, 2000), including one commercially available biochemical identification system (API 20E and API 20NE, Biomerieux, France). Antimicrobial susceptibility of all Gram-negative bacterial strains isolated either from the environmental water or from the mucus of P. motoro stingrays was determined by the standard disk diffusion method ( Bauer et al., 1966) utilizing commercially available sensitivity discs and Mueller-Hinton Agar. The results were evaluated according to the NCCLS, 2004 guidelines. The following antibiotics were tested: amikacin (AMI), amoxicillin/clavulanic acid (AMC), ampicillin (AMP), cephalotin (CFL), ceftazidime (CAZ),
ciprofloxacin (CIP), chloramphenicol (CLO), trimethoprim/sulfamethoxazole (SUT), streptomycin (EST) and tetracycline (TET). For quality control the test Quizartinib order Tanespimycin was run against the following ATCC strains: Escherichia coli 25922 and P. aeruginosa 27853. Blood-agar culture plates were prepared according to Beutin et al. (1989). Briefly, 1.5 g of TSA (Tryptic Soy Agar) re-suspended in a 10 mM solution of CaCl2 was autoclave. When the temperature of the agar fell to 45 °C, goat red cells previously washed three times in PBS pH 7.2 were then added to the agar
until a final concentration of 5% was reached. The agar was then added to petri dish plates (20 mL per plate), left to solidify and kept at 4 °C until use. Forty microliters of bacterial culture previously grown in TSB (Tryptic Soy Broth) for not 18 h at 37 °C were added in triplicates to 3 mL of TSB and incubated overnight at 37 °C. After incubation, 100 μL of each bacterial culture was added to blood-agar plates in aliquots of 10 μL each. The plates were then incubated for 18 h at 37 °C and the presence of hemolysin was determined by the formation of a halo of lysed erythrocytes around the bacterial growth. Bacterial isolates cultured in TSB were centrifuged at 12,000 g for 15 min at 4 °C and filtered
through a Millipore 0.45 μm pore-diameter syringe filter. Clarified supernatant was tested for proteolytic activity on casein agar plates. Casein agar plates consisted of 25 mM Tris (pH 7.2), 150 mM NaCl, 0.6% casein (Sigma technical grade) and 1% TSA. Aliquots (10 μL) of culture supernatants were placed in 3 mm diameter wells cut in the casein agar and incubated at 37 °C for 18 h. The plates were overlaid with 3% acetic acid, and proteolytic activities were noted as a clear zone around the sample well. Trypsin (1 μg/mL) was used as a positive control standard. Gelatinase production was determined by API 20E and API 20NE biochemical identification kit from Biomerieux, France. Forty microliters of bacterial culture previously grown in TSB at 37 °C for 18 h (106 cell/mL) were added in triplicate to 3 mL of TSB in the presence of either 5, 1 or 0.5 mg of P. motoro venom and incubated for 18 h at 37 °C. As control, the bacterial strains were grown in the presence of TSB alone.
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