Escherichia coli is a common bacterium found in the intestines of warm-blooded animals. While most strains are harmless, certain strains can cause serious illness. Observing the physical characteristics of E. coli requires specialized tools, as the human eye cannot see them. Microscopy is the foundational technique used to classify and study this organism, providing visual confirmation of its size, shape, and cellular components.
What Does E. coli Look Like Under the Microscope?
The defining characteristic of E. coli is its shape, classified as a bacillus, or a rod-shaped bacterium. These cells typically measure between 2.0 and 6.0 micrometers in length and 1.1 to 1.5 micrometers in width. They often appear as individual units, though they can sometimes be seen in pairs or short, loose chains during rapid growth phases.
The bacterium’s movement, or motility, is another notable feature visible under the microscope. Motility is powered by hair-like appendages called flagella, which are arranged all over the cell surface in a peritrichous configuration. This arrangement allows the bacterium to swim in a chaotic, tumbling motion, distinguishing it from non-motile bacteria.
The structure of the E. coli cell wall dictates its appearance after staining. E. coli is classified as Gram-negative because its cell wall possesses only a thin layer of peptidoglycan covered by an outer membrane. This structure determines the final color of the cell when subjected to common identification procedures in the microbiology laboratory.
Viewing E. coli: Staining and Preparation
Because bacterial cells are nearly transparent, they must be treated with chemical dyes to become visible under a standard light microscope. The Gram stain is the primary technique used for E. coli, exploiting differences in cell wall composition to impart a distinct color. The procedure begins with applying crystal violet, a purple primary stain that colors all cells on the slide.
Next, Gram’s iodine (a mordant) is added to form a large crystal violet-iodine complex within the cell. The differential step involves washing the slide with a decolorizer, typically an alcohol solution. Due to the thin peptidoglycan layer of Gram-negative E. coli, the complex is easily washed out, leaving the cells colorless.
Finally, a counterstain, Safranin (a pink or red dye), is applied. The colorless E. coli cells absorb this dye, causing them to appear pink-red under the microscope, confirming their Gram-negative status.
Other preparation techniques include the simple wet mount. A drop of liquid culture is placed under a coverslip to allow for the observation of living, unstained cells. This method is used to view their characteristic peritrichous motility.
Microscopy Tools Used to Study E. coli
The most frequently used instrument for observing E. coli is the compound light microscope, specifically the brightfield configuration. This tool views the results of the Gram stain, allowing identification of the rod shape, arrangement, and pink-red color. High magnification, usually 1000x using an oil immersion lens, is required. While brightfield is excellent for stained and fixed cells, it provides limited detail for live organisms.
For studying living E. coli in its natural state, phase contrast microscopy is employed. This technique enhances the contrast of structures without the need for chemical staining, which would kill the cell. This allows researchers to clearly observe the cell boundary and its movement in a wet mount. Phase contrast helps differentiate true, flagella-driven motility from random Brownian motion.
To visualize ultra-fine details, such as flagellar attachment points or internal organelles, electron microscopy is required due to its superior magnification. Transmission Electron Microscopy (TEM) provides high-resolution images of the cell’s internal architecture, revealing structures like the nucleoid and internal membranes.
Scanning Electron Microscopy (SEM), conversely, images the cell’s surface topography in three dimensions. SEM is suitable for studying external features like pili or fimbriae and the overall surface texture of the bacillus.