Rapid plate method for screening hyaluronidase and chondroitin sulfatase-producing microorganisms.

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Microbial hyaluronidase and chondroitin sulfatase have been studied because of the possible associations between these enzymes and microbial mechanisms of pathogenicity. One of the most sensitive biological assays for mucopolysaccharide-degrading enzymes is the turbidity reducing unit (TRU) method (3). In most assays for these two enzymes, measurement of activity involves the conjugation of bovine serum fractions with nondepolymerized substrate in acetic acid. The turbidity produced by the conjugate or the lack of it can be directly related to the amount of substrate depolymerized in solution. Recently, a direct localization and visualization technique to study hyaluronate lyase by agar-gel electrophoresis was reported (1). This technique was modified as a cultivable screening plate method for bacteria. The basic medium consisted of Brain Heart Infusion broth (BBL) prepared to make 100 ml, to which was added 1 g of Noble agar (Difco). The medium was autoclaved at 121 C for 15 min and cooled to 46 C. Aqueous solutions of 2 mg of umbilical sodium hyaluronidate (Sigma Chemical Co., St. Louis, Mo.) per ml, 4 mg of bovine nasal chondroitin sulfate (Pentex, Inc., Kankakee, Ill.) per ml, and 5% bovine albumin fraction V (Signa) were sterilized by filtration with 0.20-,um Nalgene filter units (Nalge Co., Inc., Rochester, N.Y.). Each substrate was added to the cooled media to give final concentrations of 400 ,Ag/ml. The bovine albumin fraction-V was then added with constant stirring to give a final concentration of 1%. The agar was poured to a depth of 3 to 4 mm. The finalpH of each medium was 6.8 0.1. After solidification, plates were tempered at 4 C to provide a firm surface for streaking or swabbing. Pure or mixed cultures