The nature and origin of the large "protoplasmic" potential in Halicystis must be studied by altering conditions, not only in external solutions, but in the sap and the protoplasm itself. Such interior alteration caused by the penetration of ammonia is described. Concentrations of NH4Cl in the sea water were varied from 0.00001 M to above 0.01 M. At pH 8.1 there is little effect below 0.0005 M NH4Cl. At about 0.001 M a sudden reversal of the potential difference across the protoplasm occurs, from about 68 mv. outside positive to 30 to 40 mv. outside negative. At this threshold value the time curve is characteristically S-shaped, with a slow beginning, a rapid reversal, and then an irregularly wavering negative value. There are characteristic cusps at the first application of the NH4Cl, also immediately after the reversal.
The application of higher NH4Cl concentrations causes a more rapid reversal, and also a somewhat higher negative value. Conversely the reduction of NH4Cl concentrations causes recovery of the normal positive potential, but the threshold for recovery is at a lower concentration than for the original reversal. A temporary overshooting or increase of the positive potential usually occurs on recovery. The reversals may be repeated many times on the same cell without injury.
The plot of P.D. against the log of ammonium ion concentration is not the straight line characteristic of ionic concentration effects, but has a break of 100 mv. or more at the threshold value. Further evidence that the potential is not greatly influenced by ammonium ions is obtained by altering the pH of the sea water. At pH 5, no reversal occurs with 0.1 M NH4Cl, while at pH 10.3, the NH4Cl threshold is 0.0001 M or less. This indicates that the reversal is due to undissociated ammonia.
The penetration of NH3 into the cells increases both the internal ammonia and the pH. The actual concentration of ammonium salt in the sap is again shown to have little effect on the P.D. The pH is therefore the governing factor. But assuming that NH3 enters the cells until it is in equilibrium between sap and sea water, no sudden break of pH should occur, pH being instead directly proportional to log NH3 for any constant (NH4) concentration. Experimentally, a linear relation is found between the pH of the sap and the log NH3 in sea water. The sudden change of P.D. must therefore be ascribed to some system in the cell upon which the pH change operates. The pH value of the sap at the NH3 threshold is between 6.0 and 6.5 which corresponds well with the pH value found to cause reversal of P.D. by direct perfusion of solutions in the vacuole.