Lab 3

Lab 3: Pre-Lab

Your task in Lab 3 will be to construct your own experimental protocol to predict the glycemic index of unknown food items, based on their molecular make-up, using chemical indicators. You may also have some time to continue the analysis you began in Lab 1.

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

1) What is a macromolecule and how can you test for its presence in food?

A marcomolecule (or polymer) is a very large molecule comprised of smaller subunits (or monomers). We tend to recognize four primary types which are highly diverse in atomic make-up and structure and thus, function. We will focus on the first three (carbs, lipids and proteins) in this lab. You should know the basic subunits of each, their shape, general function and a few examples. You need a basic understanding of dehydration synthesis & hydrolysis (or condensation reactions) which build and breakdown macromolecules. You also need to know which chemical test can be used to indicate each specific macromolecule.

Please review the slideshow. It provides an overview of macromolecules, the chemical indicators than can detect each in solution, and the affect of each macromolecule on the glycemic index score (an important dietary consideration for diabetics).

Please review THIS VIDEO on condensation reactions (<2m).

What is the glycemic index?

Remember...diabetes is a disease that occurs when blood glucose (i.e., blood sugar) is elevated because the body isn't making enough insulin or doesn’t use insulin well. The glucose then builds up in the blood, and doesn’t reach the cells where it is needed as an energy source. Over time, having too much blood glucose causes the serious and chronic health problems associated with diabetes.

Please read this quick explanation of the GI from the American Diabetes Association.

Because the blood glucose levels in diabetics are not well regulated, their diet becomes much more important. Primarily, diabetics have to be concerned with the frequency and types of carbohydrates they consume. Carbohydrates can be identified by their glycemic index (GI), which is a measure of how quickly the carbohydrate is broken down into its component parts to be used by the body.

Most simple monosaccharides and disaccharides, like glucose and fructose, are quickly broken down, rapidly increase blood sugar, have high GI scores and are especially detrimental to diabetics.
Larger, more completed polysaccharides take longer to be broken down, slowly increase blood sugar, have lower GI scores and better for diabetics.

However, keep in mind, diabetics don't just manage their diets based on the GI. They must also select foods that maintain their cholesterol and triglycerides levels, blood pressure, and weight management.

The affect of high vs. low GI foods on blood glucose levels over time.

Lab 3 will proceed in three parts:

1) The pipette challenge: You will learn the basics of measurement, including how to correctly transfer liquids using micropipettes.

If you've never used a micropipette before, try practicing a bit in this virtual lab setup.

Pipette Virtual Lab. Click to Enter.

2) Predict the GI score of unknown food items: In the lab you will be provided with

4 chemical indicators that, when added to a sample, will "indicate" the molecular contents (mono/disaccharide, polysaccharide, or protein).
A series of known positive controls for each macromolecule.
Water to use as a negative control.
Unknown food samples.
Common glassware and equipment.

You will then be asked to develop a protocol using the above materials, that might help you predict the GI score for each of the unknown food items.

Learn about lab glassware and review the metric system as needed. Make sure you understand WHY scientists use the metric system.

Review the importance of a positive and negative control.

3) Predict the GI score of unknown food items. Would any of these foods be good for a diabetic? Can you predict the GI score of a food based on its molecular composition? You will analyze your data and predictions vs. the actual GI score of the food items.

If you feel confident with this material, click the bridge icon and navigate to Blackboard to take the LABridge for Lab 3.
Be ready to submit your Notebook Entry from last week (Word.doc ONLY) and to be tested on this Pre-Lab material.

Click here to get to WKU's blackboard to take your LABridge for this week. Be sure your Notebook Entry from last lab is ready to submit!

Lab 3: 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, if time allows, you can continue analysis of the diabetes data.

Exercise I. Pipette Challenge
Exercise II. Predict the Glycemic Index
Exercise III. Continue Analysis

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

Exercise I
Exercise II
Exercise III

Exercise I. The Pipette Challenge.

At the beginning of lab, your instructor will demonstrate how to properly use a micropipette.
Watch carefully and follow along.

Review the parts of a micropipette: Diagram at right.

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.
Tip Ejector Button: SLOWLY push to eject a tip into the Bio-hazard container.
Volume Indicator: Pipettes are labeled with their capacity. 1 - 5mL for larger volumes and 100-1000uL for smaller volumes.
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.

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.

Instructions for the digital balance. Click to enlarge.

Procedure: Your task is to demonstrate your ability to accurately use a pipette.

First each group member should practice using the pipette.
Use the water in the beaker labeled "distilled water" or "Di H2O."
It's easy to test if you are using the pipette correctly. Use the digital balance to obtain the mass of your sample.
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.
After everyone has tested themselves, call over your instructor. Draw up a requested volume, predict the mass it should weigh, and test your accuracy. Once you've 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 glycemic index (GI) score of unknown food items. If you have ANY questions about what a macromolecule is, or what the GI is, please refer to the pre-lab above.

Equipment @ your station.

A good test tube set-up. Click to enlarge.

Take photos as you go!

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
Each group will be provided 5 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
Review common lab equipment.

SPECIAL NOTE: All of these tests are time-sensitive and should be read following the strict protocols in the side bar. A bright yellow/orange result on a Biuret test is common if you wait too long to read it; it is still a NEGATIVE result and is related to pH. A dark brown test result with Benedict's means it might have stayed in the water bath for too long but is a POSITIVE result.

Procedure

If anyone in your group feels uncertain about the concepts of macromolecules, chemical indicators or the GI, 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 labs in the future.
Download the Lab 3 Notebook Guide from the sidebar.
Identify the chemical indicators you will use. You may not use the SUDAN IV indicator which tests for lipids. Ask your TA to clarify.
Identify the positive (C+) controls needed for each molecular test, and answer the questions below as a group.
1. Protein Solution (Egg albumin): C+ for protein... so it's the C+ for which indicator?
2. Starch (1%): C+ for polysaccharides... so it's the C+ for which indicator?
3. Glucose (10%): C+ for monosaccharides... so it's the C+ for which indicator?
4. Oil: C+ for lipids... so it's the C+ for which indicator?
Identify your beaker of distilled water to serve as the negative control (C-) for each test.
Collect 5 unknown samples using the vials provided in each group’s tub. Your TA will show you the collection stations.
Download and follow the Lab 3 Notebook Guide from the sidebar.
As you begin to design your experiment, refer to the protocols for each indicator in the sidebar. The protocol will explain exactly how the indicator works and what the testing procedure is.
KEEP A CLEAN STATION. Before you leave it must look exactly as it does now!
Work through the first four steps in your lab notebook guide. Once you have designed your procedure move on to designing your data table. Here's an example. Your table will need to include room to test more items.
Get approval from your instructor to proceed with your tests as designed. Be sure notes are taken and data are recorded throughout. Also, take photos as you move through for reference.
Clean-up: All materials/solutions in your test tubes should be disposed of in the hazardous waste container in the back of the lab!
Once you have tested your unknown food samples, and completed your data table, provide your results in the class data table. This will either be on the board or in Excel on the presentation screen.

Indicator Info & Protocols

Lab 3 Notebook Guide. Click to download.

Clean-up: All materials/solutions in your test tubes should be disposed of in the hazardous waste container in the back of the lab!

Exercise III. Would any of these foods be good for a diabetic? Can you predict the GI score of a food based on its molecular composition?

Procedure

If you're not confident with the Glycemic Index (GI), review the "Do you know enough?" section of our Pre-Lab.
Use your test results for each unknown item to predict what the Glycemic Index score might be, step 5 in lab notebook guide.
You should decide on a single number, and a range, and order the unknowns from highest to lowest predicted GI score. Don't forget about the link between GI score and blood glucose levels!
Once complete, ask your instructor to provide you with the key, which will give you the name and GI score for each food item.
Complete your lab notebook guide.
Ensure all members have an electronic copy for reference and be ready to upload your Lab 3 Notebook Entry in the LABridge before we meet again next week.

GI range.

If you finish early, work on your report.
LAB 2 Exercise III. has all the details.

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

Faculty Spotlight: Dr. Sigrid Jacobshagen, Emeritus

Email: sigrid.jacobshagen@wku.edu

Dr. Jacobshagen is extremely interested in clocks; not the kind hanging on the wall or on your phone, but biological clocks and circadian rhythms. She explores these internal physiological regulators, like the sleep/wake cycle, using algae as a model organism. Projects in her lab center on particular types of macromolecules called, photoreceptor proteins that are able to reset these circadian clocks. She is an accomplished researcher in the field of biochemistry and teaches our BioChem courses. She is also heavily involved with the Biochemisty Major. Reach out to her if you found Lab 3 particularity interesting or if you're still trying to decide what track you'd like to pursue in our department.
Research Key Words: Circadian clocks, photoreceptor proteins, gene regulation and mathematical modeling
Recent Publication: Chlamydomonas reinhardtii strain CC-124 is highly sensitive to blue light in addition to green and red light in resetting its circadian clock, with the blue-light photoreceptor plant cryptochrome likely acting as negative modulator.

[Cells]The Public Health Epidemic of DiabetesLab 3: The Role of Food

​Lab 3: Pre-Lab