Stomata: Structure, Function and Microscopy

How Stomata Open and Close

• How does a plant open a microscopic pore without any muscles?
• First, in the presence of light, potassium ions ($K^+$) are actively pumped into the guard cells from surrounding tissues.
• Next, this lowers the internal water potential, causing water to move into the guard cells by osmosis.
• The cells become turgid (swollen) due to high turgor pressure and, because the thin outer walls stretch more than the thick inner walls, the cells bow outwards to pull the pore open.
• To close the pore during darkness or water stress, potassium ions leave the cells.
• Water then exits by osmosis, causing the guard cells to become flaccid (limp) as turgor pressure decreases, and the thick inner walls spring back together to seal the gap.

Gas Exchange and Transpiration

• Understanding stomata explains how plants manage the conflicting needs of feeding themselves and holding onto water.
• Stomata facilitate gas exchange by allowing carbon dioxide ($CO_2$) to diffuse in for photosynthesis, and oxygen ($O_2$) to diffuse out down their concentration gradients.
• When stomata are open for gas exchange, water vapour also escapes from the leaf.
• This loss of water vapour is called transpiration, and OCR examiners frequently describe it as an inevitable side-effect of opening stomata for $CO_2$.
• By closing the stomata during water stress, guard cells limit and regulate the rate of water loss, effectively managing transpiration.
• The rate of transpiration can be calculated using a potometer:

$$\text{Rate} = \frac{\text{Distance moved by air bubble}}{\text{Time}}$$

Preparing a Slide to View Stomata

• Every time a biologist wants to study leaf surfaces, they must choose the right preparation method to get a clear view.
• Method 1: The Epidermal Peel
• Step 1: Snap a leaf (like Tradescantia or lettuce) and tear it diagonally to peel away a transparent layer of the lower epidermis.
• Step 2: Use forceps to transfer the epidermal peel onto a microscope slide.
• Step 3: Add a drop of water or a stain (like Iodine or Safranin) to highlight cell structures.
• Step 4: Lower a coverslip at a 45-degree angle using a mounted needle to prevent trapping air bubbles.
• Method 2: The Nail Varnish Impression
• Step 1: Paint a thin layer of clear nail varnish on the leaf underside and let it dry for 2 to 5 minutes.
• Step 2: Press clear sticky tape over the dried varnish, peel it off to capture the surface cast, and stick it directly onto a slide (no water or coverslip is needed).

Focusing the Light Microscope

• The most common way students break microscope slides is by focusing in the wrong direction!
• Step 1: Always start by selecting the lowest power objective lens (usually $\times 4$).
• Step 2: Place the slide on the stage and use the coarse focus knob to raise the stage as close to the lens as possible while looking from the side (never through the eyepiece) to avoid crushing the slide.
• Step 3: Look through the eyepiece and slowly turn the coarse focus knob to move the stage AWAY from the lens until the specimen comes into rough focus.
• Step 4: Use the fine focus knob to sharpen the image.
• Step 5: Rotate to a high-power lens (e.g., $\times 40$) and use ONLY the fine focus knob to adjust.
• Total magnification is calculated using this formula:

$$\text{Total Magnification} = \text{Eyepiece Lens Magnification} \times \text{Objective Lens Magnification}$$

Calculating Stomatal Density

• To compare different leaves, start with a concrete measurement: stomatal density, which is the number of stomata packed into a specific area.
• Step 1: Calculate the area of the Field of View using the formula for the area of a circle:

$$\text{Area} = \pi r^2$$

• Step 2: Count the number of stomata visible within that area.

• Step 3: Divide the number of stomata by the area.

• Worked Example:

• Step 1: If the field of view has a diameter of $0.5$ mm, the radius ($r$) is $0.25$ mm.

• Step 2: Calculate Area: $\text{Area} = 3.14 \times 0.25^2 = 0.196 \text{ mm}^2$.

• Step 3: Calculate Density: If you count 20 stomata in this view:

$$\text{Density} = \frac{20}{0.196} = 102 \text{ stomata per mm}^2$$