Unit 7: Mitochondria

I. Orientation

Mitochondria are membrane-bound organelles responsible for most aerobic ATP production in eukaryotic cells. Their presence can be demonstrated in living or recently prepared cells using Janus green B, a supravital redox indicator whose colour depends on oxidation and reduction. In human cheek epithelial cells, the stain provides visible evidence of mitochondria through their oxidative activity, although the organelles are small and may be difficult to resolve individually with a light microscope.

• Organelle identity: Mitochondria are double-membrane organelles containing an outer membrane, an intermembrane space, an inner membrane, cristae, and a matrix.
• Main function: The inner mitochondrial membrane contains the electron transport chain and ATP synthase, which support oxidative phosphorylation.
• Cellular location: Mitochondria occur in the cytoplasm, often distributed around regions with high energy demand.
• Semi-autonomous features: Mitochondria contain their own circular DNA, ribosomes, and machinery for synthesizing some proteins.
• Dependence on oxygen: Aerobic respiration uses oxygen as the terminal electron acceptor in the electron transport chain.
• Janus green principle: Oxidized Janus green B is blue-green, whereas its reduced form becomes pale or colourless; active mitochondria maintain conditions that favour the coloured form.
• Interpretive convention: Blue-green cytoplasmic granules or threads are interpreted as mitochondria only when their distribution and staining behaviour are consistent with mitochondrial activity.
• Specimen characteristic: Cheek epithelial cells are flat, non-photosynthetic animal cells with cytoplasm, a nucleus, a plasma membrane, and mitochondria.

II. Determination of Mitochondria by Janus Green B Staining — supravital demonstration of oxidative activity

A. Purpose and principle

The method uses a dilute Janus green B solution to stain mitochondria in living cheek epithelial cells. The procedure is called supravital because the cells are examined while they remain alive or shortly after removal from the body.

• Purpose: To demonstrate mitochondria in human cheek epithelial cells by observing blue-green staining in the cytoplasm.
• Chemical basis: Janus green B acts as a reversible oxidation–reduction indicator:
  • Oxidized form: Blue-green colour.
  • Reduced form: Very pale or colourless form.
• Mitochondrial connection: Electron-transfer reactions associated with mitochondrial respiration help maintain the oxidized dye in and around mitochondria.
• Microscopic appearance: Mitochondria may appear as fine blue-green granules, short rods, or thread-like structures in the cytoplasm.
• Biological limitation: The stain indicates respiratory redox activity rather than revealing every mitochondrial membrane directly; a colourless region is not proof that mitochondria are absent.
• Key condition: Fresh, viable cells are essential because prolonged drying, heating, or chemical treatment can destroy mitochondrial activity and weaken the reaction.

B. Determine the presence of mitochondria in human cheek epithelial cells by Janus green staining

This procedure determines the presence of mitochondria by collecting cheek cells, exposing them to Janus green B, and examining stained cytoplasmic structures under a microscope.

• Materials: A clean glass slide, coverslip, sterile toothpick or cotton swab, physiological saline or another approved isotonic medium, dilute Janus green B solution, dropper, microscope, lens tissue, and biohazard disposal container.
• Safety: Treat saliva and used swabs as potentially infectious; wear gloves, avoid mouth contact with laboratory materials, disinfect the work area, and dispose of contaminated items according to laboratory rules.
• Cell collection: Gently scrape the inside of the cheek with a sterile swab or toothpick. Excessive scraping can cause bleeding and introduces unnecessary debris.
• Cell suspension: Transfer the collected material into a small drop of isotonic saline on the slide. A thin suspension is preferable because overlapping cells obscure the cytoplasm.
• Staining: Add the prescribed amount of dilute Janus green B and mix gently without producing bubbles. The exact concentration and exposure time should follow the laboratory protocol because commercial preparations and teaching protocols differ.
• Mounting: Lower the coverslip at an angle to reduce trapped air. Excess liquid should be removed from the coverslip edge with absorbent paper without allowing the preparation to dry.
• Microscope search: Begin with the low-power objective to locate intact, flat cells, then use high power to inspect the cytoplasm. Oil immersion should be used only if the microscope and protocol require it.
• Cell identification: A suitable cheek epithelial cell is usually polygonal or irregularly rounded, with a distinct boundary and a prominent nucleus. The cytoplasm is thin and may be nearly transparent before staining.
• Positive observation: Fine blue-green granules or short filaments distributed through the cytoplasm, but not confined to the nucleus, support the presence of mitochondria.
• Nuclear distinction: The nucleus is generally larger, centrally located, and more homogeneous than mitochondrial granules. Janus green staining is interpreted mainly in the surrounding cytoplasm.
• Observation record: Record the objective used, approximate magnification, staining time, cell condition, colour, location of stained structures, and whether the pattern is granular or filamentous.
• Suggested observation table:

• Expected result: Intact cheek epithelial cells show blue-green mitochondrial granules or threads in the cytoplasm against a comparatively pale background.
• Conclusion format: A suitable conclusion is that blue-green cytoplasmic structures consistent with mitochondria were observed after Janus green treatment, supporting the presence of mitochondria in cheek epithelial cells.
• Worked interpretation: If a cell has a blue-green nucleus but no cytoplasmic structures, the result should not immediately be called positive for mitochondria; the apparent colour may represent nonspecific staining, debris, or an overconcentrated stain. A positive result requires cytoplasmic localization and a mitochondrial-looking pattern.

C. Controls and interpretation of results

Controls distinguish a genuine mitochondrial reaction from staining artefacts, dead-cell changes, or microscope-related errors.

• Unstained control: A cheek-cell preparation without Janus green shows the natural appearance of the cells and identifies background particles that are not stain-related.
• Viability control: Fresh, intact cells should be compared with visibly damaged or dried cells. Strong mitochondrial staining is expected to be more reliable in the fresh preparation.
• Reagent control: A drop of stain without cells reveals whether the reagent itself contains precipitated particles or unusual colour.
• Positive comparison: If available, a preparation known to contain abundant mitochondria, such as a metabolically active tissue cell, demonstrates the expected Janus green reaction.
• Colour interpretation: Blue-green staining suggests oxidation of the dye; loss of colour may reflect reduction, poor viability, oxygen limitation, insufficient staining, or technical dilution.
• Distribution: Mitochondrial staining should occur in the cytoplasm. Staining restricted to the slide surface, stain precipitate, or cell exterior is not adequate evidence.
• Intensity: Darker staining does not necessarily mean more mitochondria. It may result from a thicker cell, longer exposure, higher dye concentration, or uneven illumination.
• Negative result: No visible blue-green structures may result from dead cells, excessive washing, an incorrect reagent, excessive delay before observation, poor focusing, or mitochondria below the resolving power of the microscope.
• Resolution limit: A typical light microscope may show mitochondrial clusters or threads but cannot reliably display all individual mitochondria. Electron microscopy or fluorescent mitochondrial markers would provide more detailed structural information.
• Biological qualification: Janus green detects redox-active mitochondrial regions, so the method demonstrates functional staining behaviour rather than proving the complete structure of each organelle.

D. Variables, sources of error, and limitations

Reliable results depend on preserving cell viability, controlling the stain, and distinguishing genuine structures from artefacts.

• Cell age: Cheek epithelial cells are mature, flattened cells that may contain fewer mitochondria than cells with high energy demands, such as muscle or liver cells.
• Sampling pressure: Vigorous scraping can rupture cells, release debris, and produce blood contamination that makes the preparation difficult to interpret.
• Drying: Air-drying damages membranes and changes cell shape. A dried cell may show irregular stain deposits that resemble organelles.
• Stain concentration: Excessively concentrated Janus green causes a dark background and can obscure fine cytoplasmic structures; overly dilute stain may produce a weak result.
• Exposure time: Too little time may prevent visible staining, while prolonged exposure can increase nonspecific background and reduce interpretive clarity.
• Temperature: Excessive heat accelerates cell damage and may alter redox reactions. The preparation should be kept under the conditions specified by the laboratory protocol.
• Oxygen availability: Because the reaction depends on oxidation–reduction chemistry, limited oxygen diffusion or altered cell metabolism can affect colour intensity.
• pH and medium: An unsuitable medium can damage cells or alter dye behaviour. Isotonic saline or the specified physiological medium helps preserve cell shape.
• Optical problems: Dust, air bubbles, poor illumination, or incorrect focusing can be mistaken for stained organelles. Fine focus and systematic scanning improve reliability.
• Contamination: Food particles, bacteria, mucus, and stain crystals may occur in a cheek sample. Structures should be checked for their position within intact cells.
• Interpretive limitation: Absence of visible blue-green granules is not equivalent to absence of mitochondria; it may only indicate that the reaction was unsuccessful or below microscopic detection.
• Method significance: The experiment links a cell structure to a physiological process: mitochondria are identified through oxidative activity rather than through shape alone.
• Best evidence: The strongest conclusion combines fresh intact cells, appropriate staining, cytoplasmic blue-green granules, suitable controls, and consistent observations across several cells.