Bacterial strain and culture conditions

Hemolytic activity against erythrocytes was determined using the method described by Deshpande and Khan [20], with some modifications. Horse, human, sheep, and rabbit erythrocytes were used. Human erythrocyte samples were collected from 8 volunteers (blood types: A, B, AB, and O; 2 persons each). Ethical approval was obtained from the Research Ethics Committee of the School of Dentistry, Health Sciences University of Hokkaido. Erythrocyte samples were washed 3 times with phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 and pH adjusted to 6.8) and diluted with the same buffer. Next, 1% v/v erythrocytes (cell count, 7×107/mL), 5% v/v bacterial samples, and 5% v/v inhibitor or PBS were mixed (final concentration) and incubated at 37 °C for 5 h. After centrifugation (1500×g for 5 min), the absorbance of the supernatant was measured at 540 nm by using a UV mini-1240 spectrophotometer (Shimadzu, Kyoto, Japan). One hemolytic unit (HU) was defined as the amount of hemolysin REQUIRED to release 50% hemoglobin in a standardized erythrocyte suspension.

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