Studying a live Paramecium under a microscope requires strategic preparation of the slide to counteract the organism’s rapid movement and transparency. Paramecium is a ciliate protist, a single-celled organism propelled by thousands of tiny cilia, allowing it to move quickly across the field of view. The goal of slide preparation is to gently immobilize the organism and enhance its internal features for detailed, live observation. This involves creating a stable environment, slowing the organism’s speed, and boosting the visibility of its structures.
Preparing the Basic Wet Mount
The initial step in observing Paramecium is creating a standard wet mount slide. Place a small drop of the culture medium containing the organisms onto the center of a clean glass slide. The drop must be small enough to be contained entirely by the coverslip, limiting the organisms’ free swimming space.
A coverslip is carefully lowered over the drop at a 45-degree angle to avoid trapping air bubbles, which can obscure the view. The liquid volume must be balanced: enough to fill the space between the slide and coverslip, but not so much that the Paramecium moves too quickly. Too little liquid can crush the organism, making precise volume critical for successful observation.
Methods for Slowing Movement
Since the cilia on a Paramecium propel it at a high speed, often causing it to zip out of the microscope’s field of view in seconds, immobilization is necessary. This is achieved by increasing the viscosity of the surrounding liquid, which slows the ciliary action without immediately harming the cell. The most common chemical agent used for this is methylcellulose, often sold commercially under names like Protoslo.
Methylcellulose is a non-toxic polymer that forms a thick, syrupy solution when mixed with water. When the culture is mixed with a 3% methylcellulose solution on the slide, the increased viscosity acts like a gentle, transparent gel, restricting movement. This allows the viewer to sustain focus and observe internal structures, such as the contractile vacuoles and gullet, in slow motion.
Physical Obstruction
Physical methods offer an alternative to chemical thickening agents. Introducing fine cotton or lens paper fibers into the culture drop before adding the coverslip can physically obstruct the Paramecium. The organisms become temporarily trapped or slowed by the fibers, allowing for a brief period of observation. While less consistent than viscous solutions, this method is effective for quick, non-chemical immobilization.
Improving Contrast and Detail
Once the Paramecium is immobilized, its inherent transparency makes internal organelles difficult to distinguish under brightfield microscopy. The simplest method to enhance visibility is adjusting the light passing through the specimen by manipulating the microscope’s condenser and diaphragm.
Adjusting Illumination
Closing the iris diaphragm, located beneath the stage, reduces the light aperture, increasing contrast by casting subtle shadows on clear structures. This technique, known as oblique illumination, accentuates the outlines of the organism and larger parts, such as the macronucleus and food vacuoles. Avoid closing the diaphragm excessively, as this reduces overall resolution and sharpness.
Using Vital Stains
For detailed study of specific organelles, temporary or vital stains can be introduced to the slide. The technique involves adding a small drop of stain, such as Neutral Red or Methylene Blue, to one edge of the coverslip and drawing it under using a piece of paper towel placed on the opposite edge to absorb the liquid. Neutral Red is a pH indicator that is readily taken up by the food vacuoles, causing them to turn a reddish color and making the feeding process visible. Methylene Blue is another common vital stain that helps highlight the nucleus and other cytoplasmic granules, providing a clearer view of the organism’s internal anatomy.