368 



SCIENCE 



[N. S. Vol. XLVI. No. 1189 



cytes indicates that there is rapid oxidation 

 at the surface of the cell as well as at the sur- 

 face of the nucleus. 



The objection might be made to the use of 

 indophenol reaction that the result may de- 

 pend somewhat on the manner in which the 

 reagent penetrates. If the oxidizing sub- 

 stances of the cell are largely concentrated 

 in the nucleus, those which are difFused 

 throughout the cytoplasm will first meet the 

 reagent at the cell surface and produce at 

 that point a deposit of granules of indophenol. 

 In the same manner the oxidizing substances 

 which are retained within the nucleus will 

 first meet the reagent at the surface of the 

 nucleus and produce a deposit in that region. 

 It would therefore appear that the reaction 

 might be depended on if it showed the nucleus 

 to have the greatest oxidative activity, since 

 its error would lie in the opposite direction. 

 But any conclusions drawn from it regarding 

 oxidation at the surface of the cytoplasm 

 would be of doubtful value. 



It would seem that more reliable evidence 

 can be obtained by investigating cases where 

 it is not necessary that the reagent should 

 penetrate from without owing to the fact that 

 the cell itself produces the reagent. 



The writer has studied a case of this kind 

 in the Indian Pipe {Monotropa uniflora), 

 which is extremely well suited to such investi- 

 gations, because the cells contain a colorless 

 chromogen which oxidizes and darkens very 

 rapidly upon injury. An additional advan- 

 tage is that the leaves are so thin and trans- 

 parent that they may be placed under a micro- 

 scope and the details of the cell structure 

 studied with care before the cells are injured 

 or treated with reagents. 



In a typical leaf cell the cytoplasm is trans- 

 parent and nearly colorless, with a few gran- 

 ules, while the nucleus is only slightly less 

 transparent, is finely granular and has a nu- 

 cleolus. When a leaf is mounted in a drop 

 of water under a cover glass the cells remain 

 for hours unchanged in appearance. 



If an intact portion of the leaf is cut or 

 crushed the cells in the neighborhood soon 

 In the course of five or ten minutes 



the nuclei of the cells nearest the injury as- 

 sume a more coarsely granular (or vacuo- 

 lated) appearance and soon begin to darken. 

 The darkening does not begin at the surface, 

 but appears to take place almost simultane- 

 ously throughout the whole mass of the nu- 

 cleus. Not until the nucleus has become 

 very dark (so as to stand out very conspicu- 

 ously when the preparation is viewed under 

 the low power of the microscope) does the 

 cytoplasm begin to darken perceptibly. It 

 may be several hours after the nucleus has 

 darkened perceptibly before a change of color 

 can be perceived in the cytoplasm. The 

 darkening of the cytoplasm does not seem to 

 be more rapid at the surface than elsewhere. 



That the darkening is due to oxidation is 

 shovsTi by the fact that it is retarded by the 

 partial exclusion of air^° and is inhibited 

 by the usual means employed to prevent the 

 action of oxidases. When young leaves (free 

 from discolorations) are torn^^ and placed in 

 water the torn edges become dark. This does 

 not occur in 0.1 M HCl, 0.1 M KClSr,!^ 0.1 

 M ISTaOH, or in boiling water. If the color- 

 less chromogen is extracted by 0.1 M NaOH 

 and kept in a tightly stoppered bottle so as 

 to exclude oxygen it remains pale yellow for 

 months, but if oxygen be admitted it soon 

 turns deep red. 



That the darkening of the nucleus is due 

 to oxidation taking place in the niicleus 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 



10 That the oxidation is not completely inhibited 

 by exclusion of air is doubtless due to the fact that 

 a considerable supply of combined oxygen is pres- 

 ent in the cell which can be used for oxidation of 

 the chromogen. 



11 Cutting with a knife was avoided on account 

 of the action of the metal. 



12 In 0.1 M NaOH and 0.1 M KCN the whole 

 leaf becomes pale yellow and then colorless. The 

 yellow color is doubtless due to the fact that the 

 KCN solution is alkaline. 



