1. To begin, lay your underpad down and place your dissecting tray on top of it. Lay out your dissecting tools. Be sure you have all of your safety equipment on before beginning.

2. Once prepared, gently open the bag your pig is in. Note: DO NOT destroy this bag or empty out the preserving solution within the bag, you will need it for the whole semester.

3. Lay your pig into the dissecting tray ventral (belly) side up. Slide the tied string over the dissection tray, allowing the belly of the pig to be exposed.

4. Peel back the flaps of the abdominal wall to expose the internal organs of the pig.

5. Gently push (do not remove) the intestines to one side. Leave a portion of the large intestine in place (this will be used to locate the rectum).

6. Locate the kidneys. These look like small, bean shaped organs against the dorsal wall of the body.

7. Looking at the kidney, locate the adrenal gland. This sits near the superior surface of the kidney.

8. Locate the ureter (Figure 8). This stems from the medial surface of the kidney. Near the origination of the ureter, notice the renal vein and the renal artery.

9. Follow the ureters posteriorly until you locate the urinary bladder and the urethra (Figure 9). Notice the elongation of the urinary bladder in the fetal pig. This occurs because the urinary bladder is actually connected to the umbilical cord in a fetus. If the urine generated by the fetus were to pass to the urethra as it does in adults, the amniotic sac would become toxic to the fetus. Instead, the fetus transports waste directly through the allantoic duct and through to the allantois, a small sac created specifically to handle toxic waste in a fetus, which then passes the waste onto the umbilical blood vessels. At birth, this process collapses and urine begins to flow from the urinary bladder into the urethra.

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Figure 8: Close view of the kidney and ureter.

10. Return to the kidney. Carefully make a longitudinal incision along the side of the kidney, as if you were cutting a bean in half. Gently lay the kidney open.

11. Inside, the kidney is made up of three different regions: the inner renal pelvis where the ureter begins. The darker tissue extending from the renal pelvis is known as the middle medulla. Within the middle medulla are the renal pyramids which look like triangular or cone-shaped masses. The outer portion of the kidney is called the outer cortex. Locate these three regions within the kidney (Figure 10) .

12. Notice the process in which waste is removed from the body. The process begins with blood flowing in from the renal arteries into the kidneys. As the blood flows through the kidneys, it passes through many small vessels that remove waste, water and other ions. The cleansed blood then flows out of the kidney via the renal veins. The waste removed is collected and then passes through the inner renal pelvis into the ureter, on its way to the urinary bladder. The waste again collects in the urinary bladder until it flows down the urethra and is expelled from the body.

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Figure 9: Close view of the urinary bladder.

13. Be sure that you can follow this process within the pig.

14. To finish, locate the bag the pig came in. Gently place the pig back into the bag and tightly secure the bag with a rubber band, or place in the zip-seal bag provided in the dissection box.

15. Replace the pig in a cool environment. Remember, the best place to store the pig is a cool, dark place.

16. After your pig has been put away, clean off your dissecting tray and dissection tools with soap and water. Biological scraps should not be thrown into the garbage. Securely store the biological scraps until the end of the term so they can be properly disposed of at one time.

17. Clean the area in which you worked with soap and water as well. As long as the underpad has not been damaged, keep it for future experiments.

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Figure 10: Close view of the dissected kidney.

Post-Lab Questions

1. Label the arrows in Figure 11.

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Figure 11: Kidney (sagittal-view)

2. What is the function of the urinary bladder?

3. Would you think the kidneys are highly vascularized? Why or why not?

4. What is the function of the adrenal glands?

5. Explain, in detail, the process by which urine is made.

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