The accurate and rapid identification of the bacterium Staphylococcus aureus is a significant concern in clinical medicine due to its capacity to cause a wide range of infections. This organism is a common member of the human microbiota, frequently colonizing the skin and nasal passages, but it can transition into an opportunistic pathogen responsible for conditions from skin abscesses to life-threatening sepsis. Because of its medical relevance, laboratories rely on a systematic process of selective culturing and biochemical testing to confirm its presence quickly. This identification protocol utilizes specialized culture media, known as agar plates, as the primary method for isolating and presumptively distinguishing this bacterium from a mixed sample. The distinct growth patterns and metabolic activities displayed on these media guide microbiologists toward a definitive diagnosis.
The Distinctive Profile of Staphylococcus aureus
The initial step in identification involves observing the organism’s fundamental cellular and colonial properties. Staphylococcus aureus is classified as a Gram-positive coccus, meaning its thick peptidoglycan cell wall retains the crystal violet stain, appearing purple under the microscope. These spherical cells are typically arranged in irregular, grape-like clusters, which helps distinguish them from other medically relevant cocci like Streptococcus, which form chains. Cultured on general laboratory media, S. aureus colonies are often round, raised, opaque, and sometimes exhibit a distinct yellow or golden pigmentation.
A foundational biochemical test is the Catalase test, which differentiates the genus Staphylococcus from Streptococcus. Staphylococcus species possess the enzyme catalase, which breaks down hydrogen peroxide into water and oxygen gas, producing visible bubbles. This positive result confirms the isolate belongs to the Staphylococcaceae family. This initial profile—Gram-positive cocci in clusters that are Catalase-positive—significantly reduces the number of potential pathogens, setting the stage for more specific differentiation on specialized media.
Selective Growth and Differentiation on Mannitol Salt Agar (MSA)
Mannitol Salt Agar (MSA) serves a dual purpose as both a selective and differential medium tailored to isolate and presumptively identify S. aureus. The selective power of MSA stems from its high concentration of sodium chloride, typically around 7.5%. This elevated salt level creates an osmotic pressure that inhibits the growth of most non-staphylococcal bacteria, effectively selecting for salt-tolerant species like Staphylococcus.
The differential function relies on the presence of the carbohydrate mannitol and the pH indicator phenol red. Phenol red is incorporated into the agar to monitor the metabolic activity of the growing bacteria; the medium starts with a neutral pH and a corresponding red color. Staphylococcus aureus possesses the necessary enzymes to ferment mannitol, which produces acidic byproducts. This acid production lowers the pH of the surrounding medium, causing the phenol red indicator to change from red to bright yellow.
Conversely, other Staphylococcus species that can tolerate the high salt concentration, known as Coagulase-Negative Staphylococci (CoNS), generally cannot ferment mannitol. These non-fermenting organisms will grow, but the agar surrounding their colonies will remain red. The visual presence of yellow colonies surrounded by a yellow halo on an otherwise red plate provides a strong preliminary indication of Staphylococcus aureus.
Identifying Hemolytic Activity on Blood Agar
Blood Agar (BA) is a common differential medium composed of a nutrient base enriched with 5% sheep blood. This medium is used to observe the organism’s ability to produce hemolysins, which are toxins that damage red blood cells. Staphylococcus aureus is frequently characterized by beta-hemolysis, a complete lysis and clearing of the red blood cells in the agar directly surrounding the colony.
The zone of beta-hemolysis appears as a transparent, pale yellow area where the hemoglobin has been fully degraded. This complete clearing is caused by the bacterium’s secretion of multiple hemolysins, such as alpha and delta toxins, which act to rupture the red blood cell membranes. Observing this distinct pattern helps to differentiate S. aureus from other organisms that may exhibit alpha-hemolysis (partial, greenish clearing) or gamma-hemolysis (no change). The visual evidence of a creamy, sometimes golden, colony paired with a clear hemolytic zone further strengthens the presumptive identification.
Essential Biochemical Confirmation Tests
While the growth and color changes on agar plates provide strong presumptive evidence, final confirmation of S. aureus identity requires specific biochemical tests that detect unique enzymatic activity. The most definitive test is the Coagulase test, which distinguishes S. aureus from the less pathogenic Coagulase-Negative Staphylococci. The enzyme coagulase, produced by S. aureus, acts like the enzyme thrombin, causing plasma to clot.
S. aureus produces two forms of this enzyme: bound coagulase, also called clumping factor, and free coagulase. The presence of bound coagulase can be rapidly detected using a slide test, where bacterial cells are mixed with rabbit plasma, and a positive result is indicated by the immediate clumping of the cells. The free coagulase is detected in a slower tube test, where the organism is incubated with plasma, and a positive result is the formation of a visible fibrin clot within four hours.
Modern laboratories often utilize rapid latex agglutination tests as a quicker alternative for final confirmation. These commercial kits use latex particles coated with fibrinogen and antibodies that bind to both the clumping factor and Protein A, another unique surface protein on S. aureus. A positive result in this rapid test, indicated by particle agglutination, provides a highly specific and timely confirmation of the organism’s identity.