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Decreasing the agar concentration of a counting medium from the usual 1.5% resulted in larger colonies with less interference from gas inClostridium botulinum 115B andC. sporogenes PA 3679. Optimal agar concentration was 0.65% forC. botulinum with 24-hr incubation and 0.50% forC. sporogenes with 48-hr incubation. Lower concentrations yielded growth too diffuse for counting. Motility was considered the explanation for increased colony size in softer agar. The greater the degree of motility, the greater would be the diffusibility expected, and thus the higher the agar concentration required to insure discrete colonies. For quantitating motility, evaluations were made by use of microscopic examination of liquid cultures and rate of diffusion in a semisolid medium. With both criteria, the degree of motility ofC. botulinum 115B clearly exceeded that ofC. sporogenes PA 3679. Small-colony variants ofC. botulinum in 0.65% agar yielded only small colonies on subculture, with a corresponding decrease in degree of motility of the cells by both criteria. Colony size of the nonmotileC. perfringens ATCC 3624 was unaffected by lowered agar concentrations.

Agar Concentration in Counting Clostridium Colonies