Making and observing slides Knowledge-Based Study

Study notes

1. Introduction to Slide Preparation

   In biological microscopy, most specimens must be prepared on a slide to allow light to pass through them effectively. Because standard compound light microscopes operate using transmitted light, specimens must be sufficiently thin, ideally a single cell or tissue layer thick.

   Key Tools and Materials

   • Glass Slide: A flat, rectangular piece of clear glass that supports the specimen.
   • Coverslip: A very thin, fragile square of glass or plastic placed over the specimen. It protects the microscope's objective lens from contacting the liquid, flattens the specimen to maintain a uniform focal plane, and slows down evaporation.

   Depending on the nature of the specimen, slides can be prepared as dry mounts (for dry, non-living samples like pollen or fibers) or wet mounts (for living cells, aquatic microorganisms, or fresh tissues).

   Step-by-Step Wet Mount Procedure

   A wet mount is the most common preparation method used in introductory biology to observe living cells and hydrated tissues.

   1. Collect and Position: Place a small, thin sample of the specimen in the center of a clean glass slide.
   2. Add Liquid Medium: Using a dropper or pipette, place one drop of water, physiological saline, or an appropriate liquid medium directly onto the specimen.
   3. Position the Coverslip: Hold the coverslip by its edges to prevent fingerprints. Rest one edge of the coverslip on the slide next to the liquid drop at approximately a 45-degree angle.
   4. Lower the Coverslip: Slowly and gently lower the coverslip over the drop using a mounting needle or forceps. This gradual angled lowering allows air to escape, preventing the formation of trapped air bubbles, which appear as dark-bordered circular artifacts under the microscope.
   5. Remove Excess Liquid: If excess liquid seeps out from under the coverslip, touch the edge with a piece of filter paper or paper towel to blot it away without shifting the coverslip.

   Why Staining Is Necessary

   Most biological cells are largely transparent and watery, offering poor natural contrast under brightfield illumination. Staining introduces chemical dyes that bind selectively to specific cellular components, making distinct structures readily visible.

   Common Stains in Biology

   • Methylene Blue: A basic dye that binds strongly to acidic components, particularly nucleic acids, clearly highlighting cell nuclei in animal cells.
   • Iodine (Lugol's Solution): Commonly used for plant tissues; it stains starch granules deep blue-black and improves the contrast of cell walls and nuclei.

   The Irrigation (Perfusion) Technique

   When applying a stain to an already prepared wet mount without lifting the coverslip:

   1. Place a drop of stain directly against one edge of the coverslip.
   2. Place a small piece of absorbent paper towel against the opposite edge of the coverslip.
   3. The paper towel draws the liquid out by capillary action, pulling the stain underneath the coverslip across the specimen.

   Focusing and Examining the Slide

   To examine a prepared slide safely and systematically, follow the standard focusing sequence:

   1. Mount the Slide: Secure the slide onto the stage using stage clips, ensuring the specimen is centered directly over the stage aperture where light passes through.
   2. Start with the Lowest Power Objective: Always begin observations using the lowest magnification (scanning objective). This provides the widest field of view and largest depth of field, making the specimen easy to locate.
   3. Coarse Focus: Use the coarse adjustment knob to bring the stage and objective close together while looking from the side, then slowly focus away until the specimen enters view.
   4. Fine Tuning and Higher Magnification: Use the fine adjustment knob to sharpen the image. Center the specific area of interest before rotating the revolving nosepiece to a higher magnification.

   Precaution: Never use the coarse adjustment knob when working with high-power objectives, as this risks cracking the coverslip and damaging the objective lens.