Lab 2: Protocol

In today's lab you will learn how to use a micropipette. You will set up experiments to try and predict the glycemic index of unknown food items and you  will continue analysis of the diabetes data.
​Exercise I. Pipette Challenge
Exercise II. Predict the Glycemic Index
​Exercise III. Continue Analysis 
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Lab Objectives: Following today's lab, you should be able to...

Exercise I. The Pipette Challenge.  

Please keep the following in mind to increase the likelihood of both accurate and precise measurements in lab:
  • Always record the measured value of a liquid at the bottom of the meniscus: the concave or convex surface of a liquid due to cohesion
  • You should always use the smallest possible vessel for your measurements. For example, if you need to measure 25mL of solution, you would use 50mL flask, not a 125mL or 250mL.
  • When transferring small volumes, you will usually use a micropipette. Occasionally you may be asked to use “drops” from a dropper bottle instead. 
  • For liquids: Graduated cylinders are best, second are flasks, never use beakers.
One of our most useful measuring tools is the micropipette, which you will need to use today in Exercise II. However, if this instrument is not used correctly is it neither accurate nor precise, and can be easily damaged. 
Review the parts of a micropipette:
  1. The Plunger & Volume Dial: If you SLOWLY push the plunger down, you will feel resistance. This is the first stop designed for drawing up fluids. If you continue to push the plunger, you will reach the end or second stop designed to expel fluid. If you draw fluid using the second stop, it will over-draw and your experiment will be compromised. The dial serves as volume adjustment dial. SLOWLY turn the dial to set the volume. DO NOT set a pipette past its listed capacity.
  2. Tip Ejector Button: SLOWLY push to eject a tip into the Bio-hazard container.
  3. Volume Indicator: Pipettes are labeled with their capacity. 1 - 5mL for larger volumes and 100-1000uL for smaller volumes. 
  4. Tips: Disposable tips are provided. Attach a tip by GENTLY pushing the pipette into tip and removing the tip from the box. As part of aseptic technique (methods used to prevent contamination), it is standard practice to dispose of each tips after each transfer and to keep the lid box closed at all times. 
At the beginning of lab, your instructor will demonstrate how to properly use a micropipette. Watch carefully and follow along. 
Procedure​: Your task is to demonstrate your ability to accurately use a pipette.
  1. First each group member should practice using the pipette.
  2. It's easy to test if you are using the pipette correctly. Use the digital balance to obtain the mass of your sample. 
  3. Remember! In the metric system, you can covert between types of measurement: From length to volume to mass: 1cm3 = 1mL = 1g. Ex) 4.3mL in your pipette should weigh exactly 4.3g. Ex) 150uL = 0.15mL, should weigh exactly 0.15g.
  4. After everyone has tested themselves, call over your instructor. Each of you will draw up a requested volume, predict the mass it should weigh, and test your accuracy. Once you passed this challenge to your instructor's satisfaction, you can move on!

Exercise II. Can you use chemical indicators to predict the glycemic index of unknown food items?

Your task in Exercise II is to develop a series of tests, using chemical indicators, to predict the gycelmic index score of unknown food items. ​
For shorter experiments like this one, you will be provided with an Experimental Notes and Data template. Each template is lab specific, follows the steps of the scientific method, and is meant to approximate what you would record in a lab notebook as you work through each experiment. Every lab member needs to submit one in the post-lab and every member should save them in a folder labeled BIOL 121 Lab Notebook.
Materials: Your Tool Kit
  • Chemical indicators. For a review, use the tabs in the side bar.
  • Four solutions that will test positive for each indicator: glucose (positive on Bendict's Test), starch (positive on the Iodine Test), oil (positive on the Sudan IV Test), and egg albumin (positive on the Biuret Test). These four solutions should serve as your positive control for each test.
  • Distilled water which should serve as your negative control for each test
  • ​Many unknown food items for testing.
  • Lab supplies including: test tube clamps, test tubes, sample containers, hot water bath, beakers, test tube racks, stirring rods, graduated cylinders and micropipettes
  • BIOL 121: Experiment 2 Notes & Data template
Procedure
  1. Review your group contract and roles for Lab 2. 
  2. If anyone in your group feels uncertain about the concepts of macromolecules, chemical indicators or the glycemic index, please review the pre-lab together until everyone feels confident. If you notice a lab mate falling behind, encourage them to spend more time in preparation for lab's in the future. 
  3. Download and follow the Experiment 2 Notes & Data template.
  4. Work through the the first four steps and stop to get approval. You will need to construct a data table based on your protocol. Here is an example that may helpful.
  5. Proceed with your tests as designed. Be sure notes are taken and data are recorded throughout. 
  6. Also, take photos as you move through for reference. 
  7. Once you have tested your unknown food samples, and completed your results table, use it to predict what the glycemic score of each item might be. You should decide on a single number, and a range, and order the unknowns from highest to lowest predicted GI score. 
  8. Once complete, ask your instructor to provide you with the key, which will give you the name and glycemic index score for each food item.
  9. Complete your notes and data template for experiment 2. 
  10. Save the document. Make sure every group member has a copy.
  11. Be ready to submit your notes and data in the post-lab.
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Click to review lab equipment
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Take photos of your set-up and your experiment

Exercise III. Continue Analysis

Go back to Lab 1. We are not ready for graphing yet!

You left off at this step at the end of lab 1. Let's pick back up and continue. Exercise II might have given you some new ideas?
Continue Results Analysis (from last week). Use the resources available to complete hypothesis testing for both of your diabetes data research questions. Be prepared to show your instructor Excel tabs with the data used and results for each test.
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T-test Online Calculator.
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Pearson's Correlation Online Calculator.
You need approval before moving on. Be ready with any questions you may have regarding how to troubleshoot or how to interpret your results. Proceed to next steps only AFTER everyone feels secure and confident in your results.
Procedure: Create graphs of your data & start your technical report
  1. Let's start with your "difference" hypothesis that we tested with a t-test. 
  2. The T-test told you if the means between your two groups were significantly different (p < 0.05) from each other, or not. The best way to graphically represent these data is through a bar graph.
  3. Use excel to construct a bar graph representing your data. Be sure to include the standard error associated with each mean (each bar). If you need some help with this task, please review the demo in the sidebar. Please note, there are differences between the paired and unpaired versions.
  4. Next, let's move on to your "relationship" hypothesis that we tested with a correlation.
  5. The correlation test told you if their was a significant relationship between your variables (p < 0.05), or not. The best way to graphically represent these data is through scatterplot.
  6. Use excel to construct a scatterplot representing your data. If you need some help with this task, please review the demo in the sidebar. 
  7. Once you have finished both graphs, copy paste them onto one, 1-page, Word document.
  8. Be prepared to submit this word document in the Post-Lab.
  9. Review the technical report template and rubric. We will pick up here next week. 

Lab 2 BIOL 120 CONNECTIONS
Section 2.5: Life is Carbon Based
​Chapter 3: Proteins
Chapter 5: Carbohydrates
Section 6.1: Lipid Structure & Function
Big Picture 2: The Chemistry of Life
BioSkills 1: Using the Metric System
​Bioskills 14: Reading Chemical Structures
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