PAG 01 - Microscopy

Observing Structures Using a Light Microscope

1. Calibrating the Graticule

  • Eyepiece Graticule: A ruler for measuring structures under a microscope.

  • Stage Graticule (Stage Micrometer): A slide with an accurate measuring scale used for calibration.

  • Calibration Process:

    1. Set microscope to the required magnification.

    2. Place the stage graticule on the stage.

    3. Align the two scales (eyepiece and stage graticules).

    4. Count the number of eyepiece divisions equivalent to each stage micrometer division.

    5. Calculate the length of one eyepiece division based on known values of stage micrometer divisions.

  • Important Notes:

    • If magnification changes, recalibrate the eyepiece graticule.

    • Repeat calibration for each objective lens to have a complete set of calibration factors.

2. Preparing Slides

A. Staining

  • Purpose: Provides contrast for distinguishing structures in the sample.

  • Types of Stains:

    • Acetic Orcein: Binds to DNA; stains chromosomes dark red.

    • Eosin: Stains cytoplasm dark red or pink.

    • Iodine: Stains starch blue-black (appears violet under the microscope); in potassium iodide solution, stains cellulose yellow.

    • Haematoxylin: Stains RNA/DNA purple/blue.

    • Methylene Blue: An all-purpose stain for DNA, staining it blue.

B. Mounting Techniques

  1. Wet Mount: Suitable for live specimens (e.g., aquatic animals).

    • a) Place a drop of water on the slide with a pipette.

    • b) Add the specimen using tweezers.

    • c) Carefully place a cover slip at an angle to avoid bubbles.

    • d) Add a non-toxic stain using capillary action for even distribution.

  2. Dry Mount: Used for non-liquid specimens (e.g., hairs, plant parts).

    • a) Thinly slice the specimen for light passage.

    • b) Position the specimen in the center of the slide using tweezers.

    • c) Place a cover slip on top without trapping air bubbles.

3. Using a Light Microscope

  1. Clip the slide onto the stage.

  2. Select the lowest-powered objective lens.

  3. Use the coarse adjustment knob to bring the lens above the slide.

  4. Look through the eyepiece and use the fine adjustment to focus the image clearly.

    • Note: Always move the lens away from the slide to avoid breakage.

  5. For higher magnification, switch to a higher-powered objective lens and refocus.

4. Measuring Size with a Microscope

  • Use the calibrated eyepiece graticule to measure structures.

  • To find actual length: multiply the number of eyepiece divisions by the length of one division (calculated during calibration).

  • For irregular objects, measure the greatest distance across the object.

5. Drawings

  • Guidelines for Drawings:

    • No shading; label shaded areas instead.

    • Drawing should occupy at least half of the page.

    • Label lines must be horizontal, drawn with a ruler, and accurately touch the object without overlapping.

    • Lines should connect completely, appearing neat rather than "hairy."

    • Include a scale for magnification.

    • Use pencil to create a scale that resembles the actual image.