Materials Included In Kit

Additional Materials Required

Prelab Preparation

Actively growing onion root tips are required for this activity. Allow 2–4 days for new roots to grow. You may grow the roots ahead of time or you may have students grow them as a group and complete the activity days later.

Locally obtain 5–6 onion bulbs. (Select large, non-dried onions. Do not use small green onions.) Peel off any old root growth on the bottom of the bulbs. Place each onion bulb into a plastic cup or jar of water so that only the root portion of the bulb is under water (see Figure 1).

As shown in the figure, push several toothpicks into the bulb to support the bulb on the rim of the cup. Add water to the cup during the root growing time as needed to keep the root area under water. The roots should be about 2 cm in length when they are ready to harvest. Have students remove entire lengths of root when cutting the roots from the onion. Do not have them cut just partial roots. This will prevent later students from removing a root that doesn’t have a root tip! After removing the entire root only the 1-cm tip should be cut off and used in the exercise. Several onions should provide enough root tips for an entire class.

Safety Precautions

Hydrochloric acid and acetic acid are toxic by ingestion and inhalation and severely corrosive to skin and eyes. Sodium bisulfite is slightly toxic and is a severe irritant to skin and tissue. Schiff’s Reagent Dye is a body tissue irritant. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information.

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. Chemicals used in this activity can be disposed of according to Flinn Suggested Disposal Methods as follows: hydrochloric acid, #24b; acetic acid, #24a; sodium bisulfite, #26b; and Schiff’s Reagent Dye, #26b. The small amounts rinsed off during the slide preparation can follow #26b.

Teacher Tips

• Enough materials are provided in this kit for 30 students working individually. The laboratory activity can reasonably be completed in one 50-minute class period.
• Student slides can be sealed with nail polish, wax or other slide-sealing materials for longer term use or for examination during a second laboratory period.
• An excellent supplement to this laboratory is the examination of prepared slides of plant and animal mitosis. Students can compare their staining of chromosomes with that of professionally prepared slides. Chances will be good that students will get excellent preparations on their slides.
• Other stains are also effective for staining DNA and can be used to stain actively dividing cells. Aceto-carmine, methylene blue and other orcein stains can be tried.

Answers to Questions

1. Make sketches of your best DNA/cell observations:

Student answers will vary.

2. Where in the cell is the DNA located?

The DNA is located in the nucleus.

3. How does the stained DNA compare in color to the other cellular parts?

The nucleus is typically stained darker than other organelles.

4. When in a cell’s life cycle does the DNA seem most visible?

The DNA is most visible during the S phase.

5. Why is DNA considered to be such an important molecule of life?

DNA is considered the molecule of life because it stores information in biological molecules of living organisms. It carries the information/instructions needed to guide the development of an entire organism.

Introduction

DNA (deoxyribonucleic acid) has come to symbolize modern molecular biology. It is significant for understanding molecular genetics and the critical workings of all living things. A first step in DNA study involves the isolation, staining and examination of DNA with a microscope.

Concepts

• DNA
• Biological staining
• Cell cycle

Background

Cells are very small yet very complex. It is difficult to observe many cell organelles by simply viewing them with a microscope. Most organelles do not interfere with the passage of light through the cell. Therefore, most cells are nearly transparent when viewed through a microscope. For more than 300 years, biologists have worked to develop a variety of techniques and aids for enhancing the visibility of cell structures. One technique that helps make cell structures more visible is to apply stains.

During the 19th century, the textile industry started to boom. The demand for new dyes and vibrant fabrics was high. This demand sparked a great deal of experimentation and exploration for new dye sources. As dyes were discovered and items were dyed, it was observed that many dyes showed specific affinities for specific materials. An offshoot of this was the realization that certain dyes showed specific affinities for certain cell parts. It was then learned that certain dyes and dying procedures were particularly effective in staining DNA. This lab activity will utilize one of these staining procedures often referred to as the Feulgen stain reaction for DNA. Several scientists, including Robert Feulgen, described an initial method for staining chromosome materials in 1924. It has been refined throughout the years and variations on the initial procedure have been tried and tested, but the technique still used today is very similar to the one Feulgen used in 1924.

Materials

Safety Precautions

Hydrochloric acid and acetic acid are toxic by ingestion or inhalation and severely corrosive to skin and eyes. Sodium bisulfite is slightly toxic and is a severe irritant to skin and tissue. Schiff’s Reagent is a body tissue irritant. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Grow onion root tips as directed by your instructor or secure and treat root tips that your instructor has grown for you.
2. Use a razor blade or sharp scalpel to cut an entire root from the end of an actively growing onion root tip. Then cut about 1 cm of tissue from the very tip of the root.
3. Place the root tip on a clean microscope slide.
4. Using a Beral-type pipet, cover the root tip with 3–4 drops of 1 M hydrochloric acid solution (HCl).
5. Allow the root tip to soak in the hydrochloric acid for at least three minutes.
6. Rinse the root tip thoroughly with tap water. Wash away the acid slowly by dispensing water over the top of the root tip using a pipet. Do this over a sink or a collection container. Do not rinse so vigorously that the root tip is washed away. Blot away excess water with a paper towel.
7. Add enough Schiff’s reagent dye to cover the root tip (2–3 drops). Allow it to stand for 20 minutes.
8. Use a pipet and tap water to rinse any excess Schiff’s Dye from the root tip, being careful to not lose the root tip. Blot away excess water with a paper towel.
9. Cover the root tip with sodium bisulfite solution (4–5 drops) for 2–3 minutes. This solution bleaches all the parts of the cell that do not contain the stained DNA.
10. Rinse the root tip with water one more time and then add sodium bisulfite solution (4–5 drops) again for 2–3 minutes.
11. Rinse the root tip with water again and blot away any excess water with a paper towel.
12. Move the root tip to a clean microscope slide and add 1–3 drops of acetic acid solution.
13. Place the slide with the root tip on a smooth, flat surface. Place a coverslip over the top of the root tip.
14. Fold a piece of paper toweling and slip the slide with the coverslip inside of the folded paper toweling. Press slowly and firmly straight down on the toweling and on top of the coverslip. Do not twist or grind the coverslip, but rather apply a firm and even pressure straight down on the coverslip as you “squash” the root tip.
15. Wipe off any excess liquid on the slide and examine the slide using a compound microscope. Use low power to try to identify darkly stained, deep red–purple areas of DNA. When cells are identified and DNA located, switch to high power for a more detailed view. Be sure to try various light intensities during your observations and continually focus up and down through the tissue.
16. Examine many different areas of your squashed root tip. Especially look at tissue that was near the very tip and look for cells that appear to be dividing or show the phases of mitosis. Record your observations on the DNA Staining Worksheet and then answer the questions on the worksheet.
17. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10421_Student1.pdf