OYTOLOGIOAL CHANGES PRODUCED IN DRO8ERA. 203

Further Study of Cytological Changes producedin Drosera.

Part II.

II. Huie,Physiological Laboratory, Oxford.

With Plate 22.

THIS research is an expansion of that previously undertaken,of which an account has been given in this Journal, vol. 39.

Aims.

The present aim was to ascertain—(1) Whether substances which differ chemically will produce

specific alterations in either the cytoplasm or the nucleus.(2) To what extent the cytoplasm and the nucleus are

interdependent.(3) Whether the rapidity with which changes occur depends

on the ease with which substances are absorbed.(4) The relation between cytological changes and the irri-

tability of leaves, the latter being calculated from the rate ofclosure of the tentacles.

(5) What effects can be produced by bringing healthy cellsin contact with supposed waste products of ordinary metabolicactivity.

204 LILT H. HUIK.

Methods.To deal with these questions satisfactorily necessitates that

no reliance should be placed on any single fixing method, andtherefore the whole of my previous work has been repeatedwith chemically very diverse fixatives, e.g. 5 per cent, and10 per cent, trichloracetic acid, 5 per cent, and 10 per cent,phosphotungstic acid, osmic acid vapour, picric acid, picro-corrosive sublimate with tannin added, absolute alcohol,Hermann's fluid, Flemming's strong mixture, and Flemming'sweak mixture. All these methods fully confirmed what Ihave stated in my previous paper, so that in pursuing mynew research I had every confidence in employing again themethods I formerly used, viz. Mann's aqueous picro-corrosivefixative and Mann's eosin and toluidin blue stain.

The following substances were placed on vigorous fully ex-panded leaves :—Paraffin, white of egg, Kiihne's pure ampho-peptone, Witte's peptone (albumoses), ammonium sulphate,fibrin, Griibler's fibrin peptone, globulin, casein, nuclein andnucleic acid, milk, calcium phosphate, glutin, leucin, creatin,urea.

In all cases except that of casein the species of Droseraused was D. rotundifolia, as plants reared in greenhousescannot be considered normal.

The leaves after being fed were fixed at intervals varyingfrom one minute up to the time of reopening of the leaves.As, however, in the case of white-of-egg feeding the changesproduced after one minute are so remarkable,1 shorter periodsvarying from 5 to 60 seconds were tried with white of egg andalso with pure peptone.

Unfed leaves were fixed at the same time to serve as controls,and to avoid all errors caused by irregular staining sectionsof control leaves were always mounted alongside those of fedones on the same slide.

It may be well to quote here again a definition of the termsapplied by me to the nuclear organs.

1 See previous paper.

OYTOLOGICAL CHANGES PRODUCED IN DROSERA. 205

Chromosomes are the organs which show the well-knownaffinity for alkaline dyes. They are situated at the peripheryof the nucleus, and consist of nuclein.

Nuclear plasm is the material which, in the resting state,forms the main bulk of the nucleus. It is neutrophile incharacter, and is frequently called the nuclear sap, but corre-sponds to " cedematin " globules.

Nuclear sap is the more watery fluid inside the nucleus, inwhich is suspended the nuclear plasm, the latter being pre-cipitable by HgCl2 and other reagents.

Nucleoli are the spherical organs which show a specialaffinity for acid dyes, and have a more or less central position,and they consist of Flemming's paranuclein.

Rest ing Gland Cells (fig. 1).—The gland cells in theirresting state, when fixed and stained by Mann's methods,appear as follows :

The cell wall is pale blue; the cytoplasm is arranged in anopen foam-like manner; it stains pure blue with a coarsegranulation embedded in it, which appears of a deeper blueand represents the zymogen.

The nucleus is situated just below the middle of the cell.It may be spherical or oval. It stains purplish, and is sharplydefined against the cytoplasm, partly because of the differencein tint, and also because of the peripherally placed, veryminute, dark blue chromosomes.

The iluclear plasm is granular, always very dense, and of apurplish tint. One (sometimes more) very distinct, deep rednucleolus is always present, surrounded by a narrow Frora-mann's clear zone.

Exper iments .

Paraffin (fig. 2).—The rapid changes which can be pro-duced by certain foods suggested the idea of ascertainingwhether purely physical factors, such as contact with insolublesubstances, would not also produce effects. "When pieces ofwejl-boiled cork were applied to the leaves vacuolation of the

206 LILY H. HUIB.

gland cells resulted; but as this alteration might have beendue to traces of soluble substances it was thought necessary touse paraffin of a high melting-point (58° C), which had beencarefully cleaned by boiling. Shavings of it were laid on thetentacles, and pressed into thorough contact with them bymeans of a brush. About one hour after the application ofthe paraffin the alteration in the cells reaches its height. Itconsists in a vacuolation of the cytoplasm, but with noalteration in colour reactions. The nucleus remains quiteunchanged. In the course of twenty-four hours completerecuperation has taken place.

White of Egg [stains Red] (fig. 3).—My former researchhaving dealt minutely with the changes produced in the glandcells by white of egg, I have now only to add those resultingin the exceedingly short time of five seconds. They are asfollows:

Cell wall very pale blue.Cell plasm deep purple, frequently red round the nucleus,

vacuolated in the upper third of the cell, densely aggregatedbelow.

Nuclear plasm unaltered in arrangement, but staining darkpurple or red like the cytoplasm surrounding the nucleus.

Chromosomes as in controls.Nucleolus staining somewhat less brightly.Peptones (figs. 4—9).—The experiments were made on a

large number of leaves, which were fed and fixed on theirnative moor. It is important to note this, as plants whichwere taken home did not show the characteristic precipitatesabout to be described, but only vacuolation.

Histological Changes.—In all cases the peptone ap-peared as greenish-blue droplets adhering to the exteriorsurface of the gland cells. In the cells of that layer whichin my former paper I called the third layer of gland cells,the cell sap is particularly rich in tannin, and here is formeda heavy greenish-blue precipitate encrusting the peripheralwalls. When viewed with a low power the dark blue linethus formed gives a perfectly cliaracteristic appearance to all

CYTOLOGICAL CHANGES PRODUCED IN DROSfiRA. 2 0 7

the sections of material used in this experiment. The changesin the apical gland cells are as follows:

Five Seconds after the Appl icat ion of a 10 percent. Solution (fig. 4).—A precipitate (?) resembling minuteblue droplets covers the interior of the cell walls, especially thelateral ones.

The cell walls stain very faintly blue, or show no colour.The Cell plasm stains much more deeply blue than in

controls. It appears more dense and homogeneous, beingapparently almost free from vacuoles. The reticular arrange-ment seen in controls has given place to what appears like athick, evenly distributed granular mass. Thus instead ofvacuolation there is found a direct increase in bulk. At theperiphery of the plasmic body are seen dark blue droplets orgranules resembling those on the walls.

The nucleus is normal as to position, size, shape, and clear-ness of definition.

The nuclear plasm is often also bluer than in controls,though it frequently stains normally. Its arrangement isunaffected.

The nuclear chromosomes are unchanged.The nucleolus is normal as to size, and also as to colour in

those nuclei in which the plasm stains normally. In thebluer nuclei it stains more of a purple. Frommann's zoneremains clear.

The nuclei of the third layer remain unchanged.Ten to Thir ty Seconds.—The above description holds

good. I have, however, observed a slight shortening of thenuclei of the third layer of gland cells in leaves fed for thirtyseconds.

Specimens fed for one minute require no separate detaileddescription, as they resemble the bluer types about to bedescribed.

Five Minutes after Feeding (fig. 5).—The precipitatesappear as in earlier stages. The cell walls stain blue withextreme faintness. The cytoplasm remains as shown in fig.4, or is more vacuolated, and stains less deeply (fig. 5). The

VOL. 4 2 , fABT 2.—NEW SERIES. 0

208 LILT H. EUIB.

vacuoles appear to be filled with a substance which stainspale blue. In a few tentacles there is a reddish tinge in theapical half of the cells. The nucleus is normal as to position,size, and shape. The nuclear plasm stains occasionally bluerthan in controls (fig. 4) j or again in those cells which showa tendency in the cell plasm to stain red, the nuclear plasmtakes on the same tint. There is, therefore, at this stage adecrease in the basophile affinity which was so marked acharacteristic of the earlier stages. The nuclear plasm is notdiminished in quantity or altered in arrangement.

The chromosomes are not perceptibly increased in size, butappear to be slightly more numerous. In some nuclei theyappear as if united by fine threads of a substance which alsostains dark blue. The nucleolus is normal.

The nuclei of the third layer are either normal or veryslightly sViortened.

One Hour after Feeding (figs. 6—8). — The dropletsadhering to the exterior of the tentacle and those on thelateral walls of the gland cells generally stain more faintlyand are less conspicuous than in earlier stages.

The cell walls stain extremely pale blue.The cytoplasm is paler blue and less dense than in earlier

stages, but is quite as full or fuller than in most controls, andits mass is often outlined with dark drops or granules.

The nucleus is sometimes normal in size and shape, andsometimes shrunken. The nuclear membrane has quite dis-appeared. The nuclear plasm stains like that of controls, andin those nuclei which have not shrunken it is arranged as incontrols, while in the shrunken nuclei its arrangement cannotbe made out.

The chromosomes have increased enormously. In thosennclei which have not shrunken they are seen to be ribbon-shaped. I have not found it possible to determine whether ornot they exist in any fixed number.

The nucleolus is smaller than normal. In the unshruukennuclei it is surrounded by a clear zone.

In the third layer the precipitate is as heavy as before.

CYTOLOGUCAL CHANGES PRODUCED IN DROSEEA. 2 0 9

The nuclei vary in shape from being only slightly shorter thannormal to forms such as are shown in figs. 32 and 33 of myprevious paper.

One Day after Feeding (fig. 9).—After the leaves hadbeen fed it was unfortunately necessary to remove the plantsfrom their natural habitat. They were placed in a coolgreenhouse, where the leaves that were allowed to remaingrowing reopened healthily in two to four days, but it ispossible that the transplanting may have affected the cyto-logical appearances about to be described. It is remarkablethat ther.e was nothing to be seen resembling the precipitatesof the earlier stages either externally or internally.

The cell walls are very pale blue, almost colourless.The cytoplasm is purple, very granular, greatly vacuolated

iu the apical half of the cells, dense in the basal half, thoughshowing here also small vacuoles. In the neighbourhood ofthe large vacuoles dark blue granular matter collects.

The nucleus is normal in size and shape, and is surroundedby a membrane, which facts suggest that recuperation hasbegun iu the nucleus, though not as yet in the cytoplasm.

The nuclear plasm is of the same tint as the cytoplasm, andis irregularly disposed, instead of being regularly distributedas seen in controls and in the earliest stages of peptonefeeding.

The chromosomes are large and angular, drawn out intopoints, which frequently gives them a stellate appearance. Ihave not ascertained whether their number is constant.

The nucleolus is not very much smaller than normal.The nuclei of the third layer are globular or irregular in

shape, pale pink, nearly destitute of nuclear plasm, with afew conspicuous chromatin granules and a diminished nu-cleolus. They resemble fig. 32 of my former paper.

Ammonium Sulphate .—As Kiihne's amphopeptoue isprepared with ammonium sulphate, it was thought advisable toascertain whether the precipitates formed in the cells by feed-ing with atnphopeptone might be due to traces of this salt.Ammonium sulphate in 10 per cent., 100 per cent., and 200

210 LILY H. HUIK

per cent, solutions in distilled water was therefore applied tothe leaves. No precipitates resulted, and vacuolation of thecytoplasm was the only effect produced in the cells.

Some experiments were also tried with Witte's peptone,which is very rich in albumoses. The tentacles did not closerapidly, but in from one to four hours they had all closed.The bending caused was more rapid than that induced by thefibrin peptone. In about three days the leaves reopened.The material fed with Witte's peptone was accidentallyspoiled and rendered useless for histological examination.

Before describing, however, the changes induced by fibrin-peptone it is best, for comparison, to detail alterations causedin the gland cells by Griibler's fibrin.

Fibr in (stains lied).—Maximum change shown in fig. 11.The fibrin was reduced to a powder by crushing, and was thenapplied either dry or after being moistened with distilled water.If all the tentacles were loaded they closed in about tenminutes, otherwise only those that were touched closed, anddid so more slowly. The leaf itself did not double up as afterfeeding with white of egg. Cytological changes :

Five Minutes after Feeding.—The only observablechanges are that the cytoplasm is vacuolated in the upper halfof the cell, and the nucleolus somewhat diminished.

After One Hour (fig. 10«).—The cell wall is paler bluethan normal. The cytoplasm pale blue, much vacuolated ; fre-quently the cell is almost emptied. The nucleoplasm is aggre-gated. The chromatin is normal. The nucleolus has dimin-ished to about one half its original diameter.

After One Day (fig. 10 6).—The cell wall is almost colour-less. The cytoplasm is reduced to a scanty pale blue reticulum.The nucleoplasm is normal as to colour, or redder, aggregatedand diminished in amount, showing empty spaces. The chro-mosomes have enlarged. The nucleolus is always diminished,sometimes reduced to a mere point.

After Two Days (maximum change, fig. 11).—The cellwalls are colourless or slightly pink. The cell plasm is blue,extremely scanty. The nucleoplasm is thin, pale purple. The

OTTOLOGIOAL CHANGES PRODUCED IN DBOSKRA.. 211

chromosomes are enlarged. The nucleolus is small and dullred.

After Two to Four Day s.—Recuperation takes place as de-scribed for leaves fed with white of egg, but much more slowly,and is complete in four to seven days.

Griibler's fibrin-peptone stains red and produces the maxi-mum change in one hour after feeding, as shown in fig. 12.The food appears as pink drops adhering to the outside of thegland. The cell wall is nearly colourless, or faintly greyishblue. The cytoplasm is very scanty. In different leaves itvaries in colour from pale blue to pinkish grey. The nucleiare more or less shrunken ; the nuclear plasm reduced to alittle granular cloud which stains violet. The chromatin hasincreased enormously, and forms large segments with roundedends. Whether their number is constant or not I have notdetermined. The nucleolus is small and of a dull red tint.

Milk (stains Blue).—A small drop was placed on thecentre of the leaf or drawn over the marginal rows of tentacles.In the first case the leaf itself folded up. In the second case thetentacles closed regularly inwards in ten minutes or less, andafter an hour or two the entire leaf doubled over as in the firstcase. In from one to three days the leaves reopened perfectlyuninjured unless the tentacles had been here and there gluedtogether by the milk drying. Milk may be absorbed twice orthrice and the leaf open as clean as a control.

Five Minutes after Feed ing (fig. 13).—The cell wallsare somewhat deeper blue than normal. The cytoplasm hascontracted into dense strands, appearing coarsely granular andstaining a much deeper blue than normal. The nuclear plasmis somewhat redder than in controls, and the individualgranules have fused (aggregated) to form dark masses alter-nating with clear spaces. The nuclear chromosomes arenormal. The nucleolus appears more of a purple.

One Hour after Feeding.—The cell wall is normal. Thecytoplasm disappears completely except at the periphery of thecell and round the nucleus. It stains pale blue. The nucleiare sometimes slightly shrunken. The nuclear plasm is red-

212 LILY H. HUIB.

dish and becoming more scanty. The chromosomes are normal.The nucleolus is diminished.

One to Two Days after Feeding (usual type, fig. 14).—The cell walls are very pale blue. The cytoplasm is extremelyscanty, and stains very pale blue. The nuclear membrane isindistinct. The nuclear plasm is reddish and scanty. Thechromosomes have slightly enlarged. The description justgiven is typical for the state of " maximum change " usuallyproduced by milk in one to two days. It will be seen that thenuclear changes, especially those affecting the chromatin, arecomparatively small. I have found this to be characteristic ofmilk. In a few cases, however, the nuclear changes have pre-sented the appearance shown in fig. 15, which must be regardedas exceptional. The nuclear membrane is scarcely traceable.The nuclear chromosomes are much enlarged, angular, andvery clear. They closely resemble those produced by calciumphosphate feeding (see fig. 16). The nuclear plasm has almostentirely disappeared. The nucleolus is pale red and verysmall. Recuperation proceeds in the usual way, and is verycomplete.

Calcium Phosphate (fig. 16).—The salt did not adhereto the tentacles after fixation, and so does not appear in stainedmaterial. When treated with a mixture of the two stainsemployed it seems to take up both, assuming a violet tint.The salt was applied as a dry powder. The leaves closed fairlyrapidly. In two to three days they reopened, showing stillthe white powder apparently unaffected.

Changes induced in Five Minutes.—The cell wallstains a deeper blue than normal. The cell plasm shows aslightly increased vacuolation. The nucleus is contracted toabout two thirds of its normal size. The nuclear plasm stainsdeep blue. The chromosomes and nucleolus are normal.

One Hour after Feeding.—The cell wall is blue. Thecell plasm shows increased vacuolatiou. The nucleus is con-tracted. The chromosomes are normal. The nucleolus isdiminished somewhat.

One to Two Days .after Feeding (maximum change.

CTTOLOGICAL CHANGES PRODUCED IN DROSERA. 213

fig. 16).—The cell walls are pale blue. The cytoplasm isexhausted and very pale blue. The nucleus is contractedirregularly. The nuclear plasm is scanty—very pale violet orblue. The chromosomes are greatly enlarged. The nucleolusis diminished, sometimes excessively small, and stains dull red.

The maximum changes thus described closely resemble con-ditions produced by milk (fig. 16).

In Two to Three Days.—Eecuperation occurs. Thecytoplasm remains rather poor. The nucleus becomes quitenormal.

Globulin (stains Red) (maximum change, fig. 17).—Theleaves were dusted with the dry powder and closed rapidly,i. e. seven to ten minutes. The globulin was entirely absorbed,and the leaves reopened in about three days quite clean.Some of them were fed a second time.

Cytological Changes: Five Minutes after Feeding.—The cell walls stain very pale blue or purple. The cyto-plasm is diminished in amount, and large vacuoles appear in it.It stain9 blue or slightly purple. The nuclear plasm showsslight aggregations. The chromosomes and nucleolus arenormal.

One Hour after Feeding.—The cell walls do not stainat all. The cytoplasm is grey and scanty. The nucleus losesits sharply defined contour, and its outline is only indicatedby the dark blue circle of the somewhat enlarged chromo-somes. The nuclear plasm is aggregated, and has become moreeosinophilous. The nucleolus has diminished, and stains lessbrightly.

One Day after Feeding (maximum change, fig. 17).—The food in immediate contact with the apex of the tentacleappears at this stage striated with colourless lines issuing in aradiate manner from the apical cells, and suggesting thatsecretory products pass out through the apical walls. At thesame time the food seems to be passing in between the lateralwalls of the cells, which seem to stand apart, apparently sepa-rated by the red-stained globulin. The cell walls stain red.The cytoplasm is pale blue, or grey and scanty. The nuclear

214 LILY H. HUIE.

plasm is reduced to a few purplish granules. The chromatinis greatly increased, and is distributed in a few large angularsegments, which, however, do not equal in size those resultingfrom feeding with white of egg. The nucleolus is small, andstains dull red.

In Two to Four Days.—Recuperation takes place asin leaves fed with white of egg.

Casein prepared by " H a m m e r s t e n ' s " Method(stains Red).—The experiments, owing to the time of year,had to be made on Drosera capensis kept in a greenhouse,and I therefore do not regard them as satisfactory.

The powder was dusted on the tentacles, which closed slowly,taking several hours. The maximum change observed re-sulted in two or three days, when the cytoplasm had entirelydisappeared with the exception of a very narrow red peri-pheral layer, and a scanty remnant round the nucleus. Thenucleus remained normal in size and shape, but the nuclearplasm .was red and aggregated. The chromosomes showedslight enlargement, and the nucleolus appeared to be slightlydiminished.

Nuclein (stains Violet).—Nuclein was applied to theleaves dry, and also wetted with distilled water. It caused nomovement of the tentacles and no increased secretion, i.e.apparently no effect at all.

Five Minutes after Feeding.—The cell walls stain adeep violet colour. The cell plasm is somewhat vacuolated.The vacuoles appear blue. The nuclear plasm is unaltered inarrangement, but is sometimes of a bluer tint than normal.The nuclear chromosomes and nucleolus are unchanged.

One Hour after Feeding (maximum change, fig. 18).—The cell walls stain deep violet. The cytoplasm stains verydeep blue, and forms a network of thick strands enclosinglarge vacuoles, which appear blue. The nuclear plasm stainsthe same deep blue tint as the cytoplasm. It appears to beslightly aggregated. The nuclear chromosomes are normal.The nucleolus is somewhat smaller and more purple. Nofurther changes occur, and the cells become completely normal

07T0L0GICAL CHANGES PRODUCED IN DBOSEBA. 215

in one to two days. Fig. 19 shows such a cell nearly quite re-cuperated after twenty-four hours.

Nucleic Acid (stainsBlue) (ags. 20—23).—The acid wasprepared absolutely pure by Dr. Griibler. The powder wasmade into a paste, and a piece about the size of a pin's headplaced oil the centre of a leaf which folded up and secretedviolently for one to two days. At another time dry powder,or minute particles of the paste, were dusted on the tentacles,which then bent inwards and secreted copiously for one to twodays.

In both cases the leaves commenced to reopen in one tothree days, aud were perfectly uninjured.

Five Minutes after Feeding (fig. 20).—The cell wallsstain a much deeper blue than in controls. The cytoplasmshows very great vacuolation, and has contracted into thickstrands, forming a network which stains very deep blue, andhas a coarsely granular or knotted appearance. The nucleushas shrunk considerably. The nuclear membrane and nuclearchromosomes are unchanged. The nuclear plasm stains bluerthan normal, is somewhat aggregated and less dense. Thenucleolus is normal in size, but stains purple.

One Hour after Feeding (fig. 21).—The cell walls andcytoplasm show no further change. The nucleus has becomeirregular in shape, and usually has elongated transversely tothe long axis of the cell. The nuclear periphery is distinct, themembrane staining dark blue and remaining distended. Thenuclear plasm has contracted away from the nuclear membrane,and forms a small, compact, purple sphere round the nucleolus,suspended in the nuclear cavity by fine radiating threadsattached to the nuclear membrane. A homogeneous fluidwhich takes a pale blue stain appears to fill the space be-tween nuclear plasm and membrane. The chromosomes are notdistinguishable. The nucleolus stains dark crimson. From-mann's clear zone is obliterated by the closely contractednuclear plasm.

One Day after Feeding (fig. 22, a, b).—The cell wallsStain paler blue. The cytoplasm is very pale blue, and is

216 LILT H. HU1E.

reduced to a mere vestige. The nuclei are frequently enlarged,and may be spherical, oblong, or distended irregularly. Thenuclear plasm is absent or represented by a faint cloudiness inthe homogeneous pinkish fluid (?) which fills the nuclearcavity. The chromosomes appear as extremely minute, peri-pherally placed, blue granules. In some very clear nuclei theyappear to be connected by fine threads which also stain blue.The nucleolus is slightly smaller than normal.

One to Two Days (fig. 23).—The cell walls are pale blue.The cytoplasm is almost completely exhausted, very pale blue.The nucleus is small. The nuclear plasm stains as in controls,but deeper. The nuclear membrane is ill-defined and thechromosomes are normal. Nucleolus diminished and stainingdull red.

One to Three Days.—Recuperation takes place in a per-fectly normal way.

Glutin (stains Red).—In the first experiments glutinwas crushed into a powder and moistened with distilled water.Minute particles of this were placed on the tentacles. Thetentacles closed rapidly, in about five minutes. The wholesupply of glutin given to each leaf did not exceed an ordinarypin's head in bulk. In half an hour the particles had swollenand become white and translucent like milk. Dry glutin,even if powdered, was not conveyed by the tentacles to thecentre of the leaf with anything like the same rapidity;indeed, it appeared to be indigestible.

All the tentacles and parts of the leaf which had beentouched by the moistened glutin, as just described, died, i. e.became brown and withered, though the leaf as a wholereopened. Therefore the experiments were repeated with stillsmaller quantities of glutin. It was moistened and softenedwith distilled water, and placed with great care on the tentacleheads. There resulted complete absorption, and when theleaves reopened they were perfectly clean and healthy.

These experiments show what very different results mayfollow dissimilar applications of the same stimulus.

Five Minutes after Feeding. — The cell walls are

CVTOLOGICAL CHANGES PRODUCED IN DROSERA. 2 1 7

normal. The cytoplasm is normal as to colour, much vacuo-lated in upper third. The nuclear plasm is reddish purple,aggregated. The chromosomes and nucleolus are unchanged.

One Hour after Feeding. — The cell walls are pale.The cytoplasm stains blue, and is much vacuolated. Thenuclear plasm is reddish purple aud aggregated. The chro-mosomes are more prominent than in controls. The nucleolusis duller red and smaller.

One Day.—The cell walls are colourless or slightly pink.The cytoplasm is very pale blue, extremely scanty, merelylining the cell wall and surrounding the nucleus. The nucleiare often shrunken. The nuclear plasm is almost absent, andstains faintly blue or violet. The chromosomes are more orless enlarged. The nucleolus is dull red and small. Eecu-peration is normal.

In the tentacles which died the cytological appearance wasthe same as that caused by death from feeding with urea (figs.26 and 27).

Creat in (fig. 24).—Creatin was employed dry and alsowetted with distilled water. The latter proved the betterstimulant. The tentacles bent inwards slowly. Secretionwas not profuse. Some leaves seemed quite unaffected, andtheir tentacles did nbt bend. Those that closed reopened intwo days, and the white particles of creatin were adheringto their tentacles apparently unaffected. The examinationshowed conditions as follows :

One Hour after Feeding.—The cell wall is normal; thecytoplasm slightly pink, and greatly vacuolated in the upperhalf of the cell; the nucleus unaffected.

In the course of the one or two days in which the leafremains closed the further cytological changes are not great.

The nuclear plasm diminishes in quantity. The chromo-somes in,some cases increase slightly. The nucleolus dimin-ishes to some extent (fig. 24). Recuperation is complete intwo to three days.

Leucia (fig. 25).—Did not adhere to fixed tentacles, anddoes not appear in stained sections. Ijeucin was applied

218 LILT H. HUIE.

both dry and wetted with distilled water; the former provedmost active. In about 15 per cent, of the leaves used some ofthe tentacles bent slowly, taking about twenty-four hours toclose; but as a rule the tentacles did not move at all, thoughtheir secretory activity appeared to be stimulated. Some ofthe leucin was dissolved and hung from the leaves in largedrops, which finally fell off. In some cases it was not dis-solved, and fell off or was blown away. In any case the ten-tacles were to all appearance left quite uninjured.

Leaves were fixed at the following intervals after feeding :—Five minutes, one hour, one day, two days, three days.

The cytological changes seem to be produced very tardily.I find none during the first two periods. After one day thereis considerable vacuolation of cell plasm and nuclear plasm,and slight diminution of nucleolus. In tentacles that hadclosed there is slight but quite distinct increase of chromatinthree days after feeding (fig. 25). This has not been seen tooccur in tentacles which remained unbent.

Urea did not adhere to fixed material, and so does notappear in the stained preparations.

Crystals were powdered, and dusted on the tentacles. Theurea readily melted in the secretion, and seemed to increasethe exudation, but caused no bending of the tentacles. At theend of half an hour the leaves looked as if they had beenunder a shower of rain. In twenty-four hours the leavesappeared yellow, flaccid, with weak and shrivelled tentacles.Death of the leaf invariably ensued.

One Hour after Feeding (fig. 26).—The cell walls areblue. The cytoplasm is still blue and granular in the lowerhalf of the cells, but is vacuolated in the upper half. Thenuclear membrane remains irregularly distended, but thenuclear contents have contracted to form a small darkpurple body in the centre of the nuclear cavity, in whichneither nuclear plasm, chromosomes, nor nucleolus can bedistinguished.

One to Two Days after Feeding (fig. 27).—Thewithered tentacles show the cytoplasm reduced to a scanty

OTfTOIiOGICAr, CHANGES PRODUCED IN DROSERA. 2 1 9

reticulum of threads, staining blue, purple, or red. Thenucleus has almost lost its identity, having amalgamated withits surrounding cytoplasm to form a more or less crescenticbody, which stains reddish purple, and exhibits no differentia-tion whatever.

Summary.

1. By feeding with chemically different foods very cha-racteristic alterations are obtained, both in the colour re-action and morphology of the cell. For example, in fiveseconds white of egg causes both the cytoplasm and nuclearplasm to become more eosinophile, while pure amphopeptoneincreases their affinity for blue stains. The former food quicklycauses'great impoverishment of cytoplasm and nuclear plasm,while the first effects of pure peptone are to increase theirbulk and density. Both the foods produce an enormous in-crease of the chromatin element of the nucleus, while otherfoods, e. g. nucleic and nucleic acid, produce no such result.

2. While the cytoplasm is the cell constituent most rapidlyand most constantly affected by external stimuli, the nucleusis the seat of metabolic activity, and the state of the nuclearorgans indicates whether or not the food supply was of serviceto the metabolism of the plant.

It is not surprising, therefore, to find a certain independenceof cytoplasm and nucleus in their behaviour to externalstimuli, coupled with great interdependence with respect toall resulting processes of metabolism. Thus, substances suchas paraffin and nucleic acid act merely as stimuli to the secre-tive activity of the cells; and they cannot be regarded asfoods, for they affect the nucleus only by causing a slightdrainage on the nuclear plasm and nucleolus. If the stimulusis very transient the nucleus remains unaffected. On theother hand, with highly nutritious food like egg-albumin andpeptone the nucleus is the seat of the greatest change, andthe chromosomes, i. e. the nuclein, undergo an enormous

220 LILY H. HIKE.

increase, independently of the state of the cytoplasm. Withegg-albumin we do not get these great changes in the nuclearorgans till the cell plasm is thoroughly exhausted, while in thecase of 10 per cent, amphopeptone we get them while the cellcavity is full of cytoplasm.

In all cases the process of recuperation begins in the nucleus.The nuclear plasm first becomes abundant, and restoration ofthe cytoplasm begins in contact with the nucleus, and spreadsthence to the remoter parts of the cell.

3. To answer the question, whether the rapidity with whichchanges occur depends on the ease with which substances areabsorbed, we must deal only with such changes as are charac-teristic of foods. Changes that are produced also by merecontact with insoluble substances must not be taken account of.

Changes which are produced in five seconds are rapid, yet inthis short space of time we get quite specific alterations fortwo foods so different in their diffusibility as egg-albumin andpeptone. But then only the plasm is affected, that is that cellconstituent which I have just shown to be most rapidly alteredby external stimuli. Nuclear changes, e. g. the increase ofchromatin, must depend for their rapidity on the constitutionof the food, i. e. on the series of chemical changes the lattermust undergo before it is converted into basophile chromatin.In the two cases just cited the length of time required toproduce this change differs widely, being about twenty hoursin the case of egg-albumin, while oue hour suffices for peptone ;and yet from the changes wrought in the cytoplasm in fiveseconds we might argue that both foods entered the cells withequal rapidity. We may conclude, then, that the rate ofplasmic changes depends on the rate of absorption, but thatthe rapidity of nuclear changes is commensurate only with thedigestibility of the food.

4. In determining the relation between cytological changesin the gland cells on the one hand, and the degree of irritabilityin the leaves calculated from their rate of closure on the otherhand, cytoplasmic changes only constitute our legitimatecriterion; for I have shown above that the cytoplasm is the

OYTOLOCTIGAL CHANGES PRODUCED IN DROSERA. 2 2 1

cell constituent directly influenced by external stimuli, andthat nuclear changes are the secondary results of metabolism.A careful comparison of the action of the substances appliedto the tentacles proves that there is a constant concord betweenthe rate of closure of the tentacles and the degree of vacuola-tion produced in the cytoplasm. The citation of a few caseswill illustrate this :

Paraffin and nuclein caused no closure, and very slight andtransient vacuolation.

Pure peptone caused very slow inbending, and no vacuolationfor one or two hours.

White of egg and milk caused rapid bending and rapidvacuolation.

Fibrin caused slower closure and slower vacuolation.Creatin did not induce bending of all of the tentacles to

which it was applied; and those that closed did so with extremeslowness. The vacuolation was only transient.

Leucin sometimes, but not invariably, occasioned bending,and this was extremely slow. Vacuolation of short durationensued.

Urea caused no bending, but killed the leaves. Beingtherefore a poison, its action should not be compared with thatof the other stimulants named.

5. By bringing healthy cells into contact with the wasteproducts, creatin, leucin, and urea, urea was found to act as apoison, creatin as a mild stimulus to movement, and leucincaused active secretion without much movement. These twosubstances, therefore, do not injure the leaves, and they seem tobe of some nutritive value.

At a future time I may ascertain the effects of feeding withvarious carbohydrates, should the results obtained frompreliminary experiments give promise that such an investiga-tion would be of any value.

In conclusion I desire to acknowledge the kindness ofProfessor Gotch in allowing me to work in the PhysiologicalLaboratory at Oxford, and to express my indebtedness toDr. Gustav Mann for his constant advice and help.

222 LILT H. HU1E.

DESCRIPTION OF PLATE 22,

Illustrating Lily H. Huie's paper on "Further Study ofCytological Changes produced in Drosera."

All figures were drawn with Zeiss's camera lucida, Zeiss's y j apochromaticimmersion objective, and No. 8 compensating ocular. The magnificationequals 2000 diameters. All cells represent apical gland cells.

FIG. 1.—The resting condition (control). Vide p. 205.

F I G . 2.—After paraffin shavings, one hour. Vide p. 205.

F I G . 3.—White of egg, Qve seconds. Vide p. 206.

F I G . 4.—10 per cent, araphopeptone, five seconds. Vide p. 207.

FIG. 5.—10 per cent, ampliopeptone, five minutes. Vide p. 207.

FIGS. 6, 7, and 8.—10 per cent, ampliopeptone, one hour. Vide p. 208.

F I G . 9.—10 per cent, amphopeptone, two days. Vide p. 209.

FIG. 10 a.—Fibrin, one hour. 10 b. Fibrin, one day. Vide p. 210.

F I G . 11.—Fibrin, two days. Vide p. 210.

F I G . 12.—Fibrin-peptone, one hour. Vide p. 211.

F I G . 13.—Milk, five minutes. Vide p. 211.

FIG. 14.—Milk, oue to two days, usual type. Vide p. 212.

F I G . 15.—Milk, one to two days, occasional type. Vide p. 212.

FIG. 16.—Calcium phosphate, one day. Vide p. 212.

FiG. 17.—Globulin, one day. Vide p. 213.

Fio. 18.—Nuolein, one hour. Vide p. 214.

FIG. 19.—Nuclein, recuperating one day. Vide p. 215.

FIG. 20.—Nucleic acid, five minutes. Vide p. 215.

FIG. 21.—Nucleic acid, one hour. Vide p. 215.

FIGS. 22 and 23.—Nucleic acid, one day. Vide p. 215.

FIG. 24.—Creatin, one to two days. Vide p. 217.

FIG. 25.—Leucin, three days. Vide p. 217.

FIG. 26.—Urea, one hour. Vide p. 218.

F I G . 27.—Urea, two days. Vide p. 218.

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