Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. To prepare the plant sample in Part II:

1. Soak the seeds overnight in water. Use one seed for each 10 mL of solution needed.
2. Grind the soaked seeds in a mortar and pestle using 10–15 mL of water per seed. The final mixture should have the consistency of very runny oatmeal. If desired, this step may be completed by each student group.
3. Any other experimental tissues can be likewise prepared.

2. To prepare the unknowns in Part I use the following recipes: (Increase or decrease quantities in proportion as your required number of setups dictates. The following recipes will provide enough of each solution for all five classes.)

Unknown A: Use only distilled water.
Unknown B: Mix 1 g of gelatin into 100 mL of hot distilled water.
Unknown C: Mix 1 g of soluble starch into 100 mL of hot distilled water.
Unknown D: Use mineral oil provided mixed with an equal volume of water.
Unknown E: Mix 1 g of DNA into 200 mL of distilled water.

3. The chemicals for the kit are provided in stock bottles and need to be dispensed of efficiently for student use. Beral-type pipets and labels are provided for this purpose. In advance, label beakers and pipets appropriately at chemical dispensing areas designated in the lab. This should help eliminate cross contamination of chemicals as students conduct their tests. To label the pipets, use waterproof markers and write on only one-half of the label. Fold the label in half around the pipet barrel just below the bulb as shown in Figure 2.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The test solutions from this lab should be collected and grouped by chemical test and then disposed of as follows: carbohydrate/iodine—Flinn Suggested Disposal Method #12a, protein/biuret—Flinn Suggested Disposal Method #10, Sudan III—Flinn Suggested Disposal Method #26b, and nucleic acid/diphenylamine—Flinn Suggested Disposal Method #5.

Teacher Tips

• This kit is a “super-kit” and will easily serve 5 sections of 30 students working in pairs. Microcentrifuge tubes will need to be cleaned and used from class to class.

• For short class periods, extra microcentrifuge tubes (Catalog No. FB0002) would allow students to begin with the diphenylamine test and to continue with the remaining tests while waiting for the 10–20 minute water bath to complete.
• Consider adding the diphenylamine to the tubes for the students.
• In Part I, if a positive DNA test is not readily seen, try placing the microcentrifuge tube containing the DNA and diphenylamine solution in a beaker of boiling water for 10–20 minutes. A positive color change (blue or purple color) should be seen during this time. When performing this test, be sure the top of the microcentrifuge tube stays securely fastened.
• Create your own floating microcentrifuge trays using the bottoms from Styrofoam^® cups. Remove the sides from each cup and use a cork borer or other small round tube to cut holes in the bottom. This way, if the tops of the microcentrifuge tubes pop off due to increased pressure created by heating the contents, the contents will not spill into the bath and be lost.
• Tests can be run on “known” solutions prior to doing the “unknowns” in Part I of this laboratory kit. Additional test chemicals may be required depending upon the number of additional tests conducted.
• The tubes containing mineral oil will be difficult to clean. If extra tubes are available, you may want to dispose of these used tubes.
• This activity can be taught in a more open-ended format than outlined in this activity. Consider allowing students to design their own experiments to “prove” the presence of the various chemicals in the plant tissue or other tissues of their choice. Controls become important in student designed experiments and should be emphasized in their design plans.
• The lipid and nucleic acid tests can fail entirely on mature plant samples. Young immature tissue or seed tissue works best for producing the reddish-orange and lavender colors, respectively. If there are small quantities of the substances present compared to the amount of test solution, the color change can often be very subtle and appear as swirls in the solution that may disappear when completely mixed. Careful observation is required in dilute and colored tissue material.

Sample Data

Part I. Testing Unknowns

Answers to Questions

Part I. Testing Unknowns

1. Identify the major biochemical in each unknown.

By comparing against knowns (controls) and comparing to established tests. B contains protein, C contains a carbohydrate, D contains a lipid and E contains a nucleic acid.

2. How did you determine the identity of each unknown?

By testing and comparing with known tests for the four basic chemical components.

1. How did you test for the presence of chemicals? What was your control?

A tube was left without testing for comparison. The tubes were compared with known test results.

2. Was the plant tissue composed of key cellular chemicals?

Yes. Positive tests were recorded for carbohydrates, proteins, lipids and nucleic acids.

3. Are plants made of cells? Defend your answer from your experiment.

The results of the chemical tests indicate that the seed tissue contains all the key chemicals found in cells. Chances are very likely that they are made of cells.

References

This kit was created by David Brock, Roland Park Country School, Baltimore, MD.

Introduction

The fundamental chemical components of cells can be grouped into four basic categories: carbohydrates, proteins, lipids and nucleic acids. Other chemicals such as water, salts and minerals are also present and vital to living cells. Tests have been devised by biologists for determining the presence of many of the key chemicals. Learn the chemical tests for the major chemical compounds found in cells and then use them to test for the presence of the chemicals in unknown samples.

Concepts

• Carbohydrates

• Proteins
• Lipids
• Nucleic acids
• Biochemistry
• Chemical nature of living cells

Background

Carbohydrates, proteins, fats and nucleic acids have different chemical structures and different chemical properties. A variety of simple tests has been developed to identify these nutrients based on differences in their chemical properties.

Carbohydrates are the most abundant class of organic compounds found in plants. The basic building blocks of all carbohydrates are simple sugars, such as glucose (blood sugar) and fructose (fruit sugar), and are called monosaccharides. Other more complex carbohydrates are the addition products of two, three or even thousands of simple sugars. These compounds are called disaccharides, trisaccharides and polysaccharides, respectively. Examples of disaccharides include sucrose (table sugar), lactose (milk sugar) and maltose (malt sugar). The most common polysaccharide is starch, which is composed of thousands of glucose units joined together.

The most common polysaccharides are starch and cellulose. Starch serves as an energy storage molecule in plants, where it typically clumps into visible grains. The most familiar sources of dietary starch are potatoes, beans (legumes) and cereal grains (corn, wheat, barley). Reaction with iodine (the iodine test) is used to identify the presence of starch in foods. A positive iodine test result is observed by the appearance of a dark blue color due to the formation of a starch–iodine absorption complex. Cellulose serves a structural role in plants (cell walls) and is not digested in the human stomach.

Proteins are large natural polymers composed of amino acids joined together in chain-like fashion via peptide linkages. The so-called polypeptide chains that make up all proteins can fold up on themselves to form spherically shaped globular proteins, such as enzymes or they can aggregate to form protein fibers such as collagen. Examples of food proteins include albumin (egg white) and casein (milk). Gelatin is a mixture of proteins obtained by hydrolysis of collagen in animal skin, ligaments and tendons.

The presence of proteins in foods can be identified using the biuret test. Biuret test solution contains copper sulfate dissolved in very strong base. The dissolved copper(II) ions coordinate with nitrogen and oxygen atoms in two or more adjacent peptide linkages in a protein molecule to form purple-colored complex ions. A positive biuret test result is marked by the appearance of a lavender or purple color. The intensity of the purple color depends on the nature of the protein and on how much protein is present. The overall reaction for a positive biuret test is summarized in Figure 1.

Fats and oils are members of a biological class of compounds called lipids. Lipids are classified based on a simple physical property—they are insoluble in water. This property of lipids makes them different from carbohydrates and proteins, which generally dissolve in water. The Sudan III test is a classic test for identifying lipids. Sudan III is a special dye that dissolves in nonpolar compounds, such as lipids. It does not dissolve in water. Sudan III is used as a “fat stain” to identify lipids in foods, seeds and animal tissues. The distinction between a positive and negative lipd test can be subtle. Make a positive control using corn or vegetable oil, if necessary.

All living things contain nucleic acids—either RNA or DNA and RNA. Nucleic acids ingested from food are reused in the body to form many components of new cells or to repair or replace damaged cell. Diphenylamine reacts with the nitrogenous bases of the nucleic acid to product a blue to purple-colored complex for DNA or a green-colored complex in the presence of RNA.

Experiment Overview

The purpose of this experiment is to identify the biochemical nutrients—carbohydrates, proteins, fats and nucleic acids—in a variety of foods. The classification tests (and the corresponding nutrients that give positive results) are the iodine test (for starches), the biuret test (for proteins), the Sudan III test (for fats and oils) and the diphenylamine test for nucleic acids.

Materials

Safety Precautions

Diphenylamine contains concentrated sulfuric acid and glacial acetic acid. Sulfuric acid is severely corrosive to eyes, skin and other tissue, extremely hazardous when in contact with finely divided materials, such as carbides, chlorates, nitrates and other combustible materials. Acetic acid is corrosive to skin and tissue and a moderate fire risk. It is also moderately toxic by ingestion and inhalation. Diphenylamine is moderately toxic by ingestion. Iodine solution is an eye and skin irritant. Biuret test solution is corrosive to body tissues. Sudan III solution is alcohol based and is flammable and toxic by ingestion and inhalation. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Conduct activity in a well-ventilated room or in a chemical fume hood. Follow all laboratory safety guidelines. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Procedure

Part I. Testing Unknowns

1. Label five microcentrifuge tubes A–E, respectively.
2. Add 12 drops of unknown A to tube A, 12 drops of unknown B to tube B, etc. through tube E.
3. Add 2–4 drops of iodine to each tube to test for the presence of carbohydrates.
4. Use a “+” to indicate a positive test and a “–” to indicate a negative test. Record the results on the Chemicals of Life Worksheet.
5. Remove the unknowns from the tubes and thoroughly clean and rinse the microcentrifuge tubes.
6. Repeat steps 1–5 using 12 drops of biuret test solution instead of iodine to test for proteins.
7. Repeat steps 1–5 using 5 drops of Sudan III. Mix thoroughly.
8. Repeat steps 1–3 using 12 drops of diphenylamine solution. Place the microcentrifuge tubes into a floating rack within the hot water bath for 10–20 minutes before interpreting the results.
9. Answer the questions for Part I on the Chemicals of Life Worksheet.

Part II. Testing Plant Tissue

1. Secure a sample of plant tissue from your teacher.
2. Test the plant sample for the presence of key chemicals of life. Since the tissue is not clear in color, how will this affect your tests. What can be used as a control?
3. Record your results for Part II on the Chemicals of Life Worksheet and answer the questions for Part II.

Student Worksheet PDF

10254_Student1.pdf