The microbial cultures were resuspended in physiological saline solution (PSS). The concentration of the resuspended micro organisms was estimated by measurement of the optical density of the suspension using a DU 520 UV–vis spectrophotometer (Beckmann Instruments, Palo Alto, CA, USA) operating at 550nm, according to the calibration curve which was defined by reference samples. The numbers of microorganisms in the reference samples were controlled by serial dilution and plating of 100 mL of the suspension on MH agar. The colonies were counted after the cultivation at 37C for 24h.