A2.2.2 Microscopy skills Notes

Preparing Temporary Mounts and Staining

1. When observing cells under a microscope, the first step is to prepare a slide.
2. A temporary mount enables you to examine fresh or living specimens without the need for complex preparation techniques.

How to Prepare a Temporary Mount

1. Obtain the Specimen: Select a thin layer of cells or tissue, such as the lower epidermis of a leaf or cells scraped from the inside of your cheek.
2. Place on the Slide: Lay the specimen flat on a clean microscope slide.
3. Add a Drop of Water or Stain: Use water to keep the cells hydrated, or apply a stain like methylene blue or iodine to enhance visibility by highlighting specific structures (e.g., the nucleus or starch granules).
4. Lower the Cover Slip: Using forceps, gently lower a cover slip at an angle to minimize the risk of trapping air bubbles.
5. Remove Excess Liquid: Use a paper towel to blot away any excess water or stain from the edges of the cover slip.

Focusing the Microscope

1. Focusing correctly is essential for obtaining a clear image.
2. Always start with the lowest magnification (e.g., 40×) and follow these steps:
   1. Position the Slide: Place the prepared slide on the stage and secure it with stage clips. Ensure the area of interest is centered over the light source.
   2. Coarse Focus: Use the coarse focusing knob to bring the specimen into view. Move the stage away from the objective lens to prevent accidental damage.
   3. Fine Focus: Once the image is nearly clear, switch to the fine focusing knob to sharpen the details.

Measuring Sizes Using an Eyepiece Graticule

1. It allows you to measure the size of structures in "eyepiece units" (EPU).
2. However, the actual size represented by each EPU varies with magnification, so calibration is required.

How to Calibrate the Graticule

1. Use a Stage Micrometer: Place a slide with a known scale (e.g., 1 mm divided into 100 divisions) on the stage.
2. Align the Scales: Focus the microscope and align the stage micrometer scale with the eyepiece graticule.
3. Calculate the Value of One EPU:
   1. Count how many EPUs align with a known length on the stage micrometer.
   2. Divide the known length by the number of EPUs.

Calculating Actual Size and Magnification

Key Formulae

1. Magnification:
   $$
   \text{Magnification} = \frac{\text{Size of Image}}{\text{Actual Size of Specimen}}
   $$
2. Actual Size:
   $$
   \text{Actual Size} = \frac{\text{Size of Image}}{\text{Magnification}}
   $$

Using Scale Bars

1. Scale bars provide a visual representation of size.
2. For instance, a 10 µm scale bar on a micrograph magnified 1,000× would appear as a 10 mm bar in the image.

To calculate the actual size represented by the scale bar:
$$
\text{Actual Size} = \frac{\text{Length of Scale Bar in Image}}{\text{Magnification}}
$$

Taking Photographs and Producing Drawings

1. Modern microscopes often allow you to attach a smartphone or digital camera to capture images.
2. This is useful for creating a permanent record of your observations.

Biological Drawings

1. Drawings are simplified representations of what you see under the microscope.
2. Follow these guidelines:
   1. Use clean, continuous lines (avoid shading).
   2. Label structures with straight lines and annotations.
   3. Clearly indicate the magnification.

Nature of Science: Quantitative Observations

1. Using microscopes combines qualitative observation (structure, shape, colour) with quantitative measurement (size, number).
2. Quantitative observation = objective, measurable data (e.g., cell diameter 80 µm).
3. Qualitative observation = descriptive features (e.g., cell is spherical, cytoplasm stained blue).