W. J. V. OSTERHOUT 277 



It is evident that the most satisfactory method is to place the cell 

 in a solution containing the substance whose penetration is to be 

 investigated and, after a definite time of exposure, to obtain the cell 

 sap without contamination and test it for the presence of this sub- 

 stance. Experiments of this sort have apparently not been carried 

 out, though interesting results have been obtained by Meyer,® Han- 

 sen,' Wodehouse,' and Crozier^ by comparing the cell sap of the 

 marine alga Valonia (which can be obtained without contamination) 

 with the sea water. Janse^'^ found that filaments of Spirogyra which 

 had been kept for a time in a solution of KNO3 gave a test for NO3 

 after being rinsed and caused to burst in a solution of diphenylamine. 

 In this method there is serious risk of contamination by substances 

 in and upon the cell wall (or between the cell wall and the protoplasm). 



In order to avoid this difficulty the writer has employed the large, 

 multinucleate cells of a species of Nitella, some of which reach a length 

 of 6 inches and a diameter of a thirty-second of an inch. 



Within the cell wall the protoplasm forms a thin layer in which 

 the chlorophyll bodies are imbedded. Inside this layer is the large 

 central vacuole filled with cell sap. It is possible to obtain the cell 

 sap without contamination in various ways. The writer has made 

 use of the following methods: The cells are placed for the desired 

 length of time in a solution containing the substance whose penetra- 

 tion is to be tested. They are removed, washed in running tap water 

 (followed in m.any cases by distilled water), and dried by means of 

 filter paper. The cells are so large and turgid that this manipulation 

 presents no difficulty. A cell is then placed on a piece of glass or 

 filter paper and pierced with the point of a clean capillary tube 

 (which has been drawn out to a fine tip). The cell sap is drawn up 

 into the tube (by capillary action) quite free from protoplasm or 

 chloroplasts." Another method, which is preferable in many cases, 



« Meyer, A., Ber. deutsch. hot. Ges., 1891, ix, 77. 

 'Hansen, A., Milt. Zool. Stat. Ncapel, 1893, xi, 255. 



8 Wodehouse, R. P., /. Biol. Chem., 1917, xxix, 453. 



9 Crozier, W. J., J. Gen. Physiol., 1918-19, i, 581. 



1° Janse, J. M., Versl. Meded. Kan. Akad. Wetcnsch. Afd. Natuurk. (3), 1888, 

 iv, 332. 



" During the manipulation care should be taken to prevent the sap from run- 

 ning out of the cell and coming in contact with its outer surface. 



