Relationship of free ionic copper and toxicity to bacteria in solutions of organic compounds

The complexation of curpic ions added to solutions of different organic compounds and to culture media was determined using an ion-specific electrode to ascertain the toxic forms of copper. Toxicity of Cu2+ to copper-sensitive and cooper-tolerant strains of Pseudomonas syringae was reduced in the presence of all organic compounds tested, including 100 mM solutions of glucose, fructose, sucrose, succinate, and particularly citrate. The apparent toxicity of copper solutions was reduced 30X or more in the presence of organic solutes. Copper-sensitive and copper-tolerant strains of P. syringae were killed in aqueous solutions of copper only when the molar concentration of added sodium citrate was less than that of the copper. Copper-citrate complexes had no detectable toxicity to either copper-sensitive or copper-tolerant strains of P. syringae. Concentration of free cupric ions in a complex culture medium (CYE: casitone-yeast extract-glycerol agar) increased nearly logarithmically as a function of the amount of Cu2+ added to the medium. Copper-tolerant strains of P. syringae that grew in CYE medium amended with high (>50 micrograms/ml) concentrations of Cu2+ exhibited much higher tolerance of free copper ions in distilled water relative to a sensitive strain that tolerated only about 10 micrograms/ml of added Cu2+ in CYE medium. A direct relationship was observed between the LC99 or Cu2+ to copper-sensitive and copper-tolerant strains of P. syringae when assayed in copper-amended distilled water solutions and the maximum concentration of ionic copper remiaing free in copper-amended CYE medium. These results indicate that a very high fraction (> 99%) of copper added to complex culture media is complexed, that such complexed forms of copper are not toxic to strains of P. syringae, and that these strains are sensitive only to the remaining free ionic copper.