Bacteria are ubiquitous microorganisms, invisible to the unaided eye. These single-celled life forms are too small for direct observation, yet a microscope allows us to magnify them and explore their diverse forms and behaviors. Learning to properly use a microscope unveils the unseen complexities of bacterial life.
Essential Tools and Materials
Observing bacteria requires specific equipment, primarily a compound light microscope. This microscope typically features objective lenses ranging from 4x to 100x, which, when combined with a 10x eyepiece, provide total magnifications from 40x up to 1000x. Magnifications of 400x to 1000x are generally needed to visualize bacteria, with 1000x requiring immersion oil.
Immersion oil is a specialized liquid applied between the 100x objective lens and the microscope slide. This oil reduces the refraction of light as it passes from the slide into the lens, ensuring more light enters the objective. This process significantly enhances the resolution and clarity of the magnified image. Glass slides and thin glass cover slips are also necessary, providing a flat surface to hold the sample.
Bacteria are largely transparent, making them difficult to see under a microscope without additional preparation. To address this, basic stains are employed to increase contrast and make the cells visible. Common examples include methylene blue and crystal violet. These stains are positively charged and are attracted to the negatively charged components of bacterial cells, such as cell walls and nucleic acids, thereby coloring the cells and allowing their morphology to be observed.
Collecting and Preparing Samples
Preparing a bacterial sample for microscopic viewing involves either creating a wet mount or a stained smear.
Wet Mount Preparation
For a wet mount, safe and accessible sources of bacteria include a small drop of plain yogurt, a sample of pond water, or even a cheek swab diluted in a tiny amount of water. These sources contain live bacteria that can be observed in their natural state. To make a wet mount, place a small drop of the sample onto a clean glass slide. Then, gently lower a cover slip over the liquid at a 45-degree angle to minimize air bubbles, allowing the liquid to spread evenly.
Stained Smear Preparation
For a stained smear, begin by placing a small drop of water on a clean slide if working with a solid bacterial source like yogurt. Next, use a sterile toothpick or inoculating loop to transfer a tiny amount of the sample into the water drop, spreading it thinly and evenly across a small area of the slide. If using a liquid sample, such as pond water, a small drop can be spread directly onto the slide without additional water. Allow the smear to air dry completely.
Once the smear is dry, heat-fix the sample by passing the underside of the slide quickly through a flame two or three times. This process adheres the bacteria to the slide and prepares them for staining. After heat-fixing, apply a few drops of a basic stain, such as methylene blue or crystal violet, to cover the dried smear. Let the stain sit for approximately 30 seconds to one minute, allowing the bacterial cells to absorb the color. Finally, gently rinse the stained slide with distilled water and allow it to air dry before observation.
Microscope Operation and Observation
Effective microscope use begins with proper focusing, starting with a low-power objective lens, typically 10x or 40x. Position the prepared slide on the stage and secure it with the clips. Using the coarse adjustment knob, bring the specimen into approximate focus, then refine the clarity with the fine adjustment knob. Adjust the light intensity using the rheostat and the iris diaphragm to achieve optimal illumination and contrast.
Once a general area of interest is located under lower magnification, rotate the revolving nosepiece to a higher power objective, such as 400x. Recalibrate the focus using only the fine adjustment knob, as the coarse knob can cause the objective to crash into the slide at higher magnifications. When ready for the highest magnification (1000x), apply a small drop of immersion oil directly onto the cover slip over the viewing area. Carefully switch to the 100x oil immersion objective, ensuring it makes contact with the oil. Use the fine adjustment knob to bring the bacteria into sharp focus.
Under high magnification, bacteria appear as distinct shapes. Their size typically ranges from 0.2 to 2.0 micrometers in diameter for spherical forms, and 2 to 8 micrometers in length for rod-shaped bacteria. Spirals can vary significantly, from 1 micrometer to over 100 micrometers in length. The three basic bacterial shapes are cocci (spherical or round), bacilli (rod-shaped), and spirilla (spiral or helical). Observing these characteristic shapes and sizes helps in identifying different bacterial morphologies.
Safety Precautions and Disposal
Handling biological samples, even common ones, necessitates adherence to basic safety practices. Always wash your hands thoroughly with soap and water before and after handling any samples, slides, or microscope equipment. Avoid touching your eyes, nose, or mouth while working to prevent accidental transfer of microorganisms.
After observation, properly clean all equipment. Use lens paper and a specialized lens cleaning solution to gently wipe objective lenses, eyepieces, and the condenser, removing any oil or smudges. Clean glass slides and cover slips can be washed with soap and water, rinsed, and dried for reuse, or disposed of responsibly.
For non-pathogenic bacterial samples, such as those from yogurt or pond water, prepared slides can typically be discarded in regular household trash. However, it is always prudent to disinfect slides and any contaminated surfaces with a common household disinfectant before disposal or storage.