Unit 7: Mitochondria - Subjective Questions

Mitochondria are double-membrane-bound organelles found in the cytoplasm of most eukaryotic cells. They are commonly called the powerhouses of the cell because they produce ATP through aerobic respiration. In human cheek epithelial cells, mitochondria provide energy required for cellular maintenance, active transport, repair, and other metabolic activities. Although cheek cells are relatively simple and flat, they contain mitochondria that can be demonstrated using a suitable vital stain such as Janus green B.

Janus green B is a vital stain used to demonstrate mitochondria in living cells. In the oxidized state, it appears blue or blue-green. Mitochondria contain electron-transfer enzymes that maintain the stain in its oxidized form, causing them to appear blue-green under the microscope. In regions where the dye is reduced, it becomes pale or colourless. Therefore, the appearance of distinct blue-green granules or rods in the cytoplasm indicates the presence of mitochondria.

The required materials generally include:

• A clean glass slide and coverslip
• Sterile toothpicks, cotton swabs, or a clean applicator for collecting cheek cells
• Janus green B stain in suitable dilution
• A dropper or micropipette
• Normal saline or physiological saline
• A compound light microscope
• Lens paper and tissue paper
• A waste container and suitable disinfectant

The use of clean materials and sterile collection tools helps prevent contamination and reduces the risk of transferring microorganisms.

A suitable procedure is as follows:

1. Rinse the mouth with clean water to remove food particles.
2. Gently scrape the inner surface of the cheek with a sterile toothpick or cotton swab.
3. Transfer the collected material to a clean slide containing a drop of physiological saline.
4. Spread the material gently to obtain a thin smear.
5. Add a drop of dilute Janus green B solution to the smear.
6. Place a coverslip carefully to avoid air bubbles.
7. Allow the preparation to stand briefly so that the stain can act on the cells.
8. Examine the slide first under low power and then under high power.
9. Observe the cytoplasm for small blue-green granules or short rods representing mitochondria.
10. Record the cellular features and prepare a labelled diagram if required.

Under the microscope, a cheek epithelial cell is expected to appear as a large, flat, irregularly shaped or polygonal cell. The cell membrane forms the outer boundary, while the cytoplasm appears lightly stained. A distinct nucleus may be visible as a darker, rounded structure. Mitochondria appear as numerous small blue-green granules or short rods distributed in the cytoplasm, often near regions of greater metabolic activity. The cell wall is absent because cheek epithelial cells are animal cells.

Janus green B is considered a vital stain because it can stain living cells without immediately killing them when used at an appropriate concentration. Its colour depends on its oxidation-reduction state. Active mitochondria maintain the dye in an oxidized blue-green form through their respiratory enzyme systems. This allows mitochondria to be observed in living cells and provides an indication of mitochondrial activity as well as mitochondrial location.

A positive result is indicated by the appearance of distinct blue-green granules, rods, or threads in the cytoplasm of the cheek epithelial cells. These structures are interpreted as mitochondria. A negative or inconclusive result occurs when no specific blue-green mitochondrial structures are visible. However, a negative observation does not always prove that mitochondria are absent because poor staining, old cells, excessive dye, incorrect focusing, or unsuitable illumination may prevent their detection. A proper control and repeat observation should therefore be considered.

Mitochondria contain enzymes of the electron transport chain, which carry out oxidation-reduction reactions during cellular respiration. Janus green B behaves as a redox indicator. In its oxidized state, it is blue-green, whereas in its reduced state it becomes much paler or colourless. Active mitochondria provide an oxidizing environment through respiratory activity, allowing the stain to remain blue-green in those organelles. Thus, the observed colour is related to the metabolic activity of mitochondria rather than merely to their physical presence.

In an unstained preparation, cheek epithelial cells may be faint and difficult to distinguish clearly. The cell boundary, cytoplasm, and nucleus may not be sufficiently contrasted, and mitochondria are generally not visible as distinct structures. In a Janus green B-stained preparation, the cell outline and nucleus may become easier to identify, while mitochondria appear as blue-green granules or rods in the cytoplasm. The stain therefore increases contrast and permits the location of mitochondria to be studied.

A thin smear is important because it allows:

• Even penetration of Janus green B into the cells
• Better transmission of light through the specimen
• Reduced overlapping of cells
• Clearer observation of the nucleus and cytoplasmic organelles
• More accurate focusing under high magnification

A thick or clumped smear may trap stain unevenly, produce dark regions, and make it difficult to determine whether blue-green structures are mitochondria or staining artefacts.

Important precautions include:

• Use a clean, grease-free slide and coverslip.
• Collect cells gently to avoid injury to the cheek.
• Use a sterile toothpick or swab for sampling.
• Prepare a thin, evenly spread smear.
• Use the recommended concentration of Janus green B; excessive stain can obscure cell structures.
• Avoid allowing the preparation to dry during observation.
• Place the coverslip gently to prevent air bubbles.
• Begin microscopy with low power before using high power.
• Do not confuse stain precipitates with mitochondria.
• Dispose of used biological material safely and disinfect the work area.

The nucleus is usually a single, relatively large, rounded or oval structure that appears darker and more prominent than the surrounding cytoplasm. Mitochondria are much smaller and generally occur as numerous blue-green granules, short rods, or threads distributed throughout the cytoplasm. The nucleus is not identified merely by the blue-green mitochondrial reaction. Size, number, shape, and location help distinguish the two structures: the nucleus is large and central or slightly eccentric, whereas mitochondria are small and scattered outside the nucleus.

Human cheek epithelial cells are convenient because they can be obtained non-invasively and without complicated equipment. They are large, flat, and relatively transparent, making them suitable for light microscopy. Their simple structure allows students to identify the cell boundary, cytoplasm, and nucleus while observing mitochondrial staining. However, cheek epithelial cells may contain fewer visibly active mitochondria than highly metabolic cells, so careful staining and microscopy are necessary.

Failure to observe mitochondria may result from several factors:

• An overly thick or poorly spread smear
• Improper concentration or deterioration of Janus green B
• Excessive staining or insufficient staining time
• Drying of the preparation
• Use of old or damaged cheek cells
• Inadequate illumination or incorrect focusing
• Failure to use high-power magnification
• Confusion between stain deposits and cellular structures
• Low mitochondrial activity or poor preservation of living cells

The experiment should be repeated with fresh cells, correctly prepared stain, and proper microscopy settings.

A control preparation helps determine whether the observed colour is due to mitochondria or to nonspecific staining. For example, one slide may contain cheek cells without Janus green B, while another contains cheek cells treated with the stain. The unstained slide provides information about the natural appearance of the cells. A known mitochondrial preparation, if available, can serve as a positive control. Comparing the preparations supports the conclusion that blue-green granules in the stained sample are associated with mitochondria rather than with debris or dye precipitates.

Mitochondria produce ATP, the principal immediate energy currency of the cell. Cells with high energy requirements generally contain more mitochondria or more densely packed mitochondrial structures. For example, muscle cells and secretory cells usually require greater ATP production than relatively inactive cells. Cheek epithelial cells are mainly protective and have moderate energy requirements, so the number of visibly stained mitochondria may be lower than in highly metabolic tissues. Mitochondrial abundance is therefore related to cellular function and metabolic demand.

Variation in staining can be explained by differences in:

1. Cell age: Older or damaged cells may have reduced metabolic activity.
2. Cell condition: Healthy cells retain respiratory activity more effectively.
3. Mitochondrial distribution: Organelles may not be evenly distributed in every cell.
4. Smear thickness: Thick areas receive and transmit light differently.
5. Stain penetration: Janus green B may enter some cells more effectively than others.
6. Time after collection: Prolonged exposure outside the body can reduce cell viability.
7. Microscope focus and illumination: Small structures may be missed under unsuitable settings.

Consequently, several cells should be examined before drawing a final conclusion.

The method has several limitations:

• Janus green B is not completely specific for mitochondria under all experimental conditions.
• The colour reaction depends on oxidation-reduction conditions and mitochondrial activity.
• Dead or damaged cells may not show the expected blue-green reaction.
• Stain concentration and exposure time strongly affect the result.
• Small mitochondria may be difficult to resolve with a basic light microscope.
• Dye precipitates may be mistaken for mitochondria.
• The technique usually demonstrates approximate location rather than detailed mitochondrial ultrastructure.

Therefore, the result should be interpreted alongside cell morphology, controls, and proper experimental technique.

Janus green B staining is a relatively simple, inexpensive, and rapid technique that can demonstrate mitochondria in living or recently collected cells using a light microscope. It provides information mainly about the approximate location and activity-related staining of mitochondria. Electron microscopy requires complex equipment and extensive fixation and preparation, but it provides much greater resolution. It can reveal the double membrane, cristae, matrix, and detailed internal organization of mitochondria. Thus, Janus green B is suitable for routine laboratory demonstration, whereas electron microscopy is more appropriate for ultrastructural study.

The report should include:

• The date and objective of the experiment
• The materials and concentration of stain used
• A brief description of the procedure
• The microscope magnification used
• The shape and appearance of cheek epithelial cells
• The location and colour of the nucleus
• The presence, colour, shape, and distribution of mitochondrial structures
• A labelled biological drawing or micrograph, if required
• Any unusual observations or technical difficulties
• A clear conclusion stating whether mitochondria were demonstrated

The conclusion should be based on repeated observation of several cells rather than on a single cell.