250 Pub. Puget Sound Biol. Sta. Vol. 2, No. 48 



blue was determined for several species and some data were obtained on 

 color gradients resulting from the reduction of KMnO^. In several 

 species the course of death in standing water was also observed. 



The methods of procedure have been described in earlier papers 

 (Child, 1916a, 1916c, 1917a, 1917b, 1919a) and need only brief consider- 

 ation here. The thalli, or portions of them, are simply immersed in sea 

 water solutions of the agents used and the course of the death changes 

 in the cells is followed along the axes. These death changes may be deter- 

 mined in various ways : for example, the smaller, less opaque forms may 

 first be stained to some degree with neutral red and then placed in some 

 other agent, e.g., KNC. Neutral red is orange red or light red in solu- 

 tions near neutralitj' and becomes purple in acid and yellow in alkaline 

 solution. The living cell stains light red with the dye, but as it approaches 

 death the color deepens, indicating a change toward increased acidity and 

 the coagulating masses of protoplasm may become deep blackish-purple 

 as if markedly acid, but soon after the break-up and coagulation of the 

 protoplasm the color changes to yellow, i.e., the dead cells become alkaline. 

 When the cells are killed with KNC following staining with neutral red, 

 this change occurs very rapidly, but essentially the same changes occur 

 when the cells are killed in neutral red alone, this so-called vital dye 

 being lethal in sufficient concentration and with sufficient time. The death 

 changes in neutral red and in other agents following neutral red are fully 

 described in the papers referred to above. In the use of neutral red as 

 a stain preceding other agents care must be taken not to stain too deeply 

 or too long a time, since the toxic effect of the neutral red may alter or 

 even reverse the susceptibility relations to the other agent (Child, 1916c, 

 1917a). 



In the green algae the death changes in the various agents, KNC, 

 acid, alcohol, can often be directly observed as changes in appearance and 

 color of the protoplasm, but they usually appear more clearly if the 

 plant is first stained with neutral red and then treated with some other 

 agent. When the agent is not strongly acid, death is very soon follov/ed 

 by the loss of red color, while in acid agents the cells may become opaque 

 black. 



In the brown and red algae the pigment of the plant may be used 

 as an indicator of the entrance of the agent and the death of the cell, 

 since death is accompanied by a change in color as well as by coagulatory 

 changes in the protoplasm. In the brown algae the color changes to 

 yellowish green or dull olive green, according to the species and the agent, 

 this change being evidently due to the more or less complete loss of the 

 pigment (phycophaein, phycoxanthin) from the cells, and where the 

 quantity of the pigment is sufficient, the solution may become colored by it. 



