Four strains of Streptococcus pyogenes were propagated at 37 degrees C in a reduced iron medium supplemented with Fe3+-citrate to give concentrations of 1 through 11 micrograms per milliliter, in order to observe the effects of iron on growth and on the vitro production of Streptolysin S. Both growth and hemolysin production were observed to be influenced by medium iron concentration of which 1.2 micrograms per ml of iron was critical. Hemolysin was produced during the exponential phase of the growth cycle with maximum yield as the organism entered the stationary phase. Hemolytic activity (which was accepted as the ability of the hemolysin to lyse sheep erythrocytes) fell below detectable levels as the organisms entered fully into the stationary phase (9-10 hours post incubation). Serum (bovine, human, chicken) was observed to have a high stabilizing effect on the hemolysin.

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