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Journal

OF THE

Royal Microscopical Society;

CONTAINING ITS TRANSACTIONS AND PROCEEDINGS

AND A SUMMARY OF CURRENT RESEARCHES RELATING TO

zooXiOG-^sr -A-3sriD B o T .A. nsr ^2-

(principally Invertebrata and Cryptogamia),

PRANK CRISP, LL.B., B.A.,

One of the Secretaries of the Society and a Vice-President and Treasurer of the Liftttean Society of London ;

WITH THE ASSISTANCE OF THE PUBLICATION COMMITTEE AND

A. W. BENNETT, M.A., B.Sc, I F. JEFFKEY BELL, M.A.,

Lecturer on Botany at St. Thomas's Hospital, \ Professor of Comparative Anatomy in King's College,

S. O. RIDLEY, M.A., of the British Museum, and JOHN MAYALL, JuN.,

FELLOWS OF THE SOCIETY.

.-VOL. III. TiHrtlL

PUBLISHED FOR THE SOCIETY BY

WILLIAMS & NORGATE,

LONDON AND EDINBURGH. I 885^

\jol- 3

)p BI-MONTHLY. SE^ \

Vol. III. No. 4.] AUGUST, 1880. [ "^ p^ce 487''

f

Ai.

Journal

OF THE

Royal Microscopical Society;

CONTAINING ITS TRANSACTIONS AND PROCEEDINGS,

AND A RECORD OF CURRENT RESEARCHES RELATING TO

INVERTEBRATA, CRYPTOGAMIA, MICROSCOPY, &c.

Edited, tinder the direction of the PiMication Committee, by

FRANK CRISP, LL.B., B.A., P.L.S.,

One of the Secretaries of the Society;

WITH TUB ASSISTANCE OP

A. W. BENNETT, M.A., B.Sc, j F. JEFFREY BELL, M.A,.

Lecturer on Botany at St. T/umias's Hospital, | Professor of Contparaiive Anatomy in King's College,

AND

S. O. RIDLEY, B.A., F.L.S.,

Of i/ie British Mtisctim,

\

FELLOWS OF THE SOCIETY,,.-

WILLIAMS & NORGATE,

M LONDON AND EDINBURGH. ^i^l

PRINTED BV WM. CLOWES AND SONS, UMITkD,] [STAMrORD STKBET ANf CIIARJNO CRO..S.

( 2 )

JOUENAL

OF THE

EOYAL MICEOSCOPICAL SOCIETY.

VOL. III. No. 4.

CONTENTS.

Tbansactions of the Society paob

XVI. Notes on Aoinetina : Trtchophrya epistylidis, and Podo- PHRYA QUADRiPARTiTA. By John Badcock, F.R.M.S. (Plate

XIV.) 561

XVII. On the Visibility of Minute Objects mounted in Phos- phorus, Solution of Sulphur, Bisulphide op Carbon, and other Media. By J. W. Stephenson, Treasurer R.M.S.,

F.R.A.S .. 664

XVIII. On the Development and Retrogression of Blood-vessels. By George Hoggan, M.B., and Frances Elizabeth Hoggan,

M.D. (Plate XV.) 568

XIX. On a Parabolized Gas Slide. By James Edmunds, M.D.,

M.R.C.P. Lond., F.R.M.S. (Figs. 52 and 53) .. .. 585

Record of Current Researches relating to Invertebrata,

Cryptogamia, Microscopy, &c. ,. .. .. .. 587

Zoology.

Bevelopmeut of the Vertebrate Eye 587

Embryology of Batrachiuns 587

Vital Properties of Cells 589

Coalescence of Amoeboid Cells into Plasmodia 590

Structure and Development of Dentine 590

Ovary of Mammals 591

Influence of Saline Solutions on Protoplasm 591

" Law of Association' 592

Degeneration 594

Animal Development 597

Colours of Animals 598

Organisms in Ice from Stagnant Water .. 598

Fertilization of the Ovum 599

Renal Organs of Invertebrata 600

Phylogeny of the Dibranchiate Cephalopoda COl

Aptychi of Ammonites 604

Development of the Pulmoimte Gaderopoda 005

Generative Organs of (he Young Helix aspersa 608

Gasteropoda from the Troas 608

Gasteropoda from the Auckland Islands 608

Marine Polyzoa 609

Fresh-water Polyzoa 609

Larva of Bowerhankia 611

Enldi minaria ducalis 611

( « )

Beoord op Current Kbskarohes, &c. continued.

PAQB

Nervous Collars of Arthropods 611

Nerve-endings in Muscles of Insects .. 612

Habits of Ants 613

Bespiratory and Circulatory Apparatus of Dipterous Larvx 615

Blepharoceridse 616

Tracheal System of Larval Libellulidm 61(j

Hemains of Branchias in a Libellulid: Smooth Muscle-Fibres in Insects .. 618

Metamorphosis of Prosopistoma 618

Piercing Organ of the Lepidopteran Proboscis 619

Generative Glands and Sexual Products in Bombyx mori 620

Development of Forficula 621

Adora sestuum from the Shore at Heligoland 621

Destruction of Noxious Insects by Mould 622

Development of the Araneina 622

Peculiar Modification of a Parasitic Acarian 624

Structure of Trombidium 625

Central Nervous System of the Crayfish 627

Influence of Acids and Alkalies on Crayfishes 628

Head of the Lobster ., .. 629

Shoiiened Development in Palxmon potiuna 630

Toilet-appendages of the Crustacea .. .. ,. 631

Anal Btspiration of the Copepoda 632

Parasitic Corycxidse 633

Parasite of the American Blue Pike 633

New Crustacea 634

Genital Glands and Segmental Organs of the Polychseta 635

Development of the Spermatozoa of the Earthworm 63G

Embryology of Ligula 637

Nervous System of the Trematoda 638

New Turbellarian , .. .. 640

New Nemerteans 640

New Genus of Echinoidea 641

Fossil Tertiary Echini 641

Mediterranean Echinoderms J 642

Remarkable Ophiurid 642

Intracellular Digestion in Ccelenterata 642

Nervous System of Beroe . . . . 643

Pleurobrachia pileus 644

Anatomy and Histology of tJie Actiniae 645

Structure of some Coralliaria . . . . 648

Antipatharia of the ' Blake ' Expedition 649

American Siphonophora ., 649

Origin and Development of the Ovum in Eucope before Fecundation ., ., 650

Proportion of Water in the Medusm 652

A Fresh-water Hydroid Medusa 652

Physiology of the Fresh-water Medusa 657

Sponges of the Leyden Museum 661

Structure and Affinities of the Genus Protospong id, Salter 661

Batschli's Protozoa 662

Amaebiform and other new Foraminifera ., 662

Vumpyrella lateritia ., .. .. 664

Acinetx l°65

Botany.

Disengagement of Carbonic / 'I from Boots 665

Sensitiveness in the Acacia 665

Copper in Plants 666

Action of Ozone on the Colouring-matters of Plants 667

Bed Cohuring-matter of the Leaves of tlie Virginian Creeper 667

Chemical Composition of Aleurone-grains 667

*' Ci stoma" 668

Apical Growth with several Apical Cells 668

Structure of the Fructification of Pilularia * . . . . 6(59

British Moss-Flora 670

( 4 )

Kkoobd op Current Eeskabohbs, &c. continued.

PAGE

British Characese 670

Formation of Fat in Fungi 671

Secretion from a Fungus 671

Anthracnose of the Vine 671

Urocystis Cepulx 672

Sterigmatocystis and Nematogonum 672

Mycotheca Marchica 672

Ceriomycesterrestris 673

Vine-pock , 673

Prehistoric Polyporus 673

Relationship of Ozonium to Coprinus , . . . 673-

Dieease of the Apple-tree caused by Alcoholic Fermentation 674

Saecharomyces apiculutus 674

Plasmodia of Myxomycetes 674

Epiphora 675

Lichens of Mont-Dare and Haute-Vienne .. .. 675

Morphology of Floridex 676

Bilateralness in Floridem 677

Fructification of Chsetopteris plumosa 678

Fructification of Squamariem . . 678

Fresh-water Algse of Nova Zemhla 679

Thermal Anahmna 679

Polycystis xruginosa, a cause of the Bed Colour of Drinking-water .. .. 680

Bain of Blood 680

Endochrome of Diatomacese (Fig. 54:) 680

Belgian Diatomacese , . . . . 687

New Deposit of Diutomaceous Earth 688

Preservation of Solutions of Palmelline 688

Microscopy.

Localities for Fresh-water Microscopical Organisms 689

Collection of Living Foraminifera 690

Cleaning Foraminifera 692

Wax Cells 692

Carbolic Acid for Mounting 693

Double-staining of Vegetable Tissues . . . . 693

Wickersheimer's Preservative Fluid and Vegetable Objects 696

Hardening Canada Balsam in Microscopic Preparations by Hot Steam . . 696

Ringing and Finishing Slides 696

Cleaning Cover-glasses 698

Preparing Sections of Coal 698

Cutting Bock Sections 699

Simple Mechanical Finger . . ., 700

Slides from the Naples Zoological Station 700

Homogeneous-Immersion Lenses 701

Fluid for Homogeneous Immersion .. .. 701

Errors of Refraction in the Eyes of Mieroscopists 701

Micrometre or Micromillimetre . . 702

Micrometry and Collar-adjustment .. . . 702

Zeiss' s Microspectroscope (Fig. 55) 703

Boss's Improved Microscope (Pl&te X.YI.) 704

Professor Huxley's Dissecting Microscope (Fig. 56) 705

Nachet's Chemical Microscope (Fig. 57) 707

Tiffany's Prepuce Microscope 709

Tolles-Blackham Microscope-stand .. .. ,, .. , 709

Weber-LieVs Ear Microscope (Fig. 58) 710

Trichina-Microscopes Hager's, Schmidt and Haensclis, Waechter's, and

Tescftwer's (Figs. 59-63) .. .. 711

Matthews' Improved Turntable (Figa. 64: and 65) 716

BiBIJOGRAPHY .4 .. .. .. .. .. .. .. 718

Peoobbdings of the Society .. .. .. .. .. .. 733

JO URN. R.MICR. SOC.VOL.III.pl. XIV,

(I

H.^Mmx7ar Co lith

AemetmaJriehopLrya episLylidis ^Podophrya quadripar^ita

JOURNAL

OF THE

ROYAL MICROSCOPICAL SOCIETY.

AUGUST, 1880.

TRANSACTIONS OF THE SOCIETY.

XVI. Notes on Acineiina: Trichojylirya episfyliclis, and Podo- jplirya qiuidripartita. By John Badcock, F.R.M.S.

(Bead 10th March, 1880.) Plate XIV.

Eakly in November 1879 I found on some filamentous Algae in one of the ponds in Victoria Park, a curious amoeboid form of what seemed to be an Acineton, and which I subsequently found had been originally discovered by MM. Claparede and Lachmann, and named Trichophrtja epistylidis. They found it parasitic on the Epistylis, and being struck with its singular character, con- sidered it entitled to rank as a new genus under the above name. They give a somewhat brief account of it. It is, however, very singular that those authors should have contrasted this form with Podophrya quadripartita (originally discovered by Baker, and subsequently found by Stein). Stein had argued in favour of the theory of the ^cme^a*-state in the life-history of many of the Infusoria, and among others had described P. quadripartita as the Acineta of Epnstylis plieatilis, because they were generally found together, and Claparede and Lachmann say that for the same reason T. epistylidis might be inferred to be similarly related to the Epistylis, for " The one, hke the other, seems in eflfect to lead the life of a parasite, almost exclusively on the branches of EpistyUs."

It would be a curious commentary on the disputes of those high authorities on these matters if it could be shown that Ti'i- chophrya epistylidis and Podo'phrya quadripartiia are one and the same species in different stages, and that EpistyUs has nothing to do with either. Such I believe to bo the case, as the following observations will show, if not conclusively, yet as probable in the highest degree.

I do not think that the identity of the organism which I found

* This theory has since been abandoQcd by Stein. VOL. III. 2 r

562 Transactions of the Society.

(PI. XIV., Fig. 1) with those of Claparede and Lachmann will be disputed, as both the figures and descriptions prove it, with one or two exceptions which are not essential. Thus, as to parasitism, I did not find mine on the Ejpistylis, but on filamentous algae : neither have I seen the faint outline of any embryo as described by them.

Having placed my first find in a small zoophyte trough, for the purpose of daily watching it, I soon noticed that the sides of the glass were covered with very much smaller bodies than those on the algse, and, though having the same Acineta-like character, were much more varied in form as well as being very transparent (see Figs. 2, 2a, and 3). These were very interesting objects of observation, as one could plainly see the contractile vesicles, the suctorial character of the tentacles, and their slowly spiral move- ment of protrusion and retraction. They were not of slow growth, but came suddenly as though a vesicle or similar body had been ruptured and its contents shot forth, which coming in contact with the glass would produce just the appearance noted. The contractile vesicles were similarly irregular, both as to position and number. In fact, it was impossible to find any two bodies alike in shape or organic differentiation. Only one common character pervaded them, they were all bright, shining patches, semi-fluid, transparent, and acinetiform.

As the winter advanced the pseudopodia or tentacles disap- peared, and also the contractile vesicles and other signs of active life, leaving only small lumps and patches of what may be called protoplasm. These had nothing of the appearance which death produces. They were simply bits of quiescent matter, looking more like shining crystals than anything else.

I had not expected to be able to make any further observations until another season, when the following incident attracted my attention. I had given some of the algae to my friend Mr. Cocks, with the animal forms on it in abundance, which he placed in his aquarium. This he has recently found to be covered with the very beautiful forms represented in Figs. 4 and 5, or in other words by Podophrya quadrijKcrtita. On seeing these at first, and taking note of similarity in some points notwithstanding difierences in others, my suspicions as to tlieir being the same were mate- rially strengthened, if not confirmed, by comparison with one form which I had drawn last November (Fig. 6). This was found with the others, but not presenting the same special appearance, I had not considered it in its true character ; and my view now is, that as in all forms of life some few more vigorous, or favoured by other circumstances, will remain after the majority have passed away, so these solitary individuals remained. There can be no doubt, I think, of the identity with Figs. 4 and 5.

Notes on Acinetina. By John Badcock. 563

This being so, Nos. 1, la, 2, and 3, are the immature stages in the life-history of the perfect form now recognized as Podoplirya quadripartita ; and consequently the new genus Tricliophrya of Claparede and Lachmann must be abandoned.

One of the forms here figured illustrates the so-called Acineta of Epistylis. Fig. 7 is the Ejnstylis with the Acineta here and there upon its branches, and on first observing it under the Microscope with Mr. Cocks we were inclined to think it a con- firmation of Stein's theory, when my son, whom we had asked to sketch it, remarked that it was not a portion of the Epistylis, but only attached to it. It was somewhat difficult to see the attachment, however, but we were confirmed as to its nature by subsequently seeing it on Carchesium and Ophrydiu7n, as well as by its abnormal position on the sides of the branches of Ejnstylis.

Since writing the foregoing I have been able to make some further observations of an interesting nature, which I will briefly state.

I have traced the life-history of one form with tolerable clear- ness. I had often noticed several small round ciliated bodies moving about the field of view, sometimes rapidly spinning round, and then springing with a jerking bound from place to place. On pursuing one of these bodies it was found finally to settle down on a filament of the alga, and gradually to develop a peduncle; then the ciliate character simultaneously changed to that of the Acineta, and finally it gradually branched out to the three- or four-cornered perfect form of Podojjhrya qiiadripartita*

These ciliated forms correspond to the description usually given to Megatricha imrtita, and in their further development attached and with a pedicle to Podophrya fixa. Further I have obseiTed that in the Megafrieha-stsLte they multiply by self- division. May we hazard the inference, in view of these observa- tions, that as not only these, but many other similar forms of life, pass through several life-cycles, in each of which they " increase and multiply," this peculiarity has been the fruitful cause of num- berless new genera and species having been too hastily adopted ?

* This I have seen in many instances since, and found tlicni to develop on the glass as well as on the weed.

L' P li

564 Transactions of the Society.

XVII. On the Visibilitij of Minute Objects mounted in Phos- phorus, Solution of Sulj>hur, Bisulphide of Carhon, and other Media. By J. W. Stephenson, Treasurer E.M.S., F.K.A.S,

CSead 9th June, 1880.)

The theory that there is a " loss of aperture on balsam-mounted objects " was enunciated more than twenty years ago by more than one writer, and although never accepted without question, it has been maintained with more or less frequency until a comparatively recent date, when Professor Abbe's demonstration of the theory of microscopic vision rendered it absolutely untenable.

It is not only untrue that there is a loss of aperture under such circumstances, but it is positively the reverse of the truth in every case in which it produces any effect whatever.

It has already been pointed out in the Society's Journal,* how this mistaken notion probably arose, viz. by failing to distin- guish between a diminution of angle (which of course takes place in the case of balsam-mounted objects) and a diminution of aperture, two entirely different matters, as a small angle in one medium (as oil) may be capable of embracing more diffraction spectra than a large angle in another medium (as air), the small angle having in fact the larger aperture and vice versa.

The loss of aperture by transmitted light is therefore on objects mounted in air, and this can only be prevented by mounting in balsam, or some other medium which has a refractive index equal to, or greater than, the numerical aperture of the immersion objec- tive employed.

This loss from " dry mounting," as it is called, arises in all objectives which have an equivalent angle exceeding 180°, which is the case with so many of the modern immersion objectives, and notably so in those on the homogeneous principle.

It is this fact which has induced me to bring the subject of mounting in different media before the Society this evening, as it is obviously of little use to obtain objectives of the large apertures with which we are now familiar, if by employing them on objects surrounded by air we reduce their effectiveness to the common level of 180" (= 1 n. a.).

I have said " surrounded by air " because when an object is in physical contact with the cover, the loss is, by its adhesion on one side, reduced to one-half, just as in an object mounted in balsam the whole aperture is preserved by the contact of both its sides with the medium in which it is mounted.

But in mounting diatoms (and some other objects) in Canada balsam, we find that although we have secured the full aperture of

* See this Journal, ii. (1879) p. 774.

On the Visibility, &g. By J. W. Stephenson. 565

the objective, and therefore its fall resolving power, we have done so at the expense of the visibility of the resultant image, which has become fainter by the nearer approximation to equality of the refractive indices of the diatomaceous silex and the Canada balsam in which the object is mounted ; the markings, whatever they may be, are less pronounced than they would have been in air had the structure been sufficiently coarse for resolution in that medium, a result which Professor Abbe has shown to be attributable to the paler diffraction spectra yielded by the balsam-mounted object hence we see that it may be possible to resolve an object in balsam which would be impossible in air, but that if resolvable hi hoih it would be more visible in air than in balsam.

It may be demonstrated that the visibiHty of very minute structures is ijroportional to the difference between the refractive indices of the object and the medium in which it is mounted {n-ni).

It follows from this that when this diiference = 0, or is very small, the structure is invisible. This is the case, as most of us know, when diatoms are immersed in strong suljihuric acid, and it may therefore be inferred, as was pointed out some years ago, that the refractive index of diatomaceous silex is about 1 43, which, without any pretence that it is exact, I shall assume as its true value in the following observations.

As the visibility of minute structures is proportional to the difference between the refractive indices of object and medium, it is necessary to give a short table of the refractive indices of those substances to which I shall refer, and fi'om which the differences of the indices are to be deduced.

Table of Indices.

Air =1

Water = 1-33

Diatomaceous silex and sulphuric acid = 1'43

Canada balsitni = 1'54

Bisiilpiiide of carbon = l'G8

Solution of sulphur in bisulphide of carbon (approximately) . . = 1-75

Sulphur = 2-11

Solution of phosphorus in bisulphide of carbon (approximately) =2 10

The first case we will consider is that of the visibihty of a diatom in air, which, although it is otherwise excluded from consideration in consequence of the lo.-^s of aperture involved, is nevertheless valuable as a standard of comparison.

The index of diatomaceous silex being taken as 1 43, and that of air being 1, we have as a measure of the visibility of a fine diatom in air the number "43.

Taking now the various media in succession, and connucncing with water, of which the index is I '33, the index of diatomucious

566 Transactions of the Society.

silex being, as before, 1 * 43, the difference, being tbe measure of visibility of a diatom in ivater, is represented by 'lO.

The next in order is Canada balsam, with its index of 1 ' 54 ; deducting the index of silex, 1 '43, we obtain the difference of •!!, which is the measure of visibility of the same object in balsam, and almost identical with that of water.

The next in succession is bisulphide of carbon,* index 1*68, diatomaceous silex 1 43, giving as the measure of visibility in hisuljihide of carbon ' 25, which it will be observed is about two and a half times as great as that obtainable in water or balsam.

This result may however be exceeded by dissolving sulphur in the bisulphide of carbon, although to what extent I am unable at this moment to say, but as sulphur has an index of 2* 115, and is moderately soluble, I think I am safe in assuming that the index of the solution is 1 75 ; deducting from this 1 43, we obtain 32 as the measure of visibility in solution of sul^jhur, which is nearly three times as great as that of balsam.

The last in the list is phosphorus, but as this, from its crystal- line character, cannot be conveniently used in its solid form, it is also dissolved in bisulphide of carbon, the solution being just short of that point at which crystals appear.

From the extreme inflammability of phosphorus and other diffi- culties it is very improbable that it will ever be used to any great extent, although there is to my mind great scientific interest in the experiment.

If we take the solution of phosphorus as having an index of 2*1, and deduct that of the silex, 1*43, we obtain '67 as the measure of the visibility of fine diatom markings in solution of 2)hospJiorus, which is six times as great as that of the same object in balsam, and no less than 50 per cent, higher than its visibility in air itself whilst the greater brightness of the diffraction spectra will make the more refrangible rays effective, and thus give a greater power of visual (as distinguished from photograjyhie) resolution.

Summarized we get the following results :

Table showing the Visibility of Fine Diatoms when Mounted in the FOLLOWING Media, securing the full Aperture of Objective.

Water 10

Canada balsam 11

Bisulphide of carbon 25

Solution of sulphur in bisulphide of carbon . . 32 Solution of phosphorus in bisulphide of carbon .. 67

The practical result of the investigation appears to be that it is essential, if the whole aperture of an objective is to be utilized, to mount minute structures in some medium other than air.

* Oil of cassia gives almost exactly the same result.

On the Visibility, &e. By J. W. Ste])henson. 567

That although the full aperture and resolving power are secured by mounting in balsam, it gives nevertheless nearly the faintest image of all.

That a solution of phosphorus is, as far as visibility is con- cerned, by far the most effective, but the difficulties attending its use must render it unpopular.

The next best is a solution of sulphur in bisulphide of carbon (although pure bisulphide is very good), and with these there is no technical difficulty whatever.

A ring of the aqueous solution used by Mr. Browning in making his bisulphide prisms being formed on the slip, and a drop of the sulphur solution or pure bisulphide being placed in its centre, nothing is necessary but to place over it the thin cover with its adhering diatoms, press it down on the still moist ring, running round it a somewhat copious margin of the cement, and the thing is done.

In a short time the glutinous cement sets and finally becomes dry, when, in order to protect it from the water of the ordinary immersion lenses, it is desirable to give it a coat of gold size, or shellac varnish, although for mere keeping purposes this is un- necessary.

The same course may be adopted in mounting in phosphorus, except that the solution must be run in from the edge of the thin cover to avoid the phosphoric acid which rapidly forms on its surface, and destroys the effect wherever it comes in contact with the object. I have found varnish made of the best red sealing- wax (which is better than pure shellac) as useful as Browning's aqueous cement above referred to, but as it is brittle when dry it should also be protected by a coating of gold size.

There are now on the table objects mounted in phosphorus and bisulphide of carbon, which I exhibited in 1873,* and they still remain unchanged notwithstanding the volatile nature of the materials. On that occasion I fell into the error of saying that there was a loss of aperture (instead of angle) with dry objectives on objects mounted in phosphorus and bisulphide of carbon, when in fact the aperture remained unchanged.

* See 'Mon. Micr. Journ.,' x. (1873) p. 1.

568 Transactions of the Societtj.

XVIII. On the Development and Betrogression of Blood-vessels.

By George Hoggan, M.B., and Frances Elizabeth Hoggan,

M.D.

I^Rectd Uth April, 1880.) Plate XV.

At the present day it is not necessary to hold pessimist ideas in histology in order to admit that our knowledge of the manner in which blood-vessels are formed is still unsatisfactory; and. although for the last thirty years the most eminent histologists have sought to elucidate the question, it may fairly be said that the very latest opinions enunciated, differing as they do from all previous ones, are in no way more satisfactory. Although many of these opinions appear diametrically opposed to each other, they are principally so, it seems to us, in being too exclusive in their application; and with the view of reconciling them, we desire to put on record a series of clearly ascertained facts or appearances which certain new histological processes devised by ourselves have enabled us to obtain. In our opinion, the general disagreement among histologists upon this question is caused, in the first place, by the unsuitability of the tissues in which it has been studied, and in the second place, by the mode of preparation employed. Paradoxical though it may appear, we have learnt from experience that the worst place in which to study the development of any special tissue is the em- bryo itself. There the embryonic cells are so little differentiated from each other in shape, the intercellular substance or matrix is so extremely scanty, while the process of developmental growth is so rapid, that it is almost impossible to obtain a clear demonstra- tion. The membranous expansion of the tail of a living tadpole, which has been so often employed for this kind of research, and from which so diametrically opposed views have been deduced, is espe- cially unsatisfactory, because in the living cell no nucleus is visible, and the polar star of the histological explorer being invisible, all true ideas of direction and course of development are naturally enough shrouded in obscurity. For our part, we have found nothing so suitable as the growing broad ligament of pregnant rats and mice, more especially during a first pregnancy, for there we have a fringe of developing capillaries lying in a thin, rapidly distending membrane, in which the gelatinous matrix is so plentiful and clear that every vessel-forming cell stands out in distinct relief. In that membrane, moreover, the silvei? method of fixing and marking can be applied most favourably, in order to show the junctions of the cells forming, or about to form, the blood-vessels, in the position and shape they possessed when alive.

JOURK.R.MICR. SOC.VOL m.PL.Xy.

iVtst Mtvrrnar &• Cv Uth

Develoxinient &Ket.T-»car»essi on oP Blood- vessels.

Development, &c., of Blood-vessels. By G. and F. E. Uoggan. 569

The animal (by preference a house mouse) ought to be only moderately well nourished, as both extremes of nutrition defeat our object, either by obscuring the developing vessels by fat-cells, or preventing the vessels from being formed. It ought first to be gently anaesthetized by chloroform under a jar, and as soon as it is insensible, it ought to be drenched with the anaesthetic, and then left to die. We never lose time by injecting the animal and after- wards allowing it to cool, as by that process not only do the cells alter in shape, but the injection interposes an annoying obstacle to vision when it has filled the vessels.

As soon as it is dead, we open up the abdomen along the linea alba, so as to completely expose the gravid uterus, and then seizing the uterus of one side with fine pointed forceps, we raise it out of the body cavity, so as gently to distend the membrane or broad ligament which attaches it to the abdominal wall. On one side of this membrane we place the smaller of a pair of the histological rings invented by us, and already described in this Journal ; * and without allowing it to glide or rub over the surface, we place the larger of the two rings upon the smaller. In this way a miniature tambomine is formed ; and after the two rings have been carefully jammed one on the other, by a slight circular movement, the excess of membrane can be snipped ofi" external to the rings, and a one- half per cent, solution of silver in distiUed water poured upon either or both surfaces, without preliminary washmg ; but after a few minutes exposure to a dull light, the whole may be gently washed with ordinary water.

In our piece of membrane not only are the cells fixed in their living shape, but, as the blood-vessels were full of blood when the one ring was jammed upon the other, the distending blood was thus retained within them, and the silver solution now fixes them in this condition, and makes also the outlines of the cells, which alone form them, distinctly visible. The membrane is now ready for staining, the best of all methods for this purpose, according to our experience, being the one invented and published by one of us. By this method the membranous portion of the tambourine is first soaked for a few minutes in methylated spirit, a teaspoonful in a watch-glass or small saucer being suflicient. This is then pourtxl away, and in its stead a few drops of a 2 per cent, solution of per- chloride of iron in spirit is filtered upon the membrane. After a few minutes a 2 per cent." sohition of pyroi^'allic acid in spirit is next filtered upon it, and allowed to remain tlu're from a few seconds to a few minutes, according to the depth of tint reipiireil, and then the whole is well washed with ordinary water, and tho staining process is complete. A few drops of glycerine may then be phicecl u[)on tho membrane to clarify it, and the preparation Sec vol. ii. (I87'.i) !». W.u.

570 Transactions of the Societtj.

may be studied at once under the Microscope, or mounted on a slide as a permanent preparation.

It may with equal facility be rendered transparent by alcohol and an essential oil, and mounted in balsam or copal varnish, but it then possesses all the disadvantages of a balsam preparation. Under all circumstances, the membrane must be clarified before it is excised from the rings, to prevent unequal contraction. It is easily excised by running the edge of a knife round the outer rim of the inner ring, and having prepared a slide previously with a drop of glycerine upon it, the disk of membrane remains in place when applied to it ; the glass cover may then be put on and sealed, as we do it, by hot sealing-wax dropped round the edges, and trimmed with a hot wire while the whole is compressed by a paper- clip.

We have thus a preparation mounted in glycerine, in which no undue distension has taken place, to whose surface no injury has been done during the whole course of preparation, and whose progress at every stage could be examined under the Microscope without damaging it. Moreover, when mounted in glycerine the blood leaves the vessels when the disk is excised, and is washed away at the edges with the excess of glycerine, so that all the vessels appear as rigid, hollow tubes, the thickness of whose walls and the joints and nuclei of the cells composing them, can be equally well seen by the silver and pyrogallate of iron processes we have used.

As an admirable little review of the opinions already arrived at by difterent observers on the question of the development of blood- vessels, has lately been given by Dr. George Thin in ' The Quarterly Journal of Microscopical Science ' for July, 1876, we think it inadvisable to lengthen out this paper by any recapitula- tion of them. With regard to even the latest views. Dr. Thin states : " The conclusion to which I have therefore come is, that the cellules vasoformatives of Eanvier are spaces in the omentum, to which, I submit, the term ' cell ' is not applicable. The develop- ment of blood-vessels takes place by an escape, first of fluid, and finally of the formed elements of the blood from the vascular system into these spaces. The establishment of the blood current is speedily followed by the formation of a membranous wall around the current, which is impermeable for an injection mass or the blood, and the process is complete."

We are careful to give Dr. Thin's views in his own words, as they are the latest, to our knowledge, which have appeared in English. They are opposed to the views of all previous observers, and they are equally opposed to all the facts we have ascertained and are about to state in this paper. Indeed we fail to understand how, if he has used the silver process, he has overlooked the fact

Development, dte., of Blood-vessels. By G. and F, E. Hoggan. 571

that portions of capillaries show the junction markings of the hollow cells composing them, before ever they have become con- nected with the circulation.

We have found that a new development of blood-vessels takes place solely by the aid and addition of the wandering cells.* In the membranous sheet under consideration, the only cells present, apart from the layers of endothelium covering the two surfaces of the rapidly growing tissue, are the wandering cells. They may be seen here in at least three conditions. They may be found wandering purposeless over the free surface of either layer of endo- thelium, or through the soft gelatinous matrix forming the mem- brane between these layers. If the tissue has been properly prepared, they are generally found branched in the latter locality, although on the free surfaces they have retracted into a globular or circular form, being surrounded by no matrix to retain them in the branched condition when the silver is applied to fix them. If the animal has been injected and left to cool before it is opened, and the silver solution be then applied, they will probably appear round in shape within the matrix, and very plentiful on the free surfaces in the same form ; or they may be found developing into fat-cells in the neighbourhood of the blood-vessels, in which condition they may either appear round or with matiij branches, according to the condi- tions of preparation already referred to. They may have more than one nucleus in the purely wandering condition, but they have not more than one nucleus as a rule when developing into a fat-cell. Again, they may be found placing or having placed themselves in position to form or to strengthen a blood-vessel in course of deve- lopment. The methodical manner in which this is effected would almost argue an instinct or intelligence worthy of higher animals ; and although the directions the cells move in when forming the new vessel may be manifold, they seem to follow a regular course throughout. They may either plant themselves at a point in a blood-vessel where a connection is to be formed, and prolong their protoplasmic cell substance to join hands with another cell liuk in the chain of capillary development, as at a, Fig. 9 (Plate XV.), and e, Fig. 8, or, as is more common, they may appear external to the future point of junction, and, stretching towards it their proto- plasmic arm, thus complete the connection. This peripheral position may be either in direct linear continuation of a new vessel, as at d,

Wc tliink it unnecessary that we should aguin enter at any lengtli into the rcasims we have jdready piven in our furnior nrtiolu on tlic Fut-cell, for rejecting tlic liypotliesis that tlio fixctl cc-\U of the connective tinsue have any Bliare in tlio fornialion of blood-vo8.s. Is, fat-cclLs, &c. We cannot admit tiiat any fixed cell of any tis.sue can normally devcloj) directly into the fixed cell of any other tissue. A "ciinecr cell may indeed impress its charact<r u|ion any lixed or ond)ryonic cells near it, so that these also Income cancer cells; hut, normally, fixed cells can only arise from or return to emhryouic cells.

572 Transactions of the Societtj.

Figs. 4, 5, and 6, or at right angles to it, as at a, Figs. 1 and 2, and i, Fig. 12. Strange to say, in tlie latter condition the already- existing blood-vessel or capillary seems always ready to meet such an advance half-way, and will either bend its whole tube, in the case of a capillary, or dimple its cellular wall, in the case of a larger blood- vessel, towards the vessel-forming cell, as seen in the examj)les last named.

It is also worthy of notice that, when we examine the membrane in the vicinity of such a cell, we find that no other cell is as yet in position to continue the process of development ; that, in short, the solitary vessel-forming cell has specially come to place itself in the most favourable position to enter into the continuation of the vessel peripherally, and acts there until another cell may come and place itself beyond it to continue the process. But the most wonderful instinct of all is seen when a large capillary loop is about to be formed, when several cells are seen placing themselves at considerable distances from each other in the precise line which the future vessel is to occupy. This is well seen in Fig. 12, A, which shows under a power of 100 diameters a plan of such a loop about to be formed between a and h, the nodal points in already formed capillaries, where attachments to the circulation are to be formed. In this loop or chain, independently of the cells at a, h, and c already attached to the capillaries (all the component cells of this chain are drawn separately in the same figure at a much higher power) we have four links formed, three of them consisting as yet of only single cells e, f, and h, and one link g, consisting of three cells, two of which are already vacuolating or hollowing out to form a tube before any connection is made with the comparatively distant blood-vessels. Indeed this, the most advanced link of the chain, is almost equidistant from the nodal points of junction at a and h. It will also be observed that while gig', the cells which specially hollow out to form the tube, join by overlapping their ends, or, in other words, by forming a splice, the third cell g" places itself upon the splice or junction of the cells, and therefore at the weakest point, by way of strengthening the whole. This splicing of cells and application at the point of junction of strengthening cells we have found invariably throughout, as will be seen also in all the other figures. Another point of interest at this spot is the position or presence of a fibre or fibres which seem to connect the cells together and with the nodal points, or, in other