Factors Influencing Rate of Reduction of Potassium Ferricyanide by "Resting" Escherischia coli

Abstract

It has been shown that broth cultures of Staphylococcus aureus in the maximum stationary phase reduce potassium ferricyanide at a rate in the neighborhood of 6.6 x 10-14 millimols per cell per minute. Further work with cultures of Escherischia coli (K-12) gave similar results and will be reported elsewhere. The experiments to be reported here were designed to measure quantitatively the rate of reduction of potassium ferricyanide by “resting” Esch. coli under carefully controlled environmental conditions. Forty-eight hour cultures of Esch. coli, grown on nutrient agar at 37.5°C., were suspended in saline and washed 3 times by centrifugation. The organisms were then diluted with a mixture of equal parts of M/15 phosphate buffer, pH 7.5, and physiological saline solution to a standard turbidity corresponding to a total count of approximately 20 x 109 cells per cubic centimeter. Measured volumes of this suspension of “resting” Esch coli were introduced into tubes similar to those employed in the studies with Staph. aureus and appropriate amounts of buffer, saline, and Difco peptone solutions were added. These suspensions were deaerated for one-half hour, by bubbling nitrogen through them at a constant rate, before the addition of a 0.1 molar solution of 3 parts of potassium ferricyanide to 1 part of potassium ferrocyanide. The change in the redox potential with time at 37.5°C. was followed potentiometrically at 10 minute intervals and the concentration of ferricyanide calculated directly from the potential readings. The rate of reduction of the ferricyanide increases with increase in concentration of peptone (other variables being maintained constant) up to 2.0 or 3.0% and then decreases as the concentration of peptone is further increased. The rate of reduction of ferricyanide by “resting” Esch. coli in the absence of peptone or by peptone in the absence of “resting” bacteria is negligible.