THE BEHAVIOR OF CERTAIN MICRO-ORGANISMS IN BRINE. 63 



Gelatine would not go into solution in hot brine of high specific gravity in quantities suffi- 

 cient to solidify it on cooling, but agar-agar was perfectly satisfactory and is much more 

 easily usable than silicic acid jelly. The objection to agar-agar commonly made is that 

 it filters very slowly and is not clear. Both of these objections can be readily overcome 

 by the method described by Kanthack and Drysdale/ which consists essentially in allow- 

 ing the agar-agar fiber, cut into short pieces, to soak and swell for 15 minutes in a con- 

 siderable volume of 1 per cent acetic acid, washing in ruiming water until every trace of 

 acid is gone, and dissolving in the hot brine. A proportion of 0.5 per cent agar-agar is 

 scarcely sufficient to keep the brine quite solid under the microscope at ordinary room 

 temperatures, while 1 per cent is a little more than is needed. A certain amount of water 

 will escape from the brine as steam while the agar-agar is being dissolved. In order to 

 clear and at the same time to restore the density of the brine, beat the white of an egg to 

 a froth in a volume of cool distilled water about equal to the quantity of water given off 

 in heating the brine; mix this thoroughly with the brine and allow it to settle and solidify 

 in a clean hot-water funnel, keeping the solution warm but quiet for a half hour and then 

 allowing it to cool. In this way the greater part of the cloudy material of the brine as 

 well as of the agar-agar will be carried down by the egg and may be cut off and thrown away. 

 The clearer, if not quite clear, upper part of the conical block of agar-agar may now be 

 melted again in a double boiler and poured into the test-tubes, flasks, and petri dishes 

 to be used for the cultures. No further sterilization is needed, for the brine organisms 

 have already been killed and have settled out with the frothy egg-albumen, and no other 

 organisms can live in these concentrated brines. 



In figure 1, Plate 9, are represented the three species of unicellular alga? occurring in 

 concentrated brines at Redwood. These are Dunaliella salina (a in the figure), Pyrami- 

 monassp. ? (b in the figure), and the remainder represent the two forms of D. viridis. The 

 rectangular bodies of large size are crystals of salt (NaCl). A brine of 1.190 specific gravity, 

 which I had filtered, sterilized by heating, solidified with 1 per cent agar-agar, and cooled 

 in a petri dish, was inoculated at five points on its surface with five drops of brine of the 

 same concentration but containing only the two species of Dunaliella. Their numbers 

 were very dissimilar, as figure 1 suggests. This, and the other dishes inoculated at the 

 same time, were set on a plate-glass shelf in a window, and pains were taken to keep the 

 culture dishes always in the same position relative to the direction of illumination. When 

 inoculated, all the organisms were actively moving and plainly ciliated. Figure 2, Plate 9, 

 shows {gr) a group or colony of D. viridis, formed by division of one or a few cells which 

 had come to rest. Their cilia have become invisible. One of the cells is in process of division. 

 The numerous granules in these cells are starch and testify to ample food manufacture 

 under the conditions of culture. The same figure (6r) also shows an individual D. salina, 

 also in process of division after having come to rest, and its cilia having disappeared. 

 In both D. viridis and D. salina on the surface of the agar-agar the cells have become 

 slightly flattened and broader and considerably less elongated than the pear-shaped cells 

 which, impelled by the motion of their paired cilia, were being pulled through the sirupy 

 brine in which I first saw them. Although these organisms are actively phototactic, I 

 was not able to see that the direction of their divisions could be connected definitely with 

 the direction from which the light fell most strongly upon them, namely from the window. 

 Figure 3, Plate 9, indicates this for D. salina and figures 4-8 show the same for D. viridis 

 in another culture, for here the division walls run at various angles to the incident rays of 

 light. There results from this, in the formation of the one-layered colonies on the surface of 

 agar-agar, no regular relation of the cells to the direction of illumination, their clear ends, 

 eye-spots, etc., lying generally toward the periphery of the colony but not always, as three 

 cells in figure 4a, Plate 9, show. 



" Kanthack, A. A., and Dryedale, J. H. A course of elementary practical bacteriology, p. 90. London, 1895. 



