2.2. Determination of bacterial resistance to Cr(VI)
The minimal inhibitory concentration (MIC) of chromium at which no growth occurred was determined by both the visual method (no turbidity was observed as a signal of bacterial growth inhibition) and the “spectrophotometrical” method (turbidity measured as the absorbance at 600 nm).
2.2.1. Visual method
Two or three colonies of bacterial strains were suspended in 5 mL of physiological solution to get turbidity equivalent to the test tube 1 (standard) in the Mc Farland scale [10]. MIC was determined in duplicate by inoculating 0.1 mL of the suspension in 3 mL of PYG broth (casein peptone 2.5 g/L, yeast extract 1.25 g/L, glucose 0.5 g/L)
with 0.1, 0.25, 0.50, 0.75, 1, 2 and 5 mM Cr(VI). Cultures were incubated at 32◦C for at least 1 week, checking bacterial development by comparing with the control cultures Cr(VI)-free.
2.2.2. Spectrophotometrical method
This method was used only for K. ornithinolytica 1P, because all the other strains form aggregates that interfere with the absorbance measurements at low bacterial densities. MIC was determined in duplicate in PYG broth with the same concentrations of Cr(VI)
mentioned before and the bacterial development by absorbance at 600 nm using a Turner spectrophotometer (SP 830 model) every 24 h. MIC was estimated as the first dilution which completely inhibits bacterial growth in PYG medium tubes.
2.2. Determination of bacterial resistance to Cr(VI)
The minimal inhibitory concentration (MIC) of chromium at which no growth occurred was determined by both the visual method (no turbidity was observed as a signal of bacterial growth inhibition) and the “spectrophotometrical” method (turbidity measured as the absorbance at 600 nm).
2.2.1. Visual method
Two or three colonies of bacterial strains were suspended in 5 mL of physiological solution to get turbidity equivalent to the test tube 1 (standard) in the Mc Farland scale [10]. MIC was determined in duplicate by inoculating 0.1 mL of the suspension in 3 mL of PYG broth (casein peptone 2.5 g/L, yeast extract 1.25 g/L, glucose 0.5 g/L)
with 0.1, 0.25, 0.50, 0.75, 1, 2 and 5 mM Cr(VI). Cultures were incubated at 32◦C for at least 1 week, checking bacterial development by comparing with the control cultures Cr(VI)-free.
2.2.2. Spectrophotometrical method
This method was used only for K. ornithinolytica 1P, because all the other strains form aggregates that interfere with the absorbance measurements at low bacterial densities. MIC was determined in duplicate in PYG broth with the same concentrations of Cr(VI)
mentioned before and the bacterial development by absorbance at 600 nm using a Turner spectrophotometer (SP 830 model) every 24 h. MIC was estimated as the first dilution which completely inhibits bacterial growth in PYG medium tubes.
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2.2. Determination of bacterial resistance to Cr(VI)
The minimal inhibitory concentration (MIC) of chromium at which no growth occurred was determined by both the visual method (no turbidity was observed as a signal of bacterial growth inhibition) and the “spectrophotometrical” method (turbidity measured as the absorbance at 600 nm).
2.2.1. Visual method
Two or three colonies of bacterial strains were suspended in 5 mL of physiological solution to get turbidity equivalent to the test tube 1 (standard) in the Mc Farland scale [10]. MIC was determined in duplicate by inoculating 0.1 mL of the suspension in 3 mL of PYG broth (casein peptone 2.5 g/L, yeast extract 1.25 g/L, glucose 0.5 g/L)
with 0.1, 0.25, 0.50, 0.75, 1, 2 and 5 mM Cr(VI). Cultures were incubated at 32◦C for at least 1 week, checking bacterial development by comparing with the control cultures Cr(VI)-free.
2.2.2. Spectrophotometrical method
This method was used only for K. ornithinolytica 1P, because all the other strains form aggregates that interfere with the absorbance measurements at low bacterial densities. MIC was determined in duplicate in PYG broth with the same concentrations of Cr(VI)
mentioned before and the bacterial development by absorbance at 600 nm using a Turner spectrophotometer (SP 830 model) every 24 h. MIC was estimated as the first dilution which completely inhibits bacterial growth in PYG medium tubes.
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