[Validation of TKT medium for detection of Streptococcus agalactiae in bulk milk samples]

A selective medium for quantitative determination of Streptococcus agalactiae in bulk milk samples was tested. Among the medium bases, Edwards medium (EW) supplemented with 6 to 9% (v/v) of carefully washed sheep erythrocytes was proved to be best (Tab. V). The production of sphingomyelinasis C (BHE, Tab. II) or D (COREX, Tab. I) as a supplement supporting the formation of specific hemolytic zones in the medium, was tested in four strains of Staphylococcus aureus and seven strains of Corynebacterium pseudotuberculosis. Submersion aerated cultivation was used for enzyme production; optimum cultivation time is about 100 hours (Figs. 1, 2). To determine the activity of the enzymes produced a screening method was developed applying hemolytic interactions with CAMP factor of Str. agalactiae. Method sensitivity and reproducibility are sufficiently high for the purposes of observation (Tab. III). The produced enzymes did not show any changes in their activity over the whole period of storage under all conditions of storage (32 days/7 degrees C, 48 weeks/-18 degrees C, lyophilizate 48 weeks/20 degrees C). Sphingomyelinasis C is more resistant to heat denaturation, not losing its activity even after being warmed to 85 degrees C for 5 min; sphingomyelinasis D is sensitive to temperatures higher than 50 degrees C (Fig. 3). COREX was selected (sphingomyelinasis D), produced by the strain CNTCC 18/62 of C. pseudotuberculosis. The enzyme was applied either in raw filtrate of the medium (sterilization by repeated membrane filtration) or in prepurified form obtained by cold acetone precipitation, providing the same results. The medium is highly specific and likely enough selective for Str. agalactiae, unlike BHE it does not provide any positive results with UBERIS-factor+strains of Str. uberis (Tab. V). Optimum cultivation time is 18 hours; prolongation of cultivation for more than 24 hours brings about the risk of falsely positive readings (Tab. VI) in the strains of accompanying microflora (Morganella morganii ssp. morganii, delta-hemolytic staphylococci).