Direct Detection Methods

Pus or exudates should be smeared, stained with Gram or Giemsa stain, and examined microscopically (Figure 39-1). S. moniliformis is a pleomorphic, gram-negative rod. Cells may appear straight of variable size or as long tangled chains and filaments with bulbar swellings. The cells may also appear spiral shaped and resemble a string of pearls. Direct detection of the 16sRNA gene sequence for S. moniliformis using polymerase chain reaction (PCF) analysis has been described.

Cultivation

As previously mentioned, S. moniliformis requires the presence of blood, ascitic fluid, or serum for growth. Growth occurs on blood agar, incubated in a very moist environment with 5% to 10% carbon dioxide (CO₂), usually after 48 hours of incubation at 37°C. Colonies are nonhemolytic. The addition of 10% to 30% ascitic fluid (available commercially from some media suppliers) or 20% horse serum should facilitate recovery of the organism. In broth cultures, the organism grows as “fluff balls” or “bread crumbs” near the bottom of the tube of broth or on the surface of the sedimented red blood cell layer in blood culture media. Colonies grown on brain-heart infusion agar supplemented with 20% horse serum are small, smooth, glistening, colorless or grayish and have irregular edges.

Colonies are embedded in the agar and may also have a “fried egg” appearance, with a dark center and a flattened, lacy edge. These colonies are also referred to as L-phase colonies because they have undergone spontaneous transformation to the L form. Staining of L-form colonies yields coccobacillary or bipolar-staining coccoid forms (usually a special stain, such as the Dienes stain performed by pathologists), is required. Acridine orange stain also reveals the bacteria when Gram stain fails because of lack of cell wall constituents.

As previously stated, Gram-stained organisms from standard colonies show extreme pleomorphism, with long, looped, filamentous forms, chains, and swollen cells. The club-shaped cells can be 2 to 5 times the diameter of the filament. Carbolfuchsin counterstain or Giemsa stain may be necessary for visualization (see Figure 39-1).