Unit 4: Cell Biology

Study of Plant Cell (Epidermal Peel)

Objective

To prepare a temporary mount of an epidermal peel (e.g., onion, *Rhoeo*, *Crinum*) to observe the basic structure of a plant cell.

Materials

Onion bulb, *Rhoeo* or *Crinum* leaf, slide, coverslip, water, glycerine, microscope, forceps, needle.

Procedure

1. Take an onion bulb and remove a fleshy scale.
2. Snap the scale to break it, and with forceps, peel off the thin, transparent inner epidermis.
3. (For *Rhoeo*, use the lower epidermis, which is purple).
4. Place the peel in a drop of water on a clean slide. Make sure it is flat and not folded.
5. Add a drop of glycerine (to prevent drying) or a drop of dilute iodine (to stain the nucleus).
6. Gently lower a coverslip to avoid air bubbles.
7. Observe under low power (10x) and then high power (40x).

Observations

• Cells are rectangular (onion) or polygonal (*Rhoeo*) and arranged compactly.
• A distinct, thick cell wall is visible on the outside.
• A thin cell membrane is present just inside the cell wall (though hard to see unless plasmolysed).
• A large, central vacuole occupies most of the cell volume.
• The cytoplasm is pushed to the periphery, forming a thin layer (primordial utricle).
• A prominent, dense nucleus is visible in the peripheral cytoplasm.
• In *Rhoeo*, the vacuole is filled with purple-colored cell sap (anthocyanin pigment).

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Cytochemical Staining (Feulgen & PAS)

These are techniques to stain specific chemical components within the cell.

DNA - Feulgen Stain

• Objective: To specifically stain DNA within the nucleus.
• Principle: The stain relies on acid hydrolysis (e.g., warm HCl) which selectively breaks the bonds between purine bases (A, G) and deoxyribose sugar in DNA. This exposes aldehyde groups. These aldehyde groups then react with Schiff's reagent (a colorless liquid) to produce a characteristic purple-red (magenta) color.
• Result: Only the DNA (chromatin in the nucleus) will stain magenta. Cytoplasm remains unstained.

Cell Wall - PAS (Periodic Acid-Schiff) Stain

• Objective: To stain complex carbohydrates, primarily cellulose in the cell wall.
• Principle: Periodic acid is a strong oxidizing agent. It oxidizes the 1,2-glycol groups in polysaccharides (like cellulose) to create aldehyde groups. These aldehydes then react with Schiff's reagent to produce a magenta color.
• Result: The cell wall stains a bright magenta/pink. Starch grains will also stain.

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Measurement of Cell Size (Micrometry)

Objective

To measure the dimensions (length, width) of a cell using a calibrated microscope.

Materials

Microscope, Ocular Micrometer (a ruler in the eyepiece), and a Stage Micrometer (a special slide with a known, precise ruler, e.g., 1mm = 100 divisions).

Procedure: Part 1 - Calibration (This is the most important part)

1. Insert the ocular micrometer into the microscope's eyepiece.
2. Place the stage micrometer on the stage and focus on its ruler (e.g., at 10x or 40x).
3. Rotate the eyepiece to align the ocular ruler parallel with the stage ruler.
4. Find where the "0" marks of both rulers line up.
5. Look along the rulers to find the next point where lines from both rulers align perfectly.
6. Count the number of divisions on the ocular micrometer (OD) and the corresponding number of divisions on the stage micrometer (SD) between these two points.
7. Calculate the calibration factor:
   • We know 1 SD = 0.01 mm = 10 µm (this is the most common value, check your stage micrometer).
   • Let's say 50 OD = 10 SD.
   • Then, 50 OD = 10 * 10 µm = 100 µm.
   • Therefore, 1 OD = 100 µm / 50 = 2 µm.

Procedure: Part 2 - Measurement

1. Remove the stage micrometer.
2. Place your specimen slide (e.g., onion peel) on the stage.
3. Focus on a cell using the same objective lens you calibrated.
4. Measure the length of the cell using your ocular ruler (in OD). Let's say the cell is 60 OD long.
5. Convert OD to µm using your calibration factor.
6. Calculation: Cell Length = (Measurement in OD) * (Calibration factor) = 60 * 2 µm = 120 µm.

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Counting Cells (Haemocytometer)

Objective

To determine the number of cells per unit volume (e.g., cells/mL) in a suspension (e.g., yeast, *Chlorella*).

Materials

Haemocytometer (a special thick slide with a counting grid) with coverslip, cell suspension (e.g., yeast), microscope, pipette.

Procedure

1. Place the special coverslip on the haemocytometer.
2. Prepare your cell suspension (you may need to dilute it so cells aren't overlapping).
3. Use a pipette to load a small drop of suspension into the V-shaped groove. The liquid will be pulled under the coverslip by capillary action.
4. Place the haemocytometer on the microscope stage and focus on the grid lines (use 10x).
5. The grid has 9 large squares. We typically count the cells in the 4 large corner squares and the 1 large central square (which is further divided).
6. Counting Rule: To avoid counting cells twice, only count cells that are inside a square or touching the top and left lines. Ignore cells touching the bottom and right lines.
7. Calculate the total number of cells counted (e.g., you count 150 cells in the 5 large squares).

Calculation

• The volume of 1 large square is: 1 mm (L) * 1 mm (W) * 0.1 mm (H) = 0.1 mm³ = 0.0001 mL.
• Average cells per square = (Total cells counted) / (Number of squares counted)
  e.g., 150 cells / 5 squares = 30 cells/square
• Cells per mL = (Average cells per square) * (Dilution factor) / (Volume of one square in mL)
• Simple Formula:
  Cells/mL = (Total cells counted / Squares counted) * Dilution * 10,000
  e.g., (150 / 5) * 1 (no dilution) * 10,000 = 30 * 10,000 = 300,000 cells/mL

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Study of Plasmolysis and De-plasmolysis

Objective

To observe the effect of solutions with different water potentials (tonicity) on plant cells.

Principle

• Plasmolysis: When a plant cell is placed in a hypertonic solution (a solution with a lower water potential, e.g., strong salt water), water moves by exosmosis (out of the cell). The protoplast (cell membrane + cytoplasm + vacuole) shrinks and pulls away from the rigid cell wall.
• De-plasmolysis: If the plasmolysed cell is then placed in a hypotonic solution (e.g., pure water), water moves by endosmosis (into the cell). The protoplast swells and presses against the cell wall again, and the cell becomes turgid.

Procedure

[Image of Turgid, Plasmolysed, and De-plasmolysed cells]

1. Prepare a wet mount of a *Rhoeo* epidermal peel in water (this is the hypotonic control). Observe the turgid cells. The purple cell sap fills the entire cell.
2. Prepare a new mount, or add a few drops of a 10% salt (NaCl) solution (hypertonic) to one side of the coverslip of the first mount, pulling the water out with a paper towel on the other side.
3. Wait a few minutes and observe. You will see the purple protoplast shrinking away from the cell walls. This is plasmolysis.
4. Now, "wash" the salt solution out by adding pure water to one side of the coverslip and drawing the salt solution out from the other.
5. Observe again. The protoplast will swell and refill the cell. This is de-plasmolysis.

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Effect of Solvent & Temperature on Membrane Permeability

Objective

To demonstrate that high temperatures and organic solvents damage cell membranes, increasing their permeability.

Principle

We use beetroot because its cells store a bright red pigment (betacyanin) in their vacuoles. A healthy, intact cell membrane and vacuolar membrane (tonoplast) will keep this pigment inside. If the membranes are damaged, the pigment will leak out into the surrounding water, coloring it red.

Procedure (Temperature)

1. Take several uniform-sized beetroot discs (use a cork borer). Wash them thoroughly until the water runs clear (to remove pigment from cut cells).
2. Prepare water baths at different temperatures (e.g., 10°C, 25°C, 45°C, 70°C).
3. Place a test tube with a beetroot disc and 10 mL of water in each bath for 15 minutes.
4. Remove the discs and observe the color of the water in each test tube.

Observation: Little or no color leakage at low/room temperatures. At high temperatures (e.g., 70°C), the water will be deep red. This is because heat denatures membrane proteins and melts membrane lipids, destroying the membrane's integrity.

Procedure (Organic Solvents)

1. Prepare a series of ethanol or acetone solutions in water (e.g., 0%, 10%, 25%, 50%, 75%).
2. Place a washed beetroot disc in each test tube with 10 mL of the solvent solutions.
3. Leave for 15 minutes, shaking occasionally.
4. Observe the intensity of the red color in each solution.

Observation: The 0% (control) will have no leakage. As the solvent concentration increases, the water will become redder. This is because organic solvents like ethanol dissolve the lipid bilayer of the cell membranes, causing them to leak.

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Study of Mitosis and Meiosis Stages

Objective

To identify the different stages of mitosis and meiosis from prepared permanent slides.

Material: Mitosis (Onion Root Tip - Meristem)

The root tip is a meristem, a region of active cell division (growth).

• Interphase: Nucleus is intact, chromatin is not condensed. Looks "stringy." This is most of the cells.
• Prophase: Chromatin condenses into visible, thick chromosomes (threads). Nuclear envelope may be breaking down.
• Metaphase: The "middle" stage. Chromosomes (each with 2 chromatids) are clearly visible and are all aligned in a single, neat line at the metaphase plate (the cell's equator).
• Anaphase: The "apart" stage. Sister chromatids separate and are pulled to opposite poles. You will see two groups of V-shaped chromosomes moving apart.
• Telophase: The "end" stage. Two clusters of chromosomes are at the poles. A cell plate (new cell wall) begins to form in the middle. The chromosomes start to decondense.

Material: Meiosis (e.g., Lily Anther - Pollen formation)

You will see cells undergoing Meiosis I and Meiosis II.

• Prophase I: Very complex. Look for homologous chromosomes paired up (bivalents). You might see crossing over (chiasmata).
• Metaphase I: Pairs of chromosomes (bivalents) line up at the metaphase plate (compare to single chromosomes in mitosis).
• Anaphase I: Homologous chromosomes separate and move to opposite poles. (Sister chromatids stay together! This is the key difference from mitotic anaphase).
• Telophase I: Two new cells (dyads) are formed.
• Metaphase II: Looks like mitosis. Single chromosomes line up at the metaphase plate in each of the two cells.
• Anaphase II: Sister chromatids separate and are pulled to opposite poles.
• Telophase II / Tetrads: The final stage. You will see groups of four haploid cells (a tetrad), which will become pollen grains.