THE EFFECTS OF SALTS OF AMMONIA.
Manner of performing the experiments--Action of distilled water in comparison with the solutions--Carbonate of ammonia, absorbed by the roots--The vapour absorbed by the glands--Drops on the disc--Minute drops applied to separate glands--Leaves immersed in weak solutions--Minuteness of the doses which induce aggregation of the protoplasm--Nitrate of ammonia, analogous experiments with--Phosphate of ammonia, analogous experiments with--Other salts of ammonia--Summary and concluding remarks on the action of salts of ammonia.
THE chief object in this chapter is to show how powerfully the salts of ammonia act on the leaves of Drosera, and more especially to show what an extraordinarily small quantity suffices to excite inflection. I shall, therefore, be compelled to enter into full details. Doubly distilled water was always used; and for the more delicate experiments, water which had been prepared with the utmost possible care was given me by Professor Frankland. The graduated measures were tested, and found as accurate as such measures can be. The salts were carefully weighed, and in all the more delicate experiments, by Borda's double method. But extreme accuracy would have been superfluous, as the leaves differ greatly in irritability, according to age, condition, and constitution. Even the tentacles on the same leaf differ in irritability to a marked degree. My experiments were tried in the following several ways.
[Firstly.--Drops which were ascertained by repeated trials to be on an average about half a minim, or the 1/960 of a fluid ounce (.0296 ml.), were placed by the same pointed instrument on the discs of the leaves, and the inflection of the exterior rows of tentacles observed at successive intervals of time. It was first ascertained, from between thirty and forty trials, that distilled water dropped in this manner produces no effect, except that sometimes, though rarely, two or three tentacles become inflected. In fact all the many trials with solutions which were so weak as to produce no effect lead to the same result that water is inefficient.
Secondly.--The head of a small pin, fixed into a handle, was dipped into the solution under trial. The small drop which adhered to it, and which was much too small to fall off, was cautiously placed, by the aid of a lens, in contact with the secretion surrounding the glands of one, two, three, or four of the exterior tentacles of the same leaf. Great care was taken that the glands themselves should not be touched. I had supposed that the drops were of nearly the same size; but on trial this proved a great mistake. I first measured some water, and removed 300 drops, touching the pin's head each time on blotting-paper; and on again measuring the water, a drop was found to equal on an average about the 1/60 of a minim. Some water in a small vessel was weighed (and this is a more accurate method), and 300 drops removed as before; and on again weighing the water, a drop was found to equal on an average only the 1/89 of a minim. I repeated the operation, but endeavoured this time, by taking the pin's head out of the water obliquely and rather quickly, to remove as large drops as possible; and the result showed that I had succeeded, for each drop on an average equalled 1/19.4 of a minim. I repeated the operation in exactly the same manner, and now the drops averaged 1/23.5 of a minim. Bearing in mind that on these two latter occasions special pains were taken to remove as large drops as possible, we may safely conclude that the drops used in my experiments were at least equal to the 1/20 of a minim, or .0029 ml. One of these drops could be applied to three or even four glands, and if the tentacles became inflected, some of the solution must have been absorbed by all; for drops of pure water, applied in the same manner, never produced any effect. I was able to hold the drop in steady contact with the secretion only for ten to fifteen seconds; and this was not time enough for the diffusion of all the salt in solution, as was evident, from three or four tentacles treated successively with the same drop, often becoming inflected. All the matter in solution was even then probably not exhausted.
Thirdly.--Leaves cut off and immersed in a measured quantity of the solution under trial; the same number of leaves being immersed at the same time, in the same quantity of the distilled water which had been used in making the solution. The leaves in the two lots were compared at short intervals of time, up to 24 hrs., and sometimes to 48 hrs. They were immersed by being laid as gently as possible in numbered watch-glasses, and thirty minims (1.775 ml.) of the solution or of water was poured over each.
Some solutions, for instance that of carbonate of ammonia, quickly discolour the glands; and as all on the same leaf were discoloured simultaneously, they must all have absorbed some of the salt within the same short period of time. This was likewise shown by the simultaneous inflection of the several exterior rows of tentacles. If we had no such evidence as this, it might have been supposed that only the glands of the exterior and inflected tentacles had absorbed the salt; or that only those on the disc had absorbed it, and had then transmitted a motor impulse to the exterior tentacles; but in this latter case the exterior tentacles would not have become inflected until some time had elapsed, instead of within half an hour, or even within a few minutes, as usually occurred. All the glands on the same leaf are of nearly the same size, as may best be seen by cutting off a narrow transverse strip, and laying it on its side; hence their absorbing surfaces are nearly equal. The long-headed glands on the extreme margin must be excepted, as they are much longer than the others; but only the upper surface is capable of absorption. Besides the glands, both surfaces of the leaves and the pedicels of the tentacles bear numerous minute papillae, which absorb carbonate of ammonia, an infusion of raw meat, metallic salts, and probably many other substances, but the absorption of matter by these papillae never induces inflection. We must remember that the movement of each separate tentacle depends on its gland being excited, except when a motor impulse is transmitted from the glands of the disc, and then the movement, as just stated, does not take place until some little time has elapsed. I have made these remarks because they show us that when a leaf is immersed in a solution, and the tentacles are inflected, we can judge with some accuracy how much of the salt each gland has absorbed. For instance, if a leaf bearing 212 glands be immersed in a measured quantity of a solution, containing 1/10 of a grain of a salt, and all the exterior tentacles, except twelve, are inflected, we may feel sure that each of the 200 glands can on an average have absorbed at most 1/2000 of a grain of the salt. I say at most, for the papillae will have absorbed some small amount, and so will perhaps the glands of the twelve excluded tentacles which did not become inflected. The application of this principle leads to remarkable conclusions with respect to the minuteness of the doses causing inflection.
On the Action of Distilled Water in Causing Inflection.
Although in all the more important experiments the difference between the leaves simultaneously immersed in water and in the several solutions will be described, nevertheless it may be well here to give a summary of the effects of water. The fact, moreover, of pure water acting on the glands deserves in itself some notice. Leaves to the number of 141 were immersed in water at the same time with those in the solutions, and their state recorded at short intervals of time. Thirty-two other leaves were separately observed in water, making altogether 173 experiments. Many scores of leaves were also immersed in water at other times, but no exact record of the effects produced was kept; yet these cursory observations support the conclusions arrived at in this chapter. A few of the long-headed tentacles, namely from one to about six, were commonly inflected within half an hour after immersion; as were occasionally a few, and rarely a considerable number of the exterior round-headed tentacles. After an immersion of from 5 to 8 hrs. the short tentacles surrounding the outer parts of the disc generally become inflected, so that their glands form a small dark ring on the disc; the exterior tentacles not partaking of this movement. Hence, excepting in a few cases hereafter to be specified, we can judge whether a solution produces any effect only by observing the exterior tentacles within the first 3 or 4 hrs. after immersion.
Now for a summary of the state of the 173 leaves after an immersion of 3 or 4 hrs. in pure water. One leaf had almost all its tentacles inflected; three leaves had most of them sub-inflected; and thirteen had on an average 36.5 tentacles inflected. Thus seventeen leaves out of the 173 were acted on in a marked manner. Eighteen leaves had from seven to nineteen tentacles inflected, the average being 9.3 tentacles for each leaf. Forty-four leaves had from one to six tentacles inflected, generally the long-headed ones. So that altogether of the 173 leaves carefully observed, seventy-nine were affected by the water in some degree, though commonly to a very slight degree; and ninety-four were not affected in the least degree. This amount of inflection is utterly insignificant, as we shall hereafter see, compared with that caused by very weak solutions of several salts of ammonia.
Plants which have lived for some time in a rather high temperature are far more sensitive to the action of water than those grown out of doors, or recently brought into a warm greenhouse. Thus in the above seventeen cases, in which the immersed leaves had a considerable number of tentacles inflected, the plants had been kept during the winter in a very warm greenhouse; and they bore in the early spring remarkably fine leaves, of a light red colour. Had I then known that the sensitiveness of plants was thus increased, perhaps I should not have used the leaves for my experiments with the very weak solutions of phosphate of ammonia; but my experiments are not thus vitiated, as I invariably used leaves from the same plants for simultaneous immersion in water. It often happened that some leaves on the same plant, and some tentacles on the same leaf, were more sensitive than others; but why this should be so, I do not know.
FIG. 9. (Drosera rotundifolia.) Leaf (enlarged) with all the tentacles closely inflected, from immersion in a solution of phosphate of ammonia (one part to 87,500 of water.)
Besides the differences just indicated between the leaves immersed in water and in weak solutions of ammonia, the tentacles of the latter are in most cases much more closely inflected. The appearance of a leaf after immersion in a few drops of a solution of 1 grain of phosphate of ammonia to 200 oz. of water (i.e. one part to 87,500) is here reproduced: such energetic inflection is never caused by water alone. With leaves in the weak solutions, the blade or lamina often becomes inflected; and this is so rare a circumstance with leaves in water that I have seen only two instances; and in both of these the inflection was very feeble. Again, with leaves in the weak solutions, the inflection of the tentacles and blade often goes on steadily, though slowly, increasing during many hours; and this again is so rare a circumstance with leaves in water that I have seen only three instances of any such increase after the first 8 to 12 hrs.; and in these three instances the two outer rows of tentacles were not at all affected. Hence there is sometimes a much greater difference between the leaves in water and in the weak solutions, after from 8 hrs. to 24 hrs., than there was within the first 3 hrs.; though as a general rule it is best to trust to the difference observed within the shorter time.
With respect to the period of the re-expansion of the leaves, when left immersed either in water or in the weak solutions, nothing could be more variable. In both cases the exterior tentacles not rarely begin to re-expand, after an interval of only from 6 to 8 hrs.; that is just about the time when the short tentacles round the borders of the disc become inflected. On the other hand, the tentacles sometimes remain inflected for a whole day, or even two days; but as a general rule they remain inflected for a longer period in very weak solutions than in water. In solutions which are not extremely weak, they never re-expand within nearly so short a period as six or eight hours. From these statements it might be thought difficult to distinguish between the effects of water and the weaker solutions; but in truth there is not the slightest difficulty until excessively weak solutions are tried; and then the distinction, as might be expected, becomes very doubtful, and at last disappears. But as in all, except the simplest, cases the state of the leaves simultaneously immersed for an equal length of time in water and in the solutions will be described, the reader can judge for himself.]
CARBONATE OF AMMONIA.
This salt, when absorbed by the roots, does not cause the tentacles to be inflected. A plant was so placed in a solution of one part of the carbonate to 146 of water that the young uninjured roots could be observed. The terminal cells, which were of a pink colour, instantly became colourless, and their limpid contents cloudy, like a mezzo-tinto engraving, so that some degree of aggregation was almost instantly caused; but no further change ensued, and the absorbent hairs were not visibly affected. The tentacles did not bend. Two other plants were placed with their roots surrounded by damp moss, in half an ounce (14.198 ml.) of a solution of one part of the carbonate to 218 of water, and were observed for 24 hrs.; but not a single tentacle was inflected. In order to produce this effect, the carbonate must be absorbed by the glands.
The vapour produces a powerful effect on the glands, and induces inflection. Three plants with their roots in bottles, so that the surrounding air could not have become very humid, were placed under a bell-glass (holding 122 fluid ounces), together with 4 grains of carbonate of ammonia in a watch-glass. After an interval of 6 hrs. 15 m. the leaves appeared unaffected; but next morning, after 20 hrs., the blackened glands were secreting copiously, and most of the tentacles were strongly inflected. These plants soon died. Two other plants were placed under the same bell-glass, together with half a grain of the carbonate, the air being rendered as damp as possible; and in 2 hrs. most of the leaves were affected, many of the glands being blackened and the tentacles inflected. But it is a curious fact that some of the closely adjoining tentacles on the same leaf, both on the disc and round the margins, were much, and some, apparently, not in the least affected. The plants were kept under the bell-glass for 24 hrs., but no further change ensued. One healthy leaf was hardly at all affected, though other leaves on the same plant were much affected. On some leaves all the tentacles on one side, but not those on the opposite side, were inflected. I doubt whether this extremely unequal action can be explained by supposing that the more active glands absorb all the vapour as quickly as it is generated, so that none is left for the others, for we shall meet with analogous cases with air thoroughly permeated with the vapours of chloroform and ether.
Minute particles of the carbonate were added to the secretion surrounding several glands. These instantly became black and secreted copiously; but, except in two instances, when extremely minute particles were given, there was no inflection. This result is analogous to that which follows from the immersion of leaves in a strong solution of one part of the carbonate to 109, or 146, or even 218 of water, for the leaves are then paralysed and no inflection ensues, though the glands are blackened, and the protoplasm in the cells of the tentacles undergoes strong aggregation.
[We will now turn to the effects of solutions of the carbonate. Half-minims of a solution of one part to 437 of water were placed on the discs of twelve leaves; so that each received 1/960 of a grain or .0675 mg. Ten of these had their tentacles well inflected; the blades of some being also much curved inwards. In two cases several of the exterior tentacles were inflected in 35 m.; but the movement was generally slower. These ten leaves re-expanded in periods varying between 21 hrs. and 45 hrs., but in one case not until 67 hrs. had elapsed; so that they re-expanded much more quickly than leaves which have caught insects.
The same-sized drops of a solution of one part to 875 of water were placed on the discs of eleven leaves; six remained quite unaffected, whilst five had from three to six or eight of their exterior tentacles inflected; but this degree of movement can hardly be considered as trustworthy. Each of these leaves received 1/1920 of a grain (.0337 mg.), distributed between the glands of the disc, but this was too small an amount to produce any decided effect on the exterior tentacles, the glands of which had not themselves received any of the salt.
Minute drops on the head of a small pin, of a solution of one part of the carbonate to 218 of water, were next tried in the manner above described. A drop of this kind equals on an average 1/20 of a minim, and therefore contains 1/4800 of a grain (.0135 mg.) of the carbonate. I touched with it the viscid secretion round three glands, so that each gland received only 1/14400 of a grain (.00445 mg.). Nevertheless, in two trials all the glands were plainly blackened; in one case all three tentacles were well inflected after an interval of 2 hrs. 40 m.; and in another case two of the three tentacles were inflected. I then tried drops of a weaker solution of one part to 292 of water on twenty-four glands, always touching the viscid secretion round three glands with the same little drop. Each gland thus received only the 1/19200 of a grain (.00337 mg.), yet some of them were a little darkened; but in no one instance were any of the tentacles inflected, though they were watched for 12 hrs. When a still weaker solution (viz. one part to 437 of water) was tried on six glands, no effect whatever was perceptible. We thus learn that the 1/14400 of a grain (.00445 mg.) of carbonate of ammonia, if absorbed by a gland, suffices to induce inflection in the basal part of the same tentacle; but as already stated, I was able to hold with a steady hand the minute drops in contact with the secretion only for a few seconds; and if more time had been allowed for diffusion and absorption, a much weaker solution would certainly have acted.
Some experiments were made by immersing cut-off leaves in solutions of different strengths. Thus four leaves were left for about 3 hrs. each in a drachm (3.549 ml.) of a solution of one part of the carbonate to 5250 of water; two of these had almost every tentacle inflected, the third had about half the tentacles and the fourth about one-third inflected; and all the glands were blackened. Another leaf was placed in the same quantity of a solution of one part to 7000 of water, and in 1 hr. 16 m. every single tentacle was well inflected, and all the glands blackened. Six leaves were immersed, each in thirty minims (1.774 ml.) of a solution of one part to 4375 of water, and the glands were all blackened in 31 m. All six leaves exhibited some slight inflection, and one was strongly inflected. Four leaves were then immersed in thirty minims of a solution of one part to 8750 of water, so that each leaf received the 1/320 of a grain (.2025 mg.). Only one became strongly inflected; but all the glands on all the leaves were of so dark a red after one hour as almost to deserve to be called black, whereas this did not occur with the leaves which were at the same time immersed in water; nor did water produce this effect on any other occasion in nearly so short a time as an hour. These cases of the simultaneous darkening or blackening of the glands from the action of weak solutions are important, as they show that all the glands absorbed the carbonate within the same time, which fact indeed there was not the least reason to doubt. So again, whenever all the tentacles become inflected within the same time, we have evidence, as before remarked, of simultaneous absorption. I did not count the number of glands on these four leaves; but as they were fine ones, and as we know that the average number of glands on thirty-one leaves was 192, we may safely assume that each bore on an average at least 170; and if so, each blackened gland could have absorbed only 1/54400 of a grain (.00119 mg.) of the carbonate.
A large number of trials had been previously made with solutions of one part of the nitrate and phosphate of ammonia to 43750 of water (i.e. one grain to 100 ounces), and these were found highly efficient. Fourteen leaves were therefore placed, each in thirty minims of a solution of one part of the carbonate to the above quantity of water; so that each leaf received 1/1600 of a grain (.0405 mg.). The glands were not much darkened. Ten of the leaves were not affected, or only very slightly so. Four, however, were strongly affected; the first having all the tentacles, except forty, inflected in 47 m.; in 6 hrs. 30 m. all except eight; and after 4 hrs. the blade itself. The second leaf after 9 m. had all its tentacles except nine inflected; after 6 hrs. 30 m. these nine were sub-inflected; the blade having become much inflected in 4 hrs. The third leaf after 1 hr. 6 m. had all but forty tentacles inflected. The fourth, after 2 hrs. 5 m., had about half its tentacles and after 4 hrs. all but forty-five inflected. Leaves which were immersed in water at the same time were not at all affected, with the exception of one; and this not until 8 hrs. had elapsed. Hence there can be no doubt that a highly sensitive leaf, if immersed in a solution, so that all the glands are enabled to absorb, is acted on by 1/1600 of a grain of the carbonate. Assuming that the leaf, which was a large one, and which had all its tentacles excepting eight inflected, bore 170 glands, each gland could have absorbed only 1/268800 of a grain (.00024 mg.); yet this sufficed to act on each of the 162 tentacles which were inflected. But as only four out of the above fourteen leaves were plainly affected, this is nearly the minimum dose which is efficient.
Aggregation of the Protoplasm from the Action of Carbonate of Ammonia.--I have fully described in the third chapter the remarkable effects of moderately strong doses of this salt in causing the aggregation of the protoplasm within the cells of the glands and tentacles; and here my object is merely to show what small doses suffice. A leaf was immersed in twenty minims (1.183 ml.) of a solution of one part to 1750 of water, and another leaf in the same quantity of a solution of one part to 3062; in the former case aggregation occurred in 4 m., in the latter in 11 m. A leaf was then immersed in twenty minims of a solution of one part to 4375 of water, so that it received 1/240 of a grain (.27 mg.); in 5 m. there was a slight change of colour in the glands, and in 15 m. small spheres of protoplasm were formed in the cells beneath the glands of all the tentacles. In these cases there could not be a shadow of a doubt about the action of the solution.
A solution was then made of one part to 5250 of water, and I experimented on fourteen leaves, but will give only a few of the cases. Eight young leaves were selected and examined with care, and they showed no trace of aggregation. Four of these were placed in a drachm (3.549 ml.) of distilled water; and four in a similar vessel, with a drachm of the solution. After a time the leaves were examined under a high power, being taken alternately from the solution and the water. The first leaf was taken out of the solution after an immersion of 2 hrs. 40 m., and the last leaf out of the water after 3 hrs. 50 m.; the examination lasting for 1 hr. 40 m. In the four leaves out of the water there was no trace of aggregation except in one specimen, in which a very few, extremely minute spheres of protoplasm were present beneath some of the round glands. All the glands were translucent and red. The four leaves which had been immersed in the solution, besides being inflected, presented a widely different appearance; for the contents of the cells of every single tentacle on all four leaves were conspicuously aggregated; the spheres and elongated masses of protoplasm in many cases extending halfway down the tentacles. All the glands, both those of the central and exterior tentacles, were opaque and blackened; and this shows that all had absorbed some of the carbonate. These four leaves were of very nearly the same size, and the glands were counted on one and found to be 167. This being the case, and the four leaves having been immersed in a drachm of the solution, each gland could have received on an average only 1/64128 of a grain (.001009 mg.) of the salt; and this quantity sufficed to induce within a short time conspicuous aggregation in the cells beneath all the glands.
A vigorous but rather small red leaf was placed in six minims of the same solution (viz. one part to 5250 of water), so that it received 1/960 of a grain (.0675 mg.). In 40 m. the glands appeared rather darker; and in 1 hr. from four to six spheres of protoplasm were formed in the cells beneath the glands of all the tentacles. I did not count the tentacles, but we may safely assume that there were at least 140; and if so, each gland could have received only the 1/134400 of a grain, or .00048 mg.
A weaker solution was then made of one part to 7000 of water, and four leaves were immersed in it; but I will give only one case. A leaf was placed in ten minims of this solution; after 1 hr. 37 m. the glands became somewhat darker, and the cells beneath all of them now contained many spheres of aggregated protoplasm. This leaf received 1/768 of a grain, and bore 166 glands. Each gland could, therefore, have received only 1/127488 of a grain (.00507 mg.) of the carbonate.
Two other experiments are worth giving. A leaf was immersed for 4 hrs. 15 m. in distilled water, and there was no aggregation; it was then placed for 1 hr. 15 m. in a little solution of one part to 5250 of water; and this excited well-marked aggregation and inflection. Another leaf, after having been immersed for 21 hrs. 15 m. in distilled water, had its glands blackened, but there was no aggregation in the cells beneath them; it was then left in six minims of the same solution, and in 1 hr. there was much aggregation in many of the tentacles; in 2 hrs. all the tentacles (146 in number) were affected--the aggregation extending down for a length equal to half or the whole of the glands. It is extremely improbable that these two leaves would have undergone aggregation if they had been left for a little longer in the water, namely for 1 hr. and 1 hr. 15 m., during which time they were immersed in the solution; for the process of aggregation seems invariably to supervene slowly and very gradually in water.]
Summary of the Results with Carbonate of Ammonia.--The roots absorb the solution, as shown by their changed colour, and by the aggregation of the contents of their cells. The vapour is absorbed by the glands; these are blackened, and the tentacles are inflected. The glands of the disc, when excited by a half-minim drop (.0296 ml.), containing 1/960 of a grain (.0675 mg.), transmit a motor impulse to the exterior tentacles, causing them to bend inwards. A minute drop, containing 1/14400 of a grain (.00445 mg.), if held for a few seconds in contact with a gland, soon causes the tentacle bearing it to be inflected. If a leaf is left immersed for a few hours in a solution, and a gland absorbs the 1/134400 of a grain (.0048 mg.), its colour becomes darker, though not actually black; and the contents of the cells beneath the gland are plainly aggregated. Lastly, under the same circumstances, the absorption by a gland of the 1/268800 of a grain (.00024 mg.) suffices to excite the tentacle bearing this gland into movement.
[NITRATE OF AMMONIA.
With the salt I attended only to the inflection of the leaves, for it is far less efficient than the carbonate in causing aggregation, although considerably more potent in causing inflection. I experimented with half-minims (.0296 ml.) on the discs of fifty-two leaves, but will give only a few cases. A solution of one part to 109 of water was too strong, causing little inflection, and after 24 hrs. killing, or nearly killing, four out of six leaves which were thus tried; each of which received the 1/240 of a grain (or .27 mg.). A solution of one part to 218 of water acted most energetically, causing not only the tentacles of all the leaves, but the blades of some, to be strongly inflected. Fourteen leaves were tried with drops of a solution of one part to 875 of water, so that the disc of each received the 1/1920 of a grain (.0337 mg.). Of these leaves, seven were very strongly acted on, the edges being generally inflected; two were moderately acted on; and five not at all. I subsequently tried three of these latter five leaves with urine, saliva, and mucus, but they were only slightly affected; and this proves that they were not in an active condition. I mention this fact to show how necessary it is to experiment on several leaves. Two of the leaves, which were well inflected, re-expanded after 51 hrs.
In the following experiment I happened to select very sensitive leaves. Half-minims of a solution of one part to 1094 of water (i.e. 1 gr. to 2 1/2 oz.) were placed on the discs of nine leaves, so that each received the 1/2400 of a grain (.027 mg.). Three of them had their tentacles strongly inflected and their blades curled inwards; five were slightly and somewhat doubtfully affected, having from three to eight of their exterior tentacles inflected: one leaf was not at all affected, yet was afterwards acted on by saliva. In six of these cases, a trace of action was perceptible in 7 hrs., but the full effect was not produced until from 24 hrs. to 30 hrs. had elapsed. Two of the leaves, which were only slightly inflected, re-expanded after an additional interval of 19 hrs.
Half-minims of a rather weaker solution, viz. of one part to 1312 of water (1 gr. to 3 oz.) were tried on fourteen leaves; so that each received 1/2880 of a grain (.0225 mg.), instead of, as in the last experiment, 1/2400 of a grain. The blade of one was plainly inflected, as were six of the exterior tentacles; the blade of a second was slightly, and two of the exterior tentacles well, inflected, all the other tentacles being curled in at right angles to the disc; three other leaves had from five to eight tentacles inflected; five others only two or three, and occasionally, though very rarely, drops of pure water cause this much action; the four remaining leaves were in no way affected, yet three of them, when subsequently tried with urine, became greatly inflected. In most of these cases a slight effect was perceptible in from 6 hrs. to 7 hrs., but the full effect was not produced until from 24 hrs. to 30 hrs. had elapsed. It is obvious that we have here reached very nearly the minimum amount, which, distributed between the glands of the disc, acts on the exterior tentacles; these having themselves not received any of the solution.
In the next place, the viscid secretion round three of the exterior glands was touched with the same little drop (1/20 of a minim) of a solution of one part to 437 of water; and after an interval of 2 hrs. 50 m. all three tentacles were well inflected. Each of these glands could have received only the 1/28800 of a grain, or .00225 mg. A little drop of the same size and strength was also applied to four other glands, and in 1 hr. two became inflected, whilst the other two never moved. We here see, as in the case of the half-minims placed on the discs, that the nitrate of ammonia is more potent in causing inflection than the carbonate; for minute drops of the latter salt of this strength produced no effect. I tried minute drops of a still weaker solution of the nitrate, viz. one part to 875 of water, on twenty-one glands, but no effect whatever was produced, except perhaps in one instance.
Sixty-three leaves were immersed in solutions of various strengths; other leaves being immersed at the same time in the same pure water used in making the solutions. The results are so remarkable, though less so than with phosphate of ammonia, that I must describe the experiments in detail, but I will give only a few. In speaking of the successive periods when inflection occurred, I always reckon from the time of first immersion.
Having made some preliminary trials as a guide, five leaves were placed in the same little vessel in thirty minims of a solution of one part of the nitrate to 7875 of water (1 gr. to 18 oz.); and this amount of fluid just sufficed to cover them. After 2 hrs. 10 m. three of the leaves were considerably inflected, and the other two moderately. The glands of all became of so dark a red as almost to deserve to be called black. After 8 hrs. four of the leaves had all their tentacles more or less inflected; whilst the fifth, which I then perceived to be an old leaf, had only thirty tentacles inflected. Next morning, after 23 hrs. 40 m., all the leaves were in the same state, excepting that the old leaf had a few more tentacles inflected. Five leaves which had been placed at the same time in water were observed at the same intervals of time; after 2 hrs. 10 m. two of them had four, one had seven, one had ten, of the long-headed marginal tentacles, and the fifth had four round-headed tentacles, inflected. After 8 hrs. there was no change in these leaves, and after 24 hrs. all the marginal tentacles had re-expanded; but in one leaf, a dozen, and in a second leaf, half a dozen, submarginal tentacles had become inflected. As the glands of the five leaves in the solution were simultaneously darkened, no doubt they had all absorbed a nearly equal amount of the salt: and as 1/288 of a grain was given to the five leaves together, each got 1/1440 of a grain (.045 mg.). I did not count the tentacles on these leaves, which were moderately fine ones, but as the average number on thirty-one leaves was 192, it would be safe to assume that each bore on an average at least 160. If so, each of the darkened glands could have received only 1/230400 of a grain of the nitrate; and this caused the inflection of a great majority of the tentacles.
This plan of immersing several leaves in the same vessel is a bad one, as it is impossible to feel sure that the more vigorous leaves do not rob the weaker ones of their share of the salt. The glands, moreover, must often touch one another or the sides of the vessel, and movement may have been thus excited; but the corresponding leaves in water, which were little inflected, though rather more so than commonly occurs, were exposed in an almost equal degree to these same sources of error. I will, therefore, give only one other experiment made in this manner, though many were tried and all confirmed the foregoing and following results. Four leaves were placed in forty minims of a solution of one part to 10,500 of water; and assuming that they absorbed equally, each leaf received 1/1152 of a grain (.0562 mg.). After 1 hr. 20 m. many of the tentacles on all four leaves were somewhat inflected. After 5 hrs. 30 m. two leaves had all their tentacles inflected; a third leaf all except the extreme marginals, which seemed old and torpid; and the fourth a large number. After 21 hrs. every single tentacle, on all four leaves, was closely inflected. Of the four leaves placed at the same time in water, one had, after 5 hrs. 45 m., five marginal tentacles inflected; a second, ten; a third, nine marginals and submarginals; and the fourth, twelve, chiefly submarginals, inflected. After 21 hrs. all these marginal tentacles re-expanded, but a few of the submarginals on two of the leaves remained slightly curved inwards. The contrast was wonderfully great between these four leaves in water and those in the solution, the latter having every one of their tentacles closely inflected. Making the moderate assumption that each of these leaves bore 160 tentacles, each gland could have absorbed only 1/184320 of a grain (.000351 mg.). This experiment was repeated on three leaves with the same relative amount of the solution; and after 6 hrs. 15 m. all the tentacles except nine, on all three leaves taken together, were closely inflected. In this case the tentacles on each leaf were counted, and gave an average of 162 per leaf.
The following experiments were tried during the summer of 1873, by placing the leaves, each in a separate watch-glass and pouring over it thirty minims (1.775 ml.) of the solution; other leaves being treated in exactly the same manner with the doubly distilled water used in making the solutions. The trials above given were made several years before, and when I read over my notes, I could not believe in the results; so I resolved to begin again with moderately strong solutions. Six leaves were first immersed, each in thirty minims of a solution of one part of the nitrate to 8750 of water (1 gr. to 20 oz.), so that each received 1/320 of a grain (.2025 mg.). Before 30 m. had elapsed, four of these leaves were immensely, and two of them moderately, inflected. The glands were rendered of a dark red. The four corresponding leaves in water were not at all affected until 6 hrs. had elapsed, and then only the short tentacles on the borders of the disc; and their inflection, as previously explained, is never of any significance.
Four leaves were immersed, each in thirty minims of a solution of one part to 17,500 of water (1 gr. to 40 oz.), so that each received 1/640 of a grain (.101 mg.); and in less than 45 m. three of them had all their tentacles, except from four to ten, inflected; the blade of one being inflected after 6 hrs., and the blade of a second after 21 hrs. The fourth leaf was not at all affected. The glands of none were darkened. Of the corresponding leaves in water, only one had any of its exterior tentacles, namely five, inflected; after 6 hrs. in one case, and after 21 hrs. in two other cases, the short tentacles on the borders of the disc formed a ring, in the usual manner.
Four leaves were immersed, each in thirty minims of a solution of one part to 43,750 of water (1 gr. to 100 oz.), so that each leaf got 1/1600 of a grain (.0405 mg.). Of these, one was much inflected in 8 m., and after 2 hrs. 7 m. had all the tentacles, except thirteen, inflected. The second leaf, after 10 m., had all except three inflected. The third and fourth were hardly at all affected, scarcely more than the corresponding leaves in water. Of the latter, only one was affected, this having two tentacles inflected, with those on the outer parts of the disc forming a ring in the usual manner. In the leaf which had all its tentacles except three inflected in 10 m., each gland (assuming that the leaf bore 160 tentacles) could have absorbed only 1/251200 of a grain, or .000258 mg.
Four leaves were separately immersed as before in a solution of one part to 131,250 of water (1 gr. to 300 oz.), so that each received 1/4800 of a grain, or .0135 mg. After 50 m. one leaf had all its tentacles except sixteen, and after 8 hrs. 20 m. all but fourteen, inflected. The second leaf, after 40 m., had all but twenty inflected; and after 8 hrs. 10 m. began to re-expand. The third, in 3 hrs. had about half its tentacles inflected, which began to re-expand after 8 hrs. 15 m. The fourth leaf, after 3 hrs. 7 m., had only twenty-nine tentacles more or less inflected. Thus three out of the four leaves were strongly acted on. It is clear that very sensitive leaves had been accidentally selected. The day moreover was hot. The four corresponding leaves in water were likewise acted on rather more than is usual; for after 3 hrs. one had nine tentacles, another four, and another two, and the fourth none, inflected. With respect to the leaf of which all the tentacles, except sixteen, were inflected after 50 m., each gland (assuming that the leaf bore 160 tentacles) could have absorbed only 1/691200 of a grain (.0000937 mg.), and this appears to be about the least quantity of the nitrate which suffices to induce the inflection of a single tentacle.
As negative results are important in confirming the foregoing positive ones, eight leaves were immersed as before, each in thirty minims of a solution of one part to 175,000 of water (1 gr. to 400 oz.), so that each received only 1/6400 of a grain (.0101 mg.). This minute quantity produced a slight effect on only four of the eight leaves. One had fifty-six tentacles inflected after 2 hrs. 13 m.; a second, twenty-six inflected, or sub-inflected, after 38 m.; a third, eighteen inflected, after 1 hr.; and a fourth, ten inflected, after 35 m. The four other leaves were not in the least affected. Of the eight corresponding leaves in water, one had, after 2 hrs. 10 m., nine tentacles, and four others from one to four long-headed tentacles, inflected; the remaining three being unaffected. Hence, the 1/6400 of a grain given to a sensitive leaf during warm weather perhaps produces a slight effect; but we must bear in mind that occasionally water causes as great an amount of inflection as occurred in this last experiment.]
Summary of the Results with Nitrate of Ammonia.--The glands of the disc, when excited by a half-minim drop (.0296 ml.), containing 1/2400 of a grain of the nitrate (.027 mg.), transmit a motor impulse to the exterior tentacles, causing them to bend inwards. A minute drop, containing 1/28800 of a grain (.00225 mg.), if held for a few seconds in contact with a gland, causes the tentacle bearing this gland to be inflected. If a leaf is left immersed for a few hours, and sometimes for only a few minutes, in a solution of such strength that each gland can absorb only the (1/691200 of a grain (.0000937 mg.), this small amount is enough to excite each tentacle into movement, and it becomes closely inflected.
PHOSPHATE OF AMMONIA.
This salt is more powerful than the nitrate, even in a greater degree than the nitrate is more powerful than the carbonate. This is shown by weaker solutions of the phosphate acting when dropped on the discs, or applied to the glands of the exterior tentacles, or when leaves are immersed. The difference in the power of these three salts, as tried in three different ways, supports the results presently to be given, which are so surprising that their credibility requires every kind of support. In 1872 I experimented on twelve immersed leaves, giving each only ten minims of a solution; but this was a bad method, for so small a quantity hardly covered them. None of these experiments will, therefore, be given, though they indicate that excessively minute doses are efficient. When I read over my notes, in 1873, I entirely disbelieved them, and determined to make another set of experiments with scrupulous care, on the same plan as those made with the nitrate; namely by placing leaves in watch-glasses, and pouring over each thirty minims of the solution under trial, treating at the same time and in the same manner other leaves with the distilled water used in making the solutions. During 1873, seventy-one leaves were thus tried in solutions of various strengths, and the same number in water. Notwithstanding the care taken and the number of the trials made, when in the following year I looked merely at the results, without reading over my observations, I again thought that there must have been some error, and thirty-five fresh trials were made with the weakest solution; but the results were as plainly marked as before. Altogether, 106 carefully selected leaves were tried, both in water and in solutions of the phosphate. Hence, after the most anxious consideration, I can entertain no doubt of the substantial accuracy of my results.
[Before giving my experiments, it may be well to premise that crystallised phosphate of ammonia, such as I used, contains 35.33 per cent. of water of crystallisation; so that in all the following trials the efficient elements formed only 64.67 per cent. of the salt used.
Extremely minute particles of the dry phosphate were placed with the point of a needle on the secretion surrounding several glands. These poured forth much secretion, were blackened, and ultimately died; but the tentacles moved only slightly. The dose, small as it was, evidently was too great, and the result was the same as with particles of the carbonate of ammonia.
Half-minims of a solution of one part to 437 of water were placed on the discs of three leaves and acted most energetically, causing the tentacles of one to be inflected in 15 m., and the blades of all three to be much curved inwards in 2 hrs. 15 m. Similar drops of a solution of one part to 1312 of water, (1 gr. to 3 oz.) were then placed on the discs of five leaves, so that each received the 1/2880 of a grain (.0225 mg.). After 8 hrs. the tentacles of four of them were considerably inflected, and after 24 hrs. the blades of three. After 48 hrs. all five were almost fully re-expanded. I may mention with respect to one of these leaves, that a drop of water had been left during the previous 24 hrs. on its disc, but produced no effect; and that this was hardly dry when the solution was added.
Similar drops of a solution of one part to 1750 of water (1 gr. to 4 oz.) were next placed on the discs of six leaves; so that each received 1/3840 of a grain (.0169 mg.); after 8 hrs. three of them had many tentacles and their blades inflected; two others had only a few tentacles slightly inflected, and the sixth was not at all affected. After 24 hrs. most of the leaves had a few more tentacles inflected, but one had begun to re-expand. We thus see that with the more sensitive leaves the 1/3840 of a grain, absorbed by the central glands, is enough to make many of the exterior tentacles and the blades bend, whereas the 1/1920 of a grain of the carbonate similarly given produced no effect; and 1/2880 of a grain of the nitrate was only just sufficient to produce a well-marked effect.
A minute drop, about equal to 1/20 of a minim, of a solution of one part of the phosphate to 875 of water, was applied to the secretion on three glands, each of which thus received only 1/57600 of a grain (.00112 mg.), and all three tentacles became inflected. Similar drops of a solution of one part to 1312 of water (1 gr. to 3 oz.) were now tried on three leaves; a drop being applied to four glands on the same leaf. On the first leaf, three of the tentacles became slightly inflected in 6 m., and re-expanded after 8 hrs. 45 m. On the second, two tentacles became sub-inflected in 12 m. And on the third all four tentacles were decidedly inflected in 12 m.; they remained so for 8 hrs. 30 m., but by the next morning were fully re-expanded. In this latter case each gland could have received only the 1/115200 (or .000563 mg.) of a grain. Lastly, similar drops of a solution of one part to 1750 of water (1 gr. to 4 oz.) were tried on five leaves; a drop being applied to four glands on the same leaf. The tentacles on three of these leaves were not in the least affected; on the fourth leaf, two became inflected; whilst on the fifth, which happened to be a very sensitive one, all four tentacles were plainly inflected in 6 hrs. 15m.; but only one remained inflected after 24 hrs. I should, however, state that in this case an unusually large drop adhered to the head of the pin. Each of these glands could have received very little more than 1/153600 of a grain (or .000423); but this small quantity sufficed to cause inflection. We must bear in mind that these drops were applied to the viscid secretion for only from 10 to 15 seconds, and we have good reason to believe that all the phosphate in the solution would not be diffused and absorbed in this time. We have seen under the same circumstances that the absorption by a gland of 1/19200 of a grain of the carbonate, and of 1/57600 of a grain of the nitrate, did not cause the tentacle bearing the gland in question to be inflected; so that here again the phosphate is much more powerful than the other two salts.
We will now turn to the 106 experiments with immersed leaves. Having ascertained by repeated trials that moderately strong solutions were highly efficient, I commenced with sixteen leaves, each placed in thirty minims of a solution of one part to 43,750 of water (1 gr. to 100 oz.); so that each received 1/1600 of a grain, or .04058 mg. Of these leaves, eleven had nearly all or a great number of their tentacles inflected in 1 hr., and the twelfth leaf in 3 hrs. One of the eleven had every single tentacle closely inflected in 50 m. Two leaves out of the sixteen were only moderately affected, yet more so than any of those simultaneously immersed in water; and the remaining two, which were pale leaves, were hardly at all affected. Of the sixteen corresponding leaves in water, one had nine tentacles, another six, and two others two tentacles inflected, in the course of 5 hrs. So that the contrast in appearance between the two lots was extremely great.
Eighteen leaves were immersed, each in thirty minims of a solution of one part to 87,500 of water (1 gr. to 200 oz.), so that each received 1/3200 of a grain (.0202 mg.). Fourteen of these were strongly inflected within 2 hrs., and some of them within 15 m.; three out of the eighteen were only slightly affected, having twenty-one, nineteen, and twelve tentacles inflected; and one was not at all acted on. By an accident only fifteen, instead of eighteen, leaves were immersed at the same time in water; these were observed for 24 hrs.; one had six, another four, and a third two, of their outer tentacles inflected; the remainder being quite unaffected.
The next experiment was tried under very favourable circumstances, for the day (July 8) was very warm, and I happened to have unusually fine leaves. Five were immersed as before in a solution of one part to 131,250 of water (1 gr. to 300 oz.), so that each received 1/4800 of a grain, or .0135 mg. After an immersion of 25 m. all five leaves were much inflected. After 1 hr. 25 m. one leaf had all but eight tentacles inflected; the second, all but three; the third, all but five; the fourth; all but twenty-three; the fifth, on the other hand, never had more than twenty-four inflected. Of the corresponding five leaves in water, one had seven, a second two, a third ten, a fourth one, and a fifth none inflected. Let it be observed what a contrast is presented between these latter leaves and those in the solution. I counted the glands on the second leaf in the solution, and the number was 217; assuming that the three tentacles which did not become inflected absorbed nothing, we find that each of the 214 remaining glands could have absorbed only 1/l027200 of a grain, or .0000631 mg. The third leaf bore 236 glands, and subtracting the five which did not become inflected, each of the remaining 231 glands could have absorbed only 1/1108800 of a grain (or .0000584 mg.), and this amount sufficed to cause the tentacles to bend.
Twelve leaves were tried as before in a solution of one part to 175,000 of water (1 gr. to 400 oz.), so that each leaf received 1/6400 of a grain (.0101 mg.). My plants were not at the time in a good state, and many of the leaves were young and pale. Nevertheless, two of them had all their tentacles, except three or four, closely inflected in under 1 hr. Seven were considerably affected, some within 1 hr., and others not until 3 hrs., 4 hrs. 30 m., and 8 hrs. had elapsed; and this slow action may be attributed to the leaves being young and pale. Of these nine leaves, four had their blades well inflected, and a fifth slightly so. The three remaining leaves were not affected. With respect to the twelve corresponding leaves in water, not one had its blade inflected; after from 1 to 2 hrs. one had thirteen of its outer tentacles inflected; a second six, and four others either one or two inflected. After 8 hrs. the outer tentacles did not become more inflected; whereas this occurred with the leaves in the solution. I record in my notes that after the 8 hrs. it was impossible to compare the two lots, and doubt for an instant the power of the solution.
Two of the above leaves in the solution had all their tentacles, except three and four, inflected within an hour. I counted their glands, and, on the same principle as before, each gland on one leaf could have absorbed only 1/1164800, and on the other leaf only 1/1472000, of a grain of the phosphate.
Twenty leaves were immersed in the usual manner, each in thirty minims of a solution of one part to 218,750 of water (1 gr. to 500 oz.). So many leaves were tried because I was then under the false impression that it was incredible that any weaker solution could produce an effect. Each leaf received 1/8000 of a grain, or .0081 mg. The first eight leaves which I tried both in the solution and in water were either young and pale or too old; and the weather was not hot. They were hardly at all affected; nevertheless, it would be unfair to exclude them. I then waited until I got eight pairs of fine leaves, and the weather was favourable; the temperature of the room where the leaves were immersed varying from 75o to 81o (23o.8 to 27o.2 Cent.) In another trial with four pairs (included in the above twenty pairs), the temperature in my room was rather low, about 60o (15o.5 Cent.); but the plants had been kept for several days in a very warm greenhouse and thus rendered extremely sensitive. Special precautions were taken for this set of experiments; a chemist weighed for me a grain in an excellent balance; and fresh water, given me by Prof. Frankland, was carefully measured. The leaves were selected from a large number of plants in the following manner: the four finest were immersed in water, and the next four finest in the solution, and so on till the twenty pairs were complete. The water specimens were thus a little favoured, but they did not undergo more inflection than in the previous cases, comparatively with those in the solution.
Of the twenty leaves in the solution, eleven became inflected within 40 m.; eight of them plainly and three rather doubtfully; but the latter had at least twenty of their outer tentacles inflected. Owing to the weakness of the solution, inflection occurred, except in No. 1, much more slowly than in the previous trials. The condition of the eleven leaves which were considerably inflected will now be given at stated intervals, always reckoning from the time of immersion:--
(1) After only 8 m. a large number of tentacles inflected, and after 17 m. all but fifteen; after 2 hrs. all but eight inflected, or plainly sub-inflected. After 4 hrs. the tentacles began to re-expand, and such prompt re-expansion is unusual; after 7 hrs. 30 m. they were almost fully re-expanded.
(2) After 39 m. a large number of tentacles inflected; after 2 hrs. 18 m. all but twenty-five inflected; after 4 hrs. 17 m. all but sixteen inflected. The leaf remained in this state for many hours.
(3) After 12 m. a considerable amount of inflection; after 4 hrs. all the tentacles inflected except those of the two outer rows, and the leaf remained in this state for some time; after 23 hrs. began to re-expand.
(4) After 40 m. much inflection; after 4 hrs. 13 m. fully half the tentacles inflected; after 23 hrs. still slightly inflected.
(5) After 40 m. much inflection; after 4 hrs. 22 m. fully half the tentacles inflected; after 23 hrs. still slightly inflected.
(6) After 40 m. some inflection; after 2 hrs. 18 m. about twenty-eight outer tentacles inflected; after 5 hrs. 20 m. about a third of the tentacles inflected; after 8 hrs. much re-expanded.
(7) After 20 m. some inflection; after 2 hrs. a considerable number of tentacles inflected; after 7 hrs. 45 m. began to re-expand.
(8) After 38 m. twenty-eight tentacles inflected; after 3 hrs. 45 m. thirty-three inflected, with most of the submarginal tentacles sub-inflected; continued so for two days, and then partially re-expanded.
(9) After 38 m. forty-two tentacles inflected; after 3 hrs. 12 m. sixty-six inflected or sub-inflected; after 6 hrs. 40 m. all but twenty-four inflected or sub-inflected; after 9 hrs. 40 m. all but seventeen inflected; after 24 hrs. all but four inflected or sub-inflected, only a few being closely inflected; after 27 hrs. 40 m. the blade inflected. The leaf remained in this state for two days, and then began to re-expand.
(10) After 38 m. twenty-one tentacles inflected; after 3 hrs. 12 m. forty-six tentacles inflected or sub-inflected; after 6 hrs. 40 m. all but seventeen inflected, though none closely; after 24 hrs. every tentacle slightly curved inwards; after 27 hrs. 40 m. blade strongly inflected, and so continued for two days, and then the tentacles and blade very slowly re-expanded.
(11) This fine dark red and rather old leaf, though not very large, bore an extraordinary number of tentacles (viz. 252), and behaved in an anomalous manner. After 6 hrs. 40 m. only the short tentacles round the outer part of the disc were inflected, forming a ring, as so often occurs in from 8 to 24 hrs. With leaves both in water and the weaker solutions. But after 9 hrs. 40 m. all the outer tentacles except twenty-five were inflected; as was the blade in a strongly marked manner. After 24 hrs. every tentacle except one was closely inflected, and the blade was completely doubled over. Thus the leaf remained for two days, when it began to re-expand. I may add that the three latter leaves (Nos. 9, 10, and 11) were still somewhat inflected after three days. The tentacles in but few of these eleven leaves became closelyinflected within so short a time as in the previous experiments with stronger solutions.
We will now turn to the twenty corresponding leaves in water. Nine had none of their outer tentacles inflected; nine others had from one to three inflected; and these re-expanded after 8 hrs. The remaining two leaves were moderately affected; one having six tentacles inflected in 34 m.; the other twenty-three inflected in 2 hrs. 12 m.; and both thus remained for 24 hrs. None of these leaves had their blades inflected. So that the contrast between the twenty leaves in water and the twenty in the solution was very great, both within the first hour and after from 8 to 12 hrs. had elapsed.
Of the leaves in the solution, the glands on leaf No. 1, which in 2 hrs. had all its tentacles except eight inflected, were counted and found to be 202. Subtracting the eight, each gland could have received only the 1/1552000 grain (.0000411 mg.) of the phosphate. Leaf No. 9 had 213 tentacles, all of which, with the exception of four, were inflected after 24 hrs., but none of them closely; the blade was also inflected; each gland could have received only the 1/1672000 of a grain, or .0000387 mg. Lastly, leaf No. 11, which had after 24 hrs. all its tentacles, except one, closely inflected, as well as the blade, bore the unusually large number of 252 tentacles; and on the same principle as before, each gland could have absorbed only the 1/2008000 of a grain, or .0000322 mg.
With respect to the following experiments, I must premise that the leaves, both those placed in the solutions and in water, were taken from plants which had been kept in a very warm greenhouse during the winter. They were thus rendered extremely sensitive, as was shown by water exciting them much more than in the previous experiments. Before giving my observations, it may be well to remind the reader that, judging from thirty-one fine leaves, the average number of tentacles is 192, and that the outer or exterior ones, the movements of which are alone significant, are to the short ones on the disc in the proportion of about sixteen to nine.
Four leaves were immersed as before, each in thirty minims of a solution of one part to 328,125 of water (1 gr. to 750 oz.). Each leaf thus received 1/12000 of a grain (.0054 mg.) of the salt; and all four were greatly inflected.
(1) After 1 hr. all the outer tentacles but one inflected, and the blade greatly so; after 7 hrs. began to re-expand.
(2) After 1 hr. all the outer tentacles but eight inflected; after 12 hrs. all re-expanded.
(3) After 1 hr. much inflection; after 2 hrs. 30 m. all the tentacles but thirty-six inflected; after 6 hrs. all but twenty-two inflected; after 12 hrs. partly re-expanded.
(4) After 1 hr. all the tentacles but thirty-two inflected; after 2 hrs. 30 m. all but twenty-one inflected; after 6 hrs. almost re-expanded.
Of the four corresponding leaves in water:--
(1) After 1 hr. forty-five tentacles inflected; but after 7 hrs. so many had re-expanded that only ten remained much inflected.
(2) After 1 hr. seven tentacles inflected; these were almost re-expanded in 6 hrs.
(3) and (4) Not affected, except that, as usual, after 11 hrs. the short tentacles on the borders of the disc formed a ring.
There can, therefore, be no doubt about the efficiency of the above solution; and it follows as before that each gland of No. 1 could have absorbed only 1/2412000 of a grain (.0000268 mg.) and of No. 2 only 1/2460000 of a grain (.0000263 mg.) of the phosphate.
Seven leaves were immersed, each in thirty minims of a solution of one part to 437,500 of water (1 gr. to 1000 oz.). Each leaf thus received 1/16000 of a grain (.00405 mg.). The day was warm, and the leaves were very fine, so that all circumstances were favourable.
(1) After 30 m. all the outer tentacles except five inflected, and most of them closely; after 1 hr. blade slightly inflected; after 9 hrs. 30 m. began to re-expand.
(2) After 33 m. all the outer tentacles but twenty-five inflected, and blade slightly so; after 1 hr. 30 m. blade strongly inflected and remained so for 24 hrs.; but some of the tentacles had then re-expanded.
(3) After 1 hr. all but twelve tentacles inflected; after 2 hrs. 30 m. all but nine inflected; and of the inflected tentacles all excepting four closely; blade slightly inflected. After 8 hrs. blade quite doubled up, and now all the tentacles excepting eight closely inflected. The leaf remained in this state for two days.
(4) After 2 hrs. 20 m. only fifty-nine tentacles inflected; but after 5 hrs. all the tentacles closely inflected excepting two which were not affected, and eleven which were only sub-inflected; after 7 hrs. blade considerably inflected; after 12 hrs. much re-expansion.
(5) After 4 hrs. all the tentacles but fourteen inflected; after 9 hrs. 30 m. beginning to re-expand.
(6) After 1 hr. thirty-six tentacles inflected; after 5 hrs. all but fifty-four inflected; after 12 hrs. considerable re-expansion.
(7) After 4 hrs. 30 m. only thirty-five tentacles inflected or sub-inflected, and this small amount of inflection never increased.
Now for the seven corresponding leaves in water:--
(1) After 4 hrs. thirty-eight tentacles inflected; but after 7 hrs. these, with the exception of six, re-expanded.
(2) After 4 hrs. 20 m. twenty inflected; these after 9 hrs. partially re-expanded.
(3) After 4 hrs. five inflected, which began to re-expand after 7 hrs.
(4) After 24 hrs. one inflected.
(5), (6) and (7) Not at all affected, though observed for 24 hrs., excepting the short tentacles on the borders of the disc, which as usual formed a ring.
A comparison of the leaves in the solution, especially of the first five or even six on the list, with those in the water, after 1 hr. or after 4 hrs., and in a still more marked degree after 7 hrs. or 8 hrs., could not leave the least doubt that the solution had produced a great effect. This was shown not only by the vastly greater number of inflected tentacles, but by the degree or closeness of their inflection, and by that of their blades. Yet each gland on leaf No. 1 (which bore 255 glands, all of which, excepting five, were inflected in 30 m.) could not have received more than one-four-millionth of a grain (.0000162 mg.) of the salt. Again, each gland on leaf No. 3 (which bore 233 glands, all of which, except nine, were inflected in 2 hrs. 30 m.) could have received at most only the 1/3584000 of a grain, or .0000181 mg.
Four leaves were immersed as before in a solution of one part to 656,250 of water (1 gr. to 1500 oz.); but on this occasion I happened to select leaves which were very little sensitive, as on other occasions I chanced to select unusually sensitive leaves. The leaves were not more affected after 12 hrs. than the four corresponding ones in water; but after 24 hrs. they were slightly more inflected. Such evidence, however, is not at all trustworthy.
Twelve leaves were immersed, each in thirty minims of a solution of one part to 1,312,500 of water (1 gr. to 3000 oz.); so that each leaf received 1/48000 of a grain (.00135 mg.). The leaves were not in very good condition; four of them were too old and of a dark red colour; four were too pale, yet one of these latter acted well; the four others, as far as could be told by the eye, seemed in excellent condition. The result was as follows:--
(1) This was a pale leaf; after 40 m. about thirty-eight tentacles inflected; after 3 hrs. 30 m. the blade and many of the outer tentacles inflected; after 10 hrs. 15 m. all the tentacles but seventeen inflected, and the blade quite doubled up; after 24 hrs. all the tentacles but ten more or less inflected. Most of them were closely inflected, but twenty-five were only sub-inflected.
(2) After 1 hr. 40 m. twenty-five tentacles inflected; after 6 hrs. all but twenty-one inflected; after 10 hrs. all but sixteen more or less inflected; after 24 hrs. re-expanded.
(3) After 1 hr. 40 m. thirty-five inflected; after 6 hrs. "a large number" (to quote my own memorandum) inflected, but from want of time they were not counted; after 24 hrs. re-expanded.
(4) After 1 hr. 40 m. about thirty inflected; after 6 hrs. "a large number all round the leaf" inflected, but they were not counted; after 10 hrs. began to re-expand.
(5) to (12) These were not more inflected than leaves often are in water, having respectively 16, 8, 10, 8, 4, 9, 14, and 0 tentacles inflected. Two of these leaves, however, were remarkable from having their blades slightly inflected after 6 hrs.
With respect to the twelve corresponding leaves in water, (1) had, after 1 hr. 35 m., fifty tentacles inflected, but after 11 hrs. only twenty-two remained so, and these formed a group, with the blade at this point slightly inflected. It appeared as if this leaf had been in some manner accidentally excited, for instance by a particle of animal matter which was dissolved by the water. (2) After 1 hr. 45 m. thirty-two tentacles inflected, but after 5 hrs. 30 m. only twenty-five inflected, and these after 10 hrs. all re-expanded; (3) after 1 hr. twenty-five inflected, which after 10 hrs. 20 m. were all re-expanded; (4) and (5) after 1 hr. 35 m. six and seven tentacles inflected, which re-expanded after 11 hrs.; (6), (7) and (8) from one to three inflected, which soon re-expanded; (9), (10), (11) and (12) none inflected, though observed for twenty-four hours.
Comparing the states of the twelve leaves in water with those in the solution, there could be no doubt that in the latter a larger number of tentacles were inflected, and these to a greater degree; but the evidence was by no means so clear as in the former experiments with stronger solutions. It deserves attention that the inflection of four of the leaves in the solution went on increasing during the first 6 hrs., and with some of them for a longer time; whereas in the water the inflection of the three leaves which were the most affected, as well as of all the others, began to decrease during this same interval. It is also remarkable that the blades of three of the leaves in the solution were slightly inflected, and this is a most rare event with leaves in water, though it occurred to a slight extent in one (No. 1), which seemed to have been in some manner accidentally excited. All this shows that the solution produced some effect, though less and at a much slower rate than in the previous cases. The small effect produced may, however, be accounted for in large part by the majority of the leaves having been in a poor condition.
Of the leaves in the solution, No. 1 bore 200 glands and received 1/48000 of a grain of the salt. Subtracting the seventeen tentacles which were not inflected, each gland could have absorbed only the 1/8784000 of a grain (.00000738 mg.). This amount caused the tentacle bearing each gland to be greatly inflected. The blade was also inflected.
Lastly, eight leaves were immersed, each in thirty minims of a solution of one part of the phosphate to 21,875,000 of water (1 gr. to 5000 oz.). Each leaf thus received 1/80000 of a grain of the salt, or .00081 mg. I took especial pains in selecting the finest leaves from the hot-house for immersion, both in the solution and the water, and almost all proved extremely sensitive. Beginning as before with those in the solution:--
(1) After 2 hrs. 30 m. all the tentacles but twenty-two inflected, but some only sub-inflected; the blade much inflected; after 6 hrs. 30 m. all but thirteen inflected, with the blade immensely inflected; and remained so for 48 hrs.
(2) No change for the first 12 hrs., but after 24 hrs. all the tentacles inflected, excepting those of the outermost row, of which only eleven were inflected. The inflection continued to increase, and after 48 hrs. all the tentacles except three were inflected, and most of them rather closely, four or five being only sub-inflected.
(3) No change for the first 12 hrs.; but after 24 hrs. all the tentacles excepting those of the outermost row were sub-inflected, with the blade inflected. After 36 hrs. blade strongly inflected, with all the tentacles, except three, inflected or sub-inflected. After 48 hrs. in the same state.
(4) to (8) These leaves, after 2 hrs. 30 m., had respectively 32, 17, 7, 4, and 0 tentacles inflected, most of which, after a few hours, re-expanded, with the exception of No. 4, which retained its thirty-two tentacles inflected for 48 hrs.
Now for the eight corresponding leaves in water:--
(1) After 2 hrs. 40 m. this had twenty of its outer tentacles inflected, five of which re-expanded after 6 hrs. 30 m. After 10 hrs. 15 m. a most unusual circumstance occurred, namely, the whole blade became slightly bowed towards the footstalk, and so remained for 48 hrs. The exterior tentacles, excepting those of the three or four outermost rows, were now also inflected to an unusual degree.
(2) to (8) These leaves, after 2 hrs. 40 m., had respectively 42, 12, 9, 8, 2, 1, and 0 tentacles inflected, which all re-expanded within 24 hrs., and most of them within a much shorter time.
When the two lots of eight leaves in the solution and in the water were compared after the lapse of 24 hrs., they undoubtedly differed much in appearance. The few tentacles on the leaves in water which were inflected had after this interval re-expanded, with the exception of one leaf; and this presented the very unusual case of the blade being somewhat inflected, though in a degree hardly approaching that of the two leaves in the solution. Of these latter leaves, No. 1 had almost all its tentacles, together with its blade, inflected after an immersion of 2 hrs. 30 m. Leaves No. 2 and 3 were affected at a much slower rate; but after from 24 hrs. to 48 hrs. almost all their tentacles were closely inflected, and the blade of one quite doubled up. We must therefore admit, incredible as the fact may at first appear, that this extremely weak solution acted on the more sensitive leaves; each of which received only the 1/80000 of a grain (.00081 mg.) of the phosphate. Now, leaf No. 3 bore 178 tentacles, and subtracting the three which were not inflected, each gland could have absorbed only the 1/14000000 of a grain, or .00000463 mg. Leaf No. 1, which was strongly acted on within 2 hrs. 30 m., and had all its outer tentacles, except thirteen, inflected within 6 hrs. 30 m., bore 260 tentacles; and on the same principle as before, each gland could have absorbed only 1/19760000 of a grain, or .00000328 mg.; and this excessively minute amount sufficed to cause all the tentacles bearing these glands to be greatly inflected. The blade was also inflected.]
Summary of the Results with Phosphate of Ammonia.--The glands of the disc, when excited by a half-minim drop (.0296 ml.), containing 1/3840 of a grain (.0169 mg.) of this salt, transmit a motor impulse to the exterior tentacles, causing them to bend inwards. A minute drop, containing 1/153600 of a grain (.000423 mg.), if held for a few seconds in contact with a gland, causes the tentacle bearing this gland to be inflected. If a leaf is left immersed for a few hours, and sometimes for a shorter time, in a solution so weak that each gland can absorb only the 1/9760000 of a grain (.00000328 mg.), this is enough to excite the tentacle into movement, so that it becomes closely inflected, as does sometimes the blade. In the general summary to this chapter a few remarks will be added, showing that the efficiency of such extremely minute doses is not so incredible as it must at first appear.
[Sulphate of Ammonia.--The few trials made with this and the following five salts of ammonia were undertaken merely to ascertain whether they induced inflection. Half-minims of a solution of one part of the sulphate of ammonia to 437 of water were placed on the discs of seven leaves, so that each received 1/960 of a grain, or .0675 mg. After 1 hr. the tentacles of five of them, as well as the blade of one, were strongly inflected. The leaves were not afterwards observed.
Citrate of Ammonia.--Half-minims of a solution of one part to 437 of water were placed on the discs of six leaves. In 1 hr. the short outer tentacles round the discs were a little inflected, with the glands on the discs blackened. After 3 hrs. 25 m. one leaf had its blade inflected, but none of the exterior tentacles. All six leaves remained in nearly the same state during the day, the submarginal tentacles, however, becoming more inflected. After 23 hrs. three of the leaves had their blades somewhat inflected; and the submarginal tentacles of all considerably inflected, but in none were the two, three, or four outer rows affected. I have rarely seen cases like this, except from the action of a decoction of grass. The glands on the discs of the above leaves, instead of being almost black, as after the first hour, were now after 23 hrs. very pale. I next tried on four leaves half-minims of a weaker solution, of one part to 1312 of water (1 gr. to 3 oz.); so that each received 1/2880 of a grain (.0225 mg.). After 2 hrs. 18 m. the glands on the disc were very dark-coloured; after 24 hrs. two of the leaves were slightly affected; the other two not at all.
Acetate of Ammonia.--Half-minims of a solution of about one part to 109 of water were placed on the discs of two leaves, both of which were acted on in 5 hrs. 30 m., and after 23 hrs. had every single tentacle closely inflected.
Oxalate of Ammonia.--Half-minims of a solution of one part to 218 of water were placed on two leaves, which, after 7 hrs., became moderately, and after 23 hrs. strongly, inflected. Two other leaves were tried with a weaker solution of one part to 437 of water; one was strongly inflected in 7 hrs.; the other not until 30 hrs. had elapsed.
Tartrate of Ammonia.--Half-minims of a solution of one part to 437 of water were placed on the discs of five leaves. In 31 m. there was a trace of inflection in the exterior tentacles of some of the leaves, and this became more decided after 1 hr. with all the leaves; but the tentacles were never closely inflected. After 8 hrs. 30 m. they began to re-expand. Next morning, after 23 hrs., all were fully re-expanded, excepting one which was still slightly inflected. The shortness of the period of inflection in this and the following case is remarkable.
Chloride of Ammonium.--Half-minims of a solution of one part to 437 of water were placed on the discs of six leaves. A decided degree of inflection in the outer and submarginal tentacles was perceptible in 25 m.; and this increased during the next three or four hours, but never became strongly marked. After only 8 hrs. 30 m. the tentacles began to re-expand, and by the next morning, after 24 hrs., were fully re-expanded on four of the leaves, but still slightly inflected on two.]
General Summary and Concluding Remarks on the Salts of Ammonia.--We have now seen that the nine salts of ammonia which were tried, all cause the inflection of the tentacles, and often of the blade of the leaf. As far as can be ascertained from the superficial trials with the last six salts, the citrate is the least powerful, and the phosphate certainly by far the most. The tartrate and chloride are remarkable from the short duration of their action. The relative efficiency of the carbonate, nitrate, and phosphate, is shown in the following table by the smallest amount which suffices to cause the inflection of the tentacles.
Column 1 : Solutions, how applied. Column 2 : Carbonate of Ammonia. Column 3 : Nitrate of Ammonia. Column 4 : Phosphate of Ammonia.
Placed on the glands of the disc, so as to act indirectly on the outer tentacles : 1/960 of a grain, or 0675 mg. : 1/2400 of a grain, or .027 mg. : 1/3840 of a grain, or .0169 mg.
Applied for a few seconds directly to the gland of an outer tentacle : 1/14400 of a grain, or .00445 mg. : 1/28800 of a grain, or .0025 mg. grain, 1/153600 of a grain, or .000423 mg.
Leaf immersed, with time allowed for each gland to absorb all that it can : 1/268800 of a grain, or .00024 mg. : 1/691200 of a grain, or .0000937 mg. : 1/19760000 of a grain, or .00000328 mg.
Amount absorbed by a gland which suffices to cause the aggregation of the protoplasm in the adjoining cells of the tentacles. 1/134400 of a grain, or .00048 mg.
From the experiments tried in these three different ways, we see that the carbonate, which contains 23.7 per cent. of nitrogen, is less efficient than the nitrate, which contains 35 per cent. The phosphate contains less nitrogen than either of these salts, namely, only 21.2 per cent., and yet is far more efficient; its power no doubt depending quite as much on the phosphorus as on the nitrogen which it contains. We may infer that this is the case, from the energetic manner in which bits of bone and phosphate of lime affect the leaves. The inflection excited by the other salts of ammonia is probably due solely to their nitrogen,--on the same principle that nitrogenous organic fluids act powerfully, whilst non-nitrogenous organic fluids are powerless. As such minute doses of the salts of ammonia affect the leaves, we may feel almost sure that Drosera absorbs and profits by the amount, though small, which is present in rain-water, in the same manner as other plants absorb these same salts by their roots.
The smallness of the doses of the nitrate, and more especially of the phosphate of ammonia, which cause the tentacles of immersed leaves to be inflected, is perhaps the most remarkable fact recorded in this volume. When we see that much less than the millionth* of a grain of the phosphate, absorbed by a gland of one of the exterior tentacles, causes it to bend, it may be thought that the effects of the solution on the glands of the disc have been overlooked; namely, the transmission of a motor impulse from them to the exterior tentacles. No doubt the movements of the latter are thus aided; but the aid thus rendered must be insignificant; for we know that a drop containing as much as the 1/3840 of a grain placed on the disc is only just able to cause the outer tentacles of a highly sensitive leaf to bend. It is
* It is scarcely possible to realise what a million means. The best illustration which I have met with is that given by Mr. Croll, who says, "Take a narrow strip of paper 83 ft. 4 in. in length, and stretch it along the wall of a large hall; then mark off at one end the tenth of an inch. This tenth will represent a hundred, and the entire strip a million.
certainly a most surprising fact that the 1/19760000 of a grain, or in round numbers the one-twenty-millionth of a grain (.0000033 mg.), of the phosphate should affect any plant, or indeed any animal; and as this salt contains 35.33 per cent. of water of crystallisation, the efficient elements are reduced to 1/30555126 of a grain, or in round numbers to one-thirty-millionth of a grain (.00000216 mg.). The solution, moreover, in these experiments was diluted in the proportion of one part of the salt to 2,187,500 of water, or one grain to 5000 oz. The reader will perhaps best realise this degree of dilution by remembering that 5000 oz. would more than fill a 31-gallon cask; and that to this large body of water one grain of the salt was added; only half a drachm, or thirty minims, of the solution being poured over a leaf. Yet this amount sufficed to cause the inflection of almost every tentacle, and often of the blade of the leaf.
I am well aware that this statement will at first appear incredible to almost everyone. Drosera is far from rivalling the power of the spectroscope, but it can detect, as shown by the movements of its leaves, a very much smaller quantity of the phosphate of ammonia than the most skilful chemist can of any substance.* My results were for a long time incredible
* When my first observations were made on the nitrate of ammonia, fourteen years ago, the powers of the spectroscope had not been discovered; and I felt all the greater interest in the then unrivalled powers of Drosera. Now the spectroscope has altogether beaten Drosera; for according to Bunsen and Kirchhoff probably less than one 1/200000000 of a grain of sodium can be thus detected (see Balfour Stewart, 'Treatise on Heat,' 2nd edit. 1871, p. 228). With respect to ordinary chemical tests, I gather from Dr. Alfred Taylor's work on 'Poisons' that about 1/4000 of a grain of arsenic, 1/4400 of a grain of prussic acid, 1/1400 of iodine, and 1/2000 of tartarised antimony, can be detected; but the power of detection depends much on the solutions under trial not being extremely weak.
even to myself, and I anxiously sought for every source of error. The salt was in some cases weighed for me by a chemist in an excellent balance; and fresh water was measured many times with care. The observations were repeated during several years. Two of my sons, who were as incredulous as myself, compared several lots of leaves simultaneously immersed in the weaker solutions and in water, and declared that there could be no doubt about the difference in their appearance. I hope that some one may hereafter be induced to repeat my experiments; in this case he should select young and vigorous leaves, with the glands surrounded by abundant secretion. The leaves should be carefully cut off and laid gently in watch-glasses, and a measured quantity of the solution and of water poured over each. The water used must be as absolutely pure as it can be made. It is to be especially observed that the experiments with the weaker solutions ought to be tried after several days of very warm weather. Those with the weakest solutions should be made on plants which have been kept for a considerable time in a warm greenhouse, or cool hothouse; but this is by no means necessary for trials with solutions of moderate strength.
I beg the reader to observe that the sensitiveness or irritability of the tentacles was ascertained by three different methods--indirectly by drops placed on the disc, directly by drops applied to the glands of the outer tentacles, and by the immersion of whole leaves; and it was found by these three methods that the nitrate was more powerful than the carbonate, and the phosphate much more powerful than the nitrate; this result being intelligible from the difference in the amount of nitrogen in the first two salts, and from the presence of phosphorus in the third. It may aid the reader's faith to turn to the experiments with a solution of one grain of the phosphate to 1000 oz. of water, and he will there find decisive evidence that the one-four-millionth of a grain is sufficient to cause the inflection of a single tentacle. There is, therefore, nothing very improbable in the fifth of this weight, or the one-twenty-millionth of a grain, acting on the tentacle of a highly sensitive leaf. Again, two of the leaves in the solution of one grain to 3000 oz., and three of the leaves in the solution of one grain to 5000 oz., were affected, not only far more than the leaves tried at the same time in water, but incomparably more than any five leaves which can be picked out of the 173 observed by me at different times in water.
There is nothing remarkable in the mere fact of the one-twenty-millionth of a grain of the phosphate, dissolved in above two-million times its weight of water, being absorbed by a gland. All physiologists admit that the roots of plants absorb the salts of ammonia brought to them by the rain; and fourteen gallons of rain-water contain* a grain of ammonia, therefore only a little more than twice as much as in the weakest solution employed by me. The fact which appears truly wonderful is, that the one-twenty-millionth of a grain of the phosphate of ammonia (including less than the one-thirty-millionth of efficient matter), when absorbed by a gland, should induce some change in it, which leads to a motor impulse being transmitted down the whole length of the tentacle, causing the basal part to bend, often through an angle of above 180 degrees.
Astonishing as is this result, there is no sound reason
* Miller's 'Elements of Chemistry,' part ii. p. 107, 3rd edit. 1864.
why we should reject it as incredible. Prof. Donders, of Utrecht, informs me that from experiments formerly made by him and Dr. De Ruyter, he inferred that less than the one-millionth of a grain of sulphate of atropine, in an extremely diluted state, if applied directly to the iris of a dog, paralyses the muscles of this organ. But, in fact, every time that we perceive an odour, we have evidence that infinitely smaller particles act on our nerves. When a dog stands a quarter of a mile to leeward of a deer or other animal, and perceives its presence, the odorous particles produce some change in the olfactory nerves; yet these particles must be infinitely smaller* than those of the phosphate of ammonia weighing the one-twenty-millionth of a grain. These nerves then transmit some influence to the brain of the dog, which leads to action on its part. With Drosera, the really marvellous fact is, that a plant without any specialised nervous system should be affected by such minute particles; but we have no grounds for assuming that other tissues could not be rendered as exquisitely susceptible to impressions from without if this were beneficial to the organism, as is the nervous system of the higher animals.
* My son, George Darwin, has calculated for me the diameter of a sphere of phosphate of ammonia (specific gravity 1.678), weighing the one-twenty-millionth of a grain, and finds it to be 1/1644 of an inch. Now, Dr. Klein informs me that the smallest Micrococci, which are distinctly discernible under a power of 800 diameters, are estimated to be from .0002 to .0005 of a millimetre--that is, from 1/50800 to 1/127000 of an inch--in diameter. Therefore, an object between 1/31 and 1/77 of the size of a sphere of the phosphate of ammonia of the above weight can be seen under a high power; and no one supposes that odorous particles, such as those emitted from the deer in the above illustration, could be seen under any power of the microscope.)