Escherichia coli is one of the most studied bacteria globally, serving as a fundamental model organism in biology and medical research. This rod-shaped microbe, often simply called E. coli, is typically cultured in the laboratory using agar plates, which are solid growth mediums contained within Petri dishes. Standardized techniques are used to manipulate and grow this bacterium on agar, ensuring both purity and accurate observation. These methods allow scientists to isolate the organism, quantify its presence, and analyze its metabolic capabilities.
The Foundation: Agar Media and Aseptic Handling
Culturing E. coli begins with preparing the agar medium, which provides the necessary nutrition and a solid surface for growth. Agar itself is a complex polysaccharide derived from seaweed, acting solely as a solidifying agent that remains firm at incubation temperatures. The primary growth media, such as Luria-Bertani (LB) agar, contain rich sources of protein and carbohydrates, typically in the form of tryptone and yeast extract, along with sodium chloride for osmotic balance.
Before any bacterial sample is introduced, the medium must be completely free of all other living organisms. This step is achieved through sterilization, most commonly by autoclaving, which uses high-pressure steam at temperatures around \(121^\circ\text{C}\) for a set duration. After the agar cools but before it fully solidifies, it is poured into sterile Petri dishes, creating a clean slate for the experiment.
The manipulation of the sample and the prepared media demands strict adherence to aseptic technique. This technique prevents contamination, ensuring that only the target organism, E. coli, is introduced to the plate. It involves sterilizing tools like inoculating loops by heating them until red-hot and working near a flame or in a sterile hood. Maintaining sterility is necessary because contamination from environmental microbes could invalidate the results.
Applying the Sample: Inoculation Techniques
Once the sterile medium is ready, the bacterial sample must be physically transferred and distributed onto the agar surface, a process called inoculation. The most frequent technique for achieving a pure culture from a mixed sample is the quadrant streaking method. This procedure aims to dilute the bacterial concentration across the plate until individual cells are separated enough to grow into distinct colonies.
The quadrant streak involves dividing the plate surface conceptually into four sections. A sterile inoculating loop collects the culture and spreads it over the first quadrant. The loop is sterilized, and the second quadrant is inoculated by dragging the loop through the edge of the first. This process of sterilizing the loop and overlapping the previous section is repeated for the third and fourth quadrants, progressively reducing the number of bacteria transferred.
While streaking is used for isolation, other techniques are employed when the goal is quantification. Spread plating involves applying a small volume of a liquid sample onto the agar and distributing it evenly across the entire surface using a sterile spreader. Alternatively, pour plating mixes the sample with molten agar before it solidifies, allowing colonies to grow both on the surface and embedded within the medium. These methods ensure a uniform distribution of cells, which is necessary for accurately counting the number of viable bacteria in a suspension.
Interpreting Growth: Colony Morphology and Quantification
After incubation, the E. coli cells multiply rapidly, and the results of the inoculation techniques become visible as colonies. A colony is a macroscopically visible mass of cells originating from a single bacterial cell or cluster, often referred to as a Colony Forming Unit (CFU). On a general-purpose medium like nutrient agar, E. coli typically forms large, circular colonies that are smooth, moist, and possess a grayish-white to cream color.
Scientists observe several distinct characteristics to describe a colony’s morphology, which can be an initial step in identification. These features include the overall shape (form), the appearance of the outer edge (margin), and the cross-sectional view (elevation). E. coli colonies often exhibit an entire margin and a slightly raised or low convex elevation.
Beyond visual description, quantification determines the number of viable organisms in the original sample. This process relies on counting the CFUs on a plate that contains between 30 and 300 colonies, a range that ensures statistical accuracy. To achieve this countable range, the original sample is subjected to serial dilution, where the culture is repeatedly diluted by factors of ten. The calculated number of colonies is then multiplied by the specific dilution factor and the volume plated to estimate the concentration, expressed as CFU per milliliter of the initial sample.
Beyond General Growth: Differential and Selective Media
While general media support the growth of most bacteria, specialized agars are used to make more specific observations about E. coli’s metabolic activities. These media fall into two main categories: selective and differential. Selective media contain ingredients like bile salts or specific dyes that inhibit the growth of unwanted bacteria, allowing only the target group, such as Gram-negative organisms like E. coli, to flourish.
Differential media, in contrast, contain chemical indicators that reveal metabolic differences between species, typically through a color change. MacConkey Agar is a classic example that is both selective and differential, containing bile salts and crystal violet to inhibit Gram-positive bacteria, along with the sugar lactose and a pH indicator. E. coli ferments the lactose, producing acid that lowers the medium’s pH, which causes the neutral red indicator to turn the colonies bright pink.
Another common specialized medium is Eosin Methylene Blue (EMB) Agar, which selects for Gram-negative bacteria and differentiates based on lactose fermentation. E. coli is a vigorous fermenter of the sugars in EMB, leading to a significant drop in pH. This acid production causes the Eosin and Methylene Blue dyes to precipitate. The resulting colonies have a characteristic dark center and a metallic green sheen, which helps confirm the presence of E. coli.