Studies on immunity in anthrax. IX. Effect of variations in cultural conditions on elaboration of protective antigen by strains of Bacillus anthracis.

Wright, George G. (Fort Detrick, Frederick, Md.), Milton Puziss, and W. Brook Neely. Studies on immunity in anthrax. IX. Effect of variation in cultural conditions on elaboration of protective antigen by strains of Bacillus anthracis. J. Bacteriol. 83:515-522. 1962.-Nonproteolytic and nonencapsulated avirulent mutants were isolated from six virulent strains of Bacillus anthracis and tested for elaboration of protective antigen in the chemically defined medium developed previously (599 medium). Initially the strains grew rather slowly in 599 medium; serial transfer in the medium increased the rate of growth but reduced the elaboration of antigen. Two of the strains tended to revert to the encapsulated form during serial passage in 599 medium. Modifications in the medium and cultural conditions were studied in an attempt to obtain improved growth; the most significant alterations were the addition of adenosine and l-alanine, reduction in the concentration of ferrous sulfate, and growth under anaerobic conditions with agitation. Study of amino acid utilization in the medium revealed that the concentrations of certain amino acids could be reduced. These modifications, together with substitution of more productive strains, resulted in an approximately fivefold increase in elaboration of protective antigen, and greatly simplified production of the antigen on a practical scale. Omission of bicarbonate from the medium had no significant influence on utilization of amino acids.