). Biotyping β-galactosidase,
lipase activity and hippurate hydrolysis were performed as find more described previously . Egg yolk agar plates for lipase reactions were obtained from PML Microbiologicals (PML Microbiologicals, Wilsonville, OR.) were inoculated and examined click here daily for 7 days. The presence of an oily sheen on and surrounding the bacterial growth was interpreted as a positive result for lipase activity. Staphylococcus aureus and Pseudomonas aeruginosa were used as positive controls, Lactobacillus crispatus was used as a negative control. Lipase activity using 4-methylumbelliferyl-oleate Lipase activity was also determined as described by Briselden and Hillier , using 4-methylumbelliferyl-oleate (Fluka 75164, from the Sigma Aldrich Chemical Co., St. Louis, MO.) using a spot test as previously described [6, 19]. Briefly, MUO was dissolved in absolute ethanol to a concentration of 4 mg/ml and used to soak a 60 by 6
mm (approximate) Whatman #2 filter paper strip (Whatman, Inc., Clifton, N.J.). After air drying, strips were moistened with buffer, phosphate buffered saline or ACES both pH 7.0 containing 22 mM N-octyl-β-D-glucopyranoside, and 12 mM CaCl2. Alternatively, the MUO was suspended in water and mixed with equal parts of 2 fold concentrated buffer as described above. The strips were inoculated with a loopful of bacteria, incubated at 35°C, and read under a long-wavelength (365 nm), hand-held mineral lamp as described previously . Sialidase Selleckchem Dasatinib activity using 2′(4-methylumbelliferyl) – α-D-N-acetylneuraminic acid Sialidase activity Carbohydrate was determined as described previously by Moncla et al.  using 4-methylumbelliferyl- α-D-N-acetylneuraminic acid as substrate (Sigma M8639, from the Sigma Aldrich Chemical Co., St. Louis, MO.). Stock solutions of the substrate were prepared by dissolving 1 mg in 6.6 ml distilled water, dispensing into 180 μl volumes and storing at -20°C. The stock solutions were thawed and
20 μl 1.0 M sodium acetate buffer, pH 4.8 added and mixed in. The resulting solutions were used in a filter paper strip assay as described above for the 4-methylumbelliferyl-oleate lipase assay. Results All strains survived for 7 days on GVA (Table 1) and by week three about one third of the strains were viable. We did not find any isolates representing biotypes 6 and 8. Several isolates grew little if at all on the GVA or blood agar aerobically; however, good growth was observed for all isolates when cultured anaerobically. On EY (see below), 7 of the isolates failed to grow in air plus 6% CO2 but did grow well anaerobically (Table 1). Strains grown on GVA gave identical biochemical reactions for β-galactosidase, sialidase and hippurate hydrolysis as when grown on BAP.