Lab 7: Pre-Lab

Your task in Lab 7 is to carryout your experiment regarding the rate of cellular respiration and begin to analyze your data. To prepare for Lab 7, please review this pre-lab page. Once you feel confident regarding the below topics, complete the corresponding pre-lab quiz in Blackboard.

• Introduction/Review
• Do you know enough?
• What will we do in lab?
• LABridge

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Cellular Respiration

Click to Enlarge

​Energy is the currency of life: all living organisms require energy to survive and reproduce. Metabolism is the series of reactions and processes, catalyzed by enzymes, which together maintain life. These reactions fall into two types: catabolic or anabolic. These processes are the inverse of each other and in photosynthetic organisms occur in tandem as the anabolic reactions of photosynthesis create the products that are then broken down by the catabolic reactions of cellular respiration (see figure). 
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​There are two general classes of cellular respiration that are characterized by their relative efficiency (ATP production): anaerobic (without oxygen) and aerobic (oxygenated) respiration. We are focusing on aerobic respiration in this lab, which is a highly efficient process occurring within the mitochondria of eukaryotic organisms that have higher energy requirements for survival. In a 4 step process, oxygen and glucose are used to produce energy (ATP), H2O, and CO2,.

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CONNECTION! ​​Cellular respiration is the topic of Chapter 9 in your BIOL 120 lecture. Please review your textbook as needed for this lab. 

ANAEROBIC Respiration  review  

Aerobic respiration  review        

Please review anaerobic and aerobic respiration.

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Be sure you know the basic steps of cellular respiration, and the reactants and products. Could you write the full equation? ​Be sure you understand the production of CO2 as a bi-product.

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Click to Enlarge.

Do you know enough about crayfish?

​The freshwater crayfishes (Order Decapoda) are one of the better known crustacean groups in Kentucky. Worldwide, freshwater crayfishes are represented by over 640 species (Crandall and Buhay 2008) with the southeastern United States being one of the epicenters of diversity. Three hundred sixty species are represented in the United States (Taylor et al. 2007) with 54 species in Kentucky. Modification of habitats, sedimentation, and dams are serious threats to freshwater crayfishesas is the introduction and establishment of nonnative crayfishes. Several studies have shown the displacement of native species by more aggressive or opportunistic non-native species (Capelli 1982; Taylor and Redmer 1996; Hill and Lodge 1999). Many introductions are suspected to be from fisherman dumping their purchased live crayfish into the stream at the end of the day. Nationally, about 48% of crayfish species are of conservation concern (ranging from Vulnerable to Endangered); over a third (37%) of the Kentucky fauna falls into this category (KSNPC, 2010). Cave species are particularly at-risk from upland activities that pollute groundwater flowing into cave systems; this includes issues with chemical spills, agricultural runoff, salt from roads, and siltation from poor land use. Best Management Practices are needed to guard against perturbations to groundwater.

Cellular respiration in the crayfish. Click to enlarge.

After reading this exerpt from the Kentucky Wildlife Action Plan (WAP), use the resources below to get to know our test organism for Lab 7.

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Facts on KY Crayfish

Crayfish Natural History

 What will we do in lab & how will we do it?

How will we measure the rate of cellular respiration? ...By measuring the CO2 produced as a bi-product of the metabolic process!  You will place your crayfish in an aquatic environment  and allow it to "respire." After 20m, you will remove the crayfish and analyze how much CO2 was produced as a result of cellular respiration.  Review the following steps to understand how we will quantify respiration rate. Use the links provided. You MUST understand the concepts below.

• The water you will place your crayfish in will be slightly basic and will appear pink! This is because it will contain a pH indicator called, phenolphthalein, which is pink in the presence of a base and turns clear when it approaches neutral (pH=7).
• As cellular respiration is occurring inside your crayfish, CO2  will be expelled into the solution as a bi-product. ​In an aqueous environment, this CO2 breaks down into carbonic acid (H2CO3) then into bicarbonate (HCO3-) and H+ ions.
• Increased concentrations of H+ ions increase the acidity in solution and lower the pH. As the pH decreases the solution will turn from pink (basic) to clear (neutral). BUT! We cannot wait for that to happen on its own, it would take far too long! ​
• So...after your crayfish "respires" in solution for your set period time, you will remove it. The water will still appear pink. Some respiration and CO2 production HAS occurred, but not enough to change the pH enough to see a color change. So we will use sulfuric acid as our titrant. We will add sulfuric acid (H2SO4) to the solution, via titration, until we see a color change from pick to clear. View Diagram.​
• Lastly, you will use the "amount of titrant used" to calculate the micromoles of carbonic acid (H2CO3​) produced in solution using this equation.

​If you feel confident with this material, click the bridge icon and navigate to Blackboard to take the LABridge. Be sure your Lab 6 Notebook Guide is ready to submit!

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Click here to get to WKU's blackboard to take your LABridge for this week. You will not be submitting a Lab Notebook Entry for this LABridge.

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Lab 7: Protocol

In today's lab you will work with your lab group to conduct your experiment and begin to analyze your data. 

Exercise I. Run the experiment
​Exercise II. Titrations
Exercise III. Analyze your data

Lab Objectives: Following today's lab, you should be able to...

• Exercise I
• Exercise II
• Exercise III Post-Lab

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Exercise I. Start the Experiment

​Gloves and goggles all day.
Sulfuric acid is dilute but requires caution.
​Shellfish allergies should be reported.
Any cruel treatment of animals in BIOL 121 will not be tolerated. Be careful. Be respectful.

Any cruel treatment of animals in BIOL 121 will not be tolerated. Be careful. Be respectful.
Do NOT titrate your crayfish.
Do NOT put your crayfish on its back.

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Procedure
You will determine the effect of temperature or glucose concentration (as decided as a class) on respiratory rates via titration with a basic pH indicator (phenolphthalein) to determine product creation (relative amount of H2CO3 produced). ​

You will be running one of two treatments involving different water temperatures or different glucose concentrations. Your TA will divide you up and let you know which variable to test. Your TA will also give you information regarding your necessary controls.

You will create the matching environment in your beaker labeled "experimental" Examples will include a room temperature vs. a cold environment OR no glucose vs. glucose added. ​

1. Review your provided materials. You are provided with: ​Gloves & goggles, glass stirring rods, several beakers of various sizes, graduated cylinders of various sizes and 1 burette containing 0.02 N sulfuric acid (H2SO4). If you need more sulfuric acid at anytime today, your instructor will refill your burette.
2. Download, open and save THIS SPREADSHEET entitled Lab 7 data.
3. In the spreadsheet, record the test variable your class decided on and the treatment condition you will be testing.
4. REMEBER (from the Pre-Lab): The water (stock solution) you will place your crayfish in will be slightly basic and will appear pink! This is because it will contain a pH indicator called, phenolphthalein, which is pink in the presence of a base and turns clear when it approaches neutral (pH=7). 
5. Pour 200 mL of stock solution into a 250 mL graduated cylinder. ***Check with your TA. If you are testing with glucose you will use a glucose-positive test solution. 
6. Gently place your animal into the graduated cylinder TAIL-FIRST.
7. Record its volume (rise in solution level in mL) in your spreadsheet.
8. Carefully transfer the solution into the experiment/treatment beaker.
9. Set beaker on lab bench, do not agitate further and record time.
10. Watch your beaker carefully. If at any time the pink color disappears, record the start time in your spreadsheet.
11. Wait 20 minutes. While you wait proceed to Exercise II.

Lab 7 Data Spreadsheet

Exercise II. Titration

Procedure

1. While your crayfish is respiring, we will prep for titration.
2. Watch the demos from your TA. They will conduct a titration or two to determine your control values (Nr).​
3. After the 20 minutes has elapsed, return your crayfish to the recovery tank on the front bench (you can pick it up).
4. Pour 50 mL of solution from the experimental beaker into an Erlenmeyer flask.
5. Record the initial level of titrant in your spreadsheet.
6. Use a burette to titrate with 0.02 N sulfuric acid to a colorless endpoint: add small volumes of the titrant by turning the knob on the burette until the desired color change (known as an end point) is achieved.
7. Go slow! You do not want to over-titrate. 
8. Record the final level of the titrant in your spreadsheet.
9. Determine the amount of titrant required by using the equation below (Ne).
10. Obtain the control data from your TA (Nr).
11. Calculate the micromoles (um) of carbonic acid released in the solution using the equation below. You will need the Ne, Nr, volume of your crayfish, and duration time in hours. 

I still don't understand how this works!

• As cellular respiration is occurring inside your crayfish, CO2  will be expelled into the solution as a bi-product. ​In an aqueous environment, this CO2 breaks down into carbonic acid (H2CO3) then into bicarbonate (HCO3-) and H+ ions.
• Increased concentrations of H+ ions increase the acidity in solution and lower the pH. As the pH decreases the solution will turn from pink (basic) to clear (neutral). BUT! We cannot wait for that to happen on its own, it would take far too long! ​
• So...after your crayfish "respires" in solution for your set period time, you will remove it. The water will still appear pink. Some respiration and CO2 production HAS occurred, but not enough to change the pH enough to see a color change. So we will add more acid to the solution (H2SO4), via titration, until we see a color change from pick to clear. View Diagram.​​

Exercise III. Analysis

Procedure

1. You are ready to move forward. Now, what type of statistics should we use to determine if the mean respiration rate between our 2 groups is significantly different? ...hopefully this is an easy answer. Are the mean umols carbonic acid (H2CO3) produced in your two groups the same or are they different?
2. Use the tools below to complete your Lab Notebook Guide. 
3. The demo below will take you through, step-by-step.
4. You will need to create a table in Excel, a graph in Excel, run a T-test and answer a set of questions.
5. Be sure everyone has an updated copy of your excel spreadsheet and the Lab Notebook Guide to upload it in the next LABridge.

T-Test & bar graph demo (labs 7 & 8)

T-test Online Calculator.

Lab 7 BIOL 120 CONNECTIONS
Section 1.6: Doing Biology
Big Picture 1: How to Think Like a Scientist
BioSkillls 2: Reading & Making Graphs
BioSkillls 3: Interpreting Standard Error and Using Statistical Tests
BioSkillls 4: Working with Probabilities
Chapter 9: Cellular Respiration

Faculty Spotlight: Dr. Noah Ashley

[email protected]

We are conducting experiments to identify factors that affect the physiological process of cellular respiration in crayfish. Similarly, Dr. Noah Ashley's lab works to identify physiological, immunological, and behavioral responses to various factors, like sickness and sleep loss, in mice and birds. Specifically, they are investigating the costs and benefits of the sickness response in vertebrates, the inflammatory response in sleep-deprived mice, sleep loss in migratory birds, and the sleep-wake cycle in arctic songbirds. Dr. Ashley's lab is extremely productive! His research proposals have been funded by the NSF and the NIH. Learn more here (Lab Web Page). You just might recognize one of his current graduate students!
​Research Key Words: physiology, ornithology, eco-immunology, genomics, sleep loss, arctic song birds, migrating birds
​Recent Publication:  Cooper, L. N., Mishra, I., Ashley, N.T. 2019. Short-Term Sleep Loss Alters Cytokine Gene Expression in Brain and Peripheral Tissues and Increases Plasma Corticosterone of Zebra Finch (Taeniopygia guttata). Physiological and Biochemical Zoology 92:80-91.