OSTERHOUT: THE NUCLEUS AS A CENTER OF OXIDATION 345 
The darkening which occurs is due in part to free oxygen left in the 
leaf and in part to oxygen in compounds from which it can be split 
off for the oxidation of the chromogen (analogous to anaérobic 
respiration). 
2. If leaves are torn in two or crushed at once, dropped into boiling 
water, 0.1 M HCl, 0.1 M NaOH, 0.1 M KCN, or 3 percent H2O: the 
darkening does not occur. ‘These agents are inhibitors of oxidation in 
living tissues. Hydrogen peroxide may inhibit at high concentration, 
but accelerate at low concentrations. In NaOH and KCN the leaf 
becomes pale yellow: this seems to be due to the action of hydroxyl 
ions. 
3. The chromogen may be extracted by placing stems in 0.1 M 
NaOH in a bottle completely filled (so as to exclude air) and tightly 
stoppered (with a glass stopper coated with vaseline). The solution 
becomes pale yellow (or slightly reddish) and may be kept in this 
condition for months. On opening the bottle and pouring out the 
solution into a shallow dish it at once becomes red as the result of 
oxidation. The behavior seems to be analogous to that of pyrogallol, 
which is easily oxidized by the air in alkaline solution, but not in 
neutral solution except under the influence of oxidases (from plants or 
animals) or other catalyzers. 
That the darkening of the nucleus is due to oxidation taking place 
in the nucleus itself and not to the taking up by the nucleus of a stain 
produced in the cytoplasm or vacuoles is shown by the following 
experiment. Plants were ground in a mortar and allowed to stand 
until they became black. The juice was squeezed out and centrifuged, 
giving an inky fluid. In this were placed pieces of leaves which had 
been treated with 0.1 KCN and afterward with water. ‘The solution 
was allowed to stand until it became concentrated by evaporation: 
it then appeared black. It was found that where the nuclei had been 
squeezed out of the cut cells by the knife they had taken up some stain 
but not more than the cytoplasm. In cells which were merely cut 
open there was little or no staining of the nucleus. 
We must therefore conclude that oxidation occurs more rapidly in 
the nucleus than elsewhere in the cell. The only way to escape this 
conclusion would be by assuming that at the moment of injury there 
is a sudden migration into the nucleus of some or all of the substances 
necessary for the oxidation. This is not only very improbable from a 
theoretical standpoint, but observation shows that it can not be the 
case, for in this migration the substances would mingle and produce 
the pigment either outside the nucleus or at its surface before any 
pigment appeared in the interior of the nucleus. Observation of the 
nucleus shows that the pigment appears as soon in the interior of the 
nucleus as at its surface. 
