Experiments were undertaken with the purpose of determining the type of non-nutrient fluid most suitable for the suspension of pneumococci with a minimum amount of injury. For comparative studies, dilutions in the various fluids tested were made from a standard suspension containing approximately 1,000 million pneumococci per cc. The protective properties of each solution were judged by the length of time viable organisms could be recovered (in culture) from a suspension containing 0.0000001 cc. of the standard suspension. The hydrogen ion concentration of the solutions, the temperature at which the suspensions were kept, and the culture media were carefully controlled. Furthermore, plates were made at the beginning of the experiment to determine the number of organisms present in a unit of suspension.
It was found that pneumococci suspended in Locke's solution, 0.5 and 0.9 per cent NaCl solutions, and water, remained alive for only a few hours at most. Salt solution was shown to be the least suitable of these three; not infrequently by the time the 0.000001 cc. dilution in this fluid had been reached, the suspension was sterile. The addition, however, of 0.1 per cent gelatin to the above fluids transformed all, except 0.9 per cent NaCl, into highly favorable solutions. Pneumococci suspended in gelatin-water and gelatin-Locke's solution remained alive at room temperature for 6 to 7 days; in gelatin-salt 0.5 per cent solution, for at least 2 days. It was possible to vary the H ion concentration of the suspension fluids from pH 7.0 to 8.2 without any marked effect on the results. However, a pH of 7.4 to 8.0 appeared to be the most suitable.
The nature of the protective action of gelatin was investigated. Series of plates made at frequent intervals failed to reveal any growth in suspensions of pneumococci containing this small concentration of gelatin. The beneficial effect of gelatin in the above solutions was found to lie largely in its protection of the pneumococci against the mechanical injury which occurs during the process of dilution in crystalloid solutions or water. Gelatin shows in addition a well marked preservative action, that is to say, it protected organisms against early dissolution. The nature of this preservation is uncertain.
The presence of 0.1 per cent gelatin in 0.9 per cent NaCl failed to protect pneumococci against the toxic action of Na. But it was found possible to neutralize to a considerable degree this toxic effect of sodium by the addition of a small quantity (2 per cent) of a M/15 balanced phosphate mixture or sodium phosphate. In a gelatin-salt 0.9 per cent solution buffered with phosphate, pneumococci survived for 24 to 48 hours. Even in gelatin-salt, 0.5 per cent the presence of phosphate had a definite beneficial effect.