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‘THE EFFECT OF CHLORAL HYDRATE ON THE PEANT CELL’
BY
WILLIAM CROCKER, A. B. ’o2.
THESIS FOR THE DEGREE OF MASTER OF ARTS
IN) THE
GRADUATE SCHOOL
©F PFHE
UNIVERSITY OF ILLINOIS.
1903.
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EFFECT OF CHLORAL HYDRATE ON THE PLANT CELL. 1. General.
From his study of mitosis in the epithelial cells of the salaman-— der, Rabl concluded that chromosomes never lose their individuality but remain distinct even in the reticulun of the resting nucleus, He thought that the reticulum is formed by the anastamosing of project- ing bridges of the chromosomes end thet these bridges are later drawn in and the chromosomes hold their former position in the newly orgérmn— inet spireme.
Boveri found in his studies of the abnormalities in the ages of Ascaris that the number of chromosomes that enter into a resting nu- cleus is equal to the number that reappear upon the later division of that nucleus. In cases where the two egg chromosomes were in some way sevareted each formed a nucleus of one-half the size of a normal nucleus. At the following division each nucleus showed only one chromosome.
For a review of the literature and a list of references upon the individuality of chromosomes see Wilson (The Cell in Development and inheritance, pp. 294-301).
Two facts breught out in establishing the individuality of chro- mosomes especially concern us in this naper. Any portion or any ex— cess of the number of chromosomes thet normally enter into the makeup of a nucleus (if they are in some way isolated), may form a nucleus. The nucleus thus formed varies in size with the number of chromosomes that enter into its construction; and the number of chromosomes that
appear at the following division is equal to the number that entered
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into its construction,
Strasburger believes the cytoplasm to consist of a fibrillar ac- tive portion, kKinoplasm, and an alveolar less active portion, tropho- Plasm. The cytoplasm can differentiate an additionai amount or kino- YPlasm. The kKinoplasm gives rise to the fibrillae of the achromatic figure and mscle fibers; constitutes the centrosomes, peripheral cel) layer (or dermoplast ), and the middle piece of spermatozoa; and forms the contractile vortion of cilia and flagella. Kinopiasin brings about, in the main, the more active or motor processes of the cell, while trophopl2sm is concerned with nutrition. They are distinguished morphologicaliy and by st ending reaction: kinoplasm is fibriliar and generally stained deeply by gentian violet and irom haemotoxylon; whiie trophopliasm is alvoelar and colored but siightly by these
stains.
II. Method of Experimentation. The root tip of Vicia Faba was used in these experiments. Con— siderabie time was spent in ascertaining the concentration of the so-
lution and the duration of exposure that brought about abnormalities
in the celi in greatest abundance, and yet left the celis and the en- tire root in a condition that they would continue growth when brought
back into normal surroundings. A two per cent. solution acting for
One hour kilis the root, while a one-eighth per cent. solution acting for twelve hours brings about scarcely any abnormalities, After con siderable experimenting I found that a one-half per cent. solution
acting for one and one-half to three and one-half hours produces near-
ly all abnormalities appearing in any of the cultures and produces
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The experiments from which the results are recorded were conduct- ed as follows: The seeds were soaked in water twenty-four hours, and then pianted in moist saw dust. After the roots had attained a length of 5-10 om. the beans were placed on a screen with the roots dipping into water, and left for several hours in order that the roots might adapt themselves to a water medium. At 10:50 A. M. five roots were killed and the others transferred tc a one-haif per cent. chioral hydrate solution where they remained for one and one-half heurs. At this time five more were killed and the others transferred to water to recover from the effects of the narcotic. At the expiration of every successive five hours (up to forty-five hours) five more roots were Killed. We have then in each experiment a series of preparations as shown in the following tabie:
No. 1 Fixed from water—-normal.
No. 2 i) immediately after the action of chloral hydrate.
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III. Effects on Cytoplasm.
In many ways the effects of chloral hydrate upon the cytopiasm and nucleus are so interrelated that the discussion of one almost necessitates the discussion of the other in the same connection. For convenience and order, however, the effects upon the cytoplasm wili be discussed first and those upon tne nucleus later.
in many sets of the preparations, tho not in ell, peculiar masses appear in the cytoplasm. These masses are more or less spherical and
non-granular. They stain with safranin, but far less deeply than
chromatin does. Three such masses are shown in the right of Fig. l and one in the left. The mass is often surrounded by a ring of vary- ing width which stains less deeply than the mass. The outer boundary of this less intensely stained ring is always clearly marked by a line of deeper red, as is shown in Fig. 110 the right. In the fig- ure the masses as well as the surrounding ring appear granular. They are really homogenous, but could not be so represented with the pen. This abnormality often shows a striking resemblance to the food vacuoles of Infusoria. Perheps not functionally, but direly in ap- pearance, the mass corresponds to the food particle and the surround— ing more lightly stained ring to the food vacuole. Frequentiy sever- al concentric rings centered by a mass appear. In such cases the rings are clearly bounded by darkly stained fibriller lines. Often
these bounding lines seem to be the succeeding coils of a spiral in-
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stead of a series of concentric circumferences. These masses were
found in No. 3 but never in Nos. 2 or 4. No. 2 often shows a coarse granular cytoplasmic structure. The masses, then, seem to form be- fore the roots have been out of the chloral hydrate five hours and to disappear before the roots have been out ten hours.
The cells that show these masses also show a tendency towards a coarse aiveoliar structure which is weli iliustrated in Fig. 1. It is not infrequent to find the entire cytoplasm of cells showing this
character. Of course in these cases no masses are present. There is
some evidence that leads one to believe that this alveolar structure is directly reiaited in its origin to the masses that appear in the cytoplasm, and that each alveolus results from the soiution or di- gestion of one of these masses. If this is the case, each of the
several smail alveoli in Fig. i resulted from the dissolution of a
relatively small mass, and later a larger alveolus will appear as the result of the dissolution of the large mass at the right. The as- sumption is supported by Sue fact that only small alveoli appeer in No. 3 while much larger ones are present in No. 4, and that the bounding walls of the alveoli resemble the bounding walls of the rings that surround the inasses.
If this assumption is right, between the time of Nos. 2 and 3 oc-
cur the formation of the masses and the dissolution of the smaller
ones forming small alveoli; while between the time of Nos. 3 and 4 the complete dissolution of the larger masses and the formation of larger alveoli. Perhaps far more conciusive evidence of the relation
of the masses to the alveoli would be obtained by killing several in-
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It must be mentioned in this connection that the masses and pecu- liar vacuoligation are entirely Limited to merismatic tissue and that they are most abundant in the most rapidly dividing cells. MTnis fact precludes any attempt to explain the vacuolization as a result of the aifferentiation of cells.
While the origin of the alveolar structure is not certainly es— tablished the source of the masses is perhaps yet more doubtful. If they originate from the nucleus one would expect to find some struc-— tural evidence of it, but in no case has any such morphological con- nection been found.
Dr. Hottes in his unpublished researches on the root tip of Vicia
Faba found that high temperature (438 C.) causes a great acceleration
in the activities of the cell-—especially in the growth of the spin-
Gle fibers—-and a very marked reduction in the size of the nucleoli. If, however, the roots were suddenly transferred to a low temperature and the rate of grewth thereby reduced masses appeared in the cyto- plasm, identical in appearance and staining characters to those found in chloral hydrate. In roots grown under pressure and thereby caused to do considerable work, he again found a great reduction in the size of the nucleoli. When these roots were released from pressure masses Similar to those described above appeared in the cytopiasim. These researches are very strong evidence in favor of Strasburger's theory that the nucleolus is a food organ, and they indicate strongly that
the peculiar masses in the cytoplasm are of nucleolar origin. If
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If these masses are of nucleolar origin, the substance imst be dis— solved, carried out into the cytoplasm and then precipitated.
It would seem that, if the masses are of nucleolar origin, those cells which show the masses ought to possess nucleoli that are smail- er than the nucleoli of normal cells. Such a relation is difficult to establish for two reasons: the amount of nucleolar substance re- quired to form the masses might be very slight and the variation in the size of the nucleoli in normal conditions is great. I covld de- tect no reduction in the size of the nucleoli. This matter might, however, be viewed in quite another way. Perhaps growth under high temperature, under pressure, and under normal conditions each has a rate at which the nucleolar substance is used up by the activities of | the cell. Perhaps aliso thet, if in the first two cases the roots are suddenly brought into normal conditions, the activities of the celis | ang the rate of using the nucleolar matter are lowered, and the
stream of food (less readily reduced in its rete) from the nucleolus is caused to precipitate instead of being used up by the activities of the cell. Cells of roots grown in normal conditions may likewise show a reduction in their activities and a precipitation of the nu- Cleolar focd material by being subjected to the influence of chloral hydrate, a narcotic. Such a supposition, however, has very meager experimental proof at present. After these masses have been produced in a greater variety of ways we can eliminate the unlike conditions and select the like and essential conditions with fap greater cer- tainty.
These masses are probably in no way related to the nebenkern
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certainly do not originate from remains of the spindle. It seems
very probable, however, that they are sinilar in nature to the neben-
kern found in the cells of the pancreas of Amphibia (Mathews, The Changes in Structure of the Pancreas Cell, Journal of Morphology, Vol. 15 Supplement ).
As we have seen, there is evidence that this formation of masses in the cytoplasm and its vacuolization are mtritive phenomena; thet is, effects upon the trophoplasm. There are also two very marked ef- fects of chioral hydrate upon the kinoplasmic element of the cyto- plasm. They are the tendency to bring about a lack of coordination in the action of the spindle fibers and finally the adatedetiod of the spindle.
it is evident that these effects wiii be shown only by those cells that are influenced by chlorai hydrate when they are in divi- sien. Fig. 2, which is drawn from a slide of preparation No. 5, material kilied fifteen hours waves the action of chloral hydrate, shows both these effects. This cell Was probably in division when the material was in the solution of chloral hydrate (the evidence for this will be brought out later). One-half the daughter chromosomes have been thrown to one pole in the customary way and are organizing &@ regular nucleus. The other half of the daughter chromesomes have been thrown to the other pole, but in two greups connected by a bridge of one or two chromosomes; and they are organizing a very ir- regular nucleus consisting of two larger portions connected by a nar—
row bridge. The material of the spindle is very evident between the
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two organizing nuclei; but the individual fibers are entirely broken
up. At this stage the spindle would ordinarily have gone far towards
the construction of the cell wall; but here the spindle is so badly injured that it is doubtful whether the cell wall will be constructed
at all, and certainly not in the ordinary way. Fig. 3 shows another
| cell from preparation No. 5. The spindle here has begun the forma-
tien cf the cell wall, but a central bridge of chromatin connects the
two organizing nuclei. This central bridge of chromatin probably re—
sulted from the destruction of spindle fibers at the time they had
drawn the last two chromosomes only part way to the poles. Fig. 4
shows e@ similar condition excepting that the wall formation has not
| progressed so far and the bridge of chromatin is at one side instead
of central. Fig. 5 shows two masses of chromatin organizing nuclei,
the remains of the disorganized spindle, but no sign of a cell wall.
It is probabie that this cell would later have contained two resting
nmuciei as is the case in Fig. 6. Fig. 8 represents three almost e- qual organizing nuciei in the same cell without any sign of a cell wall forming to separste them. This drawing was taken from prepara—
tien No. 7, material killed twenty-five hours after the action of
chloral hydrate. It seems to represent 2 marked case of the broken
coordination of the spindle fibers and the final destruction of the
spindle. Probably this ceil would iater have shown three resting
nuclei.
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spindle; while the other figures mentioned above show only some other
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“injury to or the destruction of the spindle. In many of the prepara-— — there are mitotic figures in which the coordination is clearly broken while the spindle still seems to be in goed condition. Fig. 7 from preparation No. 7, material killed twenty-five hours after the action of chloral hydrate, is a striking example of this. Here the chromosomes have been thrown in three directions and a typical tripo-— lar figure formed. The poles are of unequal size: the lower one seems to be One pole of a regular bipolar figure, while the other two poles seem to have originated from the division of the chromosomes that would normally have formed the other pole. Such tripolar tripolar ‘figures were not of very frequent occurence in the preparations——not
| more than a dozen were ever found in a single slide.
These figures were not found in material Killed immediately after
the action of chloral hydrate, but first appeared in material killed fifteen hours later and were most abundant in material killed twenty— five hours later.
It seems peculiar that the chromatin masses of Fig. 7, which is from material killed twenty-five hours efter the action of chloral hydrate, have not yet begun to construct nuclei; while the chromatin masses of Figures 2-5, killed fifteen hours after the action of chloral hydrate, have progressed far in the organization of nuclei.
Three explanations for the apparent slowness of the chromatin in the
tripolar figures may be suggested. These cells may have begun divi-
sion some time after the roots were transferred from chloral hydrate
to water, and thereby may have been influenced in its division by a
concentration of chloral hydrate much below one-half per cent. due ito
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= i its diffusion thru the water. In this cease we mst assume that the low percentages broke the coordination of the spindle fibers without otherwise seriously injuring the spindle. If this supposition is true lower percentages of chloral hydrate ought to _—_— tripolar figures with the spindles in good condition. A series of experiments with low percentages acting for different durations of time produced no such effects; hence this suoposition seems invalid. It may be that cells as represented in Fig. 7 were in division when the roots were in chloral hydrate, but that the chromatin was exceptionally
slow about organizing nuclei. It is peculiar, however, that a cell
so sturdy in one resvect—as is indicated by the condition of the spindie—--is so siow in its functioning in another. It might again be | supposed thet the cytoplasm retained the effect of the chloral hy-
arate and that this retained effect was sufficient to destroy the co-
Ordination of the spindle fibers without otherwise seriously injuring
the spindie. This supposition seems to agree best with the data.
Often when the figure is bipolar the chromosomes are distributed very unequally to the two poles. This is probably the explanation of
the marked difference in the size of the two nuciei in each of the
Celis of Pig. 9.
Hertwig, Galiotti, and others have produced asymuetricai mitosis by the use of poisins and other chemical substances (quinine, chlo-
ral, nicotine, etc.). Klebs, Hausemann and Caliotti have demonstrated
its frequent occurrence in abnormal growths, such as cancers and tu-
|mors. Galiot+ti produced asymmetrical figures in the epithelium of
the salamander by the use of cocaine, antepvyrin, and quinine. In eli
: ihe : ; eh,
. a
Pech ems Oke
tedi? wibiies oat Ic sot satonas Dey + wo 4 manta | _
. = ee Hen sit a ILA sehen orm
subery of fétyeo steam er be Me apesneoiend
f ae _ ' for, S e : ee
an . ws nar ed ! necnebeg etd: ;
. 7 iy 7
“ fae may) “ Hes . ‘a yithe es. epit Meem
~ » «+ * = al # " .. satel Ae 2 tteaat t 1 Pe 2 ae F , eS @ dA — hb 4 + - 4 4 Ou : eet ‘ * > ' - 1 s “a = oe & * . ‘ ry ir + a ~ a Ie Gs. vm mn PGS ' » 1 * fon : 4 r + (Q i% if i di b Ll + a ee A i: FF ata - * na “” aye ) < x + Bey os" bm + <) = R SS } fea Ee _— ° > o - 94/8 Lg = nN 4 © 7 \ ts Awe ’ Jy ic? -» 44 i why ibe os. © vi a ° P Ma ae : h i , Sas i- * * ef ,@ ~ +2 * é é = . ner? Bi x + ¢ mer the ah sii ea dal - éh . ar Ory oT. ® 4 q ;
po. toe os Segoe td sVat eteiity Bi ,tisoline, 4
Sime, ionteado -oodto. _parsiyen te sm.
CD ec mere! hawt
. +1 =
i! Oi apie wee jos sBILMOTSE iaormeane. at eon — a
, > 3o. ubthediige.~us 5 ead seine we”
hd ai on Jifap ee icy tea SHR OD ETD oe 4 av: nf t
= q >." ™
atte f
'these cases of asymmetry both bipolar figures with poles of unequal
| | size and multipolar figures were found along with a greater number of | symmetrical figures,
| Lustig and Galiotti noticed that the unequal distribution of the chromatin is always accompanied by inequality of the centrosomes, which in turn produce unequal amphiasters., They concluded from this that the unequal division of the centrosome is probably the cause of
the unequal distribution of the chromatin. Wilson believes that the
_tripolar figure is formed by one of the two centrosomes dividing anda “thereby forming three amphiasters instead of two and that quadripolar figures are formed by both centrosomes dividing and forming four am— “phiasters. Whatever value this explanaticn may have for cells that “posess centrosomes, it certainly can not hold for forms in which there is no evidence of the existence of centrosomes, as is the cese in
| Vaeta.
Por a review and list of the literature on asymmetrical and path- Veaeeies) mitosis see Wilsen (The Cell in Development and Inheritance, pp. 97-99).
| IV. Effects on the Nucleus.
It is evident that the two effects upon the kinoplasm which were just discussed bring out peculiar modifications of the nucleus and “naturally lead up to the discussion of the effects on the latter. Be— fore discussing the effects upon the nucleus, however, we must review
an article recently published by Waldemar V. Wasielewski ( Theoritische
und Experimeitalle Beitrage zur Kenntniss der Amitosis, Jahrblcher f.
wiss. Botan., Bd. XXXVIII, Heft 3, pp. 377-420) in which he claims to
a fe
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west tuum. ow , to vo ro ena ott tear ntow rte. enouiéisoom®? pines lelssWeay aera lovee eves sdent 1 mo io2otte\ ¢ettodion deta aaa 3 Oftnetasn eaidakae abe {ORS ENE ih Oe 4 TA
| -13-
have produced amitosis in the root tip of Vicia Faba by the use of ecebenen hydrate. At the time his paper appeared I had almost com- pleted my researches and had arrived at essentially my present conclu- sions so far as the features discussed by Wasielewski are concerned.
After a rather extended review of the historical and theoretical
features of the Knowledge of amitosis he tekes up the discussion of
his researches. He mentions that he started ovt in his experiments
with very definite aims; first to find a means of producing, in abur-
dance and with certainty, amitosis in the embryonic tissue of flower- ing plants (root tip of Vicia Paba); then to study the behavior of
the nucleus and cytoplasm in this process,
Follewing the suggestion of Nathansohn's work on Spirogyra ( Phys—
iologische Untersuchungen Uber amitotische Kerntheiiung, Jahrb. f.
Wiss. Botan. Bd. 35) he sought to accomplish his aims by the use of ether, but after considerable variation in the concentration of the solution and duration of exposure he was unsuccessful. Still believ—
ing that the results of Nathansohn were due to narcotic action, he
turned to chliorals and here met with success. After considerable ex—
perimenting he found that a one-half per cent. chioral hydrate solu— genes acting for one hour produced abundant amiotic divisions, if the roots were then washed in flowing water for one hour and afterwards grown in sawdust for twenty-four hours.
He says that the first sign of amitosis is the doubling of the “nucleolus. He describes this doubling as taking place in @e typical
Manner: the nucleolus becomes considerably elongated, is later re—
stricted at the equator, and finally cut into two daughter nucleoli.
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aay
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’ i 4 iia ut ia of tet? enoténese aN Jae
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— a . 4 » die ia fg - <« < k jsoege “i Jupyieab eH (26 , ba
2 ol setteeomos oft mt dolta tray oft weiterteo wo dte.
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Before the division of the nucleus is completed the daughter micleoli often begin a second division. In only one case did he find the sec- ond division taking place simultaneously with the first. By what he took to be an unbiased count he found that 1249 per cent. of the cells in the tip of a normally grown root showed double nucleoli; while 25.9 per cent. of the cells of roots exposed to one-half per. cent. chloral hydrate for one hour and then put in flowing water Tor one hour showed douwhle nucleoli. He describes the doubling of the ‘nucleolus as half completing the amitotic division of the nucleus, Aside from the doubiing of the nucleolus and a slight elongation of the nucleus an Serey eecoaiiay dividing nucleus is not at first un- like an ordinary resting nucleus. After these two initiative steps the reticulum begins to divide, generally into two equal parts, tho
sometimes into parts of very different size. In this connection he
names and describes two sorts of direct nuclear division. One he terms diaspase (distraction) of the nucleus. This, he points out, is found in Saccharomycetes and Valonia and begins, after the division
of the nucleolus, with the nuclear substance traveling to opposite
“poles and forming a dumb—beil—shaped nucleus which finaliy forms two nuclei by the gradual narrowing of the restricted boundary. He em
phasizes the fact thet the nucleolus and nuclear reticulum are active
in this process, while the nuclear membrane is comparatively inactive.
He terms the other sort of direct division diatmese (dissection) of
the nucleus and cites as an example the internodal cells of Chara. He
characterizes it as being marked by great activity or the nucleolus
and the meclear membrane, which cuts the nucleus in two, and compara—
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1 5
tive inactivity of the nuclear reticulum. In the direct division of
Spirogyra Nathansohn noticed that the nucleus is divided by a deeven-
ing restriction that extends entirely around it. In his researches
Wasielewski observed that the restriction is a half ring. He de-
scribes it as a modification of diatmese of the mucleus and thinks it the only form of direct division existing in his preparations. He mentions the difficulty of explaining why the nucleolus and nuclear membrane, which are normally inactive during division, become very active under the influence of chloral hydrate; while the nuclear re- ticulum, normally active in division, become inactive under the same influence. |
Schmitz and Fairchild described a peculiar sort of mitosis in Valonia. The nucleolus dissolves; and chromosomes are formed but
they do not split longitudinally. The nuclear substance, enclosed in
the sac-like nuclear membrane, now moves towards opposite poles and forms 2 dumb—bell-—sheped mass which finally separates into daughter nuclei. Wasielewski terms this hemimitosis. Beginning with a sort
of division that shows great activity of the mecleolus and nuclear
membrane but inactivity of the nuclear reticuium, he believes we can
pass step by step to a sort of division that shows inactivity of the
nucleolus and nuclear membrane but great activity of the nuclear re-
ticulum. He mentions the stages: diatmese, diaspase, hemimitosis,
mitosis. He claims to find forms which resemble the peculiar mitosis
in Valonia and which are intermediate between his diatmese and mitosis. He noticed that amitotically dividing celis are very slow about
building their walls. Considerable time elapses between the completed
; (ast) ,sienidekh tage to =
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atte division of the nucleus and the beginning of the cell wall. This he believes explains the existence in the preparations of many binuclear cells such és seen in Fig. 9. Indeed the wall formation is often de- layed so long that the two nuclei begin a second division (mitotic, however, now) without any sign of a wall separating them (see Fig.10). He describes this as daughter division beginning before the mother di- vision has completed itself, and believes that the delayed wall will finally be built. If this wall were not built the inner poles of the two mitotic figures in Fig. 10 would form a binuclear celi. This would reappear after every successive division and versist thruout.
He found no binuclear cells in material killed fifty-five hours after the action of chloral hydrate.
The cell wall, he found, is generally built between the two nu- Clei. Occasionally, however, it is started at the same side of both nuclei, but in such cases is never completed. In older ceils which do not contain sufficient cytoplasm to fill the entire lumen of a cell, a bridge of cytoplasm stretches entirely across the cell be—- tween the nuclei. The wall is built along this bridge of cytoplasm: sometimes at one border of the brdige; sometimes centrally thru its mass. Often the nuclei are near the place of wall construction, tho not aiways; aid the wail is often piaced in a cieft of the cytoplasn, tho frequently not. He notes that the wall begins on one border of
the cell and builds across as @ growing half ring; and puts great stress upon the fact that it resembles the method in lowly organized plants where the wall begins as a ring on the entire periphery and
continues its growth by additions to the inner edge of this ring.
4
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{iow e 20 Qpte\ ea sped? ty.
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Both amitosis and this peculiar wall rormation he believes to be atavic characters brought on by the influence of the narcotic. He thinks: the narcotic pushes the cell back myriads of generations ina few minutes; effaces those characters latest acquired, such as mito- sis; and brings into appearance the primitive ones. He compares this to the effect of an anasthetic on man. Under its influence those characters latest acquired, such as consciousness, fade away first; but the deeper-founded constitutional characters are the last to suc-— cumb.
He then asks the question: Can a nucleus that has divided ami- totically latex divide mitotically? and answers it in the affirmative. After working out two almost metaphysical reasons for this answer, -he then gives what he considers conclusive evidence. The amitoti- caliy divided nuclei can, he believes, be distinguished from those of mitotic formation by the delay in building the wall. He finds the nuclei, which are thus distinguished as of amitotic origin, later di- viding mitotically (see Fig. 10). He was in no case able to totally suppress mitotic division and have present only amitosis,
In his cultures he used one-half or three-fourths per cent. solu- tion, acting for one hour. His list of cultures are:
No. 1 killed immediately after the action of chloral hydrate,
No. 2 " IaL/2 hours)" " " " " " No. 3 . 3 " ” ” “ r) ” . No. 4 “ 7 a" tt u " tt ” " No, 5 Be 25 " " ” i" ” ” "
No. 6 Ris Bd ” " " " ” tt
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I
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i “ 4 ~ . . . ton ve vee wo 4 . . e a) ~
ao fo $Aquose
- r . “ - 12k wv atta LaF a > x ; = nk. Th4 2 ia Pr Lay s ‘ - rs - * ¢ y 4 ga d —~ — ”- “mr + > ogg a Mors “bits - an — GL A he
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No. 7 Killed 48 hours after the action of chloral hydrate.
No. 8 Be eS yess " " ” ” ” ”
He found amitosis (diatmese) first appearing in No. 4; most abun- dant in No. 5; and last present in No, 7. The abundance of amitosis, he notes, forms a very regular curve with the maximum in material Killed twenty-four hours after the action of chloral hydrete.
While he found bimiclear cells in abundence he in no case found
celis containing more than two nuclei.
There is one other point brought out in his paper, which we must mention, before beginning a discussion of the article. He noticed many nuclei of very irregular form (see Figures 11 and 12). ‘These nuclei as well as the celis that contain then are much larger than
the surrounding ones. Often, too, there are three or four nucleoli
in each nucleus. He was unable te give the significance of the large
size of the nucleus and cell or of the numerous nucleoli. He believes,
however, that in part, at least, the irregularities of the nucleus are due to amoeboid movements. He mentions this tendency towards amoeboid movements as another effect of chloral hydrate upon the nu- cleus.
Nearly all abnormalities of the nucleus mentioned in Wasielewski's article were found in preparaticns similarly treated; but there were many forms found in mine which he makes no mention of and which iead
me to quite a different interpretation of structures he explains as
amitotic. Many of these structures appear in preparations corres- ponding to which he has none, for example, material killed fifteen
hours after the action of chloral hydrate.
, : - ie bo: agile tine —- ~ 8, Fe 100g. 10 Poise fends ’
veiltld: fo tro lee ,eiaosx6e. to. om
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In my original cultures I had only three preparations: material kilied immediately after the action of chloral hydrate, material killed five hours later, and material killed twenty-four hours later. In material killed twenty—four hours after the action of chloral hy- drate I was struck by the abundance of forms resembling Fig. 15-- Wasielewski's typical diatmese of the nucleus. I at first interpreted these as amitosis, but sought to make this sure by finding the gene— sis and fate of the forms. To this end I began conducting my exper- iments as shown in the table given early in this paper. I found in material killed five and ten hours after the action of chloral hy-
drate many forms like Fig. 14, and in material Killed fifteen hours
after this action numerous forms like Figures 3, 4 and 5. It is evi-
dent that when the chromatin mass of Pig. 14 organizes a resting nu- Cleus, this nucieus will show a considerable indentation on one side not unlike that shown by the resting nucleus of Fig. 13. The seme is true of the chromatin mass of Fig. 4 which is already well started with the nuclear formation. Again I find, occasionaliy in prepara- tions killed twenty-five hours after the action of chloral hydrate and frequently in preparations killed twenty hours after this action, forms like Fig. 16. This is clearly one of the nuclei which Wasiel- ewski describes as dividing amitotically (by diatmese) in the spireme stage of mitotic division. When compared with the nuclei cf the small cell of Fig. 18 or the mononuclear cell of Fig. 17, we see that the mclei of Figures 13 and i6 are of about double size.
These facts suggest, at least, an explanation--differing radical-—
x” from Wasielerski's both as to origin and fate of the structures—
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for nuclear forms iike Fig. 15. The spindle of Pig. 14 was wrobably in the act of pulling the chromosomes to the poles when afrected by the chloral hydrate. The narcotic broke up the coordination of the spindle fibers and the simultaneous movement of the chromosomes to the poles, and finally lead to the destruction of the spindle. As a re— suit of the broken coordination and final destruction of the spindle
@ lateral bridge of chromatin connects the two larger masses that have been drawn to the poles. Such forms as Fig. 13, then, do not result directly from the effect of chloral hydrate upon the nucleus but in- directly from the effects of chiorai hydrate upon the kinoplasmic element (spindle) of the cytoplasm. These effects we have discussed in the early part of the paper. It is evident that the number of chromosomes entering into the construction of such nuclei as that of Fig. 13 is the mumber that would normally construct two nuclei. If accordance with the features of the individuality of chromosomes dis— cussed in the early part of this paper these nuclei are about double
the size of normal nuclei. For convenience they will be termed giant
My preparations lead me to believe that binuciear celis, aiso, originate from the destruction of the spindie. In preparations killed five and ten hours after the action of chlorai hydrate many cells are found in which the chromatin masses are thrown to opvosite poles with the disorganized spindle mass between them, but no sign of a cell wall. In preparations kiiied fifteen hours after the action of chioral hy- arate many forms iike Fig. 5 appear, In these the chromatin masses
have already begun the organization of nuclei. Here, toc, the disor-
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withing ganized spindle mass is still present. In Fig. 6, material killed twenty-five hours after the action of chloral hydrate, the resting nuclei are formed and the spindle has almost entirely disappeared. Fig. 10, material killed thirty-five hours aftér the action of chlo- ral hydrate, shows the two nuclei of a bimelear cell in division. Very often such binuclear cells show the wall partly built as is the case in the celis of Fig. 9. These, I believe, are identical with the partly built walls reported by Wasielewski, but of course he could not interpret them as being built by the spindle and yet main-— tain the amitotic origin of the two mclei. My preparations have led me to believe, however, that all partiaily built walls that Wasiel- ewski reports are built by the spindle in the ordinary way before its destruction, and that they are never completed, Wasielewski fails to see the connection of these partially built walls with the spindle, because he takes only end products, that is, observes the forms after the resting nuclei are fully organized and the remnants of the spin- Gle have disappeared. In Fig. 3 we find that, altho the chromosomes were not all pulled apart, the wall had progressed far in its con- struction before the spindle was destroyed. Here, toc, contrary to Wasielewski's idea, the cell wali is first constructed in the central part of the cell. I found a number of bimuclear cells already in mi- totic division which showed the cell wail between them only vartially built. Wasielewski has little if any evidence that such a wall will ever be entirely built and he has still less evidence that his "in- complete mother Givision” will ever be completed by the building of
the lacking wall. Contrary to his observations, I found binuclear
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cells in the old specialized tissue of the root in material killed sixty-five hours after the action of chloral hydrate. This indicates that his “incomplete mother division" has not yet been completed. It is my belief Chat. pamnex aun ceélis originate from the destruction of the spindle before the wall is even partly constructed.
(It must be stated in this connection thet neculiar wall forme- tions, not at all to be connected with these just discussed, are found in many of my preparations. I am at present umable to explain their significance. I have material in preparation to clear this point. )
My preparations indicate again that Wasielevski's large irregular nuclei (Figures 11 and 12) are produced by the broken coordination of the spindie fibers and the finak destruction of the spindie. In such cases Only a few of the chromosomes are thrown out from the mother aster in @ scattered manner, when the destruction of the spindle oc- curs. Since such nuclei contain the chromosomes that ordinarily form two muciei they are doubie normal size. If the spindle is destroyed before any chremosomes have been drawn from the mother aster a regu lar giant nuclei will be formed. I believe the large nucleus of Fig. 18 has originated thus.
Wasielewski mentions that he failed to produce similar abnormali- ties of the nucleus by the use of ether. Dr. Hottes in his unpub— lished researches reports similar forms produced by a ten per cent. ether solution.
Contrary to Wasielewski's resuits I found many trinuclear and a
very few quadrinuclear cells (Figures 8 and 17). We have already ex-—
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Plained that these originate by the spindle fibers throwing the chro-
mosomes in three or four different directions, forming as many dis-
tinct groups of chromatin, The average size of such nuclei is, too, it must be noted, approximately inversely as the number. This agrees with the features of the individuality of chromosomes already dis- cussed. Notice that the nuclei of the large cell of Fig. 1” average about half the size of normal nuclei.
Wasielewski has emphasized the doubling of the nucleoli as evi- dence cf amitosis. Such a relation is hard to establish, because of
the great variation in this respect even in normal material. I con-
nect the fragmentation of the micleoli with nutritive processes ( a- greeing with Strasburger's theory of the function of the nucieolus), rather than with division.
Nathansohn, in an article already cited, claims to have produced
amitosis in Spirogyra. His arguments are ably answered in an article
by Hacker (Mitosen im Gefolge amitosenahnlicher Vorgange, Anatomischer Anzeiger 1 Jamar 1900 Bd XVII), and by forthcoming works of Dr. Hottes, Hacker terms the distorted mitosis which we have described as pseudoamitosis and maintains that it has no other meaning than a distortion of the karyokenetic precess by interference with its deli- Cate machinery. V. Methods tised by Wasielewski.
Some very serious faults may be urged against Wasieleyski's meth—
od of research--—fauits which wndeubtediy go far in leading him to
misinterpret these abnormalities of the nucleus.
1. He started out with the intention of bringing about a certain
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result-—-amitosis. Under the influence of this aim anything that had the appearance of amitosis seemed to be interpreted as such without a scrutinizing test tc see if it could be interpreted otherwise.
2. He took, almost entirely, end products-—-material killed twenty-— four hours after the action of chloral hydrate.
5. He failed to make preparations that would show the genesis of these end products—-material killed fifteen hours efter the action of chloral hydrate.
4. Finaliy he ignored or failed to see forms that would clearly show the fate of these products (especially his diatmese of the nu- Cleus )—-spireme stages as shown in Figures 16 and 20.
VI. Weakness of the Hypothesis of Amitosis.
The hypothesis—-that these abnormalities of the nucleus are due to amitosis--shows some very weak points:
1. It assumes a thing which Wasielevski acknowledged is peculiar and which, it seems, is highly improbable: "Wie es Kommt, dass die Chloraibehandlung bei einem Theil des Kernes, dem Gerust, eine inac- tivirende, lahmende Wirkung hat (indem die hier sonst eintretenden Substanzsonderungen und-verschiebungen unterbleiben), bei anderen Theilen dagegen (Nucleolus und Kemmembran) eine activirende, erregende Wirkung (indem wir hier sonst nicht auftretende Bewegung sich voll- zgiehen sehen), wissen wir nicht.”
2. It gives no expianation for forms Like those shown in Figures Pe 4 ey. 8, pnd 14. i
3. It again breaks down in the presence of forms like Pigures 16
and 20.
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VII. Conclusions.
i. Chloral hydrate brings about the denosition of certain masses in the cytoplasm.
2. These masses may be of nucleolar origin and they may be ceused by the chloral hydrate lowering the cytoplasmic activities without greatly reducing the food current — the nucleolus.
5. The chloral hydrate breaks up wthe coordination in the action of the spindle fibers and finally destroys the spindie.
4. Many abnormal nuclear structures--—polynuclear cells, irregular giant nuclei, regular giant nuclei, and nuclei which Wasielewski de- scribes as dividing by diatmese--result from the effects mentioned in .&).
5. The retained effects of the chtoral hydrate often break up the coordination of the spindle fibers without destroying the spindle,
thereby producing tripolar figures with the spindle in good condition.
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