Detection and Quantification of Capsular Exopolysaccharides from Streptococcus thermophilus Using Lectin Probes

Streptococcus thermophilus MR-1C reacting with immobilized lectins on agarose beads. The bacterial cells were incubated with (A) peanut agglutinin (PNA)-immobilized agarose, (B) wheat germ agglutinin-immobilized agarose, (C) PNA-immobilized agarose in the presence of 100mM galactose, and (D) PNA-immobilized agarose after treatment at 100°C in the presence of 10% TCA (wt/vol).

Transmission electron microscopy of Strep. thermophilus MR-1C before (A) and after (B) hot TCA treatment; the arrow indicates the location of the capsular structure. The capsule was stained with ruthenium red as described by Dabour et al. (2005).

Epifluorescence microscopy of Strep. thermophilus MR-1C using fluorescein isothiocyanate-labeled peanut agglutinin (PNA) as a probe. Strain MR-1C was suspended in PBS and heated at 100°C for 5min (A); strain MR-1C was suspended in 10% TCA (wt/vol) and heated at 100°C for 1min (B) or for 5min (C); and strain MR-1C was suspended in 10% TCA (wt/vol) and kept at 20°C (D) before the lectin assay.

Epifluorescence microscopy of Strep. thermophilus MR-1C and RD534 using fluorescein isothiocyanate-labeled wheat germ agglutinin. Strain MR-1C was suspended (A) in 10% TCA (wt/vol) at 20°C or (B) in 10% TCA (wt/vol) and heated for 5min before the lectin assay. Strain Strep. thermophilus RD534 was suspended in 10% TCA (wt/vol) at 20°C (C) or heated for 5min (D) before the lectin assay.

The agglutination test for Streptococcus thermophilus MR-1C [optical density at 600nm (OD₆₀₀) = 5] in PBS (A) and in PBS containing peanut agglutinin (PNA) at 0.02μg/mL (B). Agglutination test for MR-1C (OD₆₀₀ = 0.5) in PBS containing PNA at 0.02μg/mL (C) and in PBS containing PNA at 0.02μg/mL and soluble capsular polysaccharides obtained from the hot TCA treatment of MR-1C (D).

Growth of Strep. thermophilus MR-1C at 40°C in milk, as determined by the decrease in milk pH (– * - -), and in M17 broth supplemented with lactose (LM17), as measured by the absorbance at 600nm (OD₆₀₀; - - + - -). The production of capsular polysaccharides was estimated by the relative fluorescence of cells grown in milk (– ■ –) and in LM17 (– ♦ –) using fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin and in milk (– ˙ –) using FITC-conjugated wheat germ agglutinin as a probe.