Materials Included In Kit

Additional Materials Required

Prelab Preparation

Actively growing onion root tips are required for Part II of the activity. Allow at least 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 Part II afterward.

Locally obtain 5–6 onion bulbs. Peel off any dead root growth from 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 2).

As shown in Figure 2, push toothpicks into the sides of 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 other 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 (5–6) will be required to provide enough root tips for an entire class.

Safety Precautions

Hydrochloric acid solution is toxic by ingestion or inhalation and severely corrosive to skin and eyes. Toluidine blue stain is slightly toxic by ingestion and is a permanent stain on many objects. 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. Toluidine blue solution can be disposed of according to Flinn Suggested Disposal Method #26b and hydrochloric acid according to method #24b.

Teacher Tips

• Enough materials are provided in this kit for 30 students working individually or in pairs. The entire activity (except for root growing time) can be reasonably completed in two 50-minute class periods.

• An excellent supplement to this laboratory is the examination of prepared slides of plant and animal mitosis. The comparison of animal and plant cytokinesis can be easily made with prepared slides. In addition, the various stages of mitosis can be quantified and time estimates of the length of various stages of mitosis can be made. Have students randomly sample 100 cells and determine their mitotic condition and then tally the percent of each stage. The percentage correlates with the relative amount of time cells spend in each stage of the process.
• The terms prophase, metaphase, anaphase, telophase, etc., are not included in the materials in this activity. These terms are easily added to the instruction if desired. Have students add them when discussing the drawings in this activity.
• There are many excellent video sequences available showing cells dividing. These video clips can help students visualize the actual movement of chromosomes and are highly recommended during the teaching of the cell cycle.
• The pictures in the activity can be very useful as an evaluation tool. Start with a picture other than picture B and have students sequence the pictures until it recycles back to the starting point. Alternately, leave out some pictures in the sequence and have students draw and describe the missing steps.
• The sequence of mitosis pictures and their matching descriptions should be: B–1, E–2, H–3, A–4, C–5, I–6, G–7, F–8 and D–9.

References

Campbell, N. A.; Reece, J. B. Biology, 7th ed.; Pearson Education: San Francisco, 2005; Chapter 12.

Inside the Cell; U.S. Department of Health and Human Services. National Institutes of Health. National Institute of General Medical Sciences. NIH Publication No. 05–1051, September 2005 revision. 46–51.

Introduction

Living cells are continually involved in complex reactions associated with being alive. One important sequence of events critical to the survival of a cell type is its reproductive cycle. How do cells produce more cells like themselves?

Concepts

• Cell cycle

• Mitosis
• Chromosomes
• Cytokinesis

Background

The life of a eukaryotic cell consists of a continuous sequence of events known as the cell cycle (see Figure 1).

The cell cycle begins with the formation of a new cell and continues until that cell divides into two offspring cells. Each offspring cell then begins the cycle again. The M (mitosis) phase, which features the dramatic events of cell division, is only a brief segment in the total life cycle of the cell. Most of the life of a cell is spent performing normal metabolic activities, growing and preparing the cell for its next division. Although the main sequence of phases in the life cycle of a cell is fixed, the amount of time spent in each phase varies among cells of different organisms and among different cells within an organism.

The G₁ (gap 1) phase is the time of cell growth just following cell division. New cells are metabolically very active and are synthesizing RNA and new proteins. Some cells go into an extended G₁ phase and rarely ever divide again—this is called a G₀ phase. Nerve cells, for example, are very active and important cells but tend to remain in G₀ phase for extended periods of time.

From the G₁ phase, most cells enter the S (synthesis) phase. In the S phase, an exact copy of DNA is made in the nucleus of the cell. Replication of the DNA ensures that a complete and identical set of genes is available for each new offspring cell following cell division. The S phase is typically the longest phase of interphase.

G₂ (gap 2) is the phase of growth and metabolism occurring after the S phase. The synthesis of protein, RNA and other macromolecules continues as the cell prepares to divide. (See Figure 1 and compare differences in size of each phase.) Because the DNA replicates in the S phase, a cell in G₂ has twice as much DNA in its nucleus than in G₁.

The M phase occurs after G₂ and is easily identified because it is the only phase in which the chromosomes are visible with a light microscope. Mitosis is nuclear division in which the replicated chromosomes separate to form two nuclei from one. In most cells, mitosis (nuclear division) is followed quickly by cytokinesis (cell division) of the parent cell into two offspring cells completing the M phase. After the M phase, each offspring cell enters G₁, and the cycle continues.

Materials

Safety Precautions

Toluidine blue stain is moderately toxic by ingestion and is a permanent stain on many objects. Hydrochloric acid solution is toxic by ingestion or inhalation and severely corrosive to skin and eyes. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Razor blades are extremely sharp. Use care when cutting with them. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part I. Picturing Mitosis

1. Locate a Mitosis Picture Set, a Mitosis Descriptions Worksheet and a Mitosis Study Worksheet.
2. Cut out the numbered descriptions from the Mitosis Descriptions Worksheet.
3. Spread out the mitosis pictures on the table. Read each description carefully and match it with the picture that best illustrates the description. Place the description on its matching picture. When there is consensus about the matches, use cellophane tape to tape the description to the bottom of each lettered picture.
4. Begin with picture B and place the other pictures in order of their occurrence through one cycle of cell division. Record the picture order created on Part I of the Mitosis Study Worksheet.

Part II. Onion Root Cells

1. Grow onion root tips as directed by your instructor or secure and treat root tips that your instructor has provided.
2. Cut three roots from actively growing onion root tips using a razor blade. Note: The razor blade is extremely sharp.
3. Trim the tip of each root to 1 cm. Use only the tapered end of the root tip. Discard the remainder of the root.
4. Use forceps to place 2–3 root tips (use only the 1-cm tips) on a glass microscope slide and cover them with 2–3 drops of 1 M hydrochloric acid.
5. Allow the root tips to soak in the acid for 5 minutes.
6. After 5 minutes, use a paper towel to blot away any excess hydrochloric acid.
7. Using a clean pipet, add 2–3 drops of distilled or deionized water to the root tips.
8. Blot off any excess water and repeat steps 7 and 8.
9. Add 2 drops of toluidine blue stain to the root tips. Allow them to soak for 3 minutes.
10. Use forceps to move one root tip to a clean microscope slide.
11. Apply a cover slip over the root tip. Cover the slide with a paper towel and apply thumb pressure to squash the root tissue. Apply an even downward pressure on the root preparation. Caution: Do not press so hard as to break the coverslip.
12. Using a microscope, study the slide first on low power (100X) and then on high power (400X). Study all of the squashed tissue and locate as many stages of mitosis as possible. Sketch all of the stages found in the spaces provided on the Mitosis Study Worksheet.
13. Try to locate a cell with distinctly visible chromosomes and try to determine the chromosome number for onion cells.
14. Try to find cells corresponding to every stage pictured in your picture set. What stage was found most often?
15. Answer the questions in Part II on the Mitosis Study Worksheet.
16. Consult your instructor for appropriate disposal procedures.