Pseudomonas Aeruginosa on Agar: Media, Pigments, and Growth

Pseudomonas aeruginosa is a versatile, opportunistic pathogen found in soil, water, and hospital environments. It poses a significant threat to immunocompromised individuals, contributing to infections such as pneumonia, urinary tract infections, and sepsis. Its antibiotic resistance makes identification in clinical and laboratory settings crucial for effective treatment.

When cultured on agar, P. aeruginosa exhibits distinctive growth characteristics that aid in its recognition. Understanding its preferred media, colony morphology, pigment production, and odor helps differentiate it from other bacteria.

Growth Requirements

Pseudomonas aeruginosa thrives in diverse environments due to its metabolic adaptability. As a facultative aerobe, it primarily relies on oxygen but can survive in low-oxygen conditions by using nitrate as an alternative electron acceptor. This flexibility allows it to colonize hospital surfaces, medical equipment, and natural water sources. It grows at temperatures between 4°C and 42°C, making it a persistent contaminant in clinical and industrial settings.

Its nutritional requirements are minimal, as it can metabolize various organic compounds. Unlike many pathogens that require enriched media, P. aeruginosa proliferates in minimal media containing basic carbon and nitrogen sources. This metabolic versatility, supported by its extensive genome, enables it to utilize amino acids, fatty acids, and carbohydrates. Its ability to degrade complex hydrocarbons also makes it valuable in bioremediation.

Moist environments support its growth, explaining its frequent isolation from medical equipment, sinks, and respiratory therapy devices. Biofilm formation enhances its persistence, providing protection against desiccation and antimicrobial agents. Biofilm-associated cells exhibit up to a 1,000-fold increase in antibiotic resistance compared to planktonic cells, complicating eradication in healthcare settings.

Colony Appearance

On agar, Pseudomonas aeruginosa forms colonies with distinctive traits that facilitate identification. These colonies are typically large, 2-4 mm in diameter after 24 hours at 37°C, with irregular or slightly undulating edges due to the bacterium’s motility and surface-spreading ability.

Colony texture varies with environmental conditions and media. Most appear smooth or mucoid, though some strains exhibit a wrinkled or rough morphology. The mucoid phenotype, common in cystic fibrosis patients, results from overproduction of alginate, contributing to biofilm formation and chronic infections. Non-mucoid strains tend to be dry or slightly granular, often linked to acute infections.

Colony coloration is another defining feature. The greenish hue results from pyocyanin, a blue-redox-active phenazine, and pyoverdine, a yellow-green fluorescent pigment visible under ultraviolet light. Some strains also produce pyorubin (red) or pyomelanin (brown-black), leading to variations in pigmentation. The presence and intensity of these pigments depend on iron availability, oxygen levels, and metabolic state.

Moisture and nutrient composition further influence morphology. On high-salt or nutrient-limited media, colonies may be smaller and more compact, with reduced spreading. In humid environments or on nutrient-rich agar, colonies develop a glossy, almost slimy texture due to increased exopolysaccharide production.

Culture Media Options

The selection of an appropriate medium is essential for isolating and identifying Pseudomonas aeruginosa in clinical and environmental samples. While the bacterium grows on various general and selective media, certain formulations enhance its characteristic traits, such as pigment production and colony morphology.

Cetrimide Agar

Cetrimide agar is a selective medium designed for isolating P. aeruginosa. Cetrimide, a detergent, inhibits most other bacteria by disrupting their cell membranes, while P. aeruginosa’s intrinsic resistance allows it to thrive.

This medium enhances pigment production, promoting pyocyanin and pyoverdine synthesis, resulting in colonies with a striking blue-green fluorescence under ultraviolet light. Colonies are typically smooth, slightly raised, and emit a distinctive grape-like or tortilla-like odor. Due to its high specificity, cetrimide agar is widely used in hospital laboratories for detecting P. aeruginosa in wound infections, respiratory specimens, and contaminated medical equipment.

MacConkey Agar

MacConkey agar is a differential and selective medium primarily used for isolating Gram-negative bacteria. While not specifically designed for P. aeruginosa, it supports its growth and helps differentiate it from lactose-fermenting bacteria.

On MacConkey agar, P. aeruginosa forms large, non-lactose-fermenting colonies that appear colorless or slightly translucent, distinguishing them from lactose-fermenters like Escherichia coli, which produce pink colonies. The colonies may also exhibit a slightly irregular shape with a metallic sheen due to pyocyanin production. While not the most selective medium, MacConkey agar remains useful in mixed cultures where differentiation from enteric bacteria is necessary.

Blood Agar

Blood agar is a nutrient-rich medium supporting a wide range of bacteria, including P. aeruginosa. While not selective, it provides valuable information about hemolytic activity.

P. aeruginosa typically exhibits beta-hemolysis, characterized by a clear zone around colonies due to red blood cell lysis. This hemolytic activity is linked to virulence factors such as hemolysins. Colonies on blood agar are usually large, irregularly shaped, and may display a greenish discoloration due to pigment diffusion. The distinct odor associated with P. aeruginosa is also noticeable, aiding identification. Though less selective than cetrimide agar, blood agar remains useful for assessing hemolysis and colony morphology.

Pigment And Odor

Pseudomonas aeruginosa produces various pigments that contribute to its vivid coloration on agar. Pyocyanin, a blue-green phenazine compound, generates reactive oxygen species that can damage competing microorganisms. Its synthesis is regulated by quorum sensing, a bacterial communication system responding to population density and environmental conditions. When iron is scarce, P. aeruginosa increases pyoverdine production, a yellow-green fluorescent siderophore that scavenges iron, further intensifying colony coloration.

Certain strains produce additional pigments such as pyorubin, which gives a reddish-brown tint, and pyomelanin, responsible for a brown-black pigmentation. The presence and intensity of these pigments vary based on nutrient composition, oxygen levels, and regulatory gene expression. Clinical isolates often exhibit increased pigment production compared to environmental strains, suggesting a potential link between pigmentation and virulence. Pigment diffusion into the surrounding agar can create a distinctive halo effect, especially in media that enhance secondary metabolite production.

Beyond its visual markers, P. aeruginosa is known for its characteristic odor, often resembling grapes, tortilla chips, or corn tortillas. This scent arises from volatile compounds such as 2-aminoacetophenone, a metabolic byproduct associated with quorum sensing and bacterial persistence. The intensity of this odor varies with growth conditions, with nutrient-rich environments amplifying its presence. Clinically, this distinctive smell serves as an early diagnostic clue, particularly in wound infections and respiratory specimens.