Materials Included In Kit

Additional Materials Required

Prelab Preparation

Cut off the hard end and the leafy end of each celery stalk. Precut 1" pieces of celery for each lab group right before the lab to save time during the lab period, if necessary. Be careful not to touch the celery with your hands.

Safety Precautions

Reminds students to always cut away from themselves and others when using razor blades. Sodium bisulfite is a mild body tissue irritant; avoid all contact with skin and eyes. Janus Green B stain will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please consult 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. Excess sodium bisulfite and sucrose solutions may be disposed of according to Flinn Suggested Disposal Method #26b. Excess Janus Green B staining solution may be disposed of according to Flinn Suggested Disposal Method #5. 

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. This laboratory activity can reasonably be completed in one 50-minute class period.
• For this lab to be successful, fresh celery must be used.
• Fresh dye may be added to the slide several times to observe the color change.

Teacher Tips

• This activity is intended for students who are familiar with the use of the compound microscope. An activity such as “Exploring the Compound Microscope” (Flinn Catalog No. FB1819) is great for introducing students to the parts and use of the compound microscope.

Answers to Questions

1. Describe the unstained cell slide—what organelles were visible?

   Nuclei can be seen as round white spheres, mitochondria may be visible as colorless rods, and plasmids appear round and green in color. Cytoplasmic streaming may also be observed.

2. Describe the changes that were observed on the cell slide that are minute after the Janus Green B dye was added.

   Mitochondria are visible as rod-shaped, blue-green colored organelles under 40X. There may be several visible in a single cell. Other observations include cytoplasmic streaming, nuclei, or other organelles.

3. Describe the changes that were observed several minutes after adding the Janus Green B dye to the slide.

   As the dye is reduced, the bluish color will fade to colorless.

4. What happened to the appearance of the mitochondria when the sodium bisulfite solution was added to the slide?

   The blue color reappeared as the dye is oxidized by the sodium bisulfite. The chemical reaction on the dye was reversed as it was oxidized by the sodium bisulfite.

5. What purpose did the sucrose solution serve? The Janus Green B dye?

   The sucrose solution created an isotonic environment for the celery cells, preventing the organelles from bursting in the aqueous environment created when the dye solution was added to the sample. The Janus Green B dye acted as a proton acceptor as it was reduced. This was observed as the blue-colored mitochondria faded to colorless.

Introduction

This activity provides an opportunity to view live, functioning mitochondria—organelles that are rarely seen—under the microscope!

Concepts

• Cell structure
• Enzymes
• Organelles
• Oxidation–reduction

Background

Cells, though very small, contain complex systems. They have mechanisms for obtaining and using energy, reproducing, transporting materials as well as a multitude of cellular processes. Living organisms are classified into two broad categories—prokaryotes and eukaryotes—based upon their distinctive cellular structures. Prokaryotic cells are simple cells and represent the simplest of living organisms, such as bacteria. Eukaryotic cells are larger and more complex and they have specialized cells. Plants, animals, and other organisms are composed of eukaryotic cells. Eukaryotic cells contain complex organelles which have characteristic functions similar to that of organs in the body.

Mitochondria are bean- or rod-shaped organelles that are suspended in the liquid portion of the cell, which is called cytoplasm. They are composed of two protective membranes, the inner and the outer membrane. A series of folds in the inner membrane are called cristae, and provide the ample surface area required for the energy producing reactions in the mitochondria. The open spaces in between cristae are referred to as the matrix (see Figure 1).

Mitochondria are extremely important to cells since they provide a location for cellular respiration. Also, they produce enzymes which catalyze oxidation reactions in the Krebs cycle ultimately producing energy in the form of ATP (adenosine triphosphate) for the cell to use. Due to this, the mitochondria are often considered the “powerhouse” or the “energy centers” of the cell. Glycolysis, the process by which glucose is oxidized, occurs in the cytoplasm of the cell. The end-product of glycolysis is a compound called pyruvate. Pyruvate leaves the cytoplasm and enters the mitochondria where, once converted to the appropriate intermediate compounds, the Krebs cycle begins. Depending on the type of cell and the demand for energy, there may be anywhere from a few to several thousand mitochondria in a single cell.

Although mitochondria are abundant, they are often difficult to see under the microscope. Biological stains commonly used to prepare microscope slides make the transparent mitochondria in each cell nearly impossible to see in the stained cytoplasm. Therefore an alternative procedure, such as the one described in this lab, must be used. The celery sample used in this lab must first be treated with sucrose solution to create an isotonic environment. Isotonic means the concentration of a specific molecule, sugar in this case, is equal in concentration on both sides of the cellular membrane. An isotonic environment prevents organelles from swelling in contact with water, which would cause them to burst when an aqueous solution is added to the sample. As mitochondrial enzymes remove hydrogen atoms from intermediates in the Krebs cycle within the cell, a vital stain that is easily reduced (Janus Green B) intercepts the hydrogen atoms (the dye is reduced) and the blue color of the dye fades to colorless. This provides visual evidence for the enzymatic reactions taking place in the mitochondria.

Materials

Safety Precautions

Razor blades are extremely sharp. Exercise care when working with a razor blade. Always cut away from your body, and others, when using a razor blade. Sodium bisulfite is a mild body tissue irritant; avoid all contact with skin and eyes. Janus Green B solution will stain skin and clothing. 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. Add 2 drops of 0.1 M sucrose solution onto the microscope slide.
2. Using the razor blade, carefully slice a vertical section ¼" thick by ½" long (see Figure 2).
   {10796_Procedure_Figure_2}
3. Peel off the stringy outer layer from the top of the celery piece using the razor blade if needed, and throw the outer layer away in the trash.
4. Using the razor blade, carefully cut a very thin horizontal piece only a few cell layers thick from the fleshy tissue (the thickness of cardstock or paper).
5. Use the razor blade to transfer the celery sample onto the sucrose solution on the slide. Do not touch the fleshy surface of the celery with your hands—the salt from your skin can affect cell function.
6. Cover the sample with a coverslip.
7. Place the slide onto the microscope stage with the 4X objective in position. Focus the microscope as needed and carefully switch to the 40X objective. Bring the sample into focus using the fine focusing knob only.
8. Examine the sample closely for cytoplasmic streaming. Nuclei may be visible as a colorless or white imperfect spheres, plasmids will look like small green specs, and mitochondria may appear as rod-shaped white or colorless objects that may be seen slowly moving within the cellular membrane. Locate a single cell near the edge of the section that contains a nucleus and other organelles.
9. Tear a small (1" x 2") piece of paper towel and hold it to the left side of the coverslip on the microscope slide.
10. Place three drops of Janus Green B solution on the microscope slide to the right of the coverslip. The paper towel will “wick” the staining solution, drawing it through the celery sample (see Figure 3).
    {10796_Procedure_Figure_3}
11. Carefully observe the cell. After one minute note any changes to the cell. Pay particular attention to an “particles” that appear. Janus Green Will stain mitochondria blue. Record your observations on the Mitochondria in Action Worksheet.
12. Observe the mitochondria for the next 3–5 minutes. Record any changes on the Mitochondria in Action Worksheet.
13. Place a fresh 1" x 2" piece of paper towel to the left of the coverslip on the microscope slide.
14. Add 2 drops of 0.5 M sodium bisulfite solution to the right of the coverslip on the microscope slide. Record any changes in the appearance of the mitochondria on the Mitochondria in Action Worksheet.
15. Repeat steps 9–14 again and record your observations on the Mitochondria in Action Worksheet.
16. The microscope slides containing the celery sample may be disposed of in a glass disposal container. Consult your instructor for appropriate disposal or storage procedures of razor blades.

Student Worksheet PDF

10796_Student.pdf