FACTORS LIMITING BACTERIAL GROWTH

There is as yet no agreement among bacteriologists as to the specific physical and chemical influences responsible for changes observed in the rate of growth during the life of the bacterial culture. 1 In Fig. 1 typical curves are presented, illustrating these changes as they occurred during the development of a broth culture of Bacterium coli. The plotted values were calculated by us from the data of Martin, 2 by means of the formula B-A/T for the rate of population-increase, and log B-log A/T log 2 for the rate of growth, where A represents the population at the beginning and B at the end of the observed time-interval T. It will be seen that during the period of rapid increase in population, the rate of growth shows a sharp rise, followed by a continuous decrease. It has been the purpose of our experiments to reinvestigate the causes for these changes. The possibility had presented itself that measurements of rate of oxygen-consumption per cell might be used as a convenient index of comparative growth-rates. The observations of Martin on the oxygen-uptake of Bad. coli cultures seemed to support this inference. In Fig. 1 we have plotted the rates of oxygen-consumption which he observed, using a single time-scale for these and for the corresponding growth-rates. A comparison of these curves shows that a close relation exists between the rate of growth, and the rate of oxygen-uptake per cell, throughout the duration of the experiment. The present paper deals with the influence of certain factors on the rate of oxygen-consumption per cell. Future publications will present fuller justification for extending conclusions based on these data to the question of actual growth-rates. The methods we have used were essentially the same in all experiments. Bacterial cells to be examined were centrifuged from the culture fluid, resuspended in sterile salt solution, and standardized both by photoelectric estimation of turbidity using solutions of copper sulfate as standard for comparison, and by the dilution and pourplate method to obtain the viable count. The medium used was ordinary 0.5% beef extract—1.0% peptone broth, containing sufficient phosphate buffer of pH 6.6 to give a final concentration of M/20. Whenever the culture-fluids were to be examined, they were sterilized by heat or by filtration, with or without readjustment of pH. The required amount of suspension, buffer, and saline were pipetted onto the floor of the respirometer-vessel, and peptone broth of 5 times the desired concentration was placed in a side arm. When necessary, as in the examination of culture-filtrates, this procedure was reversed, the cells being placed in the side arm and the culture-fluid in the vessel proper. Rolls of starch-free filter paper moistened with 10% KOH served to adsorb the carbondioxide liberated. The vessel was then attached to its differential manometer of the Barcroft type, together with a compensator containing the same amounts of nutrient broth, and the apparatus was allowed to reach equilibrium in a waterbath held at 37 ± .001°C, by means of a thyratronrelay. Manometric readings were begun and continued at 5-minute intervals following the introduction of the substrate from the side-arm. With the corrected values of average oxygen-uptake for the successive 5-minute intervals, curves were laid down, and extrapolated graphically to the vertical axis, where the hypothetical quantity of oxygen consumed per 5 minutes at zero time could be read off. In this way it was possible to obtain consistent values for oxygen-uptake per cell to the exclusion of complicating factors of cellular multiplication and changing composition of medium. It is evident that changes in the rate of growth may be dependent either on the physiology of the cell, on the nature of its environment, or on both. Our first experiments were directed toward separate study of changes in cellular activity, and alterations in the medium of growth in relation to the multiplication-rate observed in broth cultures of Bad. coli. The cultures for examination were prepared by inoculation of 100 ml. of infusion-broth in 500 ml. Erlenmeyer flasks with 0.1 ml of an 18-hour test-tube culture in the same medium and incubated at 37.5°C. without resort to special methods of aeration. The results obtained by the methods outlined above may be summarized very briefly for our present purpose by saying that neither the changes detected in the rate of oxygen-uptake of cells taken from cultures of different age, nor differences in the effect of the corresponding culture-filtrates upon growth and respiration, were sufficiently great to explain the divergence of growth-rates observed at different times in the parent-cultures. Some additional environmental factor apparently was involved.