Pseudomonas aeruginosa is an opportunistic pathogen frequently encountered in clinical environments, such as hospitals and burn units. Its identification is a routine process in microbiology laboratories for managing patient care and tracking potential outbreaks. Isolation begins with culturing patient samples on specialized growth media. Blood Agar is a common enriched medium used for initial isolation and distinguishing between different types of bacteria, providing the first visual clues for presumptive identification.
Understanding Blood Agar as a Medium
Blood Agar is a standard, non-selective medium that serves both an enriched and a differential function. The medium is typically formulated using a nutritious base, such as Tryptic Soy Agar, supplemented with five to ten percent sterile defibrinated animal blood, most commonly sheep blood. This addition supplies essential nutrients and growth factors. The differential function centers on observing the hemolytic reaction—the ability of a bacterium to lyse red blood cells using toxins called hemolysins. Plates are typically incubated aerobically at 37°C for 24 to 48 hours.
Visual Characteristics of P. aeruginosa Colonies
Presumptive identification relies heavily on observing a distinct set of macroscopic features. Colonies are generally large, possessing a flat profile and often exhibiting irregular or spreading edges. Some strains, particularly those isolated from chronic infections, may produce a rough, capsulated form, resulting in a highly mucoid or slimy appearance due to alginate production.
A characteristic metallic or iridescent sheen is visible on the colony surface under reflected light. This optical effect is often described as a “beaten-copper” appearance, resulting from light refraction off the smooth, flat surfaces. The colonies are often a dull gray-white with a yellowish tint, but the surrounding medium frequently displays more vibrant colors due to diffusible pigments.
P. aeruginosa produces water-soluble pigments that diffuse out and color the surrounding agar. Pyocyanin (blue) and Pyoverdin (fluorescent yellow-green) are the most common, yielding the signature blue-green color of the culture. Some strains may also produce Pyorubin (red) or Pyomelanin (brown-black).
The interaction with the red blood cells provides a key visual identifier, as most clinical isolates display a wide zone of beta-hemolysis. This appears as a complete clearing of the red color in the agar directly surrounding the colony. The zone is transparent because the hemolysins produced by the bacteria have fully lysed the red blood cells.
Confirming Identification Beyond Sight
While the visual characteristics on Blood Agar provide strong presumptive evidence, biochemical tests are necessary for confirmation. One distinctive clue is sensory, as P. aeruginosa cultures often produce a strong, characteristic scent. This odor is commonly described as fruity, sweet, or grape-like, caused by the volatile organic compound aminoacetophenone.
A mandatory step is the Oxidase test, for which P. aeruginosa yields a positive result. This test detects the presence of the enzyme cytochrome c oxidase. A positive reaction is observed as a rapid color change, typically to a deep purple, upon application of the oxidase reagent.
Further microscopic confirmation is achieved through a Gram stain, which reveals the organism as a Gram-negative rod. The cells are slender, appearing singly or in pairs. The bacterium is also actively motile. The combination of the distinctive odor, the positive oxidase result, and the Gram-negative rod morphology provides a high level of certainty in identification.