DEPARTMENT OF MARINE BIOLOGY. 129 



which equilibrium is conditioned by the presence of these products, is again 

 able to proceed. It is well known that stimulation in the case both of a 

 muscle and an egg results in an increased production of CO,. 



The problem this summer (1910) was, therefore, to test the permeability 

 of the egg for some one substance and to determine the effect of certain sub- 

 stances in changing the permeability of eggs. We do not know whether the 

 acid or alkali added to sea-water to make eggs mature, or to form membranes, 

 or to render them capable of cross-fertilization, brings about its result by 

 entering the eggs or whether the effect is a purely surface one. The same 

 statement applies to the action of ions on muscle and nerve tissue and cilia. 



I have studied the penetration of NaOH into eggs and the method em- 

 ployed is the use of neutral red as an indicator within the cell. This dye is 

 red in neutral and acid solutions and yellow in alkaline solutions. 



In echinoderm eggs (Toxopnenstes, Hipponoe, and Holothuria) neutral 

 red is taken up (combined?) by definite granules which stain red. They are 

 present in the fertilized as well as the unfertilized eggs and are characterized 

 by their high specific gravity, for they pass to the distal pole of the egg in the 

 centrifuge whether first stained and then centrifuged or first centrifuged and 

 then stained. If left in the dye for a long time other granules in the general 

 yolk mass also stain. The rate of staining of fertilized eggs (or eggs with 

 artificial membranes) and unfertilized eggs is very different, however, the 

 former staining much the more rapidly. If the unfertilized eggs are left in 

 the solution long enough, as many and as definite granules stain as in the fer- 

 tilized. This would appear to indicate a difference in permeability to neutral 

 red between the fertilized and unfertilized eggs, and I can therefore confirm 

 for neutral red what Lyon has recently found for methylene blue. 



If eggs stained in neutral red are placed in sea-water to which as much 

 alkali has been added as possible without precipitation ( 100 c.c. sea-water -f- 

 1.3 c.c. N/10 NaOH) they retain their red color for over 3 hours; but if 

 this same solution is saturated with chloroform the alkali passes in almost 

 instantly and turns the neutral red to yellow. The change from red to yel- 

 low is independent of the swelling of the egg caused by the chloroform, for 

 the penetration of alkali takes place similarly when swelling is prevented 

 (for a short time) by the addition of cane-sugar to the sea-water. Either 

 the NaOH fails to enter or only enters so slowly that it is neutralized within 

 the egg. Since the number of eggs is very small, compared with the bulk of 

 alkaline solution, and the alkali would continue to diffuse in so long as neu- 

 tralized, it would require an enormous production of acid on the part of the 

 egg to neutralize the NaOH entering. For this reason the first alternative 

 seems the more probable. 



By treating eggs stained in neutral red with stronger and stronger concen- 

 trations of NaOH in 0.6 molecular NaCl the alkali penetrates the egg more 

 and more rapidly. If a concentration of NaOH which enters the eggs (Hip- 

 ponoe) in 20 minutes be one-quarter saturated with chloroform, the NaOH 

 enters in 10 minutes. One-quarter saturated chloroform has no visible effect 

 on the eggs even after an hour. The effect of dilute solutions of chloroform 

 which fail to cytolyse is to increase the permeability of Hipponoe eggs to 

 NaOH. The above statement was found true for ether also. It is obvious 

 that the number of substances whose effect on permeability may be tested in 

 this way is limited, for most of them combine with NaOH. 



Thus chloroform, one of the most effective substances for producing arti- 

 ficial membranes, increases the permeability of the egg for alkali. It can be 

 further shown, by comparing the time it takes for the stained eggs, fertilized 



9 — YB 



