Bacteria are microscopic organisms, typically ranging from 0.2 to 2.0 micrometers in diameter, and are completely invisible to the unaided human eye. Their extremely small size requires specialized equipment and preparation methods for observation. Bacterial cells are also largely transparent, presenting almost no color or contrast when suspended in a liquid medium. This lack of inherent visual information means they cannot be distinguished from the background without magnification, fixation, and artificial coloring.
The Essential Tool: Understanding Magnification
The primary instrument used to visualize bacteria is the compound light microscope, which utilizes a system of lenses to achieve the necessary magnification. This instrument works by passing light through the specimen and then through two sets of lenses: the objective lenses and the ocular lenses. Total magnification is calculated by multiplying the magnification power of the objective lens by the magnification power of the ocular lens, which is typically \(10text{x}\).
For adequate viewing of bacteria, a total magnification of approximately \(1000text{x}\) is required, usually achieved using a \(100text{x}\) objective lens combined with a \(10text{x}\) eyepiece. At this level, oil immersion is employed, involving placing a drop of special oil between the slide and the \(100text{x}\) objective lens. This oil minimizes the refraction of light rays as they pass from the glass slide into the lens, maximizing the light captured and enhancing image clarity.
Magnification must be paired with resolution, the ability of the lens system to distinguish between two adjacent objects as separate. The practical limit of resolution for a standard light microscope is about \(0.2\) micrometers, which is just enough to visualize the morphology and arrangement of most bacterial cells. Increasing magnification beyond \(1000text{x}\) without improving resolution will not provide a clearer image, as quality is limited by the physical properties of light.
Preparing the Specimen Slide
Before observation, bacterial cells must be properly secured to a glass slide, beginning with creating a “smear.” This involves transferring a small amount of the bacterial culture onto a clean slide and mixing it with a tiny drop of water or saline to create a thin, even film. A thin film is important because a thick layer of cells prevents light from passing through, making the final image too dense to analyze.
The next step is to allow the slide to thoroughly air-dry at room temperature until all the liquid has evaporated from the smear. Rushing this step is detrimental, as residual water will boil in the subsequent stage, causing the bacterial cells to rupture and distort their natural shape. Once completely dry, the smear is “fixed” onto the glass, most commonly by quickly passing the slide through the tip of a flame several times.
Heat fixation serves two purposes: it coagulates the proteins within the bacterial cell, which kills the organism, and it adheres the cells firmly to the glass surface. Proper heat fixing ensures the thin film of bacteria will not be washed away during the next step, which involves the application of liquid dyes and rinsing. The slide must be heated just enough for the cells to adhere without overheating, which would damage or significantly distort cellular structures.
Adding Contrast: The Role of Stains
Even when successfully fixed to the slide, bacteria remain virtually transparent and blend into the bright background when viewed under the light microscope. The purpose of staining, which uses biological dyes, is to chemically add color and contrast to the specimen, making the cells visually stand out. These stains are typically basic dyes, such as methylene blue or crystal violet, which have a positive electrical charge that attracts them to the negatively charged components of the bacterial cell, like nucleic acids and cell walls.
The simplest method is a simple stain, which uses only a single dye to color all the cells uniformly, making it possible to determine the basic size, shape, and arrangement of the bacteria. Simple staining, however, does not provide information beyond the organism’s general morphology. For a more detailed analysis, a differential stain is used, employing two or more dyes to differentiate between types of bacteria or specific cellular structures.
The Gram stain is the most common example of a differential technique, separating bacteria into two large groups based on their cell wall composition. This process involves a primary stain (crystal violet), a mordant (iodine), a decolorizer (alcohol/acetone), and a counterstain (safranin). Bacteria with a thick peptidoglycan cell wall retain the primary stain and appear purple (Gram-positive), while those with a thinner wall lose the primary stain during decolorization and pick up the counterstain, appearing red or pink (Gram-negative).