ABSTRACT
The action of isotonic solutions of single salts in causing artificial parthenogenesis was first described by R. S. Lillie (1910, 1911 a, 1911 b). He found that while the salts of sodium and potassium caused development, those of calcium and magnesium were ineffective. Addition of calcium or magnesium inhibited the action of sodium or potassium. In a later paper (1916) Lillie showed that activation of the egg, whether by fertilisation or by artificial methods, is accompanied by an increase in permeability of the surface membrane of the egg to water.
It seemed desirable to repeat and extend these observations especially as, in Lillie’s experiments, the hydrogen ion concentration was uncontrolled. The eggs of Thalassema neptuni were found to provide favourable material for work on this and allied subjects. The animals are readily obtainable at Plymouth where this work was carried out and they have an unusually long breeding season, extending from the middle of July until the beginning of October.
Compared with other marine forms the number of eggs laid by Thalassema neptuni is small. A large individual probably contains not more than about 40,000 eggs, and small specimens are considerably less prolific. The ripe eggs or spermatozoa are contained in the four nephridia which have powerful muscular walls. The nephridia can usually be seen through the semi-transparent wall of the animal and the difference between the dead white of the sperm and the pale orange colour of the eggs enables the sex of the animal to be distinguished in most cases without the necessity of opening it.
The technique of handling the animals for experiment is as follows. The selected individuals are thoroughly washed in fresh water to destroy any sperm that may adhere to the surface. They are then pinned down and the body wall is slit. The contents of the body cavity are quickly washed out with fresh water and the nephridia taken out and put into sea-water. The muscular walls of the nephridia contract rhythmically and drive out the eggs or sperm.
The eggs are very tightly packed in the nephridia and, as the result of the pressure to which they are subjected, they are much distorted. Usually the eggs do not become spherical if allowed to remain unfertilised in sea-water ; if they do so at all it is only after some hours. If, however, fertilisation takes place, the spherical form is acquired in less than a minute. The eggs of Thalassema undergo this change almost as rapidly when treated with an activating agent, such as isotonic calcium chloride, as when fertilised. Lefevre (1907) has recorded a similar change occurring in the eggs of Thalassema mellita as the result of natural or artificial activation.
The eggs of Thalassema neptuni appear to be similar to those of Thalassema mellita described by Griffin (1899) and by Lefevre (1907). They are about 100 µ in diameter after fertilisation. The nucleus of the unfertilised egg is large and is usually somewhat eccentrically placed. The cytoplasm is fairly transparent and uniformly granular; there are no large yolk droplets. The egg is surrounded by a thick vitelline membrane.
Maturation does not normally begin until the egg is fertilised. A sample of eggs which has been standing, unfertilised, in sea-water for so long as eight hours may show a few in which maturation has begun, but these have rarely been observed to form more than 5 per cent.
The changes undergone by the egg after fertilisation may be briefly described. The first sign that fertilisation has occurred is that the egg becomes spherical. This process takes less than one minute, and by the time it is completed the vitelline membrane can be seen to be separating from the surface of the egg as the fertilisation membrane. The elevation of the fertilisation membrane to its final position takes several minutes to complete. During the early stages of separation of the fertilisation membrane a completely transparent gelatinous substance appears on the outside, carrying away with it the excess sperm which can now be seen as a ring situated at some distance from the surface of the egg. In this respect the behaviour of the egg of Thalassema is similar to that of Nereis according to the description of F. R. Lillie (1919) and other workers. Soon after fertilisation the nuclear membrane begins to disappear; then the two polar bodies are formed. Normally a definitive female pronucleus is not formed and maturation is directly succeeded by the processes leading to the first cleavage. Eggs which have been activated by experimental agents, however, often proceed no further than maturation. The female pronucleus is formed and the egg undergoes no further change until cytolysis occurs.
The experimental procedure consisted of treating the eggs for various times with isotonic solutions of single salts or with series of mixtures of two salts. Kahlbaum’s purest salts were employed. The chlorides of lithium, sodium, and potassium were made up in 0.6 M. solution and those of calcium and magnesium in 0.4 M. solution. These concentrations were assumed to be approximately isotonic with sea-water. The hydrogen ion concentration was kept, as nearly as possible, constant at about pH 8.2 which is equivalent to that of sea-water at Plymouth. The sodium, lithium, and potassium chloride solutions were buffered by the addition of one drop of saturated sodium bicarbonate solution to 200 c.c. All the solutions were thoroughly aerated and the final adjustment of pH was made by the addition of carbonate or saturated Ca(OH)2 solution. In the solutions where the calcium content is high, adequate buffering is impossible. In such cases the variation in pH was prevented as far as possible by filling the experimental vessel up to the top and covering with a glass plate so that practically no air was in contact with the solution. In this way a variation of pH of more than 0.3 was avoided and in many experiments the alteration was less.
The solutions with which the eggs were to be treated were contained in glass vessels with flat bottoms and vertical sides. Into each of these was fitted a small sieve formed by a ring of celluloid over which was fastened a piece of fine bolting silk with a rubber band. These sieves were found to be very convenient for handling a large series of egg samples which require to be exposed to a long set of mixtures of salts for the same length of time. With care the time error can be reduced to a few seconds. The sieves are taken to pieces and washed thoroughly after each experiment. Before being used for the first time all the size was removed from the bolting silk by repeated washing. Careful control experiments were made which showed that the eggs were not in any way influenced by the technique employed or by the materials of which the sieves were composed. As soon as the correct time of exposure of the eggs is completed the sieve is lifted out, the fluid drains off quickly and the eggs are washed off the sieve in a finger bowl containing sea-water. As soon as the eggs settle at the bottom, the supernatant fluid is poured off and the bowl is filled with fresh sea-water. All the sea-water used in these experiments was “outside water,” that is to say, water which was collected in carboys from the open sea, outside the breakwater. This had always, when used, been in the laboratory for two or three days, and consequently contained no living spermatozoa. In all experiments controls were kept of unfertilised eggs in sea-water.
In each experiment the eggs of only one individual were used. In nearly all experiments the eggs were examined alive but in a few cases the eggs were preserved in 5 per cent, formalin. Counts were made in almost every case of 300–1000 eggs, depending on the number available.
The temperature was not specially controlled. In different experiments it varied from 13° C. to 18° C. but in any one experiment the variation was less than 1° C.
It should be noticed that in the following pages for the sake of convenience the term “activation” when not otherwise qualified is used to designate the condition of those eggs which have begun maturation but which have not undergone cleavage. Maturation is assumed to begin, for purposes of description, with the complete disappearance of the membrane surrounding the oocyte nucleus and not with the earlier stages of this process. Under the heading “activation” are included also all eggs which have completed maturation and developed asters but which, because of the abnormal form or number of these structures, have failed to divide.
In the experiments to be described the eggs were only allowed to reach the earlier cleavage stages although ciliated larvae can readily be obtained as the result of artificial activation by the agents employed. When quantitative observations are being made on the effects of various agents in initiating cell division it is important that the estimates should be based on the examination of as many eggs as possible. The counts are made much more easily during the earlier stages of development when the blastomeres are large. At this stage it is also easier to distinguish true cleavage from the cases of fragmentation which are liable to occur in all experiments on artificial parthenogenesis.
THE ACTION OF ISOTONIC SOLUTIONS OF SINGLE SALTS
The salts whose action on the eggs of Thalassema has been investigated are the chlorides of sodium, lithium, potassium, and calcium. The effect of the first three of these salts on the unfertilised eggs is similar. When the eggs are exposed to the solutions of sodium, lithium, or potassium chloride at the pH of sea-water for periods up to 60 min. no visible effect is produced, either in the solution or after removal to sea-water. The eggs preserve the distorted shape which they possessed before the treatment, and there is practically no maturation. Strikingly different is the effect of calcium chloride. Shortly after immersion in this solution all the eggs have become spherical and soon maturation begins in a varying number and continues whether the eggs remain in the solution permanently or are transferred to sea-water. A certain number of the eggs removed to sea-water begin cleavage some time afterwards. The time factor is of prime importance in all these experiments in determining the proportion of maturing eggs and especially the percentage of cleavage. Cleavage may also occur among those eggs left permanently in the calcium chloride solution but the percentage is small and the dividing eggs do not usually proceed beyond the 2-cell stage. Table I illustrates the result of a typical experiment and Fig. 1 shows graphically the effect of the calcium chloride.
It is somewhat unexpected to encounter an instance such as the one here described in which calcium is an effective agent in inducing development while the alkali-metals, under the conditions of these experiments have no such action. It is true that some other cells, such as the eggs of Echinus esculentus, are remarkably little affected by pure, isotonic solutions of sodium or potassium chlorides, but calcium and magnesium chlorides have also little action under similar conditions. On the eggs of Thalassema, however, calcium has a very marked action although it is generally considered to be a substance whose effect is to decrease permeability to water and salts. The action of calcium chloride is not over-emphasised by the experiment quoted. In many cases the proportion of activation induced exceeds 90 per cent.
An experiment may be quoted which shows that pure isotonic potassium chloride is not entirely devoid of action on the eggs. The material used in this experiment was unusual in that it formed the only exception encountered to the general rule that the great majority of the eggs of this animal do not mature when left in sea-water. In the control batch of eggs left in sea-water just 50 per cent, underwent maturation but developed no further. That the eggs were otherwise normal was shown by the fact that 100 per cent, perfect larvae were obtained from another lot which were fertilised. It is possible that under natural conditions if the eggs are deposited normally by the ripe female they are capable, like those of other Annelids, of beginning maturation in the sea-water before fertilisation. If this is true it furnishes an explanation for the exceptional behaviour of an otherwise normal batch of eggs, which may have been on the point of being extruded by the female. The eggs which were used for the experiment were immersed in isotonic KCI pH 8.1. At intervals batches were removed. Some of each batch were placed in sea-water free from sperm and some were placed in another dish with sea-water and inseminated immediately. The results are shown graphically on Fig. 2. It will be seen that the potassium chloride had an immediate inhibitory effect on both the spontaneous maturation and on the capacity for fertilisation of the eggs. Two minutes’ exposure caused the proportion of maturation to fall from 50 per cent, to 14 per cent., a value which remained nearly constant even after 60 min. treatment with KCI. It may be presumed that this value represents the percentage of maturation already attained at the beginning of the experiment. The graph representing fertilisation capacity shows a sudden fall from 100 per cent, to about 80 per cent, after 2 min. exposure to the KCI. This value is maintained until after 8 min. exposure when the graph descends again to a value of 2.5 per cent, after 60 min. exposure.
The reaction of the eggs to treatment with isotonic calcium chloride is in every respect different from that described for the alkali-metal chlorides. The eggs become spherical almost at once and the fertilisation membrane begins to separate in a few minutes. After about half an hour the nuclear membrane begins to disappear. Polar bodies are formed by many eggs and probably by all which are capable of undergoing cleavage. The time at which cleavage takes place is very variable but it does not usually begin until two or three hours after activation. The period before cleavage is characterised by a considerable degree of pseudo-amoeboid activity on the part of the eggs such as has been described by various authors (e.g. F. R. Lillie, 1906) for the eggs of other animals. The cytological details of the events culminating in cleavage have yet to be worked out by means of preserved material. A few series of sections have been cut of eggs activated by means of calcium chloride and fixed in corrosive-acetic. These indicate that the period of pseudo-amoeboid activity begins when the nuclear wall is breaking down, preceding somewhat the development of asters, but the movements of the cytoplasm continue until shortly before cleavage. Excessive treatment with calcium chloride frequently inhibits cleavage through the development of too many asters (cf. Herlant, 1918). Another type of abnormality due to over-treatment is the formation of a large monaster which completely fills the uncleaved egg or the blastomeres of one which has cleaved. Frequently uncleaved eggs are found to contain several nuclei in a resting condition similar to those described by Lefevre (1907) in the artificially activated eggs of Thalassema mellita which this author suggests arise as the result of multipolar mitoses. Differentiation without cleavage has not hitherto been observed. In this respect and in the various types of abnormal behaviour of artificially activated eggs Thalassema neptuni shows a remarkably close resemblance to Thalassema mellita as described by Lefevre.
A remarkable and characteristic feature of these experiments is well shown by Table I and Fig. 1. When the eggs are treated with the activating solution for various periods of time it is found that there are two optimal times of exposure. As will be shown later, this is true not only when the activating solution is composed of pure calcium chloride but also when a mixture of two salts is used. The first maximum usually occurs at about 9 min. exposure and the second at about 30 min. or more. Discussion of this matter is reserved for a later part of this paper but it may be pointed out here that the first optimal time for cleavage as a rule slightly precedes that for activation. The second optimal time appears to be the same for both cleavage and activation but this may be due to the longer intervals in this part of the experiment.
Calcium chloride does not inhibit rapidly the capacity for fertilisation of the eggs.
Table II shows the results of an experiment made to investigate this point. The procedure was similar to that already described for the experiment demonstrating the inhibitory action of potassium chloride.
Inspection of the figures in the above table shows that the percentage of cleavage in the eggs inseminated after the treatment with calcium chloride exceeded that of the uninseminated eggs. Hence a considerable proportion of the eggs which would not otherwise undergo cleavage are capable of being fertilised and dividing. In other words, the period during which fertilisation is possible does not end when maturation begins.
THE ACTION OF ISOTONIC SOLUTIONS CONTAINING TWO SALTS
If the activating solutions contain a mixture of two salts the results are more complex than those described above. The nature of the salts and their relative concentrations are of great importance in determining the proportion of activated eggs. In general a higher percentage of cleavage is obtained as the result of treatment with the most favourable binary mixtures than with calcium chloride alone. The cleavage is also, as a rule, more normal and the embryos are consequently more healthy. The morphology of development appears, however, to be closely similar although this has yet to be confirmed in detail. Polar bodies are certainly formed in most cases. The various types of abnormalities obtained resemble those found as the result of treatment with pure calcium chloride.
The activating power of mixtures of calcium chloride with the chloride of an alkali-metal shows a remarkable variation corresponding with the relative concentrations of the two constituents. Table III, and Fig. 3, illustrate the results obtained by submitting eggs for six minutes to the action of a series of mixtures of potassium and calcium chlorides. For the sake of completeness the values for pure calcium chloride and pure potassium chloride are given in the table, and are inserted in the graph joined by broken lines although, strictly, the scale used forbids their inclusion. The results obtained in different experiments vary considerably but certain general features can be distinguished. With series of potassium-calcium mixtures ten experiments have been made. In two of these little or no development took place. In one other development occurred as the result of treatment only with mixtures in which the K/Ca ratio lay between 0.5 and 32. The remaining seven experiments gave results similar to those illustrated.
Pure calcium chloride is effective in inducing cleavage but its action is greatly inhibited by the addition of a small amount of potassium. The graph then rises to a maximum and falls sharply when the K/Ca ratio reaches the value of 1.0. As the K/Ca ratio rises still further the proportion of cleavage again reaches a maximum before decreasing again to a low value as the mixture approximates more and more closely to pure potassium chloride.
The relative ineffectiveness of mixtures in which the concentrations of calcium and of potassium ions is approximately equal is characteristic. This point is well marked in the experiment quoted. That the fall in the middle of the curve is significant can be seen by comparing the value for cleavage as the result of treatment with the mixture in which K/Ca = 1.0 with the two adjacent figures. In one case (K/Ca = 2.0) the difference is over four times the standard error of difference; in the other (K/Ca = 0.5) the difference is over seven times the standard error of difference. The position of this dip between the two principal maxima of the curve is not constant but it remains approximately central.
The graph showing the total activation is in most respects similar to that just described for cleavage. Two points may however be mentioned. There is a tendency, especially for that part of the series in which the calcium concentration is high, for the maxima of the cleavage graph to fail to coincide with the maxima of activation.
This may perhaps be compared with the somewhat similar phenomenon described for eggs treated with pure calcium chloride, although in one case the varying factor is time of exposure and in the other the proportion of potassium. The second point to be noticed is that high concentrations of calcium generally cause more activation in proportion to the cleavage induced than high concentrations of potassium. Scott (1906) concluded that in Amphitrite calcium tends to stimulate nuclear division and inhibit cleavage while potassium tends to promote cleavage.
Few experiments of this type have as yet been made with sodium and lithium. Those which have been performed indicate that sodium and lithium are less effective than potassium in promoting activation in the presence of small concentrations of calcium. The graph thus tends to show a single maximum in the region where the alkali-metal/Ca ratio is low. Lithium seems to resemble potassium more closely than does sodium.
The time factor is of great importance in determining the proportion of eggs activated by exposure to mixtures of salts. The time curve has a form closely resembling that already described for pure calcium chloride. This can be seen by inspection of Table IV and Fig. 4. Table IV gives the results of an experiment undertaken to compare the effects of potassium, lithium and sodium-calcium mixtures in which the alkali-metal/Ca ratio was high.
Table IV also illustrates the fact mentioned above that sodium and lithium are not effective activating agents when their concentration is high compared with that of calcium. In this example however the difference is more striking than is often the case. Fig. 4 shows the time curve obtained in another experiment in which a lithium-calcium mixture (Li/Ca = 0-125) was used. The similarity between this curve and the curve shown in Fig. 1 is apparent, the only difference being in detail.
DISCUSSION
Although a very large number of methods have been described for causing artificial parthenogenesis and in many cases solutions of various electrolytes have been employed, yet the action of isotonic salt solutions has received little attention. Hypertonic salt solutions have long been known to induce development of the unfertilised eggs of a considerable variety of animals.
The papers dealing with this method are too numerous and well known to be dealt with here. A comparatively small number of observations has been made on the action of salt solutions in the absence of any considerable changes in osmotic pressure. Most of these experiments, however, consisted in subjecting eggs to the action of sea-water to which had been added varying quantities of salt solutions. It is evident from the experiments described in the earlier part of this paper that the relation between the eggs and the surrounding medium may be complex even in binary salt solutions. It is, therefore, evident that the results will be very difficult to interpret if the experiment consists of the addition of salts to sea-water which is already a complex and delicately balanced mixture. Slight alterations in the composition of these salt-solution-sea-water mixtures may be expected to show great differences in effect and when the additional variable presented by the eggs of various species of animals is considered it is not difficult to see that marked qualitative differences may appear. It might be expected that the observed differences in the effect of a certain salt on the eggs of various species of animals would be quantitative rather than qualitative. Apparently qualitative differences may, however, be essentially quantitative or may be indirectly the result of quantitative alterations. The investigation of the activating effect of salt solutions on unfertilised eggs must, therefore, be based on a thorough examination of the action of single salts in isotonic solution and then of binary mixtures of these salts before the more complex conditions in mixtures of three or more salts can be understood. The work of R. S. Lillie (1910, 1911a, 1911 b) is, to the writer’s knowledge, the only systematic attempt which has been made to examine the effects of isotonic salt solutions in causing artificial parthenogenesis. He, using the eggs of Arbacia punctulata and Asterias forbesii, found that isotonic solutions of sodium and potassium salts would initiate development. Lithium chloride behaved like sodium chloride. Pure magnesium and calcium chlorides would not cause development and inhibited the action of sodium and potassium salts when mixed with them. Pure strontium chloride also failed to cause parthenogenesis. Lillie’s interpretation of these results was that sodium and potassium salts caused increased permeability while the opposite effect was produced by the alkali-earth salts. This view was supported by the diffusion of pigment from the eggs of Arbacia treated with pure sodium and potassium salts and the prevention of this process by calcium and magnesium salts. His experiments are, however, open to the objections that the hydrogen ion concentration was uncontrolled and that he employed only a limited number of mixtures of sodium or potassium with calcium or magnesium. The present writer has, moreover, shown that calcium chloride alone may be very effective in causing artificial parthenogenesis of the eggs of Asterias rubens (1927).
The eggs of Annelids seem to be more susceptible to the action of salt solutions than those of Echinoderms. Mead (1896) was the first to show that the eggs of Amphitrite could be induced to form polar bodies by the action of sea-water to which a small quantity of potassium chloride had been added. Loeb (1901) found that potassium had a specific action on the eggs of Chaetopterus; whereas other salts would cause development when added to sea-water if thereby the mixture was made hypertonic, potassium chloride was effective in the absence of any increase in the total salt concentration. Potassium chloride added to sea-water, usually in hypertonic solution, has also been employed as a means of causing artificial parthenogenesis by various other investigators such as Treadwell (1902) working with Podarke, Fischer (1903) with Nereis, Bullot (1904) with Ophelia, F. R. Lillie (1906) and Allyn (1913) with Chaetopterus and Scott (1906) with Amphitrite. Experiments have less frequently been made with calcium salts but it is noteworthy that Fischer (1902) and Scott (1906) both found that addition of normal (i.e. approximately isotonic) calcium nitrate to sea-water would cause development of the unfertilised eggs of Amphitrite. Lefevre (1907) did not obtain satisfactory results by the action of salt solutions on the eggs of Thalassema mellita.
The work of Hörstadius (1923) is also of interest. He found that the eggs of Pomatoceros normally begin maturation when they are extruded into the sea-water although the process is not completed unless fertilisation takes place. Maturation is inhibited in calcium-free sea-water and is promoted in the presence of excess calcium. The effect of potassium is opposed to that of calcium. The percentage of maturing eggs varies inversely as the concentration of potassium.
The account given above of the previous work on the action of salts on unfertilised eggs does not pretend to be exhaustive but merely indicates the main results obtained by investigators in the past. It is evident from their results that under certain conditions both potassium and calcium salts can activate unfertilised eggs, or it might be wiser to say that the addition of potassium or of calcium salts in certain amounts to sea-water may produce a mixture capable of inducing artificial parthenogenesis. Activation may depend in certain cases on the absolute concentration of a particular ion or in other cases on the relative concentrations of two or more ions to each other. Unfortunately the evidence provided by previous accounts, with a few exceptions, is too limited and unsystematic for any definite conclusions to be drawn.
Cleavage in Thalassema neptuni is directly continuous with maturation. The difference between a stimulus causing maturation only and one which causes cleavage seems to be quantitative. A similar conclusion has been reached by Allyn (1913) for Chaetopterus. Within the limits of the experiments described in the present paper the eggs could not be activated in the absence of calcium. This supports the conclusion of Hörstadius (1923) that calcium favours and is necessary for maturation. The presence of certain concentrations of an alkali metal increases the proportion of both maturation and cleavage, showing that calcium is not the only factor concerned. The discovery of two markedly separate optimum potassium-calcium mixtures suggests however that the change resulting in activation may be accomplished in different ways. If, as R. S. Lillie considers, the essential change involved in the activation of an egg is an increase in permeability to water and salts, it is evident that this is a more complex problem than has hitherto been supposed. It would not be difficult to construct a hypothesis whereby could be explained the action of excess calcium in increasing the permeability to water of the surface membrane of the egg but it would be unprofitable to do so until more data are available.
The rapid attainment of a spherical shape by the eggs of Thalassema neptuni when they are fertilised is of interest as it is probably an optical demonstration of an increase in the permeability to water of the surface membrane of the egg. This change occurs almost as rapidly after artificial activation as after fertilisation. It is conceivable, of course, that a true explanation is to be found in some other change in the physical properties of the thick vitelline membrane. Nevertheless, in view of R. S. Lillie’s (1916) experiments in which an increased permeability to water was shown to succeed natural or artificial activation in the egg of Arbacia, it seems highly probable that the interpretation suggested above is correct.
It is noteworthy that cytolysis does not take place rapidly as the result of treatment with isotonic solutions of sodium or potassium chlorides. Only after several hours is any considerable amount of cytolysis to be seen even if the eggs are allowed to remain in the solutions. This seems to show that the permeability of the egg membrane to salts is not markedly increased by this treatment.
The relation between the time of exposure of the eggs to the salt solution and the degree of activation induced presents some points of interest. An important factor is the number of accessory asters formed. Eggs treated for long with calcium chloride always develop several accessory asters and Herlant (1918) has shown that this condition leads to suppression of cleavage. It is significant that the optimum time of exposure tends to precede that for activation, in view of the similar relationship found by Herlant between cleavage and “polycentrie” in Paracentrotus.
SUMMARY
The eggs of Thalassema neptuni are stored in the nephridia and, as the result of their compression by the muscular walls of these organs, are distorted in shape. This distorted shape is maintained until maturation begins. Activation, whether normal or artificial, is accompanied by the adoption of a spherical shape by the egg. This change probably indicates an increase in the permeability of the egg membrane to water and forms a useful index of activation.
Artificial parthenogenesis of the eggs of Thalassema neptuni can be induced by means of isotonic salt solutions.
At the hydrogen ion concentration of sea-water the chlorides of sodium, lithium, and potassium are incapable of causing development of the unfertilised eggs, but calcium chloride is an efficient activating agent.
Mixtures of calcium chloride with the chloride of an alkali-metal in certain proportions cause parthenogenesis.
For series of potassium-calcium mixtures two maxima for activation are obtained, one where the calcium concentration is high and one where it is low. Sodium and lithium in presence of low concentrations of calcium are much less effective than potassium.
For all the activating solutions tested two optimal times of exposure are found at about 6–9 min. and 30 min.
ACKNOWLEDGEMENTS
I wish to thank the Royal Society and the British Association for the use of tables at the Marine Laboratory, Plymouth. I am indebted to the staff of the Laboratory for their unfailing courtesy and help. I am especially grateful to Professor J. H. Ashworth, F.R.S., and to Dr E. J. Allen, F.R.S., for their helpful interest in the work.