Materials Included In Kit

Additional Materials Required

Prelab Preparation

Actively growing onion root tips are required for Activity 1. Allow at least 2–4 days for new roots to grow. You may grow the roots ahead of time or have students grow them as a group and complete Activity 1 afterwards. To grow root tips, locally obtain 5–6 onion bulbs or green onions. Peel off any old 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 4).

As shown in Figure 4, push toothpicks into the bulb to support the bulb on the rim of the cup. Add water as needed to the cup during the root growing time to keep the root area under water. The roots should be about 2 cm in length when they are ready to harvest.

Safety Precautions

Hydrochloric acid solution is toxic by ingestion or inhalation and corrosive to skin and eyes. Methylene blue stain is a permanent stain on many objects. The scalpel is a sharp object—use care when cutting with the scalpel. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron throughout this lab. Follow all normal classroom guidelines. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. 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. Methylene blue stain can be disposed of according to Flinn Suggested Disposal Method #26b and hydrochloric acid according to method #24b. Microscope slides, cover slips and scalpels may be disposed of according to Flinn Suggested Biological Waste Disposal Method Type V. Plant material may be disposed of according to Flinn Suggested Biological Waste Disposal Method Type VI, common garbage wastes.

Lab Hints

• Ensure that students label all materials with their group number and the activity number or chemical used in the container.
• We can see mitosis in action in the root tips of sprouting onion (Allium sp.) because the chromosomes are particularly large, and the mitotic rate is high.
• Student slides can be sealed with nail polish, wax or other slide sealing materials for longer term use.
• 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.
• Prometaphase is included as a phase in mitosis since it is included in literature published by the National Institute of Health. Include prometaphase events with metaphase events if prometaphase is not included in your textbook.
• Repeat Activity 1 and 2 using a prepared slide of Whitefish blastula.
• Have students remove entire lengths of root when cutting the roots from the onion. Do not have them cut just partial roots. This may cause other students to remove a root that doesn’t have a root tip! After the entire root has been removed, only the 1-cm tip should be cut off and used in the exercise. Several onions will be required to provide enough root tips for an entire class.
• The following root tips were analyzed alfalfa, barley, grass, marigold and radish. Nuclei were visible, but the phases of mitosis were not easily differentiated.

Sample Data

Analysis, Activity 1

Analysis, Activity 2

Table 1

Answers to Questions

Activity 1

1. Explain why root tip cells are selected for the study of mitosis.

   Blastula and root tips are areas with mitosis actively occurring.

2. Describe the principal cellular “events” that occur in each phase of the cell cycle. 

   Interphase consists of the G1 stage is the cell’s primary growth stage and typically the longest time is spent in this stage. New cells are very metabolically active and are actively synthesizing RNA and new proteins. The DNA is replicated during the Synthesis phase and during the G2 stage, during which various organelles are replicated, the chromosomes start to condense and microtubules are synthesized.
   Mitosis consists of five phases:

   • Prophase—The nucleolus fades and chromatin condenses into chromosomes. Microtubules disassemble and are used to create the spindle fibers necessary for chromosome separation.
   • Prometaphase—The nuclear envelope breaks down so there is no longer a recognizable nucleus. Some spindle fibers elongate and attach to the kinetochore protein bundles located on the chromosomes. Other spindle fibers elongate but instead of attaching to chromosomes, overlap each other at the cell center.
   • Metaphase—The chromosomes reach a position midway between the poles called the metaphase plate.
   • Anaphase—The kinetochore protein bundles separate and as a result the sister chromatids separate, splitting the chromosome in half. The spindle fibers shorten dragging the attached chromatid to opposite poles of the cell.
   • Telophase—The daughter chromosomes arrive at the poles and the spindle fibers that have pulled them apart disappear. A nuclear envelope reforms around each cluster of chromosomes and these chromosomes return to their more extended form.

   Cytokinesis consists of the division of the cytoplasm and organelles to form two identical daughter cells.

3. What is the role of the kinetochore? Is it necessary for mitosis? Defend your answer.

   The kinetochore proteins act as the attachment point on the chromosome for the microtubule formed by the mitotic spindle. The kinetochore proteins separate and allow the sister chromatids to be divided evenly as they are pulled to the opposite poles of the cell for cell division.

4. How would the results for the percent of cells in each phase of the cell cycle have been different if your observations had not been restricted to the area of the root tip that is actively dividing?

   The results would have been skewed due to the larger number of cells in interphase.

5. How do the cells at the root cap differ from the mature cells away from the root cap?

   The cells at the root cap are the apical meristem, the point of active mitosis for the roots of a plant.

Activity 2

1. Based on the data in Table 1, rank the phases of the cell cycle based on the relative duration of each phase, from longest to shortest.

   Root tip cells spend most of their time in interphase and the least amount of time in telophase.

2. Draw and label a pie chart showing the relative amount of time a typical onion root tip cell spends in each phase of the cell cycle.
   {10767_Answers_Figure_6}

References

Biology: Lab Manual; College Entrance Examination Board: 2001.

Introduction

One of the tenets of the cell theory is that “all cells only arise from pre-existing cells.” In fact, new cells are formed by the process of cell division to form two genetically identical daughter cells.

Objectives
After completing this laboratory, you should be able to:

• Visually recognize the phases of mitosis in plant and animal cells
• Calculate the relative duration of each phase in the cell cycle
• Compare and contrast mitosis in plant and animal cells

Concepts

• Cell cycle
• Karyokinesis
• Cytokinesis
• Mitosis

Background

The process of growth and division in a typical eukaryotic cell is called the cell cycle and is composed of five stages—G1, S, G2, M and C (see Figure 1). The cell cycle begins with the formation of a new cell and continues until that cell divides into two offspring cells. In a mammal or higher order plant, a complete cell cycle lasts about twenty-four hours. Each offspring cell then begins the cycle again. The dramatic events of nuclear division take place during the karyokinesis or M (mitosis) stage, which represents only a brief segment, typically two to four hours, in the overall 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. These stages are collectively termed interphase and include the G1, S and G2 stages. Interphase typically take between twenty and twenty-two hours in a mammal or higher order plant cell. Although the main sequence of the phases of the life cycle of a cell is fixed, the amount of time spent in each phase varies among different organisms and among different cells within an organism. Interphase is divided into three stages. The G1 (Gap 1) stage is the cell’s primary growth stage and typically the longest time is spent in this stage. New cells, which are metabolically very active, are actively synthesizing RNA and new proteins. The G stage normally lasts about ten hours. Some cells go into an extended G1 stage and rarely ever divide again. This stage is called the G0 stage. Neurons, for example, are very active and important cells but tend to remain in the G0 stage. From the G1 stage, most cells go to the S (synthesis) stage. In the S stage, an exact copy of DNA is made in the nucleus of the cell. The S stage usually takes five to six hours to create an exact copy or replicate the DNA. During the G2 (Gap 2) stage, various organelles are replicated, the chromosomes start to condense, and microtubules are synthesized. Because the DNA replicates in the S phase, a cell in G2 has twice as much DNA in its nucleus as a cell in G1. The duration of G2 is usually short, about three to four hours on average. After these three stages of the cell cycle, G1, S and G2, are complete, the nuclear division called karyokinesis or M (mitosis) stage can begin. The M stage is easily identified because it is the only phase in which the chromosomes are visible with a light microscope. In most cells, mitosis lasts only two hours of the entire twenty-four hour cell cycle. Mitosis is followed quickly by cell division of the parent cell’s cytoplasm and organelles to produce two offspring cells in the C (cytokinesis) stage.

The length of the cell cycle is important because it determines how quickly an organism can multiply. For single-celled organisms, this rate determines how quickly the organism will reproduce new, independent organisms. For higher-order species the length of the cell cycle determines how long it takes to replace damaged cells. The duration of the cell cycle varies from organism to organism and from cell to cell. Certain simple multicellular organisms have cell cycles that last only 8 minutes. Some liver cells take up to a year to complete one cell cycle. Most of the differences in cell cycle duration among different species or different kinds of cells are found in the duration of specific cell cycle stages, generally the G1 and G2 stages. In complex organisms, early embryonic cells divide in twelve hours instead of the usual twenty-four hours, often omitting the G1 and G2 stages, and then quickly proceed through successive rounds of the S stage, M stage and C stages.

In higher plants, cell division occurs in areas called meristems. Meristems usually occur at the tips of stems or roots and are responsible for plant lengthening and enlarging as well as leaf, flower, stem, and fruit production. Instead of repairing or replacing damaged cells, plant cells create new organs, such as a leaf, at the meristems locations. In animals, cell division occurs anywhere new cells are formed or as new cells replace damaged ones. However, some tissues, like nerves, in both plant and animals rarely divide once the organism is mature.

The M stage of the cell cycle is further subdivided into five phases as shown in Figure 2. During prophase, the nucleolus fades and chromatin condenses into chromosomes. Each replicated chromosome comprises two chromatids, both with the same genetic information. Microtubules of the cytoskeleton, which is responsible for cell shape, motility, and attachment to other cells during interphase, disassemble to be used to create the spindle fibers necessary for chromosome separation. In prometaphase, the nuclear envelope breaks down so there is no longer a recognizable nucleus. Some spindle fibers elongate and attach to the kinetochore protein bundles located on the chromosomes. Other spindle fibers elongate but instead of attaching to chromosomes, they overlap each other at the cell center. During metaphase, the chromosomes reach a position called the metaphase plate, which is midway between the poles. The chromosomes are at their most compact at this time. At the onset of anaphase, the kinetochore protein bundles separate and as a result the sister chromatids also separate, splitting the chromosome in half. The spindle fibers shorten and drag the attached chromatids to opposite poles of the cell. In telophase, the daughter chromosomes arrive at the poles and the spindle fibers that have pulled them apart disappear. A nuclear envelope reforms around each cluster of chromosomes and these chromosomes return to their more extended form while cytokinesis begins. In animal cells, cytokinesis results when the membrane is pulled inward by the cytoskeleton at a point called the cleavage furrow. The pulling in of this cleavage furrow continues until the deepest parts on opposite sides meet in the center of the cell. At that point, when membrane hits membrane, the cell membrane fuses together, separating the two daughter cells. In plant cells, the rigid wall requires that a cell plate be synthesized between the two daughter cells. To do this plant cells send vesicles filled with cell wall material to their equator. When the vesicles reach the equator, they bump into other vesicles and fuse together, forming the cell plate. As more vesicles go to the equator, the cell plate expands until it bumps into the cell membrane. When the cell plate reaches the cell membrane, it fuses with it to form the complete cell wall.

Experiment Overview

Activity 1. Observing Mitosis in Plant and Animal Cells
In Activity 1, the stages of the cell cycle will be visualized with a light microscope. The relative duration of each phase of mitosis and interphase will be determined.

Activity 2. Duration of the Cell Cycle
The previously prepared root tip slide is a snapshot of the cell cycle in the apical meristems of the plant. The portion of cells in each phase of the cell cycle should correspond closely with the amount of time spent in each phase. In mammals, and both monocot and dicot plants, the time to complete one cell cycle is typically 24 hours or 1440 minutes. By counting the number of cells in each phase of the cell cycle, the relative duration of each phase can be calculated.

Materials

Safety Precautions

Hydrochloric acid solution is toxic by ingestion or inhalation and corrosive to skin and eyes. Methylene blue stain is a permanent stain on many objects. The scalpel is a sharp object—use care when cutting the root tips with the scalpel. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron throughout this lab. Wash hands thoroughly with soap and water before leaving the laboratory. Follow all normal laboratory safety guidelines.

Procedure

Activity 1. Observing Mitosis in Plant and Animal Cells

1. Grow root tips as directed by your instructor or obtain root tips from your instructor.
2. Cut three roots from actively growing plant using a scalpel. Note: The scalpel is extremely sharp.
3. Trim the tip of each root to 1 cm; use only the tapered end of the root tip.
4. Use forceps to place 2–3 root tips (use only the 1-cm tips) on a glass microscope slide.
5. Using a clean, graduated pipet, add 2–3 drops of 1 M HCl to cover the root tips on the microscope slide. Note: Hydrochloric acid is severely corrosive to skin and eyes.
6. Allow the root tips to soak in the acid for 5 minutes.
7. After 5 minutes, use a paper towel to blot away any excess HCl from the slide.
8. Using a clean, graduated pipet, add 2–3 drops of deionized water to the root tips.
9. Use a paper towel to blot away any excess water.
10. Repeat step 8 and 9.
11. Using a clean, graduated pipet, add 2–3 drops of methylene blue stain to the root tip. Note: Methylene blue stain is a permanent stain.
12. Allow the root tips to soak in the stain for 3 minutes.
13. Use a paper towel to blot away the excess methylene blue stain.
14. Add 1 drop of deionized water to the root tips.
15. Apply a cover slip over the tissue. Cover the slide with a paper towel and apply thumb pressure to squash the root tissue. Apply an even downward pressure on the root tips and coverslip but not so hard as to break the cover slip. Do not twist or grind the cover slip.

Analysis

1. Using the 4X objective on the microscope, focus on the apical meristems of the root tip. This is the area just behind the root cap as shown in Figure 3.
   {10767_Procedure_Figure_3_Onion root tip diagram}
2. Switch to the 40X objective and fine focus on the plant cells. Study all of the squashed tissue and find the area in which mitosis is occurring.
3. Locate cells in the following phases of the cell cycle: interphase, prophase, prometaphase, metaphase, anaphase and telophase. Note: All phases of mitosis may not be present within a single field of view.
4. Sketch a root cell in each phase of the cell cycle on the Mitosis Worksheet.
5. Answer the questions on the Mitosis Worksheet.

Activity 2. Duration of the Cell Cycle

1. Using the 4X objective on the microscope, focus on the apical meristem of the root tip.
2. Switch to the 40X objective and fine focus on the plant cells. Find an area in which mitosis is occurring.
3. The partner observing the slide should call out the phase of each cell that is observed, while the other partner records tally marks in Table 1 on the Cell Cycle Duration Worksheet. Count all of the cells in the field of view.
4. Have the observer and recorder switch roles, so the recorder becomes the observer and visa versa. Find another area of mitosis and count another complete field of view.
5. Count at least two full fields of view or a minimum of 200 cells, whichever is greater, by alternating the roles of recorder and observer.

Analysis

1. Count the tally marks and record the total in Table 1 on the Cell Cycle Duration Worksheet.
2. Calculate the percent of cells in each phase of the cell cycle using the following equation.
   {10767_Procedure_Equation_1}
3. It takes an average of 24 hours (or 1,440 minutes) for root tip cells to complete the cell cycle. Calculate the amount of time spent in each phase of the cell cycle from the percent of cells in that phase using the following equation. Record the results on the Duration Worksheet.
   {10767_Procedure_Equation_2}
4. Answer the questions on the Duration Worksheet.
5. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10767_Student1.pdf