In this inquiry-based lab, students are provided with a case study involving a young boy with a head injury exhibiting various symptoms, as well as simulated blood and urine samples to help diagnose the boy's disease. Throughout the course of the lab, students research, design, and conduct a series of tests culminating in a patient prognosis. All of the materials, which simulate the blood, urine, and testing compounds, are readily available at the grocery store or online. This real-world problem engages the students to think about negative feedback systems, patient symptoms, the hormones associated with blood glucose levels and urine production, as well as the detection techniques employed by physicians to diagnose patients. Diagnostic methods, testing procedures, and the disease itself make this lab extraordinarily relevant to the lives of students, as evidenced by our students’ reactions to the lab.

Introduction

It can be challenging for students to apply abstract Biology content, such as negative feedback systems and cellular communication, to real-world situations. To help rectify this issue, the Next Generation Science Standards (NRC, 2013) focus on the practices of science with the hope that exposing students to what scientists do will help clarify the relevance of science to everyday life. Immersing students in real-world problems and having them apply their knowledge to these problems also increases student engagement (Dinan, 2005). For example, students may memorize the function of antidiuretic hormone (ADH), yet fail to connect brain injuries with frequent urination.

Similarly, students learn laboratory techniques, yet cannot understand how said techniques are used outside of the teaching laboratory. Four techniques, used specifically in this lab, are simulated specific gravity, serum concentrations, urine glucose, and fluorescence (sometimes called photoluminescence). Specific gravity and serum concentrations test the density of compounds, such as proteins, and urine glucose detects concentration levels in solution. Fluorescence techniques are used to detect and quantify small amounts of molecules such as proteins and hormones. Both fluorescence and specific gravity are used by physicians to diagnose patients.

To address the aforementioned issues, a lab was generated. This practice-driven, laboratory-based activity is the collaborative product of a science educator, a high school science teacher, and a medical scientist, and follows a similar format, albeit different content, of the open source cases from the University of Buffalo (Bolognese et al., 2005). Our aim was to create a laboratory activity for our Advanced Placement (AP) Biology course that focused on hormonal feedback systems, specifically insulin and ADH, NGSS practices, (NRC, 2013), as well as some of the Big Ideas from AP Biology (College Board, 2015) (Table 1). The lab could also be used in a secondary Anatomy and Physiology course or modified for use in a secondary biology course since the activity's content spans multiple courses.

Table 1.
NGSS standards and AP Biology Big Ideas addressed by the activity.
NGSS Performance StandardsAP Big Ideas
Science and Engineering Practices
Developing and using models 
Develop and use a model based on evidence to illustrate the relationships between systems or between components of a system. (HS-LS1-2) 2.C.1: Organisms use feedback mechanisms to maintain their internal environments and respond to external environmental changes. 
Science and Engineering Practices Planning and carrying out investigations Plan and conduct an investigation individually and collaboratively to produce data to serve as the basis for evidence. (HS-LS1-3) 3.D.2: Cells communicate with each other through direct contact with other cells or from a distance via chemical signaling. 
Disciplinary Core Idea
Structure and function 
Systems of specialized cells within organisms help them perform the essential functions of life. (HS-LS1-1) 3.D.4: Changes in signal transduction pathways can alter cellular response. 
Disciplinary Core Idea
Structure and function 
Feedback mechanisms maintain a living system's internal conditions within certain limits and mediate behaviors. (HS-LS1-3)  
Connections to Nature of Science
Scientific investigations use a
variety of methods 
Scientific inquiry is characterized by a common set of values that include: logical thinking, precision, open-mindedness, objectivity, skepticism, replicability of results, and honest and ethical reporting of findings. (HS-LS1-3)  
NGSS Performance StandardsAP Big Ideas
Science and Engineering Practices
Developing and using models 
Develop and use a model based on evidence to illustrate the relationships between systems or between components of a system. (HS-LS1-2) 2.C.1: Organisms use feedback mechanisms to maintain their internal environments and respond to external environmental changes. 
Science and Engineering Practices Planning and carrying out investigations Plan and conduct an investigation individually and collaboratively to produce data to serve as the basis for evidence. (HS-LS1-3) 3.D.2: Cells communicate with each other through direct contact with other cells or from a distance via chemical signaling. 
Disciplinary Core Idea
Structure and function 
Systems of specialized cells within organisms help them perform the essential functions of life. (HS-LS1-1) 3.D.4: Changes in signal transduction pathways can alter cellular response. 
Disciplinary Core Idea
Structure and function 
Feedback mechanisms maintain a living system's internal conditions within certain limits and mediate behaviors. (HS-LS1-3)  
Connections to Nature of Science
Scientific investigations use a
variety of methods 
Scientific inquiry is characterized by a common set of values that include: logical thinking, precision, open-mindedness, objectivity, skepticism, replicability of results, and honest and ethical reporting of findings. (HS-LS1-3)  

By the conclusion of the lab, students will understand how levels of hormones, plasma proteins, negative feedback mechanisms, and blood glucose indicate diseases and help physicians properly diagnose their patients. The use of blood and urine in classrooms can be difficult because of the potential pathogen precautions required in case of a spill. To avoid these difficulties, materials readily available from the grocery store are used to simulate the blood and urine samples. Specific learning objectives for the activity can be found in Table 2.

Table 2.
Student learning objectives for the activity.
  • 1.

    Identify kidney structures and explain the functions of major parts.

 
  • 2.

    Describe how filtrate is produced and its composition.

 
  • 3.

    Describe the negative feedback mechanism of urine production.

 
  • 4.

    Describe the relationship between osmolarity and urine production.

 
  • 5.

    Explain how hormones affect rate of filtrate production.

 
  • 6.

    Explain the interconnections of the urinary and endocrine systems.

 
  • 7.

    Explain how abnormal hormone levels affect rate of urine production.

 
 
  • 1.

    Identify kidney structures and explain the functions of major parts.

 
  • 2.

    Describe how filtrate is produced and its composition.

 
  • 3.

    Describe the negative feedback mechanism of urine production.

 
  • 4.

    Describe the relationship between osmolarity and urine production.

 
  • 5.

    Explain how hormones affect rate of filtrate production.

 
  • 6.

    Explain the interconnections of the urinary and endocrine systems.

 
  • 7.

    Explain how abnormal hormone levels affect rate of urine production.

 
 

The laboratory exercise was integrated into an AP biology curriculum and is designed to be done in three 50-minute class periods, with some portions of the case completed as homework. By the conclusion of the first session, students will have ordered detection techniques to help them diagnose their patient through guiding questions. In the second session, students carry out the procedures to collect evidence and refine their initial diagnoses. The final session culminates with a final patient diagnosis based on the evidence collected, a set of questions pertaining to the physiological mechanisms involved, and a short paper about our patient, Charlie's disease.

Case Study

The case study consists of six parts, with students completing two parts per class session. Part 1 of the case study introduces students to the fictional patient, Charlie, who has been drinking an excessive amount of water and craving ice, The scenario helps students become familiar with Charlie's background, and they begin to discuss a list of facts, questions, and potential diseases based on evidence gathered from the fictitious scenario (Table 3). Part 2 helps students refine the potential diseases following a visit to Charlie's physician, and concludes with students ordering laboratory tests, which they will conduct in Part 3. All laboratory tests will need to have a valid reason for being ordered. The teacher can play the role of the insurance company to assist in guiding students to warranted laboratory tests and to deny unnecessary tests.

Table 3.
Student-generated responses after completing Part 1 of the case study. Underlined disease indicates the class consensus of Charlie's disease at this point.
What I know (The facts)What I need to know (the questions)
  • Has to pee multiple times in a short time period.

  • Sleeps a lot more than normal.

  • He is super skinny compared to dad.

  • Eats a lot of snow cones.

  • He craves water.

  • He is active and plays baseball.

  • 11 years old.

 
  • Other reasons he's drinking water?

  • Any family history of diseases?

  • How is it making him more tired?

  • Is he incontinent?

  • What are the test results?

  • What was the weather like? Cause his dehydration?

 
Potential disease at this pointKidney disease, Diabetes (type 1), dehydration, polycystic kidney failure 
What I know (The facts)What I need to know (the questions)
  • Has to pee multiple times in a short time period.

  • Sleeps a lot more than normal.

  • He is super skinny compared to dad.

  • Eats a lot of snow cones.

  • He craves water.

  • He is active and plays baseball.

  • 11 years old.

 
  • Other reasons he's drinking water?

  • Any family history of diseases?

  • How is it making him more tired?

  • Is he incontinent?

  • What are the test results?

  • What was the weather like? Cause his dehydration?

 
Potential disease at this pointKidney disease, Diabetes (type 1), dehydration, polycystic kidney failure 

On the second day of the activity, students conduct the laboratory tests. They will gather evidence from Charlie's simulated blood and urine samples, including his blood ADH and insulin levels, urine glucose levels, and urine specific gravity. Each of these tests will also include a standard to help the students identify if Charlie has high or low hormone, protein, or urine glucose levels. For ADH and insulin tests, three containers of red food coloring and water simulate blood, with the addition of a fluorescent antibody (laundry detergent) to simulate fluorescent testing, which detects the hormone levels. Urine samples are simulated by yellow food coloring, non-iodized salt, and water, and represent a specific gravity test to detect the density of Charlie's urine proteins. After the evidence is gathered, the students compile their results in Part 4 and share their results to the class for refinement of their initial list of potential diseases.

The final day of the case study begins with Part 5, in which students answer a set of questions about negative feedback systems for ADH and insulin, as well as how the evidence gathered in Part 4 pertains to their diagnosis. Students then, in Part 6, write a short paper about diabetes insipidus. The case study is available at https://drive.google.com/file/d/0BwMZCopZ32NCZjd4bmNybkF1T2hZLWxvcWQ4aWVVczJRYzJn/view.

Diabetes, ADH, and Brain Injuries

“Diabetes” is Greek for “flow through” and is used to describe some diseases where there is excess urination. With excess urination, the body will lose too much water, which triggers the thirst response. The most common form of diabetes is diabetes mellitus. “Mellitus” is Greek for “honey,” and the disease gets its name because the urine is full of sugar (sweet) in many untreated patients (American Diabetes Association, 2010). Diabetes mellitus can be caused by two different problems. One is the inability to secrete enough insulin, resulting in Type I diabetes mellitus. The other problem is a poor response to insulin, also known as insulin resistance, which is thought to be the initial cause of Type II diabetes mellitus, though in later stages insulin secretion can also decrease. In both cases, blood glucose levels are not well regulated. When blood glucose levels get very high, so much glucose is filtered by the kidney that it overwhelms the capacity of the healthy kidney to reabsorb the glucose, hence glucose remains in the tubular fluid. Normally, the collecting duct tubular fluid has no glucose, but in the case of diabetes mellitus, there can be substantial amounts of glucose in the collecting duct. This excess glucose increases the osmolarity of the collecting duct fluid, and water moves from blood to collecting duct to lower the osmolarity. This extra water movement results in excess urination.

A second, and less commonly known, form of diabetes is diabetes insipidus, which is named because the urine tastes bland. In this case, it is the system for regulating urine volume that is disrupted, not high blood glucose levels. The normal regulation of urine volume is under the control of ADH. When regulatory cells in the pituitary sense an increase in blood osmolarity, or some other change, they release ADH into the bloodstream. When ADH binds to receptors in the kidney, this signals the collecting duct cells to put water channels (aquaporins) into the cell membrane. There is now a pathway for water to move between the blood and the collecting duct tubular fluid via the channels.

When the fluid arrives in the collecting duct, it is very dilute and the blood surrounding the collecting duct is not. Thus, there is a gradient for water to move from collecting duct to blood; with high ADH, there is a pathway, and subsequently water does move. Since there is fluid in the collecting duct, it drains to the bladder and becomes urine. This movement of water from collecting duct to blood means there is less water lost in the urine.

When regulatory cells in the brain sense an increase in blood osmolarity, the thirst response is triggered. The combination of drinking water (and decreasing urine volume) will eventually result in a decrease in blood osmolarity. When blood osmolarity gets too low, regulatory cells in the pituitary gland stop releasing ADH into the bloodstream. When there is not enough ADH to bind to receptors in the kidney, the collecting duct cells remove the water channels (aquaporins) from the cell membrane, and there is no longer a pathway for water to move between blood and collecting duct tubular fluid. So even though there is a gradient, water does not move from the more dilute collecting duct tubular fluid to blood, and the excess water is lost through urination.

There are two types of diabetes insipidus. In renal diabetes insipidus, the ADH receptors or the renal water channels do not function properly, which causes the kidneys to not respond to ADH. Central diabetes insipidus is a second type, in which the pituitary gland cannot produce or release ADH. In this case, the pathway cannot be formed due to insufficient hormone production. The outcome in either type of diabetes insipidus is that more volume of urine is produced, since ADH is either not produced (central diabetes insipidus) or receptors do not bind ADH properly (renal diabetes insipidus).

In Charlie's case, the regulation of blood glucose is normal, therefore he does not lose any glucose to his urine. His problem is that a hit to the head damaged a part of his pituitary, and he cannot secrete ADH (Boughey et al., 2004). He can be treated with synthetic ADH, which could be considered analogous to a person with Type I diabetes mellitus being treated with insulin. In contrast, a person with renal diabetes insipidus is not helped by treatment with synthetic ADH. This differs from people with Type II diabetes mellitus, because in renal diabetes insipidus the receptor or water channels are completely inactive, whereas in Type II diabetes mellitus, the insulin receptors work, but are just less sensitive. It is a bit like the difference between being completely deaf (renal diabetes insipidus) and being hard of hearing (Type II diabetes mellitus).

Additionally, the students might find it useful to think about the different renal mechanisms for the excess urination in diabetes insipidus vs. diabetes mellitus, which will help review the major concept that flow requires a gradient and a pathway. In diabetes insipidus, the urine loss is because there is no flow from collecting duct to tubule, as there is no pathway for water to move. In diabetes mellitus, the water pathways are fine but the urine is decreased (less flow from collecting duct to tubule) because there is not a gradient (or too small a gradient) for water to move.

Details for the Teacher

Materials for Activity

All materials for creating the samples are available at the grocery store or online.

  • 1 25-gallon plastic Rubbermaid container with lid (used for black light apparatus)

  • 9 250-mL beakers per group

  • Tonic water

  • Dextrose

  • Electronic balance

  • Desk lamp

  • Non-ionized salt

  • Laundry detergent (we used Rit©)

  • Black light (we used a black light LED bulb available at Wal-Mart)

  • Coffee filters

  • Urine glucose strips (Rapid Response© 2-parameter urinalysis reagent strips from Amazon.com)

  • Specific gravity strips (Rapid Response© 10-parameter urinalysis reagent strips from Amazon.com)

  • Disposable pipettes

  • Red and yellow food coloring

  • Distilled water

  • Razor blade (to cut a viewing window in the black light apparatus)

Teacher Setup

Creating the Black light apparatus. The fluorescence laboratory tests need a dim or dark place to simulate a laboratory prep room for antibody fluorescence. The location does not need to be pitch black, but should be dark enough that the students can see if their samples fluoresce. In our classroom, we created a black light apparatus for ease of viewing the fluorescence, and it doubles as a storage container for the lab materials. The black light apparatus is created by cutting a square viewing window in the lid of the plastic container with the razor blade. Place the desk lamp with the black light into the plastic container so the samples can be viewed without being obstructed (Figure 1). Also, a second hole can be cut in the top of the lid to accommodate the lamp cord. Remember to make it small enough that it fits the lamp's plug-in, but not large enough to allow excess light into the apparatus. An easy way to test if the apparatus is dark enough is to test the samples as they are created. If the fluorescence is hard to see, place the container in a room with the lights off and/or a stronger sample.

Figure 1.

Completed black light apparatus. Apparatus with lid (right) and without lid (left).

Figure 1.

Completed black light apparatus. Apparatus with lid (right) and without lid (left).

Charlies’ samples and standard solutions. To create the ADH blood samples, distilled water, tonic water, laundry detergent, and red food coloring are mixed in their respective graduated cylinder until a blood-like red color is obtained. Laundry detergent and tonic water are used to mimic the fluorescent antibody bound to the hormones. Each group set needs a high standard, low standard, and Charlie's sample. Charlie's samples for both ADH and insulin have laundry detergent added to them to produce a measurable fluorescence. Tonic water does fluoresce under black light, but we added laundry detergent to our insulin samples to ensure that our students could detect the different fluorescence levels between ADH and insulin (Table 4).

Table 4.
Contents of the samples. Bold text indicates differences between each sample for a test.
TestCharlie's SampleHigh Level Standard SolutionLow Level Standard Solution
ADH Fluorescence 150 mL distilled water, 0.2 g laundry detergent, 2 drops red food coloring 150 mL tonic water, 0.5 g laundry detergent, 2 drops red food coloring 125 mL distilled water, 25 mL tonic water, 0.1 g laundry detergent, 2 drops red food coloring 
Insulin Fluorescence 150 mL distilled water, 0.2 g laundry detergent, 2 drops red food coloring. 150 mL distilled water, 2 g laundry detergent, 2 drops red food coloring 150 mL distilled water, 0.1 g laundry detergent, 2 drops red food coloring 
Urine Glucose 200 mL distilled water, 1 drop yellow food coloring 200 mL distilled water, 6.5 g dextrose, 1 drop yellow food coloring 200 mL distilled water, 1 drop yellow food coloring 
Specific Gravity 200 mL distilled water, 1 drop yellow food coloring 200 mL distilled water, 8.5 g NaCl, 1 drop yellow food coloring 200 mL distilled water, 1 drop yellow food coloring 
TestCharlie's SampleHigh Level Standard SolutionLow Level Standard Solution
ADH Fluorescence 150 mL distilled water, 0.2 g laundry detergent, 2 drops red food coloring 150 mL tonic water, 0.5 g laundry detergent, 2 drops red food coloring 125 mL distilled water, 25 mL tonic water, 0.1 g laundry detergent, 2 drops red food coloring 
Insulin Fluorescence 150 mL distilled water, 0.2 g laundry detergent, 2 drops red food coloring. 150 mL distilled water, 2 g laundry detergent, 2 drops red food coloring 150 mL distilled water, 0.1 g laundry detergent, 2 drops red food coloring 
Urine Glucose 200 mL distilled water, 1 drop yellow food coloring 200 mL distilled water, 6.5 g dextrose, 1 drop yellow food coloring 200 mL distilled water, 1 drop yellow food coloring 
Specific Gravity 200 mL distilled water, 1 drop yellow food coloring 200 mL distilled water, 8.5 g NaCl, 1 drop yellow food coloring 200 mL distilled water, 1 drop yellow food coloring 

The urine glucose samples are created by dissolving dextrose into distilled water. Use the yellow food coloring to create a urine-like appearance. Only the high standard will have dextrose added to it due to the detection level of the urine glucose strips. The measurement scale of the urine glucose strips goes only as low as 100 (+/– 5) dl, and so is not sensitive enough to detect the low levels of glucose needed to simulate Charlie's disease or the low level standard. Glucose levels would be detected in real urine.

The urine specific gravity levels are created by a mixture of yellow food coloring, non-ionized salt, and distilled water. Charlie's sample and the low standard specific gravity are a mixture of water and yellow food coloring, whereas the high standard levels are yellow food coloring, non-ionized salt, and distilled water. Abnormal levels of protein in urine are simulated by the non-ionized salt. Table 5 indicates the expected results for Charlie and the standards.

Table 5.
Expected student results. Parentheses indicate relative values from our test strips.
TestCharlie's SampleHigh Level StandardLow Level Standard
ADH Low level High level Low Level 
Insulin Low level High level Low level 
Urine Glucose Light green (100 mg/dl) Dark green (>2000 mg/dl) Low level (100 mg/dl) 
Specific Gravity Dark blue/green (1.005 g/cm3Mustard yellow (1.030 g/cm3Dark blue/green (1.005 g/cm3
TestCharlie's SampleHigh Level StandardLow Level Standard
ADH Low level High level Low Level 
Insulin Low level High level Low level 
Urine Glucose Light green (100 mg/dl) Dark green (>2000 mg/dl) Low level (100 mg/dl) 
Specific Gravity Dark blue/green (1.005 g/cm3Mustard yellow (1.030 g/cm3Dark blue/green (1.005 g/cm3

How to Test the Samples

The four tests that the students will be conducting during the lab are measuring specific gravity, insulin, and ADH levels, as well as urine glucose levels. The ADH and insulin levels will be measured using the black light apparatus, and urine glucose and specific gravity will be analyzed using the purchased test strips.

Students will need to use the coffee filters to test the fluorescence of the ADH and insulin. A few drops of each of the ADH and insulin samples and standards will be placed on two separate coffee filters, one for ADH and the other for insulin. Students can record which of the samples corresponds to the levels by using a pencil to mark directly on the coffee filter. This will ensure that they know which of the three samples are the high standard, the low standard, and Charlie's sample. Each of the filters is then placed under the backlight, and the amount of fluorescence in Charlie's sample can be compared to the standards.

The purchased test strips need to be submerged into their respective samples, and a visible color change will occur. The strips come with a scale on their container, and the color change is compared to the scale to obtain the concentration of solutes in the urine samples (Figure 2).

Figure 2.

Students reading test strips for specific gravity.

Figure 2.

Students reading test strips for specific gravity.

Student Reactions and Conclusions

Our students were excited to partake in a medical case study. Many students relish science and are considering careers in the medical field; therefore our students appreciated the opportunity to think as one in the medical field. One student stated, “We felt like we were actual doctors conducting a test instead of just students.” Our class also enjoyed the puzzle aspect of the case study: “It was interesting. We were running in every direction trying to find the right conclusion.” The scenario gave the activity a personal touch and engaged our students, as they felt like they were dealing with real people. The students even asked who the characters’ personalities were based on! The students were challenged as their task was to diagnose a patient they came to genuinely care for. Moreover, the lab component of the case study gave students hands-on experience with general scientific practice.

Our students were all enrolled in AP Biology. They were able to comprehend the case study and were quite successful; however, some guidance was needed to reach the final diagnosis. Our students stated they would have had a more difficult time had they not been taught body systems before the case study. In fact, our students liked that the case study included multiple organ systems: “parts of the body that seem unrelated play major roles together.” Based on teacherss understanding of the capabilities of their students, we suggest modifying the case study to provide additional details for the renal, nervous, and endocrine systems, since the AP biology curriculum does not specifically address each system individually. We noticed that better scaffolding of the individual systems would have helped our students diagnose Charlie. In addition, our students were unfamiliar with other types of diabetes, so a discussion of this prior to the start of the activity would have helped our students move away from a diagnosis of Type I diabetes.

The case study is advantageous in the classroom for a number of reasons. First, the entire process is engaging and focuses on class collaboration. Students must work together to diagnose a patient using evidence of the test results. The teaching strategy lends itself well to critical thinking. Second, the activity reflects the vision of the Next Generation Science Standards (NRC, 2013) and several of the AP Biology Big Ideas by its focus on scientific practices, negative feedback systems, and homeostasis. Third, the content is relevant to students’ lives because many of our students are considering entering the medical field. The process of diagnosing Charlie is similar to how physicians would interact with their patients. Lastly, using materials from the grocery store to simulate blood, ADH, and urine gives teachers safe alternatives to the use in their classroom.

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