EFFECT OF LACK OF OXYGEN.
Many observers have shown that if luminous bacteria are suspended in sea-water and the oxygen removed the light disappears and again returns when oxygen is readmitted. Will the light reappear if the cells are in the meantime cytolyzed? I have made many attempts to obtain extracts of cells broken up in absence of oxygen which would give light if oxygen were readmitted. All these efforts have failed. The result might have been anticipated by the work of MacFadyen (27), who found that luminous bacteria subjected to the action of liquid air did not phosphoresce at that low temperature, but did phosphoresce as soon as warmed again; further, that if the cells were broken up by grinding at the temperature of liquid air, there was no phosphorescence on rewarming. MacFadyen worked, however, in the presence of oxy gen and moisture, and we might suppose that a slow oxidation—too slow to produce light—went on in the material broken up at low tem peratures with consequent exhaustion of the photogenic material.
In my experiments the moist bacteria have been broken up (cyto lyzed) in absence of oxygen by (1) oxygen-free distilled water and (2) toluol. All marine cells can be cytolyzed by distilled water or fat solvents.
In the first method a dense mass of bacteria are placed in a vessel from which the air is exhausted by an air-pump (see apparatus, p. 195). The bacteria stop glowing, but reglow if air is again admitted. Then oxygen-free distilled water is allowed to flow onto the bacterial mass and it is thoroughly shaken. No light appears (indicating that the water is oxygen-free), and 5 to 10 minutes later, if oxygen is added, still no light is emitted. If there is a definite soluble photogenic sub stance in the bacterial cell it should have passed into solution in the water when the cell was cytolyzed, and, provided no decomposition took place, it should have glowed when oxygen was readmitted. Even if we assume that the cell was not completely cytolyzed, the photogen, if a stable substance, although one unable to pass the cell surface, should have glowed within the cell.
In the second method a dense emulsion of the bacteria in sea-water is rendered non-luminous by removing the oxygen. Then a drop of toluol is added without admitting oxygen (air). The emulsion is shaken and no light appears. In a few minutes air is admitted and still no light appears. Similar experiments with ether, chloroform, and carbon tetrachloride gave similar results. Thus if the cells are broken up the photogen disappears even though it has not been oxi dized, for no oxygen was present. The toluol itself does not destroy the photogenic substance, as evidenced by the treatment of dried bacteria with toluol. Luminous bacteria in oxygen-containing sea
water to which a drop of toluol, ether, chloroform, or carbon tetra chloride is added very quickly stop phosphorescing. I explain this as due to the fact that on cytolysis of the cell the oxidation processes run riot and the available store of photogen is rapidly used up. The same explanation may be applied to the loss of light in distilled water. We may compare the conditions in bacteria to the conditions in a potato cell. When the cells of the potato are crushed or when their surface is destroyed by toluol or ether or chloroform, dark melanin oxidation products are rapidly formed, but if the potato is cut and the cut cells well washed to free them of their cell-contents, no blackening occurs, although the lower intact cells at the cut surface are exposed to atmos pheric oxygen and only separated from it by their plasma membranes. A destruction of these membranes would immediately cause oxida tions within to proceed rapidly.
The conclusion drawn from the above experiments has been con firmed by allowing oxygen-free sea-water to come in contact with dried bacteria in a hydrogen atmosphere. If, after 15 minutes, oxygen is admitted, no glow is observed, although dried bacteria instantly glow for a short time if moistened with oxygenated sea-water.
All the above experiments, then, point to the conclusion that if the cell is broken up while moist or if the dead cells stand in contact with water for any length of time, even though no oxygen be present, never theless the photogenic substance undergoes decomposition, a con clusion in harmony with my work and that of McDermott on the firefly. As we have seen, extraction of the dried firefly luminous organs with oxygen-free solvents will give no phosphorescent solutions on admitting oxygen, because of this instability of the photogen (p. 196). On the other hand, the substances in Cypridina extract are stable in oxygen-free water (see p. 176).
In the normal living bacterial cell (or firefly cell) I assume the photo gen to decompose through oxidation with light-production. If the living bacteria are kept in sea-water from which all oxygen has been removed and they stop glowing, they will still glow strongly if oxygen is readmitted, even after a period of 24 hours. It is therefore obvious that the breaking-up of the photogen in absence of oxygen does not occur in the intact bacteria, but only in those whose normal "structure" has been destroyed by cytolysis. I am inclined to believe that the surface layer of the cell is the "structure" involved.