Bacteria are microscopic organisms, which makes them invisible to the unaided human eye. The smallest object a person can clearly see without magnification is roughly 200 micrometers (µm) in size. Since most bacterial cells, such as Escherichia coli, measure only about 1 to 2 µm in length, specialized optical systems are needed. Observing these cells requires high-powered light microscopy to resolve objects far smaller than the eye’s limit and see their shape and arrangement.
Magnification Levels Required
To achieve a clear view of individual bacterial cells, a total magnification of 1000x is required when using a compound light microscope. This magnification is achieved by combining a 100x objective lens with a standard 10x eyepiece lens. At this high power, the challenge is not only magnification but also resolution.
Achieving sufficient resolution at 1000x requires the use of the oil immersion technique. This method involves placing a drop of immersion oil onto the slide, creating a bridge between the slide and the tip of the 100x objective lens. Because the oil has a refractive index similar to glass, it prevents light rays from bending and scattering as they pass into the lens. This reduction in light refraction increases the numerical aperture of the objective, maximizing resolution and allowing for a sharp image of the cells.
Preparing Slides and Staining
Most bacterial cells are naturally transparent and offer little contrast against the bright background of a standard light microscope slide. To make the cells visible, a preparation step is necessary, involving either a temporary wet mount or a fixed smear. Wet mounts are prepared by mixing a sample with water or broth on a slide and covering it with a cover slip. This technique is used to observe living cells in their natural state, primarily to determine if they exhibit motility.
For detailed analysis of shape and cell wall structure, a fixed smear is prepared by gently heating the sample on the slide to adhere the cells and prevent them from being washed away during staining. Simple staining involves applying a single positively charged dye, such as crystal violet or methylene blue, which binds to the negatively charged cell components. This stains all cells the same color, allowing for a quick determination of their basic shape and arrangement.
Differential Staining
A more advanced technique is differential staining, which uses multiple reagents to distinguish between different types of bacteria based on structural components. The Gram stain is the most common example, using crystal violet as a primary stain, followed by an iodine mordant, an alcohol decolorizer, and a safranin counterstain. This multi-step process differentiates bacteria based on their cell wall composition. Gram-positive cells retain the crystal violet and appear purple, while Gram-negative cells lose the primary stain and are counterstained pink or red by the safranin.
Viewing Bacterial Shape and Arrangement
Once stained and viewed at 1000x magnification, bacteria are classified into three fundamental shapes: spherical cells called cocci, rod-shaped cells referred to as bacilli, and spiral or corkscrew-shaped cells known as spirilla.
The cells’ arrangement relative to one another is also highly informative. Cocci often form characteristic groupings, such as chains (streptococci) or grape-like clusters (staphylococci), depending on the plane of cell division. Bacilli typically appear as single rods or in short chains (streptobacilli). Viewing these arrangements helps microbiologists narrow down the possible species for further testing.
Techniques for Detailed Internal Views
While the light microscope is excellent for visualizing the shape and arrangement of a bacterial cell, its resolution limit of approximately 0.2 µm prevents a clear view of smaller structures. This limitation means that internal components, such as ribosomes, DNA material, or internal membranes, cannot be clearly resolved. To see these subcellular components, the limits of visible light must be surpassed.
Electron microscopy is required to achieve the extreme magnification necessary for viewing internal structures. The Transmission Electron Microscope (TEM) utilizes a beam of electrons instead of light, allowing for magnifications exceeding 100,000x. Samples must be ultra-thin and prepared in a vacuum, but the TEM provides detailed, two-dimensional cross-sections of the cell’s interior. The Scanning Electron Microscope (SEM) also uses an electron beam but focuses on creating a three-dimensional surface image of the bacteria, which is useful for observing external features like flagella or pili.